Synthesis and biological evaluation of bile acid dimers linked with 1,2,3-triazole and bis-β-lactam

Article abstract of DOI:10.1039/b809221d

We report herein the synthesis and biological evaluation of bile acid dimers 11-18 linked through 1,2,3-triazole and bis-β-lactam. The dimers 11-18 were synthesized using 1,3-dipolar cycloaddition reaction of diazido bis-β-lactams 3, 4 and terminal alkyne

Full text of DOI:10.1039/b809221d

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis and biological evaluation of bile acid dimers linked with
1,2,3-triazole and bis-b-lactam†
Namdev S. Vatmurge,a Braja G. Hazra,*a Vandana S. Pore,a Fazal Shirazi,b Mukund V. Deshpande,b
Sreenath Kadreppa,c Samit Chattopadhyayc and Rajesh G. Gonnaded
Received 30th May 2008, Accepted 18th July 2008
First published as an Advance Article on the web 29th August 2008
DOI: 10.1039/b809221d
We report herein the synthesis and biological evaluation of bile acid dimers 11–18 linked through
1,2,3-triazole and bis-b-lactam. The dimers 11–18 were synthesized using 1,3-dipolar cycloaddition
reaction of diazido bis-b-lactams 3, 4 and terminal alkynes 7–10 derived from cholic acid/deoxycholic
acid in the presence of Cu(I) catalyst (click chemistry). These novel molecules were evaluated in vitro for
their antifungal and antibacterial activity. Most of the compounds exhibited significant antifungal as
well as antibacterial activity against all the tested fungal and bacterial strains. Moreover, their in vitro
cytotoxicities towards HEK-293 and MCF-7 cells were also established.
anti-microbial,15 selective b3 adrenergic receptor agonist16 and
Introduction
anti-allergic.17
The azetidine-2-one (b-lactam) ring system is a common structural
feature of a number of broad spectrum b-lactam antibiotics
such as penicillins, cephalosporins, carbapenems, nocardicins and
monobactams, which have been widely used as chemotherapeutic
agents for treating microbial diseases.1–3 It also shows many other
interesting biological properties, such as cholesterol absorption
inhibitors,4 human cytomegalovirus protease inhibitors,5 throm-
bin inhibitors,6 antihyperglycemic,7 anti-tumour,8 anti-HIV,9 anti-
inflammatory and analgesic activities.10 However, microorganisms
have built up resistance against the most traditional b-lactam
antibiotics due to the widespread 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.
Azoles are the largest class of antifungal agents in clinical
use.11 1,2,3-Triazole moieties are attractive connecting units, as
they are stable to metabolic degradation and capable of hydrogen
bonding, which can be favorable in binding of biomolecular
targets and solubility.12 The b-lactam ring system in combi-
nation with the 1,2,3-triazole moiety is present in a number
of drugs such as the b-lactam antibiotic Tazobactam and the
cephalosporine Cefatrizine.13 The 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 anti-HIV,14
Bile acids and their derivatives are pharmacologically inter-
esting as potential carriers of liver specific drugs, absorption
enhancers and cholesterol lowering agents.18 A common feature
of bile acid derived antimicrobials is their potential to exhibit
facially amphiphilic nature, due to polar hydroxyl groups on
one face and a nonpolar hydrophobic methyl group on the
other.19 These hydroxyl groups can have specific interactions, such
as those found in inclusion compounds,20 organogelators21 and
receptors22 based on cholic acid. The polarity of the hydroxyl
groups can be increased to emphasize the facial amphiphilicity
of the steroid unit. The resulting facial amphiphiles have many
applications in ion transport,23 combinatorial chemistry,24 vesical
fusion25 and improvement of membrane permeability.26 This type
of amphiphilicity can also be exhibited by polyene macrolide
amphotericin B, peptide antimicrobial agent polymixin B, and
squalamine in the cyclic form, which functions as an ionophore.27
Furthermore, bile acids are imperative building blocks for the
synthesis of dimers, oligomers and colaphanes28,29 due to their rigid
framework with multiple chiral centers. The dimers, oligomers
and colaphanes were synthesised from bile acids and have a wide
range of potential applications in pharmacology,29a membrane
bilayer probes,30 and ion complexation.31 The synthesis of novel
bile acid dimers containing 1,2,3-triazole as a linker have been
reported from our laboratory.32 More recently, Regen et al. have
reported bile acid derived molecular umbrellas as anti-HIV and
anti-HSV agents and molecular umbrella-assisted transport of
an oligonucleotide across cholesterol-rich phospholipid bilayers.33
Taking advantage of the amphiphilic topology of bile acids, we
have reported the synthesis of cholic acid and deoxycholic acid
dimers, which were found to posses antifungal and antiprolifera-
tive activity.34
aDivision of Organic Chemistry, National Chemical Laboratory, Pune,
411008, India. E-mail: bg.hazra@ncl.res.in; Fax: 91-20-25902629/2624;
Tel: 91-20-25902336
bBiochemical Sciences Division, National Chemical Laboratory, Pune,
411008, India
cNational Centre for Cell Science, Pune, 411007, India
dCentre for Materials Characterization, National Chemical Laboratory,
Pune, 411008, India
In continuation of our work on bile acid dimers, we report herein
the synthesis of eight novel cholic acid and deoxycholic acid dimers
11–18 using both 1,2,3-triazole and bis-b-lactam as a linker and
† CCDC reference numbers 690111 and 690112. For crystallographic data
in CIF or other electronic format see DOI: 10.1039/b809221d
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studied their antimicrobial and cytotoxic activity. The syntheses
of these novel cholic acid, deoxycholic acid dimers 11–18 linked
with two unique pharmacophore units such as 1,2,3-triazole and
b-lactam and the bioactivity data are reported herein for the first
time.
due to the non-equivalence of the two methylenes joining the
two b-lactam rings. The structures of compounds 3 and 4 have
been further confirmed unambiguously by X-ray crystal analysis
(Fig. 1).
