Synthesis and synergistic antifungal effects of monoketone derivatives of curcumin against fluconazole-resistant Candida spp.

Article abstract of DOI:10.1039/c6md00649c

Twenty-three monoketone derivatives of curcumin were synthesized to investigate the synergy with fluconazole against fluconazole-resistant Candida spp. The minimal inhibitory concentration (MIC₈₀) and the fractional inhibitory concentration index (FICI) of the antifungal synergist fluconazole were measured against fluconazole-resistant C. albicans, C. tropicalis and C. krusei in vitro. Most of these compounds showed good synergistic activities against C. tropicalis. Among them, compound 9 exhibited significant synergistic activities against Candida spp. SARs were also discussed. In particular, a cell growth test exhibited that a combination of 1 μg ml^-1 fluconazole and 64 μg ml^-1 or 128 μg ml^-1 compound 9 showed the most potent fungicidal effect against C. tropicalis. The synergistic effect may be associated with the changes of the intracellular ATP content and cell membrane permeability. Our results provided a basis for future evaluation and development of these compounds as leads for therapeutics for fluconazole-resistant candidiasis.

Full text of DOI:10.1039/c6md00649c

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DOI: 10.1039/C6MD00649C
Synthesis and synergistic antifungal effects of monoketone derivatives of curcumin against
fluconazoleꢀresistant Candida spp. ‡
Fei Zhao1†, HuaiꢀHuai Dong1†, YuanꢀHua Wang1,2†, TianꢀYi Wang1, ZeꢀHao Yan1, Fang Yan2,
DaꢀZhi Zhang1, YingꢀYing Cao1*, YongꢀSheng Jin1*
1. School of Pharmacy, Second Military Medical University, Shanghai, China, 200433
2. School of Pharmacy, Weifang Medical University, Weifang, Shandong Province, China, 261053
[ABSTRACT] Twentyꢀthree monoketone derivatives of curcumin were synthesized to restore the
effectiveness of fluconazole against fluconazoleꢀresistant Candida spp.. The minimal inhibitory
concentration (MIC80) and the fractional inhibitory concentration index (FICI) of the antifungal
synergist fluconazole were measured against fluconazoleꢀresistant C. albicans, C. tropicalis and C.
kruseii in vitro. Most of these compounds showed good synergistic activities against C. tropicalis.
Among them, compounds 9, 14 and 15 exhibited significant synergistic activities against C. spp.
SARs were also discussed. Particularly, cell growth test exhibited that combination of 1 ꢀg/ml
fluconazole and 64 ꢀg/ml or 128 ꢀg/ml compound 9 showed the most potent fungicidal effect
against C. tropicalis. The synergistic effect may be associated with the changes of the intracellular
ATP content and cell membrane permeability. Our results provided a basis for future evaluation
and development of these compounds as leads for therapeutics for fluconazoleꢀresistant
candidiasis.
[KEY WORDS] curcumin; monoketone derivatives; fluconazoleꢀresistant Candida spp.;
synergistic antifungal effect
*Corresponding
author:
Tel:
+86ꢀ21ꢀ81871227,
Email:
ysjinsmmu@163.com,
ysjin@smmu.edu.cn (Y.ꢀS, Jin).
†The authors contributed equally.
‡The authors declare no competing interests.
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DOI: 10.1039/C6MD00649C
1. Introduction
Fungal infections pose a continuous and serious threat to human health and life especially to
immunocompromised patients. During the past decades, the incidence of systemic candidiasis has
consistently increased. Candida species such as candida albicans, candida tropicalis and candida
krusei are known as the main pathogens, which may cause superficial and systemic infections.
Antifungal therapy with topical or systemic agents can be effective in the control and treatment of
candida infection. Fluconazole (FLC) is the most commonly used drug to treat Candida spp. in
prophylaxis and therapy; however, widespread and repeated use of FLC resulted in resistance to or
failure of FLC therapy. To combat the FLCꢀresistant Candida spp., much attention has been paid
to the synergism of FLC with other agents. As synergists, they can significantly sensitize fungi
towards FLC, such as tetracyclic indoles 1 and amiodarone,2 etc.
Naturally derived products are valuable source for drugs or lead compounds.3ꢀ5 Curcumin
(CCM, 1,7ꢀbisꢀ(4ꢀhydroxyꢀ3ꢀmethoxyphenyl)ꢀ1,6ꢀheptadieneꢀ3,5ꢀdione), a naturally occurring
phenolic compound, is a dietary constituent of turmeric and is well known for its broad
bioactivities. Earlier studies have revealed that CCM and its related compounds possess multiple
9
pharmaceutical applications such as antiꢀcancer,6 antibacterial7 and antifungal8, properties.
Studies indicated CCM acts as an antifungal agent via various mechanisms, such as generation of
oxidative stress and inhibits hyphae development,10 and alteration of membraneꢀassociated
properties of ATPase activity, ergosterol biosynthesis, and proteinase secretion.8 The clinical trials
have reported that ingestion of a significant dose of CCM (12 g/day) had no side effect.11
Considering the biological importance and low toxicity of CCM, many efforts have been devoted
to CCM analogs. But, so far, there is no report about the antifungal structureꢀactivity relationship
(SAR) of CCM as well as its synergistic antifungal activity. Inspired by these, it was of our
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interest to investigate the antifungal activity of CCM and understand its preliminary SAR.
Structurally, CCM is a symmetrical methoxyphenolic dienone with an active methylene center
(Table 1). Evidences from both in vitro and in vivo studies showed that βꢀdiketone moiety is
responsible for its instability and weak pharmacokinetic.11ꢀ17 During last decades, structure
modifications of CCM to enhance its bioactivities suggested that the stability and metabolic
profile of CCM could be improved by deleting βꢀdiketone moiety.11ꢀ17 Therefore, monoketone
derivatives of CCM were synthesized in our lab and evaluated for their antifungal activity alone
and synergistic antifungal activity with fluconazole against fluconazoleꢀresistant Candida spp.,
and the structureꢀactivity relationship (SAR) is also investigated and discussed.
2. Results and Discussion
Most of the title compounds were synthesized by aldol condensation of substituted
benzaldehydes or heteroaromatic aldehydes with acetone or cyclohexanone in ethyl alcohol
catalyzed by 10ꢀ20% NaOH aqueous solution with good yields (Scheme 1). But in this condition
the benzaldehydes substituted with hydroxyl group were hard to yield corresponding products,
even no reaction occurred at all. So this kind of products were obtained by aldol condensation
catalyzed by SOCl2 in anhydrous ethyl alcohol. The synthetic method of title compounds has the
advantages of simple operation, mild condition and easy workꢀup. Structures of synthesized
1
compounds were confirmed by MS and H NMR. The Z/E configuration of compounds 1ꢀ15 can
be easily confirmed as Eꢀconfiguration from the coupling constants (about J = 15.9 Hz) in
1HꢀNMR spectra. In order to confirm the Z/E configuration of compounds 16ꢀ23, compound 20
was taken as an example to perform NOESY experiments. A clear NOESY correlations between
Hꢀ1’ and Hꢀ2”, Hꢀ6” demonstrated compound 20 was Eꢀconfiguration. So it can be concluded that
compounds 16ꢀ23 was Eꢀconfiguration.
