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Definitions

• the present invention relates to a method of inhibiting mitogen- activated protein kinase-activated protein kinase-2 (MAPKAP kinase-2, or MK-2) in a subject in need of such inhibition, and also to the prevention and treatment of TNF ⁇ mediated diseases or disorders by the administration of an MK-2 inhibitor.
• mitogen- activated protein kinase-activated protein kinase-2 mitogen- activated protein kinase-activated protein kinase-2
• MK-2 mitogen- activated protein kinase-activated protein kinase-2
• MAPKs Mitogen -activated protein kinases
• MAPKs are members of conserved signal transduction pathways that activate transcription factors, translation factors and other target molecules in response to a variety of extracellular signals.
• MAPKs are activated by phosphorylation at a dual phosphorylation motif with the sequence Thr-X-Tyr by mitogen-activated protein kinase kinases (MAPKKs).
• MAPKKs mitogen-activated protein kinase kinases
• the physiological role of MAPK signaling has been correlated with cellular events such as proliferation, oncogenesis, development and differentiation. Accordingly, the ability to regulate signal transduction via these pathways could lead to the development of treatments and preventive therapies for human diseases associated with MAPK signaling, such as inflammatory diseases, autoimmune diseases and cancer.
• the p38 MAPK pathway is potentially activated by a wide variety of stresses and cellular insults. These stresses and cellular insults include heat shock, UV irradiation, inflammatory cytokines (such as TNF and IL-1 ), tunicamycin, chemotherapeutic drugs (i.e., cisplatinum), anisomycin, sorbitol/hyperosmolarity, gamma irradiation, sodium arsenite, and ischaemia. See, Ono, K., et al, Cellular Signalling 12, 1 - 13 (2000).
• stresses and cellular insults include heat shock, UV irradiation, inflammatory cytokines (such as TNF and IL-1 ), tunicamycin, chemotherapeutic drugs (i.e., cisplatinum), anisomycin, sorbitol/hyperosmolarity, gamma irradiation, sodium arsenite, and ischaemia. See, Ono, K., e
• Activation of the p38 pathway is involved in (1) production of proinflammatory cytokines, such as TNF- ⁇ ; (2) induction of enzymes, such as Cox-2; (3) expression of an intracellular enzyme, such as iNOS, which plays an important role in the regulation of oxidation; (4) induction of adherent proteins, such as VCAM-1 and many other inflammatory-related molecules.
• proinflammatory cytokines such as TNF- ⁇
• enzymes such as Cox-2
• iNOS an intracellular enzyme
• adherent proteins such as VCAM-1 and many other inflammatory-related molecules.
• adherent proteins such as VCAM-1 and many other inflammatory-related molecules.
• the p38 kinase is an upstream kinase of mitogen-activated protein kinase-activated protein kinase-2 (MAPKAP kinase-2 or MK-2).
• MK-2 is a protein that appears to be predominantly regulated by p38 in cells.
• MK-2 was the first substrate of p38 ⁇ to be identified.
• MK-2 in vitro phosphorylation of MK-2 by p38 ⁇ activates MK-2.
• LAP1 lymphocyte-specific protein 1
• CREB cAMP response element-binding protein
• SRF serum response factor
• tyrosine hydroxylase tyrosine hydroxylase.
• the substrate of MK-2 that has been best characterized is small heat shock protein 27 (hsp27).
• the role of the p38 pathway in inflammatory-related diseases has been studied in several animal models.
• the pyridinyl imidazole compound SB203580 has been shown to be a specific inhibitor of p38 in vivo, and also has been shown to inhibit activation of MK-2, (See, Rouse, J., et al, Cell, 78:1027-1037 (1994); Cuenda, A., et al, Biochem. J.,
• SB 220025 Another p38 inhibitor that has been utilized in an animal model that is believed to be more potent than SB203580 in its inhibitory effect on p38 is SB 220025.
• a recent animal study has demonstrated that SB 220025 caused a significant dose-dependent decrease in vascular density of granulomas in laboratory rats. (See Jackson, J. R., et al, J. Pharmacol. Exp. Then, 284:687 - 692 (1998)).
• MK-2 Due to its integral role in the p38 signaling pathway, MK-2 has been used as a monitor for measuring the level of activation in the pathway. Because of its downstream location in the pathway, relative to p38, MK-2 has been measured as a more convenient, albeit indirect, method of assessing p38 activation. However, so far, research efforts exploring therapeutic strategies associated with the modulation of this pathway have focused mainly on the inhibition of p38 kinase. [0009] Several compounds that inhibit the activity of p38 kinase have been described in U.S. Patent Nos. 6,046,208, 6,251 ,914, and 6,335,340.
• MK-2-deficient mice showed increased susceptibility to Listeria monocytogenes infection, and concluded that MK-2 had an essential role in host defense against intracellular bacteria, probably via regulation of TNF and IFN-gamma production required for activation of antibacterial effector mechanisms.
• the location of MK-2 in the p38 signaling pathway at a point that is downstream of p38 offers the potential that MK-2 could act as a focal point for modulating the pathway without affecting as many substrates as would the regulation of an enzyme further upstream in the signaling cascade ⁇ such as p38 MAP kinase.
• the present invention is directed to a novel method of inhibiting mitogen activated protein kinase-activated protein kinase-2 in a subject in need of such inhibition, the method comprising administering to the subject an anminocyanopyridine MK-2 inhibiting compound, or a pharmaceutically acceptable salt thereof, the compound having the structure:
• R is selected from the group consisting of -H, C ⁇ -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, carboxy C C 4 alkyl, aryl C C 4 alkyl, amino, amino
• R 2 is selected from the group consisting of -H, C C ⁇ alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, amino, amino C 1 -C 4 alkyl, C 1 -C 4 alkylamino, aryl, heteroaryl, heterocyclyl, carboxy, carboxy C 1 -C 4 alkyl, C 1 -C 4 alkoxy, hydroxy, hydroxy C 1 -C4 alkyl, hydroxy C 1 -C 4 alkylamino, hydroxy C 1 -C 4 alkoxy, C1-C 4 alkoxy C1-C4 alkyl, C 1 -C 4 alkoxy C C 4 alkylamino, amino C C alkylamino, aryl C1-C4 alkyl, C 1 -C 4 alkylamino C 1 -C 4 alkyl, di C 1 -C4 alkylamino C1-C4 alkyl, C 1 -C 4 alky
• R 3 is selected from the group consisting of -H, CrC 6 alkyl, C 2 -C6 alkenyl, C 2 -C 6 alkynyl, cyano, amino C 1 -C 4 alkyl, amino, aryl, wherein the aryl group optionally can be substituted with one or more group selected from halogen, hydroxy, C 1 -C 4 alkoxy, C 1 -C4 alkyl, carboxy, C1-C 4 alkoxycarbonyl, carboxy C 1 -C 4 alkoxy, amino, di- C C 4 alkylamino, A/-C C- 4 alkyl- ⁇ /-cyano C 1 -C 4 alkylamino, nitro, C 1 -C 4 alkylcarbonylamino, cyano; halo C 1 -C 4 alkyl, di-halo C C 4 alkyl, tri-halo C C 4 alkyl, halo C1-C4 alkoxy, di-halo C
• R 4 is selected from the group consisting of -H, C C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, hydroxy, C 1 -C 4 alkylthio, C1-C4 alkoxy, C 1 -C 4 alkoxycarbonyl, mercapto, /V-imidazoylphenyl, , C 1 -C 4 isoalkyl, aminofluorobenzhydryl, aryl and heteroaryl, wherein the aryl and heteroaryl groups optionally can be substituted with one or more groups selected from halogen, hydroxy, C 1 -C 4 alkoxy, C 1 -C 4 alkyl, C 1 -C 4 alkylthio, C 1 -C 4 alkylsulfonyl, C 1 -C 4 alkylsulfinyl, cartoxy, carbamyl, C 1 -C 4 alkoxycarbonyl, carboxy C 1 -C 4
• R 3 and R 4 groups are such that they optionally join to form a ring system selected from:
• D, E and G are each independently selected from the group consisting of carbon, oxygen, sulfur, and nitrogen;
• R 5 is selected from the group consisting of -H, and CrC 5 alkyl; and wherein the R 1 and R 5 groups optionally join to form a piperidyl ring or oxazinyl ring;
• the invention is also direct to a novel method of inhibiting mitogen activated protein kinase-activated protein kinase-2 in a subject in need of such inhibition, the method comprising administering to the subject a compound, or a pharmaceutically acceptable salt thereof, the compound having the structure:
• G is selected from the group consisting of - O -, - S -, and -N-; when G is -O-, R 41 and R 42 are absent; when G is -S-, R 41 and R 42 are optionally absent, or are oxo; when G is -N-, R 4i is absent, and R 42 is -H or CrC -alkyl; R ⁇ R 2 , R 35 , R 36 , R 37 , R 38 , R 39 , and R 40 each is independently selected from the group consisting of hydrogen, hydroxy, amino, halo, nitro, branched or unbranched C- ⁇ -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C6 alkynyl,
• CrC 6 alkoxy hydroxy C C ⁇ alkyl, hydroxy C C ⁇ alkoxy, C ⁇ -C 6 alkoxy C C ⁇ alkoxy, C C 6 alkoxy C C ⁇ alkyl, C 2 -C 6 alkenoxy, branched or unbranched amino C ⁇ -C 6 alkyl, diamino C Z -CQ alkyl, C-r C ⁇ alkylamino CrC 6 alkyl, C ⁇ -C 6 alkylamino, di-( Ci-C ⁇ alkyl)amino, CrC 4 alkoxyarylamino, C ⁇ -C4alkoxyalkylamino, amino C C ⁇ alkoxy, di-(C C4 alkylamino, C 2 -C6 alkoxy, di-(CrC 6 alkyl)amino C C 6 alkyl, Ci-C ⁇ alkylamino C C 6 alkoxy, halo C C ⁇ alkoxy, dihalo C C ⁇ alkoxy, trihalo d-
• R 38 is other than cyano.
• the present invention is also directed to a novel method of inhibiting mitogen activated protein kinase-activated protein kinase-2 in a subject in need of such inhibition, the method comprising administering to the subject a compound, or a pharmaceutically acceptable salt thereof, the compound having the structure:
• G is selected from the group consisting of -0-, -S-, and -N-; when G is -O-, R 41 and R 42 are absent; when G is -S-, R 41 and R 42 are optionally absent, or are oxo; when G is -N-, R 41 is absent , and R 42 is -H or -CH 3 ;
• R 1 is selected from the group consisting of hydrogen, ethyl, dimethylaminoethyl, butyl, propyl, methoxyethyl, tetramethylaminoethyl, and carboxymethyl;
• R 2 is selected from the group consisting of hydrogen, hydroxyethyl, propyl, ethyl, methyl, 4-methoxyphenyl, ethoxyethyl, aminoethyl, phenylmethyl, dimethylaminoethyl, phthaloaminoethyl, butyl, methoxyethyl, tetramethylaminoethyl, and carboxymethyl;
• R 35 is selected from the group consisting of hydrogen, dicyanomethyl, 2-fluorophenyl, phenyl, and 3-fluorophenyl.
• R 36 is selected from the group consisting of hydrogen, dicyanomethyl, 2-fluorophenyl, phenyl, and 3-fluorophenyl;
• R 37 is selected from the group consisting of hydrogen, hydroxy, methoxy, bromo, and 2-pyridomethyl;
• R 38 is selected from the group consisting of hydrogen, hydroxy, methoxy, amino, carboxy, diaminoethoxy, bromo, propoxy, isobutylcarboxymethoxy, dimethylamino, nitro, phenyl, chloro, pyridylmethyl, and fluoro;
• R 39 is selected from the group consisting of hydrogen, hydroxy, methoxy, hydroxyethoxy, ethoxyethoxy, ethoxy, aminoethoxy, morpholinoethoxy, carboxymethoxy, ⁇ /-pyrrolidylethoxy, dimethylaminoethoxy, pyridylmethyl, 2-propenoxy, and isobutylcarboxymethoxy, where the R 38 and R 39 groups can join to form a six membered heterocyclic ring; and
• R 40 is selected from the group consisting of hydrogen, hydroxy, fluoro, methoxy, nitro, amino, pyrrolidylethoxy, carboxymethoxy, methyl, hydroxyethoxy, aminoethoxy, 4-pyridylmethoxy, isobutyl, ethylcarboxy, dimethylaminoethoxy, carboxy, bromo, and pyrridylmethyl.
• Figure 1 is a graph showing paw thickness as a function of time from day 0 to day 7 for MK2 (+/+) and MK2 (-/-) mice, which have received serum injection;
• Figure 2 is a bar chart showing paw thickness at seven days after injection for normal mice, MK2 (+/+) mice receiving serum, MK2 (-/-) mice receiving serum, and MK2 (+/+) mice receiving serum and anti-TNF antibody.
• Aminocyanopyridine compounds that are useful in the present method include those having the structure shown in formula I:
• R 1 is selected from the group consisting of -H, C C ⁇ alkyl, C 2 -C ⁇ alkenyl, C 2 -C 6 alkynyl, carboxy C 1 -C 4 alkyl, aryl C 1 -C 4 alkyl, amino, amino C1-C 4 alkyl, C1-C4 alkoxy, C1-C4 alkylamino, C C 4 alkyl, di-( C 1 -C4 alkyl)amino C1-C4 alkyl, C1-C4 alkyl-CrC 4 alkyl, hydroxy C1-C 4 alkyl, and aryl C 1 -C4 alkylcarbonyl;
• R 2 is selected from the group consisting of -H, C C ⁇ alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, amino, amino C 1 -C 4 alkyl, C1-C 4 alkylamino, aryl, heteroaryl, heterocyclyl, carboxy, carboxy C 1 -C 4 alkyl, C 1 -C 4 alkoxy, hydroxy, hydroxy C 1 -C 4 alkyl, hydroxy C 1 -C 4 alkylamino, hydroxy C 1 -C 4 alkoxy, C 1 -C 4 alkoxy C 1 -C4 alkyl, C1-C4 alkoxy C1-C 4 alkylamino, amino Cr C 4 alkylamino, aryl C 1 -C 4 alkyl, C C 4 alkylamino C 1 -C 4 alkyl, di C 1 -C 4 alkylamino C1-C4 alkyl, C1-C4 alky
• R 3 is selected from the group consisting of -H, C C ⁇ alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, cyano, amino C- 1 -C 4 alkyl, amino, aryl, wherein the aryl group optionally can be substituted with one or more group selected from halogen, hydroxy, C1-C 4 alkoxy, C 1 -C 4 alkyl, carboxy, C 1 -C 4 alkoxycarbonyl, carboxy C 1 -C 4 alkoxy, amino,.