Results and discussion
Chemistry
The CuI-catalysed variant of the Huisgen 1,3-dipolar cycloaddi-
tion of azide and alkynes affords regioselectively 1,4-disubstituted
1,2,3-triazoles with such efficiency and scope that the transfor-
mation has been described as “click” chemistry.35 This unique
transformation was independently discovered in 2002 by two
groups, one led by Sharpless36 in USA and the other in Denmark
by Meldal.37 Accordingly, our target molecules 11–18 were synthe-
sized using the 1,3-dipolar cycloaddition reaction of bis-b-lactams
3, 4 containing azide and cholic acids 7, 9 and deoxycholic acids
8, 10 containing terminal alkyne, in the presence of Cu(I) catalyst
(click chemistry). Diazido bis-b-lactams 3 and 4 were prepared
by the cycloaddition reaction (Staudinger)38,39 using imine 2 and
ketene derived from 1. Thus, treatment of the potassium salt of
azidoacetic acid 1 and imine 2 (prepared by using a literature39
procedure) in the presence of triphosgene and triethylamine in
anhydrous dichloromethane afforded a diastereomeric mixture
of two diazido bis-b-lactams (Scheme 1). The mixture, after
careful flash column chromatographic separation on silica gel,
furnished diastereomeric compounds 3 and 4 in 35% and 41%
yields respectively. The C2-symmetric structure for bis-b-lactam
Fig. 1 Ortep view for compounds 3 and 4.
Propargyl esters 7 and 8 were prepared by coupling propargyl
alcohol with cholic acid 5 or deoxycholic acid 6 using EDC·HCl
[1-ethyl-3-(3-dimethylaminopropyl) ◦carbodiimide hydrochloride]
as coupling agent in DMF at 0–25 C with 88% and 90% yields
respectively (Scheme 2). Using similar reaction conditions, amides
9 and 10 containing terminal alkyne functionality were prepared
by coupling propargyl amine with cholic acid and deoxycholic
acid in excellent yields.
Our next goal was to synthesise targeted cholic acid/deoxy-
cholic acid dimers 11–18 linked with 1,2,3-triazole and bis-b-
lactam. Reaction of propargyl ester 8 and bis-b-lactams 4 in
t-BuOH–H2O (7 : 3) with CuSO4·5H2O, sodium ascorbate (click
chemistry) at 70 ◦C for 32 h afforded the deoxycholic acid
dimer linked with 1,2,3-triazole and bis-b-lactam 16 in 81% yield
(Scheme 3). It has been well established that reaction under
microwave conditions substantially decreased40 the reaction time
down to a few minutes as compared to several hours refluxing
under thermal conditions. The cycloaddition reaction of propargyl
esters 7, 8 and amides 9, 10 derived from cholic acid/deoxycholic
acid with diazido bis-b-lactam 3 in DMF–H2O (7 : 3) and
CuSO4·5H2O, sodium ascorbate under microwave irradiation for
five minutes furnished a diasteriomeric mixture of novel dimers
11–14 in 93–95% yields. In a similar way, microwave irradiation
of propargyl esters 7, 8 and amides 9, 10 with diazido bis-b-
lactam 4 afforded a diasteriomeric mixture of dimeric compounds
15–18 in 92 to 94% yields. The combination of racemic core 3
and 4 with optically pure 7–10 afforded a diastereomeric mixture
of dimers 11–18 in equal amounts. These diastereomers 11–18
were inseparable by flash column chromatography and also by
crystallization.
1
1
3 was assigned from H NMR spectral analysis. The H NMR
spectrum of the compound 3 showed two doublets at d 2.86
and 3.86 with geminal coupling of 11 Hz for the protons of the
methylene groups joining two b-lactam rings. The meso structure
was assigned to the other diastereomer 4 as the 1H NMR spectrum
of this compound showed two multiplets at d 3.11 and 3.60
Scheme 1 Reagents and conditions: (a) triphosgene, Et3N, CH2Cl2,
0–25 ◦C, 15 h, (35% for 3 and 41% for 4).
Scheme 2 Reagents and conditions: (a) EDC·HCl, HOBt, propargyl alcohol/propargyl amine hydrochloride, Et3N, DMF, 0–25 ◦C, 12 h.
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Scheme 3 Reagents and conditions: (a) sodium ascorbate, CuSO4·5H2O, DMF–H2O (7 : 3), microwave (385 watt), 5 min, 92–95%.
inhibitory effect with a MIC value of 16 mg mL-1, comparable to
that of amphotericin B against F. oxysporum, whereas compounds
11, 13 and 17 also showed promising activity against F. oxysporum.
Furthermore, among the dimers 11–18, none of the compounds
showed more or comparable antibacterial activity against E. coli
than tetracycline or ampicillin; compounds 11, 13, 15 and 18 were
found to be moderately active with MIC values of 16–22 mg mL-1.
However, only compound 13 showed comparable activity to that
of tetracycline and ampicillin against S. aureus with a MIC value
of 16 mg mL-1. From the overall activity results, it was observed
that the ester or amide linkage did not affect the activity of the
compounds.
Biological evaluation
Antimicrobial activity
All the newly synthesized dimers 11–18 were tested in vitro
for antifungal and antibacterial activity. The antifungal activity
was tested using the fungal strains Candida albicans, Crypto-
coccus neoformans (human pathogen), Benjaminiella poitrasii,
Yarrowia lipolytica (saprophytes) and Fusarium oxysporum (plant
pathogen). The antibacterial activity was evaluated against Es-
cherichia coli and Staphylococcus aureus. The MIC and IC50 values
were determined using a standard broth microdilution technique
described by NCCLS.41 In comparison with the antimicrobial
activity, amphotericin B and fluconazole were used as the reference
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.
