The synthesis and synergistic antifungal effects of chalcones against drug resistant Candida albicans

Article abstract of DOI:10.1016/j.bmcl.2016.05.013

To identify effective and low toxicity synergistic antifungal compounds, 24 derivatives of chalcone were synthesized to restore the effectiveness of fluconazole against fluconazole-resistant Candida albicans. The minimal inhibitory concentration (MIC₈₀) and the fractional inhibitory concentration index (FICI) of the antifungal synergist fluconazole were measured against fluconazole-resistant Candida albicans. This was done via methods established by the clinical and laboratory standards institute (CLSI). Of the synthesized compounds, 2′-hydroxy-4′-methoxychalcone (8) exhibited the most potent in vitro (FICI = 0.007) effects. The structure activity relationship of the compounds are then discussed.

Full text of DOI:10.1016/j.bmcl.2016.05.013

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The synthesis and synergistic antifungal effects of chalcones against
drug resistant Candida albicans
Yuan-Hua Wang a,b,y, Huai-Huai Dong a,y, Fei Zhao a, Jie Wang b, Fang Yan b, Yuan-Ying Jiang a,
,
⇑
Yong-Sheng Jin a,
⇑
a School of Pharmacy, Second Military Medical University, Shanghai 200433, China
b School of Pharmacy, Weifang Medical University, Weifang, Shandong Province 261053, China
a r t i c l e i n f o
a b s t r a c t
Article history:
To identify effective and low toxicity synergistic antifungal compounds, 24 derivatives of chalcone were
synthesized to restore the effectiveness of fluconazole against fluconazole-resistant Candida albicans. The
minimal inhibitory concentration (MIC80) and the fractional inhibitory concentration index (FICI) of the
antifungal synergist fluconazole were measured against fluconazole-resistant Candida albicans. This was
done via methods established by the clinical and laboratory standards institute (CLSI). Of the synthesized
compounds, 20-hydroxy-40-methoxychalcone (8) exhibited the most potent in vitro (FICI = 0.007) effects.
The structure activity relationship of the compounds are then discussed.
Received 20 February 2016
Revised 13 April 2016
Accepted 3 May 2016
Available online xxxx
Keywords:
Candida albicans
Chalcone
Ó 2016 Elsevier Ltd. All rights reserved.
Fluconazole
Synergist
Antifungal effect
Candida albicans is the most common pathogenic fungus and is
frequent in persons with weaker immunity or bacterial flora imbal-
ance including those with cancer, chronic wasting disease, or
excessive use of broad-spectrum antibiotics, immunosuppressive
agents, and hormones. Fluconazole is the most commonly used
drug to treat C. albicans in prophylaxis and therapy; however,
widespread and repeated use of fluconazole resulted in resistance
to or failure of fluconazole therapy.1
One promising approach aimed at overcoming azole resistance
has been sensitizing C. albicans toward fluconazole via small mole-
cules such as berberine,2,3 caffeic acid amides4 and minocycline.5
Recently, natural flavonoids have been reported that have syner-
gistic antifungal activity6,7 such as baicalein and quercetin—this
encouraged us to study new flavonoids with synergistic antifungal
activity. Chalcones are naturally occurring flavonoids composed of
recent study describing new active scaffolds containing an a,b
unsaturated carbonyl group against drug-resistant C. albicans.4
Inspired by these, we designed and synthesized some chalcones—
most of which, as expected, exhibited activity. Herein, we report
the results and the SAR (structure activity relationship) is investi-
gated and discussed.
Previous studies have reported that baicalein (BE, Fig. 1) and
caffeic acid amides (CAA, Fig. 1) exert synergistic antifungal activ-
ity on fluconazole-resistant C. albicans. Bitencourt’s study reported
non-substituted chalcone (NSC, Fig. 1) that was effective against
Trichophyton rubrum. In the structures of BE, CAA and NSC, all
contain the an a,b unsaturated carbonyl and catechol moiety, but
the catechol moiety was believed to be a pan assay interference
compounds (PAINS)11 and thus should be avoided in compound
design. Thus, we designed the structure of chalcone to exclude
the catechol moiety and keep the BE moiety (1-alkoxyl and
5-hydroxyl structure (Compd 1–6, Fig. 1 and Table 1)). We also
kept only the 5-hydroxyl and/or introduced other groups (Compd
6–24, Fig. 1 and Table 1) to investigate SAR.
The synthetic route to target compounds is outlined in
Scheme 1. The chalcones were synthesized by aldol condensation
catalyzed by 30% NaOH. The 20,60-dihydroxyacetophenone was
used as a starting material for the synthesis of compounds 1–6.
First, 20,60-dihydroxyacetophenone was treated with CH3I and
K2CO3 in dry DMF to give 20-hydroxy-60-methoxyacetophenoe,
which then reacted with a series of aromatic aldehydes to achieve
two aromatic rings connected by a three-carbon unit to form an a,b
unsaturated carbonyl group. Some research has demonstrated that
chalcones have antitumor, anti-fungal and anti-inflammatory
activities,8–10 but to the best of our knowledge, no study has yet
described the synergistic antifungal activity of chalcones against
drug-resistant C. albicans. This current work is motivated by a
⇑
Corresponding authors. Tel.: +86 21 81871227.
E-mail addresses: jiangyy@smmu.edu.cn (Y.-Y. Jiang), ysjinsmmu@163.com,
ysjin@smmu.edu.cn (Y.-S. Jin).
y
The authors contributed equally.
http://dx.doi.org/10.1016/j.bmcl.2016.05.013
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Wang, Y.-H.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.05.013

