Investigating spectrum of biological activity of 4- and 5-Chloro-2-hydroxy-N-[2-(arylamino)-1-alkyl-2-oxoethyl]benzamides

Article abstract of DOI:10.3390/molecules16032414

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

Full text of DOI:10.3390/molecules16032414

Molecules 2011, 16, 2414-2430; doi:10.3390/molecules16032414
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Investigating Spectrum of Biological Activity of 4- and
5-Chloro-2-hydroxy-N-[2-(arylamino)-1-alkyl-2-
oxoethyl]benzamides
Ales Imramovsky 1,*, Matus Pesko 2, Katarina Kralova 2, Marcela Vejsova 3, Jirina
Stolarikova 4, Jarmila Vinsova 5 and Josef Jampilek 6,*
1
Institute of Organic Chemistry and Technology, Faculty of Chemical Technology, University of
Pardubice, Studentska 573, 532 10 Pardubice, Czech Republic
2
Institute of Chemistry, Faculty of Natural Sciences, Comenius University, Mlynska dolina Ch-2,
842 15 Bratislava, Slovakia
3
Department of Clinical Microbiology, Charles University Medical School and Teaching Hospital,
Sokolska 581, Hradec Kralove 500 05, Czech Republic
4
Laboratory for Mycobacterial Diagnostics and TB, Institute of Public Health in Ostrava,
Partyzanske namesti 7, 702 00 Ostrava, Czech Republic
5
Department of Inorganic and Organic Chemistry, Faculty of Pharmacy in Hradec Kralove, Charles
University in Prague, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic
6
Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical
Sciences, Palackeho 1/3, 612 42 Brno, Czech Republic
* Authors to whom correspondence should be addressed; E-Mail: ales.imramovsky@upce.cz (A.I.);
Tel.: +420466037739 (A.I.); Fax: +420466038004 (A.I.); E-Mail: josef.jampilek@gmail.com (J.J.);
Tel.: +420724139484 (J.J.).
Received: 11 January 2011; in revised form: 7 March 2011 / Accepted: 11 March 2011 /
Published: 14 March 2011
Abstract: In this study, a series of twenty-two 5-chloro-2-hydroxy-N-[2-(arylamino)-1-
alkyl-2-oxoethyl]benzamides and ten 4-chloro-2-hydroxy-N-[2-(arylamino)-1-alkyl-2-
oxoethyl]benzamides is described. The compounds were analyzed using RP-HPLC to
determine lipophilicity. Primary in vitro screening of the synthesized compounds was
performed against mycobacterial, bacterial and fungal strains. They were also evaluated for
their activity related to the inhibition of photosynthetic electron transport (PET) in spinach
(Spinacia oleracea L.) chloroplasts. The compounds showed biological activity
comparable with or higher than the standards isoniazid, fluconazole, penicillin G or

Molecules 2011, 16
ciprofloxacin. For all the compounds, the relationships between the lipophilicity and the
2415
chemical structure of the studied compounds as well as their structure-activity relationships
are discussed.
Keywords: salicylanilide derivates; lipophilicity; in vitro antimycobacterial activity; in
vitro antifungal activity; in vitro antibacterial activity; PET inhibition; spinach
chloroplasts; structure–activity relationships
1. Introduction
Salicylanilides (2-hydroxy-N-phenylbenzamides) are an important class of aromatic compounds
with a wide range of pharmacological activities. A number of them show antibacterial [1-3],
antimycobacterial [4-6] and antifungal [4,7] as well as anti-inflammatory [8] or antineoplastic
activities [9-11]. In spite of their promise as potential drugs, certain physico-chemical properties of
salicylanilides, for example low solubility, prevented their widespread use in clinical practice. Thus,
improvement of the physical-chemical properties of salicylanilides is an interesting and vital area of
research.
We have focused our efforts on design and synthesis of original O-substituted derivates of
salicylanilides. Promising results of antimycobacterial evaluation of some salicylanilides acetates [4]
inspired us to design more sophisticated derivates of salicylanilides – amino acid esters. The design
strategy of these potential antimicrobial active agents was mainly oriented towards the synthesis of a
prodrug form of most antitubercular active salicylanilides by attaching an amino acid. The phenolic
hydroxyl group of the most antitubercular active salicylanilides was esterified by several N-benzyloxy-
carbonyl α-amino acids. More lipophilic amino acids such as glycine, (R and S)-alanine,
(R and S)-valine and (R and S)-phenylalanine were chosen. The prepared N-protected esters were
tested for their antifungal and antimycobacterial activity; promising results were published recently
[5-7]. Subsequent processing using standard reactions yielded unexpected products (4-chloro- and 5-
chloro-N-salicylamides for detailed structure see Scheme 1) that were described, including proposed
reaction mechanism, and published [12-14].
It was expected that chloro-2-hydroxy-N-(arylalkyl)benzamides, compounds with a special skeleton
originating from biologically active salicylanilides, would have interesting biological properties. A
series of these compounds was prepared, and most of them were tested for their antimycobacterial,
antifungal and antibacterial activity. As the presence of an amide (-NHCO-) group in acylanilides,
phenylcarbamates, ureas, etc., is characteristic of a number of herbicides acting as photosynthesis
inhibitors [15-25], the compounds were also evaluated for their photosynthesis-inhibiting activity (the
inhibition of photosynthetic electron transport) in spinach chloroplasts (Spinacia oleracea L.). These
compounds bind reversibly to photosystem II (PS II), a membrane-protein complex in the thylakoid
membranes, which catalyses oxidation of water and reduction of plastoquinone [15], and thereby
inhibit photosynthesis [26-30]. For example, in the presence of R' substituted salicylanilides the
decreased intensity of the fluorescence emission band at 686 nm (belonging to the chlorophyll-protein
complexes mainly in PS II [31]) suggested PS II as the site of action of the studied inhibitors [24].