As seen in Table 1, most of the synthesised dimers 11–18 showed
moderate to good antifungal and antibacterial activity against all
the tested fungal and bacterial strains. The activity of compounds
13 and 17 was higher than that of fluconazole against C. albicans
with a MIC value of 16–25 mg mL-1. The compounds 13 and 15–
18 showed good antifungal activity against C. neoformans having
a MIC value of 16–36 mg mL-1 higher or comparable to that of
reference drug fluconazole. However, all the compounds 11–18
except 15 showed significant growth inhibitory activity against B.
poitrasii. Y. lipolytica was adversely affected by 12, 13 and 15–
18, and in particular, 15 and 16 were the most potent with low
MIC values of 8–10 mg mL-1. Compound 12 showed significant
Cytotoxic activity
The cytotoxicity of all the dimers 11–18 was assessed in vitro
against human embryonic kidney (HEK-293) and human mam-
mary adenocarcinoma (MCF-7) cell lines. Bile acid derivatives are
known to promote proliferation and metastasis of cells of cancer
origin and inhibit the proliferation of cells of non-cancer origin.42
Hence we tested the cytotoxicity of the synthesized compounds in
two different cell lines, one of cancer origin and the other of non-
cancer origin. HEK-293 cells are normal human embryonic kidney
cells where as MCF-7 cells are human breast cancer derived. The
cytotoxicity of the compounds 11–18 against HEK-293 and MCF-
7 cells was evaluated using MTT assay.43 The observed IC50 values
of all the evaluated compounds are shown in Table 2. Among
them, compound 11 was the most toxic to both cell lines, with IC50
values of 10 and 80 mM; the rest of the compounds did not show
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Table 1 In vitro antimicrobial activity of compounds 11–18
Inhibitory concentration/mg mL-1
Fungal strains
Bacterial strains
EC
CA
CN
BP
YL
FO
SA
b
Compound
MICa
IC50
MIC
IC50
MIC
IC50
MIC
IC50
MIC
IC50
MIC
IC50
MIC
IC50
11
32
64
16
>128
85
64
25
67
2
28
12
33
8
35
17
41
10
29
51
>128
18
>128
17
32
36
11
38
5
43
4
4
11
6
10
22
—
—
17
44
32
43
94
41
12
41
23
39
—
—
2
10
21
9
36
12
4
15
8
17
—
—
>128
14
35
>128
10
8
16
17
15
32
8
16
64
6
4
4
19
16
25
75
64
31
27
32
16
8
9
9
8
42
42
10
8
10
8
16
>128
22
67
19
47
80
10
57
9
28
7
19
29
11
—
—
3
47
37
16
>128
32
64
77
>128
—
11
17
7
12
13
14
53
16
29
22
64
—
—
8
15
16
17
18
16
27
8
18
Ampho. B
Fluconazole
Tetracycline
Ampicillin
0.5
4
—
9
—
43
78
36
—
—
4
—
—
—
—
—
—
—
—
—
—
9
16
14
—
—
—
2
1
4
CA, Candida albicans (values were recorded after 48 h); CN, Cryptococcus neoformans (values were recorded after 72 h); BP, Benjaminiella poitrasii; YL,
Yarrowia lipolytica (values were recorded after 24 h); FO, Fusarium oxysporum (values were recorded after 48 h); EC, Escherichia coli; SA, Staphylococcus
aureus (values were recorded after 24 h). Negative control, DMSO (2.5% v/v), No inhibition. “–” Not tested.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.
Table 2 Cytotoxicity of compounds 11–18
IC50/mM
acid were moderately active against E. coli. Additionally, only
compound 13 exhibited comparable activity against S. aureus to
that of reference drugs. Furthermore, except compound 11, all
other compounds (12–18) did not show any significant cytotoxicity
to the tested cell lines. The synthesis of bile acid dimers linked with
two pharmacophores, 1,2,3-triazole and b-lactam, can open up a
new horizon for the control of human and plant pathogens.
Compound
HEK-293
MCF-7
11
12
13
14
15
16
17
18
10
>1000
100
500
500
>1000
100
>1000
80
>1000
>1000
>1000
1000
>1000
>1000
>1000
Experimental section
General methods
All melting points were determined on a Yanco Micro melting
point apparatus and are uncorrected. Optical rotations were
obtained on a Bellingham and Stanly ADP-220 Polarimeter.
Reactions were monitored by thin-layer chromatography (TLC)
using TLC aluminium sheets, silica gel 60-F254 precoated, Merck,
Germany and spots were located using UV light as the visualizing
agent or spraying with ethanolic phosphomolybdic acid (PMA)
solution, followed by heating. Column chromatography was
performed on silica gel. 1H and 13C NMR spectra were recorded
on Bruker AC-200 (200 MHz) at 200.13 and 50.32 or on a
Bruker MSL-300 at 300.13 and 75.47 or on a Bruker DRX-500
spectrometer at 500.13 and 125.78 respectively. Chemical shifts are
given in d values relative to TMS (tetramethylsilane) as internal
standard. IR spectra were recorded on Schimadzu 8400 series
FTIR instrument and values are reported in cm-1 units. Specific
rotations ([a]D) are reported in deg dm-1 and the concentration
(c) is given in g/100mL in the specific solvent. Mass spectra
were recorded by either a LC-MS or MS-TOF API QSTAR
PULSAR spectrometer, samples introduced by infusion method
using electrospray ionisation technique. Elemental analyses were
performed by a CHNS-O EA 1108-Elemental analyser, Carlo Erba
Instrument (Italy) or Elementor Vario EL (Germany) and were
within 0.3% of calculated values.
any cytotoxicity up to 500 mM concentration to both cell lines.
Furthermore, it was observed that compounds 12, 13, 16 and 18
enhanced the proliferation of MCF-7 cells and not HEK-293 cells.
