Synthesis and in vitro antifungal evaluation of 2-(2,4-difluorophenyl)-1- [(1H-indol-3-ylmethyl)methylamino]-3-(1H-1,2,4-triazol-1-yl)propan-2-ols

Article abstract of DOI:10.3109/14756366.2010.503607

We extended our previous studies based on the design of 1-[(1H-indol-5-ylmethyl)amino]-2-phenyl-3-(1H-1,2,4-triazol-1-yl)propan-2-ols as antifungal agents toward the identification of new indol-3-ylmethylamino derivatives. The majority of these compounds exhibited antifungal activity against a Candida albicans strain (minimum inhibitory concentrations ranging from 199.0 to 381.0ng/mL) suggesting an inhibition of 14α-demethylase by sterol analysis studies, but are weaker inhibitors compared to their indol-5-ylmethylamino analogs.

Full text of DOI:10.3109/14756366.2010.503607

Journal of Enzyme Inhibition and Medicinal Chemistry, 2011; 26(2): 261–269
© 2011 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2010.503607
ORIGINAL ARTICLE
Synthesis and in vitro antifungal evaluation of 2-(2,4-
difluorophenyl)-1-[(1H-indol-3-ylmethyl)methylamino]-3-
(1H-1,2,4-triazol-1-yl)propan-2-ols
Rémi Guillon1, Cédric Logé1, Fabrice Pagniez2, Véronique Ferchaud-Roucher4, Muriel Duflos1,
Carine Picot2, and Patrice Le Pape2,3
1Université de Nantes, Nantes Atlantique Universités, Département de Pharmacochimie, Cibles et Médicaments des
Infections, de l’Immunité et du Cancer, IICIMED-EA 1155, UFR Sciences Pharmaceutiques, Nantes, France, 2Université de
Nantes, Nantes Atlantique Universités, Département de Parasitologie et Mycologie Médicale, Cibles et Médicaments des
Infections, de l’Immunité et du Cancer, IICIMED-EA 1155, UFR Sciences Pharmaceutiques, Nantes, France, 3Laboratoire
de Parasitologie-Mycologie, CHU de Nantes, Hôtel Dieu, Nantes, France, and 4Centre de Recherche en Nutrition
Humaine, IRT-UN, Nantes, France
Abstract
We extended our previous studies based on the design of 1-[(1H-indol-5-ylmethyl)amino]-2-phenyl-3-(1H-1,2,4-
triazol-1-yl)propan-2-ols as antifungal agents toward the identification of new indol-3-ylmethylamino derivatives.
The majority of these compounds exhibited antifungal activity against a Candida albicans strain (minimum inhibitory
concentrations ranging from 199.0 to 381.0ng/mL) suggesting an inhibition of 14α-demethylase by sterol analysis
studies, but are weaker inhibitors compared to their indol-5-ylmethylamino analogs.
Keywords: Azoles, Candida albicans, antifungal agents, indoles
Introduction
membrane permeability and inhibition of fungal growth.
Voriconazole, itraconazole, and posaconazole have good
activity against most filamentous fungi, in contrast to
fluconazole, whose activity is largely limited to yeast2,3.
However, despite the arrival of new effective drugs such as
voriconazole and posaconazole, some therapeutic prob-
lems remain, in particular variable drug bioavailability,
some toxicity, lack of either oral or intravenous prepara-
tions, significant drug interactions for some agents and
development of resistance or breakthrough infections4,
thus creating a need for new antifungal agents.
CYP51 family members were known to be membrane
bound and much more difficult to crystallize. erefore,
the rational design of new antifungal agents was done
by molecular modelling studies. Several three-dimen-
sional (3D) models of CYP51 from Candida albicans and
During the last few decades, fungi have become a major
threat to many hospitalized patients particularly for se-
verely immunocompromised hosts who are highly sus-
ceptible to systemic fungal infections, most often caused
by Candida and Aspergillus species1. Currently, four main
chemical families which have globally three cellular tar-
gets in fungal cells are used for the treatment of invasive
fungal infections: polyenes, pyrimidine analogues, azoles,
and the echinocandins. Among them, azoles are the larg-
est class of antifungal agents in clinical use (Figure 1). ey
targetmainlyP450-dependentlanosterol14α-demethylase
(CYP51), encoded by the ERG11 gene, a key enzyme in the
fungal ergosterol biosynthesis. is inhibition leads to the
depletion of ergosterol in the cell membrane and accu-
mulation of toxic intermediate sterols, causing increased
Address for Correspondence: Cédric Logé, Université de Nantes, Nantes Atlantique Universités, Département de Pharmacochimie, Cibles
et Médicaments des Infections, de l’Immunité et du Cancer, IICIMED-EA 1155, UFR Sciences Pharmaceutiques, 1 rue Gaston Veil, Nantes
F-44035 Cedex 1, France. Tel.: +33(0)240 411 108. Fax: +33(0)240 412 876. E-mail: cedric.loge@univ-nantes.fr
(Received 18 February 2010; revised 21 June 2010; accepted 22 June 2010)
261

262 R. Guillon et al.
Aspergillus fumigatus were constructed on the basis of the
crystal coordinates of water-soluble CYP51 family member
from Mycobacterium tuberculosis5,6 and interactions with
azoles have been explored by flexible molecular docking.
