2-(2-Oxo-morpholin-3-yl)-acetamide derivatives as broad-spectrum antifungal agents

Article abstract of DOI:10.1021/jm501814x

From a fungicidal screen, we identified 2-(2-oxo-morpholin-3-yl)-acetamide derivatives as fungicidal agents against Candida species, additionally characterized by antifungal activity against Aspergillus species. However, development of this series was hampered by low plasmatic stability. Introduction of a gem-dimethyl on the 6-position of the morpholin-2-one core led to considerable improvement in plasmatic stability while maintaining in vitro antifungal activity. Further optimization of the series resulted in the discovery of N-(biphenyl-3-ylmethyl)-2-(4-ethyl-6,6-dimethyl-2-oxomorpholin-3-yl)acetamide (87), which, in addition to fungicidal activity against Candida species, shows promising and broad antifungal in vitro activity against various fungi species, such as molds and dermatophytes. In vivo efficacy was also demonstrated in a murine model of systemic Candida albicans infection with a significant fungal load reduction in kidneys.

Full text of DOI:10.1021/jm501814x

Article
pubs.acs.org/jmc
2‑(2-Oxo-morpholin-3-yl)-acetamide Derivatives as Broad-Spectrum
Antifungal Agents
Dorothee Bardiot,†,○ Karin Thevissen,§,○ Katrijn De Brucker,§ Annelies Peeters,§ Paul Cos,⊥
́
Carlos P. Taborda,# Michael McNaughton,† Louis Maes,⊥ Patrick Chaltin,†,‡ Bruno P. A. Cammue,§,∥
and Arnaud Marchand*,†
†Cistim Leuven vzw, Bioincubator 2, Gaston Geenslaan 2, 3001 Leuven, Belgium
‡Centre for Drug Design and Discovery, Bioincubator 2, Gaston Geenslaan 2, 3001 Leuven, Belgium
§Centre of Microbial and Plant Genetics, CMPG, KU Leuven, Kasteelpark Arenberg 20, Box 2460, 3001 Heverlee, Belgium
∥Department of Plant Systems Biology, VIB, Technologiepark 927, 9052, Ghent, Belgium
⊥Laboratory of Microbiology, Parasitology and Hygiene (LMPH), Universiteit Antwerpen, Campus Drie Eiken, building S,
Universiteitsplein 1, 2610 Antwerp, Belgium
#Instituto de Ciencias Biomedicas, Departamento de Microbiologia, Universidade de Sao Paulo, Sao Paulo, SP 05508-900, Brazil
S
* Supporting Information
ABSTRACT: From a fungicidal screen, we identified 2-(2-oxo-morpholin-3-yl)-
acetamide derivatives as fungicidal agents against Candida species, additionally
characterized by antifungal activity against Aspergillus species. However, develop-
ment of this series was hampered by low plasmatic stability. Introduction of a gem-
dimethyl on the 6-position of the morpholin-2-one core led to considerable
improvement in plasmatic stability while maintaining in vitro antifungal activity.
Further optimization of the series resulted in the discovery of N-(biphenyl-3-
ylmethyl)-2-(4-ethyl-6,6-dimethyl-2-oxomorpholin-3-yl)acetamide (87), which, in
addition to fungicidal activity against Candida species, shows promising and broad
antifungal in vitro activity against various fungi species, such as molds and
dermatophytes. In vivo efficacy was also demonstrated in a murine model of
systemic Candida albicans infection with a significant fungal load reduction in kidneys.
effects by targeting different cell components. Polyenes, such as
amphotericin B, bind to ergosterol, resulting in disruption of
the cell membrane. In contrast, the azoles and allylamines block
ergosterol biosynthesis by inhibiting the enzymes cytochrome
P450 and squalene oxidase, respectively. Nucleoside analogues,
such as flucytosine, inhibit DNA and RNA synthesis, whereas
griseofulvin inhibits mitotic spindle formation. In addition,
echinocandins, such as caspofungin, inhibit glucan synthase,
thereby blocking cell wall synthesis.6 The limited efficacy of
standard treatments, the associated toxicity, and the increase of
fungal resistance9 have stimulated the search for new antifungal
drugs.
Fluconazole, a standard fungistatic antimycotic, exerts strong
activity by inhibiting the growth of fungal species, whereas
other drugs such as amphotericin B and caspofungin are
considered to be fungicidal agents that can kill pathogens.
When designing or screening for novel antifungal drugs,
fungicidal activity is generally preferred over fungistatic activity
because it pinpoints the inhibition of targets that are essential
for fungal growth or induction of an active cell death pathway,
INTRODUCTION
■
Invasive fungal infections are caused by yeast pathogenic
species (e.g., Candida albicans, Candida glabrata) or filamen-
tous pathogens (e.g., Aspergillus fumigatus). These infections are
an increasing cause of morbidity and mortality in hospitalized
patients. Candidemia has been reported to occur, in general, as
approximately 1 case per 1000 hospital admissions.1 The overall
mortality associated with candidemia is 30−40%.2,3 While C.
albicans remains the most common pathogen, non-albicans
Candida species, like C. glabrata and Candida krusei, with
greater resistance to triazoles, are being increasingly isolated.4
Invasive aspergillosis is an important cause of mortality in
patients with hematologic malignancies and occurs in about
25% of these patients; the associated mortality is 40−50%.
Moreover, invasive aspergillosis appears to be gaining a
foothold in the intensive care unit in patients without classical
risk factors. Approximately 80% of invasive Aspergillus
infections are caused by A. fumigatus.5 In general, the mortality
associated with fungal infections depends on the severity of the
underlying disease, the infecting species, and the timing and
choice of antifungal treatment.
Currently, several structurally distinct antifungal drug classes
Received: November 24, 2014
are available.6−8 These compound classes exert their antifungal
© XXXX American Chemical Society
A
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such as apoptosis.10 Moreover, Wong et al. recently reported11
that an ideal antifungal agent should be fungicidal in order to
avoid or minimize the emergence of resistance. The minimum
inhibitory concentration (MIC), i.e., the lowest concentration
that inhibits the fungal growth, is used to characterize in vitro
fungistatic activity. Fungicidal activity can be evaluated in vitro
by determination of the minimum fungicidal activity (MFC)
resulting in at least 99% killing of the inoculum. In order to
determine whether compounds showing a 2-log reduction in
inoculum size in vitro could translate into clear in vivo efficacy,
such fungicidal compounds should be tested extensively in vivo
to make sure that the infection could effectively be cleared.
In an effort to identify new small molecules with broad
antifungal activity (more specifically, fungicidal activity), a
screen was performed with a compound library of about 34 000
compounds, resulting in the identification of 2-(4-ethyl-2-
oxomorpholin-3-yl)-N-(4-isopropylphenyl)acetamide (com-
pound 1; Figure 1) as a promising starting point. This
a
Scheme 1. Synthesis of the Morpholine Analogue 4
a
Reagents and conditions: (i) acetaldehyde, sodium acetate,
NaBH3CN, MeOH, rt, 1 h; (ii) 1 M LiOH, dioxane, water, rt, 45
min; (iii) HATU, DIPEA, 4-isopropylaniline, DMF, rt, overnight.
converted to the desired amide derivatives 33−39 by reaction
with 4-isopropylaniline under standard HATU-coupling con-
ditions.
