Bile Acid Oligomers and Their Combination with Antibiotics to Combat Bacterial Infections

Article abstract of DOI:10.1021/acs.jmedchem.8b01433

The ever-growing risk of bacterial resistance is a critical concern. Among the various antimicrobial resistant bacterial strains, methicillin and vancomycin resistant Staphylococcus aureus are among the most dreadful, causing serious complications. On the basis of the hypothesis that microbes have reduced ability to develop resistance against membrane targeting antibiotics, bile acid oligomers having unique facially amphiphilic topologies were designed and synthesized. The oligomers with specific linkers exhibited potent and selective antibacterial activity against Gram-positive bacteria. The lead compounds also improved the efficacy of a range of known antibiotics belonging to different classes when tested in combination. The active dimers were found to be effective against antibiotic-resistant clinical isolates of S. aureus, including multidrug resistant isolates. A significant inhibitory activity against S. aureus biofilm, a highly drug-resistant bacterial phenotype often unresponsive to antibiotic therapy, was also noticed. No adverse effects were observed by these dimers in a cell viability assay against HEK293 cells.

Full text of DOI:10.1021/acs.jmedchem.8b01433

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Article
Bile Acid Oligomers and their Combination
with Antibiotics to Combat Bacterial Infections
Poonam Singla, Priyanka Dalal, Mahaldeep Kaur, Geeta Arya,
Surendra Nimesh, Rachna Singh, and Deepak B Salunke
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01433 • Publication Date (Web): 02 Nov 2018
Downloaded from http://pubs.acs.org on November 3, 2018
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Bile Acid Oligomers and their Combination with Antibiotics to
Combat Bacterial Infections
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2,
Poonam, Priyanka, Mahaldeep Kaur, Geeta Arya, Surendra Nimesh, Rachna Singh, * and
1
,
Deepak B. Salunke *
1Department of Chemistry and Centre for Advanced Studies in Chemistry, Panjab University,
Chandigarh, 160014, India.
2Department of Microbial Biotechnology, Panjab University, Chandigarh, 160014, India.
3Department of Biotechnology, Central University of Rajasthan, Ajmer, 305817, India.
*
Corresponding authors: salunke@pu.ac.in; rachna.singh@pu.ac.in
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ABSTRACT
O
OH
e
c
a
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0
f
e
c
c
r
i
i
u
c
b
b
a
e
e
S
f
o
c
o
c
r
h
a
h
a
c
i
OH
u
p
f
p
f
b
S
o
r
o
r
o
r
u
r
u
h
r
d
S
d
S
p
OH
l
a
y
y
o
o
H
H
r
P
Polar
Surfa
d
y
ce
H
OH
Bile acid
The ever-growing risk of bacterial resistance is a critical concern. Among the various
antimicrobial resistant bacterial strains, methicillin and vancomycin resistant Staphylococcus
aureus are among the most dreadful, causing serious complications. Based on the hypothesis that
microbes have reduced ability to develop resistance against membrane targeting antibiotics, bile
acid oligomers having unique facially amphiphilic topologies were designed and synthesized.
The oligomers with specific linkers, exhibited potent and selective antibacterial activity against
Gram-positive bacteria. The lead compounds also improved the efficacy of a range of known
antibiotics belonging to different classes, when tested in combination. The active dimers were
found to be effective against antibiotic-resistant clinical isolates of S. aureus, including multi-
drug resistant isolates. A significant inhibitory activity against S. aureus biofilm, a highly drug-
resistant bacterial phenotype often unresponsive to antibiotic therapy, was also noticed. No
adverse effects were observed by these dimers in a cell viability assay against HEK293 cells.
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INTRODUCTION
The rise in drug resistance amongst bacteria has led to the emergence of life-threatening
infections that are difficult to treat with the presently available antibiotics. Synthesis of new
antimicrobials with increased potency towards microbes and lower toxicity is a major challenge
these days. Staphylococcus aureus, a Gram-positive opportunistic pathogen, has remarkably
developed resistance to most of the commonly used antibiotics, such as penicillin, methicillin
and even vancomycin. The diseases caused by this organism are common in both developed and
developing countries, involving organs ranging from skin, soft tissue, respiratory system, bone,
joints to endovascular tissues.1,2 So, there is an immediate need for the development of novel
antimicrobial drugs or formulations that can work well against such drug-resistant microbes to
combat the infections caused by them.
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Membrane targeting antibiotics are generally considered favorable over the other drugs because
of the reduced ability of microbes to develop resistance mechanisms against them. These include
3
daptomycin 1, polymyxinB 2 and amphotericin B (Amp B) 3 (Figure1). The other interesting
membrane targeting molecules include Cationic Steroid Antibiotics (CSA) such as ceragenin 4 as
well as bile acid based molecular umbrellas which are known to complex with phospholipids of
the bacterial cell membranes.4-6
An inherent facial amphiphilicity is the crucial element responsible for the membrane disrupting
ability of these antimicrobials.7-10 For instance, Amp B (Figure 1), a facially amphiphilic
molecule widely used as an antifungal agent to treat systemic mycoses, is known to kill fungi by
punching holes in their cell membranes.11-15
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H3N
O
NH2
O
OH
O
H
O
O
O
H
N
H
H
N
NH
N
H
N
N
O
N
N
H
N
N
O
O
CONH2
H
N
O
NH
O
O
H
H
H
O
O
O
HN
HOOC
HOOC
NH2
O
O
NH
HN
N
N
H
NH2
NH2
O
H
H
C9H19
NH
O
O
HO
NH
O
N
NH
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COOH
O
H
N
O
N
H
O
N
H
N
O
OH
H
O
O
NH2
O
1. Daptomycin
2. Polymixin B
NH2
HO
OH
H2N
O
H
OH
N
HO
O
H
H
O
H
OH OH
OH OH
O
OH
HOOC
H
H
O
OH
H2N
O
NH2
HO
H2N
O
H
O
3
. Amphotericin B
4. Ceragenin CSA-13
Figure 1. Membrane targeting antibiotics.
The self-assembled supramolecular structures facilitated by the hydrophilic interactions in Amp
B and its specific interaction with sterols in the fungal cell membranes are responsible for the
observed activity of the Amp B.16-18 The dimeric and tetrameric structures of Amp B thus formed
orient and localize themselves in such a way that they can penetrate in to the membrane
hydrophobic core of the fungus.19,20
We earlier reported the synthesis of bile acid dimers with different amphiphilic topology having
2
1
antifungal and antiproliferative activities. A typical facial amphiphilic conformation of these
dimers forming a sandwich of hydrophilic face between two hydrophobic faces was suggested to
be responsible for the observed activity. The active dimer was found to be selectively toxic to
fungus and was not explored for its antibacterial activity. Such membrane-active compounds are
expected to show synergistic activity with antibiotics and this component was also not explored
in past. Furthermore, to understand the structure activity relationship in the lead structure,
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additional bile acid oligomers were synthesized in one step and screened for their antibacterial
activity against Gram-positive as well as Gram-negative bacteria.
RESULTS AND DISCUSSION
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We previously reported the synthesis of steroid dimers by two step procedures wherein,
activation of carboxylic acid was carried out using N-hydroxysuccinimide/DCC and the final
2
1
dimers were prepared on reaction with di- and tri-amines. In the present work (Scheme 1), bile
acid oligomers were synthesized by following a typical one step peptide coupling protocol.
