Novel macrocyclic amidinoureas: Potent non-azole antifungals active against wild-type and resistant Candida species

Article abstract of DOI:10.1021/ml400187w

Novel macrocyclic amidinourea derivatives 11, 18, and 25 were synthesized and evaluated as antifungal agents against wild-type and fluconazole resistant Candida species. Macrocyclic compounds 11 and 18 were synthesized through a convergent approach using as a key step a ring-closing metathesis macrocyclization reaction, whereas compounds 25 were obtained by our previously reported synthetic pathway. All the macrocyclic amidinoureas showed antifungal activity toward different Candida species higher or comparable to fluconazole and resulted highly active against fluconazole resistant Candida strains showing in many cases minimum inhibitory concentration values lower than voriconazole.

Full text of DOI:10.1021/ml400187w

Letter
pubs.acs.org/acsmedchemlett
Novel Macrocyclic Amidinoureas: Potent Non-Azole Antifungals
Active against Wild-Type and Resistant Candida Species
Maurizio Sanguinetti,§ Stefania Sanfilippo,† Daniele Castagnolo,†,‡ Dominique Sanglard,∥
Brunella Posteraro,§ Giovanni Donzellini,† and Maurizio Botta*,†,⊥
†Dipartimento Biotecnologie, Chimica e Farmacia, Universita
̀
di Siena, via Aldo Moro 2, 53100 Siena, Italy
‡School of Applied Sciences, Northumbria University, Ellison Building, Ellison Place, NE1 8ST, Newcastle upon Tyne, United
Kingdom
§Istituto di Microbiologia, Universita
̀
Cattolica del Sacro Cuore, Largo F. Vito 1, 00168 Rome, Italy
∥Institute of Microbiology, University of Lausanne and University Hospital Center, Lausanne, Rue du Bugnon 48, CH-1011
Lausanne, Switzerland
⊥Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology,
Temple University, BioLife Science Building, Suite 333, 1900 North 12th Street, Philadelphia, Pennsylvania 19122, United States
S
* Supporting Information
ABSTRACT: Novel macrocyclic amidinourea derivatives 11, 18, and 25 were synthesized and evaluated as antifungal agents
against wild-type and fluconazole resistant Candida species. Macrocyclic compounds 11 and 18 were synthesized through a
convergent approach using as a key step a ring-closing metathesis macrocyclization reaction, whereas compounds 25 were
obtained by our previously reported synthetic pathway. All the macrocyclic amidinoureas showed antifungal activity toward
different Candida species higher or comparable to fluconazole and resulted highly active against fluconazole resistant Candida
strains showing in many cases minimum inhibitory concentration values lower than voriconazole.
KEYWORDS: Antifungal, amidinourea, macrocyclization, ring-closing metathesis, Candida species, fluconazole,
antifungal drug-resistance
array of pathogens, fungal infections pose considerable
he epidemiology of fungal infections has been an evolving
Tissue since the late 1960s when, as a consequence of the diagnostic and therapeutic challenges. Although several species
of fungi are potentially pathogenic in humans, Candida, and in
particular Candida albicans, is the organism responsible for
most fungal diseases.2,3 Defense against Candida infections has
relied on the use of a limited number of chemotherapeutic
agents, including azoles, such as fluconazole and voriconazole,
and polyenes, such as amphotericin B.4 In particular, azoles
have been extensively used to treat a wide range of candidiasis
because of their low toxicity and high degree of bioavailability
after oral administration.5 However, despite they still constitute
the preferred first line therapy, the emergence and the spread of
drug resistant fungal species6,7 draw the line at the use of these
classical antifungal drugs. Today, fluconazole is poorly or not
development of antibiotic therapies, a drastic rise of mycoses
was observed. Today, fungal infections represent a major global
health threat and the increasing incidence of invasive and
opportunistic mycoses is often associated with excessive
morbidity and mortality.1 Fungal infections have increased in
incidence in recent decades often as a result of advanced
medical treatments and the increase in the number of
immunocompromised patients. Patients who are at risk for
the development of serious fungal infections include those
undergoing blood and solid-organ transplantation, gastro-
intestinal surgery, patients with AIDS, and premature infants.