Scheme 3 illustrates the synthesis of compounds in which the
morpholin-2-one core is substituted in the 4-position with alkyl,
cycloalkyl, phenyl, acyl, and sulfonyl groups. A synthetic route
was developed to allow introduction of N-substituents at the
last step. N-Benzylmorpholin-2-one (41) was synthesized by
alkylation of amino-alcohol 40 with tert-butyl bromoacetate
followed by lactonization14 with a catalytic amount of pTsOH.
Alkylation in the 3-position, ester cleavage, amide formation,
and benzyl removal by hydrogenation led to key intermediate
44. Reductive amination of 44 with aldehydes and ketones
provided N-alkyl derivatives 45−47 and N-cycloalkyl deriva-
tives 50 and 51. Compounds 48 and 49 were synthesized by
alkylation of 44 with 1-fluoro-2-iodoethane and 2,2,2-
trifluoroethyltrifluoromethanesulfonate, respectively. Acylation
and sulfonylation of 44 provided compounds 52 and 53. N-
Phenyl analogue 57 was obtained in four steps starting from 2-
methyl-1-(phenylamino)propan-2-ol15 in a similar strategy as
that applied for compound 43.
Figure 1. Structure and properties of hit compound 1.
To determine the effect of 4-isopropylaniline replacement by
different amines, compounds 59−87 were synthesized in two
steps from tert-butyl ester 30, as depicted in Scheme 4. The tert-
butyl group was readily removed using trifluoroacetic acid to
give carboxylic acid 58, which was converted into the desired
compounds via HATU-mediated coupling reactions with
various amines.
The preparation of compound 89 with a gem-dimethyl in α-
position of the amide was achieved following the synthetic
sequence outlined in Scheme 5, where the key step was the
alkylation of 23 using tert-butyl 2-bromoisobutyrate.
compound was characterized by good fungicidal activity against
C. albicans (MFC = 12.5 μg/mL) and an interesting inhibitory
activity against A. fumigatus (MIC = 12.5 μg/mL). Due to the
presence of the lactone moiety, we investigated the plasmatic
stability of compound 1, which was very low upon incubation
in mouse and human plasma (t1/2 = 32 and 22 min,
respectively). In this study, we describe the structural
modifications of the morpholin-2-one core to improve the
plasmatic stability of 1. In addition, we report on further
optimization of the series resulting in compounds showing in
vitro broad antifungal activity and in vivo efficacy in a mouse
candidiasis model.
RESULTS AND DISCUSSION
■
Improvement of Plasmatic Stability. Despite its
interesting in vitro fungicidal activity against C. albicans, the
development of 1 was limited due to its poor plasmatic stability
(t1/2 ≤ 32 min in human and mouse plasma). Therefore, we
first directed our chemistry toward the identification of new
compounds having improved plasmatic stability while retaining
their antifungal activity. Since we strongly suspected that the
measured plasmatic instability was due to the presence of the
lactone moiety (lactones are known to be hydrolyzed by
esterases16 contained in plasma), we decided to synthesize (i)
compound 4, which was lacking only the keto function
compared to compound 1, and (ii) a series of compounds
(33−39) substituted on the 6-position of the morpholin-2-one
core to see whether these structural modifications could lead to
the identification of active and more stable compounds. It is
indeed known that the introduction of steric hindrance close to
an ester or a lactone moiety is a well-described strategy to block
or significantly reduce hydrolysis of these functional
groups.17,18 From a C. albicans activity point of view (Table
CHEMISTRY
■
The synthesis of compound 4, in which the morpholin-2-one
core was replaced by a morpholine, was conducted from the
commercially available building block ethyl 2-(morpholin-3-
yl)acetate (2) (Scheme 1). Reductive amination with
acetaldehyde afforded N-ethyl morpholine 3. Subsequent
methyl ester hydrolysis followed by amide formation with
HATU as coupling agent provided 4.
We designed a five-step synthetic route to prepare
compounds substituted in the 5- and/or 6-position of the
morpholin-2-one core (Scheme 2). Amino-alcohols 12−18
were prepared either by reductive amination12 or by epoxide
opening13 with ethylamine. Condensation with glyoxal led to
morpholin-2-ones 19−25. Alkylation in the 3-position with α-
bromoacetate proceeded smoothly after deprotonation with
LHMDS, leading to the desired compounds as racemic
mixtures. Esters 26−32 were cleaved under acidic or basic
conditions, and the corresponding carboxylic acids were
B
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a
Scheme 2. Synthesis of Derivatives 33−39 with Substituents in the 5- and/or 6-Position on the Morpholin-2-one Core
a
Reagents and conditions: (i) acetaldehyde, NaBH4, EtOH, 0 °C, 4 h; (ii) 70% EtNH2, MeOH, sealed tube, 100 °C, overnight; (iii) 40% glyoxal,
toluene, 10 °C, 2−3 h; (iv) (a) 1 M LHMDS, THF, −70 °C, 1 h; (b) BrCH2COOEt or BrCH2COOtBu, −70 °C, 3−5 h; (v) 1N LiOH, dioxane,
water, rt, 0.5−1 h (R5 = Et) or TFA, CH2Cl2, rt, 5 h (R5 = tBu); (vi) HATU, DIPEA, 4-isopropylaniline, DMF, rt, overnight.
1), it was clear that the lactone moiety was absolutely required
since compound 4 was found to be inactive compared to 1. On
the other hand, the presence of a methyl (33 and 34), a phenyl
(35 and 36), or a gem-dimethyl (37) group on the 6-position of
the morpholin-2-one was well-tolerated, whereas introduction
of a gem-diethyl group (38) led to complete loss of activity.
Compound 39, in which an additional gem-dimethyl on the 5-
position was introduced, was also found to be inactive. From a
stereochemistry point of view, it is worth noting that the
absolute configuration of the substituents on the 6-position was
not crucial for the antifungal activity since paired compounds
(33 and 34; 35 and 36) exhibited exactly the same MFC values
(12.5 μg/mL). However, we do not know whether the chiral
center on the 3-position (where the acetamide moiety is
connected) plays a crucial role in the intrinsic activity of the
compounds since we could not separate the enantiomers by
chiral preparative HPLC (no chiral synthesis was envisaged at
that time). The human plasmatic stability of compounds that
retained fungicidal activity was assessed (Table 1). Slight
improvement in plasmatic stability was observed for com-
pounds incorporating only one substituent on the 6-position
(t1/2 < 60 min). However, compound 37, with two methyl
groups on the 6-position, showed significantly enhanced
plasmatic stability (t1/2 > 240 min). This plasma stability
increase is probably due to the formation of a steric shield by
the 2 methyl groups around the lactone that consequently
prevent its hydrolysis. On the basis of this excellent result,
compound 37 was selected as a starting point for further
development of the series.
Investigation of Structure−Activity Relationships.
Having achieved a significant improvement in plasmatic
stability compared to that of initial hit 1, we next focused on
the optimization of the series based on compound 37. First, we
investigated the role of the N-ethyl group from the morpholin-
2-one core. We therefore synthesized several derivatives bearing
modifications on this position (free NH, N-alkyl, -cycloalkyl,
-phenyl, -acyl, and -sulfonyl groups). As indicated in Table 2,
none of the synthesized compounds displayed fungicidal
activity below 25 μg/mL, providing evidence that the N-ethyl
group was essential for activity. Remarkably, even a slight
modification, such as the substitution of an hydrogen by a
fluorine on the ethyl group (48), yielded a compound devoid of
any fungicidal activity at 25 μg/mL.
