Antifungal activity of salicylanilides and their esters with 4-(trifluoromethyl)benzoic acid

Article abstract of DOI:10.3390/molecules17089426

Searching for novel antimicrobial agents still represents a current topic in medicinal chemistry. In this study, the synthesis and analytical data of eighteen salicylanilide esters with 4-(trifluoromethyl)benzoic acid are presented. They were assayed in vitro as potential antimycotic agents against eight fungal strains, along with their parent salicylanilides. The antifungal activity of the presented derivatives was not uniform and moulds showed a higher susceptibility with minimum inhibitory concentrations (MIC) ≥ 0.49 μmol/L than yeasts (MIC ≥ 1.95 μmol/L). However, it was not possible to evaluate a range of 4-(trifluoromethyl)benzoates due to their low solubility. In general, the most active salicylanilide was N-(4-bromophenyl)-4-chloro-2- hydroxybenzamide and among esters, the corresponding 2-(4-bromophenylcarbamoyl)- 5-chlorophenyl 4-(trifluoromethyl) benzoate exhibited the lowest MIC of 0.49 μmol/L. However, the esterification of salicylanilides by 4-(trifluoromethyl)benzoic acid did not result unequivocally in a higher antifungal potency.

Full text of DOI:10.3390/molecules17089426

Molecules 2012, 17, 9426-9442; doi:10.3390/molecules17089426
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Antifungal Activity of Salicylanilides and Their Esters with
4-(Trifluoromethyl)benzoic Acid
Martin Krátký and Jarmila Vinšová *
Department of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles University,
Heyrovského 1203, Hradec Králové 50005, Czech Republic; E-Mail: martin.kratky@faf.cuni.cz
* Author to whom correspondence should be addressed; E-Mail: vinsova@faf.cuni.cz;
Tel.: +420-495-067-343; Fax: +420-495-067-166.
Received: 19 June 2012; in revised form: 19 July 2012 / Accepted: 30 July 2012 /
Published: 7 August 2012
Abstract: Searching for novel antimicrobial agents still represents a current topic in
medicinal chemistry. In this study, the synthesis and analytical data of eighteen
salicylanilide esters with 4-(trifluoromethyl)benzoic acid are presented. They were assayed
in vitro as potential antimycotic agents against eight fungal strains, along with their parent
salicylanilides. The antifungal activity of the presented derivatives was not uniform
and moulds showed a higher susceptibility with minimum inhibitory concentrations
(MIC)  0.49 µmol/L than yeasts (MIC  1.95 µmol/L). However, it was not possible to
evaluate a range of 4-(trifluoromethyl)benzoates due to their low solubility. In general, the
most active salicylanilide was N-(4-bromophenyl)-4-chloro-2-hydroxybenzamide and among
esters, the corresponding 2-(4-bromophenylcarbamoyl)-5-chlorophenyl 4-(trifluoromethyl)
benzoate exhibited the lowest MIC of 0.49 µmol/L. However, the esterification of
salicylanilides by 4-(trifluoromethyl)benzoic acid did not result unequivocally in a higher
antifungal potency.
Keywords: antifungal activity; in vitro activity; salicylanilide; salicylanilide ester;
4-(trifluoromethyl)benzoic acid ester
1. Introduction
The spread of fungal infections has recently been increasing because of the eminent presence
of the predisposing risk factors, e.g., malnutrition, invasive surgical procedures, treatment with

Molecules 2012, 17
9427
broad-spectrum antibiotics, corticosteroids or other immunosuppressive agents, immunodeficiency
related to diseases like diabetes mellitus or human immunodeficiency virus infections. Mycoses may
either be superficial or systemic [1,2]. Immunocompromised patients can develop opportunistic
mycoses caused by an expanding spectrum of fungal pathogens, including those with problematic
susceptibility to current antifungal drugs [3]. Invasive fungal infections are a major cause of morbidity
and mortality in these patients. Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus
comprise the most common etiologic agents, but there is an increasing number of infections caused
by rare pathogens, including e.g., non-albicans Candida species, opportunistic yeast-like fungi like
Trichosporon spp. or non-fumigatus Aspergillus spp. [4].
Pathogenic fungi can also develop resistance to various drugs with unshared mechanisms of
action [3]. That is why the development of new antifungal agents still remains challenging, despite the
intensive searching in recent times.
Salicylanilides (2-hydroxy-N-phenylbenzamides) have displayed a wide range of potentially
interesting biological effects including those against protozoa, fungi, bacteria and mycobacteria or
viruses [5,6]. Their mechanism of the action towards microbes is believed to be multiple, with a lot of
molecular targets and effects [7].
Waisser et al. [8] reported the activity of twelve salicylanilides with various substitution patterns
towards nine fungal strains. Minimum inhibitory concentrations (MIC) for yeast strains were proven to
be 15.63 µmol/L and 3.91 µmol/L for filamentous fungi, respectively; however, some derivatives
only exhibited low activity. The most susceptible strains were determined to be Trichophyton
mentagrophytes and Microsporum gypseum. Other authors have also reported rather moderate or
mild antifungal activity of salicylanilides, e.g., towards Absidia corymbifera and Trichophyton
mentagrophytes [9], Sacharomyces cerevisiae and C. albicans (MIC of 0.3125–10 mmol/L) [10], or
from another source, 2–3.2 µmol/L [11], or 25 μg/mL against C. albicans [12]. Aspergillus sp. was
also partly susceptible [13]. 2,3-Dihydroxy-N-(substituted)phenylbenzamides and 2,6-dihydroxy-N-
(substituted)phenylbenzamide derivatives (“dihydroxysalicylanilides”) also showed a significant
antifungal potency with MIC values ranging between 3.91–250 µmol/L for Candida species and
1.95–250 µmol/L for filamentous fungi [14].
The temporary masking by an esterification of a salicylanilide free phenolic group, which is
presumed to be necessary for the antimicrobial activity, may be beneficial, e.g., due to improved
bioavailability and membrane permeability, a high activity and/or a lower toxicity [7]. A correlation
between hydrophilic/lipophilic balance (lipophilicity) necessary to penetrate through biological
barriers and the antimicrobial activity with an optimal span has been described repeatedly [15,16].
The antifungal properties of salicylanilide esters (acetates, esters with N-protected amino acids)
were summarized in our earlier review [7]; later, a mild and sporadic activity was found for
salicylanilide benzoates (MIC  3.9 µmol/L) [17] and more importantly for salicylanilide pyrazinoates
(MIC  1.95 µmol/L) [18] with a general result that salicylanilide esters are more active and probably
fungicidal against filamentous fungi than against Candida strains with only a limited fungistatic activity.
Based on these facts, we synthesised a new series of salicylanilide esters with 4-(trifluoro-methyl)
benzoic acid (illustratively, esters of imidazole derivatives with 3- and 4-(trifluoro-methyl)benzoic
acids expressed some activity against Candida strains [19]) and evaluated them as well as their parent
salicylanilides as potential antifungal agents.

