HK1101073A - Substantially pure 2-{[2-(2-methylamino-pyrimidin-4-yl)-1h-indole-5-carbonyl]-amino}-3-(phenylpyridin-2-yl-amino)-propionic acid as an ikb kinase inhibitor - Google Patents

Description

Substantially pure 2- { [2- (2-methylamino-pyrimidin-4-yl) -1H-indole-5-carbonyl ] -amino } -3- (phenylpyridin-2-yl-amino) -propionic acid as ikb kinase inhibitor

Technical Field

The present invention relates to indole derivatives, processes for their preparation, pharmaceutical compositions containing them, their use and intermediates.

Background

NF- κ B is a heterodimeric transcription factor that regulates the expression of various inflammatory genes. Expression of over 70 known proteins is transcriptionally regulated by NF-. kappa.B binding to specific sequence units in the promoter regions of these genes (Baeuerle and Baichwal, Advances in Immunology 65: 111-137, 1997). NF-. kappa.B has been implicated in a number of pathophysiological processes including angiogenesis (Koch et al, Nature 376: 517-519, 1995), atherosclerosis (Brand et al, J Clin Inv.97: 1715-1722, 1996), endotoxic shock and sepsis (Bohrer et al, J Clin. Inv.100: 972-985, 1997), inflammatory bowel disease (Panel et al, Am J physiol.269: H1955-H1964, 1995), ischemia/reperfusion injury (Zwacka et al, Nature Medicine 4: 698-704, 1998) and hypersensitivity pneumonitis (Gosset et al, Int Arch Allergy immunol.106: 69-77, 1995). Given the central role played by NF- κ B in inflammation, inhibition of NF- κ B by targeted regulatory proteins in the NF- κ B activation pathway represents an attractive approach to the generation of anti-inflammatory therapeutics.

IkB kinase (IKK) is a key regulatory signaling molecule that coordinates NF-. kappa.B activation. Two IKKs, IKK-1(IKK- α) and IKK-2(IKK- β), are structurally distinct kinases that comprise an N-terminal kinase domain with a dual serine activation loop, a leucine zipper domain, and a C-terminal helix-loop-helix domain and a serine cluster. IKK enzymes have low sequence homology to other kinases and early studies with known kinase inhibitors have not found compounds with significant potency. Kinetic analysis showed that IKK-2 binds and phosphorylates IkB α and IkB ε with high and relatively consistent affinity (Heilker et al, 1999). The recombinant IKK-2 phosphorylated I.kappa.B.alpha.peptide 26-42 has a nearly identical affinity to phosphorylated full-length I.kappa.B.alpha.but the phosphorylation of full-length I.kappa.B.alpha.by the native IKK enzyme complex is 25,000-fold more efficient, suggesting that the C-terminal domain of I.kappa.B.alpha.has important regulatory sequences or that the I.kappa.B enzyme complex has additional regulatory proteins to accelerate the rate of catalysis (Burke et al, Journal of Biological Chemistry 274: 36146-36152, 1999). Phosphorylation of I.kappa.B.alpha.occurs by a random sequential kinetic mechanism, which means that either ATP or I.kappa.B.alpha.can bind to IKK-2 first, but I.kappa.B.alpha.phosphorylation must occur after both have bound (Peet and Li, Journal of Biological Chemistry 274: 32655-32661, 1999). IKK-2 binds ATP with particularly high affinity (Ki 130nM) compared to other serine-threonine kinases such as p38 and JNK, perhaps suggesting that IKK-2 carries a specific region that binds ATP, which is manifested by poor activity of many more specific kinase inhibitors when tested with IKK-2. To date, the crystal structure of IKK-2 has not been reported. However, homology models have identified three structural functional regions, including an N-terminal kinase domain with an activation loop, a leucine zipper domain that may mediate IKK-1 and IKK-2 homo/heterodimer formation, and a C-terminal helix-loop-helix domain with a serine-rich tail. IKK-2 activation is dependent on phosphorylation of serine 177 and 181 in the activation loop or T-loop. Alanine mutations abolish activity, whereas glutamate mutations produce constitutively active enzymes (Mercurio et al, Science 278: 860-866, 1997; Delhase et al, Science 284: 30313, 1999).

IKK-1 and IKK-2 can form heterodimers and IKK-2 homodimers and can bind to the 700-and 900-kDa cytoplasmic kinase complex called the "IKK signaler" (Mercurio et al, Science 278: 860-866, 1997). Another component, IKKAP-1 or NEMO/IKK γ, although not significantly catalytic, is capable of binding directly to IKK-2 and is required for complete activation of NF-. kappa.B (Mercurio et al, Mol Cell Biol 19: 1526-1538, 1999). A number of immune and inflammatory mediators, including TNF α, Lipopolysaccharide (LPS), IL-1 β, CD3/CD28 (antigen presentation), CD40L, FasL, viral infection and oxidative stress have been shown to cause activation of NF-. kappa.B. Although receptor complexes that transmit various stimuli exhibit large differences in their protein composition, it is understood that these stimuli all cause activation of IKK and NF-. kappa.B.

The IKK complex appears to be the central integration of various inflammatory signals that trigger phosphorylation of I κ B. IKK is activated at the position of the bisserine residue by upstream kinases including NF-. kappa.B-inducible kinase, NIK (Malinin et al, Nature 385: 540-544, 1997) and MEKK-1(Yujiri et al, Science 282: 1911-1914, 1998). The activities of NIK and MEKK-1 differ, for reasons not yet ascertained, but earlier data suggest that these kinases might selectively activate IKK-1 and IKK-2, respectively. Activated IKK phosphorylates cytostatic proteins, i.e., I κ B binding NF- κ B, and thereby masks nuclear localization signals in Rel proteins (Cramer et al, Structure 7: R1-R6, 1999). IKK phosphorylates IkB at serine 32 and 36 positions, forming a structural motif recognized by the E3 ligase β TRcP (Yaron et al, Nature 396: 590-594, 1998). Binding to β TRcP results in the formation of a ligase complex that polyubiquitinates I κ B, thereby serving as a target for degradation of the 26S proteosome. The free NF-. kappa.B is then transferred into the nucleus by recognition of the nuclear transporter and binds to sequence-specific regulatory units on the gene promoter.