We also investigated alternatives to the 4-isopropylaniline
moiety from compound 37. It is indeed well-known19,20 that
some aniline-containing compounds can sometimes be
associated with (geno)toxicity21 upon metabolic oxidative
cleavage (generation of reactive metabolites). For that
particular reason and in view of improving the biological
activity of compound 37, the 4-isopropylaniline moiety was
replaced by various amines. All newly synthesized compounds
(Table 3) were tested for their fungicidal activity against C.
albicans. Among all of the derivatives with a heteroarylamine,
only compound 65 (N-methylindole) retained potency.
Replacement of the aniline by a benzylamine (67) was well-
tolerated, whereas a further elongation of the chain (phenethyl-
amine derivative 68) led to a 2-fold decrease of fungicidal
activity. Derivatives bearing alkylamines 70−72 and cycloalkyl-
amines 74 and 75 were also associated with a 2-fold decrease in
potency. Compound 76, with a morpholine, proved to be
inactive. N-Alkylation of the amide group (69 and 73) led to
complete loss of activity, pointing toward a crucial role of the
NH of the amide group and indicating that it might be engaged
in a hydrogen bond. Furthermore, introduction of a gem-
dimethyl in the α-position of the amide (89) was detrimental to
fungicidal activity.
On the basis of the interesting activity of 67, the benzylamine
group was further explored. Phenyl replacements as well as
substitutions were investigated (Table 4). Bioisosteric replace-
ment22,23 of the phenyl ring by a thienyl (77) or a furyl (78)
led to compounds showing decreased fungicidal activity.
Similarly, compound 79 with a cyclohexyl was also found to
be less active. In order to evaluate the effect of substituents on
the phenyl ring, we synthesized compounds 80−87. Comparing
the activities of the ortho-, meta-, or para-chloro derivatives,
80−82, the meta-position seemed to be preferred since
compound 81 showed a 2-fold increase in activity compared
to that of compound 67. Moreover, dichloro substitution (83)
did not result in further improvement in potency. Introduction
of other electron-withdrawing (85) or electron-donating groups
(86) in the meta-position led to reduced activity, whereas the
presence of a methyl (84) or a phenyl (87) was well-tolerated.
C
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a
Scheme 3. Synthesis of Compounds 43−53 and 57 with Different N-Substituents on the Morpholin-2-one Core
a
Reagents and conditions: (i) benzylamine, MeOH, sealed tube, 100 °C, overnight; (ii) (a) BrCH2COOEt, DIPEA, CH3CN, rt to 55 °C, 7.5 h; (b)
pTsOH, toluene, reflux; (iii) (a) 1 M LHMDS, THF, −70 °C, 1 h; (b) BrCH2COOtBu, −70 °C, 4 h; (iv) TFA, CH2Cl2, rt, 6 h; (v) HATU, DIPEA,
4-isopropylaniline, DMF, rt, overnight; (vi) 10% Pd/C, H2, methyl acetate, rt, 5 h; (vii) aldehyde or ketone, NaBH3CN, acetic acid, THF, CH3CN,
rt, 1−18 h (for R = Me, Pr, iPr, cyclopentyl) or (1-ethoxycyclopropoxy)trimethylsilane, 3 Å molecular sieves, NaBH3CN, THF, rt to reflux, 26 h (R
= cyclopropyl) or 1-fluoro-2-iodoethane, DIPEA, THF, 88 h (R = CH2CH2F) or 2,2,2-trifluoroethyltrifluoromethanesulfonate, DIPEA, THF, reflux,
66 h (R = CH2CF3); (viii) AcCl, Na2CO3, CH2Cl2, rt, 24 h; (ix) MsCl, DIPEA, CH2Cl2, 0 °C to rt, 24 h; (x) BrCH2COOEt, K2CO3, DMF, 110 °C,
7 h.
a
Broad-Spectrum Activity. Representative compounds,
selected based on their fungicidal activities against C. albicans
and chemical differences (37, 65, 82, and 87), were screened
against a panel of fungal species, including the fluconazole-
resistant pathogenic yeast C. glabrata, the filamentous
pathogens A. fumigatus and Aspergillus flavus, and dermato-
phytes (Table 5). Onychomycosis is a common nail ailment
associated with significant physical and psychological morbidity.
Dermatophytes are the most commonly implicated etiologic
agents, particularly Trichophyton rubrum and Trichophyton
mentagrophytes, followed by Candida species.24 Commonly
used oral therapeutic agents include terbinafine, fluconazole,
and itraconazole. Hence, in this context, we tested the above
selected compounds along with the reference antimycotics
terbinafine and miconazole for activity against four dermato-
phytes (Table 5). The four selected compounds showed
antifungal activity against all tested species. Overall, 87 was the
most promising compound of those selected since it could
inhibit the growth of all tested species, with a MIC value below
3 μg/mL against A. fumigatus and IC50 values between 1 and 3
μg/mL against the four tested dermatophytes. 87 was, however,
Scheme 4. Synthesis of Compounds 59−87 with
Modification on the Amide Part
a
Reagents and conditions: (i) TFA, CH2Cl2, rt, 5 h; (ii) HATU,
DIPEA, R1R2NH, DMF, rt, overnight.
a
Scheme 5. Synthesis of Compound 89
a
Reagents and conditions: (i) (a) 1 M LHMDS, THF, −70 °C, 1 h;
(b) tert-butyl 2-bromoisobutyrate, −70 °C to −15 °C, 3 h; (ii) TFA,
CH2Cl2, rt, 5 h; (iii) HATU, DIPEA, 4-isopropylaniline, DMF, rt,
overnight.
D
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by a high abundance of miconazole-tolerant persister cells, and
miconazole is not fungicidal against this isolate (MFC
miconazole > 100 μg/mL). Most importantly, persister cells
can survive high doses of an antimicrobial agent, which partly
explains the recalcitrance of chronic infections against
antimicrobial therapy. Hence, it is very important to eradicate
such isolates. Interestingly, 87 was equally fungicidal against
this high-persister clinical isolate (MFC = 12.5 μg/mL) as
compared to that against the reference C. albicans strain (MFC
= 12.5 μg/mL), indicating that 87 is also fungicidal against
high-persister clinical isolates, which is of importance to combat
antifungal drug resistance.
Table 1. Fungicidal Activity and Human Plasmatic Stability
for Compounds 1, 4, and 33−39
human plasma
a
C. albicans
stability
b
MFC
compd
R1
H
R2
H
R3
H
R4
H
(μg/mL)
t1/2 (min)
1
12.5
>50
12.5
12.5
12.5
12.5
12.5
>25
>25
1.6
22
Pharmacological Evaluation. 37 and 87 were selected as
representative derivatives of the series for a detailed evaluation
of in vitro ADMET properties (Table 6). The data illustrate that
both compounds displayed good aqueous kinetic solubility and
moderate to high permeability without severe efflux, as
determined in the Caco2 assay. Despite the presence of the
lactone moiety (sometimes easily hydrolyzable under mild-
strong acid conditions), both 37 and 87 showed good chemical
stability in simulated gastric fluid medium (contains 800 000
units/L of pepsin at pH 1.2). The compounds were also tested
in simulated intestinal fluid medium (contains 1 000 000 units/
L of pancreatin at pH 6.8) and appeared to be stable under
these conditions, although 87 was slightly more degraded than
37. In addition to the chemical stability, the good plasmatic
stability of 87 was confirmed (t1/2 > 240 min), demonstrating
the beneficial stabilizing effect of the gem-dimethyl on the
morpholin-2-one core. Neither 37 nor 87 showed significant
hERG inhibition at 10 μM (11 and 25.5, respectively).
Moreover 37 did not inhibit major cytochrome P450 enzymes
involved in drug metabolism (<40% inhibition at 10 μM for
CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4).