Molecules 2011, 16
2416
Many low molecular weight drugs cross biological membranes through passive transport, which
strongly depends on their lipophilicity, therefore this physico-chemical parameter of the prepared
compounds was determined by means of the RP-HPLC. Relationships between the structure and in
vitro antimicrobial activities or/and inhibitory activity related to inhibition of photosynthetic electron
transport (PET) in spinach chloroplasts of the new compounds are discussed.
2. Results and Discussion
2.1. Chemistry
Preparation of substituted 5-chloro-2-hydroxy-N-[2-(arylamino)-1-alkyl-2-oxoethyl]benzamides
8a-v and 4-chloro-2-hydroxy-N-[2-(arylamino)-1-alkyl-2-oxoethyl]benzamides 9a-j was carried out by
two-step synthesis with a subsequent rearrangement (Scheme 1). The first step was
dicyclocarbodiimide (DCC) mediated esterification of N-protected amino acids 3 (benzyloxycarbonyl
group as protection) with selected antimicrobial active salicylanilides 1 and 2 in dry
dimethylformamide (DMF). Esters 4 and 5 were obtained optically pure in high yields [5,7].
Scheme 1. Synthetic pathway for preparation of target substituted 5-chloro-2-hydroxy-N-
[2-(arylamino)-1-alkyl-2-oxoethyl]benzamides 8a-8v and 4-chloro-2-hydroxy-N-[2-(aryl-
amino)-1-alkyl-2-oxoethyl]benzamides 9a-9j.
O
R
Cl
N
O
O
O
O
H
R
R
Cl
OH
1
N
N
HOOC
R'
H
H
a
and/
and/or
+
NH
CBZ
Cl
or
H
H
O
N
N
R
O
CBZ
O
CBZ
3
N
R'
R'
H
4
5
O
Cl
Cl
OH
2
CBZ =
O
b
O
R'
H
N
N
R
R
H
O
O
O
O
O
R
R
OH
Cl
N
N
8
H
H
c
and/
or
and/or
R'
Cl
"rearrangement"
O
H
N
NH2*HBr
NH2*HBr
O
O
N
H
R'
R'
O
6
7
Cl
OH
9
Reagents and conditions a) -15 °C, DCC, DMF; b) i) 33% HBr/AcOH, ii) Et2O; c) Et3N
(rearrangement), anhydrous CHCl3.
Acidolysis (33% HBr in acetic acid) was used as a method for N-deprotection and this method gave
appropriate hydrobromide salts 6 and 7 in almost quantitative yields. Subsequent amino group
liberation by triethylamine in anhydrous chloroform yielded unexpected rearranged products 8 and 9

Molecules 2011, 16
2417
[12,13]. This methodology is general and is not dependent on the substitution of the aromatic
salicylanilide ring as well as on the amino acid type. A small combinatorial library of discussed
compounds 8 and 9 was recently synthesized and published [22]. The compounds could be divided
into six groups based on their chemical structure: Group 1 includes 5-Cl/3-Cl substituted diamides 8a-
8g; Group 2 contains 5-Cl/4-Cl derivatives 8h-8l; Group 3 includes 5-Cl/3,4-Cl derivatives 8m-8r;
Group 4 includes 5-Cl/4-Br derivatives 8s-8v; Group 5 contains 4-Cl/4-Cl derivatives 9a-9f; and
Group 6 is composed of 4-Cl/4-X diamides 9g-9j.
2.2. Lipophilicity
Lipophilicity is a property that has a major effect on absorption, distribution, metabolism, excretion
and toxicity properties as well as pharmacological activity, because drugs cross biological membranes
through passive transport, which strongly depends on their lipophilicity. Lipophilicity has been studied
and applied as an important drug property for decades [32].
Hydrophobicities (log P/Clog P) of compounds 8 and 9 were calculated using two commercially
available programs (ChemOffice Ultra 10.0 and ACD/LogP) and measured by means of RP-HPLC
determination of capacity factors k with subsequent calculation of log k. The procedure was performed
under isocratic conditions with methanol as an organic modifier in the mobile phase using an end-
capped non-polar C18 stationary RP column. The results are shown in Table 1 and illustrated in Figure 1.
Both used programs did not resolve lipophilicity parameters within the series of enantiomers, as
expected. The program ChemOffice did not resolve various lipophilicity values of individual
positional isomers, in particular, the same log P/Clog P data were calculated for compounds 8a-8g
from Group 1, 8h-8l from Group 2, 9h-9f from Group 5, 8s-8v from Group 4 and compound 4-Cl/4-Br
(9g) from Group 6.
The results obtained with all the compounds show that the experimentally-determined
lipophilicities (log k) of the discussed compounds are in accordance with the calculated values of
compounds 8 and 9 as shown in Figure 1. Compounds series 9 substituted by chlorine in C(4) position
showed higher lipophilicity than compounds series 8 with C(5) chlorine substitution, although program
ACD/LogP showed an opposite trend. According to the program ACD/LogP, compounds 5a-5g
substituted by chlorine in C'(3) position showed higher lipophilicity than compounds 5h-5l with C'(4)
chlorine substitution, but experimental log k for C'(4) chlorine substitution is higher. As expected, the
lipophilicity of the compounds substituted in the anilide part of the molecule increases as follows:
OCH3 < CH3 < Cl < Br < CF3 < di-Cl, although program ACD/LogP calculates higher log P for Br
derivative 9g than CF3 substituted compound 9i. The lipophilicity factor log k as well as calculated log
P (ACD/LogP) and log P/Clog P values (ChemOffice) increase, depending on amino acid type
(glycine, alanine, valine and phenylalanine), that is, the lipophilicity of the compounds substituted in
R3 increases as follows: H < CH3 < CH(CH3)2 < CH2C6H5.
Log k data specify lipophilicity within this series of the discussed compounds, and all the chiral
compounds were measured several times with the same results. The determined differences in log k
parameters for individual R/S-enantiomers cannot be explained on the basis of the results
presented here.

