Synthesis and biological evaluation of novel lipoamino acid derivatives

Article abstract of DOI:10.1016/j.bmcl.2015.10.086

Seven novel lipoamino acid conjugates were synthesized from methyl oleate and amino acids. Methyl oleate was grafted to different amino acids using thioglycolic acid as a spacer group. Seven derivatives (3a-g) were prepared and characterized by spectral data (NMR, IR and MS spectral studies). All the derivatives were studied for their antimicrobial, anti-biofilm and anticancer activities. Among all the derivatives, it was found that compound 3b was the most potent antibacterial compound which showed good activity against four Gram positive bacterial strains and also exhibited excellent antifungal activity against a fungal strain. In the anti-biofilm assay, compound 3b showed promising activity with IC₅₀ value of 2.8 μM against Bacillus subtilis MTCC 121. All the compounds showed anticancer activities with 3c showing promising anticancer activity (IC₅₀ = 15.3-22.4 μM) against the four cell lines tested.

Full text of DOI:10.1016/j.bmcl.2015.10.086

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of novel lipoamino acid
derivatives
Shiva Shanker Kaki a, Sammaiah Arukali a, Padmaja V. Korlipara a, R. B. N. Prasad a,
,
⇑
Poornachandra Yedla b, C. Ganesh Kumar b
a Centre for Lipid Research, CSIR-Indian Institute of Chemical Technology, Uppal Road, Hyderabad 500007, India
b Medicinal Chemistry and Pharmacology Division, CSIR-Indian Institute of Chemical Technology, Uppal Road, Hyderabad 500007, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Seven novel lipoamino acid conjugates were synthesized from methyl oleate and amino acids. Methyl
oleate was grafted to different amino acids using thioglycolic acid as a spacer group. Seven derivatives
(3a–g) were prepared and characterized by spectral data (NMR, IR and MS spectral studies). All the
derivatives were studied for their antimicrobial, anti-biofilm and anticancer activities. Among all the
derivatives, it was found that compound 3b was the most potent antibacterial compound which showed
good activity against four Gram positive bacterial strains and also exhibited excellent antifungal activity
against a fungal strain. In the anti-biofilm assay, compound 3b showed promising activity with IC50 value
Received 9 September 2015
Revised 26 October 2015
Accepted 29 October 2015
Available online xxxx
Keywords:
Fatty acid
Lipoamino acid
Amino acid
Antimicrobial activity
Antibiofilm activity
of 2.8
lM against Bacillus subtilis MTCC 121. All the compounds showed anticancer activities with 3c
showing promising anticancer activity (IC50 = 15.3–22.4
l
M) against the four cell lines tested.
Ó 2015 Elsevier Ltd. All rights reserved.
Emergence of antimicrobial resistance increases the number of
diseases and poses a threat in prevention and treatment of infec-
tions caused by various bacteria, viruses, fungi and parasites.1 This
is a global concern and thus necessitated the interest and search
for new and more promising potential antibiotics. Production of
novel hybrid molecules by combining two biologically important
and biocompatible molecules is a well known strategy for the syn-
thesis of novel bioactives.2,3 Fatty acids, one of the building blocks
of biomolecules are reported to assist in host defense against
pathogenic microorganisms. Fatty acids and their derivatives exhi-
bit broad spectrum of antibacterial action with potencies compara-
ble to some natural antimicrobial peptides.4,5 The broad spectrum
of bioactivity with non-specific mode of action makes them attrac-
tive substrates for antimicrobial agents for application in medicine,
agriculture, cosmetics and nutraceutical areas.
