Synthesis of bicyclic molecular scaffolds (BTAa): An investigation towards new selective MMP-12 inhibitors

Article abstract of DOI:10.1016/j.bmc.2006.07.028

Starting from 3-aza-6,8-dioxa-bicyclo[3.2.1]octane scaffold (BTAa) a virtual library of molecules was generated and screened in silico against the crystal structure of the Human Macrophage Metalloelastase (MMP-12). The molecules obtaining high score were

Full text of DOI:10.1016/j.bmc.2006.07.028

Bioorganic & Medicinal Chemistry 14 (2006) 7392–7403
Synthesis of bicyclic molecular scaffolds (BTAa): An investigation
towards new selective MMP-12 inhibitors
Claudia Mannino,a,d Marco Nievo,a Fabrizio Machetti,c Athanasios Papakyriakou,d
Vito Calderone,d Marco Fragaid,e and Antonio Guarnaa,b,
*
aDepartment of Organic Chemistry ‘‘U. Schiff’’, University of Florence, 50019 Sesto Fiorentino (FI), Italy
bLaboratory of Design, Synthesis and Study of Biologically Active Heterocycles (HeteroBioLab) Via della Lastruccia 13,
50019 Sesto Fiorentino (FI), Italy
cIstituto di chimica dei composti organometallici del CNR c/o Dipartimento di Chimica Organica ‘U. Schiff ’, Firenze, Italy
dCenter of Magnetic Resonance (CERM), University of Florence, Via Sacconi 6, 50019 Sesto Fiorentino (FI), Italy
eDepartment of Agricultural Biotechnology (DIBA), University of Florence, Via Maragliano 75, 50144 Florence, Italy
Received 6 March 2006; revised 3 July 2006; accepted 10 July 2006
Available online 8 August 2006
Abstract—Starting from 3-aza-6,8-dioxa-bicyclo[3.2.1]octane scaffold (BTAa) a virtual library of molecules was generated and
screened in silico against the crystal structure of the Human Macrophage Metalloelastase (MMP-12). The molecules obtaining high
score were synthesized and the affinity for the catalytic domain of MMP-12 was experimentally proved by NMR experiments. A
BTAa scaffold 20 having a N-hydroxyurea group in position 3 and a p-phenylbenzylcarboxy amide in position 7 showed a fair inhi-
bition potency (IC50 = 149 lM) for MMP-12 and some selectivity towards five different MMPs. These results, taken together with
the X-ray structure of the adduct between MMP-12, the inhibitor 20 and the acetohydroxamic acid (AHA), suggest that bicyclic
scaffold derivatives may be exploited for the design of new selective matrix metalloproteinase inhibitors (MMPIs).
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
it contributes to increase toxicity due to its low metal
binding selectivity.8
Matrix metalloproteinases (MMPs) are a family of zinc-
dependent, calcium containing, endopeptidases involved
in extracellular matrix degradation and, consequently,
they play a crucial role in physiological processes such
as tissue remodelling and healing of wounds.1 As the
upregulation of MMPs is involved in many inflammato-
ry, malignant and degenerative diseases,2 attempts to de-
sign and develop inhibitors that may modulate their
regulation have become of great interest.3–5
The inhibitors designed up to date usually bind the ac-
tive site on the catalytic domain providing nanomolar
dissociation constants.3,4 However, several inhibitors
exhibit a poor selectivity as a consequence of the high
structural similarity among the members of the MMP
family.9 Even if the low specificity does not prevent
the use in vivo, it raises a lot of side effects and it also
limits the dose that may be daily administered.10,11 As
a consequence, the search of new potent and selective
MMP inhibitors still represents an important pharma-
ceutical target.
The main requirement for a small molecule to be an
effective metalloproteinase inhibitor (MMPI) is the pres-
ence of both a functional group capable of chelating the
active-site0 zinc ion and a lipophilic residue able to fit
into the S1 pocket.4 Hydroxamic acid is the most popu-
lar zinc binding group (ZBG) for MMPIs even if some
authors report that it is fast metabolized in vivo6,7 and
Even if the expensive high-throughput screening of small
molecule libraries is one of the most popular approaches
in pharmaceutical research, alternative strategies, based
on docking calculation and rational drug design, may be
adopted if the 3D structure of the target is solved.12–14
A new class of molecular heterocyclic scaffolds (BTAa)
based on 3-aza-6,8-dioxa-bicyclo[3.2.1]octane skele-
ton,15 which can be decorated with a ZBG and a
Keywords: Metalloproteinases; Inhibitors; BTAa; NMR spectroscopy.
*
Corresponding author. Tel.: +39 055 4573481; fax: +39 055
4573569; e-mail: antonio.guarna@unifi.it
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.07.028

C. Mannino et al. / Bioorg. Med. Chem. 14 (2006) 7392–7403
7393
lipophilic residue, was chosen to design selective MMP-
12 inhibitors (Fig. 1).
lows to score the docking results in order to identify a
class of potential ligand.17
The main feature of these scaffolds is their tridimension-
al bicyclic skeleton generated by combination of sugar
and amino acid derivatives. Their synthesis includes
only few steps starting from commercially available
enantiopure precursors and a stereochemistry control
can be accomplished in each step of the synthesis. As
dipeptide isosteres, BTAas are compounds with poten-
tial biological activity and their use as central core of
pharmaceutical targets is convenient as a high number
of positions can be functionalized leading to molecular
diversity. Limited flexibility can be advantageous to cor-
rectly direct the pharmacophore side chains and it is also
useful for computational analysis. In addition, as amino
acids, they have full compatibility with the conditions
required for solid-phase synthesis.15,16
Moreover, the experimental assessment of the affinity of
few selected molecules outlined by docking results gives
the chance to correlate binding energies and binding
constants improving the prediction reliability. A com-
bined use of virtual screening and experimental determi-
nation of the binding affinity suggested that the BTAas
may represent a new type of scaffolds to design a new
class of MMPIs.
2. Chemistry
The synthesis of 3-biphenyl-4-ylmethyl-3-aza-6,8-dioxa-
bicyclo[3.2.1]octane-7-carboxylic acid methyl ester (p-
PhBn-BTGOMe) (8) was carried out following a proce-
dure previously reported for similar compounds and is
briefly reported below Scheme 1.12
All these properties, together with fair solubility and low
molecular weight, were considered in scaffold selection.
Reductive alkylation of aminoacetal 1 with biphenyl-4-
carbaldehyde (2) and NaBH4 afforded in good yield
the aryl aminoacetal 3 which was subsequently convert-
ed into the amide 5 by treatment with (R,R) tartaric
anhydride18 4. Crude 5 was treated with thionyl chloride
in MeOH affording cyclic acetal 6, which was submitted
to the acid-catalyzed cyclization to give 7 in 40% yield
over the four steps. Reduction of amide 7 with
BH3ꢀMe2S furnished the corresponding amine 8 in
70% yield (Scheme 1).
Diversification of BTAas by easy decoration with differ-
ent functional groups may generate virtual libraries
which may be screened in silico in order to identify
new hit compounds for further lead discovery. Even if
the binding energy provided by docking programs does
not reflect exactly the real binding affinity, its value al-
R'
Basic hydrolysis of 7 and acid-catalyzed hydrolysis of 8
gave, respectively, the free acids 9 and 10 in good yields.
5
R
O
N3
O
7
R''
X
A peculiar feature of BTAas is the demonstrated high
reactivity of their methyl ester function towards amines,
although simple esters are usually not very reactive to-
wards direct aminolysis.19 Owing to this, hydroxamic
acids 11 and 12were easily prepared by mixing the scaffold
1
BTAa(O) X=O
BTAa X = H,H
Figure 1.
O
O
O
Ref. 37
L-Tartaric acid
AcO
OAc
4
OH
OAc
O
O
O
O
ii
i
H
CHO
+
N
H2N
AcO
p-PhBn
O
p-PhBn
O
O
N
1
O
3
2
5
CH2 = p-PhBn
O
O
iv
O
O
iii
O
O
O
v
O
N
p-PhBn
O
N
O
p-PhBn
N
p-PhBn
OH
O
O
O
OH
8
7
6
Scheme 1. Reagents and conditions: (i) NaBH4, MeOH, 25 ꢁC, 16 h; (ii) CH2Cl2, 25 ꢁC, 16 h; (iii) SOCl2, MeOH, 60 ꢁC, 2 h; (iv) H2SO4/SiO2,
toluene, reflux, 20 min, 41% (over the four steps); (v) BH3ÆMe2S, THF, 25 ꢁC, 16 h, 70%.

