Synthesis and antimicrobial activity of β-lactam-bile acid conjugates linked via triazole

Article abstract of DOI:10.1016/j.bmcl.2008.01.102

Synthesis of novel 1,2,3-triazole-linked β-lactam-bile acid conjugates 17-24 using 1,3-dipolar cycloaddition reaction of azido β-lactam and terminal alkyne of bile acids in the presence of Cu(I) catalyst (click chemistry) have been realized. These molecules were evaluated in vitro for their antifungal and antibacterial activities. Most of the compounds exhibited significant antifungal and moderate antibacterial activity against all the tested strains.

Full text of DOI:10.1016/j.bmcl.2008.01.102

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2043–2047
Synthesis and antimicrobial activity of b-lactam–bile
acid conjugates linked via triazole
Namdev S. Vatmurge,a Braja G. Hazra,a,* Vandana S. Pore,a Fazal Shirazi,b
Pradnya S. Chavanb and Mukund V. Deshpandeb
aOrganic Chemistry Division, National Chemical Laboratory, Pune 411 008, India
bBiochemical Sciences Division, National Chemical Laboratory, Pune 411 008, India
Received 19 October 2007; revised 14 January 2008; accepted 28 January 2008
Available online 31 January 2008
Abstract—Synthesis of novel 1,2,3-triazole-linked b-lactam–bile acid conjugates 17–24 using 1,3-dipolar cycloaddition reaction of
azido b-lactam and terminal alkyne of bile acids in the presence of Cu(I) catalyst (click chemistry) have been realized. These mol-
ecules were evaluated in vitro for their antifungal and antibacterial activities. Most of the compounds exhibited significant anti-
fungal and moderate antibacterial activity against all the tested strains.
Ó 2008 Elsevier Ltd. All rights reserved.
b-Lactams are a large class of antibiotics characterized
by the presence of an azetidine-2-one ring, which is the
core of biological activity.1 The azetidine-2-one (b-lac-
tam) ring system is a common structural feature of a
number of broad spectrum b-lactam antibiotics, like
penicillins, cephalosporins, carbapenems, nocardicins
and monobactams, which have been widely used as che-
motherapeutic agents for treating microbial diseases.2 It
also shows many other interesting biological properties,
such as cholesterol absorption inhibitors,3 human cyto-
megalovirus protease inhibitors,4 thrombin inhibitors,5
antihyperglycemic,6 anti-tumour,7 anti-HIV,8 anti-
inflammatory, analgesic activities9 and serine-dependent
enzyme inhibitors.10 However, microorganisms have
built up resistance against the most traditional b-lactam
antibiotics due to the wide-spread overuse of antibiotics.
Therefore, the phenomenon of bacterial resistance
forces the continuous modification of structure of
known active compounds and the development of new
ones.
and capable of hydrogen bonding, which can be favor-
able in binding of biomolecular targets and solubility.12
1,2,3-Triazole moiety does not occur in nature, although
the synthetic molecules containing 1,2,3-triazole units
show diverse biological activities such as antibacterial,
herbicidal, fungicidal, antiallergic, and anti-HIV.13
Bile acids have been considered very useful in the prepara-
tion of new pharmaceutical drugs because of their inher-
ent chemical and biological properties.14 They are
pharmacologically interesting as potential carriers of li-
ver-specific drugs, absorption enhancers and cholesterol
lowering agents.15 A common feature of bile acid-derived
antimicrobials is their potential to exhibit facially amphi-
philic nature, due to polar hydroxyl groups on one face
and nonpolar hydrophobic methyl group on the other.16
This type of amphiphilicity can also be exhibited by poly-
ene macrolide amphotericin B, peptide antimicrobial
agent polymixin B, and squalamine in the cyclic form
which functions as an inophores.17 Several cholic acid-de-
rived facial amphiphiles have been reported18 to improve
the permeability of membranes including bacterial cell
wall. Isolation of two naturally occurring steroidal b-lac-
tams from the plants Pachysandra terminalis19 and Pach-
ysandra procumbens20 has been reported. Steroidal
b-lactam, isolated from P. procumbens, is known as
antiestrogen-binding site inhibitory agent. There are
two reports for synthetically prepared b-lactam on steroid
and the biological activity data of these compounds have
not been revealed.21 In our group, cholic acid- and
Azoles are the largest class of antifungal agents in clin-
ical use.11 1,2,3-Triazole moieties are attractive connect-
ing units, as they are stable to metabolic degradation
Keywords: Cholic acid; Deoxycholic acid; 1,2,3-Triazole; b-Lactam;
Click chemistry; Antimicrobial activity.
*
Corresponding author. Tel.: +91 20 25902336; fax: +91 20
25902624; e-mail: bg.hazra@ncl.res.in
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.01.102