Scheme 1. Synthesis of steroidal dimers using one step protocol.a
O
O
NH
NH
n
OH
OH
O
OH
H
R
NH
OH
HO
R
HN
OH
HO
HN
a
O
H
H
R HO
HO
R
O
H
R
HO
HO
5
6
, R = OH
, R = H
7
a, R = OH, n = 1 8a, R = OH, n = 2
7b, R = H, n = 1
8b, R = H, n = 2
9a, R = OH
9b, R = H
a
Reagents and conditions: (a) DMF, EDCI, HOBt, Et N, 25 C, 24h (for compounds 7a and 7b,
3
ethylenediamine; for 8a and 8b, 1,3-diaminopropane and for 9a and 9b, diethylenetriamine was
used in the coupling reactions).
Ethylenediamine, 1,3-diaminopropane and diethylenetriamine were selected to couple with
cholic acid 5 and deoxycholic acid 6 to form six steroidal dimers (Scheme 1). For the efficient
one step synthesis of these amphiphatic molecules, the reactions were performed in DMF using
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EDCI, HOBt as coupling reagents in the presence of triethylamine as a base to form amide
linkage between bile acids and oligoamines. The reactions were completed within 24 h and the
progress of the reaction was monitored by TLC. After the completion of reaction, chilled water
was added to the reaction mixture and the precipitated product was filtered and washed with cold
water. The crude products obtained were further purified by column chromatography to furnish
0
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9
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1
13
desired oligomers in good yields. All the products were characterized by H NMR, C NMR, IR
1
and mass spectroscopy analysis. In the newly formed steroid oligomers, distinct peaks in the H
NMR were observed in the region of δ 3.5 ppm attributed to the α-methylene groups of the
1
3
oligoamines whereas, the amide carbonyls appeared at δ 170-175 ppm in the C NMR spectra.
The formation of these compounds was further confirmed by mass spectroscopy revealing
+
+
[
M+H] and [M+Na] peak in each case. We investigated the antimicrobial properties of all the
synthesized amphiphatic oligomers as these molecules can best mimic the Amp B, ceragenins,
polyhydroxylated ionophoric steroids as well as bile acid based molecular umbrellas.4,6,8,22 We
hypothesized that the specific conformation of these oligomers in the appropriate aggregate form
(Figure 2) could be responsible for their antimicrobial activity.
O
OH
face
Sur
face
OH
face
face
Sur
Sur
Sur
OH
lar
Hydrophobic
Hydrophobic
Po
Polar
Hydrophobic
Surface
OH
Bile acid
Figure 2. A pictorial representation of facial amphiphilic conformations of bile acid oligomers.
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The antibacterial activity of all the synthesized oligomers 7-9 (Scheme 1) was explored against
representative Gram-positive (S. aureus ATCC 29213) and Gram-negative (Escherichia coli
ATCC 25922) bacterial strains by disk diffusion method. Amongst the synthesized derivatives,
the compound 9a obtained by using diethylenetriamine and cholic acid exhibited substantial
antibacterial activity against the Gram-positive strain S. aureus ATCC 29213, whereas all the
other compounds were found to be inactive. The mean zone of inhibition (ZOI) diameter after
exposure to 30 µg of compound 9a was 14±1 mm. By reviewing the structures of all the six
synthesized dimers, the appropriate chain length connecting the two cholic acid scaffolds might
be responsible for the observed activity. To confirm, one additional analogue (10, Figure 3) was
synthesized using 1,5-diaminopentane (Figure 3).
1
1
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0
O
O
NH
NH
HO
HO
HO
O
R
HO
HO
HO
N
HN
HN
HO
HO
HO
HO
HO
O
HO
O
1
1, R = CH3
10
12, R = (CH2)14CH3
1
2
Figure 3. N ,N -1,5-diaminopentanebis[cholic acid amide] 10 and N-acyl derivatives 11, 12 of
compound 9a.
Compound 10 was found to be inactive against S. aureus, suggesting a specific role of free
secondary amine functionality in the linker region for the observed activity by compound 9a. To
confirm the requirement of central free amine group for the activity, the N-acylation in
compound 9a was carried out using acetic anhydride and palmitoyl chloride respectively, to yield
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1
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2
2
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compounds 11 and 12 (Figure 3). Interestingly, both the compounds 11 and 12 were found to be
inactive, confirming the fundamental requirement of the central free amine in dimer 9a for the
desired activity.
0
1
2
3
4
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8
9
0
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9
0
Bile acid oligomers are also described as molecular umbrellas and are known to be
amphomorphic in nature. Such molecular umbrellas composed of two or more facial amphiphiles
are known to produce hydrophobic or hydrophilic exterior when exposed to a hydrophobic or
hydrophilic microenvironment. Few of the bile acid derived molecular umbrellas are capable of
transporting certain biomacromolecules across liposomal membranes and were explored as drug
4
carriers. Based on the structural similarity and to further improve the understanding of Structure
Activity Relationship (SAR) in the present investigation, additional steroidal oligomers were
synthesized using tris(2-aminoethyl)amine and 1,4,8,11-tetraazacyclotetradecane oligoamines as
linkers to furnish tri-walled oligomers 13a, 13b and tetra-walled oligomer 14, respectively
(Scheme 2). None of these molecules were found to be active against S. aureus.
Scheme 2. Synthesis of tri- and tetrameric bile acid oligomers.a
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O
HO
NH
HO
R
HO
OH
HO
HO
N
HN
R
HO
HN
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
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5
5
5
5
5
5
5
5
5
6
0
1
2
3
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5
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8
9
0
1
2
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5
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8
9
0
1
2
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8
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7
8
9
0
1
2
3
4
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7
8
9
0
O
O
13a, R = OH
13b, R = H
a
5
or 6
OH
O
O
OH
OH
HO
N
N
HO
HO
N
HO
N
HO
HO
OH
O
HO
HO
O
14
a
Reagents and conditions: (a) DMF, EDCI, HOBt, Et N, 25 C, 24h. (for compounds 13a and
3
1
3b, tris(2-aminoethyl)amine and for compound 14, 1,4,8,11-tetraazacyclotetradecane were
used).
So far, the only steroidal dimer (9a) synthesized using cholic acid as umbrella “walls” and
diethylenetriamine as “linker” was capable of showing the desired antibacterial activity. The
chain length was then increased by keeping the amine functionality in the linker region. These
desired analogs (15a and 15b) were synthesized using bis(hexamethylene)triamine via the same
one step amide coupling protocol (Scheme 3). Interestingly, the dimer synthesized using cholic
acid (15a) retained the antibacterial activity (131 mm at 30 g of disk content) and 15b was
found to be inactive. For further confirmation of the need of central free amine for the activity,
the N-acylation of 15a with another molecule of cholic acid was carried to yield oligomer 16
(
Scheme 3). As expected, the acylation of free amino functionality in 15a also abolished the
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5
5
5
5
5
5
5
6
observed activity, clearly emphasizing the importance of central secondary free amine and the
particular conformation/interaction of the overall dimer derived from cholic acid scaffold.
Scheme 3. Synthesis of bile acid derived dimers and trimer.a
0
1
2
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5
6
7
8
9
0
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0
O
HN
O
HO
HO
R
R
HN
HO
HO
HO
O
HO
HO
5
HO
5
N
a
5
or 6
NH
5
HO
NH
HO
5
HO
HN
O
O
HO
1
1
5a, R = OH
5b, R = H
HO
16
a
Reagents and conditions: (a) Bis(hexamethylene)triamine, DMF, EDCI, HOBt, Et N, 25 C,
3
2
4h.
