Patent Application
20060019971
1. Field of the Invention
The present invention concerns methods of treatment for acute coronary syndrome using a pharmaceutically effective amount of p38 MAP kinase. More specifically, the invention concerns the treatment of cardiovascular disease including atherosclerosis.
2. Background
C-reactive protein (CRP) is a substance produced in the liver when arteries become inflamed. Arterial inflammation plays a central role in atherosclerosis and its complications. See Libby P., Nature. 2002;420:868-874; Libby P, Aikawa M, Nat Med. 2002 ;8:1257-1262; Libby, Ridker, Maseri, Circulation. 2002;105:1135-1143. Review; Bhatt D L, Topol E J, Circulation. 2002;106:136-140.. It is involved in leukocyte recruitment, pro-inflammmatory cytokine expression and thrombosis that is responsible for myocardial infarction and strokes. CRP has long been considered as a marker of inflammation but recently emerged as one of the strongest prognostic markers of cardiovascular events such as atherosclerosis, myocardial infarction, stroke, and vascular death in a variety of settings. See Blake G J, Ridker P M, J Intern Med. 2002;252:283-294; Blake G J, Ridker P M, Arterioscler Thromb Vasc Biol. 2002;22:1512-1513; Saadeddin S M, Habbab M A, Ferns G A, Med Sci Monit. 2002;8:RA5-12; Benzaquen L R, Yu H, Rifai N. Crit Rev Clin Lab Sci. 2002;39:459-497. Elevated CRP levels predict poor prognosis of future cardiac events both in patients with coronary disease (Blake G J, Ridker P M, Circ Res. 2001; 89: 763-771) and in apparently healthy men and women (Benzaquen L R, Yu H, Rifai N, Crit Rev Clin Lab Sci. 2002;39:459-497). Given the association of arterial inflammation in coronary disease, particularly in patients presenting with high levels of CRP, anti-inflammatory therapy appears to have a therapeutic benefit. Conventional therapies for alleviating the inflammatory response include aspirin (Ridker P M, Cushman M, Stampfer M J, Tracy R P, Hennekens C H, N Engl J Med. 1997;336:973-979) and statins. Many of the patients admitted to cardiac units are under such therapy. However, nearly 75% of such patients revisit the cardiac units. Although the conventional therapies provide temporary benefit, they do not target the specific p38 mediated mechanisms which are implicated in “unstable” atheromatous plaques and acute coronary disease.
The present invention is directed to mechanisms by which CRP and its negative effects are mediated through the p38 MAP kinase pathway. In one embodiment, the invention is directed to a method of treating various disorders associated with the presence and/or enhanced activity of CRP wherein said method comprises the administration of a pharmaceutically effective amount of p38 MAP kinase inhibitor to a patient in need thereof.
In a preferred embodiment, the invention is directed to a method of treating cardiovascular disease wherein said method comprises the administration of a pharmaceutically effective amount of an inhibitor of p38 MAP kinase to a patient in need thereof.
In another preferred embodiment, the invention is directed to a method of treating acute coronary disease wherein said method comprises the administration of a pharmaceutically effective amount of an inhibitor of p38 MAP kinase to a patient in need thereof.
In another preferred embodiment, the invention is directed to a method of treating atherosclerosis wherein said method comprises the administration of a pharmaceutically effective amount of an inhibitor of p38 MAP kinase to a patient in need thereof.
In yet another embodiment, the invention is directed to a method of treating arterial inflammation wherein said method comprises the administration of a pharmaceutically effective amount of an inhibitor of p38 MAP kinase to a patient in need thereof.
a and 1b are respectively a Western blot and a graph summarizing the results of such blot. The effect of CRP on p38 MAP kinase activation in HPBMCs is illustrated in these figures. Also illustrated is the effect of a p38 inhibitor on p38 kinase activation by CRP. 1.5×106 HPBMCs were plated in 48-well. After 1 hour the medium was removed and cells were incubated with or without 10 μg/ml of purified human CRP in serum-free RPMI 1640. In parallel experiments, cells were treated with 10% FCS together with the CRP treatment. To demonstrate p38 antagonism, the p38 inhibitor was added to a final concentration of 1 μM in the parallel treatment. After 5 minutes cells were lysed and phspho-p38 level was determined by Western. Phospho-p38 signal was normalized by GAPDH signal.
a-c are graphs which summarize data that demonstrates CRP promotion of cytokine IL-6 secretion via p38 signaling.
a-b are graphs which summarize data that demonstrate CRP promotes chemokine IL-8 secretion via p38 signaling.
a-d. are graphs which summarize data that demonstrates CRP induces TNFα and IL-1β secretion via the p38 pathway. FIGS. 4(a) and (b) demonstrate the effects of CRP on IL-1b (a) and TNFa (b) secretion from HPBMCs and their modulation by p38 inhibitor. 5×106 HPBMCs were plated in 6-well. After 1 hour medium was removed and cells were treated with vehicle or 1μM p38 inhibitor in RPMI 1640 medium. After 1 hour cells were stimulated with 10% FCS only or 10 μg/ml CRP plus 10% FCS. After 6 hours conditioned medium was collected and IL-1b and TNFa levels were determined by ELISA. FIGS. 4(c) and (d) demonstrate the effects of CRP on cytokine gene expressions in HPBMCs and their modulation by p38 inhibitor. 5×106 HPBMCs were plated in 6-well. After I hour medium was removed and cells were treated with vehicle or 1M p38 inhibitor in RPMI 1640 medium. After 1 hour cells were stimulated with 10% FCS only or 10 μg/ml CRP plus 10% FCS. After 2 hours RNA was extracted from the cells and TNFa (c) and IL-1b (d) expression levels were determined by real-time PCR as described in the Experimental section.
Definitions:
A “pharmaceutically effective amount” is intended an amount of a compound that, when administered to a mammal for treating a condition, disorder or disease, is sufficient to elicit a cellular response that is clinically significant, without excessive levels of side effects.
“Mammal” refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, and pet companion animals such as a household pet and other domesticated animal such as, but not limited to, cattle, sheep, ferrets, swine, horses, poultry, rabbits, goats, dogs, cats and the like. Preferred companion animals are dogs and cats. Preferably, the mammal is human.
A “patient” is intended a mammal, preferably a human, in need of treatment of a condition, disorder or disease.
Description
Atherosclerosis is clinically recognized under two pathophysiologically distinct syndromes: stable coronary syndromes resulting from severe stenoses and unstable acute coronary syndromes resulting from different vascular inflammation frequently superimposed with thrombosis, which results in serious cardiac events. In the inflammation theory, the entry of inflammatory cells such as monocytes into the arterial wall plays a pivotal role in atherosclerosis. See for example Saadeddin S M, Habbab M A, Ferns G A, Med Sci Monit. 2002;8:RA5-12; and Benzaquen L R, Yu H, Rifai N., Crit Rev Clin Lab Sci. 2002;39:459-497. During inflammation, the endothelial cell lining on the arterial wall is activated and expresses adhesion molecules and chemokines. This facilitates the recruitment of circulating monocytes and other leukocytes. Once the recruited monocytes emigrate into vessel wall they become tissue macrophages, which can uptake lipid droplets to form foam cells. Continuous entry of these monocytes into the arterial wall promotes the lesion development. In the plaque, foam cells produce inflammatory mediators and growth factors, which can damage tissue and promote further inflammation and stenosis. In unstable acute coronary syndromes thin fibrous cap surrounding the lesion is prone to rupture by proteases produced by inflammatory leukocytes in the plaque or by vasoconstriction. This can expose activated macrophages and endothelial cells that express potent procoagulant tissue factor to circulating blood, causing thrombus formation leading to myocardial ishemia and other complications. Inflammatory markers such as CRP and IL-6 not only participate in lesion formation but also alter plaque architecture in favor of rupture.
Besides being recognized as a marker for disease and potential coronary problems, CRP appears to be an independent risk factor and directly promotes vascular inflammation. CRP is found in atheroscleotic plaques within foam cells and smooth muscle cells (Torzewski, 2000, Yasojima, 2001). It aids recruitment of monocytes into the arterial wall, promotes the uptake of native LDL by macrophages to form foam cells, and stimulates macrophages to produce the prothrombotic tissue factor and cytokines, TNFa, I1-1b and IL-6. CRP activates complement in atherosclerotic plaques. In endothelial cells CRP induces expression of adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1) and E-selectin (10), and chemokine MCP-1. The pro-inflammatory effects of CRP in endothelial cells are implicated in part via IL-6 and endothelin-1. CRP sensitizes endothelial cells to destruction by cytotoxic CD4+ T cells. CRP has also been implicated in enhanced vasoreactivity in unstable plaques. Therefore, through several interrelated pathways, CRP exhibits deleterious effects on the vascular system and the stability of atheroscleotic plaques.
As referenced above, monocytes play a prominent role in vascular disease. Monocytes/macrophages inflammation seems to be pivotal in all stages of athersclerosis and acute coronary syndrome. As CRP is implicated in coronary syndromes, one possible route by which it exhibits an effect is by promoting various pro-inflammatory and pro-thrombotic gene expression in monocytes.
p38 MAP kinase appears to be implicated as a mediator between CRP and monocyte activity in the disease state. Accordingly, p38 antagonism in the context of CRP and vascular disease offers a novel therapy to treat underlying arterial inflammation and the complications associated with atherosclerosis including ischemia, myocardial infarction, unstable angina, stroke and sudden death.
In evaluating the role played by p38 in acute coronary disease, purified CRP was observed as activating or phosphorylating p38 kinase. As a result of p38 activation, the production and expression of certain inflammatory mediators from human peripheral blood monocytes (HPBMC's) is promoted. These effects are demonstrated below whereby CRP is observed to directly induce p38 MAP kinase activation in monocytes. In addition, CRP was also demonstrated to invoke IL-8, ET-1, COX-2, endothelin-1 and tissue factor in monocytes. This effect seems to be modulated through p38 kinase.
Pharmaceutical compositions useful for the present invention comprise an inhibitor of p38 MAP kinase as an active ingredient, a pharmaceutically acceptable carrier and optionally other therapeutic ingredients or adjuvants. The pharmaceutical compositions may be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.
Any known route of administration may used in the present invention. The compositions or compounds useful in the present invention may be administered orally, parenterally, topically, rectally, nasally, vaginally, or via implanted reservior. Parenteral or systemic administration includes, but is not limited to, subcutaneous, intravenous, intraperitoneally, intramuscular, intra-articular, intra-synovial, intrastemol, intrathecal, intralesional, and intracranial injections. Preferably, the compositions or compounds of the present invention are administered parenterally.
The manner of administration and the formulation and dosage of the compounds useful in the invention depends on the nature of the condition, the severity of the condition, the particular subject to be treated, and the judgment of the practitioner; formulation and dosage will depend on mode of administration.
Inhibitors of p38 MAP Kinase
As used herein, the term “inhibitor” includes any suitable molecule, compound, formulation or substance that may regulate p38 MAP kinase activity. The inhibitor may be a protein or fragment thereof, a small molecule compound, or even a nucleic acid molecule. It may affect a single p38 MAP kinase isoform or more than one isoform of p38 MAP kinase. In a preferred embodiment of the invention, the inhibitor regulates the α isoform of p38 MAP kinase.
According to the present invention, the inhibitor may exhibit its regulatory effect upstream or downstream of p38 MAP kinase or on p38 MAP kinase directly. Examples of inhibitor regulated p38 activity include those where the inhibitor may decrease transcription and/or translation of p38 MAP kinase, may decrease or inhibit post-translational modification and/or cellular trafficking of p38 MAP kinase, or may shorten the half-life of p38 MAP kinase. The inhibitor may also reversibly or irreversibly bind p38 MAP kinase, inhibit its activation, inactivate its enzymatic activity, or otherwise interfere with its interaction with downstream substrates.
If acting on p38 MAP kinase directly, the inhibitor should exhibit an IC50 value of about 10 μM or less, preferably 500 nm or less, more preferably 100 nm or less. In a related embodiment, the inhibitor should exhibit an IC50 value relative to the p38 α isoform that is preferably at least ten fold less than that observed when the same inhibitor is tested against other p38 MAPK isoforms in the same or comparable assay.
