Optically active antifungal azoles: Synthesis and antifungal activity of (2R,3S)-2-(2,4-difluorophenyl)-3-(5-{2-[4-aryl-piperazin-1-yl]-ethyl} -tetrazol-2-yl/1-yl)-1-[1,2,4]-triazol-1-yl-butan-2-ol

Article abstract of DOI:10.1016/j.bmc.2004.02.014

A series of (2R,3S)-2-(2,4-difluorophenyl)-3-(5-{2-[4-aryl-piperazin-1-yl]- ethyl}-tetrazol-2-yl)-1-[1,2,4]-triazol-1-yl-butan-2-ol (11a-n) and (2R,3S)-2-(2,4-difluorophenyl)-3-(5-{2-[4-aryl-piperazin-1-yl]-ethyl} -tetrazole-1-yl)-1-[1,2,4]-triazol-1-yl-butan-2-ol (12a-n) has been synthesized. The antifungal activity of compounds was evaluated by in vitro agar diffusion and broth dilution assay. Compounds 11d and its positional isomer 12d having 3-trifluoromethyl substitution on the phenyl ring of piperazine demonstrated significant antifungal activity against variety of fungal cultures (Candida spp. C. neoformans and Aspergillus spp.). The compound 12d showed MIC value of 0.12μg/mL for C. albicans, C. albicans V-01-191A-261 (resistant strain); 0.25μg/mL for C. tropicalis, C. parapsilosis ATCC 22019 and C. krusei and MIC value of 0.5μg/mL for C. glabrata, C. krusei ATCC 6258, which is comparable to itraconazole and better than fluconazole. Further, compound 11d showed significant activity (MIC; 0.25-0.5μg/mL) against Candida spp. and strong anticryptococcal activity (MIC; 0.25μg/mL) against C. neoformans.

Full text of DOI:10.1016/j.bmc.2004.02.014

Bioorganic & Medicinal Chemistry 12 (2004) 2225–2238
Optically active antifungal azoles: synthesis and antifungal activity
of (2R,3S)-2-(2,4-difluorophenyl)-3-(5-{2-[4-aryl-piperazin-1-yl]-
ethyl}-tetrazol-2-yl/1-yl)-1-[1,2,4]-triazol-1-yl-butan-2-olq
Ram Shankar Upadhayaya,a Neelima Sinha,a Sanjay Jain,a Nawal Kishore,a
Ramesh Chandrab and Sudershan K. Aroraa,*
aMedicinal Chemistry Division, New Chemical Entity Research, Lupin Research Park, 46/47 A,
At Village Nande, Taluka Mulshi, Pune 411042, India
bBundelkhand University, Jhansi, India
Received 21 November 2003; accepted 10 February 2004
Abstract—A series of (2R,3S)-2-(2,4-difluorophenyl)-3-(5-{2-[4-aryl-piperazin-1-yl]-ethyl}-tetrazol-2-yl)-1-[1,2,4]-triazol-1-yl-butan-
2-ol (11a–n) and (2R,3S)-2-(2,4-difluorophenyl)-3-(5-{2-[4-aryl-piperazin-1-yl]-ethyl}-tetrazole-1-yl)-1-[1,2,4]-triazol-1-yl-butan-2-ol
(12a–n) has been synthesized. The antifungal activity of compounds was evaluated by in vitro agar diffusion and broth dilution
assay. Compounds 11d and its positional isomer 12d having 3-trifluoromethyl substitution on the phenyl ring of piperazine dem-
onstrated significant antifungal activity against variety of fungal cultures (Candida spp. C. neoformans and Aspergillus spp.). The
compound 12d showed MIC value of 0.12 lg/mL for C. albicans, C. albicans V-01-191A-261 (resistant strain); 0.25 lg/mL for
C. tropicalis, C. parapsilosis ATCC 22019 and C. krusei and MIC value of 0.5 lg/mL for C. glabrata, C. krusei ATCC 6258, which is
comparable to itraconazole and better than fluconazole. Further, compound 11d showed significant activity (MIC; 0.25–0.5 lg/mL)
against Candida spp. and strong anticryptococcal activity (MIC; 0.25 lg/mL) against C. neoformans.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
has excellent pharmacokinetic properties.3;10 It is effec-
tive against candidiasis after both oral administration
and injection. However, its activity against Aspergillus
seems limited. Itraconazole has an excellent and broader
antifungal spectrum.4 Newer triazole agents such as
voriconazole,5 posaconazole6 and ravuconazole,7 are
active against Aspergillus and currently under clinical
trials (Fig. 1).
The incidence of systemic fungal infections such as
Candidosis, Cryptococcosis and Aspergillosis has been
increasing recently due to an increase in the number of
immunocompromised hosts. For the treatment of these
infections, the new antifungal azoles have been devel-
oped for clinical use. Attention has been paid to triazole
derivatives because of their generally broad antifungal
spectrum and low toxicity.1 Triazole derivatives displace
lanosterol from lanosterol 14-demethylase (14 DM), a
cytochrome P-450-dependent enzyme, and block the
biosynthesis of an essential component of fungal cell
membrane, ergosterol.2 Fluconazole has relatively low
antifungal activity in vitro, but it is water soluble, and
In order to seek new triazole antifungal agents we de-
signed a series of tetrazole–triazole compounds depicted
by the formula 11 and 12. We presumed that the left half
portion of the molecule is essential to the high anti-
fungal activity. It is a common substructure seen in
many other triazole antifungals. The 1-aryl-4-(2-[1H-
tetrazol-5-yl]-ethyl)-piperazine ring was introduced in
the expectation that: (1) since salt of aryl piperazine
moieties will make molecule more water soluble than
simple hydrocarbon moieties, the compound could be
more easily delivered to the target enzyme; (2) since
many other triazole antifungal agents have heteroatoms
at the corresponding part of the molecule, complemen-
tary structure of the target enzyme would be implied;
and (3) a variety of aryl piperazines should be available
Keywords: Antifungal activity; Tetrazole derivatives; Chiral synthesis;
Tetrazole–triazole compounds.
q Taken in part from the PhD dissertation of Ram Shankar Up-
adhayaya, Bundelkhend University, 2003.
* Corresponding author. Tel.: +91-20-25126689; fax: +91-20-25126175;
e-mail addresses: sudershanarora@hotmail.com, sudershanarora@
lupinpharma.com
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.02.014

2226
R. S. Upadhayaya et al. / Bioorg. Med. Chem. 12 (2004) 2225–2238
Me
OH
Me
OH
N
N
N
N
N
N
N
N
F
N
N
F
N
Ar
Ar
N
N
N
N
N
N
N
F
F
12
11
Me
OH
Me
F
OH
OH
N
N
S
N
N
O
N
F
N
F
N
F
N
N
N
N
N
N
N
N
N
CN
F
F
F
Fluconazole
Voriconazole
Ravuconazole
O
N
O
N
Cl
N
N
O
Cl
N
N
N
Itraconazole
O
N
O
N
N
F
N
N
O
F
N
N
OH
N
Posaconazole
Cl
O
Me
OH
N
N
N
N
N
F
N
*
*
N
N
Me
I
F
Figure 1. Structures of compounds 11, 12, fluconazole, itraconazole, voriconazole, posaconazole, ravuconazole and I.
as the side chain (Fig. 1). The methyl group at the 3-
position mimics the 13b-methyl group of lanosterol.8;9
The two fluorine atoms in the benzene ring in the left
half of the molecule aim at strengthening the antifungal
activity.10;11
Similar findings have been observed by us in our other
series of azole antifungals.15
In this paper, we will present our recent findings on
synthesis and antifungal activities of the compounds 11
and 12.
Furthermore, the stereoselective interaction of the
enzyme with stereoisomeric azoles has become an
intriguing field of study. The relationship between ste-
reochemistry and antifungal activity showed that the
target enzyme, cytochrome P-45014DM, recognized the
configuration of the chiral centres: (1R,2R)-enantiomers
showed the most potent activity but findings support the
assumption that the (1R,2S) and (1S,2R) also fit in the
2. Results and discussion
2.1. Chemistry
The title compounds 11a–n and 12a–n were prepared
from the optically active key intermediate (2R,3R)-2-
(2,4-diflourophenyl)-3-methyl-2-[1,2,4]-triazol-1-yl-me-
thyl-oxirane (8),16 which was derived from ethyl (S)-
lactate in eight steps as shown in Scheme 1. The chiral
intermediate 3 was prepared by following the literature
methods.16 The intermediate 3 was then converted to the
exomethylene intermediate 4 by reacting with methyl
same pocket as the lanosterol D ring in the target en-
12;13
zyme, cytochrome P-45014DM
.
To further prove this
rule in our series and examine the influence of each
centreꢀs absolute configuration upon activity, we had
synthesized four stereoisomers of compound I (Fig. 1)
[i.e., (1R,2R), (1S,2S), (1R,2S) and (1S,2R)], and its
recemate were tested in vitro and in vivo and found that
(1R,2R) and (1S,2R) have same antifungal activity.14

R. S. Upadhayaya et al. / Bioorg. Med. Chem. 12 (2004) 2225–2238
2227
Me
H
Me
H
Me
H
Me
H
O
O
O
OTMS
OTMS
OH
OTMS
Me
H
F
N
O
N
O
1
F
a
b
c
d
O
OH
OEt
F
F
4
Ethyl(S)-lactate
2
3
Me
Me
H
Me
H
H
O
O
O
N
Me
H
N
F
OH
OH
OMs
N
g
F
F
F
e
f
h
F
F
F
F
5
6
7
8
Scheme 1. Synthesis of key intermediate 8. Reagents and conditions: (a) morpholine, 80 ꢁC, 4 days; (b) HMDS, PyÆHBr, DCM, rt; (c) 2,4-
F2C6H3MgBr, THF; (d) t-BuOK, PPh3MeI, toluene, 80 ꢁC, 2 h; (e) HCOOH, EtOH, rt, 1 h; (f) Ti(i-PrO)3,
50 ꢁC, 30 min; (h) NaH, 1,2,4-triazole, 50 ꢁC, 30 min.
L-(+)-DET, t-BuOOH; (g) MsCl, TEA,
triphenyl phosphonium iodide in presence of potassium
tert-butoxide in dry toluene. The compound 4, after
deprotection, provided the substituted allyl alcohol (5),
which was then reacted with tert-butyl hydroperoxide in
(10a–n) as given in Scheme 3. The method of prepara-
tion of 5-substituted tetrazoles is same as given in
literature (Scheme 2).18–21 The method involves the
reaction of a nitrile with an inorganic azide, using an
amine salt in an aromatic solvent in a facile work-up
procedure to produce tetrazoles (10a–n) with higher
purity in greater yield.
the presence of titanium isopropoxide and (L)-(+)-di-
ethyl tartarate, following the ꢁSharplessꢀ stereoselective
epoxidation method,16 in an aprotic solvent at a tem-
perature of )20 ꢁC to generate an asymmetric centre
with high diastereomeric yields. The compound 6 thus
produced was treated with methanesulfonyl chloride in
presence of triethylamine in DCM at 0 ꢁC to afford
methylsulfonate (7).17 This compound on reaction with
1,2,4-triazole in DMF in the presence of sodium hydride
at 55 ꢁC produced oxirane (8). The chiral intermediates
4–8 were prepared by following the literature methods
and their spectral and analytical data were also same as
given in the literature.16 Methodology to obtain each of
the above compounds 1–8 is given in experimental sec-
tion. Having obtained the oxirane (8), the next task was
to couple it with a number of substituted tetrazoles
The oxirane 8 so obtained was reacted with various
tetrazoles (10a–n) in presence of K2CO3 in aprotic co-
solvents DMF and NMP at 90 ꢁC to give the conden-
sation product (Scheme 3).22 TLC showed it to be a
mixture of two main products; one that is of lower
polarity designated as 11a–n and the other 12a–n. These
(2R,3S)-2-(2,4-difluorophenyl)-3-(5-{2-[4-aryl-piperazin-
1-yl]-ethyl}-tetrazol-2-yl)-1-[1,2,4]-triazol-1-yl-butan-2-ol
(11a–n) and (2R,3S)-2-(2,4-difluorophenyl)-3-(5-{2-[4-aryl-
piperazin-1-yl]-ethyl}-tetrazole-1-yl)-1-[1,2,4]-triazol-
1-yl-butan-2-ol (12a–n) were separated by flash column
chromatography over silica gel (230–400 mesh) using 2%
a
b
Ar
N
N
N
N
N
Ar
N
NH
Ar
N
N
CN
N
H
10a-n
9
Scheme 2. Synthesis of substituted tetrazoles 10a–n. Reagents and conditions: (a) acrylonitrile, TEA, MeOH, )5 to 10 ꢁC; (b) NaN3, TEAÆHCl,
toluene, reflux.
Me
OH
Me
OH
N
N
N
N
N
N
N
N
F
N
N
F
N
K2CO3, NMP-DMF
90 o
Ar
Ar
N
N
N
N
N
+
10a-n
8 +
C
N
N
F
F
12a-n
11a-n
Scheme 3. Synthesis of (2R,3S)-2-(2,4-difluorophenyl)-3-(5-{2-[4-aryl-piperazin-1-yl]-ethyl}-tetrazol-2-yl/1-yl)-1-[1,2,4]-triazol-1-yl-butan-2-ol
(11a–n and 12a–n).

