Design, synthesis, and structure-activity relationship studies of novel triazole agents with strong antifungal activity against Aspergillus fumigatus

Article abstract of DOI:10.1016/j.bmcl.2020.126951

The incidence of invasive fungal infections has dramatically increased for several decades. In order to discover novel antifungal agents with broad spectrum and anti-Aspergillus efficacy, a series of novel triazole derivatives containing 1,2,3-benzotriazin-4-one was designed and synthesized. Most of the compounds exhibited stronger in vitro antifungal activities against tested fungi than fluconazole. Moreover, 6m showed comparable antifungal activity against seven pathogenic strains as voriconazole and albaconazole, especially against Aspergillus fumigatus (MIC = 0.25 μg/ml), and displayed moderate antifungal activity against fluconazole-resistant strains of Candida albicans. A clear SAR study indicated that compounds with groups at the 7-position resulted in novel antifungal triazoles with more effectiveness and a broader-spectrum.

Full text of DOI:10.1016/j.bmcl.2020.126951

Journal Pre-proofs
Design, synthesis, and structure-activity relationship studies of novel triazole
agents with strong antifungal activity against Aspergillus fumigatus
Zichao Ding, Tingjunhong Ni, Fei Xie, Yumeng Hao, Shichong Yu, Xiaoyun
Chai, Yongsheng Jin, Ting Wang, Yuanying Jiang, Dazhi Zhang
PII:
S0960-894X(20)30004-4
DOI:
https://doi.org/10.1016/j.bmcl.2020.126951
BMCL 126951
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
30 October 2019
26 December 2019
1 January 2020
Please cite this article as: Ding, Z., Ni, T., Xie, F., Hao, Y., Yu, S., Chai, X., Jin, Y., Wang, T., Jiang, Y., Zhang,
D., Design, synthesis, and structure-activity relationship studies of novel triazole agents with strong antifungal
activity against Aspergillus fumigatus, Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/
10.1016/j.bmcl.2020.126951
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Design, synthesis, and structure-activity relationship
studies of novel triazole agents with strong antifungal
activity against Aspergillus fumigatus
Zichao Dinga,†,Tingjunhong Nib,†, Fei Xiea†, Yumeng Haoa, Shichong Yua,
Xiaoyun Chaia, Yongsheng Jina, Ting Wanga, Yuanying Jiangb,*, Dazhi Zhanga,*
a Department of Organic Chemistry, School of Pharmacy, Second Military Medical University, 325 Guohe Road,
Shanghai 200433(China)
b Shanghai Tenth People's Hospital, Tongji University School of Medicine, 301 Middle yanchang Road, Shanghai
200072(China)
Corresponding Authors
Jiang Y. Tel.: +86-021-81871357, E-mail address: 13761571578@163.com
Zhang D. Tel.: +86-021-81871226, E-mail address: zdzhang_yjhx@smmu.edu.cn
† The authors contributed equally.
Abstract:
The incidence of invasive fungal infections has dramatically increased for several decades. In order
to discover novel antifungal agents with broad spectrum and anti-Aspergillus efficacy, a series of
novel triazole derivatives containing 1,2,3-benzotriazin-4-one was designed and synthesized. Most
of the compounds exhibited stronger in vitro antifungal activities against tested fungi than
fluconazole. Moreover, 6m showed comparable antifungal activity against seven pathogenic strains
as voriconazole and albaconazole, especially against Aspergillus fumigatus (MIC = 0.25 μg/ml),
and displayed moderate antifungal activity against fluconazole-resistant strains of Candida albicans.
A clear SAR study indicated that compounds with groups at the 7-position resulted in novel
antifungal triazoles with more effectiveness and a broader-spectrum.
Keywords: Triazole; CYP51; Antifungal; Molecular docking; Synthesis
Over the past decades, invasive fungal infections (IFIs) have been dramatically increasing and
presenting a severe threat to human health [1-4]. Clinically, the three major pathogens, including
the Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus, account for most of
IFIs, which continue to be a major cause of morbidity and mortality in immunocompromised or
HIV-infected patients [5]. The therapeutic antifungal agents available for the treatment of IFIs can
be divided into four classes: polyenes (e.g. amphotericin B), echinocandins (e.g. caspofungin and
micafungin), nucleoside analogues (e.g. 5-fluorocytosine) and azoles (e.g. fluconazole and
itraconazole).
Among these antifungal agents, azoles exert activity by selective inhibition the lanosterol 14α-
demethylase (CYP51), the key enzyme in sterol biosynthesis of fungi [6]. Most azoles including
fluconazole, itraconazole, voriconazole, and posaconazole (Fig.1) are orally active, and show a

