Design and synthesis of fluconazole/bile acid conjugate using click reaction

Article abstract of DOI:10.1016/j.tet.2006.09.021

Novel fluconazole/bile acid conjugates were designed and their regioselective synthesis was achieved in very high yield via Cu(I) catalyzed intermolecular 1,3-dipolar cycloaddition. These new molecules showed good antifungal activity against Candida speci

Full text of DOI:10.1016/j.tet.2006.09.021

Tetrahedron 62 (2006) 11178–11186
Design and synthesis of fluconazole/bile acid conjugate
using click reaction
a
b
b
Vandana S. Pore,a, Nilkanth G. Aher, Manish Kumar and Praveen K. Shukla
*
aDivision of Organic Chemistry Synthesis, National Chemical Laboratory, Pune 411008, Maharashtra, India
bDivision of Fermentation Technology, Central Drug Research Institute, Lucknow 226001, India
Received 23 June 2006; revised 24 August 2006; accepted 8 September 2006
Available online 2 October 2006
Abstract—Novel fluconazole/bile acid conjugates were designed and their regioselective synthesis was achieved in very high yield via Cu(I)
catalyzed intermolecular 1,3-dipolar cycloaddition. These new molecules showed good antifungal activity against Candida species.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
have been shown to accept and carry a variety of analogues
that are derivatized at different positions of bile acids.7 They
have beenusedasabsorptionenhancersandasnew cholesterol
lowering agents.7a A common feature of bile acid derived an-
timicrobials is its potential to exhibit facially amphiphilic na-
ture, due to polar hydroxyl groups on one face and nonpolar
hydrophobicmethylgroupsontheotherface.8 Polyenemacro-
lide amphotericin-B, peptide antimicrobial agent polymixin
B, and squalamine in the cyclic form show such amphiphilic-
ity and function as ionophores.9
The incidence of life threatening fungal infections has tre-
mendously increased in last two decades due to greater use
of immunosuppressive drugs, prolonged use of broad-spec-
trum antibiotics, wide-spread use of indwelling catheters,
and also in cancer and AIDS patients.1 The presently
marketed antifungal drugs are either highly toxic (ampho-
tericin-B) or becoming ineffective due to appearance of resis-
tant strains (flucytosine and azoles).2 Azole antifungals are
strong inhibitors of lanosterol 14a-demethylase, which is
major component of fungal cell membrane.3 Fluconazole
(Fig. 1) is an orally effective, potent, and safe triazole based
antifungal drug, with favorable pharmacokinetic characteris-
tics and low toxicity.4 Due to the emergence of new fungal
pathogens, resistant to fluconazole, great efforts have been
made to modify the chemical structure of fluconazole, in or-
derto broaden its antifungalactivityandincrease itspotency.5
Bioconjugation has recently emerged as a fast growing tech-
nology that affects almost every discipline of life science. It
aims at the ligation of two or more molecules to form new
complexes with the combined properties of its individual
components.10 In continuation of our work on bile acids,11
we designed new bioconjugates 3 (Scheme 1) of bile acids
N
N
N
+
N3
R2
R1
OH
R1
R2
N
N
1
2
3
N
N
F
N
N
R1 = Fluconazole part
Scheme 1.
R2 = Bile acid part
F
having amphiphilic nature as amphotericin-B and pharmaco-
phore of fluconazole, linked together with 1,2,3-triazole,
which may be viewed as an isoster of one of the 1,2,4-triazole
component of fluconazole. 1,2,3-Triazole moieties are attrac-
tive connecting units, since they are stable to metabolic deg-
radation and capable of hydrogen bonding, which can be
favorable in binding of biomolecular targets and for solubil-
ity.12 1,2,3-Triazole moiety does not occur in nature,
although the synthetic molecules containing 1,2,3-triazole
unit shows diverse biological activities including antibacte-
rial, herbicidal, fungicidal, antiallergic, and anti-HIV.13
Figure 1. Fluconazole.
In recent years bile acid structures have become increasingly
importantin anumberof fields, suchas pharmacology, biomi-
metic, and supramolecular chemistry.6 Bile acid transporters
Keywords: Bile acid; Fluconazole/bile acid conjugate; Click chemistry;
Antifungal agent; 1,2,3-Triazole.
* Corresponding author. Tel.: +91 20 25902320; fax: +91 20 25902624;
e-mail: vs.pore@ncl.res.in
0040–4020/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.09.021

V. S. Pore et al. / Tetrahedron 62 (2006) 11178–11186
11179
1,3-Dipolar cycloaddition of terminal acetylene and organic
azides has been a method of choice for the synthesis of 1,2,3-
triazoles.14
presence of acetylenic group was also evident from IR spec-
trum wherein the absorption due to acetylenic group was ob-
served at 3307 cmꢁ1
.
C-24-Azido bile acid derivatives 17 and 18 were synthesized
from the corresponding mesyl compounds 1311a and 15
(Scheme 3). IR of these compounds showed absorption
due to azido group at 2100 cmꢁ1. In proton NMR spectrum,
resonance corresponding to C-24-methylene group was
observed at d 3.24 ppm. Similarly, C-3-azido bile acid deriv-
atives 21 and 22 were synthesized according to the literature
procedures16 with small modifications. The presence of azido
group of the compounds 21 and 22 was also confirmed by
IR spectrum. All the four compounds are well characterized
by 1H NMR, 13C NMR, mass, and elemental analyses. The
cycloaddition of 6 to azido compound 17 was attempted un-
der previously reported conditions11a with copper sulfate
and sodium ascorbate in t-BuOH/H2O. This reaction failed
at low temperature and at 50–60 ꢀC, the reaction was slow
and after 3 days the product 23 was obtained in 10% yield
along with starting material. Although the result was encour-
aging, the reaction condition needed to be optimized.
Among the various reaction conditions, microwave assisted
Cu(I) catalyzed reaction was found to be suitable for this
conjugation.17 Under microwave irradiation compound 6
was reacted with C-24-azide 17 in DMF/H2O using catalytic
amount of Cu(I) to give fluconazole/bile acid conjugate 23
as a diastereomeric mixture in 92% yield (Scheme 4).
2. Results and discussion
In our approach to synthesize these new molecules, we con-
sidered performing Huisgen (click) reaction to connect flu-
conazole part containing terminal alkyne 1 and bile acid
containing terminal azide 2, in the presence of Cu(I) catalyst
to form fluconazole/bile acid conjugate 3 (Scheme 1).
Accordingly, we synthesized 2-(2,4-difluorophenyl)-1-(1H-
1,2,4-triazol-1-yl)pent-4-yn-2-ol 6 by propargylation of the
corresponding ketone 55b by using propargyl bromide and
zinc dust to obtain racemic compound 6 (Scheme 2).
OH
N
N
O
N
F
N
F
F
N
N
c
a, b
F
F
6
F
5
4
Scheme 2. Reagents and conditions: (a) AlCl3, 1,2-dichloroethane, chloro-
acetyl chloride, 25 ꢀC, 7 h; (b) 1,2,4-triazole, NaHCO3, toluene, reflux, 4 h
(overall 55% in a+b step); (c) Zn, propargyl bromide, DMF/THF, 25 ꢀC, 5 h,
95%.
The compound 6 constitutes an alkyne component. In the
proton NMR spectrum of compound 6, the acetylenic
proton was identified as triplet at d 2.06 ppm and doublet
of doublet at d 2.87 ppm obtained due to b methylene. In
the 13C NMR spectrum it showed intricacies of various
19F–13C coupling as in fluconazole molecule.15 The
In the proton NMR spectrum of compound 23, resonance
corresponding to C-24-methylene protons was identified at
d 3.15 ppm and 3.48 ppm (two doublets) and the proton of
1,2,3-triazole was noticed at d 7.19 ppm. Methylene at C-4
position of 1,2,3-triazole was identified at d 4.20 ppm. In
the IR spectrum, compound 23 showed absorption due to
O
O
OH
OH
OH
OH
OMe
HO
OH
b
c
a
HO
HO
R
R
R
H
H
H
9 R = H
10 R = OH
7 R = H
8 R = OH
11 R = H
12 R = OH
OH
OH
OMs
N3
d
13/15
R1O
R
HO
R
H
17 R = H
H
13 R = H, R1 = H
14 R = H, R1 = OMs
15 R = OH, R1 = H
16 R = OH, R1 = OMs
18 R = OH
COOCH3
COOCH3
OH
OH
R
e
f
9/10
MsO
R
N3
H
H
21 R = H
19 R = H
20 R = OH
22 R = OH
Scheme 3. Reagents and conditions: (a) p-TsOH/MeOH, 25 ꢀC, 24 h, 95–96%; (b) LAH/THF, 25 ꢀC, 2 h, 93–97%; (c) (i) 11, MsCl, Et3N, CH2Cl2, 0 ꢀC,
10 min or (ii) 12, MsCl, pyridine, 0 ꢀC, 10 min; (d) NaN3, DMF, 60 ꢀC, 3 h, 93–94%; (e) MsCl, Et3N, CH2Cl2, 0 ꢀC, 10 min; (f) NaN3, DMF, 60 ꢀC, 3 h,
90–91%.

