Azole-triphenylphosphonium conjugates combat antifungal resistance and alleviate the development of drug-resistance

Article abstract of DOI:10.1016/j.bioorg.2021.104771

Azole antifungals are commonly used to treat fungal infections but have resulted in the occurrence of drug resistance. Therefore, developing azole derivatives (AZDs) that can both combat established drug-resistant fungal strains and evade drug resistance is of great importance. In this study, we synthesized a series of AZDs with a fluconazole (FLC) skeleton conjugated with a mitochondria-targeting triphenylphosphonium cation (TPP⁺). These AZDs displayed potent activity against both azole-sensitive and azole-resistant Candida strains without eliciting obvious resistance. Moreover, two representative AZDs, 20 and 25, exerted synergistic antifungal activity with Hsp90 inhibitors against C. albicans strains resistant to the combination treatment of FLC and Hsp90 inhibitors. AZD 25, which had minimal cytotoxicity, was effective in preventing C. albicans biofilm formation. Mechanistic investigation revealed that AZD 25 inhibited the biosynthesis of the fungal membrane component ergosterol and interfered with mitochondrial function. Our findings provide an alternative approach to address fungal resistance problems.

Full text of DOI:10.1016/j.bioorg.2021.104771

Bioorganic Chemistry 110 (2021) 104771
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Azole-triphenylphosphonium conjugates combat antifungal resistance and
alleviate the development of drug-resistance
,
,
,
Xin Wanga 1, Jun Liua 1, Jinyao Chenb 1, Ming Zhangc, Chuan Tiana, Xiaoping Penga,
Gang Lia, Wenqiang Changb *, Hongxiang Loua
,
,b,*
a School of Pharmacy, Qingdao University, Qingdao 266071, China
b Department of Natural Product Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Cheeloo College of
Medicine, Shandong University, Jinan 250012, China
c The Second Hospital, Cheeloo College of Medicine, Shandong University, Jinan 250012, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Azole antifungals are commonly used to treat fungal infections but have resulted in the occurrence of drug
resistance. Therefore, developing azole derivatives (AZDs) that can both combat established drug-resistant fungal
strains and evade drug resistance is of great importance. In this study, we synthesized a series of AZDs with a
fluconazole (FLC) skeleton conjugated with a mitochondria-targeting triphenylphosphonium cation (TPP+).
These AZDs displayed potent activity against both azole-sensitive and azole-resistant Candida strains without
eliciting obvious resistance. Moreover, two representative AZDs, 20 and 25, exerted synergistic antifungal ac-
tivity with Hsp90 inhibitors against C. albicans strains resistant to the combination treatment of FLC and Hsp90
inhibitors. AZD 25, which had minimal cytotoxicity, was effective in preventing C. albicans biofilm formation.
Mechanistic investigation revealed that AZD 25 inhibited the biosynthesis of the fungal membrane component
ergosterol and interfered with mitochondrial function. Our findings provide an alternative approach to address
fungal resistance problems.
Azole-resistance
Candida
Fluconazole
Mitochondria-targeting
Erg11
1. Introduction
and spread of antifungal drug resistance which poses a growing public
health problem [8]. The rise of azole resistance and multidrug resistance
Over 300 million people suffer from serious fungal diseases, which
result in over 1.6 million deaths annually [1]. Candida species are
considered the most frequent fungi isolated from cancer patients [2], the
second most common organisms to cause catheter-associated urinary
tract infections, the third most frequently pathogen responsible for pe-
diatric sepsis [3,4], and the fourth-ranked cause of hospital-acquired
fungemia with a mortality rate of approximately 40% [5]. Nosocomial
Candida bloodstream infection (candidaemia) has become one of the
most serious fungal diseases among hospitalized patients worldwide,
despite improvement in medical interventions [6].
(MDR) in Candida species has worsened the treatment of nosocomial
candidaemia [6,9]. Therefore, developing novel azole derivatives
(AZDs) that can combat current drug-resistant fungal organisms and
evade drug resistance is important to treat candidaemia.
Azole resistance in clinical Candida strains is primarily caused by the
overexpression of drug efflux pumps and upregulation or mutations of
the drug target Erg11 [10]. Gain-of-function mutations in transcrip-
tional factors of Mrr1 or Tac1 result in constitutive overexpression of the
efflux pump-encoding genes MDR1 or CDR1/CDR2, respectively,
enhancing the efflux of azole drugs and thereby promoting azole resis-
tance [11,12]. The gain-of-function mutations of Upc2 cause the upre-
gulation of ERG11, which encodes the target of azole drugs and confers
azole resistance [13]. In addition, mutations in Erg11 are another
prevalent source of clinical azole resistance [14]. At present, compro-
mising drug efflux or cellular stress responses through the addition of
efflux pump inhibitors or Tor/Hsp90 inhibitors is considered a powerful
Azole drugs, particularly fluconazole (FLC), are widely used to treat
life-threatening invasive fungal infections including candidaemia due to
their high efficacy, excellent bioavailability, low toxicity and ease of
access [7]. FLC inhibits the activity of sterol 14-demethylase (Erg11),
which is required for the biosynthesis of the fungal membrane compo-
nent ergosterol. However, the use of azoles has promoted the evolution
* Corresponding authors at: School of Pharmacy, Qingdao University, Qingdao 266071, China (H. Lou).
E-mail addresses: changwenqiang@sdu.edu.cn (W. Chang), louhongxiang@sdu.edu.cn (H. Lou).
1
Xin Wang, Jun Liu and Jinyao Chen contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2021.104771
Received 18 November 2020; Received in revised form 5 February 2021; Accepted 21 February 2021
Available online 24 February 2021
0045-2068/© 2021 Published by Elsevier Inc.

X. Wang et al.
Bioorganic Chemistry 110 (2021) 104771
strategy to abrogate azole resistance [15–18]. However, these combi-
nation treatments have not been approved for clinical use due to
concomitant host toxicity or other factors. The development of an
alternative strategy to increase the efficacy of FLC and overcome azole
resistance is still of great importance.
2. Results and discussions
2.1. Design and synthesis of AZDs
A previous study reported that alkylated piperazine-azole hybrids
displayed much better antifungal activity than FLC, particularly for non-
albicans Candida and Aspergillus strains [31]. We, therefore, designed
and synthesized a series of azole analogs by conjugation of the FLC
skeleton with TPP+ using alkyl linkers. The moieties of piperazine were
Imaging the intracellular distribution of FLC using fluorescent probes
revealed that FLC analogs primarily localize within mitochondria at
least in the first stage of drug uptake [19]. The agents berberine, tet-
randrine, isoquercitrin and some flavonoids that increase reactive oxy-
gen species (ROS) formation in mitochondria exhibited synergistic
action with FLC against azole-resistant Candida strains [20–24]. The
synergistic effect of the Hsp90 inhibitor geldanamycin and FLC against
azole-resistant Candida tropicalis was also associated with increased ROS
formation [25,26]. FLC alone elicits ROS formation to some extent in
some fungal species, such as C. tropicalis, Candida glabrata, and Crypto-
coccus neoformans [27,28]. A high dose of FLC led to the disruption of
mitochondrial homeostasis, including mitochondrial membrane depo-
larization and ROS formation, and exhibited fungicidal activity against
C. albicans [29]. These results suggest that the mitochondria may be
another important organelle targeted by FLC. We previously found that
some potential efflux pump substrates can be delivered into Candida
cells by a mitochondrial-targeting moiety TPP+ and avoid efflux by
efflux pumps to kill azole-resistant strains [30]. These findings led to the
hypothesis that azole conjugates with mitochondria-targeting moieties
may overcome fungal azole resistance and increase azole efficacy.
In this study, we developed a series of AZDs with conjugation of the
FLC skeleton and mitochondria-targeting triphenylphosphonium cation
(TPP+) through ester bonds. These AZDs were active against common
drug-resistant Candida strains and could evade drug resistance.
Furthermore, they exerted antifungal activity by inhibiting ergosterol
synthesis and interfering with mitochondrial function. AZDs 20 and 25,
with potent in vitro activities, displayed fungicidal activity against
resistant C. albicans strains with Hsp90 inhibitors. Moreover, AZD 25
with minimal cytotoxicity could effectively prevent C. albicans biofilms,
which are highly resistant to antifungal drugs.
used to replace the 1,2,4-triazole group on the
α-carbon with the
difluorophenyl ring of FLC. The synthesis started with commercially
available 2,4-difluoro-alpha-(1H-1,2,4-triazolyl) acetophenone (2), the
carbonyl group of which was epoxidized by using trimethylsulfoxonium
iodide in the presence of a strong base to generate epoxide 3. For
piperazine-substituted azole analogs, epoxide 3 was reacted with
piperazine to form intermediate 4, which was transformed to bromides
5–12 using different alkyl dibromides under basic conditions. Finally,
TPP+-conjugated products 13–20 were prepared by coupling bromides
5–12 with intermediate 1, which was synthesized by procedures
described previously [30] (Scheme 1). Compounds 22–25 with a short
carbon chain length between the ester bond and TPP+ group were then
obtained by coupling intermediate 21 with bromides 8, 10, 11 and 12
(Scheme 2). Compounds 26–33 with terminal OH groups were prepared
for comparison with TPP+ conjugations through a nucleophilic substi-
tution reaction (Scheme 3).
2.2. Evaluation of the antifungal activity of AZDs
To evaluate the antifungal activity of these synthesized AZDs, two
types of C. albicans strains were selected to test the minimal inhibitory
concentrations (MICs), including the wild-type strain SC5314 and efflux
pump Cdrs-deficient strain DSY654. FLC was used as the positive con-
trol. The results are presented in Table 1. Based on the results, we found
that the piperazine-azole hybrids (26–33) were active against DSY654
but were inert towards the wild-type strain SC5314. When these hybrids
were conjugated with TPP+ (13–20, 22–25), the resulting products
became active against the wild-type strain. However, the length of alkyl
linkers obviously affected the activity. When the carbon chain length
between the ester bond and the FLC skeleton was less than eight, the
Scheme 1. General procedures for synthesizing azole analogs by conjugation of FLC skeleton with TPP+ using long-chain alkyl linker.
2

X. Wang et al.
Bioorganic Chemistry 110 (2021) 104771
Scheme 2. General procedures for synthesizing azole analogs by conjugation of FLC skeleton with TPP+ using short-chain alkyl linker.
Scheme 3. General procedures for synthesizing azole analogs without TPP+ group.
Table 1
Table 2
MIC values (µg/ml) of synthesized AZDs against C. albicans strains.
MIC values (µg/mL) of AZDs 20 and 25 against C. albicans strains.
MIC (µg/mL)
MIC (µg/mL)
Strains a
MIC
Compound
SC5314a
DSY654b
Compound
SC5314
DSY654
20
25
FLC
FLCc
13
14
15
16
17
18
19
20
22
23
2
0.25
16
16
16
8
24
25
26
27
28
29
30
31
32
33
4
2
SC5314
ATCC10231
YEM13
YEM15
DSY654
DSY1050
28A
1
1
2
16
16
16
16
8
1
0.5
>16
>16
16
8
1
0.5
1
1
>16
>16
>16
>16
>16
>16
>16
>16
1
64
1
2
64
0.5
0.5
0.5
2
0.5
0.5
0.5
2
0.25
0.25
>128
>128
>128
>128
>128
1
4
8
4
4
4
1
4
28I
1
0.5
16
4
1
G5
2
2
16
8
0.5
F5
2
1
Gu5
4
4
CP1
1
0.5
1
a
b
c
SC5314 is the C. albicans wild-type strain.
