N-O-Isopropyl Sulfonamido-Based Hydroxamates as Matrix Metalloproteinase Inhibitors: Hit Selection and in Vivo Antiangiogenic Activity

Article abstract of DOI:10.1021/acs.jmedchem.5b00367

Matrix metalloproteinases (MMPs) have been shown to be involved in tumor-induced angiogenesis. In particular, MMP-2, MMP-9, and MMP-14 have been reported to be crucial for tumor angiogenesis and the formation of metastasis, thus becoming attractive targets in cancer therapy. Here, we report our optimization effort to identify novel N-isopropoxy-arylsulfonamide hydroxamates with improved inhibitory activity toward MMP-2, MMP-9, and MMP-14 with respect to the previously discovered compound 1. A new series of hydroxamates was designed, synthesized, and tested for their antiangiogenic activity using in vitro assays with human umbilical vein endothelial cells (HUVECs). A nanomolar MMP-2, MMP-9, and MMP-14 inhibitor was identified, compound 3, able to potently inhibit angiogenesis in vitro and also in vivo in the matrigel sponge assay in mice. Finally, X-ray crystallographic and docking studies were conducted for compound 3 in order to investigate its binding mode to MMP-9 and MMP-14.

Full text of DOI:10.1021/acs.jmedchem.5b00367

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Article
N-O-Isopropyl Sulfonamido-Based Hydroxamates as Matrix Metalloproteinase
Inhibitors: Hit Selection and in vivo Antiangiogenic Activity
Elisa Nuti, Anna Rita Cantelmo, Cristina Gallo, Antonino Bruno, Barbara Bassani, Caterina
Camodeca, Tiziano Tuccinardi, Laura Vera, Elisabetta Orlandini, Susanna Nencetti, Enrico
Adriano Stura, Adriano Martinelli, Vincent Dive, Adriana Albini, and Armando Rossello
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00367 • Publication Date (Web): 11 Aug 2015
Downloaded from http://pubs.acs.org on August 15, 2015
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N-O-Isopropyl Sulfonamido-Based Hydroxamates as Matrix Metalloproteinase
Inhibitors: Hit Selection and in vivo Antiangiogenic Activity
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Elisa Nuti, Anna Rita Cantelmo, Cristina Gallo, Antonino Bruno, Barbara Bassani, Caterina
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Camodeca, Tiziano Tuccinardi, Laura Vera, Elisabetta Orlandini, Susanna Nencetti, Enrico
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3,†
1,†,*
A. Stura, Adriano Martinelli, Vincent Dive, Adriana Albini, and Armando Rossello
1
Dipartimento di Farmacia, Università di Pisa, via Bonanno 6, 56126 Pisa, Italy.
2
Science and Technological Park, IRCCS MultiMedica, via Fantoli 16/15, 20138 Milan, Italy.
3
Laboratory of Translational Research, IRCCS Arcispedale Santa Maria Nuova, viale
Risorgimento 80, 42121 Reggio Emilia, Italy.
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Division of Immunology, Transplants and Infectious Diseases, IRCCS San Raffaele, Via
Olgettina 60, 20132 Milano, Italy
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CEA, iBiTecꢀS, Service d’Ingenierie Moleculaire des Proteines (SIMOPRO), CEꢀSaclay, 91191
Gif sur Yvette Cedex, France.
*
To whom correspondence should be addressed: Armando Rossello, Dipartimento di Farmacia,
Università di Pisa: phone: +39 050 2219562; eꢀmail: armando.rossello@farm.unipi.it.
†
These authors equally contributed to the work.
§
Current Address: Laura Vera, Paul Scherrer Institute, 5232 Villigen, Switzerland
Abstract
Matrix metalloproteinases (MMPs) have been shown to be involved in tumorꢀinduced angiogenesis.
In particular, MMPꢀ2, MMPꢀ9 and MMPꢀ14 have been reported to be crucial for tumor
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angiogenesis and the formation of metastasis, thus becoming attractive targets in cancer therapy.
Here, we report our optimization effort to identify novel Nꢀisopropoxyꢀarylsulfonamide
hydroxamates with improved inhibitory activity toward MMPꢀ2, MMPꢀ9 and MMPꢀ14 with respect
to the previously discovered compound 1. A new series of hydroxamates was designed, synthesized
and tested for their antiꢀangiogenic activity using in vitro assays with human umbilical vein
endothelial cells (HUVECs). A nanomolar MMPꢀ2, MMPꢀ9 and MMPꢀ14 inhibitor was identified,
compound 3, able to potently inhibit angiogenesis in vitro and also in vivo in the matrigel sponge
assay in mice. Finally, Xꢀray crystallographic and docking studies were conducted for compound 3
in order to investigate its binding mode to MMPꢀ9 and MMPꢀ14.
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. Introduction
Angiogenesis is the formation of new blood vessels from preexisting ones and it is an essential
feature of tissue remodeling associated with physiological (i.e. wound healing) and pathological
processes (i.e. solid tumor growth, rheumatoid arthritis, psoriasis). In particular, tumorꢀinduced
angiogenesis is considered to be important to support growth of solid tumors to the size when they
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become invasive and able to give metastasis . Without this process of neovascularization tumors
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remain in their dormant form, where proliferation is balanced with apoptosis . Tumorꢀinduced
angiogenesis requires degradation of the vascular basement membrane and remodeling of the
extracellular matrix (ECM) in order to allow endothelial cells to migrate and invade the surrounding
tissue.
Matrix metalloproteinases (MMPs) or matrixins are a family of 23 zincꢀcontaining endopeptidases
that catalyze the cleavage of various components of the ECM. Given their ability to degrade the
ECM, MMPs involvement in tumorꢀinduced angiogenesis has been extensively investigated.
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Several studies have shown that MMPs are involved in the “angiogenic switch”, one of the first
stages in tumor growth and progression, and also in the maintenance of the growing vascular bed.
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Among all MMPs, MMPꢀ2 , MMPꢀ9 and MMPꢀ14 have been shown to be crucial for tumor
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angiogenesis and the formation of metastasis. MMPꢀ14 or membraneꢀtype matrix
metalloproteinaseꢀ1 (MT1ꢀMMP) is particularly important because besides cleaving many
substrates in the ECM, including type I collagen, it is also involved in MMPꢀ2 activation on the
surface of invasive tumor cells. These actions combined make this membraneꢀbound MMP essential
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to promote pericellular proteolysis and invasion . The importance of MMPꢀ14 as target in cancer
therapy has been recently validated by a study from Dyax Corporation which used a highly
selective fully human MMPꢀ14 inhibitory antibody (DXꢀ2400) in a xenograft model of human
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breast cancer expressing high levels of MMPꢀ14 (MDAꢀMBꢀ231) . The selective inhibition of
MMPꢀ14 was able to block tumor growth, to reduce tumor vascularization and to retard the
formation of metastasis.
Synthetic MMP inhibitors (MMPIs) have been proposed as suitable antiangiogenic agents to be
used in cancer therapy, often in combination with cytotoxic drugs, over the last fifteen years. The
first clinical trials using MMPIs in cancer treatments were largely disappointing due to the fact that
most trials were conducted on patients with advanced stage disease, when the tumor vasculature
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was already wellꢀestablished, and to the fact that broadꢀspectrum MMPIs were used . Nowadays it
is widely accepted that selective inhibitors of only those MMPs validated as targets in anticancer
therapy (i.e. MMPꢀ2, MMPꢀ9 and MMPꢀ14) should be used in order to have efficacy and to avoid
side effects due to crossꢀinhibition of antiꢀtarget proteases. Successful examples of this strategy are
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represented by SBꢀ3CT , a selective MMPꢀ2 and MMPꢀ9 inhibitor able to reduce prostate cancer
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metastasis to the bone and Tꢀcell lymphoma metastasis to the liver in animal models, and cisꢀ
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ACCP a MMPꢀ2 selective inhibitor with antimetastatic activity. A peculiar case is represented by
NSC405020, an allosteric selective inhibitor of MMPꢀ14 able to bind the hemopexin domain which
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significantly inhibited tumor growth in an animal model of breast cancer .
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In our previous papers,
we described the synthesis and biological evaluation of NꢀisopropoxyꢀNꢀ
biphenylsulfonylaminobutyl hydroxamic acids as selective inhibitors of MMPꢀ2 and MMPꢀ14 for
cancer therapy. In particular, compound 1 (ARP101, Figure 1) was found to be a potent
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antiangiogenic agent, being able to block human umbilical vein endothelial cells (HUVEC)
chemoinvasion at micromolar concentrations. Moreover the same compound, tested on the human
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glioma U87MG cell line, was shown to reduce cell invasion by 48% at nanomolar concentrations .
In the present paper, we report our optimization effort to identify novel Nꢀisopropoxyꢀ
arylsulfonamide hydroxamates with improved inhibitory activity toward MMPꢀ2, MMPꢀ9 and
MMPꢀ14 with respect to 1. Our starting hypothesis was that a simultaneous inhibition of all three
MMPs involved in tumorꢀinduced angiogenesis could have an increased effect against angiogenesis
able to block tumor growth. A small series of new ethylamido αꢀsubstituted hydroxamates, 2a-g and
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(Figure 1), was designed and synthesized. These compounds were tested in vitro on recombinant
MMPs, and the most promising derivatives were tested in the chemoinvasion assay, in order to
verify the capability of endothelial cells to invade and digest a reconstituted basement membrane
(matrigel). Finally, the in vivo antiangiogenic activity of 3 was determined using the matrigel
sponge assay in mice.
FIGURE 1 HERE
2
. Results.
