Synthesis of α-trifluoromethyl-α-amino-β-sulfone hydroxamates: Novel nanomolar inhibitors of matrix metalloproteinases

Article abstract of DOI:10.1016/j.tetlet.2005.07.078

The racemic α-trifluoromethyl-α-amino-β-sulfone hydroxamates 1 were synthesized by means of a nucleophilic addition of sulfur-stabilized carbanions to a N-Cbz imine of trifluoropyruvate (4). The free amino derivative 1a was the most potent inhibitor of both MMP-3 (stromelysin-1) and MMP-9 (gelatinase-B), showing an IC₅₀ = 14 nM and 1 nM, respectively, and excellent selectivity versus MMP-1 (>5000-fold difference in inhibitory capacity). The N-Me derivative 1b was the most selective for MMP-3 with respect to MMP-9 (62-fold difference).

Full text of DOI:10.1016/j.tetlet.2005.07.078

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 6515–6518
Synthesis of a-trifluoromethyl-a-amino-b-sulfone
hydroxamates: novel nanomolar inhibitors of
matrix metalloproteinases
Roberta Sinisi,a Monica Sani,a Gabriele Candiani,b Rachele Parente,c Franc¸oise Pecker,b
Stefano Bellostac and Matteo Zandaa,*
aC.N.R.—Istituto di Chimica del Riconoscimento Molecolare, sezione ‘A. Quilico’, and Dipartimento di Chimica,
Materiali ed Ingegneria Chimica ‘G. Natta’ del Politecnico di Milano, via Mancinelli 7, I-20131 Milano, Italy
bInserm, U581; Universite´ Paris 12, Faculte´ de Me´decine; Cre´teil F-94000, France
`
Dipartimento di Scienze Farmacologiche, Universita degli Studi di Milano, via Balzaretti 9, I-20133 Milano, Italy
c
Received 9 June 2005; revised 12 July 2005; accepted 19 July 2005
Abstract—The racemic a-trifluoromethyl-a-amino-b-sulfone hydroxamates 1 were synthesized by means of a nucleophilic addition
of sulfur-stabilized carbanions to a N-Cbz imine of trifluoropyruvate (4). The free amino derivative 1a was the most potent inhibitor
of both MMP-3 (stromelysin-1) and MMP-9 (gelatinase-B), showing an IC50 = 14 nM and 1 nM, respectively, and excellent selec-
tivity versus MMP-1 (>5000-fold difference in inhibitory capacity). The N-Me derivative 1b was the most selective for MMP-3 with
respect to MMP-9 (62-fold difference).
Ó 2005 Elsevier Ltd. All rights reserved.
Matrix metalloproteinases (MMPs) are a family of zinc
metalloendopeptidases secreted by cells, which are
responsible for much of the turnover of matrix
components.1
MMPs have been reported to play a key-role, in combi-
nation with their natural tissue inhibitors (TIMPs), in
many serious diseases, such as heart failure and cancer.
Figure 1.
Progression and growth of both pathologies, mainly in
the early stages, is favoured by the proteolytic activity
of several MMPs, such as stromelysin-1 (MMP-3) and
effect observed clinically with the broad-spectrum
gelatinase-B (MMP-9). For these reasons, inhibition of
MMP inhibitor marimastat.3
MMPs is actively studied as a promising therapeutic
target for heart failure and cancer therapy.2
Whereas a large number of different alkyl and alkylaryl
residues were well tolerated as nitrogen substituents R2,
Recently, Becker et al. described a family of a-alkyl-a-
only inhibitors bearing R1 = H, CH3 or Ph were
amino-b-sulfone hydroxamates A (Fig. 1) as potent
described.
inhibitors of MMP-2, MMP-9 and MMP-13, while
exhibiting limited inhibition of MMP-1, an enzyme
Within the frame of a research project aimed at a better
thought to be responsible of the musculoskeletal side
understanding and rationalization of the Ôfluorine-effectÕ
in peptidomimetic structures, particularly those display-
ing activity as protease inhibitors,4 we decided to inves-
tigate the effect of a trifluoromethyl (Tfm) group,
Keywords: Fluorine; Matrix metalloproteinases; Inhibitors; Sulfones.
positioned as R1 substituent of structures A, on the inhi-
*
Corresponding author. Tel.: +39 0223993084; fax: +39 0223993080;
e-mail: matteo.zanda@polimi.it
bition of MMPs activity. It is in fact widely recognized
0040-4039/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2005.07.078

