Fluconazole analogues with metal-binding motifs impact metal-dependent processes and demonstrate antifungal activity in Candida albicans

JBIC Journal of Biological Inorganic Chemistry

https://doi.org/10.1007/s00775-020-01796-x

ORIGINAL PAPER

Fluconazole analogues with metal‑binding motifs impact

metal‑dependent processes and demonstrate antifungal activity

in Candida albicans

Elizabeth W. Hunsaker¹· Katherine J. McAulife¹· Katherine J. Franz¹

Received: 18 February 2020 / Accepted: 25 May 2020

Abstract

Abstract Azole antifungals are an important class of antifungal drugs due to their low cost, ability to be administered orally,

and broad-spectrum activity. However, their widespread and long-term use have given rise to adaptation mechanisms that

render these compounds less efective against common fungal pathogens, including Candida albicans. New antifungals

are desperately needed as drug-resistant strains become more prevalent. We recently showed that copper supplementation

potentiates the activity of the azole antifungal fuconazole against the opportunistic fungal pathogen C. albicans. Here, we

report eight new azole analogues derived from fuconazole in which one triazole group has been replaced with a metal-

binding group, a strategy designed to enhance potentiation of azole antifungal activity by copper. The bioactivity of all eight

compounds was tested and compared to that of fuconazole. Three of the analogues showed activity against C. albicans and

two had lower levels of trailing growth. One compound, Flu-TSCZ, was found to impact the levels, speciation, and bioavail-

ability of cellular metals.

Graphic abstract

Keywords Antifungal · Azole · Copper · Metals in medicine · Candida albicans · Homeostasis

Introduction

Electronic supplementary material The online version of this

article (https://doi.org/10.1007/s00775-020-01796-x) contains

supplementary material, which is available to authorized users.

More than 1.5 million deaths globally in 2017 were attrib-

uted to fungal infections, with another 1 billion individu-

als carrying a fungal infection in that year alone [1]. These

infections are particularly dangerous to individuals with

immune systems compromised from a variety of conditions

ranging from chemotherapy to organ transplants to asthma.

* Katherine J. Franz

katherine.franz@duke.edu

1

Department of Chemistry, French Family Science Center,

Duke University, 124 Science Drive, Durham, NC 27708,

USA

Vol.:(0123456789)

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JBIC Journal of Biological Inorganic Chemistry

The azole antifungal fuconazole has been used in the clinic

for nearly three decades to treat life-threatening fungal infec-

tions, including those caused by Candida albicans and Cryp-

tococcus neoformans. Azoles bind and inhibit cytochrome

P450 lanosterol 14⍺-demethylase (Cyp51) thereby blocking

the biosynthesis of ergosterol, an important cell membrane

component. Unfortunately, widespread and prolonged use of

these drugs has allowed pathogens to develop several modes

of resistance. These mechanisms include increased produc-

tion of Cyp51, changes in the ergosterol biosynthetic path-

way, modifcations to Cyp51 that hinder substrate binding,

and upregulation of efux pumps that reduce the intracellu-

lar concentration of the drug [2–5]. It is becoming apparent

that the use of fuconazole to treat serious infections may not

be a viable long-term option, yet development of entirely

new classes of antifungal compounds is a signifcant chal-

lenge owing to the similarity between eukaryotic pathogens

and their hosts [6]. Repurposing existing drugs is one strat-

egy to extend t he utility of the currently limited antifungal

arsenal [7–10]. Another approach is to create analogues of

antifungal compounds to take advantage of a known fungal

target while hopefully circumventing adaptation mechanisms

associated with the original drug. There have been several

reports of fuconazole analogues in which one of the triazole

side arms is modifed to accommodate features suggested

from molecular docking and structure–activity relationship

studies [11–21].

fungistatic, three analogues were active against C. albicans

at 25 µM or below and two exhibited lower trailing growth

at 48 h compared to fuconazole. A thiosemicarbazone-based

analogue, Flu-TSCZ, was capable of overriding a defect in

Cu transport, distinguishing it from fuconazole and other

analogues in its ability to supply cells with bioavailable Cu.

Overall, we identify Flu-TSCZ as a promising compound for

future development.

Materials and methods

General

Materials and general methods

Chemicals and solvents were obtained from commercial

suppliers and used as received unless otherwise noted. All

reactions were performed under N₂and anhydrous solvent

was used unless otherwise noted. All aqueous solutions

were prepared using Milli-Q water. Stock solutions were

prepared either in DMSO or Milli-Q water. Working stock

solutions for each experiment were prepared by serial dilu-

tion into growth media. Stock solutions of Mg(II) (1.0 M),

Ca(II) (1.0 M), Cu(II) (100 mM), Fe(III) (100 mM), Mn(II)

(100 mM), Zn(II) (100 mM), Ni(II) (100 mM), and Co(II)

(100 mM) were prepared from MgSO₄·7H₂O, CaCl₂·H₂O,

CuSO₄·5H₂O or CuCl₂, FeCl₃·6H₂O, MnCl₂·4H₂O,

ZnSO₄·7H₂O, NiCl₂, and CoCl₂. Working solutions of metal

salts were prepared for each experiment by serial dilution

into growth media from concentrated stock solutions pre-

pared in Milli-Q water.

Other appr oaches to extend the biological activity and

broaden the spectrum of azole drugs include taking advan-

tage of metal coordination; metal complexes of econazole

[22], ketoconazole [23–25], clotrimazole [23, 25–28],

miconazole [25, 28 ], tioconazole [28], and fluconazole

[29] have been r eported. Of particular interest was the

approximate 30% growth reduction reported for a binuclear

Cu(II)–fuconazole complex as compared to the copper-

free drug against several strains of C. albicans [29]. We

have observed Cu-potentiated fuconazole activity with no

evidence the Cu(II)–fuconazole complex forms under the

conditions of our growth assays, leading us to conclude that

complex formation is not required for Cu-potentiated biolog-

ical activity [30]. Indeed, fuconazole binds Cu in aqueous

bufer, but not in the growth medium [30], illustrating that

the presence of metal ions can impact a compound’s biologi-

cal activity even if the complex dissociates. Although com-

plex formation is not required, we reasoned that synthetically

modifying azoles to encourage Cu binding may enhance the

Cu-potentiated activity previously observed.

Compound characterization

Bulk purity was confrmed by NMR and analytical LC–MS

and elemental composition by HRMS. NMR spectra (¹H and

¹³C) were obtained on either a 400 MHz Varian INOVA or

500 MHz Varian spectrometer, as noted. High-resolution

mass spectrometry was performed on an Agilent LCMS-

TOF–DART at Duke University’s Department of Chemistry

Instrumentation Facility.

Yeast strains and culture conditions

Fungal stocks were maintained in 25% glycerol in YPD

at−80 °C. Unless otherwise noted, experiments were per-

formed with C. albicans clinical isolate SC5314, which

was obtained from the American Type Culture Collection

(ATCC). The SN152 strain and isogenic mac1Δ/Δ strain

were obtained from the Fungal Genetics Stock Center [57,

58]. The KC2 strain and isogenic crp1Δ/Δ strain were

generously provided by the Culotta Lab of Johns Hopkins

In the present study, we synthesized eight new analogues

of fuconazole by replacing one of the 1,2,4-triazole rings

with side arms that in principle should facilitate Cu binding.

We hypothesized that such analogues may enhance the Cu-

potentiated activity we have reported for fuconazole [30] or

lead to new Cu-dependent modes of action. Although still

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JBIC Journal of Biological Inorganic Chemistry

University. A second SC5314 strain and isogenic ctr1Δ/Δ

strain were kindly provided by the Brown Lab of the Uni-

versity of Exeter. The C. neoformans H99 and ctr1Δ ctr4Δ

strains were generously provided by the Thiele Lab of Duke

University. Cells were cultured at 30 °C in yeast peptone

dextrose (YPD, Gibco, catalog number A1374501) medium,

unless otherwise indicated. The SC5314 strain from the

Brown lab was used as a control in experiments involving

the ctr1Δ/Δ strain.

48-h timepoint data is reported by plotting OD600 readings

versus treatment conditions.

Metal drop‑out growth assays (defned media)

All Tris-bufered Synthetic Defned (Tris:SD) media for-

mulations were prepared from Chelex-treated Milli-Q water

with individual addition of media components to allow for

rigorous control of metal content. To deplete trace met-

als from water prior to media preparation, Milli-Q water

was treated with Chelex 100 resin 100–200 mesh via batch

method (50 g/L, Bio-Rad Laboratories). A concentrated

stock of Synthetic Defned media omitted for Cu, Fe, Mn,

and Zn (10X SD-) was prepared in the Chelex-treated Milli-

Q water by adding glucose and yeast nitrogen base (YNB)

ingredients at 10× concentrations. YNB components were

added individually to avoid trace metals present in com-

mercial YNB mixtures. To prepare working 1X Tris:SD

medium, 10× SD- was diluted into Chelex-treated water

(1:10), and Ultra-Pure Tris–HCl (VWR) was added to a fnal

concentration of 50 mM. The pH of the media was adjusted

to 7.4 using 1.0 M HCl or 1.0 M NaOH, and this media was

flter-sterilized. Finally, CuSO₄, FeCl₃, MnCl₂, and ZnSO₄

were added, as appropriate, to create individual dropout

medias for each metal. Final metal drop-out formulations

were analyzed for metal content (Tables S4–S7, ESI).

Growth assays (general)

Prior to all experiments, cells were streaked onto YPD agar

plates from frozen glycerol stocks and incubated at 30 °C

for 24 h. A single colony was used to inoculate 10 mL of

YPD medium, which was then incubated overnight (∼18 h)

at 30 °C, 200 rpm. This overnight culture was diluted to

an optical density at 600 nm (OD600) of 0.002 with fresh

YPD medium and used as the working culture. Azole com-

pounds to be tested were serially diluted twofold in YPD

medium from DMSO stocks to fnal concentrations ranging

from 0 to 100 μM, with<1% DMSO and plated in a clear,

fat-bottomed 96-well plate. For experiments in which sup-

plemental Cu was used, fresh working solutions of Cu(II)

were prepared by diluting 100 mM aqueous stocks of CuSO₄

into YPD medium, and aliquots of these working solutions

were added to appropriate wells at fnal concentrations indi-

cated in fgure legends. BCS working solutions were simi-

larly prepared. Azole working solutions were serially diluted

twofold to achieve fnal test concentrations indicated in fg-

ure legends. The working culture of C. albicans was then

aliquoted to the 96-well plate to a fnal OD600 of 0.001 and

a fnal volume of 200 μL per well. For each experiment, a

compound-free positive growth control and a cell-free, nega-

tive control were included. Plates were incubated for 48 h

at 30 °C, 200 rpm. Plates were covered with air-permeable

AeraSeal flm (Sigma) to minimize evaporation.

ctr1Δ/Δ and ctr1Δ ctr4Δ growth recovery assay

Overnight cultures of WT SC5314 and isogenic ctr1Δ/Δ

(C. albicans) or WT H99 and isogenic ctr1Δ ctr4Δ (C. neo-

formans) in YPD were washed twice with PBS, pH 7.4 and

resuspended in 10 mL YPEG medium with 2% EtOH and 3%

glycerol. Cell suspensions were diluted to OD600 of 0.002

with fresh YPEG medium. WT was aliquoted to the top half

of a 96-well plate and Cu importer deletion strain to the bot-

tom half. Test compounds were added from DMSO stock

solutions to fnal concentrations ranging from 0 to 100 μM.

