New derivatives of salicylamides: Preparation and antimicrobial activity against various bacterial species

Article abstract of DOI:10.1016/j.bmc.2013.08.029

Three series of salicylanilides, esters of N-phenylsalicylamides and 2-hydroxy-N-[1-(2-hydroxyphenylamino)-1-oxoalkan-2-yl]benzamides, in total thirty target compounds were synthesized and characterized. The compounds were evaluated against seven bacterial and three mycobacterial strains. The antimicrobial activities of some compounds were comparable or higher than the standards ampicillin, ciprofloxacin or isoniazid. Derivatives 3f demonstrated high biological activity against Staphylococcus aureus (≤0.03 μmol/L), Mycobacterium marinum (≤0.40 μmol/L) and Mycobacterium kansasii (1.58 μmol/L), 3g shows activity against Clostridium perfringens (≤0.03 μmol/L) and Bacillus cereus (0.09 μmol/L), 3h against Pasteurella multocida (≤0.03 μmol/L) and M. kansasii (≤0.43 μmol/L), 3i against methicillin-resistant S. aureus and B. cereus (≤0.03 μmol/L). The structure-activity relationships are discussed for all the compounds.

Full text of DOI:10.1016/j.bmc.2013.08.029

Bioorganic & Medicinal Chemistry 21 (2013) 6574–6581
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New derivatives of salicylamides: Preparation and antimicrobial
activity against various bacterial species
a
b,c,d
a,
e
c
⇑
ˇ
´
Karel Pauk , Iveta Zadrazilová
, Aleš Imramovsky , Jarmila Vinšová , Michaela Pokorná ,
Martina Masaríková , Alois Cízek c,d, Josef Jampílek b
c,d
ˇ
ˇ
ˇ
a Institute of Organic Chemistry and Technology, Faculty of Chemical Technology, University of Pardubice, Studentská 573, 532 10 Pardubice, Czech Republic
b Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Palackého 1/3, 612 42 Brno, Czech Republic
c Department of Infectious Diseases and Microbiology, Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences, Palackého 1/3,
612 42 Brno, Czech Republic
d CEITEC VFU, University of Veterinary and Pharmaceutical Sciences, Palackého 1/3, 612 42 Brno, Czech Republic
e Charles University, Faculty of Pharmacy, Department of Inorganic and Organic Chemistry, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 July 2013
Revised 12 August 2013
Accepted 14 August 2013
Available online 24 August 2013
Three series of salicylanilides, esters of N-phenylsalicylamides and 2-hydroxy-N-[1-(2-hydroxyphenyla-
mino)-1-oxoalkan-2-yl]benzamides, in total thirty target compounds were synthesized and character-
ized. The compounds were evaluated against seven bacterial and three mycobacterial strains. The
antimicrobial activities of some compounds were comparable or higher than the standards ampicillin,
ciprofloxacin or isoniazid. Derivatives 3f demonstrated high biological activity against Staphylococcus
aureus (60.03
l
mol/L), Mycobacterium marinum (60.40
l
mol/L) and Mycobacterium kansasii
mol/L) and Bacillus cereus
mol/L), 3h against Pasteurella multocida (60.03 lmol/L) and M. kansasii (60.43 lmol/L), 3i against
Keywords:
Salicylamide derivatives
Antibacterial
(1.58
(0.09
l
l
mol/L), 3g shows activity against Clostridium perfringens (60.03 l
methicillin-resistant S. aureus and B. cereus (60.03
cussed for all the compounds.
l
mol/L). The structure–activity relationships are dis-
Antimycobacterial
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
acquired infections and livestock associated infections with high
levels of morbidity and mortality. Methicillin resistance is con-
nected with clinically inadequate susceptibility not only to all b-
lactam antibiotics but usually also to other antimicrobial drugs
(macrolides, clindamycin, fluoroquinolones).4,5 Also veterinary cli-
nicians face bacterial pathogens causing serious diseases in ani-
mals (e.g., Clostridium perfringens as a major cause of enteritis in
livestock,6 Pasteurella multocida causing respiratory tract infec-
tions,7 etc.). All of these diseases are of economic significance, so
new, potent and fast acting antibacterial drugs could serve as a
solution not only to the economic consequences of these diseases.
Therefore there is an urgent need to develop new, potent and fast
acting anti-tuberculosis and anti-MRSA drugs.8
Variously substituted salicylanilides 3 (N-substituted hydrox-
ybenzamides), see Figure 1, are well-known organic compounds
with a wide range of pharmacological activities, including antimi-
crobial9 and antifungal10 effects that become more and more
important. They have also found use as mollusicidal11 or anthel-
mintic agents12 in human and veterinary practice. The latest stud-
ies described this compounds as inhibitors of apicomplexan
parasite Toxoplasma gondii.13,14 Mechanism of their action is still
under investigation. It is known that salicylanilides can serve as
inhibitors of protein kinase epidermal growth factor receptor
(EGFR PTK),15 further studies extended the group of inhibitors of
The increasing number of mycobacterial, bacterial, viral and
associated fungal infections underlines the importance of search-
ing for new antimicrobial chemotherapeutics with a target effect.
Tuberculosis (TB) is a common, and in many cases lethal, infectious
disease caused by various strains of mycobacteria. It is very alarm-
ing that about one-third of the world’s population (two billion peo-
ple) is infected with Mycobacterium tuberculosis (MTB) and 10% of
them will progress to the active disease. The highest evidence of
occurrence of tuberculosis disease is in India, China, Indonesia,
Nigeria, and Bangladesh.1 The treatment of this disease is mediated
by administration of various antimicrobial chemotherapeutics,
however it is generally recognized that the massive application
of these chemotherapeutics is the main reason of increased antibi-
otic resistance among bacteria.2,3 Moreover, the antibiotic resis-
tance of the important Gram-positive pathogen Staphylococcus
aureus has become one of the most challenging and persistent
worldwide health problems. Methicillin-resistant S. aureus (MRSA)
has caused life-threatening nosocomial infections for the decades
and has recently become a significant threat for community
⇑
Corresponding author. Tel.: +420 466037739; fax: +420 466038004.
