New azole antifungals with a fused triazinone scaffold

Article abstract of DOI:10.1016/j.ejmech.2020.112082

We identified a new series of azole antifungal agents bearing a pyrrolotriazinone scaffold. These compounds exhibited a broad in vitro antifungal activity against pathogenic Candida spp. (fluconazole-susceptible and fluconazole-resistant) and were 10- to 100-fold more active than voriconazole against two Candida albicans isolates with known mechanisms of azole resistance (overexpression of efflux pumps and/or specific point substitutions in the Erg11p/CYP51 enzyme). Our lead compound 12 also displayed promising in vitro antifungal activity against some filamentous fungi such as Aspergillus fumigatus and the zygomycetes Rhizopus oryzae and Mucor circinelloides and an in vivo efficiency against two murine models of lethal systemic infections caused by Candida albicans.

Full text of DOI:10.1016/j.ejmech.2020.112082

Journal Pre-proof
New azole antifungals with a fused triazinone scaffold
David Montoir, Rémi Guillon, Sophie Gazzola, Isabelle Ourliac-Garnier, Kossi
Efouako Soklou, Alain Tonnerre, Carine Picot, Aurélien Planchat, Fabrice Pagniez,
Patrice Le Pape, Cédric Logé
PII:
S0223-5234(20)30049-0
DOI:
https://doi.org/10.1016/j.ejmech.2020.112082
EJMECH 112082
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 October 2019
Revised Date: 17 January 2020
Accepted Date: 17 January 2020
Please cite this article as: D. Montoir, Ré. Guillon, S. Gazzola, I. Ourliac-Garnier, K.E. Soklou, A.
Tonnerre, C. Picot, Auré. Planchat, F. Pagniez, P. Le Pape, Cé. Logé, New azole antifungals with a
fused triazinone scaffold, European Journal of Medicinal Chemistry (2020), doi: https://doi.org/10.1016/
j.ejmech.2020.112082.
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Graphical Abstract

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New Azole Antifungals with a Fused Triazinone Scaffold
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David Montoir , Rémi Guillon , Sophie Gazzola , Isabelle Ourliac-Garnier , Kossi
Efouako Soklou a, Alain Tonnerre a, Carine Picot b, Aurélien Planchat c, Fabrice Pagniez
b, Patrice Le Pape b, Cédric Logé a,*.
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a
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Université de Nantes, Nantes Atlantique Universités, Département de Chimie
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Thérapeutique, Cibles et Médicaments des Infections et du Cancer, IICIMED- EA1155,
Institut de Recherche en Santé 2, F-44200 Nantes, France
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b Université de Nantes, Nantes Atlantique Universités, Département de Parasitologie et
Mycologie Médicale, Cibles et Médicaments des Infections et du Cancer, IICIMED-
EA1155, Institut de Recherche en Santé 2, F-44200 Nantes, France
c
Université de Nantes, Nantes Atlantique Universités, CEISAM, Chimie et
Interdisciplinarité, Synthèse, Analyse, Modélisation, UMR CNRS 6230, Faculté des
Sciences et Techniques, F-44322 Nantes, France
* Corresponding author, Département de Chimie Thérapeutique, IICIMED-EA1155,
Institut de Recherche en Santé 2, Université de Nantes, 22 boulevard Bénoni Goullin, F-
44200 Nantes, France. Tel.: +33 253009154.
E-mail address: cedric.loge@univ-nantes.fr (C. Logé).
Abstract
We identified a new series of azole antifungal agents bearing a pyrrolotriazinone
scaffold. These compounds exhibited a broad in vitro antifungal activity against
pathogenic Candida spp. (fluconazole-susceptible and fluconazole-resistant) and were
10- to 100-fold more active than voriconazole against two Candida albicans isolates
with known mechanisms of azole resistance (overexpression of efflux pumps and/or
specific point substitutions in the Erg11p/CYP51 enzyme). Our lead compound 12 also
displayed promising in vitro antifungal activity against some filamentous fungi such as
Aspergillus fumigatus and the zygomycetes Rhizopus oryzae and Mucor circinelloides

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and an in vivo efficiency against two murine models of lethal systemic infections
caused by Candida albicans.
Keywords
3H-pyrrolo[1,2-d][1,2,4]triazin-4-one;
Antifungal
agents;
Azoles;
Candida;
Filamentous fungi; Invasive fungal infections.

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1. Introduction
Invasive fungal infections (IFIs) are common and a life-threatening problem in
immunocompromised patients. While 90% of all reported fungal-related deaths result
from species that belong to one of four genera: Candida, Aspergillus, Cryptococcus and
Pneumocystis, disseminated infections due to non-Aspergillus moulds such as Rhizopus
spp., Mucor spp., Fusarium spp. and Scedosporium spp. are increasing [1,2,3]. The
antifungal armamentarium includes the polyenes (amphotericin B formulations),
fluoropyrimidine (flucytosine), echinocandins and the azoles such as fluconazole,
itraconazole (first-generation triazoles), voriconazole, posaconazole and isavuconazole
(second-generation triazoles). All these compounds differ in their spectrum of activity,
pharmacokinetics/pharmacodynamics (PK/PD) properties, indications, dose, safety
profile, and cost [4,5]. Among them, the triazoles inhibit cytochrome P450-dependent
lanosterol 14α-demethylation (CYP51) leading to a depletion of ergosterol (major
component of the fungal cell membrane) and accumulation of 14α-methylated sterols in
the fungal cell membrane. All marketed triazoles are also inhibitors and/or substrates of
off-target human cytochromes P450 (CYPs), inducing liver toxicity and potential drug-
drug interactions (DDIs) in the presence of concomitant medications [6]. Additionally,
long-term therapy of azole antifungals for the treatment of candidiasis has led to the
emergence of azole-resistant strains of Candida albicans and other Candida spp.,
showing the need to develop new more effective antifungals, in particular those able to
circumvent resistance phenomena which can be attributed to mutations in ERG11 gene
encoding Erg11p (CYP51), overexpression of ERG11 or genes encoding membrane
transport proteins (CDR1, CDR2, and MDR1), or alterations in cell wall composition
[7a-b]. The recent introduction of isavuconazole, administered as a water-soluble
prodrug isavuconazonium sulfate, displays high oral bioavailability, linear
pharmacokinetics and fewer DDIs compared to voriconazole and posaconazole, but
clinical experience is still limited [5]. Other promising investigational compounds VT-
1161 and VT-1598 (Viamet Pharmaceuticals, Inc.) bearing a tetrazole moiety with
improved fungal CYP51 selectivity compared to human hepatic CYP enzymes [8,9],
have been co-crystalized with either Candida albicans-CYP51 (CA-CYP51, pdb code:
5TZ1) [10] or Aspergillus fumigatus-CYP51B (AF-CYP51B, pdb code: 5FRB) [11].
These structures highlighted the importance of an optimized hydrogen-bonding