For each test run, a compound-free positive growth control

and a cell-free, negative control were included. Plates were

incubated at 30 °C and OD600 recorded at 0, 24, and 48 h.

All tests were performed in triplicate for each condition in a

single experiment, and two separate experiments were car-

ried out. For a single experiment, each of the three replicate

conditions were averaged and the error was calculated as

standard deviation (SD).

Fungal growth was evaluated by measuring OD600 using

a PerkinElmer Victor3 V multilabel plate reader at 0, 24,

and 48 h. For some experiments, additional timepoint data

were collected. OD600 values were adjusted by subtract-

ing the 0 h timepoint readings from other timepoint data

to remove background signal from growth media. In cases

where the OD was outside of the linear range (OD600 values

approximately 0.0–0.8), cell suspensions were diluted four-

fold in fresh media and rescanned. To calculate actual OD

values, the 0 h timepoint reading was subtracted from the

diluted readings, and this value was multiplied by four. At

least two biological replicates were performed with a mini-

mum of three technical replicates per experiment. For a sin-

gle experiment, each of the three replicate conditions were

averaged and the error was calculated as standard deviation

(SD), which is indicated by error bars in all fgures. Final

Determination of CFUs

Fungicidal assays were initiated in 96-well plates by follow-

ing the procedure described above for the growth assay for

WT C. albicans in YPD medium. Aliquots of cell suspen-

sions were removed after 48 h, diluted and plated on solid

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JBIC Journal of Biological Inorganic Chemistry

YPD medium. The number of colonies on each plate for

each condition were counted after incubating the plates at

30 °C for 24 h. All tests were performed in duplicate for each

condition in a single experiment, and two separate experi-

ments were carried out.

After digestion, samples were diluted with 1.5 mL of 1%

nitric acid.

ICP‑MS measurements

ICP-MS measurements were performed by Dr. Martina Ralle

in the OHSU Elemental Analysis Core with partial support

from the NIH instrumentation grant S10RR025512. Meas-

urements were performed using an Agilent 7700×equipped

with an ASX 500 autosampler. The system was operated at a

radio frequency power of 1550 W, an argon plasma gas fow

rate of 15 L/min, and Ar carrier gas fow rate of 0.9 L/min.

Elements were measured in kinetic energy discrimination

(KED) mode using He gas (4.3 mL/min). Data were quanti-

fed using an 11-point [0, 0.5, 1, 2, 5, 10, 20, 50, 100, 500,

1000 ppb (µg/kg)] for all elements except Mg and Ca [0,

0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 50, 100 ppm (µg /kg)] using

a multi-element standard [CEM 2, (VHG-SM70B-100)]

and single element standards for Mg (Inorganic Ventures,

CGMG-1) and Ca (Inorganic Ventures, CGCA-1). For each

sample, data were acquired in triplicates and averaged. A

coefcient of variance (CoV) was determined from frequent

measurements of a sample containing approximately 10 ppb

of all elements except Ca (1 ppm). An internal standard (Sc,

Ge, Bi) continuously introduced with the sample was used

to correct for detector fuctuations and to monitor plasma

stability. Accuracy of the calibration curve was assessed

by measuring NIST reference material (water, SRM 1643f)

twice during the measurement and found to within 4% for

all determined elements.

UV–Vis and EPR spectroscopy measurements

UV–Visible absorption spectra were collected using a Varian

Cary 50 UV–Visible spectrophotometer in quartz cuvettes

with 1 cm pathlengths. UV–Vis studies probing Cu(I) bind-

ing were conducted in air using CuCl₂with excess ascor-

bate added as a reducing agent. X-band continuous wave

(CW) EPR spectroscopy was conducted on a Bruker ESP

300 spectrometer equipped with an Oxford Instruments ESR

910 continuous helium fow cryostat. Typical experimen-

tal parameters were at 77 K, 9.37 GHz, 20 mW microwave

power, and 5 G modulation amplitude. Spectra were baseline

corrected using Bruker Xenon data processing software.

Preparation of cells for EPR spectroscopy

Overnight cultures of C. albicans in YPD medium were

inoculated into fresh YPD medium at an OD600 of 0.3 and

allowed to grow for 3 h at 30 °C with shaking at 200 rpm.

Cultures were then either left untreated or treated with

CuSO₄(10 µM), Flu-TSCZ (10, 25, or 100 µM), or both

CuSO₄and Flu-TSCZ. After 6 h of treatment, 2×10⁹cells

were harvested and washed with cold 5 mM disodium

EDTA in PBS (1 mL) followed by cold 20 mM Tris–HCl,

pH 7.4 (1 mL). The cell pellet was resuspended in 300 µL

of 20 mM Tris–HCl, pH 7.4 containing 20% glycerol. The

entire suspension was transferred to an EPR tube and cells

were fash-frozen immediately and stored at −80 °C until

EPR measurements were performed.

To obtain cellular metal concentrations (µM/cell), the

analytical level of phosphorus (P) determined for each sam-

ple by ICP-MS was converted to cell count using a calcu-

lated constant for the mol P per cell. The constant, deter-

mined to be 1.3 0.4×10^–14mol P/cell, was calculated by

dividing the analytical mol P for each sample by the theoreti-

cal number of cells for each sample (1×10⁸), then averaging

these values. This constant represents the average P content

per cell and corrects for the variation in the number of cells

submitted for analysis. For µM/cell, a cell volume of 95 fL

was used [59].

Preparation of cells for ICP‑MS

Overnight cultures of C. albicans in YPD medium were

inoculated into fresh YPD medium at an OD600 of 0.3 and

allowed to grow for 3 h at 30 °C with shaking at 200 rpm.

Cultures were then either left untreated or treated with

CuSO₄(10 µM), Flu-TSCZ (10, 25, or 100 µM), or both

CuSO₄and Flu-TSCZ. At the timepoints indicated in fgure

legends, 1×10⁸cells were harvested and placed in 15-mL

metal-free centrifuge tubes (VWR, cat. no. 89049–170).

Cell suspensions were pelleted at 4000×g and washed with

1 mL of 1 mM disodium EDTA prepared in ultrapure water

for trace metal analysis (VWR, Cat. No. 87003-236), then

with 1 mL of ultrapure water for trace metal analysis. Pel-

lets were resuspended in 100 µL of concentrated nitric acid

(Trace Metal Grade, Fisher) and heated at 90 °C for 1 h.

Synthetic procedures

2‑(2,4‑difuorophenyl)‑1‑(1H‑1,2,4‑triazol‑1‑yl)

pent‑4‑yn‑2‑ol (Flu‑alkyne, 2) [14]

To a suspension of 1 (0.1986 g, 0.890 mmol) in DMF/THF

(1:1, 2 mL), propargyl bromide (80 wt% in toluene, 0.30 mL,

3.37 mmol) was added. To this well-stirred mixture, acti-

vated zinc powder (0.1793 g, 2.74 mmol) was added. Prior

to use, Zn powder was stirred in 2% HCl, washed with

H₂O, and vacuum dried. The exothermic reaction brought

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JBIC Journal of Biological Inorganic Chemistry

itself to refux and was allowed to attain room temperature.

The reaction mixture was then stirred under N₂for 6.5 h at

room temperature. HCl (5%) was then added to the reaction

fask, and the product was extracted with DCM (3×40 mL),

washed with H₂O (2×20 mL) and brine (2×20 mL), dried

over Na₂SO₄, and fltered. The solvent was removed by

evaporation under reduced pressure, yielding a light orange

sticky solid. In most cases, the product was used without

further purifcation. The product can be recrystallized in

MeOH to give a white solid as pure product, but with highly

diminished yield. Yield (crude): 0.205 g (87%). ¹H NMR

(400 MHz, Chloroform-d) δ 7.97 (s, 1H), 7.84 (s, 1H), 7.53

(td, J=9.0, 6.4 Hz, 1H), 6.85–6.75 (m, 2H), 4.82–4.66 (m,

2H), 4.50 (s, 1H), 2.85 (qd, J =17.0, 2.7 Hz, 2H), 2.07 (t,

J=2.7 Hz, 1H). MS: m/z 264 (M+1).

Chloroform-d) δ 163.03 (dd, J = 252.5, 13.9 Hz), 158.61

(dd, J=245.9, 11.7 Hz), 151.62, 144.30, 130.19 (dd, J=9.5,

5.5 Hz), 122.78 (dd, J=13.0, 3.9 Hz), 111.98 (dd, J=21.1,

3.4 Hz), 104.32 (t, J=26.6 Hz), 76.26 (d, J=5.1 Hz), 57.01

(d, J=4.6 Hz), 54.74 (d, J=5.9 Hz). MS: m/z 281 (M+1).

1‑amino‑2‑(2,4‑difuorophenyl)‑3‑(1H‑1,2,4‑triazol‑1‑yl)

propan‑2‑ol (Flu‑amine, 5)

To a nitrogen-fushed solution of 4 (1.42 g, 5.07 mmol) in

10:1 MeOH/CHCl₃(17 mL), Pd/C (0.4468 g) was added.

The reaction fask was ftted with a balloon to allow for

gas expansion. To this suspension, triethylsilane (6.75 mL,

42.3 mmol) was slowly added dropwise, resulting in an exo-

thermic reaction. Care must be taken not to add triethylsilane

too quickly. The reaction mixture was left to stir at room

temperature for 3 h under N₂, at which point the reaction

mixture was fltered through Celite to remove Pd/C. The

product was extracted into H₂O (3 × 50 mL), the solvent

was removed by evaporation under reduced pressure, and

the product was recrystallized in CHCl₃, yielding a white

crystalline solid. Yield: 0.535 g (50%). ¹H NMR (400 MHz,

Methanol-d4) δ 8.41 (s, 1H), 7.90 (s, 1H), 7.56 (td, J=9.1,

6.5 Hz, 1H), 7.12–6.94 (m, 2H), 4.77 (s, 2H), 3.49 (dd,

J=98.9, 13.3 Hz, 2H). MS: m/z 255 (M+1).