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Scheme 1. Synthetic route for the title compounds. Reagents and conditions: (a) NaOH, EtOH, rt,
12ꢀ24 h; (b) SOCl2, anhydrous EtOH, rt, 12ꢀ24 h.
The in vitro synergistic antifungal activities of the title compounds were tested using the
microbroth dilution method according to the standards of the Clinical and Laboratory Standards
Institute, USA.14 The MIC80 of FLC against the FLCꢀresistant C. albicans (clinical isolate 103), C.
tropical (clinical isolate 087) and C. kruseii (clinical isolate 2159) were determined to be 128.0
ꢀg/ml. The MIC80 of each title compound with or without FLC (8.0 ꢀg/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—synergistic or indifferent were defined according to FICI values of ≤ 0.5 or >0.5,
respectively.18 The structures of tile compounds and their synergistic antifungal activities were
summarized in table 1.
Table 1. Structures and interaction modes of the title compounds and their MIC80 and FICI values.
MIC80 (ꢀg/ml)
C. albicans 103
Mode of
C. tropical 087
Mode of
C. kruseii 2159
Mode of
No.
Structures
with
with
FLC[a]
with
FLC[a]
FICI
FICI
FICI
Interꢀ
Interꢀ
Interꢀ
FLC[a]
action[b]
action[b]
action[b]
1
2
>64
>64
>1.06
>1.06
I
I
16
32
0.313
0.563
S
I
>64
>64
>1.06
>1.06
I
I
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3
>64
>64
>64
8
>1.06
>1.06
>1.06
0.188
>1.06
>1.06
0.125
>1.06
0.563
0.563
>1.06
>1.06
>1.06
>1.06
>1.06
>1.06
>1.06
>1.06
>1.06
>1.06
>1.06
0.125
I
I
16
32
16
>64
8
0.313
0.563
0.313
>1.06
0.188
>1.06
0.125
0.188
0.188
0.313
>1.06
0.125
>1.06
0.188
1.06
S
I
>64
>64
>64
>64
>64
>64
8
>1.06
I
I
I
I
I
I
S
I
I
S
I
I
I
I
I
I
I
I
I
I
I
I
4
>1.06
>1.06
>1.06
>1.06
>1.06
0.188
>1.06
>1.06
0.094
>1.06
>1.06
>1.06
>1.06
>1.06
>1.06
>1.06
>1.06
>1.06
>1.06
>1.06
0.563
5
I
S
I
6
S
I
7
>64
>64
4
S
I
8
I
>64
4
9
S
I
S
S
S
S
I
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
>64
32
8
>64
>64
2
I
8
32
I
16
>64
4
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
4
I
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
2
I
S
I
I
>64
8
I
S
I
I
64
8
I
0.188
1.06
S
I
I
64
64
16
>64
4
I
1.06
I
I
0.313
>1.06
0.125
0.125
S
I
I
I
S
S
S
4
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CCM
8
0.188
S
8
0.188
S
8
0.188
S
Note: The MIC80 value of all compounds alone was > 64 ꢀg/ml except for compound 9 with the
MIC80 value of 64 ꢀg/ml alone against C. albicans 103, C. tropicals and C. kruseii, and compound
24 with the MIC80 value of 4 ꢀg/ml alone against C. kruseii and MIC80 value of 64 ꢀg/ml alone
against C. albicans 103 and C. tropical.
[a] MIC80 value of compound in column 2 in combination with 8.0 ꢀg/ml fluconazole;
[b] I: indifferent; S: synergy.
Firstly, compounds 1ꢀ15 were synthesized and their synergistic antifungal activities were
tested. CCM had the synergistic antifungal activities against all the screened Candida spp. with
FICI value of 0.188. Most of title compounds exhibited selectivity against Candida spp., usually
they displayed more potent synergy against C. tropical than C. albicans and C. kruseii. For
example, except for compounds 2, 4, 6, 8, 13 and 15, all the other compounds displayed
synergistic activity against C. tropical, while only two compounds (6 and 9) showed synergy
against C. albicans and only two compounds (9 and 12) showed synergy against C. kruseii. These
results demonstrated the monoketone moiety was more beneficial for the synergy against C.
tropical, but not for C. albicans and C. kruseii.
The synergistic antifungal effects against C. tropical of these active compounds were equal or
even superior to CCM (FICI = 0.188). Compound 9 which was substituted with 3’,4’ꢀdihydroxyl
group and compound 14 which was substituted with 3’,4’ꢀdimethoxy group are the most potent
synergists (FICI = 0.125). Compounds with monohydroxyl (10) and 3’ꢀhydroxylꢀ4’ꢀmethoxyl
substitutions (11) also exhibited potent synergistic activities (FICI = 0.188). However, compounds
with halogen atoms (1, 2, 3, 8 and 15) and dimethylamine (4) showed weak or no activities, while
compounds with heteroaryl groups (5, 6) showed almost no inhibitory activity. These results
demonstrated the hydroxyl substitution was beneficial for the synergistic antifungal effects against
C. tropical.
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Although only two compounds 6 and 9 showed synergy against C. albicans and two
compounds 9 and 12 showed synergy against C. kruseii, the synergistic antifungal effects of these
active compounds were equal or superior to CCM. The FICI values of compounds 6 and 9 against
C. albicans were 0.188 and 0.125, respectively; and those of compounds 9 and 12 against C.
kruseii were 0.188 and 0.094, respectively. A preliminary result could be concluded that the
catechol moiety was favorable for the synergistic antifungal against C. albicans, C. tropical and C.
kruseii; and catechol methylene ether moiety was favorable for the synergistic antifungal against C.
kruseii. A control compound 24 (3’,4’ꢀdihydroxyl curcumin) was synthesized to further evaluated
monoketone scaffold. The FICI values of compounds 24 and 9 against C. albicans and C. tropical
were both 0.125, except that compound 24 when used alone against C. kruseii showed potent
antifungal activity with MIC80 = 4 ꢀg/ml. These results further confirmed that catechol moiety was
important for synergy, and βꢀdiketone moiety could be changed into monoketone.