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Design of novel MMPs inhibitors. As already seen the NꢀOꢀisopropyl substituent (P2’ group,
Figure 1) was important in order to achieve selectivity over MMPꢀ1, while still maintaining activity
against MMPꢀ2. For this reason we chose to maintain the NꢀOꢀisopropyl group in the new series of
sulfonamido derivatives. Moreover, the isopropyl substituent in the α position relative to the
hydroxamate (P1 group) present in 1 was replaced with several Nꢀethylamido groups because
changes at this level in similar arylsulfonamidic scaffolds had already given an improved activity
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against the target enzyme, ADAMꢀ17, in previous studies . In our hypothesis, the proper P1’
substituent together with the NꢀOꢀisopropyl group should be able to direct the selectivity of the new
compounds towards the target enzymes, while the presence of P1 substituents of variable size
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should modulate their potency exploiting small changes in the S1 pocket between the different
MMPs.
Chemistry. Optically active hydroxamates 2a-g (Scheme 1) were prepared starting from the (S)ꢀαꢀ
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hydroxyꢀtertꢀbutylꢀester 7, which was synthesized as previously described. A Mitsunobu
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condensation of sulfonamide 4ꢀ6
with the alcohol 7 in the presence of diisopropyl
azodicarboxylate (DIAD) and triphenylphosphine gave the tertꢀbutyl esters (R)ꢀ8-10. Tertꢀbutyl
esters 14a-g were obtained by Pdꢀcatalyzed hydrogenation of 8-10 followed by acylation with
commercial acyl chlorides, using N,Nꢀdiisopropylethylamine (DIPEA) as base. Acid cleavage of
esters 14a-g yielded carboxylates 15a-g which were finally converted to their corresponding
hydroxamates 2a-g by condensation with Oꢀ(tertꢀbutyldimethylsilyl)hydroxylamine followed by
acid hydrolysis with TFA.
SCHEME 1 HERE
Hydroxamic acid 3 was synthesized as reported in Scheme 2, starting from the previously described
19a
alcohol 16 . A Mitsunobu coupling of sulfonamide 5 with the alcohol 16, gave (R)ꢀtertꢀbutyl ester
7. Acid cleavage of 17 yielded carboxylic acid 18 which was converted to its Oꢀsilylate derivative
1
upon treatment with Oꢀ(tertꢀbutylꢀdimethylsilyl)hydroxylamine. Hydrolysis with trifluoroacetic
acid of tertꢀbutyl Oꢀsilylate provided hydroxamic acid 3.
SCHEME 2 HERE
Biological activity on isolated enzymes. The new hydroxamic acids synthesized (2a-g and 3) were
tested in vitro on human recombinant MMPs by a fluorometric assay, which uses a fluorogenic
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peptide as the substrate. Results are reported in Table 1, together with those already obtained for
the previously described analogue 1. The compounds were tested on MMPꢀ2, ꢀ9 and ꢀ14, as
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validated targets in cancer therapy, and MMPꢀ1, considered as antiꢀtarget whose inhibition could be
responsible for some side effects, such as musculoskeletal syndrome, clinically observed with the
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use of broad spectrum MMPIs. Selectivity over MMPꢀ3 was also evaluated because this enzyme
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was validated as potential antiꢀtarget in cancer therapy. Finally, the activity against MMPꢀ13 was
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also taken in consideration, given the recently reported role of this enzyme in promoting tumor
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angiogenesis.
The replacement of the isopropyl substituent in the α position relative to the hydroxamate (P1
group) present in 1 with several Nꢀethylamido chains (compounds 2a-d) caused a decrease of
activity against MMPꢀ14. This change did not affect activity against MMPꢀ9 but derivative 2c
bearing an Nꢀethylacetoamido group in P1 resulted 2ꢀfold more active than 1 on MMPꢀ2 (Table 1),
showing an IC =0.33 nM. Given these promising results, we chose to introduce a paraꢀmethoxy
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substituent on the biphenyl moiety (P1’group) present in 2c in order to further increase activity and
selectivity towards MMPꢀ2 (known to be a deepꢀS1’ pocket MMP). As expected, the paraꢀmethoxyꢀ
analogue of 2c, compound 2e, resulted about 6ꢀfold more active than 1 on MMPꢀ2, with an
IC50=0.13 nM. In a further attempt to expand the selectivity of action towards MMPꢀ14, as our third
target for the inhibition of tumorꢀinduced angiogenesis controlled by MMPs, we decided to verify
the effects of the introduction of a planar and lipophilic group in P1 on the scaffold of 2e in place of
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its acetamido R substituent. Therefore derivative 3, bearing an Nꢀethylphthalimido substituent in
P1, potentially able to give both hydrophobic πꢀπ stacking interactions with aromatic residues and
hydrogen bonds with the catalytic site of MMPꢀ14, was synthesized. Actually, compound 3 gave
the best inhibitory results on MMPꢀ9 (IC =0.43 nM) and MMPꢀ14 (IC =3.9 nM) still maintaining
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a subꢀnanomolar activity on MMPꢀ2 (IC =0.67 nM). The activity data on MMPꢀ14 was
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noteworthy, given the lack of nanomolar small molecule MMPꢀ14 inhibitors reported so far in
literature. Moreover, compound 3 showed a 15ꢀfold increase of activity towards MMPꢀ9 and also an
improved selectivity over MMPꢀ1 and MMPꢀ3 with respect to the reference compound 1. On the
contrary, the benzenesulfonamido derivative 2g (R=H) showed a decrease of activity, in the
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micromolar range, against all tested enzymes, probably due to the small size of its P1' group unable
to properly fit in the S1' pocket of MMPs. This is a peculiarity of our NꢀOꢀisopropyl sulfonamidoꢀ
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based hydroxamates which have already shown different profiles of potency and selectivity and
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associated lack of cell toxicity
with respect to the other well known families of sulfonamide
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hydroxamates. In particular, the use of a short aromatic sulfonamido group is detrimental for this
class of compounds (high microꢀmolar activity on all tested enzymes), while in all the other known
classical sulfonamides (e.g. tertiary and secondary sulfonamides) this type of small substituent is
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often able to maintain nanomolar activities, without selectivity of action.
The most promising compounds of this series, 2e and 3, were selected to be submitted to cellꢀbased
assays in order to prove their antiangiogenic activity, and we used 2g as a negative control.
TABLE 1 HERE
Effect of MMP inhibitors on endothelial cell migration and invasion. The effects of 2e and 3 on
human umbilical vein endothelial cell (HUVEC) migration were assessed in vitro, and compared to
those obtained using compound 2g. The assay was performed using a modified Boyden chamber
where fetal bovine serum (FBS) acted as a chemoattractant. Addition of 0.001 to 10 ꢁM of
compound 3 and 2e resulted in an inhibition of endothelial cell migration, as compared to
compound 2g (Figure 2AꢀC). The strongest and significant (***p<0.001,* p<0.05) inhibitory effect
on HUVEC migration was exerted by compound 3 in a dose dependent manner.
FIGURE 2 HERE
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The effect of 3, 2e and 2g on HUVEC invasion was evaluated using modified Boyden chambers
where 8 µm poreꢀsize polycarbonate filters, preꢀcoated with a reconstituted basement membrane
(matrigel, 10 mg/mL), were used as a barrier to invasion. Treatment with 2e and 3 significantly
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(***p<0.001, **p<0.01) reduced the FBSꢀinduced HUVEC invasion as compared to 2g, in a doseꢀ
dependent manner (Figure 3AꢀC). Interestingly, a significant inhibitory effect with 3 was observed
already at the lower concentration (0.001 ꢁM) (***p<0.001; Figure 3A).
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FIGURE 3 HERE
Effect of MMP inhibitors on endothelial morphogenesis in vitro. When cultured in a 3ꢀ
dimensional (3D) matrigel layer, HUVECs tend to organize into capillaryꢀlikeꢀstructures,
mimicking in vitro the events that occur in vivo during angiogenesis. The addition of compound 3
significantly interfered with FBSꢀinduced morphogenesis of HUVE cells in a dose dependent
manner, restraining their ability to organize in a capillaryꢀlike network, as observed following 6 h of
treatment and compared to compound 2e, and 2g (Figure 4A). Quantification of meshes, as a direct
parameter to measure HUVEC network efficiency, showed a significantly decreased number of
meshes (***p<0.001, *p<0.05) in cells treated with compound 3 as compared to 2e and 2g (Figure
4B).
FIGURE 4 HERE
Effect of MMP inhibitors on MMP-2 and MMP-9 levels by qPCR, western blot (WB) and
gelatin zymography. The ability of compound 3 to interfere with MMPꢀ2 transcript and protein
levels was assessed by qPCR and confirmed by WB. Since HUVEC constitutively expressed only
MMPꢀ2, the cells were preꢀstimulated under serumꢀfree condition, with 30 nM PMA (Phorbolꢀ12ꢀ
myristateꢀ13ꢀacetate) to promote MMPs production. After PMA preꢀtreatment, low levels of MMP9
mRNA were detectable (data not shown). Both MMPꢀ2 protein and mRNA were decreased
following 24h of treatment with compound 3 (Figure 5AꢀB).
To investigate whether compounds 3 and 2e were effectively able to inhibit MMPꢀ2 and MMPꢀ9
activity, gelatin zymography were performed on conditioned media (CM) obtained either from
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untreated HUVECs, followed by exposition to increasing concentrations of 3, 2e and 2g during the
zymography, or conditioned media from HUVE cells exposed to the compounds 3 and 2g. As
shown in Figure 5CꢀD, both compound 3 and 2e reduced MMPꢀ2 and MMPꢀ9 gelatinase activity in
a doseꢀdependent manner, as compared to 2g. Compound 3 showed a strong inhibitory effect
already at low concentrations. The inhibitory effect of compound 3 starts from 0.001 ꢁM, while for
compound 2e, which exerted a less pronounced gelatinase activity, inhibition was active from 0.1
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ꢁM. Compound 2g showed no effect on the MMPs activities. We therefore evaluated the ability of
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to inhibit MMPꢀ2 and MMPꢀ9 activity directly treating HUVE cells, using 2g as a negative
control. Cells were treated with 3 and 2g 10 ꢁM for 24 hours, using the same protocol for WB
analysis. As shown in figure 5EꢀFꢀG only compound 3 significantly (**p<0.01) affected MMP
release and production.
