Novel fluconazole derivatives with promising antifungal activity

Article abstract of DOI:10.1016/j.bmc.2017.12.018

The fungistatic nature and toxicity concern associated with the azole drugs currently on the market have resulted in an increased demand for new azole antifungal agents for which these problematic characteristics do not exist. The extensive use of azoles has resulted in fungal strains capable of resisting the action of these drugs. Herein, we report the synthesis and antifungal activity of novel fluconazole (FLC) analogues with alkyl-, aryl-, cycloalkyl-, and dialkyl-amino substituents. We evaluated their antifungal activity by MIC determination and time-kill assay as well as their safety profile by hemolytic activity against murine erythrocytes as well as cytotoxicity against mammalian cells. The best compounds from our study exhibited broad-spectrum activity against most of the fungal strains tested, with excellent MIC values against a number of clinical isolates. The most promising compounds were found to be less hemolytic than the least hemolytic FDA-approved azole antifungal agent voriconazole (VOR). Finally, we demonstrated that the synthetic alkyl-amino FLC analogues displayed chain-dependent fungal membrane disruption as well as inhibition of ergosterol biosynthesis as possible mechanisms of action.

Full text of DOI:10.1016/j.bmc.2017.12.018

Accepted Manuscript
Novel fluconazole derivatives with promising antifungal activity
Nishad Thamban Chandrika, Sanjib K. Shrestha, Huy X. Ngo, Kaitlind C.
Howard, Sylvie Garneau-Tsodikova
PII:
S0968-0896(17)31933-8
https://doi.org/10.1016/j.bmc.2017.12.018
BMC 14123
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Received Date:
Revised Date:
Accepted Date:
30 September 2017
2 December 2017
12 December 2017
Please cite this article as: Chandrika, N.T., Shrestha, S.K., Ngo, H.X., Howard, K.C., Garneau-Tsodikova, S., Novel
fluconazole derivatives with promising antifungal activity, Bioorganic & Medicinal Chemistry (2017), doi: https://
doi.org/10.1016/j.bmc.2017.12.018
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Novel fluconazole derivatives with promising antifungal activity
Nishad Thamban Chandrika,a,† Sanjib K. Shrestha,a,† Huy X. Ngo,a Kaitlind C. Howard,a and
Sylvie Garneau-Tsodikovaa,*
a University of Kentucky, Department of Pharmaceutical Sciences, Lexington, KY, 40536-0596, USA.
† These authors contributed equally to this work.
* To whom the correspondence should be addressed: E-mail: sylviegtsodikova@uky.edu; Tel: 859-218-1686.
The authors declare no conflict of interest.

Abstract
The fungistatic nature and toxicity concern associated with the azole drugs currently on the
market have resulted in an increased demand for new azole antifungal agents for which these
problematic characteristics do not exist. The extensive use of azoles has resulted in fungal strains
capable of resisting the action of these drugs. Herein, we report the synthesis and antifungal
activities of novel fluconazole (FLC) analogues with alkyl-, aryl-, cycloalkyl-, and dialkyl-amino
substituents. We evaluated their antifungal activity by MIC determination and time-kill assay as
well as their safety profile by hemolytic activity against murine erythrocytes as well as
cytotoxicity against mammalian cells. The best compounds from our study exhibited broad-
spectrum activity against most of the fungal strains tested, with excellent MIC values against a
number of clinical isolates. The most promising compounds were found to be less hemolytic than
the least hemolytic FDA-approved azole antifungal agent voriconazole (VOR). Finally, we
demonstrated that the synthetic alkyl-amino FLC analogues displayed chain-dependent fungal
membrane disruption as well as inhibition of ergosterol biosynthesis as possible mechanisms of
action.
Keywords: Azoles, Clinical isolates, Cytotoxicity, Hemolysis, Time-kill studies.

1. Introduction
The rise in the number of infections caused by fungi poses a serious threat to human health
and life.1 Fungal infections can be endogenous (e.g., Candida infections) or acquired from the
environment (e.g., Cryptococcus and Aspergillus infections). Invasive fungal infections have
become a major problem for patients with immunodeficiency syndrome (e.g., AIDS), organ
transplant patients, and patients receiving chemotherapeutic agents for cancer treatment.2-4
Clinically, candidiasis, aspergillosis, and cryptococcosis are the major infections in
immunocompromised patients.5, 6 Candida and Aspergillus species are responsible for the
majority of documented fungal infections. Recent studies indicated an epidemiological shift
towards infections caused by emerging non-albicans Candida and Aspergillus species resistant
to the current antifungal drugs.7-9 Non-albicans Candida species such as C. glabrata, C.
parapsilosis, C. tropicalis, and C. krusei are more prominent now and they account for more
incidence of invasive candidiasis such as candidemia than C. albicans.10-13 The relative presence
of these fungal strains is region dependent. For example, C. glabrata is the second most common
species after C. albicans in North America.14 Fungal strains such as C. parapsilosis and C.
tropicalis are relatively more common in Europe, Australia, Latin America, and Asia.15-17 As
resistance to the currently available antifungal agents is emerging in many of these non-albicans
Candida, there is a need for developing novel antifungals.18
Most of the current drugs on the market are either highly toxic (e.g., amphotericin B (AmB))
or becoming ineffective due to appearance of resistant fungal strains (e.g., azoles such as
fluconazole (FLC) and voriconazole (VOR)) (Fig. 1).19 Azoles are the most frequent class of
antifungals used to treat fungal infections as they are inexpensive and are available for oral
administration.20 However, there is an extensive documentation of intrinsic and developed