2
Y.-H. Wang et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
HO
HO
O
O
OH
BE
O
OH
Ar
Ar
HN
OH
R
OH
O
O
O
R
CAA
NSC
1-24
O
Figure 1. The design of title compounds.
Table 1
Structures and interaction modes of the title compounds and their MIC80 and FICI values
No.
Structure
MIC80
(l
g mLꢀ1
With FLCa
)
FICI
Mode of interaction
Alone
>64
OH
OH
OH
OH
OH
OH
O
O
S
1⁄
2⁄
3
8
0.125
0.094
0.094
0.094
0.125
0.188
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
OH
O
O
>64
>64
>64
>64
>64
4
O
O
O
O
4
O
O
4
4
O
O
O
5⁄
8
O
O
N
6
16
O
O
Br
7
64
4
0.125
Synergy
HO
OH
O
Br
O
8
>64
>64
>64
1
0.007
0.188
0.125
Synergy
Synergy
Synergy
O
OH
9
16
8
OH
O
Br
O
10
OH
OH
Please cite this article in press as: Wang, Y.-H.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.05.013

Y.-H. Wang et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
Table 1 (continued)
No.
Structure
MIC80
(l
g mLꢀ1
)
FICI
Mode of interaction
Synergy
Alone
>64
With FLCa
O
11
12
32
0.315
0.506
OH
O
F
>64
64
Indifferent
OH
O
CF3
O
S
13
14
15
16
17
18
>64
>64
>64
>64
>64
>64
64
0.506
>1.06
>1.06
0.315
>1.06
>1.06
Indifferent
Indifferent
Indifferent
Synergy
OH
O
>64
>64
32
OH
O
O
OH
O
OH
O
O
OH
O
O
>64
>64
Indifferent
Indifferent
HO
HO
O
O
O
19
20
>64
>64
8
0.125
0.506
Synergy
F
N
O
64
Indifferent
F
O
O
O
O
21
22
23
>64
>64
>64
32
0.315
>1.06
>1.06
Synergy
Cl
O
O
>64
>64
Indifferent
Indifferent
Cl
O
O
O
NH2
O
O
24
>64
32
32
4
0.315
0.188
Synergy
Synergy
NH2
O
BE
—
a
MIC80 value [lg/mL] of compound in column 1 in combination with 8.0 lg/mL fluconazole.
Please cite this article in press as: Wang, Y.-H.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.05.013