2228
R. S. Upadhayaya et al. / Bioorg. Med. Chem. 12 (2004) 2225–2238
methanol–chloroform as eluent. Compounds 11a–n
and 12a–n showed the same molecular ion peak in their
mass spectrum indicating them to be the positional
isomers.
C. glabrata and C. krusei), and C. neoformans, additionally
compounds 11d and 12d showed activity against
Aspergillus cultures. These compounds were further
tested for antifungal activity against different species of
Candida, Cryptococcus and Aspergillus by microbroth
dilution assay to determine the minimum inhibitory
values of the compounds against fungal cultures (Table
1).
The structural data of compounds 12a–n were consistent
with compounds 11a–n except that the 1H NMR spectra
of 11a–n showed up-field shifting of CHCH3 protons.
The ratio of isolated yields of the above two isomers
suggested that the (11a–n) isomers are the predominated
one. Steric factors can also play a role in the ratio of
isomers formed; such types of observation have been
reported earlier also.17;23–29
Compound 12d having 3-trifluoromethyl substitution on
the phenyl ring of piperazine was the most active of all
the compounds (Table 1) with MIC value of 0.12 lg/mL
for C. albicans, C. albicans V-01-191A-261 (resistant
strain); 0.25 lg/mL for C. tropicalis, C. parapsilosis
ATCC 22019 and C. krusei and MIC value of 0.5 lg/mL
for C. glabrata, C. krusei ATCC 6258. Compound 12d
further demonstrated similar MIC value of 0.12 lg/mL
for Cryptococcus neoformans strain LA314. This strong
wide spectrum of activity demonstrated by compound
12d was better than fluconazole against C. albicans and
better than fluconazole for resistant Candida cultures
and was equivalent to the activity of itraconazole.
Similar strong antifungal activity was also shown by its
positional isomer 11d, which has MIC value of 0.25 lg/
mL for C. albicans, C. albicans V-01-191A-261 (resistant
strain), C. tropicalis, C. krusei and C. parapsilosis ATCC
2.2. Antifungal activity
The antifungal activities of compounds 11a–n and 12a–n
were evaluated by in vitro agar diffusion and broth
dilution assay and the results of which are presented in
Table 1. Most of the compounds showed activity in the
initial screen against fungal cultures when tested at
500 lg/mL concentration of compound by agar diffusion
assay. Compounds 11d, 12d, 11m, 12m, 11g, 12g, 11l and
12l inhibited the growth of drug resistant species of
Candida (C. albicans, C. tropicalis, C. parapsilosis,
Table 1. In vitro susceptibility of compounds 11a–n and 12a–n on clinical isolates of the fungal cultures
Compd
Ar
MIC (lg/mL) of compounds and standard drugs against fungal culturea
C. a I
C. a II
C. g
C. t
C. k I
C. k II
C. p
C. n
A. f
A. n
>8.0
11a
11b
2-MeOC6H4
4-MeOC6H4
4-NO2C6H4
3-CF3C6H4
2-ClC6H4
3-ClC6H4
4-ClC6H4
CH2C6H5
4-FC6H4
8.0
16.0
2.0
8.0
16.0
2.0
>8.0
>8.0
>16.0
4.0
>8.0
>16.0
4.0
>8.0
>16.0
8.0
>8.0
>16.0
8.0
>8.0
>16.0
4.0
>8.0
>16.0
4.0
0.5
>16.0
>8.0
1.0
>16.0
>8.0
1.0
11c
11d
0.25
4.0
0.5
4.0
0.25
0.5
8.0
0.25
8.0
0.25
0.25
11e
11f
8.0
8.0
8.0
4.0
8.0
>8.0
>8.0
>16.0
8.0
8.0
4.0
>8.0
>8.0
>8.0
>16.0
>8.0
>16.0
>8.0
>8.0
>8.0
>8.0
>8.0
>16.0
>8.0
0.5
>8.0
>8.0
>8.0
>16.0
>8.0
>16.0
>8.0
>8.0
>8.0
>8.0
>8.0
>16.0
>8.0
1.0
2.0
1.0
4.0
2.0
>8.0
8.0
8.0
4.0
11g
11h
2.0
>16.0
4.0
4.0
>16.0
2.0
4.0
>16.0
2.0
16.0
2.0
16.0
2.0
>16.0
4.0
>16.0
8.0
11i
11j
2,4-(Cl)2C6H3
2-Pyridyl
CHPh2
16.0
4.0
16.0
4.0
>16.0
8.0
8.0
>16.0
8.0
8.0
>16.0
>8.0
8.0
>16.0
>8.0
8.0
>16.0
>8.0
8.0
>16.0
8.0
8.0
11k
11l
4.0
0.5
4.0
1.0
11m
11n
2-n-BuOC6H4
C6H5
0.5
8.0
0.5
8.0
2.0
1.0
1.0
1.0
8.0
8.0
4.0
8.0
4.0
>8.0
>8.0
>16.0
8.0
>8.0
>8.0
>16.0
4.0
>8.0
>8.0
>16.0
>8.0
12a
12b
2-MeOC6H4
4-MeOC6H4
4-NO2C6H4
3-CF3C6H4
2-ClC6H4
3-ClC6H4
4-ClC6H4
CH2C6H5
4-FC6H4
>8.0
>16.0
4.0
>8.0
>16.0
4.0
8.0
>16.0
4.0
16.0
2.0
16.0
2.0
12c
12d
0.12
4.0
0.12
4.0
0.5
8.0
0.25
0.5
8.0
0.25
8.0
0.25
0.12
12e
12f
8.0
8.0
8.0
>8.0
2.0
8.0
4.0
>8.0
>8.0
>8.0
>16.0
>8.0
>16.0
>8.0
>8.0
>8.0
>8.0
1.0
>8.0
>8.0
>8.0
>16.0
>8.0
>16.0
>8.0
>8.0
>8.0
>8.0
1.0
2.0
0.5
4.0
0.5
4.0
1.0
>8.0
1.0
8.0
1.0
12g
12h
1.0
>16.0
2.0
1.0
>16.0
2.0
16.0
2.0
16.0
2.0
>16.0
4.0
>16.0
4.0
>16.0
8.0
>16.0
8.0
12i
12j
2,4-(Cl)2C6H3
2-Pyridyl
CHPh2
16.0
4.0
16.0
4.0
>16.0
8.0
4.0
>16.0
8.0
4.0
>16.0
>8.0
4.0
>16.0
8.0
4.0
>16.0
>8.0
8.0
>16.0
8.0
8.0
12k
12l
4.0
0.5
4.0
0.5
12m
12n
2-n-BuOC6H4
C6H5
0.5
0.5
8.0
1.0
1.0
0.5
>8.0
0.25
8.0
8.0
8.0
0.5
>8.0
0.5
>8.0
0.5
>8.0
0.25
0.25
0.5
11dÆHCl
12dÆHCl
Fluconazole
Itraconazole
3-CF3C6H4
3-CF3C6H4
0.25
0.12
0.12
0.007
0.25
0.25
0.25
0.12
2.0
0.12
8.0
0.5
>8.0
0.25
8.0
0.5
>8.0
0.25
0.25
8.0
0.5
>8.0
1.0
>8.0
0.5
0.25
0.25
0.12
0.007
0.25
0.06
0.12
a C. a I: C. albicans, C. a II: C. albicans V-01-191A-261 (resistant strain), C. t: C. tropicalis, C. k I: C. krusei ATCC 6528, C. k II: C. krusei, C. p: C.
parapsilosis ATCC 22019, C. g: C. glabrata, C. n: Cryptococcus neoformans LA314, A. f: Aspergillus fumigatus, A. n: Aspergillus niger.