11180
V. S. Pore et al. / Tetrahedron 62 (2006) 11178–11186
N
OH
N
N
F
OH
N
6
17/18
+
HO
R
F
N
H
23 R = H
24 R = OH
N
COOCH3
OH
F
+
21/22
6
F
R
N
N
H
N
N
N
N
OH
25 R = H
26 R = OH
Scheme 4. Reagents and conditions: CuSO4$5H2O (5 mol %), sodium ascorbate (40 mol %), DMF/H2O (9:1), microwave, 5 min, 90–95%.
hydroxyl group at 3391 cmꢁ1. In addition, it gave satisfac-
tory elemental analysis and in mass spectrum it showed mo-
lecular ion peak at 667.34 (M+1).
All these fluconazole/bile acid bioconjugates showed very
good antifungal activity against Candida species (Table 1).
For the comparison of biological activity we synthesized
bile acid azoles conjugates 27a–d containing imidazole,
benzimidazole, triazole, and benzotriazole at C-24 position
from C-24-monomesyl compound 13 (Scheme 5). However,
We then extrapolated the ligation protocol successfully to
other bile acid derived azides, 18, 21, and 22 and synthesized
fluconazole/bile acid conjugates 24, 25, and 26 (Scheme 4).
Table 1. In vitro antifungal activity for compounds 23, 24, 25, 26, and 27a–d
Compound
Inhibitory concentration in mg/mL against
1
2
3
4
5
6
MIC
3.12
IC50
2.11
MIC
12.5
50
25
IC50
11.34
44.76
22.23
48.42
MIC
6.25
25
25
IC50
6.11
12.34
23.34
2.64
MIC
12.5
25
25
IC50
10.58
24.82
22.48
21.46
MIC
IC50
MIC
6.25
IC50
5.82
23
24
25
26
27a
27b
27c
27d
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
6.25
6.25
6.25
4.58
5.72
3.44
6.25
6.25
3.12
5.16
5.48
2.18
50
3.12
25
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
Amphotericin-B
Fluconazole
0.12
0.5
0.09
0.13
0.06
1.0
0.04
0.46
0.12
2.0
0.08
1.06
0.12
1.0
0.09
0.63
0.5
2.0
0.38
1.06
0.12
1.0
0.11
0.21
1. Candida albicans, 2. Cryptococcus neoformans, 3. Sporothrix schenckii, 4. Trichophyton mentagrophytes, 5. Aspergillus fumigatus, 6. Candida parapsilosis
(ATCC-22019).
OH
R
OH
OMs
HO
HO
N
H
N
N
27a. R =
27b. R =
H
13
27 (a-d)
N
N
N
27c. R =
27d. R =
N
N
N
N
Scheme 5. Reagents and conditions: RH, NaH, DMF, 25 ꢀC, 12–20 h, 75–95%.

V. S. Pore et al. / Tetrahedron 62 (2006) 11178–11186
11181
all these compounds showed very weak antifungal activity
(Table 1).
step); mp¼104–106 ꢀC (lit.5b 103–105 ꢀC); IR (cmꢁ1): 1703,
1614, 1593; 1H NMR (CDCl3, 200 MHz): d 5.59 (d,
J¼3.54 Hz, 2H), 6.93–7.10 (m, 2H), 7.99–8.11 (m, 2H), 8.21
(br s, 1H); 13C NMR (CDCl3, 50 MHz): d 58.2 (dd,
2
3. Conclusion
4JCF¼14.09 Hz), 104.8 (dd, JCF¼25.66 Hz), 112.9 (dd,
2JCF¼21.63 Hz, 4JCF¼3.02 Hz), 118.8 (dd, 2JCF¼14.09 Hz,
3
In summary we have designed and synthesized fluconazole/
bile acid conjugates at C-3 and C-24 positions of bile acids
under microwave assisted Cu(I) catalyzed cycloaddition re-
action. This reaction gave fluconazole/bile acid conjugates,
linked with 1,4-disubstituted 1,2,3-triazole regioselectively,
in excellent yield and in less reaction time. These new
molecules showed very good antifungal activity against
Candida albicans, Sporothrix schenckii, and Candida para-
psilosis having MIC ranging from 3.12 to 6.25 mg/mL. It is
thought that in this biological activity, bile acid part acts
as a drug carrier and fluconazole part acts as an inhibitor
of 14a-demethylase enzymes in fungal cell. Work to clarify
the role of bile acid is underway in our laboratory. This is the
first report of use of ‘click chemistry’ for the modification of
fluconazole.
4JCF¼3.52 Hz), 132.9 (dd, JCF¼4.52, 10.81 Hz), 144.8,
1
3
151.7, 163.0 (dd, JCF¼256.38 Hz, JCF¼13.08 Hz),
1
3
166.6 (dd, JCF¼259.9 Hz, JCF¼12.58 Hz), 187.6 (d,
3JCF¼5.53 Hz). Anal. Calcd for C10H7F2N3O: C, 53.82; H,
3.16; F, 17.03; N, 18.83. Found: C, 54.10; H, 3.02; F,
16.87; N, 18.71; MS (LC–MS) m/z: 224.57 (M+1), 246.57
(M+23 for Na).
4.2.2. 2-(2,4-Difluorophenyl)-1-(1H-1,2,4-triazole-1-yl)-
pent-4-yn-2-ol (6). The ketone 5 (0.500 g, 2.24 mmol)
and propargyl bromide (4 mL, 6.73 mmol) were dissolved
in a mixed solvent DMF/THF 1:1 (10 mL). To this well
stirred solution, activated zinc dust (washed with 2% HCl
and water and dried in vacuum) (0.439 g, 6.73 mmol) was
slowly added at room temperature.18 After 5 min exother-
mic reaction brought itself to reflux, which was allowed
to attain 25 ꢀC. The whole reaction mixture was then stirred
for 5 h at 25 ꢀC. Ice-cold HCl solution (5%) was added to
the reaction mixture and it was extracted with EtOAc,
and washed with water and brine. Solvent was evaporated
under reduced pressure to afford crude product, which
was purified by column chromatography on silica gel (5%
MeOH/DCM) to produce compound 6 (0.551 g) as a white
solid.
4. Experimental
4.1. General experimental techniques and apparatus
TLC was performed on precoated silica gel F254 plates
(0.25 mm; E. Merck) and product(s) and starting material(s)
were detected by either viewing under UV light or treating
with an ethanolic solution of phosphomolybdic acid or ani-
saldehyde spray followed by heating. Column chromato-
graphy was performed on neutral deactivated aluminum
oxide. Optical rotations were obtained on Bellingham &
Stanley ADP-220 Polarimeter. Specific rotations ([a]D) are
reported in deg/dm and the concentration (c) is given in
g/100 mL in the specified solvent. Infrared spectra were re-
corded in CHCl3 as a solvent on Schimadzu 8400 series
FTIR instrument. 1H NMR spectra were recorded on a
Brucker AC-200 and 400 spectrometers at 200.13 and
400.13 MHz and 13C NMR spectra were recorded on
a Brucker AC-200 at 50.32 MHz. The chemical shifts are
given in parts per million relative to tetramethylsilane.