In particular, the receptor-based pharmacophore model
published by Sheng et al. was established to guide the
rational optimization of the azole antifungal agents within
the channel 2 oriented to the FG loop and highlighted the
importance of Tyr118 and Ser378 residues of C. albicans
sterol 14α-demethylase P450 in the stabilization of the
inhibitors7. Tyr118, a highly conserved residue in CYP51
family, may be a potential site for the π–π interaction with
the inhibitor, while Ser378, a conserved amino acid across
the fungal CYP51 enzymes, could form specific hydrogen-
bonding interaction. e role of the Tyr118 residue in de-
termining azole susceptibility was also validated by site-
directed mutagenesis8,9.
agents, based on an indole scaffold (I, Scheme 1). ese
indol-5-ylmethylamino compounds exhibited potent
minimum inhibitory concentrations (MICs; < 65ng/mL)
against a C. albicans strain whereas no activity was demon-
strated for the A. fumigatus strain10. Interestingly, the best
compound (X=F, R=H, R1 = Boc, R3 =H, MIC80 =3.0ng/mL)
retained all the main characteristics mentioned above (an
H-bond acceptor substituent positioned on an aromatic
moiety).
In this paper, considering necessity to develop azoles
with improved profile and due to a limited range of
variations at the indole moiety, we extended the struc-
tural modification to a series of indol-3-ylmethylamino
analogs (II, Scheme 1). We focussed first on the intro-
duction of a N-methyl group in the linker since in a
precedent series an emergence of activity was observed
on a A. fumigatus strain11, but also on the effect of the
introduction of electronegative functions such as a N-
Boc protective group, a methylenedioxy substituent and
We reported earlier the synthesis and the structure-
activity relationships (SARs) of a novel series of antifungal
O
O
O
OH
N
N
N
N
N
N
N
N
N
N
N
O
N
N
N
Cl
F
F
Fluconazole
Cl
O
Itraconazole
O
CH3
O
OH
N
N
N
N
N
N
N
N
N
N
N
N
N
OH
F
F
F
F
F
Voriconazole
Posaconazole
Figure 1. Structures of some triazoles used in clinic.
N
N
R5
N
N
N
N
R3
R6
OH
OH
N
N
*
*
F
CH3
X
R
N
N
R1
R1
II
I
F
X
R1 = H or Boc
X = Cl or F
R5, R6 = OCH2O
R = H or CH3
R5 = H, R6 = H or F
R1 = H, Boc or Benzoyl
R3 = H or COCF3
Scheme 1. General structures of synthesized compounds.
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis of new antifungual agents 263
a fluorine atom, at positions 1, 5, and/or 6 of the indole
ring. We hypothesized that the use of these easily avail-
able substituents should alter the physical properties
and binding characteristics of the studied compounds
by acting as appropriate hydrogen-bond acceptors into
the active site.
1H-indole-3-carbaldehyde (2b). Yield 87%; white
powder; mp = 195–196°C; IR (KBr) cm−1: 3167 (N-H),
1
1634 (C=O), 1463, 1514 (C=C); H NMR (DMSO-d6): δ
ppm 7.25–7.29 (m, 2H), 7.54 (d, 1H, 3J = 7.0 Hz), 8.12 (dd,
1H, 3J = 8.2 Hz, 4J = 1.8 Hz), 8.32 (s, 1H), 9.97 (s, 1H), 12.17
(bs, 1H).
6-fluoro-1H-indole-3-carbaldehyde (2c). Yield 85%;
light brown powder; mp = 173–174°C; IR (KBr) cm−1:
3463 (N-H), 1638 (C=O), 1444, 1530 (C=C), 1148 (C-F);
1H NMR (DMSO-d6): δ ppm 7.08–7.17 (m, 1H), 7.35 (dd,
1H, 3JH-F = 9.7 Hz, 4JH-H = 2.1 Hz), 8.10 (dd, 1H, 3JH-H = 8.5 Hz,
4JH-F = 5.5 Hz), 8.34 (s, 1H), 9.95 (s, 1H), 12.21 (bs, 1H).
Materials and methods
Chemistry
Instrumentation. Melting points were determined using
an Electrothermal IA9300 digital melting point apparatus
and reported uncorrected. Infrared (IR) spectra were ob-
tained in KBr pellets using a Perkin–Elmer Paragon FTIR
1000 PC spectrometer (Beaconsfield Bucks, England). Only
the most significant absorption bands have been reported.
1H and 13C nuclear magnetic resonance (NMR) spectra
were recorded on a Bruker AC250 (250MHz) or on Bruker
Avance 400 spectrometer (400MHz). Chemical shifts are
expressed as δvalues (ppm) relative to tetramethylsilane as
internal standard (in NMR description, s=singlet, d=dou-
blet, t=triplet, m=multiplet and b=broad). Coupling con-
stants J are given in Hz. Electrospray mass spectrometric
analysis was performed on an Esquire-LC Ion Trap System
mass spectrometer. Elemental analyses were performed by
CEISAM UMR CNRS 6230 Laboratory (Nantes, France) and
found to be within a range of 0.4% of theoretical values.
All reactions were monitored by thin-layer chromatog-
raphy using 0.2 mm-thick silica gel plates 60F-254 (5735
Merck). Column chromatography was carried out using
silica gel 60 (70–230 Mesh, ASTM, Merck). Chemicals and
solvents used were commercially available. Compound 4
was prepared according to reported method10.