CG1
0.5
1
0.5
DSY654 is a constructed C. albicans strain with deletion of CDR1 and CDR2.
CT2
0.5
32
FLC represents Fluconazole.
a
ATCC10231 is a standard strain of C. albicans obtained from ATCC(American
Type culture collection); YEM13 is a constructed C. albicans strain with hyper-
expression of MDR1; YEM15 is a constructed C. albicans strain with hyper-
expression of both CDR1 and CDR2; DSY1050 is a constructed C. albicans strain
with deletion of CDR1, CDR2 and MDR1; 28A and 28I are clinical pan-azole-
resistant strains isolated from a cancer patient; G5 is a FLC-resistant clinical
isolate from AIDS patient G with the mutation of MRR1; F5 is a FLC-resistant
clinical isolate from AIDS patient F with the mutation of MRR1 and UPC2;
Gu5 is a FLC-resistant clinical isolate from AIDS patient Gu with the mutation of
TAC1; CP1 represents Candida Parapsilosis strain 1; CG1 represents Candida
glabrata strain 1; CT2 represents Candida tropicalis strain 2.
activity was relatively low. When the length of the carbon chain was
increased to more than eight, the activity was improved significantly.
The carbon chain length between the ester bond and TPP+ had minimal
effects on antifungal activity. Among them, AZDs 20 and 25 exhibited
the most potent antifungal activity.
So, AZDs 20 and 25 were chosen as the candidates to test the anti-
fungal activity against many other strains. Five clinical azole-resistant
strains including G5, F5, Gu5, 28A, and 28I isolated from AIDS or can-
cer patients who suffered from oropharyngeal candidiasis, two labora-
tory constructed azole-resistant strains YEM13 and YEM15, and one
azole-susceptible strain ATCC10231 were used in this study. As shown
in Table 2, they displayed potent antifungal activity against all tested
strains. Moreover, these AZDs were active against other clinically
derived Candida strains including C. tropicalis (CT2), Candida glabrata
(CG1) and Candida parapsilosis (CP1). These suggested the potential use
of AZDs 20 and 25 in treating candidiasis caused by azole-resistant
strains.
2.3. TPP+ conjugation facilitates cell entry into azole-resistant C. albicans
cells
To determine whether the discrepancy in the ability of AZDs and FLC
to combat azole-resistant strains was attributable to differential intra-
cellular accumulation, we measured the total intracellular contents of
FLC, AZD 20 and AZD 25 as two representatives in the wild-type strain
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X. Wang et al.
Bioorganic Chemistry 110 (2021) 104771
SC5314 and efflux pump-activated strains YEM13 and YEM15 by high-
performance liquid chromatography (HPLC). The average intracellular
contents of AZD 20 and AZD 25 in wild-type C. albicans strain SC5314
were 23.93
μ
g and 22.9
μ
g per 108 cells, respectively (Fig. 1), whereas
the amounts of the parent compounds without TPP+ moiety were below
the threshold of detection. Moreover, AZD 20 and AZD 25 accumulated
in the efflux pump-activated strains YEM13 and YEM15 (Fig. 1). In
contrast, we failed to detect the presence of FLC in the cells of YEM13
and YEM15. This suggests that the activity of AZDs against azole-
resistant strains was attributed to their ability to bypass active expul-
sion by efflux pumps. The ability of AZDs 20 and 25 to overcome the
barrier of drug efflux pumps is greatly meaningful to address the azole-
resistance problem because overexpression of efflux pumps dominate
the azole-resistant mechanisms.
Fig. 2. The potential of tested agents for developing drug resistance. C. albicans
ATCC10231 was repeated treated with a sub-lethal concentration of FLC, 20
and 25 for 40 passages. Each datum point represents one passage.
2.4. AZDs have a low tendency to induce drug resistance
C. albicans can readily develop FLC resistance [32]. To evaluate the
ability of C. albicans to develop resistance to AZDs, we exposed
C. albicans ATCC10231 to sub-MIC levels of AZD 20, AZD 25 and FLC for
40 days using a serial passage method in a 96-well plate. The MICs of
AZD 20 and AZD 25 increased 2-fold and 4-fold, respectively,
throughout 40 passages, whereas the MIC value of FLC increased by 64-
combination was restricted to only wild-type or resistant strains without
activated efflux pumps. Strains with activated efflux pumps, such as
YEM13 and YEM15, were still resistant to the combination treatment of
FLC and Hsp90 inhibitors. Here, we found that AZD 20 or 25 alone had
no obvious fungicidal activity, but the combination of the Hsp90 in-
hibitor geldanamycin (GA) and AZDs exhibited killing activity against
C. albicans strains with activated efflux pumps (Table 3). Transforming
fungistatic agents into fungicidal will benefit in treating candidaemia.
AZD 20 or 25 may be used in the future to eliminate resistant C. albicans
strains through combination with fungal selective Hsp90 inhibitors.
fold from 2 μg/mL to 128 μg/mL (Fig. 2).
We further used a rapid selection regime to select resistant mutants
in which large numbers of cells were plated onto medium containing
high concentrations of the tested agents. Three different strains,
including the wild-type strain SC5314 and efflux pump-deficient strains
DSY654 and DSY1050, were used in this assay. When large numbers of
SC5314 cells were plated on YPD medium containing FLC, many col-
2.6. Cytotoxicity assay
onies were recovered, even at 100
were plated on medium containing 20 or 25, no resistant colonies were
recovered when the concentrations were above 12.5 g/mL (Fig. S1). A
μg/mL (Fig. S1). However, when cells
We then evaluated the safety profile by testing hemolytic activity
against murine erythrocytes as well as cytotoxicity against mammalian
μ
cells. The hemolysis assay revealed that AZD 20 at 32 μg/mL and AZD
similar phenomenon was observed when strains DSY654 and DSY1050
were used (Fig. S1). These results demonstrate that AZDs 20 and 25
have a low probability of inducing resistance in C. albicans. The devel-
opment of drug resistance is an important factor that results in treatment
failure in managing fungal infections. These findings suggested that
TPP+ conjugation with azole drugs may not only address the problem of
acquired azole-resistance but also avoid the occurrence of drug
resistance.
25 at 64 μg/mL displayed < 5% hemolysis (Fig. 3). The MTT assay using
cell lines A549 and PC3 showed that AZD 25 had relatively low cell
cytotoxicity (Table S1). The hemolysis activity and cytotoxicity of AZD
25 were 32– or 64-fold higher than its overall MIC values. These results
suggest the potential in vivo use of AZD 25.
2.7. AZD 25 is active in preventing C. albicans biofilm formation
C. albicans biofilm formation on implanted medical devices is
increasingly recognized as a key mediator of fungal infections in the host
[37,38]. The formation of biofilms is associated with antifungal resis-
tance and increased pathogenicity [39]. We utilized both microscopic
inspection and XTT reduction assays to evaluate the ability of AZD 25 to
prevent biofilm formation. The microscopic images showed that
C. albicans formed a robust biofilm structure in the nondrug-treated
group. In contrast, C. albicans failed to form a biofilm structure in the
2.5. AZDs exhibited synergistic toxicity with GA against resistant C.
Albicans
Heat shock protein 90 (Hsp90) or its client protein calcineurin is
essential for the survival of C. albicans in the presence of FLC [33]. The
combination of FLC and inhibitors of calcineurin or Hsp90 exhibited
toxicity against C. albicans [34–36]. We found that the efficacy of this
Table 3
Synergistic antifungal action of FLC/AZDs and GA against C. albicans.
Strain
Compds.
Alone
Combination with GA
MIC
MFC
MIC
MFC
(µg/
(µg/
(µg/mL)
(µg/mL)
mL)
mL)
SC5314
YEM13
YEM15
FLC
20
2
>64
>64
>64
>64
4
0.5 + GA(1.25)
0.5 + GA(1.25)
0.25 + GA(1.25)
32 + GA(5)
0.5 + GA(1.25)
0.5 + GA(1.25)
0.5 + GA(1.25)
32 + GA(5)
1
25
1
FLC
20
64
1
0.5 + GA(5)
0.25 + GA(5)
32 + GA(5)
1 + GA(5)
25
1
16
0.5 + GA(5)
>64 + GA(5)
32 + GA(5)
FLC
20
64
1
>64
64
Fig. 1. The total intracellular contents of FLC, 20 and 25 in wild type strain
SC5314 and efflux pumps activated strains YEM13 and YEM15 measured
by HPLC.
1 + GA(5)
25
2
>64
0.5 + GA(5)
8 + GA(5)
4

X. Wang et al.
Bioorganic Chemistry 110 (2021) 104771
Fig. 3. Hemolytic activity against murine erythrocytes of AZDs 20 and 25.
presence of AZD 25, even at a dose of 0.25 μg/mL (Fig. 4). The CCK-8
reduction assay revealed that C. albicans biomass was dose-
dependently reduced under AZD 25 treatment. These results suggest
that AZD 25 can effectively prevent C. albicans biofilm formation.
2.8. AZD 25 still inhibits ergosterol synthesis
Based on these promising results, we needed to determine whether
the target of AZD 25 was still Erg11. We extracted and analyzed the
fungal sterols when cells were treated with AZD 25. GC–MS analysis
revealed that AZD 25 could almost completely block the synthesis of
ergosterol, similar to the results with FLC-treated samples (Fig. 5).
Due to the mitochondrial guidance role of TPP+, we inferred that
AZDs might affect normal mitochondrial function. We examined the
mitochondrial membrane potential (MMP) and ROS levels in two wild-
type C. albicans strains in the presence of AZD 25 or FLC (Fig. 6). The
data showed that MMP and ROS contents were significantly elevated in
cells challenged with AZD 25 or FLC. However, MMP in FLC-treated
cells was stable as the dose increased. In contrast, MMP in 25-treated
cells was dose-dependently reduced, suggesting the potential effect of
25 on MMP or mitochondrial function. These suggested that conjugation
with TPP+ enabled azole drugs to overcome antifungal resistance by
simultaneously avoiding active efflux and disturbing the mitochondrial
function of Candida cells.
Fig. 5. Analysis of the total sterols of C. albicans SC5314. Total intracellular
sterols were extracted using the alcoholic KOH method. The extracts were
analyzed by gas chromatography-mass spectrometry (GC–MS) (a: ergosterol; b:
β-sitosterol; c: lanosterol).
3. Conclusions
In summary, we synthesized two series of AZDs by conjugation of the
FLC skeleton and TPP+ using different alkyl linkers. The AZDs with over
eight carbons in the linker displayed potent activity against both wild-
type and azole-resistant Candida strains. Moreover, these AZDs had a
low probability of developing drug resistance compared with FLC. AZDs
20 and 25, as two representative agents with the most potent antifungal
activity, exerted synergistic antifungal activity with Hsp90 inhibitors
against C. albicans strains resistant to the combination treatment of FLC
and Hsp90 inhibitors. AZD 25 with minimal cytotoxicity blocked the
development of C. albicans biofilms in vitro. Moreover, AZD 25 could
interfere with normal mitochondrial function, probably due to
Fig. 4. The inhibitory effect of 25 on C. albicans biofilm information. C. albicans cells of SC5314, YEM13 and YEM15 were incubated with different concentrations of
25. After incubation of 24 h, the biofilm was imaged by a microscope (Scale bars, 100 µm) (A). Meanwhile, the biofilm mass was evaluated using a CCK-8 reduction
assay. The color change of biofilm was taken by a camera (B) and the OD450 of each well was measured by a microplate reader (C).
