WO2012041524A1

WO2012041524A1 - Hydrazonopyrazolones as protein tyrosine phosphatase inhibitors

Info

Publication number: WO2012041524A1
Application number: PCT/EP2011/004935
Authority: WO
Inventors: Stefanie Grosskopf; Jörg Rademann; Chris Eckert; Walter Birchmeier
Original assignee: Max-Delbrück-Centrum Für Molekulare Medizin (Mdc); Forschungsverbund Berlin E.V.
Priority date: 2010-10-01
Filing date: 2011-10-04
Publication date: 2012-04-05

Abstract

This invention relates to novel compounds useful in inhibiting protein tyrosine phosphatase activity, especially' Shp2 protein tyrosine phosphatase activity, pharmaceutical compositions comprising at least one of said compounds and their use for treating phosphatase-mediated diseases, especially Shp2- mediated diseases. These compounds act as protein tyrosine phosphatase (FTP) inhibitors, preferably as selective Shp2 inhibitors, and can therefore be used for the treatment of conditions, where protein tyrosine phosphatases are implicated, including cardiovascular, immunological, infectious, neurological, metabolic diseases, and cancer. Compounds of the invention are also useful as research tools for investigating the role of PTPs in signal transduction.

Description

HYDRAZONOPYRAZOLONES AS PROTEIN TYROSINE PHOSPHATASE INHIBITORS

Field of the Invention

This invention relates to novel compounds useful in inhibiting protein tyrosine phosphatase activity, especially Shp2 protein tyrosine phosphatase activity, pharmaceutical compositions comprising at least one of said compounds and their use for treating phosphatase-mediated diseases, especially Shp2- mediated diseases. These compounds act as protein tyrosine phosphatase (PTP) inhibitors, preferably as selective Shp2 inhibitors, and can therefore be used for the treatment of conditions, where protein tyrosine phosphatases are implicated, including cardiovascular, immunological, infectious, neurological, metabolic diseases, and cancer. Compounds of the invention are also useful as research tools for investigating the role of PTPs in signal transduction.

Background of the Invention

The function of many proteins, particularly those involved in signal transduction pathways, is dependent upon their tyrosine phosphorylation status, which is finely regulated by the action of protein tyrosine kinases and protein tyrosine phosphatases. Many protein tyrosine kinases such as Bcr-Abl, c- kit, ErbB, and VEGFR are validated drug targets for cancer therapy (Bridges AJ. Chem. Rev. 2001, 101 , 2541 -2571). Accordingly, development of protein tyrosine phosphatase inhibitors as an alternative strategy to modulate key target protein phosphorylation states has attracted attention (Bialy L and Waldmann H, Angew. Chem. -Int. Ed. 2005, 44, 3814-3839; Jiang Z-X and Zhang Z-Y, Cancer Metast. Rev. 2008, 27, 263-272; Tautz L, et al., Expert Opin. Ther. Targets 2006, 10, 157-177; Dewang P et al., Curr. Med. Chem. 2005, 12, 1 -22; Alonso A et al., Cell 2004, 1 17, 699-71 1).

Shp2, encoded by the PTPN11 gene, is a 68 kD non-receptor PTP with two Src homology-2 (SH2) domains (N-SH2, C-SH2), (Alonso A et al., Cell 2004, 1 17, 699-71 1 ; Neel BG et al., Trends Biochem Sci. 2003, 2S, 284-293) located in the N-terminal region and two potential Grb2 SH2 domain binding sites located in the C-terminal region. It is also known as SHPTP2, Syp, PTP1D and PTP2C. Shp2 mediates activation of Erkl and Erk2 (Erkl/2, Erk) MAP kinases by receptor tyrosine kinases such as ErbBl , ErbB2, and c-Met (Nishida K, Hirano, T, Cancer Sci. 2003, 94, 1029-33; Gu H, Neel BG, Trends Cell Biol. 2003, 13, 122-30; Deb TB et al., J Biol Chem 1998, 273, 16643-6; Cunnick JM et al., J. Biol. Chem. 2000, 275, 13842-8; Maroun CR et al., Mol. Cell Biol. 2000, 20, 8513-25; Furge KA et al., Oncogene 2000, 19, 5582-9). Shp2 is basally inactive due to auto-inhibition by its N-SH2 domain (Hot P et al., Cell 1998, 92, 441 - 450). Shp2 is activated by interaction with a variety of components and acts as a positive regulator of cell proliferation. In growth factor- and cytokine-stimulated cells, Shp2 binds to tyrosine- phosphorylated docking proteins through its SH2 domains, resulting in its activation (Cunnick JM et al., J Biol. Chem. 2001 , 276, 24380-24387). Shpl is mostly expressed in hematopoietic and epithelial cells and functions as a negative regulator of signaling pathways in lymphocytes (Neel B, Tonks N, Curr. Opin. Cell Biol. 1997, 9, 193-204; Poole A, Jones M, Cell. Signalling 2005, 17, 1323-1332). The crystal structure of ligand-free Shp 1 shows a similar arrangement of tandem SH2 domains that adopt a conformation blocking the PTP catalytic site (Yang J et al., J. Biol. Chem. 2003, 278, 6516-20). Shp2 has been shown to bind Gabl (or Gab2) in cells stimulated with EGF, HGF, or interleukin-6 (Cunnick JM et al., J Biol. Chem. 2001 , 276, 24380-24387; Gu H and Neel BG, Trends Cell. Biol. 2003, 73, 122-130; Maroun CR et al., Mol. Cell. Biol. 2000, 20, 8513-8525; Nishida K and Hirano T, Cancer Sci. 2003, 94, 1029- 1033). Gabl-Shp2 interaction as well as Shp2 PTP activity are necessary for Erkl/2 activation by these growth factors (Cunnick et al., 2002; Neel et al., 2003; Cunnick JM et al., J Biol. Chem. 2002, 277, 9498-9504). The primary function of Gabl association with an activated Shp2 in MAP kinase activation includes targeting of the activated Shp2 to the membrane. Association of Gabl with Shp-2 (but not with PI3K, CRKL or Grb2) was essential to induce a branching morphogenesis program (Schaeper et al., J Cell Biol 2000, 149, 1419-1432). It could be shown that Shp2 is an important downstream signaling molecule of Met/Gab 1 for the activation of the mitogen activated protein (MAP) kinase pathway which can lead to cell transformation, a prerequisite for the development of cancer. Though growth factor-activation of Shp2 has been elucidated, the mechanisms by which Shp2 produces downstream signals, like activation of Ras-Erkl/2 MAP kinase pathway, are less clear (Mohi MG et al., Cancer Cell 2005, 7, 179-191 ). Because the Shp-2 phosphatase is a positive mediator of growth factor signaling, especially in the Ras pathway, its inhibition should be of therapeutic benefit. Similarly, specific Shp-2 inhibitors should be an excellent means for the treatment of EGF/EGFR dependent tumors of epithelial origin which represent the vast majority of cancers (e. g. breast and prostate carcinomas).

Hepatocyte growth factor/scatter factor (HGF/SF), its receptor, the tyrosine kinase Met, and the downstream signaling adapter Gabl are important mediators of invasive growth which comprises increased proliferation, cell-cell dissociation and motility, matrix degeneration and survival of epithelial cells. HGF/SF is a strong inducer of angiogenesis in vivo, a process that is also important for blood vessel formation in rumors. All these processes become important during metastasis (Birchmeier et al., Nat Rev Mol Cell Biol 2003, 4, 915-925). Under physiological conditions, the formation and patterning of an embryo, wound healing, axon guidance or organ regeneration is adjusted by the HGF Met/Gabl system. The importance of these proteins in development is shown by the embryonic lethality of HGF/SF, Met or Gabl null-mutations. Nearly identical phenotypes verify these factors as key components of Met signal transduction. The Met receptor was originally identified in a constitutively active mutant form (Tpr-Met) as an oncogene. A variety of human solid tumors (e. g. bladder, breast, non-small lung cell, renal, thyroid, prostate, hepatocellular, colorectal and gastric carcinoma) demonstrate aberrant Met activity due to HGF-dependent autocrine loop, Met overexpression, gene amplification or gene mutations. Experimentally, expression of HGF and Met or mutant Met conferred malignant properties on normal cells (motility, invasion, tumorigenicity) (Jeffers et al., Proc Natl Acad Sci USA 1998, 95, 14417-14422). Clinically, HGF/Met overexpression has been shown to correlate with poor prognosis in several types of cancer. Therefore, the Met signaling system has been regarded as a promising therapeutic target in the treatment of cancer and especially metastases. The development of specific Shp-2 inhibitors should therefore allow blocking or reducing aberrant Met activity and provide an excellent means for the treatment of HGF Met- dependent metastases.

Metastases are defined as distant settlements of primary tumor cells. Sooner or later during the development of most types of human cancer, primary tumor masses generate pioneer cells that move out, invade adjacent tissues, and travel to distant sites where they may succeed in founding new colonies. The capability for invasion and metastasis enables cancer cells to escape the primary tumor mass and colonize new terrain in the body where, at least initially, nutrients and space are not limiting. Metastases are one of the greatest sources of pain in late stage cancer.

