Discovery of novel indazole-linked triazoles as antifungal agents

Article abstract of DOI:10.1016/j.bmcl.2007.03.074

The in vitro and in vivo activities of a series of (2R, 3R)-2-(2,4-difluorophenyl)-3-(substituted indazol-1-yl)-1-(1H-1,2,4-triazol-1-yl)butan-2-ol as potential antifungal agents are described. In particular, the analog 12j having 5-bromo substitution on the indazole ring exhibited significant antifungal activity against a variety of fungal cultures (Candida spp. and Aspergillus spp.). In addition, oral administration of 12j showed its excellent efficacy against Candida albicans in a murine infection model and the significantly improved survival rates of the infected mice.

Full text of DOI:10.1016/j.bmcl.2007.03.074

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3486–3490
Discovery of novel indazole-linked triazoles as antifungal agents
Joon Seok Park,a Kyung A Yu,b Tae Hee Kang,a Sunghoon Kima and Young-Ger Suha,*
aCollege of Pharmacy, Seoul National University, San 56-1 Shinrim-Dong, Kwanak-Gu, Seoul 151-742, Republic of Korea
bPharmaceutical Research Institute, Daewoong Pharmaceutical Co., Ltd, 501-2, Samgye-ri, Pogok-myun,
Kyunggi-do 449-814, Republic of Korea
Received 9 February 2007; revised 20 March 2007; accepted 23 March 2007
Available online 27 March 2007
Abstract—The in vitro and in vivo activities of a series of (2R,3R)-2-(2,4-difluorophenyl)-3-(substituted indazol-1-yl)-1-(1H-1,2,4-
triazol-1-yl)butan-2-ol as potential antifungal agents are described. In particular, the analog 12j having 5-bromo substitution on the
indazole ring exhibited significant antifungal activity against a variety of fungal cultures (Candida spp. and Aspergillus spp.). In addi-
tion, oral administration of 12j showed its excellent efficacy against Candida albicans in a murine infection model and the signifi-
cantly improved survival rates of the infected mice.
ꢀ 2007 Elsevier Ltd. All rights reserved.
The incidence of systemic fungal infections such as can-
didosis, cryptococcosis, and aspergillosis has rapidly
been increasing due to an increase of the immunocom-
promised hosts. Thus, the development of new and clin-
ically useful antifungal azoles has been considered as
one of the most attractive approaches for the treatment
of these infections. In particular, attention has been paid
to the triazole derivatives because of their broad anti-
fungal spectrum and low toxicity.1 Triazoles are known
to displace lanosterol from lanosterol 14-demethylase
(14 DM), a cytochrome P-450-dependent enzyme and
block the biosynthesis of ergosterol, an essential compo-
nent of fungal cell membrane.2
terol,9,10 and the fluorobenzene moieties for high anti-
fungal activity,11,12 our structural modification focused
on the variation of the arylalkyl moiety. Thus, our pre-
liminary work on the development of new antifungal
triazoles included an introduction of a variety of linker
moiety such as hydrazine, semicarbazide, amide, urea,
and amine. Among these, the hydrazine-linked triazole
analogs, which have not been reported yet, were found
to have the potent antifungal activity and the substituted
benzyl-hydrazine analog (5) turned out to be most ac-
tive. However, we extended the structural modification
to a series of the indazole analogs depicted as 11 and
12, which are the cyclic hydrazine fused with benzyl
groups, because the hydrazine moiety was known as
an inappropriate drug moiety in terms of stability.
Fluconazole (1) is an antifungal agent of choice for the
treatment of infections by Candida albicans and Crypto-
coccus neoformans due to its potent activity, excellent
safety profile, and favorable pharmacokinetic character-
istics.3,4 However, its poor efficacy against Aspergillus
infections5 prompted the developments of new triazoles
such as voriconazole (2),6 albaconazole (3),7 and ravuco-
nazole (4),8 which are therapeutically useful for the
treatment of Aspergillus infection or under clinical trials
(Fig. 1).
In this paper, we describe synthesis and antifungal activ-
ity of the novel antifungal agents 11 and 12.
The title compounds 11a–j, 12b, 12d, and 12h–j were
prepared from the optically active oxirane intermediate
(10),13 which was conveniently derived from methyl
(R)-lactate in eight steps as shown in Scheme 1. The
amide intermediate 6 was prepared from methyl (R)-lac-
tate by amidation with morpholine followed by THP
protection. The amide 6 was then reacted with phenyl-
magnesium bromide, followed by epoxidation (Corey’s
reagent, DMSO) to afford the oxirane intermediate 7
as a diastereomeric mixture in favor of the desired dia-
stereomer (10R,2R)-7 (4:1). The diastereomeric mixture
was reacted with 1H-1,2,4-triazole in the presence of so-
Considering necessity of the C3-methyl, which functions
as an equivalent of the 13b-methyl group of lanos-
Keywords: Triazoles; Antifungal agents; Infection.
*
Corresponding author. Tel.: +82 2 880 7875; fax: +82 2 888 0649;
e-mail: ygsuh@snu.ac.kr
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.03.074