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interaction with the fungal-specific residue His377 of CA-CYP51 or the corresponding
His374 of AF-CYP51B, respectively.
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Previously, we have already described the design of a broad-spectrum antifungal agent
bearing a linear thiazoloquinazolinone scaffold (compound I) [12]. This compound
exhibited high in vitro activity against pathogenic Candida species and filamentous
fungi and showed in vivo antifungal e cacy in a mice model of systemic candidiasis.
To further investigate structure-activity relationships (SAR) in this series, the central
scaffold was modified by a fused pyrrolotriazinone moiety, that allows us to increase
the size of our molecules by the introduction of a benzene moiety at position 7 (Fig. 1).
We suppose that para-substitution with hydrogen bond acceptors (HBA) should target
the histidine fungal-specific residue, enhance binding for fungal CYP51 and also
compensate mutations near the heme site that confer resistance to compact triazoles.
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2. Chemistry
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The synthesis of newly azoles 6-12 was accomplished as depicted in Scheme 1, starting
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from 1H-pyrrole-2-carboxaldehyde
1
which can be brominated with N-
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bromosuccinimide affording 4-bromo-1H-pyrrole-2-carboxaldehyde 2 [13]. According
to the literature, pyrrolo[1,2-d][1,2,4]triazin-4-one 3 can be prepared either by
condensation of 1-carbethoxy-2-formylpyrrole with formylhydrazine to give a
hydrazone and cyclisation under acidic conditions but with low yield [14], or by
condensation of carbethoxyhydrazine with pyrrole-2-carboxaldehyde to give a
carbethoxyhydrazone and cyclisation in the presence of sodium propoxide in propanol
under reflux conditions for 24h [15]. For this latter condition, Sakai et al. also used a
sub-stoichiometric amount of sodium hydride using N,N-dimethylformamide (DMF) at
100°C for 15h, because the liberated alkoxide ion functioned as a base in the reaction
[16]. We applied a similar strategy for the synthesis of compounds 3 and 4, while
exploring the use of microwave activation for our key intermediate 7-bromo derivative
(4) by changing the solvents (absolute ethanol instead of DMF for the hydrazone
synthesis and 1,4-dioxane instead of DMF in the cyclization step), adding a drying work
up and changing the amount of sodium hydride (1.1 eq. instead of sub-stoichiometric
amount). These modifications resulted in a reduced overall synthesis time (from 15-24 h
to less than 2 h), an improved yield (quantitative) and a cleaner reaction. Both these
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scaffolds were then condensed into the previously described chiral oxirane 5
(synthesized in 8 steps from (R)-methyl lactate) [17] in presence of K2CO3 in N-methyl-
2-pyrrolidone (NMP) at 80°C, to afford compounds 6 and 7 in 49% and 44% yield,
respectively. Finally, starting from compound 7, aromatic moieties were introduced at
position 7 under a microwave-assisted Suzuki cross-coupling reactions with various
boronic acids. The structure of 9 was confirmed by a single-crystal X-ray diffraction
(Fig. 2).
3. Results and discussion
3.1. Biological assays
3.1.1. In vitro antifungal activity
The in vitro antifungal activities of compounds 6-12 were compared with those of
fluconazole, voriconazole, itraconazole, posaconazole, and amphotericin B against
pathogenic Candida species (fluconazole-susceptible and fluconazole-resistant) and
some filamentous fungi (Aspergillus and non-Aspergillus moulds). The minimum
inhibitory concentration (MIC) values are shown in Tables 1 and 2.
Against the fluconazole-susceptible Candida albicans isolate (CAAL93) (Table 1), all
these compounds exhibited a high level of activity with MIC values generally below
0.001 µg.mL-1 (highest MIC value at 0.048 µg.mL-1), comparable to that observed for
voriconazole. Against the two fluconazole-resistant Candida albicans isolates with
identified mechanisms of resistance (for DSY735: upregulation of the ERG11 and ABC
transporters CDR1 and CDR2 genes; for DSY292: mutations G464S, R467K, Y132H in
the encoded drug target enzyme Erg11p (CYP51) and overexpression of the multidrug
resistance MDR1 gene) [18,19], compounds bearing 7-aryl substituents (8-12) were the
most active with MIC values 10- to 100-fold lower than that of voriconazole, while the
more compact unsubstituted molecule 6 and, to a lesser extent, its bromo derivative 7,
did not significantly improve the antifungal activity. These results demonstrate that our
extended compounds are able to bypass the main mechanisms of fluconazole resistance
in C. albicans (overexpression of efflux pumps and Erg11p (CYP51) as well as certain
specific mutations close to the heme site reducing binding affinity for azole). In

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addition, a broad-spectrum antifungal activity was also observed more particularly for
compounds 8-12 against two intrinsically fluconazole-resistant C. krusei isolates
(CAKR7 and CAKR8) and against a less fluconazole-sensitive C. glabrata isolate
(CAGL2) with MIC values ranging from 0.008 (for CAGL2) to 0.101 µg.mL-1 (for
CAKR8).
Among 8 clinical filamentous fungi isolates (Table 2), these compounds showed
variable antifungal activities with the lowest MIC values observed for the 7-aryl
substituents (9-12) against some zygomycetes (Rhizopus orizae RHPOR1 and RHPOR2
(MIC, 0.0625 to 0.25 µg.mL-1) and Mucor circinelloides MURI1 (MIC, 0.19 to 0.75
µg.mL-1)), similar to those observed for amphotericin B (the first-line treatment for
mucormycosis) [20] and posaconazole, whereas voriconazole lacked any useful activity
(MIC, ≥ 8 µg.mL-1). Against some Aspergillus fumigatus isolates (itraconazole-
susceptible, ASFU7 and ASFU76 or -resistant, ASFU77), the antifungal activity of
compounds 9 and 12 was less effective than that seen against Mucorales with MIC
values on the µg.mL-1 range, even if these compounds are only 10-fold less active than
voriconazole (usually the first-line agent for the treatment of invasive aspergillosis)
[21]. Against a clinical isolate of Scedosporium apiospermum (SCAP1), only
compounds 7 and 12 displayed moderate MIC values on the µg.mL-1 range, but
nevertheless close to those observed for itraconazole and voriconazole (MIC, 0.5
µg.mL-1) which typically have the lowest MIC value among the antifungal drugs [22].
Finally, none of these compounds showed significant antifungal activities against a
clinical isolate of a Fusarium sp. (FU sp.), a species which often display high levels of
resistance to existing antifungal agents and are some of the more difficult fungi to treat
[23]. Based on all these biological results, compound 12 was selected for further testing.
3.1.2. Sterol analysis
The mechanism of action of this series was investigated by studying inhibition of C.
albicans CAAL93 ergosterol biosynthesis after treatment by compound 12. As shown in
Table 3, 32% of ergosterol biosynthesis inhibition was obtained with a concentration of
1.0 ng.mL-1 corresponding to the MIC value. At a 10-fold higher concentration, a dose-
dependent effect was observed causing 53% ergosterol biosynthesis inhibition. This
significant reduction of ergosterol and the concomitant accumulation of 14α-methylated