1‑((2‑(2,4‑difuorophenyl)oxiran‑2‑yl)methyl)‑1H‑1,2,4‑tria‑

zole (Flu‑epoxide, 3) [60]

To a solution of 1 (0.5710 g, 2.56 mmol) in toluene (6 mL),

trimethylsulfoxonium iodide (0.6851 g, 3.11 mmol) was

added, followed by sodium hydroxide (20%, 0.6 mL). Tri-

methylsulfoxonium iodide is light-sensitive and was han-

dled in the dark. The reaction mixture was heated to 60 °C

and stirred under N₂in the dark for 4.5 h. The product

was extracted into toluene (3×50 mL), and the combined

organic layers were washed with H₂O (2×25 mL) and brine

(2 × 25 mL), dried over Na₂SO₄, and fltered. The solvent

was removed by evaporation under reduced pressure, yield-

4‑(chloromethyl)‑1H‑imidazole (10) [61]

1

ing a sticky orange solid. Yield: 0.387 g (64%). H NMR

To a suspension of 4(5)-hydroxymethyl imidazole (0.5934 g,

6.04 mmol) in ACN (7 mL), a catalytic amount of DMF

(0.3 mL) was added, followed by thionyl chloride (0.75 mL,

10.28 mmol). The exothermic reaction brought itself to

refux and was allowed to return to room temperature. After

2.5 h, the solvent and remaining thionyl chloride were

removed by evaporation under reduced pressure, yielding

a brown solid. Yield: 0.617 g (88%). ¹H NMR (400 MHz,

Acetonitrile-d3) δ 13.76 (s, 1H), 8.56 (s, 1H), 7.45 (s, 1H),

4.78 (s, 2H). MS: m/z 117 (M+1).

(400 MHz, Chloroform-d) δ 7.99 (s, 1H), 7.76 (s, 1H), 7.08

(td, J = 8.4, 6.2 Hz, 1H), 6.72 (dddd, J = 12.6, 10.4, 8.2,

2.5 Hz, 2H), 4.58 (dd, J = 127.3, 14.9 Hz, 2H), 2.82 (dd,

J=28.0, 4.7 Hz, 2H). MS: m/z 238 (M+1).

1‑azido‑2‑(2,4‑difuorophenyl)‑3‑(1H‑1,2,4‑triazol‑1‑yl)

propan‑2‑ol (Flu‑azide, 4) [12]

To a solution of 3 (0.6873 g, 2.90 mmol) in MeOH (15 mL),

ammonium chloride (0.1906 g, 3.56 mmol) was added fol-

lowed by sodium azide (0.5685 g, 8.74 mmol). The reaction

mixture was heated to refux under N₂. After 6 h, the mixture

was diluted with 5% NaHCO₃. The product was extracted

into EtOAc (3× 50 mL), and the combined organic layers

were washed with H₂O (1×25 mL) and brine (1×25 mL),

dried over Na₂SO₄, and fltered. The compound was puri-

fed by column chromatography on silica gel using EtOAc/

BuOH/AcOH/H₂O (80:10:5:5) as eluent. The solvent was

removed by evaporation under reduced pressure, yielding a

light-yellow oil. Yield: 0.542 g (67%). ¹H NMR (500 MHz,

Chloroform-d) δ 7.94 (s, 1H), 7.76 (s, 1H), 7.49 (td, J=9.0,

6.4 Hz, 1H), 6.82 – 6.71 (m, 2H), 5.80 (s, 1H), 4.74–4.61 (m,

2H), 3.59 (dd, J=86.2, 12.9 Hz, 2H); ¹³C NMR (125 MHz,

4‑(azidomethyl)‑1H‑imidazole (6a) [61]

To a solution of 10 (0.3648 g, 3.13 mmol) in DMF (11 mL),

sodium azide (0.4070 g, 6.26 mmol) was added. After stir-

ring at room temperature for 19 h, the reaction mixture was

diluted with H₂O, and the product was extracted into EtOAc

(3×25 mL). The combined organic layers were washed with

H₂O (2×25 mL), dried over Na₂SO₄, fltered, and concen-

trated under reduced pressure, yielding a brown crystalline

solid. Yield: 0.153 g (40%). ¹H NMR (400 MHz, Acetoni-

trile-d3) δ 11.74 (s, 1H), 7.69 (s, 1H), 7.14 (s, 1H), 4.29 (s,

2H). MS: m/z 124 (M+1).

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JBIC Journal of Biological Inorganic Chemistry

2‑azidophenol (6b) [62]

which time a white precipitate formed. The precipitate was

fltered, washed with cold H₂O, and recrystallized from

DCM to give a white crystalline solid. Yield: 4.155 g (29%).

¹H NMR (400 MHz, Chloroform-d) δ 8.36 (s, 1H), 4.69 (s,

2H), 2.66 (s, 3H).

2-aminophenol (0.7642 g, 7.00 mmol) was dissolved in

HCl (2.0 M, 10 mL) at -4 °C to give a light pink solution.

A separate solution was prepared by dissolving sodium

nitrite (0.5878 g, 8.52 mmol) in H₂O (2 mL) at 0 °C, and

this solution was added dropwise to the fask containing

2-aminophenol over a period of fve minutes. After an addi-

tional 5 minutes, urea (0.0522 g, 0.869 mmol) was added

to consume excess nitrous acid. In a separate fask, sodium

azide (0.9136 g, 14.05 mmol) was added to a solution of

sodium acetate (0.0028 g, 0.034 mmol) in H₂O (10 mL) at

−3 °C. To this solution, the previously prepared diazonium

salt solution was added dropwise, and the reaction mixture

was stirred at −3 °C for 2 h. The product was extracted into

diethyl ether (3×25 mL), and the combined organic layers

were washed with H₂O (1×25 mL), dried over Na₂SO₄, and

fltered. Solvent was removed by evaporation under reduced

(E)‑4‑((2‑(methylcarbamothioyl)hydrazineylidene)methyl)

benzoic acid (8a) [65]

A solution of 4-formylbenzoic acid (0.4590 g, 3.06 mmol)

and 11 (0.3566 g, 3.39 mmol) was refuxed in EtOH (6 mL)

for 1.5 h, during which time a solid precipitated. The pre-

cipitate was isolated via vacuum fltration, and triturated

with EtOH (3×10 mL), yielding a white crystalline solid.

1

Yield: 0.572 g (79%). H NMR (400 MHz, DMSO-d6) δ

13.07 (s, 1H), 11.64 (s, 1H), 8.65–8.60 (m, 1H), 8.09 (s,

1H), 7.98–7.89 (m, 4H), 3.02 (d, J=4.5 Hz, 3H). MS: m/z

238 (M+1).

1

pressure, giving a dark red oil. Yield: 0.934 g (99%). H

NMR (400 MHz, DMSO- d6) δ 10.05 (s, 1H), 7.03–6.73

(m, 4H). MS: m/z 134 (M—1).

1‑(1‑((1H‑imidazol‑4‑yl)methyl)‑1H‑1,2,3‑triazol‑4‑yl)‑2‑(

2,4‑difuorophenyl)‑3‑(1H‑1,2,4‑triazol‑1‑yl)propan‑2‑ol

(Flu‑Im, I‑a)

N,N‑bis(pyridin‑2‑ylmethyl)prop‑2‑yn‑1‑amine

(DPA‑alkyne, 7a) [63]

To a mixture of 2 (0.6703 g, 2.55 mmol) and 6a (0.2980 g,

2.42 mmol) in tert-butanol and H₂O (1:1, 35 mL),

(+)-sodium l-ascorbate (7.203 g, 36.4 mmol) was added,

followed by copper (II) sulfate pentahydrate (0.9095 g,

3.64 mmol). The reaction mixture was stirred at room

temperature for 20 h before being diluted with a solution

of ethanolamine and water (25 mL/100 mL). The product

was extracted into EtOAc (5 × 25 mL), and the combined

organic layers were washed with H₂O (1 × 25 mL), dried

over Na₂SO₄, and fltered. The solvent was removed by

evaporation under reduced pressure, yielding an of-white

To a solution of bis(2-picolyl)amine (1.000 g, 5.02 mmol)

in toluene (50 mL), propargyl bromide (80 wt% in tolu-

ene, 0.70 mL, 7.86 mmol) and triethylamine (0.75 mL,

5.38 mmol) were added. The reaction mixture was refuxed

for 23 h, during which time a brown solid precipitated. The

solid was removed via vacuum fltration, and the fltrate

was concentrated under reduced pressure. The compound

was purifed by column chromatography on silica gel using

DCM/ACN/NH₄OH (5:5:0.1) as eluent. The solvent was

removed by evaporation under reduced pressure, yielding a

dark brown oil. Yield: 0.766 g (64%). ¹H NMR (400 MHz,

Chloroform-d) δ 8.27 (d, J = 4.9 Hz, 2H), 7.40–7.31 (m,

2H), 7.24 (d, J=7.8 Hz, 2H), 6.90–6.81 (m, 2H), 3.65 (s,

4H), 3.17–3.12 (m, 2H), 2.10 (q, J=2.0 Hz, 1H). MS: m/z

238 (M+1).

1

solid. Yield: 0.083 g (9%). H NMR (400 MHz, DMSO-

d6) δ 12.06 (s, 1H), 8.28 (s, 1H), 7.75 (s, 1H), 7.61 (s, 1H),

7.50 (s, 1H), 7.22–7.09 (m, 2H), 7.05 (s, 1H), 6.82 (td,

J=8.5, 2.6 Hz, 1H), 5.97 (s, 1H), 5.30 (s, 2H), 4.72–4.46

(m, 2H), 3.38–3.29 (d, 1H), 3.11 (d, J=14.8 Hz, 1H). ¹³

C

NMR (125 MHz, DMSO-d6) δ 160.22 (ddd, J = 354.2,

246.3, 12.4 Hz), 150.56, 144.93, 141.53, 129.92 (dd,

J = 9.6, 6.1 Hz), 123.26, 110.59 (dd, J = 20.5, 3.1 Hz),

103.84 (t, J = 26.8 Hz), 73.70 (d, J = 5.0 Hz), 56.88 (d,

J=4.6 Hz), 47.02, 34.56 (d, J=4.5 Hz). ESI-HRMS calcd.

for C₁₇H₁₆F₂N₈O [M+H]⁺=387.1488, found 387.1489.

Methyl hydrazinecarbodithioate (11) [64]

To an ice-cold solution of potassium hydroxide (6.554 g,

117 mmol) in H₂O (12.5 mL), 2-propanol (10 mL) was

added, and this solution was maintained at 0 °C. Next,

hydrazine hydrate (80% solution, 7.3 mL, 117 mmol) was

added with stirring. Ice-cold carbon disulfide (7.0 mL,

117 mmol) was added dropwise to this solution over 1.5 h

while maintaining 0 °C. The bright yellow mixture obtained

was further stirred for 2.5 h, and then, ice-cold iodomethane

(7.4 mL, 117 mmol) was added drop wise over a period of

2 h. Stirring was continued for an additional 1.5 h during

2‑(4‑(2‑(2,4‑difuorophenyl)‑2‑hydroxy‑3‑(1H‑1,2,4‑tria‑

zol‑1‑yl)propyl)‑1H‑1,2,3‑triazol‑1‑yl)phenol (Flu‑Phenol,

I‑b)

To a solution of 2 (0.3127 g, 1.19 mmol) and 6b (0.2200 g,

1.63 mmol) in a mixture of tert-butanol and H₂O (1:1,

8 mL), (+)-sodium L-ascorbate (0.0622 g, 0.314 mmol) was

1 3

JBIC Journal of Biological Inorganic Chemistry

added, followed by copper (II) acetate monohydrate (0.0261,

0.131 mmol). The reaction mixture was allowed to stir for

5 days at room temperature. The product was extracted into

EtOAc (3×25 mL), and the combined organic layers were

washed with H₂O (1×25 mL) and brine (1×25 mL), dried

over Na₂SO₄, and fltered. The compound was purifed by

column chromatography on silica gel using 5% MeOH in

DCM as eluent. The solvent was removed by evaporation

under reduced pressure, yielding an off-white powder.

