WO1997008934A2 - Derives d'acide aryl-acrylique convenant comme inhibiteurs de proteine-tyrosine-phosphatase - Google Patents

Derives d'acide aryl-acrylique convenant comme inhibiteurs de proteine-tyrosine-phosphatase Download PDF

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WO1997008934A2
WO1997008934A2 PCT/US1996/018401 US9618401W WO9708934A2 WO 1997008934 A2 WO1997008934 A2 WO 1997008934A2 US 9618401 W US9618401 W US 9618401W WO 9708934 A2 WO9708934 A2 WO 9708934A2
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alkyl
group
protein tyrosine
tyrosine phosphatase
formula
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PCT/US1996/018401
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WO1997008934A3 (fr
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Adnan Mjalli
Xiaodong Cao
Edmund J. Moran
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Ontogen Corporation
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Priority claimed from US08/543,630 external-priority patent/US5770620A/en
Application filed by Ontogen Corporation filed Critical Ontogen Corporation
Priority to EP96940489A priority Critical patent/EP0833629A4/fr
Priority to JP9511473A priority patent/JPH11508919A/ja
Priority to AU77358/96A priority patent/AU713863B2/en
Publication of WO1997008934A2 publication Critical patent/WO1997008934A2/fr
Publication of WO1997008934A3 publication Critical patent/WO1997008934A3/fr

