Identification of inhibitors of drug-resistant Candida albicans strains from a library of bicyclic peptidomimetic compounds

Article abstract of DOI:10.1021/jm901734u

The screening of a library of small molecule peptidomimetics toward secreted aspartic proteinase-2 (SAP2) of Candida albicans allowed us to identify two compounds that showed in vitro inhibitory potency comparable to pepstatin A. In an experimental model of vaginal candidiasis, the two candidate compounds were as active as a therapeutic dose of fluconazole. Importantly, this activity was fully preserved when the challenger was a fluconazole-resistant strain of the fungus. Altogether, our data demonstrate SAP2 as a valid C. albicans target for the development of new drugs against this important human pathogen.

Full text of DOI:10.1021/jm901734u

2502 J. Med. Chem. 2010, 53, 2502–2509
DOI: 10.1021/jm901734u
Identification of Inhibitors of Drug-Resistant Candida albicans Strains from a Library of Bicyclic
Peptidomimetic Compounds
Andrea Trabocchi,† Claudia Mannino,† Fabrizio Machetti,‡ Flavia De Bernardis,§ Silvia Arancia,§ Roberto Cauda,
Antonio Cassone,*,§ and Antonio Guarna*,†
†Department of Chemistry “Ugo Schiff”, University of Florence, Polo Scientifico e Tecnologico, Via della Lastruccia 13, 50019 Sesto Fiorentino,
Florence, Italy, ‡ICCOM- CNR, c/o Department of Chemistry “Ugo Schiff”, University of Florence, Polo Scientifico e Tecnologico,
Via della Lastruccia 13, 50019 Sesto Fiorentino, Florence, Italy, §Department of Infectious, Parasitic, and Immune-Mediated Diseases,
Istituto Superiore di Sanitaꢀ, Viale Regina Elena 299, 00161 Rome, Italy, and Department of Infectious Diseases,
Catholic University of the Sacred Heart, Largo A. Gemelli 8, 00168 Rome, Italy
Received November 23, 2009
The screening of a library of small molecule peptidomimetics toward secreted aspartic proteinase-2
(SAP2) of Candida albicans allowed us to identify two compounds that showed in vitro inhibitory
potency comparable to pepstatin A. In an experimental model of vaginal candidiasis, the two candidate
compounds were as active as a therapeutic dose of fluconazole. Importantly, this activity was fully
preserved when the challenger was a fluconazole-resistant strain of the fungus. Altogether, our data
demonstrate SAP2 as a valid C. albicans target for the development of new drugs against this important
human pathogen.
Introduction
C. albicans expresses 10 distinct SAP genes (SAP1-10) in
vitro and in vivo, but there is evidence that different SAP
isoenzymes have different functions and can be differently
expressed in different infections (e.g., mucosal versus systemic)
or even at different stages of the same infection.7 In particular,
SAP2 is one of the most expressed enzymes implicated in host
invasion. It has been recognized as a crucial virulence factor
for vaginal infection,8 and both reversible and irreversible
inhibitors have been reported.9
With the aim of discovering new molecules active against
drug-resistant C. albicans, we envisaged the possibility of
targeting SAP2 with small-molecule peptidomimetic inhibi-
tors. An efficient method for the generation of a library of
peptidomimetics based on the 6,8-dioxa-3-azabicyclo[3.2.1]-
octane scaffold was recently reported by our group. This
method consists of a collection of amides obtained by the
parallel combination of bicyclic methyl esters with various
amines through a solvent-free synthetic reaction.10 The avail-
ability of a wide variety of primary and secondary amines
allowed us to synthesize a large and highly diverse small-
molecule library based on this bicyclic scaffold. Dipeptide
isosteres are particularly attractive because of the bicyclic
acetal portion acting as a potential transition state analogue
in the interaction with enzyme active sites.
Candida albicans is an opportunistic fungal pathogen that
causes severe mucosal and systemic infections especially in
immunocompromised subjects.1 Although a certain number
of antifungal agents are available, the need for new drugs
against C. albicans is escalating because of both the wide-
spread occurrence of mucosal infections caused by this fungus
and the development of resistance against available drugs.2 In
fact, despite drug availability, C. albicans ranks as a highly
frequent cause of morbidity, cost of hospitalization, and
mortality.3 Although the ability to cause disease is likely a
complex process involving multiple interactions between
Candida and the host, secretory aspartic proteinases (SAPsa)
appear to be a major determinant in the virulence of this
fungus, allowing it to adhere and invade host tissues.4 There-
fore, this family of enzymes offers a potential target for drug
intervention in infections, mostly where current treatments
fail owing to resistance to available drugs.5 The recognized
role of aspartic proteinase inhibitors for AIDS treatment and
the homology between HIV and Candida proteinases6 add
further interest to this approach.
*To whom correspondence should be addressed. For A.C.: phone,
þ39 06 49387113; fax, þ39 06 49387183; e-mail, antonio.cassone@
iss.it. For A.G.: phone, þ39 055 4573481; fax, þ39 055 4573569; e-mail,
antonio.guarna@unifi.it.
