Diversity-oriented synthesis of pyrazoles derivatives from flavones and isoflavones leads to the discovery of promising reversal agents of fluconazole resistance in Candida albicans

Article abstract of DOI:10.1016/j.bmcl.2018.03.066

Diversity-oriented synthesis of derivatives of natural products is an important approach for the discovery of novel drugs. In this paper, a series of novel 3,4-diaryl-1H-pyrazoles and 3,5-diaryl-1H-pyrazoles derivatives were synthesized through the one-po

Full text of DOI:10.1016/j.bmcl.2018.03.066

Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Diversity-oriented synthesis of pyrazoles derivatives from flavones and
isoflavones leads to the discovery of promising reversal agents of
fluconazole resistance in Candida albicans
Chang-Yi Cui a, Jun Liu a, Hong-Bo Zheng a, Xue-Yang Jin a, Xiao-Yu Zhao a, Wen-Qiang Chang a,
Bin Sun a,b, , Hong-Xiang Lou a,
⇑
⇑
a School of Pharmaceutical Sciences, Shandong University, Jinan 250012, PR China
b National Glycoengineering Research Center, Shandong University, Jinan 250012, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Diversity-oriented synthesis of derivatives of natural products is an important approach for the discovery
of novel drugs. In this paper, a series of novel 3,4-diaryl-1H-pyrazoles and 3,5-diaryl-1H-pyrazoles
derivatives were synthesized through the one-pot reaction of flavones and isoflavones with the hydrazine
hydrate and substituted hydrazine hydrate. Some of these novel compounds exhibited antifungal effects
against Candida albicans SC5314, and displayed more potent inhibitory activities against the efflux-pump-
deficient strain DSY654. In addition, compounds 25, 28 and 32a displayed outstanding reversal activity of
azole resistance against clinical azole-resistant Candida albicans in combination with fluconazole (FLC),
with FICI values ranging from 0.012 to 0.141. The preliminary structure-activity relationship (SAR) of
these compounds was also discussed. In conclusion, this study provides several novel agents that dis-
played potent antifungal activities alone or together with fluconazole, which makes progress for devel-
opment of antifungal drugs.
Received 10 February 2018
Revised 19 March 2018
Accepted 23 March 2018
Available online xxxx
Keywords:
Candida albicans
3,4-Diaryl pyrazoles
3,5-Diaryl pyrazoles
Antifungal
Resistance
Ó 2018 Published by Elsevier Ltd.
Candida albicans, isolated from human oral, gastrointestinal,
vaginal, cutaneous and mucosal surfaces, is one of the most com-
mon human opportunistic fungal pathogen, causing high mortality
in nosocomial bloodstream infections. Currently, azole antifungal
agents are widely used as first-line antifungal therapy by inhibiting
antifungal activity, which could be used as the precursor for the
discovery of novel antifungal agents.7–10 In addition, Furlan and
Gu have revealed a strategy to generate chemically engineered
extracts through chemical diversification of natural product mix-
tures.11,12 Specifically, the extract of flavones was reacted with
hydrazine monohydrate, and the following bioactivity-guided frac-
tionation of the semisynthetic mixture led to the isolation of 3,5-
diaryl-1H-pyrazole 3, which displayed excellent antifungal activity
(Fig. 2).11 Moreover, it was reported that the isoflavones could also
react with hydrazine to generate 3,4-diaryl-1H-pyrazoles in one
step with good-to-excellent yields.13,14 Furthermore, pyrazole
was considered as an important antifungal pharmacophore, and
many pyrazole derivatives have been reported to exhibit effective
antifungal activity.15,16,3 All these findings motivated us to prepare
a series of 3,4-diaryl- and 3,5-diaryl-pyrazole derivatives by the
reaction of flavones and isoflavones with hydrazine hydrate and
substituted hydrazine hydrate, and these novel pyrazole deriva-
tives were then evaluated for their antifungal activity.
fungal lanosterol 14a-demethylase, and fluconazole (FLC) is the
most commonly used azole drug to treat C. albicans in prophylaxis
and therapy. Although contemporary antifungal medications are
still effective, the usefulness of these drugs is compromised by
the frequent emergence of high-grade resistance.1–6 This acquired
drug resistance has been rapidly increasing worldwide and posed a
grave threat to human health. Therefore, the development of new
and more potent antifungal drugs becomes even more urgent.
Natural products are considered as great sources for the devel-
opment of novel pharmaceuticals and their scaffolds are also rec-
ognized as ‘‘privileged structures” for further modifications.