Although both kinases can phosphorylate I.kappa.B in vitro, early studies with genetic mutants showed that the activation of NF-. kappa.B by pro-inflammatory stimuli (such as IL-1. beta. and TNF. alpha.) was key to IKK-2 rather than IKK-1. In addition, only catalytically inactive mutants of IKK-2 block the expression of genes regulated by NF-. kappa.B, such as single-cell chemotactic protein (MCP-1) and intercellular adhesion molecule (ICAM-1) (Mercurio et al, Science 278: 860-866, 1997). IKK-1 and IKK-2 studies in knockout animals confirmed these early findings (Hu et al, Science 284: 316-320, 1999; Li et al, Genes&Development 13: 1322-1328, 1999; li et al, Science 284: 321-324, 1999; takeda et al, Science 84: 313-; tanaka et al, Immunity 10: 421-429, 1999). IKK-1^-/-Animals die within hours after birth. Puppies develop skin abnormalities due to defective proliferation and differentiation, but do not show major defects in cytokine-induced activation of NF-. kappa.B. In contrast, IKK-2^-/-Embryos died at 14-16 days of gestation due to loss of liver function and apoptosis, which is very similar to that of Rel A knockout animals (Beg et al, Nature 376: 167-170, 1995). Further, IKK-2^-/-The activation of NF-kappa B of animal embryonic fibroblasts after being stimulated by cytokines is obviously reduced, and IKK-1^-/-The animal does not have this movement.

Thus, cellular and animal experiments have shown that IKK-2 is a central regulator of the pro-inflammatory effects of NF-. kappa.B, where IKK-2 is activated in response to immune and inflammatory stimuli as well as signaling pathways. Many immune and inflammatory mediators, including IL-1 β, LPS, TNF α, CD3/CD28 (antigen presentation), CD40L, FasL, viral infections and oxidative stress, play an important role in respiratory diseases. In addition, the ubiquitous expression of NF-. kappa.B and the response to various stimuli has suggested that almost all cell types in the lung can serve as potential targets for anti-NF-. kappa.B/IKK-2 therapy. These cells include alveolar epithelial cells, mast cells, fibroblasts, vascular endothelial cells, and infiltrating leukocytes (including neutrophils, macrophages, lymphocytes, eosinophils, and basophils). It is believed that IKK-2 inhibitors may exhibit broad anti-inflammatory activity by inhibiting the expression of genes such as cyclooxygenase-2 and 12-lipoxygenase (synthesis of inflammatory mediators), TAP-1 peptide transporter (antigen processing), MHC class I H-2K and class II stable chains (antigen presentation), E-selectins and vascular cell adhesion molecules (leukocyte aggregation), interleukin-1, 2, 6, 8 (cytokines), panates, eotaxin, GM-CSF (chemokines), and superoxide dismutase and NADPH quinone oxidoreductase (reactive oxygen species).

Patent application WO 94/12478 (the contents of which are incorporated herein by reference) describes indole derivatives that inhibit platelet aggregation. Patent applications WO 01/00610 and WO 01/30774 (the contents of which are incorporated herein by reference) describe indole and benzimidazole derivatives capable of modulating NF-. kappa.B. As described above, NF-. kappa.B is a heterodimeric transcription factor capable of activating a large number of genes encoding proinflammatory cytokines such as IL-1, IL-2, TNF. alpha. or IL-6. NF-. kappa.B is present in the cytosol, where it forms a complex with the natural inhibitor I.kappa.B. Stimulation of cells, such as by cytokines, causes phosphorylation and subsequent proteolysis of I κ B. This proteolysis results in activation of NF-. kappa.B, which then migrates into the nucleus where it activates a number of pro-inflammatory genes.

NF- κ B is activated to an excessive degree in diseases such as rheumatoid arthritis, osteoarthritis, asthma, Chronic Obstructive Pulmonary Disease (COPD), rhinitis, multiple sclerosis, myocardial infarction, Alzheimer's disease, type II diabetes, inflammatory bowel disease or arteriosclerosis. Inhibition of NF-. kappa.B has been reported to be useful as a stand-alone means or in combination with cytostatic therapy for the treatment of cancer. It has been demonstrated that inhibition of various links in the NF- κ B activation signal chain or direct interference of gene transcription by glucocorticoids, salicylates or gold salts can be used as a therapeutic measure for rheumatism.

The first step in the signaling cascade described above is the breakdown of I.kappa.B. This phosphorylation process is regulated by specific I κ B kinases. Hitherto, known I κ B kinase inhibitors generally have the disadvantage of not being able to specifically inhibit only one type of kinase. For example, most I κ B kinase inhibitors may inhibit multiple different kinases simultaneously because the catalytic domains of these kinases are structurally similar to each other. Thus, such inhibitors act in an undesirable manner on a wide variety of enzymes, including those with important functions.

Chronic Obstructive Pulmonary Disease (COPD) is a wasting inflammatory disease of the lungs characterized by progressive development of airflow obstruction that is not completely reversible (Pauwels et al, 2001). Airflow obstruction is associated with an abnormal inflammatory response of the lungs to toxic particles or gases, which is mainly caused by smoking. Although COPD affects the lungs, it also produces significant systemic consequences. The term COPD includes chronic obstructive bronchitis with obstruction of the small airways and emphysema with increased air space and destruction of the soft tissue of the lungs, loss of elasticity of the lungs and closure of the small airways. While chronic bronchitis is defined as a disease in which expectoration (due to mucus hypersecretion) occurs for more than three consecutive months with episodes occurring for two consecutive years. Some epidemiological evidence suggests that excessive mucus secretion is often accompanied by airflow obstruction, possibly due to obstruction of the peripheral airways. The majority of patients with chronic obstructive pulmonary disease have three conditions: chronic obstructive bronchitis, emphysema and mucus obstruction, but the severity of emphysema and obstructive bronchitis varies from person to person, Vestbo et al, 1996; barnes, 2004a, Barnes, 2004 b; hogg, 2004.

In industrialized countries, the majority of cases of chronic obstructive pulmonary disease result from smoking; but in developing countries other environmental pollutants, especially sulphur dioxide and particulate matter, as well as certain occupational chemicals such as cadmium, are important causes of chronic obstructive pulmonary disease. Passive smoking is also a causative agent.

Patients with chronic obstructive pulmonary disease are prone to deterioration, i.e. the respiratory symptoms are exacerbated dramatically. Exacerbations are the natural progression of chronic obstructive pulmonary disease characterized by changes in the patient's baseline dyspnea, cough and/or sputum levels beyond daily changes to a degree sufficient to require changes in treatment regimen (Rodriguez-Roisin, 2000; Burge and Wedzicha, 2003).

Bronchial infections are considered to be a common cause of exacerbations of chronic obstructive pulmonary disease, but controversy remains as to the nature of the source of infection and its actual role (Wedzicha, 2002; White et al, 2003). In addition, the exacerbation of chronic obstructive pulmonary disease is closely related to the levels of inhalable particles and environmental air pollutants, which in turn are related to the rate of admission (Rennard and Farmer, 2004).