4
Y.-H. Wang et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
OH
O
O
OH
O
OH
O
O
K2CO3
30% (aq) NaOH
Ar
1-6
CH3I
+
+ Ar-CHO
EtOH, reflux
MeOH
rt, 24-48 h
OH
O
O
O
O
Br
Br
Br2
30% (aq) NaOH
+
CH3COOH
5 C
MeOH
rt, 24-48 h
HO
OH
HO
OH
HO
OH
Br
Br
7
O
O
30% (aq) NaOH
Ar
8-24
Ar-CHO
+
R
R
MeOH
rt, 24-48 h
Scheme 1. The synthetic route for the title compounds.
compounds 1–6. The 30,50-dibromo-20,40-dihydroxyacetophenone—
an intermediate for the synthesis of compound 7—was obtained by
20,40-dihydroxyacetophenone react with Br2/AcOH at 5 °C. Com-
pounds 1, 2 and 5 are reported for the first time. The structures
of all compounds were confirmed by 1H NMR, ESI-MS.
The in vitro synergistic antifungal activities of the title com-
pounds were tested using the microbroth dilution method accord-
ing to the standards of the Clinical and Laboratory Standards
Institute, USA.12 The MIC80 of fluconazole (FLC) against the flucona-
zole-resistant C. albicans (clinical isolate 103) was determined to
baicalein,6,13,14 quercetin, and catechin.7 Cao and Dai et al.6,14
reported that baicalein could dramatically decrease biofilm pro-
duction by C. albicans and induce apoptosis in C. albicans cells.
The apoptosis was associated with a breakdown of mitochondrial
membrane potential. However, we have been investigating their
antifungal mechanism and target based on our studies on biofilms
and apoptosis in C. albicans, but have not achieved any clear results
thus far. Bitencourt et al.15 reported that the trans-chalcone
showed antifungal activity against T. rubrum and reduced ergos-
terol levels and modulated the expression of FAS1 and ERG6. Lacka
et al.16 reported that the oxathiolone-fused chalcone derivative
AMG-148 showed antifungal activity against several strains of C.
be 128.0
lg/ml. The MIC80 of each title compound when used alone
and when combined with fluconazole (8.0
lg/ml) are described in
Table 1. Furthermore, the fractional inhibitory concentration index
(FICI) of each agent was calculated by adding the ratios of the
MIC80 (with FLC)/MIC80 (used alone). The interaction modes—syn-
ergistic or indifferent—were defined according to FICI values of
60.5 or >0.5, respectively.4
Several chalcones displayed synergistic antifungal activity in
the range of FICI from 0.007 to 0.315 as we expected (Table 1). This
indicated that they have synergistic antifungal activity with FLC.
However, the MIC80 (used alone) values of title compounds are
all P64—this suggested that they have no antifungal activity
themselves, but that they can restore the sensitivity of the flucona-
zole-resistant C. albicans to fluconazole. In a series of 20-hydroxyl
and 60-menthoxyl substituted chalcones, all compounds (1–6)
showed synergistic antifungal activity. Moreover, when the B ring
was a heterocyclic ring (1–5), the synergistic effect was better than
phenyl ring (6).
albicans with MIC values from 1 to 16 l
g mLꢀ1 in vitro. This was
an inhibitor of beta (1?3)glucan synthase of fungi, which may
be helpful for us to investigate their mechanism against drug-resis-
tant fungi. Thus, these findings suggest that chalcone is a new syn-
ergist of fluconazole against fluconazole-resistant C. albicans.
In this study, 24 chalcones have been synthesized, and the
bioassay results showed most of the compounds displayed syner-
gistic antifungal effects. Compound
8 was the most potent
(FICI = 0.0781). The SAR study indicates that the 20-hydroxyl-60-
menthoxyl or 20-hydroxyl-40-menthoxyl are important groups on
chalcone because of their synergistic activity with fluconazole
against fluconazole-resistant C. albicans. These results are impor-
tant for the discovery of fluconazole synergists against flucona-
zole-resistant C. albicans. Further studies on structural
optimization are in progress in our laboratory.
Of the series of 20-hydoxychalcons (7–16) and 40-hydroxychal-
cones (17, 18), only compounds 7, 8, 9, 10, 11 showed synergistic
effects—of these compound 8 was the most potent with a FICI of
0.0781 in all synthetic compounds. Moreover, compounds substi-
tuted by fluoro (19, 20), chloro (21, 22) or amino (23, 24) at the
20-position (except for compound 19) showed synergistic effects.
All others had no effect. These results show that the chalcones with
only one substituent at the 20-position or the 40-position were not
conducive to antifungal or synergistic effects. The 20-hydroxyl-60-
menthoxyl and 20-hydroxy-40-methoxyl chalcones had good
effects. Our results are mainly consistent with Dai’s4 in terms of
Acknowledgement
This work was supported by the grant from the Key Program of
National Natural Science Foundation of China (81330083).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.05.
013.
References and notes
structure and FICI. This indicates that the a,b unsaturated carbonyl
moiety is critical pharmacophores of chalcone for synergistic func-
tion with fluconazole against fluconazole-resistant C. albicans.
Natural flavonoids have been reported for their antibacterial,
antifungal, and synergistic antifungal effects including
1. White, T. C.; Holleman, S.; Dy, F.; Mirels, L. F.; Stevens, D. A. Antimicrob. Agents
Chemother. 2002, 46, 1704.
2. Li, D. D.; Xu, Y.; Zhang, D. Z.; Quan, H.; Mylonakis, E.; Hu, D. D.; Li, M. B.; Zhao, L.
X.; Zhu, L. H.; Wang, Y.; Jiang, Y. Y. Antimicrob. Agents Chemother. 2013, 57,
6016.
Please cite this article in press as: Wang, Y.-H.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.05.013