• ring substituent groups that join to form additional ring structures adjacent the substituted ring can be described with reference to chemical formulas that show wavy lines to indicate that a partial molecule is shown.
• the wavy lines cut through the ring to which the substituents are joined (in this case, the pyridine ring of formula I), rather than across the bond joining the substituent group to the ring.
• the partial ring that is shown is the ring to which the substituent groups are shown as being bonded in the general formula.
• R 4 is selected from the group consisting of -H, Ci-C ⁇ alkyl, C 2 -C ⁇ alkenyl, C 2 -C 6 alkynyl, hydroxy, C ⁇ -C alkylthio, C 1 -C 4 alkoxy, CrC 4 alkoxycarbonyl, mercapto, V-imidazoylphenyl, , C 1 -C 4 isoalkyl, aminofluorobenzhydryl, aryl and heteroaryl, wherein the aryl and heteroaryl groups optionally can be substituted with one or more groups selected from halogen, hydroxy, C 1 -C 4 alkoxy, C 1 -C 4 alkyl, C 1 -C 4 alkylthio, C1-C 4 alkylsulfonyl, C 1 -C 4 alkylsulfinyl, cartoxy, carbamyl, C C 4 alkoxycarbonyl, carboxy C 1 -C4 alkyl, carboxy C 1
• R 3 and R 4 groups are such that they optionally join to form a ring system selected from: D, E and G are each independently selected from the group consisting of carbon, oxygen, sulfur, and nitrogen;
• R 5 is selected from the group consisting of -H, and C 1 -C 5 alkyl; and wherein the R 1 and R 5 groups can join to form a piperidyl ring or an oxazinyl ring;
• R 6 R 7 R 8 p9 R 10 R 11 p12 p13 p 14 R 15 R 16 p17 R 18 p19 p20 ri , ri , ri , ri , ri , ri , ri , ri , ri , ⁇ , ri , ri , ri , ri , ri , p21 R 22 p23 n 24 R 25 p26 p27 p28 p29 p30 R 31 R 32 R 33 p34 p35 R 36 ri , ri , ri , ri , ri , ri , ri , ri , ri , ri , ri , ri , ⁇ , ri , ri , ri , ri , ri , p37 p 38 R 39 R 40 R
• R 3 is other than cyano.
• R ⁇ R 2 , R 3 , R 4 , and R 5 is other than hydrogen.
• R , R 3 and R 5 are hydrogen:
• R 2 is other than alkenyl, alkyl, alkynyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heterocycle, heterocyclealkyl, heterocyclealkylcarbonyl, (NZ ⁇ Z 2 )alkyl, or -R A R B ; where Z ⁇ and Z 2 are each independently selected from the group consisting of hydrogen, alkoxycarbonyl, alkyl, alkylcarbonyl, benzyl, benzyloxycarbonyl, and formyl;
• R A is selected from the group consisting of aryl and arylalkyl
• R B is selected from the group consisting of aryl, arylalkoxy, arylalkyl, aryloxy, heterocycle, and heterocyclealkyl; and R 4 is other than alkenyl, alkoxyalkynyl, alkyl, alkynyl, cycloalkyl, aryl, arylalkyl, heterocycle, heterocyclealkyl, or -RCRDRE," where R c is selected from the group consisting of aryl, arylalkyl, heterocycle and heterocyclealkyl;
• R D is selected from the group consisting of aryl, arylalkoxy, arylalkoxyimino, arylalkyl, aryloxy, heterocycle, heterocyclealkoxy, heterocyclealkyl, heterocyclecarbonyl, heterocycleimino, heterocycleoxy, heterocycleoxyalkyl, heterocycleoxyimino, heterocycleoxyiminoalkyl, and heterocyclesulfonyl; and
• RE is absent or selected from the group consisting of aryl, arylalkoxy, arylalkoxyimino, arylalkyl, aryloxy, heterocycle, heterocyclealkoxy, heterocyclealkyl, heterocyclecarbonyl, heterocycleimino, heterocycleoxy, heterocycleoxyalkyl, heterocycleoxyimino, heterocycleoxyiminoalkyl, and heterocyclesulfonyl.
• alkyl alone or in combination, means an acyclic alkyl radical, linear or branched, which, unless otherwise noted, preferably contains from 1 to about 10 carbon atoms and more preferably contains from 1 to about 6 carbon atoms.
• Alkyl also encompasses cyclic alkyl radicals containing from 3 to about 7 carbon atoms, preferably from 3 to 5 carbon atoms.
• the alkyl radicals can be optionally substituted with groups as defined below. Examples of such alkyl radicals include methyl, ethyl, chloroethyl, hydroxyethyl, n-propyl, isopropyl, n-butyl, cyanobutyl, isobutyl, sec-butyl, tert-butyl, pentyl, aminopentyl, iso-amyl, hexyl, octyl, and the like.
• alkenyl refers to an unsaturated, acyclic hydrocarbon radical, linear or branched, in so much as it contains at least one double bond. Unless otherwise noted, such radicals preferably contain from 2 to about 6 carbon atoms, preferably from 2 to about 4 carbon atoms, more preferably from 2 to about 3 carbon atoms.
• the alkenyl radicals may be optionally substituted with groups as defined below.
• alkenyl radicals examples include propenyl, 2- chloropropylenyl, buten-1yl, isobutenyl, penten-1yl, 2-methylbuten-1 -yl, 3- methylbuten-1 -yl, hexen-1-yl, 3-hydroxyhexen-1 -yl, hepten-1 -yl, octen-1-yl, and the like.
• alkynyl refers to an unsaturated, acyclic hydrocarbon radical, linear or branched, in so much as it contains one or more triple bonds, such radicals preferably containing 2 to about 6 carbon atoms, more preferably from 2 to about 3 carbon atoms.
• the alkynyl radicals may be optionally substituted with groups as described below.
• alkynyl radicals examples include ethynyl, proynyl, hydroxypropynyl, butyn-1 -yl, butyn-2-yl, pentyn-1 -yl, pentyn-2-yl, 4- methoxypentyn-2-yl, 3-methylbutyn-1-yl, hexyl-1 -yl, hexyn-2-yl, hexyn-3-yl, 3,3-dimethylbutyn-1-yl radicals, and the like.
• alkoxy includes linear or branched oxy-containing radicals, each of which has, unless otherwise noted, alkyl portions of 1 to about 6 carbon atoms, preferably 1 to about 4 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, isobutoxy radicals, and the like.
• alkoxyalkyl also embraces alkyl radicals having one or more alkoxy radicals attached to the alkyl radical, that is, to form monoalkoxyalkyl and dialkoxyalkyl radicals.
• radicals examples include methoxyalkyls, ethoxyalkyls, propoxyalkyls, isopropoxyalkyls, butoxyalkyls, tert-butoxyalkyls, and the like.
• the "alkoxy" radicals may be further substituted with one or more halo atoms, such as fluoro, chloro, or bromo, to provide "haloalkoxy” radicals.
• halo atoms such as fluoro, chloro, or bromo
• examples of such radicals included fluoromethoxy, chloromethoxy, trifluoromethoxy, difluoromethoxy, trifluoroethoxy, fluoroethoxy, tetrafluoroethoxy, pentafluoroethoxy, fluoropropoxy, and the like.
• alkylthio embraces radicals containing a linear or branched alkyl radical, preferably, unless otherwise noted, of from 1 to about 6 carbon atoms, attached to a divalent sulfur atom.
• lower alkylthio is methylthio (CH 3 -S-).
• alkylthioalkyl embraces alkylthio radicals, attached to an alkyl group.
• An example of such radicals is methylthiomethyl.
• halo means radicals comprising halogens, such as fluorine, chlorine, bromine, or iodine.
• heterocyclyl means a saturated or unsaturated mono- or multi-ring carbocycle wherein one or more carbon atoms is replaced by N, S, P, or O. This includes, for example, structures such as:
• Z, Z 1 , Z 2 , or Z 3 is C, S, P, O, or N, with the proviso that one of Z, Z 1 , Z 2 , or Z 3 is other than carbon, but is not O or S when attached to another Z atom by a double bond or when attached to another O or S atom.
• the optional substituents are understood to be attached to Z, Z 1 , Z 2 , or Z 3 only when each is C.
• heterocycle also includes fully saturated ring structures, such as piperazinyl, dioxanyl, tetrahydrofuranyl, oxiranyl, aziridinyl, morpholinyl, pyrrolidinyl, piperidinyl, thiazolidinyl, and others.
• heteroaryl means a fully unsaturated heterocycle, which can include, but is not limited to, furyl, thenyl, pyrryl, imidazolyl, pyrazolyl, pyridyl, thiazolyl, quinolinyl, isoquinolinyl, benzothienyl, and indolyl.
• the point of attachment to the molecule of interest can be at the heteroatom or elsewhere within the ring.
• cycloalkyl means a mono- or multi-ringed carbocycle wherein each ring contains three to about seven carbon atoms, preferably three to about six carbon atoms, and more preferably three to about five carbon atoms. Examples include radicals, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloalkenyl, and cycloheptyl.
• cycloalkyl additionally encompasses spiro systems wherein the cycloalkyl ring has a carbon ring atom in common with the seven-membered heterocyclic ring of the benzothiepine.
• oxo means a doubly-bonded oxygen.
• aryl means a fully unsaturated mono- or multi-ring carbocycle, including, but not limited to, substituted or unsubstituted phenyl, naphthyl, or anthracenyl.
• the present aminocyanopyridine compounds inhibit the activity of the MK-2 enzyme. When it is said that a subject compound inhibits MK- 2, it is meant that the MK-2 enzymatic activity is lower in the presence of the compound than it is under the same conditions in the absence of such compound.
• MK-2 inhibitor is to measure the "IC 50 " value of the compound.
• the IC50 value of an MK-2 inhibitor is the concentration of the compound that is required to decrease the MK-2 enzymatic activity by one-half. Accordingly, a compound having a lower IC 50 value is considered to be a more potent inhibitor than a compound having a higher IC 50 value.
• aminocyanopyridine compounds that inhibit MK-2 can be referred to as aminocyanopyridine MK-2 inhibitors, or aminocyanopyridine MK-2 inhibiting compounds or MK-2 inhibiting agents. [00043] Examples of aminocyanopyridine compounds that are suitable for use as MK-2 inhibitors in the present invention are shown in Table I. Table I: Aminocyanopyridine MK-2 Inhibitors
• the aminocyanopyridine compound may be shown with a solvent, such as, for example, trifluoroacetate, with which it can form a salt. Both the salt and acid forms of the aminocyanopyridine compound are included in the present invention.
• b Compound names generated by ACD/Name software.
• the method of the present invention comprises the administering to the subject an aminocyanopyridine compound having the structure shown in formula I, where:
• R is selected from the group consisting of -H, methyl, ethyl, propyl, butyl, -(CH 2 )COOH, phenyl, pyridyl, dimethylaminoethyl, methoxyethyl, tetramethylaminoethyl, carboxymethyl, and phenylacetyl;
• R 2 is selected from the group consisting of -H, methyl, ethyl, propyl, butyl, amino, phenyl, methoxy, carboxy, carboxymethyl, hydroxyethylamino, propylamino, ethylamino, methylamino, methoxyethyl, ethoxyethylamino, aminoethylamino, benzylamino, dimethylaminoethylamino, phthaloaminoethyl, fluorophenyl, difluorophenyl, chlorophenyl, bromophenyl, furyl, carbamylpyrryl, methyl-1 ,3-isodiazoyl, 1 ,3-isodiazoyl, 1 ,3,4-triazoyl, methoxyphenyl, -S(CH3), tetramethylaminoethyl, acetylaminophenyl, methoxyphenyla
• R 3 is selected from the group consisting of -H, methyl, ethyl, propyl, isopropyl, cyano, aminomethyl, phenyl, fluorophenyl, and amino; wherein the R 2 and R 3 groups are such that they optionally join to form a ring system selected from the group consisting of:
• R is selected from the group consisting of -H, methyl, ethyl, propyl, hydroxy, furyl, methylfuryl, methylimidazolyl, phenyl, hydroxyphenyl, carboxyphenyl, pyrazolyl, hydroxy, dihydroxyphenyl, methoxyphenyl, chlorophenyl, bromophenyl, fluorophenyl, dichlorophenyl, dihydroxyborophenyl, thienyl, pyrryl, ⁇ /-methylpyrryl, pyridyl, methylthio, methylsulfonylphenyl, carboethoxyphenyl, methoxy, carbamylphenyl, mercapto, /V-isoimidazoylphenyl, isopropyl, amino, hydroxynaphthyl, thiazoyl, carboxymethylphenyl, trifluoromethylphenyl, methylphenyl, cyanophenyl
• R 3 and R 4 groups are such that they optionally join to form a ring system selected from the group consisting of:
• D, E and G are each independently selected from the group consisting of carbon, oxygen, sulfur, and nitrogen;
• R 5 is selected from the group consisting of -H, and C1-C 5 alkyl; and wherein the R and R 5 groups can join to form a piperidyl ring; c p6 p7 D 8 p9 D 10 R 11 p12 p13 p 14 R 15 p16 p17 p18 p19 p20 o ri , ⁇ , ⁇ , ⁇ , ri , ⁇ , li , li , H , n , ri , n , ri , ri , ri , ri , p 21 p22 p23 p 24 D 25 p26 p27 p 28 p29 p30 p31 p32 p33 p 34 p35 D 36 ri , ri , ri , ri , ri , ri , ri , ri , ri ,
• R 69 , R 70 R 71 , R 72 , R 73 , R 74 , R 75 , and R 76 are each optionally present (for 0 example, they can be present when required to balance the valence of the atom to which they are shown as being bound) and are each independently selected from the group consisting of -H, methyl, ethyl, propyl, butyl, isobutyl, amino, nitro, hydroxy, methoxy, ethoxy, propoxy, 2- propenoxy, oxo, carboxy, bromo, chloro, fluoro, trifluoromethyl, 5 chloromethyl, hydroxymethyl, dicyanomethyl, 2-fluorophenyl, 3- fluorophenyl, hydroxyethoxy, ethoxyethoxy, -(CH 2 )-0-(C 6 H 4 )-O-(CH 3 ), carboxymethoxy, isopropylcarboxymethoxy, isobutylcarboxymeth
• the present method can be practiced by the administration of an aminocyanopyridine compound that provides an IC- 50 of less than about 200 ⁇ M, in an in vitro assay of MK-2 inhibitory activity.