The incidence of exacerbations is related to the severity of chronic obstructive pulmonary disease. Exacerbations can adversely affect the natural course of these diseases, perhaps because it can accelerate the deterioration of lung function, spread the disease throughout the body and cause premature death of the patient. Unfortunately, to date, no generally accepted definition has been made for the characterization of exacerbations of chronic obstructive pulmonary disease (Rodriguez-Roisin, 2000). How much the intensity and duration of an exacerbation should be to constitute an exacerbation is difficult to define. In fact, several definitions coexist and many clinical trials employ widely differing standards or are vague describing definitions used to diagnose exacerbations. The most commonly used clinical criteria to characterize acute exacerbations of chronic obstructive pulmonary disease are those described by Anthonisen et al. In this study, exacerbations were classified into three categories: type 1 exacerbations are characterized by an exacerbation of dyspnea, an increase in sputum volume, and an exacerbation of sputum purulence and purulence; type 2 exacerbations include any two of the above symptoms; category 3 exacerbations include any of the above symptoms with at least one additional symptom including sore throat or runny nose, fever of unknown origin, increased wheezing, increased cough, or a 20% increase in respiratory rate or heart rate over baseline levels over the first 5 days. These criteria have been used as benchmarks since their establishment and all proposals to worsen the aetiology need to be linked to these critical features.

It has also been proposed that inhibition of NF- κ B may be used as a stand-alone means or in combination with cytostatic therapy to treat proliferative diseases, such as tumors and leukemias. It has been demonstrated that inhibition of various links in the NF-. kappa.B activation signal chain or direct interference of gene transcription by glucocorticoids, salicylates or gold salts can be used to treat rheumatism.

Patent application WO 01/30774 discloses indole derivatives and U.S. application Ser. No. 10/642,970 discloses indole derivatives and benzimidazole derivatives which regulate NF-. kappa.B and exhibit strong inhibitory effects on I.kappa.B kinase. U.S. application serial No. 10/642,970 discloses, inter alia, indole and benzimidazole derivatives of formula (I), methods of preparing them, pharmaceutical compositions containing them, and methods of preventing and treating diseases associated with increased I κ B kinase activity comprising administering them.

In addition, U.S. application Ser. No. 10/642,970 discloses compounds represented by the following formulae (B), (C) and (D):

(B) (Compound (43) in the presence of a catalyst,

(C) (Compound 32); and(D) (Compound 48).

However, U.S. application Ser. No. 10/642,970 does not specifically disclose compounds of formula (I) wherein M is N, R1 is hydrogen, R2 is carboxyl (-COOH), R3 is methyl, R4 is pyridin-2-yl, R11 is hydrogen, and X is CH.

As noted above, there is a need for an I κ B kinase inhibitor, particularly an IKK-2 inhibitor, that acts by selectively inhibiting IKK. The inhibitor should also have a local rather than systemic effect. The inhibitors should be useful in the treatment of IKK-2 mediated pathological diseases or conditions, such as asthma or Chronic Obstructive Pulmonary Disease (COPD), which are alleviated by targeted administration of the inhibitor.

Summary of The Invention

The present invention relates to compounds having IkB kinase (IKK), particularly IKK-2 inhibitor activity, preferably selective inhibitor activity, as well as pharmaceutical compositions containing the compounds and methods of use.

In particular, the present invention relates to substantially pure compounds of formula (a):

or a pharmaceutically acceptable salt or solvate of said compound.

In addition, the invention relates to a pharmaceutical composition comprising a pharmaceutically effective amount of a compound of formula (a) and a pharmaceutically acceptable carrier.

Furthermore, the invention relates to the use of the compounds of formula (a) as I κ B kinase inhibitors.

Drawings

FIG. 1 photograph of a mouse imaging study in a NF-. kappa.B-luciferase reporter mouse model, in which animals received the following treatments: vehicle/PBS solution (negative control animals), NF-. kappa.B activation induced by IL-1. beta. but no compound [ vehicle/IL-1. beta.) or dose escalating (0.3mpk, 1mpk, 3mpk, and 10mpk) compound (A) or compound (B).

FIG. 2 is a graph of the results of a mouse imaging study in a NF- κ B-luciferase reporter mouse model showing the bioluminescence levels of animals that received the following treatments: vehicle/PBS solution (negative control animals), NF-. kappa.B activation induced by IL-1. beta. but no compound [ vehicle/IL-1. beta.) or dose escalating (0.3mpk, 1mpk, 3mpk, and 10mpk) compound (A) or compound (B).

FIG. 3 left panel: the content of compound (A) in lung and plasma tissues after injection of compound (A) at a dose of 0.3mg/kg into the trachea; right panel: the contents of compound (A) and compound (B) in lung and plasma tissues after injection of compound (B) at a dose of 0.3mg/kg into the trachea.

FIG. 4 is a graph showing the concentration of compound (A) in the lung after administration of increasing doses of compound (A) (0.01mpk, 0.03mpk, 0.10mpk, and 0.30 mpk); the right panel shows the concentration of compound (A) in plasma after administration of increasing doses of compound (A) (0.01mpk, 0.03mpk, 0.10mpk, and 0.30 mpk).

FIG. 5 is a graph on the left showing the concentrations of compounds (A) and (B) in the lung after administration of increasing doses of compound (B) (0.01mpk, 0.03mpk, 0.10mpk, and 0.30 mpk); the right panel shows the concentration of compounds (A) and (B) in plasma after administration of increasing doses of compound (B) (0.01mpk, 0.03mpk, 0.10mpk, and 0.30 mpk).

Detailed Description

Abbreviations

The following abbreviations referred to above and throughout the specification are to be understood as having the following meanings, unless otherwise indicated:

Boc₂di-tert-butyl O dicarbonate

DIEA N, N-diisopropylethylamine

DMAP 4-dimethylaminopyridine

DMF dimethyl formamide

DMSO dimethyl sulfoxide

ESI-MS electrospray ionization mass spectrometry

FAB-MS fast atom bombardment mass spectrometry

HATU O- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate

Acid salts

HPLC high performance liquid chromatography

PBS phosphate buffer solution

in the nasal cavity

PO for oral administration

i.p. intraperitoneal

i.t. intratracheal

Microcystin-LR hepatotoxins produced by certain cyanobacteria of the Anabaena and Oscillatoria genera

mbr mbar

mpk mg/kg

HEPES 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid

DTT dithiothreitol

ATP adenosine triphosphate

streptavidin-HRP streptavidin protein-horseradish peroxidase conjugate

TMB tetramethyl benzidine

pfu spot-forming Unit

MDI metered dose inhaler

DPI dry powder inhaler

Definition of

The following terms mentioned above and throughout the present specification should be understood to have the following meanings unless otherwise indicated.

"Compound of the invention" and similar expressions refer to a compound of formula (A) as described above, including pharmaceutically acceptable salts and solvates, such as hydrates, of the compound. By analogy, the intermediates mentioned herein, whether or not they are claimed per se, also include the salts and solvates of these intermediates, as far as the text is concerned. For the sake of clarity, specific examples are set forth herein where the text so permits, but these examples are purely for the purpose of reference and do not exclude other examples.

