JP2001517631A - Proteasome inhibitors, ubiquitin pathway inhibitors, or agents that interfere with NF-κB activation via the ubiquitin proteasome pathway for treating inflammatory and autoimmune diseases - Google Patents

Abstract

(57) Abstract: The present invention relates to inflammatory and autoimmune by administering a proteasome inhibitor, a ubiquitin pathway inhibitor, an agent that blocks the activation of NF-κB via the ubiquitin-proteasome pathway, or a mixture thereof. It is directed to the treatment of sexual diseases. Further, the present invention provides for administering an effective combination of a glucocorticoid with a proteasome inhibitor, a ubiquitin pathway inhibitor, an agent that blocks NF-κB activation via the ubiquitin-proteasome pathway, or a mixture thereof. It is aimed at treating inflammatory and autoimmune diseases. Pharmaceutical compositions comprising a combination of a glucocorticoid with a proteasome inhibitor, a ubiquitin pathway inhibitor, an agent that blocks NF-κB activation via the ubiquitin-proteasome pathway, or a mixture thereof, are also within the spirit of the invention. Is intended.

Description

DETAILED DESCRIPTION OF THE INVENTION

TECHNICAL FIELD The present invention relates to compositions and methods for treating inflammatory and autoimmune diseases.

BACKGROUND OF THE INVENTION Eukaryotic cells contain a number of proteolytic systems, including lysosomal proteases, calpains, ATP-ubiquitin-proteasome-dependent pathways, and ATP-independent non-lysosomal processes. The major central proteolytic activities in the cytosol and nucleus are the proteasome, 20S (70), which has multiple peptidase activities.
0 kDa) particles. The 20S complex is the proteolytic core of a 26S (1500 kDa) complex that degrades or processes ubiquitin-conjugated proteins. Ubiquitination marks the protein for hydrolysis by the 26S proteasome complex. Many abnormal or short-lived normal polypeptides are ubiquitin-
Degraded by the proteasome-dependent pathway. Aberrant peptides are proteins induced by oxidants (e.g., those with oxidized disulfide bonds), products of immature translation arrest (e.g., those with exposed hydrophobic groups recognized by the proteasome), and stress-induced Modified, damaged or damaged proteins (stresses are induced, for example, by changes in pH or temperature or exposure to metals). The proteasome is involved in the rapid elimination of proteins involved in cell regulation (eg, cell cycle, gene transcription and metabolic pathways) and post-translational processing, intercellular communication and immune responses (eg, antigen presentation).

[0003] The transcription factor NF-κB is a member of the Rel protein family. The Rel family of transcription activator proteins can be divided into two groups. The first group involves p105 and p100 which require proteolytic processing and are processed to p50 and p52, respectively. The second group does not require proteolytic processing and includes p65 (Rel A), Rel (c-Rel), and Rel B. NF-κB contains two subunits, p50 and additional members of the Rel gene family, such as p65. The unprocessed p105 is p
65 and other members of the Rel family. In most cells, the p50-p65 heterodimer binds to IκB-α and exists in an inactive form in the cytoplasm. The ternary complex can be activated by dissociation and disruption of IκB-α, while the p65 / p105 heterodimer can be activated by processing p105.

[0004] The ubiquitin-proteasome pathway plays an essential role in regulating NF-κB activity, which is responsible for both processing of p105 to p50 and degradation of the inhibitor protein IκB-α. To target for proteasome degradation,
IκB-α must first undergo selective phosphorylation of serine residues 32 and 36, followed by ubiquitination. (Chen et al. Genes & Development (1995)
9: 1586; Chen et al. Cell (1996) 84: 853; Brockman et al. Mol. Cell. Biol
. (1995) 15: 2809; Browm et al. Science (1995) 267: 1485;)

When activated, NF-κB translocates to the nucleus where it plays a central role in regulating a very diverse set of genes involved in the immune and inflammatory responses (Grilli et al.
International Review of Cytology (1993) 143: 1-62). For example, NF-κB is composed of the TMF-α gene and the cell adhesion molecules E-selectin, P-selectin, I-selectin.
It is required for the expression of many genes involved in the inflammatory response, such as CAM and VCAM (Collins, T., Lab. Invest. (1993) 68: 499). NF-κB is required for the expression of many cytokine genes, such as IL-2, IL-6 granulocyte colony stimulating factor, and IFN-β. Inducible nitric oxide synthetase is under the regulatory control of NF-κB. Proteasome inhibitors block the degradation of IκB-α and the activation of NF-κB (Palombella et al. WO 95/25533, published September 28, 1995; Treanckner, et al. EMBO. J. (1994 13: 5433). Proteasome inhibitors also include leukocyte adhesion molecules E-selectin, VCAM-1 and IC
Block TNF-α induced expression of AM-1 (Read, et al. Immunity (1995)
2: 493).

[0006] Cyclin is a protein involved in cell cycle braking. The proteasome is involved in the degradation of sanculin. Cyclin degradation allows cells to exit one cell cycle stage (eg, mitosis) and enter another stage (eg, division). There is evidence that cyclins are converted to a form that is weaker to ubiquitin ligase, or that cyclin-specific ligase is activated during mitosis (Ciecha
nover Cell (1994) 79:13). Inhibition of the proteasome inhibits cyclin degradation,
Therefore, it inhibits cell proliferation (Kumatori et al. Proc. Natl. Acad. Sci. USA (
1990) 87: 7071).

[0007] Continuous turnover of cellular proteins by the ubiquitin-proteasome pathway is used by the immune system to screen for the presence of abnormal intracellular proteins (Go
ldberg and Rock Nature (1993) 357: 375). In this process, lymphocytes continuously monitor small fragments of cellular proteins present in Class I major histocompatibility complex (MHC) molecules. The proteasome first degrades proteins into small peptides, most of which are rapidly hydrolyzed to amino acids by cytosolic exopeptidases. However, some of these peptides are transported to the endoplasmic reticulum where they bind to MHC molecules and are transported to the cell surface in a process known as antigen presentation. If the peptide is abnormal (eg, if it is derived from a viral protein), it will induce cell destruction by cytotoxic T cells. Inhibitors that interfere with proteasome function have been shown to block the production of most peptides present on MHC class I molecules (Rock
, et al. Cell (1994) 78:76).

[0008] Multiple sclerosis (MS) is a non-curable neurological disorder that often causes chronic disability. MS is the most common demyelinating disease of the human central nervous system and typically affects women rather than young people and men. Clinically, the disease is characterized by a relapsing-remitting or chronic progressive stage, but there are more precisely defined stages for research purposes. It tends to follow an almost unpredictable course, leading to chronic and sometimes catastrophic destruction. MS is the result of an autoimmune disorder in genetically affected individuals mediated by autoreactive T cells that migrate to the CNS and initiate inflammatory demyelinating lesions.

[0009] Airway hyperresponsiveness to various protoplasms characterized by eosinophilia and lung inflammation is a pathology characteristic of asthma (Beasely, et al. Am. Rev. Resp. Dis
(1989) 139: 806). Asthma is a chronic disease of the airways that involves the release of many mediators and neurotransmitters that have multiple effects on many types of inflammatory cells as well as various target cells in the airways. The extent and spread of inflammation in the airway walls is widely related to the clinical severity of asthma. The inflammatory response of asthma consists of activation of mast cells present in the respiratory tract, an increase in the number of lymphocytes (mainly CD4 ⁺ T lymphocytes), and infiltration of eosinophils that appear to degranulate. There is a need in the art for effective therapies for the treatment of multiple sclerosis or asthma.

DISCLOSURE OF THE INVENTION The present invention is selected from the group consisting of an effective amount of a proteasome inhibitor, a ubiquitin pathway inhibitor, an agent that interferes with NF-κB activation via the ubiquitin-proteasome pathway, and mixtures thereof. A method for treating a patient suffering from multiple sclerosis or asthma. In certain embodiments of the invention, the agent is a proteasome inhibitor. Preferably, the proteasome inhibitor is peptidyl aldehyde, boronic acid (boroni
c acid), boronic esters, lactacystin and lactacystin analogs. In a preferred embodiment, the proteasome inhibitor is lactacystin or a lactacystin analog, more preferably lactacystin, crust-lactacystin β-lactone, 7-ethyl-crust-lactacystin β-lactone, 7-n-lactone. Propyl-crust-lactacystin β-lactone or 7-n-butyl-crust-lactacystin β-lactone. Most preferably, the proteasome inhibitor is 7-n-propyl-crust-lactacystin β-lactone.

[0011] In another embodiment of the invention, the agent is a ubiquitin pathway inhibitor. In yet another embodiment of the invention, the agent is an agent that interferes with the activation of NF-κB by the ubiquitin-proteasome pathway. Preferably, the agent that interferes with NF-κB activation is an agent that inhibits IκB-α phosphorylation. The present invention further provides an agent selected from the group consisting of glucocorticoids, proteasome inhibitors, ubiquitin pathway inhibitors, agents that interfere with NF-κB activation via the ubiquitin-proteasome pathway, and mixtures thereof. A method for treating a patient suffering from asthma.

[0012] In a preferred embodiment, the combination comprises an agent in an amount sufficient to reduce the required dose or frequency of treatment of the glucocorticoid. In certain preferred embodiments, the combination comprises an amount of glucocorticoid that is less than its standard recommended dose. In another preferred embodiment, the combination comprises a dose required for the agent or an amount of glucocorticoid sufficient to reduce the frequency of treatment.

In one preferred embodiment, the glucocorticoid is selected from the group consisting of flunisolide, triamcinolone acetonide, beclomethasone dipropionate, sodium dexamethasone phosphate, fluticasone propionate, budesonide, hydrocortisone, prednisone, prednisolone, mometasone, tipredan and butyxicort.

[0014] In another preferred embodiment, the combination used to treat a patient with asthma comprises a glucocorticoid and a proteasome inhibitor. More preferably, the proteasome inhibitor is lactacystin or a lactacystin analog. More preferably, the combination comprises 7-n-propyl-crust-lactacystin β-lactone and budesonide.

The present invention is further selected from the group consisting of glucocorticoids and proteasome inhibitors, ubiquitin pathway inhibitors, agents that interfere with NF-κB activation via the ubiquitin-proteasome pathway, or mixtures thereof. Pharmaceutical compositions comprising combinations with the agents. In certain embodiments, the pharmaceutical composition is provided in a unit dosage form. Preferably, the unit dosage form contains glucocorticoid in an amount less than its standard recommended dosage.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention relates to compositions and methods for treating inflammatory and autoimmune diseases. All patent applications, patents, and references cited herein are hereby incorporated by reference in their entirety. In the case of inconsistency, the present disclosure will prevail.

The present invention provides an effective amount of an agent selected from the group consisting of a proteasome inhibitor, a ubiquitin pathway inhibitor, an agent that inhibits NB-κB activation via a ubiquitin-proteasome inhibitor, and a mixture thereof. Is administered to a patient suffering from multiple sclerosis or asthma. It has now unexpectedly been found that the ubiquitin-proteasome pathway is a target for treating multiple sclerosis, asthma and rheumatoid arthritis. The following definitions are used in this specification. By “treating” is meant reducing any symptoms following administration of any proteasome inhibitor, ubiquitin pathway inhibitor, or agent that interferes with NF-κB activation via a ubiquitin proteasome inhibitor. .

“Ubiquitin pathway inhibitor” means any substance that specifically inhibits ubiquitination or transport of ubiquitin to a protein. "Proteasome inhibitor" means any substance that specifically inhibits the proteasome or its activity. “Agents that interfere with NF-κB activation by the ubiquitin-proteasome pathway”
Are 1) specifically inhibiting the proteasome or its activity; 2) IκB-α
Or 3) any substance that specifically inhibits ubiquitination of p105; or 3) specifically inhibits phosphorylation of IκB-α or p105.

“Specifically inhibits” refers to a protein that mediates its biological function at an inhibitor concentration that is lower than that required to produce another untreated biological effect. Means to interfere with ability. Preferably, the concentration of the inhibitor required for such interference is at least 2 times lower, more preferably at least 5 times lower, even more preferably at least 10 times lower than the concentration required to produce an irrelevant biological effect, Most preferably it is at least 20 times lower. Such inhibitors include, but are not limited to, interacting with another protein, substrate, or cofactor, either by interference with the active site or conformation of the protein, by effects on the protein itself or on other proteins. It can act by any of a variety of mechanisms, including interfering with the ability of the protein to act, and altering the microenvironment in which the biological function of the protein occurs normally.

In a first aspect, the present invention provides a method for treating multiple sclerosis. Multiple sclerosis (MS) is an incurable neurological disorder that frequently causes chronic disability. MS is widely believed to be the result of an autoimmune disorder in genetically affected individuals mediated by autoreactive T cells that migrate to the CNS and initiate inflammatory demyelinating lesions. The observation that MS is an autoimmune disease stems in part from the systemic abnormalities of immune function observed in patients with the disease, and experimental autoimmune cerebrospinal cord acting as a model for human disease. Partially derived through similarity to inflammation (EAE) (Kennedy et al., J. Neuroimmunol. (1987) 16: 345; Amason et al., Neurol. Clin. (1983) 1: 765; van der Veen et al. J. Neuroimmunol. (1989
48: 213; Immunol. Today (1986) 7: 121 by Gonatas et al .; Acta by Wckerle.
Neurol. (1991) 13: 197).

EAE is an inflammatory, autoimmune, demyelinating disease mediated by T cells of the CNS.
You. The disease may include whole brain homogenate in adjuvant, myelin basic protein
(MBP) or by injection of proteolipoprotein (PLP)
Thus, it can be induced in various laboratory animals including primates. EAE is T
-Cell mediated disease, passive migration of MBP- or PLP-reactive T cells
Induces the disease. Relapsing-remitting experimental autoimmune encephalomyelitis (R-EAE)
, Immunodominant epidemic on proteolipid protein (PLP139-151)
Either by immunization with toep or PLP139-151-specific CD4 ⁺ Induced in SJL / J mice by adoptive transfer of T cells (J. Ney by McRae et al.
uroimmunol. (1992) 38: 229). Clinical illness is characterized by recovery following an acute paralytic phase and secondary relapse. Occurs over a period of weeks or months
This pattern of recurrence and spontaneous recovery in laboratory animal models has been
Closely resembles the clinical signs of disease found in multiple sclerosis (MS) patients
You.

