Osteoarthritis (OA) is a progressive degenerative joint disease which affects a large part of the elderly population seriously impacting the quality of life. OA is characterised by pathological changes that occur in the articular cartilage, synovium and subchondral bone leading to pain and loss of articular function. Rheumatoid arthritis (RA) is a systemic inflammatory disease characterised by articular synovitis leading to cartilage degradation, bone erosion and pain. Although arthritis (OA and RA) are defined as a diseases affecting the joints, the primary feature is chronic pain. Accordingly, the most of the currently used pharmacological treatment for arthritis is largely confined to analgesics, steroidal and especially non-steroidal anti-inflammatory drugs (NSAIDs). These agents impact the symptoms of the disease (mainly pain) rather than the underlying process, in addition, though these drugs have provided an important mean of controlling inflammation and pain in arthritis, their application has been overshadowed by the gastrointestinal tract side-effects, when considering classical NSAIDs drugs (By Sweet et al., Am. J. Health Syst. Pharm., 61, 18, 1917-21, 2004) and other side-effects when considering COX-2 inhibitors (Mamdani M. et al., Lancet, 363, 1751-6, 2004). Therefore, the development of new therapeutic agents able to prevent or counteract cartilage degradation in arthritis along with the related pain is essential, since OA and RA affect million people all over the world and its incidence is expected to increase with the increase of the population average age.
The degradation of cartilage that occurs in these diseases is the result of enzymatic cleavage of its structural components. Cartilage is constituted of chondrocytes and an extra-cellular matrix that consists of proteoglycans (mainly aggrecan), collagen and water. The interaction between proteoglycans and collagen provides unique structural and physiological properties for cartilage to function in weight bearing and joint motion. Cartilage proteoglycans consist of a protein core with glycosaminoglycan (GAG) side chains; GAG components absorb water and provide to the cartilage its characteristic resistance to mechanical stress and constitute a protective layer essential to the joint function. Healthy cartilage maintains a dynamic equilibrium between processes that produce and processes that degrade the matrix components; in pathological conditions this equilibrium is altered leading to the prevalence of the degenerative process, which causes matrix degradation, and hence cartilage roughening and fissuring which at the end could result in erosion of the subchondral bone and synovial inflammation.
The large aggregating proteoglycan, called aggrecan, forms aggregates that bind hyaluronic acid (HA) and together with type II collagen is responsible for the biomechanical properties of cartilage. Thus aggrecan interacting in a complex network with HA and type II collagen, enables the tissue to bear load resisting to mechanical compression and endows the cartilage with those biomechanical characteristics necessary to joint functionality. Aggrecan protein consists of three globular regions termed G1, G2 and G3 (P. J. Roughley, European Cells & Material, 2006, 12, 92-101). The G1 and G2 regions are separated by a short interglobular domain (IGD) while the G2 and G3 regions are separated by a long GAG attachment region. The G1 domain is at the amino-terminus of the protein and through an ancillary protein it constitutes the binding region of aggrecan to HA. Aggrecan molecules are not isolated within the extra-cellular matrix but form aggregates composed of a central HA filament with up to 100 aggrecan molecules radiating from it. The GAG-attachment region of aggrecan provides the high anionic charge density needed for binding water and conferring to the cartilage the unique osmotic properties necessary to guarantee its functionality.
Loss of cartilage integrity in arthritis is associated with impaired aggrecan integrity due to proteolytic cleavage of the protein. Two sites located in the IGD of aggrecan have been identified as the major targets of proteolytic aggrecan attack the Asn341-Phe342 bond has been shown to be mainly cleaved by several Matrix Metalloproteases (MMPs) and the Glu373-Ala374 bond which is the site of cleavage in pathological conditions as resulted by analysis of synovial fluids of patients (L. S. Lohmander et al., Arthritis & Rheum., 1993, 36, 1214-1222; J. D. Sandy, J. Clin. Invest. 1992, 89, 1412. B) or in widely reported laboratory studies, where this site resulted the major aggrecan cleavage site in chondrocytes cultures stimulated by cytokines (the cytokines: IL-1, TNF-α, IL-6, IL-8 stimulate the chondrocytes to produce an increased amount of matrix degrading enzymes, while IL-4 inhibits this process). Two enzymes, Aggrecanase 1 and 2, which cleave aggrecan at Glu373-Ala374 bond but not at the MMP site, have been identified and cloned. Both these enzymes belong to the ADAMTS (a disintegrin-like and metalloproteinase domain with thrombospondin type 1 motifs) family of proteases and are named ADAMTS-4 and ADAMTS-5 respectively. ADAMTS family represents a group of zinc metalloproteases belonging to reprolysin subfamily (related to the snake venom toxin reprolysin; C. G. Jones et al., Arthritis Res. Ther, 2005, 7, 160-69; C. G. Jones et al., Biochem J., 2005, 386, 15-27).