Combination of fatty acids with natural amino acids produces
amphiphilic lipoamino acids with combined properties of both
lipids and amino acids.6 Lipoamino acid derivatives are reported
to be employed in the preparation of lipid core peptide systems
for application in the delivery of genes, drugs and vaccines.7 The
lipoamino acid derivatives are expected to possess membrane like
properties which could help in the passage of drugs through the
biological membranes.8 Studies of biological activities of
N-palmitoylated amino acids and N-stearyl amino acids showed
that these compounds function as potential biostatic additives.9,10
Therefore, conjugation of amino acids with a fatty acid derivative
changes the lipophilicity which influences the membrane perme-
ability of the synthetic molecule. These types of lipoamino acid
conjugates are used to enhance the oral absorption of drugs like
peptides, b-lactam antibiotics and alkaloid-based molecules.11
Moreover, products like lipoaminoacids and derivatives have been
reported to be biodegradable and can find wide range of applica-
tions in food, pharmaceutical and cosmetic formulations.12
In the present study, we describe the functionalization of
methyl oleate with thioglycolic acid group by thiol-ene coupling,
followed by condensation of the carboxylic acid moiety with amine
group of amino acid esters to produce novel lipoamino acid deriva-
tives. Synthesis was carried out as shown in Scheme 1. Initially
methyl oleate was functionalized with thioglycolic acid based on
a reported method via thiol-ene coupling reaction using AIBN as
initiator to give the corresponding product in 92.5% yield.13 The
second step was condensation of the acid group with amine group
of different amino acid esters catalyzed by EDC.HCl (N-(3-dimethy-
laminopropyl)-N0-ethylcarbodiimide hydrochloride) and HOBt
(1-hydroxybenzotriazole hydrate) reagent which resulted in the
final products (3a–g) in yields ranging from 78% to 82%.
Biological activities: All the prepared compounds were tested
for biological activities such as antimicrobial, anti-biofilm and
cytotoxic activities.
⇑
Corresponding author. Tel./fax: +91 40 27193370.
E-mail address: rbnprasad@iict.res.in (R.B.N. Prasad).
http://dx.doi.org/10.1016/j.bmcl.2015.10.086
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Kaki, S. S.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.10.086

2
S. S. Kaki et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
O
O
Compound 3c showed moderate antibacterial activity (MIC value
of 31.2 g/mL) against both M. luteus MTCC 2470 and S. aureus
MTCC 96. Compound 3g showed promising antibacterial activity
(MIC value of 3.9 g/mL) against both M. luteus MTCC 2470 and
B. subtilis MTCC 121.
Based on the preliminary antibacterial activity data screening,
further studies on minimum bactericidal concentration (MBC)
and biofilm inhibition assay was conducted on four bacterial
strains.15 The compounds which showed antibacterial activity
were tested for determining the MBC and it was found that com-
pound 3b was the most potent compound showing very good
activity against all the four Gram positive microbes, namely
M. luteus MTCC 2470, S. aureus MTCC 96, S. aureus MLS-16 MTCC
2940 and B. subtilis MTCC 121.
AIBN
O
O
l
S
2
HS
COOH
COOH
1
reflux
l
O
(3a) R = H
EDC.HCl
HOBt
DMAP
H2N
O
(3b) R = CH3
R
R
(3c) R = CH(CH3)CH2CH3
(3d) R = CH2CH(CH3
O
)
2
O
S
(3e) R =
(3f) R =
O
NH
O
3 (a-g)
O
NH
NH
(3g) R =
N
The results of the MBC data are presented in Table 2. All the
compounds showed activity against M. luteus MTCC 2470 with
compounds 3b and 3g being the most potent compounds, both
Scheme 1. Synthetic route for lipoamino acid conjugates.
Antimicrobial activity: The antimicrobial activities of the novel
showing MIC value of 7.8
promising bactericidal activity against B. subtilis MTCC 121 with
MIC value of 3.9 g/mL. However, the values were than the stan-
dard reference drug, Ciprofloxacin which had MIC values ranging
between 0.9 and 1.9 g/mL.