[0022] The method according to this aspect of the invention is selected from the group consisting of proteasome inhibitors, ubiquitin pathway inhibitors, agents that interfere with NF-κB activation via the ubiquitin-proteasome pathway, and mixtures thereof. Administering an effective amount of the agent to a patient suffering from MS. In a preferred embodiment, the agent is administered in an amount sufficient to reduce the frequency or severity of the disease recurrence. When administered during the remission phase at doses of 0.3 or 1.0 mg / kg, proteasome inhibitor 3b reduced the rate and severity of relapse in the R-EAE model (FIG. 1-2).

[0023] In a second aspect, the invention provides a method for treating asthma. Asthma is an obstructive pulmonary disease characterized by an airway hypersensitivity response, a severe form of airway narrowing in response to many different stimuli such as histamine, exercise, cold and allergens. Because of temporary bronchoconstriction, treatment is based in part on bronchodilation with β-adrenergic agonists. However, more recently, asthma has been correctly identified as a chronic condition of the airways involving the release of many mediators and neurotransmitters that have multiple effects on many types of inflammatory cells as well as various target cells in the airways. It is becoming possible. The severity and extent of inflammation in the airway walls is widely related to the clinical severity of asthma. The inflammatory response of asthma consists of activation of mast cells present in the respiratory tract, an increase in the number of lymphocytes (mainly CD4 ⁺ T lymphocytes), and infiltration of eosinophils, which manifests as degranulation. Increases in total eosinophil counts in peripheral blood are almost unchanged without suppression by corticoids or sympathomimetics. Eosinophils also appear on sputum examination.

Several animal models have been developed to study pulmonary inflammation with a characteristic appearance of airway eosinophils. One of the frequently used animal models is ovalbumin-sensitized guinea pigs (Am. Rev. Respir. Dis. (1990) 142: 6 by Dunn et al.).
80; Br. J. Pharmacol. (1990) 99: 679 by Sanjar et al., Am. R by Gulbenkian et al.
ev. Respir. Dis. (1990) 142: 680). Selective accumulation of both neutrophils and eosinophils has also been described in acutely sensitized Brown-Norway rats (Kips et al., Am. Rev. Respir. Dis. (1992) 145: 1306; Agents Actions by Richards et al., Supplement 34 (1991) 34: 359; Am. J. Resp. Crit. Ca by Chapman et al.
re Med. (1996) 153: A219). Allergen-induced pulmonary eosinophilia in effectively sensitized Brown-Norway rats is inhibited by the steroid, dexamethasone. Glucocorticoid therapy remains one of the most effective anti-inflammatory treatments available, and these drugs have been shown to reduce pulmonary eosinophilia in asthmatics (Holgate et al. Int.Arch.Allergy.Appl.Immunol
(1991) 94: 210).

[0025] The method according to this aspect of the invention is selected from the group consisting of proteasome inhibitors, ubiquitin pathway inhibitors, agents that interfere with activation of NF-κB via the ubiquitin-proteasome pathway, and mixtures thereof. Administering an effective amount of the agent to a patient suffering from asthma. In a preferred embodiment, the agent is administered in an amount sufficient to reduce the frequency or severity of the onset of asthma.

When administered intratracheally 1 hour, 24 hours and 48 hours after allergen challenge, 3b (0.1 or 0.3 mg / kg) increased eosinophils in effectively sensitized Brown Norway rats Disease was inhibited (Fig. 3-4).

It is further contemplated within the spirit of the present invention to treat asthma with combination administration with another agent or group of agents. Recently, the therapy approved per asthma, cromoglycate (cromoglycate), nedocromil (nedocromil), theophylline (theophylline), short - and long - acting beta ₂ - adrenergic receptor agonists, and inhaled or oral glucocorticoids Is included. More recently developed therapies include inhibitors of leukotriene biosynthesis, leukotriene receptor antagonists, and thromboxane antagonists. Anti-IL-
5 and anti-IgE antibodies have been developed (Science (1997) 276: 1643), and antisense approaches have also been studied (Nyce and Metzger, Nature (199).
7) 383: 721). In one preferred embodiment, the agents of the invention are used in an amount sufficient to reduce the required dose or frequency of treatment of the other agent or agents. In another preferred embodiment, the other agent or agents are used in an amount sufficient to reduce the frequency or frequency of treatment required for the agents of the present invention. The agent can be administered simultaneously with the other agent or agents, or can be administered at a different time.

[0028] Steroid therapy is particularly effective in treating asthma and is an essential course of therapy for severe asthma. Unfortunately, however, bone growth suppression, Addison's disease,
Many complications, including Cushing's syndrome, cataracts, immunosuppression, and excessive bruising, result from prolonged steroid use. Many of these side effects can be minimized by topical administration of the drug to the lungs by inhalation. However, high doses, such as those required for severe cases, generate significant systemic exposure and increase the associated side effects. Thus, drugs that tolerate lower steroid doses ("steroid-thrift") offer very real clinical benefit.

Surprisingly, 3b (0.03 or 0.1 mg / kg) in combination with the glucocorticoid, budesonide (0.1 mg / kg) 1 hour before and 24 and 48 hours after allergen challenge Was found to inhibit eosinophilia in effectively sensitized Brown Norway rats (FIG. 5-6).
Strikingly, neither drug was effective when administered alone at those doses, suggesting a synergistic effect of the two drugs.

In a third aspect, the present invention provides a method for treating glucocorticoid with NF-κ via a proteasome inhibitor, a ubiquitin pathway inhibitor, a ubiquitin-proteasome pathway.
There is provided a method of treating a patient suffering from asthma, comprising administering to a patient a combination of an agent that interferes with the activation of B and an agent selected from the group consisting of mixtures thereof. The glucocorticoid and agent can be administered simultaneously or at different times on the same or different days, and at the same or different frequencies. Preferably,
The dose of each agent is spaced such that a combined physiological effect is achieved. The glucocorticoid is preferably between 0 minutes and about 1 month before or after the agent of the invention, more preferably between 0 minutes and about 1 week before or after the agent of the invention, most preferably the invention. Is administered between 0 minutes and 24 hours before or after.

The glucocorticoid used in the present invention is not limited, but
Flunisolide, triamcinolone acetonide, beclomethasone dipropionate
ate), dexamethasone sodium phosphate, fluticasone propionate (fluticasone propionate), budesonide (
budesonide), hydrocortisone (hydrocortisone), prednisone (predonis)
one), predonisolone, mometasone, tipredane and butixicort. Preferably, the glucocorticoid is budesonide. Suitable formulations, dosages, and routes of administration for glucocorticoids are known in the art (Physician's Desk®
eference, 51st Edition, 1997, Medical Economics: Montvale, NJ).

In certain preferred embodiments, the agents of the present invention are administered in a dose required for glucocorticoids or in an amount sufficient to decrease the frequency of treatment. Preferably, the amount of glucocorticoid administered does not exceed the standard recommended dosage, more preferably
The amount of glucocorticoid administered is less than the standard recommended dosage of the drug when administered alone.

In certain preferred embodiments, the amount of glucocorticoid administered is less than a proteasome inhibitor, a ubiquitin pathway inhibitor, an agent that blocks activation of NF-κB via the ubiquitin-proteasome pathway, and mixtures thereof. The dose required for an agent selected from the group and the frequency of treatment are sufficient. Most preferably, a combination of a glucocorticoid with an agent selected from the group consisting of proteasome inhibitors, ubiquitin pathway inhibitors, agents that interfere with NF-κB activation via the ubiquitin-proteasome pathway, and mixtures thereof. Treatment of patients suffering from asthma with. Produces efficiencies with fewer or less severe side effects or toxicity than treatment with either agent alone.

In a fourth aspect, the present invention provides a method for treating glucocorticoids with NF-κ via a proteasome inhibitor, a ubiquitin pathway inhibitor, a ubiquitin-proteasome pathway.
There is provided a pharmaceutical composition comprising a combination with an agent that interferes with the activation of B, and an agent selected from the group consisting of mixtures thereof, which is further contemplated within the spirit of the invention. The pharmaceutical compositions of the present invention can be provided in unit dosage form. In a preferred embodiment, the unit dosage form contains an amount of glucocorticoid that is less than its standard recommended dosage when administered as such. The following description of non-limiting examples of suitable proteasome inhibitors, ubiquitin pathway inhibitors, and agents that interfere with NF-κB activation via the ubiquitin-proteasome pathway applies to pharmaceutical formulations and methods according to the present invention. Is done.

Non-limiting examples of proteasome inhibitors for use in the present invention include peptidyl aldehyde (US Pat. No. 5,580,854 to Orlowski et al .; WO 9 to Stein et al.).
5/24914; WO 91/13904 by Siman et al .; J. Med. Chem., 38:22 by Iqbal et al.
76-2277 (1995)), boronic acid peptidyl (WO 96/13266 by Adams et al .; WO 91/13904 by Siman et al.), And other peptidyl derivatives having proteasome inhibitory activity (by Iqbal et al.). U.S. Patent No. 5,614,649; Iqbal et al. U.S. Patent No. 5,550,262; Spaltenstein et al., Tetrahedron Letters, 1
996, 37, 1343), and lactacystin and lactacystin analogs (Fen
Proc. Natl. Acad. Sci. USA (1994) 91: 3358 by teany et al .; WO by Fenteany et al.
No. 96/32105; U.S. patent application Ser. No. 08 / 912,111 by Soucy et al.
Is included. The agents disclosed herein can be administered by any route including intradermal, intraperitoneal, intranasal, intratracheal, subcutaneous, oral or intravenous. For asthma indications, administration is preferably by the inhalation route.

The peptide aldehyde proteasome inhibitors used in the present invention are preferably those described in WO 95/24914 by Stein et al. Published on September 21, 1995 or by Siman et al. Published on September 19, 1991. These are disclosed in WO 91/13904 (both are explicitly referred to and regarded as part of the present specification).

The boronic acid or boronic acid ester compound used in the present invention includes:
Preferably WO 96/13266 by Adams et al., WO 91/13904 by Siman et al. Or
And US Pat. No. 5,614,649 to Iqbal et al.
The source is explicitly identified and considered as part of the present specification).

In certain preferred embodiments, the boronic acid compound used in the present invention is: N-acetyl-L-leucine-β- (1-naphthyl) L-alanine-L-leucine boronic acid, β- (1- Naphthyl) -L-alanine-L-leucine boronic acid, N- (4-morpholine) carbonyl-β- (1-naphthyl) -L-alanine-L-leucine boronic acid, N- (8-quinoline) sulfonyl -Β- (1
-Naphthyl) -L-alanine-L-leucine boronic acid, N- (2-pyrazine) carbonyl-L-phenylalanine-L-leucine boronic acid, and N-
It is selected from the group consisting of (4-morpholine) carbonyl- [O- (2-pyridylmethyl)]-L-tyrosine-L-leucineboronic acid.

Lactacystin and lactacystin analogs used in the present invention include:
Preferably, those disclosed in WO 96/32105 by Fenteany et al. Or in U.S. patent applications by Soucy et al. Is considered part of the book). In certain preferred embodiments, the lactacystin analog compound is lactacystin, crust-lactacystin β-lactone, 7-ethyl-crust-lactacystin β-lactone, 7-lactone.
It is selected from the group consisting of n-propyl-crust-lactacystin β-lactone, and 7-n-butyl-crust-lactacystin β-lactone. These compounds can be prepared as shown in Schemes 1 and 2. Most preferably, the lactacystin analog compound is 7-n-propyl-crust-lactacystin β-lactone (3b (Scheme 2)).

In a preferred embodiment, the agent used to treat a patient with multiple sclerosis or asthma is a proteasome inhibitor. Preferably, the proteasome inhibitor is lactacystin or a lactacystin analog, more preferably 7-
n-propyl-crust-lactacystin β-lactone. Preferably,
The combination used to treat a patient suffering from asthma comprises a glucocorticoid and a proteasome inhibitor. More preferably, the proteasome inhibitor is lactacystin or a lactacystin analog. Most preferably, the combination comprises 7-n-propyl-crust-lactacystin β-lactone and budesonide.

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[0044] Non-limiting examples of ubiquitin pathway inhibitors include Biochem. 35 (5) by Berleth et al.
: 1664-1671 (1996). Inhibitors of IκB-α phosphorylation are also well known (Chen, Cell, 84: 853 (1996); Chen, U.S. Patent Application No. 08 / 825,559).

The concentration of the disclosed compounds in the pharmaceutically acceptable mixture depends on several factors, including the dose of the compound to be administered, the pharmacokinetic characteristics of the compound (s) used, and the route of administration. Will change. An effective amount of an agent for treating multiple sclerosis, asthma or rheumatoid arthritis is about 10 μg / Kg body weight of the recipient mammal.
It will be in a wide range between g and about 50 mg. The agents can be administered in a single dose or in multiple doses. The treatment can be administered daily or more frequently, depending on the patient's age and overall health, and on the factors selected, including the formulation and route of administration of the compound (s) selected. Other factors to consider in determining a dose include the type of treatment, if any, the frequency of treatment and the nature of the effect desired; extent of tissue damage; gender; duration of symptoms; If the opposite adaptation (counter indica
option); as well as other variables that are evaluated by individual physicians.

In certain preferred embodiments, the concentration of the proteasome inhibitor determines the proteasome activity after administering the proteasome inhibitor to a mammal ex vivo (
ex vivo). Such measurements include obtaining one or more test biological samples from a mammal at one or more specific times after administering a proteasome inhibitor; measuring proteasome activity in the test biological sample or samples. Determining the amount of proteasome activity in the test biological sample or group of samples; and comparing the amount of proteasome activity in the test biological sample with that in the reference biological sample obtained from the mammal not receiving the proteasome inhibitor. Including comparing.