resistance to azole drugs among C. albicans and non-albicans Candida species. As the frequency
of occurrence of infections caused by non-albicans Candida species is increasing in clinical
settings, there is currently a need to improve on the existing azole scaffolds to develop novel
antifungals. Various studies were reported by our and other groups, which illustrated the role of
alkylation on different drug scaffolds resulting in promising antifungal activity.21-27 There are
examples of 2,4-difuoro-2-(1H-1,2,4-triazo-1-yl)acetophenone analogues with linear C5-C8 alkyl
chains25 and of an n-alkylated ebsulfur derivative with a linear C5 alkyl chain, which displayed
strong antifungal activity.24 Similarly, examples of aminoglycosides (e.g., kanamycin B (KANB)
and tobramycin (TOB)) with linear alkyl chains with 12 and 14 carbons (C12 and C14) displaying
antifungal activity were also reported.21, 22
Fig. 1. Structures of all antifungal agents used as controls in this study.

Based on the information provided above and the promise shown by these small molecules,
herein, we decided to generate novel FLC derivatives in which the triazole ring on the carbon
alpha to the dihalophenyl ring of FLC was displaced by various linear alkyl-, aryl-, dialkyl-, and
cycloalkyl-amino substituents. We report the synthesis of twelve novel FLC derivatives (Fig. 2)
and their antifungal activity against a variety of C. albicans, non-albicans Candida, Aspergillus,
and Cryptococcus strains as established by in vitro MIC determination as well as by time-kill
studies. We explore the hemolytic activity as well as cytotoxicity of these compounds against
murine erythrocytes and mammalian cell lines, respectively. Finally, we investigate the potential
mechanism of action of selected compounds by probing their ability to disrupt fungal membrane.
Boc2O, Et3N,
A
OH
OH
H2N
BocHN
CH2Cl2, rt, 12 h
1: (79%)
I
NaH, DMF,
0 °C to rt, 24 h
4
TFA, CH2Cl2,
rt, 2 h
O
N
O
H2N
BocHN
4
4
3: (90%)
2: (36%)
trimethylsulfoxonium
iodide, NaOH,
B
O
N
N
O
N
N
N
cetyl bromide,
toluene, 60 °C, 1 h
F
F
F
F
4: (68%)
CH3(CH2)nNH(CH2)nCH3, EtOH,
Et3N, 80 °C, 12 h, n = 5 and 7
or
H2N
Y
n
cycloctyl amine, EtOH, Et3N,
80 °C, 12 h
EtOH, Et3N,
rt or 80 °C, 2-12 h
or
Y
4-isopropylaniline,
EtOH, Et3N, 80 °C, 12 h
n
HN
N
HO
N
N
R1
R2
N
N
N
HO
N
F
F
5: Y = Me, n = 5 (14%)
6: Y = Me, n = 7 (70%)
7: Y = Me, n = 9 (91%)
8: Y = Me, n = 11 (59%)
9: Y = Me, n = 13 (94%)
10: Y = Me, n = 15 (91%)
11: Y = OH, n = 6 (51%)
F
F
13: R1 = R2 = n-hexyl (57%)
14: R1 = R2 = n-octyl (65%)
15: R1 = cyclooctyl, R2 = H (57%)
16: R1 = p-iPrPh, R2 = H (57%)
O
12: Y =
4 , n = 6 (39%)

Fig. 2. Synthetic schemes for the preparation of A. amine derivative 3, and B. novel azole
analogues 5-16.
2. Results and discussion
2.1. Design and synthesis of antifungal agents 5-16. We synthesized the alkyl-/aryl- and
cycloalkyl-amino FLC derivatives 5-16 in two steps by using the commercially available
fluorinated compound 2,4-difuoro-2-(1H-1,2,4-triazo-1-yl)acetophenone as a starting material
(Fig. 2B). We first converted the carbonyl group of 2,4-difuoro-2-(1H-1,2,4-triazo-1-
yl)acetophenone to an epoxide by using trimethylsulfoxonium iodide in the presence of a strong
base and a surfactant to yield the oxirane intermediate 4, which we then reacted with various
amines (all commercially available, with the exception of amine 3 used in the synthesis of
derivative 12) under mild basic conditions to afford derivatives 5-16. The amine 3 used for the
synthesis of derivative 12 was prepared in three steps (Fig. 2A). The amino group of 6-
aminohexanol was protected with Boc to yield compound 1, which was then subjected to
nucleophilic substitution reaction with 1-iodopentane. The deprotection of the Boc group of
intermediate 2 yielded the desired amine 3.
2.2. Antifungal activity and structure-activity-relationship (SAR) analysis. We first
evaluated the antifungal activity of the newly prepared FLC derivatives 5-16 against a panel of
seven C. albicans (ATCC 10231 (A), ATCC 64124(R) (B), ATCC MYA-2876(S) (C), ATCC
90819(R) (D), ATCC MYA-2310(S) (E), ATCC MYA-1237(R) (F), and ATCC MYA-1003(R)
(G)), three non-albicans Candida (C. glabrata ATCC 2001 (H), C. krusei ATCC 6258 (I), and
C. parapsilosis ATCC 22019 (J)), and three Aspergillus (A. flavus ATCC MYA-3631 (K), A.