Molecules 2012, 17
9428
2. Results and Discussion
2.1. Chemistry
Salicylanilides and their esters with 4-(trifluoromethyl)benzoic acid were obtained by two
procedures used previously by our group [5,20]. Firstly, salicylanilides 1 were prepared by the reaction
of appropriate salicylic acids and anilines in the presence of PCl3 in chlorobenzene (Ph-Cl) under
microwave irradiation (Scheme 1). Then they were esterified by 4-(trifluoromethyl)benzoic acid via
N,N′-dicyclohexylcarbodiimide (DCC) coupling in dry N,N-dimethylformamide (DMF), see Scheme 2.
Yields of salicylanilides ranged within 75–90%, of esters within 49–86%.
Scheme 1. Synthesis of salicylanilides.
R1 = H, 4-Cl, 5-Cl, 5-Br; R2 = H, 3-Cl, 4-Cl, 3,4-diCl, 3-Br, 4-Br, 3-F, 4-F, 3-CF3, 4-CF3.
Scheme 2. Synthesis of salicylanilide 4-(trifluoromethyl)benzoates.
R1 of esters 2 = H, 4-Cl, 4-Br, 5-Cl; R2 = H, 3-Cl, 4-Cl, 3,4-diCl, 3-Br, 4-Br, 3-F, 4-F, 3-CF3, 4-CF3.
2.2. In Vitro Antifungal Evaluation
In vitro antifungal properties of thirty seven salicylanilide derivatives were assayed against four
Candida strains (Candida albicans ATCC 44859, Candida tropicalis 156, Candida krusei E28, and
Candida glabrata 20/I), Trichosporon asahii 1188 and three filamentous fungi (Aspergillus fumigatus
231, Absidia corymbifera 272, and Trichophyton mentagrophytes 445).
Although it was our intention, the sharply increased lipophilicity of salicylanilide 4-(trifluoro-methyl)
benzoates (e.g., ClogP of 4.49 for 1n vs. 6.02 for 2n) caused problems with the biological assessment.
In contrast to the parent salicylanilides, some of the esters (ten derivatives: 2a, 2b, 2d, 2g, 2i, 2l, 2m,
2n, 2o, 2s) did not dissolve very well in the testing medium or they precipitated after a very short
period; therefore it was not possible to assay their MIC values. Tables 1 and 2 overview the antifungal
properties, and Table 1 for yeasts, Table 2 for filamentous fungi. 4-(Trifluoro-methyl)benzoic acid
itself was completely inactive for all species under our conditions.

Molecules 2012, 17
9429
Table 1. In vitro activity of salicylanilides 1 and their 4-(trifluoromethyl)benzoates 2 towards yeasts.
MIC/IC80 [mol/L
Candida
albicans
Candida
tropicalis
Candida
krusei
Candida
glabrata
Trichosporon
asahii
Code
1a
2a
1b
2b
1c
2c
1d
2d
1e
2e
1f
R1
R2
3-Cl
3-Cl
3-Cl
3-Cl
4-Cl
4-Cl
4-Cl
ClogP
5.85
7.02
5.18
6.71
5.50
6.68
4.83
6.37
6.51
7.69
5.84
7.38
5.92
7.09
5.25
6.87
5.88
7.05
5.21
6.74
5.20
6.37
4.53
6.06
24 h
>125
ND
>125
ND
48 h
>125 >125
ND ND
>125 >125
ND ND
24 h
48 h
>125
ND
24 h
>125
ND
>125
ND
48 h
>125 >125
ND ND
>125 >125
ND ND
24 h
48 h
>125
ND
24 h
>125
ND
48 h
>125
ND
5-Cl
4-Cl
4-Cl
5-Cl
5-Cl
4-Cl
4-Cl
5-Cl
5-Cl
4-Cl
4-Cl
5-Cl
5-Cl
4-Cl
4-Cl
5-Cl
5-Cl
4-Cl
4-Cl
5-Cl
5-Cl
4-Cl
4-Cl
5-Cl
>125
ND
>125
ND
>125
ND
>125
ND
>125
62.5
>125
ND
>125
>500
>125
>125
>125
ND
>125 >125
>250 >250
>125 >125
ND
>125 >125
>500 7.81
>125 >125
>125 >125
>125 >125
>125
>250
>125
ND
>125
250
>125
ND
>125 >125
>250 >250
>125 >125
>125
>250
>125
ND
>125
250
>125
ND
>125
>250
>125
ND
4-Cl
ND
ND
ND
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3-Br
3-Br
3-Br
3-Br
4-Br
4-Br
4-Br
4-Br
3-F
>125
31.25
>125
>125
>125
ND
>125
7.81
>125
62.5
>125
ND
>125
125
>500
ND
7.81
31.25
>125
>250
>125
ND
>125 >125
31.25 >500
>125 >125
>125 >125
>125 >125
ND
>125 >125
250 >500
>500 >500
ND ND
>125 >125
62.5
>125
>250
>125
ND
>125
>500
>125
>125
>125
ND
>125
>500
3.9
31.25
15.62
ND
>125
>500
15.62
>125
62.5
ND
2f
1g
2g
1h
2h
1i
2i
1j
2j
ND
ND
ND
>125
125
>500
ND
7.81
15.62
>125
250
>125
ND
>125 >125
>500 >500
>500 >500
ND
>125 >125
31.25 500
>125 >125
>250 >250
>125 >125
>125
>500
>500
ND
>125
>500
>500
ND
1.95
62.5
3.9
62.5
250
3.9
ND
ND
ND
>125
>500
>125
>250
>125
ND
>125
>500
>125
>250
>125
ND
1.95
15.62
>125
>250
>125
ND
7.81
31.25
>125
>250
>125
ND
500
>125
>250
>125
ND
1k
2k
1l
3-F
3-F
3-F
2l
ND
ND

Molecules 2012, 17
9430
Table 1. Cont.
MIC/IC80 [mol/L
Candida
albicans
Candida
tropicalis
Candida
krusei
Candida
glabrata
Trichosporon
asahii
Code
1m
2m
1n
2n
1o
R1
5-Cl
4-Cl
4-Cl
5-Cl
5-Cl
4-Cl
4-Cl
5-Cl
5-Cl
4-Cl
4-Cl
5-Cl
5-Br
4-Br
H
R2
4-F
4-F
4-F
ClogP
5.16
6.33
4.49
6.02
6.06
7.24
5.39
6.93
5.72
6.90
5.05
6.59
5.49
6.50
3.27
24 h
>125
ND
>125
ND
48 h
>125 >125 >125
ND ND ND
>125 >125 >125
ND ND ND
>125 >125 >125
ND ND ND
24 h
48 h
24 h
>125
ND
>125
ND
>125
ND
48 h
>125 >125
ND ND
>125 >125
ND ND
>125 >125
24 h
48 h
>125
ND
24 h
>125
ND
48 h
>125
ND
>125
ND
>125
ND
>125
ND
4-F
3-CF3
3-CF3
3-CF3
3-CF3
4-CF3
4-CF3
4-CF3
4-CF3
4-CF3
4-CF3
H
>125
ND
>125
ND
>125
ND
>125
ND
2o
ND
ND
500
1p
2p
1q
2q
1r
2r
1s
2s
1t
>500
>500
>125
>125
>125
62.5
>125
ND
>500 >500 >500
>500 >500 >500
>125 >125 >125
>125 >125 >125
>125 >125 >125
>500
>500
>125
>125
>125
31.25
>125
ND
>500
>500
>500
>125
>125
>125
>500
>125
ND
250
500
>500 >500
>125 >125
>125 >125
>125 >125
62.5
>125 >125
>500
>125
>125
1.95
15.62
1.95
ND
>500
>125
>125
15.62
62.5
7.81
ND
500
>500 >500
>500
>125 >125 >125
ND
500
ND
250
ND
500
ND
500
ND
250
250
250
250
250
500
UND
FLU
>500
1.0
>500 >500 >500
2.0 3.0 5.0
>500
>50
>500 >500
>50 22.0
>500
>50
250
4.00
>500
9.00
The best MIC values are highlighted. UND: undecylenic acid; FLU: fluconazole.