51.27, 30.96, 24.85. ESI-HRMS calcd. for C₁₉H₂₀F₂N₆O₅

[M+H]⁺=451.1536, found 451.1539.

2‑(2,4‑difuorophenyl)‑1‑(4‑(pyridin‑2‑yl)‑1H‑1,2,3‑tria‑

zol‑1‑yl)‑3‑(1H‑1,2,4‑triazol‑1‑yl)propan‑2‑ol (Flu‑Pyr, II‑b)

To a solution of 4 (0.2850 g, 1.02 mmol) and 2-ethynylpyr-

idine (0.1470 g, 1.43 mmol) in a mixture of tert-butanol

and H₂O (1:1, 8 mL), (+)-sodium l-ascorbate (0.0433 g,

0.219 mmol) was added, followed by copper (II) sulfate pen-

tahydrate (0.0272, 0.109 mmol). The reaction mixture was

allowed to stir for 21 h at room temperature. The product

was extracted into EtOAc (3 × 25 mL), and the combined

organic layers were washed with H₂O (1×25 mL) and brine

(1×25 mL), dried over Na₂SO₄, and fltered. The compound

was purifed by column chromatography on silica gel using

5% MeOH in DCM as eluent. The solvent was removed by

evaporation under reduced pressure, yielding a light pink

crystalline solid. Yield: 0.178 g (46%). ¹H NMR (500 MHz,

Chloroform-d) δ 8.50 (ddd, J=4.9, 1.8, 0.9 Hz, 1H), 8.25

(s, 1H), 8.05 (dt, J=7.9, 1.1 Hz, 1H), 7.99 (s, 1H), 7.77 (s,

1H), 7.72 (td, J=7.8, 1.8 Hz, 1H), 7.42 (td, J=9.1, 6.3 Hz,

1H), 7.19 (ddd, J=7.6, 4.9, 1.2 Hz, 1H), 6.79 (ddd, J=12.0,

8.3, 2.5 Hz, 1H), 6.72 (tdd, J=8.7, 2.5, 0.8 Hz, 1H), 5.68 (s,

1H), 4.91–4.77 (m, 3H), 4.37 (d, J=14.3 Hz, 1H). ¹³C NMR

(125 MHz, Acetonitrile-d3) δ 165.78–158.69 (m), 152.30,

151.01, 150.53, 148.48, 146.06, 137.95, 130.91, 125.21,

123.88, 123.59, 120.56, 112.07, 105.00, 75.87, 56.78, 55.87.

ESI-HRMS calcd. for C₁₈H₁₅F₂N₇O [M+H]⁺=384.1379,

found 384.1385.

1

Yield: 0.305 g (65%). H NMR (500 MHz, DMSO-d6) δ

10.49 (s, 1H), 8.31 (d, J=1.1 Hz, 1H), 8.03 (s, 1H), 7.78 (d,

J=1.1 Hz, 1H), 7.55 (dt, J=8.0, 1.5 Hz, 1H), 7.31–7.21 (m,

2H), 7.18 (ddd, J=12.0, 9.1, 2.3 Hz, 1H), 7.06 (dt, J=8.3,

1.3 Hz, 1H), 6.97–6.91 (m, 1H), 6.86 (td, J = 8.5, 2.7 Hz,

1H), 6.05 (s, 1H), 4.75–4.55 (m, 2H), 3.46 (d, J=14.9 Hz,

1H), 3.25 (d, J = 14.8 Hz, 1H). ¹³C NMR (125 MHz,

DMSO-d6) δ 160.32 (ddd, J = 351.8, 246.6, 12.7 Hz),

150.61, 149.02, 144.98, 141.44, 130.05 (t, J = 8.0 Hz),

129.71, 125.84–125.66 (m), 124.93, 124.55, 124.44, 119.60,

117.17, 110.72 (d, J = 20.3 Hz), 103.93 (t, J = 27.0 Hz),

73.79 (d, J = 5.0 Hz), 56.97, 34.62. ESI-HRMS calcd. for

C₁₉H₁₆F₂N₆O₂[M+H]⁺=399.1376, found 399.1378.

Dimethyl 2‑((1‑(2‑(2,4‑difuorophenyl)‑2‑hydroxy‑3‑(1H

‑1,2,4‑triazol‑1‑yl)propyl)‑1H‑1,2,3‑triazol‑4‑yl)methyl)

malonate (Flu‑DME, II‑a)

To a solution of 4 (0.305 g, 1.09 mmol) and dimethyl pro-

pargylmalonate (0.2 mL, 1.31 mmol) in tert-butanol and

H₂O (1:1, 20 mL), (+)-sodium l-ascorbate (0.0488 g,

0.246 mmol) was added, followed by copper (II) acetate

monohydrate (0.0311 g, 0.156 mmol). The reaction mixture

was stirred at room temperature for 21 h. The product was

extracted into EtOAc (3×25 mL), and the combined organic

layers were washed with a saturated solution of disodium

EDTA (1×25 mL) and H₂O (1×25 mL), dried over Na₂SO₄,

and fltered. The solvent was removed by evaporation under

reduced pressure, yielding a yellow oil, which was purifed

by column chromatography on silica gel using 5% MeOH in

DCM with 1% triethylamine as eluent. Fractions containing

product were combined and further purifed via HPLC on a

C18 column using a linear gradient running from 95%–5%

H₂O/ACN over 40 min, giving a white shiny solid. Yield:

0.130 g (27%). ¹H NMR (400 MHz, Chloroform-d) δ 7.98

(s, 1H), 7.79 (s, 1H), 7.45 (s, 1H), 7.37 (td, J=8.6, 6.0 Hz,

1H), 6.82–6.71 (m, 2H), 5.43 (s, 1H), 4.83 (d, J=14.3 Hz,

1H), 4.70 (q, J = 14.3 Hz, 2H), 4.25 (d, J = 14.3 Hz, 1H),

3.79 (td, J = 7.5, 1.6 Hz, 1H), 3.76–3.62 (m, 6H), 3.24

(d, J = 7.5 Hz, 2H). ¹³C NMR (125 MHz, Chloroform-d)

δ 168.96, 165.39–1154.29 (m), 151.95, 144.63, 143.83,

130.10, 124.22, 121.95, 112.08 (d, J = 20.8 Hz), 104.39

(t, J=26.9 Hz), 75.27 (d, J=4.9 Hz), 55.87, 54.63, 52.72,

1‑(4‑((bis(pyridin‑2‑ylmethyl)amino)methyl)‑1H‑1,2,3‑tri‑

azol‑1‑yl)‑2‑(2,4‑difuorophenyl)‑3‑(1H‑1,2,4‑triazol‑1‑yl)

propan‑2‑ol (Flu‑DPA, II‑c)

To a solution of 4 (0.286 g, 1.02 mmol) and 7a (0.300,

1.26 mmol) in MeOH (7 mL), (+)-sodium l-ascorbate

(0.0422 g, 0.213 mmol) was added, followed by copper (II)

acetate monohydrate (0.0225 g, 0.113 mmol). The reaction

mixture stirred at room temperature for 24 h. The prod-

uct was extracted into EtOAc (5 × 20 mL), and the com-

bined organic layers were washed with disodium EDTA

(3×30 mL), then H₂O (1×20 mL) and brine (1×20 mL),

dried over Na₂SO₄, and fltered. The solvent was removed by

evaporation under reduced pressure to aford a metallic dark

brown oil as crude product. Crude product was purifed via

HPLC on a C18 column using a linear gradient running from

95%–5% H₂O/ACN over 40 min, giving a light brown oil.

Yield: 0.098 g (19%). ¹H NMR (400 MHz, Chloroform-d) δ

8.53 (dd, J=4.9, 1.8 Hz, 2H), 8.02 (s, 1H), 7.83 (s, 1H), 7.67

(s, 1H), 7.64 (dd, J=7.7, 1.9 Hz, 1H), 7.50 (d, J=7.8 Hz,

2H), 7.38 (td, J=9.0, 6.3 Hz, 1H), 7.18–7.12 (m, 2H), 6.71

(dtd, J = 16.3, 8.3, 4.2 Hz, 2H), 5.40 (s, 1H), 4.87–4.30

1 3

JBIC Journal of Biological Inorganic Chemistry

(m, 4H), 3.80 (s, 2H), 3.72 (s, 4H). ¹³C NMR (125 MHz,

Chloroform-d) δ 164.77–156.89 (m), 159.15, 152.11,

149.23, 144.64 (d, J=23.0 Hz), 136.63, 130.20 (dd, J=9.6,

5.4 Hz), 125.35, 123.34, 122.23, 112.20 (dd, J = 20.9,

3.3 Hz), 105.18–103.90 (m), 75.52 (d, J= 4.7 Hz), 59.52,

55.98 (d, J = 4.8 Hz), 54.77 (d, J = 5.7 Hz), 48.49, 41.12.

ESI-HRMS calcd. for C₂₆H₂₅F₂N₉O [M+H]⁺=518.2223,

found 518.2224.

washed with 5% LiCl (3×150 mL), NaHCO₃(1×200 mL),

and H₂O (2×200 mL), dried over Na₂SO₄, and fltered. Sol-

vent was removed by evaporation under reduced pressure,

and crude product was purifed by column chromatography

on silica gel using 100% EtOAc as eluent to give the pure

compound as a light-yellow powder. Yield: 0.424 g (72%).

¹H NMR (400 MHz, DMSO-d6) δ 11.61 (s, 1H), 8.59 (t,

J = 6.1 Hz, 2H), 8.34 (s, 1H), 8.06 (s, 1H), 7.90–7.74 (m,

5H), 7.41 (td, J=9.0, 6.7 Hz, 1H), 7.18 (ddd, J=11.9, 9.1,

2.6 Hz, 1H), 6.93 (td, J = 8.5, 2.6 Hz, 1H), 6.27 (s, 1H,

OH), 4.78–4.51 (m, 2H), 3.79 (d, J=6.0 Hz, 2H), 3.02 (d,

J = 4.5 Hz, 3H, CH₃). ¹³C NMR (125 MHz, DMSO-d6) δ

177.83, 167.15, 163.71–156.77 (m), 150.53, 144.99, 140.46,

137.14, 134.42, 130.00 (d, J = 9.9 Hz), 127.62, 126.90,

124.77 (d, J=12.6 Hz), 110.70 (d, J=20.2 Hz), 103.94 (t,

J = 27.1 Hz), 75.17 (d, J = 5.2 Hz), 55.11 (d, J = 5.8 Hz),

46.66, 30.86. ESI-HRMS calcd. for C₂₁H₂₁F₂N₇O₂S

[M+H]⁺=474.1518, found 474.1520.

2‑amino‑3‑(1‑(2‑(2,4‑difuorophenyl)‑2‑hydroxy‑3‑(1H‑1,2

,4‑triazol‑1‑yl)propyl)‑1H‑1,2,3‑triazol‑4‑yl)propanoic acid

(Flu‑APA, II‑d)

To a solution of 4 (0.2106 g, 0.752 mmol) and DL-prop-

argylglycine (0.0908 g, 0.803 mmol) in a mixture of tert-

butanol and H₂O (1:1, 6 mL), (+)-sodium l-ascorbate

(0.0382 g, 0.193 mmol) was added, followed by copper (II)

acetate monohydrate (0.0207, 0.104 mmol). The reaction

mixture was allowed to stir for 14 h at room temperature.

