JPH10175863A - Pharmaceutical composition and method for modulating signal transmission - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a specific pharmaceutical composition that modulates and/or controls signal transmission, and inhibits protein tyrosine phosphatase activity. SOLUTION: This pharmaceutical composition is composed of a compound of the formula [Z and Q are each a (substituted) nitrogen-containing heterocyclic ring; T<1> and T<2> are each a (substituted) alkyl, (substituted) cycloalkyl, (substituted) aryl, etc.; (q) is 1-3; (p) and (r) are each 0, 1, 2], for example, 3-[(5- nitrothiazol-2-il)mercapto]-5-phenyl-1,2,4-triazole, etc., or its pharmaceutically permissible salt, and administered preferably in a dose of 0.2-0.5 mg/kg/clay for systemic administration. This compound is obtained preferably by using, as a starting material, 2-bromo-5-nitrothiazole, which is produced be treating 2-amino-5-nitrothiazole with sodium nitrite and hydrogen bromide.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to compounds capable of modulating and / or modulating the activity of phosphotyrosine phosphatase, which modulates signal transduction. In particular, the invention relates to the use of such compounds for the treatment of diseases caused by dysfunctional signaling.

[0002]

[Prior art]

2.1. Signaling Cellular signaling is a fundamental mechanism by which external stimuli that regulate various cellular processes are relayed inside the cell. Biochemical pathways by which signals are transmitted into cells include the circuitry of interacting proteins that are directly or functionally related. One of the major biochemical mechanisms of signal transduction involves reversible phosphorylation of tyrosine residues on proteins. The phosphorylation state of a protein can affect its cellular location as well as its conformation and / or enzymatic activity. The phosphorylation state of protein is determined by various specific tyrosine residues
(PTK) and protein tyrosine phosphatase (PTP)
Are changed by the opposing actions of 2.2. A common mechanism by which protein tyrosine kinases and phosphatase receptors regulate cell function is inducible tyrosine kinase activity conferred by other proteins that are endogenous to the receptor or become associated with the receptor (Darnell et al., 1994,
Science 264: 1415-1421; Heldin, 1995, Cell 80: 213-2
23; Pawson, 1995, Nature 373: 573-580).

[0003] Protein tyrosine kinases include a large family of transmembrane receptors and intracellular enzymes with multifunctional domains (Taylor et al., 1992 Ann. Rev. Cell Biology.
ol. 8: 429-62). Ligand binding transmits the signal across the cell membrane in an allosteric manner, where the cytoplasmic portion of the PTK initiates a cascade of molecular interactions that propagate the signal into the cell and into the nucleus. Many receptor protein tyrosine kinases (RPTKs), such as epidermal growth factor receptor
(EGFR) and platelet-derived growth factor receptor (PDGFR) undergo oligomerization after ligand binding, and the receptor undergoes phosphorylation at certain tyrosine residues in the cytoplasmic portion of the receptor (autophosphorylation or (By phosphoryl transfer) (S
chlessinger and Ullrich, 1992, Neuron, 9: 383-91,
Heldin, 1995, Cell 80: 213-223). Cytoplasmic protein tyrosine kinases (CPTK), such as Janus kinases (eg, JKA1, JKA2, TYK2), Src kinases (eg, sr
c, lck, fyn) are cytokines (eg, IL-2, IL-3, I
L-6, erythropoietin) and interferon and antigen receptors. Also, while these receptors have tyrosine residues that become oligomerized and become phosphorylated during activation, the receptor polypeptides themselves do not have kinase activity.

[0004] Like PTKs, protein tyrosine phosphatases (PTPs) include a family of transmembrane and cytoplasmic enzymes. This enzyme has a consensus motif [I / V
Has a catalytic domain of at least about 230 amino acids including a highly conserved active site with HCXAGXXR [S / T] G. PTK substrate is PTK with phosphotyrosine residue
Alternatively, it may be a substrate of PTK (Hunter, 1989, Cell
58: 1013-16; Fischer et al., 1991, Science 253: 401-6; S
aito & Streuli, 1991, Cell Growth and Differentiati
on 2: 59-65; Pot and Dixon, 1992, Biochem.Biophy
s. Acta, 1136: 35-43).

[0005] Transmembrane or receptor-like PTP (RPTP) has an extracellular domain, a single transmembrane domain, and one or two catalytic domains followed by a short cytoplasmic tail. The extracellular domains of these RPTPs can be small glycosylated segments (eg, RPTPα, RPTPε), immunoglobulin-like domains and / or fibronectin.
Repeated sequence (tandem) of type III domains (eg, LAR)
repeat) or carbonic anhydrase-like domains (eg, RPTPγ, RPTPβ) and are very diverse. These extracellular features may suggest that these RPTPs function as receptors on the cell surface, and their enzymatic activity may be modulated by ligands. Intracellular or cytoplasmic PTPs (CPTPs), eg, PTP1C, PTP1D, typically contain a single catalytic domain flanked by several types of modular conserved domains. For example, PTP1C, a hematopoietic cell CPTP, is characterized by two Src-homology 2 (Src-homology 2; SH2) domains that recognize a short peptide motif with phosphotyrosine (pTyr).

[0006] In general, these modular conserved domains affect the intracellular localization of proteins. SH2-containing proteins are found in activating receptors and cytoplasmic phosphoproteins.
Can bind to the pTyr site. Another conserved domain, known as SH3, binds proteins that have regions rich in proline residues. Plextrin-homology
A third type, known as the (pleckstrin-homology; PH) domain, has also been identified. These modular domains include both CPTK and CPTP, as well as non-catalytic adapter molecules, such as Grb (Growth factor Receptor Bou
nd), which mediate protein-protein interactions between components of the signaling pathway (Skolnik et al., 1991, Ce
ll 65: 83-90; Pawson, 1995, Nature 373: 573-580).

[0007] Multiprotein signaling complexes, including receptor subunits, kinases, phosphatases and adapter molecules, form subcellular compartmentalities through specific and dynamic interactions between these domains with their binding motifs. Assembled inside. Such signaling complexes accumulate extracellular signals from ligand-coupled receptors and relay the signals to other downstream signaling proteins or complexes located elsewhere in the cell or nucleus ( Koch et al., 1991,
Science 252: 668-674; Pawson, 1994, Nature 373: 573-
580; Mauro et al., 1994, Trends Biochem Sci 19: 151-155;
Cohen et al., 1995, Cell 80: 237-248). 2.3. Aberrant signal transmission in human disease The level of tyrosine phosphorylation required for normal cell growth and differentiation at any time is achieved by the coordinated action of PTKs and PTPs. Depending on the context of the cell, these two types of enzymes may antagonize or coordinate with each other during signaling. Imbalances between these enzymes can impair normal cell function, resulting in metabolic disease and cell transformation.

[0008] For example, insulin binding to the insulin receptor, PTK, induces various metabolic and growth promoting effects such as glucose transport, glycogen and fat biosynthesis, DNA synthesis, cell division and differentiation. . Diabetes mellitus, which is characterized by a deficiency or deficiency of insulin signaling, can be caused by abnormalities at any step along the insulin signaling pathway (Ole
fsky, 1988, “CecilTextbook of Medicine”, Part 18,
2: 1360-81).

[0009] For example, overexpression of PTK such as HER2 may play a decisive role in the development of cancer (Slamon
1987, Science 235: 77-82), and that antibodies capable of blocking the activity of this enzyme can stop tumor growth (Drebin et al., 1988, Oncogene 2: 387-394). . Tyrosine kinases, such as Flk-1 and
Blocking the signaling capacity of the PDGF receptor has been shown to block tumor growth in animal models (Mil
lauer et al., 1994, Nature 367: 577; Ueno et al., Science, 2
52: 844-848).

[0010] The relative role of tyrosine phosphatase in signal transduction is relatively unknown.
PTP may play a role in human disease. For example, RP
Ectopic expression of TPα results in a transformed phenotype in embryonic fibroblasts (Zheng et al., Nature 359: 336-339)
Overexpression of RPTPα in embryonal carcinoma cells causes the cells to differentiate into cell types with a neuronal phenotype (den Hertog
EMBO J 12: 3789-3798). The gene for human RPTPγ has been localized to chromosome 3p21, a highly mutated segment in renal and small cell lung cancer. Mutations may occur in the extracellular segment of RPTPγ, which may render RPTP no longer responsive to external signals (L
aForgia et al., Wary et al., 1993, Cancer Res 52: 478-482).
Mutations in the gene encoding PTP1C (HCP, also known as SHP) cause a motheaten phenotype in mice suffering from severe immunodeficiency and a systemic autoimmune disease with macrophage hyperproliferation (Schul
tz et al., 1993, Cell 73: 1445-1454). PTP1D (Syp or
(Also known as PTP2C) is located at the site of phosphorylation in PDGFR, EGFR and insulin receptor substrate 1 (IRS-1).
It has been shown to bind through the SH2 domain. Anti
Reduced PTP1D activity by microinjection of PTP1D antibodies has been shown to block insulin or EGF-induced mitogenesis
(Xiao et al., 1994, J Biol Chem 269: 21244-21248).

It has been reported that some of the biological effects of insulin can be mimicked by vanadium salts such as vanadate and pervanadate. Vanadate and pervanadate are known to be non-specific phosphatase inhibitors. However, this class of compounds is toxic. Since each compound contains a heavy metal (US Pat. No. 5,155,031;
Fantus et al., 1989, Biochem., 28: 8864-71; Swarup et al., 19
82, Biochem. Biophys. Res. Commun. 107: 1104-9).

[0012]

SUMMARY OF THE INVENTION The present invention relates to the use of organic molecules that can modulate and / or modulate signal transduction. Furthermore, the invention relates to the use of a compound for inhibiting the activity of protein tyrosine phosphatase (PTP). Thus, the present invention includes a method of inhibiting protein tyrosine phosphatase activity by contacting a cell with an effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof. Further, the present invention includes methods of treating disease states in mammals, including humans, which are ameliorated by modulating and / or modulating signaling by inhibiting protein tyrosine phosphatase activity. Such disease states or disorders include, but are not limited to, diabetes and cancer.

The compounds of the present invention have the formula I:

[0014]

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(Wherein Z and Q may be the same or different and each represents an atom necessary to complete an unsubstituted or substituted nitrogen-containing heterocycle, and T ₁ and T ₂ are
May be the same or different, and each is alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, aryloxy, halogen, cyano, hydroxy, carboxyl, sulfo, carbamoyl, acyl, acylamino, thioacylamino, sulfamoyl Or a sulfonamide, wherein q is 1, 2,
Or 3 and p and r are 0, 1 or 2). Formula I
Wherein the nitrogen-containing heterocyclic nucleus denoted by the term "Q" is preferably nitropyridine or nitrothiazole.
In addition, the present invention includes pharmaceutically acceptable salts or analogs of the above compounds.

[0016]

DETAILED DESCRIPTION OF THE INVENTION Preferred compounds of the present invention have the formula II:

[0017]

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Wherein Z, T ₁ and p are as defined above. In another embodiment of the present invention, the compound of the present invention has formula III:

[0019]

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Wherein A is (i) 1 to 4 heterocyclic atoms (at least one of which is nitrogen, the rest of which are nitrogen,
A substituted or unsubstituted monocyclic 5- or 6-membered ring having a ring selected from oxygen or sulfur), for example, pyridine, pyrrole, imidazole, thiazole, isothiazole, isoxazole, furazane, pyrrolidine, piperidine,
Imidazolidine, piperazine, oxazole, tetrazole, pyrazole, triazole, oxadiazole,
A thiodiazole; (ii) a substituted or unsubstituted monocyclic or fused bicyclic 6 having 1 to 4 heterocyclic atoms, one of which is nitrogen, the rest being selected from nitrogen, oxygen or sulfur. Membered to 10-membered ring, for example, indole, quinoxaline, quinazoline, quinoline, isoquinoline, purine;
Or (iii) represents a substituted or unsubstituted monocyclic or fused polycyclic saturated or unsaturated ring having 3 to 15 atoms, which are carbon, sulfur, nitrogen or oxygen. You.

The heterocycle as defined above may be saturated or unsaturated. The unsaturated ring or heteroaromatic group may optionally have one or more substituents that do not substantially adversely affect the activity of the compound of Formula I. Examples of such substituents include alkyl, alkoxy, phenoxy, alkenyl, alkynyl, phenylalkyl, hydroxyalkyl, haloalkyl, aryl, arylalkyl, alkyloxy, alkylthio, alkenylthio, phenylalkylthio, hydroxyalkyl-thio, alkylthiocarba Examples include millthio, phenyl, cyclohexyl, pyridyl, piperidinyl, alkylamino, amino, nitro, mercapto, cyano, hydroxyl, halogen, oxygen (forming ketone or N-oxide) or sulfur (forming thione). Can be 3.1 Definitions As used herein, the term “alkyl” refers to
A straight or branched chain saturated hydrocarbon group having 20 carbon atoms, for example, methyl, ethyl, isopropyl, n-butyl, s-butyl, t-butyl, n-amyl, isoamyl, n-hexyl, n- Octyl and n-decyl are meant. The terms "alkenyl" and "alkynyl" have 2 to 10 carbon atoms and are unsaturated by a double or triple bond, respectively, straight or branched chain hydrocarbon groups such as vinyl, allyl, propargyl, 1-methylvinyl, 1-butenyl, 2-butenyl, 2-butynyl, 1-methyl-2-butenyl, 1-pentenyl, 3-
Used to mean pentenyl, 3-methyl-1-butynyl, 1,1-dimethylallyl, 2-hexenyl and 1-methyl-1-ethylallyl. The term "phenylalkyl" refers to the above alkyl groups substituted by a phenyl group, for example, benzyl, phenethyl, phenopropyl, 1-benzylethyl, phenobutyl and 2
-Means benzylpropyl. As used herein, the term "aryl" is meant to include a monocyclic or bicyclic ring, wherein at least one ring comprises an aromatic or hetero-aromatic hydrocarbon It is aromatic. The term "hydroxy-alkyl" refers to an alkyl group as described above substituted by a single hydroxyl group, e.g., 2-hydroxyethyl, 2-hydroxypropyl,
3-hydroxypropyl, 4-hydroxybutyl, 1-
Means hydroxybutyl and 6-hydroxyhexyl. As used herein, "alkylthio, alkenylthio, alkynylthio, alkylthio, hydroxy-
The term "alkylthio and phenyl-alkylthio" refers to an alkyl group attached to the group R via a sulfur atom,
It means an alkenyl group, an alkynyl group, a hydroxy-alkyl group and a phenyl-alkyl group.