Molecules 2011, 16
Table 1. Comparison of calculated lipophilicities (log P/Clog P) with determined log k
2418
values, Hammett's parameter (σ) and bulk parameter (MR, reflecting bulkiness).
3
O
R
H
N
N
1
2
1
R
R
H
O
2
OH
log P/Clog P
ChemOffice
log P
σR2
MRR3
[33]
0
Comp.
R1
R2
R3
log k
ACD/LogP
4.36 ± 0.47
4.71 ± 0.48
4.71 ± 0.48
5.59 ± 0.48
5.59 ± 0.48
6.64 ± 0.49
6.64 ± 0.49
4.64 ± 0.47
5.52 ± 0.48
5.52 ± 0.48
6.57 ± 0.49
6.57 ± 0.49
5.20 ± 0.49
5.55 ± 0.50
6.43 ± 0.50
6.43 ± 0.50
7.48 ± 0.51
7.48 ± 0.51
5.76 ± 0.54
5.76 ± 0.54
6.81 ± 0.55
6.81 ± 0.55
4.54 ± 0.47
4.54 ± 0.47
5.41 ± 0.48
5.41 ± 0.48
6.47 ± 0.49
6.47 ± 0.49
5.65 ± 0.54
5.46 ± 0.51
4.91 ± 0.46
4.46 ± 0.48
[33]
8a
8b
8c
8d
8e
8f
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
3-Cl
3-Cl
H
0.2439
0.4166
0.4153
0.6124
0.6114
0.7098
0.7066
0.4308
0.6371
0.6125
0.7394
0.7329
0.4971
0.6698
0.8623
0.8614
0.9485
0.9408
0.6709
0.6646
0.7779
0.7673
0.4934
0.4921
0.6839
0.6832
0.7540
0.7479
0.7353
0.7892
0.5902
0.3623
2.42 / 4.33156
2.91 / 4.64056
2.91 / 4.64056
3.80 / 5.56856
3.80 / 5.56856
4.58 / 6.05856
4.58 / 6.05856
2.91 / 4.64056
3.80 / 5.56856
3.80 / 5.56856
4.58 / 6.05856
4.58 / 6.05856
2.98 / 5.01472
3.47 / 5.32372
4.35 / 6.25172
4.35 / 6.25172
5.14 / 6.74172
5.14 / 6.74172
4.07 / 5.71856
4.07 / 5.71856
4.86 / 6.20856
4.86 / 6.20856
2.91 / 4.64056
2.91 / 4.64056
3.80 / 5.56856
3.80 / 5.56856
4.58 / 6.05856
4.58 / 6.05856
4.07 / 5.71856
4.16 / 5.93176
3.72 / 5.09696
3.11 / 4.67336
0.373
0.373
0.373
0.373
0.373
0.373
0.373
0.227
0.227
0.227
0.227
0.227
0.600
0.600
0.600
0.600
0.600
0.600
0.232
0.232
0.232
0.232
0.227
0.227
0.227
0.227
0.227
0.227
0.232
0.740
-0.170
-0.270
(S)-CH3
(R)-CH3
4.7
3-Cl
4.7
3-Cl
(S)-CH(CH3)2
(R)-CH(CH3)2
(S)-CH2C6H5
(R)-CH2C6H5
(R)-CH3
14.0
14.0
29.0
29.0
4.7
3-Cl
3-Cl
8g
8h
8i
3-Cl
4-Cl
4-Cl
(S)-CH(CH3)2
(R)-CH(CH3)2
(S)-CH2C6H5
(R)-CH2C6H5
H
14.0
14.0
29.0
29.0
0
8j
4-Cl
8k
8l
4-Cl
4-Cl
8m
8n
8o
8p
8q
8r
8s
8t
3,4-Cl
3,4-Cl
3,4-Cl
3,4-Cl
3,4-Cl
3,4-Cl
4-Br
(S)-CH3
4.7
(S)-CH(CH3)2
(R)-CH(CH3)2
(S)-CH2C6H5
(R)-CH2C6H5
(S)-CH(CH3)2
(R)-CH(CH3)2
(S)-CH2C6H5
(R)-CH2C6H5
(S)-CH3
14.0
14.0
29.0
29.0
14.0
14.0
29.0
29.0
4.7
4-Br
8u
8v
9a
9b
9c
9d
9e
9f
4-Br
4-Br
4-Cl
4-Cl
(R)-CH3
4.7
4-Cl
(S)-CH(CH3)2
(R)-CH(CH3)2
(S)-CH2C6H5
(R)-CH2C6H5
(R)-CH(CH3)2
(S)-CH(CH3)2
(S)-CH(CH3)2
(S)-CH(CH3)2
14.0
14.0
29.0
29.0
14.0
14.0
14.0
14.0
4-Cl
4-Cl
4-Cl
9g
9h
9i
4-Br
4-CF3
4-CH3
4-OCH3
9j

Molecules 2011, 16
Figure 1. Relationship between calculated log P data (ACD/LogP) and experimentally
2419
found log k values. If both enantiomers were prepared, only (S)-enantiomers are illustrated
due to similar log k values.
1.0
8q
8o
9h
0.8
8u
9g
8s
8k
8f
9e
9c
8n
8i
9i
0.6
0.4
0.2
8d
y = 0.1772x - 0.3625
R2 = 0.7810
9a
8m
8h
8b
9j
8a
4.5
4.0
5.0
5.5
6.0
6.5
7.0
7.5
8.0
log P [ACD/LogP]
2.3. Biological activities
The compounds showed a wide range of biological activities, and some noteworthy structure-
activity relationships were observed. All the results are shown in Table 2. Generally, all the discussed
compounds (especially compounds 8b, 8q, 8r) exhibited lower solubility in comparison with the initial
esters [5,7].
2.3.1. Antimycobacterial screening
The discussed amides were tested against three Mycobacteria strains. According to the results of in
vitro evaluation (see Table 2), all compounds demonstrated noteworthy effects. Activities of some
benzamides, especially against M. avium and M. kansasii, considerably exceeded the activity of
isoniazid (INH) used as an internal standard. The discussed compounds can be divided according to
the activities against individual Mycobacteria strains, and structure-activity relationships within
Groups 1, 2 and 4 can be discussed. Compound 8b showed the lowest activity due to precipitation in
the testing medium, contrary to its (R)-enantiomer 8c. Also compounds 8q and 8r precipitated from
testing medium, therefore 8b, 8q and 8r were excluded from the below SAR discussion.
It can be concluded that compound N-{(1R)-1-benzyl-2-[(4-chlorophenyl)amino]-2-oxoethyl}-5-
chloro-2-hydroxybenzamide (8l) and N-{(1R)-1-benzyl-2-[(4-chlorophenyl)amino]-2-oxoethyl}-4-
chloro-2-hydroxybenzamide (9f) showed the highest antitubercular/antimycobacterial activity, see Table 2.