Gain-of-function Shp2 mutants are found in childhood hematological malignancies such as juvenile myelomonocytic leukemia (JMML), some cases of solid tumors, and are associated with 50% cases of Noonan syndrome (Bentires-Alj M et al., Nat. Med. 2006, 12, 283-285; Bentires-Alj M et al., Cancer Res. 2004, 64, 8816-8820; Tartaglia M and Gelb BD, Eur. J. Med. Genet. 2005, 4S, 81 -96). JMML is a progressive myelodysplastic/myeloproliferative disorder characterized by overproduction of tissue- infiltrating myeloid cells. Somatic mutations in PTPN11 account for about 35% of JMML patients, (Kratz CP et al., Blood 2005, 706, 2183-2185) and recent reports indicated JMML-associated Shp2 mutants can transform murine bone marrow and fetal liver cells (Chan RJ et al., Blood 2005, 705, 3737- 3742; Mohi MG et al., Cancer Cell 2005, 7, 179-191 ; Schubbert S et al., Blood 2005, 706, 311 - 317). Noonan syndrome is a developmental disorder characterized by facial anomalies, short stature, heart disease, skeletal defects, and hematological disorders (Tartaglia M and Gelb BD, Eur. J. Med. Genet. 2005, 4S, 81-96), with about 50% of cases caused by germline PTPN11 mutations. All Shp2 mutants found in Noonan syndrome and JMML are gain-of-function mutations, mostly resulting from weaker autoinhibition of the N-SH2 domain (Fragale A et al., Hum. Mutat. 2004, 23, 267-277). Wang et al. (J Biol Chem 2009, 284, 913-920) confirmed the positive role of Shp2 in cell motility and showed that Noonan syndrome/leukemia-associated gain-of-function mutations in Shp2 enhance cell migration and angiogenesis. Gain-of-function mutations in several types of leukemia define Shp2 as a bona fide oncogene. It has also been reported that Shp2 is a key mediator of the oncogenic CagA protein of Helicobacter pylori, which causes gastric cancer (Higashi H et al., Science 2002, 295, 683- 6; Meyer-ter-Vehn T et al., J. Biol. Chem. 2000, 275, 16064-72). A survey of the role of Shp2 in development and cancer can be found in Grossmann et al., Adv Cancer Res 2010, 106, 53-89.

PTP inhibitor development is an emerging area in the field of drug development (Bialy L and Waldmann H, Angew Chem. Int. Ed. Engl. 2005, 44, 3814-3839). Several compounds have been reported to non-selectively inhibit Shp2, with most efforts of PTP inhibitor discovery and design focused on PTP1 B, a common drug target in diabetes type II (insulin resistance), and Cdc25 inhibitors (Lazo JS et al., Mol. Pharmacol. 2002, 67, 720-728; Zhang ZY, Ann. Rev. Pharmacol. Toxicol. 2002, 42, 209-234; Huijsduijnen RHv et al., J Med Chem 2004, 47, 4142-4146).

The development of a Shp2-specific inhibitor that does not cross-inhibit Shpl is important for development of effective treatment modalities. Developing a Shp2-specific inhibitor is, however, complicated by the similarity between Shpl and Shp2, which share 60% overall sequence identity and approximately 75% similarity in their PTP domains. However, Shpl and Shp2 catalytic domains have different substrate specificity (Tenev T et al., J. Biol. Chem. 1997, 272, 5966-73; O'Reilly A, Neel B, Mol. Cell Biol. 1998, 18, 161-77) suggesting that the catalytic cleft is not identical. Furthermore, the surface electrostatic potential of the catalytic cleft is much more positive in human Shp2 than in human Shpl. (Yang, J., et al., J. Biol. Chem. 1998, 273, 28199-207). The PTP catalytic cleft consists of a base and four sides in the 3D structures (Hof P, et al. Cell 1998, 92, 441-450; Yang J et al., J. Biol. Chem. 2003, 278, 6516-6520). Although amino acid residues present at the base of Shpl and Shp2 PTP catalytic clefts are identical, all four sides of the catalytic cleft contain one or more residues that are different between Shpl and Shp2.

PTP1 B inhibitors that cross-inhibit Shp2 have been described (Huang P et al., Bioorg. Med. Chem. 2003, 77, 1835-1849; Shen K et al., J. Biol. Chem. 2001, 276, 47311 -47319), but none of them has demonstrated in vivo activity in cell cultures.

Sulfhydantoins have been disclosed as uncharged small molecule inhibitors of Shp-2 in WO 2004/062664 Al (Vertex Pharmaceuticals Inc.). However, no cellular activity of these Shp-2 inhibitors is described. WO 2006/128909 discloses small molecule Shp2 inhibitors, pharmaceutical compositions comprising them and their use for treating phosphatase-mediated diseases.

Hellmuth et al. (Proc Natl Acad Sci USA, 2008, 105, 7275-7280) disclose phenylhydrazonopyrazolone sulfonates as specific inhibitors of Shp2 PTP.

Oxindole scaffold Shp2 inhibitors NSC-87877, NSC-117199, analogues thereof and their use for the treatment of cancer are disclosed in US 11/733,023, WO 2007/117699, WO 2009/135000, and Chen L et al., Mol Pharmacol 2006, 70, 562-570.

Zhang X et al., J Med Chem 2010, 53, 2482-2493 disclose a salicylic acid based small molecule Shp2 inhibitor as a potential lead compound for development of more potent and selective Shp2 inhibitors, which may be used as therapeutics for the treatment of Noonan syndrome, various kinds of leukemias, and solid tumors.

New agents for the treatment or prevention of conditions and diseases associated with gain-of-function Shp2 mutations, e.g. Noonan syndrome, JMML, HGF/Met-dependent metastases, various kinds of cancers, especially solid tumors, and leukemias, are of considerable interest as these conditions account for a significant number of death in patients and administration of many of the presently employed drugs is associated with complex drug interactions and many adverse side effects.

Additionally, the use of Shp2 inhibitors as therapeutics is so far, for example, hampered by their insufficient cell permeability, target specificity, or solubility.

Therefore, the problem underlying the present invention is to provide novel compounds useful in inhibiting protein tyrosine phosphatase activity, especially Shp2 protein tyrosine phosphatase activity. Advantageously, the compounds of the invention possess improved properties, e.g. pharmacokinetic properties, solubility, and target specificity. .

Description of the Invention

The present invention was made in view of the prior art and the needs described above. The aspects of the present invention will become apparent upon reference to the following detailed description and definitions.

The present invention relates to a compound of general formula (I-a):

R¹ is -NH-(CO)-CH₃, -N0₂, -OH, or -S0₂NH₂;

R² and R⁴ are each and independently of each other selected from hydrogen atom, halogen atom, cyano, nitro, and amino group;

R³ is -SO₃H or -S0₂NH₂; and

wherein

R^a is nitro, Me, Et, t-butyl, or alkoxy;

R^b is hydrogen atom, halogen atom, cyano, nitro, or amino group;

R^c and R^d are each and independently of each other selected from halogen atom, cyano, nitro, amino group, alkyl, and alkoxy;

R^e is alkyl, alkoxy, -alkylaryl, or -alkenylaryl;

R^f is -alkylaryl, or -alkenylaryl; if R¹ is -N0₂ or -OH, and both R² and R⁴ are hydrogen atom;

(ii) aryl, heteroaryl, aralkyl or heteroaralkyl, wherein said aryl, heteroaryl, aralkyl or heteroaralkyl can be independently substituted with from 1 to 4 substituents which substituents are independently of each other selected from halogen atom, hydroxy, nitro, amino, cyano, =0, =S, =NH, sulfo, alkoxy, carboxyl, unsubstituted or substituted Ci-C₆alkyl, unsubstituted or substituted C₂- Qalkenyl, unsubstituted or substituted C₂-C₆alkynyl, unsubstituted or substituted C2-C₉heteroalkyl, unsubstituted or substituted C₇-Ci₂aralkyl, unsubstituted or substituted C₆-C ^heteroaralkyl; if R¹ is -NH-(CO)-CH₃ or -S0₂ H₂; or

(iii) aryl, heteroaryl, aralkyl or heteroaralkyl, wherein said aryl, heteroaryl, aralkyl or heteroaralkyl can be independently substituted with from 1 to 4 substituents which substituents are independently of each other selected from halogen atom, hydroxy, nitro, amino, cyano, =0, =S, =NH, sulfo, alkoxy, carboxyl, unsubstituted or substituted C|-C₆alkyl, unsubstituted or substituted C₂- C₆alkenyl, unsubstituted or substituted C₂-C₆alkynyl, unsubstituted or substituted C₂-C₉heteroalkyl, unsubstituted or substituted C₇-Ci₂aralkyl, unsubstituted or substituted C₆-Ci₂heteroaralkyl; if R² and/or R⁴ is/are not hydrogen atom.

Compounds are generally described herein using standard nomenclature, unless otherwise specified, and may be obtained according to methods described in the literature (e.g. WO 2006/128909; Hellmuth et al., Supporting Information 10.1073/pnas.0710468105; and El-Dahshan et al., J. Org. Lett. 2007, 9, 949 ) or exemplified herein. For compounds having asymmetric centers, it should be understood that, unless otherwise specified, all of the optical isomers and mixtures thereof are encompassed. Compounds with two or more asymmetric elements can also be present as mixtures of diastereomers. In addition, compounds with carbon-carbon double bonds may occur in Z- and E- forms, with all isomeric forms of the compounds being included in the present invention unless otherwise specified. Where a compound exists in various tautomeric forms, a recited compound is not limited to any one specific tautomer, but rather is intended to encompass all tautomeric forms. Recited compounds are further intended to encompass compounds in which one or more atoms are replaced with an isotope, i.e., an atom having the same atomic number but a different mass number. By way of general example, and without limitation, isotopes of hydrogen include tritium and deuterium and isotopes of carbon include ⁿC, ¹³C, and ¹⁴C.

Compounds according to the formulas provided herein, which have one or more stereogenic center(s), have an enantiomeric excess of at least 50%. For example, such compounds may have an enantiomeric excess of at least 60%, 70%, 80%, 85%, 90%, 95%, or 98%. Some embodiments of the compounds have an enantiomeric excess of at least 99%. It will be apparent that single enantiomers (optically active forms) can be obtained by asymmetric synthesis, synthesis from optically pure precursors or by resolution of the racemates. Resolution of the racemates can be accomplished, for example, by conventional methods such as crystallization in the presence of a resolving agent, or chromatography, using, for example, a chiral HPLC column.

Compounds herein may also be described using a general formula that includes variables such as, e.g. R'-R⁵^³^, Y, etc. Unless otherwise specified, each variable within such a formula is defined independently of any other variable, and any variable that occurs more than one time in a formula is defined independently at each occurrence. Thus, for example, if a group is shown to be substituted with 0-2 R^*, the group may be unsubstituted or substituted with up to two R^* groups and R^* at each occurrence is selected independently from the definition of R^*. Also, combinations of substituents and/or variables are permissible only if such combinations result in stable compounds, i.e. compounds that can be isolated, characterized and tested for biological activity.