Having observed the specific need of amino functionality for the biological activity, we were
keen to explore the effect of additional amino functionality in the linker region on the observed
activity. Accordingly, the synthesis of cholic acids dimer 17 was achieved using
triethylenetetramine (Scheme 4). The newly synthesized dimer 17 was found to be significantly
active against Gram-positive bacterial strain. The mean zone of inhibition (ZOI) diameter after
exposure to 30 µg of compound 17 was 11±1 mm.
Scheme 4. Synthesis of triethylenetetramine derived steroidal dimers.a
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8
9
O
O
NH
NH
HO
HO
O
HO
HO
HO
HO
HN
O
N
O
a
b
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
NH
N
O
HN
HN
HO
HO
HO
HO
HO
O
HO
O
17
18
a
Reagents and conditions: (a) Triethylenetetramine, DMF, EDCI, HOBt, Et N, 25 C, 24h. (b)
3
Boc O, DMF, Et N, 25 C, 30 min.
2
3
Further masking of the free amino functionalities in compound 17 using Boc O resulted in
2
compound 18 (Scheme 4), which was found to be inactive. Thus, it can be concluded from the
above SAR studies that the basic requirement for the desired activity is the free amine group in
the linker region and particular conformation adopted by the oligomer 9a, 15a and 17
synthesized using cholic acid. The specific conformational flexibility by the cholic acid dimer 9a
was recently described by Zhu and coworkers by studying the behavior of three guest molecules
of different polarity. Using molecular modeling it was suggested that the molecular
conformations and interactions with the guest molecule may depend on the length and the
functionality of the linker between the two cholic acid moieties in the dimer.23
The complete SAR study also demands the analysis for the necessity of two cholic acid moieties
along with the appropriate linker containing free amine. The need of cholic acid scaffold to adopt
specific conformation leading to the biological activity was confirmed by synthesizing analogue
2
2 wherein one of the cholic acid moiety was replaced by palmitoyl group. The desired analogue
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
was synthesized using multistep synthetic procedure starting from diethylenetriamine (Scheme
5
).
Scheme 5. Stepwise synthesis of asymmetric bile acid derived palmitamide.a
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
O
O
O
O
O
O
O
H
a
b
N
N
N
H N
2
NH2
O
N
NH2
O
N
N
H
14
H
H
19
20
H
N
N
N
O
H
H
H
14
c
d
N
H N
2
N
H
14
OH
21
22
OH
OH
a
Reagents and conditions: (a) i. F CCOOEt, MeOH, 0 C, 1h. ii. Boc O, 0 C to rt, 1h. iii. Aq.
3
2
MeOH, NH , 25 C, 15h; (b) palmitoyl chloride, DCM, Et N, 30 min; (c) TFA:DCM (1:1), 25
3
3
C, 10 min; (d) Compound 5, EDCI, HOBt, Et N, 25 C, 8h.
3
For the synthesis of compound 22, firstly one of the primary amino group of diethylenetriamine
was selectively protected with ethyl trifluoroacetate (Scheme 5).24 The reaction led to the
formation of mono-trifluoroacetamide as the major product. Boc O was added to this reaction
2
mixture to protect the remaining two free amino functionalities which was monitored by TLC.
After completion of the reaction, concentrated aqueous ammonia was added to the reaction
mixture until the pH was increased to 11 to cleave the trifluoroacetate protecting group to yield
intermediate 19. The synthesized compound 19 was isolated, column purified and confirmed by
+
MS analysis (304.22 corresponding to the [M+H] was observed). The primary amine group of
compound 19 was allowed to react with palmitoyl chloride in the presence of triethylamine as
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1
base to afford the compound 20 (confirmed by H NMR and MS analysis). Compound 20 was
reacted with TFA:DCM (1:1) for the deprotection of Boc groups to afford the compound 21
which on further coupling reaction with cholic acid resulted in desired analogue 22. The
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
formation of compound 22 was confirmed using H NMR and mass spectroscopy analysis. The
inactivity of compound 22 against S. aureus bacterial strain confirmed the requirement of two
cholic acid scaffolds for the activity. Conclusively, the bis-cholic amide with linker containing
free amine is the fundamental requirement for the desired activity.
None of the synthesized oligomers showed any inhibitory activity against E. coli ATCC 25922.
To further confirm the specificity of these compounds against Gram-positive bacteria, the
compounds were also screened against another Gram-negative bacterium Pseudomonas
aeruginosa and no antibacterial activity was noted.
The selective antibacterial activity of compounds 9a, 15a and 17 against Gram-positive but not
against Gram-negative bacteria may be attributed to the differences in their cell-wall architecture
and composition. Apart from containing peptidoglycan that is a cell-wall constituent in all
2
5
bacteria, Gram-negative bacteria also harbor a layer of outer membrane. This outer membrane
comprises of lipopolysaccharide and serves as an additional barrier to the entry of compounds
into the bacterial cell, thereby conferring increased protection to drug penetration and efficacy.26
Apart from testing the antibacterial activity, all the synthesized compounds were also screened
for antifungal activity. Only the compounds 9a and 15a were found to be active against fungi by
disk diffusion, albeit to a lesser extent than bacteria. The mean ZOI diameters obtained after
exposure of C. albicans to 30 μg of these compounds were 10 ± 1 and 7 ± 0 mm respectively.
These results further demonstrate that differences in cell-wall structure and composition are
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9
1
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2
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2
2
2
3
3
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3
3
3
4
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4
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5
5
5
5
5
5
5
5
5
5
6
likely to influence the penetrability and thereby the activity of the tested compounds against
different microbial cell types.
The minimum inhibitory concentrations (MIC) of compounds 9a, 15a and 17 against S. aureus
0
1
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3
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ATCC 29213 were found to be 4, 4 and 16 µg/mL, respectively. Their IC values were 2.87,
5
0
2
.36 and 12.08 µg/mL, respectively. Based on the results obtained, compound 9a and compound
1
5a were selected for further study against S. aureus. Scanning electron micrographs revealed
bacterial cell disruption upon exposure to these compounds, possibly by damage to the cell
surface. Morphological alterations such as bleb formation and shriveling were observed (Figure
4
).
Figure 4. Scanning electron micrographs of S. aureus ATCC 29213 treated with compounds 9a
and 15a at 0.5  MIC (2 μg/mL) and 1  MIC (4 μg/mL), respectively. A, Untreated control; B
and C, Cells treated with Compound 9a; D, Cells treated with Compound 15a.
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The compounds were also found to be effective against drug-resistant clinical isolates of S.
aureus by both disk diffusion and broth microdilution assay (Table 1).
Table 1: Susceptibility of drug-resistant clinical isolates of S. aureus to compounds 9a and 15a
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Strain
Compound 9a
Compound 15a
ZOI
MIC
IC50
ZOI
MIC
IC50
(mm)
13±1
13±1
(µg/mL) (µg/mL)
(mm)
10±1
11±1
(µg/mL)
(µg/mL)
5.32
S. aureus 1704
S. aureus 2673
8
4
4.36
2.18
8
8
3.75
The interaction of the active compounds 9a and 15a with four commonly used antibiotics,
belonging to different classes, namely cefotaxime (beta-lactams), amikacin (aminoglycosides),
vancomycin (glycopeptides) and ciprofloxacin (fluoroquinolones) was tested by broth
microdilution checkerboard assay at concentrations ranging from 0.03 to 32 µg/mL. An additive
effect of both the compounds was observed with all the four classes of antibiotics (Table 2), with
Fractional Inhibitory Concentration (FIC) indices ranging from 0.51 to 1. At 2 µg/mL of the test
compound, the MIC of cefotaxime, amikacin and ciprofloxacin was appreciably reduced (Table
2
). A 33-fold reduction in the MIC of cefotaxime and 67-fold reduction in the MIC of amikacin
was observed when combined with 2 µg/mL of the test compound 9a. The interaction appeared
to be weaker for vancomycin. This may be attributed to its bigger size (1449.265 g/mol) and
incompatibility to be accommodated in the hydrophilic cavity formed by the steroid dimers.
