Synthesis of chimeric tetrapeptide-linked cholic acid derivatives: Impending synergistic agents

Article abstract of DOI:10.1016/j.bmcl.2008.09.013

Tetrapeptides derived from glycine and β-alanine were hooked at the C-3β position of the modified cholic acid to realize novel linear tetrapeptide-linked cholic acid derivatives. All the synthesized compounds were tested against a wide variety of microorganisms (Gram-negative bacteria, Gram-positive bacteria and fungi) and their cytotoxicity was evaluated against human embryonic kidney (HEK293) and human mammary adenocarcinoma (MCF-7) cell lines. While relatively inactive by themselves, these compounds interact synergistically with antibiotics such as fluconazole and erythromycin to inhibit growth of fungi and bacteria, respectively, at 1-24 μg/mL. The synergistic effect shown by our novel compounds is due to their inherent amphiphilicity. The fractional inhibitory concentrations reported are comparable to those reported for Polymyxin B derivatives.

Full text of DOI:10.1016/j.bmcl.2008.09.013

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5512–5517
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of chimeric tetrapeptide-linked cholic acid derivatives: Impending
synergistic agents
a,
b
Sudhir N. Bavikar a, , Deepak B. Salunke a,b, , Braja G. Hazra a, , Vandana S. Pore , Robert H. Dodd ,
*
*
Josiane Thierry b, Fazal Shirazi c, Mukund V. Deshpande c, Sreenath Kadreppa d, Samit Chattopadhyay d
a Organic Chemistry Synthesis Division, National Chemical Laboratory, Dr. Homi Bhabha Road, Pune, Maharashtra 411 008, India
b Institut de Chimie des Substances Naturelles, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France
c Biochemical Sciences Division, National Chemical Laboratory, Pune, India
d National Centre for Cell Science, Pune, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 May 2008
Revised 2 September 2008
Accepted 4 September 2008
Available online 9 September 2008
Tetrapeptides derived from glycine and b-alanine were hooked at the C-3b position of the modified cholic
acid to realize novel linear tetrapeptide-linked cholic acid derivatives. All the synthesized compounds
were tested against a wide variety of microorganisms (Gram-negative bacteria, Gram-positive bacteria
and fungi) and their cytotoxicity was evaluated against human embryonic kidney (HEK293) and human
mammary adenocarcinoma (MCF-7) cell lines. While relatively inactive by themselves, these compounds
interact synergistically with antibiotics such as fluconazole and erythromycin to inhibit growth of fungi
and bacteria, respectively, at 1–24 lg/mL. The synergistic effect shown by our novel compounds is due to
their inherent amphiphilicity. The fractional inhibitory concentrations reported are comparable to those
reported for Polymyxin B derivatives.
Keywords:
Cholic acid
Tetrapeptide
Synergism
Antimicrobial activity
Cytotoxicity
Ó 2008 Elsevier Ltd. All rights reserved.
The dramatically rising prevalence over the past few decades of
multidrug-resistant microbial infections has become a serious
health problem. In order to circumvent this situation, there is an
urgent need to develop new antimicrobial therapeutics. The class
of membrane-disrupting drugs is ideal as antimicrobial agents be-
cause microbes are unlikely to develop resistance to them.1,2 As the
outer membrane or cell wall of microbes provides a protective bar-
rier against many types of antibiotics,3 amphipathic molecules that
can act synergistically with various hydrophobic antibiotics as out-
er membrane permeabilizers may represent a new class of antibi-
otic agents.
