Design, synthesis, and evaluation of caffeic acid amides as synergists to sensitize fluconazole-resistant Candida albicans to fluconazole

Article abstract of DOI:10.1016/j.bmcl.2014.11.022

A series of caffeic acid amides were designed, synthesized, and their synergistic activity with fluconazole against fluconazole-resistant Candida albicans was evaluated in vitro. The title caffeic acid amides 3-30 except 26 exhibited potent activity, and the subsequent SAR study was conducted. Compound 3, 5, 21, and 34c, at a concentration of 1.0 μg/ml, decreased the MIC₈₀ of fluconazole from 128.0 μg/ml to 1.0-0.5 μg/ml against the fluconazole-resistant C. albicans. This result suggests that the caffeic acid amides, as synergists, can sensitize drug-resistant fungi to fluconazole. The SAR study indicated that the dihydroxyl groups and the amido groups linking to phenyl or heterocyclic rings are the important pharmacophores of the caffeic acid amides.

Full text of DOI:10.1016/j.bmcl.2014.11.022

Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, and evaluation of caffeic acid amides as synergists
to sensitize fluconazole-resistant Candida albicans to fluconazole
Li Dai a, , Chengxu Zang a, , Shujuan Tian a, , Wei Liu b, Shanlun Tan a, Zhan Cai a, Tingjunhong Ni a,
Maomao An b, Ran Li a, Yue Gao a, Dazhi Zhang a, , Yuanying Jiang a,b,
⇑
⇑
a School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, PR China
b Department of Pharmacology, Tongji University School of Medicine, 1239 Siping Road, Shanghai 200092, PR 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 caffeic acid amides were designed, synthesized, and their synergistic activity with fluconazole
against fluconazole-resistant Candida albicans was evaluated in vitro. The title caffeic acid amides 3–30
except 26 exhibited potent activity, and the subsequent SAR study was conducted. Compound 3, 5, 21,
Received 12 August 2014
Revised 15 October 2014
Accepted 7 November 2014
Available online xxxx
and 34c, at a concentration of 1.0
lg/ml, decreased the MIC80 of fluconazole from 128.0 lg/ml to
1.0–0.5 g/ml against the fluconazole-resistant C. albicans. This result suggests that the caffeic acid
l
amides, as synergists, can sensitize drug-resistant fungi to fluconazole. The SAR study indicated that
the dihydroxyl groups and the amido groups linking to phenyl or heterocyclic rings are the important
pharmacophores of the caffeic acid amides.
Keywords:
Caffeic acid
Synergistic activity
Fluconazole-resistant Candida albicans
Structure–activity relationship
Ó 2014 Elsevier Ltd. All rights reserved.
Fluconazole is the most commonly used drug to treat Candida
albicans (C. albicans) in the prophylaxis and therapy, however, wide-
spread and repeated use of fluconazole resulted in resistance to or
failure of fluconazole therapy.1 To combat the fluconazole-resistant
C. albicans, much attention has been paying to the synergism of
fluconazole with other agents, as synergists, which can significantly
sensitize fungi towards fluconazole,2,3 for instance, tetracyclic
indoles,4 piperazinyl quinolines,5 amiodarone,6 allicin,7 sulfated
Sterols.8
Since our previous study indicated that berberine 1 has potent
synergistic activity with fluconazole against fluconazole-resistant
C. albicans,9 we have been focusing on the structure modification
and deconstruction of berberine, which led us to reconstruct a
new active scaffold 2.10 Inspired by scaffold hopping from the struc-
ture of 1–2, we designed and prepared caffeic acid amides 3–13, as
shown in Fig. 1, and their synergistic activity with fluconazole
against fluconazole-resistant C. albicans was evaluated in our lab.
As we expected, most of them exhibited potent activity. Herein
we report the result and the SAR is investigated and discussed.
Caffeic acid amides 3–30 were synthesized by coupling caffeic
acid with a series of amines using dicyclohexylcarbodiimide
(DCC) as coupling reagent, as shown in Scheme 1. Compounds
16, 18, 22, 27–30 are novel and characterized by H NMR and MS.