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Definitions

  • the present invention provides novel protein tyrosine phosphatase modulating compounds, compositions comprising the compounds, and methods of making and using the same.
  • Reversible phosphoryiation and dephosphorylation of proteins comprises a prevalent biological mechanism for modulation of enzymatic activity in living organisms. Tonks et ol., J. Biol. Chem., 263(14) -.6722-30 ( 1988) . Such reversible phosphoryiation generally is thought to require both a protein kinase (PK), to phosphorylate a protein at a particular site, and a protein phosphatase (PP) , to remove the phosphate moieties.
  • PK protein kinase
  • PP protein phosphatase
  • PK's PP's the protein serine /threonine kinases/phosphatases - have been shown to play critical roles in the regulation of metabolism. See generally, Cohen, Trends Biochem. Set, 17:408-413 ( 1992) ; Shenolikar, Ann. Rev. Cell Biol, 10:55-86 ( 1994); Bollen et d., Crit. Rev. Biochem. Mol. Biol, 27:227-81 ( 1992) .
  • protein serine /threonine kinases and phosphatases phosphorylate and dephosphorylate serine or threonine moieties of stbstrate proteins.
  • Inhibitors of protein serine /threonine phosphatases and kinases have been described. See, e.g, MacKintosh and MacKintosh, TIBS, 19:444-448 ( 1994) .
  • the protein tyrosine kinases/phosphatases comprise a second, distinct family of PK/PP enzymes of significant interest, having been implicated in the control of normal and neoplastic cell growth and proliferation. See Fisher et d., Science, 253:401-406 ( 1991) . Protein tyrosine kinase (PTK) genes are ancient in evolutionary origin and share a high degree of inter-species conservation. See generally Hunter and Cooper, Ann. Rev. Biochem., 54:897-930 ( 1985) . PTK enzymes exhibit high specificity for tyrosine, and ordinarily do not phosphorylate serine, threonine, or hydroxyproline .
  • PTPases Protein tyrosine phosphatases
  • CD45 expressed exclusively on hemopoietic cells, is one of the most abundant of the cell surface glycoproteins and functions as a critical component in lymphocyte signal transduction. See generally Trowbridge and Thomas, Ann. Rev. Immunol, 12:85-116 ( 1994) . In particular, evidence suggests that CD45 plays a pivotal role in antigen-stimulated proliferation of both T and B lymphocytes. Trowbridge and Thomas, supra Kishihara et d., Cell 74:143-56 ( 1993) . In a study employing CD45-deficient mice, CD45 was shown to be essential for the antibody- ( I gE-) mediated degranulation of mast cells. Berger et d., J. Exp. Med ⁇ 180:471-6 ( 1994) .
  • HePTP lymphoid-specific protein tyrosine phosphatase designated HePTP has also been implicated in the immune response. HePTP is expressed in both resting T and B lymphocytes, but not non-hem opoie tic cells. Upon stimulation of these cells, HePTP mRNA levels increase 10-15 fold. Zanke et d blunt Eur. J. Immunol, 22:235- 239 ( 1992) . The activity of a number of other newly discussed phosphatases are currently under investigation. Two of these: PTP-1C and Syp/PTP1D/SHPTP2/PTP2C have recently been implicated in the activation of Platelet Derived Growth F ctor and Epidermal Growth Factor induced responses.
  • PTPases appear to be required for the mitogenic effects of certain cytokines (e.g., lLr-4) and interferons. See Burke et d., Biochem and Biophys Res. Comm., 204(1) :129-34 ( 1994) .
  • PTPase Yop2b is an essential virulence determinant in the pathogenic bacterium Yersinia responsible for bubonic plague. Bliska et d., Proc. Natl Acad Sci. USA, 88:1187-91 ( 1991) .
  • PTPase protein is essential to the viriience of pathogenic Yersinia strains, an antibiotic indication exists for PTPase inhibitor compounds, too.
  • the antibiotic suramin which also appears to possess anti-neoplastic indications, has recently been shown to be a potent irreversible, non-competitive inhibitor of CD45. See Ghosh and Miller, Biochem. Biophys. Res. Comm. 194:36-44 ( 1993) .
  • PTPases have been implicated in diabetic conditions.
  • Experiments with one family of PTPase inhibitors, vanadium derivatives indicate a therapeutic utility for such compounds as oral adjuvants or as alternatives to insulin for the treatment of hyperglycemia See Posner et d., J.
  • certain organic phosphotyrosine mimetics are reportedly capable of competitively inhibiting PTPase molecules when such mimetics are incorporated into polypeptide artificial PTPase substrates of 6-11 amino acid residues.
  • a "natural" (phosphorylated tyrosine) PTPase substrate which maybe depicted by the formula
  • hexapeptide inhibitors nonetheless possess drawbacks for PTPase modulation in vivo. More particularly, the hexapeptide inhibitors described by Burke et d. are sufficiently large and anionic to potentially inhibit efficient migration across cell membranes, for interaction with the catalytic domains of transmembrane and intracellular PTPase enzymes which lie within a cell membrane. A need exists for small, organic- molecule based PTPase inhibitors having fewer anionic moieties, to facilitate migration across cell membranes.
  • the invention provides aryl aciylic acid compounds and derivatives thereof useful for modulating, and especially inhibiting, the phosphatase activity of one or more protein tyrosine phosphatase (PTPase) enzymes such as PTP-IB and/or HePTP.
  • PTPase protein tyrosine phosphatase
  • the inventions further provides salts, esters, solvates, and the like of the compounds, and compositions comprising the compounds.
  • the present invention is based on the discovery that aryl acrylic acid compounds of the formula (A) below and their derivatives inhibit protein tyrosine phosphatase activity:
  • n 0 or 1
  • X is CH or Nitrogen (i.e., the aryl group is a phenyl or pyridyl group, respectively)
  • X is oxygen or sulfur (i.e., the aryl group is a furyl or thiophe ⁇ yl (thienyl) group, respectively).
  • derivatives is meant: aryl acrylic acids of formula (A) having substitution (with, e.g., hydroxy, halo, amino, carboxy, nitro, cyano, methoxy, etc.) at one or more atoms of the aromatic ring, as set forth in greater detail below, in Table 4, and in Example 1 1.
  • “derivatives” includes compounds of the formula (A) having substitution at the alkene carbons with, e.g. an electron withdrawing group (e.g, Cl, F, Br, CF 3 , phenyl) or an electron donating group (e.g. CH 3 , alkoxy) ; and compounds of formula (A) wherein the carboxylic acid group is replaced with, e.gr_ atetrazolyl group, or esterified with, e.g., an imidazole.
  • an electron withdrawing group e.g, Cl, F, Br, CF 3 , phenyl
  • an electron donating group e.g. CH 3 ,
  • the invention includes protein tyrosine phosphatase modulating compounds having the formula (AA) :
  • compositions of the invention include compositions comprising such compounds (or pharmaceutically acceptable salts, esters, or solvates of the compounds) in admixture with a pharmaceutically acceptable dilusnt, adjuvent, or carrier.
  • Preferred protein tyrosine phosphatase activity- modulating aryl acrylic acid derivative compounds according to the invention have the following general structural formula (I) :
  • n 0 or 1
  • R j , R ⁇ and R 3 are independently selected from the group consisting of (i) hydrogen, C,. u alkyl,
  • aryl of (ii), (iii), (iv), (v) , and (vi) are independently selected from the group consisting of phenyl, naphthyl, pyridyl, furyl, pyrryl, thienyl, isothiazolyl, imidazolyl, benzimidazolyl, tetrazolyl, pyrazinyl, pyrimidyl, quinolyl, isoquinolyl, benzofuryl, benzothienyl, pyrazolyl, indolyl, benzodioxolyl, piperonyl, isoindolyl, puri ⁇ yl, carbazolyl, isoxazolyl, thiazolyl, oxazolyl, benzthiazolyl, and benzoxazolyl; and (c) wherein when R 3 of Formula (I) has the formula