broad-spectrum against most yeasts and filamentous fungi [7]. Due to their broader antifungal
spectrum, higher efficacy and lower toxicity, azole drugs have been used as first-line antifungal
agents.
Unfortunately, the increasing number of invasive aspergillosis among immunocompromised
patients has increased dramatically [8]. Pulmonary infections with Aspergillus fumigatus can cause
progressive dysphagia in severely ill patients [9]. However, this pathogen is intrinsically resistant
to fluconazole, which is the most widely used azole in clinic. Therefore, it is urgent to develop novel
antifungal azoles with excellent activity against pathogenic fungi, particularly to further enhance
the anti-Aspergillus efficacy.
Albaconazole is a new oral triazole antifungal agent with good pharmacokinetics and excellent
oral bioavailability [10,11]. It has demonstrated high in vitro activity against pathogenic yeasts and
filamentous fungi, especially Aspergillus fumigatus, and has been shown to be effective in animal
models [11]. Albaconazole has been studies in several clinical trials, including phase I and II studies
on candidal vulvovaginitis, tinea pedis, and onychomycosis and no side effects have been published
[12,13]. However, fewer literatures of structural modification to albaconazole have been reported
than that to fluconazole.
As part of our research program focused on synthesis novel broad-spectrum and anti-
Aspergillus antifungal agents, we aimed to develop a series of structural analogue of albaconazole.
A quinazolinone unit is the structure characteristic of albaconazole and is the bioisostere of 1,2,3-
benzotriazin-4-one. 1,2,3-benzotriazin-4-one is an important component of nitrogen-containing
heterocycle and has attracted much attention in both the medicinal and agrochemical fields [14-16].
For example, many pharmacological compounds have been reported, including agents with
antitumor, antiarthritic, diuretic and antitubercular activities [17-20]. Inspired by the bioisosteric
replacement of quinazolinone moiety with 1,2,3-benzotriazin-4-one, a series of novel albaconazole
analogues was designed and synthesized.

O
OH
O
O
N
N
N
N
N
N
N
N
N
F
N
N
O
N
Cl
N
N
F
Cl
Fluconazole
Itraconazole
F
O
OH
O
O
N
N
N
N
N
N
N
N
N
N
OH
N
F
F
N
N
F
F
Posaconazole
Voriconazole
O
O
OH
OH
N
N
Follow-up
Strategy
N
F
N
N
F
N
N
R
N
N
N
Cl
N
F
Albaconazole
F
target compounds
Fig. 1. Structures of clinic triazole antifungal agents and the designed compounds
All compounds in this study were prepared following the general strategies illustrated in Fig.
2. Key intermediate 1 was prepared following the procedure established in the literature [21]. Then,
triazolyldiol 1 was formed methanesulfonate and then eliminated to yield epoxide 2. The ring-
opening reaction of epoxide 2 with NaN3 followed by catalytic hydrogenation to give amino 4,
which was then subjected to the amidation reaction conditions with commercially available
substituted anthranilic acid. Finally, the diazotization on the amino group was coupled to the amide
to form the target compounds 6a-z.
OH
OH
O
N
N
N
N
OH
F
N
N3
F
N
i
ii
iii
F
N
N
N
MeOOC
OH
F
1
F
3
F
2
O
R
O
R
OH
OH
OH
N
N
N
N
N
H
F
N
N
F
N
NH2
F
N
N
N
H N
2
iv
N
v
N
F
F
4
F
5a-z
6a-z
Fig. 2. Synthesis of the target compounds. (i) a) MsCl, Et3N, DCM, 0 ℃, 30 min; b) NaOH, H2O,