Table 2. Additive interaction of the test compounds 9a and 15a with known antibiotics against S.
aureus ATCC 29213.
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Antibiotic
Antibiotic Antibiotic MIC (µg/mL) with 2 FIC index
MIC
(µg/mL)
1
µg/mL of test compound*
Compound 9a Compound 15a Compound 9a Compound 15a
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Cefotaxime
Amikacin
0.03
33)
0.06
67)
0.03
(33)
0.5
(8)
0.53
0.51
0.56
1
0.56
0.56
1
(
4
(
Ciprofloxacin 0.5
0.03
(17)
1
0.25
(2)
Vancomycin
2
2
1
(2)
(1)
*values in parenthesis indicate fold change in the MIC of the antibiotic when used with 2 µg/mL
of the test compounds. The MIC of both compounds 9a and 15a against S. aureus ATCC 29213
was 4 µg/mL.
Most of the bacterial infections involve formation of thick, multilayered biofilms, which are
highly hydrated cell clusters irreversibly associated with a biotic (e.g. damaged tissue) or abiotic
surface (e.g. indwelling medical devices like catheters) and encased in a self-produced
extracellular polymeric substance matrix. These biofilms are highly resistant to the action of the
most of the commercially available antibiotics; consequently, biofilm-associated infections are
27
difficult to treat and often exhibit relapsing symptoms. The activity of compounds 9a and 15a
was therefore evaluated against S. aureus biofilms, a highly drug-resistant bacterial phenotype
commonly implicated in device-associated and chronic infections that are often unresponsive to
antibiotic therapy. The steroid oligomers 9a and 15a demonstrated a significant inhibitory
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activity against S. aureus biofilm where the minimum biofilm inhibitory concentrations (MBIC)
were found to be 32 µg/mL and 64 µg/mL, respectively. At these concentrations, complete
inhibition of biofilm growth (MBIC ) was observed. In contrast, the known antibiotics tested
1
00
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(cefotaxime, amikacin, ciprofloxacin and vancomycin) were ineffective and were unable to cause
even a 50% of reduction in biofilm growth at concentration as high as 128 µg/mL.
The in-vitro cell viability of all the synthesized compounds was estimated via alamar blue assay
using HEK293 cells which is one of the most sensitive and preferred assay and have no toxic
2
8
effect on cells itself. It was evident from the results (Figure 5) that the synthesized compounds
did not show any appreciable cytotoxicity and cells were significantly viable even after 24 h of
treatment with test compounds as compared to the control. Only the compound 16 was found to
be slightly toxic with 35% decrease in viability at very high concentration (200 µM) in which
more than 92% of cells were viable at lowest concentration (1 µM).
16 (15a) 14 13b 13a (9a) 8b 8a 7b 7a
100
8
6
4
2
0
0
0
0
0
control
1µM
5µM
10µM
50µM
100µM 200µM
Concentration
Figure 5. Cell viability assay via Alamar Blue against HEK293 cells with different
concentrations of test compounds and untreated cells as positive control.
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9
1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
CONCLUSION
A series of steroid oligomers having facially amphiphilic topologies were designed and
synthesized. Among all the synthesized compounds, three dimers 9a, 15a and 17 having free
amine in the linker displayed antibacterial activity against Gram-positive bacterium i.e. S. aureus
and towards its antibiotic-resistant strains. No antibacterial activity was observed against Gram
negative bacteria i.e. E. coli and P. aeruginosa. The lead compounds 9a and 15a also showed
additive effect with four known antibiotics. A 33-fold reduction in the MIC of cefotaxime and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
7-fold reduction in the MIC of amikacin was observed when combined with 2 µg/mL of the test
compound 9a. Interestingly, the lead compounds also demonstrated biofilm inhibitory activity. A
complete inhibition of biofilm growth was observed at 32 µg/mL concentration of compound 9a.
The known antibiotics were found to be ineffective at this concentration and were unable to
cause even 50% reduction in biofilm growth at concentration as high as 128 µg/mL. A very
specific Gram-positive antibacterial activity of the lead bile acid dimers against S. aureus, the
additive effect with known antibiotics and the ability of these compounds to inhibit the biofilm
formation along with their non-cytotoxic nature against HEK293 cell lines demonstrate
therapeutic potential of these amphiphilic steroids to be used either alone or in combination with
known antibiotics to treat deadly infections. The exact mechanism of action of these steroid
oligomers is still not clear and work is in progress to understand the exact mechanism, structural
requirements for the biological activity and preparation of mixed formulations with known
antibiotics.
EXPERIMENTAL SECTION
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Chemistry. All the chemicals, reagents and solvents were purchased from commercial
suppliers. The bulk solvents (Hexane, DCM, MeOH) were distilled before use. The organic
solvents were dried by literature procedures wherever necessary. The reactions were performed
under nitrogen atmosphere. All the reactions were monitored by thin-layer chromatography
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(TLC) carried out on Merck silica gel aluminium sheets and visualized with different stains such
as phosphomolybdic acid and ninhydrin made in ethanol. Column chromatography was carried
1
out using 60-120, 230-400 mesh silica gel or neutral alumina as per the requirement. H NMR
were recorded on Bruker 300 MHz or Bruker Advance II 400 MHz or JEOL JNM ECS400 or
JEOL 500 MHz spectrometer with DMSO-d , CDCl or CD OD as solvents and TMS as internal
6
3
3
standard. The splitting patterns in NMR are designated as s, singlet; bs, broad singlet; d, doublet;
t, triplet; m, multiplet. The chemical shift values were reported in units of δ. The mass spectra
were recorded on Waters Q-TOF or Thermo LTQ-XL Mass spectrometer. Reverse Phase High
Performance Liquid Chromatography (RP-HPLC) was performed on Waters HPLC system
attached to waters 1525 binary HPLC pump linked with Waters 2424 ELSD detector to
determine the purity of active compound. Elution was carried on C18 column by isocratic
conditions for 30 minutes where methanol:water (1:1) were used as eluents. The HPLC data
displayed >95% purity of the active compound.
Experimental Details and Analytical Data for Compounds 7-22.