R. S. Upadhayaya et al. / Bioorg. Med. Chem. 12 (2004) 2225–2238
2229
Table 2. Comparison with standards
Compd
MIC (lg/mL) of 11d and 12d and standard drugs against fungal culturea
C. a I
C. a II
C. g
C. t
C. k I
C. k II
C. p
C. n
A. f
1.0
A. n
1.0
11d
12d
0.25
0.12
0.12
0.007
0.015
0.25
0.03
0.5
0.5
0.5
0.25
0.5
0.5
0.25
0.25
0.5
0.25
0.25
8.0
0.25
0.12
2.0
0.12
8.0
0.25
8.0
0.5
>8.0
1.0
>8.0
0.5
Fluconazole
Itraconazole
Voriconazole
Posaconazole
Ravuconazole
>8.0
>8.0
0.25
0.5
0.25
0.5
0.25
0.12
0.12
0.12
0.007
0.5
0.5
0.25
0.25
0.12
0.5
0.06
0.25
1.0
0.12
0.25
0.5
0.12
0.5
0.5
1.0
0.25
0.03
0.5
0.5
0.06
16
0.5
0.12
>16
>16
a C. a I: C. albicans, C. a II: C. albicans V-01-191A-261 (resistant strain), C. t: C. tropicalis, C. k I: C. krusei ATCC 6528, C. k II: C. krusei, C. p: C.
parapsilosis ATCC 22019, C. g: C. glabrata, C. n: Cryptococcus neoformans LA314, A. f: Aspergillus fumigatus, A. n: Aspergillus niger.
22019 and MIC of 0.5 lg/mL against C. glabrata, C.
krusei ATCC 6258 (Table 1). Against Cryptococcus
neoformans strain LA314 the compound 11d had MIC
value of 0.25 lg/mL. The antifungal activity of 11d and
12d was comparable and better than fluconazole against
resistant Candida species. These compounds (11d and
12d) also showed significant inhibitory activity against
A. fumigatus (MIC; 0.5–1.0 lg/mL) and A. niger 1.0 lg/
mL.
activity of 2.0–4.0 lg/mL against Cryptococcus neofor-
mans strain LA314. In addition moderate to weak
activity was also shown by compounds 11a,f,k,l,n and
12a,f,k,l,n against fungal cultures (Table 1) MIC value
of 2.0–4.0 lg/mL for compounds 11f,k,n and 12f,k,n;
and 4.0–8.0 lg/mL for compounds 11a,n and 12a,n
against C. albicans, C. albicans V-01-191A-261 (resistant
strain); 4.0–8.0 lg/mL for C. tropicalis, C. glabrata and
C. krusei ATCC 6528 by compounds 11f,k,l and 12f,k,l
and 8.0–>8.0 lg/mL for C. krusei, and C. parapsilosis
ATCC 22019 for compounds 11a,f,k,l,n and 12a,f,k,l,n.
These compounds inhibited the growth of Cryptococcus
neoformans strain LA314 at concentration of 4.0–
>8.0 lg/mL.
Compounds 11m and 12m having 2-butoxy group on the
phenyl ring of piperazine (Table 1) also showed signifi-
cant activity against all the fungal cultures. A MIC value
of 0.5 lg/mL for C. albicans, C. albicans V-01-191A-261
(resistant strain), C. glabrata and C. tropicalis; 1.0 lg/
mL for C. krusei and, against C. parapsilosis ATCC
22019 and 2.0 lg/mL for C. krusei ATCC 6528, was
shown by both the compounds. The compounds also
inhibited the growth of (MIC; 0.25 lg/mL) Cryptococcus
neoformans strain LA314. This activity of the com-
pounds 11m and 12m was better than fluconazole and
comparable to itraconazole against resistant Candida
cultures. Compound 12g having 4-Cl group on the
phenyl ring of piperazine demonstrated good activity
against fungal cultures as is evident from (Table 1) MIC
value of 0.5 lg/mL for C. albicans, C. albicans V-01-
191A-261 (resistant strain), 1.0 lg/mL for C. tropicalis,
C. glabrata, C. krusei ATCC 6528, C. krusei; 2.0 lg/mL
for C. parapsilosis ATCC 22019. The compounds also
showed moderate activity of 2.0 lg/mL against Crypto-
coccus neoformans strain LA314. Similar activity was
seen with compound 11g having 4-Cl group on the
phenyl ring of piperazine against fungal cultures (Table
1) MIC value of 1.0 lg/mL for C. albicans; 2.0 lg/mL for
C. albicans V-01-191A-261 (resistant strain), and C.
glabrata; 4.0 lg/mL against C. tropicalis, C. krusei;
8.0 lg/mL for C. krusei ATCC 6528, and >8.0 lg/mL for
C. parapsilosis ATCC 22019. The compounds had a
MIC value of activity of 4.0 lg/mL against Cryptococcus
neoformans strain LA314.
These structure activity relationship clearly suggest that
3-trifluoromethyl group in compounds 11d and 12d is
responsible for broad-spectrum antifungal activity. The
presence of the groups like 2-butoxy (11m and 12m), 4-
Cl (11g and 12g), 4-NO2 (11c and 12c) and 4-F (11i and
12i) are also play a significant role in imparting anti-
fungal activity to the compounds.
2.3. Comparison with reference compounds
Table 2 shows the in vitro activity of compounds 11d
and 12d against Candida, Aspergillus and Cryptococcus
species compared to those of fluconazole, itraconazole,
voriconazole, posaconazole and ravuconazole. Com-
pounds 11d and 12d had comparable low MICs and
broad spectra of activity against most of the fungal
pathogen tested. They are active against all species of
Candida, Aspergillus and Cryptococcus.
3. Conclusion
In conclusion, a novel series of antifungal agents has
been designed and synthesized, that demonstrated sig-
nificant wide spectrum activity against Candida, Asper-
gillus and Cryptococcus species. These compounds
demonstrated activity against fluconazole resistant
Candida species. The antifungal activity of these com-
pounds was better than fluconazole and was equivalent
to itraconazole, voriconazole, posaconazole and rav-
uconazole.
Compounds 11i, 12i, 11c and 12c having 4-F and 4-NO2
group on the phenyl ring of piperazine also demon-
strated mild activity against fungal cultures (Table 1)
with a MIC value of 2.0 lg/mL for C. albicans, C.
albicans V-01-191A-261 (resistant strain) and C. tropi-
calis; 4.0 lg/mL for C. glabrata, and C. krusei ATCC
6528; and 8.0 lg/mL for C. krusei, and C. parapsilosis
ATCC 22019. These compounds also showed moderate

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R. S. Upadhayaya et al. / Bioorg. Med. Chem. 12 (2004) 2225–2238
4. Experimental
was added dropwise to the reaction mixture. The reac-
tion mixture was then stirred at ambient temperature for
4 h, then a saturated aqueous solution of NH4Cl
(100 mL) and water (100 mL) were added, and the
resulting mixture was extracted with ethyl acetate
(2 · 500 mL). The combined organic phase was washed
with water (2 · 50 mL) and brine (50 mL), dried over
anhydrous Na2SO4 and filtered. The filtrate was evap-
orated under reduced pressure to give crude product.
The crude product was purified by column chromato-
graphy over silica gel (100–200 mesh) using hexane–
ethyl acetate (15:75) as eluent to give 40.83 g (84.80%) 3
Melting points were taken in an electrically heated
instrument and are uncorrected. Compounds were rou-
tinely checked for their purity on silica gel 60 F254 TLC
plates and their spots were visualized by exposing them
to iodine vapour or UV lamp or by spraying the plates
with Dragendorff or KMnO4 reagents. IR spectra (kmax
in cmꢀ1) were recorded either on Perkin–Elmer spec-
1
trum RX FT-IR model and H NMR spectra were re-
corded on Bruker Advance DRX 200 MHz instrument
as solutions in CDCl3 otherwise mentioned, using TMS
as internal reference and chemical shifts values are ex-
pressed in d units. Mass spectra were run on Applied
Biosystems API 3000 instrument using direct inlet sys-
tem. Elemental analyses were carried out with a Perkin–
Elmer 2400 analyzer and the values found were within
0.4% of theoretical values.
1
as a pale yellow oil. H NMR (CDCl3): d 0.18 (s, 9H),
1.34 (d, J ¼ 7:0 Hz, 3H), 4.45–4.53 (m, 1H), 6.68–7.05
(m, 2H), 7.84–8.00 (m, 1H). MS: m=z 259 (M+1).
4.4. 1-[(1S)-2-(2,4-Difluorophenyl)-1-methylprop-2-enyl-
oxy]-1,1-dimethyl-1-silaethane (4)
4.1. (2S)-2-Hydroxy-1-morpholin-4-yl-propan-1-one (1)
To a solution of trimethylsulfonium methyl iodide
(29.70 g, 73.64 mmol) in toluene (160 mL) was added
potassium tert-butoxide (10.72 g, 95.73 mmol) under
nitrogen over a period of 2 h. To this solution was added
3 (18.9 g, 73.64 mmol) in dry toluene (30 mL). The
reaction mixture was again heated at 80 ꢁC for 1 h. After
completion of the reaction, product was extracted with
toluene (3 · 50 mL). The combined organic layer was
concentrated under reduced pressure to afford crude
product as oil. Hexane (120 mL) was added and stirred
for 4 h, followed by cooling to )30 ꢁC under stirring.
Filtered the solid thus separated out and washed with
chilled hexane. Organic layer was concentrated under
reduced pressure to obtain compound 4 as oil. Yield:
A mixture of ethyl (S)-lactate (40 g, 338 mmol) and
morpholine (100 mL, 1017 mmol) was heated at 80 ꢁC
for 4 days. The reaction mixture was evaporated under
reduced pressure to obtain crude product. The crude
product was purified by column chromatography over
silica gel (100–200 mesh). Elution with hexane–ethyl
acetate (1:1) gave 1 as pale yellow oil 30.5 g (55%). IR
.
(neat): m 1635 cmꢀ1 1H NMR (CDCl3): d 1.34 (d,
J ¼ 6:5 Hz, 3H), 2.43 (t, J ¼ 5:0 Hz, 2H), 3.55–3.80 (m,
6H), 3.82 (d, J ¼ 7:5 Hz, 1H), 4.44–4.53 (m, 1H). MS:
m=z 160 (M+1).
1
17.83 g (95.0%). H NMR (CDCl3): d 0.16 (s, 9H), 1.34
4.2. (2S)-2-(1,1-Dimethyl-1-silaethoxy)-1-morpholin-4-yl-
propan-1-one (2)
(d, J ¼ 6:5 Hz, 3H), 4.40–4.50 (m, 1H), 5.13–6.15 (m,
2H), 6.82–7.00 (m, 2H), 7.84–8.00 (m, 1H). MS: m=z 257
(M+1).
To a mixture of 1 (30.5 g, 191.8 mmol) and pyridinium
hydrobromide (3.84 g, 23.98 mmol) in CH2Cl2 (180 mL),
hexamethyldisilazane (0.07 g, 124.68 mmol) was added
at ambient temperature, the mixture was stirred over a
period of 3 h. After completion of reaction, the organic
layer was washed with water (2 · 100 mL), brine
(20 mL), dried over anhydrous Na2SO4 and filtered. The
filtrate was concentrated under reduced pressure to yield
4.5. (2S)-3-(2,4-Difluorophenyl)-but-3-en-2-ol (5)
To a solution of 4 (17.83 g, 69.65 mmol) in ethanol
(50 mL) was added formic acid (4.67 mL, 123.98 mmol)
dropwise over a period of 0.5 h and reaction mixture was
neutralized with aqueous solution of sodium bicarbon-
ate (pH ¼ 8.0). The aqueous layer was extracted with
DCM (2 · 100 mL). The combined organic layer was
dried over anhydrous Na2SO4 and filtered. The filtrate
was concentrated under reduced pressure to obtain
crude product. The crude product was purified over
silica gel (100–200 mesh) using hexane–ethyl acetate
(9:1) as eluent to obtain 5 as oil. Yield: 7.0 g (54.60%).
1H NMR (CDCl3): d 1.32 (d, J ¼ 6:5 Hz, 3H), 4.42–4.28
(m, 1H), 4.94 (br s, 1H), 5.10–5.60 (m, 2H), 6.80–6.95
(m, 2H), 7.90–8.00 (m, 1H). MS: m=z 185 (M+1).
the 2 as oil. Yield: 43.2 g (97.5%). IR (neat): m 1662 cmꢀ1
.
1H NMR (CDCl3): d 0.18 (s, 9H), 1.34 (d, J ¼ 6:5 Hz,
3H), 3.43–3.80 (m, 8H), 4.44–4.53 (m, 1H). MS: m=z 232
(M+1).
4.3. (2S)-1-(2,4-Difluorophenyl)-2-(1,1-dimethyl-1-sila-
ethoxy)-propan-1-one (3)
To
a suspension of magnesium turnings (4.94 g,
205.7 mmol) in dry THF (110 mL), 1-bromo-2,4-diflu-
orobenzene (39.7 g, 25.7 mmol) was added dropwise
under vigorous stirring at such a rate that temperature
of the reaction mixture was maintained at 35–40 ꢁC.
After completion of addition, the reaction mixture was
stirred at ambient temperature for 1 h to get the 2,4-di-
fluorobenzene magnesium bromide and then cooled to
20 ꢁC. A solution of 2 (43.2 g, 18 mmol) in THF (40 mL)
4.6. 1-[(2R)-2-(2,4-Difluorophenyl)-oxirane-2-yl]-(1S)-
ethan-1-ol (6)
ꢀ
To a mixture of molecular sieves (4 A, 15 g) in anhy-
drous DCM (200 mL), titanium isopropoxide (2.70 mL,
9.07 mmol) was added at )20 ꢁC. Then
L-(+)-diethyl