7394
C. Mannino et al. / Bioorg. Med. Chem. 14 (2006) 7392–7403
idazole (CDI) and treated with O-benzylhydroxylamine,
O
O
N
O
p-PhBn
OH
as a free base, providing the substituted N-hydroxyurea
19 in 60% yield after crystallization.21 Hydrogenolytic
cleavage of the benzyl group over 5% Pd/C afforded the
desired final compound 20 (Scheme 4).
O
X = O
9
i
O
O
X = H,H
ii
O
N
X
O
p-PhBn
O
N
p-PhBn
p-PhBn
OH
3. Results and discussion
O
O
10
The availability of high-resolution structure of the
MMP-12 catalytic domain22,23 provided the possibility
to employ a structure-based approach to design and
screen a virtual library.
7 X = O
8 X = H,H
iii
O
H
O
N
X
N
OH
O
11 X = O
12 X = H,H
A biphenylic moiety and a hydroxamic (or carboxylic)
acid were chosen as good substituents24 to be introduced
in different position of BTAa and hundreds of structures
were so generated and tested in silico for their affinity to
the catalytic domain of MMP-12.
Scheme 2. Reagents and conditions: (i) LiOH, MeOH/THF, 25 ꢁC,
12 h, 80%; (ii) HCl 4 M, 25 ꢁC, 12 h, 90%; (iii) NH2OHÆHCl, NEt3,
CHCl3, 25 ꢁC, 48 h, 21% (11), 26% (12).
The hits with the highest score were selected and, among
them, compounds 9-12 were synthesized due to their
higher feasibility. In this class of molecules the scaffold
supports the biphenylic moiety in front of the ZBG.
The Autodock binding model predicted a deep insertion
of the biphenyl group into the S01 cavity though the coor-
dination geometry of the ZBG appeared not to be
optimized.
with an excess of hydroxylamine hydrochloride (5 equiv)
and NEt3 in CHCl3 at room temperature (Scheme 2).
To prepare the model compound 15 having a N-hy-
droxyurea as zinc-binding group, piperidine 13 was
treated with triphosgene and then with benzyl-hydroxyl-
amine and NEt3 affording compound 14. The debenzyl-
ated product 15 was readily obtained by mild
hydrogenolytic cleavage of the protecting group over
5% Pd/C at 1 atm (Scheme 3).20
Dissociation constants (KD) of compounds 9–12 were
calculated through N–1H HSQC experiments.25 In
15
addition, compounds 21–24, already present in our
chemical library, were submitted to the same experi-
ments in order to compare the results and support the
docking model.
A new BTAa scaffold 20 having the N-hydroxyurea
group in position 3 and the p-phenylbenzylcarboxy
amide in position 7 was then prepared. Due to the high
reactivity of ester 16, the biphenyl derivative 17 was easily
and quantitatively prepared by stirring the starting mate-
rial at 60 ꢁC with a neat excess of amine. Debenzylation
was carried out by refluxing a solution of 17 in MeOH
in the presence of ammonium formate and catalytic Pd/
C. Then, compound 18 was activated with carbonyldiim-
Moreover, the inhibitory activity (IC50) of compounds
9–12 was measured by enzymatic assays.26
The values of KD and IC50 for the tested compounds
9–12 and 20–24 are reported in Table 1.
O
O
O
O
O
iii
i, ii
N
N
HN
HN
OEt
HN
OEt
OEt
OBn
OH
14
13
15
Scheme 3. Reagents and conditions: (i) (CCl3O)2CO, CH2Cl2, from 0 ꢁC to 25 ꢁC, 12 h; (ii) Bn–ONH2ÆHCl, NEt3, CH2Cl2, 25 ꢁC, 12 h, 40%; (iii) H2
(1 atm), 5% Pd/C, AcOEt, 25 ꢁC, 12 h, quantitative.
ii
O
O
O
i
H
H
O
O
O
BnN
BnN
HN
O
N
N
N
p-PhBn
O
p-PhBn
O
O
O
16
17
18
O
O
O
iii
H
N
H
O
iv
O
N
N
p-PhBn
p-PhBn
HN
HN
O
O
OBn
OH
19
20
Scheme 4. Reagents and conditions: (i) p-PhBn–NH2, 60 ꢁC, 12 h, 93%; (ii) HCO2NH4, 10% Pd/C, MeOH, reflux, 2 h, 64%; (iii) BnONH2, CDI,
THF, 25 ꢁC, 12 h, 60%; (iv) H2 (1 atm), 5% Pd/C, THF, 25 ꢁC, 12 h, 68%.