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N. S. Vatmurge et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2043–2047
deoxycholic acid-based antimicrobials have been pre-
pared as bile acid amides derived from chiral amino alco-
hols.22 Flucanazole/bile acid conjugates have been
reported recently from our laboratory with good anti-
fungal activity.23 In continuation of our recent work on
bile acids,22,24 we report herein the synthesis of 1,2,3-tria-
zole-linked b-lactam-cholic acid/deoxycholic acid conju-
gates 17–24 and have studied their antibacterial and
antifungal properties. This is the first report of synthesis
and biological activity of the triazole-linked b-lactam on
cholic acid/deoxycholic acid.
ethyl azidoacetate 2. Subsequent saponification of ester
functionality of compound 2 with KOH in methanol
gave the potassium salt of azidoacetic acid 3 with 92%
overall yield in two steps. The azido b-lactams 9 and
10 were prepared by using the ketene–imine cycloaddi-
tion (Staudinger)25 reaction of 3 and imines 7 or 8 (pre-
pared from the aldehydes 4 or 5 with the amine 6 in
excellent yields) in the presence of triphosgene and tri-
ethylamine in anhydrous dichloromethane with 81%
and 77% yields, respectively. Compounds 9 and 10 were
obtained as racemic mixture and no attempt was made
to resolve these in optically pure form.
Target molecules 17–24 were synthesized using 1,3-dipo-
lar cycloaddition reaction of b-lactams containing azide
9 and 10 and bile acids containing terminal alkyne 13–
16, in the presence of Cu(I) catalyst (click chemistry).
Accordingly, the synthesis of azido b-lactams 9 and 10
was started from ethyl bromoacetate 1 (Scheme 1).
Compound 1 on treatment with sodium azide afforded
Propargyl esters 13 and 14 were prepared by coupling
propargyl alcohol with cholic acid 11 or deoxycholic
acid 12 using EDCÆHCl [1-ethyl-3-(3-dimethylamino-
propyl) carbodiimide hydrochloride] as coupling agent
in DMF at 0–25 °C with 88% and 90% yields, respec-
tively (Scheme 2). Using similar reaction conditions,
O
O
O
b
a
N3
N3
Br
OK
OEt
OEt
3
1
2
c
R1
CHO
R1
CH N
+
H2N
OMe
OMe
4, R1 = H
5, R1 = Cl
7, R1 = H
8, R1 = Cl
6
N3
H
O
d
H
N
3 + 7/ 8
OMe
9, R1 = H
10, R1 = Cl
R1
( )
Scheme 1. Reagents and conditions: (a) NaN3, Bu4NBr, CH2Cl2/H2O (1:1), 25 °C, 36 h, 98%; (b) KOH, MeOH, 25 °C, 4 h, 94%; (c) anhydrous
MgSO4, CH2Cl2, 25 °C, 12 h, (97% for 7 and 95% for 8); (d) Triphosgene, Et3N, CH2Cl2, 0–25 °C, 15 h, (81% for 9 and 77% for 10).
O
O
OH
OH
OH
O
a
HO
R2
HO
R2
H
H
11, R2 = OH (cholic acid)
13, R2 = OH
12, R2 = H (deoxycholic acid)
14, R2 = H
O
O
OH
OH
OH
N
H
b
HO
R2
HO
R2
H
H
11, R2 = OH
15, R2 = OH
16, R2 = H
12, R2 = H
Scheme 2. Reagents and conditions: (a) EDCÆHCl, HOBt, Propargyl alcohol, DMF, 0–25 °C, 12 h, 88% for 13 and 90% for 14; (b) EDCÆHCl, HOBt,
Propargyl amine hydrochloride, Et3N, DMF, 0–25 °C, 11 h, 89% for 15 and 92% for 16.

