WO1997040017A2 - Modulators of molecules with phosphotyrosine recognition units - Google Patents

Modulators of molecules with phosphotyrosine recognition units Download PDF

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WO1997040017A2
WO1997040017A2 PCT/DK1997/000166 DK9700166W WO9740017A2 WO 1997040017 A2 WO1997040017 A2 WO 1997040017A2 DK 9700166 W DK9700166 W DK 9700166W WO 9740017 A2 WO9740017 A2 WO 9740017A2
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Henrik Sune Andersen
Niels Peter Hundahl Møller
Peter Madsen
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Novo Nordisk A/S
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    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/38—Phosphonic acids RP(=O)(OH)2; Thiophosphonic acids, i.e. RP(=X)(XH)2 (X = S, Se)
    • C07F9/3804—Phosphonic acids RP(=O)(OH)2; Thiophosphonic acids, i.e. RP(=X)(XH)2 (X = S, Se) not used, see subgroups
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    • C07F9/3882—Arylalkanephosphonic acids

Definitions

  • the present invention relates to novel organic compounds, to methods for their preparation, to compositions containing them, to their use for treatment of human and animal disorders, to their use for purification of proteins or glycoproteins, and to their use in diagnosis.
  • the invention relates to modulation of the activity of molecules with phospho-tyrosine recognition units, including protein tyrosine phosphatases (PTPases) and proteins with Src-homology-2 domains, in in vitro systems, micro-organisms, eukaryotic cells, whole animals and human beings.
  • PTPases protein tyrosine phosphatases
  • PTKs protein tyrosine kinases
  • PTPases protein tyrosine phosphatases
  • the protein phosphatases are composed of at least two separate and distinct families (Hunter, T., Cell 58: 1013-1016 (1989)) the protein serine/threonine phosphatases and the PTPases.
  • the PTPases are a family of enzymes that can be classified into two groups: a) intracellular or nontransmembrane PTPases and b) receptor-type or transmembrane PTPases.
  • Intracellular PTPases All known intracellular type PTPases contain a single conserved catalytic phosphatase domain consisting of 220-240 amino acid residues. The regions outside the PTPase domains are believed to play important roles in localizing the intracellular PTPases subcellularly (Maura, LJ. and Dixon, J.E. TIBS 19: 151-155 (1994)). The first intracellular PTPase to be purified and characterized was PTP1 B which was isolated from human placenta (Tonks etal., J. Biol. Chem. 263: 6722-6730 (1988)). Shortly after, PTP1B was cloned (Charbonneau et al., Proc. Natl.
  • intracellular PTPases include (1) T-cell PTPase (Cool et al. Proc. Natl. Acad. Sci. USA 86: 5257-5261 (1989)), (2) rat brain PTPase (Guan ef al., Proc. Natl. Acad. Sci. USA 87:1501-1502
  • LMW-PTPase Low molecular weight phosphotyrosine-protein phosphatase shows very little sequence identity to the intracellular PTPases described above.
  • this enzyme belongs to the PTPase family due to the following characteristics: (i) it possesses the PTPase active site motif: Cys-Xxx-Xxx-Xxx-Xxx-Xxx-Arg (Cirri etal, Eur. J. Biochem. 214: 647-657 (1993)); (ii) this Cys residue forms a phospho-intermediate during the catalytic reaction similar to the situation with 'classical' PTPases (Cirri et al., supra; Chiarugi etal, FEBS Lett.
  • Receptor-type PTPases consist of a) a putative ligand-binding extracellular domain, b) a transmembrane segment, and c) an intracellular catalytic region.
  • the structures and sizes of the putative ligand-binding extracellular domains of receptor-type PTPases are quite divergent.
  • the intracellular catalytic regions of receptor-type PTPases are very homologous to each other and to the intracellular PTPases.
  • Most receptor-type PTPases have two tandemly duplicated catalytic PTPase domains.
  • the first receptor-type PTPases to be identified were (1) CD45/LCA (Ralph, S.J., EMBO J. 6: 1251-1257 (1987)) and (2) LAR (Streuli et al., J. Exp. Med. 168: 1523-1530 (1988)) that were recognized to belong to this class of enzymes based on homology to PTP1B (Charbonneau etal, Proc. Natl. Acad. Sci. USA 86: 5252-5256 (1989)).
  • CD45 is a family of high molecular weight glycoproteins and is one of the most abundant leukocyte cell surface glycoproteins and appears to be exclusively expressed upon cells of the hematopoietic system (Trowbridge and Thomas, Ann. Rev. Immunol. 12: 85-116 (1994)).
  • PTP-U2/GLEPP1 (Seimiya etal., Oncogene 10: 1731-1738 (1995); (Thomas ef al., J. Biol. Chem. 269: 19953-19962 (1994)), and (14) DEP-1; (IV) PTP ⁇ ,_PTP ⁇ . All receptor-type PTPases except Type IV contain two PTPase domains. Novel PTPases are continously identified, and it is anticipated that more than 500 different species will be found in the human genome, i.e. close to the predicted size of the protein tyrosine kinase superfamily (Hanks and Hunter, FASEB J. 9: 576-596 (1995)).
  • PTPases are the biological counterparts to protein tyrosine kinases (PTKs). Therefore, one important function of PTPases is to control, down-regulate, the activity of PTKs.
  • PTKs protein tyrosine kinases
  • a more complex picture of the function of PTPases now emerges.
  • Several studies have shown that some PTPases may actually act as positive mediators of cellular signalling.
  • the SH2 domain-containing PTP1D seems to act as a positive mediator in insulin- stimulated Ras activation (Noguchi etal, Mol. Cell. Biol. 14: 6674-6682 (1994)) and of growth factor-induced mitogenic signal transduction (Xiao ef al, J. Biol. Chem.
  • PTPases as positive regulators has been provided by studies designed to define the activation of the Src-family of tyrosine kinases. In particular, several lines of evidence indicate that CD45 is positively regulating the activation of hematopoietic cells, possibly througn dephosphorylation of the C-terminal tyrosine of Fyn and Lck (Chan et al., Annu. Rev. Immunol. 12: 555-592 (1994)).
  • Dual specificity protein tyrosine phosphatases define a subclass within the PTPases family that can hydrolyze phosphate from phosphortyrosine as well as from phosphor-serine/threonine.
  • dsPTPases contain the signature sequence of PTPases: His- Cys-Xxx-Xxx-Gly-Xxx-Xxx-Arg. At least three dsPTPases have been shown to dephosphorylate and inactivate extracellular signal-regulated kinase (ERKs)/mitogen- activated protein kinase (MAPK): MAPK phosphatase (CL100, 3CH134) (Charles etal, Proc. Natl. Acad.
  • dsPTPases Transcription of dsPTPases are induced by different stimuli, e.g. oxidative stress or heat shock (Ishibashi et al., J. Biol. Chem. 269: 29897-29902 (1994); Keyse and Emslie, Nature 359: 644-647 (1992)).
  • stimuli e.g. oxidative stress or heat shock (Ishibashi et al., J. Biol. Chem. 269: 29897-29902 (1994); Keyse and Emslie, Nature 359: 644-647 (1992)).
  • cdc25 Millar and Russell, Cell 68: 407-410 (1992)
  • KAP Hannon etal, Proc. Natl. Acad. Sci. USA 91: 1731- 1735 (1994)
  • tyrosine dephosphorylation of cdc2 by a dual specific phosphatase, cdc25 is required for induction of mitosis in yeast (review by Walton and Dixon, Annu. Rev. Biochem. 62: 101-120 (1993)).
  • Hormones, growth factors, cytokines, antigens, extracellular matrix components as well as molecules positioned at the cell surface induce signal transduction by binding to specific cell surface structures or receptors on target cells (reviewed in Pawson, Nature 373: 573-580 (1995)).
  • the resulting cellular signal is often mediated through a series of phosphorylation and dephosphorylation reactions on tyrosine residues of signalling molecules.
  • pTyr phosphotyrosine
  • SH2 domains and PTB domains primarily act as docking molecules with little or no catalytic activity.
  • tyrosine phosphorylated proteins have the capacity to bind other proteins containing SH2 domains or PTB domains thereby controlling the subcellular location of signalling molecules.
  • SH2 domains from the Src kinase family bind the peptide pTyr-Glu-Glu-lle in a relatively selective manner, whereas the PTPD1 seems to recognize at least five, primarily hydrophobic residues C-terminal to the pTyr (Pawson, supra).
  • Inhibition of signal transduction processes could, in principle, be achieved by using non-hydrolyzable pTyr-containing peptides with preferential affinity for specific PTPases, SH2 domains or PTB domains.
  • pTyr-containing peptides with preferential affinity for specific PTPases, SH2 domains or PTB domains.
  • Such selective compounds can either initiate, increase or decrease defined signal transduction processes.
  • Vanadate was found to inhibit protein-tyrosine phosphatases in mammalian cells with a concomitant increase in the level of phosphotyrosine in cellular proteins leading to transformation (Klariund, Cell 41: 707-717 (1985)).
  • Vanadium-based phosphatase inhibitors are relatively unspecific. Therefore, to assess the importance of specific structures on PTPase activity more selective inhibitors are needed.
  • One possibility for obtaining selective PTPase inhibitors would be through design of different ancillary ligands for peroxovanadium-based compounds (Posner et al., J. Biol. Chem. 269: 4596-4604 (1994)).
  • PTPases the insulin receptor signalling pathway/diabetes
  • Insulin is an important regulator of different metabolic processes and plays a key role in the control of blood glucose. Defects related to its synthesis or signalling lead to diabetes mellitus. Binding of insulin to its receptor causes rapid (auto) phosphorylation of several tyrosine residues in the intracellular part of the ⁇ -subunit. Three closely positioned tyrosine residues (the tyrosine-1150 domain) must all be phosphorylated to obtain full activity of the insulin receptor tyrosine kinase (IRTK) which transmits the signal further downstream by tyrosine phosphorylation of other cellular substrates, including insulin receptor substrate-1 (IRS-1) (Wilden et al, J. Biol. Chem.
  • IRTK appears to be tightly regulated by PTP- mediated dephosphorylation in vivo (Khan et al., J. Biol. Chem. 264: 12931-12940 (1989); Faure et al., J. Biol. Chem. 267: 11215-11221 (1992); Rothenberg et al, J. Biol. Chem. 266: 8302-8311 (1991)).
  • PTPases ⁇ and ⁇ While it appears that the target of the negative regulatory activity of PTPases ⁇ and ⁇ is the receptor itself, the downmodulating effect of the intracellular TC-PTP seems to be due to a downstream function in the IR-activated signal. Although PTP1B and TC-PTP are closely related, PTP1B had only little influence on the phosphorylation pattern of insulin-treated cells. Both PTPases have distinct structural features that determine their subcellular localization and thereby their access to defined cellular substrates (Frangione ef al., Cell 68: 545-560 (1992); Faure and Posner, Glia 9: 311- 314 (1993)).
  • the tack of activity of PTP1B and TC-PTP towards the IRTK may, at least in part, be explained by the fact that they do not co-localize with the activated insulin receptor.
  • PTP1B and TC-PTP have been excluded as candidates for the IR-associated PTPases in hepatocytes based on subcellular localization studies (Faure etal, J. Biol. Chem. 267: 11215-11221 (1992)).
  • the transmembrane PTPase CD45 which is believed to be hematopoietic cell-specific, was in a recent study found to negatively regulate the insulin receptor tyrosine kinase in the human multiple myeloma cell line U266 (Kulas ef al, J. Biol. Chem. 271: 755-760 (1996)).
  • Somatostatin inhibits several biological functions including cellular proliferation (Lamberts ef al, Molec. Endocrinol. 8: 1289-1297 (1994)). While part of the antiproliferative activities of somatostatin are secondary to its inhibition of hormone and growth factor secretion (e.g. growth hormone and epidermal growth factor), other antiproliferative effects of somatostatin are due to a direct effect on the target cells. As an example, somatostatin analogs inhibit the growth of pancreatic cancer presumably via stimulation of a single PTPase, or a subset of PTPases, rather than a general activation of PTPase levels in the cells (Liebow ef a/., Proc. Natl.
  • PTPases the immune svstem/autoimmunity
  • CD45 a lymphocyte-specific member of the Src family protein-tyrosine kinase (Mustelin et al, Proc. Natl. Acad. Sci. USA 86: 6302-6306 (1989); Ostergaard etal, Proc. Natl. Acad. Sci. USA 86: 8959-8963 (1989)).
  • Fyn another member of the Src family protein-tyrosine kinases, Fyn, seems to be a selective substrate for CD45 compared to Lck and Syk (Katagiri et al., J. Biol. Chem. 270: 27987- 27990 (1995)).
  • HePTP a hematopoietic cell specific PTPase
  • PTPase a hematopoietic cell specific PTPase
  • the hematopoietic cell specific PTP1C seems to act as a negative regulator and play an essential role in immune cell development
  • selective PTPase inhibitors may be attractive drug candidates both as immunosuppressors and as immunostimulants.
  • PTPase inhibitors as immunmodulators by demonstrating the capacity of the vanadium- based PTPase inhibitor, BMLOV, to induce apparent B cell selective apoptosis compared to T cells (Schieven etal, J. Biol. Chem. 270: 20824-20831 (1995)).
  • BMLOV vanadium- based PTPase inhibitor
  • Focal adhesion plaques an in vitro phenomenon in which specific contact points are formed when fibroblasts grow on appropriate substrates, seem to mimic, at least in part, cells and their natural surroundings.
  • Several focal adhesion proteins are phosphorylated on tyrosine residues when fibroblasts adhere to and spread on extracellular matrix (Gumbiner, Neuron 11, 551-564 (1993)).
  • Aberrant tyrosine phosphorylation of these proteins can lead to cellular transformation.
  • the intimate association between PTPases and focal adhesions is supported by the finding of several intracellular PTPases with ezrin-like N-terminal domains, e.g. PTPMEG1 (Gu ef a/., Proc. Natl. Acad. Sci.
  • PTPH1 Yang and Tonks, Proc. Natl. Acad. Sci. USA 88: 5949-5953 (1991)
  • PTPD1 Yang and Tonks, Proc. Natl. Acad. Sci. USA 88: 5949-5953 (1991)
  • PTPD1 M ⁇ ller et al, Proc. Natl. Acad. Sci. USA 91: 7477-7481 (1994)
  • the ezrin-like domain show similarity to several proteins that are believed to act as links between the cell membrane and the cytoskeleton.
  • PTPD1 was found to be phosphorylated by and associated with c-src in vitro and is hypothesized to be involved in the regulation of phosphorylation of focal adhesions (M ⁇ ller et al., supra).
  • PTPases may oppose the action of tyrosine kinases, including those responsible for phosphorylation of focal adhesion proteins, and may therefore function as natural inhibitors of transf ormation.
  • TC-PTP and especially the truncated form of this enzyme (Cool et al, Proc. Natl. Acad. Sci. USA 87: 7280-7284 (1990)), can inhibit the transforming activity of v- erb and (Lammers ef al., J. Biol. Chem. 268: 22456-22462 (1993); Zander et al, Oncogene 8: 1175-1182 (1993)).
  • PTP1B The expression level of PTP1B was found to be increased in a mammary cell line transformed with neu (Zhay ef al., Can ⁇ rRes. 53: 2272-2278 (1993)).
  • the intimate relationship between tyrosine kinases and PTPases in the development of cancer is further evidenced by the recent finding that PTP ⁇ is highly expressed in murine mammary tumors in transgenic mice over-expressing c-neu and v-Ha-ras, but not c-myc or int-2 (Elson and Leder, J. Biol. Chem. 270: 26116-26122 (1995)).
  • PTPK and PTP ⁇ Two closely related receptor-type PTPases, PTPK and PTP ⁇ , can mediate homophilic cell- cell interaction when expressed in non-adherent insect cells, suggesting that these PTPases might have a normal physiological function in cell-to-cell signalling (Gebbink et al, J. Biol. Chem. 268: 16101-16104 (1993); Brady-Kalnay et al., J. Cell Biol. 122: 961-972 (1993); Sap etal, Mol. Cell. Biol. 14: 1-9 (1994)).
  • PTPK and PTP ⁇ do not interact with each other, despite their structural similarity (Zondag etal, J. Biol. Chem.
  • PTPases may play an important role in regulating normal cell growth.
  • PTPases may also function as positive mediators of intracellular signalling and thereby induce or enhance mitogenic responses. Increased activity of certain PTPases might therefore result in cellular transformation and tumor formation.
  • over-expression of PTP ⁇ was found to lead to transformation of rat embryo fibroblasts (Zheng, supra).
  • SAP-1 a novel PTP, SAP-1, was found to be highly expressed in pancreatic and colorectal cancer cells.
  • SAP-1 is mapped to chromosome 19 region q13.4 and might be related to carcinoembryonic antigen mapped to 19q13.2 (Uchida et al, J. Biol. Chem. 269: 12220-12228 (1994)). Further, the dsPTPase, cdc25, dephosphorylates cdc2 at Thr14/Tyr-15 and thereby functions as positive regulator of mitosis (reviewed by Hunter, Cell 80: 225-236 (1995)). Inhibitors of specific PTPases are therefore likely to be of significant therapeutic value in the treatment of certain forms of cancer.
  • PTPases platelet aggregation
  • PTPases are centrally involved in platelet aggregation.
  • Agonist- induced platelet activation results in calpain-catalyzed cleavage of PTP1 B with a concomitant 2-fold stimulation of PTPase activity (Frangioni et al, EMBO J. 12: 4843-4856 (1993)).
  • the cleavage of PTP1 B leads to subcellular relocation of the enzyme and correlates with the transition from reversible to irreversible platelet aggregation in platelet-rich plasma.
  • the rate of bone formation is determined by the number and the activity of osteoblasts, which in term are determined by the rate of proliferation and differentiation of osteoblas progenitor cells, respectively. Histomo ⁇ hometric studies indicate that the osteoblast number is the primary determinant of the rate of bone formation in humans (Gruber ef a/., Mineral Electrolyte Metab. 12: 246-254 (1987); reviewed in Lau ef a/., Biochem. J. 257: 23-36 (1989)). Acid phosphatases/PTPases may be involved in negative regulation of osteoblast proliferation. Thus, fluoride, which has phosphatase inhibitory activity, has been found to increase spinal bone density in osteoporotics by increasing osteoblast proliferation (Lau et al., supra).
  • OST-PTP parathyroid regulated, receptor-like PTPase
  • PTPases microorganisms
  • the inventors have identified a novel class of compounds that has the capacity to modulate the activity of molecules with tyrosine recognition units, including PTPases, preferably a selective modulation.
  • the present invention relates to novel organic compounds thereof of general formula (I)
  • the present invention relates to novel organic compounds thereof of formula (I)
  • n 1 , 2, 3, 4, or 5 and (L) lake represents up to five (5) substituents which independentiy of each other are hydrogen, d -6 -alkyl, C 1-6 -alkoxy, C ⁇ alkylthio, hydroxy, halogen, tirihalogenomethyl, hydroxy-C L ⁇ -alkyl, amino-C ⁇ -alkyl, -COR 2 , -NO 2 , -CN, -CHO, -C ⁇ .
  • R 2 is Ci-e-alkyl, aryl optionally substituted, aralkyl optionally substituted, -OH, -NR 3 R 4 wherein R 3 and R 4 independently of each other are hydrogen, Ci-e-alkyl. aryl optionally substituted, aralkyl optionally substituted;
  • R 5 and R 6 are independently of each other hydrogen or Ci-e-alkyl, aryl optionally substituted, aralkyl optionally substituted or-COZi wherein Zi is d ⁇ -alkyl, aryl optionally substituted, aralkyl optionally substituted;
  • L is A ⁇ -Y ⁇ -(W ⁇ )-X-(W 2 )-Y 2 - wherein X is a chemical bond, -CO, -CONR 7 , -NR 7 CO, -NR 7 ,-
  • Yi and Y 2 are independently a chemical bond, -O-, -S-, or -NR 7 ;
  • R 7 is hydrogen, Ci- ⁇ -alkyl, aryl optionally substituted, aralkyl optionally substituted, heteroaryl optionally substituted, -COZ 2 wherein Z 2 is Ci-e-alkyl, aryl optionally substituted, aralkyl optionally substituted;
  • Wi and W 2 are independently a chemical bond or saturated or unsaturated Ci- ⁇ -alkylene
  • Ai is aryl optionally substituted, heteroaryl optionally substituted, biaryl optionally substituted, arylheteroaryl optionally substituted, -NR 8 Rg wherein R 8 and Rg independently are hydrogen, Ci-e-alkyl, aryl optionally substituted, aralkyl optionally substituted, heteroaryl optionally substituted, -COZ 3 wherein Z 3 is Ci-e-alkyl, aryl optionally substituted, aralkyl optionally substituted, heteroaryl optionally substituted or when R 8 and R 9 together with the nitrogen atom forms a ring system
  • Ai is a saturated or partially saturated heterocyclic ring system optionally substituted with Ci-e-alkyl, aryl optionally substituted, aralkyl optionally substituted, heteroaryl optionally substituted, -OH, d-e-alkoxy, Ci-e-alkylthio, hydroxy-d-e-alkyl, amino-C ⁇ .
  • A is -PO(OR ⁇ 8 )(OR, 9 ), -NH-SO3H, -NH-SO2-CH3, -NH-S0 2 -CF 3 , -CO-NH-OH or a heterocycle as shown in scheme 1 wherein the point of attachment is indicated with a I
  • R 20 is hydrogen, -OH, d-e-alkoxy, -SH, d-e-alkylthio, Ci ⁇ -alkylcarbonyloxy , -COR 21 , -SOR22,
  • R21, R22, and R 23 are -OR 2 e, Ci-e-alkyl, -NR24R25, trihalogenomethyl;
  • R 24 and R 25 independently are hydrogen, Ci-e-alkyl, -SO 2 R 2 6, -COZ 5 wherein Zs is
  • R 2 e is hydrogen, Ci- ⁇ -alkyl, trihalogenomethyl; nn is 1 or 2; and An is aryl or heteroaryl;
  • aryl, heteroaryl, An or Ai are exemplified by the following examples.
  • Specific examples of the aryl and biaryl residues include phenyl, biphenyi, indenyl, fluorenyl, naphthyl (1-naphthyl, 2-naphthyl), anthracenyl (1-anthracenyl, 2-anthracenyl, 9- anthracenyl).
  • heteroaryl examples include pyrrolyl (2-pyrrolyl), pyrazolyl (3- pyrazolyl), imidazolyl (1 -imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl), triazolyl (1,2,3- triazol-1-yl, 1 ,2,3-triazol-2-yt 1,2,3-triazo!-4-yl, 1,2,4-triazol-3-yl), oxazolyl (2-oxazolyl, 4- oxazolyl, 5-oxazolyl), isoxazolyl (3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl), thiazolyl (2-thiazolyl, 4-thiazolyl, 5-thiazolyl), thiophenyl (2-thiophenyl, 3-thiophenyl), pyridyl (2-pyridyl, 3-pyridyl, 4- pyridyl, 2-
  • arylheteroaryl residue examples include phenylpyridyl (2-phenylpyridyl, 3- phenylpyridyl, 4-phenylpyridyl), phenylpyrimidinyl (2-phenylpyrimidinyl, 4-phenylpyrimidinyl, 5- phenylpyrimidinyl, 6-phenylpyrimidinyl), phenylpyrazinyl, phenylpyridazinyl (3-phenyl- pyridazinyl, 4-phenylpyridazinyl, 5-phenylpyridazinyl).
  • the Ci-e-alkyl residues include aliphatic hydrocarbon residues, unsaturated aliphatic hydrocarbon residues, alicyclic hydrocarbon residues.
  • Examples of the aliphatic hydrocarbon residues include saturated aliphatic hydrocarbon residues having 1 to 6 carbon atoms such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec.butyl, tertbutyl, n-pentyl, isopentyl, neopentyl, tertpentyl, n-hexyl, isohexyl.
  • Example of the unsaturated aliphatic hydrocarbon residues include those having 2 to 6 carbon atoms such as ethenyl, 1-propenyl, 2-propenyl, 1- butenyl, 2-butenyl, 3-butenyl, 2-methyl-1-propenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4- pentenyl, 3-methyl-2-butenyl, 1-hexenyl, 3-hexenyl, 2,4-hexadienyl, 5-hexenyl, ethynyl, 1-propionyl, 2-propionyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexynyl, 3-hexynyl, 2,4-hexadiynyl, 5-hexynyl.
  • Examples of the alicyclic hydrocarbon residue include
  • C 5 C 5 . s unsaturated alicyclic hydrocarbon residues having 5 to 6 carbon atoms such as 1- cyclopentenyl, 2-cyclopentenyl, 3-cyclopentenyl, 1 -cyclohexenyl, 2-cyclohexenyl, 3-cyclohexenyl.
  • the Ci -e-alkoxy residues include aliphatic hydrocarbon residues connected to an oxygene atom.
  • the aliphatic hydrocarbon residues include saturated aliphatic hydrocarbon residues having 1 to 6 carbon atoms such as methoxy, ethoxy, propoxy, iso ⁇ propoxy, butoxy, isobutoxy, sec.butoxy, tertbutoxy, pentoxy, isopentoxy, neopentoxy, tert.pentoxy, hexyloxy, isohexyloxy.
  • the Ci-e-alkylthio residues include aliphatic hydrocarbon residues connected to an sulphur atom.
  • the aliphatic hydrocarbon residues include saturated aliphatic hydrocarbon residues having 1 to 6 carbon atoms such as methythio, ethylthio, propoylthio, iso-propylthio, butylthio, isobutylthio, sec.butylthio, tert.butylthio, pentylthio, isopentylthio, neopentylthio, tert. pentylthio, hexylylthio, isohexylythio.
  • the C ⁇ .6-alkoxycarbonyl residues include a Ci-e-alkoxy residue connected to a carbonyl residue such as methoxycarbonyl, ethoxy-carbonyl, propoxycarbonyl, and tert- butoxyca ⁇ onyl.
  • the Ci-e-alkylcarbonyloxy residues include a Ci-e-alkyl residue connected to a carbonyloxy residue such as acetic acid, propionic acid, butyric acid.