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R. Sinisi et al. / Tetrahedron Letters 46 (2005) 6515–6518
Scheme 1.
in medicinal chemistry that the Tfm group is a substitu-
ent of distinctive qualities. It is at the same time highly
hydrophobic and sterically demanding.5 Moreover, its
high electron density could provide additional interac-
tions within the MMPs active sites, possibly including
hydrogen bonding.6 In addition, the Tfm has been used
to replace both methyl and phenyl groups.5
acceptable yields (Scheme 2).11 The resulting N-
methyl-a-amino ester 8 was submitted to saponification,
but the reaction was surprisingly slow and after three
days a complex mixture of products was formed. Differ-
ent basic conditions were attempted (such as LiOH and
KOH), but no significant improvement could be
achieved. We thus decided to hydrogenolyze first the
Cbz group of 8, which produced the expected secondary
amine 9. Unfortunately, also in this case we were unable
to perform the final saponification step.
We first undertook the synthesis of the hydroxamic acid
1a, having a free quaternary amino group (Scheme 1).
The intermediate sulfone 3 was synthesized by Pd-cata-
lyzed reaction of phenol with the p-Br derivative 2.7
Lithiation of 3, followed by nucleophilic addition to
the N-Cbz imine of trifluoropyruvate 48 afforded the
a-Tfm a-amino acid derivative 5 in fair yields.9 Saponi-
fication of the ester function delivered the carboxylic
acid 6, that was subjected to condensation with O-Bn-
hydroxylamine affording the hydroxamate 7 (oxazolin-
5-one B was formed as co-product, in ca. 20% yield, in
the condensation), which was hydrogenolyzed to the tar-
get molecule 1a.10
We therefore decided to re-investigate the synthetic pro-
tocol on the sulfanyl analogues of 8, which were synthe-
sized as portrayed in Scheme 3. To this end, the sulfanyl
diaryl ether 11 was prepared from phenol and 10 accord-
ing to a Ullmann-type reaction,12 then oxidized to the
sulfoxide 12. Lithiation and Mannich-type reaction with
4 afforded a nearly equimolar mixture of sulfoxide dia-
stereomers 13, which were deoxygenated to the racemic
sulfide 14 according to the Drabowicz and Oae
protocol.13
With 1a in hand, we next addressed the synthesis of its
N-alkyl derivatives. The challenging N-methylation of
the a-Tfm-substituted compound 5 was achieved in
N-Alkylation occurred in good to excellent yields afford-
ing the corresponding sulfides 15b–d. Due to the pres-
ence of the sulfide function, which could interfere with
Scheme 2.