General procedure for the preparation of compounds 3a-c
To a solution of an appropriate indolecarbaldehyde 2a-c
(1.32 mmol) in 4 mL of acetonitrile was added di-tert-
butyl dicarbonate (374 mg, 1.71 mmol) followed by ad-
dition of 4-dimethylaminopyridine (DMAP; 16 mg, 0.13
mmol). e mixture was stirred at room temperature for
2 h. Water was added (20 mL) and product was extracted
with dichloromethane (3 × 20 mL). Organics layers were
combined, dried over anhydrous sodium sulfate, filtered
and concentrated in vacuo.
5-(tert-butoxycarbonyl)-1,3-dioxolo[4,5-f ]indole-
7-carbaldehyde (3a). Yield 94%; light brown powder;
mp = 149–150°C; IR (KBr) cm−1: 1740 (C=O), 1670 (C=O),
1563 (C=C); 1H NMR (DMSO-d6): δ ppm 1.68 (s, 9H), 6.13
(s, 2H), 7.54 (s, 1H), 7.60 (s, 1H), 8.52 (s, 1H), 10.02 (s,
1H).
1-(tert-butoxycarbonyl)-1H-indole-3-carbaldehyde
(3b). Yield 98%; white powder; mp = 125–126°C; IR (KBr)
cm−1: 1745 (C=O), 1679 (C=O), 1454, 1559 (C=C), 1363
1
(C-N), 1154 (C-O); H NMR (DMSO-d6): δ ppm 1.71 (s,
9H), 7.39–7.53 (m, 2H), 8.18 (m, 2H), 8.71 (s, 1H), 10.12
General procedure for the preparation of compounds 2a-c
To a three-necked round flask was introduced anhydrous
N,N-dimethylformamide (1 mL, 12.92 mmol) at 0°C
followed by slow addition of phosphorus oxychloride
(0.190 mL, 2.05 mmol). Solution was mixed at 0°C for
40 min. A solution of an appropriate indole 1a-c (1.86
mmol) in 1 mL of N,N-dimethylformamide was slowly
added maintaining the temperature below 10°C. e mix-
ture was stirred at this temperature for 40 min and then
at 35°C for additional 40 min. Pilled ice was added to the
flask and a solution of sodium hydroxide (931mg, 23.27
mmol dissolved in 2.5 mL of water) was introduced drop-
wise. Solution was vigorously stirred during the addition,
then heated to 100°C for 30 min and left to reach room
temperature. Mixture was diluted with water (100 mL)
and product was extracted with ethyl acetate (3 × 30 mL).
Organics layers were combined, dried over anhydrous
sodium sulphate, filtered and concentrated in vacuo.
5H-1,3-dioxolo[4,5-f]indole-7-carbaldehyde (2a). Yield
80%; light brown powder; mp = 217–218°C; IR (KBr) cm−1:
3195 (N-H), 1632 (C=O), 1442 (C=C), 1179 (C-O); 1H NMR
[dimethylsulfoxide d6 (DMSO-d6)]: δ ppm 6.05 (s, 2H),
(s, 1H).
1-(tert-butoxycarbonyl)-6-fluoro-1H-indole-3-
carbaldehyde (3c). Yield 99%; light yellow powder;
mp = 136–137°C; IR (KBr) cm−1: 1746 (C=O), 1670 (C=O),
1554 (C=C), 1157 (C-F); 1H NMR (DMSO-d6): δ ppm 1.70
3
(s, 9H), 7.27–7.35 (m, 1H), 7.86 (dd, 1H, JH-F = 10.3 Hz,
4JH-H = 2.1 Hz), 8.17 (dd, 1H, JH-H = 8.8 Hz, JH-F = 5.8 Hz),
3
4
8.71 (s, 1H), 10.10 (s, 1H).
General procedure for the preparation of compounds 5a-c
To a solution of 4 (278 mg, 1.04 mmol) in 5 mL of metha-
nol and 0.1 mL of acetic acid was added the correspond-
ing N-Boc indoles 3a-c (1.04 mmol) at room temperature
under argon. en sodium cyanoborohydride (78 mg,
1.24 mmol) was added and the mixture was stirred for
24 h. Mixture was diluted with water (20 mL) and product
was extracted with dichloromethane (3 × 20 mL). Organic
layers were combined, washed with saturated sodium
bicarbonate (20 mL), dried over anhydrous sodium sul-
fate, filtered and concentrated in vacuo. e residue was
purified on silica gel column chromatography (dichlo-
romethane and dichloromethane/ ethanol, 99:1).
3
7.07 (s, 1H), 7.50 (s, 1H), 8.11 (d, 1H, J= 2.7 Hz), 9.87 (s,
1H), 11.97 (bs, 1H).
1-({[5-(tert-butoxycarbonyl)-1,3-dioxolo[4,5-f]indol-
7-yl]methyl}methylamino)-2-(2,4-difluorophenyl)-3
© 2011 Informa UK, Ltd.

264 R. Guillon et al.
-(1H-1,2,4-triazol-1-yl)propan-2-ol (5a). Yield 59%;
white powder; mp = 108–109°C; IR (KBr) cm−1: 3445
(O-H), 1729 (C=O), 1406, 1466, 1620 (C=C and C=N),
1288 (C-O), 1163 (C-F); 1H NMR (DMSO-d6): δ ppm 1.64
(s, 9H), 2.11 (s, 3H), 2.69 (d, 1H, 2J = 13.8 Hz), 3.14 (d, 1H,
2J = 13.8 Hz), 3.45 (d, 1H, 2J = 13.2 Hz), 3.64 (d, 1H, 2J = 13.2
not visible); MS (ES+) m/z 516.1 (M + H)+. Anal. Calcd.
for C26H28F3N5O3: C, 60.58; H, 5.47; N, 13.58; found: C,
60.12; H, 5.48; N, 13.23%.
