Stereochemically pure α-trifluoromethyl-malic hydroxamates: synthesis and evaluation as inhibitors of matrix metalloproteinases

Article abstract of DOI:10.1016/j.tet.2006.08.036

The synthesis of trifluoromethyl (Tfm) analogs of known nanomolar matrix metalloproteinases (MMPs) inhibitors has been performed. The synthetic protocol is based on a moderately stereoselective aldol reaction of trifluoropyruvate with an N-acyl-oxazolidin

Full text of DOI:10.1016/j.tet.2006.08.036

Tetrahedron 62 (2006) 10171–10181
Stereochemically pure a-trifluoromethyl-malic hydroxamates:
synthesis and evaluation as inhibitors of matrix metalloproteinases
a
c
Margherita Moreno,a,y Monica Sani,b,y Guido Raos,a, Stefano V. Meille, Dorina Belotti,
*
Raffaella Giavazzi,c Stefano Bellosta,d Alessandro Volonterioa and Matteo Zandab,
*
aDipartimento di Chimica, Materiali ed Ingegneria Chimica ‘G. Natta’ del Politecnico di Milano, via Mancinelli 7,
I-20131 Milano, Italy
bC.N.R.—Istituto di Chimica del Riconoscimento Molecolare, via Mancinelli 7, I-20131 Milano, Italy
cDipartimento di Oncologia, Istituto di Ricerche Farmacologiche Mario Negri, via Gavazzeni 11, 24125 Bergamo, Italy
dDipartimento di Scienze Farmacologiche, Universitaꢀ degli Studi di Milano, via Balzaretti 9, I-20133 Milano, Italy
Received 21 March 2006; revised 25 July 2006; accepted 10 August 2006
Available online 1 September 2006
Abstract—The synthesis of trifluoromethyl (Tfm) analogs of known nanomolar matrix metalloproteinases (MMPs) inhibitors has been per-
formed. The synthetic protocol is based on a moderately stereoselective aldol reaction of trifluoropyruvate with an N-acyl-oxazolidin-2-thione
for the construction of the core a-Tfm-malic unit. Both the diastereomeric forms of the target a-Tfm-malic hydroxamates showed micromolar
inhibitory potency toward MMP-2 and 9, according to zymographic tests, with a substantial drop with respect to the parent unfluorinated
compounds. We also report some molecular modeling results, which provide a rationale for the experimental findings.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
work evidenced the importance of MMPs inhibitors sparing
MMP-1, an enzyme thought to be responsible of the mus-
culoskeletal side effect observed clinically with the broad-
spectrum MMP inhibitor marimastat.4
Incorporation of fluorine into organic molecules is an effec-
tive strategy for improving and modifying their biological
activity.1 In particular, the trifluoromethyl group is recog-
nized in medicinal chemistry as a substituent of distinctive
qualities. It is, in fact, at the same time highly hydrophobic,
electron rich, sterically demanding, moreover it can provide
high in vivo stability, and is good mimic of several naturally
occurring residues such as methyl, isopropyl, phenyl, etc.2
Some years ago, Jacobson and co-workers described a new
family of potent peptidomimetic hydroxamate inhibitors A
(Fig. 1) of MMP-1, -3, and -9, bearing a quaternary a-methyl-
alcoholic moiety at P1 position, and several different R1
groups at P10.5 Interestingly, the other stereoisomers, includ-
ing the epimers at the quaternary carbinol function, showed
much lower activity, as the authors demonstrated that the
hydroxamic binding function was moved away from the cat-
alytic Zn2+ center. The crystal structure of the inhibitor A
(R¼CH3) with MMP-3 (see Fig. 3) reveals several interesting
features, including the presence of a hydrogen bond between
the quaternary hydroxyl (H-bond donor) of A and the
Glu-202 residue of the MMP-3 active site.6
Matrix metalloproteinases (MMPs) are zinc (II)-dependent
proteolytic enzymes involved in the degradation of the extra-
cellular matrix.3 More than 25 human MMPs have been iden-
tified so far. Loss in the regulation of their activity can result
in the pathological destruction of connective tissue, a process
associated with a number of severe diseases, such as cancer
and arthritis. The inhibition of various MMPs has been envis-
aged as a strategy for the therapeutic intervention against
such pathologies. To date, however, a number of drawbacks
have hampered the successful exploitation of MMPs as
pharmacological targets. In particular, the toxicity demon-
strated by many MMPs’ inhibitors in clinical trials has
been ascribed to nonspecific inhibition. For example, recent
P1
O
R2
HO R
H
N
NHR3
P3'
HO
N
H
R1
O
O
P1'
P2'
* Corresponding authors. Tel.: +39 02 23993084; fax: +39 02 23993080
(M.Z.); e-mail addresses: guido.raos@polimi.it; matteo.zanda@polimi.it
These authors contributed equally to the work.
A
R = CH3;
1
R = CF3
y
Figure 1.
0040–4020/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.08.036

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M. Moreno et al. / Tetrahedron 62 (2006) 10171–10181
Within the framework of a project aimed at studying the
‘fluorine effect’ in peptides and identifying selective fluori-
nated inhibitors of aspartic proteinases and MMPs,8 we de-
cided to explore the effect of the replacement of the
quaternary a-methyl group in A with a trifluoromethyl
(Tfm) group, with the hope of (1) increasing the affinity of
the a-Tfm malic inhibitors with MMPs by reinforcing the
a-OH hydrogen bonding, thanks to the increased acidity of
the carbinolic function bearing the electron-withdrawing
a-Tfm group; (2) improving the selectivity in favor of
MMP-3 and -9 through the increased stereo-electronic
demands of the Tfm group.
produced a switch of diastereocontrol affording a 1.6:1.0
mixture in favor of 5, in overall 74% yield. However, due
to the cost of (ꢀ)-sparteine and the absence of a favorable
scale-up effect, the TiCl4/DIPEA system was always em-
ployed for conducting the reaction on a multigram scale.
S
O F3C
OH
O
N
CO2Et
S
O
4
Bn
O
N
Ph
Major
Ph
TiCl4 (2 equiv)
+
Bn
DIPEA (2 equiv)
2
S
+
O F3C
In this paper we describe in full detail the synthesis of the
Tfm-analogs 1 (Fig. 1) of A, the effect of the replacement
of the a-CH3 group with a CF3 on the inhibition of MMP-
3 and 9, and an attempt to rationalize the experimental find-
ings through molecular modeling.9
CH2Cl2 (dry)
OH
CO2Et
CF3
O
N
0°C, then -70°C,
then rt
O
CO2Et
5
Bn
Minor
d.r. 2.7:1.0
3
Ph
yield 72% (hundreds
mgs scale), 90% (10
grams scale)
2. Results and discussion
2.1. Synthesis
Scheme 1. The aldol reaction to form the a-Tfm-malic framework.
We decided to concentrate our efforts on the substrates 1
having R1¼(CH2)4Ph, whose analogs A were reported to
be very active. The a-Tfm-malic unit of 1 was recently
obtained by our group via titanium (IV) catalyzed aldol
reaction of trifluoropyruvic esters with enantiopure N-acyl
oxazolidin-2-ones.10 Although this reaction was per se satis-
factory, the subsequent exocyclic cleavage of the oxazolidin-
2-one auxiliary could not be performed, despite intensive
efforts. We therefore decided to exploit the potential of ox-
azolidin-2-thiones,11 whose cleavage has been reported to
occur much more smoothly.12
Cleavage of the oxazolidin-2-thione was found to be consid-
erably more challenging than expected. In fact, under the
standard conditions reported in the literature (BnOH, cat.
DMAP, DCM, rt) the reaction on 4 was very slow,12 afford-
ing modest conversion to the corresponding Bn-ester 6
(60%) after one week at reflux (Scheme 2), with partial
(17%) a-epimerization to give ent-7. Even less effectively,
the same reaction performed on 5 gave 56% yield of the
diastereomeric Bn-ester 7, containing 33% of the a-epimer
ent-6. Although the unreacted starting materials 4 and 5
could be recovered unchanged in good yields, we felt that
more efficient conditions were needed in order to complete
the synthesis. Disappointingly, exploration of several differ-
ent combinations of alcohols, solvents, and bases did not
improve the situation.