J. S. Park et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3486–3490
3487
N
N
N
N
OH
N
OH
N
OH
N
O
N
N
N
F
N
N
N
N
F
F
F
N
N
Cl
F
F
F
1
2
3
N
N
N
N
OH
N
OH
N
OH
N
N
N
N
R1
5
N
N
NH2
N
CN
F
F
S
F
3
4
R2
F
F
F
11 (N1)
12 (N2)
5
4
Figure 1. Structures of fluconazole (1), voriconazole (2), albaconazole (3), ravuconazole (4), and the novel triazole analogs (5, 11, and 12).
N
N
OH
H
Me
N
Me
H
Me
O
OTHP
O
H
*
Me
OTHP
OTHP
vi
i,
iii, iv
F
v
*
ii
O
N
OH
F
OMe
O
F
F
Methyl (R)-lactate
7
8
6
N
N
N
N
N
OH
N
O
N
OH
N
OH
R1
N
Me
N
Me
N
N
N
R1
N
+
OH
N
N
vii, viii
H
ix
F
F
F
F
R2
R2
F
F
F
F
11
12
9
10
Scheme 1. Reagents and conditions: (i) Morpholine, 85 ꢁC, 88%; (ii) 3,4-dihydro-2H-pyran, p-TsOH, CH2Cl2, 0 ꢁC, 85%; (iii) 2,4-F2C6H3MgBr,
THF, 88%; (iv) (CH3)3S+OIꢀ, NaH, DMSO; (v) 1,2,4-triazole, NaH, DMF, 80 ꢁC, 61% for two steps; (vi) PPTS, EtOH, 55 ꢁC, then recrystallization
(IPE), 54%; (vii) MsCl, EtOAc–CH2Cl2, 0 ꢁC; (viii) NaOMe, MeOH, 0 ꢁC, 80% for two steps; (ix) indazoles, K2CO3, DMF, 120 ꢁC, 25–47%.
dium hydride to give 8. The THP group of 8 was re-
moved with pyridinium p-toluenesulfonate and the
resulting diol was recrystallized to give the diastereomer-
ically pure diol (2R,3R)-9. The optically active diol 9
was converted to the oxirane (2R,3S)-10 by selective
mesylation and sodium methoxide-assisted epoxidation.
Finally, the oxirane 10 was reacted with various indaz-
oles in the presence of K2CO3 in DMF to give the con-
densation products14 as a single product or a mixture of
11 and 12, which were separated by flash column chro-
tio of isolated yields of the above two isomers suggested
that the 11b, 11d, and 11h–j isomers are the predomi-
nated one. The steric and electronic factors of substitu-
ents seem to affect the ratio of the produced isomers as
reported earlier.17
The antifungal activities of 11a–j, 12b, 12d, and 12h–j
were evaluated by in vitro broth microdilution assay
and their MIC80s are summarized in Table 1. Generally,
the indazole analogs 11 and 12 were more potent than
the hydrazine analog 5. The C5-nitro indazole analog
(11c) exhibited the most potent activity as well as the
broad spectrum compared to other analogs, which have
1
matography or preparative HPLC. The H NMR spec-
tra showed the characteristic resonance for C3-CH3 of
11 in more upfield compared to those of 12.15,16 The ra-