As used herein, the term "substituted" refers to the group in question, such as alkyl, aryl, etc., where halogen, hydroxy, cyano, amino, nitro, mercapto, carboxy and those skilled in the art are known. Mean that it may have one or more substituents, including but not limited to other substituents. As used herein, the term "saturated" refers to an organic compound that does not have any double or triple bonds. The term "unsaturated", as used herein, refers to an organic compound that contains either double or triple bonds.

[0023] relates to the use of 5. DETAILED DESCRIPTION OF THE INVENTION The present invention can modulate or regulate signal transduction in normal or diseased cells in the compound. The present invention also relates to a protein tyrosine phosphatase for modulating or inducing signaling.
(PTP). The use of a compound capable of inhibiting the activity of (PTP). Furthermore, the present invention relates to the modulation of cellular processes controlled by signal transduction through suppression of PTP activity by compounds. Further, the invention provides the use of such compounds in the treatment of a patient having a disease caused by dysfunctional signaling.

In one embodiment of the invention, the compounds of the invention inhibit the activity of protein tyrosine phosphatases, which are transmembrane or intracellular and may have one or more characteristic catalytic domains. be able to. The amino acid sequence of PTP in the catalytic domain is [I / V] HCXA
GXXR [S / T] G (one-letter amino acid notation; X is any amino acid), but is not limited thereto. In addition, PTPs may have one or more modular conserved domains, including but not limited to SH2, SH3 and PH domains. In a particular embodiment of the invention, the compound of the invention is PTP1B (Charbonneau).
Acad Sci USA, 86: 5252-5256), 1989, Proc. Natl. Acad Sci USA.
T-cell PTP (Cool et al., 1989, Proc. Natl. Acad Sci USA,
86: 5257-5261), PTP1C (Shen et al., 1991, Nature, 352:
736-739), PTP1D (Vogel et al., 1993, Science 259: 1611-1)
614), RPTPα, RPTPβ, RPTPγ (Kaplan et al., 1990, Pro
c. Natl. Acad Sci USA, 87: 7000-7004), RPTPσ (Yan
1993, J Biol Chem 268: 24880-24886), RPTPκ (Ji
ang et al., 1993, Mol Cell Biol, 13: 2942-2951) and CD
45 (Charbonneau et al., 1988, Proc. Natl. Acad Sci USA,
85: 7182-7186) can be used to inhibit phosphatase activity. Preferred PTPs according to the invention are of human origin. Suppression of phosphatase activity that is substantially specific for PTP or a set of PTPs in the signaling pathway is preferred. Inhibition of phosphatase activity is believed to be the mechanism of action of the compounds of the present invention for their ability to modulate and / or modulate signaling, but other mechanisms have not been excluded.

The term "signaling" as used herein is not limited to transmembrane signaling, but also includes multiple pathways that branch through cells and reach the nucleus. Such signaling pathways include the Ras pathway (Schlessi
nger, 1994, Curr Opin Genet Dev 4: 25-30), JAK / STAT
Pathway (Sadowski et al., 1994, Science 261: 1739-1744), the phosphoinositide 3-kinase pathway and the phospholipase C-γ pathway, but are not limited thereto. “Modulate” or “modulate” as used herein refers to up-regulation and down-regulation of a signaling pathway. Cell processes under the control of signal transduction include the transcription of certain genes, normal cell function, such as metabolism, proliferation, differentiation, adhesion, apoptosis and survival, as well as abnormal processes, such as transformation, differentiation. Blocking and transfer include, but are not limited to.

A signal is triggered by the binding of a ligand to its receptor on the cell surface, and the signal is converted and propagated by phosphorylation or dephosphorylation of specific tyrosine residues on various substrates within the cell. Certain interactions between PTKs, PTPs and their substrates may involve the formation of transient or stable multimolecular complexes in the plasma membrane lining or other subcellular compartments, including the nucleus. The substrate may include one or more tyrosine residues that are phosphorylated or dephosphorylated by PTK or PTP in the signaling pathway. Such substrates include the receptor and its subunits, molecules associated with or recruited to the receptor,
For example, it may contain cytoplasmic kinases, cytoplasmic phosphatases, adapter molecules, cytoskeletal proteins and transcription factors. The term receptor as used herein refers to the insulin receptor, a member of the insulin-like growth factor receptor family, the epidermal growth factor receptor family, the fibroblast growth factor receptor family, the hepatocyte growth factor receptor family. Vascular endothelial growth factor receptor family, neurotropin receptor (trk) family, T cell receptor, B
Cell receptors and members of the type I-IV cytokine receptor family (Heldin, 1995, Cell. 80: 213-223; Taniguchi, 199
5, Science, 268: 251-255), but is not limited thereto. Adapter molecules that are substrates include Grb protein, IRS-1, Zap-70 and Shc (Pawson et al., 1995, Nature 373: 573-580).
Cytoskeletal proteins such as actin and transcription factors such as STAT proteins (Ihle et al., Trends Biochem Sci, 19: 222-227) can also be used as substrates. The term ligand, as used herein, is synonymous with extracellular signaling molecule and refers to a growth factor such as insulin, EGF
, PDGF, fibroblast growth factor, vascular endothelial growth factor, and neurotropin, and cytokines such as, but not limited to, growth hormone, erythropoietin, tumor necrosis factor, interleukins and interferons. The term ligand is not limited to soluble molecules and includes, for example, extracellular matrix proteins, cell adhesion molecules, as well as antigen proteins associated with major histocompatibility complex proteins on the surface of antigen presenting cells.

In one embodiment of the present invention, the compounds of the present invention can be used to induce or up-regulate signal transduction in a cell, such that the effect of ligand binding to the receptor is enhanced, or If not, a pseudo effect is obtained. These compounds are
It exerts its effect by suppressing or reducing the activity of phosphatases in signaling pathways that normally act on members for signal transduction. One mechanism by which PTP usually downregulates signaling involves the dephosphorylation of specific phosphotyrosine residues (pTyr) of PTKs and their substrates. What a lot of PT
This is because K requires phosphorylation of some of its own tyrosine residues to be optimally active in the signaling pathway. The compounds of the present invention can be used to block the dephosphorylation of pTyr residues of receptors or their subunits that normally become phosphorylated after ligand binding, thereby reducing the extent and duration of PTK phosphorylation. Can be enhanced. Also, the compounds of the present invention can be used to block the dephosphorylation of PTKs, in which case tyrosine residues are autophosphorylated or transphosphorylated by their basic activity. In these PTKs, a signal can be triggered by the compounds of the present invention, even in the absence of ligand binding. This is because the basic activity of PTK is sufficient to enhance the signal when constitutive PTP activity is suppressed or reduced by the compounds of the present invention.

A preferred embodiment of the present invention relates to a method for inducing, enhancing or sustaining insulin receptor signaling by inhibiting constitutive dephosphorylation of the pTyr site of the activated insulin receptor. This leaves the insulin receptor phosphorylated, thus enhancing or sustaining the insulin signal. Furthermore, since the insulin receptor has been shown to be phosphorylated at low levels even in the absence of insulin (Goldstein, 1992, J. Cell Biol.
l., 48: 33-42), compounds of the present invention elicit a signal even in the absence of insulin by allowing tyrosine residues on the receptor to become autophosphorylated Can be used to

Another mechanism by which PTPs can exert a member's effect on signal transduction is by dephosphorylation of specific pTyr sites where SH2-containing molecules bind during signal transduction. Absence of such a pTyr site would prevent recruitment of SH2-containing molecules to specific subcellular compartments to form a multiprotein signaling complex, thereby preventing further propagation of the signal . Thus, the compounds of the present invention provide a substrate protein that normally acts as a binding site for SH2-containing proteins that promote signal transduction.
By blocking the dephosphorylation of the pTyr site, it can be used to up regulate or prolong signaling. In another embodiment of the invention, the compounds of the invention are used to prevent the dephosphorylation of a particular pTyr residue of any substrate, which pTyr residue is essential for signal transduction or propagation. Is also good. Further, the compounds of the present invention may be used to block the dephosphorylation of certain pTyr residues of a substrate, which pTyr residues are inhibitory to signal transduction.

Also, the compounds of the present invention can be used to suppress or down regulate signaling in cells, thereby abolishing or reducing the effect of ligand binding to the receptor. These compounds can inhibit phosphatases in signaling pathways that normally act positively for signaling. For example, PTP is Sr of PTK
Promotes signaling through activation of c-family members. Src family PTKs have a phosphorylation inhibitory site in their carboxy terminus and enhance kinase activity by dephosphorylation. Thus, the compounds of the present invention can be used to block the dephosphorylation of an inhibitory pTyr at the carboxy terminus of a kinase that normally functions to promote signal transduction. As Src family PTKs, Src, Fyn,
Lck, Lyn, Blk, Hck, Fgr and Yrk. Other kinases that can also be regulated by phosphatases include Fak and Csk (Taniguc
hi, 1995, Science 268: 251-255).

The ability of the compounds of the present invention to inhibit protein tyrosine phosphatase activity and to induce or upregulate cellular processes controlled by signal transduction is demonstrated in the examples below. 5.1. Assays for Measuring Inhibitory Activity of Compounds Various procedures known in the art can be used to identify, evaluate or analyze the inhibition of protein tyrosine phosphatase activity by compounds of the present invention. Generally, this assay involves contacting a target cell in culture with a compound;
Analyzing the cell lysate biochemically for levels and / or identity of tyrosine phosphorylated proteins, or
(b) scoring for a phenotypic or functional change in the treated cells compared to control cells that have not been contacted with the test substance.

When identifying or evaluating mimics of a natural ligand for a signaling receptor, cells are contacted with a compound of the invention, a positive control contacted with the natural ligand only, and a compound of the invention or natural ligand. Compare to the non-contacted member control. For receptors known to be phosphorylated at basal levels in the absence of the natural ligand, for example, the insulin receptor, the assay may be performed in the absence of the ligand. When identifying or assessing an inhibitor or enhancer of ligand-induced signaling, cells are contacted with a compound of the invention in the presence of a natural ligand and compared to a control not contacted with a compound of the invention.

The assay described below can be used as a primary screen to evaluate the phosphatase inhibitory activity of the compounds of the present invention. Also, these assays are tested in a range of concentrations, for example, 100 μM to 1 pM, and the amount of phosphorylation or signaling is compared to controls.
By calculating the concentration (IC50) that decreases or increases by 50%, it can be used to assess the relative potency of the compound.

5.1.1. Biochemical Assay Target cells having a substrate molecule whose tyrosine residues are phosphorylated or dephosphorylated during signal transduction are contacted with a compound of the invention and a radiolabeled phosphate, followed by lysis. As a result, cell components containing the target substrate are released.
The substrate is separated from the protein component of the cell lysate using one- or two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) technology, and
It can be analyzed by detecting the presence of phosphorylated proteins by contacting with a line film. In a similar technique that does not use a radioactive label, the protein components separated by SDS-PAGE are transferred to a nitrocellulose membrane and the presence of pTyr is detected using an anti-phosphotyrosine (anti-pTyr) antibody. Alternatively, the substrate of interest is first incubated with a cell lysate with a substrate-specific anchoring antibody bound to the solid support, followed by washing away unbound cell components and removing the solid support with an anti-pTyr antibody. Preferably, it is isolated by assessing the presence or absence of the above pTyr. This preferred method can be easily performed in a microtiter plate format by an automated robotic system, allowing the testing of large numbers of samples within a fairly short time frame. Compounds of the invention have been identified and evaluated by this preferred method as described in the following section.

The anti-pTyr antibody can be detected by labeling it with a radioactive substance which facilitates its detection by autoradiography. Further, the anti-pTyr antibody is bound to an enzyme such as horseradish peroxidase, and can be detected by subsequently adding a substrate for colorimetric measurement of the enzyme. Yet another method involves detecting by reacting a secondary antibody that recognizes the anti-pTyr antibody with the anti-pTyr antibody, wherein the secondary antibody is labeled with a radioactive substance or enzyme as previously described. You. Other methods of detecting antibodies known in the art may be used.

The above method may also be used in a cell-free system, in which case a cell lysate containing a signaling substrate molecule and a phosphatase is mixed with a compound of the present invention and a kinase. Its substrate is adenosine triphosphate (AT
It is phosphorylated by initiating the kinase reaction with the addition of P). To evaluate the activity of the compound,
The reaction mixture may be analyzed by the SDS-PAGE technique, or it may be added to a substrate-specific anchoring antibody bound to a solid support, and the detection operation described above may be separated or trapped on the substrate. To assess the presence or absence of pTyr. The results are compared with those obtained with the reaction mixture without addition of the compound. The cell-free system does not require recognition of the natural ligand or its identity. For example, Posner et al. (US Pat. No. 5,155,031)
It describes the use of insulin receptor as a substrate and rat adipocytes as target cells to demonstrate the ability of pervanadate to inhibit PTP activity. As another example, Burke et al. (1994, Biochem Biophys Res Comm 204: 1
29-134) are autophosphorylated insulin receptors and recombinant PTP1 in assessing the inhibitory activity of phosphotyrosyl mimetics.
It describes the use of B.

In addition to measuring the phosphorylation or dephosphorylation of substrate proteins, it also activates or modulates second messenger production, changes in cell ion levels,
The association, dissociation or translocation of signaling molecules, gene induction or transcription or translation of a particular gene may also be monitored. These biochemical assays can be performed using conventional techniques developed for these purposes.