attention because of its potent antimicrobial activity against a
broad spectrum of microorganisms8,9 (Fig. 1). Soon after the iso-
lation of squalamine, Regen reported10 rapid construction of
squalamine mimic 2 which displays extraordinary antimicrobial
properties. Naturally occurring steroid-amino acid conjugates
such as bufotoxin 3 and polymastiamide A 4, exhibit in vitro
antimicrobial activity.11 Several peptides have been identified
that increase the permeability of the outer membranes of
Gram-negative bacteria and sensitize these organisms to hydro-
phobic antibiotics.12 The best studied of these peptides are the
polymyxin
B (PMB) 5
derivatives. Savage and co-workers13
Design of novel amphipathic molecules: A literature survey of
antimicrobial steroids reveals that several amino cholesterol
derivatives exhibit profound antimicrobial activity.4 The in vitro
antibacterial properties of bile acids against certain Gram-posi-
tive microorganisms are well known.5 Consequently, the prepa-
ration of various bile acid-based aminosterols was reported
with a view to examine their activity as anti-microbial agents.6
A recent approach to combat against pathogens is to introduce
a polycationic chain onto a steroid scaffold. One such chimeric
natural product namely squalamine7 1 has attracted considerable
designed a class of cationic steroid antibiotics (CSA) 6 as PMB
mimics which display antibacterial activities comparable or
superior to that of Squalamine 1, bufotoxin 3 or PMB 5.
A number of intuitive assumptions can be drawn from this
wide-ranging literature survey.
ꢀ A common feature of steroid derived antimicrobials is their
potential to exhibit facially amphiphilic conformations contain-
ing polar and hydrophobic surfaces.14
ꢀ The overall amphiphilicity of the molecule plays a key role in
determining the level of antimicrobial activity.
ꢀ A generic structure with fine-tuning of the molecular amphiph-
ilicity may lead to novel molecules capable of selectively perme-
abilizing the microbial membranes.
* Corresponding author. Tel.: +91 20 25902336; fax: +91 20 25902629/25902624.
E-mail address: bg.hazra@ncl.res.in (B.G. Hazra).
These authors contributed equally in this work.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.09.013

S. N. Bavikar et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5512–5517
5513
O
H
N
O
O
O
O
NH
S
O
O
OH
NH3
1
H
S
N
HN
O
H3N
O
2
O
O
O
COOH
N
O
H
O
OMe
OH
HO3SO
H
NH
NH2
O
H
4
H
N
N
O
H
COOH
O
O
3
NH2
OH
O
N
O
H
H
H
O
N
N
N
O
N
N
N
H
O
H
H
O
NH2
O
O
NH2
O
O
NH
NH2
NH2
HN
NH2
O
NH2
O
NH2
H
6
N
NH
NH2
N
5
H
O
OH
NH2
Figure 1. Squalamine 1, steroid polyamine conjugate 2, steroid-amino acids conjugates 3 and 4, polymyxin B 5, and cationic steroid antibiotics 6.
With this in view, novel cholic acid-tetrapeptide conjugates of
glycine and b-alanine were synthesized and tested against a wide
variety of microorganisms. Cholic acid has been chosen because
of its natural amphiphathic nature.15 It differs from the conven-
tional head-to-tail amphiphiles because the polar and non-polar
domains are separated along the longitudinal axis of the molecule,
which gives rise to distinct polar and non-polar faces.16 A polypep-
tide segment has been introduced on to the cholic acid to have
combination of a hydrophilic functional moiety as well as a hydro-
phobic carrier in the same molecule. Synthetic variations of the
amino acid residues can produce a library of compounds with var-
iable amphiphilicity.
using 2M HCl:Et2O to afford the desired peptides 23 and 24 in
96% and 97% yield, respectively. Following the modified procedure
(using EDCI and HOBt), we have drastically reduced the reaction
time (7–8 h) compared to the earlier reported method, which re-
quired 3 days for the reaction to complete.18
Synthesis of the cholic acid intermediates, which are the build-
ing blocks for realization of the desired conjugates, is depicted in
Scheme 2. 3b-Amino methyl cholate 26 was synthesized from cho-
lic acid 25 in four steps in a straightforward way with an overall
yield of 77%.19 The amine functionality in compound 26 was pro-
tected using Boc anhydride to afford compound 27 in excellent
yield (Scheme 2).
