Synthesis and biological evaluation of a new series of N-acyldiamines as potential antibacterial and antifungal agents

Article abstract of DOI:10.1016/j.bmcl.2014.08.047

In continuation of our efforts to find new antimicrobial compounds, series of fatty N-acyldiamines were prepared from fatty methyl esters and 1,2-ethylenediamine, 1,3-propanediamine or 1,4-butanediamine. The synthesized compounds were screened for their antibacterial activity against Gram-positive bacteria (Staphylococcus aureus, Staphylococcus epidermidis), Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa) and for their antifungal activity against four species of Candida (C. albicans, C. tropicalis, C. glabrata and C. parapsilosis). Compounds 5a (N-(2-aminoethyl)dodecanamide), 5b (N-(2-aminoethyl)tetracanamide) and 6d (N-(3-aminopropyl)oleamide) were the most active against Gram-positive bacteria, with MIC values ranging from 1 to 16 μg/mL and were evaluated for their activity against 21 clinical isolates of methicillin-resistant S. aureus. All the compounds exhibited good to moderate antifungal activity. Compared to chloramphenicol, compound 6b displayed a similar activity (MIC₅₀= 16 μg/mL). A positive correlation could be established between lipophilicity and biological activity.

Full text of DOI:10.1016/j.bmcl.2014.08.047

Accepted Manuscript
Synthesis and biological evaluation of a new series of N-acyldiamines as po-
tential antibacterial and antifungal agents
Bianca da S. Ferreira, Angelina M. de Almeida, Thiago C. Nascimento, Pedro
P. de Castro, Vania L. Silva, Claúdio G. Diniz, Mireille Le Hyaric
PII:
S0960-894X(14)00891-9
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.08.047
BMCL 21945
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Accepted Date:
11 July 2014
21 August 2014
Please cite this article as: Ferreira, B.d.S., de Almeida, A.M., Nascimento, T.C., de Castro, P.P., Silva, V.L., Diniz,
C.G., Le Hyaric, M., Synthesis and biological evaluation of a new series of N-acyldiamines as potential antibacterial
and antifungal agents, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.
2014.08.047
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Synthesis and biological evaluation of a new series of N-
acyldiamines as potential antibacterial and antifungal agents
Bianca da S. Ferreiraa, Angelina M. de Almeidaa, Thiago C. Nascimentob, Pedro P. de
Castrob, Vania L. Silvab, Claúdio G. Dinizb, Mireille Le Hyarica.
aDepartamento de Química, ICE, Universidade Federal de Juiz de Fora, 36036-330
Juiz de Fora, MG, Brazil
bDepartamento de Parasitologia, Microbiologia e Imunologia, ICB, Universidade
Federal de Juiz de Fora, 36036-330 Juiz de Fora, MG, Brazil
In continuation of our efforts to find new antimicrobial compounds, series of fatty N-
acyldiamines were prepared from fatty methyl esters and 1,2-ethylenediamine, 1,3-
propanediamine or 1,4- butanediamine. The synthesized compounds were screened
for their antibacterial activity against Gram-positive bacteria (S. aureus, S. epidermidis),
Gram-negative bacteria (E. coli, P. aeruginosa) and for their antifungal activity against
four species of Candida (C. albicans, C. tropicalis, C. glabrata and C. parapsilosis).
Compounds 5a (N-(2-aminoethyl)dodecanamide), 5b (N-(2-aminoethyl)tetracanamide)
and 6d (N-(3-aminopropyl)oleamide) were the most active against Gram-positive
bacteria, with MIC values ranging from 1 to 16 µg/mL and were evaluated for their
activity against 21 clinical isolates of methicillin-resistant S. aureus. All the compounds
exhibited good to moderate antifungal activity. Compared to chloramphenicol,
compound 6b displayed a similar activity (MIC50= 16 µg/mL). A positive correlation
could be established between lipophilicity and biological activity.
Keywords: N-acyldiamines, antibacterial, antifungal, MRSA, Lipophilicity
There is no doubt about the success of antimicrobial therapy throughout the world, but
the indiscriminate use of antibiotics for the prevention and treatment of diseases has
accelerated the emergence of drug-resistant strains. Antimicrobial resistance has
become one of the most serious health threats, resulting in an urgent need to find new
antimicrobial compounds.
The antimicrobial properties of fatty acids and their derivatives are well known.1 Free
fatty acids are potent enzyme inhibitors, and unsaturated fatty acids are usually more
active than the saturated ones.2,3 The polyunsaturated linoleic acid displays antifungal
activity against several plant pathogenic fungi4 and arachidonic acid may increase