The TiCl4 catalyzed reaction of the N-acyl-oxazolidin-2-thi-
one 2 (Scheme 1) with ethyl trifluoropyruvate 3 afforded the
two diastereomeric adducts 4 and 5, out of four possible, in
low diastereomeric ratio. It is worth noting that the reaction
features a favorable scale-up effect, affording ca. 70% yield
on a hundred-milligram scale, and 90% on a ten-gram scale
(the reaction was repeated many times on both scales). Sev-
eral alternative conditions were explored, but no improve-
ment in terms of diastereocontrol could be achieved. For
example, with Sn(OTf)2/NEt3 and Bu2BOTf/NEt3 no reac-
tion was observed, whereas LDA afforded a 4:5 ratio¼2.6/
1.0 (48% overall). However, the use of TiCl4/(ꢀ)-sparteine
However, we were glad to find that solid K2CO3 in moist
dioxane (rt, 10–12 h) was able to produce directly the key
carboxylic acid intermediates 8 and 9 (Scheme 3), from 4
and 5, respectively, in satisfactory yields and with very
low a-epimerization (2% for 8, 9% for 9).
In order to assess the stereochemistry of these compounds,
the diastereomer 8 was coupled with L-Ala-NH(CH2)2Ph
F3C
F3C
S
O F3C
OH
CO2Et
OH
CO2Et
OH
CO2Et
BnO2C
BnO2C
BnOH (2 equiv)
O
N
+
DCM, reflux
1 week (60%)
6
ent-7
4
Bn
Ph
Ph
Major
Ph
4.8:1.0
F3C
S
N
F3C
O F3C
OH
CO2Et
OH
CO2Et
OH
CO2Et
BnO2C
ent-6
BnO2C
BnOH (2 equiv)
DCM, reflux
O
+
7
5
1 week
(56%)
Bn
Minor
Ph
Ph
2:1
Ph
Scheme 2. Attempted oxazolidin-2-thione cleavage with BnOH.

M. Moreno et al. / Tetrahedron 62 (2006) 10171–10181
10173
S
O F3C
F3C
OH
OH
CO2Et
HO2C
K2CO3 (1.5 equiv)
O
O
N
CO2Et
moist dioxane
10 hours
4
8
(71%)
Bn
Major
Ph
Ph
S
N
F3C
OH
O F3C
OH
CO2Et
HO2C
K2CO3 (1.5 equiv)
CO2Et
moist dioxane
12 hours
9
(69%)
5
Bn
Minor
Ph
Ph
Scheme 3. Cleavage of the oxazolidin-2-thione auxiliary with K2CO3 in
moist dioxane.
Figure 2. ORTEP view of 8x.
(Scheme 4), affording the crystalline dipeptide 8x, whose
structure was determined by X-ray diffraction (Fig. 2).13
by hydrogenolysis, that provided the hydroxamates 14a–c
in good yields.
Coupling of the acid 8 with a-amino acid amides 10a–c was
achieved in good yields with the HOAt/HATU system
(Scheme 5).14 The resulting peptidomimetic esters 11a–c
were submitted to saponification, affording the acids 12a–
c in high yields. The subsequent coupling of 12a–c with
O-Bn hydroxylamine proved to be extremely challenging,
owing to the low reactivity and high steric hindrance of
the carboxylic group bound the quaternary a-Tfm carbinolic
center. A number of ‘conventional’ coupling agents for pep-
tides15 were tested (among them DCC/DMAP, EDC/HOBt,
DIC/HOBt, HATU/HOAt, PyBroP/DIPEA) but no trace of
the target O-Bn hydroxamates 13a–c could be obtained. Fi-
nally, we found that freshly prepared BrPO(OEt)2 was able
to promote the coupling in reasonable yields (32–61%).16
With 13a–c in hand we addressed the final O-Bn cleavage
Since 14a–c are the ‘wrong’ diastereomers with respect to A,
we deemed it necessary to synthesize at least one analog hav-
ing the correct stereochemistry, in order to have a complete
set of biological data on the effect of the introduction of
the Tfm group. However, a tailored synthetic protocol had
to be developed ex-novo, because the minor diastereomer 9
(Scheme 6) featured a dramatically different reactivity in
the key steps of the synthesis. First of all, we noticed that
the coupling of 9 and 10a with HATU/HOAt gave rise to
relevant amounts of the b-lactone 15, which had to be pro-
cessed separately, besides the expected coupling product 16.
Thus, we decided to first prepare the intermediate 15 (72%),
which could be purified by short flash chromatography (FC).
CF3
HO
EtO2C
CF3
O
HO
H
N
CO2H
H
EtO2C
N
H
Ph
HOAt/HATU
N
H2N
Ph
+
O
TMP, DMF
76%
Ph
8x
O
8
Ph
Scheme 4. Synthesis of the dipeptide 8x for X-ray diffraction.
CF3
HO
EtO2C
CO2H
R2
CF3
O
R2
CF3
O
HO
HO
KOH 0.5 N
NHCH3
8
NHCH3
MeOH/H2O 7:3
(92-96%)
EtO2C
N
HOAt/HATU
TMP, DMF
HO2C
N
H
H
Ph
R2
O
O
+
Ph
Ph
12a-c
11a R2 = t-Bu (71%)
11b R2 = p-MeO-Bn (65%)
NHCH3
11c R2 = (CH2)3NHBoc (68%)
H2N
O
10a-c
CF3
O
R2
HO
CF3
O
R2
HO HN
NHCH3
HO
BnONH2
N
NHCH3
H2/Pd(OH)2
MeOH, rt
H
BrPO(OEt)2
BnOHNOC
N
H
O
O
O
TMP, DMF
CH2Cl2
Ph
Ph
13a R2 = t-Bu (61%)
14a R2 = t-Bu (87%)
13b R2 = p-MeO-Bn (60%)
14b R2 = p-MeO-Bn (79%)
13c R2 = (CH2)3NHBoc (32%)
14c R2 = (CH2)3NHBoc (60%)
Scheme 5. Synthesis of the peptidomimetics 14 from the major diastereomer 8.

10174
M. Moreno et al. / Tetrahedron 62 (2006) 10171–10181
F3C
O
CF3 O
OH
CO2Et
HO
CF3
CO2Et
KOH 0.5 N
O
HO2C
HOAt/HATU
TMP, DMF
10a, TMP
NHCH3
MeOH/H2O 7:3
EtO2C
N
H
DMF
O
9
15
Ph
Ph
16
(89%)
(72%)
Ph
CF3
CF3
O
HO
NHCH3
HO2C
N
H
CF3
O
1. BnONH2, BrPO(OEt)2,
TMP, DMF, CH2Cl2
O
HO
Ph
17
H2/Pd(OH)2
MeOH, rt
NHCH3
+
BnOHNOC
N
H
2. Flash chromatogr.
O
Ph
19
(52%)
O
HO
NHCH3
HO2C
N
H
O
18
CF3
O
Ph
HO
O
HO HN
NHCH3
3:1 mixture (90% overall)
N
H
O
(83%)
20
Ph
Scheme 6. Synthesis of the target peptidomimetic 20 from the minor diastereomer 9.
The latter was reacted with free 10a, affording the desired
molecule 16 in high yields.17
2.2. Simulations
In an attempt to explain the different activities of the hydro-
genated (A) and the fluorinated (1) compounds, we first
performed molecular dynamics (MD) simulations of a few
protein-inhibitor complexes. The experimental X-ray struc-
tures5 of the complexes of 1 with MMP-3 (Fig. 3) provided
a very convenient starting point for these simulations. How-
ever we found that these simulations could not clearly dis-
criminate between the different inhibitors, since both the
hydrogenated and the fluorinated ligands remained coordi-
nated to the active site within the time of the simulation.
The likely reason for this failure is the limited time span,
which could be explored by MD with current computer re-
sources (a few nanoseconds). Therefore, instead of studying
the whole protein–ligand complexes, we decided to factor
the problem and investigate separately the effect of fluorina-
tion on (a) the coordinating strength of the hydroxamate
group and (b) non-bonded interactions and the conforma-
tional equilibrium of the ligands.
Saponification of the ester 16 occurred effectively, but disap-
pointingly a partial epimerization of the [Ph(CH2)3]-stereo-
center occurred, affording a 3:1 mixture of diastereomers 17
and 18 under optimized conditions. Since their chromato-
graphic separation proved to be difficult, 17 and 18 were
subjected together to coupling with BnONH2 under the pre-
viously optimized conditions. The resulting diastereomeric
O-Bn hydroxamates could be separated by FC, affording
pure 19 (52%), that was hydrogenated to the target free
hydroxamate 20 in 83% yield.