However, microsomal stability appeared to be a major concern
for the series. While 37 presented reasonable human micro-
somal stability (t1/2 = 29 min), 87 was rapidly metabolized, as
demonstrated by its short half-life (t1/2 = 6 min). In view of
performing a mouse in vivo efficacy study, the stability of 37 and
87 was also assessed in mouse liver microsomes (MLM).
Similar to the human data, 37 appeared to be more stable than
87, but both compounds were rapidly metabolized in the MLM
assay (t1/2 = 6 and 1 min for compounds 37 and 87,
respectively). Some additional compounds within the series
were profiled in the MLM assay (data not shown), but,
unfortunately, all of them were also metabolized very rapidly
(t1/2 < 10 min). In order to better understand this observation,
we conducted a metabolite identification study in MLM with
compound 37. After incubation of 37 (10 μM) in MLM for 90
min, the sample was analyzed by LC-MS/MS, which led to the
identification of four major putative metabolites. Two oxidative
metabolites at 349 m/z (+16 m/z difference from parent
compound) and 365 m/z (+32 m/z difference from parent
compound) were observed. Although the data did not allow for
definitive determination of the structures, the MS/MS analysis
led us to believe that these hydroxylations were most likely
occurring on the substituted aniline moiety. The metabolite
observed at 305 m/z (−28 m/z difference from the parent)
clearly seemed to be a result of N-deethylation. Finally, the
metabolite at 331 m/z (−2 m/z difference from parent
compound) was likely a result of a dehydrogenation, but it was
impossible to conclude from the product ion spectrum where
this dehydrogenation occurred. Taking into account these
results as well as the available structure−activity relationship
c
4
ND
33
34
35
36
37
38
39
Me
H
H
H
H
57
Me
H
H
H
55
Ph
H
H
H
49
Ph
Me
Et
H
H
31
Me
Et
H
H
>240
ND
ND
ND
ND
H
H
Me
Me
Me
Me
d
AmB
e
Fluc
>100
a
b
Candida albicans SC5314 strain. MFC, minimal fungicidal
c
concentration that results in 99% killing of the inoculum. ND, not
determined. AmB, amphotericin B. Fluc, fluconazole.
d
e
Table 2. Fungicidal Activity of Compounds 37, 43−53, and
57
a
b
compd
R
C. albicans MFC (μg/mL)
37
43
44
45
46
47
48
49
50
51
52
53
57
ethyl
12.5
>50
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>50
benzyl
H
methyl
propyl
i-propyl
2-fluoroethyl
trifluoroethyl
cyclopropyl
cyclopentyl
acetyl
mesyl
phenyl
a
b
Candida albicans SC5314 strain. MFC, minimal fungicidal
concentration that results in 99% killing of the inoculum.
modestly active against A. flavus (MIC = 25 μg/mL), similar to
the other compounds except 37, which showed a 2-fold
increase in MIC. In addition to this broad-spectrum antifungal
evaluation, the cytotoxicity of compounds 37, 65, 82, and 87
was assessed against the MRC-5 cell line (see Biological
Methods section). None of the compounds showed significant
cytotoxicity at the highest tested concentration (CC50 > 20 μg/
mL). However, to have an exact indication of their therapeutic
window, higher concentrations of the compounds should be
tested with regard to cytotoxicity. Next, we assessed the
fungicidal activity of 87 against the CA2 high-persister C.
albicans clinical isolate.25 This C. albicans isolate is characterized
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Table 3. Fungicidal Activity of Compounds 37, 59−76, and 89
a
b
Candida albicans SC5314 strain. MFC, minimal fungicidal concentration that results in 99% killing of the inoculum.
might be synthesized, although the generated data point toward
a sharp SAR at this position (see Table 2).
Table 4. Fungicidal Activity of Compounds 67 and 77−87
Despite the fact that 37 was rapidly metabolized in mouse
microsomes, but taking into account its fungicidal activity, we
wondered whether it could demonstrate some in vivo efficacy in
a murine model of systemic candidiasis. Therefore, BALB/c
mice were infected intravenously with C. albicans SC5314 and
were treated intraperitoneally over 5 days, starting 16 h after
the infection, with (i) compound 37 at a dose of 10 mg/kg/day,
(ii) fluconazole at a dose of 10 mg/kg/day, and (iii) vehicle. To
determine the efficacy of therapy in murine models, a
determination of renal fungal burden as CFU is commonly
used. In a recent study, using bioluminescent C. albicans
reporter strains aiming at real-time noninvasive imaging to
monitor infection in vivo, the kidneys were confirmed to be the
main target organ.26 Hence, at the end of the study, mice were
sacrificed, and tissue burden of infection in the kidneys was
assessed. As shown in Figure 2, both fluconazole (10 mg/kg)
and 37 (10 mg/kg) yielded a significantly lower fungal load in
the kidneys (p < 0.05) compared to that from treatment with
vehicle. Encouraged by these promising results, we also decided
to assess the in vivo activity of another compound from the
series. Hence, compound 87 was tested in the same murine
model of systemic C. albicans infection with a daily intra-
peritoneal injection of 10 mg/kg. A positive control group
(fluconazole 10 mg/kg) and a vehicle control group were also
added to the study. After 5 days of treatment, compound 87
a
b
Candida albicans SC5314 strain. MFC, minimal fungicidal
concentration that results in 99% killing of the inoculum.
(SAR), one of the best options to improve the MLM stability
may be to block the oxidative hot spots from the aromatic
amides (ortho, meta, and para substitutions are tolerated, as can
be seen from Tables 3 and 4). Alternatively, some additional
compounds bearing modifications of the N-ethyl substituent
F
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Table 5. Antifungal and Fungicidal Activity of a Selection of Compounds against Yeast, Filamentous Molds, and Dermatophytes
strain
C. albicans
C. glabrata
A. fumigatus
A. flavus
T. mentagrophytes
T. rubrum
cd
M. canis
cd
S. schenckii
cd
a
a
b
b
,
,
,
MFC
MFC
MIC
MIC
IC50
IC50
IC50
cd
,
compd
(μg/mL)
(μg/mL)
(μg/mL)
(μg/mL)
IC50 (μg/mL)
(μg/mL)
(μg/mL)
(μg/mL)
37
12.5
12.5
12.5
12.5
25
1.6
12.5
25
10.6
11
6.3
6.6
11.8
10
65
50
3.1
11
6.2
82
12.5
12.5
6.2
25
2.7
2.0
2.0
2.2
87
≤3.1
25
3.0
0.70
0.02
ND
2.4
2.7
terbinafine
miconazole
0.02
ND
ND
0.02
ND
0.12
a
b
MFC, minimal fungicidal concentration that results in 99% killing of inoculum. MIC, minimal inhibitory concentration that inhibits growth of the
c
d
fungus. IC50, minimal concentration that inhibits growth for 50% compared to nontreated controls ND, not determined
Table 6. ADMET Properties of Compounds 37 and 87
37
87
kinetic solubility at pH 7.4 (μM)
permeability [Papp (10−6 cm s−1)]
50
50
65 (A−B)
35 (B−A)
93.9
22 (A−B)
13 (A−B)
99.7
human protein plasma binding (% bound)
chemical stability
a
SGF [half-life (min)]
>240
>240
>240
29
>240
223
>240
6
b
SIF [half-life (min)]
human plasma stability [half-life (min)]
human liver microsomal stability
[half-life (min)]
mouse liver microsomal stability
[half-life (min)]
6
1
hERG (% inhibition at 10 μM)
11
25.5
a
b
SGF, simulated gastric fluid. SIF, simulated intestinal fluid.