Conclusion
A series of novel bile acid dimers 11–18 linked through 1,2,3-
triazole and bis-b-lactam have been synthesized using a Cu(I)
catalysed cycloaddition reaction (click chemistry) of diazido bis-
b-lactams 3–4 and terminal alkynes 7–10 derived from cholic
acid/deoxycholic acid in excellent yield. These novel dimers 11–
18 were evaluated for antifungal as well as antibacterial and
cytotoxic activities. Most of the compounds demonstrated potent
antimicrobial activity against all the strains tested. Among them,
compounds 13 and 17 showed significant antifungal activity
against human pathogen, C. albicans and compounds 13, 15 and
18 showed appreciable antifungal activity against C. neoformans
than reference drug fluconazole. In the case of plant pathogen F.
oxysporum, compound 12 showed comparable inhibitory activity
to amphotericin B. In particular, compounds 15 and 16 exhibited
the most significant activity against Y. lipolitica with MIC values
of 8–10 mg mL-1. Compounds 11, 13 and 15 derived from cholic
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Procedure for the synthesis of b-lactams 3 and 4
transmission 0.9296 and 0.9878; maximum and minimum residual
-3
˚
electron densities 0.149 and -0.128 e A .
A solution of triphosgene (0.741 g, 2.5 mmol), in anhydrous
CH2Cl2 (20 mL), was added slowly to a mixture of the potassium
salt of azidoacetic acid 1 (0.695 g, 5 mmol), imine 2 (0.472 g,
2 mmol) and triethylamine (2 mL, 15 mmol) in anhydrous CH2Cl2
(20 mL) at 0 ◦C. After the addition, the reaction mixture was
allowed to warm up to room temperature (25 ◦C) and was stirred
for 15 h. The reaction mixture was then washed with water (25 mL),
saturated sodium bicarbonate solution (2 ¥ 20 mL) and brine
(20 mL). The organic layer was dried over anhydrous sodium sul-
fate and concentrated to get the crude product as a diastereomeric
mixture. Flash column chromatographic purification over silica gel
using ethyl acetate–petroleum ether (12 : 88) as an eluent afforded
pure b-lactam 3 (0.281 g, 35%). Further elution with same solvent
system yielded pure b-lactam 4 (0.329 g, 41%).
All the data were corrected for Lorentzian, polarization and
absorption effects using Bruker’s SAINT and SADABS programs.
SHELX-97 (G. M. Sheldrick, SHELX-97 program for crystal
structure solution and refinement, University of Gottingen, Ger-
many, 1997) was used for structure solution and full-matrix least-
squares refinement on F2. Hydrogen atoms for both the structures
were placed in a geometrically idealized position with C–H =
˚
˚
0.93 A (for Ar–H), C–H= 0.98 A (for methyne-H) and C–H =
˚
0.97 A (for methylene-H) and constrained to ride on their parent
atoms with Uiso(H) = 1.2Ueq(C).
General procedure for the synthesis of alkyne compounds 7–10
EDC·HCl (1.5 equiv) and HOBt (0.5 equiv) were added to a
solution of cholic acid/deoxycholic acid (1 equiv) and propargyl
alcohol/propargyl amine (1.1 equiv) in dry DMF (5 mL) under
argon at 0 ◦C. After the addition, the reaction mixture was allowed
to warm up to 25 ◦C and was stirred for 12 h. The reaction
was quenched by adding crushed ice and extracted three times
with ethyl acetate. The combined extracts were washed with
water and brine, dried over anhydrous sodium sulfate, filtered and
concentrated in vacuo. The resulting crude product was purified
by column chromatography to give pure alkyne compounds 7–10.
1,2-Bis[3¢-azido-4¢-phenylazetidin-2¢-one-1¢-yl]ethane 3. Mp
123–124 ◦C; (Found: C, 59.87; H, 4.62; N, 28.09. Calc.
for C20H18N8O2: C, 59.69; H, 4.51; N, 27.85%.); IR nmax
(CHCl3)/(cm-1) 2115 (N3), 1762 (CO); 1H NMR (CDCl3, 200
MHz) d 7.46–7.21 (10H, m, Ar-H), 5.17 (2H, d, J = 5.1), 4.91
(2H, d, J = 5.1), 3.84 (2H, d, J = 11.2), 2.86 (2H, d, J = 11.2);
13C NMR (CDCl3, 50 MHz) d 165.8, 132.6, 129.1, 128.8, 127.7,
68.6, 59.9, 37.8. MS (LCMS) m/z: 425 (M + Na)+.
1-[3¢-Azido-4¢-phenylazetidin-2¢-one-1¢-yl]-2-[3¢¢-azido-4¢¢-phenyl-
azetidin-2¢¢-one-1¢¢-yl]ethane 4. Mp 162–163 ◦C; (Found: C,
59.57; H, 4.29; N, 28.13. Calc. for C20H18N8O2: C, 59.69; H, 4.51;
Propargyl 3a,7a,◦12a-trihydroxy-5b-cholan-24-oate 7. Yield:
88%; mp 112–114 C; [a]2D7 +26.15 (c 1.3, CHCl3); (Found: C,
72.35; H, 9.23. Calc. for C27H42O5: C, 72.61; H, 9.48%.); IR nmax
(CHCl3)/(cm-1) 3396, 3307, 1737; 1H NMR (CDCl3, 200 MHz) d
4.68 (2H, d, J = 2.5 Hz, OCH2), 3.96 (1H, bs, CH-12), 3.84 (1H,
bs, CH-7), 3.45 (1H, m, CH-3), 2.47 (1H, t, J = 2.5 Hz, alkyne
CH), 0.98 (3H, d, J = 5.8 Hz, CH3-21), 0.89 (3H, s, CH3-19), 0.68
(3H, s, CH3-18); 13C NMR (CDCl3, 75 MHz) d 173.4, 77.7, 74.7,
73.0, 71.7, 68.3, 51.6, 46.8, 46.3, 41.4, 39.3, 35.1, 34.7, 30.9, 30.6,
30.2, 29.6, 28.0, 27.4, 26.1, 23.1, 22.3, 17.2, 12.3; MS (LCMS) m/z:
469 (M + Na)+.