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precursors lanosterol and eburicol, in a dose-dependent manner, indicated that
compound 12 interfered with sterol 14α-demethylase cytochrome P450 (CYP51).
3.1.3. Acute oral toxicity study in rats
Treatment of rats with compound 12 by an oral route at a dose level of 300 mg/kg bw
did not cause mortality during the 14 days of observation. This dose level caused
hunched back and slightly decreased activity in all the experimental animals but all of
them were symptom free the next day after treatment. In addition, this study showed no
indication of a test item-related effect concerning body weight gain and was not
associated with any macroscopic findings. The method used was not intended to allow
the calculation of a precise LD50 value, but according to the Globally Harmonized
Classification System (GHS) criteria [24], compound 12 can be ranked as "Category 4"
for acute oral exposure.
3.1.4. In vivo antifungal activity
Two murine models of lethal systemic Candida infection were developed to evaluate
the in vivo efficacy of antifungal compound 12 (Fig. 4). After a tail injection of C.
albicans blastoconidia with the fluconazole-susceptible isolate (CAAL93), all control
mice died within 7 days while the groups treated with 10 mg/kg fluconazole or
compound 12 showed 100% survival after 14 days (Fig. 4A). A significant reduction in
kidney fungal burden (Log(CFU)/gram of tissue) compared to control was also
observed for fluconazole and compound 12 (p<0.0001). In the disseminated Candida
infection model caused by a fluconazole- and voriconazole-resistant isolate (DSY292)
(Fig. 4B), all control mice or those treated with 15 mg/kg fluconazole died within 5
days (p=0.4110) or within 8 days for those treated with voriconazole (p=0.0054), while
the group treated with 15 mg/kg compound 12 were still alive 10 days post infection
(p<0.0001). However, these mice were suddenly in a moribund state and, due to ethical
rules, required euthanasia to avoid animal suffering. Furthermore, in this assay, kidneys
of mice treated with compound 12 demonstrated a significant decrease of fungal burden
compared to control, fluconazole and voriconazole groups (p<0.0001). Although a
significant increase of survival and a significant decrease of tissue burden was observed,
further optimization concerning pharmacokinetic/pharmacodynamic (PK/PD)

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characteristics of compound 12 could be performed by increasing doses or prolonging
the duration of exposure to this antifungal.
3.2. Molecular modeling
Docking analysis of compound 12 into the active site pocket of CA-CYP51 (Fig. 5)
showed a hydrogen-bonding interaction between the imidazole side chain of fungal-
specific residue His377 and the methoxy group of the molecule, in addition to many
other van der Waals / aromatic interactions including Tyr118, Leu121, Phe126, Ile131,
Tyr132, Phe228, Phe233, Pro230, Leu376, Phe380 and Met508. Since all compounds
bearing 7-aryl substituents (8-12) are strongest C. albicans CYP51 inhibitors against
isolate DSY292 which have mutations near the heme site, compared to the more
compact molecules 6, voriconazole and fluconazole (Table 1), we can support the
importance of such hydrophobic extended side chains in the stabilization of the
inhibitors with the protein moieties. The importance of residue His377 is not clear as
equivalent biological activities were observed in all C. albicans isolates, even if such an
interaction may contribute to reinforce both affinity and specificity of these antifungals
within the active site.
4. Conclusion
In summary, we have synthesized a new series of triazole antifungal agents with a
pyrrolotriazinone scaffold. Our lead compound 12 significantly reduced mortality rates
and kidney fungal burden in two murine models of lethal systemic infections due to
fluconazole-susceptible or fluconazole- and voriconazole-resistant isolates and showed
promising in vitro antifungal activity against some filamentous fungi such as
Aspergillus fumigatus and the zygomycetes Rhizopus oryzae and Mucor circinelloides.
The mechanism of action for this antifungal against C. albicans was confirmed by sterol
analyses and is characteristic of an in vitro CYP51 inhibition. Further studies on this
series will focus on developing murine models of invasive pulmonary aspergillosis
(IPA) and mucormycosis, which are among the most common life-threatening mould
complications in immunocompromised patients, but also on the determination of their

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binding affinity with human CYP enzymes, as drug-drug interactions (DDIs) still
represent a major “Achilles’ heel” for most azoles in clinical use.
5. Experimental section
5.1. General remarks
Melting points were determined using an Electrothermal IA9300 digital melting point
apparatus and reported uncorrected. IR absorption spectra were recorded on a MIRacle
Shimatzu spectrometer. NMR spectra were recorded on Bruker Avance 400
spectrometer (1H NMR at 400 MHz and 13C NMR at 100 MHz). Chemical shifts are
expressed as δ values (ppm) relative to tetramethylsilane as internal standard (in NMR
description, s = singlet, d = doublet, t = triplet, q = quadruplet, m = multiplet and bs =
broad signal). Coupling constants J are given in Hertz (Hz). All reactions were
monitoring by thin layer chromatography (TLC) using commercially available plates
Alugram® Xtra SIL G/UV254 (0.20 mm). Focused microwave irradiations were carried
out with a CEM DiscoverTM focused microwave reactor (300W, 2455 MHz, monomode
system). Silica gel column chromatography was carried out with Macherey-Nagel Silica
gel 60 (0.063-0.2 mm). Reverse-phase flash column chromatography was performed on
a BUCHI Grace Reveleris® X2 apparatus using Chromabond® flash RS 120 C18
cartridges. Electrospray mass spectrometric analyses were performed on a Waters
Acquity UPLC system ZQ 2000 single quadrupole spectrometer. Formic acid (0.1%)
was added in diluent to improve ionization. All tested compounds displayed more than
97% purity as determined using this analytical LC-MS system (TIC and UV). High
resolution mass spectra were obtained by ESI/TOF. The optical rotations were recorded
with a polarimeter Schmidt-Haensch Polartronic NH8. Chemicals and solvents used
were commercially available. Optically oxirane (2R,3S) 5 was synthesized according to
protocol described in Ref. [17] ([α]20D = -9.0 (c = 1.0 in MeOH) (lit: [α]20D = -8.3)).
5.2. Chemistry

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5.2.1. 4-Bromo-1H-pyrrole-2-carboxaldehyde (2).
To a stirred solution of 1H-pyrrole-2-carboxaldehyde 1 (1 g, 10.52 mmol) in acetonitrile
(10 mL) at 0°C was added N-bromosuccinimide (1.872 g, 10.52 mmol) and the solution
was stirred at 0°C for 15 minutes. Water was added and the resulting mixture was
extracted with diethyl ether. Organic layers were dried over anhydrous Na2SO4, filtered
and concentrated in vacuo. Crystallization of the crude mixture using
cyclohexane/ethanol afforded compound 2 as white crystals (1.125 g, 60%): Rf=0.22
(CH2Cl2); mp: 121-122 °C (lit.[13] mp: 120°C); 1H NMR (400 MHz, DMSO-d6): δ 7.12
(m, 1H), 7.41 (m, 1H), 9.48 (s, 1H), 12.51 (bs, 1H); 13C NMR (100 MHz, DMSO-d6): δ
97.0, 120.8, 126.5, 132.9, 179.1; IR (cm-1): 3238, 3108, 2926, 2860, 1655, 1380, 1357,
1147, 1104, 920, 827, 771, 744, 598; MS (ESI) m/z 173.9 (95) 79Br[M+H]+; 175.9 (95)
81Br[M+H]+.
5.2.2. 3H-Pyrrolo[1,2-d][1,2,4]triazin-4-one (3).
To a stirred solution of ethyl carbazate (241 mg, 2.31 mmol) in N,N-dimethylformamide
(5 mL) was added 1H-pyrrole-2-carboxaldehyde (1) (200 mg, 2.10 mmol) and the
solution was stirred at 90°C for 24 h. The solution was cooled to 0°C, sodium hydride
(60% dispersion in mineral oil) (42 mg, 1.05 mmol) was added and the reaction mixture
was stirred at 90°C for 24 h. Water was added and the resulting mixture was extracted
with ethyl acetate. Organic layers were washed with brine, dried over anhydrous
Na2SO4, filtered and concentrated in vacuo. The residue was purified on silica gel
column chromatography (petroleum ether/diethyl ether 7:3) to yield compound 3 as a
white powder (172 mg, 60%): Rf=0.31 (Petroleum ether/Et2O 1:1); mp: 160-161 °C
1
(lit.[15] mp: 169°C); H NMR (400 MHz, DMSO-d6): δ 6.86 (m, 2H), 7.79 (m, 1H),
13
8.28 (s, 1H), 12.29 (bs, 1H); C NMR (100 MHz, DMSO-d6): δ 109.1, 115.4, 116.2,
125.6, 132.4, 144.7; IR (cm-1): 3238, 3200, 3100, 2996, 2935, 1718, 1602, 1438, 1421,
1352, 1313, 1260, 1215, 1163, 1081, 875, 762, 732, 626; MS (ESI) m/z 135.8 (100)
[M+H]+.
5.2.3. 7-Bromo-3H-pyrrolo[1,2-d][1,2,4]triazin-4-one (4).
To a stirred solution of ethyl carbazate (329 mg, 3.16 mmol) in absolute ethanol (17
mL) was added 1H-pyrrole-2-carboxaldehyde (1) (500 mg, 2.87 mmol) and the solution

