Synthesis and in vitro and in vivo antifungal activity of the hydroxy metabolites of saperconazole

Article abstract of DOI:10.1002/cmdc.201000040

Herein we describe the scalable diastereoselective and enantio-selective syntheses of eight enantiomers of hydroxy metabolites of saperconazole. The in vitro antifungal activity of the eight stereoisomers (compounds 1-8) was compared against a broad panel

Full text of DOI:10.1002/cmdc.201000040

DOI: 10.1002/cmdc.201000040
Synthesis and in vitro and in vivo Antifungal Activity of
the Hydroxy Metabolites of Saperconazole
Lieven Meerpoel,*[a] Jan Heeres,[a] Leo J. J. Backx,[a] Louis J. E. Van der Veken,[a]
Rob Hendrickx,[a] David Corens,[a] Alex De Groot,[a] Stef Leurs,[a] Luc Van der Eycken,[a]
Johan Weerts,[a] Paul Luyts,[a] Frans Van Gerven,[a] Filip A. A. Woestenborghs,[a]
Andre Van Breda,[a] Michel Oris,[a] Pascal van Dorsselaer,[a] Gustaaf H. M. Willemsens,[a]
Danny Bellens,[a] Patrick J. M. G. Marichal,[a] Hugo F. Vanden Bossche,[a] and Frank C. Odds*[b]
Herein we describe the scalable diastereoselective and enantio-
selective syntheses of eight enantiomers of hydroxy metabo-
lites of saperconazole. The in vitro antifungal activity of the
eight stereoisomers (compounds 1–8) was compared against a
broad panel of Candida spp. (n=93), Aspergillus spp. (n=10),
Cryptococcus spp. (n=19), and dermatophytes (n=27). The
four 2S isomers 1–4 of the new agent were generally slightly
more active than the four 2R isomers 5–8. All eight isomers
were tested in a model of experimental A. fumigatus infection
in guinea pigs by intravenous inoculation of the fungal coni-
dia. Treatment doses were 1.25 mgkgꢀ1 and 2.5 mgkgꢀ1 per
day. Infection severity was measured in terms of mean survival
time (MST) after infection and mean tissue burdens in brain,
liver, spleen, and kidney at postmortem examination. Among
the eight isomers, the 2S diastereomers 1–4 showed a general-
ly higher level of activity than the 2R diastereomers 5–8, re-
vealing compounds 1 and 4 as the most potent overall in
eradicating tissue burden and MST. Compared with reference
compounds itraconazole and saperconazole, the hydroxy iso-
mers 1–8 are less potent inhibitors of the growth of A. fumi-
gatus in vitro and of ergosterol biosynthesis in both A. fumi-
gatus and C. albicans.
Introduction
Systemic fungal infections (SFI) are life-threatening conditions
that most commonly affect patients with decreased immunity,
which often results from therapeutic interventions to treat ma-
lignant diseases.[1–10] The number of various fungi that have
been involved in SFI is large and still growing. Despite many
cases of invasive candidiasis and aspergillosis, there has been
an increased incidence of infections due to other molds such
as Scedosporium apiospermum, Fusarium spp., and Zygomy-
cetes, Rhizopus and Mucor spp.[9–11] Therefore, effective thera-
peutic agents to treat all these infections must have a very
broad spectrum of activity. Over the past few decades itraco-
nazole,[12–14] fluconazole,[15] ketoconazole,[16] and intravenous or
liposomal amphotericin B[17] have been used to treat SFI, and
all these agents have their limitations with regard to spectrum,
safety, or ease of administration.
Of all antimycotic agents, azoles still represent a unique class
of compounds that display the broadest antifungal spectrum
via inhibition of 14-a-demethylase, an enzyme essential for er-
gosterol biosynthesis in fungi.[27]
Taking itraconazole as a starting point for further structural
optimization, we have from previous work that replacement of
the 2,4-dichlorophenyl moiety with
a 2,4-difluorophenyl
moiety (see saperconazole, Figure 1) results in significant im-
provement of potency and spectrum, especially against Asper-
gillus spp.[28,29] Moreover, strategies to enhance the aqueous
solubility of poorly soluble drugs often rely on prodrug
design.[30] The prodrug design we envisaged started from the
identification of hydroxyitraconazole as a major antifungal-
[a] Dr. L. Meerpoel, Dr. J. Heeres, L. J. J. Backx, L. J. E. Van der Veken,
R. Hendrickx, Dr. D. Corens, Dr. A. De Groot, Dr. S. Leurs, L. Van der Eycken,
J. Weerts, P. Luyts, F. Van Gerven, F. A. A. Woestenborghs, A. Van Breda,
M. Oris, P. van Dorsselaer, G. H. M. Willemsens, D. Bellens,
Dr. P. J. M. G. Marichal, H. F. Vanden Bossche
More recently, a third generation of azoles has been investi-
gated and introduced to the market (Figure 1), improving the
treatment options in intensive care units. Voriconazole
(VfendTM [18,19] and posaconazole (NoxofilTM [20–23]
) have shown
)
Johnson & Johnson Pharmaceutical Research & Development, a division of
Janssen Pharmaceutica N.V., Turnhoutseweg 30, 2340 Beerse (Belgium)
Fax. (+32)14-60-53-44
much improvement in the treatment of life-threatening inva-
sive SFI such as candidiasis, aspergillosis, and infections due to
Fusarium spp. at clinically relevant dosages. Moreover, posaco-
nazole shows efficacy against infections caused by emerging
species of Zygomycota.[24] Echinocandins such as anidulafungin,
caspofungin, and micafungin, which are noncompetitive inhibi-
tors of 1,3-b-glucan synthesis in fungal cell walls, display high
efficacy against Candida and Aspergillus spp., but have no ac-
tivity against Cryptococcus, Fusarium, or Zygomycetes spp.[25,26]
E-mail: lmeerpoe@its.jnj.com
[b] Prof. F. C. Odds
The Old Mill, North Coldstream, Drumoak, AB31 5EP (UK)
E-mail: f.odds@abdn.ac.uk
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201000040: Individual dose–response
curves for the in vitro antifungal activity of compounds 1–8 against a
broad range of yeasts and moulds.
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L. Meerpoel, F. C. Odds, et al.
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pure cis-dioxolanes 17a (2S)
and 18 (2R), which were previ-
ously described,[42] and the phe-
nols 12a–d in dry DMF in the
presence of sodium hydroxide
pellets (see Scheme 3 in the Ex-
perimental Section below). An
improved yield (91%) and a
scalable method for compound
1 was worked out by changing
the solvent to DMA and by op-
timizing the workup method
without the need for chromato-
graphic purification of target
product 1, by starting from di-
oxolane 17b and phenol 12a.
The key step for this synthe-
sis, however, was the diastereo-
selective and enantioselective
syntheses of compounds 12a–
Figure 1. Antifungal azoles for the treatment of systemic fungal infections (SFI).
d. The diastereoselective syn-
thesis started from a selective
reduction of ketone 11, easily
active metabolite of itraconazole.[31–35] A similar metabolite is
generated in vivo from saperconazole. The hydroxy metabo-
lites 1–8 (Figure 2) have four chiral centers each, resulting in
16 possible enantiomers. Fortunately the trans-dioxolane iso-
mers[36,37] proved to be far less active than the cis isomers,
which left us with only eight isomers to be evaluated for their
antifungal spectrum and potency.
The antifungal activity of racemic hydroxyitraconazole[33] has
been published, but there are no reports that describe the ac-
tivity of the individual isomers, or that of hydroxysapercona-
zole metabolites. Moreover, posaconazole is a single enantio-
meric hydroxy metabolite of the investigational drug Sch-
51048.[38,39] The enantioselective synthesis and antifungal activi-
ty of the various enantiomers have been reviewed.[40] In addi-
tion, others have described an enantioselective synthesis of
(2R,4S,2’S,3’R)-hydroxyitraconazole without discussing its anti-
fungal spectrum.[41]
obtainable from alkylation of triazolone 9 and O-demethylation
(Scheme 1). According to Cram’s rule,[43–45] chelating conditions
would result in a selective formation of the SR/RS 12c,d
isomer, and nonchelating conditions would give more SS/RR
12a,b. Ideally this would require nonpolar solvents such as
ethers; however, the poor solubility of ketone 11 in THF and
Et2O required a co-solvent such as DMF. Thus reduction of
ketone 11 with K-selectride in a mixture of THF/DMF (1.5:5) at
ꢀ108C gave a 90:10 diastereomeric mixture of 12a,b/12c,d,
which was purified by chiral HPLC to give 12a and 12b in 31
and 34% yields, respectively. Despite chelating conditions
using Zn(BH4)2 in a Et2O/DMF (1.6:1) mixture at 08C, the eryth-
ro diastereomeric selectivity was poor 12a,b/12c,d (70:30).
The fact that we had to use DMF as a co-solvent, which proba-
bly hampered the chelating properties of the zinc ion, explains
this disappointing selectivity. Chiral separation of the diastereo-
meric mixture gave 12c and 12d in 14 and 13% yields, respec-
tively (Scheme 1).
Herein we describe the diastereoselective and enantioselec-
tive synthesis of eight hydroxy metabolites of saperconazole
(compounds 1–8; Figure 2), their antifungal spectrum against a
broad panel of Candida spp. (n=93), Cryptococcus spp. (n=
19), Aspergillus spp. (n=10), and dermatophytes (n=27). We
also discuss the results of a treatment of an experimentally dis-
seminated A. fumigatus-induced infection in guinea pigs with
the various hydroxy isomers 1–8, and the mode of action of
these compounds against A. fumigatus and C. albicans.
The need for a scalable method that would give access to
all four enantiomers prompted us to look into enantioselective
syntheses for alcohols 12a–d (Scheme 2). Nucleophilic ring
opening of meso-(4R,5R)-4,5-dimethyl-1,2,3-dioxathiolane-2,2-
dioxide with triazolone 9 gave enantiomer 13c in good yield
(84%). This method, although independently discovered, was
previously reported for the synthesis of itraconazole hydroxy
metabolites.[41] Demethylation using a 1:1 mixture of 48%
aqueous HBr in acetic acid saturated with HBr gas at 808C
gave 12c in moderate yield (72%). The optical rotation of the
purified and re-crystallized sample of 12c corresponds very
well to the analytical data obtained for 12c after diastereose-
lective reduction of 11 and chiral separation: for example,
Results and Discussion
Chemistry
D
A convergent synthesis was used to prepare target com-
pounds 1–8 (Figure 2). The eight diastereomers 1–8 were ob-
tained in moderate to good yields starting from the optically
½aꢁ20 =ꢀ7.66 (c=0.430 w/v% in DMF; from Scheme 2) and
D
½aꢁ20 =ꢀ7.07 (c=0.976 w/v% in DMF; from Scheme 1). Inver-
sion of the alcohol of 13c under Mitsunobu conditions gave
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Antifungal Activity of Saperconazole Metabolites
Figure 2. The eight hydroxy metabolites of saperconazole and a summary of their syntheses.
