Bulky 1,4-benzoxazine derivatives with antifungal activity

Article abstract of DOI:10.1016/j.bmc.2009.04.051

For many years the development of new azole antifungals has been quite empirically based. More recently, the publication of the crystal structure of CYP51 of Mycobacterium tuberculosis (MT-CYP51) provided new opportunities to rationalize the knowledge abo

Full text of DOI:10.1016/j.bmc.2009.04.051

Bioorganic & Medicinal Chemistry 17 (2009) 3838–3846
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Bulky 1,4-benzoxazine derivatives with antifungal activity
a
a
b
a
Renata Fringuelli a, , Nicola Giacchè , Lara Milanese , Elio Cenci , Antonio Macchiarulo ,
*
Anna Vecchiarelli b, Gabriele Costantino a, , Fausto Schiaffella a
a Dipartimento di Chimica e Tecnologia del Farmaco, Via del Liceo 1, Università di Perugia, 06100 Perugia, Italy
b Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Via del Giochetto, Università di Perugia, 06100 Perugia, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
For many years the development of new azole antifungals has been quite empirically based. More
recently, the publication of the crystal structure of CYP51 of Mycobacterium tuberculosis (MT-CYP51) pro-
vided new opportunities to rationalize the knowledge about antifungal action of this class of compounds.
Recent studies reported that a ‘channel 2 opened’ conformation of the enzyme could better explain the
interaction with ketoconazole (KTZ)-like drugs. Conformational changes were made on our model of Can-
dida albicans CYP51 (CA-CYP51) previously reported and docking experiments were performed. The
results allowed new KTZ analogues to be designed, by predicting that the 1,4-benzoxazine moiety could
replace the KTZ aryl-piperazinyl chain. The synthesis of derivatives 12 and 13 was planned.
The in vitro antifungal activity was evaluated against different Candida species and low and high capsu-
lated strains of Cryptococcus neoformans. Since the in vitro activity do not necessarily correlate with the
in vivo antifungal activity the newly synthesized compounds were also tested in a murine model of sys-
temic C. albicans infection. The therapeutic effect was evaluated in terms of animal survival and of fungal
growth in the kidneys, the target organ in systemic candidiasis.
Received 17 December 2008
Revised 16 April 2009
Accepted 17 April 2009
Available online 3 May 2009
Keywords:
Azole antifungals
Ketoconazole-like drugs
1,4-Benzoxazine
CA-CYP51 inhibitors
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
Therefore, new effective anti-Candida agents are needed to
combat the drug-resistant strains and widespread diffusion of C.
In recent years, the incidence of fungal infections has markedly
increased particularly in high risk units in which hospitalized pa-
tients are treated with immunosuppressive agents, anticancer,
anti-AIDS drugs, or broad-spectrum antibiotics.1,2 Amongst the dif-
ferent pathogens responsible for opportunistic fungal infections
are yeasts belonging to the genera Candida and Cryptococcus. In
particular, Candida albicans is a ubiquitous mucosal commensal
in equilibrium with the microbial flora and the host immune sys-
tem, but is also responsible for a wide range of infections in immu-
nocompromised persons. A major obstacle in the treatment of
Candida infections is the spread of antifungal drug resistance, par-
ticularly in patients subjected to prolonged antimycotic therapy.3
Polyenic and azole antifungal agents are usually used to treat Can-
dida infections, but despite the good antifungal activities observed
in vitro, their therapeutic efficacy is variable, so that candidemia is
still a major cause of death.4 Moreover, antimycotic therapy is usu-
ally marred by drug solubility problems as well as by a number of
adverse side effects.5
albicans.
For many years the development of new azole antifungals has
been quite empirically based. More recently, the publication of
the crystal structure of CYP51 of Mycobacterium tuberculosis (MT-
CYP51, pdb code: 1ea1, 1e9x), both alone and co-crystallized with
fluconazole (FLU),6 provided new opportunities to rationalize the
knowledge about antifungal action of this class of compounds.
In a program aimed at the design and synthesis of novel azole
inhibitors of C. albicans CYP51 (CA-CYP51), a series of azole 1,4-ben-
zothiazines (1,4-BTs) and 1,4-benzoxazines (1,4-BOs) were recently
synthesized.7–13 To better understand the SAR of these derivatives,
and to hypothesize the modes of interactions between the synthe-
sized compounds and the target enzyme, a 3D homology model of
CYP51 of C. albicans was built on the basis of the homology relation-
ship existing between the amino acidic sequences of CA-CYP51 and
MT-CYP51 (40% similarity, 22% sequence identity).11
Docking studies were performed on the model using FLU and
representative 1,4-BTs and 1,4-BOs chosen from those previously
synthesized. The best docking solutions showed that FLU and the
considered compounds adopt similar binding modes inside the cat-
alytic site of CA-CYP51. For this purpose, the attention was focused
on portions of the molecule that had not been investigated previ-
ously. To better characterize the putative interaction with the en-
zyme of bulky molecules similar to ketoconazole (KTZ), and
* Corresponding author. Tel.: +39 0755855134; fax: +39 0755855161.
E-mail address: fringuel@unipg.it (R. Fringuelli).
Present address: Dipartimento Farmaceutico, Via GP Usberti 27/A, Università di
Parma, 43100 Parma, Italy.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.04.051

R. Fringuelli et al. / Bioorg. Med. Chem. 17 (2009) 3838–3846
3839
Figure 1. Homology model of CA-CYP51. The two channels controlling the access to the catalytic site are labeled and indicated with the arrows. Channel 1 runs parallel to the
plane of the heme group whereas channel 2 is perpendicular to that plane. The principal secondary structure elements of the enzyme are also indicated.
compound 1 previously synthesized in our laboratory14 further
analyses on several CYP51 X-ray structures were carried out.15,16
Results of these studies highlighted the presence of a second sub-
strate access channel (channel 2), almost perpendicular to the
heme, apparently closed in the MT-CYP51 published crystal.