C. Mannino et al. / Bioorg. Med. Chem. 14 (2006) 7392–7403
Table 1. Calculated MMP-12 binding affinity (KD) and inhibition potency (IC50
7395
)
R'
N
R
O
O
R''
X
c
d
Compound
X
R
R0
R00
MMP-12 KD
MMP-12 IC50 (lM)
9
10
O
p-PhBn
p-PhBn
p-PhBn
p-PhBn
HONHCO
Bn
H
COOH
COOH
P1 mM
P1 mM
954
835
425
399
149
—
H, H
O
H
11
12
H
CONHOH
CONHOH
CONH–p-PhBn
COOH
P500 lM
P500 lM
154 lM
P10 mM
P10 mM
P10 mM
not detec.b
H, H
H, H
O
H
20
H
21a
22a
23a
24a
H
H, H
O
H, H
Bn
Bn
H
COOH
CONHOH
H
—
—
H
COOH
p-PhBn
—
O
N
HO NH
O
3.4 mM
—
OEt
15
a Compounds previously described.15,29,30
b No detectable interaction found by NMR.
c Dissociation constants measured through 15N–1H HSQC NMR experiments.
d Inhibition potency measured by fluorescence enzymatic assays.
Figure 2. (A) Surface model of the MMP-12 showing the catalytic zinc and the S01 pocket. (B) Residues of the MMP-12 affected by chemical shift
perturbation upon the addition of 12.
Since the identification of the amino acids involved in
the binding may be crucial to prove a direct interaction
with the catalytic site and, consequently, for the optimi-
zation of the process, NMR-based strategies were also
used to localize the binding site on the target.27 The
identification of the binding site was easily performed
analyzing the protein resonance chemical shifts in
1H–15N HSQC spectra, recorded either in presence or
in absence of the ligand.28
members of this class. In particular, the NH cross-peak
of His218 was only weakly shifted by lactams 9 and 11
with respect to compound 12.
The analysis of the results summarized in Table 1 sug-
gests that the bicyclic skeleton of BTAa should be com-
patible with the metalloelastase catalytic domain, but
the activity is strongly modulated by the type and
position of the substituents. Hydroxamic acids 11, 12
(IC50 = 425 lM, 399 lM, respectively) are more potent
than the carboxylic ones 9, 10 (IC50 = 954 lM,
835 lM, respectively). The affinity observed within this
series seemed to be highly dependent on the presence
of the biphenylic moiety: the replacement of the biphe-
nylmethyl with a benzylic-protecting group resulted in
a loss of binding affinity, so compounds 21 and 22
(KD P 10 mM) are almost inactive; and the introduc-
tion of the hydroxamic moiety in position 7 did not
restore the activity (see compound 23). Otherwise, the
presence of the ZBG and the lipophilic residue in vicinal
position decreases the binding affinity as demonstrated
The pattern of the shifts was similar for all ligands. Sev-
eral residues forming the S01 cavity, one or more zinc
binding histidines and the neighbouring amino acids
are strongly influenced by the presence of this class of
molecules. As example, a ribbon representation of
MMP-12 is shown in Figure 2. The NHs showing signif-
icant chemical shift perturbation upon the addition of
compound 12 are highlighted. The affected amino acids
nicely define the ligand binding site that is the same for
all the designed compounds in agreement with the dock-
ing results. Only few differences were found among the

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C. Mannino et al. / Bioorg. Med. Chem. 14 (2006) 7392–7403
by compound 24 for which no detectable interactions
were found by NMR.
With these results in our hands we decided to focus our
efforts on the design of new derivatives with improved
affinity. A deeper penetration in the S01 cavity was possi-
ble moving the biphenyl group quite far from the scaf-
fold. Due to the high reactivity of the BTAa methyl
ester, it was synthetically very easy to introduce the
biphenylic moiety in 7 through an amide bond. As a
consequence, the best anchoring point for the ZBG
resulted in the N-3 giving rise to a N-hydroxyurea (com-
pound 20). The use of this functional group as metal
chelator in MMP inhibitors has been previously report-
ed6 and recently validated by a X-ray structure.31
In order to determine the affinity of the N-hydroxyurea
moiety, the model compound 15 was tested in silico and
synthesized (see Scheme 3).
Figure 3. Expansion of 1H–15N HSQC spectrum showing the shift of
the His218 backbone NH upon the addition of increasing concentra-
tion of compound 15.
The in silico model suggested that the interaction of 15
with the active site is limited to the catalytic zinc and
to the entrance into the S01 subsite. The structure of
the adduct shows the same coordination scheme of the
hydroxamic acid which is able both to chelate the
active-site zinc ion and to provide a hydrogen bond
interaction with the enzyme backbone. The protonated
oxygen atom (O4) is involved in a strong hydrogen bond
with carboxylate Oe2 of Glu219, while the NH shows
weaker electrostatic interaction with the Ala181 carbon-
yl oxygen.
MMP-8, MMP-13), matrilysin 1 (MMP-7) and strom-
elysin 2 (MMP-10) and the corresponding data are sum-
marized in Table 2. Compound 20 is a fairly selective
MMP-12 inhibitor since it possesses limited activity
against the other MMPs especially the MMP-1 and
MMP-7. These two enzymes differ from other MMP
family members since they have a relatively small S10
pocket which presumably cannot accommodate the
large biaryl substituent.
To determine the binding affinity for the MMP-12 cata-
lytic domain, we analyzed the alteration of the chemical
1
shifts induced on 2D H–15N HSQC upon the titration
Although compound 20 exhibits an improved affinity
towards MMP-12, nevertheless the micromolar value
of KD demonstrates that all the planned interactions
are not optimized. The X-ray structure of the MMP-
12–20 complex (PDB code: 2HU6) provided a direct
inspection on ligand–protein interactions (Fig. 5A).
As correctly predicted by the NMR studies the mole-
cule binds the metalloelastase at the catalytic site, fit-
ting the biaryl moiety into the S01 pocket. The
electron density of the inhibitor is well defined because
it is held in place by two hydrogen bonds with Pro238
and Leu181 of the enzyme backbone. However, the
contribution of the hydroxyurea to the binding is abso-
lutely negligible since it points towards the exterior of
the active site and does not interact with the catalytic
zinc ion.
with the N-hydroxyurea derivative 15. The fit of the
experimental data provided a dissociation constant of
3.4 mM. The binding mode predicted in silico is sup-
ported by the pattern of resonance frequencies shifted
as a consequence of the interaction. The NH signal cor-
responding to His218 is strongly affected by millimolar
concentration of the ligand (Fig. 3), while only few
residues forming the S01 cavity are weakly influenced.
Since the metal binding capability of the N-hydroxyurea
was confirmed, the new scaffold 20 was synthesized. (see
Scheme 4)
1
The H–15N HSQC spectra for 20 showed a pattern of
shifts similar to compounds 9–12 indicating for all these
molecules the same binding site (Fig. 4).
Noteworthy, the zinc ion is coordinated by the weak
inhibitor acetohydroxamic acid (AHA) present, as stabi-
lizer, in the crystallization buffer. This would suggest that
the lack of interaction between the hydroxyurea and the
catalytic zinc, evidenced in the crystal structure, is due
to the competition with the high concentration of AHA.
A different effect on the zinc binding histidine with re-
spect to the N-hydroxyurea prototype is probably pres-
ent since the shift perturbation involved mainly His222
instead of His218. The fit of Dd as a function of the
ligand concentration provided a KD of 154 lM, in good
agreement with the IC50 value of 149 lM obtained by
the enzymatic assay (Fig. 4).
In order to investigate this point, an energy minimiza-
tion of the ligand was performed after removing AHA
from the PDB and blocking the biaryl group in the crys-
tal structure conformation. The AMBER-8 minimized
model indicates that the hydroxyurea function could
To probe the selectivity towards MMP-12, N-hydroxy-
urea 20 was also tested by fluorescence enzymatic exper-
iments for the inhibition of collagenases (MMP-1,