13a in 71% yield, and subsequent demethylation with saturat-
ed hydrobromic acid in acetic acid gave 12a in 63% yield. The
optical rotation of 12a, obtained via this enantioselective
Biological activity
The in vitro activity of the various antifungal triazoles 1–8 was
tested against several pathogenic fungi. The test strains includ-
ed 20 isolates each of Candida albicans, C. glabrata, and
C. krusei, 12 each of C. lusitaniae and C. parapsilosis, 9 of C. tro-
picalis, 19 of Cr. neoformans, 10 isolates of A. fumigatus, and 9
isolates each of Microsporum canis, Trichophyton mentagro-
phytes, and T. rubrum. The activity of the reference compound
saperconazole and the test results for compounds 1–4 (2S iso-
mers) and 5–8 (2R isomers) are summarized in Table 1. Against
A. fumigatus and dermatophyte species, saperconazole showed
a more potent in vitro activity than itraconazole; the opposite
situation applied for Candida spp.[28,29]
D
method, was ½aꢁ20 =ꢀ10.43 (c=0.987 w/v% in DMF) and corre-
sponded very well to the previously obtained results for 12a
by selective reduction and chiral chromatography (Scheme 1)
D
½aꢁ20 =ꢀ10.81 (c=1.008 w/v% in DMF). The moderate yield of
the Mitsunobu reaction of 13c was caused mainly by elimina-
tion of the 4-nitrobenzoate to give compound 16. The deme-
thylation reaction could be further optimized during scale-up,
as formation of acetate 15 was observed as the main side
product, which could be easily hydrolyzed upon treatment
with aqueous sodium hydroxide.
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L. Meerpoel, F. C. Odds, et al.
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the 2R diastereomers 5–8, but
within each species tested, dif-
ferences in activity of the eight
compounds were minor.
To evaluate possible activity
differentials in vivo, a dissemi-
nated aspergillosis model in
guinea pigs was chosen as a
suitable test system, as such in-
fections are not easy to treat
and are therefore likely to reveal
clearer differences in activity
among the test compounds 1–
8. The test compounds were for-
mulated in 20% hydroxypropy-
11-b-cyclodextrin and were ad-
ministered into guinea pigs in-
travenously by a permanently
implanted catheter connected
to an infusion pump via a pro-
prietary swivel apparatus de-
vised to allow the animal full
mobility within the cage. The
catheter was kept patent with a
continuous flush of physiologi-
cal saline when no treatment
was administered. The activities
of the placebo solution (20%
hydroxypropy-11-b-cyclodextrin)
and the test compounds 1–8
were compared those of itraco-
nazole and saperconazole. To
Scheme 1. Reagents and conditions: a) 3-chloro-2-butanone, K2CO3, DMF/toluene (1:1), 1108C, 16 h, 97%; b) HBr(aq)
(48%), NaHSO3, reflux, 5 h, 84%; c) K-selectride (1m), THF/DMF (1.5:5), ꢀ108C!RT, 2 h, (12a,b/12c,d 90:10) Chir-
alpac AD: 31% 12a, 34% 12b; d) Zn(BH4)2, Et2O/DMF (1.6:1), 08C!RT, 2 h, (12a,b/12c,d 70:30) Chiralpac AD:
14% 12c, 13% 12d.
The 2S isomers 1–4 showed broadly equivalent inhibitory
potency against most of the fungal species tested (Table 1), al-
though compounds 3 and 4 were less potent than compounds
1 and 2 against C. tropicalis isolates. Compounds 1 and 2 were
conspicuously more potent inhibitors than the two other iso-
mers 3 and 4, and saperconazole against C. glabrata.
minimize the number of animals involved, experimentation to
evaluate the method was held to a minimum (the model with-
out the indwelling intravenous catheters has been described
before[29]). For similar reasons only two doses of the test
agents 1–8 and itraconazole and saperconazole, 1.25 and
2.5 mgkgꢀ1 per day, were found necessary to evaluate their ac-
tivities (Tables 2 and 3). These doses were selected on the
basis of previous experience obtained with itraconazole and
saperconazole as reference substances.
Among the 2R isomers of the new triazoles 5–8 no major
differences in potency were apparent against A. fumigatus and
Cr. neoformans (Table 1). Compounds 5 and 6 were more
potent inhibitors of C. glabrata than the two other 2R isomers
7 and 8 and saperconazole racemate. These two compounds
were also the most potent inhibitors of the dermatophyte iso-
lates tested (Table 1). Further interspecies variations in suscept-
ibility were apparent at the level of 2S isomers 1–4 versus 2R
isomers 5–8. Against Candida spp. and A. fumigatus (Table 1)
the 2S isomers showed a slightly higher level of activity than
the 2R isomers in terms of IC50 values. However, against
Cr. neoformans the 2R isomers showed a trend toward lower
IC50 and IC90 values than the 2S isomers. Compounds 1 and 2
both showed a good in vitro activity against Candida spp. and
A. fumigatus, although they were less active than sapercona-
zole against Cr. neoformans and dermatophyte fungi.
The indwelling catheter as a means of intravenous adminis-
tration of test agents was the only difference from previous
work with this model;[29] nevertheless, the evaluation of stereo-
isomers of antifungal agents on the basis of the relatively few
results presented in this report requires a realistic appreciation
of the limitations of the data. The highly complex nature of
the surgical preparation for catheter insertion, the specially de-
signed apparatus, and the requirement for a continuous infu-
sion pump for each animal tested precluded repetition of the
experiments with greater numbers of animals. Despite this
caveat, the experiments described did show a number of con-
sistent features that suggest superior efficacy among some of
the isomers tested. The infection parameters in placebo-treat-
ed guinea pigs showed that a reasonably consistent degree of
infection was achieved in terms of mean survival time and in
tissue burdens of A. fumigatus measured in the liver and
Among the eight stereoisomers of the novel triazole antifun-
gal agents studied in vitro, there was an overall modest trend
toward higher activity among the 2S diastereomers 1–4 than
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Antifungal Activity of Saperconazole Metabolites
Scheme 2. Reagents and conditions: a) 1. tBuOK, THF/DMA, 608C, 2 h, 2. H2SO4, 608C, 16 h, 84%; b) 1. HBr(aq) (48%), CH3COOH (HBr-satd.), 808C, 24 h,
2. NaOH; c1) PPh3, p-NO2(C6H4)COOH, THF/DMA, DEAD, RT!508C, 2.5 h; c2) NaOH (1n), 508C, 71%.
spleen (see the Experimental Section below and Tables 2 and
3).
an in vivo efficacy profile that similar to that of saperconazole.
Among the 2R series 5–8, isomer 5 was notably the least effec-
tive and compound 6 the most potent, showing a prolonged
mean survival time (MST) to eight days at 2.5 mgkgꢀ1 and a
tissue burden decrease similar to or slightly better than that of
saperconazole. Compounds 1 and 4 prolonged the MST to
seven or more days at at least one of the concentrations
tested, and both eradicated A. fumigatus by 70% or more in
liver, brain, kidney, and/or spleen samples at at least one of the
concentrations tested (Tables 2 and 3).
From these data the superior anti-A. fumigatus potency of
saperconazole over itraconazole was apparent in the data for
mean survival times and CFUgꢀ1 in the organs examined.[28,29]
The data for itraconazole at both doses showed no efficacy at
all in terms of prolongation of survival of the infected animals
over controls and only modest decreases in the fungal tissue
burdens. Neither compound was particularly effective in elimi-
nating A. fumigatus from the liver, spleen, or kidney of infected
animals, however a higher proportion of fungus-negative
organs was observed in saperconazole-treated animals as com-
pared with itraconazole-treated animals (Table 2 and Table 3).
Among the test compounds 1–8, the activity of the 2S com-
pounds 1–4 in vivo was once again generally superior to that
of the 2R compounds 5–8 as previously noted in vitro
(Table 1). However, differences in antifungal efficacy under the
test conditions used were evident within these groups of iso-
mers (Tables 2 and 3). Among the 2S series 1–4, isomer 2 was
markedly less active than the other three by all parameters
measured. Compound 1 was the most active of the four 2S iso-
mers in terms of all parameters measured and was clearly
more potent than saperconazole. Compounds 3 and 4 showed
The mode of action of azole antifungal agents has been well
established as inhibition of the P450 14-a-demethylase (CYP51,
Erg11) of the ergosterol biosynthetic pathway.[46,47] Itraconazole
and saperconazole have been reported as potent inhibitors of
ergosterol biosynthesis in A. fumigatus and C. albicans.[14,46,48,49]
In order to identify the mode of action of the novel triazoles, a
study was designed to examine the interaction of compounds
1–8 with ergosterol biosynthesis in fungal cells. The results ob-
tained with these compounds were compared with itracona-
zole and saperconazole as internal references in the same ex-
periment. The effects of the compounds tested on the growth
of A. fumigatus, and the incorporation of [14C]acetate into ergo-
sterol by C. albicans and A. fumigatus were measured as de-
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L. Meerpoel, F. C. Odds, et al.
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scribed previously.[48] The effects of azole antifungal agents on
the growth of A. fumigatus, listed in Table 4, confirm those ob-
tained previously with itraconazole and saperconazole.[48,49]
Both triazole derivatives are potent inhibitors of the growth of
A. fumigatus. The hydroxy isomers 1–8 were generally 4- to 19-
fold less active than the parent compound saperconazole. The
most potent enantiomers were compounds 3, 8, 7, 4, and 1.
These results are based on 50% growth inhibition of a single
A. fumigatus strain, whereas the results in Table 1 are based on
growth inhibition of a panel of 10 A. fumigatus isolates, and
therefore give a more comprehensive picture of breadth of
spectrum, which is clinically more relevant. Despite this caveat,
the experimental data listed in Table 4 do confirm a trend for
higher potency with the 2S isomers 1–4 on growth inhibition
in A. fumigatus relative to the 2R isomers 5–8: for example, 1
and 3 were better than their 2R enantiomers 5 and 7, and 2
and 4 were similar to 6 and 8. The effects on ergosterol bio-
synthesis in A. fumigatus and C. albicans were measured after
16 h incubation of the fungi in the presence of itraconazole,
saperconazole, or the hydroxy isomers 1–8, according to ex-
perimental procedures previously described.[48] Inhibition (IC50
values) of ergosterol biosynthesis from [14C]acetate was
reached at 0.02 and 0.04 mm for saperconazole and itracona-
zole, respectively. The hydroxy isomer showed a consistently
lower potency in this assay relative to saperconazole and itra-
conazole, with compounds 2 and 4 being the most potent.