R. Sinisi et al. / Tetrahedron Letters 46 (2005) 6515–6518
6517
Scheme 3.
the hydrogenolysis, the Cbz was cleaved with HBr,14
affording the secondary amines 16b–d in nearly quanti-
tative yields. To our satisfaction, the ÔdifficultÕ ester
saponification step could be performed smoothly,
affording the carboxylic acids 17b–d in good to excellent
yields.15 Coupling of 17b–d with O-Bn-hydroxylamine
occurred smoothly affording the sulfanyl hydroxamates
18b–d, which were oxidized to sulfones 19b–d.16 The tar-
get hydroxamic acids 1b–d were obtained in fair yields
by hydrogenolysis with the Pearlman catalyst.
excellent selectivity versus MMP-1 (>5000-fold). We
tested the effect of the primary a-amino hydroxamate
1a also on the gelatinolytic activity of full length
MMP-9 secreted by macrophages in culture.18 Interest-
ingly, even in these different experimental conditions,
the compound showed an inhibitory activity. These
results show that a Tfm group can be successfully used
as a substituent in protease inhibitors,19 and is very well
tolerated by the enzymes. On the other hand, previous
results from our group showed that replacement of an
a-methyl by Tfm group in another structural class of
hydroxamate inhibitors of MMPs was responsible for
a dramatic loss of inhibitory potency.20 It was also
shown that the final outcome of Tfm incorporation is
strongly dependent on the whole structure of the
inhibitor.21
With racemic 1a–d in hand, we next addressed the inhi-
bition tests on the catalytic domains of MMP-1, MMP-3
and MMP-9.17
The primary a-amino hydroxamate 1a is the most po-
tent compound (Table 1), but it is worth noting that
all of them are nanomolar inhibitors of MMP-3 and
MMP-9, with the exception of the N-Me derivative 1b
which showed good selectivity for MMP-3 (ca. 62-fold
selective vs MMP-9). Even more importantly, 1a showed
We are currently investigating the synthesis and the
inhibitory activity of the single enantiomers of 1 on a
wider range of MMPs, in order to have a more complete
picture of the effect of fluorine in terms of potency and
selectivity in this particular class of protease inhibitors.
Attempts to obtain X-ray data on the co-crystals of 1
with MMP catalytic domains are also in progress, in
order to have more detailed structural information
about the role of the Tfm group in the binding process.
Table 1. Effect of the compounds on different MMPsÕ proteolytic
activity
Compound
IC50/MMP-3
(nM)
IC50/MMP-9
(nM)
IC50/MMP-1
(nM)
1a
1b
1c
1d
14
32
28
53
1
1982
63
>5000
n.a.
n.a.
Acknowledgements
59
n.a.
We thank the European Commission (IHP Network
grant ÔFLUOR MMPIÕ HPRN-CT-2002-00181), MIUR
n.a. = not available.

