Synthesis, optimization, antifungal activity, selectivity, and cyp51 binding of new 2-aryl-3-azolyl-1-indolyl-propan-2-ols

Article abstract of DOI:10.3390/ph13080186

A series of 2-aryl-3-azolyl-1-indolyl-propan-2-ols was designed as new analogs of fluconazole (FLC) by replacing one of its two triazole moieties by an indole scaffold. Two different chemical approaches were then developed. The first one, in seven steps, involved the synthesis of the key intermediate 1-(1H-benzotriazol-1-yl)methyl-1H-indole and the final opening of oxiranes by imidazole or 1H-1,2,4-triazole. The second route allowed access to the target compounds in only three steps, this time with the ring opening by indole and analogs. Twenty azole derivatives were tested against Candida albicans and other Candida species. The enantiomers of the best anti-Candida compound, 2-(2,4-dichlorophenyl)-3-(1H-indol-1-yl)-1-(1H-1,2,4-triazol-1-yl)-propan-2-ol (8g), were analyzed by X-ray diffraction to determine their absolute configuration. The (?)-8g enantiomer (Minimum inhibitory concentration (MIC) = IC₈₀ = 0.000256 μg/mL on C. albicans CA98001) was found with the S-absolute configuration. In contrast the (+)-8g enantiomer was found with the R-absolute configuration (MIC = 0.023 μg/mL on C. albicans CA98001). By comparison, the MIC value for FLC was determined as 0.020 μg/mL for the same clinical isolate. Additionally, molecular docking calculations and molecular dynamics simulations were carried out using a crystal structure of Candida albicans lanosterol 14α-demethylase (CaCYP51). The (?)-(S)-8g enantiomer aligned with the positioning of posaconazole within both the heme and access channel binding sites, which was consistent with its biological results. All target compounds have been also studied against human fetal lung fibroblast (MRC-5) cells. Finally, the selectivity of four compounds on a panel of human P450-dependent enzymes (CYP19, CYP17, CYP26A1, CYP11B1, and CYP11B2) was investigated.

Full text of DOI:10.3390/ph13080186

pharmaceuticals
Article
Synthesis, Optimization, Antifungal Activity,
Selectivity, and CYP51 Binding of
New 2-Aryl-3-azolyl-1-indolyl-propan-2-ols
Nicolas Lebouvier 1,2 , Fabrice Pagniez 3 , Young Min Na 1, Da Shi 4 , Patricia Pinson 1,
Mathieu Marchivie 5 , Jean Guillon 6 , Tarek Hakki 7, Rita Bernhardt 7 , Sook Wah Yee 8,
Claire Simons 8 , Marie-Pierre Lézé 1,9, Rolf W. Hartmann 9 , Angélique Mularoni 10
,
Guillaume Le Baut 1, Isabelle Krimm 11,12 , Ruben Abagyan 4 , Patrice Le Pape 3,
*
and
Marc Le Borgne 1,10,
*
1
EA1155—IICiMed, Institut de Recherche en Santé 2, Département de Pharmacochimie,
Nantes Atlantique Universités, Université de Nantes, F-44200 Nantes, France;
nicolas.lebouvier@univ-nc.nc (N.L.); ymna@joowonpat.com (Y.M.N.); patricia.pinson@univ-nantes.fr (P.P.);
mpl_leze@yahoo.fr (M.-P.L.); guillaume.lebaut@wanadoo.fr (G.L.B.)
Institut des Sciences Exactes et Appliquées (ISEA) EA 7484, Université de la Nouvelle-Calédonie, 98851
Noumea CEDEX, New Caledonia
EA1155—IICiMed, Institut de Recherche en Santé 2, Département de Parasitologie et Mycologie Médicale,
Nantes Atlantique Universités, Université de Nantes, F-44200 Nantes, France;
fabrice.pagniez@univ-nantes.fr
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, CA 92093,
USA; das046@ucsd.edu (D.S.); ruben@ucsd.edu (R.A.)
ICMCB CNRS-UPR 9048, Université de Bordeaux, F-33608 Pessac CEDEX, France;
mathieu.marchivie@icmcb.cnrs.fr
ARNA Laboratory, INSERM U1212, UMR CNRS 5320, UFR des Sciences Pharmaceutiques,
Université de Bordeaux, F-33076 Bordeaux, France; jean.guillon@u-bordeaux.fr
Department of Biochemistry, Saarland University, Campus B2.2, 66123 Saarbrücken, Germany;
t.hakki@dr-hakki.de (T.H.); ritabern@mx.uni-saarland.de (R.B.)
School of Pharmacy & Pharmaceutical Sciences, Cardiff University, King Edward VII Avenue,
Cardiff CF10 3NB, UK; sookwah.yee@ucsf.edu (S.W.Y.); SimonsC@cardiff.ac.uk (C.S.)
Department of Pharmacy, Pharmaceutical and Medicinal Chemistry, Saarland University, Campus E8.1,
66123 Saarbrücken, Germany; Rolf.Hartmann@helmholtz-hzi.de
2
3
4
5
6
7
8
9
10
EA 4446 Bioactive Molecules and Medicinal Chemistry, Faculté de Pharmacie-ISPB,
SFR Sant
CEDEX 8, F-69373 Lyon, France; angelique.mularoni@univ-lyon1.fr
Centre de RMN Tr s Hauts Champs, CNRS, ENS, Universit Claude Bernard Lyon 1, Universit
5 rue de la Doua, F-69100 Villeurbanne, France; isabelle.krimm@univ-lyon1.fr
Centre de Recherche en Cancérologie de Lyon, INSERM 1052, CNRS 5286, Centre Léon Bérard,
Université Claude Bernard Lyon 1, Université de Lyon, F-69008 Lyon, France
é
Lyon-Est CNRS UMS3453-INSERM US7, Universit
é
Claude Bernard Lyon 1, Universit
é
de Lyon,
de Lyon,
11
12
à
è
é
é
*
Correspondence: Patrice.Le-Pape@univ-nantes.fr (P.L.P.); marc.le-borgne@univ-lyon1.fr (M.L.B.);
Tel.: +33-240-084-072 (P.L.P.); +33-478-777-082 (M.L.B.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 15 July 2020; Accepted: 5 August 2020; Published: 8 August 2020
Abstract: A series of 2-aryl-3-azolyl-1-indolyl-propan-2-ols was designed as new analogs of
fluconazole (FLC) by replacing one of its two triazole moieties by an indole scaffold. Two different
chemical approaches were then developed. The first one, in seven steps, involved the synthesis of
the key intermediate 1-(1H-benzotriazol-1-yl)methyl-1H-indole and the final opening of oxiranes by
imidazole or 1H-1,2,4-triazole. The second route allowed access to the target compounds in only three
steps, this time with the ring opening by indole and analogs. Twenty azole derivatives were tested
against Candida albicans and other Candida species. The enantiomers of the best anti-Candida compound,
2-(2,4-dichlorophenyl)-3-(1H-indol-1-yl)-1-(1H-1,2,4-triazol-1-yl)-propan-2-ol (8g), were analyzed
Pharmaceuticals 2020, 13, 186; doi:10.3390/ph13080186
www.mdpi.com/journal/pharmaceuticals

Pharmaceuticals 2020, 13, 186
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by X-ray diffraction to determine their absolute configuration. The (
−
)-8g enantiomer (Minimum
inhibitory concentration (MIC) = IC80 = 0.000256
the S-absolute configuration. In contrast the (+)-8g enantiomer was found with the R-absolute
configuration (MIC = 0.023 g/mL on C. albicans CA98001). By comparison, the MIC value for
FLC was determined as 0.020 g/mL for the same clinical isolate. Additionally, molecular docking
calculations and molecular dynamics simulations were carried out using a crystal structure of
Candida albicans lanosterol 14 -demethylase (CaCYP51). The ( )-(S)-8g enantiomer aligned with
µg/mL on C. albicans CA98001) was found with
µ
µ
α
−
the positioning of posaconazole within both the heme and access channel binding sites, which was
consistent with its biological results. All target compounds have been also studied against human
fetal lung fibroblast (MRC-5) cells. Finally, the selectivity of four compounds on a panel of human
P450-dependent enzymes (CYP19, CYP17, CYP26A1, CYP11B1, and CYP11B2) was investigated.
Keywords: azoles; antifungal agents; indole; microwave irradiation; X-ray crystallography;
Candida species; docking; CYP51; selectivity; cytochromes P450
1. Introduction
Invasive fungal infections are related to a high mortality rate despite the availability of several
antifungal drugs. Fungi species that belong to one of these four genera (Cryptococcus, Candida,
Aspergillus, and Pneumocystis) kill about one and a half million people every year [1,2]. These infections
have increased in frequency and severity over the last two decades as a result of an increasing number
of immunocompromised hosts due to cancer chemotherapy, organ and bone marrow transplantation,
human immunodeficiency virus, or therapy against autoimmune and inflammatory diseases especially
with TNF inhibitors [3]. Candida albicans (C. albicans) is the major opportunistic pathogen of fungal
infections and Candida spp. are the fourth most common nosocomial bloodstream pathogens in the
USA with a mortality rate of 40% [1,2].
For many years, the polyene amphotericin B has been the only available antifungal drug for
invasive fungal infections despite nephrotoxicity, infusion-related reactions, and other adverse effects [
To this day, approved antifungal agents belong to only four drug classes: Polyenes, azoles, echinocandins,
and anti-metabolites [ ]. Introduction of triazoles (e.g., fluconazole (FLC), itraconazole) offered new
4].
5
treatment options with excellent antifungal activity and a good safety profile. Prophylactic use of FLC has
been developed to reduce the risk of Candida colonization and infection in high-risk patients with minimal
toxicity [
6
,
7
]. Nevertheless, the emergence of C. krusei and C. glabrata infections in patients with bone
marrow transplantation or neutropenia receiving FLC prophylaxis has been reported [8,9
]. Development of
Candida spp. resistance has also commonly been observed in HIV-infected patients who received prolonged
FLC treatment for oropharyngeal candidiasis [2,10,11].
Fourth generation azoles (e.g., voriconazole, posaconazole, ravuconazole, isavuconazole) and
echinocandins (e.g., caspofungin, micafungin, anidulafungin) approved for use or under clinical trials
have proven to be less toxic than conventional amphotericin B, resulting in a significant advance in
treatment of serious fungal infections. However, voriconazole interferes with many drugs and its
prolonged use may expose severely immunocompromised patients to the risk of zygomycosis [12],
while echinocandins are not available orally [13]. Consequently, most patients requiring therapy
for invasive candidiasis are still placed on the safe, inexpensive, and orally available fluconazole.
Despite recent advances in antifungal chemotherapy, the mortality rates of invasive fungal infections
have remained unchanged and there is a necessity for the development of new antifungal compounds.
Aiming at new and effective antifungal drugs, our attention was focused on the azole derivatives because
of their low toxicity, broad spectrum of activity, and favorable pharmacokinetic properties [10,11,14].
Antifungal azoles (Figure 1) target the P450-dependent enzyme lanosterol 14
α-demethylase
(CYP51), preventing the production of ergosterol, a key component in the fungal cell membrane [15].

Pharmaceuticals 2020, 13, 186
3 of 32
Depletion of ergosterol and accumulation of 14
α
-methylated sterols alter membrane fluidity, increase
permeability, and reduce the activity of membrane-associated enzymes. Azoles bind to the iron of
the protoporphyrin unit located in the active site of CYP51, preventing the access of the natural
substrate lanosterol [16
,17]. Azoles are also used for the treatment of estrogen-dependent disease
by inhibition of aromatase (CYP19) in breast cancer therapy [18
,19], and the inhibition of human
P450-dependent enzymes is an unwanted side effect that could lead to impediments for safe therapeutic
profile, including toxicity and undesirable drug interactions [20]. Selectivity of antifungal agents is
therefore crucial because of the high levels of similarity between the eukaryotic fungal pathogens
and the human hosts, and must be considered as a top priority in the development of new antifungal
azoles [5,10,21].
N
N
Cl
N
F
N
N
HO
F
OH
N
N
N
Cl
H
N
O
F
O
N
N
N
N
O
F
N
N
O
N
N
N
N
F
F
itraconazole
fluconazole
voriconazole
CN
CN
F
O
O
N
N
N
S
N
N
N
S
O
H
N
N
N
HO
OH
N
HO
N
N
N
N
N
F
posaconazole
F
N
F
F
ravuconazole
Figure 1. Structures of the main azole antifungal agents.
isavuconazole
Our research group has developed several potent series of antifungal compounds based on an
indole scaffold with better in vitro antifungal activities against C. albicans compared with FLC [22 27].
–
To continue this pharmacomodulation strategy, we describe in the present paper the synthesis
of 2-phenyl-1-(1H-indol-1-yl)-3-(azolyl)propan-2-ols using two different synthetic routes and their
anti-Candida activities. The safety profile of target compounds was also evaluated by a cytotoxicity
assay on human fetal lung fibroblast (MRC-5) cells, and by selectivity assessment using a panel
of human P450-dependent enzymes. Additionally, separation of the two enantiomers of the most
potent compound, 2-(2,4-dichlorophenyl)-3-(1H-indol-1-yl)-1-(1H-1,2,4-triazol-1-yl)-propan-2-ol 8g
was performed and the determination of their absolute configuration was achieved. A molecular
modeling study was finally performed to assess the binding feasibility of (R)-8g and (S)-8g for lanosterol
14α-demethylase (CYP51).
,
2. Results and Discussion
2.1. Chemistry
Two synthetic routes were designed to obtain the 2-phenyl-3-(1H-indol-1-yl)-1-(azol-1-yl)propan-2-ol
derivatives 8a–g and 11a–l.
The first pathway strategy (route 1) involved the synthesis of the key intermediate
1-(1H-benzotriazol-1-yl)methyl-1H-indole 4 (Scheme 1). According to this scheme, hydroxymethylation of

Pharmaceuticals 2020, 13, 186
4 of 32
benzotriazole
, which was converted into 1-chloromethyl-1H-benzotriazole
was then alkylated by compound in the presence of sodium hydride (NaH) in dimethyl sulfoxide (DMSO)
to give the intermediate . Lithiation of with n-butyllithium at
78 ◦C in tetrahydrofuran (THF) followed
by addition of the appropriate esters gave the target -benzotriazolyl ketones 5a . Debenzotriazolylation
of 5a occurred in the presence of zinc and acetic acid in a mixture of THF/EtOH (1:1, v/v) under
ultrasonic activation. The corresponding ketones 6a were then converted into oxiranes 7a by the
Corey–Chaykovsky epoxidation using trimethylsulfoxonium iodide (TMSOI) and an aqueous solution of
sodium hydroxide in dichloromethane under reflux. The ring opening of 7a with 1H-1,2,4-triazole or
1
using an aqueous solution of formaldehyde quickly gave 1-hydroxymethyl-1H-benzotriazole
2
3
using thionyl chloride at 0 ◦C. The indole
3
4
4
−
α
–f
–f
–f
–f
–
f
imidazole in the presence of potassium carbonate (K2CO3) at reflux in acetonitrile (CH3CN) gave the target
2-aryl-3-(1H-indol-1-yl)-1-(azol-1-yl)propan-2-ol derivatives 8a–g.
N
N
N
N
N
N
a
c
b
N
N
N
NH
N
N
Cl
N
HO
1
2
3
4
d
N
N
N
N
e
g
O
f
N
O
O
OH
Y
X
N
X
A
N
N
X
X
N
Y
Y
Y
7a-f
6a-f
: X = H, Y = F
5a-f
: X = H, Y = F, A = CH
: X = H, Y = Cl, A = CH
: X = H, Y = Br, A = CH
: X = F, Y = F, A = CH
: X = Cl, Y = Cl, A = CH
8a
8b
8c
8d
8e
5a, 6a, 7a
5b, 6b, 7b
5c, 6c, 7c
5d, 6d, 7d
: X = H, Y = Cl
: X = H, Y = Br
: X = H, Y = CF
3
: X = H, Y = CF , A = N
8f
: X = F, Y = F
5e, 6e, 7e
3
: X = Cl, Y = Cl, A = N
: X = Cl, Y = Cl
5f, 6f, 7f
8g
Scheme 1. Synthesis of compounds 8a
temperature (rt), 5 min; (
derivatives, THF,
78 ◦C to rt, 12 h; (
CH2Cl2, reflux, 48 h; (g) K2CO3, imidazole or 1H-1,2,4-triazole, CH3CN, reflux, 7 h.
–
g
(Route 1). Reagents and conditions: (
) NaH, 1H-indole, DMSO, rt, 12 h; ( ) n-BuLi, ethylbenzoate
) AcOH, Zn, THF, EtOH, ultrasound, 35 ◦C, 5 h; (
) TMSOI, NaOHaq
a) HCHOaq, room
b
) SOCl2, reflux, 1 h; (
c
d
−
e
f
,
In order to reduce the number of reaction steps, we investigated another synthetic pathway called
route 2 (Scheme 2). First, imidazole and 1H-1,2,4-triazole were alkylated by commercially available
halogenoacetophenones using K2CO3 in CH3CN under microwave heating (85 ◦C, 50 W) to give the
target ketones 9a
–f, which were converted to oxiranes 10a–f in the presence of TMSOI and an aqueous
solution of sodium hydroxide in toluene under microwave heating (80 ◦C, 50 W). The reaction times of