Surprisingly, compound 1 (IC50 =0.20 mm) appeared to be the
least potent of all 2S isomers. Head-to-head comparison of the
enantiomeric pairs, however, again revealed a higher potency
for the 2S diastereomers 1–4 in comparison with their 2R
counterparts 5–8. For the inhibition of ergosterol biosynthesis
in C. albicans, however, we observed a consistently higher po-
tency for the 2R isomers: for example, 1<5, 2<6, 3~7, and
4<8.
The weak trend for 2S-dioxolane isomers 1–4 to be more
active than the 2R isomers 5–8 is in contrast with the much
more significant difference between the 5S dystomer and 5R
eutomer[50,51] of the tetrahydrofuran series related to posacona-
zole.[40] Given the strong similarity between the posaconazole
series and compounds 1–8, it is not surprising that compound
1, one of the most potent among the series tested in vivo, has
the same absolute configuration as posaconazole (Figure 1).
The design and synthesis of the hydroxy derivatives 1–8
were based on the assumption of a similar metabolism of sa-
perconazole compared with itraconazole, namely hydroxy me-
tabolite formation at the sec-butyl substituent of the triazolone
group.[31,32] However, equal formation and distribution of the
individual hydroxy metabolites of saperconazole in vivo is un-
likely to be the case. A more detailed study on the metabolism
of itraconazole was recently published.[35] It shows a stereose-
lective metabolism of itraconazole in vitro and in vivo. Only
the 2R isomers of itraconazole were metabolized by CYP3A4
into their corresponding hydroxy metabolites. The 2S isomers,
independent of the stereochemistry of the sec-butyl group, ap-
peared to be very stable upon incubation with CYP3A4. Similar
observations were also made in healthy volunteers after single
and multiple doses, in which a higher Cmax value was measured
Table 1. Activity of saperconazole and eight stereoisomers against vari-
ous fungal pathogens in vitro.
Test species
n
Compound
MIC data [mgmLꢀ1
]
[a]
[a]
Range
IC50
IC90
All Candida spp.
93 saperconazole ꢂ0.05–>25
0.10 >25
0.20 >25
0.40 >25
0.20 >25
0.40 >25
0.80 >25
0.40 >25
0.80 >25
0.40 >25
1
2
3
4
5
6
7
8
ꢂ0.05–>25
ꢂ0.05–>25
ꢂ0.05–>25
ꢂ0.05–>25
ꢂ0.05–>25
ꢂ0.05–>25
ꢂ0.05–>25
ꢂ0.05–>25
C. glabrata
20 saperconazole
0.8–>25
0.4–>25
0.4–>25
0.4–>25
0.8–>25
0.4–>25
0.4–>25
3.2–>25
0.8–>25
>25
6.3
25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
1
2
3
4
5
6
7
8
6.3
>25
>25
>25
C. tropicalis
Cr. neoformans
A. fumigatus
9
saperconazole
0.05–>25
ꢂ0.05–>25
ꢂ0.05–>25
ꢂ0.05–>25
ꢂ0.05–>25
0.05–>25
0.05–>25
0.05–>25
0.05–>25
13
>25
1
2
3
4
5
6
7
8
0.40 >25
0.20 >25
6.3
13
13
0.80 >25
0.80 >25
1.6
>25
>25
>25
>25
19 saperconazole ꢂ0.05–0.40
0.20
0.20
0.20
0.20
0.20
0.20
0.10
0.20
0.20
0.40
0.80
0.80
1.60
0.80
0.40
0.80
0.40
0.40
1
2
3
4
5
6
7
8
ꢂ0.05–0.80
ꢂ0.05–0.80
ꢂ0.05–1.6
ꢂ0.05–0.80
ꢂ0.05–0.40
0.05–0.80
ꢂ0.05–0.80
ꢂ0.05–0.80
10 saperconazole ꢂ0.05–0.20
0.10
0.10
0.20
0.10
0.10
0.40
0.40
0.20
0.20
0.20
0.20
0.40
0.20
0.20
0.80
0.80
0.40
0.40
1
2
3
4
5
6
7
8
0.05–0.20
ꢂ0.05–0.40
ꢂ0.05–0.20
ꢂ0.05–0.20
0.10–0.80
ꢂ0.05–0.20
ꢂ0.05–0.05
ꢂ0.05–0.40
All dermatophytes 27 saperconazole ꢂ0.05–0.80
0.05
ꢂ0.05
ꢂ0.05
ꢂ0.05
ꢂ0.05
0.05
0.05
0.10
0.10
0.05
0.10
0.20
0.10
0.10
0.10
1
2
3
4
5
6
7
8
ꢂ0.05–0.40
ꢂ0.05–0.40
ꢂ0.05–0.10
ꢂ0.05–0.40
ꢂ0.05–0.40
ꢂ0.05–0.40
ꢂ0.05–0.40
ꢂ0.05–0.10
0.10
ꢂ0.05
ꢂ0.05
[a] IC50 and IC90 values indicate the concentrations required to inhibit 50
and 90% of the test isolates, respectively.
762
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ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2010, 5, 757 – 769

Antifungal Activity of Saperconazole Metabolites
2R isomers 5–8 could explain
the higher potency of the 2S
isomers over the 2R isomers in
the experimental A. fumigatus
infection in guinea pigs. Com-
pounds 1 and 4 appeared to be
the most potent overall in erad-
icating tissue burden and in
prolonging MST.
Table 2. Results of treatment of experimental disseminated A. fumigatus infection in guinea pigs.[a]
Treatment No. animals Dose MST [days][b] Geometric mean log CFU(g tissue)ꢀ1
ꢃ
Negative tissues[c]
[mgkgꢀ1
]
SD in:
Liver Spleen
Brain
Kidney
No. [/n]
[%]
placebo
itraconazole
24
5
5
5
5
–
5.2
4.8
5.2
7.4
8.4
1.9ꢃ1.7 4.0ꢃ1.1 4.3ꢃ0.8 3.3ꢃ1.5
2.5+2.3 4.3ꢃ0.2 4.4ꢃ0.4 2.1ꢃ2.0
0.0ꢃ0.0 3.7ꢃ0.5 4.0ꢃ0.6 2.7ꢃ1.8
1.7ꢃ2.3 2.6ꢃ1.5 2.9ꢃ1.7 1.1ꢃ1.5
0.7ꢃ1.4 2.2ꢃ1.4 2.8ꢃ1.7 0.0ꢃ0.0
14/96
4/20
6/20
8/20
11/20
15
20
30
40
55
1.25
2.5
1.25
2.5
saperconazole
1
2
3
4
5
5
5
4
4
4
4
4
1.25
2.5
1.25
2.5
1.25
2.5
1.25
2.5
9.0
7.8
5.2
4.8
7
8.2
6.8
7
0.5ꢃ1.1 0.8ꢃ1.8 0.7ꢃ1.6 0.5ꢃ1.0
0.0ꢃ0.0 1.6ꢃ2.2 1.5ꢃ2.0 0.4ꢃ1.0
2.3ꢃ2.1 3.9ꢃ0.9 4.4ꢃ1.3 3.6ꢃ0.6
1.6ꢃ1.8 1.8ꢃ2.2 2.6ꢃ2.0 0.7ꢃ1.4
0.7ꢃ1.4 1.7ꢃ2.0 2.8ꢃ2.1 1.0ꢃ2.0
1.3ꢃ1.5 1.8ꢃ2.0 2.5ꢃ1.8 0.6ꢃ1.1
0.7ꢃ1.3 0.9ꢃ1.7 1.6ꢃ1.9 0.7ꢃ1.4
0.0ꢃ0.0 1.4ꢃ1.7 1.0ꢃ2.0 0.6ꢃ1.1
16/20
15/20
2/20
4/16
9/16
8/16
11/16
12/16
80
75
10
25
56
50
69
75
Conclusions
In summary, the goal of this
study was to identify the most
active hydroxy metabolite of sa-
perconazole in experimental
fungal infection as a potential
starting point for prodrug
design. Scaleable diastereose-
lective and enantioselective syn-
theses of hydroxy metabolites
of saperconazole were de-
scribed. The activities of eight
diastereomers (compounds 1–8)
5
6
7
8
5
5
4
3
4
4
5
5
1.25
2,5
1.25
2.5
1.25
2.5
1.25
2.5
5
1.3ꢃ1.8 3.2ꢃ1.8 3.3ꢃ1.9 2.1ꢃ1.2
1.8ꢃ1.7 4.0ꢃ1.1 4.4ꢃ0.7 2.7ꢃ1.5
2.3ꢃ2.7 3.2ꢃ0.5 2.7ꢃ2.0 2.8ꢃ0.7
0.0ꢃ0.0 1.3ꢃ2.3 1.9ꢃ1.8 0.8ꢃ1.3
1.7ꢃ2.0 4.0ꢃ0.8 4.2ꢃ0.8 1.6ꢃ1.8
0.0ꢃ0.0 1.8ꢃ2.2 3.0ꢃ2.2 1.0ꢃ2.0
0.7ꢃ1.5 2.1ꢃ1.9 1.9ꢃ1.8 0.9ꢃ1.2
1.3ꢃ1.7 1.7ꢃ2.3 3.3ꢃ1.2 1.7ꢃ1.7
6/20
3/20
3/16
8/12
4/16
10/16
11/20
12/20
30
15
19
67
25
63
55
60
6.8
6.5
8
5
7.8
6.4
7.6
[a] Animals were treated with two intravenous infusions (formulation in 20% hydroxypropy1-1-b-cyclodextrin)
daily for a total of 9.5 days. [b] Mean survival time. [c] No fungus isolated by culture of tissue homogenates.
of
a novel antifungal agent
were evaluated against a panel
of fungal isolates tested in vitro.
The four 2S diastereomers 1–4
of the new agent were generally
slightly more active than the
four 2R diastereomers 5–8, al-
though none of the isomers
matched the activity of the ref-
erence compounds itraconazole
and saperconazole against der-
matophytes and Cr. neoformans
in vitro. Compounds 1 and 2
were, by a small margin, the
two best inhibitors of fungal
growth among the eight iso-
mers, showing efficacy similar to
that of itraconazole against a
panel of 93 Candida isolates rep-
resenting six pathogenic spe-
cies, and activity similar to that
of saperconazole against 10 iso-
lates of A. fumigatus.