• an aminocyanopyridine compound that provides an IC- 50 of less than about 200 ⁇ M
• examples of such compounds comprise the compound shown in formula I, where:
• R 1 is selected from the group consisting of -H, methyl, ethyl, - (CH 2 )COOH, and phenyl;
• R 2 is selected from the group consisting of -H, methyl, ethyl, amino, phenyl, methoxy, carboxy, hydroxyethylamino, propylamino, ethylamino, methylamino, methoxyethyl, ethoxyethylamino, aminoethylamino, benzylamino, dimethylaminoethylamino, fluorophenyl, difluorophenyl, chlorophenyl, bromophenyl, furyl, carbamylpyrryl, methyl- 1 ,3-isodiazoyl, 1 ,3-isodiazoyl, 1 ,3,4-triazoyl, methoxyphenyl, -S(CH3), acetylaminophenyl, methoxyphenylamino, carboxyphenyl, cyanophenyl, cyclopropyl, phenoxyphenyl, pyridy
• R 3 is selected from the group consisting of -H, methyl, ethyl, propyl, isopropyl, cyano, and aminomethyl; wherein the R 2 and R 3 groups are such that they optionally join to form a ring system selected from the group consisting of:
• R 4 is selected from the group consisting of -H, methyl, ethyl, propyl, hydroxy, furyl, indolyl, methylfuryl, methylimidazolyl, phenyl, hydroxyphenyl, carboxyphenyl, pyrazolyl, hydroxy, dihydroxyphenyl, methoxyphenyl, chlorophenyl, dichlorophenyl, dihydroxyborophenyl, thienyl, pyrryl, ⁇ /-methylpyrryl, pyridyl, methylthio, methylsulfonylphenyl, carboethoxyphenyl, methoxy, carbamylphenyl, ⁇ /-isoimidazoylphenyl, amino, hydroxynaphthyl, thiazoyl, carboxymethylphenyl, aminosulfonylphenyl, and
• R 3 and R 4 groups are such that they optionally join to form a ring system selected from the group consisting of:
• D, E and G are each independently selected from the group consisting of carbon, oxygen, sulfur, and nitrogen;
• R 5 is selected from the group consisting of -H, and C 1 -C 5 alkyl; p6 R 7 R 8 R 9 p10 R 11 p12 p13 pi 4 p15 p16 p17 p18 R 19 R 20 r , rt , ri , ri , ri , ri , ri , ⁇ , ri , ri , ri , ⁇ , ri , ri , ri , ⁇ , ri ,
• R 71 , R 72 , R 73 , R 74 , R 75 , and R 76 are each optionally present (such as when required to balance the valence of the atom to which they are shown as being bound) and are each independently selected from the group consisting of - H, methyl, ethyl, butyl, amino, nitro, hydroxy, methoxy, ethoxy, oxo, 2-propenoxy, carboxy, bromo, chloro, fluoro, trifluoromethyl, chloromethyl, hydroxymethyl, dicyanomethyl, hydroxyethoxy, ethoxyethoxy, -(CH 2 )-0-(C 6 H 4 )-O-(CH 3 ), carboxymethoxy, isopropylcarboxymethoxy, methylamino, dimethylamino, aminoethoxy, diaminoethoxy, cyanomethoxymethyl, methoxymethyl, isopropoxymethyl, ethoxymethyl, -(CH 2
• the present method can be practiced by the administration of an aminocyanopyridine compound that provides an IC 50 of less than about 100 ⁇ M, in an in vitro assay of MK-2 inhibitory activity.
• an aminocyanopyridine compound that provides an IC 50 of less than about 100 ⁇ M
• examples of such compounds comprise the compound shown in formula I, where:
• R 1 is selected from the group consisting of -H, methyl, and ethyl
• R 2 is selected from the group consisting of -H, methyl, amino, phenyl, methoxy, hydroxyethylamino, propylamino, ethylamino, methylamino, methoxyethyl, ethoxyethylamino, aminoethylamino, benzylamino, dimethylaminoethylamino, fluorophenyl, difluorophenyl, chlorophenyl, bromophenyl, furyl, carbamylpyrryl, methyl-1 ,3-isodiazoyl, 1 ,3-isodiazoyl, 1 ,3,4-triazoyl, methoxyphenyl, -S(CH 3 ), acetylaminophenyl, methoxyphenylamino, carboxyphenyl, cyanophenyl, cyclo
• R 3 is selected from the group consisting of -H, methyl, ethyl, propyl, isopropyl, and cyano; wherein the R 2 and R 3 groups are such that they optionally join to form a ring system selected from the group consisting of :
• R 4 is selected from the group consisting of -H, methyl, ethyl, propyl, hydroxy, furyl, indolyl, methylfuryl, methylimidazolyl, phenyl, hydroxyphenyl, carboxyphenyl, pyrazolyl, hydroxy, dihydroxyphenyl, methoxyphenyl, chlorophenyl, dichlorophenyl, dihydroxyborophenyl, thienyl, pyrryl, ⁇ /-methylpyrryl, pyridyl, methylthio, methylsulfonylphenyl, carboethoxyphenyl, methoxy, carbamylphenyl, amino, and aminosulfonylphenyl; wherein the R 3 and R 4 groups are such that they optionally join to form a ring system selected from:
• D, E and G are each independently selected from the group consisting of carbon, oxygen, sulfur, and nitrogen; R 5 is -H;
• R 35 , R 36 , R 37 , R 38 , R 39 , R 40 , R 41 , R 42 , R 71 , R 72 , R 73 , R 74 , R 75 , and R 76 are each optionally present (such as when required to balance the valence of the atom to which they are shown as being bound) and are each independently selected from the group consisting of - H, methyl, ethyl, butyl, amino, nitro, hydroxy, methoxy, ethoxy, oxo, 2-propenoxy, carboxy, bromo, fluoro, trifluoromethyl, chloromethyl, dicyanomethyl, hydroxyethoxy, ethoxyethoxy, -(CH 2 )-0-(C 6 H 4 )-O-(CH 3 ), carboxymethoxy, isopropylcarboxymethoxy, methylamino, dimethylamino, aminoethoxy, diaminoethoxy, phenyl
• the present method can be practiced by the administration of an aminocyanopyridine compound that provides an IC-50 of less than about 50 ⁇ M, in an in vitro assay of MK-2 inhibitory activity.
• an aminocyanopyridine compound that provides an IC-50 of less than about 50 ⁇ M
• examples of such compounds comprise the compound shown in formula I, where:
• R 1 is selected from the group consisting of -H, methyl, and ethyl
• R 2 is selected from the group consisting of -H, methyl, amino, phenyl, methoxy, hydroxyethylamino, propylamino, ethylamino, methylamino, methoxyethyl, ethoxyethylamino, aminoethylamino, benzylamino, dimethylaminoethylamino, fluorophenyl, difluorophenyl, chlorophenyl, bromophenyl, furyl, carbamylpyrryl, methyl-1 , 3-isodiazoyl, 1 ,3-isodiazoyl, 1 ,3,4-triazoyl, methoxyphenyl, -S(CH 3 ), acetylaminophenyl, methoxyphenylamino, carboxyphenyl, and
• R 3 is selected from the group consisting of -H, methyl, ethyl, propyl, and isopropyl; wherein the R 2 and R 3 groups are such that they optionally join to form a ring system consisting of :
• R 4 is selected from the group consisting of -H, methyl, ethyl, propyl, furyl, indolyl, methylfuryl, methylimidazolyl, phenyl, hydroxyphenyl, carboxyphenyl, pyrazolyl, hydroxy, dihydroxyphenyl, methoxyphenyl, chlorophenyl, dichlorophenyl, dihydroxyborophenyl, thienyl, pyrryl, N- methylpyrryl, pyridyl, methylthio, methylsulfonylphenyl, carboethoxyphenyl, and aminosulfonylphenyl; wherein the R 3 and R 4 groups are such that they optionally join to form a ring system selected from:
• D, E and G are each independently selected from the group consisting of carbon, oxygen, sulfur, and nitrogen; R 5 is -H;
• R 6 p7 p8 R 9 p10 p11 D 12 p35 p36 R 37 p38 p39 p40 R 41 p42 ri , ri , ri , ri , ri , ri , ri , ri , ri , ri , ri , ri , ri , n , ri ,
• R 71 , R 72 , R 73 , R 74 , R 75 , and R 76 are each optionally present (such as when required to balance the valence of the atom to which they are shown as being bound) and are each independently selected from the group consisting of - H, methyl, ethyl, butyl, amino, nitro, hydroxy, methoxy, ethoxy, oxo, 2-propenoxy, carboxy, bromo, fluoro, trifluoromethyl, chloromethyl, dicyanomethyl, hydroxyethoxy, ethoxyethoxy, carboxymethoxy, isopropylcarboxymethoxy, methylamino, dimethylamino, aminoethoxy, diaminoethoxy, morpholinylethoxy, pyrrolidylethoxy, N- pyrrolidylethoxy, and pyridylmethyl, and wherein R 38 and R 39 are such that they optionally join to form a ring
• the present method can be practiced by the administration of an aminocyanopyridine compound that provides an IC- 50 of less than about 20 ⁇ M, in an in vitro assay of MK-2 inhibitory activity.
• an aminocyanopyridine compound that provides an IC- 50 of less than about 20 ⁇ M
• examples of such compounds comprise the compound shown in formula I, where:
• R 1 is -H
• R 2 is selected from the group consisting of amino, phenyl, fluorophenyl, difluorophenyl, furyl, carbamylpyrryl, methyl-1 ,3-isodiazoyl, 1 ,3-isodiazoyl, 1 ,3,4-triazoyl, methoxyphenyl, acetylaminophenyl, methoxyphenylamino, and carboxyphenyl;
• R 3 is selected from the group consisting of -H, methyl, ethyl, and propyl;
• R 4 is selected from the group consisting of methyl, ethyl, propyl, furyl, phenyl, hydroxyphenyl, carboxyphenyl, pyrazolyl, hydroxy, dihydroxyphenyl, methoxyphenyl, chlorophenyl, dihydroxyborophenyl, and aminosulfonylphenyl;
• R 3 and R 4 groups are such that they optionally join to form a ring system selected from the group consisting of:
• D, E and G are each independently selected from the group consisting of carbon, oxygen, sulfur, and nitrogen;
• R 5 is -H
• R 6 R 7 R8 R 9 R 10 R 11 R 12 R35 p36 R 37 p38 R 39 p40 R 41 p 42 ri , ri , ri , ri , ri , ri , ri , ri , li , ri , ri , ri , ri , ri , ri , ri , ri ,
• R 71 , R 72 , R 73 , R 74 , R 75 , and R 76 are each optionally present (such as when required to balance the valence of the atom to which they are shown as being bound) and are each independently selected from the group consisting of - H, amino, nitro, hydroxy, methoxy, ethoxy, oxo, 2- propenoxy, carboxy, bromo, fluoro, trifluoromethyl, chloromethyl, dicyanomethyl, hydroxyethoxy, ethoxyethoxy, carboxymethoxy, isopropylcarboxymethoxy, methylamino, dimethylamino, aminoethoxy, diaminoethoxy, morpholinylethoxy, pyrrolidylethoxy, and pyridylmethyl, and wherein R 38 and R 39 are such that they optionally join to form a ring system consisting of:
• the present method can be practiced by the administration of an aminocyanopyridine tricyclic compound having the structure shown in formula II:
• G is selected from the group consisting of - O -, - S -, and -N-; when G is -O-, R 41 and R 42 are absent; when G is -S-, R 41 and R 42 are optionally absent, or are oxo; when G is -N-, R 41 is absent, and R 42 is -H or CrC 4 -alkyl; each of Ft 1 , R 2 , R 35 , R 36 , R 37 , R 38 , R 39 , and R 40 is independently selected from the group consisting of hydrogen, hydroxy, amino, halo, nitro, branched or unbranched C ⁇ -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C C ⁇ alkoxy, hydroxy CrC 6 alkyl, hydroxy C ⁇ alkoxy, CrC 6 alkoxy C-p C 6 alkoxy, CrC 6 alkoxy C C 6 alkyl, CrC 6 alkenoxy,
• alkyl, alkenyl, alkynyl, alkoxy, alkoxyalkyl, haloalkoxy, halo, alkylthio, alkylthioalkyl, heterocyclyl, cyclyl, aryl, heteroaryl, cycloaryl, and oxo have the same meanings as described above.
• the tricyclic aminocyanopyridine compounds that are useful in the present invention include benzonapthyridines, pyridochromanes, and pyridothiochromanes.
• Examples of tricyclic aminocyanopyridine compounds that are useful as MK-2 inhibitors in the present method are shown in Table II: TABLE 2: Tricyclic Aminocyanopyridine MK-2 Inhibitors
• a The aminocyanopyridine compound may be shown with a solvent, such as, for example, trifluoroacetate, with which it can form a salt. Both the salt and acid forms of the aminocyanopyridine compound are included in the present invention.
• b Compound names generated by ACD/Name software.
