Synthesis and SAR studies of biaryloxy-substituted triazoles as antifungal agents

Article abstract of DOI:10.1016/j.bmcl.2008.04.056

A series of 1-(substituted biaryloxy)-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl) propan-2-ol were synthesized and their antifungal activities were evaluated against eight human pathogenic fungi in vitro. Seventeen compounds showed activity 4- to 64-fold higher than voriconazole against Candida albicans. SAR clearly suggested that introduction of a biaryloxy side chain greatly enhanced the antifungal activity of triazole analogs against Candida species.

Full text of DOI:10.1016/j.bmcl.2008.04.056

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 3261–3265
Synthesis and SAR studies of biaryloxy-substituted triazoles
as antifungal agents
Ping Liu,a,b Shaolong Zhu,a Peng Li,a Weijie Xie,a Yongsheng Jin,a Qingyan Sun,a
Qiuye Wu,a Peng Sun,a Yingjun Zhang,a Xiaohong Yang,b,*
Yuanying Jiangc,* and Dazhi Zhanga,*
aDepartment of Organic Chemistry, School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433,
PR China
bDepartment of Medicinal Chemistry, School of Pharmacy, Jilin University, Changchun 130021, PR China
cDepartment of Pharmacology, School of Pharmacy, Second Military Medical University, Shanghai 200433, PR China
Received 7 December 2007; revised 30 March 2008; accepted 21 April 2008
Available online 25 April 2008
Abstract—A series of 1-(substituted biaryloxy)-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl) propan-2-ol were synthesized and
their antifungal activities were evaluated against eight human pathogenic fungi in vitro. Seventeen compounds showed activity 4-
to 64-fold higher than voriconazole against Candida albicans. SAR clearly suggested that introduction of a biaryloxy side chain
greatly enhanced the antifungal activity of triazole analogs against Candida species.
Ó 2008 Elsevier Ltd. All rights reserved.
Systemic fungal infections are life-threatening and have
become increasingly common in the immunocompro-
OH
R
O
X
N
N
mised hosts.1 Currently, triazole drugs (fluconazole, itr-
aconazole, voriconazole, and posaconazole) are the
most frequently used antifungals in clinic.2 However,
resistance to azoles is emerging and may pose a serious
health problem in the future.3 In addition, triazole drugs
are often associated with hepatotoxicity and limited
antifungal spectrum.4,5 Consequently, it remains attrac-
tive to develop new triazole derivatives possessing
broader antifungal spectra and higher therapeutic
indexes.
N
Y
X = Cl or F
Y = CH, N
X
Figure 1. Structure of biaryloxy triazole derivatives.
membrane.8 Therefore, current researches are mainly fo-
cused on the optimization of the side chain attached to
the pharmacophore. Optimization of the side chain
has led to new compounds with better biological and/
or pharmacological properties.6,9,10
In the past three decades, SAR of antifungal triazoles
has been extensively studied.6,7 These studies have re-
vealed a pharmacophore, bolded in Figure 1, which con-
tains a triazole ring linked to a dihalophenyl ring
through a two carbon chain. In addition, the carbon al-
pha to the phenyl ring bears a hydroxyl group. The
pharmacophore is critical for the binding of triazoles
with the active site of CYP51 (lanosterol 14-demethyl-
ase), the enzyme which is required for the biosynthesis
of ergosterol, an essential component of fungal cell
The side chains in many triazole antifungals bear one or
two 1,4-disubstituted aryl substituents for improved
antifungal activity.6,7 However, the lipophilicity of these
aryl substitutents, in addition to the hydrophobic dihal-
ophenyl group in the triazole pharmacophore, could
give rise to poor water solubility of the antifungals to
prevent their clinical applications. Herein, we wish to re-
port the synthesis and SAR of a series of triazole deriv-
atives bearing a variety of biaryloxy side chains. These
compounds were designed by introducing a variety of
*
Corresponding authors. Tel.: +86 21 2507 4595; e-mail: zhangdzh@
hotmail.com
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.04.056

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P. Liu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3261–3265
hydrophilic substituents into the biphenyloxy or 4-(pyri-
din-3-yl)phenyloxy side chain to improve their solubility
and antifungal activity.
with HBr. Reaction of 9 with compound 1 in the pres-
ence of K2CO3 in DMF afforded amine 10. The target
compounds 11a–f were obtained by reductive amination
of 10 with the corresponding aldehydes by NaBH3CN.