N. S. Vatmurge et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2043–2047
2045
amides 15 and 16 containing terminal alkyne functional-
ity were prepared by coupling propargyl amine with
cholic acid and deoxycholic acid in excellent yields.
against Escheirchia coli and Staphylococcus aureus.
The MIC and IC50 values were determined using stan-
dard broth microdilution technique described by
NCCLS.28 In comparison with the antimicrobial activ-
ity, amphotericin B and fluconazole were used as the ref-
erence antifungal agents, while tetracycline and
ampicillin were used as the reference antibacterial
agents. All the biological data of the tested compounds
are depicted in Table 1 as MIC and IC50 values.
Our next target was to synthesise 1,2,3-triazole-linked b-
lactam–bile acid conjugates 17–24. The cycloaddition
reaction of propargyl esters 13 and 14 with azido b-lac-
tams 9 and 10 in the presence of Cu(I) catalyst (click
chemistry)23 under microwave irradiation furnished dia-
stereomeric mixture of novel conjugates 17–20 in excel-
lent yields (Scheme 3). In a similar way, exposure of
propargyl amides 15 and 16 with azido b-lactams 9
and 10 afforded diastereomeric mixture of hitherto un-
known compounds 21–24 with 95–97% yields. The com-
bination of racemic core 9 and 10 with optically pure
13–16 afforded diastereomeric mixture of triazole-linked
b-lactam–bile acid conjugates 17–24 in equal amounts.
These diastereomers were inseparable by flash column
chromatography and also by crystallization.
From the biological data (Table 1), it was observed that
azido b-lactams 9, 10 and steroidal alkynes 13–16 were
almost inactive against all the tested strains. The MIC
value for all these compounds was >128 lg/mL. As seen
in Table 1 most of the b-lactam–bile acid conjugates 17–
24 generally showed potent antifungal and antibacterial
activity against all the tested fungal and bacterial
strains. The activity of compounds 20–22 and 24 was
higher or comparable to that of fluconazole against C.
albicans with MIC value of 16–32 lg/mL. The com-
pounds 17, 19, 20, 22 and 23 showed good antifungal
activity against C. neoformans having MIC value of
32 lg/mL comparable to that of reference drug fluconaz-
ole. However, the growth inhibitory activity of com-
pound 24 was more potent than the reference drug
amphotericin B and fluconazole against B. poitrasii,
and also the compounds 17–19 and 23 showed signifi-
cant activity against B. poitrasii with MIC value of
32 lg/mL. Y. lipolytica was adversely affected by 18,
19, 21, 23 and 24, and in particular, 18 was the most po-
tent with a low MIC value of 4 lg/mL. The compounds
18 and 20 showed significant inhibitory effect with MIC
value of 16 lg/mL comparable to that of amphotericin B
against F. oxysporum. Furthermore, compounds 19 and
22 showed good antibacterial activity against E. coli
having MIC value of 16 lg/mL. The compounds 17,
All the newly synthesized azido b-lactams 9, 10, steroi-
dal alkynes 13–16 and 1,2,3-triazole-linked b-lactam–
bile acid conjugates 17–24 were tested in vitro for anti-
fungal and antibacterial activity. The antifungal activity
was tested using NCL isolate fungal strains Candida
albicans, Cryptococcus neoformans (human pathogen),
Benjaminiella poitrasii, Yarrowia lipolytica (saprophytes)
and Fusarium oxysporum (plant pathogen). Most of the
pathogen fungi viz C. albicans are dimorphic in nature.
However, their use as model faces a number of problems
of slow growth rate and difficulties in getting synchro-
nous growth.26 Therefore nonpathogenic dimorphic
fungus B. poitrasii was used as a model which exhibits
a rapid and simple one-step process of yeast–mycelium
transition in response to temperature and/or glucose
change.27 The antibacterial activity was evaluated
O
O
N3
H
O
OH
H
O
N
N
a
N
N
H
N
13/14
+
H
OMe
OMe
HO
R2
R1
R1
H
+
_
( )
17, R1 = H, R2 = OH
18, R1 = H, R2 = H
19, R1 = Cl, R2 = OH
20, R1 = Cl, R2 = H
9, R1 = H
10, R1 = Cl
17-20 are mixture of two diastereomers
O
O
OH
H
N
N
N
H
N
N
a
15/16
9/10
OMe
+
H
HO
R2
R1
H
21, R1 = H, R2 = OH
22, R1 = H, R2 = H
23, R1 = Cl, R2 = OH
24, R1 = Cl, R2 = H
21-24 are mixture of two diastereomers
Scheme 3. Reagents: (a) Sodium ascorbate, CuSO4Æ5H2O, DMF/H2O (7:3), microwave (385 W), 5 min, 95–97%.