Molecules 2012, 17
9431
Table 2. In vitro activity of salicylanilides 1 and their 4-(trifluoromethyl)benzoates 2 towards moulds.
MIC/IC80/IC50 [mol/L
Aspergillus
fumigatus
Absidia
corymbifera
Trichophyton
mentagrophytes
Code
1a
2a
1b
2b
1c
R1
R2
3-Cl
24 h
48 h
>125
ND
24 h
7.81
ND
48 h
62.5
ND
72 h
1.95
ND
150 h
1.95
ND
5-Cl
4-Cl
4-Cl
5-Cl
5-Cl
4-Cl
4-Cl
5-Cl
5-Cl
4-Cl
4-Cl
5-Cl
5-Cl
4-Cl
4-Cl
5-Cl
5-Cl
4-Cl
4-Cl
5-Cl
5-Cl
4-Cl
4-Cl
5-Cl
5-Cl
4-Cl
4-Cl
5-Cl
5-Cl
4-Cl
4-Cl
5-Cl
5-Cl
4-Cl
4-Cl
5-Cl
5-Br
4-Br
H
>125
ND
3-Cl
3-Cl
3-Cl
4-Cl
4-Cl
4-Cl
4-Cl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3-Br
3-Br
3-Br
3-Br
4-Br
4-Br
4-Br
4-Br
3-F
3-F
3-F
3-F
4-F
4-F
4-F
4-F
3-CF3
3-CF3
3-CF3
3-CF3
4-CF3
4-CF3
4-CF3
4-CF3
4-CF3
4-CF3
H
>125
ND
>125
>250
>125
ND
>125
>500
>125
>125
>125
ND
>125
>500
>500
ND
>125
15.62
>125
>250
>125
ND
>125
ND
>125
ND
>125
ND
>125
>250
>125
ND
>125
>500
>125
>125
>125
ND
>125
>500
>500
ND
>125
31.25
>125
>250
>125
ND
>125
ND
>125
ND
125
ND
125
250
>125
ND
>125
250
>125
ND
1.95
ND
1.95
15.62
1.95
ND
31.25
15.62
3.9
>125
1.95
ND
1.95
0.98
0.98
ND
1.95
0.49
3.9
62.5
3.9
ND
7.81
ND
62.5
ND
3.9
ND
>500
15.62
3.9
3.9
3.9
1.95
ND
1.95
15.62
1.95
ND
31.25
125
3.9
>125
1.95
ND
1.95
1.95
0.98
ND
1.95
0.98
3.9
62.5
3.9
2c
1d
2d
1e
2e
1f
15.62
ND
>125
>500
3.9
>125
15.62
ND
>125
250
15.62
ND
7.81
7.81
>125
250
>125
ND
>125
ND
>125
>500
7.81
>125
31.25
ND
>125
>500
31.25
ND
7.81
15.62
>125
>250
>125
ND
>125
ND
>125
ND
3.9
ND
>500
>500
>125
>125
3.9
15.62
7.81
ND
2f
1g
2g
1h
2h
1i
2i
1j
2j
1k
2k
1l
2l
ND
7.81
ND
62.5
ND
3.9
1m
2m
1n
2n
1o
2o
1p
2p
1q
2q
1r
>125
ND
3.9
>125
ND
>125
ND
ND
ND
>500
>500
>125
>125
>125
15.62
>125
ND
>500
>500
>125
>125
>125
15.62
>125
ND
>500
>500
>125
>125
3.9
15.62
3.9
ND
>500
31.25
7.81
15.62
3.9
1.95
7.81
ND
2r
1s
2s
1t
0.49
7.81
ND
31.25
250
500
>500
>50
500
>500
>50
250
>500
>50
500
>500
>50
125
250
26.0
UND
FLU
17.0
The best MIC values are highlighted. UND: undecylenic acid; FLU: fluconazole.

Molecules 2012, 17
9432
None of the compounds in this study inhibited Candida glabrata at 125 µmol/L, while for other
strains some activity was found. Four esters (2c, 2h, 2j, 2r) and two salicylanilides (1j, 1t) affected
Candida albicans up to the concentration of 500 µmol/L with 1j (7.81 µmol/L) and 2j showing
superiority. Only ester 2e exhibited a significant activity towards C. tropicalis at low concentrations
(7.81/31.25 µmol/L). C. crusei was inhibited by seven molecules (2e, 2f, 2h, 1j, 2j, 2r, 1t) from
7.81 µmol/L. Fourteen salicylanilide derivatives inhibited Trichosporon asahii, the most susceptible
yeast with MIC values ranging between 1.95–500 µmol/L. None of the presented derivatives reached
the activity of fluconazole against C. albicans, C. tropicalis and C. glabrata, whereas four showed for
C. krusei a lower MIC than fluconazole. Additionally, four derivatives surpassed the MIC of
fluconazole for T. asahii after 24 h of incubation (1h, 1j, 1r, 1s; 1f and 1i were comparable) and three
salicylanilides after 48 h (1i, 1j, 1s). Undecylenic acid, an established non-specific antimycotic agent for
topical usage [21], exhibited mostly MIC values  500 µmol/L (except 250 µmol/L for T. asahii).
Based on earlier findings [5,16,18], we expected a higher activity of the salicylanilide scaffold-based
molecules towards moulds. However, only two esters (2j, 2r) inhibited the growth of A. fumigatus at
15.62–31.25 µmol/L. Seventeen compounds affected A. corymbifera with MICs of 3.9–500 µmol/L
(salicylanilides 1a, 1b, 1c, 1d, 1f, 1g, 1i, 1j, 1o, 1r, 1s, 1t and five esters 2c, 2h, 2j, 2k, 2r); only one
ester 2f and one salicylanilide 1p did not exhibit any activity towards Trichophyton mentagrophytes.
This strain was assayed as being the most susceptible (MIC  0.49 µmol/L) from the evaluated panel
of fungi. Moreover, based on a comparison of MIC values, the mechanism of the action seems to be
fungicidal like against A. corymbifera and in contrast to Candida strains. Twenty derivatives (1a, 1b,
1c, 1d, 1f, 1g, 1h, 2h, 1i, 1j, 2j, 1k, 1l, 1m, 1o, 1q, 2q, 1r, 2r, 1s) surpassed the activity of fluconazole
towards T. mentagrophytes with 1i and 2j showing superiority (0.49–0.98 µmol/L). Undecylenic acid
stopped the growth of T. mentagrophytes at 250 µmol/L.
In general, the most antifungal active salicylanilide assayed was N-(4-bromophenyl)-4-chloro-2-
hydroxybenzamide (1j). Bromine and 4-trifluoromethyl moiety-containing salicylanilides expressed
mostly a significant activity towards T. asahii, A. corymbifera and T. mentagrophytes, while
dichlorosalicylanilides were only active against the last strain. It is difficult to postulate distinct
structure-activity relationships due to a lot of partially inactive derivatives; for some strains the activity
is only sporadic.
Similarly, the fact that for most of the esters it was not possible to determine their MIC values,
complicates the systematic evaluation of how the esterification by 4-(trifluoromethyl)benzoic acid
influenced the activity of the parent salicylanilides. In some cases the esterification improved the
activity (e.g., 1e vs. 2e and 1r vs. 2r for Candida sp., 1j vs. 2j for filamentous fungi, 1p vs. 2p for
T. mentagrophytes), for others the impact was the reverse (e.g., 1j vs. 2j for yeasts, 1c vs. 2c for
moulds, 1f vs. 2f, 1k vs. 2k). However, only the esters inhibited A. fumigatus, C. tropicalis and also
C. krusei (additionally with 1j) at low MIC values. Additionally, some 4-(trifluoromethyl)benzoates
(2h, 2j, 2r) share submicromolar MICs for T. mentagrophytes after 72 h of incubation.
Although lipophilicity is generally presumed to belong to the factors influencing antifungal activity,
there is no sharp correlation in this series, as demonstrated above for the pairs of salicylanilide vs. its
ester. The most lipophilic salicylanilide 1e was not the unambiguously most active derivative; its MIC
values were even below-average. Similarly, the least lipophilic fluorinated salicylanilides 1k, 1l, 1m
and 1n showed a better activity in some cases than more lipophilic molecules like 1e or 1p.