Molecules 2011, 16
Table 2. Antimycobacterial activity (MIC/IC90) of compounds in comparison with
2420
isoniazid (INH) standard; in vitro antifungal and antibacterial activity (MIC/IC50 or IC90)
of compounds compared with fluconazole (FLU), penicillin G (PEN) and ciprofloxacin
(CPF) standards; and IC50 values of discussed compounds related to photosynthetic
electron transport (PET) inhibition in spinach chloroplasts in comparison with 3-(3,4-
dichlorophenyl)-1,1-dimethylurea
(DCMU)
standard.
(MIC-minimum
inhibitory
concentration, ND-not determined, MTB-Mycobacterium tuberculosis, MA-M. avium, MK-M.
kansasii, CT-Candida tropicalis, CK-C. krusei, TM-Trichophyton mentagrophytes, SA-S. aureus,
MRSA-methicilin resistant Staphylococcus aureus, SE-S. epidermidis, EF-Enterococcus sp.)
MIC [µmol/L]
PET
IC50
MTBa
14h
21h
MAa
14h
21h
MKa
14h
21h
CTb
24h
48h
CKb
24h
48h
TMc
72h
SAd
24h
48h
MRSAd
24h
48h
SEd
24h
48h
EFd
24h
48h
Comp.
[μmol/L]
378.6
41.7
ND
120h
125
125
250
>500
62.5
125
62.5
62.5
500
>500
125
250
250
250
500
>500
62.5
62.5
125
125
125
125
125
125
>250
>250
>250
>250
>500
>500
>125
>125
15.62
15.62
>500
>500
>500
>500
>500
>500
7.81
>250
>250
>250
>250
>500
>500
>125
>125
31.25
31.25
>500
>500
>500
>500
>500
>500
15.62
15.62
>250
>250
>250
>250
>500
>500
>125
>125
31.25
31.25
>500
>500
>500
>500
>500
>500
31.25
250
>250
>250
>250
>250
>500
>500
>125
>125
62.5
8a
>125
>125
31.25
125
>125
>125
31.25
62.5
125
125
8be
8c
8d
8e
8f
0.49
0.49
32
62.5
32
62.5
62.5
125
125
125
62.5
125
125
250
62.5
125
62.5
125
62.5
125
>125
>125
15.62
62.5
31.25
125
>125
>125
3.9
>125
>125
125
13.8
20.4
12.3
ND
125
125
125
250
7.81
1.95
3.9
>500
>500
>500
>500
>500
>500
31.25
150
62.5
62.5
32
32
62.5
62.5
31.25
125
125
125
1.95
3.9
8g
8h
8i
125
125
125
250
>125
>125
>125
>125
>125
>125
125
125
125
>125
>125
>125
>125
62.5
8
16
32
32
ND
62.5
62.5
62.5
62.5
>125
>125
62.5
125
ND
15.62
32
32
62.5
62.5
62.5
62.5
31.25
125
250
500
7.81
7.81
7.81
7.81
ND
8j
62.5
125
125
32
62.5
>500
>500
>125
>125
>125
>125
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>125
>125
>125
>125
>125
>125
3.9
>500
>500
>125
>125
>125
>125
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>125
>125
>125
>125
>125
>125
7.81
>500
>500
>125
>125
>125
>125
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>125
>125
>125
>125
>125
>125
62.5
>500
>500
>125
>125
>125
>125
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>125
>125
>125
>125
>125
>125
62.5
16
16
32
32
29.8
33.7
ND
8k
8l
>125
125
>125
125
>125
125
125
16
16
125
500
32
32
62.5
500
32
32
125
500
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
500
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
500
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
500
8m
8n
8o
8p
8qe
8re
8s
32
32
32
62.5
62.5
500
ND
32
32
62.5
125
32
62.5
ND
16
32
125
>1000
125
>1000
62.5
62.5
62.5
125
125
>1000
125
>1000
62.5
62.5
32
62.5
250
>1000
250
>1000
62.5
125
ND
74.2
ND
26.2
ND
62.5
62.5
62.5
62.5
62.5
125
8t
32
32
32
32
250
250
250
250
62.5
125
32
32
31.2
ND
8u
8v
>500
500
>500
>500
500
>500
500
125
62.5
15.62
15.62
>500
>500