These four compounds were also tested for biofilm inhibition
assay according to a reported protocol.16 The bacteria which
caused biofilm formation were reported to tolerate antibiotics
which posed a major threat in treatment of bacterial infections.17
The compounds prepared in the present study are derived from
amino acids and fatty acid and therefore can be projected as new
molecules which can prevent the formation of biofilm. Accordingly,
the compounds which showed activity in the preliminary antimi-
crobial assay were screened for biofilm inhibition assay and the
results to this regard are presented in Table 3. It was observed that
compound 3g showed excellent anti-biofilm activity against
B. subtilis MTCC 121 and M. luteus MTCC 2470 with IC50 values of
lg/mL. Compounds 3a and 3g showed
lipoamino acid esters were determined using well diffusion
method14 against different pathogenic strains. The synthesized
compounds were evaluated for antimicrobial activity against seven
bacterial organisms, namely Micrococcus luteus (M. luteus) MTCC
2470, Staphylococcus aureus (S. aureus) MTCC 96, S. aureus MLS-
16 MTCC 2940, Bacillus subtilis (B. subtilis) MTCC 121, Escherichia
coli (E. coli) MTCC 739, Pseudomonas aeroginosa (P. aeroginosa)
MTCC 2453 and Klebsiella planticola (K. planticola) MTCC 530 and
also against a fungal strain, Candida albicans (C. albicans) MTCC
3017. The MIC (Minimum inhibitory concentration) was deter-
mined by comparing the standard Ciprofloxacin and Miconazole
as reference drugs for evaluating the antibacterial and antifungal
activities, respectively. Out of the compounds tested, two com-
pounds showed both antibacterial and antifungal activity and
two compounds showed only antibacterial activity. The results of
antimicrobial evaluation are presented in Table 1. The compounds
l
l
whose MIC was less than 125
antimicrobial compounds and were taken up for further
evaluation.
l
g/mL were considered as active
1.9 and 2.1
activity against all the strains with IC50 values ranging from 2.8
to 9.3 M. Compound 3a showed moderate activity against
lM, respectively. Compound 3b showed anti-biofilm
l
From the antimicrobial activity data, it was observed that four
compounds showed activity out of the total seven compounds
screened. Among the four active compounds, compounds 3a and
3b exhibited both antibacterial and antifungal activity with com-
pound 3b being more potent than 3a. Compound 3b exhibited
good antimicrobial activity with MIC value of 7.8, 15.6, 7.8 and
M. luteus MTCC 2470 and B. subtilis MTCC 121, whereas compound
3c showed lowest activity among all the compounds tested for bio-
film inhibition assay.
From a structure–activity relationship perspective, the prepared
lipoamino acid ester derivatives (3a–g) indicated that compound
3b derived from alanine showed promising activity with respect
to the evaluation of antimicrobial and anti-biofilm activities. The
lipoamino acid from glycine (3a) and alanine (3b) were found to
3.9 lg/mL against M. luteus MTCC 2470, S. aureus MTCC 96,
S. aureus MLS-16 MTCC 2940 and B. subtilis MTCC 121, respectively.
Table 1
Antimicrobial activity of the lipoamino acid conjugates 3 (a–g)
S. No
Test compounds
Minimum inhibitory concentration (lg/ml)
M ia
S ab
S ac
B sd
E ce
P af
K pg
C ah
1
2
3
4
5
6
7
3a
3b
3c
3d
3e
3f
15.6
7.8
>125
15.6
31.2
>125
>125
>125
>125
0.9
>125
7.8
7.8
3.9
>125
>125
>125
>125
>125
>125
>125
0.9
>125
>125
>125
>125
>125
>125
>125
0.9
>125
>125
>125
>125
>125
>125
>125
0.9
31.2
7.8
31.2
>125
>125
>125
3.9
>125
>125
>125
>125
>125
0.9
>125
>125
>125
>125
3.9
>125
>125
>125
>125
>125
>125
7.8
3g
Ciprofloxacin (Standard)
Miconazole (Standard)
0.9
>125
0.9
>125
>125
>125
>125
>125
>125
a
b
c
M. luteus MTCC 2470.
S. aureus MTCC 96.
S. aureus MLS-16 MTCC 2940.
B. subtilis MTCC 121.
E. coli MTCC 739.
P. aeruginosa MTCC 2453.
K.planticola MTCC 530.