(4R,4'R)-N,N'-(ethane-1,2-diyl)bis(4-((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-
trihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-
yl)pentanamide) (7a). To a solution of cholic acid 5 (500 mg, 1.22 mmol) in dry DMF (10 mL),
ethylenediamine (41µL, 0.61 mmol), HOBt (83 mg, 0.61 mmol) and triethylamine (341 µL, 2.45
o
o
mmol) were added at 0 C under nitrogen atmosphere. After 10 minutes of stirring at 0 C, EDCI
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9
1
1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
352 mg, 1.83 mmol) was added to the reaction mixture. The reaction mixture was allowed to
warm to room temperature and stirred for overnight and monitored by TLC. After completion of
the reaction, water was added to the reaction mixture and the precipitated crude product was
collected through filtration. The crude solid obtained was further purified by column
chromatography on silica gel using methanol/DCM as eluents to obtain compound 7a as White
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
-1
1
solid. Yield: 78%; IR (neat, cm ) υ: 3279 (m), 1629 (m). H NMR (400 MHz, DMSO-d ) δ 7.76
6
(m, 2H), 3.77 (m, 2H), 3.60 (m, 2H), 3.15-3.20 (m, 2H), 3.05 (m, 4H), 0.91 (d, J = 6.3 Hz, 6H),
1
3
0
6
.79 (s, 6H), 0.56 (s, 6H). C NMR (75 MHz, DMSO-d ) δ 172.9(2C), 71.0(2C), 70.5(2C),
6
6.3(2C), 46.1(2C), 45.8(4C), 41.5(2C), 41.4(2C), 38.4(2C), 35.3(2C), 35.1(2C), 34.9(2C),
34.4(4C), 32.6(2C), 31.6(2C), 30.4(2C), 28.6(2C), 27.3(2C), 26.1(2C), 22.8(2C), 22.6(2C),
+
+
1
8
7.1(2C), 12.3(2C). (HRMS) m/z calculated for C H N O [M+H] : 841.6300, found:
50 85 2 8
41.6333. The H NMR and 13C NMR spectra were consistent with that reported in the
1
literature.21
(4R,4'R)-N,N'-(ethane-1,2-diyl)bis(4-((3R,5R,8R,9S,10S,12S,13R,14S,17R)-3,12-dihydroxy-
1
0,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamide) (7b). To a
solution of deoxycholic acid 6 (500 mg, 1.27 mmol) in dry DMF (10 mL), ethylenediamine (43
µL, 0.64 mmol), HOBt (86 mg, 0.64 mmol) and triethylamine (351 µL, 2.54 mmol) were added
o
o
at 0 C under nitrogen atmosphere. After 10 minutes of stirring at 0 C, EDCI (366 mg, 1.91
mmol) was added to the reaction mixture. The reaction mixture was allowed to warm to room
temperature and stirred for overnight and monitored by TLC. The follow up of the reaction was
done using the similar procedure as described for compound 7a to obtain compound 7b as white
-1
1
solid. Yield 83%; IR (neat, cm ) υ: 3309 (m), 1634 (m). H NMR (400 MHz, DMSO-d ) δ 7.66
6
(m, 2H), 4.36 (m, 2H), 4.01 (m, 2H), 3.79 (m, 2H), 3.09 (m, 4H), 0.92 (d, J = 5.8 Hz, 6H), 0.83
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13
(
s, 6H), 0.58 (s, 6H). C NMR (75 MHz, DMSO-d ) δ 172.8(2C), 71.0(2C), 69.9(2C), 47.5(2C),
6
4
6.2(2C), 46.0(2C), 41.6(2C), 38.4(2C), 36.3(2C), 35.7(2C), 35.1(2C), 35.1(2C), 33.8(2C),
32.9(2C), 32.6(2C), 31.6(2C), 30.2(2C), 28.6(2C), 27.2(2C), 26.9(2C), 26.1(2C), 23.5(2C),
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
+
2
8
3.1(2C), 17.1(2C), 12.4(2C). (ESI-MS) m/z calculated for C H N O [M+H] : 809.64, found:
50 85 2 6
+
1
13
09.46, calculated for [M+Na] : 831.62, found: 831.36. The H NMR and C NMR spectra were
consistent with that reported in the literature.21
(
4R,4'R)-N,N'-(propane-1,3-diyl)bis(4-((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-
trihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-
yl)pentanamide) (8a). The synthesis of compound 8a was achieved using the same protocol as
described for compound 7a except the amine used was 1,3-diaminopropane. White solid; Yield:
-1
1
7
4%; IR (neat, cm ) υ: 3292 (m), 1628 (m). H NMR (400 MHz, DMSO-d ) δ 7.76 (t, J = 5.6
6
Hz, 2H), 4.37 (d, J = 4.2 Hz, 2H), 4.12 (d, J = 3.5 Hz, 2H), 4.03 (d, J = 3.3 Hz, 2H), 3.77 (m,
2H), 3.60 (m, 2H), 3.18 (m, 2H), 2.98-3.02 (m, 4H), 0.91 (d, J = 6.4 Hz, 6H), 0.80 (s, 6H), 0.56
13
(
s, 6H). C NMR (100 MHz, DMSO-d ) δ 172.5(2C), 71.0(2C), 70.4(2C), 66.2(2C), 46.1(2C),
6
4
3
1
5.7(4C), 41.5(2C), 41.3(2C), 36.2(2C), 35.3(2C), 35.1(2C), 34.8(2C), 34.3(4C), 32.5(2C),
1.7(2C), 30.4(2C), 29.3(1C), 28.5(2C), 27.3(2C), 26.2(2C), 22.8(2C), 22.6(2C), 17.0(2C),
+
+
2.3(2C). (ESI-MS) m/z calculated for C H N O [M+H] : 855.64, found: 855.59, calculated
5
1
87
2
8
+
for [M+Na] : 877.62, found: 877.50.
(
4R,4'R)-N,N'-(propane-1,3-diyl)bis(4-((3R,5R,8R,9S,10S,12S,13R,14S,17R)-3,12-
dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-
yl)pentanamide) (8b). The synthesis of compound 8b was achieved using the same protocol as
described for compound 7b except the amine used was 1,3-diaminopropane. White solid; yield
-1
1
79%; IR (neat, cm ) υ: 3292 (m), 1652 (m). H NMR (400 MHz, DMSO-d ) δ 7.73 (t, J = 5.6
6
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1
1
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1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
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Hz, 2H), 4.47 (d, J = 4.3 Hz, 2H), 4.19 (d, J = 4.1 Hz, 2H), 3.78 (m, 2H), 2.98-3.03 (m, 4H),
0
.91 (d, J = 6.4 Hz, 6H), 0.84 (s, 6H), 0.58 (s, 6H). 13C NMR (100 MHz, DMSO-d ) δ
6
172.5(2C), 71.0(2C), 69.9(2C), 47.5(2C), 46.2(2C), 45.9(2C), 41.6(2C), 36.3(2C), 36.2(2C),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
2
5.7(2C), 35.1(2C), 34.9(2C), 33.8(2C), 32.9(2C), 32.6(2C), 31.7(2C), 30.2(1C), 29.4(2C),
8.6(2C), 27.2(2C), 26.9(2C), 26.1(2C), 23.5(2C), 23.1(2C), 17.1(2C), 12.4(2C). (ESI-MS) m/z
+
+
+
+
calculated for C H N O [M+H] : 823.66, found: 823.67 [M+H] , calculated for [M+Na] :
51 87
2
6
8
45.63, found: 845.68.