Compounds 12a–e were prepared by reacting 10 with
the corresponding acyl chlorides in pyridine.14 Com-
pounds 13a–f were prepared by reacting the chloride
intermediate 12a or 12b with the corresponding amines
in acetonitrile, respectively (Scheme 3). All the new com-
pounds (3–6, 10–14) described above were characterized
by IR, HRMS, and NMR spectroscopic analysis.15
Synthesis of the biphenyloxy derivatives 4–14 from oxi-
rane 1 was accomplished using chemistry illustrated in
Scheme 1. Oxirane 1 was allowed to react with 4-bromo-
phenol in the presence of K2CO3 in DMF to provide
compound 2. Suzuki coupling of 2 with 4-form-
ylphenylboronic acid afforded the aldehyde intermediate
3.11,12 The target compounds 4a–f and 5a–c were ob-
tained by reductive amination of aldehyde 3 with the
corresponding amines by NaBH3CN in methanol.13
Compounds 6a and 6b were obtained by the NaBH4
reduction or oximination of the aldehyde 3, respectively.
The in vitro antifungal activities of the 30 novel biaryl-
oxy-substituted triazole derivatives were evaluated by
the standard broth microdilution method of the
NCCLS.16 The tested fungi species included four yeast
species (Candida albicans, Candida tropicalis, Candida
parapsilosis, and Cryptococcus neoformans) and four
mold species (Aspergillus fumigatus, Trichophyton ru-
brum, Microsporum gypseum, and Fonsecaea compacta).
The positive controls included four different classes of
Synthesis of the 4-(pyridine-3-yl)phenyloxy derivatives
11a–f and 12a–e is outlined in Scheme 2. Suzuki cou-
pling of 4-methoxyphenylboronic acid (7) with 5-
bromopyridin-2-amine afforded the biaryl intermediate
8. The methyl group of 8 was removed by refluxing 8
O
OH
OH
N
N
O
F
CHO
O
F
Br
N
N
N
N
CH3SO3H
i
ii
N
N
N
F
.
3
2
1
F
F
F
OH
F
6a: R = OH
6b: R = =NOH
4a: R = NHCH3
5a: R =
5b: R =
N
4b: R = N(CH3)2
4c: R = NHC2H5
4d: R = NHCH(CH3)2
O
F
N
N
CH3
N
R
iii for 4a-f and 5a-c
iv for 6a
NH
4e: R = NHCH2CH2OH
v for 6b
4a-f
Cl
5a-c
6a-b
NH
COOH
5c: R =
4f: R =
N
O
Scheme 1. Synthesis of the biphenyloxy derivatives 4a–f, 5a–c, and 6a–b. Reagents and conditions: (i) 4-bromophenol, K2CO3, DMF, 90 °C, 65%; (ii)
4-formylphenylboronic acid, Pd(OAc)2, PPh3, Na2CO3, 2-propanol, reflux, 57%; (iii) RH, NaBH3CN, MeOH, 33–56%; (iv) 10%Pd/C, H2, MeOH,
85%; (v) NH2OHÆHCl, MeOH, 97%.
OH
i
ii
HO
NH2
B
CH3O
NH2
H3CO
N
OH
OH
N
9
7
8
OH
F
R
N
N
O
F
NH
N
N
O
F
NH2
N
N
iv
iii
N
N
11a
11b
11c
: R = C6H5
11a-f
10
: R = 2-(OH)C6H5
: R = 4-(OH)C6H4
F
11d: R = 3-ClC6H5
11e: R = CH3
v
O
OH
11f: R = n-C6H13
R
N
N
O
NH
12a
12b
12c
: R = 4-(ClCH2)C6H4
N
F
N
: R = CH2Cl
: R = 4-(CH3)C6H4
12d: R = 3-(CH3)C6H4
12e: R = 4-(OCH3)C6H4
12a-e
F
Scheme 2. Synthesis of the 4-(pyridine-3-yl)phenyloxy derivatives 11a–f and 12a–e. Reagents and conditions: (i) 5-bromopyridin-2-amine, Pd(OAc)2,
PPh3, Na2CO3 (2 N), 2-propanol, reflux, 63%; (ii) HBr, reflux, 98%; (iii) compound 1, K2CO3, DMF, 90 °C, 47%; (iv) RCHO, NaBH3CN, MeOH,
37–70%; (v) RCOCl, Pyr, 0 °C, 42–64%.