"treating" refers to preventing, partially alleviating, or curing a disease. The compounds and pharmaceutical compositions of the invention are useful in the treatment of diseases characterized by NF-. kappa.B activation and/or increased levels of cytokines and mediators (including but not limited to TNF. alpha. and IL-1. beta.) regulated by NF-. kappa.B. Inhibition or suppression of NF-. kappa.B and/or genes regulated by NF-. kappa.B, such as TNF. alpha., may be effected locally, for example in a certain tissue of a patient, or more generally in the general body of a patient suffering from a disease. Inhibition or suppression of NF-. kappa.B and/or genes regulated by NF-. kappa.B, such as TNF. alpha., may occur by one or more mechanisms, e.g., inhibition or suppression of any step of the signaling pathway, such as inhibition of IKK. The term "NF-. kappa.B-associated disorder" refers to a disease characterized by activation of NF-. kappa.B in the cytoplasm (e.g., phosphorylation of I.kappa.B). The term "TNF α -related disorder" refers to a disease characterized by an increase in TNF α levels. In the present specification, NF-. kappa.B-associated disorders include but are not limited to those associated with TNF α, since NF-. kappa.B is also associated with upregulation of the activity and activity of other pro-inflammatory proteins and genes. The term "inflammatory or immune disease" as used herein includes disorders associated with NF-. kappa.B as well as disorders associated with TNF α, e.g., any disorder or disease associated with the release of NF-. kappa.B and/or elevated levels of TNF α, including the disorders described herein.

"patient" includes humans and other mammals.

"pharmaceutically effective amount" refers to an amount of a compound, composition, drug, or other active ingredient that is effective to produce a desired therapeutic effect.

By "substantially pure" is meant that the compound is substantially free of, e.g., is separated from a biological or chemical composition with, other biological or chemical components, and preferably has an analytical purity of at least 70%. More preferably it has an analytical purity of at least 90%, still more preferably it has an analytical purity of at least 95%; in addition, "substantially pure" also means that the compound is substantially free of its prodrug, such as compound (B).

The invention also relates to a process for the preparation of the compound of formula (a), as shown in the following figure.

The starting compounds for this chemical reaction are known or can be prepared conveniently by methods known in the literature. U.S. application serial No. 10/642,970 describes the preparation of the indole carboxylic acid intermediate (compound 8) used in coupling step (vi) above. Compounds of formulae (B), (C) and (D) are prepared according to the method disclosed in U.S. application Ser. No. 10/642,970, incorporated herein by reference.

The condensation of the compounds can be carried out by coupling methods known to the person skilled in the art in peptide chemistry (see Houben-Weyl, Methoden der Organischen Chemie, methods of organic chemistry, volumes 15/1 and 15/2, Georg Thieme Verlag, Stuttgart, 1974, the contents of which are incorporated herein by reference). Suitable condensing or coupling agents are carbonyldiimidazole, carbodiimides such as dicyclohexylcarbodiimide or Diisopropylcarbodiimide (DIC), O- ((cyano (ethoxycarbonyl) methylene) -amino) -N, N' -tetramethyluronium tetrafluoroborate (TOTU) or polyphosphoric acid (PPA).

The condensation reaction can be carried out under standard conditions. In the condensation, it is necessary to protect an amino group which does not participate in the reaction with a reversible protecting group. The same applies to carboxyl groups which do not participate in the reaction, these groups preferably being chosen from (C) in the condensation reaction₁-C₆) In the form of alkyl, benzyl or tert-butyl esters. If the amino group is still present in the form of a precursor, such as nitro or cyano, and is not hydrogenated to amino after the condensation reaction, it is not necessary to protect the amino group. After the condensation reaction, the protecting group can be removed in an appropriate manner. Removal of NO, for example, by hydrogenation₂Base (guanidino protection of amino acids), benzyloxycarbonyl in benzyl ester, and benzyl. The tert-butyl-like protecting group can be removed under acidic conditions and the 9-fluorenylmethoxy-carbonyl group can be removed with a secondary amine.

The invention also relates to pharmaceutical compositions comprising a pharmaceutically effective amount of a compound of formula (a) and a pharmaceutically acceptable carrier.

Detailed description of the preferred embodiments

Due to their pharmacological profile the compounds of the invention are suitable for the treatment of diseases which can be alleviated by targeted administration of I κ B kinase inhibitors, by injection of the drug into the body part of the patient or the patient at risk, since for such diseases as asthma or Chronic Obstructive Pulmonary Disease (COPD) the effect of the local action of the drug is higher than the effect of the systemic action.

In clinical practice, the compounds of the present invention may be administered to humans and other animals in pharmaceutically acceptable dosage forms by means of local or systemic administration, including oral, inhalation, rectal, nasal, buccal, sublingual, vaginal, colonic, parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intraspinal and epidural), intracisternal and intraperitoneal. It will be appreciated that the optimal route of administration will depend, for example, on the condition of the patient.

Nasal, intratracheal or inhalation administration as well as aerosol administration are particular methods of administering the compounds of the present invention.

The drug combination therapy is more effective than increasing the single drug dose and can also reduce side effects. The IKK inhibitor may be used in combination with bronchodilators including, but not limited to, short-acting β 2 agonists; long-acting β 2 agonists such as salmeterol and formoterol; anticholinergic agents such as ipratropium bromide and tiotropium bromide. The IKK inhibitor may also be used in combination with a methylxanthine such as theophylline.

IKK2 inhibitors can be combined with several anti-inflammatory therapies including, but not limited to, immunomodulators directed at various stages of the inflammatory cascade and immunomodulators used to alleviate the course of inflammation. These therapies include, but are not limited to:

(A) inhibitors of cell aggregation and toxic inflammatory mediators, including but not limited to phosphodiesterase-4 inhibitors; inhibitors of protein kinases activated by p38 mitogen; biological agents such as anti-tumor necrosis factor alpha, anti-interleukin-8, and anti-monocyte chemotactic protein-1; adhesion molecules and chemokine inhibitors; and molecules that interfere with cell survival, clearance/apoptosis;

(B) proteolytic enzyme inhibitors, including but not limited to inhibitors of neutrophil-derived serine proteases (such as neutrophil elastase); and inhibitors of matrix metalloproteinases (MMPs, such as MMP-2, MMP-9, and MMP-12);

(C) antioxidants, including but not limited to N-acetyl cysteine and active oxygen species inhibitors or scavengers; and toxic peptides such as defensins which can directly damage cells;

(D) mucus secretion inhibitors, including but not limited to mucosal gene inhibitors; and mucus scavengers such as expectorants, sputum lysing agents and sputum mobilizing agents; and

(E) antibiotic therapy, such as the use of ketolides, e.g., Ketek .

The pharmaceutical combinations of the present invention may be administered to cells or cell populations or human patients by administering each compound simultaneously or sequentially in a pharmaceutically acceptable single component formulation, or in a multi-component formulation or a combination of a single component formulation and multiple agent formulations. Regardless of the mode of administration, the combination of drugs should have a pharmaceutically effective amount.