C. Mannino et al. / Bioorg. Med. Chem. 14 (2006) 7392–7403
7397
1
Figure 4. (A) H–15N HSQC spectrum of MMP-12 catalytic domain in absence (black) and in presence (light grey) of compound 20 (500 lM). (B)
Expansion of 1H–15N HSQC spectrum showing the shift of the His222 backbone NH upon the addition of increasing concentration of compound 20.
Table 2. MMP enzyme inhibition potency of compound 20 measured by enzymatic assay
Compound
IC50 (lM)
MMP-1
1180
MMP-7
1510
MMP-8
513
MMP-10
865
MMP-12
149
MMP-13
778
20
Figure 5. (A) Crystal structure of MMP-12–20 adduct; the zinc ion is coordinated by AHA (Green). (B) AMBER-8-minimized model of MMP-12–20
adduct.
coordinate the zinc ion, even if the hydrogen bond inter-
actions with the zinc binding site are not well defined as
in the case of AHA (Fig. 5B).
fold functionalized with a biphenyl moiety and with a
ZBG.
The adducts provided by docking calculation enabled
the identification of the catalytic pocket of MMP-12 as
the binding site for this class of compounds. The inspec-
tion of the residues showing a significant chemical shift
perturbation supports this picture, with the lipophilic
moiety fitted into S01 cavity and the bicyclic skeleton
located outside, in front of the catalytic zinc.
Therefore, these data suggest that the anchoring point
and/or the length of the ZBG are not optimized and fur-
ther studies have to be carried out in order to design a
nanomolar inhibitor.
4. Conclusion
Among the synthesized compounds, the scaffold 20 hav-
ing the N-hydroxyurea group in position 3 and the
p-phenylbenzylcarboxy amide in position 7 showed an
improved affinity (KD = 154 lM and IC50 149 lM) for
MMP-12 and some selectivity towards the other MMPs.
New compounds with affinity for the catalytic domain
of the MMP-12 were designed using an integrated
approach based on virtual screening and on NMR
analysis. The molecules are built around a BTAa scaf-

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C. Mannino et al. / Bioorg. Med. Chem. 14 (2006) 7392–7403
The X-ray structure of the catalytic domain of Human
Macrophage Metalloelastase in the presence of hydroxy-
urea derivative 20 gave more insight into the ligand–en-
zyme interaction and appeared as a new starting point
for further MMP-12 inhibitors’ designing.
MMP-1 (Val101-Pro269), MMP-7 (Tyr100-Lys272),
MMP-8 (Met100-Gly262), MMP-10 (Phe99-Gly263,
F170N mutant), MMP-13 (Tyr104-Pro269) were ex-
pressed, purified and refolded according to the protocol
already described for MMP-12, with minor
modifications.
New fragments may be easily inserted in the bicyclic
scaffold to better interact with the enzyme backbone
and to selectively accommodate into the binding pocket
of MMP-12. Therefore, the synthetic versatility of
BTAas, taken together with dissociation constant in
micromolar range and fair selectivity, candidate these
molecules as new compound guides to develop more po-
tent and selective inhibitors for MMP-12.
1H–15N HSQC experiments, implemented with the sen-
sitivity enhancement scheme,35 were recorded at 298 K
on a Bruker DRX 700 operating at proton nominal fre-
quency of 700.21 MHz. The NMR experiments were
performed on samples containing 0.1 mM of 15N-
enriched MMP-12 in 10 mM Tris–HCl buffer, 10 mM
CaCl2, 0.1 mM ZnCl2, 0.3 M NaCl and 0.2 M acetohy-
droxamic acid at pH 7.2.
5. Methods
The compounds were evaluated for their ability to inhib-
it the hydrolysis of fluorescence-quenched peptide
Docking calculations were performed using the
Lamarckian genetic algorithm (LGA) of the program
Autodock 3.032 which, using principles of natural evolu-
tion mutations and cross-over, probes the designed mol-
ecules on the three-dimensional structure of the target
macromolecule. In this work, the crystal structure of
the catalytic domain of MMP-12 (PDB code: 1RMZ),
processed by AutoDockTools, was used as target pro-
tein. The three-dimensional structures of the designed li-
gands, generated by Chem3D Pro program, were
minimized by semiempirical calculations and the Gastei-
ger-Marsili charges33 assigned by the program Babel. In
substrate
Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2
(Biomol, Inc.). The assays were performed in 50 mM
HEPES buffer, containing 10 mM CaCl2, 0.05% Brij-
35, at pH 7, using 1 nM of proteolytic enzyme (catalytic
domains of MMP-1, MMP-7, MMP-8, MMP-10,
MMP-12, MMP-13) and 1 lM of peptide. The enzyme
was incubated at 25 ꢁC with increasing concentration
of inhibitor and the fluorescence (excitationmax
328 nm; emissionmax 393 nm) was measured for 3 min
after the addition of the substrate using a Varian
Eclipse fluorimeter. Fitting of rates as a function of
inhibitor concentration provided IC50 values (see Table
1). The inhibitor N-isobutyl-N-[4-methoxyphenylsulfo-
nyl]glycyl hydroxamic acid (Biomol, Inc.) was used as
control.
order to include all the active site,
˚
a box of
3
19.75 · 19.75 · 19.75 A , centred near the catalytic zinc
and with a grid spacing of 0.275 A, was selected as dock-
˚
ing space. A total of 50 runs were performed for each
ligand and the results were ranked according to the
docking energy.
Crystals of human MMP-12, already containing AHA
from the refolding process, grew at 20 ꢁC from a
0.1 M Tris–HCl, 30% PEG 6000, 200 mM AHA,
1.0 M LiCl2 solution at pH 8.0 using the vapour diffu-
sion technique. The final protein concentration was
about 10 mg/ml. Soaking procedure was implemented
in order to allow the inhibitor binding; 20 was added
in the powdered form directly into the drop using a nee-
dle and was left incubating for a few days. The data were
measured exploiting synchrotron radiation at the beam-
line ID23-1 (ESRF, Grenoble, France). The dataset was
collected at 100 K and the crystal used for data collec-
tion was cryo-cooled without any cryo-protectant treat-
The cDNA, encoding the fragment Gly106-Gly263
(F171D mutant)34 of the macrophage metalloelastase,
was cloned into the pET21 vector (Novagen) using NdeI
and BamHI as restriction enzymes and then transfected
into E. coli strain BL21 Codon Plus cells. Uniform 15N-
labelled protein was expressed by induction with
0.5 mM IPTG at 37 ꢁC for 4 h in M9 minimal media
containing 15 mM (15NH4)2SO4.
The inclusion bodies, containing the protein, were solu-
bilized in a buffered solution with 20 mM Tris–HCl and
8 M urea at pH 8. The protein was then purified with a
size-exclusion chromatography (Pharmacia HiLoad
Superdex 75 16/60) in 6 M urea and 50 mM sodium ace-
tate. A second step of purification was performed on
cation exchange column Mono-S (Pharmacia) using a
linear gradient of NaCl up to 0.5 M.
˚
ment. The crystal diffracted up to 1.3 A resolution and
˚
belongs to space group C2 (a = 51.25 A, b = 60.18 A,
˚
˚
c = 53.97 A, a = c = 90ꢁ, b = 114.59ꢁ) with one molecule
in the asymmetric unit, a solvent content of about 50%
and a mosaicity of 0.7ꢁ. The data were processed in all
cases using the program MOSFLM36 and scaled using
the program SCALA37 with the TAILS and SECOND-
ARY corrections on (the latter restrained with a TIE
SURFACE command) to achieve an empirical absorp-
tion correction.
The protein was refolded by using a multi-step dialysis
against solution containing 50 mM Tris–HCl (pH 7.2),
10 mM CaCl2, 0.1 mM ZnCl2, 0.3 M NaCl and decreas-
ing concentrations of urea (from 4 M up to 2 M). The
last two dialyses were performed against solution
containing 20 mM Tris–HCl (pH 7.2), 5 mM
CaCl2, 0.1 mM ZnCl2, 0.3 M NaCl and 200 mM of
hydroxamic acid (AHA). The catalytic domains of
Table 3 shows the data collection and processing statis-
tics for all datasets. The structure was solved using the
molecular replacement technique; the model used was
that of the MMP-12–AHA adduct (1Y93) from where
the inhibitor, all the water molecules and ions were

