Salicylanilide esters of N-protected amino acids as novel antimicrobial agents

Article abstract of DOI:10.1016/j.bmcl.2008.11.080

A series of novel, highly antimicrobial salicylanilide esters of N-protected amino acids were synthesized and characterized. Their in vitro antimicrobial activity against eight fungal strains and Mycobacterium tuberculosis was determined. The compounds had the highest level of activity against Aspergillus fumigatus, Absidia corymbifera and Trichophyton mentagrophytes, and these levels were higher than that of the standard drug fluconazole. In addition, three compounds showed interesting antituberculosis activity, with inhibition ranging from 89% to 99%. (S)-4-Chloro-2-(4-trifluoromethylphenylcarbamoyl)-phenyl 2-benzyloxy-carbonylamino-propionate had the highest level of both antifungal and antimycobacterial activity. The structure-activity relationships of the new compounds are discussed.

Full text of DOI:10.1016/j.bmcl.2008.11.080

Bioorganic & Medicinal Chemistry Letters 19 (2009) 348–351
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Salicylanilide esters of N-protected amino acids as novel antimicrobial agents
a,b,
a,
a
c
d,e
*
*
´
Aleš Imramovsky
, Jarmila Vinšová , Juana Monreal Férriz , Vladimír Buchta , Josef Jampílek
a Department of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles University, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic
b Institute of Organic Chemistry and Technology, Faculty of Chemical Technology, University of Pardubice, nam. Cs. Legii 565, 532 10 Pardubice, Czech Republic
c Faculty of Medicine and University Hospital, Charles University, Sokolska 581, 500 05 Hradec Kralove, Czech Republic
d Zentiva a.s., U kabelovny 130, 102 37 Prague, Czech Republic
e Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Palackeho 1/3, 612 42 Brno, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel, highly antimicrobial salicylanilide esters of N-protected amino acids were synthesized
and characterized. Their in vitro antimicrobial activity against eight fungal strains and Mycobacterium
tuberculosis was determined. The compounds had the highest level of activity against Aspergillus fumiga-
tus, Absidia corymbifera and Trichophyton mentagrophytes, and these levels were higher than that of the
standard drug fluconazole. In addition, three compounds showed interesting antituberculosis activity,
with inhibition ranging from 89% to 99%. (S)-4-Chloro-2-(4-trifluoromethylphenylcarbamoyl)-phenyl
2-benzyloxy-carbonylamino-propionate had the highest level of both antifungal and antimycobacterial
activity. The structure–activity relationships of the new compounds are discussed.
Received 9 September 2008
Revised 20 November 2008
Accepted 21 November 2008
Available online 27 November 2008
Keywords:
N-protected amino acids esters
Salicylanilides derivates
Antifungal activity
Ó 2008 Elsevier Ltd. All rights reserved.
Antimycobacterial activity
Salicylanilides (2-hydroxy-N-phenylbenzamides) have been re-
ported as a class of compounds with a wide variety of interesting
biological activities, including antimycobacterial and antifungal ef-
fects.1–5 Although the antibacterial activity involves multiple
mechanisms, these compounds have been shown to be inhibitors
of the two-component regulatory systems (TCS) of bacteria.6,7
The most recent studies identified them as selective inhibitors of
interleukin-12p40 production8 and inhibitors of the protein kinase
epidermal growth factor receptor (EGFR PTK).9,10
A dramatic rise in the incidence of life-threatening systemic
fungal infections has been observed over the past two decades.11
This can be ascribed to an increase in the number of immuno-com-
promised patients due to the growth of the number of HIV-infected
individuals, cases of cancer chemotherapy and the indiscriminate
use of antibiotics. The majority of antifungal agents in clinical
use suffer from various drawbacks in terms of toxicity, efficacy
and cost, and their frequent use has led to the emergence of resis-
tant strains. Hence, there is a great demand for new antifungal
agents belonging to a wide range of structural classes, acting selec-
tively on novel targets and with fewer unwanted side-effects.12
No new drug class has been introduced in the past 50 years for
the treatment of tuberculosis (TB).13 Currently, patients require 6–
9 months of treatment. This long period leads to the lack of
compliance, which in turn can be responsible for the relapse and
incidence of MDR-TB strains.14 There is an urgent need to develop
a new potent and fast-acting antituberculosis drug with a low tox-
icity profile. The new drug must be suitable for use in conjunction
with the drugs currently used for the treatment of HIV infection,
which are active against both growing and latent infections of TB.