E-mail address: Ales.Imramovsky@upce.cz (A. Imramovsky´).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.08.029

K. Pauk et al. / Bioorg. Med. Chem. 21 (2013) 6574–6581
6575
Figure 1. Discussed salicylanilide-like compounds 3, 4, 6 and partial numbering of their skeleton.
the new dual inhibitors of HDAC-EGFR.16 They are generally de-
signed to compete with ATP for binding in catalytic domain of tyr-
osin kinase.17,18 The latest studies specified them also as selective
inhibitors of interleukin-12p40 production that plays a specific
role in initiation, expansion and control of cellular response to
tuberculosis.19,20 It was also proved that salicylanilides inhibit bac-
3 by using N,N-dicyclohexylcarbodiimide activation.36 Prepared
esters25 4a–l were treated by solution of hydrobromic acid in ace-
tic acid (33%) for smooth deprotection of the N-benzyloxycarbonyl
group. The classical deprotection using catalytical hydrogenation
was also tested, but this method failed. Desired 2-hydroxy-N-
[(2S)-1-oxo-1-(phenylamino)alkan-2-yl]benzamides 6a–i were ob-
tained by stirring hydrobromic salt 5 in chloroform in the presence
of a base via the rearrangement described in literature.25,37
terial sortase A,21 -alanine ligase22 and transglykosylas-
D-alanine–D
es,23 that is, enzymes participating in secretion of various proteins
or synthesis of cell wall. Recently it was described that salicylani-
lides-like derivatives inhibit essential mycobacterial enzymes
methionine aminopeptidase catalyzing a key step of the posttrans-
lational modification of nascent proteins and isocitrate lyase that is
indispensable for the metabolism of fatty acids.24 In spite of the
promise of salicylanilides as potential drugs, their widespread
use in clinical practice was prevented by their physico-chemical
properties, such as low solubility. Therefore, improvement of the
physico-chemical properties of salicylanilides is an interesting
and vital area of research.
An interesting and unprecedented attribute of N-protected ami-
no esters of N-phenylsalicylamides 4, see Figure 1, was discovered
during investigation of salicylanilides modification. An unexpected
rearrangement of esters of N-phenylsalicylamides to 2-hydroxy-N-
[1-(2-hydroxyphenylamino)-1-oxoalkan-2-yl]benzamides 625 was
observed, see Figure 1. Due to their characteristic skeleton were
these compounds called ‘diamides’. The ‘diamides’ were published
within preparation of a combinatorial library.26 More recently, our
research group has found them to be potential antimicrobial
agents,27 presenting in vitro antimycobacterial activity against M.
tuberculosis and against some nontuberculous strains, such as M.
avium and M. kansasii. A number of derivatives were prepared
and their antimycobacterial activity was investigated. The activity
of these derivatives was found to be comparable or higher than
that of the starting salicylanilide compounds.28–33
2.2. Biological activities
As discussed above, the compounds under investigation could
be divided into three groups based on their chemical structure:
Group I includes anilides 3a–i; Group II contains esters 4a–l; and
Group III includes diamides 6a–i. All compounds showed a wide
range of antimicrobial activities, and some interesting structure–
activity relationships were observed. Biological activity against
various mycobacterial strains was published in many arti-
cles,9,10,29,35 but the absolute novelty is the biological activity of
salicylanilides and their derivatives against chosen bacterial spe-
cies (S. aureus, Bacillus cereus, P. multocida, C. perfringens). All the
results of antimicrobial screening are summarized in Tables 1
and 2.
2.2.1. In vitro antibacterial activity
Most of compounds were tested for their in vitro antibacterial
activity against six Gram-positive bacterial strains such as S. aureus
ATCC 29213 (SA), methicillin-resistant S. aureus (MRSA 63718, SA
630, SA 3202), B. cereus ATCC 14579 (BC) and C. perfringens CNCTC
5770 (CP) and against one Gram-negative bacterial strain P. multo-
cida (PM). The results of the screening are summarized in Table 1.
The screened salicylanilides and their derivatives (esters and diam-
ides) showed very interesting biological activity against the
mentioned strains; particularly the activity of compounds 3f–i,
4g, 4h, 4j–l, 6f, 6h, 6i was comparable or higher than that of the
standards. In general it can be concluded that antibacterial activity
decreases as follows: Group I (salicylanilides) > Group II (esters of
salicylanilides) > Group III (diamides).
This study is a follow-up paper to recently published arti-
cles.27,30–33 A series of salicylanilide derivatives with general struc-
ture 3, 4 or 6 were synthesized and evaluated against bacterial and
mycobacterial strains. The structure–activity relationships
between the structure and in vitro biological activities of all the
evaluated compounds are discussed.
Within Group I (salicylanilides) a significant antimicrobial
activity was exhibited, especially by compounds 3e–h and 3i that
contain a halogen group on the aniline ring. The presence of halo-
gen seems to be crucial for high biological activity. These deriva-
tives, especially compounds 3i, 3g and 3f, showed activity
against all tested strains. These facts support the hypothesis about
the necessary presence of the halogen or trifluoromethyl moiety on
the aniline ring; in the case of 3g and 3i the aniline ring contains
2. Results and discussion
2.1. Chemistry
The synthetic approach to preparation of 2-hydroxy-N-[(2S)-1-
oxo-1-(phenylamino)alkan-2-yl]benzamides 6a–i was mediated
via the known rearrangement that was described by Imramovsky
et al.25,26 The synthetic pathway begins from substituted salicylic
acid 1 and appropriate anilines 2, see Scheme 1. This step was car-
ried out in a microwave reactor using phosphorus trichloride in
chlorobenzene34 to give salicylanilides 3a–i in very good yields
(70–85%); these results were comparable with classical synthesis
described by Waisser et al.35 N-Benzyloxycarbonyl amino acids
(N-Cbz-AA) were further esterified with prepared salicylanilides
0
two chlorines in positions C(3) and C(4)0, in the case of 3f it contains
CF3 in position C(4)0. Based on the results of antimicrobial effect of
3i and 3g it also seems that the substitution of C(4) position in the
salicylic ring is more advantageous than that of C(5) position. It can
be generally stated (see Table 1) that the activity within Group I in-
creases with the increase of lipophilicity (see Fig. 2) and electron-
withdrawing properties (expressed as Hammett’s
r parameters) of
R2 substituents (see Fig. 3). In Figure 2 the dependence of

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K. Pauk et al. / Bioorg. Med. Chem. 21 (2013) 6574–6581
Scheme 1. Synthesis of 2-hydroxy-N-[(2S)-1-oxo-1-(phenylamino)alk-2-yl]benzamides. Reagents and conditions: (a) PCl3, PhCl, MW, 400 W, 20 min; (b) DCC, DMF, ꢀ10 °C,
2 h; N-Cbz-AA (c) 33% HBr/AcOH, RT, 30 min; (d) TEA, CHCl3, RT, 30 min.