Pharmaceuticals 2020, 13, 186
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these two steps (alkylation and epoxidation) are greatly reduced by using microwave heating as we
have already reported [27]. Finally, the ring opening of 10a with indole using NaH in DMSO gave
the derivatives 8g and 11a . A similar ring opening of the intermediates 9d and 9e by indoles with
methyl groups in position 2 and/or 3 furnished the target derivatives 11f . In the case of the synthesis
of compound 8g, route 2 gave a great improvement of the overall yield (route 1: 5% vs. route 2: 29%).
–
f
–e
–
k
X
O
X
O
A
X
O
a
b
c
N
N
Z
Y
A
Y
Y
N
N
9a-f
10a-f
X = H, Y = F, Z = Cl
: X = H, Y = F, A = N
: X = H, Y = Cl, A = N
: X = H, Y = Br, A = N
: X = F, Y = F, A = N
: X = Cl, Y = Cl, A = N
9a, 10a
9b, 10b
9c, 10c
9d, 10d
9e, 10e
R2
X = H, Y = Cl, Z = Cl
X = H, Y = Br, Z = Cl
X = F, Y = F, Z = Cl
X = Cl, Y = Cl, Z = Cl
X = H, Y = CF3, Z =Br
R1
N
OH
X
A
N
: X = H, Y = CF , A =CH
N
9f, 10f
3
Y
,
11a-k 8g
: X = H, Y = F, A = N, R = H, R = H
11a
11b
11c
11d
1
2
: X = H, Y = Cl, A = N, R = H, R = H
1
2
: X = H, Y = Br, A = N, R = H, R = H
1
2
: X = F, Y = F, A = N, R = H, R = H
1
2
: X = Cl, Y = Cl, A = N, R = H, R = H
8g
1
2
: X = H, Y = CF , A = CH, R = H, R = H
11e
3
1
2
: X = F, Y = F, A = N, R = Me, R = H
11f
1
2
: X = Cl, Y = Cl, A = N, R = Me, R = H
11g
11h
1
2
: X = F, Y = F, A = N, R = H, R = Me
1
2
: X = Cl, Y = Cl, A = N, R = H, R = Me
11i
11j
11k
1
2
: X = F, Y = F, A = N, R = Me, R = Me
1
2
: X = Cl, Y = Cl, A = N, R = Me, R = Me
1
2
Scheme 2. Synthesis of compounds 8g and 11a
–
k
(Route 2). Reagents and conditions: (
a
) K2CO3,
imidazole or 1H-1,2,4-triazole, CH3CN, MW 85 ◦C, 50 W, 50 min; (
reflux, 72 h; (c) NaH, indole derivative, DMSO, rt, 12 h.
b) NaOHaq 20%, TMSOI, CH2Cl2,
To observe the influence of the nitrogen atom position in the azole ring, the effective route 2
was used to synthesize two other 2-(2,4-dichlorophenyl)-3-(1H-indol-1-yl)-1-(azol-1-yl)propan-2-ol
derivatives 11l and 11m with azole rings 1H-1,2,3-triazole and 2H-1,2,3-triazole (Scheme 3). Alkylation
of 1H-1,2,3-triazole by 2,20,40-trichloroacetophenone gave a mixture of N-1 9g and N-2 9h alkylation
products. This alkylation by conventional heating provided a ratio of 61/39 N-1 and N-2 products
while a microwave activation at the same temperature gave a 49/51 mixture. Epoxidation and indole

Pharmaceuticals 2020, 13, 186
6 of 32
alkylation from 9g and 9h under the same conditions as in route 2 gave the final products 11l and 11m
(Scheme 3).
Cl
O
B
Cl
O
B
Cl
O
a or b
c
d
N
N
N
Cl
Cl
N
Cl
OH
Cl
Cl
N
A
B
N
A
N
A
9g/9h
condition a: 49/51
condition b: 61/39
Cl
9g: A = N, B = CH
9h: A = CH, B = N
10g: A = N, B = CH
10h: A = CH, B = N
11l: A = N, B = CH
11m: A = CH, B = N
Scheme 3. Synthesis of compounds 11l and 11m. Reagents and conditions: (
CH3CN, MW 85 ◦C, 50 W, 50 min; (
TMSOI, CH2Cl2, reflux, 72 h; (d) NaH, indole, DMSO, rt, 12 h.
a
) K2CO3, 1H-1,2,3-triazole,
b
) K2CO3, 1H-1,2,3-triazole, CH3CN, 85 ◦C, 8 h; (
c) NaOHaq 20%,
2.2. X-ray Structural Studies
In order to confirm the absolute configuration of the synthesized compounds 8g, X-ray structure
analyses for the two derivatives were performed. The atom labeling and thermal ellipsoids of (+)-8g
and (
found in the asymmetric crystallographic unit of (+)-8g and (
)-8g was determined by observing and calculating the F(+)/F(
F value of each independent reflection. Based on the results, the absolute configuration at C-9 and C-59
in (+)-8g was determined to be R. The ( )-8g enantiomer was found with the S-absolute configuration.
−
)-8g are shown in Figures 2 and 3. Two independent molecules, designated as A and B, were
)-8g. The configuration of (+)-8g and
) ratios of Bijvoet pairs with the mean
−
(−
−
−
Figure 2. View of the crystal structure of (R)-8g with our numbering scheme, displacement ellipsoids
are drawn at the 30% probability level.

Pharmaceuticals 2020, 13, 186
7 of 32
Figure 3. View of the crystal structure of (S)-8g with our numbering scheme, displacement ellipsoids
are drawn at the 30% probability level.
2.3. Biological Results
2.3.1. In Vitro Anti-Candida Activity
All anti-Candida activities of propanol derivatives 8a
Concerning the expression of the results during the evaluation of the compounds, we deliberately chose
to calculate a more selective minimal inhibitory concentration (MIC = IC80, g/mL) than an IC50 in order
to highlight some very active compounds. Concerning the 1H-1,2,4-triazole sub-series, substitutions of
–g and 11a–m are presented in Table 1.
µ
the phenyl ring by halogens greatly impacted anti-C. albicans activities. The 4-trifluoromethylated 8f
,
4-chlorinated 11b, 4-brominated 11c, and 2,4-difluorinated 11d compounds had activities comparable
with FLC (MIC = 0.020
µg/mL on C. albicans CA98001), while the 4-fluorinated derivative 11a was less
active (MIC = 0.210 g/mL). The most favorable substitution corresponded to the 2,4-dichlorinated
µ
phenyl ring for compound 8g. The asymmetric carbon of the propanol chain also played a very
important role in the activity against C. albicans. Indeed, activity of the (+)-(R)-enantiomer was similar
to FLC (MIC = 0.023
racemic mixture (MIC values of 0.000256 and 0.000259 µg/mL, respectively).
µ
g/mL), whereas the ( )-(S)-enantiomer had an action comparable with the
−
In the imidazole sub-series, there was little difference in activity depending on the substitution of the
phenyl ring because all compounds had activities similar to that of FLC except for the 2,4-difluorinated
derivative 8d, which was 57 times more active (MIC = 0.00035
the imidazole and 1H-1,2,4-triazole series according to the substitution of the phenyl ring. Compounds
of 1H-1,2,3-triazole and 2H-1,2,3-triazole series with 2,4-dichlophenyl substitution 11l were totally
µg/mL). There was no correlation between
–m
inactive. This pharmacomodulation of the azolyl unit highlighted the necessity of the 1H-1,2,4-triazole
or imidazole moiety to promote activity against C. albicans. The inactivity of 1H-1,2,3-triazole and
2H-1,2,3-triazole derivatives could be explained by the impossibility of these rings to coordinate with
the iron atom of CYP51.

Pharmaceuticals 2020, 13, 186
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Table 1. Anti-Candida activity of propanol derivatives 8a–g and 11a–m.
R2
R1
N
OH
X
D
N
C
A
B
Y
MIC 1 (µg/mL) C.
albicans CA98001
Compound
A
B
C
D
R1
R2
X
Y
8a
8b
8c
8d
8e
11e
11a
11b
11c
11d
8g
CH
CH
CH
CH
CH
CH
N
N
N
N
N
N
N
N
N
N
N
N
N
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
Cl
H
H
H
H
F
Cl
Cl
Cl
H
F
Cl
F
Cl
F
Cl
Cl
Cl
F
Cl
Br
F
Cl
CF3
F
Cl
Br
F
Cl
Cl
Cl
CF3
F
Cl
Cl
Cl
F
Cl
Cl
Cl
0.03
0.02
0.02
0.00035
0.062
0.23
0.21
0.024
0.027
0.0198
0.000259
0.023
0.000256
0.009
0.022
0.0058
0.0011
0.007
0.157
1.246
>100
(+)-(R)-8g
(−)-(S)-8g
8f
11f
CH3
CH3
H
11g
11h
11i
11j
11k
11l
11m
KTC
FLC
CH3
CH3
CH3
CH3
H
H
CH3
CH3
H
N
N
N
CH
H
H
>100
0.005
0.02
1
Minimum inhibitory concentration (MIC = IC80, µg/mL) was expressed as the mean of triplicate measures.
The last work of pharmacomodulation focused on the presence of the methyl group in position 2
and/or 3 of the indole ring. With a 2,4-difluorinated phenyl group, the presence of a methyl at position
2 on the indole ring did not change the activity (11f, MIC = 0.022 µg/mL); on the other hand, the
introduction of a methyl at position 3 enhanced the activity level (11h, MIC = 0.011
two methyl groups at positions 2 and 3, the activity was reduced (11j, MIC = 0.157
µ
g/mL). In the case of
µg/mL). The activities
of 2,4-dichloro derivatives were generally better than those encountered with 2,4-difluorinated
products. However, none of the proposed pharmacomodulations retained the activity obtained with
the unsubstituted indole. In this case, the inactivity of the 2,3-dimethylindole unit was even more
pronounced. The conjugated steric effect of the methyl groups on positions 2 and 3 on the indole ring
may have been the cause for this decrease in activity, by imposing an unfavorable position of the indole
in the active site of the enzyme.
Overall, it emerged that the synthesized compounds showed activity against C. albicans comparable
with or greater than fluconazole. For example, difluoro derivatives (8d and 11h) and dichloro derivatives
(
8g and 11i) were the most active derivatives. In the particular case of compound 8g, ( )-8g and its
±
(−)-(S) enantiomer were ≈80 times more active than FLC against C. albicans CA98001.
The activity spectrum on Candida species (Table 2) shows that compound 8g and more particularly
its (S)-enantiomer maintained a better activity than FLC against the various strains tested, especially
on those naturally resistant to FLC. For example, on C. krusei, the MIC values of (S)-8g were between
0.167 and 0.039 µg/mL.

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Table 2. MIC 1 (µg/mL) spectrum of compounds 8a–g, 11a–k, ketoconazole (KTC), and FLC.
Compound ATCC2091 CA98002 CA98005 ATCC6258 CK98002 CK98003
CL98001
ATCC90018
8a
8b
8c
8d
8e
11e
11a
11b
11c
11d
8g
0.131
0.215
0.357
0.726
0.19
0.205
0.165
0.25
0.609
0.251
0.32
0.148
0.106
0.21
>33.5
>35.2
>39.6
3.863
1.2
22.121
19.24
2.434
1.51
12.761
2.723
5.076
5.692
1.8
18.306
21.426
12.666
2.463
1.55
2.788
22.074
0.022
10.557
7.589
>40
28.597
>35.183
36.565
5.205
0.28
16.225
18.668
2.434
2.503
24.05
3.563
19.363
0.039
3.068
5.452
>40
0.231
0.222
0.25
2.11
1.3
1.84
0.614
0.24
0.439
0.274
0.258
0.437
0.222
0.286
0.064
<0.004
2.707
0.026
0.749
0.516
0.269
0.265
0.03
0.068
0.116
0.173
0.259
0.208
0.215
0.021
<0.004
9.682
0.024
0.199
0.232
0.181
0.023
0.025
0.256
2.866
0.007
0.55
2.12
2.466
2.523
2.046
2.026
0.201
0.15
>38.726
0.027
2
2.505
>40
0.309
0.245
4.015
9.76
0.437
0.258
0.477
0.021
<0.004
1.007
<0.039
0.247
0.199
0.526
0.136
0.026
0.256
2.741
0.008
0.07
0.404
0.261
0.338
0.026
<0.004
2.75
<0.039
0.233
0.302
0.321
0.033
0.044
0.382
1.121
0.009
1
2.181
0.08
(+)-(R)-8g
(−)-(S)-8g
8f
24.398
0.167
19.091
17.572
>40
1.4
3.01
17.97
25.293
0.079
7.5
11f
11g
11h
11i
11j
11k
KTC
FLC
0.81
0.7
0.803
11.472
20.226
0.37
1.685
13.92
>41.532
0.43
2.409
2.99
0.006
0.57
0.064
0.6
2
6.7
1
MIC (IC80, µg/mL) was expressed as the mean of triplicate measures. ATCC strains are reference strains (American
Type Culture Collection). ATCC2091 is a Candida albicans strain. ATCC6258 is a Candida krusei strain. ATCC90018 is
a Candida parapsilosis strain. CA = Candida albicans. CK = Candida krusei. CL = Candida lusitaniae.
2.3.2. Toxicity of Selected Compounds on Human Fetal Lung Fibroblast (MRC-5) Cells
The in vitro toxicity of compounds 8a
shown in Table 3. In the imidazole sub-series (compounds 8a
was approximatively 1.4 to 2.2 times higher than that of ketoconazole (KTC) (IC50 = 69.1
of this sub-series with the 1,2,4-triazole sub-series (unsubstituted indole derivatives 11a
has shown that the triazole moiety decreased significantly the cytotoxicity (IC50 > 100
–
g
and 11a
–
k
was evaluated on MRC-5 cells. The results are
and 11e), the cytotoxicity (IC50 = 31–50 M)
M). Comparison
11b, and 11d
M), except for
M), 8g, and its enantiomers. Indeed, the cytotoxicity values of 8g,
–e
µ
µ
,
µ
)
the 4-bromo derivative 11c (IC50 = 38
(+)-(R)-8g, and ( )-(S)-8g (IC50 = 35
µ
−
µ
M [28], 32, and 30 µM, respectively) were two times higher than that
of KTC and at least three times higher than those of FLC (IC50 > 100 µM).
The presence of a methyl group in position 2 and/or 3 on the indole ring (derivatives 11f
–
k
) was
190 M),
the presence of a methyl at position 2 did not change the cytotoxicity on MRC-5 cells when compared to
compound 11d 197 M). On the contrary, with a 2,4-dicholorinated 11g phenyl ring (= 157 M), a methyl
group at position 2 decreased the cytotoxicity on MRC-5 cells when compared to compound 8g (= 35 M).
On the other hand, the presence of a methyl group at position 3 led to a two-fold increase in cytotoxicity for
11h (2,4-difluoro derivative, IC50 = 105.4 M) and for 11i as well (2,4-dichloro derivative, IC50 = 19 M).
In the sub-series of 2,3-dimethyl derivatives, the cytotoxicity was increased with 11j (2,4-difluoro derivative,
IC50 = 25 M) compared with compound 11d 197 M). In the case of 11k (2,4-dichloro derivative,
IC50 97 M), the cytotoxicity on MRC-5 cells was decreased compared with compound 8g (= 35 M).
compared with the unsubstituted indole derivatives. With a 2,4-difluorinated 11f phenyl ring (
≈
µ
(≈
µ
µ
µ
µ
µ
µ
(≈
µ
≈
µ
µ
While the presence of a methyl group on the indole ring globally increased the cytotoxicity of the 2,4-difluoro
derivatives 11h and 11j, the introduction of a methyl group in position 2 into 2,4-dichlorinated products
seemed to give less cytotoxic compounds (e.g. 11g, 11k).