To determine whether a candidate is an inhibitor useful for the invention, an evaluation can be done on its p38 MAP kinase activity as well as its relative IC50 value. This evaluation can be accomplished through a variety of convential in vitro assays. Such assays include those that assess inhibition of kinase or ATPase activity of activated p38 MAP kinase. The assays may also assess the ability of the inhibitor to bind p38 MAP kinase or to reduce or block an identified downstream effect of activated p38 MAP kinase, e.g., cytokine secretion.
For example, conventional binding assays are fairly inexpensive and simple to run. As previously mentioned, binding of a molecule to p38 MAP kinase, in and of itself, may be inhibitory, due to steric, allosteric or charge-charge interactions. A binding assay can be performed in solution or on a solid phase using p38 MAP kinase or a fragment thereof as a target. By using this as an initial screen, one can evaluate libraries of compounds for potential p38 regulatory activity.
The target may be either free in solution, fixed to a support, expressed in or on the surface of a cell. A label (ie. radioactive, fluorescent, quenching, et cetera.) can be placed on the target, compound, or both to determine presence or absence of binding. This approach can also be used to conduct a competitive binding assay to assess the inhibition of binding of a target to a natural or artificial substrate or binding partner. In any case, one may measure, either directly or indirectly, the amount of free label versus bound label to determine binding. There are many known variations and adaptations of this approach to minimize interference with binding activity and optimize signal.
For purposes of in vitro cellular assays, the compounds that represent potential inhibitors of p38 MAP kinase function can be administered to a cell in any number of ways. Preferably, the compound or composition can be added to the medium in which the cell is growing, such as tissue culture medium for cells grown in culture. The compound is provided in standard serial dilutions or in an amount determined by analogy to known modulators. Alternatively, the potential inhibitor may be encoded by a nucleic acid that is introduced into the cell wherein the cell essentially produces the potential inhibitor itself.
Alternative assays involving in vitro analysis of potential inhibitors include those where cells (HeLa) transfected with DNA coding for relevant kinases can be activated with substances such as sorbitol, IL-1, TNF, or PMA (phorbol myristate acetate). After immunoprecipitation of cell lysates, equal aliquots of immune complexes of the kinases are pre-incubated for an adequate time with a specific concentration of the potential inhibitor followed by addition of kinase substrate buffer mix containing labeled ATP and GST-ATF2 or MBP. After incubation, kinase reactions are ceased by the addition of SDS loading buffer. Phosphorylated substrate is resolved through SDS-PAGE and visualized and quantitated in a phosphorimager. Both p38 regulation, in terms of phosphorylation, and IC50 values can be determined by quantitation. See, for example Kumar,S., McDonnell, P., Gum, R., Hand, A., Lee, J., and Young, P. (1997) Biochem. Biophys. Res. Commun. 235, 533-538.
Other in vitro assays may also assess the production of TNF-A as a correlate to p38 MAP kinase activity. One such example is a human whole blood assay. In this assay, venous blood is collected from healthy male volunteers into a heparinized syringe and is used within 2 hours of collection. Test compounds are dissolved in 100% DMSO and 1 μl aliquots of drug concentrations ranging from 0 to 1 mM are dispensed into quadruplicate wells of a 24-well microtiter plate (Nunclon Delta SI, Applied Scientific, So. San Francisco, Calif.). Whole blood is added at a volume of 1 ml/well and the mixture is incubated for 15 minutes with constant shaking (Titer Plate Shaker, Lab-Line Instruments, Inc., Melrose Park, Ill.) at a humidified atmosphere of 5% CO2 at 37° C. Whole blood is cultured either undiluted or at a final dilution of 1:10 with RPMI 1640 (Gibco 31800 +NaHCO3, Life Technologies, Rockville, Md. and Scios, Inc., Sunnyvale, Calif.). At the end of the incubation period, 10 μl of LPS (E. coli 0111:B4, Sigma Chemical Co., St. Louis, Mo.) is added to each well to a final concentration of 1 or 0.1 μg/ml for undiluted or 1:10 diluted whole blood, respectively. The incubation is continued for an additional 2 hours. The reaction is stopped by placing the microtiter plates in an ice bath and plasma or cell-free supernates are collected by centrifugation at 3000 rpm for 10 minutes at 4° C. The plasma samples are stored at −80° C. until assayed for-TNF-α levels by ELISA, following the directions supplied by Quantikine Human TNF-α assay kit (R&D Systems, Minneapolis, Minn.). IC50 values are calculated using the concentration of inhibitor that causes a 50% decrease as compared to a control.
A similar assay is an enriched mononuclear cell assay. The enriched mononuclear cell assay, begins with cryopreserved Human Peripheral Blood Mononuclear Cells (HPBMCs) (Clonetics Corp.) that are rinsed and resuspended in a warm mixture of cell growth media. The resuspended cells are then counted and seeded at 1×106 cells/well in a 24-well microtitre plate. The plates are then placed in an incubator for an hour to allow the cells to settle in each well. After the cells have settled, the media is aspirated and new media containing 100 ng/ml of the cytokine stimulatory factor lipopolysaccharide (LPS) and a test chemical compound is added to each well of the microtiter plate. Thus, each well contains HPBMCs, LPS and a test chemical compound. The cells are then incubated for 2 hours, and the amount of the cytokine Tumor Necrosis Factor Alpha (TNF-α) is measured using an enzyme linked immunosorbent assay (ELISA). One such ELISA for detecting the levels of TNF-α is commercially available from R&D Systems. The amount of TNF-α production by the HPBMCs in each well is then compared to a control well to determine whether the chemical compound acts as an inhibitor of cytokine production.
IC50 values are calculated using the concentration of inhibitor that causes a 50% decrease as compared to a control.
Exemplary Inhibitors
Compounds useful in the practice of the present invention include, but are not limited to, compounds of formula:
wherein
R1 is a heteroaryl ring selected from 4-pyridyl, pyrimidinyl, quinolyl, isoquinolinyl, quinazolin-4-yl, 1-imidazolyl, 1-benzimidazolyl, 4-pyridazinyl, and a 1,2,4-triazin-5-yl ring, which heteroaryl ring is substituted one to three times with Y, N(R10)C(O)Rb, a halo-substituted mono- or di-C1-6 alkyl-substituted amino, or NHRa and which ring is further optionally substituted with C1-4 alkyl, halogen, hydroxyl, optionally-substituted C1-4 alkoxy, optionally-substituted C1-4 alkylthio, optionally-substituted C1-4 alkylsulfinyl, CH2OR12, amino, mono- and di-C1-6 alkyl-substituted amino, NHFa, N(R10)C(O)Rb, N(R10)S(O)2Rd, or an N-heterocyclyl ring which has from 5 to 7 members and optionally contains an additional heteroatom selected from oxygen, sulfur or NR15;
Y is X1-Ra;
X1 is oxygen or sulfur;
Ra is C1-6 alkyl, aryl, arylC1-6 alkyl, heterocyclic, heterocyclylC1-6 alkyl, heteroaryl, or heteroarylC1-6 alkyl, wherein each of these moieties can be optionally substituted;
Rb is hydrogen, C1-6 alkyl, C3-7 cycloalkyl, aryl, arylC1-4 alkyl, heteroaryl, heteroarylC1-4 alkyl, heterocyclyl, or heterocyclylC1-4 alkyl;
Rd is C1-6 alkyl, C3-7 cycloalkyl, aryl, arylC1-4 alkyl, heteroaryl, heteroarylC1-4 alkyl, heterocyclyl, or heterocyclylC1-4 alkyl;
R3 is hydrogen;
R4 is phenyl, naphth-1-yl, naphth-2-yl, or a heteroaryl, which is optionally substituted by one or two substituents, each of which is independently selected, and which, for a 4-phenyl, 4-naphth-1-yl, 5-naphth-2-yl or 6-naphth-2-yl substituent, is halogen, cyano, nitro, —C(Z)NR7R17, —C(Z)OR16, —(CR10R20)vCOR12, —SR5, —SOR5, —OR12, halo-substituted-C1-4 alkyl, C1-4 alkyl, —ZC(Z)R12, —NR10C(Z)R16, or —(CR10R20)vNR10R20 and which, for other positions of substitution, is halogen, cyano, —C(Z)NR13R14, —C(Z)ORf, —(CR10R20)m″CORf, —S(O)mRf, —ORf, —OR12, halo-substituted C1-4 alkyl, C1-4 alkyl, —(CR10R20)m″NR10C(Z)Rf, —NR10S(O)m′R8, —NR10S(O)m′NR7R17, —ZC(Z)Rf, —ZC(Z)R12, or —(CR10R20)m″NR13R14;
Rf is heterocyclyl, heterocyclylC1-10 alkyl or R8;
Z is oxygen or sulfur;
v is 0,1, or 2;
m is 0, 1, or 2;
m′ is 1 or 2;
m″ is 0, 1,2,3,4, or 5;
R2 is C1-10 alkyl N3, —(CR10R20)n′OR9, heterocylyl, heterocycylC1-10 alkyl, C1-10 alkyl, halo-substituted C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C3-7 cycloalkyl, C3-7 cycloalkylC1-10 alkyl, C5-7 cycloalkenyl, C5-7cycloalkenylC1-10 alkyl, aryl, arylC1-10 alkyl, heteroaryl, heteroarylC1-10 alkyl, (CR10R20)nOR11, (CR10R20)nS(O)mR18, (CR10R20)nNHS(O)2R18, (CR10R20)nNR13R14, (CR10R20)nNO2, (CR10R20)nCN, (CR10R20)n′SO2R18, (CR10R20)nS(O)m′NR13R14, (CR10R20)nC(Z)R11, (CR10R20)nOC(Z)R11, (CR10R20)nC(Z)OR11, (CR10R20)nC(Z)NR13R14, (CR10R20)nC(Z)NR11OR9, (CR10R20)nNR10C(Z)R11, (CR10R20)nNR10C(Z)NR13R14, (CR10R20)nN(OR6)C(Z)NR13R14, (CR10R20)nN(OR6)C(Z)R11, (CR10R20)nC(═NOR6)R11, (CR10R20)nNR10C(═NR19)NR13R14, (CR10R20)nOC(Z)NR13R14, (CR10R20)nNR10C(Z)NR13R14, (CR10R20)nNR10C(Z)OR10, 5-(R18)-1,2,4-oxadiazol-3-yl or 4-(R12)-5-(R18R19)-4,5-dihydro-1,2,4-oxadiazol-3-yl; wherein the aryl, arylalkyl, heteroaryl, heteroaryl alkyl, cycloalkyl, cycloalkyl alkyl, heterocyclic and heterocyclic alkyl groups can be optionally substituted;
n is an integer having a value of 1 to 10;
n′ is 0, or an integer having a value of 1 to 10;
R5 is hydrogen, C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl or NR7R17, excluding the moieties ‥SR5 being —SNR7R17 and —S(O)R5 being —SOH;
R6 is hydrogen, a pharmaceutically-acceptable cation, C1-10 alkyl, C3-7 cycloalkyl, aryl, arylC1-4 alkyl, heteroaryl, heteroarylC1-10 alkyl, heterocyclyl, aroyl, or C1-10 alkanoyl;