Table 1
Structure of discussed compounds, their calculated lipophilicity (logP) and in vitro antibacterial activity (MIC) of compounds compared to ampicillin (APC) and ciprofloxacin
(CPX) standards
Comp.
R1
R2
R3
logPa
MIC (lmol/L)
SA
MRSA 63718
SA 630
SA 3202
BC
CP
PM
3a
3b
3c
3d
3e
3f
3g
3h
3i
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
4-Cl
H
—
—
—
—
—
—
—
—
—
4.24
4.72
4.20
4.82
5.00
5.08
5.43
3.91
5.64
16.15
>978
>921
28.35
0.76
60.03
0.79
0.85
32.30
>978
>921
56.71
0.76
0.10
0.77
1.71
0.05
16.15
>978
>921
28.35
0.76
0.20
0.77
0.85
60.03
16.15
>978
>921
56.71
0.76
0.20
0.77
1.71
0.05
32.30
30.56
>921
14.17
0.38
0.10
0.09
0.85
60.03
>1034
>978
>921
14.17
3.06
1.58
60.03
1.71
1.01
1.91
28.80
3.54
0.19
0.20
0.19
60.03
0.20
4-CH3
4-OCH3
4-Cl
4-Br
4-CF3
3,4-Cl
4-NO2
3,4-Cl
60.03
0.20
4a
4b
4c
4d
4e
4f
4g
4h
4i
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
5-Cl
5-Cl
5-Cl
4-H
(S)-iso-Pr
(S)-Me
(S)-iso-Pr
(S)-Bn
(R)-CH2-indolyl
(S)-iso-Pr
(S)-CH2-cHx
(S)-iso-Pr
(S)-iso-Pr
(S)-iso-Pr
(S)-iso-Pr
(S)-Bn
5.21
4.53
5.16
6.57
6.50
5.27
7.20
5.84
5.03
5.81
6.42
7.20
>517
34.26
32.31
29.46
27.48
>501
3.25
3.64
3.80
3.57
1.81
>517
>548
129
>517
>548
129
>517
>548
64.63
29.46
54.97
>501
1.62
0.91
3.80
1.78
0.22
>517
7.13
32.31
29.46
27.48
250
1.62
0.91
7.60
1.78
0.45
0.41
>517
>548
>517
29.46
109
>501
3.25
1.82
3.80
3.57
0.90
1.67
>517
2.14
4.04
7.36
6.87
62.62
0.40
1.82
0.95
0.89
0.90
0.83
4-CH3
4-CH3
4-CH3
4-CH3
4-OCH3
4-Br
4-CF3
4-NO2
4-Br
58.93
54.97
>501
1.62
0.91
3.80
3.57
0.22
0.20
29.46
54.97
>501
1.62
0.91
3.80
1.78
0.45
0.20
4j
4k
4l
3,4-Cl
3,4-Cl
1.67
0.20
6a
6b
6c
6d
6e
6f
6g
6h
6i
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
4-Cl
4-Cl
4-Cl
4-CH3
4-CH3
4-CH3
4-CH3
4-OCH3
4-CF3
4-Br
(S)-Me
(S)-iso-Pr
(S)-Bn
(R)-CH2-indolyl
(S)-iso-Pr
(S)-iso-Pr
(S)-iso-Pr
(S)-iso-Pr
(S)-Bn
3.88
4.45
5.25
5.31
4.16
4.98
5.24
5.72
5.89
>769
88.68
>626
>571
>679
4.82
9.39
2.40
1.07
>769
>709
>626
>571
>679
9.64
18.79
4.81
2.15
>769
>709
>626
>571
>679
4.82
9.39
2.40
2.15
>769
88.68
>626
>571
>679
4.82
9.39
2.40
1.07
>769
22.17
>626
>571
>679
2.41
2.34
1.20
1.07
>769
88.68
>626
>571
>679
4.82
9.39
38.48
34.50
>769
88.68
>626
>571
>679
>617
>601
>615
>552
3,4-Cl
3,4-Cl
APC
CPX
—
—
—
—
—
—
—
—
5.72
>48.29
>45.79
>48.29
>45.79
>48.29
>45.79
>48.29
>45.79
—
<0.72
—
1.43
—
a
Calculated using the sw. ACD/Percepta ver. 2012; SA = Staphylococcus aureus ATCC 29213, MRSA = methicillin-resistant S. aureus 63718 or SA 630 or SA 3202 (National
Institute of Public Health, Prague, Czech Republic), BC = Bacillus cereus ATCC 14579, CP = Clostridium perfringens CNCTC 5770, PM = Pasteurella multocida (clinical isolate).
antibacterial activity against S. aureus (2A), against B. cereus (2B)
and against C. perfringens (2C) expressed as log (1/MIC [mol/L])
on lipophilicity is illustrated. Compound 3h was excluded due to
the different character of the nitro moiety in comparison with
the halogen moieties. Figure 2A and B illustrate typical activity of
lipophilicity, while in Figure 2C a linear curve with lipophilicity
optimum at logP ca. 5.4 is shown.
Within Group II (esters of salicylanilides 4) more complex rela-
tionships can be found. The basic requirement for antimicrobial
activity is the substitution of the salicylic ring (R1) by a lipophilic
moiety. If R2 = 4-CF3 substitution of compound 4h is considered
bioisosteric to R2 = 3,4-Cl substitution of compound 4k, that is,
both compounds differ from each other only by the position of R1
substitution, it can be stated that compounds substituted by chlo-
rine in C(5) position of the salicylic ring have higher antibacterial
potential than substituted by chlorine in C(4) position of the sali-
cylic ring. The same observation was mentioned above. Also the
R2 substituent has substantial influence. As discussed above, the
activity is increased especially by the lipophilicity of R2 moiety
and its electron-withdrawing properties. R3 substituent can be
considered as the last structure parameter influencing activity
within Group II. This substitution increases the overall lipophilicity
and bulkiness of the target compounds. R3 is probably responsible
for the increase of activity within the series of 4-Cl/4-CH3

K. Pauk et al. / Bioorg. Med. Chem. 21 (2013) 6574–6581
6577
Table 2
In vitro antimycobacterial activity (MIC) of selected the compounds in comparison
with isoniazid (INH) standard
Comp.
MIC (lmol/L)
Figure 3. Increase of general antimicrobial activity within group I in dependence on
MS
MM
MK
Hammett’s
r parameter (calculated by ACD/Percepta ver. 2012).