C. Mannino et al. / Bioorg. Med. Chem. 14 (2006) 7392–7403
7399
Table 3. Data collection and anisotropic refinement statisticsa
Preparation of acid silica gel (SiO2/H2SO4): SiO2 (29 g)
was suspended in CH2Cl2 (400 ml) with 12 g of H2SO4
and left under vigorous magnetic stirring for 40 min.
The suspension was then concentrated at low pressure.
Space group
C2
A = 51.25, b = 60.18,
˚
Cell dimensions (A,ꢁ)
c = 53.97, b = 114.59
49.10–1.32
34825 (5291)
99.0 (98.8)
5.3 (27.6)
3.1 (2.9)
8.6 (2.5)
10.55
˚
Resolution (A)
Unique reflections
Overall completeness (%)
Rsym(%)
6.1. Biphenyl-4-yl-methyl-(2,2-dimethoxy-ethyl)-amine
(3)
Multiplicity
I/(rI)
Wilson plot B-factor (A )
Rcryst/Rfree (%)
A
solution containing 2,2-dimethoxy-ethylamine
2
(14.4 g, 137 mmol) and biphenyl-4-carbaldehyde
(25 g, 137 mmol) in MeOH (300 ml) was stirred at
room temperature for 2.5 h. NaBH4 was then slowly
added at 0 ꢁC and the reaction mixture was stirred
at room temperature overnight. Once the reaction
was completed MeOH was evaporated in vacuo and
the crude residue was suspended in ethyl acetate,
washed with water and dried over Na2SO4. The result-
ing product was used in next step without further
purification. Purification by column chromatography
using ethyl acetate/petroleum ether (1:1) as eluent
afforded the pure 3 as a colourless oil.
˚
16.2/18.7
1238
Protein atoms (excluding hydrogens)
Ions
Ligand atoms
5
33
Water molecules
rmsd bond lengths (A)
300
0.006
˚
rmsd bond angles (ꢁ)
Mean B-factor (A )
1.0
13.90
2
˚
a The numbers in parentheses refer to values in the highest resolution
shell.
omitted. The correct orientation and translation of the
molecule within the crystallographic unit cell was deter-
mined with standard Patterson search techniques38,39 as
implemented in the program MOLREP.40,41 The aniso-
tropic refinement was carried out using REFMAC5.42
In between the refinement cycles the model was subject-
ed to manual rebuilding by using XtalView.43 The same
program was used to model the inhibitors. Water mole-
cules were added by using the standard procedures with-
in the ARP/WARP suite.44 The stereochemical quality
of the refined models was assessed using the program
Procheck.45 The Ramachandran plot is of very good
quality.
1H NMR (CDCl3): d 7.62–7.26 (m, 9H), 4.53 (t, 1H,
J = 5.5 Hz), 3.86 (s, 2H), 3.39 (s, 6H), 2.80 (d, 2H,
J = 5.6 Hz); 13C (CDCl3): d 140.9, 139.9, 139.1 (s),
128.7–127.0 (d), 104.0 (d), 54.10 (t), 53.68 (t), 50.70
(q); MS m/z 271 (M+, 15), 167 (100); IR (CHCl3)
3323, 2829 cmꢁ1; Anal. Calcd for C16H19NO2 (257.1):
C, 74.68; H, 7.44; N, 5.44. Found: C, 74.75; H, 7.52;
N, 5.65.
6.2. (2R,6R/S)-(4-Biphenyl-4-yl-methyl-6-methoxy-3-
oxo-morpholin-2-yl)-(R)-hydroxyacetic acid methyl ester
(6)
To a suspension of (R,R)-2,3-di-O-acetyltartaric anhy-
dride (4) (30 g, 137 mmol) in dry DCM (120 ml) was
added, at 0 ꢁC and under N2 atmosphere, a solution
of 3 (37 g, 137 mmol) in dry DCM (60 ml). The reac-
tion mixture was stirred at room temperature over-
night. After evaporation of the solvent, the crude
product was dissolved in MeOH (230 ml) and thionyl
chloride (8.5 ml, 116 mmol) was added dropwise at
0 ꢁC. The mixture was then allowed to reach 60 ꢁC
and stirred for 2 h. The solvent was removed and
the crude product was isolated as a yellow oil and
used without further purification in the next step.
Purification of the crude product by column chroma-
tography (ethyl acetate/petroleum ether 2:1) afforded
6 as a white solid.
6. Experimental
Melting points are uncorrected and were measured on
microscope RCH Kofler apparatus. Chromatographic
purifications were recorded with silica gel 60 (0.040–
0.063 mm), unless otherwise stated, using flash column
techniques; all TLC development was performed on
silica gel coated (Merck Silica gel 60 F254, 0.25 mm)
sheets. IR spectra were recorded on Perkin-Elmer
881 spectrophotometer. 1H and 13C NMR spectral
analyses were obtained on a Varian Gemini 200
(200 MHz for H and 50.33 MHz for 13C) instrument
1
or a Varian Mercury 400 (400 MHz for 1H and
100 MHz for 13C) instrument using tetramethylsilane
as internal standard and the chemical shifts were
reported in d (ppm) units. EI mass spectral analyses
were carried out at 70 eV ionizing voltage by direct
introduction on a QMD 1000 Carlo Erba instrument,
whereas ESI-MS spectra were carried out on LTQ
Thermo Finnigan instrument. Microanalyses were car-
ried out on Perkin Elmer 240 C elemental analyser.
Optical rotation measurements were performed on
JASCO DIP-370 digital polarimeter. All reactions
requiring anhydrous conditions were carried out under
N2 atmosphere and were performed in oven-dried
glassware.
26
D
1
Mp 115–120 ꢁC; ½aꢂ +111.1 (c 1.3, CHCl3); H NMR
(CDCl3): d 7.60–7.26 (m, 9H), 4.96 (s, 1H), 4.87 (s,
1H), 4.84 (d, 1H, J = 14 Hz), 4.64 (s, 1H), 4.54 (d, 1H,
J = 14 Hz), 3.85 (s, 3H), 3.58 (dd, 1H, J1 = 14 Hz,
J2 = 3.2 Hz), 3.39 (s, 3H), 3.16 (d, 1H, J = 14 Hz); 13C
NMR (CDCl3): d 172.4 (s), 165.4 (s), 140.6, 140.4,
134.5 (s), 128.7, 128.2, 127.4, 127.2, 127.0 (d), 95.95
(d), 72.28 (d), 71.60 (d), 55.15 (q), 52.89 (q), 49.65 (t),
49.55 (t); MS m/z 385 (M+, 58), 167 (100); IR (CHCl3)
3545, 1745, 1654 cmꢁ1; Anal. Calcd for C21H23NO6
(385.2): C, 65.44; H, 6.02; N, 3.63. Found: C, 65.03;
H, 6.22; N, 3.27.