In addition, cancer chemotherapy and allogeneic bone marrow
transplantation are often associated with fungal disease, and up
to 30% of patients with acute leukemia experience invasive
fungal infections. Given the complexity of the population of
patients suffering from a mycosis and the diverse and increasing
Received: May 15, 2013
Accepted: July 22, 2013
© XXXX American Chemical Society
A
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ACS Medicinal Chemistry Letters
Letter
effective against mutant Candida species making the develop-
ment of new and more active drugs a priority.8 Recently, we
reported the discovery and synthesis of a new class of potent
antifungal agents, namely, guanidines and macrocyclic
amidinoureas,9−13 which proved to be strongly active against
Candida species. Macrocycle 1, bearing a crotyl moiety on the
alkylguanidine side chain, emerged as the most interesting
compound with a biological activity higher than fluconazole
against several Candida spp.14,15 Only few structure−activity
relationships (SAR) arose from our previous studies, indicating
that the nature of the alkyl group bound to the guanidine
moiety plays a key role in the biological activity.
However, no investigations were carried out on the
macrocyclic core, both in terms of size as well as regarding
the effects that the incorporation of polar/apolar moieties in to
the ring might have on the antifungal activity. Hence, with the
aim to identify new macrocyclic amidinoureas active both on
wild-type as well as mutant fungal species and to further
investigate their SAR, we planned the synthesis of two series of
amidinourea derivatives A and B, both preserving some
structural characteristics of the lead 1 (Figure 1). The first
latter compounds were refluxed in THF together with 6
affording amidinoureas 7a−c. Secondary amine 6 was
synthesized in a two amidation−reduction sequence starting
from Cbz-aminooctanoic acid 5, which was first coupled with
allylamine followed by reduction of the resulting amide with
diisobutylaluminium hydride (DIBAL-H). Macrocyclization of
7a−c was then carried out using Grubbs’ catalyst second
generation at 40−80 °C leading to macrocycles 8a−c in good-
high yields,17,21 as a mixture of E/Z isomers. Hydrogenation of
8a−c allowed the reduction of the double bond and the Cbz
cleavage in one single step, affording amines 9a−c. Primary
amines were then guanylated with N-crotyl-S-Me-isothiourea-
N,N′-diBoc and then treated with trifluoroacetic acid (TFA),
leading to desired compounds 11a−c as trifluoroacetic salts. A
similar approach was used for the synthesis of derivatives 18a−
b bearing an ester moiety on the macrocyclic ring. Beta-alanine
12 was first guanylated in the presence of trimethylsilyl chloride
(TMSCl) to protect in situ the carboxylic moiety,22 and the
resulting acid was esterified using the appropriate alcohol to
afford guanidines 13a−b. Allylamine 6 was then reacted with
13a−b in refluxing tetrahydrofuran (THF) leading to dienes
14a−b in high yields. RCM macrocyclization of these latter
compounds was accomplished with Grubbs’ catalyst in DCM at
40 °C, affording a mixture of E/Z alkenes 15a−b. Hydro-
genation of 15a−b followed by guanylation and TFA mediated
Boc deprotection led finally to desired derivatives 18a−b. In
our previous paper, we described the synthesis of lead 1 starting
from the commercially available 1,17-diamino-9-azaheptade-
cane. However, because of the removal of this building block
from the market, we were forced to design a new synthetic
pathway. Thus, macrocyclic derivatives 25a−b were synthesized
as outlined in Scheme 2. The coupling between amine 19 and
acid 20 led to the formation of amide 21, which was in turn
reduced with DIBAL-H affording the secondary amine 22.23
Refluxing of 22 in THF led to the formation of the desired
macrocycle 23 in good yield. The Cbz protecting group was
then cleaved by hydrogenation affording the corresponding
primary amine. This latter compound was then guanylated with
two different S-Me-alkyl-thioureas 26a−b leading, after Boc
cleavage, to the desired derivatives 25a−b. Compounds 11a−c,
18a−b, and 25a−b were then assayed against a total of 116
clinical isolates of seven different wild-type Candida species
(oral, vaginal, anorectal, urine, stool, blood, central venous
catheter, and respiratory tract specimen with each strain
representing a single isolate from a patient). In Table 1, the
MIC90 medium values for each species of Candida were
reported. Macrocycles 25a and 25b proved to be the most
active compounds with MIC values better than fluconazole
against most of the Candida strains.