Chemistry: We undertook the synthesis and evaluation of a new
family of cholic acid-polypeptide conjugates by exploiting a con-
vergent approach, using classical solution phase synthesis. Accord-
ing to the literature procedures,17 suitably protected monomers 9,
11 and 10, 12 were synthesized from glycine 7 and b-alanine 8,
respectively. The stepwise elongation was performed in dichloro-
methane (DCM) using 1-(3-dimethylaminopropyl)-3-ethylcarbodi-
imide hydrochloride (EDCI) activation to furnish Boc-(Gly)2-OBn
13 in 76% yield and Boc-(b-Ala)2-OBn 14 in 74% yield (Scheme 1).
Removal of the tert-butoxycarbonyl (Boc) group from peptides 13
and 14 was performed in trifluoroacetic acid (TFA)/DCM, to afford
compounds 15 and 16 in quantitative yield. On the other hand, cat-
alytic hydrogenolysis of the benzyl (Bn) group from peptides 13
and 14, furnished compounds 17 and 18 in excellent yields. Com-
pounds 19 and 20 were obtained by fragment condensation of 15
with 17 and 16 with 18 in DMF using EDCI activation in the pres-
ence of 1-hydroxybenzotriazole (HOBt) as catalyst. These tetrapep-
tides were purified by column chromatography to furnish
compounds 19 and 20 as white solids in 73% and 70% yields,
respectively.
Reduction of the methyl ester of compound 27 was carried out
using LAH to afford C-24 hydroxy compound 28 in 74% yield. Acyl-
ation of the C-7 and C-24 hydroxyl groups of compound 28 was
carried out using acetic anhydride and a catalytic amount of N,N-
dimethylaminopyridine (DMAP) to furnish compound 29 in 87%
yield. The protected C-3b amino functionality of compound 29
was unmasked using TFA to afford compound 30 in almost quanti-
tative yield.
Coupling of 3b-amino cholic acid intermediates 26 and 30 with
Boc-(Gly)4-OH 21 and Boc-(b-Ala)4-OH 22 under mild condition
using EDCI/HOBt and Et3N in DMF provided compounds 31, 32 in
74% and 71% yield and compounds 37, 38 in 75% and 74% yield,
respectively (Scheme 3). Subsequent hydrolysis of the methyl ester
functionality in compound 32 and C-7 and C-24 acetates in com-
pound 38 (LiOH 2M, CH3OH) and aqueous work-up provided the
corresponding acid 35, and hydroxy compound 40 in 89% and
84% yield, respectively. Cleavage of the Boc group in compounds
31, 32, 35, 37 and 40 was accomplished with 2M HCl:Et2O to afford
free amino compounds 33, 34, 36, 39, and 41, respectively, in yields
ranging from 89–96%. It is worth mentioning here that all the com-
pounds 31–41 (Scheme 3) are new and characterized fully by IR, 1H
NMR and 13C NMR spectroscopy.
The Benzyl groups of Boc-(Gly)4-OBn 19 and Boc-(b-Ala)4-OBn
20 were removed by a similar catalytic hydrogenation reaction to
furnish compounds 21 and 22 in 98% and 97% yield, respectively.
Finally, the Boc groups of compounds 21 and 22 were removed
Antimicrobial activity: Tetrapeptides 19–24, Cholic acid 25, and
cholic acid-tetrapeptide conjugates 31–41 were examined for

5514
S. N. Bavikar et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5512–5517
respectively, to find out the minimum inhibitory concentration
a
b
OH
OBn
-
SO3
H2N
+H3N
x
O
x
O
(MIC) values (Table 1). These compounds were also tested for their
ability to permeabilize the outer membrane of Gram-negative bac-
teria such as E. coli causing sensitization to hydrophobic antibiotics
that inefficiently cross the outer membrane. We also demonstrated
such permeabilization by cholic acid derivatives with C. albicans, a
pathogenic fungus (Table 2).
