Design, synthesis, and microbiological evaluation of new Candida albicans CYP51 inhibitors

Article abstract of DOI:10.1021/jm050685j

In a program aimed at the design and synthesis of novel azole inhibitors of Candida albicans CYP51 (CA-CYP51), a series of azole 1,4-benzothiazines (BT) and 1,4-benzoxazines (BO) were recently synthesized. A morphological study of the enzyme active site h

Full text of DOI:10.1021/jm050685j

7658
J. Med. Chem. 2005, 48, 7658-7666
Design, Synthesis, and Microbiological Evaluation of New Candida albicans
CYP51 Inhibitors
Fausto Schiaffella,† Antonio Macchiarulo,† Lara Milanese,† Anna Vecchiarelli,‡ Gabriele Costantino,†
Donatella Pietrella,‡ and Renata Fringuelli*,†
Department of Drug Chemistry and Technology and Department of Experimental Medicine and Biochemical Sciences,
University of Perugia, Perugia, Italy
Received July 19, 2005
In a program aimed at the design and synthesis of novel azole inhibitors of Candida albicans
CYP51 (CA-CYP51), a series of azole 1,4-benzothiazines (BT) and 1,4-benzoxazines (BO) were
recently synthesized. A morphological study of the enzyme active site highlighted a hydrophobic
access channel, and a docking study pointed out that the C-2 position of the BT or BO nucleus
was oriented toward the access channel. Here, we report the design, synthesis, and
microbiological evaluation of C-2-alkyl BT and BO compounds. In both series, introduction of
the alkyl chain maintained and in some cases improved the anti-Candida in vitro activity;
however, there was not always a strict correlation between in vitro and in vivo activity for
several compounds.
Chart 1
Introduction
In recent years, fungal infections have increased
markedly and one of the main agents is the op-
portunistic pathogen Candida albicans. A major ob-
stacle in the treatment of C. albicans infections is the
spread of antifungal drug resistance mainly in patients
chronically subjected to antimycotic therapy, i.e., those
treated with broad-spectrum antibiotics, immunosup-
pressive agents, anticancer, and anti-AIDS drugs.1,2
Azole and non-azole antifungal agents are usually
used to treat Candida infections, but despite the good
antifungal activities observed in vitro, candidemia is
still a major cause of death.1 Many valid therapy
programs fail because of widespread secondary C.
albicans infections. New effective anti-Candida agents
are needed to combat the drug-resistant strains and
widespread diffusion of C. albicans.
Up to only 30 years ago, the choice of systemically
available antimycotics was almost exclusively am-
phothericin B with all of its adverse side effects. Then
a series of inhibitors of the biosynthesis of ergosterol,
the major sterol of the fungal cell membrane, were found
that had excellent antifungal activity and were safer;
some were also active after oral or parenteral applica-
tion.3 Starting with miconazole and ketoconazole, and
improved with fluconazole, itraconazole, and voricona-
zole, the azoles have been a mainstay of the antifungal
armamentarium (Chart 1). These drugs inhibit the
synthesis of ergosterol by binding to the heme cofactor
located in the active site of the P450-dependent enzyme
lanosterol 14R-demethylase (CYP51). It has been pro-
posed that ergosterol depletion, coupled with the ac-
cumulation of methylated sterol precursors, affects both
membrane-bound proteins, including chitin synthase.
The net result is an inhibition of fungal growth.4
In a program aimed at the design and synthesis of
novel azole inhibitors in order to evaluate the effect of
replacing the aromatic ring present in the most com-
monly used drugs with 1,4-benzothiazine (BT) nucleus
that, in itself, shows some antifungal activity,5 a series
of azole BT, structurally correlated with fluconazole and
econazole, were recently synthesized.6
In the framework of this research project, we have
also reported the construction of a 3D model of CYP51
of C. albicans (CA-CYP51) and docking experiments of
a series of BT and 1,4-benzoxazine (BO) imidazole
derivatives.7 In addition, a morphological study of the
CA-CYP51 active site highlighted a hydrophobic access
channel and a docking study pointed out that for all the
best docking solutions the C-2 position of the BT or BO
nucleus was oriented toward the access channel and the
distance between C-2 and the channel entry was esti-
mated to be about 6-7 Å. This suggested that the
introduction of an alkyl chain on the nucleus base might
stabilize the enzyme-drug complex by interacting with
* To whom correspondence should be addressed. Phone: +39 075
5855134. Fax: +39 075 5855161. E-mail: fringuel@unipg.it.
† Department of Drug Chemistry and Technology.
‡ Department of Experimental Medicine and Biochemical Sciences.
10.1021/jm050685j CCC: $30.25 © 2005 American Chemical Society
Published on Web 11/01/2005

Evaluation of CA-CYP51 Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7659
Figure 1.
the residues delimiting the channel and improve lipo-
philicity and fungal membrane crossing.
In the synthesis, butyl and O-butyl chains were
introduced in C-2 of the BT and BO nuclei to furnish
compounds 5-12 (Figure 1). A docking study was
performed for these compounds, and results showed that
the introduction of the C-2 alkyl chain really stabilizes
the drug-enzyme complex and significantly improves
the octanol/water partition coefficient.
Figure 2. 3D model of CA-CYP51 used in this study. The BC
loop (127-139) is highlighted in yellow. Channel 1 (white dots)
extends from the surface to the catalytic site (orange dots) of
heme (cyan CPK).
(Figure 2). This latter site consists of a chamber of about
1500 Å3. The dome of the chamber is 13 Å above the
heme group.
The open conformation of channel 1 in the 3D model
of CA-CYP51 to date has been an unexploited feature
in the design of novel BT and BO imidazole inhibitors.