In order to further study SARs, some monoketone analogs (16ꢀ23) of CCM which were
incorporated with a fiveꢀmembered ring were synthesized and evaluated their synergistic
antifungal activity. We hope this modification could help to enhance activity by restricting the
conformation of the diarylpentanoid system to increase the rigidity of molecules. But to our
disappointment, this modification resulted in the loss of activity for all compounds 16ꢀ23 against
C. albicans and C. kruseii. As for C. tropical, only four compounds (16, 18, 21 and 23) showed
synergistic antifungal activity. Among them, compound 18 substituted with free hydroxyl showed
potent synergy (MIC = 8 ꢀg/ml, FICI = 0.188) and compound 23 substituted with
5’-hydroxymethyl furan was the most active (MIC = 4 ꢀg/ml, FICI = 0.125), even compared with
CCM. These results demonstrated the introduction of a fiveꢀmembered ring would not help to
improve the activity in general; and the free hydroxyl or hydroxymethyl furan moiety is beneficial
for the synergistic against C. tropical.
To visualize and further confirm the synergistic effect of the most active 9 with FLC, the
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antifungal susceptibility assays were conducted by turbidity observation method and cell growth
test (growth curves). The results were summarized in Fig. 1 and Fig. 2. The suspension of cells
treated with 9 alone at the concentration of 64 ꢀg/ml showed a markedly reduced turbidity (Fig. 1).
While the suspension was thick when cells were treated with FLC at the maximum concentration
of 1 ꢀg/ml. But in combination with FLC, 9 exhibited antifungal effect for FLCꢀresistant C.
tropicals 087 at the concentration of 4 ꢀg/ml. This result suggested 9 showed strong antifungal
effect at the concentration of 64 ꢀg/ml alone or 4 ꢀg/ml in combination with FLC against FLC
ꢀresistant C. tropical 087. In the growth curves (Fig. 2), the synergistic fungicidal effect of 9 with
fluconazole was further confirmed. As shown in Fig. 2A, 9 at the concentration of 64 ꢀg/ml
exhibited the same inhibition effect as that of FLC at 0.5 ꢀg/ml. When concentration of 9 reached
to 128 ꢀg/ml, cells were killed or cell apoptosis was promoted. The synergistic effect of 9 on
growth could be obviously seen from other curves. When treated with the combination of 9 and
FLC at low concentrations, C. tropical 087 were significantly grown at lower rates. Especially, the
combination of 1 ꢀg/ml FLC and 9 (64 ꢀg/ml or 128 ꢀg/ml) showed the most potent antifungal
effect (Fig. 2B). Together, the results suggested 9 showed strong synergistic fungicidal effect
against FLCꢀresistant C. tropical 087.
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Fig. 1. Antifungal susceptibility assays of FLC alone or in combination with compound 9.
FLCꢀresistant C. tropical 087 were treated with or without different concentrations of compound 9
(0, 4, 8, 16, 32, 64, 128 ꢀg/ml) and FLC (0, 0.25, 0.5, 1 ꢀg/ml) alone or the combinations of FLC
and compound 9 in a shaking incubator. The turbidity of suspension was observed and pictures
were taken after 16 h incubation.
A
B
C
D
Fig. 2. Growth curves of cells with different treatments. (A) Timeꢀgrowth curve for C.
tropical 087 treated with compound 9 (64, 128 ꢀg/ml) or FLC (0.25, 0.5 ꢀg/ml) alone in RPMI
1640 medium at 30 °C for 48 h. OD was measured at 630 nm. (B) Timeꢀgrowth curve for C.
tropical 087 treated with FLC (1 ꢀg/ml) or in combination with compound 9 (64, 128 ꢀg/ml). (C)
Timeꢀgrowth curve for C. tropical 087 treated with FLC (0.5 ꢀg/ml) or in combination with
compound 9 (64, 128 ꢀg/ml). (D) Timeꢀgrowth curve for C. tropical 087 treated with FLC (0.25
ꢀg/ml) or in combination with compound 9 (64, 128 ꢀg/ml). FLC, fluconazole; compd. 9,
compound 9. Data were shown as the mean ± SD from three independent experiments.
In order to explore the synergistic antifungal mechanism of 9, we measured intracellular ATP
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levels and membrane permeability as reported before.19, 20 As showed in Fig. 3A, CCM inhibited
intracellular ATP production alone or in combination with FLC obviously. However, a significant
difference was not detected between CCM alone and in combination with FLC. Whereas the
combination of compound 9 and FLC could obviously decrease the intracellular ATP content
significantly compared with the compound 9 or FLC used alone (P < 0.01, Fig. 3B). The results
indicated that the reduction intracellular ATP was not the synergistic mechanism but an individual
antifungal mechanism for CCM. Nevertheless, the difference between the intracellular ATP
contents when used compound 9 alone and in combination with FLC suggested the synergy of
compound 9 may be associated with the inhibition on ATP production.
Since the changes of cell membrane permeability were targets for many antifungal agents,
cell membrane permeability assay was carried out to analyze the membrane damage caused by
compound 9 and fluconazole. PI (Propidium iodide) is a kind of nucleic acid staining dye and can
only enter into the cells which have permeable membranes. When cell membranes changed, the
dye would bind to nucleic acid and showed a red fluorescence. As showed in Fig 4A1, we noticed
that rare PI fluorescence was observed in untreated cells. But increased PI was observed when
cells were incubated with 32 ꢀg/ml compound 9 alone (Fig 4A2). And fluconazole, for 2 ꢀg/ml,
could have an effect on permeability of cell membranes (Fig 4A3). Significantly, much more PI
fluorescence was caught in the combinationꢀtreated (32 ꢀg/ml compound 9 plus 2 ꢀg/ml
fluconazole) cells (Fig 4A4). The changes and difference could also be seen in Fig 4B. In
summary, our results suggested that the synergistic effect may be also associated with the changes
of cell membrane permeability.
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A
B
Fig. 3. Intracellular ATP content in C. tropicalis 087 treated with different treatments. (A)
FLC (4 ꢀg/ml), CCM (32, 64, 128 ꢀg/ml) or the combination for 8 hours. (B) C. tropicalis 087
were treated with fluconazole (4 ꢀg/ml), compound 9 (32, 64, 128 ꢀg/ml) or the combination for 8
hours. Fluorescence intensities were determined on a TD 20/20 luminometer (Turner Biosystem,
Sunnyvale, CA). Each experiment was independently repeated for three times. * indicates P <
0.05 compared with drugꢀfree control, ** indicates P < 0.01 compared with drugꢀfree control.