Results and Discussion
a Abbreviations: SAP, secreted aspartic proteinase; AIDS, acquired
immune deficiency syndrome; HIV, human immunodeficiency virus;
BTK, bicycles from L-tartaric acid and amino ketones; BTG, bicycles
from L-tartaric acid and glycine; BtG, bicycles from D-tartaric acid and
glycine; BTF, bicycles from L-tartaric acid and L-phenylalanine (the O in
parentheses refers to the presence of a carbonyl group at the C-2 position
of the bicyclic scaffold); OD, optical density; DMF, dimethylforma-
mide; DCM, dichloromethane; BSA, bovine serum albumin; SD, stan-
dard deviation; YPD, yeast peptone dextrose; CFU, colony forming
unit.
For the first series of experiments, 17 pools, each consisting
of 2-5 compounds, were selected to screen for in vitro
inhibition of SAP2 activity. The pools were assembled in a
way such that each pool contained similar scaffolds (Chart 1)
decorated with different substituents at position 7 (Chart 2).
The selected scaffolds differed either by the presence of the
carbonyl group at position 2 or by hydrophobic substituents
at the nitrogen atom and at positions 4 and 5.
r
pubs.acs.org/jmc
Published on Web 02/25/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2503
Table 1. Selected Pools for the First Screening to SAP2
Chart 1. Selected Pools of Compounds Containing Different
Bicyclic Scaffolds
no. of
elements
%
inhibitiona
normalized %
inhibitionb
pool
compd
1a, 1i, 1k
1m, 1s, 1t, 1o
2-NH2, 2b, 2c, 2j, 2h
2p, 2s
I
II
III
IV
V
VI
VII
VIII
IX
3
4
5
2
3
4
4
4
5
3
2
3
5
4
4
2
2
0
19
18
19
92
87
13
6
0
5
4
10
31
22
3
2m, 2n, 2t
2c, 2a, 2e, 2k
3d, 3f, 3g, 3h
3l, 3m, 3n, 3y
4g, 4c, 4a, 4e, 4k
4m, 4n, 4t
2
42
40
73
87
87
12
23
80
57
8
X
XI
13
36
29
17
3
4d, 4i
4f, 4h, 4b
4p, 4o, 4s, 4y, 4x
5e, 5d, 5g, 5c
5l, 5n, 5o, 5y
6m, 6q
XII
XIII
XIV
XV
XVI
6
40
28
XVII 6r, 6z
a The inhibitory potency was determined using the ΔOD value,
compared with the difference between ΔOD control proteinase -
ΔOD (control proteinase þ peptsatin A), which was taken as the
100% inhibition value. The concentration of each compound in the
mixtures was 10 μM. b This value corresponds to the % inhibition
normalized to the value of a single compound at 10 μM.
Chart 2. Primary and Secondary Amines Used as the X Com-
ponent
Scheme 1. Synthesis of Leucine-Derived Peptidomimetics of
Pools XVI and XVII
XV. Compounds of pools III and IV showed moderate to low
inhibitory potency, whereas pools V and VI, containing the
same scaffold, showed good inhibitory activity. These results
suggested that the above scaffold could be a good template for
ligand discovery. They also indicated the C-7 substituents in
pools V and VI as good variants for further analysis. The lack
of efficacy of pools VII and VIII, in conjunction with the
inactivity of pools XIV and XV, provided preliminary evi-
dence that the carbonyl group at C-2 of the scaffold is crucial
for SAP2 binding affinity. Pools IX and X-XIII contain the
BTF(O) scaffold, which is derived from the combination of
phenylalanine and tartaric acid derivatives. These pools
showed interesting inhibitory potency, suggesting the compat-
ibility of the phenyl ring at position 4 of the scaffold.
The variation of the substituent at the nitrogen atom was
taken into account in generating the pools XVI and XVII with
a Leu isostere at position 3 of the BTG(O) scaffold (Table 1).
The fair inhibitory activity of these pools indicated the
possibility of the modulation at the nitrogen atom of the
bicyclictemplatewithother nonpolar substituents. The results
obtained with pools V and VI and with pools X and IX (which
Compounds belonging to pools XVI and XVII (Table 1)
were prepared using L-leucine methyl ester as the starting
material to build the bicyclic scaffold (Scheme 1). Briefly,
leucine methyl ester hydrochloride (7) was treated with bro-
moacetaldehyde dimethylacetal at 120 °C in DMF and in the
presence of Et3N and KI. Amine 8 was then converted into
amide 9 through a coupling reaction with (2S,3S)-di-O-acet-
yltartaric anhydride, which was subsequently treated with
thionyl chloride in MeOH to give the cyclic acetal 10. Final
trans-acetalization to give the bicyclic scaffold 11 was
achieved by treating 10 in refluxing toluene for 30 min in the
presence of H2SO4 over silica gel. Amides for pools XVI and
XVII were synthesized by heating methyl ester 22 in the
presence of neat amine at 60 °C for 18 h.
As reported in Table 1, pools I and II, differing by the
substitution level at C-7, did not show any relevant inhibitory
potency with respect to pepstatin A, indicating a detrimental
effect of the phenyl ring at position 5 of the bicyclic scaffold.
This detrimental effect was also noticed for pools XIV and

2504 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6
Trabocchi et al.
contained, respectively, a different template but similar sub-
stituents) indicated that the scaffold BTG(O), together with
the selected functional groups at position 7 of the molecule, is
endowed with better structural requisites. Specifically, pool
V (inhibition of 31%) showed better activity than pool
X (inhibition of 13%), and pool VI showed higher potency
(inhibition of 22%) as compared to pool IX (inhibition
of 8%).
