Novel Carboline Fungal Histone Deacetylase (HDAC) Inhibitors for Combinational Treatment of Azole-Resistant Candidiasis

Article abstract of DOI:10.1021/acs.jmedchem.0c01763

Due to the evolution and development of antifungal drug resistance, limited efficacy of existing drugs has led to high mortality in patients with serious fungal infections. To develop novel antifungal therapeutic strategies, herein a series of carboline fungal histone deacetylase (HDAC) inhibitors were designed and synthesized, which had potent synergistic effects with fluconazole against resistant Candida albicans infection. In particular, compound D12 showed excellent in vitro and in vivo synergistic antifungal efficacy with fluconazole to treat azole-resistant candidiasis. It cooperated with fluconazole in reducing the virulence of C. albicans by blocking morphological mutual transformation and inhibiting biofilm formation. Mechanism studies revealed that the reversion of drug resistance was due to downregulation of the expression of the azole target gene ERG11 and efflux gene CDR1. Taken together, fungal HDAC inhibitor D12 offered a promising lead compound for combinational treatment of azole-resistant candidiasis.

Full text of DOI:10.1021/acs.jmedchem.0c01763

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Novel Carboline Fungal Histone Deacetylase (HDAC) Inhibitors for
Combinational Treatment of Azole-Resistant Candidiasis
Zhuang Li, Jie Tu, Guiyan Han, Na Liu,* and Chunquan Sheng*
Cite This: J. Med. Chem. 2021, 64, 1116−1126
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ABSTRACT: Due to the evolution and development of antifungal drug resistance, limited efficacy of existing drugs has led to high
mortality in patients with serious fungal infections. To develop novel antifungal therapeutic strategies, herein a series of carboline
fungal histone deacetylase (HDAC) inhibitors were designed and synthesized, which had potent synergistic effects with fluconazole
against resistant Candida albicans infection. In particular, compound D12 showed excellent in vitro and in vivo synergistic antifungal
efficacy with fluconazole to treat azole-resistant candidiasis. It cooperated with fluconazole in reducing the virulence of C. albicans by
blocking morphological mutual transformation and inhibiting biofilm formation. Mechanism studies revealed that the reversion of
drug resistance was due to downregulation of the expression of the azole target gene ERG11 and efflux gene CDR1. Taken together,
fungal HDAC inhibitor D12 offered a promising lead compound for combinational treatment of azole-resistant candidiasis.
modification, regulated by HDACs, plays a vital role in stress-
signaling responses, which is related to fungal adaptation to
various environmental stresses including drug stress.4,11 Fungal
HDACs participate in fungal infection and resistance through
various ways, including limiting fungal development, reducing
fungal virulence, and preventing its dissemination. It is
reported that the combination of HDAC inhibitors and azole
drugs has synergistic antifungal effects against azole-resistant C.
albicans.12−15 However, human HDAC inhibitors are mostly
developed as antitumor agents, which hamper their direct use
as synergistic antifungal agents. Thus, there is an urgent need
to develop fungal HDAC inhibitors with improved selectivity
and synergistic potency and reduced cytotoxicity.
INTRODUCTION
■
Invasive fungal infections (IFIs) have been increasingly
realized as a lethal threat to immunocompromised and critical
patients in clinic. It is estimated that the number of deaths
caused by IFIs reaches 1.5 to 2 million per year.1 About 17%
intensive care unit (ICU) patients suffer from candidiasis.2
Due to the high survival ability in the bloodstream, Candida
albicans is the most common fungal pathogen.1−4 Currently,
only four types of antifungal drugs (azoles, polyenes,
echinocandins, and flucytosine) are approved to treat fungal
infections, whose therapeutic effects are limited by low potency
and high toxicity.5,6 A single type of antifungal agent could
easily lead to the rapid evolution and development of drug
resistance, and in most cases, higher mortality is closely
associated with the drug-resistant fungal infection.7,8 There-
fore, there is an urgent need to develop novel antifungal agents
or drug combinations with synergistic effects to expand current
therapeutic strategies.
In our previous studies, we designed lanosterol 14α-
demethylase (CYP51)-HDAC16 and Janus kinase 2 (JAK2)-
HDAC617 dual inhibitors for the treatment of resistant
Received: October 7, 2020
Published: December 27, 2020
Combinational drug therapy is one of the promising
strategies that could enhance the efficacy of first-line drugs
and block the emergence of drug resistance.9,10 Drug resistance
of fungi involves transcriptional regulation, in which chromatin
kinetics and histone modification play a major role.4 Histone
https://dx.doi.org/10.1021/acs.jmedchem.0c01763
© 2020 American Chemical Society
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Figure 1. Design of carboline compounds as novel fungal HDAC inhibitors. The fraction inhibitory concentration index (FICI) is defined as the
sum of the ratios that divide the MIC value of each drug used in combination by the value of the drug used alone. Synergism and antagonism were
defined by FICIs of ≤0.5 and >4, respectively. An FICI > 0.5 but <4 is considered irrelevant. The subfigure at the bottom right is the antifungal
activity in the form of a heat map measured by the checkerboard microdilution assay. The relative growth of fungi (0−1.5) changes from green to
black (the greener the color, the stronger the inhibition).
a
Scheme 1. Chemical Synthesis of Carboline Compounds
a
Reagents and conditions: (a) K2CO3, CH3OH, yield 64%; (b) NaH, dimethylformamide (DMF), yield 24−85%; (c) trifluoroacetic acid (TFA),
CH2Cl2; and (d) KOH, NH2OH·HCl, and CH3OH, yield 31−84%.
candidiasis. In particular, the HDAC6/JAK2 dual inhibitors
showed excellent synergistic effects with fluconazole (FLC) to
treat resistant C. albicans infections. Inspired by the results, we
further assayed the synergistic effects of tubastatin A (TBA), a
selective HDAC6 inhibitor, with azole antifungal agents. The
results revealed that TBA synergized with FLC, voriconazole
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(VRC), and itraconazole (ITC) against resistant C. albicans
isolates (Table S1 in the Supporting Information). Thus,
HDAC6 inhibitor TBA offered a lead compound for
discovering novel synergistic antifungal agents. To further
improve the antifungal activity and clarify the structure−
activity relationships (SARs), herein a series of carboline
compounds were designed and synthesized on the basis of our
previously identified carboline antifungal scaffold18 (Figure 1).
Most compounds were able to potentiate the antifungal activity
of FLC against FLC-resistant C. albicans cells. In particular, the
combination of compound D12 and FLC effectively blocked
the morphological changes and biofilm formation of C. albicans
and showed potent in vivo efficacy in a model of resistant
candidiasis.