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Table 3. Cytotoxicity activity on MRC-5 cells of propanol derivatives 8a–g and 11a–k.
1
Compound
IC50 (µM)
8a
8b
50
37
8c
31
8d
34
8e
31
11e
36
11a
11b
11c
>100
>100
38
11d
8g
(+)-(R)-8g
(−)-(S)-8g
8f
197.2
35.0 2
32
30
76
11f
11g
11h
11i
190.7
157
105.4
19
11j
25
11k
KTC
FLC
97.3
69.1
>100
1
2
IC50 (half-maximal inhibitory concentration) values (µM) were expressed as the mean of triplicate measures.
Cytotoxicity activity of 8g previously determined [28].
2.3.3. Inhibitory Activity on Cytochrome P450 Enzymes
Triazole derivatives 11d (2,4-difluoro) and 8g (2,4-dichloro) were further investigated for their
potential to inhibit diverse cytochrome P450 enzymes. Among our panel, four steroidogenic enzymes
involved in the biosynthesis of active steroid hormones were selected. This includes 11
β-hydroxylase
(CYP11B1), aldosterone synthase (CYP11B2), 17 -hydroxylase/17,20-lyase (CYP17), and aromatase
α
(CYP19). Another enzyme, retinoic acid 4-hydroxylase (CYP26A1), was also used to check the selectivity
of compounds 11d and 8g. Due to the presence of 1H-1,2,4-triazole, both compounds could coordinate
their N-4 atom with the heme iron of these enzymes. In addition, it is the reason that the majority of
reference compounds used in our assays (Table 4) were azole agents such as anastrozole, letrozole,
liarozole, BW19 (4-(6-methoxy-1-methyl-3,4-dihydro-naphthalen-2-ylmethyl)-1H-imidazole) [29],
and KTC. Aminoglutethimide (AG) and fadrozole possess other nitrogen moieties such as amino and
nitrile groups, respectively.
First, compounds 11d
(IC50 3.58 M), 8g, and its enantiomers (+)-(R) and (
were weak inhibitors of CYP19. Some studies described inhibitory activities of FLC against CYP19
(IC50 = 26.8, >140 or = 300 M, depending on the method used [30 31]). Comparing ( )-(S)-8g with
,
8g, and its enantiomers were tested against CYP19 at 36
µM. Only 11d
µ
−
)-(S) (% inhibition of 27, 51, and 72, respectively)
µ
,
−
letrozole, the most potent non-steroidal inhibitor (IC50 = 0.025 nM), there was no significant activity for
our antifungal agent against CYP19. In the opposite way, all works undertaken around the indole and
the design of non-steroidal aromatase inhibitors [18
azolyl-substituted indoles [22,23].
,19,32–36] helped us to design our first antifungal

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Table 4. Selectivity of compounds 11d, 8g, and its enantiomers on a panel of human P450-dependent
enzymes (CYP19, CYP17, CYP26A1, CYP11B1, and CYP11B2).
CYP19 1 IC50 (µM)
(% Inhibition)
CYP17 2 IC50 (µM)
(% Inhibition)
CYP26A1 3
IC50 (µM)
CYP11B1 4 IC50 (µM) CYP11B2 4 IC50 (µM)
Compound
11d
(Inhibition Effect)
(Inhibtion Effect)
3.58
(89)
-
-
-
-
-
(no inhibition)
8g
-
-
-
-
(27)
-
(51)
-
(72)
29.75
0.030
0.163
0.025
-
(<10%)
no inhibition
slight inhibition
(+)-(R)-8g
-
34
-
18
-
-
-
-
-
7
-
10
-
-
(<5% inhibition)
no inhibition
slight inhibition
(−)-(S)-8g
-
-
-
(<5% inhibition)
no inhibition
no inhibition
AG
fadrozole
anastrozole
letrozole
liarozole
BW19
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
not active
-
-
-
-
-
0.15
4.5
KTC
1
Values are the mean of at least two experiments performed in duplicate. % inhibition was determined at
2
36
2.5
µM. Values are the mean of two experiments performed in duplicate. % inhibition was determined at
4
µ
M. 3 The IC50 values are the mean (
5%) of two experiments. The results were obtained from three to four
±
independent experiments.
For CYP17, all compounds tested (11d
weak inhibitors. For example, 8g gave an inhibitory effect below 10% at 2.5
,
8g, and its enantiomers) were either inactive or very
M. The range was very
µ
significant with BW19 and KTC, with IC50 values of 0.15 and 4.5 µM, respectively.
The enantiomers of 8g were evaluated for inhibitory activity against retinoic acid 4-hydroxylase
(CYP26A1) using a cellular assay. The standards for comparison in the CYP26A1 assay were the
broad spectrum CYP inhibitor KTC and liarozole. The (+)-(R)-8g was a very weak inhibitor of
CYP26A1 (IC50 = 34
µ
M), while the ( )-(S)-8g enantiomer also displayed weak inhibitory activity
−
against CYP26A1 (IC50 = 18 µM) compared with KTC (IC50 = 10 µM) and liarozole (IC50 = 7 µM).
Racemic compound 8g and its enantiomers were also tested for their potential inhibitory effect
against CYP11B1 and CYP11B2. 8g showed no inhibitory activity against CYP11B1 and only a slight
inhibitory effect against CYP11B2 (at high concentrations). Both enantiomers showed no inhibitory
activity against CYP11B1. As mentioned before [37], KTC was a strong inhibitor of CYP11B1; on the
other hand, FLC was a minor inhibitor. Only (+)-(R)-8g showed a slight inhibitory effect against
CYP11B2 (at high concentrations) while KTC exerted 100% inhibition in another similar study [38].
2.4. Molecular Modeling Studies
2.4.1. Molecular Docking
The predicted binding pose of (S)-8g (Figure 4) was to some degree consistent with the binding
conformation of co-crystallized posaconazole in the Protein Data Bank (PDB) structure (PDB ID: 5FSA [39])
with the nitrogen atom in the triazole ring interacting with the Fe atom in the heme group. However,
the predicted binding pose of (R)-8g (Figure 5) was not consistent with the posaconazole pose in its co-crystal
structure where the triazole ring was more parallel with the heme group, rather than perpendicular to the
heme, that is, a less favorable angle and subsequently a weaker binding interaction would be expected.
Therefore, the (S)-8g generated a better docking score and more credible binding pose compared with (R)-8g
,
which is consistent with the biological experiments described in 2.3.1.

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Figure 4. Predicted binding pose of (S)-8g (light green) using CaCYP51.
Figure 5. Predicted binding pose of (R)-8g (cream) using CaCYP51.
2.4.2. Molecular Dynamics Simulation
In order to confirm the first results obtained by molecular docking, a molecular dynamics (MD)
approach was also applied to CaCYP51-azole complexes.
The (R)- and (S)-enantiomers of compound 8g were docked with the CaCYP51 crystal structure
(PDB 5FSA [39]) using molecular operating environment (MOE) software [40], and the resulting
CaCYP51 (R)-8g and (S)-8g ligand-protein complexes subjected to 100 ns MD simulations using the
Desmond programme of Maestro [41].
Both enantiomers formed primarily hydrophobic interactions including Tyr118, Phe126, and
Tyr132 for both enantiomers, Ile304 for (R)-8g, and Leu376 for (S)-8g (Figures 6 and 7). In both
enantiomers the dichlorophenyl ring forms a
π–π stacking interaction, with Phe126 and Tyr132 for the
(R)-8g and (S)-8g enantiomer, respectively. Of note is the interaction of the triazole N with the heme

Pharmaceuticals 2020, 13, 186
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iron, which occurred for 75% of the simulation time for (R)-8g and 100% of the simulation time for
(S)-8g, suggesting improved binding of the (S)-enantiomer with the heme active site.
(R)-8g
(S)-8g
Figure 6. A schematic of detailed ligand atom interactions with the protein residues. Interactions
that occur more than 30.0% of the simulation time in the selected trajectory (0.00 through 100.00 ns)
are shown.
(R)-8g
(S)-8g
Figure 7. A schematic of detailed ligand atom interactions of (R)-8g and (S)-8g with the protein residues
of wild-type CaCYP51 protein. Interactions that occur more than 30.0% of the simulation time in the
selected trajectory (0 through 100 ns) are shown. (Hydrophobic (purple), water bridges (blue), H-bonds
(green), ionic (pink)).
The reason for the reduced binding interaction of the (R)-enantiomer of 8g can be seen from
the 3D image after MD simulation (Figure 8). For optimal binding of the triazole nitrogen with the
heme iron, the triazole should have been positioned perpendicular to the heme as observed for the
(S)-8g enantiomer; however, the triazole of the (R)-8g enantiomer was positioned parallel to the heme,
reducing optimal binding. The unfavorable position of the (R)-enantiomer triazole also increased the
distance between the triazole N and the heme iron to 3.11 Å compared with 2.43 Å for the optimally
positioned (S)-enantiomer.

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(R)-8g
(S)-8g
Figure 8. 3D images of (R)-8g (cyan) and (S)-8g (magenta) ligand-CaCYP51 protein complexes after MD
simulation, showing key binding amino acids and positioning of the triazole ring above the heme (orange).
The overlap of both enantiomers of 8g and posaconazole shows that the (S)-enantiomer aligned
with the positioning of posaconazole within both the heme and access channel binding sites, whereas for
the (R)-enantiomer the dichlorophenyl was outside the small aryl binding pocket and the indole was
directed away from the access channel (Figure 9).
Figure 9. CaCYP51-posaconazole complex (PDB 5FSA) with posaconazole (yellow) positioned along
the access channel and above the heme (orange). (R)-8g (cyan) and (S)-8g (magenta) are aligned after
MD simulations.

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3. Materials and Methods
3.1. Chemistry
The microwave reactor was a monomode system (Discover, CEM) with focused waves operating
at 2.45 GHz. Melting points were determined on an Electrothermal IA9300 melting point digital
apparatus and were reported uncorrected. Infrared (IR) spectra were obtained in KBr pellets or neat
1
liquid films with a Perkin–Elmer Paragon FTIR 1000 PC spectrometer. H and 13C-NMR spectra were
recorded on a Bruker AC 250 or AVANCE 400 spectrometer in DMSO-d6 as solvent. Chemical shifts (δ)
were reported in part per million (ppm) relative to tetramethylsilane as internal standard. The splitting
pattern abbreviations are as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; dd,
doublet of doublet; td, triplet of doublet. Coupling constants J were given in Hz. Mass spectral (MS)
analysis was performed on a quadrupole H.P. 5889A instrument using EI mode at 70 eV. Microanalyses
were performed on a Perkin–Elmer CHN 240 apparatus. All reactions were monitored by TLC, using
0.25 mm-thick precoated silica gel plates (E. Merck, Darmstadt, Germany). Compounds were purified
by column chromatography using silica gel 60 as a stationary phase. All common chemicals and
solvents utilized were reagent grade and purchased from Sigma–Aldrich (Saint Quentin, France).
The synthesis of key intermediates 9a
–
e
and 10a
–
2
e
was previously described by IICiMed
), 1-chloromethyl-1H-benzotriazole ( ),
) were previously published [42 43] and some spectral
(Nantes) [27].
1-Hydroxymethyl-1H-benzotriazole (
3
and 1-(1H-benzotriazol-1-yl-methyl)-1H-indole (
data were added to be as precise as possible.
4
,
3.2. Synthesis of Compounds 8a–g (Route 1)
3.2.1. Procedure for the Synthesis of 1-Hydroxymethyl-1H-benzotriazole (2)
A solution of 1H-benzotriazole ( ) (10 g, 83.94 mmol) in formaldehyde (37% in aqueous solution,
6.81 mL, 83.94 mmol) was stirred at rt for 5 min. The precipitate was filtered and recrystallized from
THF. 1-Hydroxymethyl-1H-benzotriazole ( ) was obtained (94% yield): White powder; Rf = 0.45
(dichloromethane /EtOH: 19/1); mp: 148–150 ◦C (148–151 ◦C, lit. [42]); IR (KBr):
3192 (s, OH), 1622,
1510 cm−1; 1H NMR (DMSO-d6):
8.11 (d, 1H, J = 8.3 Hz, H4), 7.95 (d, 1H, J = 8.3 Hz, H7), 7.46–7.62
1
2
ν
δ
(m, 2H, H5, H6), 7.27 (t, 1H, J = 7.7 Hz, OH), 6.07 (d, 2H, J = 7.7 Hz, CH2).
3.2.2. Procedure for the Synthesis of 1-Chloromethyl-1H-benzotriazole (3)
1-Hydroxymethyl-1H-benzotriazole (2
) (8.91 g, 59.7 mmol) was cooled at 0 ◦C and thionyl chloride
(26 mL, 360 mmol) was added dropwise. Then the mixture was stirred and refluxed for 1 h and the
volatile fraction was evaporated under reduced pressure. The residue was dissolved in MeOH and
filtered at rt. 1-Chloromethyl-1H-benzotriazole (
3
) was obtained (93% yield): White powder; Rf = 0.74
1612, 1506, 1095 cm−1
δ 8.19 (d, 1H, J = 8.3 Hz, H4), 8.06 (d, 1H, J = 8.4 Hz, H7), 7.52–7.76 (m, 2H, H5,
(dichloromethane /EtOH: 19/1); mp: 136–138 ◦C (136–138 ◦C, lit. [42]); IR (KBr):
ν
;
1H NMR (DMSO-d6):
H6), 6.93 (s, 2H, CH2).
3.2.3. Procedure for the Synthesis of 1-(1H-Benzotriazol-1-yl-methyl)-1H-indole (4)
Sodium hydride (0.73 g, 30.3 mmol) was dissolved in DMSO (30 mL) and 1H-indole (3.23 g, 27.6 mmol)
was added portionwise and the mixture was stirred at rt for 1 h. Then 1-chloromethyl-1H-benzotriazole (
(4.62 g, 27.6 mmol) was added and the reaction mixture stirred for 2 h. At the end of this period, the mixture
was diluted with H2O (30 mL) and extracted with EtOAc (3 30 mL). The organic layer was washed with
3)
×
brine (30 mL), dried over anhydrous sodium sulfate, and the volatile fraction was evaporated under reduced
pressure. The residue was crystallized from diisopropyl ether. 1-(1H-Benzotriazol-1-yl-methyl)-1H-indole
(
4
) was obtained (80% yield): White powder; Rf = 0.35 (dichloromethane); mp: 176–178 ◦C (176–178 ◦C,
lit. [43]); IR (KBr):
ν
1616, 1511, 1349 cm−1; 1H NMR (DMSO-d6):
δ
8.15 (d, 1H, J = 8.4 Hz, H4 ), 8.07 (d, 1H,
0
0
J = 8.4 Hz, H7 ), 7.86 (d, 1H, J = 7.2 Hz, H4), 7.85 (d, 1H, J = 3.3 Hz, H2), 7.56 (d, 1H, J = 7.9 Hz, H7), 7.41–7.70