C. albicans MTCC 3017.
d
e
f
g
h
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S. S. Kaki et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
Table 2
Table 4
Minimum bactericidal concentration data
Antifungal activity of compound 3b
Test compounds
Minimum bactericidal concentration
g/mL)
S
No
Test organism
MIC (
Miconazole 3b
7.8 7.8 15.6
l
g/mL)
MFC (lg/mL)
(
l
3b
Miconazole
M la
S ab
S ac
B sd
1
2
3
4
5
6
7
8
9
C. albicans MTCC 183
C. albicans MTCC 227
C. albicans MTCC 854
C. albicans MTCC 1637
C. albicans MTCC 3018
C. albicans MTCC 3958
C. albicans MTCC 4748
C. albicans MTCC 7315
C. parapsilosis MTCC 1744
C. aaseri MTCC 1962
C. glabrata MTCC 3019
C. krusei MTCC 3020
Issatchenika hanoiensis
MTCC 4755
7.8
3a
3b
3c
3g
31.2
7.8
31.2
7.8
—
—
15.6
—
—
1.9
3.9
7.8
—
3.9
1.9
15.6 7.8
31.2 7.8
31.2 7.8
15.6 7.8
7.8 7.8
15.6 7.8
15.6 7.8
7.8 7.8
15.6 7.8
31.2 7.8
15.6 7.8
15.6 7.8
31.2 15.6
62.5 7.8
31.2 15.6
31.2
15.6
15.6
31.2 15.6
7.8 7.8
31.2 15.6
31.2
31.2
—
7.8
7.8
7.8
Ciprofloxacin (Standard)
0.9
1.9
a
b
c
M. luteus MTCC 2470.
S. aureus MTCC 96.
S. aureus MLS-16 MTCC 2940.
B. subtilis MTCC 121.
10
11
12
13
d
31.2
15.6
31.2
7.8
7.8
7.8
exhibit antifungal activities too. It was interesting to note that
compound 3e bearing phenylalanine did not show antimicrobial
activity which suggests that the phenyl moiety could be affecting
the bioactivity. However, compound 3g derived from histidine
showed excellent activity against M. luteus MTCC 2470 and
showed antifungal activity against C. parapsilosis MTCC 1744 with
MFC value of 7.8 g/mL which was on par with Miconazole.
l
B. subtilis MTCC 121 with MIC value of 3.9
biofilm activity, compound 3g showed promising activity against
B. subtilis MTCC 121 and M. luteus MTCC 2470 with IC50 values of
l
g/mL. In the anti-
It is know that many antifungal drugs currently available for
treatment of Candida infections target the ergosterol biosynthetic
pathway or its end product, ergosterol.18 Considering this fact,
we examined the compound 3b in comparison with the standard
miconazole drug to delineate the mode of action in the ergosterol
biosynthetic pathway against one of the highly susceptible strain
of C. parapsilosis MTCC 1744 and the results to this regard are
shown in Table 5.
From the ergosterol biosynthesis inhibition assay, it was
observed that the ergosterol content decreased significantly with
an increase in the concentration of test compound 3b. Similarly,
dose-dependent decrease in ergosterol content was observed when
the C. parapsilosis MTCC 1744 was cultured in presence of micona-
zole. Our findings suggest that the lipoamino acid conjugate (3b)
alters the sterol profile and thus exerts its antifungal activity
through inhibition of ergosterol biosynthesis. The selective cyto-
toxic behavior of this compound suggests that it has affinity to
the specific target site in the ergosterol biosynthetic pathway.
The fungicidal activity of the compound 3b may also be due to
the cell membrane permeability and subsequent cell death. It
was also earlier reported that lipid derivatives disorganized the
cytoplasm by disintegrating the plasma membrane which could
be the plausible reason for effective antifungal activity.19 Also the
strong lipophilic character of the derivative could have helped it
to affect the cellular membrane and the peptide linkage would
have exhibited the selective antimicrobial activity.
1.9 and 2.1 lM, respectively. However, compound 3b was observed
to exhibit biofilm inhibition against all the four bacterial strains
tested. It can be observed that all the compounds showed lower
activity as compared with the reference drug, Ciprofloxacin in
the antibacterial and biofilm inhibition assays studied. Based on
the structural characteristics, it is interesting to note that
compounds 3b and 3e derived from alanine and phenylalanine,
respectively, showed considerable differences in the activities
studied in the present investigation.