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was exposed to microwave irradiation for 5 min at 105°C using maximal microwave
power up to 200 W. After completion, the solvent was evaporated to dryness; the
residue was dissolved in ethyl acetate (20 mL), dried over Na2SO4, filtered and the
filtrate was evaporated to dryness. Sodium hydride (60% dispersion in mineral oil)
(128.7 mg, 3.22 mmol) was added portionwise to a solution of the obtained residue in
anhydrous dioxane (17 mL) and the mixture was stirred at room temperature for 45 min.
Afterwards the mixture was irradiated for 1 h at 140°C using microwave system
operating at maximal microwave power up to 200W. Solvent was evaporated to dryness
and the residue was quenched with a saturated aqueous solution of NH4Cl (55 mL),
water (5 mL) and extracted with ethyl acetate (3×120 mL). The aqueous phase was
extracted with ethyl acetate (2×120 mL) after saturation with NaCl (crystals). Organic
layers were dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The
residue was purified on silica gel column chromatography (CH2Cl2/EtOH 99:1) to yield
compound 4 as a grey powder (424 mg, 69%): Rf=0.24 (CH2Cl2/EtOH 99:1); mp: 187-
188 °C; 1H NMR (400 MHz, DMSO-d6): δ 6.95 (d, 1H, 4J=1.6 Hz), 7.93 (d, 1H, 4J=1.6
Hz), 8.21 (s, 1H), 12.44 (bs, 1H); 13C NMR (100 MHz, DMSO-d6): δ 104.4, 110.6,
115.8, 126.3, 131.3, 143.3; IR (cm-1): 3203, 3151, 3102, 1717, 1533, 1474, 1409, 1383,
1327, 1302, 1147, 891, 825, 727, 628; MS (ESI) m/z 213.9 (66) 79Br[M+H]+; 215.9
(51) 81Br[M+H]+; UPLC purity 99%.
5.2.4.
3-[(1R,2R)-2-(2,4-Difluorophenyl)-2-hydroxy-1-methyl-3-(1,2,4-triazol-1-yl)
propyl]-3H-pyrrolo[1,2-d][1,2,4]triazin-4-one (6).
To a stirred solution of (2R,3S)-2-(2,4-difluorophenyl)-3-methyl-2-[(1H-1,2,4-triazol-1-
yl)methyl]oxirane 5 (279 mg, 1.11 mmol) in N-methyl-2-pyrrolidone (3 mL) was added
K2CO3 (113 mg, 0.81 mmol) and 3H-pyrrolo[1,2-d][1,2,4]triazin-4-one 3 (100 mg, 0.74
mmol). The solution was stirred at 80°C for 24 h. Mixture was diluted with water and
product was extracted with ethyl acetate. Organic layers were dried over anhydrous
Na2SO4 and concentrated in vacuo. The residue was purified on silica gel column
chromatography (Et2O) to yield compound 6 as a white powder (140 mg, 49%): Rf=0.10
1
(Et2O); mp: 131-132 °C; [α]20 = +30.0 (c = 0.1 in MeOH); H NMR (400 MHz,
D
3
2
2
DMSO-d6): δ 1.20 (d, 3H, J=6.8 Hz), 4.46 (d, 1H, J=14.4 Hz), 4.90 (d, 1H, J=14.4
Hz), 5.61-5.68 (m, 2H), 6.89-6.98 (m, 3H), 7.23 (m, 1H), 7.33 (m, 1H), 7.58 (s, 1H),

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7.87 (s, 1H), 8.27 (s, 1H), 8.47 (s, 1H); C NMR (100 MHz, DMSO-d6): δ 14.1, 54.7,
55.5, 77.4, 103.9 (dd, JC-F=2JC-F=28.0 Hz), 109.6, 110.9 (d, JC-F=20.0 Hz), 116.0,
117.5, 124.0 (d, JC-F=13.0 Hz), 125.0, 130.0 (dd, JC-F=3JC-F=7.0 Hz), 132.7, 144.7,
145.0, 150.2, 158.7 (d, JC-F=245.0 Hz), 161.9 (d, JC-F=245.0 Hz); IR (cm-1): 3398,
3126, 1706, 1676, 1617, 1598, 1499, 1448, 1424, 1344, 1272, 1164, 1139, 963, 732,
682; MS (ESI) m/z 387.1 (100) [M+H]+; UPLC purity 100%. HRMS (ESI): calcd. for
C18H16N6O2F2 [M+H]+ 387.1377, found: 387.1376.
2
2
2
3
1
1
5.2.5. 7-Bromo-3-[(1R,2R)-2-(2,4-difluorophenyl)-2-hydroxy-1-methyl-3-(1,2,4-triazol-
1-yl)propyl]-3H-pyrrolo[1,2-d][1,2,4]triazin-4-one (7).
According to the synthesis of 3-[(1R,2R)-2-(2,4-difluorophenyl)-2-hydroxy-1-methyl-3-
(1H-1,2,4-triazol-1-yl)propyl]pyrrolo[1,2-d][1,2,4]triazin-4-one 6 starting from (2R,3S)-
2-(2,4-difluorophenyl)-3-methyl-2-[(1H-1,2,4-triazol-1-yl)methyl]oxirane 5 (880 mg,
3.51 mmol) and 7-bromo-3H-pyrrolo[1,2-d][1,2,4]triazin-4-one 4 (500 mg, 2.34 mmol).
The residue was purified by reverse-phase flash column chromatography
(water/acetonitrile 98:2 + formic acid 0.1%
‰ 10 mn ‰ acetonitrile + formic acid
0.1%) to yield compound 7 as a beige powder after lyophilization and trituration in
diisopropyl ether (480 mg, 44%): Rf=0.27 (CH2Cl2/EtOH 98:2); mp: 142-143 °C; [α]20
= +20.0 (c = 0.1 in MeOH); 1H NMR (400 MHz, DMSO-d6): δ 1.22 (d, 3H, 3J=7.2 Hz),
D
2
2
4.50 (d, 1H, J=14.4 Hz), 4.89 (d, 1H, J=14.4 Hz), 5.50-5.70 (m, 2H), 6.96 (ddd, 1H,
3JH-F=3JH-H=8.4 Hz, JH-H=2.6 Hz), 7.06 (d, 1H, J=1.2 Hz), 7.24 (ddd, 1H, JH-F=3J’H-
4
4
3
4
4
F=9.2 Hz, JH-H=2.6 Hz), 7.34 (m, 1H), 7.60 (s, 1H), 8.05 (d, 1H, J=1.2 Hz), 8.27 (s,
1H), 8.43 (s, 1H); 13C NMR (100 MHz, DMSO-d6): δ 14.1, 54.7, 55.9, 77.4, 103.9 (dd,
2JC-F=2JC-F=28.0 Hz), 104.8, 111.0, 111.1, 117.0, 124.0, 125.7, 130.0 (dd, JC-F=3JC-
3
1
1
F=9.0 Hz), 131.5, 143.7, 144.7, 150.2, 158.7 (d, JC-F=245.0 Hz), 161.9 (d, JC-F=245.0
Hz); IR (cm-1): ν=3376, 3159, 1706, 1617, 1499, 1458, 1401, 1329, 1294, 1272, 1173,
1133, 1043, 961, 852, 727, 670; MS (ESI) m/z 465.0 (94) 79Br[M+H]+; 466.9 (100)
81Br[M+H]+; UPLC purity 100%. HRMS (ESI): calcd. for C18H15N6O2F2Br [M+H]+
465.0492, found: 465.0481.
5.2.6.
3-[(1R,2R)-2-(2,4-Difluorophenyl)-2-hydroxy-1-methyl-3-(1,2,4-triazol-1-
yl)propyl]-7-phenyl-3H-pyrrolo[1,2-d][1,2,4]triazin-4-one (8).