Mass spectra were recorded on LC–MS/MS-TOF API
QSTAR PULSAR spectrometer, and samples were intro-
duced by infusion method using Electrospray Ionisation
Technique. Elemental analyses were performed by CHNS-
O EA 1108-Elemental analyser, Carloerba Instrument (Italy)
or Elementar vario EL (Germany) and were within ꢂ0.4%
of calculated values. Melting points were determined on
a Thermonik Campbell melting point apparatus and were
uncorrected. Microwave irradiation was carried out in an
open glass vessel using a domestic microwave oven (800 W,
BPL-make). Standard work up: after extraction of all the re-
actions, the organic extracts were washed with water and
brine and dried over anhydrous Na2SO4 and concentrated
in vacuum.
1
Yield 95%; mp¼145–146 ꢀC; IR (cmꢁ1): 3272, 3137; H
NMR (CDCl3, 200 MHz): d 2.06 (t, J¼2.65 Hz, 1H), 2.86
(dd, J¼16.01, 2.65 Hz, 1H ABX pattern), 2.92 (dd, J¼18.01,
2.65 Hz, 1H ABX pattern), 4.13 (br s, 1H, OH), 4.72 and
4.82 (two d, J¼14.01 Hz, 2H AB pattern), 6.73–6.87 (m,
2H), 7.50–7.59 (m, 1H), 7.87 (s, 1H), 8.20 (s, 1H); 13C NMR
4
(CDCl3+CD3OD, 50 MHz): d 29.1 (d, JCF¼5.03 Hz),
4
3
56.3 (d, JCF¼5.03 Hz), 71.9, 73.4 (d, JCF¼4.53 Hz),
2
78.1, 103.9 (dd, JCF¼26.17, 27.17 Hz), 111.1 (dd,
2JCF¼20.63 Hz, JCF¼3.52 Hz), 124.2 (dd, JCF¼13.08 Hz,
4
2
4JCF¼3.52 Hz), 129.6 (dd, JCF¼6.04, 9.56 Hz), 144.2,
3
1
3
150.4, 158.6 (dd, JCF¼246.58 Hz, JCF¼12.08 Hz), 162.6
1
3
(dd, JCF¼249.59 Hz, JCF¼12.08 Hz). Anal. Calcd for
C13H11F2N3O: C, 59.31; H, 4.21; F, 14.43; N, 15.96. Found:
C, 59.45; H, 4.13; F, 14.31; N, 15.87; MS (LC–MS) m/z:
264.06 (M+1), 286.05 (M+23 for Na).
4.3. Synthesis of terminal azide
4.3.1. Synthesis of methyl 3a,12a-dihydroxy-5b-cholane-
24-oate (9) and methyl 3a,7a,12a-trihydroxy-5b-
cholane-24-oate (10). Compounds 9 and 10 were synthe-
sized in overall good yield starting from cholic acid 7 and
deoxycholic acid 8 using the literature procedure.11e
4.3.1.1. Methyl 3a,12a-dihydroxy-5b-cholane-24-oate
(9). White solid; mp¼82–105 ꢀC (lit.19a 70–108 ꢀC); IR
1
4.2. Synthesis of terminal acetylene
(cmꢁ1): 3385, 1728; H NMR (CDCl3, 500 MHz): d 0.68
(s, 3H, CH3-18), 0.91 (s, 3H, CH3-19), 0.98 (d,
J¼6.36 Hz, 3H, CH3-21), 3.62 (m, 1H, CH-3), 3.67 (s,
3H), 3.98 (br s, 1H, CH-12); 13C NMR (CDCl3, 50 MHz):
d 12.6, 17.1, 23.0, 23.6, 26.0, 27.1, 27.4, 28.4, 29.6, 30.1,
4.2.1. 1-(2,4-Difluorophenyl)-2-(1H-1,2,4-triazol-1-yl)
ethanone (5). Compound 5 was synthesized from 4 using lit-
erature procedure.5b White solid, yield 55% (overall in a+b

11182
V. S. Pore et al. / Tetrahedron 62 (2006) 11178–11186
30.8, 31.0, 33.4, 34.0, 35.2, 35.9, 36.2, 42.0, 46.3, 47.0, 48.0,
51.4, 71.4, 72.9, 174.7.
d 0.68 (s, 3H, CH3-18), 0.92 (s, 3H, CH3-19), 1.00 (d,
J¼6.26 Hz, 3H, CH3-21), 3.00 (s, 3H), 3.01 (s, 3H), 4.00
(s, 1H, CH-12), 4.21 (t, J¼6.66 Hz, 2H), 4.65 (m, 1H,
CH-3); 13C NMR (CDCl3, 50 MHz): d 12.6, 17.4, 22.7,
23.4, 25.7, 25.8, 26.6, 27.4, 27.5, 28.5, 31.2, 33.1, 33.4,
33.7, 34.7, 34.9, 35.7, 37.2, 38.7, 41.9, 46.3, 47.1, 47.9,
70.6, 72.7, 82.7. Anal. Calcd for C26H46O7S2: C, 58.39; H,
8.67; S, 11.99. Found: C, 58.22; H, 8.39; S, 12.16; MS
(LC–MS) m/z: 557.32 (M+23 for Na).
4.3.1.2. Methyl 3a,7a,12a-trihydroxy-5b-cholane-
24-oate (10). White solid; mp¼155–156 ꢀC (lit.19b
1
155–156 ꢀC); IR (cmꢁ1): 3376, 1731; H NMR (CDCl3,
200 MHz): d 0.68 (s, 3H, CH3-18), 0.89 (s, 3H, CH3-19),
0.98 (d, J¼5.69 Hz, 3H, CH3-21), 3.57 (m, 1H, CH-3),
3.67 (s, 3H), 3.89 (br s, 1H, CH-7), 4.01 (br s, 1H, CH-12);
13C NMR (CDCl3, 50 MHz): d 12.8, 17.7, 22.8, 23.6, 26.6,
27.7, 28.5, 30.0, 31.4, 31.5, 35.2, 35.2, 35.7, 35.8, 39.9,
39.9, 42.0, 42.0, 46.8, 47.3, 51.8, 68.8, 72.2, 73.4, 175.2.
4.3.4. Synthesis of 3a,7a,12a-trihydroxy 24-mesyloxy-
5b-cholane (15) and 3a,24-dimesyloxy-7a,12a-dihydroxy-
5b-cholane (16). To a solution of 12 (0.394 g, 1 mmol) in
dry pyridine (5 mL), methane sulfonyl chloride (0.12 mL,
1.5 mmol) was added to the reaction mixture. After 10 min,
ice was added and it was extracted with EtOAc. Organic layer
was washed with cold water, cold-HCl (5%), water, and brine
and dried over Na2SO4. Solvent was evaporated under reduced
pressure and the crude product was purified by column
chromatography (10% MeOH/CH2Cl2) to obtain pure 15
(0.329 g) and pure 16 (0.125 g).
4.3.2. Synthesis of 3a,12a,24-trihydroxy-5b-cholane (11)
and 3a,7a,12a,24-tetrahydroxy-5b-cholane (12). Com-
pounds 11 and 12 were synthesized in overall good yield
starting from methyl esters of cholic acid 9 and deoxycholic
acid 10 using the literature procedure.19c
4.3.2.1. 3a,12a,24-Trihydroxy-5b-cholane (11). White
solid; mp¼123–124 ꢀC (lit.19d 107–114 ꢀC, lit.19e 123 ꢀC);
1
IR (cmꢁ1): 3257; H NMR (CDCl3, 200 MHz): d 0.69 (s,
3H), 0.91 (s, 3H), 0.99 (d, J¼6.87 Hz, 3H), 3.61 (m, 3H),
4.00 (br s, 1H); 13C NMR (CDCl3, 50 MHz): d 12.7, 17.7,
23.1, 23.7, 26.2, 27.2, 27.6, 28.5, 29.4, 30.4, 31.8, 33.6,
34.1, 35.3, 35.4, 36.1, 36.4, 42.1, 46.5, 47.5, 48.2, 63.4,
71.8, 73.3.