5.1.2. Biological Assays The ability of the compounds of the invention to modulate the activity of PTP to regulate signaling can also be measured by scoring for morphological or functional changes associated with ligand binding. . Qualitative or quantitative techniques known in the art can be applied to observe and measure cellular processes that fall under the control of phosphatases in signal transduction pathways. Such cellular processes include anabolic and catabolic processes, cell proliferation, cell differentiation, cell adhesion,
Cell migration as well as cell killing include, but are not limited to.

The techniques used to examine the various biological effects of vanadate as phosphatase inhibitors may be suitable for use with the compounds of the present invention. For example, vanadate has been shown to activate an insulin-sensitive enhanced transport system for glucose and glucose analogs in rat adipocytes (Dub
yak et al., 1980, J Biol Chem 256: 5306-5312). Activity of the compounds of the present invention, glucose analog in rat adipocytes that have been contacted with the compound, for example, 2-deoxy - ³ H-
It can be assessed by measuring the increase in the rate of glucose transport. Vanadate also shows an insulin mimic effect on glucose oxidation in rat adipocytes (Shechter et al., 1980, Nature 284: 556-558). Compounds of the invention can be tested for stimulation of glucose oxidation by measuring the conversion of ¹⁴ C-glucose to ¹⁴ CO ₂ . In addition, the effect of sodium orthovanadate on erythropoietin-mediated cell growth was measured by cell cycle analysis based on DNA content as estimated by incorporation of tritiated thymidine during DNA synthesis (Spi
vak et al., 1992, Exp Hematol, 20: 500-504). Similarly, the activity of the compounds of the invention with respect to phosphatases, which play a role in cell proliferation, can be analyzed by cell cycle analysis.

The activity of the compounds of the present invention can also be evaluated in animals using an experimental model of a disease caused by dysfunctional signaling or a disease related thereto. For example, the activity of a compound can be measured in non-obese diabetic mice (Lund et al.,
1990, Nature 345: 727-729), BB Wistar rats and streptozotocin-induced diabetic rats (Solomon et al., 198
9, Am J Med Sci 297: 372-376) for its effect on insulin receptor signaling. Also, the activity of these compounds can be evaluated in animal carcinogenesis experiments. Phosphatase can play an important role in dysfunctional signaling leading to cell transformation. For example, the phosphatase inhibitor okadaic acid has been shown to promote tumor formation in mouse skin (Suganuma
Et al., 1988, Proc Natl Acad Sci 85: 1768-1771).

The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for human use. The dosage of the compounds of the invention should be within a range of circulating concentrations that cause little or no toxicity. Dosages may vary within this range depending on the dosage form employed and the route of administration. The above assays are exemplary and are not intended to limit the scope of the present invention. One skilled in the art will appreciate that this assay can be modified to develop an equivalent assay that achieves the same result.

5.2. Phosphatase Inhibitors The present invention relates to compounds capable of modulating and / or modulating signal transduction, including but not limited to inhibiting the activity of protein tyrosine phosphatases. Including. More specifically, the present invention includes compounds that can inhibit protein tyrosine phosphatase activity. These compounds are generally referred to herein as "phosphatase inhibitors", even if these compounds up-regulate or down-regulate cellular processes that are controlled by signal transduction. Generally, the compounds of the present invention are nitrothiazole compounds or derivatives thereof.

More specifically, the compounds of the present invention have the following general formula I:

[0044]

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Wherein Z and Q may be the same or different and each represents an atom necessary to complete an unsubstituted or substituted nitrogen-containing heterocycle, and T ₁ and T ₂ are
May be the same or different, and each is alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, aryloxy, halogen, cyano, hydroxy, carboxyl, sulfo, carbamoyl, acyl, acylamino, thioacylamino, sulfamoyl Or a sulfonamide, wherein q is 1, 2,
Or 3 and p and r are 0, 1 or 2).
In formula I, the nitrogen-containing heterocyclic nucleus denoted by the term "Q" is preferably nitropyridine or nitrothiazole. In addition, the present invention includes pharmaceutically acceptable salts or analogs of the above compounds.

As a preferred compound of the present invention, a compound of formula II:

[0047]

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Wherein Z, T ₁ and p are as defined above, and pharmaceutically acceptable salts thereof. In another embodiment of the present invention, the compound of the present invention has formula III:

[0049]

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Wherein A is (i) 1 to 4 heterocyclic atoms (at least one of which is nitrogen, the rest of which are nitrogen,
A substituted or unsubstituted monocyclic 5- or 6-membered ring having a ring selected from oxygen or sulfur), for example, pyridine, pyrrole, imidazole, thiazole, isothiazole, isoxazole, furazane, pyrrolidine, piperidine,
Imidazolidine, piperazine, oxazole, tetrazole, pyrazole, triazole, oxadiazole,
A thiodiazole; (ii) a substituted or unsubstituted monocyclic or fused bicyclic 6 member having 1-4 heterocyclic atoms, one of which is nitrogen, the rest being nitrogen, oxygen or sulfur. ~ 10 membered rings, for example, indole, quinoxaline,
Quinoline, isoquinoline, quinazoline, purine; or
(iii) described by substituted or unsubstituted monocyclic or fused polycyclic saturated or unsaturated rings having 3 to 15 atoms, which are carbon, sulfur, nitrogen or oxygen. . The present invention further includes pharmaceutically acceptable salts of the above compounds. Examples of the structure contained in the group (i) include, for example,

[0051]

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(Wherein R is hydrogen, halogen, cyano, nitro, amino, amide, carboxy, acylamino, hydroxy, alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, benzyl arylalkyl etc., aryloxy or sulfur, nitrogen or a substituted or unsubstituted heterocyclic 5-membered ring system or or 6-membered ring system heteroatoms containing 0-3 oxygen, such as phenoxy, R ₂ Is hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, arylalkyl,
Substituted arylalkyl, and n is 0-5).

Preferred structures in the above group (i) include:

[0054]

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(Wherein R ₂ is hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl,
Substituted cycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, wherein R ₃ is hydrogen, halogen, cyano, nitro, hydroxy, alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, aryl, Substituted aryl, arylalkyl, substituted arylalkyl, carboxy, amide, amino, acylamino, sulfonyl,
A sulfonamide, an aminosulfone, or a substituted or unsubstituted 5- or 6-membered ring system containing one or two heteroatoms which are sulfur, nitrogen or oxygen)
Is mentioned.

The structure contained in the above group (ii) includes, for example,

[0057]

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And the like. The structures contained in the group (iii) include cyclopentyl, cyclohexyl, adamantyl, tetrahydroquinoline, tetrahydropyrazole, and substituted derivatives thereof. As specific examples of preferred compounds within the scope of the present invention, compounds of formula (IV):

[0059]

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(Wherein R ₂ and R ₃ are as defined in Table 1 below), but are not limited thereto.

[0061]

[Table 1]

Additional compounds within the scope of the present invention are found in the examples. Generally, the compounds of the present invention are accepting compounds, some of which have been reported as photosensitizers for photographic materials. Compounds within the scope of the present invention are described in U.S. Pat.
No. 3, No. 3,870,725 and No. 3,850,939, which are incorporated herein by reference.

The compounds within the scope of the present invention include the following compounds and their pharmaceutically acceptable salts.

[0064]

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[0065]

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[0066]

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[0067]

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[0068]

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[0069]

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[0070]

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[0071]

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[0072]

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[0073]

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[0074]

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[0075]

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[0076]

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[0077]

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[0078]

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[0079]

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[0080]

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[0081]

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[0082]

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In addition, the sulfur bond between the nitrothiazole ring and the adjacent ring has the formula V

[0084]

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Wherein A is as defined above and R ′ is hydrogen, C ₁ -C ₄ alkyl and substituted C ₁ -C
It has been found that the compound can be substituted by an amino bond, for example, -NR'-, as in the compound represented by formula ( ₄ ).
Such compounds also have potent activity in inhibiting or promoting phosphatase activity. Accordingly, the present invention includes the above compounds wherein the thio bond is replaced by an amino bond (see Formulas I, II, III and IV).

The present invention further relates to a pharmaceutical composition comprising a pharmaceutically effective amount of the above compound and a pharmaceutically acceptable carrier or excipient. Such compositions are believed to inhibit the activity of protein tyrosine phosphatase, which may be useful in treating diseases associated with dysfunctional signaling, including diabetes and cancer. Also, such compositions can act directly on cells causing the disease (eg, tumor cells). More particularly, the compounds of the present invention are useful, among other diseases, for diabetic retinopathy, glioma, melanoma, caposi sarcoma, hemangiomas and ovarian cancer,
It may be included in a method for treating breast cancer, lung cancer, pancreatic cancer, prostate cancer, colon cancer and epidermoid cancer.

Finally, the invention also relates to diabetes, diabetic retinopathy, rheumatoid arthritis, hemangiomas and cancers, more particularly those associated with solid cell tumor growth (eg glioblastomas, melanomas and The present invention relates to a method for treating (including but not limited to) Kaposi's sarcoma and diseases including ovarian cancer, lung cancer, breast cancer, prostate cancer, pancreatic cancer, colon cancer and epidermoid cancer.

5.2.1. Analogs and / or Salts As used herein, a “pharmaceutically acceptable salt” refers to a compound that retains the biological effectiveness and properties of the compound, and which contains an inorganic acid or base, For example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-
A salt obtained by a reaction with toluenesulfonic acid, salicylic acid, or the like.

In addition to the above compounds and their pharmaceutically acceptable salts, the present invention further provides, where appropriate, solvated or unsolvated forms which have the ability to modulate and / or modulate phosphatase activity ( Hydrated form). The compounds can be prepared by any method known to be applicable to the preparation of chemically related compounds. The preferred method is illustrated by the following representative example. The necessary starting materials are obtained by the usual procedures in organic chemistry.

5.3. Pharmaceutical Formulations and Routes of Administration The compounds identified are Kaposi's sarcoma, glioblastoma,
And various diseases including ovarian, lung, breast, prostate, pancreatic, colon and epidermoid carcinoma, including diabetes, melanoma, diabetes, diabetic retinopathy, hemangiomas and rheumatoid arthritis. It can be administered to human patients alone at therapeutic or ameliorating doses or as a pharmaceutical composition in admixture with a suitable carrier or excipient. Further, a therapeutically effective dose refers to that amount of the compound that is sufficient to ameliorate the symptoms of uncontrolled angiogenesis and angiogenesis. Techniques for formulating and administering the compounds of the present application are described in “Remington's Pharmaceutical Sciences”, Mack Pub.
lishing Co., Easton, PA.

The formulations of the present invention are usually mixed with a carrier, or diluted with a carrier, or sealed with an ingestible carrier in the form of a capsule, sachet, cachet, paper or other container, or with a disposable container such as an ampoule. Or at least one encapsulated compound of formula I. The carrier or diluent may be a solid, semi-solid or liquid material, which may include a vehicle for the active therapeutic substance,
Available as an excipient or vehicle.

Some examples of diluents or carriers that may be used in the pharmaceutical compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, propylene glycol, liquid paraffin, white petrolatum, kaolin,
Microcrystalline cellulose, calcium silicate, silica, polyvinylpyrrolidone, cetostearyl alcohol, starch, gum arabic, calcium phosphate, cacao butter, theobroma
(theobroma) oil, arachis oil, alginate, tragacanth, gelatin, syrup BP, methylcellulose, polyoxyethylene sorbitan monolaurate, ethyl lactate and propyl hydroxybenzoate, sorbitan trioleate, sorbitan sesquioleate and oleyl alcohol It is.

5.3.1. Routes of Administration Suitable routes of administration include, for example, oral, rectal, transmucosal or intestinal administration; intramuscular, subcutaneous, intramedullary and intrathecal injection, direct injection Intraventricular injection,
Parenteral delivery, including intravenous, intraperitoneal, intranasal, or intraocular injection; transdermal, topical, vaginal, and the like. Dosage forms include, but are not limited to, tablets, troches, dispersions, suspensions, suppositories, solutions, capsules, creams, patches, minipumps, and the like.

The compounds may also be administered in a local rather than systemic manner, for example, by injection of the compound directly into a solid tumor, often as a depot or sustained release formulation. Further, the agent may be administered in a targeted drug delivery system, for example, a liposome coated with a tumor-specific antibody. Liposomes target the tumor and will be selectively absorbed by the tumor.

5.3.2. Composition / Formulation The pharmaceutical composition of the present invention can be prepared by a method known per se, for example, a usual mixing method, dissolution method, granulation method, dragee production method, milling method, emulsification method. It can be manufactured by methods, encapsulation, confinement or lyophilization. Thus, the pharmaceutical compositions for use in the present invention utilize one or more physiologically acceptable carriers that comprise excipients and auxiliaries that facilitate the processing of the active compounds in pharmaceutically usable formulations. It can be formulated in the usual way. Proper formulation is dependent upon the route of administration chosen.

For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. For oral administration, the compounds can be formulated readily by mixing the active compounds with pharmaceutically acceptable carriers known in the art. With such carriers, the compounds of the invention may be formulated as tablets, pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions, etc. for oral ingestion by the patient to be treated. be able to. Pharmaceutical preparations for oral use obtain solid excipients, optionally pulverize the resulting mixture,
If desired, tablets or dragee cores can be obtained by processing the mixture of granules, after adding the appropriate auxiliaries. Suitable excipients are, in particular, fillers, for example,
Sugars containing lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP)
It is. If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose, a concentrated sugar solution may be used,
Gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, a lacquer, and optionally a suitable organic solvent or solvent mixture may be included. Dyes or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. Push-fit capsules can contain the active ingredient in admixture with fillers, for example, a binder such as lactose, starch, and / or a lubricant such as talc or magnesium stearate, and, optionally, a stabilizer. In soft capsules, the active compounds are suitable liquids,
For example, they may be dissolved or suspended in fatty oils, liquid paraffin, or liquid polyethylene glycol. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.

For sublingual administration, the compositions may take the form of tablets or lozenges formulated in conventional manner. For inhaled administration, the compounds for use in the present invention may be prepared in a pressurized pack or nebulizer using a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. And is conveniently delivered in the form of an aerosol spray. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to dispense a metered amount. A compound and a suitable powder base,
For example, capsules and cartridges of, for example, gelatin, may be formulated for use in an inhaler or insufflator, including powder mixtures with lactose or starch.