Figure 1. Structure of hit compound 1.
series of derivatives A was planned with the aim to modulate
the antifungal activity introducing an ester moiety (polar
group) or fusing a benzene (apolar group) with the macrocyclic
core, thus potentially favoring the interaction of novel
compounds with target enzymes through H-bond or π-
interactions. Moreover, the synthesis of 14- and 15-membered
ring amidinoureas was also planned in order to further explore
the chemical space and evaluate the effects that the macrocycle
size might have on the biological activity.
The synthesis of two additional derivatives B bearing
different alkyl substituents on the guanidine moiety was also
planned with the aim to evaluate the importance of an
unsaturated double bond for the biological activity. Com-
pounds 11a−c and 18a−b were synthesized through a
convergent approach using as a key step a ring-closing
metathesis (RCM) macrocyclization,16−20 as described in
Scheme 1. Aldehyde 2 was first O-alkylated with the
appropriate alkenylbromide and in turn condensed with
NH2OH. The resulting oximes were then reduced in the
presence of Zn affording the free amines 3a−c, which were
guanylated to give the guanidine building blocks 4a−c. These
In particular, 25b showed to be strongly active against C.
krusei, a species resistant to common azole drugs, and against C.
tropicalis. Compounds 11 and 18 retain antifungal activity as
well, in many cases with MIC values better than fluconazole. In
general, compounds 11a−c possessing an aromatic substitution
on the macrocyclic core proved to be more active when
compared to 18a−b bearing an ester moiety. The increase of
the ring size in compounds 11 from 13 to 15 carbon atoms
evidenced an enhancement of activity toward all Candida
species. However, it is noteworthy that the 14-membered ring
11b showed the highest activity toward C. albicans, C.
guillermondii, and C. parapsilosis, while the larger macrocycle
11c proved to be more active against C. tropicalis and C. kefyr.
In all cases, 11b−c resulted more than or as active as
B
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ACS Medicinal Chemistry Letters
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a
Scheme 1. Synthesis of Derivatives 11a−c and 18a−b
a
Reagents and conditions: (i) Br(CH2)nCHCH2, K2CO3, DCM, reflux; (ii) NH2OH, Py, EtOH, reflux; (iii) Zn, HCl, THF, reflux; (iv)
(BocNH)2CNTf, Et3N, DCM; (v) AllylNH2, EDC, HOBt, DIPEA, DMF; (vi) DIBAL-H, DCM, r.t.; (vii) THF, reflux, 12 h; (viii) Grubbs’ Cat.
second gen., toluene or DCM 2−10 mM, 40−80 °C; (ix) H2, Pd/C, EtOH; (x) CrotylNBoc(CNBoc)SMe, THF, reflux, 12 h; (xi) TMSCl, Et3N,
DCM, reflux; (xii) DMAP, DCC, HO(CH2)nCHCHCH3, DCM, rt 24 h; (xiii) 6, THF, reflux, 12 h; (xiv) Grubbs’ Cat. second gen., DCM 2 mM,
40 °C; (xv) H2, Pd/C, EtOH; (xvi) CrotylNBoc(CNBoc)SMe, THF, reflux, 12 h.
a
analogue 25. In general, the introduction of an apolar moiety
leads to the decrease of the antifungal activity, with the
exception of compound 11b, which showed a stronger activity
against C. glabrata, a species resistant to common azoles, with a
minimum inhibitory concentration (MIC) = 8 μg/mL.
Compounds 18a−b present a lower activity than 25 and 11
toward all Candida species. Again the larger macrocycle 18b
resulted more active than 18a. As a general rule, the
introduction of a substituent on the macrocyclic ring results
in a slight decrease of antifungal activity. However, the presence
of a fused aromatic ring seems to be more beneficial than the
introduction of an ester moiety while larger rings lead to an
increase of biological activity. Azole drugs are known to act as
antifungals inhibiting the fungal enzyme 14α-demethylase,
which produces ergosterol, an important component of the
fungal plasma membrane. At the molecular level, different
mechanisms contribute to resistance against azole agents.24 The
principal mechanism regards the alterations of 14-α-demethy-
lase due to the overexpression and mutations of the gene
ERG11 coding for the enzyme. As a consequence, a higher
intracellular azole concentration is needed to complex the
increased number of demethylase enzymes present in the fungal
cells.25 In addition, it has been demonstrated that mutations in
ERG11 prevents binding of azoles to the enzymatic site. A
second major mechanism leading to azole resistance is the
overexpression of plasma membrane efflux pumps. This
Scheme 2. Synthesis of Derivatives 25a−b
a
Reagents and conditions: (i) EDC, HOBt, DIPEA, DMF; (ii)
DIBAL-H, DCM, r.t.; (iii) THF, reflux, 12 h; (iv) H2, Pd/C, EtOH;
(v) 26a−b, THF, reflux, 12 h.