Table 1. In Vitro Antifungal Activity of Compounds Used
Alone or in Combination with FLC against the C. albicans
Strain (0304103) by the Checkerboard Microdilution Assay
compounds
alone
FLC
synergetic
a
synergetic
alone
FICI
D1
D2
D3
D4
D5
D6
D7
D8
>64
>64
>64
>64
>64
64
>64
>64
>64
64
4
2
4
2
2
2
4
8
2
2
2
2
8
16
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
0.5
0.5
0.25
2
1
0.25
1
0.5
1
1
0.5
0.25
025
2
0.070
0.039
0.066
0.062
0.047
0.035
0.078
0.13
0.047
0.047
0.039
0.035
0.13
D9
D10
D11
D12
SAHA
TBA
RESULTS AND DISCUSSION
■
>64
>64
>64
>64
Design of Carboline HDAC Inhibitors. The pharmaco-
phores of typical HDAC inhibitors can be divided into three
parts: a cap group, a linker (usually hydrophobic), and a zinc-
binding group (ZBG). In particular, ZBG (hydroxamic acid or
benzamide group) binds to the zinc ion and plays a crucial role
in the inhibition of zinc-dependent HDACs.19 Typically, TBA
consists of a carboline cap group, a benzyl linker, and a
hydroxamic acid ZBG group. On the basis of our previous
work, the β-carboline ring was proven to be a potent antifungal
scaffold,18 which was selected as the cap group and the
hydroxamic acid ZBG necessary for HDAC binding was
retained. Considering the structural features of TBA and
carboline lead compounds, the propoxy phenyl group was
designed as the linker and various substitutions were
introduced on the carboline scaffold (D1−12, Figure 1).
Chemistry. The chemical synthesis of carboline com-
pounds is described in Scheme 1. Intermediates 4a−m were
synthesized according to our previous methods by using
various substituted indoles as starting materials.18 A sub-
stitution reaction between intermediate 3 and intermediates
4a−m was performed in the presence of NaH to give products
5a−m. Target compounds (D1−12) were obtained via two
steps including the removal of the t-butoxycarbonyl protective
group in intermediates 5a−m and the nucleophilic addition
reaction with hydroxylamine.
0.28
a
Synergism and antagonism were defined by FICIs of ≤0.5 and >4,
respectively. An FICI > 0.5 but < 4 is considered irrelevant.
synergistic effect and could be considered as a potential lead
compound for the combinational treatment of candidiasis.
Inspired by the synergistic antifungal effect of compound
D12 and FLC, a series of clinically isolated strains were
selected to further verify whether D12 also has a broad
synergic activity against various types of Candida spp. As
shown in Table 2, compound D12 showed excellent synergistic
antifungal activity against all of these strains when used in
combination with FLC (FICI range: 0.035−0.25). Compared
with TBA, compound D12 had a better synergistic effect
against all of the tested strains. It is worth noting that TBA had
no synergistic effect on Candida krusei, while compound D12
had an FICI of 0.25 (Table 2 and Figure S1 in the Supporting
Information). The synergistic activities of compound D12 in
combination with VRC and ITC against other clinical strains
such as Candida auris, Candida glabrata (Cornus glabrata),
Candida tropicalis, and Cryptococcus neoformans were further
tested. The results revealed that compound D12 had
synergistic effects with azoles against most Candida species,
whereas the synergism was lost against C. neoformans (Table
S3 in the Supporting Information). Therefore, compound D12
represents a lead compound for potential antifungal combina-
tion therapy, whose in vitro and in vivo antifungal activities and
antiresistance mechanisms were further investigated.
Compound D12 Blocked the Emergence of Fungal
Resistance Combined with FLC and the Synergism
Mainly Depended on Compound D12 Rather than FLC.
To validate the synergistic effect of compound D12 more
intuitively, its ability to block the emergence of azole resistance
was tested. It was observed that drug resistance appeared in a
high concentration of FLC (32 μg/mL) or a medium
concentration of compound D12 (8 μg/mL). But no colony
occurred on the medium containing both FLC and compound
D12 at the same concentration (Figure 2A). In Figure 2B, it
could be observed that compound D12 could enhance the
antifungal effects of FLC in a dose-dependent manner by
comparing the size of the inhibition ring. Thus, the
combination of compound D12 and FLC blocked the
emergence of FLC resistance and enhanced the efficacy of
FLC. However, it still remains unclear what the relationship
Carboline Derivatives in Combination with FLC
Inhibited Azole-Resistant C. albicans. The antifungal
activities of TBA and carboline derivatives used alone or in
combination with FLC are listed in Table 1. The minimum
concentration of 80% inhibition (MIC80) is defined as the
lowest concentration of a drug alone or synergistically that
inhibits fungal cell growth by 80%. Carboline derivatives
generally were inactive in inhibiting the growth of FLC-
resistant C. albicans (MIC80 ≥ 64 μg/mL) when used alone. In
contrast, they showed synergistic effects with FLC and the
antifungal activities of compounds D1−12 in combination
with FLC were significantly enhanced. The MIC80 values were
reduced from >64 μg/mL to 0.25−8 μg/mL with the FICI
values ranging from 0.035 to 0.13. Compounds D1−12 were
also tested for the synergistic effects with other azoles (VRC
and ITC) against azole-resistant C. albicans, which also showed
excellent synergistic activities (FICI = 0.035−0.13, Table S2 in
the Supporting Information). Thus, carboline compounds
could enhance the efficacy of azole drugs against azole-resistant
C. albicans and reverse the development of antifungal drug
resistance. Among them, compound D12 showed the best
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Table 2. In Vitro Synergistic Effect of Compound D12 When Used in Combination with FLC against Various C. albicans
Strains and C. krusei Strains
MIC80 (μg/mL)
alone
synergetic
FLC
alone
synergetic
a
strains
D12
FLC
D12
FICI
TBA
FLC
TBA
FLC
FICI
9172
9884
10061
4108
>64
64
>64
64
>64
>64
>64
>64
64
2
2
2
2
8
0.5
0.5
2
0.25
8
0.039
0.039
0.062
0.035
0.25
>64
>64
>64
>64
>64
>64
>64
>64
>64
64
16
4
2
8
64
0.5
1
0.5
0.5
32
0.26
0.078
0.039
0.13
1.5
10153
64
a
C. albicans strains include 9172, 9884, 10061, and 4108. The C. krusei strain includes 10153.
between FLC and compound D12 is, and which is more
important in suppressing the emergence of fungal resistance.
Therefore, the relationship between the synergism and the
concentration of the two drugs was investigated. Single use of
compound D12 (32 μg/mL) or FLC (64 μg/mL) led to no or
weak antifungal activity. In contrast, the growth of fungi was
inhibited markedly when the two drugs were used in
combination (Figure 2C). In addition, after fixing FLC to a
specific concentration (4, 16, or 64 μg/mL), the inhibitory
effect was dependent on the concentration of compound D12.