• the present method can be practiced by administering aminocyanopyridine compounds comprising the compound shown in formula II, where:
• G is selected from the group consisting of - O -, - S -, and -N-; when G is -0-, R 41 and R 42 are absent; when G is -S-, R 41 and R 42 are optionally absent, or are oxo; when G is -N-, R 41 is absent, and R 42 is -H or d-C 4 -alkyl;
• R 1 is selected from the group consisting of hydrogen, branched or unbranched alkyl, alkenyl, alkynyl, alkoxy, alkylaryl, arylalkyl, carboxy, carboxyalkyl, hydroxyalkyl, alkylcarboxy, aryl, amino, aminoalkyl, alkylamino, halo, alkylaminoalkyl, alkoxy, alkoxyalkyl, monocyclyl, bicyclyl, polycyclyl, and heterocyclyl;
• R 2 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, hydroxyalkyl, alkylaryl, arylalkyl, alkoxyaryl, aminoalkyl, alkylaminoalkyl, arylaminoalkyl, alkoxyalkyl, alkylcarboxy, and carboxyalkyl;
• R 35 is selected from the group consisting of hydrogen, dicyanoalkyl, and substituted or unsubstituted heterocyclyl and cyclyl, where substituents, if any, comprise halo moieties
• R 36 is selected from the group consisting of hydrogen, dicyanoalkyl, and substituted or unsubstituted heterocyclyl and cyclyl, where substituents, if any, comprise halo moieties;
• R 37 is selected from the group consisting of hydrogen, alkoxy, halo, alkyl, alkenyl, alkylyl, arylalkyl, or alkylaryl;
• R 38 is selected from the group consisting of hydrogen, hydroxy, alkoxy, alkyl, alkenyl, alkynyl, amino, alkylamino, arylamino, alkylaminoalkyl, carboxy, aminoalkoxy, halo, alkylcarboxyalkyl, alkylamino, aminoalkyl, nitro, aryl, arylalkyl, alkylaryl, or arylamino;
• R 39 is selected from the group consisting of hydrogen, hydroxy, alkoxy, alkenoxy, hydroxyalkoxy, alkoxyalkoxy, aminoalkoxy, heterocyclylalkyl, heterocyclylalkoxy, carboxyalkoxy, alkylaminoalkoxy, and alkylcarboxyalkoxy; ⁇ - - - /
• R 38 and R 39 groups can join to form a six membered heterocyclic ring
• R 40 is selected from the group consisting of hydrogen, hydroxy, halo, nitro, amino, alkyl, alkoxy, heterocyclylalkoxy, carboxyalkoxy, pyrrolidylethoxy, carboxymethoxy, hydroxyalkoxy, aminoalkoxy, alkylcarboxy, alkylaminoalkyl, carboxy, and heterocyclylalkyl.
• the present method can be practiced by the administration of an aminocyanopyridine compound comprising the compound shown in formula II, where: G is selected from the group consisting of -O-, -S-, and -N-; when G is -0-, R 41 and R 42 are absent; when G is -S-, R 41 and R 42 are optionally absent, or are oxo; when G is -N-, R 4 is absent, and R 42 is -H or -CH 3 ;
• R 1 is selected from the group consisting of hydrogen, ethyl, dimethylaminoethyl, butyl, propyl, methoxyethyl, tetramethylaminoethyl, and carboxymethyl;
• R 2 is selected from the group consisting of hydrogen, hydroxyethyl, propyl, ethyl, methyl, 4-methoxyphenyl, ethoxyethyl, aminoethyl, phenylmethyl, dimethylaminoethyl, phthaloaminoethyl, butyl, methoxyethyl, tetramethylaminoethyl, and carboxymethyl;
• R 35 is selected from the group consisting of hydrogen, dicyanomethyl, 2-fluorophenyl, phenyl, and 3-fluorophenyl.
• R 36 is selected from the group consisting of hydrogen, dicyanomethyl, 2-fluorophenyl, phenyl, and 3-fluorophenyl;
• R 37 is selected from the group consisting of hydrogen, hydroxy, methoxy, bromo, and 2-pyridomethyl;
• R 38 is selected from the group consisting of hydrogen, hydroxy, methoxy, amino, carboxy, diaminoethoxy, bromo, propoxy, isobutylcarboxymethoxy, dimethylamino, nitro, phenyl, chloro, pyridylmethyl, and fluoro;
• R 39 is selected from the group consisting of hydrogen, hydroxy, methoxy, hydroxyethoxy, ethoxyethoxy, ethoxy, aminoethoxy, ⁇ - - - - - /
• R 40 is selected from the group consisting of hydrogen, hydroxy, fluoro, methoxy, nitro, amino, pyrrolidylethoxy, carboxymethoxy, methyl, hydroxyethoxy, aminoethoxy, 4-pyridylmethoxy, isobutyl, ethylcarboxy, dimethylaminoethoxy, carboxy, bromo, and pyrridylmethyl.
• the present method can be practiced by the administration of an aminocyanopyridine compound that provides an IC 5 o of less than about 200 ⁇ M, in an in vitro assay of MK-2 inhibitory activity.
• an aminocyanopyridine compound that provides an IC 5 o of less than about 200 ⁇ M
• examples of such compounds comprise the compound shown in formula II, where:
• G is selected from the group consisting of -O- and -S-; - when G is -S-, R 41 and R 42 are optionally absent, or are oxo; when G is -O-, R 41 and R 42 are absent;
• R 1 is selected from the group consisting of hydrogen, and C1-C2 alky
• R 2 is selected from the group consisting of hydrogen, C1-C3 alkyl, hydroxy C 1 -C 2 alkyl, CrC 2 alkoxyphenyl, C ⁇ -C 2 alkoxy CrC 2 alkyl, amino
• R 35 and R 36 are each independently selected from the group consisting of hydrogen, dicyano C1-C 2 alkyl, and halophenyl;
• R 37 is selected from the group consisting of hydrogen, and hydroxy; R is selected from the group consisting of hydrogen, hydroxy, Ci -
• R 39 is selected from the group consisting of hydrogen, hydroxy, Ci - C3 alkoxy, hydroxy Ci - C 2 alkoxy, Ci - C 2 alkoxy Ci - C 2 alkoxy, amino Ci - C 2 alkoxy, morpholino Ci - C 2 alkoxy, carboxyl Ci - C 2 alkoxy, pyrrolidyl
• Ci - C 2 alkoxy di Ci - d alkylamino Ci - C 2 alkoxy, pyrrolidyl C - C 2 alkyl, iso C 3 - C 4 alkylcarboxy Ci - C 2 alkoxy, and 2-propenoxy, where the R 38 and R 39 groups optionally join to form a six membered heterocyclic ring; and
• R 40 is selected from the group consisting of hydrogen, hydroxy, halo, C 1 -C2 alkyl, C 1 -C 2 alkoxy, nitro, amino, pyrrolidyl C C 2 alkoxy, carboxy d-C 2 alkoxy, hydroxy C C 2 alkoxy, and amino C ⁇ -C 2 alkoxy.
• the present method can be practiced by the administration of an aminocyanopyridine compound that provides an IC 50 of less than about 100 ⁇ M, in an in vitro assay of MK-2 inhibitory activity.
• an aminocyanopyridine compound that provides an IC 50 of less than about 100 ⁇ M
• examples of such compounds comprise the compound shown in formula II, where:
• G is selected from the group consisting of -O- and -S-; when G is sulfur, R 41 and R 42 are optionally absent, or are oxo; when G is -O-, R 41 and R 42 are absent;
• R 1 is hydrogen
• R 2 is selected from the group consisting of hydrogen, Ci - C 3 alkyl, hydroxy Ci - d alkyl, Ci - C 2 alkoxyphenyl, Ci - C2 alkoxy Ci - C 2 alkyl, amino Ci - C 2 alkyl, phenyl C - C 2 alkyl, and di Ci - C 2 alkylamino Ci - C 2 alkyl;
• R 35 and R 36 are each independently selected from the group consisting of hydrogen, and dicyano Ci - C 2 alkyl.
• R 37 is selected from the group consisting of hydrogen, and hydroxy
• R 38 is selected from the group consisting of hydrogen, hydroxy, d- C 2 alkoxy, amino, carboxy, nitro, diamino C 1 -C 2 alkoxy, halo, 2-propenoxy, iso C 3 -C 4 alkylcarboxy C 1 -C 2 alkoxy, di C 1 -C2 alkylamino, and phenyl;
• R 39 is selected from the group consisting of hydrogen, hydroxy, Ci -
• the present method can be practiced by the administration of an aminocyanopyridine compound that provides an IC 50 of less than about 50 ⁇ M, in an in vitro assay of MK-2 inhibitory activity.
• an aminocyanopyridine compound that provides an IC 50 of less than about 50 ⁇ M
• examples of such compounds comprise the compound shown in formula II, where:
• G is selected from the group consisting of -O- and -S-; when G is sulfur, R 41 and R 42 are optionally absent, or are oxo; when G is -0-, R 41 and R 42 are absent;
• R 1 is hydrogen
• R 2 is selected from the group consisting of hydrogen, C 1 -C 3 alkyl, hydroxy C C 2 alkyl, C ⁇ -C 2 alkoxyphenyl, C1-C 2 alkoxy CrC 2 alkyl, amino C 1 -C 2 alkyl, and phenyl d-d alkyl;
• R 35 and R 36 are each independently selected from the group consisting of hydrogen, and dicyano d-C alkyl.
• R 37 is selected from the group consisting of hydrogen, and hydroxy
• R 38 is selected from the group consisting of hydrogen, hydroxy, d- C 2 alkoxy, amino, carboxy, diamino C ⁇ -C 2 alkoxy, halo, 2-propenoxy, iso
• R 39 is selected from the group consisting of hydrogen, hydroxy, C - C 2 alkoxy, hydroxy d-C 2 alkoxy, C1-C 2 alkoxy C1-C2 alkoxy, amino d-C alkoxy, morpholino C1-C2 alkoxy, carboxyl C 1 -C 2 alkoxy, pyrrolidyl C 1 -C 2 alkoxy, di C ⁇ C 2 alkylamino C 1 -C 2 alkoxy, pyrrolidyl C ⁇ -C 2 alkyl, iso C 3 -C 4 alkylcarboxy C 1 -C 2 alkoxy, and 2-propenoxy; where the R 38 and R 39 groups optionally join to form a six membered heterocyclic ring; and
• R 40 is selected from the group consisting of hydrogen, hydroxy, halo, C1-C2 alkoxy, nitro, amino, and pyrrolidyl C 1 -C 2 alkoxy.
• the present method can be practiced by the administration of an aminocyanopyridine compound that provides an IC50 of less than about 20 ⁇ M, in an in vitro assay of MK-2 inhibitory activity.
• an aminocyanopyridine compound that provides an IC50 of less than about 20 ⁇ M
• examples of such compounds comprise the compound shown in formula II, where:
• G is selected from the group consisting of -O- and -S-; when G is sulfur, R 41 and R 42 are optionally absent, or are oxo; when G is -0-, R 41 and R 42 are absent;
• R 1 is hydrogen
• R 2 is selected from the group consisting of hydrogen, C 1 -C 3 alkyl, hydroxy C ⁇ -C alkyl, d-C 2 alkoxyphenyl, C 1 -C2 alkoxy d-C 2 alkyl, and amino C1-C2 alkyl;
• R 35 and R 36 are each independently selected from the group consisting of hydrogen, and dicyanoethyl
• R 37 is selected from the group consisting of hydrogen, and hydroxy
• R 38 is selected from the group consisting of hydrogen, hydroxy, C 1 - C alkoxy, amino, carboxy, diamino d- alkoxy, halo, 2-propenoxy, iso
• R 39 is selected from the group consisting of hydrogen, hydroxy, d- C alkoxy, hydroxy C ⁇ -C 2 alkoxy, d-C 2 alkoxy d-d alkoxy, amino d-C 2 alkoxy, morpholino C 1 -C 2 alkoxy, carboxyl C1-C2 alkoxy, pyrrolidyl d-C 2 alkoxy, di C ⁇ -C 2 alkylamino C ⁇ -C 2 alkoxy, pyrrolidyl C1-C2 alkyl, iso C 3 -C4 alkylcarboxy C -C 2 alkoxy, and 2-propenoxy; where the R 38 and R 39 groups optionally join to form a six membered heterocyclic ring; and
• R 40 is selected from the group consisting of hydrogen, hydroxy, halo, methoxy, nitro, and amino.
• aminocyanopyridine MK-2 inhibitor compounds that can be used in the present method include, without limitation, the following:
• Aminocyanopyridine MK-2 inhibiting compounds of the type shown in formula II, above include tricyclic aminocyanopyridine MK-2 inhibiting compounds, such as benzonapthyridines, pyridochromanes, and pyridothiochromanes.
• tricyclic aminocyanopyridine MK-2 inhibiting compounds such as benzonapthyridines, pyridochromanes, and pyridothiochromanes.
• a general method for the synthesis of these tricyclic aminocyanopyridines is shown in Scheme 1 , below: Scheme 1 :
• a substituted benzaldehyde is reacted with a tricarbonitrile, preferably 2-amino-1-propene-1 ,1 ,3-tricarbonitrile.
• the reaction can be carried out by heating the reactants to reflux in a solution of acetic acid and ethanol.
• the reaction product can be concentrated in vacuo and dissolved in trifluoroacetic acid.
• Triethylsilane is added and the mixture is stirred. In a preferred method, the mixture is stirred for about 1 hour at 0°C.
• Dichloromethane is then added and solids are collected. The solids can be collected by filtration, and can be washed with dichloromethane and ether.
• the solids comprise the desired tricyclic aminocyanopiyridine MK-2 inhibiting compound of the type including benzonapthyridines, pyridochromanes, and pyridothiochromanes.
• Z can be OH, SH, or NR a Y, where Y is a protecting group for nitrogen.
• the Y group can be benzyl, allyl, an alkyl carbamate, or a benzyl carbamate.
• Other nitrogen protecting groups are know to persons having skill in the art of organic synthesis.
• a perferred protecting group is tert- butylcarbamate.
• R a can be an alkyl group, an aryl group, or a heteroaryl group.
• the benzene ring of the benzaldehyde can be further substituted by one, two, three, or four additional R groups at carbons 3, 4, 5, or 6.
• Each R can independently be hydrogen; alkyl; aryl; a heteroatom, such as O, N, or S, substituted with hydrogen, d-C 6 alkyl, C ⁇ -C 6 branched alkyl, aryl, heteroaryl (wherein the heteroaryl can include, but is not limited to, pyrazolyl, inidizolyl, pyrryl, pyridyl, thiophyl, furyl and pyrimidyl), ester and amido.