0 ℃, 2 h; (ii) NaN3, NH4Cl, DMF, 100 ℃, 8 h; (iii) H2, Pd/C, MeOH, rt, 4 h; (iv) Substututed
anthranilic acids, DIEA, PyBOP, DMF, rt, 2 h; (v) Tertbutyl nitrite, MeCN, 0 ℃, 2 h.
The in vitro antifungal activities of target compounds 6a-z were evaluated according to the
protocols from the NCCLS [22,23]. The broth microdilution method was used to determine the
minimum inhibitory concentration (MIC) of the target compounds in 96-well microtest plates.
Fluconazole, voriconazole and albaconazole were used as reference drugs. Previous experiments
showed that DMSO has no influence on the growth of the fungi tested under the test conditions.
As shown in Table 1, most of the target compounds showed strong activities against Candida
albicans and Cryptococcus neoformans with MIC values in the range of 0.0156 to 2.0 μg/ml. What
is important is that compounds 6l-s, 6w and 6z exhibited good activities against Aspergillus
fumigatus. Subsequently, compounds 6l-s, 6w and 6z were selected out in order to further evaluate
the antifungal spectrum as showed in Table 2. According to the results, these compounds possessed
excellent antifungal activities against all strains tested. Among these compounds, 7-Cl substituted
analogue 6m displayed the most remarkable in vitro activity against all of the tested strains,
especially with the MIC value of 0.25 μg/ml against Aspergillus fumigatus and Microsporum
gypseum which was superior to fluconazole and was comparable to voriconazole and albaconazole.
Table 1. In vitro antifungal activity of the target compounds against three pathogenic fungi
(MIC, μg/ml)
Compd.
6a
6b
6c
R
5-F
C.alb. SC5314
0.5
C.neo. 32605
0.0625
0.0625
0.0625
0.25
A.fum. 7544
>64.0
32.0
32.0
16.0
>64.0
>64.0
32.0
32.0
>64.0
>64.0
16.0
1.0
5-Cl
0.25
5-Br
0.25
6d
6e
6-F
0.0625
0.0313
0.125
0.125
0.0625
0.25
6-Cl
0.5
6f
6-Br
2.0
6g
6h
6i
6-I
0.5
6-OCH3
6-CF3
6-NO2
6-CH3
7-F
1.0
2.0
6j
0.5
2.0
6k
6l
0.0625
0.0156
0.0156
0.0156
0.0156
0.0156
0.0156
0.0156
0.0156
0.5
0.25
0.25
6m
6n
6o
6p
6q
6r
7-Cl
0.0313
0.0625
0.125
0.125
0.125
0.25
0.25
0.5
7-Br
7-I
1.0
7-OCH3
7-CF3
7-NO2
7-CH3
8-F
2.0
4.0
4.0
6s
0.125
0.0313
0.25
2.0
6t
>64.0
>64.0
>64.0
2.0
6u
6v
6w
8-Cl
0.25
8-Br
0.5
0.25
6,7-2F
0.0313
0.5