FIGURE 5 HERE
Effects of compound 3 on endothelial cell viability and induction of apoptosis. We explored the
possibility that compound 3, the most promising compound of this series, could exert nonꢀselective
toxicity or induce endothelial cell death. We analyzed proliferation by MTT (3ꢀ[4,5ꢀ
dimethylthiazolꢀ2ꢀyl]ꢀ2,5 diphenyl tetrazolium bromide) assays and by staining with Annexin V and
7ꢀaminoꢀactinomycin D followed by flow cytometry analysis. No significant inhibition of
endothelial cell growth was observed at different doses of compound 3 (Figure 6). Hydrogen
peroxide (200 µM) induced apoptosis in approximately 20% of the cells (both Annexin V and 7ꢀ
aminoꢀactinomycin D positive; Figure 7). Cells treated with the inhibitor, even at highest
concentrations, showed a high percentage (about 85%) of viable endothelial cells and no apoptosis
(Figure 7). These data suggest that compound 3 does not exert toxicity or induce apoptosis in
endothelial cells.
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FIGURE 6 HERE
FIGURE 7 HERE
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Effects of compound 3 on angiogenesis in vivo. We used the matrigel sponge assay model as a
rapid and quantitative system for measuring the in vivo the antiꢀangiogenic activity of compound 3.
Subcutaneous injection of matrigel produces a threeꢀdimensional pellet that, when associated to
angiogenic factors, becomes rapidly vascularized. VEGF, TNFꢀα, and heparin were used as
angiogenic/inflammatory stimuli mixed with the matrigel, inducing an intense angiogenic reaction,
as indicated indirectly by hemoglobin content. Addition of increasing concentrations of 3 to this
mixture inhibited the in vivo angiogenic response (Figure 8A) decreasing hemoglobin content. To
more precisely quantify the effects of compound 3 on angiogenesis, we quantified the number of
ꢀ
+
endothelial cells in the pellets (identified as CD45 /CD31 cells) by flow cytometry. The plugs
excised from mice treated with compound 3 at 0.1ꢀ1ꢀ10 µM, both by adding the compound in the
plugs and by IP injection (starting at day 0 of matrigel injection), showed significantly (*p<0.05)
ꢀ
+
decreased number of CD45 /CD31 cells in a dose dependent manner, as compared with VTH alone
Figure 8 B).
(
FIGURE 8 HERE
X-ray Crystallography and Molecular Modeling. To get an insight into the binding mode of
compound 3 into the MMPꢀ9 active site, Xꢀray crystallography studies were performed in
comparison with the reference compound 1.
The structures of three polymorphs of MMPꢀ9 with 1 have been analyzed (Figure 9A). The trigonal
crystal form (PDB code 4XCT) has not been reported before. The structure has been solved at 1.3 Å
resolution, with a good refinement statistic (Table 1S, Supp. Information). The other crystal forms
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with two molecules in the MMPꢀ9 asymmetric unit have anisotropy problems not reported for other
inhibitor complexes. Despite refinement problems, the three polymorphs show that the binding of 1
is well defined, except variability in the propyl and oxypropyl moieties (Figure 9B). The structure
of the MMPꢀ9ꢀ3 complex (PDB code 4WZV) was determined in the monoclinic space group P2
.65 Å resolution, with similar cell parameters as one of the structures determined for 1. The
refinement statistics are reported in Table 1S. The ligand is positioned in strong electron density
Figure 9C). The binding in the S1' pocket for the two ligands is identical and the two biphenyl
1
at
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1
(
rings overlap. Excluding the oxypropyl which in the MMP9 catalytic site, for 1 adopts a different
orientation from that for 3, there is also significant variation in the positioning of the sulfur oxygens
between 1 and 3 (Figure 9D). The interaction that the sulfonamide oxygen makes for 1 with the
protein backbone are at 2.90, 3.43 and 3.11 Å from the amide of Leu 188 and the carbonyl and
amide of Ala 189, while the distances improve for 3 to 2.75, 3.4 and 3.13, respectively. Only a
single additional direct hydrogen bond (2.97 Å) is observed between one of the phthaloyl carboxyl
of 3 and the amide nitrogen of Ala 191 that in the 1 complex is made with a water molecule. The
second phthaloyl carboxyl of 3 participates in promoting MMPꢀ9 homodimerization, through an
interaction with Tyr 420 via a water molecule (Figure 9E). The more precise positioning of 3 is
likely to contribute to its higher affinity for MMPꢀ9.
FIGURE 9 HERE
Since compound 3 showed a high inhibition activity also against MMPꢀ2 and MMPꢀ14, its
interactions and binding disposition into these two MMPs were evaluated by means of docking
studies followed by molecular dynamic simulations. The docking results highlighted that the
binding orientation of the ligand into both MMPs was almost identical to that observed into the
MMP9ꢀ3 Xꢀray structure. Then, with the aim of verifying the stability of the inhibitor−enzyme
interactions, the MMPꢀ2ꢀ, MMPꢀ9ꢀ, and MMPꢀ14ꢀ3 complexes were subjected to 5 ns of molecular
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dynamic simulations (Figure 10). The ligand binding disposition suggested by the crystallographic
structure was very stable for all the three MMPs with an average RMSD of about 1.2 Å, and with
the maintenance of the lipophilic interactions of the biphenyl moiety inside the S1’ cavity and the
hydrogen bonds of the sulfonamide portion with the nitrogen backbone of L199 and A200 (MMPꢀ9
residue number). Differently from that observed from the MMP9ꢀ3 Xꢀray structure, the
phthalimidic scaffold did not show Hꢀbonds but in all three complexes showed a highly stable πꢀπ
interaction with the catalytic H243, that should be responsible for the high activity of this
compound.
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FIGURE 10 HERE
3
. Discussion and Conclusions.
In this study, a new series of NꢀOꢀisopropyl sulfonamidoꢀbased hydroxamates with improved
inhibitory activity toward MMPꢀ2, MMPꢀ9 and MMPꢀ14 with respect to the previously discovered
compound 1 was designed and synthesized. Among these compounds, two promising derivatives,
2
e and 3, were identified by fluorometric assays on isolated enzymes and were chosen to be
submitted to cellꢀbased assays on HUVECs in order to assess their antiangiogenic activity. 2e was a
nanomolar inhibitor of MMPꢀ2 and MMPꢀ9, while 3 was a subꢀnanomolar inhibitor of MMPꢀ2 and
MMPꢀ9 but also a nanomolar inhibitor of MMPꢀ14 (IC50= 3.9 nM) selective over MMPꢀ1 and
MMPꢀ3. Xꢀray crystallographic and docking studies validated the importance of the Nꢀ
ethylphthalimido group for a proper positioning of 3 in the catalytic site of MMPꢀ9 and MMPꢀ14.
First, the ability of the two compounds to inhibit gelatinase activity was evaluated on HUVEC
conditioned medium derived either from untreated cells, further subjected to MMPI exposition or
on directly MMPIꢀtreated cells, by gelatin zymography and their efficacy was verified in the
nanomolar range. Then, these two compounds were tested in the chemoinvasion assay, an in vitro
model of angiogenesis able to quantify the invasive potential of endothelial cells, and in a
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chemotaxis assay, used to measure the effect of 2e and 3 on HUVEC migration. Compound 2g was
used as negative control in all these biological assays to prove our starting hypothesis that a
simultaneous inhibition of all three MMPs involved in tumorꢀinduced angiogenesis could have an
increased effect against angiogenesis. In fact 2g is MMPꢀ2 inhibitor unable to inhibit MMPꢀ9 and
MMPꢀ14 in the nanomolar range. From this screening, compound 3 emerged as the most active one,
being able to block angiogenesis in vitro already at nanomolar concentrations while 2g resulted
inactive in all assays at the concentrations used. Moreover, 3 confirmed its activity in the matrigel
sponge model in mice, without exerting toxicity or inducing apoptosis in endothelial cells. Taken
together, these results supported our starting hypothesis that the simultaneous inhibition of all three
MMPs (MMPꢀ2, ꢀ9 and ꢀ14), sparing the antiꢀtargets MMPꢀ1 and ꢀ3, can represent an effective way
to increase the antiangiogenic potential of MMP inhibitors to be exploited for cancer therapy, alone
or in combination with cytotoxic compounds. Further studies will be necessary to demonstrate the
efficacy of compound 3 to block tumor growth in human cancer models.
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4. Experimental section
1
Chemistry. Melting points were determined on a Kofler hotstage apparatus and are uncorrected. H
13
and C NMR spectra were determined with a Varian Gemini 200 MHz spectrometer or a Bruker
Avance III HD 400 MHz spectrometer. Chemical shifts (δ) are reported in parts per million
downfield from tetramethylsilane and referenced from solvent references. Coupling constants J are
13
reported in hertz; C NMR spectra were fully decoupled. The following abbreviations are used:
singlet (s), doublet (d), triplet (t), doubleꢀdoublet (dd), broad (br) and multiplet (m).
Chromatographic separations were performed on silica gel columns by flash column
chromatography (Kieselgel 40, 0.040–0.063 mm; Merck) or using ISOLUTE Flash Si II cartridges
(Biotage). Reactions were followed by thinꢀlayer chromatography (TLC) on Merck aluminum silica
gel (60 F254) sheets that were visualized under a UV lamp and hydroxamic acids were visualized
with FeCl aqueous solution. Evaporation was performed in vacuo (rotating evaporator). Sodium
3
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sulfate was always used as the drying agent. Commercially available chemicals were purchased
from SigmaꢀAldrich. Combustion analyses on target compounds were performed by our Analytical
Laboratory in Pisa. All compounds showed >95% purity. See the Supporting Information for
compound purity analysis data for final compounds.