  • R 2 may further be of the formula (B)
  • R 5 and Rg are independently selected from the group consisting of (b) (i) and (b) (ii) .
  • R 3 of Formula (I) has the formula
  • PTPase modulating compounds of Formula (II) are para- substituted cinnamic acid derivative compounds of the general formula (IIA)
  • alkyl includes branched or straight chain and cyclic (e.g., cycloalkyl) saturated aliphatic hydrocarbon groups having the specified number of carbon atoms, e.g., methyl (Me), ethyl (Et) , propyl (Pr) , butyl (Bu) , pentyl, hexyl, heptyl, octyl, nonyl, decyl, isopropyl (i-Pr) , isobutyl (i-Bu) , tert-butyl ( t-Bu), sec-butyl ( s-Bu), isopentyl, cyclohexyl, and the like.
  • alkyloxy or “alkoxy” represent an alkyl group as defined above having the indicated number of carbon atoms attached through an oxygen bridge , e.g., methoxy, ethoxy, propyloxy, and the like.
  • alkylthio and “alkylamino” represent an alkyl group as defined above having the indicated number of carbon atoms attached through a sulfur bridge or an amine bridge, respectively. The amine bridge may itself be substituted with an alkyl or aryl group.
  • the term “amino” is intended to include alkyl substituted amino groups.
  • amino alkyl (e.g., "amino C,. 6 alkyl) represents an amino group as defined above attached throtgh an alkyl group as defined above having the indicated number of carbon atoms, the amino group being attached to any carbon of the alkyl group.
  • alkylcarbonyl represents an alkyl group as defined above having the indicated number of carbon atoms attached through a carbonyl group.
  • alkylcarbonylamino represents an alkylcarbonyl group as defined above wherein the carbonyl is in turn attached through the nitrogen atom of an amino group as defined above.
  • alkylaminocarbonyl represents an alkyl group as defined above having the indicated number of carbon atoms attached through the nitrogen atom of an amino group as defined above, the nitrogen atom in turn being attached through a carbonyl group.
  • carboxy alkyl e.g., “carboxy C, 6 alkyl”
  • carboxy group represents a carboxy group attached through an alkyl group as defined above having the indicated number of carbon atoms, the carboxy group being attached to any carbon of the alkyl group.
  • aryl includes the indicated monocyclic polycyclic, and heterocyclic aromatic groups covalently attached at any ring position capable of forming a stable covalent bond with the aryl group, certain preferred points of attachment being apparent to those skilled in the art (eg., 3- indolyl, 4-imidazolyl) .
  • alkyl aryl represents an alkyl group as defined above having the indicated number of carbon atoms attached through an aryl group as defined above, the alkyl group being attached at any position of the aryl group capable of forming a stable covalent bond with the alkyl group.
  • phenyl alkyl (e.g., "phenyl C j .g alkyloxy,” “phenyl C j . 6 alkylthio,” “phenyl C ⁇ _ 6 alkylamino”) represents a phenyl group attached through an alkyl group as defined above having the indicated number of carbon atoms, the phenyl group being attached to any carbon of the alkyl group (the alkyl group in turn attached through an oxygen bridge, a sulfur bridge, an amine bridge, etc.) .
  • Preferred protein tyrosine phosphatase activity modulating /inhibiting compounds of the invention are carboxyl substituted cinnamic acids wherein the carboxyl group ring substituent is derivatized to form an amide.
  • the amide linkage joins the carboxyl-substituted cinnamic acid moiety to a dipeptide or tripeptide moiety structurally resembling or identical to a dipeptide or tripeptide of naturally-occurring amino acids.
  • novel aryl acrylic acid compounds having phosphatase modulating activity as well as new phosphatase activity modulating uses for previously known aryl acrylic acid compounds.
  • compositions comprising PTPase modulating compounds of the invention suitable for administration to a mammalian host.
  • the invention further provides methods for making compounds of the present invention having PTPase- modulatory inhibitory activity.
  • compounds of the invention are synthesized in a multi- component combinatorial array, which permits rapid synthesis of numerous, structurally related compounds for subsequent evaluation.
  • the acrylic acid moiety of a compound is protected during synthesis by, e.g. esterification with a t-butyl protecting group.
  • a preferred method of making compounds of the invention comprises use of a protected acrylic acid reagent and removal of the protective group by, e.g., treatment of a precursor ester compound with acid.
  • such a method includes further esterifying or salifying the acrylic acid product thereby obtained.
  • FIGURE 1 depicts a phosphotyrosyl structure and a cinnamic acid derivative structure according to the present invention.
  • FIGURE 2 depicts the four-component Ugi Reaction synthesis of compounds of the present invention.
  • FIGURE 3 depicts the synthesis of aryl acrylate reagents, which reagents were utilized to synthesize PTPase activity- modulating compounds of the invention.
  • FIGURE 4 depicts the four component Ugi Reaction synthesis of compounds of the present invention, wherein a carboxylic acid t-butyl cinnamate reagent is employed.
  • FIGURE 5 depicts the inhibitory activity, expressed as % inhibition at lOmM concentrations, of tripeptide amide cinnamic acid derivatives of the invention. Individual tri ⁇ peptide amide compounds are depicted by the letter C (for p- carboxycinnamate) followed by a three-letter tri-peptide designation.
  • a phosphotyrosyl residue has a phenyl component (boxed) covalently linked to a phosphono component (circled) through an oxygen atom.
  • This oxygen atom is believed to be a locus of chemical interaction between a phosphotyrosyl residue and the catalytic domain(s) of a protein tyrosine phosphatase ⁇ n2yme.
  • an ar l acrylic acid derivative such as para-substituted cinnamic acid, has a phenyl component (boxed) covalently linked to a carboxylic acid component (circled) through an alkene group. It is believed that the phosphono and corresponding carboxylic acid component, the corresponding aromatic components, and the oxygen and corresponding alkene components of these moieties depicted in Fig.
  • aryl acrylic acid PTPase inhibitors were further characterized by assaying derivatives of such molecules for PTPase activity. Importantly, it was determined that esterification of the carboxylic acid, and /or reduction of the alkene bond, substantially eliminates PTPase inhibitory activity of these compounds. Equally importantly, it was determined that a measure of variability is permissible with respect to the aryl substituent of the PTPase inhibitors of the invention. Thus.
  • PTPase compounds of formula (I) are synthesized using a four-component combinatorial array Ugi Reaction synthesis as described in International Patent Application No. PCT /US94/08141 . filed July 15, 1994 and published January 26, 1995 (Publication No. WO 95/02566) . See also Ugi et d., “Multicomponent Reactions In Organic Chemistry," in Endeavor, New Series. Vol. 18, No. 3, Elsevier Science Ltd., Great Britain ( 1994) .
  • Each component in the multicomponent synthesis comprises a series of reagent compounds sharing one or more common functional groups, e.g., an amine, an aldehyde, etc.