5

X. Wang et al.
Bioorganic Chemistry 110 (2021) 104771
Fig. 6. Analysis of mitochondria membrane potential and ROS of C. albicans. (A-B) The mitochondria membrane potential (A) and ROS (B) were detected using
Rh123 and ROS probe DCFHDA, respectively, after C. albicans SC5314 cells were treated with AZD 25 or FLC. The fluorescence intensity was detected by
flow cytometry.
mitochondrial targeting, while still retaining the ability to inhibit
ergosterol biosynthesis. These findings open a new avenue for
combating fungal resistance and treating azole-resistant candidaemia.
were added at room temperature. The reaction mixture was stirred at
60 ◦C for 4 h, and the reaction was monitored by TLC. When finished, the
reaction was quenched with water, and extrated with dichloromethane.
The combined extracts were washed with brine, dried over MgSO4,
filtered and concentrated under reduced pressure to give the crude
product, which was purified by flash chromatography to provide com-
pound 3 (3.60 g, 68.0%) as a yellow gummy oil.
4. Experimental section
4.1. General material and methods for chemistry
All commercially available reagents were used without further pu-
rification. Anhydrous solvents were dried through routine protocols. All
reactions were carried out under a nitrogen atmosphere in dry glassware
with magnetic stirring. Column chromatography was carried out on 200
– 300 mesh silica gel (Qingdao Haiyang Chemical, China). Analytical
TLC was carried out by employing 0.25 mm silica gel plates (HSGF254)
and visualization under UV light. The NMR spectra were recorded on a
Bruker 400 (1H, 400; 13C, 100 MHz) or Bruker 600 (1H, 600 MHz; 13C,
150 MHz) spectrometer. Chemical shifts were expressed in ppm, and J
values were given in Hz. High-resolution mass spectra were measured
using a Thermo Fisher Finnigan LTQ Orbitrap Elite mass spectrometer.
Ionization was achieved using the positive mode. All compounds eval-
uated for biological effects were > 95% pure.
4.1.3. 2-(2,4-difluorophenyl)-1-(piperazin-1-yl)-3-(1H-1,2,4-triazol-1-yl)
propan-2-ol (4)
To a stirred solution of compound 3 (1.00 g, 4.22 mmol) in EtOH
(10.0 mL) was added piperazine (3.60 g, 42.2 mmol) and Et3N (0.80 mL,
5.77 mmol) at room temperature. The reaction mixture was stirred at
80 ◦C for 12 h. After TLC analysis indicated the consumption of starting
material, the reaction was quenched with water and extracted with
dichloromethane. The combined extracts were washed with brine, dried
over MgSO4, filtered and concentrated under reduced pressure to give
the crude product, which was purified by flash chromatography to
provide compound 4 (1.05 g, 77.1%) as a yellow oil. 1H NMR (500 MHz,
CDCl3) δ 8.05 (s, 1H), 7.80 (s, 1H), 7.50 (dd, J = 15.6, 8.4 Hz, 1H), 6.90 –
6.72 (m, 2H), 4.56 (s, 2H), 3.06 (d, J = 13.6 Hz, 1H), 3.03 – 2.90 (m,
4H), 2.70 (d, J = 13.7 Hz, 1H), 2.68 – 2.55 (m, 4H). ESI-MS m/z
calculated for C15H20F2N5O+ [M+H]+ 324.16, found 324.16.
4.1.1. (7-carboxyheptyl)triphenylphosphonium bromide (1)
To a stirred solution of triphenylphosphine (5.00, 19.1 mmol) in
anhydrous toluene (30.0 mL) was added 8-bromooctanoic acid (3.00 g,
13.4 mmol) at room temperature. Then, the mixture was allowed to stir
under reflux for 12 h. After cooling to room temperature, the white
precipitate was filtered and washed with an additional 20.0 mL of
toluene. The white precipitate was dried in vaccum to afford compound
1, which was used directly without further purification.
4.1.4. (8-(3-bromopropoxy)-8-oxooctyl)triphenylphosphonium bromide
(5)
To a stirred solution of compound 4 (50.0 mg, 0.155 mmol) in DMF
(5.00 mL) was added 1,3-dibromopropane (217 μL, 1.55 mmol),and
K2CO3 (42.8 mg, 0.310 mmol) at room temperature. The reaction
mixture was stirred at room temperature for 24 h. After this time, the
reaction mixture was diluted with dichloromethane and washed with
H2O and brine, dried over MgSO4, filtered and concentrated under
reduced pressure to give the crude product, which was purified by flash
chromatography to provide compound 5 (33.9 mg, 49.3%) as a colorless
oil.
4.1.2. 1-((2-(2,4-difluorophenyl)oxiran-2-yl)methyl)-1H-1,2,4-triazole
(3)
To a stirred solution of 2,4-difuoro-2-(1H-1,2,4-triazo-1-yl)acetophe-
none (5.00 g, 22.4 mmol) in toluene (30.0 mL), trimethylsulfoxonium
iodide (5.40 g, 26.9 mmol), hexadecyltrimethylammonium bromide
(815 mg, 2.25 mmol), and 7.50 mL of 20.0% (v/v) sodium hydroxide
6

X. Wang et al.
Bioorganic Chemistry 110 (2021) 104771
4.1.5. (8-(4-bromobutoxy)-8-oxooctyl)triphenylphosphonium bromide (6)
To a stirred solution of compound 4 (50.0 mg, 0.155 mmol) in DMF
found 515.20.
(5.00 mL) was added 1,4-dibromobutane (184
μ
L, 1.55 mmol), and
4.1.10. 1-(4-(9-bromononyl)piperazin-1-yl)-2-(2,4-difluorophenyl)-3-
(1H-1,2,4-triazol-1-yl)propan-2-ol (11)
K2CO3 (42.8 mg, 0.310 mmol). The reaction mixture was stirred at room
temperature for 24 h. The reaction mixture was diluted with dichloro-
methane and washed with H2O and brine, dried over MgSO4, filtered
and concentrated under reduced pressure to give the crude product,
which was purified by flash chromatography to provide compound 6
(34.2 mg, 48.3%) as a colorless oil.
To a stirred solution of compound 4 (50.0 mg, 0.155 mmol) in DMF
(5.00 mL) was added 1,9-dibromononane (312 μL, 1.55 mmol), and
K2CO3 (42.8 mg, 0.310 mmol). The reaction mixture was stirred at room
temperature for 24 h. The reaction mixture was diluted with dichloro-
methane and washed with H2O and brine, dried over MgSO4, filtered
and concentrated under reduced pressure to give the crude product,
which was purified by flash chromatography to provide compound 11
(38.0 mg, 46.6%) as a colorless oil. 1H NMR (500 MHz, CDCl3) δ 8.00 (d,
J = 4.3 Hz, 1H), 7.82 (d, J = 2.8 Hz, 1H), 7.50 (s, 1H), 6.82 (s, 1H), 4.63
– 4.56 (m, 1H), 3.40 (dd, J = 8.0, 5.6 Hz, 1H), 3.29 (t, J = 13.6 Hz, 1H),
3.10 (dd, J = 20.2, 13.3 Hz, 1H), 2.89 – 2.78 (m, 1H), 2.73 – 2.63 (m,
1H), 1.86 (s, 1H), 1.42 (s, 1H), 1.29 (d, J = 11.1 Hz, 1H). ESI-MS m/z
calculated for C24H37BrF2N5O+ [M+H]+ 529.21, found 529.21.
4.1.6. (8-((5-bromopentyl)oxy)-8-oxooctyl)triphenylphosphonium
bromide (7)
To a stirred solution of compound 4 (50.0 mg, 0.155 mmol) in DMF
(5.00 mL) was added 1,5-dibromopentane (208 μL, 1.55 mmol), and
K2CO3 (42.8 mg, 0.310 mmol). The reaction mixture was stirred at room
temperature for 24 h. When finished, the reaction mixture was diluted
with dichloromethane and washed with H2O and brine, dried over
MgSO4, filtered and concentrated under reduced pressure to give the
crude product, which was purified by flash chromatography to provide
compound 7 (36.9 mg, 49.8%) as a colorless oil.
4.1.11. 1-(4-(10-bromodecyl)piperazin-1-yl)-2-(2,4-difluorophenyl)-3-
(1H-1,2,4-triazol-1-yl)propan-2-ol (12)
To a stirred solution of compound 4 (50.0 mg, 0.155 mmol) in DMF
4.1.7. 1-(4-(6-bromohexyl)piperazin-1-yl)-2-(2,4-difluorophenyl)-3-(1H-
1,2,4-triazol-1-yl)propan-2-ol (8)
(5.00 mL) was added 1,10-dibromodecane (350 μL, 1.55 mmol), and
K2CO3 (42.8 mg, 0.310 mmol). The reaction mixture was stirred at room
temperature for 24 h. The reaction mixture was diluted with dichloro-
methane and washed with H2O and brine, dried over MgSO4, filtered
and concentrated under reduced pressure to give the crude product,
which was purified by flash chromatography to provide compound 12
(27.9 mg, 33.3%) as a colorless oil. 1H NMR (500 MHz, CDCl3) δ 8.05 (s,
1H), 7.80 (s, 1H), 7.47 (dd, J = 15.7, 9.0 Hz, 1H), 6.79 (dd, J = 14.5, 7.7
Hz, 2H), 4.61 (s, 2H), 3.48 (s, 2H), 3.31 (dd, J = 26.1, 11.9 Hz, 2H), 3.12
(t, J = 11.5 Hz, 2H), 2.91 – 2.74 (m, 6H), 1.76 (dt, J = 13.3, 6.8 Hz, 4H),
1.48 (dt, J = 15.3, 7.9 Hz, 2H), 1.43 – 1.23 (m, 12H). ESI-MS m/z
calculated for C25H39BrF2N5O+ [M+H]+ 543.22, found 543.23.
To a stirred solution of compound 4 (50.0 mg, 0.155 mmol) in DMF
(5.00 mL) was added 1,6-dibromohexane (235 μL, 1.55 mmol), and
K2CO3 (42.8 mg, 0.310 mmol). The reaction mixture was stirred at room
temperature for 24 h. When finished, the reaction mixture was diluted
with dichloromethane and washed with H2O and brine, dried over
MgSO4, filtered and econcentrated under reduced pressure to give the
crude product, which was purified by flash chromatography to provide
1
compound 8 (23.0 mg, 30.6%) as a colorless oil. H NMR (500 MHz,
CDCl3) δ 8.09 (s, 1H), 7.75 (s, 1H), 7.47 (dd, J = 15.5, 8.6 Hz, 1H), 6.77
(dd, J = 12.3, 6.8 Hz, 2H), 4.56 (s, 2H), 3.38 (t, J = 6.8 Hz, 3H), 2.99 –
2.53 (m, 12H), 1.93 – 1.75 (m, 2H), 1.74 – 1.59 (m, 2H), 1.26 (m, 6H).