Pharmaceuticals 2020, 13, 186
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0
0
(m, 2H, H5 , H6 ), 7.28 (s, 2H, CH2), 7.24 (dd, 1H, J = 7.9 Hz, J = 7.2 Hz, H6), 7.09 (dd, 1H, J = J = 7.2 Hz, H5),
6.56 (d, 1H, J = 3.3 Hz, H3).
3.2.4. General Procedure for the Synthesis of Benzotriazole Derivatives 5a–f
1-(1H-Benzotriazol-1-yl-methyl)-1H-indole (4) (1.88 g, 7.57 mmol) in THF (50 mL) was cooled
to –78 ◦C under argon, and n-BuLi (1.6 M in THF, 5.67 mL, 9.1 mmol) was added dropwise, then the
mixture was stirred at –78 ◦C for 1 h. At the end of this period, the corresponding ethyl benzoate
(9.46 mmol) was added dropwise and the mixture was slowly warmed to rt over a period of 12 h.
Then saturated aqueous ammonium chloride solution (35 mL) was added, the mixture was diluted
with H2O (30 mL), and extracted with diethyl ether (3
brine (40 mL), dried over anhydrous sodium sulfate, and the volatile fraction was evaporated under
reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane).
×
40 mL). The organic layer was washed with
1-[(1H-Benzotriazol-1-yl)(4-fluorobenzoyl)methyl]-1H-indole (5a). White powder (61% yield); Rf = 0.29
1
(dichloromethane); mp: 121–123 ◦C; IR (KBr):
ν
1701 (s, C=O), 1594, 1304 cm−1; H NMR (DMSO-d6):
0 0
9.45 (s, 1H, CH), 8.09–8.12 (m, 1H, H4 ), 8.03–8.07 (m, 2H, H2”, H6”), 8.01 (d, 1H, J = 8.2 Hz, H7 ), 7.90
δ
0
(d, 1H, J = 8.2 Hz, H4), 7.60–7.67 (m, 1H, H6 ), 7.56–7.59 (m, 1H, H7), 7.56 (d, 1H, J = 3.4 Hz, H2), 7.44–7.50
0
(m, 1H, H5 ), 7.35–7.42 (m, 2H, H3”, H5”), 7.22 (dd, 1H, J = 8.2 Hz, J = 7.1 Hz, H6), 7.12 (dd, 1H, J = 8.2 Hz,
J = 7.1 Hz, H5), 6.60 (d, 1H, J = 3.4 Hz, H3).
1-[(1H-Benzotriazol-1-yl)(4-chlorobenzoyl)methyl]-1H-indole (5b). White powder (61% yield);
Rf = 0.30 (dichloromethane); mp: 184–185 ◦C; IR (KBr):
ν
1712 (s, C=O), 1587, 1323 cm−1; 1H NMR
0 0
δ 9.46 (s, 1H, CH), 8.11 (d, 1H, J = 8.1 Hz, H4 ), 8.00 (d, 1H, J = 8.2 Hz, H7 ), 8.00 (d, 2H,
(DMSO-d6):
J = 8.5 Hz, H2”, H6”), 7.90 (d, 1H, J = 8.3 Hz, H4), 7.64 (d, 2H, J = 8.5 Hz, H3”, H5”), 7.60–7.64 (m, 1H,
0
0
H5 ), 7.56–7.62 (m, 1H, H7), 7.57 (d, 1H, J = 3.1 Hz, H2), 7.47 (dd, 1H, J = 8.2 Hz, J = 7.9 Hz, H6 ), 7.26
(dd, 1H, J = 7.6 Hz, J = 7.3 Hz, H6), 7.13 (dd, 1H, J = 8.3 Hz, J = 7.3 Hz, H5), 6.61 (d, 1H, J = 3.1 Hz, H3).
1-[(1H-Benzotriazol-1-yl)(4-bromobenzoyl)methyl]-1H-indole (5c). White powder (50% yield);
Rf = 0.40 (dichloromethane); mp: 156–157 ◦C; IR (KBr):
ν
1710 (s, C=O), 1583, 1323 cm−1; 1H NMR
0 0
δ 9.46 (s, 1H, CH), 8.12 (d, 1H, J = 8.3 Hz, H4 ), 8.03 (d, 1H, J = 8.4 Hz, H7 ), 7.92 (d, 2H,
(DMSO-d6):
J = 8.6 Hz, H2”, H6”), 7.89 (d, 1H, J = 7.2 Hz, H4), 7.78 (d, 2H, J = 8.6 Hz, H3”, H5”), 7.65 (dd, 1H, J = 8.4
0
Hz, J = 8.0 Hz, H6 ), 7.57 (d, 1H, J = 3.3 Hz, H2), 7.56–7.60 (m, 1H, H7), 7.47 (dd, 1H, J = 8.3 Hz, J = 8.0
0
Hz, H5 ), 7.26–7.30 (m, 1H, H6), 7.16 (dd, 1H, J = J = 7.2 Hz, H5), 6.61 (d, 1H, J = 3.3 Hz, H3).
1-[(1H-Benzotriazol-1-yl)(4-trifluoromethylbenzoyl)-methyl]-1H-indole (5d). Yellow powder (75%
yield); mp: 175–177 ◦C; IR (KBr):
ν δ 9.55 (s, 1H,
1711 (s, C=O), 1598, 1330 cm−1; 1H NMR (DMSO-d6):
0
0
CH), 8.21 (d, 2H, J = 7.9 Hz, H2”, H6”), 8.12 (d, 1H, J = 8.6 Hz, H4 ), 8.08 (d, 1H, J = 8.6 Hz, H7 ), 7.96 (d,
0
2H, J = 7.9 Hz, H3”, H5”), 7.93–7.96 (m, 1H, H4), 7.67 (dd, 1H, J = J = 8.6 Hz, H6 ), 7.58 (d, 1H, J = 3.1
0
Hz, H2), 7.58–7.61 (m, 1H, H7), 7.48 (dd, 1H, J = J = 8.6 Hz, H5 ), 7.28 (dd, 1H, J = J = 7.0 Hz, H6), 7.14
(dd, 1H, J = J = 7.0 Hz, H5), 6.62 (d, 1H, J = 3.1 Hz, H3).
1-[(1H-Benzotriazol-1-yl)(2,4-difluorobenzoyl)methyl]-1H-indole (5e). White powder (41% yield);
Rf = 0.26 (dichloromethane); mp: 111–113 ◦C; IR (KBr):
ν
1705 (s, C=O), 1580, 1334 cm−1; 1H NMR
0
δ 9.02 (s, 1H, CH), 8.26–8.77 (m, 1H, H6”), 8.10 (d, 1H, J = 8.5 Hz, H4 ), 8.02 (d, 1H, J = 8.5
(DMSO-d6):
0
0
Hz, H7 ), 7.78 (d, 1H, J = 8.6 Hz, H4), 7.62–7.68 (m, 1H, H6 ), 7.61 (d, 1H, J = 3.1 Hz, H2), 7.57 (d, 1H,
0
J = 7.9 Hz, H7), 7.44–7.53 (m, 1H, H5 ), 7.44–7.47 (m, 1H, H3”), 7.41–7.44 (m, 1H, H5”), 7.22 (dd, 1H,
J = 7.9 Hz, J = 7.6 Hz, H6), 7.11 (dd, 1H, J = 8.6 Hz, J = 7.6 Hz, H5), 6.59 (d, 1H, J = 3.1 Hz, H3).
1-[(1H-Benzotriazol-1-yl)(2,4-dichlorobenzoyl)methyl]-1H-indole (5f). White powder (51% yield);
Rf = 0.34 (dichloromethane); mp: 141–142 ◦C; IR (KBr):
ν
1714 (s, C=O), 1582, 1375 cm−1; 1H NMR
0 0
δ 9.34 (s, 1H, CH), 8.14 (d, 1H, J = 8.3 Hz, H4 ), 8.09 (d, 1H, J = 8.3 Hz, H7 ), 7.85 (d, 1H,
(DMSO-d6):
J = 8.2 Hz, H4), 7.71–7.76 (m, 1H, H6”), 7.68–7.71 (m, 1H, H6 ), 7.59 (d, 1H, J = 3.1 Hz, H2), 7.41–7.50 (m,
0
0
2H, H7, H5 ), 7.31–7.39 (m, 1H, H3”), 7.26 (dd, 1H, J = 7.6 Hz, J = 7.3 Hz, H6), 7.05–7.15 (m, 1H, H5”),
6.99 (dd, 1H, J = 8.2 Hz, J = 7.3 Hz, H5), 6.44 (d, 1H, J = 3.1 Hz, H3).

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3.2.5. General Procedure for the Synthesis of N-Substituted-1H-indoles 6a–f
The corresponding 1-[(1H-benzotriazol-1-yl)(halogenobenzoyl)methyl]-1H-indole 5a–f (3.00
mmol) in ethanol (15 mL) and THF (15 mL) was stirred at rt. Then acetic acid (3 mL) and zinc
(0.98 g, 15 mmol) were added, and the reaction mixture was stirred at 35 ◦C for 5 h in an ultrasonic
bath. The mixture was filtered on Celite 545 and the filtrate was evaporated under reduced pressure.
The residue was diluted with H2O (40 mL) and extracted with EtOAc (3
×
40 mL), then the organic
layer was washed with H2O (40 mL), dried over anhydrous sodium sulfate, and the volatile fraction
was evaporated under reduced pressure. The residue was crystallized from diisopropyl ether.
N-(4-Fluorobenzoylmethyl)-1H-indole (6a). White powder (57% yield); Rf = 0.61 (dichloromethane);
0
mp: 153–154 ◦C; IR (KBr):
ν δ 8.19–8.24 (m, 2H, H2 ,
1691 (s, C=O), 1597, 1325 cm−1; 1H NMR (DMSO-d6):
0
0
0
H6 ), 7.60 (d, 1H, J = 7.4 Hz, H4), 7.48–7.51 (m, 2H, H3 , H5 ), 7.37–7.44 (m, 2H, H2, H7), 7.02–7.13 (m, 2H, H5,
H6), 6.52 (d, 1H, J = 3.1 Hz, H3), 5.93 (s, 2H, CH2).
N-(4-Chlorobenzoylmethyl)-1H-indole (6b). White powder (66% yield); Rf = 0.62 (dichloromethane);
mp: 156–157 ◦C; IR (KBr):
ν δ 8.12–8.15 (m, 1H, H4),
1692 (s, C=O), 1586, 1399 cm−1; 1H NMR (DMSO-d6):
0
0
0
0
7.72 (d, 2H, J = 8.6 Hz, H2 , H6 ), 7.58–7.60 (m, 1H, H7), 7.37 (d, 2H, J = 8.6 Hz, H3 , H5 ), 7.35–7.40 (m, 1H,
H2), 7.04–7.11 (m, 2H, H5, H6), 6.50 (d, 1H, J = 3.1 Hz, H3), 5.93 (s, 2H, CH2).
N-(4-Bromobenzoylmethyl)-1H-indole (6c).
White powder (66% yield); Rf = 0.60
(dichloromethane); mp: 185–186 ◦C; IR (KBr): 1689 (s, C=O), 1582, 1384 cm−1; 1H NMR (DMSO-d6):
ν
0 0 0 0
8.07 (d, 2H, J = 8.5 Hz, H2 , H6 ), 7.87 (d, 2H, J = 8.5 Hz, H3 , H5 ), 7.60 (d, 1H, J = 7.3 Hz, H4), 7.40
δ
(d, 1H, J = 8.2 Hz, H7), 7.35 (d, 1H, J = 3.1 Hz, H2), 7.12 (dd, 1H, J = 8.2 Hz, J = 7.3 Hz, H6), 7.05 (dd, 1H,
J = J = 7.3 Hz, H5), 6.52 (d, 1H, J = 3.1 Hz, H3), 5.94 (s, 2H, CH2).
N-(4-Trifluoromethylbenzoylmethyl)-1H-indole (6d). White powder (45% yield); mp: 182–185 ◦C;
IR (KBr):
1690 (s, C=O), 1580, 1382 cm−1; 1H NMR (DMSO-d6):
0 0
ν δ 8.33 (d, 2H, J = 7.6 Hz, H2 , H6 ),
0
0
8.03 (d, 2H, J = 7.6 Hz, H3 , H5 ), 7.61 (d, 1H, J = 8.0 Hz, H4), 7.43 (d, 1H, J = 8.0 Hz, H7), 7.38 (d, 1H,
J = 3.1 Hz, H2), 7.13 (dd, 1H, J = J = 8.0 Hz, H5), 6.93 (dd, 1H, J = J = 8.0 Hz, H6), 6.54 (d, 1H, J = 3.1 Hz,
H3), 6.02 (s, 2H, CH2).
N-(2,4-Difluorobenzoylmethyl)-1H-indole (6e).
White powder (60% yield); Rf = 0.39
(dichloromethane); mp: 101–103 ◦C; IR (KBr): 1698 (s, C=O), 1587, 1379 cm−1; 1H NMR (DMSO-d6):
ν
0 0
8.03–8.12 (m, 1H, H6 ), 7.58–7.60 (m, 1H, H4), 7.43 (d, 1H, J = 7.3 Hz, H7), 7.35–7.41 (m, 2H, H2, H5 ),
δ
0
7.31–7.35 (m, 1H, H3 ), 7.02–7.11 (m, 2H, H5, H6), 6.51–6.52 (m, 1H, H3), 5.79 (s, 2H, CH2).
N-(4-Dichlorobenzoylmethyl)-1H-indole (6f). White powder (46% yield); Rf = 0.64 (dichloromethane);
mp: 98–99 ◦C; IR (KBr):
ν δ 8.04 (d, 1H, J = 8.5 Hz,
1704 (s, C=O), 1580, 1367 cm−1; 1H NMR (DMSO-d6):
0
0
0
H6 ), 7.83 (d, 1H, J = 1.4 Hz, H3 ), 7.66–7.70 (m, 1H, H5 ), 7.60 (d, 1H, J = 7.3 Hz, H4), 7.42 (d, 1H, J = 7.9 Hz,
H7), 7.38 (d, 1H, J = 3.1 Hz, H2), 7.16 (dd, 1H, J = 7.9 Hz, J = 7.0 Hz, H5), 7.07 (dd, 1H, J = 8.5, J = 7.0 Hz, H6),
6.52 (d, 1H, J = 3.1 Hz, H3), 5.77 (s, 2H, CH2).
3.2.6. General Procedure for the Synthesis of Oxiranes 7a–f
Trimethylsulfoxonium iodide (0.44 g, 2.02 mmol) and sodium hydroxide (6 g, 150 mmol, 48% in
aqueous solution) were added to a solution of the corresponding N-substituted indole 6a
in dichloromethane (5 mL). Then the reaction mixture was stirred and refluxed for 48 h. At the end of
this period, the mixture was diluted with H2O (10 mL) and extracted with dichloromethane (3 10 mL).
–f (1.41 mmol)
×
The organic layer was washed with H2O (15 mL), dried over anhydrous sodium sulfate, and the
volatile fraction was evaporated under reduced pressure. The residue was purified by silica gel column
chromatography (dichloromethane/hexane 1:1, v/v).
2-(4-Fluorophenyl)-3-(1H-indol-1-yl)-1,2-epoxypropane (7a). Yellow oil (40% yield); Rf = 0.68
(dichloromethane); IR (NaCl):
H4”), 7.49–7.52 (m, 1H, H7”), 7.44–7.49 (m, 2H, H2 , H6 ), 7.27 (d, 1H, J = 3.1 Hz, H2”), 7.10–7.17 (m, 3H,
ν δ 7.59 (d, 1H, J = 7.6 Hz,
1620, 1366, 1289 cm−1; 1H NMR (DMSO-d6):
0
0
0
0
H3 , H5 , H6”), 6.98–7.04 (m, 1H, H5”), 6.40 (d, 1H, J = 3.1 Hz, H3”), 5.07 (d, 1H, J = 15.7 Hz, H3), 4.57
(d, 1H, J = 15.7 Hz, H3), 2.81 (d, 1H, J = 4.9 Hz, H1), 2.93 (d, 1H, J = 4.9 Hz, H1).

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2-(4-Chlorophenyl)-3-(1H-indol-1-yl)-1,2-epoxypropane (7b). Colorless oil (75% yield); Rf = 0.68
(dichloromethane); IR (NaCl): 7.58–7.61 (m, 1H, H4”),
1598, 1315, 1270 cm−1; 1H NMR (DMSO-d6):
ν
δ
0
0
0
0
7.48–7.51 (m, 1H, H7”), 7.43–7.46 (m, 2H, H3 , H5 ), 7.36 (d, 2H, J = 8.6 Hz, H2 , H6 ), 7.26 (d, 1H, J = 3.1
Hz, H2”), 7.10–7.16 (m, 1H, H6”), 6.97–7.03 (m, 1H, H5”), 6.38 (d, 1H, J = 3.1 Hz, H3”), 5.09 (d, 1H,
J = 15.6 Hz, H3), 4.54 (d, 1H, J = 15.6 Hz, H3), 2.95 (d, 1H, J = 4.9 Hz, H1), 2.80 (d, 1H, J = 4.9 Hz, H1).
2-(4-Bromophenyl)-3-(1H-indol-1-yl)-1,2-epoxypropane (7c). Colorless oil (37% yield); Rf = 0.62
(dichloromethane); IR (NaCl):
ν δ 7.59–7.62 (m, 1H, H4”),
1600, 1320, 1266 cm−1; 1H NMR (DMSO-d6):
0
0
0
0
7.51 (d, 2H, J = 8.5 Hz, H3 , H5 ), 7.42–7.44 (m, 1H, H7”), 7.40 (d, 2H, J = 8.5 Hz, H2 , H6 ), 7.29 (d, 1H,
J = 3.1 Hz, H2”), 7.14 (dd, 1H, J = 8.2 Hz, J = 7.0 Hz, H6”), 6.97–7.05 (m, 1H, H5”), 6.39 (d, 1H, J = 3.1 Hz,
H3”), 5.11 (d, 1H, J = 15.6 Hz, H3), 4.55 (d, 1H, J = 15.6 Hz, H3), 2.96 (d, 1H, J = 4.9 Hz, H1), 2.81 (d, 1H,
J = 4.9 Hz, H1).
2-(4-Trifluoromethylphenyl)-3-(1H-indol-1-yl)-1,2-epoxypropane (7d). Yellow oil (62% yield); IR
(NaCl):
1592, 1312, 1271 cm−1; 1H NMR (DMSO-d6):
0 0 0 0
ν δ 7.68 (s, 4H, H2 , H3 , H5 , H6 ), 7.64 (d, 1H,
J = 8.6 Hz, H4”), 7.51 (d, 1H, J = 8.6 Hz, H7”), 7.29 (d, 1H, J = 4.0 Hz, H2”), 7.15 (dd, 1H, J = J = 8.6 Hz,
H5”), 7.02 (dd, 1H, J = J = 8.6 Hz, H6”), 6.40 (d, 1H, J = 4.0 Hz, H3”), 5.20 (d, 1H, J = 15.3 Hz, H3), 4.59
(d, 1H, J = 15.3 Hz, H3), 3.03 (d, 1H, J = 4.9 Hz, H1), 2.85 (d, 1H, J = 4.9 Hz, H1).
2-(2,4-Difluorophenyl)-3-(1H-indol-1-yl)-1,2-epoxypropane (7e). Yellow oil (59% yield); Rf = 0.59
(dichloromethane); IR (NaCl):
H4”), 7.52 (d, 1H, J = 7.6 Hz, H7”), 7.43 (d, 1H, J = 7.9 Hz, H6 ), 7.18 (d, 1H, J = 3.1 Hz, H2”), 7.11–7.15
ν δ 7.56 (d, 1H, J = 7.6 Hz,
1589, 1369, 1290 cm−1; 1H NMR (DMSO-d6):
0
0
0
(m, 1H, H3 ), 7.07–7.10 (m, 1H, H5 ), 6.98–7.04 (m, 1H, H6”), 6.91–6.98 (m, 1H, H5”), 6.40 (d, 1H, J = 3.1
Hz, H3”), 4.81 (d, 1H, J = 15.5 Hz, H3), 4.53 (d, 1H, J = 15.5 Hz, H3), 2.98 (d, 1H, J = 4.9 Hz, H1), 2.91
(d, 1H, J = 4.9 Hz, H1).
2-(2,4-Dichlorophenyl)-3-(1H-indol-1-yl)-1,2-epoxypropane (7f). Green oil (61% yield); Rf = 0.64
(dichloromethane); IR (NaCl):
ν
1612, 1371, 1314 cm−1; 1H NMR (DMSO-d6):
δ
7.68 (d, 1H, J = 1.8 Hz,
0
H3 ), 7.52 (d, 1H, J = 7.3 Hz, H4”), 7.40 (d, 1H, J = 7.9 Hz, H7”), 7.17 (d, 1H, J = 3.1 Hz, H2”), 7.09–7.15
0
0
(m, 1H, H6”), 7.07–7.11 (m, 1H, H5 ), 7.01–7.04 (m, 1H, H6 ), 6.98–7.04 (m, 1H, H5”), 6.41 (d, 1H, J = 3.1
Hz, H3”), 4.90 (d, 1H, J = 15.6 Hz, H3), 4.51 (d, 1H, J = 15.6 Hz, H3), 3.01 (d, 1H, J = 4.6 Hz, H1), 2.90
(d, 1H, J = 4.6 Hz, H1).
3.2.7. General Procedure for the Synthesis of Imidazole Derivatives 8a–e
Potassium carbonate (0.25 g, 1.83 mmol) and 1H-imidazole (0.12 g, 1.82 mmol) were added to
a solution of the corresponding oxirane 7a
reaction mixture was stirred and refluxed for 7 h. At the end of this period, the mixture was diluted
with H2O (20 mL) and extracted with EtOAc (3 40 mL). The organic layer was washed with brine
–e (0.64 mmol) in dimethylformamide (20 mL). Then the
×
(40 mL), dried over anhydrous sodium sulfate, and the volatile fraction was evaporated under reduced
pressure. The residue was purified by silica gel column chromatography (dichloromethane/ethanol
19:1, v/v).
2-(4-Fluorophenyl)-1-(1H-imidazol-1-yl)-3-(1H-indol-1-yl)propan-2-ol (8a). White powder (50%
yield); Rf = 0.13 (dichloromethane /EtOH: 19/1); mp: 166–167 ◦C; IR (KBr):
ν
3438 (w, OH), 1607, 1329
cm−1; 1H NMR (DMSO-d6):
δ 7.44–7.49 (m, 2H, H4 ”, H7 ”), 7.35 (s, 1H, H2”), 7.15 (d, 1H, J = 3.1 Hz,
0
0
0
0
0
0
0
0
0
H2 ”), 6.87 (s, 1H, H4”), 6.72 (s, 1H, H5”), 6.97–7.09 (m, 6H, H5 ”, H6 ”, H2 , H3 , H5 , H6 ), 6.37 (d, 1H,
0
J = 3.1 Hz, H3 ”), 5.94 (s, 1H, OH), 4.63 (d, 1H, J = 14.4 Hz, H3), 4.57 (d, 1H, J = 14.1 Hz, H1), 4.43 (d, 1H,
J = 14.1 Hz, H1), 4.29 (d, 1H, J = 14.4 Hz, H3); MS m/z: 335 (M+), 240, 130 (100), 81. Anal. Calcd. for
C20H18FN3O (335.37): C: 71.62; H: 5.37; N: 12.52; found: C: 71.61; H: 5.36; N: 12.50%.
2-(4-Chlorophenyl)-1-(1H-imidazol-1-yl)-3-(1H-indol-1-yl)propan-2-ol (8b). White powder (60%
yield); Rf = 0.15 (dichloromethane /EtOH: 19/1); mp: 201–202 ◦C; IR (KBr):
ν
3120 (w, OH), 1609, 1317
cm−1; 1H NMR (DMSO-d6):
δ
7.51 (d, 2H, J = 8.5 Hz, H3 , H5 ), 7.49 (d, 1H, J = 7.5 Hz, H4 ”), 7.48 (d, 1H,
0
0
0
0
0
0
0
J = 8.0 Hz, H7 ”), 7.35 (s, 1H, H2”), 7.30 (d, 2H, J = 8.5 Hz, H2 , H6 ), 7.16 (d, 1H, J = 3.1 Hz, H2 ”), 7.08
0
0
(dd, 1H, J = 8.0 Hz, J = 7.0 Hz, H6 ”), 6.98 (dd, 1H, J = 7.5 Hz, J = 7.0 Hz, H5 ”), 6.89 (s, 1H, H4”), 6.72
0
(s, 1H, H5”), 6.37 (d, 1H, J = 3.1 Hz, H3 ”), 5.99 (s, 1H, OH), 4.63 (d, 1H, J = 14.3 Hz, H3), 4.59 (d, 1H,