During the course of treatment, the treatment regimen/dosage schedule may be varied appropriately so that the minimum amount of each pharmaceutically effective amount of the compounds which exhibits satisfactory therapeutic efficacy when combined, and so that the duration of use of such combined pharmaceutically effective amounts of the compounds is minimized to the extent necessary to effect successful healing of the patient.

The pharmaceutical compositions of the present invention are preferably prepared and administered in the form of dosage units, each containing a specific dose of the compound (active ingredient). Pharmaceutically acceptable salts of the compounds of formula (a) are also within the scope of the invention. The term "salt" refers to an acid or base addition salt formed with an acid or base. In addition, "salts" also include salts of zwitterionic (inner) salts, i.e. compounds of formula (a) containing both a basic group, such as an amine, pyridine or imidazole ring, and an acidic group, such as a carboxylic acid. Pharmaceutically acceptable salts (i.e., non-toxic, physiologically acceptable salts), such as metal and amine salts whose cations do not significantly affect the toxicity and biological activity of the salt, are preferred. However, other salts may be used in the separation and purification steps of, for example, the manufacturing process, and such salts are also within the scope of the present invention. Salts of the compounds of formula (a) may be formed, for example, by reacting a compound of formula (a) with an amount (e.g., an equivalent amount) of an acid or base in a medium which allows the salt to form a precipitate or in an aqueous medium followed by lyophilization.

Acid addition salts are formed from compounds of the present invention which bear basic groups such as imino nitrogens, amino groups, or mono-or di-substituted groups. A particular acid addition salt should be a pharmaceutically acceptable acid addition salt, i.e. a salt whose anion is non-toxic to the patient at the pharmaceutical dose of the salt, so that the beneficial effects inherent in the compound in free form are not altered by side effects of the anion. The salt selected should be compatible with conventional pharmaceutical carriers and be suitable for the corresponding mode of administration. Acid addition salts of the compounds of the invention may be formed by reacting a free molecule bearing a basic group with an appropriate acid in a known suitable manner. For example, acid addition salts of the compounds of the present invention may be formed as follows: dissolving the free molecules with basic groups in water or aqueous alcoholic solution or other suitable solvent containing suitable acid, and evaporating the solution to separate the salt; or reacting the free molecule bearing the basic group with an acid in an organic solvent, in which case the salt can be isolated directly or obtained by concentrating the solution. Suitable acids for preparing the acid addition salts of the present invention are hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, various organic carboxylic and sulfonic acids such as acetic acid, citric acid, propionic acid, succinic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, ascorbic acid, malic acid, methanesulfonic acid, toluenesulfonic acid, fatty acids, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, cyclopentanepropionic acid, diglucosic acid, dodecylsulfuric acid, bisulfate, butyric acid, lactic acid, lauric acid, lauryl sulfuric acid, maleic acid, hydroiodic acid, 2-hydroxy-ethanesulfonic acid, glycerophosphoric acid, picric acid, trimethylacetic acid, palmitic acid, pectic acid, persulfuric acid, 3-phenylpropionic acid, thiocyanic acid, 2-naphthalenesulfonic acid, undecanoic acid, nicotinic acid, hemisulfuric acid, heptanoic acid, hexanoic acid, camphoric acid, camphorsulfonic acid, and the like.

Acid addition salts of the compounds of the present invention may be reduced to the parent compound of the present invention using known or modified methods. For example, an acid addition salt can be treated with a base (e.g., aqueous sodium bicarbonate or ammonia) to form the parent compound of the invention.

Base addition salts are formed from compounds of the present invention having a carboxyl group. A particular base addition salt is a pharmaceutically acceptable base addition salt, i.e. a salt whose cation is non-toxic to the patient at a pharmaceutical dose of the salt, so that the beneficial effects inherent in the compound in free form are not altered by side effects of the cation. The salt selected should be compatible with conventional pharmaceutical carriers and be suitable for the corresponding mode of administration. Base addition salts of the compounds of the invention can be formed by reacting free molecules bearing acidic groups with appropriate bases in a manner known to be appropriate. For example, base addition salts of the compounds of the present invention may be formed as follows: dissolving the free molecules with acidic groups in water or aqueous alcoholic solution or other suitable solvent containing a suitable base, and evaporating the solution to separate the salt; or reacting the free molecules bearing acidic groups with an acid in an organic solvent, in which case the salt can be isolated directly or by concentrating the solution. Suitable bases for the preparation of base addition salts are bases or amines derived from alkali metals and alkaline earth metals, such as: sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide, ammonia, ethylenediamine, N-methyl-glucamine, lysine, arginine, ornithine, choline, N' -dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris (hydroxymethyl) -aminomethane, tetramethylammonium hydroxide, and the like.

The base addition salts of the present invention may be reduced to the parent compounds of the present invention using known or modified methods. For example, base addition salts can be treated with an acid (e.g., hydrochloric acid) to form the parent compounds of the invention.

In practice, the compounds of the invention are administered to the patient in appropriate formulations to localize their effect. It will be appreciated that the preferred mode of administration will depend on the site of the condition to which it is administered.

Pharmaceutically acceptable dosage forms refer to dosage forms of the compounds of the invention, including, for example, powders, suspensions, sprays, inhalants, tablets, emulsions, or solutions, especially dosage forms suitable for inhalation. Reference may be made to the latest versions of Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.

If more effective delivery of the agent is desired, the compounds of the invention may also be microencapsulated or otherwise attached to slow release or targeted delivery systems, such as biocompatible, biodegradable polymer matrices (e.g., poly (d, l-lactide/glycolide copolymers), liposomes and microspheres, for subcutaneous or intramuscular injection in a so-called subcutaneous or intramuscular depot process, allowing the compounds to be released slowly and continuously over a period of two weeks or more.

The compounds of the present invention may also be sterilized, for example, by filtration through a sterile filter, or by incorporating sterilizing agents into sterile solid compositions which may be dissolved in sterile water or other sterile medium immediately prior to use.

Formulations suitable for nasal or tracheal administration are those suitable for administration to a patient by nasal or inhalation. The formulation may contain a powdered carrier having a particle size of, for example, between 1 and 500 microns (including particle sizes between 20 and 500 microns in 5 micron increments, e.g., 30 microns, 35 microns, etc.). Suitable formulations in which the carrier is a liquid for administration in the form of a nasal spray or nasal drops include aqueous or oily solutions of the active ingredient. Formulations suitable for spray administration may be prepared according to conventional methods and may be administered with other therapeutic agents. MDIs and DPIs are viable methods for administering doses of the compounds of the present invention to patients by inhalation.

The actual amount of active ingredient in the pharmaceutical compositions of the present invention may be varied to obtain a pharmaceutically effective amount of the active ingredient which will achieve the desired therapeutic response for a particular composition and method of administration. Thus, the selected dosage size for any particular patient will depend upon a variety of factors including the desired therapeutic effect, the route of administration, the course of treatment, the etiology and severity of the disease, the physical condition of the patient, body weight, sex, diet and age, the type and effect of each active ingredient, the rate of absorption, metabolism and/or excretion and other factors.