On the basis of initial evidence that a specific set of scaffolds
may express a fair inhibitory activity, single compounds were
screened to identify the best flanking units for scaffold
decoration. This step also served to obtain more insights into
the structure-activity relationship, hence facilitating the se-
lection of the best hit compounds for in vivo experiments. The
molecules possessing the N-benzyl-BTG(O) scaffold 2 were
thus selected for the screening of single compounds and for
assessing the preferred substituents at position 7 (Figure 1 and
Table 2).
13, respectively. Furthermore, the presence of hydroxylic
groups around position 7 indicated a favorable effect, as
shown by the remarkable inhibitory potency of 14 and 15,
containing the glycinol and leucinol moiety, respectively. The
discrepant inhibitory activities of compounds 14-23, which
contain diverse amino alcohol derived substituents, suggested
that the inhibitory potency could be affected by the hydro-
phobic content. The stereochemistry of the substituents did
not influence the inhibitory potency, as suggested by the
comparable inhibition values found for compounds 18, 19
and 21, 22, corresponding to both enantiomers of proline and
phenylalanine, respectively. More interestingly, N-benzyla-
tion of the leucinol derivative caused a significant loss of
inhibition, as shown by the comparison of the activity of 23
(35%) with that of 15 (71%).
The systematic evaluation of the library members for SAP2
inhibition resulted in the identification of compounds 2s, 13,
14, and 15 as those possessing an inhibitory potency above
70% at 20 μM, thus being comparable to pepstatin A activity.
The quantification of the inhibitory potency of two selected
compounds indicated molecule 13 as the compound posses-
sing slightly better binding capability than compound 15,
both expressing an IC50 value in the low micromolar range
(Table 3).
All compounds showed moderate to good inhibitory po-
tency, compared to pepstatin A. Among secondary amines,
N-carboethoxypiperazine- and piperidine-derived compounds
showed the highest inhibitory potency, as found for 2s and
Finally, the stereochemical scanningof all the stereoisomers
of compounds 13 and 15 indicated a slight modulation of
SAP2 inhibition by the stereochemical arrangement of the
pharmacophoric elements, since moderate to good inhibition
Figure 1. Selected scaffold for SAP2 inhibition.
Table 2. Array of Bn-BTG(O)-X Scaffolds toward SAP2
a The inhibitory potency was determined using the ΔOD value, compared with the difference between ΔOD control proteinase - ΔOD (control
proteinase þ peptsatin A), which was taken as the 100% inhibition value. The compounds were screened at 20 μM.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2505
Table 3. Concentration-Dependent Inhibitory Potency of 13 and 15
toward SAP2
to draw a preliminary structure-activity relationship of
BTAa scaffolds, thus identifying some elements favoring or
disfavoring SAP2 inhibitory activity. Specifically, it was
found that the CO group at position 2 of the scaffold is crucial
for binding activity, whereas position 7 can lead to a modula-
tion of the inhibitory potency. The two enantiomeric BTG(O)
and BtG(O) scaffolds did not determine dramatic differences
in enzyme recognition, as demonstrated by the good inhibi-
tion expressed by pools XVI andXVII containing the enantio-
meric BtG(O) scaffold. This suggests a “pseudosymmetric”
interaction in the catalytic site of SAP2. BTF(O) scaffold
showed some inhibitory potency, though highly influenced by
the substituents at position 7 of the scaffold, thus indicating
the possibility of placing a hydrophobic group at position
4-exo of the scaffold. On the contrary, the phenyl ring at the
vicinal position 5 determined a loss of activity, suggesting a
strict topological requirement for the aromatic group.
% inhibitiona
concn, μM
13
80
15
20
10
74
61
67
50
19
0
2
6.9
0.2
0.02
1.4
a The inhibitory potency was determined using the ΔOD value,
compared with the difference between ΔOD control proteinase - ΔOD
(control proteinase þ peptsatin A), which was taken as the 100%
inhibition value.
Table 4. Stereochemical Scanning of 13 and 15 toward SAP2a
In vivo experiments performed with the rat model of
estrogen-dependent C. albicans vaginitis showed that the
two hit compounds were the most effective inhibitors of the
experimental infection. In particular, they caused a rapid
decay of vaginal C. albicans burden. The kinetics of fungus
clearance in rats intravaginally treated with compounds 13
and 15 was similar to the kinetics of the fungus clearance in
rats treated with the comparator SAP2 inhibitor, pepstatin A.
It was also similar to the kinetics of infection in rats treated
with fluconazole (Table 5 and Figure 2). More importantly,
the two compounds showed marked acceleration of fungus
clearance in rats challenged with the fluconazole-resistant
AIDS68 strain of C. albicans, similar to the activity
shown against the fluconazole-susceptible strain of the fungus
(Table 6 and Figure 3).
Conclusions
In conclusion, the screening of a library of bicyclic pepti-
domimetics for inhibition of SAP2 enzyme allowed us toselect
two hit compounds possessing in vitro activity comparable to
pepstatin A, a prototypal inhibitor of aspartic proteinases. In
a model of rat vaginal infection caused by fluconazole-sus-
ceptible or -resistant C. albicans strains, the two leads were
highly effective, demonstrating the potential of the two pepti-
domimetics to cure both drug-susceptible and drug-resistant
infection.