In the preliminary bioevaluation, tetrapeptides 19–24 as well as
cholic acid 25 did not show any appreciable antifungal or antibac-
terial effect. In comparison the tetrapeptide-linked cholic acid
derivatives 31–41 showed good to moderate activity against C.
albicans, B. poitrasii and F. oxysporum (columns A, C and E) whereas
these compounds were less active towards bacteria (columns F and
G) than fungi. The antifungal activity of most of the compounds
9, X = 1, 90%
10, X = 2, 92%
7, X = 1, Glycine
8, X = 2, β-Alanine
O
OH
O
N
x
H
O
11, X = 1, 97%
12, X = 2, 98%
O
O
H
c
N
(9 + 11) / (10 + 12)
O
N
OBn
x
O
H
x
13, X = 1, 76%
14, X = 2, 74%
O
O
H
was found to be similar to that of fluconazole (MIC, 32 lg/mL).
N
d
+H3N
OBn
O
F3C
O-
13 / 14
x
O
The difference in the toxicity of the synthesized compounds
against a wide variety of microorganisms can be attributed to the
differences in their cell wall/cell membrane compositions which
affect the passage of these compounds through cell wall/cell
membrane.20
To characterize synergism of 19–25 and 31–41 with fluconazole
(an antifungal agent) and erythromycin (a hydrophobic antibacte-
rial agent), we determined the concentrations of these compounds
x
15, X = 1, 98%
16, X = 2, 99%
O
O
H
N
e
13 / 14
N
OH
x
x
H
O
17, X = 1, 98%
18, X = 2, 97%
necessary to lower the MIC values of the antibiotics to 1 lg/mL (a
O
O
O
H
H
N
c
N
concentration at which many clinically useful antibiotics are acti-
ve).12b This measurement entailed incubating a known population
of yeast suspension (C. albicans) for 48 h in YPG and bacterial cells
(E. coli) for 24 h in a nutrient broth with fluconazole and erythro-
mycin, respectively, with incrementally varied concentrations of
the synthesized compounds as described by Savage et al.12b Tetra-
peptides 19–24 and cholic acid 25 did not showed any appreciable
synergism, on the other hand, almost all the cholic acid conjugates
exhibited very good synergism. This suggests that the synergism
effect shown by our novel compounds is due to their inherent
amphiphilicity.
To quantify the synergistic behaviour of our compounds with
fluconazole and erythromycin, fractional inhibition concentration
(FIC) values were also calculated.21 Synergism between antibiotics
is indicated by FIC values of less than 0.5. In the present case all the
synthesised compounds 31–41 displayed FIC values of less than 0.5
with both fluconazole and erythromycin and many of the FICs
shown in Table 2 are comparable to those reported for PMB
derivatives.12a
(15 + 17) / (16 + 18)
O
O
N
H
N
OBn
x
O
x
O
x
H
x
19, X = 1, 73%
20, X = 2, 70%
O
O
H
H
N
e
N
19 / 20
O
N
H
N
OH
x
O
x
O
x
x
H
21, X = 1, 98%
22, X = 2, 97%
O
O
H
H
N
f
N
21 / 22
H3N
N
O
x
O
x
O
x
x
H
23, X = 1, 96%
24, X = 2, 97%
Scheme 1. Synthesis of polypeptide chains. Reagents and conditions: (a) benzyl
alcohol, pTSA, toluene, reflux, 4 h; (b) Boc anhydride, 1 N NaOH, dioxane:water,
0–25 °C, 30 min; (c) EDCI, HOBt, Et3N, DCM, 0–25 °C, 6 h; (d) TFA, DCM, 0–25 °C,
2 h; (e) H2, Pd-C, MeOH, 25 °C, 1 h; (f) 2M HCl:Et2O, 0–25 °C, 1.5 h.
in vitro antifungal as well as antibacterial activity against the fun-
gal strains viz., Candida albicans, Cryptococcous neoformans, Benja-
miniella poitrasii, Yarrowia lipolytica, Fusarium oxysporum strains
and bacterial strains Escherichia coli, and Staphylococcus aureus,
Antiproliferative activity: Bile acids are known to promote prolif-
eration and metastasis of cells of cancer origin and inhibit the pro-
liferation of cells of non-cancer origin.22 They are also known to be
extremely toxic at high doses, presumably damaging cell mem-
O
O
O
OH
OH
OH
OH
ref. 19
77%
OMe
OMe
a
b
74%
98%
HO
H2N
BocHN
OH
OH
OH
H
H
H
25
27
26
OH
OH
OH
OAc
OH
OAc
c
d
99%
87%
BocHN
BocHN
OH
OAc
H2N
OAc
H
H
H
29
30
28
Scheme 2. Synthesis of steroid backbone. Reagents and conditions: (a) Boc anhydride, Et3N, dioxane:water, 25 °C 6 h; (b) LAH, THF, 0–25 °C, 1 h; (c) Ac2O, DMAP, Et3N, DCM,
25 °C, 8 h; (d) TFA, DCM, 0–25 °C, 2 h.