6x
6y
6-F-7-Cl
0.125
0.125
0.0313
0.5
0.25
0.5
8.0
>64.0
4.0
Naphthalene
6z
H
/
0.25
FCZ
VCZ
ALB
0.5
>64.0
0.25
0.25
/
0.0313
0.0313
0.0625
0.0313
/
Abbreviations: C.alb.: Candida albicans; C.neo.: Cryptococcus neoformans; A.fum.: Aspergillus fumigatus; FCZ: fluconazole;
VCZ: voriconazole; ALB: albaconazole
Table 2. In vitro antifungal activity of the selected target compounds against other four
pathogenic strains (MIC, μg/ml)
Compd.
6l
R
7-F
C.alb. Y0109
0.0156
0.0156
0.0156
0.0156
0.0156
0.0156
0.0156
0.0156
0.0156
0.0156
0.5
C.par. 22019
0.0313
0.0156
0.0313
0.0625
0.0313
0.0313
0.0313
0.0313
0.0313
0.0313
1.0
C.gla. 537
0.0313
0.0156
0.0156
0.0313
0.0156
0.0313
0.0313
0.0156
0.0625
0.0625
2.0
M.gyp. cmcc
2.0
6m
6n
7-Cl
7-Br
7-I
0.25
0.5
6o
1.0
6p
7-OCH3
7-CF3
7-NO2
7-CH3
6,7-2F
H
2.0
6q
2.0
6r
4.0
6s
2.0
6w
1.0
6z
2.0
FCZ
VCZ
ALB
/
32.0
0.25
1.0
/
0.0156
0.0156
0.0156
0.0156
0.0156
0.0625
/
Abbreviations: C.alb.: Candida albicans; C.par.: Candida parapsilosis; C.gla.: Candida glabrata; M.gyp.: Microsporum gypseum;
FCZ: fluconazole; VCZ: voriconazole; ALB: albaconazole
Due to the widespread treatment use of fluconazole in the clinic, fluconazole-resistance for
treating fungal infections was seriously increasing. Based on the results of in vitro antifungal
activities assays. Compound 6l-o were further evaluated against fluconazole-resistant strains of
Candida albicans 100 and 103. As shown in Table 3, compound 6m showed moderate antifungal
activity with MIC values in the range of 2.0 to 8.0 μg/ml. This result was comparable to the
albaconazole, and was superior to fluconazole and voriconazole.
Table 3. In vitro antifungal activity of the target compounds against fluconazole-resistant strains
(MIC, μg/ml)
Compd.
6l
R
7-F
7-Cl
7-Br
7-I
/
C.alb. 100
32.0
C.alb. 103
64.0
6m
8.0
2.0
6n
16.0
32.0
6o
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
FCZ
VCZ
/

ALB
/
8.0
4.0
Abbreviations: C.alb. 100: The fluconazole-resistant Candida albicans isolate 100; C.alb. 103: The fluconazole-resistant Candida
albicans isolate 103; FCZ: fluconazole; VCZ: voriconazole; ALB: albaconazole
The structure-activity relationship between various substituents on the 1,2,3-benzotriazin-4-
one was investigated. In the overall view, compounds with groups at the 7-position exhibited more
potent and broader-spectrum antifungal activities than that with groups at the 5-, 6- and 8- position.
Furthermore, compounds with electron-withdrawing groups at the 7-position such as –NO2 (6r) and
–CF3 (6q) resulted in a decrease against Aspergillus fumigatus and Microsporum gypseum. In
addition, compounds 6l-o and 6w with halogens showed higher antifungal activities than those with
electron-withdrawing groups. Moreover, Compound 6y which replaced 1,2,3-benzotriazin-4-one
with 1,2,3-naphthotriazin-4-one showed no advantage than 6z. Finally, 7-Cl substituted analogue
6m displayed the most remarkable in vitro activity against all of the tested strains, especially with
the MIC value of 0.25 μg/ml against Aspergillus fumigatus and Microsporum gypseum.
In order to understand the binding mode of compound 6m, docking simulations were carried
out using the Surflex-Dock program in the SYBYL-X 2.0 software. The published crystal structures
of C. albicans CYP51 (PDB ID: 5TZ1) and A. fumigatus CYP51 (PDB ID: 4UYM) served as a
useful template for generating binding modes [24,25]. Images depicting the proposed binding modes
were generated using PyMOL. As showed in Fig.3A, the triazole ring and difluorophenyl group of
6m formed a coordination bond and hydrophobic interactions with heme group and Phe-126 and
Tyr-132, respectively. The 1,2,3-benzotriazin-4-one side chain extended into the CYP51 channel to
form favorable hydrophobic interactions and van der Waals with the surrounding residues such as
Tyr-118, Leu-121, Phe-233, Phe-380 and Met-508. The binding mode of 6m with A. fumigatus
CYP51 was similar to that with C. albicans CYP51 (Fig. 3B).
Fig.3. The binding mode of compound 6m in the active site of (A) C. albicans CYP51 (PDB
ID:5TZ1) and (B) A. fumigatus CYP51 (PDB ID:4UYM)
The incidence of invasive fungal infections has dramatically increased for several decades,
especially the increasing number of invasive aspergillosis among immunocompromised patients.

