Bioorganic & Medicinal Chemistry Letters 27 (2017) 2171–2173

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Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier.com/locate/bmcl

Design, synthesis, and in vitro evaluation of novel antifungal triazoles

Fei Xie a,d, Tingjunhong Ni a,d, Jing Zhao b,d, Lei Pang b, Ran Li a, Zhan Cai a, Zichao Ding a, Ting Wang a,
Shichong Yu a, Yongsheng Jin a, Dazhi Zhang a,⇑, Yuanying Jiang c,⇑
a
Department of Organic Chemistry, School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, China
b
School of Pharmacy, Fujian University of Traditional Chinese Medicine, 1 Yueyang Road, Fuzhou 350112, China
c
Center of New Drug Research, School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, China

a r t i c l e i n f o a b s t r a c t

Article history: Twenty-nine novel triazole analogues of ravuconazole and isavuconazole were designed and synthesized.
Received 30 November 2016 Most of the compounds exhibited potent in vitro antifungal activities against 8 fungal isolates. Especially,
Revised 8 March 2017 compounds a10, a13, and a14 exhibited superior or comparable antifungal activity to ravuconazole
Accepted 22 March 2017
against all the tested fungi. Structure-activity relationship study indicated that replacing 4-
Available online 23 March 2017
cyanophenylthioazole moiety of ravuconazole with fluorophenylisoxazole resulted in novel antifungal
triazoles with more effectiveness and a broader-spectrum.
Keywords:
Ó 2017 Elsevier Ltd. All rights reserved.
Isoxazole
Triazole
Antifungal activity
Structure-activity relationship
Synthesis

Global mortality from fungal infection is over 1.35 million peo-
ple per annum, far more than are killed by malaria (0.63 mil-
lion).1 The high rate of mortality associated with invasive fungal
infections often exceeds 50%.2 Triazole antifungals still keep the
leading and progressive position in antifungal drug research and
development. The increased fungal resistance triazole drugs3
makes it urgent to develop novel antifungal azoles with more
effectiveness, lower toxicity and broader-spectrum.
It is widely recognized that the azole antifungal agents bind
with the active site of CYP51 (lanosterol 14a-demethylase) and
block the synthesis of ergosterol.4 SAR studies5–7 have revealed a
pharmacophore of antifungal triazoles, which contains a triazole
ring linked to a difluorophenyl through a two-carbon chain, as
shown in Fig. 1. Optimization research on triazoles has been
mainly focused on their side chains which play important roles
to their drug likeness by affecting their target binding and physic-
ochemical properties.8
In 2015, isavuconazonium sulfate was first launched in the U.S.
for the oral and intravenous treatment of invasive aspergillosis and
invasive mucormycosis. Another new drug candidates, ravucona-
zole, is similar to isavuconazole with the difference of the fluoro
⇑ Corresponding authors.
E-mail addresses: zdzhang_yjhx@smmu.edu.cn (D. Zhang), jiangyy@smmu.edu.
cn (Y. Jiang).
d
These authors contributed equally to this work.
substitution on the phenyl ring (Fig. 1). Both agents have a
cyanophenylthioazole moiety as the side chain. Inspired by the
bioisosteric replacement of their thiazole moiety with is oxazole,
a series of novel ravuconazole analogues a1–21 and isavuconazole
analogues b1–8 were designed and synthesized (Fig. 1).
We constructed the isoxazolemoiety of the target compound
(a1–21, b1–8) via 1,3-dipolar cycloaddition9 of alkynes 7 with var-
ious substituted N-hydroxybenzimidoyl chloride 3 in presence of
triethylamine and ZnCl2 in tetrahydrofuran as shown in Scheme 1.
The intermediates 3 were prepared by chlorination of benzalde-
hyde oximes 2, which was obtained by hydroxylamination of var-
ious substituted benzaldehydes 1. The alkynes 7 were synthesized
starting from 2-chloro-1-(2,4-difluorophenyl)ethanone 4 for target
compounds a1–21, and from 2-chloro-1-(2,5-difluorophenyl)etha-
none 4 for compounds b1–8, respectively. Both the starting mate-
rials 4 were treated with (R)-but-3-yn-2-yl-methanesulfonate 5 in
the presence of Pd(CH3CN)2Cl2, ZnEt2, and PPh3, to give intermedi-
ate 6, which underwent alkylation reaction with 1,2,4-triazole to
give the alkynes 7. Details of all step and analysis data are
described in Supporting information. X-ray crystallographic analy-
sis clearly supports the chiral structure of the intermediate alkynes
7a containing 2,4-difluorophenyl to construct a1–21. (Fig. 2,
Table S1 in the Supplementary material).
The in vitro antifungal activities of compounds a1–21 and b1–8
were evaluated against eight human pathogenic fungi including
Candida albicans Y0109, Candida albicans SC5314, Candida parap-