  • the Ci-e-alkanoyloxy residues include a acyl residue connected to an oxygen atom wherein the acyl residue is an aliphatic hydrocarbon residues connected to an carbonyl residue such as acetyloxy, propionyloxy, isopropionyloxy.
  • the aralkyl residue include an aryl residue connected to an Ci-e-alkyl residue e.g. phenyl alkyls having 7 to 9 carbon atoms such as benzyl, phenethyl, 1-phenylethyl, 3-phenylpropyl, 2-phenyipropyl and 1-phenylpropyl; and naphthyl alkyl having 11 to 13 carbon atoms such as 1-naphthylmethyl, 1-naphthylethyl, 2-naphthylmethyl, and 2-naphthylethyl.
  • phenyl alkyls having 7 to 9 carbon atoms such as benzyl, phenethyl, 1-phenylethyl, 3-phenylpropyl, 2-phenyipropyl and 1-phenylpropyl
  • naphthyl alkyl having 11 to 13 carbon atoms such as 1-naphthylmethyl, 1-naphth
  • Aryloxy include an aryl connected to an oxygen atom such as phenyloxy, naphthyloxy.
  • Aralkyloxy include an aralkyl connected to an oxygen atom such as benzyloxy, phenethyloxy, naphthylmethyloxy.
  • Biaryl include an aryl connected to an aryl residue such as biphenyi, 1-phenylnaphthyl, 2- phenylnaphthyl .
  • Biaryloxy include an biaryl connected to an oxygen atom such as biphenyloxy, 4-(naphthalene-1 -yl)phenoxy, 4-(naphthalene-2-yl)phenoxy.
  • the heteroaryl residue is a 5- or 6-membered aromatic ring, which can be fused to one or more phenyl rings and contains, besides carbon atoms, 1 to 4 atoms selected from N, O, and S as atoms constituting the ring, which is bonded through ca ⁇ on atoms such as defined above.
  • the halogen residue include fluorine, chlorine, bromine, and iodine.
  • optionally substituted means an aryl residue, a heteroaryl residue, or a Ci-6-alkyl residue that may be unsubstituted or may have 1 or more preferably 1 to 5 substituents, which are the same as or different from one another.
  • substituents include, halogen (fluorine, chlorine, bromine, iodine), hydroxyl, cyano, nitro, trifluoromethyl, carbamoyl, d ⁇ -acy! (e.g. acetyl, propionyl, isopropionyl), Ci-e-alkoxy (e.g.
  • Ci-e-alkyl e.g. methyl, ethyl, propyl, cyclopropyl, isopropyl, butyl, and tert.butyl
  • Ci- ⁇ -alkoxycarbonyl e.g. ones having 2 to 6 carbon atoms such as methoxycarbonyl, ethoxycarbonyl, and propoxycarbonyl
  • C 1 - 6 - alkanoyloxy e.g. ones having 2 to 6 carbon atoms such as acetyloxy, propionyloxy, isopropionyloxy
  • C ⁇ _ 4 -alky!thio e.g.
  • the compounds of formula (I) may exist as geometric and optical isomers and all isomers and mixtures thereof are included herein. Isomers may be separated by means of standard methods such as chromatographic techniques or fractionated crystallisation of e.g. suitable salts. It is to be understood that the heterocyclic moieties depicted throughout this application is capable of undergoing tautomerisation. Example of tautomerisation is given by the following example, thus:
  • the compounds according to the invention may optionally exist as pharmaceutically acceptable salts comprising acid addition salts or metal salts or - optionally alkylated - ammonium salts.
  • salts include the alkali metal or amine salts of 1 H- or 2H-tetrazoles of this invention, such as the sodium, potassium, Ci- ⁇ -alkylamine, di (Ci-e-alkyl) amine, tri (Ci-e-alkyl) amine and the four (4) corresponding omega-hydroxy analogues (e.g.
  • tetrazole derivatives and their salts thus obtained can be isolated and purified by known means of separation and purification such as concentration, concentration under reduced pressure, crystallisation, recrystaliisation, extraction and chromatography.
  • nitrile derivatives (II) used as starting materials in the method A of this invention can be produced by, for example, the following manner.
  • NMP N-methyl pyrrolidone
  • DMF dimethylformamide
  • THF tetrahydrofuran
  • acetone dibutyl ether, 2-butanone, methyl tert-butyl ether, methyl ethyl ketone, ethyl acetate or toluene
  • a base e.g. potassium carbonate or sodium hydride
  • a catalyst e.g. an alkali metal iodide, copper or a copper salt e.g. (CuCl, CuBr, Cul, or Cu 2 O) or in the case of a Mitsunobu reaction (for a review see, O.
  • a solvent such as triethylamine (TEA), methanol, ethanol or dimethylsulfoxid (DMSO) in the presence of a palladium catalyst, e.g. (Pd/C, Pd/AI 2 O 3 , Pd/BaSO 4 , Pd/Si0 2 or Pd(OAc) 2 (a Heck reaction) ) and a triaryl-phosphine catalyst as e.g. (triphenyl-phosphine or tri-o-tolyl-phosphine) at temperatures ranging from 50 °C to 150 °C for 1 to 60 hours.
  • a palladium catalyst e.g. (Pd/C, Pd/AI 2 O 3 , Pd/BaSO 4 , Pd/Si0 2 or Pd(OAc) 2 (a Heck reaction)
  • a triaryl-phosphine catalyst as e.g. (triphenyl-phosphine or tri-o-tolyl-phosphin
  • reaction may be carried out in a solvent such as methanol, ethanol, tetrahydrofuran (THF). toluene, N,N-dimethylfo ⁇ mamide (DMF) or dimethylsulfoxid (DMSO) in the presence of a base such as triethylamine, pyridine, piperidine, sodium hydride, sodium methoxide, sodium ethoxide, potassium tert-butoxide, lithium diisopropylamide at temperatures ranging from -50 °C to 150 °C for 1 to 60 hours.
  • a solvent such as methanol, ethanol, tetrahydrofuran (THF). toluene, N,N-dimethylfo ⁇ mamide (DMF) or dimethylsulfoxid (DMSO)
  • a base such as triethylamine, pyridine, piperidine, sodium hydride, sodium methoxide, sodium ethoxide, potassium tert-butoxide, lithium diis
  • the tetrazole group can, for example, be protected by a trityl group.
  • Introduction and removal of such groups is e.g. described in "Protective Groups in Organic Synthesis” T.W. Greene and P.G.M. Wuts, ed. Second edition (1991).
  • the compounds of the invention modulate the activity of protein tyrosine phosphatases or other molecules with phosphotyrosine recognition unit(s).
  • the compounds of the invention act as inhibitors of PTPases, e.g. protein tyrosine phosphatases involved in regulation of tyrosine kinase signalling pathways.
  • PTPases e.g. protein tyrosine phosphatases involved in regulation of tyrosine kinase signalling pathways.
  • Preferred embodiments include modulation of receptor-tyrosine kinase signalling pathways via interaction with regulatory PTPases, e.g. the signalling pathways of the insulin receptor, the IGF-I receptor and other members of the insulin receptor family, the EGF- receptor family, the platelet-derived growth factor receptor family, the nerve growth factor receptor family, the hepatocyte growth factor receptor family, the growth hormone receptor family and members of other receptor-type tyrosine kinase families.
  • regulatory PTPases e.g. the signalling pathways of the insulin receptor, the IGF-I receptor and other members of the insulin receptor family, the EGF- receptor family, the platelet-
  • Further preferred embodiments of the inventions is modulation of non-receptor tyrosine kinase signalling through modulation of regulatory PTPases, e.g. modulation of members of the Src kinase family.
  • PTPases e.g. modulation of members of the Src kinase family.
  • One type of preferred embodiments of the inventions relate to modulation of the activity of PTPases that negatively regulate signal transduction pathways.
  • Another type of preferred embodiments of the inventions relate to modulation of the activity of PTPases that positively regulate signal transduction pathways.
  • the compounds of the invention act as modulators of the active site of PTPases.
  • the compounds of the invention modulate the activity of PTPases via interaction with structures positioned outside of the active sites of the enzymes, preferably SH2 domains.
  • Further preferred embodiments include modulation of signal transduction pathways via binding of the compounds of the invention to SH2 domains or PTB domains of non-PTPase signalling molecules.
  • Other preferred embodiments include use of the compounds of the invention for modulation of cell-cell interactions as well as cell-matrix interactions.
  • the present invention include within its scope pharmaceutical compositions comprising, as an active ingredient, at least one of the compounds of formula (I) in association with a pharmaceutical carrier or diluent.
  • the pharmaceutical composition can comprise at least one of the compounds of formula (I) combined with compounds exhibiting a different activity, e.g. an antibiotic or other pharmacologically active material.
  • the compounds of the invention may be used as therapeuticals to inhibit of PTPases involved in regulation of the insulin receptor tyrosine kinase signalling pathway in patients with type I diabetes, type II diabetes, impaired glucose tolerance, insulin resistance, and obesity.
  • Further preferred embodiments include use of the compounds of the invention for treatment of disorders with general or specific dysfunction's of PTPase activity, e.g. proliferarive disorders such as psoriasis and neoplastic diseases.
  • the compounds of the invention may be used in pharmaceutical preparations for treatment of osteoporosis.
  • Preferred embodiments of the invention further include use of compound of formula (I) in pharmaceutical preparations to increase the secretion or action of growth hormone and its analogous or somatomedins including IGF-1 and IGF-2 by modulating the activity of PTPases or other signal transduction molecules with affinity for phosphotyrosine involved controlling or inducing the action of these hormones or any regulating molecule.
  • compounds of the invention can be administered for pu ⁇ oses of stimulating the release of growth hormone from the pituitary or increase its action on target tissues thereby leading to similar effects or uses as growth hormone itself.
  • growth hormone may be summarized as follows: stimulation of growth hormone release in the elderly; prevention of catabolic side effects of glucocorticoids; treatment of osteoporosis, stimulation of the immune system; treatment of retardation, acceleration of wound healing; accelerating bone fracture repair; treatment of growth retardation; treating renal failure or insufficiency resulting in growth retardation; treatment of physiological short stature including growth hormone deficient children and short stature associated with chronic illness; treatment of obesity and growth retardation associated with obesity; treating growth retardation associated with the Prader-Willi syndrome and Turner's syndrome; accelerating the recovery and reducing hospitalization of bum patients; treatment of intrauterine growth retardation, skeletal dysplasia, hypercortisolism and Cushings syndrome; induction of pulsatile growth hormone release; replacement of growth hormone in stressed patients; treatment of osteochondro-dysplasias, Noonans syndrome, schizophrenia, depressions, Alzheimer's disease, delayed wound healing and psychosocial deprivation; treatment of pulmonary dysfunction and ventilator dependency; attenuation
  • the compounds of the invention may be used in pharmaceutical preparations for treatment of various disorders of the immune system, either as a stimulant or suppressor of normal or perturbed immune functions, including autoimmune reactions. Further embodiments of the invention include use of the compounds of the invention for treatment of allergic reactions, e.g. asthma, dermal reactions, conjunctivitis.
  • allergic reactions e.g. asthma, dermal reactions, conjunctivitis.
  • compounds of the invention may be used in pharmaceutical preparations for prevention or induction of platelet aggregation.
  • compounds of the invention may be used in pharmaceutical preparations for treatment of infectious disorders.
  • the compounds of the invention may be used for treatment of infectious disorders caused by Yersinia and other bacteria as well as disorders caused by viruses or other micro-organisms.
  • Compounds of the invention may additionally be used for treatment or prevention of diseases in animals, including commercially important animals.
  • Also included in the present invention is a process for isolation of PTPases via affinity purification procedures based on tine use of immobilised compounds of the invention using procedures well-known to those skilled in the art.
  • the invention is further directed to a method for detecting the presence of PTPases in cell or in a subject comprising:
  • the invention further relates to analysis and identification of the specific functions of certain PTPases by modulating their activity by using compounds of the invention in cellular assay systems or in whole animals.
  • Signal transduction is a collective term used to define all cellular processes that follow the activation of a given cell or tissue.
  • Examples of signal transduction which are not intended to be in any way limiting to the scope of the invention claimed, are cellular events that are induced by polypeptide hormones and growth factors (e.g. insulin, insulin-like growth factors I and II, growth hormone, epidermal growth factor, platelet-derived growth factor), cytokines (e.g. inter-leukins), extracellular matrix components, and cell-cell interactions.
  • polypeptide hormones and growth factors e.g. insulin, insulin-like growth factors I and II, growth hormone, epidermal growth factor, platelet-derived growth factor
  • cytokines e.g. inter-leukins
  • Phosphotyrosine recognition units/tyrosine phosphate recognition units/pTyr recognition units are defined as areas or domains of proteins or glycoproteins that have affinity for molecules containing phosphorylated tyrosine residues (pTyr).
  • Examples of pTyr recognition units which are not intended to be in any way limiting to the scope of the invention claimed, are: PTPases, SH2 domains and PTB domains.
  • PTPases are defined as enzymes with the capacity to dephosphorylate pTyr-containing proteins or glycoproteins.
  • Examples of PTPases which are not intended to be in any way limiting to the scope of the invention claimed, are: 'classical' PTPases (intracellular PTPases (e.g. PTP1B, TC-PTP, PTP1C, PTP1D, PTPD1, PTPD2) and receptor-type PTPases (e.g. PTP ⁇ , PTP ⁇ , PTP ⁇ , PTP ⁇ , CD45, PTPK, PTP ⁇ ), dual specificty phosphatases (VH1, VHR, cdc25), LMW-PTPases or acid phosphatases.
  • intracellular PTPases e.g. PTP1B, TC-PTP, PTP1C, PTP1D, PTPD1, PTPD2
  • receptor-type PTPases e.g. PTP ⁇ , PTP ⁇ , PTP ⁇ , P
  • Modulation of cellular processes is defined as the capacity of compounds of the invention to 1) either increase or decrease ongoing, normal or abnormal, signal transduction, 2) initiate normal signal transduction, and 3) initiate abnormal signal transduction.
  • Modulation of pTyr-mediated signal transduction/modulation of the activity of molecules with pTyr recognition units is defined as the capacity of compounds of the invention to 1) increase or decrease the activity of proteins or glycoproteins with pTyr recognition units (e.g. PTPases, SH2 domains or PTB domains) or to 2) decrease or increase the association of a pTyr-containing molecule with a protein or glyco-protein with pTyr recognition units either via a direct action on the pTyr recognition site or via an indirect mechanism.
  • proteins or glycoproteins with pTyr recognition units e.g. PTPases, SH2 domains or PTB domains
  • Examples of modulation of pTyr-mediated signal transduction/modulation of the activity of molecules with pTyr recognition units are: a) inhibition of PTPase activity leading to either increased or decreased signal transduction of ongoing cellular processes; b) inhibition of PTPase activity leading to initiation of normal or abnormal cellular activity; c) stimulation of PTPase activity leading to either increased or decreased signal transduction of ongoing cellular processes; d) stimulation of PTPase activity leading to initiation of normal or abnormal cellular activity; e) inhibition of binding of SH2 domains or PTB domains to proteins or glycoproteins with pTyr leading to increase or decrease of ongoing cellular processes; f) inhibition of binding of SH2 domains or PTB domains to proteins or glycoproteins with pTyr leading to initiation of normal or abnormal cellular activity.
  • a subject is defined as any mammalian species, including humans.
  • dosage will vary depending on the compound of formula (I) employed, on the mode of administration and on the therapy desired. However, in general, satisfactory results are obtained with a dosage of from about 0.5 mg to about 1000 mg, preferably from about 1 mg to about 500 mg of compounds of formula (I), conveniently given from 1 to 5 times daily, optionally in sustained release form.
  • dosage forms suitable for oral administration comprise from about 0.5 mg to about 1000 mg, preferably from about 1 mg to about 500 mg of the compounds of formula (I) admixed with a pharmaceutical carrier or diluent.
  • the compounds of formula (I) may be administered in a pharmaceutically acceptable acid addition salt form or where possible as a metal or a Ci-e-alkylammonium salt. Such salt forms exhibit approximately the same order of activity as the free acid forms.
  • This invention also relates to pharmaceutical compositions comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and, usually, such compositions also contain a pharmaceutical earner or diluent.
  • the compositions containing the compounds of this invention may be prepared by conventional techniques and appear in conventional forms, for example capsules, tablets, solutions or suspensions.
  • the pharmaceutical earner employed may be a conventional solid or liquid carrier.
  • solid carriers are lactose, terra alba, sucrose, talc, gelatine, agar, pectin, acacia, magnesium stearate and stearic acid.
  • liquid carriers are syrup, peanut oil, olive oil and water.
  • the carrier or diluent may include any time delay material known to the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax.
  • the preparation can be tabletted, placed in a hard gelatine capsule in powder or pellet form or it can be in the form of a troche or lozenge.
  • the amount of solid earner will vary widely but will usually be from about 25 mg to about 1 g.
  • the preparation may be in the form of a syrup, emulsion, soft gelatin capsule or sterile injectable liquid such as an aqueous or non-aqueous liquid suspension or solution.
  • the compounds of this invention are dispensed in unit dosage form comprising 10- 200 mg of active ingredient in or together with a pharmaceutically acceptable carrier per unit dosage.
  • the dosage of the compounds according to this invention is 1-500 mg/day, e.g. about 100 mg per dose, when administered to patients, e.g. humans, as a drug.
  • a typical tablet which may be prepared by conventional tabletting techniques contains Core:
  • Active compound (as free compound 100 mg or salt thereof)
  • the route of administration may be any route which effectively transports the active compound to the appropriate or desired site of action, such as oral or parenteral e.g. rectal, transdermal, subcutaneous, intranasal, intramuscular, topical, intravenous, intraurethral, ophthalmic solution or an ointment, the oral route being preferred.
  • oral or parenteral e.g. rectal, transdermal, subcutaneous, intranasal, intramuscular, topical, intravenous, intraurethral, ophthalmic solution or an ointment, the oral route being preferred.
  • reaction mixture was quenched with water (250 ml) and extracted with diethyl ether (2 x 100 ml). The combined organic extracts were washed with saturated aqueous sodium chloride (2 x 80 ml), dried (MgSO 4 ), filtered and evaporated in vacuo affording 5.2 g (84%) of naphthalene-2-carbonyl isothiocyanate.
  • the cooled reaction mixture was quenched by addition of 1 N sodium hydroxide (150 ml) and diluted by ad ⁇ dition of diethyl ether.
  • the precipitate was filtered off, washed with water (2 x 10 ml) and diethyl ether (2 x 10 ml) and dried at 50 °C which af- forded 0.25 g (39%) of the title compound as a solid.
  • the solid was first washed with a mixture of ethyl acetate, heptane and dichloromethane (1 :1:6, 40 ml) and then washed with dichloromethane (20 ml). Drying in vacuo at 50 °C afforded 1.88 g (28%) of the titie compound as a solid.
  • Carbazole (8.25 g, 49 mmol) was dissolved in N,N'-dimethylformamide (100 ml). Under a atmosphere of nitrogen was added sodium hydride (2.56 g, 64 mmol of a 60% suspen- sion in mineral oil) in portion during 15 minutes. The mixture was then stirred at room temperature for 0.5 h. To the resulting mixture was added 4-phenylbenzylchloride (10 g, 49 mmol) in portions during 10 minutes. Then additional N,N'-dimethylformamide (100 ml) was added and the mixture was stirred at room temperature for 3.5 h.
  • Diethylaminosulfur trifluonde (2 2 ml, 8 2 mmol) was dissolved in dichloromethane (25 ml) and the solution was cooled to -70 °C and added dropwise to a solution of the above hydroxymethylphosphonic acid di-tert-butyl ester (4.0 g, 8 3 mmol) in dichloromethane (15 ml) at -70 °C. The mixture was stirred at -70 °C for 3 h and at room temperature for 20 h.
  • EXAMPLE 25 The PTP1B and PTP ⁇ cDNA was obtained by standard polymerase chain reaction technique using the Gene Amp Kit according to the manufacturer's instructions (Perkin Elmer/Cetus).
  • the oligonucleotide primers were designed according to published sequences (Chernoff et al., Proc. Natl. Acad. Sci. U.S.A. 87: 2735-2739 (1990); Krueger et al.EMBO J. 9: 3241-3252 (1990)) including convenient restriction nuclease sites to allow cloning into expression vectors.
  • the cDNA corresponding to the full-length sequence of PTP1B and the intracellular part of PTP ⁇ were introduced into the insect cell expression vector pVL1392.
  • the proteins were expressed according to standard procedures.
  • PTP1 B was semi-purified by ion exchange chromatography, and PTP ⁇ was purified to apparent homogeneity using a combination of ion exchange chromatography and gel filtration techniques using standard procedures.
  • TC-PTP and LAR domain 1 were obtained from New England Biolabs.
  • Yersinia PTP was a kind gift from J.E. Dixon, The University of Michigan, Ann Arbor, USA p- Nitrophenyl phosphate was purchased from Sigma and used without further purification.
  • p-Nitrophenyl phosphate is a general phosphatase substrate including a substrate for PTPases.
  • pNPP colourless
  • pNPP hydrolysed by a phosphatase to phosphate and p- nitrophenolate (yellow in alkaline solutions)
  • the enzyme reaction can be followed by measuring the optical density at 410 nm after adjusting the pH appropriately.
  • pNPP was used as general substrate to analyse the PTPase inhibitory capacity of the compounds of the invention.
  • the inhibiting effect of a compound is given by its Kj value, which expresses the concentration of inhibitor ( ⁇ M) in the reaction mixture necessary for a 50 percent reduction of the enzyme activity.
  • the Kj may be determined by a titration curve using several appropriately diluted solutions of the inhibitor or by using the following more simple formula, when the concentration of inhibitor is in large excess of the enzyme concentration:
  • Ki lo x E/(Eo - E) where l 0 is the concentration of inhibitor ( ⁇ M) added to the reaction mixture, E is the activity of the enzyme in the reaction mixture containing the inhibitor, and Eo is the enzyme activity in a corresponding control reaction mixture without the inhibitor.
  • the Kj values of inhibitors towards PTP1B were measured as follows. In all cases the inhibiting effects were determined at pH 5.5 and at 37 °C with a reaction time of 60 minutes.
  • reaction mixtures were:
  • the substrate solution contained 0.2 M acetate buffer, pH 5.5, 11 mM p-nitrophenyl phosphate, 5.5 mM dithiotreitol.
  • the reaction was stopped by addition of 4 ml 0.2 N NaOH, and the enzyme activity was determined by measuring the release of p-nitrophenol at 410 nm.
  • the inhibiting effect was calculated as shown above.
  • the Kj values of inhibitors towards TdPTP, LAR domainl, PTP ⁇ domain 1+2, and Yersinia PTP were measured essentially as described for PTP1B with the exception that all reactions were carried out in 96-wells microtiter plates. In all cases the inhibiting effects were determined at pH 5.5 and at room temperature with a reaction time of 15 minutes.
  • the reaction mixtures were:
  • the final concentrations 0.2 M acetate buffer, pH 5.5, 5 mM p-nitrophenyl phosphate, 5 mM dithiotreitol.
  • the reaction was stopped by addition of 100 ⁇ l 0.4 N NaOH, and the enzyme activity was determined by measuring the release of p-nitrophenol at 405 nm. The inhibiting effect was calculated as shown above.