The hydroxamates 14a–c and 20 were tested for their ability
to inhibit MMP-2 and MMP-9 activity using zymographic
analysis. The four hydroxamates inhibited, in a dose-depen-
dent manner, the gelatinolytic bands at 92 and 72 kDa,
corresponding respectively to pro-MMP-9 and pro-MMP-2
released in the conditioned medium by human melanoma
cells WM983A. The IC50 values (mM) portrayed in Table
1 show that diastereomers 14a–c displayed low inhibitory
activity, in line with the parent CH3 compounds. Disappoint-
ingly, 20 showed a much lower activity than the exact CH3-
analog A, that was reported to be a low nanomolar inhibitor
of MMP-9.
The effect of fluorination on the coordinating strength of the
ligands was investigated by ab initio B3LYP/6-31G* calcu-
lations on the reactants and products of the exchange reac-
tion shown in Figure 4 (see Section 3). After energy
minimization, we computed the net reaction energy as DE¼
E(products)ꢀE(reactants)¼+8.8 kJ/mol. Therefore the re-
placement of –CH3 by –CF3 reduces the coordinating ability
of the hydroxamate, presumably because of the electron-
withdrawing effect of the latter.
Table 1. IC50 (mM) of the target Tfm-hydroxamates
Compound
MMP-2
MMP-9
14a
14b
14c
20
156
407
722
23
121
84
23
15
Inspection of the conformation adopted by inhibitors A
within the active site of MMP-3 reveals that the closest
non-bonded contact formed by the methyl in P1 position is
an intramolecular one, with the aromatic ring of the
pseudo-tyrosine at the P20 position (see Fig. 5). Replacement
of this methyl by the more sterically demanding and elec-
tron-rich Tfm might produce a change in the intramolecular
conformational equilibria of the ligand, affecting as a side
It is also worth noting that 14a and 20 showed little selectiv-
ity, whereas 14b and 14c showed a better affinity for MMP-
9, in comparison with MMP-2.

M. Moreno et al. / Tetrahedron 62 (2006) 10171–10181
10175
H3CO
O
CH
HO
3H
H
N
N
S
H3C
N
H
OH
O
O
A
Figure 3. X-ray structure of the inhibitor A [R1¼(CH2)3Ph, R2¼4-CH3O–C6H4CH2, R3¼CH3] bound to MMP-3 (courtesy of Bristol–Myers Squibb). The Zn2+
ion in the active site, the inhibitor, the three coordinating histidines, and the Glu-202 residue have been highlighted. Image produced with VMD.7
H
N
H
N
H
N
H
N
OH
CH3
CH3
OH
CF3
CH3
H
H
O
O
H
N
H
N
N
N
OH
CF3
CH3
OH
CH3
CH3
H
H
O
O
Zn2+
Zn2+
O
O
+
+
N
N
N
N
O
O
N
H
N
H
N
H
N
H
Figure 4. Exchange reaction between models of the hydrogenated and fluorinated ligands at the active site, investigated by ab initio calculations.
effect its ability to fit inside the protein active site. Therefore,
we decided to carry out MD simulations of the inhibitors in
water to test this hypothesis.
shows that the resulting torsion free energies are very similar,
except for one important detail: the conformation at 300ꢁ,
which is the one adopted by the inhibitor in the active site,
is slightly destabilized by the introduction of the –CF3 group.
We identify this destabilization with the unfavorable inter-
action between the electron-rich Tfm and aromatic group.
The lowest-energy conformation (by 2.5 kJ/mol) now corre-
sponds to the state at 60ꢁ. As can be seen in Figure 5, this
produces major change in the overall molecular shape.
The analysis of a long (12 ns) MD simulation of the inhibitors
in water (see Section 3 for details) produced very similar his-
tograms for the populations of almost every torsion angle,
indicating that they are mostly unaffected by fluorine substi-
tution. However, we observed a certain difference in the pop-
ulations of the C–C bond connecting the pseudo-tyrosine
residue to the backbone (highlighted in yellow in Fig. 5).
Thereforewe decided to investigate further this point by com-
puting the free energy profile of the molecules along this
particular degree of freedom (see again Section 3). Figure 6
A (-CH3)
1 (-CF3)
25
20
15
10
5
0
0
60
120
180
240
300
360
Figure 5. Minimum free energy conformations of A (left) and 1 (right), as
obtained by MD simulations in water. With reference to Figure 1,
R1¼CH2CH(CH3)2, R2¼4-CH3O–C6H4CH2, R3¼CH3. The conformation
of A coincides with the experimental conformation within the active site.
The broken yellow line indicates the torsion angle sampled in the free energy
calculations. Images produced with VMD.7
Torsion angle (°)
Figure 6. Free energy curves of A and 1 in water, corresponding to the
torsion about the C–C bond connecting the pseudo-tyrosine residue to the
backbone. The arrow indicates the conformation adopted inside the protein
active site.

10176
M. Moreno et al. / Tetrahedron 62 (2006) 10171–10181
In summary, our molecular modeling study has allowed to
identify two concurrent reasons for the reduced activity of
the fluorinated inhibitors: (a) reduced coordinating strength
of the neighboring hydroxamate group, and (b) the need of
the fluorinated molecule to adopt within the binding site
a conformation which does not coincide with its mini-
mum-energy conformation in solution. Assuming additivity
of these effects, we estimate that the overall binding energy
of the fluorinated inhibitor to the active site is reduced by
approximately 11.3 kJ/mol, compared to the original one.
This results, at room temperature, in the reduction in the
binding constant by two order of magnitude ½KF=KH ¼
expfꢀ11:3=ð0:00831 ꢂ 298Þg¼ 0:010ꢃ. This result roughly
corresponds to the experimental observation.
(s, 3F); MS (DIS EI 70 eV) m/z (%): 524 [M+H+] (36),
330 (62), 194 (100); HRMS m/z 523.1630 (calculated
523.1633, C26H28F3NO5S).
Compound 5: yellow oil; Rf 0.36 (EtOAc/n-Hex 2:8); [a]D23
+37.3 (c 1.6, CHCl3); FTIR (film) nmax: 3467, 1747, 1693,
1
1498 cmꢀ1; H NMR (250 MHz, CDCl3) d: 7.47–7.02 (m,
10H), 6.34 (dd, J¼10.4, 3.7 Hz, 1H), 4.95 (m, 1H), 4.44–
4.14 (m, 5H), 3.29 (dd, J¼13.1 and 3.1 Hz, 1H), 2.67 (dd,
J¼13.1 and 10.4 Hz, 1H), 2.65–2.53 (m, 2H), 2.11–1.99
(m, 1H), 1.85–1.45 (m, 3H), 1.32 (t, J¼6.9 Hz, 3H); 13C
NMR (62.9 MHz, CDCl3) d: 184.7, 174.3, 167.1, 141.2,
134.9, 129.3, 129.0, 128.3, 127.5, 123.2 (q, J¼288.5 Hz),
79.1 (q, J¼29.6 Hz), 69.8, 63.7, 59.9, 42.7, 37.0, 35.0,
28.8, 28.3, 13.8; 19F NMR (235.3 MHz, CDCl3) d: ꢀ75.6
(s, 3F); MS (DIS EI 70 eV) m/z (%): 524 [M+H+] (13),
330 (38), 194 (100).
3. Experimental
3.1. General details
3.3. Cleavage of the oxazolidin-2-thione
Commercially available reagent-grade solvents were em-
ployed without purification. All reactions where an organic
solvent was employed were performed under nitrogen atmo-
sphere, after flame-drying of the glass apparatus. Melting
points (mp) are uncorrected and were obtained on a capillary
apparatus. TLC were run on silica gel 60 F254 Merck. Flash
chromatographies (FC) were performed with silica gel 60
3.3.1. A—Cleavage with BnOH. General procedure. To
a solution of 4 (102 mg, 0.19 mmol) in CH2Cl2 (5 mL),
neat BnOH (40 mL, 0.38 mmol) and DMAP (4.2 mg,
0.038 mmol) were added. The solution was refluxed for
one week. The solvent was removed in vacuo and the crude
was purified by FC (n-Hex/EtOAc 9:1), affording 30 mg of
6, 5 mg of ent-7, and 15 mg of their mixture (60% overall
yield).