Molecules 2011, 16
2421
Table 2. Cont.
62.5
62.5
32
62.5
62.5
62.5
125
125
125
125
62.5
125
125
125
125
125
62.5
62.5
>250
>250
250
>250
>250
250
>250
>250
250
250
500
500
500
250
250
500
500
500
61.5
ND
9a
7.81
7.81
>500
>500
>500
>500
>250
>250
>500
>500
ND
7.81
7.81
>500
>500
>500
>500
>250
>250
>500
>500
ND
1.95
1.95
>500
>500
>500
>500
>250
>250
>500
>500
ND
9b
250
250
250
500
>125
>125
>125
>125
125
>125
>125
>125
>125
125
>125
>125
7.81
7.81
125
125
>500
>500
>500
>500
>500
>500
>500
>500
ND
23.8
ND
9c
62.5
62.5
62.5
62.5
62.5
62.5
9d
32
32
32
62.5
32
62.5
ND
9e
125
125
32
32
>125
>125
ND
ND
ND
>125
>125
ND
ND
ND
>125
>125
ND
ND
ND
32
32
32
32
23.5
9f
ND
ND
ND
ND
0.5
ND
ND
ND
ND
>250
>250
ND
ND
ND
ND
>250
>250
24.0
14.2
37.3
35.7
9g
9h
9i
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
9j
–
–
–
–
–
–
–
–
–
–
–
–
–
–
INH
0.5
0.12
>125
3.91
15.62
1.95
3.91
–
–
–
–
–
–
–
–
FLU
PEN
0.24
0.24
0.98
0.98
–
125
125
500
500
–
31.62
125
250
250
–
7.81
15.62
0.98
0.98
–
–
–
–
–
–
–
–
–
–
–
–
–
CPF
1.9
DCMU
a
The MIC determinations were performed according to the CLSI reference protocol: mycobacteria
b
c
d
(IC90 value), M27-A2 for yeasts (IC80 value), M38-A for moulds-fungi (IC50 value) and bacteria
(IC90 value), ecompounds precipitated during the experiments.
Compounds 8h and 8k/l (Group 2) exhibited the highest activity against M. tuberculosis. Based on
these observations, it can be concluded that chlorination in the C(4) position results in less active
compounds, substitution in the position C'(4) of the anilide part is essential for high activity, and 4-Cl
(Group 2) is more advantageous than 4-Br (Group 4). In general, compounds within Group 1 (3-Cl
substituted) showed negligible activity. The influence of chlorine in the position C'(4) of the anilide part
seems to be dominant in Group 3 (series 5-Cl/3,4-Cl), see activity in Table 2. Lipophilicity seems to be
only a secondary parameter; in the same way, the use of electronic parameter σ of R2 substituent does
not improve the results of SAR discussion. High activity of 8h cannot be explained on the basis of
simple pattern.
Compounds 8k/l and 8g (Group 2 and Group 1) and 9e/f (Group 5) showed the highest activity
against M. avium and other compounds 8i/j, 8a, 8s and 9d demonstrated medium activity. Based on
these facts, it can be assumed that the substitution of R3, especially by benzyl moiety, as well as
preferential substitution by chlorine, especially in C'(4) position of the anilide part of a molecule, is
important for high activity. Chlorination of C(4) or C(5) position is only a secondary parameter.
Substitution in C'(4) by bromine (Group 4) or disubstitution in C'(3,4) (Group 3) by chlorine causes
activity decrease compared with Group 2. Lipophilicity seems to be also one of the parameters
affecting antimycobacterial activity. The most active compounds from all groups show log k about
0.70. Stereoisomerism considerably influences antimycobacterial activity against M. avium as
discussed below.

Molecules 2011, 16
2422
Compounds 8l, 8v and 9f (Group 2, Group 4 and Group 5) showed the highest antimycobacterial
activity against M. kansasii, and other compounds, 8k, 9e, 8i/j, 8g and 8c, expressed substantial
activity as well. Based on these facts, it can be concluded that chlorination of C(4) or C(5) position is
only a secondary parameter, while substitution of C'(4) position is more advantageous for high activity,
and substitution by chlorine is preferred. Disubstitution of C'(3,4) position is connected with activity
decrease. The substitution of R3, particularly by benzyl or isopropyl moiety, is associated with higher
activity, which can be positively influenced by lipophilicity increase. Similarly, based on Tables 1 and
2 it can be considered that antimycobacterial activity against M. kansasii is also partly affected by bulk
parameters expressed as MR (reflecting bulkiness) of R3 substituents [33]. It seems that
antimycobacterial activity is increased by the increase of the bulkiness of individual R3 substituents in
the molecule.
From the set of 28 tested compounds the dependence of activity (log 1/MIC, 14 h experiment) on
log k related to antimycobacterial activity against M. kansasii (see Figure 2) was linear for 24
compounds (compounds 8b/c and 8q/r were excluded from the correlation). Introduction of σ
parameter did not improve the results of statistical analysis:
log(1/MIC) = 3.186(±0.091) + 1.574(±0.136) log k
r = 0.926, s = 0.092, F = 132.07, n = 24
(1)
(2)
log (1/MIC) = 3.232(±0.095) +1.594(±0.135) log k – 0.185(±0.135) σ
r = 0.933, s = 0.090, F = 70.12, n = 24
Figure 2. Dependence of antimycobacterial activity against M. kansasii (log 1/MIC
[mol/L], 14 h experiment) on compounds lipophilicity expressed as log k.
4.6
9k
9l
8l
8p
8o
8k
8v
8u
4.4
4.2
4.0
3.8
3.6
3.4
3.2
8e
8d
8j 8i
8f
8t
9c
8s 8n 8g
9d
8c
8h
8m
9a
9b
8a
8r 8q
8b
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
log k
Group 1 Group 2 Group 3 Group 4 Group 5
A very important parameter influencing activity is stereoisomerism, because individual enantiomers
demonstrate considerable difference in their antituberculotic/antimycobacterial activity, e.g.
enantiomers 8b/c, 8f/g, 8k/l, 8o/p, 8u/v, 9c/d and 9e/f showed different antimycobacterial activity.
These differences are especially evident for antimycobacterial activity against the atypical strains M.