[0047] Biological samples obtained from mammals can include, but are not limited to, blood, urine, organ and tissue samples. In certain preferred embodiments, the biological sample is a blood sample, more preferably a blood sample from which leukocytes are isolated prior to measuring proteasome activity. Methods for fractionating blood cells are known in the art and are further described in the Examples. Hemoglobin strongly interferes with fluorescence measurements,
Therefore, when fluorescence measurement is used for proteasome activity, it is preferably excluded from the test sample. Once fractionated, leukocytes are lysed by standard techniques. Variation in lysis efficiency determines the total protein content in the sample using standard techniques,
It can be corrected by normalizing the proteasome activity measured in the sample to the protein content in the sample.

Preferably, a substrate having a detectable label is provided to the reaction mixture and proteolytic cleavage of the substrate is monitored, followed by disappearance of the substrate or appearance of the cleaved protein. Detection of the label can be performed, for example, by a fluorescence, colorimetric or radioactivity assay. More preferably, the substrate is a peptide substrate and the reaction mixture further comprises a 20S proteasome activator. Preferably, the activator is that taught by Coux et al. (Ann. Rev. Bioc.
hem. 65: 801-847 (1995)), more preferably PA28 or sodium dodecyl sulfate (SDS). Preferably, the peptide substrate contains a cleavable fluorescent label, and the release of the label is monitored by a fluorescence assay. More preferably,
The substrate is N-succinylleucylleucylvalyltyrosyl 7-amino-4-methylcoumarin (Suc-Leu-Leu-Val-Tyr-AMC).

The activity measured in the biological sample is compared to that measured in a reference biological sample obtained from a mammal not receiving the inhibitor. In certain preferred embodiments, the test biological sample and the reference biological sample each comprise a plurality of samples, each separately stored from a group of mammals, preferably mice, being treated. In other preferred embodiments, the test biological sample and the reference biological sample each comprise a single sample obtained from an individual mammal. Individual sample assays are currently preferred, except where impractical due to the small size of the mammal. In some preferred embodiments, the statistical sample is obtained by storing data from an individual test biological sample or from an individual reference biological sample.

Daily variability in assays can result from factors such as differences in buffers, operator variability, variability in instrument performance, and temperature variability. Such variability can be minimized by normalizing proteasome activity in both the biological sample and the reference sample relative to a standard proteasome sample. In certain preferred embodiments, the standard sample comprises purified 20S proteasome, more preferably, purified 20S proteasome from rabbit reticulocytes.

[0051] In certain preferred embodiments, the reference sample is obtained from a treated mammal prior to initiating treatment. This embodiment is currently preferred for higher mammals in order to minimize the effects of inter-mammal variability. Clinical monitoring of drug action now preferably includes this embodiment of the invention, wherein each patient serves as his or her own baseline control.

A decrease in inhibitor activity in a biological sample relative to a reference sample is indicative of the in vivo effect of the inhibitor at the time the biological sample was obtained. In certain preferred embodiments, the biological sample is obtained at multiple time points after administering the inhibitor. In these embodiments, measurement of proteasome activity in the biological sample provides an indication of the extent and duration of the inhibitor's in vivo effect. In certain other preferred embodiments, multiple biological samples are obtained from a single mammal at one or more time points. In these embodiments, measurement of proteasome activity in the biological sample provides an indication of the distribution of the inhibitor in the mammal.

[0053] In certain preferred embodiments, the dosage and frequency of administration of the proteasome inhibitor is selected to avoid excessive proteasome inhibition. In certain embodiments, excessive proteasome inhibition produces a toxic effect, including, but not limited to, vomiting, diarrhea, hypovolemia, hypotension, and lethality. Preferably, the dose and frequency of administration of the proteasome inhibitor is selected such that proteasome inhibition in any future biological sample does not exceed about 95%.

[0054] In certain other preferred embodiments, the dosage and frequency of administration of the proteasome inhibitor is selected to achieve clinically useful proteasome inhibition. Preferably, clinically useful proteasome inhibition results in a clinically beneficial anti-tumor, anti-inflammatory, anti-viral or anti-parasitic effect. Preferably, the dosage and frequency of administration of the proteasome inhibitor will be at least about 15%, preferably about 20%, more preferably about 30%, although in some instances proteasome inhibition as high as 95% may be preferred. Even more preferably about 40%, and even more preferably about 50%, and most preferably about 50% to about 80%
Is selected to be achieved in the future biological sample.

The agent used in the present invention can be prepared by any method well known in the pharmaceutical field, for example, Remington's Pharmaceutical Sciences (Mack Pub. Co., Easton, PA,
1980) can be prepared for administration. Preparations may be in the form of a liquid or liquid suspension for use in parenteral administration; in the form of tablets or capsules for oral administration; powders, gels, oily solutions, nasal drops for intranasal or intratracheal administration It can be prepared in the form of an agent, an aerosol or a mixture. Formulations for parenteral administration may include conventional excipients sterile water or sterile saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalene, and the like. In part, by using lactide biocompatible, biodegradable polymers, and lactide / glycolide or polyoxyethylene / polyoxypropylene copolymers, controlled release of the agent can be obtained. Additional parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps,
Includes implantable infusion systems and liposomes. Formulations for inhaled administration may include lactose, polyoxyethylene-9-lauryl ether, glycocholate, or deoxycholic acid.

For the treatment of asthma, administration by the inhalation route is preferred to minimize potential side effects or toxicity resulting from systemic exposure to the agent. According to the invention, an "effective amount" of an agent is an amount sufficient to produce an improvement in any of the symptoms or signs of the disease (Basic & Clinica by Stites et al.).
l Immunology Lange Medical Publications, Los Altos, California, 1982).

The use of any of the agents disclosed herein in combination with another agent or group of agents for use in treating multiple sclerosis or asthma is further contemplated within the spirit of the invention. The invention is further illustrated by the following non-limiting examples:

EXAMPLES Example 1 Relapse-Remission Experimental Autoimmune Encephalomyelitis Materials and Methods Mice. Female SJL / J mice, 6 weeks old, were purchased from Harlan Laboratories (Indianapol, Inc.).
is, IN), housed in a Northwestern animal care facility, and maintained on free standard laboratory food and water. Paralyzed mice had easier access to food and water. Peptide. PLP139-151 (HSLGKWLGHHPDKF) is Peptides
Purchased from International (Louisville, KY). Amino acid composition was confirmed by mass spectrometry, and purity (> 98%) was evaluated by HPLC.

Induction of R-EAE. As described previously (J. Neuroimmune by McRae et al.
ol. (1992) 38: 229), PLP13 in Freund's complete adjuvant (CFA).
Mice were immunized by subcutaneous injection of 9-151. Each mouse received 50 μg of PLP139-151 distributed over two sites outside each hind leg. Drug treatment. Starting on day 22, animals (10 per group) were dosed ip (5 mL / kg) once daily with vehicle or 3b (0.3 or 1.0 mg / kg).
Processing continued until day 40.

Clinical evaluation. Mice were observed daily for clinical signs of disease. Mice were scored according to the following clinical severity: Grade 0, no abnormalities; Grade 1, weak tail; Grade 2, weak tail and hind leg weakness (sloppy walking); Partial hind leg paralysis; grade 4, complete hind leg paralysis; and grade 5, moribund.

Results Data were plotted as mean daily clinical scores for all animals in a particular treatment group (FIG. 1). Recurrence is indicated if the animal has at least a full clinical sc
ore) was defined as at least one full grade increase in clinical score after previously improving and stabilizing. Animals treated with 3b (both dose groups) showed a reduced clinical score compared to vehicle-treated animals. The incidence of relapse was 5/10 for the 0.3 mg / kg group and 2/10 for the 1.0 mg / kg group compared to 6/10 for the vehicle-treated group. .

Data were also plotted as mean daily recurrence rates for all animals in a particular treatment group (FIG. 2). The average maximum clinical score per group is also provided as an indicator of disease severity. Animals treated with 3b (both dose groups) exhibited reduced disease incidence and reduced disease severity compared to vehicle-treated animals.

Example 2 Effectively Sensitized Allergens in Brown-Norwegian Rats
Effect of treatment with 3b on induced lung leukocytes Materials and Methods Rat. Male Brown-Norway rats are from Harlan Olac Limited (Biceste
r, Oxon, UK) and collected in the 180-200 g weight range. After acclimation for at least 5 days, the animals were effectively sensitized for 3 weeks and were within the 250-300 g body weight range at the time of allergen exposure. Food and water were provided ad libitum.

Sensitization. Ovalbumin (OA) mixed with aluminum hydroxide gel (10 mg)
; 10 μg) were injected (0.5 mL, ip) into Brown-Norway rats and repeated 7 and 14 days later. Drug treatment. On day 21, sensitized rats were anesthetized (halothane in O ₂ ) and 1 hour before OA exposure, 3b, dexamethasone or vehicle (lactose) was instilled via a cannula placed directly in the trachea. . This operation was repeated 24 and 48 hours after OA exposure. attack. After recovery, sensitized animals were restrained in plastic tubes and exposed to OA (10 mg / mL) aerosol using a nasal limited exposure system (60 min).
. Animals were sacrificed 72 hours later with pentobarbital (250 mg / kg, ip).

Analysis. 3 aliquots (4 mL) of Hanks solution (HBSS × 10, 100 mL; EDTA 100 mM, 100 mL; HEPES 1 M, 10 mL with water 11 m
The collected cells were stored and the total volume of the collected fluid was adjusted to 12 mL by adding Hank's solution. Total cells were counted (Sysmex Microcell Counter F-500, TOPA Medical Electronics Ltd., Ja
pan). The harvested fluid was diluted (to approximately 10 ⁶ cells / mL) and centrifuged (Cytospin,
Smears were generated by spinning an aliquot (100 μL) in Shandon, UK). Air dry the smear and fast green (2 mg / mL) in methanol to identify eosinophils, neutrophils, macrophages and lymphocytes
Fix for 5 seconds with eosin G (5 seconds) and thiazine (5 seconds) (Diff
-Quik, Baxter Dade Ltd., Switzerland). A total of 500 cells per smear were counted by light microscopy (x1000) under oil immersion.

Results As a result of ovalbumin challenge, B from actively sensitized (AS) Brown Norway rats was compared to naive (naked) (N) rats.
Eosinophils, neutrophils and total leukocytes in AS fluid were significantly increased. Treatment with dexamethasone (0.1 mg / kg it) prevented this increase. At doses of 0.1 and 0.3 mg / kg, 3b reduced the inward flow of eosinophils and whole leukocytes. Also, a significant increase in lymphocyte count was observed at all doses (
(Fig. 3-4)

Conclusion Compound 3b is effective in preventing leukocyte inflow after allergen challenge in an animal model of asthma.

Example 3 Effect of Treatment with a Combination of 3b and Budesonide on Allergen-Induced Pulmonary Leukocyte Accumulation in Actively Sensitized Brown Norway Rats Materials and Methods Rats. Male Brown Norway is Harlan Olac Limi
supplied by Ted (Bicester, Oxon-UK), 180-20
It was delivered within a 0 g body weight range. Following at least 5 days of acclimation, animals were actively sensitized for 3 weeks and 250-3 at the time of allergen exposure.
It was within the 00 g body weight range. Food and water were available ad libitum.

[0069] Sensitization. Ovalbumin (OA; mixed with aluminum hydroxide gel (10 mg)
10 μg) were injected into Brown Norway rats (0.5 ml, it.
), Repeated after 7 and 12 days. Drug treatment. Day 21, were anesthetized sensitized rats (O ₂ in halothane 5%), Bihiku Le (group V; lactose, 1 mg), budesonide (group C, 0.1 mg / kg; group F
, 0.5 mg / kg), 3b (Group A, 0.03 mg / kg; Group B, 0.1 mg / kg)
kg) or a mixture of budesonide and 3b (Group D, 0.1 / 0.03 mg /
kg; group E, 0.1 / 0.1 mg / kg; group G, 0.5 / 0.03 mg / kg;
Groups H, 0.5 / 0.1 mg / kg) were intratracheally treated (it) one hour prior to OA exposure. The drug was dropped via a cannula directly into the trachea. This procedure was repeated 24 and 48 hours after OA exposure.

Attack. After recovery, the sensitized animals were restrained in plastic tubes (60 minutes) and exposed to an aerosol of OA (10 mg / ml) using a nose-only exposure system (6).
0 minutes). After 72 hours sacrificed with pentobarbital (250 mg / ip). analysis. Hanks' solution (HBSS × 10, 100 ml; EDTA 100 mM,
Lungs were flushed with 3 aliquots (4 ml) of 100 ml; HEPS 1N, 10 ml, 11 ml with water); collected cells were pooled and the total lysis of collected fluid was determined by addition of Hanks' solution. Adjusted to 12 ml. All cells were counted (Sysnex Microcell Counter F-500, T
(ORA medical electronics LTD, Japan). The collected fluid was diluted (to approximately 10 ⁶ cells / ml) and a smear was created by spinning an aliquot (100 μl) in a centrifuge tube (Cytospin Shandon, UK). The smear was air dried and a dark green solution in methanol (2 ml /
L) for 5 seconds, eosin G (5 seconds) and thiazine (5 seconds) (
(Diff-Quik, Base Dade Ltd, Switzerland) to differentiate eosinophils, neutrophils, macrophages and lymphocytes. A total of 500 cells per smear were counted by light microscopy under oil immersion (x1000)
.

Results Eosinophils, neutrophils and BAS fluids from actively sensitized vehicle-treated (V) Brown Norway rats as a result of ovalbumin challenge as compared to naive untreated (N) rats Total leukocytes increased significantly. 0.03mg / kg (
At doses of A) and 0.1 mg / kg (B), 3b failed to prevent this increase. At a dose of 0.1 mg / kg (C), budesonide had no effect when administered alone. However, 0.1 mg / kg budesonide and 0 mg
. Combination with 03 mg / kg (D) or 0.1 mg / kg (E) resulted in a significant decrease in eosinophil count. A statistically significant reduction in neutrophil counts was estimated to be .0.
Achieved only in the 1 / 0.03 mg / kg (D) group. High dose (0.5mg
/ Kg, group F), budesonide treatment alone was effective in preventing eosinophil, neutrophil and total leukocyte proliferation, with budesonide (0.5 mg / kg) and 0.03
/ Kg (G) or 0.1 mg / kg (H) in combination with 3b was also effective. (FIG. 5-6).