However, this pathogen is intrinsically resistant to fluconazole. A major focus of our optimization
effort was to broaden antifungal spectrum and increase the anti-Aspergillus efficacy. In this report
we have designed and synthesized a series of novel triazole derivatives containing 1,2,3-
benzotriazin-4-one and their in vitro antifungal activities were evaluated. Most of the compounds
exhibited stronger antifungal activities against Candida albicans and Cryptococcus neoformans
with MIC values in the range of 0.0156 to 2.0 μg/ml than fluconazole. Moreover, 6m showed
comparable antifungal activity against seven pathogenic strains as voriconazole and albaconazole,
especially against Aspergillus fumigatus (MIC = 0.25 μg/ml), and displayed moderate antifungal
activity against fluconazole-resistant strains of Candida albicans. A clear SAR study indicated that
compounds with groups at the 7-position resulted in novel antifungal triazoles with more
effectiveness and a broader-spectrum. The SARs and binding mode established in this study will be
useful for further lead optimization and pharmacokinetic evaluation.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (No. 81673280;
81830106), National Mega-project for Innovative Drugs (2019ZX09721001) and Science and
Technology Commission of Shanghai Municipality (NO. 19431901300).
Reference
[1] Kathiravan M K, Salake A B, Chothe A S, et al. The biology and chemistry of antifungal agents: A
review. Bioorg Med Chem. 2012, 20(19): 5678-5698.
[2] Groll A H, Lumb J. New developments in invasive fungal disease. Future Microbiol. 2012, 7(2): 179-
184.
[3] Turel O. Newer antifungal agents, Expert Rev. Anti-infect Ther. 2011,9: 325-338.
[4] Sorbera L A, Aravamudan J, Rosa E. Therapeutic target for candidiasis. Drugs Future. 2011,36: 627-
630.
[5] Latge J P. Aspergillus fumigatus and aspergillosis. Clin Microbiol Rev. 1999,12: 310-350.
[6] Yuzo Y, Yuri A. Yeast Cytochrome P-450 Catalyzing Lanosterol 14a-Demethylation. J Biol Chem.
1984, 259(3): 1661-1666.
[7] Xie, F, et al. Design, synthesis, and in vitro evaluation of novel antifungal triazoles. Bioorg Med
Chem Lett. 2017, 27(10): 2171-2173.
[8] Hadrich I, MakniF, Neji S, et al. Invasive aspergillosis: resistance to antifungal drugs.
Mycopathologia.2012,12: 131-141.
[9] Ueda Y, Matiskella J D, Golik J, et al. Phosphonooxymethyl prodrugs of the broad spectrum
antifungal azole, ravuconazole: synthesis and biological properties. Bioorg Med Chem Lett. 2003, 13:
3669−3672.
[10] Bartroli J, Turmo E, Alquero, M, et al. New azole antifungals: 3. Synthesis and antifungal activity
of 3-substituted- 4(3H)-quinazolinones. J Med Chem. 1998, 41: 1869−1882.
[11] Bartroli J, Merlos M, Sisniega H. Overview of albaconazole. Eur. Infect. Dis. 2011, 5: 88−91.
[12] Guillon R, Pagniez F, Picot C, et al. Discovery of a Novel Broad-Spectrum Antifungal Agent
Derived from Albaconazole. ACS Med Chem Lett. 2013, 4(2): 288-292.
[13] Cao X F, Sun Z S, et al. Design, Synthesis, and Structure−Activity Relationship Studies of Novel
Fused Heterocycles-Linked Triazoles with Good Activity and Water Solubility. J Med Chem. 2014, 57:

3687-3706
[14] Hunt J C, Briggs E, Clarke E D, et al. Synthesis and SAR studies of novel antifungal 1,2,3-triazines.
Bioorg Med Chem Lett. 2007, 17: 5222-5226.
[15] Migawa M T, Townsend B L. Synthesis and Unusual Chemical Reactivity of Certain Novel 4,5-
Disubstituted 7-Benzylpyrrolo[2,3-d][1,2,3]triazines. J Org Chem. 2001, 66: 4776-4782.
[16] Migawa M T, Drach J C, Townsend L B. Design, Synthesis and Antiviral Activity of Novel 4,5-
Disubstituted 7-(β-d-Ribofuranosyl)pyrrolo[2,3-d][1,2,3]triazines and the Novel 3-Amino-5-methyl-1-
(β-d-ribofuranosyl)- and 3-Amino-5-methyl-1-(2-deoxy-β-d-ribofuranosyl)-1,5-dihydro-1,4,5,6,7,8-
hexaazaacenaphthylene as Analogues of Triciribine. J Med Chem. 2005, 48: 3840-3851.
[17] Gadekar S M, Ross E. Notes. Some Halogenated 1,2,3-Benzotriazin-4(3H)ones. J Org Chem. 1961,
26: 613-615.
[18] Gadekar S M, Frederick J L. 1,2,3-Benzotriazine Sulfonamides. A New Class of Oral Diuretic
Agents. J Org Chem. 1962, 27: 1383-1386.
[19] Caliendo G, Fiorino F, Grieco P, et al. Preparation and local anaesthetic activity of benzotriazinone
and benzoyltriazole derivatives. Eur J Med Chem. 1999, 34: 1043-1051.
[20] Kumar K S, Sandra R S, Rambabu D, et al. Cu-mediated N-arylation of 1,2,3-triazin-4-ones:
Synthesis of fused triazinone derivatives as potential inhibitors of chorismate mutase. Bioorg Med Chem
Lett. 2012, 22: 1146-1150
[21] Jaan P, Chen C K, et al. The Process Development of Ravuconazole: An Efficient Multikilogram
Scale Preparation of an Antifungal Agent. Org Process Res Dev. 2009, 13: 716-728.
[22] Clinical and Laboratory Standards Institute/National Committee for Clinical Laboratory Standards,
Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeast, third ed., Clinical and
Laboratory Standards Institute, Wayne, PA, 2009. Approved Standard, Document M27ea3.
[23] Clinical and Laboratory Standards Institute/National Committee for Clinical Laboratory Standards,
Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeast, third ed., Clinical and
Laboratory Standards Institute, Wayne, PA, 2008. Approved Standard, Document M38eA2.
[24] Hargrove T Y, Friggeri L, Warwzak Z, et al. Structural analyses of Candida albicans sterol 14 alpha-
demethylase complexed with azole drugs address the molecular basis of azole-mediated inhibition of
fungal sterol biosynthesis. J Biol Chem. 2017, 292: 6728-6743.
[25] Hargrove T Y, Warwzak Z, Lamb D C, et al. Structure-Functional Characterization of Cytochrome
P450 Sterol Alpha-Demethylase (Cyp51B) from Aspergillus Fumigatus and Molecular Basis for the
Development of Antifungal Drugs. J Biol Chem. 2015, 290: 23916-23934.
Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which
may be considered as potential competing interests:
Design, synthesis, and structure-activity relationship
studies of novel triazole agents with strong antifungal
activity against Aspergillus fumigatus
Zichao Dinga,†, Tingjunhong Nib,†, Fei Xiea,†, Yumeng Haoa, Shichong Yua,
Xiaoyun Chaia, Yongsheng Jina, Ting Wanga, Yuanying Jiangb,*, Dazhi Zhanga,*
a Department of Organic Chemistry, School of Pharmacy, Second Military Medical University, 325 Guohe Road,
Shanghai 200433 (China)
b Shanghai Tenth People's Hospital, Tongji University School of Medicine, 301 Middle yanchang Road, Shanghai
200072 (China)
Corresponding Authors
Jiang Y. Tel.: +86-021-81871357, E-mail address: 13761571578@163.com
Zhang D. Tel.: +86-021-81871226, E-mail address: zdzhang_yjhx@smmu.edu.cn
† The authors contributed equally.

O
N
O
N
OH
OH
N
N
Follow-up
Strategy
N
F
N
N
F
N
N
R
N
N
Cl
F
Albaconazole
F
target compounds