Molecules 2012, 17
9433
Similarly, compounds with a comparable calculated logP showed non-uniform MIC values
(e.g., 1b vs. 1j, 2h vs. 2p). Moreover, if the antifungal properties depend only on the lipophilicity, the
salicylanilides derived from 5-chlorosalicylic acid and 3-substituted anilines should be unanimously
more active than those synthesized from 4-chlorosalicylic acid and 4-substituted anilines; but this clear
relationship was not observed and N-(4-bromophenyl)-4-chloro-2-hydroxybenzamide (1j) revealed the
most significant in vitro antifungal features. However, the logP values presented here are only
calculated values, not experimentally found; therefore their significance should be viewed with caution.
Similarly, it was shown that the individual biological impacts of related compounds with various
substitution patterns may be in part often influenced by the volume of the substituents. This bulkiness
can be expressed, e.g., by a bulk parameter like MR (molar refractivity) [9,22]. The esterification of
salicylanilides by 4-(trifluoromethyl)benzoic acid led to the larger molecules with a calculated
increment of MR of 34.74. However, as pointed out previously, this modification did not improve the
activity unequivocally. Similarly, bulkier derivatives 1e, 1f, 2e and 2f with two chlorines on the aniline
ring (MR for these atoms together = 9.6) did not present uniformly superior activity, and only some
MICs are excellent, when the remainder are average, weak or comparatively high. On the other hand,
the replacement of chlorine (MR = 4.8) by a bulkier bromine (MR = 7.6) on the salicylic acid ring
resulted in the improvement of the activity towards T. asahii and A. corymbifera (1q vs. 1s), while for
other strains this brought no additional benefit. Similarly, anilines containing bromine (MR = 7.6)
showed a higher antifungal activity than those substituted by chlorine (4.8), fluorine (−0.4) and
it is slightly superior to a trifluoromethyl group (MR = 4.0). These data indicate, analogously to
lipophilicity, that there is no simple linear correlation between biological activity and steric parameter MR.
When compared to other recently evaluated salicylanilide esters, 4-(trifluoromethyl)benzoates did
not surpass the activity of salicylanilide acetates [16], although some MICs are comparable. Contrarily,
benzoates [17] are much less potent in vitro than the here presented esters. The comparison with
salicylanilide pyrazinoates [18] and esters with N-benzyloxycarbonyl amino acids [23] is not
unambiguous, while these derivatives showed more consistent activity; however, MICs of many
4-(trifluoromethyl)benzoates exceeded them. Esters with N-acetyl-L-phenylalanine showed less and
less uniform antifungal potency [5]. In some cases, the esterification of parent salicylanilides by
various carboxylic acids led to a decreased activity.
3. Experimental
3.1. General Methods
All the reagents and solvents were purchased from Sigma-Aldrich (Seelze, Germany) or Penta
Chemicals (Prague, Czech Republic) and they were used as received. Reactions and purity of products
were monitored by thin layer chromatography with a toluene/ethyl-acetate 4:1 mixture as eluent for
salicylanilides and a toluene/methanol 9:1 mixture as eluent for esters; plates were coated with 0.2 mm
Merck 60 F254 silica gel and were visualized by UV irradiation (254 nm). Melting points were
determined on a Büchi Melting Point machine B-540 apparatus using open capillaries and the reported
values are uncorrected.
Elemental analysis (C, H, N) were performed on an automatic microanalyser CHNS-O CE
instrument (FISONS EA 1110, Milano, Italy). Infrared spectra (ATR) were recorded on FT-IR

Molecules 2012, 17
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spectrometer Nicolet 6700 FT-IR in the range of 400–4,000 cm−1. The NMR spectra were recorded on
1
13
a Varian Mercury-Vxbb 300 (300 MHz for H and 75.5 MHz for C; Varian, Inc., Palo Alto, CA,
USA) at ambient temperature using deuterated dimethylsulfoxide (DMSO-d6) solutions of the samples.
The chemical shifts δ are given in ppm, with respect to tetramethylsilane as an internal standard. The
coupling constants (J) are reported in Hz.
The calculated logP values (ClogP), that are the logarithms of the partition coefficients for octan-1-
ol/water, were determined using the program ACD/ChemSketch (Freeware) version 12.01 (Advanced
Chemistry Development, Inc., Toronto, ON, Canada); while the program CS ChemOffice Ultra version
12.0 (CambridgeSoft, Cambidge, MA, USA) did not allow us to distinguish lipophilicity for particular
position isomers providing the same logP data. Only the mean values without deviations are
reported in this work. The increment of molar refractivity due to esterification of salicylanilides by
4-(trifluoromethyl)benzoic acid was calculated using the program ACD/ChemSketch (Freeware)
version 12.01.
3.2. Synthesis of Salicylanilides
A substituted salicylic acid and corresponding aniline (both 0.002 mol) were suspended in
chlorobenzene (20 mL) and PCl3 (0.001 mol) was added. The reaction was carried out with vigorously
stirring in a microwave reactor (530 W, 600 rpm, MicroSYNTH Milestone) for 20 min to refluxing.
The reaction mixture was filtered while hot, let stand at 20 °C and then at +4 °C for 24 h. The crude
product was filtered off and recrystallized from boiling 96% ethanol to obtain the pure product, in all
cases white crystals. Salicylanilides, whose physical and spectral properties are not presented here,
were synthesized and reported previously [24,25].
N-(3-Bromophenyl)-4-chloro-2-hydroxybenzamide (1h). Yield 79%; m.p.: 224.5–226.5 °C. IR: 3307,
1608 (CO amide), 1547, 1497, 1425, 1376, 1228, 1204, 1093, 1070, 927, 861, 848, 777, 762, 717, 677.
1H-NMR: δ 11.87 (1H, s, OH), 10.42 (1H, bs, NH), 8.04 (1H, s, H2′), 7.88 (1H, d, J = 8.3 Hz, H6),
7.67–7.60 (1H, m, H6′), 7.32–7.29 (2H, m, H4′, H5′), 7.05–7.00 (2H, m, H3, H5). 13C-NMR: δ 165.6,
158.7, 140.0, 137.6, 131.2, 130.9, 126.9, 123.1, 121.7, 119.6, 119.5, 117.8, 116.9. Anal. Calcd. for
C13H9BrClNO2 (326.57): C, 47.81; H, 2.78; N, 4.29. Found: C, 47.99; H, 2.83; N, 4.10.
4-Chloro-2-hydroxy-N-[3-(trifluoromethyl)phenyl]benzamide (1p). Yield 85%; m.p.: 193–195 °C.
IR: 3322, 3057, 1633, 1614 (CO amide), 1603, 1656, 1494, 1423, 1334, 1313, 1223, 1170, 1124, 1099,
1
1072, 923, 891, 864, 794, 711, 698. H-NMR: δ 11.83 (1H, s, OH), 10.56 (1H, bs, NH), 8.20 (1H, s,
H2′), 7.94–7.87 (2H, m, H6, H6′), 7.59 (1H, t, J = 8.0 Hz, H5′), 7.46 (1H, d, J = 7.8 Hz, H4′),
7.05–7.01 (2H, m, H3, H5). 13C-NMR: δ 165.9, 158.8, 139.2, 137.7, 131.2, 130.1, 129.7 (q, J = 31.8 Hz),
124.4, 124.3 (q, J = 272.6 Hz), 120.6 (q, J = 3.8 Hz), 119.5, 117.8, 116.9 (q, J = 3.8 Hz). Anal. Calcd.
for C14H9ClF3NO2 (315.67): C, 53.27; H, 2.87; N, 4.44. Found: C, 53.55; H, 2.70; N, 4.55.
3.3. Synthesis of Salicylanilide 4-(Trifluoromethyl)benzoates
4-(Trifluoromethyl)benzoic acid and appropriate salicylanilide (both 0.001 mol) were dissolved in
dry DMF (15 mL), the solution was cooled to −20 °C and DCC in a mild excess (0.0012 mol) was
added in three portions during 1 h. Next the mixture was stirred for 3 h at the same temperature and