Figure 3. In vivo efficacy of compound 87 in a candidiasis mouse
model (C. albicans SC 5314). CFU from kidneys (K), spleen (S), and
liver (L) of BALB/c infected mice (n = 10) after 5 days of
intraperitoneal administration at 10 mg/kg/day of 87 or vehicle alone
(DMSO/0.5% methylcellulose, 5:95) or 10 mg/kg/day intraperitoneal
fluconazole. *** p < 0.0001.
activities remain modest compared to that of marketed
fungicidal compounds such as amphotericin B. Furthermore,
the overall metabolic stability of the series is low both in mouse
and human microsomes and should therefore be optimized. To
this end, one option may be to reduce or block the N-
deethylation cleavage as well as the oxidation sites on the
aromatic amide moiety observed in the metabolite identi-
fication study. Additionally, it will be interesting to separate the
pure enantiomers of compounds 37 and 87 and see whether
the chiral center on the 3-position plays a role on the in vitro
activities as well as on the overall ADMET properties.
Mode of Action Study. In order to start unravelling the
exact mechanism of action of these compounds, we assessed
potential antagonism and synergy between compound 87 and a
selection of 96 chemical compounds affecting diverse cellular
processes. To this end, we used a phenotype microarray assay
and tested 87 at two concentrations, namely, 12.5 μg/mL
(which equals the MFC toward C. albicans) and 50 μg/mL.
Both concentrations resulted in a significant growth reduction
of the model yeast Saccharomyces cerevisiae (data not shown).
We found that sodium cyanide (respiration inhibitor), sodium
thiosulfate (calcium remover), chlortetracycline hydrochloride
(calcium binder), potassium chromate (oxidative agent), or
monothioglycerol (respiration inhibitor) antagonized the
antifungal activity of compound 87 at 12.5 and 50 μg/mL
against S. cerevisiae. These data point toward the fact that 87
Figure 2. In vivo efficacy of compound 37 in a candidiasis mouse
model (C. albicans SC 5314). Colony forming units (CFU) from
kidneys (K) of BALB/c infected mice (n = 5) after 5 days of
intraperitoneal treatment with fluconazole (10 mg/kg), 37 (10 mg/
kg), and vehicle alone (DMSO/0.5% methylcellulose, 5:95). * p <
0.05.
exhibited a marked in vivo antifungal effect similar to the effect
of fluconazole and seemed to be effective in controlling the
fungal infection. The fungal load in the kidneys was significantly
reduced, and, as expected, no fungi were detected in liver and
spleen (Figure 3). Despite their low mouse microsomal
stability, both 37 and 87 showed very good in vivo efficacy in
our C. albicans infection mouse model. Although these results
clearly demonstrate the potential for this new chemical series,
more medicinal chemistry efforts are necessary to bring it to a
further stage of development. Indeed, the in vitro fungicidal
G
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To a solution of the carboxylic acid (0.199 mmol) in DMF (1.5
mL) cooled at 0 °C were added HATU (0.140 g; 0.369 mmol) and
DIPEA (0.103 mL; 0.590 mmol). After 1 h at 0 °C, 4-isopropylaniline
(0.034 mL; 0.249 mmol) was added, and the reaction mixture was
stirred overnight at room temperature. The reaction mixture was
partitioned between CH2Cl2 and saturated NaHCO3. The phases were
separated. The organic phase was washed with water and brine, dried
over Na2SO4, filtered, and concentrated under reduced pressure. The
residue was purified by flash chromatography on silica gel using a
gradient of MeOH (0 to 10%) in CH2Cl2 to yield 0.016 g (28%) of the
title compound as a yellow oil. ESI/APCI(+): 291 (M + H); 313 (M +
Na). 1H NMR (DMSO-d6, 300 MHz) δ 10.55 (1H, br s), 7.44 (2H, d,
J = 8.4 Hz), 7.18 (2H, d, J = 8.5 Hz), 3.85 (2H, m), 3.65 (2H, m),
2.8−3.1 (5H, m), 2.25−2.5 (3H, m), 1.23 (1H, d, J = 6.9 Hz), 1.17
(3H, t, J = 7.2 Hz).
requires calcium and a functional respiratory chain to exert its
antifungal activity against S. cerevisiae. However, the precise
mechanism of action could not be determined yet and therefore
additional studies will be required in the future.
CONCLUSIONS
■
In summary, we discovered and optimized a new class of
morpholin-2-one derivatives as antifungal agents. Hit 1,
identified from a screening campaign, showed interesting
fungicidal properties against C. albicans, but its further
development was limited by its plasma instability. By
introducing a gem-dimethyl on the 6-position of the
morpholin-2-one core, we succeeded in obtaining a compound
that combined both fungicidal activity and excellent plasmatic
stability. Synthetic routes were developed to allow introduction
of structural modifications at the last step of the sequence.
Extensive SAR revealed that the aniline moiety could be
replaced by various amines, from which meta-substituted
benzylamines turned out to be the best. However, all of the
modifications to the ethyl residue led to inactive compounds,
showing that this N-ethyl substituent was crucial for fungicidal
activity. Compounds from this new chemical series also showed
broad-spectrum in vitro activity against resistant Candida
isolates and against various fungal species, including dermato-
phytes. The preliminary mechanism of action study demon-
strated that compound 87 requires calcium and a functional
respiratory chain to exert its antifungal activity against S.
cerevisiae. Finally, compounds 37 and 87 demonstrated clear in
vivo efficacy when evaluated in a murine systemic candidiasis
model.
General Procedure for Epoxide Opening with an Amine.
Exemplified for 1-(Ethylamino)-2-methylpropan-2-ol (16). To a
solution of isobutylene oxide (3.5 mL, 39.7 mmol) in MeOH (60 mL)
in a sealed tube was added a 70% ethylamine solution in water (7.2
mL; 104 mmol). The reaction mixture was heated overnight at 100 °C.
The reaction mixture was concentrated under reduced pressure.
Purification by distillation under reduced pressure furnished 4.42 g
(95%) of the title compound as a colorless liquid. ESI/APCI(+): 118
1
(M + H). H NMR (CDCl3, 300 MHz) δ 2.70 (2H, q, J = 7.5 Hz),
2.53 (2H, s), 1.17 (6H, s), 1.11 (3H, t, J = 7.5 Hz).
General Procedure for Morpholin-2-one Formation. Exem-
plified for 4-Ethyl-6,6-dimethylmorpholin-2-one (23). To a
mixture of a 40% glyoxal solution in water (4.5 mL; 39.2 mmol) in
toluene (20 mL) cooled at 10 °C was added a solution of 16 (4.4 g;
37.7 mmol) in toluene (11 mL). After 2 h at 10 °C, the reaction
mixture was refluxed to azeotrope out solvents. Purification by
distillation under reduced pressure furnished 4.72 g (80%) of the title
compound as a colorless liquid. ESI/APCI(+): 158 (M + H); 180 (M
+ Na). 1H NMR (CDCl3, 300 MHz) δ 3.21 (2H, s), 2.47 (2H, s), 2.44
(2H, q, J = 7.3 Hz), 1.43 (6H, s), 1.09 (3H, t, J = 7.3 Hz). 13C NMR
(CDCl3, 300 MHz) δ 168.5, 99.8, 81.5, 59.3, 54.6, 51.0, 26.9, 11.7.