ESI-MS m/z calculated for C21H31BrF2N5O+ [M+H]+ 487.17, found
487.17.
4.1.12. (8-(3-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-
1-yl)propyl) piperazin-1-yl)propoxy)-8-oxooctyl)triphenylphosphonium
bromide (13)
4.1.8. 1-(4-(7-bromoheptyl)piperazin-1-yl)-2-(2,4-difluorophenyl)-3-(1H-
1,2,4-triazol-1-yl)propan-2-ol (9)
To a stirred solution of compound 1 (13.0 mg, 0.040 mmol) in DMF
(3.00 mL) was added compound 5 (30.0 mg, 0.050 mmol), K2CO3 (11.1
mg, 0.080 mmol) and tetrabutylammonium iodide (3.0 mg, 0.008
mmol). The reaction mixture was then stirred at room temperature for
24 h, after which TLC analysis indicated the consumption of the starting
material. The reaction mixture was diluted with dichloromethane and
washed with H2O and brine, dried over MgSO4, filtered and concen-
trated under reduced pressure to give the crude product, which was
purified by flash chromatography to provide compound 13 (16.2 mg,
38.5%) as a colorless oil. 1H NMR (500 MHz, CD3OD) δ 8.34 (s, 1H), 7.89
(dd, J = 7.5, 6.8 Hz, 3H), 7.83 – 7.70 (m, 13H), 7.46 (dd, J = 15.6, 8.7
Hz, 1H), 6.97 – 6.89 (m, 1H), 6.88 – 6.82 (m, 1H), 4.72 – 4.61 (m, 2H),
4.08 (t, J = 6.2 Hz, 2H), 3.44 – 3.36 (m, 2H), 3.05 – 2.55 (m, 12H), 1.63 –
1.53 (m, 6H), 1.33 (s, 8H). 13C NMR (125 MHz, CD3OD) δ 175.2, 136.3
(d, J = 2.8 Hz), 134.8 (d, J = 9.9 Hz), 131.5 (d, J = 12.6 Hz), 129.8,
112.0 (d, J = 86.3 Hz), 111.9, 104.9, 69.1, 63.2, 55.7, 54.3, 53.9, 40.2,
34.8, 33.1, 29.7, 29.47, 26.9, 25.7, 14.4. HR-ESI-MS m/z calculated for
To a stirred solution of compound 4 (50.0 mg, 0.155 mmol) in DMF
(5.00 mL) was added 1,7-dibromoheptane (260 μL, 1.55 mmol), and
K2CO3 (42.8 mg, 0.310 mmol). The reaction mixture was stirred at room
temperature for 24 h. The reaction mixture was diluted with dichloro-
methane and washed with H2O and brine, dried over MgSO4, filtered
and concentrated under reduced pressure to give the crude product,
which was purified by flash chromatography to provide compound 9
(33.0 mg, 42.7%) as a colorless oil. 1H NMR (500 MHz, CDCl3) δ 8.05 (s,
1H), 7.81 (s, 1H), 7.48 (dd, J = 15.6, 9.1 Hz, 1H), 6.83 – 6.76 (m, 2H),
4.58 (s, 2H), 3.39 (t, J = 6.7 Hz, 2H), 2.95 (dd, J = 158.4, 13.7 Hz, 8H),
1.88 – 1.71 (m, 6H), 1.46 – 1.30 (m, 8H). ESI-MS m/z calculated for
C
22H33BrF2N5O+ [M+H]+ 501.17, found 501.18.
4.1.9. 1-(4-(8-bromooctyl)piperazin-1-yl)-2-(2,4-difluorophenyl)-3-(1H-
1,2,4-triazol-1-yl)propan-2-ol (10)
To a stirred solution of compound 4 (50.0 mg, 0.155 mmol) in DMF
C
44H53F2N5O3P+ [M - Br]+ 768.3849, found 768.3845.
(5.00 mL) was added 1,8-dibromooctane (283
μL, 1.55 mmol), and
K2CO3 (42.8 mg, 0.310 mmol). The reaction mixture was stirred at room
temperature for 24 h. The reaction mixture was diluted with dichloro-
methane and washed with H2O and brine, dried over MgSO4, filtered
and concentrated under reduced pressure to give the crude product,
which was purified by flash chromatography to provide compound 10
(45.0 mg, 56.7%) as a colorless oil. 1H NMR (500 MHz, CDCl3) δ 8.03 (s,
1H), 7.81 (s, 1H), 7.48 (dd, J = 15.7, 8.8 Hz, 1H), 6.80 (dd, J = 14.4, 7.9
Hz, 2H), 4.63 – 4.54 (m, 2H), 3.40 (d, J = 6.7 Hz, 2H), 2.90 (dt, J = 58.3,
29.1 Hz, 10H), 1.87 – 1.79 (m, 4H), 1.41 (dt, J = 13.5, 6.9 Hz, 2H), 1.32
(m, 8H). ESI-MS m/z calculated for C23H35BrF2N5O+ [M+H]+ 515.19,
4.1.13. (8-(4-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-
1-yl)propyl) piperazin-1-yl)butoxy)-8-oxooctyl)triphenylphosphonium
bromide (14)
To a stirred solution of compound 1 (25.0 mg, 0.080 mmol) in DMF
(3.00 mL) was added compound 6 (40.0 mg, 0.060 mmol), K2CO3 (18.0
mg, 0.130 mmol) and tetrabutylammonium iodide (6.0 mg, 0.016
mmol). The reaction mixture was then stirred at room temperature for
24 h, after which TLC analysis indicated the consumption of the starting
material. The reaction mixture was diluted with dichloromethane and
washed with H2O and brine, dried over MgSO4, filtered and
7

X. Wang et al.
Bioorganic Chemistry 110 (2021) 104771
concentrated under reduced pressure to give the crude product, which
was purified by flash chromatography to provide compound 14 (10.5
mg, 18.8%) as a colorless oil. 1H NMR (500 MHz, CD3OD) δ 8.38 (s, 1H),
7.89 (t, J = 7.4 Hz, 3H), 7.78 (dq, J = 10.9, 8.0 Hz, 13H), 7.47 (dd, J =
15.6, 8.6 Hz, 1H), 6.97 – 6.90 (m, 1H), 6.90 – 6.83 (m, 1H), 4.74 – 4.66
(m, 2H), 4.08 (d, J = 6.2 Hz, 2H), 3.45 – 3.37 (m, 2H), 3.06 – 2.66 (m,
12H), 1.56 (dq, J = 14.9, 7.6 Hz, 6H), 1.29 (s, 10H).13C NMR (126 MHz,
CD3OD) δ 175.4, 136.25 (d, J = 2.7 Hz), 134.8 (d, J = 10.0 Hz), 131.5 (d,
J = 12.5 Hz), 112.0 (d, J = 86.3 Hz), 112.1 (dd, J = 21.2, 3.3 Hz), 104.9,
75.8 (d, J = 5.5 Hz), 64.1 (d, J = 3.5 Hz), 57.9, 57.3 (d, J = 4.4 Hz), 53.5,
53.3, 34.8 33.0, 29.7, 29.5, 27.1, 25.7, 23.4, 22.4, 14.4. HR-ESI-MS m/z
calculated for C45H55F2N5O3P+ [M - Br]+ 782.4005, found 782.4008.
mmol). The reaction mixture was then stirred at room temperature for
24 h, after which TLC analysis indicated the consumption of the starting
material. The reaction mixture was diluted with dichloromethane and
washed with H2O and brine, dried over MgSO4, filtered and concen-
trated under reduced pressure to give the crude product, which was
purified by flash chromatography to provide compound 17 (13.0 mg,
45.0%) as a colorless oil. 1H NMR (500 MHz, CD3OD) δ 8.37 (s, 1H), 7.89
(t, J = 7.1 Hz, 3H), 7.84 – 7.74 (m, 13H), 7.47 (dd, J = 15.7, 8.7 Hz, 1H),
6.98 – 6.91 (m, 1H), 6.87 (td, J = 8.7, 1.8 Hz, 1H), 4.69 (s, 2H), 4.04 (t, J
= 6.6 Hz, 2H), 3.44 – 3.37 (m, 2H), 2.92 (ddd, J = 81.6, 59.4, 32.8 Hz,
12H), 1.59 – 1.51 (m, 6H), 1.37 (s, 6H), 1.29 (s, 10H).13C NMR (125
MHz, CD3OD) δ 175.3, 136.1 (d, J = 3.1 Hz), 134.4 (d, J = 9.9 Hz), 131.4
(d, J = 12.6 Hz), 119.4 (d, J = 86.3 Hz), 112.0 (d, J = 24.6 Hz), 104.8,
75.6 (d, J = 5.5 Hz), 67.0, 65.2, 64.0, 57.1 (dd, J = 4.8, 3.2 Hz), 53.0,
52.5, 29.5 (d, J = 3.6 Hz), 29.3 (d, J = 3.1 Hz), 27.4, 26.5, 25.5, 23.2,
22.9, 22.5. HR-ESI-MS m/z calculated for C48H61F2N5O3P+ [M - Br]+
824.4475, found 824.4467.
4.1.14. (8-((5-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-
1-yl)propyl) piperazin-1-yl)pentyl)oxy)-8-oxooctyl)triphenylphosphonium
bromide (15)
To a stirred solution of compound 1 (12.0 mg, 0.040 mmol) in DMF
(3.00 mL) was added compound 7 (30.0 mg, 0.050 mmol), K2CO3 (11.1
mg, 0.080 mmol) and tetrabutylammonium iodide (3.0 mg, 0.008
mmol). The reaction mixture was then stirred at room temperature for
24 h, after which TLC analysis indicated the consumption of the starting
material. The reaction mixture was diluted with dichloromethane and
washed with H2O and brine, dried over MgSO4, filtered and concen-
trated under reduced pressure to give the crude product, which was
purified by flash chromatography to provide compound 15 (12.3 mg,
30.1%) as a colorless oil. 1H NMR (500 MHz, CD3OD) δ 8.34 (s, 1H), 7.89
(td, J = 7.2, 1.8 Hz, 3H), 7.84 – 7.74 (m, 13H), 7.47 (td, J = 9.0, 6.7 Hz,
1H), 6.94 (ddd, J = 11.6, 8.8, 2.4 Hz, 1H), 6.87 (td, J = 8.5, 2.6 Hz, 1H),
4.70 (t, J = 8.7 Hz, 3H), 4.06 (t, J = 6.5 Hz, 2H), 3.65 (d, J = 2.4 Hz, 1H),
3.44 – 3.37 (m, 2H), 3.35 (s, 4H), 3.24 – 2.56 (m, 8H), 1.62 – 1.52 (m,
6H), 1.37 – 1.32 (m, 12H).13C NMR (125 MHz, DMSO) δ 175.5, 151.2,
146.1, 136.3 (d, J = 3.0 Hz), 134.8 (d, J = 9.9 Hz), 131.5 (d, J = 12.5
Hz), 112.0 (d, J = 89.5 Hz), 112.1 (d, J = 24.1 Hz), 104.9, 76.2 (d, J =
5.6 Hz), 69.1, 65.0, 57.9, 57.2 (d, J = 4.8 Hz), 53.3, 52.7, 40.2, 33.0,
31.6, 29.8, 29.5, 29.2, 28.1, 24.9, 24.2, 14.4.HR-ESI-MS m/z calculated
for C46H57F2N5O3P+ [M - Br]+ 796.4162, found 796.4156.