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General procedure for the preparation of (R)-tert-butyl esters 8-10. Diisopropyl
azodicarboxylate (DIAD) (1.28 mL, 6.52 mmol) was added dropwise to a solution containing (S)ꢀ
tertꢀbutyl 4ꢀ(benzyloxycarbonylamino)ꢀ2ꢀhydroxybutanoate 7 (0.8 g, 2.61 mmol), the appropriate
sulfonamide 4-6 (3.91 mmol) and triphenylphosphine (2.05 g, 7.83 mmol) in anhydrous THF (45
mL) under nitrogen atmosphere at 0 ºC. The resulting solution was stirred for 5h at rt and
evaporated under reduced pressure to afford a crude product, which was purified by flash
chromatography on silica gel.
(R)-tert-Butyl
8-(biphenyl-4-ylsulfonyl)-10-methyl-3-oxo-1-phenyl-2,9-dioxa-4,8-
diazaundecane-7-carboxylate (8). The title compound was prepared from Nꢀisopropoxyꢀ
sulfonamide 4 following the general procedure. The crude was purified by flash chromatography on
1
silica gel (nꢀhexane/EtOAc 5:1) to give a yellow oil (79% yield). HꢀNMR (CDCl
3
) δ: 1.22ꢀ1.25
(
m, 15H); 2.04 (m, 2H); 3.22ꢀ3.37 (m, 2H); 4.12 (dd, J= 7.14 Hz, J= 14.29 Hz, 1H); 4.43 (septet,
J= 6.2 Hz, 1H); 5.08 (s, 2H); 7.34 (m, 5H); 7.42ꢀ7.53 (m, 3H); 7.55ꢀ7.61 (m, 2H); 7.7ꢀ7.74 (m, 2H);
.94ꢀ7.98 (m, 2H).
(R)-tert-Butyl 8-(4'-methoxybiphenyl-4-ylsulfonyl)-10-methyl-3-oxo-1-phenyl-2,9-dioxa-4,8-
7
diazaundecane-7-carboxylate (9). The title compound was prepared from Nꢀisopropoxyꢀ
sulfonamide 5 following the general procedure. The crude was purified by flash chromatography on
1
silica gel (nꢀhexane/EtOAc 5:2) to give a yellow oil (74% yield). HꢀNMR (CDCl ) δ: 1.21ꢀ1.32 (m,
3
1
5H); 1.90ꢀ2.10 (m, 2H); 3.10ꢀ3.50 (m, 2H); 3.86 (s, 3H); 4.06ꢀ4.16 (m, 1H); 4.43 (septet, J= 6.2
Hz, 1H); 5.08 (s, 2H); 6.97ꢀ7.02 (m, 2H); 7.34 (m, 5H); 7.51ꢀ7.56 (m, 2H); 7.65ꢀ7.70 (m, 2H);
.90ꢀ7.94 (m, 2H).
7
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(R)-tert-Butyl
8-(4-bromophenylsulfonyl)-10-methyl-3-oxo-1-phenyl-2,9-dioxa-4,8-
diazaundecane-7-carboxylate (10). The title compound was prepared from Nꢀisopropoxyꢀ
sulfonamide 6 following the general procedure. The crude was purified by flash chromatography on
1
silica gel (nꢀhexane/EtOAc 8:3) to give a yellow oil (87% yield). HꢀNMR (CDCl
3
) δ: 1.19ꢀ1.30 (m,
10
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5H); 1.90ꢀ2.10 (m, 2H); 3.10ꢀ3.50 (m, 2H); 4.01ꢀ4.12 (m, 1H); 4.43 (septet, J= 6.2 Hz, 1H); 5.10
(s, 2H); 7.36 (m, 5H); 7.64ꢀ7.68 (m, 2H); 7.73ꢀ7.77 (m, 2H).
General procedure for the preparation of (R)-tert-butyl esters 11-13. A solution of the
appropriate Cbzꢀderivative 8-10 (1.27 mmol) in MeOH (80 mL) was stirred under hydrogen
atmosphere in the presence of 10% PdꢀC (0.20 g) and glacial acetic acid (80 mL) for 17 h at rt. The
resulting mixture was filtered on celite and the filtrate was evaporated under reduced pressure to
give the deprotected salt.
(R)-4-tert-Butoxy-3-(N-isopropoxybiphenyl-4-ylsulfonamido)-4-oxobutan-1-aminium acetate
(11). The title compound was prepared from Cbzꢀderivative 8 following the general procedure.
1
Brownish oil, 93% yield. HꢀNMR (CDCl
3
) δ: 1.10 (brs, 9H); 1.20 (t, J= 4.4Hz, 6H); 2.16ꢀ2.30 (m,
2H); 3.16 (m, 2H); 4.34ꢀ4.46 (m, 2H); 7.40ꢀ7.51 (m, 3H); 7.56ꢀ7.59 (m, 2H); 7.71ꢀ7.75 (m, 2H);
13
8
1
.00ꢀ8.04 (m, 2H). CꢀNMR (CDCl ) δ: 21.15; 21.22; 27.72; 36.70; 62.97; 80.03; 82.49; 127.03;
3
27.45; 127.59; 127.76; 128.67; 129.14; 130.49; 133.60; 139.30; 146.89.
(R)-4-tert-Butoxy-3-(N-isopropoxy-4'-methoxybiphenyl-4-ylsulfonamido)-4-oxobutan-1-
aminium acetate (12). The title compound was prepared from Cbzꢀderivative 9 following the
1
general procedure. Brownish oil, 95% yield. HꢀNMR (CDCl
3
) δ: 1.15ꢀ1.27 (m, 15H); 2.04ꢀ2.10
(m, 2H); 3.06 (m, 2H); 3.86 (s, 3H); 4.27 (m, 1H); 4.41 (septet, J= 6.2 Hz, 1H); 6.98ꢀ7.02 (m, 2H);
7
.52ꢀ7.56 (m, 2H); 7.68ꢀ7.72 (m, 2H); 7.95ꢀ7.99 (m, 2H).
(R)-4-tert-Butoxy-3-(N-isopropoxyphenylsulfonamido)-4-oxobutan-1-aminium acetate (13).
The title compound was prepared from Cbzꢀderivative 10 following the general procedure. Brown
1
foam, 87% yield. HꢀNMR (CDCl
3
) δ: 1.17ꢀ1.33 (m, 15H); 2.17ꢀ2.40 (m, 2H); 3.22 (m, 2H); 4.33ꢀ
4
.50 (m, 1H); 5.04 (septet, J= 6.2 Hz, 1H); 7.53ꢀ7.66 (m, 3H); 7.96ꢀ8.00 (m, 2H).
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General procedure for the preparation of (R)-tert-butyl esters 14a-g. A solution of the
appropriate aminium acetate 11-13 (0.59 mmol) in dry DMF (6 mL) was treated with the
appropriate acyl chloride (0.70 mmol) and i-Pr
was stirred at rt for 17h, then was diluted with AcOEt, washed with H
evaporated. The crude was purified by flash chromatography on silica gel.
2
NEt (0.20 mL, 1.18 mmol). The reaction mixture
2
O, dried over Na SO and
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(R)-tert-Butyl 4-benzamido-2-(N-isopropoxybiphenyl-4-ylsulfonamido)butanoate (14a). The
title compound was prepared from aminium acetate 11 and benzoyl chloride following the general
procedure. The crude was purified by flash chromatography on silica gel (nꢀhexane/EtOAc 2.5:1) to
1
give 14a as a yellow oil (34% yield). HꢀNMR (CDCl
3
) δ: 1.13 (brs, 9H); 1.23 (d, J= 5.1 Hz, 3H);
1.26 (d, J= 4.4Hz, 3H); 2.10ꢀ2.21 (m, 2H); 3.34ꢀ3.50 (m, 1H); 3.80ꢀ3.92 (m, 1H); 4.23 (t, J= 7.1
Hz, 1H); 4.45 (septet, 1H); 7.39ꢀ7.59 (m, 10H); 7.69ꢀ7.73 (m, 2H); 7.93ꢀ7.98 (m, 2H).
(R)-tert-Butyl
4-methanesulfonamido-2-(N-isopropoxybiphenyl-4-ylsulfonamido)butanoate
(14b). A solution of 11 (0.30 g, 0.60 mmol) in dry THF (3 mL) was treated with methanesulfonyl
chloride (0.05 mL, 0.60 mmol) and Nꢀmethylmorpholine (0.13 mL, 1.2 mmol). The reaction
mixture was stirred at rt overnight, then was diluted with AcOEt, washed with H O, dried over
Na SO and evaporated. The crude was purified by flash chromatography (nꢀhexane/AcOEt = 3:2),
2
2
4
1
to give 14b (100 mg, 32 % yield) as a yellow oil. HꢀNMR (CDCl ) δ: 1.14 (brs, 9H); 1.20ꢀ1.26 (m,
3
6
H); 2.04 (brs, 2H); 2.95 (s, 3H); 3.29 (brs, 2H); 4.25 (t, J= 7.1Hz, 1H); 4.42 (septet, 1H); 7.43ꢀ7.54
(m, 3H); 7.58ꢀ7.62 (m, 2H); 7.74ꢀ7.78 (m, 2H); 7.96ꢀ8.00 (m, 2H).
R)-tert-Butyl 4-acetamido-2-(N-isopropoxybiphenyl-4-ylsulfonamido)butanoate (14c). The
(
title compound was prepared from aminium acetate 11 and acetyl chloride following the general
procedure. The crude was purified by flash chromatography on silica gel (nꢀhexane/EtOAc 1:1) to
1
give 14c as a yellow oil (22% yield). HꢀNMR (CDCl
3
) δ: 1.19ꢀ1.24 (m, 15H); 1.90ꢀ2.01 (m, 5H);
3.13ꢀ3.23 (m, 1H); 3.53 (m, 1H); 4.12 (m, 1H); 4.40 (septet, 1H); 6.11 (brs, 1H); 7.41ꢀ7.52 (m, 3H);
13
7
3
.57ꢀ7.61 (m, 2H); 7.72ꢀ7.76 (m, 2H); 7.94ꢀ7.98 (m, 2H). CꢀNMR (CDCl ) δ: 21.17; 23.48;
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27.76; 36.17; 63.59; 79.80; 82.25; 127.39; 127.52; 128.72; 129.16; 130.14; 133.80; 139.19; 146.84;
1
70.22.