S. N. Bavikar et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5512–5517
5515
O
O
H
H
O
N
N
HO
HO
O
OMe
OMe
HO
HO
x
x
HN
HN
a
b
21/22 + 26
O
O
O
O
x
x
H
HN
NH3
x
HN
N
O
xN
xN
x
H
H
O
O
O
33, X = 1, 95%
34, X = 2, 94%
31, X = 1, 74%
32, X = 2, 71%
O
O
H
H
N
O
N
O
HO
HO
HO
O
OH
HO
b
c
32
HN
O
HN
O
91%
89%
H
H
N
H
H
H
N
NH3
N
N
N
O
O
O
O
O
O
35
36
H
N
H
O
N
HO
HO
O
AcO
AcO
x
OAc
OAc
37
HN
HN
b
a
21/22 + 30
O
O
O
O
x
H
96%
HN
NH3
HN
N
O
N
H
xN
H
x
O
O
O
39, X = 1
37, X = 1, 75%
38, X = 2, 74%
H
N
H
N
O
HO
HO
O
HO
HO
c
38
b
OH
OH
84%
89%
HN
O
HN
O
H
N
H
N
H
H
H
N
N
O
N
NH3
O
O
O
O
O
41
40
Scheme 3. Syntheses of cholic acid-tetrapeptide conjugates. Reagents and conditions: (a) EDCI, HOBt, Et3N, DMF, 0–25 °C, 6 h; (b) 2 M HCl:Et2O, 0- 25 °C, 1.5 h; (c) 2M LiOH,
MeOH, 25 °C, 12 h.
Table 1
Minimum inhibitory concentration (MIC) of modified steroids
Entry
Compound
Antimicrobial activity MIC (lg/mL)
Fungal strains
Bacterial strains
A
B
C
D
E
F
G
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
25
31
32
33
34
35
36
37
38
39
40
41
>128
16
32
32
128
32
32
64
32
64
128
64
2
32
NT
NT
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
16
128
64
16
64
24
48
24
32
64
16
>128
64
16
32
>128
>128
96
>128
>128
>128
>128
>128
96
>128
>128
>128
16
>128
64
64
128
32
64
64
64
>128
32
64
>128
16
8
NT
NT
>128
96
>128
>128
96
>128
32
64
>128
>128
>128
32
NT
NT
>128
32
>128
96
64
>128
>128
>128
32
64
>128
>128
NT
NT
16
AmpB
Fluconazole
Tetracycline
Erythromycin
32
NT
NT
64
NT
NT
NT
NT
8
64
32
A, C. albicans; B, C. neoformans; C, B. poitrasii; D, Y. lipolytica; E, F. oxysporum; F, E. coli; G, S. aureus; NT, Not Tested.

5516
S. N. Bavikar et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5512–5517
Table 2
MIC, Permeabilization and FIC Data for 19–25, and 31–41 with C. albicans and E. coli
Entry
Compound
a (lg/ml)
a0
(
lg/ml)
a00
b (
lg/ml)
b0
(l
g/ml)
b00
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
31
32
33
34
35
36
37
38
39
40
41
>128
>128
>128
>128
128
>128
>128
16
32
32
128
32
32
128
128
64
1.03
1.03
0.53
0.53
1.03
1.03
0.53
0.53
0.22
0.22
0.055
0.41
0.062
0.22
0.28
0.125
0.125
0.125
>128
>128
>128
>128
128
>128
>128
96
>128
>128
96
>128
32
128
64
128
>128
64
>128
64
24
16
12
8
16
4
24
10
8
1.03
0.53
1.03
1.03
0.53
1.03
0.52
0.265
0.14
0.11
0.099
0.14
0.14
0.39
0.093
0.078
0.172
0.265
64
>128
128
64
8
6
6
3
12
1
12
8
6
64
32
64
128
64
64
>128
>128
>128
32
12
6
20
8
a: MIC of the synthesized compounds against C. albicans.