No precise insight into the mechanism of inhibition has
been addressed here; therefore, it remains unknown whether
our peptidomimetic inhibitors of SAP2 would express similar
inhibitory activity against other members of SAP family.13
However, some degree of inhibition of at least SAP1 and
SAP3 activity is likely, given: (i) the high similarity of SAP2 to
SAP1 and SAP3; (ii) the dominant role, as virulence factor,
played by the latter two SAPs, together with SAP2, in our rat
vaginits model.13
Overall, the results of this library screening allowed us to
identify a new scaffold that is of interest in the research of
novel aspartic proteinase inhibitors. The two peptidomimetics
identified here as inhibitors of fluconazole-resistant C. albi-
cans strains are being considered for subsequent trials in the
drug development process.
a The inhibitory potency was determined using the ΔOD value,
compared with the difference between ΔOD control proteinase -
ΔOD (control proteinase þ peptsatin A), which was taken as the
100% inhibition value. The compounds were screened at 20 μM.
was observed with all the compounds (Table 4). The two hit
compounds 13 and 15 identified in the first run of screening
were confirmed as the best stereoisomers in the hit identifica-
tion for subsequent in vivo trials, as demonstrated by inhibi-
tion values of 24 compared to 13 and of 25-27 compared
to 15.
Experimental Section
Chemistry. Pools of bicyclic compounds were prepared fol-
lowing the reported methods for the generation of BTK and
BTAa scaffolds.11 Libraries from libraries of peptidomimetics
The optimization of enzymatic assays carried out on dif-
ferent pools of structurally analogous compounds allowed us

2506 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6
Trabocchi et al.
Table 5. Acceleration of Candida SA40 Clearance in Rats Intravaginally Treated with 13 and 15 after Challenge (1, 24, 48 h)a
days
SA40 þ 15
>100
SA40 þ 13
100 ( 0
SA40 þ pepstatin A
>100
SA40
SA40 þ fluconazole
100 ( 0
0
>100
>100
>100
1
70.0 ( 1.3
57.6 ( 1.4
39.2 ( 3
30.6 ( 1.8
14.4 ( 1.6
8.0 ( 1.5
1.2 ( 0.7
61 ( 2.6
58 ( 1.8
35.8 ( 2.2
28.8 ( 1.2
14.0 ( 2.3
4.2 ( 2.1
2.2 ( 0.9
56.8 ( 2
51.0 ( 1.2
32.4 ( 2.5
28 ( 1.5
9.4 ( 1.4
5.0 ( 1.3
0
53.5 ( 4.4
49.7 ( 4.1
25.7 ( 1.7
11.7 ( 0.8
5.0 ( 2.4
1.25 ( 0.9
0 ( 0
2
5
80.0 ( 2.6
66.0 ( 2.1
26.2 ( 1.8
12.8 ( 1.2
5.8 ( 1.6
7
14
21
28
a All values are CFU/mL ꢀ1000. SA40: untreated control. Starting day 1, all differences between 13- and 15-treated and untreated control are statisti-
cally significant (P < 0.01, Mann-Withney U test). No statistically significant difference is detected between pepstatin A- and 13- or 15-treated rats.
removed, and the crude 10 was isolated as a yellow oil and used
without further purification in the next step. A solution of 10
(1.63 g, 4.7 mmol) in toluene (8 mL) was quickly added to a
refluxing suspension of SiO2/H2SO4 (1 g), prepared as re-
ported,12 in toluene (12 mL). The mixture was allowed to react
for 30 min, and then one-third of the solvent was distilled off.
The hot reaction mixture was filtered through a pad of NaHCO3
and after evaporation of the solvent, the crude product was
purified by flash chromatography (silica gel, EtOAc/petroleum
ether 1:2), affording 11 as a white solid (730 mg, 50% yield over
three steps). [R]2D4 þ22.0 (c 1.0, MeOH). 1H NMR (CDCl3,
200 MHz): δ 5.88 (d, J = 2 Hz, 1 H), 5.09 (t, J = 8 Hz, 1 H), 4.87
(s, 1 H), 4.59 (s, 1 H), 3.72 (s, 3 H), 3.64 (s, 3 H), 3.50 (dd, J =
12.0, 2.0 Hz, 1 H), 3.11 (dd, J = 12.0, 2.0 Hz, 1 H), 1.67-1.60 (m,
2 H), 1.46-1.32 (m, 1 H), 0.88 (s, 3 H), 0.84 (s, 3 H). 13C NMR
(CDCl3, 200 MHz): δ 170.8 (s), 168.7 (s), 165.6 (s), 100.0 (d), 77.8
Figure 2. Vaginal infection with C. albicans SA40 in rats intravag-
inally treated with 13 and 15 after challenge (1, 24, 48 h): ([) SA40 þ
15; (9) SA40 þ 13; (2) SA40 þ pepstatin A; (ꢀ) SA40; (/) SA40 þ
fluconazole.
(d), 77.3 (d), 52.8 (d), 52.4 (q), 52.3 (q), 48.1 (t), 36.6 (t), 24.7 (d),
23.3 (q), 21.3 (q). MS m/z 315 (11), 256 (100), 240 (4). Anal.
Calcd for C14H21NO7 (315.33): C, 53.33; H, 6.71; N, 4.44.
Found: C, 53.09; H, 6.58; N, 4.59.