Our previous results pinpointed that BT and BO
derivatives adopt similar binding modes inside the
catalytic site of CA-CYP51 with subtle differences in the
interactions. Furthermore, the log P values of these
compounds strongly affect their in vitro antifungal
activity with the optimal value of the water/octanol
partition coefficient being >2.
On the basis of the above observations and results,
we designed BT and BO derivatives with the insertion
of alkyl or O-alkyl chains at the C-2 position of the BT
and BO rings. This insertion would have the 2-fold
benefit of improving the interaction energy of the
inhibitors with the enzyme by filling channel 1 and
increasing the log P value of the compounds.
Molecular Modeling
We previously reported the construction of a 3D model
of CYP51 of C. albicans and docking experiments of a
series of 1,4-BT and 1,4-BO imidazole derivatives.7 In
particular, the CA-CYP51 model was constructed by
exploiting the sequence homology relationship and the
crystal structure of the CYP51 of Mycobacterium tuber-
culosis (MT-CYP51, PDB codes 1ea1, 1e9x)8 as template.
Although the available P450 structures show a con-
served overall fold,9 there are variable structural fea-
tures, some of which resemble protein conformational
changes in response to recognition and binding of
structurally diverse substrates and/or inhibitors. Thus,
depending on the structural template used, these fea-
tures can be exploited to construct different conforma-
tions of CA-CYP51 and aid the design of novel inhibi-
tors.10
In our study, MT-CYP51 exhibits the P450 fold but
contains structural differences that define the active site
access channel. In contrast to other P450 crystal struc-
tures, the BC loop (Gly127-Leu139) adopts an open
conformation in MT-CYP51. This feature gives rise to
the opening of a channel (channel 1) endowed with a
large mouth of about 20 Å × 10 Å that extends from
the surface to the catalytic site.
Docking experiments of each stereoisomer of com-
pounds 5-12 were carried out in the catalytic site of
CA-CYP51. Binding energies were calculated as ex-
plained in the Experimental Section and are reported
in Table 1.
Although the calculated binding energies may suffer
from technical (poor parametrization of the force field)
and/or methodological approximations (treatment of the
enzyme as a rigid body), some interesting observations
can be made. Compared to their related unsubstituted
analogues (1-4), these compounds show an improved
binding energy and better log P values. Interestingly,
the best interacting stereoisomers of compounds 5-12
show an inverted preference on the first chiral center,
being shifted from 1-S to 1-R. This behavior is explained
by the accommodation of the alkyl/O-alkyl chains into
the catalytic site that forces the BT and BO rings to
adopt a slightly different pose that is best fitted by the
1-R stereoisomers.
Interestingly, this channel runs almost parallel to the
heme in contrast to other P450 folds, such as P450BM3
and P450cam,11 where another substrate access channel
has been identified that runs perpendicular to the heme
(channel 2). Although channel 2 is also present in MT-
CYP51, it is closed by the interaction between helix A
and the FG loop. Thus, channel 1 is the only substrate/
inhibitor accessible pathway to the heme in the enzy-
matic conformation crystallized in MT-CYP51.
Constructed on the crystal structure of MT-CYP51,
our model of CA-CYP51 contains the above feature that
connects the surface of the enzyme to the catalytic site

7660 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
Schiaffella et al.
Scheme 1a
Table 1. Binding Energies and the log P Values of Different
Compoundsa
compd
binding energy (kcal/mol)
log P
aS-1
-30.95b
-31.80b
-32.51b
-30.81b
-61.83
-62.84
-84.68
-71.23
-66.76
-59.33
-66.03
-52.71
-29.10b
0.50
3.07
0.16
2.73
2.30
4.87
2.02
4.60
1.96
4.53
1.68
4.25
1.58
aS-2
aS-3
aS-4
a Reagents: (a) 2-Br-hexanoic acid, 5% NaOH aqueous solution;
(b) 3 N HCl; (c) t-BuOK, MeI.
(aR,bS)-5
(aR,bS)-6
(aR,bR)-7
(aR,bR)-8
(aR,bS)-9
(aR,bS)-10
(aR,bR)-11
(aR,bR)-12
FLU
Scheme 2a
a Only the best fitting stereoisomer is reported for each com-
pound. b Data are taken from ref 7.
a Reagents: (a) SO2Cl2; (b) BuOH, SiO2.
configurations at the chiral center that are different
from those predicted.
Chemistry
The intermediate 6-acetyl-2-butyl-4-methyl-3,4-dihy-
dro-2H-1,4-benzothiazin-3-one (14) was prepared start-
ing from 3-amino-4-mercaptoacetophenone.12 The con-
densation with racemic 2-bromohexanoic acid in aqueous
NaOH followed by acidification with 3 N HCl afforded
13, which was methylated with MeI and t-BuOK in dry
DMF to furnish 14 (Scheme 1).
Starting from 6-acetyl-4-methyl-3,4-dihydro-2H-1,4-
benzothiazin-3-one,13 6-acetyl-2-butoxy-4-methyl-3,4-di-
hydro-2H-1,4-benzothiazin-3-one (15) was prepared by
chlorination with sulfuryl chloride in dry benzene
followed by reaction with butyl alcohol (Scheme 2).
Figure 3. Binding mode of (aR,bS)-6 (A) and (aR,bR)-8 (B)
into the catalytic site of CA-CYP51.
Scheme 3 outlines the synthesis of BO intermediates
17 and 23 starting from 3-amino-4-hydroxyacetophe-
none.14 2-Bromohexanoyl chloride in CHCl3 was added
dropwise to a cooled mixture of starting acetophenone,
benzyltriethylammonium chloride (TEBA), and NaH-
CO3 in CHCl3. The successive methylation afforded 17.
On the other hand, 3-amino-4-hydroxyacetophenone was
first reacted with dichloroacetyl chloride to give 18,
which was cyclized with NaHCO3 to furnish 19. The
alcoholic group was protected with tert-butyl(dimethyl)-
silyl chloride (TBSCl), then methylated and deprotected
under acidic conditions to afford 22, and finally alky-
lated with butyl iodide to furnish the intermediate 23.