  • the synthesis is conducted under conditions such that the common functional groups of the compounds of the various components react with one another to form product compounds having a common core structure.
  • the series of reagents that comprise each individual component are added to a plurality of reaction vessels "combinatorially" to form an array, such that each reaction vessel of the array contains a unique combination of individual reagents.
  • the reagent components are added under appropriate conditions such that a unique product, sharing the common core structure of interest, forms in each reaction vessel.
  • the total number of analogs synthesized is equal to the product of the number of structural variants of each component in the array.
  • the multicomponent combinatorial array synthesis provides a relatively simple and rapid means for synthesis of a library of numerous structural variants sharing a core structure of interest. More particularly, to synthesize compounds of formula (I), an Ugi Reaction as depicted in Fig. 2 is conducted, wherein the four reagent components of a given reaction are (a) an aldehyde of formula 3; (b) a commercially available Rink resin or other suitable solid support resin of formula 2; (c) an isocyanide of formula 4; and (d) an acid of formula ⁇ . In a first stage, the four components are reacted to yield a compound of formula 6.
  • the compound of Formula 6 is reacted with an acid, .g. 10% trifluoroacetic acid (TEA) in CH 2 C1 2 , to yield a compound of formula (I) .
  • an acid .g. 10% trifluoroacetic acid (TEA) in CH 2 C1 2
  • TAA trifluoroacetic acid
  • the compound is then esterified, salified, or otherwise formulated to form compositions as described herein.
  • the invention provides in vitro procedures for screening the compounds for PTPase inhibitory activity. The preferred screening procedure is rapid and can be performed with micrornolar quantities of the compounds of interest.
  • PTPase inhibition assays are described herein with respect to two particular PTPase molecules (PTP- IB and HePTP) , it is understood that numerous additional PTPase enzymes have been characterized, and that other PTPase molecules are expected to be identified and characterized.
  • the assays described herein and other in vitro and in vivo PTPase inhibition assays may be conducted with such other enzymes, to demonstrate the PTPase modulating properties of compounds of the invention.
  • the following examples further illustrate aspects of the invention.
  • Example 1 the synthesis of aryl acrylate reagents is described, which reagents are useful for Ugi Reaction synthesis of compounds of the invention.
  • Example 2 the use of such aryl acrylate reagents to synthesize cinnamic acid derivative compounds of the invention is described.
  • Examples 3 through 5 pertain to protocols for assaying compounds of the invention for PTPase modulating activity.
  • Example 3 describes the purification of a truncated PTP-I B fusion protein possessing PTPase activity, which fusion protein was employed to assay PTPase inhibitory activity of compounds of the invention.
  • Example 4 describes the purification of a second recombinant PTPase enzyme, HePTP, which also was employed to assay the PTPase modulating activity of compounds of the invention.
  • Example 5 describes with particularity the PTPase inhibition assays conducted with PTP- I B and HePTP.
  • Examples 6 through 8 describe the synthesis and characterization of additional cinnamic acid derivative compounds of the invention.
  • Example 9 describes the synthesis and characterization of a number of furylacrylic acid derivative compounds of the invention.
  • Example 10 describes a procedure whereby the
  • Example 1 1 describes a library of 125 cinnamic acid derivative compounds synthesized using known protocols.
  • the library comprises a family of cinnamic acid derivatives having tripeptide amide substituents of considerable functional variation.
  • small compound libraries designed in this manner and screened for PTPase modulating activity against a selected enzyme provide useful information for designing PTPase modulating compounds with enhanced activity against the selected enzyme.
  • Example 12 describes a whole cell growth- inhibition assay in which compounds of the invention demonstrated cell growth inhibitory activity.
  • n and X are defined as described above for formula (IR).
  • the R 3 substituent was provided using aryl acrylate reagents, which reagents were synthesized as follows. Referring to Fig. 3, a mixture of approx. 10 mmol of an aryl bromide compound of general formula ( 1) , 12.5 mmol of t-butyl acrylate, 20 mmol of dry triethylamine, 0.1 mmol of palladium acetate, and 0.4 mmol of tri-o-tofylphosphine is prepared in a flask.
  • the flask is blown out with argon and heated at 100 ° C. This reaction may be monitored using thin layer chromotography, which rereads that, after about 2 hours, all of the aryl bromide compound has reacted, forming a solid product.
  • the solid product is extracted with EtOAc and water and then adjusted to pH 3 using 1 N HCl.
  • the organic phase is filtered, dried over sodium sulfate, and further dried by evaporation.
  • the reaction product of general formula (2) is hydrolized to the desired acid reagent of general formula (3) , using 0.5 M LiOH in a 1:1 mixture of 1 ,4- dioxanerwater.
  • the reagent of general formula (3) is dried and its identity confirmed by NMR and mass spectrometry.
  • the dried reagent of general formula (3) is suitable for use in a carboxylic acid reagent in an Ugi Reaction synthesis as described herein. Exemplary NMR and mass spectrometry data is provided below for selected compounds:
  • Cinnamic Acid Derivative Synthesis and Initial Screening for PTPase Inhibitory Activity Cinnamic acid derivative compounds were synthesized in a combinatorial Ugi Reaction and initially screened for protein tyrosine phosphatase modulating activity as set forth below.
  • the four reagent components of a given reaction are (a) an aldehyde of formula
  • a 0.5 M solution of each component is prepared, e. _ by dissolving 0.05 mMoles of the component in 100 ml of an appropriate preferred solvent set as forth in Table 1 :
  • Ortho-, meta-, and para- carboxylic acid t-butyl cinnamate was synthesized as described in the previous example to provide the acid reagents of Formula 5.
  • the multi-component combinatorial synthesis reactions were performed using Ontogen (Carlsbad CA) 96-well reaction plates (2.0 ml/well capacity) and the multisynthesizer described in co-owned, co-pending U.S. Patent Application Serial No. 08/422,869 (Attorney Docket No. 8140-008-999) , filed April 18, 1995 (John Cargill and Romaine R. Maiefsky, inventors) entitled "Methods and Apparatus for the Generation of Chemical Libraries.”
  • Distribution of aldehydes, isocyanides, and acids in "combinatorial fashion” involves distribution of distinct aldehydes (e. _ having different R, groups) , distinct isocyanides (e.g_ having different RJ J groups) and distinct acids (e.g- ortho-, met , or para substituted) in a three-dimensional array on a series of plates, such that a unique compound is synthesized in each well of each plate. For example, eight distinct aldehydes, ten distinct isocyanides, and three distinct acid components maybe combined combinatorialiy on three plates (80 wells/plate) to produce at least 240 distinct compounds (ignoring, e.g., stereochemistry) within the genus of Formula II .