40-Methyl-(20S)-20-[(1R,5S,7S)-7-(morpholine-4-carbonyl)-2-
oxo-6,8-dioxa-3-azabicyclo[3.2.1]oct-3-yl]pentanoic Acid Methyl
Ester (6m). Compound 11 (100 mg, 0.32 mmol) and morpholine
(0.55 mL, 6.3 mmol) were stirred at 60 °C overnight. The
reaction mixture was then diluted with methanol and eluted
through Amberlyst 15. After solvent evaporation, the crude
product was purified by column chromatography (silica gel,
DCM/MeOH 20:1) giving pure 6m (95 mg, 65% yield) as a
yellow oil. [R]D22 þ29.0 (c 1.0, CHCl3). 1H NMR (CDCl3,
200 MHz): δ 5.86 (d, J = 2 Hz, 1 H), 5.16 (s, 1 H), 5.06 (dd,
J = 8 Hz, 1 H), 4.76 (s, 1 H), 3.70 (s, 3 H), 3.67-3.52 (m, 9 H),
3.15 (d, J = 12 Hz, 1 H), 1.75-1.67 (m, 2 H), 1.53-1.43 (m, 1 H),
0.94 (d, J = 6 Hz, 3 H), 0.92 (d, J = 6 Hz, 3 H). 13C NMR
(CDCl3, 200 MHz): δ 170.8 (s), 99.8 (d), 84.6 (d), 78.0 (d), 66.8
(t), 66.6 (t), 52.8 (q), 52.5 (d), 48.6 (t), 46.0 (t), 42.7 (t), 36.8 (t),
24.8 (d), 23.3 (q), 21.6 (q). MS m/z 370 (14), 311 (60), 283 (19),
168 (100). IR (CHCl3) 2932, 1735, 1668 cm-1. Anal. Calcd for
C17H26N2O7 (370.41): C, 55.13; H, 7.08; N, 7.56. Found: C,
55.17; H, 7.12; N, 7.52.
achieved by means of functionalization of the carboxylic group
at position 7 have been reported.10 Combustion analysis was
used to determine purity. All tested compounds were >95%
pure.
2-(2,2-Dimethoxyethylamino)-4(S)-methylpentanoic Acid
Methyl Ester (8). A solution containing L-leucine methyl ester
hydrochloride 7 (2.9 g, 16 mmol), bromoacetaldehyde dimethy-
lacetal (1.9 mL, 2.7 g, 16 mmol), Et3N (6.7 mL, 48 mmol), and a
catalytic amount of KI in DMF (190 mL) was stirred at 120 °C
for 3 days. The reaction mixture was concentrated under
reduced pressure, diluted with water, and extracted with
DCM. The organic layer was then washed with brine, dried
over Na2SO4, and evaporated. The crude product was purified
by column chromatography (silica gel, EtOAc/petroleum ether
1:1) to afford compound 8 as a yellow oil (1.2 g, 32% yield). [R]D24
1
-3.32 (c 1.0, CHCl3). H NMR (CDCl3, 200 MHz): δ 4.38 (t,
J = 6 Hz, 1 H), 3.65 (s, 3 H), 3.30 (s, 3 H), 3.29 (s, 3 H), 3.24 (t,
J = 6 Hz, 1 H), 2.68 (dd, J = 6 Hz, 1 H), 2.52 (dd, J = 6 Hz,
1 H), 1.71-1.55 (m, 2 H), 1.44-1.37 (m, 2 H), 0.86 (d, J = 4 Hz,
3 H), 0.83 (d, J = 4 Hz, 3 H). 13C NMR (CDCl3, 200 MHz): δ
175.9 (s), 103.6 (d), 59.9 (d), 54.0 (q), 53.1 (q), 51.7 (q), 49.3 (t),
42.8 (t), 25.0 (d), 22.8 (q), 22.5 (q). MS m/z 233 (0.5), 202 (7.2),
174 (33), 158 (14), 75 (100). IR (CHCl3) 2915, 1729, 1130, 1065
cm-1. Anal. Calcd for C11H23NO4 (233.30): C, 56.63; H, 9.94;
N, 6.00. Found: C, 57.09; H, 9.90; N, 6.14.