Table 3. Numbers and percentages of individual tissues that were culture-negative for A. fumigatus.
Treatment
No. animals
Dose [mgkgꢀ1
]
Brain
Liver
No.
Spleen
Kidney
No.
[%]
[%]
No.
[%]
No.
[%]
placebo
itraconazole
24
5
5
5
5
–
10
2
5
3
4
42
40
100
60
1
0
0
1
1
4
0
0
20
20
0
0
0
1
1
0
0
0
20
20
3
2
1
3
5
13
40
20
60
100
1.25
2.5
1.25
2.5
saperconazole
80
1
2
3
4
5
5
5
4
4
4
4
4
1.25
2.5
1.25
2.5
1.25
2.5
1.25
2.5
4
5
2
2
4
3
3
4
80
100
40
50
100
75
4
3
0
1
2
3
3
2
80
60
0
25
50
75
75
50
4
3
0
0
1
1
2
3
80
60
0
4
4
0
1
3
3
3
3
80
80
0
25
75
75
75
75
0
25
25
50
75
75
100
5
6
7
8
5
5
4
3
4
4
5
5
1.25
2.5
1.25
2.5
1.25
2.5
1.25
2.5
3
2
2
3
2
4
4
3
60
40
50
100
50
100
80
1
0
0
2
0
2
2
3
20
0
0
67
0
50
40
60
1
0
1
1
0
1
2
0
20
0
25
33
0
25
40
0
1
1
0
2
2
3
3
2
20
20
0
67
50
75
60
40
Compared with itraconazole
and saperconazole, the hydroxy
isomers 1–8 are less potent in-
hibitors of the growth of A. fu-
60
for the 2S isomers relative to the 2R isomers.[35] Although a sys-
tematic in vivo pharmacokinetic evaluation of the new azole
compounds 1–8 has not been done, one may assume a similar
trend for the in vivo pharmacokinetics of the hydroxy deriva-
tives 1–8. Better bioavailability for the 2S isomers 1–4 over the
migatus and of ergosterol biosynthesis in both A. fumigatus
and C. albicans. Among the enantiomers, compound 4 is the
most potent inhibitor of ergosterol biosynthesis in A. fumi-
gatus, whereas compound 8 is the best inhibitor of ergosterol
biosynthesis in C. albicans.
ChemMedChem 2010, 5, 757 – 769
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
763

L. Meerpoel, F. C. Odds, et al.
MED
azol-3-one (1): According to the synthesis of compound 2 (see
below) starting from dioxolane 17b (5.4 g, 0.012 mol) and phenol
12a (4.5 g, 0.0109 mol) to yield compound 1 as a white solid
Table 4. Effects on the growth of A. fumigatus and ergosterol biosynthe-
sis in A. fumigatus and C. albicans.
D
(4.7 g, 62%); mp=189.48C (MeOH); ½aꢁ20 =ꢀ20.15 (c=1.00 w/v%
Compounds
IC50 [mm]
1
in DMF); H NMR (360 MHz, CDCl3): d=1.25 (d, J=6.4 Hz, 3H), 1.46
A. fum. Growth A. fum. Erg. Synth. C. alb. Erg. Synth.
(d, J=6.9 Hz, 3H), 3.17 (d, J=8.1 Hz, 1H), 3.21–3.25 (m, 4H), 3.34–
3.39 (m, 4H), 3.49 (dd, J=9.7, 6.3 Hz, 1H), 3.78 (dd, J=9.7, 4.7 Hz,
1H), 3.82 (dd, J=8.4, 5.2 Hz, 1H), 3.98 (dd, J=8.4, 6.6 Hz, 1H),
3.97–4.05 (m, 1H), 4.25 (qd, J=6.9, 5.6 Hz, 1H), 4.37–4.46 (m, J=
6.4, 6.4, 5.2, 4.7 Hz, 1H), 4.68 (d, J=14.7 Hz, 1H), 4.73 (d, J=
14.7 Hz, 1H), 6.80 (d, J=9.0 Hz, 2H), 6.85–6.92 (m, 2H), 6.94 (d, J=
9.0 Hz, 2H), 7.03 (d, J=8.9 Hz, 2H), 7.41 (d, J=8.9 Hz, 2H), 7.50 (td,
J=8.9, 6.4 Hz, 1H), 7.66 (s, 1H), 7.89 (s, 1H), 8.23 (s, 1H); Anal.
calcd for C35H38F2N8O5: C 61.04, H 5.56, N 16.27, found: C 61.04, H
5.47, N 16.33.
1
2
3
4
5
6
7
8
0.08
0.12
0.04
0.07
0.19
0.16
0.07
0.06
0.2
1.1
0.08
0.11
0.06
0.45
0.43
0.13
0.13
0.02
0.04
0.73
0.48
0.64
0.4
0.39
0.58
0.21
0.07
0.05
saperconazole 0.01
itraconazole 0.008
Scale-up optimized procedure for compound 1: Phenol 12a
(378.3 g, 0.93 mol), dioxolane 17b (416.1 g, 1.108 mol), and NaOH
pellets (44.3 g, 1.108 mol) were added to a 5 L round-bottomed
flask, and after purging with N2 gas, DMA (1390 mL) was added,
followed by vigorous stirring. Methylisobutyl ketone (744 mL) was
then added, whereupon the reaction mixture was heated to 808C
under vigorous stirring. The initial viscous mixture became homo-
genous from 508C onward with the exception of the NaOH pellets.
After stirring for 10 h at 808C, the reaction mixture was allowed to
cool to room temperature, and stirring was continued for an addi-
tional 16 h. Ice-cooled H2O (1.5 L) was added carefully over a
period of 30 min (exothermic!), followed by stirring for 2 h at room
temperature. The resulting precipitate was filtered off, washed
with H2O (1 L), MIK (1 L), and finally dried in vacuo at 408C to yield
compound 1 (584 g, 91.2%). Compound 1 (435 g) was dissolved 1-
methoxy-2-propanol (4.3 L) at reflux, filtered warm, and the filtrate
was stirred for 16 h at room temperature. The precipitate was fil-
tered off, washed with 2-propanol (200 mL), and dried in vacuo at
508C for 16 h to yield compound 1 (339 g). A second crop with
the desired purity was obtained from the filtrate to yield 51 g of
compound 1, giving a total yield of 89% for the re-crystallization
procedure.
In vitro activity against a broad panel of Candida and Asper-
gillus spp., and good activity against disseminated aspergillosis
in guinea pigs was the basis for selection of compound 1 as a
suitable starting point for the design of derived prodrugs with
improved solubility. Additional work on the design and anti-
fungal activity of these prodrugs will be published in a forth-
coming paper.
Experimental Section
Chemistry
General methods: Melting points were measured in open capilla-
ries on a Bꢁchi B545 instrument and are uncorrected. 1H and
13C NMR spectra were recorded on Bruker DPX 360 and Avance
600 spectrometers. CDCl3 was used as solvent, unless otherwise
mentioned. Chemical shifts (d) are expressed in parts per million
(ppm) with (CH3)4Si as an internal standard. Elemental analyses
were performed with a Carlo–Erba EA1110 analyzer. Mass spectra
were obtained with a Waters-Micromass ZQ mass spectrometer
with an electrospray ionization source operating in positive and
negative ionization modes. Mass spectra were acquired by scan-
ning in an area starting from 100 to 1000 mass units in 1 s using a
dwell time of 0.1 s. The capillary needle voltage was 3 kV, and the
source temperature was maintained at 1408C; N2 was used as the
nebulizer gas. Cone voltage was 10 V for positive ionization mode
and 20 V for negative ionization mode. Data acquisition was per-
formed with a Waters-Micromass MassLynx-Openlynx data system.
Silica gel thin-layer chromatography (TLC) was performed on pre-
coated Kieselgel 60 F254 plates (E. Merck, AG Darmstadt, Germany).
Silica gel column chromatography was performed with Kieselgel 60
(0.063–0.200 mm) (E. Merck, AG Darmstadt, Germany). Chiral HPLC
purification was performed on a Daicel cellulose normal-phase
Chiracelꢂ ODtm HPLC column, purchased from Chiral Technologies
Europe. Optical rotations were performed on a polarimeter (Perki-
nElmer). All commercial reagents and solvents were used without
any pretreatment. All reactions were carried out under an atmos-
phere of dry N2 gas. Brine is a saturated solution of NaCl in H2O;
80% NaH (Sigma–Aldrich) was washed free of oil with hexanes
prior to use. DEAD=diethylazodicarboxylate, DMA=N,N-dimethy-
lacetamide, DMF=N,N-dimethylformamide, THF=tetrahydrofuran,
MIK=4-methyl-2-pentanone, DIPE=diisopropyl ether.