An "inhibitor" as used herein is a compound that binds to its target, e.g. Shp2 FTP, and decreases its activity. The binding of an inhibitor can stop a substrate from entering the target enzyme's active site and/or hinder the enzyme from catalysing its reaction. Inhibitor binding is either reversible or irreversible. Irreversible inhibitors usually react with the enzyme and change it chemically. In contrast, reversible inhibitors bind non-covalently. There are four kinds of reversible enzyme inhibitors, namely competitive inhibitors, uncompetitive inhibitors, mixed-type inhibitors, and non-competitive inhibitors. The different types of inhibition produced depend on whether these inhibitors bind the enzyme, the enzyme-substrate complex, or both. An inhibitor is often judged by its specificity (its lack of binding to other proteins) and its potency (its dissociation constant, which indicates the concentration needed to inhibit the enzyme). A high specificity and potency ensure that an inhibitor, which is used as a drug will have few side effects and thus low toxicity.

A "pharmaceutically acceptable salt" of a compound disclosed herein preferably is an acid or base salt that is generally considered in the art to be suitable for use in contact with the tissues of human beings or animals without excessive toxicity or carcinogenicity, and preferably without irritation, allergic response, or other problem or complication. Such salts include mineral and organic acid salts of basic residues such as amines, as well as alkali or organic salts of acidic residues such as carboxylic acids.

Suitable pharmaceutical salts include, but are not limited to, salts of acids such as hydrochloric, phosphoric, hydrobromic, malic, glycolic, fumaric, sulfuric, sulfamic, sulfanilic, formic, toluenesulfonic, methanesulfonic, benzene sulfonic, ethane disulfonic, 2-hydroxyethylsulfonic, nitric, benzoic, 2-acetoxybenzoic, citric, tartaric, lactic, stearic, salicylic, glutamic, ascorbic, pamoic, succinic, fumaric, maleic, propionic, hydroxymaleic, hydroiodic, phenylacetic, alkanoic such as acetic, HOOC-(CH₂)„-COOH where n is any integer from 0 to 4, i.e., 0, 1, 2, 3, or 4, and the like. Similarly, pharmaceutically acceptable cations include, but are not limited to sodium, potassium, calcium, aluminum, lithium and ammonium. Those of ordinary skill in the art will recognize further pharmaceutically acceptable salts for the compounds provided herein. In general, a pharmaceutically acceptable acid or base salt can be synthesized from a parent compound that contains a basic or acidic moiety by any conventional chemical method. Briefly, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two. Generally, the use of nonaqueous media, such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile, is preferred.

It will be apparent that each compound of formula (Γ) may, but need not, be present as a hydrate, solvate or non-covalent complex. Γη addition, the various crystal forms and polymorphs are within the scope of the present invention, as are prodrugs of the compounds of formula (I) provided herein.

A "prodrug" is a compound that may not fully satisfy the structural requirements of the compounds provided herein, but is modified in vivo, following administration to a subject or patient, to produce a compound of formula (I) provided herein. For example, a prodrug may be an acylated derivative of a compound as provided herein. Prodrugs include compounds wherein hydroxy, carboxy, amine or sulfhydryl groups are bonded to any group that, when administered to a mammalian subject, cleaves to form a free hydroxy, carboxy, amino, or sulfhydryl group, respectively. Examples of prodrugs include, but are not limited to, acetate, formate, phosphate and benzoate derivatives of alcohol and amine functional groups within the compounds provided herein. Prodrugs of the compounds provided herein may be prepared by modifying functional groups present in the compounds in such a way that the modifications are cleaved in vivo to generate the parent compounds.

A "substituent," as used herein, refers to a molecular moiety that is covalently bonded to an atom within a molecule of interest, e.g. to a compound of formula (I)or a prodrug thereof. For example, a "ring substituent" may be a moiety such as a halogen atom, alkyl group, haloalkyl group or other substituent described herein that is covalently bonded to an atom, preferably a carbon or nitrogen atom, that is a ring member. The term "substituted," as used herein, means that any one or more hydrogens on the designated atom is replaced with a selection from the indicated substituents, provided that the designated atom's normal valence is not exceeded, and that the substitution results in a stable compound, i.e., a compound that can be isolated, characterized and tested for biological activity. When a substituent is oxo, i.e., =0, then 2 hydrogens on the atom are replaced. An oxo group that is a substituent of an aromatic carbon atom results in a conversion of -CH- to -C(=0)- and a loss of aromaticity. For example a pyridyl group substituted by oxo is a pyridone.

The expression alkyl preferably refers to a saturated, straight-chain or branched hydrocarbon group that contains from 1 to 20 carbon atoms, preferably from 1 to 12 carbon atoms, more preferably from 1 to 6 carbon atoms, for example a methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert- butyl, n-pentyl, n-hexyl, n-heptyl, 2,2-dimethylbutyl, n-octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl, or dodecyl group. The expressions alkenyl and alkynyl refer to at least partially unsaturated, straight-chain or branched hydrocarbon groups that contain from 2 to 20 carbon atoms, preferably from 2 to 12 carbon atoms, more preferably from 2 to 6 carbon atoms, for example an ethenyl, allyl, acetylenyl, propargyl, isoprenyl or hex-2-enyl group. Preferably, alkenyl groups have one or two, more preferably one, double bond(s) and alkynyl groups have one or two, more preferably one, triple bond(s).

The expression heteroalkyl preferably refers to an alkyl, alkenyl or alkynyl group, for example heteroalkenyl, heteroalkynyl, in which one or more, preferably 1, 2 or 3 carbon atoms have been replaced each independently of the others by an oxygen, nitrogen, phosphorus, boron, selenium, silicon or sulphur atom, preferably oxygen, sulphur or nitrogen. The expression heteroalkyl, for example, encompasses an alkoxy group. An alkoxy group denotes an alkyl group linked to oxygen thus: -O-alkyl. Furthermore, heteroalkyl preferably refers to a carboxylic acid or to a group derived from a carboxylic acid such as, for example, acyl, acylalkyl, alkoxycarbonyl, acyloxy, acyloxyalkyl, carboxyalkylamide, alkylcarbamoylalkyl, alkylcarbamoyloxyalkyl, alkylureidoalkyl, or alkoxycarbonyloxy.

Examples of heteroalkyl groups are groups of formulas -S-Y^a-L, -S-Y^a-CO-NR^aR^b, -Y^a'-NR^c'-CO-NR^a'R^b', -Y^a'-NR^c'-CO-0-R^d', -Y^-NR'-COR^11', -Y^a'-NR^c'-CO-NR^d'-L,

-Y^-NR^-CS-NR^-L, -Y^-O-CO-NR^R"^', -Y^a-CO-NR^aR^b, -0-Y^a'-CO-NR^aR^b', -Y^-NR^-CO-L, -Y^a'-0-CO-0-R^c', -Y^a'-0-CO-R^c', -Y^a'-0-R^c', -Y^-CO-L, -Y^a-NR^aR^b, R^c'-S-Y^a'-, R^a-N(R^b)-Y^a-, R^c'-CO-Y^a'-, R'-O-CO-Y ^'-, R^c'-CO-0-Y^a'-, R^c'-CO-N(R^b')-Y^a'-, R^a'-N(R^b')-CO-Y^a'-, R'-SO-Y^3'-, R^c'-S0₂-Y^a'-, -Y^a'-NR^c'-S0₂-NR^aR^b', -Y^a'-S0₂-NR^a'R^b', -Y^a'-NR^c'-S0₂-R^d', R^a'-0-CO-N(R^b')-Y^a'-, R^a'-N(R^b,)-C(=NR^d')-N(R^c')-Y^a'-, R^-S-CO-Y^3'-, R'-CO-S-Y^3'-, R^c'-S-CO-N(R^b')-Y^3'-, R^a'-N(R^b')-CO-S-Y^a'-, R^c-S-CO-0-Y^a-, R^c-0-CO-S-Y^3'-, R^c'-S-CO-S-Y^3'-; wherein R^a' being a hydrogen atom, a Ci-C₆alkyl, a C₂-C₆alkenyl, a C₂-C₆alkynyl , or is joined to R^b to form a 4- to 10- membered cycloalkyl or heterocycloalkyl; R^b being a hydrogen atom, a Ci-Cealkyl, a C₂-C₆alkenyl or a C₂-C₆alkynyl , or taken together with R^a to form a 4- to 10-membered cycloalkyl or heterocycloalkyl; R^c being a hydrogen atom, an optionally substituted CVCgalkyl, an optionally substituted Q-Cgalkenyl or an optionally substituted C₂-Cgalkynyl ; R^d being a hydrogen atom, optionally substituted C Cgalkyl, optionally substituted C₂-C₈alkenyl or optionally substituted C₂- Cgalkynyl; L being a cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, aralkyl, or heteroaralkyl; and Y³ being a bond, a Ci- C₆alkylene, a C₂-C₆alkenylene or a C₂-C₆alkynylene group. Specific examples of heteroalkyl groups are methoxy, trifluoromethoxy, ethoxy, n-propyloxy, isopropyloxy, ier/-butyloxy, methoxymethyl, ethoxymethyl, methoxyethyl, methylamino, ethylamino, dimethylamino, diethylamino, isopropyl- ethylamino, methylaminomethyl, ethylaminomethyl, diisopropylaminoethyl, enol ether, dimethyl- aminomethyl, dimethylaminoethyl, acetyl, propionyl, butyryloxy, acetyloxy, methoxycarbonyl, ethoxycarbonyl, isobutyrylamino-methyl, N-ethyl-N-methylcarbamoyl and N-methylcarbamoyl. Further examples of heteroalkyl groups are nitrile, isonitrile, cyanate, thiocyanate, isocyanate, isothiocyanate and alkylnitrile groups. An example of a heteroalkylene group is a group of formulas -CH₂CH(OH)- or -CONH-.