antifungal susceptibility of biofilms formed by Candida species5 to antibacterial
compounds. Fatty amides derived from anthranilic acid have been shown to exhibit
Gram-positive growth6 and unsaturated fatty esters are active against oral
microorganisms.7
We showed in a previous work that N-acyldiamines inhibited the growth of several
strains of Gram-positive and Gram-negative bacterial strains.8 It is believed that these
compounds act as non-ionic surfactants: the hydrophobic portion of the molecule is
able to penetrate the lipid bilayer structure of the bacterial membrane, disturbing its
permeability and causing the cell death.
In continuation to our ongoing efforts to find new antimicrobial,9-11 the present study
reports the preparation and in vitro evaluation of the antimicrobial activities against
Gram-positive and Gram-negative bacteria and fungi of a series of N-acyldiamines
synthesized by the reaction of long chain saturated fatty acids (12-16 carbons) or oleic
acid with 1,2-ethylendiamine, 1,3-propanediamine and 1,4-butanediamine.
The synthesis of the compounds was performed according to literature procedure.8
Briefly, the methyl fatty esters were treated with an excess of the diamines (1,2-
ethylenediamine, 1,3-propanediamine, 1,4-butanediamine in methanol under reflux
(Scheme 1), leading to the expected amides in good yields. All the compounds were
purified by recrystallization from methanol, except compounds 5d, 6d and 7d which
were purified by column chromatography on silica gel. All the compounds were
characterized by IR and NMR spectroscopy. The spectroscopic data (supplementary
material) were in full agreement with those expected.
Scheme 1. Synthesis of N-acyl diamines 5a-d, 6a-d and 7a-d.
The antibacterial activity of the synthesized compounds was assessed in vitro against
two Gram-positive bacterial strains (Staphylococcus aureus ATCC 29213 and
Staphylococcus epidermidis ATCC 12228), two Gram-negative bacteria (Escherichia
coli ATCC 11229 and Pseudomonas aeruginosa ATCC 27853) and 21 clinical isolates
of MRSA obtained from the culture collection at the Laboratory of Bacterial Physiology
and Molecular Genetics, Federal University of Juiz de Fora. The minimal inhibitory
concentration (MIC) was determined by the broth dilution method in Mueller Hinton
medium (DIFCO Laboratories, Detroit, MI, USA), according to the CLSI.12

Chloramphenicol and vancomycin were used as reference antimicrobials considering
the bacterial groups evaluated.
For the determination of the bactericidal concentrations of the compounds, selected
aliquots (two dilutions below the MIC and two dilutions higher than the MIC) were
transferred with the aid of a sterile swab to a Petri dish containing Mueller Hinton Agar
(MHA, Himedia Laboratories, Mumbai, India AMH). The plates were incubated under
aerobic conditions at 37°C for 24 h. The minimum bactericidal concentration (MBC)
was determined considering the solution of the lower concentration of tested compound
which resulted in no microbial growth after culture in MHA. If bacterial growth was
observed at all concentrations studied, the compound was classified as
bacteriostatic.13,14
The antifungal activities of the synthesized compounds were evaluated against four
strains of Candida (Candida albicans ATCC 18804, Candida tropicalis ATCC 750,
Candida glabrata ATCC 90030, Candida parapsilosis ATCC 22019). Antifungal
susceptibility testing was performed using the broth microdilution method according to
CLSI guidelines for antifungigram (CLSI) document M27-A3.15 Itraconazole was used
as reference antifungal agent.
The MIC values are shown in Table 2. With the exception of compounds 5c and 6b, all
of the N-acyldiamines showed good to moderate activity against at least one the
bacteria tested. In general they were more active against Gram- positive bacteria than
against Gram- negative strains. Amides 6c and 7c, both carrying a saturated
hexadecanoyl moiety were active against two of the bacteria strain tested. This was
expected: considering the cell wall complexicity of Gram-negative bacteria, it is
expected to be less sensitive to amphiphilic compounds than Gram-positive bacteria.
The three dodecanoic acid derivatives 5a, 6a and 7a were active against the four
bacteria tested, with MIC values ranging from 8-256 µg/mL. The amide 5a exhibited a
higher growth inhibitory potency when compared to the positive control
chloramphenicol. The best MIC values, ranging were obtained for the N-oleylamine 6d,
against S. aureus, S. epidermidis and E. coli (2 µg/mL, 1 µg/mL and 4 µg/mL,
respectively).
These results show that, if lipophilicity is important for the activity, the late one
decreases with the increase of the carbon chain, as evidenced by the loss of activity
observed comparing the MIC values obtained for the tetradecanoyl series (compounds
5a, 6a, 7a) to those obtained for the hexadecanoyl series (compounds 5c, 6c and 7c).