There is a need for antibacterial agents with a novel mechanism
of action and the salicylanilides are promising candidates. An elec-
tron-withdrawing group on the salicyloyl ring and hydrophobic
groups on the anilide moiety, as well as the 2-hydroxy group, are
essential for their optimal antimicrobial effect. The salicylanilides
substituted with halogens in both parts meet the requirements
and form the most active derivatives possessing antifungal and anti-
tuberculosis activity against some atypical strains of mycobacteria.1
Their unsuitable physical properties led us to modify the struc-
ture by esterification of amino acids with suitable salicylanilides. In
addition, amino acids facilitate the possibility of targeting drugs as
a drug delivery system. Prepared esters can be considered as pro-
drug forms of salicylanilides with better bioavailability and, due
to the higher degree of liphophilicity, more efficient transport
through the mycobacterial cell membrane.
Here, we report the synthesis, experimental determination of
liphophilicity by reversed phase high performance liquid chroma-
tography (RP-HPLC), antifungal and antimycobacterial activity, as
well as SAR of a series of salicylanilide derivatives.
The starting salicylanilide compounds were obtained by the
reaction of 5-chlorosalicylic acid and appropriate anilines in a
microwave reactor in the presence of PCl3 and chlorobenzene as
a solvent. A method using dicyclohexycarbodiimide (DCC) in anhy-
drous DMF was chosen for esterification of the N-benzyloxycar-
bonyl amino acids (Scheme 1).15
* Corresponding authors. Tel.: +420 495 067 343; fax: +420 495 506 166.
E-mail addresses: ales.imramovsky@faf.cuni.cz (A. Imramovsky´), jarmila.vinsova
@faf.cuni.cz (J. Vinšová).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.080

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A. Imramovsky et al. / Bioorg. Med. Chem. Lett. 19 (2009) 348–351
Scheme 1. Synthesis of N-protected amino acid esters. Reagents: (a) PCl3, chlorobenzene, microwave reactor; (b) DCC, DMF.
All the substituted salicylanilide esters of amino acids were
analyzed for lipophilicity using RP-HPLC. The procedure was per-
formed under isocratic conditions with methanol as an organic
modifier in the mobile phase using an end-capped non-polar C18
stationary RP column.
The hydrophobicity (logP/ClogP data) of each of the compounds
4a–4j were calculated using two commercially available programs
(CS ChemOffice 9.0 and ACD/LogP 1.0) and measured by RP-HPLC
determination of the capacity factor k with the subsequent calcu-
lation of log k. The results are shown in Table 1(A).
vidual R/S-enantiomers cannot be explained on the basis of the re-
sults presented here.
As expected, the lipophilicity of compounds 4a–j substituted
in the R2 position increased in the order: R2 = H (compounds 4a,
4d, 4h and 4j) < R2 = CH3 (compounds 4b/4c and 4i) < R2 =
CH(CH3)2 (compounds 4e/4f) < R2 = CH2C6H5 (compound 4g). The
dependence between logk and the alkyl/phenylalkyl substituents
in the individual series of the compounds (R2 = H, –CH3,
–
CH(CH3)2, –CH2C6H5) is approximately linear, as shown in
Figure 1.
Logk data specify the lipophilicity within this series of com-
pounds. Neither of the computer programs resolved lipophilicity
parameters within the series of enantiomers; nevertheless, the chi-
ral compounds 4b, 4c, 4e–4g and 4i were measured several times
with the same results. The differences in logk parameters for indi-
All the N-benzyloxycarbonyl amino acid esters of salicylanilides
were tested for their in vitro antifungal and antituberculosis
activity.
The in vitro antifungal screening was used for the eight strains
at concentrations in the range 62.5–1.95 lmol/L.