2046
N. S. Vatmurge et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2043–2047
Table 1. In vitro antimicrobial activity of compounds 9, 10 and 13–24
Compound
Inhibitory concentration (lg/mL)
Fungal strains
BP
Bacterial strains
EC SA
CA
IC50
CN
YL
FO
b
MICa
MIC
IC50
MIC
IC50
MIC
IC50
MIC
IC50
MIC
IC50
64
MIC
IC50
9
10
>128
>128
>128
128
>128
>128
64
64
64
>128
>128
>128
>128
>128
>128
32
>128
64
>128
64
>128
128
16
32
8
>128
>128
>128
>128
>128
>128
32
>128
>128
>128
128
>128
128
8
>128
>128
>128
>128
>128
>128
64
128
>128
64
128
64
>128
16
2
>128
>128
>128
>128
>128
>128
64
64
64
128
64
128
64
32
8
>128
>128
>128
>128
>128
>128
64
>128
>128
>128
>128
>128
>128
32
>128
>128
128
128
64
128
128
128
128
>128
32
8
13
14
128
32
15
16
>128
64
8
128
16
17
18
128
128
32
64
16
8
>128
32
32
32
32
16
17
4
16
32
16
>128
32
64
16
19
20
8
64
4
>128
16
32
64
8
8
8
>128
64
64
32
32
32
4
64
32
32
16
8
128
32
32
16
21
22
32
32
16
16
32
8
128
32
16
8
16
16
16
32
8
32
8
128
16
32
16
8
64
16
32
128
64
32
32
23
24
64
16
32
64
8
32
128
32
16
32
—
—
4
16
8
8
16
4
32
16
64
>128
—
—
64
Ampho. B
Fluconazole
Tetracycline
Ampicillin
2
32
0.5
4
16
32
8
16
8
16
8
8
—
—
—
—
16
—
—
32
64
32
—
—
4
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
8
16
16
8
2
1
4
CA, Candida albicans (NCL1); CN, Cryptococcus neoformans (NCL2); BP, Benjaminiella poitrasii (NCL3); YL, Yarrowia lipolytica (NCL4); FO,
Fusarium oxysporum (NCL5); EC, Escherichia coli (NCIM No. 2574); SA, Staphylococcus aureus (NCIM No. 2122).
Negative control, DMSO, no inhibition.
a MIC (minimum inhibitory concentration) was determined as 90% inhibition of growth with respect to the growth control.
b IC50 was the concentration at which 50% growth inhibition was observed.
19, 21 and 23 derived from cholic acid having 7-hydroxy
showed moderate antibacterial activity against S. aur-
eus. However, the compounds 18, 20, 22, and 24 derived
from deoxycholic acid in the absence of 7-hydroxy were
less active against S. aureus with MIC value of P128 lg/
mL. From the overall activity results, it was observed
that the ester or amide linkage and chloro substituent
on phenyl ring of b-lactam part did not affect the activ-
ity of the compounds.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2008.01.102.
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In conclusion, a series of novel 1,2,3-triazole-linked
b-lactam–bile acid conjugates were synthesized using
Cu(I) catalyzed cycloaddition reaction of azido b-lac-
tams and terminal alkynes derived from cholic acid/
deoxycholic acid in excellent yields and their antimicro-
bial activities were evaluated. Compounds 17–24 dem-
onstrated potent antimicrobial activity against all the
strains tested. The compound 24 showed very good anti-
fungal activity with MIC value of 16 lg/mL against
C. albicans and 8 lg/mL against B. poitrasii. In particu-
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Acknowledgments
N.S.V. thanks CSIR, New Delhi, for the award of a Se-
nior Research Fellowship. Emeritus Scientist Scheme
awarded to B.G.H. by the CSIR, New Delhi, is grate-
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