Recent studies, carried on by Xiao and coworkers, reported that
a ‘channel 2 opened’ conformation of the enzyme could better ex-
plain the interaction with KTZ-like drugs.17 (Fig. 1)
BO derivatives bound to the ketal moiety through the position C-
6 of the nucleus-base.
The synthesis of derivatives 12 and 13 was so planned taking
into account that the alkylation of the C-2 position was favorable
in other series of compounds12 (Fig. 2).
2. Docking experiments
According to this methodology, we have modified our model of
CA-CYP51 in order to reproduce the ‘channel 2 opened’ conforma-
tion, and new docking experiments were thus performed on this
new model. As a result, we show here that new KTZ analogues
can be designed, by replacing the KTZ aryl-piperazinyl chain with
1,4-BO. Indeed, several structures were docked into the model,
and those that showed the best binding energy values were 1,4-
According to the hypothesis of Xiao et al.,17 the conformation of
the side chain of two amino acids (Tyr118 and Met508) was man-
ually changed in order to open channel 2 in our homology model of
CA-CYP51.11 In particular, this was accomplished by rotating of
about 60° the dihedral angle defined by the atoms N, C , Cb, C of
a
c
Tyr118 and Met508. To evaluate the interaction energy between
O
O
O
N
N
O
N
N
Cl
O
O
N
O
O
N
S
Cl
O
N
Me
N
N
Ketoconazole
1
O
R
O
O
N
Cl
Me
O
O
N
Cl
12 R = H
13 R = nBu
N
Figure 2.

3840
R. Fringuelli et al. / Bioorg. Med. Chem. 17 (2009) 3838–3846
Table 1
then the R isomer. The adopted binding pose of compounds 13S
and 12, while in agreement with that of FLU, allows the benzox-
azine moiety to form hydrogen bonding with the backbone of
Tyr505 and Met508. Finally, the inspection of the Alog P values (Ta-
ble 1) reveals a good balance of hydrophilic and hydrophobic prop-
erties of these compounds for crossing the fungi membrane.11
Binding energies and Alog P values of ketoconazole (KTZ) and 1,4-benzoxazine
derivatives with best docking results
Compound
Ebin (kcal/mol)
Alog P
KTZ
12
(S)-13
(R)-13
À28.616
À35.000
À35.784
À7.846
4.480
3.660
5.459
5.459
3. Chemistry
The synthesis and determination of the configuration of O-p-tol-
uenesulfonyl (Ts) ketal precursors, cis- and trans-8, were per-
formed as described by Rotstein and coworkers.18 Since 1,4-BO of
the 2-butyl serie did not react with the Ts-ketals with satisfactory
yields, the corresponding methanesulfonyl (Ms) derivative 9 was
also prepared. In this case, it was not possible to separate the enan-
tiomeric couples by chromatography, so the successive reactions
were performed on the whole mixture and the separation was car-
ried out on the final products.
the ligands and the enzyme, we performed automated docking
experiments of the inhibitors as reported in Section 5.
Docking results presenting the binding pose of the inhibitors
with the long side chain stretched out in the channel 2 were con-
sidered as ‘productive’ and stored for further analysis. The binding
energies (Ebin, Table 1) were calculated using a knowledge-based
strategy considering the protein as a rigid-body and removing all
the constrains used during docking experiments.11
All compounds adopt a similar interaction pattern which is
schematically represented in the following Figure 3.
As far as it concerns compound 13, docking experiments reveal
that the S isomer fits better the enzymatic site along channel 2
For the synthesis of the 1,4-BO moiety the key step was to ob-
tain the 1,4-BO intermediates with a reactive function in the C-6
position: compound 6 was obtained by the selective 6-hydroxyl-
ation of 3,4-dihydro-2H-1,4-benzoxazin-3-one performed with
Figure 3. Best binding poses resulting from docking experiments of KTZ (a), NN compound (b) and MM compound (c, d). (a–c) were drawn using the MOE Ligand Interaction
tool (Molecular Operating Environment, 2006.08 ed.; Chemical Computing Group, Inc. Montreal, Quebec, Canada.). (d) shows the binding pose of the compound MM (green
sticks) highlighting the hydrogen bonds (green lines) with Tyr505 and Met508. In magenta are shown the 2 residues (Met508 and Tyr118) whose conformation was modified
in order to open channel 2. In blue is shown the cavity of channel 2.

R. Fringuelli et al. / Bioorg. Med. Chem. 17 (2009) 3838–3846
3841
O
R
O
O
R
O
O
R
O
MeO
N
HO
N
HO
N
H
Me
Me
2 R = H
3 R = nBu
4 R = H
5 R = nBu
6 R = H
7 R = nBu
OR'
Cl
O
O
Br
Cl
8 R’ = Ts
9 R’ = Ms
O
N
R
O
O
R
O
N
O
O
Cl
O
Cl
Me
Me
O
O
O
12 R = H
13 R = nBu
10 R = H
11 R = nBu
N
Br
Cl
Cl
N
Scheme 1. Reagents: (a) tBuOK, MeI; (b) 48% HBr; (c) NaH; (d) K2CO3, 1H-imidazole.
[bis(trifluoroacetoxy)iodo]benzene (PIFA),19 and the same reaction
gave good yields of the 2-butyl derivative 7 starting from 2-butyl-
3,4-dihydro-2H-1,4-benzoxazin-3-one.20 The hydroxylated inter-
mediates thus obtained were methylated (4 and 5) and the fenolic
function was restored refluxing with 48% HBr. Compound 6 was
then condensed with both cis- and trans-8, while derivative 7 re-
acted with the corresponding Ms-ketal 9. Finally, imidazole was
introduced thus obtaining the desired products 12 and 13 (Scheme
1).
As far as compound 13 concerns, since the ketal moiety em-
ployed in the synthesis was composed of all four diastereomers,
and the 1,4-BO moiety introduces a third stereogenic center, our
product was a mixture of eight isomers. This mixture was split into
two fractions by flash chromatography. Examination of their 1H
NMR spectra, by analogy with data previously reported, has rea-
sonably permitted to suppose that the less polar fraction is com-
posed of stereoisomers with a syn configuration on the ketal
moiety, cis-13, while the second fraction, trans-13, is the mixture
of the four anti isomers.