R. S. Upadhayaya et al. / Bioorg. Med. Chem. 12 (2004) 2225–2238
2231
tartarate in anhydrous DCM (300 mL) was added under
vigorous stirring. To this, tert-butylhydroperoxide
(2.62 mmol, 3.2 molar solution) in toluene (82 mL) was
added dropwise over a period of 1 h, followed by 5
(16.70 g, 90.76 mmol) in DCM (75 mL). The reaction
mixture was stirred for 2 h at )20 ꢁC and then for 6 h at
)10 ꢁC. The reaction mixture was quenched with 100 mL
of water at 0 ꢁC and stirred for 1 h at ambient temper-
ature. Sodium hydroxide solution (30%, 80 mL) was
added to hydrolyze the tartarate and the solid separated
out was filtered off by passing through a Celite pad.
Filtrate was washed with brine (200 mL). The organic
layer was dried over anhydrous Na2SO4 and filtered.
The filtrate was concentrated under reduced pressure to
obtain crude product. The crude product was purified
over silica gel (100–200 mesh) using dichloromethane–
hexane (1:1) as eluent to obtain 6 as oil. Yield: 10.91 g
reduced pressure and the residue was dissolved in DCM
(100 mL). The organic phase was washed with water
(2 · 20 mL), brine (20 mL), dried over anhydrous
Na2SO4 and filtered. The filtrate was concentrated under
1
reduced pressure to give 9a as oil. Yield: 84.30%. H
NMR (CDCl3): d 2.57–2.70 (m, 8H), 3.03–3.11 (m, 4H),
3.74 (s, 3H), 6.61–6.85 (m, 2H), 6.94–6.99 (m, 2H). MS:
m=z 246 (M+1).
The other 3-(4-aryl-piperazin-1-yl)-propionitrile (9b–n)
were also prepared by the same procedure as described
above and their spectral and analytical data are given
below.
4.9. 3-[4-(4-Methoxyphenyl)-piperazin-1-yl]-propionitrile
(9b)
1
(60%). H NMR (CDCl3): d 1.16 (d, J ¼ 6:5 Hz, 3H),
2.69 (br s, 1H), 2.88–2.96 (m, 1H), 3.27–3.30 (m, 1H),
4.08–4.12 (m, 1H), 6.79–6.94 (m, 2H), 7.32–7.44 (m,
1H). MS: m=z 201 (M+1).
This was obtained in 86.56% yield as oil. 1H NMR
(CDCl3): d 2.57–2.70 (m, 8H), 3.09–3.17 (m, 4H), 3.76
(s, 3H), 6.61–6.71 (m, 4H). MS: m=z 246 (M+1).
4.7. (2R,3R)-2-(2,4-Difluorophenyl)-3-methyl-2-[1,2,4]-
triazol-1-yl-methyl-oxirane (8)
4.10. 3-[4-(4-Nitrophenyl)-piperazin-1-yl]-propionitrile
(9c)
To a solution of 6 (2.0 g, 10 mmol), methanesulfonyl
chloride (1.52 g, 15.0 mmol) in DCM (40 mL) was added
triethylamine (1.52 g, 15 mmol) at 5 ꢁC under stirring.
The stirring was continued at same temperature for 1 h.
Water (20 mL) was added to the reaction mixture and
washed the organic layer with a saturated NaHCO3
solution (20 mL). The organic layer was dried over
anhydrous sodium sulfate and filtered. The filtrate was
concentrated at reduced pressure to get 7. In another
100 mL round bottom flask NaH (1.44 g, 60.0 mmol)
pre-washed with hexane was taken in DMF (50 mL) and
1,2,4-triazole (2.76 g, 69.0 mmol) was added at 0 ꢁC un-
der stirring. The stirring was continued for 15 min fol-
lowed by the addition of the compound 7. The reaction
mixture was stirred for 20 min at 0 ꢁC and then at 50–
55 ꢁC for 1 h. The reaction mixture was taken in ethyl
acetate (100 mL), washed with water (2 · 25 mL). The
organic layer was dried over anhydrous Na2SO4 and
filtered. The filtrate was concentrated under reduced
pressure to obtain the (2R,3R)-2-(2,4-difluorophenyl)-3-
methyl-2-[1,2,4]-triazol-1-yl-methyl-oxirane (8) as oil.
This was obtained in 91.04% yield as oil. 1H NMR
(CDCl3): d 2.55–2.72 (m, 8H), 3.10–3.18 (m, 4H), 6.58–
6.62 (d, 2H), 8.04–8.10 (d, 2H). MS: m=z 261 (M+1).
4.11. 3-[4-(3-Trifluoromethylphenyl)-piperazin-1-yl]-pro-
pionitrile (9d)
This was obtained in 89.43% yield as oil. 1H NMR
(CDCl3): d 2.57–2.73 (m, 8H), 3.08–3.16 (m, 4H), 6.03–
6.86 (m, 1H), 7.00–7.13 (m, 1H), 7.31–7.55 (m, 2H). MS:
m=z 284 (M+1).
4.12. 3-[4-(2-Chlorophenyl)-piperazin-1-yl]-propionitrile
(9e)
This was obtained in 89.40% yield as oil. 1H NMR
(CDCl3): d 2.56–2.70 (m, 8H), 3.07–3.15 (m, 4H), 6.46–
6.80 (m, 3H), 7.50–7.54 (m, 1H). MS: m=z 250 (M+1).
1
Yield: 2.04 g (81.27%). H NMR (CDCl3): d 1.62 (d,
J ¼ 5:5 Hz, 3H), 3.16 (q, J ¼ 5:5 Hz, 1H), 4.39 (d,
J ¼ 15:0 Hz, 1H), 4.62 (d, J ¼ 15:0 Hz, 1H), 6.63–6.80
(m, 2H), 6.91–7.03 (m, 1H), 8.01 (s, 1H), 8.15 (s, 1H).
MS: m=z 252 (M+1).
4.13. 3-[4-(3-Chlorophenyl)-piperazin-1-yl]-propionitrile
(9f)
This was obtained in 85.20% yield as oil. 1H NMR
(CDCl3): d 2.55–2.70 (m, 8H), 3.09–3.17 (m, 4H), 6.48–
6.83 (m, 3H), 7.52–7.55 (m, 1H). MS: m=z 250 (M+1).
4.8. 3-[4-(2-Methoxyphenyl)-piperazin-1-yl]-propionitrile
(9a)
4.14. 3-[4-(4-Chlorophenyl)-piperazin-1-yl]-propionitrile
(9g)
To a solution of 1-(2-methoxyphenyl)-piperazine (1.67 g,
8.7 mmol) in methanol (10 mL) was added TEA
(1.33 mL, 9.1 mmol) at 0 ꢁC followed by the addition of
acrylonitrile (0.5 mL, 8.74 mmol). After complete addi-
tion, the reaction mixture was stirred at ambient tem-
perature for 24 h. Excess TEA was removed under
This was obtained in 84.23% yield as oil. 1H NMR
(CDCl3): d 2.55–2.70 (m, 8H), 3.09–3.17 (m, 4H), 6.46
(d, J ¼ 8:2 Hz, 2H), 6.89 (d, J ¼ 8:2 Hz, 2H). MS: m=z
250 (M+1).

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R. S. Upadhayaya et al. / Bioorg. Med. Chem. 12 (2004) 2225–2238
4.15. 3-[4-Benzylpiperazin-1-yl]-propionitrile (9h)
33.46 mmol) in toluene (50 mL) was refluxed at boiling
point. After 48 h, the reaction mixture was cooled and
toluene was removed under reduced pressure. The resi-
due was dissolved in water (20 mL), acidified with
aqueous HCl (10%) and washed it with chloroform
(2 · 20 mL). The chloroform layer was discarded and the
aqueous layer was basified with solid NaHCO3 (pH 10),
washed it with ether (2 · 20 mL). The aqueous layer was
concentrated under reduced pressure to get a solid. The
solid was taken in MeOH–CHCl3 (1:9, 50 mL) and
stirred for 1 h, then filtered off, residue was washed with
CHCl3 and filtrate was evaporated to get 10a. Yield:
This was obtained in 89.46% yield as oil. 1H NMR
(CDCl3): d 2.30–3.12 (m, 12H), 3.52 (s, 2H), 7.24–7.27
(m, 5H). MS: m=z 230 (M+1).
4.16. 3-[4-(4-Fluorophenyl)-piperazin-1-yl]-propionitrile
(9i)
This was obtained in 92.30% yield as oil. 1H NMR
(CDCl3): d 2.58–2.72 (m, 8H), 3.09–3.18 (m, 4H), 6.50–
6.73 (m, 4H). MS: m=z 234 (M+1).
1
82.00%, oil. H NMR (CDCl3): d 2.57–2.67 (m, 8H),
3.00–3.11 (m, 4H), 3.64 (s, 3H), 6.14–6.62 (m, 4H), 7.90
(br s, 1H). MS: m=z 401 (M+1).
4.17. 3-[4-(2,4-Dichlorophenyl)-piperazin-1-yl]-propio-
nitrile (9j)
The other 1-aryl-4-[2-(1H-tetrazol-5-yl)-ethyl]-pipera-
zines (10b–n) were also prepared by the same procedure
as described above and their spectral and analytical data
are given below.
This was obtained in 90.24% yield as oil. 1H NMR
(CDCl3): d 2.55–2.70 (m, 8H), 3.07–3.15 (m, 4H), 6.91–
7.02 (m, 2H), 7.36–7.39 (m, 2H). MS: m=z 284 (M+1),
286 (M+3).
4.23. 1-(4-Methoxyphenyl)-4-[2-(1H-tetrazol-5-yl)-ethyl]-
piperazine (10b)
4.18. 3-[4-(2-Pyridyl)-piperazin-1-yl]-propionitrile (9k)
This was obtained in 84.20% yield as oil. 1H NMR
(CDCl3): d 2.57–2.65 (m, 8H), 3.09–3.17 (m, 4H), 3.62
(s, 3H), 6.22 (d, J ¼ 8:0 Hz, 2H), 6.60 (d, J ¼ 8:0 Hz,
2H), 7.90 (br s, 1H). MS: m=z 289 (M+1).
This was obtained in 84.60% yield as oil. 1H NMR
(CDCl3): d 2.45–2.62 (m, 8H), 3.24–3.32 (m, 4H), 6.46
(m, 2H), 7.54–7.57 (m, 1H), 8.08 (m, 1H). MS: m=z 217
(M+1).
4.24. 1-(4-Nitrophenyl)-4-[2-(1H-tetrazol-5-yl)-ethyl]-
piperazine (10c)
4.19. 3-[4-Diphenylmethyl-piperazin-1-yl]-propionitrile
(9l)
This was obtained in 86.32% yield as oil. 1H NMR
(CDCl3): d 2.58–2.74 (m, 8H), 3.10–3.17 (m, 4H), 7.10
(d, J ¼ 8:0 Hz, 2H), 7.78 (d, J ¼ 8:0 Hz, 2H), 7.90 (br s,
1H). MS: m=z 304 (M+1).
This was obtained in 89.32% yield as oil. 1H NMR
(CDCl3): d 2.42–3.15 (m, 12H), 4.18 (s, 1H), 7.12–7.23
(m, 10H). MS: m=z 306 (M+1).
4.20. 3-[4-(2-Butoxyphenyl)-piperazin-1-yl]-propionitrile
(9m)
4.25. 1-(3-Trifluoromethylphenyl)-4-[2-(1H-tetrazol-5-yl)-
ethyl]-piperazine (10d)
This was obtained in 90.60% yield as oil. 1H NMR
(CDCl3): d 0.97 (t, J ¼ 7:0 Hz, 3H), 1.58–1.65 (m, 4H),
2.57–2.70 (m, 8H), 3.03–3.11 (m, 4H), 4.03 (t,
J ¼ 7:0 Hz, 2H), 6.60–6.83 (m, 2H), 6.90–7.12 (m, 2H).
MS: m=z 288 (M+1).
This was obtained in 89.20% yield as a solid, mp 183–
185 ꢁC (lit.18 mp 184–186 ꢁC). 1H NMR (CDCl3): d
2.58–2.66 (m, 8H), 3.09–3.18 (m, 4H), 6.68–6.86 (m,
4H), 7.90 (br s, 1H). MS: m=z 327 (M+1).
4.26. 1-(2-Chlorophenyl)-4-[2-(1H-tetrazol-5-yl)-ethyl]-
piperazine (10e)
4.21. 3-[4-(Phenyl)-piperazin-1-yl]-propionitrile (9n)
This was obtained in 93.00% yield as a solid, mp 70–
72 ꢁC (lit.21 mp 71.3–72.3 ꢁC). 1H NMR (CDCl3): d
2.56–2.71 (m, 8H), 3.12–3.20 (m, 4H), 6.77–7.21 (m,
5H). MS: m=z 216 (M+1).
This was obtained in 88.20% yield as oil. 1H NMR
(CDCl3): d 2.57–2.74 (m, 8H), 3.06–3.14 (m, 4H), 6.50–
6.95 (m, 4H), 7.90 (br s, 1H). MS: m=z 293 (M+1), 295
(M+3).
4.22. 1-(2-Methoxyphenyl)-4-[2-(1H-tetrazol-5-yl)-ethyl]-
piperazine (10a)
4.27. 1-(3-Chlorophenyl)-4-[2-(1H-tetrazol-5-yl)-ethyl]-
piperazine (10f)
A mixture of 9a (2.0 g, 8.3 mmol), triethylamine hydro-
chloride (4.60 g, 33.46 mmol) and sodium azide (2.17 g,
This was obtained in 86.30% yield as a solid, mp 163–
164 ꢁC (lit.18 mp 165–166 ꢁC). 1H NMR (CDCl3): d