A series of amines reacted with (E)-3-(3,4-dimethoxyphenyl)
acrylic acid, and cinnamic acid in the presence of oxalyl chloride
and TEA in dichloromethane to give compounds 31a–c, and
32a–i, respectively, as shown in Scheme 2. Compounds 31a–c were
treated with Lawesson’s Reagent in toluene under reflux to give
33a–c,11 which were then demethylated with boron tribromide
to afford the corresponding phenol 34a–c.12 Compounds 32h,
33a–c, 34a–c are novel and characterized by H NMR and 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.13 The MIC80 of fluconazole against the fluconazole-
resistant C. albicans (clinical isolate 103) was determined to be
128.0
lg/ml, while the MIC80 values of each title compound used
alone, and combined with fluconazole (8.0
lg/ml), were deter-
mined as shown in Table 1. Furthermore, the fractional inhibitory
concentration index (FICI) of each agent was calculated by summing
up the ratios of the MIC80 (with FLC)/MIC80 (used alone). The inter-
action modes, synergistic or indifferent, were defined according to
FICI values of 60.5 or >0.5, respectively.9
At the beginning of this study, caffeic acid anilides 3–13 were
designed through scaffold hopping based on active compounds 2
and berberine. As shown in Table 1, all the caffeic acid anilides
3–13, at concentrations ranging from 0.5 to 8.0
lg/ml, decreased
the MIC80 of fluconazole from 128.0 to 8.0 g/ml. Their FICI values
range from 0.070–0.125, which indicated that they have potent
l
⇑
Corresponding authors. Tel.: +84 21 81871226 (D.Z.), +84 21 81871357 (Y.Y.).
E-mail addresses: zdzhang_yjhx@smmu.edu.cn (D. Zhang), jiangyy@smmu.edu.
cn (Y. Jiang).
synergistic activity with fluconazole. However, the MIC80 (used
The authors contributed equally.
http://dx.doi.org/10.1016/j.bmcl.2014.11.022
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Dai, L.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.11.022

2
L. Dai et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
O
O
Cl-
H
O
N
O
O
N
HO
HO
N
H
R
O
OCH3
OCH3
R
2
3-13
berberine
1
Figure 1. Scaffold hopping design.
O
O
HO
HO
HO
OH
amine
(a)
R
HO
3 30
-
24: R=N
17:
R=HN
10:
RHN=
F
3:
R=HN
O
11: R=HN
12: R=HN
Br
25: R=N
26: R=N
18:
19:
R=HN
R=HN
4:
5:
R=HN
R=HN
Br
F
Cl
O
Cl
O
27: R=HN
28: R=HN
20: R=HN
21: R=HN
S
O
O
6:
7:
R=HN
R=HN
13: R=HN
O
O
O
OH
N
Br
O
14: R=HN
O
HO
N
29: R=HN
30: R=N
Cl
F
22: R=HN
23: R=HN
OEt
8:
9:
R=HN
R=HN
N
15: R=HN
16: R=HN
O
O
Scheme 1. Reagents and conditions: (a) DCC, THF, refluxing, 3–6 h.
O
O
R3
R4
R1
R3
R4
R1
R2
(a)
H2N
N
H
OH
n
n
+
R2
1
2
1
2
32a-i
31a-c
: R ,R =H
: R ,R =OCH3
R
R
S
S
HO
HO
O
O
(c)
(b)
N
N
H
H
34a c
-
33a c
-
3
4
31a: n=0 R3=H R4=Cl
33a: R=Cl
33b
32d
: n=1 R =H R =H
3
4
: R=F
31b
32e: n=1 R3=H R4=Cl
: n=0 R =H R =F
33c: R=CH3
3
4
31c: n=0 R3=H R4=CH3
: n=1 R =H R =Br
32f
32g
34a
34b
: R=Cl
3
4
3
4
32a
32b
: n=0 R =H R =CH3
: n=2 R =H R =H
: R=F
32h: n=2 R3=H R4=Br
34c: R=CH3
3
4
: n=0 R =H R =Br
3
32c: n=0 R3=H R4=OCH3
: n=2 R =OCH3 R4=OCH3
32i
Scheme 2. Reagents and conditions: (a) Oxalyl chloride, TEA, CH2Cl2, rt, 3–6 h; (b) Lawesson’s reagent, toluene, refluxing; (c) BBr3, CH2Cl2, 0 °C.