The reaction mixture was diluted with H₂O (20 mL), and

unreacted 4 was extracted into DCM (3 × 25 mL), while

the product remained in the water layer. The solvent was

removed by evaporation under reduced pressure, yielding

a yellow sticky solid as crude product. The crude product

was purifed via HPLC on a C18 column using a linear gra-

dient running from 95%–5% H₂O/ACN over 40 min, giv-

ing a shiny cream-colored solid as clean product. Yield:

N‑(2‑(2,4‑difuorophenyl)‑2‑hydroxy‑3‑(1H‑1,2,4‑tria‑

zol‑1‑yl)propyl)‑8‑hydroxyquinoline‑7‑carboxamide

(Flu‑8HQ, III‑b)

To a solution of 5 (0.43 g, 1.69 mmol) and 8-hydroxy-

7-quinoline carboxylic acid (0.321 g, 1.697 mmol) in DMF

(15 mL), 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline

(EEDQ, 0.466 g, 1.88 mmol) was added. The reaction mix-

ture was stirred at room temperature for 18 h, at which point

still no reaction had occurred. The reaction mixture was

heated to 60 °C for 3 days, and still no reaction occurred.

Additional EEDQ was added (0.160 g, 0.647 mmol), and the

reaction mixture was stirred for an additional 24 h. Solvent

was removed by evaporation under reduced pressure, and the

crude products were reconstituted in EtOAc (50 mL). The

organic layer was washed with a 10% solution of citric acid

(4×40 mL), H₂O (1×25 mL), and brine (1×25 mL), then

dried over Na₂SO₄and fltered. Crude product was purifed

by HPLC on a C18 column using a linear gradient running

from 95–5% H2O/ACN over 40 min, giving a bright yellow

1

0.105 g (33%). H NMR (400 MHz, DMSO-d6) δ 8.79

(s, 1H), 8.27 (s, 1H), 8.24 (d, J=11.2 Hz, 1H), 7.72–7.57

(m, 2H), 7.33 (td, J = 8.5, 8.1, 3.6 Hz, 1H), 7.06 (s, 1H),

5.40 (dd, J=14.3, 5.0 Hz, 1H), 5.18 (dd, J=27.3, 14.4 Hz,

2H), 5.01 (d, J=14.4 Hz, 1H), 4.57 (dd, J=10.0, 5.5 Hz,

1H), 3.64 (s, 2H), 3.57 (td, J = 15.6, 13.9, 6.4 Hz, 2H).

¹³C NMR (125 MHz, DMSO-d6) δ 169.99 (d, J=7.1 Hz),

163.97–157.05 (m), 150.88, 145.22, 140.43, 129.91, 124.97

(d, J=12.5 Hz), 123.14, 110.98 (d, J=20.2 Hz), 104.00 (t,

J = 26.8 Hz), 73.76 (d, J = 4.9 Hz), 55.38 (d, J = 5.9 Hz),

54.80, 52.02 (d, J = 9.6 Hz), 26.15 (d, J = 5.6 Hz). ESI-

HRMS calcd. For C₁₆H₁₇F₂N₇O₃[M + H]⁺= 394.1434,

found 394.1440.

1

solid. Yield: 0.040 g (6%). H NMR (500 MHz, Chloro-

form-d) δ 8.81 (dd, J=4.4, 1.5 Hz, 1H), 8.38 (t, J=6.0 Hz,

1H), 8.19–8.13 (m, 2H), 8.07 (d, J =8.9 Hz, 1H), 7.83 (s,

1H), 7.64 (td, J=9.2, 6.5 Hz, 1H), 7.53 (ddd, J=8.2, 4.2,

1.0 Hz, 1H), 7.35 (d, J=8.8 Hz, 1H), 6.80 (dtd, J=10.7, 8.3,

2.6 Hz, 2H), 6.41 (s, 2H), 4.71–4.62 (m, 2H), 4.13–3.92 (m,

2H), 2.17 (d, J=1.1 Hz, 1H). ¹³C NMR (125 MHz, Chloro-

form-d) δ 168.12, 163.04 (d, J=249.6 Hz), 151.77, 151.62,

148.69, 144.80, 138.26, 136.38, 130.83–130.50 (m), 130.36,

127.54, 124.07 (d, J = 4.0 Hz), 123.82, 118.07, 112.97,

111.87 (dd, J=20.6, 3.5 Hz), 104.21 (t, J=26.6 Hz), 76.75

(d, J=5.1 Hz), 56.36 (d, J=4.6 Hz), 48.06 (d, J=4.7 Hz).

ESI-HRMS calcd. for C₂₁H₁₇F₂N₅O₃[M+H]⁺=426.1372,

found 426.1372.

(E)‑N‑(2‑(2,4‑difuorophenyl)‑2‑hydroxy‑3‑(1H‑1,2,4‑tria‑

zol‑1‑yl)propyl)‑4‑((2‑(methylcarbamothioyl)hydraziney‑

lidene)methyl)benzamide (Flu‑TSCZ, III‑a)

To a solution of 8a (0.3058 g, 1.29 mmol) in dry DMF

(6.5 mL), N-methylmorpholine (50% in DMF, 0.5 mL,

2.27 mmol) was added followed by HBTU (0.5337 g,

1.41 mmol). This mixture was allowed to stir under N₂

for 15 min. Then, compound 5 (0.3573 g, 1.41 mmol) was

added. The reaction mixture was allowed to stir at room tem-

perature under N₂for 18 h. The product was extracted into

EtOAc (3×50 mL), and the combined organic layers were

1 3

JBIC Journal of Biological Inorganic Chemistry

activity. Specifcally, thiosemicarbazones (TSCZs) have

known antimicrobial properties that are often enhanced

by metal-binding [31], making them an attractive class of

compounds for our studies, and 8-hydroxyquinoline was

investigated on the basis of the Cu-dependent fungicidal

activity observed previously in C. neoformans [32, 33].

In addition to alpha-numerical labeling, each compound

was assigned an abbreviated nickname that refects both its

fuconazole origin and its new metal-binding motif: Flu-

Imidazole (Flu-Im, I-a), Flu-Phenol (I-b), Flu-Dimethy-

lester (Flu-DME, II-a), Flu-Pyridyl (Flu-Pyr, II-b), Flu-

Dipicolylamine (Flu-DPA, II-c), Flu-Aminopropanoic acid

(Flu-APA, II-d), Flu-Thiosemicarbazone (Flu-TSCZ, III-

a), and Flu-8-Hydroxyquinoline (Flu-8HQ, III-b).

Results

Synthesis of fuconazole analogues

with copper‑binding side arms

As shown in Scheme 1, eight analogues of fuconazole

were synthesized from a single commercially available

building block in a way that retains the difuorophenyl

and hydroxyl group pharmacophores of fuconazole, as

well as one of its two triazole rings, as only one is needed

for binding the heme Fe of Cyp51 [13]. The other triazole

was replaced with side arms rich in nitrogen, sulfur, and

oxygen donors that were selected due to their predilection

for binding Cu(II) and/or Cu(I). Synthetic intermediates in

Scheme 1 are labeled with Arabic numerals, whereas fnal

compounds are given a classifcation I, II, or III depend-

ing on which of three fuconazole-based scafolds were

used to prepare them: either an azide, alkyne, or amine

handle for conjugating with an alkyne-, azide-, or carbox-

ylic acid-bearing side arm, respectively. The commercial

availability or ease of synthesis was also considered, and

some side arms were selected based on known biological

Beginning from commercially available ketone 1, the

alkyne or azide versions of fuconazole (compounds 2 and

4, respectivel y) were synthesized using previously reported

methods [12, 13]. Reduction of 4 via Pd/C-catalyzed hydro-

genation wit h triethylsilane [34] yielded compound 5, the

amine version of fuconazole. Analogues were synthesized

from one of these three scafolds via Cu-catalyzed azide-

alkyne cycloaddition (CuAAC) starting from 2 and 4 or

HBTU- or EEDQ-mediated coupling starting from 5. These

Flu-Im

Flu-Phenol

Flu-DME

Flu-Pyr

Flu-DPA

Flu-APA

Flu-TSCZ

Flu-8HQ

Scheme 1. Synthesis of fuconazole analogues. Commercially avail-

able ketone 1 was converted to alkyne-bearing fuconazole structure

2, which was then reacted with azide-bearing side arm 6 via CuAAC

(route A) to obtain compounds with structure I. Alternatively, the

ketone was converted to epoxide 3 via a Corey–Chaykovsky reaction.

Epoxide 3 then underwent a ring-opening reaction with sodium azide

to give azide-bearing fuconazole structure 4. Azide 4 then underwent

CuAAC (route B) with alkyne-bearing side arm 7 to aford analogues

with structure II or was reduced to the corresponding amine (5) via

Pd/C catalyzed hydrogenation. Amine 5 was coupled to carboxylic

acid-bearing side arm 8 via HBTU- or EEDQ-mediated coupling

(route C) to yield analogues with structure III

1 3

JBIC Journal of Biological Inorganic Chemistry

conjugation strategies are favorable because they result in

the formation of groups that could in principle participate

in metal binding: a 1,2,3-triazole from CuAAC or an amide

group from HBTU/EEDQ-mediated coupling.

d). Potentiation by Cu was still evident for these analogues,

though it was less dramatic due to the reduction in trailing

growth observed for the compound alone.

Of note, there were two batches of YPD medium (lots

2044606 and 2101669) in which trailing growth was not

evident at 48 h following exposure to the three active ana-

logues or fuconazole (Fig. S2, ESI). We have previously

observed minor fuctuations in the degree of 48-h trailing

growth between batches of YPD medium received from a

commercial supplier [30]. Trailing growth may not be evi-

dent at 48 h if the onset of tolerance is delayed due to envi-

ronmental conditions (e.g. diferent nutrients in diferent

batches of medium). Indeed, trailing growth does eventu-

ally occur when the growth assay is carried out to 72 h (Fig.

S3a, ESI). We hypothesized that limiting Cu availability

may restore 48-h trailing growth in this batch of YPD. To

test this idea, the growth medium was supplemented with

the membrane-impermeable extracellular Cu(I) chelator

bathocuproinedisulfonic acid (BCS). Indeed, supplement-

ing analogue-treated cells with BCS partially rescued growth

for all three compounds (Fig. S2, ESI). The return of 48-h

trailing growth upon supplementation with BCS bolsters the

conclusion that Cu availability infuences tolerance to the

azole analogues.