General Procedure for Morpholin-2-one Alkylation in 3-
Position. Exemplified for tert-Butyl 2-(4-Ethyl-6,6-dimethyl-2-
oxomorpholin-3-yl)acetate (30). To a solution of 24 (2.0 g; 12.7
mmol) in THF (80 mL) cooled at −70 °C was added dropwise a 1 M
LHMDS solution in THF (12.7 mL; 12.7 mmol). After 1 h at −70 °C,
tert-butyl bromoacetate (2.1 mL; 14.1 mmol) was added dropwise, and
the reaction mixture was stirred at −70 °C for 4 h. The reaction was
quenched by addition of saturated NH4Cl. After warming to room
temperature, the solids were filtered, and the filtrate was concentrated
under reduced pressure. The residue was taken up with EtOAc, dried
over Na2SO4, filtered, and concentrated under reduced pressure.
Purification by flash chromatography on silica gel using a gradient of
EtOAc (10 to 40%) in heptane furnished 2.61 g (75%) of the title
compound as a white solid. ESI/APCI(+): 272 (M + H); 294 (M +
EXPERIMENTAL SECTION
■
Chemistry. All reagents and solvents were purchased from
commercial sources and used without further purification. Flash
chromatography purifications were performed on Biotage prepacked
silica gel columns using Biotage Isolera or SP4 instruments. TLC was
carried out with Macherey-Nagel Alugram Sil G/UV254 plates. TLC
plates were revealed with UV light, KMnO4, p-anisaldehyde, or
1
ninhydrine solutions. H NMR spectra were recorded on a 300 MHz
Bruker spectrometer. Proton chemical shifts are reported in parts per
million (δ) using TMS as a standard. Electrospray mass (ESI)
measurements were obtained on a Bruker Esquire 6000 mass
spectrometer. The purity of all compounds screened in biological
assays was >95%. Purity was determined by LC-MS recorded on a
system consisting of a Dionex Ultimate 3000 HPLC equipped with a
PDA detector and a Bruker Esquire 6000 mass spectrometer, using a
C-18 column (SunFire C18, 3.5 μm, 3.0 × 100 mm or XBridge C18,
3.5 μm, 3.0 × 100 mm).
Ethyl 2-(4-Ethylmorpholin-3-yl)acetate (3). A mixture of ethyl
2-(morpholin-3-yl)acetate (2) (0.300 g; 1.73 mmol), sodium acetate
(0.226 g; 2.75 mmol), and acetaldehyde (0.291 mL; 5.19 mmol) in
MeOH (2 mL) was stirred at room temperature for 1 h. The reaction
mixture was concentrated under reduced pressure. The residue was
taken up with EtOAc, dried over Na2SO4, filtered, and concentrated
under reduced pressure. The residue was purified by flash
chromatography on silica gel using a gradient of MeOH (0 to 10%)
in CH2Cl2 to yield 0.256 g (73%) of the title compound as a yellow oil.
ESI/APCI(+): 202 (M + H).
1
Na). H NMR (CDCl3, 300 MHz) δ 3.32 (1H, m), 3.00 (1H, m),
2.75−2.85 (3H, m), 2.30−2.40 (2H, m), 1.50 (3H, s), 1.46 (9H, s),
1.38 (3H, s), 1.05 (3H, t, J = 7.1 Hz). 13C NMR (CDCl3, 300 MHz) δ
170.4, 170.0, 81.0, 80.4, 60.9, 57.5, 47.2, 36.5, 28.1, 27.3, 26.4, 11.1.
General Procedure for tert-Butyl Ester Cleavage. Exempli-
fied for 2-(4-Ethyl-6,6-dimethyl-2-oxomorpholin-3-yl)acetic
Acid (58). To a solution of 30 (0.300 g; 1.11 mmol) in CH2Cl2 (5
mL) was added TFA (1.7 mL). After 5 h at room temperature, the
reaction mixture was concentrated and coevaporated with toluene to
give quantitatively the title compound under its trifluoroacetate salt
form, which was used in the next step without further purification.
General Procedure for Amide Formation. Exemplified for 2-
(4-Ethyl-6,6-dimethyl-2-oxomorpholin-3-yl)-N-(4-
isopropylphenyl)acetamide (37). To a solution of 58 (0.442
mmol) in DMF (2.9 mL) were added DIPEA (0.385 mL; 2.20 mmol)
and HATU (0.203 g; 0.534 mmol). After 30 min at room temperature,
4-isopropylaniline (0.074 mL; 0.541 mmol) was added, and the
reaction mixture was stirred overnight at room temperature. The
solvent was evaporated under reduced pressure. The residue was
partitioned between CH2Cl2 and saturated NaHCO3. The phases were
separated. The organic phase was washed with water and brine, dried
2-(4-Ethylmorpholin-3-yl)-N-(4-isopropylphenyl)acetamide
(4). To a solution of 3 (0.040 g; 0.199 mmol) in a mixture of dioxane/
water (2 mL; 1:1) was added 1 N LiOH (0.400 mL; 0.400 mmol).
After 45 min at room temperature, the reaction mixture was acidified
(pH 5) with 1 N HCl, concentrated under reduced pressure, and
coevaporated with toluene to give the carboxylic acid, which was used
without further purification.
H
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over Na2SO4, filtered, and concentrated under reduced pressure.
Purification by flash chromatography on silica gel using a gradient of
EtOAc (30 to 80%) in heptane furnished 0.132 g (90%) of the title
compound as a beige powder. ESI/APCI(+): 333 (M + H); 355 (M +
stirred overnight at room temperature. Water was added, and the
phases were separated. The organic phase was washed with brine,
dried over Na2SO4, filtered, and concentrated under reduced pressure.
Purification by flash chromatography on silica gel using a gradient of
MeOH (0 to 8%) in CH2Cl2 followed by recrystallization from EtOAc
furnished 0.053 g (58%) of the title compound as a white powder.
ESI/APCI(+): 347 (M + H); 369 (M + Na); 715 (2M + Na). ESI/
APCI(−): 345 (M + H). 1H NMR (CDCl3, 300 MHz) δ 7.69 (1H, br
s), 7.39 (2H, d, J = 8.4 Hz), 7.16 (2H, d, J = 8.4 Hz), 4.90 (1H, t, J =
3.8 Hz), 4.11 (1H, m), 3.55 (1H, m), 3.31 (2H, m), 2.86 (1H, m),
2.14 (3H, s), 1.50 (3H, s), 1.37 (3H, s), 1.21 (6H, d, J = 7.0 Hz). 13C
NMR (CDCl3, 300 MHz) δ 169.8, 168.6, 168.0, 145.2, 135.2, 126.9,
120.4, 81.0, 51.9, 50.6, 38.6, 33.6, 26.0, 24.8, 24.0, 21.7.
1
Na). ESI/APCI(−): 331 (M − H). H NMR (CDCl3, 300 MHz) δ
8.49 (1H, br s), 7.40 (2H, d, J = 8.4 Hz), 7.16 (2H, d, J = 8.4 Hz), 3.41
(1H, m), 2.8−3.1 (5H, m), 2.4−2.55 (2H, m), 1.47 (3H, s), 1.41 (3H,
s), 1.22 (6H, d, J = 6.8 Hz), 1.11 (3H, t, J = 7.1 Hz). 13C NMR
(CDCl3, 300 MHz) δ 171.1, 167.9, 144.9, 135.6, 126.8, 120.1, 81.0,
61.3, 57.6, 47.4, 37.6, 33.6, 27.2, 26.7, 24.0, 10.9.
