1. Field of the Invention
The previous invention related generally to compounds effective as inhibitors of phosphatase enzymes and metallophosphatases, and specifically to chemical compounds that are inhibitors of phosphatase enzymes, their method of manufacture; and, particularly use of a class of chemical compounds as inhibitors of phosphatase enzymes including metallophosphatases. The present invention relates to thiophosphonates as inhibitors of phosphatases and particularly metallophosphatases, and a method of preparation of these thiophosphonate inhibitors.
2. Description of Related Art
Phosphate esters are ubiquitous in living organisms. Recently, the key role of the phosphorylation and dephosphorylation of proteins in the control of a host of biochemical processes within living organisms, and especially hunans, have been appreciated. The chemistry of phosphate esters makes them particularly suited for the basis of a regulatory mechanism and therefore provides a potential tool for biological analysis and treatment.
While the hydrolysis of a phosphate monoester, such as serine phosphate or methyl phosphate, is thermodynamically favorable, the activation barrier is formidable. Under physiological conditions (neutral pH, 25° C.) methyl phosphate has an estimated half-life for unanalyzed hydrolysis of about 500,000 years. The relative inertness of phosphate esters in the absence of enzymatic catalysis makes them a logical bases for a control mechanism and therefore as possible mechanism in the treatment of certain types of disease or conditions.
Protein kinases and phosphatases are now known to be the key cellular players in the process of signaling and the balance between phosphorylation and dephosphorylation of proteins. They have been shown to regulate a wide range of biochemical processes, including metabolism, DNA transcription and replication, cell differentiation, and immune responses.
Protein targets are phosphorylated at specific sites by one or more protein kinases, and the reverse reactions (hydrolysis of the phosphate esters) are catalyzed by protein phosphatases. Despite the fact that this phosphorylation/dephosphorylation process often takes place far from active sites, these processes function as a control between active and inactive states for many enzymes. The activation effects can be dramatic, often resulting in rate accelerations of 102 to 105-fold. The critical role played in regulating metabolism by phosphorylation and the counterbalancing roles played by kinases and phosphatases is well established (see Cohen, P. “Signal integration at the level of protein kinases, protein phosphatases and their substrates” Trends Biochem Sci 17, (1992) 408-13. and Cohen, P. “The Croonian Lecture 1998.; Identification of a protein kinase cascade of major importance in insulin signal transduction” Philos Trans R Soc Lond B Biol Sci 354, (1999) 485-95.)
In addition, the reversible phosphorylation of proteins is the chemical basis of a major mechanism of signal transduction in both prokaryotes and eukaryotes. These signaling pathways are used for transmitting signals across the cytoplasm to the nucleus, to induce the transcription of particular genes via the phosphorylation and activation of transcription factors. The signal cascade is activated following the binding of ligands, such as growth factors and cytokines to cell surface receptors, or in response to cellular stresses.
Thus, phosphatase enzymes are critical in the catalysis of the transfer of a phosphoryl group from a phosphomonoester or a phosphoanhydride to water, producing inorganic phosphate. The Ser/Thr phosphatases possess binuclear metal centers (metallophosphates) and transfer the phosphoryl group directly to a metal-coordinated hydroxide molecule. These enzymes dephosphorylate both phosphoserine and phosphothreonine amino acids in proteins. The protein-tyrosine phosphatases (PTPases) do not contain metal ions but use a cysteine nucleophile to form a phosphorylated cysteine intermediate.
Metaliophosphatases, as indicated above, are a subfamily of phosphatases and include all of the alkaline phosphatases, and all members of the Serine/Threonine family of protein phosphatases (calcineurin, lambda, and PP2C as used herein). These enzymes and a number of others have been structurally characterized by X-ray crystallography. They are shown to have two metal ions at the active site that is used in catalysis. In metallophosphatases distances that vary in a narrow range separate the metal ions. The identities of the metal ions vary. Alkaline phosphatases contain two zinc ions. Calcineurin contains a ferric ion (Fe in the +3 state) and a zinc ion (zinc +2). The physiological metal ions utilized by other metallophosphatases are uncertain. The enzymes function with a variety of divalent metal ions, and most typically they are studied using manganese ions (Mn+2) or, less commonly, magnesium.
Phosphatases are, therefore, essential enzymes in the regulation of many biochemical pathways in living organisms. They have been identified as essential virulence factors in a number of pathogenic bacteria that are crucial in the development of diabetes as well as the rejection of transplanted organs. Consequently, phosphatase enzyme inhibitors have useful applications as biochemical research tools in the study of the roles of these enzymes. As pharmaceuticals phosphatase enzyme inhibitors have potential use in the treatment of a wide variety of diseases, such as diabetes, autoimmune diseases, cancer, and viral infection, such as HIV.