R7 and R17 are each independently selected from hydrogen or C1-4 alkyl, or R7 and R17 together with the nitrogen to which they are attached form a heterocyclic ring of 5 to 7 members which ring optionally contains an additional heteroatom selected from oxygen, sulfur or NR15;
R8 is C1-10 alkyl, halo-substituted C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C3-7 cycloalkyl, C5-7 cycloalkenyl, aryl, arylC1-10 alkyl, heteroaryl, heteroarylC1-10 alkyl, (CR10R20)nOR11, (CR10R20)nS(O)mR18, (CR10R20)nNHS(O)2R18, or (CR10R20)nNR13R14, wherein the aryl, arylalkyl, heteroaryl, and heteroaryl alkyl can be optionally substituted;
R9 is hydrogen, —C(Z)R11, optionally-substituted C1-10 alkyl, S(O)2R18, optionally-substituted aryl or optionally-substituted arylC1-4 alkyl;
R10 and R20 are each independently selected from hydrogen or C1-4 alkyl;
R11 is hydrogen, C1-10 alkyl, C3-7 cycloalkyl, heterocyclyl, heterocyclylC1-10 alkyl, aryl, arylC1-10 alkyl, heteroaryl or heteroarylC1-10 alkyl;
R12 is hydrogen or R16;
R13 and R14 are each independently selected from hydrogen or optionally-substituted C1-4 alkyl, optionally-substituted aryl or optionally-substituted arylC1-4 alkyl, or together with the nitrogen to which they are attached form a heterocyclic ring of 5 to 7 members which ring optionally contains an additional heteroatom selected from oxygen, sulfur or NR9;
R15 is R10 or C(Z)C1-4 alkyl;
R16 is C1-4 alkyl, halo-substituted C1-4 alkyl, or C3-7 cycloalkyl;
R18 is C1-10 alkyl, C3-7 cycloalkyl, heterocyclyl, aryl, arylC1-10 alkyl, heterocyclyl, heterocyclylC1-10 alkyl, heteroaryl or heteroarylC1-10 alkyl; and
R19 is hydrogen, cyano, C1-4 alkyl, C3-7 cycloalkyl or aryl;
or a pharmaceutically-acceptable salt thereof,
or wherein
R1, Y, X1, Ra, Rb, Rd, V, m, m′, m″, Z, n, n′, and R5 are defined as above, and
R2 is hydrogen, C1-10 alkyl, halo-substituted C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C3-7 cycloalkyl, C3-7 cycloalkylC1-10 alkyl, C5-7 cycloalkenyl, aryl, arylC1-10 alkyl, heteroaryl, heteroarylC1-10 alkyl, heterocyclyl, heterocyclylC1-10 alkyl, (CR10R28)nOR12, (CR10R28)n′OR13, (CR10R28)n′S(O)mR25, (CR10R28)nS(O)2R25, (CR10R28)n′NHS(O)2R25, (CR10R28)n′NR8R9, (CR10R28)n′NO2, (CR10R28)n′CN, (CR10R28)n′S(O)mNR8R9, (CR10R28)n′C(Z)R13, (CR10R28)n′C(Z)OR13, (CR10R28)n′C(Z)NR8R9, (CR10R28)n′C(Z)NR13OR12, (CR10R28)n′NR10C(Z)R13, (CR10R28)n′NR10C(Z)NR8R9, (CR10R28)n′N(OR21)C(Z)NR8R9, (CR10R28)n′N(OR21)C(Z)R13, (CR10R28)n′C(═NOR21)R13, (CR10R28)n′NR10C(═NR27)NR8R9, (CR10R28)n′OC(Z)NR8R9, (CR10R28)n′NR10C(Z)OR10, (CR10R28)n′NR10C(Z)OR10, 5-(R25)-1,2,4-oxadiazol-3-yl or 4-(R12)-5-(R18R19)-4,5-dihydro-1,2,4-oxadiazol-3-yl; wherein the cycloalkyl, cycloalkyl alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, or heterocyclylalkyl moieties can be optionally substituted;
R3 is hydrogen or Q-(Y1)t;
Q is an aryl or heteroaryl group;
t is 1, 2, or 3;
Y1 is independently selected from hydrogen, C1-5 alkyl, halo-substituted C1-5 alkyl, halogen, or —(CR10R20)nY2;
Y2 is OR8, NO2, S(O)m″R11, SR8, S(O)m″OR8, S(O)mNR8R9, NR8R9, O(CR10R20)n′NR8R9, C(O)R8, CO2R8, CO2(CR10R20)n′CONR8R9, ZC(O)R8, CN, C(Z)NR8R9, NR10C(Z)R8, C(Z)NR8OR9, NR10C(Z)NR8R9, NR10S(O)m″R11, N(OR21)C(Z)NR8R9, N(OR21)C(Z)R, C(═NOR21)R8, NR10C(═NR15)SR11, NR10C(═NR15)NR8R9, NR10C(═CR14R24)SR11, NR10C(═CR14R24)NR8R9, NR10C(O)C(O)NR8R9, NR10C(O)C(O)OR10, C(═NR13)NR8R9, C(═NOR13)NR8R9, C(═NR13)ZR11, OC(Z)NR8R8, NR10S(O)m″CF3,NR10C(Z)OR10, 5-(R18)-1,2,4-oxadiazol-3-yl or 4-(R12)-5-(R18R19)-4,5-dihydro-1,2,4-oxadiazol-3-yl;
R4 is phenyl, naphth-1-yl or naphth-2-yl which is optionally substituted by one or two substituents, each of which is independently selected, and which, for a 4-phenyl, 4-naphth-1-yl or 5-naphth-2-yl substituent, is halo, nitro, cyano, C(Z)NR7R17, C(Z)OR23, (CR10R20)vCOR36, SR5, SOR5, OR36, halo-substituted-C1-4 alkyl, C1-4 alkyl, ZC(Z)R36, NR10C(Z)R23, or (CR10R20)vNR10R20 and which, for other positions of substitution, is halo, nitro, cyano, C(Z)NR16R26, C(Z)OR8, (CR10R20)m″COR8, S(O)mR8, OR8, halo-substituted-C1-4 alkyl, C1-4 alkyl, (CR10R20)m″NR10C(Z)R8, NR10S(O)m′R11, NR10S(O)m′NR7R17, ZC(Z)R8 or (CR10R20)m″NR16R26;
R7 and R17 are each independently selected from hydrogen or C1-4 alkyl, or R7 and R17 together with the nitrogen to which they are attached form a heterocyclic ring of 5 to 7 members, which ring optionally contains an additional heteroatom selected from oxygen, sulfur or NR22;
R8 is hydrogen, heterocyclyl, heterocyclylalkyl or R11;
R9 is hydrogen, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C3-7 cycloalkyl, C5-7 cycloalkenyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl, or R8 and R9 can together with the nitrogen to which they are attached form a heterocyclic ring of 5 to 7 members, which ring optionally contains an additional heteroatom selected from oxygen, sulfur or NR12;
R10 and R20 are each independently selected from hydrogen or C1-4 alkyl;
R11 is C1-10 alkyl, halo-substituted C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C3-7 cycloalkyl, C5-7 cycloalkenyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl;
R12 is hydrogen, —C(Z)R13 or optionally-substituted C1-4 alkyl, optionally-substituted aryl, optionally-substituted arylC1-4 alkyl, or S(O)2R25;
R13 is hydrogen, C1-10 alkyl, C3-7 cycloalkyl, heterocyclyl, heterocyclylClilo alkyl, aryl, arylC1-10 alkyl, heteroaryl or heteroaryl C1-10 alkyl, wherein all of these moieties can be optionally substituted;
R14 and R24 are each independently selected from hydrogen, alkyl, nitro or cyano;
R15 is hydrogen, cyano, C1-4 alkyl, C3-7 cycloalkyl or aryl;
R16 and R26 are each independently selected from hydrogen or optionally-substituted C1-4 alkyl, optionally-substituted aryl or optionally-substituted arylC1-4 alkyl, or together with the nitrogen to which they are attached form a heterocyclic ring of 5 to 7 members, which ring optionally contains an additional heteroatom selected from oxygen, sulfur or NR12;
R18 and R19 are each independently selected from hydrogen, C1-4 alkyl, substituted alkyl, optionally-substituted aryl, optionally-substituted arylalkyl, or together denote an oxygen or sulfur;
R21 is hydrogen, a pharmaceutically-acceptable cation, C1-10 alkyl, C3-7 cycloalkyl, aryl, arylC1-4 alkyl, heteroaryl, heteroarylalkyl, heterocyclyl, aroyl, or C1-10 alkanoyl;
R22 is R10 or C(Z)—C1-4 alkyl;
R23 is C1-4 alkyl, halo-substituted-C1-4 alkyl, or C3-5 cycloalkyl;
R25 is C1-10 alkyl, C3-7 cycloalkyl, heterocyclyl, aryl, arylalkyl, heterocyclyl, heterocyclylC1-10 alkyl, heteroaryl or heteroarylalkyl;
R27 is hydrogen, cyano, C1-4 alkyl, C3-7 cycloalkyl, or aryl;
R28 is hydrogen, C1-6 alkyl, C3-7 cycloalkyl, aryl, arylC1-4 alkyl, heteroaryl, heteroarylC1-4alkyl, heterocyclyl, or heterocyclylC1-4 alkyl moiety, all of which can be optionally substituted; and
R36 is hydrogen or R23;
or a pharmaceutically acceptable salt thereof
Exemplary compounds of the above formula include but are not limited to:
Compounds of a different scaffold that are also useful in the practice of the present invention include, but are not limited to, compounds of formula:
wherein
R1 is hydrogen, C1-5 alkyl, halogen, C1-5 alkoxy, or arylC1-5 alkyl;
R2 and R4 are independently hydrogen, C1-5 alkyl, aryl, arylC1-5 alkyl, heteroaryl, heteroarylC1-5 alkyl, heterocyclic, or heterocyclicC1-5 alkyl; and
R3 is hydrogen or C1-3 alkyl; or a pharmaceutically-acceptable salt thereof.
Another class of compounds that are useful in the practice of the present invention include, but are not limited to, compounds of formula:
wherein
X is O, CH2, S or NH, or the moiety X—R1 is hydrogen;
R1 is hydrogen, C1-6 alkyl, C3-7 cycloalkyl, aryl, arylC1-6 alkyl, heterocyclyl, heterocyclylC1-6 alkyl, heteroaryl, or heteroarylC1-6 alkyl, any of which, except for hydrogen, can be optionally substituted;
V is CH or N;
Ar is an aryl or heteroaryl ring, either of which can be optionally substituted;
one of X1 and X2 is N, and the other is NR15, wherein R15 is hydrogen, C1-6 alkyl, or arylC1-6 alkyl;
X3 is a covalent bond or C(R2)(R3);
R2 and R3 independently represent optionally substituted C1-6 alkyl, or R2 and R3 together with the carbon atom to which they are attached form an optionally substituted C3-7 cycloalkyl, C3-7 cycloalkenyl, or 5- to 7-membered heterocyclyl ring containing up to three heteroatoms independently selected from N, O, and S;
n is 0,1,2,3, or 4;
Y is NR10R11, NR10C(Z)NR10R11, NR10COOR11, NR10SO2R11, or C(O)NR4R5;
R4 and R5 independently represent hydrogen, C1-6 alkyl, C3-7 cycloalkyl, aryl, arylC1-6 alkyl, heteroaryl, heteroarylC1-6 alkyl, heterocyclyl, or heterocyclylC1-6 alkyl, any one of which, except hydrogen, can be optionally substituted, or R4 and R5 together with the nitrogen atom to which they are attached form a 4- to 10-membered optionally-substituted monocyclic or bicyclic ring;
R13 is hydrogen, X—R1, halogen, optionally-substituted C1-6 alkylsulfinyl, CH2OR14, di-C1-6 alkylamino, N(R6)C(O)R7, N(R6)S(O)2R8, or a 5- to 7-membered N-heterocyclyl ring which optionally contains an additional heteroatom selected from O, S, and NR9;
R14 is hydrogen, —C(Z)R12 or optionally-substituted C1-6 alkyl, optionally-substituted aryl, optionally-substituted arylC1-6 alkyl or S(O)2R8;
R6 is hydrogen or C1-6 alkyl;
R7 is hydrogen, C1-6 alkyl, C3-7 cycloalkyl, aryl, arylC1-6 alkyl, heteroaryl, heteroarylC1-6 alkyl, heterocyclyl or heterocyclylC1-4 alkyl;
R8 is C1-6 alkyl, C3-7 cycloalkyl, aryl, arylC1-6 alkyl, heteroaryl, heteroarylC1-6 alkyl, heterocyclyl or heterocyclylC1-6 alkyl;
R9 is hydrogen, cyano, C1-4 alkyl, C3-7 cycloalkyl or aryl;
R10, R11 and R12 are independently selected from hydrogen, C1-6 alkyl, C3-7 cycloalkyl, heterocyclyl, heterocyclylC1-6 alkyl, heterocyclylC2-6 alkenyl, aryl, arylC1-6 alkyl, arylC2-6 alkenyl, heteroaryl, heteroarylC1-6 alkyl and heteroarylC2-6 alkenyl, any of which can be optionally substituted; or NR10R11 can represent a 5- to 7-membered heterocyclyl ring optionally containing an additional heteroatom selected from O, N and S; and
Z is oxygen or sulfur;
or a pharmaceutically-acceptable salt thereof.
