Toward a fragment-based approach to MMPs inhibitors: An expedite and efficient synthesis of N-hydroxylactams

Article abstract of DOI:10.1016/j.tetlet.2012.05.124

Matrix metalloproteinases (MMPs), a class of zinc-enzymes over-activated in many pathologies, such as arthritis and cancer, can be efficiently inhibited by a variety of molecules bearing zinc-binding groups (ZBGs). The hydroxamic acid moiety represents one of the most potent and widely exploited ZBG but the poor target selectivity and in vivo toxicity have tempered the initial enthusiasm for this class of potential therapeutics. These drawbacks might be circumvented, at least in part, by increasing the structural constraints around the hydroxamic moiety. Following this strategy we designed and prepared N-hydroxylactam molecules of different size through a synthetic protocol based on a ring closing metathesis amenable to a fragment-based approach potentially leading to a large molecular diversity.

Full text of DOI:10.1016/j.tetlet.2012.05.124

Tetrahedron Letters 53 (2012) 4114–4116
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Toward a fragment-based approach to MMPs inhibitors: an expedite
and efficient synthesis of N-hydroxylactams
Francesco Leonetti a, , Giovanni Muncipinto b, Angela Stefanachi a, Orazio Nicolotti a, Saverio Cellamare a,
⇑
Marco Catto a, Leonardo Pisani a, Giovanni Pellegrino a, Angelo Carotti a,
⇑
a Dipartimento Farmaco-Chimico, Università degli Studi di Bari ‘Aldo Moro’, Via Orabona 4, 70125 Bari, Italy
b Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02142, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Matrix metalloproteinases (MMPs), a class of zinc-enzymes over-activated in many pathologies, such as
arthritis and cancer, can be efficiently inhibited by a variety of molecules bearing zinc-binding groups
(ZBGs). The hydroxamic acid moiety represents one of the most potent and widely exploited ZBG but
the poor target selectivity and in vivo toxicity have tempered the initial enthusiasm for this class of
potential therapeutics. These drawbacks might be circumvented, at least in part, by increasing the struc-
tural constraints around the hydroxamic moiety. Following this strategy we designed and prepared N-
hydroxylactam molecules of different size through a synthetic protocol based on a ring closing metath-
esis amenable to a fragment-based approach potentially leading to a large molecular diversity.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 16 March 2012
Revised 23 May 2012
Accepted 25 May 2012
Available online 30 May 2012
Keywords:
Ring closing metathesis
N-Hydroxylactams
Metalloproteinase inhibitors
Zinc-enzymes
Metal ions play a relevant role in several physiological and
pathological processes,1 being involved, chiefly as enzyme cofac-
tors,2 in a variety of biochemical transformations3 including redox
reactions triggering oxidative stress in many neurological inflam-
matory disorders.4 An increased level of copper and iron in well
localized tissues and cellular brain districts has been observed in
Parkinson’s and Alzheimer’s diseases, two widespread neurological
disorders characterized by a metal-catalyzed formation of toxic
radical species and metal-promoted aggregation of amyloid pep-
tides5 and alpha-synuclein.6
Matrix metalloproteinases (MMPs), are zinc-containing proteo-
lytic enzymes with potent zinc-mediated endopeptidase activity
against several protein components of both extracellular matrix
and basal membrane.7 The activity of MMPs is fundamental for
maintaining a number of key physiologic processes such as tissue
turn-over and embryonic development. In addition, over-expres-
sion of MMPs is responsible for many pathologies such as arthritis
and cancer.3,8 For this reason, great efforts have been made to set
molecular chemical strategies for an effective and stable coordina-
tion of zinc ion to inhibit specific MMPs and possibly block cancer
and associated metastatic processes triggered by their over-activa-
tion.9,10 In view of this, the identification of suitable scaffold mole-
cules bearing zinc-binding groups (ZBGs), such as hydroxamic acid
and thiol units, is a well established approach for the development
of potent MMPs and histone deacetylase (HDACs) inhibitors.11,12
Unfortunately, the lack of target selectivity of many hydroxamic
acid derivatives along with their pronounced toxicity has to some
extent discouraged further clinical developments of this class of
compounds. Since these negative features might also be associated
with a high conformational mobility of most of the explored
hydroxamic acid derivatives, we planned the synthesis of a series
of molecules containing the hydroxamic acid moiety constrained
in a cycle. The general structures I and II of the designed rigid mol-
ecules are illustrated in Chart 1. Five- and six-membered N-
hydroxylactams were exploratively synthesized. Structures II can
allocate a double bond either in the a,b- or in the b,c-position.