alone) of the title compounds are >64.0 or 64.0
lg/ml, which sug-
0.5 to 4.0
lg/ml. However, the MIC80 values of caffeic acid, used
gests that they have no antifungal activity themselves, but they can
sensitize the fluconazole-resistant C. albicans to fluconazole. More-
over, the anilino moiety in compounds 3–13 was replaced: (1) by
benzyl and phenylethyl amino groups in compounds 14–23; (2)
by alkyl amino groups in compound 24–26; (3) by heterocyclic
or heterocyclic alkyl amino groups in compounds 27–30; respec-
tively. All the above-mentioned analogues, except compound 26,
showed good activity with MIC80 (with FLC) value ranging from
alone and with fluconazole, were determined to be >64.0
l
g/ml,
which indicates it has neither antifungal nor synergistic activity
with fluconazole. The results suggested the amide moieties are
important to their activity.
The MIC80 values (with FLC) of compounds 31a–c, 32a–i, and
33a–c are all >64.0 lg/ml, and their FICI are all >0.5, thus indicates
they totally lost the synergistic activity. The key point in their struc-
tures is that the dihydroxyl groups of caffeic acid moiety were
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L. Dai et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
3
Table 1
Table 2
The MIC80, FICI and interaction mode of the title compounds
Checkerboard microdilution assay of the title compounds and fluconazole
Compound
MIC80
Alone
(l
g/ml)
FICI
Mode of
interaction
Compound
MIC80
g/ml)a
MIC80 (with FLC,
g/ml)b
FICI
Mode of
interaction
(
l
l
With FLCa
3
64.0
8.0(0.5)
4.0(0.5)
2.0(0.5)
1.0(1.0)
8.0(0.5)
4.0(0.5)
2.0(0.5)
1.0(1.0)
8.0(1.0)
4.0(0.5)
2.0(1.0)
1.0(1.0)
8.0(0.5)
4.0(0.5)
2.0(0.5)
1.0(0.5)
8.0(0.25)
4.0(0.5)
2.0(0.5)
1.0(0.5)
0.129
0.066
0.035
0.023
0.066
0.035
0.020
0.016
0.070
0.035
0.023
0.016
0.066
0.035
0.020
0.012
0.252
0.129
0.066
0.035
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
64.0
64.0
64.0
>64.0
>64.0
>64.0
64.0
>64.0
64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
64.0
>64.0
>64.0
>64.0
>64.0
>64.0
64.0
0.5
0.5
0.5
4.0
1.0
8.0
1.0
1.0
1.0
4.0
4.0
0.5
1.0
1.0
4.0
1.0
1.0
1.0
1.0
1.0
4.0
4.0
4.0
>64.0
4.0
0.070
0.070
0.070
0.094
0.070
0.125
0.078
0.070
0.078
0.094
0.094
0.066
0.070
0.070
0.094
0.070
0.070
0.070
0.070
0.078
0.094
0.094
0.094
>0.5
0.094
0.125
0.094
0.125
>0.5
>0.5
>0.5
>0.5
>0.5
>0.5
>0.5
>0.5
>0.5
>0.5
>0.5
>0.5
>0.5
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
Indifferent
Synergy
5
>64.0
>64.0
>64.0
32.0
21
34c
Berberine
a
The fluconazole-resistant C. albicans isolate 103.
The corresponding MIC80 of fluconazole is in parentheses.
b
4.0
4.0
4.0
Synergy
Synergy
Synergy
Supporting information. Four MIC80 values and FICI values were
obtained for each compound as shown in Table 2. The MIC80 values
of each compound and fluconazole, which gave the lowest FICI, are
their best combination concentrations. The lowest FICI values of
compound 3, 5, 21, and 34c are 0.023, 0.016, 0.016, and 0.012
respectively, meanwhile their best MIC80 values, with that of fluco-
nazole in the parentheses, are 1.0(1.0), 1.0(1.0), 1.0(1.0) and
1.0(0.5)
concentration of 1.0
from 128.0 to 1.0–0.5
albicans. Berberine, which is found to be one of the strongest
synergist of fluconazole in our lab, was set to be positive control.