nidulans ATCC 38163 (L), and A. terreus ATCC MYA-3633 (M)) strains using a concentration
range of 0.03-31.3 µg/mL (Tables 1 and S1). We used the commercially available antifungal
agents such as AmB, caspofungin (CAS), FLC, and VOR as positive controls for comparison.
For derivatives 5-16 as well as the reference drugs AmB and CAS, we reported MIC-0 values,
which correspond to no visible growth. We reported MIC-2 values (i.e., 50% growth inhibition)
for FLC and VOR against all fungal strains tested with the exception of strain A by VOR. We
defined antifungal activity as excellent (0.03-1.95 µg/mL), good (3.9 µg/mL), moderate (7.8-
15.6 µg/mL), or poor (≥31.3 µg/mL) based on MIC values. In this manuscript, we performed all
activity comparisons by using the MIC values reported in µg/mL (Note: the corresponding MIC
values are also provided in µM into parentheses in Tables 1, 2, S1, and S2).
Table 1: MIC valuesa (in µg/mL) (the corresponding values in µM are provided in Table S1) determined for compounds 5-16 and for four control
antifungal agents (AmB, CAS, FLC, and VOR) against various yeast strains and filamentous fungi. Each experiment was performed in triplicate.
Yeast strains
F
Filamentous fungi
Cpd #
5
A
B
C
D
E
G
H
I
J
K
L
M
7.8
>31.3
>31.3
15.6
15.6
31.3
3.9
31.3
7.8
31.3
>31.3
15.6
7.8
31.3
31.3
7.8
15.6
7.8
7.8
0.975
0.06
0.03
0.48
0.48
0.48
>31.3
0.24
31.3
0.975
3.9
>31.3
>31.3
7.8
>31.3
1.95
0.975
1.95
3.9
>31.3
15.6
3.9
0.975
0.48
0.975
1.95
>31.3
>31.3
3.9
31.3
0.975
0.48
0.975
3.9
6
3.9
7.8
0.975
0.975
0.975
0.975
>31.3
15.6
>31.3
31.3
31.3
7.8
7
15.6
3.9
1.95
7.8
15.6
7.8
15.6
31.3
>31.3
>31.3
7.8
7.8
3.9
8
>31.3
>31.3
>31.3
>31.3
>31.3
>31.3
31.3
>31.3
31.3
31.3
31.3
>31.3
31.3
>31.3
>31.3
1.95
0.06
15.6
1.95
31.3
>31.3
>31.3
>31.3
3.9
>31.3
>31.3
>31.3
>31.3
>31.3
>31.3
31.3
7.8
9
31.3
31.3
3.9
3.9-31.3
>31.3
15.6
3.9
3.9
>31.3
>31.3
31.3
>31.3
>31.3
>31.3
15.6
3.9
10
>31.3
1.95
>31.3
31.3
31.3
>31.3
3.9
>31.3
7.8
11
12
>31.3
31.3
31.3
>31.3
3.9
>31.3
31.3
31.3
31.3
0.975
0.12
>125
1.95
>31.3
>31.3
3.9-31.3
31.3
>31.3
31.3
31.3
31.3
3.9
>31.3
>31.3
>31.3
31.3
3.9
>31.3
15.6
>31.3
7.8
13
14
15
>31.3
3.9
7.8
31.3
16
1.95
1.95
0.06
>31.3
0.06
1.95
1.95
1.95
<0.03
15.6
3.9
AmB
CAS
FLC
VOR
0.975
62.5
0.24
0.24
0.12
0.24
62.5
7.8
0.48
62.5
1.95
0.48
>31.3
0.12
>31.3
62.5
>31.3
62.5
0.12
>31.3
62.5
0.12
>125
3.9
>125
0.975
0.24
Yeast strains: A = Candida albicans ATCC 10231, B = C. albicans ATCC 64124, C = C. albicans ATCC MYA-2876(S), D = C. albicans
ATCC 90819(R), E = C. albicans ATCC MYA-2310(S), F = C. albicans ATCC MYA-1237(R), G = C. albicans ATCC MYA-1003(R), H =
Candida glabrata ATCC 2001, I = Candida krusei ATCC 6258, J = Candida parapsilosis ATCC 22019. NOTE: Here, the (S) and (R) indicate
that ATCC reports these strains to be susceptible (S) and resistant (R) to ITC and FLC.
Filamentous fungi: K = Aspergillus flavus ATCC MYA-3631, L = Aspergillus nidulans ATCC 38163, M = Aspergillus terreus ATCC MYA-
3633.
Known antifungal agents: AmB = amphotericin B, CAS = caspofungin, FLC = fluconazole, VOR = voriconazole.
a
For yeast strains: MIC-0 values are reported for FLC analogues 5-16, AmB, and CAS, whereas MIC-2 values are reported for azoles. For
filamentous fungi, MIC-0 values are reported for all compounds.