4. Results and discussion
The antifungal activity of the synthesized compounds was eval-
uated against different Candida species and low and high capsu-
lated strains of Cryptococcus neoformans. The results, reported in
Table 2, indicate that all compounds exhibited good antifungal
activity on C. albicans strain CA-6, with MIC equal to 0.49
after 24 and 48 h incubation. The MIC of KTZ, used as reference,
was <0.0076 g/mL. Interestingly, only cis-13 exhibited low (MIC
125 g/mL) but appreciable activity against the low virulent C.
lg/mL
l
l
albicans strain PCA-2 unable to undergo mycelial transition, all
other compounds being totally ineffective. As regards Candida
krusei, Candida glabrata and Candida parapsilosis, all compounds
showed no or modest antifungal activity, with MIC ranging from
3.90 to >500 lg/mL.
When assayed on C. neoformans, the compounds showed a good
antifungal activity, particularly against the high capsulated strain
CAP-67, with MIC ranging from 0.0019 for cis-12 to 3.90
for trans-13.
lg/mL
Table 2
In vitro antifungal activity of different synthesized compounds
MICa
C. glabrata
24 h 48 h
15.60 >500 7.80 >500 250
15.60 >500 15.60 250
(
l
g/mL)
C. albicans strain CA-6 C. albicans strain PCA-2
C. krusei
24 h 48 h
C. parapsilosis
C. neoformans strain 6995 C. neoformans strain CAP67
24 h
0.49
trans-12 0.49
cis-13 0.49
48 h
24 h
48 h
24 h
48 h
48 h
72 h
48 h
72 h
cis-12
0.49
0.49
0.49
>500
>500
125
>500
>500
125
>500
>500
31.25
500
0.24
0.97
1.95
7.80
0.24
1.95
7.80
7.80
0.0019
0.03
0.97
0.0019
0.03
1.95
>500
3.90
3.90
0.49
250
3.90
7.80
0.97
>500 125
>500 >500
31.25 125
62.50 >500 125
31.25
trans-13 0.49
0.49
>500
15.60
>500
>500
>500
15.60
>500
>500
3.90
3.90
KTZb
FLUc
<0.0076
<0.0076
<0.0076
>500
0.12
1.95
250
<0.00006 0.00095 0.00095
7.80
0.00095
3.90
0.00012
0.49
0.00012
0.49
<0.0076
15.60
>500
1.95
Diluentd >500
>500
>500 >500
>500
>500
>500
>500
a
MIC: minimum inhibitory concentration.
KTZ: ketoconazole.
FLU: fluconazole.
b
c
d
Diluent: EtOH/H2O 1:4.

3842
R. Fringuelli et al. / Bioorg. Med. Chem. 17 (2009) 3838–3846
resulting in 75% of animals surviving until the end of the observa-
tion period.
In parallel experiments, evaluation of fungal burden in the kid-
neys 7 days post infection, showed a significant reduction in CFU
recovery in cis-13 and trans-13 treated mice. Remarkably, FLU trea-
ted mice almost completely cleared the infection (Fig. 5).
In the same experimental conditions, histological analysis of
PAS-stained kidneys sections showed extensive fungal growth
with multiple inflammatory foci in renal parenchima of C. albicans
infected animals (Fig. 6A), while treatment with cis-13, trans-13, or
FLU resulted in rare inflammatory foci with a small number of fun-
gal cells (Fig. 6B–D).
To further assess the effect of synthesized compounds in mur-
ine systemic candidiasis, in selected experiments serum collected
from mice 1 h after inoculation with cis-13, trans-13, FLU, or 2%
Figure 4. Percent survival of C. albicans infected mice. Mice were infected at day 0
with C. albicans CA-6 (7 Â 105/mouse, i.v.) and treated 2 h before and once a day for
6 consecutive days after the infection with cis-13, trans-13, KTZ, FLU (all 10 mg/kg,
i.p.) or vehicle (0.2 ml 2% EtOH in saline, i.p.). : p < 0.05, treated versus untreated.
*
Collectively, the results demonstrate that synthesized com-
pounds are active against Candida and Cryptococcus in vitro. Inter-
estingly, data indicate that maximum activity is shown against
Candida isolates capable of hypha transition like C. albicans CA6
and C. krusei rather than C. albicans PCA-2 and C. glabrata, both
agerminative, suggesting that process leading to hypha formation
could be targeted by the compounds.
Since the in vitro activity does not necessarily correlate with the
in vivo antifungal activity21–23 the newly synthesized compounds
were also tested in a murine model of systemic C. albicans infec-
tion. To this purpose, mice were infected with C. albicans CA-6
and inoculated with the different compounds. Preliminary experi-
ments indicated that treatment with cis-12 and trans-12 was inef-
fective (not shown), therefore they were not included in
subsequent experiments. The therapeutic effect was evaluated in
terms of animal survival and of fungal growth in the kidneys, the
target organ in systemic candidiasis. Survival data, reported in Fig-
ure 4, indicate that treatment of infected mice with both cis-13 and
trans-13 resulted in significant outcome improvement. In particu-
lar, treatment with trans-13 resulted in 50% of mice surviving the
full 30 days observation period, while treatment with cis-13 re-
sulted in significantly higher survival when compared to untreated
controls (animals dying in 23 and 17 days, respectively). Notewor-
thy, treatment with trans-13 resulted in improved survival rates
when compared to KTZ treatment; of course (obviously), in the
same experimental setting, FLU significantly improved outcome,
Figure 6. Histological analysis of kidneys from C. albicans infected mice. Mice were
infected at day 0 with C. albicans CA-6 (7 Â 105/mouse, i.v.) and treated 2 h before
and once a day for 6 consecutive days after the infection with cis-13, trans-13 or FLU
(all 10 mg/kg). 7 days after infection, kidneys were excised and fixed in formalin.