Scheme 4 summarizes the common reactions that lead
to final products 5-12 starting from key intermediates
14, 15, 17, and 23. Through the formation of bromo
derivatives 24-27, the reaction with 1H-imidazole
supplied derivatives 28-31, and the reduction with
NaBH4 led to carbinols 5, 7, 9, and 11 that were
converted into ether derivatives 6, 8, 10, and 12 by
reaction with p-chlorobenzyl chloride.
The insertion of alkyl/O-alkyl chains gives rise to a
second chiral center. Depending upon the kind of
substituent, the preferred configuration at the second
chiral center is different in docked poses. In particular,
the insertion of a butyl chain shows a 2-S configuration
preference (Figure 3A).
In the docked pose, the butyl chain folds over the
scaffold of the inhibitor and occupies the dome of the
catalytic site. Conversely, upon the insertion of the one-
atom-longer O-butyl chain, the isomer R shows a better
binding energy. Docking experiments show that the
longer chain does not fill the dome because of the
presence of steric clashes but runs along channel 1
(Figure 3B).
Since the above compounds were first synthesized and
tested as racemic mixtures, we cannot prove the con-
figuration of the eutomer. It should be mentioned,
however, that neglected conformational changes of the
enzyme may play a role in making room to host the
substituted BT and BO rings, which in turn would favor

Evaluation of CA-CYP51 Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7661
Scheme 3a
a Reagents: (a) NaHCO3, TEBA, 2-Br-hexanoyl chloride; (b) NaH, MeI; (c) Cl2HCOCl; (d) NaHCO3; (e) TBSCl, 1H-imidazole; (f) K2CO3,
MeI; (g) 1 N HCl; (h) K2CO3, BuI.
Scheme 4a
Table 2. In Vitro Antifungal Activities of Different
Compoundsa
MIC (µg/mL)
compd
C. albicans CA-6
C. krusei 6258
diluent
250
250
1
125-62.5
<1.8
250
2
31.2-15.6
31.2
15.6
250
3
250
4
15.6-7.8
62.5-31.2
<1.8
5
6
<1.8
125
7
62.5-31.2
1.8-3.9
125
8
15.6
125
9
10
11
12
FLU
7.8
7.8
125
31.2
3.9
125
<1.8
62.5
a The data represent the MIC (minimum inhibitory concentra-
tion) of various compounds against C. albicans or C. krusei. The
test was performed by the microdilution method as specified in
the Experimental Section.
a Reagents: (a) Br2; (b) 1H-imidazole; (c) NaBH4; (d) NaH, p-Cl-
benzyl chloride.
Results and Discussion
penetration into the cytosol. The importance of the
hydrophobic/hydrophilic balance is further confirmed by
the poor in vitro activity of compounds 5, 7, 9, and 11.
Even though these compounds are endowed with good
interaction energies, an increasing hydrophilic character
negatively affects their ability to cross the membrane
of the fungi.
The weaker MIC of compounds 10 and 12, which are
the BO analogues of compounds 6 and 8, is linked to
their lower interaction energies.
The best compound seemed to be the BT ether
derivative 6, which showed an MIC value comparable
to that of fluconazole on C. albicans and interesting
activity on C. krusei (<1.8 µg/mL), partially resistant
to fluconazole, on which the corresponding alcohol 5 was
ineffective. The derivative that was not substituted at
C-2 only had a slight activity (compare 6 with 2).
In the BO series, the butyl chain at C-2 (9 and 10)
did not affect the C. albicans inhibition profile with
In vitro anti-Candida activity was determined ac-
cording to the NCCLS guidelines.15 Compounds were
tested on C. albicans CA-6, clinically isolated and
identified according to taxonomical criteria, and on C.
krusei 6258, coming from the American Type Culture
Collection. Results of preliminary studies are shown in
Table 2. As a general consideration, introduction of the
butyl chain retained or slightly improved the anti-
Candida in vitro activity, in both the BT and BO series.
The presence of the secondary alcohol negatively
affected the activity possibly because the low atom-
based approach of log P (A log P) values were not
influenced enough by the insertion of the alkyl chain.
Alkylation of the carbinol to p-chlorobenzyl ether proved
to be favorable, particularly with regard to anti-C. krusei
activity.
The good in vitro activity of compounds 6 and 8 is
ascribed to their strong binding energies coupled with
a high lipophilic character, which allows an optimal

7662 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
Schiaffella et al.
Table 3. In Vivoa Antifungal Activities of Different
Compounds against C. albicans CA-6
range survival
CFU (×103)b
196 ( 18
110 ( 9
78 ( 6
143 ( 12
127 ( 10
230 ( 16
85 ( 9
186 ( 10
227 ( 19
0
MSTc
(day)
D/Td
diluente
12
14
3-12
9-10
10/10
10/10
8/10
5
6
12
2 f 30
9-10
7
14
10/10
10/10
8/10
8
14
3-17
9
14
9 f 30
6 f 30
4-12
10
11
12
FLU
12
8/10
12
10/10
10/10
2/10
14
8-10
23 f 30
>30
a Groups of 10 mice were inoculated iv with 1 × 105 cells of C.
albicans CA6. Diluent, azoles, and fluconazole were given ip at a
dose of 14 mg/kg, 2 h before and once daily for 8 consecutive days
after infection. b Data represent the mean ( SD of colony forming
units (CFU) recovered from the kidneys of three mice sacrificed
11 days after infection. c Median survival time (days). d Number
of dead mice at 30 days over total number of animals tested.
e Diluent ) DMSO/H2O (1:4).