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J = 14.4 Hz, H1), 4.44 (d, 1H, J = 14.4 Hz, H1), 4.29 (d, 1H, J = 14.3 Hz, H3); MS m/z: 351, 353 (M+), 240,
130 (100), 81. Anal. Calcd. for C20H18ClN3O (351.83): C: 68.27; H: 5.12; N: 11.94; found: C: 68.26; H:
5.11; N: 11.92%.
2-(4-Bromophenyl)-1-(1H-imidazol-1-yl)-3-(1H-indol-1-yl)propan-2-ol (8c). White powder (21%
yield); Rf = 0.13 (dichloromethane /EtOH: 19/1); mp: 134–135 ◦C; IR (KBr):
ν 3054 (w, OH), 1602, 1313
cm−1; 1H NMR (DMSO-d6):
0 0 0 0 0
δ 7.62 (s, 1H, H2”), 7.45–7.51 (m, 5H, H2 , H3 , H5 , H6 , H4 ”), 7.45–7.48 (m,
0
0
0
0
1H, H7 ”), 7.19 (d, 1H, J = 3.1 Hz, H2 ”), 7.06–7.12 (m, 1H, H6 ”), 6.99 (dd, 1H, J = J = 7.0 Hz, H5 ”), 6.96
0
(s, 1H, H4”), 6.87 (s, 1H, H5”), 6.40 (d, 1H, J = 3.1 Hz, H3 ”), 6.09 (s, 1H, OH), 4.70 (d, 1H, J = 14.3 Hz,
H3), 4.60 (d, 1H, J = 14.4 Hz, H1), 4.48 (d, 1H, J = 14.4 Hz, H1), 4.32 (d, 1H, J = 14.3 Hz, H3); MS m/z:
395, 397 (M+), 240, 130 (100), 81. Anal. Calcd. for C20H18BrN3O (396.28): C: 60.61; H: 4.54; N: 10.60;
found: C: 60.58; H: 4.55; N: 10.58%.
2-(2,4-Difluorophenyl)-1-(1H-imidazol-1-yl)-3-(1H-indol-1-yl)propan-2-ol (8d). White powder
(50% yield); Rf = 0.17 (dichloromethane /EtOH: 19/1); mp: 166–168 ◦C; IR (KBr):
ν 3424 (w, OH), 1616,
1317 cm−1; 1H NMR (DMSO-d6):
δ
7.49 (d, 1H, J = 7.6 Hz, H4 ”), 7.42 (s, 1H, H2”), 7.39–7.42 (m, 1H,
0
0
0
0
0
H7 ”), 7.25–7.28 (m, 1H, H6 ), 7.20–7.21 (m, 2H, H2 ”, H4”), 7.07–7.13 (m, 1H, H6 ”), 6.98–7.01 (m, 1H,
0
0
0
0
H5 ), 6.89–6.95 (m, 1H, H3 ), 6.83–6.89 (m, 1H, H5 ”), 6.73 (s, 1H, H5”), 6.39–6.40 (m, 1H, H3 ”), 6.24
(s, 1H, OH), 4.68 (d, 1H, J = 14.4 Hz, H3), 4.68 (d, 1H, J = 14.3 Hz, H1), 4.49 (d, 1H, J = 14.3 Hz, H1), 4.30
(d, 1H, J = 14.4 Hz, H3); MS m/z: 353 (M+), 240, 130 (100), 81. Anal. Calcd. for C20H17F2N3O (353.37):
C: 67.97; H: 4.81; N: 11.89; found: C: 67.95; H: 4.79; N: 11.86%.
2-(2,4-Dichlorophenyl)-1-(1H-imidazol-1-yl)-3-(1H-indol-1-yl)propan-2-ol (8e). White powder
(70% yield); Rf = 0.16 (dichloromethane /EtOH: 19/1); mp: 212–213 ◦C; IR (KBr):
ν
3408 (w, OH), 1609,
1329 cm−1; 1H NMR (DMSO-d6):
δ
7.98 (s, 1H, H2”), 7.57–7.61 (m, 1H, H3 ), 7.44–7.48 (m, 1H, H4 ”),
0
0
0
0
0
0
7.36 (d, 1H, J = 8.5 Hz, H7 ”), 7.23 (d, 1H, J = 3.1 Hz, H2 ”), 7.16–7.19 (m, 2H, H4 , H5 ), 7.05–7.11 (m, 1H,
0
0
0
H6 ”), 6.91 (s, 1H, H4”), 6.87–6.91 (m, 1H, H5 ”), 6.68 (s, 1H, H5”), 6.35 (d, 1H, J = 3.1 Hz, H3 ”), 6.01
(s, 1H, OH), 5.15 (d, 1H, J = 14.4 Hz, H3), 4.96 (d, 1H, J = 14.3 Hz, H1), 4.75 (d, 1H, J = 14.3 Hz, H1), 4.47
(d, 1H, J = 14.4 Hz, H3); MS m/z: 385, 387, 389 (M+), 240, 130 (100), 81. Anal. Calcd. for C20H17Cl2N3O
(386.27): C: 62.18; H: 4.40; N: 10.87; found: C: 62.20; H: 4.43; N: 10.88%.
3.2.8. General Procedure for the Synthesis of Triazole Derivatives 8f and 8g
Potassium carbonate (0.25 g, 1.83 mmol) and 1,2,4-1H-triazole (0.13 g, 1.82 mmol) were added to
a solution of the corresponding oxirane 7d,f (0.64 mmol) in dimethylformamide (20 mL). Then the
reaction mixture was stirred and refluxed for 7 h. At the end of this period, the mixture was diluted
with H2O (20 mL) and extracted with EtOAc (40 mL). The organic layer was washed with brine (40 mL),
dried over anhydrous sodium sulfate, and the volatile fraction was evaporated under reduced pressure.
The residue was purified by silica gel column chromatography (dichloromethane/ethanol 19:1, v/v).
1-(1H-Indol-1-yl)-3-(1,2,4-1H-triazol-1-yl)-2-(4-trifluoromethyl-phenyl)propan-2-ol (8f). White
powder (86% yield); mp: 165–168 ◦C; IR (KBr):
ν
3420 (w, OH), 1613, 1307 cm−1; 1H NMR (DMSO-d6):
0
0
δ
8.32 (s, 1H, H3”), 7.88 (s, 1H, H5”), 7.68 (d, 2H, J = 8.0 Hz, H3”, H5 ”), 7.58 (d, 2H, J = 8.0 Hz, H2”, H6 ”),
0
0
0
7.48 (d, 1H, J = 7.6 Hz, H4 ), 7.40 (d, 1H, J = 7.6 Hz, H7 ), 7.29 (d, 1H, J = 2.8 Hz, H2 ), 7.03 (dd, 1H,
0
0
0
J = 7.6 Hz, H6 ), 6.96 (dd, 1H, J = 7.6 Hz, H5 ), 6.41 (d, 1H, J = 2.8 Hz, H3 ), 6.16 (s, 1H, OH), 4.92 (d, 1H,
J = 14.9 Hz, H1), 4.64 (d, 1H, J = 14.9 Hz, H1), 4.61 (s, 2H, H3); MS m/z: 386 (M+), 241, 130 (100), 82.
Anal. Calcd. for C20H17F3N4O (386.37): C: 62.17; H: 4.40; N: 14.49; found: C: 62.12; H: 4.41; N: 14.47%.
2-(2,4-Dichlorophenyl)-3-(1H-indol-1-yl)-1-(1H-1,2,4-triazol-1-yl)-propan-2-ol (8g). White powder
(45% yield); Rf = 0.11 (AcOEt/hexane: 1/1); mp: 161–162 ◦C; IR (KBr):
1H NMR (DMSO-d6):
ν
3423 (w, OH), 1585, 1273 cm−1
;
0
0
0
δ
8.32 (s, 1H, H3 ”), 7.79 (s, 1H, H5 ”), 7.63 (d, 1H, J = 2.1 Hz, H3 ), 7.50 (d, 1H,
0
J = 8.2 Hz, H6 ), 7.50 (d, 1H, J = 7.9 Hz, H4”), 7.42 (d, 1H, J = 7.9 Hz, H7”), 7.23 (dd, 1H, J = 8.2 Hz,
0
J = 2.1 Hz, H5 ), 7.21–7.23 (m, 1H, H2”), 7.13 (dd, 1H, J = J = 7.9 Hz, H6”), 7.01 (dd, 1H, J = J = 7.9 Hz,
H5”), 6.40 (d, 1H, J = 2.7 Hz, H3”), 6.36 (s, 1H, OH), 5.31 (d, 1H, J = 14.3 Hz, H3), 4.88 (d, 1H, J = 15.0
Hz, H1), 4.78 (d, 1H, J = 15.0 Hz, H1), 4.55 (d, 1H, J = 14.3 Hz, H3); 13C NMR (DMSO-d6): 50.8, 53.4,
76.7, 101.2, 110.1, 118.2, 120.2, 121.1, 127.0, 127.6, 129.6, 129.9, 131.2, 131.3, 133.1, 136.9, 137.0, 145.4,

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151.2; MS m/z: 386, 388, 390 (M+), 241, 130 (100), 82. Anal. Calcd. for C19H16Cl2N4O (387.26): C: 58.92;
H: 4.13; N: 14.46; found: C: 58.89; H: 4.11; N: 14.45%.
3.3. Synthesis of Compounds 8g and 11a–k (Route 2)
3.3.1. Synthesis of 2-(1H-Imidazol-1-yl)-1-(4-trifluoromethylphenyl)ethanone (9f)
To a solution of 2-bromo-40-trifluoromethylacetophenone (2.99 g, 11.19 mmol) in acetonitrile (40 mL)
was added 1H-imidazole (1.52 g, 22.37 mmol) and K2CO3 (3.09 g, 22.37 mmol). The reaction mixture was
irradiated for 50 min in a microwave oven, programmed to obtain reflux with a maximum power output
of 50 W. After cooling, the mixture was filtered and evaporated under reduce pressure. The residue
was diluted with H2O (40 mL) and extracted with dichloromethane (3 x 40 mL), then the organic layer
was washed with brine (40 mL), dried over anhydrous sodium sulfate, and the volatile fraction was
evaporated under reduced pressure. The residue was purified by silica gel column chromatography
(ethanol/dichloromethane 1:10, v/v). 2-(1H-Imidazol-1-yl)-1-(4-trifluoromethylphenyl)ethanone (9f
)
was obtained (80% yield): White powder; Rf = 0.80 (EtOH/dichloromethane: 1/10); mp: 115–116 ◦C; IR
(KBr):
(d, 2H, J = 8.2 Hz, H2 , H6 ); 7.63 (s, 1H, H2”), 7.17 (s, 1H, H4”), 6.97 (s, 1H, H5”), 5.84 (s, 2H, H2). 13C
1702 (s, C=O), 1603, 1228 cm−1; 1H NMR (DMSO-d6):
0 0
ν δ 8.26 (d, 2H, J = 8.2 Hz, H3 , H5 ); 8.02
0
0
NMR (DMSO-d6):
δ
53.3, 121.2, 124.2 (q, 1JCF = 287.1 Hz), 126.3 (q, 2C, 3JCF = 3.8 Hz), 129.2, 129.4,
133.4 (q, 2JCF = 32.0 Hz), 137.4, 138.1, 193.6.
3.3.2. Synthesis of 3-(1H-imidazol-1-yl)-2-(4-trifluoromethylphenyl)-1,2-epoxypropane (10f)
Trimethylsulfoxonium iodide (4.59 g, 20.85 mmol) and sodium hydroxide (4.17 g, 104.26 mmol, 20% in
aqueous solution) were added to a solution of 2-(1H-imidazol-1-yl)-1-(4-trifluoromethylphenyl)ethanone
(
9f) (2.65 g, 10.43 mmol) in dichloromethane (50 mL). Then the reaction mixture was stirred and
refluxed for 72 h. At the end of this period, the mixture was diluted with H2O (50 mL) and
extracted with dichloromethane (3 50 mL). The organic layer was washed with H2O (50 mL), dried
×
over anhydrous sodium sulfate, and the volatile fraction was evaporated under reduced pressure.
The residue was purified by silica gel column chromatography (dichloromethane/ethanol 10:1, v/v).
3-(1H-Imidazol-1-yl)-2-(4-trifluoromethylphenyl)-1,2-epoxypropane (10f) was obtained (53% yield): Red
oil; Rf = 0.91 (EtOH/dichloromethane: 1/10); IR (NaCl):
ν δ
1541, 1328, 1073 cm−1; 1H NMR (DMSO-d6):
0
0
0
0
7.75 (d, 2H, J = 7.9 Hz, H3 , H5 ); 7.64 (d, 2H, J = 7.9 Hz, H2 , H6 ); 7.56 (s, 1H, H2”); 7.10 (s, 1H, H4”); 6.85
(s, 1H, H5”); 5.04 (d, 1H, J = 15.0 Hz, H3); 4.38 (d, 1H, J = 15.0 Hz, H3); 3.10 (d, 1H, J = 4.9 Hz, H1); 2.90
(d, 1H, J = 4.9 Hz, H1). 13C NMR (DMSO-d6):
δ
49.6, 53.8, 59.1, 118.2, 124.1 (q, 1JCF = 267.9 Hz), 125.3 (q, 2C,
3JCF = 3.8 Hz), 126.7, 128.3, 128.5 (q, 2JCF = 31.9 Hz), 138.1, 141.6.
3.3.3. General Procedure for the N-Alkylation of Indole Derivatives 8g and 11a–k
Sodium hydride (0.06 g, 2.61 mmol) was dissolved in DMSO (20 mL) and the corresponding
indole derivative (2.61 mmol) was added portionwise, then the mixture was stirred at rt for 1 h under
argon. After this period, the corresponding oxirane derivative 10a
was added and the mixture was stirred for 12 h. At the end of this period, the mixture was diluted with
H2O (20 mL) and extracted with ethyl acetate (3 40 mL). The organic layer was washed with brine
–f (2.61 mmol) in DMSO (10 mL)
×
(40 mL), dried over anhydrous sodium sulfate, and the volatile fraction was evaporated under reduced
pressure. The residue was purified by silica gel column chromatography (EtOAc/hexane 1:1, v/v).
2-(4-Fluorophenyl)-3-(1H-indol-1-yl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (11a). White powder
(57% yield); Rf = 0.08 (EtOAc/hexane: 1/1); mp: 190–191 ◦C; IR (KBr):
ν ;
3408 (w, OH), 1602, 1217 cm−1
1H NMR (DMSO-d6):
J = 7.0 Hz, H4”), 7.27 (d, 1H, J = 3.1 Hz, H2”), 7.05 (dd, 1H, J = J = 7.0 Hz, H6”), 7.04 (dd, 2H, J = J = 8.9
δ
0 0 0 0
8.27 (s, 1H, H3 ”), 7.90 (s, 1H, H5 ”), 7.44–7.49 (m, 3H, H2 , H6 , H4”), 7.40 (d, 1H,
0
0
Hz, H3 , H5 ), 6.97 (dd, 1H, J = J = 7.0 Hz, H5”), 6.39 (d, 1H, J = 3.1 Hz, H3”), 6.00 (s, 1H, OH), 4.82
(d, 1H, J = 14.3 Hz, H3), 4.60 (d, 1H, J = 15.3 Hz, H1), 4.54 (d, 1H, J = 15.3 Hz, H1), 4.53 (d, 1H, J = 14.3
Hz, H3); 13C NMR (DMSO-d6):
δ
53.6, 56.1, 75.9, 100.8, 110.3, 114.3 (d, 2C, 2JCF = 21.5 Hz), 118.7, 120.0,