Table 1
Lipophilicity and antimicrobial evaluation of the new compounds
Compound
R1
R2
logk
logP/ClogP (ChemOffice)
logP (ACD/logP)
(A) Comparison of the logk values with the calculated lipophilicity of the new compounds
4a
4b
4c
4d
4e
4f
4g
4h
4i
3-Cl
3-Cl
3-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-CF3
4-CF3
3,4-Cl
H
0.6119
0.6995
0.6990
0.6367
0.9421
0.9417
1.0971
0.7292
0.8149
0.8639
4.71/5.14625
5.20/5.45525
5.20/5.45525
4.71/5.14625
6.09/6.38325
6.09/6.38325
6.88/6.87325
5.07/5.50945
5.57/5.81845
5.27/5.7738
5.04 0.48
5.39 0.48
5.39 0.48
5.00 0.47
6.23 0.48
6.23 0.48
7.28 0.40
5.03 0.51
7.28 0.40
5.91 0.50
(S)-CH3
(R)-CH3
H
(S)-CH(CH3)2
(R)-CH(CH3)2
(S)-CH2C6H5
H
(S)-CH3
H
4j
MIC/IC80
(l
mol/L)
M. tbc H37Rv
CA
CT
CK
CG
TB
AF
AC
TM
MIC (l
g/mL)
Inh. (%)
24h
48h
24h
48h
24h
48h
24h
48h
24h
48h
24h
48h
24h
48h
72h
120h
(B) The in vitro antifungal and antituberculotic activity of N-benzyloxycarbonyl amino acids esters of salicylanilides
4a
4b
4c
>62.5
>62.5
31.25
>62.5
62.5
>62.5
62.5
62.5
>62.5
>62.5
>62.5
>62.5
62.5
>62.5
>62.5
>62.5
>62.5
62.5
>62.5
31.25
31.25
>62.5
>62.5
>62.5
>62.5
31.25
31.25
125
>125
500
500
>62.5
>62.5
0.12
>125
—
62.5
62.5
62.5
>62.5
31.25
>62.5
62.5
62.5
62.5
62.5
>62.5
>62.5
>62.5
>62.5
31.25
31.25
31.25
>125
125
250
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
62.5
>62.5
>62.5
31.25
>62.5
>62.5
31.25
62.5
>62.5
>62.5
62.5
62.5
31.25
31.25
7.81
7.81
7.81
>6.25
3.13
7
97
36
16
38
77
16
89
99
0
62.5
15.63
15.63
31.25
31.25
31.25
62.5
>62.5
>62.5
>62.5
>62.5
15.63
15.63
31.25
31.25
31.25
62.5
31.25
31.25
31.25
31.25
31.25
31.25
31.25
15.63
31.25
31.25
15.63
15.63
31.25
31.25
7.81
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
125
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
3.13
7.81
4d
4e
4f
15.63
15.63
15.63
15.63
15.63
15.63
7.81
4g
4h
4i
62.5
62.5
62.5
62.5
7.81
31.25
31.25
62.5
15.63
15.63
1.95
>125
7.81
250
31.25
>62.5
>62.5
3.91
15.62
—
250
15.62
>62.5
>62.5
>125
>125
—
1.95
4j
>62.5
>62.5
0.06
0.12
—
>62.5
>62.5
0.24
0.48
—
62.5
>62.5
>125
>125
—
31.25
31.25
1.95
3.91
—
>6.25
—
FLU
INH
0.98
3.91
—
—
0.025–0.216
—
CA, Candida albicans ATCC 44859; CT, Candida tropicalis 156; CK, Candida krusei E28; CG, Candida glabrata 20/I; TB, Trichosporon beigelii 1188; AF, Aspergillus fumigatus 231; AC,
Absidia corymbifera 272 and TM, Trichophyton mentagrophytes 445.

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A. Imramovsky et al. / Bioorg. Med. Chem. Lett. 19 (2009) 348–351
350
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
The levels of antituberculosis activity of compounds 4a–4j gi-
ven in Table 1(B) suggest that lipophilicity is again a secondary
parameter for antituberculosis activity, and there is no correlation
between logk and the antituberculosis activity of these com-
pounds. Only the range of preferable hydrophobicity from 0.7 to
0.8 can be observed.
The type and the position of substitution are of great impor-
0
tance. The most appropriate is substitution on the C(4) position
by the CF3 moiety.
The values of individual substituent electron-deficiency ex-
pressed as Hammett’s parameter
r are connected with the
4a
4d
4b
4c
4h
4i
4j
4e
4f
4g
above-mentioned facts. Hammett’s constants rm for meta and rp
for the para positions of the individual substituents were taken
from the literature.19
Compounds
log k log P [ChemOffice] Clog P [ChemOffice] log P [ACD/LogP]
The electron-withdrawing effect caused by 4-CF3 substitution
(rp = 0.54 for 4i, 4h) and/or 3-Cl substitution (rm = 0.37 for 4b)
seems to be the most important for antituberculosis activity. The
optimum of electron deficiency, which corresponds to a higher le-
vel of antifungal activity, is approximately 0.5 and the range is 0.4–
0.6. Any value outside that range causes a decrease in the level of
Figure 1. Comparison of logP/ClogP data calculated using the two computer
programs with the experimentally determined logk values. The compounds are
ordered according to the increase in logk values in the series of compounds 4a–4j.
All the esters had the highest level of activity against Trichophy-
ton mentagrophytes 445. Most had a higher level of activity than
fluconazole, especially against Aspergillus fumigatus 231 and Absi-
dia corymbifera 272. For the results of the antifungal assays, see Ta-
ble 1(B).