Conclusions The combination of compound 3b with the glucocorticoid budesonide is effective in preventing leukocyte inflow after an allergen challenge in animal models of asthma at doses where neither drug alone has any effect.

Example 4, Preparation of Formylamide 14 (Reaction Scheme 1) Acyloxazolidinone 9b (R = n-Pr) (S)-(-)-4-benzyl-2-oxazolidinone (4) in 75 ml of anhydrous THF (2.0 g, 22.6 mmol) of a cooled (-78 ° C) solution was treated with a 2.5 M solution of n-BuLi in hexane (9.1 ml, 22.6 mmol) for 15 minutes. Five minutes later, pure valeryl chloride (2.95 ml, 24.9 mmol) was added dropwise, and the mixture was stirred at -78 ° C for another 45 minutes. The mixture was then allowed to reach room temperature, stirred for a further 90 minutes, and then treated with 50 ml of a saturated NH ₄ Cl solution. Then dichloromethane (50 mL) was added and the organic layer was washed with brine (2 × 30 ml), dried over MgSO ₄ and concentrated in vacuo. This gave 5.94 g (100%) of the desired acyloxazolidinone 9b as a clear, colorless oil. ¹ H NMR (300 MHz, CDCl ₃ ) δ7.36-7.20 (m, 5H), 4.71-4.64 (m, 1H), 4.23-4.14 (m, 1
H), 3.40 (dd, J = 13.3, 3.2 Hz, 1H), 3.04-2.84 (m, 2H), 2.77 (dd, J = 13.3, 9.6
Hz, 1H), 1.74-1.63 (m, 2H), 1.46-1.38 (m, 2H), 0.96 (t, J = 7.3 Hz, 3H).

Acyl Oxazolidinone 9a (R = Et) The lithium anion of (S)-(−)-4-benzyl-2-oxazolidinone is obtained by the same procedure as described for preparing acyl oxazolidinone 9b. Treatment with butyryl chloride provided the acyl oxazolidinone 9a in 94% yield. ¹ H NMR (300 MHz, CDCl ₃ ) δ7.37-7.20 (m, 5H), 4.68 (ddd, J = 13.1, 7.0, 3.4
Hz, 1H), 4.23-4.13 (m, 2H), 3.30 (dd, J = 13.3, 9.6 Hz, 1H), 3.02-2.82 (m,
2H, 2.77 (dd, J = 13.3, 9.6 Hz, 1H), 1.73 (q, J = 7.3 Hz, 2H), 1.01 (t, J =
(7.3 Hz, 3H).

Acyloxazolidinone 9c (R = n-Bu) The lithium anion of (S)-(−)-4-benzyl-2-oxazolidinone is chlorinated by a procedure similar to that described for preparing acyloxazolidinone 9b. Treatment with hexanoyl provided the acyloxazolidinone 9a in 96% yield. ¹ H NMR (300 MHz, CDCl ₃ ) δ7.36-7.20 (m, 5H), 4.68 (m, 1H), 4.23-4.14 (m, 2
H), 3.30 (dd, J = 13.3, 3.3Hz, 1H), 3.02-2.83 (m, 2H), 2.76 (dd, J = 13.3, 9
.6 Hz, 1H), 1.70 (m, 2H), 1.43-1.34 (m, 4H), 0.92 (t, J = 3.3 Hz, 3H).

4-Methylvaleryl chloride 4-Methylvaleryl chloride was prepared from commercially available 4-methylvaleric acid as follows: 4-Methylvaleryl chloride in 50 ml anhydrous CH ₂ Cl ₂ containing 10 ml DMF. Herbic acid (
1.85 mL, 15.0 mmol) of cold (0 ° C.) was treated with 1.95 μL of oxalyl chloride (22.5 mmol). The mixture was then stirred at room temperature for 3 hours, concentrated in vacuo and filtered to give 1.65 g (100%) of the desired acid chloride as a colorless liquid.

Acyloxazolidinone 9d (R = i-Bu) The lithium anion of (S)-(−)-4-benzyl-2-oxazolidinone is chlorinated by a procedure similar to that described for preparing acyloxazolidinone 9b. Treatment with 4-methylvaleryl provided the acyloxazolidinone 9d in 85% yield. ¹ H NMR (300 MHz, CDCl ₃ ) δ7.37-7.20 (m, 5H), 4.70-4.63 (m, 1H), 4.23-4.15
(m, 2H), 3.30 (dd, J = 13.2, 3.2 Hz, 1H), 2.98-2.90 (m, 2H), 2.76 (dd, J = 13
.3, 9.6 Hz, 1H), 1.68-1.54 (m, 3H), 0.94 (d, J = 6.2 Hz, 3H).

Acyloxazolidinone 9e (R = CH ₂ Ph) The lithium anion of (S)-(−)-4-benzyl-2-oxazolidinone is chlorinated by a procedure similar to that described for preparing acyloxazolidinone 9b. Treatment with hydrocinnamoyl provided the acyl oxazolidinone 9e in 82% yield. _{^{1 H NMR (300MHz, CDCl 3}} ) δ7.35-7.16 (m, 10H), 4.70-4.63 (m, 1H), 4.21-4.14 (m, 2H), 3.38-3.19 (m, 3H), 3.08-2.98 (m, 2H), 2.75 (dd, J = 13.4, 9.5 Hz
, 1H).

Acyloxazolidinone 10b (R = n-Pr) 110 mL of anhydrous CH_TwoCl_TwoAcyl oxazolidinone 9b (5.74 g,
22.0 mmol) in 2.52 mL of TiCl_Four(23.1 mmol) resulting in the formation of a rich precipitate. After 5 minutes, diisopro
Pyrethylamine (4.22 mL, 24.2 mmol) was added slowly to give
The resulting dark brown solution was stirred at room temperature for 35 minutes. Benzyl chloromethyl ether (
6.0 ml, 44.0 mmol) were added rapidly and the mixture was stirred at room temperature for 5 minutes.
Was. 50 mL CH_TwoCl_TwoAnd 75 ml of 10% NH_FourAs a result of the Cl aqueous solution, a yellow gum was formed. After vigorously stirring the suspension for 10 minutes, the supernatant was separated.
Transfer to funnel and remove gummy residue with 100 ml of 1: 1 10% NH_FourCl / CH_TwoCl _Two Take up in aqueous solution. Next, 1N hydrochloric acid and saturated NaCHO were sequentially added to the combined organic layers._Three And washed with brine, MgSO_FourDried on and concentrated in vacuo. The crude solid material was recrystallized from EtOAc / hexane to give 6.80 g of the desired acyl oxalate.
Zolidinone 10b was obtained as a white solid at 81% yield.¹ H NMR (300MHz, CDCl_Three) δ7.34-7.18 (m, 10H), 4.77-4.69 (m, 1H), 4.55 (s, 2
H), 4.32-4.23 (m, 1H), 4.21-4.10 (m, 2H), 3.80 (t, J = 9.0 Hz, 1H), 3.65 (d
d, J = 9.0, 5.0 Hz, 1H), 3.23 (dd, J = 13.5, 3.3 Hz, 1H), 2.69 (dd, J = 13.5,
9.3 Hz, 1H), 1.74-1.64 (m, 1H), 1.54-1.44 (m, 1H), 1.40-1.28 (m, 2H), 0.9
1 (t, J = 7.3 Hz, 3H) .LRMS (FAB) m / e382 (M + H⁺)

Acyloxazolidinone 10a (R = Et) Acyloxazolidinone 10a was obtained in 80% yield by a procedure similar to that described for the preparation of acyloxazolidinone 10b. ¹ H NMR (300 MHz, CDCl ₃ ) δ7.36-7.18 (m, 10H), 4.55 (s, 2H), 4.21-4.11 (m,
3H), 3.81 (t, J = 9.0 Hz, 1H), 3.66 (dd, J = 9.0, 5.0 Hz, 1H), 3.23 (dd, J = 1
3.5, 3.2 Hz, 1H), 2.70 (dd, J = 13.5, 9.3 Hz, 1H), 1.78-1.57 (m, 2H), 0.94
(t, J = 7.5 Hz, 3H).

Acyloxazolidinone 10c (R = n-Bu) Acyloxazolidinone 10c was obtained in 91% yield by a procedure similar to that described for the preparation of acyloxazolidinone 10b. ¹ H NMR (300 MHz, CDCl ₃ ) δ7.38-7.17 (m, 10H), 4.72 (m, 1H), 4.54 (s, 2H),
4.27-4.10 (m, 2H), 3.79 (t, J = 8.7 Hz, 1H), 3.65 (dd, J = 9.1, 5.0 Hz, 1H),
3.23 (dd, J = 13.5 3.3 Hz, 1H), 2.68 (dd, J = 13.5, 9.3 Hz, 1H), 1.75-1.68
(m, 1H), 1.31-1.26 (m, 4H), 0.87 (t, J = 6.8 Hz, 3H).

Acyloxazolidinone 10d (R = i-Bu) Acyloxazolidinone 10d was obtained in 98% yield by a procedure similar to that described for the preparation of acyloxazolidinone 10b. ¹ H NMR (300 MHz, CDCl ₃ ) δ7.38-7.17 (m, 10H), 4.75-4.67 (m, 1H), 4.57 (d,
J = 12.0 Hz, 1H), 4.51 (d, J = 12.0 Hz, 1H), 4.41-4.36 (m, 1H), 4.20-4.09 (
m, 2H), 3.74 (t, J = 9.0 Hz, 1H), 3.65 (dd, J = 9.0, 5.1 Hz, 1H), 3.23 (dd,
J = 13.5, 3.2 Hz, 1H), 2.63 (dd, J = 13.5, 9.5 Hz, 1H), 1.74-1.52 (m, 2H), 1
.35 (dd, J = 13.1, 6.1 Hz, 1H), 0.92 (d, J = 2.9 Hz, 3H), 0.90 (d, J = 2.9 Hz,
3H).

Acyloxazolidinone 10e (R = CH ₂ Ph) Acyloxazolidinone 10e was obtained in 84% yield by a procedure similar to that described for the preparation of acyloxazolidinone 10e. ¹ H NMR (300 MHz, CDCl ₃ ) δ7.38-7.15 (m, 15H), 4.62-4.50 (m, 4H), 4.03 (dd, J = 9.0, 2.7 Hz, 1H), 3.93-3.82 (m, 2H ), 3.66 (dd, J = 9.2, 4.8 Hz, 1H), 3
.19 (dd, J = 13.5, 3.2 Hz, 1H), 2.98 (dd, J = 13.4, 8.2 Hz, 1H), 2.88 (dd, J
= 13.4, 7.3 Hz, 1H), 2.68 (dd, J = 13.5, 9.3 Hz, 1H).

Carboxylic acid 11b (R = n-Pr) 6.60 g (17.3 mmol) of a cold (0 ° C.) solution in 320 ml of THF / H ₂ O are successively treated with 6.95 ml of 35% H _2. Treated with a solution of lithium hydroxide monohydrate (1.46 g, 34.6 mmol) in aqueous O ₂ and 20 ml of H ₂ O. The mixture was stirred at 0 ° C. for 16 h, then NaSO ₃ (1 in 55 ml H ₂ O)
0.5 g) solution, carefully treated with a solution of NaHCO ₃ (4.35 g) in 100 ml H ₂ O. The mixture was stirred at room temperature for 30 minutes and concentrated in vacuo to remove THF. The resulting aqueous mixture was then washed with CH ₂ Cl ₂ (4 × 75 ml),
Cool to 0 ° C., acidify with 6N hydrochloric acid, and add CH ₂ Cl ₂ (1 × 200 ml and 3 ×
100 ml). The combined organic layers were then dried over MgSO ₄ and concentrated in vacuo to give 3.47 g (90%) of the desired acid 11b as a clear, colorless oil. 'H NMR (300MHz, CDCl ₃ ) δ7.38-7.26 (m, 5H), 4.55 (s, 2H), 3.67 (m, 1H), 3
.57 (dd, J = 9.2, 5.2 Hz, 1H), 2.75 (m, 1H), 1.72-1.31 (m, 4H), 0.93 (t, J
= 7.2 Hz, 3H). LRMS (FAB) m / e223 (M + H ⁺ )

Carboxylic acid 11a (R = Et) Acyloxazolidinone 11a was obtained in 48% yield by a procedure similar to that described for preparing acyloxazolidinone 11b. ¹ H NMR (300 MHz, CDCl ₃ ) δ7.36-7.27 (m, 5H), 4.55, (s, 2H), 3.68 (dd, J = 9.
2, 7.0 Hz, 1H), 3.59 (dd, J = 9.2, 5.4 Hz, 1H), 2.68-2.65 (m, 1H), 1.71-1.
62 (m, 2H), 0.97 (t, J = 7.5 Hz, 3H).

Carboxylic acid 11c (R = n-Bu) Acyloxazolidinone 11c was obtained in 96% yield by a procedure similar to that described for preparing acyloxazolidinone 11b. ¹ H NMR (300 MHz, CDCl ₃ ) δ7.37-7.28 (m, 5H), 4.55 (s, 2H), 3.67 (dd, J = 9.1
, 8.1 Hz, 1H), 3.57 (dd, J = 9.2, 5.3 Hz, 1H), 2.72 (m, 1H), 1.67-1.51 (m,
2H), 1.36-1.27 (m, 4H), 0.89 (t, J = 6.9 Hz, 3H).

Carboxylic acid 11d (R = i-Bu) Acyloxazolidinone 11d was obtained in 80% yield by a procedure similar to that described for preparing acyloxazolidinone 11b. ¹ H NMR (300 MHz, CDCl ₃ ) δ7.37-7.28 (m, 5H), 4.55 (s, 2H), 3.64 (t, J = 9.1,
Hz, 1H), 3.54 (dd, J = 9.1, 5.1 Hz, 1H), 2.81 (m, 1H), 1.68-1.54 (m, 2H),
1,36-1.27 (m, 1H), 0.92 (d, J = 4.9 Hz, 3H), 0.90 (d, J = 4.9 Hz, 3H).