6518
R. Sinisi et al. / Tetrahedron Letters 46 (2005) 6515–6518
62.5, 52.5, 13.7, the CF3 signal was obscured; 19F NMR
(235.3 MHz, CDCl3) d: À74.5 (s, 3F); MS (DIS EI 70 eV)
m/z (%): 551 [M+] (4), 233 (23), 108 (82), 91 (100).
(Cofin 2004, Project ÔPolipeptidi Bioattivi e Nano-
strutturatiÕ), Politecnico di Milano, and C.N.R. for eco-
nomic support. We thank Professor Dario Neri (ETH
Zurich, Switzerland) and Professor Wolfram Bode (Max
Planck Institute fur Biochemie, Martinsried, Germany)
for their help with MMP-3 and MMP-9 production.
10. Synthesis of 1a: to a solution of 7 (100 mg, 0.16 mmol) in
MeOH (3 mL), a catalytic amount of Pd(OH)2/C was
added and the reaction mixture was kept vigorously
stirred under hydrogen atmosphere at room temperature
for 5 h. The catalyst was filtered over a Celite pad, and
washed with MeOH. The solvent was removed in vacuo
and the residue was purified by FC (CHCl3–MeOH, 97:3),
affording 60 mg of 1a (93% yield); Rf: 0.17 (CHCl3–
MeOH, 95:5); FT-IR (film) mmax 3011.3, 1688.8,
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1
1584.3 cmÀ1; H NMR (250 MHz, CDCl3) d: 10.21–9.38
(br s, 1H), 7.90 (d, 2H, J = 8.5 Hz), 7.58–7.21 (m, 4H),
7.18–7.02 (m, 3H), 6.03 (s, 1H), 4.22 (d, 1H, J = 14.8 Hz),
4.10–3.80 (br s, 2H), 3.48 (d, 1H, J = 14.8 Hz); 13C NMR
(250 MHz, CDCl3) d: 162.9, 154.5, 132.8, 130.7, 130.5,
125.1, 123.3 (q, J = 280.4 Hz), 62.7 (q, J = 29.2 Hz), 57.8;
19F NMR (235.3 MHz, CDCl3) d: À74.4 (s, 3F); MS (DIS
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sulfanyl protons.
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modest to fair yields, as a likely consequence of side
reactions involving the hydroxamate moiety and/or the
electron-rich aryl groups. In fact, several unidentified by-
products were detected by TLC monitoring.
17. Full length MMP-1 was purchased from Biomol, and the
activity of 1a measured by collagen gel zymography as
previously described (see Ref. 18). The catalytic domains
of MMP-3 and MMP-9 enzymes were produced in E. coli,
transfected with cDNAs corresponding to the respective
human sequences. Proteins were purified by affinity
chromatography and the inhibitory potencies of racemic
1 and single enantiomers were assayed with synthetic,
general MMP fluorescent substrate (Mca-PLGLDpaAR,
Tebu-bio) using a FL600 Avantec fluorimeter. For MMP-
3 see: (a) Ye, Q. Z.; Johnson, L. L.; Hupe, D. J.; Baragi, V.
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Biochemistry 1995, 34, 4702–4708.
18. The effect on full length MMP-9 activity was evaluated as
previously described: Inhibition of metalloproteinase-9
activity and gene expression by polyphenolic compounds
isolated from the bark of Tristaniopsis calobuxus (Myrt-
aceae): Bellosta, S.; DellÕAgli, M.; Canavesi, M.; Mitro,
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TLC monitoring.; (b) Synthesis of 5: to a stirred solution
of DIPEA (1.75 mL, 12.5 mmol) in dry THF (20 mL),
cooled at À74 °C and under nitrogen, a 2.5 M solution of
n-BuLi (5 mL, 12.5 mmol) was added and the solution was
allowed to warm at 0 °C. The resulting solution was
cooled at À78 °C, and a solution of sulfone 3 (2.4 g,
9.6 mmol) in dry THF (5 mL) was added. After 10 min, a
solution of the crude imino derivative 4 (12.5 mmol) was
added dropwise. After stirring for 2 h at À70 °C the
reaction mixture was quenched with saturated aqueous
NH4Cl. The layers were separated and the aqueous phase
was extracted with AcOEt. The collected organic phases
were dried over anhydrous Na2SO4 and the solvent was
removed in vacuo. The residue was purified by flash
chromatography (n-hexane–AcOEt, 9:1), affording 2.12 g
of 5 (40%); Rf: 0.43 (n-hexane–AcOEt, 8.2); FT-IR (film)
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Danishefsky, S. J. J. Am. Chem. Soc. 2004, 126, 10913–
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;
mmax 3403.0, 2986.7, 1732.6, 1488.6 cmÀ1 1H NMR
(250 MHz, CDCl3) d: 7.81 (d, 2H, J = 8.9 Hz), 7.47–7.12
(m, 8H), 7.08–6.92 (m, 4H), 6.23 (s, 1H), 5.11 (d, 1H,
J = 14.1 Hz), 4.95 (d, 1H, J = 12.4 Hz), 4.62 (d, 1H,
J = 12.4 Hz), 4.45 (m, 2H), 3.96 (d, 1H, J = 14.1 Hz), 1.40
(t, 3H, J = 7.3 Hz); 13C NMR (250 MHz, CDCl3) d: 164.3,
162.8, 154.5, 153.3, 135.2, 132.9, 130.8, 130.2, 128.6, 128.4,
127.9, 125.3, 120.4, 117.2, 67.1, 64.8, 62.7 (q, J = 29.6 Hz),
21. Sani, M.; Candiani, G.; Pecker, F.; Zanda, M. Tetra-
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