The expression cycloalkyl preferably refers to a saturated or partially unsaturated cyclic group that contains one or more rings, preferably 1 or 2, containing from 3 to 14 ring carbon atoms, preferably from 3 to 10, more preferably 3, 4, 5, 6 or 7, ring carbon atoms. In an embodiment a partially unsaturated cyclic group has one, two or more double bonds, such as a cycloalkenyl group. Examples of a cycloalkyl group are a cyclopropyl, cyclobutyl, cyclopentyl, spiro[4,5]decanyl, norbornyl, cyclo- hexyl, cyclopentenyl, cyclohexadienyl, decalinyl, bicyclo[4.3.0]nonyl, tetralin, cyclopentylcyclohexyl, fluorocyclohexyl or cyclohex-2-enyl group.

The expression heterocycloalkyl preferably refers to a cycloalkyl group as defined above in which one or more, preferably 1, 2 or 3, ring carbon atoms have been replaced each independently of the others by an oxygen, nitrogen, silicon, selenium, phosphorus or sulphur atom, preferably oxygen, sulphur or nitrogen. A heterocycloalkyl group has preferably 1 or 2 ring(s) containing from 3 to 10, more preferably 3, 4, 5, 6 or 7, ring atoms. Examples are a piperidyl, piperazinyl, morpholinyl, urotropinyl, pyrrolidinyl, tetrahydrothiophenyl, tetrahydropyranyl, tetrahydrofuryl or 2-pyrazolinyl group and also a lactam, a lactone, a cyclic imide and a cyclic anhydride.

The expression alkylcycloalkyl preferably refers to a group containing both cycloalkyl and also an alkyl, alkenyl or alkynyl group in accordance with the above definitions, for example alkylcycloalkyl, cycloalkylalkyl, alkylcycloalkenyl, alkenylcycloalkyl and alkynylcycloalkyl groups. An alkylcycloalkyl group preferably contains a cycloalkyl group that contains one or two ring systems having from 3 to 10, preferably 3, 4, 5, 6 or 7, carbon atoms, and one or two alkyl, alkenyl or alkynyl groups having 1 or 2 to 6 carbon atoms, the cyclic groups being optionally substituted.

The expression heteroalkylcycloalkyl preferably refers to alkylcycloalkyl groups as defined above in which one or more, preferably 1, 2 or 3, carbon atoms have been replaced each independently of the others by an oxygen, nitrogen, silicon, selenium, phosphorus or sulphur atom, preferably oxygen, sulphur or nitrogen. A heteroalkylcycloalkyl group preferably contains 1 or 2 ring systems having from 3 to 10, preferably 3, 4, 5, 6 or 7, ring atoms, and one or two alkyl, alkenyl, alkynyl or heteroalkyl groups having from 1 or 2 to 6 carbon atoms. Examples of such groups are alkylhetero- cycloalkyl, heterocycloalkylalkyl, alkylheterocycloalkenyl, alkenylheterocycloalkyl, alkynylhetero- cycloalkyl, heteroalkylcycloalkyl, heteroalkylheterocycloalkyl and heteroalkylheterocycloalkenyl, the cyclic groups being optionally substituted and saturated or mono-, di- or tri-unsaturated.

The expression aryl or Ar preferably refers to an aromatic group that contains one or more rings containing from 6 to 14 ring carbon atoms, preferably from 6 to 10, more preferably 6, ring carbon atoms. Examples are a phenyl, naphthyl, biphenyl, or anilinyl group.

The expression heteroaryl preferably refers to an aromatic group that contains one or more rings containing from 5 to 14 ring atoms, preferably from 5 to 10, more preferably 5 or 6, ring atoms, and contains one or more, preferably 1, 2, 3 or 4, oxygen, nitrogen, phosphorus or sulphur ring atoms, preferably O, S or N. Examples are 4-pyridyl, 2-imidazolyl, 3-phenylpyrrolyI, thiazolyl, oxazolyl, triazolyl, tetrazolyl, isoxazolyl, indazolyl, indolyl, benzimidazolyl, pyridazinyl, quinolinyl, purinyl, carbazolyl, acridinyl, pyrimidyl, 2,3'-bifuryl, 3-pyrazolyl and isoquinolinyl.

The expression aralkyl preferably refers to a group containing both aryl and also alkyl, alkenyl, alkynyl and/or cycloalkyl groups in accordance with the above definitions, such as, for example, aryl- alkyl, arylalkenyl, arylalkynyl, arylcycloalkyl, arylcycloalkenyl, alkylarylcycloalkyl and alkylarylcycloalkenyl groups. Specific examples of aralkyls are toluene, xylene, mesitylene, styrene, benzyl, lH-indene, tetralin, dihydronaphthalene, phenylcyclopentyl, cumene, cyclohexylphenyl, fluorene and indan. An aralkyl group preferably contains one or two aromatic ring systems, 1 or 2 rings, containing from 6 to 10 carbon atoms and one or two alkyl, alkenyl and/or alkynyl groups containing from 1 or 2 to 6 carbon atoms and/or a cycloalkyl group containing 5 or 6 ring carbon atoms.

The expression heteroaralkyl preferably refers to an aralkyl group as defined above in which one or more, preferably 1, 2, 3 or 4, carbon atoms have been replaced each independently of the others by an oxygen, nitrogen, silicon, selenium, phosphorus, boron or sulphur atom, preferably oxygen, sulphur or nitrogen, that is to say to groups containing both aryl or heteroaryl and also alkyl, alkenyl, alkynyl and/or heteroalkyl and/or cycloalkyl and/or heterocycloalkyl groups in accordance with the above definitions. A heteroaralkyl group preferably contains one or two aromatic ring systems, 1 or 2 rings, containing from 5 or 6 to 10 ring carbon atoms and one or two alkyl, alkenyl and/or alkynyl groups containing 1 or 2 to 6 carbon atoms and/or a cycloalkyl group containing 5 or 6 ring carbon atoms, 1 , 2, 3 or 4 of those carbon atoms having been replaced each independently of the others by oxygen, sulphur or nitrogen atoms.

Examples of heteroaralkyl groups are aryloxy, arylheteroalkyl, arylheterocycloalkyl, arylheterocyclo- alkenyl, arylalkylheterocycloalkyl, arylalkenylheterocycloalkyl, arylalkynylheterocycloalkyl, arylalkylheterocycloalkenyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heteroarylhetero- alkyl, heteroarylcycloalkyl, heteroarylcycloalkenyl, heteroarylheterocycloalkyl, hetero- arylheterocycloalkenyl, heteroarylalkylcycloalkyl, heteroarylalkylheterocycloalkenyl, heteroaryl- heteroalkylcycloalkyl, heteroarylheteroalkylcycloalkenyl, heteroalkylheteroarylalkyl and hetero- arylheteroalkylheterocycloalkyl groups, the cyclic groups being saturated or mono-, di- or tri- unsaturated. Specific examples are a tetrahydroisoquinolinyl, benzoyl, 2- or 3-ethylindolyl, 4-methyl- pyridino, 2-, 3- or 4-methoxyphenyl, 4-ethoxyphenyl, 2-, 3- or 4-carboxyphenylalkyl group.

The expression "substituted" or "optionally substituted" as used herein in connection with any group refers to a group in which one or more hydrogen atoms have been replaced each independently of the others by fluorine, chlorine, bromine or iodine atoms or by OH, =0, SH, =S, NH₂, = H, CN or N0₂ groups. Examples of substituted alkyl are 2,2,2-trichloroethyl or trifluoromethyl, and examples of substituted aryl (or substituted Ar) are 2-fluorophenyl, 3-nitrophenyl or 4-hydroxyphenyl. The expression "substituted" refers furthermore to a group in which one or more hydrogen atoms have been replaced each independently of the others by an unsubstituted Ci-C₆alkyl, unsubstituted C₂- C₆alkenyl, unsubstituted C₂-C₆alkynyl, unsubstituted Ci-Ceheteroalkyl, unsubstituted C₃-C₁₀cyclo- alkyl, unsubstituted C₂-C₉heterocycloalkyl, unsubstituted C₆-Ci₀aryl, unsubstituted C C₉heteroaryl, unsubstituted C₇-C₎₂aralkyl or unsubstituted C₂-C_n heteroaralkyl group.

The expression "halogen" or "halogen atom" as preferably used herein means fluorine, chlorine, bromine, iodine.

As used herein a wording defining the limits of a range of length such as, e. g., "from 1 to 5" means any integer from 1 to 5, i. e. 1, 2, 3, 4 and 5. In other words, any range defined by two integers explicitly mentioned is meant to comprise and disclose any integer defining said limits and any integer comprised in said range.

The present invention, moreover, relates to a compound of general formula (I-b):

wherein

R , R , R , R , and R are defined as in general formula (I-a).

The present invention also relates to a compound of general formula (I):

or a pharmacologically acceptable salt, solvate, or hydrate thereof, wherein

R¹ is -N0₂ or -S0₂NH₂;

R³ is -SO₃H or -S0₂NH₂; and

R is selected from (i)

wherein

R^a is nitro, Me, Et, t-butyl, or alkoxy;

R^b is hydrogen atom, halogen atom, cyano, nitro, or amino group;

R^e is alkyl, alkoxy, -alkylaryl, or -alkenylaryl;

R^f is -alkylaryl, or -alkenylaryl; if R¹ is -NO₂, and both R² and R⁴ are hydrogen atom;

(ii) aryl, heteroaryl, aralkyl or heteroaralkyl, wherein said aryl, heteroaryl, aralkyl or heteroaralkyl can be independently substituted with from 1 to 4 substituents which substituents are independently of each other selected from halogen atom, hydroxy, nitro, amino, cyano, =0, =S, =NH, sulfo, alkoxy, carboxyl, unsubstituted or substituted Ci-C₆alkyl, unsubstituted or substituted C₂- C₆alkenyl, unsubstituted or substituted C2-C₆alkynyl, unsubstituted or substituted C₂-C₉heteroalkyl, unsubstituted or substituted C₇-Ci₂aralkyl, unsubstituted or substituted C6-Ci₂heteroaralkyl; if R¹ is -S0₂NH₂; or

(iii) aryl, heteroaryl, aralkyl or heteroaralkyl, wherein said aryl, heteroaryl, aralkyl or heteroaralkyl can be independently substituted with from 1 to 4 substituents which substituents are independently of each other selected from halogen atom, hydroxy, nitro, amino, cyano, =0, =S, =NH, sulfo, alkoxy, carboxyl, unsubstituted or substituted Ci-C₆alkyl, unsubstituted or substituted C₂- Qalkenyl, unsubstituted or substituted C₂-C₆alkynyl, unsubstituted or substituted C₂-C₉heteroalkyl, unsubstituted or substituted C₇-Ci₂aralkyl, unsubstituted or substituted C₆-Ci₂heteroaralkyl; if R² and/or R⁴ is/are not hydrogen atom.