1
N, 27.85%.); IR nmax (CHCl3)/(cm-1) 2115 (N3), 1762 (CO); H
NMR (CDCl3, 200 MHz) d 7.48–7.28 (10H, m, Ar-H), 4.79 (2H,
d, J = 4.9), 4.74 (2H, d, J = 4.9,), 3.55–3.69 (2H, m), 3.04–3.18
(2H, m); 13C NMR (CDCl3, 50 MHz) d 164.7, 132.3, 129.3, 128.9,
127.9, 68.1, 60.9, 38.6. MS (LCMS) m/z: 425 (M + Na)+.
X-Ray crystal structure determination for 3 and 4
Single crystals of compounds 3 and 4 were obtained from an
ethyl acetate–light petroleum ether mixture (30 : 70). X-Ray
diffraction data were collected on a Bruker SMART APEX
CCD diffractometer with graphite-monochromatized (Mo Ka =
Propargyl 3a,12a-dihydroxy-5b-cholan-24-oate 8. Yield: 90%;
◦
mp 160–161 C; [a]2D7 +43.5 (c 1.5, CHCl3); (Found: C, 75.11;
˚
0.71073 A) radiation at room temperature (297 K).†
H, 9.62. Calc. for C27H42O5: C, 75.31; H, 9.83%.); IR nmax
(CHCl3)/(cm-1) 3382, 3307, 1737; 1H NMR (CDCl3, 300 MHz) d
4.68 (2H, d, J = 2.2 Hz, OCH2), 3.98 (1H, bs, CH-12), 3.55–3.64
(1H, m, CH-3), 2.47 (1H, t, J = 2.2 Hz, alkyne CH), 0.97 (3H, d,
J = 5.9 Hz, CH3-21), 0.91 (3H, s, CH3-19), 0.67 (3H, s, CH3-18);
13C NMR (CDCl3, 75 MHz) d 173.2, 77.7, 74.6, 72.9, 71.5, 51.6,
48.1, 47.1, 46.4, 42.0, 36.3, 35.9, 35.2, 35.1, 34.0, 33.5, 30.9, 30.7,
30.2, 28.5, 27.4, 27.1, 26.06, 23.6, 22.9, 17.1, 12.6; MS (LCMS)
m/z: 453.14 (M + Na)+.
Crystallographic data for 3. (C20H18N8O2): M = 402.42, crystal
dimensions 0.58 ¥ 0.32 ¥ 0.17 mm3, monoclinic, space group
˚
P 21/c, a = 13.838(11), b = 6.873(6), c = 22.068(19) A, b =
◦
96.710(15) ; V = 2085(3) A ; Z = 4; rcalcd = 1.282 gcm-3, m (Mo-
3
˚
Ka) = 0.089 mm-1, F(000) = 840, 2qmax = 50.00◦, 9849 reflections
collected, 3667 unique, 2786 observed (I > 2s (I)) reflections,
298 refined parameters, R value 0.0541, wR2 = 0.1327 (all data
R = 0.0699, wR2 = 0.1433), S = 1.068, minimum and maximum
transmission 0.9503 and 0.9851; maximum and minimum residual
N-Propargyl-3a,7a,12a-trihydroxy-5b-cholan-24-amide 9. Yi-
eld: 89%; mp 276–278 ◦C; [a]D27 +22.9 (c 1.31, CHCl3); (Found: C,
72.93; H, 9.51; N, 2.92. Calc. for C27H43NO4: C, 72.77; H, 9.73;
N, 3.14%.); IR nmax (CHCl3)/(cm-1) 3309, 1652; 1H NMR (CDCl3,
200 MHz) d 6.43 (1H, t, J = 5.2 Hz, CONH), 4.02–4.06 (2H, m,
NH-CH2), 3.97 (1H, bs, CH-12), 3.84 (1H, bs, CH-7), 3.38–3.51
(1H, m, CH-3), 2.84 (3H, s, OH), 2.24 (1H, t, J = 2.5 Hz, alkyne
CH), 0.99 (3H, d, J = 5.8 Hz, CH3-21), 0.89 (3H, s, CH3-19), 0.68
(3H, s, CH3-18); 13C NMR (CDCl3, 50 MHz) d 174.2, 80.0, 73.1,
71.8, 71.1, 68.4, 46.2, 41.4, 39.3, 35.3, 35.2, 34.7, 34.6, 32.4, 31.4,
-3
˚
electron densities 0.155 and -0.142 e A .
Crystallographic data for 4. (C20H18N8O2): M = 402.42, crystal
dimensions 0.78 ¥ 0.15 ¥ 0.13 mm3, monoclinic, space group
˚
P 21/c, a = 13.196(8), b = 5.751(4), c = 13.632(8) A, b =
◦
109.453(9) ; V = 975.4(10) A ; Z = 2; rcalcd = 1.370 gcm-3, m (Mo-
3
˚
Ka) = 0.095 mm-1, F(000) = 420, 2qmax = 50.00◦, 8813 reflections
collected, 1715 unique, 1538 observed (I > 2s (I)) reflections,
136 refined parameters, R value 0.0359, wR2 = 0.0896 (all data
R = 0.0399, wR2 = 0.0930), S = 1.070, minimum and maximum
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30.2, 28.9, 28.0, 27.5, 26.2, 23.2, 22.4, 17.4, 12.4; MS (LCMS) m/z:
500 MHz) d 7.53 (2H, s, triazole-H), 7.21 (10H, bs, Ar-H), 6.12
(2H, bs, unresolved splitting), 5.55 (2H, s, unresolved splitting),
4.19 (4H, s, -NHCH2), 4.08 (2H, t, J = 9.8 Hz, N-CH2), 3.94
(2H, bs, CH-12), 3.83 (2H, bs, CH-7), 3.42–3.37 (2H, m, CH-3,),
3.17–3.14 (2H, m, N-CH2), 0.96 (6H, d, J = 5.5 Hz, CH3-21),
0.89 (6H, s, CH3-19), 0.66 (6H, s, CH3-18); 13C NMR (CDCl3, 125
MHz) d 174.6, 163.5, 144.7, 130.8, 128.7, 128.2, 126.7, 123.2, 72.6,
71.2, 68.7, 67.8, 60.6, 46.1, 45.9, 41.2, 41.1, 39.0, 38.7, 38.6, 35.0,
34.9, 34.3, 34.1, 33.8, 32.3, 31.2, 29.4, 27.6, 27.1, 25.9, 22.8, 22.0,
16.7, 12.0; MS (LCMS) m/z: 1316 (M + Na)+.
