Design, synthesis and biological evaluation of novel non-azole derivatives as potential antifungal agents

Article abstract of DOI:10.1016/j.cclet.2015.04.030

A series of 3-substituted quinazolinones, 2-substituted quinoxalines and 2-substituted benzopyrans were synthesized and evaluated for their antifungal activity in vitro. The new compounds revealed excellent in vitro antifungal activity with broad spectrum. The structure-activity relationships (SARs) of the derivatives were analyzed. Compound 9A2 exhibits better antifungal activity against 5 tested fungi in vitro than fluconazole, especially against Trichophyton rubrum and Microsporum gypseum. This study provides a series of novel lead compounds for the development of non-azole antifungal agents.

Full text of DOI:10.1016/j.cclet.2015.04.030

G Model
CCLET 3297 1–5
Chinese Chemical Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
1
2
Original article
3
4
Design, synthesis and biological evaluation of novel non-azole
derivatives as potential antifungal agents
a,1
b,1
a
a
a
a,
5
Q1 Hui Tang , Juan Wu , Wen Zhang , Lei Zhao , Ya-Hui Zhang , Cheng-Wu Shen *
a
6
7
Q2 Department of Pharmacy, Shandong Provincial Hospital Affiliated to Shandong University, Jinan 250021, China
b
Department of Pharmacy, General Hospital of PLA Chengdu Military Area Command, Chengdu 610083, China
A R T I C L E I N F O
A B S T R A C T
Article history:
A series of 3-substituted quinazolinones, 2-substituted quinoxalines and 2-substituted benzopyrans
were synthesized and evaluated for their antifungal activity in vitro. The new compounds revealed
excellent in vitro antifungal activity with broad spectrum. The structure–activity relationships (SARs) of
the derivatives were analyzed. Compound 9A2 exhibits better antifungal activity against 5 tested fungi in
vitro than fluconazole, especially against Trichophyton rubrum and Microsporum gypseum. This study
provides a series of novel lead compounds for the development of non-azole antifungal agents.
ß 2015 Cheng-Wu Shen. Published by Elsevier B.V. on behalf of Chinese Chemical Society and Institute of
Materia Medica, Chinese Academy of Medical Sciences. All rights reserved.
Received 5 January 2015
Received in revised form 27 January 2015
Accepted 3 April 2015
Available online xxx
Keywords:
Non-azole
Biological evaluation
Structure–activity relationships
Synthesis
Antifungal agents
8
9
1. Introduction
enzymes [10]. Therefore, the search for new non-azole CYP51 29
inhibitors is important, and this area has been a focus of our 30
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
During the past several years, the incidence of life-threatening
invasive and systemic fungal infections has increased dramatically
due to an increase in the number of immunocompromised hosts,
such as patients undergoing anticancer chemotherapy or organ
transplants and patients with AIDS [1,2]. Several antifungal drugs
such as azoles, amphotericin B [3], 5-fluorocytosine [4], and
caspofungin [5] have been developed to reduce the impact of
fungal diseases (Fig. 1). Azoles, especially triazole agents, are used
widely and effectively as first-line agents for the treatment of fungal
infections in the clinic. However, the increasing administration of
antifungal agents has led to fungal resistance [6,7], and the currently
available antifungal drugs do not meet the requirements of clinical
treatments for complex infections. Therefore, new antifungal drugs
are a major focus of drug development.
research in recent years. Three-dimensional models of CYP51 have 31
been constructed using the homology modeling technique [11– 32
13]. Among our reported non-azole lead compounds, the 33
molecules based on the isoquinoline scaffold showed potent 34
antifungal activity, and L-6 was the most potent compound that 35
interacted with the residues of fungal CYP51 apoprotein without 36
binding to the heme (Fig. 1) [14,15]. A series of compounds were 37
designed and synthesized to investigate the structure–activity 38
relationships of non-azole antifungal compounds. First, we 39
replaced the isoquinoline scaffold with quinazolinone, benzopyran 40
or quinoxaline in order to investigate the antifungal activity of the 41
core scaffold structure. A long alkyl side chain was introduced at 42
the position 3 of the quinazolinone, the position 2 of the 43
quinoxaline or the benzopyran ring, in order to interact with 44
the hydrophobic S3 pocket of CYP51. As a result, a series of 3- 45
substituted quinazolinone compounds, 2-substituted quinoxaline 46
compounds and 2-substituted benzopyran compounds (Fig. 2) 47
were designed and synthesized, and their antifungal activity was 48
Lanosterol 14a-demethylase (CYP51) is a key enzyme in fungal
sterol biosynthesis [8]. Azole antifungals are competitive inhibi-
tors of CYP51 that are used clinically [9]; however, cases of fatal
hepatotoxicity with azole drugs have been reported due to the
coordination of azoles with the heme groups of cytochrome P450
evaluated in vitro.
49
2. Experimental
50
*
Corresponding author.
The chemical synthesis of heterocyclic compounds is outlined 51
in Schemes 1–3. First, treatment of substituted 2-aminobenzoic 52
E-mail address: scw810@163.com (C.-W. Shen).
These authors contributed equally to this work.
1
http://dx.doi.org/10.1016/j.cclet.2015.04.030
001-8417/ß 2015 Cheng-Wu Shen. Published by Elsevier B.V. on behalf of Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical
1
Sciences. All rights reserved.
Please cite this article in press as: H. Tang, et al., Design, synthesis and biological evaluation of novel non-azole derivatives as potential
antifungal agents, Chin. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.cclet.2015.04.030