A
(1) Untreated
(2) Compound.9
(3) Fluconazole
(4) Combination
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B
Fig. 4. Membrane permeability changes induced by compound 9 and fluconazole in C.
tropicals 087. A: PI staining for detection of disruption of membrane permeability in C.
tropicals 087, (1) cells were treated no agents, (2) cells were treated with compound 9 for 32
ꢀg/ml, (3) cells were treated with fluconazole for 2 ꢀg/ml and (4) cells were treated with the
combination of compound 9 (32 ꢀg/ml) and fluconazole (2 ꢀg/ml). B: PI staining for detection of
increase in membrane permeability treated with compounds in C. tropicals 087. (black, cells were
treated no agents; yellow, cells were treated with compound 9 for 32 ꢀg/ml alone; blue, cells were
treated with fluconazole for 2 ꢀg/ml alone; red, cells were treated with the combination of
fluconazole and compound 9).
Further cytotoxicity activities of active compounds 6, 7, 9, 10, 12, 14, 16, 18, 23 and CCM
were investigated on human umbilical vein endothelial cells (HUVECs) by the MTT assay. The
results are reported in Table 2. Curcumin exhibited significantly stronger cytotoxic effect at the
concentration of 32 ꢀg/ml with a viability of 22.0%. This is in agreement with previous research.21
However, compounds 6, 7, 9, 10, 12, 14, 16, 18, and 23 showed moderate cytotoxic effect at 32
ꢀg/ml with viabilities over 50%. Meanwhile, compounds 6, 9, 10, 12, and 16 possessed lower
cytotoxic effects than curcumin at the concentration of 16 ꢀg/ml. Among these synergistic
compounds, compound 9 showed the lowest cytotoxicity against HUVECs. Taken together, our
results suggested that these monoketone derivatives of CCM exhibited lower cytotoxicity than
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CCM.
Table 2. Cytotoxic activity of curcumin and its analogues on HUVEC.
Viability [%][a]
Compd.
32 ꢀg/ml
16 ꢀg/ml
CCM
6
22 ± 1.8
49 ± 2.5
43 ± 1.0
75 ± 1.6
75 ± 2.7
71 ± 2.9
55 ± 2.6
69 ± 2.6
60 ± 0.3
57 ± 4.0
80 ± 1.7
84 ± 1.1
64 ± 1.1
91 ± 2.5
87 ± 4.2
88 ± 4.0
67 ± 3.0
86 ± 4.0
69 ± 1.8
68 ± 0.3
7
9
10
12
14
16
18
23
[a] Data were shown as the mean ± SD from three independent experiments.
3. Conclusion
So our study may be also useful to find more advanced FLC synergists against FLCꢀresistant
C. spp.. Our results showed monoketone derivatives of CCM displayed selectivity against C.
albicans, C. tropical and C. kruseii, and most of the compounds displayed good synergistic
antifungal effects against C. tropical and no effects against C. albicans 103 and C. kruseii 2159.
The SAR study indicates that substituted hydroxyl or methoxyl are important groups in
monoketone derivatives. Compound 9 was the most potently synergistic effect against C. tropical
087 and C. albicans 103 (FICI = 0.188) in combination with FLC in vitro. And this results were
further confirmed by turbidity observation method and growth curves. The synergistic antifungal
effect may be associated with the changes of the intracellular ATP content and the changes of
membrane permeability. The synergistic antifungal mechanism of curcumin had been reported that
could be due to modulate ABC multidrug transporters22 and the overexpression of the MFS pump
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CaMdr1p23, but to the best of our knowledge, this study reports for the first time the ability of
compound 9, a curcumin derivative, to change the intracellular ATP content and membrane
permeability. Therefore, we propose that compound 9 be considered a potential candidate in the
development of a novel synergistic antimicrobial agent on account of its significant antimicrobial
activity.
4. Experimental
4.1 General
Reagents were purchased from common commercial suppliers, and were used without further
purification. Analytical TLC was carried out on silica gel F254 precoated (0.2 mm thickness)
plastic TLC sheets. The TLC plates were spotted with samples using a fine glass capillary tube
and developed in a chromatographic tank saturated with solvent vapor at room temperature.
1
Melting points were determined in open capillary tubes and are uncorrected. H NMR and 2D
NMR spectra were recorded on a Bruker Ac 300 or 600 MHz spectrometer in DMSOꢀd6 or CDCl3
solution. Chemical shifts were recorded in δ ppm relative to internal TMS. Mass spectra (MS and
HRMS) were obtained by electron spray ionization (ESI) in positive or negative mode using an
LCQ DECA XP LCꢀMS and Agilent UHPLC 1290 and QꢀTOF 6538 MSꢀMS spectrometer.
4.2. Experimental procedures
4.2.1 General procedure for the synthesis of compounds 1-8 and 12-23
To a stirring solution of appropriate ketone (1 mmol, 1 equiv) and corresponding aromatic
aldehyde (2 mmol, 2 equiv) in ethanol (10 ml) at room temperature was added 10% NaOH
(aqueous) solution (4 mmol, 4 equiv) slowly and starring continued at room temperature. Upon
completion, monitored by TLC, the mixture was poured into an excessive amount of ice ꢀ water.
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Then filtrated, dried and recrystallized from ethanol or DMF to give the title compounds.
4.2.2 General procedure for the synthesis of compounds 9-11
To a stirring solution of appropriate ketone (1 mmol, 1 equiv) and corresponding aromatic
aldehyde containing hydroxyl group (2 mmol, 2 equiv) in anhydrous ethanol (10 ml) at room
temperature was added SOCl2 (2 mmol, 2 equiv) slowly and starring continued at room
temperature. Upon completion, monitored by TLC, the mixture was poured into an excessive
amount of ice ꢀ water. Then filtrated, dried and recrystallized from ethanol or DMF to give the title
compounds.
4.2.3 (1E,4E)-1,5-bis(2,4-dichlorophenyl)penta-1,4-dien-3-one (1)
Yield 77%, mp: 207ꢀ210 °C; 1H NMR (300 MHz, CDCl3) δ 8.07 (2H, d, J = 18.0 Hz, Hꢀβ),
7.67 (2H, d, J = 6.0 Hz, ArꢀH ), 7.50 (2H, d, J = 3.0 Hz, ArꢀH), 7.35 (2H, dd, J1 = 6.0 Hz, J2 =
6.0Hz, ArꢀH ), 7.05 (2H, d, J = 18.0 Hz, Hꢀα ). 13C NMR (75 MHz, CDCl3) δ 188.2, 138.3 (× 2),
136.7 (× 2), 136.0 (× 2), 131.5 (× 2), 130.2 (× 2), 128.4 (× 2), 127.7 (× 4). HRMS (ESI): [M+H]+
= 370.9564 (calcd. for C17H11Cl4O 370.9559).