4.1.17. (8-((8-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-
1-yl)propyl) piperazin-1-yl)octyl)oxy)-8-oxooctyl)triphenylphosphonium
bromide (18)
To a stirred solution of compound 10 (20.0 mg, 0.040 mmol) in DMF
(3.00 mL) was added compound 1 (39.0 mg, 0.080 mmol), K2CO3 (11.0
mg, 0.080 mmol),and tetrabutylammonium iodide (3.0 mg, 0.008
mmol). The reaction mixture was then stirred at room temperature for
24 h, after which TLC analysis indicated the consumption of the starting
material. The reaction mixture was diluted with dichloromethane and
washed with H2O and brine, dried over MgSO4, filtered and concen-
trated under reduced pressure to give the crude product, which was
purified by flash chromatography to provide compound 18 (17.0 mg,
47.2%) as a colorless oil.1H NMR (500 MHz, CD3OD) δ 8.42 (s, 1H), 7.89
(td, J = 7.2, 1.5 Hz, 3H), 7.79 (dtd, J = 11.5, 8.3, 5.4 Hz, 13H), 7.47 (td,
J = 8.9, 6.8 Hz, 1H), 6.93 (ddd, J = 11.6, 8.9, 2.4 Hz, 1H), 6.86 (td, J =
8.4, 2.3 Hz, 1H), 4.72 (s, 2H), 4.03 (t, J = 6.6 Hz, 2H), 3.47 – 3.39 (m,
2H), 2.97 (ddd, J = 90.9, 60.2, 45.9 Hz, 12H), 1.62 – 1.53 (m, 6H), 1.40
– 1.27 (m, 18H).13C NMR (125 MHz, CD3OD) δ 175.5, 151.0, 136.2 (d, J
= 2.9 Hz), 134.8 (d, J = 10.0 Hz), 131.5 (d, J = 12.5 Hz), 112.0 (d, J =
86.2 Hz), 112.1 (dd, J = 21.4, 2.4 Hz), 104.9 (t, J = 26.8 Hz), 76.1 (d, J
= 5.5 Hz), 65.4, 64.0 (d, J = 3.7 Hz), 58.0, 57.3 (d, J = 5.1 Hz), 53.2,
52.7, 34.9, 30.0, 29.7, 29.4, 27.6, 26.8, 25.8, 25.1, 23.5, 22.8, 22.4. HR-
ESI-MS m/z calculated for C49H63F2N5O3P+ [M - Br]+ 838.4631, found
838.4626.
4.1.15. (8-((6-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-
1-yl)propyl) piperazin-1-yl)hexyl)oxy)-8-oxooctyl)triphenylphosphonium
bromide (16)
To a stirred solution of compound 8 (10.0 mg, 0.020 mmol) in DMF
(3.00 mL) was added compound 1 (11.0 mg, 0.04 mmol), K2CO3 (5.50
mg, 0.04 mmol), and tetrabutylammonium iodide (2.00 mg, 0.004
mmol). The reaction mixture was then stirred at room temperature for
24 h, after which TLC analysis indicated the consumption of the starting
material. The reaction mixture was diluted with dichloromethane and
washed with H2O and brine, dried over MgSO4, filtered and concen-
trated under reduced pressure to give the crude product, which was
purified by flash chromatography to provide compound 16 (9.00 mg,
50.1%) as a colorless oil. 1H NMR (500 MHz, CD3OD) δ 8.33 (s, 1H), 7.89
(td, J = 7.2, 1.7 Hz, 3H), 7.83 – 7.73 (m, 13H), 7.51 – 7.44 (m, 1H), 6.94
(ddd, J = 11.6, 8.9, 2.4 Hz, 1H), 6.87 (td, J = 8.6, 2.3 Hz, 1H), 4.72 –
4.63 (m, 2H), 4.04 (t, J = 6.6 Hz, 2H), 3.40 (td, J = 13.5, 8.4 Hz, 2H),
2.85 (ddd, J = 74.8, 56.0, 27.7 Hz, 12H), 1.55 (dt, J = 15.6, 7.9 Hz, 6H),
1.29 (s, 14H). 13C NMR (125 MHz, CDCl3) δ 173.9, 150.8, 145.1, 135.2,
133.7 (d, J = 9.8 Hz), 130.7 (d, J = 12.4 Hz), 118.4 (d, J = 85.8 Hz),
111.5 (d, J = 20.7 Hz), 64.2, 62.6, 57.5, 56.1, 52.3, 34.4, 32.0, 29.8,
28.9, 28.5, 27.3, 26.7, 25.8, 25.0, 22.8. HR-ESI-MS m/z calculated for
4.1.18. (8-((9-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-
1-yl)propyl) piperazin-1-yl)nonyl)oxy)-8-oxooctyl)triphenylphosphonium
bromide (19)
To a stirred solution of compound 11 (50.0 mg, 0.090 mmol) in DMF
(3.00 mL) was added compound 1 (91.0 mg, 0.200 mmol), K2CO3 (25.0
mg, 0.180 mmol), and tetrabutylammonium iodide (6.0 mg, 0.018
mmol). The reaction mixture was then stirred at room temperature for
24 h, after which TLC analysis indicated the consumption of the starting
material. The reaction mixture was diluted with dichloromethane and
washed with H2O and brine, dried over MgSO4, filtered and concen-
trated under reduced pressure to give the crude product, which was
purified by flash chromatography to provide compound 19 (27.3 mg,
30.9%) as a colorless oil. 1H NMR (500 MHz, CD3OD) δ 8.37 (s, 1H), 7.89
(td, J = 7.2, 1.6 Hz, 3H), 7.84 – 7.75 (m, 12H), 7.47 (td, J = 8.9, 6.8 Hz,
1H), 6.93 (ddd, J = 11.6, 8.9, 2.4 Hz, 1H), 6.86 (td, J = 8.4, 2.4 Hz, 1H),
4.73 – 4.66 (m, 2H), 4.03 (t, J = 6.7 Hz, 2H), 3.46 – 3.38 (m, 2H), 2.92
(ddd, J = 73.4, 59.2, 31.2 Hz, 12H), 1.61 – 1.55 (m, 6H), 1.42 – 1.22 (m,
20H). 13C NMR (125 MHz, CD3OD) δ 175.5, 151.1, 136.2 (d, J = 2.8 Hz),
134.8 (d, J = 10.0 Hz), 131.5 (d, J = 12.6 Hz), 112.0 (d, J = 86.3 Hz),
112.1 (d, J = 17.8 Hz), 104.9 (t, J = 26.8 Hz), 76.1, 65.5, 64.0, 58.2,
57.2, 53.3, 52.9, 34.9, 30.3, 30.2, 29.7, 29.4, 27.7, 26.9, 25.8, 25.3,
23.5, 22.8, 22.4. HR-ESI-MS m/z calculated for C50H65F2N5O3P+ [M -
Br]+ 852.4788, found 852.4772.
C
47H59F2N5O3P+ [M - Br]+ 810.4318, found 810.4305.
4.1.16. (8-((7-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-
1-yl)propyl) piperazin-1-yl)heptyl)oxy)-8-oxooctyl)triphenylphosphonium
bromide (17)
To a stirred solution of compound 9 (16.0 mg, 0.032 mmol) in DMF
(3.00 mL) was added compound 1 (18.0 mg, 0.040 mmol), K2CO3 (8.29
mg, 0.060 mmol),and tetrabutylammonium iodide (3.0 mg, 0.006
8

X. Wang et al.
Bioorganic Chemistry 110 (2021) 104771
4.1.19. (8-((10-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-
triazol-1-yl)propyl) piperazin-1-yl)decyl)oxy)-8-oxooctyl)
chromatography to provide compound 23 (10.0 mg, 28.9%) as a
colorless oil. 1H NMR (500 MHz, CD3OD) δ 8.36 (s, 1H), 7.91 (t, J = 7.0
Hz, 3H), 7.80 (ddd, J = 11.0, 9.8, 5.5 Hz, 13H), 7.48 (dd, J = 15.4, 8.7
Hz, 1H), 6.95 (ddd, J = 11.7, 9.0, 2.3 Hz, 1H), 6.91 – 6.86 (m, 1H), 4.69
(s, 2H), 4.10 – 4.05 (m, 2H), 3.72 (dd, J = 12.3, 6.0 Hz, 4H), 3.12 – 2.70
(m, 9H), 1.60 (d, J = 7.3 Hz, 6H), 1.33 (s, 8H). 13C NMR (125 MHz,
CD3OD) δ 171.8 (d, J = 14.9 Hz), 136.5 (d, J = 2.9 Hz), 134.9 (d, J =
10.1 Hz), 131.6 (d, J = 12.7 Hz), 130.8, 119.4 (d, J = 87.2 Hz), 67.2,
66.7 (d, J = 5.9 Hz), 63.9 (d, J = 3.3 Hz), 61.4, 58.1, 57.3 (d, J = 4.1 Hz),
53.3, 52.8 (d, J = 2.6 Hz), 30.5, 30.3, 28.1, 26.9, 23.7, 14.4. HR-ESI-MS
m/z calculated for C44H53F2N5O3P+ [M - Br]+ 768.3849, found
768.3861.
triphenylphosphonium bromide (20)
To a stirred solution of compound 12 (50.0 mg, 0.090 mmol) in DMF
(3.00 mL) was added compound 1 (54.4 mg, 0.110 mmol), K2CO3 (24.9
mg, 0.180 mmol), and tetrabutylammonium iodide (6.0 mg, 0.018
mmol). The reaction mixture was then stirred at room temperature for
24 h, after which TLC analysis indicated the consumption of the starting
material. The reaction mixture was diluted with dichloromethane and
washed with H2O and brine, dried over MgSO4, filtered and concen-
trated under reduced pressure to give the crude product, which was
purified by flash chromatography to provide compound 20 (34.6 mg,
40.3%) as a colorless oil. 1H NMR (500 MHz, CD3OD) δ 8.34 (s, 1H), 7.89
(td, J = 7.0, 1.5 Hz, 3H), 7.83 – 7.75 (m, 13H), 7.46 (td, J = 8.9, 6.8 Hz,
1H), 6.93 (ddd, J = 11.6, 8.9, 2.4 Hz, 1H), 6.86 (td, J = 8.4, 2.3 Hz, 1H),
4.70 (d, J = 14.3 Hz, 1H), 4.65 (d, J = 14.3 Hz, 1H), 4.03 (dd, J = 9.0,
4.3 Hz, 2H), 3.44 – 3.37 (m, 2H), 3.07 – 2.65 (m, 12H), 1.56 (dd, J =
15.0, 7.5 Hz, 6H), 1.31 (dd, J = 14.8, 9.2 Hz, 22H). 13C NMR (125 MHz,
CD3OD) δ 174.1, 134.8 (d, J = 2.9 Hz), 133.4 (d, J = 10.0 Hz), 130.1 (d,
J = 12.6 Hz), 118.6(d, J = 86.4 Hz), 110.7 (d, J = 24.2 Hz), 103.5, 64.1,
62.7, 57.1, 55.9, 53.4, 52.0 (d, J = 18.2 Hz), 33.5, 29.1 (d, J = 6.6 Hz),
28.9, 28.3, 28.0, 26.5, 25.6, 24.4, 22.0, 21.4, 21.0. HR-ESI-MS m/z
calculated for C51H67F2N5O3P+ [M - Br]+ 866.4944, found 866.4937.