fluconazole, while the 13-membered ring 11a proved to be less
active. However, compounds 11 bearing a benzene fused with
the macrocyclic core resulted less active than the unsubstituted
C
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ACS Medicinal Chemistry Letters
Letter
a
Table 1. Antifungal Activity (MIC90, μg/mL)
b
c
species (No. strains tested)
F
11a
11b
11c
18a
18b
25a
25b
1
C. albicans (22)
C. guillermondii (10)
C. krusei (13)
2
16
16
32
8
4
2
8
2
2
4
8
4
32
32
64
32
32
32
64
16
8
1
1
2.5
n.d.
5
4
2
2
2
256
0.5
2
4
32
8
4
0.5
4
C. parapsilosis (24)
C. tropicalis (11)
C. kefyr (10)
2
2
5
8
1
16
16
32
0.5
4
0.5
4
1.25
n.d.
20
1
4
2
C. glabrata (26)
16
64
16
16
32
a
b
c
MIC90 values were determined at 24 h both visually and spectrophotometrically. Fluconazole. Activity values expressed as MIC50.
Table 2. Antifungal Activity on C. albicans and C. glabrata Fluconazole Resistant Strains
a
MIC90 (μg/mL)
b
c
strains
species
resistance mechanism
F
VOR
4
11a 11b 11c 18a 18b 25a 25b
DSY284
C. albicans mutation in the ERG11 gene; increased expression of the CDR1
256
128
256
32
16
16
8
2
4
2
2
9
16
16
8
8
1
2
1
2
1
2
1
1
and CDR2 genes
DSY296
DSY289
DSY348
C. albicans mutation in the ERG11 gene; increased expression of the CDR1
8
10
5
16
8
and CDR2 genes
C. albicans mutation in the ERG11 gene; increased expression of the CDR1
8
and CDR2 genes
C. albicans mutation in the ERG11 gene; increased expression of the CDR1
0.25
8
5
8
8
and CDR2 genes
DSY292
DSY735
DSY775
C. albicans mutation in the ERG11 gene; increased expression of the MDR1
C. albicans increased expression of the CDR1 and CDR2 genes
64
2
2
8
16
8
4
2
4
10
5
16
8
16
16
16
2
1
2
2
1
2
64
C. albicans mutation in the ERG11 gene; increased expression of the CDR1
128
16
10
16
and CDR2 genes
DSY751
C. albicans increased expression of the MDR1 gene; mutation in the ERG11
256
0.25
16
4
10
16
16
2
gene
DSY530
DSY754
DSY756
C. glabrata increased expression of the CgCDR1 genes
C. glabrata increased expression of the CgCDR1 genes
64
0.5
2
64
64
64
8
36
40
36
64
32
32
64
16
16
16
16
32
16
64
16
8
64
C. glabrata increased expression of the CgCDR1, CgCDR2, and CgSNQ2
128
4
128
genes
DSY2254
DSY2271
C. glabrata increased expression of the CgCDR1 and CgCDR2 genes
128
64
2
64
64
16
8
40
36
128
64
64
32
32
16
32
32
C. glabrata increased expression of the CgCDR2 genes
0.125
a
b
c
MIC values were determined at 24 h both visually and spectrophotometrically. Fluconazole. Voriconazole.
Table 3. Antifungal Activity on Amphotericin Susceptible and Resistant Candida Strains
a
MIC90 (μg/mL)
b
strains
species
AmB
11a
11b
11c
18a
18b
25a
25b
ATCC 200955
ATCC 200950
ATCC 200951
ATCC 200952
ATCC 200953
ATCC 24348
ATCC 60247
ATCC 200956
C. albicans
2
1
1
2
1
8
1
2
2
2
4
2
4
4
5
8
4
1
4
2
1
2
2
4
2
1
2
2
2
4
1
2
1
C. lusitaniae
C. lusitaniae
C. lusitaniae
C. lusitaniae
C. lusitaniae
C. lusitaniae
C. tropicalis
8
5
8
8
8
5
4
4
8
5
4
4
16
8
5
8
8
0.125
0.125
8
5
4
8
16
16
5
4
4
10
16
16
a
b
MIC values were determined at 24 h both visually and spectrophotometrically. Amphotericin B.