By a survey of the data reported in Table 1, the following observations could rapidly be
made. The introduction of a side-chain comprising (i) a terminal hydroxyl group as in compound
11, (ii) a dialkyl-amino moiety as in derivatives 13 and 14, (iii) a cycloalkyl-amino group as in
compound 15, and (iv) an aryl-amino functionality as in compound 16, resulted in all cases in
molecules that were generally poor antifungals. A few exceptions were noted: compounds 14 and
15 displayed excellent (0.975 µg/mL) and good (3.9 µg/mL) activity against the C. parapsilosis
strain J. In contrary, we found that mono-alkylation resulted in much better antifungals. For
derivatives 5-10, we generally observed better activity against non-albicans Candida and
Aspergillus strains than against C. albicans. More specifically, when exploring the data for
strains H-M, we found that compounds 7 and 8 displayed excellent (0.03-1.95 µg/mL) activity
against the non-albicans Candida strains H, I, and J, as well as against the Aspergillus strain L.
Additionally, both compounds 7 and 8 exhibited moderate (7.8 µg/mL) and good (3.9 µg/mL)
activity against the Aspergillus strains K and M, respectively. Compounds 5, 6, 9, and 10,
displayed excellent activity (0.06-1.95 µg/mL) against strains J, (I, J and L), (H and J), and (H
and J), respectively. Compounds 5, 6, and 9 displayed moderate activity (7.8-15.6 µg/mL)
against strains (H and I), (H and M), and M, respectively. In addition, derivatives 9 and 10
showed good (3.9 µg/mL) activity against strains I and L. When assessing the data for the C.
albicans strains A-G, we found that compounds 6-9 exhibited excellent activity (0.48-1.95
µg/mL) against strain A. We also observed that derivatives 7 and 8 generally displayed good to
moderate (3.9-15.6 µg/mL) activity against strains B-G, with the exception of compound 8
displaying excellent (1.95 µg/mL) activity against strain D. In addition, compounds 7 and 8
displayed excellent to good antifungal activity against most strains tested and derivative 9
displayed strong activity against non-albicans Candida and Aspergillus strains. These data

indicated that the optimal chain lengths for maximal antifungal activity were C10 and C12, and the
general trend for activity versus chain length was C10 > C12 > C8 > C14 > C16 = C6. Finally, we
observed that replacing one of the carbon atom in the side-chain by an oxygen as in compound
12 was detrimental as its activity against all strains was generally lower (higher MIC values)
than that of its counterpart 8.
Having established that derivatives 5-16 displayed excellent antifungal activity against non-
albicans Candida strains, we further evaluated these compounds against three clinical strains of
C. glabrata (CG1, CG2, and CG3) and C. parapsilosis (CP1, CP2, and CP3), as well as three
Cryptococcus neoformans (CN1, CN2, and CN3) clinical isolates (Table 2). The trends observed
in Table 2 correlated perfectly to those described for the data presented in Table 1. Compounds
11 and 13 were inactive against all nine clinical isolates tested, whereas compounds 14-16
exhibited excellent to good (0.975-3.9 µg/mL) activity against C. parapsilosis CP1, CP2, and
CP3. For derivatives 5-10, we generally observed excellent activity against most C. glabrata, C.
parapsilosis, and C. neoformans clinical isolates. More precisely, we found that compound 8
displayed excellent (0.06-1.95 µg/mL) activity against all clinical isolates tested. Compounds 7,
9, and 10 displayed excellent (0.03-1.95 µg/mL) activity against all isolates, with the exception
of C. glabrata CG3. In addition, compound 6 displayed excellent (0.12-1.95 µg/mL) activity
against CG2, CP1, CP2, CP3, and CN1, whereas compound 5 was found to display excellent
(0.975 µg/mL) activity against C. parapsilosis CP1, CP2, and CP3 isolates. Overall, compounds
6-10 displayed better activity against the clinical isolates presented in Table 2 than they did
against the commercially available strains for which the data are presented in Table 1. In general,
we found that the three most active compounds synthesized (based on the data from Tables 1 and
2), 7-9, displayed better activity than FLC and similar or better activity than AmB against most