The compounds may be formulated for parenteral administration by injection, eg, by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, eg, in ampoules or in multi-dose containers, with an added preservative.
The compositions may take such forms as suspensions, solvents or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. .

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions are substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose.
It may contain sorbitol or dextran. If desired, suspensions may also contain agents or suitable stabilizers which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

The active ingredient may also be in powder form for constitution with a suitable vehicle, eg, sterile pyrogen-free water, before use. The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, eg, containing conventional suppository bases such as cocoa butter or other glycerides. In addition to the above formulations, the compounds may also be formulated as a depot. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compound may be formulated with a suitable polymeric or hydrophobic material (eg, as an emulsion in an acceptable oil) or an ion exchange resin, or a sparingly soluble derivative, such as a sparingly soluble salt It may be formulated as

Pharmaceutical carriers for hydrophobic compounds include benzyl alcohol, non-polar surfactants, water-miscible organic polymers,
And a cosolvent system containing an aqueous phase.
The co-solvent system may be a VPD co-solvent system.
VPD is 3% w /
v benzyl alcohol, 8% w / v non-polar surfactant polysorbate 80, and 65% w / v polyethylene glycol 300 solution. VPD cosolvent type (VPD: 5W)
Is VP diluted 1: 1 with 5% dextrose in aqueous solution
Consists of D This cosolvent system dissolves hydrophobic compounds well and as such has low toxicity after systemic administration. Of course,
The proportions of a cosolvent system can be varied considerably without compromising its solubility and toxicity properties. Further, the identity of the co-solvent components can vary. For example, other low toxicity non-polar surfactants may be used in place of polysorbate 80. The fraction size of the polyethylene glycol can vary. Other biocompatible polymers, for example,
Polyvinylpyrrolidone may be replaced by polyethylene glycol, and other sugars or polysaccharides may be replaced by dextrose.

Further, other delivery systems for hydrophobic pharmaceutical compounds may be used. Liposomes and emulsifiers are known examples of delivery vehicles or carriers for hydrophobic drugs. Also, some organic solvents, such as dimethyl sulfoxide, usually at the expense of greater toxicity.
May be used. Further, the compound may be a sustained release system, such as
Delivery may be accomplished using a semi-permeable matrix of a solid hydrophobic polymer containing the therapeutic agent. Various sustained-release materials have been established and are known by those skilled in the art. Sustained-release capsules
Depending on their chemistry, compounds may be released over a few weeks to over 100 days. Depending on the chemical nature and biological stability of the therapeutic reagent, additional strategies for protein stabilization may be used.

The pharmaceutical compositions may also contain suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols. In addition to the common dosage forms described above, the compounds of the present invention may also include Alza
It may be administered by controlled release means and / or delivery devices, including Alzet® osmotic pumps available from Corporatin. A preferred delivery device is U.S. Pat.
Nos. 845,770, 3,916,899, 3,536,809, 3,598,123, 3,944,064 and 4,008,71
No. 9 and these disclosures are incorporated herein by reference in their entirety.

Many of the phosphatase modulating compounds of the present invention can be provided as salts with pharmaceutically compatible counterions. Pharmaceutically compatible salts are hydrochloric, sulfuric, acetic, lactic,
It can be produced with many acids including, but not limited to, tartaric acid, malic acid, succinic acid, and the like. Salts tend to be more soluble in aqueous or other protic solvents, which are in the corresponding free base form.

5.3.3. Effective Dose Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount effective to prevent the onset of the patient being treated or to ameliorate its existing symptoms. Determination of an effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

For the compounds used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. For example, the dose may be an IC50 as measured in cell culture.
(I.e., the concentration of test compound that achieves a half-maximal inhibition of PTP activity) can be formulated in animal models to achieve a circulating concentration range that includes it. Such information can be used to more accurately determine useful doses in humans.

A therapeutically effective dose refers to that amount of a compound that ameliorates a patient's symptoms or prolongs survival. The toxicity and therapeutic efficacy of such compounds can be determined, for example, by the LD50 (population
It can be determined by routine pharmaceutical procedures in cell culture or experimental animals to determine the 50% lethal dose and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50. Compounds that exhibit high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending on the dosage form used and the route of administration used. The exact dosage form, route of administration, and dosage can be selected by the individual physician in view of the patient's condition (eg,
ngl et al., 1975, "Pharmacological basis of therapeutic agents", Ch.

Dosage amount and interval may be adjusted individually to provide phosphatase modulating effects, or plasma levels of the active moiety that are sufficient to maintain a minimum effective concentration (MEC). The MEC will vary for each compound, but can be estimated from in vitro data, for example, the concentration required to obtain 50-90% inhibition of phosphatase using the assays described herein. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to measure plasma concentrations.

Dosage intervals can also be determined using MEC value.
You. The compound increases plasma levels by 10-90% of the time, preferably
From MEC over 30-90%, most preferably 50-90%
Should be administered using a program to maintain on
You. Normal patient dose for systemic administration is 1-2000 m
g / day, usually 1-250 mg / day, typically 10-150 mg / day.
It is an enclosure. In terms of patient weight, the usual dose is
0.02-25 mg / kg / day, usually 0.02-3 mg / kg / day, typically
The range is 0.2-1.5 mg / kg / day. The patient's body surface area
The usual dose is 0.5-1200mg / m ^Two/ Day, normal
0.5 to 150 mg / m^Two/ Day, typically 5-100 mg / m^Two/ Day range
It is an enclosure. Typical average plasma levels are 50-5000 μg / ml,
Maintain 50-1000 μg / ml, typically 100-500 μg / ml
It should be.

For local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration. The amount of composition administered will, of course, vary with the subject being treated,
It will depend on the patient's weight, the severity of the affliction, the manner of administration and the judgment of the referring physician. The desired blood level is HP
It can be maintained by continuous infusion of the compound, as confirmed by plasma levels measured by LC. It should be noted that the attending physician would know how and when to terminate, discontinue, or adjust to a lower dose due to toxicity or bone marrow failure, liver failure, or renal failure. Conversely,
If the clinical response is not appropriate (except for toxicity), the attending physician will know to bring the treatment to a higher level.

The degree of prophylactic or therapeutic dose of a compound in the acute or chronic treatment of a disease will vary with the severity of the condition to be treated and the route of administration. Again, it should be noted that the clinician or physician should know when to discontinue treatment and / or adjust the therapeutic dose due to toxicity or bone marrow failure, liver failure or renal failure. Dosage, and perhaps dosage frequency, will also vary according to the age, weight, and response of the individual patient. Generally, as described above, the daily dose range for the total compound will be from about 0.02 to about 25 mg / kg patient for most of the diseases described herein. Preferably, a daily dose range is from about 0.02 to about 3 mg / kg, but most preferably, a daily dose range is from about 0.2 to about 1.5 mg / kg / day. In addition, infants, toddlers, and patients over the age of 65, as well as patients with impaired renal or hepatic function, should be administered initially at low doses and determined based on individual clinical responses and blood levels It is recommended that As will be apparent to those skilled in the art, in some cases it may be necessary to use doses outside these ranges.

5.3.4. Packaging compositions may, if desired, be presented in a pack or dispenser which may contain one or more unit dosage forms containing the active ingredient. The pack may comprise metal or plastic foil, for example, a blister pack. The pack or dispenser may be accompanied by instructions for administration. A composition comprising a compound of the invention formulated in a compatible pharmaceutical carrier is also prepared, and placed in a suitable container.
Labels may be added for treatment of the indicated conditions. Suitable conditions indicated on the label are tumor, e.g.,
It may also include treatment of glioma or glioblastoma and suppression of angiogenesis.

5.4. Methods of Treatment Any compound of the invention that inhibits or reduces PTP activity in the signaling pathway can be used in the methods of treatment of the invention. In a preferred embodiment, the activity of the compound is sufficiently specific for PTP in its pathway, so that the compound does not interfere with the function of other phosphatases in the cell. Compounds of the present invention can be identified and evaluated, for example, by the methods described in Section 7 below.

The compounds and pharmaceutical compositions of the present invention can be used to treat diabetes mellitus. The etiology of diabetes is generally accompanied by insufficient insulin signaling or its complete lack. Lack or absence of the insulin signal can be caused by a variety of reasons, such as insulin deficiency, loss of binding affinity, defective receptor or under-expression of the receptor. Insulin receptor activity can be modulated by inhibiting phosphatases during signaling using the compounds of the present invention. Unlike currently available therapeutic modalities based on the insulin receptor, insulin signals can be restored or stimulated in cells via suppression of dephosphorylation activity, even in the absence of insulin. The example of diabetes mellitus illustrates the principles of therapeutic application of the present invention, which may be applied to other diseases that signal signaling by phosphotyrosine phosphatase.

The compounds and pharmaceutical compositions of the present invention can be used to treat an immune disorder in which cytokine signaling is insufficient. Cytokines play an important role not only in hematopoiesis but also in coordinating the immune response and the inflammatory response. The compounds are used to replace or enhance the activity of cytokines in signaling the differentiation and proliferation of hematopoietic cells as well as B and T cells in response to antigenic stimulation, thus treating anemia and immunodeficiency It is beneficial.
Also, the compounds of the present invention may be used as anti-inflammatory agents for treating diseases such as rheumatoid arthritis. Also, the compounds of the present invention may be therapeutically useful for treating neurodegenerative diseases by stimulating neuronal proliferation and differentiation regulated by neurotropin-mediated signaling.

In another embodiment of the present invention, the compounds and pharmaceutical compositions of the present invention comprise a cancer, eg, a glioma,
It may be used to treat melanoma, Kaposi's sarcoma, hemangiomas and ovarian cancer, breast cancer, lung cancer, pancreatic cancer, liver cancer, prostate cancer, colon cancer and epidermoid cancer, where the malignant cells are mediated by growth factors Proliferate and / or metastasize as a result of uncontrolled signaling. For example, overexpression of PTKs such as HER2 has been shown to correlate with abnormal growth characteristics of tumor cells. Also,
Angiogenesis and / or angiogenesis that promote tumor growth may be inhibited by the compound. The compounds modulate signaling in these tumor cells, thereby restoring normal growth characteristics. Also, the compounds may be beneficial in treating psoriasis caused by excessive epidermal growth factor-mediated signaling.

Having generally described the invention, the invention will be more readily understood with reference to the following examples. These examples are given by way of illustration,
It does not limit the invention. 6. Examples: Compound Synthesis As described above, the compounds of the present invention may be synthesized from readily available materials using conventional organic synthetic chemistry techniques. For example, the compounds of the present invention are disclosed in U.S. Pat.
Nos. 3,870,725 and 3,850,939, which are incorporated herein by reference. Still other preparation routes can be found in the literature and related art.

Examples of compound synthesis are provided here for illustrative purposes only. 6.1. First Embodiment 3-[(5-Nitrothiazol-2-yl) mercapto] -5-phenyl-1,2,4-triazole (compound 7) 2-Amino-5-nitrothiazole (Aldrich) is treated with sodium nitrite and hydrogen bromide. The starting material, 2-bromo-5-nitrothiazole, was prepared by treatment (Fr.
mande 2,015,434, 1970). First, benzoyl chloride is reacted with thiosemicarbazide in pyridine at 0 ° C. to give benzoylthiosemicarbazide, thereby obtaining 3-phenyl-1,2,4-triazole-5-thione (E.
J. Chem. Soc. (1949) 1163) was prepared. Benzoyl thiosemicarbazide was treated with potassium hydroxide in ethanol to give 3-phenyl-1,2,4-triazole-5-thione. Next, 3-phenyl-1,2,4-triazole-5-thione (1.77 g) was dissolved in 50 mL of methanol, and 9
Treat with 0.57 g of 5% sodium methoxide, then add 2%
-Bromo-5-nitrothiazole (2.09 g).
The mixture was stirred at room temperature for 2 hours and the precipitated sodium bromide was removed by filtration. The methanol is evaporated and the product is crystallized from ethanol and water to give 3-[(5-nitrothiazol-2-yl) mercapto] -5-phenyl-1,2,4-triazole (white solid, mp 155-150).
157 ° C.).

6.2. Second Embodiment 2-[(5-nitrothiazol-2-yl) mercapto] -5-t-butyl-1,
2,4-Triazole (Compound 2) The title compound was prepared by the method described in Example 1. After obtaining pivaloyl thiosemicarbazide using pivaloyl chloride instead of benzoyl chloride used in Example 1, 3
-Tert-Butyl-1,2,4-triazole-5-thione was obtained. 1.79 g of the sodium salt of thione were reacted with 2.09 g of 2-bromo-5-nitrothiazole as in Example 1 to give 2-[(5-nitrothiazol-2-yl).
1 g of [mercapto] -5-t-butyl-1,2,4-triazole (yellow solid, melting point 219-221 ° C.) was obtained.

6.3. Third Embodiment 3-[(5-nitrothiazol-2-yl) mercapto] -5- (thien-2-yl) -1,2,4-triazole (Compound 3) The title compound was prepared by the method described in Example 1. . Thiophene-2 was used in place of the benzoyl chloride used in Example 1.
-Using carboxylic acid acid chloride (prepared from the acid and oxalyl chloride) to obtain thiosemicarbazide of thiophene-2-carboxylic acid followed by 3- (thien-2-yl)
-1,2,4-Triazole-5-thione was obtained. 1.73 g of the sodium salt of thione were added as in Example 1.
-Bromo-5-nitrothiazole 2.09 g,
3-[(5-nitrothiazol-2-yl) mercapto]
1 g of -5- (thien-2-yl) -1,2,4-triazole (orange solid, mp 179-181 ° C) was obtained.