Sections of paraffin-embedded tissues were stained using the periodic acid-Schiff
(PAS). Histologic evaluation of kidneys from mice infected with C. albicans shows
extensive fungal growth, associated with the presence of numerous foci of
inflammatory reaction throughout the parenchyma (A). Note the reduced fungal
growth and few pathologic lesions in the kidneys of C. albicans infected mice treated
with cis-13 (B), trans-13 (C), or FLU (D). Boxes indicate the areas magnified.
Figure 5. Fungal burden in the kidneys of C. albicans infected mice. Mice were
infected at day 0 with C. albicans CA-6 (2.5 Â 105/mouse, i.v.) and treated 2 h before
and once a day for 6 consecutive days after the infection with cis-13, trans-13, FLU
(all 10 mg/kg, i.p.) or vehicle (0.2 ml 2% EtOH in saline, i.p.). CFU recovery from
*
kidneys was determined 7 days after infection. : p < 0.05, treated versus untreated.

R. Fringuelli et al. / Bioorg. Med. Chem. 17 (2009) 3838–3846
3843
ethanol solution, was passively transferred into recipient mice sub-
sequently challenged with C. albicans CA-6. Recovery of CFU in the
kidneys, 7 days after challenge, showed that inoculation of serum
from treated animals resulted in significantly lower fungal burden
in kidneys (Fig. 7).
This result confirms that the synthesized compounds are indeed
distributed by blood circulation to the different organs. Moreover,
the high effect shown in Figure 7 could suggest that a sort of mod-
ification or activation of this compound would occur soon after
inoculation in vivo.
rated solution of NaHCO3, then extracted with EtOAc. The organic
phase was dried, evaporated, and purified by chromatography elut-
ing with cyclohexane/EtOAc 40:60 to give 3 (0.73 g, 67%) as a white
solid, mp 191–193 °C, 1H NMR (DMSO-d6) d: 0.88 (3H, t, J = 7.0 Hz,
CH2CH3), 1.20–2.00 (6H, m, CH2CH2CH2CH3), 4.40 (1H, m, CHO),
6.15–6.85 (3H, m, aromatic H), 9.18 (H, br s, NH), 10.52 (H, br s,
OH). Anal. Calcd for C12H15NO3: C, 65.14; H, 6.83; N, 6.33. Found:
C, 65.03; H, 6.84; N, 6.31.
5.2. 6-Methoxy-4-methyl-3,4-dihydro-2H-1,4-benzoxazin-3-
We conclude that it may be feasible to modify the structure of
KTZ to render more active the compound in vivo. Furthermore,
we demonstrated that these conformational modifications lead to
powerful antifungal activity in vivo, possibly due to shedding of
potent antifungal molecules, as documented by protective effects
of passive transfer of serum from trans-13 treated mice into C. albi-
cans infected animals.
one (4)
t-BuOK (1.68 g, 15.00 mmol) was added to a solution of 6-hydro-
xy-3,4-dihydro-2H-1,4-benzoxazin-3-one19 (2) (1.00 g, 6.06 mmol)
in dry DMF (30 mL). The mixture was stirred at room temperature
for 15 min, and then MeI (2.13 g, 15.00 mmol) in dry DMF (10 mL)
was added dropwise. After being stirred for 1 h, the mixture was
poured into ice-chilled water and extracted with EtOAc. The residue
was chromatographed, eluting with cyclohexane/EtOAc 65:35.
Compound 4 (0.47 g, 40%) was obtained as a white solid, mp 70.6–
71.8 °C, 1H NMR (CDCl3) d 3.33 (3H, s, NCH3), 3.80 (3H, s, OCH3),
4.55 (2H, s, OCH2), 6.45–6.60 (2H, m, H-5 and H-8), 6.90 (1H, dd,
J = 8.0 and 2.5 Hz, H-7). Anal. Calcd for C10H11NO3: C, 62.17; H,
5.74; N, 7.25. Found: C, 62.24; H, 5.74; N, 7.24.
5. Experimental
Melting points determined in capillary tubes (Electrothermal,
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 val-
ues. 1H NMR and 13C NMR spectra were recorded at 200 MHz (Bru-
ker AC-200 spectrometer) with Me4Si as internal standard.
Chemical shifts are given in ppm (d), and the spectral data are con-
sistent with the assigned structures. HPLC-MS was checked
employing an Agilent 1100 binary HPLC system (Agilent Technolo-
gies, CA, USA) interfaced with an API 150EX single quadrupole MS
spectrometer (Applied Biosystem/MDS Sciex, CA, USA) equipped
with an Electrospray (ESI) Ion Source. Reagents and solvents were
purchased from common commercial suppliers and used as re-
ceived. Column chromatography separations were carried out on
Merck Silica Gel 60 (mesh 230–400). Yields of purified products
were not optimized. All starting materials were commercially
available unless otherwise indicated.
5.3. 2-Butyl-6-methoxy-4-methyl-3,4-dihydro-2H-1,4-
benzoxazin-3-one (5)
It was synthesized starting from 3 using the same procedure de-
scribed for 4. Oil, 70% yield. 1H NMR (CDCl3) d 0.93 (3H, t, J = 7.0 Hz,
CH2CH3), 1.20–1.60 (4H, m, CH2CH2CH2CH3), 1.60–2.00 (2H, m,
CH2CH2CH2CH3), 3.35 (3H, s, NCH3), 3.81 (3H, s, OCH3), 4.53 (1H,
m, CHO), 6.40–7.00 (3H, m, aromatic H). Anal. Calcd for C14H19
NO3: C, 67.45; H, 7.68; N, 5.62. Found: C, 67.24; H, 7.70; N, 5.64.