4.2.4 (1E,4E)-1,5-bis(4-bromophenyl)penta-1,4-dien-3-one (2):
1
Yield 82%, mp: 146ꢀ148 °C; H NMR (300 MHz, CDCl3) δ 7.69 (2H, d, J = 15.9 Hz, Hꢀβ),
7.57 (4H, d, J = 8.4 Hz, ArꢀH), 7.49 (4H, d, J = 8.4 Hz, ArꢀH), 7.06 (2H, d, J = 15.9 Hz, Hꢀα). 13
C
NMR (75 MHz, CDCl3) δ 188.3, 142.2 (× 2), 133.6 (× 2), 132.3 (× 4), 129.8 (× 4), 125.8 (× 2),
124.8 (× 2). HRMS (ESI): [M+H]+ = 390.9333 (calcd. for C17H13Br2O 390.9333).
4.2.5 (1E,4E)-1,5-bis(4-chlorophenyl)penta-1,4-dien-3-one (3):
Yield 79%, mp: 192ꢀ194 °C; 1H NMR (300 MHz, CDCl3) δ 7.70 (2H, d, J = 15.9 Hz, Hꢀβ),
7.56 (4H, d, J = 8.4 Hz, ArꢀH), 7.41 (4H, d, J = 8.4 Hz, ArꢀH), 7.05 (2H, d, J = 15.9 Hz, Hꢀα). 13
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NMR (75 MHz, CDCl3) δ 188.4, 142.1 (× 2), 136.5 (× 2), 133.2 (× 2), 129.6 (× 4), 129.3 (× 4),
125.7 (× 2). HRMS (ESI): [M+H]+ = 303.0343 (calcd. for C17H13Cl2O 303.0343).
4.2.6 (1E,4E)-1,5-bis(4-(dimethylamino)phenyl)penta-1,4-dien-3-one (4):
Yield 83%, mp:188ꢀ190 °C; 1H NMR (300 MHz, CDCl3) δ 7.71 (2H, d, J = 15.9 Hz, Hꢀβ),
7.54 (4H, d, J = 8.7 Hz, ArꢀH), 6.91 (2H, d, J = 15.9 Hz, Hꢀα), 6.71 (4H, d, J = 8.7 Hz, ArꢀH),
3.06 (12H, s, NCH3). 13C NMR (75 MHz, CDCl3) δ 188.9, 151.8 (× 2), 143.0 (× 2), 130.1 (× 4),
122.9 (× 2), 121.3 (× 2), 111.9 (× 4), 40.2 (× 4). HRMS (ESI): [M+H]+ = 321.1965 (calcd. for
C21H25N2O 321.1967).
4.2.7 (1E,4E)-1,5-di(furan-2-yl)penta-1,4-dien-3-one (5):
Yield 65%, mp:58ꢀ60 °C; 1H NMR (300 MHz, CDCl3) δ 6.51 (2H, m, ArꢀH), 6.71 (2H, d, J
= 3.6 Hz, ArꢀH), 6.93 (2H, d, J = 15.6 Hz, Hꢀα), 7.50 (2H, overlapped, ArꢀH), 7.50 (2H, d, J =
15.9 Hz, Hꢀβ). 13C NMR (75 MHz, CDCl3) δ 188.2, 151.5 (× 2), 145.0 (× 2), 129.3 (× 2), 123.2
(× 2), 116.0 (× 2), 112.7 (× 2). HRMS (ESI): [M+H]+ = 215.0711 (calcd. for C13H11O5
215.0708).
4.2.8 (1E,4E)-1,5-di(thiophen-2-yl)penta-1,4-dien-3-one (6):
Yield 70%, mp:112ꢀ114 °C; 1H NMR (300 MHz, CDCl3) δ 7.86 (2H, d, J = 15.6 Hz, Hꢀβ),
7.43 (2H ,d, J = 5.1 Hz, ArꢀH ), 7.35 (2H, d, J = 3.6 Hz, ArꢀH ), 7.10 (2H, t, J1 = 4.8 Hz, J2 = 8.7
Hz, ArꢀH), 6.84 (2H, d, J = 15.6 Hz, Hꢀα). 13C NMR (75 MHz, CDCl3) δ 187.7, 140.3 (× 2), 135.6
(× 2), 131.9 (× 2), 128.9 (× 2), 128.4 (× 2), 124.4 (× 2). HRMS (ESI): [M+H]+ = 247.0249 (calcd.
for C13H11OS2 247.0251).
4.2.9 (1E,4E)-1,5-bis(4-fluorophenyl)penta-1,4-dien-3-one (7):
Yield 63%, mp: 150ꢀ152 °C; 1H NMR (300 MHz, CDCl3) δ 7.72 (2H, d, J = 15.9 Hz, Hꢀβ),
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7.63 (4H, dd, J1 = 5.4, J2 = 8.7 Hz, ArꢀH), 7.13 (4H, t, J1 = 8.7 Hz, J2 = 17.4 Hz, ArꢀH ), 7.01 (2H,
1
d, J = 15.9Hz, Hꢀα). 13C NMR (75 MHz, CDCl3) δ 188.5, 165.7ꢀ162.4 (× 2, d, JCF = 250.2 Hz),
142.1 (× 2), 130.9 (× 2, d, JCF = 3.2 Hz), 130.4 (×4, d, JCF = 8.7 Hz), 125.1, 125.0, 116.2 (× 4, d,
JCF = 21.8 Hz). HRMS (ESI): [M+H]+ = 271.0936 (calcd. for C17 H13 F2O 271.0934).
4.2.10 (1E,4E)-1,5-bis(2,6-dichlorophenyl)penta-1,4-dien-3-one (8):
Yield 78%, mp: 144ꢀ146 °C; 1H NMR (300 MHz, CDCl3) δ 7.84 (2H, d, J = 16.5 Hz, Hꢀβ),
7.40 (4H, d, J = 7.8Hz, ArꢀH), 7.23 (2H, d, J = 16.5 Hz, Hꢀα), 7.23 (2H, d, J1 = 12.3 Hz, J2 = 16.2,
ArꢀH). 13C NMR (75 MHz, CDCl3) δ 188.9, 137.4 (× 2), 135.2 (× 2), 133.1 (× 2), 132.3 (× 4),
130.0 (× 2), 128.9 (× 4). HRMS (ESI): [M+H]+ = 370.9559 (calcd. for C17H11Cl4O 370.9564).
4.2.11 (1E,4E)-1,5-bis(3,4-dihydroxyphenyl)penta-1,4-dien-3-one (9):
Yield 60%, mp: 151ꢀ153 °C; 1H NMR (300 MHz, DMSOꢀd6) δ 9.63 (2H, s), 9.15 (2H, s),
7.57 (2H, d, J = 15.9 Hz, Hꢀβ), 7.11 (4H, t, J1 = 15.9 Hz, J2 = 24.0Hz, ArꢀH), 7.00 (2H, d, J = 15.9
Hz, Hꢀα), 6.80 (2H, d, J = 8.1 Hz, ArꢀH). 13C NMR (75 MHz, MeOH) δ 190.2, 148.5 (× 2), 145.4
(× 2), 144.2 (× 2), 126.9 (× 2), 122.2 (× 2), 122.0 (× 2), 115.2 (× 2), 114.2 (× 2). HRMS (ESI):
[M+H]+ = 299.0919 (calcd. for C17H17O5 299.0920).