Abstract

The present invention relates to novel organic compounds, to methods for their preparation, to compositions containing them, to their use for treatment of human and animal disorders, to their use for purification of proteins or glycoproteins, and to their use in diagnosis. The invention relates to modulation of the activity of molecules with phospho-tyrosine recognition units, including protein tyrosine phosphatases (PTPases) and proteins with Src-homology-2 domains, in in vitro systems, micro-organisms, eukaryotic cells, whole animals and human beings. The novel organic compounds are compounds of the general formula (I) (L)n--Ar1--R1--A, wherein (L)n, n, Ar1, R1 and A are defined as defined in the application.

Description

MODULATORS OF MOLECULES WITH PHOSPHOTYROSINE RECOGNITION UNITS
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to novel organic compounds, to methods for their preparation, to compositions containing them, to their use for treatment of human and animal disorders, to their use for purification of proteins or glycoproteins, and to their use in diagnosis. The invention relates to modulation of the activity of molecules with phospho-tyrosine recognition units, including protein tyrosine phosphatases (PTPases) and proteins with Src-homology-2 domains, in in vitro systems, micro-organisms, eukaryotic cells, whole animals and human beings.
Background of the invention
Phosphorylation of proteins is a fundamental mechanism for regulation of many cellular processes. Although protein phosphorylation at serine and threonine residues is quantitatively dominating in eukaryotic cells, reversible tyrosine phosphorylation seems to play a pivotal role in regulation of cell growth and differentiation as well as in neoplastic transformation (Hunter, Cell 80: 225-236 (1995); Schlessinger and Ullrich, Neuron 9: 383- 391 (1992); Cantleyet al., Ce//64: 281-302 (1991); Ullrich and Schlessinger, Cell 61 :203- 212 (1990); Hunter, Curr. Opin. Cell. Biol. 1: 1168-1181 (1989)); Hunter and Cooper, Annu. Rev. Biochem. 54: 897-930 (1985)).
The regulation of protein tyrosine phosphorylation in vivo is mediated by the opposing actions of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPases). The level of protein tyrosine phosphorylation of cellular proteins is determined by the balanced activities of PTKs and PTPase (Hunter, 1995, supra). PTPases - an overview
The protein phosphatases are composed of at least two separate and distinct families (Hunter, T., Cell 58: 1013-1016 (1989)) the protein serine/threonine phosphatases and the PTPases.
The PTPases are a family of enzymes that can be classified into two groups: a) intracellular or nontransmembrane PTPases and b) receptor-type or transmembrane PTPases.
Intracellular PTPases: All known intracellular type PTPases contain a single conserved catalytic phosphatase domain consisting of 220-240 amino acid residues. The regions outside the PTPase domains are believed to play important roles in localizing the intracellular PTPases subcellularly (Maura, LJ. and Dixon, J.E. TIBS 19: 151-155 (1994)). The first intracellular PTPase to be purified and characterized was PTP1 B which was isolated from human placenta (Tonks etal., J. Biol. Chem. 263: 6722-6730 (1988)). Shortly after, PTP1B was cloned (Charbonneau et al., Proc. Natl. Acad. Sci. USA 86: 5252-5256 (1989); Chemoff et al., Proc. Natl. Acad. Sci. USA 87: 2735-2789 (1989)). Other examples of intracellular PTPases include (1) T-cell PTPase (Cool et al. Proc. Natl. Acad. Sci. USA 86: 5257-5261 (1989)), (2) rat brain PTPase (Guan ef al., Proc. Natl. Acad. Sci. USA 87:1501-1502
(1990)), (3) neuronal phosphatase STEP (Lombroso etal., Proc. Natl. Acad. Sci. USA 88: 7242-7246 (1991)), (4) ezrin-domain containing PTPases: PTPMEG1 (Guef al, Proc. Natl. Acad. Sci. USA 88: 5867-57871 (1991)), PTPH1 Yang and Tonks, Proc. Natl. Acad. Sci. USA 88: 5949-5953 (1991), PTPD1 and PTPD2 (Møller ef al., Proc. Natl. Acad. Sci. USA 91: 7477-7481 (1994)), FAP-1/BAS (Sato ef al., Science 268: 411-415 (1995); Banville ef al., J. Biol. Chem. 269: 22320-22327 (1994); Maekawa et al, FEBS Letters 337: 200-206 (1994)), and SH2 domain containing PTPases: PTP1C/SH-PTP1 (Plutzky ef al., Proc. Natl. Acad. Sci. USA 89: 1123-1127 (1992); Shen et al., Nature Lond. 352: 736-739 (1991)) and PTP1D/Syp/SH-PTP2 (Vogel etal., Science 259: 1611-1614 (1993); Feng et al., Science 259: 1607-1611 (1993); Bastein et al., Biochem. Biophys. Res. Comm. 196: 124-133 (1993)). Low molecular weight phosphotyrosine-protein phosphatase (LMW-PTPase) shows very little sequence identity to the intracellular PTPases described above. However, this enzyme belongs to the PTPase family due to the following characteristics: (i) it possesses the PTPase active site motif: Cys-Xxx-Xxx-Xxx-Xxx-Xxx-Arg (Cirri etal, Eur. J. Biochem. 214: 647-657 (1993)); (ii) this Cys residue forms a phospho-intermediate during the catalytic reaction similar to the situation with 'classical' PTPases (Cirri et al., supra; Chiarugi etal, FEBS Lett. 310: 9-12 (1992)); (iii) the overall folding of the molecule shows a suφrising degree of similarity to that of PTP1B and Yersinia PTP (Su et al., Nature 370: 575-578 (1994)).
Receptor-type PTPases consist of a) a putative ligand-binding extracellular domain, b) a transmembrane segment, and c) an intracellular catalytic region. The structures and sizes of the putative ligand-binding extracellular domains of receptor-type PTPases are quite divergent. In contrast, the intracellular catalytic regions of receptor-type PTPases are very homologous to each other and to the intracellular PTPases. Most receptor-type PTPases have two tandemly duplicated catalytic PTPase domains.
The first receptor-type PTPases to be identified were (1) CD45/LCA (Ralph, S.J., EMBO J. 6: 1251-1257 (1987)) and (2) LAR (Streuli et al., J. Exp. Med. 168: 1523-1530 (1988)) that were recognized to belong to this class of enzymes based on homology to PTP1B (Charbonneau etal, Proc. Natl. Acad. Sci. USA 86: 5252-5256 (1989)). CD45 is a family of high molecular weight glycoproteins and is one of the most abundant leukocyte cell surface glycoproteins and appears to be exclusively expressed upon cells of the hematopoietic system (Trowbridge and Thomas, Ann. Rev. Immunol. 12: 85-116 (1994)).
The identification of CD45 and LAR as members of the PTPase family was quickly followed by identification and cloning of several different members of the receptor-type PTPase group. Thus, 5 different PTPases, (3) PTPα, (4) PTPβ, (5) PTPδ, (6) PTPε, and (7) PTPζ, were identified in one early study (Krueger ef al., EMBO J. 9: 3241-3252 (1990)). Other examples of receptor-type PTPases include (8) PTPγ (Barnea ef al., Mol. Cell. Biol. 13: 1497-1506 (1995)) which, like PTPζ (Krueger and Saito, Proc. Natl. Acad. Sci. USA 89: 7417-7421 (1992)) contains a carbonic anhydrase-like domain in the extracellular region, (9) PTPμ (Gebbink ef al., FEBS Letters 290: 123-130 (1991), (10) PTPK (Jiang et al., Mol. Cell. Biol. 13: 2942-2951 (1993)). Based on structural differences the receptor-type PTPases may be classified into subtypes (Fischer ef al., Science 253: 401-406 (1991)): (I) CD45; (II) LAR, PTPδ, (11) PTPσ ; (III) PTPβ, (12) SAP-1 (Matozaki et al., J. Biol. Chem. 269: 2075-2081 (1994)), (13) PTP-U2/GLEPP1 (Seimiya etal., Oncogene 10: 1731-1738 (1995); (Thomas ef al., J. Biol. Chem. 269: 19953-19962 (1994)), and (14) DEP-1; (IV) PTPα,_PTPε. All receptor-type PTPases except Type IV contain two PTPase domains. Novel PTPases are continously identified, and it is anticipated that more than 500 different species will be found in the human genome, i.e. close to the predicted size of the protein tyrosine kinase superfamily (Hanks and Hunter, FASEB J. 9: 576-596 (1995)).
PTPases are the biological counterparts to protein tyrosine kinases (PTKs). Therefore, one important function of PTPases is to control, down-regulate, the activity of PTKs. However, a more complex picture of the function of PTPases now emerges. Several studies have shown that some PTPases may actually act as positive mediators of cellular signalling. As an example, the SH2 domain-containing PTP1D seems to act as a positive mediator in insulin- stimulated Ras activation (Noguchi etal, Mol. Cell. Biol. 14: 6674-6682 (1994)) and of growth factor-induced mitogenic signal transduction (Xiao ef al, J. Biol. Chem. 269: 21244- 21248 (1994)), whereas the homologous PTP1C seems to act as a negative regulator of growth factor-stimulated proliferation (Bignon and Siminovitch, Clin. Immunol. Immunopathol. 73: 168-179 (1994)). Another example of PTPases as positive regulators has been provided by studies designed to define the activation of the Src-family of tyrosine kinases. In particular, several lines of evidence indicate that CD45 is positively regulating the activation of hematopoietic cells, possibly througn dephosphorylation of the C-terminal tyrosine of Fyn and Lck (Chan et al., Annu. Rev. Immunol. 12: 555-592 (1994)).
Dual specificity protein tyrosine phosphatases (dsPTPases) define a subclass within the PTPases family that can hydrolyze phosphate from phosphortyrosine as well as from phosphor-serine/threonine. dsPTPases contain the signature sequence of PTPases: His- Cys-Xxx-Xxx-Gly-Xxx-Xxx-Arg. At least three dsPTPases have been shown to dephosphorylate and inactivate extracellular signal-regulated kinase (ERKs)/mitogen- activated protein kinase (MAPK): MAPK phosphatase (CL100, 3CH134) (Charles etal, Proc. Natl. Acad. Sci. USA 90: 5292-5296 (1993)); PAC-1 (Ward et al., Nature 367: 651-654 (1994)); rVH6 (Mourey ef al, J. Biol. Chem. 271: 3795-3802 (1996)). Transcription of dsPTPases are induced by different stimuli, e.g. oxidative stress or heat shock (Ishibashi et al., J. Biol. Chem. 269: 29897-29902 (1994); Keyse and Emslie, Nature 359: 644-647 (1992)). Further, they may be involved in regulation of the cell cycle: cdc25 (Millar and Russell, Cell 68: 407-410 (1992)); KAP (Hannon etal, Proc. Natl. Acad. Sci. USA 91: 1731- 1735 (1994)). Interestingly, tyrosine dephosphorylation of cdc2 by a dual specific phosphatase, cdc25, is required for induction of mitosis in yeast (review by Walton and Dixon, Annu. Rev. Biochem. 62: 101-120 (1993)).
PTPase specificity
Several studies have addressed the question of PTPase specificity using synthetic peptides and provided important insight with respect to primary structural sequence requirements for substrate recognition (Ramachandran etal., Biochemistry 31: 4232-4238 (1992); Cho, H. et al., Biochemistry 31: 133-138 (1992); Zhang, Z.-Y etal, Proc. Natl. Acad. Sci. USA 90: 4446-4450 (1993); Zhang, Z.-Y. et al, Biochemistry 33: 2285-2290 (1994)). However, an obvious limitation of this approach is the lack of defined three-dimensional structure of the peptide analogs. Likewise, the PTPases utilized for these analyses are removed from their natural environment. Since at least part of the PTPase specificity seems to be conveyed by a defined subcellular localization (Mauro and Dixon, TIBS 19: 151-155 (1994)), it is essential that such studies are complemented with measurements of PTPase activity towards cellular substrates in intact cells.
Phosphotyrosine recognition in signal transduction
Hormones, growth factors, cytokines, antigens, extracellular matrix components as well as molecules positioned at the cell surface induce signal transduction by binding to specific cell surface structures or receptors on target cells (reviewed in Pawson, Nature 373: 573-580 (1995)). The resulting cellular signal is often mediated through a series of phosphorylation and dephosphorylation reactions on tyrosine residues of signalling molecules. To allow efficient and selective signalling, several recognition units for phosphotyrosine (pTyr) have developed during evolution: a) PTPases; b) Src-homology-2 (SH2) domains; c) pTyr-binding (PTB) domains. As described above, the recognition of pTyr by PTPases leads to dephosphorylation with concommitant dissociation from the molecular target. Dephosphorylation may either lead to upregulation or downregulation of the signal. In contrast, SH2 domains and PTB domains primarily act as docking molecules with little or no catalytic activity. In other words, tyrosine phosphorylated proteins have the capacity to bind other proteins containing SH2 domains or PTB domains thereby controlling the subcellular location of signalling molecules. There appears to be a significant degree of selectivity in SH2 domain recognition of pTyr and their surroundings. Thus, SH2 domains from the Src kinase family bind the peptide pTyr-Glu-Glu-lle in a relatively selective manner, whereas the PTPD1 seems to recognize at least five, primarily hydrophobic residues C-terminal to the pTyr (Pawson, supra). Certain PTPase domains, in particular the C-terminal domain of some receptor-type PTPases, seem to have little or no catalytic activity. It may be hypothesized that these domains have a function as pTyr recognition units similar to SH2 domains and PTB domains. Inhibition of signal transduction processes could, in principle, be achieved by using non-hydrolyzable pTyr-containing peptides with preferential affinity for specific PTPases, SH2 domains or PTB domains. However, due to the lack of efficient bioavailabihty of peptides there is a need for development of either peptidomimetics or novel small molecules with preferential binding to pTyr recognition units of specific cellular targets. Such selective compounds can either initiate, increase or decrease defined signal transduction processes.
PTPases: Inhibitors
In an early study, vanadate was found to inhibit protein-tyrosine phosphatases in mammalian cells with a concomitant increase in the level of phosphotyrosine in cellular proteins leading to transformation (Klariund, Cell 41: 707-717 (1985)). Vanadium-based phosphatase inhibitors are relatively unspecific. Therefore, to assess the importance of specific structures on PTPase activity more selective inhibitors are needed. One possibility for obtaining selective PTPase inhibitors would be through design of different ancillary ligands for peroxovanadium-based compounds (Posner et al., J. Biol. Chem. 269: 4596-4604 (1994)). Another avenue taken by several investigators has been to incoφorate nonhydrolyzable tyrosine phosphate analogs into specific peptide substrates: (1) phosphonomethyl phenylalanine (Zhang ef al, Biochemistry 33: 2285-2290 (1994)); (2) difluorophosphono- methyl phenylalanine Burk etal., Synthesis 11: 1019-1020 (1991)); (3) L-O-malonyltyrosine (Kole et al., Biochem. Biophys. Res. Commun. 209: 817-822 (1995)); (4) cinnamic acid (Moran et al, J. Am. Chem. Soc. 117: 10787-10788 (1995); Cao ef al, Bioorganic Med. Chem. Lett. 5: 2953-2958 (1995)); (5) sulfotyrosyl (Liotta etal, J. Biol. Chem. 269: 22996- 23001 (1994)). A suφrising degree of selectivity is observed with simple peptide analogs containing phosphonodifluoromethyl phenylalanine as a substitute for tyrosine (Chen etal, Biochem. Biophys. Res. Commun. 216: 976-984 (1995)). Important information has further been obtained with synthetic peptides containing sulfotyrosyl residues. A synthetic peptide corresponding to the amino acid sequence of a defined loop of the insulin receptor tyrosine kinase, Thr-Arg-Asp-lle-Xxx-Glu-Thr-Asp-Xxx-Xxx-Arg-Lys (where Xxx denotes sulfotyrosyl), acts as a PTPase inhibitor (Liotta etal, 1994, supra). More importantly, this peptide, when tagged with stearic acid can penetrate cells, and stimulate the action of insulin (Liotta et al, 1994, supra).
PTPases: the insulin receptor signalling pathway/diabetes
Insulin is an important regulator of different metabolic processes and plays a key role in the control of blood glucose. Defects related to its synthesis or signalling lead to diabetes mellitus. Binding of insulin to its receptor causes rapid (auto) phosphorylation of several tyrosine residues in the intracellular part of the β-subunit. Three closely positioned tyrosine residues (the tyrosine-1150 domain) must all be phosphorylated to obtain full activity of the insulin receptor tyrosine kinase (IRTK) which transmits the signal further downstream by tyrosine phosphorylation of other cellular substrates, including insulin receptor substrate-1 (IRS-1) (Wilden et al, J. Biol. Chem. 267: 16660-16668 (1992); Myers and White, Diabetes 42: 643-650 (1993); Lee and Pilch, Am. J. Physiol. 266: C319-C334 (1994); White et al, J. Biol. Chem. 263: 2969-2980 (1988)). The structural basis for the function of the tyrosine- triplet has been provided by recent X-ray crystallographic studies of IRTK that showed tyrosine-1150 to be autoinhibitory in its unphosphorylated state (Hubbard ef a/., Nature 372: 746-754 (1994)).
Several studies clearly indicate that the activity of the auto-phosphorylated IRTK can be reversed by dephosphorylation in vitro (reviewed in Goldstein, Receptor 3: 1-15 (1993); Mooney and Anderson, J. Biol. Chem. 264: 6850-6857 (1989)), with the tri-phosphorylated tyrosine-1150 domain being the most sensitive target for protein-tyrosine phosphatases (PTPases) as compared to the di- and mono- phosphorylated forms (King et al., Biochem. J. 275: 413-418 (1991)). It is, therefore, tempting to speculate that this tyrosine-triplet functions as a control switch of IRTK activity. Indeed, the IRTK appears to be tightly regulated by PTP- mediated dephosphorylation in vivo (Khan et al., J. Biol. Chem. 264: 12931-12940 (1989); Faure et al., J. Biol. Chem. 267: 11215-11221 (1992); Rothenberg et al, J. Biol. Chem. 266: 8302-8311 (1991)). The intimate coupling of PTPases to the insulin signalling pathway is further evidenced by the finding that insulin differentially regulates PTPase activity in rat hepatoma cells (Meyerovitch et al., Biochemistry 37: 10338-10344 (1992)) and in livers from alloxan diabetic rats (Boylan et al, J. Clin. Invest. 90: 174-179 (1992)).
Relatively little is known about the identity of the PTPases involved in IRTK regulation. However, the existence of PTPases with activity towards the insulin receptor can be demonstrated as indicated above. Further, when the strong PTPase-inhibitor pervanadate is added to whole cells an almost full insulin response can be obtained in adipocytes (Fantus ef a/., Bi∞hemistry 28: 8864-8871 (1989); Eriksson et al, Diabetologia 39: 235-242 (1995)) and skeletal muscle (Leighton ef al., Bi∞hem. J. 276: 289-292 (1991)). In addition, recent studies show that a new class of peroxovanadium compounds act as potent hypoglycemic compounds in vivo (Posner ef al.,supra). Two of these compounds were demonstrated to be more potent inhibitors of dephosphorylation of the insulin receptor than of the EGF-receptor.
It was recently found that the ubiquitously expressed SH2 domain containing PTPase, PTP1D (Vogel etal., 1993, supra), associates with and dephosphorylates IRS-1, but apparently not the IR itself (Kuhne ef al., J. Biol. Chem. 268: 11479-11481 (1993); (Kuhne et al., J. Biol. Chem. 269: 15833-15837 (1994)). Previous studies suggest that the PTPases responsible for IRTK regulation belong to the class of membrane-associated (Faure etal., J. Biol. Chem. 267: 11215-11221 (1992)) and glycosylated molecules (Haring et al, Biochemistry 23: 3298-3306 (1984); Sale, Adv. Prot. Phosphatases 6: 159-186 (1991)). Hashimoto etal. have proposed that LAR might play a role in the physiological regulation of insulin receptors in intact cells (Hashimoto et al, J. Biol. Chem. 267: 13811-13814 (1992)). Their conclusion was reached by comparing the rate of dephosphorylation/inactivation of purified IR using recombinant PTP1B as well as the cytoplasmic domains of LAR and PTPα. Antisense inhibition was recently used to study the effect of LAR on insulin signalling in a rat hepatoma cell line (Kulas et al, J. Biol. Chem. 270: 2435-2438 (1995)). A suppression of LAR protein levels by about 60 percent was paralleled by an approximately 150 percent increase in insulin-induced auto-phosphorylation. However, only a modest 35 percent increase in IRTK activity was observed, whereas the insulin- dependent phosphatidylinositol 3-kinase (PI 3-kinase) activity was significantly increased by 350 percent. Reduced LAR levels did not alter the basal level of IRTK tyrosine phosphorylation or activity. The authors speculate that LAR could specifically dephosphorylate tyrosine residues that are critical for PI 3-kinase activation either on the insulin receptor itself or on a downstream substrate.
While previous reports indicate a role of PTPα in signal transduction through src activation (Zheng ef al, Nature 359: 336-339 (1992); den Hertog ef a/., EMBO J. 12: 3789-3798
(1993)) and interaction with GRB-2 (den Hertog etal, EMBO J. 13: 3020-3032 (1994); Su ef al, J. Biol. Chem. 269: 18731-18734 (1994)), a recent study suggests a function for this phosphatase and its close relative PTPε as negative regulators of the insulin receptor signal (Møller et al, 1995 supra). This study also indicates that receptor-like PTPases play a significant role in regulating the IRTK, whereas intracellular PTPases seem to have little, if any, activity towards the insulin receptor. While it appears that the target of the negative regulatory activity of PTPases α and ε is the receptor itself, the downmodulating effect of the intracellular TC-PTP seems to be due to a downstream function in the IR-activated signal. Although PTP1B and TC-PTP are closely related, PTP1B had only little influence on the phosphorylation pattern of insulin-treated cells. Both PTPases have distinct structural features that determine their subcellular localization and thereby their access to defined cellular substrates (Frangione ef al., Cell 68: 545-560 (1992); Faure and Posner, Glia 9: 311- 314 (1993)). Therefore, the tack of activity of PTP1B and TC-PTP towards the IRTK may, at least in part, be explained by the fact that they do not co-localize with the activated insulin receptor. In support of this view, PTP1B and TC-PTP have been excluded as candidates for the IR-associated PTPases in hepatocytes based on subcellular localization studies (Faure etal, J. Biol. Chem. 267: 11215-11221 (1992)).
The transmembrane PTPase CD45, which is believed to be hematopoietic cell-specific, was in a recent study found to negatively regulate the insulin receptor tyrosine kinase in the human multiple myeloma cell line U266 (Kulas ef al, J. Biol. Chem. 271: 755-760 (1996)).
PTPases: somatostatin
Somatostatin inhibits several biological functions including cellular proliferation (Lamberts ef al, Molec. Endocrinol. 8: 1289-1297 (1994)). While part of the antiproliferative activities of somatostatin are secondary to its inhibition of hormone and growth factor secretion (e.g. growth hormone and epidermal growth factor), other antiproliferative effects of somatostatin are due to a direct effect on the target cells. As an example, somatostatin analogs inhibit the growth of pancreatic cancer presumably via stimulation of a single PTPase, or a subset of PTPases, rather than a general activation of PTPase levels in the cells (Liebow ef a/., Proc. Natl. Acad. Sci. USA 86: 2003-2007 (1989); Colas etal., Eur. J. Biochem. 207: 1017-1024 (1992)). In a recent study it was found that somatostatin stimulation of somatostatin receptors SSTR1, but not SSTR2, stably expressed in CHO-K1 cells can stimulate PTPase activity and that this stimulation is pertussis toxin-sensitive. Whether the inhibitory effect of somatostatin on hormone and growth factor secretion is caused by a similar stimulation of PTPase activity in hormone producing cells remains to be determined.
PTPases: the immune svstem/autoimmunity
Several studies suggest that the receptor-type PTPase CD45 plays a critical role not only for initiation of T cell activation, but also for maintaining the T cell receptor-mediated signalling cascade. These studies are reviewed in: Weiss A., Ann. Rev. Genet. 25: 487-510 (1991); Chan et al, Annu. Rev. Immunol. 12: 555-592 (1994); Trowbridge and Thomas, Annu. Rev. Immunol. 12: 85-116 (1994).
The exact function of CD45 in lymphocyte activation is currently under intense investigation in many laboratories. Several studies suggest that the PTPase activity of CD45 plays a role in the activation of Lck, a lymphocyte-specific member of the Src family protein-tyrosine kinase (Mustelin et al, Proc. Natl. Acad. Sci. USA 86: 6302-6306 (1989); Ostergaard etal, Proc. Natl. Acad. Sci. USA 86: 8959-8963 (1989)). These authors hypothesized that the phosphatase activity of CD45 activates Lck by dephosphorylation of a C-terminal tyrosine residue, which may, in turn, be related to T-cell activation. In a recent study it was found that recombinant p56fck specifically associates with recombinant CD45 cytoplasmic domain protein, but not to the cytoplasmic domain of the related PTPα (Ng et al, J. Biol. Chem. 271: 1295-1300 (1996)). The p56fck-CD45 interaction seems to be mediated via a nonconventional SH2 domain interaction not requiring phosphotyrosine. In immature B cells, another member of the Src family protein-tyrosine kinases, Fyn, seems to be a selective substrate for CD45 compared to Lck and Syk (Katagiri et al., J. Biol. Chem. 270: 27987- 27990 (1995)).
HePTP, a hematopoietic cell specific PTPase, is induced after activation of resting T cells and may play a role in late T cell activation or as a negative regulator of T cell responses (Zanke et al., Eur. J. Immunol. 22: 235-239 (1992)). Likewise, the hematopoietic cell specific PTP1C seems to act as a negative regulator and play an essential role in immune cell development In accordance with the above-mentioned important function of CD45, HePTP and PTP1C, selective PTPase inhibitors may be attractive drug candidates both as immunosuppressors and as immunostimulants. One recent study illustrates the potential of PTPase inhibitors as immunmodulators by demonstrating the capacity of the vanadium- based PTPase inhibitor, BMLOV, to induce apparent B cell selective apoptosis compared to T cells (Schieven etal, J. Biol. Chem. 270: 20824-20831 (1995)). PTPases: cell-cell interactions/cancer
Focal adhesion plaques, an in vitro phenomenon in which specific contact points are formed when fibroblasts grow on appropriate substrates, seem to mimic, at least in part, cells and their natural surroundings. Several focal adhesion proteins are phosphorylated on tyrosine residues when fibroblasts adhere to and spread on extracellular matrix (Gumbiner, Neuron 11, 551-564 (1993)). However, aberrant tyrosine phosphorylation of these proteins can lead to cellular transformation. The intimate association between PTPases and focal adhesions is supported by the finding of several intracellular PTPases with ezrin-like N-terminal domains, e.g. PTPMEG1 (Gu ef a/., Proc. Natl. Acad. Sci. USA 88: 5867-5871 (1991)), PTPH1 (Yang and Tonks, Proc. Natl. Acad. Sci. USA 88: 5949-5953 (1991)) and PTPD1 (Møller et al, Proc. Natl. Acad. Sci. USA 91: 7477-7481 (1994)). The ezrin-like domain show similarity to several proteins that are believed to act as links between the cell membrane and the cytoskeleton. PTPD1 was found to be phosphorylated by and associated with c-src in vitro and is hypothesized to be involved in the regulation of phosphorylation of focal adhesions (Møller et al., supra).