Molecules 2012, 17
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stored at +4 °C for 48 h. After this time, the precipitated by-product N,N′-dicyclohexylurea was filtered
off and the solvent was evaporated in vacuo. The rest was dissolved in a small amount of ethyl-acetate
and the insoluble portion (another N,N′-dicyclohexylurea) was filtered off. The filtrate was
again evaporated in vacuo. The crude product was purified by the repeated crystallization from
ethyl-acetate/hexane; all esters are white solids.
4-Chloro-2-(3-chlorophenylcarbamoyl)phenyl-4-(trifluoromethyl)benzoate (2a). Yield 63%; m.p.:
184–186 °C. IR (ATR): 3308, 2934, 2858, 1718 (CO ester), 1666, 1650, 1588, 1522, 1479, 1452,
1425, 1410, 1323, 1291, 1271, 1205, 1167, 1131, 1113, 1088, 1066, 1015, 861, 766, 693, 680.
1H-NMR: δ 10.71 (1H, bs, NH), 8.26 (2H, d, J = 8.1 Hz, H2′′, H6′′), 7.94 (2H, d, J = 8.3 Hz, H3′′,
H5′′), 7.87 (1H, d, J = 2.6 Hz, H3), 7.77–7.71 (3H, m, H5, H6, H2′), 7.54 (1H, d, J = 8.7 Hz, H6′), 7.30
13
(1H, t, J = 8.1 Hz, H5′), 7.11 (1H, ddd, J = 0.9 Hz, J = 2.1 Hz, J = 8.0 Hz, H4′). C-NMR: δ 163.3,
162.9, 146.9, 140.2, 133.7 (q, J = 32.1 Hz), 133.1, 132.4, 131.9, 130.9, 130.8, 130.7, 130.6, 129.3, 126.2
(q, J = 3.7 Hz), 125.6, 123.8, 123.7 (q, J = 272.9 Hz), 119.5, 118.4. Anal. Calcd. for C21H12Cl2F3NO3
(454.23): C, 55.53; H, 2.66; N, 3.08. Found: C, 55.27; H, 2.80; N, 3.22.
5-Chloro-2-(3-chlorophenylcarbamoyl)phenyl-4-(trifluoromethyl)benzoate (2b). Yield 83%; m.p.:
165.5–168 °C. IR (ATR): 3284, 2933, 2857, 1738 (CO ester), 1650, 1602, 1593, 1543, 1483, 1451,
1
1413, 1323, 1259, 1242, 1168, 1130, 1112, 1068, 1016, 860, 699, 673. H-NMR: δ 10.67 (1H, bs,
NH), 8.27 (2H, d, J = 8.1 Hz, H2′′, H6′′), 7.94 (2H, d, J = 8.3 Hz, H3′′, H5′′), 7.82 (1H, d, J = 8.3 Hz,
H3), 7.73–7.71 (2H, m, H6, H2′), 7.60 (1H, dd, J = 8.3 Hz, J = 2.1 Hz, H4), 7.50 (1H, dd, J = 8.3 Hz,
J = 2.0, H6′), 7.30 (1H, t, J = 8.1 Hz, H5′), 7.11 (1H, m, H4′). 13C-NMR: δ 163.4, 163.2, 148.8, 140.3,
136.0, 133.8 (q, J = 32.1 Hz), 133.1, 132.4, 131.1, 130.9, 130.6, 128.3, 126.7, 126.2 (q, J = 3.6 Hz),
124.0, 123.9 (q, J = 272.9 Hz), 123.8, 119.4, 118.4. Anal. Calcd. for C21H12Cl2F3NO3 (454.23): C,
55.53; H, 2.66; N, 3.08. Found: C, 55.34; H, 2.85; N, 3.01.
4-Chloro-2-(4-chlorophenylcarbamoyl)phenyl-4-(trifluoromethyl)benzoate (2c). Yield 49%; m.p.:
170–172.5 °C. IR (ATR): 3322, 3073, 2934, 1719 (CO ester), 1667, 1625, 1601, 1547, 1491, 1420, 1399,
1324, 1291, 1244, 1221, 1172, 1134, 1112, 1095, 1067, 1013, 822, 705, 656. 1H-NMR: δ 10.67 (1H, bs,
NH), 8.25 (2H, d, J = 8.2 Hz, H2′′, H6′′), 7.93 (2H, d, J = 8.3 Hz, H3′′, H5′′), 7.86 (1H, d, J = 2.6 Hz,
H3), 7.73 (1H, dd, J = 8.8 Hz, J = 2.1 Hz, H5), 7.62 (1H, d, J = 8.9 Hz, H6), 7.54–7.40 (2H, m, H2′,
13
H6′), 7.34–7.31 (2H, m, H3′, H5′). C-NMR: δ 163.3, 162.7, 146.9, 137.8, 133.7 (q, J = 32.0 Hz),
133.2, 130.9, 130.8, 130.3, 129.2, 128.8, 128.0, 126.2 (q, J = 3.6 Hz), 125.6, 123.9 (q, J = 272.8 Hz), 121.6.
Anal. Calcd. for C21H12Cl2F3NO3 (454.23): C, 55.53; H, 2.66; N, 3.08. Found: C, 55.40; H, 2.51; N, 3.34.
5-Chloro-2-(4-chlorophenylcarbamoyl)phenyl-4-(trifluoromethyl)benzoate (2d). Yield 86%; m.p.:
170.5–173 °C. IR (ATR): 3305, 2933, 2858, 1737 (CO ester), 1699, 1650, 1601, 1544, 1492, 1452,
1
1408, 1325, 1258, 1240, 1182, 1157, 1128, 1111, 1097, 1068, 1016, 829, 769, 698, 669. H-NMR: δ
10.63 (1H, bs, NH), 8.26 (2H, d, J = 8.1 Hz, H2′′, H6′′), 7.95 (2H, d, J = 8.3 Hz, H3′′, H5′′), 7.82 (1H,
d, J = 8.3 Hz, H3), 7.72 (1H, d, J = 2.0 Hz, H6), 7.54–7.40 (3H, m, H4′, H2′, H6′), 7.34–7.30 (2H, m,
13
H3′, H5′). C-NMR: δ 163.6, 163.2, 148.9, 136.1, 133.7 (q, J = 32.0 Hz), 132.4, 131.1, 130.8, 128.1,
127.9, 126.7, 126.2 (q, J = 3.6 Hz), 125.5, 124.2, 123.9 (q, J = 272.9 Hz), 119.8. Anal. Calcd. for
C21H12Cl2F3NO3 (454.23): C, 55.53; H, 2.66; N, 3.08. Found: C, 55.69; H, 2.47; N, 2.86.