The lowest FICI of berberine is 0.035. By comparison of the lowest
FICI values, it can be seen that compounds 3, 5, 21, and 34c showed
higher synergistic activity than berberine.
Natural products bearing caffeoyl group have been reported to
have antifungal and antibacterial activity.14,15 Fu et al.16 reported
antifungal activity of caffeic acid amides against drug-sensitive C.
albicans. In their report, compounds 4, 5, and 9–12, used alone,
>64.0
64.0
31a
31b
31c
32a
32b
32c
32d
32e
32f
32g
32h
32i
33a
33b
33c
34a
34b
34c
Caffeic acid
Berberine
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
32.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
1.0
Indifferent
Indifferent
Indifferent
Indifferent
Indifferent
Indifferent
Indifferent
Indifferent
Indifferent
Indifferent
Indifferent
Indifferent
Indifferent
Indifferent
Indifferent
Synergy
l
g/ml. This result means that the tested compounds, at a
g/ml, can decrease the MIC80 of fluconazole
g/ml against the fluconazole-resistant C.
l
l
>0.5
>0.5
0.070
0.070
0.066
>0.5
1.0
0.5
>64.0
1.0
Synergy
Synergy
Indifferent
Synergy
0.094
showed no antifungal activity (MIC >50.0
and 6 showed weak antifungal activity (MIC = 42.8
research result is mostly consistent to ours (MIC80 >64.0
l
g/ml) and compound 3
g/ml). Their
g/ml),
a
The concentration of fluconazole (FLC) is 8.0
nazole used alone is 128.0 g/ml.
lg/ml, and the MIC80 of fluco-
l
l
l
although the tested C. albicans in our research is fluconazole-
resistant clinical isolates. However, we have been investigating
their antifungal mechanism and target based on our studies on
Reactive oxygen species (ROS) and efflux in C. albicans, but got no
clear results so far. Sandai group reported caffeic acid can inhibit
the isocitrate lyase (ICL1) involving the glyoxylate cycle in
C. albicans.17 Ma et al. reported their caffeic acid amides inhibited
1,3-b-glucan synthase of C. albicans,18 which may be helpful for us
to investigate their mechanism against drug-resistant fungi.
Therefore, these finds would provide caffeic acid amides a new kind
of chemosensitizers of fluconazole against fluconazole-resistant
C. albicans.
replaced by methoxyl groups or hydrogen atoms. Neither lengthen-
ing the amide chain by one or two methylene groups in 32d–i, nor
replacing oxygen atom by sulfur atom in the amido bond of 33a–c,
could recover their activity at all. However, compounds 34a–c,
afforded by demethylation of 33a–c, exhibited potent activity.
Moreover, replacing the oxygen atom of amido bond with sulfur
atom led to hardly no change in synergic activity, since compounds
5, 10, and 9 showed comparable MIC80 values (with FLC) to that of
34a, 34b, and 34c, both ranging from 0.5 lg/ml to 1 lg/ml.
In summary, SAR study indicates that both the dihydroxyl groups
in caffeic acid moiety and the amide moiety are important pharma-
cophores of the caffeic acid amides for their synergisitic activity
with fluconazole against fluconazole-resistant C. albicans. This
result is consistent to our previous SAR study on compounds 2.10
For the best combination concentrations of caffeic acid amides
and fluconazole, the active compounds 3, 5, 21, and 34c were
subjected to checkerboard microdilution assay, as described in
Acknowledgments
This research was supported by the National Natural Science
Fund of China (No. 21072226, 21272270, 30772644, 90913008),
Shanghai Pujiang Program (No. 05PJ14003).
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4
L. Dai et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
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Supplementary data
2009, 72, 1524.
9. 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.
10. Liu, H.; Wang, L.; Li, Y.; Liu, J.; An, M. M.; Zhu, S. L.; Cao, Y. B.; Jiang, Z. H.; Zhao,
M. Z.; Cai, Z.; Dai, L.; Ni, T.; Liu, W.; Chen, S. M.; Wei, C.; Zang, C. X.; Tian, S. J.;
Yang, J. Y.; Wu, C. F.; Zhang, D. Z.; Liu, H.; Jiang, Y. Y. ChemMedChem 2014, 9,
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Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.11.022.
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