  • each well contains a compound covalently linked to the rink resin as depicted in Formulae (Fig. 4) .
  • the Rink resin polymer and t-b ⁇ iyl protective group were removed by treatment with dry acid.
  • a solution of 10% triflouroacetic acid (TFA) in DCM (600 ul) was added to each well and the plates were shaken for twenty minutes on the orbital shaker.
  • the solvent in the wells (in which the compounds of interest were dissolved) was then drained and the wells were rinsed again with the TFA solution (300 ul) .
  • the TFA solution was then removed by evaporation in a vacuum oven to provide one compound of Formula II per reaction (approx. 0.025 mmol compound per reaction) .
  • reaction products from the combinatorial synthesis were resuspended in DMSO and screened for PTPase inhibitory activity using the in vitro PTPase inhibition assays described in Example 5, below. More particularly, based on an assumption that each synthesis yielded approximately 0.025 mmol of the desired reaction product, the compounds were screened for PTP-IB or HePTP inhibitory activity at an effective concentration of 10 mM. Compounds exhibiting at least about 50 percent inhibition against at least one of these enzymes were selected for further analysis.
  • PTP-IB Gene Cloning and Protein Purification The following procedure was conducted for recombinant production and purification of protein tyrosine phosphatase PTP-IB, for use as a substrate in PTPase inhibition assays.
  • a h nan placental cDNA library was synthesized in a 50 ml reaction containing 1 mg human placental poly(A) + mRNA (Clontech, Palo Alto, CA) , 4 ml random hexamer primers, 8 ml of lOmM dNTPs (Pharmacia, Piscataway N J) , l ml (200 U/ml) Moloney murine leukemia virus reverse transcript ⁇ ee (Gibco-BRL Canada) , 05 ml (26 U/ml) RNAsin (Promega, Madison WI) , and 12 ml 5x buffer (Gibco-BRL) .
  • the synthesis reaction was incubated at 37 ° C for one hour and then heat inactivated at 95 ° C for five minutes.
  • a PTP- IB cDNA was amplified, using polym erase chain reaction (PCR) , from the cDN As synthesized as described above. More particularly, based on the published sequence of PTB-IB, two PCR primers were synthesized to amplify a portion of the PTP-IB coding sequence known to encode a 321 amino acid fragment containing the PTP- IB catalytic domain and having PTPase activity. See Hoppe et d., Eur. J. Biochem., 223:1069-77 ( 1994) ; Barford, D., et d., J. Molec. Biol, 239.726-730 ( 1994) ; Chernoff et d., Proc.
  • PCR polym erase chain reaction
  • PTP-1B-A(5') (SEQ ID NQ 1) 5' 0_ _ACTGGAT(XTCATGGAGATGGAAAAGG 3'
  • the first primer which hybridizes to the non-coding strand, corresponds to the 5' portion of the PTP- IB coding sequence and encodes a BamH I restriction site upstream of the initiation codon, to facilitate cloning.
  • the second primer which hybridizes to the coding strand, corresponds to the 3' portion of the PTB- IB fragment of interest, and encodes a stop codon and an EcoR I restriction site downstream from the stop codon.
  • the PCR reaction product (992 bp) was digested with B ⁇ mH I and EcoR I (New England Biolabs, Beverly MA) to yield a 975 bp product encoding the 321 amino acid PTP-IB protein fragment, and having "sticky ends" to facilitate cloning.
  • the 975 bp PTP- IB partial cDNA was purified by agarose gel electrophoresis and ligated into a B ⁇ mH I/EcoR I- digested pGEX-3X plasm id vector (Pharmacia, Piscataway, NJ) .
  • the pGEX vector is designed to produce a fusion of glutathione-S-transferase (GST) to a protein encoded by another DNA fragment inserted into the vector's cloning site.
  • GST glutathione-S-transferase
  • E. coli strain DH5a (Gibco-BRL) was transformed with plasmid pGEX-3X-PTP- 1 B following the supplier s transformation protocol and grown at 37 ° C with vigorous shaking in Luria-Bertani broth supplemented with 100 mg/ml ampicillin. When the cultures reached an O .go o of 0.7- 1 , production of the GST /PTP- I B fusion protein was induced with 0.1 mM IPTG dsopropyl b-D-Thiogalactoside) . After 3 additional hours of culturing at 37 ° C, the bacteria were pelleted by centrifugation
  • the bacterial pellet was resuspended in l Ox (w/v) lysis buffer consisting of 12.5 mM HEPES, 2 mM EDTA pH 7.0, 15 mM b-mercaptoethanol (bme) and 1 mM PMSF.
  • the lysate was sonicated (on ice) until slight clearing was observed (approx. three min.) and then centrifuged at 10,000 revolutions per minute (RPM) for 10 min.
  • the supernatant was diluted 1 :4 with buffer A (25 mM HEPES, pH 7.0, and 15 mM bme).
  • Fractions containing PTPase activity were pooled, diluted 1 :4 with NET buffer (20 mM Tris, pH 8.8, 100 mM NaCl, 1 mM EDTA and 15 M bme) , and loaded onto a 10 ml GST-Sepharose 4B column (Pharmacia) . After loading, the column was washed first with 3 bed volumes of NET buffer + 1% NP40 (Sigma Chemical Co., St. Louis, MO) , then with NET buffer until O.D. at 280 nm was basal. The GST /PTP- 1 B fusion protein was eluted from the column using 10 mM glutathione in 33 mM Tris, pH 8.8.
  • NET buffer 20 mM Tris, pH 8.8, 100 mM NaCl, 1 mM EDTA and 15 M bme
  • GST-Sepharose 4B purification were pooled , concentrated into a final storage buffer (0.2 M NaCl, 25 mM HEPES, 1 mM EDTA, and 5 mM DTT, pH 7.0) using a 1 ml Hi-Trap Q column (pre ⁇ packed, Pharmacia) , and stored at -80C (final concentration of 0.52 mg/ml).
  • the foregoing procedure yielded approximately 5mg of PTP-IB fusion protein per 500 ml of cultured cells, purified to substantial homogeneity as assessed by SDS-PAGE.
  • the protein concentration of the PTP-I B enzyme preparation was determined using the Bio-Rad Protein Assay kit (Bio-Rad, Hercules CA) . An aliquot from each sample was taken and diluted to 2 mg protein/ml using activity assay buffer ( 100 mM Sodium Acetate, pH 6.0, 1 mM EDTA, 0.1%o TX-100 (International Biotechnologies, Inc.) and 15 mM bME) to form a PTP-IB stock solution.
  • activity assay buffer 100 mM Sodium Acetate, pH 6.0, 1 mM EDTA, 0.1%o TX-100 (International Biotechnologies, Inc.) and 15 mM bME
  • a 100 ml reaction mixture was prepared containing 10 ml of the PTP- 1 B stock solution, 10 ml of 9 mM p-nitrophenylphosphate ((pNPP) , Sigma Chemical Co.. St. Louis MO) , and 80 ml of activity assay buffer. Reactions were mixed gently and incubated at 37 ° C for 65 minutes. Enzymatic cleavage of phosphate from pNPP (a tyrosine phosphate analog) is marked by a colorimetric change in this substrate. See, e.gu Imbert et d., Biochem J., 297:163-173 ( 1994) ; Ghosh and Miller, Biochem. Biophys. Res. Comm., 194:36-44 ( 1993) ; Zanke et d., Eur. J. Immunol, 22.235-39 ( 1992) .
  • pNPP 9 mM p-nitrophenylphosphate
  • HePTP Gene Cloning and Protein Purification The following procedure was conducted to recombinantiy produce and purify protein tyrosine phosphatase HePTP, for use as a substrate in PTPase inhibition assays.
  • HePTP cDNA is produced as described in the preceding example, using, e.g., 100 mg poly(A) + RNA and the primers described below for PCR amplification.
  • a HePTP cDNA isolated by these or other means is used to recombinantiy produce HePTP as described herein.
  • Zanke was amplified using PCR. Two PCR primers were synthesized to amplify the HePTP coding sequence, based on the published DNA sequence of this gene. Zanke et d. ( 1992) , supra: NDE2760.3(N 3 ) (SEQ ID NO: 3)
  • BAM2760.2(B,) (SEQ ID NO: 4) 5' TTATGGAT(XAGGGTGGCAGGGGTCAGG 3'