6.4. Fourth Embodiment 3- (4-Chlorophenyl) -5-[(5-nitrothiazol-2-yl) mercapto] -1,2,4-triazole (Compound 8) The title compound was prepared by the method described in Example 1. After using 4-chlorobenzoyl chloride in place of benzoyl chloride used in Example 1 to obtain 4-chlorobenzoylthiosemicarbazide, then 3- (4-chlorophenyl) -1,2,4-triazole-5 was obtained. -Thion was obtained.
3- (4-chlorophenyl)-as in Example 1
Sodium salt of 1,2,4-triazole-5-thione
2.34 g was reacted with 2.09 g of 2-bromo-5-nitrothiazole to give 3- (4-chlorophenyl) -5-[(5-
Nitrothiazol-2-yl) mercapto] -1,2,4
Triazole (light brown solid, melting point 181-184 ° C) 1.
5 g were obtained.

6.5. Embodiment 5 3-hydroxy-5
[(5-nitrothiazol-2-yl) mercapto] -4
-Phenyl-1,2,4-triazole (compound 5) The title compound was prepared by the general method described by Potts, KT (1961) Chem. Rev. 61:87. 4-phenyl-3
-Thiosemicarbazide (4.18 g) (Aldrich) in 50 mL of pyridine
And treated at 0 ° C. with 2.71 g of ethyl chloroformate.
The reaction was stirred for 2 hours and then refluxed for 18 hours. The solvent was evaporated and triturated with water to give 3-hydroxy-5.
2.5 g of -mercapto-4-phenyl-1,2,4-triazole were obtained. 3-hydroxy-5-mercapto-4-
Phenyl-1,2,4-triazole (1.93 g) was stirred with 10 equivalents of a solution of potassium carbonate in 10 mL of ethanol for 1 hour in 10 mL of ethanol, then 2-bromo-5-nitrothiazole was used as in Example 1. Reacted with 2.09 g. Crystallization with ethanol and water gives 3-hydroxy-
5-[(5-nitrothiazol-2-yl) mercapto]
0.6 g of -4-phenyl-1,2,4-triazole (dark yellow solid, mp 188-190 DEG C.) was obtained.

6.6. Embodiment 6 4-cyclohexyl-3
-Hydroxy-5-[(5-nitrothiazol-2-yl) mercapto] -1,2,4-triazole (Compound 4) The title compound was prepared in a manner similar to that described in Example 5. Cyclohexyl isothiocyanate (3.53 g)
Was added to a solution of hydrazine (0.8 g) in 20 mL of acetonitrile over a period of 30 minutes. The reaction was further stirred for 2 hours, and evaporated to dryness to obtain 4.4 g of 4-cyclohexylcarbonyl-3-thiosemicarbazide. 4-Cyclohexylcarbonyl-3-thiosemicarbazide (2.02 g) was treated as in Example 5 with ethyl chloroformate (1.08 g). Reaction product 3-hydroxy-5
-Mercapto] -4-cyclohexyl-1,2,4-triazole (1.0 g) was prepared as in Example 5 using 2-bromo-
It was reacted with 1.05 g of 5-nitrothiazole. Crystallization with ethanol and water gives 3-hydroxy-5-[(5-nitrothiazol-2-yl) mercapto] -4-cyclohexyl-1,2,4-triazole (yellow solid, melting point
(237-239 ° C) 0.3 g was obtained.

6.7. Embodiment 7 4-Benzyl-3-hydroxy-5-[(5-nitrothien-2-yl) mercapto] -1,2,4-triazole (Compound 9) The title compound is described in Example 5 starting from benzyl isothiocyanate. Prepared in a manner similar to that described. Intermediate 4-benzyl-3-thiosemicarbazide (1.8
1g) as in Example 5 ethyl chloroformate (1.09g)
Processed. Reaction product 4-benzyl-3-hydroxy-5-mercapto-1,2,4-triazole (1.04 g)
Was reacted with 1.05 g of 2-bromo-5-nitrothiazole as in Example 5. Crystallized from ethanol and water, 0.3 g of 4-benzyl-3-hydroxy-5-[(5-nitrothien-2-yl) mercapto] -1,2,4-triazole (yellow solid, mp 221-224 ° C.) I got

6.8. Embodiment 8 3-hydroxy-5
[(5-nitrothiazol-2-yl) mercapto] -4
-[2- (trifluoromethyl) phenyl] -1,2,2
4-Triazole (compound 6) The title compound was prepared in a similar manner to that described in Example 5, starting from 2- (trifluoromethyl) phenylisothiocyanate. The intermediate 4- [2- (trifluoromethyl) phenyl] -3-thiosemicarbazide (2.04 g) was converted to ethyl chloroformate as in Example 5.
(1.09 g). Reaction product 3-hydroxy-5-
Mercapto-4- [2- (trifluoromethyl) phenyl] -1,2,4-triazole (0.78 g) was reacted as in Example 5 with 0.63 g of 2-bromo-5-nitrothiazole. Crystallization with ethanol and water gave 3-hydroxy-5-[(5-nitrothiazol-2-yl) mercapto] -4- [2- (trifluoromethyl) phenyl] -1,2,4-triazole (yellow solid , Melting point 18
0.3-185 ° C).

6.9. Embodiment 9 3- (1-ethyl-3-
Methylpyrazol-5-yl) -4- (3-methoxy-n
-Propyl) -5-[(5-nitrothiazol-2-yl) mercapto] -1,2,4-triazole (Compound 1) The title compound was prepared in a similar manner to that described in Example 1. 3-Methoxy-n-propylisothiocyanate is prepared from 3-methoxy-n-propylamine and thiophosgene at elevated temperature and then reacted with hydrazine in pyridine to give the intermediate 4- (3-methoxy-n-propyl)- 3-thiosemicarbazide was obtained. 4- (3-methoxy-n-propyl) -3-thiosemicarbazide (1.64
g) with 1-ethyl-3-methylpyrazole-5-carboxylic acid chloride (1.73 g, prepared from the acid and oxalyl chloride) to give 1- (1-ethyl-3-methylpyrazole-5-carbonyl) -4- (3-methoxy-n-
2 g of propyl) -3-thiosemicarbazide were obtained. 1-
(1-Ethyl-3-methylpyrazole-5-carbonyl) -4- (3-methoxy-n-propyl) -3-thiosemicarbazide is treated with potassium hydroxide in ethanol to give 3- (1-ethyl-3). -Methylpyrazol-5-yl) -5-mercapto-4- (3-methoxy-n-propyl) -1,2,4-triazole was obtained. 3- (1-
Ethyl-3-methylpyrazol-5-yl) -5-mercapto-4- (3-methoxy-n-propyl) -1,2,2
The sodium salt of 4-triazole (0.73 g) was treated as in Example 1 with 2-bromo-5-nitrothiazole (0.52 g).
To give crude 3- (1-ethyl-3-methylpyrazol-5-yl) -4- (3-methoxy-n-propyl)-
5-[(5-nitrothiazol-2-yl) mercapto]
-1,2,4-Triazole was obtained. Crystallization with ethanol and water gives 3- (1-ethyl-3-methylpyrazol-5-yl) -4- (3-methoxy-n-propyl)-.
5-[(5-nitrothiazol-2-yl) mercapto]
-1,2,4-triazole (light brown solid, melting point 117
~ 118 ° C).

6.10. Example 10. 3- (4-Chlorophenyl) -5-[(5-nitrothiazol-2-yl) amino] -1,2,4-triazole (compound 13) 3-amino-5- ( 4-chlorophenyl) -1,2,4
-Triazole is heated with 2-bromo-5-nitrothiazole in refluxing tetrahydrofuran, followed by silica gel column chromatography with a solvent mixture of dichloromethane and methanol to give 3- (4-chlorophenyl) -5-[(5-nitrothiazol-2. The title compound was prepared in a similar manner to that described in Example 1 by obtaining -yl) amino] -1,2,4-triazole.

6.11. Example 11. 4-Allyl-3-hydroxy-5-[(5-nitrothien-2-yl) mercapto] -1,2,4-triazole (compound 14) Starting from allyl isothiocyanate The title compound was prepared in a similar manner to that described in Example 5. 4-allyl-3-hydroxy-5-mercapto-
The 1,2,4-triazole was converted to 2-
Reacted with bromo-5-nitrothiazole. Crystallization with ethanol and water gives 4-allyl-3-hydroxy-
5-[(5-nitrothien-2-yl) mercapto]-
1,2,4-Triazole was obtained as a yellow solid.

6.12. Example 12. 3-[(5-Nitrothiazol-2-yl) mercapto] -5-phenyl-1,
2,4-Triazole (Compound 7) The main starting material 2-bromo-5-nitrothiazole was prepared by treating 2-amino-5-nitrothiazole (Aldrich) with sodium nitrite and hydrogen bromide (F
r.Demande 2,015,434, 1970). First, 3-phenyl-1,2,4-triazole-5-thione (E. Hogard) was prepared by reacting benzoyl chloride with thiosemicarbazide in pyridine at 0 ° C. to give benzoylthiosemicarbazide.
th, J. Chem. Soc. (1949) 1163) was prepared. Benzoyl thiosemicarbazide is treated with potassium hydroxide in ethanol to give 3-phenyl-1,2,4-triazole-5.
-Thion was obtained. Next, 3-phenyl-1,2,4-triazole-5-thione (1.77 g) was dissolved in 50 mL of methanol, treated with 0.57 g of 95% sodium methoxide, and then treated with 2-bromo-5-nitrothiazole ( 2.09g). The mixture was stirred at room temperature for 2 hours and the precipitated sodium bromide was removed by filtration. The methanol was evaporated and the product crystallized from ethanol and water to give 3-
[(5-nitrothiazol-2-yl) mercapto] -5
1.5 g of -phenyl-1,2,4-triazole (orange solid) were obtained.