4-[4-[4-[4-[[(2S,4R)-2-(2,4-difluorophenyl)-2-(1H-1,2,4-triazol-1-yl-
methyl)-1,3-dioxolan-4-yl]methoxy]phenyl]-1-piperazinyl]phen-
yl]-2,4-dihydro-2-[(1R,2R)-2-hydroxy-1-methylpropyl]-3H-1,2,4-
triazol-3-one (2): Compound 17a (5.4 g, 0.012 mol), phenol 12b
(4.5 g, 0.0109 mol), and NaOH pellets (0.44 g, 0.011 mol) were
added to a round-bottomed flask, and after purging with N2 gas,
DMF (150 mL) was added under vigorous stirring. The reaction mix-
ture was stirred overnight at 708C, followed by cooling and subse-
quently poured out into an ice–H2O mixture. The resulting precipi-
tate was filtered off and dried to yield compound 2 (7 g). This frac-
tion was purified by HPLC over Silica Motrex Amicon (20–45 mm;
eluent: 1,1,2-trichloroethane/EtOH 90:10). The pure fractions were
collected, and after evaporation of the solvent, the residue was tri-
turated in warm MeOH, cooled, filtered off, and dried in vacuo at
D
408C to yield compound 2 as a white solid (5.3 g, 70%); ½aꢁ20
=
1
ꢀ7.71 (c=0.97 w/v% in DMF); H NMR (360 MHz, CDCl3): d=1.25
(d, J=6.4 Hz, 3H), 1.47 (d, J=6.9 Hz, 3H), 3.04 (d, J=8.5 Hz, 1H),
3.20–3.27 (m, 4H), 3.34–3.40 (m, 4H), 3.48 (dd, J=9.7, 6.3 Hz, 1H),
3.79 (dd, J=9.7, 4.7 Hz, 1H), 3.82 (dd, J=8.5, 5.2 Hz, 1H), 3.96–4.05
(m, 2H), 4.26 (qd, J=6.9, 5.6 Hz, 1H), 4.38–4.45 (m, J=6.4, 6.4, 5.2,
4.7 Hz, 1H), 4.68 (d, J=14.7 Hz, 1H), 4.74 (d, J=14.7 Hz, 1H), 6.80
(d, J=9.0 Hz, 2H), 6.85–6.91 (m, 2H), 6.92–6.97 (m, 2H), 7.04 (d, J=
8.9 Hz, 2H), 7.42 (d, J=8.9 Hz, 2H), 7.50 (td, J=8.9, 6.4 Hz, 1H),
7.66 (s, 1H), 7.89 (s, 1H), 8.22 (s, 1H); Anal. calcd for C35H38F2N8O5:
4-[4-[4-[4-[[(2S,4R)-2-(2,4-difluorophenyl)-2-(1H-1,2,4-triazol-1-yl-
methyl)-1,3-dioxolan-4-yl]methoxy]phenyl]-1-piperazinyl]phen-
yl]-2,4-dihydro-2-[(1S,2S)-2-hydroxy-1-methylpropyl]-3H-1,2,4-tri-
764
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ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2010, 5, 757 – 769

Antifungal Activity of Saperconazole Metabolites
C 61.04, H 5.56, N 16.27, found: C 61.10, H 5.46, N 16.24; MS (ESI)
J=21.2, 3.4 Hz, 1C), 115.2 (s, 2C), 116.51 (s, 2C), 118.39 (s, 2C),
121.66 (dd, J=13.0, 3.7 Hz, 1C), 123.69 (s, 2C), 125.29 (s, 1C), 129.18
(dd, J=9.7, 4.6 Hz, 1C), 134.25 (s, 1C), 144.81 (s, 1C), 145.93 (s, 1C),
150.71 (s, 1C), 151.26 (s, 1C), 152.05 (s, 1C), 152.56 (s, 1C), 160.36
(dd, J=252.7, 12.2 Hz, 1C), 163.63 (dd, J=251.6, 11.7 Hz, 1C); Anal.
calcd for C35H38F2N8O5: C 61.04, H 5.56, N 16.27, found: C 61.05, H
5.46, N 16.26; MS (ESI) m/z 689 ([M+H]+).
m/z 689 ([M+H]+).
The procedure for the preparation of compound 2 was used as a
general method for the synthesis of compounds 1–8 (see
Scheme 3).
4-[4-[4-[4-[[(2R,4S)-2-(2,4-difluorophenyl)-2-(1H-1,2,4-triazol-1-yl-
methyl)-1,3-dioxolan-4-yl]methoxy]phenyl]-1-piperazinyl]phen-
yl]-2,4-dihydro-2-[(1R,2R)-2-hydroxy-1-methylpropyl]-3H-1,2,4-
triazol-3-one (5): According to the synthesis of compound 2 start-
ing from dioxolane 18 (5.4 g, 0.012 mol) and phenol 12b (4.5 g,
0.0109 mol) to yield compound 5 as a white solid (4.8 g, 64%);
D
mp=190.38C (MeOH); ½aꢁ20 =+19.49 (589 nm, c=1.03620 w/v%,
1
DMF, 208C); H NMR (360 MHz, CDCl3): d=1.25 (d, J=6.4 Hz, 3H),
Scheme 3. Reagents and conditions: a) 17a or 18: DMF, NaOH, 60–808C;
b) 17b, DMF or DMA, NaOH, 808C, 7–16 h.
1.46 (d, J=6.9 Hz, 3H), 3.17 (d, J=8.1 Hz, 1H), 3.21–3.25 (m, 4H),
3.34–3.39 (m, 4H), 3.49 (dd, J=9.7, 6.3 Hz, 1H), 3.78 (dd, J=9.7,
4.7 Hz, 1H), 3.82 (dd, J=8.4, 5.2 Hz, 1H), 3.98 (dd, J=8.4, 6.6 Hz,
1H), 3.97–4.05 (m, 1H), 4.25 (qd, J=6.9, 5.6 Hz, 1H), 4.37–4.46 (m,
J=6.4, 6.4, 5.2, 4.7 Hz, 1H), 4.68 (d, J=14.7 Hz, 1H), 4.73 (d, J=
14.7 Hz, 1H), 6.80 (d, J=9.0 Hz, 2H), 6.85–6.92 (m, 2H), 6.94 (d, J=
9.0 Hz, 2H), 7.03 (d, J=8.9 Hz, 2H), 7.41 (d, J=8.9 Hz, 2H), 7.50 (td,
J=8.9, 6.4 Hz, 1H), 7.66 (s, 1H), 7.89 (s, 1H), 8.23 (s, 1H); Anal.
calcd for C35H38F2N8O5: C 61.04, H 5.56, N 16.27, found: C 61.11, H
5.51, N 16.27; MS (ESI) m/z 689 ([M+H]+).
4-[4-[4-[4-[[(2S,4R)-2-(2,4-difluorophenyl)-2-(1H-1,2,4-triazol-1-yl-
methyl)-1,3-dioxolan-4-yl]methoxy]phenyl]-1-piperazinyl]phen-
yl]-2,4-dihydro-2-[(1S,2R)-2-hydroxy-1-methylpropyl]-3H-1,2,4-tri-
azol-3-one (3): According to the synthesis of compound 2 starting
from dioxolane 17b (5.4 g, 0.012 mol) and phenol 12c (4.5 g,
0.0109 mol) to yield compound 3 as a white solid (4.4 g, 59%);
D
mp=210.18C (MeOH); ½aꢁ20 =ꢀ17.79 (589 nm, c=0.99500 w/v%,
1
DMF, 208C); H NMR (360 MHz, CDCl3): d=1.25 (d, J=6.4 Hz, 3H),
1.43 (d, J=6.9 Hz, 3H), 3.21–3.26 (m, 4H), 3.34–3.41 (m, 4H), 3.49
(dd, J=9.7, 6.3 Hz, 1H), 3.69 (d, J=2.7 Hz, 1H), 3.79 (dd, J=9.7,
4.7 Hz, 1H), 3.82 (dd, J=8.5, 5.2 Hz, 1H), 3.98 (dd, J=8.4, 6.5 Hz,
1H), 4.17–4.25 (m, J=6.4, 6.4, 6.4, 2.9, 2.7 Hz, 1H), 4.28 (qd, J=6.9,
2.9 Hz, 1H), 4.36–4.46 (m, J=6.4, 6.4, 5.2, 4.7 Hz, 1H), 4.68 (d, J=
14.7 Hz, 1H), 4.74 (d, J=14.7 Hz, 1H), 6.80 (d, J=8.9 Hz, 2H), 6.85–
6.91 (m, 2H), 6.94 (d, J=8.9 Hz, 2H), 7.04 (d, J=8.9 Hz, 2H), 7.40
(d, J=8.9 Hz, 2H), 7.50 (td, J=8.9, 6.7 Hz, 1H), 7.64 (s, 1H), 7.89 (s,
1H), 8.22 (s, 1H); 13C NMR (151 MHz): d=12.48 (s, 1C), 19.36 (s, 1C),
49.04 (s, 2C), 50.49 (s, 2C), 54.43 (d, J=3.7 Hz, 1C), 57.28 (s, 1C),
67.54 (s, 1C), 67.59 (s, 1C), 69.83 (s, 1C), 74.90 (s, 1C), 105.12 (t, J=
25.9 Hz, 1C), 106.69 (d, J=4.0 Hz, 1C), 111.18 (dd, J=20.9, 3.4 Hz,
1C), 115.22 (s, 2C), 116.53 (s, 2C), 118.41 (s, 2C), 121.67 (dd, J=13.0,
3.7 Hz, 1C), 123.70 (s, 2C), 125.30 (s, 1C), 129.19 (dd, J=9.7, 4.6 Hz,
1C), 134.26 (s, 1C), 144.83 (s, 1C), 145.95 (s, 1C), 150.73 (s, 1C),
151.27 (s, 1C), 152.06 (s, 1C), 152.57 (s, 1C), 160.37 (dd, J=252.7,
12.2 Hz, 1C), 163.64 (dd, J=251.9, 10.9 Hz, 1C); Anal. calcd for
C35H38F2N8O5: C 61.04, H 5.56, N 16.27, found: C 60.68, H 5.44, N
16.17; MS (ESI) m/z 689 ([M+H]+).
4-[4-[4-[4-[[(2R,4S)-2-(2,4-difluorophenyl)-2-(1i-1,2,4-triazol-1-yl-
methyl)-1,3-dioxolan-4-yl]methoxy]phenyl]-1-piperazinyl]phen-
yl]-2,4-dihydro-2-[(1S,2S)-2-hydroxy-1-methylpropyl]-3H-1,2,4-tri-
azol-3-one (6): According to the synthesis of compound 2 starting
from dioxolane 18 (5.4 g, 0.012 mol) and phenol 12a (4.5 g,
0.0109 mol) to yield compound 6 as a white solid (4.3 g, 57%);
½aꢁ20 =+7.13 (c=0.99540 w/v% in DMF); H NMR (360 MHz, CDCl3):
d=1.25 (d, J=6.4 Hz, 3H), 1.45 (d, J=6.9 Hz, 3H), 3.20–3.27 (m,
4H), 3.28 (d, J=7.8 Hz, 1H), 3.34–3.40 (m, 4H), 3.48 (dd, J=9.7,
6.3 Hz, 1H), 3.79 (dd, J=9.7, 4.7 Hz, 1H), 3.82 (dd, J=8.5, 5.2 Hz,
1H), 3.96–4.05 (m, 2H), 4.26 (qd, J=6.9, 5.6 Hz, 1H), 4.38–4.45 (m,
J=6.4, 6.4, 5.2, 4.7 Hz, 1H), 4.68 (d, J=14.7 Hz, 1H), 4.74 (d, J=
14.7 Hz, 1H), 6.80 (d, J=9.0 Hz, 2H), 6.85–6.91 (m, 2H), 6.92–6.97
(m, 2H), 7.04 (d, J=8.9 Hz, 2H), 7.42 (d, J=8.9 Hz, 2H), 7.50 (td, J=
8.9, 6.4 Hz, 1H), 7.66 (s, 1H), 7.89 (s, 1H), 8.22 (s, 1H); Anal. calcd
for C35H38F2N8O5: C 61.04, H 5.56, N 16.27, found: C 60.80, H 5.33, N
16.16; MS (ESI) m/z 689 ([M+H]+).