-
5.4. 6-Hydroxy-4-methyl-3,4-dihydro-2H-1,4-benzoxazin-3-
one (6)
A solution of 4 (1.00 g, 5.18 mmol) in 48% HBr (15 mL) was re-
fluxed for 2 h, then was poured into ice-chilled water and extracted
with EtOAc. The organic phase was dried and evaporated in vacuo
to give 6 (0.87 g, 93%) as a pale yellow solid, mp 207.7–208.5 °C,
which was used without further purification. 1H NMR (CDCl3) d
3.37 (3H, s, NCH3), 4.60 (2H, s, OCH2), 6.49 (1H, dd, J = 8.5 and
2.7 Hz, H-7), 6.57 (1H, d, J = 2.7 Hz, H-5), 6.89 (1H, d, J = 8.5 Hz,
H-8), 8.13 (1H, s, OH). Anal. Calcd for C9H9NO3: C, 60.33; H, 5.06;
N, 7.82. Found: C, 60.33; H, 5.07; N, 7.79.
5.1. 2-Butyl-6-hydroxy-3,4-dihydro-2H-1,4-benzoxazin-3-one (3)
[Bis(trifluoroacetoxy)iodo]benzene (PIFA) (2.50 g, 5.81 mmol)
in 10 mL of trifluoroacetic acid (TFA) was added to a refluxing solu-
tion of 2-butyl-3,4-dihydro-2H-1,4-benzoxazin-3-one20 (1.00 g,
4.88 mmol) in TFA (10 mL). After refluxing for 5 min, the solution
was concentrated at half of its volume, neutralized with a satu-
5.5. 2-Butyl-6-hydroxy-4-methyl-3,4-dihydro-2H-1,4-
benzoxazin-3-one (7)
It was synthesized starting from 5 using the same procedure de-
scribed for 6. White solid, mp 80–82 °C, 70% yield. 1H NMR (ace-
tone-d6) d 0.93 (3H, t, J = 7.0 Hz, CH2CH3), 1.25–1.65 (4H, m,
CH2CH2CH2CH3), 1.65–1.90 (2H, m, CH2CH2CH2CH3), 3.31 (3H, s,
NCH3), 4.44 (1H, dd, J = 13.1 and 4.8 Hz, CHO), 6.51 (1H, dd,
J = 8.6 and 2.6 Hz, H-7), 6.62 (1H, d, J = 2.6 Hz, H-5), 6.84 (1H, d,
J = 8.6 Hz, H-8), 8.26 (1H, s, OH). Anal. Calcd for C13H17NO3: C,
66.36; H, 7.28; N, 5.95. Found: C, 66.30; H, 7.30; N, 5.93.
5.6. cis-6-{[2-(Bromomethyl)-2-(2,4-dichlorophenyl)-1,3-
dioxolan-4-yl]methoxy}-4-methyl-3,4-dihydro-2H-1,4-
benzoxazin-3-one (cis-10)
Figure 7. Fungal clearance after passive transfer of serum in C. albicans infected
mice. Mice were infected with C. albicans CA-6 (7 Â 105/mouse, i.v.) after
intraperitoneal inoculation with 0.3 ml of serum recovered from mice 1 h after
treatment with cis-13, trans-13, FLU (all 10 mg/kg, i.p.) or vehicle (0.2 ml 2% EtOH in
saline, i.p.). CFU recovery from kidneys was determined 7 days after C. albicans
A solution of 6 (0.26 g, 1.44 mmol) in dry DMSO (3 mL) was
*
infection. : p < 0.05, treated versus untreated.
added dropwise to a suspension of NaH (60% mineral oil disper-

3844
R. Fringuelli et al. / Bioorg. Med. Chem. 17 (2009) 3838–3846
sion, 0.063 g, 1.58 mmol) in dry DMSO (2 mL). After stirring at
room temperature for 1 h, a solution of cis-[2-(bromomethyl)-2-
(2,4-dichlorophenyl)-1,3-dioxolan-4-yl]methyl 4-methylbenzene-
sulfonate18 (cis-8) (0.50 g, 1.01 mmol) in DMSO (3 mL) was added
dropwise. The mixture was then heated to 80 °C for 4 h, poured
into ice-chilled water and extracted with EtOAc. The residue was
chromatographed, eluting with cyclohexane/EtOAc 80:20, thus
giving cis-10 (0.40 g, 79%), as a white solid that was crystallized
from cyclohexane/EtOAc, mp 97.9–99.4 °C, 1H NMR (CDCl3) d
3.39 (3H, s, NCH3), 3.91 and 4.02 (each 1H, d, J = 11.4 Hz, CH2Br),
4.02–4.32 (4H, m, CH2CHCH2OAr), 4.41–4.88 (1H, m,
CH2CHCH2OAr), 4.61 (2H, s, OCH2CO), 6.55–6.75 (2H, m, benzox-
azine H-5 and H-7), 6.96 (1H, d, J = 8.5 Hz, benzoxazine H-8),
7.33 (1H, dd, J = 9.0 and 2.0 Hz, 2,4-dichlorophenyl H-5), 7.48
(1H, d, J = 2.0 Hz, 2,4-dichlorophenyl H-3), 7.70 (1H, d, J = 9.0 Hz,
2,4-dichlorophenyl H-6). Anal. Calcd for C20H18BrCl2NO5: C,
47.74; H, 3.61; N, 2.78. Found: C, 47.80; H, 3.60; N, 2.78.
(2H, m, 2,4-dichlorophenyl H-6 and imidazolic H); 13C NMR
(CDCl3) d: 28.37 (NCH3), 51.30 (CH2N), 67.00 (OCH2CO), 67.52
(CH2CHCH2OAr), 67.58 (CH2CHCH2OAr), 75.00 (CH2CHCH2OAr),
104.70 (benzoxazine C-5), 107.94 (OCO), 111.40 (benzoxazine C-
8), 111.85 (benzoxazine C-7), 121.10 (NCHCHN), 127.20 (2,4-
dichorophenyl C-5), 128.50 (NCHCHN), 129.45 (2,4-dichorophenyl
C-6), 130.20 (benzoxazine C-10), 131.30 (2,4-dichorophenyl C-3),
133.15 (2,4-dichorophenyl C-2), 134.48 (2,4-dichorophenyl C-1),
136.00 (2,4-dichorophenyl C-4), 138.60 (NCHN), 139,05 (benzox-
azine C-9), 157.60 (benzoxazine C-6), 165.50 (OCH2CO); HPLC-
MS: 490.0 (M+). Anal. Calcd for C23H21Cl2N3O5: C, 56.34; H, 4.32;
N, 8.57. Found: C, 56.48; H, 4.31; N, 8.55.
