HK1211927B - Aggrecanase inhibitors - Google Patents

Description

Inhibitors of proteoglycan enzymes

Connective tissue is a desirable component of all mammals. Which provides rigidity, differentiation, adhesion, and in some cases elasticity. Connective tissue components include, for example, collagen, elastin, proteoglycans, fibronectin, and laminin. These biochemicals make up (or are components of) structures such as skin, bone, teeth, tendons, cartilage, basement membranes, blood vessels, cornea, and vitreous humor. Arthritis is a form of joint disorder involving inflammation of the connective tissue of one or more joints. Regardless of the type of arthritis, common symptoms of all arthritic disorders include varying degrees of pain, swelling, joint stiffness, and sometimes recurrent pain around the joints.

Half of the human patients evaluated 65 years and older and essentially every 75 years and older had osteoarthritis. There are a number of treatments for Osteoarthritis (OA), ranging from tylonox to opioids (for managing OA pain), and in extreme cases total joint replacement surgery. Currently, there is no approved treatment that has been shown to affect OA progression.

Arthritis is much more common in dogs than other domestic pets. Arthritis is a serious disease because it causes pain and restricts movement in animals. Any dog may have arthritis, although older dogs and larger breeds may be more susceptible. Active dogs, such as working dogs or hunting dogs, may also be at greater risk because of their increased activity levels.

Biochemical characterization of cartilage in arthritic joints shows severe loss of two key matrix components, collagen, particularly type II collagen and aggrecan. Aggrecan degradation is one of the early changes observed in cartilage erosion, particularly Osteoarthritis (OA). Studies have shown that two extracellular matrix proteases identified as proteoglycans catabolize aggrecan. Two proteoglycan enzyme proteases, ADAMTS-4 (a thrombospondin-motif-bearing depolymerin and metalloprotease, proteoglycan enzyme 1) and ADAMTS-5 (proteoglycan enzyme 2), were identified as being particularly effective in catabolizing aggrecan. There is a need to provide more effective treatment of arthritis, particularly treatment of OA.

Degradation or erosion of joints occurs in a variety of diseases including rheumatoid arthritis, psoriatic arthritis, osteoarthosis, excessive swelling arthritis and osteoarthritis. In addition, acute inflammation of arthritis may be accompanied by cartilage destruction. Examples of diseases involving acute joint inflammation are yersinia paraphragmatis arthritis, pyrophosphate arthritis, gout arthritis and septic arthritis. In addition, another element that may lead to the destruction or degeneration of cartilage is treatment with cortisone.

The present invention provides compounds that can be used to treat arthritis, particularly osteoarthritis, as well as inhibit cartilage erosion. The compounds of the present invention exhibit an effect on ADAMTS4 and/or ADAMTS 5.

The present invention provides a compound having the formula:

formula I

Wherein R1 is selected from the group consisting of methyl, ethyl, propyl, dimethyl, and cyclopropyl. As can be seen in formula I, the compounds of the present invention have two chiral centers, or one chiral center when R1 is dimethyl:

the present invention provides compounds having the formula:

formula Ia

Wherein R1 is selected from the group consisting of methyl, ethyl, propyl, and cyclopropyl, or

Formula Im

When R1 is dimethyl.

The present invention provides compounds having the formula:

formula Ib

Formula Ic

Formula Id

Formula Ie

Formula If

Formula Ig

Formula Ih

Formula Ii

Formula Ij

Formula Ik

Formula Il

The formula Im.

In another aspect, the present invention provides a pharmaceutical composition comprising a compound according to the present invention, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier, excipient or diluent.

In another aspect, the present invention provides a method of inhibiting cartilage erosion, such as seen in osteoarthritis, in a patient in need thereof, comprising administering to said patient an effective amount of a compound according to the present invention, or a pharmaceutically acceptable salt thereof.

In another aspect, the present invention provides a method of treating arthritis in a patient in need thereof, comprising administering to said patient an effective amount of a compound according to the present invention, or a pharmaceutically acceptable salt thereof.