mechanism is mediated by two types of transporters, the major
facilitators (encoded by MDR genes in C. albicans) and the
ABC transporters (encoded by CDR genes in C. albicans).26
Upregulation of the CDR genes confers resistance to multiple
azoles in C. albicans, whereas upregulation of MDR1 alone
leads to fluconazole resistance exclusively. Compounds 11, 18,
and 25 were thus assayed against mutant C. albicans and C.
glabrata strains, and voriconazole, an azole active on
fluconazole resistant strains, was chosen as the reference
compound. All the tested derivatives proved to be active against
C. albicans and C. glabrata fluconazole-resistant strains (Table
2). Among them macrocycles 25a−b showed better activity
values against resistant C. albicans strains than voriconazole
(MIC = 1−2 μg/mL) with the exception of DSY348 and
DSY751 strains. Compound 11b resulted more active than
voriconazole itself against mutant strains DSY289, DSY296,
DSY284, and DSY775 as well as 11c against DSY289.
Derivatives 18 were more active than fluconazole but in
general less active than 11 and voriconazole, except for DSY289
strain. Analogues 11a, 11c, and 18a−b resulted inactive against
C. glabrata resistant strains. However, the 14-membered
macrocycle 11b, as well as compounds 25a−b, showed a
good activity proving to be more active than fluconazole against
all C. glabrata strains and showing a MIC value against DSY756
close to voriconazole. In addition all these compounds are fully
D
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ACS Medicinal Chemistry Letters
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(6) Ahmad, A.; Khan, A.; Manzoor, N. Reversal of efflux mediated
antifungal resistance underlies synergistic activity of two monoterpenes
with fluconazole. Eur. J. Pharm. Sci. 2013, 48, 80−86.
(7) Klepser, M. E. Candida resistance and its clinical relevance.
Pharmacotherapy 2006, 26, 68S−75S.
active against Candida isolates reported as resistant to
amphotericin B (Table 3).27
In conclusion, novel macrocyclic amidinoureas 11a−c, 18a−
b, and 25a−b were synthesized and biologically assayed as
antifungal agents. Derivatives 11 and 18 were obtained through
an innovative convergent synthetic pathway whose key step was
represented by a RCM macrocyclization. All the new
macrocycles showed potent antifungal activity against different
wild-type Candida species and fluconazole- or Amphotericin B-
resistant Candida strains. In particular, 25a−b and 11b resulted
in the most interesting potential antifungal agents. Macrocyclic
amidinoureas proved to be excellent non-azole lead compounds
able to act both on classic as well as resistant fungal infections.
Because of their innovative structure, it is reasonable to
hypothesize for macrocyclic amidinoureas a mechanism of
action different from that of classical azole and polyenic drugs.
Further studies to disclose the mechanism of action are
currently in progress in our laboratories.
(8) Casalinuovo, I. A.; Di Francesco, P.; Garaci, E. Fluconazole
resistance in Candida albicans: a review of mechanisms. Eur. Rev. Med.
Pharmacol. Sci. 2004, 8, 69−77.
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triamines, and polyamines: chemistry and biological properties. Chem.
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(10) Dreassi, E.; Zizzari, A. T.; D’Arezzo, S.; Visca, P.; Botta, M.
Analysis of guazatine mixture by LC and LC−MS and antimycotic
activity determination of principal components. J. Pharm. Biomed. Anal.
2007, 43, 1499−1506.
(11) Raffi, F.; Corelli, F.; Botta, M. Efficient synthesis of
iminoctadine, a potent antifungal agent and polyamine oxidase
inhibitor (PAO). Synthesis 2007, 19, 3013−3016.
(12) Castagnolo, D.; Raffi, F.; Giorgi, G.; Botta, M. Macrocyclization
of di-Boc-guanidino-alkylamines related to guazatine components:
discovery and synthesis of innovative macrocyclic amidinoureas. Eur. J.
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(13) Castagnolo, D. New Strategies in Chemical Synthesis and
Catalysis; Wiley-VCH Verlag GmbH & Co. KGaA: Berlin, Germany,
2012; pp 97−126.