5.10. trans-6-{[2-(2,4-Dichlorophenyl)-2-(1H-imidazol-1-
ylmethyl)-1,3-dioxolan-4-yl]methoxy}-4-methyl-3,4-dihydro-
2H-1,4-benzoxazin-3-one (trans-12)
It was synthesized starting from trans-10, using the same proce-
dure described for cis-12. Amorphous solid, 68% yield. 1H NMR
(CDCl3) d 3.27 (3H, s, NCH3), 3.65–3.97 (4H, m, CH2CHCH2OAr),
4.08–4.22 (1H, m, CH2CHCH2OAr), 4.44 (2H, s, CH2N), 4.54 (2H, s,
OCH2CO), 6.26 (1H, d, J = 2.5 Hz benzoxazine H-5), 6.30 (1H, dd,
J = 8.6 and 2.5 Hz, benzoxazine H-7), 6.83 (1H, d, J = 8.6 Hz, benzox-
azine H-8), 7.01, 7.05 and 7.63 (each 1H, br s, imidazolic H), 7.20
(1H, dd, J = 8.4 and 2.0 Hz, 2,4-dichlorophenyl H-5), 7.42 (1H, d,
J = 2.0 Hz, 2,4-dichlorophenyl H-3), 7.58 (1H, d, J = 8.4 Hz, 2,4-
dichlorophenyl H-6); 13C NMR (CDCl3) d: 28.37 (NCH3), 51.70
(CH2N), 67.00 (OCH2CO), 67.55 (CH2CHCH2OAr), 75.49 (CH2CH-
CH2OAr), 104.70 (benzoxazine C-5), 108.10 (OCO), 111.42 (benzox-
azine C-8), 111.85 (benzoxazine C-7), 121.10 (NCHCHN), 127.20
(2,4-dichorophenyl C-5), 128.50 (NCHCHN), 129.45 (2,4-dichor-
ophenyl C-6), 130.20 (benzoxazine C-10), 131.30 (2,4-dichorophenyl
C-3), 133.15 (2,4-dichorophenyl C-2), 134.45 (2,4-dichorophenyl
C-1), 136.00 (2,4-dichorophenyl C-4), 138.50 (NCHN), 139.00 (ben-
zoxazine C-9), 157.50 (benzoxazine C-6), 165.55 (OCH2CO); HPLC-
MS: 490.0 (M+). Anal. Calcd for C23H21Cl2N3O5: C, 56.34; H, 4.32; N,
8.57. Found: C, 56.40; H, 4.30; N, 8.58.
5.7. trans-6-{[2-(Bromomethyl)-2-(2,4-dichlorophenyl)-1,3-
dioxolan-4-yl]methoxy}-4-methyl-3,4-dihydro-2H-1,4-
benzoxazin-3-one (trans-10)
It was synthesized starting from 6 and ketal trans-[2-(bromo-
methyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl]methyl 4-meth-
ylbenzenesulfonate18 (trans-8), using the same procedure
described for cis-10. Amorphous solid, 59% yield, 1H NMR (CDCl3)
d 3.29 (3H, s, NCH3), 3.85 and 4.00 (3H, m, CHHCHCH2OAr), 3.89
(2H, s, CH2Br), 4.46 (1H, dd, J = 8.2 and 6.5 Hz, CHHCHCH2OAr),
4.55 (2H, s, OCH2CO), 4.70–4.87 (1H, m, CH2CHCH2OAr), 6.30
(1H, d, J = 2.6 Hz, benzoxazine H-5), 6.37 (1H, dd, J = 8.5 and
2.6 Hz, benzoxazine H-7), 6.85 (1H, d, J = 8.5 Hz, benzoxazine H-
8), 7.24 (1H, dd, J = 8.5 and 2.1 Hz, 2,4-dichlorophenyl H-5), 7.39
(1H, d, J = 2.1 Hz, 2,4-dichlorophenyl H-3), 7.68 (1H, d, J = 8.5 Hz,
2,4-dichlorophenyl H-6). Anal. Calcd for C20H18BrCl2NO5: C,
47.74; H, 3.61; N, 2.78. Found: C, 47.84; H, 3.61; N, 2.77.
5.8. 6-{[2-(Bromomethyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-
4-yl]methoxy}-2-butyl-4-methyl-3,4-dihydro-2H-1,4-
benzoxazin-3-one (11)
5.11. 2-Butyl-6-{[2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-
ylmethyl)-1,3-dioxolan-4-yl]methoxy}-4-methyl-3,4-dihydro-
2H-1,4-benzoxazin-3-one (13)
It was synthesized using the same procedure described for cis-
10 starting from 7 and [2-(bromomethyl)-2-(2,4-dichlorophenyl)-
1,3-dioxolan-4yl]methymethanesulfonate24 (9), the latter as the
cis/trans diastereomeric mixture. The mixture of the eight isomers
of 11 thus obtained (overall yield 48%) was used for the following
condensation reaction. 1H NMR according to diastereomeric
mixture.