3488
J. S. Park et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3486–3490
Table 1. In vitro antifungal activity of R1-substituted analogs
Compound
R1
R2
MIC80 (lg/mL) of compounds and standard drugs against fungal culturea
CA
CG
CK
CN
CL
CT
CP
AF
Af
AT
AN
5
11a
1
nd
2
nd
nd
nd
nd
nd
nd
2
nd
1
nd
2
nd
4
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
60.015
60.015
60.015
60.015
60.015
0.25
0.5
1
0.063
0.063
0.125
0.063
0.031
0.031
60.015
60.015
2
11b
11c
4-NO2
5-NO2
6-NO2
7-NO2
5-NH2
5-N(CH3)2
5-F
2
0.031
0.063
0.25
60.015
60.015
60.015
60.015
0.25
60.015
0.031
60.015
60.015
1
1
2
1
2
4
0.25
0.5
0.5
8
1
1
2
2
11d
11e
8
2
2
2
4
4
0.125
1
0.25
2
1
2
4
11f
11g
>8
8
nd
1
nd
1
nd
2
nd
2
0.063
1
0.063
0.5
0.5
11h
11i
60.015
60.015
60.015
60.015
0.063
4
1
0.063
0.031
60.015
0.063
0.5
60.015
60.015
60.015
0.125
0.25
0.5
0.125
0.125
0.125
0.25
4
4
4
8
5-C1
5-Br
4
2
2
4
4
8
11j
12b
4
2
1
2
4
4
4
4-NO2
6-NO2
5-F
>8
>8
4
4
>8
1
1
2
1
4
12d
12h
0.5
8
0.25
32
0.25
0.25
0.25
>128
32
0.5
0.5
0.5
>128
128
0.5
0.5
0.5
>128
64
1
60.015
60.015
60.015
2
0.5
0.125
0.125
0.063
0.015
60.015
8
60.015
60.015
60.015
8
0.125
0.031
60.015
>8
0.031
60.015
60.015
>8
12i
12j
5-C1
5-Br
4
1
2
>8
1
>128
1 (Fluconazole)
>8
nd, not determined.
a CA, C. albicans ATCC 36082, CG, C. glabrata, ATCC 34138. CK, C. krusei ATCC 6528, CN, Cryptococcus neoformans ATCC 24065, CL, Candida
lusitanea ATCC 42720, CT, Candida tropicalis ATCC 750, CP, Candida parapsilosis ATCC 21019, AF, Aspergillus fumigatus ATCC 16424, Af,
Aspergillus flavus ATCC MYA-1004, AT, Aspergillus terreus ATCC 28301, AN, Aspergillus niger ATCC 9042.
Table 2. In vitro antifungal activity of R1 and R2-substituted compounds
Compound
R1
R2
MIC80 (lg/mL) of compounds and standard drugs against fungal culture
CA
CG
CK
CN
CL
CT
CP
AF
Af
AT
AN
11c
11k
111
11h
11m
11n
11o
11p
11q
11r
5-NO2
5-NO2
5-NO2
5-F
H
60.015
0.015
4
0.25
4
0.063
0.25
nd
60.015
0.063
nd
0.031
4
0.031
0.125
nd
1
1
16
2
2
16
Me
Ph
H
8
4
8
2
nd
4
nd
1
nd
0.25
4
nd
4
nd
4
nd
4
nd
60.015
0.031
0.063
0.5
60.015
0.125
0.063
0.125
0.25
0.125
0.5
8
16
5-F
5-F
Me
Et
8
4
4
8
4
60.015
60.015
0.031
4
2
0.125
0.125
0.5
2
0.125
0.25
0.5
16
16
16
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
4-F
6-F
Me
Me
Me
Me
8
1
1
>8
4
8
>8
1
5-C1
5-CF3
60.015
60.015
2
0.25
1
60.015
0.25
0.125
1
8
4
>8
nd, not determined.
nitro-substituent at other position. In particular, the ut-
most of 11c in antifungal activity was shown against
Aspergillus spp.
Candida glabrata and Candida krusei, known as the
fluconazole-resistant species, as the size of halogen in-
creases. Introduction of free amines at C5-position
(11f) almost eliminated the antifungal activity. However,
the N,N-dimethyl amine-substituted analog (11g)
showed an antifungal activity although it was less potent
against Candida spp. than the analogs, which possess the
electron-withdrawing substituent such as the nitro
group. The N-2 regioisomers 12h–j having halogen
groups were generally more active than the N-1 isomers
Thus, other substituents (11f–11j) were introduced at the
C5-position of the indazole ring and the antifungal activ-
ities of the corresponding analogs were evaluated. The
analogs with halogens at C5-position (11h–j) showed
remarkable antifungal activities against Aspergillus
spp. Generally, the potency decreased especially against
Table 3. In vitro antifungal activity of 12j
Compound
MIC80 (lg/mL) of 12j and standard drugs against fungal culture
CA
CG
CK
0.125
CN
CL
CT
CP
AF
Af
AT
AN
12j
<0.015
2
2
>8
4
60.015
8
60.015
8
60.015
>8
0.25
60.015
>8
0.25
0.25
>128
0.25
0.5
>128
0.5
0.5
1
>128
Fluconazole
Voriconazole
Albaconazole
Ravuconazole
Amphotericin B
>8
1
>128
60.015
60.015
60.015
nd
0.125
60.015
60.015
nd
0.063
60.015
60.015
nd
1
1
1
2
1
8
0.25
0.125
nd
0.031
60.015
nd
60.015
60.015
nd
0.5
0.125
1
0.5
0.5
4
2
nd
0.5
2
0.5
nd, not determined.