Further compounds useful in the practice of the present invention also include, but are not limited to, compounds of formulas:
wherein
R1 is a heteroaryl selected from 4-pyridyl, 4-pyrimidinyl, 4-quinolyl, 6-isoquinolinyl, quinazolin-4-yl, 1-imidazolyl, 1-benzimidazolyl, 4-pyridazinyl, and a 1,2,4-triazin-5-yl ring, which heteroaryl ring is substituted one to three times with Y, NHRa, optionally-substituted C1-4 alkyl, halogen, hydroxyl, optionally-substituted C1-4 alkoxy, optionally-substituted C1-4 alkylthio, optionally-substituted C1-4 alkylsulfinyl, CH2OR12, amino, mono- and di-C1-6 alkyl-substituted amino, N(R10)C(O)Rb, N(R10)S(O)2Rd, or an N-heterocyclyl ring which has from 5 to 7 members and optionally contains an additional heteroatom selected from oxygen, sulfur or NR15;
Y is X1-Ra;
X1 is oxygen or sulfur;
Ra is C1-6 alkyl, aryl, arylC1-6 alkyl, heterocyclic, heterocyclylC1-6 alkyl, heteroaryl, or heteroarylC1-6 alkyl, wherein each of these moieties can be optionally substituted;
Rb is hydrogen, C1-6 alkyl, C3-7 cycloalkyl, aryl, arylC1-4 alkyl, heteroaryl, heteroarylC1-4 alkyl, heterocyclyl, or heterocyclylC1-4 alkyl;
Rd is C1-6 alkyl, C3-7 cycloalkyl, aryl, arylC1-4 alkyl, heteroaryl, heteroarylC1-4 alkyl, heterocyclyl, or heterocyclylC1-4 alkyl;
R4 is phenyl, naphth-1-yl, naphth-2-yl, a heteroaryl or a fused phenyl-containing ring system, which is optionally substituted by one or two substituents, each of which is independently selected, and which, for a 4-phenyl, 4-naphth-1-yl, 5-naphth-2-yl or 6-naphth-2-yl substituent, is halogen, cyano, nitro, —C(Z)NR7R17, —C(Z)OR16, —(CR10R20)vCOR12, —SR5, —SOR5, —OR12, halo-substituted-C1-4 alkyl, C1-4 alkyl, —ZC(Z)R12, —NR10C(Z)R16, or —(CR10R20)vNR10R20 and which, for other positions of substitution, is halogen, cyano, nitro, phenyl, —C(Z)NR13R14, —C(Z)ORf, —(CR10R20)m″CORf, —S(O)mRf, —ORf, halo-substituted C1-4 alkyl, C1-10 alkyl, —ZC(Z)Rf, optionally-substituted phenyl, —(CR10R20)m″NR10C(Z)Rf, —NR10S(O)m′R8, —NR10S(O)m′NR7R17, —ZC(Z)R12, or —(CR10R20)m″NR13R14;
Rf is heterocyclyl, heterocyclylC1-10 alkyl or R8;
v is 0, 1, or 2;
m is 0, 1, or 2;
m′ is 1 or 2;
m″ is 0, 1, 2, 3, 4, or 5;
R2 hydrogen, —(CR10R23)nOR9, heterocylyl, heterocyclylC1-10 alkyl, C1-10 alkyl, halo-substituted C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C3-7 cycloalkyl, C3-7 cycloalkylC1-10 alkyl, C5-7 cycloalkenyl, C5-7cycloalkenylC1-10 alkyl, aryl, arylC1-10 alkyl, heteroaryl, heteroarylC1-10 alkyl, (CR10R23)nOR11, (CR10R23)nS(O)mR18, (CR10R23)nNHS(O)2R18, (CR10R23)nNR13R14, (CR10R23)nNO2, (CR10R23)nCN, (CR10R23)nS(O)m′NR13R14, (CR10R23)nC(Z)R11, (CR10R23)nOC(Z)R11, (CR10R23)nC(Z)OR11, (CR10R23)nC(Z)NR13R14, (CR10R23)nC(Z)NR11OR9, (CR10R23)nNR10C(Z)R11, (CR10R23)nNR10C(Z)NR13R14, (CR10R23)nN(OR6)C(Z)NR13R14, (CR10R23)nN(OR6)C(Z)R11, (CR10R23)nC(═NOR6)R11, (CR10R23)nNR10C(═NR19)NR13R14, (CR10R23)nOC(Z)NR13R14, (CR10R23)nNR10C(Z)NR13R14, (CR10R23)nNR10C(Z)OR10, 5-(R18)-1,2,4-oxadiazol-3-yl or 4-(R12)-5-(R18R19)-4,5-dihydro-1,2,4-oxadiazol-3-yl; wherein the aryl, arylalkyl, heteroaryl, heteroaryl alkyl, cycloalkyl, cycloalkyl alkyl, heterocyclic and heterocyclic alkyl groups can be optionally substituted;
n is 0, or an integer having a value of 1 to 10;
Z is oxygen or sulfur;
R5 is hydrogen, C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl or NR7R17, excluding the moieties —SR5 being —SNR7R17 and —S(O)R5 being —SOH;
R6 is hydrogen, a pharmaceutically-acceptable cation, C1-10 alkyl, C3-7 cycloalkyl, aryl, arylC1-4 alkyl, heteroaryl, heteroarylC1-4 alkyl, heterocyclyl, aroyl, or C1-10 alkanoyl;
R7 and R17 are each independently selected from hydrogen or C1-4 alkyl, or R7 and R17 together with the nitrogen to which they are attached form a heterocyclic ring of 5 to 7 members which ring optionally contains an additional heteroatom selected from oxygen, sulfur or NR15;
R8 is C1-10 alkyl, halo-substituted C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C3-7 cycloalkyl, C5-7 cycloalkenyl, aryl, arylC1-10 alkyl, heteroaryl, heteroarylC1-10 alkyl, (CR10R20)nOR11, (CR10R20)nS(O)mR18, (CR10R20)nNHS(O)2R18, or (CR10R20)nNR13R14, wherein the aryl, arylalkyl, heteroaryl, and heteroaryl alkyl can be optionally substituted;
R9 is hydrogen, —C(Z)R11, optionally-substituted C1-10 alkyl, S(O)2R18, optionally-substituted aryl or optionally-substituted arylC1-4 alkyl;
R10 and R20 are each independently selected from hydrogen or C1-4 alkyl;
R11 is hydrogen, C1-10 alkyl, C3-7 cycloalkyl, heterocyclyl, heterocyclylC1-10 alkyl, aryl, arylC1-10 alkyl, heteroaryl or heteroarylC1-10 alkyl, wherein the aryl, arylalkyl, heteroaryl, heteroaryl alkyl, heterocyclyl or heterocyclylalkyl can be optionally substituted;
R12 is hydrogen or R16;
R13 and R14 are each independently selected from hydrogen or optionally-substituted C1-4 alkyl, optionally-substituted aryl or optionally-substituted arylC1-4 alkyl, or together with the nitrogen to which they are attached form a heterocyclic ring of 5 to 7 members which ring optionally contains an additional heteroatom selected from oxygen, sulfur or NR9;
R15 is hydrogen, C1-4 alkyl or C(Z)—C1-4 alkyl;
R16 is C1-4 alkyl, halo-substituted C1-4 alkyl, or C3-7 cycloalkyl;
R18 is C1-10 alkyl, C3-7 cycloalkyl, heterocyclyl, aryl, arylC1-10 alkyl, heterocyclyl, heterocyclylC1-10 alkyl, heteroaryl or heteroarylC1-10 alkyl, wherein the aryl, arylalkyl, heteroaryl, heteroaryl alkyl, heterocyclyl or heterocyclylalkyl can be optionally substituted;
R19 is hydrogen, cyano, C1-4 alkyl, C3-7 cycloalkyl or aryl; and
R23 is hydrogen, C1-6 alkyl, C3-7 cycloalkyl, aryl, arylC1-4 alkyl, heteroaryl, heteroarylC1-4 alkyl, heterocyclyl, or heterocyclylC1-4 alkyl, all of which can be optionally substituted;
or a pharmaceutically-acceptable salt thereof.
Exemplary compounds of this class include but are not limited to:
Similar compounds that are also useful in the practice of the present invention include, but are not limited to, compounds of formulas:
wherein
R1 is 4-pyridyl or 4-pyrimidinyl ring, which ring is optionally substituted one or more times with Y, C1-4 alkyl, halogen, hydroxyl, C1-4 alkoxy, C1-4 alkylthio, C1-4 alkylsulfinyl, CH2OR12, amino, mono- and di-C1-6 alkyl-substituted amino, N(R10)C(O)Rb, or an N-heterocyclyl ring which has from 5 to 7 members and optionally contains an additional heteroatom selected from oxygen, sulfur or NR15;
Y is X1—Ra;
X1 is oxygen, sulfur, or NH;
Ra is C1-6 alkyl, aryl, arylC1-6 alkyl, heterocyclic, heterocyclylC1-6 alkyl, heteroaryl, or heteroarylC1-6 alkyl, wherein each of these moieties can be optionally substituted;
Rb is hydrogen, C1-6 alkyl, C3-7 cycloalkyl, aryl, arylC1-4 alkyl, heteroaryl, heteroarylC1-4 alkyl, heterocyclyl, or heterocyclylC1-4 alkyl, wherein each of these moieties can be optionally substituted;
R4 is phenyl, naphth-1-yl, naphth-2-yl, or a heteroaryl, which is optionally substituted by one or two substituents, each of which is independently selected, and which, for a 4-phenyl, 4-naphth-1-yl, 5-naphth-2-yl or 6-naphth-2-yl substituent, is halogen, cyano, nitro, —C(Z)NR7R17, —C(Z)OR16, —(CR10R20)vCOR12, —SR5, —SOR5, —OR12, halo-substituted-C1-4 alkyl, C1-4 alkyl, —ZC(Z)R12, —NR10C(Z)R16, or —(CR10R20)vNR10R20 and which, for other positions of substitution, is halogen, cyano, —C(Z)NR13R14, —C(Z)ORf, —(CR10R20)m″CORf, —S(O)mRf, —ORf, halo-substituted C1-4 alkyl, C1-4 alkyl, —ZC(Z)Rf, —(CR10R20)m″NR10C(Z)Rf, —NR10S(O)m′R8, —NR10S(O)m″NR7R17, or —(CR10R20)m″NR13R14;
Rf is heterocyclyl, heterocyclylC1-10 alkyl or R8;
v is 0, 1, or 2;
m is 0, 1, or 2;
m′ is 1 or 2;
m″ is 0,1,2,3,4, or 5;
R2 hydrogen, C(HOURS)(A)(R22), —(CR10R23)nOR9, heterocylyl, heterocyclylC1-10 alkyl, C1-10 alkyl, halo-substituted C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C3-7 cycloalkyl, C3-7 cycloalkylC1-10 alkyl, C5-7 cycloalkenyl, C5-7cycloalkenylC1-10 alkyl, aryl, arylC1-10 alkyl, heteroaryl, heteroarylC1-10 alkyl, (CR10R23)nOR11, (CR10R23)nS(O)mR18, (CR10R23)nNHS(O)2R18, (CR10R23)nNR13R14, (CR10R23)nNO2, (CR10R23)nCN, (CR10R23)nS(O)m′NR13R14, (CR10R23)nC(Z)R11, (CR10R23)nOC(Z)R11, (CR10R23)nC(Z)OR11, (CR10R23)nC(Z)NR13R14, (CR10R23)nC(Z)NR11OR9, (CR10R23)nNR10C(Z)R11, (CR10R23)nNR10C(Z)NR13R14, (CR10R23)nN(OR6)C(Z)NR13R14, (CR10R23)nN(OR6)C(Z)R11, (CR10R23)nC(═NOR6)R11, (CR10R23)nNR10C(═NR19)NR13R14, (CR10R23)nOC(Z)NR13R14, (CR10R23)nNR10C(Z)NR13R14, (CR10R23)nNR10C(Z)OR10, 5-(R18)-1,2,4-oxadiazol-3-yl or 4-(R12)-5-(R18R19)-4,5-dihydro-1,2,4-oxadiazol-3-yl; wherein the aryl, arylalkyl, heteroaryl, heteroaryl alkyl, cycloalkyl, cycloalkyl alkyl, heterocyclic and heterocyclic alkyl groups can be optionally substituted;
A is an optionally-substituted aryl, heterocyclyl or heteroaryl ring, or A is a substituted C1-10 alkyl;
n is 0, or an integer having a value of 1 to 10;
Z is oxygen or sulfur;
R5 is hydrogen, C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl or NR7R17, excluding the moieties —SR5 being —SNR7R17 and —S(O)R5 being —SOH;
R6 is hydrogen, a pharmaceutically-acceptable cation, C1-10 alkyl, C3-7 cycloalkyl, aryl, arylC1-4 alkyl, heteroaryl, heteroarylC1-4 alkyl, heterocyclyl, aroyl, or C1-10 alkanoyl;
R7 and R17 are each independently selected from hydrogen or C1-4 alkyl, or R7 and R17 together with the nitrogen to which they are attached form a heterocyclic ring of 5 to 7 members which ring optionally contains an additional heteroatom selected from oxygen, sulfur or NR15;
R8 is C1-10 alkyl, halo-substituted C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C3-7 cycloalkyl, C5-7 cycloalkenyl, aryl, arylC1-10 alkyl, heteroaryl, heteroarylC1-10 alkyl, (CR10R20)nOR11, (CR10R20)nS(O)mR18, (CR10R20)nNHS(O)2R18, or (CR10R20)nNR13R14, wherein the aryl, arylalkyl, heteroaryl, and heteroaryl alkyl can be optionally substituted;
R9 is hydrogen, —C(Z)R11, optionally-substituted C1-10 alkyl, S(O)2R18, optionally-substituted aryl or optionally-substituted arylC1-4 alkyl;
R10 and R20 are each independently selected from hydrogen or C1-4 alkyl;
R11 is hydrogen, C1-10 alkyl, C3-7 cycloalkyl, heterocyclyl, heterocyclylC1-10 alkyl, aryl, arylC1-10 alkyl, heteroaryl or heteroarylC1-10 alkyl, wherein the aryl, arylalkyl, heteroaryl, heteroaryl alkyl, heterocyclyl or heterocyclylalkyl can be optionally substituted;
R12 is hydrogen or R16;
R13 and R14 are each independently selected from hydrogen or optionally-substituted C1-4 alkyl, optionally-substituted aryl or optionally-substituted arylC1-4 alkyl, or together with the nitrogen to which they are attached form a heterocyclic ring of 5 to 7 members which ring optionally contains an additional heteroatom selected from oxygen, sulfur or NR9;
R15 is R10 or C(Z)C1-4 alkyl;
R16 is C1-4 alkyl, halo-substituted C1-4 alkyl, or C3-7 cycloalkyl;
R18 is C1-10 alkyl, C3-7 cycloalkyl, heterocyclyl, aryl, arylC1-10 alkyl, heterocyclyl, heterocyclylC1-10 alkyl, heteroaryl or heteroarylC1-10 alkyl;
R19 is hydrogen, cyano, C1-4 alkyl, C3-7 cycloalkyl or aryl; and
R23 is hydrogen, C1-6 alkyl, C3-7 cycloalkyl, aryl, arylC1-4 alkyl, heteroaryl, heteroarylC1-4 alkyl, heterocyclyl, or heterocyclylC1-4 alkyl, all of which can be optionally substituted;
or a pharmaceutically-acceptable salt thereof.