The mixture of the eight isomers of 11 was treated as described
for the synthesis of cis-12. The residue obtained after the extraction
was chromatographed eluting with EtOAc, thus obtaining cis-13
(36% yield), and trans-13 (25.3% yield), as amorphous solids.
cis-13: 1H NMR (CDCl3) d 0.88 (3H, t, J = 6.8 Hz, CH2CH3), 1.15–
1.65 (4H, m, CH2CH2CH2CH3), 1.65–1.95 (2H, m, CH2CH2CH2CH3),
3.10–3.25 (1H, m, CHHCHCH2OAr), 3.33 (3H, s, NCH3), 3.55–3.95
(3H, m, CHHCHCH2OAr), 4.25–4.57 (4H, m, OCHCO, CH2CHCH2OAr
and CH2N), 6.35–6.50 (2H, m, benzoxazine H-5 and H-7), 6.86 (1H,
d, J = 8.4 Hz, benzoxazine H-8), 6.98 (2H, br s, imidazolic H), 7.25
(1H, dd, J = 8.4 and 2.0 Hz, 2,4-dichlorophenyl H-5), 7.45 (1H, d,
J = 2.0 Hz, 2,4-dichlorophenyl H-3), 7.54 (1H, br s, imidazolic H),
7.58 (1H, d, J = 8.4 Hz, 2,4-dichlorophenyl H-6); 13C NMR (CDCl3)
d: 14.10 (CH2CH2CH2CH3), 21.50 (CH2CH2CH2CH3), 26.00
(CH2CH2CH2CH3), 28.70 (NCH3), 30.33 (CH2CH2CH2CH3), 51.40
(CH2N), 67.48 (CH2CHCH2OAr), 67.70 (CH2CHCH2OAr), 75.00
(CH2CHCH2OAr), 81.38 (OCHCO), 104.40 (benzoxazine C-5),
108.00 (OCO), 111.00 (benzoxazine C-8), 111.40 (benzoxazine C-
7), 121.00 (NCHCHN), 127.40 (2,4-dichorophenyl C-5), 128.00
(NCHCHN), 129.50 (2,4-dichorophenyl C-6), 129.75 (benzoxazine
C-10), 131.05 (2,4-dichorophenyl C-3), 133.24 (2,4-dichorophenyl
C-2), 133.60 (2,4-dichorophenyl C-1), 135.96 (2,4-dichorophenyl
C-4), 138.63 (NCHN), 140.00 (benzoxazine C-9), 157.70 (benzox-
azine C-6), 168.50 (OCH2CO); HPLC-MS: 546.1 (M+). Anal. Calcd
5.9. cis-6-{[2-(2,4-Dichlorophenyl)-2-(1H-imidazol-1-
ylmethyl)-1,3-dioxolan-4-yl]methoxy}-4-methyl-3,4-dihydro-
2H-1,4-benzoxazin-3-one (cis-12)
A mixture of cis-10 (0.40 g, 0.79 mmol), 1H-imidazole (0.27 g,
3.98 mmol) and K2CO3 (0.55 g, 3.98 mmol) in DMA (5 mL) was re-
fluxed for 6 h, then cooled, diluted with water, and extracted with
EtOAc. The residue obtained after evaporation, was chromato-
graphed eluting with CHCl3 furnishing cis-12 (0.25 g, 65%) as a
white solid that was crystallized from cyclohexane/EtOAc, mp
160.2–160.8 °C, 1H NMR (CDCl3) d 3.25 (1H, dd, J = 9.4 and 6.8 Hz,
CHHCHCH2OAr), 3.41 (3H, s, NCH3), 3.65–4.00 (3H, m, CHHCH-
CH2OAr), 4.30–4.45 (1H, m, CH2CHCH2OAr), 4.46 and 4.58 (each
1H, d, J = 14.5 Hz, CH2N), 4.60 (2H, s, OCH2CO), 6.43 (1H, dd,
J = 8.6 and 2.7 Hz, benzoxazine H-7), 6.55 (1H, d, J = 2.7 Hz, benzox-
azine H-5), 6.93 (1H, d, J = 8.6 Hz, benzoxazine H-8), 7.06 (2H, br s,
imidazolic H), 7.32 (1H, dd, J = 8.4 and 2.0 Hz, 2,4-dichlorophenyl
H-5), 7.55 (1H, d, J = 2.0 Hz, 2,4-dichlorophenyl H-3), 7.60–7.70

R. Fringuelli et al. / Bioorg. Med. Chem. 17 (2009) 3838–3846
3845
for C27H29Cl2N3O5: C, 59.35; H, 5.35; N, 7.69. Found: C, 59.38; H,
5.36; N, 7.71.
virulent echinocandin resistant strain PCA-2 was obtained from D.
Kerridge, Department of Biochemistry, University of Cambridge.
The low and high capsulated C. neoformans strains (6995 and
CAP-67, respectively) were obtained from the The Centraal Bureau
voor Schimmelcultures (Utrecht, The Netherlands). All fungal cul-
tures were maintained by serial passages on Sabouraud agar (Bio-
Mèrieux, Lyon, France). The cells were harvested by suspending a
single colony in sterile saline, washed twice, counted in a hemocy-
tometer and adjusted to the desired concentration.
trans-13: 1H NMR (CDCl3) d 0.89 (3H, t, J = 6.7 Hz, CH2CH3),
1.23–1.60 (4H, m, CH2CH2CH2CH3), 1.70–1.92 (2H, m,
CH2CH2CH2CH3), 3.26 and 3.29 (1.92 and 1.08H, respectively, s,
NCH3), 3.60–3.95 (3H, m, CHHCHCH2OAr), 4.07–4.23 (1H, m,
CH2CHCH2OAr), 4.37–4.55 (1H, m, OCHCO), 4.44 (2H, s, CH2N),
6.21–6.38 (2H, m, benzoxazine H-5 and H-7), 6.78 and 6.83 (0.36
and 0.64H, respectively, d, J = 8.6 Hz, benzoxazine H-8), 7.01 and
7.04 (each 1H, br s, imidazolic H), 7.20 (1H, dd, J = 8.5 and 2.0 Hz,
2,4-dichlorophenyl H-5), 7.42 (1H, d, J = 2.0 Hz, 2,4-dichlorophenyl
H-3), 7.60 (1H, d, J = 8.5 Hz, 2,4-dichlorophenyl H-6), 7.61 (1H, br s,
imidazolic H); 13C NMR (CDCl3) d: 14.10 (CH2CH2CH2CH3), 21.55
(CH2CH2CH2CH3), 26.00 (CH2CH2CH2CH3), 28.70 (NCH3), 30.33
(CH2CH2CH2CH3), 51.84 (CH2N), 67.48 (CH2CHCH2OAr), 75.50
(CH2CHCH2OAr), 81.38 (OCHCO), 104.40 (benzoxazine C-5),
108.20 (OCO), 111.05 (benzoxazine C-8), 111.40 (benzoxazine C-
7), 120.90 (NCHCHN), 127.40 (2,4-dichorophenyl C-5), 128.00
(NCHCHN), 129.50 (2,4-dichorophenyl C-6), 129.70 (benzoxazine
C-10), 131.05 (2,4-dichorophenyl C-3), 133.30 (2,4-dichorophenyl
C-2), 133.50 (2,4-dichorophenyl C-1), 136.01 (2,4-dichorophenyl
C-4), 138.55 (NCHN), 140.00 (benzoxazine C-9), 157.70 (benzox-
azine C-6), 168.50 (OCH2CO); HPLC-MS: 546.1 (M+). Anal. Calcd
for C27H29Cl2N3O5: C, 59.35; H, 5.35; N, 7.69. Found: C, 59.40; H,
5.35; N, 7.67.