PTPases may oppose the action of tyrosine kinases, including those responsible for phosphorylation of focal adhesion proteins, and may therefore function as natural inhibitors of transf ormation. TC-PTP, and especially the truncated form of this enzyme (Cool et al, Proc. Natl. Acad. Sci. USA 87: 7280-7284 (1990)), can inhibit the transforming activity of v- erb and (Lammers ef al., J. Biol. Chem. 268: 22456-22462 (1993); Zander et al, Oncogene 8: 1175-1182 (1993)). Moreover, it was found that transformation by the oncogenic form of the HER2/neu gene was suppressed in NIH 3T3 fribroblasts overexpressing PTP1 B (Brown-Shimer ef al, Cancer Res. 52: 478-482 (1992)).
The expression level of PTP1B was found to be increased in a mammary cell line transformed with neu (Zhay ef al., Can∞rRes. 53: 2272-2278 (1993)). The intimate relationship between tyrosine kinases and PTPases in the development of cancer is further evidenced by the recent finding that PTPε is highly expressed in murine mammary tumors in transgenic mice over-expressing c-neu and v-Ha-ras, but not c-myc or int-2 (Elson and Leder, J. Biol. Chem. 270: 26116-26122 (1995)). Further, the human gene encoding PTPγ was mapped to 3p21, a chromosomal region which is frequently deleted in renal and lung carcinomas (LaForgia et al, Proc. Natl. Acad. Sci. USA 88: 5036-5040 (1991)).
In this context, it seems significant that PTPases appear to be involved in controlling the growth of fibroblasts. In a recent study it was found that Swiss 3T3 cells harvested at high density contain a membrane-associated PTPase whose activity on an average is 8-fold higher than that of cells harvested at low or medium density (Pallen and Tong, Proc. Natl. Acad. Sci. USA 88: 6996-7000 (1991)). It was hypothesized by the authors that density- dependent inhibition of cell growth involves the regulated elevation of the activity of the PTPase(s) in question. In accordance with this view, a novel membrane-bound, receptor- type PTPase, DEP-1, showed enhanced (>=10-fold) expression levels with increasing cell density of WI-38 human embryonic lung fibroblasts and in the AG1518 fibroblast cell line (Ostman etal., Proc. Natl. Acad. Sci. USA 91: 9680-9684 (1994)).
Two closely related receptor-type PTPases, PTPK and PTPμ, can mediate homophilic cell- cell interaction when expressed in non-adherent insect cells, suggesting that these PTPases might have a normal physiological function in cell-to-cell signalling (Gebbink et al, J. Biol. Chem. 268: 16101-16104 (1993); Brady-Kalnay et al., J. Cell Biol. 122: 961-972 (1993); Sap etal, Mol. Cell. Biol. 14: 1-9 (1994)). Interestingly, PTPK and PTPμ do not interact with each other, despite their structural similarity (Zondag etal, J. Biol. Chem. 270: 14247-14250 (1995)). From the studies described above it is apparent that PTPases may play an important role in regulating normal cell growth. However, as pointed out above, recent studies indicate that PTPases may also function as positive mediators of intracellular signalling and thereby induce or enhance mitogenic responses. Increased activity of certain PTPases might therefore result in cellular transformation and tumor formation. Indeed, in one study over-expression of PTPα was found to lead to transformation of rat embryo fibroblasts (Zheng, supra). In addition, a novel PTP, SAP-1, was found to be highly expressed in pancreatic and colorectal cancer cells. SAP-1 is mapped to chromosome 19 region q13.4 and might be related to carcinoembryonic antigen mapped to 19q13.2 (Uchida et al, J. Biol. Chem. 269: 12220-12228 (1994)). Further, the dsPTPase, cdc25, dephosphorylates cdc2 at Thr14/Tyr-15 and thereby functions as positive regulator of mitosis (reviewed by Hunter, Cell 80: 225-236 (1995)). Inhibitors of specific PTPases are therefore likely to be of significant therapeutic value in the treatment of certain forms of cancer.
PTPases: platelet aggregation
Recent studies indicate that PTPases are centrally involved in platelet aggregation. Agonist- induced platelet activation results in calpain-catalyzed cleavage of PTP1 B with a concomitant 2-fold stimulation of PTPase activity (Frangioni et al, EMBO J. 12: 4843-4856 (1993)). The cleavage of PTP1 B leads to subcellular relocation of the enzyme and correlates with the transition from reversible to irreversible platelet aggregation in platelet-rich plasma. In addition, the SH2 domain containing PTPase, PTP1C/SH-PTP1, was found to translocate to the cytoskeleton in platelets after thrombin stimulation in an aggregation-dependent manner (Li et al, FEBS Lett. 343: 89-93 (1994)).
Although some details in the above two studies were recently questioned there is over-all agreement that PTP1 B and PTP1C play significant functional roles in platelet aggregation (Ezumi ef a/., J. Biol. Chem. 270: 11927-11934 (1995)). In accordance with these observations, treatment of platelets with the PTPase inhibitor pervanadate leads to significant increase in tyrosine phosphorylation, secretion and aggregation (Pumiglia et al, Biochem. J. 286: 441-449 (1992)).
PTPases: osteoporosis
The rate of bone formation is determined by the number and the activity of osteoblasts, which in term are determined by the rate of proliferation and differentiation of osteoblas progenitor cells, respectively. Histomoφhometric studies indicate that the osteoblast number is the primary determinant of the rate of bone formation in humans (Gruber ef a/., Mineral Electrolyte Metab. 12: 246-254 (1987); reviewed in Lau ef a/., Biochem. J. 257: 23-36 (1989)). Acid phosphatases/PTPases may be involved in negative regulation of osteoblast proliferation. Thus, fluoride, which has phosphatase inhibitory activity, has been found to increase spinal bone density in osteoporotics by increasing osteoblast proliferation (Lau et al., supra). Consistent with this observation, an osteoblastic acid phosphatase with PTPase activity was found to be highly sensitive to mitogenic concentrations of fluoride (Lau ef al, J. Biol. Chem. 260: 4653-4660 (1985); Lau et al, J. Biol. Chem. 262: 1389-1397 (1987); Lau et al, Adv. Protein Phosphatases 4: 165-198 (1987)). Interestingly, it was recently found that the level of membrane-bound PTPase activity was increased dramatically when the osteoblast-like cell line UMR 106.06 was grown on collagen type-l matrix compared to uncoated tissue culture plates. Since a significant increase in PTPase activity was observed in density-dependent growth arrested fibroblasts (Pallen and Tong, Proc. Natl. Acad. Sci. 88: 6996-7000 (1991)), it might be speculated that the increased PTPase activity directly inhibits cell growth. The mitogenic action of fluoride and other phosphatase inhibitors (molybdate and vanadate) may thus be explained by their inhibition of acid phosphatases/PTPases that negatively regulate the cell proliferation of osteoblasts. The complex nature of the involvement of PTPases in bone formation is further suggested by the recent identification of a novel parathyroid regulated, receptor-like PTPase, OST-PTP, expressed in bone and testis (Mauro etal, J. Biol. Chem. 269: 30659-30667 (1994)). OST-PTP is up-regulated following differentiation and matrix formation of primary osteoblasts and subsequently down-regulated in the osteoblasts which are actively mineralizing bone in culture. It may be hypothesized that PTPase inhibitors may prevent differentiation via inhibition of OST-PTP or other PTPases thereby leading to continued proliferation. This would be in agreement with the above- mentioned effects of fluoride and the observation that the tyrosine phosphatase inhibitor orthovanadate appears to enhance osteoblast proliferation and matrix formation (Lau et al, Endocrinology 116: 2463-2468 (1988)). In addition, it was recently observed that vanadate, vanadyl and pervanadate all increased the growth of the osteoblast-like cell line UMR 106. Vanadyl and pervanadate were stronger stimulators of cell growth than vanadate. Only vanadate was able to regulate the cell differentiation as measured by cell alkaline phosphatase activity (Cortizo et al, Mol. Cell. Biochem. 145: 97-102 (1995)).
PTPases: microorganisms
Dixon and coworkers have called attention to the fact that PTPases may be a key element in the pathogenic properties of Yersinia (reviewed in Clemens etal. Molecular Microbiology 5: 2617-2620 (1991)). This finding was rather suφrising since tyrosine phosphate is thought to be absent in bacteria. The genus Yersinia comprises 3 species: V. pestis (responsible for the bubonic plague), Y. pseudoturberculosis and Y. enterocolitica (causing enteritis and mesenteric lymphadenitis). Interestingly, a dual-specificity phosphatase, VH1 , has been identified in Vaccinia virus (Guan et al., Nature 350: 359-263 (1991)). These observations indicate that PTPases may play critical roles in microbial and parasitic infections, and they further point to PTPase inhibitors as a novel, putative treatment principle of infectious diseases.
SUMMARY OF THE INVENTION
The inventors have identified a novel class of compounds that has the capacity to modulate the activity of molecules with tyrosine recognition units, including PTPases, preferably a selective modulation. In one aspect, the present invention relates to novel organic compounds thereof of general formula (I)
Figure imgf000018_0001
(l) wherein
(L)n, n, An, Ri and A are defined as below.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to novel organic compounds thereof of formula (I)
Figure imgf000018_0002
(I) wherein
n is 1 , 2, 3, 4, or 5 and (L)„ represents up to five (5) substituents which independentiy of each other are hydrogen, d-6-alkyl, C1-6-alkoxy, C^alkylthio, hydroxy, halogen, tirihalogenomethyl, hydroxy-CLβ-alkyl, amino-C^-alkyl, -COR2, -NO2, -CN, -CHO, -Cι.6-alkanoyloxy, carbamoyl, -NR5R6l aryloxy optionally substituted; R2 is Ci-e-alkyl, aryl optionally substituted, aralkyl optionally substituted, -OH, -NR3R4 wherein R3 and R4 independently of each other are hydrogen, Ci-e-alkyl. aryl optionally substituted, aralkyl optionally substituted;
R5 and R6 are independently of each other hydrogen or Ci-e-alkyl, aryl optionally substituted, aralkyl optionally substituted or-COZi wherein Zi is d^-alkyl, aryl optionally substituted, aralkyl optionally substituted;
or L is Aι-Yι-(Wι)-X-(W2)-Y2- wherein X is a chemical bond, -CO, -CONR7 , -NR7CO, -NR7 ,-
O-, -S-, -SO, or-SO2;
Yi and Y2 are independently a chemical bond, -O-, -S-, or -NR7;
R7 is hydrogen, Ci-β-alkyl, aryl optionally substituted, aralkyl optionally substituted, heteroaryl optionally substituted, -COZ2 wherein Z2 is Ci-e-alkyl, aryl optionally substituted, aralkyl optionally substituted;
Wi and W2 are independently a chemical bond or saturated or unsaturated Ci-β-alkylene;
Ai is aryl optionally substituted, heteroaryl optionally substituted, biaryl optionally substituted, arylheteroaryl optionally substituted, -NR8Rg wherein R8 and Rg independently are hydrogen, Ci-e-alkyl, aryl optionally substituted, aralkyl optionally substituted, heteroaryl optionally substituted, -COZ3 wherein Z3 is Ci-e-alkyl, aryl optionally substituted, aralkyl optionally substituted, heteroaryl optionally substituted or when R 8 and R9 together with the nitrogen atom forms a ring system Ai is a saturated or partially saturated heterocyclic ring system optionally substituted with Ci-e-alkyl, aryl optionally substituted, aralkyl optionally substituted, heteroaryl optionally substituted, -OH, d-e-alkoxy, Ci-e-alkylthio, hydroxy-d-e-alkyl, amino-Cι.6-alkyl, -COZi wherein 2\ is -OH, d-e-alkyl, -NRioRn wherein R10 and Ru independently are hydrogen, Ci. e-alkyl; Ri is a linker selected from a chemical bond, -Ci-e-alkyl-, -0(CH2)m-, -NR12-, -CONR12-, - NR13CO-, -SO2NRι4-, -NR15SO2-, -CRιe=CRιr, -CH= -, -CHRι7, -CHr, -CHF-, -CF2-, -SO2-; R12, R13, Ru, R15 Rιe and Ri7 are hydrogen, Ci_6-alkyl, aralkyl and m is 1,2, or 3;
A is -PO(ORι8)(OR,9), -NH-SO3H, -NH-SO2-CH3, -NH-S02-CF3, -CO-NH-OH or a heterocycle as shown in scheme 1 wherein the point of attachment is indicated with a I
(single bond) or || (double bond)
Scheme 1
Figure imgf000021_0001
Figure imgf000021_0002
Figure imgf000021_0003
Figure imgf000021_0004
Figure imgf000021_0005
optionally substituted by hydrogen, halogen, d-s-alkyi optionally substituted by phenyl optionally substituted by Cι.6-alkoxy, Ci-β-alkylthio; -COOX1 wherein X1 is Ci-e-alkyl optionally substituted by phenyl or benzyl optionally substituted; Ria, and Ri9 independently are hydrogen, Ci-e-alkyl, phenyl, benzyl;
R20 is hydrogen, -OH, d-e-alkoxy, -SH, d-e-alkylthio, Ci ^-alkylcarbonyloxy , -COR21, -SOR22,
-SO2R23, -NR24R2s, -NHCN, halogen, trihalogenomethyl;
R21, R22, and R23 are -OR2e, Ci-e-alkyl, -NR24R25, trihalogenomethyl;
R24 and R25 independently are hydrogen, Ci-e-alkyl, -SO2R26, -COZ5 wherein Zs is
Ci-e-alkyl, trihalogenomethyl
R2e is hydrogen, Ci-β-alkyl, trihalogenomethyl; nn is 1 or 2; and An is aryl or heteroaryl;
or a pharmaceutically acceptable salt thereof.
In the above-mentioned formula (I) aryl, heteroaryl, An or Ai are exemplified by the following examples. Specific examples of the aryl and biaryl residues include phenyl, biphenyi, indenyl, fluorenyl, naphthyl (1-naphthyl, 2-naphthyl), anthracenyl (1-anthracenyl, 2-anthracenyl, 9- anthracenyl). Specific examples of the heteroaryl include pyrrolyl (2-pyrrolyl), pyrazolyl (3- pyrazolyl), imidazolyl (1 -imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl), triazolyl (1,2,3- triazol-1-yl, 1 ,2,3-triazol-2-yt 1,2,3-triazo!-4-yl, 1,2,4-triazol-3-yl), oxazolyl (2-oxazolyl, 4- oxazolyl, 5-oxazolyl), isoxazolyl (3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl), thiazolyl (2-thiazolyl, 4-thiazolyl, 5-thiazolyl), thiophenyl (2-thiophenyl, 3-thiophenyl), pyridyl (2-pyridyl, 3-pyridyl, 4- pyridyl), pyrimidinyl (2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl), pyrazinyl, pyridazinyl (3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl), quinolyl (2-quinolyl, 3-quinolyl, 4- quinolyl, 5-quinolyl, 6-quinolyl, 7-quinolyl, 8-quinolyl), isoquinolyl (1-isoquinolyl, 3-isoquinolyl, 4-isoquinolyl, 5-isoquinolyl, 6-isoquinolyl, 7-isoquinolyl, 8-isoquinolyl), benzo[b]furanyl (2-benzo[b]furanyl, 3-benzo[b]furanyl, 4-benzo[b]furanyl, 5-benzo[b]furanyl,
6-benzo[b]furanyl, 7-benzo[b]furanyl), 2,3-dihydro-benzo[b]furanyl (2-(2,3-dihydro- benzo[b]furanyl), 3-(2,3-dihydro-benzo[b]furanyl), 4-(2,3-dihydro-benzo[b]furanyl), 5-(2,3- dihydro-benzo[b]furanyl), 6-(2,3-dihydro-benzo[b]furanyl), 7-(2,3-dihydro-benzo[b]furanyl), benzo[b]thiophenyl (2-benzo[b]thiophenyl, 3-benzo[b]thiophenyl, 4-benzo[b]thiophenyl, 5-benzo[b]thiophenyl,
6-benzo[b]thiophenyl, 7-benzo[b]thiophenyl), 2,3-dihydro-benzo[b]thiophenyl (2-(2,3-dihydro- benzo[b]thiophenyl), 3-(2,3-dihydro-benzo[b]thiophenyl), 4-(2,3-dihydro-benzo[b]thiophenyl), 5-(2,3-dihydro-benzo[b]thiophenyl), 6-(2,3-dihydro-benzo[b]thiophenyl), 7-(2,3-dihydro- benzo[b]thiophenyl), indolyl (1 -indolyl, 2-indolyl, 3-indolyl, 4-indolyl, 5-indolyl, 6-indolyl, 7-indolyl), indazolyl (1-indazolyl, 3-indazolyl, 4-indazolyl, 5-indazolyl, 6-indazolyl, 7-indazolyl), benzimidazolyl (1-benzimidazolyl, 2-benzimidazolyl, 4-benzimidazolyl, 5-benzimidazolyl, 6- benzimidazolyl, 7-benzimidazolyl, 8-benzimidazolyl), benzoxazolyl (1-benzoxazolyl, 2- benzoxazolyl), benzothiazolyl (1-benzothiazolyl, 2-benzothiazolyl, 4-benzothiazolyl, 5-benzothiazolyl, 6-benzothiazolyl, 7-benzothiazolyl), carbazolyl (1-caΦazolyl, 2-carbazolyl, 3-carbazolyl, 4-carbazolyl), 5H-dibenz[b,f]azepine (5H-dibenz[b,fjazepin-1-yl, 5H-dibenz[b,f]azepine-2-yl, 5H-dibenz[b,f]azepine-3-yl, 5H-dibenz[b,f]azepine-4-yl, 5H- dibenz[b,fJazepine-5-yl), 10,11-dihydro-5H-dibenz[b,fJazepine (10,11-dihydro-5H- dibenz[b,f]azepine-1-yl, 10,11-dihydro-5H-dibenz[b,f]azepine-2-yl, 10,11-dihydro-5H- dibenz[b,f]azepine-3-yl, 10,11-dihydro-5H-dibenz[b,f]azepine-4-yl, 10,11-dihydro-5H- dibenz[b,f]azepine-5-yl), piperidinyl (2-piperidinyl, 3-piperidinyl, 4-piperidinyl), pyrrolidinyl (1- pyrrolidinyl, 2-pyrrolidinyl, 3-pyrrolidinyl), moφholiπyl (1-moφholinyl, 2-moφholinyl), piperazinyl (1-piperazinyl).
Specific examples of the arylheteroaryl residue include phenylpyridyl (2-phenylpyridyl, 3- phenylpyridyl, 4-phenylpyridyl), phenylpyrimidinyl (2-phenylpyrimidinyl, 4-phenylpyrimidinyl, 5- phenylpyrimidinyl, 6-phenylpyrimidinyl), phenylpyrazinyl, phenylpyridazinyl (3-phenyl- pyridazinyl, 4-phenylpyridazinyl, 5-phenylpyridazinyl).
The Ci-e-alkyl residues include aliphatic hydrocarbon residues, unsaturated aliphatic hydrocarbon residues, alicyclic hydrocarbon residues. Examples of the aliphatic hydrocarbon residues include saturated aliphatic hydrocarbon residues having 1 to 6 carbon atoms such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec.butyl, tertbutyl, n-pentyl, isopentyl, neopentyl, tertpentyl, n-hexyl, isohexyl. Example of the unsaturated aliphatic hydrocarbon residues includ those having 2 to 6 carbon atoms such as ethenyl, 1-propenyl, 2-propenyl, 1- butenyl, 2-butenyl, 3-butenyl, 2-methyl-1-propenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4- pentenyl, 3-methyl-2-butenyl, 1-hexenyl, 3-hexenyl, 2,4-hexadienyl, 5-hexenyl, ethynyl, 1-propionyl, 2-propionyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexynyl, 3-hexynyl, 2,4-hexadiynyl, 5-hexynyl. Examples of the alicyclic hydrocarbon residue include saturated alicyclic hydrocarbon residues having 3 to 6 carbon atoms such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl; and
C5.s unsaturated alicyclic hydrocarbon residues having 5 to 6 carbon atoms such as 1- cyclopentenyl, 2-cyclopentenyl, 3-cyclopentenyl, 1 -cyclohexenyl, 2-cyclohexenyl, 3-cyclohexenyl.
The Ci -e-alkoxy residues include aliphatic hydrocarbon residues connected to an oxygene atom. Examples of the aliphatic hydrocarbon residues include saturated aliphatic hydrocarbon residues having 1 to 6 carbon atoms such as methoxy, ethoxy, propoxy, iso¬ propoxy, butoxy, isobutoxy, sec.butoxy, tertbutoxy, pentoxy, isopentoxy, neopentoxy, tert.pentoxy, hexyloxy, isohexyloxy.
The Ci-e-alkylthio residues include aliphatic hydrocarbon residues connected to an sulphur atom. Examples of the aliphatic hydrocarbon residues include saturated aliphatic hydrocarbon residues having 1 to 6 carbon atoms such as methythio, ethylthio, propoylthio, iso-propylthio, butylthio, isobutylthio, sec.butylthio, tert.butylthio, pentylthio, isopentylthio, neopentylthio, tert. pentylthio, hexylylthio, isohexylythio.
The Cι.6-alkoxycarbonyl residues include a Ci-e-alkoxy residue connected to a carbonyl residue such as methoxycarbonyl, ethoxy-carbonyl, propoxycarbonyl, and tert- butoxycaΦonyl.
The Ci-e-alkylcarbonyloxy residues include a Ci-e-alkyl residue connected to a carbonyloxy residue such as acetic acid, propionic acid, butyric acid.
The Ci-e-alkanoyloxy residues include a acyl residue connected to an oxygen atom wherein the acyl residue is an aliphatic hydrocarbon residues connected to an carbonyl residue such as acetyloxy, propionyloxy, isopropionyloxy.
The aralkyl residue include an aryl residue connected to an Ci-e-alkyl residue e.g. phenyl alkyls having 7 to 9 carbon atoms such as benzyl, phenethyl, 1-phenylethyl, 3-phenylpropyl, 2-phenyipropyl and 1-phenylpropyl; and naphthyl alkyl having 11 to 13 carbon atoms such as 1-naphthylmethyl, 1-naphthylethyl, 2-naphthylmethyl, and 2-naphthylethyl.
Aryloxy include an aryl connected to an oxygen atom such as phenyloxy, naphthyloxy.
Aralkyloxy include an aralkyl connected to an oxygen atom such as benzyloxy, phenethyloxy, naphthylmethyloxy.
Biaryl include an aryl connected to an aryl residue such as biphenyi, 1-phenylnaphthyl, 2- phenylnaphthyl .
Biaryloxy include an biaryl connected to an oxygen atom such as biphenyloxy, 4-(naphthalene-1 -yl)phenoxy, 4-(naphthalene-2-yl)phenoxy.
The heteroaryl residue is a 5- or 6-membered aromatic ring, which can be fused to one or more phenyl rings and contains, besides carbon atoms, 1 to 4 atoms selected from N, O, and S as atoms constituting the ring, which is bonded through caΦon atoms such as defined above.
The halogen residue include fluorine, chlorine, bromine, and iodine.
The term "optionally substituted" means an aryl residue, a heteroaryl residue, or a Ci-6-alkyl residue that may be unsubstituted or may have 1 or more preferably 1 to 5 substituents, which are the same as or different from one another. Examples of these substituents include, halogen (fluorine, chlorine, bromine, iodine), hydroxyl, cyano, nitro, trifluoromethyl, carbamoyl, d^-acy! (e.g. acetyl, propionyl, isopropionyl), Ci-e-alkoxy (e.g. methoxy, ethoxy, propoxy, isopropoxy, butoxy, and tertbutoxy), Ci-e-alkyl (e.g. methyl, ethyl, propyl, cyclopropyl, isopropyl, butyl, and tert.butyl), Ci-β-alkoxycarbonyl (e.g. ones having 2 to 6 carbon atoms such as methoxycarbonyl, ethoxycarbonyl, and propoxycarbonyl), C1-6- alkanoyloxy (e.g. ones having 2 to 6 carbon atoms such as acetyloxy, propionyloxy, isopropionyloxy), Cι_4-alky!thio (e.g. ones having 1 to 4 carbon atoms such as methylthio, ethylthio, propylthio, and isopropylthio), Cι.4-alkylamino (e.g. one having 1 to 4 carbon atoms such as methylamino, ethylamino, dimethylamino, and 1 -pyrrolidinyl), heteroaryl (as exemplified above), aryloxy (e.g. phenyloxy), and a aralkyloxy (e.g. benzyloxy).
The compounds of formula (I) may exist as geometric and optical isomers and all isomers and mixtures thereof are included herein. Isomers may be separated by means of standard methods such as chromatographic techniques or fractionated crystallisation of e.g. suitable salts. It is to be understood that the heterocyclic moieties depicted throughout this application is capable of undergoing tautomerisation. Example of tautomerisation is given by the following example, thus:
Figure imgf000026_0001
The compounds according to the invention may optionally exist as pharmaceutically acceptable salts comprising acid addition salts or metal salts or - optionally alkylated - ammonium salts.
Examples of such salts include the alkali metal or amine salts of 1 H- or 2H-tetrazoles of this invention, such as the sodium, potassium, Ci-β-alkylamine, di (Ci-e-alkyl) amine, tri (Ci-e-alkyl) amine and the four (4) corresponding omega-hydroxy analogues (e.g. methylamine, ethylamine, propylamine, dimethylamine, diethylamine, dipropylamine, trimethylamine, triethylamine, tripropylamine, di(hydroxyethyl)amine, and the like; inorganic and organic acid addition salts such as hydrochloride, hydrobromide, sulphate, phosphate, acetate, fumarate, maleate, citrate, lactate, tartrate, oxalate or similar pharmaceutically acceptable inorganic or organic acid addition salts, and include the pharmaceutically acceptable salts listed in Journal of Pharmaceutical Science 66: 2 (1977) which are hereby incoφorated by reference. The compounds of formula (I) may be prepared by art-recognised procedures from known compounds or readily preparable intermediates. An exemplary general procedure is as follows:
Method A:
(L)n-Ari Rf-≡Ξ N * MN3 ► (I)
(II) (III)
By allowing a compound of formula (II) wherein (L)„, Arit Ri, and n are as defined above to react with an azide of formula (111) wherein M is an alkali metal (J. Am. Chem. Soc. 80: 3908 (1958)), trialkyltin Sn(Rι8)3 wherein Ri8 is C^-alkyl (J. Org. Chem. 56: 2395 (1991)), or trialkyisilyl Si(R19)3 wherein Ri9 is d-4-alkyl (Tetrahedron Lett. 34: 8011 (1993)), a tetrazole derivative of formula (I) wherein A is a tetrazol is produced. These cyclisation reactions may be earned out in a solvent such as dimethylformamide (DMF), tetrahydrofuran (THF) or toluene at temperatures ranging from 80 °C to 150 °C for 1 to 60 hours.
The tetrazole derivatives and their salts thus obtained can be isolated and purified by known means of separation and purification such as concentration, concentration under reduced pressure, crystallisation, recrystaliisation, extraction and chromatography.
The nitrile derivatives (II) used as starting materials in the method A of this invention can be produced by, for example, the following manner.
A, -w,— x + Y-W2 — Ar, — R, — ≡ N ► (ii)
(IV) (V)
A, — W,— Y + X -W2 Ar,~ Rn -≡ N ► (II)
(VI) (VII)
By allowing a compound of formula (IV) wherein Ai and Wt are as defined above, X is OH, SH, and NHR7 wherein R7 is as defined above to react with a compound of formula (V) wherein W2 and An are as defined above and Y is a suitable leaving group such as halogen, p-toluene sulphonate, mesylate or hydroxy; or by allowing a compound of formula (VI) wherein Ai and Wi are as defined above and Y is a suitable leaving group such as halogen, p-toluene sulphonate, mesylate or hydroxy to react with a compound of formula (VII) wherein W2 and An are as defined above, X is OH, SH, and NHR7 wherein R7 is as defined above. These reactions may be earned out in a solvent such as N-methyl pyrrolidone (NMP), dimethylformamide (DMF), tetrahydrofuran (THF), acetone, dibutyl ether, 2-butanone, methyl tert-butyl ether, methyl ethyl ketone, ethyl acetate or toluene in the presence of a base e.g. potassium carbonate or sodium hydride and a catalyst, e.g. an alkali metal iodide, copper or a copper salt e.g. (CuCl, CuBr, Cul, or Cu2O) or in the case of a Mitsunobu reaction (for a review see, O. Mitsunobu, Synthesis, 1 (1981)) in the presence of e.g. diethyl azodicarboxylate and triphenylphosphine at temperatures ranging from -10 °C to 200 °C for 1 to 60 hours.
Method B:
(L)n- Ar1 - X1 + R, -A ► (I)
(VIII) (IX) By allowing a compound of formula (VIII), wherein (L)n, n, and An are as defined above and Xi is a suitable leaving group such as bromo, iodo or trifiuoromethane sulfonyloxy to react with a compound of formula (IX) wherein Ri is CH2=CH and A are as defined above.
These reactions may be earned out in a solvent such as triethylamine (TEA), methanol, ethanol or dimethylsulfoxid (DMSO) in the presence of a palladium catalyst, e.g. (Pd/C, Pd/AI2O3, Pd/BaSO4, Pd/Si02 or Pd(OAc)2 (a Heck reaction) ) and a triaryl-phosphine catalyst as e.g. (triphenyl-phosphine or tri-o-tolyl-phosphine) at temperatures ranging from 50 °C to 150 °C for 1 to 60 hours.
Method C:
(D - A^ -CHO + l^-CH2-A ► (|)
(X) (XI)
By allowing a compound of formula (X), wherein (L)n, n, and An are as defined above to react with a compound of formula (XI) wherein A is as defined above and U is trimethylsilyl (a Peterson reaction), triphenyl-phosphonium (a Wittig reaction), diethyl phosphate (a modified Wittig reaction) or carbonyloxyCι-6-alkyl (e.g. -COOEt or -COOMe) or,
by allowing a compound of formula (XII), wherein (L)n, n, and An are as defined above and U is trimethylsilyl (a Peterson reaction), triphenyl-phosphonium (a Wittig reaction), diethyl phosphate (a modified Wittig reaction) or carbonyloxyCi-e-alkyl (e.g. -COOEt or -COOMe) to react with a compound of formula (XIII) wherein A is as defined above.
Figure imgf000029_0001
These reactions may be carried out in a solvent such as methanol, ethanol, tetrahydrofuran (THF). toluene, N,N-dimethylfoπmamide (DMF) or dimethylsulfoxid (DMSO) in the presence of a base such as triethylamine, pyridine, piperidine, sodium hydride, sodium methoxide, sodium ethoxide, potassium tert-butoxide, lithium diisopropylamide at temperatures ranging from -50 °C to 150 °C for 1 to 60 hours.
Method D:
(L)„— (I)
Figure imgf000030_0001
(XIV)
By allowing a compound of foπnula (XIV), wherein (L)n, n and An are as defined above and Hal is chloro or bromo and R is Ci-e-alkyl to react with a compound of formula (XV) wherein X is O or S whereby a compound of formula (I) is produced wherein Ri is CF2 and A are 2,4- dihydroxy-oxazolidin-5-yl or 2,4-dihydroxy-thiazolidin-5-yl;
Compounds of formula (II), (IV) to (XV) may be prepared by methods familiar to those skilled in the art.