Molecules 2011, 16
2423
avium and M. kansasii. More often (R)-enantiomers showed higher activity. The determined
differences in antimycobacterial activity for individual R/S-enantiomers cannot be explained on the
basis of the results presented here. Due to these facts it can be assumed that stereospecific bond is
formed between the compound and an enzyme in Mycobacterium sp. with subsequent enzyme inhibition.
2.3.2. In vitro antifungal susceptibility testing
The evaluation of in vitro antifungal activity of the synthesized compounds was performed against
eight fungal strains, but only activity against Candida tropicalis 156 (CT), C. krusei ATCC 6258 (CK)
and against Trichophyton mentagrophytes 445 (TM) is shown in Table 2. Generally, all the compounds
expressed only moderate antifungal activity, which was caused by low solubility of compounds in the
testing medium and their precipitation during the incubation period, therefore no definite structure-
activity relationships could be established. Only four compounds from Group 1, 8f/g, 8e and 8b/c,
showed very high antifungal activity comparable with or higher than the standard fluconazole (FLU)
against C. krusei and against T. mentagrophytes 445. More often (R)-enantiomers showed higher
activity, e.g. 8c/b, 8e/d, 9d/c, nevertheless, the determined differences in antibacterial activity for
individual R/S-enantiomers cannot be explained on the basis of the results presented here.
Generally, according to the results from Table 2, it can be concluded that chloration of C(4) position
or disubstitution R2 = 3,4-Cl considerably decreases solubility, and therefore it is not advantageous.
The substitution R2 = 3-Cl is essential for high activity. The substitution of R3 modulates lipophilicity,
and it seems to be an important parameter. Based on Table 2 it can be concluded that a lipophilicity
decrease results in a decrease of antifungal activity: log k = 0.70 (8f/g) > 0.61 (8e) > 0.41 (8b/c).
When the dependence of activity (log 1/MIC, 24 h experiment) on log k related to antifungal
activity of tested compounds against C. krusei (which is the most inhibited fungi strain) was studied,
the dependence was linear only for Group 1 (3-Cl substituted derivatives):
log (1/MIC) = 2.901(±0.085) + 3.986(±0.149) log k
r = 0.998, s = 0.061, F = 711.38, n = 5
(3)
The above discussed antimycobacterial activity against M. kansasii for the whole series of the
tested compounds is mainly dependent on lipophilicity. In contrast to the antimycobacterial activity,
the effect of σ parameter of R2 substituent on the activity of evaluated compounds against C. krusei
was much more expressive than that of log k:
log (1/MIC) = 1.732(±0.780) + 8.863(±2.575) σ
r = 0.754, s = 0.614, F = 11.84, n = 11
(4)
(5)
log (1/MIC) = 4.276 (±1.070) + 0.102 (±1.683) log k
r = 0.203, s = 0.934, F = 0.004, n = 11
Multilinear correlation was found to fit the biological activity of compounds as a function of
compound lipophilicity log k and σ constant of R2 substituent, and the results of statistical analysis
showed that the activity was affected by both parameters.
log (1/MIC) = -0.038 (±1.185) + 1.986 (±1.077) log k + 10.743(±2.507) σ
r = 0.834, s = 0.546, F = 9.19, n = 11
(6)

Molecules 2011, 16
2424
2.3.3. In vitro antibacterial susceptibility testing
Twenty-eight benzamides were tested for their in vitro antibacterial activity against four Gram
positive bacterial strains Staphylococcus aureus CCM 4516/08 (SA), methicilin resistant S. aureus H
5996/08 (MRSA), S. epidermidis H 6966/08 (SE) and Enterococcus sp. J 14365/08 (EF). The results
are shown in Table 2. Generally, all the compounds expressed only moderate antibacterial activity
caused by the low solubility of compounds in the testing medium and their precipitation during the
incubation period, as it was mentioned above. In summary, five compounds 8v, 8j, 8e and 9b showed
notable antibacterial activity comparable with or higher than the both standards penicillin G and
ciprofloxacin against S. aureus methicilin resistant and S. epidermidis.
Based on the results from Table 2, especially halogenation of C'(4) position (R2 = 4-Br or 4-Cl) seem
to be important for antibacterial activity. Chloration of C(4) position decreased solubility compared
with chloration of C(5) position. Stereoisomerism is probably also an important parameter influencing
the activity, because individual enantiomers demonstrate considerable difference in their antibacterial
activity, e.g., enantiomers 8u/v, 8d/e, 8i/j and 9a/b. More often (R)-enantiomers showed higher
activity (the same as M. kansasii). R3 substitution modulates physico-chemical properties of the
discussed compounds. According to Table 2, a bulkier substituent is probably more advantageous. A
more detailed SAR study cannot be established due to the small number of effective compounds.
2.3.4. Inhibition of photosynthetic electron transport (PET) in spinach chloroplasts
Seventeen compounds were tested for their PET in spinach (Spinacia oleracea L.) chloroplasts, and
they showed some wide-range activity, see Table 2. Compounds 8f (IC50 = 12.3 µmol/L), 8e
(IC50 = 13.8 µmol/L) and 9h (IC50 = 14.2 µmol/L) were the most effective inhibitors of the tested
compounds, which PET-inhibiting activity in spinach chloroplasts was measured. The activity of the
rest of the studied compounds was moderate or low relative to the standard.
Due to the fact that PET-inhibiting data are available only from Group 1 and Groups 5 + 6, SAR
study involves the first of these series, but a general SAR hypothesis may be proposed. Generally, it
can be concluded that C(4) substitution is more advantageous then C(5) substitution. In the C(5) series
C´(3) substitution is important for PET activity. Compounds within the C(5) series with substitution in
C´(4) by Cl or Br exhibit similar activity, which is lower than that of C´(3) substituted compounds, but
solubility in the testing medium is drastically reduced in the case of C´(4) substitution. Disubstitution in
C´(3,4) by Cl also causes lower solubility. Bulkiness and branching of R3 substitution are also
important. It can be noticed that mainly (S)-enantiomers are active in PET assay (compare 8d/e or
8k/l) and only 8e, 9f, 9g are slightly active (R)-enantiomers. The correlations between PET-inhibiting
activity and lipophilicity could be expressed by equation:
log (1/IC50) = 1.925(±0.427) + 8.302(±1.497) log k – 6.270(±1.266) (log k)2
r = 0.856, s = 0.191, F = 19.17, n =17
(7)
For PET inhibition of tested compounds the lipophilicity was determinant because the introduction
of σ parameter did not improve the results of statistical analysis:
log (1/IC50) = 1.903(±0.460) + 8.365(±1.595) log k – 6.345(±1.383) (log k)2 + 0.0476(±0.279) σ
r = 0.856, s = 0.198, F = 11.91, n = 17
(8)