The introduction of a strong structural constrain as a cyclic moi-
ety, was pursued to bias molecular selectivity, which is still the
main drawback of the majority of the already published linear cong-
eners carrying the hydroxamic acid moiety. On the other hand, the
intentionally reduced conformational flexibility, in addition to a
suitable substitution pattern ‘clock’ around the N-hydroxylactam
scaffold was instead conceived to enable the selective inhibition
R
R
N
O
N
O
HO
OH
⇑
Corresponding authors. Tel.: +39 080 544 2779; fax: +39 080 544 2230 (F.L.);
I
II
tel.: +39 080 544 2782; fax: +39 080 544 2230 (A.C.).
E-mail addresses: fleonetti@farmchim.uniba.it (F. Leonetti), carotti@farmchim.
uniba.it (A. Carotti).
Chart 1. General structures of the targeted N-hydroxylactams.
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.05.124

F. Leonetti et al. / Tetrahedron Letters 53 (2012) 4114–4116
4115
of MMPs as well as to control the toxicity profile often associated
with more flexible MMP inhibitors. Furthermore, as showed in
Chart 2, the proposed chemical pathway might enable the design
of focused libraries of potential MMPs inhibitors through a frag-
ment-based approach.13
ONH2
O
O
a
Cl
+
ONH
97%
The general retrosynthetic protocol envisioned for the synthesis
of compounds with general structures I and II is depicted in Chart 2,
where PG stands for protecting group. It is based on the coupling of
differently substituted olefins with a suitable O-protected hydrox-
ylamine, such as O-(p-methoxybenzyl)hydroxylamine (PMB-
ONH2), followed by a final ring closing metathesis (RCM) reaction
catalyzed by the second generation Grubbs catalyst.14 Although
the synthetic strategy showed in Chart 2 could be in principle ap-
plied to the preparation of variously sized cycles, in this Letter we
focused our attention on the synthesis of compounds bearing only
five- and six-membered N-hydroxylactam rings. The synthesis of
these derivatives is summarized in Schemes 1–4.
PMB
2
3
OCH3
1
95%
b
O
O
c, d
85%
ON
N
PMB
HO
5
4
Scheme 1. Synthesis of five-membered ring derivative 5. Reagents and conditions:
(a) TEA, DCM, 0 °C; (b) allyl bromide, K2CO3, CH3CN, reflux, (c) Grubbs II catalyst,
toluene, 60 °C, 1 h; (d) TFA, triethylsilane, DCM, 0 °C.
The preparation of five-membered ring derivatives (Scheme 1)
was accomplished in three chemical steps starting from PMB-
ONH2 (1), which was reacted with acryloyl chloride in dichloro-
methane (DCM), using triethylamine (TEA) as a base.
Under these experimental conditions, intermediate 3 was ob-
tained in 97% yield. This intermediate was alkylated with allyl bro-
mide in CH3CN using K2CO3 as a base to afford 4, in 95% yield, which
was then cyclized using the second generation Grubbs catalyst un-
O
O
a
ONH2
PMB
HO
ONH
+
94%
PMB
1
6
7
der RCM conditions to give after TFA deprotection the N-hydroxy-c-
lactam 5 in 85% yield over two steps. The synthesis of compounds of
general structure II was carried out following two different ap-
proaches based on the position of the endocyclic double bond.
27%
b
O
O
The synthesis of b,c-unsaturated N-hydroxy-d-lactam 9 (Scheme
c, d
31%
ON
2) was performed starting from 1, which was coupled with but-3-
enoic acid using diisopropylcarbodiimmide (DIC) and N-hydroxy-
succinimide (NHS) in dry DCM.