D
1
4-[4-[4-[4-[[(2R,4S)-2-(2,4-difluorophenyl)-2-(1H-1,2,4-triazol-1-yl-
methyl)-1,3-dioxolan-4-yl]methoxy]phenyl]-1-piperazinyl]phen-
yl]-2,4-dihydro-2-[(1R,2S)-2-hydroxy-1-methylpropyl]-3H-1,2,4-tri-
azol-3-one (7): According to the synthesis of compound 2 starting
from dioxolane 18 (5.4 g, 0.012 mol) and phenol 12d (4.5 g,
0.0109 mol) to yield compound 7 as a white solid 6.3 g (84%);
4-[4-[4-[4-[[(2S,4R)-2-(2,4-difluorophenyl)-2-(1H-1,2,4-triazol-1-yl-
methyl)-1,3-dioxolan-4-yl]methoxy]phenyl]-1-piperazinyl]phen-
yl]-2,4-dihydro-2-[(1R,2S)-2-hydroxy-1-methylpropyl]-3H-1,2,4-
triazol-3-one (4): According to the synthesis of compound 2 start-
ing from dioxolane 17b (5.4 g, 0.012 mol) and phenol 12d (4.5 g,
0.0109 mol) to yield compound 4 as a white solid (5.9 g, 79%);
D
mp=211.18C (MeOH); ½aꢁ20 =+17.79 (c=0.99520 w/v% in DMF);
D
mp=199.98C (MeOH); ½aꢁ20 =ꢀ9.36 (c=1.01540 w/v% in DMF);
1H NMR (360 MHz, CDCl3): d=1.25 (d, J=6.4 Hz, 3H), 1.43 (d, J=
6.9 Hz, 3H), 3.21–3.26 (m, 4H), 3.34–3.41 (m, 4H), 3.49 (dd, J=9.7,
6.3 Hz, 1H), 3.69 (d, J=2.7 Hz, 1H), 3.79 (dd, J=9.7, 4.7 Hz, 1H),
3.82 (dd, J=8.5, 5.2 Hz, 1H), 3.98 (dd, J=8.4, 6.5 Hz, 1H), 4.17–4.25
(m, J=6.4, 6.4, 6.4, 2.9, 2.7 Hz, 1H), 4.28 (qd, J=6.9, 2.9 Hz, 1H),
4.36–4.46 (m, J=6.4, 6.4, 5.2, 4.7 Hz, 1H), 4.68 (d, J=14.7 Hz, 1H),
4.74 (d, J=14.7 Hz, 1H), 6.80 (d, J=8.9 Hz, 2H), 6.85–6.91 (m, 2H),
6.94 (d, J=8.9 Hz, 2H), 7.04 (d, J=8.9 Hz, 2H), 7.40 (d, J=8.9 Hz,
2H), 7.50 (td, J=8.9, 6.7 Hz, 1H), 7.64 (s, 1H), 7.89 (s, 1H), 8.22 (s,
1H); Anal. calcd for C35H38F2N8O5: C 61.04, H 5.56, N 16.27, found: C
61.23, H 5.49, N 16.28; MS (ESI) m/z 689 ([M+H]+).
1H NMR (360 MHz, [D6]DMSO): d=0.98 (d, J=6.2 Hz, 3H), 1.34 (d,
J=6.8 Hz, 3H), 3.11–3.22 (m, 4H), 3.26–3.36 (m, 4H), 3.65–3.83 (m,
4H), 3.91–4.00 (m, 2H), 4.35–4.43 (m, J=5.7, 5.7, 5.7, 5.7 Hz, 1H),
4.66–4.76 (m, 2H), 4.94 (d, J=5.9 Hz, 1H), 6.84 (d, J=8.7 Hz, 2H),
6.96 (d, J=8.7 Hz, 2H), 7.03–7.13 (m, 3H), 7.31 (ddd, J=11.3, 9.1,
2.5 Hz, 1H), 7.44 (td, J=8.9, 6.8 Hz, 1H), 7.50 (d, J=8.6 Hz, 2H),
7.87 (s, 1H), 8.33 (s, 1H), 8.41 (s, 1H); 13C NMR (151 MHz): d=12.49
(s, 1C), 19.35 (s, 1C), 49.03 (s, 2C), 50.47 (s, 2C), 54.41 (d, J=3.4 Hz,
1C), 57.26 (s, 1C), 67.53 (s, 1C), 67.57 (s, 1C), 69.81 (s, 1C), 74.89 (s,
1C), 105.11 (t, J=25.7 Hz, 1C), 106.68 (d, J=4.2 Hz, 1C), 111.17 (dd,
ChemMedChem 2010, 5, 757 – 769
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
765

L. Meerpoel, F. C. Odds, et al.
MED
4-[4-[4-[4-[[(2R,4S)-2-(2,4-difluorophenyl)-2-(1H-1,2,4-triazol-1-yl-
methyl)-1,3-dioxolan-4-yl]methoxy]phenyl]-1-piperazinyl]phen-
yl]-2,4-dihydro-2-[(1S,2R)-2-hydroxy-1-methylpropyl]-3H-1,2,4-tri-
azol-3-one (8): According to the synthesis of compound 2 starting
from dioxolane 18 (5.4 g, 0.012 mol) and phenol 12c (4.5 g,
0.0109 mol) to yield compound 8 as a white solid (6.6 g, 88%);
2,4-dihydro-2-[(1S,2S)-2-hydroxy-1-methylpropyl]-4-[4-[4-(4-hy-
droxyphenyl)-1-piperazinyl]phenyl]-3H-1,2,4-triazol-3-one (12a):
D
mp=201.28C (MeOH); ½aꢁ20 =ꢀ10.81 (c=1.01 w/v% in DMF);
1H NMR (360 MHz, [D6]DMSO): d=1.11 (d, J=6.3 Hz, 3H), 1.24 (d,
J=7.0 Hz, 3H), 3.04–3.13 (m, 4H), 3.26–3.33 (m, 4H), 3.75–3.84 (m,
J=7.2, 6.3, 6.3, 6.3, 5.2 Hz, 1H), 3.98–4.06 (m, J=7.2, 6.9, 6.9,
6.9 Hz, 1H), 4.73 (d, J=5.2 Hz, 1H), 6.68 (d, J=9.0 Hz, 2H), 6.85 (d,
J=9.0 Hz, 2H), 7.09 (d, J=9.1 Hz, 2H), 7.49 (d, J=9.1 Hz, 2H), 8.30
(s, 1H), 8.89 (s, 1H); Anal. calcd for C22H27N5O3: C 64.53, H 6.65, N
17.10, found: C 64.77, H 6.51, N 17.09; MS (ESI) m/z 410 ([M+H]+).
D
mp=199.38C (MeOH); ½aꢁ20 =+9.22 (c=1.03040 w/v% in DMF);
1H NMR (360 MHz, CDCl3): d=1.24 (d, J=6.4 Hz, 3H), 1.43 (d, J=
6.9 Hz, 3H), 3.20–3.26 (m, 4H), 3.34–3.40 (m, 4H), 3.49 (dd, J=9.7,
6.3 Hz, 1H), 3.75 (d, J=2.7 Hz, 1H), 3.78 (dd, J=9.7, 4.7 Hz, 1H),
3.82 (dd, J=8.4, 5.2 Hz, 1H), 3.98 (dd, J=8.4, 6.6 Hz, 1H), 4.17–4.24
(m, J=6.4, 6.4, 6.4, 2.9, 2.7 Hz, 1H), 4.28 (qd, J=6.9, 2.9 Hz, 1H),
4.38–4.46 (m, J=6.4, 6.4, 5.2, 4.7 Hz, 1H), 4.68 (d, J=14.7 Hz, 1H),
4.73 (d, J=14.7 Hz, 1H), 6.80 (d, J=9.0 Hz, 2H), 6.85–6.91 (m, 2H),
6.94 (d, J=9.0 Hz, 2H), 7.03 (d, J=8.9 Hz, 2H), 7.40 (d, J=8.9 Hz,
2H), 7.50 (td, J=8.9, 6.4 Hz, 1H), 7.64 (s, 1H), 7.89 (s, 1H), 8.23 (s,
1H); Anal. calcd for C35H38F2N8O5: C 61.04, H 5.56, N 16.27, found: C
61.00, H 5.30, N 16.15; MS (ESI) m/z 689 ([M+H]+).
2,4-dihydro-2-[(1R,2R)-2-hydroxy-1-methylpropyl]-4-[4-[4-(4-hy-
droxyphenyl)-1-piperazinyl]phenyl]-3H-1,2,4-triazol-3-one (12b):
D
mp=200.48C (MeOH); ½aꢁ20 =+10.35 (c=0.98 w/v% in DMF);
1H NMR (360 MHz, [D6]DMSO): d=1.11 (d, J=6.3 Hz, 3H), 1.24 (d,
J=7.0 Hz, 3H), 3.04–3.13 (m, 4H), 3.26–3.33 (m, 4H), 3.75–3.84 (m,
J=7.2, 6.3, 6.3, 6.3, 5.2 Hz, 1H), 3.98–4.06 (m, J=7.2, 6.9, 6.9,
6.9 Hz, 1H), 4.73 (d, J=5.2 Hz, 1H), 6.68 (d, J=9.0 Hz, 2H), 6.85 (d,
J=9.0 Hz, 2H), 7.09 (d, J=9.1 Hz, 2H), 7.49 (d, J=9.1 Hz, 2H), 8.30
(s, 1H), 8.89 (s, 1H); Anal. calcd for C22H27N5O3: C 64.53, H 6.65, N
17.10, found: C 63.26, H 6.66, N 16.70; MS (ESI) m/z 410 ([M+H]+).
2,4-dihydro-4-[4-[4-(4-methoxyphenyl)-1-piperazinyl]phenyl]-2-
(1-methyl-2-oxopropyl)-3H-1,2,4-triazol-3-one (10): A mixture of
triazolone
9 (61.5 g, 0.175 mol), 3-chlorobutan-2-one (23.6 g,
Synthesis of 12c and 12d: Zn(BH4)2 was prepared according to
the following procedure: ZnCl2 (40 g, 0.45 mol) was stirred in dry
Et2O (500 mL) at reflux until all ZnCl2 was dissolved. This solution
was added dropwise to a suspension of NaBH4 (27 g, 0.17 mol) in
dry Et2O (300 mL), and the mixture was stirred overnight at room
temperature. After filtration, the Zn(BH4)2/Et2O solution was added
dropwise to an ice-cooled (08C) stirred solution of compound 11
(40.7 g, 0.1 mol) in dry DMF (500 mL). The reaction mixture was al-
lowed to warm slowly to room temperature over a period of 3 h,
and was poured into ice–H2O. The resulting precipitate was filtered
off and dried in vacuo to give 12a,b and 12c,d (40 g, 70:30 respec-
tive mixture). The reaction was repeated at the same amount and
both crops were combined to give 12a,b/12c,d (77g), which was
purified by HPLC over Amicon silica (eluent: CH2Cl2/EtOAc/MeOH
66:30:4). The desired erythro fractions were collected, and the sol-
vent was evaporated to give 12c,d 27.5 g (33%). This fraction was
separated into its enantiomers over Chiralpac AD (eluent: 100%
MeOH). Two pure fractions were collected and evaporated: first
fraction yielding 12c (11.5 g, 14%); second fraction 12d (10.9 g,
13%). Both fractions were crystallized from MeOH, filtered, and
dried in vacuo at 408C.