General procedure for the preparation of compounds 6a-c
To a solution of an appropriate azole 5a-c (0.54 mmol)
in 10 mL of dichloromethane was added zinc bromide
(724 mg, 3.21 mmol) at room temperature under argon.
e solution was stirred 24 h. en water (55 mL) was
added and the mixture was stirred for 2h. Product was
extracted with dichloromethane (3 × 30 mL), organic
layers were combined, dried over anhydrous sodium
sulfate, filtered and concentrated in vacuo. e residue
was purified on silica gel column chromatography (di-
chloromethane and dichloromethane/ethanol, 99:1).
1-{[(5H-1,3-dioxolo[4,5-f ]indol-7-yl)methyl]
methylamino}-2-(2,4-difluorophenyl)-3-(1H-1,2,4-
triazol-1-yl)propan-2-ol (6a). Yield 21%; grey powder;
mp = 137–138°C; IR (KBr) cm−1: 3422 (O-H and N-H),
1472, 1503, 1619 (C=C and C=N), 1312 (C-O), 1185
2
2
Hz), 4.48 (d, 1H, J = 14.0 Hz), 4.54 (d, 1H, J = 14.0 Hz),
3
5.79 (s, 1H), 6.04 (d, 2H, J = 3.6 Hz), 6.69 (s, 1H), 6.96
3
4
(ddd, 1H, JH-F = 3JH-H = 8.4 Hz, JH-H = 2.4 Hz), 7.08 (ddd,
3
4
1H, JH-F = 3J′H-F = 9.2 Hz, JH-H = 2.4 Hz), 7.30 (s, 1H), 7.43
(ddd, 1H, 3JH-H = 8.4 Hz, 4JH-F = 4J′H-F = 6.8 Hz), 7.50 (s, 1H),
7.79 (s, 1H), 8.28 (s, 1H); 13C NMR (DMSO-d6): δ ppm
27.8 (3C, CH3), 43.9 (CH3), 53.7 (CH2), 56.2 (CH2), 63.1
(CH2), 75.3 (Cq), 83.8 (Cq), 96.0 (CH), 99.3 (CH), 101.2
(CH2), 103.8 (CH), 110.9 (CH), 118.4 (CH), 123.1 (Cq),
124.2 (Cq), 126.3 (Cq), 130.0 (CH), 130.1 (Cq), 143.9 (Cq),
145.0 (CH), 145.8 (Cq), 149.1 (C=O), 150.6 (CH), (CF not
visible); MS (ES+) m/z 542.3 (M + H)+. Anal. Calcd. for
C27H29F2N5O5: C, 59.88; H, 5.40; N, 12.93; found: C, 59.69;
H, 5.45; N, 12.67%.
1
1-{[(1-tert-butoxycarbonylindol-3-yl)methyl]
methylamino}-2-(2,4-difluorophenyl)-3-(1H-1,2,4-
triazol-1-yl)propan-2-ol (5b). Yield 47%; white powder;
mp = 120–121°C; IR (KBr) cm−1: 3446 (O-H), 1733 (C=O),
1451, 1497, 1621 (C=C and C=N), 1262 (C-O), 1154
(C-F); H NMR (DMSO-d6): δ ppm 2.09 (s, 3H), 2.78 (d,
2
2
1H, J = 13.7 Hz), 3.04 (d, 1H, J = 13.7 Hz), 3.53 (d, 1H,
2J = 13.2 Hz), 3.60 (d, 1H, 2J = 13.2 Hz), 4.48 (d, 1H, 2J = 14.6
Hz), 4.53 (d, 1H, J = 14.6 Hz), 5.72 (s, 1H), 5.93 (s, 2H),
2
6.71 (s, 1H), 6.85 (s, 1H), 6.99 (m, 2H), 7.13 (ddd, 1H,
1
(C-F); H NMR (DMSO-d6): δ ppm 1.66 (s, 9H), 2.12 (s,
3JH-F = 3J′H-F = 9.2 Hz, 4JH-H = 2.4 Hz), 7.43 (ddd, 1H, 3JH-H = 8.4
2
2
4
3H), 2.75 (d, 1H, J = 13.6 Hz), 3.15 (d, 1H, J = 13.6 Hz),
Hz, JH-F = 4J′H-F = 6.8 Hz), 7.77 (s, 1H), 8.29 (s, 1H), 10.67
2
2
3.56 (d, 1H, J = 13.4 Hz), 3.71 (d, 1H, J = 13.4 Hz), 4.49
(bs, 1H); 13C NMR (DMSO-d6): δ ppm 43.7 (CH3), 54.3
(CH2), 56.2 (CH2), 62.6 (CH2), 74.9 (Cq), 92.1 (CH), 97.8
(CH), 100.1 (CH2), 103.7 (CH), 110.5 (CH), 111.9 (Cq),
121.3 (Cq), 123.2 (CH), 126.4 (Cq), 130.1 (CH), 131.2 (Cq),
141.8 (Cq), 143.9 (Cq), 144.3 (CH), 150.6 (CH), (CF not
visible); MS (ES+) m/z 442.2 (M + H)+. Anal. Calcd. for
C22H21F2N5O3: C, 59.86; H, 4.80; N, 15.86; found: C, 59.59;
H, 4.79; N, 15.58%.