Carboxylic acid 11e (R = CH ₂ Ph) Acyloxazolidinone 11e was obtained in 92% yield by a procedure similar to that described for preparing acyloxazolidinone 11b. ¹ H NMR (300 MHz, CDCl ₃ ) δ7.38-7.16 (m, 10H), 4.53 (d, J = 12.1 Hz, 1H), 4.5
0 (d, J = 12.1 Hz, 1H), 3.68-3.57 (m, 2H), 3.09-2.85 (m, 3H).

Diethylamide 12b (R = n-Pr) Diethylamine (2.36 ml, 23.0 mmol) and 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetrafluoroborate 1: 1 containing methyluronium (TBTU, 5.89 g, 18.4 mmol)
Carboxylic acid 11b (3.40 g, 1 .4 in MeCN / CH ₂ Cl ₂ (150 ml))
(5.3 mmol) was treated with diisopropylethylamine (6.7 ml, 38.2 mmol) over 1.5 h (syringe pump).
The mixture was then concentrated in vacuo and partitioned between ether (200ml) and H ₂ O (10 0ml). The aqueous layer was extracted with more ether (2 × 100 ml) and the combined organic layers were washed with 1N hydrochloric acid (3 × 50 ml), saturated aqueous NaHCO ₃ and brine, dried over MgSO ₄ and concentrated in vacuo did. Of chromatography purification: by (230-400 mesh SiO _2, 1 elution with 3 AsOEt / hexane) to give the diethylamide 12b of 4.24 g (97%) as a clear colorless oil. _{^{1 H NMR (300MHz, CDCl 3}} ) δ7.35-7.23 (m, 5H), 4.52 (d, J = 12.0 Hz, 1H), 4.44
(d, J = 12.0 Hz, 1H), 3.67 (t, J = 8.6 Hz, 1H), 3.51 (dd, J = 8.7, 5.5 Hz, 1H
), 3.46-3.27 (m, 4H) 2.96 (m, 1H), 1.67-1.57 (m, 1H), 1.48-1.22 (m, 4H),
1.20-1.10 (m, 6H), 0.90 (t, J = 7.2 Hz, 3H). LRMS (FAB) m / e278 (M + H ⁺ )

Diethylamide 12a (R = Et) By a procedure similar to that described for preparing diethylamide 12b, diethylamide 12a was obtained in 73% yield. ¹ H NMR (300 MHz, CDCl ₃ ) δ7.33-7.26 (m, 5H), 4.52 (d, J = 12.0 Hz, 1H), 4.44
(d, J = 12.0 Hz, 1H), 3.68 (t, J = 8.6 Hz, 1H), 3.53-3.33 (m, 5H), 2.90 (m,
1H), 1.75-1.50 (m, 2H), 1.18 (t, J = 7.1 Hz, 3H), 1.13 (t, J = 7.1 Hz, 3H),
0.89 (t, J = 7.4 Hz, 3H).

Diethylamide 12c (n = Bu) By a procedure similar to that described for preparing diethylamide 12c, diethylamide 12c was obtained in 94% yield. ¹ H NMR (300 MHz, CDCl ₃ ) δ7.35-7.25 (m, 5H), 4.51 (d, J = 12.0 Hz, 1H), 4.44
(d, J = 12.0 Hz, 1H), 3.67 (t, J = 8.6 Hz, 1H), 3.51 (dd, J = 8.8, 5.5Hz, 1H)
, 3.46-3.29 (m, 1H) 2.94 (m, 1H), 1.66-1.62 (m, 2H), 1.33-1.10 (m, 9H), 0
.85 (t, J = 7.0 Hz, 3H).

Diethylamide 12d (R = i-Bu) By a procedure similar to that described for preparing diethylamide 12d, diethylamide 12d was obtained in 95% yield. _{^{1 H NMR (300MHz, CDCl 3}} ) δ7.35-7.23 (m, 5H), 4.51 (d, J = 12.0 Hz, 1H), 4.44
(d, J = 12.0 Hz, 1H), 3.65 (t, J = 8.7 Hz, 1H), 3.54-3.28 (m, 5H), 3.03 (m,
1H), 1.63-1.49 (m, 2H), 1.33-1.24 (m, 1H), 1.18 (t, J = 7.1 Hz, 3H), 1.12
(t, J = 7.1 Hz, 3H), 0.90 (t, J = 6.4 Hz 3H).

Diethylamide 12e (R = CH ₂ Ph) By a procedure similar to that described for preparing diethylamide 12b, diethylamide 12e was obtained in 89% yield. ¹ H NMR (300 MHz, CDCl ₃ ) δ7.35-7.16 (m, 10H), 4.53 (d, J = 12.1 Hz, 1H), 4.4
7 (d, J = 12.1 Hz, 1H), 3.77 (t, J = 8.5 Hz, 1H), 3.59 (dd, J = 8.8, 5.7 Hz, 1
H), 3.40 (m, 1H), 3.22-2.89 (m, 5H), 2.79 (dd, J = 13.0, 5.1Hz, 3H), 1.01 (
t, J = 7.1 Hz, 3H), 0.85 (t.J = 7.2 Hz, 3H).

Alcohol 13b (R = n-Pr) To a solution of diethylamide 12b (4.08 g, 14.7 mmol) in 140 ml of MeOH was added 20% Pd (OH) ₂ / C (400 mg) and suspended. The liquid was hydrogenated at room temperature for 15 hours at atmospheric pressure. Filtration of the catalyst and concentration of the filtrate in vacuo gave 2.84 g (100%) of the desired primary alcohol 13b. ¹ H NMR (300 MHz, CDCl ₃ ) δ3.74 (br.d, J = 4.2 Hz, 1H), 3.61-3.15 (m, 5H), 2.7
1 (m, 1H), 1.69-1.24 (m, 4H), 1.20 (t, J = 7.1 Hz, 3H), 1.12 (t, J = 7.1 Hz
, 3H), 0.92 (t, J = 7.2 Hz, 3H). LRMS (FAB) m / e188 (M + H ⁺ )

Alcohol 13a (R = Et) Alcohol 13a was obtained in 100% yield by a procedure similar to that described for preparing alcohol 13b. ¹ H NMR (300 MHz, CDCl ₃ ) δ 3.76 (m, 2H), 3.58-3.19 (m, 4H), 2.64 (m, 1H), 1
.71-1.65 (m, 2H), 1.21 (t, J = 7.1 Hz, 3H), 1.13 (t, J = 7.1 Hz, 3H), 0.96 (
t, J = 7.4 Hz, 3H).

Alcohol 13c (R = n-Bu) Alcohol 13c was obtained in 100% yield by a procedure similar to that described for preparing alcohol 13b. ¹ H NMR (300 MHz, CDCl ₃ ) δ 3.76 (d, J = 4.5 Hz, 2H), 3.58-3.19 (m, 4H), 2.72
-2.65 (m, 2H), 1.68-1.55 (m, 2H), 1.40-1.24 (m, 4H), 1.20 (t, J = 7.1 Hz,
3H), 1.12 (t, J = 7.1 Hz, 3H), 0.90 (t, J = 6.9 Hz, 3H).

Alcohol 13d (i = Bu) Alcohol 13d was obtained in 100% yield by a procedure similar to that described for preparing alcohol 13b. _{^{1 H NMR (300MHz, CDCl 3}} ) δ3.78-3.68 (m, 2H), 3.57-3.15 (m, 4H), 2.81-2.73
(m, 1H), 1.70-1.60 (m, 2H), 1.40-1.28 (m, 1H), 1.21 (t, J = 7.1 Hz, 3H), 1
.12 (t, J = 7.1 Hz, 3H), 0.92 (m, 6H).

Alcohol 13e (R = CH ₂ Ph) Alcohol 13e was obtained in 100% yield by a procedure similar to that described for preparing alcohol 13b. _{^{1 H NMR (300MHz, CDCl 3}} ) δ7.29-7.16 (m, 5H), 3.81-3.71 (m, 2H), 3.61-3.50
(m, 1H), 3.15-2.87 (m, 6H), 1.05 (t, J = 7.1 Hz, 3H), 0.98 (t, J = 7.1 Hz,
3H).

Alcohol 13b (2.34 g, 25 ml, prepared by stirring CH ₂ Cl ₂ and water and separating the organic layer) in aldehyde 14b (R = n-Pr) wet CH ₂ Cl ₂ (125 mL, 12.7 mmol) in a solution of Dess-Martin periodinane (
8.06 g, 19.0 mmol) were added. The mixture was stirred at room temperature for 40 minutes and then poured into a mixture of a 5% aqueous solution of Na ₂ S ₂ O ₃ (250 mL) containing 5.2 g of NaHCO ₃ and ether (200 mL). 5 biphasic mixtures
Stir vigorously for 5 min and extract the aqueous layer with 15% CH ₂ Cl ₂ / Et ₂ O (2 × 100 mL). The combined organic layers were then washed with H ₂ O (3 × 75 mL) and brine, dried over MgSO ₄ , filtered, and concentrated in vacuo to 2.06 g (
88%) of the desired aldehyde 14b as a colorless and transparent oil. ¹ H NMR (300 MHz, CDCl ₃ ) δ 9.60 (d, J = 3.5 Hz, 1H), 3.49-3.30 (m, 5H), 1.96-1.85 (m, 2H), 1.39-1.31 (m, 2H), 1.19 (t, J = 7.1 Hz, 3H), 1.13 (t, J = 7.1H)
z, 3H), 0.95 (t, J = 7.3 Hz, 3H).

Aldehyde 14a (R = Et) Aldehyde 14a was obtained in 80% yield by a procedure similar to that described for the preparation of alcohol 14b. ¹ H NMR (300 MHz, CDCl ₃ ) δ 9.61 (d, J = 3.6 Hz, 1H), 3.48-3.29 (m, 5H), 2.02-1.90 (m, 2H), 1.19 (t, J = 7.1 Hz, 3H), 1.14 (t, J = 7.1 Hz, 3H), 0.96 (t, J = 7
.4Hz, 3H)

Aldehyde 14c (R = n-Bu) Aldehyde 14c was obtained in 98% yield by a procedure similar to that described for the preparation of alcohol 14b. ¹ H NMR (300 MHz, CDCl ₃ ) δ 9.59 (d, J = 3.6 Hz, 1H), 3.48-3.29 (m, 5H), 1.97-1.87 (m, 2H), 1.39-1.22 (m, 4H), 1.18 (t, J = 7.2 Hz, 3H), 1.13 (t, J = 7.2H
z, 3H), 0.90 (t, J = 7.0 Hz, 3H).

Aldehyde 14d (R = i-Bu) By a procedure similar to that described for the preparation of alcohol 14b, aldehyde 14d was obtained in 96% yield. ¹ H NMR (300 MHz, CDCl ₃ ) δ 9.57 (d, J = 3.7 Hz, 1H), 3.51-3.27 (m, 5H), 1.83 (t, J = 7.1 Hz, 3H) ), 1.66-1.55 (m, 1H), 1.20 (t, J = 7.1 Hz, 3H), 1.13 (t, J = 7)
.1Hz, 3H), 0.93 (d, J = 6.6Hz, 6H).

Aldehyde 14e (R = CH ₂ Ph) Aldehyde 14e was obtained in 97% yield by a procedure similar to that described for the preparation of alcohol 14b. ¹ H NMR (300 MHz, CDCl ₃ ) δ 9.69 (d, J = 2.9 Hz, 1H), 7.29-7.16 (m, 5H), 3.65 (m, 1H), 3.53- 3.42 (m, 1H), 3.30 (dd, J = 13.5, 9.3 Hz, 1H), 3.23-3.13 (m, 2H), 3.06-2.91 (m , 2H), 1.04 (t, J = 7.1 Hz, 3H), 0.93 (t, J = 7.1 Hz, 3H).

Example 5: Preparation of β-lactone 3 (Scheme 2) Aldol 5b (R = n-Pr) To a cold (-78 ° C) solution of trans-oxazoline 4 in ether (35 mL) was added lithium bis ( Trimethylsilyl) amide (1M solution in hexane, 2.17, 2.17 mmol) was added. After 30 minutes, the organic solution was treated dropwise with a 1 M solution of dimethylaluminum chloride in hexane (4.55 mL, 4.55 mmol) and the mixture was allowed to cool to -85 ° C (liquid N ₂ was dried ice / acetate). Stirring for an additional 60 minutes before adding to the bath. A solution of aldehyde 14b (420 mg, 2.27 mmol) in ether (4 mL) was added over 10 minutes over the flask wall. The mixture was then allowed to warm to −40 ° C. for 2.5 hours, and then quenched by adding 35 mL of saturated aqueous NH ₄ Cl and 25 mL of AcOEt. Then enough 2N HCl was added until 2 clear phases were obtained (approximately 15 mL was added).
The aqueous layer was extracted with AcOEt (2 × 20 mL) and the combined organic layers were 0.5N diluted hydrochloric acid (20 mL), H ₂ O (20 mL), 0.5 M aqueous NaHSO ₃ (2 × 15 mL), NaHCO ₃ Wash sequentially with saturated aqueous solution and finally with brine, then dry over Na ₂ SO _{4 and} concentrate under vacuum to give 879 mg (> 100%) of crude aldol product 5b, which is used directly in the next step Was pure enough. ¹ H NMR (300 MHz, CDCl ₃ ) δ 8.02-7.97 and 7.53-
7.39 (m, 5H), 6.58 (d, J = 9.9 Hz, 1H), 4.82 (d, J = 2.4 Hz, 1H), 3.73 (s, 3H), 3. 69-3.61 (m, 2H), 3.49-3
.39 (m, 2H), 3.24-3.16 (m, 1H), 3.05 (m, 1H), 2.89 (m, 1H), 2.28-2.23 (m, 1H) ), 1.98-1.91 (m, 1H), 1.37-1.20 (m, 6H), 1.19-1.06 (m, 6H), 0.87 (t, J = 7) .
1 Hz, 3H), 0.70 (d, J = 6.7 Hz, 3H).

The aldol product 5b was also prepared by a procedure similar to that described above except that cis-oxazoline 21 (see below) was used instead of trans-oxazoline 4.
Obtained in 00% yield.