  • the first primer which hybridizes to the non-coding strand, corresponds to the 5' portion of the HePTP coding sequence and encodes a Nde I restriction site upstream of the initiation codon, to facilitate cloning.
  • the second primer which hybridizes to the coding strand, corresponds to the 3' portion of the HePTP coding sequence and encodes a B ⁇ mH I restriction site downstream from the HePTP termination codon.
  • the resultant 1 1 15 base pair PCR product was digested with BamH I and Nde I (New England Holabs), gel- purified, and ligated into a B ⁇ mH l/Nde I - digested pET12a expression vector (Novagen) .
  • BamH I and Nde I New England Holabs
  • Nde I New England Holabs
  • ligated into a B ⁇ mH l/Nde I - digested pET12a expression vector Novagen
  • E. coli strain BL21 (DE3) (Novagen, Madison WI) was transformed with plasmid pET 12a-HePTP and grown at 37 ° C with vigorous shaking in Luria-Bertani broth supplemented with 100 mg/ml ampicillin. When the cultures reached an O.O. W0 of 0.7- 1 , production of HePTP protein was induced with 0.1 mM IPTG. After 3 additional hours of culturing at 37 ° C, the bacteria were pelleted. The bacterial pellet was resuspended in l Ox (w/v) lysis buffer consisting of 12.5 mM HEPES, 2 mM EDTA pH 7.0, 15 mM bme, and 1 mM PMSF.
  • the lysate was sonicated (on ice) until slight clearing was observed (approx three minutes) and then centrifuged at 10,000 RPM for 10 min.
  • the supernatant was diluted 1 :4 with buffer A (25 mM HEPES, pH 7.0, and 15 mM bme) .
  • Fractions containing PTPase activity were pooled, diluted 1 :4 with TB buffer (20 mM Tris, pH 8, and 15 mM bme), and loaded into a 10 ml Q-Sepharose column (Pharmacia) for further purification. After loading, the column was washed with 3 bed volumes of TB buffer. HePTP protein was eluted from the column using a linear gradient of Buffer A and Buffer B as previously described, assaying fractions for protein content and PTPase activity as previously described.
  • HePTP-conta ning fractions were pooled, concentrated into a final storage buffer (0.2 M Nad, 25 mM HEPES, 1 mM EDTA, and 5 mM DTT, pH 7.0) using a 1 ml Hi-Trap Q column (pre ⁇ packed, Pharmacia) , and stored at -80 ° C (final concentration 3.127 mg/ml) .
  • the procedure yielded approx. 5 mg HePTP protein per 2 liters of culture.
  • HePTP enzymatic activity was assayed as described above for PTP- IB, except that 20 mM pNPP was substituted for the 9 mM pNPP employed in the PTP- IB reactions.
  • 0.001 mmol of the cinnamic acid derivative (or other PTPase inhibitor compound) was dissolved in 100 ul of DMSO to create a 10 mM stock solution.
  • the 10 mM stock solution was used to add varying concentrations ( 100 uM, 33 uM, 10 uM, 3 uM, 1 uM, 0.3 uM , 0.1 uM, 0.03 uM , 0.01 uM or 0.003 uM) of the inhibitor compound to a series of otherwise identical PTPase activity assay reactions ( 100 ul final volume in microtiter wells).
  • each 100 ul reaction contained 10 ul PTPase enzyme stock solution (final PTP- I B or HePTP concentration of 20 ng/well) , 70 ul activity assay buffer, 10 ul pNPP stock solution (final pNPP concentration of .9 mM for F P- IB assay and 2 mM for HePTP assay) , and 10 ul of the diluted inhibitor compound in DMSO.
  • the GST/PTP- l B or HePTP enzyme was added to the reaction mixtures to begin the reactions, which were incubated at 37 ° C for 65 min., stopped, and colorimetrically analyzed as described above.
  • reactions were performed containing 10 ul DMSO with no inhibitor compound, or containing the known PTPase inhibitor vanadate (final concentration .5 mM) substituted for the inhibitor compound of the invention.
  • the concentration of inhibitor compound required to inhibit 50% of the PTPase activity was determined as follows. First, absorbance readings from the vanadate control reactions were treated as a baseline and subtracted from the absorbance readings of the experimental reactions. Then, for each reaction, a percent inhibition was calculated using the following formula
  • IC50 concentration was calculated from a best-fit computer analysis of the calculated percent inhibition for the various dilutions of the compound.
  • Inhibitor compounds having an IC50 less than 10 uM (an optimally less than 5uM) for a particular PTPase were scored as highly effective inhibitors of that PTPase enzyme, and are preferred inhibitors of the present invention.
  • Table 2 reports the IC50 concentration for a number of cinnamic acid derivative compounds of Formula II (Hg. 4) of the present invention.
  • Example 6
  • Example 7 Seven of the eight commercially available isocyanide reagents described in Example 2 were employed ( 1 , 1 ,3,3- tetramethylbutyl isocyanide was not selected) . Meta- and para- carboxylic acid t-butyl cinnamate were synthesized as described in Example 1 to provide the acid reagent of Formula 5. The synthesized compounds of Formulall were screened for PTP-IB or HePTP inhibitory activity at an effective concentration of 10 mM, as described in Example 2. Since none of these compounds exhibited substantial PTPase inhibitory activity against these two enzymes in this initial screening, the reaction products were not further characterized. Example 7
  • the synthesized compounds were screened for HePTP inhibitory activity at an effective concentration of 10 mM, as described in Example 2, and a number of the compounds exhibited substantial HePTP inhibitory activity.
  • At least one compound was selected for further analysis, re synthesized, and confirmed by mass spectrometry and NMR:
  • HePTPase inhibitory activity for this compound was determined as described in Example 5, and is reported in
  • Example 2 except that the following furylacrylic acid derivative, synthesized as described in Example 1 , was employed as the carboxylic acid reagent:
  • the synthesized compounds were screened at 5.0 mM concentrations for PTP- 1 B or HePTP inhibitory activity as described previously.
  • the results of the screening assay, set forth in Table 3, provide an indication that furylacrylic acid derivatives are effective PTPase inhibitor compounds.
  • X and n are defined as in formula (A) , supra wherein m is an integer from 0 to 4, and wherein R', R" and R'" represent hydrogen(s) and/or one or more independently selected appropriate substituents (eg, substituents comprising electron withdrawing and/or donating groups, polar groups, cationic and/or anionic groups, acidic and/or basic groups, hydrophilic and/or hydrophobic groups, hydrogen bond donors or acceptors, aromatic groups, etc.) of the aromatic ring (pyridyl, furyl, phenyl, thiophenyl) or alkene carbons selected to optimize the potency, selectivity, specificity, etc. of such inhibitor compounds towards different PTPase enzymes. See generally, Roberts and Price, The Role of Organic Chemistry in Drug Research, Academic Press ( 1985) : Silverman, R.B., The Organic Chemistry of Drug Design and Drug Action. Academic Press, 1992.
  • substituents comprising electron withdrawing and/or donating groups, polar groups,
  • Tripeptide Amide Cinnamic Acid Derivatives A library of tripeptide amide cinnamic acid derivatives was synthesized and assa ⁇ d for FTPase inhibitory activity.
  • the library compounds are oi the formula (AA) , supra wherein one R' substituent is a tripeptide amide and the remaining R', R", and R"' are hydrogens.
  • the library compounds are of the formula (I) , supra wherein R 3 of formula (I) has the formula (IR) and R. and R, are independently- selected as described, supra Amino acid substituents of the tripeptide amide were selected to create a library of compounds having a variety of side chain functionality.