3a
3b
3d
3e
3f
64.60
>978
28.35
24.49
12.67
13.67
12.64
>548
259
64.60
61.13
7.08
12.24
60.40
3.42
3.16
68.53
259
16.15
30.56
7.08
6.12
1.58
60.43
3.16
17.13
129
58.93
13.74
62.62
13.03
7.28
7.60
14.29
7.27
13.38
88.68
38.57
75.17
76.97
34.50
3h
3i
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
6b
6f
6g
6h
6i
Figure 4. Increase of general antimicrobial activity owing to R3 substituent in
dependence on distributive parameter
and molar volume MV (cm3) (both
calculated using ACD/Percepta ver. 2012).
p
236
110
251
118
110
251
26.06
29.14
30.42
57.16
29.10
26.76
>709
77.14
150
26.06
14.57
7.60
14.29
14.55
26.76
355
77.14
150
76.97
138
2.2.2. In vitro antimycobacterial activity
The genus Mycobacterium consists of a closely related group of
fast and slow-growing species. M. tuberculosis causes one of the
most serious human infections, tuberculosis. Difficulties should
be considered while studying M. tuberculosis—especially a slow
growth rate and the requirement of working in high containment
biosafety facilities. To lower risks and make manipulation in the
laboratory easier, surrogate model pathogens for M. tuberculosis
can be used in laboratory studies. M. smegmatis is an ideal repre-
sentative of a fast-growing nonpathogenic microorganism partic-
ularly useful in studying basic cellular processes of special
relevance to pathogenic mycobacteria. M. marinum is very closely
related to M. tuberculosis and is the cause of TB-like infections in
poikilothermic organisms, especially frogs and fish. M. marinum is
a good model for studying especially because of lower risk for
laboratory workers, genetic relatedness and similar pathology to
human TB.38–40 However, because of M. tuberculosis, the patho-
genic role of nontuberculous mycobacteria (NTM) in humans
was overshadowed for a long time. M. kansasii, the most virulent
of the NTM, causes nontuberculous mycobacterial lung infections
which are very common nowadays and can be indistinguishable
from tuberculosis.41 That is the reason why M. smegmatis, M. mar-
inum and M. kansasii were chosen as model species for screening
of prospective antimycobacterial drugs to control mycobacterial
diseases.
76.97
69.00
INH
117
467
29.17
MS = Mycobacterium smegmatis ATCC 700084, MM = M. marinum CAMP 5644,
MK = M. kansasii DSM 44162.
derivatives (4b–e); with the increase of lipophilicity/bulkiness the
activity increases, see Table 2. Based on this observation, bulky
substituents (benzyl, methylindolyl, methylcyclohexyl) seem to
be advantageous, see Figure 4. Probably the methylcyclohexyl frag-
ment increases the antimicrobial activity of compound 4g in com-
parison with compound 4j.
It can be stated that diamides (Group III) possessed the lowest
activity within the discussed groups. Compounds 6h and 6i were
found as the most effective within Group III, which underlines
the significance of the substitution of C(4) position of the salicylic
ring in comparison with C(5) position for high antibacterial activity,
compare 6h and 6f. As mentioned above, the decrease of lipophil-
icity and electron-withdrawing properties of the R2 substituent
causes the decrease of activity: 4-CF3 (6f) >>> 4-CH3 (6b) >
4-OCH3 (6e). Lipophilic and bulky R3 substituents optimize proper-
ties, and consequently antimicrobial activity is increased, compare
6h and 6i. Generally it can be concluded that antimicrobial activity
increases with increasing lipophilicity as is illustrated in Figure 5A
and B. Only for C. perfringens the dependence of activity on lipo-
philicity can be observed with an optimum at logP ca. 5, that is,
activity decreases with a decrease or an increase of lipophilicity,
see Figure 5C.
The evaluation of the in vitro antimycobacterial activity of the
compounds was performed against M. smegmatis ATCC 700084
(MS), M. marinum CAMP 5644 (MM) and M. kansasii DSM 44162
(MK). All the results are shown in Table 2. Most of the tested com-
pounds showed antimycobacterial activity against the three men-
tioned strains comparable or higher than the standard isoniazid.
Compound 3f demonstrated the highest activity against M. mari-
num (60.40
l
mol/L), and compound 3h showed the highest activ-
mol/L). The activity of compounds
ity against M. kansasii (60.43
l
from Group III was lower than the activity of compounds from
A
B
C
Figure 2. Dependence of in vitro antibacterial activity log (1/MIC [mol/L]) against S. aureus (A), against B. cereus (B) and against C. perfringens (C) on lipophilicity (logP
calculated by ACD/Percepta ver. 2012) of studied compounds of group I, exclusive of 3h (rhombs = 5-Cl, triangle = 4-Cl).

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K. Pauk et al. / Bioorg. Med. Chem. 21 (2013) 6574–6581
C
A
B
Figure 5. Dependence of in vitro antibacterial activity log (1/MIC [mol/L]) against S. aureus (A), against B. cereus (B) and against C. perfringens (C) on lipophilicity (logP
calculated by ACD/Percepta ver. 2012) of studied compounds of group III, exclusive of 3h (rhombs = 5-Cl, trianglev4-Cl).
Group II, which in its turn was lower than that of Group I.
Compounds 3f, 3h, 3i and 4i demonstrated the highest activity
against all the three strains.
4. Experimental
4.1. General
Within Group I the following SAR can be found for all the
three mycobacterial strains: the activity is significantly influenced
by R2 substituents with electron-withdrawing effect (4-CF3, 4-
NO2, 3,4-Cl), while lipophilicity plays only a secondary role. It
seems that C(5) position of chlorine in the salicylic ring is more
advantageous than C(4) position contrary to the results of antibac-
terial screening.
It is important to note that within Group II compounds substi-
tuted by chlorine in C(4) position of the salicylic ring showed more
significant activity than C(5) positional isomers. Nevertheless, as
mentioned above, the electron-withdrawing effect of R2 substitu-
ents seems to be more important than lipophilicity. A bulky and/
or lipophilic moiety as R3 substituent (methylindolyl, meth-
ylcyclohexyl) is more advantageous than the benzyl ring.
Based on the presented antimycobacterial data it is not possible
to determine whether C(4) or C(5) substitution of the position of the
salicylic ring is more advantageous within Group III. Antimycobac-
terial activity is significantly influenced by electron-withdrawing
substituents (4-CF3). Both isopropyl and benzyl are convenient R3
substituents.