2 - ( 6 , 6 - D i m e t h y l - 2 - o x o m o r p h o l i n - 3 - y l ) - N - ( 4 -
isopropylphenyl)acetamide (44). To a mixture of 2-(4-benzyl-6,6-
dimethyl-2-oxomorpholin-3-yl)-N-(4-isopropylphenyl) acetamide
(43) (0.500 g; 1.27 mmol) in methyl acetate (20 mL) was added
10% Pd/C (0.500 g). The reaction mixture was stirred at room
temperature for 5 h under a hydrogen atmosphere and then was
filtered through Celite. The filtrate was concentrated under reduced
pressure. Purification by flash chromatography on silica gel using a
gradient of MeOH (3 to 8%) in CH2Cl2 furnished 0.345 g (89%) of
the title compound as a white foam. ESI/APCI(+): 305 (M + H); 327
(M + Na); 609 (2M + H); 631 (2M + Na). ESI/APCI(−): 303 (M −
2-(6,6-Dimethyl-4-(methylsulfonyl)-2-oxomorpholin-3-yl)-N-
(4-isopropylphenyl)acetamide (53). To a solution of 44 (0.080 g;
0.263 mmol) and DIPEA (0.092 mL; 0.527 mmol) in CH2Cl2 (5 mL)
cooled at 0 °C was added mesyl chloride (0.031 mL; 0.401 mmol).
After 4 h at 0 °C, DIPEA (0.092 mL; 0.527 mmol) and mesyl chloride
(0.031 mL; 0.401 mmol) were added again. The reaction mixture was
stirred at 0 °C for 4 h and overnight at room temperature. The
reaction mixture was diluted with CH2Cl2 and washed with saturated
NaHCO3. The organic phase was washed with water and brine, dried
over Na2SO4, filtered, and concentrated under reduced pressure.
Purification by flash chromatography on silica gel using a gradient of
EtOAc (20 to 60%) in heptane furnished 0.076 g (76%) of the title
compound as a white foam. ESI/APCI(+): 383 (M + H); 405 (M +
Na); 787 (2M + Na). ESI/APCI(−): 381 (M − H). 1H NMR
(CDCl3, 300 MHz) δ 7.39 (1H, br s), 7.35 (2H, d, J = 8.5 Hz), 7.18
(2H, d, J = 8.5 Hz), 4.78 (1H, t, J = 4.0 Hz), 3.60 (2H, m), 3.21 (2H,
m), 3.06 (3H, s), 2.87 (1H, m), 1.49 (3H, s), 1.43 (3H, s), 1.22 (6H,
d, J = 7.1 Hz). 13C NMR (CDCl3, 300 MHz) δ 168.0, 167.3, 145.7,
134.8, 127.0, 120.5, 82.5, 52.5, 50.1, 41.7, 39.7, 33.6, 25.9, 25.5, 24.0.
Biological Methods. Microorganisms and Materials. The
yeast strains used in this study were S. cerevisiae BY4741 (Euroscarf,
Germany), C. albicans strain SC5314,27 and C. glabrata strain BG2.28
The fungal strains used in this study were A. fumigatus (CBS 117202),
A. flavus (CBS111.45), T. rubrum (B68183), T. mentagrophytes
(B70554), Microsporus canis (B68128), and Sporothrix schenkii
(B62482). Spore suspensions were prepared as previously described.29
The fungal isolates were obtained from the Scientific Institute of
Public Health (IHEM, Brussels, Belgium) and cultivated on Sabouraud
dextrose agar (SDA) (Oxoid). PBS consists of 8 g/L NaCl, 0.2 g/L
KCl, 1.44 g/L Na2HPO4, and 0.24 g/L KH2PO4 (pH 7.4).
Methylcellulose 0.5% was from Sigma (St. Louis, MO, USA). YPD
consists of YPD 10 g/L yeast extract, 20 g/L peptone, and 20 g/L
glucose).
1
H). H NMR (CDCl3, 300 MHz) δ 7.61 (1H, br s), 7.38 (2H, d, J =
8.4 Hz), 7.17 (2H, d, J = 8.4 Hz), 3.80 (1H, m), 2.8−3.1 (6H, m), 1.52
(3H, s), 1.40 (3H, s), 1.22 (6H, d, J = 6.7 Hz).
General Procedure for Reductive Amination. Exemplified
for N-(4-Isopropylphenyl)-2-(4,6,6-trimethyl-2-oxomorpholin-
3-yl)acetamide (45). To a solution of 44 (0.080 g; 0.263 mmol) in
THF (1.4 mL) and CH3CN (3.8 mL) was added a 37% formaldehyde
solution in water (0.098 mL; 1.34 mmol). After 15 min at room
temperature, NaBH3CN (0.033 g; 0.525 mmol) was added
portionwise. The reaction mixture was stirred for an additional 15
min, and acetic acid (0.031 mL; 0.542 mmol) was added. The reaction
mixture was stirred at room temperature for 1 h. The reaction was
quenched by addition of saturated NaHCO3, and the reaction mixture
was diluted with EtOAc. The phases were separated. The organic
phase was washed with water and brine, dried over Na2SO4, filtered,
and concentrated under reduced pressure. Purification by flash
chromatography on silica gel using a gradient of EtOAc (30 to
80%) in heptane furnished 0.045 g (54%) of the title compound as a
white foam. ESI/APCI(+): 319 (M + H); 341 (M + Na); 659 (2M +
1
Na). ESI/APCI(−): 317 (M − H). H NMR (CDCl3, 300 MHz) δ
8.21 (1H, s), 7.40 (2H, d, J = 8.4 Hz), 7.15 (2H, d, J = 8.4 Hz), 3.05−
3.15 (2H, m), 2.80−2.95 (3H, m), 2.49 (1H, m), 2.47 (3H, s), 1.49
(3H, s), 1.39 (3H, s), 1.21 (6H, d, J = 7.4 Hz). 13C NMR (CDCl3, 300
MHz) δ 170.5, 167.7, 144.9, 135.5, 126.8, 120.1, 80.8, 63.8, 62.4, 43.4,
37.5, 33.6, 27.1, 26.9, 24.0.
Antifungal and Fungicidal Activity Tests. The fungicidal
activity of the compounds against C. albicans and C. glabrata was
determined in PBS, and the MFC for each compound was calculated
according to the definition of Thevissen and co-workers.30 To this end,
overnight cultures of C. albicans or C. glabrata in YPD (1% yeast
extract, 2% peptone, and 2% glucose) were 1:200 and 1:400 diluted in
PBS, respectively, and treated with the compounds or DMSO (2.5% as
solvent control) for 24 h at 37 °C. After 24 h, the MFC was calculated
by counting CFUs.31 Antifungal activity of protein samples against the
filamentous fungi was assayed by microspectrophotometry of liquid
cultures grown in microtiter plates as described previously.29 To
determine the MIC of a compound against A. fumigatus or A. flavus, a
2-fold dilution series of the compound was incubated with the
corresponding spore suspension in PDB (potato dextrose broth,
Difco) (2 × 104 spores/mL). After 24 h incubation at 25 °C, MIC was
determined as the minimal concentration that inhibits growth of the
fungus.