Recently, it has been reported that flavones and isoflavones, such
as flavone (1) and formononetin (2) (Fig. 1) exhibited moderate
Firstly, each of the natural isolated flavones 1, 10, 11, 13, 14 and
isoflavones 2, 4, 5, 12 was reacted with hydrazine monohydrate,
respectively, under reflux in ethanol. Interestingly, only four
pyrazoles (compounds 6–9) were obtained (Scheme 1), and the
starting material bearing 5-hydroxyl or 8-hydroxyl group
⇑
Corresponding authors at: National Glycoengineering Research Center,
Shandong University, Jinan 250012, PR China (B. Sun).
E-mail addresses: sunbin@sdu.edu.cn (B. Sun), louhongxiang@sdu.edu.cn
(H.-X. Lou).
https://doi.org/10.1016/j.bmcl.2018.03.066
0960-894X/Ó 2018 Published by Elsevier Ltd.
Please cite this article in press as: Cui C.-Y., et al. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2018.03.066

2
C.-Y. Cui et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
resulted in the formation of pyrazoles derivatives 25–29 in satis-
fied yield (Scheme 2).17 The proposed reaction mechanism of fla-
vones and isoflavones with hydrazine is illustrated in Fig. 3.
Nucleophilic attack of hydrazine at C-2 of flavone or isoflavone fol-
lowed by ring-opening afforded the intermediate VI. The further
nucleophilic attack of the second nitrogen atom at the carbonyl
carbon of VI and subsequent dehydration could lead to the forma-
tion of the pyrazole ring. The obtained pyrazoles may exist as mix-
tures of OHAN and NHAO tautomers.
Fig. 1. The chemical structures of flavone (1) and formononetin (2).
Secondly, to prepare more structurally diversed pyrazoles
derivatives and define their antifungal structure-activity relation-
ship (SAR), the flavones and isoflavones 1, 2, 4 and 5 as well as
the methylated intermediates 15–17 and 19 were then reacted
with substituted hydrazines containing electron-donating groups,
such as ethyl, phenyl, p-methoxyphenyl, 2-hydroxy ethyl etc, and
twenty-five more novel pyrazoles derivatives were obtained
(Scheme 3). However, when the substituted hydrazines bearing
electron-withdrawing groups, the reaction did not work, and these
observations indicated that the nucleophilic potency of hydrazine
derivatives was essential for this reaction.
Fig. 2. Synthesis of diarylpyrazole 3.
In addition, as shown in Scheme 4, we also studied the reaction
of flavones with ethylenediamine. The compounds 1, 14, 15, 18,
and 19 were treated with ethylenediamine under reflux in ethanol,
and the novel 5,7-diaryl-2,3-dihydro-1,4-diazepine derivatives 47–
51 were then obtained in satisfied yield.18,19
All the synthetic pyrazoles and diazepines derivatives were
evaluated for antifungal activities against C. albicans SC5314 (the
wild-type strain) and DSY654 (the Cdr1, Cdr2 efflux pumps defi-
cient strain), and fluconazole served as the positive antifungal
agent.20 The minimum inhibitory concentrations (MICs) of active
compounds are summarized in Table 1. Compounds 6, 20 and 29
exhibited antifungal activity against the SC5314 with MIC80 values
ranging from 8 to 16
and 43a displayed very strong inhibitory activity against DSY654
with MIC80 values ranging from 1 to 4 g/mL, and compound 20
was found to be the most potent antifungal agent (MIC80 = 1 g/
lg/mL. Surprisingly, compounds 20, 21, 40b
l
Scheme 1. Synthesis of pyrazoles derivatives 6–9. Reagents and conditions: (a)
EtOH, 90 °C, reflux for 8–12 h.
l
mL). However, the growth inhibitory activity of all the synthesized
compounds against C. albicans SC5314 and DSY654 was less potent
than the reference drug FLC, with MIC80 values of 2 and 0.25 lg/
mL, respectively. Among all the derivatives, compound 6 displayed
moderate antifungal activity against SC5314 and DSY654, with
(compounds 10–14) could not react with hydrazine under this con-
dition, which might because of the intramolecular hydrogen bond
impacting the ring-open process. Compounds 10–14 were then
methylated by CH3I, and the resulting intermediates 15–19 reacted
with hydrazine hydrate to provide the pyrazoles 20–24 as we
expected. The following demethylation by boron tribromide
MIC80 values of 16 and 8
methoxyl groups at C-40 and C-60 on arene A led to the most potent
compound 20 (MIC80 = 8 and 1 g/mL for SC5314 and DSY654,
lg/mL, respectively. Introduction of two
l
Scheme 2. Synthesis of pyrazoles derivatives 20–29. Reagents and conditions: (a) acetone, 60 °C, reflux for overnight; (b) EtOH, 90 °C, reflux for 8–12 h; (c) DCM, -78 °C,
reflux for 1 h, then reflux for overnight at rt.
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C.-Y. Cui et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
3
Fig. 3. Proposal of mechanism for the reaction of methylated 5, 7-dihydroxyl flavones and 5, 7-dihydroxyl isoflavones with hydrazine.