R)-tert-Butyl
14d). The title compound was prepared from aminium acetate 11 and 2ꢀphenylacetyl chloride
(
(
2-(N-isopropoxybiphenyl-4-ylsulfonamido)-4-(2-phenylacetamido)butanoate
10
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following the general procedure. The crude was purified by flash chromatography on silica gel (nꢀ
1
hexane/EtOAc 2:1) to give 14d as a yellow oil (20% yield). HꢀNMR (CDCl ) δ: 1.10ꢀ1.22 (m,
3
1
5H); 1.81ꢀ2.05 (m, 2H); 3.00ꢀ3.21 (m, 1H); 3.50ꢀ3.60 (m, 3H); 3.96 (t, J= 7.5Hz, 1H); 4.38
septet, 1H); 7.28ꢀ7.37 (m, 5H); 7.43ꢀ7.54 (m, 3H); 7.57ꢀ7.62 (m, 2H); 7.68ꢀ7.73 (m, 2H); 7.80ꢀ
.84 (m, 2H).
(R)-tert-Butyl 4-acetamido-2-(N-isopropoxy-4'-methoxybiphenyl-4-ylsulfonamido)butanoate
14e). The title compound was prepared from aminium acetate 12 and acetyl chloride following the
(
7
(
general procedure. The crude was purified by flash chromatography on silica gel (nꢀhexane/EtOAc
1
2
:3) to give 14e as a yellow oil (42% yield). HꢀNMR (CDCl
3
) δ: 1.19ꢀ1.25 (m, 15H); 1.90ꢀ2.04 (m,
5
H); 3.13ꢀ3.50 (m, 2H); 3.87 (s, 3H); 4.12 (m, 1H); 4.41 (septet, 1H); 6.99ꢀ7.03 (m, 2H); 7.53ꢀ7.57
(m, 2H); 7.69ꢀ7.73 (m, 2H); 7.91ꢀ7.95 (m, 2H).
R)-tert-Butyl 2-(N-isopropoxy-4'-methoxybiphenyl-4-ylsulfonamido)-4-(2-
phenylacetamido)butanoate (14f). The title compound was prepared from aminium acetate 12 and
(
2
ꢀphenylacetyl chloride following the general procedure. The crude was purified by flash
1
chromatography on silica gel (nꢀhexane/EtOAc 3:2) to give 14f as a yellow oil (24% yield). Hꢀ
NMR (CDCl
3
) δ: 1.16ꢀ1.25 (m, 15H); 1.90ꢀ2.05 (m, 2H); 3.12 (m, 2H); 3.57 (s, 2H); 3.87 (s, 3H);
.94 (t, J= 7.5Hz, 1H); 4.37 (septet, 1H); 6.98ꢀ7.04 (m, 2H); 7.31ꢀ7.37 (m, 5H); 7.52ꢀ7.57 (m, 2H);
.64ꢀ7.68 (m, 2H); 7.77ꢀ7.81 (m, 2H).
3
7
(R)-tert-Butyl 4-acetamido-2-(N-isopropoxyphenylsulfonamido)butanoate (14g). The title
compound was prepared from aminium acetate 13 and acetyl chloride following the general
procedure. The crude was purified by flash chromatography on silica gel (nꢀhexane/EtOAc 3:1) to
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give 14g as a yellow oil (55% yield). HꢀNMR (CDCl ) δ: 1.16ꢀ1.23 (m, 15H); 1.94 (s, 3H); 3.10ꢀ
3
3
.50 (m, 2H); 4.08 (m, 1H); 4.36 (septet, 1H); 7.49ꢀ7.69 (m, 3H); 7.87ꢀ7.92 (m, 2H).
General procedure for the preparation of (R)-carboxylic acids 15a-g. Trifluoroacetic acid
(
0.9 mL, 57.00 mmol) was added dropwise to a stirred solution of the appropriate tertꢀbutyl ester
14a-g (0.21 mmol) in freshly distilled CH Cl (1.0 mL), cooled to 0°C. The solution was stirred for
h at 0°C and the solvent was removed in vacuo to give a crude that was purified by trituration
with Et O/nꢀhexane.
R)-4-Benzamido-2-(N-isopropoxybiphenyl-4-ylsulfonamido)butanoic acid (15a). The title
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0
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2
5
2
(
compound was prepared from tertꢀbutyl ester 14a following the general procedure. Yellow oil, 99%
1
yield. HꢀNMR (CDCl ) δ: 1.20 (t, J= 5.1 Hz, 6H); 1.80ꢀ2.28 (m, 2H); 3.40ꢀ3.55 (m, 1H); 3.60ꢀ3.82
3
(
m, 1H); 4.30ꢀ4.50 (m, 2H); 6.56 (brs, 1H); 7.43ꢀ7.58 (m, 10H); 7.66ꢀ7.69 (m, 2H); 7.91ꢀ7.94 (m,
2
H).
(R)-4-Methanesulfonamido-2-(N-isopropoxybiphenyl-4-ylsulfonamido)butanoic acid (15b).
The title compound was prepared from tertꢀbutyl ester 14b following the general procedure. White
1
solid, 79% yield. HꢀNMR (CDCl ) δ: 1.22 (d, J= 6.2 Hz, 6H); 2.06ꢀ2.17 (m, 2H); 2.91 (s, 3H); 3.21
3
(
brs, 2H); 4.34ꢀ4.44 (m, 2H); 7.42ꢀ7.53 (m, 3H); 7.61ꢀ7.66 (m, 2H); 7.74ꢀ7.78 (m, 2H); 7.95ꢀ7.99
m, 2H).
(
(R)-4-Acetamido-2-(N-isopropoxybiphenyl-4-ylsulfonamido)butanoic acid (15c). The title
compound was prepared from tertꢀbutyl ester 14c following the general procedure. White solid,
1
8
0% yield. HꢀNMR (CDCl ) δ: 1.16 (d, J= 2.01 Hz, 3H); 1.19 (d, J= 2.01 Hz, 3H); 1.92ꢀ2.10 (m,
3
5
H); 3.15ꢀ3.60 (m, 2H); 4.20ꢀ4.39 (m, 2H); 6.73 (brs, 1H); 7.42ꢀ7.52 (m, 3H); 7.59ꢀ7.63 (m, 2H);
7.73ꢀ7.77 (m, 2H); 7.93ꢀ7.97 (m, 2H); 10.24 (brs, 1H).
R)-2-(N-isopropoxybiphenyl-4-ylsulfonamido)-4-(2-phenylacetamido)butanoic acid (15d). The
(
title compound was prepared from tertꢀbutyl ester 14d following the general procedure. White
1
solid, 70% yield. HꢀNMR (CDCl ) δ: 1.14 (d, J= 3.4 Hz, 3H); 1.17 (d, J= 3.4 Hz, 3H); 1.93ꢀ2.10
3
(
m, 2H); 3.14 (m, 1H); 3.47 (m, 1H); 3.61 (s, 2H); 4.13 (t, J= 7.3 Hz, 1H); 4.33 (septet, 1H); 5.32
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(brs, 1H); 6.14 (brs, 1H); 7.22ꢀ7.26 (m, 1H); 7.30ꢀ7.37 (m, 4H); 7.42ꢀ7.53 (m, 3H); 7.58ꢀ7.63 (m,
2
H); 7.67ꢀ7.73 (m, 2H); 7.82ꢀ7.86 (m, 2H).
(R)-4-Acetamido-2-(N-isopropoxy-4'-methoxybiphenyl-4-ylsulfonamido)butanoic acid (15e).
The title compound was prepared from tertꢀbutyl ester 14e following the general procedure. White
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14
15
16
17
18
19
20
21
22
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solid, 67% yield. HꢀNMR (CDCl
3
) δ: 1.15ꢀ1.22 (m, 6H); 1.88ꢀ2.05 (m, 5H); 3.19ꢀ3.35 (m, 2H);
3.84 (s, 3H); 4.24 (t, 1H); 4.37 (septet, 1H); 6.30 (brs, 1H); 6.96ꢀ7.00 (m, 2H); 7.53ꢀ7.58 (m, 2H);
.67ꢀ7.71 (m, 2H); 7.89ꢀ7.94 (m, 2H).
R)-2-(N-isopropoxy-4'-methoxybiphenyl-4-ylsulfonamido)-4-(2-phenylacetamido)butanoic
7
(
acid (15f). The title compound was prepared from tertꢀbutyl ester 14f following the general
1
procedure. White solid, 70% yield. HꢀNMR (CDCl
3
) δ: 1.15 (d, J= 1.8 Hz, 3H); 1.18 (d, J= 1.8Hz,
3
6
H); 1.93ꢀ2.10 (m, 2H); 3.10 (m, 2H); 3.56 (s, 2H); 3.86 (s, 3H); 4.15 (m, 1H); 4.37 (septet, 1H);
.98ꢀ7.02 (m, 2H); 7.30ꢀ7.39 (m, 5H); 7.54ꢀ7.58 (m, 2H); 7.63ꢀ7.67 (m, 2H); 7.80ꢀ7.84 (m, 2H).
(
R)-4-Acetamido-2-(N-isopropoxyphenylsulfonamido)butanoic acid (15g). The title compound
1
was prepared from tertꢀbutyl ester 14g following the general procedure. White solid, 94% yield. Hꢀ
NMR (CDCl
H); 3.15ꢀ3.50 (m, 2H); 4.24 (m, 1H); 4.37 (m, 1H); 6.05 (brs, 1H); 7.52ꢀ7.72 (m, 3H); 7.90ꢀ7.94
m, 2H).