Examples of this class of compounds include but are not limited to:
Yet another class of compounds that are useful in the practice of the present invention includes compounds of formula:
and the pharmaceutically acceptable salts thereof, or a pharmaceutical composition thereof, wherein
represents a single or double bond;
one Z2 is CA or CR8A and the other is CR1, CR12, NR6 or N wherein each R1, R6 and R8 is independently hydrogen or noninterfering substituent;
A is —CO(X)jY wherein Y is COR2 or an isostere thereof and R2 is hydrogen or a noninterfering substituent, X is a spacer preferably of 2-6 Å, and j is 0 or 1;
Z3 is NR7 or O;
each R3 is independently a noninterfering substituent;
n is 0-3;
each of L1 and L2 is a linker;
each R4 is independently a noninterfering substituent;
m is 0-4;
Z1 is CR5 or N wherein R5 is hydrogen or a noninterfering substituent;
each of l and k is an integer from 0-2 wherein the sum of l and k is 0-3;
Ar is an aryl group substituted with 0-5 noninterfering substituents, wherein two noninterfering substituents can form a fused ring; and the distance between the atom of Ar linked to L2 and the center of the α ring is preferably less than 24 Å. As provided above, certain positions of the molecule are described as permitting “noninterfering substituents.” This terminology is where there is a wide variety of substituents possible in these positions as determined by those skilled in the art with the proviso however that the substituents must not impart a detrimental effect on the essential activity or application of the molecule when taken as a whole.
Exemplary Compounds of this formula include but are not limited to those in the following Table A
Compounds useful in the practice of the present invention also include, but are not limited to, compounds of formulas:
pharmaceutically acceptable salts thereof, wherein
HET is a 5-7 membered heterocycle with 1 to 4 N, S or O atoms, which heterocycle is substituted with 1 to 3 C1-C4 branched or straight chain alkyl groups. HET can optionally be substituted with halo, cyano, N(R′)2, OR′, CO2R′, CON(R′)2, and SO2N(R2)2;
X is O or NR′;
n is 1 to 3;
R′ is selected from hydrogen, (C1-C3)-alkyl, (C2-C3)-alkenyl or alkynyl, phenyl or phenyl substituted with 1 to 3 substituents independently selected from halo, methoxy, cyano, nitro, amino, hydroxy, methyl or ethyl; or a 5-6 membered heterocyclic ring system optionally substituted with 1 to 3 substituents independently selected from halo, methoxy, cyano, nitro, amino, hydroxy, methyl or ethyl;
R1 is selected from hydrogen, (C1-C3)-alkyl, hydroxy, or (C1-C3)-alkoxy;
R2 is selected from hydrogen, (C1-C3)-alkyl, or (C1-C3)-alkenyloxy; each optionally substituted with —N(R′)2, —OR′, —SR′, —C(O)—N(R′)2, —S(O2)—N(R′)2, —C(O)—OR′, or R3; and
R3 is selected from 5-6 membered aromatic carbocyclic or heterocyclic ring systems.
Compounds useful in the practice of the present invention also include, but are not limited to, compounds of formulas:
wherein
R1 is an aryl or heteroaryl ring, which ring is optionally substituted;
R2 is hydrogen, C1-10 alkyl, C3-7 cycloalkyl, C3-7 cycloalkylC1-10 alkyl, aryl, arylC1-10 alkyl, heteroaryl, heteroarylC1-10 alkyl, heterocyclic, or a heterocyclylC1-10 alkyl moiety; and wherein each of these moieties, excluding hydrogen, are optionally substituted;
R3 is a C1-10 alkyl, C3-7cycloalkyl, C3-7 cycloalkylC1-10alkyl, arylC1-10alkyl, heteroaryl C1-10alkyl, or heterocyclylC1-10 alkyl moiety; and wherein each of these moieties are optionally substituted;
X is R2, OR2, S(O)mR2 or (CH2)nNR4R14, or (CH2)nNR2R4;
n is 0 or an integer having a value of 1 to 10;
m is 0 or an integer having a value of 1 or 2;
R4 and R14 are each independently selected from hydrogen, optionally substituted C1-14 alkyl, optionally substituted aryl, or an optionally substituted arylC1-4alkyl, or R4 and R14 together with the nitrogen to which they are attached form a heterocyclic ring of 5 to 7 members, which ring optionally contains an additional heteroatom selected from oxygen, sulfur or NR9, and which ring can be optionally substituted;
R6 is hydrogen, C1-10 alkyl, C3-7 cycloalkyl, heterocyclyl, heterocyclylC1-10alkyl, aryl, arylC1-10 alkyl, heteroaryl or a heteroarylC1-10 alkyl moiety; and wherein each of these moieties, excluding hydrogen, can be optionally substituted;
R9 is hydrogen, C(Z)R6, optionally substituted C1-10 alkyl, optionally substituted aryl or optionally substituted arylC1-4 alkyl;
Z is oxygen or sulfur;
or a pharmaceutically acceptable salt thereof.
Compounds useful in the practice of the present invention also include, but are not limited to, compounds of formulas:
or pharmaceutically acceptable salts thereof, wherein
each of Q1 and Q2 are independently selected from 5-6 membered aromatic carbocyclic or heterocyclic ring systems, or 8-10 membered bicyclic ring systems comprising aromatic carbocyclic rings, aromatic heterocyclic rings or a combination of an aromatic carbocyclic ring and an aromatic heterocyclic ring;
the rings that make up Q1 are substituted with 1 to 4 substituents, each of which is independently selected from halo; C1-C3 alkyl optionally substituted with NR′2, OR′, CO2R′ or CONR′2; (C1-C3)-alkoxy optionally substituted with NR′2, OR′, CO2R′ or CONR′2; NR′2; OCF3; CF3; NO2; CO2R′; CONR′; SR′; S(O2)N(R′)2; SCF3; CN; N(R′)C(O)R4; N(R′)C(O)OR4; N(R′)C(O)C(O)R4; N(R′)S(O2)R4; N(R′)R4; N(R4)2; OR4; OC(O)R4; OP(O)3H2; or N═C—N (R′)2;
the rings that make up Q2 are optionally substituted with up to 4 substituents, each of which is independently selected from halo; C1-C3 straight or branched alkyl optionally substituted with NR′2, OR′, CO2R′, S(O2)N(R′)2, N═C—N(R′)2, R3, or CONR′2; (C1-C3)-alkoxy optionally substituted with NR′2, OR′, CO2R′, S(O2)N(R′)2, N═C—N(R′)2, R3, or CONR′2; NR′2, OCF3; CF3; NO2; CO2R′; CONR′; R3; OR3; NR3; SR3; C(O)R3; C(O)N(R′)R3; C(O)OR3; SR′; S(O2)N(R′)2; SCF3; N═C—N(R′)2; or CN;
R′ is selected from hydrogen, (C1-C3)-alkyl; (C2-C3)-alkenyl; (C2-C3) alkynyl; phenyl substituted with 1 to 3 substituents independently selected from halo, methoxy, cyano, nitro, amino, hydroxy, methyl or ethyl;
R3 is selected from 5-6 membered aromatic carbocyclic or heterocyclic ring systems;
R4 is (C1-C4)-alkyl optionally substituted with N(R′)2, OR′, CO2R′, CON(R′)2, or SO2N(R2)2; or a 5-6 membered carbocyclic or heterocyclic ring system optionally substituted with N(R′)2, OR′, CO2R′, CON(R′)2, or SO2N(R2)2; X, if present, is selected from —S—, —O—, —S(O2)—, —S(O)—, —S(O2)—N(R2)—, —N(R2)—S(O2)—, —N(R2)—C(O)O—, —O—C(O)—N(R2), —C(O)—, —C(O)O—, —O—C(O)—, —C(O)—N(R2)—, —N(R2)—C(O)—, —N(R2)—, —C(R22—, or —C(OR2)2—;
each R is independently selected from hydrogen, —R2, —N(R2)2, —OR2, SR2, —C(O)—N(R2)2, —S(O2)—N(R2)2, or —C(O)—OR2, wherein two adjacent R are optionally bound to one another and, together with each Y to which they are respectively bound, form a 4-8 membered carbocyclic or heterocyclic ring;
R2 is selected from hydrogen, (C1-C3)-alkyl, or (C1-C3)-alkenyl; each optionally substituted with —N(R′)2, —OR′, SR′, —C(O)—N(R′)2, —S(O2)—N(R′)2, —C(O)—OR′, or R3;
Y is N or C;
Z, if present, is N, NH, or, if chemically feasible, O;
A, if present, is N or CR′;
n is 0 or 1; and
R1 is selected from hydrogen, (C1-C3)-alkyl, hydroxy, or (C1-C3)-alkoxy. Compounds useful in the practice of the present invention also include, but are not limited to, compounds of formula:
wherein A is
wherein
R3′, R4′, R5′ are each independently HOURS, C1-10-alkyl, optionally substituted by halogen up to perhalo, C1-10 alkoxy, optionally substituted by halogen, up to perhaloalkoxy, halogen; NO2 or NH2;
R6′ is HOURS, C1-10-alkyl, C1-10 alkoxy, —NHCOR1; —NR1COR1; NO2;
one of R4′, R5′, or R6′ can be —X—Y; or
2 adjacent R4′—R6′ can together be an aryl or heteroaryl ring with 5-12 atoms, optionally substituted by C1-10-alkyl, C1-10 alkoxy, C3-10 cycloalkyl, C2-10 alkenyl, C1-10 alkanoyl, C6-12 aryl, C5-12 heteroaryl or C6-12 arakyl;
R1 is C1-10-alkyl optionally substituted by halogen, up to perhalo;
X is —CH2—, —S—, —N(CH3)—, —NHC(O)—, —CH2—S—, —S—CH2—, —C(O)—, or —O—;
X is additionally a single bond where Y is pyridyl;
Y is phenyl, pyridyl, naphthyl, pyridone, pyrazine, benzodioxane, benzopyridine, pyrimidine or benzothiazole, each optionally substituted by C1-10-alkyl, C1-10-alkoxy, halogen, OH, —SCH3 or NO2 or, where Y is phenyl, by
or a pharmaceutically-acceptable salt thereof;
wherein
R1 is selected from the group consisting of C3-C10 alkyl, C3-C10 cycloalkyl, up to per-halo substituted C1-C10 alkyl and up to per-halosubstituted C3-C10 cycloalkyl; and
R2 is C6-C14 aryl, C3-C14 heteroaryl, substituted C6-C14 aryl or substituted C3-C14 heteroaryl;
wherein if R2 is a substituted group, it is preferably substituted by one or more substituents independently selected from the group consisting of halogen, up to per-halosubstitution, and Vn, where n=0-3 and each V is independently selected from the group consisting of —CN, —OC(O)NR5R5′, —CO2R5, —C(O)NR5R5′, —OR5, —SR5, —NR5R5′, —C(O)R5, —NR5C(O)OR5′, —SO2R5—SOR5, —NR5C(O)R5′, —NO2, C1-C10 alkyl, C3-C10 cycloalkyl, C6-C14 aryl, C3-C13 heteroaryl, C7-C24 alkaryl, C4-C24 alkheteroaryl, substituted C1-C10 alkyl, substituted C3-C10 cycloalkyl, substituted C6-C14 aryl, substituted C3-C13 heteroaryl, substituted C7-C24 alkaryl and substituted C4-C24 alkheteroaryl;