Scheme 3. Synthesis of pyrazoles derivatives 30a–39a, 41a–46a, 34b, 35b, 37b–40b, 42b–44b. Reagents and conditions: (a) EtOH, 90 °C, reflux for 48–60 h.
respectively). This indicated that the methoxyl groups on arene A
were very important for the antifungal activity. Interestingly, when
the substitutive groups were introduced to the ring B, the inhibi-
tory activity against SC5314 was diminished and even lost, while
the antifungal potency against DSY654 was maintained or
improved (30a, 31a, 40b, 43a and 46a). In addition, the replace-
ment of pyrazole ring by the dihydrodiazepine moiety resulted in
an obviously decreased potency (20 vs. 48 and 29 vs. 51). Further-
more, the derivatives bearing the 3,5-diaryl-pyrazole skeleton dis-
played more potent antifungal activity than the 3,4-diaryl-pyrazole
compounds.
The in vitro synergistic antifungal activities of the most potent
compound 20 were then tested using the broth microdilution
checkboard assay, and the MIC80 of 20 and FLC alone or in combi-
nation against three clinically isolated FLC-resistant C. albicans
strains (28A, 28I, and 28J) were summarized in Table 2. The FICI
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C.-Y. Cui et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Scheme 4. Synthesis of diarylbenzodiazepines 47–51. Reagents and conditions: (a) ethylenediamine, EtOH, 90 °C, reflux for 2–12 h.
Table 1
In vitro antifungal effects of thirteen active compounds against Candida albicans SC5314 and DSY654.a
Compounds
SC5314
DSY654
Compounds
SC5314
DSY654
6
9
16
>128
8
64
8
8
16
1
2
8
40b
43a
46a
48
49
51
>128
>128
>128
128
>128
>128
2
4
4
8
8
16
32
0.25
20
21
29
30a
31a
>128
128
8
8
FLC
a
Mean values based on three independent experiments; all data are minimum inhibitory concentrations (MIC80), given as lg/mL.
Table 2
The susceptibility test of compound 20 alone and in combination with FLC against three FLC-resistant Candida albicans strains.
Strains
MIC80 (mg/mL)
Alone
In combination
FICI
Interpretationa
20
FLC
20
FLC
28A
28I
28J
32
32
32
>128
>128
>128
8
8
8
0.25
0.25
0.5
0.252
0.252
0.254
SYN
SYN
SYN
MIC, minimum inhibitory concentration; FICI, fraction inhibited concentration index.
a
SYN, synergism. SYN was defined as a FICI of ꢀ0.5, antagonism was defined as a FICI of >4.0, and Indifference was defined as a FICI of >0.5–4.
values of 20 for strains 28A, 28I, and 28J are 0.252, 0.252 and 0.254,
respectively, which indicated that pyrazole derivative 20 displayed
potent synergistic activity with FLC. In addition, the data of the
synergistic effect of 20 against FLC-resistant C. albicans strain 28A
was displayed as a heat map using HemI 1.0.3.7 (Fig. 4). These
encouraging results motivated us to evaluate all the synthetic
derivatives for their synergistic antifungal activity with FLC to dis-
cover more potent reversal agents of drug resistance. Compounds
6, 20, 21, 25, 28, 32a, 34b, 40b and 48 displayed excellent synergis-
tic activity, with FICI values ranging from 0.012 to 0.266, and com-
pound 28 was the most potent agent with FICI value of 0.012
(Table 3). Compound 6, bearing the 3,5-diaryl-1H-pyrazole skele-
ton, exhibited excellent synergistic antifungal activity, with a FICI
value of 0.266. Introduction of methoxyl groups to the arene A
led to compounds 20, 21 and 40b with FICI values ranging from
0.252 to 0.266, which are comparable to that of compound 6. This
suggests that the methoxyl groups on arene A do not provide any
Fig. 4. Compound 20 enhances the efficacy of fluconazole against FLC-resistant
Candida albicans strain 28A. A dose response matrix was performed in RPIM 1640
medium with gradients of FLC and 20. Date was analyzed after 48 h at 35 °C and
displayed as a heat map using HemI 1.0.3.7, see color bar.
obvious effect on the activity. Compound 25, with three hydroxyl
groups at C-20, C-40 and C-60 of arene A, exhibited very potent syn-
ergistic antifungal activity with FICI value of 0.075. Surprisingly,
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C.-Y. Cui et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
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Table 3
In vitro susceptibilities of pyrazoles derivatives and FLC acting alone and in combination against clinical FLC-resistant Candida albicans 28A.