Under certain circumstances it may be necessary to protect the intermediates used in the above methods. The tetrazole group can, for example, be protected by a trityl group. Introduction and removal of such groups is e.g. described in "Protective Groups in Organic Synthesis" T.W. Greene and P.G.M. Wuts, ed. Second edition (1991).
In preferred embodiments, the compounds of the invention modulate the activity of protein tyrosine phosphatases or other molecules with phosphotyrosine recognition unit(s).
In one preferred embodiment the compounds of the invention act as inhibitors of PTPases, e.g. protein tyrosine phosphatases involved in regulation of tyrosine kinase signalling pathways. Preferred embodiments include modulation of receptor-tyrosine kinase signalling pathways via interaction with regulatory PTPases, e.g. the signalling pathways of the insulin receptor, the IGF-I receptor and other members of the insulin receptor family, the EGF- receptor family, the platelet-derived growth factor receptor family, the nerve growth factor receptor family, the hepatocyte growth factor receptor family, the growth hormone receptor family and members of other receptor-type tyrosine kinase families. Further preferred embodiments of the inventions is modulation of non-receptor tyrosine kinase signalling through modulation of regulatory PTPases, e.g. modulation of members of the Src kinase family. One type of preferred embodiments of the inventions relate to modulation of the activity of PTPases that negatively regulate signal transduction pathways. Another type of preferred embodiments of the inventions relate to modulation of the activity of PTPases that positively regulate signal transduction pathways.
In a preferred embodiment the compounds of the invention act as modulators of the active site of PTPases. In another preferred embodiment the compounds of the invention modulate the activity of PTPases via interaction with structures positioned outside of the active sites of the enzymes, preferably SH2 domains. Further preferred embodiments include modulation of signal transduction pathways via binding of the compounds of the invention to SH2 domains or PTB domains of non-PTPase signalling molecules.
Other preferred embodiments include use of the compounds of the invention for modulation of cell-cell interactions as well as cell-matrix interactions.
As a preferred embodiment, the present invention include within its scope pharmaceutical compositions comprising, as an active ingredient, at least one of the compounds of formula (I) in association with a pharmaceutical carrier or diluent. Optionally, the pharmaceutical composition can comprise at least one of the compounds of formula (I) combined with compounds exhibiting a different activity, e.g. an antibiotic or other pharmacologically active material.
As a preferred embodiment, the compounds of the invention may be used as therapeuticals to inhibit of PTPases involved in regulation of the insulin receptor tyrosine kinase signalling pathway in patients with type I diabetes, type II diabetes, impaired glucose tolerance, insulin resistance, and obesity. Further preferred embodiments include use of the compounds of the invention for treatment of disorders with general or specific dysfunction's of PTPase activity, e.g. proliferarive disorders such as psoriasis and neoplastic diseases. As another embodiment, the compounds of the invention may be used in pharmaceutical preparations for treatment of osteoporosis.
Preferred embodiments of the invention further include use of compound of formula (I) in pharmaceutical preparations to increase the secretion or action of growth hormone and its analogous or somatomedins including IGF-1 and IGF-2 by modulating the activity of PTPases or other signal transduction molecules with affinity for phosphotyrosine involved controlling or inducing the action of these hormones or any regulating molecule.
To those skilled in the art, it is well known that the current and potential uses of growth hormone in humans are varied and multi-tudinous. Thus, compounds of the invention can be administered for puφoses of stimulating the release of growth hormone from the pituitary or increase its action on target tissues thereby leading to similar effects or uses as growth hormone itself. The uses of growth hormone may be summarized as follows: stimulation of growth hormone release in the elderly; prevention of catabolic side effects of glucocorticoids; treatment of osteoporosis, stimulation of the immune system; treatment of retardation, acceleration of wound healing; accelerating bone fracture repair; treatment of growth retardation; treating renal failure or insufficiency resulting in growth retardation; treatment of physiological short stature including growth hormone deficient children and short stature associated with chronic illness; treatment of obesity and growth retardation associated with obesity; treating growth retardation associated with the Prader-Willi syndrome and Turner's syndrome; accelerating the recovery and reducing hospitalization of bum patients; treatment of intrauterine growth retardation, skeletal dysplasia, hypercortisolism and Cushings syndrome; induction of pulsatile growth hormone release; replacement of growth hormone in stressed patients; treatment of osteochondro-dysplasias, Noonans syndrome, schizophrenia, depressions, Alzheimer's disease, delayed wound healing and psychosocial deprivation; treatment of pulmonary dysfunction and ventilator dependency; attenuation of protein catabolic responses after major surgery; reducing cachexia and protein loss due to chronic illness such as cancer or AIDS; treatment of hyperinsulinemia including nesidio-blastosis; Adjuvant treatment for ovulation induction; stimulation of thymic development and prevention the age-related decline of thymic function; treatment of immunosuppressed patients; improvement in muscle strength, mobility, maintenance of skin thickness, metabolic homeostasis, renal hemeostasis in the frail elderly; stimulation of osteoblasts, bone remodelling and cartilage growth; stimulation of the immune system in companion animals and treatment of disorder of aging in companion animals; growth promotant in livestock and stimulation of wool growth in sheep.
The compounds of the invention may be used in pharmaceutical preparations for treatment of various disorders of the immune system, either as a stimulant or suppressor of normal or perturbed immune functions, including autoimmune reactions. Further embodiments of the invention include use of the compounds of the invention for treatment of allergic reactions, e.g. asthma, dermal reactions, conjunctivitis.
In another embodiment compounds of the invention may be used in pharmaceutical preparations for prevention or induction of platelet aggregation.
In yet another embodiment, compounds of the invention may be used in pharmaceutical preparations for treatment of infectious disorders. In particular, the compounds of the invention may be used for treatment of infectious disorders caused by Yersinia and other bacteria as well as disorders caused by viruses or other micro-organisms.
Compounds of the invention may additionally be used for treatment or prevention of diseases in animals, including commercially important animals.
Also included in the present invention is a process for isolation of PTPases via affinity purification procedures based on tine use of immobilised compounds of the invention using procedures well-known to those skilled in the art.
The invention is further directed to a method for detecting the presence of PTPases in cell or in a subject comprising:
(a) contacting said cell or an extract thereof with labelled compounds of the invention. (b) detecting the binding of the compounds of the invention or measuring the quantity bound, thereby detecting the presence or measuring the quantity of certain PTPases. The invention further relates to analysis and identification of the specific functions of certain PTPases by modulating their activity by using compounds of the invention in cellular assay systems or in whole animals.
DEFINITIONS
Signal transduction is a collective term used to define all cellular processes that follow the activation of a given cell or tissue. Examples of signal transduction, which are not intended to be in any way limiting to the scope of the invention claimed, are cellular events that are induced by polypeptide hormones and growth factors (e.g. insulin, insulin-like growth factors I and II, growth hormone, epidermal growth factor, platelet-derived growth factor), cytokines (e.g. inter-leukins), extracellular matrix components, and cell-cell interactions.
Phosphotyrosine recognition units/tyrosine phosphate recognition units/pTyr recognition units are defined as areas or domains of proteins or glycoproteins that have affinity for molecules containing phosphorylated tyrosine residues (pTyr). Examples of pTyr recognition units, which are not intended to be in any way limiting to the scope of the invention claimed, are: PTPases, SH2 domains and PTB domains.
PTPases are defined as enzymes with the capacity to dephosphorylate pTyr-containing proteins or glycoproteins. Examples of PTPases, which are not intended to be in any way limiting to the scope of the invention claimed, are: 'classical' PTPases (intracellular PTPases (e.g. PTP1B, TC-PTP, PTP1C, PTP1D, PTPD1, PTPD2) and receptor-type PTPases (e.g. PTPα, PTPε, PTPβ, PTPγ, CD45, PTPK, PTPμ), dual specificty phosphatases (VH1, VHR, cdc25), LMW-PTPases or acid phosphatases.
Modulation of cellular processes is defined as the capacity of compounds of the invention to 1) either increase or decrease ongoing, normal or abnormal, signal transduction, 2) initiate normal signal transduction, and 3) initiate abnormal signal transduction. Modulation of pTyr-mediated signal transduction/modulation of the activity of molecules with pTyr recognition units is defined as the capacity of compounds of the invention to 1) increase or decrease the activity of proteins or glycoproteins with pTyr recognition units (e.g. PTPases, SH2 domains or PTB domains) or to 2) decrease or increase the association of a pTyr-containing molecule with a protein or glyco-protein with pTyr recognition units either via a direct action on the pTyr recognition site or via an indirect mechanism. Examples of modulation of pTyr-mediated signal transduction/modulation of the activity of molecules with pTyr recognition units, which are not intended to be in any way limiting to the scope of the invention claimed, are: a) inhibition of PTPase activity leading to either increased or decreased signal transduction of ongoing cellular processes; b) inhibition of PTPase activity leading to initiation of normal or abnormal cellular activity; c) stimulation of PTPase activity leading to either increased or decreased signal transduction of ongoing cellular processes; d) stimulation of PTPase activity leading to initiation of normal or abnormal cellular activity; e) inhibition of binding of SH2 domains or PTB domains to proteins or glycoproteins with pTyr leading to increase or decrease of ongoing cellular processes; f) inhibition of binding of SH2 domains or PTB domains to proteins or glycoproteins with pTyr leading to initiation of normal or abnormal cellular activity.
A subject is defined as any mammalian species, including humans.
Pharmacological Compositions
For the above indications the dosage will vary depending on the compound of formula (I) employed, on the mode of administration and on the therapy desired. However, in general, satisfactory results are obtained with a dosage of from about 0.5 mg to about 1000 mg, preferably from about 1 mg to about 500 mg of compounds of formula (I), conveniently given from 1 to 5 times daily, optionally in sustained release form. Usually, dosage forms suitable for oral administration comprise from about 0.5 mg to about 1000 mg, preferably from about 1 mg to about 500 mg of the compounds of formula (I) admixed with a pharmaceutical carrier or diluent.
The compounds of formula (I) may be administered in a pharmaceutically acceptable acid addition salt form or where possible as a metal or a Ci-e-alkylammonium salt. Such salt forms exhibit approximately the same order of activity as the free acid forms. This invention also relates to pharmaceutical compositions comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and, usually, such compositions also contain a pharmaceutical earner or diluent. The compositions containing the compounds of this invention may be prepared by conventional techniques and appear in conventional forms, for example capsules, tablets, solutions or suspensions.
The pharmaceutical earner employed may be a conventional solid or liquid carrier. Examples of solid carriers are lactose, terra alba, sucrose, talc, gelatine, agar, pectin, acacia, magnesium stearate and stearic acid. Examples of liquid carriers are syrup, peanut oil, olive oil and water.
Similarly, the carrier or diluent may include any time delay material known to the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax.
If a solid carrier for oral administration is used, the preparation can be tabletted, placed in a hard gelatine capsule in powder or pellet form or it can be in the form of a troche or lozenge. The amount of solid earner will vary widely but will usually be from about 25 mg to about 1 g. If a liquid earner is used, the preparation may be in the form of a syrup, emulsion, soft gelatin capsule or sterile injectable liquid such as an aqueous or non-aqueous liquid suspension or solution.
Generally, the compounds of this invention are dispensed in unit dosage form comprising 10- 200 mg of active ingredient in or together with a pharmaceutically acceptable carrier per unit dosage.
The dosage of the compounds according to this invention is 1-500 mg/day, e.g. about 100 mg per dose, when administered to patients, e.g. humans, as a drug.
A typical tablet which may be prepared by conventional tabletting techniques contains Core:
Active compound (as free compound 100 mg or salt thereof)
Colloidal silicon dioxide (Areosil®) 1.5 mg Cellulose, microcryst. (Avicel®) 70 mg
Modified cellulose gum (Ac-Di-Sol®) 7.5 mg
Magnesium stearate
Coating: HPMC approx. 9 mg
'Mywacett® 9-40 T approx. 0.9 mg
Acylated monoglyceride used as plasticiser for film coating.
The route of administration may be any route which effectively transports the active compound to the appropriate or desired site of action, such as oral or parenteral e.g. rectal, transdermal, subcutaneous, intranasal, intramuscular, topical, intravenous, intraurethral, ophthalmic solution or an ointment, the oral route being preferred.
EXAMPLES
The process for preparing compounds of formula (I) and preparations containing them is further illustrated in the following examples, which, however, are not to be construed as limiting.
Hereinafter, TLC is thin layer chromatography, CDCI3 is deuterio chloroform and DMSO-d6 is hexadeuterio dimethylsulfoxid. The structures of the compounds are confirmed by either elemental analysis or NMR, where peaks assigned to characteristic protons in the title compounds are presented where appropriate. 1H NMR shifts (δH) are given in parts per million (ppm) down field from tetramethylsilane as internal reference standard. M.p. is melting point and is given in °C and is not corrected. Column chromatography was earned out using the technique described by W.C. Still etal., J. Org. Chem. 43: 2923 (1978) on Merck silica gel 60 (Art. 9385). HPLC analyses were performed using 5μm C18 4 x 250 mm column eluted with various mixtures of water and acetonitrile, flow = 1 ml/min, as described in the experimental section.
Compounds used as starting material are either known compounds or compounds which can readily be prepared by methods known p_er se.
EXAMPLE 1
Figure imgf000038_0001
5-Naphthalen-2-yl-3H-[1,3,4]oxadiazole-2-thione
To a solution of 2-naphthyl carboxylic acid ethyl ester (2.0 g, 9.99 mmol) in absolute et¬ hanol (30 ml) was added hydrazine hydrate (4.85 ml, 99.9 mmol) and the reaction mixtu¬ re was heated at reflux temperature for 72 h. The reaction mixture was cooled and the precipitate was filtered off, washed with 96 % ethanol (2 x 10 ml) and diethyl ether (3 x 10 ml), dried in vacuo at 50 °C which afforded 0.9 g (48%) of naphthalene-2-carboxylic acid hydrazide as a solid.
To a stirred solution of the above hydrazide (1.0 g, 5.37 mmol) in methanol (20 ml) was added potassium hydroxide (0.33 g, 5.88 mmol) and carbondisulfide (0.94 g, 12.35 mmol) at 0 °C. The reaction mixture was stirred at reflux temperature for 7 h, cooled and quenched with water (100 ml). The resultant mixture was washed with diethyl ether (50 ml) and acidified to pH = 1 with 1 N hydrochloric acid. The precipitate was filtered off, washed with water (2 x 20 ml) and heptane (2 x 20 ml) and dried in vacuo at 50 °C which afforded 0.77 g (63%) of the tjtie compound as a solid. Calculated for Cι2H8N2OS:
C, 63.14 %; H, 3.53 %; N, 12.27 %. Found
C, 62.86 %; H, 3.47 %; N, 12.17 %.
EXAMPLE 2
Figure imgf000039_0001
Naphthalene-2-carboxylic acid (3-oxo-2,3-dihydro-[1 ,2,4]thiadiazol-5-yl)-amide
To a mixture of 2-naphthyl carboxylic acid (5.0 g, 29.0 mmol) and 2 drops of N,N'- dimethylformamide in dry tetrahydrofuran (50 ml) was added dropwise thionyl chloride (6.3 ml, 87 mmol) and the resulting reaction mixture was stirred at reflux temperature for 3 h. The volatiles were evaporated in vacuo and the solid residue was redissolved in dry tetrahydrofuran (30 ml) and added dropwise to a solution of potassium thiocyanate (2.9 g, 30 mmol) in acetone (40 ml). The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was quenched with water (250 ml) and extracted with diethyl ether (2 x 100 ml). The combined organic extracts were washed with saturated aqueous sodium chloride (2 x 80 ml), dried (MgSO4), filtered and evaporated in vacuo affording 5.2 g (84%) of naphthalene-2-carbonyl isothiocyanate.
To a solution of the above isothiocyanate (5.0 g, 23 mmol) in acetone (100 ml) was ad¬ ded urea (1.44 g, 24 mmol) and the resulting mixture was heated at reflux temperature for 4 h. An additional portion of urea (0.8 g, 13.3 mmol) was added and the reaction mixture was heated at reflux temperature for 17 h. The cooled reaction mixture was qu¬ enched by addition of water (150 ml) and stirred for 15 min. The precipitate was filtered off and washed with water (2 x 25 ml), dried in vacuo at 50 °C affording 5.1 g (80%) of naphthalene-2-carboxylic acid ureidocarbothioyl-amide as a solid. To a stirred solution of the above ureidocarbothioyl-amide (4.5 g, 0.017 mol) in ethanol (40 ml) at 35 °C was added dropwise a 1 N solution of bromine in dichloromethane (17 ml) during 10 min. The resulting reaction mixture was stirred for 0.5 h at room tempera¬ ture. Water (50 ml) was added and the precipitate was filtered off, washed with water (2 x 50 ml) and diethyl ether (2 x 50 ml) and dried in vacuo at 50 °C affording 3.1 g (69%) of the title compound as a solid.
m.p.: > 250 °C.
Calculated for C13H9N3O2S:
C, 57.56 %; H, 3.34 %; N, 15.59 %. Found
C, 57.59 %; H, 3.34 %; N, 15.07 %.
EXAMPLE 3
Figure imgf000040_0001
5-Naphthalen-2-yl-[1 ,3,4]oxadiazol-2-ylamine
To a stirred solution of naphthalene-2-carboxylic acid hydrazide (0.6 g, 3.22 mmol, pre¬ pared as described in example 1) in dioxane (20 ml) was added a solution of sodium hy- drogen carbonate (0.27 g, 3.22 mmol) in water (15 ml) and the resulting mixture was stir¬ red for 5 min. To the reaction mixture was added cyanogen bromide (0.35 g, 3.3 mmol) and the mixture was stirred for 3 h at room temperature. The precipitate was filtered off and washed with diethyl ether (2 x 15 ml) and dried in vacuo at 50 °C affording 0.4 g of crude product which was suspended in absolute ethanol (15 ml) and stirred at reflux temperature for 0.5 h. The cooled suspension was filtered and the filter cake was dried in vacuo at 50 °C affording 130 mg (20%) of the title compound as a solid. m.p.: 245-247 °C
Calculated for C12H9N3O, 0.1x H20: C, 67.66 %; H, 4.35 %; N, 19.73 %. Found C, 67.56 %; H, 4.21 %; N, 19.84 %.
EXAMPLE 4
Figure imgf000041_0001
5-Naphthalen-2-yl-3H-[1 ,3,4]oxadiazol-2-one
To a stirred mixture of naphthalene-2-carboxylic acid hydrazide (2.0 g, 10.7 mmol, pre¬ pared as described in example 1) and triethylamine (1.1 g, 10.7 mmol) in dry tetrahy¬ drofuran (40 ml) was added carbonyl diimidazole (2.2 g, 13.4 mmol) at 0 °C. The reacti¬ on mixture was stirred at 0 °C for 1 h and at room temperature for 17 h. The resulting reaction mixture was evaporated in vacuo and to the residue was added water (50 ml) and ethyl acetate (50 ml). The phases were separated and the organic phase was washed with saturated aqueous sodium chloride (2 x 25 ml), dried (MgSO4) and filtered and evaporated in vacuo affording 2.2 g of crude product which was recrystallised from a mixture of ethyl acetate and heptane 1:1 (60 ml) affording after drying in vacuo at 50 °C 1.2 g (52%) of the title compound as a solid.
m.p.: 197-199 °C Calculated for Ci2H8N2O2: C, 67.92 %; H, 3.80 %; N, 13.20 %. Found C, 67.92 %; H, 3.73 %; N, 13.04 %. EXAMPLE 5
Figure imgf000042_0001
5-(2-Naphthalen-2-yl-vinyl)-1 ,3,4-oxadiazole-2(3H)-thione
To a stirred solution of 2-naphthyl acrylic acid (5.0 g, 25.0 mmol) in dry tetrahydrofuran (100 ml) and N, N-dimethylf ormamide (0.2 ml) was added dropwise at 0 °C oxalyl chlori¬ de (4.8 g, 38.0 mmol). The mixture was stirred for 1 h at room temperature and the sol¬ vent was evaporated jni vacuo affording crude acid chloride. To a solution of tert-butyl carbazate (6.7 g, 50.0 mmol) in dry tetrahydrofuran (80 ml) was added dropwise a solu¬ tion of the acid chloride in dry tetrahydrofuran (50 ml) at 0 °C. The resulting reaction mixture was stirred for 17 h at room temperature and the solvent was evaporated in vacuo. To the residue was added water (200 ml) and ethyl acetate (200 ml) and the phases were separated. The organic phase was washed with 0.1 N sodium hydroxide (2 x 100 ml), dried (MgSO4), filtered and evaporated in vacuo. The solid residue was sus¬ pended in heptane (50 ml), filtered off and dried in vacuo at 50 °C which afforded 4.7 g (59%) of N-(3-naphthalen-2-yl-acryloyl)hydrazine carboxylic acid tert butyl ester as a so¬ lid.
To a solution of the above hydrazine carboxylic acid tert butyl ester (4.5 g, 14.4 mmol) in ethanol (25 ml) was added 2 N hydrochloric acid and the mixture was refluxed for 1 h. The solvent was evaporated in vacuo and the residue was dissolved in water (100 ml) and made alkaline to pH = 9 with 1 N sodium hydroxide. The precipitated was filtered off and washed with water (2 x 30 ml) and heptane (2 x 30 ml) and dried in vacuo at 50 °C which afforded 2.8 g (92%) of 3-naphthalen-2-yl-acrylic acid hydrazide as a solid. To a stirred solution of the above acrylic acid hydrazide (1.5 g, 7.05 mmol) in absolute ethanol (15 ml) was added potassium hydroxide (0.40 g, 7.05 mmol) and carbondisulfide (1.50 g, 16.25 mmol) at 0 CC. After stimng for 1 ,5 h at 0 °C absolute ethanol (50 ml) was added and the reaction mixture was reflux temperature for 4 h. The reaction mixture was evaporated in vacuo, and to the residue was added water (100 ml). The resultant mixtu¬ re was acidified to pH = 1 with concentrated hydrochloric acid followed by addition of ethyl acetate (50 ml). The mixture was stirred for 0.5 h and the precipitate was filtered off, washed with water (2 x 10 ml) and diethyl ether (2 x 10 ml) and dried jn vacuo at 50 °C which afforded 0.31 g (17%) of the title compound as a solid.
m.p.: > 250 °C
Calculated for Cι4Hι0N2OS: xθ.75 H2O C, 60.09 %; H, 3.85 %; N, 10.01 %. Found C, 60.06 %; H, 3.24 %; N, 9.85 %.
EXAMPLE 6
Figure imgf000043_0001
5-(2-Naphthalen-2-yl-vinyl)-1,3,4-oxadiazol-2(3H)-one
To a stirred suspension of 3-naphthalen-2-yl-acrylic acid hydrazide (1.0 g, 4.71 mmol, prepared as described in example 5) and triethylamine (0.64 g, 6.28 mmol) in dry tetra¬ hydrofuran (15 ml) was added carbonyl diimidazole (1.02 g, 6.28 mmol) at 0 °C. The re¬ action mixture was stirred at room temperature for 1 h. The resulting reaction mixture was evaporated in vacuo and to the residue was added water (25 ml). The precipitate was filtered off and washed with water (2 x 10 ml) and diethyl ether (2 x 10 ml) and re- crystallised from ethyl acetate (100 ml) affording after drying in vacuo at 50 °C 0.5 g (45%) of the title compound as a solid.
m.p.: 252-254 °C
Calculated for Cι4HιoN2O2:
C, 70.58 %; H, 4.23 %; N, 11.76 %. Found
C, 70.87 %; H, 4.22 %; N, 11.61 %.
EXAMPLE 7
Figure imgf000044_0001
5-Naphthalen-2-yl-2H-pyrazol-3-ol
To a solution of diethyl carbonate (18 ml) and sodium hydride (5.9 g, 0.15 mol, 60% dis¬ persion in mineral oil) in dry toluene (60 ml) was added dropwise a solution of methyl 2- naphthyl ketone (10.0 g, 0.06 mol) in diethyl carbonate (7.4 g, 0.06 mol) and the resul¬ ting reaction mixture was slowly heated to 80 °C (exothermic) and diluted with toluene (50 ml, do to heavy precipitation). The reaction mixture was stirred and heated at 80 °C for 1 h. The cooled reaction mixture was quenched by carefully addition of water (100 ml) followed by addition of diethyl ether (100 ml). The phases were separated and the aqueous phase extracted with diethyl ether (100 ml). The combined organic phases we¬ re washed with water (100 ml) and saturated aqueous sodium chloride (100 ml), dried (MgSO4), filtered and evaporated in vacuo. The residue (15 g) was purified by column chromatography on silicagel (900 ml) using a mixture of ethyl acetate and heptane (1:10) as eluent. This afforded 10.1 g (71%) of 3-(2-naphthyl)-3-oxo-propionic acid ethyl ester as an oil. A mixture of the above β-keto ester (1.0 g, 4.13 mmol) and hydrazine hydrate (0.41 g, 8.25 mmol) in ethanol (15 ml) was stirred at reflux temperature for 17 h. The reaction mixture was cooled and the precipitate was filtered off and washed with ethanol (2 x 10 ml), dried in vacuo at 50 °C affording 0.3 g which was dissolved in a mixture of ethyl acetate (50 ml) and water (50 ml). 1 N hydrochloric acid was added to pH = 1 and the aqueous phase was separated off. The organic phase was washed with saturated aqueous sodium chloride (2 x 25 ml), dried (MgS04), filtered and evaporated in vacuo affording 75 mg (9%) of the title compound as a solid.
m.p.: 186 - 188 °C
Calculated for Cι3Hι0N2O: xθ.2 H20 C, 73.02 %; H, 4.90 %; N, 13.10 %. Found C, 72.95 %; H, 4.78 %; N, 12.96 %.
EXAMPLE 8
Figure imgf000045_0001
Naphthalene-2-carboxylic acid [1,3,4]thiadiazol-2-yl amide
To a stirred solution of 2-naphthyl carboxylic acid (76.5 g, 0.45 mol) in dichloromethane (500 ml) was added thionyl chloride (38.7 ml, 0.53 mol) and the mixture was heated at reflux temperature for 48 h. The volatiles were evaporated in vacuo affording 80 g (94%) of 2-naphthoyl chloride.
To a stirred solution of 2-amino-1,3,4-thiadiazol (0.84 g, 8.0 mmol) in pyridine (5 ml) was added 2-naphthoyl chloride (1.9 g, 10 mmol). The resulting mixture was stirred at reflux temperature for 15 min. cooled and quenched with water (100 ml). The precipitate was filtered off, washed with water (2 x 10 ml) and heptane (2 x 10 ml) and dried in vacuo affording 0.80 g (38%) of the title compound as a solid.
m.p.: 197 - 199 °C
Calculated for Cι3H9N3OS:
C, 61.16 %; H, 3.55 %; N, 16.46 %. Found
C, 61.49 %; H, 3.53 %; N, 16.52 %.
EXAMPLE 9
Figure imgf000046_0001
Naphthalene-2-carboxylic acid (5-amino-2H-[1 ,2,4]triazol-3-yl)-amide