wherein if V is a substituted group, it is substituted by one or more substituents independently selected from the group consisting of halogen, up to per-halosubstitution, —CN, —CO2R5, —C(O)R5, —C(O)NR5R5′, —NR5R5′, —OR5, —SR5, —NR5C(O)R5′, —NR5C(O)OR5′ and —NO2; and
R5 and R5′ are independently selected form the group consisting of HOURS, C1-C10 alkyl, C3-C10 cycloalkyl, C6-C14 aryl, C3-C13 heteroaryl, C7-C24 alkaryl, C4-C23 alkheteroaryl, up to per-halosubstituted C1-C10 alkyl, up to per-halosubstituted C3-C10 cycloalkyl, up to per-halosubstituted C6-C14 aryl and up to per-halosubstituted C3-C13 heteroaryl;
or a pharmaceutically-acceptable salt thereof;
or
(c) a substituted moiety of up to 40 carbon atoms of the formula: —L-(M-L1)q, where L is a 5- or 6-membered cyclic structure bound directly to D, L1, comprises a substituted cyclic moiety having at least 5 members, M is a bridging group having at least one atom, q is an integer of from 1-3; and each cyclic structure of L and L1 contains 0-4 members of the group consisting of nitrogen, oxygen and sulfur;
L1 is substituted by at least one substituent selected from the group consisting of —SO2Rx, —C(O)Rx and —C(NRy)Rz;
Ry is hydrogen or a carbon-based moiety of up to 24 carbon atoms optionally containing heteroatoms selected from N, S and O and optionally halosubstituted, up to perhalo;
Rz is hydrogen or a carbon-based moiety of up to 30 carbon atoms optionally containing heteroatoms selected from N, S and O and optionally substituted by halogen, hydroxy and carbon-based substituents of up to 24 carbon atoms, which optionally contain heteroatoms selected from N, S and O and are optionally substituted by halogen; and
Rx is Rz or NRaRb where Ra and Rb are
i) independently hydrogen,
ii) Ra and Rb together form a 5-7 member heterocyclic structure of 1-3 heteroatoms selected from N, S and O, or a substituted 5-7 member heterocyclic structure of 1-3 heteroatoms selected from N, S and O, substituted by halogen, hydroxy or carbon-based substituents of up to 24 carbon atoms, which optionally contain heteroatoms selected from N, S and O and are optionally substituted by halogen; or
iii) one of Ra or Rb is —C(O)—, a C1-C5 divalent alkylene group or a substituted C1-C5 divalent alkylene group bound to the moiety L to form a cyclic structure with at least 5 members, wherein the substituents of the substituted C1-C5 divalent alkylene group are selected from the group consisting of halogen, hydroxy, and carbon-based substituents of up to 24 carbon atoms, which optionally contain heteroatoms selected from N, S and O and are optionally substituted by halogen;
or a pharmaceutically-acceptable salt thereof; and
B is an unsubstituted or substituted, up to tricyclic, aryl or heteroaryl moiety with up to 30 carbon atoms with at least one 5- or 6-membered aromatic structure containing 0-4 members of the group consisting of nitrogen, oxygen and sulfur;
wherein if B is substituted, it is substituted by one or more substituents selected from the group consisting of halogen, up to per-halo, and Wn, wherein n is 0-3 and each W is independently selected from the group consisting of —CN, —CO2R7, —C(O)NR7R7, —C(O)R7, —NO2, —OR7, —SR7, —NR7R7, —NR7C(O)OR7, —NR7C(O)R7, C1-C10 alkyl, C2-10-alkenyl, C1-10-alkoxy, C3-C10 cycloalkyl, C6-C14 aryl, C7-C24 alkaryl, C3-C13 heteroaryl, C4-C23 alkheteroaryl, substituted C1-C10 alkyl, substituted C2-10-alkenyl, substituted C1-10-alkoxy, substituted C3-C10 cycloalkyl, substituted C4-C23 alkheteroaryl and —Q—Ar;
wherein if W is a substituted group, it is substituted by one or more substituents independently selected from the group consisting of —CN, —CO2R7, —C(O)NR7R7, —C(O)R7, —NO2, —OR7, —SR7, —NR7R7, —NR7C(O)OR7, —NR7C(O)R7 and halogen up to per-halo;
wherein each R7 is independently selected from HOURS, C1-C10 alkyl, C2-10-alkenyl, C3-C10 cycloalkyl, C6-C14 aryl, C3-C13 heteroaryl, C7-C24 alkaryl, C4-C23 alkheteroaryl, up to per-halosubstituted C1-C10 alkyl, up to per-halosubstituted C2-10-alkenyl, up to per-halosubstituted C3-C10 cycloalkyl, up to per- halosubstituted C6-C14 aryl and up to per-halosubstituted C3-C13 heteroaryl;
wherein Q is —O—, —S—, —N(R)7, —(CH2)—m, —C(O)—, —CH(OH)—, —NR7C(O)NR7R7—, —NR7C(O)—, —C(O)NR7—, —(CH2)mO—, —(CH2)mS—, —(CH2)mN(R7)—, —O(CH2)m—, —CHXa, —CXa2—, —S—(CH2)m— and —N(R7)(CH2)m—, where m =1-3, and Xa is halogen; and
Ar is a 5-10 member aromatic structure containing 0-4 members of the group consisting of nitrogen, oxygen and sulfur, which is unsubstituted or substituted by halogen up to per-halosubstitution and optionally substituted by Zn1, wherein n1 is 0 to 3 and each Z substituent is independently selected from the group consisting of —CN, —CO2R7, —C(O)NR7R7, —C(O)—NR7, —NO2, —OR7, —SR7, —NR7R7, —NR7C(O)OR7, C(O)R7, —NR7C(O)R7, C1-C10 alkyl, C3-C10 cycloalkyl, C6-C14 aryl, C3-C13 heteroaryl, C7-C24 alkaryl, C4-C23 alkheteroaryl, substituted C1-C10 alkyl, substituted C3-C10 cycloalkyl, substituted C7-C24 alkaryl and substituted C4-C23 alkheteroaryl; wherein the one or more substituents of Z are independently selected from the group consisting of —CN, —CO2R7, —C(O)NR7R7, —OR7, —SR7, —NO2, —NR7R7, —NR7C(O)R7 and —NR7C(O)OR7;
Exemplary compounds of these formulas include:
Such compounds are described in published PCT applications WO 96/21452, WO 96/40143, WO 97/25046, WO 97/35856, WO 98/25619, WO 98/56377, WO 98/57966, WO 99/32110, WO 99/32121, WO 99/32463, WO 99/61440, WO 99/64400, WO 00/10563, WO 00/17204, WO 00/19824, WO 00/41698, WO 00/64422, WO 00/71535, WO 01/38324, WO 01/64679, WO 01/66539, and WO 01/66540, each of which is herein incorporated by reference.
In all instances herein where there is an alkenyl or alkynyl moiety as a substituent group, the unsaturated linkage, i.e., the vinylene or acetylene linkage, is preferably not directly attached to the nitrogen, oxygen or sulfur moieties, for instance in ORf, or for certain R2 moieties.
As used herein, “optionally substituted” unless specifically defined shall mean such groups as halogen, such as fluorine, chlorine, bromine or iodine; hydroxy; hydroxy-substituted C1-10-alkyl; C1-10 alkoxy, such as methoxy or ethoxy; S(O)m alkyl, wherein m is 0, 1 or 2, such as methyl thio, methylsulfinyl or methyl sulfonyl; amino, mono and di-substituted amino, such as in the NR7R17 group; or where the R7R17 can together with the nitrogen to which they are attached cyclize to form a 5- to 7-membered ring which optionally includes an additional heteroatom selected from O, N, and S; C1-10 alkyl, cycloalkyl, or cycloalkyl alkyl group, such as methyl, ethyl, propyl, isopropyl, t-butyl, etc. or cyclopropyl methyl; halo-substituted C1-10 alkyl, such as CF3; an optionally substituted aryl, such as phenyl, or an optionally substituted arylalkyl, such as benzyl or phenethyl, wherein these aryl moieties can also be substituted one to two times by halogen; hydroxy; hydroxy-substituted alkyl; C1-10 alkoxy; S(O)m alkyl; amino, mono- and di-substituted amino, such as in the NR7R17 group; alkyl, or CF3.
Inhibitors useful in the present invention can be used with any pharmaceutically acceptable salt. The term “pharmaceutically acceptable salts” refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids. When the compound utilized by the present invention is acidic, its corresponding salt can be conveniently prepared from pharmaceutically acceptable non-toxic bases, including inorganic bases and organic bases. Salts derived from such inorganic bases include aluminum, ammonium, calcium, copper (ic and ous), ferric, ferrous, lithium, magnesium, manganese (ic and ous), potassium, sodium, zinc and the like salts. Particularly preferred are the ammonium, calcium, magnesium, potassium and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, as well as cyclic amines and substituted amines such as naturally occurring and synthesized substituted amines. Basic salts of inorganic and organic acids also include as hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, methane sulphonic acid, ethane sulphonic acid, acetic acid, malic acid, tartaric acid, citric acid, lactic acid, oxalic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, salicylic acid, phenylacetic acid and mandelic acid. In addition, pharmaceutically-acceptable salts of the above-described compounds can also be formed with a pharmaceutically-acceptable cation, for instance, if a substituent group comprises a carboxy moiety. Suitable pharmaceutically-acceptable cations are well known to those skilled in the art and include alkaline, alkaline earth, ammonium and quaternary ammonium cations.
Other pharmaceutically acceptable organic non-toxic bases from which salts can be formed include ion exchange resins such as, for example, arginine, betaine, caffeine, choline, N,N-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like.