The chemicals were purchased from commercial sources (Sigma
Aldrich, Acros Organics, TCI, Merck). Commercial grade reagents
were used without further purification. Reactions were monitored
by means of thin layer chromatography plates coated with 0.2 mm
Silica Gel 60 F254 (Merck). TLC plates were visualized by UV irra-
diation (254 nm). The products were purified by crystallization, by
means of column chromatography employing Silica Gel 60
(Merck). All melting points were determined on Melting Point B-
540 apparatus (Buchi, Germany) and are uncorrected. NMR spectra
were measured in DMSO-d6 solutions (if not specified otherwise)
on a Bruker Avance 400 apparatus at 400.13 MHz (1H) and
100.62 MHz (13C). The chemical shifts d are given in ppm, relating
to tetramethylsilane (TMS) as an internal standard. The coupling
constants (J) are reported in hertz (Hz). Elemental analysis (C, H,
N) was performed on a Thermo Scientific Flash 2000 Organic ele-
mental analyser.
4.2. General procedures
4.2.1. Preparation of salicylamides (3a–i)
A mixture of substituted salicylic acid 1 (0.1 mol), appropriate
amine 2 (0.1 mol) and phosphorus trichloride (0.05 mol) in chlo-
robenzene (250 ml) was stirred under microwave irradiation in
the microwave reactor (400 W input power) for 20 min. Reaction
mixture was then left in the fridge overnight. The precipitate
was collected by filtration and recrystallized from ethanol to
give salicylamide in sufficient purity.
3. Conclusion
Thirty-two new salicylanilides, esters of N-phenylsalicyla-
mides and 2-hydroxy-N-[1-(2-hydroxyphenylamino)-1-oxoalkan-
2-yl]benzamides, were prepared. The compounds were evaluated
against seven bacterial and three mycobacterial strains. The
antimicrobial activities of some compounds were comparable
or higher than the standards ampicillin, ciprofloxacin or isonia-
zid. Compounds 3f–i, 4g, 4h, 4k, 4l, 6i demonstrated the highest
antibacterial activity against six Gram-positive/negative bacterial
strains, and compounds 3f, 3h, 3i and 4i showed the highest
activity against all three mycobacterial strains. Compound 3f
demonstrated high biological activity against S. aureus
4.2.2. Preparation of amino acid salicylanilide esters (4a–l)
N-Benzyloxycarbonyl protected amino acid (0.01 mol) and
substituted salicylanilide 3 (0.01 mol) were dissolved in dry DMF
(45 mL). The solution was cooled to—10 °C, and N,N-dicyclohexyl-
carbodiimide (0.011 equiv) was added in three portions for more
than 1 h. The mixture was then stirred for 3 h at the same temper-
ature and stored at 4 °C for 20 h. Precipitated N,N-dicyclohexylurea
was removed by filtration, and the solvent was evaporated in va-
cuo. The crude product was crystallized from ethyl acetate–
hexane.
(60.03
(1.58
eus (0.09
(60.03 mol/L) and M. kansasii (60.43
3i against methicillin-resistant S. aureus and B. cereus
(60.03 mol/L). The structure–activity relationships are dis-
l
mol/L), M. marinum (60.40
mol/L); 3g against C. perfringens (<0.03
mol/L); compound 3h against P. multocida
mol/L); and compound
l
mol/L) and M. kansasii
l
lmol/L) and B. cer-
l
l
l
l
4.2.3. Preparation of diamides (6a–i) via rearrangement
cussed for all the compounds. As regards antibacterial activity,
generally it can be stated that the R1 substitution in the para po-
sition to the carboxamide moiety is important together with an
R2 lipophilic and electron-withdrawing moiety and a bulky R3
substituent. For antimycobacterial activity especially R1 substitu-
tion in the meta position to the carboxamide moiety is important
for high activity. Also the electron-withdrawing substituent, such
as R2 = NO2, CF3 or 2Cl, and the bulky R3 substituent are
important.
A 33% solution of hydrogen bromide in acetic acid (10 ml)
was slowly added to N-benzyloxycarbonyl-protected ester
4
(1 mmol) while stirring. The suspension was stirred at room
temperature for 30 min. During this time, the suspension turned
into a clear brown solution, and evolution of carbon dioxide was
observed. When the gas evolution ceased, dry diethyl ether
(50 ml) was added. The precipitate was collected by filtration,
washed with diethyl ether and dried. Obtained hydrobromide

K. Pauk et al. / Bioorg. Med. Chem. 21 (2013) 6574–6581
6579
salts 5 were suspended in dry chloroform (10 ml), and triethyl-
4.5. Characterization of prepared compounds
amine (0.95 mmol) was added at room temperature. After
30 min of stirring, insoluble material was filtered off, and the fil-
trate was purified by column chromatography (ethyl acetate–
hexan 1:1).
4.5.1. 4-Chloro-2-(phenylcarbamoyl)phenyl (2S)-2-
{[(benzyloxy)carbonyl]amino}-3-methylbutanoate (4a)
White solid, yield 34%, mp 125–127 °C. 1H NMR (400 MHz,
DMSO-d6): d 10.48 (1H, br s, NH), 7.86 (1H, d, J = 8.38 Hz, CH-
NH), 7.73–7.62 (4H, m, Ar-H), 7.36–7.30 (7H, m, Ar-H), 7.19–7.15
(1H, m, Ar-H), 7.09–7.06 (1H, m, Ar-H), 5.04 (2H, s, OCH2), 4.18–
4.12 (1H, m, CH-NH), 2.18–2.12 (1H, m, CH(CH3)2), 0.80–0.70
(6H, m, (CH3)2). 13C NMR (100 MHz, DMSO-d6): d 170.8, 163.1,
157.1, 146.7, 139.5, 137.4, 132.7, 131.5, 130.7, 129.2, 129.1,
129.0, 128.5, 128.5, 125.6, 124.4, 120.2, 66.3, 60.5, 29.9, 19.6,
18.4. Anal. Calcd for C26H25ClN2O5 (480.94): C, 64.93; H, 5.24; N,
5.82. Found: C, 64.89; H, 5.19; N, 5.75.
4.3. In vitro antibacterial susceptibility testing
The synthesized compounds were evaluated for in vitro anti-
bacterial activity against Gram-positive strains (B. cereus ATCC
14579, C. perfringens CNCTC 5770), one Gram-negative strain
(clinical isolate of P. multocida) and representatives of multi-
drug-resistant bacteria (clinical isolates of methicillin-resistant
S. aureus (MRSA) 63718, SA 630 and SA 3202 obtained from
the National Institute of Public Health, Prague, Czech Republic).