A mixture of 2-naphthyl carboxylic acid (2.0 g, 11.6 mmol) and N.N'-carbonyldiimidazole (2.07 g, 12.8 mmol) in dry tetrahydrofuran (50 ml) was stirred at reflux temperature for 1 h. To the cooled reaction mixture was added 3,5-diamino-1,2,4-triazole (1.15 g, 11.6 mmol) and the resulting mixture was refluxed for 3 h. The cooled reaction mixture was quenched with water (75 ml) and extracted with diethyl ether (2 x 75 ml). The combined organic extracts were washed with saturated aqueous sodium chloride (2 x 50 ml), dried (MgSO4), filtered and evaporated in vacuo. The residue was crystallised from diethyl et- her (20 ml), filtered off and washed with diethyl ether (2 x 10 ml), dried in vacuo affor¬ ded 0.95 g (32%) of the title compound as a solid.
m.p.: 192 - 194 °C
Calculated for C13H11N5O: C, 61.65 %; H, 4.38 %; N, 27.65 %. Found C, 61.96 %; H, 4.39 %; N, 27.11 %.
EXAMPLE 10
Figure imgf000047_0001
Naphthalene-2-carboxylic acid (5-trifluoromethyl-[1 ,3,4]thiadiazol-2-yl)-amide
A mixture of 2-naphthyl carboxylic acid (2.0 g, 11.6 mmol) and N.N'-carbonyldiimidazole (2.07 g, 12.8 mmol) in dry tetrahydrofuran (50 ml) was stirred at reflux temperature for 1 h. To the cooled reaction mixture was added 2-amino-5-trifluoromethyl-1,2,4-thiadiazole (2.0 g, 11.6 mmol) and the resulting mixture was refluxed for 3 h. The cooled reaction mixture was quenched with water (100 ml) and extracted with ethyl acetate (2 x 50 ml). The combined organic extracts were washed with water (2 x 50 ml), saturated aqueous sodium chloride (2 x 50 ml), dried (MgSO4), filtered and evaporated in vacuo. The resi¬ due was crystallised from diethyl ether (20 ml), filtered off and washed with diethyl ether (2 x 15 ml) and heptane (2 x 15 ml), dried in vacuo at 50 °C afforded 1.4 g (37%) of the title compound as a solid.
m.p.: 249 - 251 °C
Calculated for Ci4H8N3F3OS:
C, 52.01 %; H, 2.49 %; N, 13.00 %. Found C, 51.90 %; H, 2.40 %; N, 13.20 %. EXAMPLE 11
Figure imgf000048_0001
Naphthalene-2-carboxylic acid (4H-[1 ,2,4]triazol-3-yl)-amide
A mixture of 2-naphthyl carboxylic acid (3.0 g, 17 mmol) and N.N'-carbonyldiimidazole (3.1 g, 19 mmol) in dry tetrahydrofuran (75 ml) was stirred at reflux temperature for 1 h. To the cooled reaction mixture was added 3-amino-1,2,4-triazole (1.5 g, 17 mmol) and the resulting mixture was refluxed for 2.5 h. The cooled reaction mixture was quenched with water (150 ml) and the precipitate was filtered off and washed with water (2 x 20 ml), heptane (2 x 20 ml) and diethyl ether (2 x 20 ml), dried in vacuo at 50 °C affording 2.55 g (61%) of the title compound as a solid.
m.p.: 191 - 192 °C
Calculated for Cι3H10N4O:
C, 65.54 %; H, 4.23 %; N, 23.52 %. Found C, 65.30 %; H, 4.20 %; N, 23.61 %.
EXAMPLE 12
Figure imgf000048_0002
5-Naphthalen-2-yl-2,3-dihydro-[1,3,4]oxadiazol-2-yl-cyanamide
To a stirred solution of naphthalene-2-carboxylic acid hydrazide (0.7 g, 3.76 mmol) in is¬ opropanol (40 ml) was added triethylamine (630 μl, 4.51 mmol) and diphenyl cyanocar- bonimidate (0.99 g, 4.14 mmol) and the resulting reaction mixture was stirred at room temperature for 1.5 h. The volatiles were evaporated in vacuo and to the residue was added water (50 ml) and diethyl ether (50 ml). The organic phase was separated and the aqueous phase was acidified to pH = 1 with concentrated hydrochloric acid. The preci¬ pitate was filtered off and washed with water (3 x 10 ml) and diethyl ether (2 x 10 ml) and dried in vacuo at 50 °C which afforded 0.45 g (51%) of the title compound as a so¬ lid.
m.p.: > 250 °C
Calculated for C13H10N4O:
C, 66.10 %; H, 3.41 %; N, 23.72 %. Found
C, 65.81 %; H, 3.33 %; N, 23.54 %.
EXAMPLE 13
Figure imgf000049_0001
5-Naphthalen-2-yl-3H-[1 ,3,4]thiadiazole-2-thione
To a stirred solution of 1-naphthyl acrylic acid (20.0 g, 0.12 mol) in dry tetrahydrofuran (200 ml) and N,N-dimethylformamide (2 ml) was added dropwise at 0 °C oxalyl chloride (21.5 ml, 0.256 mol). The mixture was stirred for 1 h at room temperature and the sol¬ vent was evaporated in vacuo affording crude acid chloride. To a solution of 25% aqueous ammonium hydroxide (33 ml) in tetrahydrofuran (100 ml) was added dropwise a solution of the acid chloride in dichloromethane (150 ml) at 0 °C. The resulting reaction mixture was stirred for 1 h at room temperature. To the reaction mixture was added diethyl ether (200 ml) and water (200 ml). The precipitate was filtered off and washed with water (2 x 75 ml) and diethyl ether (2 x 75 ml), dried in vacuo at 50 °C affording 20.0 g (100%) of naphthalene-2-carboxylic acid amide as a solid.
To a solution of the above amide (10.0 g, 58 mmol) in dry tetrahydrofuran (250 ml) was added [2,4-bis-(4-methoxyphenyl)-1,3-dithia-2,4-diphosphenate-2,4-disulfide] (Lawesson's reagent) (16.5 g, 41 mmol) and the resulting mixture was stirred at room temperature for 48 h. The volatiles were evaporated in vacuo and the residue was dis¬ solved in ethyl acetate (100 ml) and filtered through silicagel (100 ml) using ethyl acetate as eluent. The solvent was evaporated in vacuo and to the residue was added a mixture of ethyl acetate (25 ml) and heptane (25 ml). The precipitate was filtered off and washed with heptane (40 ml), dried in vacuo at 50 °C which afforded 9.0 g (83%) of naphthale- ne-2-carbothioic acid amide as a solid.
To a solution of the above carbothioic acid amide (8.0 g, 42 mmol) in methanol (200 ml) was added dropwise hydrazine monohydrate (3.3 ml, 68 mmol). The resulting reaction mixture was stirred for 17 h at room temperature. The reaction mixture was evaporated in vacuo to 1/3 of is volume and purified by column chromatography on silicagel (1 I) using first ethyl acetate and later on a mixture of ethyl acetate and ethanol (1:1) as eluents. This afforded 4.4 g (56%) of naphthalene-2-carboximidic acid hydrazide as a solid.
To a solution of the above hydrazide (4.0 g, 22 mmol) in methanol (100 ml) was added dropwise carbon disulphide (3.4 ml, 56 mmol). The resulting reaction mixture was stirred for 4 h at room temperature. The precipitate was filtered off and washed with water (2 x 15 ml) and ethyl acetate (2 x 15 ml) and dried in vacuo at 50 °C yielding 1.7 g of crude product which was recrystallised from ethyl acetate (20 ml) affording after drying in vacuo at 50 °C 1.3 g (85%) of the title compound as a solid. m.p.: 254 - 256 °C
Calculated for Ci2H8N2S2: C, 58.99 %; H, 3.30 %; N, 11.47 %. Found C, 59.10 %; H, 3.21 %; N, 11.35 %.
EXAMPLE 14
Figure imgf000051_0001
2-Methylsulfanyl-5-naphthalen-2-yl-[1,3,4]thiadiazole
To a solution of 5-naphthalen-2-yl-3H-[1 ,3,4]thiadiazole-2-thione
(2.8 g, 11.5 mmol) in methanol (100 ml) was added dropwise 1 N sodium hydroxide (.12 ml, 12 mmol) at 0 °C. After stirred for 10 min. at 0 °C iodomethane (2.0 g, 13.8 mmol) was added dropwise and stirring was continued at 0 °C for 5 min. and at room tempera¬ ture for 2 h. The volatiles were evaporated in vacuo and to the residue was added water (100 ml). The precipitate was filtered off and dried in vacuo at 50 °C. The dried com¬ pound (2.4 g) was recrystallised from ethyl acetate (60 ml) affording after drying in vacuo at 50 °C 0.7 g (23%) of the title compound as a solid.
m.p.: 133 - 135 °C
Calculated for Cι3HιoN2S2:
C, 60.44 %; H, 3.90 %; N, 10.84 %. Found
C, 60.47 %; H, 3.89 %; N, 10.66 %. EXAMPLE 15
Figure imgf000052_0001
2-Methanesulfinyl-5-naphthalen-2-yl-[1 ,3,4]thiadiazole
To a solution of 2-methylsulfanyl-5-naphthalen-2-yl-[1,3,4]thiadiazole (1.3 g, 5.03 mmol) in dichloromethane (50 ml) was added 50% moisten 3-chloro peroxy- benzoic acid (1.9 g, 5.53 mmol) at 0 °C. After stirred for 1 h at 0 °C the reaction was di¬ luted with dichloromethane (50 ml) and quenched with addition of saturated aqueous sodium hydrogen carbonate (50 ml). The organic phase was separated and washed with water (50 ml), dried (MgSO4), filtered and evaporated in vacuo. The residue (1.4 g) was purified by column chromatography on silicagel (400 ml) using a mixture of ethyl acetate and heptane (1:1) as eluent. This afforded after drying in vacuo at 50 °C 0.9 g (65%)-of the title compound as a solid.
m.p.: 148 - 150 °C
Calculated for Cι3HιoN2OS2:
C, 56.91 %; H, 3.67 %; N, 10.21 %. Found
C, 57.07 %; H, 3.71 %; N, 9.92 %.
EXAMPLE 16
Figure imgf000052_0002
5-Naphthalen-2-yl-2,3-dihydro-[1,3,4]thiadiazol-2-yl-cyanamide
To a stirred solution of potassium tert-butoxide (0.46 g, 4.08 mmol) in tert-butanol (25 ml) was added cyanamide (0.32 g, 7.65 mmol) and the resulting mixture was stirred for 15 min. at room temperature. To this mixture was added 2-methanesulfinyl-5- naphthalen-2-yl-[1 ,3,4]thiadiazole (0.7 g, 2.55 mmol) and the mixture was heated at re¬ flux temperature for 15 min. followed by addition of an additional portion of cyanamide (0.2 g, 4.76 mmol). Heating was continued for an additional 2 h. The cooled reaction mixture was quenched by addition of 1 N sodium hydroxide (150 ml) and diluted by ad¬ dition of diethyl ether. The organic phases were separated and the aqueous phase was acidified to pH = 1 with concentrated hydrochloric acid. The precipitate was filtered off, washed with water (2 x 10 ml) and diethyl ether (2 x 10 ml) and dried at 50 °C which af- forded 0.25 g (39%) of the title compound as a solid.
m.p.: > 260 °C
Calculated for Ci3Hi0N4S: xθ.25 H2O C, 60.80 %; H, 3.34 %; N, 21.82 %. Found
C, 61.10 %; H, 3.07 %; N, 21.77 %.
EXAMPLE 17
r>
5-(2-Naphtylmethyl)-1H-tetrazole
2-Bromomethylnaphthalene (5.00 g, 23 mmol) was dissolved in N.N'-dimethylformamide (50 ml) and potassium cyanide (2.95 g, 45 mmol) was added and the resulting mixture was stirred at room temperature for 16 hours. The supernatant was decanted and parti- tioned between water (100 ml) and diethyl ether (2 x 75 ml). The combined organic pha¬ ses were washed with water (100 ml), dried (MgS04), filtered and evaporated in vacuo affording 2.53 g (67%) of 2-naphtylacetonitrile as a solid.
m.p.: 84 - 85 °C.
Rf = 0.12 (SiO2: Ethyl acetate/heptane = 1:10)
A mixture of the above acetonitrile (2.50 g, 15 mmol), ammonium chloride (1.60 g, 30 mmol) and sodium azide (1.94 g, 30 mmol) in N.N'-dimethylformamide (25 ml) was stir- red at 125 °C for 15 hours. The cooled reaction mixture was poured into water (300 ml) and acidified with 1 N hydrochloric acid, stirred at room temperature for 2 hours. The precipitate was filtered off and washed successively with water, a 1 :1 mixture of diethyl ether and heptane and finally with heptane. The solid was dried by suction affording 1.69 g (53%) of the title compound as a solid.
m.p.: 153 - 156 °C.
From the mother liquor further 1.05 g (33%) of the title compound was isolated giving a total yield of 86%.
EXAMPLE 18
Figure imgf000054_0001
5-(1 -Naphtylmethyl)-1 H-tetrazole
1-Chloromethylnaphthalene (5.00 ml, 33 mmol) was dissolved in N,N'- dimethylformamide (50 ml) and potassium cyanide (4.31 g, 66 mmol) and potassium io- dide (0.1 g) were added and the resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was partitioned between water (150 ml) and diethyl ether (2 x 100 ml). The combined organic phases were washed with water (100 ml), dried (MgSO4), filtered and concentrated in vacuo affording 5.41 g (98%) of 1- naphtylacetonitrile as an oil.
TLC: Rr = 0.14 (Si02: Ethyl acetate/heptane = 1:10)
A mixture of the above acetonitrile (5.40 g, 32 mmol), ammonium chloride (2.59 g, 48 mmol) and sodium azide (3.15 g, 48 mmol) in N,N'-dimethylformamide (100 ml) was stir- red at 125 °C for 16 hours. After cooling the mixture was poured into water (300 ml) and extracted with ethyl acetate (2 x 150 ml) The combined organic phases were washed with water (100 ml) and evaporated in vacuo. The residue was crystallised from diethyl ether (20 ml), filtered off and washed with diethyl ether affording 1.86 g (27%) of the title compound as a solid.
m.p.: 157 - 159 °C.
EXAMPLE 19
Figure imgf000055_0001
3-(Biphenyl-4-yloxymethyl)-N-(3-hydroxy-[1 ,2,4]thiadiazol-5-yl)-benzamide
To a stirred solution of 3-bromomethyl-benzoic acid methyl ester (30.9 g, 0.13 mol) and 4-phenylphenole (20.15, 0.12 mol) in dry N,N'-dimethylf ormamide (250 ml) was added potassium carbonate (48.1 g, 0.35 mol) and the resulting mixture was stirred for 20 h. The reaction mixture was poured on to water (600 ml) followed by addition of ethyl ace¬ tate (200 ml). The precipitate was filtered off and washed with water (2 x 50 ml) and dried in vacuo at 50 °C which afforded 30.2 g (85%) of 3-(biphenyl-4-yloxymethyl)- benzoic acid methyl ester as a solid.
To the above benzoic acid methyl ester (12.3 g, 40.0 mmol) suspended in a mixture of water (125 ml) and ethanol (125 ml) was added sodium hydroxide (4.80 g, 0.12 mol) and the reaction mixture was heated at 60 °C for 20 h. The volatiles were evaporated in vacuo and to the residue was added water (50 ml) followed by concentrated hydrochloric acid to pH = 1. The resulting mixture was stirred at room temperature for 20 h and the precipitate was filtered off and washed with water (3 x 25 ml), suspended in diethyl ether (100 ml) and stirred for 2 h. The precipitate was filtered off and dried in vacuo at 50 °C which afforded 10.23 g (81%) of 3-(biphenyl-4-yloxymethyl)benzoic acid as a solid.
To a mixture of the above benzoic acid (4.72 g, 15.0 mmol) and 2 drops of N,N'- dimethylformamide in dry tetrahydrofuran (50 ml) was added dropwise thionyl chloride (3.3 ml, 45 mmol) and the resulting reaction mixture was stirred at reflux temperature for 3 h. The volatiles were evaporated in vacuo and the solid residue was redissolved in dry tetrahydrofuran (30 ml) and added dropwise to a solution of potassium thiocyanate (1.53 g, 15.3 mmol) in acetone (40 ml). The reaction mixture was stirred at room temperature for 16 h. filtered and evaporated in vacuo. To a solution of the residue in acetone (50 ml) was added urea (0.92 g, 15,3 mmol) and the resulting mixture was heated at reflux tem¬ perature for 4 h. The cooled reaction mixture was evaporated in vacuo and the residue was stirred for 0.5 h with ice water (100 ml). The precipitate was filtered off and washed with water (2 x 25 ml), dried in vacuo at 50 °C. The crude product (6.03 g) was recrystal¬ lised from acetonitrile (750 ml) affording 3.17 g (52%) of 3-(biphenyl-4-yloxymethyl)-N- ureidocarbothioyl-benzamide as a solid.
To a stirred solution of the above ureidocarbothioyl-benzamide (3.17 g, 7.8 mmol) in et¬ hanol (30 ml) at 35 °C was added dropwise a 1 N solution of bromine in dichlorometha¬ ne (7.8 ml, 7.8 mmol)) during 10 min. The resulting reaction mixture was stirred for 0.5 h at room temperature. The precipitate was filtered off, washed with diethyl ether (2 x 15 ml) and recrystallised from a mixture of N,N'-dimethylf ormamide and acetone (1 :2) which afforded after washing with acetone (10 ml) and diethyl ether (20 ml) and drying in vacuo at 50 °C 1.73 g (55%) of the title compound as a solid.
Calculated for C22Hι7N3O3S: C, 65.49 %; H, 4.25 %; N, 10.42 %. Found C, 65.40 %; H, 4.34 %; N, 10.10 %.
EXAMPLE 20
Figure imgf000057_0001
3-(Biphenyl-4-yloxymethyl)-N-(1H-tetrazol-5-yl)benzamide
To a mixture of 3-(biphenyl-4-yloxymethyl)benzoic acid (2.36 g, 7.5 mmol, prepared as described in example 20) and 2 drops of N,N'-dimethylformamide in dry tetrahydrofuran (25 ml) was added dropwise thionyl chloride (1.7 ml, 22.5 mmol) and the resulting reac¬ tion mixture was stirred at reflux temperature for 3 h. The volatiles were evaporated in vacuo and the solid residue was dissolved in dichloromethane (20 ml) and added dropwise to a stirred suspension of 5-amino-tetrazole monohydrate (0.86 g, 8.3 mmol) and triethylamine (3.2 ml, 22.5 mmol) in dichloromethane (10 ml). After the addition was complete pyridine (5 ml) and 4-dimethylaminopyridine (10 mg) were added and the re¬ sulting mixture was stirred at room temperature for 48 h. The volatiles were evaporated in vacuo and the residue was suspended in water (100 ml) and acidified to pH = 3 with concentrated hydrochloric acid. Ethyl acetate (100 ml) was added and the mixture was stirred for 0.5 h. The precipitate was filtered off and washed with water (2 x 10 ml) and dried in vacuo at 50 °C afforded 1.39 g (50%) of the titie compound as a solid.
Calculated for C2ιHι7NsO2: xθ.1 triethylamine C, 68.00 %; H, 4.89 %; N, 18.72 %. Found C, 67.88 %; H, 4.68 %; N, 18.23 %.
EXAMPLE 21
Figure imgf000058_0001
5-(3-(Biphenyl-4-ylmetoxy)benzylidene)-2,4-thiazolidinedione
A mixture of 3-hydroxybenzaldehyde (6.02 g, 49 mmol), 4-phenyl-benzylchloride (10 g, 49 mmol) and potassium carbonate (20 g, 148 mmol) in N.N'-dimethylformamide (100 ml) was stirred at room temperature for 16 h. The mixture was poured into water (500 ml) and stirred for 1 h. The solid formed was filtered off, washed with water (2 x 200 ml) and heptane (2 x 75 ml) and dried in vacuo at 50 °C for 16 h affording 12.3 g (87%) of 3- (biphenyl-4-ylmetoxy)benzaldehyde as a solid. TLC showed presence of unchanged 4- phenylbenzylchloride.
TLC: Rf = 0.28 (SiO2: Ethyl acetate/heptane = 1 :10) A mixture of the above benzaldehyde (5.00 g, 17 mmol), 2,4-thiazolidinedione (3.03 g, 26 mmol) and piperidine (0.35 ml, 3.5 mmol) in ethanol (75 ml) was stirred at reflux tem¬ perature for 16 h. The reaction mixture was cooled and the precipitated was filtered off and washed thoroughly with ethanol and dried in vacuo at 50 °C. The solid was first washed with a mixture of ethyl acetate, heptane and dichloromethane (1 :1:6, 40 ml) and then washed with dichloromethane (20 ml). Drying in vacuo at 50 °C afforded 1.88 g (28%) of the titie compound as a solid.
m.p.: 224 - 226 °C. EXAMPLE 22
Figure imgf000059_0001
5-((9-(4-Phenylbenzyl)-9H-carbazol-3-yl)-methylidene)-2,4-thiazolidinedione
Carbazole (8.25 g, 49 mmol) was dissolved in N,N'-dimethylformamide (100 ml). Under a atmosphere of nitrogen was added sodium hydride (2.56 g, 64 mmol of a 60% suspen- sion in mineral oil) in portion during 15 minutes. The mixture was then stirred at room temperature for 0.5 h. To the resulting mixture was added 4-phenylbenzylchloride (10 g, 49 mmol) in portions during 10 minutes. Then additional N,N'-dimethylformamide (100 ml) was added and the mixture was stirred at room temperature for 3.5 h. Water (125 ml) was added and the mixture was stirred vigorously at room temperature for 0.5 h, the solid formed was filtered off, washed with water (2 x 100 ml) and with hexane (2 x 100 ml). Drying in vacuo at 50 °C for 16 h afforded 15.9 g (97%) of 9-(biphenyl-4-ylmethyl)- 9H-carbazole as a solid. TLC: Rf = 0.46 (SiO2, ethyl acetate/heptane = 1:10).
Under a atmosphere of nitrogen at 0 °C phosphorous oxychloride (3.0 ml, 33 mmol) was added dropwise to N.N'-dimethylformamide (1.2 ml, 15.8 mmol). After the addition was complete, the mixture was stirred at 0 °C for 1 h and heated to 45 °C. At 45 °C the above carbazole (5.00 g, 15 mmol) was added during 15 minutes. The solid reaction mixture was then heated at 95 °C for 16 h. To the cooled reaction mixture water (125 ml) was added and the mixture was stirred vigorously at room temperature for 4 h. The solid for¬ med was filtered off, washed with water and dried in vacuo at 50 °C affording almost quantitatively 9-(4-phenylbenzyl)-9H-carbazole-3-carboxaldehyde as a solid. TLC: Rf = 0.19 (SiO2: ethyl acetate/heptane = 1:10)
A mixture of the above carboxaldehyde (2.00 g, 5.5 mmol), 2,4-thiazolidinedione (0.97 g, 8.3 mmol) and piperidine (0.11 ml, 1.1 mmol) in ethanol (50 ml) was stirred at reflux temperature for 4 days. After cooling the precipitate was filtered off and washed tho¬ roughly with ethanol and dried in vacuo at 50 °C. The solid was washed with dichloro¬ methane (75 ml), and dried in vacuo at 50 °C which afforded 0.22 g (9%) of the title compound as a solid.
m.p.: >250 °C.
Additional 1.4 g of the titie compound was isolated from the mother liquor.
EXAMPLE 23
Figure imgf000060_0001
(3-(Naphthalen-2-ylmethoxy)phenyl)phosphonic acid
2-Bromomethylnaphthalene (5.00 g, 22.6 mmol) was dissolved in N,N'- dimethylformamide (100 ml) and potassium carbonate (9.4 g, 68 mmol) and 3- bromophenol (3.91 g, 22.6 mmol) were added. The mixture was stirred vigorously for 16 h at room temperature and then poured into water (700 ml). The precipitate was filtered off, washed with water and dried in vacuo at 50 °C for 16 h to give 6.17 g (87%) of 2-(3- bromophenoxymethyl)naphthalene as a solid.
m.p.: 109 - 112 °C The above naphthalene (5.50 g, 17.6 mmol) was dissolved in toluene (50 ml), and di- ethylphosphite (2.50 ml, 19.4 mmol), triethylamine (2.9 ml, 21.1 mmol) and tetra- kis(triphenylphosphine)palladium(0) (1.02 g, 0.88 mmol) were added and the mixture was stirred at reflux temperature for 16 h. The mixture was concentrated in vacuo and the residue was purified by column chromatography on silica gel eluting with a mixture of ethyl acetate, heptane and triethylamine (50:50:1). This afforded 5.11 g (78%) of (3- (naphthalen-2-ylmethoxy)phenyl]phosphonic acid diethyl ester as a solid.
m.p.: 55 - 57 °C.
The above phosphonic acid diethyl ester (4.43 g, 12 mmol) was dissolved in acetonitrile (50 ml) and bromotrimethylsilane (3.5 ml, 26 mmol) was added. The resulting mixture was stirred at room temperature for 48 h. The slightly turbid mixture was filtered and the solvent evaporated in vacuo. The residue was dissolved in diethyl ether (100 ml) and methanol (6 ml) was added. The mixture was stirred at room temperature for 16 h and the precipitate filtered off, washed with diethyl ether and dried in vacuo at 50 °C for 16 h which afforded 3.51 g (93%) the title compound as a solid.
m.p.: 131 - 134 °C.
EXAMPLE 24
Figure imgf000061_0001
((3-(Biphenyl-4-ylmethoxy)-phenyl)fluoromethyl)phosphonic acid
3-(Biphenyl-4-ylmetoxy)benzaldehyde (5.0 g, 17 mmol, prepared as described in exam¬ ple 22) was mixed with di-tert-butyl phosphite (3.4 g, 17 mmol) and caesium fluoride (3.2 g, 21 mmol) was added and the mixture was stirred at room temperature for 5 h. The re- action mixture was diluted with dichloromethane (50 ml), filtered and concentrated in vacuo The residue was crystallised from heptane, filtered and washed with heptane which afforded 7 20 g (86%) of ((3-(biρhenyl-4- ylmethoxy)phenyl)hydroxymethyl)phosphonιc acid di-tert-butyl ester as a solid
TLC. Rf = 0.24 (SiO2, ethyl acetate/heptane = 1.1).
Diethylaminosulfur trifluonde (2 2 ml, 8 2 mmol) was dissolved in dichloromethane (25 ml) and the solution was cooled to -70 °C and added dropwise to a solution of the above hydroxymethylphosphonic acid di-tert-butyl ester (4.0 g, 8 3 mmol) in dichloromethane (15 ml) at -70 °C. The mixture was stirred at -70 °C for 3 h and at room temperature for 20 h. With stirring the mixture was poured into 1 N aqueous potassium hydroxide (200 ml) and the mixture was extracted with dichloromethane (1 x 300 ml) and (1 x 100 ml) The combined organic extracts were washed with saturated aqueous sodium chloride (100 ml), dried (MgSO4), filtered and evaporated in vacuo affording 1.43 g (36%) of ((3- (biphenyl-4-ylmethoxy)-phenyl)fluoromethyl)phosphonιc acid di-tert-butyl ester as a solid
1H-NMR (200 MHz, CDCI3). δH = 1 45 (18H, d), 5 12 (2H, s), 5.5 (1H, dd), 6 95-7.65 (13H, m)
The above fluoromethylphosphonic acid di-tert-butyl ester (1 29 g, 2.7 mmol) was dissol¬ ved in dichloromethane (10 ml) and trifluoroacetic acid (2.5 ml) was added and the mix¬ ture was stirred at room temperature for 16 h The mixture was filtered and the filtrate was evaporated in vacuo. The residue was partitioned between ethyl acetate (100 ml) and water (50 ml) and a solid was formed in the aqueous phase This was filtered off and dried in vacuo at 50 °C affording 55 mg (6%) of the titie compound as a solid,
m.p.: 216 °C (dec).
EXAMPLE 25 The PTP1B and PTPα cDNA was obtained by standard polymerase chain reaction technique using the Gene Amp Kit according to the manufacturer's instructions (Perkin Elmer/Cetus). The oligonucleotide primers were designed according to published sequences (Chernoff et al., Proc. Natl. Acad. Sci. U.S.A. 87: 2735-2739 (1990); Krueger et al.EMBO J. 9: 3241-3252 (1990)) including convenient restriction nuclease sites to allow cloning into expression vectors. The cDNA corresponding to the full-length sequence of PTP1B and the intracellular part of PTPα were introduced into the insect cell expression vector pVL1392. The proteins were expressed according to standard procedures. PTP1 B was semi-purified by ion exchange chromatography, and PTPα was purified to apparent homogeneity using a combination of ion exchange chromatography and gel filtration techniques using standard procedures. TC-PTP and LAR domain 1 were obtained from New England Biolabs. Yersinia PTP was a kind gift from J.E. Dixon, The University of Michigan, Ann Arbor, USA p- Nitrophenyl phosphate was purchased from Sigma and used without further purification.
Methods
p-Nitrophenyl phosphate (pNPP) is a general phosphatase substrate including a substrate for PTPases. When pNPP (colourless) is hydrolysed by a phosphatase to phosphate and p- nitrophenolate (yellow in alkaline solutions) the enzyme reaction can be followed by measuring the optical density at 410 nm after adjusting the pH appropriately. pNPP was used as general substrate to analyse the PTPase inhibitory capacity of the compounds of the invention.
The inhibiting effect of a compound is given by its Kj value, which expresses the concentration of inhibitor (μM) in the reaction mixture necessary for a 50 percent reduction of the enzyme activity.
The Kj may be determined by a titration curve using several appropriately diluted solutions of the inhibitor or by using the following more simple formula, when the concentration of inhibitor is in large excess of the enzyme concentration:
Ki = lo x E/(Eo - E) where l0 is the concentration of inhibitor (μM) added to the reaction mixture, E is the activity of the enzyme in the reaction mixture containing the inhibitor, and Eo is the enzyme activity in a corresponding control reaction mixture without the inhibitor.
The Kj values of inhibitors towards PTP1B were measured as follows. In all cases the inhibiting effects were determined at pH 5.5 and at 37 °C with a reaction time of 60 minutes.
The reaction mixtures were:
1) 25 μl enzyme solution
25 μl inhibitor solution in DMSO 500 μl substrate solution
or
2) 25 μl enzyme solution
25 μl DMSO 500 μl substrate solution
The substrate solution contained 0.2 M acetate buffer, pH 5.5, 11 mM p-nitrophenyl phosphate, 5.5 mM dithiotreitol.
The reaction was stopped by addition of 4 ml 0.2 N NaOH, and the enzyme activity was determined by measuring the release of p-nitrophenol at 410 nm. The inhibiting effect was calculated as shown above.
The Kj values of inhibitors towards TdPTP, LAR domainl, PTPα domain 1+2, and Yersinia PTP were measured essentially as described for PTP1B with the exception that all reactions were carried out in 96-wells microtiter plates. In all cases the inhibiting effects were determined at pH 5.5 and at room temperature with a reaction time of 15 minutes. The reaction mixtures were:
1) 5 μl enzyme solution
5 μl inhibitor solution in DMSO (final concentration 100 μM) 90 μl substrate solution or
2) 5 μl enzyme solution 5 μl DMSO
90 μl substrate solution
The final concentrations: 0.2 M acetate buffer, pH 5.5, 5 mM p-nitrophenyl phosphate, 5 mM dithiotreitol.
The reaction was stopped by addition of 100 μl 0.4 N NaOH, and the enzyme activity was determined by measuring the release of p-nitrophenol at 405 nm. The inhibiting effect was calculated as shown above.
Results
Using the above assay systems we have demonstrated that compounds of the invention are PTPase inhibitors.