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4-Chloro-2-(3,4-dichlorophenylcarbamoyl)phenyl-4-(trifluoromethyl)benzoate (2e). Yield 73%; m.p.:
156–158.5 °C. IR (ATR): 3313, 3079, 2933, 2858, 1718 (CO ester), 1671, 1650, 1627, 1580, 1518,
1478, 1451, 1384, 1323, 1278, 1249, 1204, 1174, 1134, 1107, 1087, 1066, 1016, 862, 823, 767, 696.
1H-NMR: δ 10.80 (1H, bs, NH), 8.25 (2H, d, J = 8.1 Hz, H2′′, H6′′), 7.94 (2H, d, J = 8.3 Hz, H3′′,
H5′′), 7.90–7.87 (2H, m, H2′, H3), 7.75 (1H, dd, J = 2.6 Hz, J = 8.7 Hz, H5), 7.61 (1H, d, J = 8.8 Hz,
H6), 7.56–7.52 (2H, m, H5′, H6′). 13C-NMR: δ 163.3, 162.9, 146.9, 138.9, 133.7 (q, J = 32.1 Hz),
132.4, 132.1, 131.1, 130.9, 130.8, 130.7, 130.6, 129.3, 126.2 (q, J = 3.7 Hz), 125.7, 125.6, 123.9 (q,
J = 272.8 Hz), 121.2, 120.0. Anal. Calcd. for C21H11Cl3F3NO3 (488.67): C, 51.61; H, 2.27; N, 2.87.
Found: C, 51.77; H, 2.41; N, 2.64.
5-Chloro-2-(3,4-dichlorophenylcarbamoyl)phenyl-4-(trifluoromethyl)benzoate (2f). Yield 74%; m.p.:
144–146 °C. IR (ATR): 3306, 29333, 2858, 1748 (CO ester), 1699, 1649, 1601, 1581, 1525, 1477,
1452, 1409, 1379, 1325, 1261, 1250, 1194, 1164, 1130, 1120, 1068, 1016, 864, 769, 698. 1H-NMR: δ
10.77 (1H, bs, NH), 8.25 (2H, d, J = 8.1 Hz, H2′′, H6′′), 7.94 (2H, d, J = 8.3 Hz, H3′′, H5′′), 7.91 (1H,
s, H2′), 7.82 (1H, d, J = 8.3 Hz, H3), 7.74 (1H, d, J = 2.0 Hz, H6), 7.61 (1H, dd, J = 2.0 Hz, J = 8.5 Hz,
H4), 7.57–7.52 (2H, m, H5′, H6′). 13C-NMR: δ 163.4, 163.2, 148.8, 138.9, 136.1, 133.7 (q, J = 31.8 Hz),
132.4, 131.1, 131.0, 130.9, 130.8, 127.9, 126.8, 126.2 (q, J = 3.7 Hz), 125.6, 124.0, 123.8 (q, J = 272.3 Hz),
121.1, 120.0. Anal. Calcd. for C21H11Cl3F3NO3 (488.67): C, 51.61; H, 2.27; N, 2.87. Found: C, 51.48;
H, 2.11; N, 2.98.
2-(3-Bromophenylcarbamoyl)-4-chlorophenyl-4-(trifluoromethyl)benzoate (2g). Yield 49%; m.p.:
196–198 °C. IR (ATR): 3319, 2932, 2853, 1717 (CO ester), 1666, 1650, 1625, 1573, 1521, 1478,
1451, 1410, 1323, 1291, 1271, 1241, 11167, 1132, 1112, 1088, 1066, 1016, 893, 860, 766, 696, 686.
1H-NMR: δ 10.68 (1H, bs, NH), 8.26 (2H, d, J = 8.1 Hz, H2′′, H6′′), 7.95 (2H, d, J = 8.2 Hz, H3′′,
H5′′), 7.88–7.85 (2H, m, H2′, H3), 7.75 (1H, dd, J = 2.6 Hz, J = 8.7 Hz, H5), 7.63 (1H, d, J = 8.0 Hz,
13
H6), 7.54 (1H, d, J = 8.7 Hz, H6′), 7.26–7.23 (2H, m, H4′, H5′). C-NMR: δ 163.3, 162.8, 146.9,
140.4, 133.7 (q, J = 32.1 Hz), 132.4, 131.9, 130.9, 130.9, 130.8, 130.7, 129.3, 126.8, 126.2 (q, J = 3.7 Hz),
125.6, 123.8 (q, J = 268.2 Hz), 122.4, 121.6, 118.8. Anal. Calcd. for C21H12BrClF3NO3 (498.68): C,
50.58; H, 2.43; N, 2.81. Found: C, 50.72; H, 2.19; N, 2.55.
2-(3-Bromophenylcarbamoyl)-5-chlorophenyl-4-(trifluoromethyl)benzoate (2h). Yield 85%; m.p.:
166–168.5 °C. IR (ATR): 3305, 2933, 2857, 1738 (CO ester), 1650, 1589, 1537, 1479, 1452, 1411,
1323, 1258, 1242, 1169, 1130, 1111, 1068, 1016, 861, 770, 698, 675. 1H-NMR: δ 10.65 (1H, bs, NH),
8.26 (2H, d, J = 7.9 Hz, H2′′, H6′′), 7.94 (2H, d, J = 7.9 Hz, H3′′, H5′′), 7.85 (1H, s, H2′), 7.82 (1H, d,
J = 8.3 Hz, H3), 7.72 (1H, d, J = 1.9 Hz, H6), 7.65–7.53 (2H, m, H4, H6′), 7.31 (1H, m, H5′), 7.23
(1H, m, H4′). 13C-NMR: δ 163.4, 163.2, 148.8, 140.4, 136.0, 133.7 (q, J = 32.3 Hz), 131.1, 130.9,
130.8, 128.2, 127.9, 126.7, 126.6, 126.2 (q, J = 3.6 Hz), 123.9, 123.4 (q, J = 272.9 Hz), 122.3, 121.6,
118.8. Anal. Calcd. for C21H12BrClF3NO3 (498.68): C, 50.58; H, 2.43; N, 2.81. Found: C, 50.46; H, 2.58;
N, 2.71.
2-(4-Bromophenylcarbamoyl)-4-chlorophenyl-4-(trifluoromethyl)benzoate (2i). Yield 53%; m.p.:
173–175.5 °C. IR (ATR): 3329, 2932, 2857, 1717 (CO ester), 1672, 1650, 1523, 1487, 1451, 1394,
1
1323, 1277, 1242, 1203, 1172, 1133, 1112, 1088, 1066, 1016, 860, 817, 767, 695. H-NMR: δ 10.66
(1H, bs, NH), 8.25 (2H, d, J = 8.1 Hz, H2′′, H6′′), 7.94 (2H, d, J = 8.3 Hz, H3′′, H5′′), 7.86 (1H, d,

Molecules 2012, 17
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J = 2.6 Hz, H6), 7.73 (1H, dd, J = 2.6 Hz, J = 8.7 Hz, H5), 7.63 (1H, d, J = 8.1 Hz, H3), 7.58-7.52
(2H, m, H2′, H6′), 7.47-7.44 (2H, m, H3′, H5′). 13C-NMR: δ 163.3, 162.7, 146.9, 138.2, 133.6 (q,
J = 32.0 Hz), 132.5, 131.8, 131.7, 130.9, 130.8, 130.6, 129.2, 126.2 (q, J = 3.7 Hz), 125.6, 123.8 (q,
J = 273.0 Hz), 121.9, 115.8. Anal. Calcd. for C21H12BrClF3NO3 (498.68): C, 50.58; H, 2.43; N, 2.81.
Found: C, 50.40; H, 2.67; N, 2.64.
2-(4-Bromophenylcarbamoyl)-5-chlorophenyl-4-(trifluoromethyl)benzoate (2j). Yield 72%; m.p.:
152.5–154.5 °C. IR (ATR): 3291, 2933, 1737 (CO ester), 1650, 1602, 1544, 1489, 1449, 1406, 1325,
1257, 1241, 1182, 1156, 1128, 1111, 1069, 1015, 893, 861, 824, 770, 698, 667. 1H-NMR: δ 10.63 (1H,
bs, NH), 8.25 (2H, d, J = 8.1 Hz, H2′′, H6′′), 7.94 (2H, d, J = 8.3 Hz, H3′′, H5′′), 7.81 (1H, d, J = 8.3 Hz,
H3), 7.71 (1H, d, J = 2.1 Hz, H6), 7.64–7.55 (3H, m, H4, H2′, H6′), 7.46–7.43 (2H, m, H3′, H5′).
13C-NMR: δ 163.2, 162.7, 148.8, 138.3, 135.9, 133.7 (q, J = 32.1 Hz), 132.4, 131.7, 131.0, 130.9,
128.3, 126.7, 126.2 (q, J = 3.7 Hz), 123.9, 123.8 (q, J = 272.8 Hz), 121.9, 115.7. Anal. Calcd. for
C21H12BrClF3NO3 (498.68): C, 50.58; H, 2.43; N, 2.81. Found: C, 50.36; H, 2.65; N, 2.94.
4-Chloro-2-(3-fluorophenylcarbamoyl)phenyl-4-(trifluoromethyl)benzoate (2k). Yield 76%; m.p.:
151–153.5 °C. IR (ATR): 3317, 2933, 2857, 1721 (CO ester), 1699, 1650, 1543, 1444, 1410, 1323,
1272, 1236, 1167, 1154, 1130, 1115, 1089, 1065, 1020, 859, 844, 766, 686. 1H-NMR: δ 10.73 (1H, bs,
NH), 8.26 (2H, d, J = 7.9 Hz, H2′′, H6′′), 7.94 (2H, d, J = 7.9 Hz, H3′′, H5′′), 7.86 (1H, s, H3), 7.73
(1H, d, J = 8.5 Hz, H5), 7.64–7.49 (2H, m, H6, H6′), 7.39–7.29 (2H, m, H2′, H5′), 6.88 (1H, t,
13
J = 8.3 Hz, H4′). C-NMR: δ 163.3, 162.8, 162.1 (d, J = 241.5 Hz), 146.9, 140.5 (d, J = 11.1 Hz),
133.7 (q, J = 31.7 Hz), 132.5, 131.9, 130.9, 130.7, 130.5 (d, J = 9.4 Hz), 129.2, 127.9, 126.2 (q, J = 3.6 Hz),
125.6, 123.8 (q, J = 273.0 Hz), 115.8 (d, J = 2.8 Hz), 110.6 (d, J = 21.2 Hz), 106.7 (d, J = 26.2 Hz).
Anal. Calcd. for C21H12ClF4NO3 (437.77): C, 57.62; H, 2.76; N, 3.20. Found: C, 57.88; H, 2.99; N, 3.03.
5-Chloro-2-(3-fluorophenylcarbamoyl)phenyl-4-(trifluoromethyl)benzoate (2l). Yield 55%; m.p.:
169–171.5 °C. IR (ATR): 3320, 2932, 2852, 1739 (CO ester), 1651, 1624, 1602, 1569, 1548, 1491,
1
1414, 1325, 1259, 1242, 1200, 1172, 1138, 1122, 1070, 1016, 861, 770, 700, 669. H-NMR: δ 10.70
(1H, bs, NH), 8.26 (2H, d, J = 8.0 Hz, H2′′, H6′′), 7.94 (2H, d, J = 8.1 Hz, H3′′, H5′′), 7.82 (1H, d,
J = 8.3 Hz, H3), 7.72 (1H, d, J = 1.8 Hz, H6), 7.59 (1H, dd, J = 1.7 Hz, J = 8.3 Hz, H4), 7.52 (1H, d,
J = 11.4 Hz, H6′), 7.38–7.28 (2H, m, H2′, H5′), 6.87 (1H, t, J = 8.4 Hz, H4′). 13C-NMR: δ 163.4,
163.2, 162.1 (d, J = 241.5 Hz), 148.8, 140.6 (d, J = 10.9 Hz), 135.9, 133.7 (q, J = 32.1 Hz), 132.4,
131.0, 130.9, 130.5 (d, J = 9.4 Hz), 128.3, 126.7, 126.2 (q, J = 3.7 Hz), 124.0, 123.8 (q, J = 272.8 Hz),
115.7 (d, J = 2.8 Hz), 110.5 (d, J = 21.0 Hz), 106.7 (d, J = 26.2 Hz). Anal. Calcd. for C21H12ClF4NO3
(437.77): C, 57.62; H, 2.76; N, 3.20. Found: C, 57.39; H, 3.02; N, 3.47.
4-Chloro-2-(4-fluorophenylcarbamoyl)phenyl-4-(trifluoromethyl)benzoate (2m). Yield 70%; m.p.:
181–183.5 °C. IR (ATR): 3329, 2933, 2858, 1721 (CO ester), 1665, 1650, 1572, 1530, 1508, 1479,
1451, 1408, 1323, 1279, 1235, 1167, 1132, 1114, 1087, 1065, 1016, 825, 767, 695. 1H-NMR: δ 10.58
(1H, bs, NH), 8.25 (2H, d, J = 7.8 Hz, H2′′, H6′′), 7.94 (2H, d, J = 7.9 Hz, H3′′, H5′′), 7.85 (1H, d,
J = 2.4 Hz, H3), 7.77–7.73 (1H, dd, J = 2.4 Hz, J = 8.7 Hz, H5), 7.64–7.51 (3H, m, H6, H2′, H6′),
7.13–7.09 (2H, m, H3′, H5′). 13C-NMR: δ 163.3, 162.5, 158.5 (d, J = 240.6 Hz), 146.9, 135.2 (d,
J = 2.6 Hz), 133.6 (q, J = 32.0 Hz), 132.5, 131.7, 131.0, 130.9, 130.6, 129.2, 126.2 (q, J = 3.8 Hz),