The specific roles of only a few of the many known phosphatases are clearly understood. PP2B, also known as calcineurin, has an essential role in the production of T cells in the immune response pathway. (Rusnak, F. and Mertz, P. “Calcineurin: form and function” Physiol Rev 80, (2000) 1483-521.) Calcineurin is the target of the immunosuppressive drugs FK506 and cyclosporin A. The protein-tyrosine phosphatase PP-1B regulates the production of insulin, and is currently a major target of studies aimed at developing a selective inhibitor that might prove a viable treatment for diabetes. However the physiological roles of only a very small percentage of the many known phosphatases are understood.
A major hindrance to studies that would aid in this understanding has been the lack of selective inhibitors. In cellular or animal studies selectively inhibiting the enzyme and observing the biochemical results can be used to study the role of an enzyme in a biochemical pathway. There are several phosphatase inhibitor “cocktails” that are commercially available for this purpose, which contain a mixture of compounds that typically give only very broad specificity. For instance, two are available from Sigma-Aldrich, one of which will inhibit all protein-tyrosine phosphatase as well as alkaline phosphatases; the other inhibits all serine/threonine phosphatases plus alkaline phosphatases.
Human placental alkaline phosphatase (PLAP) is one of three tissue-specific human alkaline phosphatases extensively studied because of its expression in tumors and it is a well-known tumor marker.
This lack of selective inhibitors of phosphatases, and more importantly, specific subfamilies of phosphatases impedes biochemical studies aimed at determining the biological roles of specific phosphatases, as well as the development of effective treatment regimes and pharmaceuticals.
Many avenues have been pursued to create specific, effective phosphatase enzyme inhibitors, but none to date have been overly successful. One such phosphatase inhibitor, that has been extensively pursued, is the phosphonates. The phosphonate group is similar to the phosphate group that is present in the natural substrates of phosphatases and some appear to exhibit slight inhibition of a few protein phosphatases (Kole et. Al., Biochem. Journal, 1995, 311, 1025-1031; Taing et al., Biochemistry, 1999, 38, 3793-3803; Burke et al., Biochemistry, 35, 15989-15996.) The synthesis of a number of phosphonic acid inhibitors and their inhibition constants for several phosphatase enzymes were reported by Myers, Antonelli and Widlanski (Journal of the American Cancer Society, 1997, 119, 3163-3164.)
Additionally, heteroaromatic phosphonates with a thio ether linkage have been prepared previously as inhibitors of the enzyme fructose 1,6-bisphosphatase. In these compounds the heteroaromatic moiety is intended to mimic the fructose portion of the natural substrate. Several aryloxymethylphosphonates were prepared previously and evaluated as inhibitors of protein tyrosine phosphatases, and were found to be weak inhibitors with Ki values in the millimolar range (Ibrahimi et al., Bioorg. Med. Chem. Lett., 2000, 10: 457-460). However, none of the phosphatase inhibitors produced to date have inhibited phosphatase enzymes to a significant level. This has been due to many factors. One important factor is that binding of the phosphatase inhibitor has not interfered with the active region of the phosphatase enzyme. (Kantrowitz et al., Protein Science, 2000, 9:907-915.) In addition, the bond that has occurred between the phosphatase inhibitors produced to date and the phosphatase enzymes has been very weak.
Thus, phosphatase inhibitors that are specific and demonstrate high degrees of phosphate inhibition are needed to further study phosphate enzyme chemistry, as well as to treat and/or retard a wide variety of health conditions and diseases. In particular, phosphatase inhibitors that are not hydrolyzed by the phosphatase enzyme and exhibit a high degree of interference with the active region of the enzyme are needed.
In particular compounds which function, as inhibitors of metallophosphatases are important to these studies as well as to treat and/or retard a wide variety of health conditions and diseases. In our prior application, a specific class of compounds was discovered that exhibited a high degree of interference with the active region of the phosphatase enzyme and particularly metallophosphatase enzymes. The enzyme did not hydrolyze these compounds, thus providing an effective phosphatase enzyme inhibitor. The effectiveness of these compounds was attributable in part to their molecular footprint, which mimics the transition state of the phosphate ester hydrolysis reaction.
The class of compounds of that application were generally ethers represented by the general formula R—X—R′—PO3−2 wherein X is selected from the group consisting of O, NR″, or S where R″ is H or an organic moiety of from about 1 to about 100 carbon atoms; R′ is a non hydrolysable moiety providing a bond spacing between P and X of from about 2.5 to about 4.5, and preferably from about 3 to about 4 Å; and, R is, a moiety that does not interfere with the bond spacing between P and X.