J. S. Park et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3486–3490
Table 4. In vitro hepatocyte toxicity of the selected analogs
3489
11a
12j
11o
11r
Ketoconazole
51.0 1.6
CC50 (lM)
599.0 124.4
141.3 4.3
8.1 2.31a
2.9 2.8a
a % Inhibition at 50 lM. Values are means of two or three (for CC50) experiments.
Table 5. In vivo efficacy (mean survival days) in Candida albicans
infection model in mice
promising leads for antifungal therapy. These analogs
showed potent antifungal activity, broad spectrum, sig-
nificantly low hepatocyte toxicity, and good oral efficacy.
Mean survival days
Control
11a
11c
12j
6.1
3.5
2.0
7.2
12.4a
13.5a
Acknowledgment
nd, not determined.
a P < 0.05 versus each vehicle control.
We thank the Pharmacology and Toxicology Research
group of Daewoong Pharm. Co., Ltd for their kind sup-
port for this work.
11h–j. However, The N-2 regioisomers 12b, d having ni-
tro groups were quite less active than the N-1 isomers
11b–e.
References and notes
1. Sheehan, D. J.; Hitchcock, C. A.; Sibley, C. M. Clin.
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Next, we investigated the C3-substituent (R2 group) ef-
fect of indazole ring on the antifungal activity, as shown
in Table 2. The antifungal activity decreased as the size
of the R2 increases. Especially, the phenyl derivative 11l
was almost devoid of antifungal activity. In addition,
the MICs of the analogs 11m, 11o, and 11p possessing
methyl as R2 and fluorine as R1 at various positions
were compared. Again, the 5-fluoro analog 11m turned
out to be the most potent compound among the C3-
methyl (R2) series. The other C3-methyl analogs 11q
and 11r were almost inactive against Aspergillus spp.
2. (a) Vanden Bossche, H. In Curr. Top. Med. Mycol.;
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Table 3 shows the in vitro activity of 12j against Candida
spp. and Aspergillus spp. The analog 12j exhibited an
excellent antifungal activity and broad spectra against
most of the fungal pathogens tested, which is superior
or comparable to those of the currently available anti-
fungal agents.
The hepatotoxic potential of the analogs 11a, 12j, 11o,
and 11r was determined in human liver cancer cell
HepG2 by the neutral red uptake assay according to
the protocol described previously.18,19 Ketoconazole
was used as a reference antifungal agent. The CC50 (half
concentration of cytotoxicity) value of ketoconazole
(51 lM) was quite close to the CC50 which was previ-
ously reported. The hepatotoxic potentials of all
the tested analogs were lower than that of ketoconazole
(Table 4). In particular, the CC50s of 11a and 12j were
599 lM and 141 lM, respectively.
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In vivo efficacy of the selected analogs, which exhibited
excellent in vitro antifungal activities, was evaluated in
the systemic C. albicans infection model in mice.20 The
results are summarized in Table 5. Each analog was
compared to the untreated control group. The analogs
11c and 12j showed good efficacies on oral administra-
tion and the significantly improved survival rates of
the infected mice.
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In conclusion, the indazole analogs with C3-H and C5-
halogen substituent, respectively, were identified as the

3490
J. S. Park et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3486–3490
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15. Unambiguous assignments of the structures for N-1 and
N-2 isomer were obtained by a 1D NOE experiment.
Irradiation of the signal for the C-3 proton on indazole
ring of one isomer resulted in an enhancement of the
signals for the protons attached to C3-CH3 and C3-H,
consistent with the N-2 isomer. As required, the corre-
sponding experiment with the N-1 isomer led to the quite
low enhancement of the signal for the proton attached to
C3-CH3 only.
16. Isin, E.; de Jonge, M.; Castagnoli, N., Jr. J. Org. Chem.
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17. Souers, A.; Gao, J.; Brune, M.; Bush, E.; Wodka, D.;
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20. SPF (specific pathogen-free) female ICR mice were used in
these studies. Mice were infected systemically by intrave-
nous injection of C. albicans ATCC 36082 (inoculum size
about 5 · 105 per animal). Treatment of the selected
analogs, which were orally administered, was started after
2 h post-infection and the antifungal agents were given
once a day for 7 consecutive days. All the analogs were
administered with 20 mg/kg of once daily doses. Efficacy
was determined by mean of survival days for 21 or 28 days
after infections.