http://dx.doi.org/10.1016/j.bmcl.2017.03.062
0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
2172 F. Xie et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 2171–2173
Fig. 1. Structures of triazole antifungal drugs and the designed compounds.
silosis 22019, Cryptococcus neoformans 32605, Candida glabrata 537,
Aspergillus fumigatus 7544, Trichophyton rubrum cmccftla and
Microsporum gypseum cmccftla. Antifungal drugs ketoconazole, flu-
conazole and ravuconazole were used as controls. The MIC80 values
of these compounds were determined by the standard micro broth
dilution method of the NCCLS.10
As shown in Table 1, compounds a11, a17, and b8 against Can-
dida glabrata, and a12 against Candida albicans, exhibited the low-
est MIC80 value of 0.0156 lg/mL. Compounds a1, a2, a9, a10, a11,
a12, a14, exhibited MIC80 value of 0.0313 lg/mL against 4 fungal
isolates: Candida albicans Y0109, Candida albicans SC5314, Crypto-
coccus neoformans, and Candida glabrata, respectively. This concen-
tration is lower than the MIC80 values of the three control drugs.
The MIC80 values of compounds a10, a13, and a14, bearing 2-
flourophenyl-, 2,3-diflourophenyl, and 2,4-diflourophenyl isoxa-
zole, respectively, are equal to or lower than that of ravuconazole
against the tested 8 fungal isolates. Moreover, the other fluo-
rophenylisoxazole compound a15 (2,5-diF), a16 (2,6-diF), a20
(2,3,6-triF), and a21 (2,4,6-triF) also showed very good activity in
Scheme 1. Reagents and conditions:(i) NH2OH
HCl, NaHCO3, H2O, methanol, r.t., 2 h; (ii) N-chlorosuccinimide, dimethylformamide, 35 °C, 2 h; (iii) Pd(CH3CN)2Cl2, THF, ZnEt2,
PPh3, r.t., 1 h; (iv) DMSO, NaOH, 1H-1,2,4-triazole, 70 °C, 4 h; (v) THF, triethylamine, ZnCl2, 35 °C, 16–20 h.
Fig. 2. The single-crystal structure of alkynes 7a.
general. On the contrary, the compounds without fluoro substitu-
tion on the phenyl ring, such as a3 (4-CH3), a5 (4-NO2), a6 (4-SO2-
CH3), and a7 (4-CN), shows lower activity than the compounds
with fluorophenyl group. These results give us a clear conclusion
that fluorophenyl group is quite favorable to the antifungal
activity.
Since compounds a10, a2, a13–16, a11, and a17 in series a1–21
showed very good antifungal activity, these active compounds
were reconstructed by replacing their 2,4-diflourophenyl group
with 2,5-diflourophenyl group to give the series b1–8, respectively,
inspired by the structure of isavuconazole.
Compounds b1–8 exhibited excellent activity against Cryptococ-
cus neoformans and Candida glabrata with MIC80 values ranging
from 0.0156 to 0.125 lg/mL, which are much lower than that of
ravuconazole and fluconazole. However, most of the series b1–8
exhibited lower activity against other 6 tested fungal isolates than
ravuconazole. Even worse, most of their MIC80 values against
Aspergillus fumigatus and Microsporum gypseum are more than
8.0 lg/mL. Thus indicated that 2,4-diflourophenyl group is more
favorable to antifungal activity than 2,5-diflourophenyl group.
In summary, we have synthesized a series of novel isoxazole-
containing triazole analogues of ravuconazole and isavuconazole.
Most of the designed compounds exhibited potent in vitro antifun-
gal activities against 8 fungal isolates, especially, compounds a10,
a13, and a14 exhibited comparable or superior antifungal activity
to ravuconazole. SAR study gave us a clear conclusion that replac-
ing 4-cyanophenylthioazole of ravuconazole with fluo-
rophenylisoxazole resulted in more active and spectrum-
widening antifungal agents.
 F. Xie et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 2171–2173 2173

Table 1
In vitro antifungal activity of the target compounds.