(14) Botta, M.; Raffi, F.; Visca, P. Linear and cyclic guanidine
derivatives as antifungal agents and their method of preparation. US
patent WO2009113033A2, 2009.
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthetic methods and characterization of compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*(M.B.) Phone: +39 0577234306. E-mail: botta.maurizio@
gmail.com.
■
(15) Manetti, F.; Castagnolo, D.; Raffi, F.; Zizzari, A. T.; Rajamaki, S.;
D’Arezzo, S.; Visca, P.; Cona, A.; Fracasso, M. E.; Doria, D.; Posteraro,
B.; Sanguinetti, M.; Fadda, G.; Botta, M. Synthesis of new linear
guanidines and macrocyclic amidinourea derivatives endowed with
high antifungal activity against Candida spp. and Aspergillus spp. J. Med.
Chem. 2009, 52, 7376−7379.
Notes
The authors declare no competing financial interest.
(16) Furstner, A.; Langemann, K. Conformationally unbiased
macrocyclization reactions by ring closing metathesis. J. Org. Chem.
1996, 61, 3942−3943.
ACKNOWLEDGMENTS
■
(17) Han, S.-Y.; Chang, S. Handbook of Metathesis; Wiley-VCH
Verlag GmbH: Berlin, Germany, 2003; pp 5−127.
Bakker Medical S.r.l. and University of Siena are gratefully
acknowledged for economical support and technical assistance.
Dr. A. Vivi is acknowledged for NMR technical assistance.
(18) Furstner, A.; Davies, P. W. Alkyne metathesis. Chem. Commun.
2005, 42, 2307−2320.
(19) Gradillas, A.; Perez-Castells, J. Macrocyclization by ring-closing
metathesis in the total synthesis of natural products: reaction
conditions and limitations. Angew. Chem., Int. Ed. 2006, 45, 6086−
6101.
(20) Kulkarni, A. A.; Diver, S. T. Ring synthesis by stereoselective,
methylene-free enyne cross metathesis. J. Am. Chem. Soc. 2004, 126,
8110−8111.
(21) The 13-membered macrocycle 8a was obtained in toluene (10
mM solution) at 80 °C. When the RCM was performed at lower
temperature (DCM, 40 °C) compound 8a was not formed and diene
7a was entirely recovered. Macrocycles 8b and 8c were obtained in
DCM (2 mM solution) at 40 °C
ABBREVIATIONS
■
D C M , d i c h l o r o m e t h a n e ; E D C , 1 - e t h y l - 3 - ( 3 -
dimethylaminopropyl)carbodiimide; HOBt, hydroxybenzotria-
zole; DIPEA, N,N-diisopropylethylamine; DIBAL-H, diisobu-
tylaluminium hydride; TMS, trimethylsilyl chloride; DMAP, 4-
dimethylaminopyridine; TFA, trifluoroacetic acid; DCC,
dicyclohexylcarbodiimide; Cbz, benzyl carbamate; Boc, tert-
butoxy carbamate; MIC, minimum inhibitory concentration;
MDR, multidrug resistance; CDR, Candida drug resistance;
RCM, ring closing metathesis; gen, generation
(22) Lal, B.; Gangopadhyay, A. K. A practical synthesis of free and
protected guanidino acids from amino acids. Tetrahedron Lett. 1996,
37, 2483−2486.
(23) Attempts to reduce the amide using BH3·SMe2, BH3−THF, or
NaBH4/TiCl4 led to the cleavage of Boc protecting groups.
(24) Peman, J.; Canton, E.; Espinel-Ingroff, A. Antifungal drug
resistance mechanisms. Expert Rev. Anti-Infect. Ther. 2009, 7, 453−460.
(25) Sanglard, D.; Odds, F. C. Resistance of Candida species to
antifungal agents: molecular mechanisms and clinical consequences.
Lancet Infect. Dis. 2002, 2, 73−85.
(26) Perea, S.; Lopez-Ribot, J. L.; Kirkpatrick, W. R.; McAtee, R. K.;
Santillan, R. A.; Martinez, M.; Calabrese, D.; Sanglard, D.; Patterson,
T. F. Prevalence of molecular mechanisms of resistance to azole
antifungal agents in Candida albicans strains displaying high-level
fluconazole resistance isolated from human immunodeficiency virus-
infected patients. Antimicrob. Agents Chemother. 2001, 45, 2676−2684.
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