446.19 (M + 1)+, 468.16 (M + Na)+.
N-Propargyl-3a,12a-dihydroxy-5b-cholan-24-amide 10. Yield:
92%; mp 184 ◦C; [a]D27 +51.76 (c 0.97, CHCl3); (Found: C, 75.26;
H, 9.87; N, 3.45. Calc. for C27H43NO3: C, 75.48; H, 10.09; N,
3.26%.); IR nmax (CHCl3)/(cm-1) 3475, 3446, 3238, 3209, 1633; 1H
NMR (CDCl3 + DMSOD6, 200 MHz) d 6.80 (1H, bs, CONH),
3.88–3.92 (2H, m, NH-CH2), 3.85 (1H, bs, CH-12), 3.41–3.55 (1H,
m, CH-3), 2.14 (1H, t, J = 2.5 Hz, alkyne CH), 0.87 (3H, d, J =
6.1 Hz, CH3-21), 0.79 (3H, s, CH3-19), 0.56 (3H, s, CH3-18); 13C
NMR (CDCl3 + DMSOD6, 50 MHz) d 174.5, 79.3, 72.7, 71.0,
70.7, 47.7, 46.3, 46.0, 41.8, 35.6, 35.0, 34.9, 33.8, 33.2, 32.3, 31.2,
29.4, 28.4, 28.2, 27.2, 26.8, 25.8, 23.4, 22.7, 16.6, 12.3; MS (LCMS)
m/z: 430.21 (M + 1)+, 452.19 (M + Na)+.
Dimer 14. Mp 177–178 ◦C; (Found:C, 70.37;H, 8.08; N, 11.29.
Calc. for C74H104N10O8: C, 70.45; H, 8.31; N, 11.10%.); IR nmax
1
(CHCl3)/(cm-1) 3392, 1766, 1658; H NMR (CDCl3 + CD3OD,
500 MHz) d 7.21 (10H, bs, Ar-H), 6.21 (2H, bs, unresolved
splitting), 5.56 (2H, bs, unresolved splitting), 4.10 (4H, s, -
NHCH2), 3.96 (2H, s, CH-12), 3.58 (2H, bs, CH-3), 3.14–3.00
(4H, m, -NCH2), 0.96 (6H, bs, CH3-21), 0.90 (6H, s, CH3-19),
0.67 (6H, s, CH3-18); 13C NMR (CDCl3 + CD3OD 125 MHz): d
163.7, 130.8, 128.8, 128.3, 126.7, 72.6, 71.0, 70.0, 59.9, 47.7, 46.4,
46.1, 41.7, 38.5, 35.7, 35.6, 35.1, 34.9, 33.8, 33.1, 31.2, 29.5, 28.2,
27.2, 26.8, 25.8, 23.4, 22.7, 16.8, 12.3; MS (LCMS) m/z: 1284
(M + Na)+, 1285 (M + Na + 1)+.
General procedure for the synthesis of dimers 11–18
The alkynes of cholic acid/deoxycholic acid (1 equiv) and the
diazido bis-b-lactams 3 or 4 (0.5 equiv) were dissolved in DMF–
H2O 7 : 3 (10 mL). To this solution, CuSO4·5H2O (0.05 equiv) and
sodium ascorbate (0.5 equiv) were added. The reaction mixture
was placed in a domestic microwave reactor and irradiated for
5 min at 385 watt. It was then cooled to room temperature,
quenched with crushed ice and extracted with ethyl acetate.
The extract was washed with water and brine, and dried over
anhydrous sodium sulfate. The solvent was evaporated under
reduced pressure and the crude product was purified by column
chromatography to obtain dimers 11–18.
Dimer 15. Mp 173–175 ◦C; (Found: C, 68.74; H, 7.83; N, 8.57.
Calc. for C74H102N8O12: C, 68.60; H, 7.94; N, 8.65%.); IR nmax
(CHCl3)/(cm-1) 3454, 1770, 1731; 1H NMR (CDCl3, 400 MHz) d
7.67 (1H, s, triazole-H), 7.49 (1H, s, triazole-H), 7.30–7.27 (10H,
m, Ar-H), 6.23–6.16 (2H, m), 5.38 (2H, bs, unresolved splitting),
5.0 (4H, bs, -OCH2), 3.95 (2H, bs, CH-12), 3.84 (2H, bs, CH-7),
3.76–3.47 (6H, m, 2 ¥ CH-3, 2 ¥ -NCH2), 0.95 (6H, bs, CH3-21),
0.88 (6H, s, CH3-19), 0.67 (6H, s, CH3-18); 13C NMR (CDCl3,
125 MHz): d 173.8, 163.2, 131.1, 129.2, 128.6, 127.0, 72.7, 71.4,
68.7, 68.1, 61.7, 56.7, 2 ¥ 46.6, 46.1, 41.3, 40.3, 39.2, 35.1, 34.5,
34.3, 30.8, 30.5, 29.8, 27.8, 27.3, 26.0, 22.8, 22.2, 17.0, 12.2; MS
(LCMS) m/z: 1317 (M + Na)+.
Dimer 11. Mp 167–168 ◦C; (Found: C, 68.45; H, 7.71; N, 8.89.