• Advantages of this method include that it is a general method that can be used to produce various types of the tricyclic compounds of formula II depending upon the types of reactants used. It is also an easy and straightforward synthesis method that can be carried out in a single vessel.
• a tricyclic aminocyanopyridine MK-2 inhibiting compound can be prepared by reacting a substituted benzaldehyde having the structure:
• Z is selected from the group consisting of -OH, -SH, and -NR a Y;
• R a is selected from the group consisting of alkyl, aryl, and heteroaryl;
• Y is a protecting group for nitrogen.
• nitrogen protecting groups include benzyl, allyl, alkyl carbamates and benzyl carbamates.
• G is selected from the group consisting of -O-, -S-, and -NR X -; R x is alkyl;
• R b is selected from the group consisting of furyl and -NH-R 2 ;
• R 2 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, hydroxyalkyl, alkylaryl, arylalkyl, alkoxyaryl, aminoalkyl, alkylaminoalkyl, arylaminoalkyl, alkoxyalkyl, alkylcarboxy, and carboxyalkyl;
• R 3 and R 4 are each independently selected from the group consisting of hydrogen, dicyanoalkyl, and substituted or unsubstituted heterocyclyl and cyclyl, where substituents, if any, comprise halo moieties;
• R 5 , R 6 , R 7 and R 8 are each independently selected from the group consisting of hydrogen, hydroxy, alkoxy, halo, alkyl, alkenyl, alkylyl, arylalkyl, alkylaryl, amino, alkylamino, arylamino, alkylaminoalkyl, carboxy, aminoalkoxy, alkylcarboxyalkyl, alkylamino, aminoalkyl, nitro, aryl, arylamino, alkenoxy, hydroxyalkoxy, alkoxyalkoxy, heterocyclylalkyl, heterocyclylalkoxy, carboxyalkoxy, alkylaminoalkoxy, alkylcarboxyalkoxy, pyrrolidylethoxy, hydroxyalkoxy, and alkylcarboxy, where R 6 and R 7 are such that they optionally join to form a six membered heterocyclic ring.
• R 2 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, hydroxyalkyl, alkylaryl, arylalkyl, alkoxyaryl, aminoalkyl, alkylaminoalkyl, arylaminoalkyl, alkoxyalkyl, alkylcarboxy, and carboxyalkyl;
• R 3 and R 4 are each independently selected from the group consisting of hydrogen, dicyanoalkyl, and substituted or unsubstituted heterocyclyl and cyclyl, where substituents, if any, comprise halo moieties;
• R 5 is selected from the group consisting of hydrogen, alkoxy, halo, alkyl, alkenyl, alkylyl, arylalkyl, or alkylaryl;
• R 6 is selected from the group consisting of hydrogen, hydroxy, alkoxy, alkyl, alkenyl, alkynyl, amino, alkylamino, arylamino, alkylaminoalkyl, carboxy, aminoalkoxy, halo, alkylcarboxyalkyl, alkylamino, aminoalkyl, nitro, aryl, arylalkyl, alkylaryl, or arylamino;
• R 7 is selected from the group consisting of hydrogen, hydroxy, alkoxy, alkenoxy, hydroxyalkoxy, alkoxyalkoxy, aminoalkoxy, heterocyclylalkyl, heterocyclylalkoxy, carboxyalkoxy, alkylaminoalkoxy, and alkylcarboxyalkoxy; where the R 6 and R 7 groups can join to form a six membered heterocyclic ring; and
• R 8 is selected from the group consisting of hydrogen, hydroxy, halo, nitro, amino, alkyl, alkoxy, heterocyclylalkoxy, carboxyalkoxy, pyrrolidylethoxy, carboxymethoxy, hydroxyalkoxy, aminoalkoxy, alkylcarboxy, alkylaminoalkyl, carboxy, and heterocyclylalkyl.
• the substituted benzaldehyde comprises salicaldehyde and the tricarbonitrile comprises 2- amino-1 -propene-1 ,1 ,3-tricarbonitrile.
• the nitrogen protecting group "Y" comprises tert-butylcarbamate.
• Z is selected from the group consisting of -OH, -SH, and -NR a Y;
• R a is selected from the group consisting of alkyl, aryl, and heteroaryl;
• Y is a protecting group for nitrogen that is selected from the group consisting of benzyl, allyl, alkyl carbamates and benzyl carbamate;
• G is selected from the group consisting of -O-, -S-, and -NR X -;
• Rx is C ⁇ -C 6 alkyl
• R b is selected from the group consisting of furyl and -NH-R 2 ;
• R 2 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, hydroxyalkyl, alkylaryl, arylalkyl, alkoxyaryl, aminoalkyl, alkylaminoalkyl, arylaminoalkyl, alkoxyalkyl, alkylcarboxy, and carboxyalkyl;
• R 3 and R 4 are each independently selected from the group consisting of hydrogen, dicyanoalkyl, and substituted or unsubstituted heterocyclyl and cyclyl, where substituents, if any, comprise halo moieties;
• R 5 , R 6 , R 7 and R 8 are each independently selected f om the group consisting of: hydrogen, hydroxy, amino, halo, nitro, branched or unbranched d-C 6 alkyl, C 2 -C 6 alkenyl, d-d alkynyl, C ⁇ -C 6 alkoxy, hydroxy Ci-Ce alkyl, hydroxy d-d alkoxy, d-C 6 alkoxy d- d alkoxy, d-C 6 alkoxy d- alkyl, C 2 -C 6 alkenoxy, branched or unbranched amino C ⁇ -C 6 alkyl, diamino - alkyl, C ⁇ - d alkylamino Ci-Ce alkyl, d-C 6 alkylamino, di-( d- alkyl)amino, d-C 4 alkoxyarylamino, Cr alkoxyalkylamino, amino d- alkoxy, di-(
• alkyl, alkenyl, alkynyl, alkoxy, alkoxyalkyl, haloalkoxy, halo, alkylthio, alkylthioalkyl, heterocyclyl, cyclyl, aryl, heteroaryl, cycloaryl, and oxo have the same meanings as described above.
• a general method for the synthesis of aminocyanopyridine MK- 2 inhibitors that are not tricyclic benzonapthyridines, pyridochromanes, and pyridothiochromanes can be found in Kambe, S. et al, Synthesis 5:366 - 368 (1980). Further details of the synthesis of aminocyanopyridines are provided in the examples.
• the MK-2 inhibiting activity of an aminocyanopyridine compound can be determined by any one of several methods that are well known to those having skill in the art of enzyme activity testing. One such method is described in detail in the general methods section of the examples.
• an aminocyanopyridine MK-2 inhibiting compound in therapeutic applications can be determined by testing for inhibition of TNF ⁇ production in cell culture and in animal model assays.
• the aminocyanopyridine MK-2 inhibiting compounds of the present invention be capable of inhibiting the production and/or the release of TNF ⁇ in cell cultures and in animal models.
• the aminocyanopyridine MK-2 inhibitor compounds that are described herein can be used as inhibitors of MAPKAP kinase-2. When this inhibition is for a therapeutic purpose, one or more of the present compounds can be administered to a subject that is in need of MK-2 inhibition.
• a subject in need of MK-2 inhibition is a subject who has, or who is at risk of contracting a TNF ⁇ mediated disease or disorder.
• TNF ⁇ mediated diseases and disorders are described in more detail below.
• a subject in need of prevention or treatment of a TNF ⁇ mediated disease or disorder is treated with one or more of the present aminocyanopyridine compounds.
• the subject is treated with an effective amount of the aminocyanopyridine MK-2 inhibitor compound.
• the effective amount can be an amount that is sufficient for preventing or treating the TNF ⁇ mediated disease or disorder.
• aminocyanopyridine compound that is used in the subject method can be any aminocyanopyridine compound that is described above.
• the aminocyanopyridine MK-2 inhibitor compound can be used in any amount that is an effective amount. It is preferred, however, that the amount of the aminocyanopyridine compound that is administered is within a range of about 0.1 mg/day per kilogram of the subject to about 150 mg/day/kg. It is more preferred that the amount of the aminocyanopyridine compound is within a range of about 0.1 mg/day/kg to about 20 mg/day/kg. An amount that is within a range of about 0.1 mg/day/kg to about 10 mg/day/kg, is even more preferred.
• a pharmaceutical composition that contains one or more of the aminocyanopyridine MK-2 inhibitors can be administered to a subject for the prevention or treatment of a TNF ⁇ mediated disease or disorder.
• the pharmaceutical composition includes a aminocyanopyridine MK-2 inhibitor of the present invention and a pharmaceutically acceptable carrier.
• kits can be produced that is suitable for use in the prevention or treatment of a TNF ⁇ mediated disease or disorder.
• the kit comprises a dosage form comprising an aminocyanopyridine MK-2 inhibitor in an amount which comprises a therapeutically effective amount.
• an "effective amount” means the dose or effective amount to be administered to a patient and the frequency of administration to the subject which is readily determined by one of ordinary skill in the art, by the use of known techniques and by observing results obtained under analogous circumstances.
• the dose or effective amount to be administered to a patient and the frequency of administration to the subject can be readily determined by one of ordinary skill in the art by the use of known techniques and by observing results obtained under analogous circumstances.
• a number of factors are considered by the attending diagnostician, including but not limited to, the potency and duration of action of the compounds used, the nature and severity of the illness to be treated, as well as the sex, age, weight, general health and individual responsiveness of the patient to be treated, and other relevant circumstances.
• the phrase "therapeutically-effective” indicates the capability of an agent to prevent, or improve the severity of, the disorder, while avoiding adverse side effects typically associated with alternative therapies.
• the phrase “therapeutically-effective” is to be understood to be equivalent to the phrase “effective for the treatment, prevention, or inhibition”, and both are intended to qualify the amount of an agent for use in therapy which will achieve the goal of improvement in the severity of pain and inflammation and the frequency of incidence, while avoiding adverse side effects typically associated with alternative therapies.
• dosages may also be determined with guidance from Goodman & Goldman's The Pharmacological Basis of Therapeutics. Ninth Edition (1996), Appendix II, pp. 1707-1711.
• the frequency of dose will depend upon the half-life of the active components of the composition. If the active molecules have a short half life (e.g. from about 2 to 10 hours) it may be necessary to give one or more doses per day. Alternatively, if the active molecules have a long half-life (e.g. from about 2 to about 15 days) it may only be necessary to give a dosage once per day, per week, or even once every 1 or 2 months.
• a preferred dosage rate is to administer the dosage amounts described above to a subject once per day. [00083] For the purposes of calculating and expressing a dosage rate, all dosages that are expressed herein are calculated on an average amount-per-day basis irrespective of the dosage rate.
• one 100 mg dosage of an aminocyanopyridine MK-2 inhibitor taken once every two days would be expressed as a dosage rate of 50 mg/day.
• the dosage rate of an ingredient where 50 mg is taken twice per day would be expressed as a dosage rate of 100 mg/day.
• the weight of a normal adult human will be assumed to be 70 kg.
• the pharmaceutical compositions that are described above can be formed.
• Pharmaceutically acceptable carriers include, but are not limited to, physiological saline, Ringer's, phosphate solution or buffer, buffered saline, and other carriers known in the art.
• Pharmaceutical compositions may also include stabilizers, anti-oxidants, colorants, and diluents.
• Pharmaceutically acceptable carriers and additives are chosen such that side effects from the pharmaceutical compound are minimized and the performance of the compound is not canceled or inhibited to such an extent that treatment is ineffective.
• pharmacologically effective amount shall mean that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by a researcher or clinician. This amount can be a therapeutically effective amount.
• compositions include metallic ions and organic ions. More preferred metallic ions include, but are not limited to, appropriate alkali metal salts, alkaline earth metal salts and other physiological acceptable metal ions. Exemplary ions include aluminum, calcium, lithium, magnesium, potassium, sodium and zinc in their usual valences.
• Preferred organic ions include protonated tertiary amines and quaternary ammonium cations, including in part, trimethylamine, diethylamine, ⁇ /,/V-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (/V-methylglucamine) and procaine.
• Exemplary pharmaceutically acceptable acids include, without limitation, hydrochloric acid, hydroiodic acid, hydrobromic acid, phosphoric acid, sulfuric acid, methanesulfonic acid, acetic acid, formic acid, tartaric acid, maleic acid, malic acid, citric acid, isocitric acid, succinic acid, lactic acid, gluconic acid, glucuronic acid, pyruvic acid oxalacetic acid, fumaric acid, propionic acid, aspartic acid, glutamic acid, benzoic acid, and the like.
• isomeric forms and tautomers and the pharmaceutically-acceptable salts of the aminocyanopyridine MK-2 inhibitors are included in the invention.
• Illustrative pharmaceutically acceptable salts are prepared from formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, mesylic, stearic, salicylic, p-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, toluenesulfonic, 2-hydroxyethanesulfonic, sulfanilic, cyclohexylaminosulfonic, algenic, ⁇ -hydroxybutyric, galactaric
• Suitable pharmaceutically-acceptable base addition salts of compounds of the present invention include metallic ion salts and organic ion salts. More preferred metallic ion salts include, but are not limited to, appropriate alkali metal (Group la) salts, alkaline earth metal (Group Ila) salts and other physiological acceptable metal ions. Such salts can be made from the ions of aluminum, calcium, lithium, magnesium, potassium, sodium and zinc.
• Preferred organic salts can be made from tertiary amines and quaternary ammonium salts, including in part, trifluoroacetate, trimethylamine, diethylamine, ⁇ /./V-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N- methylglucamine) and procaine. All of the above salts can be prepared by those skilled in the art by conventional means from the corresponding compound of the present invention.
• the method of the present invention is useful for, but not limited to, the prevention and treatment of diseases and disorders that are mediated by TNF ⁇ .
• the aminocyanopyridine MK-2 inhibitors of the invention would be useful to treat arthritis, including, but not limited to, rheumatoid arthritis, spondyloarthopathies, gouty arthritis, osteoarthritis, systemic lupus erythematosus and juvenile arthritis.