Compounds
MIC80 (mg/mL)
Alone
In combination
Pyrazoles
FICI
Interpretationa
Pyrazoles
FLC
FLC
6
9
20
21
25
28
30a
31a
32a
34b
40b
43a
46a
48
64
>128
32
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
16
>128
8
16
8
2
0.266
2
SYN
IND
SYN
SYN
SYN
SYN
IND
IND
SYN
SYN
SYN
IND
IND
SYN
IND
IND
>128
0.25
2
0.252
0.266
0.078
0.012
2
1.016
0.141
0.266
0.266
2
64
>128
>128
>128
128
>128
>128
>128
>128
>128
128
2
1
0.5
>128
2
2
2
>128
128
16
32
32
>128
>128
32
>128
>128
2
>128
>128
2
>128
>128
2
0.266
2
2
49
51
>128
>128
MIC, minimum inhibitory concentration; FICI, fraction inhibited concentration index.
a
SYN, synergism; IND, indifference. SYN was defined as a FICI of ꢀ0.5, antagonism was defined as a FICI of >4.0, and IND was defined as a FICI of >0.5–4.
the further introduction of phenolic group at C-400 of arene C led to
the most potent compound 28. These findings indicated that the
hydroxyl groups on arene A and C is critical for the improved syn-
ergistic activity.
sized compounds) associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmcl.2018.03.066.
References
In summary, we have described an efficient synthesis of a series
of pyrazole and dihydrodiazepine derivatives in one-pot by the
reaction of flavones and isoflavones with substituted hydrazines
and ethylenediamine. Some of these novel compounds exhibited
antifungal effects against Candida albicans SC5314, and displayed
more potent inhibitory activities against the efflux-pump-deficient
strain DSY654. Moreover, compounds 25, 28 and 32a displayed
outstanding reversal activity of drug resistance against clinical
drug-resistant Candida albicans in combination with FLC, and 28
was proved to be the most potent agent with FICI value of 0.012.
The preliminary SAR study indicated that the 3,5-diaryl-pyrazole
skeleton was preferable for antifungal activity, and the methoxyl
and hydroxyl groups on arene A also played critical role in promot-
ing the potency. Further studies on structural optimization are in
progress in our group.
1. Le Borgne Marc, Lebouvier Nicolas. Bioorg Med Chem Lett. 2007;17:3686.
2. Bhat Manoj K, Aher Nilkanth G, Pore Vandana S. Bioorg Med Chem Lett.
2009;19:759.
3. Menozzi Giulia, Merello Luisa, La Colla Paolo. Bioorg Med Chem. 2004;12:5465.
4. Xiang Mi-Jie, Liu Jin-Yan, Shi Ce. Res Microbiol. 2015;166:153.
5. Jin Yo-Sheng, Wang Yu-Hua, Dong Hu-Huai. Bioorg Med Chem Lett.
2016;26:3098.
6. Vukic0evic Rastko D, Damljanovic Ivan. Bioorg Med Chem Lett. 2009;19:1093.
7. Rner Martin Weidenbo, Jha Hem Chandra. Mycol Res. 1997;101:733.
8. das Neves Michelline Viviane Marques, de Jesus Oliveira Eduardo. Braz
Microbiol. 2016;47:159.
J
9. Ashok D, Ravi S, Ganesh Arram, Vijaya Lakshmi B, Adam Shaik. Med Chem Res.
2016;25:909.
10. Ji Zhiqin, Wei Shaopeng, Wenjun Wu. Int J Mol Sci. 2012;13:7375.
11. Furlan Ricardo LE, López Silvia N. PNAS. 2007;104:441.
12. Gu Qiong, Wu Taizong. J Nat Prod. 2015;78:1593.
13. Zhang Zun-Ting, Tan Da-jin, Xue Dong. Helv Chim Acta. 2007;11:2096.
14. Jeko József, Lévai Albert, Artur M, Silva S. Aust J Chem. 2007;60:905.
15. Chimenti Franco, Fioravanti Rossella, Turini Paola, Ortuso Francesco. Eur J Med
Chem. 2008;43:2262.
16. Zhang Jin, Tan Da-Jin, Zhang Zun-Ting. J Mol Struct. 2017;1149:235.
17. Botting Nigel P, Marshall Laura J, Cable Karl M. J Labelled Compd Radiopharm.
2010;53:601.
18. Menezes Júlio César L, Vaz Luana Beatriz A, Taylor Jason G. Molecules.
2015;20:43.
Acknowledgments
This work was financially supported by grant from the National
Natural Science Foundation (NNSF) of China (No. 81630093). We
thank Dr. Shuqi Wang for NMR measurements.
19. Ramajayam R, Giridhar Rajani, Gamyyadav Mange. J Enzyme Inhib Med Chem.
2007;22:716.
20. Li Ying, Chang Wenqiang, Lou Hongxiang. FEMS Yeast Res. 2015;15:2.
A. Supplementary data
Supplementary data (general methods for key intermediates,
typical reaction procedure, and experimental data for the synthe-
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