Aldol 5a (R = Et) By a procedure similar to that described for the preparation of aldol 5b, the lithium anion of trans-oxazoline 4 was treated sequentially with dimethylaluminum chloride and aldehyde 14a to give 95% yield of aldol 5a. Rate obtained. ¹ H NMR (300 MHz, CDCl ₃ ) δ 8.00-7.97 and 7.51-
7.39 (m, 5H), 6.50 (d, J = 9.9 Hz, 1H), 4.80 (d, J = 2.4 Hz, 1H), 3.81-3.64 (m, 2H) ), 3.74 (s, 3H), 3.45 (m
, 2H), 3.19 (m, 2H), 2.93-2.84 (m, 2H), 2.24 (m, 1H), 1.89 (m, 1H), 1.73-1. 64 (m, 4H), 1.29 (t, J = 7.2 Hz, 3H), 1.12 (d, J = 6.9 Hz, 3H), 1.07 (d, J = 7.2 Hz, 3H), 0.70 (d, J = 6.7 Hz, 3H).

Aldol 5c (R = n-Bu) By a procedure similar to that described for the preparation of aldol 5b, the lithium anion of trans-oxazoline 4 was treated sequentially with dimethylaluminum chloride and aldehyde 14c to give aldol 5c 100 % Yield. ¹ H NMR (300 MHz, CDCl ₃ ) δ 8.02-7.98 and 7.53-
7.33 (m, 5H), 6.57 (d, J = 10.0 Hz, 1H), 4.81 (d, J = 2
.3Hz, 1H), 3.73 (s, 3H), 3.68-3.60 (m, 2H), 3.49-3.17 (m, 2H), 3.00 (m, 1H), 2.90 (m, 1H), 1.98-1.87 (m, 2H), 1.38-0.83 (m, 16H), 0.70 (d, J = 6.7 Hz, 3
H).

Aldol 5d (R = i-Bu) By a procedure similar to that described for the preparation of aldol 5b, the lithium anion of trans-oxazoline 4 was treated sequentially with dimethylaluminum chloride and aldehyde 14d to give aldol 5d 100 % Yield. ¹ H NMR (300 MHz, CDCl ₃ ) δ 8.01-7.80 and 7.55-
7.20 (m, 5H), 4.87 (d, J = 2.3 Hz, 1H), 3.73 (s, 3H), 3.69-3.58 (m, 2H), 3.51- 3.32 (m, 2H), 2.98-2.87
(M, 1H), 2.33-2.24 (m, 1H), 2.12-2.02 (m, 1H), 1.83 (t, J = 7.1 Hz, 1H), 1.35 (T, J = 7.1 Hz, 3H), 1.25-
1.05 (m, 5H), 0.93 (d, J = 6.6 Hz, 3H), 0.89 (d, J = 6.5 Hz, 3H), 0.80 (d, J = 6.5 Hz) , 3H), 0.69 (d, J = 6.7 Hz)
, 3H).

Aldol 5e (R = CH ₂ Ph) In a procedure similar to that described for the preparation of aldol 5b, the lithium anion of trans-oxazoline 4 was treated sequentially with dimethylaluminum chloride and aldehyde 14e to give 100% yield. Gave aldol 5e. ¹ H NMR (300 MHz, CDCl ₃ ) δ 8.01-7.93 and 7.54-
7.10 (m, 10H), 4.71 (d, J = 2.5 Hz, 1H), 3.73 (s, 3H)
, 3.68-3.58 (m, 2H), 3.48-2.79 (m, 6H), 2.17 (m, 1H), 1.12-0.91 (m, 9H), 0.68 (d, J = 6.7 Hz, 3H).

Γ-lactam 7b (R = n-Pr) To a solution of aldol 5b (4.72 g, 10.9 mmol) in 100 mL of 1: 9 AcOH / MeOH was added 4.8 g of 20% Pd (OH ₂ ) / C was added and shaken vigorously under 55 psi H _{2 for} 60 hours. The mixture was allowed to come to room temperature before being filtered and concentrated under vacuum. The resulting solid was purified by flash chromatography (SiO ₂ , eluting with 1: 1 AcOEt / 1% AcOH in hexane) to give 2.23 g (75%) of the desired γ-lactam 7b as a white solid Obtained as a product. ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.89 (br.s, 1H), 4.77
(Br.d, J = 11.5 Hz, 1 H), 4.47 (dd, J = 11.5, 5.6 Hz, 1 H), 4.08 (dd, J = 9.4, 5.0 Hz, 1H), 3.83 (s, 3H), 2.93
(M, 1H), 1.78-1.39 (m, 6H), 1.02-0.88 (m, 9H).

Aldol 5a is hydrogenated at 55 psi for 48 hours to give γ-lactam 7a by a procedure similar to that described for the preparation of γ-lactam 7a (R = Et) %
Obtained in yield. ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.79 (br.s, 1H), 4.62
(Br.d, J = 11.2 Hz, 1H), 4.51 (dd, J = 11.2, 5.4 Hz, 1H
), 3.83 (s, 3H), 2.85 (m, 1H), 1.77-1.64 (m, 3H), 1.
01 (t, J = 7.4 Hz, 3H), 0.98 (d, J = 6.9 Hz, 3H), 0.95 (
d, J = 6.9 Hz, 3H).

Γ-lactam 7c (R = n-Bu) To a solution of aldol 5c (361 mg, 0.80 mmol) in 6 mL of 1: 9 AcOH / MeOH was added 250 mg of 20% Pd (OH) ₂ / C. Was added and it was shaken vigorously under 50 psi H _{2 for} 24 hours. Then additional catalyst (100 mg) was added and the mixture was again shaken at 50 psi for another 24 h, after which it was allowed to return to room temperature before filtration. The filtrate was then heated at reflux for 30 minutes, cooled to room temperature and concentrated in vacuo. The solid obtained was azeotroped once with toluene and purified by flash chromatography (SiO ₂ , eluting with 4% MeOH / CHCl ₃ ) to give 140 mg (61%) of the desired γ-lactam 7c. Obtained as a white solid. ¹ H NMR (300 MHz, CDCl ₃ ) δ 8.02 (br.s, 1H), 4.93
(Br.d, J = 11.3 Hz, 1H), 4.46 (dd, J = 11.3, 5.5 Hz, 1H), 4.15-4.08 (m, 1H), 3.83 (S, 3H), 2.94-2.87 (m
, 1H), 1.80-1.34 (m, 6H), 0.94 (d, J = 6.9 Hz, 3H), 0.9.
89 (t, J = 7.2 Hz, 3H).

Aldole 5d is hydrogenated at 50 psi for 40 hours by a procedure similar to that described for the preparation of γ-lactam 7d (R = i-Bu) at 50 psi, then heated to reflux for 30 minutes. γ
-Lactam 7d was obtained in 61% yield. ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.92 (br.s, 1H), 4.81
(Br.d, J = 11.5 Hz, 1H), 4.46 (m, 1H), 4.09 (m, 1H), 3.83 (s, 3H), 3.04-2.98 (m , 1H), 1.78-1.73 (m, 2H
), 1.66-1.47 (m, 3H), 1.00-0.90 (m, 12H).

Aldole 5e was hydrogenated at 50 psi for 24 hours and then heated to reflux for 30 minutes by a procedure similar to that described for the preparation of γ-lactam 7e (R = CH ₂ Ph). γ
-Lactam 7e was obtained in 71% yield. ¹ H NMR (300 MHz, CDCl ₃ ) δ 8.01 (br.s, 1H), 7.35
−7.15 (m, 5H), 5.02 (br.d, J = 11.7 Hz, 1H), 4.40-4
.34 (m, 1H), 4.06-4.01 (m, 1H), 3.84 (s, 3H), 3.34 -3.27 (m, 1H), 3.10-3.04 (M, 2H), 1.84-1.72 (m, 1
H), 0.98 (d, J = 6.7 Hz, 3H), 0.93 (d, J = 6.9 Hz, 3H).

Β-lactone 3b (R = n-Pr; 7-n-propyl-crust-lactacystin β-lactone) Cooling of γ-lactam 7b (2.20 g, 8.06 mmol) in EtOH (100 mL) (0 ° C.) To the solution was added 0.1N aqueous NaOH (100 mL, 10.0 mmol). The mixture was stirred for 15 hours at room temperature, followed by the addition of H ₂ O (50 mL) and AcOEt (100 mL). Then, the aqueous layer was AcOE
Washed with t (2 × 50 mL), acidified with 6N diluted hydrochloric acid and concentrated in vacuo to a volume of approximately 60 mL. The solution was then frozen and lyophilized.
The resulting solid was suspended in THF, filtered to remove sodium chloride, and concentrated in vacuo to give 2.05 g (98%) of the desired dihydroxy acid as a white solid. ¹ H NMR (300 MHz, CD ₃ OD) δ 4.42 (d, J = 5.8 Hz, 1H), 3.90 (d, J = 6.5 Hz, 1H), 2.84 (m, 1H), 1 .70-1.24 (m, 6H), 0.95-0.84 (m, 9H).

Dihydroxy acid (1.90 g, 7.33 mmol) in anhydrous THF (36 mL)
To a solution of 2- (1H-benzotriazole-1 in anhydrous MeCN (36 mL).
-Yl) -1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU, 2.59, 8.06 mmol) followed by the addition of triethylamine (0.72 mL, 22.0 mmol). . After stirring at room temperature for 70 minutes, some toluene was added and the mixture was concentrated in vacuo and azeotroped twice with toluene. Purification by flash chromatography (SiO ₂ , eluting with 2: 3 AcOEt / hexane) provided 1.44 g (81%) of the desired β-lactone 3b as a white solid. ¹ H NMR (300 MHz, CDCl ₃ ) δ 6.07 (br.s, 1H), 5.26
(D, J = 6.1 Hz, 1H), 3.97 (dd, J = 6.4, 4.4 Hz, 1H), 2.7
0-2.63 (m, 1H), 2.03 (d, J = 6.4 Hz, 3H), 1.93-1.44
(M, 5H), 1.07 (d, J = 7.0 Hz, 3H), 0.99 (d, J = 7.3 Hz, 3H), 0.91 (d, J = 6.7 Hz, 3H) . LRMS (FAB) m / e 242 (M + H ⁺ )

Hydrolysis of β-lactone 3a (R = Et; 7-ethyl-crust-lactacystin β-lactone) 7b as described above gave the corresponding dihydroxy acid in 100% yield. ¹ H NMR (300 MHz, CD ₃ OD) δ 4.45 (d, J = 5.8 Hz, 1H), 3.90 (d, J = 6.4 Hz, 1H), 2.74 (m, 1H), 1 0.71-1.53 (m, 3H), 0.94 (t, J = 7.4 Hz, 3H), 0.92 (d, J = 6.8 Hz, 3H), 0.88 (d, J = 6.8Hz, 3H).

A procedure similar to that described for the preparation of β-lactone 3b provided β-lactone 3a in 79% yield. ¹ H NMR (300 MHz, CDCl ₃ ) δ 6.17 (br.s, 1H), 5.30
(D, J = 6.0 Hz, 1H), 3.98 (dd, J = 6.4, 4.4 Hz, 1H), 2.6
0 (m, 1H), 2.08 (d, J = 6.4 Hz, 3H), 1.97 (m, 2H), 1.75 (m, 1H), 1.12 (t, J = 7 0.5Hz, 3H), 1.07 (d, J = 6.8Hz
, 3H), 0.92 (d, J = 6.8 Hz, 3H).

Β-lactone 3c (R = n-Bu; 7-n-butyl-crust-lactacystin β-lactone) 7c is hydrolyzed as described above for 7b to give the corresponding dihydroxy acid in 100% yield. Obtained. ¹ H NMR (300 MHz, CD ₃ OD) δ 4.42 (d, J = 5.8 Hz, 1H), 3.90 (d, J = 6.4 Hz, 1H), 2.86-2.79 (m, 1H), 1.70-1.24 (m, 8H), 0.97-0.86 (m, 9H).

By a procedure similar to that described for the preparation of β-lactone 3b, β-lactone 3c was obtained in 40% yield. ¹ H NMR (300 MHz, CDCl ₃ ) δ 6.14 (br.s, 1H), 5.27
(D, J = 6.1 Hz, 1H), 3.97 (d, J = 4.4 Hz, 1H), 2.68-2.61 (m, 1H), 1.94-1.86 (m, 2H), 1.72-1.36 (m, 7H),
1.07 (d, J = 7.0 Hz, 3H), 0.93 (t, J = 7.1 Hz, 3H), 0.91 (d, J = 6.8 Hz, 3H). LRMS (FAB) m / e 256 (M + H ⁺ )

Β-lactone 3d (R = i-Bu; 7-i-butyl-crust-lactacystin β-lactone) 7b is hydrolyzed as described above to give the corresponding dihydroxy acid in 100% yield. Obtained. ¹ H NMR (300 MHz, CD ₃ OD) δ 4.50 (d, J = 5.8 Hz, 1H), 4.00 (d, J = 6.5 Hz, 1H), 3.09-3.02 (m, 1H), 1.90-1.61 (m, 3H), 1.49-1.40 (m, 2H), 1.02 (d, J = 6.7H).
z, 3H), 0.98 (d, J = 6.5 Hz, 3H), 0.97 (d, J = 6.7 Hz, 3H).

By a procedure similar to that described for the preparation of β-lactone 3b, β-lactone 3d was obtained in 62% yield. ¹ H NMR (300 MHz, CDCl ₃ ) δ 6.16 (br.s, 1H), 5.25
(D, J = 6.1 Hz, 1H), 3.97 (d, J = 4.4 Hz, 1H), 2.71 (dd)
, J = 15.1, 6.2 Hz, 1H), 1.95-1.66 (m, 5H), 1.08 (d, J
= 6.9 Hz, 3H), 0.99 (d, J = 6.3 Hz, 3H), 0.98 (d, J = 6.3 Hz, 3H), 0.92 (d, J = 6.7 Hz, 3H). LRMS (FAB) m / e 256 (M + H ⁺ )

Hydrolysis of β-lactone 3e (R = CH ₂ Ph; 7-benzyl-crust-lactacystin β-lactone) as described above for 7b gave the corresponding dihydroxy acid in 88% yield. . ¹ H NMR (300 MHz, CD ₃ OD) δ 7.25-7.04 (m, 5H), 4.
29 (d, J = 5.7 Hz, 1H), 3.83 (d, J = 6.4 Hz, 1H), 3.01-
2.82 (m, 3H), 1.65 (m, 1H), 0.90 (d, J = 6.6 Hz, 3H), 0.86 (d, J = 6.8 Hz, 3H).