2
2
(d, 1H, J = 14.2 Hz), 4.59 (d, 1H, J = 14.2 Hz), 5.78 (s,
1H), 6.97 (ddd, 1H, JH-F = 3JH-H = 8.4 Hz, JH-H = 2.4 Hz),
3
4
7.11–7.16 (m, 2H), 7.28–7.32 (m, 2H), 7.40–7.46 (m, 2H),
3
7.79 (s, 1H), 8.02 (d, 1H, J = 8.4 Hz), 8.29 (s, 1H); 13C
NMR (DMSO-d6): δ ppm 27.9 (3C, CH3), 43.9 (CH3), 53.5
(CH2), 56.2 (CH2), 63.2 (CH2), 75.2 (Cq), 83.8 (Cq), 103.8
(CH), 110.8 (CH), 114.7 (CH), 118.2 (Cq), 120.2 (CH),
122.4 (CH), 124.4 (CH), 124.5 (CH), 126.3 (Cq), 130.1
(Cq), 130.2 (CH), 135.1 (Cq), 145.0 (CH), 149.2 (C=O),
150.6 (CH), (CF not visible); MS (ES+) m/z 498.1 (M +
H)+. Anal. Calcd. for C26H29F2N5O3: C, 62.77; H, 5.88; N,
14.08; found: C, 62.62; H, 5.91; N, 13.79%.
1-{[(1-tert-butoxycarbonyl-6-fluoroindol-3-yl)meth-
yl]methylamino}-2-(2,4-difluorophenyl)-3-(1H-1,2,4-
triazol-1-yl)propan-2-ol (5c). Yield 68%; white powder;
mp = 149–150°C; IR (KBr) cm−1: 3444 (O-H), 1734 (C=O),
1494, 1616 (C=C and C=N), 1274 (C-O), 1157 (C-F);
1H NMR (DMSO-d6): δ ppm 1.66 (s, 9H), 2.12 (s, 3H),
2-(2,4-difluorophenyl)-1-[(1H-indol-3-ylmethyl)
methylamino]-3-(1H-1,2,4-triazol-1-yl)-propan-2-ol
(6b). Yield 64%; white powder; mp = 73–74°C; IR (KBr)
cm−1: 3435 (O-H and N-H), 1492, 1625 (C=C and C=N),
1183 (C-F); 1H NMR (DMSO-d6): δ ppm 2.11 (s, 3H), 2.84
(d, 1H, 2J = 13.6 Hz), 3.04 (d, 1H, 2J = 13.6 Hz), 3.64 (d, 1H,
2J = 13.2 Hz), 3.68 (d, 1H, 2J = 13.2 Hz), 4.48 (d, 1H, 2J = 14.3
2
Hz), 4.53 (d, 1H, J = 14.3 Hz), 5.72 (s, 1H), 6.93 (dd, 1H,
3J = 3J′= 7.6Hz),6.98(ddd,1H,3JH-F = 3JH-H = 8.4Hz,4JH-H = 2.4
Hz), 7.07 (dd, 1H, 3J = 3J′= 7.6 Hz), 7.13–7.19 (m, 2H), 7.33
(m, 2H), 7.43 (ddd, 1H, 3JH-H = 8.4 Hz, 4JH-F = 4J′H-F = 6.8 Hz),
7.76 (s, 1H), 8.28 (s, 1H), 10.89 (bs, 1H); 13C NMR (DMSO-
d6): δ ppm 43.7 (CH3), 54.2 (CH2), 56.2 (CH2), 63.0 (CH2),
74.9 (Cq), 103.9 (CH), 110.8 (CH), 111.4 (CH), 111.6 (Cq),
118.5 (CH), 119.1 (CH), 121.1 (CH), 124.8 (CH), 126.7
(Cq), 127.5 (Cq), 130.0 (CH), 136.5 (Cq), 145.0 (CH), 150.6
(CH), (CF not visible); MS (ES+) m/z 398.3 (M + H)+.
Anal. Calcd. for C21H21F2N5O: C, 63.47; H, 5.33; N, 17.62;
found: C, 63.25; H, 5.48; N, 17.48%.