P. Liu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3261–3265
3263
R
O
N
N
13a: R =
13b: R =
OH
i
NH
N
N
O
F
12a
N
N
13a-c
13c: R =
N
O
F
O
R
OH
F
14a: R = NHCH3
NH
N
N
i
O
F
N
N
14b: R =
14c: R =
12b
N
14a-c
N
NH
Scheme 3. Synthesis of the 4-(pyridine-3-yl)phenyloxy derivatives 13a–c and 14a–c. Reagents and conditions: (i) RH, CH3CN, reflux, 55–81%.
Table 1. In vitro antifungal activity of biaryloxy triazole derivatives
a
Compound
MIC80 (lg/mL)
Yeastsb
Moldsb
C. albicans
C. parapsilosis
>64
0.25
C. tropicalis
C. neoformans
A. fumigatus
T. rubrum
M. gypseum
F. compacta
3
0.0625
0.25
0.25
1
0.25
0.25
1
64
4
>64
>64
64
1
0.25
0.25
4
4a
4b
4c
4d
4e
4f
0.0625
0.0625
0.25
0.25
1
1
1
0.25
0.25
0.0625
0.25
4
16
16
64
>64
>64
>64
>64
>64
>64
16
1
1
1
0.0625
1
0.0625
1
1
4
4
16
4
16
4
0.0625
1
0.0625
1
0.25
0.0625
0.25
0.25
0.25
0.25
5a
5b
5c
6a
6b
1
>64
>64
1
16
1
>64
>64
1
16
>64
1
0.25
0.0156
0.0156
0.0039
0.0625
0.0625
0.0039
0.0039
0.0156
0.0156
0.25
1
>64
>64
1
16
1
1
1
1
10
0.0625
0.25
0.25
0.25
0.25
64
1
4
0.0156
0.25
0.0625
0.25
0.0625
11a
11b
11c
11d
11e
11f
12a
12b
12c
12d
12e
13a
13b
13c
14a
14b
14c
0.0625
0.0625
0.0625
0.25
4
64
>64
4
0.25
1
0.0625
0.0625
4
0.0625
0.0156
0.25
>64
>64
4
0.0625
>64
>64
>64
>64
>64
>64
>64
64
>64
4
1
4
>64
4
0.0625
0.25
0.0625
1
0.0625
0.25
16
1
>64
1
1
0.25
1
0.0156
0.25
0.0625
0.25
0.0625
0.25
0.25
1
0.25
0.25
0.0625
1
1
1
1
1
0.0625
0.25
0.0625
0.25
0.25
0.0625
0.25
0.25
0.25
0.25
1
0.25
1
1
1
0.0625
0.0625
0.0625
0.0625
0.0625
0.0625
16
64
0.25
1
1
0.25
0.25
1
0.25
0.0625
0.0625
0.0625
0.25
4
>64
4
1
0.0625
0.0625
0.25
1
0.25
1
4
16
>64
4
>64
1
>64
1
16
1
0.0625
4
0.0156
1
1
64
>64
>64
0.25
1
0.25
1
1
1
FCZc
VCZc
ICZc
TBLc
KCZc
AMBc
16
4
1
0.25
1
>64
0.25
1
1
1
16
0.25
0.5
1
0.0625
0.0312
0.25
0.0625
1
0.0156
0.25
0.25
0.0625
4
0.0156
0.0156
0.0625
0.25
0.0156
0.0625
0.0156
0.25
1
0.0625
0.0625
0.0625
0.25
1
0.25
0.0156
1
0.0625
1
16
0.25
0.25
16
16
a The tested concentration ranges were from 0.00024 to 64 lg/mL. The given data are mean values of three parallel experiments.
b Candida albicans ATCC 76615, Cryptococcus neoformans ATCC 32609. The other tested organisms are clinic isolates obtained from Chinese
Changhai Hospital’s Fungi Culture Collection.
c Fluconazole (FCZ), voriconazole (VCZ), itraconazole(ICZ), terbinafine (TBL), ketoconazole (KCZ), and amphotericin B (AMB) were used as the
positive controls.