R. S. Upadhayaya et al. / Bioorg. Med. Chem. 12 (2004) 2225–2238
2233
2.58–2.74 (m, 8H), 3.10–3.18 (m, 4H), 6.60–6.93 (m,
4H), 7.89 (br s, 1H). MS: m=z 293 (M+1), 295 (M+3).
2.58–2.72 (m, 8H), 3.12–3.20 (m, 4H), 4.04 (t,
J ¼ 7:0 Hz, 2H), 6.38–6.46 (m, 2H), 6.66–6.91 (m, 3H),
7.91 (br s, 1H). MS: m=z 331 (M+1).
4.28. 1-(4-Chlorophenyl)-4-[2-(1H-tetrazol-5-yl)-ethyl]-
piperazine (10g)
4.35. 1-Phenyl-4-[2-(1H-tetrazol-5-yl)-ethyl]-piperazine
(10n)
This was obtained in 83.26% yield as a solid, mp 218–
221 ꢁC (lit.18 mp 220–221 ꢁC). 1H NMR (CDCl3): d
2.58–2.72 (m, 8H), 3.10–3.18 (m, 4H), 6.71 (d,
J ¼ 8:0 Hz, 2H), 6.80 (m, J ¼ 8:0 Hz, 2H), 7.90 (br s,
1H). MS: m=z 293 (M+1), 295 (M+3).
This was obtained in 86.80% yield as a solid, mp 200–
203 ꢁC (lit.18 mp 200–201 ꢁC). 1H NMR (CDCl3): d
2.57–2.72 (m, 8H), 3.10–3.17 (m, 4H), 6.82–7.22 (m,
5H), 7.90 (br s, 1H). MS: m=z 259 (M+1).
4.29. 1-Benzyl-4-[2-(1H-tetrazol-5-yl)-ethyl]-piperazine
(10h)
4.36. (2R,3S)-2-(2,4-Difluorophenyl)-3-(5-{2-[4-(2-meth-
oxyphenyl)-piperazin-1-yl]-ethyl}-tetrazol-2-yl)-1-[1,2,4]-
triazol-1-yl-butan-2-ol (11a) and (2R,3S)-2-(2,4-difluoro-
phenyl)-3-(5-{2-[4-(2-methoxyphenyl)-piperazin-1-yl]-
ethyl}-tetrazol-1-yl)-1-[1,2,4]-triazol-1-yl-butan-2-ol
(12a)
This was obtained in 87.90% yield as oil. 1H NMR
(CDCl3): d 2.32–2.40 (m, 8H), 2.56–2.67 (m, 4H), 3.52
(s, 2H), 6.70–6.81 (m, 5H), 7.09–7.28 (br s, 1H). MS: m=z
273 (M+1).
A mixture of 8 (0.50 g, 1.99 mmol), K2CO3 (1.37 g,
9.96 mmol), 10a (0.55 g, 2.29 mmol) in NMP (5 mL) and
DMF (4 mL) was stirred at 90 ꢁC for 10 h. The reaction
mixture was cooled to room temperature and poured
into water (20 mL). The water layer was extracted with
ethyl acetate (50 mL), washed with water (2 · 20 mL),
brine (2 · 10 mL), dried over anhydrous Na2SO4 and
filtered. The filtrate was concentrated under reduced
pressure to give crude product. The crude product was
purified by column chromatography over silica gel (230–
400 mesh) using MeOH–CHCl3 (2:98) as eluent to
obtain 11a and 12a.
4.30. 1-(4-Fluorophenyl)-4-[2-(1H-tetrazol-5-yl)-ethyl]-
piperazine (10i)
This was obtained in 88.60% yield as a solid, mp 193–
195 ꢁC (lit.18 mp 194–196 ꢁC). 1H NMR (CDCl3): d
2.58–2.67 (m, 8H), 3.09–3.17 (m, 4H), 6.48 (d,
J ¼ 8:0 Hz, 2H), 6.88 (d, J ¼ 8:0 Hz, 2H), 7.90 (br s,
1H). MS: m=z 277 (M+1).
4.31. 1-(2,4-Dichlorophenyl)-4-[2-(1H-tetrazol-5-yl)-
ethyl]-piperazine (10j)
This was obtained in 88.30% yield as a solid, mp 206–
208 ꢁC (lit.18 mp 207–209 ꢁC). 1H NMR (CDCl3): d
2.56–2.72 (m, 8H), 3.09–3.17 (m, 4H), 6.92–7.03 (m,
2H), 7.36–7.39 (m, 1H), 7.90 (br s, 1H). MS: m=z 327
(M+1), 329 (M+3).
4.37. (2R,3S)-2-(2,4-Difluorophenyl)-3-(5-{2-[4-(2-meth-
oxyphenyl)-piperazin-1-yl]-ethyl}-tetrazol-2-yl)-1-[1,2,4]-
triazol-1-yl-butan-2-ol (11a)
Yield: 40.80%, oil. 1H NMR (CDCl3): d 1.02 (d,
J ¼ 7:0 Hz, 3H), 2.60–2.70 (m, 4H), 2.90–2.95 (m, 2H),
3.02–3.11 (m, 2H), 3.30–3.41 (m, 4H), 3.70 (s, 3H), 3.95
(q, J ¼ 7:0 Hz, 1H), 4.28 (d, J ¼ 14:0 Hz, 1H), 4.46 (d,
J ¼ 14:0 Hz, 1H), 5.45 (br s, 1H), 6.20–6.51 (m, 4H),
6.90–6.96 (m, 2H), 7.93–7.98 (m, 1H), 8.06 (s, 1H), 8.22
(s, 1H). MS: m=z 540 (M+1). Anal. Calcd for
C26H31F2N9O2: C, 57.87; H, 5.79; N, 23.36. Found: C,
57.99; H, 5.84; N, 23.49.
4.32. 1-(2-Pyridyl)-4-[2-(1H-tetrazol-5-yl)-ethyl]-pipera-
zine (10k)
This was obtained in 87.40% yield as oil. 1H NMR
(CDCl3): d 2.58–2.72 (m, 8H), 3.24–3.32 (m, 4H), 6.46–
6.51 (m, 2H), 7.56–7.58 (m, 1H), 7.91 (br s, 1H), 8.00–
8.08 (m, 1H). MS: m=z 260 (M+1).
4.33. 1-Diphenylmethyl-4-[2-(1H-tetrazol-5-yl)-ethyl]-
piperazine (10l)
4.38. (2R,3S)-2-(2,4-Difluorophenyl)-3-(5-{2-[4-(2-meth-
oxyphenyl)-piperazin-1-yl]-ethyl}-tetrazol-1-yl)-1-[1,2,4]-
triazol-1-yl-butan-2-ol (12a)
This was obtained in 86.29% yield as oil. 1H NMR
(CDCl3): d 2.57–2.74 (m, 8H), 2.34–2.42 (m, 4H), 4.18
(s, 1H), 7.03–7.20 (m, 10H), 7.90 (br s, 1H). MS: m=z 345
(M+1).
Yield: 13.50%, oil. 1H NMR (CDCl3): d 0.97 (d,
J ¼ 7:0 Hz, 3H), 2.60–2.70 (m, 4H), 2.90–2.95 (m, 2H),
3.02–3.11 (m, 2H), 3.30–3.41 (m, 4H), 3.71 (s, 3H), 4.08
(q, J ¼ 7:0 Hz, 1H), 4.32 (d, J ¼ 14:0 Hz, 1H), 4.53 (d,
J ¼ 14:0 Hz, 1H), 5.45 (br s, 1H), 6.22–6.54 (m, 4H),
6.97–7.03 (m, 2H), 7.99–8.03 (m, 1H), 8.06 (s, 1H), 8.21
(s, 1H). MS: m=z 540 (M+1). Anal. Calcd for
C26H31F2N9O2: C, 57.87; H, 5.79; N, 23.36. Found: C,
58.03; H, 5.96; N, 23.55.
4.34. 1-(2-Butoxyphenyl)-4-[2-(1H-tetrazol-5-yl)-ethyl]-
piperazine (10m)
This was obtained in 83.70% yield as oil. 1H NMR
(CDCl3): d 0.98 (t, J ¼ 7:0 Hz, 3H), 1.59–1.61 (m, 4H),