Figure 4. 3D model of CA-CYP51 in complex with (aR,bR)-
8. Naturally occurring mutations in azole-resistant CA-CYP51
are divided into four hotspots on the basis of their structural
localization. They are labeled respectively in blue, red, orange,
and green. It is worth noting the proximity of the blue hotspot
to the entrance of channel 1.
respect to the corresponding nonsubstituted compounds
3 and 4. The alcohol derivatives were not active, while
the corresponding p-chlorobenzyl ethers showed moder-
ate activity. Derivative 12 showed good inhibitory
activity on C. krusei, with an MIC value of 3.9 µg/mL.
Since the in vitro activity did not necessarily correlate
with the in vivo activity,16 the newly synthesized
compounds were also tested in a murine model of
systemic C. albicans infection. To this end, mice were
treated intraperitoneally with various compounds 1 day
before systemic challenge with C. albicans and once
daily for 14 consecutive days. The results reported in
Table 3 show that there is not a strict correlation
between in vitro and in vivo activity for several com-
pounds. In particular, 8 and 12, which exhibited an
antifungal effect in vitro, did not show any beneficial
effect in systemic candidiasis, while a trend of correla-
tion between anticandidal activity in vitro and beneficial
effect in vivo was observed for 6 and 10. However,
despite differences in survival range, no significant
difference in median survival time (MST) was observed
for all groups.
It is noteworthy that an increase of survival in terms
of survival range is correlated with a drastic decrease
of fungal load (colony forming unit, CFU) in kidneys
that represent the main target organ for C. albicans.
This indicates that variation of dose, schedule, or route
of administration could lead to optimization of the
beneficial effect of different compounds on survival. In
addition, another consideration emerging from these
results is that compounds 5-12 could endow fungistatic
activity in vivo, facilitating the clearance but not
eradication of the fungus from the organs. This hypoth-
esis is consistent with a significant reduction of C.
albicans observed in the kidneys.
between the inhibitor and the enzyme channel and to
the increase of the hydrophobic/hydrophilic balance.
Furthermore, the presence of alkyl/O-alkyl substituents
on the BT and BO rings allow an interesting surface
region of the enzyme which is located at the mouth of
channel 1 to be reached (Figure 4).
Several naturally occurring CYP51 mutations of
azole-resistant C. albicans have been reported in this
region and are clustered around the BC loop whose
open/closed conformation is the gate of the above chan-
nel. The physical-chemical properties of these mutant
residues can be exploited in the future to design novel
BT and BO derivatives endowed with inhibitor activity
toward C. albicans resistant strains.
Experimental Section
Melting points determined in capillary tubes (Electrother-
mal, model 9100, melting point apparatus) were uncorrected.
Elemental analysis was performed on a Carlo Erba element
analyzer 1106, and the data for C, H, and N are within (0.4%
of the theoretical values. 1H NMR spectra were recorded at
200 MHz (Bruker AC-200 spectrometer) with Me4Si as internal
standard. Chemical shifts are given in ppm (δ), and the
spectral data are consistent with the assigned structures.
Reagents and solvents were purchased from common com-
mercial suppliers and used as received. Column chromatog-
raphy separations were carried out on Merck silica gel 60
(mesh 230-400). Yields of purified products were not opti-
mized. Fluconazole was purchased from Pfizer (New York). All
starting materials were commercially available unless other-
wise indicated.
6-Acetyl-2-butyl-3,4-dihydro-2H-1,4-benzothiazin-3-
one (13). 3-Amino-4-mercaptoacetophenone (18.00 g, 107.78
mmol) was suspended in 5% NaOH aqueous solution, and a
mixture of 2-bromohexanoic acid (21.02 g, 107.78 mmol),
NaOH (5.60 g, 140.12 mmol), and H2O (200 mL) was added.
The mixture was then heated to 70-80 °C for 20 min, then
cooled at room temperature and acidified with 3 N HCl. The
brown precipitate thus obtained was collected and chromato-
graphed, eluting with CHCl3/MeOH 98:2 to give 13 (10.21 g,
Conclusion
The presence of a thus far unexploited hydrophobic
channel (channel 1) in the 3D model of CA-CYP 51
prompted us to design 2-substituted BT and BO. The
insertion of alkyl and O-alkyl chains at position 2 of the
BT and BO rings yielded an overall improvement of the
in vitro activity. As expected, this is ascribed to both
the resulting extension of the interaction surfaces
1
36%), mp 125-127 °C. H NMR (CDCl3) δ: 0.92 (3H, t, J )
7.0 Hz, CH2CH3), 1.25-1.75 (5H, m, SCHCHHCH2CH2CH3),
1.80-2.10 (1H, m, SCHCHHCH2), 2.65 (3H, s, COCH3), 3.50

Evaluation of CA-CYP51 Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7663
(1H, dd, J ) 8.4 and 6.4 Hz, SCHCH2), 7.40-7.75 (3H, m,
(1H, d, J ) 8.5 Hz, H-3), 7.73 (1H, dd, J ) 8.5 and 2.0 Hz,
H-4), 8.57 (1H, d, J ) 2.0 Hz, H-6), 10.00 (1H, bs, OH), 11.22
(1H, bs, NH).
aromatic H), 9.10 (1H, bs, CONH).
6-Acetyl-2-butyl-4-methyl-3,4-dihydro-2H-1,4-benzothi-
azin-3-one (14). t-BuOK (2.52 g, 22.57 mmol) was added to a
solution of 13 (3.30 g, 12.54 mmol) in dry DMF (33 mL). The
mixture was stirred at room temperature for 15 min, and then
MeI (2.13 g, 15.04 mmol) in dry DMF (6 mL) was added
dropwise. After being stirred for 4 h, the mixture was poured
into ice-chilled water and extracted with EtOAc. The residue
was chromatographed, eluting with cyclohexane/EtOAc 85:15.