of the fungal strains tested, as well as better activity than CAS against the three Aspergillus
strains tested. When examining the data obtained with clinical isolates of C. glabrata, C.
parapsilosis, and C. neoformans (Table 2), we observed that compounds 7-9 displayed similar or
better activity than both CAS and FLC. When comparing compounds 7-9 to VOR, we found
them to display stronger activity against some of the clinical strains tested.
Table 2: MIC valuesa (in µg/mL) (the corresponding values in µM are provided in Table S2) determined for compounds 5-16 and for two control
antifungal agents (FLC and VOR) against various non-albicans Candida and Cryptococcus neoformans clinical isolates. Each experiment was
performed in triplicate.
Yeast strains
Cpd #
H
7.8
CG1
31.3
7.8
CG2
7.8
CG3
31.3
15.6
3.9
J
CP1
0.975
0.48
0.06
0.06
0.48
0.975
31.3
0.12
>31.3
CP2
0.975
0.12
0.12
0.24
0.975
0.48
31.3
0.12
>31.3
CP3
0.975
0.24
0.24
0.12
0.48
0.975
31.3
0.12
>31.3
CN1
7.8
CN2
>31.3
3.9
CN3
31.3
3.9
0.975
0.06
0.03
0.48
0.48
0.48
>31.3
0.24
31.3
5
0.975
0.48
0.975
1.95
>31.3
>31.3
3.9
0.975
0.48
1.95
0.24
0.24
0.12
0.48
>31.3
0.48
>31.3
6
1.95
1.95
1.95
1.95
>31.3
15.6
>31.3
0.24
0.24
0.24
0.48
>31.3
1.95
>31.3
0.48
0.48
0.48
0.48
>31.3
1.95
15.6
7
0.975
1.95
1.95
3.9
8
9
0.975
>31.3
1.95
7.8
10
11
12
13
>31.3
31.3
>31.3
>31.3
>31.3
31.3
31.3
>31.3
0.06
62.5
0.24
15.6
31.3
7.8
7.8
31.3
0.975
3.9
0.975
0.975
3.9
1.95
1.95
1.95
3.9
>31.3
7.8
>31.3
>31.3
31.3
>31.3
15.6
14
15.6
7.8
31.3
0.975
3.9
15
7.8
7.8
7.8
>31.3
15.6
16
1.95
>31.3
0.975
0.24
>31.3
3.9
0.975
>31.3
>31.3
1.95
1.95
<0.03
0.48
0.48
0.48
0.975
<0.03
15.6
7.8
31.3
CAS
FLC
VOR
0.975
<0.03
0.975
<0.03
>31.3
0.975
>31.3
0.12
0.24
Yeast strains: H = Candida glabrata ATCC 2001, CG1-CG3 = C. glabrata clinical isolates, J = Candida parapsilosis ATCC 22019, CP1-CP3
= C. parapsilosis clinical isolates, CN1-CN3 = Cryptococcus neoformans clinical isolates.
Known antifungal agents: CAS = caspofungin, FLC = fluconazole, VOR = voriconazole.
a For these yeast strains: MIC-0 values are reported for FLC analogues 5-16, whereas MIC-2 values are reported for azoles.
The antifungals currently on the market are known to bind to proteins and be less efficient in
intracellular matrices. For this reason, we tested the three best compounds, 7-9, against three
representative strains, the C. albicans A, the non-albicans Candida (C. parapsilosis) J, and the
Aspergillus L, in presence and absence of fetal bovine serum (FBS) (Table 3). We found that the
alkyl-amino azole analogue 7 retained its full antifungal activity (only 1 double dilution
difference in some cases) against all three strains tested in the presence of FBS. Compound 8
retained its full activity against strain J and experienced a 2- and 4-fold decrease in activity in
the presence of FBS against strains A and L, respectively. Compound 9 displayed a 2- to 8-fold

decrease in activity against the strains tested. Even though there was a small loss in activity in
some instances, analogues 7-9 still remained good antifungal with the exception of compound 9
against strain L.
Table 3: MIC valuesa (in µg/mL) determined for compounds 7-9 and for two control antifungal agents (AmB and VOR) against various yeast
strains and filamentous fungi in the absence or presence of FBS. Each experiment was performed in triplicate.
Yeast strains
Candida albicans Candida albicans Candida
Filamentous fungi
Candida parapsilosis Aspergillus nidulans Aspergillus nidulans
Cpd #
ATCC 10231 (A) ATCC 10231 (A) parapsilosis ATCC ATCC 22019 (J)
(no FBS)
0.48
ATCC 38163 (L) (no ATCC 38163 (L) (+10%
(+10% FBS)
0.48
22019 (J) (no FBS) (+10% FBS)
FBS)
0.975
1.95
3.9
FBS)
1.95
7.8
0.03
0.48
0.48
1.95
0.015
0.06
7
0.975
1.95
1.95
0.24
8
7.8
0.975
15.6
31.2
15.6
0.12
9
3.9
7.8
3.9
AmB
VOR
0.48
0.48
0.015
0.12
2.3. Time-kill studies. The information regarding the rate and extend of fungicidal activity can
be gathered by time-kill assays. To determine the fungistatic or fungicidal nature of the
compounds generated, we performed time-kill assays over a 24-h period with one of the best
FLC derivatives, compound 7, and one of the good ones, compound 6. These compounds and
VOR (positive control) were tested against fungal strains C. albicans ATCC 10231 (A) and C.
parapsilosis ATCC 22019 (J) (Fig. 3). At 4× their respective MIC values, when tested against
strain A, compounds 6 and 7 were found to be fungistatic and to be better than the control drug
VOR. When tested against strain J, both compounds also displayed fungistatic activity at up to
4× their MIC values. However, against strain J, compound 6 displayed lower reduction in fungal
growth than VOR, but compound 7 displayed activity equal to VOR. Overall, the compounds 6
and 7 performed better in time-kill studies than the control drug VOR.