S. aureus ATCC 29213 was used as a reference and quality con-
trol strain. Ampicillin (Sigma, Germany) and ciprofloxacin (Sig-
ma, Germany) were used as the standards. Prior to testing,
each strain was cultured on nutrient agar (Oxoid, UK) with
5% of ovine blood, and bacterial inocula were prepared by sus-
pending a small portion of the bacterial colony in sterile phos-
phate buffered saline (pH 7.2–7.3). The cell density was
adjusted to 0.5 McFarland units using a densitometer (Densi-
La-Meter, LIAP, Latvia). The final inoculum was made by 1:20
dilution of the suspension with the Mueller–Hinton broth
(MH broth). The compounds were dissolved in DMSO (Sigma,
Germany), and the final concentration of DMSO in the MH
broth (Oxoid, UK) did not exceed 2.5% of the total solution
composition. The broth dilution micro-method modified accord-
ing to NCCLS guidelines42,43 in MH broth was used to deter-
mine the minimum inhibitory concentration (MIC). Drug-free
controls, sterility controls and controls consisted of MH broth
and DMSO alone were included. The determination of results
was performed visually after 24 h of static incubation in the
darkness at 37 °C in an aerobic atmosphere (anaerobic atmo-
sphere for C. perfringens). The MICs were defined as the lowest
concentration of the compound at which no visible bacterial
growth was observed.
4.5.2. 4-Chloro-2-[(4-methylphenyl)carbamoyl]phenyl (2S)-2-
{[(benzyloxy)carbonyl]amino}-propanoate (4b)
White solid, yield 50%, mp 121–125 °C. 1H NMR (400 MHz,
DMSO-d6): d 8.16 (1H, s, NH), 7.99 (1H, d, J = 2.4 Hz, Ar-H), 7.45
(2H, m, CH-NH, Ar-H), 7.38–7.30 (6H, m, Ar-H), 7.12–7.06 (3H, m,
Ar-H), 5.38 (1H, d, J = 8.4 Hz, Ar-H), 5.09–5.12 (1H, m, OCHH),
5.00–4.96 (1H, m, OCHH), 4.44–4.38 (1H, m, CH-NH), 2.30 (3H, d,
J = 6.5 Hz, CH3), 2.27 (3H, s, CH3). 13C NMR (100 MHz, DMSO-d6):
d 170.4, 162.1, 156.4, 145.6, 135.9, 134.9, 134.5, 132.0, 131.5,
130.4, 129.6, 129.4, 128.5, 128.2, 128.0, 124.3, 120.4, 67.2, 59.5,
30.5, 20.9. Anal. Calcd for C25H23ClN2O5 (466.91): C, 64.31; H,
4.97; N, 6.00. Found: C, 64.25; H, 5.10; N, 5.88.
4.5.3. 4-Chloro-2-[(4-methylphenyl)carbamoyl]phenyl (2S)-2-
{[(benzyloxy)carbonyl]amino}-3-methylbutanoate (4c)
White solid, yield 40%, mp 129–132 °C. 1H NMR (400 MHz,
DMSO-d6): d 8.16 (1H, s, NH), 7.99 (1H, d, J = 2.4 Hz, Ar-H), 7.47–
7.43 (2H, m, CH-NH, Ar-H), 7.38–7.30 (6H, m, Ar-H), 7.15–7.04
(3H, m, Ar-H), 5.38 (1H, d, J = 8.4 Hz, Ar-H), 5.09–5.05 (1H, m,
OCHH), 5.00–4.96 (1H, m, OCHH), 4.45–4.39 (1H, m, CH-NH),
2.35–2.29 (1H, m, CH(CH3)2), 2.27 (3H, s, CH3), 0.97 (3H, d,
J = 6.8 Hz, CH3), 0.86 (3H, d, J = 6.7 Hz, CH3). 13C NMR (100 MHz,
DMSO-d6): d 170.4, 162.1, 156.4, 145.6, 135.9, 134.9, 134.5,
132.0, 131.5, 130.4, 129.6, 129.4, 128.5, 128.2, 128.0, 124.3,
120.4, 67.2, 59.5, 30.5, 20.9, 19.2, 17.3. Anal. Calcd for C27H27
ClN2O5 (494.97): C, 65.52; H, 5.50; N, 5.66. Found: C, 65.40; H,
5.36; N, 5.50.
4.4. In vitro antimycobacterial susceptibility testing
The evaluation of in vitro antimycobacterial activity of the
compounds was performed against M. smegmatis ATCC 700084,
M. marinum CAMP 5644 and M. kansasii DSM 44162. The broth
dilution micro-method in Middlebrook 7H9 medium (Difco,
USA) supplemented with ADC Enrichment (BBL, USA) was used
to determine the minimum inhibitory concentration (MIC) as
previously described.44 The tested compounds were dissolved as
described above in Section 4.3. Isoniazid (Sigma, Germany) was
used as a reference antibacterial drug. Bacterial inocula were pre-
pared by transferring colonies from culture to sterile water. The
cell density was adjusted to 0.5 McFarland units using a densi-
tometer (Densi-La-Meter, LIAP, Latvia). The final inoculum was
made by 1:1000 dilution of the suspension with sterile water.
Drug-free controls, sterility controls and controls consisted of
medium and DMSO alone were included. The determination of
results was performed visually after 3 days of static incubation
in the darkness at 37 °C in an aerobic atmosphere for M. smegma-
tis, after 7 days of static incubation in the darkness at 37 °C in an
aerobic atmosphere for M. kansasii and after 21 days of static
incubation in the darkness at 28 °C in an aerobic atmosphere
for M. marinum. The MICs were defined as the lowest concentra-
tion of the compound at which no visible bacterial growth was
observed.
4.5.4. 4-Chloro-2-[(4-methylphenyl)carbamoyl]phenyl (2S)-2-
{[(benzyloxy)carbonyl]amino}-3-phenylpropanoate (4d)
White solid, yield 16%, mp 145–147 °C. 1H NMR (400 MHz,
DMSO-d6): d 10.42 (1H, br s, NH), 8.02 (1H, d, J = 8.08 Hz, CH-
NH), 7.75 (1H, d, J = 2.55 Hz, Ar-H), 7.66–7.64–7.57 (3H, m, Ar-H),
7.32–7.19 (11H, m, Ar-H), 7.15–7.09 (2H, m, Ar-H), 4.96 (2H, s,
OCH2), 4.47–4.43 (1H, m, CH-NH), 3.25–3.17 (1H, m, CHH), 2.95–
2.85 (1H, m, CHH), 2.24 (3H, s, CH3). 13C NMR (100 MHz, DMSO-
d6): d 170.7, 162.8, 156.6, 146.9, 137.9, 137.4, 136.9, 133.5, 132.3,
131.7, 130.8, 129.7, 129.4, 128.9, 128.8, 128.4, 128.2, 128.0,
127.1, 125.5, 120.5, 66.1, 56.3, 36.5, 21.1. Anal. Calcd for
C31H27ClN2O5 (543.00): C, 68.57; H, 5.01; N, 5.16. Found:
C, 68.40; H, 4.99; N, 5.03.