The inhibitors of p38 MAP kinase can be used as single therapeutic agents or in combination with other therapeutic agents. Drugs that could be usefully combined with these compounds include monoclonal antibodies targeting cells of the immune system, antibodies or soluble receptors or receptor fusion proteins targeting immune or non-immune cytokines, and small molecule inhibitors of cell division, protein synthesis, or mRNA transcription or translation, or inhibitors of immune cell differentiation, activation, or function (e.g., cytokine secretion).
Other compounds useful in the practice of the present invention include, but are not limited to, the compounds shown in Table B, below.
All compounds provided herein are intended for guidance and exemplary purposes only. It should be understood that any inhibitor of p38 MAP kinase is useful for the invention provided that it exhibits activity relative to p38 to the extent adequate to exhibit a therapeutic effect in a reasonable dosage and concentration.
Formulations and Methods of Administration
A pharmaceutical composition useful in the present invention comprises a p38 MAP kinase inhibitor (such as those described above) and a pharmaceutically acceptable carrier, excipient, diluent and/or salt.
Pharmaceutically acceptable carrier, diluent, excipient, and/or salt means that the carrier, diluent, excipient and/or salt must be compatible with the other ingredients of the formulation, does not adversely affect the therapeutic benefit of the p38 MAP kinase inhibitor, and is not deleterious to the recipient thereof.
Administration of the compounds or pharmaceutical compositions thereof for practicing the present invention can be by any method that delivers the compounds systemically and/or locally (e.g., at the site of the disease, intravascular, etc.). These methods include oral routes, parenteral routes, intraduodenal routes, and the like. Preferably, the compositions are delivered parenterally.
For topical applications, the compound or pharmaceutical composition thereof can be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax, sugars such as lactose and water. Alternatively, the pharmaceutical compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
Depending on the particular condition, disorder or disease to be treated, additional therapeutic agents can be administered together with the p38 MAP kinase inhibitors. Those additional agents can be administered sequentially in any order, as part of a multiple dosage regimen, from the p38 MAP kinase inhibitor-containing composition (consecutive or intermittent administration). Alternatively, those agents can be part of a single dosage form, mixed together with the p38 MAP kinase inhibitor in a single composition (simultaneous or concurrent administration).
For oral administration, a pharmaceutical composition useful in the invention can take the form of solutions, suspensions, tablets, pills, capsules, powders, granules, semisolids, sustained release formulations, elixirs, aerosols, and the like. Tablets containing various excipients such as sodium citrate, calcium carbonate and calcium phosphate are employed along with various disintegrants such as starch, preferably potato or tapioca starch, and certain complex silicates, together with binding agents such as polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often very useful for tabletting purposes. Solid compositions of a similar type are also employed as fillers in soft and hard-filled gelatin capsules; preferred materials in this connection also include lactose or milk sugar as well as high molecular weight polyethylene glycols. When aqueous suspensions and/or elixirs are desired for oral administration, the compounds of this invention can be combined with various sweetening agents, flavoring agents, coloring agents, emulsifying agents and/or suspending agents, as well as such diluents as water, ethanol, propylene glycol, glycerin and various like combinations thereof.
The choice of formulation depends on various factors such as the mode of drug administration (e.g., for oral administration, formulations in the form of tablets, pills or capsules are preferred) and the bioavailability of the drug substance. Recently, pharmaceutical formulations have been developed especially for drugs that show poor bioavailability based upon the principle that bioavailability can be increased by increasing the surface area i.e., decreasing particle size. For example, U.S. Pat. No. 4,107,288 describes a pharmaceutical formulation having particles in the size range from 10 to 1,000 nm in which the active material is supported on a crosslinked matrix of macromolecules. U.S. Pat. No. 5,145,684 describes the production of a pharmaceutical formulation in which the drug substance is pulverized to nanoparticles (average particle size of 400 nm) in the presence of a surface modifier and then dispersed in a liquid medium to give a pharmaceutical formulation that exhibits remarkably high bioavailability.
The term “parenteral” as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, intramedullary and intraarticular injection and infusion. Parenteral delivery is a preferred route of delivery for purposes of the present invention. A pharmaceutical composition for parenteral injection can comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal injection purposes. In this connection, the sterile aqueous media employed are all readily obtainable by standard techniques well-known to those skilled in the art. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
The pharmaceutical compositions useful in the present invention can also contain adjuvants such as, but not limited to, preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, such as for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.
In some cases, in order to prolong the effect of the drugs, it is desirable to slow the absorption from subcutaneous or intramuscular injection. This can be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, can depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide, polyglycolide, and polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.
The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use..
Suspensions, in addition to the active compounds, can contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.
For purposes of transdermal (e.g., topical) administration, dilute sterile, aqueous or partially aqueous solutions (usually in about 0.1% to 5% concentration), otherwise similar to the above parenteral solutions, are prepared.
The pharmaceutical compositions useful in the invention can also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and can be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.
In nonpressurized powder compositions, the active ingredients in finely divided form can be used in admixture with a larger-sized pharmaceutically acceptable inert carrier comprising particles having a size, for example, of up to 100 μm in diameter. Suitable inert carriers include sugars such as lactose. Desirably, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 μm.
Alternatively, the composition can be pressurized and contain a compressed gas, such as, e.g., nitrogen, carbon dioxide or a liquefied gas propellant. The liquefied propellant medium and indeed the total composition are preferably such that the active ingredients do not dissolve therein to any substantial extent. The pressurized composition can also contain a surface active agent. The surface active agent can be a liquid or solid non-ionic surface active agent or can be a solid anionic surface active agent. It is preferred to use the solid anionic surface active agent in the form of a sodium salt.
Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of the invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the drugs.
The compositions useful in the present invention can also be administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. The present compositions in liposome form can contain, in addition to the compounds of the invention, stabilizers, preservatives, excipients, and the like. The preferred lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art (see e.g., Prescott, E., Meth. Cell Biol. 14:33 (1976)).
Other pharmaceutically acceptable carrier includes, but is not limited to, a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type, including but not limited to ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
Solid pharmaceutical excipients include, but are not limited to, starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk and the like. Liquid and semisolid excipients can be selected from glycerol, propylene glycol, water, ethanol and various oils, including those of petroleum, animal, vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc. Preferred liquid carriers, particularly for injectable solutions, include water, saline, aqueous dextrose, and glycols.
Methods of preparing various pharmaceutical compositions with a certain amount of active ingredient are known, or will be apparent in light of this disclosure, to those skilled in this art. Other suitable pharmaceutical excipients and their formulations are described in Remington's Pharmaceutical Sciences, edited by E. W. Martin, Mack Publishing Company, 19th ed. (1995).
Pharmaceutical compositions useful in the present invention can contain 0.1%-95% of the compound(s) of this invention, preferably 1%-70%. In any event, the composition or formulation to be administered will contain a quantity of a compound(s) according to this invention in an amount effective to treat the condition, disorder or disease of the subject being treated.
One of ordinary skill in the art will appreciate that pharmaceutically effective amounts of the p38 MAP kinase inhibitor can be determined empirically and can be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt, ester or prodrug form. The agents can be administered to a patient as pharmaceutical compositions in combination with one or more pharmaceutically acceptable excipients. It will be understood that, when administered to, for example, a human patient, the total daily usage of the agents or composition of the present invention will be decided within the scope of sound medical judgment by the attending physician. The specific pharmaceutically effective dose level for any particular patient will depend upon a variety of factors: the type and degree of the cellular response to be achieved; activity of the specific agent or composition employed; the specific agents or composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the agent; the duration of the treatment; drugs used in combination or coincidental with the specific agent; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the agents at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosages until the desired effect is achieved.
For example, satisfactory results are obtained by oral administration of the compounds at dosages on the order of from 0.05 to 500 mg/kg/day, preferably 0.1 to 100 mg/kg/day, more preferably 1 to 50 mg/kg/day, administered once or, in divided doses, 2 to 4 times per day. On administration parenterally, for example, by i.v. bolus, drip or infusion, dosages on the order of from 0.01 to 1000 mg/kg/day, preferably 0.05 to 500 mg/kg/day, and more preferably 0.1 to 100 mg/kg/day, can be used. Suitable daily dosages for patients are thus on the order of from 2.5 to 500 mg p.o., preferably 5 to 250 mg p.o., more preferably 5 to 100 mg p.o., or on the order of from 0.5 to 250 mg i.v., preferably 2.5 to 125 mg i.v. and more preferably 2.5 to 50 mgi.v.
Dosaging can also be arranged in a patient specific manner to provide a predetermined concentration of the agents in the blood, as determined by techniques accepted and routine in the art (HPLC is preferred). Thus patient dosaging can be adjusted to achieve regular on-going blood levels, as measured by HPLC, on the order of from 50 to 5000 ng/ml, preferably 100 to 2500 ng/ml.
Kits
The invention also relates to combining separate pharmaceutical compositions in kit form. The kit can have a carrier means being compartmentalized in close confinement to receive two or more container means therein, having (1) a first container means containing a pharmaceutically effective amount of a p38 MAP kinase inhibitor and (2) a second container means containing a pharmaceutically effective amount of carrier, excipient or diluent. Optionally, the kit can have additional container mean(s) containing a pharmaceutically effective amount of additional agents.
The kit comprises a container for containing the separate compositions such as a divided bottle or a divided foil packet, however, the separate compositions can also be contained within a single, undivided container. Typically the kit comprises directions for administration of the separate components. The kit form is particularly advantageous when the separate components are preferably administered in different dosage forms (e.g., oral and parenteral) or at different dosage intervals, or when titration of the individual components of the combination is desired by the prescribing physician.
An example of such a kit is a so-called blister pack. Blister packs are well known in the packaging industry and are being widely used for the packaging of pharmaceutical unit dosage forms (tablets, capsules, and the like). Blister packs generally consist of a sheet of relatively stiff material covered with a foil of a preferably transparent plastic material. During the packaging process, recesses are formed in the plastic foil. The recesses have the size and shape of the tablets or capsules to be packed. Next, the tablets or capsules are placed in the recesses and the sheet of relatively stiff material is sealed against the plastic foil at the face of the foil which is opposite from the direction in which the recesses were formed. As a result, the tablets or capsules are sealed in the recesses between the plastic foil and the sheet. Preferably the strength of the sheet is such that the tablets or capsules can be removed from the blister pack by manually applying pressure on the recesses whereby an opening is formed in the sheet at the place of the recess. The tablet or capsule can then be removed via said opening.
It can be desirable to provide a memory aid on the kit, e.g., in the form of numbers next to the tablets or capsules whereby the numbers correspond with the days of the regimen which the dosage form so specified should be ingested. Another example of such a memory aid is a calendar printed on the card e.g., “First Week, Monday, Tuesday . . . Second Week, Monday, Tuesday. . .” etc. Other variations of memory aids will be readily apparent. A “daily dose” can be a single tablet or capsule or several tablets or capsules to be taken on a given day. Also, a daily dose of the compound, a prodrug thereof, or a pharmaceutically acceptable salt of the compound can consist of one tablet or capsule while a daily dose of the second compound can consist of several tablets or capsules and vice versa. The memory aid should reflect this.
It will be readily apparent to one of ordinary skill in the relevant art that other suitable modifications and adaptations to the methods and applications described herein can be made without departing from the scope of the invention or any embodiment thereof.
The following examples are offered to illustrate but not to limit the invention.
It is contemplated that a variety of compounds will exhibit an effect for the treatment of acute coronary syndrome. For example, in Table A provided above, compounds 2, 3, 15, 33, 57, 67, 84, 92, 96, 141, 154, 162, or 169 generally exhibit p38 MAP kinase activity as observed in an assay similar to the phosphorylation assay described above (see Kumar).
Cryopreserved human peripheral blood monocytes (HPBMCs, from Biowitker, Walkersvill, Md.) were thawed and washed twice in RPMI 1640. Cells were plated in tissue culture plates and incubated in 5% CO2 at 37 C. After 1 hr media were removed and cells were treated with various treatments. After 2 to 20 hours conditioned media and/or cell lysates were collected for the analyses of secreted cytokines, protein or mRNA expression. 10 μg/ml of purified human CRP (Calbiochem, San Diego, Calif.) was used in most of the treatments. In some experiments fetal calf serum (FCS from ATCC) was added to a final concentration of 10% together with CRP. In the examples provided herein, including those assessing the effects of p38 antagonism, cells were pre-treated with SD, a p38 small molecule inhibitor (Compound 162 in Table A) for 1 hour prior to CRP treatment as described above. In a parallel series of experiments, the effect of vehicle (DMSO) was tested. For TaqMan analyses of various gene expression cells were lysed in ice-cold RLT lysis buffer (Qiagen, Valencia, Calif.) and the lysates were kept in −80° C. until the analyses. For Western analyses cells were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40) containing phosphatase inhibitor cocktails and protease inhibitor cocktail (Promega, Madison, Wis.).