• aminocyanopyridine MK-2 inhibitor compounds of the invention would be useful in the treatment of asthma, bronchitis, menstrual cramps, tendinitis, bursitis, connective tissue injuries or disorders, and skin related conditions such as psoriasis, eczema, burns and dermatitis.
• the aminocyanopyridine MK-2 inhibitor compounds that are useful in the method of the invention also would be useful to treat gastrointestinal conditions such as inflammatory bowel disease, gastric ulcer, gastric varices, Crohn's disease, gastritis, irritable bowel syndrome and ulcerative colitis and for the prevention or treatment of cancer, such as colorectal cancer.
• Such aminocyanopyridine MK-2 inhibiting compounds would be useful in treating inflammation in diseases and conditions such as herpes simplex infections, HIV, pulmonary edema, kidney stones, minor injuries, wound healing, vaginitis, candidiasis, lumbar spondylanhrosis, lumbar spondylarthrosis, vascular diseases, migraine headaches, sinus headaches, tension headaches, dental pain, periarteritis nodosa, thyroiditis, aplastic anemia, Hodgkin's disease, sclerodoma, rheumatic fever, type I diabetes, myasthenia gravis, multiple sclerosis, sarcoidosis, nephrotic syndrome, Behcet's syndrome, polymyositis, gingivitis, hypersensitivity, swelling occurring after injury, myocardial ischemia, and the like.
• diseases and conditions such as herpes simplex infections, HIV, pulmonary edema, kidney stones, minor injuries, wound healing, vagin
• aminocyanopyridine MK-2 inhibitors would also be useful in the treatment of ophthalmic diseases, such as retinitis, retinopathies, conjunctivitis, uveitis, ocular photophobia, and of acute injury to the eye tissue. These compounds would also be useful in the treatment of pulmonary inflammation, such as that associated with viral infections and cystic fibrosis. The compounds would also be useful for the treatment of certain central nervous system disorders such as cortical dementias including Alzheimer's disease. [00093] As used herein, the terms "TNF ⁇ mediated disease or disorder" are meant to include, without limitation, each of the symptoms or diseases that is mentioned above.
• treating or “to treat” mean to alleviate symptoms, eliminate the causation either on a temporary or permanent basis, or to prevent or slow the appearance of symptoms.
• treatment includes alleviation, elimination of causation of or prevention of pain and/or inflammation associated with, but not limited to, any of the diseases or disorders described herein.
• the subject compounds are also useful for treatment of mammals, including horses, dogs, cats, rats, mice, sheep, pigs, etc.
• the term "subject" for purposes of treatment includes any human or animal subject who is in need of the prevention of or treatment of any one of the TNF ⁇ mediated diseases or disorders.
• the subject is typically a mammal.
• "Mammal”, as that term is used herein, refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cattle, etc., Preferably, the mammal is a human.
• the subject is any human or animal subject, and preferably is a subject that is in need of prevention and/or treatment of a TNF ⁇ mediated disease or disorder.
• the subject may be a human subject who is at risk of obtaining a TNF ⁇ mediated disease or disorder, such as those described above.
• the subject may be at risk due to genetic predisposition, sedentary lifestyle, diet, exposure to disorder- causing agents, exposure to pathogenic agents and the like.
• the subject pharmaceutical compositions may be administered enterally and parenterally. Parenteral administration includes subcutaneous, intramuscular, intradermal, intramammary, intravenous, and other administrative methods known in the art.
• Enteral administration includes solution, tablets, sustained release capsules, enteric coated capsules, and syrups.
• the pharmaceutical composition may be at or near body temperature.
• the pharmaceutical compositions of the present invention can be administered orally, for example, as tablets, coated tablets, dragees, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs.
• compositions intended for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions 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 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, maize 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 adsorption 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 may be employed.
• Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredients are mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredients are present as such, or mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil.
• an inert solid diluent for example, calcium carbonate, calcium phosphate or kaolin
• an oil medium for example, peanut oil, liquid paraffin, or olive oil.
• Aqueous suspensions can be produced that contain the aminocyanopyridine MK-2 inhibitors in admixture with excipients suitable for the manufacture of aqueous suspensions.
• excipients are suspending agents, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, sodium alginate, polyvinylpyrrolidone gum tragacanth and gum acacia; dispersing or wetting agents may be naturally-occurring phosphatides, for example lecithin, or 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 heptadecaethyleneoxycetanol, or 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
• 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, or one or more sweetening agents, such as sucrose or saccharin.
• preservatives for example, ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, or one or more sweetening agents, such as sucrose or saccharin.
• Oily suspensions may be formulated by suspending the active ingredients in an omega-3 fatty acid, 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 may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.
• Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent and one or more preservatives.
• a dispersing or wetting agent e.g., a suspending agent
• Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above.
• Additional excipients for example sweetening, flavoring and coloring agents, may also be present.
• Syrups and elixirs containing the novel compounds may be formulated with sweetening agents, for example glycerol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents.
• compositions can also be administered parenterally, either subcutaneously, or intravenously, or intramuscularly, or intrasternally, or by infusion techniques, in the form of sterile injectable aqueous or olagenous suspensions.
• sterile injectable aqueous or olagenous suspensions Such suspensions may be formulated according to the known art using those suitable dispersing of wetting agents and suspending agents which have been mentioned above, or other acceptable agents.
• the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally- acceptable diluent or solvent, for example as a solution in 1 ,3-butanediol.
• Suitable vehicles and solvents that may be 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 di-, glycerides.
• n-3 polyunsaturated fatty acids may find use in the preparation of injectables.
• compositions can also be administered by inhalation, in the form of aerosols or solutions for nebulizers, or rectally, in the form of suppositories prepared by mixing the drug with a suitable non- irritating excipient which is solid at ordinary temperature 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 temperature but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
• Such materials are cocoa butter and poly-ethylene glycols.
• the novel compositions can also be administered topically, in the form of creams, ointments, jellies, collyriums, solutions or suspensions.
• Daily dosages can vary within wide limits and will be adjusted to the individual requirements in each particular case.
• a daily dosage for administration to adults, an appropriate daily dosage has been described above, although the limits that were identified as being preferred may be exceeded if expedient.
• the daily dosage can be administered as a single dosage or in divided dosages.
• Various delivery systems include capsules, tablets, and gelatin capsules, for example.
• Varian Unity Innova 400 a Varian Unity Innova 300 a Varian Unity 300, a Bruker AMX 500 or a Bruker AV-300 spectrometer. Chemical shifts are given in ppm ( ⁇ ) and coupling constants, J, are reported in Hertz. Tetramethylsilane was used as an internal standard for proton spectra and the solvent peak was used as the reference peak for carbon spectra.
• Mass spectra were obtained on a Perkin Elmer Sciex 100 atmospheric pressure ionization (APCI) mass spectrometer, a Finnigan LCQ Duo LCMS ion trap electrospray ionization (ESI) mass spectrometer, a PerSeptive Biosystems Mariner TOF HPLC-MS (ESI), or a Waters ZQ mass spectrometer (ESI).
• APCI atmospheric pressure ionization
• ESI Finnigan LCQ Duo LCMS ion trap electrospray ionization
• ESI PerSeptive Biosystems Mariner TOF HPLC-MS
• Waters ZQ mass spectrometer ESI
• Compounds are evaluated as potential inhibitors of the MK2 kinase by measuring their effects on MK2 phosphorylation of the peptide substrate.
• Compounds may be screened initially at two concentrations prior to determination of IC 50 values. Screening results are expressed as percent inhibition at the concentrations of compound tested. For IC 50 value determinations, compounds are tested at six concentrations in ten-fold serial dilutions with each concentration tested in triplicate. Results are expressed as Ido values in micromolar. The assay is performed at a final concentration of 2% DMSO.
• Preferred aminocyanopyridine MK-2 inhibiting compounds of the present invention provide Ido values for MK-2 inhibition of below 200 ⁇ M.
• One method that can be used for determining the MK-2 inhibition I o value is that described just above. More preferred aminocyanopyridine
• MK-2 inhibiting compounds have the capability of providing MK-2 inhibition I o values of below 100 ⁇ M, yet more preferred of below 50 ⁇ M, even more preferred of below 20 ⁇ M, yet more preferred of below 10 ⁇ M, and even more preferred of below 1 ⁇ M.
• the human monocyte-like cell line, U937 (ATCC #CRL-1593.2), is cultured in RPM 11640 media with 10% heat-inactivated fetal calf serum (GIBCO), glutamine and pen/strep at 37°C and 5% CO 2 .
• GEBCO heat-inactivated fetal calf serum
• glutamine glutamine
• pen/strep at 37°C and 5% CO 2 .
• Differentiation of U937 to monocytic/macrophage-like cells is induced by the addition of phorbol12-myristate 13-acetate (Sigma) at final concentration of 20 ng/ml to a culture of U937 cells at -0.5 million cells/ml and incubated for 24 hrs.
• the cells are centrifuged, washed with PBS and resuspended in fresh media without PMA and incubated for 24 hrs.
• Cells adherent to the culture flask are harvested by scraping, centrifugation, and resuspended in fresh media to 2 million cells/ml, and 0.2 ml is aliquoted to each of 96 wells in flat-bottom plate. Cells are then incubated for an additional 24 hrs to allow for recovery. The media is removed from the cells, and 0.1 ml of fresh media is added per well. 0.05 ml of serially diluted compound or control vehicle (Media with DMSO) is added to the cells. The final DMSO concentration does not exceed 1%.
• U937 cell TNF ⁇ ELISA [000123] ELISA plates (NUNC-lmmunoTM Plate MaxisorbTM Surface) were coated with purified mouse monoclonal lgG1 anti-human TNF ⁇ antibody (R&D Systems #MAB610; 1.25 ug/ml in sodium bicarbonate pH 8.0, 0.1 ml/well) and incubated at 4°C. Coating solution was aspirated the following day and wells were blocked with 1 mg/ml gelatin in PBS (plus 1x thimerasol) for 2 days at 4°C. Prior to using, wells were washed 3x with wash buffer (PBS with 0.05% Tween).
• PBS wash buffer
• EIA buffer 5 mg/ml bovine ⁇ -globulin, 1 mg/ml gelatin, 1 ml/l Tween-20, 1 mg/ml thimerasol in PBS
• EIA buffer 5 mg/ml bovine ⁇ -globulin, 1 mg/ml gelatin, 1 ml/l Tween-20, 1 mg/ml thimerasol in PBS
• Wells 0.1 ml/well
• a mixture of rabbit anti-human TNF ⁇ polyclonal antibodies (1 :400 dilution of Sigma #T8300, and 1 :400 dilution of Calbiochem #654250 was added for 1 hr at 37°C.
• Preferred aminocyanopyridine MK-2 inhibiting compounds of the present invention provide TNF ⁇ release Ido values of below 200 ⁇ M in an in vitro cell assay.
• One method that can be used for determining TNF ⁇ release IC 5 o in an in vitro cell assay is that described just above.
• More preferred aminocyanopyridine MK-2 inhibiting compounds have the capability of providing TNF ⁇ release IC 50 values of below 50 ⁇ M, yet more preferred of below 10, and even more preferred of below 1.0 ⁇ M.
• LPS Lipopolvsaccharide
• Compounds were prepared as a suspension in a vehicle consisting of 0.5% methylcellulose, 0.025% Tween-20 in PBS. Compounds or vehicle were orally administered in a volume of 1 ml using an 18 gauge gavage needle.
• LPS E. coli serotype 0111 :B4, Lot #39H4103, Cat. # L-2630, Sigma
• Plasma was administered 1 -4 hr later by injection into the penile vein at a dose of 1 mg/kg in 0.5 ml sterile saline.
• Blood was collected in serum separator tubes via cardiac puncture 1.5 hr after LPS injection, a time point corresponding to maximal TNF ⁇ production. After clotting, serum was withdrawn and stored at -20°C until assay by ELISA (described below).
• ELISA plates (NUNC-lmmunoTM Plate MaxisorbTM Surface) were coated with 0.1 ml per well of an Protein G purified fraction of a 2.5 ug/ml of hamster anti-mouse/rat TNF ⁇ monoclonal antibody TN 19.12 (2.5 ug/ml in PBS, 0.1 ml/well).
• the hybridoma cell line was kindly provided by
• TNF levels in serum were quantitated from a recombinant rat TNF ⁇ (BioSource International, Cat. #PRC3014.) standard curve using a quadratic parameter fit generated by SoftMaxPRO software. ELISA sensitivity was approximately 30 pg TNF/ml. Results are expressed in percent inhibition of the production of TNF ⁇ as compared to blood collected from control animals dosed only with vehicle.
• Preferred aminocyanopyridine MK-2 inhibiting compounds of the present invention are capable of providing some degree of inhibition of TNF ⁇ in animals. That is, the degree of inhibition of TNF ⁇ in animals is over 0%.
• One method for determining the degree of inhibition of TNF ⁇ is the rat LPS assay that is described just above.
• More preferred aminocyanopyridine MK-2 inhibiting compounds have the capability of providing rat LPS TNF ⁇ inhibition values of at least about 25%, even more preferred of above 50%, yet more preferred of above 70%, and even more preferred of above 80%.
• Synthesis of aminocyanopyridine compounds [000132] A general method for the synthesis of aminocyanopyridines described in Examples 1 - 213 can be found in Kambe, S. et al, "A simple method for the preparation of 2-amino-4-aryl-3-cyanopyridines by the condensation of malononitrile with aromatic aldehydes and alkyl ketones in the presence of ammonium acetate", Synthesis 5:366 - 368 (1980). Further details of the synthesis of aminocyanopyridines of the present invention are provided below.
• EXAMPLE 2 This example illustrates the production of 2-amino-4-(2- fluorophenyl)-6-(2-furyl)nicotinonitrile trifluoroacetate.
• 2-Fluorobenzaledhyde (2 mmol, 1.0 equiv., 210 ⁇ L)
• malononitrile (2 mmol, 1.0 equiv., 126 ⁇ L) were combined in toluene (3 mL) and heated to 50°C for 0.5 hours.
• Step 1 Production of 2-(1 H-imidazol-5- ylmethylene)malononitrile.
• malononitrile (20 mmol, 1.0 equiv., 1.26mL) were combined in trimethylorthoformate (30 mL) and triethylamine (7mL).