4.5.5. 4-Chloro-2-[(4-methylphenyl)carbamoyl]phenyl (2S)-2-
[(tert-butoxycarbonyl)amino]-3-(1H-indol-2-yl)propanoate
(4e)
White solid, yield 40%, mp 162–164 °C. 1H NMR (400 MHz,
DMSO-d6): d 10.85 (1H, br s, NH), 10.44 (1H, br s, Ar-NH), 7.75
(1H, d, J = 2.36 Hz, CH-NH), 7.66–7.61 (3H, m, Ar-H), 7.48–7.32
(3H, m, Ar-H), 7.21–7.04 (5H, m, Ar-H), 6.91 (1H, m, Ar-H), 4.38–
4.32 (1H, m, CH-NH), 3.30 (1H, m, CHH), 3.01 (1H, m, CHH), 4.15

6580
K. Pauk et al. / Bioorg. Med. Chem. 21 (2013) 6574–6581
(1H, m, CH-NH), 2.25 (3H, s, CH3), 1.32 (9H, m, (CH3)3). 13C NMR
(100 MHz, DMSO-d6): d 171.3, 162.9, 156.1, 147.0, 136.9, 136.6,
133.5, 132.4, 131.6, 130.7, 129.7, 129.3, 127.5, 125.5, 124.4,
121.6, 120.5, 119.0, 118.6, 112.0, 110.3, 79.0, 55.4, 28.3, 26.6,
21.1. Anal. Calcd for C30H30ClN3O5 (548.03): C, 65.75; H, 5.52; N,
7.67. Found: C, 65.66; H, 5.49; N, 7.58.
ClN3O7 (525.94): C, 59.38; H, 4.60; N, 7.99. Found: C, 59.22; H,
4.51; N, 7.89.
4.5.10. 5-Chloro-2-[(4-bromophenyl)carbamoyl]phenyl (2S)-2-
{[(benzyloxy)carbonyl]amino}-3-methylbutanoate (4j)
White solid, yield 34%, mp 160–163 °C. 1H NMR (400 MHz,
DMSO-d6): d 10.59 (1H, br s, NH), 7.87 (1H, d, J = 8.38 Hz, CH-
NH), 7.70–7.65 (3H, m, Ar-H), 7.51–7.49 (3H, m, Ar-H), 7.36–7.28
(6H, m, Ar-H), 5.04 (2H, s, OCH2), 4.17–4.14 (1H, m, CH-NH),
2.16–2.16 (1H, m, CH(CH3)2), 0.88–0.83 (6H, m, (CH3)2). 13C NMR
4.5.6. 4-Chloro-2-[(4-methoxyphenyl)carbamoyl]phenyl (2S)-2-
{[(benzyloxy)carbonyl]amino}-3-methylbutanoate (4f)
White solid, yield 36%, mp 107.5–108.7 °C. 1H NMR (400 MHz,
DMSO-d6): d 8.06 (1H, s, NH), 7.75 (1H, d, J = 2.0 Hz, Ar-H), 7.50–
7.46 (2H, m, CH-NH, Ar-H), 7.42–7.36 (1H, m, Ar-H), 7.38–7.26
(5H, m, Ar-H), 7.10–7.06 (1H, m, Ar-H), 6.85–6.79 (2H, m, Ar-
H), 5.32 (1H, d, J = 8.4 Hz, Ar-H), 5.10–5.06 (1H, m, OCHH),
5.01–4.97 (1H, m, OCHH), 4.44–4.38 (1H, m, CH-NH), 3.76 (3H,
s, CH3), 2.28–2.24 (1H, m, CH(CH3)2), 0.99 (3H, d, J = 6.4 Hz,
CH3), 0.88 (3H, d, J = 6.4 Hz, CH3). 13C NMR (100 MHz, DMSO-
d6): d 170.5, 162.0, 156.7, 156.4, 135.9, 132.1, 131.6, 130.5,
130.3, 129.8, 128.5. 128.2. 128.1. 124.3. 122.2, 114.1, 67.3,
59.6, 55.4, 30.5, 24.9, 19.2, 17.4. Anal. Calcd for C27H27ClN2O6
(510.97): C, 63.47; H, 5.33; N, 5.48. Found: C, 63.40; H, 5.25;
N, 5.38.
(100 MHz, DMSO-d6):
d 170.6, 163.6, 157.1, 148.5, 138.9,
137.4, 135.6, 132.1, 131.0, 139.9, 129.0, 128.5, 128.4, 126.9,
123.7, 122.1, 116.0, 66.3, 60.5, 29.9, 19.6, 18.4. Anal. Calcd for C26-
H24BrClN2O5 (559.84): C, 55.78; H, 4.32; N, 5.00. Found: C, 55.69;
H, 4.25; N, 4.15.
4.5.11. 5-Chloro-2-[(3,4-dichlorophenyl)carbamoyl]phenyl (2S)-
2 {[(benzyloxy)carbonyl]amino}-3-methylbutanoate (4k)
White solid, yield 18.4%, mp 132–134 °C. 1H NMR (400 MHz,
DMSO-d6): d 10.74 (1H, br s, NH), 8.06 (1H, s, Ar-H), 7.87 (1H, d,
J = 7.56 Hz, CH-NH), 7.70 (1H, d, J = 8.82, Ar-H), 7.59 (2H, s, Ar-H),
7.54–7.52 (1H, m, Ar-H), 7.35–7.30 (6H, m, Ar-H), 5.03 (2H, s,
OCH2), 4.18–4.15 (1H, m, CH-NH), 2.17–2.13 (1H, m, CH(CH3)2),
0.89–0.83 (6H, m, (CH3)2). 13C NMR (100 MHz, DMSO-d6): d
170.6, 164.0, 157.1, 148.6, 139.5, 137.4, 135.9, 131.5, 131.2,
131.0, 129.6, 128.9, 128.5, 128.4, 127.0, 125.9, 123.7, 121.5,
120.3, 66.3, 60.5, 29.9, 19.6, 18.4. Anal. Calcd for C26H23Cl3N2O5
(549.83): C, 56.80; H, 4.22; N, 5.09. Found: C, 56.75; H, 4.19; N,
4.03.