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To a 10 mL vial were added under argon 7-bromo-3-[(1R,2R)-2-(2,4-difluorophenyl)-2-
hydroxy-1-methyl-3-(1,2,4-triazol-1-yl)propyl]-3H-pyrrolo[1,2-d][1,2,4]triazin-4-one 7
(200 mg, 0.43 mmol), benzeneboronic acid (61 mg, 0.43 mmol), Pd(PPh3)4 (25 mg, 0.05
mmol), 2M Na2CO3 aqueous solution (2 mL) and acetonitrile (2 mL). The resulting
mixture was exposed to microwave irradiation for 10 min at 120°C using maximal
microwave power up to 100 W. The reaction mixture was cooled to room temperature
and diluted with water. Product was extracted with methylene chloride, organic layers
were dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was purified
on silica gel column chromatography (CH2Cl2 and CH2Cl2/EtOH 99:1) to yield
compound 8 as a green powder (119 mg, 60%): Rf=0.29 (CH2Cl2/EtOH 98:2); mp: 146-
1
147 °C; [α]20D = +80.0 (c = 0.1 in MeOH); H NMR (400 MHz, DMSO-d6): δ 1.20 (d,
3H, 3J=6.8 Hz), 4.48 (d, 1H, 2J=14.4 Hz), 4.89 (d, 1H, 2J=14.4 Hz), 5.60-5.67 (m, 2H),
6.92 (ddd, 1H, 3JH-F=3JH-H=8.4 Hz, 4JH-H=2.4 Hz), 7.21 (m, 1H), 7.27-7.37 (m, 3H), 7.43
3
3
3
(dd, 2H, JH-H=7.2 Hz; JH-H=8.0 Hz), 7.56 (s, 1H), 7.85 (d, 2H, JH-H=7.2 Hz), 8.26 (s,
1H), 8.33 (s, 1H), 8.44 (s, 1H); 13C NMR (100 MHz, DMSO-d6): δ 14.1, 54.8, 55.5,
2
2
77.4, 104.0 (dd, JC-F=2JC-F=28.0 Hz), 106.7, 111.0 (d, JC-F=21.0 Hz), 113.9, 124.1 (d,
2JC-F=16.0 Hz), 125.9, 126.0 (2C), 127.6, 129.0 (2C), 130.1, 130.7, 132.5, 132.7, 144.7,
1
1
144.8, 150.2, 158.7 (d, JC-F=245.0 Hz), 161.9 (d, JC-F=245.0 Hz); IR (cm-1): ν=3403,
1696, 1617, 1500, 1458, 1408, 1272, 1210, 1133, 1045, 964, 851, 758, 728, 677, 511;
MS (ESI) m/z 463.1 (100) [M+H]+; UPLC purity 100%. HRMS (ESI): calcd. for
C24H20N6O2F2 [M+H]+ 463.1687, found: 463.1689.
5.2.7.
7-(4-Chlorophenyl)-3-[(1R,2R)-2-(2,4-difluorophenyl)-2-hydroxy-1-methyl-3-
(1,2,4-triazol-1-yl)propyl]-3H-pyrrolo[1,2-d][1,2,4]triazin-4-one (9).
According to the synthesis of 3-[(1R,2R)-2-(2,4-difluorophenyl)-2-hydroxy-1-methyl-3-
(1,2,4-triazol-1-yl)propyl]-7-phenyl-3H-pyrrolo[1,2-d][1,2,4]triazin-4-one
8
starting
from 7-bromo-3-[(1R,2R)-2-(2,4-difluorophenyl)-2-hydroxy-1-methyl-3-(1,2,4-triazol-
1-yl)propyl]-3H-pyrrolo[1,2-d][1,2,4]triazin-4-one 7 (200 mg, 0.43 mmol) and 4-
chlorobenzeneboronic acid (61 mg, 0.43 mmol) to yield compound 9 as a light yellow
powder (98 mg, 46%): Rf=0.25 (CH2Cl2/EtOH 98:2); mp: 204-205 °C; [α]20D = +30.0 (c
1
3
= 0.1 in MeOH); H NMR (400 MHz, DMSO-d6): δ 1.20 (d, 3H, J=6.8 Hz), 4.48 (d,
2
2
1H, J=14.4 Hz), 4.88 (d, 1H, J=14.4 Hz), 5.55-5.67 (m, 2H), 6.93 (m, 1H), 7.22 (m,