1
(60–200 mm, Merck). H, 13C, and 19F NMR spectra were
run at 250, 400 or 500 MHz. Chemical shifts are expressed
in parts per million (d), using tetramethylsilane (TMS) as
Compound 6: colorless oil; Rf 0.25 (EtOAc/n-Hex 1:9); [a]D23
+7.9 (c 0.9, CHCl3); FTIR (film) nmax: 3741, 1748,
1
internal standard for H and 13C nuclei (dH and dC¼0.00),
whileC6F6 wasusedasexternalstandard(dF ꢀ162.90)for19F.
1456 cmꢀ1; H NMR (250 MHz, CDCl3) d: 7.41–6.99 (m,
1
10H), 5.08 (s, 2H), 4.23–3.99 (m, 3H), 3.26 (dd, J¼9.4,
4.5 Hz, 1H), 2.64–2.53 (m, 2H), 2.02–1.46 (m, 4H), 1.24
(t, J¼6.6 Hz, 3H); 13C NMR (62.9 MHz, CDCl3) d: 171.1,
168.9, 141.7, 134.9, 128.6, 128.5, 128.3, 123.1 (q,
J¼288.4 Hz), 78.1 (q, J¼29.6 Hz), 67.1, 63.8, 47.7, 35.6,
29.2, 25.8, 13.6; 19F NMR (235.3 MHz, CDCl3) d: ꢀ75.1
(s, 3F); MS (DIS EI 70 eV) m/z (%): 439 [M+H+] (54),
117 (78), 91 (100); HRMS m/z 438.1649 (calculated
438.1647, C23H25F3O5).
3.2. Synthesis of aldol adducts 4 and 5
To a solution of N-acyl-oxazolidin-2-thione 2 (107 mg,
0.30 mmol) in dry CH2Cl2 (4 mL), cooled at 0 ꢁC and under
nitrogen atmosphere, a 1 M solution of TiCl4 in toluene
(600 mL, 0.60 mmol) was added. The solution was stirred
for 5 min, then neat DIPEA (103 mL, 0.60 mmol) was added.
The dark red solution of titanium enolate was stirred for
20 min at 0 ꢁC, then cooled at ꢀ70 ꢁC and neat ethyl tri-
fluoropyruvate 3 (100 mL, 0.75 mmol) was added dropwise.
The resulting mixture was stirred for 1 h at ꢀ70 ꢁC, then
warmed to rt. The reaction was quenched with a saturated
aqueous NH4Cl solution. The layers were separated and
the aqueous phase was extracted with CH2Cl2. The collected
organic phases were dried over anhydrous Na2SO4, filtered
and the solvent was removed in vacuo. The residue was
purified by FC (n-Hex/EtOAc 9:1), affording 59 mg of 4,
33 mg of 5, and 21 mg of their mixture (72% overall yield).
Compound 7: colorless oil; Rf 0.20 (EtOAc/n-Hex 1:9); [a]D23
ꢀ3.8 (c 1.4, CHCl3); FTIR (film) nmax: 3467, 1746,
1
1456 cmꢀ1; H NMR (250 MHz, CDCl3) d: 7.29–6.99 (m,
10H), 5.21 (d, J¼12.0 Hz, 1H), 5.11 (d, J¼12.0 Hz, 1H),
4.47 (s, 1H), 4.35–4.20 (m, 2H), 3.21 (dd, J¼11.7, 3.4 Hz,
1H), 2.56 (m, 2H), 2.07–1.87 (m, 1H), 1.65–1.29 (m, 3H),
1.24 (t, J¼6.8 Hz, 3H); 13C NMR (62.9 MHz, CDCl3) d:
171.9, 167.2, 141.2, 134.9, 128.6, 128.3, 128.2, 125.9,
123.1 (q, J¼288.5 Hz), 78.5 (q, J¼27.7 Hz), 67.4, 63.7,
46.9, 35.1, 28.6, 27.1, 13.8; 19F NMR (235.3 MHz,
CDCl3) d: ꢀ76.9 (s, 3F); MS (DIS EI 70 eV) m/z (%): 439
[M+H+] (59), 421 (100).
Compound 4: yellow oil; Rf 0.44 (EtOAc/n-Hex 2:8); [a]D23
+88.9 (c 1.4, CHCl3); FTIR (film) nmax: 3462, 1748, 1691,
1
1348 cmꢀ1; H NMR (250 MHz, CDCl3) d: 7.48–7.00 (m,
10H), 6.11 (dd, J¼7.3, 5.8 Hz, 1H), 4.76 (m, 1H), 4.44–
4.14 (m, 4H), 4.11–4.00 (m, 1H), 3.23 (dd, J¼13.1,
3.1 Hz, 1H), 2.71–2.49 (m, 3H), 2.12–1.94 (m, 2H), 1.89–
1.51 (m, 2H), 1.31 (t, J¼7.3 Hz, 3H); 13C NMR
(62.9 MHz, CDCl3) d: 184.5, 174.4, 168.3, 141.4, 134.9,
129.3, 129.1, 128.3, 127.5, 125.9, 123.0 (q, J¼286.7 Hz),
78.6 (q, J¼29.6 Hz), 69.7, 63.7, 60.5, 43.8, 37.1, 35.9,
28.4, 27.3, 13.9; 19F NMR (235.3 MHz, CDCl3) d: ꢀ75.4
3.3.2. B—Cleavage with K2CO3. General procedure. To
a solution of 4 (91 mg, 0.17 mmol) in moist dioxane
(2 mL), K2CO3 (70 mg, 0.51 mmol) was added. The result-
ing mixture was stirred for 10 h. The solvent was removed in
vacuo and the crude was dissolved in EtOAc. A 1 M solution
of HCl was added until pH 1–2 was reached. The layers were
separated and the aqueous phase was extracted with EtOAc.
The collected organic phases were dried over anhydrous

M. Moreno et al. / Tetrahedron 62 (2006) 10171–10181
10177
Na2SO4, filtered and the solvent was removed in vacuo to
give 42 mg of 8 (71% yield).
3H), 2.63–2.46 (m, 2H), 1.86–1.65 (m, 2H), 1.52–1.39 (m,
2H), 1.28 (t, J¼7.2 Hz, 3H); 13C NMR (100.6 MHz,
CDCl3) d: 171.2, 170.7, 169.2, 158.8, 141.5, 130.1, 128.4,
128.3, 128.2, 125.9, 122.8 (q, J¼287.5 Hz), 114.3, 78.5 (q,
J¼29.3 Hz), 64.0, 55.2, 54.8, 49.3, 37.3, 35.5, 28.6, 26.2,
13.8; 19F NMR (235.3 MHz, CDCl3) d: ꢀ74.3 (s, 3F); MS
(DIS EI 70 eV) m/z (%): 539 [M+H+] (34), 191 (100);
HRMS m/z 538.2281 (calculated 538.2282, C27H33F3N2O6).
Compound 8: colorless oil; Rf 0.48 (CHCl3/MeOH 8:2);
[a]2D3 +16.5 (c 1.4, MeOH); FTIR (film) nmax: 3466.6,
1732.9, 1454.5 cmꢀ1 1H NMR (400 MHz, CD3OD) d:
;
7.29–7.05 (m, 5H), 4.25 (q, J¼7.5 Hz, 2H), 3.13 (m, 1H),
2.71–2.53 (m, 2H), 1.95–1.52 (m, 4H), 1.27 (t, J¼7.5 Hz,
3H); 13C NMR (100.6 MHz, CD3OD) d: 177.0, 168.9,
141.5, 128.3, 128.2, 125.9, 122.9 (q, J¼288.5 Hz), 77.9 (q,
J¼29.6 Hz), 64.2, 47.8, 35.6, 29.2, 25.7, 13.6; 19F NMR
(235.5 MHz, CD3OD) d: ꢀ75.6 (s, 3F); MS (DIS EI
70 eV) m/z (%): 349 [M+H+] (65), 330 (60), 91 (100);
HRMS m/z 348.1200 (calculated 348.1198, C16H19F3O5).