The total daily dose of the compounds of the invention (taken in one or several divided doses) is about 1000mg, in particular from 50mg to 300mg, in more particular from 10mg to 100 mg. However, doses above or below this daily dose may also be suitable. The daily dose may be administered in one dose unit or in several smaller dose units at one time or divided into several portions to be administered at predetermined time intervals. The percentage of active ingredient in the pharmaceutical composition may vary so long as an appropriate dosage is provided. It will be apparent that multiple unit dosage forms may be administered at substantially the same time. The frequency of administration is determined as needed to achieve the desired therapeutic effect. Some patients may respond rapidly to higher or lower doses, and require lower maintenance doses. For other patients, long-term treatment may be necessary, depending on their physiological needs, with a frequency of administration of 1 to 4 doses per day. Of course, for some patients, the daily dose should not exceed 1 or 2 doses.

The pharmaceutical formulations may be presented in unit dosage form by any of the methods known in the art of pharmacy. These methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more additional ingredients. In general, the formulations are prepared by: the active ingredient is uniformly and intimately associated with liquid carriers and/or finely divided solid carriers, and the product is then shaped, if necessary.

Experiment of

The analysis was carried out by mass spectrometry (FAB-MS, ESI-MS). The temperature units are centigrade; RT means room temperature (from 22 ℃ to 26 ℃). Abbreviations used herein have been set forth or are otherwise conventional in the art.

The invention is illustrated by the following examples.

Examples

Preparation example 1

Synthesis of methyl (3- (N-phenyl-N-2-pyridyl) amino) -2- (di-t-butoxycarbonylamino) propionate

(scheme 1, Compound 3)

Scheme 1

Methyl 2- (di-tert-butoxycarbonylamino) acrylate (scheme 1, Compound2)

50g (0.228mol) of (tert-butoxycarbonyl) serine (A)1) Dissolved in 300mL of acetonitrile. 107g (0.493mol) of di-tert-butyl dicarbonate and 2.64g (22mmol) of 4- (dimethylamino) pyridine (DMAP) are added. The mixture was stirred at room temperature overnight, the solvent was removed under reduced pressure, and the residue was extracted with 500mL of ethyl acetate. The organic phase was washed with 500mL of 1N HCl, dried over magnesium sulfate and the organic solvent was removed under reduced pressure. The residue is dissolved in 200mL of heptane, crystallized at-30 ℃ and filtered off with suction to give 23g of the acrylate2. The mother liquor was concentrated and the residue was dissolved in 140mL of acetonitrile. 31g (0.142mol) of di-tert-butyl dicarbonate and 1.26g (10mmol) of DMAP are added. The mixture was heated at 50 ℃ for 8 hours, the solvent was removed in vacuo, and the residue was extracted with 500mL of ethyl acetate. The organic phase is washed with 400mL of 1N HCl and dried over magnesium sulfate. After removal of the solvent in vacuo, crystallization from heptane gave in addition 31.5g of acrylate 2. 54.5g (0.181mol) of the product are obtained in 79% yield. Experimental formula is C₁₄H₂₃NO₆；M.W.＝301.34；MS((2M^*)+Na+)625.7。¹H NMR(DMSO-d₆)1.40(s，18H)，3.74(s，3H)，5.85(s，1H)，6.28(s，1H)。

(3- (N-phenyl-N-2-pyridyl) amino) -2- (di-tert-butoxycarbonylamino) propionic acid methyl ester (scheme 1, Compound 3)

4.96g (16.5mmol) of acrylate2With 5.6g (33mmol) of 2-anilinopyridine and 32.16g (98.7mmol) of cesium carbonate. 50mL of acetonitrile was added and the mixture was stirred at 45 ℃ for 2 days. The mixture was filtered through celite with suction to filter off the solid material, then washed 3 times with 100mL each time of acetonitrile. The organic phases were combined and the solvent was evaporated and the residue was chromatographed on silica gel with 1: 1 heptane/diethyl ether to give 5.66g (73%) of the ester3. Experimental formula is C₂₅H₃₃N₃₃O₆(ii) a Molecular weight 471.56; MS (M + H) 472.2.

Separation of enantiomers (scheme 1, Compound)3(S)And compounds3(R))

Racemic amino esters3From the corresponding acrylates2Preparation and subsequent resolution into the enantiomers 3(S) and 3(R) by preparative HPLC using a chiral stationary phase (e.g., Chiralpak AD (Daicel) 100X 380 at room temperature at a flow rate of 300 mL/min). The enantiomeric purity was determined by analytical grade HPLC (e.g., Chiralpak-AD-H (Daicel) 4.6X 250, 30 ℃, flow rate 1mL/min, room temperature).

Preparation example 2

2- (2-methylaminopyrimidin-4-yl) -1H-indole-5-carboxylic acid(s) ((R))8) Synthesis of (2)

(scheme 2, Compound8)

Scheme 2

1-dimethylamino-4, 4-dimethoxypent-1-en-3-one (scheme 2, compound6)

100g (0.76mol) of 3, 3-dimethoxy-2-butanone (b)4) With 90.2g (0.76mol) of N, N-dimethylformamide dimethyl acetal (5) Mix and stir at 120 ℃ for 48 hours. Methanol formed by the reaction is continuously removed from the reaction solution by distillation. Crystals spontaneously formed as the solution cooled and a small amount of heptane was added to complete the crystallization. 128.24g of crude product were obtained6(yield 90%) and the product was used in the next reaction without purification. The product has the empirical formula C₉H₁₇NO₃(ii) a Molecular weight 187.24; MS (M + H) 188.2.¹H NMR(DMSO-d₆)1.22(s，3H)，2.80(s，3H)，3.10(s，9H)，5.39(d，J＝15Hz，1H)，7.59(d，J＝15Hz，1H)。

[4- (1, 1-Dimethoxyethyl) pyrimidin-2-yl group]Methylamine (scheme 2, compound7)

1.22g (53mmol) of sodium were dissolved in 100mL of absolute ethanol. To this solution were added 5.8g (53mmol) of methyl guanidine hydrochloride and 10g (53mmol) of 1-dimethylamino-4, 4-dimethoxypent-1-en-3-one (g)6) The mixture was heated under reflux for 4 hours while stirring. The reaction was terminated by evaporating ethanol. Obtaining the product7The product was used in the next reaction without purification. Yield: 11.5g (58mmol, quant.); experimental formula is C₉H₁₅N₃O₂(ii) a Molecular weight 197.24; MS (M + H) 198.2.¹H NMR(DMSO-d₆)1.45(s，3H)，2.78(s，3H)，3.10(s，6H)，6.75(d，J＝3Hz，1H)，7.0-7.1(s(b)，1H)，8.30(d，J＝3Hz，1H)。

2- (2-methylaminopyrimidin-4-yl) -1H-indole-5-carboxylic acid (FIG. 2, Compound)8)