Y.-H. Wang et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
5
3. Quan, H.; Cao, Y. Y.; Xu, Z.; Zhao, J. X.; Gao, P. H.; Qin, X. F.; Jiang, Y. Y.
Antimicrob. Agents Chemother. 2006, 50, 1096.
4. Dai, L.; Zang, C.; Tian, S.; Liu, W.; Tan, S.; Cai, Z.; Ni, T.; An, M.; Li, R.; Gao, Y.;
Zhang, D.; Jiang, Y. Bioorg. Med. Chem. Lett. 2015, 25, 34.
10. Pinho-Ribeiro, F. A.; Hohmann, M. S.; Borghi, S. M.; Zarpelon, A. C.; Guazelli, C.
F.; Manchope, M. F.; Casagrande, R.; Verri, W. A., Jr. Chem. Biol. Interact. 2015,
228, 88.
11. Baell, J.; Walters, M. A. Nature 2014, 513, 481.
5. Jesus, F. P.; Ferreiro, L.; Loreto, E. S.; Pilotto, M. B.; Ludwig, A.; Bizzi, K.; Tondolo,
J. S.; Zanette, R. A.; Alves, S. H.; Santurio, J. M. Antimicrob. Agents Chemother.
2014, 58, 5621.
12. Wayne, P. A. Reference Method for Broth Dilution Antifungal Susceptibility Testing
of Yeasts, Approved Standard M27–A3, 3rd ed; Clinical and Laboratory Standards
Institute (CLSI): USA, 2008.
6. Cao, Y.; Dai, B.; Wang, Y.; Huang, S.; Xu, Y.; Cao, Y.; Gao, P.; Zhu, Z.; Jiang, Y. Int.
J. Antimicrob. Agents 2008, 32, 73.
13. Serpa, R.; Franca, E. J.; Furlaneto-Maia, L.; Andrade, C. G.; Diniz, A.; Furlaneto,
M. C. J. Med. Microbiol. 2012, 61, 1704.
7. da Silva, C. R.; de Andrade Neto, J. B.; de Sousa Campos, R.; Figueiredo, N. S.;
Sampaio, L. S.; Magalhaes, H. I.; Cavalcanti, B. C.; Gaspar, D. M.; de Andrade, G.
M.; Lima, I. S.; de Barros Viana, G. S.; de Moraes, M. O.; Lobo, M. D.; Grangeiro, T.
B.; Nobre Junior, H. V. Antimicrob. Agents Chemother. 2014, 58, 1468.
8. Mishra, P. K.; Sarma, B. K.; Singhai, P. K.; Singh, U. P. Mycobiology. 2007, 35, 72.
9. Leao, M.; Soares, J.; Gomes, S.; Raimundo, L.; Ramos, H.; Bessa, C.; Queiroz, G.;
Domingos, S.; Pinto, M.; Inga, A.; Cidade, H.; Saraiva, L. Life Sci. 2015, 142, 60.
14. Dai, B. D.; Cao, Y. Y.; Huang, S.; Xu, Y. G.; Gao, P. H.; Wang, Y.; Jiang, Y. Y. J.
Microbiol. Biotechnol. 2009, 19, 803.
15. Bitencourt, T. A.; Komoto, T. T.; Massaroto, B. G.; Miranda, C. E.; Beleboni, R. O.;
Marins, M.; Fachin, A. L. BMC Complement Altern Med. 2013, 13, 229.
16. Lacka, I.; Konieczny, M. T.; Bulakowska, A.; Rzymowski, T.; Milewski, S. Mycoses
2011, 54, e407.
Please cite this article in press as: Wang, Y.-H.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.05.013