Calc. for C74H102N8O12: C, 68.60; H, 7.94; N, 8.65%.); IR nmax
(CHCl3)/(cm-1) 3411, 1766, 1731; 1H NMR (CDCl3, 400 MHz) d
7.50 (1H, s, triazole-H), 7.47 (1H, s, triazole-H), 7.21 (10H, bs, Ar-
H), 6.16 (1H, d, J = 4.8, Hz), 6.13 (1H, d, J = 5.0, Hz), 5.56 (2H,
d, J = 4.8 Hz), 4.97 (4H, s, -OCH2), 4.11 (2H, t, J = 9.3 Hz), 3.94
(2H, bs, CH-12), 3.84 (2H, d, J = 13.6 Hz, CH-7), 3.40 (2H, bs,
CH-3,), 3.13 (2H, t, J = 11.8 Hz), 0.94 (6H, s, CH3-19), 0.86 (6H,
d, J = 7.8 Hz, CH3-21), 0.64 (6H, s, CH3-18); 13C NMR (CDCl3,
125 MHz) d 173.7, 163.4, 142.4, 130.9, 128.8, 128.4, 126.6, 124.0,
72.6, 71.3, 68.7, 67.9, 60.4, 56.5, 46.4, 46.0, 41.1, 39.0, 38.9, 38.6,
34.9, 34.4, 34.2, 30.7, 30.3, 29.6, 27.7, 27.2, 25.9, 22.8, 22.1, 16.8,
12.1; MS (LCMS) m/z: 1295 (M + 1)+, 1318 (M + Na)+.
Dimer 16. Mp 145–146 ◦C; (Found: C, 70.26; H, 8.11; N, 8.96.
Calc. for C74H102N8O10: C, 70.34; H, 8.14; N, 8.87%.); IR nmax
(CHCl3)/(cm-1) 3442, 1766, 1731; 1H NMR (CDCl3, 200 MHz) d
7.62 (2H, s, triazole-H), 7.32–7.22 (10H, m, Ar-H), 6.15–6.13 (2H,
m), 5.37–5.33 (2H, m), 5.0 (4H, s, -OCH2), 3.97 (1H, bs, CH-12),
3.83–3.43 (6H, m, 2 ¥ CH-3, 2 ¥ -NCH2), 0.95 (6H, bs, CH3-21),
0.91 (6H, s, CH3-19), 0.67 (6H, s, CH3-18); 13C NMR (CDCl3,
100 MHz): d 173.6, 163.1, 142.5, 131.2, 129.1, 128.6, 127.0, 124.1,
72.3, 71.3, 68.7, 61.8, 56.8, 47.9, 46.8, 46.3, 41.9, 40.3, 36.2, 35.8,
35.1, 35.0, 33.9, 33.3, 30.8, 30.5, 30.1, 28.5, 27.3, 27.0, 26.0, 23.5,
22.9, 17.0, 12.5; MS (LCMS) m/z: 1285 (M + Na)+.
Dimer 12. Mp 157–159 ◦C; (Found: C, 70.07; H, 7.94; N, 9.02.
Calc. for C74H102N8O10: C, 70.34; H, 8.14; N, 8.87%.); IR nmax
1
(CHCl3)/(cm-1) 3434, 1768, 1730; H NMR (CDCl3, 200 MHz):
d 7.43 (2H, s, Ar-H), 7.20–7.25 (10H, m, Ar-H), 6.15–6.12 (2H,
m), 5.55 (2H, d, J = 5.1 Hz), 4.97 (4H, s, -OCH2), 4.12 (2H, d,
J = 11.1 Hz), 3.97 (1H, bs, CH-12), 3.66–3.55 (2H, m, CH-3), 3.13
(2H, d, J = 11.3 Hz), 2.35–2.16 (4H, m, -CH2CO), 0.94 (6H, d,
J = 5.8 Hz, CH3-21), 0.91 (6H, s, CH3-19), 0.66 (6H, s, CH3-18);
13C NMR (CDCl3, 100 MHz): d 173.7, 163.6, 142.7, 131.2, 129.1,
128.7, 126.8, 123.9, 72.9, 71.5, 69.1, 60.6, 56.9, 48.1, 47.0, 46.4,
42.0, 38.7, 36.3, 35.9, 35.2, 35.0, 34.0, 33.5, 31.0, 30.9, 30.6, 30.3,
28.6, 27.4, 27.0, 26.1, 23.6, 23.0, 17.2, 12.6; MS (LCMS) m/z: 1286
(M + Na)+.
Dimer 17. Mp199–200 ◦C; (Found:C, 68.46;H, 8.31; N, 10.54.
Calc. for C74H104N10O10: C, 68.70; H, 8.10; N, 10.83%.); IR nmax
(CHCl3)/(cm-1) 3500, 1758, 1650; 1H NMR (CDCl3 + DMSOD6,
400 MHz) d 7.60 (1H, s, triazole-H), 7.51 (1H, s, triazole-H),
7.27–7.25 (10H, m, Ar-H), 6.17–6.13 (2H, m), 5.38–5.35 (2H, m),
4.21 (4H, bs, -NHCH2), 3.90 (1H, bs, CH-12), 3.82–3.77 (4H, m,
2 ¥ CH-7, N-CH2), 3.48–3.43 (2H, m, CH-3), 3.39–3.33 (2H, m,
N-CH2), 0.98 (6H, d, J = 5 Hz, CH3-21), 0.88 (6H, s, CH3-19),
0.66 (6H, s, CH3-18); 13C NMR (CDCl3 + CD3OD, 125 MHz): d
174.5, 163.2, 144.6, 131.1, 129.0, 128.4, 127.0, 123.2, 72.7, 71.3,
68.5, 67.9, 61.6, 46.0, 41.3, 39.9, 39.1, 35.1, 34.5, 34.3, 32.3, 31.3,
Dimer 13. Mp 195–196 ◦C;(Found:C, 68.41;H, 8.23;N, 10.66.
Calc. for C74H104N10O10: C, 68.70; H, 8.10; N, 10.83%.); IR nmax
1
(Nujol)/(cm-1) 3369, 1758, 1650; H NMR (CDCl3 + CD3OD,
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29.7, 29.4, 27.8, 27.2, 26.0, 22.9, 22.2, 17.0, 12.1; MS (LCMS) m/z:
Antiproliferative activity. Materials and methods: cell culture
1316 (M + Na)+.