4.1.23. (3-((9-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-
1-yl)propyl) piperazin-1-yl)nonyl)oxy)-3-oxopropyl)triphenylphosphonium
bromide (24)
To a stirred solution of compound 11 (35.0 mg, 0.066 mmol) in DMF
(3.00 mL) was added compound 21 (52.0 mg, 0.132 mmol), K2CO3
(18.2 mg, 0.132 mmol), and tetrabutylammonium iodide (5.0 mg,0.013
mmol). The reaction mixture was then stirred at room temperature for
24 h, after which TLC analysis indicated the consumption of the starting
material. The reaction mixture was diluted with DCM and washed with
H2O and brine, dried over MgSO4, filtered and concentrated under
reduced pressure to give the crude product, which was purified by flash
chromatography to provide compound 24 (24.6 mg, 43.1%) as a
colorless oil. 1H NMR (400 MHz, DMSO) δ 8.36 (d, J = 4.3 Hz, 1H), 7.90
(t, J = 7.1 Hz, 3H), 7.87 – 7.73 (m, 13H), 7.38 (dd, J = 16.1, 8.6 Hz, 1H),
7.15 (t, J = 10.5 Hz, 1H), 6.95 (t, J = 8.4 Hz, 1H), 4.63 – 4.52 (m, 2H),
3.89 – 3.77 (m, 2H), 3.53 (d, J = 19.8 Hz, 2H), 2.77 (ddd, J = 35.0, 27.2,
12.1 Hz, 12H), 1.58 (s, 2H), 1.52 – 1.42 (m, 2H), 1.22 (s, 10H). 13C NMR
(101 MHz, DMSO‑d6) δ 166.6 (dd, J = 737.9, 14.9 Hz), 159.0 (dd, J =
261.6, 14.6 Hz), 150.4, 145.0, 135.1 (d, J = 2.5 Hz), 133.7 (d, J = 10.3
Hz), 130.3 (d, J = 12.6 Hz), 118.0 (d, J = 86.5 Hz), 110.8 (dd, J = 20.1,
2.7 Hz), 103.8 (t, J = 27.0 Hz), 74.9 (d, J = 5.0 Hz), 64.9, 62.7 (d, J =
16.1 Hz), 60.7, 55.6 (d, J = 7.7 Hz), 51.0 (d, J = 13.5 Hz), 32.5, 31.3,
28.0, 26.1, 25.3, 14.0.HR-ESI-MS m/z calculated for C45H55F2N5O3P+
[M - Br]+ 782.4005, found 782.3993.
4.1.20. (2-carboxyethyl)triphenylphosphonium bromide (21)
To a stirred solution of triphenylphosphine (5.00 g, 19.1 mmol) in
anhydrous toluene (30.0 mL) was added 3-bromopropanoic acid (2.00 g,
12.7 mmol). Then, the mixture was allowed to stir under reflux for 12 h.
After cooling to room temperature, the white precipitate was filtered
and washed with an additional 20.0 mL of toluene. The precipitate was
dried under vaccum to afford compound 21, which was used directly
without further purification.
4.1.21. (3-((6-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-
1-yl)propyl) piperazin-1-yl)hexyl)oxy)-3-oxopropyl)triphenylphosphonium
bromide (22)
To a stirred solution of compound 8 (10.0 mg, 0.020 mmol) in DMF
(3.00 mL) was added compound 21 (8.00 mg, 0.024 mmol), K2CO3
(5.53 mg, 0.040 mmol), and tetrabutylammonium iodide (2.00 mg,
0.004 mmol). The reaction mixture was then stirred at room tempera-
ture for 24 h, after which TLC analysis indicated the consumption of the
starting material. The reaction mixture was diluted with DCM and
washed with H2O and brine, dried over MgSO4, filtered and concen-
trated under reduced pressure to give the crude product, which was
purified by flash chromatography to provide compound 22 (7.0 mg,
41.4%) as a colorless oil. 1H NMR (500 MHz, CD3OD) δ 8.35 (s, 1H), 7.91
(dd, J = 7.3, 5.8 Hz, 3H), 7.80 (ddd, J = 10.9, 7.8, 5.4 Hz, 13H), 7.48
(dd, J = 15.6, 8.9 Hz, 1H), 6.99 – 6.92 (m, 1H), 6.88 (td, J = 8.7, 2.2 Hz,
1H), 4.69 (s, 2H), 4.23 (dd, J = 13.5, 6.2 Hz, 2H), 3.11 – 2.53 (m, 14H),
1.63 – 1.51 (m, 6H), 1.41 (s, 4H).13C NMR (126 MHz, CD3OD) δ 175.7,
170.6, 167.9, 135.6 (d, J = 3.0 Hz), 133.9 (dd, J = 10.1, 5.9 Hz), 130.0
(d, J = 3.9 Hz), 117.8 (d, J = 86.7 Hz), 68.3, 65.7, 64.8, 64.0, 61.2, 56.9
(d, J = 12.1 Hz), 29.8, 29.5, 27.4, 22.8, 14.3.HR-ESI-MS m/z calculated
for C42H49F2N5O3P+ [M - Br]+ 740.3536, found 740.3535.
4.1.24. (3-((10-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-
triazol-1-yl)propyl) piperazin-1-yl)decyl) oxy)-3-oxopropyl)
triphenylphosphonium bromide (25)
To a stirred solution of compound 12 (50.0 mg, 0.092 mmol) in DMF
(3.00 mL) was added compound 21 (36.9 mg, 0.110 mmol), K2CO3
(25.4 mg, 0.184 mmol) and tetrabutylammonium iodide(7.00 mg,
0.020 mmol). The reaction mixture was then stirred at room tempera-
ture for 24 h, after which TLC analysis indicated the consumption of the
starting material. The reaction mixture was diluted with DCM and
washed with H2O and brine, dried over MgSO4, filtered and concen-
trated under reduced pressure to give the crude product, which was
purified by flash chromatography to provide compound 25 (15.4 mg,
15.9%) as a colorless oil. 1H NMR (500 MHz, CDCl3) δ 8.36 (s, 1H), 7.82
– 7.64 (m, 16H), 7.49 (dd, J = 15.7, 8.7 Hz, 1H), 6.73 (dd, J = 12.8, 8.0
Hz, 2H), 4.68 (d, J = 14.2 Hz, 1H), 4.58 (d, J = 14.2 Hz, 1H), 3.80 (t, J =
6.6 Hz, 2H), 2.99 – 2.89 (m, 6H), 2.82 – 2.64 (m, 8H), 1.65 (s, 2H), 1.45
– 1.39 (m, 2H), 1.22 (s, 14H). 13C NMR (125 MHz, CDCl3) δ 170.5 (d, J
= 9.2 Hz), 163.6 (d, J = 12.7 Hz), 160.0 (d, J = 12.1 Hz), 158.0 (d, J =
12.1 Hz), 135.4 (d, J = 2.6 Hz), 133.8 (d, J = 10.1 Hz), 130.7 (d, J =
12.6 Hz), 117.8 (d, J = 86.5 Hz), 111.4 (dd, J = 20.4, 2.7 Hz), 104.0,
74.0 (d, J = 4.9 Hz), 65.9, 62.6, 57.6, 56.0 (d, J = 3.4 Hz), 52.2, 29.8,
28.3, 27.3, 26.9, 25.7, 14.2. HR-ESI-MS m/z calculated for
4.1.22. (3-((8-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-
1-yl)propyl) piperazin-1-yl)octyl)oxy)-3-oxopropyl)triphenylphosphonium
bromide (23)
To a stirred solution of compound 10 (21.0 mg, 0.040 mmol) in DMF
(3.00 mL) was added compound 21 (32.5 mg, 0.080 mmol), K2CO3
(11.1 mg, 0.080 mmol) and tetrabutylammonium iodide (3.0 mg, 0.008
mmol). The reaction mixture was then stirred at room temperature for
24 h, after which TLC analysis indicated the consumption of the starting
material. The reaction mixture was diluted with DCM and washed with
H2O and brine, dried over MgSO4, filtered and concentrated under
reduced pressure to give the crude product, which was purified by flash
C
46H57F2N5O3P+ [M - Br]+ [M - Br]+ 796.4162, found 796.4147.
4.1.25. 3-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)
propyl) piperazin-1-yl)propan-1-ol(26)
To a stirred solution of compound 4 (30.0 mg, 0.090 mmol) in DMF
(3.00 mL) was added 3-bromopropan-1-ol (10.0 μL, 0.110 mmol) and
9

X. Wang et al.
Bioorganic Chemistry 110 (2021) 104771
K2CO3 (25.7 mg, 0.180 mmol). The reaction mixture was stirred at room
temperature for 24 h. After that, the mixture was diluted with DCM and
washed with H2O and brine, dried over MgSO4, filtered and concen-
trated under reduced pressure to give the crude product, which was
purified by flash chromatography to provide compound 26 (14.7 mg,
41.0%) as a colorless oil. 1H NMR (500 MHz, CD3OD) δ 8.33 (s, 1H), 7.76
(s, 1H), 7.50 – 7.43 (m, 1H), 6.92 (dd, J = 11.3, 9.5 Hz, 1H), 6.85 (t, J =
8.3 Hz, 1H), 4.68 (q, J = 14.0 Hz, 2H), 3.53 (td, J = 6.6, 1.8 Hz, 2H),
3.03 (t, J = 14.3 Hz, 4H), 2.87 (t, J = 9.0 Hz, 3H), 2.73 (s, 4H), 1.54 –
1.48 (m, 2H). 13C NMR (125 MHz, CD3OD) δ 151.2, 146.1, 131.0 (dd, J
= 16.8, 10.8 Hz), 76.0 (d, J = 5.2 Hz), 64.0 (d, J = 2.9 Hz), 63.0, 58.3,
57.2 (d, J = 2.9 Hz), 53.4, 53.1, 27.8. ESI-MS m/z calculated for
2.70 (d, J = 13.7 Hz, 1H), 2.56 (t, J = 27.0 Hz, 10H), 1.62 – 1.52 (m,
4H), 1.39 (dd, J = 14.9, 7.6 Hz, 2H), 1.24 (s, 2H). 13C NMR (125 MHz,
CDCl3) δ 162.9 (dd, J = 250.0, 12.4 Hz), 158.9 (dd, J = 246.9, 11.9 Hz),
151.3, 144.7, 129.5 (dd, J = 9.1, 6.0 Hz), 125.8 (dd, J = 12.6, 2.7 Hz),
111.7 (dd, J = 20.5, 2.6 Hz), 104.4 (t, J = 26.3 Hz), 73.0 (d, J = 3.6 Hz),
62.4, 57.9, 56.2 (d, J = 4.9 Hz), 53.0 (d, J = 24.0 Hz), 32.1, 29.8, 25.2,
23.4. ESI-MS m/z calculated for C21H32F2N5O+2 [M+H]+ 424.25, found
424.25.