4.3.4.1. 3a,7a,12a-Trihydroxy 24-mesyloxy-5b-chol-
ane (15). White solid; mp¼79–81 ꢀC; [a]D26 +68.49 (c 0.9,
1
CHCl3); IR (cmꢁ1): 3408; H NMR (CDCl3, 200 MHz):
d 0.68 (s, 3H, CH3-18), 0.89 (s, 3H, CH3-19), 0.99 (d,
J¼6.44 Hz, 3H, CH3-21), 3.01 (s, 3H), 3.44 (m, 1H,
CH-3), 3.84 (s, 1H, CH-7), 3.97 (s, 1H, CH-12), 4.21 (t,
J¼6.57 Hz, 2H); 13C NMR (CDCl3, 50 MHz): d 12.3,
17.5, 20.8, 22.3, 23.1, 25.8, 26.2, 27.5, 28.0, 29.6, 30.1,
31.3, 34.5, 34.7, 35.1, 37.4, 39.2, 39.3, 41.3, 41.5, 46.3,
46.9, 68.4, 70.8, 71.9, 73.1. Anal. Calcd for C25H44O6S: C,
63.52; H, 9.38; S, 6.78. Found: C, 63.21; H, 9.12; S, 6.53;
MS (LC–MS) m/z: 473.91 (M+1), 495.89 (M+23 for Na).
4.3.2.2. 3a,7a,12a,24-Tetrahydroxy-5b-cholane (12).
1
Mp¼236–238 ꢀC (lit.19c 236.5–238 ꢀC); IR, H NMR, and
13C NMR spectroscopic data are consistent that reported in
literature.19c
4.3.3. Synthesis of 3a,12a-dihydroxy 24-mesyloxy-5b-
cholane (13) and 3a,24-dimesyloxy-12a-hydroxy-5b-
cholane (14). To a solution of 11 (2.000 g 5.28 mmol) in
dry CH2Cl2 (20 mL) was added triethylamine (1.5 mL,
0.56 mmol) at 0 ꢀC. Methane sulfonyl chloride (0.53 mL,
6.86 mmol in 10 mL CH2Cl2) was added dropwise in
10 min at 0 ꢀC, and ice was added to the reaction mixture im-
mediately after addition was complete. The reaction mixture
was extracted with CH2Cl2. Organic layer was washed with
NaHCO3, water, and brine. Solvent was evaporated under
reduced pressure. The crude product was purified by col-
umn chromatography (0.5% MeOH/CH2Cl2) to obtain pure
products 13 (1.805 g) and 14 (0.615 g).
4.3.4.2. 3a,24-Dimesyloxy-7a,12a-dihydroxy-5b-chol-
ane (16). White solid; mp¼82–84 ꢀC; [a]D25 +29.85 (c 0.8,
1
CHCl3); IR (cmꢁ1): 3434; H NMR (CDCl3, 200 MHz):
d 0.69 (s, 3H, CH3-18), 0.91 (s, 3H, CH3-19), 0.99 (d,
J¼6.44 Hz, 3H, CH3-21), 2.99 (s, 3H), 3.01 (s, 3H), 3.87
(br s, 1H), 4.00 (s, 1H, CH-12), 4.21 (t, J¼6.69 Hz, 2H),
4.51 (m, 1H, CH-3); 13C NMR (CDCl3, 50 MHz): d 12.3,
17.5, 22.1, 22.7, 23.0, 26.3, 27.4, 27.8, 28.0, 31.3, 34.2,
34.4, 34.7, 35.1, 35.9, 37.2, 38.7, 39.2, 41.3, 41.6, 46.3,
47.1, 68.0, 70.7, 72.8, 83.0. Anal. Calcd for C26H46O8S2:
C, 56.70; H, 8.42; S, 11.64. Found: C, 56.84; H, 8.26; S,
11.81; MS (LC–MS) m/z: 573.51 (M+23 for Na).
4.3.3.1. 3a,12a-Dihydroxy 24-mesyloxy-5b-cholane
(13). White solid; mp¼78 ꢀC; [a]D26 +93.04 (c 0.2, CHCl3);
4.3.5. Synthesis of 3a,12a-dihydroxy 24-azido-5b-chol-
ane (17) and 3a,7a,12a-trihydroxy 24-azido-5b-cholane
(18). To a solution of 13 (0.300 g, 0.66 mmol) in dry DMF
(10 mL), sodium azide (0.214 g, 3.28 mmol) was added
and stirring was continued at 60–65 ꢀC for 3–5 h. The re-
action mixture was allowed to cool to room temperature. It
was then poured into ice-cold water (30 mL) and extracted
with EtOAc. The organic extract was washed with cold
water and brine. Solvent was evaporated under reduced
pressure to afford crude product 17, which was purified by
column chromatography on silica gel (10% EtOAc/hexane)
to produce pure compound 17 as a white solid (0.247 g).
IR (cmꢁ1): 3419, 1416, 1448 cmꢁ1 1H NMR (CDCl3,
;
300 MHz): d 0.68 (s, 3H, CH3-18), 0.91 (s, 3H, CH3-19),
0.99 (d, J¼6.26 Hz, 3H, CH3-21), 3.00 (s, 3H), 3.60 (m,
1H, CH-3), 3.97 (s, 1H, CH-12), 4.19 (t, J¼6.66 Hz, 2H);
13C NMR (CDCl3, 50 MHz): d 12.6, 17.4, 23.0, 23.6, 25.9,
26.0, 27.0, 27.5, 28.6, 30.3, 31.4, 33.5, 34.0, 35.0, 35.2,
35.9, 36.3, 37.3, 42.0, 46.4, 47.3, 48.1, 70.5, 71.6, 73.0.
Anal. Calcd for C25H44O5S: C, 65.75; H, 9.71; S, 7.02.
Found: C, 65.45; H, 9.53; S, 7.28; MS (LC–MS) m/z:
457.19 (M+1), 479.13 (M+23 for Na).
4.3.3.2. 3a,24-Dimesyloxy-12a-hydroxy-5b-cholane
(14). White solid; mp¼67–68 ꢀC; [a]D27 +43.25 (c 2.7,
4.3.5.1. 3a,12a-Dihydroxy 24-azido-5b-cholane (17).
White solid, yield 94%; mp¼126 ꢀC; [a]D28 +40.57 (c 1.4,
1
CHCl3); IR (cmꢁ1): 3566; H NMR (CDCl3, 300 MHz):

V. S. Pore et al. / Tetrahedron 62 (2006) 11178–11186
11183
CHCl3); IR (cmꢁ1): 2090, 3409; 1H NMR (CDCl3,
200 MHz): d 0.69 (s, 3H, CH3-18), 0.91 (s, 3H, CH3-19),
0.99 (d, J¼6.65 Hz, 3H, CH3-21), 3.24 (t, J¼7.05 Hz, 2H),
3.62 (m, 1H, CH-3), 4.00 (br s, 1H, CH-12); 13C NMR
(CDCl3, 50 MHz): d 12.7, 17.6, 23.1, 23.6, 25.6, 26.1,
27.1, 27.5, 28.7, 30.5, 32.9, 33.7, 34.1, 35.3, 36.1, 36.5,
42.2, 46.5, 47.5, 48.3, 51.9, 71.7, 73.2. Anal. Calcd for
C24H41N3O2: C, 71.42; H, 10.24; N, 10.41. Found: C,
71.19; H, 10.46; N, 10.38; MS (LC–MS) m/z: 404.88
(M+1), 426.83 (M+23 for Na).
4.3.7.1. Methyl-3b-azido-12a-hydroxy-5b-cholane-
24-oate (21). White solid, yield 91%; mp¼127–128 ꢀC
(lit.16a 128 ꢀC); [a]D27 +41.34 (c 0.8, CHCl3); IR (cmꢁ1):
1
1728, 2102, 3503; H NMR (CDCl3, 200 MHz): d 0.68 (s,
3H), 0.94 (s, 3H), 0.97 (d, J¼6.20 Hz, 3H), 3.66 (s, 3H),
3.94 (br s, 1H), 3.99 (br s, 1H); 13C NMR (CDCl3,
50 MHz): d 12.7, 17.3, 23.5, 23.5, 24.5, 25.9, 26.4, 27.4,
28.8, 30.1, 30.5, 30.8, 31.0, 33.2, 34.4, 35.0, 35.8, 37.2,
46.5, 47.3, 48.3, 51.4, 58.7, 73.1, 174.6. Anal. Calcd for
C25H41N3O3: C, 69.57; H, 9.57; N, 9.74. Found: C, 69.47;
H, 9.90; N, 9.66; MS (LC–MS) m/z: 432.26 (M+1), 454.25
(M+23 for Na).