HO
N
PMB
8
9
Under these experimental conditions, compound 7 was ob-
tained in 94% yield. The subsequent alkylation of 7 under the same
reaction conditions reported in Scheme 1 gave rise to a remarkable
reduction of yield (27% vs 95%) due to the isomerization of termi-
nal alkene double bond. Key intermediate 8 was cyclized under
RCM conditions and then deprotected by TFA affording the final
product 9 in 31% yield over two steps. Unlike compound 9, the
Scheme 2. Synthesis of six-membered ring b,c-unsaturated derivative 9. Reagents
and conditions: (a) DIC, NHS, dry DCM, room temperature; (b) allyl bromide, K2CO3,
CH3CN, reflux; (c) Grubbs II, toluene, 60 °C, 1 h; (d) TFA, triethylsilane, DCM, 0 °C.
O
Br
O
a
R1
+
ONH
9%
a,b-unsaturated isomer was prepared following a diverse chemical
ON
PMB
PMB
3
10
approach (Scheme 3).
R1
b
The reaction of intermediate 3 with 1-(1-bromobut-3-en-1-yl)-
4-nitrobenzene 10 under SN conditions resulted in a disappointing
9% yield. The subsequent RCM followed by TFA deprotection of the
4-nitrophenyl derivative 12 gave the expected N-hydroxylactam
13 in good yield (97% over the two steps).15 In order to improve
the yield of the critical SN alkylation reaction, we used an Ag2O-
based coupling reaction16 (Scheme 4), resulting in a significant
increase of yields (63% vs 9%).17
11
R1 = 4-NO2-C6H4
98%
O
N
O
c
99%
O
N
PMB
HO
The synthetic approach proposed herein allowed the facile and
R1
12
R1
13
versatile construction of N-hydroxy-c and d-lactam derivatives.
Moreover, our reaction pathway may enable the introduction of a
range of chemically diverse substituents on the carbon directly
bound to the nitrogen atom of the hydroxamate unit as depicted
in Chart 2. Consequently, the potential for a fragment-based ap-
proach for the design and synthesis of libraries of MMP inhibitors
appears clear and feasible. As discussed above, the key step of our
synthetic pathway is the RCM reaction of the diene intermediates
to obtain the corresponding cyclic N-hydroxylactams. The only
concern of our synthetic approach is the alkylation reaction be-
cause of the occurrence of competitive side reactions. One of these
is the isomerization of the terminal double bond, as for compound
8 or the HBr elimination for reagent 10, used in the synthesis of the
Scheme 3. Synthesis of six-membered ring a,b-unsaturated derivative 13. Reagents
and conditions: (a) 1-(1-bromobut-3-en-1-yl)-4-nitrobenzene, K2CO3, CH3CN,
reflux; (b) Grubbs II catalyst, toluene, 60 °C, 1 h; (c) TFA, triethylsilane, DCM, 0 °C.
O
O
Br
a
R1
+
ONH
ON
63%
PMB
PMB
3
14
R1 = 4-Cl-C6H4
R1
15
a,b-unsaturated six-membered ring in 13. To prevent the latter
Scheme 4. Ag2O-mediated coupling. Reagents and conditions: Ag2O, MgSO4, Et2O,
side reaction, we utilized an alternative chemical approach based
room temperature.

4116
F. Leonetti et al. / Tetrahedron Letters 53 (2012) 4114–4116
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PGO NH2
PGO
+
m
N
H
Y
m
X
Y
m= 0, 1, ...
Y= absent, CHR
X= OH, Cl
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Stefanachi, A.; Carotti, A. PLoS ONE 2011, 6, e25597.
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O
Y
O
PGO
HO
N
N
Y
m
m
Z
Z
n
n
Z= absent, n= 1, ...
Z= CHR1, n= 0, 1, ...
15. Synthesis of compound 13
To a solution of 1 (6 mmol, 0.92 g) in CH2Cl2 (40 mL) and Et3N (10 mmol,
1.4 mL), cooled at 0 °C by an external ice-bath, acryloyl chloride (2 mmol,
0.16 mL) was added dropwise over ten minutes. The reaction mixture was
stirred at 0 °C for 15 min, then extracted with a 2 N solution of HCl (3 Â 10 mL),
washed with brine (3 Â 10 mL), dried over anhydrous Na2SO4 and finally
concentrated to dryness under vacuum. The oily residue was purified by
chromatography on silica gel using hexane/EtOAC 85/15 as the eluent affording
0.40 g (97%) of pure 3. The latter (1.9 mmol, 0.39 g) was dissolved in anhydrous
CH3CN (20 mL) and reacted with 10 (2.1 mmol, 0.53 g) for 4 h at reflux
temperature under magnetic stirring using K2CO3 (2.6 mmol, 0.42 g) as a base.