7400
C. Mannino et al. / Bioorg. Med. Chem. 14 (2006) 7392–7403
6.3. (1S,5S,7R)-3-Biphenyl-4-ylmethyl-2-oxo-3-aza-6,8-
dioxa-bicyclo[3.2.1]octane-7-carboxylic acid methyl ester
(7)
and the resulting organic phase was dried over Na2SO4
and concentrated under reduced pressure to afford 9
(65 mg, 80%) as a white solid.
A solution of 6 (52 g, 137 mmol) in toluene (200 ml) was
quickly added to a refluxing suspension of H2SO4/SiO2
(29 g) in toluene (400 ml). The mixture was allowed to
react for 20 min and one-third of the solvent was dis-
tilled off. The hot reaction mixture was filtered through
a short layer of NaHCO3 and, after evaporation of the
solvent, the crude product was purified by column chro-
matography using ethyl acetate/petroleum ether (2:1) as
eluent and affording 7 (20 g, 41% over four steps) as a
white solid.
Mp 186–195 ꢁC; 1H NMR (DMSO): d 7.66–7.27 (m,
9H), 5.95 (s, 1H), 4.89 (s, 1H), 4.80 (s, 1H), 4.60–4.42
(AB system, 2H), 3.38 (d, 1H, J = 14 Hz), 3.10 (d, 1H,
J = 14); 13C NMR (DMSO): d 170.6 (s), 165.7 (s),
140.1, 139.7, 135.9 (s), 129.3, 128.7, 127.8, 127.4, 127.0
(d), 99.85 (d), 77.62 (d), 77.50 (d), 51.64 (t), 47.39 (t);
MS m/z 339 (M+, 17), 167 (100); Anal. Calcd for
C19H17NO5 (339.1): C, 67.25; H, 5.05; N, 4.13. Found:
C, 66.86; H, 5.06; N, 4.04.
6.6. (1S,5S,7R)-3-Biphenyl-4-ylmethyl-3-aza-6,8-dioxa-
bicyclo[3.2.1]octane-7-carboxylic acid (10)
25
1
Mp 160–165 ꢁC; ½aꢂ ꢁ40.5 (c 1.0, CHCl3); H NMR
D
7.60–7.26 (m, 9H), 5.89 (d, 1H,
(CDCl3):
d
J = 2.2 Hz), 5.01 (s, 1H), 4.79 (s, 1H), 4.66–4.52 (Ab sys-
tem, 2H), 3.81 (s, 3H), 3.42 (dd, 1H, J1 = 2.2 Hz,
J2 = 12 Hz), 3.17 (d, 1H, J = 12 Hz); 13C NMR
(CDCl3): d 169.0 (s), 165.4 (s), 140.9, 140.4, 134.2 (s),
128.7, 128.4, 127.6, 127.4, 127.0 (d), 100.1 (d), 77.83
(d), 77.73 (d), 52.93 (q), 51.23 (t), 48.21 (t); MS m/z
A solution containing 8 (80 mg, 0.23 mmol) and aq. HCl
4 M (0.800 ml) was stirred overnight at room tempera-
ture. The product was then freeze-dried affording 10
(75 mg, 90%) as a white solid.
1
Mp 158–165 ꢁC; H NMR (DMSO): d 11.18 (br s, 1H),
353 (M+, 11), 167 (100); IR (CHCl3) 1756, 1674 cmꢁ1
;
7.72–7.67 (m, 5H), 7.48–7.35 (m, 4H), 5.83 (s, 1H), 5.45
(s, 1H), 4.97 (s, 1H), 4.34 (m, 2H), 3.60–3.48 (m, 1H),
3.42–3.22 (m, 1H), 3.20–2.96 (m, 2H); 13C NMR
(DMSO) (carboxylic carbon not detected): d 139.7,
132.8 (s), 129.4–127.1 (d), 101.0 (d), 75.03 (d), 74.31
(d), 59.47 (t), 53.19 (t), 52.86 (t); MS m/z 325 (M+,
21), 167 (100); IR (KBr) 3406, 2355, 1741 cmꢁ1; Anal.
Calcd for C19H20ClNO4 (361.11): C, 63.07; H, 5.57; N,
3.87. Found: C, 62.88; H, 5.71; N, 3.75.
Anal. Calcd for C20H19NO5 (353.1): C, 67.98; H, 5.42;
N, 3.96. Found: C, 67.54; H, 5.42; N, 3.69.
6.4. (1S,5S,7R)-3-Biphenyl-4-ylmethyl-3-aza-6,8-dioxa-
bicyclo[3.2.1]octane-7-carboxylic acid methyl ester (8)
BH3ÆSMe2 (0.403 ml, 4.2 mmol) was added dropwise to
a cooled (0 ꢁC) solution of 7 (1 g, 2.8 mmol) in dry
THF under N2 atmosphere and the resulting mixture
was stirred at room temperature overnight. Once the
reaction was completed EtOH (2 ml) and H2O were add-
ed and the resulting mixture was washed with OEt2. The
organic layer was dried over Na2SO4 and concentrated
in vacuo. The crude product was purified by column
chromatography using ethyl acetate/petroleum ether
(3:1) as eluent to afford 8 (670 mg, 70%) as a white solid.
6.7. (1S,5S,7R)-3-Biphenyl-4-ylmethyl-2-oxo-3-aza-6,8-
dioxa-bicyclo[3.2.1]octane-7-carboxylic acid hydroxy-
amide (11)
A solution containing 7 (100 mg, 0.28 mmol), hydroxyl-
amine hydrochloride (97 mg, 1.42 mmol) and NEt3
(236 ll, 1.7 mmol) in CHCl3 (500 ll) was stirred for 5
days at room temperature. The resulting suspension
was then filtered and the organic solvents were evaporat-
ed in vacuo. The crude product was purified by column
chromatography, using ethyl acetate as eluent and
affording 11 (21 mg, 21%) as a white solid.
24
D
1
Mp 130–134 ꢁC; ½aꢂ ꢁ57.0 (c 1.0, CHCl3); H NMR
(CDCl3): d 7.63–7.36 (m, 9H), 5.66 (s, 1H), 4.86 (s,
1H), 4.66 (s, 1H), 3.78 (s, 3H), 3.70–3.50 (AB system,
2H), 2.87 (pseudo t, 2H, J = 11.6 Hz), 2.56 (dd, 1H,
J1 = 1.6, J2 = 11.2 Hz), 2.35 (d, 1H, J = 12 Hz); 13C
NMR (CDCl3): d 171.4 (s), 140.7, 140.2, 136.3 (s),
129.2, 128.7, 127.2, 127.1, 127.0 (d), 101.4 (d), 77.00
(d), 76.07 (d), 61.16 (t), 56.44 (t), 54.85 (t), 52.54 (q);
MS m/z 339 (M+, 23), 167 (100); IR (CHCl3) 1753,
1733 cmꢁ1; Anal. Calcd for C20H21NO4 (339.2): C,
70.78; H, 6.24; N, 4.13. Found: C, 70.34; H, 6.41; N,
4.00.
1
Mp 197–203 ꢁC; H NMR (DMSO): d 10.74 (br s, 1H),
9.00 (br s, 1H), 7.71–7.17 (m, 9H), 5.92 (s, 1H), 4.69 (s,
2H), 4.49 (s, 2H), 3.39 (d, 1H, J = 12 Hz), 3.07 (d, 1H,
J = 12 Hz); 13C NMR (DMSO): d 165.9 (s), 165.2 (s),
140.1, 139.7, 135.9 (s), 129.3, 128.7, 127.8, 127.4, 127.0
(d), 99.84 (d), 78.84 (d), 77.46 (d), 51.60 (t), 47.41 (t);
MS m/z 354 (M+, 2), 167 (100); Anal. Calcd for
C19H18N2O5 (354.1): C, 64.40; H, 5.12; N, 7.91. Found:
C, 64.12; H, 5.42; N, 7.53.
6.5. (1S,5S,7R)-3-Biphenyl-4-ylmethyl-2-oxo-3-aza-6,8-
dioxa-bicyclo[3.2.1]octane-7-carboxylic acid (9)
6.8. (1S,5S,7R)-3-Biphenyl-4-ylmethyl-3-aza-6,8-dioxa-
bicyclo[3.2.1]octane-7-carboxylic acid hydroxyamide (12)
A solution containing 7 (85 mg, 0.24 mmol) and LiOH
(25 mg, 0.6 mmol) in a mixture of MeOH and THF in
a 2:1 ratio (5 ml) was stirred overnight at room temper-
ature. The reaction mixture was then acidified with 5%
HCl and the organic solvents were removed in vacuo.
The remaining aqueous layer was washed with DCM
A solution containing 8 (98 mg, 0.29 mmol), hydroxyl-
amine hydrochloride (97 mg, 1.42 mmol) and NEt3
(242 ll, 1.7 mmol) in CHCl3 (500 ll) was stirred for 5
days at room temperature. The resulting suspension