Compound 11c: white solid; Rf 0.19 (AcOEt/n-Hex 1:1);
[a]2D3 +13.9 (c 0.84, CHCl3); FTIR (microscope) nmax
:
3284, 1750, 1639, 1531 cmꢀ1
;
1H NMR (250 MHz,
CDCl3) d: 7.43 (br s, 1H), 7.35–7.08 (m, 5H), 7.02 (br s,
1H), 5.01 (br s, 1H), 4.59 (br s, 1H), 4.38–4.19 (m, 2H),
3.46–3.25 (m, 1H), 3.18 (dd, J¼10.8, 4.2 Hz, 1H), 3.13–
2.95 (m, 1H), 2.74 (d, J¼4.4 Hz, 3H), 2.69–2.54 (m, 2H),
2.03–1.70 (m, 4H), 1.69–1.56 (m, 2H), 1.46 (s, 9H), 1.31
(t, J¼6.8 Hz, 3H); 13C NMR (62.9 MHz, CDCl3) d: 171.9,
171.6, 168.8, 156.7, 141.5, 128.4, 128.2, 125.7, 122.9 (q,
J¼286.7 Hz), 78.9 (q, J¼29.6 Hz), 63.2, 48.3, 35.5, 28.6,
28.3, 25.9, 13.7; 19F NMR (235.3 MHz, CDCl3) d: ꢀ74.2
(s, 3F); MS (DIS EI 70 eV) m/z (%): 576 [M+H+] (20),
474 (30), 430 (60), 57 (100); HRMS m/z 575.2811 (calcu-
lated 575.2808, C27H40F3N3O7).
Compound 9: colorless oil; Rf 0.46 (CHCl3/MeOH 8:2);
[a]2D3 +11.9 (c 0.9, MeOH); FTIR (film) nmax: 3476, 1748,
1454 cmꢀ1 1H NMR (400 MHz, CD3OD) d: 7.28–7.09
;
(m, 5H), 4.29–4.15 (m, 2H), 3.05 (dd, J¼11.7, 3.2 Hz,
1H), 2.61 (dt, J¼7.2, 2.0 Hz, 2H), 1.97–1.85 (m, 1H),
1.76–1.59 (m, 2H), 1.44–1.32 (m, 1H), 1.21 (t, J¼7.2 Hz,
3H); 13C NMR (100.6 MHz, CD3OD) d: 174.8, 168.7,
141.8, 129.4, 126.9, 124.8 (q, J¼282.6 Hz), 80.4 (q,
J¼27.7 Hz), 64.1, 49.2, 36.2, 30.2, 27.8, 14.2; 19F NMR
(235.3 MHz, CD3OD) d: ꢀ74.1 (s, 3F); MS (DIS EI 70 eV)
m/z (%): 349 [M+H+] (5), 330 (18), 159 (40), 91 (100).
Compound 8x: white solid; Rf 0.29 (AcOEt/n-Hex 4:6); [a]D23
ꢀ10.4 (c 1.2, CHCl3); mp¼93–97 ꢁC (EtOAc/n-Hex);
3.4. Synthesis of peptidomimetics esters 11a–c and 8x.
General procedure
FTIR (microscope) nmax: 3280, 1752, 1641, 1533.5 cmꢀ1
;
1H NMR (250 MHz, CDCl3) d: 7.37–7.11 (m, 10H), 6.53
(d, J¼7.9 Hz, 1H), 5.96 (br t, J¼5.3 Hz, 1H), 4.36–4.22
(m, 3H), 3.59–3.43 (m, 2H), 3.04 (dd, J¼11.2, 5.3 Hz, 1H),
3.87–2.77 (m, 2H), 2.73–2.57 (m, 2H), 1.94–1.82 (m, 2H),
1.79–1.68 (br s, 1H), 1.67–1.57 (m, 2H), 1.33 (t, J¼7.2 Hz,
3H), 1.27 (d, J¼6.6 Hz, 3H); 13C NMR (62.9 MHz,
CDCl3) d: 171.4, 170.7, 168.8, 141.5, 138.4, 128.7,
128.6, 128.3, 128.2, 126.5, 125.9, 123.0 (q, J¼286.7 Hz),
78.6 (q, J¼29.5 Hz), 63.6, 48.7, 40.7, 35.5, 35.3, 28.7,
25.8, 18.4, 13.8; 19F NMR (235.3 MHz, CDCl3) d: ꢀ73.8
(s, 3F); MS (DIS EI 70 eV) m/z (%): 576 [M+H+] (20), 474
(30), 430 (60), 57 (100). Anal. Calcd for C27H33F3N2O5:
C, 62.06; H, 6.37; N, 5.36. Found: C, 61.98; H, 6.44;
N, 5.31.
To a solution of 8 (198 mg, 0.57 mmol) and 10a (98 mg,
0.68 mmol) in dry DMF (5 mL), HOAt (77 mg,
0.57 mmol), HATU (217 mg, 0.57 mmol), and TMP
(150 mL, 1.14 mmol) were added. The solution was stirred
for 2 h, then diluted with H2O. The resulting mixture was ex-
tracted with Et2O and the organic phase was washed with
a 1 M solution of HCl and with brine. After drying over
anhydrous Na2SO4, and filtration, the solvent was removed
in vacuo, and the residue was purified by FC (n-Hex/EtOAc
6:4) to give 192 mg of 11a (71% yield), as a white solid: Rf
0.67 (EtOAc/n-Hex 6:4); [a]2D3 +4.5 (c 1.8, CHCl3); FTIR
(microscope) nmax: 3307, 1754, 1647, 1535 cmꢀ1; 1H NMR
(400 MHz, CDCl3) d: 7.29–7.19 (m, 2H), 7.18–7.07 (m,
3H), 6.70 (br d, J¼9.3 Hz, 1H), 6.03, (br s, 1H), 4.92 (br s,
1H), 4.33–4.17 (m, 2H), 4.14 (d, J¼9.3 Hz, 1H), 3.08 (dd,
J¼9.3, 5.9 Hz, 1H), 2.72 (d, J¼4.6 Hz, 3H), 2.70–2.52 (m,
2H), 1.93–1.79 (m, 2H), 1.61 (m, 2H), 1.27 (t, J¼7.2 Hz,
3H), 0.94 (s, 9H); 13C NMR (100.6 MHz, CDCl3) d: 171.5,
170.5, 168.7, 141.5, 128.4, 128.2, 125.9, 122.9 (q, J¼
285.9 Hz), 78.9 (q, J¼28.9 Hz), 63.6, 60.8, 49.1, 35.7,
34.5, 28.7, 26.6, 26.4, 26.1, 13.8; 19F NMR (235.3 MHz,
CDCl3) d: ꢀ74.5 (s, 3F); MS (DIS EI 70 eV) m/z (%): 474
[M+] (8), 416 (18), 86 (100); HRMS m/z 474.2340 (calcu-
lated 474.2333, C23H33F3N2O5).
3.5. Synthesis of acids 12a–c. General procedure
To a solution of 11a (123 mg, 0.26 mmol) in a MeOH/H2O
7:3 mixture (5 mL), a 0.5 M aqueous solution of KOH
(1.00 mL, 0.52 mmol) was added. The reaction was stirred
for 2 h, then the MeOH was removed in vacuo. A 1 M solu-
tion of HCl was added until pH 1–2 was reached, and the re-
action mixture was extracted with EtOAc. The organic phase
was dried over anhydrous Na2SO4, filtered and the solvent
was removed in vacuo to give 111 mg of 12a (96% yield),
as a white solid: Rf 0.54 (CHCl3/MeOH 7:3); [a]2D3 +1.3 (c
1.2, MeOH); FTIR (microscope) nmax: 3453, 1749, 1654,
Compound 11b: white solid; Rf 0.42 (EtOAc/n-Hex 6:4);
[a]2D3 +2.9 (c 1.2, in CHCl3); FTIR (microscope) nmax
1488 cmꢀ1 1H NMR (400 MHz, CD3OD) d: 7.30–7.06
;
:
(m, 5H), 4.19 (s, 1H), 3.23 (dd, J¼10.3, 4.7 Hz, 1H), 2.64
(s, 4H), 2.59–2.50 (m, 1H), 1.85–1.74 (m, 2H), 1.68–1.51
(m, 2H), 0.97 (s, 9H); 13C NMR (100.6 MHz, CD3OD) d:
174.1, 172.6, 170.8, 141.5, 128.4, 128.2, 125.9, 122.9 (q,
J¼285.9 Hz), 78.9 (q, J¼28.9 Hz), 63.6, 35.7, 34.5, 28.7,
26.6, 26.4, 26.1, 13.8; 19F NMR (250 MHz, CD3OD) d:
ꢀ74.5 (s, 3F); MS (DIS EI 70 eV) m/z (%): 447 [M+H+]
3300, 1745, 1650, 1517.1 cmꢀ1 1H NMR (400 MHz,
;
CDCl3) d: 7.29–7.21 (m, 2H), 7.19–7.00 (m, 6H), 6.79 (d,
J¼8.8 Hz, 2H), 6.68 (br d, J¼7.7 Hz, 1H), 5.80 (br s, 1H),
4.47 (q, J¼7.2 Hz, 1H), 4.33–4.19 (m, 2H), 3.72 (s, 3H),
3.04 (dd, J¼10.9, 4.4 Hz, 1H), 2.90 (dd, J¼13.7, 7.2 Hz,
1H), 2.88 (dd, J¼13.7, 7.2 Hz, 1H), 2.67 (d, J¼4.9 Hz,

10178
M. Moreno et al. / Tetrahedron 62 (2006) 10171–10181
(8), 388 (20), 91 (40), 86 (100); HRMS m/z 446.2018 (calcu-
lated 446.2021, C21H29F3N2O5).