Compound 14 (1.04 g, 30%) was obtained as an amorphous
6-Acetyl-2-hydroxy-3,4-dihydro-2H-1,4-benzoxazin-3-
one (19). NaHCO3 (0.07 g, 0.76 mmol) was added to a
suspension of 18 (0.10 g, 0.38 mmol) in H2O (4 mL). The
mixture was heated to 70 °C, stirred for 2 h, then cooled to
room temperature, neutralized with 3 N HCl, and extracted
with EtOAc. The combined organic layers were evaporated to
dryness, and the residue was purified by chromatography,
eluting with CHCl3/MeOH 96:4 to give 19 (0.06 g, 76%) as a
white solid, mp 196-198 °C. 1H NMR (DMSO-d6) δ: 2.54 (3H,
s, COCH3), 5.55-5.60 (1H, m, CHOH), 7.15 (1H, d, J ) 8.3
Hz, H-8), 7.54 (1H, d, J ) 1.0 Hz, H-5), 7.66 (1H, dd, J ) 8.3
and 1.0 Hz, H-7), 8.25 (1H, bs, CHOH), 11.02 (1H, s, NH).
6-Acetyl-2-{[tert-butyl(dimethyl)silyl]oxy}-3,4-dihydro-
2H-1,4-benzoxazin-3-one (20). 1H-Imidazole (0.25 g, 3.67
mmol) and TBSCl (0.43 g, 2.89 mmol) were added to a solution
of 19 (0.50 g, 2.41 mmol) in dry DMF (5 mL). The mixture
was stirred for 2 h at room temperature and then poured into
H2O. The precipitate was collected and purified by chroma-
tography, eluting with cyclohexane/EtOAc 80:20 and thus
obtaining 20 (0.50 g, 65%) as a white solid, mp 150 °C. 1H NMR
(CDCl3) δ: 0.21 and 0.25 (each 3H, s, CH3SiCH3), 0.88 (9H, s,
SiC(CH3)3), 2.63 (3H, s, COCH3), 5.70 (1H, s, CHO), 7.12 (1H,
d, J ) 8.4 Hz, H-8), 7.60 (1H, d, J ) 1.8 Hz, H-5), 7.69 (1H,
dd, J ) 8.4 and 1.8 Hz, H-7), 8.61 (1H, s, NH).
6-Acetyl-2-{[tert-butyl(dimethyl)silyl]oxy}-4-methyl-
3,4-dihydro-2H-1,4-benzoxazin-3-one (21). 20 (0.10 g, 0.31
mmol) was added to a suspension of K2CO3 (0.09 g, 0.65 mmol)
in dry DMF (2 mL). The mixture was stirred at room
temperature for 10 min, and then MeI (0.12 g, 0.37 mmol) was
added. After being stirred for 3 h, the mixture was poured into
ice/H2O and the precipitated was collected. 21 (0.09 g, 90%)
was obtained as a white solid, mp 82-83 °C. 1H NMR (CDCl3)
δ: 0.19 and 0.22 (each 3H, s, CH3SiCH3), 0.86 (9H, s, SiC-
(CH3)3), 2.63 (3H, s, COCH3), 3.50 (3H, s, NCH3), 5.72 (1H, s,
CHO), 7.11 (1H, d, J ) 8.0 Hz, H-8), 7.65-7.75 (2H, m, H-5
and H-7).
6-Acetyl-2-hydroxy-4-methyl-3,4-dihydro-2H-1,4-ben-
zoxazin-3-one (22). A solution of 21 (0.20 g, 0.60 mmol) in
MeOH (10 mL) was cooled to 0-5 °C, and then 1 N HCl (2
mL) was added. The solution was stirred at room temperature
for 72 h, neutralized with a saturated solution of NaHCO3,
and evaporated to dryness. The residue, chromatographed and
eluting with cyclohexane/EtOAc with gradual increase of the
concentration of EtOAc, furnished 22 (0.08 g, 66%) as a white
solid, mp 217-220 °C. 1H NMR (DMSO-d6) δ: 2.61 (3H, s,
COCH3), 3.40 (3H, s, NCH3), 5.70 (1H, d, J ) 5.7 Hz, CHOH),
7.20 (1H, d, J ) 8.1 Hz, H-8), 7.65-7.80 (2H, m, H-5 and H-7),
8.27 (1H, d, J ) 5.7 Hz, CHOH).
6-Acetyl-2-butoxy-4-methyl-3,4-dihydro-2H-1,4-benzox-
azin-3-one (23). K2CO3 (0.37 g, 2.71 mmol) was added to a
solution of 22 (0.10 g, 0.45 mmol) in dry CH3CN (15 mL). After
the mixture was heated to 60 °C and was stirred for 15 min,
BuI (0.18 g, 0.97 mmol) was added dropwise. The mixture was
heated to reflux for 3 h, then evaporated to dryness, resus-
pended in H2O, acidified with 3 N HCl, and extracted with
EtOAc. The combined organic layers, evaporated to dryness,
were chromatographed, eluting with cyclohexane/EtOAc 85:
15 and thus giving 23 (0.09 g, 75%) as an amorphous solid.
1H NMR (CDCl3) δ: 0.87 (3H, t, J ) 7.3 Hz, CH2CH3), 1.10-
1.30 and 1.40-1.60 (each 2H, m, CH2CH2CH3), 2.64 (3H, s,
COCH3), 3.49 (3H, s, NCH3), 3.65-3.90 (2H, m, OCH2), 5.49
(1H, s, CHO), 7.14 (1H, d, J ) 8.8 Hz, H-8), 7.65-7.70 (2H,
m, H-5 and H-7).
General Method for the Preparation of Bromo De-
rivatives 24-27. This preparation is illustrated by the
synthesis of 6-(bromoacetyl)-2-butyl-4-methyl-3,4-dihydro-2H-
1,4-benzothiazin-3-one (24).