5g (25mmol) of [4- (1, 1-dimethoxyethyl) pyrimidin-2-yl are stirred]Methylamine (a)7) And 3.85g of 4-hydrazinobenzoic acid to 150mL of 50% sulfuric acid, and the mixture was heated at 130 ℃ for 4 hours. Methanol formed by the reaction is continuously removed from the reaction solution by distillation. After the mixture was cooled to 10 ℃, it was poured into 200mL of ice and the pH was then adjusted to around 5.5 with concentrated sodium hydroxide solution. The precipitated mixture of sodium sulfate and product was filtered off and the residue was then extracted several times with methanol. Concentrating the combined methanol extracts to obtain the product8It was purified by flash chromatography (dichloromethane/methanol 9: 1). Obtaining 0.76g (11%) of product; experimental formula is C₁₄H₁₂N₄O₂(ii) a Molecular weight 268.28; MS (M + H) 269.1.¹H NMR(DMSO-d₆)2.95(s，3H)，6.90-7.10(s(b)，1H)，7.18(d，J＝3Hz，1H)，7.4(s，1H)，7.58(d，J＝4.5Hz，1H)，7.80(d，J＝4.5Hz，1H)，8.30(s，1H)，8.38(d，J＝3Hz，1H)，11.85(s，1H)，12.40-12.60(s(b)，1H)。

Example 1

2- { [2- (2-methylamino-pyrimidin-4-yl) -1H-indole-5-carbonyl ] -amino } -3- (phenyl-pyridin-2-yl-amino

(ii) yl) -propionic acidA) Synthesis of (2)

Scheme 3

2- { [2- (2-methylamino-pyrimidin-4-yl) -1H-indole-5-carbonyl]-amino } -3- (phenyl-pyridin-2-yl-amino) -propionic acid methyl ester (scheme 3, compound10)

And (2) mixing.9g of the S enantiomer of 3: (3(S)) Dissolved in 30mL of dioxane and the solution cooled to 0 ℃.30 mL of 4N HCl in dioxane were added and the mixture was warmed to room temperature and then stirred for 12 hours. The solvent was removed in vacuo. The residue was extracted with 30mL of DMF (solution A). 2.47g (9.2mmol) of acid8Dissolved in 50mL of DMF and the mixture was cooled to 0 ℃. 4.21g of HATU and 6.4mL of DIEA were added. The mixture was stirred at 0 ℃ for 45 minutes, allowed to warm to room temperature, and then solution a was added. The mixture was stirred at room temperature for 12 hours. The solvent was removed in vacuo and the residue was taken up in 300mL of saturated NaHCO₃The solution was partitioned with 300mL ethyl acetate. The aqueous phase is extracted 3 times with 100mL of ethyl acetate and the organic phases are combined and washed with 400mL of saturated NaCl solution. The organic phase is dried over magnesium sulfate, the solvent is then removed under reduced pressure and the residue is purified by chromatography on silica gel (1: 3 heptane/ethyl acetate). 1.78g (55%) of the ester are obtained10. Experimental formula is C₂₉H₂₇N₇O₃(ii) a (ii) a Molecular weight 521.58; MS (M + H) 522.2.

2- { [2- (2-methylaminopyrimidin-4-yl) -1H-indole-5-carbonyl]-amino } -3- (phenylpyridin-2-yl-amino) -propionic acid (scheme 3, compoundA)

2.0g (3.8mmol) of methyl ester10Dissolved in 200mL of methanol. 1mL of a 2N aqueous NaOH solution was added, and the mixture was stirred at room temperature for 12 hours. After evaporation of the solvent, the residue was dissolved in water and saturated NaH was used₂PO₄The solution was adjusted to a pH of about 5. The precipitate formed is filtered off and washed with water. Drying at 40 ℃ under reduced pressure (about 1mbar) gave 1.95g (quantitative yield) of acidA. Experimental formula is C₂₈H₂₅N₇O₃(ii) a Molecular weight 507.56; MS (M + H) 508.3.¹H NMR(DMSO-d₆)2.95(s，3H)，4.22-4.50(m，2H)，4.65-4.72(m，1H)，6.29-6.36(d，1H)，6.70-6.79(m，1H)，6.90-7.10(sb，1H)，7.13-7.19(m，1H)，7.22-7.38(m，5H)，7.40-7.48(m，3H)，7.50-7.55(m，1H)，7.57-7.60(m，1H)，7.96(bs，1H)，8.34-8.40(m，2H)，8.80-8.90(d，1H)，11.80(s，1H)。

In vitro test method

IKK enzyme ELISA

The composition of the assay buffer was as follows: 50mM HEPES, 10mM MgCl₂10mM beta-glycerophosphate, 2. mu.M Microcytin-LR, 0.01% NP-40, and 5mM DTT.

IKK enzyme preparation was diluted 50-fold (from stock) and the test compound was dissolved in DMSO (final concentration in well: 2%).

The test procedure was as follows:

incubate enzyme and compound for 30 minutes;

adding 1mM ATP or 50. mu.M ATP;

pSer36-IkB peptide (substrate): 40 mu M;

incubation for 45 min, followed by addition of anti-pSer 32-pSer36-IkB peptide antibody;

after incubation for 45 minutes, transfer to plates coated with G protein coating;

after incubation for 90 minutes, 3 times of washing;

adding streptavidin-HRP, incubating for 45 min, and washing for 6 times;

adding TMB and incubating for 15 minutes; then the

The reaction was terminated and the reading of the luminometer was taken.

The in vitro test results are shown in table 1.

TABLE I

Compound (I)	IC50(nM)1mM ATP	IC50(nM)50μM ATP
			A	0.08	0.4
B	56.4	0.8
			C	378	16.8
D	3.8	-

IkB kinase IC of the Compound of formula (A) in the IKK enzyme ELISA described above, when ATP is 1mM₅₀705, 4725 and 47.5 times as much as the compounds of formulae (B), (C) and (D), respectively. This data shows that the activity of the compound of formula (a) is unexpectedly much higher than that of compounds (B), (C) and (D).

In vivo test comparison of Compound A and Compound B

Gene expression induced by NF-. kappa.B is closely related to inflammatory diseases such as asthma and arthritis. IkB kinase (IKK) is a central activation of NF-kB by various inflammatory agonists.

IKK is a complex of many subunits, including two catalytic subunits, IKK-1 (also known as IKK- α) and IKK-2 (also known as IKK- β), and a regulatory subunit, IKK- γ. Gene knockout studies have clearly shown that the IKK-2 or IKK- β subunits of the IKK complex are required for NF-. kappa.B activation by all known pro-inflammatory stimuli, including Lipopolysaccharide (LPS) and IL-1 β. Thus, IKK- β deficient cells are unable to activate IKK and NF- κ B in response to tumor necrosis factor α (TNF α) or interleukin-1 β (IL-1 β).