General Procedure This general procedure was performed using the starting mercaptan identified.
The following compounds are prepared by using Tetrahi
Dissolve in drofuran or ethanol or a mixture of both
2-bromo-5-nitrothiazole and the corresponding
Of mercaptans (thiols)
Is stirred at room temperature for 24 hours. Starting material still present
In this case, the reaction is carried out by heating at 50 ° C. for 24 hours. Then the mixture
Diluted with ethyl acetate and dilute sodium carbonate solution
You. Subsequently, the organic extract is washed with water and saline, and then washed with sodium sulfate.
Dry with lium and filter. After concentration, the crude product
Purify by column chromatography or crystallization.
Preparation of starting substituted mercaptans (thiols) according to literature
Or use a commercial product. 6.13. Example 13 1-methyl-2-[(5-nitrothiazol-2-yl) mercap
G] Imidazole Starting mercaptan: 1-methyl-2-mercaptoimidazo
6.14. Example 14 2-Amino-5-[(5-nitrothiazol-2-yl) mercap
G] -1,3,4-thiadiazole Starting mercaptan: 2-amino-5-mercapto-1,3,4-thiophene
Adiazole 6.15. Example 15 1-Methyl-2-[(5-nitrothiazol-2-yl) mercap
G] tetrazole Starting mercaptan: 1-methyl-2-mercaptotetrazo
6.16. Example 16 1-benzyl-2-[(5-nitrothiazol-2-yl) mercap
G] Imidazole Starting mercaptan: 1-benzyl-2-mercaptoimidazo
6.17. Example 17 1-Tosyl-2-[(5-nitrothiazol-2-yl) mercap
G] Benzimidazole Starting mercaptan: 1-tosyl-2-mercaptobenzimi
Dazol 6.18. Example 18 1-ethylaminocarbonyl-5-nitro-2-[(5-nitrothi
Azol-2-yl) mercapto] Benzimidazole Starting mercaptan: 1-ethylaminocarbonyl-2-mer
Capto-5-nitrobenzimidazole 6.19. Example 19 3-bromo-2-methyl-6-[(5-nitrothiazol-2-yl)
Mercapto] imidazo [4,5-b] pyridine Starting mercaptan: 3-bromo-6-mercaptan-2-methyl
Louisimidazo [4,5-b] pyridine 6.20. Example 20 6-[(5-Nitrothiazol-2-yl) mercapto] imidazo
[4,5-b] pyridine Starting mercaptan: 6-mercaptoimidazo [4,5-b] pyri
Gin 6.21. Example 21 2- [N-phenyl-N- [3- (trifluoromethyl) phenyl
Aminocarbonylmethyl] amino] -5-[(5-nitrothiazo
Ru-2-yl) mercapto] -1,3,4-thiadiazole
Lcaptan: 2- [N-phenyl-N- [3- (trifluoromethyl
Phenylaminocarbonyl-methyl] amino] -5-meth
Lcapto-1,3,4-thiadiazole 6.22. Example 22 2-formamide-5-[(5-nitrothiazol-2-yl) mel
Capto] -1,3,4-thiadiazole Starting mercaptan: 2-formamide-5-mercapto-1,
3,4-thiadiazole 6.23.Example 23 2-n-butylmercapto-5-[(5-nitrothiazol-2-a
M) mercapto] -1,3,4-thiadiazole Starting mercaptan: 2-n-butylmercapto-5-mercapto
G-1,3,4-thiadiazole 6.24. Example 24 2-[(5-nitrothiazol-2-yl) mercapto] -5-[(fe
Nonoxycarbonyl) methylmercapto] -1,3,4-thiadia
Sol Starting mercaptan: 2-mercapto-5-[(phenoxycal
Bonyl) methylmercapto] -1,3,4-thiadiazole 6.25. Example 25 2-[(ethoxycarbonyl) methylmercapto] -5-[[2-
[(5-nitrothiazol-2-yl) mercapto] -1,3,4-thia
Diazol-5-yl] mercapto] -1,3,4-thiadiazole Starting mercaptan: 2-[(ethoxycarbonyl) methylmeth
Rucapto] -5-mercapto-1,3,4-thiadiazol-5-a
[Mercapto] -1,3,4-thiadiazole 6.26. Example 26 2- (2-chloroethylmercapto) -5-[[2-[(5-nitrothia
Sol-2-yl) mercapto] -1,3,4-thiadiasol-5-a
[Mercapto] -1,3,4-thiadiazole Starting mercaptan: 2- (2-chloroethylmercapto) -5-
Mercapto-1,3,4-thiadiazol-5-yl] mercapto
G] -1,3,4-thiadiazole 6.27. Example 27 2- (2,5-dihydroxyphenylmercapto) -5-[[2-[(5-
Nitrothiazol-2-yl) mercapto] -1,3,4-thiadia
Sol-5-yl] mercapto] -1,3,4-thiadiazole Starting mercaptan: 2- (2,5-dihydroxyphenylmer
Capto) -5-Mercapto-1,3,4-thiadiazol-5-yl]
Mercapto] -1,3,4-thiadiazole 6.28. Example 28 2-Ethylmercapto-5-[(5-nitrothiazol-2-yl)
Mercapto] -1,3,4-thiadiazole Starting mercaptan: 2-ethylmercapto-5-mercapto
-1,3,4-thiadiazole 6.29. Example 29 1- (4-aminophenyl) -5-[(5-nitrothiazol-2-a
Le) mercapto] tetrazole starting mercaptan: 1- (4-aminophenyl) -5-mercap
6.30. Example 30 1-allyl-5-[(5-nitrothiazol-2-yl) mercap
G] tetrazole Starting mercaptan: 1-allyl-5-mercaptotetrazo
6.31.Example 31 1- (4-acetamidophenyl) -5-[(5-nitrothiazol-2
-Yl) mercapto] tetrazole starting mercaptan: 1- (4-acetamidophenyl) -5-meth
Lcaptotetrazole 6.32.Example 32 1- (4-aminophenyl) -5-[(5-nitrothiazol-2-a
Le) mercapto] tetrazole starting mercaptan: 1- (4-aminophenyl) -5-mercap
Example 33 1- (4-Aminosulfonylphenyl) -5-[(5-nitrothiazo
Ru-2-yl) mercapto] tetrazole starting mercaptan: 1- (4-aminosulfonylphenyl)-
Example 34 1-Ethyl-5-[(5-nitrothiazol-2-yl) mercap
G] tetrazole Starting mercaptan: 1-ethyl-5-mercaptotetrazo
6.35. Example 35 2-[(5-nitrothiazol-2-yl) mercapto] -4-quinazo
Ron Starting mercaptan: 2-mercapto-4-quinazolone 6.36. Example 36 1,3-dimethyl-5-[(5-nitrothiazol-2-yl) merca
[Pto] imidazo [4,5-d] pyrimid-2-one Starting mercaptan: 1,3-dimethyl-5-mercaptoimid
Zo [4,5-d] pyrimid-2-one 6.37. Example 37 4,6-Dihydroxy-2-[(5-nitrothiazol-2-yl) meth
Lcapto] imidazo [4,5-d] pyrimidine Starting mercaptan: 4,6-dihydroxy-2-mercaptoy
Midazo [4,5-d] pyrimidine 6.38. Example 38 2-Amino-5-[(5-nitrothiazol-2-yl) mercap
G] -1,3,4-thiadiazole Starting mercaptan: 2-amino-5-mercapto-1,3,4-thiophene
Adiazole 6.39. Example 39 5,6-Dichloro-2-[(5-nitrothiazol-2-yl) merca
] Cycloheximidazole Starting mercaptan: 5,6-dichloro-2-mercaptocyclo
Heximidazole 6.40. Example 40 4-bromo-2-[(5-nitrothiazol-2-yl) mercap
G] -6- (trifluoromethyl) benzimidazole Starting mercaptan: 4-bromo-2-mercapto-6- (trif
Fluoromethyl) benzimidazole 6.41. Example 41 4-Chloro-2-[(5-nitrothiazol-2-yl) mercap
To] -6- (trifluoromethyl) benzimidazole Starting mercaptan: 4-chloro-2-mercapto-6- (trif
(Ruolomethyl) benzimidazole 6.42. Example 424-Methoxycarbonyl-3-methyl-2-
[(5-Nitrothiazol-2-yl) mercapto] imidazo
Starting mercaptan: 4-methoxycarbonyl-3-methyl-2
-Mercaptoimidazole 6.43. Example 43 4-ethoxycarbonyl-2-[(5-nitrothiazol-2-a
Le) mercapto] imidazole Starting mercaptan: 4-ethoxycarbonyl-2-mercap
Toimidazole 6.44. Example 44 4-ethoxycarbonyl-3-methyl-2-[(5-nitrothiazo
Ru-2-yl) mercapto] imidazole Starting mercaptan: 4-ethoxycarbonyl-3-methyl-2
-Mercaptoimidazole 6.45. Example 45 5-Methoxy-2-[(5-nitrothiazol-2-yl) mercap
G] Benzimidazole starting mercaptan: 5-methoxy
-2-mercaptobenzimidazole 6.46.Example 46 4,7-diethoxy-2-[(5-nitrothiazol-2-yl) mel
Capto] Benzimidazole Starting mercaptan: 4,7-diethoxy-2-mercaptoben
6.47.Example 47 1-Cyclohexyl-4,5-di (ethoxycarbonyl) -2-
[(5-Nitrothiazol-2-yl) mercapto] imidazo
Le Starting mercaptan: 1-cyclohexyl-4,5-di (ethoxy
6.48.Example 48 4,5-di (ethoxycarbonyl) -2-[(5-nitrothiazol-2)
-Yl) mercapto] imidazole Starting mercaptan: 4,5-di (ethoxycarbonyl) -2-me
6.49.Example 49 3,7-dihydroxy-5-[(5-nitrothiazol-2-yl) meth
Lcapto] -4-propylimidazo [4,5-d] pyrazine Starting mercaptan: 3,7-dihydroxy-5-mercapto-4
-Propylimidazo [4,5-d] pyrazine 6.50. Example 50 5-Methyl-2-[(5-nitrothiazol-2-yl) mercap
G] Benzimidazole Starting mercaptan: 5-methyl-2-mercaptobenzimi
Dazol 6.51. Example 51 2-[(5-nitrothiazol-2-yl) mercapto] -5-sulfo
Benzimidazole Starting mercaptan: 2-mercapto-5-sulfobenzimi
Dazol 6.52. Example 52 5-chloro-1-isopropyl-2-[(5-nitrothiazol-2-
Yl) mercapto] benzimidazole Starting mercaptan: 5-chloro-1-isopropyl-2-mer
Captobenzimidazole 6.53. Example 53 7-Methyl-3- (trifluoromethyl) -6-[(5-nitrothiazo
Ru-2-yl) mercapto] imidazo [4,5-b] pyridine Starting mercaptan: 7-methyl-3-trifluoromethyl-6
-Mercaptoimidazo [4,5-b] pyridine 6.54. Example 54 1- (4-Fluorophenyl) -5-[(5-nitrothiazol-2-a
Le) mercapto] tetrazole starting mercaptan: 1- (4-fluorophenyl) -5-merca
Example 55 1- (4-hydroxyphenyl) -5-[(5-nitrothiazol-2-
Yl) mercapto] tetrazole starting mercaptan: 1- (4-hydroxyphenyl) -5-mer
Captotetrazole 6.56. Example 56 1-Methyl-5-[(5-nitrothiazol-2-yl) mercap
G] tetrazole Starting mercaptan: 1-methyl-2-mercaptoimidazo
Example 57 1-Ethyl-5-[(5-nitrothiazol-2-yl) mercap
G] tetrazole Starting mercaptan: 1-ethyl-5-mercaptotetrazo
6.58.Example 58 1-Carboxymethyl-5-[(5-nitrothiazol-2-yl)
Mercapto] tetrazole starting mercaptan: 1-carboxymethyl-5-mercapto
Example 59 2-Amino-5-[(5-nitrothiazol-2-yl) mercap
G] -1,3,4-thiadiazole Starting mercaptan: 2-amino-5-mercapto-1,3,4-thiophene
Adiazole 6.60. Example 60 2-Methyl-5-[(5-nitrothiazol-2-yl) mercap
G] -1,3,4-thiadiazole Starting mercaptan: 2-methyl-5-mercapto-1,3,4-thiophene
Adiazole 6.61. Example 61 2-Methylmercapto-5-[(5-nitrothiazol-2-yl)
Mercapto] -1,3,4-thiadiazole Starting mercaptan: 2-methylmercapto-5-mercapto
-1,3,4-thiadiazole 6.62. Example 62 2-Cyclopropylmethylmercapto-5-[(5-nitrothia
Sol-2-yl) mercapto] -1,3,4-thiadiazole Starting mercaptan: 2-cyclopropylmethylmercapto
-5-mercapto-1,3,4-thiadiazole 6.63. Example 63 2-[(5-nitrothiazol-2-yl) mercapto] -5- [3- (to
Trifluoromethyl) benzylmercapto] -1,3,4-thiazi
Azole Starting mercaptan: 2-mercapto-5- [3- (trifluoro
Methyl) benzylmercapto] -1,3,4-thiadiazole 6.64. Example 64 2- (2,4-dinitrobenzyl) -5-[(5-nitrothiazol-2-
Yl) mercapto] -1,3,4-thiadiazole Starting mercaptan: 2- (2,4-dinitrobenzyl) -5-mer
Capto-1,3,4-thiadiazole 6.65. Example 65 2-benzoylmercapto-5-[(5-nitrothiazol-2-a
Le) mercapto] -1,3,4-thiadiazole Starting mercaptan: 2-benzoylmercapto-5-merca
Pt-1,3,4-thiadiazole 6.66. Example 66 2-Methyl-5-[(5-nitrothiazol-2-yl) mercap
G] -1,3,4-thiadiazole Starting mercaptan: 2-methyl-5-mercapto-1,3,4-thiophene
Azizole 6.67. Example 67 4- [4- (4,5-dichloroimidazol-1-yl) phenyl] -2-
[(5-Nitrothiazol-2-yl) mercapto] pyrimidine Starting mercaptan: 4- [4- (4,5-dichloroimidazole-1-
Yl) phenyl] -2-mercapto-pyrimidine 6.68. Example 68 2-[(5-nitrothiazol-2-yl) mercapto] cyclohe
Xylpyrimid-5-one Starting mercaptan: 2-mercaptocyclohexylpyrimi
6.69.Example 69 2- (4-methylphenylamino) -5-[(5-nitrothiazol-2
-Yl) mercapto] -1,3,4-thiadiazole Starting mercaptan: 2- (4-methylphenylamino) -5-meth
Lcapto-1,3,4-thiadiazole 6.70. Example 70 3- (2,6-dimethylphenylamino) -6-[(5-nitrothiazo
Ru-2-yl) mercapto] -1,2,5-thiadiazine Starting mercaptan: 3- (2,6-dimethylphenylamino)-
6-Mercapto-1,2,5-thiadiazine 6.71. Example 71 2- (2,4-dimethylphenylamino) -5-[(5-nitrothiazo
Ru-2-yl) mercapto] -1,3,4-thiadiazole Starting mercaptan: 2- (2,4-dimethylphenylamino)-
6.72. Example 72 2-[(5-nitrothiazol-2-yl) mercapto] -5- (2,4,6
-Trimethylphenylamino) -1,3,4-thiadiazole Starting mercaptan: 2-mercapto-5- (2,4,6-trimethyl
Phenylamino) -1,3,4-thiadiazole 6.73. Example 73 2-[(5-nitrothiazol-2-yl) mercapto] -5-phenyl
L-amino-1,3,4-thiadiazole Starting mercaptan: 2-mercapto-5-phenylamino-
1,3,4-thiadiazole 6.74. Example 74 2-[(5-nitrothiazol-2-yl) mercapto] -5 (2,4,5-
Trimethylphenylamino) -1,3,4-thiadiazole Starting mercaptan: 2-mercapto-5 (2,4,5-trimethyl
Phenylamino) -1,3,4-thiadiazole 6.75. Example 75 2-cyclohexylamino-5-[(5-nitrothiazol-2-a
M) mercapto] -1,3,4-thiadiazole Starting mercaptan: 2-cyclohexylamino-5-merca
Pt-1,3,4-thiadiazole 6.76. Example 76 2- (2-methoxyphenylamino) -5-[(5-nitrothiazole
-2-yl) mercapto] -1,3,4-thiadiazole Starting mercaptan: 2- (2-methoxyphenylamino) -5-
Mercapto-1,3,4-thiadiazole 6.77. Example 77 2- (5-chloro-2-methylphenylamino) -5-[(5-nitro
Thiazol-2-yl) mercapto] -1,3,4-thiadiazole Starting mercaptan: 2- (5-chloro-2-methylphenyla)
Mino) -5-Mercapto-1,3,4-thiadiazole 6.78. Example 78 2- (2-Nitrofuran-5-yl) -5-[(5-nitrothiazol-2
-Il) mercapto] -1,2,4-triazole Starting mercaptan: 2- (2-nitrofuran-5-yl) -5-meth
Lcapto-1,2,4-triazole 6.79. Example 79 2-[(5-nitrothiazol-2-yl) mercapto] benzi
Midazole Starting mercaptan: 2-mercaptobenzimidazole 6.80. Example 80 1-benzyl-5-[(5-nitrothiazol-2-yl) mercap
G] tetrazole Starting mercaptan: 1-benzyl-5-mercaptotetrazo
Example 81 2- (2-Chlorophenylamino) -5-[(5-nitrothiazol-2
-Yl) mercapto] -1,3,4-thiadiazole Starting mercaptan: 2- (2-chlorophenylamino) -5-meth
Lcapto-1,3,4-thiadiazole 6.82. Example 82 1-Cyclohexyl-5-[(5-nitrothiazol-2-yl) meth
Lecapto] tetrazole starting mercaptan: 1-cyclohexyl-5-mercaptothe
6.83. Example 83 2-[(5-nitrothiazol-2-yl) mercapto] -5- (tri
Fluoromethyl) pyrimidine Starting mercaptan: 2-mercapto-5- (trifluoromethyl
6.84. Example 84 2-[(5-bromothiazol-2-yl) mercapto] -1- (2,4-
Dichlorophenyl) imidazole Starting mercaptan: 2-mercapto-1- (2,4-dichlorophene)
Nil) Imidazole 6.85. Example 85 2-[(5-bromothiazol-2-yl) mercapto] -1- (2,3-
Dichlorophenyl) imidazole Starting mercaptan: 2-mercapto-1- (2,3-dichlorophene)
Example 86 2- [2-Methyl-4- (trifluoromethyl) pyrid-3-i-yl) imidazole 6.86.
Le] -5-[(5-Nitrothiazol-2-yl) mercapto] -1,3,
4-oxadiazole Starting mercaptan: 2- [2-methyl-4- (trifluoromethyl
Le) pyrid-3-yl] -5-mercapto-1,3,4-oxadia
Sol 6.87. Example 87 2-[(5-nitrothiazol-2-yl) mercapto] imidazo
Starting mercaptan: 2-mercaptoimidazole 6.88. Example 88 1- (3-hydroxyphenyl) -5-[(5-nitrothiazol-2-
Yl) mercapto] tetrazole starting mercaptan: 1- (3-hydroxyphenyl) -5-mer
Captotetrazole 6.89. Example 89 1- [4- (n-Hexanoylamino) phenyl] -5-[(5-nitro
Thiazol-2-yl) mercapto] tetrazole Starting mercaptan: 1- [4- (n-hexanoylamino) phenyl
Nyl] -5-mercaptotetrazole 6.90. Example 90 7-[(5-nitrothiazol-2-yl) mercapto] -benzi
Midazo [5,6-b] 1,4-dioxane Starting mercaptan: 7-mercaptobenzimidazo [5,6-
b] 1,4-dioxane 6.91. Example 91 5-[(5-nitrothiazol-2-yl) mercapto] -1-phenyl
Letetrazole Starting mercaptan: 5-mercapto-1-phenyltetrazo
6.92. Example 92 2-[(5-nitrothiazol-2-yl) mercapto] -5-propa
Lugyl mercapto-1,3,4-thiadiazole Starting mercaptan: 2-mercapto-5-propargylmer
Capto-1,3,4-thiadiazole 6.93. Example 93 2-[(5-nitrothiazol-2-yl) mercapto] -1- (pyrro
L-3-yl) imidazole Starting mercaptan: 2-mercapto-1- (pyrrol-3-yl)
Example 94 1-Isopropyl-2-[(5-nitrothiazol-2-yl) mel
Capto] Benzimidazole Starting mercaptan: 1-isopropyl-2-mercaptoben
Example 95 1-Isopropyl-2-[(5-nitrothiazol-2-yl) mel
Capto] -5- (trifluoromethyl) benzimidazole Starting mercaptan: 1-isopropyl-2-mercapto-5-
(Trifluoromethyl) benzimidazole 6.96. Example 96 5-Chloro-1-isopropyl-2-[(5-nitrothiazol-2-
Yl) mercapto] benzimidazole Starting mercaptan: 5-chloro-1-isopropyl-2-mer
6. CaptobenzimidazoleDemonstration of phosphatase inhibitory activity of compounds 7.1.Phosphotyrosine enzyme-linked immunosorbent assay
(ELISA) In this example, the compound of the present invention was administered with insulin.
Dephosphorylation of phosphotyrosine (pTyr) residues on the receptor (IR)
The inhibitory ability is described. Any compound of the present invention
It can be used for this assay. The skilled person is different
By using target cells and anchor antibodies,
Plate-derived growth factor receptor and other substrate molecules
You can recognize that it can be used. This app
Say uses different substrate molecules to achieve different
Compounds of the Invention for Rotate Tyrosine Phosphatase
The activity of the product can be evaluated. In the case of IR, intrinsic
Sex kinase activity is low even without insulin binding.
Active. Therefore, stimulates phosphorylation of IR
To do so, insulin is not needed. Contact with compound
After the cell lysate is prepared, the anti-insulin receptor
Add to microtiter plate coated with antibody
Added. Level of phosphorylation of entrapped insulin receptor
Is detected using an anti-pTyr antibody and an enzyme-linked secondary antibody.
did. 7.1.1.Materials and methods 1. The cell line used for the IR assay was human IR (H25 cells).
NIH3T3 cells (A
TCC # CRL 1658). The growth medium for these cells is
10% fetal bovine serum, 1% L-glutamine, and 20 mM H
DMEM (Gibco) containing epes. 2. The anchor antibody used is the extracellular domain of human IR.
Recognized BBE, Enzymology Laboratories, Sugen
 Inc. purified. 3. PBS (Gibco): 0.20 g / L KH_TwoPO_Four, 2.16 g / L K_TwoHP
O_Four, 0.20 g / L KCl, 8.00 g / L NaCl (pH 7.2). 4. Rabbit polyclonal anti-phosphotyrosine antibody (anti-
(pTyr) is manufactured by Enzymology Laboratories, Sugen, Inc.
Was done. 5. Goat anti-rabbit IgG POD conjugate (Tago, Burlingame,
 CA, Cat. No. 6430) was used as the secondary antibody. 6. TBST buffer: 50 mM Tris-HCl, 150 mM NaCl
PH 0.1 with 10N HCl containing 0.1% Triton X-100
Adjustment. 7. Blocking buffer: PBS + 5% milk (car
Nation instant skim milk powder) 8.5 x HNTG buffer: 100 mM HEPES, 750 mM NaCl,
Contains 50% glycerol, 0.5% Triton X-100 (pH 7.5)
 . 9. ABTS solution: 100 mM citric acid, 250 mM Na_TwoHPO_Four, 0.5m
g / ml ABTS (2,2'-azinobis (3-ethylbenzthiazuri
Sulfonic acid) and adjusted to pH 4.0 with 1N HCl. Ten. Cell lysis buffer: 1 mM Na_ThreeVO_Four(To aliquot
0.5M solution stored separately as a 100-fold stock at -80 ° C
Solution), 5 mM NaP_TwoO₇And HNTG containing 5mM EDTA
Adjust before use and store on ice until use. 11． Hydrogen peroxide: 30% solution 7.1.2.Preparation of assay plate Microtiter plate (96 well, Easy Wash ELIS
A plate, Corning 25805-96) in 100 μL of PBS.
Solution containing 0.5 μg / well anchor antibody, at least
Were also coated for 2 hours at room temperature or overnight at 4 ° C.
Prior to use, add 100 μL block of coating buffer.
Replace with King buffer and pre-coated assay plate
The rate was shaken at room temperature for 30 minutes. Before adding lysate
Next, the wells were washed three times with water and once with TBST buffer.
Was. 7.1.3.Seeding of cells Cells were plated in DMEM containing 10% fetal bovine serum (FBS).
80-90 cm culture dish (Corning 25020-100)
% Confluence. Cells were trypsin-EDTA (0.25
%, 0.5 mL, Gibco), 10% FBS, 1% L
-Resuspend in fresh medium containing glutamine and Hepes
96-well round bottom tissue culture plates (Corning 25806-9
In 6), 25,000 cells / well were transferred at 100 μL / well. Fine
Bubble at 37 ° C, 5% CO_TwoAnd incubated for 24 hours.
Change the medium by inverting the plate, and add 0.5% FBS and Hepe
and a DMEM medium containing Cells are further incubated at 37 ° C, 5
% CO _TwoAnd incubated. 7.1.4.Assay procedure Assays were performed in 96-well tissue culture plates
Was. Remove medium from wells before adding compound to cells.
The serum DMEM medium was replaced with 90 μL / well. Positive controls include
80 μL of DMEM was added. 90 μL DMEM for negative control
Was added. The compound of the present invention is diluted 1:10 with DMEM and diluted.
Diluted test substance at 10 μL / well to cells in each well
Transfer to a final concentration of 1: 100. Positive control and negative pair
10 μL / well dimethyl sulfoxide (DMSO)
In addition, the final concentration was 1%. Additional positive control wells
0.1M Na_ThreeVO_Four To a final concentration of 10 mM by adding 10 μL / well.
did. Tissue culture plate at 37 ° C, 5% CO_TwoInk in
Shake for 1 minute before incubation. 90 minutes incube
After plating, invert the plate and remove the medium,
μL / well of cell lysis buffer was added to the cells. set
Shake the tissue culture plate for 5 minutes and then place on ice for 10 minutes.
Was. Repeat cells to homogenize cells,
To the corresponding wells of the assay plate pre-coated with
Moved.