4.2.12 (1E,4E)-1,5-bis(4-hydroxyphenyl)penta-1,4-dien-3-one (10):
1
Yield 70%, mp: 234ꢀ237 °C; H NMR (300 MHz, DMSOꢀd6) δ 10.03 (2H, s, ꢀOH), 7.66
(2H, d, J = 16.2 Hz, Hꢀβ), 7.63 (4H, d, J = 8.4 Hz, ArꢀH), 7.10 (2H, d, J = 16.2 Hz, Hꢀα), 6.84 (4H,
d, J = 8.4 Hz, ArꢀH). 13C NMR (75 MHz, MeOH) δ 190.3, 160.2 (× 2), 143.8 (× 2), 130.3 (× 4),
126.3 (× 2), 122.0 (× 2), 115.5 (× 4). HRMS (ESI): [MꢀH]ꢀ = 265.0867 (calcd. for C17 H13O3
265.0865).
4.2.13 (1E,4E)-1,5-bis(3-hydroxy-4-methoxyphenyl)penta-1,4-dien-3-one (11):
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1
Yield 78%, mp: 195ꢀ197 °C; H NMR (300 MHz, DMSOꢀd6) δ 9.64 (2H, s, ꢀOH), 7.66
(2H, d, J = 15.9 Hz, Hꢀβ), 7.38 (2H, d, J = 3.0 Hz, ArꢀH), 7.21 (2H, d, J = 8.1 Hz, ArꢀH), 7.15 (2H,
d, J = 15.9 Hz, Hꢀα), 6.84 (2H, d, J = 8.1 Hz, ArꢀH), 3.86 (6H, s, OCH3). 13C NMR (75 MHz,
DMSOꢀd6) δ 188.5, 149.9 (× 2), 148.4 (× 2), 143.2 (× 2), 126.8 (× 2), 123.8 (× 2), 123.5 (× 2),
116.1 (× 2), 111.8 (× 2), 56.2 (× 2). HRMS (ESI): [MꢀH]ꢀ = 325.1075 (calcd. for C19H17O5
325.1076).
4.2.14 (1E,4E)-1,5-bis(benzo[d][1,3]dioxol-5-yl)penta-1,4-dien-3-one (12):
Yield 79%, mp:183ꢀ185 °C; 1H NMR (300 MHz, CDCl3) δ 7.66 (2H, d, J = 15.6 Hz, Hꢀβ),
7.14 (2H, s), 7.11 (2H, d, J = 8.1Hz), 6.90 (2H, d, J = 15.6 Hz, Hꢀα), 6.85 (2H, d, J = 8.1 Hz), 6.04
(4H, s, ꢀOCH2Oꢀ). 13C NMR (75 MHz, CDCl3) δ 188.6, 149.8 (× 2), 148.4 (× 2), 142.9 (× 2),
129.3 (× 2), 125.1 (× 2), 123.8 (× 2), 108.7 (× 2), 106.6 (× 2), 101.6 (× 2). HRMS (ESI): [M+H]+
= 323.0921 (calcd. for C19H15O5 323.0920).
4.2.15 (1E,4E)-1,5-bis(4-methoxyphenyl)penta-1,4-dien-3-one (13):
Yield 82%, mp: 129ꢀ131 °C; 1H NMR (300 MHz, CDCl3) δ 7.72 (2H ,d, J = 16.2 Hz, Hꢀβ),
7.59 (4H, d, J = 8.7 Hz, ArꢀH), 7.00 (2H, d, J = 16.2 Hz, Hꢀα), 6.94 (4H, d, J1 = 5.7 Hz, J2 = 8.7
Hz, ArꢀH), 3.87 (6H, s, OCH3). 13C NMR (75 MHz, CDCl3) 188.9, 161.5 (× 2), 142.7 (× 2), 130.1
(× 4), 127.6 (× 2), 123.5 (× 2), 114.4 (× 4), 55.4 (× 2). HRMS (ESI): [M+H]+ = 295.1323 (calcd.
for C19H19O3 295.1334).
4.2.16 (1E,4E)-1,5-bis(3,4-dimethoxyphenyl)penta-1,4-dien-3-one (14):
1
Yield 83%, mp: 82ꢀ84 °C; H NMR (300 MHz, CDCl3) δ 7.71 (2H, d, J = 15.9Hz, Hꢀβ),
7.22 (2H , dd, J1 = 1.5Hz, J2 = 8.1 Hz, ArꢀH), 7.16 (s, 2H), 6.97 (2H, d, J = 15.9Hz, Hꢀα), 6.91
(2H, d, J = 8.4Hz), 3.96 (6H, s, OCH3), 3.95 (6H, s, OCH3). 13C NMR (75 MHz, CDCl3) δ 188.7,
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151.3 (× 2), 149.2 (× 2), 143.1 (× 2), 127.8 (× 2), 123.6 (× 2), 123.1 (× 2), 111.1 (× 2), 109.8 (× 2),
56.0 (× 2), 55.9 (× 2). HRMS (ESI): [M+H]+ = 355.1543 (calcd. for C21H23O5 355.1546).
4.2.17 (1E,4E)-1,5-bis(3,4-dichlorophenyl)penta-1,4-dien-3-one (15):
1
Yield 70%, mp: 191ꢀ193 °C; H NMR (300 MHz, CDCl3) δ 7.70 (2H, s), 7.62 (2H, d, J =
15.6 Hz, Hꢀβ). 7.48 (4H, m, ArꢀH), 7.02 (2H, d, J = 15.6 Hz, Hꢀα). 13C NMR (75 MHz, CDCl3) δ
187.7, 141.0 (× 2), 134.6 (× 2), 133.4 (× 2), 131.0 (× 2), 129.8 (× 2), 129.2 (× 2), 127.5 (× 2),
126.5 (× 2). HRMS (ESI): [M+H]+ = 370.9563 (calcd. for C17H11Cl4O 370.9564).
4.2.18 (2E,5E)-2,5-bis(furan-2-ylmethylene)cyclopentan-1-one (16):
Yield 65%, mp:166ꢀ168 °C; 1H NMR (300 MHz, CDCl3) δ 7.62 (2H, d, J = 1.8 Hz, ArꢀH),
7.38 (2H, s, ArꢀH), 6.72 (2H, d, J = 3.3 Hz, ArꢀH), 6.57 (2H, m, ArꢀH), 3.10 (4H, s, ꢀCH2CH2ꢀ).