2234
R. S. Upadhayaya et al. / Bioorg. Med. Chem. 12 (2004) 2225–2238
The other compounds of this series (11b–n) and (12b–n)
were also prepared by the same procedure as described
above and their spectral and analytical data are given
below.
Calcd for C25H28ClF2N9O: C, 55.20; H, 5.19; N, 23.17.
Found: C, 55.49; H, 5.37; N, 23.44.
4.43. (2R,3S)-2-(2,4-Difluorophenyl)-3-(5-{2-[4-(3-chloro-
phenyl)-piperazin-1-yl]-ethyl}-tetrazol-2-yl)-1-[1,2,4]-
triazol-1-yl-butan-2-ol (11f)
4.39. (2R,3S)-2-(2,4-Difluorophenyl)-3-(5-{2-[4-(4-meth-
oxyphenyl)-piperazin-1-yl]-ethyl}-tetrazol-2-yl)-1-[1,2,4]-
triazol-1-yl-butan-2-ol (11b)
This was obtained in 36.60% yield as oil. 1H NMR
(CDCl3): d 1.02 (d, J ¼ 7:0 Hz, 3H), 2.60–2.70 (m, 4H),
2.90–2.95 (m, 2H), 3.02–3.11 (m, 2H), 3.30–3.41 (m,
4H), 3.99 (q, J ¼ 7:0 Hz, 1H), 4.27 (d, J ¼ 14:0 Hz, 1H),
4.50 (d, J ¼ 14:0 Hz, 1H), 5.42 (br s, 1H), 6.25–6.50 (m,
4H), 7.01–7.10 (m, 2H), 7.85–7.89 (m, 1H), 8.05 (s, 1H),
8.21 (s, 1H). MS: m=z 544 (M+1), 546 (M+3). Anal.
Calcd for C25H28ClF2N9O: C, 55.20; H, 5.19; N, 23.17.
Found: C, 55.57; H, 5.37; N, 23.22.
This was obtained in 38.00% yield as oil. 1H NMR
(CDCl3): d 1.03 (d, J ¼ 7:0 Hz, 3H), 2.60–2.70 (m, 4H),
2.90–2.95 (m, 2H), 3.02–3.11 (m, 2H), 3.30–3.41 (m,
4H), 3.72 (s, 3H), 4.00 (q, J ¼ 7:0 Hz, 1H), 4.30 (d,
J ¼ 14:0 Hz, 1H), 4.52 (d, J ¼ 14:0 Hz, 1H), 5.50 (br s,
1H), 6.20 (d, J ¼ 8:0 Hz, 2H), 6.55 (d, J ¼ 8:0 Hz, 2H),
6.85–6.90 (m, 2H), 7.90–7.99 (m, 1H), 8.10 (s, 1H), 8.23
(s, 1H). MS: m=z 540 (M+1). Anal. Calcd for
C26H31F2N9O2: C, 57.87; H, 5.79; N, 23.36. Found: C,
58.11; H, 6.04; N, 23.81.
4.44. (2R,3S)-2-(2,4-Difluorophenyl)-3-(5-{2-[4-(4-chloro-
phenyl)-piperazin-1-yl]-ethyl}-tetrazol-2-yl)-1-[1,2,4]-
triazol-1-yl-butan-2-ol (11g)
4.40. (2R,3S)-2-(2,4-Difluorophenyl)-3-(5-{2-[4-(4-nitro-
phenyl)-piperazin-1-yl]-ethyl}-tetrazol-2-yl)-1-[1,2,4]-tria-
zol-1-yl-butan-2-ol (11c)
This was obtained in 38.90% yield as oil. 1H NMR
(CDCl3): d 1.04 (d, J ¼ 7:0 Hz, 3H), 2.60–2.70 (m, 4H),
2.90–2.95 (m, 2H), 3.02–3.11 (m, 2H), 3.30–3.41 (m,
4H), 4.00 (q, J ¼ 7:0 Hz, 1H), 4.26 (d, J ¼ 14:0 Hz, 1H),
4.49 (t, J ¼ 14:0 Hz, 1H), 5.45 (br s, 1H), 6.19 (d,
J ¼ 8:0 Hz, 2H), 6.66 (d, J ¼ 8:0 Hz, 2H), 6.93–6.96 (m,
2H), 7.99–8.03 (m, 1H), 8.11 (s, 1H), 8.22 (s, 1H). MS:
m=z 544 (M+1), 546 (M+3). Anal. Calcd for
C25H28ClF2N9O: C, 55.20; H, 5.19; N, 23.17. Found: C,
55.32; H, 5.29; N, 23.33.
This was obtained in 36.70% yield as oil. 1H NMR
(CDCl3): d 1.04 (d, J ¼ 7:0 Hz, 3H), 2.60–2.70 (m, 4H),
2.90–2.95 (m, 2H), 3.02–3.11 (m, 2H), 3.30–3.41 (m,
4H), 3.96 (q, J ¼ 7:0 Hz, 1H), 4.26 (d, J ¼ 14:0 Hz, 1H),
4.47 (d, J ¼ 14:0 Hz, 1H), 5.52 (br s, 1H), 6.67–6.82 (m,
4H), 7.38–7.42 (m, 1H), 8.05 (s, 1H), 8.15 (s, 1H), 8.20
(d, J ¼ 9:0 Hz, 2H). MS: m=z 555 (M+1). Anal. Calcd
for C25H28F2N10O3: C, 54.15; H, 5.09; N, 25.26. Found:
C, 54.39; H, 5.24; N, 25.64.
4.45. (2R,3S)-2-(2,4-Difluorophenyl)-3-(5-{2-[4-benzyl-
piperazin-1-yl]-ethyl}-tetrazol-2-yl)-1-[1,2,4]-triazol-1-yl-
butan-2-ol (11h)
4.41. (2R,3S)-2-(2,4-Difluorophenyl)-3-(5-{2-[4-(3-trifluo-
romethylphenyl)-piperazin-1-yl]-ethyl}-tetrazol-2-yl)-1-
[1,2,4]-triazol-1-yl-butan-2-ol (11d)
This was obtained in 35.50% yield as oil. 1H NMR
(CDCl3): d 1.00 (d, J ¼ 7:0 Hz, 3H), 2.60–2.70 (m, 4H),
2.90–2.95 (m, 2H), 3.02–3.11 (m, 2H), 3.30–3.41 (m,
4H), 3.52 (s, 2H), 3.96 (q, J ¼ 7:0 Hz, 1H), 4.28 (d,
J ¼ 14:0 Hz, 1H), 4.50 (d, J ¼ 14:0 Hz, 1H), 5.50 (br s,
1H), 6.30–6.61 (m, 5H), 6.90–6.95 (m, 2H), 7.93–7.96
(m, 1H), 8.10 (s, 1H), 8.21 (s, 1H). MS: m=z 524 (M+1).
Anal. Calcd for C26H31F2N9O: C, 59.64; H, 5.97; N,
24.08. Found: C, 59.97; H, 6.24; N, 24.41.
This was obtained in 32.70% yield as oil. 1H NMR
(CDCl3): d 1.03 (d, J ¼ 7:0 Hz, 3H), 2.60–2.70 (m, 4H),
2.90–2.95 (m, 2H), 3.02–3.11 (m, 2H), 3.30–3.41 (m,
4H), 3.98 (q, J ¼ 7:0 Hz, 1H), 4.28 (d, J ¼ 14:0 Hz, 1H),
4.48 (d, J ¼ 14:0 Hz, 1H), 5.50 (br s, 1H), 6.30–6.55 (m,
4H), 6.90–6.95 (m, 2H), 7.90–7.98 (m, 1H), 8.03 (s, 1H),
8.22 (s, 1H). MS: m=z 578 (M+1). Anal. Calcd for
C26H28F5N9O: C, 54.07; H, 4.89; N, 21.83. Found: C,
54.44; H, 5.14; N, 22.12.
4.46. (2R,3S)-2-(2,4-Difluorophenyl)-3-(5-{2-[4-(4-fluoro-
phenyl)-piperazin-1-yl]-ethyl}-tetrazol-2-yl)-1-[1,2,4]-
triazol-1-yl-butan-2-ol (11i)
4.42. (2R,3S)-2-(2,4-Difluorophenyl)-3-(5-{2-[4-(2-chloro-
phenyl)-piperazin-1-yl]-ethyl}-tetrazol-2-yl)-1-[1,2,4]-
triazol-1-yl-butan-2-ol (11e)
This was obtained in 41.60% yield as oil. 1H NMR
(CDCl3): d 1.05 (d, J ¼ 7:0 Hz, 3H), 2.60–2.70 (m, 4H),
2.90–2.95 (m, 2H), 3.02–3.11 (m, 2H), 3.30–3.41 (m,
4H), 3.96 (q, J ¼ 7:0 Hz, 1H), 4.28 (d, J ¼ 14:0 Hz, 1H),
4.47 (d, J ¼ 14:0 Hz, 1H), 5.55 (br s, 1H), 6.30 (d,
J ¼ 8:0 Hz, 2H), 6.64 (d, J ¼ 8:0 Hz, 2H), 6.98–7.04 (m,
2H), 7.90–7.97 (m, 1H), 8.09 (s, 1H), 8.21 (s, 1H). MS:
m=z 528 (M+1). Anal. Calcd for C25H28F3N9O: C, 56.92;
H, 5.35; N, 23.90. Found: C, 57.19; H, 5.54; N, 24.14.
This was obtained in 40.60% yield as oil. 1H NMR
(CDCl3): d 1.00 (d, J ¼ 7:0 Hz, 3H), 2.60–2.70 (m, 4H),
2.90–2.95 (m, 2H), 3.02–3.11 (m, 2H), 3.30–3.41 (m,
4H), 4.00 (q, J ¼ 7:0 Hz, 1H), 4.29 (d, J ¼ 14:0 Hz, 1H),
4.47 (d, J ¼ 14:0 Hz, 1H), 5.48 (br s, 1H), 6.25–6.55 (m,
4H), 6.94-6.98 (m, 2H), 7.98–8.02 (m, 1H), 8.11 (s, 1H),
8.24 (s, 1H). MS: m=z 544 (M+1), 546 (M+3). Anal.