The in vitro susceptibility screens with dermatophytes were
performed as previously described.32 Briefly, 10 μL of prediluted
compound solution was spotted onto 96-well plates (U-bottom;
Greiner Bio-One), with 64 μM as the highest concentration; 103 CFU
in 200 μL of RPMI-MOPS was added to each well. After incubation,
growth inhibition was measured after adding 10 μL/well 0.005% (w/
v) resazurin (Sigma), allowing fluorimetric reading (λex, 550 nm; λem,
General Procedure for N-Alkylation. Exemplified for 2-(4-(2-
Fluoroethyl)-6,6-dimethyl-2-oxomorpholin-3-yl)-N-(4-
isopropylphenyl)acetamide (48). To a solution of 44 (0.059 g;
0.194 mmol) in THF (3 mL) heated at 80 °C were added
progressively DIPEA (0.266 mL; 1.52 mmol) and 1-fluoro-2-
iodoethane (0.266 g; 1.53 mmol) over 6 days. The reaction mixture
was concentrated under reduced pressure. The residue was dissolved
in EtOAc and washed with saturated NaHCO3. The organic phase was
washed with water and brine, dried over Na2SO4, filtered, and
concentrated under reduced pressure. Purification by flash chromatog-
raphy on silica gel using a gradient of EtOAc (20 to 80%) in heptane
furnished 0.022 g (32%) of the title compound as a white foam. ESI/
1
APCI(+): 351 (M + H); 373 (M + Na); 723 (2M + Na). H NMR
(CDCl3, 300 MHz) δ 7.99 (1H, br s), 7.39 (2H, d, J = 8.4 Hz), 7.16
(2H, d, J = 8.4 Hz), 4.71 (1H, m), 4.55 (1H, m), 3.58 (1H, m), 2.7−
3.2 (6H, m), 2.65 (1H, m), 1.46 (3H, s), 1.40 (3H, s), 1.22 (6H, d, J =
7.1 Hz). 13C NMR (CDCl3, 300 MHz) δ 170.9, 167.8, 145.0, 135.4,
126.8, 120.2, 83.5, 81.2, 61.4, 59.1, 53.4, 38.4, 33.6, 26.9, 26.4, 24.0.
2-(4-Acetyl-6,6-dimethyl-2-oxomorpholin-3-yl)-N-(4-
isopropylphenyl)acetamide (52). To a mixture of 44 (0.080 g;
0.263 mmol) and Na2CO3 (0.084 g; 0.793 mmol) in CH2Cl2 (5 mL)
was added acetyl chloride (0.028 mL; 0.394 mmol). After 3 h at room
temperature, Na2CO3 (0.042 g; 0.396 mmol) and acetyl chloride
(0.028 mL; 0.394 mmol) were added again. The reaction mixture was
I
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Article
590 nm). Activity is expressed as IC50, i.e., the concentration that
inhibits growth by 50% compared to nontreated controls. Miconazole
and terbinafine were included as reference antifungals, and they were
purchased from Sigma. Three independent replicates were performed
for each observation.
ASSOCIATED CONTENT
* Supporting Information
Experimental and analytical data of all intermediates and final
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
■
S
Cytotoxicity Assay.32 MRC-5 SV2 cells, human fetal lung
fibroblasts, were cultivated in MEM, supplemented with L-glutamine
(20 mM), 16.5 mM sodium hydrogen carbonate, and 5% FCS, at 37
°C and 5% CO2. For the assay, 104 MRC-5 cells/well were seeded
onto the test plates containing the prediluted compounds and
incubated at 37 °C and 5% CO2 for 72 h. After 72 h of incubation,
parasite growth was assessed fluorimetrically by adding resazurin for 24
h at 37 °C. Fluorescence was measured using a GENios Tecan
fluorimeter (λex, 530 nm; λem, 590 nm). CC50 values are calculated
from duplicate determinations, with relative difference below 25%.
ADMET Assays. The chemical stability (SIF and SGF medium),
plasma stability, microsomal stability, and plasma protein binding
assays were performed by Anthem Biosciences (http://www.
anthembio.com/). The CYP inhibition, hERG inhibition, and Caco2
permeability assays and the metabolite identification study were
performed by CEREP (www.cerep.fr).
AUTHOR INFORMATION
Corresponding Author
*Tel: +3216852604; Fax: +3216852601; E-mail: arnaud.
marchand@cistim.be.
■
Author Contributions
○D.B. and K.T. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
K.T. and P.C. acknowledge the receipt of a postdoctoral
fellowship from the Industrial Research Fund (KU Leuven).
Furthermore, the research leading to these results received
funding from the INTERREG IV-project Incubatorennet-
werk(t) (IVA-VLANED-1.54). We thank Els Meert for
Mouse Systemic Candidiasis Model. BALB/c, 6 to 8 week-old
male mice, were bred at the University of Sao Paulo animal facility
̃
under specific pathogen-free conditions. All animals were handled in
accordance with good animal practice as defined by the relevant
national animal welfare bodies, and all in vivo testing was approved by
the Institutional Animal Care and Use Committee of the University of
̀
technical assistance and Agnes Calleja for analytical support.
ABBREVIATIONS USED
■
MFC, minimal fungicidal concentration; MIC, minimal
inhibitory concentration; HATU, O-(7-azabenzotriazol-1-yl)-
N,N,N′,N′-tetramethyluronium hexafluorophosphate; LHMDS,
lithium hexamethyldisilazide; pTsOH, p-toluene sulfonic acid;
DIPEA, N,N-diisopropylethylamine; CFU, colony forming unit;
QD, quaque die (once a day); BID, bis in die (twice a day);
PM, phenotype microarray
Sao Paulo. BALB/c mice were treated during the experiment with 100
̃
mg/kg cyclophosphamide (Sigma), at 4 days and 24 h before onset of
infection, and an additional dose at 3 days postinfection. To establish
the C. albicans infection, 2 × 103 C. albicans SC5314 cells from an
overnight culture in brain heart infusion (Difco) at 37 °C were
suspended in 100 μL of sterile saline and injected intravenously in
BALB/c mice. Compound 37 or 87 formulated in DMSO/0.5%
methylcellulose in water (5:95) was administered by intraperitoneal
injection at 10 mg/kg/day. As a control, mice were treated by IP
injection with fluconazole (10 mg/kg/day) formulated in water or
with vehicle alone [DMSO/0.5% methylcellulose in water (5:95)].
Administration of the compounds/vehicle started 16 h after the
challenge with C. albicans and was continued daily for 5 days.
To evaluate the fungal burden, kidney, spleen, and liver of the mice
were dissected aseptically on day 7 after infection, weighed, and
homogenized in 1 mL of PBS. Aliquots of the homogenate (100 μL)
were seeded in brain heart infusion (BHI, Difco) containing 2% agar.
After incubation for 18 h at 37 °C, the number of CFUs was
determined. The effectiveness of different treatments was determined
by considering the number of CFUs per gram of tissue of treated
animals compared with the number of CFUs per gram of tissue of
animals treated with vehicle alone. ANOVA with the post-Tukey test
was used to evaluate the statistical significance of results obtained in all
experiments. The differences between the results obtained by
treatment with the compounds compared to the control were
considered statistically significant when the p value was less than 0.05.
Phenotype Microarray (PM) Assay. The PM22-PM25 chemical
sensitivity test microplates for fungi were used to compare the cellular
phenotypes of S. cerevisiae BY4741 treated with or without compound
87 under 96 different conditions. The layout and contents of these
plate set can be viewed at http://www.biolog.com/pdf/pm_lit/PM21-
PM25.pdf. Preparation of the different IF (inoculating fluids;
proprietary formulation supplied by BIOLOG, Hayward, CA, USA)
solutions and inoculation of the PM plates was performed according to
the BIOLOG PM protocol for yeast/fungi, using an overnight S.
cerevisiae culture grown in YPD (final culture dilution was 1:500 in the
plates). Plates were incubated at 37 °C for 24 h in the Omnilog plate
reader (BIOLOG).
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