4.3.5.2. 3a,7a,12a-Trihydroxy 24-azido-5b-cholane
(18). Compound 18 was prepared by similar procedure
from compound 15. White solid, yield 93%; mp¼160 ꢀC;
4.3.7.2. Methyl-3b-azido-7a,12a-dihydroxy-5b-chol-
ane-24-oate (22). White solid, yield 90%; mp¼169–
170 ꢀC (lit.16b 157 ꢀC); [a]2D7 +22.45 (c 1.16, MeOH)
(lit.16b +23.7); IR (cmꢁ1): 1728, 2098, 3439; 1H NMR
(CDCl3, 200 MHz): d 0.70 (s, 3H), 0.93 (s, 3H), 0.97 (d,
J¼6.06 Hz, 3H), 3.67 (s, 3H), 3.86–3.89 (br s, 2H), 3.99
(br s, 1H); 13C NMR (CDCl3, 50 MHz): d 12.4, 17.2, 22.7,
23.2, 24.5, 26.0, 27.4, 28.3, 30.4, 30.8, 31.0, 33.0, 34.2,
35.1, 35.2, 36.7, 39.3, 41.7, 46.5, 47.2, 51.5, 58.7, 68.4,
73.0, 174.7. Anal. Calcd for C25H41N3O4: C, 67.08; H,
9.23; N, 9.39. Found: C, 67.21; H, 9.18; N, 9.31; MS
(LC–MS) m/z: 448.24 (M+1), 470.22 (M+23 for Na).
1
[a]2D5 +31.46 (c 0.8, CHCl3); IR (cmꢁ1): 2098, 3410; H
NMR (CDCl3, 200 MHz): d 0.68 (s, 3H), 0.89 (s, 3H),
0.99 (d, J¼6.32 Hz, 3H), 3.25 (t, J¼6.57 Hz, 2H), 3.52 (m,
1H), 3.87 (br s, 1H), 4.00 (br s, 1H), 4.50 (br s, 3H, OH);
13C NMR (CDCl3, 50 MHz): d 12.4, 17.6, 22.4, 23.1, 25.6,
26.2, 27.6, 28.1, 30.2, 32.8, 34.6, 34.7, 35.3, 35.3, 39.3,
39.4, 41.4, 41.5, 46.3, 47.1, 51.9, 68.4, 71.8, 73.1. Anal.
Calcd for C24H41N3O3: C, 68.70; H, 9.85; N, 10.01. Found:
C, 68.65; H, 9.69; N, 9.94; MS (LC–MS) m/z: 420.24 (M+1),
442.23 (M+23 for Na).
4.3.6. Synthesis of methyl-3a-mesyloxy-12a-hydroxy-
5b-cholane-24-oate (19) and methyl-3a-mesyloxy-7a-
12a-dihydroxy-5b-cholane-24-oate (20). Compounds
19 and 20 were prepared from compounds 9 and 10 by
using similar procedure as used for the preparation of com-
pound 13.
4.4. General procedure for cycloaddition (23–26)
The alkyne 6 (1 equiv) and the azide 17, 18, 21, or 22
(1.3 equiv) were dissolved in DMF/H2O 4:1 (5 mL). To
this solution, CuSO4$5H2O (0.05 equiv) and sodium ascor-
bate (0.40 equiv) were added. The reaction mixture was
placed in a domestic microwave reactor and irradiated for
5 min at 415 W. The reaction mixture was cooled, ice was
added, and it was then extracted with EtOAc. The extract
was washed with water and brine. Solvent was evaporated
under reduced pressure and crude product was purified
by column chromatography on silica gel using 5%
MeOH/CH2Cl2 system to obtain fluconazole/bile acid con-
jugates 23, 24, 25, or 26 linked with 1,4-disubstituted
1,2,3-triazole.
4.3.6.1. Methyl-3a-mesyloxy-12a-hydroxy-5b-chol-
ane-24-oate (19). White solid, yield 89%; mp¼62–63 ꢀC;
1
[a]2D7 +45.77 (c 4.1, CHCl3); IR (cmꢁ1): 1728, 3549; H
NMR (CDCl3, 200 MHz): d 0.67 (s, 3H), 0.91 (s, 3H),
0.97 (d, J¼6.06 Hz, 3H), 2.99 (s, 3H), 3.65 (s, 3H), 3.98
(br s, 1H), 4.64 (m, 1H); 13C NMR (CDCl3, 50 MHz):
d 12.3, 16.8, 22.5, 23.3, 25.6, 26.5, 27.1, 27.3, 28.3, 30.5,
30.6, 32.9, 33.1, 33.5, 34.5, 34.8, 35.5, 38.4, 41.7, 46.1,
46.7, 47.6, 51.1, 72.3, 82.5, 174.3. Anal. Calcd for
C26H44O6S: C, 64.43; H, 9.15; S, 6.62. Found: C, 64.58;
H, 9.02; S, 6.73; MS (LC–MS) m/z: 485.23 (M+1), 507.22
(M+23 for Na).
4.4.1. 3a,12a-Dihydroxy-24-[(4-(2-(2,4-difluorophenyl)-
2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)-1H-1,2,3-
triazol-1-yl)]-5b-cholane (23). White solid, yield 95%;
1
4.3.6.2. Methyl-3a-mesyloxy-7a-12a-dihydroxy-5b-
cholane-24-oate (20). White solid, yield 87%; mp¼83–
85 ꢀC; [a]D28 +29.98 (c 0.9, CHCl3); IR (cmꢁ1): 1728,
3460; 1H NMR (CDCl3, 200 MHz): d 0.69 (s, 3H), 0.91 (s,
3H), 0.99 (d, J¼6.06 Hz, 3H), 2.99 (s, 3H), 3.67 (s, 3H),
3.88 (br s, 1H), 4.00 (br s, 1H), 4.51 (m, 1H); 13C NMR
(CDCl3, 50 MHz): d 12.4, 17.2, 22.1, 23.0, 26.3, 27.4,
27.8, 28.0, 30.7, 30.9, 34.1, 34.4, 34.7, 35.1, 35.9, 38.7,
39.3, 41.3, 41.6, 46.4, 47.0, 51.4, 68.0, 72.8, 82.9, 174.7.
Anal. Calcd for C26H44O7S: C, 62.37; H, 8.86; S, 6.40.
Found: C, 62.23; H, 8.92; S, 6.23; MS (LC–MS) m/z:
501.07 (M+1), 523.17 (M+23 for Na).
mp¼169–171 ꢀC; IR (cmꢁ1): 1597, 1618, 3391; H NMR
(CDCl3, 200 MHz): d 0.65 (s, 3H), 0.90–0.93 (6H), 3.15
and 3.48 (two doublets, J¼14.90 Hz, 2H, adjacent to C-4
end of 1,2,3-triazole), 3.62 (m, 1H), 3.95 (br s, 1H), 4.20
(t, J¼6.82 Hz, 2H), 4.53–4.74 (two doublets, J¼14.15 Hz,
2H), 5.55 (1H, OH), 6.70–6.80 (m, 2H), 7.19 (br s, 1H),
7.33–7.46 (m, 1H), 7.81 (br s, 1H), 8.16 (br s, 1H). Anal.
Calcd for C37H52F2N6O3: C, 66.64; H, 7.86; F, 5.70; N,
12.60. Found: C, 66.81; H, 7.77; F, 5.51; N, 12.55; MS
(LC–MS) m/z: 667.34 (M+1).