(2S)-2-[(1R,5S,7S)-7-(4-Phenylethylpiperazine-1-carbonyl)-2-
oxo-6,8-dioxa-3-azabicyclo[3.2.1]oct-3-yl]-4-methylpentanoic Acid
Methyl Ester (6q). Compound 6q was prepared as reported for
6m starting from compound 11 (100 mg, 0.32 mmol) and 1-
phenylethylpiperazine (1.2 mL, 6.3 mmol), giving the pure
product (89 mg, 59% yield) as a yellow oil. [R]2D5 þ21.3 (c 0.9,
1
CHCl3). H NMR (CDCl3, 200 MHz): δ 7.33-7.18 (m, 5 H),
(1S,5R,7S)-3-(1-Methoxycarbonyl-3(S)-methylbutyl)-2-oxo-6,
8-dioxa-3-azabicyclo[3.2.1]octane-7-carboxylic Acid Methyl Es-
ter (11). To a suspension of (2S,3S)-2,3-di-O-acetyltartaric
anhydride (1 g, 4.7 mmol) in dry DCM (4.5 mL) was added, at
0 °C and under a nitrogen atmosphere, a solution of 8 (1 g, 4.7
mmol) in dry DCM (2.5 mL). The reaction mixture was stirred at
room temperature overnight. After evaporation of the solvent,
crude 9 was dissolved in MeOH (8 mL), and thionyl chloride
(292 μL, 4 mmol) was added dropwise at 0 °C. The mixture was
then allowed to reach 60 °C and stirred for 2 h. The solvent was
5.88 (d, J = 2 Hz, 1 H), 5.17 (s, 1 H), 5.09 (dd, J = 8.0, 6.0 Hz,
1 H), 4.81 (s, 1 H), 3.72 (s, 3 H), 3.78-3.63 (m, 4 H), 3.57 (dd,
J = 12.0, 2.0 Hz, 1 H), 3.18 (d, J = 12.0 Hz, 1 H), 2.88-2.80 (m,
2 H), 2.70-2.58 (m, 6 H), 1.78-1.70 (m, 2 H), 1.53-1.25 (m,
1 H), 0.98 (d, J = 6.0 Hz, 3 H), 0.94 (d, J = 6.0 Hz, 3 H). 13
C
NMR (CDCl3, 200 MHz): δ 170.6 (s), 166.5 (s), 164.8 (s), 138.5
(s), 128.4 (d), 128.3 (d), 126.1 (d), 99.5 (d), 77.7 (d), 76.9 (d), 59.5
(t), 52.6 (q), 52.4, 52.2 (t), 51.9 (d), 48.2, 44.3 (t), 41.3 (t), 36.5 (t),
32.4 (t), 24.4 (d), 22.8 (q), 21.2 (q). MS m/z 414 (1), 382 (95),
56(100). IR (CHCl3) 2923, 1740, 1672 cm-1. Anal. Calcd. for

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2507
Table 6. Acceleration of Candida AIDS68 Clearance in Rats Intravaginally Treated with 13 and 15 after Challenge (1, 24, 48 h)a
days
AIDS68 þ 15
AIDS68 þ 13
AIDS68 þ pepstatin A
AIDS68 þ fluconazole
AIDS68
0
>100 ( 0
71.8 ( 1.3
62.6 ( 1.5
40.6 ( 1.4
23.2 ( 1.4
12.8 ( 1.2
3.4 ( 1.7
0 ( 0
100 ( 0
>100 ( 0
58.4 ( 1.0
52.0 ( 1.3
37.2 ( 1.6
30.0 ( 1.2
19.8 ( 0.8
3.8 ( 1.9
0 ( 0
>100 ( 0
100 ( 0
>100 ( 0
100 ( 0
100 ( 0
71.0 ( 1.6
50.0 ( 3.5
25.0 ( 1.6
10.7 ( 1.6
7.7 ( 3
1
61.6 ( 1.2
55.4 ( 1.7
26.8 ( 1.0
22.8 ( 2.2
11.2 ( 1.1
2.2 ( 0.9
0 ( 0
2
93.0 ( 4.3
61.0 ( 2.5
44.0 ( 2.9
18.7 ( 3.8
11.7 ( 0.7
0 ( 0
5
7
14
21
28
a All values are CFU/mL ꢀ1000. AIDS68: untreated control. Starting day 1, all differences between 13- and 15-treated and untreated control are
statistically significant (P < 0.01, Mann-Withney U test). Also, starting from day 1 postchallenge, all values of 13-, 15-, and pepstatin A-treated rats
denote statistically significantdifferences compared to same-day values of AIDS68-treated rats. No statistically significant difference is detected between
pepstatin A- and 13- or 15-treated rats.
36.8 (t), 24.8 (d), 23.3 (q), 21.6 (q). MS m/z 459 (10), 400 (1), 330
(1), 175 (19), 91 (100). IR (CHCl3) 2940, 1740, 1672 cm-1. Anal.
Calcd for C24H33N3O6 (459.55): C, 62.73; H, 7.24; N, 9.14.
Found: C, 62.80; H, 7.32; N, 9.50.
(1S,5R,7R)-3-Benzyl-2-oxo-6,8-dioxa-3-azabicyclo[3.2.1]octane-
7-carboxylic Acid Benzyl-(1-hydroxymethyl-3-methylbutyl)amide
(23). Amide 23 was prepared as reported,10 starting from the
corresponding (1S,5R,7R)-bicyclic ester (40 mg, 0.144 mmol)
and N-benzyl-L-leucinol (300 mg, 1.44 mmol), giving the pure
product after flash chromatography (EtOAc/petroleum ether
2:1). [R]2D4 þ12.8 (c 1.2, CHCl3). 1H NMR (CDCl3, 200 MHz): δ
7.30-7.11 (m, 10 H), 6.57 (d, J = 2.1 Hz, 1 H), 4.94 (s, 1 H), 4.64
(s, 1 H), 4.47 (s, 2 H), 3.97-3.25 (m, 8 H), 3.02 (d, J = 12.5 Hz,
1 H), 1.55 (m, 1 H), 1.30 (m, 2 H), 0.82-0.80 (m, 6 H). 13C NMR
(CDCl3, 50 MHz): δ 168.8 (s), 165.6 (s), 135.2 (s), 129.0, 128.7,
128.2, 127.8, 99.8 (d), 79.4 (d), 77.7 (d), 65.2 (t), 61.5 (t), 50.9 (t), 50.0
(d), 48.2 (t), 40.0 (t), 25.0 (d), 23.2 (q), 22.2 (q). MS m/z452 (11), 219
(12), 105 (45), 91 (100). Anal. Calcd for C26H32N2O5 (452.54): C,
69.01; H, 7.13; N, 6.19. Found: C, 68.96; H, 7.08; N, 6.11.