2
2
2.73 (d, 1H, J = 13.6 Hz), 3.15 (d, 1H, J = 13.6 Hz), 3.54
2
2
(d, 1H, J = 13.4 Hz), 3.70 (d, 1H, J = 13.4 Hz), 4.48 (d,
2
2
1H, J = 14.3 Hz), 4.54 (d, 1H, J = 14.3 Hz), 5.79 (s, 1H),
6.97–7.02 (m, 2H), 7.11 (ddd, 1H, JH-F = 3J′H-F = 9.2 Hz,
3
4JH-H = 2.4 Hz), 7.28 (dd, 1H, JH-H = 8.8 Hz, JH-F = 5.6 Hz),
7.40–7.46 (m, 2H), 7.73 (dd, 1H, 3JH-F = 10.4 Hz, 4JH-H = 1.6
Hz), 7.79 (s, 1H), 8.29 (s, 1H); 13C NMR (DMSO-d6): δ
ppm 27.8 (3C, CH3), 44.0 (CH3), 53.5 (CH2), 56.2 (CH2),
63.1 (CH2), 75.3 (Cq), 84.3 (Cq), 101.6 (CH), 103.8 (CH),
110.3 (CH), 110.9 (CH), 118.2 (Cq), 121.4 (CH), 124.9
(CH), 126.3 (Cq), 126.7 (Cq), 130.1 (CH), 135.2 (Cq),
145.0 (CH), 149.0 (C=O), 150.6 (CH), 160.2 (CF), (CF
3
4
2-(2,4-difluorophenyl)-1-[(6-fluoro-1H-indol-3-ylm-
ethyl)methylamino]-3-(1H-1,2,4-triazol-1-yl)-propan-2-ol
(6c). Yield 76%; white powder; mp=77–78°C; IR (KBr)
cm−1: 3419 (O-H and N-H), 1456, 1503, 1622 (C=C and
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis of new antifungual agents 265
C=N), 1275 (C-N), 1137 (C-F); 1H NMR (DMSO-d6): δ ppm
to 100mL with 100% ethanol). en suspension was vor-
texed for 1min and incubated at 80°C for 60min. Sterols
were then extracted by addition of a mixture of 1mL of
distilled water and 4mL of n-hexane (Merck, Darmstadt,
Germany). Hexane extract was then evaporated.
2
2
2.11 (s, 3H), 2.80 (d, 1H, J=13.6 Hz), 3.05 (d, 1H, J=13.6
Hz), 3.60 (d, 1H, 2J=13.2 Hz), 3.66 (d, 1H, 2J=13.2 Hz), 4.47
2
2
(d, 1H, J=14.0 Hz), 4.53 (d, 1H, J=14.0 Hz), 5.75 (s, 1H),
6.77 (m, 1H), 6.98 (ddd, 1H, JH-F = 3JH-H =8.4 Hz, JH-H =2.4
3
4
3
4
Samples were derivatized with 100 µL of silylating
mixture (Fluka, Saint Quentin Fallavier, France) at room
temperature for 30 min, evaporated and diluted in 500
µL of n-hexane. Two microliters were injected into a
gas chromatograph (model 6890N, Agilent Technolo-
gies, Palo Alto, CA) coupled with a quadrupole mass
spectrometer (5973i, Agilent Technologies, Palo Alto,
CA). Analyses were carried out in a splitless mode with
helium as carrier gas (constant rate of 1.2 mL/min) and
the injector temperature was 250°C. e transfer line be-
tween gas chromatograph and mass spectrometer was
operated at 290°C and the EI source was held at 280°C.
e gas chromatography (GC) capillary column was an
HP-5MS (30 m × 0.25 mm ID, 0.25 µm film thickness,
Agilent Technologies). e GC oven was programmed
as follows: initial temperature 150°C, hold for 0.5 min,
ramp to 280°C at 40°C/min, and ramp to 300°C at 5°C/
min, hold for 6 min. Sterols of interest were identified
by their mass spectrum. In view to study the treatment
influence on sterol abundance, the area under curve
(AUC) of each peak was used to calculate a ratio: sterol
AUC/sum of sterols AUC.
Hz), 7.11 (dd, 1H, JH-F =10.4 Hz, JH-H =2.4 Hz), 7.13–7.18
(m, 2H), 7.26 (dd, 1H, 3JH-H =8.8 Hz, 4JH-F =5.6 Hz), 7.43 (ddd,
1H, JH-H =8.4 Hz, JH-F = 4J′H-F =6.8 Hz), 7.76 (s, 1H), 8.29 (s,
1H), 10.95 (bs, 1H); 13C NMR (DMSO-d6): δ ppm 43.7
(CH3), 54.1 (CH2), 56.2 (CH2), 63.0 (CH2), 75.0 (Cq), 97.3
(CH), 103.7 (CH), 106.8 (CH), 110.8 (CH), 111.9 (Cq), 120.2
(CH), 124.3 (Cq), 125.3 (CH), 126.5 (Cq), 130.1 (CH), 136.3
(Cq), 145.0 (CH), 150.6 (CH), (CF not visible); MS (ES+)
m/z 416.4 (M + H)+. Anal. Calcd. for C21H20F3N5O: C, 60.72;
H, 4.85; N, 16.86; found: C, 60.42; H, 5.01; N, 16.56%.
3
4
Antifungal activity
All these compounds were screened for their antifungal
activity against C. albicans CA98001 and A. fumigatus
AF98003 strains. Inhibition growth was measured as pre-
viously described12. Fluconazole and itraconazole were
used as positive controls. e MIC80 values (in ng/mL)
are presented in Table 1.
Sterol extraction and analysis
To study sterol synthesis, C. albicans CA98001 cells were
incubated in 50mL Sabouraud broth medium (Sigma–
Aldrich, Munich, Germany) during 18h at 35°C with stir-
ring. Compound 5c was introduced into culture medium
before incubation. Cells were collected by centrifugation
at 1500g. Pellet was suspended in 3mL of saponification
medium (25g of KOH, 36mL of distilled water and brought
Homology model of CYP51-C. albicans
Molecular modelling studies were performed using
SYBYL software version 8.013 running on a dell precision
T3400 workstation. e structure of CYP51 from M.
Table 1. In vitro antifungal activity of indol-3-ylmethylamino derivatives.