Molecules 2011, 16
2425
3. Experimental
3.1. Synthesis
The studied compounds are shown in Scheme 1 and listed in Table 1. Synthesis and
characterization of the discussed chloro-2-hydroxy-N-[2-(arylamino)-1-alkyl-2-oxoethyl]benzamides
were described in reference [14].
3.2. Lipophilicity determination by HPLC (capacity factor k/calculated log k)
A Waters Alliance 2695 XE HPLC separation module and a Waters Photodiode Array Detector
2996 (Waters Corp., Milford, MA, USA) were used. A Symmetry® C18 5 μm, 4.6 × 250 mm, Part No.
WAT054275 (Waters Corp., Milford, MA, USA) chromatographic column was used. The HPLC
separation process was monitored by Empower™ 2 Chromatography Data Software, Waters 2009
(Waters Corp., Milford, MA, USA). A mixture of MeOH p.a. (70%) and H2O-HPLC – Mili-Q Grade
(30%) was used as a mobile phase. The total flow of the column was 1.0 mL/min, injection volume,
30 μL, column temperature, 30 °C and sample temperature, 10 °C. The detection wavelength of
210 nm was chosen. The KI methanolic solution was used for the dead time (tD) determination.
Retention times (tR) were measured in minutes. The capacity factors k were calculated using the
Empower™ 2 Chromatography Data Software according to formula k = (tR - tD)/tD, where tR is the
retention time of the solute, whereas tD denotes the dead time obtained using an unretained analyte.
Log k, calculated from the capacity factor k, is used as the lipophilicity index converted to log P scale.
The log k values of the individual compounds are shown in Table 1.
3.3. Lipophilicity calculations
Log P, i.e. the logarithm of the partition coefficient for n-octanol/water, was calculated using the
programs CS ChemOffice Ultra ver. 10.0 (CambridgeSoft, Cambridge, MA, USA) and ACD/LogP ver.
1.0 (Advanced Chemistry Development Inc., Toronto, Canada). Clog P values (the logarithm of
n-octanol/water partition coefficient based on established chemical interactions) were generated by
means of CS ChemOffice Ultra ver. 10.0 (CambridgeSoft, Cambridge, MA, USA) software. The
results are shown in Table 1.
3.4. In vitro antimycobacterial evaluation
The in vitro antimycobacterial activity of all prepared compounds was evaluated against
Mycobacterium tuberculosis MTB CNCTC My 331/88 (identical with H37RV and ATCC 27294,
dilution of the strain was 10−3 μmol/L), Mycobacterium avium MA CNCTC My 330/88 (identical with
ATCC 25291, dilution of the strain was 10−5 μmol/L) and Mycobacterium kansasii MK CNCTC My
235/80 (identical with ATCC 12478, dilution of the strain was 10−4 μmol/L) in the Laboratory for
mycobacterial diagnostics and TB, Institute of Public Health in Ostrava, Czech Republic. All strains
were obtained from the Czech National Collection of Type Cultures (CNCTC). Antimycobacterial
activities were determined in the Sula semisynthetic medium (Sevac, Prague, Czech Republic). Each
strain was simultaneously inoculated into a Petri plates containing the Lowenstein-Jensen medium for

Molecules 2011, 16
2426
the control of sterility of the inoculum and its growth. The tested compounds were added to the
medium as DMSO solutions. The following concentrations were used: 250, 125, 62, 32, 16, 8, 4, 2,
and 1 μmol/L. Inoculated plates kept in microtone bags were incubated at 37 °C. Reading was carried
out on a stand with a bottom magnifying mirror, macroscopically, with the use of magnifying glass.
The growth in plates was evaluated after 14 and 21 days of the incubation. The growth of the colonies
in a control well plate, corresponding to the growth into 100 colonies in the control Lowenstein-Jensen
medium was considered as an optimum dilution for the evaluation of the results. In the course of the
test evaluation, the MIC (μmol/L) definition was considered as the lowest substance concentration at
which the inhibition of growth of mycobacteria occurred. [34] Isoniazid (INH) was used as the
standard as it is a clinically used anti-mycobacterial drug. The results are shown in Table 2. The MIC
for mycobacteria was defined as a 90% or greater (IC90) reduction of growth in comparison with the
control. The MIC/IC90 value is routinely and widely used in bacterial assays and is a standard
detection limit according to the Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS,
www.clsi.org/).
3.5. In vitro antifungal susceptibility testing
The broth microdilution test [35-37] was used for the assessment of in vitro antifungal activity of
the synthesized compounds against Candida albicans ATCC 44859, C. tropicalis 156, C. krusei E28,
C. glabrata 20/I, Trichosporon asahii 1188, Trichophyton mentagrophytes 445, Aspergillus
fumigantus 231 and Absidia corymbifera 272. Fluconazole (FLU) was used as a standard, since it is a
clinically used antimycotic drug. The procedure was performed with a two-fold dilution of the
compounds in RPMI 1640 (Sevapharma a.s., Prague, Czech Republic) buffered to pH 7.0 with
0.165 mol of 3-morpholino-propane-1-sulfonic acid (MOPS, Sigma, Germany). The final
concentrations of the compounds ranged from 500 to 0.975 μmol. Drug–free controls were included.
The MIC determination was performed according to the CLSI reference protocol M27-A2 for yeasts
(IC80 value) and M38-A for moulds (IC50 value). IC50 values were defined as a 50% reduction of
growth in comparison with the control, and IC80 values were defined as a 80% reduction of growth in
comparison with the control. The trays were incubated at 35 °C, and MICs were read visually for
filamentous fungi and photometrically for yeasts as absorbance at 540 nm after 24 h and 48 h. The
MIC values for the dermatophytic strain (T. mentagrophytes) were determined after 72 h and 120 h.
MICs were determined twice and in duplicate. The results are summarized in Table 2.
3.6. In vitro antibacterial susceptibility testing
The synthesized compounds were evaluated for in vitro antibacterial activity against
Staphylococcus aureus CCM 4516/08, methicilin resistant Staphylococcus aureus H 5996/08,
Staphylococcus epidermidis H 6966/08 and Enterococcus sp. J 14365/08. Penicillin G (PEN) and
ciprofloxacin (CPF) were used as standards since they are clinically used antibacterial drugs. All
strains were sub-cultured on nutrient agar (HiMedia) and maintained on the same medium at 4 °C.
Prior to testing, each strain was passaged onto nutrient agar and bacterial inocula were prepared by
suspending a small portion of bacterial colony in sterile 0.85% saline. The cell density was adjusted to
0.5 McFarland units using a densitometer (Densi-La-Meter, PLIVA Lachema Diagnostika, Czech