Compd R MIC80 (lg/mL)

C.alb Y0109 C.alb SC5314 C.par 22019 C.neo 32605 C.gla 537 A.fum 7544 T.rub cmccftla M.gyp cmccftla
a1 4-Cl 0.0313 0.125 0.125 0.125 0.125 4.0 0.125 1.0
a2 4-F 0.125 0.25 0.0625 0.125 0.0313 2.0 0.0625 0.5
a3 4-CH3 0.25 0.0625 0.125 0.125 0.25 8.0 0.25 2.0
a4 4-CF3 0.125 0.5 1.0 0.125 1.0 >8.0 2.0 2.0
a5 4-NO2 0.125 0.125 0.5 0.125 0.5 >8.0 1.0 2.0
a6 4-SO2CH3 0.125 0.125 0.125 0.25 0.25 8.0 1.0 4.0
a7 4-CN 0.25 0.25 0.125 0.125 0.0625 8.0 0.125 1.0
a8 4-OCHF2 0.0625 0.0625 0.125 0.125 0.0625 8.0 0.125 1.0
a9 4-OCF3 0.0313 0.125 0.5 0.125 0.0625 8.0 0.125 4.0
a10 2-F 0.0625 0.0313 0.125 0.125 0.0313 2.0 0.0625 0.5
a11 2-F-4-Cl 0.125 0.125 0.125 0.0313 0.0156 8.0 0.25 2.0
a12 3-F-4-Cl 0.0156 0.0313 0.5 0.25 0.25 >8.0 0.5 4.0
a13 2,3-diF 0.0625 0.0625 0.25 0.125 0.125 2.0 0.125 0.5
a14 2,4-diF 0.0625 0.0625 0.125 0.125 0.0313 2.0 0.0625 0.5
a15 2,5-diF 0.0625 0.0625 0.125 0.125 0.0625 4.0 0.125 1.0
a16 2,6-diF 0.0625 0.125 0.125 0.125 0.0625 2.0 0.125 1.0
a17 3,4-diF 0.125 0.125 0.125 0.125 0.0156 8.0 0.5 4.0
a18 3,5-diF 0.0625 0.125 0.25 0.125 0.0625 4.0 0.25 2.0
a19 3,4-diCl 0.0625 0.25 0.5 0.25 0.25 >8.0 0.5 4.0
a20 2,3,6-triF 0.125 0.25 0.125 0.0625 0.0625 2.0 0.0625 0.5
a21 2,4,6-triF 0.125 0.25 0.125 0.0625 0.0625 2.0 0.5 1.0
b1 2-F 0.5 0.5 1.0 0.0625 0.0625 >8.0 2.0 >8.0
b2 4-F 0.125 0.125 0.25 0.0625 0.0625 >8.0 1.0 >8.0
b3 2,3-diF 0.5 0.5 2.0 0.125 0.125 >8.0 8.0 >8.0
b4 2,4-diF 0.125 0.25 1.0 0.0625 0.0625 >8.0 1.0 4.0
b5 2,5-diF 0.5 1.0 1.0 0.125 0.125 >8.0 8.0 >8.0
b6 2,6-diF 1.0 0.5 1.0 0.0625 0.125 >8.0 4.0 >8.0
b7 2-F-4-Cl 0.25 0.5 1.0 0.125 0.0313 >8.0 1.0 8.0
b8 3,4-diF 0.125 0.125 0.25 0.125 0.0156 >8.0 1.0 >8.0
KCZ / 0.25 0.125 0.5 0.0625 0.0625 4.0 1.0 1.0
FCZ / 0.25 0.5 2.0 0.5 2.0 >64.0 8.0 32.0
RCZ / 0.0625 0.0625 0.25 0.125 0.125 2.0 0.125 1.0

Abbreviations: C.alb: Candida albicans; C.par: Candida parapsilosis; C.neo: Cryptococcus neoformans; C.gla: Candida glabrata; A.fumi: Aspergillus fumigatus; T.rub: Trichophyton
rubrum; M.gyp: Microsporum gypseum; KCZ: ketoconazole; FCZ: fluconazole; RCZ: ravuconazole.

Acknowledgments References

This work was supported by the National Natural Science Foun-
dation of China (No. 81673280; 21272270; 21072226; 815734738)
and Science and Technology Commission of Shanghai Municipality
(No. 15431901800).
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