In another aspect, the present invention provides the use of a compound according to the present invention, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament, in particular a medicament for the treatment of arthritis or for inhibiting cartilage erosion.

In another aspect, the invention provides a compound according to the invention, or a pharmaceutically acceptable salt thereof, for use in therapy.

In another aspect, the present invention provides a compound according to the present invention, or a pharmaceutically acceptable salt thereof, for use in the treatment of arthritis or inhibition of cartilage erosion.

In another aspect, the present invention provides a compound, method, use or composition with one or more other active agents according to the present invention.

As noted above, preferred compounds of the present invention exhibit improved binding to and inhibit the activity of proteoglycans enzymes, particularly ADAMTS 4/5. Thus, these compounds can inhibit degradation of aggrecan. Inhibition of aggrecan degradation in cartilage can be used to treat arthritis, preferably OA, and/or its pathological sequelae or symptoms.

By "patient" is meant a mammal, including humans, other primates (e.g., monkeys, chimpanzees, etc.), companion animals (e.g., dogs, cats, horses, etc.), livestock (e.g., goats, sheep, pigs, cows, etc.), laboratory animals (e.g., mice, rats, etc.), and wild and zoo animals (e.g., wolves, bears, deer, etc.). The term "patient" also refers to a mammal, particularly a human and/or companion animal such as dogs and cats or a domestic animal such as a horse, suffering from the adverse pathological effects of cartilage erosion, arthritis, and/or osteoarthritis.

By "effective amount" is meant an amount of a compound of the present invention, or a pharmaceutically acceptable salt thereof, effective to treat arthritis or osteoarthritis, and/or one or more of the sequelae of arthritis or osteoarthritis. For use on or in a mammal, ranges for the method include 0.01 to 1000 mg/kg, more desirably 0.1 to 100 mg/kg of the mammal's body weight. The frequency of administration also depends on several factors and can be a single dose administered once a day, once a week, or once a month, for a duration determined by the attending physician or veterinarian. Additional active agents may be administered using the compounds of the present invention.

As used herein, "pharmaceutically acceptable," e.g., with reference to salts and formulation components such as carriers, refers to those salts and components that are not deleterious to the patient and are compatible with other ingredients, active agents, salts, or components. Pharmaceutically acceptable includes "veterinary acceptable" and thus independently includes human and non-human mammalian applications.

By "inhibit" is meant the generally accepted meaning which includes prophylactically treating a patient suffering from cartilage erosion and arresting and/or treating cartilage erosion present in the patient. Thus, the present methods include medical treatment and/or prophylactic treatment, as the case may be.

The term "administering" as used herein refers to administering an effective amount of a compound of the present invention to a patient. Administration can be by various means including oral administration, parenteral administration such as injection (intramuscular, subcutaneous, intravenous, intraperitoneal) and the like; or topical administration, e.g., with or without transdermal penetration.

The compounds of the invention inhibit the activity of proteoglycans and are therefore preferably advantageous for use in the treatment of arthritis, in particular for the treatment of OA. The term "effective amount" as used herein refers to an amount of a compound of the present invention, i.e., formula I, which is capable of or effective for treating or alleviating the symptoms of the various pathological conditions described herein. It is understood that the amount of the compound actually administered is determined by a physician taking into account the relative circumstances and circumstances of the patient, such as age, weight, progression and severity of the disease. The compounds of the present invention are preferably formulated into pharmaceutical compositions for administration by various routes. Most preferably, such compositions are for oral administration, for example in the form of tablets, capsules, solutions, or suspensions.