Although oleic acid has a longer carbon chain (18 carbon atoms) its derivatives 6d and
7d showed high antibacterial activities against Gram-positive bacteria and against E.
coli, with MIC values ranging from 1 to 16 µg/mL. Thus, it seems that not only the size
of the lipohilic chain is important, but also the presence of insaturations. Others works
described the higher potency of unsaturated fatty acids versus saturated acid.
Regarding to the diamine moiety, it appears from the comparison of the MIC values
obtained for compounds 5-7a, or for compounds 5-7b, that ethylenediamine provides a
better antibacterial activity than 1,3-propylenediamine or 1,4-butylamine. This could not
be observed for the oleyl series (compounds 5-7d) in which the highest MIC is of the
1,3-propanediamine derivative 7d.
Table 1. Minimum Inhibitory Concentrations (MIC, µg/mL)
Table 2. Minimal Bactericidal Concentrations (µg/mL)
Compounds 5a, 5b and 6d evaluated for their antibacterial activity against 21 clinical
isolates of methicillin- resistant S. aureus (MRSA) isolated from patients of the
University Hospital Juiz de Fora, between the years 2005-2010. The tests were
performed using the broth dilution method as described above to determine the
minimum bactericidal activity of the compounds (Table 3). The activities are also
presented in terms of inhibitory potencies (minimum inhibitory concentration at which
50 or 90% of strains tested were inhibited) and variation of MICs (Table 4).
The results show that the compounds 5a, 5b and 6d were active against almost all of
the MRSA isolates. The oleyl derivative 6d showed bactericidal activity against all the
tested clinical samples, with MIC values ranging from 4 to 1024 µg.mL-1. Furthermore
this compound presented the same antibacterial potency in vitro than chloramphenicol,
with MIC50 =16 µg.mL-1.
Table 3. Minimum bactericidal activity(µg/mL) of compounds 5a, 5b and 6d against
MRSA.
Table 4. MIC50 and MIC90
From the results shown in Table 5, it is possible to observe that the N-acyldiamines
displayed significant antifungal activity, and in most cases the MIC values were lower

than the values found for the reference drug. The most active compounds were the
oleyl derivatives 6d and 7d. Free fatty acids are known to have antifungal properties,
as the aliphatic chain can insert into the cell membrane and disturb it. The higher
antifungal activity of unsaturated fatty acids is due to the greater cross section they
occupy in the membrane and possibly to the increased oxidative stress they cause.
Table 5. Antifungal activity of N-acylated diamines (MIC µg/mL).
In an attempt to correlate the lipophilicity to the antifungal activity of the tested
compounds, the MIC values expressed as mmol.L-1 were converted to pMIC and logP
was calculated using ChemDraw Ultra (CambridgeSoft). P is an important
physicochemical parameter that reflects the ability of the compound to penetrate
biological membranes, has a great influence on the bioavailability of the drug, and is
often correlated with its biological activity. The coefficient is expressed as logP and can
be determined experimentally or calculated. The data were subjected to linear
regression analysis using Origin 8.0 software (Scheme 2). A high collinearity, indicated
by the coefficient of pearson (r> 0.9), was found for two series of N-acyldiamines 6a-d
and 7a-d, indicating that in these series, the antifungal activity depends on lipophilicity
of compounds.
6a-d C. parapisilosis ATCC 22019: pMIC = 0.5816log(P) + 1.9702 r = 0.97
6a-d C. albicans ATCC 18804: pMIC = 0.69404log(P) + 1.29526 r = 0.95
7a-d C. albicans ATCC 18804: pMIC = 0.95094log(P) – 0.02844 r = 0.92
Scheme 2. Linear regression analysis for antifungal activity
The N-acyldiamines were synthesized in good yields from fatty acids and showed good
in vitro antibacterial and antifungal activities against ATCC isolates of bacteria (Gram-
positive and Gram-negative), clinical isolates of MRSA
and fungus. N-(2-
aminoethyl)dodecanamide 5a showed the best activity against the ATCC bacteria
tested, and N-(3-aminopropyl)oleamide 6d was the most active against MRSA clinical
isolates. Compound 6d and N-(4-aminobutyl)oleamide 7d where the most active
against Candida strains, and it was possible to establish a correlation between their
lipophilicity and antifungal activity.
Acknowledgments