Human embryonic kidney (HEK-293) and human mammary
adenocarcinoma (MCF-7) cell lines were grown in a mono-
layer in nutrient media DMEM (Dulbecco’s Modified Eagle’s
Media) supplemented with fetal bovine serum (10%), penicillin
(100 U mL-1), and streptomycin (100 mg mL-1) (all from Invitrogen
Life Technologies, MD). The cells were grown at 37 ◦C in the
presence of 5% CO2.
Dimer 18. Mp 180–181 ◦C; (Found: C, 70.61; H, 8.15; N,
11.22. Calc. for C74H104N10O8: C, 70.45; H, 8.31; N, 11.10%.);
IR nmax (CHCl3)/(cm-1) 3373, 1768, 1658; 1H NMR (CDCl3, 400
MHz) d 7.58 (1H, s, triazole-H), 7.47 (1H, s, triazole-H), 7.27–
7.24 (10H, m, Ar-H), 6.72–6.63 (2H, m, -NH), 6.12 (2H, bs,
unresolved splitting), 5.34 (2H, bs, unresolved splitting), 4.27 (4H,
bs, -NHCH2), 3.96 (1H, bs, CH-12), 3.77 (2H, bs, N-CH2), 3.59–
3.45 (4H, m, 2 ¥ CH-3, N-CH2), 0.95 (6H, bs, CH3-21), 0.91 (6H, s,
CH3-19), 0.67 (6H, s, CH3-18); 13C NMR (CDCl3, 125 MHz): d
174.3, 163.0, 144.7, 131.0, 128.9, 128.3, 126.9, 123.1, 72.6, 71.0,
68.6, 61.4, 47.6, 46.3, 46.1, 41.7, 39.9, 35.8, 35.6, 35.1, 33.8, 33.1,
31.2, 29.7, 28.2, 27.2, 26.9, 25.9, 23.4, 22.8, 16.9, 12.3; MS (LCMS)
m/z: 1284 (M + Na)+.
MTT cell proliferation assay
HEK-293 and MCF-7 cells were plated at a density of 104 cells per
well in 96-well tissue culture plates. Cells were allowed to adhere
for 24 h at 37 ◦C. Stock solutions of all the compounds were
prepared in DMSO at a concentration of 10 mM and diluted to
the required concentration. The 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) was dissolved (5 mg mL-1)
in DMEM (without phenol red) and filtered through a 0.22 mm
filter before use. The cells were treated with various concentrations
(0, 1, 10, 100 and 1000 mM) of compounds dissolved in DMSO
for an additional 48 h, in triplicate. In the control wells, nutrient
medium with a corresponding concentration of DMSO only was
added to the cells. Thereafter, the drug containing medium was
replaced with 50 mL media containing 1 mg mL-1 MTT and
Antimicrobial activity: materials and methods
Human pathogens C. albicans and C. neoformans; saprophytes B.
poitrasii and Y. lipolytica were maintained on YPG (yeast extract,
0.3%; peptone, 0.5%; and glucose, 1%) agar slants. F. oxysporum
(plant pathogen) was maintained on PDA (potato, 20%; dextrose,
◦
2%) agar slants at 28 C. E. coli and S. aureus were maintained
◦
incubated for 4 h at 37 C. Medium was then aspirated off from
on nutrient agar (NA; beef extract, 0.3%; peptone, 0.5%; sodium
chloride, 0.5%) agar slants. Strains of C. albicans, C. neoformans,
Y. lipolytica and B. poitrasii were inoculated in YPG broth. C.
albicans, C. neoformans and Y. lipolytica were incubated at 28 ◦C
whereas B. poitrasii was incubated at 37 ◦C for 24 h. F. oxysporum
was inoculated in potato dextrose and incubated at 28 ◦C for 48 h
whereas bacterial strains E. coli and S. aureus were inoculated in
NA broth for 24 h. Compounds 11–18 were solubilized in DMSO
(2.5% v/v) and stock solutions of 1.28 mg mL-1 were prepared.
Amphotericin B, fluconazole, tetracycline and ampicillin were also
dissolved in DMSO, and were used as a positive control.
the formazan crystals, which were then solubilized in 100 mL of
acidified isopropanol. The optical density was read on a microplate
reader at 570 nm using 630 nm as a reference filter against a blank
prepared from cell free wells. Absorbance given by cells treated
with the carrier DMSO alone was taken as 100% cell growth. All
assays were performed in triplicate.
Acknowledgements
NSV thanks CSIR, New Delhi, for the award of a Senior Research
Fellowship. An Emeritus Scientist Scheme awarded to BGH by the
CSIR, New Delhi is gratefully acknowledged.
MIC and IC50 determination
In vitro antifungal and antibacterial activity of the newly synthe-
sized compounds were studied against the fungal strains viz., C.
albicans, C. neoformans, B. poitrasii, Y. lipolytica, F. oxysporum
strains and bacterial strains E. coli, and S. aureus, respectively to
find out MIC (minimum inhibitory concentration) and IC50 (50%,
inhibition of growth). All the experiments were done in triplicate
under similar experimental conditions. MIC and IC50 of the
synthesized compounds were determined according to a standard
broth microdilution technique as per NCCLS guidelines. Testing
was performed in U bottom 96 well tissue culture plates in YPG,
PD broth for fungal strains and nutrient broth for bacterial strains.
The concentration range of tested compounds and standard was
0.25 to 128 mg mL-1. The plates were incubated at 28 ◦C for all the
microorganisms (except at 37 ◦C for B. poitrasii). Absorbances at
600 nm were recorded to assess the inhibition of cell growth after
24 h for B. poitrasii and Y. lipolytica, 48 h for C. albicans and F.
oxysporum, 72 h for C. neoformans and 24 h for bacterial cultures.
MIC was determined as 90% inhibition of growth with respect to
the growth control and IC50 was the concentration at which 50%
growth inhibition was observed.
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