A solution of 14 (0.80 g, 2.89 mmol) in AcOH (5 mL) was
heated to 40 °C, and then Br2 (0.55 g, 3.46 mmol) in AcOH (8
mL) was added dropwise. After being stirred for 1 h, the
mixture was evaporated to dryness and the residue was
1
solid. H NMR (CDCl3) δ: 0.90 (3H, t, J ) 6.9 Hz, CH2CH3),
1.20-1.70 (5H, m, SCHCHHCH2CH2CH3), 1.75-2.00 (1H, m,
SCHCHHCH2), 2.65 (3H, s, COCH3), 3.47 (1H, dd, J ) 8.1 and
6.4 Hz, SCHCH2), 3.54 (3H, s, NCH3), 7.48 (1H, d, J ) 8.0 Hz,
H-8), 7.63 (1H, dd, J ) 8.0 and 1.6 Hz, H-7), 7.72 (1H, d, J )
1.6 Hz, H-5).
6-Acetyl-2-butoxy-4-methyl-3,4-dihydro-2H-1,4-benzothi-
azin-3-one (15). SO2Cl2 (0.30 g, 2.26 mmol) diluted in dry
benzene (3 mL) was added dropwise over 30 min to a solution
of 6-acetyl-4-methyl-3,4-dihydro-2H-1,4-benzothiazin-3-one (0.50
g, 2.26 mmol) in dry benzene (10 mL) heated to 40 °C. After
being stirred at this temperature for 30 min, the mixture was
gradually heated to 75 °C over 30 min, then stirred for further
30 min and evaporated to dryness. The crude residue was
resuspended in dry BuOH (10 mL), and SiO2 in a catalytic
amount was added. The mixture was heated in a bain-marie
for 2 h and evaporated to dryness, and the residue was
chromatographed, eluting with cyclohexane/EtOAc 86:14 and
thus obtaining 15 (0.38 g, 57.5%) as a white solid, mp 81-83
°C. 1H NMR (CDCl3) δ: 0.81 (3H, t, J ) 7.3 Hz, CH2CH3),
1.10-1.20 and 1.35-1.55 (each 2H, m, CH2CH2CH3), 2.66 (3H,
s, COCH3), 3.20-3.55 and 3.70-3.80 (each 1H, m, CHOCH2),
3.60 (3H, s, NCH3), 5.14 (1H, s, SCHO), 7.49 (1H, d, J ) 8.0
Hz, H-8), 7.66 (1H, dd, J ) 8.0 and 1.5 Hz, H-7), 7.80 (1H, d,
J ) 1.5 Hz, H-5).
6-Acetyl-2-butyl-3,4-dihydro-2H-1,4-benzoxazin-3-
one (16). 3-Amino-4-hydroxyacetophenone (0.50 g, 3.33 mmol)
was added to a mixture of TEBA (0.75 g, 3.29 mmol), NaHCO3
(1.13 g, 13.29 mmol), and dry CHCl3 (18 mL), and the mixture
was cooled to 0-5 °C. A solution of 2-bromohexanoyl chloride17
(1.70 g, 7.94 mmol) in dry CHCl3 (5 mL) was added dropwise.
The mixture was heated to 55 °C and stirred for 3 h, then
cooled at room temperature, stirred for 24 h, and evaporated
to dryness. The crude residue was purified by chromatography,
eluting with cyclohexane/EtOAc 65:35 to give 16 (0.33 g, 40%)
as a white solid, mp 109-110 °C. 1H NMR (CDCl3) δ: 0.96
(3H, t, J ) 7.1 Hz, CH2CH3), 1.25-1.70 (4H, m, CH2CH2CH3),
1.75-2.10 (2H, m, CHCH2), 2.61 (3H, s, COCH3), 4.71 (1H,
dd, J ) 8.1 and 4.9 Hz, CHO), 7.06 (1H, d, J ) 8.4 Hz, H-8),
7.57 (1H, d, J ) 1.9 Hz, H-5), 7.65 (1H, dd, J ) 8.4 and 1.9
Hz, H-7), 9.10 (1H, bs, NH).
6-Acetyl-2-butyl-4-methyl-3,4-dihydro-2H-1,4-benzox-
azin-3-one (17). NaH (60% mineral oil dispersion, 0.05 g, 1.21
mmol) was added to a solution of 16 (0.20 g, 0.81 mmol) in
dry DMF (2 mL). The mixture was stirred at room temperature
for 30 min, and then MeI (0.17 g, 1.21 mmol) in dry DMF (1
mL) was added dropwise. After being stirred for 2 h, the
mixture was poured into ice-chilled water and the precipitated
was collected and chromatographed, eluting with cyclohexane/
EtOAc 80:20 to furnish 17 (0.09 g, 47%) as a yellow oil. 1H
NMR (CDCl3) δ: 0.95 (3H, t, J ) 7.0 Hz, CH2CH3), 1.25-2.10
(6H, m, CH2CH2CH2CH3), 2.63 (3H, s, COCH3), 3.45 (3H, s,
NCH3), 4.70 (1H, dd, J ) 8.2 and 4.7 Hz, CHO), 7.07 (1H, d,
J ) 8.7 Hz, H-8), 7.60-7.75 (2H, m, H-5 and H-7).
N-(5-Acetyl-2-hydroxyphenyl)-2,2-dichloroacetamide
(18). Dichloroacetyl chloride (1.07 g, 7.28 mmol) was slowly
added to a solution of 3-amino-4-hydroxyacetophenone (1.10
g, 7.28 mmol) in dry THF (15 mL). After the mixture was
stirred for 1 h at room temperature, a solid was filtered off.