In contrast, changing the concentration of FLC with a specific
value of compound D12 (8, 16, or 32 μg/mL) had no
significant impact on the antifungal activity. These results
indicated that compound D12 not only enhanced the activity
of FLC, but also played a major role in the synergistic effects.
For further investigating the synergistic antifungal mechanism,
the fungicidal effect of the combination was tested. The results
showed that the combination of compound D12 and FLC had
a fungicidal effect at high concentrations (Figure 2D).
Compound D12 Exhibited Selective Inhibitory Effect
on Fungal HDACs. Due to limited knowledge of fungal
HDACs, there lacks well-established assays for the evaluations
of fungal HDAC inhibitors. Using our previously developed
fungal whole cell HDAC enzymatic assay,16 the inhibitory
activity of compound D12 against fungal and human total
HDACs was determined (Figure 3). As compared with TBA,
compound D12 generally showed improved inhibitory activity
against fungal HDACs and decreased activity against human
HDACs. Compound D12 inhibited fungal HDACs stronger
Figure 2. Compound D12 cooperates with FLC to block the
emergence of drug resistance and plays a major role in the synergistic
process. (A) Compound D12 blocks the emergence of FLC resistance
in C. albicans (0304103). In total, 1 × 104 cells were plated on the
Sabouraud Dextrose Agar (SDA) medium containing no drugs
(Control), compound D12 (8 μg/mL), FLC (32 μg/mL), or the
combination. Plates were photographed 48 h after incubation at 35
°C. (B) FLC and compound D12 are synergistic against C. albicans.
Disks containing compound D12 were plated on the plate containing
FLC (32 μg/mL). The doses of compound D12 on the disks were 32,
8, 2, and 0 μg from the top to the bottom. Experiments could be
duplicated biologically. (C) Time growth curves of the C. albicans
strain 0304103 under different concentrations of compounds. The
initial concentration of C. albicans was 1.5 × 106 cells/mL. The
number of cells was counted at a specific time point (0, 6, 12, 24, 48
h) and the concentration was calculated. (D) Time kill curves of the
C. albicans strain 0304103 when compound D12 (32 μg/mL) was
used in combination with different concentrations of FLC. Three
independent experiments were performed.
Figure 3. Inhibitory activity of compound D12 and TBA against
fungal (C. albicans 0304103) and human (HUVEC) total HDACs.
Three independent experiments were performed.
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than TBA at the same concentration. In contrast, compound
D12 was less active in inhibiting human HDACs than TBA.
Moreover, compound D12 also exhibited weaker inhibitory
activity against human-derived HDAC1 (IC50 = 1034 nM) and
HDAC6 (IC50 = 549 nM) than TBA and SAHA (Table 3). As
combination was determined by the real-time reverse tran-
scriptase polymerase chain reaction (RT-PCR). The over-
expression (about 100-fold increase compared with the
control) of ERG11 could be detected after treated with FLC,
which might be the main reason for the FLC resistance.
Interestingly, the expression of ERG11 was decreased to the
basal levels after treated with the combination of FLC and
compound D12. As for efflux pump genes, FLC upregulated
the expression of CDR1, whereas the expression was
downregulated to normal levels when FLC was used in
combination with compound D12. FLC alone has no effect on
the expression of CDR2 and MDR1. On the contrary,
compound D12 alone upregulated the two genes. The
upregulation of CDR2 could be recovered to normal levels
when compound D12 was used in combination with FLC.
However, the drug combination enhanced the MDR1 levels by
8-fold. It is demonstrated that the combination of compound
D12 and FLC recovered azole sensibility in resistant C.
albicans by regulating the azole target gene ERG11 and efflux
gene CDR1 (Figure 4).
Combination of Compound D12 and FLC Blocks the
Morphological Changes of C. albicans. Pathogenic C.
albicans has various morphological states, termed fungal
polymorphism,21 including budded (yeasty) forms and hyphal
forms.22 The budded-to-hyphal transition process is crucial to
fungi virulence23 and the conversion exists during the whole
process of C. albicans tissue invasion.21 Photomicrographs of
cells treated with different drugs are shown in Figure 5.
Morphological conversion from yeast to hypha was still
observed after the treatment with FLC or compound D12
alone. In the group treated with FLC and compound D12 in
combination, only yeast fungi were observed. Thus, compound
D12 successfully assisted FLC to block the morphological
transition of fungi and reduce the invasion ability of C. albicans.
Table 3. Human HDAC1/6 Enzyme Inhibition (IC50, nM)
compounds
HDAC1
HDAC6
D12
TBA
SAHA
2226 373.4
752 70.6
22.7 0.9
459 56
1.9 0.6
28
5
a result, the enhanced synergistic effect of compound D12
might be associated with the increased selectivity of fungal
HDACs over human HDACs. Furthermore, the cytotoxicity of
compound D12 alone and in combination with FLC on human
normal cells was also tested. The result revealed that
compound D12 showed a relatively low toxicity to human
umbilical vein endothelial cells (HUVEC, IC50 = 9.64 μM) and
human normal liver cells (L02, IC50 = 11.46 μM, Table S4 in
the Supporting Information). The combination of compound
D12 and FLC also revealed relatively low cytotoxicity
(HUVEC, IC50 = 12.2 μM, Table S5 in the Supporting
Information).
Combination of Compound D12 and FLC Reversed
Azole Resistance in C. albicans by Regulating the Azole
Target Gene (ERG11) and Efflux gene (CDR1). Previous
research confirmed that azole resistance in C. albicans was
correlated with the overexpression of ERG11, CDR, and
MDR1.20 ERG11 is the target gene of azoles, and the efflux
genes associated with azole resistance mainly include CDR1,
CDR2, MDR1, and FLU1. To clarify the mechanism of the
carboline derivative in reversing resistance, expression of
several resistant genes treated with drugs alone or in
Figure 4. Expression of resistant genes of C. albicans. (A) Difference of ERG11 expressed in each group. (B) Difference of MDR1 expressed in each
group. (C) Difference of CDR1 expressed in each group. (D) Difference of CDR2 expressed in each group. C. albicans (0304103) solution with
initial concentrations of 1 × 106 cells/mL was divided into four groups, and, respectively, treated with DMSO (control), FLC (32 μg/mL), D12
(32 μg/mL), and FLC + D12 (32 + 32 μg/mL). RT-PCR was used to detect differences in the gene expression. Three independent experiments
were performed.
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combination. The rod-shaped fungi (hyphal forms) observed
in the drug-free group and single-drug group verified the
morphological changes of budded-to-hyphal transition (200
fold) in mammals (Figure 7B). Thus, the combination of
compound D12 and FLC could reduce the colonization and
dissemination of fungi in tissue and attenuate the virulence of
fungi to the host by inhibiting the transition of C. albicans to
the hyphal state.