(1S,5S,7S)-3-Benzyl-7-(piperidine-1-carbonyl)-6,8-dioxa-3-aza-
bicyclo[3.2.1]octan-2-one (24). Amide 24 was prepared as re-
ported for the enantiomer 13, starting from the corresponding
(1R,5S,7S)-bicyclic ester (40 mg, 0.144 mmol) and piperidine
(143 μL, 1.44 mmol), giving the pure product after flash
chromatography (EtOAc/petroleum ether 2:1). [R]2D4 -44.6 (c
1.3, CHCl3). 1H NMR (CDCl3, 200 MHz): δ 7.28-7.14 (m, 5 H),
5.77 (d, J = 1.8 Hz, 1H), 5.07 (s, 1 H), 4.86 (s, 1 H), 4.60 (part A
of AB system, J = 15.0 Hz, 1 H), 4.36 (part B of AB system, J =
15.0 Hz, 1 H), 3.43 (m, 3 H), 3.32 (dd, J = 12.1, 1.8 Hz, 1 H), 3.04
(d, J = 12.1 Hz, 1 H), 2.85 (d, J = 15.3 Hz, 1 H), 1.56 (m, 6 H).
Anal. Calcd for C18H22N2O4 (330.38): C, 65,44; H, 6,71; N, 8,48.
Found: C, 65.38; H, 6.65; N, 8.41.
(1R,5S,7S)-3-Benzyl-2-oxo-6,8-dioxa-3-azabicyclo[3.2.1]octane-
7-carboxylic Acid [1-Hydroxymethyl-3(S)-methylbutyl]amide (25).
Compound 25 was prepared as reported for the diastereomer 15,
starting from the corresponding (1R,5S,7S)-bicyclic ester
(40 mg, 0.144 mmol) by aminolysis with (S)-leucinol (190 μL,
1.44 mmol), giving the pure compound after flash chromatog-
raphy (EtOAc/petroleum ether 2:1). [R]2D3 -19.9 (c 1.3, CHCl3).
1H NMR (CDCl3, 200 MHz): δ 7.30-7.14 (m, 5 H), 6.52 (d, J =
7.7 Hz, 1 H), 5.80 (d, J = 2.2 Hz, 1 H), 4.99 (s, 1 H), 4.62 (s, 1 H),
4.50 (AB system, J = 15.1 Hz, 2 H), 3.97 (m, 1 H), 3.63 (dd, J =
11.0, 2.9 Hz, 1 H), 3.50 (dd, J = 11.0, 5.1 Hz, 1 H), 3.36 (d, J =
2.2 Hz, 1 H), 2.00 (br, 1 H), 1.56 (m, 1 H), 1.40 (m, 2 H), 0.89 (d,
J = 2.2 Hz, 3 H), 0.86 (d, J = 1.8 Hz, 3 H). 13C NMR (CDCl3,
50 MHz): δ 168.6 (s), 165.6 (s), 135.2 (s), 128.7 (d, 2 C), 127.8 (d,
2 C), 127.7 (d), 99.8 (d), 79.3 (d), 77.5 (d), 65.3 (t), 50.9 (t), 49.9
(d), 48.2 (t), 40.1 (t), 24.9 (d), 23.0 (q), 22.2 (q). MS m/z 362 (12),
332 (55), 219 (67), 92 (100). Anal. Calcd for C19H26N2O5
(362.42): C, 62.97; H, 7.23; N, 7.73. Found: C, 62.85; H, 7.28;
N, 7.68.
Figure 3. Vaginal infection with C. albicans AIDS68 in rats intra-
vaginally treated with 13 and 15 after challenge (1, 24, 48 h): ([)
AIDS68 þ 15; (9) AIDS68 þ 13; (2) AIDS68 þ pepstatin A; (ꢀ)
AIDS68; (/) AIDS68 þ fluconazole.
C25H35N3O6 (473.57): C, 63.41; H, 7.45; N, 8.87. Found: C,
63.28; H, 7.41; N, 8.79.
(2S)-2-[(1R,5S,7S)-7-(4-Methylpiperazine-1-carbonyl)-2-oxo-
6,8-dioxa-3-azabicyclo[3.2.1]oct-3-yl]-4-methylpentanoic Acid
Methyl Ester (6r). Compound 6r was prepared as reported for
6m starting from compound 11 (150 mg, 0.48 mmol) and 1-
methylpiperazine (1.06 mL, 9.5 mmol) to give the pure product
(128 mg, 72% yield) as a yellow oil. [R]2D5 þ28.1 (c 0.9, CHCl3).
1H NMR (CDCl3, 200 MHz): δ 5.85 (s, 1 H), 5.12 (s, 1 H), 5.05
(t, J = 8.0 Hz, 1 H), 4.77 (s, 1 H), 3.68 (s, 3 H), 3.62-3.51 (m,
5 H), 3.14 (d, J = 12.0 Hz, 1 H), 2.42-2.33 (m, 4 H), 2.72 (s, 3 H),
1.73-1.65 (m, 2 H), 1.49-1.42 (m, 1 H), 0.92 (d, J = 6.0 Hz,
3 H), 0.90 (d, J = 4.0 Hz, 3 H). 13C NMR (CDCl3, 200 MHz): δ
170.8 (s), 166.8 (s), 165.0 (s), 99.7 (d), 78.0 (d), 76.4 (d), 55.0, 54.6
(t), 52.8 (q), 52.5 (d), 48.6 (t), 46.1 (q), 45.4 (t), 42.3 (t), 36.9 (t),
24.8 (d), 23.3 (q), 21.6 (q). MS m/z 383 (23), 352 (2.4), 324 (9), 99
(55), 70 (100). IR (CHCl3) 2866, 1738, 1670 cm-1. Anal. Calcd.
for C18H29N3O6 (383.44): C, 56.38; H, 7.62; N, 10.96. Found: C,
56.22; H, 7.58; N, 11.01.