(4R,4'R)-N,N'-(azanediylbis(ethane-2,1-diyl))bis(4-((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-
3
,7,12-trihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-
yl)pentanamide) (9a). The synthesis of compound 9a was achieved using the same protocol as
described for compound 7a except the amine used was diethylenetriamine. White solid; yield:
-1
1
7
7%; IR (neat, in cm ) υ: 3292 (m), 1632 (m). H NMR (400 MHz, DMSO-d ) δ 7.72 (t, J = 5.5
6
Hz, 2H), 4.35 (m, 2H), 4.11 (m, 2H), 4.02 (m, 2H) 3.77 (m, 2H), 3.60 (m, 2H), 3.18 (m, 2H),
13
3
.06-3.09 (m, 4H), 0.91-0.92 (d, J = 6.4 Hz, 6H), 0.80 (s, 6H), 0.57 (s, 6H). C NMR (75 MHz,
DMSO-d ) δ 173.2(2C), 71.5(2C), 70.9(2C), 66.7(2C), 48.7(2C), 46.6(2C), 46.2(4C), 42.0(2C),
6
4
2
1.8(2C), 38.8(2C), 35.8(2C), 35.6(2C), 35.4(2C), 34.9(4C), 33.0(2C), 32.1(2C), 30.9(2C),
9.0(2C), 27.8(2C), 26.7(2C), 23.3(2C), 23.1(2C), 17.4(2C), 12.8(2C). (ESI-MS) m/z calculated
+
+
+
for C H N O [M+H] : 884.67. found: 884.62, calculated for [M+Na] : 906.65, found:
5
2
90
3
8
+
+
9
06.54. (HRMS) m/z calculated for C H N O [M+H] : 884.6738, found: 884.6722, calculated
52 90 3 8
+
1
13
for [M+Na] : 906.6490, found: 906.6542. The H NMR, C NMR spectra were consistent with
that reported in the literature.23
(4R,4'R)-N,N'-(azanediylbis(ethane-2,1-diyl))bis(4-((3R,5R,8R,9S,10S,12S,13R,14S,17R)-
3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-
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yl)pentanamide) (9b). The synthesis of compound 9b was achieved using the same protocol as
described for compound 7b except the amine used was diethylenetriamine. White solid; Yield:
-1
1
83%; IR (neat, cm ) υ: 3273 (m), 1646 (m). H NMR (400 MHz, DMSO-d ) δ 7.70 (t, J = 5.5
6
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Hz, 2H), 4.47 (m, 2H), 4.19 (m, 2H), 3.77 (m, 2H), 3.04-3.09 (m, 4H), 2.52-2.53 (m, 4H), 0.90
1
3
(
d, J = 6.4 Hz, 6H), 0.83 (s, 6H), 0.57 (s, 6H). C NMR (100 MHz, DMSO-d ) δ 172.6(2C),
6
7
3
2
1.0(2C), 69.9(2C), 48.4(2C), 47.4(2C), 46.2(2C), 45.9(2C), 41.6(2C), 38.6(2C), 36.2(2C),
5.6(2C), 35.1(2C), 35.1(2C), 33.8(2C), 32.9(2C), 32.5(2C), 31.6(2C), 30.2(2C), 28.6(2C),
7.2(2C), 26.9(2C), 26.1(2C), 23.5(2C), 23.1(2C), 17.0(2C), 12.4(2C). (HRMS) m/z calculated
+
+
for C H N O [M+H] : 852.6824, found: 852.6857.
52 90
3
6
(4R,4'R)-N,N'-(pentane-1,5-diyl)bis(4-((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-
trihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-
yl)pentanamide) (10). The synthesis of compound 10 was achieved using the same protocol as
described for compound 7a except the amine used was 1,5-diaminopentane. White solid; yield
-1
1
8
2
3
2%; IR (neat, cm ) υ: 3292 (m), 1627 (m). H NMR (400 MHz, DMSO-d ) δ 7.75 (t, J = 5.4 Hz
6
H), 4.48 (m, 2H), 4.10 (m, 2H), 4.01 (m, 2H), 3.77 (m, 2H), 3.60 (m, 2H), 3.16 (m, 2H), 2.95-
1
3
.00 (m, 4H), 0.91 (d, J = 6.3 Hz, 6H), 0.79 (s, 6H), 0.56 (s, 6H). C NMR (100 MHz, DMSO-
d6) δ 172.5(2C), 70.9(2C), 70.4(2C), 66.2(2C), 46.1(2C), 45.7(4C), 41.5(2C), 41.3(2C),
3
2
8.2(2C), 35.3(2C), 35.1(2C), 34.8(2C), 34.3(4C), 32.5(2C), 31.7(2C), 30.3(2C), 28.8(2C),
8.5(2C), 27.3(2C), 26.2(2C), 23.6(1C), 22.8(2C), 22.6(2C), 17.1(2C), 12.3(2C). (ESI-MS) m/z
+
+
calculated for C H N O Na [M+Na] : 905.65, found: 905.58.
5
3
90
2
8
N,N-bis(2-((R)-4-((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10,13-
dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)ethyl)acetamide
(11). To a solution of 9a (320 mg, 0.36 mmol) in dry DMF (1 mL), acetic anhydride (34 µL, 0.36
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2
2
2
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mmol), and triethylamine (51 µL, 0.36 mmol) were added at room temperature and stirred for 1 h
and monitored by TLC. After completion of the reaction, solvent was evaporated under vacuum
and the crude product obtained was column purified using DCM/MeOH as eluents. White solid;
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
-1
1
Yield: 81%; IR (neat, cm ) υ: 3275 (m), 1624 (m). H NMR (400 MHz, CD OD-d ) δ 3.94 (m,
3
4
2
0
1
4
3
H), 3.79 (m, 2H), 3.44-3.47 (m, 4H), 3.34-3.37 (m, 6H), 2.11 (s, 3H), 1.02 (d, J = 6.2 Hz, 6H),
.90 (s, 6H), 0.71 (s, 6H). 13C NMR (100 MHz, , CD OD-d ) δ 177.2 (1C), 177.1(1C),
3
4
73.9(1C), 74.0(2C), 72.9(2C), 69.0(2C), 49.9(1C), 48.0(1C), 47.9(1C), 47.5(2C), 46.5(1C),
3.2(2C), 43.0(2C), 41.0(2C), 40.5(2C), 38.7(1C), 38.3(1C), 36.9(2C), 36.5(2C), 35.9(4C),
4.1(1C), 34.0(1C), 33.2(2C), 31.2(2C), 29.6(2C), 28.7(2C), 27.9(2C), 24.3(2C), 23.2(2C),
+
+
21.5(1C), 17.7(2C), 13.1(2C). (ESI-MS) m/z calculated for C H N O [M+H] : 926.68, found:
5
4
92
3
9
+
9
26.75, calculated for [M+Na] : 948.66, found: 948.73.
N,N-bis(2-((R)-4-((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10,13-
dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-
yl)pentanamido)ethyl)palmitamide (12). To a solution of compound 9a (200 mg, 0.23 mmol)
in dry DMF (1 mL), palmitoyl chloride (62 mg, 0.23 mmol), and triethylamine (68 µL, 0.23
mmol) were added at room temperature and stirred for 1 h and monitored by TLC. After
completion of the reaction, solvent was evaporated under vacuum and the crude product obtained
-1
was column purified using DCM/MeOH as eluents. White solid; Yield: 86%; IR (neat, cm ) υ:
1
3
273 (m), 1642 (m). H NMR (400 MHz, DMSO-d ) δ 7.95 (t, J = 5.9 Hz, 1H), 7.77 (t, J = 5.6
6
Hz, 1H), 4.32 (m, 2H), 4.09 (m, 2H), 4.00 (m, 2H), 3.77 (m, 2H), 3.60 (m, 2H), 3.19 (m, 2H),
13
3
.10 (m, 8H), 0.91 (d, J = 6.2 Hz, 6H), 0.85 (t, J = 6.8 Hz, 3H), 0.80 (s, 6H), 0.57 (s, 6H). C
NMR (75 MHz, DMSO-d ) δ 173.6(1C), 173.3(1C), 172.9(1C), 71.5(2C), 70.9(2C), 66.7(2C),
6
46.6(2C), 46.2(2C), 41.9(2C), 41.8(2C), 35.8(2C), 35.5(2C), 35.3(2C), 34.8(2C), 32.9(2C),
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2.0(3C), 31.8(2C), 30.8(2C), 29.6(10C), 29.2(2C), 28.9(2C), 27.7(1C), 26.6(2C), 25.5(1C),
3.3(3C), 23.0(4C), 22.6(2C), 17.5(3C), 14.4(2C), 12.8(2C). (ESI-MS) m/z calculated for
+
+
+
C H N O [M+H] : 1122.90, found: 1123.01, calculated for [M+Na] : 1144.88, found:
6
8
120
3
9
1
1
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1
1
1
1
1
1
1
2
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2
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0
1
2
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5
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
8
9
0
1
2
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4
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7
8
9
0
1
145.06.