The authors gratefully acknowledge CAPES, CNPq and FAPEMIG for
fellowships and financial support
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Table 1
Gram-positive
Gram-negative
E. coli P. aeruginosa
Compound
S. aureus
S. epidermidis
5a
16
8
16
16
>1024
8
8
64
8
5b
1024
>1024
>1024
64
5c
>1024
>1024
256
>1024
>1024
2
>1024
>1024
128
>1024
32
5d
6a
32
1024
4
6b
1024
>1024
>1024
32
6c
6d
1
4
7a
64
16
16
16
4
64
7b
7c
16
256
1024
8
1024
>1024
>1024
32
512
16
7d
Chloramphenicol
Vancomycin
16
32
-
16
0.5
-
-

Table 2. Minimal Bactericidal Concentrations (µg/mL)
Compound
S. aureus
S. epidermidis
E. coli
P. aeruginosa
5a
5b
5c
16 µg/mL
128 µg/mL
-
16 µg/mL
**
16 µg/mL
16 µg/mL
**
**
-
-
-
5d
6a
-
**
-
-
**
32 µg/mL
**
512 µg/mL
256 µg/mL
6b
-
-
**
6c
-
64 µg/mL
16 µg/mL
16 µg/mL
32 µg/mL
128 µg/mL
128 µg/mL
128 µg/mL
**
-
6d
128 µg/mL
**
64 µg/mL
1024 µg/mL
1024 ug/mL
**
-
7a
512 µg/mL
32 µg/mL
7b
1024 µg/mL
1024 µg/mL
7c
7d
**
**
**
-
**
**
Chloramphenicol
**
**Bacteriostatic

Table 3. Minimum bactericidal activity(µg/mL) of compounds 5a, 5b and 6d against
MRSA.
Clinical
isolates of
5a
5b
6d
Chloramphenicol Vancomycin
MRSA
a
1
1024
256
1024
256
32
-
16
16
16
512
16
64
16
16
16
8
64
4
1
1
2
512
512
512
512
512
3
4
0.5
1
4
8
5
4
1
6
32
8
1
a
-
7
32
64
64
8
1
a
-
8
16
0.5
1
a
-
9
32
10
11
12
13
14
15
16
17
18
19
32
512
64
64
64
64
64
32
32
4
1
a
>1024
>1024
32
-
16
16
64
1024
16
8
1
a
-
1
a
-
1
>1024
1024
1024
256
32
128
64
1
1
a
-
1
128
128
16
8
0.5
1
8
a
-
128
4
128
0.5

a
20
256
-
1024
512
4
8
0.5
0.5
21
>1024
128
-a bacteriostatic

Table 4. MIC50 and MIC90
Compound
MIC (µg/mL)
90%
50%
32
Range
16– >1024
32 – 256
4– 1024
8 - 128
5a
>1024
256
5b
6d
32
16
512
Chloramphenicol
16
32

Table 5. Antifungal activity of N-acylated diamines (MIC µg/mL).
Compound
C. albicans
C. tropicalis
C. glabrata
C. parapisilosis
ATCC 18804
ATCC 750
ATCC 90030
ATCC 22019
5a
128
4
16
16
16
16
8
16
32
5b
5c
4
16
4
5d
128
256
32
64
2
128
128
32
64
32
64
8
128
64
6a
6b
16
6c
16
8
6d
4
4
4
7a
256
16
8
128
64
1024
32
32
2
256
128
128
1
7b
7c
64
7d
2
4
Itraconazole
NT: Not tested
0.5
>512
256
>512