CONCLUSIONS
■
In summary, novel carboline fungal HDAC inhibitors were
designed, which provide a promising therapeutic strategy for
combinational treatment of IFIs caused by azole-resistant C.
albicans. In particular, compound D12 had an excellent
synergism with FLC against resistant Candida spp. The
inhibition of hyphae and biofilm formation also demonstrated
that the combination of compound D12 and FLC could
reduce fungal virulence. Compound D12 reversed FLC
resistance in C. albicans and enhanced efficacy of FLC
probably because of downregulating the expression of the
target gene EGR11 and efflux gene CDR1. The in vivo
antifungal potency was further validated in a mice candidiasis
model, in which compound D12 synergized with FLC to
effectively clear the C. albicans burden in kidneys. Taken
together, the fungal HDAC inhibitor D12 represents a drug-
like lead compound in the combinational treatment of
candidiasis. Further research for fungal HDAC subtype
selectivity, antifungal mechanisms, and lead optimization is
proceeding.
Figure 5. Hypha formation of C. albicans treated with D12 and FLC
alone or in combination. The initial concentration of C. albicans was 2
× 106 cells/mL. The Spider medium was used to induce the hyphae
formation. The samples were observed and photographed after 3 h of
incubation at 37 °C. Three independent experiments were performed.
Combination of Compound D12 and FLC Inhibits
Fungal Biofilm Formation and Mature Biofilms. There is
an intimate connection between fungal filamentation and
biofilm formation because the hypha is an important part of
mature fungal biofilm.24 Biofilm formation is considered as a
fungal survival mechanism related to drug resistance and
immune escape.25,26 The synergism of compound D12 and
FLC on C. albicans biofilm formation and disruption of
preformed biofilms was investigated. The results showed that
FLC or compound D12 used alone could not inhibit fungal
biofilm formation. Compound D12 and FLC used in
combination blocked the formation of fungal biofilms
significantly at 64 μg/mL of compound D12, while the
synergistic inhibition effect was not influenced by the
concentrations of FLC (Figure 6A). The result is consistent
with the time growth curves (Figure 2C). The disruption of
preformed biofilms by the combination was also investigated
(Figure 6B). Compound D12 could cooperate with FLC to
destroy mature biofilms only at a high concentration (64 μg/
mL).
Compound D12 Enhanced the In Vivo Therapeutic
Effect of FLC. The above results demonstrated the synergism
of compound D12 used in combination with FLC in vitro.
Then, the in vivo antifungal potency of the drug combination
was investigated. The mice models of candidiasis were
established via tail vein injection of C. albicans. As shown in
Figure 7A, compound D12 was inactive, and FLC had a weak
therapeutic effect (P < 0.05). In the group of drug
combination, the antifungal potency was significantly enhanced
(P < 0.0001). The combinational use of compound D12 (10
mg/kg) and FLC (1 mg/kg) effectively reduced the kidney
fungal burden in the infected mice. The fungal invasions in
tissues were further monitored through the tissue section of
the kidney to clearly understand the status of the fungal
infection in mice and the effect of drug treatment. In the
control group, a large number of fungal clusters stained deeply
with amaranth (rod-shaped and spherical) were observed in
the renal pelvis (arrow 1) but not obvious in the renal
parenchyma cortex and medulla. When compound D12 or
FLC was used alone, there were also small clusters of fungal
infiltration in the renal pelvis (arrow 2 and 3). In contrast, the
fungal clusters were cleared in the renal pelvis or medulla in
the group treated with compound D12 and FLC in
EXPERIMENTAL SECTION
■
1
Chemistry. General Methods. H NMR and 13C NMR spectra
were recorded on Bruker AVANCE600 spectrometers (Bruker
Company, Germany), using tetramethylsilane (TMS) as an internal
standard and CDCl3 or DMSO-d6 as solvents. Chemical shifts are
given in ppm (δ). The mass spectra were recorded on an Esquire
3000 LC-MS mass spectrometer. Silica gel thin-layer chromatography
was performed on precoated plates GF-254 (Qingdao Haiyang
Chemical, China). All solvents and reagents were analytically pure,
and no further purification was needed. All starting materials were
commercially available. The purities of the compounds were
determined by high-performance liquid chromatography (HPLC,
Agilent 1260), and all final compounds exhibited purities greater than
95%.
Methyl 2-(4-(3-Bromopropoxy)phenyl)acetate (3). To a solution
of methyl 2-(4-hydroxyphenyl)acetate (2, 5 g, 30.11 mmol) and
K2CO3 (8.31 g, 60.22 mmol) in MeOH (150 mL) was added 1,3-
dibromopropane (1, 9.12 g, 45.16 mmol) dropwise with stirring at
room temperature. The reaction mixture was refluxed at 80 °C for 4 h.
The solid was removed by filtration after the reaction mixture was
cooled to room temperature. Then, the filtrate was concentrated
under reduced pressure, H2O (20 mL) was added, and the reaction
mixture was extracted with EtOAc (40 mL × 3). The combined
organic layer was washed with H2O (30 × 3) and saturated NaCl (30
mL), dried over Na2SO4, filtered, and concentrated under reduced
pressure. The residue was purified by column silica gel chromatog-
raphy (hexane/EtOAc = 5/1) to give target compound 3 (5.5 g,
64.00%) as a colorless transparent liquid. 1H NMR (600 MHz,
DMSO-d6): δ 7.17 (d, J = 8.6 Hz, 1H), 6.89 (d, J = 8.6 Hz, 1H), 4.06
(t, J = 6.0 Hz, 1H), 3.66 (t, J = 6.6 Hz, 1H), 3.61−3.57 (m, 2H), 2.23
(p, J = 6.3 Hz, 1H).
tert-Butyl 8-Chloro-9-(3-(4-(2-methoxy-2-oxoethyl)phenoxy)-
propyl)-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole-2-carboxylate
(5l). To a solution of tert-butyl 8-chloro-1,3,4,9-tetrahydro-2H-
pyrido[3,4-b]indole-2-carboxylate (4l, 0.24 g, 0.77 mmol) in dry
DMF (3 mL), NaH (55 mg, 2.3 mmol) was added slowly at 0 °C.
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Figure 6. Inhibition of biofilm formation and disruption of mature biofilms treated with D12 and FLC alone or in combination. (A) Inhibition of
biofilm formation of C. albicans treated with D12 and FLC alone or in combination. (B) Disruption of preformed biofilms of C. albicans treated
with D12 and FLC alone or in combination. The initial concentration of C. albicans solution was 2 × 106 cells/mL. The comparison between the
two components was completed by the t-test. *** P < 0.001 compared to the control group. Three independent experiments were performed.