0.22 mol), and K2CO3 (20 g) in DMF (400 mL) and toluene (400 mL)
was stirred overnight at 1108C with azeotropic removal of H2O.
The reaction mixture was cooled, and H2O (500 mL) and DIPE
(500 mL) were added. The product was crystallized from the reac-
tion mixture, filtered off, and washed with H2O and a small amount
of MeOH. Crystallizing the product from MIK, collecting and drying
in vacuo at 608C gave pure compound 10 (71 g, 97%) as a white
solid; mp=1728C (MIK); Anal. calcd for C23H27N5O3: C 65.54, H 6.46,
N 16.62, found: C 65.20, H 6.24, N 16.65.
2,4-dihydro-4-[4-[4-(4-hydroxyphenyl)-1-piperazinyl]phenyl]-2-
(1-methyl-2-oxopropyl)-3H-1,2,4-triazol-3-one (11): A mixture of
compound 10 (15 g, 0.035 mol) and NaHSO3 (4 g) in 48% aqueous
HBr (200 mL) was stirred and held at reflux for 5 h. The product
was then crystallized from the cooled reaction mixture, and the
solid was filtered off and washed with acetone. The resulting HBr
salt was stirred in a mixture of an aqueous NaHCO3 solution (~
300 mL) and a solution of CH2Cl2/n-butanol (90:10, ~300 mL). The
layers were separated, and the organic layer was washed with H2O,
brine, dried (MgSO4), filtered, and the solvent was evaporated. The
residue was triturated in MeOH, and the product was subsequently
collected by suction and dried in vacuo at 508C to yield compound
11 (12 g, 84%) as white crystals; mp=193.88C (MeOH); 1H NMR
(360 MHz, [D6]DMSO): d=1.47 (d, J=7.2 Hz, 3H), 2.10 (s, 3H), 3.06–
3.16 (m, 4H), 3.28–3.33 (m, 4H), 4.91 (q, J=7.2 Hz, 1H), 6.68 (d, J=
8.8 Hz, 2H), 6.86 (d, J=8.8 Hz, 2H), 7.11 (d, J=8.9 Hz, 2H), 7.51 (d,
J=8.9 Hz, 2H), 8.43 (s, 1H), 8.88 (s, 1H); Anal. calcd for C22H25N5O3:
C 64.85, H 6.18, N 17.19, found: C 64.78, H 6.06, N 17.35.
2-((1S,2R)-2-hydroxy-1-methylpropyl)-4-{4-[4-(4-hydroxyphenyl)-
piperazin-1-yl]phenyl}-2,4-dihydro-3H-1,2,4-triazol-3-one (12c):
D
mp=221.98C (MeOH); ½aꢁ20 =ꢀ7.07 (c=0.97600 w/v% in DMF);
1H NMR (360 MHz, [D6]DMSO): d=0.97 (d, J=6.2 Hz, 3H), 1.34 (d,
J=6.8 Hz, 3H), 3.04–3.15 (m, 4H), 3.24–3.34 (m, 4H), 3.71–3.81 (m,
J=7.7, 6.2, 6.2, 6.2, 5.9 Hz, 1H), 3.89–3.99 (m, J=7.7, 6.8, 6.8,
6.8 Hz, 1H), 4.92 (d, J=5.9 Hz, 1H), 6.68 (d, J=8.6 Hz, 2H), 6.85 (d,
J=8.6 Hz, 2H), 7.08 (d, J=8.7 Hz, 2H), 7.48 (d, J=8.7 Hz, 2H), 8.32
(s, 1H), 8.89 (s, 1H); Anal. calcd for C22H27N5O3: C 64.53, H 6.65, N
17.10, found: C 64.37, H 6.70, N 17.11; MS (ESI) m/z 410 ([M+H]+).
Synthesis of 12a and 12b: A stirred mixture of compound 11
(25 g, 0.06 mol) in dry DMF (500 mL) was purged with N2 and
cooled to ꢀ108C. Potassium tri(sec-butyl)borohydride (K-selectride;
150 mL of a 1m solution in THF) was added dropwise. The mixture
was allowed to warm to room temperature, followed by pouring
out into ice–H2O. The resulting precipitate was filtered off, washed
with a small amount of MeOH, and crystallized from MeOH. The
crystals were collected and dried in vacuo to yield 18.9 g of a mix-
ture (90:10) of 12a,b and 12c,d. The mixture was purified by HPLC
over Chiralpac AD (eluent: EtOH). Two pure fractions were collect-
ed (compound 12b was eluted first followed by 12a), and evapo-
rated until dry. Each residue was triturated in MeOH, subsequently
filtered off, and dried in vacuo at 408C to yield compound 12a
(7.3 g, 30%) and compound 12b (8 g, 32.5%).
2-((1R,2S)-2-hydroxy-1-methylpropyl)-4-{4-[4-(4-hydroxyphenyl)-
piperazin-1-yl]phenyl}-2,4-dihydro-3H-1,2,4-triazol-3-one (12d):
D
mp=224.38C (MeOH); ½aꢁ20 =+6.86 (c=0.99160 w/v% in DMF);
1H NMR (360 MHz, [D6]DMSO): d=0.97 (d, J=6.2 Hz, 3H), 1.34 (d,
J=6.8 Hz, 3H), 3.04–3.15 (m, 4H), 3.24–3.34 (m, 4H), 3.71–3.81 (m,
J=7.7, 6.2, 6.2, 6.2, 5.9 Hz, 1H), 3.89–3.99 (m, J=7.7, 6.8, 6.8,
6.8 Hz, 1H), 4.92 (d, J=5.9 Hz, 1H), 6.68 (d, J=8.6 Hz, 2H), 6.85 (d,
J=8.6 Hz, 2H), 7.08 (d, J=8.7 Hz, 2H), 7.48 (d, J=8.7 Hz, 2H), 8.32
(s, 1H), 8.89 (s, 1H); Anal. calcd for C22H27N5O3: C 64.53, H 6.65, N
17.10, found: C 64.41, H 6.67, N 17.09; MS (ESI) m/z 410 ([M+H]+).
766
www.chemmedchem.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2010, 5, 757 – 769

Antifungal Activity of Saperconazole Metabolites
D
2,4-dihydro-2-[(1S,2S)-2-hydroxy-1-methylpropyl]-4-[4-[4-(4-me-
thoxyphenyl)-1-piperazinyl]phenyl]-3H-1,2,4-triazol-3-one (13a):
A mixture of intermediate 13c (1.38 g, 0.00327 mol), PPh3 (2.1 g,
0.00806 mol) and p-nitrobenzoic acid (1.2 g, 0.00717 mol) in a dry
mixture of THF/DMA (3:2, 50 mL) was heated until complete disso-
lution, followed by cooling to room temperature, whereupon di-
ethyl azodicarboxylate (1.4 g, 0.00806 mol) was added dropwise.
The mixture was then stirred at room temperature for 90 min and
for an additional 1 h at 508C, followed by the addition of NaOH
(1n, 10 mL) at 508C. The mixture was then poured out into a mix-
ture of H2O (100 mL) and 1n NaOH (90 mL), followed by stirring.
The resulting precipitate was filtered off and recrystallized from 2-
propanol (60 mL). After 48 h, the precipitate was filtered off and
dried in vacuo at 408C to yield compound 13a (0.98 g, 71%),
which was used in the next step without further purification (the
preparation contained traces of compound 16).
tical rotation of ½aꢁ20 =ꢀ6.59 (c=0.500 w/v% in DMF) correspond-
ing to 12c.
Biological assays
In vitro antifungal activities (results listed in Table 1): The com-
pounds were dissolved in DMSO up to
a concentration of
1.5 mgmLꢀ1. These stock solutions were stored at ꢀ208C for up to
six months. For susceptibility determinations, doubling dilutions of
the stock solutions were prepared in DMSO and these were further
diluted in sterile H2O to threefold the desired final test concentra-
tion. DMSO alone was diluted in H2O for the preparation of drug-
free controls. The antifungal dilutions and control solutions were
dispensed in 50 mL volumes in microplate wells: the plates were
stored at ꢀ208C for up to six months before use.
The test strains included 20 clinical isolates each of C. albicans,
C. glabrata and C. krusei, 12 each of C. lusitaniae and C. parapsilosis,
9 of C. tropicalis, 19 of Cr. neoformans, 10 isolates of A. fumigatus,
and 9 isolates each of M. canis, T. mentagrophytes and T. rubrum.
The fungi, which were stored in suspensions at ꢀ708C with 10%
glycerol as cryoprotectant were re-grown on Sabouraud glucose
agar (Oxoid, UK) at appropriate incubation temperatures. Inocula
of yeasts were prepared by growing the organisms to a concentra-
tion of 4ꢃ107 cellsmLꢀ1 at 308C with rotation at 20 rpm in CYGi
broth.[52,53] Inocula of moulds were prepared on Sabouraud agar at
25–308C. The growth was flooded with sterile 0.05% SDS and agi-
tated to provide a suspension with a turbidity OD value of ~1 at
530 nm. Mould inocula contained predominantly conidia or pre-
dominantly hyphal fragments, according to the level of sporulation
achieved by the isolate concerned.
Synthesis of 12a from 13a: Compound 13a (0.98 g, 0.0023 mol)
was heated at 908C for 5 h in a mixture of HBr (48%, 25 mL) and
HBr-saturated acetic acid (25 mL) in the presence of a small
amount of NaHSO3 (1 g). The mixture was stirred overnight at
room temperature. The precipitate was filtered off, suspended in
H2O (200 mL), and after addition of 50% NaOH (20 mL) stirring was
continued for another 2 h. After acidification to pH 4 with concen-
trated HCl and subsequent neutralization with an aqueous saturat-
ed solution of NaHCO3, the precipitate was filtered off and purified
by chromatography on silica gel (2% MeOH in CH2Cl2). The desired
D
fraction was re-crystallized from MeOH to give (0.6 g, 63%): ½aꢁ20
=
ꢀ10.2 (c=0.995 w/v% in DMF) corresponding to 12a, confirmed
by chiral HPLC Chiralpac AD (eluent: EtOH).