13C NMR (75 MHz, CDCl3) δ 195.4, 152.7 (× 2), 145.1 (× 2), 135.9 (× 2), 119.8 (× 2), 116.0 (× 2),
112.7 (× 2), 25.8 (× 2). HRMS (ESI): [M+H]+ = 241.0859 (calcd. for C15H13O3 241.0865).
4.2.19 2,5-bis((E)-4-methoxybenzylidene)cyclopentan-1-one (17):
Yield 77%, mp: 212ꢀ214 °C; 1H NMR (300 MHz, CDCl3) δ 7.60 (6H, m), 6.99 (4H, d, J = 8.7
Hz, ArꢀH), 3.89 (6H, s, OCH3), 3.10 (4H, s, ꢀCH2CH2ꢀ). 13C NMR (75 MHz, CDCl3) δ 196.3,
160.5 (× 2), 135.3 (× 2), 133.3 (× 2), 132.5 (× 4), 128.8 (× 2), 114.3 (× 4), 55.4 (× 2), 26.5 (× 2).
HRMS (ESI): [M+H]+ = 321.1485 (calcd. for C21H21O3 321.1491).
4.2.20 2,5-bis((E)-4-hydroxybenzylidene)cyclopentan-1-one (18):
Yield 67%, mp: >300 °C;1H NMR (300 MHz, DMSOꢀd6) δ 10.09 (2H, s), 7.56 (4H, d, J =
8.4 Hz), 7.36 (2H, s), 6.90 (4H, d, J = 8.1 Hz), 3.03 (4H, s, ꢀCH2CH2ꢀ). 13C NMR (75 MHz,
MeOD) δ 199.3, 163.6 (× 2), 139.2 (× 2), 137.4 (× 4), 137.1 (× 2), 131.3 (× 2), 120.6 (× 4), 30.6
(× 2). HRMS (ESI): [M+H]+ = 293.1172 (calcd. for C19 H17O3 293.1178).
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4.2.21 2,5-bis((E)-4-(dimethylamino)benzylidene)cyclopentan-1-one (19):
Yield 82%, mp:118ꢀ120 °C; 1H NMR (300 MHz, CDCl3) δ 7.57 (6H, m), 6.77 (4H, d, J =
8.4 Hz), 3.10 (4H, s, ꢀCH2CH2ꢀ), 3.07 (12H, s, OCH3). 13C NMR (75 MHz, CDCl3) δ 196.1, 150.7
(× 2), 133.5 (× 2), 132.6 (× 2), 132.6 (× 4), 124.3 (× 2), 111.9 (× 4), 40.1 (× 2), 26.7 (× 4). HRMS
(ESI): [M+H]+ = 347.2123 (calcd. for C23H27N2O 347.2123).
4.2.22 (2E,5E)-2,5-bis(benzo[d][1,3]dioxol-5-ylmethylene)cyclopentan-1-one (20):
Yield 78%, mp:247ꢀ249 °C; 1H NMR (600 MHz, CDCl3) δ 7.50 (2H, s, ArꢀH), 7.12 (2H, dd,
J1 = 8.4 Hz, J2 = 1.8 Hz, ArꢀH), 7.10 (2H, d, J = 1.8 Hz, ArꢀH), 6.87 (2H, d, J = 8.4 Hz, ArꢀH),
6.02 (4H, s,ꢀOCH2Oꢀ), 3.06 (4H, s, ꢀCH2CH2ꢀ). 13C NMR (75 MHz, CDCl3) δ 196.1, 148.7 (× 2),
135.5 (× 2), 133.6 (× 2), 130.3 (× 2), 126.7 (× 2), 109.7 (× 2), 108.8 (× 2), 101.5 (× 2), 29.7 (× 2),
26.5 (× 2). HRMS (ESI): [M+H]+ = 349.1080 (calcd. for C21H17O5 349.1076).
4.2.23 (2E,5E)-2,5-bis(3,4-dimethoxybenzylidene)cyclopentan-1-one (21):
Yield 80%, mp:155ꢀ157 °C; 1H NMR (300 MHz, CDCl3) δ 7.58 (2H, s), 7.28 (2H, m), 7.17
(2H, s), 6.97 (2H, d, J = 8.4 Hz), 3.97 (12H, s), 3.15 (4H, s, ꢀCH2CH2ꢀ). 13C NMR (75 MHz,
CDCl3) δ 196.1, 150.2 (× 2), 148.9 (× 2), 135.4 (× 2), 133.7 (× 2), 129.0 (× 2), 124.6 (× 2), 113.4
(× 2), 111.1 (× 2), 56.0 (× 2), 55.9 (× 2), 26.5 (× 2). HRMS (ESI): [M+H]+ = 381.1698 (calcd. for
C23H25O5 381.1702).
4.2.24 (2E,5E)-2,5-bis(2,3-dimethoxybenzylidene)cyclopentanone (22):
Yield 77%, mp:140ꢀ142 °C; H NMR(600 MHz, CDCl3): 7.92 (1H × 2, s, ꢀCH=), 7.15 (1H ×
2, dd, J1 = 7.8 Hz, J2 = 1.2 Hz, ArꢀH), 7.08 (1H × 2, m, ArꢀH), 6.94 (1H × 2, dd, J1 = 7.8 Hz, J2 =
1.2 Hz, ArꢀH), 3.88 (6H × 2, s, ꢀOCH3), 3.02 (4H, s, ꢀCH2CH2ꢀ). 13C NMR (75 MHz, CDCl3) δ
196.3, 153.0 (× 2), 149.2 (× 2), 138.6 (× 2), 130.2 (× 2), 128.2 (× 2), 123.8 (× 2), 121.6 (× 2),
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113.4 (× 2), 61.5 (× 2), 55.9 (× 2), 26.8 (× 2). HRMS (ESI): [M+H]+ = 381.1700 (calcd. for
C23H25O5 381.1702).
4.2.25 (2E,5E)-2,5-bis((5-(hydroxymethyl)furan-2-yl)methylene)cyclopentan-1-one (23):
Yield 65%, mp: 151ꢀ153 °C; 1H NMR (300 MHz, DMSOꢀd6) δ 7.18 (2H, s), 6.94 (2H, d, J
= 3.3 Hz), 6.56 (2H, d, J = 3.3Hz), 5.44 (2H, m), 4.50 (4H, d, J = 4.2 Hz), 3.02 (4H, s). 13C NMR
(75 MHz, DMSO) δ 194.4, 159.3 (× 2), 151.6 (× 2), 135.9 (× 2), 119.3 (× 2), 118.1 (× 2), 110.7 (×
2), 56.4 (× 2), 25.9 (× 2). HRMS (ESI): [M+H]+ = 301.1072 (calcd. for C17H17O5 301.1076).