The reaction mixture was stirred at 0 °C for 5 min and then kept at
room temperature for 30 min. Methyl 2-(4-(3-bromopropoxy)-
phenyl) acetate (3, 0.33 g, 1.15 mmol) was added into the mixture
and stirred at room temperature for 1 h. The reaction mixture was
added dropwise into H2O (10 mL) and extracted with EtOAc (20 mL
× 3). The combined organic layer was washed with saturated NaCl
(15 × 6), dried over Na2SO4, filtered, and concentrated under
reduced pressure. The residue was purified by column silica gel
chromatography (hexane/EtOAc = 15/1) to give target compound 5l
(0.31 g, 85.30%) as a transparent oil. 1H NMR (600 MHz, CDCl3): δ
7.34 (d, J = 7.5 Hz, 1H), 7.20−7.14 (m, 2H), 7.11 (d, J = 7.6 Hz,
1H), 6.98 (t, J = 7.7 Hz, 1H), 6.84−6.80 (m, 2H), 4.64 (s, 2H),
4.55−4.51 (m, 2H), 3.90 (t, J = 5.7 Hz, 2H), 3.68 (s, 3H), 3.67−3.63
(m, 2H), 3.57−3.53 (m, 2H), 2.73 (s, 2H), 2.28−2.22 (m, 2H), 1.49
(s, 9H).
2-(4-(3-(8-Chloro-1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indol-9-yl)-
propoxy)phenyl)-N-hydroxyacetamide (D12). To a solution of tert-
butyl 8-chloro-9-(3-(4-(2-methoxy-2-oxoethyl)phenoxy)propyl)-
1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole-2-carboxylate (5l, 0.30 g,
0.59 mmol) in dry dichloromethane (DCM, 7 mL), TFA (3.5 mL)
was added dropwise with stirring at 0 °C. The reaction mixture was
allowed to stir for 30 min to remove the protection of tert-
butoxycarbonyl. Saturated NaHCO3 was added dropwise into the
mixture with stirring violently for 30 min after the addition of EtOAc
(20 mL). The mixture was extracted with EtOAc (20 × 2) and the
combined organic layer was washed with saturated NaCl, dried over
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Figure 7. Combination of compound D12 and FLC exhibits excellent therapeutic effects in mice candidiasis models. (A) Fungal burden in mice
kidneys after treatment with different drugs. The infection model was established using 5 × 105 cells of C. albicans (0.04103) and the mice were
divided into the control group, FLC group (1 mg/kg), D12 group (10 mg/kg), and the combination group (FLC + D12, 1+10 mg/kg). The fungal
burden of kidneys was determined after 5 days of treatment. The difference between the groups was compared by the t-test (* p < 0.05, ** p < 0.01,
*** p < 0.001, **** p <0.0001). (B) Kidney tissue sections after drug treatment in mouse models of candidiasis. (-) means treated with the vehicle,
and the treatments were consistent with the kidney fungal burden experiment. The tissue was observed and photographed with a biological
microscope under 40× and 200× fields of view, respectively.
Na2SO4, filtered, and concentrated under reduced pressure. The
residue was used for the next reaction without further purification.
A solution of KOH (2.80 g, 200 mmol) in MeOH (7 mL) was
added dropwise into a solution of NH2OH·HCl (2.32 g, 33.5 mmol)
in MeOH (12 mL) at 0 °C. Then, MgSO4 (1 g, 8.31 mmol) was
added into the reaction mixture and stirred at 0 °C for 30 min. The
mixture was filtered to get a solution of NH2OH. Methyl 2-(4-(3-
(1,2,3,4-tetrahydro-9H-pyrido[3,4-b] indol-9-yl)propoxy)phenyl)-
acetate obtained by the above reaction (0.27 g) was added into the
solution of NH2OH (17 mL, freshly prepared). The reaction was
stirred at 45 °C for 2 h. The solvent was removed under reduced
pressure. H2O (3 mL) and 1N HCl were added into it to adjust the
pH = 7−8. Then, the mixture was filtered and the residue was purified
by HPLC to obtain target compound D12 (0.12 g, 49%) as a white
solid. 1H NMR (600 MHz, DMSO-d6): δ 10.57 (s, 1H), 7.35 (d, J =
7.4 Hz, 1H), 7.15 (d, J = 8.6 Hz, 2H), 7.07 (d, J = 7.4 Hz, 1H), 6.95
(t, J = 7.7 Hz, 1H), 6.82 (d, J = 8.6 Hz, 2H), 4.44 (t, J = 7.2 Hz, 2H),
3.92 (t, J = 5.9 Hz, 2H), 3.87 (s, 2H), 3.18 (s, 2H), 2.90 (t, J = 5.6 Hz,
2H), 2.56 (t, J = 5.4 Hz, 2H), 2.13−2.06 (m, 2H). 13C NMR (151
MHz, DMSO-d6) δ 167.8 (s), 157.5 (s), 137.8 (s), 131.1 (s), 130.8
(s), 130.4 (s), 128.6 (s), 122.7 (s), 120.1 (s), 117.1 (s), 115.5 (s),
114.7 (s), 108.8 (s), 64.9 (s), 43.4 (s), 42.0 (s), 41.6 (s), 39.0 (s),
32.0 (s), 22.4 (s). ESI-MS (m/z): 414.52 [M + 1]. ESI-HRMS: calcd
for C22H24ClN3O3 m/z: [M + H]+ = 414.1579; found [M + H]+ =
414.1588, HPLC purity: 99.47%.
2.02 (m, 2H). 13C NMR (151 MHz, DMSO) δ 167.8 (s), 157.4 (s),
157.4 (d, J = 213.8 Hz), 135.0 (s), 133.0 (s), 130.8 (s), 130.4 (s),
128.7 (s), 127.2 (d, J = 9.9 Hz), 114.8 (s), 110.7 (d, J = 9.7 Hz),
109.0 (d, J = 25.8 Hz), 107.2 (d, J = 4.0 Hz), 103.1 (d, J = 23.2 Hz),
64.8 (s), 42.9 (s), 41.3 (s), 40.5 (s), 38.9 (s), 29.8 (s), 21.1 (s). ESI-
HRMS: calcd for C22H24FN3O4 m/z: [M + H]+ = 398.1874, [M −
H]− = 396.1729; found [M + H]+ = 398.1878 m/z, [M − H]−
=
396.1751.
2-(4-(3-(6-Bromo-1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indol-9-yl)-
propoxy)phenyl)-N-hydroxyacetamide (D6). A light yellow solid,
1
0.18 g, yield 64%. H NMR (600 MHz, DMSO-d6) δ 10.60 (s, 1H),
7.57 (d, J = 1.9 Hz, 1H), 7.38 (d, J = 8.7 Hz, 1H), 7.18−7.13 (m,
3H), 6.84 (d, J = 8.6 Hz, 2H), 4.18 (t, J = 6.9 Hz, 2H), 3.96 (s, 2H),
3.87 (t, J = 5.9 Hz, 2H), 3.20 (s, 2H), 2.99 (t, J = 5.6 Hz, 2H), 2.62 (t,
J = 5.3 Hz, 2H), 2.10−2.03 (m, 2H). ESI-HRMS: calcd for
C22H24BrN3O3 m/z: [M + H]+ = 460.1053, [M − H]− = 458.0908;
found [M + H]+ = 460.1063, [M − H]− = 458.0938.