Claims

1. A compound of formula (I)
(L)— Arn— FL,— A
(0 wherein
n is 1 , 2, 3, 4, or 5 and (L)n represents up to five (5) substituents which independently of each other are hydrogen, Ci-e-alkyl, Ci-e-alkoxy, hydroxy, halogen, trihalogenomethyl, hydroxy-Ci- e-alkyl, amino-Ci-e-alkyl, -COR2, -N02, -CN, -CHO, Cι.6-alkanoyloxy, caΦamoyl, -NR5R6, aryloxy optionally substituted;
R2 is Ci-e-alkyl, aryl optionally substituted, aralkyl optionally substituted, -OH, -NR3R4 wherein R3 and R4 independently of each other are hydrogen, Ci-e-alkyl, aryl optionally substituted, aralkyl optionally substituted;
R5 and R6 are independently of each other hydrogen or Ci-e-alkyl, aryl optionally substituted, aralkyl optionally substituted or -COZι wherein Zi is Ci-e-alkyl, aryl optionally substituted, aralkyl optionally substituted;
or L is
Figure imgf000066_0001
wherein X is a chemical bond, -CO, -CONR7 , -NR7CO, -NR7 ,- 0-, -S-, -SO, or-SO2;
Yi and Y2 are independently a chemical bond, -O-, -S-, or -NR7; R7 is hydrogen, Ci-e-alkyl, aryl optionally substituted, aralkyl optionally substituted, heteroaryl optionally substituted, -COZ2 wherein Z2 is Ci-e-alkyl, aryl optionally substituted, aralkyl optionally substituted;
Wi and W2 are independently a chemical bond or saturated or unsaturated Cι.β-alkylene;
Ai is aryl optionally substituted, heteroaryl optionally substituted, biaryl optionally substituted, arylheteroaryl optionally substituted, -NR8R9 wherein R8 and R9 independently are hydrogen, Ci-6-alkyl, aryl optionally substituted, aralkyl optionally substituted, heteroaryl optionally substituted, -COZ3 wherein Z3 is Ci-e-alkyl, aryl optionally substituted, aralkyl optionally substituted, heteroaryl optionally substituted or when R8 and R9 together with the nitrogen atom forms a ring system Ai is a saturated or partially saturated heterocyclic ring system optionally substituted with
Cι-6-alkyl, aryl optionally substituted, aralkyl optionally substituted, heteroaryl optionally substituted, -OH, Ci-e-alkoxy, hydroxy-Cι.6-alkyl, amino-Ci-e-alkyl, -COZ4 wherein Z, is -OH,
Ci-e-alkyl, NRι0Rn wherein R10 and Rn independently are hydrogen, Cι.6-alkyl;
Ri is a linker selected from a chemical bond, -Cι.6-aikyl-, -0(CH2)m-, -NRιr, -CONR12-, - NR13CO-, -SO2NRι4-, -NR15SOr, -CRι6=CRι7-, -CH= -, -CHR17, -CH2-, -CHF-, -CF2-, -S02-; R12, R13, Ru, Rιs Rιβ and Rι7 are hydrogen, Cι.6-aikyl, aralkyl and m is 1,2, or 3;
A is -PO(ORι8)(ORι9), -NH-SO3H, -NH-SO2-CH3, -NH-SO2-CF3, -CO-NH-OH or a heterocycle as shown in scheme 1 wherein the point of attachment is indicated with a I
(single bond) or || (double bond)
Scheme 1
Figure imgf000068_0001
Figure imgf000068_0002
Figure imgf000068_0003
Figure imgf000068_0004
Figure imgf000068_0005
optionally substituted by hydrogen, halogen, Ci-e-alkyl optionally substituted by phenyl optionally substituted by Ci-e-alkoxy, Ci-e-alkylthio; -COOX1 wherein X1 is Ci-e-alkyl optionally substituted by phenyl or benzyl optionally substituted; Rι8, and R19, are Ci-e-alkyl, phenyl, benzyl;
R20 is hydrogen, -OH, Ci-e-alkoxy, -SH, Ci-e-alkylthio, -COR21, -SOR22, -SO2R23, -NR24R25, -NHCN, halogen, trihalogenomethyl;
R21, R∑∑.and R23 are -OR26, Ci-e-alkyl, -NR24R25, trihalogenomethyl; R24 and R25 independently are hydrogen, Ci-e-alkyl, -S02R26, -COZ5 wherein Z5 is Ci-e-alkyl, trihalogenomethyl R26 is hydrogen, Ci-e-alkyl, trihalogenomethyl; nn is 1 or 2; and An is aryl or heteroaryl;
or a pharmaceutically acceptable salt thereof.
2. A compound according to the preceding claim: wherein
L is ArYι-(Wι)-X-(W2)-Y2 wherein X is a chemical bond, -CO, -CONR7 , -NR7CO, -NR7 ,-0-, - S-, -SO, or -SO2;
Y1 and Y2 are independently a chemical bond, -O-, -S-, or -NR7; R7 is hydrogen, Ci-e-alkyl, aryl optionally substituted, aralkyl optionally substituted, heteroaryl optionally substituted, -COZ2 wherein Z2 is Cι-6-alkyl, aryl optionally substituted, aralkyl optionally substituted;
W1 and W2 are independently a chemical bond or saturated or unsaturated Cι.6-alkylene;
Ai is aryl optionally substituted, heteroaryl optionally substituted, biaryl optionally substituted, arylheteroaryl optionally substituted, -NR8R9 wherein R8 and R9 independently are hydrogen, Ci-e-alkyl, aryl optionally substituted, aralkyl optionally substituted, heteroaryl optionally substituted, -COZ3 wherein Z3 is Cι-6-alkyl, aryl optionally substituted, aralkyl optionally substituted, heteroaryl optionally substituted or when R 8 and R9 together with the nitrogen atom forms a ring system Ai is a saturated or partially saturated heterocyclic ring system optionally substituted with Ci-e-alkyl, aryl optionally substituted, aralkyl optionally substituted, heteroaryl optionally substituted, -OH, Ci- e-alkoxy, hydroxy-Cι.6-alkyl, amino-Ci-e-alkyl, -COZi wherein Z4 is -OH, Ci-e-alkyl, -NRι0Rn wherein Rι0 and Rn independently are hydrogen, Ci-e-alkyl;
Ri is a linker selected from a chemical bond, -Ci-e-alkyl-, -O(CH2)m-, -NR12-, -CONR12-, - NR13CO-, -S02NRn-, -NRisSOr, -CR1β=CRιr, -CH= -, -CHR,7, -CH2-, -CHF-, -CF2-, -S02-; R12, Ri3> Ru, R15 Rιe and R17 are hydrogen, Ci-e-alkyl, aralkyl and m is 1,2, or 3;
A is -PO(ORιβ)(ORιβ), -NH-SO3H, -NH-SO2-CH3, -NH-S02-CF3, -CO-NH-OH or a heterocycle as shown in scheme 1 wherein the point of attachment is indicated with a | (single bond) or || (double bond)
Scheme 1
Figure imgf000071_0001
Figure imgf000071_0002
Figure imgf000071_0003
Figure imgf000071_0004
Figure imgf000071_0005
optionally substituted by hydrogen, Ci-e-alkyl optionally substituted by phenyl optionally substituted by Ci-e-alkoxy, Ci-e-alkylthio; -COOX1 wherein X1 is Ci-e-alkyl optionally substituted by phenyl or benzyl optionally substituted; R18, and Ri9 independently are hydrogen, Ci-e-alkyl, phenyl, benzyl; R20 is hydrogen, -OH, Ci-e-alkoxy, -SH,-Cι.6-alkylthio, -COR2ι, -SOR22, -S02R23, -NR24R25,
-NHCN, halogen, trihalogenomethyl;
R21, R22, and R23 are -OR2e, Ci-e-alkyl, -NR24R25, trihalogenomethyl;
R24 and R25 independently are hydrogen, Cι-6-alkyl, -SO2R26, -COZ5 wherein Z5 is
Ci-e-alkyl, trihalogenomethyl
R26 is hydrogen, Ci-e-alkyl, trihalogenomethyl,
An is aryl or heteroaryl; nn is 1 or 2 and n is preferably 1, 2 or 3.
or a pharmaceutically acceptable salt thereof.
3. A compound according to any one of the preceding claims wherein Ar! is an optionally substituted phenyl, naphthyl or heteroaryl.
4. A compound according to any one of the preceding claims wherein Ri is ethylene (-CH=CH-).
5. A compound according to claim 4: wherein the double bond configuration is trans (E).
6. A compound according to any one of the preceding claims wherein Ri is -CONH- or-NHCO-.
7. A compound according to any one of the preceding claims wherein A is -NH-SO3H, -NH-SO2-CH3 or -NH-SO2-CF3.
8. A compound according to any one of the claims 1-6: wherein A is -PO(OH)2.
9. A compound according to any one of the preceding claims wherein A is:
2-Hydroxy-1 H-imidazol-4(5)-yl; 4(5)-Hydroxy-1 H-imidazol-2-yl; 3-Hydroxy-1 H-pyrazol-1-yl; 3-Hydroxy-1 H-pyrazol-3-yl; 3-Hydroxy-1 H-pyrazol-5-yl;
3-Hydroxy-4H-1 ,2,4-triazol-5-yl;
3-Hydroxy-1 ,2,4-oxadiazol-5-yl;
3-Hydroxy-1 ,2,4-thiadiazol-5-yl; 2-Hydroxy-oxazol-4-yl;
2-Hydroxy-thiazol-4-yl;
4-Hydroxy-thiazol-2-yl;
5-Hydroxy-1 ,2,4-oxadiazol-3-yl;
5-Hydroxy-1 ,2,4-thiadiazol-3-yl; o 5-Hydroxy-1 ,2,5-thiadiazol-4-yl;
3-Hydroxy-isoxazol-5-yl;
3-Hydroxy-isothiazol-5-yl;
5-Hydroxy-isoxazol-3-yl;
1-Oxo-5-hydroxy-2,3-dihydro-1,2,4-thiadiazol-3-yl; 5 1 -Oxo-5-hydroxy-2,3-dihydro-1 ,2,4-thiadiazol-3-ylidene;
4-Hydroxy-1 ,2,3-triazol-2-yl;
4-Hydroxy-1 ,2,3-triazol-1-yl;
2,4-Dihydroxy-imidazol-5-yl;
2-Hydroxy-4-oxo-imidazol-5-ylidene; 0 2,4-Dihydroxy-thiazol-5-yl;
2-Hydroxy-4-oxo-thiazol-5-ylidene;
2,4-Dihydroxy-oxazoI-5-yl;
2-Hydroxy-4-oxo-oxazol-5-ylidene;
3-Oxo-5-hydroxy-1,2,4-thiazolidin-2-yl; 5 1 , 1-Dioxo-3-oxo-[1 ,2,5]thiadiazolidin-2-yl;
1 , 1-Dioxo-3-oxo-[1 ,2,5]thiadiazolidin-5-yl;
2-Oxo-3,4-dihydroxy-5H-furan-5-yl;
2-Oxo-4-hydroxy-5H-furan-3-yl;
Tetrazol-5-yl; 0 2,5-Dioxo-pyrrol-3-yl; or tautomers thereof.
10. A compound according to any one of the claims 1-6: wherein Ri is -CF2- or -CHF- and A is selected from the following: 2,4-Dihydroxy-thiazol-5-yl; 2,4-Dihydroxy-oxazol-5-yl.
11. A compound according to any one of the preceding claims selected from the following:
5-Naphthalen-2-yl-3H-[1,3,4]oxadiazole-2-thione;
Naphthalene-2-carboxylic acid (3-oxo-2,3-dihydro-[1 ,2,4]thiadiazol-5-yl)-amide;
5-Naphthalen-2-yl-[1 ,3,4]oxadiazol-2-ylamine;
5-Naphthalen-2-yl-3H-[1 ,3,4]oxadiazol-2-one; 5-(2-Naphthalen-2-yl-vinyl)-1,3,4-oxadiazole-2(3H)-thione;
5-(2-Naphthalen-2-yl-vinyl)-1 ,3,4-oxadiazol-2(3H)-one;
5-Naphthalen-2-yl-2H-pyrazol-3-ol;
Naphthalene-2-carboxylic acid [1 ,3,4]thiadiazol-2-yl amide;
Naphthalene-2-carboxylic acid (5-amino-2H-[1 ,2,4]triazol-3-yl)-amide; Naphthalene-2-carboxylic acid (5-trifluoromethyl-[1,3,4]thiadiazol-2-yl)-amide;
Naphthalene-2-carboxylic acid (4H-[1,2,4]triazol-3-yl)-amide;
5-Naphthalen-2-yl-2,3-dihydro-[1 ,3,4]oxadiazol-2-yl-cyanamide;
5-Naphthalen-2-yl-3H-[1,3,4]thiadiazole-2-thione;
2-Methylsulfanyl-5-naphthalen-2-yl-[1 ,3,4]thiadiazole; 2-Methanesulfinyl-5-naphthalen-2-yl-[1 ,3,4]thiadiazole;
5-Naphthalen-2-yl-2,3-dihydro-[1 ,3,4]thiadiazol-2-yl-cyanamide;
Naphthalene-2-carboxylic acid (3-hydroxy-isoxazol-5-yl)-amide;
((3-(Biphenyl-4-ylmethoxy)-phenyl)fluoromethyl)phosphonic acid
N-(3-Hydroxy-(1,2,4-thiadiazol-5-yl)-4-methoxy-benzamide; 3-(Biphenyl-4-yloxymethyl)-N-(3-hydroxy-(1,2,4-thiadiazol-5-yl)benzamide;
9-Bipheπyl-4-ylmethyl-9H-carbazol-3-carboxylic acid (3-hydroxy-(1 ,2,4-thiadiazol-5-yl) amide;
5-(2-(3-(Biphenyl-4-yloxymethyl)-phenyl)vinyl)-1H-tetrazole;
2-(3-(Biphenyl-4-yloxymethyl)-phenyl)vinyl phosphonic acid; 5-(Difluoro-(4-(2-(methyl-pyridin-2-yl-amino)ethoxy)-phenyl)-methyl)-thiazolidine-2,4-dione;
5-((4-(2-(5-Ethyl-pyridin-2-yl)-ethoxy)-phenyl)-difluoro-methyl)-thiazolidine-2,4-dione;
5-((2-benzyl-chroman-6-yl)-difluoro-methyl)-thiazolidine-2,4-dione;
5-(Difluoro-(4-(3-(5-methyl-2-phenyl-oxazol-4-yl)-propionyl)-phenyl)-methyl)-thiazolidine-2,4- dione; 5-(Difluoro-(4-(2-hydroxy-2-(5-methyl-2-phenyl-oxazol-4-yl)-ethoxy)-phenyl)-methyl)- thiazolidine-2,4-dione; or 5-(Difluoro-(4-(6-hydroxy-2,5,7,8-tetramethyl-chroman-2-yl-methoxy)-phenyl)-methyl)- thiazolidine-2,4-dione. 5-(2-Naphtylmethyl)-1 H-tetrazole;
5-(1-Naphtylmethyl)-1 H-tetrazole;
3-(Biphenyl-4-yloxymethyl)-N-(1H-tetrazol-5-yl)benzamide;
5-(3-(Biphenyl-4-ylmetoxy)benzylidene)-2,4-thiazolidinedione;
5-((9-(4-Phenylbenzyl)-9H-carbazol-3-yl)-methylidene)-2,4-thiazolidinedione; (3-(Naphthalene-2-ylmethoxy)phenyl)phosphonic acid; or a pharmaceutically acceptable salt thereof.
12. A method of preparing a compound according to any one of the preceding compounds claims, characterised in
(L)n-Arι R<— ≡ N + MN3 ► (I)
(II) (III)
allowing a compound of formula (II) wherein (L)n, Ar1 ( Ri, and n are as defined above to react with an azide of formula (III) wherein M is defined as above in order to obtain the compounds of formula (I) wherein A is a 5-substituted tetrazole; or
(L)n— ArrX, + R, -A ► (I)
(VI") (IX)
allowing a compound of formula (VIII), wherein (L)n, n, An and Xi are as defined above to react with a compound of formula (IX) wherein Ri is CH2=CH and A is as defined above in order to obtain the compounds of formula (I); or
(LJ - A^ -CHO + L^-CH2- A ► (|)
(X) (XI) allowing a compound of formula (X), wherein (L)n, n, and An are as defined above to react with a compound of formula (XI) wherein A and U, are as defined above in order to obtain the compounds of formula (I); or
(LJ - A^ -CHj— |^ + OHO- A ► (|)
(XII) (XI")
allowing a compound of formula (XII), wherein (L)n, n, An and L, are as defined above to react with a compound of formula (XIII) wherein A is as defined above in order to obtain the compounds of formula (I); or
Figure imgf000076_0001
(XIV)
allowing a compound of formula (XIV), wherein (L)„, n, and An are as defined above and X is a suitably leaving group and R is Ci-e-alkyl to react with a compound of formula (XV) wherein X is O or S whereby a compound of formula (I) is produced wherein Ri is CF2 and A are 2,4-dihydroxy-oxazolidin-5-yl or 2,4-dihydroxy-thiazolidin-5-yl;
13. A pharmaceutical composition comprising as active component a compound according to any one of the preceding compound claims together with a pharmaceutically acceptable earner or diluent.
14. A pharmaceutical composition suitable for modulating the activity of PTPases or other molecules with tyrosine phosphate recognition unit(s) comprising an effective amount of a compound according to any one of the preceding compound claims together with a pharmaceutically acceptable caπier or diluent.
15. The pharmaceutical composition according to any one of claims 13 or 14 suitable for treating or preventing type I diabetes, type ll diabetes, impaired glucose tolerance, insulin resistance, obesity, immune dysfunction's including autoimmunity and AIDS, diseases with dysfunction's of the coagulation system, allergic diseases, osteoporosis, proliferative disorders including cancer and psoπasis, diseases with decreased or increased synthesis or effects of growth hormone, diseases with decreased or increased synthesis of hormones or cytokines that regulate the release of/or response to growth hormone, diseases of the brain including Alzheimer's disease and schizophrenia, and infectious diseases.
16 The pharmaceutical composition according to any one of the claims 13, 14 or 15 compπsing between 0 5 mg and 1000 mg of a compound according to any one of the preceding compound claims per unit dose
17 A method of modulating the activity of PTPases or other molecules with phosphotyrosine recognition unιt(s) in a subject in need of such treatment compπsing administeπng to said subject an effective amount of a compound or composition according to any one of the preceding compound or composition claims
18 The use of a compound according to any one of the preceding compound claims for prepaπng a medicament
19 The use of a compound according to any one of the preceding compound claims for prepaπng a medicament for modulating the activity of PTPases or other molecules with tyrosine phosphate recognition unιt(s)
20 The use of a compound according to any one of the preceding compound claims for prepanng a medicament for treating or preventing type I diabetes, type II diabetes, impaired glucose tolerance, insulin resistance, obesity, immune dysfunction's including autoimmunity and AIDS, diseases with dysfunction's of the coagulation system, allergic diseases, osteoporosis, proliferative disorders including cancer and psoπasis, diseases with decreased or increased synthesis or effects of growth hormone, diseases with decreased or increased synthesis of hormones or cytokines that regulate the release of/or response to growth hormone, diseases of the brain including Alzheimer's disease and schizophrenia, and infectious diseases
21. The use of a compound according to any one of the preceding compound claims for preparing a medicament for treating a subject in need of such treatment.
22. The use of a compound according to any one of the preceding compound claims for preparing a medicament for use as an immunosuppressant.
23. An immobilised compound comprising a suitable solid-phase coupled with a compound according to any one of the preceding compound claims.
24. A method for coupling a compound according to any one of the preceding compound claims to a suitable solid-phase matrix.
25. A method for isolating a protein or a glycoprotein with affinity for a compound according to any one of the preceding compound claims from a biological sample, comprising
- contacting an immobilised compound according to claim 23 with said biological sample in order for said immobilised compound to form a complex by binding said protein or glycoprotein
- removing unbound material from said biological sample and isolating said complex - extracting said protein or glycoprotein from said complex.
26. A method for isolating a protein-tyrosine phosphatase with affinity for a compound according to any one of the preceding compound claims from a biological sample, comprising - contacting an immobilised compound according to claim 23 with said biological sample in order for said immobilised compound to form a complex by binding said protein-tyrosine phosphatase
- removing unbound material from said biological sample and isolating said complex
- extracting said protein-tyrosine phosphatase.
27. A method for isolating a Src-homology 2 domain containing protein or a phosphotyrosine binding domain containing protein with affinity for a compound according to any one of the preceding compound claims from a biological sample, comprising - contacting an immobilised compound according to claim 23 with said biological sample in order for said immobilised compound to form a complex by binding said Src-homology 2 domain containing protein or a phosphotyrosine binding domain containing protein
- removing unbound material from said biological sample and isolating said complex
- extracting said Src-homology 2 domain containing protein or a phosphotyrosine binding domain containing protein from said complex.
28. A compound according to any one of the preceding compound claims coupled to a fluorescent or radioactive molecule.
29. A method for coupling a fluorescent or radioactive molecule to a compound according to any one of the preceding compound claims comprising
- contacting said compound with said fluorescent or radioactive molecule in a reaction mixture to produce a complex - removing uncomplexed material and isolating said complex from said reaction mixture.
30. A method for detecting protein-tyrosine phosphatase or other molecules with phosphotyrosine recognition unit(s) in a cell or in a subject using a compound according to claim 28 comprising - contacting said cell or an extract thereof or a biological sample from said subject or by injecting said compound into said subject in order for said compound to produce a complex with said protein-tyrosine phosphatase or said molecules with phosphotyrosine recognition unit(s)
- detecting said complex, thereby detecting tiie presence of said protein tyrosine phosphatase or said other molecules with phosphotyrosine recognition unit(s).
31. A method for quantifying the amount of protein-tyrosine phosphatases or other molecules with phosphotyrosine recognition unit(s) in a cell or in a subject using a compound according to claim 28 comprising - contacting said cell or an extract thereof or a biological sample from said subject or by injecting said compound into said subject in order for said compound to produce a complex with said protein-tyrosine phosphatase or said molecules with phosphotyrosine recognition unit(s)
- measuring the amount of said complex, thereby detecting the presence of said protein tyrosine phosphatase or said molecules with phosphotyrosine recognition unit(s).
32. A method for determining the function of a given protein-tyrosine phosphatase or group of protein-tyrosine phosphatases or said molecules with phosphotyrosine recognition unit(s) in a cell or a subject using a compound according to claim 28 comprising - contacting said cell or an extract thereof or a biological sample from said subject or by injecting said compound into said subject in order for said compound to produce a complex with said protein-tyrosine phosphatase or said molecules with phosphotyrosine recognition unit(s) measuring the biological effects induced by said complex.
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Cited By (88)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999058519A1 (en) * 1998-05-12 1999-11-18 American Home Products Corporation Phenyl oxo-acetic acids useful in the treatment of insulin resistance and hyperglycemia
WO2000017211A1 (en) * 1998-09-21 2000-03-30 Merck Frosst Canada & Co. Phosphonic acids derivatives as inhibitors of ptp-1b
WO2000043006A1 (en) * 1999-01-19 2000-07-27 Sankyo Company, Limited Inhibitor for nerve cell death due to glutamic acid cytotoxicity
US6166069A (en) * 1998-05-12 2000-12-26 American Home Products Corporation Phenyl oxo-acetic acids useful in the treatment of insulin resistance and hyperglycemia
WO2001017516A2 (en) * 1999-09-10 2001-03-15 Novo Nordisk A/S Method of inhibiting protein tyrosine phosphatase 1b and/or t-cell protein tyrosine phosphatase and/or other ptpases with an asp residue at position 48
WO2001046203A1 (en) * 1999-12-22 2001-06-28 Merck Frosst Canada & Co. Phosphonic acid biaryl derivatives as inhibitors of protein tyrosine phosphatase 1b (ptp-1b)
US6365592B1 (en) 1999-05-14 2002-04-02 Merck Frosst Canada & Co. Phosphonic and carboxylic acid derivatives as inhibitors of protein tyrosine phosphatase-1B (PTP-1B)
US6376524B1 (en) 2000-06-21 2002-04-23 Sunesis Pharmaceuticals, Inc. Triphenyl compounds as interleukin-4 antagonists
US6448429B1 (en) 1999-12-22 2002-09-10 Merck Frosst Canada & Co. Protein tyrosine phosphatase 1B (PTP-1B) inhibitors containing two ortho-substituted aromatic phosphonates
US6465444B2 (en) 2000-03-22 2002-10-15 Merck Frosst Canada & Co. Aryldifluoromethylphosphonic acids with sulfur-containing substituents as PTP-1B inhibitors
US6486142B2 (en) 1999-12-22 2002-11-26 Merck Frosst Canada & Co. Phosphonic acid derivatives as inhibitors of protein tyrosine phosphatase 1B (PTP-1B)
US6503935B1 (en) 1998-08-07 2003-01-07 Abbott Laboratories Imidazoles and related compounds as α1A agonists
WO2003043997A1 (en) * 2001-11-15 2003-05-30 Eli Lilly And Company Peroxisome proliferator activated receptor alpha agonists
US6583126B2 (en) 1999-12-22 2003-06-24 Merck Erosst Canada & Co. Phosphonic acid derivatives as inhibitors of protein tyrosine phosphatase 1B (PTP-1B)
US6593326B1 (en) 1998-12-24 2003-07-15 Astrazeneca Ab 2,4-diamino pyrimidine compounds having anti-cell proliferative activity
US6613903B2 (en) 2000-07-07 2003-09-02 Novo Nordisk A/S Modulators of protein tyrosine phosphatases (PTPases)
WO2003082841A1 (en) * 2002-04-03 2003-10-09 Novartis Ag 5-substituted 1,1-dioxo-`1,2,5!thiazolidine-3-one derivatives as ptpase 1b inhibitors
JP2003532708A (en) * 2000-05-11 2003-11-05 コンセホ・スペリオル・インヴェスティガシオンズ・シエンティフィカス Heterocyclic inhibitors of glycogen synthase kinase GSK-3
US6649608B2 (en) 2000-03-01 2003-11-18 Astrazeneca Ab 2,4-di(hetero-)arylamino (oxy)-5-substituted pyrimidines as antineoplastic agents
US6710052B2 (en) 2000-03-01 2004-03-23 Astrazeneca Pyrimidine compounds
US6713473B1 (en) 1999-04-20 2004-03-30 Meiji Seika Kaisha, Ltd. Tricyclic compounds
WO2004041799A1 (en) * 2002-11-07 2004-05-21 Astrazeneca Ab 5- (substituted phenyl) -thiadiazolidine-3-ones and their use as ptp1b
WO2004050646A1 (en) * 2002-11-29 2004-06-17 Astrazeneca Ab 1, 2, 5-thiadiazolidin-3-one 1, 1 dioxide derivatives as inhibitors of protein tyrosine phosphatase ptp1b
US6777433B2 (en) 1999-12-22 2004-08-17 Merck Frosst Canada & Co. Protein tyrosine phosphatase 1B (PTP-1B) inhibitors containing two ortho-substituted aromatic phosphonates
WO2004071448A2 (en) * 2003-02-12 2004-08-26 Transtech Pharma Inc. Substituted azole derivatives as inhibitors of protein tyrosine phosphatases
US6838464B2 (en) 2000-03-01 2005-01-04 Astrazeneca Ab 2,4-Di(hetero-)arylamino(-oxy)-5-substituted pyrimidines as antineaoplastic agents
US6844341B2 (en) 2001-02-17 2005-01-18 Astrazeneca Ab Pyrimidine derivatives for inhibition of cell proliferation
WO2005012256A1 (en) 2003-07-22 2005-02-10 Astex Therapeutics Limited 3, 4-disubstituted 1h-pyrazole compounds and their use as cyclin dependent kinases (cdk) and glycogen synthase kinase-3 (gsk-3) modulators
US6855719B1 (en) 1999-08-21 2005-02-15 Astrazeneca Ab Imidazo[1,2-A]pyridine and pyrazolo[2,3-A]pyridine derivatives
US6906065B2 (en) 2000-03-28 2005-06-14 Astrazeneca Ab 4-Amino-5-cyano-2-anilino-pyrimidine derivatives and their use as inhibitors of cell-cycle kinases
US6908920B2 (en) 2000-07-11 2005-06-21 Astrazeneca Ab Pyrimidine derivatives
US6933272B1 (en) 1998-09-22 2005-08-23 Erik Helmerhorst Use of non-peptidyl compounds for the treatment of insulin related ailments
US6939872B2 (en) 2001-05-30 2005-09-06 Astrazeneca Ab 2-anilino-pyrimidine derivatives as cyclin dependent kinase inhibitors
US6951878B2 (en) 1998-03-12 2005-10-04 Novo Nordisk A/S Benzo[b]thiophenyl or tetrahydro-benzo[b]thiophenyl modulators of protein tyrosine phosphatases (PTPases)
US6969714B2 (en) 2000-09-05 2005-11-29 Astrazeneca Ab Imidazolo-5-YL-2-anilino-pyrimidines as agents for the inhibition of the cell proliferation
WO2005114197A2 (en) * 2004-04-15 2005-12-01 Albert Einstein College Of Medicine Of Yeshiva University Activity-based probes for protein tyrosine phosphatases
WO2006058648A2 (en) * 2004-12-03 2006-06-08 F. Hoffmann-La Roche Ag Biaryloxymethylarene carboxylic acids
WO2006077428A1 (en) 2005-01-21 2006-07-27 Astex Therapeutics Limited Pharmaceutical compounds
WO2006077424A1 (en) 2005-01-21 2006-07-27 Astex Therapeutics Limited Pharmaceutical compounds
US7115624B1 (en) 1999-09-10 2006-10-03 Novo Nordisk A/S Method of inhibiting protein tyrosine phosphatase 1B and/or T-cell protein tyrosine phosphatase 4 and/or other PTPases with an Asp residue at position 48
US7141596B2 (en) 2003-10-08 2006-11-28 Incyte Corporation Inhibitors of proteins that bind phosphorylated molecules
US7153964B2 (en) 2000-03-01 2006-12-26 Astrazeneca Ab Pyrimidine compounds
WO2007039177A2 (en) 2005-09-29 2007-04-12 Sanofi-Aventis Phenyl- and pyridinyl- 1, 2 , 4 - oxadiazolone derivatives, processes for their preparation and their use as pharmaceuticals
US7208509B2 (en) 2003-03-24 2007-04-24 Roche Palo Alto Llc Non-nucleoside reverse transcriptase inhibitors
WO2007059359A2 (en) * 2005-11-21 2007-05-24 Biogen Idec Ma Inc. Substituted pyrazalones
WO2007115058A2 (en) * 2006-03-31 2007-10-11 Novartis Ag Thiadiazolidinone inhibitors of ptpase
WO2007119600A1 (en) * 2006-03-23 2007-10-25 Otsuka Pharmaceutical Co., Ltd. Carbazole compound
US7355049B2 (en) 2003-06-24 2008-04-08 Hoffmann-La Roche Inc. Biaryloxymethylarenecarboxylic acids as glycogen synthase activator
US7427626B2 (en) 2003-05-16 2008-09-23 Astrazeneca Ab 2-Anilino-4-(imidazol-5-yl)-pyrimidine derivatives and their use as cdk (cdk2) inhibitors
US7442697B2 (en) 2002-03-09 2008-10-28 Astrazeneca Ab 4-imidazolyl substituted pyrimidine derivatives with CDK inhibitory activity
US7446105B2 (en) 2002-03-09 2008-11-04 Astrazeneca Ab Pyrimidine compounds
US7465728B2 (en) 2002-03-09 2008-12-16 Astrazeneca Ab Derivatives of 4-(imidazol-5-yl)-2-(4-sulfoanilino)pyrimidine with CDK inhibitory activity
WO2008157407A2 (en) * 2007-06-13 2008-12-24 University Of Virginia Patent Foundation Thiadiazole, oxadiazole and triazole derivatives for treating leukemia
US7485638B2 (en) 2002-03-09 2009-02-03 Astrazeneca Ab Pyrimidine compounds
US7521473B2 (en) 2004-02-25 2009-04-21 Wyeth Inhibitors of protein tyrosine phosphatase 1B
WO2009080223A1 (en) 2007-12-26 2009-07-02 Sanofi-Aventis Cyclic pyridyl-n-(1,3,4)-thiadiazol-2-yl-benzene sulfonamides, processes for their preparation and their use as pharmaceuticals
US7579344B2 (en) 2003-05-16 2009-08-25 Astrazeneca Ab Pyrimidine derivatives possessing cell-cycle inhibitors activity
EP2107054A1 (en) * 2008-04-01 2009-10-07 Università Degli Studi Di Milano - Bicocca Antiproliferative compounds and therapeutic uses thereof
US7655652B2 (en) 2004-02-03 2010-02-02 Astrazeneca Ab Imidazolo-5-yl-2-anilinopyrimidines as agents for the inhibition of cell proliferation
US7666891B2 (en) 2004-07-27 2010-02-23 Roche Palo Alto Llc Heterocyclic antiviral compounds
US7745428B2 (en) 2005-09-30 2010-06-29 Astrazeneca Ab Imidazo[1,2-A]pyridine having anti-cell-proliferation activity
US7851493B2 (en) 2005-09-29 2010-12-14 Sanofi-Aventis Phenyl-1,2,4-oxadiazolone derivatives with phenyl group, processes for their preparation and their use as pharmaceuticals
US7906540B2 (en) 2007-03-29 2011-03-15 Roche Palo Alto Llc Non-nucleoside reverse transcriptase inhibitors
WO2011057959A1 (en) 2009-11-11 2011-05-19 F. Hoffmann-La Roche Ag Indole and indazole derivatives as glycogen synthase activators
WO2011058154A1 (en) 2009-11-16 2011-05-19 F. Hoffmann-La Roche Ag Biphenyl carboxylic acids and bioisosteres as glycogen synthase activators
WO2011057993A1 (en) 2009-11-11 2011-05-19 F. Hoffmann-La Roche Ag Indazolone derivatives as glycogen synthase activators
WO2011057956A1 (en) 2009-11-11 2011-05-19 F. Hoffmann-La Roche Ag Benzisoxazole analogs as glycogen synthase activators
WO2011058122A1 (en) 2009-11-16 2011-05-19 F. Hoffmann-La Roche Ag Piperidine analogs as glycogen synthase activators
WO2011067266A1 (en) 2009-12-04 2011-06-09 F. Hoffmann-La Roche Ag Carboxylic acid analogs as glycogen synthase activators
WO2011067174A1 (en) 2009-12-01 2011-06-09 F. Hoffmann-La Roche Ag Aniline analogs as glycogen synthase activators
US8012988B2 (en) 2005-04-11 2011-09-06 Novartis Ag N-(2,4-dioxo-6-(tetrahydrofuran-2-yl)-7-(trifluoromethyl)-1,4-dihydro-2H-quinazolin-3-yl)methanesulfonamide
US8106070B2 (en) 2006-10-20 2012-01-31 Merck Sharp & Dohme Corp. Substituted imidazoles as bombesin receptor subtype-3 modulators
US8183275B2 (en) 2006-10-20 2012-05-22 Merck Sharp & Dohme Corp. Substituted imidazoles as bombesin receptor subtype-3 modulators
US8193228B2 (en) 2006-10-20 2012-06-05 Merck Sharp & Dohme Corp. Substituted imidazole as bombesin receptor subtype-3 modulators
US8293778B2 (en) 2004-07-27 2012-10-23 Roche Palo Alto Llc Heterocyclic antiviral compounds
US8299085B2 (en) 2004-07-27 2012-10-30 Novartis Ag Quinazoline derivatives
CN102834392A (en) * 2010-02-17 2012-12-19 詹森药业有限公司 Aminothiazolones as estrogen related receptor-alpha modulators
US8357711B2 (en) 2007-03-23 2013-01-22 Pfizer Limited Heterocyclic sulfonamides as inhibitors of ion channels
US9340503B2 (en) 2009-01-28 2016-05-17 Karus Therapeutics, Limited Scriptaid isosteres and their use in therapy
US9505728B2 (en) 2012-03-09 2016-11-29 Inception 2, Inc. Triazolone compounds and uses thereof
US9676754B2 (en) 2012-12-20 2017-06-13 Inception 2, Inc. Triazolone compounds and uses thereof
US9676765B2 (en) 2012-11-07 2017-06-13 Karus Therapeutics Limited Histone deacetylase inhibitors and their use in therapy
US9776976B2 (en) 2013-09-06 2017-10-03 Inception 2, Inc. Triazolone compounds and uses thereof
US9862685B2 (en) 2013-05-10 2018-01-09 Karus Therapeutics Limited Histone deacetylase inhibitors
US10407435B2 (en) 2014-10-29 2019-09-10 Karus Therapeutics Limited Diheteroaryl histone deacetylase inhibitors and their use in therapy
US10533003B2 (en) 2014-10-29 2020-01-14 Karus Therapeutics Limited Polyheteroarl histone deacetylase inhibitors and their use in therapy
US11578066B1 (en) 2019-12-20 2023-02-14 Tenaya Therapeutics, Inc. Fluoroalkyl-oxadiazoles and uses thereof
US11938134B2 (en) 2017-03-10 2024-03-26 Eikonizo Therapeutics, Inc. Metalloenzyme inhibitor compounds