4.2.26 (1E,4Z,6E)-1,7-bis(3,4-dihydroxyphenyl)-5-hydroxyhepta-1,4,6-trien-3-one (24)
A suspension of curcumin (368 mg, 1 mmol) in 20 mL of anhydrous dichloromethane was
cooled to −20 °C under nitrogen atmosphere, and 6.5 mL of 1M boron tribromide solution in
dichloromethan (6.5 equiv) was slowly added this stirred suspension. After 1 h, reaction proceed
at room tepreture for 24 h. The reaction mixture was carefully poured into saturated sodium
bicarbonate solution with stirring. Water and dichloromethane layers were separated and the water
layer was extracted three times with ethyl acetate. Combined the dichloromethane layer and ethyl
acetate layer were dried over anhydrous sodium sulfate for 12 h, after filtration, then removed by
rotary evaporator and the resulting solid was purified by silica gel column chromatography
(CH2Cl2/ MeOH; 40:1 – 5:1%) and Sephadex LHꢀ20.
Yield 55%, mp: > 200 °C ; 1H NMR (300 MHz, DMSOꢀd6) δ 6.08 (1H, s), 6.58 (2H, d, J =
15.6 Hz, Hꢀα), 6.79 (2H, d, J = 8.1 Hz), 7.04 (4H, m), 7.46 (2H, d, J = 15.6 Hz, Hꢀβ), 9.20 ( 2H, s),
9.65 (2H, s).13C NMR (75 MHz, DMSOꢀd6) δ 183.6, 148.8 (× 2), 146.1 (× 2), 141.2 (× 2), 126.7
(× 2), 122.1 (× 2), 121.1 (× 2), 116.3 (× 2), 115.1 (× 2), 101.4 (× 2). HRMS (ESI): [M+H]+ =
341.1023 (calcd. for C19H17O6 341.1025).
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4.4 Biological activity assays
4.4.1 Antifungal activity (broth microdilution method)
The clinical isolates of fluconazole resistant C. albicans 103, C. tropical 087 and C. kruseii
2159 (MIC80 =128.0 ꢀg/ml) were used in this study, and C. albicans ATCC 90028 was used as a
quality control. The antifungal properties of title compounds were evaluated by the broth
microdilution method according to the NCCLS reference document M27ꢀA2 14. Curcumin was
used as positive controls. The MIC80 of FLC against the FLCꢀresistant C. albicans (clinical isolate
103) was determined to be 128.0 ꢀg/ml. The MIC80 of each title compound when used alone and
when combined with FLC (8.0 ꢀg/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—synergistic or indifferent—were
defined according to FICI values of ≤ 0.5 or >0.5, respectively.
4.4.2 Antifungal susceptibility assays (turbidity observation method)
Antifungal susceptibility assays were done according to Zhao et al 24 with little modification.
The initial concentration of fluconazoleꢀresistant C. tropicals 087 suspension was 104 CFU/ml in
RPMI 1640 medium in glass tubes. Different concentrations of compound 9 (0, 4, 8, 16, 32, 64,
128 ꢀg/ml) and fluconazole (0, 0.25, 0.5, 1, 2 ꢀg/ml) alone or in combination were added to the
tubes. Test tubes were incubated at 30 °C, 200 rpm, for 16 h, the turbidity was monitored and
pictures were taken.
4.4.3 Cell growth and cell death measurement (growth curves)
24
Growth curves was conducted as previously described with little modification. Cells were
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incubated in RPMI 1640 medium with increasing concentrations of fluconazole (0.25, 0.5, 1 ꢀg/ml)
alone or in combination with compound 9 (64, 128 ꢀg/ml). Growth condition was measured at
OD630 for an interval of 8 hours. Curves were generated using Prism 5.0 (GraphꢀPad Software,
Inc.)
4.4.4 Measurements of Intracellular ATP Levels
Measurement of intracellular ATP levels were performed as reported before19. C.tropicalis
087 were treated with fluconazole (4 ꢀg/ml), curcumin (32, 64, 128 ꢀg/ml) and compound 9 (32,
64, 128 ꢀg/ml) or the combination for 8 hours and then adjusted to 1×107 CFU/ml in PBS
(phosphate buffer saline). Intracellular ROS of C. tropicalis 087 were observed with confocal
scanning laser microscope (excitation, 485 nm, emission, 538 nm; Leica TCS sp2). For the
measurement of ATP level, a total volume of 100 ꢀl BacTiterꢀGlo reagent (Promega Corparation,
Madison, WI) were mixed completely with the same volume of cell suspension and incubated for
10 minutes at 30 °C. Fluorescence intensities were determined on a TD 20/20 luminometer
(Turner Biosystem, Sunnyvale, CA) and ATP content were calculated based on the ATP
increments standard curve (1 ꢀM to 10 pM). Each experiment was independently repeated for
three times.
4.4.5 Cell membrane permeability assay
Membrane permeability was performed according to a previous research.20 C. tropicals 087
(1×106) were suspended in RPMI 1640 medium and treated with fluconazole (2 ꢀg/ml), compound
9 (32 ꢀg/ml) for 12 hours at 30 °C. After the treatment, cells were incubated in water with 1.49
ꢀM (PI) at 30 °C for 50 minutes. C. tropicals 087 were harvested by centrifugation and
resuspended in PBS (phosphate buffer saline). Flow cytometric analysis was detected by a FACS
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Calibur flow cytometer (BectonꢀDickꢀinson, Franklin Lakes, NJ, USA).
4.4.6 Cell culture and MTT cytotoxicity assay
Human umbilical vein endothelial cells (HUVECs) were cultured in DMEM medium (Gibco,
USA) with 10% FCS (Gibco, CA, USA) and 100 ꢀg/ml streptomycin in 5% CO2 at 37 °C. The
cytotoxicity assay of curcumin and its analogues was based on the reduction of
3ꢀ(4,5ꢀdimethylꢀthiazolꢀ2yl)ꢀ2,5ꢀdiphenyl tetrazolium bromide (MTT, sigma, USA) as previously
described.25 To determine the cytotoxicity, cells were exposed to different concentrations of
compounds (16 and 32 ꢀg/ml) for 24 h. After the treatment, cells were incubated with MTT
solution (5 mg/ml) for 3 h. The optical density of different samples was measured with a
microplate reader (Thermo, MA, USA) at 570 nm. The cytotoxic activities were compared with
drugꢀfree control and expressed as the relative viability percentages.
Acknowledgements
This work was supported by the grant from the Key Program of National Natural Science
Foundation of China (81330083) and Undergraduates Innovation Practice Base of Second Military
Medical University (YJꢀ2015ꢀ08, YJꢀ2015ꢀ07).
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