Molecules 2012, 17
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125.6, 123.8 (q, J = 272.9 Hz), 121.9 (d, J = 7.9 Hz), 115.5 (d, J = 22.3 Hz). Anal. Calcd. for
C21H12ClF4NO3 (437.77): C, 57.62; H, 2.76; N, 3.20. Found: C, 57.95; H, 2.52; N, 3.26.
5-Chloro-2-(4-fluorophenylcarbamoyl)phenyl-4-(trifluoromethyl)benzoate (2n). Yield 72%; m.p.:
176.5–179 °C. IR (ATR): 3294, 2933, 2857, 1737 (CO ester), 1646, 1602, 1556, 1508, 1452, 1413,
1
1328, 1260, 1234, 1192, 1156, 1137, 1126, 1111, 1069, 1016, 861, 833, 771, 699, 669. H-NMR: δ
10.55 (1H, bs, NH), 8.26 (2H, d, J = 7.8 Hz, H2′′, H6′′), 7.94 (2H, d, J = 7.8 Hz, H3′′, H5′′), 7.81 (1H,
d, J = 8.2 Hz, H3), 7.71 (1H, d, J = 1.9 Hz, H6), 7.64–7.59 (3H, m, H4, H2′, H6′), 7.13–7.08 (2H, m,
H3′, H5′). 13C-NMR: δ 163.2, 163.0, 158.5 (d, J = 240.5 Hz), 148.8, 135.7, 135.2 (d, J = 2.5 Hz),
133.7 (q, J = 32.2 Hz), 132.5, 131.0, 130.9, 128.5, 126.7, 126.2 (q, J = 3.7 Hz), 123.9, 123.8 (q,
J = 272.9 Hz), 121.8 (d, J = 7.9 Hz), 115.5 (d, J = 22.2 Hz). Anal. Calcd. for C21H12ClF4NO3 (437.77):
C, 57.62; H, 2.76; N, 3.20. Found: C, 57.94; H, 2.59; N, 2.94.
4-Chloro-2-(3-(trifluoromethyl)phenylcarbamoyl)phenyl-4-(trifluoromethyl)benzoate (2o). Yield 68%;
m.p.: 158.5–160.5 °C. IR (ATR): 3306, 2933, 2858, 1719 (CO ester), 1699, 1670, 1650, 1543, 1450,
1409, 1323, 1276, 1236, 1168, 1131, 1108, 1066, 1020, 844, 766, 696, 687. 1H-NMR: δ 10.84 (1H, bs,
NH), 8.26 (2H, d, J = 8.1 Hz, H2′′, H6′′), 7.94–7.84 (4H, m, H3, H2′, H3′′, H5′′), 7.76 (1H, dd, J = 8.7 Hz,
J = 2.5 Hz, H5), 7.63 (1H, d, J = 8.0 Hz, H6′), 7.56 (1H, d, J = 8.7 Hz, H6), 7.52 (1H, t, J = 7.9 Hz,
13
H5′), 7.40 (1H, d, J = 7.8 Hz, H4′). C-NMR: δ 163.3, 163.0, 146.9, 139.5, 133.7 (q, J = 32.1 Hz),
132.4, 132.0, 130.9, 130.8, 130.7, 130.2, 129.8 (q, J = 29.8 Hz), 129.3, 126.1 (q, J = 3.7 Hz), 125.5,
124.4 (q, J = 263.2 Hz), 123.8 (q, J = 272.2 Hz), 123.6, 120.5 (q, J = 3.7 Hz), 116.2 (q, J = 3.7 Hz).
Anal. Calcd. for C22H12ClF6NO3 (487.78): C, 54.17; H, 2.48; N, 2.87. Found: C, 54.02; H, 2.57; N, 2.99.
5-Chloro-2-(3-(trifluoromethyl)phenylcarbamoyl)phenyl-4-(trifluoromethyl)benzoate (2p). Yield 50%;
m.p.: 160–162.5 °C. IR (ATR): 3290, 2934, 2858, 1739 (CO ester), 1699, 1650, 1601, 1558, 1543,
1
1492, 1413, 1322, 1259, 1246, 1164, 1133, 1067, 1016, 884, 862, 802, 771, 697. H-NMR: δ 10.80
(1H, bs, NH), 8.27 (2H, d, J = 8.1 Hz, H2′′, H6′′), 7.95–7.83 (5H, m, H3, H2′, H6′, H3′′, H5′′), 7.73
(1H, d, J = 2.1 Hz, H6), 7.60 (1H, dd, J = 8.3 Hz, J = 2.0 Hz, H4), 7.51 (1H, t, J = 8.0 Hz, H5′), 7.38
(1H, d, J = 7.8 Hz, H4′). 13C-NMR: δ 163.5, 163.2, 148.8, 139.6, 136.1, 133.7 (q, J = 32.1 Hz), 132.3,
131.1, 130.9, 130.1, 129.8 (q, J = 30.7 Hz), 128.2, 126.8, 126.1 (q, J = 3.7 Hz), 124.1 (q, J = 272.3 Hz),
123.9, 123.8 (q, J = 272.9 Hz), 123.5, 120.4 (q, J = 3.9 Hz), 116.1 (q, J = 4.1 Hz). Anal. Calcd. for
C22H12ClF6NO3 (487.78): C, 54.17; H, 2.48; N, 2.87. Found: C, 54.46; H, 2.65; N, 2.67.
4-Chloro-2-(4-(trifluoromethyl)phenylcarbamoyl)phenyl-4-(trifluoromethyl)benzoate (2q). Yield 75%;
m.p.: 166.5–169 °C. IR (ATR): 3330, 2933, 2857, 1717 (CO ester), 1676, 1650, 1527, 1478, 1452,
1
1409, 1322, 1280, 1257, 1237, 1168, 1126, 1110, 1088, 1066, 1017, 861, 843, 767, 697. H-NMR: δ
10.89 (1H, bs, NH), 8.25 (2H, d, J = 8.1 Hz, H2′′, H6′′), 7.92 (2H, d, J = 8.2 Hz, H3′′, H5′′), 7.90 (1H,
d, J = 2.6 Hz, H3), 7.81 (2H, d, J = 8.5 Hz, H2′, H6′), 7.76 (1H, dd, J = 2.6 Hz, J = 8.7 Hz, H5),
13
7.64 (2H, d, J = 8.7 Hz, H3′, H5′), 7.55 (1H, d, J = 8.7 Hz, H6). C-NMR: δ 163.3, 163.1, 147.0,
142.4, 133.7 (q, J = 32.1 Hz), 132.4, 132.0, 130.9, 130.8, 130.7, 129.3, 126.2 (m), 125.6, 124.5 (q,
J = 271.4 Hz), 124.4 (q, J = 31.8 Hz), 123.9 (q, J = 272.9 Hz), 119.9. Anal. Calcd. For C22H12ClF6NO3
(487.78): C, 54.17; H, 2.48; N, 2.87. Found: C, 54.42; H, 2.71; N, 3.14.