Then the solution was evaporated to dryness, and the residue
was chromatographed, eluting with CHCl3/MeOH 99:1 to give
18 (1.53 g, 80%) as a white solid, mp 196.5-197.5 °C. 1H NMR
(DMSO-d6) δ: 2.51 (3H, s, COCH3), 3.37 (1H, s, CHCl2), 7.02

7664 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
Schiaffella et al.
chromatographed, eluting with cyclohexane/EtOAc 94:6 to give
imidazole ring at 2.37 Å from the heme iron along the line
orthogonal to the heme plane. The value of 2.37 Å is the
average distance of Fe-N coordination bonds observed in
crystallized complexes of MT-CYP51 with fluconazole and
4-phenylimidazole. Starting from this docking pose, a reduced
conformational profile of compounds was explored using a
random sampling protocol as implemented in the Cerius-2
software package. In particular, all torsional angles of azole
inhibitors, except the Fe-N coordination bond, were varied
randomly in a window of (5°, generating 1000 conformations.
For each enzyme-inhibitor complex, the resulting conformers
were clustered on the basis of the rmsd using the autocluster
protocol implemented in the conformer analysis module of
Cerius-2. The center of each cluster, representing a docking
solution, was submitted to a geometry optimization protocol.
During the minimization, the protein was treated as a rigid
body by applying fixed constraints on the coordinates of its
atoms. The limitations and advantages of such a procedure
were discussed in our previous work. Energy minimization was
performed using the universal force field, version 1.2 software,
and the Smart Minimizer protocol of the Open Force Field
module (OFF). The value of the dielectric constant of the
protein surrounding the binding site of the heme group was
set to a value of 6.5 as reported elsewhere.21 Conversely, the
conformational strain energy cost of the inhibitors to reach
the bioactive conformation from the global minimum was
determined using a dielectric constant of 80 according to the
hypothesis that this process occurs in the water. The refined
docking solutions were ranked according to their binding
energy. For each enzyme-inhibitor complex, only the best
solution, in terms of binding energy, was stored and used for
further analysis. The binding energies were calculated using
1
24 (0.47 g, 45%) as an oil. H NMR (CDCl3) δ: 0.91 (3H, t, J
) 7.2 Hz, CH2CH3), 1.25-1.70 (5H, m, SCHCHHCH2CH2CH3),
1.80-2.00 (1H, m, SCHCHHCH2), 3.49 (1H, dd, J ) 8.1 and
6.2 Hz, SCHCH2), 3.55 (3H, s, NCH3), 4.45 (2H, s, CH2Br),
7.50 (1H, d, J ) 8.0 Hz, H-8), 7.65 (1H, dd, J ) 8.0 and 1.6
Hz, H-7), 7.75 (1H, d, J ) 1.6 Hz, H-5).
General Method for the Preparation of Imidazole
Derivatives 28-31. This preparation is illustrated by the
synthesis of 2-butyl-6-(1H-1-imidazolylacetyl)-4-methyl-3,4-
dihydro-2H-1,4-benzothiazin-3-one (28).
1H-Imidazole (0.27 g, 3.96 mmol) was added to a solution
of 24 (0.47 g, 1.32 mmol) in CHCl3 (25 mL) and stirred at room
temperature for 5 h. The mixture was evaporated to dryness,
and the residue was chromatographed, eluting with CHCl3/
MeOH 97:3 to give 28 (0.35 g, 77%) as an amorphous solid.
1H NMR (CDCl3) δ: 0.91 (3H, t, J ) 6.9 Hz, CH2CH3), 1.20-
1.70 (5H, m, SCHCHHCH2CH2CH3), 1.80-2.00 (1H, m,
SCHCHHCH2), 3.45-3.65 (4H, m, NCH3 and SCHCH2), 5.53
(2H, s, CH2N), 7.04, 7.22 and 7.83 (each 1H, bs, imidazolic H),
7.54 (1H, d, J ) 8.0 Hz, H-8), 7.63 (1H, dd, J ) 8.0 and 1.3
Hz, H-7), 7.72 (1H, d, J ) 1.3 Hz, H-5).
General Method for the Preparation of Carbinols 5,
7, 9, and 11. This preparation is illustrated by the synthesis
of 2-butyl-6-[1-hydroxy-2-(1H-1-imidazolyl)ethyl]-4-methyl-3,4-
dihydro-2H-1,4-benzothiazin-3-one (5).
NaBH4 (0.078 g, 2.04 mmol) was added to a solution of 28
(0.35 g, 1.02 mmol) in dry MeOH (10 mL) over 1 h. The mixture
was then evaporated to dryness, and the residue was chro-
matographed, eluting with CHCl3/MeOH 90:10 to furnish 5
1
(0.30 g, 85%) as an amorphous solid. H NMR (DMSO-d6) δ:
0.84 (3H, t, J ) 7.0 Hz, CH2CH3), 1.10-1.60 (5H, m,
SCHCHHCH2CH2CH3), 1.65-1.80 (1H, m, SCHCHHCH2),
3.33 (3H, s, NCH3), 3.55 (1H, dd, J ) 7.7 and 6.4 Hz, SCHCH2),
4.00-4.30 (2H, m, CH2N), 4.80-5.00 (1H, m, CHOH), 5.86 (1H,
bs, CHOH), 6.85 and 7.48 (each 1H, s, imidazolic H), 7.00-
7.25 (3H, m, aromatic H and imidazolic H), 7.37 (1H, dd, J )
7.8 and 1.2 Hz, H-7). Anal. (C18H23N3O2S) C, H, N.
Ebin ) Eint + Econf
(1)
where the energy of binding (Ebin) is obtained from the sum of
the interaction energy (Eint) and the conformational strain
energy (Econf). The interaction energy (Eint) is calculated with
General Method for the Preparation of Ether Deriva-
tives 6, 8, 10, and 12. This preparation is illustrated by the
synthesis of 2-butyl-6-{1-[(4-chlorobenzyl)oxy]-2-(1H-1-imida-
zolyl)ethyl}-4-methyl-3,4-dihydro-2H-1,4-benzothiazin-3-one (6).