1e+8
1e+7
1e+6
1e+5
1e+4
1e+9
1e+8
1e+7
A
B
No drug ctrl
Cpd 6 (1x MIC)
Cpd 6 (2x MIC)
Cpd 6 (4x MIC)
VOR (4x MIC)
1e+6
1e+5
1e+4
1e+3
0
5
10
15
20
25
0
5
10
15
20
25
Time (h)
Time (h)
1e+8
1e+7
1e+6
1e+5
1e+4
1e+3
1e+2
1e+9
D
C
No drug ctrl
Cpd 7 (1x MIC)
Cpd 7 (2x MIC)
Cpd 7 (4x MIC)
VOR (4x MIC)
1e+8
1e+7
1e+6
1e+5
1e+4
1e+3
0
5
10
15
20
25
5
10
15
20
25
Time (h)
Time (h)
Fig. 3. Representative time-kill curves for compounds 6 and 7 against C. albicans ATCC 10231
(strain A; panels A and C, respectively) and C. parapsilosis ATCC 22019 (strain J; panels B and
D, respectively). Fungal strains were treated with no drug (black circles), 1× MIC (white circles),
2× MIC (inverted black triangles), or 4× MIC (white triangles) of compounds 6 or 7, or with 4×
MIC (black squares) of VOR. The experiments were performed in duplicate.
2.4. Hemolysis assay. The promising antifungal activity shown by the synthetic analogues
demanded further safety analysis for these compounds. We tested compounds 6-10 for their
hemolytic activity against murine red blood cells (mRBCs) (Fig. 4 and Table S3). Compounds 6
(C8) and 7 (C10) displayed <10% and <40% hemolysis at concentrations of 31.3 µg/mL (1- to
512-fold of its overall MIC values) and 15.6 µg/mL (1- to 512-fold of its overall MIC values),

respectively. Similarly, compound 8 (C12) induced only 21% lysis at 7.8 µg/mL (1- to 218-fold
of its overall MIC values). In addition, compounds 9 (C14) and 10 (C16) displayed <50% and
<10% hemolysis at concentrations of 31.3 µg/mL (1- to 256-fold of its overall MIC values) and
62.5 µg/mL (2- to 128-fold of its overall MIC values), respectively. By comparing the hemolysis
of compounds 6-10 with their corresponding MIC values against the non-albicans Candida, A.
nidulans, and C. neoformans strains tested, we concluded that all of these compounds displayed
minimal hemolytic activity. Overall, compounds 6 and 10 displayed the lowest hemolytic
activity. Importantly, some of the newly synthesized compounds displayed less hemolytic effect
than the FDA-approved control drug VOR.
100
80
60
62.5
31.3
40
15.6
7.8
3.9
20
1.95
0.975
0.48
0
6
7
8
Compound
9
10
VOR
Fig. 4. 3D bar graph depicting the dose-dependent hemolytic activity of azole derivatives 6-10
and VOR against mRBCs, which were treated and incubated for 1 h at 37 °C with each

compound tested at concentrations ranging from 0.48-62.5 µg/mL. Triton X-100® (1% v/v) was
used as a positive control (100% hemolysis, not shown).
2.5. In vitro cytotoxicity assay. Another crucial parameter to consider when developing
antifungal drugs is their selectivity for fungal over mammalian cells. We tested our active
compounds 6-10 against three different cell lines, HEK-293, BEAS-2B, and A549, along with
the FDA-approved antifungal agent VOR as a positive control (Fig. 5). Against HEK-293,
compounds 6, 7, and 10 exhibited no toxicity up to 31 µg/mL (1- to 512-fold of its overall MIC
values), 7.8 µg/mL (1- to 256-fold of its overall MIC values), and 7.8 µg/mL (1- to 16-fold of its
overall MIC values), respectively. However, compounds 8 and 9 exhibited some toxicity (>50%
cell survival) at a concentration of 3.9 µg/mL (1- to 64-fold of its overall MIC values) against
HEK-293. Interestingly, compounds 8-10 were non-toxic to both BEAS-2B and A549 at up to
7.8 µg/mL (1- to 128-fold of its overall MIC values). In the case of compound 6, no toxicity was
observed against BEAS-2B (at 31 µg/mL) (1- to 512-fold of its overall MIC values) and A549
(at 15.5 µg/mL) (1- to 256-fold of its overall MIC values). With compound 10 we basically
observed no toxicity against BEAS-2B and A549 at 7.8 µg/mL (1- to 16-fold of its overall MIC
values). A general trend of greater toxicity with respect to longer chain substitution was observed
against the three cell lines. Compounds 6 and 7 exhibited better overall safety profiles than
compounds 8-10. When considering the very low MIC values for these analogues against clinical
isolates, these cytotoxicity data provide us with a reasonable therapeutic window.

A
B
C
Triton X (TX)
No drug
6
7
8
9
10
VOR
120
100
80
60
40
20
0
TX
TX
TX
0
0
0
0.13 0.25 0.5 1.0 1.9 3.9 7.8 15.5 31
Co
ncentrati
on (
µ
g/m
L)
120
100
80
60
40
20
0
0.13 0.25 0.5 1.0 1.9 3.9 7.8 15.5 31
C
oncentra
tion (
µg/
L
m )
120
100
80
60
40
20
0
0.13 0.25 0.5 1.0 1.9 3.9 7.8 15.5 31
Conce
ntration (
µg/mL)
Fig. 5. Representative cytotoxicity assays of compounds 6-10 against three mammalian cell
lines: A. HEK-293, B. BEAS-2B, and C. A549. Cells were treated with various concentrations of
compounds 6 (yellow), 7 (orange), 8 (turquoise), 9 (blue), 10 (pink), and VOR (purple). The
positive control consisted of cells treated with Triton X-100® (TX, 20% v/v, pink). The negative
control consisted of cells treated with DMSO (no drug, green). The experiments were performed
in duplicate.