Pharmaceuticals 2020, 13, 186
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120.7, 127.5, 129.8, 128.0 (d, 2C, 3JCF = 8.1 Hz), 137.0, 137.5 (d, 4JCF = 3.3 Hz), 145.0, 150.6, 161.3 (d,
1JCF = 242.7 Hz); MS m/z 336 (M+), 241, 130 (100), 82. Anal. Calcd. for C19H17FN4O (336.36): C: 67.84;
H: 5.05; N: 16.65; found: C: 67.87; H: 5.03; N: 16.61%.
2-(2,4-Dichlorophenyl)-3-(1H-indol-1-yl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (8g). White powder
(38% yield); all physicochemical and spectral data were similar to 8g obtained by route 1.
2-(4-Chlorophenyl)-3-(1H-indol-1-yl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (11b). White powder
(89% yield); Rf = 0.77 (EtOH/dichloromethane: 1/10); mp: 188–189 ◦C; IR (KBr):
ν
3408 (w, OH), 1596,
1269 cm−1; 1H NMR (DMSO-d6):
δ 8.28 (s, 1H, H3 ”), 7.89 (s, 1H, H5 ”), 7.49 (d, 1H, J = 7.0 Hz, H4”),
0
0
0
0
0
0
7.47 (d, 2H, J = 8.9 Hz, H2 , H6 ), 7.44 (d, 1H, J = 7.0 Hz, H7”), 7.28 (d, 2H, J = 8.9 Hz, H3 , H5 ), 7.27
(d, 1H, J = 2.7 Hz, H2”), 7.06 (dd, 1H, J = J = 7.0 Hz, H6”), 6.98 (dd, 1H, J = J = 7.0 Hz, H5”), 6.40 (d, 1H,
J = 2.7 Hz, H3”), 6.05 (s, 1H, OH), 4.84 (d, 1H, J = 14.0 Hz, H3), 4.62 (d, 1H, J = 15.0 Hz, H1), 4.60 (d, 1H,
J = 14.0 Hz, H3), 4.54 (d, 1H, J = 15.0 Hz, H1); 13C NMR (DMSO-d6):
δ 53.5, 56.0, 76.0, 100.9, 110.4, 118.7,
119.9, 120.7, 120.8, 125.1, 127.5 (2C), 127.9 (2C), 129.8, 137.0, 140.4, 145.0, 150.6; MS m/z: 352 (M+), 354
(M+2), 241, 130 (100), 82. Anal. Calcd. for C19H17ClN4O (352.82): C: 64.68; H: 4.82; N: 15.87; found: C:
64.64; H: 4.79; N: 15.89%.
2-(4-Bromophenyl)-3-(1H-indol-1-yl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (11c). White powder
(45% yield); Rf = 0.09 (EtOAc/hexane: 1/1); mp: 186–187 ◦C; IR (KBr):
ν ;
3406 (w, OH), 1587, 1271 cm−1
1H NMR (DMSO-d6):
0 0
δ 8.27 (s, 1H, H3 ”), 7.88 (s, 1H, H5 ”), 7.48 (d, 1H, J = 7.0 Hz, H4”), 7.44 (d, 1H,
0
0
0
0
J = 7.0 Hz, H7”), 7.41 (s, 4H, H2 , H3 , H5 , H6 ), 7.26 (d, 1H, J = 3.1 Hz, H2”), 7.06 (dd, 1H, J = J = 7.0
Hz, H6”), 6.98 (dd, 1H, J = J = 7.0 Hz, H5”), 6.40 (d, 1H, J = 3.1 Hz, H3”), 6.04 (s, 1H, OH), 4.84 (d, 1H,
J = 14.3 Hz, H3), 4.61 (d, 1H, J = 15.0 Hz, H1), 4.57 (d, 1H, J = 14.3 Hz, H3), 4.54 (d, 1H, J = 15.0 Hz,
H1); 13C NMR (DMSO-d6):
δ 53.5, 55.9, 76.0, 100.9, 110.4, 118.7, 120.0, 120.5, 120.8, 127.5, 128.3 (2C),
129.8, 130.5 (2C), 137.0, 140.9, 145.0, 150.6; MS m/z: 396, 398 (M+), 241, 130 (100), 82. Anal. Calcd. for
C19H17BrN4O (397.27): C: 57.44; H: 4.28; N: 14.10; found: C: 57.40; H: 4.25; N: 14.08%.
2-(2,4-Difluorophenyl)-3-(1H-indol-1-yl)-1-(1H-1,2,4-triazol-1-yl)-propan-2-ol (11d). White powder
(41% yield); Rf = 0.08 (EtOAc/hexane: 1/1); mp: 119–120 ◦C; IR (KBr):
ν
3206 (w, OH), 1615, 1313 cm−1; 1H
0 0
δ 8.34 (s, 1H, H3 ”), 7.82 (s, 1H, H5 ”), 7.49 (d, 1H, J = 7.6 Hz, H4”), 7.39 (d, 1H, J = 7.6 Hz,
NMR (DMSO-d6):
0
0
H7”), 7.25 (d, 1H, J = 3.1 Hz, H2”), 7.17–7.29 (m, 2H, H3 , H5 ), 7.10 (dd, 1H, J = J = 7.6 Hz, H6”), 6.99 (dd, 1H,
0
J = J = 7.6 Hz, H5”), 6.86 (td, 1H, J = 8.6Hz, J = 2.4 Hz, H2 ), 6.40 (d, 1H, J = 3.1 Hz, H3”), 6.29 (s, 1H, OH), 4.90
(d, 1H, J = 14.3 Hz, H3), 4.68 (d, 1H, J = 15.0 Hz, H1), 4.57 (d, 1H, J = 15.0 Hz, H1), 4.56 (d, 1H, J = 14.3 Hz,
H3); 13C NMR (DMSO-d6):
δ
52.1, 54.8, 75.2 (d, 3JCF = 5.2 Hz), 100.9, 103.9 (dd, 2JCF = 2JCF = 26.2 Hz), 111.0
(dd, 2JCF = 20.5 Hz, 4JCF = 2.9 Hz), 111.3, 118.7, 120.0, 120.8, 124.0 (dd, 2JCF = 13.4 Hz, 4JCF = 3.8 Hz), 125.1,
129.6, 130.1 (dd, 3JCF = 10.0 Hz, 3JCF = 6.7 Hz), 136.9, 145.0, 150.7, 158.9 (dd, 1JCF = 247.0 Hz, 3JCF = 12.4
Hz), 162.5 (dd, 1JCF = 246.1 Hz, 3JCF = 12.9 Hz); MS m/z: 354 (M+), 241, 130 (100), 82. Anal. Calcd. for
C19H16F2N4O (354.35): C: 64.40; H: 4.52; N: 15.80; found: C: 64.43; H: 4.50; N: 15.84%.
1-(1H-Imidazol-1-yl)-3-(1H-indol-1-yl)-2-(4-trifluoromethylphenyl)propan-2-ol (11e). Orange
powder (9% yield); Rf = 0.61 (EtOAc/hexane: 1/1); mp: 246–247 ◦C; IR (KBr):
ν
3421 (w, OH), 1610,
1328 cm−1; 1H NMR (DMSO-d6):
δ
7.73 (d, 2H, J = 8.2 Hz, H3 ”, H5 ”), 7.60 (d, 2H, J = 8.2 Hz, H2 ”
,
0
0
0
0
0
H6 ”), 7.48 (d, 1H, J = 7.9 Hz, H4”), 7.45 (d, 1H, J = 7.9 Hz, H7”), 7.38 (s, 1H, H2 ), 7.18 (d, 1H, J = 3.1 Hz,
0
H2”), 7.07 (dd, 1H, J = J = 7.9 Hz, H6”), 6.94 (dd, 1H, J = J = 7.9 Hz, H5”), 6.91 (s, 1H, H5 ), 6.72 (s, 1H,
0
H4 ), 6.38 (d, 1H, J = 3.1 Hz, H3”), 6.16 (s, 1H, OH), 4.72 (d, 1H, J = 14.3 Hz, H3), 4.63 (d, 1H, J = 14.7 Hz,
H1), 4.48 (d, 1H, J = 14.7 Hz, H1), 4.35 (d, 1H, J = 14.3 Hz, H3); 13C NMR (DMSO-d6):
δ 53.6, 56.9, 76.8,
100.5, 110.4, 117.4, 119.1, 119.9, 120.6, 124.2 (q, 2C, 3JCF = 5.7 Hz), 124.8 (q, 1JCF = 263.4 Hz), 127.2 (2C),
127.4, 128.3, 128.9 (q, 2JCF = 26.7 Hz), 130.6, 138.1, 138.6, 139.9; MS m/z: 385 (M+), 240, 130 (100), 81.
Anal. Calcd. for C21H18F3N3O (385.38): C: 65.44; H: 4.67; N: 10.90; found: C: 65.41; H: 4.64; N: 10.87%.
2-(2,4-Difluorophenyl)-3-(2-methyl-1H-indol-1-yl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol
(11f).
Orange powder (34% yield); Rf = 0.17 (EtOAc/hexane: 1/10); mp: 125–126 ◦C; IR (KBr):
ν
3113 (w, OH),
1612, 1270 cm−1; 1H NMR (DMSO-d6):
δ
8.38 (s, 1H, H3 ”), 7.82 (s, 1H, H5 ”), 7.27–7.42 (m, 2H, H3 ,
0
0
0
0
H5 ), 7.41 (d, 1H, J = 7.0 Hz, H4”), 7.25 (d, 1H, J = 7.0 Hz, H7”), 6.96–7.04 (m, 2H, H5”, H6”), 6.93 (td, 1H,
0
J = 8.2 Hz, J = 2.4 Hz, H6 ), 6.22 (s, 1H, H3”), 6.09 (s, 1H, OH), 4.96 (d, 1H, J = 14.4 Hz, H3), 4.61 (d, 1H,

Pharmaceuticals 2020, 13, 186
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J = 15.3 Hz, H1), 4.59 (d, 1H, J = 14.4 Hz, H3), 4.46 (d, 1H, J = 15.3 Hz, H1), 2.22 (s, 3H, CH3); 13C NMR
(DMSO-d6):
13.0, 52.1, 54.8, 75.2 (d, 3JCF = 5.2 Hz), 100.9, 103.9 (dd, 2JCF = 2JCF = 26.2 Hz), 111.0
δ
(dd, 2JCF = 20.5 Hz, 4JCF = 2.9Hz), 111.3, 118.7, 120.0, 120.8, 124.0 (dd, 2JCF = 3.4 Hz, 4JCF = 3.8 Hz),
125.1, 129.6, 130.1 (dd, 3JCF = 10.0 Hz, 3JCF = 6.7 Hz), 136.9, 145.0, 150.7, 158.9 (dd, 1JCF = 247.0 Hz,
3JCF = 12.4 Hz), 162.5 (dd, 1JCF = 246.1 Hz, 3JCF = 12.9 Hz); MS m/z: 368 (M+), 255, 144 (100), 82. Anal.
Calcd. for C20H18F2N4O (368.38): C: 65.20; H: 4.89; N: 15.20; found: C: 65.18; H: 4.91; N: 15.17%.
2-(2,4-Dichlorophenyl)-3-(2-methyl-1H-indol-1-yl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (11g). White
powder (15% yield); Rf = 0.30 (EtOAc/hexane: 1/1); mp: 170–171 ◦C; IR (KBr):
ν
3422 (w, OH), 1582, 1271
cm−1; 1H NMR (DMSO-d6):
δ
8.40 (s, 1H, H3 ”), 7.82 (s, 1H, H5 ”), 7.70 (d, 1H, J = 2.1 Hz, H3 ), 7.49 (d, 1H,
0
0
0
0
0
J = 7.2 Hz, H6 ), 7.42 (d, 1H, J = 8.3 Hz, H4”), 7.31 (d, 1H, J = 7.2 Hz, H5 ), 7.23 (dd, 1H, J = 8.3 Hz, J = 2.1
Hz, H7”), 6.95–7.03 (m, 2H, H5”, H6”), 6.24 (s, 1H, H3”), 6.06 (s, 1H, OH), 5.36 (d, 1H, J = 14.3 Hz, H3), 4.88
(d, 1H, J = 15.4 Hz, H1), 4.71 (d, 1H, J = 14.3 Hz, H3), 4.55 (d, 1H, J = 15.4 Hz, H1), 2.25 (s, 3H, CH3); 13
C
NMR (DMSO-d6):
δ 12.9, 48.2, 53.5, 76.6, 100.7, 110.2, 118.9, 119.0, 120.0, 127.2, 127.8, 130.1, 131.1, 131.6, 133.3,
137.9, 138.2 (2C), 145.9, 150.8; MS m/z: 400, 402, 404 (M+), 255, 144 (100), 82. Anal. Calcd. for C20H18Cl2N4O
(401.29): C: 59.86; H: 4.49; N: 13.95; found: C: 59.84; H: 4.44; N: 13.91%.
2-(2,4-Difluorophenyl)-3-(3-methyl-1H-indol-1-yl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol
(11h).
White powder (35% yield); Rf = 0.12 (EtOAc/hexane: 1/10); mp: 112–113 ◦C; IR (KBr):
ν
3421 (w, OH),
1617, 1272 cm−1; 1H NMR (DMSO-d6):
δ
0 0 0 0
8.33 (s, 1H, H3 ”), 7.80 (s, 1H, H5 ”), 7.18–7.30 (m, 2H, H3 , H5 ),
7.44 (d, 1H, J = 7.3 Hz, H4”), 7.32 (d, 1H, J = 7.3 Hz, H7”), 7.12 (dd, 1H, J = 7.3 Hz, H6”), 7.07 (s, 1H, H2”),
0
6.99 (dd, 1H, J = 7.3 Hz, H5”), 6.85 (td, 1H, J = 7.9 Hz, J = 2.3 Hz, H6 ), 6.25 (s, 1H, OH), 4.89 (d, 1H,
J = 14.3 Hz, H3), 4.54 (s, 2H, H1), 4.51 (d, 1H, J = 14.3 Hz, H3), 2.23 (s, 3H, CH3); 13C NMR (DMSO-d6):
δ
9.4, 52.0, 54.7, 75.2, 103.9 (dd, 2JCF = 2JCF = 25.8 Hz), 109.2, 109.5, 110.9 (d, 2JCF = 20.5 Hz), 118.2,
118.3, 121.0, 124.3 (dd, 2JCF = 12.9 Hz, 4JCF = 3.3 Hz), 127.3, 127.9, 130.1 (dd, 3JCF = 3JCF = 6.2 Hz),
137.2, 145.0, 150.6, 159.1 (dd, 1JCF = 246.5 Hz, 3JCF = 12.4 Hz), 160.5 (dd, 1JCF = 246.5 Hz, 3JCF = 12.9
Hz); MS m/z: 368 (M+), 255, 144 (100), 82. Anal. Calcd. for C20H18F2N4O (368.38): C: 65.20; H: 4.89; N:
15.20; found: C: 65.18; H: 4.91; N: 15.22%.
2-(2,4-Dichlorophenyl)-3-(3-methyl-1H-indol-1-yl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (11i). Red
powder (24% yield); Rf = 0.65 (EtOAc/hexane: 1/10); mp: 161–162 ◦C; IR (KBr):
ν
3399 (w, OH), 1586,
1275 cm−1; 1H NMR (DMSO-d6):
δ
8.31 (s, 1H, H3 ”), 7.77 (s, 1H, H5 ”), 7.62 (d, 1H, J = 2.1 Hz, H3 ), 7.46
0
0
0
0
(d, 1H, J = 7.5 Hz, H7”), 7.44 (d, 1H, J = 7.5 Hz, H4”), 7.42 (d, 1H, J = 8.9 Hz, H6 ), 7.25 (dd, 1H, J = 8.9
0
Hz, J = 2.1 Hz, H5 ), 7.13 (dd, 1H, J = J = 7.5 Hz, H5”), 7.04 (s, 1H, H2”), 7.01 (dd, 1H, J = J = 7.5 Hz,
H5”), 6.33 (s, 1H, OH), 5.31 (d, 1H, J = 14.3 Hz, H3), 4.73 (s, 2H, H1), 4.50 (d, 1H, J = 14.3 Hz, H3), 2.22 (s,
3H, CH3); 13C NMR (DMSO-d6):
δ 9.4, 50.7, 53.3, 76.7, 109.4, 109.9, 118.3 (2C), 121.2, 127.0, 127.4, 128.0,
129.9, 131.2, 131.4, 133.1, 137.0, 137.4, 145.1, 150.6; MS m/z: 400, 402, 404 (M+), 255, 144 (100), 82. Anal.
Calcd. for C20H18Cl2N4O (401.29): C: 59.86; H: 4.49; N: 13.95; found: C: 59.83; H: 4.52; N: 13.98%.
2-(2,4-Difluorophenyl)-3-(2,3-dimethyl-1H-indol-1-yl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (11j). Red
powder (22% yield); Rf = 0.16 (EtOAc/hexane: 1/10); mp: 102–103 ◦C; IR (KBr): ν 3384 (w, OH), 1614, 1272
cm−1; 1H NMR (DMSO-d6):
0 0 0 0
δ 8.38 (s, 1H, H3 ”), 7.79 (s, 1H, H5 ”), 7.20–7.31 (m, 2H, H3 , H5 ), 7.39 (d, 1H,
0
J = 6.4 Hz, H4”), 7.36 (d, 1H, J = 6.4 Hz, H7”), 7.09 (dd, 1H, J = J = 6.4 Hz, H6”), 6.86–7.01 (m, 2H, H6 ,
H5”), 6.06 (s, 1H, OH), 4.96 (d, 1H, J = 14.5 Hz, H3), 4.60 (d, 1H, J = 14.5 Hz, H1), 4.55 (d, 1H, J = 14.5 Hz,
H3), 4.43 (d, 1H, J = 15.6 Hz, H1), 2.18 (s, 6H); 13C NMR (DMSO-d6): 8.8, 10.2, 49.9, 54.8, 75.6, 104.1 (dd,
2JCF = 2JCF = 25.7 Hz), 106.1, 109.7, 111.0 (dd, 2JCF = 20.5 Hz, 4JCF = 2.9 Hz), 117.3, 118.5, 120.2, 124.9 (dd,
3JCF = 9.1 Hz, 3JCF = 12.9 Hz), 128.3, 130.1 (dd, 2JCF = 22.4 Hz, 4JCF = 3.8 Hz), 133.8, 137.2, 145.2, 150.6,
159.3 (dd, 1JCF = 254.6 Hz, 3JCF = 11.4 Hz), 160.8 (dd, 1JCF = 246.5 Hz, 3JCF = 8.5 Hz); MS m/z: 382 (M+),
269, 158 (100), 82. Anal. Calcd. for C21H20F2N4O (382.41): C: 65.95; H: 5.23; N: 14.64; found: C: 65.98; H:
5.25; N: 14.62%.
2-(2,4-Dichlorophenyl)-3-(2,3-dimethyl-1H-indol-1-yl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (11k).
White powder (15% yield); Rf = 0.17 (EtOAc/hexane: 1/1); mp: 123–124 ◦C; IR (KBr):
ν
3429 (w, OH),
1584, 1273 cm−1; 1H NMR (DMSO-d6):
δ 8.39 (s, 1H, H3 ”), 7.80 (s, 1H, H5 ”), 7.57 (d, 1H, J = 1.8 Hz,
0
0
0
0
H3 ), 7.51 (d, 1H, J = 7.9 Hz, H4”), 7.40 (d, 1H, J = 8.3 Hz, H6 ), 7.37 (d, 1H, J = 7.9 Hz, H7”), 7.27 (dd, 1H,