2-(4-(3-(7-Fluoro-1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indol-9-yl)-
propoxy)phenyl)-N-hydroxyacetamide (D9). A light yellow solid,
1
0.24 g, yield 66%. H NMR (600 MHz, DMSO-d6) δ 10.63 (s, 1H),
7.40−7.36 (m, 1H), 7.27 (d, J = 8.9 Hz, 1H), 7.19−7.14 (m, 2H),
6.87−6.80 (m, 3H), 4.16 (t, J = 6.8 Hz, 2H), 3.97 (s, 2H), 3.88 (t, J =
5.8 Hz, 2H), 3.20 (s, 2H), 3.02 (t, J = 5.3 Hz, 2H), 2.69−2.61 (m,
2H), 2.09−2.03 (m, 2H). 13C NMR (151 MHz, DMSO) δ 167.8 (s),
159.1 (d, J = 233.9 Hz), 157.4 (s), 136.3 (d, J = 12.2 Hz), 133.8 (s),
130.8 (s), 130.4 (s), 128.7 (s), 123.8 (s), 118.9 (d, J = 10.3 Hz),
114.7 (s), 107.2 (d, J = 29.9 Hz), 96.5 (d, J = 26.4 Hz), 64.8 (s), 42.9
(s), 41.3 (s), 40.5 (s), 38.9 (s), 29.6 (s), 21.2 (s). ESI-HRMS: calcd
for C22H24FN3O3 m/z: [M + H]+ = 398.1874, [M − H]− = 396.1729;
found [M + H]+ = 398.1888, [M − H]− = 396.1741.
2-(4-(3-(7-Chloro-1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indol-9-yl)-
propoxy)phenyl)-N-hydroxyacetamide (D10). A light yellow solid,
0.17 g, yield 78%. 1H NMR (600 MHz, DMSO-d6): δ 10.59 (s, 1H),
7.50 (d, J = 1.7 Hz, 1H), 7.38 (d, J = 8.3 Hz, 1H), 7.16 (d, J = 8.5 Hz,
2H), 6.97 (dd, J = 8.3, 1.8 Hz, 1H), 6.85 (d, J = 8.6 Hz, 2H), 4.17 (t, J
= 6.9 Hz, 2H), 3.90−3.86 (m, 4H), 3.20 (s, 2H), 2.94 (t, J = 5.6 Hz,
2H), 2.59 (t, J = 5.4 Hz, 2H), 2.09−2.04 (m, 2H). 13C NMR (151
MHz, DMSO) δ 167.7 (s), 157.4 (s), 136.58 (s), 136.4 (s), 130.4 (s),
128.7 (s), 126.1 (s), 125.7 (s), 119.1 (s), 114.8 (s), 109.6 (s), 107.9
(s), 64.9 (s), 43.4 (s), 41.9 (s), 38.9 (s), 29.7 (s), 22.3 (s). MS (ESI
positive) m/z: 414.39 [M + 1]. ESI-HRMS: calcd for C22H24ClN3O3
m/z: [M + H]+ = 414.1579 m/z; found [M + H]+ = 414.1579.
Biology: Strains, Culture, and Agents. All of the strains used in
this study are listed in Tables 1, 2, and S3 and derived from clinical
isolates that were identified by standard mycological and molecular
tests. The commonly used media are mainly as follows: the yeast
The synthesis of compounds 5a−m and D1−D11 was similar to
the methods of 5l and D12, respectively.
2-(4-(3-(5-Chloro-1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indol-9-yl)-
propoxy)phenyl)-N-hydroxyacetamide (D2). A light yellow solid,
0.091 g, yield 54%. 1H NMR (600 MHz, DMSO-d6) δ 10.58 (s, 1H),
7.35 (dd, J = 7.7, 0.9 Hz, 1H), 7.16 (d, J = 8.6 Hz, 2H), 6.99−6.93
(m, 2H), 6.87−6.82 (m, 2H), 4.18 (t, J = 6.9 Hz, 2H), 3.90−3.83 (m,
4H), 3.20 (s, 2H), 2.94−2.87 (m, 4H), 2.09−2.03 (m, 2H). 13C NMR
(151 MHz, DMSO) δ 167.8 (s), 157.4 (s), 137.3 (s), 136.9 (s), 130.4
(s), 128.65 (s), 124.7 (s), 124.3 (s), 121.4 (s), 119.3 (s), 114.7 (s),
108.8 (s), 107.3 (s), 64.8 (s), 43.7 (s), 42.0 (s), 38.9 (s), 29.6 (s),
24.4 (s). MS (ESI positive) m/z: 414.57 [M + 1]. ESI-HRMS: calcd
for C22H24ClN3O3 m/z: [M + H]+ = 414.1579; found [M + H]+ =
414.1585.
2-(4-(3-(6-Fluoro-1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indol-9-yl)-
propoxy)phenyl)-N-hydroxyacetamide (D4). A light yellow solid,
0.096 g, yield 84%. 1H NMR (600 MHz, DMSO-d6) δ 10.61 (s, 1H),
7.41 (dd, J = 8.9, 4.4 Hz, 1H), 7.19 (dd, J = 9.8, 2.5 Hz, 1H), 7.16 (d,
J = 8.6 Hz, 2H), 6.90 (td, J = 9.2, 2.5 Hz, 1H), 6.85 (d, J = 8.6 Hz,
2H), 4.20 (t, J = 6.9 Hz, 2H), 4.07 (s, 2H), 3.88 (t, J = 5.9 Hz, 2H),
3.20 (s, 2H), 3.10 (t, J = 5.7 Hz, 2H), 2.69 (t, J = 5.5 Hz, 2H), 2.12−
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extract peptone dextrose (YEPD) liquid medium (1% yeast extract,
2% peptone, and 2% dextrose), RPMI 1640 medium (buffered with
0.165 M MOPS, 0.2% NaHCO3, and 0.27% NaOH as the testing
medium), and SDA medium (1% peptone, 4% glucose, and 2% agar).
Strains were routinely resurrected in YEPD medium at 30 °C with
continuous shaking. Compounds and FLC were diluted into 2 mg/
mL solution using DMSO.
In Vitro Antifungal Activity Assay. In vitro antifungal activity of
the two compounds in synergy was measured by the checkerboard
microdilution method in 96-well microtest plates. The assay was
analyzed according to the methods described by the Clinical &
Laboratory Standards Institute (CLSI, M27-A3) with a few
modifications.3 Briefly, exponentially grown C. albicans cells were
harvested and resuspended in a fresh RPMI 1640 medium to a
concentration of 103 cells/mL. The final concentrations for FLC and
tested compounds ranged from 0.25 to 64 μg/mL and 2 to 64 μg/mL,
respectively. Fungi were incubated at 35 °C for 48 h. The optical
density at 630 nm (OD630) was determined using a microtiter plate
reader while subtracting the background OD of each well. Synergy
and antagonism were defined by FICIs of ≤0.5 and >4, respectively.