The substrate in the cell lysate was shaken at room temperature for 1 hour to bind to the anchor antibody. The lysate was then removed and the assay plate was washed. Washing of all ELISA plates was performed by rinsing three times with water followed by one rinse with TBST. The plate was patted dry on a paper towel and dried. 100 μL / well of anti-pTyr antiserum diluted 1: 3000 in TBST was added to the wells,
Incubation with shaking at room temperature for 30 minutes detected phosphotyrosine. The unbound excess anti-pTyr antiserum was then removed and the assay plate was washed as described above. Secondary antibody diluted 1: 3000 in TBST was added to the wells and incubated for 30 minutes at room temperature with shaking. Next, the secondary antibody was removed, the plate was washed, and fresh ABTS / H
₂ O ₂ (0.5 mg / mL in 100 mM citric acid, 250 mM Na ₂ HPO ₄
2,2-azinobis (3-ethylbenzthiazulin) in 10 mL of sulfonic acid containing 1.2 μL of 30% H ₂ O ₂ , pH 4.0)
Was added for color development. After 10 minutes, 100 μL / well of 0.
The reaction was stopped by adding 2 M HCl and incubating for 1 minute with shaking. The absorbance at 410 nm was measured with an ELISA plate reader (Dynatec MR5000). 7.1.5. Experimental Results The results for some compounds of the present invention are shown in Table 1 above. Compound activity was expressed as the concentration of compound that resulted in an increase in the indicated percentage of phosphotyrosine compared to the vanadate control (see Table 1).

If a compound was shown to be active in the assay, a range of concentrations of the compound was used for kinetic experiments. As shown in FIG. 1, (2-[(5
-Nitrothiazol-2-yl) mercapto] -3-cyano-5
-Compound 10, which is acetoxy-6-dimethoxymethyl-pyridine, produces pT on the insulin receptor for over 90 minutes.
Greatly increased yr levels. The increase in the level of pTyr depends on the dose of compound 10 in the range 15.6 μM to 250 μM. The kinetics of inhibition of dephosphorylation by low doses of Compound 10 were similar to that of 10 mM vanadate.

The above results indicate that the compounds of the present invention can increase the amount of phosphotyrosine on the insulin receptor. This assay may also be used to test the ability of the compounds of the invention to inhibit the dephosphorylation of other substrate molecules such as insulin-like growth factor 1 receptor (IGF-1R) or epidermal growth factor receptor (EGFR). Can be used for When assaying the effect of the compounds of the present invention on dephosphorylation of IGF-1R, NIH3T3 / IGF-1R cells expressing IGF-1R starved in serum-free medium were cultured for 2 hours.
Each well of a tissue culture plate was seeded at a density of 0,000 cells / well. The wells of the ELISA plate were coated with anti-IGF-1R antibody. For assay of effect on EGFR, NIH3T3 / EGFR cells expressing EGFR were grown in medium containing 0.5% for 40 hours at a density of 10,000 cells / well in 96-well tissue culture plate wells. Sown inside.
The wells of the ELISA plate were coated with anti-EGFR antibody.

The results for some other compounds are shown in Table 2. The activity of these compounds was expressed as the concentration (μM) of the compound that caused a 50% increase in the amount of phosphotyrosine compared to the control.

[0137]

[Table 2] Table 3 below shows the test results of still another compound of the present invention.
It was shown to. The activity of these compounds is determined by the concentration of compound that causes a 50% increase in the amount of phosphotyrosine (μ
M).

[0138]

[Table 3] 7.2. Glucose transport assay This assay is used to evaluate the ability of compounds of the invention to inhibit phosphatase activity. This phosphatase activity is involved in a signaling pathway that controls the insulin-induced facilitated transport of glucose into adipocytes. Incubation of isolated adipocytes with vanadate has been shown to result in a dose-dependent increase in glucose uptake. All compounds of the invention can be tested in this assay. 7.2.1.Materials and Methods The cell line used for the glucose transport assay was a preadipocyte cell line overexpressing the insulin receptor, 3T3-
L1 (American Type Culture Collection CCL92.1). 3T3-L1 cells were first prepared with 10% fetal bovine serum (FBS)
The cells were differentiated by treating them in DMEM containing 0.5 mM 3-isobutyl-1-methyl-xanthine, 5 μg / mL porcine insulin, and 250 mM dexamethasone in a confluent growth state for 2 days. . The cells were then grown for 2 days in DMEM containing 10% fetal bovine serum (FBS) and 5 μg / mL porcine insulin, followed by 10% FB
The cells were cultured in DMEM containing only S.

The cells used for the assay were initially 1%
The cells were grown overnight in DMEM containing FBS at 37 ° C. with 5% CO ₂ . Two hours before use, the medium used for overnight growth was 5 mM
Was replaced with a serum-free medium containing glucose. After washing the cells twice with phosphate buffered saline (PBS), the compound of the present invention, diluted 1: 100 in DMEM, is serially diluted so that the final concentration range is 0.1 μM to 500 μM. Added to each well. Negative control wells received only DMEM. The cells were incubated with the test compound for 1-4 hours at 37 ° C., 5% CO ₂ . 15 minutes before each measurement time, 2-deoxy - ³ H- glucose to a final concentration of 50μM and 0.5
It was added so as to be μCi / mL. At the end, the compounds were removed and the wells were washed twice with PBS containing 10 mM glucose. Cells were lysed with 50 μL of 0.5 N NaOH, and the cell lysate was transferred to a scintillation vial and mixed with 5.2 μL of glacial acetic acid. The wells were washed with 200 μL of PBS and the washes were transferred to the corresponding scintillation vials. ³ H
Was counted with a Beckman counter. 7.2.2. Experimental Results All compounds tested in this assay (Table 1) increased glucose uptake into these cells in the absence of insulin.