G Model
CCLET 3297 1–5
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H. Tang et al. / Chinese Chemical Letters xxx (2015) xxx–xxx
Fig. 1. Antifungal drugs.
Fig. 2. The targeted compounds.
53
54
55
56
57
58
59
60
61
62
63
acid 1A–1B with propionic acid anhydride 2 afforded compounds
3A–3B [16–18]. Then, intermediates 3A–3B were reacted with
different alkylamines in dry dimethylformamide (DMF) to afford
the targeted compounds 5A–5B. Compounds 8A1–8A5 were
synthesized according to a reported procedure [19,20] by heating
ethanolic mixtures to 160–170 8C or using microwave (MW)
irradiation for 1 h in the presence of diisopropylamine (DIPA) as a
base. Reduction of 8A1–A5 with lithium aluminum hydride
11 in good yields [21]. A good method for preparing the quinoxaline-
2-carboxaldehyde 12 was found in carrying out the oxidation with
selenium dioxide of compound 11 in aqueous dioxane at moderate
temperatures [22]. Condensation of compound 12 with hydrazines
resulted in corresponding hydrazone compounds 13A1–13A4 [23].
All chemical structures were confirmed by electrospray ionization
mass spectrometry and nuclear magnetic resonance spectroscopic
analyses.
64
65
66
67
68
69
70
71
72
73
74
(LiAlH
4
) in an ice bath yielded the targeted compounds 9A1–A5.
In vitro antifungal activity was measured by means of the
MIC using the serial dilution method in 96-well microtest plates.
Test fungal strains were obtained from the ATCC. The MIC
In order to generate useful intermediates, condensation of 1,2-
diaminobenzene 10 with pyruvaldehyde gave 2-methylquinoxaline
O
O
R1
COOH
a
R
1
b
R1
R2
O
O
N
NH2
O
O
N
N
1
A
R1=CH
2
3A
3B
R1=CH3
R =C H
2 5
3
5A1-5A4
5B1-5B4
1B
R =C H
1
2
5
1
0
Scheme 1. The synthesis route of the target compounds. Reagents and conditions: (a) 120 8C, reflux, yield 61.6%; (b) alkylamine, N,N -dicyclohexylcarbodiimide (DCC),
dimethylformamide (DMF), 16 h, yield 30–60%.
Please cite this article in press as: H. Tang, et al., Design, synthesis and biological evaluation of novel non-azole derivatives as potential
antifungal agents, Chin. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.cclet.2015.04.030