The inorganic residue was filtered-off and the filtrate concentrated under
reduced pressure and purified by chromatography on silica gel using hexane/
EtOAC 90/10 as eluent to afford 65 mg (9%) of 11 as a light brown oil.
N-[(4-methoxybenzyl)oxy]-N-[1-(4-nitrophenyl)but-3-enyl]acrylamide 11
1H NMR (CDCl3) d: 8.04 (d, 2H, J = 8.8 Hz); 7.27 (d, 2H, J = 8.8 Hz); 7.14 (d, 2H,
J = 8.8 Hz); 6.83 (d, 2H, J = 8.8 Hz); 6.07 (dd, 1H, J = 17.3, 10.7 Hz); 5.85 (dd, 1H,
J = 17.3, 1.6 Hz); 5.75 (t, 1H, J = 6.6 Hz); 5.71–5.57 (m, 1H); 5.41 (dd, 1H,
J = 10.4, 1.6 Hz); 5.04–4.96 (m, 2H); 4.86–4.73 (m, 2H); 3.82 (s, 3H); 2.74–2.64
(m, 1H); 2.56–2.46 (m, 1H). ESI-MS for C21H22N2O5: m/z [M+Na]+ = 405.
Sixty milligrams (0.16 mmol) of 11 were dissolved in toluene (2.5 mL) and
treated with II generation Grubbs catalyst (32 mg) at 60 °C under magnetic
stirring for 20 min. The mixture was concentrated under reduced pressure then
purified on silica gel using hexane/EtOAC 80/20 as the eluent to give 55 mg
(98%) of 12. The latter was dissolved in CH2Cl2 (0.5 mL) and treated with TFA
(0.05 mL) and triethylsilane (0.005 mL) under magnetic stirring for 20 min. The
solvent was removed under reduced pressure and the oily residue was treated
with a few drops of Et2O affording the final product 13 in quantitative yield as a
pale yellow solid.
Chart 2. Retrosynthetic scheme.
on the use of Ag2O to perform the coupling between the O-pro-
tected hydroxamic acid intermediate and the suitable bromoalkyl
derivatives. The use of such reaction conditions afforded a signifi-
cant improvement of the yield.
The versatility of the present approach is witnessed also by the
chance of obtaining six- and theoretically highly membered rings
bearing the endocyclic double bound at diverse positions. It goes
without saying that the double bond may be conveniently reduced
to obtain the corresponding fully saturated derivatives.
Acknowledgments
Apulia Region and MIUR are kindly thanked for their financial
support (Neurobiotech PS126 and PRIN 20085HR5JK_005 grants
respectively). The authors thank the University of Bari ‘Aldo Moro’
(Progetto IDEA-Giovani Ricercatori 2008) for financial support.
References and notes
1-Hydroxy-6-(4-nitrophenyl)-5,6-dihydropyridin-2(1H)-one 13
Mp 66–68 °C. 1H NMR (CDCl3) d: 8.27 (d, 2H, J = 8.6 Hz); 7.62 (d, 2H, J = 8.6 Hz);
7.10 (br s, 1H); 6.30–6.40 (m, 1H); 6.20–6.14 (m, 1H); 5.30 (dd, 1H, J = 9.9,
4.4 Hz); 2.70–2.50 (m, 2H). ESI-MS for C11H10N2O4: m/z [MÀH]À = 233.
16. Aurich, H. G.; Biesemeier, F.; Boutahar, M. Chem. Ber. 1991, 124, 2329–2334.
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To a solution of 3 (1.9 mmol, 0.39 g) in Et2O (20 mL) were added 14 (6.4 mmol,
1.6 g), Ag2O (1.9 mmol, 1.4 g), and MgSO4 (40 mg). After stirring at room
temperature for 12 h, the mixture was concentrated under reduced pressure
and then purified on silica gel using hexane/EtOAC 80/20 as the eluent to afford
0.47 g (63%) of 15 as light brown oil.
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