The present invention preferably relates to a compound or salt of formula (I), wherein R¹ is -N0₂; R² and R⁴ are hydrogen atom, R³ is -S0₃H or -S0₂NH₂; and R⁵ is selected from

wherein

R^a is nitro, Me, Et, t-butyl, or alkoxy;

R^b is hydrogen atom, halogen atom, cyano, nitro, or amino group; R^c and R^d are each and independently of each other selected from halogen atom, cyano, nitro, amino group, alkyl, and alkoxy;

R^e is alkyl, alkoxy, -alkylaryl, or -alkenylaryl;

R^f is -alkylaryl, or -alkenylaryl.

Preferably, the present invention relates to a compound or salt of formula (I), wherein R¹ is -N0₂; R² and R⁴ are hydrogen atom, R³ is -S0₃H; and R⁵ is selected from

Further, the present invention preferably relates to a compound or salt of formula (I-a) or (I-b), wherein R¹, if present, is -N0₂; R² and R⁴ are hydrogen atom, R³ is -S0₃H; R⁵ is selected from

nitro, Me, Et, t-butyl, or alkoxy; R^b is hydrogen atom, halogen atom, cyano, nitro, or amino group;

R^e is alkyl, alkoxy, -alkylaryl, or -alkenylaryl; and

R^f is -alkylaryl, or -alkenylaryl.

Further preferred is a compound or salt of formula (I), wherein R¹ is -S0₂NH₂, and R⁵ is selected from aryl, heteroaryl, aralkyl or heteroaralkyl, wherein said aryl, heteroaryl, aralkyl or heteroaralkyl can be independently substituted with from 1 to 4 substituents which substituents are independently of each other selected from halogen atom, hydroxy, nitro, amino, cyano, =0, =S, =NH, sulfo, alkoxy, carboxyl, unsubstituted or substituted Ci-C₆alkyl, unsubstituted or substituted C₂-C₆alkenyl, unsubstituted or substituted C2-C₆alkynyl, unsubstituted or substituted C₂-C₉heteroalkyl, unsubstituted or substituted C₇-Ci₂aralkyl, unsubstituted or substituted C₆-C₁₂heteroaralkyl, with the proviso that a compound, wherein R² and R⁴ are hydrogen atom, R³ is -S0₃H, and R⁵ is phenyl is excluded.

Also preferred is a compound or salt of formula (I), wherein R² and R⁴ are each and independently of each other selected from hydrogen atom, halogen atom, cyano, nitro, and amino group; and R⁵ is selected from aryl, heteroaryl, aralkyl or heteroaralkyl, wherein said aryl, heteroaryl, aralkyl or heteroaralkyl can be independently substituted with from 1 to 4 substituents which substituents are independently of each other selected from halogen atom, hydroxy, nitro, amino, cyano, =0, =S, =NH, sulfo, alkoxy, carboxyl, unsubstituted or substituted Ci-C₆alkyl, unsubstituted or substituted C₂- Qalkenyl, unsubstituted or substituted C₂-C₆alkynyl, unsubstituted or substituted C₂-C₉heteroalkyl, unsubstituted or substituted C₇-Ci₂aralkyl, unsubstituted or substituted Ce-Cnheteroaralkyl, with the proviso that at least one of R² or R⁴ is not hydrogen atom.

Also preferred is a compound or salt of formula (I-a) or (I-b), wherein R² and R⁴ are each and independently of each other selected from hydrogen atom, halogen atom, cyano, nitro, and amino group; and R⁵ is selected from aryl, heteroaryl, aralkyl or heteroaralkyl, wherein said aryl, heteroaryl, aralkyl or heteroaralkyl can be independently substituted with from 1 to 4 substituents which substituents are independently of each other selected from halogen atom, hydroxy, nitro, amino, cyano, =0, =S, =NH, sulfo, alkoxy, carboxyl, unsubstituted or substituted Ci-C₆alkyl, unsubstituted or substituted C₂-C₆alkenyl, unsubstituted or substituted C₂-C₆alkynyl, unsubstituted or substituted C₂- C₉heteroalkyl, unsubstituted or substituted C₇-Ci₂aralkyl, unsubstituted or substituted C₆- Ci₂heteroaralkyl, with the proviso that at least one of R² or R⁴ is not hydrogen atom.

Preferably, in the compound or salt of formula (I) R¹ is -N0₂. Similarly preferred is a compound or salt of formula (I-b), wherein R¹ is -N0₂.

Also preferred is a compound or salt of formula (I), wherein R¹ is -S0₂NH₂.

Also preferred is a compound or salt of formula (I) or (I-b), wherein R¹ is -NH-(CO)-CH₃.

Also preferred is a compound or salt of formula (I) or (I-b), wherein R¹ is -OH.

Further preferred is a compound or salt of formula (I), wherein both R² and R⁴ are hydrogen atom. Similarly preferred is a compound or salt of formula (I-a) or (I-b), wherein both R² and R⁴ are hydrogen atom.

Further preferred is a compound or salt of formula (I), wherein R² is halogen atom and R⁴ is hydrogen atom. Preferabyl, in the compound of formula (I-a) or (I-b), R² is halogen atom and R⁴ is hydrogen atom.

Also preferred is a compound or salt of formula (Γ), wherein both R² and R⁴ are halogen atom, preferably chlorine or fluorine. Particularly preferred is a compound or salt of formula (I-a), (I-b) or (I), wherein both R² and R⁴ are chlorine atom.

Further preferred is a compound or salt of formula (I), wherein R³ is -S0₃H. Also preferred is a compound or salt of formula (I-a) or (I-b), wherein R³ is -S0₃H.

Also preferred is a compound or salt of formula (I), wherein R³ is -S0₂NH₂.

The present invention preferably relates to a compound or salt of formula (I-a) or (I-b), wherein R¹, if present, is -N0₂;

R² is halogen atom;

R⁴ is hydrogen atom or halogen atom; and

R⁵ is selected from

hydrogen atom, nitro, Me, Et, t-butyl, or alkoxy; R^b is hydrogen atom, halogen atom, cyano, nitro, or amino group;

R^e is alkyl, alkoxy, -alkylaryl, or -alkenylaryl; and

R^f is -alkylaryl, or -alkenylaryl.

Preferably, in the compound of formula (I-a) or (I-b), R⁵ is:

Further preferred is a compound or salt of formula (I), wherein R⁵ is:

Also preferred is a compound or salt of formula (I), wherein R⁵ is:

More preferred is a compound or salt of formula (I), wherein R¹ is -N0₂; R² and R⁴ are hydrogen atom; R³ is -S0₃H; and R⁵ is

Further preferred is a compound or salt of formula (I), wherein R¹ is -N0₂; R² and R⁴ are hydrogen atom; R³ is -S0₃H; and R⁵ is

Further preferred is a compound or salt of formula (I), wherein R¹ is -N0₂; R² and R⁴ are chlorine atom; R³ is -S0₃H; and R⁵ is phenyl.

It is to be noted that the present invention preferably encompasses all possible combinations of all preferred embodiments.

The compound or salt of formula (I) according to the present invention has preferably one or more improved properties, especially, improved activity or specificity, low toxicity, low drug drug interaction, improved bioavailability, especially with regard to oral administration, improved metabolic stability, and improved solubility.

The compounds of formula (I) provided herein exhibit high activity on proteins selected from the group comprising phosphatases, Src homology 2 (SH2) and phosphotyrosine binding (PTB) domains containing proteins and phosphotyrosine-binding proteins. Preferably, the compounds according to the invention inhibit PTPs, more preferably, Shp2 PTP or an oncogenic variant or point mutant of Shp2. The compounds can also exhibit a high activity on proteins of species other than human, e.g. rat, mouse, gerbil, guinea pig, rabbit, dog, cat, pig, or cynomolgus monkey.

The activity and more specifically pharmacological activity of the compounds according to the present invention can be assessed using appropriate in vitro or in vivo assays, which are standard assays and known to those skilled in the art, e.g. from the art discussed above. For instance, IC₅₀ values of the compounds according to the present invention for PTPs may be determined by measuring phosphate release via complexation of the released phosphate (see, for example, Baykov et al., Anal Biochem 1988, 171, 266-270) or by determining dephosphorylation of the substrate, e.g. p-NPP or DiFMUP (see, for example, Blaskovich MA, Curr Med Chem 2009, 16, 2095-2176; Welte et al., Anal Biochem 2005, 338, 32-38; and WO 2006/128909). Examples of such assays are also provided below. Preferred compounds of the invention have an IC5₀ (half-maximal inhibitory concentration) of about less than 10 micromolar (uM), preferably an IC₅₀ of 5 μΜ or less, still more preferably of about 500 nanomolar (nM) or less, or even 50 nM or less, even more preferably about 10 nM or less, and most preferably 1 nM or less in the assays mentioned above.