A procedure similar to that described for the preparation of β-lactone 3b provided β-lactone 3e in 77% yield. ¹ H NMR (300 MHz, CDCl ₃ ) δ7.36-7.20 (m, 5H), 6.
57 (br.s, 1H), 5.08 (d, J = 5.4 Hz, 1H), 3.94 (d, J = 4
2.5 Hz, 1H), 3.25 (d, J = 10.1 Hz, 1H), 3.01-2.89 (m, 2H), 1.92-1.81 (m, 1H), 1.05 (D, J = 6.9 Hz, 3H), 0.86 (d, J = 6.7 Hz, 3H). LRMS (FAB) m / e 290 (M + H ⁺ )

Example 6: Pharmacokinetics of N- (pyrazine) carbonyl-L-phenylalanine-L-leucine boronic acid (1) in rats and primates N- (pyrazine) carbonyl-L-phenylalanine-L-leucineboro Single dose intravenous pharmacokinetic experiments with nicked acid (1) were performed on Sprague-Dawley rats (140-280 g). The animals were divided into three groups (6 / sex in groups 1 and 2; 9 / sex in group 3). Animals in groups 1, 2 and 3 include:
Each received 1 of 0.03, 0.1 or 0.3 mg / kg in the same dose volume.

Before dosing and approximately 10 and 30 minutes and 1, 3, and 24 after dosing on day 1
After an hour, a blood sample (approximately 1.0 mL) was drawn from the jugular vein of the animal. The samples were assayed one by one using the chromatography / mass spectrometry (LC / MS / MS) method. The lowest limit for analytical quantification was established at 2.5 ng / mL for 1 in rat plasma and whole blood.

According to a single intravenous dose, one plasma or whole blood level was measured only at the 0.3 mg / kg dose level. Recognized C_maxOccurs at the first time; therefore, the peak concentration (T_max) Was estimated to be less than 10 minutes in both male and female rats. Generally, males have slightly higher peak concentrations (C _max ) And concentration-time curves (AUC_0-1) Area below the value. In male
C in plasma and whole blood_maxThe values were 51.8 and 22.7 ng / mL, respectively, for females, 36.9 and 19.1 ng / mL, respectively. Male
AUC in plasma and whole blood_0-1The values were 14.0 and 18.6 ng · h / mL, respectively, and 12.9 and 17.7 ng · h / mL for females, respectively.
Emission half-life (t_1/2Estimates were not possible due to one level of instability during the final period. These findings suggest that 1 rapidly disappears from the blood.

Example 7: Preparation of a blood sample for in vitro measurement of 20S proteasome activity This preparation technique applies to blood samples collected from mammals, especially human subjects, cynomolgus monkeys, rats and mice. Peripheral leukocytes were separated from blood samples at the time of blood collection and stored at about -70 ° C until tested. To prevent interference with the fluorescence assay,
It is important that the sample preparation is free of all hemoglobin.

Procedure The required amount of blood was collected in tubes containing the anticoagulant. Approximately 5 mL of blood is required for human subjects and primates; approximately 4 m for rats
L blood was required; for mice, approximately 1 mL of blood was required from each of 5 mice, and 5 blood samples were stored to obtain approximately 5 mL.

The blood samples were diluted 1: 1 (v / v) with sterile saline and the blood-saline mixture was added to 14 (〜1 mL Nyco in 75 mm polystyrene test tubes).
It was laminated on a prep ^™ separation medium (GIBCO BRL Products). 500 samples
(G, centrifuged at room temperature for approximately 30 minutes. The top layer was removed, leaving a 2-3 mm cell band between the top and bottom layers. The remaining cell band was pipetted into a clean centrifuge tube. The cell band was washed with 3 mL of cold phosphoric acid-buffered saline and centrifuged at 400 (g, 4 ° C. for 5 minutes.
Resuspended in 1 mL of cold phosphate-buffered saline. The suspension was transferred to a 1.5 mL Eppendorf tabletop centrifuge tube and centrifuged at 6600 (g, 4 ° C for approximately 10 minutes. The supernatant was aspirated off and the cell pellet was stored at -70 ° C.

Example 8: Assay for measuring 20S proteasome activity in peripheral leukocytes The assay is based on SDS-induced chymotrypsin-like activity of free 20S particles. It uses fluorescence to measure the rate at which 20S proteasome hydrolyzes amide bonds in small peptide substrates. By measuring this rate in the absence and presence of the inhibitor, one can determine how the enzyme is bound by the inhibitor. This assay was used to measure 20S proteasome activity in peripheral leukocytes in mammals, especially humans, cynomolgus monkeys, rats and mice.

Abbreviations: AMC 7-amino-4-methylcoumarin DMF dimethylformamide BSA bovine serum albumin DMSO dimethylsulfoxide DTT dithiothreitol EDTA disodium ethylenediaminetetraacetate HEPES N- (2-hydroxyethyl) piperazine-N- ( Hgb hemoglobin SDS Any sodium dodecyl sulfate-SDS-grade: 99% sodium dodecyl sulfate Lauryl grade: ~ 70% sodium dodecyl sulfate, balance tetradecyl and hexadecyl sulfate TMB 3 , 3,5,5-tetramethylbenzidine WBC leukocyte Ys substrate N-succinylleucylleucylvalyltyrosyl 7-amino-4
-Methyl coumarin (Suc-Leu-Leu-Val-Tyr-AMC)

Procedure Leukocytes prepared as described in Example 1 are lysed by adding 200 μL of 5 mM EDTA to each sample. The sample is left on ice for at least 1 hour. The Ys substrate is dissolved in DMSO. Purified 20S proteasome standard from rabbit reticulocytes was prepared using 20 mM HEPES / 0.5 mM EDTA (pH 7.8).
To 1: 9 (v / v). The Ys substrate buffer contains 20 mM HEPES, 0.5 mM EDTA, 0.35% SDS and 60 μM Ys substrate. Coomassie protein assays (measuring total protein content) and hemoglobin assays are performed on test samples according to standard procedures using commercially available kits. The amount of leukocyte protein is calculated by the following formula:

[0134]

(Equation 1)

The 20S proteasome standard is 20 mM HEPES / 0.5 mM EDTA (
Dilute 1:10 (v / v) with pH 7.8) to form a 12 μg / mL stock solution and place on ice. Add 10 μl of the standard 20S proteasome solution to the cuvette and run the reaction for 10 minutes. The maximum linear slope is measured with a fluorimeter to provide a measure of standard proteasome activity.

5 μL of test sample is added to a cuvette containing 2 mL of Ys substrate buffer at 37 ° C. and the reaction is performed for 10 minutes. Full activation of the 20S proteasome is achieved over 10 minutes. Consistent results were obtained after 3 minutes from readings taken at 10 minutes. The maximum linear slope for the data for at least 1 minute is measured with a fluorimeter. If the rate is less than 1 picomole AMC / sec, the measurement is repeated with 10 μL of test sample.

The amount of 20S proteasome activity in a test sample is calculated according to the following formula:

(Equation 2)

Example 9: Proteasome Activity Levels in Peripheral Leukocytes of Human Volunteers Blood samples (about 2 ml each) were obtained five times from five human volunteers over a period of 10 weeks. After collection, leukocytes were isolated from individual blood samples using Nycoprep ^™ . The resulting pellets were stored in a freezer set to maintain at -60C to -80C until the day of testing. Samples taken each time were tested together and each sample was tested in duplicate.

The 20S proteasome activity measures the rate of proteolytic hydrolysis of a fluorescent (AMC) tagged peptide substrate by a sample, as described in Example 2 above,
The activity was determined by normalizing the activity to the amount of cell-specific protein present in the lysate. The 20S proteasome activity in a sample is determined by the formula:

(Equation 3) [Where C = conversion factor that corrects the amount of fluorescence to the concentration of free AMC (FU / picomolar AMC)].

Results and Discussion Individual test data and mean +/- mean standard error (SEM) for each sample are presented in Appendix A. The mean 20S proteasome activity values found for each human volunteer ranged from 10.73 to 14.79 pmol AMC / sec / mg protein (Table 1 and FIG. 1). The average 20S proteasome activity found in the population was 12.12 +/- 0.81 picomolar AMC / sec / mg protein with individual observed values ranging from 53% to 165% of the average.

A duplicate test was performed on each sample. Variation in test duplicates on day 5 of test (S
EM%) were 9.9%, 11.9%, 10.3%, 8.2% and 9.2%. In duplicates of the individual tests, this ranged from 0.8% to 22.9%. The average variation between duplicates of the test over the four days of the test was 10.0%. Further test development to reduce variability is ongoing.

[0142]

[Table 1]

[0143]

[Table 2]

Example 10: N- (Pyrazine) carbonyl-L-phenylalanine-L-leucine
-Transient 20S proteasome activity in isolated leukocytes and tissues following administration of -boronic acid (1)

General Procedure One dosing formulation was prepared daily during the study. Dilutions were prepared from stock solutions. One stock solution was made with 98% saline (0.9%), 2% ethanol with 0.1% ascorbic acid. Stock dilutions were made in the same vehicle.

Female CD2-F1 mice (18-20 g), female BALB / c mice (18-20 g), female Wistar rats (150-200 g) and male Sprague Dawley rats (250-450 g) were purchased from Taconic Farms (Germa
ntown, NY). Animals were observed for at least one week and examined for general health before the start of the study. The animals used in these studies were asymptomatic. Mice were housed in polycarbonate cages with 5 rats per cage and 3 rats per cage. Corn Cob bedding (AND-1005; Farmers Exchenge, Framingham, MA) was used during observation and testing. The fluorescent light was controlled to automatically provide an alternating light and dark cycle of about 12 hours. Temperature and humidity were centrally controlled and recorded daily, and readings ranged between 21 +/- 2 ° C and 45 +/- 5 ° C, respectively.
Pellets of standard rodent chow (# 5001, Purina, St. Louis, MO) were available ad libitum during observation and testing. Cambridge city tap water was provided ad libitum by water bottles. No food and water contamination expected to interfere with the test is known.

The drug was administered intravenously in vehicle using a dose of 100 μL per mouse or 1.0 mL / kg for rats. The control group consisted of vehicle (98% saline [0.9)
%], 2% ethanol, 0.1% ascorbic acid). 1 for animals
One was administered as a single bolus, either one or multiple times. Animals showing moribund activity were euthanized with CO ₂ inhalation. Following IV administration at 1, blood was collected at various time points and peripheral leukocytes were isolated. Tissues collected were brain, colon, liver, muscle (gastrocnemius), prostate and testis.

Ex vivo measured in peripheral leukocytes of mice after a single intravenous dose of 1
20S Proteasome Activity In two combination studies, female CD2-F1 mice (18-20 g) and female BALB / c mice (18-20 g) received one intravenous dose (0.1-3 in a 100 μL dose). 0.0 mg / kg). The vehicle was 98% saline [0.9%], 2% ethanol, 0.1% ascorbic acid. Blood samples were taken at 1.0 and 24 hours after dosing. Due to the blood volume required in the 20S proteasome activity assay, groups of 5 mice were sacrificed at the same time and their blood pooled to generate a single data point.

There was a significant (p <0.05) dose-related decrease in 20S proteasome activity for all dose groups 1.0 hour after intravenous administration of 1 (FIG. 1), which recovered at 24 hours. Get started (Figure 2).

Ex vivo measured in peripheral leukocytes of rats after a single intravenous dose of 1
20S Proteasome Activity In a four-combination study, female Wistar rats (150-200 g) received one single intravenous dose (0.03-0.3 mg in a 1.0 mL / kg dose).
/ kg / day). Vehicle is 0.1% ascorbic acid / 2% ethanol
/ 98% saline (0.9%). Blood samples were taken at 1.0, 24 and 48 hours after one dose.

There was a significant (p <0.05) dose-related decrease in 20S proteasome activity 1.0 hour after intravenous administration of 1 (FIG. 3). At 24 hours post-dose, the dose-related decrease in 20S proteasome activity was small, but still significant (p <0.05) in the high dose group (0.2 mg / kg, FIG. 4). At 48 hours after administration, the 20S proteasome activity was no longer significantly reduced (FIG. 5). These studies showed a dose-dependent and reversible inhibition of 20S proteasome activity in rat peripheral leukocytes following a single intravenous dose of one. 20S
A slow rate of return to baseline for proteasome activity levels is observed in rats, possibly indicating one fast metabolism in mice.

Ex vivo measured in peripheral leukocytes of rats after one repeated intravenous administration
20S Proteasome Activity When 1 was administered intravenously daily for 7 days, a dose-related decrease in 20S proteasome activity was observed 24 hours after the last dose. 0.05 mg / significant inhibition
kg was observed at the dose. Observed 24 hours after daily intravenous administration for 7 days 2
The magnitude of the OS proteasome inhibition is greater than that observed 24 hours after a single intravenous dose, probably reflecting the cumulative effect of daily administration of one on its biological target, the proteasome. .

A significant dose-related decrease in 20S proteasome activity was observed 24 hours after the last dose for intravenous administration every 2 days for 14 days. The dose-related decrease in 20S proteasome activity was significant (p <0.05) in the 0.2 mg / kg dose group. Also, a significant (p <0.05) dose-related decrease in 20S proteasome activity was observed 24 hours after once weekly intravenous administration for 8 weeks. The dose-related decrease in 20S proteasome activity was significant (p <0.05) in the> 0.1 mg / kg dose group.

In the four repeated dose study, male Sprague Dawley rats (250-450 g; n = 6 per group) were administered for 1 week (0.01-0.1 in a 1.0 mL / kg dose) for 2 weeks. (35 mg / kg / day) twice weekly intravenously. The vehicle was 0.1% ascorbic acid / 2% ethanol / 98% saline (0.9%). Blood samples were collected 1.0 hours after the last dose for evaluation of 20S proteasome activity.