4.4.2. 3a,7a,12a-Trihydroxy-24-(4-(2-(2,4-difluoro-
phenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)-1H-
1,2,3-triazol-1-yl)-5b-cholane (24). White solid, yield
4.3.7. Synthesis of methyl-3b-azido-12a-hydroxy-5b-
cholane-24-oate (21) and methyl-3b-azido-7a,12a-
dihydroxy-5b-cholane-24-oate (22). The compounds 19
and 20 were reacted with NaN3 (3 equiv) in DMF for 4 h
at 80–90 ꢀC to give compounds 21 and 22.20
93%; mp¼118–121 ꢀC; IR (cmꢁ1): 1597, 1618, 3404; H
1
NMR (CDCl3, 400 MHz): d 0.65 (s, 3H), 0.88 (s, 3H),
0.93 (d, J¼6.52 Hz, 3H), 3.17 (d, J¼14.15 Hz, 1H adjacent
to 1,2,3-triazole), 3.42–3.49 (m, 2H, C3-H and 1H adjacent

11184
V. S. Pore et al. / Tetrahedron 62 (2006) 11178–11186
to 1,2,3-triazole), 3.83 (br s, 1H), 3.94 (br s, 1H), 4.19 (m,
2H), 4.58 and 4.72 (two doublets, J¼14.06 Hz, 2H, adjacent
to 1,2,4-triazole), 6.69–6.78 (m, 2H), 7.20 (br s, 1H), 7.42
(m, 1H), 7.83 (br s, 1H), 8.20 (br s, 1H). Anal. Calcd for
C37H52F2N6O4: C, 65.08; H, 7.68; F, 5.56; N, 12.31. Found:
C, 64.93; H, 7.66; F, 5.39; N, 12.45; MS (LC–MS) m/z:
683.29 (M+1), 705.27 (M+23 for Na).
4.5.2. 3a,12a-Dihydroxy-24-(1H-benzo[d]imidazol-1-yl)-
5b-cholane (27b). White solid, yield 91%; mp¼118 ꢀC;
[a]2D7 +42.77 (c 1.0, CHCl3); IR (cmꢁ1): 3346, 1643, 1616;
1H NMR (CDCl3, 300 MHz): d 0.65 (s, 3H), 0.90 (s, 3H),
0.95 (d, J¼6.60 Hz, 3H), 3.60 (m, 1H), 3.96 (br s, 1H),
4.13 (t, J¼6.60 Hz, 2H), 7.26–7.32 (m, 2H), 7.38–7.41 (m,
1H), 7.79–7.82 (m, 1H), 7.90 (s, 1H); 13C NMR (CDCl3,
50 MHz): d 12.5, 17.4, 23.0, 23.5, 26.0, 26.4, 27.0, 27.4,
28.7, 30.4, 32.9, 33.5, 34.0, 35.1, 35.2, 35.9, 36.3, 42.0,
45.4, 46.3, 47.0, 48.1, 71.4, 72.8, 109.5, 120.1, 121.9,
122.7, 133.7, 142.7, 143.5. Anal. Calcd for C31H46N2O2: C,
77.78; H, 9.69; N, 5.85. Found: C, 77.64; H, 9.54; N, 5.77;
MS (LC–MS) m/z: 479.26 (M+1), 501.20 (M+23 for Na).
4.4.3. Methyl-3b-[(4-(2-(2,4-difluorophenyl)-2-hydroxy-
3-(1H-1,2,4-triazol-1-yl)propyl)-1H-1,2,3-triazol-1-yl)]-
12a-hydroxy-5b-cholane-24-oate (25). White solid, yield
1
90%; mp¼183–185 ꢀC; IR (cmꢁ1): 1618, 1728, 3362; H
NMR (CDCl3, 200 MHz): d 0.67 (s, 3H), 0.80 (s, 3H),
0.96 (d, J¼5.93 Hz, 3H), 3.19 and 3.54 (two doublets,
J¼16.29 Hz, 2H, adjacent to 1,2,3-triazole), 3.65 (s, 3H),
4.00 (br s, 1H), 4.54 (br s, 1H), 4.73 (br s, 2H, adjacent to
1,2,4-triazole), 6.63–6.80 (m, 2H), 7.23–7.35 (m, 2H),
7.86 (br s, 1H), 8.46 (br s, 1H). Anal. Calcd for
C38H52F2N6O4: C, 65.68; H, 7.54; F, 5.47; N, 12.09. Found:
C, 65.43; H, 7.67; F, 5.24; N, 11.93; MS (LC–MS) m/z:
695.35 (M+1), 717.31 (M+23 for Na).
4.5.3. 3a,12a-Dihydroxy-24-(1H-1,2,4-triazol-1-yl)-5b-
cholane (27c). White solid, yield 95%; mp¼196–197 ꢀC;
1
[a]D26 +45.33 (c 0.7, CHCl3); IR (cmꢁ1): 3421; H NMR
(CDCl3, 200 MHz): d 0.66 (s, 3H), 0.90 (s, 3H), 0.98 (d,
J¼6.26 Hz, 3H), 3.61 (m, 1H), 3.97 (br s, 1H), 4.13 (t,
J¼6.65 Hz, 2H), 7.94 (s, 1H), 8.05 (s, 1H); 13C NMR
(CDCl3, 50 MHz): d 12.4, 17.1, 22.8, 23.4, 25.9, 26.2,
26.9, 27.4, 28.3, 26.6, 32.2, 33.3, 33.8, 35.0, 35.0, 35.7,
35.7, 41.8, 46.1, 46.8, 47.8, 50.1, 71.2, 72.7, 142.6, 151.0.
Anal. Calcd for C26H43N3O2: C, 72.68; H, 10.09; N, 9.78.
Found: C, 72.49; H, 10.00; N, 9.67; MS (LC–MS) m/z:
430.49 (M+1), 452.44 (M+23 for Na).
4.4.4. Methyl-3b-[(4-(2-(2,4-difluorophenyl)-2-hydroxy-
3-(1H-1,2,4-triazol-1-yl)propyl)-1H-1,2,3-triazol-1-yl)]-
7a-12a-dihydroxy-5b-cholane-24-oate (26). White solid,
yield 91%; mp¼172–174 ꢀC; IR (cmꢁ1): 1618, 1730,
3420; 1H NMR (CDCl3, 400 MHz): d 0.69 (s, 3H), 0.79 (s,
3H), 0.99 (d, J¼6.27 Hz, 3H), 3.12 and 3.51 (two doublets,
J¼14.35 Hz, 2H, adjacent to 1,2,3-triazole), 3.66 (s, 3H),
3.87 (br s, 1H), 4.00 (br s, 1H), 4.46 (br s, 1H, C3-H),
4.63–4.72 (two doublets, J¼14.30 Hz, 2H, adjacent to
1,2,4-triazole), 6.66–6.75 (m, 2H), 7.21 (br s, 1H), 7.36
(br s, 1H), 7.83 (br s, 1H), 8.30 (br s, 1H). Anal. Calcd for
C38H52F2N6O5: C, 64.21; H, 7.37; F, 5.35; N, 11.82. Found:
C, 64.10; H, 7.29; F, 5.19; N, 11.77; MS (LC–MS) m/z:
711.29 (M+1), 733.26 (M+23 for Na).
4.5.4. 3a,12a-Dihydroxy-24-(1H-benzo[d][1,2,3]triazol-
1-yl)-5b-cholane (27d). White solid, yield 92%; mp¼98–
1
101 ꢀC; [a]D26 +47.82 (c 0.5, CHCl3); IR (cmꢁ1): 3417; H
NMR (CDCl3, 300 MHz): d 0.65 (s, 3H), 0.90 (s, 3H),
0.95 (d J¼6.59 Hz, 3H), 3.61 (m, 1H), 3.95 (br s, 1H),
4.61 (t, J¼7.33 Hz, 2H), 7.34–7.40 (m, 1H), 7.46–7.55
(m, 2H), 8.06 (d, J¼8.06 Hz, 1H); 13C NMR (CDCl3,
50 MHz): d 12.6, 17.5, 23.0, 23.6, 26.1, 26.4, 27.1, 27.4,
28.6, 30.4, 32.8, 33.6, 34.0, 35.0, 35.2, 36.0, 36.4, 42.1,
46.4, 47.2, 48.1, 48.6, 71.6, 73.0, 109.2, 119.9, 126.0,
127.0, 132.9, 144.2. Anal. Calcd for C30H45N3O2: C,
75.11; H, 9.46; N, 8.76. Found: C, 75.26; H, 9.39; N, 8.71;
MS (LC–MS) m/z: 480.53 (M+1), 502.49 (M+23 for Na).