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0
J = 8.3 Hz, J = 1.8 Hz, H5 ), 7.11 (dd, 1H, J = J = 7.9 Hz, H6”), 7.00 (dd, 1H, J = J = 7.9 Hz, H5”), 6.03
(s, 1H, OH), 5.39 (d, 1H, J = 14.3 Hz, H3), 4.90 (d, 1H, J = 15.3 Hz, H1), 4.66 (d, 1H, J = 14.3 Hz, H3), 4.51
(d, 1H, J = 15.3 Hz, H1), 2.21 (s, 6H); 13C NMR (DMSO-d6): 10.3, 13.9, 48.2, 53.4, 76.7, 106.1, 109.4, 117.3,
118.5, 120.1, 127.2, 127.6, 130.0, 131.1, 131.2, 133.2, 137.3, 137.8, 140.4, 146.2, 150.6; MS m/z: 414, 416, 418
(M+), 269, 158 (100), 82. Anal. Calcd. for C21H20Cl2N4O (415.32): C: 60.73; H: 4.82; N: 13.48; found: C:
60.69; H: 4.79; N: 13.52%.
3.4. Synthesis of Compounds 11l and 11m (Route 2)
3.4.1. Procedure for the N-1 and N-2-alkylation of 1H-1,2,3-Triazole
1-(2,4-Dichlorophenyl)-2-(1H-1,2,3-triazol-1-yl)ethanone (9g) and 1-(2,4-dichlorophenyl)-2-(2H-
1,2,3-triazol-2-yl)ethan-1-one (9h) were synthesized according to methods a and b.
Method a: To a solution of 2-chloro-20,40-dichloroacetophenone (2.50 g, 11.19 mmol) in acetonitrile
(40 mL) 1H-1,2,3-triazole (1.55 g, 22.37 mmol) and K2CO3 (3.09 g, 22.37 mmol) were added. The reaction
mixture was irradiated for 50 min in a microwave oven (Discover, CEM) and programmed to obtain a
temperature of 85 ◦C with a maximum power output of 50 W. After cooling, the mixture was filtered
and the volatile fraction was evaporated under reduced pressure. The residue was diluted with H2O
(40 mL) and extracted with EtOAc (3
×
80 mL), then the organic layer was washed with brine (80 mL),
dried over anhydrous sodium sulfate, and the volatile fraction was evaporated under reduced pressure.
1H NMR (DMSO-d6) of the residue gave a ratio 49/51 of compounds 9g/9h. The residue was purified
by silica gel column chromatography (dichloromethane).
1-(2,4-Dichlorophenyl)-2-(1H-1,2,3-triazol-1-yl)ethanone (9g). White powder (49% yield); Rf = 0.06
(dichloromethane); mp: 118–120 ◦C; IR (KBr):
ν
1717 (s, C=O), 1585, 1215 cm−1; 1H NMR (DMSO-d6):
0
0
δ
8.16 (s, 1H, H5”); 8.05 (d, 1H, J = 8.5 Hz, H6 ); 7.88 (d, 1H, J = 2.1 Hz, H3 ); 7.84 (s, 1H, H4”); 7.70 (dd,
2H, J = 8.5 Hz, J = 2.1 Hz, H5 ); 6.11 (s, 2H, CH2); 13C NMR (DMSO-d6): 57.5, 126.4, 127.7, 130.6, 131.5,
0
132.1, 133.3, 133.4, 137.5, 192.9.
1-(2,4-Dichlorophenyl)-2-(2H-1,2,3-triazol-2-yl)ethan-1-one (9h). White powder (50%); Rf = 0.29
(dichloromethane); mp: 100–101 ◦C; IR (KBr):
ν
1717 (s, C=O), 1580, 1204 cm−1; 1H NMR (DMSO-d6):
0
0
δ
7.99 (d, 1H, J = 8.5 Hz, H6 ); 7.91 (s, 2H, H3”, H4”); 7.84 (d, 1H, J = 2.1 Hz, H3 ); 7.65 (dd, 1H, J = 8.5
Hz, J = 2.1 Hz, H5 ); 6.14 (s, 2H, CH2); 13C NMR (DMSO-d6): 62.1, 127.6, 130.5, 131.3, 132.0, 133.6, 135.3
0
(2C), 137.3, 193.5.
Method b: To a solution of 2-chloro-20,40-dichloroacetophenone (2.50 g, 11.19 mmol) in acetonitrile
(40 mL) 1H-1,2,3-triazole (1.55 g, 22.37 mmol) and K2CO3 (3.09 g, 22.37 mmol) were added. The reaction
mixture was heated at 85 ◦C for 8 h. After cooling, the mixture was filtered and the volatile fraction
was evaporated under reduced pressure. The residue was diluted with H2O (40 mL) and extracted
with EtOAc (3 x 80 mL), then the organic layer was washed with brine (80 mL), dried over anhydrous
1
sodium sulfate, and the volatile fraction was evaporated under reduced pressure. H NMR (DMSO-d6)
of the residue gave a ratio 61/39 of compounds 9g/9h.
3.4.2. General Procedure for the Synthesis of Oxiranes 10g and 10h
A procedure [27] similar to that for obtaining 10a was used to prepare compounds 10g and 10h.
2-(2,4-Dichlorophenyl)-3-(1H-1,2,3-triazol-1-yl)-1,2-epoxypropane (10g). Orange oil (99% yield);
Rf = 0.65 (EtOH/dichloromethane: 1/10); IR (NaCl):
(s, 1H, H5”); 7.73 (s, 1H, H4”); 7.72 (d, 1H, J = 2.1 Hz, H3 ); 7.37 (dd, 1H, J = 8.2 Hz, J = 2.1 Hz, H5 ); 7.09
ν δ 8.05
1583, 1276 cm−1; 1H NMR (DMSO-d6):
0
0
0
(d, 1H, J = 8.2 Hz, H6 ); 5.12 (d, 1H, J = 14.7 Hz, H3); 4.77 (d, 1H, J = 14.7 Hz, H3); 3.20 (d, 1H, J = 4.9
Hz, H1); 3.00 (d, 1H, J = 4.9 Hz, H1); 13C NMR (DMSO-d6): 51.9, 52.9, 58.5, 125.8, 127.3, 128.7, 130.8,
133.1, 133.4, 133.5, 134.0.
2-(2,4-Dichlorophenyl)-3-(2H-1,2,3-triazol-2-yl)-1,2-epoxypropane (10h). Orange oil (86% yield);
Rf = 0.74 (EtOH/dichloromethane: 1/10); IR (NaCl):
ν
1584, 1256 cm−1; 1H NMR (DMSO-d6):
δ
7.76 (s,
0
0
2H, H3”, H4”); 7.67 (d, 1H, J = 2.1 Hz, H3 ); 7.33 (dd, 1H, J = 8.2 Hz, J = 2.1 Hz, H5 ); 7.08 (d, 1H, J = 8.2

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0
Hz, H6 ); 5.15 (d, 1H, J = 14.7 Hz, H3); 4.78 (d, 1H, J = 14.7 Hz, H3); 3.25 (d, 1H, J = 4.6 Hz, H1); 2.98
(d, 1H, J = 4.6 Hz, H1); 13C NMR (DMSO-d6): 51.4, 58.0, 62.1, 127.2, 128.5, 130.9, 133.8 (2C), 134.8 (3C).
3.4.3. General Procedure for the Sysnthesis of Triazole Derivatives 11l and 11m
A procedure similar to 11a was used to prepare compounds 11l and 11m.
2-(2,4-Dichlorophenyl)-3-(1H-indol-1-yl)-1-(1H-1,2,3-triazol-1-yl)propan-2-ol (11l). White powder
(52% yield); Rf = 0.11 (EtOAc/hexane: 1/1); mp: 210–211 ◦C; IR (KBr):
ν
3166 (w, OH), 1585, 1214 cm−1
;
1H NMR (DMSO-d6):
δ
7.87 (s, 1H, H5 ”), 7.64 (d, 1H, J = 2.1 Hz, H3 ), 7.58 (s, 1H, H4 ”), 7.53 (d, 1H,
0
0
0
0
J = 7.0 Hz, H4”), 7.51 (d, 1H, J = 7.0 Hz, H7”), 7.36 (d, 1H, J = 8.9 Hz, H6 ), 7.23 (d, 1H, J = 2.8 Hz, H2”),
0
7.21 (dd, 1H, J = 8.9 Hz, J = 2.1 Hz, H5 ), 7.14 (dd, 1H, J = J = 7.0 Hz, H6”), 7.01 (dd, 1H, J = J = 7.0 Hz,
H5”), 6.47 (s, 1H, OH), 6.41 (d, 1H, J = 2.8 Hz, H3”), 5.61 (d, 1H, J = 13.7 Hz, H3), 4.90 (d, 1H, J = 14.4
Hz, H1), 4.83 (d, 1H, J = 14.4 Hz, H1), 4.65 (d, 1H, J = 13.7 Hz, H3); 13C NMR (DMSO-d6): 50.8, 53.7,
76.8, 101.2, 110.1, 118.9, 120.1, 121.1, 125.8, 126.9, 127.6, 129.6, 129.9, 131.2 (2C), 132.6, 133.1, 136.4, 137.0;
MS m/z: 386, 388, 390 (M+), 241, 130 (100), 82. Anal. Calcd. for C19H16Cl2N4O (387.26): C: 58.92; H:
4.13; N: 14.46; found: C: 58.88; H: 4.10; N: 14.43%.
2-(2,4-Dichlorophenyl)-3-(1H-indol-1-yl)-1-(2H-1,2,3-triazol-2-yl)propan-2-ol (11m). White powder
(56% yield); Rf = 0.59 (EtOAc/hexane: 1/1); mp: 159–161 ◦C; IR (KBr):
ν
3434 (w, OH), 1589, 1215 cm−1; 1H
0 0 0
δ 7.67 (s, 2H, H3 ”, H4 ”), 7.61 (d, 1H, J = 2.1 Hz, H3 ), 7.53 (d, 1H, J = 7.6 Hz, H4”), 7.48
NMR (DMSO-d6):
0
(d, 1H, J = 7.6 Hz, H7”), 7.32 (d, 1H, J = 8.5 Hz, H6 ), 7.20 (d, 1H, J = 3.1 Hz, H2”), 7.17 (dd, 1H, J = 8.5 Hz,
0
J = 2.1 Hz, H5 ), 7.09 (dd, 1H, J = J = 7.6 Hz, H6”), 6.98 (dd, 1H, J = J = 7.6 Hz, H5”), 6.36 (d, 1H, J = 3.1 Hz,
H3”), 6.28 (s, 1H, OH), 5.56 (d, 1H, J = 14.0 Hz, H3), 4.99 (d, 1H, J = 15.0 Hz, H1), 4.83 (d, 1H, J = 14.0 Hz, H3),
4.72 (d, 1H, J = 15.0 Hz, H1); 13C NMR (DMSO-d6): 50.8, 58.4, 77.1, 101.0, 110.2, 118.8, 120.1, 120.9, 126.8,
127.5, 129.5, 129.7, 131.3, 131.5, 132.9, 134.2 (2C); 137.0 (2C); MS m/z: 386, 388, 390 (M+), 241, 130 (100), 82.
Anal. Calcd. for C19H16Cl2N4O (387.26): C: 58.92; H: 4.13; N: 14.46; found: C: 58.90; H: 4.15; N: 14.41%.
3.5. Chiral HPLC Chromatography
The chromatography system consisted of SpectraSystem (Thermo Electron S.A., Waltham, MA,
USA) P1000-010XR2 isocratic pump and a dual wavelength SpectraSystem UV2000 detector. T data
acquisition system was performed with an IBM PC/computer using Azur 3.0 (Datalys, Saint-Martin
d’H
column (250
coated by cellulose tris(3,5-dimethylphenylcarbamate). A Rheodyne 9125 injector with a 20
é
res, France) as chromatography sofware. Chromatography was performed on the Chiralcel OD-H
4.6 mm, Daicel Chemical Industries Ltd, Tokyo, Japan) packed with 5 m silica gel
L sample
×
µ
µ
loop was used. The mobile phases used were: (A) Acetonitrile, 100; (B) acetonitrile/diethylamine,
100:0.1 (v/v); (C) ethanol/diethylamine, 100:0.1 (v/v); (D) methanol/diethylamine, 100:0.1 (v/v);
(E) n-hexane/ethanol/diethylamine, 60:40:0.1 (v/v/v); (F) n-hexane/methanol/ethanol/diethylamine,
75:15:10:0.1 (v/v/v/v). Solvents were of HPLC quality (Carlo Erba, Val de Reuil, France). The flow
rate was 1.1 mL/min and the injection volume was 20 µL. The detection wavelength was 250 nm.
The column temperature was at 25–30 ◦C. The sample concentration was 10
µg/mL in mobile phase.
This method was used to separate the enantiomers of compound 8g.
3.6. X-ray Crystallography Studies
The structure of compounds (+)-8g and (
and 3) [44]. A colorless single crystal (0.25
evaporation from methanol/chloroform (30:70, v/v) solution: Monoclinic, space group P21, a = 6.707(2)
Å, b = 32.174(4) Å, c = 10.080(2) Å,
= 90.0◦, = 109.40(2)◦, = 90.0◦, V = 2051.7(8) Å3, Z = 2,
(calcd) = 1.254 Mg.m-3, FW = 774.52 for C38H32Cl4N8O2, F(000) = 800. A colorless single crystal
(0.25 0.10 )-8g was obtained by slow evaporation from methanol/chloroform
0.07 mm3) of (
(30:70, v/v) solution: Monoclinic, space group P21, a = 6.681(3) Å, b = 32.142(4) Å, c = 10.111(4) Å,
= 90.0◦, = 109.40(2)◦, = 90.0◦, V = 2048.0(13) Å3, Z = 2, (calcd) = 1.256 Mg.m−3, FW = 774.52 for
C38H32Cl4N8O2, F(000) = 800. The unit cell dimensions were determined using the least-squares fit from
−
)-8g was established by X-ray crystallography (Figures 2
×
0.10
×
0.05 mm3) of (+)-8g was obtained by slow
α
β
γ
δ
×
×
−
α
β
γ
δ