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1H), 7.28-7.35 (m, 2H), 7.48 (d, 2H, 3JH-H=8.8 Hz), 7.56 (s, 1H), 7.89 (d, 2H, 3JH-H=8.8
13
Hz), 8.25 (s, 1H), 8.38 (s, 1H), 8.44 (s, 1H); C NMR (100 MHz, DMSO-d6): δ 14.1,
54.8, 55.8, 77.5, 103.9 (dd, 2JC-F=2JC-F=27.0 Hz), 106.7, 111.0 (d, 2JC-F=18.0 Hz), 114.3,
3
124.1 (d, JC-F=9.0 Hz), 126.0, 126.7, 127.8 (2C), 128.9 (2C), 129.4, 130.1, 131.7,
1
1
132.1, 132.5, 144.7, 150.2, 158.7 (d, JC-F=245.0 Hz), 161.9 (d, JC-F=245.0 Hz); IR
(cm-1): ν=3386, 1707, 1599, 1500, 1386, 1293, 1269, 1179, 1138, 1091, 825, 677, 519;
MS (ESI) m/z 497.1 (100) 35Cl[M+H]+, 499.1 (39) 37Cl[M+H]+; UPLC purity 97%.
HRMS (ESI): calcd. for C24H19N6O2F2Cl [M+H]+ 497.1313, found: 497.1299.
5.2.8.
3-[(1R,2R)-2-(2,4-Difluorophenyl)-2-hydroxy-1-methyl-3-(1,2,4-triazol-1-
yl)propyl]-7-(4-nitrophenyl)-3H-pyrrolo[1,2-d][1,2,4]triazin-4-one (10).
According to the synthesis of 3-[(1R,2R)-2-(2,4-difluorophenyl)-2-hydroxy-1-methyl-3-
(1,2,4-triazol-1-yl)propyl]-7-phenyl-3H-pyrrolo[1,2-d][1,2,4]triazin-4-one
8
starting
from 7-bromo-3-[(1R,2R)-2-(2,4-difluorophenyl)-2-hydroxy-1-methyl-3-(1,2,4-triazol-
1-yl)propyl]-3H-pyrrolo[1,2-d][1,2,4]triazin-4-one 7 (200 mg, 0.43 mmol) and 4-
nitrobenzeneboronic acid (72 mg, 0.43 mmol). The residue was purified on silica gel
column chromatography (petroleum ether/ethyl acetate 60:40) followed by reverse-
phase flash column chromatography (water/acetonitrile 98:2 + formic acid 0.1%
‰ 10
mn ‰ acetonitrile + formic acid 0.1%) to yield compound 10 as a brown powder after
lyophilization and trituration in diisopropyl ether (51 mg, 47%): Rf=0.25 (CH2Cl2/EtOH
98:2); mp: 130-131 °C; [α]20D = +50.0 (c = 0.1 in MeOH); 1H NMR (400 MHz, DMSO-
d6): δ 1.20 (d, 3H, 3J=7.2 Hz), 4.50 (d, 1H, 2J=14.4 Hz), 4.88 (d, 1H, 2J=14.4 Hz), 5.57-
5.70 (m, 2H), 6.94 (ddd, 1H, 3JH-F=3JH-H=8.4 Hz, 4JH-H=2.4 Hz), 7.22 (m, 1H), 7.31 (m,
3
1H), 7.46 (s, 1H), 7.56 (s, 1H), 8.16 (d, 2H, JH-H=8.8 Hz), 8.24 (s, 1H), 8.26 (d, 2H,
3JH-H=8.8 Hz), 8.48 (s, 1H), 8.59 (s, 1H); 13C NMR (100 MHz, DMSO-d6): δ 14.0, 54.7,
2
2
55.8, 77.4, 103.9 (dd, JC-F=2JC-F=27.0 Hz), 107.0, 111.0 (d, JC-F=20.0 Hz), 115.9,
124.0, 124.2 (2C), 126.3, 126.9 (2C), 128.3, 130.1, 132.6, 139.6, 144.6, 144.7, 146.4,
150.2, 158.6 (dd, 1JC-F=245.0 Hz, 3JC-F=12.0 Hz), 161.9 (dd, 1JC-F=245.0 Hz, 3JC-F=12.0
Hz); IR (cm-1): ν=3448, 1701, 1509, 1342, 1212, 1132, 964, 852, 752; MS (ESI) m/z
508.0 (100) [M+H]+; UPLC purity 98%.

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5.2.9.
4-{3-[(1R,2R)-2-(2,4-Difluorophenyl)-2-hydroxy-1-methyl-3-(1,2,4-triazol-1-
yl)propyl]-4-oxo-3,4-dihydro-pyrrolo[1,2-d][1,2,4]triazin-7-yl}benzonitrile (11).
According to the synthesis of 3-[(1R,2R)-2-(2,4-difluorophenyl)-2-hydroxy-1-methyl-3-
(1,2,4-triazol-1-yl)propyl]-7-phenyl-3H-pyrrolo[1,2-d][1,2,4]triazin-4-one
8
starting
from 7-bromo-3-[(1R,2R)-2-(2,4-difluorophenyl)-2-hydroxy-1-methyl-3-(1,2,4-triazol-
1-yl)propyl]-3H-pyrrolo[1,2-d][1,2,4]triazin-4-one 7 (200 mg, 0.43 mmol) and 4-
cyanobenzeneboronic acid (63 mg, 0.43 mmol). The residue was purified on silica gel
column chromatography (methylene chloride/ethanol 99:1 and diethyl ether/ethyl
acetate 80:20) to yield compound 11 as a light yellow powder (105 mg, 50%): Rf=0.23
(CH2Cl2/EtOH 98:2); mp: 201-202 °C; [α]20 = +20.0 (c = 0.1 in MeOH); H NMR
(400 MHz, DMSO-d6): δ 1.20 (d, 3H, 3J=6.8 Hz), 4.48 (d, 1H, 2J=14.6 Hz), 4.88 (d, 1H,
2J=14.6 Hz), 5.50-5.72 (m, 2H), 6.92 (m, 1H), 7.20 (m, 1H), 7.31 (m, 1H), 7.42 (s, 1H),
1
D
3
3
7.55 (s, 1H), 7.88 (d, 2H, JH-H=8.0 Hz), 8.07 (d, 2H, JH-H=8.0 Hz), 8.24 (s, 1H), 8.45
13
(s, 1H), 8.53 (s, 1H); C NMR (100 MHz, DMSO-d6): δ 14.1, 54.7, 55.8, 77.4, 103.9
2
2
(dd, JC-F=2JC-F=28.0 Hz), 106.9, 109.8, 111.0 (d, JC-F=22.0 Hz), 115.5, 118.9, 124.0,
126.2, 126.7 (2C), 128.8, 130.1, 132.6, 132.9 (2C), 137.6, 144.7, 150.2, 157.4, 160.8
(1C not visible); IR (cm-1): ν=3447, 2221, 1699, 1500, 1388, 1293, 1138, 1047, 966,
804; MS (ESI) m/z 488.1 (100) [M+H]+; UPLC purity 100%.
5.2.10.
3-[(1R,2R)-2-(2,4-Difluorophenyl)-2-hydroxy-1-methyl-3-(1,2,4-triazol-1-
yl)propyl]-7-(4-methoxyphenyl)-3H-pyrrolo[1,2-d][1,2,4]triazin-4-one (12).
According to the synthesis of 3-[(1R,2R)-2-(2,4-difluorophenyl)-2-hydroxy-1-methyl-3-
(1,2,4-triazol-1-yl)propyl]-7-phenyl-3H-pyrrolo[1,2-d][1,2,4]triazin-4-one
8
starting
from 7-bromo-3-[(1R,2R)-2-(2,4-difluorophenyl)-2-hydroxy-1-methyl-3-(1,2,4-triazol-
1-yl)propyl]-3H-pyrrolo[1,2-d][1,2,4]triazin-4-one 7 (350 mg, 0.75 mmol) and 4-
methoxybenzeneboronic acid (114 mg, 0.75 mmol). The residue was purified on silica
gel column chromatography (dichloromethane/ethyl acetate 96:4), then triturated in
diethyl ether to yield after filtration compound 12 as a yellow powder (218 mg, 59%):
Rf=0.29 (CH2Cl2/EtOH 98:2); mp: 172-173 °C; [α]20 = +20.0 (c = 0.1 in MeOH); H
1
D
3
NMR (400 MHz, DMSO-d6): δ 1.19 (d, 3H, J=6.8 Hz), 3.78 (s, 3H), 4.47 (d, 1H,
2J=14.6 Hz), 4.88 (d, 1H, J=14.6 Hz), 5.55-5.70 (m, 2H), 6.92 (m, 1H), 6.98 (d, 2H,
3JH-H=8.8 Hz), 7.21 (m, 1H), 7.25 (d, 1H, JH-H=1.6 Hz), 7.30 (m, 1H), 7.55 (s, 1H),
2
4