  • leucine (L) was selected for lypophilicity, tyrosine (Y) for aromatic structure, glutamate (E) for anionic charge, lysine (K) for cationic character, and glycine ( G) for conformational flexibility.
  • the library contained all 125 unique tripeptide amide compounds that could be synthesized using the five selected amino acids.
  • the 125 compound library was assayed for PTP- I B inhibitory activity as described herein, using 10 mM final concentrations of each tripeptide amide compound, and the compounds were rank-ordered based upon their observed inhibitory activity.
  • Fig. 5 depicts the percent inhibition of the twelve best and worst PTB- IB inhibitors of the library. The most potent inhibition of PTP- IB was observed for the compounds Qnn-GEL (96%) and Cinn-GEE (95%) , the structures of which are depicted below:
  • MLR Cell Growth Inhibition Assay The immunomodulatory properties of compounds of the present invention were demonstrated iBing the Mixed Lymphocyte Reaction (MLR) Assay.
  • MLR Mixed Lymphocyte Reaction
  • Spleens were removed from Balb C and C57BL6 mice, two distinct, inbred strains. In a tissue culture hood, the spleens were gently mashed through a fine screen into Hanks Balanced Saline Solution (Bio Whittaker 10-5088) media in a 10 cm petri dish. The red blood cells were lysed from the preparations by hypotonic shock, and then 10X concentrated PBS solution was added to restore osmolarity, followed by 4 ml of Hank's Balanced Saline Solution (BSS) . The cell preparations were then pipetted up and down; dead red cells were allowed to stick to the side of the pipette. Cells were then washed twice in BSS.
  • BSS Hank's Balanced Saline Solution
  • Balb C cells for background control
  • all C57BL6 cells were irradiated with 2000 rads, to extinguish cell proliferation.
  • These damaged cells (the stimulator cells) and the non-irradiated Balb C cells (the responder cells) were each resuspended in Iscove's media with 10% fetal calf serun, 1% L-glutamine, 10 mM bme and 100 mg/ml each of penicillin/streptomycin.
  • MLR reactions final volume - 150 ml
  • PTPase modulating compounds of the present invention have asymmetric centers and may occur as racemates, racemic mixtures, and as individual enantiomers or diastereoisomers, with all isomeric forms being included in the present invention as well as mixtures thereof.
  • the invention includes within its scope pharmaceutically acceptable salts, particularly where a basic or acidic group is present in a compound according to invention therein.
  • pharmaceutically acceptable salts particularly where a basic or acidic group is present in a compound according to invention therein.
  • an acidic substituent such as - COOH
  • ammonium, sodium, potassium, calcium and the like salts are contemplated as preferred embodiments for administration to a biological host.
  • an acidic salt such as hydrochloride, hydrobromide, acetate, maleate, pamoate, phosphate, methanesulfonate, p-toluenesulfonate, and the like, is contemplated as a preferred form for administration to a biological host.
  • esters of the compound e.g., methyl, tert-butyl, pivaloyloxym ethyl, succinyl, and the like
  • esters being known in the art for modifying solubility and/or hydrolysis characteristics for use as sustained release or prodrug formulations.
  • some of the phosphatase modulating compounds of the instant invention may form solvates with water or common organic solvents. Such solvates are encompassed within the scope of the invention.
  • compositions comprising phosphatase modulating compounds of the invention.
  • pharmaceutical composition is meant a composition that maybe administered to a mammalian host, e.g. orally, topically, parenterally, by inhalation spray, or rectally, in unit dosage formulations containing conventional non-toxic carriers, diluents, adjuvants, vehicles and the like.
  • parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intracistemal injection or infusion techniques.
  • compositions containing a PTPase modiiating compound maybe in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs.
  • Compositions intended for oral use maybe prepared according to any known method, and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets may contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.
  • excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia and lubricating agents, for example 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.
  • a time delay material such as glyceryl monostearate or glyceryl distearate maybe employed. They may also be coated by the techniques described in the US. Patents 4,256, 108; 4, 166,452; and 4,265,874 to form osmotic therapeutic tablets for controlled release.
  • Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelating capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil.
  • Aqueous suspensions may contain the active compounds in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients are suspending agents, for example sodium carboxymethylcellulose, methyl cellulose, hydroxy- propylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents maybe a naturally-occurring phosphatide, for example lecithin.
  • condensation products of an alkylene oxide with fatty acids for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyl-eneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate.
  • the aqueous suspensions may also contain 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.
  • preservatives for example ethyl, or n-propyl, p-hydroxybenzoate
  • coloring agents for example ethyl, or n-propyl, p-hydroxybenzoate
  • coloring agents for example ethyl, or n-propyl, p-hydroxybenzoate
  • flavoring agents for example ethyl, or n-propyl, p-hydroxybenzoate
  • sweetening agents such as sucrose or saccharin.
  • Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.
  • the oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents maybe added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active compound in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives.
  • a dispersing or wetting agent e.g., glycerol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerin, glycerin, glycerin, glycerin, glycerin, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerol
  • the pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions.
  • the oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these.
  • Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally- occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate.
  • the emulsions may also contain sweetening and flavoring agents.
  • Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose.
  • Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents.
  • the pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable dilusnt or solvent, for example as asolution in 1 ,3-butane diol.
  • acceptable vehicles and solvents that maybe employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or dig cerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • the compositions may also be in the form of suppositories for rectal administration of the PTPase modulating compound.
  • compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • suitable non-irritating excipient are cocoa butter and polyethylene glycols, for example.
  • topical application creams, ointments, jellies, solutions of suspensions, etc., containing the PTPase modulating compounds are contemplated.
  • topical application shall include mouth washes and gargles.