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P. Liu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3261–3265
antifungal drugs currently used in clinic, including
triazoles (fluconazole, voriconazole, and itraconazol),
allylamine (terbinafine), imidazole (ketoconazole), and
polyene (amphotericin B). The minimum 80% inhibi-
tory concentration (MIC80) values are summarized in
Table 1.
morpholinyl group in 4f is similar to the 4-hydrocarbon-
yl-phenyl group in 5c, both having two polar groups (O
and NH) connected through a hydrophobic linker.
Given that both compounds have higher antifungal
activity than their parallel analogs, respectively, this
structural similarity between 4f and 5c suggests the pres-
ence of a corresponding hydrogen bonding site and a
hydrophobic region in the CYP51 substrate binding site.
The MIC80 values indicate that nearly all the biaryloxy
derivatives showed excellent antifungal activities against
the three yeast Candida species including C. albicans,
C. parapsilosis, and C. tropicalis. Noticeably, more than
half (18) of the 30 compounds showed higher activity
against C. albicans than the tested six conventional
drugs. Compared to voriconazole, one of the most pre-
scribed antifungals in clinic, these compounds were 4- to
64-fold more effective (MIC80 = 0.0156–0.0625 lg/mL).
These results clearly suggested that introduction of a
biaryloxy side chain to the triazole pharmacophore
greatly enhanced the antifungal activity of these analogs
against Candida species. The yeast species C. neoformans
was found to be much less sensitive to these biaryloxy
derivatives, as indicated by their MIC80 values showing
moderate to no activity.
Replacing the amino groups with a hydroxyl in 6a or
oxime group in 6b resulted in enhanced activity. The
hydroxyl compound 6a showed not only activity
comparable to 5c against Candida but also activity
against C. neoformans which is absent in the latter.
The oxime derivative 6b was found to be the most
effective compound against Candida among the tested
30 compounds. Its activity was 64- and 16-fold
higher than that of voriconazole against C. albicans
and C. parapsilosis, respectively. The significance of
the discovery of 6b is reinforced by the fact that C. albi-
cans is the most frequently encountered Candida species
and responsible for most serious invasive fungal
infections in clinic.18
Compared to their strong activities against yeast species,
the MIC80 values indicate that the activities of these bia-
ryloxy derivatives against mold fungi are much lower.
Nearly all the 30 compounds showed no activity against
A. fumigatus and majority showed only moderate activ-
ity against M. gypseum and F. compacta. However, these
compounds showed superior activity against T. rubrum,
with the lowest MIC80 value (0.0156 lg/mL for Com-
pound 10) same as that of voriconazole and itraconoz-
ole. The inactivity of these biaryloxy derivatives
against A. fumigatus was not a surprise because it has
been known that this mold species possesses an intrinsic
mechanism resistant to triazole antifungals.17
In the 4-(pyridin-3-yl)phenyloxy series, compounds
11a–f with amino substitutents showed similar activities
as compounds 12a–e with amides. Compared to the
non-substituted compound 10, most of the N-substi-
tuted compounds showed slightly higher antifungal
activity against yeasts, suggesting that the activity may
be benefited from additional substituents. However,
compared to compound 10, nearly all the N-substituted
compounds suffered activity loss against mold fungi.
Interestingly, compounds (4f, 5a–c) in the biphenyloxy
series with bulky substitutents are also the least effective
compounds against molds. These results indicated that
additional bulky substituents deteriorated the antifungal
activity of these triazole analogs against molds. The
4-(chloromethyl)benzamide 12a was found to be the
most effective compound among the 4-(pyridin-3-yl)phe-
nyloxy series against yeast fungi, 4-fold more effective
than voriconazole against C. albicans. The least effective
compound was found to be 14c which had a piperazine
substitutent. Comparison between 13c and 14c, 13b–c
suggests that the amino group at the side chain terminal
is not favored by the enzyme.
Of the 30 biaryloxy derivatives, only compounds 10 and
11b are effective against both Candida and mold species.
Their broad antifungal spectra are comparable to that of
voriconazole, itraconozole, and terbinafine, and more
than that of ketoconazole and amphotericin B. Interest-
ingly, both compounds are 4-(pyridin-3-yl)phenyloxy
derivatives. This result suggests that the antifungal spec-
trum of the biaryloxy triazole analogs benefits more
from 4-(pyridin-3-yl)phenyloxy than the biphenyloxy
substituent. The superiority of the former could be
attributed to its higher hydrophilicity given by the pyri-
dine ring, subsequently benefiting the in vivo absorption
and distribution of the analog.