The therapeutic use of compounds of general formula (I), their pharmacologically acceptable salts, solvates or hydrates and also formulations and pharmaceutical compositions containing the same are also within the scope of the present invention. The present invention also relates to the use of those compounds of general formula (I) as active ingredients in the preparation or manufacture of a medicament.

The pharmaceutical composition according to the present invention comprises comprises one or more compound(s) of formula (I) and, optionally, at least one carrier substance, excipient and/or adjuvant. Pharmaceutical compositions may additionally comprise, for example, one or more of water, buffers {e.g., neutral buffered saline or phosphate buffered saline), ethanol, mineral oil, vegetable oil, dimethylsulfoxide, carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, adjuvants, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione and/or preservatives. Furthermore, one or more other active ingredients may (but need not) be included in the pharmaceutical compositions provided herein. For instance, the compounds of the invention may advantageously be employed in combination with an antibiotic, anti-fungal, or antiviral agent, an anti histamine, a non-steroidal anti-inflammatory drug, a disease modifying antirheumatic drug, a cytostatic drug, a drug with smooth muscle activity modulatory activity or mixtures of the aforementioned.

Pharmaceutical compositions may be formulated for any appropriate manner of administration, including, for example, topical (e.g., transdermal or ocular), oral, buccal, nasal, vaginal, rectal or parenteral administration. The term parenteral as used herein includes subcutaneous, intradermal, intravascular (e.g., intravenous), intramuscular, spinal, intracranial, intrathecal, intraocular, periocular, intraorbital, intrasynovial and intraperitoneal injection, as well as any similar injection or infusion technique. In certain embodiments, compositions in a form suitable for oral use are preferred. Such forms include, for example, tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs. Within yet other embodiments, compositions provided herein may be formulated as a lyophilizate. Preferably, the pharmaceutical composition is formulated as an aerosol, a cream, a gel, a pill, a capsule, a syrup, a solution, a transdermal patch or a pharmaceutical delivery device. Compositions intended for oral use may further comprise one or more components such as sweetening agents, flavoring agents, coloring agents and/or preserving agents in order to provide appealing and palatable preparations. Tablets contain the active ingredient in admixture with physiologically acceptable excipients that are suitable for the manufacture of tablets. Such excipients include, for example, inert diluents such as, e.g., calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate, granulating and disintegrating agents such as, e.g., corn starch or alginic acid, binding agents such as, e.g., starch, gelatin or acacia, and lubricating agents such as, e.g., magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monosterate or glyceryl distearate may be employed.

Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent such as, e.g., calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium such as,e.g., peanut oil, liquid paraffin or olive oil.

Suitable excipients include suspending agents such as, e.g., sodium carboxymethylcellulose, methylcellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; and dispersing or wetting agents such as, e.g., naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with fatty acids such as polyoxyethylene stearate, condensation products of ethylene oxide with long chain aliphatic alcohols such as heptadecaethyleneoxycetanol, condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides such as polyethylene sorbitan monooleate. Aqueous suspensions may also comprise one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

For the prevention and/or treatment of diseases mediated by PTPs, especially Shp2 or an oncogenic variant or point mutant thereof, the dose of the biologically active compound according to the invention may vary within wide limits and may be adjusted to individual requirements. Active compounds according to the present invention are generally administered in a therapeutically effective amount. Preferred doses range from about 0.1 mg to about 140 mg per kilogram of body weight per day, about 0.5 mg to about 7 g per patient per day. The daily dose may be administered as a single dose or in a plurality of doses. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. Dosage unit forms will generally contain between from about 1 mg to about 500 mg of an active ingredient.

According to the invention, the compounds of the invention can be used in the pharmaceutical preparation in a quantity comprised between 0.01 mg and 2 g, preferably from 1 mg to 1 g, very preferably from 10 mg to 500 mg. More particularly, the compounds can be administered in doses comprised between 0.1 mg/kg and 500 mg/kg, preferably 1 mg kg and 100 mg/kg, very preferably 10 mg/kg and 50 mg/kg.

It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination, i.e. other drugs being used to treat the patient, and the severity of the particular disease undergoing therapy.

The compound or pharmaceutical composition according to the invention can be used as a medicament. In particular, the compound or pharmaceutical composition according to the invention can be used in the treatment and/or prevention of a Shp2 mediated disease or a Shp2 mediated condition.

The term "Shp2-mediated disease" or "Shp-2 mediated condition", as used herein means any disease or other deleterious condition in which Shp2 is known to play a role. Such conditions include, without limitation, genetic disorders, autoimmune diseases, proliferative diseases, angiogenic disorders, and cancers.

Genetic disorders which can be treated or prevented by compounds of formula (Γ) or the pharmaceutical composition of the invention include, but are not limited to Noonan syndrome (NS) or Leopard syndrome (LS).

A proliferative disease which can be treated or prevented by the compounds of formula (Γ) or the pharmaceutical composition of the invention is a neoplasia, which includes, but is not limited to leukemias, lymphomas, sarcomas, carcinomas, neural cell tumors, undifferentiated tumors, sensinomas, melanomas, neuroblastomas, multiple myeloma, mixed cell tumors, metastatic neoplasia and neoplasia due to pathogenic infections. Leukemias include acute myelogenous leukemia, chronic myelogenous leukemia and juvenile myelomonocytic leukemia.

Angiogenic disorders which can be treated or prevented by the compounds of formula (I) or the pharmaceutical composition of the invention include, but are not limited to ocular neovasculization and infantile haemangiomas.

Autoimmune diseases which can be treated or prevented by the compounds of formula (I) or the pharmaceutical composition of the invention include, but are not limited to inflammatory reactions, diabetes, obesity, and diseases associated with cell division.

Various cancers, especially solid tumors, such as, for example, bladder, breast, renal, non-small lung cell, thyroid, prostate, hepatocellular, colorectal and gastric carcinoma and their metastases, can be treated or prevented by the compounds of formula (I) or the pharmaceutical composition of the invention. Beside the treatment of gastric carcinoma the compounds of formula (I) or the pharmaceutical composition of the invention are also suitable for treating helicobacter pylori infections and gastric ulcers which may be caused by such infections.

Inflammations in the meaning of the invention are reactions of the organism, mediated by the connective tissue and blood vessels, to an external or internally triggered inflammatory stimulus, with the purpose of eliminating or inactivating the latter and repairing the tissue lesion caused by said stimulus.

Autoimmune diseases in the meaning of the invention are diseases entirely or partially due to the formation of autoantibodies and their damaging effect on the overall organism or organ systems, i.e., due to autoaggression.

The term "angiogenesis" refers to the generation of new blood vessels into cells, tissue, organs or tumors.

The term "metastasis" refers to the process by which tumor cells are spread to distant parts of the body. The term is also used herein to refer to a tumor that develops through the metastatic process. The compounds of formula (I) or the pharmaceutical composition of the invention are preferably administered to a patient orally or parenterally, and are present within at least one body fluid or tissue of the patient.

Accordingly, the present invention further provides methods for preventing or treating patients suffering from a Shp-2 mediated disease or a Shp-2 mediated condition.

As used herein, the term "treatment" encompasses both disease-modifying treatment and symptomatic treatment, either of which may be prophylactic, i.e., before the onset of symptoms, in order to prevent, delay or reduce the severity of symptoms, or therapeutic, i.e., after the onset of symptoms, in order to reduce the severity and/or duration of symptoms.

Preferably, the method of preventing or treating a Shp-2 mediated disease or a Shp-2 mediated condition according to the invention comprises administering to a subject in need thereof an effective amount of at least one compound of formula (I).

As used herein, the term "effective amount" refers to a quantity that is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit risk ratio when used in the manner of this invention.

It is also within the present invention that the compounds according to the invention are used as PTP inhibitor, preferably Shp2 inhibitor, for investigating cellular processes in biological in vitro, in vivo or ex vivo systems, e.g. cell cultures. For instance, a compound of formula (I) may be used as diagnostic agent, whereby such diagnostic agent is for the diagnosis of the diseases and conditions which can be addressed by the compounds of the present invention for therapeutic purposes as disclosed herein. Alternatively, compounds of formula (I) can be used in cell culture to interfere with phosphatase dependent functions such as cell proliferation, cell-cycle progression, cell-cell dissociation, cell polarity, cell motility, matrix degeneration, invasion and stem cell renewal to thereby allow, for example, to elucidate signalling pathways or mechanisms by which PTPs contribute to the onset of diseases such as those discussed above. For these purposes, the compounds of the invention can be labelled by isotopes, fluorescence or luminescence markers, antibodies or antibody fragments, any other affinity label like nanobodies, aptamers, peptides etc., enzymes or enzyme substrates. These labelled compounds of this invention are, for example, useful for mapping the location of tumor cells in vivo, ex vivo, in vitro and in situ such as, e.g. in tissue sections via autoradiography and as radiotracers for positron emission tomography (PET) imaging, single photon emission computerized tomography (SPECT) and the like to characterize those cells in living subjects, cell culture or other materials. The labelled compounds according to the present invention may be used in therapy, diagnosis and other applications such as research tools in vivo and in vitro, in particular the applications disclosed herein.

It is especially preferred to combine the preferred embodiments of the present invention in any possible manner.

The compounds of the present invention can be prepared in a number of ways well known to those skilled in the art of organic synthesis. For example, the compounds of the present invention can be synthesized according to the following reaction scheme 1 , together with synthetic methods known in the art of synthetic organic chemistry, or variations thereon as appreciated by those skilled in the art.