An FICI > 0.5 but <4 was considered irrelevant.
Suspensions of C. albicans (0304103) cells (1.0 × 106 CFU/mL in the
RPMI 1640 medium) were added into a 96-well microtest plate (100
μL per well) and incubated at 37 °C for 24 h to form a mature
biofilm. The following operation was similar to the method of testing
biofilm formation. Three independent experiments were performed.
In Vitro Hyphal Formation Assay. The experimental procedure
was performed according to reported methods with a few
modifications.29 Exponentially grown C. albicans cells resuspended
in the Spider culture medium (10% Nutrient broth, 2% NaHCO3, 2%
K2HPO4·2H2O,) to 2 × 106 cells/mL were transferred to 12-well
plates while each well was added with 1 mL of the suspension. Drugs
used alone or in combination were added, and cells treated with
DMSO were used as the control group. Cells were observed and
photographed with an inversed fluorescence microscope after 3 h of
incubation at 37 °C.
Inhibition of Human HDAC1/6. The assay was performed by
quantitatively measuring the fluorescence intensity following an
enzymatic reaction according to the reported method.30 The dilution
of compounds (5 μL) was added to a 40 μL of the reaction mixture
(pH = 8.0) containing 25 mM Tris, 0.1 mM MgCl2, 0.1 mg/mL BSA,
137 mM NaCl, 2.7 mM KCl, and HDAC6 (15 ng per well), so that
the concentration of compounds ranged from 100 mM to 10 nM (3-
fold dilution). The mixture was preincubated at room temperature for
5 min. Finally, the enzyme substrate (10 μM) was added, and the
enzymatic reactions were conducted at 37 °C for 30 minutes in a final
volume of 50 μL. The reactions were terminated by adding the
termination solution (50 μL) for 30 min at room temperature. The
IC50 values were calculated by measuring the fluorescence intensity at
an excitation wavelength of 360 nm and an emission wavelength of
460 nm.
Inhibition of Fungal and Human Total HDACs. The fungal
protoplast was extracted by the rapid yeast genomic DNA isolation kit
(Sangon Biotech). About 100 mg of wet weight C. albicans cells
exponentially grown were harvested and resuspended in 3 mL of
snailase reaction buffer. Mercaptoethanol (2.4 μL) and snailase (50
μL, dissolved in snailase storage buffer to a concentration of 120 mg/
mL) were added into the reaction buffer, and the sample was then
incubated at 37 °C for 3 h. The lysate was centrifuged and the
supernatant-removed protoplast was resuspended in 20 mL of PBS.
The suspension and compounds were added into the 96-well
microtest plates (100 μL for per well). The final concentration of
tested compounds ranged from 1 to 100 μM. HDAC substrate Boc-
Lys(Ac)-AMC was added into the plates at a concentration of 33 μM
after the plates were incubated at 35 °C for 12 h. Then, the plates
were incubated at 37 °C for another 6 h, and the reaction was
terminated by adding 100 μL of the termination solution (2 mg/mL
trypsin, 1% Nonidet P-40, 50 mM Tris-HCl (pH = 8), 137mM NaCl,
2.7 mM KCl, 1 mM MgCl2) to each well and allowing the reaction to
continue at 37 °C for 2 h. The reaction solution was transferred to a
black 96-well plate, and the fluorescence intensity was measured at an
excitation wavelength of 360 nm and an emission wavelength of 460
nm. The inhibition rate was calculated by the ratio of the fluorescence
intensity of the compound group to that of the control group after
subtracting the blank. Three independent experiments were
performed. The human total HDAC Enzymatic assay was performed
using HUVEC cells. Different from fungi, there was no need to
prepare protoplasts, and about 15000 cells per well were seeded into
96-well transparent plates. After being incubated at 37 °C and 5%
CO2 atmosphere for 24 h, the compounds were added into the plates
and the remaining operations were similar to the fungal HDAC
enzymatic assay.
Disk Diffusion Assay. About 1 × 104 cells were plated on the
SDA medium or on SDA medium containing FLC (32 μg/mL) or
D12 (8 μg/mL), or both of them. Plates were incubated at 35 °C for
48 h and photographed to intuitively reflect the drug resistance of C.
albicans (0304103) and the synergism of compound D12 and FLC.
Disk diffusion testing of drugs in combination was performed as
follows. SDA plates (90 mm diameter) containing FLC (32 μg/mL)
were used in this study. The surface of the medium was coated with
approximately 105 C. albicans (0304103) cells by using a glass
spreading rod. Disks containing different doses of compound D12 (0,
2, 8, and 32 μg) were equidistantly placed onto the surface of the agar
plate inoculated with fungi, and the plate was photographed 48 h after
being incubated at 35 °C.
Growth Curve Assay. Growth curve assay was performed
according to a previous method with a few modifications.27 The
exponentially grown C. albicans strain 0304103 was washed with PBS
(phosphate buffer saline, 3 × 1 mL) and then resuspended in 5 mL of
the RPMI 1640 medium to an initial concentration of 1.5 × 106 cells/
mL and divided into different bottles. Samples added to different
concentrations of compound D12 and FLC were incubated in a
shaking incubator (200 rpm) at 30 °C and counted at a specific time
point (0, 6, 12, 24, and 48 h). Three independent experiments were
performed.
Time Kill Curve Assay. The sample preparation was the same as
that in the growth curve assay. The initial fungal concentration was
1.5 × 106 cells/mL and the samples treated with drugs alone or in
combination were incubated in a shaking incubator (200 rpm) at 30
°C. Samples were taken at a specific time point (0, 6, 12, 24, 36, and
48 h), diluted and coated on the SDA medium, and cultured at 35 °C
for 48 h. The fungal colonies in the medium were counted and the
number of viable fungi was calculated. Three independent experi-
ments were performed.
In Vitro Biofilm Formation Assay. The experimental procedure
was performed according to our previous methods with a few
modifications.28 Suspensions of C. albicans (0304103) cells (1.0 × 106
CFU/mL in the RPMI 1640 medium) were added into a 96-well
microtest plate (100 μL per well) and incubated at 37 °C for 90 min
for adhesion. The adherent cells were washed with PBS (3 × 100 μL)
after the upper culture medium was removed. A fresh RPMI 1640
medium with or without drugs was then added to adherent cells. The
final concentrations for FLC and tested compounds ranged from 2 to
32 μg/mL and 0.12 to 64 μg/mL, respectively. Then, the plates were
incubated at 37 °C for another 24 h. A semiquantitative measure of
biofilm formation was calculated by using the method of the XTT
reduction assay. The optical density at 490 nm (OD490) was
determined using a microtiter plate reader and three independent
experiments were performed.