Internal biological imaging data show that administration of dominant negative IKK beta (Adv-IKK-2DN) inhibits IL-1 beta-induced NF- κ B activity in the lung. Therefore, selective IKK- β inhibitors may not only serve as potential anti-inflammatory agents, but may also help us understand the mechanisms by which these inflammatory agonists modulate NF- κ B activation.

A. Mouse NF-kB-luciferase reporter gene model

Mouse imaging method to study compounds a and B.

SUMMARY

A suspension of nanoscale compound particles in 0.2% Tween-80 in PBS was administered by the intranasal route.

Administration of intranasal drugs and inflammatory stimulators: mice were anesthetized with oxygen containing 4% isoflurane. Mu.l of the drug was injected into each nostril and the mice were allowed to inhale the suspension.

The tested mice are Balb/c female mice with the age of 6-8 weeks, and the lung of the tested mice is injected with an AdV-NF kappa B luciferase reporter gene. For imaging, mice were anesthetized with 4% isoflurane/oxygen. Fluorescein was injected into the abdominal cavity at a dose of 150 mg/kg. 10 minutes after the injection of fluorescein, mice were imaged with the IVIS200 system (Xenogen) and the bioluminescent exposure was 1 minute. Another method is to rapidly kill the mice painlessly 10-15 minutes after the injection of fluorescein, and then to excise the internal tissues for in vitro imaging.

B. Dose response

1-2X 10 days before (in the nasal cavity) stimulation with inflammatory stimulators (IL-1. beta. or LPS)⁸pfu of adenovirus-nfkb-luciferase was injected into the nasal cavity. 0.3-10mg/kg of the compound was injected into the nasal cavity 30 min to 1h before challenge with 0.5. mu.g LPS or 50ng IL-1 β. Imaging is performed 1 to several times during 1 to 24 hours after administration of the inflammatory stimulus.

The in vivo test results are shown below.

The effects of compound (A) and compound (B) on IL-1. beta. induced NF- κ B activation are shown in FIGS. 1 and 2. Although both compounds inhibited NF-. kappa.B activity in a dose-dependent manner, Compound (A) was more potent and its ED₅₀Is estimated to be about 1 mg/kg.

C. Pharmacokinetic study method

Male Hartley guinea pigs (body weight 450-550g) previously sensitized with ovalbumin were used to determine the levels of compound in the lungs and plasma. The suspensions of nanoscale particles of compound (a) and compound (B) were administered by intratracheal infusion at doses of 0.01, 0.03, 0.1 and 0.3 mg/kg. 1 hour after administration, animals were euthanized (Euthasol), and 1mL of blood was taken by puncturing the heart with a heparin-coated injection needle cannula. The plasma and blood cells were separated by centrifugation and then stored at-80 ℃ for analysis. Guinea pigs were excised, dipped dry, weighed and stored individually in 20-25mL glass vials at-80 ℃ for compound content analysis.

The major difference in pharmacokinetics between compound (A) and compound (B) is shown in FIG. 3 (results for the highest dose group, i.e., 0.3mg/kg dose).

As shown in fig. 3, after injecting compound (a) or compound (B) into the trachea, the content of compound (B) in the lungs is relatively lower than that of compound (a), which indicates that compound (B) can be rapidly absorbed from the lungs.

As shown in fig. 3 to 5, compound (B) is less advantageous as a candidate inhalation drug because 1) it is rapidly evacuated to the whole body after intratracheal administration; 2) compound (B) is a prodrug of compound (A), the latter being present in varying amounts; and 3) Compound (B) reduces the pulmonary content of Compound (A) (relative to the direct administration of Compound (A).

Compound (a) has a greater advantage as a candidate inhalant than compound (B) because 1) the systemic content of compound (a) is lower after intratracheal and oral administration; and 2) the retention time of the compound (A) in the lung is long.

In addition, there is evidence that compound (B) has high systemic dispersibility after oral administration.

The content ratio of compound (a) in lungs and plasma was 143 to 284 (depending on the dose), while the content ratio of compound (B) in lungs and plasma was 13 to 44 (depending on the dose). These ratios are obtained by dividing the pulmonary content of the compound by its corresponding plasma content at the same dose.

Claims (14)

1. Substantially pure compounds of formula (A)

Or a pharmaceutically acceptable salt or solvate thereof.

2. A pharmaceutical composition comprising a pharmaceutically effective amount of a compound of formula (a) and a pharmaceutically acceptable carrier.

3. A method of treating a patient having a disorder ameliorated by the inhibition of IKK-2, comprising administering to said patient a pharmaceutically effective amount of a compound according to claim 1.

4. The method of claim 3, wherein the administration is performed to produce a local activity.

5. The method of claim 3, wherein the disorder is asthma, rhinitis, chronic obstructive pulmonary disease, or chronic obstructive pulmonary exacerbations.

6. The method of claim 3, wherein the administration is intratracheal, intranasal, inhalation, or nebulization.

7. A method of treating a patient suffering from asthma, comprising administering to said patient a pharmaceutically effective amount of a compound of claim 1.

8. A method of treating a patient suffering from rhinitis comprising administering to the patient a pharmaceutically effective amount of a compound of claim 1.

9. A method of treating a patient suffering from chronic obstructive pulmonary disease comprising administering to the patient a pharmaceutically effective amount of a compound of claim 1.

10. A method of treating a patient suffering from chronic obstructive pulmonary exacerbations disorder comprising administering to the patient a pharmaceutically effective amount of a compound of claim 1.

11. The pharmaceutical composition of claim 2, further comprising a pharmaceutically effective amount of a compound selected from the group consisting of: bronchodilators, long-acting beta 2 agonists, anticholinergics, methylxanthines, and anti-inflammatory drugs.

12. The pharmaceutical composition of claim 11, wherein the bronchodilator is a short-acting β 2 agonist; the long-acting beta 2 agonist is selected from salmeterol and formoterol; the anticholinergic agent is selected from ipratropium bromide and tiotropium bromide; methylxanthines are theophylline; the anti-inflammatory agent is selected from the group consisting of inhibitors of cell aggregation and toxic inflammatory mediators, inhibitors of proteolytic enzymes, antioxidants, inhibitors of mucus secretion, and antibiotics.

13. The method of treatment of claim 3, further comprising administering a pharmaceutically effective amount of a compound selected from the group consisting of: bronchodilators, long-acting beta 2 agonists, anticholinergics, methylxanthines, and anti-inflammatory drugs.

14. The method of treatment according to claim 13, wherein the bronchodilator is a short-acting β 2 agonist; the long-acting beta 2 agonist is selected from salmeterol and formoterol; the anticholinergic agent is selected from ipratropium bromide and tiotropium bromide; methylxanthines are theophylline; the anti-inflammatory agent is selected from the group consisting of inhibitors of cell aggregation and toxic inflammatory mediators, inhibitors of proteolytic enzymes, antioxidants, inhibitors of mucus secretion, and antibiotics.