• Step 2 Production of 2-[(1 - ⁇ [2-(trimethylsilyl)ethoxy]methyl ⁇ -1 H- inidazol-5-yl)methylene)malononitrile;
• Step 3 Production of 2-amino-6-(4-hydroxyphenyl)-4-(1 H- imidazol-5-yl)nicotinonitrile trifluoroacetate.
• EXAMPLE 7 This illustrates the production of 2-amino-6-(2-furyl) ⁇ 4-(1- methyl-1 H-imidazol-4-yl)nicotinonitrile bis(trifluoroacetate).
• 2-acetylfuran 1.0 mmol, 1.0 equiv., 100 ⁇ L
• ammonium acetate 1.5 mmol, 1.5 equiv., 115mg
• EXAMPLE 59 This illustrates the production of 4-[2-amino-3-cyano-6-(2- furyl)pyridin-4-yl]-1 H-pyrrole-2-carboxamide.
• EXAMPLE 76 This illustrates the production of 2-amino-6-(2-furyl)-4-(1 H- imidazol-5-yl)nicotinonitrile trifluoroacetate. [000163] StegJ . : Production of 2-amino-6-(2-furyl)-4-(1 - ⁇ [2-
• Step 2 Production of 2-amino-6-(2-furyl)-4-(1 H-imidazol-5- yl)nicotinonitrile trifluoroacetate.
• EXAMPLE 78 This illustrates the production of 4-[6-amino-5-cyano-4-(2- fluorophenyl)pyridin-2-yl]benzoic acid trifluoroacetate.
• Step 1 Production of 1 -(1 H-pyrazol-5-yl)-1 -ethanone.
• MNNG 1 -Methyl-3-1- nitrosoguanidine
• Step 2 Production of 2-amino-4-(2-furyl)-6-(1 H-pyrazol-3- yl)nicotinonitrile trifluoroacetate.
• EXAMPLE 92 This illustrates the production of 2-amino-4-(2-furyl)-8-hydroxy- 5, 6-dihydrobenzo[h]quinoline-3-carbonitrile trifluoroacetate.
• a glass vial was charged with 6-hydroxy-2-tetralone (0.49 g, 3 mmol), malononitrile, (0. g, 3 mmol), ammonium acetate (0. g, 6 mmol), furaldehyde (0. g, 3 mmol) and a magnetic stirring bar.
• Benzene (6 mL) was added to the vial, which was capped and heated to 80 degrees Celsius for 18 hours.
• the resin was washed sequentially with a 1 :1 mixture of methanol and dichloromethane (2X15 mL), 2 M ammonia in methanol (2X15 mL), and a 1 :1 mixture of methanol and dichloromethane (2X15 mL).
• the combined filtrates were concentrated in vacuo, and the residue was purified by reverse phase chromatography.
• the product was isolated as a tan solid (10.4 mg, 1 % yield).
• EXAMPLE 97 This illustrates the production of 2-amino-3-cyano-4-(4H-1 ,2,4- triazol-3-yl)-5,6-dihydrobenzo[h]quinoline-8-carboxylic acid bis (trif I uo roacetate) .
• This material was prepared in a manner similar to that used to produce 2-amino-4-(2-furyl)-8-hydroxy-5,6-dihydrobenzo[h]quinoline-3- carbonitrile trifluoroacetate, as described in Example 92. The product was isolated as a tan solid (29.4 mg, 4% yield).
• EXAMPLE 98 This illustrates the production of 2-amino-4-(2-furyl)-5,6- dihydro-1 ,8-phenanthroline-3-carbonitrile bis(trifluoroacetate).
• EXAMPLE 99 This illustrates the production of 2-amino-4-(2-fluorophenyl)-6,8 ⁇ dihydro-5H-pyrazolo[3,4-h]quinoline-3-carbonitrile bis(trifluoroacetate).
• 2-amino-4-(2-fluorophenyl)-6,8-dihydro-5H-pyrazolo[3,4- h]quinoline-3-carbonitrile bis(trifluoroacetate) was prepared in a manner similar to that used to produce 2-amino-4-(2-furyl)-8-hydroxy-5,6- dihydrobenzo[h]quinoline-3-carbonitrile trifluoroacetate, as described in
• Example 92 The product was isolated as a yellow solid (173.7 mg, 17% yield).
• EXAMPLE 101 [000196] This illustrates the production of 2-amino-3-cyano-4-(2-furyl)- 5,6-dihydrobenzo[h]quinoline-8-carboxyIic acid trifluoroacetate.
• Step- 1 (Preparation of 5-oxo-5,6,7,8-tetrahydronaphthalene-2- yl-trifluoromethanesulfonate) -
• 6-hydroxy-1 -tetralone 7.87 g, 48.5 mmol
• pyridine 97 mL
• triflic anhydride 8.24 mL, 49 mmol
• Step 2 (Preparation of methyl 5-0X0-5,6,7,8- tetrahydronaphthalene-2-carboxylate) - A three-necked round bottomed flask was charged with 5-oxo-5,6,7,8-tetrahydronaphthalene-2-yl- trifluoromethanesulfonate, prepared as described in Step 1 , (9.98 g, 33.9 mmol), bis(diphenylphosphonyl)propane (0.42 4, 1 mmol), palladium acetate (0.23 g, 1 mmol), methanol (34 mL), dimethylformamide (68 mL), triethylamine (9.5 mL, 68.3 mmol) and a magnetic stirring bar.
• the flask was fitted with a condenser and septa, then carbon monoxide was bubbled through the solution for 15 minutes.
• the flask was placed under a nitrogen atmosphere and heated to 70 degrees Celsius for 8 hours.
• the mixture was diluted with ethyl acetate (200 mL) and washed with water (1X100 mL), 5% aqueous hydrogen chloride (2X200 mL) and brine (1X100 mL).
• the organic layer was dried over magnesium sulfate and concentrated in vacuo.
• the residue was purified by flash column chromatography (0-30% ethyl acetate/hexane) to give 4.08 g of product as a yellow solid (59% yield).
• Step 3 (Preparation of 2-amino-3-cyano-4-(2-furyl)-5,6- dihydrobenzo[h]quinoline-8-carboxylic acid trifluoroacetate) -
• a glass vial was charged with methyl 5-oxo-5,6,7,8-tetrahydronaphthalene-2- carboxylate, as prepared in Step 2, above, (1.03 g, 5.06 mmol), malononitrile (0.363, 5.5 mmol), 2-furaldehyde (0.42 mL, 5.07 mmol), ammonium acetate (0.794 g, 10.3 mmol), toluene (10 mL) and a magnetic stirring bar.
• the vial was capped and heated to 80 degrees Celsius for 24 hours.
• the vial was cooled to room temperature, then the reaction mixture was diluted with a 1 :1 mixture of dichloromethane/methanol (20 mL), and amberlyst resin (20 g) was added to the flask.
• the slurry was agitated for 72 hours at room temperature, then the resin was collected by vacuum filtration and washed with dichloromethane (3x30 mL).
• the resin was then combined with 2 M ammonia in methanol and agitated for 4 hours at room temperature.
• the resin was filtered and washed with a 1 :1 mixture of dichloromethane/2M ammonia in methanol (6X30 mL).
• the combined filtrates were concentrated in vacuo.
• EXAMPLE 102 This illustrates the preparation of 2-amino-4-(2,3- difluorophenyl)-6,7-dihydro-5H-pyrazolo[3,4-h]quinoline-3-carbonitrile bis(trifluoroacetate).
• Example 106 The product was isolated as a yellow solid (205.7 mg, 17% yield).
• 1 H NMR 400 MHz, DMSO
• ⁇ 2.55-2.60 m, 2H
• 2.72-2.80 m, 2H
• 6.81 br s, 1 H
• 7.25-7.32 m, 1 H
• 7.38-7.46 m, 1 H
• 7.58-7.68 m, 1 H
• 7.97 s, 1 H
• m/z 324 (M+H) HRMS (M+H) calculated for C17H1 2 F2N 5 : 324.1055, found 324.1030.
• EXAMPLE 103 This illustrates the preparation of 2-amino-4-(2,4- difluorophenyl)-6,7-dihydro-5H-pyrazolo[3,4-h]quinoline-3-carbonitrile bis(trifluoroacetate).
• EXAMPLE 105 This illustrates the preparation of 8-amino-6-(2-furyl)-4,5- dihydro-1 H-pyrazolo[4,3-h]quinoline-7-carbonitrile.
• 8-amino-6-(2-furyl)-4,5-dihydro-1 H-pyrazolo[4,3-h]quinoline-7- carbonitrile was prepared in a manner similar to that used to produce 2- amino-4-(2-furyl)-8-hydroxy-5,6-dihydrobenzo[h]quinoline-3-carbonitrile trifluoroacetate, as described in Example 92. The product was isolated as a yellow solid (51 mg, 8% yield).
• EXAMPLE 107 This illustrates the preparation of 8-amino-6-(2-furyl)-4,5- dihydro-1 H-pyrazolo[4,3-h]quinoline-7-carbonitrile trifluoroacetate.
• 8-amino-6-(2-f uryl)-4,5-dihydro-1 H-pyrazolo[4,3-h]quinoline-7- carbonitrile trifluoroacetate was prepared in a manner similar to that used to produce 2-amino-4-(2-furyl)-8-hydroxy-5,6-dihydrobenzo[h]quinoline-3- carbonitrile trifluoroacetate, as described in Example 92.
• EXAMPLE 175 This illustrates the preparation of 4-[6-amino-5-cyano-4-(2- furyl)pyridin-2-yl]benzoic acid trifluoroacetate.
• a glass vial was charged with 4-acetylbenzoic acid (0.33 g, 2 mmol), malononitrile, (0.12 g, 3 mmol), ammonium acetate (0.23 g, 6 mmol), furaldehyde (0.19 g, 3 mmol) and a magnetic stirring bar. Toluene (3 mL) was added to the vial, which was capped and heated to 80 degrees Celsius for 18 hours.
• the resin was washed sequentially with a 1 :1 mixture of methanol and dichloromethane (2X15 mL), 2 M ammonia in methanol (2X15 mL), and a 1 :1 mixture of methanol and dichloromethane (2X15 mL).
• the combined filtrates were concentrated in vacuo, and the residue was purified by reverse phase chromatography.
• the product was isolated as a tan solid (9.1 mg, 1 % yield).
• EXAMPLE 215 This illustrates the production of 2,4-diamino-10-methyl-5,10- dihydrobenzo[b]-1 ,8-naphthyridine-3-carbonitrile trifluoroacetate.
• Step 1 (synthesis of f-Butyl 2-bromophenyl(methyl)carbamate)
• 2-bromoaniline 25 mmol, 1.0 equiv. 4.3g
• THF 150 mL
• Sodium hydride 60% in mineral oil, 1.1g
• a solution of di-f-butyl-dicarbonate in THF 1.0M, 30 mmol, 1.2 equiv., 30 mL
• sodium hydride 1.1 g
• Step 2 (synthesis of 2,4-diamino-10-methyl-5, 0- dihydrobenzo[b]-1 ,8-naphthyridine-3-carbonitrile trifluoroacetate) [000224] f-Butyl 2-bromophenyl(methyl)carbamate (2.65 mmol, 1.0 equiv., 759 mg) was dissolved in THF (20 mL).
• reaction mixture was quenched with sat. aq. NH 4 CI, and partitioned between ether and water. The organic layer was washed with water and dried over MgSO 4 , filtered and evaporated to get 820 mg of a yellow oil. This oil was carried on immediately without purification or characterization. The resulting oil was treated with 2-amino-1-propene-1 ,1 ,3-tricarbonitrile
• EXAMPLE 216 This illustrates the production of 2,4-diamino-8-ethoxy-7- hydroxy-5H-chromeno[2,3-b]pyridine-3-carbonitrile.
• EXAMPLE 217 [000227] This illustrates the production of 2,4-diamino-8-(2- ethoxyethoxy)-7-hydroxy-5H-chromeno[2,3-b]pyridine-3-carbonitrile.
• 2,4-diamino-8-(2-ethoxyethoxy)-7-hydroxy-5H-chromeno[2,3- b]pyridine-3-carbonitrile was prepared from 2,4-diamino-8-hydroxy-5H- chromeno[2,3-b]pyridine-3-carbonitrile in the same method as described above in Example 216, using 2-bromoethyl-ethylether in lieu of 2- bromoethyl-ethylether.
• EXAMPLE 220 [000231] This illustrates the production of 2,4-diamino-7,8-dihydroxy-5H- chromeno[2,3-b]pyridine-3-carbonitrile.
• EXAMPLE 221 This illustrates the production of 2,4-diamino-8-hydroxy-5H- chromeno[2,3-b]pyridine-3-carbonitrile.
• 2,4-Dihydroxy-benzaldehyde (43.4mmol, 6.0g), 2-amino-1- propene-1 ,1 ,3-tricarbonitrile (43.4mmol, 5.74g), acetic acid (13.0mL), and ethanol (125.0mL) were combined and heated to reflux for 2 hours.
• the reaction slurry was concentrated in vacuo and then dissolved in trifluoroacetic acid (160.0mL) at 0°C.
• Triethylsilane (0.28mol, 32.76g,
• EXAMPLE 222 [000235] This illustrates the production of 8,10-diamino-2,3-dihydro-11 H- [1 ,4]dioxino[2',3':6,7]chromeno[2,3-b]pyridine-9-carbonitrile.
• EXAMPLE 224 This illustrates the production of 2,4-diamino-8-(2-pyrrolidin-1 - ylethoxy)-5H-chromeno[2,3-b]pyridine-3-carbonitrile .
• 2,4-diamino-8-(2-pyrrolidin-1 -ylethoxy)-5H-chromeno[2,3- b]pyridine-3-carbonitrile was prepared from 2,4-diamino-8-hydroxy-5H- chromeno[2,3-b]pyridine-3-carbonitrilein the same manner as described in Example 223, using 1 -(2-chloroethyl)pyridine in lieu of 2-bromoethyl- ethylether.
• EXAMPLE 225 [000241] This illustrates the production of 2,4-diamino-8-(2- aminoethoxy)-5H-chromeno[2,3-b]pyridine-3-carbonitrile.