4.1.29. 7-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)
propyl) piperazin-1-yl)heptan-1-ol(30)
To a stirred solution of compound 4 (50.0 mg, 0.150 mmol) in DMF
C
18H26F2N5O+2 [M+H]+ 382.20, found 382.20.
(3.00 mL) was added 7-bromoheptan-1-ol (28.0 μL, 0.190 mmol) and
K2CO3 (42.8 mg, 0.310 mmol). The reaction mixture was stirred at room
temperature for 24 h. The reaction mixture was diluted with DCM and
washed with H2O and brine, dried over MgSO4, filtered and concen-
trated under reduced pressure to give the crude product, which was
purified by flash chromatography to provide compound 30 (38.0 mg,
56.2%) as a colorless oil. 1H NMR (500 MHz, CD3OD) δ 8.11 (s, 1H), 7.77
(s, 1H), 7.51 (dd, J = 15.7, 8.3 Hz, 1H), 6.79 (t, J = 9.9 Hz, 2H), 4.56 –
4.48 (m, 2H), 3.60 (t, J = 6.4 Hz, 2H), 3.06 (d, J = 13.6 Hz, 1H), 2.67 (d,
J = 13.6 Hz, 1H), 2.40 (dd, J = 29.0, 22.3 Hz, 10H), 1.52 (dd, J = 13.1,
6.4 Hz, 4H), 1.40 – 1.11 (m, 8H). 13C NMR (126 MHz, CD3OD) δ 162.8
(dd, J = 250.0, 12.2 Hz), 159.0 (dd, J = 246.9, 11.9 Hz), 151.2, 144.7,
129.4 (dd, J = 9.2, 5.9 Hz), 126.0 (dd, J = 12.8, 3.0 Hz), 111.7 (dd, J =
20.6, 3.1 Hz), 104.4 (t, J = 26.4 Hz), 72.5 (d, J = 3.8 Hz), 62.7, 62.4 (d,
J = 3.5 Hz), 58.2, 56.3 (d, J = 4.8 Hz), 53.6, 53.0, 32.6, 29.8, 27.0, 26.0,
25.5. ESI-MS m/z calculated for C22H34F2N5O+2 [M+H]+ 438.26, found
438.27.
4.1.26. 4-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)
propyl) piperazin-1-yl)butan-1-ol (27)
To a stirred solution of compound 4 (30.0 mg, 0.090 mmol) in DMF
(3.00 mL) was added 4-bromobutan-1-ol (15.0 μL, 0.110 mmol) and
K2CO3(25.7 mg, 0.180 mmol). The reaction mixture was stirred at room
temperature for 24 h. The reaction mixture was diluted with DCM and
washed with H2O and brine, dried over MgSO4, filtered and concen-
trated under reduced pressure to give the crude product, which was
purified by flash chromatography to provide compound 27 (22.9 mg,
62.1%) as a colorless oil. 1H NMR (500 MHz, CDCl3) δ 8.33 (s, 1H), 7.75
(s, 1H), 7.47 (dd, J = 15.7, 8.7 Hz, 1H), 6.93 (t, J = 9.8 Hz, 1H), 6.85 (t,
J = 8.4 Hz, 1H), 4.65 (dd, J = 37.5, 14.3 Hz, 2H), 3.59 (t, J = 6.1 Hz,
2H), 3.01 (d, J = 13.8 Hz, 1H), 2.80 (d, J = 13.8 Hz, 1H), 2.53 (d, J = 7.3
Hz, 10H), 1.76 – 1.68 (m, 2H), 1.31 (d, J = 15.6 Hz, 2H). 13C NMR (125
MHz, CDCl3) δ 162.7 (dd, J = 247.8, 12.4 Hz), 159.3 (dd, J = 246.5,
12.3 Hz), 149.7, 144.6, 129.4 (dd, J = 9.5, 6.2 Hz), 125.6 (dd, J = 13.0,
3.4 Hz), 110.6 (dd, J = 20.9, 2.9 Hz), 103.5, 73.8 (d, J = 5.5 Hz), 62.9 (d,
J = 3.6 Hz), 60.1, 56.0 (d, J = 4.7 Hz), 55.3, 53.5, 52.7, 28.2. ESI-MS m/
z calculated for C19H28F2N5O+2 [M+H]+ 396.22, found 396.22.
4.1.30. 8-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)
propyl) piperazin-1-yl)octan-1-ol (31)
To a stirred solution of compound 4 (30.0 mg, 0.090 mmol) in DMF
(3.00 mL) was added 8-bromooctan-1-ol (19.0 μL, 0.110 mmol) and
4.1.27. 5-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)
propyl) piperazin-1-yl)pentan-1-ol (28)
K2CO3 (25.7 mg, 0.190 mmol). The reaction mixture was stirred at room
temperature for 24 h. The reaction mixture was diluted with DCM and
washed with H2O and brine, dried over MgSO4, filtered and concen-
trated under reduced pressure to give the crude product, which was
purified by flash chromatography to provide compound 31 (17.4 mg,
41.7%) as a colorless oil. 1H NMR (500 MHz, CDCl3) δ 8.31 (s, 1H), 7.77
(s, 1H), 7.47 (td, J = 8.9, 6.7 Hz, 1H), 6.97 – 6.91 (m, 1H), 6.86 (td, J =
8.4, 2.4 Hz, 1H), 4.67 (q, J = 14.3 Hz, 2H), 3.53 (t, J = 6.6 Hz, 2H), 3.08
– 2.64 (m, 12H), 1.67 – 1.60 (m, 2H), 1.52 (p, J = 6.6 Hz, 2H), 1.32 (d, J
= 39.7 Hz, 8H). 13C NMR (125 MHz, CDCl3) δ 151.2, 146.1, 131.0 (dd, J
= 9.6, 6.6 Hz), 116.2 (d, J = 2.1 Hz), 112.1 (d, J = 20.6 Hz), 104.9 (t, J
= 26.8 Hz), 75.9 (d, J = 5.2 Hz), 64.3, 64.0 (d, J = 3.7 Hz), 62.8, 58.4,
57.3 (d, J = 4.6 Hz), 53.4 (d, J = 28.7 Hz), 33.4, 30.1, 27.8, 26.7, 25.6.
ESI-MS m/z calculated for C23H36F2N5O+2 [M+H]+ 452.28, found
452.28.
To a stirred solution of compound 4 (30.0 mg, 0.090 mmol) in DMF
(3.00 mL) was added 5-bromopentan-1-ol (15.0 μL, 0.110 mmol) and
K2CO3 (25.7 mg, 0.180 mmol). The reaction mixture was stirred at room
temperature for 24 h. The reaction mixture was diluted with DCM and
washed with H2O and brine, dried over MgSO4, filtered and concen-
trated under reduced pressure to give the crude product, which was
purified by flash chromatography to provide compound 28 (8.7 mg,
22.9%) as a colorless oil. 1H NMR (500 MHz, CDCl3) δ 8.09 (s, 1H), 7.75
(s, 1H), 7.49 (dd, J = 15.7, 8.1 Hz, 1H), 6.78 (t, J = 8.5 Hz, 2H), 4.53 –
4.46 (m, 2H), 3.50 (s, 2H), 3.05 (d, J = 13.5 Hz, 1H), 2.65 (d, J = 13.6
Hz, 1H), 2.39 (d, J = 29.4 Hz, 10H), 1.60 (s, 4H), 1.23 (s, 2H). 13C NMR
(125 MHz, CDCl3) δ 162.8 (dd, J = 249.9, 12.0 Hz), 159.0 (dd, J = 247.3,
11.8 Hz), 151.1, 144.7, 129.4 (dd, J = 9.5, 5.8 Hz), 126.0 (dd, J = 12.9,
3.8 Hz), 111.6 (dd, J = 20.6, 3.2 Hz), 105.2 – 103.9 (m), 72.4 (d, J = 5.5
Hz), 62.5, 62.2 (d, J = 3.9 Hz), 58.3, 56.3 (d, J = 5.2 Hz), 53.6 (d, J =
1.5 Hz), 52.9, 32.2, 24.9. ESI-MS m/z calculated for C20H30F2N5O2+
[M+H]+ 410.23, found 410.24.
4.1.31. 9-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)
propyl) piperazin-1-yl)nonan-1-ol (32)
To a stirred solution of compound 4 (30.0 mg, 0.090 mmol) in DMF
(3.00 mL) was added 9-bromononan-1-ol (25.1 mg, 0.112 mmol) and
K2CO3(25.7 mg, 0.190 mmol). The reaction mixture was stirred at room
temperature for 24 h. The reaction mixture was diluted with DCM and
washed with H2O and brine, dried over MgSO4, filtered and concen-
trated under reduced pressure to give the crude product, which was
purified by flash chromatography to provide compound 32 (18.1 mg,
36.8%) as a colorless oil. 1H NMR (500 MHz, CDCl3) δ 8.10 (s, 1H), 7.76
(s, 1H), 7.50 (dd, J = 15.7, 8.7 Hz, 1H), 6.78 (dd, J = 12.9, 7.0 Hz, 2H),
4.56 – 4.47 (m, 2H), 3.59 (t, J = 6.6 Hz, 2H), 3.06 (d, J = 13.6 Hz, 1H),
2.68 (d, J = 13.7 Hz, 1H), 2.44 (d, J = 51.5 Hz, 10H), 1.51 (dd, J = 13.4,
6.6 Hz, 4H), 1.28 (d, J = 19.3 Hz, 10H). 13C NMR (125 MHz, CDCl3) δ
162.8 (dd, J = 249.9, 12.3 Hz), 159.0 (dd, J = 246.7, 11.8 Hz), 151.2,
144.7, 129.4 (dd, J = 9.2, 5.8 Hz), 126.0 (dd, J = 13.1, 3.5 Hz), 111.7
4.1.28. 6-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)
propyl) piperazin-1-yl)hexan-1-ol (29)
To a stirred solution of compound 4 (30.0 mg, 0.090 mmol) in DMF
(3.00 mL) was added 6-bromohexan-1-ol (24.0 μL, 0.110 mmol) and
K2CO3 (25.7 mg, 0.180 mmol). The reaction mixture was stirred at room
temperature for 24 h. The reaction mixture was diluted with DCM and
washed with H2O and brine, dried over MgSO4, filtered and concen-
trated under reduced pressure to give the crude product, which was
purified by flash chromatography to provide compound 29 (11.3 mg,
28.8%) as a colorless oil. 1H NMR (500 MHz, CDCl3) δ 8.10 (s, 1H), 7.77
(s, 1H), 7.50 (dd, J = 15.7, 8.7 Hz, 1H), 6.79 (dd, J = 13.0, 7.1 Hz, 2H),
4.57 – 4.49 (m, 2H), 3.61 (t, J = 6.3 Hz, 2H), 3.07 (d, J = 13.7 Hz, 1H),
10

X. Wang et al.
Bioorganic Chemistry 110 (2021) 104771
(dd, J = 20.6, 2.5 Hz), 104.4 (t, J = 26.3 Hz), 72.6 (d, J = 5.6 Hz), 62.9,
62.3 (d, J = 3.4 Hz), 58.3, 56.3 (d, J = 4.6 Hz), 53.5 (d, J = 2.2 Hz), 53.0,
32.8, 29.8, 29.3, 27.3, 25.7. ESI-MS m/z calculated for C24H38F2N5O2+
[M+H]+ 466.29, found 466.30 [M+H]+.
of colony forming units (CFUs) was counted after incubation of the
◦
plates at 35 C for 48 h. MFC was defined as the lowest drug concen-
tration at which 99.9% of the inoculum was killed. The results are an
average of at least two independent experiments, and each experiment
was performed in triplicate.