2.6. Membrane permeabilization assay. Some amphiphilic molecules have been shown to
cause membrane disruption and fungal cell death. Therefore, we decided to study the potential
effect of compounds 8 and 9 with C12 and C14 linear alkyl chains to determine the impact of chain
length on membrane disruption (Fig. 6). The control drug VOR and the KANB (C14) derivative
with a 14-carbon linear alkyl chain, were used as negative and positive controls, respectively.
The propidium iodide (PI) dye was used as a probe as it can only enter cells with compromised
membrane and bind to nucleic acid to emit fluorescence, which can be observed under a
fluorescence microscope. At 4× MIC, the positive control KANB (C14) significantly increased PI
dye uptake by C. albicans ATCC 10231 (strain A), whereas the negative control VOR (at 4×
MIC) did not allow for PI uptake by fungal cells. Compound 8 with a C12 linear alkyl chain (at
4× MIC) induced cellular uptake of PI dye into C. albicans ATCC 10231 (strain A), whereas
compound 9 (with a C14 linear alkyl chain) did not cause cell membrane disruption. Interestingly,
the chain length played a crucial role on membrane disruption of C. albicans ATCC 10231
(strain A), surprisingly with C12 being more membrane disrupting than C14. In order to
completely understand the effect of chain length on membrane disruption, we additionally
performed membrane permeabilization with compounds 5, 6, and 7 (C6 -C10 linear alkyl chains)
at 4× MIC. None of these compounds induced cellular uptake of the PI drug. From this study, we
can conclude that one of the possible mechanisms of action for compound 8 is membrane
disruption. Interestingly, any other linear chains beside the C12 displayed no membrane
disruption.

Bright field
Fluorescent
Merge
Bright field
Fluorescent
Merge
Control
(no drug)
9
(1x MIC)
VOR
9
(2x MIC)
(0.975 µg/mL)
(4x MIC)
negative
control
KANB (C14
)
9
(4x MIC)
(31.3 µg/mL)
(4x MIC)
positive
control
8
(1x MIC)
5
(4x MIC)
8
(2x MIC)
6
(4x MIC)
8
(4x MIC)
7
(4x MIC)
Fig. 6. Effect of the controls VOR (negative) and KANB (C14) (positive) as well as compounds
5-9 on the cell membrane integrity of C. albicans ATCC 10231 (strain A). For the three columns
on the left from top to bottom: Propidium iodide (PI) dye uptake by yeast cells without drug,
with VOR (0.975 µg/mL), KANB (C14) (31.3 µg/mL), and compound 8 (1×, 2×, and 4× MIC).
For the three columns on the right from top to bottom: Propidium iodide (PI) dye uptake by yeast
cells with compound 9 (1×, 2×, and 4× MIC), as well as compound 5, 6, and 7 at 4× MIC. The
experiments were performed in duplicate.
2.7. Sterol profile by GC-MS. Since out of our compounds displaying antifungal activity only
compound 8 caused membrane disruption of C. albicans ATCC 10231 (strain A) in the
membrane permeabilization assay (section 2.6), we decided to explore by using gas

chromatography-mass spectrometry (GC-MS) the effect of compounds 8 and 9 on sterol
composition during ergosterol biosynthesis (the mechanism of action of other conventional
azoles) (Fig. 7). We evaluated the effect of the compounds 8 and 9 on sterol composition in C.
albicans ATCC 10231 (strain A) at sub-MIC levels of 0.48 µg/mL (Fig. 7D) and 0.975 µg/mL
(Fig. 7E), respectively. We also used VOR at 0.12 µg/mL and no drug control for comparison
(Fig. 7B and 7A, respectively). In the absence of drug, strain A accumulated 100% ergosterol
(2), suggesting that the sterol biosynthesis was fully functional in this fungal strain. When
treating strain A with VOR, we detected a lower amount of ergosterol (2, 50.80%), and an
increased amount of lanosterol (1, 15.21%) and eburicol (3, 1.73%). However, when strain A
was treated with compound 8, we observed a relatively low amount of ergosterol (2, 36.62%)
compared to VOR, but observed lower amounts of lanosterol (1, 10.03%), eburicol (3, 0.57%),
and the fungistatic metabolite 14α-methyl ergosta-8,24(28)-diene-3β,6α-diol (4, 4.69%) with
respect to VOR. Interestingly, when strain A was treated with compound 9, we observed similar
reduction in the amount of ergosterol (2, 36.43%) along with a related increase in lanosterol (1,
15.56%), eburicol (3, 1.16%) and the fungistatic metabolite 14α-methyl ergosta-8,24(28)-diene-
3β,6α-diol (4, 15.56%). From these experiments, we can conclude that our compounds inhibit
ergosterol biosynthesis similarly to the azole drug control VOR.