These data indicate that the compounds of the present invention can exert an effect similar to that of insulin in increasing the rate of glucose uptake into adipocytes in the absence of insulin. It is obvious for a person skilled in the art that the present invention can be improved and changed based on the above disclosure. Such modifications are to be understood as being limited only by the appended claims and within the spirit and scope of the invention as defined. 8. Early Adipocyte Glucose Uptake Assay This assay is used to assess the effect of the compounds of the invention on early adipocyte insulin-mediated signaling as quantified by glucose uptake by cells. All compounds of the invention can be tested in this assay. 8.1. Materials and Methods 8.1.1. Reagents The following buffers and solutions are used in the early adipocyte glucose uptake assay: mixed salt 76.74 g NaCl 3.51 g KCl 3.06 g MgSO ₄ -7H ₂ O 3.63 g CaCl ₂ -2H ₂ O (2.74 g CaCl ₂ ) was diluted with distilled water to a volume of 1 L. HEPES buffer 23.8 g HEPES 3.42 g NaH ₂ was PO ₄ -H ₂ O are dissolved in about 600mL of _{H 2 O (NaH 2 PO 4} -2H 2 O in 3.87 g). The pH of this solution was adjusted to 7.6 and the volume was adjusted to 1 L with distilled water. Albumin collagenase buffer 44.8mL of distilled water 10.0 mL HEPES buffer 10.0 mL mixed salt 35.0 mL 10% BSA 0.2 mL glucose (300 mM) 100 mg collagenase transport buffer 48mL distilled water 8mL mixed salt 8mL HEPES buffer 16 mL 10% bovine serum albumin 8.1.2. Testicular Removal of Upper Body Fat Pads Early adipocytes used in this assay were obtained from euthanized male rats weighing 200-250 g (Sprague-Dawley or other suitable strains). Older, heavier rats were not used because they may be resistant to insulin and not respond well. 1 to 1.5 g from each animal
Fat was expected to be obtained. In order to perform 40 reactions using 20 samples and the corresponding 20 LDH samples in a glucose uptake assay, about 40 reactions were required.
You need 2.5g of fat.

Using sterile techniques, a midline incision was made in the skin of the abdominal cavity and a 4-6 cm peritoneum was incised. The fat pad adjacent to the testis was identified by following the vas deferens to the testis. Fat pads were carefully excised from the epididymis and testis, and from innervating vessels. Weigh the extracted fat pad, shred it,
Digestion was performed with 5 mL of collagenase buffer at 37 ° C. for 1 hour. The digested material was then strained through a 250 micron nylon mesh sieve. The cells floating on the surface were collected and washed three times with a transport buffer. Cell concentration
It was determined by one of the following methods. (1) Cells as a percentage of the solution: 50 cells in suspension
Centrifuged at 0 xg for 5-10 minutes in blood collection tubes. The total length of the liquid column and the column length of the "white" cellular material at the bottom of the blood collection tube were measured in mm. Cell concentrations were estimated as a percentage of the column length of the cells relative to the total length. Approximately 2-3% of cells in the final reaction volume were used for glucose uptake assays. For a reaction volume of 500 μL, the adipocyte stock solution was diluted to 25% cells with transport buffer and a 50 μL aliquot was added to each sample. (2) Number of cells: Cells were first fixed with osmium tetroxide in collidine buffer so that adipocytes were sedimented in suspension. The fixed cells were centrifuged to remove osmium tetroxide, and then counted in a Coulter counter. After determining the cell number, the cell concentration was adjusted and a 50 μL aliquot of cells was added to each sample. 8.1.3. Early Adipocyte Glucose Uptake Assay In this assay, adipocytes collected from rats
The compounds of the invention were contacted in the absence or presence of saturable levels of insulin. Usually insulin-
¹⁴ C that must be taken up by cells via an induction mechanism
-Labeled glucose was added to the cells. The amount of radioactive glucose retained in the treated cells was measured and compared to evaluate the activity of the test compound.

A typical assay can be set up as follows: sample buffer compound DMSO cells ¹⁴ C glucoseブ ラ ン ク Blank 447.5 2.5 50 Base 397.5 2.5 50 50 Insulin 347.5 2.5 50 50 Sample 397.5 2.5 50 50 Sample 2 397.5 2.5 50 50 Reaction vial containing appropriate buffer and compound (polyethylene scintillation vial) , 17 mm) was set up during cell preparation. Represents saturable level
50 μL of 80 nM insulin was added to the appropriate samples just before adding the adipocytes. Lower concentrations of insulin can also be used in the assay. Dimethyl sulfoxide (DMSO; 0.5% or less) was used as a vehicle for the compounds of the present invention. Depending on solubility in DMSO
Test compounds (about 50 μM) were used at 200 ×. Adding adipocytes in a 50 μL aliquot to the reaction vial;
Incubate for 30 minutes at 37 ° C. with gentle shaking. ¹⁴ C glucose was then added to each sample further incubated at 37 ° C. for 60 minutes with shaking.

The cells can be separated from the reaction buffer by centrifugation. The amount of glucose uptake by cells can be measured by a standard scintillation counting method. In this example, cells (two samples per point) were centrifuged in a small-bore microcentrifuge tube (5.8 × 47.5 mm, capacity 0.4 mL) containing 100 μL of dimethylsilicon solution (SF96 / 50). separated.
Stir each sample to ensure a uniform distribution of cells, part 2
00 μL was transferred to a small diameter microcentrifuge tube. Mix 1300
Centrifuged at 0 rpm for 10 minutes. After centrifugation, the cells floating on the surface were separated at the silicon liquid interface. The upper layer was then transferred into a borosilicate vial containing 7-10 mL of scintillation fluid and counted for about 10 minutes. 50 μL of ¹⁴ C was used as a control for the total amount of radioactivity in each reaction. 8.2. Results A total of 109 compounds of the invention were tested in the early adipocyte glucose uptake assay using a submaximal concentration of insulin (final concentration = 100 pM) as described in section 8.1. Test compounds were used at a concentration of 50 mM. The results obtained from the sample containing the test compound and no insulin were compared with the results obtained from the sample containing only insulin and used as an insulin control.

The following compounds identified by the example numbers (see Table 3) responded in the early adipocyte glucose uptake assay to more than 125% of the insulin control: 16, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 2
8, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 4
0, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 5
2, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 6
4, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 7
6, 77, 78, 79, 80 and 81.

The following compounds, identified by the example number, were tested in this assay and did not show a response equal to or greater than the insulin control: 13, 14, 15, 86, 9, 2, 3,
6, 5, 8, 4, 94, 95, 96 and 7.

[Brief description of the drawings]

FIG. 1: Phosphotyrosine on the insulin receptor over time
Dose-dependent effect of compound 10 on the level of (pTyr) residues.

────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] May 26, 1997

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Name of invention

[Correction method] Change

[Correction contents]

Title: Pharmaceutical compositions and methods for modulating signal transduction

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI C07D 417/14 C07D 417/14

Claims (19)

[Claims] 1. An effective amount of the formula (I): Wherein Z and Q may be the same or different and represent the atoms necessary to complete an unsubstituted or substituted nitrogen-containing heterocycle, respectively, and T ₁ and T ₂ may be the same or different Well, respectively, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, aryloxy, halogen, cyano, hydroxy, carboxyl, sulfo, carbamoyl, acyl, acylamino, thioacylamino,
A compound represented by sulfamoyl or sulfonamide, q is 1, 2, or 3, and p and r are 0, 1, or 2) or a pharmaceutically acceptable salt thereof is administered to a mammal. A method for inhibiting protein tyrosine phosphatase activity, comprising: 2. An effective amount of formula (I): Wherein Z and Q may be the same or different and represent the atoms necessary to complete an unsubstituted or substituted nitrogen-containing heterocycle, respectively, and T ₁ and T ₂ may be the same or different Often, each represents alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, aryloxy, halogen, cyano, hydroxy, carboxyl, sulfo, carbamoyl, acyl, acylamino, sulfamoyl, or sulfonamide, where q is 1 Wherein p and r are 0, 1 or 2) or a pharmaceutically acceptable salt thereof, wherein said effective amount is a protein. A method of modulating signal transduction, wherein the amount is sufficient to modulate tyrosine phosphatase activity. 3. An effective amount of formula (I): Wherein Z and Q may be the same or different and represent the atoms necessary to complete an unsubstituted or substituted nitrogen-containing heterocycle, respectively, and T ₁ and T ₂ may be the same or different Often, respectively, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, aryloxy, halogen, cyano, hydroxy, carboxyl, sulfo, carbamoyl, acyl, acylamino, sulfamoyl, or sulfone Represents an amide, q is 1, 2, or 3, and p and r are 0, 1, or 2) or a pharmaceutically acceptable salt thereof. The amount is sufficient to modulate protein tyrosine phosphatase activity, Method of treating a disease state caused by potential failure signaling. 4. The method according to claim 3, wherein the disease state is selected from the group consisting of glioma, melanoma, kaposi sarcoma, hemangiomas, ovarian cancer, breast cancer, lung cancer, pancreatic cancer, prostate cancer, colon cancer, and epidermoid cancer. the method of. 5. The method of claim 3, wherein said disease state is diabetes mellitus. 6. An effective amount of formula (I): Wherein Z and Q may be the same or different and represent the atoms necessary to complete an unsubstituted or substituted nitrogen-containing heterocycle, respectively, and T ₁ and T ₂ may be the same or different Well, respectively, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, aryloxy, halogen, cyano, hydroxy, carboxyl, sulfo, carbamoyl, acyl, acylamino, thioacylamino,
A compound represented by sulfamoyl or sulfonamide, q is 1, 2, or 3, and p and r are 0, 1, or 2) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable salt thereof. A pharmaceutical composition comprising a carrier to be used. 7. An effective amount formula: Wherein R ₂ is hydrogen, halogen, cyano, amino, nitro, amide, carboxy, acylamino, hydroxy, alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, arylalkyl, R ₃ is hydrogen, alkyl, substituted alkyl, or substituted arylalkyl or a substituted or unsubstituted 5- or 6-membered ring system containing one or two heteroatoms which are sulfur, nitrogen or oxygen; , Alkoxy, substituted alkoxy, cycloalkyl,
A method of inhibiting protein tyrosine phosphatase activity, comprising administering to a mammal a compound represented by the formula: (substituted cycloalkyl, aryl, substituted aryl, arylalkyl, or substituted arylalkyl) or a pharmaceutically acceptable salt thereof. . 8. An effective amount of the formula: Wherein R ₂ is hydrogen, halogen, cyano, amino, nitro, amide, carboxy, acylamino, hydroxy, alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, arylalkyl, R ₃ is hydrogen, alkyl, substituted alkyl, or substituted arylalkyl or a substituted or unsubstituted 5- or 6-membered ring system containing one or two heteroatoms which are sulfur, nitrogen or oxygen; , Alkoxy, substituted alkoxy, cycloalkyl,
Or a pharmaceutically acceptable salt thereof, wherein the compound is a substituted cycloalkyl, aryl, substituted aryl, arylalkyl, or substituted arylalkyl. A method of modulating signal transduction, which is in an amount sufficient to modulate activity. 9. An effective amount of the formula: Wherein R ₂ is hydrogen, halogen, cyano, amino, nitro, amide, carboxy, acylamino, hydroxy, alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, arylalkyl, R ₃ is hydrogen, alkyl, substituted alkyl, or substituted arylalkyl or a substituted or unsubstituted 5- or 6-membered ring system containing one or two heteroatoms which are sulfur, nitrogen or oxygen; , Alkoxy, substituted alkoxy, cycloalkyl,
Or a pharmaceutically acceptable salt thereof, to a human, wherein the amount of the protein tyrosine phosphatase activity is a substituted cycloalkyl, aryl, substituted aryl, arylalkyl, or substituted arylalkyl. A method for treating a disease state caused by dysfunctional signaling that is in an amount sufficient to regulate dysfunction. 10. The method according to claim 9, wherein the disease state is selected from the group consisting of glioma, melanoma, kaposi's sarcoma, hemangiomas, ovarian cancer, breast cancer, lung cancer, pancreatic cancer, prostate cancer, colon cancer or epidermoid cancer. the method of. 11. The method of claim 9, wherein said disease state is diabetes mellitus. 12. An effective amount formula: Wherein R ₂ is hydrogen, halogen, cyano, amino, nitro, amide, carboxy, acylamino, hydroxy, alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, arylalkyl, R ₃ is hydrogen, alkyl, substituted alkyl, or substituted arylalkyl or a substituted or unsubstituted 5- or 6-membered ring system containing one or two heteroatoms which are sulfur, nitrogen or oxygen; , Alkoxy, substituted alkoxy, cycloalkyl,
Which is a substituted cycloalkyl, aryl, substituted aryl, arylalkyl or substituted arylalkyl) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. 13. A compound of the formula: Wherein A is (i) a substituted or unsubstituted monocyclic 5-membered member having 1 to 4 heterocyclic atoms (at least one of which is nitrogen and the rest is selected from nitrogen, oxygen or sulfur) Or a 6-membered ring, (ii) 1 to 4 heterocyclic atoms, one of which is nitrogen and the rest of which are nitrogen, oxygen or sulfur
Substituted or unsubstituted monocyclic or fused bicyclic 6 having
Membered to 10-membered ring, or (iii) a substituted or unsubstituted monocyclic or fused polycyclic saturated or unsaturated ring having 3 to 15 atoms, which are carbon, sulfur, nitrogen or oxygen. Or a pharmaceutically acceptable salt thereof. 14. A pharmaceutical composition comprising an effective amount of a compound according to claim 13 and a pharmaceutically acceptable carrier. 15. A method of inhibiting phosphatase activity in a cell, comprising administering to a mammal an effective amount of the compound of claim 13. 16. A method of modulating signaling comprising administering to a mammal an effective amount of a compound according to claim 13, wherein said amount is sufficient to modulate protein tyrosine phosphatase activity. . 17. A method according to claim 15, comprising administering to a human an effective amount of a compound according to claim 13, wherein said amount is sufficient to modulate protein tyrosine phosphatase activity. How to treat the resulting disease state. 18. The method according to claim 17, wherein the disease state is selected from the group consisting of glioma, melanoma, kaposi's sarcoma, hemangiomas, ovarian cancer, breast cancer, lung cancer, pancreatic cancer, prostate cancer, colon cancer or epidermoid cancer. the method of. 19. The method of claim 17, wherein said disease state is diabetes mellitus.