R. S. Upadhayaya et al. / Bioorg. Med. Chem. 12 (2004) 2225–2238
2235
4.47. (2R,3S)-2-(2,4-Difluorophenyl)-3-(5-{2-[4-(2,4-di-
chlorophenyl)-piperazin-1-yl]-ethyl}-tetrazol-2-yl)-1-
[1,2,4]-triazol-1-yl-butan-2-ol (11j)
4.51. (2R,3S)-2-(2,4-Difluorophenyl)-3-(5-{2-[4-phenyl-
piperazin-1-yl]-ethyl}-tetrazol-2-yl)-1-[1,2,4]-triazol-1-yl-
butan-2-ol (11n)
This was obtained in 41.70% yield as oil. 1H NMR
(CDCl3): d 1.03 (d, J ¼ 7:0 Hz, 3H), 2.60–2.70 (m, 4H),
2.90–2.95 (m, 2H), 3.02–3.11 (m, 2H), 3.30–3.41 (m,
4H), 3.98 (q, J ¼ 7:0 Hz, 1H), 4.32 (d, J ¼ 14:0 Hz, 1H),
4.45 (d, J ¼ 14:0 Hz, 1H), 5.55 (br s, 1H), 6.25–6.45 (m,
3H), 6.94–6.99 (m, 2H), 7.90–7.95 (m, 1H), 8.04 (s, 1H),
8.22 (s, 1H). MS: m=z 578 (M+1). Anal. Calcd for
C25H27Cl2F2N9O: C, 51.91; H, 4.70; N, 21.79. Found: C,
52.09; H, 4.85; N, 21.91.
This was obtained in 43.80% yield as oil. 1H NMR
(CDCl3): d 1.06 (d, J ¼ 7:0 Hz, 3H), 2.60–2.70 (m, 4H),
2.90–2.95 (m, 2H), 3.02–3.11 (m, 2H), 3.30–3.41 (m,
4H), 4.05 (q, J ¼ 7:0 Hz, 1H), 4.35 (d, J ¼ 14:0 Hz, 1H),
4.65 (d, J ¼ 14:0 Hz, 1H), 5.45 (br s, 1H), 6.15–6.56 (m,
5H), 6.85–6.93 (m, 2H), 7.80–7.92 (m, 1H), 8.04 (s, 1H),
8.16 (s, 1H). MS: m=z 510 (M+1). Anal. Calcd for
C25H29F2N9O: C, 58.93; H, 5.74; N, 24.74. Found: C,
59.17; H, 5.94; N, 24.86.
4.48. (2R,3S)-2-(2,4-Difluorophenyl)-3-(5-{2-[4-(2-pyr-
idyl)-piperazin-1-yl]-ethyl}-tetrazol-2-yl)-1-[1,2,4]-triazol-
1-yl-butan-2-ol (11k)
4.52. (2R,3S)-2-(2,4-Difluorophenyl)-3-(5-{2-[4-(4-meth-
oxyphenyl)-piperazin-1-yl]-ethyl}-tetrazol-1-yl)-1-[1,2,4]-
triazol-1-yl-butan-2-ol (12b)
This was obtained in 39.70% yield as oil. 1H NMR
(CDCl3): d 1.04 (d, J ¼ 7:0 Hz, 3H), 2.60–2.70 (m, 4H),
2.90–2.95 (m, 2H), 3.02–3.11 (m, 2H), 3.30–3.41 (m,
4H), 4.00 (q, J ¼ 7:0 Hz, 1H), 4.28 (d, J ¼ 14:0 Hz, 1H),
4.51 (d, J ¼ 14:0 Hz, 1H), 5.40 (br s, 1H), 6.26–6.44 (m,
3H), 6.94–6.99 (m, 2H), 7.90–7.97 (m, 2H), 8.09 (s, 1H),
8.23 (s, 1H). MS: m=z 511 (M+1). Anal. Calcd for
C24H28F2N10O: C, 56.46; H, 5.53; N, 27.44. Found: C,
56.77; H, 5.91; N, 27.71.
This was obtained in 12.60% yield as oil. 1H NMR
(CDCl3): d 0.98 (d, J ¼ 7:0 Hz, 3H), 2.60–2.70 (m, 4H),
2.90–2.95 (m, 2H), 3.02–3.11 (m, 2H), 3.30–3.41 (m,
4H), 3.72 (s, 3H), 4.00 (q, J ¼ 7:0 Hz, 1H), 4.35 (d,
J ¼ 14:0 Hz, 1H), 4.55 (d, J ¼ 14:0 Hz, 1H), 5.37 (br s,
1H), 6.20 (d, J ¼ 8:0 Hz, 2H), 6.55 (d, J ¼ 8:0 Hz, 2H),
6.92–6.97 (m, 2H), 7.96–7.99 (m, 1H), 8.06 (s, 1H), 8.20
(s, 1H). MS: m=z 540 (M+1). Anal. Calcd for
C26H31F2N9O2: C, 57.87; H, 5.79; N, 23.36. Found: C,
58.01; H, 5.93; N, 23.55.
4.49. (2R,3S)-2-(2,4-Difluorophenyl)-3-(5-{2-[4-(diphen-
ylmethyl)-piperazin-1-yl]-ethyl}-tetrazol-2-yl)-1-[1,2,4]-
triazol-1-yl-butan-2-ol (11l)
4.53. (2R,3S)-2-(2,4-Difluorophenyl)-3-(5-{2-[4-(4-nitro-
phenyl)-piperazin-1-yl]-ethyl}-tetrazol-1-yl)-1-[1,2,4]-tria-
zol-1-yl-butan-2-ol (12c)
This was obtained in 44.00% yield as oil. 1H
NMR (CDCl3): d 1.06 (d, J ¼ 7:0 Hz, 3H), 2.60–2.70
(m, 4H), 2.90–2.95 (m, 2H), 3.02–3.11 (m, 2H),
3.30–3.41 (m, 4H), 4.00 (q, J ¼ 7:0 Hz, 1H), 4.20 (s, 1H),
4.30 (d, J ¼ 14:0 Hz, 1H), 4.50 (d, J ¼ 14:0 Hz, 1H),
5.55 (br s, 1H), 6.25–6.62 (m, 10H), 6.85–6.90 (m, 2H),
8.00–8.03 (m, 1H), 8.09 (s, 1H), 8.23 (s, 1H). MS:
m=z 600 (M+1). Anal. Calcd for C32H35F2N9O: C, 64.09;
H, 5.88; N, 21.02. Found: C, 64.27; H, 6.05; N,
21.19.
This was obtained in 14.80% yield as oil. 1H NMR
(CDCl3): d 0.99 (d, J ¼ 7:0 Hz, 3H), 2.60–2.70 (m, 4H),
2.90–2.95 (m, 2H), 3.02–3.11 (m, 2H), 3.30–3.41 (m,
4H), 4.10 (q, J ¼ 7:0 Hz, 1H), 4.31 (d, J ¼ 14:0 Hz, 1H),
4.52 (d, J ¼ 14:0 Hz, 1H), 5.40 (br s, 1H), 6.75–6.82 (m,
4H), 7.40–7.44 (m, 1H), 8.05 (s, 1H), 8.18 (s, 1H), 8.22
(d, J ¼ 9:0 Hz, 2H). MS: m=z 555 (M+1). Anal. Calcd
for C25H28F2N10O3: C, 54.15; H, 5.09; N, 25.26. Found:
C, 54.42; H, 5.37; N, 25.41.
4.50. (2R,3S)-2-(2,4-Difluorophenyl)-3-(5-{2-[4-(2-but-
oxyphenyl)-piperazin-1-yl]-ethyl}-tetrazol-2-yl)-1-[1,2,4]-
triazol-1-yl-butan-2-ol (11m)
4.54. (2R,3S)-2-(2,4-Difluorophenyl)-3-(5-{2-[4-(3-trifluo-
romethylphenyl)-piperazin-1-yl]-ethyl}-tetrazol-1-yl)-1-
[1,2,4]-triazol-1-yl-butan-2-ol (12d)
This was obtained in 42.30% yield as oil. 1H NMR
(CDCl3):
d
0.93 (t, J ¼ 6:0 Hz, 3H), 1.03 (d,
This was obtained in 13.80% yield as oil. 1H
NMR (CDCl3): d 0.98 (d, J ¼ 7:0 Hz, 3H), 2.60–2.70
(m, 4H), 2.90–2.95 (m, 2H), 3.02–3.11 (m, 2H), 3.30–
3.41 (m, 4H), 4.04 (q, J ¼ 7:0 Hz, 1H), 4.33
(d, J ¼ 14:0 Hz, 1H), 4.53 (d, J ¼ 14:0 Hz, 1H), 5.42 (br
s, 1H), 6.28–6.60 (m, 4H), 6.95–6.99 (m, 2H), 7.96–8.00
(m, 1H), 8.04 (s, 1H), 8.20 (s, 1H). MS: m=z 578
(M+1). Anal. Calcd for C26H28F5N9O: C, 54.07;
H, 4.89; N, 21.83. Found: C, 54.32; H, 5.09; N,
22.21.
J ¼ 7:0 Hz, 3H), 1.72–1.77 (m, 4H), 2.60–2.70 (m, 4H),
2.90–2.95 (m, 2H), 3.02–3.11 (m, 2H), 3.30–3.41 (m,
4H), 3.97 (q, J ¼ 7:0 Hz, 1H), 4.04–4.11 (m, 2H), 4.30
(d, J ¼ 14:0 Hz, 1H), 4.48 (d, J ¼ 14:0 Hz, 1H), 5.52
(br s, 1H), 6.20–6.43 (m, 4H), 7.32–7.36 (m, 2H), 7.90–
7.95 (m, 1H), 8.08 (s, 1H), 8.21 (s, 1H). MS: m=z
582 (M+1). Anal. Calcd for C29H37F2N9O2: C, 59.88;
H, 6.41; N, 21.67. Found: C, 60.12; H, 6.79; N,
21.91.

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R. S. Upadhayaya et al. / Bioorg. Med. Chem. 12 (2004) 2225–2238
4.55. (2R,3S)-2-(2,4-Difluorophenyl)-3-(5-{2-[4-(2-chloro-
phenyl)-piperazin-1-yl]-ethyl}-tetrazol-1-yl)-1-[1,2,4]-
triazol-1-yl-butan-2-ol (12e)
4.59. (2R,3S)-2-(2,4-Difluorophenyl)-3-(5-{2-[4-(4-fluoro-
phenyl)-piperazin-1-yl]-ethyl}-tetrazol-1-yl)-1-[1,2,4]-
triazol-1-yl-butan-2-ol (12i)
This was obtained in 17.60% yield as oil. 1H
NMR (CDCl3): d 0.95 (d, J ¼ 7:0 Hz, 3H), 2.60–2.70
(m, 4H), 2.90–2.95 (m, 2H), 3.02–3.11 (m, 2H),
3.30–3.41 (m, 4H), 4.12 (q, J ¼ 7:0 Hz, 1H), 4.30 (d,
J ¼ 14:0 Hz, 1H), 4.50 (d, J ¼ 14:0 Hz, 1H), 5.55 (br s,
1H), 6.25–6.60 (m, 4H), 6.90–6.94 (m, 2H), 7.92–8.01
(m, 1H), 8.09 (s, 1H), 8.22 (s, 1H). MS: m=z 544
(M+1), 546 (M+3). Anal. Calcd for C25H28ClF2N9O: C,
55.20; H, 5.19; N, 23.17. Found: C, 55.41; H, 5.39; N,
23.39.
This was obtained in 14.80% yield as oil. 1H
NMR (CDCl3): d 1.00 (d, J ¼ 7:0 Hz, 3H), 2.60–2.70
(m, 4H), 2.90–2.95 (m, 2H), 3.02–3.11 (m, 2H), 3.30–
3.41 (m, 4H), 4.15 (q, J ¼ 7:0 Hz, 1H), 4.34 (d,
J ¼ 14:0 Hz, 1H), 4.50 (d, J ¼ 14:0 Hz, 1H), 5.50 (br s,
1H), 6.28 (d, J ¼ 8:0 Hz, 2H), 6.60 (d, J ¼ 8:0 Hz, 2H),
6.98–7.02 (m, 2H), 7.92–7.97 (m, 1H), 8.10 (s, 1H), 8.23
(s, 1H). MS: m=z 528 (M+1). Anal. Calcd for
C25H28F3N9O: C, 56.92; H, 5.35; N, 23.90. Found: C,
57.27; H, 5.69; N, 24.09.
4.56. (2R,3S)-2-(2,4-Difluorophenyl)-3-(5-{2-[4-(3-chloro-
phenyl)-piperazin-1-yl]-ethyl}-tetrazol-1-yl)-1-[1,2,4]-
triazol-1-yl-butan-2-ol (12f)
4.60. (2R,3S)-2-(2,4-Difluorophenyl)-3-(5-{2-[4-(2,4-di-
chlorophenyl)-piperazin-1-yl]-ethyl}-tetrazol-1-yl)-1-
[1,2,4]-triazol-1-yl-butan-2-ol (12j)
This was obtained in 18.30% yield as oil. 1H
NMR (CDCl3): d 0.96 (d, J ¼ 7:0 Hz, 3H), 2.60–2.70
(m, 4H), 2.90–2.95 (m, 2H), 3.02–3.11 (m, 2H), 3.30–
3.41 (m, 4H), 4.14 (q, J ¼ 7:0 Hz, 1H), 4.30 (d,
J ¼ 14:0 Hz, 1H), 4.55 (d, J ¼ 14:0 Hz, 1H), 5.50 (br s,
1H), 6.25–6.50 (m, 4H), 7.01–7.09 (m, 2H), 7.90–7.93
(m, 1H), 8.07 (s, 1H), 8.23 (s, 1H). MS: m=z 544
(M+1), 546 (M+3). Anal. Calcd for C25H28ClF2N9O: C,
55.20; H, 5.19; N, 23.17. Found: C, 55.29; H, 5.32; N,
23.35.
This was obtained in 18.20% yield as oil. 1H
NMR (CDCl3): d 0.98 (d, J ¼ 7:0 Hz, 3H), 2.60–
2.70 (m, 4H), 2.90–2.95 (m, 2H), 3.02–3.11 (m, 2H),
3.30–3.41 (m, 4H), 4.08 (q, J ¼ 7:0 Hz, 1H), 4.34 (d,
J ¼ 14:0 Hz, 1H), 4.50 (d, J ¼ 14:0 Hz, 1H), 5.50 (br
s, 1H), 6.20–6.50 (m, 3H), 6.90–6.98 (m, 2H), 7.93–
7.98 (m, 1H), 8.06 (s, 1H), 8.20 (s, 1H). MS: m=z
578 (M+1). Anal. Calcd for C25H27Cl2F2N9O: C,
51.91; H, 4.70; N, 21.79. Found: C, 52.17; H, 4.97; N,
21.98.
4.57. (2R,3S)-2-(2,4-Difluorophenyl)-3-(5-{2-[4-(4-chloro-
phenyl)-piperazin-1-yl]-ethyl}-tetrazol-1-yl)-1-[1,2,4]-
triazol-1-yl-butan-2-ol (12g)
4.61. (2R,3S)-2-(2,4-Difluorophenyl)-3-(5-{2-[4-(2-pyr-
idyl)-piperazin-1-yl]-ethyl}-tetrazol-1-yl)-1-[1,2,4]-triazol-
1-yl-butan-2-ol (12k)
This was obtained in 19.30% yield as oil. 1H NMR
(CDCl3): d 1.00 (d, J ¼ 7:0 Hz, 3H), 2.60–2.70 (m, 4H),
2.90–2.95 (m, 2H), 3.02–3.11 (m, 2H), 3.30–3.41 (m,
4H), 4.13 (q, J ¼ 7:0 Hz, 1H), 4.30 (d, J ¼ 14:0 Hz, 1H),
4.54 (d, J ¼ 14:0 Hz, 1H), 5.54 (br s, 1H), 6.15 (d,
J ¼ 8:0 Hz, 2H), 6.70 (d, J ¼ 8:0 Hz, 2H), 6.90–6.94 (m,
2H), 7.99–8.04 (m, 1H), 8.08 (s, 1H), 8.24 (s, 1H). MS:
m=z 544 (M+1), 546 (M+3). Anal. Calcd for
C25H28ClF2N9O: C, 55.20; H, 5.19; N, 23.17. Found: C,
55.41; H, 5.37; N, 23.29.
This was obtained in 20.00% yield as oil. 1H
NMR (CDCl3): d 1.00 (d, J ¼ 7:0 Hz, 3H), 2.60–2.70
(m, 4H), 2.90–2.95 (m, 2H), 3.02–3.11 (m, 2H),
3.30–3.41 (m, 4H), 4.11 (q, J ¼ 7:0 Hz, 1H), 4.30 (d,
J ¼ 14:0 Hz, 1H), 4.55 (d, J ¼ 14:0 Hz, 1H), 5.45 (br s,
1H), 6.23–6.40 (m, 3H), 6.94–6.99 (m, 2H), 7.96–
8.00 (m, 2H), 8.08 (s, 1H), 8.21 (s, 1H). MS: m=z
511 (M+1). Anal. Calcd for C24H28F2N10O: C, 56.46;
H, 5.53; N, 27.44. Found: C, 56.63; H, 5.79; N,
27.59.
4.58. (2R,3S)-2-(2,4-Difluorophenyl)-3-(5-{2-[4-benzyl-
piperazin-1-yl]-ethyl}-tetrazol-1-yl)-1-[1,2,4]-triazol-1-yl-
butan-2-ol (12h)
4.62. (2R,3S)-2-(2,4-Difluorophenyl)-3-(5-{2-[4-(diphen-
ylmethyl)-piperazin-1-yl]-ethyl}-tetrazol-1-yl)-1-[1,2,4]-
triazol-1-yl-butan-2-ol (12l)
This was obtained in 19.80% yield as oil. 1H
NMR (CDCl3): d 0.97 (d, J ¼ 7:0 Hz, 3H), 2.60–2.70
(m, 4H), 2.90–2.95 (m, 2H), 3.02–3.11 (m, 2H), 3.30–
3.41 (m, 4H), 3.50 (s, 2H), 4.12 (q, J ¼ 7:0 Hz, 1H), 4.34
(d, J ¼ 14:0 Hz, 1H), 4.50 (d, J ¼ 14:0 Hz, 1H), 5.45
(br s, 1H), 6.32–6.56 (m, 5H), 6.90–6.95 (m, 2H),
7.90–7.94 (m, 1H), 8.08 (s, 1H), 8.23 (s, 1H). MS:
m=z 524 (M+1). Anal. Calcd for C26H31F2N9O: C,
59.64; H, 5.97; N, 24.08. Found: C, 59.89; H, 6.13; N,
24.24.
This was obtained in 17.30% yield as oil. 1H
NMR (CDCl3): d 0.99 (d, J ¼ 7:0 Hz, 3H), 2.60–2.70
(m, 4H), 2.90–2.95 (m, 2H), 3.02–3.11 (m, 2H), 3.30–
3.41 (m, 4H), 4.09 (q, J ¼ 7:0 Hz, 1H), 4.21 (s, 1H),
4.32 (d, J ¼ 14:0 Hz, 1H), 4.53 (d, J ¼ 14:0 Hz, 1H),
5.40 (br s, 1H), 6.20–6.60 (m, 10H), 6.94–6.97 (m,
2H), 8.02–8.03 (m, 1H), 8.10 (s, 1H), 8.23 (s, 1H). MS:
m=z 600 (M+1). Anal. Calcd for C32H35F2N9O: C,
64.09; H, 5.88; N, 21.02. Found: C, 64.37; H, 5.99; N,
21.31.