a0: Concentration of the synthesized compounds required to lower the MIC of fluconazole from 32 to 1
lg/mL (a concentration at which many clinically useful antibiotics are
active).
a00: FIC values with fluconazole.
b: MIC of the synthesized compounds against E. coli.
b0: Concentration of the synthesized compounds required to lower the MIC of erythromycin from 64 to 1
b00: FIC values with erythromycin.
lg/mL.
branes and mitochondrial membranes.23 At low doses, bile acids
stimulate the cell-signalling effects involving various pathways.24
Hence we tested the cytotoxicity of the synthesized compounds
in two different cell lines, one of cancer origin (human mammary
carcinoma: MCF-7) and the other of non-cancer origin (human
embryonic kidney: HEK293). Concentration of compounds needed
to reduce the population growth of HEK293 and MCF-7 cells by
50% (IC50) in vitro was evaluated using the MTT assay25 (Table 3).
Graphical representation of antiproliferative activities of all the
compounds is included in the Supporting information.
fluconazole and erythromycin to inhibit growth of fungi and bacte-
ria, respectively, at 1–24 g/mL.
l
In conclusion, a generic structure wherein fine-tuning of the
molecular amphiphilicity is possible, have been designed utilizing
amphiphilic nature of cholic acid. We have observed that, incorpo-
ration of linear tetrapeptide fragment on the cholic acid skeleton
not only reduced its toxicity to the tested cell lines but also dem-
onstrated very good synergism effect with the known antibiotics.
The synergism of the most active compounds 36 (1 and 4
lg/mL)
and 41 (6 and 8 g/mL) greatly improves the activity of fluconazole
l
Of all the cholic acid-tetrapeptide conjugates tested, none
showed any substantial cytotoxicity up to 80 lM concentration.
and erythromycin against C. albicans and E. coli, respectively. To
conclude, we have demonstrated that molecular amphiphilicity is
one of the important factor which accounts for the easy transport
of the molecules through the membranes.
Incorporation of peptide residues on the cholic acid derivatives
drastically reduced its cytotoxicity towards HEK293 cell line.
Moreover, it was found that all the compounds except 33 and 40
enhanced the proliferation of MCF-7 cells and not HEK293 cells.
This confirms the observation that cholic acid and its derivatives
promote the proliferation of cells from cancerous origin and not
normal cells. This study also confirm that the synthesized cholic
acid-peptide conjugates are not toxic to the cell lines tested at con-
Acknowledgments
SNB thanks University Grant Commission (UGC, New Delhi) for
the Senior Research Fellowship. D.B.S. thanks Embassy of France in
India, New Delhi for Sandwich Thesis Scholarship. Emeritus Scien-
tist Scheme awarded to B.G.H. by the CSIR, New Delhi is gratefully
acknowledged. V.S.P. thanks Director NCL for In-House funding
(MLP013126).
centrations up to 80 lV. While relatively inactive by themselves,
these compounds interact synergistically with antibiotics such as
Supplementary data
Table 3
Cytotoxicity of modified steroids
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.09.013.
Entry
Compound
IC50 (lM)
HEK293
MCF-7
1
2
3
4
5
6
7
8
9
25
31
32
33
34
35
36
37
38
39
40
41
50
300
100
500
600
>1000
200
500
150
>1000
600
>1000
80
References and notes
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1998, 120, 2961.
3. (a) Hancock, R. E. Annu. Rev. Microbiol. 1984, 38, 237; (b) Labischinski, G.;
Bradaczek, H.; Naumann, D.; Rietschel, D. T.; Giesbrecht, P. J. Bacteriol. 1985,
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