Quantification of the Gene Expression by Real-Time RT-
PCR. Real-Time RT-PCR was performed according to the previous
work with a few modifications.31 Primers were synthesized by Sangon
Biotech (Shanghai) Co., Ltd. and listed in Table S6 in the Supporting
Information. C. albicans cells treated with different drugs alone or in
combination were incubated in the YEPD medium at 35 °C for 24 h.
For extraction of the total RNA of collected fungi, the Column Fungal
RNAout kit (Tiandz, Inc.) was used according to the instructions.
Reverse transcription was performed by using the PrimeScript RT
Disruption Assay of Preformed Biofilms. The experimental
procedure was similar to the methods of the biofilm formation assay.
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Master Mix kit (TaKaRa) through two steps (1. 37 °C for 15 min; 2.
85 °C for 5 s). The qRT−PCR was performed in the LightCycler
Real-Time PCR system (Roche diagnostics, GmbH Mannheim,
Germany), using SYBR Green I. Amplification of cDNA was carried
out according to the established procedures: 95 °C for 30 s and then
40 cycles of 95 °C for 5 s, 60 °C for 34 s, 95 °C for 30 s, 60 °C for 60
s, and 95 °C for 15 s. Monitoring the fluorescence during cycling
measures the threshold cycle. Fold changes in the gene expression was
analyzed using the 2−ΔΔCt method, with ACT1 as the internal control.
Murine Model of Candidiasis. Female ICR (Institute of Cancer
Research) mice (20−25 g each mouse) purchased from JOINN
Laboratories (Certificate SCXK-2018-0006) were used in this
experimentation. Four mice in each group and 5 × 105 cells C.
albicans (0304103) were used to infect mice via lateral tail vein
injection. Mice were then treated with the vehicle (1.5% glycerin and
0.5% Tween 80), 10 mg/kg compound D12, 1 mg/kg FLC, or the
combination (D12 + FLC) via intraperitoneal (IP) injections 24 h
later. Treatments were administered continuously for 5 days after
infection. The animal laboratory meets IACUC standards (Certificate
SYXK-2017-0004). The kidneys were removed and homogenized.
Diluent suspensions (1000-fold) were plated on the SDA medium
containing chloramphenicol and incubated at 30 °C for at least 48 h
to count the number of colonies and calculate the total fungal burden.
The differences between groups were analyzed by analysis of variance
(ANOVA).
0001-9489-804X; Phone: +86 21 81871201;
Email: shengcq@smmu.edu.cn
Authors
Zhuang Li − Department of Medicinal Chemistry, School of
Pharmacy, Second Military Medical University, Shanghai
200433, China
Jie Tu − Department of Medicinal Chemistry, School of
Pharmacy, Second Military Medical University, Shanghai
200433, China
Guiyan Han − Department of Medicinal Chemistry, School of
Pharmacy, Second Military Medical University, Shanghai
200433, China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01763
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
Pathological Sections of Murine Kidneys. Two mice in each
group and the infection and treatment of mice were consistent with
the methods used in the murine model of candidiasis as mentioned
above. Groups of mice were sacrificed 5 days after treatment with
drugs alone or in combination. Fixed with 4% paraformaldehyde,
sampled conventionally, dehydrated, embedded with paraffin, sliced
(4 μm thick), and stained with hematoxylin−eosin (HE), the renal
tissues removed from mice were observed (NIKON Eclipse Ci) and
photographed (40× and 200×).
ACKNOWLEDGMENTS
■
This work was supported by the National Natural Science
Foundation of China (Grant 81973175 to N.L., Grant
81725020 to C.S.) and the Innovation Program of Shanghai
Municipal Education Commission (Grant 2019-01-07-00-07-
E00073 to C.S.).
ABBREVIATIONS LIST
■
HDAC, histone deacetylase; C. albicans, Candida albicans; IFIs,
invasive fungal infections; ICU, intensive care unit; CYP51,
lanosterol 14α-demethylase; JAK2, Janus kinase; FLC,
fluconazole; TBA, tubastatin A; VRC, voriconazole; ITC,
itraconazole; SARs, structure−activity relationships; DMF,
dimethylformamide; TFA, trifluoroacetic acid; ZBG, zinc-
binding group; FICI, fractional inhibitory concentration index;
MIC80, minimum concentration of 80% inhibition; C. krusei,
Candida krusei; Candida auris, C. auris; Candida glabrata, C.
glabrata; Candida tropicalis, C. tropicalis; Cryptococcus neofor-
mans, C. neoformans; SDA, Sabouraud Dextrose Agar; YEPD,
Yeast extract Peptone Dextrose; CLSI, Clinical & Laboratory
Standards Institute; PBS, phosphate buffer saline; ICR,
Institute of Cancer Research; OD, optical density; IP,
intraperitoneal; HE, hematoxylin−eosin
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01763.
■
sı
Molecular formula strings of the target compounds
(CSV);
synergism of TBA in combination with different first-line
azole drugs; synergism of compounds D1−12 in
combination with azoles (VRC and ITC) against C.
albicans (0304103); synergism of compound D12 in
combination with azoles (VRC and ITC) against various
clinically isolated C. albicans, C. krusei, C. auris, C.
glabrata, C. tropicalis, and C. neoformans; cytotoxicity of
compound D12 and SAHA alone or in combination
with FLC; sequence of primers; the relative growth of
various clinically isolated strains (Table 2) presented by
the form of heat maps; protocols of the cytotoxicity
assay; structural characterization; spectral data of target
compounds; and HPLC spectra (PDF)
REFERENCES
■
(1) Denning, D. W.; Bromley, M. J. Infectious Disease. How to
bolster the antifungal pipeline. Science 2015, 347, 1414−1416.
(2) Gullo, A. Invasive fungal infections: the challenge continues.
Drugs 2009, 69, 65−73.
(3) Li, D. D.; Zhao, L. X.; Mylonakis, E.; Hu, G. H.; Zou, Y.; Huang,
T. K.; Yan, L.; Wang, Y.; Jiang, Y. Y. In vitro and in vivo activities of
pterostilbene against Candida albicans biofilms. Antimicrob. Agents
Chemother. 2014, 58, 2344−2355.
AUTHOR INFORMATION
Corresponding Authors
■
Na Liu − Department of Medicinal Chemistry, School of
Pharmacy, Second Military Medical University, Shanghai
200433, China; orcid.org/0000-0002-3920-1008;
Phone: +86 21 81871230; Email: 285939784@qq.com
Chunquan Sheng − Department of Medicinal Chemistry,
School of Pharmacy, Second Military Medical University,
Shanghai 200433, China; School of Medicine, Tongji
University, Shanghai 200072, China; orcid.org/0000-
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