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0193256A1 (en) * 1985-01-19 1986-09-03 Takeda Chemical Industries, Ltd. Thiazolidinedione derivatives, their production and use
EP0339549A2 (en) * 1988-04-25 1989-11-02 F. Hoffmann-La Roche Ag Tyr-peptide analogs
WO1991012003A1 (en) * 1990-02-09 1991-08-22 The Upjohn Company Use of insulin sensitizing agents to treat hypertension
EP0500337A1 (en) * 1991-02-22 1992-08-26 University Of Colorado Foundation, Inc. Composition containing aryl-substituted rhodanine derivatives for the treatment of type I diabetes
WO1995007694A1 (en) * 1993-09-15 1995-03-23 Sankyo Company, Limited Use of thiazolidinediones to prevent or delay onset of niddm
WO1996040113A2 (en) * 1995-06-07 1996-12-19 Sugen, Inc. Phosphatase inhibitors
EP0764632A2 (en) * 1995-09-21 1997-03-26 Eli Lilly And Company Selective beta3 adrenergic agonists

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0193256A1 (en) * 1985-01-19 1986-09-03 Takeda Chemical Industries, Ltd. Thiazolidinedione derivatives, their production and use
EP0339549A2 (en) * 1988-04-25 1989-11-02 F. Hoffmann-La Roche Ag Tyr-peptide analogs
WO1991012003A1 (en) * 1990-02-09 1991-08-22 The Upjohn Company Use of insulin sensitizing agents to treat hypertension
EP0500337A1 (en) * 1991-02-22 1992-08-26 University Of Colorado Foundation, Inc. Composition containing aryl-substituted rhodanine derivatives for the treatment of type I diabetes
WO1995007694A1 (en) * 1993-09-15 1995-03-23 Sankyo Company, Limited Use of thiazolidinediones to prevent or delay onset of niddm
WO1996040113A2 (en) * 1995-06-07 1996-12-19 Sugen, Inc. Phosphatase inhibitors
EP0764632A2 (en) * 1995-09-21 1997-03-26 Eli Lilly And Company Selective beta3 adrenergic agonists

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, No. 125:195654; & JP,A,08 157 461 (18-06-96). *
STN INTERNATIONAL, File CAPLUS, Accession No. 1996:543848, YANAGISAWA HIROAKI et al., "Preparation of (Azolylphenoxy)alkyl Aryl or Heterocyclyl Sulfone Derivatives Having Aldose Reductase-Inhibitory Activity as Hypolipidemics, Hypoglycemics and Antiobesity Agents". *
TETRAHEDRON LETTERS, Volume 33, No. 29, 1992, MARK S. SMYTH et al., "A General Method for the Preparation of Benzylic x,x-Difluorophosphonic Acids; Non-Hydrolyzable Mimetics of Phosphotyrosine", pages 4137-4140. *
TETRAHEDRON, Volume 52, No. 30, 1996, BIN YE et al., "Synthesis of a Difluorophosphonomethyl-Containing Phosphatase Inhibitor Designed from the X-Ray Structure of a PTP1B-Bound Ligand", pages 9963-9970. *

Cited By (126)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6951878B2 (en) 1998-03-12 2005-10-04 Novo Nordisk A/S Benzo[b]thiophenyl or tetrahydro-benzo[b]thiophenyl modulators of protein tyrosine phosphatases (PTPases)
US6166069A (en) * 1998-05-12 2000-12-26 American Home Products Corporation Phenyl oxo-acetic acids useful in the treatment of insulin resistance and hyperglycemia
WO1999058519A1 (en) * 1998-05-12 1999-11-18 American Home Products Corporation Phenyl oxo-acetic acids useful in the treatment of insulin resistance and hyperglycemia
US6503935B1 (en) 1998-08-07 2003-01-07 Abbott Laboratories Imidazoles and related compounds as α1A agonists
US6174874B1 (en) 1998-09-21 2001-01-16 Merck Frosst Canada & Co. Phosphonic acids derivatives as inhibitors of protein tyrosine phosphate 1B (PTP-1B)
WO2000017211A1 (en) * 1998-09-21 2000-03-30 Merck Frosst Canada & Co. Phosphonic acids derivatives as inhibitors of ptp-1b
US6933272B1 (en) 1998-09-22 2005-08-23 Erik Helmerhorst Use of non-peptidyl compounds for the treatment of insulin related ailments
US6593326B1 (en) 1998-12-24 2003-07-15 Astrazeneca Ab 2,4-diamino pyrimidine compounds having anti-cell proliferative activity
WO2000043006A1 (en) * 1999-01-19 2000-07-27 Sankyo Company, Limited Inhibitor for nerve cell death due to glutamic acid cytotoxicity
US6713473B1 (en) 1999-04-20 2004-03-30 Meiji Seika Kaisha, Ltd. Tricyclic compounds
US6365592B1 (en) 1999-05-14 2002-04-02 Merck Frosst Canada & Co. Phosphonic and carboxylic acid derivatives as inhibitors of protein tyrosine phosphatase-1B (PTP-1B)
US6855719B1 (en) 1999-08-21 2005-02-15 Astrazeneca Ab Imidazo[1,2-A]pyridine and pyrazolo[2,3-A]pyridine derivatives
WO2001017516A2 (en) * 1999-09-10 2001-03-15 Novo Nordisk A/S Method of inhibiting protein tyrosine phosphatase 1b and/or t-cell protein tyrosine phosphatase and/or other ptpases with an asp residue at position 48
US7115624B1 (en) 1999-09-10 2006-10-03 Novo Nordisk A/S Method of inhibiting protein tyrosine phosphatase 1B and/or T-cell protein tyrosine phosphatase 4 and/or other PTPases with an Asp residue at position 48
WO2001017516A3 (en) * 1999-09-10 2001-11-08 Novo Nordisk As Method of inhibiting protein tyrosine phosphatase 1b and/or t-cell protein tyrosine phosphatase and/or other ptpases with an asp residue at position 48
US6777433B2 (en) 1999-12-22 2004-08-17 Merck Frosst Canada & Co. Protein tyrosine phosphatase 1B (PTP-1B) inhibitors containing two ortho-substituted aromatic phosphonates
US6486142B2 (en) 1999-12-22 2002-11-26 Merck Frosst Canada & Co. Phosphonic acid derivatives as inhibitors of protein tyrosine phosphatase 1B (PTP-1B)
US6583126B2 (en) 1999-12-22 2003-06-24 Merck Erosst Canada & Co. Phosphonic acid derivatives as inhibitors of protein tyrosine phosphatase 1B (PTP-1B)
US6448429B1 (en) 1999-12-22 2002-09-10 Merck Frosst Canada & Co. Protein tyrosine phosphatase 1B (PTP-1B) inhibitors containing two ortho-substituted aromatic phosphonates
WO2001046203A1 (en) * 1999-12-22 2001-06-28 Merck Frosst Canada & Co. Phosphonic acid biaryl derivatives as inhibitors of protein tyrosine phosphatase 1b (ptp-1b)
US6486141B2 (en) 1999-12-22 2002-11-26 Merck Frosst Canada & Co. Phosphonic acid biaryl derivatives as inhibitors of protein tyrosine phosphatase 1B (PTP-1B)
US7153964B2 (en) 2000-03-01 2006-12-26 Astrazeneca Ab Pyrimidine compounds
US6649608B2 (en) 2000-03-01 2003-11-18 Astrazeneca Ab 2,4-di(hetero-)arylamino (oxy)-5-substituted pyrimidines as antineoplastic agents
US6710052B2 (en) 2000-03-01 2004-03-23 Astrazeneca Pyrimidine compounds
US7067522B2 (en) 2000-03-01 2006-06-27 Astrazeneca Ab 2,4,DI (hetero-) arylamino (-oxy)-5-substituted pyrimidines as antineoplastic agents
US6838464B2 (en) 2000-03-01 2005-01-04 Astrazeneca Ab 2,4-Di(hetero-)arylamino(-oxy)-5-substituted pyrimidines as antineaoplastic agents
US6498151B2 (en) 2000-03-22 2002-12-24 Merck Frosst Canada & Co. Aryldifluoromethylphosphonic acids with sulfur-containing substituents as PTP-1B inhibitors
US6465444B2 (en) 2000-03-22 2002-10-15 Merck Frosst Canada & Co. Aryldifluoromethylphosphonic acids with sulfur-containing substituents as PTP-1B inhibitors
US6906065B2 (en) 2000-03-28 2005-06-14 Astrazeneca Ab 4-Amino-5-cyano-2-anilino-pyrimidine derivatives and their use as inhibitors of cell-cycle kinases
JP2003532708A (en) * 2000-05-11 2003-11-05 コンセホ・スペリオル・インヴェスティガシオンズ・シエンティフィカス Heterocyclic inhibitors of glycogen synthase kinase GSK-3
JP4897179B2 (en) * 2000-05-11 2012-03-14 コンセホ・スペリオル・デ・インヴェスティガシオネス・シエンティフィカス Heterocyclic inhibitors of glycogen synthase kinase GSK-3
US6376524B1 (en) 2000-06-21 2002-04-23 Sunesis Pharmaceuticals, Inc. Triphenyl compounds as interleukin-4 antagonists
US6613903B2 (en) 2000-07-07 2003-09-02 Novo Nordisk A/S Modulators of protein tyrosine phosphatases (PTPases)
US6908920B2 (en) 2000-07-11 2005-06-21 Astrazeneca Ab Pyrimidine derivatives
US6969714B2 (en) 2000-09-05 2005-11-29 Astrazeneca Ab Imidazolo-5-YL-2-anilino-pyrimidines as agents for the inhibition of the cell proliferation
US6844341B2 (en) 2001-02-17 2005-01-18 Astrazeneca Ab Pyrimidine derivatives for inhibition of cell proliferation
US6939872B2 (en) 2001-05-30 2005-09-06 Astrazeneca Ab 2-anilino-pyrimidine derivatives as cyclin dependent kinase inhibitors
WO2003043997A1 (en) * 2001-11-15 2003-05-30 Eli Lilly And Company Peroxisome proliferator activated receptor alpha agonists
US7205321B2 (en) 2001-11-15 2007-04-17 Eli Lilly And Company Peroxisome proliferator activated receptor alpha agonists
US7446105B2 (en) 2002-03-09 2008-11-04 Astrazeneca Ab Pyrimidine compounds
US7442697B2 (en) 2002-03-09 2008-10-28 Astrazeneca Ab 4-imidazolyl substituted pyrimidine derivatives with CDK inhibitory activity
US7485638B2 (en) 2002-03-09 2009-02-03 Astrazeneca Ab Pyrimidine compounds
US7465728B2 (en) 2002-03-09 2008-12-16 Astrazeneca Ab Derivatives of 4-(imidazol-5-yl)-2-(4-sulfoanilino)pyrimidine with CDK inhibitory activity
EP2341049A1 (en) * 2002-04-03 2011-07-06 Novartis AG 5-substituted 1,1-dioxo-[1,2,5]thiazolidine-3-one derivatives as PTPASE 1B inhibitors
WO2003082841A1 (en) * 2002-04-03 2003-10-09 Novartis Ag 5-substituted 1,1-dioxo-`1,2,5!thiazolidine-3-one derivatives as ptpase 1b inhibitors
KR100868587B1 (en) * 2002-04-03 2008-11-12 노파르티스 아게 5-substituted 1,1-dioxo-1,2,5-thiazolidine-3-one derivatives as ptpase 1b inhibitors
US7291635B2 (en) 2002-04-03 2007-11-06 Novartis Ag 5-substituted 1,1-dioxo-1,2,5,-thiadiazolidin-3-one derivatives
WO2004041799A1 (en) * 2002-11-07 2004-05-21 Astrazeneca Ab 5- (substituted phenyl) -thiadiazolidine-3-ones and their use as ptp1b
WO2004050646A1 (en) * 2002-11-29 2004-06-17 Astrazeneca Ab 1, 2, 5-thiadiazolidin-3-one 1, 1 dioxide derivatives as inhibitors of protein tyrosine phosphatase ptp1b
WO2004071448A3 (en) * 2003-02-12 2004-10-14 Transtech Pharma Inc Substituted azole derivatives as inhibitors of protein tyrosine phosphatases
WO2004071448A2 (en) * 2003-02-12 2004-08-26 Transtech Pharma Inc. Substituted azole derivatives as inhibitors of protein tyrosine phosphatases
US7348345B2 (en) 2003-03-24 2008-03-25 Roche Palo Alto Llc Nonnucleoside reverse transcriptase inhibitors
US7208509B2 (en) 2003-03-24 2007-04-24 Roche Palo Alto Llc Non-nucleoside reverse transcriptase inhibitors
US7579344B2 (en) 2003-05-16 2009-08-25 Astrazeneca Ab Pyrimidine derivatives possessing cell-cycle inhibitors activity
US7427626B2 (en) 2003-05-16 2008-09-23 Astrazeneca Ab 2-Anilino-4-(imidazol-5-yl)-pyrimidine derivatives and their use as cdk (cdk2) inhibitors
US7842825B2 (en) 2003-06-24 2010-11-30 Hoffmann-La Roche Inc. Biaryloxymethylarenecarboxylic acids as glycogen synthase activator
US7355049B2 (en) 2003-06-24 2008-04-08 Hoffmann-La Roche Inc. Biaryloxymethylarenecarboxylic acids as glycogen synthase activator
US7700632B2 (en) 2003-06-24 2010-04-20 Hoffmann-La Roche Inc. Biaryloxymethylarenecarboxylic acids as glycogen synthase activator
EP2256106A1 (en) 2003-07-22 2010-12-01 Astex Therapeutics Limited 3,4-disubstituted 1H-pyrazole compounds and their use as cyclin dependent kinases (CDK) and glycogen synthase kinase-3 (GSK-3) modulators
WO2005012256A1 (en) 2003-07-22 2005-02-10 Astex Therapeutics Limited 3, 4-disubstituted 1h-pyrazole compounds and their use as cyclin dependent kinases (cdk) and glycogen synthase kinase-3 (gsk-3) modulators
US7141596B2 (en) 2003-10-08 2006-11-28 Incyte Corporation Inhibitors of proteins that bind phosphorylated molecules
US7655652B2 (en) 2004-02-03 2010-02-02 Astrazeneca Ab Imidazolo-5-yl-2-anilinopyrimidines as agents for the inhibition of cell proliferation
US7521473B2 (en) 2004-02-25 2009-04-21 Wyeth Inhibitors of protein tyrosine phosphatase 1B
US7736911B2 (en) 2004-04-15 2010-06-15 Albert Einstein College Of Medicine Of Yeshiva University Activity-based probes for protein tyrosine phosphatases
WO2005114197A2 (en) * 2004-04-15 2005-12-01 Albert Einstein College Of Medicine Of Yeshiva University Activity-based probes for protein tyrosine phosphatases
WO2005114197A3 (en) * 2004-04-15 2006-04-27 Einstein Coll Med Activity-based probes for protein tyrosine phosphatases
US8299085B2 (en) 2004-07-27 2012-10-30 Novartis Ag Quinazoline derivatives
US8293778B2 (en) 2004-07-27 2012-10-23 Roche Palo Alto Llc Heterocyclic antiviral compounds
US7666891B2 (en) 2004-07-27 2010-02-23 Roche Palo Alto Llc Heterocyclic antiviral compounds
WO2006058648A2 (en) * 2004-12-03 2006-06-08 F. Hoffmann-La Roche Ag Biaryloxymethylarene carboxylic acids
US7524870B2 (en) 2004-12-03 2009-04-28 Hoffmann-La Roche Inc. Biaryloxymethylarenecarboxylic acids as glycogen synthase activators
WO2006058648A3 (en) * 2004-12-03 2006-12-28 Hoffmann La Roche Biaryloxymethylarene carboxylic acids
KR100955110B1 (en) * 2004-12-03 2010-04-28 에프. 호프만-라 로슈 아게 Biaryloxymethylarene carboxylic acids
WO2006077428A1 (en) 2005-01-21 2006-07-27 Astex Therapeutics Limited Pharmaceutical compounds
WO2006077424A1 (en) 2005-01-21 2006-07-27 Astex Therapeutics Limited Pharmaceutical compounds
US8513268B2 (en) 2005-04-11 2013-08-20 Novartis Ag 1H-quinazoline-2,4-diones processes for their production, pharmaceutical compositions, and treatment for epilepsy
US8012988B2 (en) 2005-04-11 2011-09-06 Novartis Ag N-(2,4-dioxo-6-(tetrahydrofuran-2-yl)-7-(trifluoromethyl)-1,4-dihydro-2H-quinazolin-3-yl)methanesulfonamide
US7709481B2 (en) 2005-09-29 2010-05-04 Sanofi-Aventis Phenyl-1,2,4-oxadiazolone derivatives, processes for their preparation and methods for their use as pharmaceuticals
US7851493B2 (en) 2005-09-29 2010-12-14 Sanofi-Aventis Phenyl-1,2,4-oxadiazolone derivatives with phenyl group, processes for their preparation and their use as pharmaceuticals
WO2007039177A2 (en) 2005-09-29 2007-04-12 Sanofi-Aventis Phenyl- and pyridinyl- 1, 2 , 4 - oxadiazolone derivatives, processes for their preparation and their use as pharmaceuticals
US7745428B2 (en) 2005-09-30 2010-06-29 Astrazeneca Ab Imidazo[1,2-A]pyridine having anti-cell-proliferation activity
WO2007059359A3 (en) * 2005-11-21 2007-12-21 Biogen Idec Inc Substituted pyrazalones
WO2007059359A2 (en) * 2005-11-21 2007-05-24 Biogen Idec Ma Inc. Substituted pyrazalones
WO2007119600A1 (en) * 2006-03-23 2007-10-25 Otsuka Pharmaceutical Co., Ltd. Carbazole compound
US8084448B2 (en) 2006-03-31 2011-12-27 Novartis Ag Organic compounds
JP2009532379A (en) * 2006-03-31 2009-09-10 ノバルティス アクチエンゲゼルシャフト Piaase thiadiazolidinone inhibitor
WO2007115058A3 (en) * 2006-03-31 2008-03-20 Novartis Ag Thiadiazolidinone inhibitors of ptpase
WO2007115058A2 (en) * 2006-03-31 2007-10-11 Novartis Ag Thiadiazolidinone inhibitors of ptpase
US8193228B2 (en) 2006-10-20 2012-06-05 Merck Sharp & Dohme Corp. Substituted imidazole as bombesin receptor subtype-3 modulators
US8183275B2 (en) 2006-10-20 2012-05-22 Merck Sharp & Dohme Corp. Substituted imidazoles as bombesin receptor subtype-3 modulators
US8106070B2 (en) 2006-10-20 2012-01-31 Merck Sharp & Dohme Corp. Substituted imidazoles as bombesin receptor subtype-3 modulators
US8741934B2 (en) 2007-03-23 2014-06-03 Pfizer Limited Inhibitors of ion channels
US8357711B2 (en) 2007-03-23 2013-01-22 Pfizer Limited Heterocyclic sulfonamides as inhibitors of ion channels
US7906540B2 (en) 2007-03-29 2011-03-15 Roche Palo Alto Llc Non-nucleoside reverse transcriptase inhibitors
WO2008157407A3 (en) * 2007-06-13 2009-02-19 Univ Virginia Thiadiazole, oxadiazole and triazole derivatives for treating leukemia
WO2008157407A2 (en) * 2007-06-13 2008-12-24 University Of Virginia Patent Foundation Thiadiazole, oxadiazole and triazole derivatives for treating leukemia
US8344004B2 (en) 2007-12-26 2013-01-01 Sanofi Cyclic pyridyl-N-[1,3,4]-thiadiazol-2-yl-benzene sulfonamides, processes for their preparation and their use as pharmaceuticals
WO2009080223A1 (en) 2007-12-26 2009-07-02 Sanofi-Aventis Cyclic pyridyl-n-(1,3,4)-thiadiazol-2-yl-benzene sulfonamides, processes for their preparation and their use as pharmaceuticals
WO2009121535A3 (en) * 2008-04-01 2010-01-07 Universita`Degli Studi Di Milano - Bicocca Antiproliferative compounds and therapeutic uses thereof
EP2107054A1 (en) * 2008-04-01 2009-10-07 Università Degli Studi Di Milano - Bicocca Antiproliferative compounds and therapeutic uses thereof
WO2009121535A2 (en) * 2008-04-01 2009-10-08 Universita`Degli Studi Di Milano - Bicocca Antiproliferative compounds and therapeutic uses thereof
US9340503B2 (en) 2009-01-28 2016-05-17 Karus Therapeutics, Limited Scriptaid isosteres and their use in therapy
WO2011057993A1 (en) 2009-11-11 2011-05-19 F. Hoffmann-La Roche Ag Indazolone derivatives as glycogen synthase activators
US7947728B1 (en) 2009-11-11 2011-05-24 Hoffmann-La Roche Inc. Indole and indazole analogs as glycogen synthase activators
WO2011057956A1 (en) 2009-11-11 2011-05-19 F. Hoffmann-La Roche Ag Benzisoxazole analogs as glycogen synthase activators
WO2011057959A1 (en) 2009-11-11 2011-05-19 F. Hoffmann-La Roche Ag Indole and indazole derivatives as glycogen synthase activators
WO2011058122A1 (en) 2009-11-16 2011-05-19 F. Hoffmann-La Roche Ag Piperidine analogs as glycogen synthase activators
WO2011058154A1 (en) 2009-11-16 2011-05-19 F. Hoffmann-La Roche Ag Biphenyl carboxylic acids and bioisosteres as glycogen synthase activators
US8039495B2 (en) 2009-11-16 2011-10-18 Hoffman-La Roche Inc. Biphenyl carboxylic acids and bioisosteres as glycogen synthase activators
WO2011067174A1 (en) 2009-12-01 2011-06-09 F. Hoffmann-La Roche Ag Aniline analogs as glycogen synthase activators
WO2011067266A1 (en) 2009-12-04 2011-06-09 F. Hoffmann-La Roche Ag Carboxylic acid analogs as glycogen synthase activators
CN102834392A (en) * 2010-02-17 2012-12-19 詹森药业有限公司 Aminothiazolones as estrogen related receptor-alpha modulators
US9505728B2 (en) 2012-03-09 2016-11-29 Inception 2, Inc. Triazolone compounds and uses thereof
US10150763B2 (en) 2012-11-07 2018-12-11 Karus Therapeutics Limited Histone deacetylase inhibitors and their use in therapy
US9676765B2 (en) 2012-11-07 2017-06-13 Karus Therapeutics Limited Histone deacetylase inhibitors and their use in therapy
US9676754B2 (en) 2012-12-20 2017-06-13 Inception 2, Inc. Triazolone compounds and uses thereof
US10568871B2 (en) 2012-12-20 2020-02-25 Tempest Therapeutics, Inc. Triazolone compounds and uses thereof
US11666557B2 (en) 2012-12-20 2023-06-06 Tempest Therapeutics, Inc. Triazolone compounds and uses thereof
US9862685B2 (en) 2013-05-10 2018-01-09 Karus Therapeutics Limited Histone deacetylase inhibitors
US10870624B2 (en) 2013-05-10 2020-12-22 Karus Therapeutics Limited Histone deacetylase inhibitors
US9776976B2 (en) 2013-09-06 2017-10-03 Inception 2, Inc. Triazolone compounds and uses thereof
US10407435B2 (en) 2014-10-29 2019-09-10 Karus Therapeutics Limited Diheteroaryl histone deacetylase inhibitors and their use in therapy
US10533003B2 (en) 2014-10-29 2020-01-14 Karus Therapeutics Limited Polyheteroarl histone deacetylase inhibitors and their use in therapy
US11938134B2 (en) 2017-03-10 2024-03-26 Eikonizo Therapeutics, Inc. Metalloenzyme inhibitor compounds
US11578066B1 (en) 2019-12-20 2023-02-14 Tenaya Therapeutics, Inc. Fluoroalkyl-oxadiazoles and uses thereof
US11926622B2 (en) 2019-12-20 2024-03-12 Tenaya Therapeutics, Inc. Fluoroalkyl-oxadiazoles and uses thereof

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WO1997040017A3 (en) 1997-12-11
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AU2381397A (en) 1997-11-12

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