G Model
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H. Tang et al. / Chinese Chemical Letters xxx (2015) xxx–xxx
3
O
O
O
O
O
R2
H
a
b
O
R2
O
O
R2
OH
6
7A1-7A5
9A1-9A5
8
A1-8A5
Scheme 2. The synthesis route of the target compounds. Reagents and conditions: (a) appropriate aldehyde, diisopropylamine (DIPA), ethanol (EtOH), microwave (MW), 160–
70 8C, 1 h, 22–33%; (b) lithium aluminum hydride (LiAlH ), tetrahydrofuran (THF), ice bath, yield 65–78%.
1
4
H3C
NH2
NH2
H3C
N
N
H3C
N
N
H3C
N
a
b
c
N
R3
CHO
N
N
H
1
0
11
12
13A1-13A4
2 2 5 2
Scheme 3. The synthesis route of the target compounds. Reagents and conditions: (a) 2-Oxo-propionaldehyde, sodium pyrosulfite (Na S O ); (b) selenium dioxide (SeO ),
dioxane, reflux; (c) hydrazine, ethanol (EtOH), 60–70 8C.
75
76
77
78
79
80
81
82
83
84
85
determination was performed according to the National Commit-
tee for Clinical Laboratory Standards (NCCLS) recommendations
with RPMI 1640 (Sigma) buffered with 0.165MMOPS (Sigma) as
the test medium [24]. The MIC value was defined as the lowest
concentration of test compounds that resulted in a culture with
turbidity less than or equal to 80% inhibition when compared with
the growth of the control. Test compounds were dissolved in DMSO
serially diluted in growth medium. The yeasts were incubated at
35 8C and the dermatophytes at 28 8C Growth MIC was determined
at 24 h for Candida species, at 72 h for Cryptococcus neoformans,
and at 7 days for filamentous fungi.
broad potency against Candida tropicalis, Trichophyton rubrum and 90
Microsporum gypseum. Compounds 8, 9 and 10 were 2 to 60-folds 91
more potent against C. tropicalis than fluconazole with their MIC80 92
values in the range of 0.24–7.21
mmol/L. Compound 9A2 showed a 93
better antifungal activity against the 5 tested fungi in vitro than 94
fluconazole, especially against T. rubrum and M. gypseum. On the 95
C. neoformans strain, compounds 8A1, 8A2, 8A4, 8A5, 9A1–9A4 and 96
13A3 were more active than fluconazole. Compound 5 exhibited 97
weaker antifungal activity than the other compounds against the five 98
tested fungi. Compound 5A2 was active against C. tropicalis at a 99
concentration of 8.72
mmol/L. Compounds 8A and 9A were more 100
active than fluconazole against the 5 tested fungi except Candida 101
albicans. Compound 9A2 was the most potent compound (minimum 102
8
6
3. Results and discussion
inhibitory concentration for 80%, MIC80 0.24
mmol/L) against 103
87
88
89
The antifungal activity of targeted compounds was listed in
Table 1. Most targeted compounds exhibited antifungal activity
against the five tested fungi. Compounds 8, 9 and 10 showed
C. tropicalis. Compound 13A showed moderate inhibitory activity 104
against the five tested fungi. However, they were not very sensitive 105
against C. albicans and C. neoformans and only 13A3 showed 106
Table 1
a
In vitro antifungal activity of the target compounds (MIC80
,
m
mol/L).
Comp.
R
1
R
2
R
3
C. alb.
C .neo.
C. tro.
8.27
T. rub.
M. gyp.
5
5
5
5
5
5
5
5
8
8
8
8
8
9
9
9
9
9
1
1
1
1
A1
A2
A3
A4
B1
CH
CH
CH
CH
3
3
3
3
(CH
(CH
(CH
(CH
(CH
(CH
(CH
(CH
(CH
(CH
(CH
(CH
2
2
2
2
2
2
2
2
2
2
2
2
)
)
)
)
)
)
)
)
)
)
)
)
4
5
6
7
4
5
6
7
4
5
6
7
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
3
3
3
3
3
3
3
3
3
3
3
3
–
>16.54
>17.44
>18.35
>19.24
>15.88
>18.33
>19.21
>20.11
15.87
4.19
>16.54
>17.44
9.17
>16.54
>17.44
>18.35
>19.24
>15.88
18.33
>19.21
>20.11
7.93
>16.54
>17.44
>18.35
>19.24
>15.88
>18.33
>19.21
>20.11
7.93
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
8.72
9.17
9.62
9.62
C
C
C
C
–
–
–
–
–
–
–
–
–
–
–
–
–
–
2
2
2
2
H
H
H
H
5
5
5
5
>15.88
>18.33
9.60
>15.88
>18.33
>19.21
>20.11
1.98
B2
B3
B4
>20.11
1.98
A1
A2
A3
A4
A5
A1
A2
A3
A4
A5
3A1
3A2
3A3
3A4
2.09
1.05
2.09
2.09
8.83
4.41
4.41
1.10
4.41
18.56
4.22
2.32
2.32
9.28
9.28
CH
2
O(CH
2
)
3
CH
3
2.11
1.05
4.22
8.44
(CH
(CH
(CH
(CH
2
2
2
2
)
4
)
5
)
6
)
7
CH
3
3
3
3
7.48
1.87
1.87
7.48
1.87
CH
CH
CH
3.97
0.99
0.24
0.96
0.96
4.19
1.05
2.10
8.41
2.10
8.83
2.21
2.21
8.83
2.21
CH
–
2
O(CH
2
)
3
CH
3
3.98
3.98
1.99
3.98
7.96
CH
CH
3
12.81
13.70
7.31
12.81
6.85
6.40
6.40
12.81
6.85
–
2
CH
CH
3
1.71
1.71
–
(CH
2
)
2
3
1.82
1.82
1.82
0.91
–
CH(CH
3
)
2
>14.61
19.55
3.27
>14.61
4.88
7.31
14.61
9.78
>14.61
4.88
L-6
19.55
13.06
FCZ
–
–
–
3.27
>208.97
>208.97
a
Abbreviations: Comp.: compound; C. alb.: Candida albicans; C. neo.: Cryptococcus neoformans; C.tro.: Candida tropicalis; T. rub.: Trichophyton rubrum; M. gyp.: Microsporum
gypseum; FCZ: fluconazole.
Please cite this article in press as: H. Tang, et al., Design, synthesis and biological evaluation of novel non-azole derivatives as potential
antifungal agents, Chin. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.cclet.2015.04.030