Reaction scheme 1:

All starting materials and reagents are of standard commercial grade, and are used without further purification, or are readily prepared from such materials by routine methods. The four step synthesis depicted in the above reaction scheme 1 can, for example, be carried out as follows: step (a) acylation of the methyl 2-(triphenylphosphoranylidene)-acetate under basic conditions to yield methyl 3-oxo-2- triphenylphosphoranylidene-propanoate, e.g. R^xCOOH (1.2 eq), l-(2-mesitylenesulfonyl)-3-nitro-lH- 1 ,,2,4-triazol (MSNT; 1.2 eq), lutidine (1.4 eq), dichloromethane (DCM), RT, 16 h or R^xCOCl (1.2 eq), lutidine (1.4 eq), DCM, RT, 16 h; step (b) oxidation of the methyl 3-oxo-2- triphenylphosphoranylidene-propanoate with dimethyldioxirane (DMDO) to yield methyl 2,2- dihydroxy-3-oxo-propanoate, e.g. DMDO in acetone (3 eq), DCM, RT, lh; steps (c) and (d) one-pot reaction (condensation with hydrazine derivatives), e.g. (c): R²R³R PhNHNH2, HC1 (kat.), 85 °C, 16 h. (d): R⁵NHNH2, 85 °C, 16 h. Those skilled in the art of organic synthesis will recognize that starting materials and reaction conditions may be varied including additional steps employed to produce compounds encompassed by the present invention.

For instance, those skilled in the art of organic synthesis will recognize that R can also be selected

Brief Description of the Figures

Figure 1: HGF/SF-stimulated cell dissociation of MDCK-C cells (Madin Darby Canine Kidney, canine epithelial cells). As can be seen, the tested compounds no. 49, 50 and 39 (for details of the compounds, see examples below) inhibit cell dissociation of MDCK-C cells in a concentration dependent manner; cell dissociation plays an important role in the development of malignant tumors.

Figure 2: HGF/SF-stimulated cell dissociation of human pancreas tumor cells, HPAF Π. As can be seen, the tested compounds no. 49, 50 and 39 inhibit the HGF/SF induced cell dissociation of HPAF H cells in a concentration dependent manner.

Figure 3: Inhibition of proliferation of non-small lung cell carcinoma cell lines LXFA 526L and LXFL 1647L by test compound GS462 (herein referred to as compound no. 50) (left panel) in comparison to taxol (right panel). Figure 4: In vivo assessment of tolerance and toxicity of test compounds no. 49 and 50 in the NMRI nu/nu nude mouse animal model. Test compounds were biologically active, were readily tolerated and showed no toxicity. A daily dose of 3 ml/kg was supplied.

Figure 5: In vivo antitumoral activity of test compound no. 39 in the NMRI nu/nu nude mouse animal model with implanted LXFA 526L adenocarcinoma of the lung. Experiment according to Roth, T., Burger, A.M., Dengler, W., Willmann, H., Fiebig, H.H., Human tumor cell lines demonstrating the characteristics of patient tumors as useful models for anticancer drug screening, [ed.] H.H. Burger, A.M. Fiebig. Relevance of Tumor Models for Anticancer Drug Development. Basel : Karger, 1999, Vol. 54, 145-156. As can be taken from the graph, test compound no. 39 markedly reduced tumor growth of LXFA 526L adenocarcinoma of the lung (T/C-value of 42.6 %).

Figure 6: Fig. 6 shows the result from testing siRNA interfering with the expression of Shp2 gene and compounds no. 39 and 50 for their impact on soft agar growth of LXFA 526L human lung adenocarcinoma cell line. The soft agar assay shows that tumor growth is dependent on Shp2, since reduced expression of Shp2 resulted in a significant reduced number of tumor colonies (37 %) compared to the control (untransfected tumor cell colonies). The inhibitory activity of compound no. 39 and 50 is similar to that observed with siRNA. Compound no. 50 reduced the number of tumor colonies to 20%, and Compound No. 39 reduced the number of tumor colonies to 32%.

Figure 7: Fig. 7 shows the effect of compound no. 39 (referred to as GS-493) on group median body weights of LXFA 526L tumor xenograft-bearing nude mice.

The present invention is now further illustrated by the following examples, which are not construed to limit the broad aspects of the invention disclosed herein.

Examples

The compounds shown in the following Table 1 are representative examples of compounds of formula (I), (I-a), or (I-b) according to the present invention.

Table 1 :

Compound No. R¹ R² R³ R⁴ R⁵

9 -S0₂NH₂ H -SO₃H H -Ph

21 -N0₂ H -SO₃H CI -Ph

22 -N0₂ CI -SO₃H CI -Ph

24 -N0₂ H -SO₃H H -Ph-4-Me

26 -N0₂ H -SO₃H H -Ph-4-t-Butyl

28 -N0₂ H -SO₃H H -Benzyl-3-OH

31 -N0₂ H -SO₃H H -Ph-OMe

39 (GS-493) -N0₂ H -SO₃H H -Ph-4-N0₂

40 -N0₂ H -SO₃H H -Ph-3,4-diCI

41 -N0₂ H -SO₃H H -Ph-3-CI-4-OMe

42 -N0₂ H -SO₃H H -Ph-3,4-dimethoxy

43 -N0₂ H -SO₃H H -Ph-2-CF₃-3-CI-5-CI

45 -N0₂ H -SO₃H H

48 -N0₂ H -SO₃H H

49 -N0₂ H -SO₃H H

Example 1 : Determination of IC₅₀ Shp2

Enzymatic assays were used for the determination of Shp2 FTP activity of the compounds of the invention. In these assays, the catalytic domain of Shp2 (amino acids 225-541) and Shp2 mutant E76K were employed, respectively. Shp2 enzyme activity was assessed by (i) measuring the absorption generated by the dephosphorylation of 4-nitrophenylphosphate (pNPP) yielding the yellow product 4- nitrophenol (pNP) or (ii) measuring the fluorescence generated by the dephosphorylation of 6,8- difluoro-4-methyl-umbelliferylphosphate (DiFMUP) yielding 6,8-difluoro-4-methylumbelliferone (DiFMU). The assays are carried out in 96-well microtiter plates.

1. Enzymatic pNPP assay Test compounds are dissolved in dimethylsulfoxide (DMSO), and assay is carried out at a final concentration of below 1 % DMSO. pNPP assay buffer contains a final concentration of 25 mM Tris (pH 7.0), 50 mM NaCl, 0.01 % Brij35, 10 % Glycerol, 1 mM dithiothreitol (DTT) and 25 nM Shp2, the final assay volume is 50 μΐ. The reaction is started by adding /?NPP to a final concentration of 10 mM and incubated at 37°C. The absorption at 405 nm (A405; filter: 400 nm, band width 35 nm) is monitored for 60 min in 1 min intervalls. A405 is plotted versus time, and the slope in the linear part of the curve is determined (mOD/min). Measurements were performed in triplicate. IC₅₀-values were calculated with Origin v6.0 (Microcal Software Inc., Northampton, USA) by using a sigmoidal curve fit.

2. Enzymatic DiFMUP assay

Test compounds are dissolved in dimethylsulfoxide (DMSO) at a concentration of 10 mM or 100 mM, and assay is carried out at a final concentration of below 1 % DMSO. DiFMUP assay buffer contains a final concentration of 50 mM TrisBis (pH 6.5), 10 mM NaCl, 0.03 % Tween 20, 0.1 % BSA, 25 mM dithiothreitol (DTT) and 25 nM Shp2, the final assay volume is 30 μΐ. Enzyme and test compound in buffer solution are incubated for 1 h at RT. The reaction is started by adding DiFMUP. Excitation wavelength: 360 nm (bandwidth 20 nm); Emission wavelength 460 nm (bandwidth 20 nm); number of reads: 8; integration time: 40 μβ; number of kinetic cycles: 5; kinetic intervall: 135 s; total kinetic run time: 9 min. Measurements were performed in triplicate. ICs₀-values were calculated with Prism 5 (for Windows, Version 5.01, Graph Pad Software, Inc.).

Both assays yielded similar (identical) IC₅₀-values for tested compounds. Table 2 shows IC₅₀-values of tested compounds.

Table 2: IC₅₀-values of test compounds in relation to PTP Shp2

Compound No. IC_tt-value [μΜ] Compound No. IC₅₀-value [μΜ]

9 5 41 2

22 4 48 1

26 2 49 0.6

31 2 50 0.6

39 0.08 51 1 Compound No. ICso-value [μΜ] Compound No. IC5o-value [μΜ]

57 2 62 3

58 2

Example 2: Determination of Specificity for Shp2

In order to determine target specificity of the test compounds according to the invention an enzymatic DiFMUP assay was used. In this assay enzyme activity against related PTPs, such as Shpl, PTPIB and MptpA was assessed. The results are shown in Table 3.

Table 3: IC₅₀- values of test compounds in relation to 4 different PTPs

nd: not determined

As can be seen from Table 3, all tested compounds show ICso-values within the micromolecular range against Shp2. None of the test compounds shows a higher inhibitory activity against any of the related PTPs. Thus, the tested compounds are highly selective for Shp2.

Example 3: Comparison between PHPS1 and compounds according to the invention An enzymatic DiFMUP assay (see Example 1 above) was used for the determination of Shp2 PTP, Shpl PTP, and PTPIB activity. Test compounds were dissolved in dimethylsulfoxide (DMSO) at a concentration of 10 mM or 100 mM, and the assay was carried out at a final concentration of below 1 % DMSO. DiFMUP assay buffer contains a final concentration of 50 mM MOPSO (pH 6.5), 200 mM NaCl, 0.03 % Tween 20, 0.1 % BSA, 1 μΜ dithiothreitol (DTT, freshly added prior to each measurement) and 0,003 ng/μΐ Shp2 (final concentration), the final assay volume is 30 ul. Enzyme and test compound in buffer solution were incubated for 1 h at RT. The reaction was started by adding DiFMUP. Measurements were performed on a Genius Pro Reader (S AFIRE Π, Instrument serial number: 512000014) with the following settings: Measurement mode: Fluorescence Top; Excitation wavelength: 360 nm (bandwidth 20 nm); Emission wavelength 460 nm (bandwidth 20 nm); Gain (manual): 60; Number of reads: 8; FlashMode: High sensitivity; Integration time: 40 μβ; Lag time: 0 μβ; Z-position (manual): 13900 μιη; Number of kinetic cycles: 5; Kinetic intervall: 135 s; total kinetic run time: 9 min. Measurements were performed in triplicate. IC₅₀-values were calculated with Prism 5 (for Windows, Version 5.01, Graph Pad Software, Inc.).