Compounds 4i, 4g and 4b had the highest level of activity. On
the basis of this fact (dependence of antifungal activity/lipophilic-
ity) it can be supposed that hydrophobicity is only a secondary
parameter, see Table 1. Compounds 4a and 4j had considerably dif-
ferent degrees of lipophilicity but similar levels of activity. The
same was observed for compounds 4d, 4h, 4e/4f and 4c/4b, 4g,
where the effect of various substituents, including CF3, Cl and/or
H, CH3, CH(CH3)2, CH2C6H5, with different degrees of hydrophobic-
ity on antifungal activity was minor.
activity; for example, 4-Cl rp = 0.23 for 4f and 3,4-Cl rp+m = 0.60 for
4j.
Low-bulky substituent, that is, methyl, or absence of R2-substi-
tution is another parameter for high antituberculosis activity. Sub-
stitution by a bulkier moiety than methyl, that is, isopropyl or
benzyl, was associated with a significantly decreased level of
activity.
A very important parameter influencing the activity is stereo-
isomerism, because individual enantiomers demonstrate consider-
able difference in their antituberculosis activity, for example,
(S)-enantiomer 4e showed much lower activity than (R)-enantio-
mer 4f and on the contrary (S)-enantiomer 4b showed much higher
activity than (R)-enantiomer 4c. The determined differences in
antituberculosis activity for individual R/S-enantiomers cannot be
explained on the basis of the results presented here. These facts
are under intensive investigation.
When the position and the identity of individual substitutions
0
were compared, substitution at the C(4) position seemed to be
more important to increase the antifungal activity than substitu-
tion at the C(3) position of the anilide ring. R2-substitution by a
0
methyl or a benzyl moiety seemed to be better than that by an iso-
propyl moiety. The benzyl derivative 4g may be considered as a
phenyl cyclic analogue of compound 4i with a similar level of
activity or a benzyl analogue of compound 4d but with a higher le-
vel of activity. Branching of the methyl moiety is unlikely and
would cause a decreased level of activity. Similar structure–activity
relationships among alkyl analogues (a decreased level of biologi-
cal activity due to branching of the methyl moiety) were reported
by Vinsova et al.17 Unfortunately, compound R1 = 4-Cl, R2 = CH3,
which was expected to be highly active, was not prepared due to
unexpected cyclization during the synthesis.15
On the basis of the facts discussed above, it can be assumed that
the stereospecific bond can be probably formed between the com-
pound and enzyme in M. tuberculosis with subsequent enzyme
inhibition.
In summary, a new type of salicylanilide pro-drug was designed
and several representatives were synthesized. The series was
screened for antituberculosis and antifungal activity. Most of the
tested compounds possessed a high level of in vitro antituberculo-
sis activity. An antifungal assay showed levels of activity against
Aspergillus fumigatus, Absidia corymbifera and Trichophyton mentag-
rophytes similar to that of fluconazole. Relationships between
structure and biological activity, including the experimentally
determined degree of liphophilicity, are discussed.
All the compounds prepared in this study were screened at the
Tuberculosis Antimicrobial Acquisition and Coordinating Facility
(TAACF) run by The National Institute of Health of the US govern-
ment.18 The primary screening was conducted at 6.25
lg/mL (or
Acknowledgments
molar equivalent of the highest molecular weight compound in a
series of congeners) against Mycobacterium tuberculosis H37Rv
(ATCC 27294) in BACTEC 12B medium using the Microplate Alamar
Blue Assay (MABA). The compounds exhibiting fluorescence were
tested in the BACTEC 460-radiometric system. The compounds
This study was supported by MSM 0021620822. Antimycobac-
terial data were provided by the Tuberculosis Antimicrobial Acqui-
sition and Coordinating Facility (TAACF) through a research and
development contract with the U.S. National Institute of Allergy
and Infectious Diseases (the TAACF Contract No. N01-AI-95364).
showing <90% inhibition in the primary screen (MIC > 6.25
mL) were not generally evaluated further.
lg/
Compounds 4i, 4b and 4h showed interesting antituberculosis
activity with inhibition levels in the range 89–99%, and the results
are given in Table 1(B). The highest levels of activity against
M. tuberculosis H37Rv were found for (S)-4-chloro-2-(4-trifluorom-
ethylphenylcarbamoyl)-phenyl 2-benzyloxy-carbonylamino-pro-
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.11.080.
pionate (4i, 99% inhibition, MIC = 3.13
(3-chlorophenyl-carbamoyl)-phenyl 2-benzyloxycarbonylamino-
propionate (4b, 97% inhibition, MIC = 3.13 g/mL).
lg/mL) and (S)-4-chloro-2-
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