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H. Tang et al. / Chinese Chemical Letters xxx (2015) xxx–xxx
Table 2
activity of compounds 13A3 with three carbon atoms of
N-substituted alkyl side chain, was higher than that of the other
compounds in 13A. Compound 13A4 with an N-isopropyl
hydrazine was much lower than compound 13A3 with an N-propyl
hydrazine. The SAR analysis above provided a basis for the
modification of heterocyclic derivatives in the future.
To explain the results of the assays for antifungal activity, we
proposed a binding mode for 9A2 in the active site of CYP51 from C.
albicans using the affinity module within the Insight II 2000
software package [25]. As shown in Fig. 3, the benzopyran ring of
9A2 did not coordinate with the heme group, unlike the azoles.
Moreover, the benzopyran ring was located in a hydrophobic
pocket lined with VAL510, LEU511, TYR118, and HIS377. The alkyl
side chains of the compounds were oriented into the pocket, where
they formed hydrophobic and van der Waals interactions with
GLY309, LEU301, ILE302, TRY132, CYS134, and LEU305. Compound
9A2, with a methoxy group at the position 6 of the benzopyran
ring, exhibited reasonable antifungal activity in subsite S4, which
suggested that hydrogen-bond donors and acceptors of the
position 6 of the molecule influenced its antifungal activity.
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
The calculated interaction energies of the target compounds.
Compounds
E
vdw
E
elec
E
total
5
8
9
1
A2
ꢀ45.85
ꢀ47.47
ꢀ55.52
ꢀ46.24
ꢀ3.34
ꢀ6.87
ꢀ7.55
ꢀ5.13
ꢀ49.19
ꢀ54.34
ꢀ63.07
ꢀ51.37
A2
A2
3A3
Calculated interaction energies (kcal/mol) for the complexes of target compounds
with the active site of CYP51 from C. albicans.
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
moderate inhibitory activity (MIC80 = 7.31
respectively).
mmol/L, 1.82 mmol/L,
When the quinazolinone (compound 5A) was replaced by with
a benzopyran (compound 8A or 9A) or a quinoxaline (compound
13A), the antifungal activity increased. Meanwhile, the antifungal
activity of compound 8A or 9A was better than that of compound
13A. From the calculated interaction energies, compound 9A
bound to CYP51 with the highest value of interaction energy
among all compounds tested (Table 2). Compound 5A, with a
methyl group on the benzene ring, had potent antifungal activity,
while the replacement of the N-methyl group of compound 5A by
an ethyl group (compounds 5B) seemed not to be effective.
Compounds substituted with a carbonyl group at position 4, such
as 8A1–8A5 exhibited lower antifungal activity than those with a
hydrogen group, such as 9A1–9A5. Compounds 8A2 and 9A2,
containing an alkyl group with 6 carbon atoms, were most
effective, suggesting that the length of the alkyl group was an
important determinant of antifungal activity. The antifungal
4. Conclusion
145
In response to the need for novel heterocyclic antifungal agents,
we designed, synthesized a series of heterocyclic compounds and
their antifungal activity was evaluated in vitro. The results
indicated that the targeted compounds 8A, 9A and 13A had
potent antifungal effects. Compound 9A2 exhibits best antifungal
146
147
148
149
150
Fig. 3. The docking model for compound 9A2 and CYP51.
Please cite this article in press as: H. Tang, et al., Design, synthesis and biological evaluation of novel non-azole derivatives as potential
antifungal agents, Chin. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.cclet.2015.04.030

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H. Tang et al. / Chinese Chemical Letters xxx (2015) xxx–xxx
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Please cite this article in press as: H. Tang, et al., Design, synthesis and biological evaluation of novel non-azole derivatives as potential
antifungal agents, Chin. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.cclet.2015.04.030