(4R,4'R,4''R)-N,N',N''-(nitrilotris(ethane-2,1-diyl))tris(4-
((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10,13-dimethylhexadecahydro-
1
H-cyclopenta[a]phenanthren-17-yl)pentanamide) (13a). To a solution of cholic acid 5 (500
mg, 1.22 mmol) in dry DMF (10 mL), tris(2-aminoethyl)amine (61 µL, 0.40 mmol), HOBt (83
o
mg, 0.61 mmol) and triethylamine (511 µL, 3.67 mmol) were added at 0 C under nitrogen
o
atmosphere. After 10 minutes of stirring at 0 C, EDCI (352 mg, 1.83 mmol) was added to the
reaction mixture. The reaction mixture was allowed to warm to room temperature and stirred for
overnight and monitored by TLC. The follow up of the reaction was done using the similar
procedure described for compound 7a to obtain compound 13a as white solid. Yield 68%; IR
-1
1
(neat, cm ) υ: 3308 (m), 1627 (m). H NMR (400 MHz, DMSO-d ) δ 7.68 (m, 3H), 4.32 (d, J =
6
4
.3 Hz, 3H), 4.05 (d, J = 3.5 Hz, 3H), 3.96 (d, J = 3.3 Hz, 3H), 3.78 (m, 3H), 3.61 (m, 3H), 3.18
1
3
(m, 3H), 3.06 (m, 6H), 2.45 (m, 6H), 0.91 (d, J = 6.3 Hz, 9H), 0.80 (s, 9H), 0.57 (s, 9H). C
NMR (100 MHz, DMSO-d ) δ 172.7(3C), 71.0(3C), 70.5(3C), 66.3(3C), 53.6(3C), 46.3(3C),
6
4
5.8(6C), 41.5(3C), 41.4(3C), 36.9(3C), 35.3(3C), 35.2(3C), 34.9(3C), 34.4(6C), 32.7(3C),
1.7(3C), 30.4(3C), 28.6(3C), 27.3(3C), 26.2(3C), 22.9(3C), 22.6(3C), 17.1(3C), 12.4(3C). (ESI-
3
+
+
MS) m/z calculated for C H N O
12
[M+H] , 1317.99, found: 1317.76, calculated for
7
8
133
4
+
1
13
[M+Na] : 1339.97, found: 1339.74. The H NMR and C NMR spectra were consistent with
that reported in the literature.29
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5
5
6
(
4R,4'R,4''R)-N,N',N''-(nitrilotris(ethane-2,1-diyl))tris(4-
((3R,5R,8R,9S,10S,12S,13R,14S,17R)-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-
cyclopenta[a]phenanthren-17-yl)pentanamide) (13b). To a solution of deoxycholic acid 6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(500 mg, 1.27 mmol) in dry DMF (10 mL), tris(2-aminoethyl)amine (63 µL, 0.42 mmol), HOBt
o
(86 mg, 0.64 mmol) and triethylamine (533 µL, 3.82 mmol) were added at 0 C under nitrogen
o
atmosphere. After 10 minutes of stirring at 0 C, EDCI (366 mg, 1.91 mmol) was added to the
reaction mixture. The reaction mixture was allowed to warm to room temperature and stirred for
overnight and monitored by TLC. The follow up of the reaction was done using the similar
procedure described for compound 7a to obtain compound 13b as white solid. Yield 71%; IR
-1
1
(neat, in cm ) υ: 3313 (m), 1634 (m). H NMR (400 MHz, DMSO-d ) δ 7.68 (t, J = 5.3 Hz, 3H),
6
4
6
.48 (d, J = 4.2 Hz, 3H), 4.18 (m, 3H), 3.77 (m, 3H), 3.05 (m, 6H), 2.43 (m, 6H), 0.91 (d, J =
1
3
.3 Hz, 9H), 0.84 (s, 9H), 0.58 (s, 9H). C NMR (100 MHz, DMSO-d ) δ 172.5(3C), 71.0(3C),
6
70.0(3C), 53.7(3C), 47.4(3C), 46.3(3C), 46.0(3C), 41.6(3C), 37.0(3C), 36.3(3C), 35.7(3C),
3
2
5.2(6C), 33.8(3C), 32.9(3C), 32.6(3C), 31.7(3C), 30.2(3C), 28.6(3C), 27.2(3C), 27.0(3C),
+
6.2(3C), 23.6(3C), 23.1(3C), 17.1(3C), 12.5(3C). (ESI-MS) m/z calculated for C H N O
9
7
8
133
4
+
+
1
[M+H] : 1270.00, found: 1270.63 [M+H] . The H NMR spectra was consistent with that
reported in the literature.29
(
4R,4'R,4''R,4'''R)-1,1',1'',1'''-(1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetrayl)tetrakis(4-
(3R,5S,7R,8R,9R,10S,12S,13R,14R,17R)-3,7,12-trihydroxy-8,9,10,13,14-
(
pentamethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentan-1-one) (14). To a
solution of cholic acid 5 (500 mg, 1.22 mmol) in dry DMF (10 mL), 1,4,8,11-
tetraazacyclotetradecane (61 mg, 0.31 mmol), HOBt (83 mg, 0.61 mmol) and triethylamine (682
o
µL, 4.89 mmol) were added at 0 C under nitrogen atmosphere. After 10 minutes of stirring at 0
26
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2
3
4
5
6
7
8
9
oC, EDCI (352 mg, 1.83 mmol) was added to the reaction mixture. The reaction mixture was
allowed to warm to room temperature and stirred for overnight and monitored by TLC. The
follow up of the reaction was done using the similar procedure described for compound 7a to
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
-1
1
obtain compound 14 as white solid. Yield: 67%; IR (neat, cm ) υ: 3350 (m), 1622 (m). H NMR
(
400 MHz, DMSO-d ): 4.36 (m, 4H),4.12 (m, 4H), 4.02 (m, 4H), 3.78 (m, 4H), 3.60 (m, 4H),
6
1
3
3
1
4
2
.16 (m, 2H), 0.93 (m, 12H), 0.79 (s, 12H), 0.58 (s, 12H). C NMR (100 MHz, DMSO-d ) δ
6
72.8(4C), 71.1(4C), 70.5(4C), 66.3(4C), 55.0(4C), 46.7(4C), 46.4(4C), 45.7(8C), 41.6(4C),
1.4(4C), 35.8(4C), 35.4(4C), 35.2(4C), 34.9(4C), 34.4(12C), 30.4(4C), 28.5(4C), 27.5(4C),
+
6.2(6C), 22.9(4C), 22.6(4C), 17.3(4C), 12.4(4C). ESI-MS calculated for C H N O m/z
106 177
4
16
+
+
1762.31 [M+H] , found: 1762.28, calculated for [M+Na] : 1784.29, found: 1785.26.