4.1.32. 10-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-
yl)propyl) piperazin-1-yl)decan-1-ol (33)
4.2.3. Measurement of intracellular drug contents
To a stirred solution of compound 4 (30.0 mg, 0.090 mmol) in DMF
(3.00 mL) was added 10-bromodecan-1-ol (26.0 mg, 0.110 mmol) and
K2CO3 (25.7 mg, 0.190 mmol). The reaction mixture was stirred at room
temperature for 24 h. The reaction mixture was diluted with DCM and
washed with H2O and brine, dried over MgSO4, filtered and concen-
trated under reduced pressure to give the crude product, which was
purified by flash chromatography to provide compound 33 (15.2 mg,
34.2%) as a colorless oil. 1H NMR (500 MHz, CD3OD) δ 8.10 (s, 1H), 7.76
(s, 1H), 7.50 (dd, J = 15.7, 8.7 Hz, 1H), 6.78 (dd, J = 12.8, 7.0 Hz, 2H),
4.51 (s, 2H), 3.59 (t, J = 6.6 Hz, 2H), 3.06 (d, J = 13.6 Hz, 1H), 2.68 (d,
J = 13.7 Hz, 1H), 2.45 (d, J = 52.9 Hz, 10H), 1.51 (dd, J = 13.7, 6.7 Hz,
4H), 1.35 – 1.20 (m, 12H). 13C NMR (125 MHz, CD3OD) δ 162.8 (dd, J =
250.1, 12.0 Hz), 158.9 (dd, J = 246.9, 12.0 Hz), 151.2, 144.7, 129.4 (dd,
J = 9.5, 6.0 Hz), 125.9 (d, J = 10.8 Hz), 111.7 (dd, J = 20.6, 3.2 Hz),
104.4 (t, J = 26.3 Hz), 72.7 (d, J = 6.5 Hz), 62.9, 62.3 (d, J = 3.9 Hz),
58.3, 56.3 (d, J = 4.7 Hz), 52.9, 32.8, 29.4, 29.4, 29.3, 27.3, 25.8. ESI-
MS m/z calculated for C25H40F2N5O+2 [M+H]+ 480.31, found 480.32.
Exponential-phase SC5314 cells were diluted in RPMI 1640 medium
to 1 × 108 cells/mL. Cells were treated with 80 µg/mL of the tested
compounds for 4 h at 35 ◦C and were then collected and washed twice
with PBS. The cell pellet was dissolved in 250 μL of water and then
subjected to three freeze–thaw cycles of 3 min in liquid nitrogen fol-
lowed by 3 min in a water bath at 65 ◦C. Methanol (250
μL) was then
added to the suspension, which was disrupted with glass beads in a
Precellys 24 homogenizer (Bertin Technologies, Montigny le Bre-
tonneux, France) four times at a speed of 2348 × g (5000 rpm) for 20 s
with 10 s intervals, and the supernatant was collected. Another 500 μL of
methanol was added to the tube for the second round of cell lysis. The
supernatant was removed and combined with the previously collected
supernatant for freeze-drying. The dried sample was then dissolved in
100 μL of methanol and analyzed by HPLC as follows. Chromatographic
separation was achieved on a Phenomenex Luna C18 column (250 mm
× 4.6 mm i.d., 5
× 3.0 mm i.d., 5
μ
m) with a SecurityGuard C18 guard column (4.0 mm
μ
m; Phenomenex, Torrance, CA) using mobile phase A,
which was composed of 0.5% formic acid (FA) in water, and mobile
phase B, which was composed of acetonitrile, at a flow rate of 1.0 mL/
min. The HPLC gradient system started with 10% B and linearly
increased to 100% B over 30 min, followed by a decrease to 10% B over
2 min prior to column re-equilibration.
4.2. General material and methods for biology
4.2.1. Strains and culture conditions
The wild-type C. albicans strain SC5314, the constructed C. albicans
strains YEM13 and YEM15 with hyperexpression of MDR1 or hyper-
expression of both CDR1 and CDR2, seven clinical isolates of C. albicans
(28A, 28I, DSY294, DSY296, G5, F5 and Gu5), and clinical isolates of
C. glabrata (CG1), C. tropicalis (CT2) and C. parapsilosis (CP1) were used
in this study. The clinical C. albicans strains 28A and 28I were donated
by Professor Qingguo Qi of Shandong University of China. Other clinical
C. albicans strains were kindly donated by Professor Joachim
4.2.4. Drug resistance study
The drug resistance study was performed by treating C. albicans
repeatedly with the tested antifungal agents using broth microdilution
assays [27]. Specifically, the MIC values of compounds 20 and 25 and
FLC against C. albicans ATCC10231 were determined with the broth
microdilution method described above. C. albicans cells taken from
duplicate test tubes at a concentration of 0.5 × MIC were adjusted to 1 ×
103 cells/mL with RPMI 1640 medium as the inoculum for MIC mea-
surement of the next passage. After incubation with the tested antifungal
compounds for 24 h at 35 ◦C, the new MIC values were measured. The
process was repeated for 40 passages. All measurements were performed
with biological replicates.
¨
¨
Morschhauser of Universitat Würzburg in Germany. The isolates of
C. glabrata, C. tropicalis, and C. parapsilosis were kindly provided by the
Central Hospital of Jinan City. The clinical strains were identified as
Candida by CHROMagar based on color change and colony morphology
and further confirmed by DNA sequencing. All isolates were stored in
◦
physiological saline supplemented with 20% glycerol at ꢀ 80 C and
subcultured twice on YPD agar plates (2% tryptone, 1% yeast extract,
2% glucose and 2% agar) for 24 h at 30 ◦C. Prior to each experiment, the
cells were cultured in liquid YPD medium (2% tryptone, 1% yeast
extract, 2% glucose) overnight at 30 ◦C and 200 rpm. Stock solutions of
AZDs (1 or 10 mg/mL) and FLC (1 or 10 mg/mL, Solarbio) were pre-
pared in dimethyl sulfoxide (DMSO) and stored at ꢀ 20 ◦C until use. In
each assay, the DMSO content was below 1%.
4.2.5. Effect of drug resistance on plating medium
Strains were streaked from glycerol stocks onto YPD agar plates and
incubated for 24 h at 30 ◦C. Colonies were suspended in PBS solution,
and 2 × 104 cells were spread onto YPD agar plates containing serial
concentrations of compounds 20, 25 and FLC. The plates were incubated
◦
at 30 C for 72 h. After incubation, images of plates were taken by a
Gel Imager System.
4.2.2. Broth microdilution assays
4.2.6. The antifungal interaction of FLC/AZDs and Hsp90 inhibitors
The interaction of FLC/AZDs and Hsp90 inhibitors against C. albicans
isolates was assessed using the broth microdilution checkerboard assay.
Briefly, the culture was diluted to a final concentration of 0.5–2.5 × 103
cells/mL with RPMI 1640 medium. In a 96-well plate, 2-fold dilutions of
Hsp90 inhibitor geldanamycin (GdA), along the x-axis, and 2-fold di-
lutions of FLC or AZDs 20 or 25, along the y-axis, were mixed for a total
The minimal inhibitory concentrations (MICs) and the minimal
fungicidal concentrations (MFCs) of the tested agents were determined
by the broth microdilution procedure following the CLSI M27-A3
guideline. Briefly, overnight grown C. albicans cells were collected and
diluted to 1 × 103 cells/mL with RPMI 1640 medium. A 100
μL aliquot
of inoculum was inoculated into the wells of 96-well microtiter plates.
The tested compounds were added into the wells with a serial of drug
volume of 100 μL per well. The concentrations of the Hsp90 inhibitor
concentrations ranging from 0.25 μg/mL to 128 μg/mL, with the first
GdA ranged from 1.25
AZDs ranged from 0.5
μ
g/mL to 5
μg/mL. The concentrations of FLC and
well without drug as the negative control. The microplates were incu-
bated at 35 ◦C for 24 h. The OD600 values of each well were determined
with a plate reader, and the MIC endpoints were defined as the lowest
concentration at which at least 80% of cell growth was inhibited
compared with the negative control.
μ
g/mL to 64 μg/mL. The plates were incubated at
35 ◦C for 24 h, and the MIC and MFC were determined by using the broth
microdilution method as described above.
4.2.7. Hemolytic activity assays
To obtain the minimal fungicidal concentration (MFC), 100 µL ali-
quots were taken from every well and spread on YPD agar. The number
Blood samples were withdrawn from mice according to the protocol
11

X. Wang et al.
Bioorganic Chemistry 110 (2021) 104771
approved by the Animal Care and Use Committee at Qingdao University.
Murine red blood cells (mRBCs) were isolated by centrifugation, washed
three times in PBS by resuspending 1 mL of mRBCs in 5 mL of PBS and
centrifuged at 1000 g for 10 min to collect mRBCs. Subsequently, the
mRBCs were suspended in PBS to prepare an 8% v/v suspension. The
samples were dissolved in DMSO and then diluted in PBS to prepare two-
Acknowledgment
¨
¨
We thank Professor Joachim Morschhauser of Universitat Würzburg
for providing the C. albicans strains with Mrr1 or Tac1 mutations, Pro-
fessor Kim Lewis at Northeastern University for providing the efflux
pump-related strains. This work was supported by grants from the Na-
tional Natural Science Foundations of China (Nos. 81630093,
81773786, and 82003622), the Natural Science Foundation of Shan-
dong Province (ZR2019BH073), the Scientific Research Foundation of
Qingdao University, Fund for Innovative Team of Shandong University
to H.L., the Natural Science Fund for Excellent Young Scholars of
Shandong Province of China (ZR2020YQ63) and Young Scholars Pro-
gram of Shandong University (2017WLJH41).
fold serial dilutions. These different concentrations of samples (100
μL)
were added to each well of a 96-well plate containing 100
μL of mRBCs,
and the plate was incubated for 1 h at 37 ◦C without agitation. One
hundred microliters of 1% Triton X was used as the positive control, and
PBS was the negative control. After incubation, the plate was centri-
fuged at 1500 g for 15 min. The supernatant from each sample was
transferred to a new plate, and the absorbance was measured at 450 nm.
The percent hemolysis was calculated using the following equation: %
hemolysis = [(absorbance of sample) – (absorbance of negative con-
trol)] × 100/[(absorbance of positive control) - (absorbance of negative
control)].
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104771.
4.2.8. Anti-biofilm assay
To assess the ability of AZD 25 to prevent C. albicans biofilm for-
mation, overnight-grown C. albicans cells of SC5314, YEM13 and YEM15
were collected and diluted with RPMI1640 to 1 × 106 cells/mL. The
yeast suspension was added to the 96-well plate, and the tested agent
was added to the suspension to reach the final concentrations (0, 0.25,
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Declaration of Competing Interest
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