4.5. General procedure for 27a–d
Azole compound RH (2 mmol) and NaH (3 mmol) were
stirred in dry DMF (3 mL) at 0 ꢀC for 20 min. To this mix-
ture compound 13 (1 mmol) in DMF (2 mL) was added
dropwise at 0 ꢀC. The reaction mixture was allowed to
warm to 25 ꢀC and stirred for 24 h at this temperature. Ice
was added to the reaction mixture and it was extracted
with EtOAc. The extract was washed with water and brine,
and solvent was evaporated under reduced pressure to afford
crude product, which was purified by column chromato-
graphy on silica gel (5% MeOH/CH2Cl2) to produce
compounds 27a–d in 75–95% yield.
4.6. Biological evaluation procedure
4.6.1. MIC and IC50 determination. Minimum inhibitory
concentration of compounds was tested according to stan-
dard microbroth dilution technique as per NCCLS guide-
lines.21 Briefly, testing was performed in flat bottom 96
well tissue culture plates (CELLSTARÒ Greiner bio-one
GmbH, Germany) in RPMI 1640 medium buffered with
MOPS (3-[N-morpholino]propanesulfonic acid) (Sigma
Chem. Co., MO, USA) for fungal strains and in Muller
Hinton broth (Titan Biotech Ltd, India) for bacterial strains.
The concentration range of tested compounds was 50–0.36
and 32–0.0018 mg/mL for standard compounds. The plates
were incubated in a moist chamber at 35 ꢀC and absorbance
at 492 nm was recorded on VersaMax microplate reader
(Molecular Devices, Sunnyvale, USA) after 48 h for C. albi-
cans and C. parapsilosis, 72 h for Aspergillus fumigatus,
S. schenckii, and Cryptococcus neoformans, and 96 h for
Trichophyton mentagrophytes. MIC was determined as
90% inhibition of growth with respect to the growth control
and IC50 was the concentration at which 50% growth
4.5.1. 3a,12a-Dihydroxy-24-(1H-imidazol-1-yl)-5b-chol-
ane (27a). White solid, yield 75%; mp¼215–216 ꢀC; [a]D25
1
+50.30 (c 0.7, CHCl3); IR (cmꢁ1): 3300, 1510; H NMR
(CDCl3, 200 MHz): d 0.67 (s, 3H), 0.91 (s, 3H), 0.97 (d,
J¼6.26 Hz, 3H), 3.62 (m, 1H), 3.90 (t, J¼7.44 Hz, 2H), 3.98
(br s, 1H), 6.91 (br s, 1H), 7.06 (br s, 1H), 7.48 (br s, 1H);
13C NMR (CDCl3+CD3OD, 50 MHz): d 12.3, 17.1, 22.7,
22.7, 23.4, 25.9, 26.9, 27.3, 27.4, 28.4, 29.6, 32.4, 33.3, 33.9,
35.0, 35.0, 35.8, 41.9, 46.7, 47.4, 47.7, 48.6, 71.1, 72.6,
118.8, 128.1, 136.5. Anal. Calcd for C27H44N2O2: C, 75.65;
H, 10.35; N, 6.54. Found: C, 75.77; H, 10.21; N, 6.43; MS
(LC–MS) m/z: 429.30 (M+1), 451.26 (M+23 for Na).

V. S. Pore et al. / Tetrahedron 62 (2006) 11178–11186
11185
Pannecouque, C.; Witvrouw, M.; Hartman, T. L.; Turpin,
J. A.; Buckheit, R. W., Jr.; Cushman, M. Tetrahedron 2001,
57, 9385–9391; (c) Van Geldern, T. W.; Tu, N.; Kym, P. R.;
Link, J. T.; Jae, H.-S.; Lai, C.; Apelqvist, T.; Rhonnstad, P.;
Hagberg, L.; Koehler, K.; Grynfarb, M.; Goos-Nilsson, A.;
¨
€
Sandberg, J.; Osterlund, M.; Barkhem, T.; Hoglund, M.;
Wang, J.; Fung, S.; Wilcox, D.; Nguyen, P.; Jakob, C.;
inhibition was observed by using SOFTmax Pro 4.3 Soft-
ware (Molecular Devices, Sunnyvale, USA).
Acknowledgements
V.S.P. thanks Director NCL for in-house project funding.
N.G.A. thanks CSIR-UGC, New Delhi, for award of Junior
Research Fellowship.
¨
€
˚
Hutchins, C.; Farnegardh, M.; Kauppi, B.; Ohman, L.;
Jacobson, P. B. J. Med. Chem. 2004, 47, 4213–4230; (d)
Anelli, P. L.; Lattuada, L.; Lorusso, V.; Lux, G.; Morisetti,
A.; Morosini, P.; Serleti, M.; Uggeri, F. J. Med. Chem. 2004,
47, 3629–3641; (e) Solaja, B. A.; Terzic, N.; Pocsfalvi, G.;
Gerena, L.; Tinant, B.; Opsenica, D.; Milhous, W. K. J. Med.
Chem. 2002, 45, 3331–3336; (f) Bhat, L.; Jandeleit, B.; Dias,
T. M.; Moors, T. L.; Gallop, M. A. Bioorg. Med. Chem. Lett.
2005, 15, 85–87; (g) Scozzafava, A.; Supuran, C. T. Bioorg.
Med. Chem. Lett. 2002, 12, 1551–1557; (h) B€ulb€ul, M.;
Supplementary data
1H NMR, 13C NMR, DEPT, and spectral chart of compounds
5, 6, 13–26, and 27a–d, and LC–MS of compounds 23–26.
Supplementary data associated with this article can be found
in the online version, at doi:10.1016/j.tet.2006.09.021.
_
¨
˘ ˘
Saracoglu, N.; K€ufrevioglu, O. I.; Ciftci, M. Bioorg. Med.
Chem. 2002, 10, 2561–2567.
8. Stein, T. M.; Gellman, S. H. J. Am. Chem. Soc. 1992, 114,
3943–3950.
References and notes
9. Savage, P. B. Expert Opin. Investig. Drugs 2000, 9, 263–271.
10. Hermanson, G. T. Bioconjugate Techniques; Academic: San
Diego, 1996.
1. (a) Richardson, M. D. J. Antimicrob. Chemother. 1991, 28,
1–11; (b) Ablordeppey, S. Y.; Fan, P.; Ablordeppey, J. H.;
Mardenborough, L. Curr. Med. Chem. 1999, 6, 1151–1196;
(c) Fridkin, S. K.; Jarvis, W. R. Clin. Microbiol. Rev. 1996, 9,
499–511; (d) Larocco, M. T.; Burgert, S. J. Clin. Microbiol.
Rev. 1997, 10, 277–297; (e) Patel, R.; Paya, C. V. Clin.
Microbiol. Rev. 1997, 10, 86–124.
2. (a) Perfect, J. R.; Lindsay, M. H.; Drew, R. H. Drug Saf. 1992,
7, 323–363; (b) Johnson, E. M.; Warnock, D. W.; Luker, J.;
Porter, S. R.; Scully, C. J. Antimicrob. Chemother. 1995, 35,
103–114; (c) Rex, J. H.; Rinaldi, M. G.; Pfallar, M. A.
Antimicrob. Agents Chemother. 1995, 39, 1–8.
3. (a) Ghannoum, M. A. Dermatol. Ther. 1997, 3, 104–111; (b)
Asai, K.; Tsuchimori, N.; Okonogi, K.; Perfect, J. R.; Gotoh,
O.; Yoshida, Y. Antimicrob. Agents Chemother. 1999, 43,
1163–1169.
4. (a) Zervos, M.; Meunier, F. Int. J. Antimicrob. Agents 1993, 3,
147–170; (b) Karyotakis, N. C.; Anaissie, E. J. Curr. Opin.
Infect. Dis. 1994, 7, 658–666; (c) Kauffman, C. A.; Carver,
P. L. Drugs 1997, 53, 539–549; (d) Andriole, V. T.
J. Antimicrob. Chemother. 1999, 44, 151–162.
5. (a) Dickinson, R. P.; Bell, A. S.; Hitchcock, C. A.;
Narayanaswami, S.; Ray, S. J.; Richardson, K.; Troke, P. F.