C. Mannino et al. / Bioorg. Med. Chem. 14 (2006) 7392–7403
7401
was then filtered and the organic solvents were evaporat-
ed in vacuo. The crude product was purified by column
chromatography, using ethyl acetate as eluent, affording
12 (26 mg, 26%) as a white solid.
2.03–1.82 (m, 2H), 1.78–1.53 (m, 2H), 1.24 (t, 3H,
J = 7.16); 13C NMR (CDCl3): d 171.8 (s), 158.2 (s),
58.02 (t), 40.14 (t), 37.91 (d), 24.81 (t), 11.44 (q); ESI
MS m/z 217 (M+1); IR (CHCl3) 3682, 1724, 1668,
1218 cmꢁ1; Anal. Calcd for C9H16N2O4 (216.11): C,
49.99; H, 7.46; N, 12.96. Found: C, 49.55; H, 7.45; N,
12.89.
1
Mp 185–189 ꢁC; H NMR (DMSO): d 10.55 (s, 1H),
8.80 (s, 1H), 7.67–7.34 (m, 9H), 5.56 (s, 1H), 4.52 (s,
1H), 4.46 (s, 1H), 3.56 (s, 2H), 2.75 (pseudo t, 2H,
J = 10 Hz), 2.42 (d, 1H, J = 11.4 Hz), 2.18 (d, 1H,
J = 11.6 Hz); 13C NMR (DMSO): d 167.3 (s), 140.3,
137.1 (s), 129.7, 129.3, 129.2, 127.7, 127.0 (d), 100.5
(d), 76.92 (d), 76.73 (d), 60.66 (t), 56.42 (t), 55.28 (t);
ESI MS m/z 341 (M+1); IR (KBr) 3317, 3146,
1683 cmꢁ1; Anal. Calcd for C19H20N2O4 (340.1): C,
67.05; H, 5.92; N, 8.23. Found: C, 66.66; H, 5.45; N,
8.61.
6.11. (1S,5S,7R)-3-Benzyl-3-aza-6,8-dioxa-bicy-
clo[3.2.1]octane-7-carboxylic acid (biphenyl-4-ylmethyl)-
amide (17)
A
mixture containing BnBTGOMe (16) (314 mg,
1.19 mmol) and biphenyl-4-yl-methylamine (1 g,
4.6 mmol) was allowed to reach 60 ꢁC and was stirred
at the same temperature overnight. Once the reaction
was completed the crude product was purified by col-
umn chromatography using ethyl acetate/petroleum
ether (1:2) as eluent and affording 17 (460 mg, 93%) as
a white solid.
6.9. 1-Benzyloxycarbamoyl-piperidine-4-carboxylic acid
ethyl ester (14)
A solution of piperidine-4-carboxylic acid ethyl ester
(13) (300 mg, 1.91 mmol) in dry DCM (5 ml) was added
to a solution of triphosgene (187 mg, 0.63 mmol) in dry
DCM (4 ml) at 0 ꢁC and under inert atmosphere. The
resulting solution was allowed to reach room tempera-
ture and stirred for 1 day.
23
D
1
Mp 98–101 ꢁC; ½aꢂ +23.7 (c 0.5, CH2Cl2); H NMR
(CDCl3): d 7.58–7.19 (m, 14H), 6.97 (br s, 1H), 5.59 (s,
1H), 4.79 (s, 1H), 4.69 (s, 1H), 4.65–4.37 (m (ABX sys-
tem), 2H), 3.57 (m, 2H), 2.87 (d, 2H, J = 10.6 Hz), 2.55
(d, 1H, J = 11.8 Hz), 2.34 (d, 1H, J = 11 Hz); 13C NMR
(CDCl3): d 171.1 (s), 140.6, 140.4, 136.7 (s), 128.8, 128.7,
128.4, 128.0, 127.4, 127.3, 127.3, 127.0 (d), 101.2 (d),
77.44 (d), 77.38 (d), 61.54 (t), 56.30 (t), 55.02 (t), 42.87
(t); MS m/z 414 (M+, 7), 91 (100); IR (CHCl3) 3418,
1674, 1521, 1106 cmꢁ1; Anal. Calcd for C26H26N2O3
(414.19): C, 75.34; H, 6.32; N, 6.76. Found: C, 74.96;
H, 6.25; N, 6.37.
To the reaction mixture were then added O-benzyl-hy-
droxylamine hydrochloride (336 mg, 2.1 mmol) and
NEt3 (850 ll, 6.1 mmol) and the resulting solution was
stirred for 2 h. The reaction mixture was then concen-
trated under reduced pressure, diluted with DCM,
washed with H2O and brine, dried over Na2SO4 and
concentrated in vacuo. The crude product was purified
by column chromatography (Aluminium oxide, ethyl
acetate/petroleum ether 2:1 to clean up impurities; ethyl
acetate to isolate the final product) to afford 14 (238 mg,
40%) as a pale yellow oil.
6.12. (1S,5S,7R)-3-Aza-6,8-dioxa-bicyclo[3.2.1]octane-7-
carboxylic acid (biphenyl-4-ylmethyl)-amide (18)
A solution of 17 (438 mg, 1.06 mmol) and ammonium
formate (300 mg, 4.76 mmol) in MeOH (8 ml) and over
10% Pd/C was refluxed for 2 h under N2 atmosphere.
After separation of the catalyst by filtration over a
pad of Celite, the solvent was evaporated to afford 18
(220 mg, 64%) as a white solid.
1H NMR (CDCl3): d 7.54–7.25 (br s, 5H), 7.11 (br s,
1H), 4.84 (s, 2H), 4.14 (q, 2H, J = 7.07 Hz), 3.95–3.78
(m, 2H), 3.02–2.79 (m, 2H), 2.57–2.35 (m, 1H), 1.99–
1.80 (m, 2H), 1.77–1.51 (m, 2H), 1.25 (t, 3H,
J = 7.11 Hz); 13C NMR (CDCl3): d 174.2 (s), 158.8 (s),
135.9 (s), 129.1, 128.5, 128.5 (d), 77.94 (t), 60.61 (t),
43.32 (t), 40.79 (d), 27.70 (t), 14.17 (q); MS m/z 306
22
D
Mp 89–92 ꢁC; ½aꢂ +11.8 (c 1.0, CHCl3); 1H NMR
(CDCl3): d 7.59–7.26 (m, 9H), 6.98 (br s, 1H), 5.57 (s,
1H), 4.79 (s, 1H), 4.68 (s, 1H), 4.52 (m, 2H), 3.29 (d,
1H, J = 13.2 Hz), 2.98 (d, 2H, J = 13.6 Hz), 2.80 (d,
1H, J = 13.6 Hz); 13C NMR (CDCl3): d 171.0 (s),
140.6, 136.7 (s), 128.7, 128.0, 127.4, 127.3, 127.0 (d),
101.1 (d), 77.42 (d), 54.21 (t), 53.84 (t), 42.86 (t); MS
m/z 324 (M+, 5), 167 (100); IR (CHCl3) 3420, 1673,
(M+, 1), 91 (100); IR (CHCl3) 1724, 1676, 1222 cmꢁ1
;
Anal. Calcd for C16H22N2O4 (306.16): C, 62.73; H,
7.24; N, 9.14. Found: C, 61.65; H, 7.74; N, 8.94.
6.10. 1-Hydroxycarbamoyl-piperidine-4-carboxylic acid
ethyl ester (15)
1527, 1110 cmꢁ1
;
Anal. Calcd for C19H20N2O3
A stirred solution of 14 (150 mg, 0.5 mmol) in AcOEt
was submitted to hydrogenolysis over 10% Pd/C at
1 atm overnight. After separation of the catalyst by cen-
trifugation, the solvent was evaporated and the crude
residue washed with Et2O affording 15 (108 mg, quanti-
tative) as a white solid.
(324.15): C, 70.35; H, 6.21; N, 8.64. Found: C, 70.25;
H, 5.91; N, 8.90.
6.13. (1S,5S,7R)-3-Aza-6,8-dioxa-bicyclo[3.2.1]octane-
3,7-dicarboxylic acid 3-(benzyloxy-amide) 7-[(biphenyl-4-
ylmethyl)-amide] (19)
1
Mp 101–104 ꢁC; H NMR (CDCl3): d 6.46 (br s, 1H),
O-Benzyl-hydroxylamine16 (69 mg, 0.56 mmol) was dis-
solved in anhydrous THF (1.6 ml) and added dropwise
to a cooled solution of carbonyldiimidazole (91 mg,
4.13 (q, 2H, J = 7.09 Hz), 3.86 (dt, 2H, J1 = 13.45,
J2 = 3.69 Hz), 3.05–2.81 (m, 2H), 2.48 (m, 1H),