Flu‑Pyr, Flu‑Phenol, and Flu‑TSCZ have Cu‑potenti‑

ated antifungal activity against C. albicans

Racemic mixtures of the eight fuconazole derivatives were

tested for the ability to inhibit growth of C. albicans in liquid

culture. As shown in Fig. 1, three of these compounds—

Flu-Pyr, Flu-Phenol, and Flu-TSCZ—inhibited growth at

a concentration of 25 µM or below either with or without

supplemental Cu. Flu-8HQ and Flu-Im did not show inhibi-

tory activity below 100 µM and were therefore considered

inactive (Fig. S1, ESI). Flu-DME, Flu-APA, and Flu-DPA

were also found to be inactive, exhibiting no activity up to

100 µM (Fig. S1, ESI).

Fluconazole is a fungistatic drug to which C. albicans

can develop tolerance that leads to trailing growth, defned

as residual growth at a concentration above the minimal

inhibitory concentration (MIC) [35]. We have found that Cu

supplementation during treatment with fuconazole delays

the onset of trailing growth in C. albicans [30, 36]. Simi-

lar to fuconazole (Fig. 1a), Flu-Pyr demonstrated trailing

growth even at a concentration of 100 µM, but trailing was

abrogated by addition of 10 µM Cu (Fig. 1b). Notably, Flu-

Phenol and Flu-TSCZ gave rise to lower levels of trailing

growth than fuconazole in the absence of added Cu (Fig. 1c,

Compounds that are fungicidal rather than fungistatic are

desirable because they reduce the risk of the development

of resistance [37]. To determine whether Flu-Phenol, Flu-

Pyr, or Flu-TSCZ are fungicidal against C. albicans, colony

forming units (CFUs) were determined following exposure

to these agents with and without supplemental Cu in liquid

culture. Fluconazole was also tested as a control. Colony

growth, quantifed as CFUs per mL, was observed for all

treatment conditions, illustrating that the analogues are not

fungicidal (Fig. 2a). For most conditions, the number of

CFUs increased by approximately two orders of magnitude

above that of the original inoculum, indicating that some

trailing growth had occurred. Cu supplementation did not

impact the number of CFUs for cells treated with fucona-

zole, Flu-Phenol, or Flu-Pyr. Treatment with Flu-TSCZ in

the presence of supplemental Cu was the only condition in

which no trailing growth was evident. This result makes

sense, given that the experiment was performed in a batch

of YPD medium that exhibited low levels of trailing growth

at 48 h. The OD600 at 48 h is low for all of these conditions,

but cells treated with Flu-TSCZ and Cu had the lowest level

of growth by OD600 at 48 h (Fig. S3b, ESI). To investigate

whether Cu concentration impacts the growth of cells treated

with Flu-TSCZ, CFUs were determined for cultures exposed

to Flu-TSCZ in the presence of a range of Cu concentrations.

As shown in Fig. 2b, supplementation with very low (1 µM)

or very high (1000 µM) Cu did not reduce CFUs relative to

cells treated with Flu-TSCZ alone (0 µM supplemental Cu).

However, supplementing with 10 µM Cu did reduce CFUs

(a)

(b)

2.5

2.0

1.5

1.0

0.5

0.0

2.5

2.0

1.5

1.0

0.5

0.0

No Cu

10 µM Cu

No Cu

10 µM Cu

[Flu], µM

[Flu-Pyr], µM

(c)

(d)

2.5

2.0

1.5

1.0

0.5

0.0

2.5

2.0

1.5

1.0

0.5

0.0

No Cu

10 µM Cu

No Cu

10 µM Cu

[Flu-Phenol], µM

[Flu-TSCZ], µM

Fig. 1 Flu-Pyr, Flu-Phenol, and Flu-TSCZ exhibit Cu-potentiated

growth inhibition against C. albicans. Fluconazole is shown for com-

parison. Conditions: Growth at 30 °C in YPD (lot 2005064, metal

analysis presented in Table S1, ESI) monitored at OD600 after 48 h.

Data are reported as means with error bars representing standard

deviation of three replicate conditions

1 3

JBIC Journal of Biological Inorganic Chemistry

No Cu

C. albicans SC5314 (WT)

C. albicans ctr1 Δ/Δ

⁶

⁵

C. neoformans H99 (WT)

C. neoformans ctr1Δ ctr4Δ

⁷

(a)

(b)

1×10

(a)

(b)

1.0

0.5

0.0

10 µM Cu

3

2

1

0

1×10

⁶

1×10

⁵

1×10

⁴

1×10

⁴

³

1×10

³

1×10

[CuSO ], µM

[Flu-TSCZ], µM

[Flu-TSCZ], μM

₄

(50 µM Flu-TSCZ)

Fig. 3 Flu-TSCZ recovers growth of cells lacking Cu import machin-

ery. 48-h growth of C. albicans (a) or C. neoformans (b) WT (black

bars) and Cu import mutants (green bars) treated with 0–100 μM

Flu-TSCZ in YPEG medium. Dose dependent growth rescue was

observed for cells lacking Cu import genes under these conditions.

Data are reported as means with error bars representing standard

deviation of three replicate conditions

Fig. 2 Colony forming units (CFUs) after exposure to analogues. a

CFUs/mL were determined following 48 h of exposure to 100 µM

fuconazole (purple bars), Flu-Pyr (magenta bars), Flu-Phenol (green

bars), or Flu-TSCZ (dark blue bars) in the absence (solid bars) or

presence (striped bars) of 10 µM supplemental CuSO₄in YPD

medium (lot 2044606). The number of CFUs/mL in the original inoc-

ulum is shown in light blue. b A Cu-dependent reduction in CFUs

was observed for cells treated with 50 µM Flu-TSCZ. CFUs/mL

were determined following 48 h of exposure to 50 µM Flu-TSCZ and

0–1000 µM supplemental CuSO₄. Data are reported as means with

individual data points indicating replicates within a single experiment

activity against C. albicans and rescued growth of the strains

lacking Cu import machinery, we chose to focus our atten-

tion on this compound for further characterization.

and supplementing with 100 µM Cu reduced CFUs even

further, relative to cells treated with Flu-TSCZ alone. The

number of CFUs per mL did not drop below the original

inoculum, indicating the combination treatment is still fungi-

static and not fungicidal. However, it is notable that there is

a Cu-dependent decrease in the number of CFUs per mL for

cells treated with 50 µM Flu-TSCZ. Cells treated with the

inhibitory but lower concentration of 25 µM Flu-TSCZ did

not exhibit a Cu-dependent decrease in CFUs (Fig. S4, ESI).

Flu‑TSCZ binds Cu(II)

Analogues were endowed with side arms containing multi-

ple donor atoms in order to promote Cu binding and, in turn,

enhance potentiation of azole activity by Cu. As shown in

Fig. 4a, Flu-TSCZ exhibits a characteristic absorption band

(a) 0.8

0.6

2

1

(b)

Flu-TSCZ

Flu-TSCZ + CuSO

₄

Flu-TSCZ + CuSO + Gly

₄

Flu‑TSCZ provides bioavailable Cu to C. albicans

and C. neoformans

Flu-TSCZ + CuSO + His

₄

0

0.4

-1

-2

-3

0.2

CuSO

To determine whether the active analogues are capable

of shuttling Cu into cells, they were tested for their abil-

ity to rescue growth of C. albicans and C. neoformans

strains in which the Cu importers had been deleted. When

grown in yeast peptone ethanol glycerol (YPEG) medium,

cells are forced to respire, therefore requiring Cu for use in

cytochrome c oxidase. C. albicans relies on a single high

afnity Cu(I) importer Ctr1 and C. neoformans has two Cu

importers, Ctr1 and Ctr4. As shown in Fig. 3, wild-type

cells grow in YPEG while C. albicans ctr1Δ/Δ and C. neo-

formans ctr1Δ ctr4Δ cells do not. Flu-TSCZ was the only

compound tested that rescued growth of both deletion strains

(Fig. 3), indicating that Flu-TSCZ can deliver bioavailable

Cu to C. albicans and C. neoformans while other compounds

cannot (Fig. S5, ESI). This result distinguishes Flu-TSCZ

from fuconazole, which does not rescue growth of either

deletion strain [30]. Because Flu-TSCZ displayed antifungal

₄

CuSO + Flu-TSCZ

₄

0.0

2500 3000 3500 4000

Field (G)

Wavelength (nm)

Fig. 4 Flu-TSCZ binds Cu(II) in HEPES bufer but not in YPD

medium. a UV–Vis spectra of Flu-TSCZ Cu in 50 mM HEPES

bufer, pH 7.4. Addition of Cu (blue trace) to Flu-TSCZ (purple trace)

decreased the intensity of the absorption band at 321 nm and gave

rise to a shoulder, indicating Cu complex formation. Addition of gly-

cine (Gly, green trace) to the Cu(II)–Flu-TSCZ complex gave rise to

a new feature at 413 nm, suggesting formation of a ternary complex,

but addition of histidine (His, red trace) resulted in disappearance of

the shoulder, indicating histidine competes with Flu-TSCZ for Cu(II).

Conditions: [Flu-TSCZ]=50 µM, [CuSO₄]=25 µM, [Gly]=1 mM,

[His]=1 mM. b EPR spectra of Flu-TSCZ Cu in YPD medium.

Addition of Cu to YPD gives rise to an EPR spectrum (solid blue

trace) that is unchanged by addition of Flu-TSCZ (dotted red trace).

Conditions: [Flu-TSCZ]=300 µM, [CuSO₄]=150 µM in YPD with

20% glycerol. Data collected at 77 K

1 3

JBIC Journal of Biological Inorganic Chemistry

2.5

at 321 nm in HEPES bufer that decreases in intensity upon

addition of Cu(II), indicating formation of a Cu(II)–Flu-

TSCZ complex. Similar spectral shifts were observed for

other analogues, confrming that they also bind Cu(II) in

HEPES bufer (Fig. S6, ESI).

(a)

(b)

Flu-TSCZ

2.0

+ Cu(II)

+ Asc

1.5

+ Fz

1.0

0.5

0.0

+ BCA

The ability of Flu-TSCZ to supply bioavailable Cu to the

Cu import mutants raised the question of whether this com-

pound may bind Cu in the growth medium. In YPD medium

there is approximately 3 mM free glycine [38], which forms

a 2:1 complex with Cu(II) (log β₂=15.6 for Cu(Gly)₂) [39,

40]. Thus, the ability of a compound to bind Cu in the pres-

ence of glycine indicates the likelihood that the complex will

form in the growth medium during biological assays. We

have previously shown that glycine outcompetes fuconazole

for Cu(II) binding [30]. To determine whether glycine also

competes with Flu-TSCZ for Cu(II), glycine was added to

the preformed Cu(II)–Flu-TSCZ complex in HEPES bufer.

As shown in Fig. 4a, glycine addition partially restored the

intensity of the absorption band at 321 nm and gave rise to a

new feature at 413 nm, suggesting the formation of a ternary

complex. To determine whether another amino acid would

have a diferent efect on the stability of Cu(II)–Flu-TSCZ in

solution, a separate competition experiment was performed

with L-histidine, which has a higher afnity than glycine for

Cu(II) (log β₂=18.6) [41, 42]. Like glycine, free histidine is

present in YPD but at a lo wer concentration (approximately

0.5 mM) [38]. Upon addition of histidine to the Cu(II)–Flu-

TSCZ complex, the ligand signal at 321 nm was almost fully

restored and the shoulder unique to the Cu(II)–Flu-TSCZ

complex disappeared (Fig. 4a), suggesting that histidine

pulls Cu(II) away from Flu-TSCZ.