4.5.7. 4-Chloro-2-[(4-chlorophenyl)carbamoyl]phenyl (2S)-2-
{[(benzyloxy)carbonyl]amino}-3-cyclohexylpropanoate (4g)
White solid, yield 43%, mp 140–142 °C. 1H NMR (400 MHz,
DMSO-d6): d 10.62 (1H, br s, NH), 7.89 (1H, d, J = 7.08 Hz, CH-
NH), 7.74–7.62 (4H, m, Ar-H), 7.55–7.47 (2H, m, Ar-H), 7.37–7.20
(6H, m, Ar-H), 5.04 (2H, s, OCH2), 4.26–4.22 (1H, m, CH-NH),
1.59–1.43 (7H, m, cyclohexyl), 1.28–1.01 (4H, m, cyclohexyl),
0.63 (2H, m, CHCH2). 13C NMR (100 MHz, DMSO-d6): d 171.8,
163.1, 156.8, 138.8, 137.5, 132.4, 132.1, 131.7, 130.8, 129.1,
128.9, 128.5, 128.3, 125.5, 122.1, 116.1, 66.2, 52.3, 38.0, 33.7,
4.5.12. 5-Chloro-2-[(3,4-dichlorophenyl)carbamoyl]phenyl (2S)-
2 {[(benzyloxy)carbonyl]amino}-3-phenylpropanoate (4l)
White solid, yield 36%, mp 145–147 °C. 1H NMR (400 MHz,
DMSO-d6): d 10.73 (1H, br s, NH), 8.09 (1H, s, Ar-H), 8.02 (1H, d,
J = 7.66 Hz, CH-NH), 7.74 (1H, d, J = 8.08 Hz, Ar-H), 7.64–7.53 (3H,
m, Ar-H), 7.31–7.22 (11H, m, Ar-H), 4.94 (2H, s, OCH2), 4.52–4.46
(1H, m, CH-NH), 3.22–3.19 (1H, m, CHH), 2.95–2.89 (1H, m,
CHH). 13C NMR (100 MHz, DMSO-d6): d 170.6, 163.9, 156.5,
148.8, 139.5, 137.8, 137.4, 136.1, 131.6, 131.2, 129.7, 129.1,
128.9, 128.9, 128.4, 127.8, 127.2, 127.0, 125.9, 123.7, 121.7,
120.5, 66.1, 56.2, 36.5. Anal. Calcd for C30H23Cl3N2O5 (597.87): C,
60.27; H, 3.88; N, 4.69. Found: C, 60.20; H, 3.70; N, 4.66.
33.4, 31.7, 26.5, 26.2, 25.9. Anal. Calcd for
C30H30BrClN2O5
(613.93): C, 58.69; H, 4.93; N, 4.56. Found: C, 58.59; H, 4.53; N,
4.44.
4.5.8. 4-Chloro-2-[(4-trifluoromethylphenyl)carbamoyl]phenyl
(2S)-2-{[(benzyloxy)carbonyl]amino}-3-methylbutanoate (4h)
White solid, yield 22%, mp 135–137.4 °C. 1H NMR (400 MHz,
DMSO-d6): d 10.84 (1H, s, NH), 7.89 (2H, d, J = 8.6 Hz, Ar-H),
7.85 (1H, d, J = 7.5 Hz, Ar-H), 7.69 (2H, d, J = 8.5, Ar-H), 7.77
(1H, d, J = 2.6 Hz, CH-NH), 7.35–7.32–7.28 (6H, m, Ar-H), 7.20
(1H, d, J = 8.9 Hz, Ar-H), 5.02 (2H, s, OCH2), 4.15 (1H, t,
J = 7.0 Hz, CH-NH), 2.15–2.11 (1H, m, CH(CH3)2), 0.86 (3H, d,
J = 6.9 Hz, CH3), 0.84 (3H, d, J = 6.7 Hz, CH3). 13C NMR
(100 MHz, DMSO-d6): d 170.2, 163.1, 156.5, 146.1, 142.4, 136.9,
131.7, 131.3, 130.2, 128.6, 128.4, 128.9, 128.1, 127.9, 126.0,
125.1, 123.8, 119.6, 65.8, 60.0, 29.4, 19.0, 17.9. Anal. Calcd for
Acknowledgements
This work was financially supported by the Ministry of Educa-
tion, Youth and Sports of the Czech Republic (Project SGFChT 02
UPa), by IGA VFU Brno, Project No. 65/2012/FVL, and by the project
CEITEC (Central European Institute of Technology) (CZ.1.05/1.1.00/
02.0068) from the European Regional Development Fund.
C27H24F3ClN2O5 (548.94): C, 59.08; H, 4.41; N, 5.10. Found: C,
58.99; H, 4.40; N, 5.03.
Supplementary data
4.5.9. 4-Chloro-2-[(4-nitrophenyl)carbamoyl]phenyl (2S)-2-
{[(benzyloxy)carbonyl]amino}-3-methylbutanoate (4i)
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.08.029.
Yellowish solid, yield 36%, mp 135.6–137.4 °C. 1H NMR
(400 MHz, DMSO-d6): d 11.09 (1H, s, NH), 8.24 (2H, d, J = 9.0 Hz,
Ar-H), 7.93 (2H, d, J = 9.3 Hz, Ar-H), 7.86 (1H, d, J = 7.9, NH-CH),
7.80 (1H, d, J = 2.5 Hz, Ar-H), 7.71–7.65 (1H, m, Ar-H), 7.35–7.31
(5H, m, Ar-H), 7.22 (1H, d, J = = 8.7 Hz, Ar-H), 5.03 (2H, s, OCH2),
4.15 (1H, t, J = 7.4 Hz, CH-NH), 2.15–2.12 (1H, m, CH(CH3)2), 0.87
(3H, d, J = 6.8 Hz, CH3), 0.84 (3H, d, J = 6.9 Hz, CH3). 13C NMR
(100 MHz, DMSO-d6): d 170.3, 163.3, 156.4, 146.1, 145.0, 142.7,
131.5, 130.4, 128.7, 128.4, 127.8. 125.1. 124.9. 124.9, 120.1,
119.4, 99.6, 65.7, 59.9, 29.4, 19.1, 17.9. Anal. Calcd for C26H24
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