16 (89% yield) as a colorless oil: Rf 0.61 (EtOAc/n-Hex 1:1);
[a]2D3 ꢀ0.5 (c 1.1, MeOH); FTIR (film) nmax: 3317, 1750,
1
1654, 1545 cmꢀ1; H NMR (400 MHz, CD3OD) d: 7.27–
Compound 12b: white solid; Rf 0.45 (CHCl3/MeOH 7:3);
[a]2D3 +4.2 (c 0.3, MeOH); FTIR (microscope) nmax: 3371,
7.05 (m, 5H), 4.24–4.19 (m, 3H), 3.17 (dd, J¼11.4,
3.5 Hz, 1H), 2.65 (s, 3H), 2.47–2.54 (m, 2H), 1.96–1.74
(m, 1H), 1.59 (m, 2H), 1.38–1.28 (m, 1H), 1.22 (t,
J¼6.9 Hz, 3H), 0.98 (s, 9H); 13C NMR (100.6 MHz,
CD3OD) d: 173.9, 172.7, 168.4, 142.9, 129.5, 129.3,
126.9, 125.1 (q, J¼287.9 Hz), 80.5 (q, J¼28.1 Hz), 64.0,
62.1, 47.9, 36.3, 35.2, 29.6, 29.1, 27.1, 25.9, 14.2; 19F
NMR (235.3 MHz, CD3OD) d: ꢀ73.9 (s, 3F); MS (DIS EI
70 eV) m/z (%): 475 [M+H+] (28), 416 (20), 86 (100);
HRMS m/z 474.2330 (calculated 474.2333, C23H33F3N2O5).
1
1743, 1654, 1434 cmꢀ1; H NMR (400 MHz, CD3OD) d:
7.29–7.19 (m, 2H), 7.17–7.03 (m, 5H), 6.81 (d, J¼8.9 Hz,
2H), 4.51 (t, J¼7.2 Hz, 1H), 3.70 (s, 3H), 3.06–2.98 (m,
2H), 2.86 (dd, J¼14.1, 7.9 Hz, 1H), 2.64 (s, 3H), 2.61–
2.41 (m, 2H), 1.79–1.60 (m, 2H), 1.48–1.36 (m, 2H); 13C
NMR (100.6 MHz, CD3OD) d: 173.6, 170.6, 169.9, 158.9,
141.2, 130.2, 128.4, 128.3, 127.6, 126.0, 123.0 (q,
J¼284.7 Hz), 114.3, 77.6 (q, J¼28.9 Hz), 55.8, 55.3, 48.1,
37.7, 35.2, 28.4, 27.4, 26.2; 19F NMR (235.3 MHz,
CD3OD) d: ꢀ72.5 (s, 3F); MS (DIS EI 70 eV) m/z (%):
511 [M+H+] (15), 209 (20), 191 (100); HRMS m/z
510.1965 (calculated 510.1970, C25H29F3N2O6).
3.8. Synthesis of acids 17 and 18
To a solution of 16 (278 mg, 0.59 mmol) in a MeOH/H2O
7:3 mixture (5 mL), a 0.5 M aqueous solution of KOH
(3.5 mL, 1.77 mmol) was added. The reaction was stirred
for 20 h, then the MeOH was removed in vacuo. A 1 M so-
lution of HCl was added until pH 1–2 was reached, and the
reaction mixture was extracted with EtOAc. The organic
phase was dried over anhydrous Na2SO4, filtered and the sol-
vent was removed in vacuo to give 237 mg of a mixture of 17
and 18 (90% overall yield) as a white solid: Rf 0.37 (CHCl3/
MeOH 8:2); [a]2D3 ꢀ3.9 (c 0.8, MeOH); FTIR (microscope)
Compound 12c: white solid; Rf 0.27 (CHCl3/MeOH 8:2);
[a]2D3 ꢀ1.9 (c 1.1, MeOH); FTIR (microscope) nmax: 3302,
1
1746, 1656, 1522 cmꢀ1; H NMR (250 MHz, CD3OD) d:
7.30–7.07 (m, 5H), 4.38–4.25 (m, 1H), 3.16–2.98 (m, 3H),
2.68 (s, 3H), 2.65–2.48 (m, 2H), 1.89–1.55 (m, 8H), 1.42
(s, 9H); 13C NMR (62.9 MHz, CD3OD) d: 174.9, 171.8,
159.5, 144.1, 130.4, 130.2, 127.7, 125.8 (q, J¼286.7 Hz),
80.7 (q, J¼29.6 Hz), 62.4, 55.2, 41.5, 37.4, 31.1, 29.7,
28.6, 28.3, 27.2; 19F NMR (235.3 MHz, CD3OD) d: ꢀ72.5
(s, 3F); MS (DIS EI 70 eV) m/z (%): 548 [M+H+] (5), 430
(60), 41 (100); HRMS m/z 547.2499 (calculated 547.2496,
C25H36F3N3O7).
1
nmax: 3450, 1748, 1656, 1544 cmꢀ1; H NMR (400 MHz,
CD3OD) d: 7.28–7.08 (m, 5H), 4.19 (s, 1H), 3.23–3.16 (m,
1H), 2.71 (s, 3H), 2.68–2.50 (m, 2H), 1.89–1.53 (m, 4H),
0.97 (s, 9H); 13C NMR (62.9 MHz, CD3OD) d: 175.0,
172.3, 169.1, 143.0, 129.3, 126.6, 125.2 (q, J¼286.6 Hz),
79.8 (q, J¼27.3 Hz), 62.2, 36.5, 35.9, 35.1, 30.6, 29.9,
26.9, 26.0; 19F NMR (235.3 MHz, CD3OD) d: ꢀ72.9 (s,
3F); MS (DIS EI 70 eV) m/z (%): 447 [M+H+] (25), 388
(30), 91 (40), 86 (100).
3.6. Synthesis of lactone 15
To a solution of 9 (50 mg, 0.14 mmol) in DMF (2 mL), HOAt
(20 mg, 0.15 mmol), HATU (57 mg, 0.15 mmol), and TMP
(37 mL, 0.28 mmol) were added. The solution was stirred
for 2 h, then diluted with H2O. The resulting mixture was ex-
tracted with Et2O and the organic phase was washed with
a 1 M solution of HCl and with brine. After drying over an-
hydrous Na2SO4, and filtration, the solvent was removed in
vacuo, and the residue was purified by FC (n-Hex/(iPr)2O
9:1) to give 33 mg of 15 (72% yield) as a colorless oil: Rf
0.55 (EtOAc/n-Hex 1:9); [a]2D3 +6.8 (c 0.8, CHCl3); FTIR
3.9. Synthesis of peptidomimetics 13a–c and 19.
General procedure
To a solution of 12a (58 mg, 0.13 mmol) in a CH2Cl2/DMF
2:1 mixture (3 mL), cooled at 0 ꢁC, a solution of
BrPO(OEt)2 (35 mg, 0.16 mmol) in CH2Cl2 (0.5 mL) and
neat TMP (34 mL, 0.26 mmol) were added dropwise. The re-
sulting solution was stirred for 45 min at 0 ꢁC, then a solution
of BnONH2 (32 mg, 0.26 mmol) in CH2Cl2 (0.5 mL) and
neat TMP (34 mL, 0.26 mmol) were added. The reaction
was allowed to warm to rt and stirred for 5 h. The solvent
was removed in vacuo and the crude material was dissolved
in a EtOAc/Et2O 1:1 mixture. The resulting mixture was
washed with a 1 M solution of HCl and with brine, then
the organic phase was dried over anhydrous Na2SO4, filtered
and the solvent was removed in vacuo. The residue was pu-
rified by FC (n-Hex/EtOAc 6:4) to give 44 mg of 13a (61%
yield) as a white solid: Rf 0.26 (EtOAc/n-Hex 3:7); [a]D23
ꢀ23.9 (c 1.9, CHCl3); FTIR (microscope) nmax: 3228,
1
(film) nmax: 1870, 1752, 1454 cmꢀ1; H NMR (400 MHz,
CDCl3) d: 7.35–7.09 (m, 5H), 4.31 (q, J¼7.2 Hz, 2H), 3.99
(m, 1H), 2.73–2.58 (m, 2H), 1.90–1.69 (m, 4H), 1.30 (t,
J¼7.2 Hz, 3H); 13C NMR (100.6 MHz, CDCl3) d: 165.5,
162.4, 140.7, 128.6, 128.4, 121.9 (q, J¼281.4 Hz), 76.5 (q,
J¼33.7 Hz), 63.6, 58.0, 35.2, 28.1, 24.8, 13.9; 19F NMR
(235.3 MHz, CDCl3) d: ꢀ77.1 (s, 3F); MS (DIS EI 70 eV)
m/z (%): 330 [M+] (17), 159 (40), 104 (100); HRMS m/z
330.1079 (calculated 330.1074, C16H17F3O4).