While enzymes in ADAMTS family are usually involved in protein turnover and tissue remodelling, ADAMTS-4 and ADAMTS-5 are considered to be largely responsible for the cartilage aggrecan catabolism observed during the development of OA and RA, therefore inhibition of these enzymes may represent a therapeutic strategy for these diseases (S. S. Glasson, Current Drug Targets, 2007, 8, 2, 367-376). Though synthetic MMPs inhibitors have been clinically investigated as a means to block tissue destruction in arthritis and have been proved unsuccessful, it should be pointed out that these inhibitors were directed against MMPs which cleaves mainly collagen and interact for a minor extent with aggrecan. Conversely, aggrecanases which cleaves with high efficiency aggrecan within the IGD site, give rise to the removal of a large part of the protein from its binding site to HA thus leading to the breakage of that complex network of interactions fundamental to cartilage integrity and functions.
In addition, several members of the ADAMTS family including ADAMTS-4 and ADAMTS-5 have been found over-expressed in tumour cell lines. It should be pointed out that a prerequisite for invasiveness in cancer is cell migration based on increased expression of proteases digesting the extra-cellular matrix, the same process of extra-cellular matrix remodelling mediated by metalloproteases is also essential in angiogenesis, the process by which new blood vessels are formed from pre-existing vasculature. Angiogenesis has also been identified as a contributing factor in cancer where it is a rate-limiting step during tumour progression. Accordingly, in addition to arthritis ADAMT-4/-5 inhibitors could be useful therapeutic agents for the treatment of cancer.
The 2-phenyl-4H-1-benzopyran-4-one nucleus is well known in nature, since flavonoids form a class of benzopyran-4-one derivatives which are ubiquitous in plants as secondary metabolites. Flavonoids such as flavones and flavonols are present in a great variety of food, and especially in fruits and vegetables. Among them, Quercetin [2-(3,4-dihydroxyphenyl)-3,5,7-hydroxy-4H-1-benzopyran-4-one)] is the main flavonoid occurring in the food and is present at an average level of 10 mg/Kg (in onion its concentration is 300 mg/kg). Quercetin is a very effective antioxidant and appeared to be active in treating several disease such as cardiovascular, neurodegenerative and cancer. Further investigations highlighted how the proprieties of the 4H-1-benzopyran-4-one scaffold also known as chromenone, are not confined to Quercetin only but can be efficiently used as structural scaffolds for drug design.
Several 6-amino-2-aryl-4H-1-benzopyran-4-one derivatives are known in literature: 6-amino-2-phenyl-4H-1-Benzopyran-4-one (RN: 4613-53-0) has been reported along with other 26 flavonoids as anti-proliferative agents acting on cell cycle (Haddad, A. Q.; Prostate Cancer and Prostatic Diseases (2006), 9(1), 68-76), antispasmodic and antihistaminic activity has been reported for another group of 2-aryl-6-amino-chromen-4-one derivatives (P. K. Jesthi et al., Journal of the Institution of Chemists (India) (1981), 53(5), 234-6), inhibition of several protein kinases was reported for a group of aminoflavones (M. Cushman et al, Journal of Medicinal Chemistry (1994), 37(20), 3353-62), as well as inhibition of CDK kinase for a wide group of flavonoids including amino derivatives (PCT Int. Appl. (2000), WO 2000012496 A1 20000309). N-carbamoylderivatives of a group of aminoflavones have been reported as acetylcholine esterase inhibitors (PCT Int. Appl. (2002), WO 2002024677 A1 20020328). Though an amidine of a flavanone derivative has been reported as furamidine analogue (RN: 849368-08-7,2-[4-[amino[(1-methylethyl)imino]methyl]phenyl]-N-(1-methylethyl)-4H-1-Benzopyran-4-oxo-6-carboximidamide, H Goeker et al., Bioorganic & Medicinal Chemistry, 2005, 13(5), 1707-1714), exhibiting antimicrobial activity, none of these flavanone amino or amidinoderivatives has never been reported neither as aggrecanase inhibitor nor as analgesic.