Another group of compounds that have been the subject of a number of patents and publications as antiviral compounds are the thiophosphonates, or phosphonothioic acids and their salts (See for example, Borecka, B., Chojnowski, J., Cypryk, M. and Zielinska, J. “Synthetic and mechanistic aspects of the reaction of trialkylsilyl halides with thio and seleno esters of phosphorus” J. Organometallic Chem. 171, (1979) 17-34; Piettre, S. R. and Raboisson, P. “Easy and general access to α,α-difluoromethylene phosphonothioic acids (See, Aladzheva, I. M., Odinets, I. L., Petrovskii, P. V., Mastryukova, T. A. and Kabachnik, M. I. “Phase-transfer catalytic alkylation of hydrothiophosphoryl compounds. IV. Reactions with primary alkyl halides.” Russ. J. Gen. Chem. 63, (1993) 431-437) A new class of compounds.” Tetrahedron Letters 37, (1996) 2229-2232; Yokomatsu, T., Takechi, H., Murano, T. and Shibuya, S. “Synthesis of aryldifluoromethylphsophonothioic acids from O,O-diethyl aryldifluoromethylphosphonothioates” J. Org. Chem. 65, (2000) 5858-5861 and Ladzheva, I. M., Odinets, I. L., Petrovskii, P. V., Mastryukova, T. A. and Kabachnik, M. J. “Phase-transfer catalytic alkylation of hydrothiophosphoryl compounds. IV. Reactions with primary alkyl halides.” Russ. J. Gen. Chem. 63, (1993) 431-437). There is also evidence that this class of compounds has been investigated as plant growth regulators and as inhibitors of a number of different enzymes as well as lubricants and polymers.
While it has been unexpectedly discovered that certain of these compounds are effective as inhibitors of phosphatases and specifically metallophosphatases in accordance with the instant invention, as set forth below, current methods of preparation have heretofore not yielded satisfactory compounds on an acceptable yield bases.
Thus, while many known methods are suitable for the production of small amounts of very specific compounds they often utilize reaction conditions that would preclude large-scale production and/or the synthesis of derivatives bearing a variety of functional groups that would be useful. For example, methyl and, to a lesser extent, ethyl esters have often been used as protecting groups during syntheses of phosphates and phosphonates. However, while typically dimethyl or diethyl groups of phosphonate esters can be removed by treatment with trimethylsilyliodide or trimethylsilylbromide, this method works sparingly and results in less than satisfactory yields of thio derivatives. (See, McKenna, C. E., Li, Z. -M., Ju, J. -Y., Pham, P. -T., Kilkuskie, R., Loo, T. L. and Straw, J. “Simple and conjugate bifunctional thiophosphonates: synthesis and potential as anti-viral agents” Phosphorus, Sulfur and Silicon 74, (1993) 469-470.)
In addition, deprotection of the dibenzyl esters of α, α-difluoromethylenephosphonothioic acids has traditionally been accomplished by the cumbersome process of utilizing sodium in liquid ammonia. (See, Kabachnik, I., Mastryukova, T. A., Kurochkin, N. I., Rodionova, N. P. and Popov, E. M. “Reactivity of akali salts of acid esters of alkylthiophosphonic acids. Reactions of acylation and alkylation.” Zhur. Obschei Khim. 26, (1956) 2228-2233.) Recently another method was reported that utilized a thionothiolo rearrangement followed by Pd-catalyzed deallylation in order to accomplish deprotection of diethyl α,α-difluoromethylenephosphonothioic (Zabirov, N. G., Cherkasov, R. A. and Pudovik, A. N. “Lawesson's reagent in the synthesis of organophosphorus compounds. I. Preparation of dialkyl thiophosphites and alkyl thiophosphonites.” Russ. J. Gen. Chem. 56, (1986) 1047-1048.) Neither of these methods, however, has been found satisfactory for the production of large quantities of thiophosphonates.
All of these reported methodologies are time consuming, use very specific ester protecting groups, and are not applicable to large-scale production of phosphonothioic acids and/or the production of phosphonothioic acids with a wide range of functional groups. These prior methods for producing thiophosphonates, or phosphonothioic acids have shortcomings. First, the prior syntheses do not use methyl protecting groups, but instead use various other protecting groups that are particularly suited to the specific compound being synthesized, and which require methods for their removal that preclude general use. For example, one prior method uses benzyl ester protection, and removal using sodium in liquid ammonia. These are strong reductive conditions that would result in unwanted alterations in many functional groups if they were present in the compound.
Although, generally methyl-protecting groups are the standard in phosphate ester synthesis because they are easy to remove, using the prior art methods of thio derivative synthesis, the methyl groups are difficult to remove. Specifically, in the prior art synthesis removal of the first methyl group is relatively easy, but the second is recalcitrant. Further, the prior published synthesis can only be used for un reactive alkyl substituents. Additionally, the prior art methods require complex purification steps, which are cumbersome, and reduce yields.
It would therefore be advantageous to have a synthetic approach for producing thiophosphonates, or phosphonothioic acids by a facile method that involves only minimal purification procedures, and which is much more general than previous synthetic approaches. It would also be advantageous to have a synthetic approach for producing thiophosphonates, or phosphonothioic acids applicable to the preparation of a wide variety of these compounds bearing a variety of reactive functional groups, while requiring only a minimal purification of intermediates, and a final product purification and recovery by a simple crystallization of the pure form of the product compound.
Citation of the above documents is only a discussion of related art and not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on subjective characterization of information available to the Applicants, and does not constitute any admission as to the accuracy of the dates or contents of these documents.