300 400 500 600 700

Wavelength (nm)

Fig. 5 Flu-TSCZ competes with the chelator ferrozine (Fz), but not

bicinchoninate anion (BCA), for Cu(I) as determined by UV–Vis

spectroscopy. a Flu-TSCZ pulls Cu(I) away from the pre-formed

[Cu^I(Fz)₂]³⁻complex. b Presence of Flu-TSCZ in solution prevents

formation of the [Cu^I(Fz)₂]³⁻complex. CuCl₂was used as the source

of Cu(II), and ascorbate (Asc) was added to generate Cu(I) in situ.

Conditions: [Fz]=100 μM, [CuCl₂]=40 μM, [Ascorbate]=1 mM,

[Flu-TSCZ]=100 µM, [BCA]=100 µM in 50 mM HEPES bufer, pH

7.4

Cu(I)–Flu-TSCZ complex. As shown in Fig. 5b, addition of

the reducing agent ascorbate to Cu(II)-Flu-TSCZ caused a

slight shift in the absorption signal, indicating reduction of

the complex to Cu(I). Addition of ferrozine to this solution

did not change the absorption spectrum of this species and

the [Cu^I(Fz)₂]³⁻signal was not observed. To further probe

the afnity of Flu-TSCZ for Cu(I), the higher afnity Cu(I)

chelator bicinchoninate anion (BCA, log β₂=17.2) [47, 48]

was tested for its ability to bind Cu(I) in the presence of Flu-

TSCZ. Addition of BCA gave rise to the characteristic signal

at 562 nm, indicating formation of the [Cu^I(BCA)₂]³⁻com-

plex (Fig. 5b). Taken together, the competition data with

these two Cu(I) probes [45] benchmark the afnity of Flu-

TSCZ for Cu(I) in the picomolar range and suggest Flu-

TSCZ may be a better ligand for Cu(I) than Cu(II). It is

therefore possible that Flu-TSCZ rescues growth of cells

lacking their Cu importers through redistribution of intracel-

lular Cu(I) as opposed to shuttling in extracellular Cu(II).

To test directly whether Flu-TSCZ binds Cu(II) under

the conditions of our growth assays, we compared the EPR

spectrum of Cu alone in YPD to that of Flu-TSCZ and Cu in

YPD. There was no change to the EPR spectrum of Cu(II)

in YPD upon addition of Flu-TSCZ, suggesting that the

Cu(II)–Flu-TSCZ complex does not form under these con-

ditions in YPD medium (Fig. 4b). Considering the data from

the histidine competition, it is reasonable that amino acids

present in YPD prevent formation of a stable Cu(II)–Flu-

TSCZ complex.

Potentiation of Flu‑TSCZ activity is Cu specifc

Although analogues were designed with the intention of

strengthening the Cu potentiation observed for fuconazole,

their efcacy could be afected by the availability of other

metal ions in the growth medium. To determine the impact

of varying the availability of individual biometals, growth

assays were performed in Tris-bufered synthetic defned

(Tris:SD) drop-out media rigorously controlled for metal

content. Each medium was prepared from Chelex-treated

water to remove trace metals and then nutrients were replen-

ished to levels found in commercial SD media formulations

[49]. Individual drop-out media were prepared by omitting

Cu, Fe, Mn, or Zn, and assays were performed by titrating

each omitted metal back to obtain a stepwise gradient of

Flu‑TSCZ binds Cu(I)

Given that C u(I) is the primary oxidation state of intracel-

lular Cu [43, 44], a competitive binding assay was performed

with the Cu(I) chelator ferrozine (log β₂=15.1) [45, 46] to

predict the ability of Cu(I)–Flu-TSCZ to form intracellularly.

As shown in Fig. 5a, the absorption band for the pre-formed

[Cu^I(Fz)₂]³⁻complex disappeared upon addition of Flu-

TSCZ, suggesting that Flu-TSCZ pulls Cu(I) away from this

competing ligand. Furthermore, the [Cu^I(Fz)₂]³⁻complex

does not form upon addition of ferrozine to the pre-formed

1 3

JBIC Journal of Biological Inorganic Chemistry

that metal. As shown in Fig. 6, growth of untreated con-

trol cells improved slightly as the dropped-out metal was

returned, most clearly for Fe and Mn. Supplemental levels

of Fe, Mn, and Zn were not inhibitory up to 50 µM, but

50 µM Cu inhibited growth by approximately 50%. Nota-

bly, increasing the levels of Cu (Fig. 6a), but not Fe, Mn, or

Zn (Fig. 6b–d) potentiated the activity of Flu-TSCZ. In the

Cu drop-out medium, 25–50 µM Flu-TSCZ was required to

fully inhibit growth without Cu supplementation. By con-

trast, only 13 µM or 6 µM Flu-TSCZ was required for full

inhibition when the growth medium was supplemented with

3 µM or 12.5 µM Cu, respectively.

Candida albicans strains defective in Cu transport

have increased tolerance to Flu‑TSCZ

Cu

Fe

The observation that levels of Cu uniquely impact the ef-

cacy of Flu-TSCZ led us to test how deletion of Cu transport

genes impacts C. albicans’ tolerance to Flu-TSCZ. As shown

in Fig. 7a, deletion of Cu exporter CRP1 increased the toler-

ance of C. albicans to Flu-TSCZ, demonstrating that cells

are better equipped to adapt to Flu-TSCZ stress when they

lack the Cu exporter. Interestingly, deletion of Cu importer

CTR1 also increased tolerance, with even 100 µM Flu-TSCZ

failing to fully inhibit growth (Fig. 7b). Of note, we have

observed ctr1Δ/Δ cells grow in clumps in the overnight cul-

ture (Fig. S7a, ESI). The reason is unknown and there is no

correlation between the size of the colony used to inoculate

and whether cultures grew as planktonic cells or in clumps.

Despite these morphological diferences, similar results were

obtained when Flu-TSCZ was tested against ctr1Δ/Δ cells

growing in clumps (Fig. S7b, ESI) and planktonic ctr1Δ/Δ

cells (Fig. 7b). Deletion of transcription factor MAC1, which

controls the cellular response to Cu defciency, including

regulation of CTR1, did not afect tolerance (Fig. 7c). The

antifungal activity of Flu-TSCZ against the CTR1, CRP1,

and MAC1 deletion strains and their parent strains was not

dramatically impacted by supplementation with 10 µM Cu

(Fig. S8, ESI). Growth of ctr1Δ/Δ cells improved slightly

upon Cu supplementation, consistent with these cells lack-

ing high afnity Cu import capabilities. Growth inhibition

of crp1Δ/Δ cells and parent strain KC2 was potentiated

by Cu at high concentrations of Flu-TSCZ. In SC5314, Cu

1.5

1.0

0.5

0.0

1.5

1.0

0.5

0.0

(a)

(b)

[Flu-TSCZ], µM

0

6

[total Fe], µM

[total Cu], µM

13

25

50

100

Zn

Mn

1.5

1.0

0.5

0.0

(c)

(d)

1.5

1.0

0.5

0.0

[total Mn], µM

[total Zn], µM

Fig. 6 Cu availability specifcally has a signifcant impact on growth

of Flu-TSCZ-treated cells. 48-h growth of untreated (dark purple

lines) or Flu-TSCZ-treated C. albicans cells as a function of total

Cu (a), Fe (b), Mn (c), and Zn (d) in the respective metal drop-out

Tris:SD media. The lowest concentration of each metal represents

actual trace levels of the dropped-out metal, analyzed by ICP-MS.

The remaining concentration gradients represent theoretical metal

concentrations based on what was added on top of trace levels.

Shown are representative plots from n=2 biologically independ-

ent experiments. Full metal analyses for each media formulation are

reported in Tables S4-7, ESI

SC5314 (WT)

ctr1Δ/Δ

(a)

(b)

(c)

150

100

50

150

100

50

150

100

50

SN152 (WT)

KC2 (WT)

mac1Δ/Δ

crp1Δ/Δ

0

[Flu-TSCZ], µM

Fig. 7 C. albicans strains lacking genes involved in Cu transport have

reduced susceptibility to Flu-TSCZ. C. albicans deletion strains were

treated with 0–100 µM Flu-TSCZ in YPD medium at 30 °C for 48 h;

then growth was determined by measuring OD600. Deletion of Cu

exporter CRP1 (a) or Cu importer CTR1 (b) rendered C. albicans

more tolerant to Flu-TSCZ. Deletion of Cu-regulated transcription

factor MAC1 (c) did not impact the tolerance of C. albicans to Flu-

TSCZ. Data are normalized to the untreated control for each strain

and reported as means with error bars representing standard deviation

of three replicate conditions

1 3

JBIC Journal of Biological Inorganic Chemistry

abrogated activity at 12.5 µM Flu-TSCZ, a concentration on

the cusp of the MIC.

impact on these levels (Fig. 8b, c). Zn was the only metal

tested that decreased in response to treatment with Flu-TSCZ

(Fig. 8d), but these levels were also not impacted by Cu sup-

plementation. Interestingly, in the absence of supplemental

Cu, increasing the concentration of Flu-TSCZ from 25 to

100 µM did not further increase cell-associated levels of Cu,

Fe, or Mn or further decrease levels of Zn. In the presence

of supplemental Cu, however, increasing the concentration

of Flu-TSCZ from 25 to 100 µM caused cells to accumu-

late additional Cu. This result suggests that Flu-TSCZ can

increase cellular Cu in a dose-dependent manner, provided

ample Cu is available. Without extra Cu in the growth envi-

ronment, there seems to be a limit to the increase in cellular

Cu Flu-TSCZ can provide. Treatment with 10 µM Flu-TSCZ

did not impact metal levels, regardless of Cu supplementa-

tion (Fig. S10, ESI), indicating that the minimal concentra-

tion of Flu-TSCZ required to alter the metal status of C.

albicans is between 10 and 25 µM.

In contrast to the results obtained for Flu-TSCZ, we have

previously found that both the ctr1Δ/Δ and mac1Δ/Δ strains

are less tolerant than wild-type cells to fuconazole [50].

Thus, replacement of the triazole side arm of fuconazole

with the thiosemicarbazone motif alleviates the sensitivity of

the mac1Δ/Δ strain and fully reverses the sensitivity of the

ctr1Δ/Δ strain. By intentionally incorporating metal-binding

motifs, we have impacted the ability of strains defective in

Cu homeostasis to tolerate azole stress.