In conclusion, a series of novel biaryloxy-substituted tri-
azole antifungal agents were synthesized and their anti-
fungal activities were screened for both yeast and mold
fungi species. These analogs showed improved in vitro
antifungal activities especially against Candida species.
This research has led to the discovery of compounds
5c, 10, and 12a for further optimizations.
Among the 12 biphenyloxy derivatives, compounds 4a–e
with aliphatic amino substituents showed mainly moder-
ate antifungal activities (MIC80 = 0.25–1 lg/mL) against
C. albicans. Replacing the amino group with a 2,6-di-
methyl-morpholinyl group in compound 4f gave rise to
a 4- to 16-fold increase on the activity. Compounds 5a
and 5b with aromatic amino substituents showed mod-
erate activity against all three Candida species. Introduc-
tion of a carboxylic group in para to the amino group in
compound 5c gave rise to a significant 4- to 64-fold
increase on the activity. Interestingly, the 2,6-dimethyl-
Acknowledgments
This work was supported by the National Natural Sci-
ence Foundation of China (Nos. 30672533 and
30772644). We thank Dr. Yongying Jiang, Dr. G. Kart-
hikeyan, and Professor Tingsen Zhou for their advice
and discussions.

P. Liu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3261–3265
3265
7.51–7.48 (m, 4H), 7.36 (d, 2H, J = 7.8 Hz), 6.94–6.92 (m,
2H), 6.88–6.78 (m, 2H), 4.87 (dd, 2H, J = 14.4, 23.4 Hz),
4.60 (s, 1H), 4.29 (q, 2H, J = 9.6 Hz), 3.72–3.69 (m, 2H),
3.50 (s, 2H), 2.73 (d, 2H, J = 10.2 Hz), 1.77 (t, 2H,
J = 10.8 Hz), 1.15 (d, 6H, J = 19.8 Hz); IR (cmÀ1, KBr):
3126, 2972, 2931, 2870, 1616, 1500, 1457, 1422, 1273, 1243,
1141, 1082, 966, 851, 824, 678, 517; HRMS (m/z): calcd for
C30H33F2N4O3: 535.2521; found: 535.2616.
References and notes
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Compound 11a: 1H NMR (CDCl3, 300 MHz) d 8.22 (d,
1H, J = 1.8 Hz), 8.05 (s, 1H), 7.86 (s,1H), 7.48–6.81 (m,
14H), 6.47 (d, 1H, J = 8.7 Hz), 4.88 (d, 2H, J = 4.5 Hz),
4.55 (d, 2H, J = 6.0 Hz), 4.28 (d, 2H, J = 4.2 Hz); IR
(KBr, cmÀ1): 3234, 3121, 3029, 2925, 2849, 1605, 1512,
1421, 1388, 1279, 1245, 1138, 1059, 1028, 968, 814, 701,
678, 548; HRMS (m/z): calcd for C29H26F2N5O2:
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Compound 12a: 1H NMR (DMSO-d6, 300 MHz) d 8.67 (d,
1H, J = 2.4 Hz), 8.37 (s, 1H), 8.26 (d, 1H, J = 8.7 Hz), 8.11
(dd, 1H, J = 2.7, 8.7 Hz), 8.04 (d, 2H, J = 8.1 Hz), 7.81 (s,
1H), 7.68 (d, 2H, J = 8.7 Hz), 7.59 (d, 2H, J = 8.4 Hz),
7.51 (dd, 1H, J = 7.2, 9.0 Hz), 7.25–7.17 (m, 1H), 7.08–
7.02 (m, 3H), 4.85 (s, 2H), 4.75 (d, 2H, J = 4.2 Hz), 4.40
(d, 1H, J = 10.2 Hz), 4.26 (d, 1H, J = 10.5 Hz); IR (KBr,
cmÀ1): 3387, 3130, 2960, 2876, 1674, 1615, 1501, 1422,
1378, 1303, 1272, 1244, 1140, 1056, 967, 828, 711, 679, 523;
HRMS (m/z): calcd for C30H25ClF2N5O3: 576.1614;
found: 576.1595.
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´
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600 MHz) d 8.04 (s, 1H), 7.83 (s, 1H), 7.65–7.61 (m, 1H),
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