3
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484
7.77 (d, 2H, JH-H=8.8 Hz), 8.22 (s, 1H), 8.25 (s, 1H), 8.41 (s, 1H); 13C NMR (100
MHz, DMSO-d6): δ 14.1, 54.8, 55.2 (2C), 77.4, 103.9, 106.5, 111.1, 113.0, 114.4,
124.2, 125.2, 125.8 (2C), 127.3 (2C), 130.0, 130.6, 132.4, 144.7, 150.2, 158.9 (1C +
2CF not visible); IR (cm-1): ν=3447, 1700, 1616, 1500, 1428, 1407, 1387, 1298, 1250,
1180, 1134, 1029, 959, 808, 723; MS (ESI) m/z 493.3 (100) [M+H]+; UPLC purity
100%. HRMS (ESI): calcd. for C25H22N6O3F2 [M+H]+ 493.1805, found: 493.1794.
5.3. Crystallographic analysis
Single crystals of compound 9 suitable for X-ray diffraction were prepared by slow
diffusion of diethyl ether through a saturated solution of the product in
dichloromethane. The crystal was mounted at the tip of Lindemann capillary by means
of a solvent free glue. The intensity measurement was carried out at 293 K on a Bruker-
Nonius Kappa CCD diffractometer, using graphite-monochromatized MoK-L2,3
radiation (λ
= 0.71073 Å) up to a resolution of (sin θ/λ)max = 0.64 Å–1. The structure
was solved using the charge flipping algorithm [25] implemented in the Superflip
program [26] and refined with JANA2006 program [27] against F2 for all reflections.
Non-hydrogen atoms were refined with anisotropic displacement parameters. All H
atoms were introduced in geometrically optimized positions and refined with a riding
model, except for alcohol H atom, which position was determined by difference Fourier.
CCDC-1857098 contains the supplementary crystallographic data. These data can be
obtained free of charge from the Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.
Crystal data: C24H19Cl1F2N6O2, Mr = 496.9, tetragonal, P43212, a = 11.4927(10), b =
11.4927 (10), c = 34.328(5) Å, V = 4534.1(9) Å3, Z =8, ρcalcd = 1.4559 g.cm-3, µ =
0.221 mm-1, F(000) = 2048, prism, 0.22 × 0.21 × 0.08 mm3, T = 293 K, 25530
reflections, 4865 unique (99% completeness), 320 parameters, GOF = 1.51, wR2 =
0.1659, R = 0.0919 for 2431 reflections with I > 2σ(I).
485
486
487
488
5.4. Biology

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5.4.1. Antifungal drugs
Fluconazole, voriconazole, itraconazole, posaconazole and amphotericin B powders
were purchased from Sigma-Aldrich, St Quentin Fallavier, France.
5.4.2. In vitro antifungal activity
Candida spp.
Candida spp. suspensions were prepared in RPMI 1640 medium (Sigma, Saint Quentin
Fallavier, France) supplemented with 0.165 M morpholinopropanesulphonic acid
(MOPS, Sigma), 2% glucose and antibiotics and adjusted to give a final concentration
of 103 cells mL−1. A 96-well microplate (Nunc, Dutscher SA, France) was seeded with
100 µL of Candida suspension. Molecules were first dissolved in dimethylsulfoxide
(except fluconazole in water) and then diluted in culture medium. Each concentration of
molecule (100 µL) to be tested was added and plates were incubated at 37 °C for 24 h.
The cellular viability was evaluated spectrofluorometrically with an excitation at 550
nm and an emission at 590 nm after a 4 h incubation with 10 µL of resazurin (Interchim,
Montluçon, France) [28]. The minimal inhibitory concentration (MIC) is the
concentration that inhibited 50% of the cell growth. MICs were determined by linear
regression analysis.
Aspergillus fumigatus
Conidia of A. fumigatus were microscopically counted and diluted in RPMI 1640
medium (Sigma) supplemented with 0.165 M morpholinopropanesulphonic acid
(MOPS, Sigma), 2% glucose and antibiotics. One hundred microliters of a 104 cells mL-
1
suspension were inoculated in a 96-well microplate (Nunc). Drugs were prepared as
described for Candida spp. evaluations and 100 µL of the drug dilutions were added to
the cell suspension. After an incubation time of 48 h at 37 °C, the cellular viability was
evaluated as in Candida assay after an incubation time of 20h with resazurin [28].
Activity of the studied and reference molecules was expressed as the MIC. The MIC is
the concentration that inhibited 80% of the cell growth. MICs were determined by linear
regression analysis.
Other filamentous fungi

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552
The broth microdilution test was done in accordance with the CLSI guidelines for
filamentous fungi (CLSI document M38) [29] using RPMI 1640 medium (Sigma)
buffered to pH 7.0 with MOPS (Sigma). Drugs were diluted in DMSO and further in
culture medium. Final drug range was 0.032-16 mg/L. Stock inoculum suspensions
were prepared from 7-day-old cultures grown on potato dextrose agar following the
CLSI guidelines (document M38-A3). Stock suspensions contained conidia or
sporangiospores. The diluted (2×) inoculum sizes ranged from 0.9×104 to 4.7×104
CFU/mL. Drug-free and cell-free controls were included. The microdilution plates were
incubated at 35°C and read after 48 h except for the Zygomycetes, which were read at
24 h. The MIC endpoints were read visually with the aid of a reading mirror and
expressed as the lowest drug concentration that prevented 100% growth.
5.4.3. Sterol extraction and analysis
To study sterol synthesis, C. albicans CAAL93 blastospores were incubated in 50 mL
Sabouraud broth medium (Sigma-Aldrich) during 18 hours at 35° C with stirring.
Fluconazole and compound 12 were introduced into culture medium before incubation.
Cells were collected by centrifugation at 1500 g. Pellet was suspended in 3 mL of fresh
ethanolic potassium hydroxide solution (25 g of KOH, 36 mL of distilled water and
brought to100 mL with 100 % ethanol). Saponification was performed at 80°C for 60
min. Sterols were extracted by addition of 3 mL of hexane and the organic phase was
then washed by adding 1 mL of sterile water. Final organic phase was transferred to a
new collection tube after 5 min centrifugation step at 2000 rpm. An equivalent of 50 mg
mycelia was transferred into a glass tube and 2.5 µg of cholesterol was added as internal
standard. Hexane was evaporated by heating at 80°C. Sterols were derivatized with 100
µL of N-Methyl-N-(trimethylsilyl)trifluoroacetamide (TMS) (Sigma-Aldrich) during 30
min at room temperature. The solvent was then evaporated at 80°C. Dried sterols were
solubilized with 200 µL dichloromethane and stored at -20°C until analysis.
Sterols as TMS derivatives were analyzed by GC-MS using an Agilent 7890A GC
system, with a SLB-5TM column (30 m × 0.25 mm, 0.25 µm, Sigma-Aldrich, France)
coupled with a mass detector (Agilent 5975C inert MSD – E.I. 70 eV). One microliter
of sample was injected in splitless mode at 250 °C. The carrier gas was helium at a flow