• TNF ⁇ release assay IC 50 6.9 ⁇ M; Rat LPS Assay 88% inhibition at 20 mpk (IP). '
• EXAMPLE 227 [000245] This illustrates the production of 2,4-diamino-8-(2- hydroxyethoxy)-5H-chromeno[2,3-b]pyridine-3-carbonitrile.
• EXAMPLE 228 [000247] This illustrates the production of 2,4-diamino-8-(2-morpholin-4- ylethoxy)-5H-chromeno[2,3-b]pyridine-3-carbonitrile.
• EXAMPLE 236 [000251] This illustrates the production of 2,4-diamino-9-methoxy-5H- chromeno[2,3-b]pyridine-3-carbonitrile bis(trifluoroacetate).
• EXAMPLE 238 [000255] This illustrates the production of 2,4-diamino-5H-chromeno[2,3- b]pyridine-3-carbonitrile Bis(trifluoroacetate).
• 2,4-diamino-5H-chromeno[2,3-b]pyridine-3-carbonitrile was prepared in the same manner as described in Example 236 except that salicyaldehyde was used in place of methoxysalicyaldehyde. The product was isolated as a light tan solid (1.26 g, 33% yield).
• EXAMPLE 240 [000259] This illustrates the production of 2,4-diamino-9-hydroxy-8- methoxy-5H-chromeno[2,3-b]pyridine-3-carbonitrile trifluoroacetate.
• EXAMPLE 241 This illustrates the production of 2,4-diamino-9-hydroxy-5H- chromeno[2,3-b]pyridine-3-carbonitrile trifluoroacetate.
• 2,3-dihydroxybenzaldehyde (5 mmol, 691 mg), 2-amino-1 - propene-1 ,1 ,3-tricarbonitrile (5 mmol, 661 mg), acetic acid (1.2 mL), and ethanol (20 mL) were combined and heated to reflux overnight. The reaction slurry was concentrated in vacuo and then dissolved in trifluoroacetic acid (20 mL) at 0°C.
• Step 1 Preparation of 2,4-diamino-7-nitro-5H-chromeno[2,3- b]pyridine-3-carbonitrile: 5-nitrosalicylaldehyde (132 mmol, 22.00 g), 2- amino-1 -propene-1 ,1 ,3-tricarbonitrile (132 mmol, 17.39 g), acetic acid (31 mL), and ethanol (500 mL) were combined and heated to reflux overnight. The resulting slurry was concentrated in vacuo and then dissolved in trifluoroacetic acid (350 mL) at 0°C. Triethylsilane (1.40 mol, 162 g, 225 mL) was added.
• Step 2 A mixture of 2,4-diamino-7-nitro-5H-chromeno[2,3- b]pyridine-3-carbonitrile, produced as described above, (0.55 mmol, 155 mg) and palladium on carbon (Pd/C) (35 mg, 10% on activated carbon) in DMF (15 mL) was stirred under an atmosphere of hydrogen (balloon) for
• EXAMPLE 243 This illustrates the production of 2,4-diamino ⁇ 9-fluoro-5H- chromeno[2,3-b]pyridine-3-carbonitrile trifluoroacetate.
• 3-Fluoro-2-hydroxybenzaldehyde (3.45 mmol, 484 mg)
• 2- amino-1-propene-1 ,1 ,3-tricarbonitrile (3.50 mmol, 463 mg)
• acetic acid 0.9 mL
• ethanol 27 mL
• Triethylsilane (43mmol, 4.97 g, 6.9 mL) was added via syringe. The reaction was heated to reflux for 5 hours. Dichloromethane (50 mL) was added to the reaction and the solid formed was collected via filtration and washed with methanol. The product was isolated as a white solid (377 mg, 30% yield).
• 1 H NMR 500 MHz, DMSO- d 6 ): ⁇ 7.25-7.19 (m, 1 H), 7.15-7.08 (m, 1 H), 7.00-6.96 (m, 1 H), 6.70 (bs, 2H), 6.51 (bs, 2H), 3.75 (S, 2H); m/z 257 (M+H).
• EXAMPLE 244 This illustrates the production of 2,4-diamino-3-cyano-5H- chromeno[2,3-b]pyridine-7-carboxylic acid Bis(trifluoroacetate).
• 5-Carboxysalicyaldehyde (3 mmol, 500 mg)
• 2-amino-1 - propene-1 ,1 ,3-tricarbonitrile (3 mmol, 396 mg)
• acetic acid 1.2 mL
• ethanol 15 mL
• Triethylsilane (62 mmol, 7.2g, 10 mL) was added via syringe. The reaction was stirred for 4 hours at 50 °C and then was stirred overnight at room temperature. Dichloromethane (20 mL) was added to the reaction and the solid formed was collected via filtration and washed with dichloromethane (2x). The product was isolated as a yellow solid (560 mg, 36% yield).
• EXAMPLE 265 [000274] This illustrates the production of 2,4-diamino ⁇ 7-nitro-5H- thiochromeno[2,3-b]pyridine-3-carbonitrile.
• Step 1 Production of 5-Nitrothiosalicylaldehyde: A mixture of 2- chloro-5-nitrobenzaldehyde (2g, 11 mmol) and lithium sulfide (0.54 g, 11.7 mmol) in 30 L of anhydrous DMSO was stirred under nitrogen at room temperature overnight. The solution was then added to a mixture of ice- water, acidified with 2N HCl and extracted with ether three times.
• Step 2 A solution of the crude 5-nitro-2-thiosalicylaldehyde (1.3g, 7.1 mmol), 2-amino-1-propene-1 ,1 ,3-tricarbonitrile (7.6 mmol, 1 g), acetic acid (2.5 mL) in 70 mL of ethanol was heated at 76°C under nitrogen overnight. The reaction mixture was cooled to room temperature and filtered.
• Step 3 A reaction mixture of the aforementioned tricyclic intermediate (1.2 g, 4 mmol) and triethylsilane (15 mL) in 100 mL of trifluoroacetic acid was heated at between 60-65°C under nitrogen for 2 hours. After that, the solution was cooled to room temperature and concentrated in vacuo. Ether was added to the residue. The solid was filtered, washed with additional ether to give 2,4-diamino-7-nitro-5H- thiochromeno[2,3-b]pyridine-3-carbonitrile as an orange powder (0.9 g, 75% yield).
• EXAMPLE 266 This illustrates the production of 2,4,7 ⁇ triamino-5H- thiochromeno[2,3-b]pyridine-3-carbonitrile trifluoroacetate.
• EXAMPLE 267 This illustrates the production of 2,4-diamino-7-nitro-5H- thiochromeno[2,3-b]pyridine-3-carbonitrile 10,10-dioxide.
• EXAMPLE 269 This illustrates the production of 2,4-diamino-7-f!uoro-5H- thiochromeno[2,3-b]pyridine-3-carbonitrile.
• Example 271 and 0.6 mL of boron tribromide (6.4 mmol) in 30 mL of methylene chloride was stirred at room temperature for 18 h. After that, the solid was filtered, washed with methylene chloride, water and methanol. The methanol filtrate was concentrated to give a solid, which was washed with water, acetonitrile and ether to give the desired product as a red solid (54 mg, 33.6% yield).
• 1 H NMR 400 MHz, DMSO + D 2 0) ⁇ 9.520 (s, 1 H), 8.111 (d, 1 H), 7.561 (d, 1 H), 7.522 (s, 2H); m/z 271 (M+H).
• EXAMPLE 273 This illustrates the production of 2,4-diamino-7-nitro-5H- thiochromeno[2,3-b]pyridine-3-carbonitrile 10,10-dioxide (an alternative procedure).
• EXAMPLE 276 [000298] This illustrates the production of 2,4-diamino-7-methoxy ⁇ 5H- thiochromeno[2,3-b]pyridine-3-carbonitrile 10,10-dioxide.
• Example 280 2,4-bis ⁇ [2-(dimethylamino)ethyl]amino ⁇ -7,8-dimethoxy-5H- chromeno[2,3-b]pyridine-3-carbonitrile
• Example 281 2-amino-4-[(2-aminoethyl)amino]-7,8-dimethoxy-5H- chromeno[2,3-b]pyridine-3-carbonitrile
• Example 282 2-amino-4- ⁇ [2-(1 ,3-dioxo-1 ,3-dihydro-2H-isoindol-2- yl)ethyl]amino ⁇ -7,8-dimethoxy-5H-chromeno[2,3-b]pyridine-3-carbonitrile,
• Example 283 2-amino-7,8-dimethoxy-4-[(2-pyrrolidin-1 -ylethyl)amino]-5H- chromeno[2,3-b]pyridine-3-carbonitrile,
• Example 284 7,8-dimethoxy-2,4-bis[(2-pyrrolidin-1 -ylethyl)amino]-5H- chromeno[2,3-b]pyridine-3-carbonitrile
• Example 285 2,4-bis(glycinyl)-7,8-dimethoxy-5H-chromeno[2,3-b]pyridine-
• Example 286 ⁇ /-(2-amino-3-cyano-7,8-dimethoxy-5H-chromeno[2,3- b]pyridin-4-yl)glycine
• Example 287 7,8-dimethoxy-2,4-bis[(2-methoxyethyl)amino]-5H- chromeno[2,3-b]pyridine-3-carbonitrile
• Example 291 7,8-dimethoxy-2,4-bis(propylamino)-5H-chromeno[2,3- b]pyridine-3-carbonitrile
• Example 292 2-amino-7,8-dimethoxy-4-(propylamino)-5H-chromeno[2,3- b]pyridine-3-carbonitrile
• Example 294 2-amino-4-(ethylamino)-7,8-dimethoxy-5H-chromeno[2,3- b]pyridine-3-carbonitrile.
• EXAMPLE 296 [000310] This illustrates the production of 2-amino-7,8-dihydroxy-4- (propylamino)-5H-chromeno[2,3-b]pyridine-3-carbonitrile.
• EXAMPLE 297 This illustrates the production of 2-amino-7,8-dihydroxy-4-[(2- hydroxyethyl)amino]-5H-chromeno[2,3-b]pyridine-3-carbonitrile.
• Example 299 2,4-diamino-9-(2-aminoethoxy)-5H-chromeno[2,3- b]pyridine-3-carbonitrile
• Example 300 (2,4-diamino-3-cyano-5H-chromeno[2,3-b]pyridin-9- yl)oxy]acetic acid
• Example 305 2,4-diamino-9-hydroxy-6,8-bis(piperidin-1 -ylmethyl)-5H- chromeno[2,3-b]pyridine-3-carbonitrile
• Example 306 2,4-diamino-9-hydroxy-8-(piperidin-1 -ylmethyl)-5H- chromeno[2,3-b]pyridine ⁇ 3-carbonitrile, were produced starting with 2,4- diamino-9-hydroxy-5H-chromeno[2,3-b]pyridine-3-carbonitrile, produced as described in Examples 299 - 304, and
• Example 307 2,4-diamino-8-hydroxy-7,9-bis(piperidin-1 -ylmethyl)-5H- chromeno[2,3-b]pyridine-3-carbonitrile, was produced starting with 2,4- diamino-8-hydroxy-5H-chromeno[2,3-b]pyridine-3-carbonitrile, produced as described in Example 221.
• aminocyanopyridine compounds of the present invention can be produced by the same general method, and are shown in the table below along with NMR parameters, which were determined as described above.
• EXAMPLE 334 [000325] This illustrates the production of 2,4-diamino-7,8-dimethoxy-5H- chromeno[2,3-b]pyridine-3-carbonitrile.
• EXAMPLE 336 This illustrates the production of 2(2,4-diamino-3-cyano-7- bromo-5H-chromeno[2,3-b]pyridin-5-yl)malononitrile.
• EXAMPLE 337 This illustrates the production of 2(2,4-diamino-3-cyano-7- methoxy-5H-chromeno[2,3-b]pyridin-5-yl)malononitrile.
• 2-hydroxy-5-methoxybenzaldehyde (10mmol, 1.52g) and malononitrile (40mmol, 2.64g) in ethanol (350mL) was added six drops of piperidine and the mixture was stirred at room temperature for 18 hours. The resultant precipitate was collected by filtration, successively washed with ethanol and ether and and and air-dried to give the product as a grey solid (1.42g, 43% yield).
• EXAMPLE 339 - 348 This illustrates the production of certain aminocyanopyridine compounds of the present invention.
• EXAMPLE 349 This example illustrates that MK2 knock-out mice (MK2 (-/-)) are resistant to the formation of K/BN serum-induced arthritis.
• MK2 (-/-) MK2 knock-out mice
• a strain of mice has been reported that develops symptoms similar to human rheumatoid arthritis.
• the mice were designated K/BxN mice. See, Wipke, B. T. and P. M. Allen, J. of Immunology, / 57. 601 - 1608 (2001). Serum from the mice can be injected into host animals to provoke a typical RA response. The progression of the RA symptoms in the mice is measured by measuring paw thickness as a function of time.
• mice having normal MK-2 production were genetically altered by disabling the gene encoding MK-2 to produce mice having no capability of endogenous synthesis of active MK-2 (MK2 (-/-)).
• Normal host mice MK2 (+/+)
• MK-2 knock-out mice MK2 (-/-)
• All groups of mice were treated similarly, except that one group (Normal), composed of MK2 (+/+) mice that served as the control group, was not injected with serum from K/BxN mice, while the other three groups were injected with K/BxN serum at day 0.
• the other three groups of mice were MK2 (+/+),
• FIG. 1 is a graph that shows paw thickness as a function of time from day 0 to day 7 for MK2 (+/+) and MK2 (-/-) mice, which have received serum injection. It can be seen that paw thickness increased significantly for MK2(+/+) mice, whereas there was substantially no increase in paw thickness for MK2 knock-out mice.
• MK2 regulatory system This illustrates the utility of the MK2 regulatory system as a potential control point for the modulation of TNF production, and indicates that such regulation could serve as a treatment for inflammation -- such as that caused by arthritis, for example. It further shows that MK2 inhibition can have a beneficial effect on inflammation, and indicates that administration of an MK2 inhibitor can be an effective method of preventing or treating TNF modulated diseases or disorders.

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