N
R5
N
N
OH
R6
N
*
F
CH3
N
R1
5, 6
F
MIC80 values (ng/mL)a
Aspergillus
Candida albicans
fumigatus
AF98003
R1
R5
R6
Compounds
CA98001
5a
Boc
Boc
Boc
H
O-CH2-O
> 541.0
na
5b
H
H
H
F
373.0 ( 10.0)
381.0 ( 51.0)
264.0 ( 17.0)
258.0 ( 4.00)
199.0 ( 29.0)
190.0 ( 6.0)
—
na
5c
na
6a
O-CH2-O
na
6b
H
H
H
H
F
na
6c
H
na
—
Fluconazole
Itraconazole
420.0 ( 40.0)
MIC, minimum inhibitory concentration; na, not active.
aValues are means of triplicate, standard deviation is given in parentheses (MIC80 > 30000.0ng/mL).
© 2011 Informa UK, Ltd.

266 R. Guillon et al.
tuberculosis complexed with fluconazole (PDB code,
1EA1)14 was used as the template for the construc-
tion of our house-made model of CYP51-C. albicans15.
Multiple alignment of the CYP51-M. tuberculosis se-
quence with those of CYP51-C. albicans (PIR code,
P10613) and CYP51-A. fumigatus (PIR code, Q9P8R0)
was performed using ClustalW16. is alignment was
further checked by comparing a secondary structure
elements prediction for CYP51-C. albicans, obtained
through the PSIPRED protein structure prediction
server17, with the experimental secondary structure
assignments for CYP51-M. tuberculosis deduced from
the PDB file. e 3D model of CYP51-C. albicans was
then constructed by the Nest program from the protein
structure modelling package JACKAL18. e resulting
model was further subjected to an energy minimiza-
tion using Powell’s method available in Maximin2
procedure with the MMFF94 force field and a dielec-
tric constant of 4.0 until the gradient value reached 0.1
kcal/mol/Å. Energy minimization was started with the
core side chains, then the core main chains. During
the optimization procedure, the model quality was as-
sessed periodically by Verify-3D19 and Ramachandran
plots20. If present, improper geometries were manually
corrected and the structure minimized again with the
above procedure. Haeme and fluconazole extracted
from the CYP51-M. tuberculosis structure was added
to the model, and residues neighbouring haeme were
minimized to avoid steric conflicts. As in the crystal
structure of CYP51 from M. tuberculosis, the entrance
of channel 2 is closed from the surface by interaction
between the A′ helix and FG loop14, modifications of
some residues were applied to leave enough space in
the pocket for the binding of azoles with extended side
chains, according to Xiao et al5.
in three steps from heliotropin22. Vilsmeier–Haack re-
action under standard conditions followed by N-Boc
protection23 with di-tert-butyl dicarbonate and 4-dim-
ethylaminopyridine (DMAP) in acetonitrile afforded
derivatives 3a-c. Products 5a-c were synthesized from
previously described10 key intermediate 4 by reductive
amination with 3a-c. Finally, removal of the Boc protec-
tive group with zinc bromide in methylene chloride led
to compounds 6a-c24.
Antifungal activity
On C. albicans strain, except for compound 5a, our
compounds exhibited high antifungal activity with MIC
values ranging from 199.0 to 381.0 ng/mL, comparable
to that of fluconazole (Table 1). Compound 5a, bear-
ing a N-Boc protective group on the indole moiety and
an additional bulky methylenedioxy group showed a
decrease in activity (MIC80 > 541.0 ng/mL) leading to
the assumption that steric factors could affect the bio-
logical result. It is noteworthy that compared to their
previously described indol-5-ylmethylamino analogs
(I, Scheme 1)10, both compounds 5b and 6b are weaker
inhibitors with MIC values approximatively 7-fold less
active, confirming that isomerization from the 5- to the
3-position of the indole ring is more sensitive to steric
and/or electrostatic effects. Furthermore, the degree of
inhibition was not significantly influenced neither by
compound 6a, or compound 6c, with only H-bond ac-
ceptor substituents at positions 5 and/or 6 of the indole
ring. Once again, all these compounds were ineffective
against the A. fumigatus strain.
Sterol analysis
In view to confirm the mechanism of action of this
series of compounds, we have investigated the inhi-
bition of ergosterol biosynthesis after treatment by
compound 5c. As shown in Table 2, compound 5c
strongly inhibited ergosterol biosynthesis while lanos-
terol accumulation did occurred. At concentrations of
0.25 and 0.5 µg/mL, surrounding the MIC (0.381 µg/
mL), this effect was maximal. About 50% of ergosterol
biosynthesis inhibition was obtained with a lower con-
centration of 0.125 µg/mL. These results show that in-
hibition of ergosterol biosynthesis by compound 5c is
dose-dependent and reached his maximum at the MIC
concentration. Otherwise, methylated sterols at the
C14 position did also appeared (14-methylfecosterol,
14-methylepisterol, eburicol and 14-methyl-3,6-diol)
in a dose-dependent manner. Accumulation of lanos-
terol and production of 14-methylated sterols are typi-
cally a result of a 14α-demethylase blockage by triaz-
ole derivatives. Depletion of ergosterol, accumulation
of lanosterol and production of 14-methylated sterols
lead to membrane instability and cell death. Thus,
antifungal activity of compound 5c is due to inhibi-
tion of ergosterol biosynthesis by inhibition of 14α-
demethylase, suggesting that all these compounds act
in a similar fashion.