P38 MAP Kinase Activation
Level of activated p38 MAP kinase was determined by Western analysis of phosphorylated p38 MAP kinase. HPBMCs (1.5×106 cell suspension in RPMI 1640) were plated in each 48-well tissue culture well. After 1 hr media were replaced with RPMI 1640 medium, medium containing 10 μg/ml of purified human CRP, 10% FCS or 10 μg/ml of purified human CRP plus 10% FCS. After 5 minutes media were removed from the wells and cells lysed in 100 μl of RIPA buffer. Insoluble materials were precipitated by centrifugation at 14,000 rpm for 10 minute in microfuge and supernatants were saved for Western analyses. Protein concentrations of the lysates were determined by BCA assay (Promega, Madison, Wis.). 10 μg total proteins were resolved on LDS-PAGE (In Vitrogen, Carlsbad, Calif.), and Western analyses for phospho-p38 MAP kinase was performed employing antibodies directed to diphospho-p38 (Biosource International, Camarillo, Calif.). For normalization, GAPDH levels were used. GAPDH level was determined by Western using antibody toward GAPDH (Biogenesis, Kingston, N.H.). Goat anti-rabbit IgG-HRP and goat anti-mouse IgG-HRP were from Cell Signaling (Beverly, Mass.).
Cytokine ELISA.
Conditioned media from the treated cells were collected (6 to 20 hrs), and the level of secreted interleukin 6 (IL-6), interleukin 1 beta (IL-1 β), tumor necrosis factor alpha (TNFα), and interleukin 8 (IL-8) was determined by ELISA according to the manufacturer's instruction (Biosource International, Camarillo, Calif.). All determinations were performed in duplicates.
Real-time PCR
RNA was extracted from the cells using Qiagen RNA extraction kit according to the manufacturer's instruction. The expression of pro-inflammatory genes IL-1β, IL-6, IL-8, TNFα, COX-2, ET-1, TF were assessed by real-time PCR. All determinations were performed in triplicates.
Real-time RT-PCR was performed in a two-step manner. cDNA synthesis and real-time detection were carried out in a PTC-100™ Thermal Cycler (MJ Research Inc, Waltham, Mass.) and an ABI Prism™ 7700 Sequence Detection System (Applied Biosystems, Foster City, Calif.), respectively. Random hexamers (Qiagen, Valencia, Calif.) were used to generate cDNA from 200 ng RNA as described in Applied Biosystems User Bulletin #2. TaqMan™ PCR Core Reagent Kit or TaqMan™ Universal PCR Master Mix (Applied Biosystems) were used in subsequent PCR reactions according to the manufacturer's protocols. Relative quantitation of gene expression was performed using the relative standard curve method.
Sequence specific primers and probes were designed using Primer Express Version 2 software (Applied Biosystems). Sequences of primers and probes can be found in Table 1. Expression levels were normalized to 18S rRNA.
Tissue Factor Western
HPBMCs were treated as described above (p38 MAP kinase activation). After 16 hours media were removed and cells lysed as described. 10 μg total proteins were resolved on LDS-PAGE (In Vitrogen, Carlsbad, Calif.), and tissue factor protein expression was determined by Western using antibody against human tissue factor (American Diagnostica, Stamford, Conn.). Signals were normalized by GAPDH Western signal.
Results
CRP Activates p38 MAP Kinase.
CRP was found to directly activate the p38 MAP kinase. As demonstrated in
CRP Promotes Cytokine IL-6 Secretion via p38 Signaling.
IL-6 is the major cytokine that stimulates hepatic production of CRP upon acute inflammation setting. CRP in turn has been reported to induce IL-6 in monocytes and endothelial cells. Increased production of IL-6 represents a positive feedback mechanism for the production of CRP from the liver. IL-6 has been implicated in the pathogenesis and clinical course of atherosclerotic vascular disease.
IL-6 increases adhesion molecules and chemokines by the endothelium, increases hepatic release of fibrogen and has procoagulant effects on platelets. IL-6, by binding to sIL-6R, exerts effects in cells that lack the IL-6R per se and potentiate its own biological activity. The IL-6/sIL-6R complex stimulates leukocyte recruitment and promotes endothelial cell inflammatory responses. CRP is a physiological regulator of sIL-6R shedding in human neutrophils and markedly increases the formation of the sIL-6R/IL-6 complex.
IL-6 has been reported to mediate the CRP augmented macrophage uptake of LDL.
As part of the studies described herein, an investigation was done as to whether the CRP invoked IL-6 secretion from HPBMCs is dependent on p38 MAP kinase signaling. This was accomplished through the use of the p38 selective small molecule inhibitor SD. The effect of addition of 10% fetal calf serum (FCS) during the CRP treatment on IL-6 secretion was also assessed. As shown in
CRP Promotes Chemokine IL-8 Secretion via p38 Signaling.
Elevated IL-8 has been reported in macrophage rich plaque area of atheroma. This chemokine may recruit further leukocytes to the plaques area, amplifying the local inflammation in affected area. IL-8 also has been shown to promote firm adhesion of monocytes to endothelial cells. CRP exerts similar effects on IL-8 secretion in HPBMCs as on IL-6. As shown in
CRP Induces TNFα and IL-1β Secretion via p38 Pathway.
TNFα and IL-β are initial inflammatory cytokines produced when monocytes are activated during infalmmation. These cytokines activate variety of cells to produce further pro-inflammatory mediators and growth factors, amplifying the inflammation cascade. TNFα and IL-1 β are able to regulate the hepatic synthesis of CRP and induce synthesis and secretion of IL-6. These cytokines are implicated in the inflammatory process in atherosclerotic plaque and in acute coronary syndromes. Elevated CRP, TNFα and IL-1β have been reported in plaque area of atheroma. CRP has been reported to induce these cytokines in vitro. As pert of the studies referenced herein, an investigation as to whether CRP induced TNFα and IL-1β in HPBMCs are regulated via p38 MAP kinase signaling. HPBMCs were incubated with either 10% FCS or CRP plus 10% FCS for 6 hours and secreted cytokines TNFα and IL-1β were measured by ELISA. For p38 antagonism HPBMCs were treated with 1 μM SD prior to CRP treatment.
CRP Augments COX-2 Gene Expression via p38 Signaling.
COX-2 is important inflammatory mediator. COX-2 mRNA levels are reported to be higher in plaque tissue than in normal artery. Selective COX-2 inhibition seems to improve endothelium-dependent vasodilation and reduces low-grade chronic inflammation and oxidative stress in coronary artery disease. Potential benefits of selective COX-2 inhibitor in patients with acute coronary syndromes have been reported.
To investigate whether CRP promotes COX-2 expression in monocytes and the expression is mediated via p38 MAP kinase signaling HPBMCs were incubated either with 10% FCS or CRP plus 10% FCS in the presence or absence of SD, and the COX-2 mRNA level assessed by real-time PCR. At 6 hours, addition of CRP augmented COX-2 gene expression in HPBMCs (
CRP Augments Potent Vasoactive Gene Endothelin-1 Expression via p38 Signaling.
ET-1 is one of the most potent endogenous vasoconstrictors and mediates a host of responses, including endothelial dysfunction, vasomotor contraction, leukocyte and platelet activation, and cellular proliferation. Additionally, it augments the vascular actions of other vasoactive substances, such as A-II, norepinephrine, and serotonin.
In endothelial cells, CRP is reported to partially exert its inflammatory function via endothelin, which can induce IL-6 secretion, amplifying the inflammatory cascade. Antagonism of ET-1 and IL-6 attenuates the expression of vascular cell adhesion moecule (VCAM-1), intracellular adhesion molecule (ICAM-1) and MCP-1. ET-1 has been reported to mediate the CRP augmented macrophage uptake of LDL.
To investigate whether CRP can induce endothelin-1 in monocytes and whether it is mediated via p38 MAP kinase signaling HPBMCs were treated with CRP in the presence or absence of 1 μM SD, and ET-1 mRNA level was determine by real-time PCR. As shown in
CRP Augments Prothrombotic Tissue Factor Expression via p38 Signaling.
The acute inflammatory response is frequently accompanied by serious thrombotic events. Tissue factor activates extrinsic coagulation pathway and thus may lead to thrombosis, vascular occlusion and myocardial infarction. Tissue factor level is increased in plasma from patients with acute coronary syndrome and TF protein is found in atheromatous plaques. In patients with disseminate intravasular coagulation TF mRNA in leukocytes is significantly higher in patients with higher CRP. In vitro CRP induces procoagulant activity via tissue factor expression in monocytes.
An evaluation was done as to whether this CRP invoked tissue factor expression is mediated via p38 MAP kinase signaling in HPBMCs. As shown in
Results
The results from the examples above suggest that CRP induces pro-inflammatory and prothrombotic responses in HPBMCs via p38 signaling, and p38 regulates these mediator expressions at transcriptional and/or post-transcriptional level. They demonstrate that CRP directly activates p38 MAP kinase, promotes the production and expression of several pro-inflammatory mediators that are important in coronary vascular disease, and the process is mediated by p38 MAP kinase signaling. CRP-invoked activation of p38 MAP kinase in HPBMCs was rapid. Activation of p38 MAP kinase has been closely linked to variety of inflammatory cascade in monocytes. Also demonstrated is that CRP directly facilitated the production of cytokines IL-6, IL-1β, and TNFα and chemokine IL-8 from HPBMCs via p38 MAP kinase signaling. CRP showed great synergy with FCS in the production of IL-6 and IL-8. Additional activation of p38 MAP kinase by CRP in the presence of FCS cannot explain dramatic increase in the level of IL-6 and IL-8 production. This suggests that CRP not only simply activates p38 MAP kinase but also amplifies inflammation by promoting synergy with other unknown factors. Synergy between CRP and other factors has been shown in various settings of inflammation.
In monocytes CRP showed a synergistic effect on LPS-dependent IL-1β production. CRP also acts synergistically with lipopolysaccharide and interferon γ to induce tissue factor production by monocytes. CRP also acts synergistically with lipopolysaccharide in the activation of endothelial cells.
In the clinical setting enhanced response of blood monocytes, when challenged with LPS in vitro, was observed in patients with recurrent unstable angina with elevated CRP. This suggests that CRP may amplify the intensity of the inflammatory response by further inducing proinflammatory mediators and working synergistically with other mediators.
As shown herein, CRP augmented potent prothrombotic protein TF expression in HPBMCs and TF protein and mRNA expressions were blocked by SD. CRP augmented other inflammatory genes COX-2 and ET-1 expression in HPBMCs. Both of them are known to promote inflammation and vasoconstriction. Constriction of the vessel may render the vulnerable plaque to rupture. These gene expressions were almost completely inhibited by SD. CRP, by increasing TF expression and increasing the risk of plaque rupture via TNFα, IL-1β, IL-6, Cox-2 and ET-1, can greatly increase the risk of thrombosis that can be catastrophic. Increased thrombosis after arterial injury has been observed in transgenic mice that express human CRP (Danenberg, 2003). P38 inhibition seems to control the underlying inflammation invoked by CRP at multiple levels.
CRP directly facilitates expressions of cytokines IL-6, IL-8, IL-1β and TNFα, and COX-2 and vasoactive gene ET-1 and prothrombotic gene TF in HPBMCs. These effects seem to occur via p38 signaling and are attenuated during pharmacological intervention with SD. The data presented herein is a novel demonstration that CRP induced inflammation in HPBMC cells involve p38 MAP kinase signaling. Understanding the mechanisms and mediators of the pro-inflammatory activities of CRP suggest a novel therapeutic strategy. p38 inhibition offers such an opportunity for the treatment of cardiovascular disease.
This application claims priority from U.S. patent application Ser. No. 60/507,768 filed 30 Sep. 2003. The contents of that document are incorporated herein for reference.
| Number | Date | Country | |
|---|---|---|---|
| 60507768 | Sep 2003 | US |