NaH (60% mineral oil dispersion, 0.042 g, 1.74 mmol) was
added to a solution of 5 (0.20 g, 0.58 mmol) in dry DMF (5
mL), and then p-chlorobenzyl chloride (0.37 g, 2.31 mmol) in
dry DMF (2 mL) was added dropwise. The mixture was stirred
at room temperature for 1 h, then suspended in H2O and
extracted with EtOAc. The combined organic layers were
evaporated to dryness to afford a crude residue that was
purified by chromatography, eluting with CHCl3/MeOH 99:1
to give 6 (0.20 g, 73%) as an oil. 1H NMR (CDCl3) δ: 0.91 (3H,
t, J ) 7.0 Hz, CH2CH3), 1.25-1.70 (5H, m, SCHCHHCH2CH2-
CH3), 1.75-2.10 (1H, m, SCHCHHCH2), 3.30-3.50 (4H, m,
NCH3 and SCHCH2), 4.21 (2H, d, J ) 5.6 Hz, CH2N), 4.25 and
4.55 (each 1H, d, J ) 12.0 Hz, CH2Ph), 4.58 (1H, t, J ) 5.6
Hz, CHO), 6.80-7.70 (10H, m, aromatic H and imidazolic H).
Anal. (C25H28ClN3O2S) C, H, N.
Eint ) Ecomplex - (Ecompound + Eprotein
)
(2)
from the docking solutions of each compound. Here, Ecomplex is
the energy of the complex, Ecompound is the energy of the docked
compound, and Eprotein is the energy of the enzyme.
The conformational strain energy (Econf) is calculated as the
difference between the energy of the docked conformation and
the energy of the global minimum for each compound:
Econf ) Ecompound - Eglobal
(3)
The water/octanol partition coefficient (log P) was calculated
using the atom-based approach (A log P) as implemented in
Cerius-2.24 All calculations were carried out on SGI O2 R5000
and R10000 workstations.
Mycology. Mice. Female CD1 mice (8-10 weeks old,
weighing 25-30 g) were obtained from Charles River Breeding
Laboratories (Calco, Lecco, Italy).
Computational Method. The model of cytochrome P450
14R-sterol demethylase of Candida albicans (CA-CYP51) was
constructed using the crystal structure of cytochrome P450 of
Mycobacterium tuberculosis (MT-CYP51, PDB codes 1ea1 and
1e9x) as previously reported by us.7
Azole inhibitors were built using the sketch module of
Cerius-2.18 Atomic charges were calculated using the semiem-
pirical Mopac/AM1 method. Each compound was submitted to
an energy minimization protocol using the universal force field,
version 1.2 software,19 and the Smart Minimizer algorithm of
the Open Force Field module (OFF). Docking experiments were
performed using a knowledge-based strategy.20 Briefly, azole
antifungals bind to MT-CYP51 through the coordination of the
ring nitrogen atom (N-4 of triazole and N-3 of imidazole) to
the sixth coordination position of the iron atom of the heme
group. Accordingly, all compounds were manually docked in
CA-CYP51 by placing the nitrogen atom in position 3 of the
C. albicans CA-6, used in this study, was isolated from a
vaginal swab and identified according to the taxonomic criteria
of van Uden and Buckley.22 C. krusei strain 6258 (American
Type Culture Collection, Rockville, MD) was used in this study.
The yeasts were grown at 28 °C in Sabouraud dextrose agar.
Under these conditions the organisms grew as a pure yeast-
phase population. Before use, yeast cells were harvested from
a 24 h culture, suspended in pyrogen-free saline, washed twice,
quantified by hemocytometry, and adjusted to the desired
concentration.
Systemic Candidiasis Model. Mice were infected intra-
venously (iv) with 1 × 105 C. albicans blastoconidia via the
lateral tail vein. Diluent (DMSO/H2O ratio of 1:4), chemicals,
and fluconazole were administered intraperitoneally (ip) at a
dose of 10 mg/kg of body weight 2 h before infection and then
once daily for 14 consecutive days. The dose and administra-
tion schedules used in this study were selected from previously

Evaluation of CA-CYP51 Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7665
reported data6a and based on dose and treatment schedules
reported in the literature for a novel azole with a broad
antifungal spectrum, such as ER-30346.23 For survival studies,
mice were observed for 30 days. Three mice per group were
killed by CO2 asphyxiation 11 days after infection for quan-
titative culture of both kidneys.
Quantification of C. albicans in the Kidneys. The
kidneys of mice were aseptically removed and homogenized
with 3 mL of sterile distilled water. The number of colony
forming units (CFU) was determined by a plate dilution
method. Colonies of C. albicans cells were counted after 48 h
of incubation at room temperature, and results were expressed
as the number of CFU per organ.
Susceptibility Testing. Susceptibility testing was per-
formed by the M27-A microdilution method of the National
Committee for Clinical Laboratory Standards in 0.165 M
MOPS (morpholinepropanesulfonic acid)-buffered (pH 7) RPMI
1640 medium (Gibco BRL, Paisley, U.K.). The activity of
compounds against C. albicans and C. krusei was tested using
serial dilutions ranging from 0.9 to 500 µg/mL. The MIC was
the lowest concentration of chemical that produced an 80%
reduction in the turbidity compared to chemical-free normal
subjects.
Statistical Analysis. Differences in median survival time
were determined by the Mann-Whitney U test. Student’s t
test was used to evaluate the significance of all other data.
Each experiment was repeated three to five times.
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Acknowledgment. This work was supported by the
Italian Ministry of Education, Universities and Re-
search, Grant COFIN 2003068044_006.
Supporting Information Available: Characterization
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