Docking of azoles
e 3D structures of azoles were constructed using the
standard sketch procedure of SYBYL and their geom-
etries were subsequently optimized using the Tripos
force field including the electrostatic term calculated
from Gasteiger and Huckel atomic charges. Powell’s
method available in Maximin2 procedure was used
for energy minimization until the gradient value was
smaller than 0.001 kcal/mol/Å. Flexible docking of
azoles into the enzyme active site was performed us-
ing GOLD software21. A distance constraint was applied
from N-4 of the triazole ring to the haeme iron (2.0 < d
< 2.4 Å). For each compound, the most stable docking
model was selected according to the best scored confor-
mation predicted by the GoldScore scoring function.
Results and discussion
Chemistry
Scheme 2 outlines the synthesis of compounds 5a-c and
6a-c. Indoles 1b and 1c were commercially available
whereas 5H-1,3-dioxolo[4,5-f]indole 1a was prepared
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis of new antifungual agents 267
© 2011 Informa UK, Ltd.

268 R. Guillon et al.
undergo optimal stacking interactions with the phenol
moiety of key residue Tyr118 and/or the class-specific
residue Phe380.
SAR study
In attempts to understand the observed SARs, we
performed the docking of one of the most active
compound (6b, MIC = 258.0 ng/mL) in our model of
CYP51-C. albicans (Figure 2). Interestingly, the indole
nitrogen is in close proximity to the backbone and the
propionate side chain of the haeme, suggesting that
sterically encumbering compounds would be expected
to encounter unfavourable steric interactions with the
enzyme. e reported weak activities for compound 5a
bearing a N-Boc protective group at the 1-position of
the indole ring are consistent with that expectation. Al-
though compounds 5b-c share the same group, we can
speculate that the lack of bulky substituents at positions
5- and 6- of the indole ring allows greater flexibility
into the active site. Moreover, the decrease of activity
observed for compound 6b compared to its indol-5-
ylmethylamino analog could be explained by the abil-
ity of the benzo and pyrrolo portions of indole ring to
e complete lack of activity against A. fumigatus
strain may be due to steric hindrance within the active
site induced by the bulky indole ring or a lack of per-
meability of these compounds inside this filamentous
pathogen. However, only results on the isolated CYP51
could confirm our hypotheses. In addition, despite an
explicit information about the enzyme binding site of
pathogenic fungi, the successful design of an optimum
inhibitor using 3D models of CYP51 seems to be dif-
ficult. Recently, many crystal structures of CYP51 from
human and Trypanosomatidae in complex with azole
antifungal inhibitors revealed profound conforma-
tional changes both in organization of the active site
cavity and in the substrate access channel location
which is different from that observed in M. tuberculo-
sis CYP5125,26. erefore, particular attention must be
given to the choice of the target in a structure-based
design strategy, keeping in mind as the ideal azole an-
tifungals are those which react strongly with fungal cy-
tochrome P450s and have weak or no activities against
the mammalian enzyme variants.
Table 2. Effect of compound 5c on the sterol composition of
Candida albicans CA98001.
5c (µg/mL)
Sterols
control
1.8
0.125
28.4
0.25
42.4
35.9
0.5
44.6
39.0
Lanosterol
Eburicol
27.9
Conclusion
Zymosterol
traces
11.2
We studied the utility of original indole-containing
azole compounds as antifungal agents. ese molecules
exhibited antifungal activity against a C. albicans strain
by inhibition of 14α-demethylase but were ineffective
against a A. fumigatus strain. However, in order to de-
velop broad-spectrum compounds, the choice of a side
chain bearing a bulky and rigid indole moiety attached
to the C-3 of antifungal agents does not seem to be
useful scaffold for further optimization.
Fecosterol
14-Methylfecosterol
Episterol
0.5
2.7
1.3
1.5
1.4
14-Methylepisterol
14-Methyl-3,6-diol
Ergosterol
5.2
0.1
7.5
0.1
7.3
85.6
40.5
15.1
Sterols of interest were identified by their mass spectrum. e
area under curve (AUC) of each peak was used to calculate a
ratio: sterol AUC/sum of sterols AUC.
Cys470
Ser378
Hip377
Phe145
Tyr118
Tyr64
Phe380
Phe228
Phe126
Leu121
Figure 2. Proposed binding solution of compound (S)-6b (magenta) in the supposed active site pocket (channel 2) of CYP51-Candida
albicans. Hip377 is the protonated form of histidine residue.
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis of new antifungual agents 269
3-(1H-1,2,4-triazol-1-yl)propan-2-ols as antifungal agents. Bioorg
Med Chem Lett 2008; 18:1820–1824.
Declaration of interest
12. Pagniez F, Le Pape P. New fluorometric screening test for possible
antifungal drugs. J Mycol Med 2001;11:73–78.
e authors report no conflicts of interest.
13. Tripos International. SYBYL 8.0. 1699 South Hanley Road, St.
Louis, MO 63144, USA.
14. Podust LM, Poulos TL, Waterman MR. Crystal structure
of cytochrome P450 14α-sterol demethylase (CYP51) from
Mycobacterium tuberculosis in complex with azole inhibitors. Proc
Natl Acad Sci USA 2001;98:3068–73.
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Universités, France, October 2007.
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17. Jones DT. Protein secondary structure prediction based on
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18. Petrey D, Xiang Z, Tang CL, Xie L, Gimpelev M, Mitros T, Soto CS,
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