When 1 was administered twice weekly for 2 weeks (4 administrations total), a dose-related decrease in 20S proteasome activity was observed 1.0 hour after the last dose (FIG. 6). The dose-related decrease in 20S proteasome activity was 0.03 mg / mg for all dose groups.
It was significant (p <0.05) for kg. The results indicate that one repeated dose administration induces a dose-related decrease in 20S proteasome activity in rat leukocytes. The magnitude of inhibition of 20S proteasome activity is greater than that seen after a single dose when 1 is administered daily or every other day. If the interval between one dose is increased to allow recovery (ie, a weekly method), the degree of inhibition is equal to one single dose. This pharmacokinetic profile favors twice weekly dosing at 1, where transient inhibition is observed.

Ex vivo 20S Proteasome Activity Measured in Rat Tissue After One Repeated Intravenous Administration In two studies, female Wistar rats (150-200 g)
Intravenous doses (0.03, 0.1 and 3.0 mg / kg in a 1.0 mL / kg dose) were administered. Vehicle is 0.1% ascorbic acid / 2% ethanol /
98% physiological saline [0.9%]. Tissue samples were collected from liver and brain at 1.0, 24 and 48 hours post-dose for assessment of 20S proteasome activity.

There was a significant (p <0.05) dose-related decrease in 20S proteasome activity 1.0 hour after intravenous administration of 1. At 24 hours post-dose, the dose-related decrease in 20S proteasome activity was small but still significant (p <0.05) in the high dose group at 0.3 mg / kg. At 48 hours post-dose, 20S proteasome activity in rat liver returned to baseline. The magnitude of 20S proteasome inhibition in the liver returned to baseline levels faster than that observed for peripheral leukocytes. 20S proteasome inhibition was not observed in the brain, reflecting the lack of 1 penetration into this tissue.

In a third study, male Sprague Dawley rats (250-450)
g) is one single intravenous dose (0.1 and 0.3 m in a dose of 1.0 mL / kg).
g / kg). Vehicle is 0.1% ascorbic acid / 2% ethanol /
98% physiological saline [0.9%]. Blood and tissue samples were collected 1.0 hours post-dose for evaluation of 20S proteasome activity. Tissues collected were brain, colon, liver, muscle (gastrocnemius), prostate and testis.

A significant (p <0.05) dose-related decrease in 20S proteasome activity was observed in peripheral leukocytes, colon, liver, muscle (gastrocnemius), prostate and testis 1.0 hours after intravenous administration of 1. Was observed. 20S proteasome inhibition was not observed in brain and testis, reflecting the lack of 1 penetration into these tissues. Except for brain and testis, 20S proteasome inhibition in tissues 1.0 hour after intravenous dose administration was similar to that observed for peripheral leukocytes.

Ex vivo 20S proteasome activity measured in primates following a single intravenous dose of 1 Male and female cynomolgus monkeys (2.2 to 3.5 kg) were assigned to 4 groups (5 animals)
/ Gender / group). Each group received 1 0 (vehicle control) as a single intravenous injection at a dose of 0.3 mL / kg twice weekly for 4 weeks (days 1, 5, 8, 12, 15, 19, 22, and 26). ), 0.045, 0.067 or 0.100 mg / kg / dose. Vehicle was 0.1% ascorbic acid / 2% ethanol / 98% saline [0.9%
]Met.

The control, 3 males from the low and medium dose groups, 2 high dose males, and 3 females / group were sacrificed at the end of day 27 treatment. Two animals / sex / group were designated as recovery animals and received 4 weeks of treatment followed by 2 weeks of recovery; they were killed on day 41. Blood was obtained 1.0 hours post-dose on days 1, 8, 15, and 22; 1.0 hours pre-dose on days 5, 12, 19, and 26; and 31, 34, 38. And on day 41 (animals recovered and killed), they were harvested for 20S proteasome activity measurement prior to treatment. Blood was also collected from high-dose males on day 26 prior to moribund killing after 8 doses for determination of 20S proteasome activity.

Measurement of leukocyte 20S proteasome activity 1.0 hour post-dose revealed a significant and dose-related decrease in restored enzyme activity prior to subsequent dosing (FIG. 7).
And FIG. 8). The moribund animal has a low residual 2 in its leukocytes when killed on day 26.
It was found to have 0S proteasome levels. These data indicate that 20S proteasome levels are restored during administration,
We support twice weekly treatment for 1.

Although the foregoing invention has been described in some detail for purposes of clarity and understanding, various modifications in form and detail will occur to those skilled in the art from an interpretation of this disclosure, as well as the true scope and spirit of the invention. It will be appreciated that this can be done without departing from the scope of the appended claims.

[Brief description of the drawings]

FIG. 1 is a graphical representation of the average clinical score as a function of time in an experimental autoimmune encephalomyelitis model. These data indicate that 3b (7-n-propyl-crust-lactacystin β-lactone) treatment causes a reduction in recurrence rates and mean clinical scores compared to vehicle-treated animals.

FIG. 2 is a graphical representation of the recurrence rate as a function of time in an experimental autoimmune cerebral spinal cord model. These data indicate that 3b treatment causes a reduction in recurrence rate and severity.

FIG. 3. Naive (N) or actively sensitized (AS) Brown Norwa 72 hours after exposure to aerosolized ovalbumin (10 mg / ml).
6 is a graphical representation of the white blood cell count in bronchoalveolar lavage fluid from y. Treatment with 3b causes a dose-dependent decrease in leukocyte inflow.

FIG. 4 shows naive (N) or actively sensitized (AS) Brown Norwa 72 hours after exposure to aerosolized ovalbumin (10 mg / ml).
Figure 5 is a graphical representation of eosinophil counts in bronchoalveolar lavage fluid from y rats. 3b
Treatment causes a dose-dependent inhibition of eosinophilia in this model.

FIG. 5 shows naive untreated (N) 72 hours after exposure to aerosolized ovalbumin (10 mg / ml); actively sensitized vehicle-treated (V); or actively sensitized. Figure 2 is a graphical representation of leukocyte counts in bronchoalveolar lavage fluid from drug-treated (A-H) Brown Norway rats. Budesonide alone (
Treatment with 0.1 mg / kg) or 3b alone (0.03 or 0.01 mg / kg) was not effective. However, budesonide (0.1 mg / kg) and 3
Combination with b (0.03 or 0.1 mg / kg) causes a decrease in leukocyte inflow in this model. High dose budesonide (0.5 mg / kg) is effective with or without 3b.

FIG. 6 shows naive untreated (N); actively sensitized vehicle-treated (V); or actively sensitized 72 hours after exposure to aerosolized ovalbumin (10 mg / ml). FIG. 6 is a graphical representation of eosinophil counts in bronchoalveolar lavage fluid from allowed drug-treated (AH) Brown Norway rats. Treatment with budesonide alone (0.1 mg / kg) or 3b alone (0.03 or 0.1 mg / kg) was not effective. However, the combination of budesonide (0.1 mg / kg) and 3b (0.03 or 0.1 mg / kg) causes a decrease in eosinophilia in this model. High dose budesonide (0.5 mg / kg) is effective with or without 3b.

[Procedure amendment]

[Submission date] March 13, 2001 (2001.3.13)

[Procedure amendment 1]

[Document name to be amended] Drawing

[Correction target item name] All figures

[Correction method] Change

[Correction contents]

FIG.

FIG. 2

FIG. 3

FIG. 4

FIG. 5

FIG. 6

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // (A61K 31/407 (A61K 31/407 31: 573) 31: 573) (31) Priority number 60 / 074,887 (32) Priority date February 17, 1998 (Feb. 17, 1998) (33) Priority country United States (US) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU) , TJ, TM), AL, AM AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID , IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor Louis Plummondon United States 03472 −4922 Robins Road, Watertown, Mass., No. 144 F-term (reference) 4C084 AA17 AA19 AA20 MA02 MA13 MA17 MA23 MA35 MA37 MA43 MA52 MA59 NA05 ZA022 ZA592 ZB022 ZC202 4C086 AA01 AA02 CB22 DA10 DA12 MA01 MA02 MA04 NA05 ZA59 ZB02 ZC20

Claims (37)

[Claims] Claims 1. An agent selected from the group consisting of an effective amount of a proteasome inhibitor, a ubiquitin pathway inhibitor, an agent that interferes with the activation of NF-κB via the ubiquitin-proteasome pathway, and an agent selected from the group consisting of a mixture thereof. A method for treating a patient suffering from sclerosis, which is administered to the patient. 2. The method of claim 1, wherein said agent is administered in an amount sufficient to reduce the frequency or severity of relapse. 3. The method according to claim 1, wherein said agent is a proteasome inhibitor. 4. The method according to claim 3, wherein said proteasome inhibitor is lactacystin or a lactacystin analog compound. 5. The method of claim 1, wherein the lactacystin analog compound is lactacystin, clast-lactacystin β-lactone, 7-ethyl-crust-lactacystin β-lactone, 7-n-propyl-clast-lactacystin β-lactone, 5. The method of claim 4, wherein the method is selected from the group consisting of and 7-n-butyl-crust-lactacystin? 6. The method of claim 5, wherein said lactacystin analog compound is 7-n-propyl-crust-lactacystin β-lactone. 7. An effective amount of an agent selected from the group consisting of a proteasome inhibitor, a ubiquitin pathway inhibitor, an agent that interferes with the activation of NF-κB via the ubiquitin-proteasome pathway, and a mixture thereof, A method for treating a patient suffering from the above. 8. The method of claim 7, wherein said agent is administered in an amount sufficient to reduce the frequency or severity of asthmatic attacks. 9. The method according to claim 7, wherein said agent is a proteasome inhibitor. 10. The method according to claim 9, wherein said proteasome inhibitor is lactacystin or a lactacystin analog compound. 11. The lactacystin analog compound, wherein the lactacystin analog compound is:
Clast-lactacystin β-lactone, 7-ethyl-clast-lactacystin β-lactone, 7-n-propyl-crust-lactacystin β-lactone, and 7-n-butyl-clast-lactacystin β-lactone 11. The method of claim 10, wherein the method is selected from a group. 12. The compound of claim 7, wherein said lactacystin analog compound is 7-n-propyl-
12. The method according to claim 11, which is clast-lactacystin β-lactone. 13. An agent selected from the group consisting of glucocorticoids, proteasome inhibitors, ubiquitin pathway inhibitors, agents that interfere with NF-κB activation via the ubiquitin-proteasome pathway, and mixtures thereof. A method for treating a patient suffering from asthma, the method comprising administering an effective combination to the patient. 14. The method of claim 13, wherein said combination is administered in an amount sufficient to reduce the frequency or severity of asthmatic attacks. 15. The method of claim 13, wherein said glucocorticoid and said agent are administered simultaneously. 16. The method of claim 13, wherein said glucocorticoid and said agent are administered at different times. 17. The method of claim 13, wherein said combination comprises an amount of glucocorticoid that is less than its standard recommended dose. 18. The method of claim 13, wherein said combination comprises a dose required for said glucocorticoid or an amount of agent sufficient to reduce the frequency of treatment. 19. The method of claim 13, wherein said combination comprises an amount of glucocorticoid sufficient to reduce the dose required for the agent or the frequency of treatment. 20. The method according to claim 13, wherein said agent is a proteasome inhibitor. 21. The method of claim 20, wherein said proteasome inhibitor is lactacystin or a lactacystin analog compound. 22. The lactacystin analog compound, wherein the lactacystin analog compound comprises:
Clast-lactacystin β-lactone, 7-ethyl-clast-lactacystin β-lactone, 7-n-propyl-crust-lactacystin β-lactone, and 7-n-butyl-clast-lactacystin β-lactone 22. The method of claim 21, wherein the method is selected from a group. 23. The lactacystin analog compound is 7-n-propyl-
23. The method according to claim 22, which is clast-lactacystin β-lactone. 24. The glucocorticoid wherein flunisolide, triamcinolone acetonide, beclomethasone dipropionate, dexamethasone phosphate
Sodium, fluticasone propionate, budesonide, hydrocortisone,
14. The method of claim 13, wherein the method is selected from the group consisting of prednisone, prednisolone, mometasone, tipredan, and butixicort. 25. The method of claim 24, wherein said glucocorticoid is budesonide. 26. The method according to claim 26, wherein the agent is 7-n-propyl-crust-lactacystin β.
14. The method of claim 13, wherein the lactone is glucocorticoid is budesonide. 27. A glucocorticoid with an agent selected from the group consisting of proteasome inhibitors, ubiquitin pathway inhibitors, agents that interfere with NF-κB activation via the ubiquitin-proteasome pathway, and mixtures thereof. A pharmaceutical composition comprising an effective combination. 28. The composition of claim 27, wherein said composition is provided in a unit dosage form. 29. The composition of claim 28, wherein said unit dosage form comprises an amount of glucocorticoid that is less than its standard recommended dosage. 30. The composition of claim 27, wherein said composition comprises an agent in an amount sufficient to reduce the dose or frequency of treatment required for said glucocorticoid. 31. The composition according to claim 27, wherein said agent is a proteasome inhibitor. 32. The composition of claim 31, wherein said proteasome inhibitor is lactacystin or a lactacystin analog compound. 33. The lactacystin analog compound, wherein the lactacystin is
Clast-lactacystin β-lactone, 7-ethyl-clast-lactacystin β-lactone, 7-n-propyl-crust-lactacystin β-lactone, and 7-n-butyl-clast-lactacystin β-lactone 33. The composition of claim 32 selected from the group. 34. The lactacystin analog compound is 7-n-propyl-
34. The composition of claim 33 which is clast-lactacystin [beta] -lactone. 35. The glucocorticoid is selected from the group consisting of flunisolide, triamcinolone acetonide, beclomethasone dipropionate, dexamethasone sodium phosphate, fluticasone propionate, budesonide, hydrocortisone, prednisone, prednisolone, mometasone, tipredan and butyxicolt. Composition. 36. The composition of claim 35, wherein said glucocorticoid is budesonide. 37. The method according to claim 37, wherein the agent is 7-n-propyl-crust-lactacystin β.
28. The composition of claim 27, which is a lactone, wherein said glucocorticoid is budesonide.