7.3. Antifungal susceptibility testing
Broth microdilution testing was performed according to NCCLS
document M27-A.28 Briefly, serial twofold dilutions of synthetic
compounds, FLU, or KTZ were done in RPMI 1640 with morphol-
inepropanesulfonic acid (MOPS) (Sigma Chemical Co., St. Louis,
Mo.) buffered to pH 7.0. Dilutions were prepared in 96 well U-bot-
tom microplates ranging from 1 mg to 0.00006 lg. Yeasts, sus-
pended in RPMI 1640/MOPS, were added at the concentration of
5 Â 104 CFU. The plates were incubated at 37 °C and the minimum
inhibitory concentration (MIC), defined as the lowest concentra-
tion of the compound that completely inhibited the growth of
the yeast, was determined by visual reading at 24 and 48 h.
7.4. Mice and Infection
6. Molecular modeling
Female, 8–10 weeks old, outbred CD1 mice were obtained from
Harlan Nossan Laboratories (Milan, Italy) and housed at the Animal
Facilities of the University of Perugia, Perugia, Italy. Procedures
involving animals and their care were conducted in conformity
with national and international laws and policies. For systemic
The inhibitors were built using the sketch module of Cerius-2,25
and minimized using the Universal force-field v.1.226 adopting the
Smart Minimizer protocol of the Open Force Field module (OFF).
Atomic charges were calculated using the semi-empirical Mopac/
AM1 method. Docking experiments were carried out using the Li-
gand Fit module of Cerius-2. During these calculations, the atomic
coordinates of the imidazole ring of all compounds were con-
strained to the relative coordinates of the imidazole ring as ob-
served in the crystal structure of MT-CYP51 in complex with
fluconazole (pdb code: 1ea1).
The Universal force-field 1.2 and the Smart Minimizer protocol
of the Open Force Field module (OFF) were used to optimize the
geometry of the complexes resulting from docking experiments.
During the minimization protocol, the High Convergence option
was used as criteria to stop the minimization cycles. After the opti-
mization of the binding geometry, all compounds showed a con-
served distance of 2.4 Å between the N-3 of the imidazole group
and the iron of the heme which is in agreement with the relative
distance (2.3 Å) observed in the crystal structure of MT-CYP51
bound to FLU.
infection, mice were infected intravenously on day
0 with
2.5 Â 105 blastospores of the highly virulent C. albicans CA-6 strain
and untreated or treated 2 h before challenge and once a day for 6
consecutive days after challenge with the synthetic compounds,
KTZ or FLU (all at 10 mg/kg body weight, i.p.). 2% ethanol solution
(0.2 mL, i.p.) was used as a negative control. In selected experi-
ments mice were inoculated with serum (0.3 mL/mouse, i.p.) from
mice untreated or treated for 1 h with cis-13, trans-13, FLU, or 2%
ethanol solution, 1 h before C. albicans systemic challenge
(7 Â 105/mouse). Infected animals were monitored for survival
and organ clearance. Quantification of fungal growth, 7 days after
infection, was assessed by counting the number of colony forming
units recovered after plating serial dilutions of kidney homoge-
nates onto Sabouraud agar. The experiments were repeated three
times by using 4 animals/experimental group.
7.5. Histological analysis
7. Micology
For histology, kidneys were excised 7 days after infection and
immediately fixed in formalin. Sections (1.5 lm) of paraffin-embed-
7.1. Reagents
ded tissues were stained using the periodic acid-Schiff (PAS) proce-
dure, which detects neutral and acid polysaccharides. On PAS
stained sections, C. albicans blastospores and hyphae stain red.
The liophylized synthetic compounds were dissolved in 20%
ethanol to the concentration of 10 mg/ and stored at À20 °C until
use. For in vitro and in vivo experiments, thawed compounds were
further diluted to the desired concentration. FLU and KTZ were ob-
tained from Pfizer (Pfizer Italia S.r.l., Latina, Italy).
7.6. Statistical analysis
Student’s t test was used to determine the statistical signifi-
cance of differences in organ clearance assays. Survival data were
analyzed by the Mantel-Cox logrank test. A value of p < 0.05 was
taken as significant.
7.2. Microorganisms
The different Candida species (C. albicans, C. krusei, C. glabrata
and C. parapsilosis) were isolated from clinical specimens in the
laboratories of the Microbiology Section, University of Perugia.
For experimental systemic candidiasis, mice were inoculated with
the highly virulent C. albicans strain CA-6.27 The agerminative, low
Acknowledgements
We acknowledge Claudia Silva and Federica Vacondio, Univer-
sity of Parma, for technical support in analytical determination.

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15. Rupp, B.; Raub, S.; Marian, C.; Höltje, H. D. J. Comput. Aided Mol. Des. 2005, 19,
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