General procedure for the preparation of (R)-hydroxamic acids 2a-g. 1ꢀ[3ꢀ
(Dimethylamino)propyl]ꢀ3ꢀethyl carbodiimide hydrochloride (EDC) was added portionwise (25 mg,
.13 mmol) to a stirred and cooled solution (0 °C) of the appropriate carboxylic acid 15a-g (0.087
mmol) and Oꢀ(tertꢀbutyldimethylsilyl)hydroxylamine (12.8 mg, 0.087 mmol) in dry CH Cl (2.2
3
) δ: 1.18 (d, J= 2.01 Hz, 3H); 1.21 (d, J= 2.01 Hz, 3H); 1.63ꢀ1.80 (m, 2H); 1.97 (s,
3
(
0
2
2
mL). After stirring at room temperature overnight, the mixture was washed with water and the
organic phase was dried and evaporated in vacuo.
2 2
Silyl precursors (0.05 mmol) were then dissolved in dry CH Cl (0.4 mL) and TFA (0.22 mL, 2.8
mmol) was added dropwise at 0 °C. After 5 h of stirring, TFA was evaporated. The crude products
were purified by trituration with nꢀhexane/Et O to give the desired hydroxamates 2a-g.
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(R)-N-(4-(Hydroxyamino)-3-(N-isopropoxybiphenyl-4-ylsulfonamido)-4-oxobutyl)benzamide
(2a). The title compound was prepared from carboxylic acid 15a following the general procedure.
1
White solid, 60% yield. Mp: 125ꢀ127 °C; HꢀNMR (CDCl
3
) δ: 1.24 (t, J= 6.5Hz, 6H); 1.44ꢀ1.69 (m,
1
7
H); 2.05ꢀ2.21 (m, 1H); 3.10ꢀ3.50 (m, 2H); 4.20ꢀ4.50 (m, 2H); 7.10 (brs, 1H); 7.30ꢀ7.55 (m, 10H);
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3
3
.59ꢀ7.63 (m, 2H); 7.86ꢀ7.90 (m, 2H). CꢀNMR (CDCl ) δ: 21.10; 27.21; 36.80; 60.90; 80.50;
127.40; 127.65; 128.89; 129.04; 129.14; 129.50; 129.87; 129.96; 132.61; 134.45; 138.80; 146.90;
67.50; 168.80.
R)-4-Methanesulfonamido-N-hydroxy-2-(N-isopropoxybiphenyl-4-
1
(
ylsulfonamido)butanamide (2b). The title compound was prepared from carboxylic acid 15b
1
following the general procedure. White solid, 60% yield. HꢀNMR (CDCl
3
) δ: 1.24 (t, J= 6.4Hz,
6
7
2
1
H); 2.01ꢀ2.18 (m, 2H); 2.88 (s, 3H); 3.00ꢀ3.20 (m, 2H); 4.40ꢀ4.48 (m, 2H); 4.88 (brs, 1H); 7.42ꢀ
13
.53 (m, 3H); 7.62ꢀ7.66 (m, 2H); 7.78ꢀ7.83 (m, 2H); 7.95ꢀ7.99 (m, 2H). CꢀNMR (CDCl ) δ:
3
1.18; 27.15; 40.03; 40.25; 60.02; 80.64; 127.46; 127.92; 128.86; 129.13; 129.91; 130.06; 132.40;
38.82; 147.40; 167.35.
(R)-4-Acetamido-N-hydroxy-2-(N-isopropoxybiphenyl-4-ylsulfonamido)butanamide (2c). The
title compound was prepared from carboxylic acid 15c following the general procedure. Yellow oil,
1
7
0% yield. HꢀNMR (CDCl ) δ: 1.23 (d, J= 6.7 Hz, 6H); 1.90ꢀ2.08 (m, 5H); 3.04ꢀ3.35 (m, 2H); 4.22
3
(m, 1H); 4.40 (septet, 1H); 6.73 (brs, 1H); 7.42ꢀ7.52 (m, 3H); 7.59ꢀ7.63 (m, 2H); 7.76ꢀ7.80 (m,
1
3
2H); 7.92ꢀ7.96 (m, 2H). CꢀNMR (CDCl
3
) δ: 21.06; 23.18; 27.02; 36.89; 60.05; 80.72; 128.13;
1
29.36; 129.70; 130.57; 133.29; 139.74; 147.82; 166.70; 171.80.
(R)-N-Hydroxy-2-(N-isopropoxybiphenyl-4-ylsulfonamido)-4-(2-phenylacetamido)butanamide
(2d). The title compound was prepared from carboxylic acid 15d following the general procedure.
1
White solid, 68% yield. Mp= 65ꢀ68 °C; HꢀNMR (CDCl
3
) δ: 1.18ꢀ1.32 (m, 6H); 1.83ꢀ2.22 (m, 2H);
3.10ꢀ3.28 (m, 2H); 3.48 (s, 2H); 4.11 (m, 1H); 4.39 (septet, 1H); 6.23 (brs, 1H); 7.16ꢀ7.36 (m, 5H);
13
7
3
.43ꢀ7.47 (m, 3H); 7.57ꢀ7.61 (m, 2H); 7.67ꢀ7.72 (m, 2H); 7.86ꢀ7.90 (m, 2H). CꢀNMR (CDCl ) δ:
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21.08; 27.05; 36.72; 43.39; 61.03; 80.04; 127.41; 127.67; 128.86; 129.04; 129.12; 129.48; 129.86;
1
29.98; 132.63; 134.46; 138.82; 147.24; 167.20; 172.38.
(R)-4-Acetamido-N-hydroxy-2-(N-isopropoxy-4'-methoxybiphenyl-4-
ylsulfonamido)butanamide (2e). The title compound was prepared from carboxylic acid 15e
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43
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60
1
following the general procedure. White solid, 80% yield. Mp= 83ꢀ85 °C; HꢀNMR (CDCl
3
) δ: 1.20ꢀ
1.26 (m, 6H); 1.94ꢀ2.04 (m, 5H); 3.02ꢀ3.40 (m, 2H); 3.86 (s, 3H); 4.22 (m, 1H); 4.45 (septet, 1H);
13
6
.09 (brs, 1H); 6.98ꢀ7.02 (m, 2H); 7.55ꢀ7.59 (m, 2H); 7.71ꢀ7.75 (m, 2H); 7.89ꢀ7.94 (m, 2H). Cꢀ
NMR (CDCl ) δ: 21.12; 27.20; 37.16; 55.41; 60.86; 80.42; 114.59; 127.03; 128.58; 129.84; 129.99;
31.08; 131.66; 146.80; 160.38; 167.72; 172.94.
3
1
(R)-N-Hydroxy-2-(N-isopropoxy-4'-methoxybiphenyl-4-ylsulfonamido)-4-(2-
phenylacetamido)butanamide (2f). The title compound was prepared from carboxylic acid 15f
1
following the general procedure. White solid, 77% yield. Mp= 78ꢀ80 °C; HꢀNMR (CDCl ) δ: 1.19ꢀ
3
1
4
.22 (m, 6H); 1.94ꢀ1.98 (m, 2H); 3.00ꢀ3.20 (m, 2H); 3.51 (s, 2H); 3.86 (s, 3H); 4.10ꢀ4.16 (m, 1H);
.41 (septet, 1H); 5.89 (brs, 1H); 6.97ꢀ7.01 (m, 2H); 7.21ꢀ7.32 (m, 5H); 7.53ꢀ7.57 (m, 2H); 7.65ꢀ
13
7.69 (m, 2H); 7.83ꢀ7.87 (m, 2H). CꢀNMR (CDCl
3
) δ: 21.14; 26.90; 36.94; 43.28; 55.43; 60.83;
0.45; 114.59; 127.04; 127.53; 128.59; 129.11; 129.55; 129.85; 129.99; 131.08; 131.78; 134.18;
46.85; 160.41; 167.62; 172.84.
8
1
(
R)-4-Acetamido-N-hydroxy-2-(N-isopropoxyphenylsulfonamido)butanamide (2g). The title
compound was prepared from carboxylic acid 15g following the general procedure. White
1
semisolid, 65% yield. HꢀNMR (CDCl
3
) δ: 1.24 (d, J= 6.4 Hz, 6H); 1.70ꢀ1.85 (m, 2H); 1.95 (s, 3H);
3
.01ꢀ3.18 (m, 2H); 4.10ꢀ4.20 (m, 1H); 4.43 (m, 1H); 6.00 (brs, 1H); 7.55ꢀ7.74 (m, 3H); 7.88ꢀ7.92
13
(
m, 2H). CꢀNMR (CDCl ) δ: 21.09; 23.12; 26.90; 37.16; 60.02; 80.60; 127.03; 129.84; 131.80;
3
1
47.20; 164.70; 170.91.
(R)-tert-Butyl
ylsulfonamido)butanoate (17). Diisopropyl azodicarboxylate (DIAD) (0.4 mL, 2.0 mmol) was
4-(1,3-dioxoisoindolin-2-yl)-2-(N-isopropoxy-4'-methoxybiphenyl-4-
added dropwise to
a
solution containing (S)ꢀtertꢀButyl 4ꢀ(1,3ꢀdioxoisoindolinꢀ2ꢀyl)ꢀ2ꢀ
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hydroxybutanoate 16 (250 mg, 0.82 mmol), sulfonamide 5 (400 mg, 1.24 mmol) and
triphenylphosphine (645 mg, 2.46 mmol) in anhydrous THF (12 mL) under nitrogen atmosphere at
0
ºC. The resulting solution was stirred for 5h at rt and evaporated under reduced pressure to afford
a crude product, which was purified by flash chromatography on silica gel (nꢀhexane/AcOEt = 2:1),
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9
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4
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0
1
to give the desired product as a pale yellow oil (217 mg, 43% yield). HꢀNMR (CDCl
3
) δ: 1.24ꢀ1.29
(m, 15H); 2.10ꢀ2.30 (m, 2H); 3.51ꢀ3.74 (m, 2H); 3.87 (s, 3H); 4.06ꢀ4.23 (m, 1H); 4.46 (septet, 1H);
.98ꢀ7.03 (m, 2H); 7.52ꢀ7.88 (m, 10H).