Flu‑TSCZ treatment alters cell‑associated metal

levels

The ability of Flu-TSCZ to recover growth of strains lacking

Cu importers suggested this compound may increase levels

of cell-associated Cu, despite the lack of conclusive spec-

troscopic evidence for Cu complex formation in the growth

medium. To determine whether Flu-TSCZ alters cellular

metal status, metal content was determined for cells treated

with 10, 25, or 100 µM Flu-TSCZ with and without sup-

plemental Cu over a 12-h period via inductively coupled

plasma-mass spectrometry (ICP-MS). As we have observed

previously for untreated C. albicans cells [36], Cu levels

remained steady, Fe and Mn levels decreased, and Zn levels

increased over time (Fig. 8). We found that treatment with 25

or 100 µM Flu-TSCZ did indeed increase cell-associated Cu

above untreated levels, most notably at 6 h (Fig. 8a, Fig. S9,

ESI). Supplementing these cells with 10 µM Cu increased

these levels even further, such that cellular Cu concentrations

in co-treated cells exceeded those of cells treated only with

Cu. Levels of Fe and Mn were also elevated in cells treated

with Flu-TSCZ, but Cu supplementation had no additional

Flu‑TSCZ depletes EPR‑detectable Fe and increases

EPR‑detectable Mn

The subcellular speciation of metals detected by bulk ICP-

MS analysis was probed by analyzing cells treated with

Flu-TSCZ by electron paramagnetic resonance (EPR) spec-

troscopy. We have previously demonstrated that fuconazole

treatment attenuates the signal at g=4.3 [50], which repre-

sents the kinetically exchangeable, or labile, Fe pool [51].

Fluconazole treatment also modulated the multiline signal at

g=2 [50], taken to be from cellular Mn(II) [51]. As shown

in Fig. 9a, treatment with Cu alone did not alter the EPR

spectrum relative to the spectrum of untreated control cells.

Treating with 10 µM Flu-TSCZ, which is below the mini-

mal concentration tested that fully inhibited growth in YPD

(a)

(b)

(c)

(d)

Cu

Fe

Mn

Zn

300

200

100

0

1500

1000

500

0

100

50

0

1500

1000

500

0

9 12

0

3

6

9

12

0

3

6

9

12

0

3

6

9 12

0

3

6

Time (h)

NT

Cu

25 µM Flu-TSCZ

25 µM Flu-TSCZ + Cu

100 µM Flu-TSCZ

100 µM Flu-TSCZ + Cu

Fig. 8 Metal content of C. albicans cells reported as cellular con-

centration (µM). Cu levels (a) increased during treatment with 25

or 100 µM Flu-TSCZ (red triangles and dark blue circles, respec-

tively) relative to untreated control cells (green circles), with the

most prominent diference at 6 h. Supplementing cells with Cu alone

(light blue squares) caused Cu levels to increase steadily over time.

Co-treatment with Flu-TSCZ and Cu (purple inverted triangles and

orange squares) caused higher accumulation of Cu than Cu treatment

alone. Treatment with Flu-TSCZ also increased levels of Fe (b) and

Mn (c) with Cu supplementation having no additional efect. Zn lev-

els (d) were lower in cells treated with Flu-TSCZ. Cells were grown

in YPD medium for time indicated in fgure legends. Cell-associated

metal levels were analyzed by ICP-MS and cellular metal concentra-

tions were calculated as described in Methods. Data are reported as

mean SEM, n=3 biologically independent samples per timepoint

1 3

JBIC Journal of Biological Inorganic Chemistry

(a) (b)

(c)

1.0

(d)

g = 4.3

g = 2

g = 4.3

g = 2

g = 4.3

g = 2

1.0

0.5

0.0

1.0

0.5

0.0

0.5

0.0

-0.5

-1.0

-0.5

-1.0

-0.5

-1.0

NT

Cu

100 µM Flu-TSCZ

100 µM Flu-TSCZ + Cu

25 µM Flu-TSCZ

25 µM Flu-TSCZ + Cu

1000 2000 3000 4000

Field (G)

1000 2000 3000 4000

Field (G)

1000 2000 3000 4000

Field (G)

Fig. 9 Flu-TSCZ depletes EPR-detectable Fe and increases EPR-

detectable Mn. a Cu supplementation alone (red trace) did not impact

the EPR signal at g=4.3 relative to the untreated control cells (teal

trace). b Treatment with 10 µM Flu-TSCZ with (red trace) or without

(teal trace) Cu supplementation was not sufcient to impact the signal

at g=4.3. Increasing the concentration of Flu-TSCZ to 25 µM (c) or

100 µM (d) resulted in an attenuation of the g=4.3 signal by approx-

imately half and changed the signals in the g=2 region, which are

taken to be from Mn. Conditions: [Flu-TSCZ] is indicated in fgure

legends, [CuSO₄]=10 µM. Cells were treated for 6 h in YPD medium

before analysis

(12.5 µM), also did not impact the EPR spectrum, regardless

of whether cells were also supplemented with Cu (Fig. 9b).

Increasing the concentration of Flu-TSCZ to 25 µM attenu-

ated the signal at g=4.3 by approximately half and gave rise

to a six-line pattern centered at g=2 (Fig. 9c), which is char-

acteristic of Mn(II) ions. Further increasing the Flu-TSCZ

concentration to 100 µM did not result in any additional

change to the g=4.3 signal, but it did signifcantly increase

the intensity of the signal centered at g=2 (Fig. 9d).

exhibited fungicidal activity, cells treated with Flu-Phenol

or Flu-TSCZ had a reduced level of trailing growth com-

pared to those treated with fuconazole. It is possible that the

chemical structures of these analogues have been sufciently

modifed such that cells do not enact the same adaptative

mechanisms associated with fuconazole. By contrast, cells

treated with Flu-Pyr exhibited the same extent of trailing

growth as those treated with fuconazole, suggesting this

analogue is recognized in a way that causes a subpopulation

of cells to adapt and tolerate this compound. Another pos-

sibility is that Flu-Pyr accumulates in cells at a lower con-

centration than Flu-Phenol and Flu-TSCZ. Trailing growth

has been linked to a low intracellular concentration of fu-

conazole, although it does not result from lower uptake or

higher efux rates and the cause of the lower concentration

is unknown [35]. Fluconazole is achiral, but modifcation

of one of the side arms introduces a chiral center. All of

the analogues were tested for antifungal activity as racemic

mixtures (or a mixture of four diastereomers in the case of

Flu-APA). It is possible that separation and testing of indi-

vidual stereoisomers would reveal a preferable isomer and

therefore a lower minimal inhibitory concentration, as has

been previously reported for analogues of fuconazole [53].

Flu-TSCZ stood out among the active analogues due to its

unique ability to partially rescue growth of the C. albicans

ctr1Δ/Δ and C. neoformans ctr1Δ ctr4Δ strains, suggest-

ing this compound is capable of providing bioavailable Cu

for use in cytochrome c oxidase. This fnding distinguishes

Flu-TSCZ from fuconazole, which does not rescue growth

of these strains [30]. Interestingly, both fuconazole [36]

and Flu-TSCZ increase total levels of cell-associated Cu,

yet only Flu-TSCZ rescues growth of the Cu import mutants,

indicating that the speciation and/or location of cellular Cu

pools difer when treated with Flu-TSCZ versus fuconazole.

It was surprising that Flu-TSCZ did not show evidence of

Discussion

The development of new strategies to combat invasive fun-

gal infections is crucial as pathogens evolve to survive in

the presence of existing frontline antifungal drugs. Herein,

we report eight new derivatives of fuconazole created by

installing Cu binding sites in place of one of the triazole

arms, a strategy designed to capitalize on the Cu potentia-

tion we have observed for fuconazole [30]. Although sev-

eral analogues of fuconazole have been reported, this work

provides a unique approach by focusing on structural motifs

likely to interact with metal ions to impact metal homeosta-

sis pathways. The idea of dual function azole compounds has

also been explored by Papadopoulou and coworkers, who

synthesized dual-function derivatives of fuconazole with

the aim of inhibiting both Cyp51 and type I nitroreductase

of protozoan parasite Trypanosoma cruzi [52].

Fluconazole is a fungistatic drug that inhibits cell growth

but does not kill cells. C. albicans can therefore develop tol-

erance to fuconazole, a phenomenon in which a stable sub-

population of cells grow slowly during drug stress, despite

the drug still interacting with the target [35]. Fungicidal

drugs, on the other hand, result in cell death and do not

give rise to trailing growth. Though none of our analogues

1 3

JBIC Journal of Biological Inorganic Chemistry

Cu(II) binding in YPD medium, given that ionophores that

rescue growth of Cu import mutants are thought to do so by

acquiring Cu(II) from the growth medium [54]. The ability

of Flu-TSCZ to outcompete ferrozine for Cu(I) raises the

possibility that Flu-TSCZ rescues growth of the Cu import

mutant strains by mobilizing Cu(I). However, the mecha-

nism by which cells accumulate higher levels of total Cu

during azole treatment remains unexplained.

Incorporating metal binding moieties into known antifungal

compounds is an avenue worth additional exploration.

Acknowledgements We thank Prof. Val Culotta (Johns Hopkins Uni-

versity, Baltimore, MD) for providing the two strains of C. albicans

(KC2 and crp1Δ/Δ), Prof. Alistair Brown (University of Exeter, Exeter,

UK) for providing the SC5314 and ctr1Δ/Δ strains of C. albicans, and

Prof. Dennis Thiele (Duke University, Durham, NC) for providing the

two strains of C. neoformans (WT H99 and ctr1Δ ctr4Δ). This work

was supported by the National Institutes of Health (Grant GM084176).

E. W. H. acknowledges support from the United States Department of

Education GAANN Fellowship (Award No. P200A150114).

Attenuation of the EPR signal at g=4.3 upon treatment

with Flu-TSCZ is consistent with an increased need for cel-

lular Fe to accommodate production of hemeprotein Cyp51

following inhibition by azoles [55]. We have reported a simi-

lar efect in the EPR spectrum of fuconazole-treated cells

[36]. A notable diference is that 10 µM fuconazole is suf-

f cient to deplete the signal but 10 µM Flu-TSCZ is not. This

diference likely stems from the higher MIC of Flu-TSCZ

and suggests that antifungal activity (i.e. sufcient inhibition

of Cyp51 to cause cell stress) is necessary for cells to mobi-

lize Fe from labile pools. Indeed, upon increasing the con-

centration of Flu-TSCZ from 10 to 25 µM, changes to bulk

cellular metal levels and the whole cell EPR spectra were

observed, further supporting the idea that there is a threshold

Flu-TSCZ concentration required to impact metal homeo-

stasis. Increasing the concentration of Flu-TSCZ from 25 to

100 µM did not further attenuate the g=4.3 signal, revealing

that mobilization of labile Fe pools is a set response, not

one impacted by the amount of Flu-TSCZ. Interestingly, the

signal at g=2 does increase in magnitude with increasing

Flu-TSCZ concentration, despite the fact that cell-associated

levels of Cu, Fe, Mn, and Zn are the same whether cells

were treated with 25 µM or 100 µM Flu-TSCZ. Together,

these observations suggest that increasing the concentration

of Flu-TSCZ causes metal redistribution without a change

to total cellular metal levels. The number of paramagnetic

metal species that are EPR detectable in the g = 2 region,

including Fe-S clusters [56] and Mn species [51], make it

difcult to defnitively assign signals in this region for whole

cell samples. However, given the six-line pattern of the sig-

nal, we hypothesize it arises primarily from Mn.

Compliance with ethical standards

Conflict of interest The authors declare that they have no confict of

interest.

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Article Doi

Fluconazole analogues with metal-binding motifs impact metal-dependent processes and demonstrate antifungal activity in Candida albicans

DOI: 10.1007/s00775-020-01796-x

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