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25 reflections (25◦ <
using the CuK
θ
< 35◦). Intensities were collected with an Enraf–Nonius CAD-4 diffractometer
α
radiation and a graphite monochromator up to θ
= 55◦. No intensity variation of 2
standard reflections monitored every 90 min was observed. The data were corrected for Lorentz and
polarization effects and for empirical absorption correction [45]. The structure was solved by direct
methods Shelx 86 [46] and refined using Shelx 93 [47] suite of programs.
3.7. Biological Assays
3.7.1. Anti-Candida In Vitro Assay
Test fungal strains were obtained from the American Type Culture Collection (ATCC) or were
clinical isolates from the Laboratory of Parasitology and Medical Mycology, Centre Hospitalier
Universitaire (CHU) of Nantes. The strains were maintained on Sabouraud agar slants and were
subcultured 24 h before used. The activity of the target compounds 8a–g and 11a–m was determined by
the method previously described [48]. Briefly, molecules were diluted in RPMI 1640 medium
supplemented with 0.165 M morpholinopropanesulphonic acid (Sigma-Aldrich, Saint-Quentin
Fallavier, France), 2% glucose, and antibiotics. Candida suspensions were prepared in RPMI 1640
medium (Sigma-Aldrich, Saint-Quentin Fallavier, France) adjusted to give a final concentration of 103
cells/mL, and a 96-well microplate (Nunc, D. Dutscher, Brumath, France) was seeded with 100
Each concentration of molecules (100 L) to be tested was added (in triplicate) and plates were
incubated at 37 ◦C for 24 h. The cellular viability was evaluated on the Fluorolite 1000 (Dynatech,
µL.
µ
France) with an excitation at 550 nm and an emission at 590 nm after a 4 h incubation with 10 µL of
Alamar Blue®. The minimal inhibitory concentration (MIC) is the concentration that inhibited 80% of
the cell growth and was determined by linear regression analysis. MIC was expressed as the mean of
the triplicate values. KTC and FLC were used as standards.
3.7.2. MRC5 Toxicity Assay
The cytotoxicity of compounds was studied with human fibroblast (MRC5). Cells were grown in
RPMI 1640 medium (Sigma-Aldrich, Saint-Quentin Fallavier, France) supplemented with 10% fetal
bovin serum (Sigma–Aldrich). Drugs were tested at three concentrations (100, 10, and 1 µM) in
triplicate. After a 96-h incubation time, cytotoxicity was measured on the Fluorolite 1000 (Dynatech,
Guyancourt, France) after a 4-h incubation time with Uptiblue® (Interchim, Montluçon, France).
Inhibitory concentration 50 is a mean of triplicate values. A toxicity index was determined as follow:
IC50 against MRC5/Geometric mean of the MICs against Candida spp. [28].
3.7.3. In Vitro Inhibition of Aromatase
The assay was performed according to previously described methods [49
enzyme activity by measuring the 3H2O formed from [1 -3H]androstenedione during aromatization.
In brief, the reaction mixture, containing [1 Ci, 15 nM), unlabeled
-3H]androstenedione (0.08
M), and
–51] by monitoring the
β
β
µ
androstenedione (485 nM), the NADPH-generating system (2 mM), the inhibitor (0–100
µ
phosphate buffer (0.05 M, pH 7.4), was preincubated for 5 min at 30 ◦C. Microsomal protein (0.1 mg)
◦
was added to start the reaction. After incubation of 15 min at 30 C, the reaction was stopped
by adding 200 µL of a cold HgCl2 solution (1 mM). After the addition of 200 µL of an aqueous
dextran-coated charcoal (DCC) suspension (2%), the vials were shaken then centrifuged to separate
the charcoal-absorbed steroids. Aliquots of the supernatant were assayed for 3H2O by counting in a
scintillation mixture using a LKB-Wallac β-counter.
3.7.4. In Vitro Inhibition of 17α-Hydroxylase/17,20-Lyase
The assay was performed similar to described methods [29,52]. In summary, the reaction
mixture containing progesterone (1.25 mM), NADPH (125 nmol), the inhibitor, and phosphate buffer
(pH 7.4) was preincubated for 5 min at 32 ◦C. Microsomal protein was added to start the reaction.

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26 of 32
After incubation for 20 min at 32 ◦C, the reaction was stopped by adding 50
µL of 1 M aqueous HCl
solution. After the addition of 1 mL EtOAc, the vials were shaken then centrifuged. The organic phase
was removed, vortexed with phosphate buffer (250 L), 1 M aqueous HCl (50 L), and then dried.
Aliquots of 25 L methanol, containing 250 pmol of fluorocortisol acetate as internal standard, were
added to the extracts. The samples (20 L) were submitted to HPLC (RP-8 column, methanol/water
µ
µ
µ
µ
1:1, v/v) and measured using UV (240 nm). For compounds causing an inhibition over 80%, IC50 values
were determined.
3.7.5. MCF-7 Cell Culture and In Vitro Assay for Inhibition of Metabolism of All-trans Retinoic
Acid (ATRA)
Human MCF-7 breast cancer cells were cultured in phenol red-free RPMI 1640 medium
supplemented with 5% (v/v) charcoal free fetal calf serum, antibiotics (penicillin and streptomycin),
and fungizone at the same concentration of 10 iU/mL (all reagents from Gibco Europe Ltd., Scotland,
UK). Cells were grown in a humidified incubator (5% CO2, 95% air) at 37 ◦C.
[11,12-3H]All-trans-retinoic acid (37 MBq/mL) was purchased from PerkinElmer Life Science Ltd. (USA).
All-trans retinoic acid, NADPH, butylated hydroxyanisole, and KTC were obtained from Sigma–Aldrich
(Gillingham, UK). Liarozole was a gift from Stiefel Laboratories, High Wycombe, UK. MCF-7 cells were
seeded in 12-well cell culture plates (Cornings Inc., New York, USA) at 2.5
volume of 1.5 mL. Cells were allowed to adhere to the well for 24 h. After 24 h, the medium from each
well was removed, washed once with phosphate buffer saline (PBS), and replaced by fresh medium plus
×
105 cells per well in a total
10
and 0.1
Each treatment was performed in duplicate. The incubation was stopped by addition of 1 % acetic acid
(100 L/well), the medium was removed into separate glass tubes. Distilled water (200 L) was added to
each well and the cells scrapped off and the contents added to the appropriate glass tube. This procedure
was repeated with a further 400 L water but without scraping. Ethyl acetate containing 0.05 % (w/v)
butylated hydroxyanisole (2 2 mL) was added to each tube. After vortexing for 15 s, the tubes were spun
µ
L inhibitor/solvent (acetonitrile) and 10
µ
L of ATRA (to give a final concentration of 1
×
10−7 M ATRA
µ
Ci [11,12-3H]all-trans-retinoic acid). The plates were foil wrapped and incubated at 37 ◦C for 9 h.
µ
µ
µ
×
down at 3000 rpm for 15 min. The organic layer was then evaporated using a Christ centrifuge connected to
a vacuum pump and a multitrap at −80 ◦C.
The HPLC system was equipped with a high-pressure pump (Milton–Roy pump, Stone, UK),
injector with a 50
computer running Laura® data acquisition and analysis software. This enabled online detection and
quantification of radioactive peaks. The 10 300 mm HPLC column (Waters,
M C18 µBondapak™ 3.9
µ
L loop connected to a beta-RAM radioactivity detector, connected to a Compaq™
µ
×
UK) was used for the all-trans retinoic acid metabolism studies. The HPLC column operating at an
ambient temperature was used to separate the metabolites, which were eluted with acetonitrile/1%
ammonium acetate in water/acetic acid (75:25:0.1, v/v/v) at a flow rate of 1.9 mL/min. The Ecoscint
™
was used as the flow scintillation fluid.
The separated [3H]-metabolites were quantitatively calculated from the areas under the curves.
Using a control with acetonitrile instead of inhibitor, these results were expressed as the “percentage
inhibition relative to control” = 100[(metabolites (control)—metabolites (inhibitor)/(metabolites
control)]%. KTC and liarozole were used as standards [53].
3.7.6. In Vitro Inhibition of CYP11B1 and CYP11B2
For the CYP11B1 assay, 11-deoxycortisol (RSS) was used as substrate, whereas 11-deoxycorticosterone
(DOC) was used for the CYP11B2 assay. These non-radioactive steroids were purchased from Sima
(Deisenhofen, Germany). Radioactive steroids were obtained from Amersham Pharmacia Biotech (Freiburg,
Germany). Two Schizosaccharomyces pombe strains were used, SZ1 expressing CYP11B1 and MB164 expressing
CYP11B2. The assays were performed as described previously by Bureik et al. [54]. Exponentially growing
fission yeast cells were cultivated by shaking and with good aeration at 30 ◦C in 500
Medium Glutamate (EMMG) containing supplements as required. Inhibitors were dissolved in ethanol
µL Edinburgh Minimal

Pharmaceuticals 2020, 13, 186
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at different concentrations, and equal volumes of ethanolic solutions were used in all cases (including
controls). Final concentrations of inhibitors ranged from 100 nM to 5 M. Cells were preincubated with
µ
the respective inhibitor solutions for 15 min prior to the addition of steroid substrates (100 nM DOC or
100 nM RSS, respectively). For detection of steroids, 1% of the total substrate amount was radioactively
labeled as [3H]RSS, [3H]DOC, or [14C]DOC. After 4 h (MB164) or 24 h (SZ1) cultures were extracted with
chloroform and dried under vacuum. The residues were dissolved in 10 µL chloroform and spotted onto
glass-backed silica-coated HPTLC plates (Kieselgel 60 F254, Merck; Darmstadt, Germany). In addition,
small amounts of non-radioactive steroids were spotted as references. The HPTLC was developed twice in
chloroform/methanol/water (300:20:1, v/v/v) and steroids identified after exposure to Fuji imaging plates and
quantitated on a phosphoimager (BAS-2500, Fuji; Stamford, CT, USA).
3.8. Computational Methods
3.8.1. Molecular Docking
First molecular docking was performed for the compound (R)-8g and (S)-8g to assess their binding
feasibility and difference. A six-step procedure was applied. (1) Three crystal structures of C. albicans
CYP51 (PDB code: 5TZ1, 5FSA, 5V5Z) were taken from Pocketome [55], in which different PDB
structures of the same protein were superimposed by the binding sites to take the induce-fit effect
into consideration. (2) All structures were combined to calculate grid potential ensembles for the
docking of (R)-8g and (S)-8g. The grid maps were calculated on a 0.5 Å 3D grid, containing: (i) van der
Waals potentials; (ii) electrostatic potentials; (iii) hydrogen bonding potentials; and (iv) hydrophobic
potential grids. (3) The structures of ligands were taken from crystallography [43]. (4) Four distinctive
starting poses were generated for sampling. (5) The ligand was then sampled in the pre-generated grid
potential ensembles through biased probability Monte Carlo sampling method [56] to optimize the
positional variables of the ligand. (6) After the sampling, the ligand conformation was re-scored with
the full-atom ICM scoring function [57]. All docking and scoring were performed in ICM-Pro v3.8-7c.
3.8.2. Molecular Dynamics Simulation
Molecular dynamics simulation was performed as previously described [21]. Briefly, docking studies
were performed using MOE [40] and CaCYP51 (PDB 5FSA [39]) to generate pdb files of the CaCYP51
crystal structure complexed with (R)-8g and (S)-8g. Molecular dynamics simulations were run on the
CaCYP51-ligand complexes with the pdb files first optimized with protein preparation wizard in Maestro
by assigning bond orders, adding hydrogen, and correcting incorrect bond types. A default quick relaxation
protocol was used to minimise the MD systems with the Desmond program [41]. Force-field parameters for
the complexes were assigned using the OPLS_ 2005 forcefield, that is, a 100 ns molecular dynamic run in the
NPT ensemble (T = 300 K) at a constant pressure of 1 bar. Energy and trajectory atomic coordinate data
were recorded at each 1.2 ns.
4. Conclusions
A series of 2-aryl-3-azolyl-1-indolyl-propan-2-ols was designed as new analogs of FLC by replacing
one of its two triazole moieties by an indole scaffold. A first chemical approach was developed in
seven steps, involving the synthesis of the key intermediate 1-(1H-benzotriazol-1-yl)methyl-1H-indole
4
and the final opening of oxiranes 7 by imidazole or 1H-1,2,4-triazole. A shorter process was also
developed to access the target compounds in only three steps, this time with the ring opening by
indole and analogs. Twenty azole derivatives were synthesized and tested against C. albicans and other
Candida species. Thirteen compounds demonstrated a high level of activity against C. albicans CA98001,
with MIC < 0.027
µg/mL, by comparison with the MIC value of FLC (MIC = 0.020 µg/mL). The in vitro
cytotoxicity of compounds 8a–g and 11a–k was further evaluated on MRC-5 cells.
The enantiomers of the best anti-Candida compound, 2-(2,4-dichlorophenyl)-3-(1H-indol-1-yl)-1-
(1H-1,2,4-triazol-1-yl)-propan-2-ol 8g, were analyzed by X-ray diffraction to determine their absolute

Pharmaceuticals 2020, 13, 186
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configuration. The (
−
)-8g enantiomer (MIC = 0.000256
µ
g/mL against C. albicans CA98001) was
found with the S-absolute configuration. Additionally, molecular docking and MD simulation were
performed and confirmed that the (S)-enantiomer aligned with the positioning of posaconazole within
both the heme and access channel binding sites. These in silico results are consistent with the biological
results presented. The selectivity of compounds 11d, 8g, and its enantiomers was also investigated
against five human P450-dependent enzymes (CYP19, CYP17, CYP26A1, CYP11B1, and CYP11B2).
Overall, (S)-8g had a pharmacological profile to pursue further biological investigations such as a
murine candidiasis assay and cytochrome P450 inhibition assays (e.g. CYP3A4, CYP2D6). Recently,
we published extended biological exploration of 8g (in vitro and in vivo assays) and then we confirmed
the full potential of this molecule [28]. Another major challenge is the emergence of resistance to azole
antifungals among Candida species [58]. Then a multi-disciplinary approach with joint expertise and
networks is crucial to assist efficiently medicinal chemists for new optimizations. According to the 3D
structures of CaCYP51 and our results obtained (Figure 9), further pharmacomodulation works should
be performed to explore both the position of the azolyl chain on indole scaffold and the introduction
of a long chain to occupy the space of the access channel (as does the N-arylpiperazine chain of
posaconazole). In this regard, NMR will be of special interest for studying ligand access channels in
cytochrome P450 enzymes [59–62].
A further biological investigation could be performed by testing a selection of our best
2-aryl-3-azolyl-1-indolyl-propan-2-ols (e.g. 8d
Candida auris strain.
,
8f, ( )-(S)-8g, 11g, 11h, 11i) against the emerging
−
The last point is that the antifungal azoles targeting CaCYP51 could be a starting point for
developing new treatments in some particular diseases such as human infections with protozoa
(Trypanosomatidae) [63] and primary amoebic meningoencephalitis (PAM) [64]. The needs for new
treatments are enormous in the field of parasitic diseases, and CYP51 is a promising target for further
drug development.
Author Contributions: Conceptualization, P.L.P. and M.L.B.; methodology, Y.M.N., N.L., F.P., M.-P.L., P.P., S.W.Y.,
D.S., T.H., M.M., and J.G.; software, R.A.; validation, R.W.H., R.B., C.S., R.A., P.L.P., and M.L.B.; investigation,
G.L.P., P.L.P., and M.L.B.; resources, P.L.P. and R.W.H.; data curation, A.M., P.L.P., and M.L.B.; writing—original
draft preparation, N.L., F.P., M.-P.L., R.B., J.G., A.M., and C.S.; writing—review and editing, N.L., I.K., P.L.P. C.S.,
and M.L.B.; visualization, J.G., D.S., and R.A.; supervision, G.L.B., P.L.P., and M.L.B.; project administration,
G.L.B., P.L.P., and M.L.B.; funding acquisition, G.L.B. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was financially supported by Yang Ji Chemical Company of South Korea.
Acknowledgments: A doctoral scholarship from the Research Association AGISMED to Y.-M. Na and N. Lebouvier
are gratefully acknowledged. We are also grateful to Le Floch (Yanikem s.a.r.l.) for their continued support.
A doctoral fellowship from the « Ministère de l’Enseignement Supérieur et de la Recherche » to M.-P. Lézé is
gratefully acknowledged. Molecular dynamics simulations were undertaken using the supercomputing facilities
at Cardiff University operated by Advanced Research Computing at Cardiff (ARCCA) on behalf of the Cardiff
Supercomputing Facility and the HPC Wales and Supercomputing Wales (SCW) projects. We acknowledge
the support of the latter, which is part-funded by the European Regional Development Fund (ERDF) via the
Welsh Government.
Conflicts of Interest: The authors declare no conflict of interest.
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