2-((1S,2R)-2-hydroxy-1-methylpropyl)-4-{4-[4-(4-methoxyphenyl)-
piperazin-1-yl]phenyl}-2,4-dihydro-3H-1,2,4-triazol-3-one (13c):
tBuOK (15.8 g, 0.165 mol) was added to a stirred mixture of com-
pound 9[37] (52.6 g, 0.15 mol) in dry DMA (500 mL) under a flow of
N2 gas. The mixture was stirred for 1 h at 1008C and then cooled
to 508C. meso-(4R,5R)-4,5-Dimethyl-1,2,3-dioxathiolane-2,2-diox-
ide[41] (25.1 g, 0.165 mol) was added dropwise. The mixture was
stirred for 2 h at 50–608C, followed by dropwise addition of con-
centrated H2SO4 (20 mL), and stirring was continued at 608C for
another 2 h. H2O (20 mL) was then added, and stirring was contin-
ued for 20 h at 608C. Then the reaction mixture was cooled,
poured out into ice–H2O (1000 mL), and made alkaline (pH 8) with
NaOH (50% w/v) under stirring. The resulting precipitate was fil-
tered off, washed with H2O, and dried in vacuo at 408C, followed
by purification via column chromatography over silica gel (eluent:
CH2Cl2/MeOH 99:1). The pure fractions were collected and evapo-
rated to dryness. The residue was triturated in CH2Cl2 (100 mL) and
DIPE (50 mL), filtered off, and dried in vacuo at 408C to yield com-
Susceptibility tests with all yeasts and moulds was done with the
same medium: RPMI 1640 broth with extra glucose (final concen-
tration 20 gLꢀ1) and buffered to pH 7.0 with 0.165m MOPS. This
medium was prepared at 1.5ꢃ final concentration. Fungal inocu-
lum suspensions were diluted into the medium (20 mL per 10 mL)
and 100 mL volumes of the inoculated broths were added to the
50 mL of drug dilution already present in the microdilution trays,
thus bringing all components to the correct final concentration.
The plates were sealed with adhesive stickers. Pathogenic yeasts
were incubated at 378C; moulds at appropriate temperatures, usu-
ally 308C. The incubation time was 48 h for Candida spp. For all
other isolates, the plates were inspected at intervals, and incuba-
tion was terminated when control wells contained richly turbid
growth.
The turbidity of growth in the wells was estimated as OD405nm by
means of a microplate spectrophotometer. Growth in the presence
of test compounds was expressed as a percentage of control
growth (with experimental and control readings corrected for
background OD). The MIC of each compound for each isolate was
defined as the IC50 (lowest concentration of test substance that de-
creased growth below 50% of control) and was estimated from
dose–response curve data. Isolates for which control OD405nm read-
ings were <0.15 were excluded from analysis.
D
pound 13c (53.7 g, 84%) as a white solid; ½aꢁ20 =ꢀ5.448 (c=
0.9735 w/v% DMF); Anal. calcd for C23H29N5O3: C 65.23, H 6.90, N
16.54, found: C 65.37, H 7.16, N 16.90.
Synthesis of 12c from 13c: Compound 13c (5 g, 0.0118 mol) was
heated at 908C for 5 h in a mixture of HBr (48%, 25 mL) and HBr-
saturated acetic acid (25 mL) in the presence of NaHSO3 (1 g). The
mixture was stirred at room temperature overnight. The precipitate
was filtered off, suspended in H2O (200 mL), supplied with 50%
NaOH (20 mL) and DMF (50 mL) and stirred for another 2 h. This
mixture was acidified to pH 4 with concentrated HCl and subse-
quently neutralized with an aqueous saturated solution of NaHCO3.
The resulting precipitate was filtered off and purified by silica gel
chromatography (2% MeOH in CH2Cl2). The desired fraction was
re-crystallized from MeOH to give a solid (3 g, 62%) having an op-
In vivo antifungal activity by intravenous infusion in experimen-
tal aspergillosis (results listed in Tables 2 and 3): For intravenous
infusion the compounds were prepared as solutions in 20% hy-
droxypropy1-1-b-cyclodextrin.
Fungus and inoculum preparation: A. fumigatus strain B 19119,
originally isolated from a pulmonary infection, was used through-
out the experiments. The fungus was grown on potato glucose
agar (Difco) for six days at 358C and conidia harvested in a solution
ChemMedChem 2010, 5, 757 – 769
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
767

L. Meerpoel, F. C. Odds, et al.
MED
containing 20 mL Tween 80 per 100 mL physiological saline. The
suspension was filtered through sterile gauze, centrifuged at
3000 g for 30 min, and re-suspended in sterile physiological saline/
Tween 80. Sterile glycerol was then added to the suspension to a
final concentration of 10% (v/v), and the suspension was distribut-
ed in 1 mL volumes in cryotubes. The tubes of inoculum suspen-
sion were stored at ꢀ708C, and a fresh tube was thawed and used
for inoculation in each animal experiment. The number of viable
A. fumigatus colony-forming units (CFU) per mL suspension was
determined to be 1.75ꢃ109.
Table 5. Tissue burdens of A. fumigatus in organs removed from placebo-
treated guinea pigs.
log10 CFUgꢀ1 in five organs of guinea pigs[a]
Brain
Liver
Spleen
Kidney
Lung
0
0
4.6
3.7
3
4
0
4.5
3.7
3.9
3.7
2.4
4.6
3
4.3
4.4
0
2.7
3.2
2.9
0
0
0
0
0
0
0
0
4.2
3.4
0
3.3
3
5.2
3
Animals and their inoculation: Animal experiments were ap-
proved by the Ethics Committee on Animal Experimentation at
Johnson & Johnson Pharmaceutical Research & Development, a di-
vision of Janssen Pharmaceutica N.V. This animal research facility,
located Turnhoutseweg 30, 2340 Beerse (Belgium) is permitted to
perform animal experiments under national license number
LA1100119.
3.9
2.4
3.7
0
0
3
3.8
2.2
0
4.6
5.5
5.4
4
4.3
4.4
5.8
3.9
3.9
5.1
4.1
4.7
3.5
3.7
3.8
4.6
5
4.5
2.9
4.2
4.3
4
4.2
3.5
3.6
3.3
2.2
2.2
4.6
0
0
0
2.6
0
3.4
0
0
0
0
0
0
2.3
0
2.7
0
0
4.6
4.8
3.8
4.3
3.6
3.9
3.5
4.3
4.1
4.2
4.4
4.1
4.1
Specific pathogen-free (SPF) guinea pigs with a weight of ~400–
500 g were used in all experiments. A catheter was placed into the
left jugular vein of each animal, the vein ligated, and the catheter
connected (via a proprietary swivel device to provide free move-
ment for the animal) to a microprocessor-controlled infusion pump
(Greasby Medical) from which a continuous infusion of physiologi-
cal saline was delivered. The animals were infected via the implant-
ed catheter with a volume of inoculum calculated to deliver 4000
A. fumigatus CFU per gram body weight.
4
3.2
2.9
0
3.1
0
4.6
5.2
5.1
4.2
3.4
5
3.9
4.5
0
0
0
[a] 0 indicates that no A. fumigatus was isolated.
Treatment protocols: The first intravenous treatments began 1 h
after infection of the animals. The test formulations were then ad-
ministered on subsequent days as two 1 h infusions daily, separat-
ed by a period of 5 h, for a total of 9.5 days (19 infusions in total).
On day 10, surviving animals were sacrificed. These, and animals
that died before the end of treatment, were autopsied. The place-
ment of the catheters and local reactions to catheter placement
were assessed grossly at autopsy. Data from animals for which
there was postmortem evidence of catheter misplacement or
severe local reaction that might have interfered with intravenous
administration of the test substances were excluded from analysis.
Effects on the growth of A. fumigatus and ergosterol biosynthe-
sis in A. fumigatus and C. albicans: The effects of the compounds
tested on growth of A. fumigatus, and the incorporation of
[14C]acetate into ergosterol by C. albicans and A. fumigatus were
measured as described previously.[48]
Azoles and [14C]acetate were added to cultures of A. fumigatus im-
mediately after inoculation. Cells were collected immediately after
16 h of growth, homogenized, protein content (a measure of
growth; IC50 values: concentrations needed to achieve 50% de-
crease in protein content) determined, and lipids extracted. Sterols
were separated by HPLC. C. albicans was first grown for 16 h (in-
cluding 8 h in a reciprocal shaker) at 308C in polypeptone/yeast
extract/glucose (10:10:40 gLꢀ1) medium, then washed and resus-
pended in a 0.1m potassium phosphate buffer containing 56 mm
glucose (pH 6.5). [14C]Acetate, azole antifungal agents, and/or
DMSO were added, and the cell suspensions were incubated for
2 h at 308C. Sterols were extracted and separated by TLC. IC50
values: concentrations needed to inhibit incorporation of the 14C
label into ergosterol by 50%.
Samples of lung, brain, liver, spleen, and left kidney were removed
with aseptic precautions, weighed, homogenized, and samples of
homogenate were cultured on Sabouraud agar for enumeration of
A. fumigatus CFU. Lung samples were so rarely positive for A. fumi-
gatus that they were not included in the analysis of results.
Reproducibility of infection in placebo-treated animals: The in-
trajugular route of inoculation, continuous intravenous infusion of
fluids, and the duration of treatments used in these experiments
were all entirely novel and previously untried. Table 5 lists the data
obtained for tissue burdens of A. fumigatus in the organs sampled
postmortem to indicate the severity of infection achieved by intra-
jugular inoculation of the fungal conidia at the dose selected. The
mean survival time (MST) for placebo-treated animals was ~5 days,
with some variation between experiments (lowest MST was
4.8 days; highest was 5.8 days). The data in Table 3 show that only
four of 24 animals suffered dissemination of the A. fumigatus to
the lungs, whereas in 14 of 24, the infection disseminated to the
brain. The liver, spleen, and kidney were the organs most severely
affected, with few A. fumigatus-negative tissues among the place-
bo-treated guinea pigs. Quantitatively, the spleen was the most se-
verely infected organ, with tissue burdens ranging from 2.4 to 5.8
Acknowledgements
The authors thank Ms. Ann Dierckx for technical assistance.
Keywords: antifungal agents · disseminated aspergillosis ·
metabolites · saperconazole · triazoles
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Received: January 30, 2010
Revised: March 4, 2010
Published online on April 8, 2010
ChemMedChem 2010, 5, 757 – 769
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
769