(4R,4'R)-N,N'-(azanediylbis(hexane-6,1-diyl))bis(4-((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-
3
,7,12-trihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-
yl)pentanamide) (15a). The synthesis of compound 15a was achieved using the same protocol
as described for compound 7a except the amine used was bis(hexamethylene)triamine. White
-1
1
solid; yield: 72%; IR (neat, cm ) υ: 3296 (m), 1635 (m). H NMR (400 MHz, DMSO-d ) δ 7.72
6
(t, J = 5.2 Hz, 2H), 4.35 (m, 2H), 4.11 (m, 2H), 4.03 (m, 2H), 3.77 (m, 2H), 3.60 (m, 2H), 3.16
1
3
(
m, 2H), 2.99 (m, 4H), 2.46 (m, 4H), 0.91 (d, J = 6.2 Hz, 6H), 0.80 (s, 6H), 0.57 (s, 6H). C
NMR (100 MHz, DMSO-d ) δ 172.6(2C), 71.0(2C), 70.5(2C), 66.3(2C), 49.3(2C), 46.2(2C),
6
4
5.7(4C), 41.5(2C), 41.4(2C), 38.3(2C), 35.3(2C), 35.1(2C), 34.9(2C), 34.4(4C), 32.6(2C),
31.8(2C), 30.4(2C), 29.3(2C), 29.2(2C), 28.6(2C), 27.3(2C), 26.6(2C), 26.4(2C), 26.2(2C),
2.8(2C), 22.6(2C), 17.1(2C), 12.3(2C). (ESI-MS) m/z calculated for C H N O [M+H]+:
+
2
6
0
106
3
8
+
+
9
96.79, found: 996.54 [M+H] , calculated for [M+Na] : 1018.77, found: 1018.63. (HRMS) m/z
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Page 28 of 43
1
2
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5
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
+
calculated for C H N O [M+H] : 996.7974, found: 996.7908, calculated for [M+Na] :
6
0
105
3
8
1
018.7794, found: 1018.7691.
(4R,4'R)-N,N'-(azanediylbis(hexane-6,1-diyl))bis(4-((3R,5R,8R,9S,10S,12S,13R,14S,17R)-
,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
yl)pentanamide) (15b). The synthesis of compound 15b was achieved using the same protocol
as described for compound 7b except the amine used was bis(hexamethylene)triamine. White
-1
1
solid; Yield: 79%; IR (neat, cm ) υ: 3276 (m), 1637 (m). H NMR (400 MHz, DMSO-d ) δ 7.72
6
(
t, J = 5.6 Hz, 2H), 4.49 (m, 2H), 4.20 (m, 2H), 3.78 (m, 2H), 2.99 (m, 4H), 2.43 (t, J = 7.0 Hz,
1
3
4
H), 0.91 (d, J = 6.2 Hz, 6H), 0.84 (s, 6H), 0.58 (s, 6H). C NMR (100 MHz, DMSO-d ) δ
6
172.4(2C), 71.1(2C), 70.0(2C), 49.4(2C), 47.5(2C), 46.3(2C), 46.0(2C), 41.6(2C), 38.3(2C),
3
2
6.3(2C), 35.7(2C), 35.2(2C), 35.0(2C), 33.8(2C), 32.9(2C), 32.6(2C), 31.8(2C), 30.2(2C),
9.6(2C), 29.2(2C), 28.6(2C), 27.2(2C), 27.0(2C), 26.7(2C), 26.4(2C), 26.2(2C), 23.5(2C),
+
+
23.1(2C), 17.1(2C), 12.4(2C). (HRMS) m/z calculated for C H N O [M+H] : 964.8076,
6
0
106
3
6
found: 964.8113.
(
R)-4-((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10,13-
dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-N,N-bis(6-((R)-4-
(3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10,13-dimethylhexadecahydro-
H-cyclopenta[a]phenanthren-17-yl)pentanamido)hexyl)pentanamide (16). The synthesis of
compound 16 was achieved using the same protocol as described for compound 13a except the
(
1
-1
amine used was bis(hexamethylene)triamine. White solid; yield: 62%; IR (neat, cm ) υ: 3297
1
(
m), 1622 (m). H NMR (400 MHz, DMSO-d ) δ 7.73 (m, 2H), 4.34 (d, J = 4.2 Hz, 3H), 4.11 (m,
6
3
H), 4.02 (m, 3H), 3.77 (m, 3H), 3.60 (m, 3H), 3.17 (m, 7H), 2.99 (m, 4H), 0.92 (m, 9H), 0.80
1
3
(s, 9H), 0.56 (s, 9H). C NMR (100 MHz, DMSO-d ) δ 172.44 (1C), 172.40(1C), 171.9(1C),
6
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7
3
1.0(3C), 70.4(3C), 66.3(3C), 46.2(3C), 45.8(3C), 45.7(3C), 41.5(3C), 41.4(3C), 38.3(3C),
5.3(3C), 35.1(3C), 34.9(3C), 34.4(6C), 32.6(3C), 31.8(3C), 30.4(3C), 29.2(3C), 28.8(1C),
28.6(3C), 27.4(3C), 26.2(3C), 26.1(2C), 22.8(3C), 22.6(3C), 17.2(3C), 17.1(3C), 12.3(3C). (ESI-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
+
MS) m/z calculated for C H N O
12
[M+H] : 1387.07, found: 1386.84, Calculated for
8
4
144
3
+
[M+Na] : 1409.05, found: 1408.81.
(4R,4'R)-N,N'-((ethane-1,2-diylbis(azanediyl))bis(ethane-2,1-diyl))bis(4-
((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10,13-dimethylhexadecahydro-
1
H-cyclopenta[a]phenanthren-17-yl)pentanamide) (17). The synthesis of compound 17 was
achieved using the same protocol as described for compound 7a except the amine used was
-1
1
triethylenetetramine. White solid; Yield: 61%; IR (neat, cm ) υ: 3273 (m), 1642 (m). H NMR
(400 MHz, DMSO-d ) δ 7.73 (t, J = 5.2 Hz, 2H), 4.34 (m, 2H), 4.10 (m, 2H), 4.02 (m, 2H), 3.77
6
(m, 2H), 3.60 (m, 2H), 3.06-3.22 (m, 10H), 2.66 (m, 1H), 2.53 (m, 4H), 0.91 (d, J = 6.3 Hz, 6H),
1
3
0.80 (s, 6H), 0.57 (s, 6H). C NMR (75 MHz, DMSO-d ) δ 172.7(2C), 71.1(2C), 70.5(2C),
6
6
3
2
9
6.3(2C), 48.8(4C), 46.2(2C), 45.8(4C), 41.6(2C), 41.4(2C), 38.8(2C), 35.3(2C), 35.2(2C),
4.9(2C), 34.4(4C), 32.6(2C), 31.7(2C), 30.4(2C), 28.6(2C), 27.3(2C), 26.2(2C), 22.8(2C),
+
+
2.6(2C), 17.1(2C), 12.4(2C). (ESI-MS) m/z calculated for C H N O [M+H] : 927.71, found:
54 95
4
8
27.69.
di-tert-butyl
ethane-1,2-diylbis((2-((R)-4-((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-
trihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-
yl)pentanamido)ethyl)carbamate) (18). Compound 17 (500 mg, 0.54 mmol) was dissolved in
1
.5 mL of DMF, to it triethylamine (151 µL, 1.08 mmol) and Boc O (371 µL, 1.62 mmol) were
2
added and reaction was stirred for 1 h. Completion of the reaction was monitored by TLC. The
solvent was evaporated under vacuum. The crude product obtained was purified by column
29
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