A. Simplified ergosterol biosynthetic pathway and products resulting from inhibition of ERG11:
ERG11
sterol 14α-demethylase
Lanosterol (1)
Ergosterol (2)
4,4-dimethyl cholesta 8,14,24-trienol
Eburicol (3)
14α-methyl ergosta-8,24(28)-dien-3β,6α-diol (4)
azole inhibition
B
C
No drug
VOR
2
2
5.0e+6
4.0e+6
3.0e+6
2.0e+6
1.0e+6
0
8.0e+6
6.0e+6
4.0e+6
2.0e+6
0
1
4
3
11.0
11.5
12.0
12.5
13.0
13.5
11.0
11.5
12.0
12.5
13.0
13.5
Retention time (min)
Retention time (min)
D
E
2
2
Cpd 9
Cpd 8
3.0e+6
2.0e+6
1.0e+6
0
3.0e+6
2.0e+6
1
1
4
1.0e+6
0
4
3
3
11.0
11.5
12.0
12.5
13.0
13.5
11.0
11.5
12.0
12.5
13.0
13.5
Retention time (min)
Retention time (min)
F. Sterol composition of untreated and VOR-, cpd 8-, and cpd 9-treated C. albicans ATCC 10231 (strain A):
Sterol composition (%)
Sterol
No drug
VOR
Cpd 8
Cpd 9
Lanosterol (1)
Ergosterol (2)
Eburicol (3)
ND
100.00
ND
15.21
50.80
1.73
10.03
36.62
0.57
15.56
36.43
1.16
14α-methyl ergosta-8,24(28)-dien-3β,6α-diol (4) ND
9.86
4.69
8.33
ND = not detected
Fig. 7. A. A simplified ergosterol biosynthetic pathway and products resulting from inhibition of
ERG11. B-E. GC-MS chromatograms of the sterols extracted from untreated and antifungal-
treated C. albicans ATCC 10231 (strain A). The fungal strain treated with DMSO (no drug
control, panel B), VOR at 0.12 µg/mL (panel C), compound 8 at 0.48 µg/mL (panel D), and
compound 9 at 0.975 µg/mL (panel E). The peaks in these chromatograms are for lanosterol (1),
ergosterol (2), eburicol (3), 14α-methyl ergosta-8,24(28)-dien-3β,6α-diol (4). F. A table
summarizing the percentage of each sterol from panels B-E.

3. Conclusions
In summary, we have synthesized novel FLC derivatives in which the triazole ring on the carbon
alpha to the dihalophenyl ring of FLC was displaced by various linear alkyl-, aryl-, dialkyl-, and
cycloalkyl-amino substituents. We did not detect any antifungal activity with the aryl- and
cycloalkyl-amino substituted FLC analogues. We observed that the antifungal activity of the
alkyl-amino FLC derivatives depends on the length of the alkyl chains. Compounds 6-9 were
identified as promising antifungal agents with low hemolytic activity and low cytotoxicity. These
analogues displayed great activity against some of the C. albicans, non-albicans Candida, and
Aspergillus strains, and, in addition they were particularly excellent against the clinical strains of
C. glabrata, C. parapsilosis, as well as C. neoformans tested. These compounds also exhibited
superior activity against the clinical strains when compared to the control drugs CAS, FLC, and
VOR. The possible mechanism of action for these FLC analogues was identified as membrane
disruption with compound 8 with a C12 alkyl chain being more membrane disrupting than
compound 9 with a C14 alkyl chain. Additionally, they were found to inhibit ergosterol
biosynthesis. In the future, outside of the scope of this work, it will be interesting to see how
these promising molecules will fair in the drug development process.
Acknowledgments
This work was supported by startup funds from the University of Kentucky (to S.G.-T.) and by
NIH grant AI090048 (to S.G.-T.). H.X.N. was in part supported by a Pharmaceutical Sciences
Excellence in Graduate Achievement Fellowship from the College of Pharmacy at the University
of Kentucky.

Author Contributions
N.T.C., S.K.S, and S.G.-T. design the study, analyzed the data, wrote the manuscript and
supporting information, and made figures. N.T.C. and K.C.H. synthesized all compounds used in
this study. H.X.N. performed the cytotoxicity assays and time-kill studies and generated Figs. 2
and 4. S.K.S. performed all other biochemical/biological experiments. N.T.C., S.K.S., H.X.N.,
and S.G.-T. reviewed the manuscript and supporting information. All authors approved the
manuscript and supporting information.
Supporting Information Available: The supporting information includes experimental
procedures for the chemistry and biological work performed. More specifically, it includes: (i)
materials and instrumentation, (ii) protocols for the synthesis of compounds 1-16, (iii) a list of
antifungal agents used, (iv) a list of organisms against which compounds 5-16 were tested along
with their culture conditions, and experimental procedures for (v) MIC value determination, (vi)
time-kill studies, (vii) hemolytic activity, (viii) in vitro cytotoxicity assay, (xi) membrane
permeabilization assay, and (x) sterol profile by GC-MS. 1H and 13C NMR spectra (Fig. S1-S32)
of all compounds generated in this study are also presented. The MIC values in µM are provided
in Tables S1 and S2. The exact percentages with SDEV that were used to prepare Fig. 4 are
provided in Table S3.
Corresponding Author Information

Sylvie Garneau-Tsodikova E-mail: sylviegtsodikova@uky.edu, Phone: 859-218-1686\
Abbreviations
AmB, amphotericin B; ATCC, American Type Culture Collection; CAS, caspofungin; CLSI,
Clinical and Laboratory Standards Institute; FBS, fetal bovine serum; FLC, fluconazole; KANB,
kanamycin B; mRBCs, murine red blood cells; PI, propidium iodide; SAR, structure-activity-
relationship; TOB, tobramycin; VOR, voriconazole.
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Image for Table of Content
R1
R2
N
12 new azole
O
N
N
N
N
HO
N
N
ANTIFUNGAL
derivatives
F
F
F
F
R1/R2 = alkyl or aryl
Less toxic/hemolytic
than FDA-approved azoles
Membrane disruptors