R. S. Upadhayaya et al. / Bioorg. Med. Chem. 12 (2004) 2225–2238
2237
4.63. (2R,3S)-2-(2,4-Difluorophenyl)-3-(5-{2-[4-(2-but-
oxyphenyl)-piperazin-1-yl]-ethyl}-tetrazol-1-yl)-1-[1,2,4]-
triazol-1-yl-butan-2-ol (12m)
References and notes
1. Sheehan, D. J.; Hitchcock, C. A.; Sibley, C. M. Clin.
Microbiol. Rev. 1999, 12, 40.
This was obtained in 15.70% yield as oil. 1H NMR
(CDCl3): d 0.94 (t, J ¼ 6:0 Hz, 3H), 0.99 (d, J ¼ 7:0 Hz,
3H), 1.70–1.74 (m, 4H), 2.60–2.70 (m, 4H), 2.90–2.95
(m, 2H), 3.02–3.11 (m, 2H), 3.30–3.41 (m, 4H), 4.05–
4.10 (m, 2H), 4.12 (q, J ¼ 7:0 Hz, 1H), 4.38 (d,
J ¼ 14:0 Hz, 1H), 4.52 (d, J ¼ 14:0 Hz, 1H), 5.48 (br s,
1H), 6.22–6.40 (m, 4H), 7.38–7.45 (m, 2H), 7.92–7.98
(m, 1H), 8.09 (s, 1H), 8.23 (s, 1H). MS: m=z 582 (M+1).
Anal. Calcd for C29H37F2N9O2: C, 59.88; H, 6.41; N,
21.67. Found: C, 60.07; H, 6.59; N, 21.72.
2. (a) Vanden Bossche, H. In Current Topics in Medical
Mycology; McGuinnis, M. R., Ed.; Springer: Berlin, 1985;
Vol. 1, pp 313–351; (b) Aoyama, Y.; Yoshida, Y.; Sato, R.
J. Biol. Chem. 1984, 259, 1661; (c) Trzaskos, J. M.; Bowen,
W. D.; Shafiee, A.; Fischer, R. T.; Gaylor, J. L. J. Biol.
Chem. 1984, 259, 13402.
3. Richardson, K.; Brammer, K. W.; Marriott, M. S.;
Troke, P. F. Antimicrob. Agents Chemother. 1985, 27,
832.
4. Van Cutsem, J.; Van Gerven, F.; Janssen, P. A. J. In
Recent Trends in the Discovery, Development, and Evalu-
ation of Antifungal Agents; Fromtling, R. A., Ed.; Prous:
Barcelona, 1987; pp 177–192.
5. Dickinson, R. P.; Bell, A. S.; Hitchcock, C. A.; Naya-
yanaswami, S.; Ray, S. J.; Richardson, K.; Troke, P. F.
Bioorg. Med. Chem. Lett. 1996, 6, 2031.
6. (a) Formtling, R. A. Drugs Future 1996, 21, 160; (b)
Saksena, A. K.; Girijavallabhan, V. M.; Wang, H.; Liu,
Y.-T.; Pike, R. E.; Ganguly, A. K. Tetrahedron Lett. 1996,
37, 5657.
7. (a) Hata, K.; Kimura, J.; Miki, H.; Toyosawa, T.;
Moriyama, M.; Katsu, K. Antimicrob. Agents Chemother.
1996, 40, 2237; (b) Hata, K.; Kimura, J.; Miki, H.;
Toyosawa, T.; Moriyama, M.; Katsu, K. Antimicrob.
Agents Chemother. 1996, 40, 2243; (c) Kaku, Y.; Tsu-
ruoka, A.; Kakinuma, H.; Tsukada, I.; Yanagisawa, M.;
Naito, T. Chem. Pharm. Bull. 1998, 46, 1125.
8. Konosu, T.; Tajima, Y.; Oida, S. Chem. Pharm. Bull. 1991,
39, 2241.
4.64. (2R,3S)-2-(2,4-Difluorophenyl)-3-(5-{2-[4-phenyl-
piperazin-1-yl]-ethyl}-tetrazol-1-yl)-1-[1,2,4]-triazol-1-yl-
butan-2-ol (12n)
This was obtained in 17.90% yield as oil. 1H NMR
(CDCl3): d 1.04 (d, J ¼ 7:0 Hz, 3H), 2.50–2.57 (m, 4H),
2.76–2.82 (m, 4H), 3.00–3.30 (m, 4H), 4.00 (q,
J ¼ 7:0 Hz, 1H), 4.30 (d, J ¼ 14:0 Hz, 1H), 4.58 (d,
J ¼ 14:0 Hz, 1H), 5.40 (br s, 1H), 6.15–6.60 (m, 5H),
6.89–6.94 (m, 2H), 7.85–7.94 (m, 1H), 8.00 (s, 1H), 8.14
(s, 1H). MS: m=z 510 (M+1). Anal. Calcd for
C25H29F2N9O: C, 58.93; H, 5.74; N, 24.74. Found: C,
59.21; H, 5.89; N, 24.95.
9. Konosu, T.; Tajima, Y.; Takeda, N.; Miyaoka, T.;
Kasahara, M.; Yasuda, H.; Oida, S. Chem. Pharm. Bull.
1990, 38, 2476.
10. Richardson, K.; Cooper, K.; Marriott, M. S.; Tarbit, M.
H.; Troke, P. F.; Whittle, P. J. Ann. N.Y. Acad. Sci. 1988,
544, 6.
11. Tasaka, A.; Tsuchimori, N.; Kitazaki, T.; Hiroe, K.;
Hayashi, R.; Okonogi, K.; Itoh, K. Chem. Pharm. Bull.
1995, 43, 441.
12. Konosu, T.; Tajima, Y.; Takeda, N.; Miyaoka, T.;
Kasahara, M.; Yasuda, H.; Oida, S. Chem. Pharm. Bull.
1991, 39, 2581.
4.65. Hydrochloride salt of compound 11d
To a solution of 11d (144.3 mg, 0.25 mmol) in abso-
lute ethanol (2 mL) was added 1 M ethanolic-HCl
(9.13 mg, 0.25 mmol) at 5 ꢁC under stirring. The stirring
was continued at same temperature for 1 h. the solid
separated out was filtered through suction, washed
with diethyl ether (2 · 5 mL), dried under reduced pres-
sure to give a off-white solid in quantitative yield. MS:
m=z 578 (M+1). Anal. Calcd for C26H28F5N9OÆHCl: C,
50.86; H, 4.76; N, 20.53. Found: C, 51.23; H, 5.09; N,
20.82.
13. Bartroli, J.; Turmo, E.; Alguero, M.; Boncompte, E.;
Vericat, M. L.; Conte, L.; Ramis, J.; Merlos, M.;
Garcia-Rafanell, J.; Forn, J. J. Med. Chem. 1998, 41,
1869.
14. Verma, A. K.; Arora, S. K.; Arora, J. S.; Rattan, A. PCT
Application, WO 0166551, September 13, 2001.
15. Arora, S. K.; Sinha, N.; Jain, S.; Upadhayaya, R. S.;
Kishore, N. Unpublished results.
16. Singh, I. P.; Sidhu, I.; Palak, B. U.S. Patent 6133485,
October 17, 2000.
Using the same method as described above the hydro-
chloride salt of compound 12d was also prepared. MS:
m=z 578 (M+1). Anal. Calcd for C26H28F5N9OÆHCl: C,
50.86; H, 4.76; N, 20.53. Found: C, 50.93; H, 5.14; N,
20.66.
17. Kitazaki, T.; Tamura, N.; Tasaka, A.; Matsushita, Y.;
Hayashi, R.; Okongi, K.; Itoh, K. Chem. Pharm. Bull.
1996, 44, 314.
18. Hyao, S.; Havera, H. J.; Strycker, W. G.; Leipzig, T. J.;
Rodriguez, R. J. Med. Chem. 1967, 10, 400.
19. Strycker, W. J.; Hayao, S. U.S. Patent 3231574, January
25, 1966.
Acknowledgements
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Matsushita, Y.; Hayashi, R.; Okonogi, K.; Itoh, K. Chem.
Pharm. Bull. 2000, 48, 1935.
We wish to express our thanks to microbiology
department of New Chemical Entity Research, Lupin
Research Park, Pune for biological screening and
analytical chemistry department for 1H NMR, mass
spectroscopy, elemental analyses of compounds
synthesized.
23. Huff, L.; Henry, R. A. J. Med. Chem. 1970, 13, 777.

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