Abstract

La présente invention concerne de nouveaux composés modulant la protéine-tyrosine-phosphatase, lesquels composés présentent une structure d'acide aryl-acrylique. L'invention concerne également des compositions comprenant ces composés, ainsi que des procédés de fabrication et d'utilisation de ces composés.
PCT/US1996/018401 1995-06-19 1996-06-19 Derives d'acide aryl-acrylique convenant comme inhibiteurs de proteine-tyrosine-phosphatase WO1997008934A2 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP96940489A EP0833629A4 (fr) 1995-06-19 1996-06-19 Derives d'acide aryl-acrylique convenant comme inhibiteurs de proteine-tyrosine-phosphatase
JP9511473A JPH11508919A (ja) 1995-06-19 1996-06-19 蛋白質チロシンホスファターゼ阻害剤として有用なアリールアクリル酸誘導体
AU77358/96A AU713863B2 (en) 1995-06-19 1996-06-19 Aryl acrylic acid derivatives useful as protein tyrosine phosphatase inhibitors

Applications Claiming Priority (4)

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US49226495A 1995-06-19 1995-06-19
US08/543,630 1995-10-16
US08/492,264 1995-10-16
US08/543,630 US5770620A (en) 1995-06-19 1995-10-16 Aryl acrylic acid derivatives useful as protein tyrosine phosphatase inhibitors

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WO1997008934A2 true WO1997008934A2 (fr) 1997-03-13
WO1997008934A3 WO1997008934A3 (fr) 1997-04-24

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WO1997039748A1 (fr) * 1996-04-19 1997-10-30 Novo Nordisk A/S Modulateurs de molecules possedant des unites de reconnaissance de la phosphotyrosine
WO1999047497A2 (fr) * 1998-03-13 1999-09-23 Merck Frosst Canada & Co. Acides carboxyliques et acylsulfonamides, compositions contenant ces composes et methodes de traitement
WO1999061410A1 (fr) * 1998-05-12 1999-12-02 American Home Products Corporation Diphenyles 2,3,5-substitues utiles pour le traitement de la resistance insulinique et de l'hyperglycemie
US6043247A (en) * 1996-04-19 2000-03-28 Novo Nordisk A/S Modulators of molecules with phosphotyrosine recognition units
US6242493B1 (en) 1998-03-13 2001-06-05 Merck Frosst Canada & Co. Carboxylic acids and acylsulfonamides, compositions containing such compounds and methods of treatment
US6451827B2 (en) 1998-05-12 2002-09-17 Wyeth 2,3,5-substituted biphenyls useful in the treatment of insulin resistance and hyperglycemia
US6498182B2 (en) 2000-09-26 2002-12-24 Biovitrum Ab Compounds
US6613903B2 (en) 2000-07-07 2003-09-02 Novo Nordisk A/S Modulators of protein tyrosine phosphatases (PTPases)
US7230133B2 (en) 2003-05-01 2007-06-12 Bristol-Myers Squibb Company Malonamides and malonamide derivatives as modulators of chemokine receptor activity

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CA2469228A1 (fr) 2001-12-03 2003-06-12 Japan Tobacco Inc. Compose azole et utilisation medicinale de celui-ci
JP4822312B2 (ja) * 2005-05-13 2011-11-24 学校法人慶應義塾 ナフトキノン誘導体化合物

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997039748A1 (fr) * 1996-04-19 1997-10-30 Novo Nordisk A/S Modulateurs de molecules possedant des unites de reconnaissance de la phosphotyrosine
US6043247A (en) * 1996-04-19 2000-03-28 Novo Nordisk A/S Modulators of molecules with phosphotyrosine recognition units
US6242493B1 (en) 1998-03-13 2001-06-05 Merck Frosst Canada & Co. Carboxylic acids and acylsulfonamides, compositions containing such compounds and methods of treatment
WO1999047497A2 (fr) * 1998-03-13 1999-09-23 Merck Frosst Canada & Co. Acides carboxyliques et acylsulfonamides, compositions contenant ces composes et methodes de traitement
WO1999047497A3 (fr) * 1998-03-13 1999-10-28 Merck Frosst Canada Inc Acides carboxyliques et acylsulfonamides, compositions contenant ces composes et methodes de traitement
WO1999061410A1 (fr) * 1998-05-12 1999-12-02 American Home Products Corporation Diphenyles 2,3,5-substitues utiles pour le traitement de la resistance insulinique et de l'hyperglycemie
US6214877B1 (en) 1998-05-12 2001-04-10 John A. Butera 2,3,5-substituted biphenyls useful in the treatment of insulin resistance and hyperglycemia
US6451827B2 (en) 1998-05-12 2002-09-17 Wyeth 2,3,5-substituted biphenyls useful in the treatment of insulin resistance and hyperglycemia
US6765021B2 (en) 1998-05-12 2004-07-20 Wyeth 2,3,5-substituted biphenyls useful in the treatment of insulin resistance and hyperglycemia
US7008636B2 (en) 1998-05-12 2006-03-07 Wyeth 2,3,5-substituted biphenyls useful in the treatment of insulin resistance and hyperglycemia
US6613903B2 (en) 2000-07-07 2003-09-02 Novo Nordisk A/S Modulators of protein tyrosine phosphatases (PTPases)
US6498182B2 (en) 2000-09-26 2002-12-24 Biovitrum Ab Compounds
US7230133B2 (en) 2003-05-01 2007-06-12 Bristol-Myers Squibb Company Malonamides and malonamide derivatives as modulators of chemokine receptor activity
US7468440B2 (en) 2003-05-01 2008-12-23 Bristol-Myers Squibb Company Malonamides and malonamide derivatives as modulators of chemokine receptor activity

Also Published As

Publication number Publication date
AU713863B2 (en) 1999-12-09
CA2224874A1 (fr) 1997-03-13
JPH11508919A (ja) 1999-08-03
EP0833629A4 (fr) 1998-09-16
WO1997008934A3 (fr) 1997-04-24
EP0833629A2 (fr) 1998-04-08
AU7735896A (en) 1997-03-27

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