Table 4: Comparison of IC₅₀- values of test compounds and PHPS 1

Compound R¹ R² R³ R⁴ R⁵

Shp2 Shp1 PTP1B

57 -N0₂ H -SO₃H H 0.43 8.52 5.80

58 -N0₂ H -SO₃H H 0.30 3.73 3.04

As can be taken from Table 4, all tested compounds show a higher inhibitory acticivity against Shp2 than PHPS1. Thus, the tested compounds more effectively inhibit the biological activity of Shp2 PTP than PHPS 1. Moreover, none of the test compounds shows a higher inhibitory acticivity against the related Shpl PTP and PTPIB, respectively. Thus, the tested compounds are also highly selective for Shp2.

Example 4: Soft Agar Assay (Tumor Colony Assay)

A soft agar assay as described by Hamburger and Salmon (Science 1977, 197, 461-463) was used to determine the anti-tumorigenic effects of Compounds No. 39 and 50 on cells isolated from lung adenocarcinoma xenograft LXFA 526L. In brief, the method can be summarized as follows: Cells are seeded in semi-solid agar medium in 24-well plates. Tumor cells form multi-cellular colonies within a couple of days which is measured by determining cell viability using a fluorescent dye. Addition of inhibitors after cell seeding allows for the analyses of anti-tumorigenic effects. As a control, siRNA (Qiagen: Hs_PTPNl l_l; SI00044002) interfering the expression of Shp2 and the known cytostatic agent 5-fluorouracil (5-FU) was used.

Specifically, tumor cells were derived from NMRI nu/nu nude mouse animal model with implanted LXFA 526L adenocarcinoma of the lung. For this, tumors were extracted from the NMRI nu/nu nude mice under steril conditions, crushed mechanically and then incubated with an enzyme mixture (Collagenase Typ IV (41 U/ml), DNase I (125 U/ml), Hyaluronidase (100 U/ml), Dispase Π (1.0 U/ml) in RPMI 1640-medium (25 mM HEPES buffer incl. L-glutamine)) at 37 °C for 45min. Subsequently, the cells were further separated by sieving (mesh sizes: 200 μπι and 50 μπι); washed twice with sterile PBS. Viable cells were determined using trypan blue staining and a Neubauer counting chamber. The soft agar assay was performed in 24-well plates according to Hamburger and Salmon (ibid.): cell medium: IMDM (Iscove's modified Dulbeccos" Medium) incl. 20 % (v/v) fetal calf serum and 0,01 % (w/v) gentamicin; bottom layer: 0.75 % (w/v) Bacto Agar in cell medium; intermediate layer with cells: 4 10⁴ cells in 0.2 ml cell medium incl. 0.4 % (w/v) Bacto Agar; top layer with test compound: test compound (3-times concentration) in cell medium; 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl- tetrazoliumchloride: 1 mg/ml, 100 μΐ/well. 200 μΐ per well were platted as bottom layer and the cell layer was applied on this layer. Compound No. 39 and 50 in DMSO were added to the medium after 24 h (final concentration: 40 μΜ). The plates were incubated for up to 20 days at 37 °C and 7.5% C0₂. During this period an in vitro colony growth of >50 μπι in diameter was observered. After the maximum colony formation, the number of colonies was determined using an automatic image analysis system (BIOREADER 5000 PRO-XI, Biosys GmbH). 24 h prior to evaluation, live colonies were stained using a sterile aqueous solution of 2-(4-iodophenyl)-3-(4-nitrophenyl)-5 -phenyl - tetrazoliumchloride (Alley et al., "Improved detection of drug cytotoxicity in the soft agar colony formation assay through use of a metabolizable tetrazolium salt" Life Sci. 1982, 31, 3071-3078).

The assay was considered to be valid if the following criteria were met: the control shows > 20 colonies each having a diameter of > 50 μιη; the coefficient of variation of control wells/plate is < 50 % and the positive control 5-fluorouracil (Heidelberger et al., "Fluorinated pyrimidines, a new class of tumour-inhibitory compounds" Nature 1957, 179, 663-666) shows a reduction in colony number of < 30 % compared to the untreated control.

The results are shown in Fig. 6. As can be taken from Fig. 6, the inhibitory activity of compound 39 and 50 is similar to that observed with siRNA, where the expression of Shp2 is significantly reduced.

Claims

1. A compound of general formula (I-a):

R¹ is -NH-(CO)-CH₃₎ -N0₂, -OH, or -S0₂NH₂; R² and R⁴ are each and independently of each other selected from hydrogen atom, halogen atom, cyano, nitro, and amino group;

R³ is -SOjH or -S0₂NH₂; and

R⁵ is selected from

(i)

wherein

R^a is nitro, Me, Et, t-butyl, or alkoxy;

R^b is hydrogen atom, halogen atom, cyano, nitro, or amino group;

R^e is alkyl, alkoxy, -alkylaryl, or -alkenylaryl;

(ii) aryl, heteroaryl, aralkyl or heteroaralkyl, wherein said aryl, heteroaryl, aralkyl or heteroaralkyl can be independently substituted with from 1 to 4 substituents which substituents are independently of each other selected from halogen atom, hydroxy, nitro, amino, cyano, =0, =S, =NH, sulfo, alkoxy, carboxyl, unsubstituted or substituted Ci-Cealkyl, unsubstituted or substituted C₂- C₆alkenyl, unsubstituted or substituted C₂-C₆alkynyl, unsubstituted or substituted C₂-C₉heteroalkyl, unsubstituted or substituted C7-Ci₂aralkyl, unsubstituted or substituted C₆-Ci₂heteroaralkyl; if R' is -NH-(CO)-CH₃ or -S0₂NH₂; or (iii) aryl, heteroaryl, aralkyl or heteroaralkyl, wherein said aryl, heteroaryl, aralkyl or heteroaralkyl can be independently substituted with from 1 to 4 substituents which substituents are independently of each other selected from halogen atom, hydroxy, nitro, amino, cyano, =0, =S, =NH, sulfo, alkoxy, carboxyl, unsubstituted or substituted Ci-C₆alkyl, unsubstituted or substituted C₂- C₆alkenyl, unsubstituted or substituted C₂-C₆alkynyl, unsubstituted or substituted C₂-C₉heteroalkyl, unsubstituted or substituted C₇-Ci₂aralkyl, unsubstituted or substituted C₆-Ci₂heteroaralkyl; if R² and/or R⁴ is/are not hydrogen atom.

2. The compound or salt according to claim 1, wherein the compound is represented by general formula (I-b):

and R¹, R², R³, R⁴, and R⁵ are defined as in claim 1.

3. The compound or salt according to claim 2, wherein R¹ is N0₂.

4. The compound or salt according to any one of claims 1 to 3, wherein both R² and R⁴ are hydrogen atom.

5. The compound or salt according to any one of claims 1 to 3, wherein R² is halogen atom and R⁴ is hydrogen atom.

6. The compound or salt according to any one of claims 1 to 3, wherein both R² and R⁴ are halogen atom.

7. The compound or salt according to any one of claims 1 to 6, wherein R³ is -S0₃H.

8. The compound or salt according to any one of claims 1 to 4 and 7, wherein R⁵ is

9. The compound or salt according to any one of claims 1, 2 and 7, wherein R¹, if present, is -N0₂;

R² is halogen atom;

R⁴ is hydrogen atom or halogen atom; and

R⁵ is selected from

R^a is hydrogen atom, nitro, Me, Et, t-butyl, or alkoxy; R^b is hydrogen atom, halogen atom, cyano, nitro, or amino group;

R^e is alkyl, alkoxy, -alkylaryl, or -alkenylaryl; and

R^f is -alkylaryl, or -alkenylaryl.

10. The compound or salt according to any one of claims 1, 2, 6, 7 and 9, wherein both R² and R⁴ are chlorine atom.

11. The compound or salt according to claim 2, wherein

R¹ is -N0₂; R² and R⁴ are hydrogen atom; R³ is -S0₃H; and

R⁵ is

12. A pharmaceutical composition that comprises one or more compound(s) according to any one of claims 1 to 11 and, optionally, at least one carrier substance, excipient and/or adjuvant.

13. The pharmaceutical composition according to claim 12, wherein the pharmaceutical composition is formulated as an aerosol, a cream, a gel, a pill, a capsule, a syrup, a solution, a transdermal patch or a pharmaceutical delivery device.

14. Compound or pharmaceutical composition according to any one of claims 1 to 13 for use as a medicament.

15. Compound or pharmaceutical composition according to any one of claims 1 to 13 for use in the treatment and/or prevention of a Shp-2 mediated disease or a Shp-2 mediated condition.

16. The compound or pharmaceutical composition according to claim 15, wherein the Shp-2 mediated disease or condition is a proliferative disease, genetic disorder, autoimmune disease, angiogenic disorder or cancer.

17. The compound or pharmaceutical composition according to claim 15 or 16, wherein

(i) the proliferative disease is a neoplasia selected from the group consisting of leukemias, lymphomas, sarcomas, carcinomas, neural cell tumors, undifferentiated tumors, sensinomas, melanomas, neuroblastomas, multiple myeloma, mixed cell tumors, metastatic neoplasia and neoplasia due to pathogenic infections;

(ii) the autoimmune disease is diabetes, preferably diabetes mellitus;

(iii) the disorder is Noonan syndrome or juvenile myelomonocytic leukaemia (JMML);

(iv) the carcinoma is bladder, breast, renal, non-small lung cell, thyroid, prostate, hepatocellular, colorectal or gastric carcinoma; and metastases thereof.

18. Use of at least one compound according to any one of claims 1 to 11 as protein tyrosine phosphatase inhibitor, preferably Shp-2 inhibitor, for investigating cellular processes in biological in vitro, in vivo or ex vivo systems.

19. A method of preventing or treating a Shp-2 mediated disease or a Shp-2 mediated condition, said method comprising administering to a subject in need thereof an effective amount of at least one compound according to any one of claims 1 to 11.