7402
C. Mannino et al. / Bioorg. Med. Chem. 14 (2006) 7392–7403
0.56 mmol) in 5 ml of dry THF under N2 atmosphere.
After being stirred for 30 min at room temperature,
the resulting solution was added to the neat 18
(181 mg, 0.56 mmol) and the reaction mixture was stir-
red overnight. The solution was then diluted with
AcOEt, washed with water and brine, dried over
Na2SO4 and evaporated in vacuo. Crystallization at
room temperature from ethyl acetate afforded the pure
19 (160 mg, 60%) as a white solid.
Mrs. B. Innocenti and Mr. M. Passaponti for technical
assistance.
References and notes
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26
D
1
Mp 110–114 ꢁC; ½aꢂ +29.6 (c 0.77, CDCl3); H NMR
(CDCl3): d 7.60–7.26 (m, 14H), 6.90 (br s, 1H), 5.62 (s,
1H), 4.85 (s, 2H), 4.76 (s, 1H), 4.60–4.48 (m, 3H), 3.89
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;
Anal. Calcd for C27H27N3O5 (473.20): C, 68.48; H,
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3-hydroxyamide (20)
A stirred solution of 19 (120 mg, 0.25 mmol) in THF
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1 atm, at room temperature, overnight. After separation
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solvent was evaporated and the crude residue washed
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26
D
Mp 75–80 ꢁC; ½aꢂ +36.6 (c 0.75, CDCl3); 1H NMR
(CDCl3): (Mixture of rotamers): d 11.58 (br s, 1H),
8.41 (br s, 1H), 7.62–7.20 (m, 9H), 7.13–6.90 (m, 1H),
5.65 (s, 1H, minor rotamer), 5.60 (s, 1H, major rot-
amer), 4.78 (s, 1H, minor rotamer), 4.73 (s, 1H, major
rotamer), 4.66–4.57 (m, 1H), 4.55–4.38 (m, 2H), 4.02–
3.84 (m, 1H), 3.80–3.62 (m, 1H), 3.41–3.24 (m, 1H),
3.18–2.96 (m, 1H); 13C NMR (CDCl3): (mixture of rota-
mers): d 170.8 (s, major rotamer), 170.5 (s, minor rot-
amer), 161.5 (s, major r.), 159.0 (s, minor r.), 140.6,
140.5, 136.5 (s), 128.8–127.0 (d), 99.79 (d), 77.22 (d, min-
or r.), 76.93 (d, major r.), 75.68 (d), 47.46 (t, minor r.),
46.97 (t, major r.), 46.48 (t, minor r.), 45.88 (t, major
r.), 42.80 (t); MS m/z 383 (M+, 0.2), 324 (7), 167 (100);
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;
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The authors thank Ministero della Ricerca Scientifica e
Tecnologica,
RBNE01TTJW, COFIN 2003–2005 and COFIN
Roma;
MIUR
for
contracts
`
2004–2006. GenExpress (Universita di Firenze), Ente
Cassa di Risparmio di Firenze, Marie Curie Training
Sites (HPMT-CT-2000-00137) and CINMPIS are also
acknowledged for financial support. C. M. thanks
CERM for a PhD fellowship. The authors also thank

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