Recently Synthesized Class of Vinylphosphonates as Potent Matrix Metalloproteinase MMP-2 Inhibitors.
код для вставкиСкачать76 Srebnik et al. Abed Al-Aziz Al-Quntara, Ofir Bauma, Reuven Reichb,* Morris Srebnika* Arch. Pharm. Pharm. Med. Chem. 2004, 337, 76−80 Recently Synthesized Class of Vinylphosphonates as Potent Matrix Metalloproteinase (MMP-2) Inhibitors a Department of Medicinal Chemistry and Natural Products, Hebrew University in Jerusalem, Jerusalem, Israel b Department of Pharmacology, School of Pharmacy, Hebrew University in Jerusalem, Jerusalem, Israel An in vitro evaluation of MMP-2 inhibitors for a series of novel vinylphosphonic acids and phosphonic esters that contain various functional groups, shows that various types exhibit excellent efficiency, and points towards potent, promising compounds. Other types displayed relatively weak activity. Keywords: Vinylphosphonates; Phosphonates; Matrix Metallo-Proteinase; Structure⫺activity relationships; Cancer Received: July 18, 2003; Accepted: September 9, 2003 [FP828] DOI 10.1002/ardp.200300828 Introduction Matrix metalloproteinases (MMPs) are a family of structurally related enzymes. In addition to the presence of zinc at their active site, they contain calcium for enzyme stability. MMPs, mainly MMP-2 which is a type IV collagenase (gelatinase) play an important role in the degradation of extracellular matrix proteins that constitute cellular connective tissue and are strongly involved in both normal and pathological tissue remodeling [1⫺5]. The degradative activity of MMPs is tightly controlled both by the latency of the secreted enzymes as well as by the presence of naturally occurring inhibitors including general plasma proteinase inhibitors and tissue inhibitors of metalloproteinases. Imbalance between the levels of activated enzymes and their inhibitors causes a breakdown of the extracellular matrix [6, 7]. It has been shown that MMPs play a role in primary tumor growth [8]. In addition, they mediate invasion and metastasis, which are the processes that lethally spread cancer cells through the body [9, 10]. First, cancer cells detach from the primary site (which is often in an epithelial tissue) and breach the basement membrane separating them from other tissue layers [6,11]. Some of these invasive cells can penetrate the basement membrane surrounding a blood vessel, as well as the layer of endothelial cells lining it. The cells are then free to circulate via the bloodstream. Eventually, a cancer cell may lodge in a capillary. If it then adheres to, and penetrates the capillary wall again, it can create a secondary tumor which may survive to colonize another tissue. The mechanism for the degradation of the proteins is based on the scissile amide carbonyl coordination to Correspondence: Morris Srebnik, Department of Medicinal Chemistry and Natural Products, Hebrew University in Jerusalem, POB 12065, Jerusalem 91120, Israel. Phone: +972 2 675-7301; Fax: +972 2 675-8201; e-mail: msrebni@md2.huji.ac.il the active site zinc(II) ion. Simultaneously, it is attacked by a water molecule that is both hydrogen bonded to glutamic acid and coordinated to the zinc(II) ion. Then the water donates a proton to the scissile amide. Finally, the peptide bond is cleaved after the remaining proton from the water molecule is shuttled to the nitrogen of the scissile amide [12]. Enhanced gelatinase activity is associated with a higher tumor grade in breast [13], bladder [14], and gastric cancers [15]. It also correlates with many types of inflammations [16], including arthritis [17], restenosis [18], multiple sclerosis [19], and others [20, 21]. In order to maintain the balance of MMPs in these pathological processes, native inhibitors, such as TIMP-1 [22] and TIMP-2 [23], have been considered for therapeutic aims. Synthetic inhibitors have been developed, such as succinyl hydroxamates; these compounds are accompanied by various problems, such as low water solubility and toxicity [24], carboxylic acids [15], thiols [25], and more favorable phosphorous containing compounds [26]. The requirement for a molecule to be an effective inhibitor of MMPs is a functional group capable of chelating the active-site zinc(II) ion. In addition, functional groups which provide a hydrogen bond interaction with the enzyme, and side chains which undergo effective van der Waals-interactions, are required [12]. In this investigation, new classes of vinylphosphonic acids that have been recently prepared in our lab have been tested for the first time as MMP-2 inhibitors. Chemistry cis-Vinylphosphonate 1 was prepared by addition of 1hexynylphosphonate to the Negishi reagent, followed * M. S.a and R. R.b are affiliated with the Bloom Center for Pharmacy at the Hebrew University. 