(2S)-2-[(1R,5S,7S)-7-(4-Benzylpiperazine-1-carbonyl)-2-oxo-
6,8-dioxa-3-azabicyclo[3.2.1]oct-3-yl]-4-methylpentanoic Acid Methyl
Ester (6z). Compound 6z was prepared as reported for 6m
starting from compound 11 (100 mg, 0.32 mmol) and 1-benzyl-
piperazine (1.1 mL, 6.3 mmol) to give the pure product (106 mg,
72% yield) as a yellow oil. [R]2D3 þ20.1 (c 1.1, CHCl3). 1H NMR
(CDCl3, 200 MHz): δ 7.42-7.27 (m, 5 H), 5.88 (s, 1 H),
5.25-5.05 (m, 2 H), 4.79 (s, 1 H), 3.71 (s, 3 H), 3.63-3.53 (m,
7 H), 3.16 (d, J = 11.6 Hz, 1 H), 2.51-2.45 (m, 4 H), 1.76-1.68
(m, 2 H), 1.55-1.25 (m, 1 H), 0.96 (d, J = 5, 3 H), 0.93 (d, J =
6.2 Hz, 3 H). 13C NMR (CDCl3, 200 MHz): δ 170.8 (s), 166.8 (s),
165.0 (s), 129.1 (d), 128.3 (d), 127.3 (d), 99.7 (d), 78.0 (d), 76.4
(d), 62.9 (t), 52.9 (q), 52.7, 52.7 (t), 52.5 (d), 48.5 (t), 45.5, 42.4 (t),
(1S,5R,7R)-3-Benzyl-2-oxo-6,8-dioxa-3-azabicyclo[3.2.1]octane-
7-carboxylic Acid [1-Hydroxymethyl-3(R)-methylbutyl]amide
(26). Compound 26 was prepared as reported for 25 starting

2508 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6
Trabocchi et al.
from the corresponding (1S,5R,7R)-bicyclic ester (45 mg, 0.16
mmol) and (R)-leucinol (210 μL, 1.60 mmol), giving the pure
compound after flash chromatography (EtOAc/petroleum ether
2:1) with the same spectroscopic data as for 27. [R]2D4 þ15.2
(c 1.2, CHCl3). Anal. Calcd for C19H26N2O5 (362.42): C, 62.97;
H, 7.23; N, 7.73. Found: C, 62.67; H, 7.18; N, 7.63.
(1R,5S,7S)-3-Benzyl-2-oxo-6,8-dioxa-3-azabicyclo[3.2.1]octane-
7-carboxylic Acid [1-Hydroxymethyl-3(R)-methylbutyl]amide
(27). Compound 27 was prepared as reported10 for the enantio-
mer 15, starting from the corresponding (1R,5S,7S)-bicyclic
ester (36 mg, 0.13 mmol) by aminolysis with (R)-leucinol
(168 μL, 1.30 mmol), giving the pure compound after flash
chromatography (EtOAc/petroleum ether 2:1) with the same
spectroscopic data as for 15. [R]2D4 þ16.7 (c 1.5, CHCl3). Anal.
Calcd for C19H26N2O5 (362.42): C, 62.97; H, 7.23; N, 7.73.
Found: C, 63.01; H, 7.26; N, 7.64.
Biology. The enzymatic screening of peptidomimetic li-
braries was carried out by an established spectrophotometric
method of measuring BSA hydrolysis by SAP2 and calculating
the inhibition percentage with respect to the reference SAP2
inhibitor pepstatin A, at a fixed concentration, as detailed
below.13
Proteinase Enzyme Assay. Spectrophotometric Method. The
inhibition of proteinase activity by the various compounds of
pool I was measured by a spectrophotometric assay in a
comparison with pepstatin A activity, at the same concentra-
tion. Each assay contained 50 μL of sample in 0.4 mL of 1%
(w/v) BSA in 50 mM sodium citrate, pH 3.2, and 50 μL of
protease solution (1 μg/mL). After 30 min at 37 °C, 1 mL of 10%
(w/v) trichloroacetic acid was added. The tubes were stored in
ice for 30 min and then centrifuged (3000g) for 10 min. The
absorbance of the supernatant was read at 280 nm. The control
was 1% BSA in citrate buffer. One unit of the enzyme catalyzed
a ΔA280 of 1 min-1. With the pure proteinase the assay was
Acknowledgment. The financial support received from the
ꢀ
Fondazione Roma, MIUR, Universita degli Studi di Firenze,
CINMPIS, Istituto Superiore di Sanita, and Programma
ꢀ
Nazionale AIDS (Ministero della Salute-Istituto Superiore
ꢀ
di Sanita) is gratefully acknowledged. The authors are also
grateful to the editorial staff of the Journal of Medicinal
Chemistry for helpful suggestions about paper presentation.
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