Based on the preliminary antimicrobial evaluation data, it was
found that compounds 3a and 3b exhibited antifungal activity
against C. albicans MTCC 3017 with MIC values of 31.2 and
7.8 lg/mL, respectively. From the observed preliminary antimicro-
bial activity evaluation, compound 3b was further screened for
antifungal activity against several fungal strains and the results
to this regard are presented in Table 4. Based on the MIC values,
it was observed that compound 3b was equipotent to Miconazole
showing excellent activity against C. albicans MTCC 183, C. albicans
MTCC 3958 and C. parapsilosis MTCC 1744 with MIC value of
7.8
lg/mL similar to Miconazole. It showed good activity against
other fungal strains with MIC values of 15.6 and 31.2
lg/mL. To
further asses the antifungal activity, minimum fungicidal concen-
tration (MFC) was determined against the fungal strains. It was
interesting to observe that compound 3b showed excellent anti-
fungal activity against most of the Candida strains with MFC values
All the prepared compounds were further evaluated for in vitro
cytotoxicity by MTT assay20 against four different cell lines and the
data of this regard is presented in Table 6. It can be observed that
compounds 3a and 3c exhibited good cytotoxic activities as
compared to other compounds. According to structure–activity
relationship between these two derivatives, compound 3c derived
from isoleucine showed excellent activity against HepG2 and
ranging from 7.8 to 62.5 lg/mL. In particular, compound 3b
Table 3
SKOV3 cell lines with IC50 values of 15.3 and 16.4 lM, respectively.
Anti-biofilm activity of the selected compounds
Compounds 3f and 3g bearing heterocyclic amino acid moiety did
not show any activity against the tested cell lines. It was interest-
ing to note that among compounds 3b and 3e, the compound 3e
derived from phenylalanine showed better activity as compared
to 3b. In general, the synthesized compounds showed good to
moderate cytotoxicity as compared to the reference drug,
Doxorubicin.
Test compounds
IC50 values in (lM)
M la
S ab
S ac
B sd
3a
3b
3c
3g
8.2 0.22
4.1 0.31
—
—
4.4 0.21
9.3 0.11
3.2 0.26 2.8 0.31
16.8 0.44 21.5 0.42
—
—
—
2.1 0.28
—
1.9 0.12
Ciprofloxacin (Standard) 0.5 0.08
0.3 0.11
0.4 0.09 0.5 0.10
Novel lipoamino conjugates involving amino acids and fatty
acid were synthesized employing reported protocols. All the pro-
duct structures were characterized by 1H NMR, 13C NMR, FT-IR
and MS spectral data. Among the prepared derivatives, compounds
a
b
c
M. luteus MTCC 2470.
S. aureus MTCC 96.
S. aureus MLS-16 MTCC 2940.
B. subtilis MTCC 121.
d
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S. S. Kaki et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Table 5
Ergosterol biosynthesis inhibition of the lipoamino acid conjugate (3b)
Test compound
Mean ergosterol content of cells grown with compounds at a concentration (lg/ml)
0
2
4
8
16
3b
1.62 0.08
1.62 0.08
1.41 0.11
1.28 0.09
0.63 0.11
0.54 0.07
0.38 0.08
0.24 0.05
0.11 0.04
0.09 0.02
Miconazole
Supplementary data
Table 6
Anticancer activity of lipoamino acid conjugates (3a–g)
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.10.
086.
Test compound
IC50 values in (lM)
DU145
HepG2
SKOV3
MDA-MB
231
3a
3b
30.8 0.23
29.6 0.29
25.2 0.52
31.4 0.13
References and notes
154.4 0.44 102.5 0.25 113.6 0.36 98.7 045
3c
3d
3e
3f
19.0 0.22
214.9 0.52
49.1 0.39
—
15.3 0.18
—
33.6 0.32
—
16.4 0.24
—
42.5 0.16
—
22.4 0.11
152.1 0.48
38.6 0.29
—
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Acknowledgements
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Please cite this article in press as: Kaki, S. S.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.10.086