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Arch. Pharm. Pharm. Med. Chem. 2004, 337, 76−80 Vinylphosphonates as MMP-2 inhibitors 77 Scheme 3. Scheme 1. The 3-aminovinylphosphonates, compounds 24⫺29, were synthesized by addition of imines to the alkynylphosphonate titanium(II) complexes (Scheme 3) [32]. by hydrolysis of the ester with Me3SiBr in CH2Cl2 [27]. Compounds 2⫺7 2-(hydroxymethyl)vinylphosphonates were synthesized by the insertion of cyclic ketones into three membered ring zirconacycles (Scheme 1). The free phosphonic acid compounds 5⫺7 were obtained by the hydrolysis of the phosphonate esters by Me3SiBr in CH2Cl2. The other types of 2-(hydroxymethyl)vinylphosphonic acids 8⫺14 were obtained by insertion of aldehydes or ketones into the zirconacycle (Scheme 1) and, similarly, were hydrolyzed [28, 29]. The 3-oxovinylphosphonic acids 15⫺18 were prepared by insertion of acid chlorides into zirconacycles (Scheme 1), followed by similar hydrolysis with Me3SiBr [30]. The preparation of di- and tri-substituted vinylphosphonic acids 19⫺23 was accomplished by the tuning of the addition of acid chlorides and Grignards to the three membered ring titanacycle (Scheme 2) followed by the usual hydrolysis to yield the free acid [31]. Biology Cell culture Human HT-1080 fibro sarcoma cells from ATCC were maintained in Minimal Essential Medium, supplemented with calf serum, 10%. Glutamine, pyruvate, nonessential amino acids, vitamins, and antibiotics (Biological Industries, Kibbutz Beth HaEmek, Israel) are added as additional supplements. Basement membrane invasion Boyden chamber chemo invasion assays were performed [10]. This assay is completely dependent on the expression of MMP by the cells. Matrigel (25 µg) was dried on a polycarbonated filter (PVP free, Nucleopore, Whatman). Fibroblast conditioned medium (obtained from confluent NIH-3T3 cells cultured in serum free DMEM) was used as the chemo attractant. Cells were harvested by brief exposure to 1 mM EDTA, washed with DMEM containing 0.1% bovine serum albumin, and added to the Boyden chamber (200,000 cells). The chamber was incubated in a humidified incubator at 37 sC in 5% CO2/95% air atmosphere for 6 h. The cells which traversed the Matrigel layer and attached to the lower surface of the filter were stained with Diff Quick (American Scientific Products, AHS del Caribe, Inc., Aguada, Puerto Rico) and counted. Chemotaxis Scheme 2. Chemotaxis evaluations were performed in a similar way to basement membrane invasion, with the exception that the filters were coated with 5 µg collagen IV 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 78 Srebnik et al. Arch. Pharm. Pharm. Med. Chem. 2004, 337, 76−80 led us to a classification of these compounds into different categories. First, cis-vinylphosphonic acid 1 showed relatively weak activity as a MMP-2 inhibitor (Figure 1). Second, unlike compounds 2, 3, 4 which did not posses any satisfactory activity as the esters, compounds 5⫺7 showed very promising activity. Specifically, compound 5 was the most active in this series (Figure 2). Figure 1. MMP-2 inhibitors of compounds 1⫺4. Error bars represent the standard deviation; the experiment was repeated three times. 5 showed enhanced activity even at low micromolar range. This is supported by results from the literature which showed that the introduction of a cyclohexyl substituent in the carboxylic acid compounds improved the inhibition significantly [33]. The activity of compounds 8⫺14 varied from moderate to interesting. It can also be observed that the presence of phenyl groups in compounds 8, 13, 14 does not improve inhibition (Figure 3) [34]. Figure 2. MMP-2 inhibition of compounds 5⫺7. Error bars represent the standard deviation; the experiment was repeated three times. instead of Matrigel. This amount of collagen does not form a barrier to the migrating cells, but rather an attachment substratum. Results and discussion Various new types of vinylphosphonates and vinylphosphonic acids, compounds 1⫺29, were tested in vitro for inhibition of MMP-2 protease. A study of the results Figure 3. MMP-2 inhibitors of compounds 8⫺14. Error bars represent the standard deviation; the experiment was repeated three times. On the other hand, increasing the chain length slightly increased the inhibition, as seen for compound 12 [35]. The introduction of a carbonyl group together with the phosphonic acid dramatically enhanced the inhi- 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Arch. Pharm. Pharm. Med. Chem. 2004, 337, 76−80 Vinylphosphonates as MMP-2 inhibitors 79 olar range, particularly compounds 27, 28, and 29 (Figure 6). Figure 4. MMP-2 inhibitors of compounds 15⫺18. Error bars represent the standard deviation; the experiment was repeated three times. Figure 6. MMP-2 inhibition of compounds 24⫺29. Error bars represent the standard deviation; the experiment was repeated three times. Conclusions Figure 5. MMP-2 inhibition of compounds 19⫺23, (error bars represent the std. deviation, and the experiment was repeated three times). bition of MMP-2 protease, 15⫺18, particularly compounds 15, 16, (Figure 4). This is consistent with the mechanism stating that the active site of zinc(II) first coordinates to the carbonyl group of the scissile amide [12]. The di- and tri-substituted phosphonic acid compounds, 19⫺23, are very potent as MMP-2 protease inhibitors (Figure 5). As phosphonic esters, the 3-aminovinylphosphonates, 24⫺29, are among the most potent and effective MMP-2 protease inhibitors, even at a very low microm- Various new classes of substituted vinylphosphonates were recently synthesized in our lab and have been investigated as MMP-2 inhibitors. Cyclic hydroxy-vinylphosphonates as the free phosphonic acids have shown very potent activity compared to the (cyclic hydroxyl)vinylphosphonate esters which exhibited relatively weak activity. (Hydroxymethyl)vinylphosphonic acids showed moderate inhibiton. Vinylphosphonic acid containing carbonyl groups and various other diand tri-substituted vinylphosphonic acids were very potent compounds. 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