3.7. Synthesis of peptidomimetic ester 16
1
To a solution of 15 (139 mg, 0.42 mmol) and 10a (121 mg,
0.84 mmol) in DMF (5 mL), TMP (111 mL, 0.84 mmol)
was added. The solution was stirred for 6 h, then diluted
with H2O. The resulting mixture was extracted with Et2O
and the organic phase was washed with a 1 M solution of
HCl, then with brine. After drying over anhydrous Na2SO4
and filtration, the solvent was removed in vacuo, and the resi-
due was purified by FC (n-Hex/EtOAc 7:3) to give 177 mg of
1692, 1646, 1537 cmꢀ1; H NMR (400 MHz, CDCl3) d:
9.02 (br s, 1H), 7.46–6.96 (m, 11H), 5.84 (d, J¼4.6 Hz,
1H), 5.69 (br s, 1H), 4.86 (s, 2H), 4.14 (d, J¼9.6 Hz, 1H),
3.10 (dd, J¼10.8, 3.9 Hz, 1H), 2.67 (d, J¼4.6 Hz, 3H),
2.60–2.43 (m, 2H), 1.81–1.61 (m, 2H),1.58–1.39 (m, 2H),
0.96 (s, 9H); 13C NMR (100.6 MHz, CDCl3) d: 172.9,
170.2, 163.8, 141.5, 134.8, 129.2, 128.8, 128.6, 128.3,
125.9, 123.2 (q, J¼286.7 Hz), 78.3, 77.8 (q, J¼28.9 Hz),

M. Moreno et al. / Tetrahedron 62 (2006) 10171–10181
10179
61.3, 48.2, 35.4, 34.5, 28.5, 27.1, 26.5, 26.0; 19F NMR
(235.3 MHz, CDCl3) d: ꢀ76.4 (s, 3F); MS (DIS EI 70 eV)
m/z (%): 552 [M+H+] (12), 370 (19), 91 (99), 86 (100);
HRMS m/z 551.2599 (calculated 551.2598, C28H36F3N3O5).
(c 1.3, MeOH); 1H NMR (400 MHz, CD3OD) d: 7.26–7.09
(m, 5H), 4.20 (s, 1H), 3.28 (m, 1H), 2.62 (s, 4H), 2.58–
2.48 (m, 1H), 1.81–1.71 (m, 2H), 1.68–1.46 (m, 2H), 0.97
(s, 9H); 13C NMR (100.6 MHz, CD3OD) d: 175.2, 173.6,
166.9, 144.1, 130.2, 130.1, 127.7, 125.8 (q, J¼287.6 Hz),
80.5 (q, J¼28.1 Hz), 64.9, 39.2, 37.5, 36.0, 30.8, 28.9,
28.0, 26.7; 19F NMR (235.3 MHz, CD3OD) d: ꢀ73.0 (s,
3F); MS (DIS EI 70 eV) m/z (%): 462 [M+H+] (10), 91
(30), 86 (100); HRMS m/z 461.2131 (calculated 461.2130,
C21H30F3N3O5).
Compound 13b: white solid; Rf 0.44 (EtOAc/n-Hex 1:1);
[a]2D3 ꢀ14.8 (c 1.6, CHCl3); FTIR (microscope) nmax
:
3245, 1657, 1567, 1423 cmꢀ1
;
1H NMR (400 MHz,
CDCl3) d: 11.39 (br s, 1H), 7.48–7.02 (m, 13H), 6.84 (d,
J¼8.6 Hz, 2H), 5.49 (br s, 1H), 5.28 (br s, 1H), 4.93 (d,
J¼12.4 Hz, 1H), 4.87 (d, J¼12.4 Hz, 1H), 4.06 (m, 1H),
3.77 (s, 3H), 3.05–2.93 (m, 2H), 2.81 (dd, J¼13.7, 9.2 Hz,
1H), 2.60 (d, J¼4.8 Hz, 3H), 2.58–2.47 (m, 2H), 1.82–
1.57 (m, 2H), 1.55–1.43 (m, 2H); 13C NMR (100.6 MHz,
CDCl3) d: 172.6, 170.3, 163.5, 158.9, 141.5, 134.8, 130.3,
129.3, 128.9, 128.6, 128.4, 128.3, 128.2, 125.9, 123.1 (q,
J¼287.1 Hz), 114.3, 78.4 (q, J¼27.8 Hz), 55.6, 55.3, 48.4,
37.6, 35.5, 28.8, 26.9, 26.2; 19F NMR (235.3 MHz,
CDCl3) d: ꢀ76.9 (s, 3F); MS (DIS EI 70 eV) m/z (%): 616
[M+H+] (18), 191 (100); HRMS m/z 615.2551 (calculated
615.2547, C32H36F3N3O6).
Compound 14b: white solid; Rf 0.31 (EtOAc/n-Hex 7:3);
[a]2D3 ꢀ17.4 (c 0.9, MeOH); FTIR (microscope): nmax¼3215,
1654, 1546 cmꢀ1; 1H NMR (400 MHz, CDCl3) d: 7.27–7.06
(m, 7H), 6.85 (d, J¼8.7 Hz, 2H), 4.48 (t, J¼7.4 Hz, 1H),
3.71 (s, 3H), 3.08 (dd, J¼10.4, 4.1 Hz, 1H), 2.99 (dd,
J¼13.9, 7.7 Hz, 1H), 2.88 (dd, J¼13.9, 7.4 Hz, 1H), 2.59
(s, 3H), 2.57–2.46 (m, 2H), 1.74–1.64 (m, 2H), 1.54–1.43
(m, 2H); 13C NMR (100.6 MHz, CDCl3) d: 174.4, 173.9,
166.6, 160.7, 143.7, 131.8, 130.7, 129.9, 129.8, 127.4,
125.5 (q, J¼285.9 Hz), 115.5, 80.1 (q, J¼27.1 Hz), 57.2,
56.2, 38.6, 37.1, 30.9, 30.4, 28.3, 26.7; 19F NMR
(235.3 MHz, CDCl3) d: ꢀ73.4 (s, 3F); MS (DIS EI 70 eV)
m/z (%): 526 [M+H+] (5), 191 (100); HRMS m/z 525.2082
(calculated 525.2079, C25H30F3N3O6).
Compound 13c: white solid; Rf 0.24 (EtOAc/n-Hex 1:1);
[a]2D3 ꢀ27.4 (c 1.9, CHCl3); FTIR (microscope) nmax
:
3368, 1688, 1653, 1412 cmꢀ1
;
1H NMR (400 MHz,
CDCl3) d: 11.29 (br s, 1H), 7.68 (br s, 1H), 7.48–7.04 (m,
10H), 6.55 (br s, 1H), 5.61 (br s, 1H), 4.91 (m, 2H), 4.57
(br s, 1H), 4.12 (m, 1H), 3.23 (m, 1H), 3.09 (m, 1H), 2.97
(m, 1H), 2.73 (s, 3H), 2.63–2.49 (m, 2H), 1.71–1.55 (m,
7H), 1.47 (s, 10H); 13C NMR (100.6 MHz, CDCl3) d:
172.9, 171.6, 163.9, 156.8, 141.5, 134.5, 129.2, 128.5,
128.2, 125.8, 123.2 (q, J¼288.5 Hz), 79.2, 77.5, 77.1 (q,
J¼28.5 Hz), 51.8, 47.3, 38.7, 35.2, 29.6, 29.3, 28.5, 28.3,
25.9, 25.8; 19F NMR (235.3 MHz, CDCl3) d: ꢀ76.3 (s,
3F); MS (DIS EI 70 eV) m/z (%): 653 [M+H+] (5), 553
(40), 91 (100); HRMS m/z 652.3078 (calculated 652.3073,
C32H43F3N4O7).