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rate of 1 mL/min. The oven was set at 150°C for 0.5 min and then raised to 290 °C at
50 °C/min and from 290 °C to 305 °C at 2 °C/min. Sterols were identified via their
electron ionization fragmentation pattern, compared to published data. Results are
expressed as percent area of total sterols. Relative Retention Time are expressed as the
ratio between retention of sterol and retention time of cholesterol.
5.4.4. Acute oral toxicity study
Evaluation of acute oral toxicity of compound 12 was conducted by CiToxLab Hungary
Ltd. (study code: 15/276-001P) according to the acute toxic class method (OECD 423
and Commission Regulation (EC) No 440/2008 of 30 May 2008, B.1.Tris) [30,31] in
two groups of three female CRL:(WI) rats. The test substance, synthesized by
Diverchim (Roissy, France; DIV07169 (i.e. compound 12), batch No. 404-193-Es2,
>99% purity UV), was administered by gavage at a dose level of 300 mg/kg bw (Group
1 and Group 2). General behavioral observations, body weight changes, signs of
toxicity, and mortality were recorded at 30 minutes, 1, 2, 4 and 6 hours after dosing and
daily for 14 days thereafter. All animals were sacrificed by exsanguination under
pentobarbital anesthesia. After examination of the external appearance, the cranial,
thoracic and the abdominal cavities were opened and the organs and the tissues were
observed. Macroscopic abnormalities were recorded.
5.4.5. Animal use and care
Swiss mice (Janvier Labs, Le Genest-Saint-Isle, France) with a body weight of 25 g
were obtained and allowed to acclimate for 7 days prior to use. Environmental controls
for the animal room were set to maintain a temperature of 16 to 22°C, a relative
humidity of 30 to 70%, and a 12:12 hourly light-dark cycle. All the experimental
protocols were prior approved by the Ethic Committee on Animal Testing (comité
d’éthique en expérimentation animale N°006) and was authorized by the French
Ministry of Higher Education, Research and Innovation (APAFIS≠9710-
2017042410186554 v4).
5.4.6. Systemic C. albicans infection models

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Mice were immunodepressed by subcutaneous injection of 30 mg/kg prednisolone
(Hydrocortancyl®) one day before challenge. On day 0, mice were infected
intravenously (100 µL) with a suspension of fluconazole susceptible C. albicans
(CAAL93) or both fluconazole- and voriconazole-resistant C. albicans (DSY292)
blastoconidia (5.106 yeast/mL). One hour after infection, mice were treated per os once
daily with 10 mg/kg (for CAAL93) or 15 mg/kg (for DSY292) body weight of
compound 12, fluconazole or voriconazole solubilized in 40% DMSO, 25% tween80
and 35% olive oil for 5 consecutive days. The control group received 100 µL of 40%
DMSO, 25% tween80 and 35% olive oil. To prevent rapid clearance of the molecules,
all animals were given 50% grapefruit juice (ad libitum) instead of water 5 days before
infection until the end of the treatment period [32]. Survival was monitored for 14 days
post inoculation. Differences in cohorts were analyzed by the log-rank Mantel Cox test.
Kidneys of mice euthanized on ethically considerations or at the end of the assay (day
14) were excised and weighed. Tissues were homogenized and serially diluted 10- to
1000-fold in sterile saline, then plated onto Sabouraud dextrose agar and incubated for
48 h to determine the number of colony-forming units (CFUs). Tissue fungal burden
was expressed as average Log(CFU)/gram of tissue. Differences in mean CFUs in
kidneys were compared with the vehicle control using a one-way ANOVA with a post-
hoc Tukey test. For both tests, survival and tissue burden, a P value of <0.05 was
considered statistically significant.
5.5. Molecular modeling
Molecular modeling studies were performed using SYBYL-X 1.3 software [33] running
on a Dell precision T3400 workstation. The three-dimensional structure of compound
12 was built from a standard fragments library and optimized using the Tripos force
field [34] including the electrostatic term calculated from Gasteiger and Hückel 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). The crystal
structure of sterol 14-alpha demethylase from C. albicans (CA-CYP51) in complex with
posaconazole at 2.86 Å resolution (pdb code: 5FSA) [10] was used as template for
docking. Water molecules were removed from the coordinates set since no information

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about conserved water molecules is known for this chemical series in CA-CYP51.
Flexible docking of compound 12 into the binding site was performed using GOLD
software [35]. The most stable docking model was selected according to the best scored
conformation predicted by the GoldScore scoring function. Finally, the complexe was
energy-minimized using Powell’s method available in Maximin2 procedure with the
Tripos force field and a dielectric constant of 4.0, until the gradient value reached 0.1
kcal/mol.Å. Biovia Discovery Studio Visualizer [36] was used for graphical display.
Declaration of interest
The authors declare the following financial interests/personal relationships which may
be considered as potential competing interests: C. Logé, R. Guillon, D. Montoir, F.
Pagniez and P. Le Pape have a patent granted (WO 2017/020944 A1), “Novel fused
pyrimidinone and triazinone derivatives, their process of preparation and their therapeutic
uses as antifungal and/or antiparasitic agents” published on February 9th, 2017.
Acknowledgments
We thank Pr. Dominique Sanglard (Institute of Microbiology, University of Lausanne,
Switzerland) for providing strains DSY735 and DSY292 used in this study, and Dr.
Catherine Roullier (University of Nantes, France) for the HRMS measurements. This
work was supported by a grant from SATT Ouest Valorisation.
Supplementary materials
Electronic Supplementary Information (ESI) available: spectroscopic data (UPLC-MS,
HRMS, 1H and 13C) for representative compounds.
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Fig. 1. General structures of synthesized compounds.
(R)
(R)
(R)
(R)
(H-bond acceptor)
compound
I
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Fig. 2. ORTEP view of compound 9.
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Scheme 1. Synthesis of compounds 6-12. Reagents and conditions: (i) NBS (1 eq),
CH3CN, 0°C, 15 min, 60%; (ii) For R=H: a) H2NNHCOOEt (1.1 eq), DMF, 90°C, 24 h;
b) NaH (60% dispersion in mineral oil, 0.5eq), DMF, 90°C, 24 h, 60%; For R=Br: a)
H2NNHCOOEt (1.1 eq), EtOH, 105°C (MW), 5’ then drying work up; b) NaH (60%
dispersion in mineral oil, 1.1 eq), 1,4-dioxane (anhydrous), rt, 45 min, then 140°C
(MW), 1h, quantitative; (iii) oxirane (2R,3S) 5 (1.5 eq), K2CO3 (1.1 eq), NMP, 80°C, 24
h, 44-49%; (iv) starting from cpd 7: Ar-B(OH)2 (1 eq), Pd(PPh3)4 (0.12 eq), ACN,
Na2CO3 (2M), 120°C (MW), 10’, 46-60%.
(R)
ii
iii
(R)
R = H
R = H
R = Br
R = H
R = Br
1
2
3
4
6
7
i
R = Br
iv
(S)
(R)
(R)
(R)
R' = H
R' = Cl
R' = NO2
8
9
10
oxirane (2R,3S)
5
R' = CN
R' = OCH3
11
12
771
772
773
774