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5-Chloro-2-(4-(trifluoromethyl)phenylcarbamoyl)phenyl-4-(trifluoromethyl)benzoate (2r). Yield 70%;
m.p.: 142–143.5 °C. IR (ATR): 3346, 2932, 2856, 1719 (CO ester), 1671, 1649, 1602, 1524, 1485,
1451, 1407, 1321, 1281, 1251, 1179, 1165, 1120, 1089, 1064, 1017, 863, 843, 824, 761, 697.
1H-NMR: δ 10.85 (1H, bs, NH), 8.25 (2H, d, J = 8.1 Hz, H2′′, H6′′), 7.93 (2H, d, J = 8.2 Hz, H3′′,
H5′′), 7.85 (1H, d, J = 8.3 Hz, H3), 7.80 (2H, d, J = 8.3 Hz, H2′, H6′), 7.73 (1H, d, J = 2.0 Hz, H6),
13
7.63 (2H, d, J = 8.6 Hz, H3′, H5′), 7.60 (1H, dd, J = 2.1 Hz, J = 8.3 Hz, H4). C-NMR: δ 163.6,
163.2, 148.9, 142.5, 136.1, 133.7 (q, J = 32.1 Hz), 132.4, 131.1, 130.9, 128.1, 127.8, 126.7, 126.1 (m),
124.4 (q, J = 271.3 Hz), 124.0 (q, J = 32.0 Hz), 123.8 (q, J = 273.0 Hz), 119.8. Anal. Calcd. for
C22H12ClF6NO3 (487.78): C, 54.17; H, 2.48; N, 2.87. Found: C, 54.33; H, 2.65; N, 2.69.
4-Bromo-2-(4-(trifluoromethyl)phenylcarbamoyl)phenyl-4-(trifluoromethyl)benzoate (2s). Yield 67%;
m.p.: 162–163.5 °C. IR (ATR): 3325, 2933, 2858, 1717 (CO ester), 1699, 1674, 1650, 1528, 1452,
1
1408, 1322, 1279, 1236, 1167, 1129, 1110, 1089, 1066, 1017, 844, 766, 687. H-NMR: δ 10.88 (1H,
bs, NH), 8.25 (2H, d, J = 8.1 Hz, H2′′, H6′′), 8.03 (1H, d, J = 2.4 Hz, H3), 7.92 (2H, d, J = 8.3 Hz,
H3′′, H5′′), 7.88 (1H, dd, J = 2.6 Hz, J = 8.8 Hz, H5), 7.80 (2H, d, J = 8.5 Hz, H2′, H6′), 7.66–7.61
(2H, m, H3′, H5′), 7.48 (1H, d, J = 8.7 Hz, H6). 13C-NMR: δ 163.3, 163.0, 147.4, 142.4, 135.0, 133.7
(q, J = 32.3 Hz), 132.4, 132.0, 131.0, 130.9, 126.2 (m), 125.9, 124.4 (q, J = 32.0 Hz), 124.8 (q,
J = 274.1 Hz), 123.5 (q, J = 268.6 Hz), 119.9, 118.8. Anal. Calcd. for C22H12BrF6NO3 (532.23): C,
49.65; H, 2.27; N, 2.63. Found: C, 49.43; H, 2.12; N, 2.54.
3.4. Antifungal Activity Determination
The antifungal properties of all synthesized compounds were evaluated in vitro against four
Candida strains (Candida albicans ATCC 44859, Candida tropicalis 156, Candida krusei E28, and
Candida glabrata 20/I), Trichosporon asahii 1188 and three filamentous fungi (Aspergillus fumigatus
231, Absidia corymbifera 272, and Trichophyton mentagrophytes 445).
The microdilution broth method was used according to the CLSI M27-A3 and M38-A2 guidelines
for yeasts [26] and moulds [27] in RPMI 1640 with glutamine (KlinLab, Prague, Czech Republic)
buffered to pH 7.0 with 0.165 mol of 3-morpholino-propane-1-sulphonic acid (Sigma-Aldrich, Seelze,
Germany). DMSO served as a diluent for all compounds. In the yeast, the final size of the inoculum
was 5 × 103 ± 0.2 CFU/mL. For the moulds “Aspergillus, Trichophyton and Absidia corymbifera”, the
spores were harvested after the cultivation of a given fungal strain grown on Sabouraud agar from 3 to
10 days. The final size of the inoculum was 0.5–5 ×104 CFU/mL. The sizes of the fungal inocula were
checked using a Bürker’s chamber. Fluconazole was used as a reference specific drug and undecylenic
acid as a non-specific one. The minimum inhibitory concentrations (MIC) were assayed as an 80%
(IC80) or higher reduction of growth in comparison to the control (for salicylanilides and for yeasts in
the case of 4-(trifluoromethyl)benzoates) or as an 50% (IC50) or higher reduction of growth (for the
activity of 4-(trifluoromethyl)benzoates towards filamentous fungi) in comparison to the control. The
determination of results was performed visually and spectrophotometrically (at 540 nm). The MIC
values were determined after 24 and 48 h of incubation in the dark at 35 °C (±0.1) in a humid
atmosphere; only for T. mentagrophytes the final MIC were determined after 72 and 120 h of
incubation. MICs were determined twice and in duplicate.

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4. Conclusions
In summary, we have synthesized nineteen salicylanilides and eighteen corresponding esters with
4-(trifluoromethyl)benzoic acid. New compounds were characterised and all derivatives were
evaluated as potential antimycotic agents towards eight fungal strains. It was not possible to determine
MICs of a range of the esters because of a low solubility and/or a precipitation in the testing medium.
Salicylanilides and their esters affected the fungal growth from 0.49 µmol/L; however, their activity is
not uniform and especially against Candida spp. it seems to be most likely sporadic. However, some of
the here described compounds may represent derivatives with a promising in vitro antifungal properties.
Acknowledgements
This work was financially supported by GAUK 27610/2010, IGA NT 13346 (2012). The
publication is a result of the project implementation: Support of establishment, development, and
mobility of quality research teams at the Charles University, project number CZ.1.07/2.3.00/30.0022,
supported by The Education for Competitiveness Operational Programme (ECOP) and co-financed
by the European Social Fund and the state budget of the Czech Republic. We want to thank to Ida
Dufková for the excellent performance of antibiotic susceptibility tests and the staff of the Department
of Inorganic and Organic Chemistry, Faculty of Pharmacy, for the technical assistance and
J. Urbanová, M.A., for the language help.
Conflict of Interest
The authors declare no conflict of interest.
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© 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).