Compound 14c: white solid; Rf 0.22 (EtOAc/n-Hex 7:3);
1
[a]2D3 ꢀ25.2 (c 1.9, MeOH); H NMR (400 MHz, CD3OD)
d: 7.28–7.07 (m, 5H), 4.38–4.27 (m, 1H), 3.20 (dd,
J¼10.9, 3.7 Hz, 1H), 3.11–2.96 (m, 2H), 2.66 (s, 3H),
2.64–2.51 (m, 2H), 1.85–1.55 (m, 6H), 1.47 (m, 2H), 1.42
(s, 9H); 13C NMR (62.9 MHz, CD3OD) d: 174.7, 174.4,
166.7, 158.8, 143.5, 129.7, 129.6, 127.1, 124.4 (q,
J¼286.7 Hz), 80.1, 79.7 (q, J¼29.6 Hz), 62.4, 54.4, 40.9,
36.8, 30.5, 30.3, 29.1, 27.7, 27.4, 26.5; 19F NMR
(235.3 MHz, CD3OD) d: ꢀ72.9 (s, 3F); MS (DIS EI
70 eV) m/z (%): 563 [M+H+] (5), 114 (45), 91 (100);
HRMS m/z 562.2600 (calculated 562.2605, C25H37F3N4O7).
Compound 19: white solid; Rf 0.30 (EtOAc/n-Hex 1:1); [a]D23
+11.7 (c 0.9, CHCl3); FTIR (microscope): nmax¼3379, 1687,
Compound 20: white solid; Rf 0.24 (EtOAc/n-Hex 7:3); [a]D23
+1.8 (c 0.9, MeOH); 1H NMR (400 MHz, CD3OD) d: 7.29–
7.07 (m, 5H), 4.15 (s, 1H), 2.86 (dd, J¼11.4, 3.0 Hz, 1H),
2.70 (s, 3H), 2.69–2.62 (m, 2H), 2.61–1.51 (m, 4H), 0.97
(s, 9H); 13C NMR (100.6 MHz, CD3OD) d: 172.4, 170.2,
168.3, 142.9, 129.4, 129.3, 126.8, 124.9 (q, J¼286.7 Hz),
79.7 (q, J¼26.9 Hz), 63.2, 46.7, 36.5, 35.5, 30.1, 27.4,
27.2, 26.1; 19F NMR (235.3 MHz, CD3OD) d: ꢀ73.5 (s,
3F); MS (DIS EI 70 eV) m/z (%): 462 [M+H+] (8), 446
(18), 99 (40), 86 (100).
1
1650, 1535 cmꢀ1; H NMR (400 MHz, CDCl3) d: 8.10 (d,
J¼8.6 Hz, 1H), 7.43–7.05 (m, 11H), 6.07 (br s, 1H), 6.01
(d, J¼4.1 Hz, 1H), 4.81 (d, J¼10.6 Hz, 1H), 4.75 (d,
J¼10.6 Hz, 1H), 4.05 (d, J¼8.9 Hz, 1H), 3.01 (dd,
J¼11.3, 3.4 Hz, 1H), 2.56 (m, 5H), 2.02–1.50 (m, 4H),
0.99 (s, 9H); 13C NMR (100.6 MHz, CDCl3) d: 170.3,
169.7, 168.1, 141.4, 134.6, 129.3, 128.9, 128.6, 128.4,
128.3, 126.0, 123.1 (q, J¼287.1 Hz), 79.2 (q, J¼27.7 Hz),
78.5, 62.2, 44.2, 35.6, 34.6, 29.7, 28.9, 26.7, 26.1; 19F
NMR (235.3 MHz, CDCl3) d: ꢀ74.3 (s, 3F); MS (DIS EI
70 eV) m/z (%): 552 [M+H+] (100).
3.11. X-ray structure analysis of 8x
3.10. Synthesis of hydroxamates 14a–c and 20.
General procedure
A colorless crystal with approximate dimensions 0.25ꢂ0.3ꢂ
0.4 mmwas used. Hexagonal, spacegroupP65, a¼13.038(1),
3
˚
˚
b¼13.038(1), c¼28.066(4) A, V¼4131.7(7) A , Z¼6, Dc¼
1.260 g cmꢀ3, m¼0.844 mmꢀ1, Bruker P4 diffractometer
with graphite monochromated Cu Ka radiation (l¼
To a solution of 13a (50 mg, 0.09 mmol) in MeOH (5 mL),
a catalytic amount of Pd(OH)2/C was added and the reaction
mixture was vigorously stirred under hydrogen atmosphere
at rt for 1 h. The palladium powder was filtered over a Celite
pad and the residue was washed with MeOH. The solvent
was removed in vacuo and the residue was purified by FC
(CHCl3/MeOH 97:3), affording 36 mg of 14a (87% yield),
as a white solid: Rf 0.44 (EtOAc/n-Hex 1:1); [a]2D3 ꢀ29.6
˚
1.54179 A), u/2q scan technique, room temperature, a total
of reflections 6037 (2541 unique, Rint¼0.072) collected up
to 2q¼134.0ꢁ. The structure was solved by direct methods18
and refined19 against F2, the amide and the hydroxyl hydro-
gen atoms were determined from Fourier difference maps
and refined, as the other H atoms, in riding mode.

10180
M. Moreno et al. / Tetrahedron 62 (2006) 10171–10181
R1¼0.0414 (Rw¼0.098) for 1952 observed reflections
[Iꢄ2s(I)], 336 parameters refined, R1¼0.0585 (Rw¼
0.1074) for all data, goodness of fit¼1.012, residual electron
index20 is 0.0(3) for space group P65 and 0.2(3) for space
group P61. Although hardly significant it points to the correct
enantiomer that can be readily identified since L-alanine was
used in the synthesis of 8x.
constant 0.035 kJ/(mol deg2), 1 ns MD simulations for
each harmonic window].
density of 0.164 and ꢀ0.118 e Aꢀ3. The value of the Flack
˚
Acknowledgements
We are grateful to Giorgio Colombo (CNR Milano) and
Martin J. Field (IBS Grenoble) for discussions on molecular
modeling. We thank the European Commission (IHP
Network grant ‘FLUOR MMPI’ HPRN-CT-2002-00181,
Integrated Projects ‘STROMA’ LSHC-CT-2003-503233
and ‘CANCERDEGRADOME’ LSHC-CT-2003-503297),
MIUR (Cofin 2002, Project ‘Peptidi Sintetici Bioattivi’),
Politecnico di Milano, and C.N.R. for economic support.
Part of the computer time for the molecular modeling studies
was made available by Cilea (Milano).
3.12. Biological assays
3.12.1. Zymographic analysis. Tfm-hydroxamates 14a–c
and 20 were dissolved in EtOH to yield 10ꢀ1 M stock and
further diluted in test solutions. Subconfluents human mela-
noma cells WM983A were incubated in serum free medium
for 24 h. The conditioned medium containing pro-MMP-9
and pro-MMP-2 was then analyzed by zymography. Sam-
ples in 70 mM Tris–HCl pH 6.8, 10% glycerol, 2% SDS,
and 0.01% bromophenol blue were applied to SDS–poly-
acrylamide (8%) gels co-polymerized with 1 mg/mL gelatin.
After electrophoresis, gels were washed three times for
20 min with 2.5% Triton X-100 at room temperature and
incubated overnight in 50 mM Tris–HCI, pH 7.5, 5 mM
CaCl2, 150 mM NaCl, and 0.02% Brij-35 at 37 ꢁC in the
presence or not of Tfm-hydroxamates. Gels were then
stained with 0.5% Coomassie blue in 25% methanol and
10% acetic acid, and destained in the same solution without
Coomassie blue. Gel images were acquired with a Duoscan
T1200 scanner (AGFA), and the levels of MMPs were
quantified by the Image-Pro Plus 4.1 program. The results
were expressed in arbitrary units (IOD) and the IC50 was
calculated.
Supplementary data
Supplementary data associated with this article can be found
in the online version, at doi:10.1016/j.tet.2006.08.036
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