Molecules 2011, 16
2427
Republic). The final inoculum was made by 1:20 dilution of the suspension with the test medium
(Mueller-Hinton broth). The compounds were dissolved in DMSO and the anti-bacterial activity was
determined using Mueller-Hinton broth (MH broth, HiMedia, pH 7.0 ± 0.2). Controls consisted of MH
broth and DMSO alone. The final concentration of DMSO in the MH broth did not exceed 1% (v/v) of
the total solution composition. The activity of the studied compounds was determined as the minimal
inhibition concentration (MIC) according to NCCLS guidelines [38] using broth microdilution test.
The MICs were defined as 90% inhibition of bacterial growth compared to the control and were
determined after 24 and 48 h of static incubation at 37 °C. After incubation MICs were read visually as
an absorbance at 540 nm. The results are shown in Table 2.
3.7. Study of inhibition photosynthetic electron transport (PET) in spinach chloroplasts
Chloroplasts were prepared from spinach (Spinacia oleracea L.) according to Masarovicova and
Kralova [39]. The inhibition of photosynthetic electron transport (PET) in spinach chloroplasts was
determined spectrophotometrically (Genesys 6, Thermo Scientific, USA), using an artificial electron
acceptor 2,6-dichlorophenol-indophenol (DCIPP) according to Kralova et al. [40], and the rate of
photosynthetic electron transport was monitored as a photoreduction of DCPIP. The measurements
were carried out in phosphate buffer (0.02 mol/L, pH 7.2) containing sucrose (0.4 mol/L), MgCl2
(0.005 mol/L) and NaCl (0.015 mol/L). The chlorophyll content was 30 mg/L in these experiments and
the samples were irradiated (~100 W/m2) from 10 cm distance with a halogen lamp (250 W) using
a 4 cm water filter to prevent warming of the samples. The studied compounds were dissolved in
DMSO due to their limited water solubility. The applied DMSO concentration (up to 4%) did not
affect the photochemical activity in spinach chloroplasts. The inhibitory efficiency of the studied
compounds was expressed by IC50 values, i.e. by molar concentration of the compounds causing 50%
decrease in the oxygen evolution rate relative to the untreated control. The comparable IC50 value for a
selective herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea, DCMU (Diurone®) was about 1.9 μmol/L
[41]. The results are summarized in Table 2.
4. Conclusions
A series of twenty-two 5-chloro-2-hydroxy-N-[2-(arylamino)-1-alkyl-2-oxoethyl]benzamides and
ten 4-chloro-2-hydroxy-N-[2-(arylamino)-1-alkyl-2-oxoethyl]benzamides is described. Their
lipophilicity was determined using a well established RP-HPLC method. The prepared compounds
were tested for their antimycobacterial, antifungal and antibacterial activity as well as for their ability
to inhibit photosynthetic electron transport (PET) in spinach chloroplasts (Spinacia oleracea L.). For
all the compounds the relationships between their lipophilicity and chemical structure as well as
structure-activity relationships were discussed. N-{(1R)-1-benzyl-2-[(4-chlorophenyl)amino]-2-oxo-
ethyl}-5-chloro-2-hydroxybenzamide (8l, 32 μmol/L) and N-{(1R)-1-benzyl-2-[(4-chlorophenyl)-
amino]-2-oxoethyl}-4-chloro-2-hydroxybenzamide (9f, 32 μmol/L) showed the highest antitubercular
activity and higher antimycobacterial effect than the standard isoniazid. N-{(1S/R)-1-benzyl-2-[(3-
chlorophenyl)amino]-2-oxoethyl}-5-chloro-2-hydroxybenzamide (8f, 8g, 3.9 μmol/L) and 5-chloro-N-
[(1R)-1-{[(3-chlorophenyl)amino]-carbonyl}-2-methylpropyl)]-2-hydroxybenzamide
(8e,
7.81 μmol/L) demonstrated higher antifungal activity against Candida krusei and 5-chloro-N-{(1R)-2-

Molecules 2011, 16
2428
[(3-chlorophenyl)amino]-1-methyl-2-oxoethyl}-2-hydroxybenzamide (8c, 0.49 μmol/L) showed higher
antifungal activity against Trichophyton mentagrophytes than the standard fluconazole. 5-Chloro-N-
[(1S/R)-1-{[(4-chlorophenyl)amino]carbonyl}-2-methylpropyl)]-2-hydroxybenzamide (compound 8j,
7.81 μmol/L) exhibited higher activity against methicillin-resistant Staphylococcus aureus than
penicillin G and ciprofloxacin. 4-Chloro-N-{(1R)-2-[(4-chlorophenyl)-amino]-1-methyl-2-oxoethyl}-
2-hydroxy benzamide (9b, 7.81 μmol/L and 1.95 μmol/L) showed higher activity against methicillin-
resistant Staphylococcus aureus and S. epidermidis than penicillin G and ciprofloxacin. N-{(1S)-1-
benzyl-2-[(3-chlorophenyl)amino]-2-oxoethyl}-5-chloro-2-hydroxybenz-amide (8f, 12.3 μmol/L) and
5-chloro-N-[(1S)-1-{[(3-chlorophenyl)amino]-carbonyl}-2-methylpropyl)]-2-hydroxybenzamide (8d,
13.8 μmol/L) showed the highest PET-inhibition activity among the discussed compounds. It can be
concluded that (R)-enantiomers demonstrated higher antimicrobial activity compared with (S)-
enantiomers, while (S)-enantiomers showed higher inhibition of photosynthetic electron transport in
spinach chloroplasts.
Acknowledgements
This study was supported by the IGA NS 10367-3, the Ministry of Education of the Czech Republic
(MSM 0021627501 and MSM 0021620822) and by Sanofi-Aventis Pharma Slovakia.
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Sample Availability: Samples of the compounds are available from the authors.
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