R)-4-(1,3-Dioxoisoindolin-2-yl)-2-(N-isopropoxy-4'-methoxybiphenyl-4-
ylsulfonamido)butanoic acid (18). Trifluoroacetic acid (1.5 mL, 18.8 mmol) was added dropwise
to a stirred solution of tertꢀbutyl ester 17 (205 mg, 0.33 mmol) in freshly distilled CH Cl (2.0 mL),
cooled to 0°C. The solution was stirred for 5 h at 0 °C and the solvent was removed in vacuo to give
6
(
2
2
2
a crude that was purified by trituration with Et O/nꢀhexane. White solid, 135 mg, 74% yield. Mp=
1
1
85ꢀ187 °C; HꢀNMR (CDCl
3
) δ: 1.21 (d, J= 6.2Hz, 3H); 1.27 (d, J= 6.2Hz, 3H); 2.10ꢀ2.28 (m,
2H); 3.61 (m, 2H); 3.87 (s, 3H); 4.33 (m, 1H); 4.46 (septet, 1H); 6.98ꢀ7.02 (m, 2H); 7.50ꢀ7.54 (m,
H); 7.65ꢀ7.85 (m, 6H).
R)-4-(1,3-Dioxoisoindolin-2-yl)-N-hydroxy-2-(N-isopropoxy-4'-methoxybiphenyl-4-
ylsulfonamido)butanamide (3). 1ꢀ[3ꢀ(Dimethylamino)propyl]ꢀ3ꢀethyl carbodiimide hydrochloride
EDC) was added portionwise (65 mg, 0.34 mmol) to a stirred and cooled solution (0 °C) of
carboxylic acid 18 (130 mg, 0.23 mmol) and Oꢀ(tertꢀbutyldimethylsilyl)hydroxylamine (51 mg,
.34 mmol) in dry CH Cl (7.0 mL). After stirring at room temperature overnight, the mixture was
washed with water and the organic phase was dried and evaporated in vacuo.
Silyl precursor (100 mg, 0.14 mmol) was then dissolved in dry CH Cl (1.0 mL) and TFA (0.6 mL,
7.9 mmol) was added dropwise at 0 °C. After 5 h of stirring, TFA was evaporated. The crude
4
(
(
0
2
2
2
2
product was purified by trituration with nꢀhexane/Et
2
O to give the desired hydroxamate 3 as a white
) δ: 1.23 (d, J= 6.2Hz, 3H); 1.30 (d, J=
1
solid (61 mg, 77% yield). Mp= 75ꢀ76 °C; HꢀNMR (CDCl
3
6
.2Hz, 3H); 2.12ꢀ2.32 (m, 2H); 3.40ꢀ3.53 (m, 2H); 3.88 (s, 3H); 4.02ꢀ4.20 (m, 1H); 4.46 (septet, J=
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6.2Hz, 1H); 6.98ꢀ7.02 (m, 2H); 7.29ꢀ7.39 (m, 2H); 7.42ꢀ7.46 (m, 2H); 7.63 (m, 4H); 7.72ꢀ7.76 (m,
1
3
2
1
H). CꢀNMR (CDCl ) δ: 21.15; 26.80; 34.75; 55.44; 60.72; 80.08; 114.50; 123.29; 127.03;
3
28.63; 129.88; 131.32; 131.81; 132.01; 133.94; 134.23; 146.81; 160.30; 166.85; 168.23.
MMP inhibition assays. Recombinant human progelatinase A (proꢀMMPꢀ2), B (proꢀMMPꢀ9), and
MMPꢀ14 catalytic domain were a kind gift of Prof. Gillian Murphy (Department of Oncology,
University of Cambridge, UK). ProꢀMMPꢀ1, proꢀMMPꢀ13 and proꢀMMPꢀ3 were purchased from
Calbiochem. Proenzymes were activated immediately prior to use with pꢀaminophenylmercuric
acetate (APMA 2 mM for 1 h at 37 °C for MMPꢀ2, 1 mM for 1 h at 37 °C for MMPꢀ9, 2 mM for 2
h at 37 °C for MMPꢀ1, 1 mM for 35 min at 37 °C for MMPꢀ13). ProꢀMMPꢀ3 was activated with
trypsin (4.4 ꢁg/mL) for 30 min at 37 °C followed by addition of soybean trypsin inhibitor (61.5
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ꢁg/mL). For assay measurements, the inhibitor stock solutions (DMSO, 10 mM) were further
diluted in the fluorimetric assay buffer (FAB: Tris 50 mM, pH = 7.5, NaCl 150 mM, CaCl 10 mM,
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Brij 35 0.05% and DMSO 1%). Activated enzyme (final concentration 0.5 nM for MMPꢀ2, 1.3 nM
for MMPꢀ9, 1.0 nM for MMPꢀ14cd, 2.0 nM for MMPꢀ1, 0.66 nM for MMPꢀ13 and 5 nM for MMPꢀ
3) and inhibitor solutions were incubated in the assay buffer for 4 h at 25 °C. After the addition of
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00 ꢁM solution of the fluorogenic substrate McaꢀLysꢀProꢀLeuꢀGlyꢀLeuꢀDap(Dnp)ꢀAlaꢀArgꢀNH2
(Bachem) in DMSO (final concentration 2 ꢁM), the hydrolysis was monitored every 10 s for 20 min
recording the increase in fluorescence (λex = 325 nm, λem = 395 nm) using a Molecular Devices
SpectraMax Gemini XS plate reader. The assays were performed in a total volume of 200 ꢁL per
well in 96ꢀwell microtitre plates (Corning, black, NBS). Control wells lack inhibitor. The MMP
inhibition activity was expressed in relative fluorescent units (RFU). Percent of inhibition was
calculated from control reactions without the inhibitor. IC was determined using the formula: v /v
o
5
0
i
=
1/(1 + [I]/ IC50), where v
i
is the initial velocity of substrate cleavage in the presence of the
is the initial velocity in the absence of the inhibitor. Results
inhibitor at concentration [I] and v
o
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were analyzed using SoftMax Pro software and GraFit software.
Cell cultures
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Human umbilical vein endothelial cells (HUVECs) were purchased from Promo Cell (Heidelberg,
Germany) and grown on 1% gelatinꢀcoated tissue culture plates in Medium 199 (Sigma Aldrich
Milano), supplemented with 10% heatꢀinactivated fetal bovine serum (FBS) (Euroclone, Milano),
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factor plus 1ꢁg/ 100mL basicꢀfibroblast growth factor, PeproTech London UK), epidermal growth
factor (1ꢁg/100mL PeproTech London UK), heparin (10mg/100mL, Sigma Aldrich Milano) and
hydrocortisone (0.1mg/100mL Sigma Aldrich Milano) at 37 °C in 5 % CO . Cells were used
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between the third and sixth passage in vitro.
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x10 cells were seeded in a sixꢀwell plate and after one day of attachment, cells were starved 6 h in
serumꢀfree medium with BSA 2%. Cells were then preꢀtreated for 3 h with 30 nM PMA, followed
by exposition to compound 3 or 2g. DMSO was used as vehicle in untreated cells. After 24 hours,
conditioned media were collected for zymography and cells were processed both for RNA and
proteins extraction.
Chemotaxis and chemoinvasion assays. Migration and invasion assays were performed using
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modified Boyden chambers . Human endothelial cells (HUVEC) (5x10 ) were washed with PBS,
resuspended in serumꢀfree medium and placed in the upper compartment of the chamber, in
presence/absence of different concentrations of inhibitors. Chemoattractant (10% FBS) or serum
free medium were added in the lower compartment. Poreꢀsize polycarbonate filters (8 ꢁM) were
preꢀcoated with collagen (50 µg/mL) or matrigel (1 mg/mL) and used as the interface between the
two chambers compartments. Following 6 h (migration) or overnight (invasion) incubation, the
filters were recovered, cells on the upper surface mechanically removed using a cotton swab; cells
migrated or invaded to the lower filter surface fixed with absolute ethanol and stained with
Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA). Cells were counted in
a doubleꢀblind manner in eight consecutive fields each with a light microscope. All experiments
were performed three times in duplicate. Statistical significance was determined by One way
ANOVA using GraphPad Prism.
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Matrigel morphogenesis assay. The in vitro morphogenesis assay was performed in presence of
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0% FBS and different concentration of inhibitors. Briefly, a 24ꢀmicrowell plate, preꢀchilled at ꢀ20
°C, was carefully filled with 300 ꢁL/well of liquid matrigel (10 mg/mL) at 4 °C with a preꢀchilled
pipette, avoiding bubbles. The matrigel was then polymerized for 1 h at 37 °C, and HUVEC
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50x10 cells/well) were suspended in 1 mL of complete medium in the absence or presence of
different concentrations of inhibitors and carefully layered on the top of the polymerized matrigel.
After 6 h incubation on matrigel, the dimensional organization of the cells was examined under an
inverted microscope (Zeiss, Oberkochen, Germany), equipped with CCD optics and a digital
analysis system. The number of meshes where measured, as a direct parameter for HUVEC
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tubulogenic efficiency, using the Angiogenesis tool and ImageJ software. Statistical significance
was determined by Oneway ANOVA using GraphPad Prism.
Quantitative reverse trascription-PCR. Total RNA was extracted from cells using the RNeasy
Protect Mini Kit (Qiagen, Hilden, Germany) according to the recommendation of the manufacturer
and reverse transcribed as above with oligo dT primers in 20µL final volume. All primers for the
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genes tested were designed using primer3 software with a Tm optimum of approximately 60 °C
and a product length of 100ꢀ150nt. Real time PCR was performed on a LightCycler480 (Roche,
Penzberg, Germany) using Light Cycler 480 SYBR Green I Master (Roche), 3 µL of cDNA (10x
diluted), 3 pmol sense and antiꢀsense primers in a final reaction volume of 10 µL. After an initial
denaturation step of 5 min during which the well factor was measured, 45 cycles of 10 s at 95 °C
followed by 15 s at 60 °C and 15 s at 72 °C were performed. Fluorescence was measured during the
annealing step in each cycle. After amplification melting curves with 80 steps of 15 s and 0.5 °C
increase were performed to monitor amplicon identity. Expression data were normalized on the
mean of the expression values for three housekeeping genes: cyclophilin A, 16 s rRNA (ribosomal
RNA) and GUSB (βꢀglucuronidase). Relative expression values with standard errors and statistical
comparisons (unpaired twoꢀtailed tꢀtest) were obtained using Qgene software.