Bioorg. Med. Chem. Lett. 1996, 6, 2031–2036; (b)
Upadhayaya, R. S.; Jain, S.; Sinha, N.; Kishore, N.; Chandra,
R.; Arora, S. K. Eur. J. Med. Chem. 2004, 39, 579–592; (c)
Nam, N. H.; Sardari, S.; Selecky, M.; Parang, K. Bioorg.
Med. Chem. 2004, 12, 6255–6269; (d) Gao, P.-H.; Cao, Y.-B.;
Xu, Z.; Zhang, J.-D.; Zhang, W.-N.; Wang, Y.; Gu, J.; Cao,
Y.-Y.; Li, R.; Jia, X.-M.; Jiang, Y.-Y. Biol. Pharm. Bull. 2005,
28, 1414–1417 and references cited therein.
11. (a) Aher, N. G.; Pore, V. S. Synlett 2005, 2155–2158; (b)
Salunke, D. B.; Hazra, B. G.; Pore, V. S.; Bhat, M. K.;
Nahar, P. B.; Deshpande, M. V. J. Med. Chem. 2004, 47,
1591–1594; (c) Hazra, B. G.; Pore, V. S.; Dey, S. K.; Datta,
S.; Darokar, M. P.; Saikia, D.; Khanuja, S. P. S.; Thakur,
A. P. Bioorg. Med. Chem. Lett. 2004, 14, 773–777; (d)
Salunke, D. B.; Hazra, B. G.; Gonnade, R. G.; Bhadbhade,
M. M.; Pore, V. S. Tetrahedron 2005, 61, 3605–3612; (e)
Salunke, D. B.; Ravi, D. S.; Pore, V. S.; Mitra, D.; Hazra,
B. G. J. Med. Chem. 2006, 49, 2652–2655.
12. (a) Dalvie, D. K.; Kalgutkar, A. S.; Khojasteh-Bakht, S. C.;
Obach, R. S.; O’Donnell, J. P. Chem. Res. Toxicol. 2002,
15, 269–299; (b) Horne, W. S.; Yadav, M. K.; Stout,
C. D.; Ghadiri, M. R. J. Am. Chem. Soc. 2004, 126,
15366–15367.
13. (a) Genin, M. J.; Allwine, D. A.; Anderson, D. J.; Barbachyn,
M. R.; Emmert, D. E.; Garmon, S. A.; Graber, D. R.; Grega,
K. C.; Hester, J. B.; Hutchinson, D. K.; Morris, J.; Reischer,
R. J.; Ford, C. W.; Zurenko, G. E.; Hamel, C. J.; Schaadt,
R. D.; Stapert, D.; Yagi, B. H. J. Med. Chem. 2000, 43, 953–
970; (b) Wamhoff, H. Comprehensive Heterocyclic Chemistry;
Katritzky, A. R., Rees, C. W., Eds.; Pergamon: Oxford, 1984;
pp 669–732; (c) Buckle, D. R.; Rockell, C. J. M.; Smith, H.;
Spicer, B. A. J. Med. Chem. 1986, 29, 2262–2267; (d)
Alvarez, R.; Velazquez, S.; San-Felix, A.; Aquaro, S.; De
Clercq, E.; Perno, C. F.; Karlsson, A.; Balzarini, J.; Camarasa,
M. J. J. Med. Chem. 1994, 37, 4185–4194.
14. (a) Huisgen, R.; Szeimies, G.; Moebius, L. Chem. Ber. 1967,
100, 2494–2507; (b) Rostovtsev, V. V.; Green, L. G.; Fokin,
V. V.; Sharpless, K. B. Angew. Chem., Int. Ed. 2002, 41,
6. (a) Davis, A. P. Chem. Soc. Rev. 1993, 243–253; (b) Li, Y.; Dias,
J. R. Chem. Rev. 1997, 97, 283–304; (c) Wallimann, P.; Marti, T.;
´
2596–2599; (c) Tornøe, C. W.; Christensen, C.; Meldal, M.
€
Furer, A.; Diederich, F. Chem. Rev. 1997, 97, 1567–1608; (d)
J. Org. Chem. 2002, 67, 3057–3064; (d) Bock, V. D.;
Hiemstra, H.; Van Maarseveen, J. H. Eur. J. Org. Chem.
2006, 51–68.
Kolehmainen, E.; Tamminen, J. Molecules 2001, 6, 21–46; (e)
Salunke, D. B.; Hazra, B. G.; Pore, V. S. ARKIVOC 2003,
ix, 115–125; (f) Salunke, D. B.; Hazra, B. G.; Pore, V. S.
Curr. Med. Chem. 2006, 13, 813–847; (g) Virtanen, E.;
Kolehmainen, E. Eur. J. Org. Chem. 2004, 3385–3399; (h)
Zhu, X.-X.; Nichifor, M. Acc. Chem. Res. 2002, 35, 539–546.
7. (a) Enhsen, A.; Kramer, W.; Wess, G. Drug Discov. Today
1998, 3, 409–418; (b) Kannan, A.; De Clercq, E.;
15. Cyr, T. D.; Dawson, B. A.; Neville, G. A.; Shurvell, H. F.
J. Pharm. Biomed. Anal. 1996, 14, 247–255.
16. (a) Chen, A. K.; Kakarla, R.; Liu, D.; Sofia, M. J.; Zebovitz, T.
C. Patent 1996, WO 9600230 A1; (b) Anelli, P. L.; Lattuada,
L.; Uggeri, F. Synth. Commun. 1998, 28, 109–117.

11186
V. S. Pore et al. / Tetrahedron 62 (2006) 11178–11186
17. (a) Rijkers, D. T. S.; Van Esse, G. W.; Merkx, R.; Brouwer,
A. J.; Jacobs, H. J. F.; Pieters, R. J.; Liskamp, R. M. J. Chem.
Commun. 2005, 4581–4583; (b) Appukkuttan, P.; Dehaen,
W.; Fokin, V. V.; Van Der Eycken, E. Org. Lett. 2004, 6,
4223–4225.
21. (a) National Committee for Clinical Laboratory Standard.
Reference Method for Broth Dilution Antifungal
Susceptibility Testing of Yeast, Approved Standard. Document
M27-A; National Committee for Clinical Laboratory
Standards: Wayne, PA, USA, 1997; (b) National Committee
for Clinical Laboratory Standard. Reference Method for
Broth Dilution Antifungal Susceptibility Testing of Conidium
Forming Filamentous Fungi: Proposed Standard. Document
M38-P; National Committee for Clinical Laboratory
Standard: Wayne, PA, USA, 1998; (c) National Committee
for Clinical Laboratory Standards. Methods for Dilution
Antimicrobial Susceptibility Tests for Bacteria that Grow
Aerobically. Approved Standard, 5th ed.; NCCLS: Villanova,
PA, 2000; pp M7–A5; (d) Yamamotoa, N.; Fujita, J.;
Shinzato, T.; Higa, F.; Tateyamaa, M.; Tohyamaa, M.;
Nakasone, I.; Yamaneb, N. Int. J. Antimicrob. Agents 2006,
27, 171–173.
18. Wu, W.-L.; Yao, Z.-J.; Li, Y.-L.; Li, J.-C.; Xia, Y.; Wu, Y.-L.
J. Org. Chem. 1995, 60, 3257–3259.
19. (a) Chang, F. C.; Wood, N. F.; Holton, W. G. J. Org. Chem.
1965, 30, 1718–1723; (b) Cortese, F. J. Am. Chem. Soc.
1937, 59, 2532–2534; (c) Li, C.; Peters, A. S.; Meredith,
E. L.; Allman, G. W.; Savage, P. B. J. Am. Chem. Soc. 1998,
120, 2961–2962; (d) Blickenstaff, R. T.; Chang, F. C. J. Am.
Chem. Soc. 1959, 81, 2835–2838; (e) Ruzicka, L.; Goldberg,
M. W. Monatsh. Chem. 1951, 82, 437.
20. Sofia, M. J.; Kakarla, R.; Kagan, N.; Dulina, R.; Hui, Y. W.;
Hatzenbuhler, N. T.; Lui, D.; Chen, A.; Wagler, T. Bioorg.
Med. Chem. Lett. 1997, 7, 2251–2254.