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Western Blot. HUVE cells were grown as indicated and collected by brief trypsinization. Total
lysates were prepared using cell lysis buffer (Cell Signaling Technology, Beverly, MA). Protein
concentrations were evaluated by the DC Protein Assay (BioꢀRad, Hercules, CA). Equal amounts of
proteins for each sample were resolved on 10% sodium dodecyl sulfate–polyacrylamide gel
electrophoresis and blotted onto polyvinylidene fluoride membranes (Amersham Biosciences,
Otelfingen, CH). Following blocking with 5% nonꢀfat milk powder (wt/vol) in Trisꢀbuffered saline
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(10 mM Tris–HCl, pH 7.5, 100 mM NaCl, 0.1% Tweenꢀ20) for 1 h at room temperature,
membranes were incubated with primary antibodies directed against the following human antigens:
MMPꢀ2, MMPꢀ9 (all purchased from Cell Signaling Technology, Danvers, MA) and βꢀactin (Sigma
Aldrich, Milan). The antibodies were diluted in 2% bovine serum albumin–Trisꢀbuffered saline–
0
.1% Tween according to the manufacturer’s instructions. The bound antibodies were visualized by
horseradishꢀperoxidaseꢀ conjugated secondary antibodies and an enhanced chemiluminescence
detection system from Amersham Biosciences (Pittsburg, PA).
Zymographic Analysis. The effects of compounds on gelatinases activity was determined by gel
zymography either on MMPs derived from notꢀtreated HUVEC media, further exposed to MMP
inhibitors or by using CM directly derived from MMPꢀinhibitors treated HUVE cells. Without
heating the samples, 20 ꢁL of untreated HUVEC conditioned medium or an equal amounts of
protein derived from treated HUVECs were loaded 10% polyacrylamide additioned of 0.1% gelatin,
in presence of SDS. Following electrophoresis, the gels were incubated for 30 min at room
temperature with gentle agitation in Renaturing Buffer (Invitrogen, Eugene, OR, USA). For
gelatinase inhibition assays of notꢀtreated HUVEC, the different inhibitors were added in the
Developing Buffer (Invitrogen, Eugene, OR, USA) over night at 37 °C; the gel slab was cut into
slices corresponding to the lanes and then put in different tanks containing the stated concentrations
of inhibitors. For MMPI treated HUVEC, gels were directly incubated over night at 37 °C in
Developing Buffer. Gels were then stained for 15 min with Coomassie Brillant Blue Rꢀ250 stain
and then deꢀstained with 10% acetic acid and 30% methanol until sharp bands were visualized.
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Band intensities were quantified by densitometric analysis using ImageJ software (National
Institutes of Health).
MTT assay. Human umbilical vein endothelial cell viability was evaluated using the MTT assay.
Cells (1000/well) were seeded into 96ꢀmultiwell plates in complete medium and, after complete
adhesion, the medium was replaced with fresh medium with or without different concentrations of
compound 3 (10 nMꢀ10 µM). After different periods of incubation (24, 48, 72 and 96ꢂ h), plates
were processed and absorbance read at 570ꢂ nm.
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Assessment of apoptosis by Flow Cytometry. Human umbilical vein endothelial cells (1x10 )
were plated onto sixꢀwell plates and allowed to adhere overnight. The next day, cells were treated in
the growth medium with increasing concentration of the inhibitor (10 nMꢀ 10 µM). Growth medium
alone (NT) and hydrogen peroxide (100ꢀ 200 µM) were used as controls. After overnight, cells were
recovered, washed with PBS and transferred to test tubes. Cells were pelleted and resuspended in
Annexin Vꢀbinding buffer (0.01 M HEPES (4ꢀ(2ꢀhydroxyethyl)ꢀ1ꢀpiperazineethanesulfonic acid)
2
pH 7.4; 0.14 M NaCl; 2.5 mM CaCl ). Fluorescein isothiocyanate Annexin V and 7ꢀaminoꢀ
actinomycin D (BD Biosciences) were added to each test tube and incubated for 15 min at room
temperature in the dark. Cells were then washed in PBS, supernatants discarded and resuspended in
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00 mL of binding buffer. Samples were acquired by flow fluorocytometry using a FACSCanto
(BD Biosciences) and analyzed using FACSDiva Software 6.1.2.
29
Matrigel sponge assay (in vivo angiogenesis). The assay was performed as already described .
VTH mixture (100 ng/mL VEGFꢀA, 2 ng/mL TNFꢀα, and 25 UI/mL heparin), either alone or in
combination with compound 3 at 0.1ꢀ1ꢀ10 µM, was added to unpolymerized liquid matrigel at 4 °C
and the mixture was brought to a final volume of 0.6 mL. The matrigel suspension was then slowly
injected subcutaneously into the flanks of C57/BL6 male mice (Charles River, Calco [Lecco], Italy)
with a cold syringe. At body temperature in vivo, the matrigel quickly polymerizes to form a solid
gel. In some experiments the mice were also treated with 0.1ꢀ1ꢀ10 µM of compound 3 by
intraperitoneal (i.p.) injection, at day 0. Groups of 8 mice were used for each treatment. Four days
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after injection, the gels were recovered, weighted, minced and diluted in PBS to measure the
hemoglobin content with a Drabkin reagent kit (Sigma, St Louis, MO, USA). Part of the pellets
recovered were assessed for endothelial cell infiltrate quantification by multicolor flow cytometry.
Briefly, pellets were digested for 1 h in 1 mg/mL Collagenase IV. The cell suspension obtained was
stained with antiꢀmurine mab as follows: APCꢀCD45, FITCꢀCD31, PerCPꢀCD3, finally analyzed by
flow cytometry. Endothelial cells in the total cell suspension, gated on FSC/SSCꢀselected viable
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cells, were quantified as the % of CD31 cells, on CD45 CD3 gated cells.
Statistical analysis. Results are showed as mean ± SEM. The significance of differences was
evaluated with a twoꢀtailed tꢀtest or One way ANOVA test using Prism software (GraphPad
Software for Science, Inc., San Diego, CA, USA).
Docking Calculations. The crystal structure of MMPꢀ2 (PDB code 1QIB), MMPꢀ9 (PDB code
34
4
WZV), and MMPꢀ14 (PDB code 3MA2) was taken from the Protein Data Bank. After adding
3
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hydrogen atoms the three proteins were minimized using Amber 11 software and parm03 force
field at 300 K. The three proteins were placed in a rectangular parallelepiped water box, an explicit
solvent model for water, TIP3P, was used and the complexes were solvated with a 10 Å water cap.
Sodium ions were added as counter ions to neutralize the system. Two steps of minimization were
then carried out; in the first stage, we kept the protein fixed with a position restraint of 500
2
kcal/mol•Å and we solely minimized the positions of the water molecules. In the second stage, we
minimized the entire system through 5000 steps of steepest descent followed by conjugate gradient
(CG) until a convergence of 0.05 kcal/Å•mol. Automated docking was carried out by means of the
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AUTODOCK 4.0 program; Autodock Tools was used in order to identify the torsion angles in
the ligand, add the solvent model and assign the Kollman atomic charges to the protein. The ligand
charge was calculated using the Gasteiger method.
A grid spacing of 0.375 Å and a distanceꢀdependent function of the dielectric constant were used
for the energetic map calculations. Using the Lamarckian Genetic Algorithm, the docked compound
was subjected to 100 runs of the Autodock search, using 500000 steps of energy evaluation and the
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default values of the other parameters. Cluster analysis was performed on the results using an RMS
tolerance of 2.0 Å and the best docked conformation was taken into account.
MD Simulations.
All simulations were performed using AMBER, version 11. MD simulations were carried out using
the parm03 force field at 300 K. The complex was placed in a rectangular parallelepiped water box.
An explicit solvent model for water, TIP3P, was used, and the complex was solvated with a 10 Å
water cap. Sodium ions were added as counterions to neutralize the system. Prior to MD
simulations, two steps of minimization were carried out using the same procedure described above.
Particle mesh Ewald (PME) electrostatics and periodic boundary conditions were used in the
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simulation. The MD trajectory was run using the minimized structure as the starting conformation.
The time step of the simulations was 2.0 fs with a cutoff of 10 Å for the nonbonded interaction, and
SHAKE was employed to keep all bonds involving hydrogen atoms rigid. Constantꢀvolume
periodic boundary MD was carried out for 500 ps, during which the temperature was raised from 0
to 300 K. Then 4.5 ns of constant pressure periodic boundary MD was carried out at 300 K using
the Langevin thermostat to maintain constant the temperature of our system. All the α carbons of
2
the protein were blocked with a harmonic force constant of 10 kcal/mol•Å . General Amber force
field (GAFF) parameters were assigned to the ligand, while partial charges were calculated using
the AM1ꢀBCC method as implemented in the Antechamber suite of AMBER 11. The final structure
of the complex was obtained as the average of the last 3.0 ns of MD minimized by the CG method
2
until a convergence of 0.05 kcal/mol•Å . The average structure was obtained using the ptraj
program implemented in AMBER 11.
Crystallization and Structure determination. The expression and purification of MMPꢀ9 was
3
9
carried out as previously described. In brief, MMPꢀ9 comprises residues Met109–Gly215 and
Gln391–Gly444, without the additional fibronectin domains. Plasmids were propagated in the
Escherichia coli strain XL1ꢀBlue and the recombinant catalytic domains expressed in E. coli cells
BL21 (DE3 star). The protein was refolded from inclusion bodies, and purified as previously
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