Thus, another aspect of the present invention is a pharmaceutical composition comprising an effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof. Examples of pharmaceutically acceptable Salts can be found in S.M. Berge, et al, "Pharmaceutical Salts," J.Phar. Sci., 66: 1-19 (1977) and "A Handbook of Pharmaceutical Salts Properties, Selection, and Use", Wermuth, C.G. and Stahl, P.H. (eds.) VerlagHelvtica Chimica Acta, 2002. For example, the compounds of the present invention may be formulated with pharmaceutically acceptable excipients, diluents, or carriers, and formed into tablets, capsules, suspensions, solutions, powders, and the like. Pharmaceutical compositions and methods for preparing them are known in The art, and examples may be found in Remington, "The Science and practice of Pharmacy" (a. Gennaro, et al, 19 th edition. Mack Publishing co. 1995), which is incorporated herein by reference. The formulations can be administered by various means including oral administration, parenteral administration such as injection (intramuscular, subcutaneous, intravenous, intraperitoneal) and the like; or topical administration, with or without transdermal penetration. Additional active agents may be included in formulations containing the compounds of the present invention.

In addition to pharmaceutically acceptable salts, other salts may be included in the present invention. They may serve as intermediates in the purification of compounds or in the preparation of other pharmaceutically acceptable salts, or may be used for identification, characterization or purification.

The compounds of the invention may find advantageous use for the treatment of arthritis and the accompanying sequelae. The term arthritis as used herein includes, but is not limited to, Rheumatoid Arthritis (RA), juvenile rheumatoid arthritis, Systemic Lupus Erythematosus (SLE), gout, scleroderma, psoriatic arthritis, ankylosing spondylitis, Osteoarthritis (OA), and reiter's syndrome (reactive arthritis). The compounds of the invention may find particularly advantageous use in the treatment of Osteoarthritis (OA).

When used in combination with another active agent, such as an anti-inflammatory agent or a pain-relieving agent, the active agent may be a steroidal or non-steroidal agent. The compound of the invention and the other active agent may be administered simultaneously in a single formulation or in separate formulations. Alternatively, the compounds of the invention and other agents may be administered to the patient sequentially or as needed.

As used herein, the following terms have the indicated meanings: "n-BuLi" means n-butyllithium; "DCM" means dichloromethane; "Dibal-H" refers to diisobutylaluminum hydride; "DMF" refers to N, N-dimethylformamide; "DMSO" refers to dimethylsulfoxide; "EDTA" means ethylenediaminetetraacetic acid; "EtOAc" refers to ethyl acetate; "Et₂O "means diethyl ether; "EtOH" refers to ethanol; "Ex" refers to examples; "IPA" refers to isopropanol; "LDA" refers to lithium diisopropylamide; "MeOH" refers to methanol; "Prep" refers to preparative examples; "t-boc or boc" refers to tert-butoxycarbonyl; TFA means trifluoroacetic acid; "THF" refers to tetrahydrofuran; "X" as used herein refers to halogen, i.e., I, Br, Cl, or F; ' IC₅₀"refers to the concentration of an agent that produces 50% of the maximal inhibitory response for that agent.

The compounds according to the invention can be prepared according to the reactions described in the examples below.

Preparation example 1

(5R) -5- (aminomethyl) -5-cyclopropyl-imidazolidine-2, 4-bisKetohydrochlorides

Step 1N- [2- (methoxy (methyl) amino) -2-oxo-ethyl]Synthesis of tert-butyl carbamate

To a solution of Boc-Gly-OH (4250g, 24.26 mol), N, O-dimethylhydroxylamine-HCl (2839g, 29.10 mol) and DMAP (297g, 2.43 mol) in dichloromethane (36L) was added triethylamine (5.54L) followed by EDC hydrochloride (5674g, 29.60 mol) over a period of 90 min at 0 ℃. The mixture was stirred at 0 ℃ for 1 hour, then warmed to room temperature for 24 hours. The reaction mixture was cooled to 0 ℃, quenched to pH3 to 4 with 1.0M HCl, stirred at room temperature for 20min, then allowed to stand and separate. The organic phase was washed successively with 1.0M HCl (15L), water (15.0L) and brine (8.0L) over Na₂SO₄Dried and filtered. The filtrate was concentrated under reduced pressure to give the title compound as a white solid (4985 g; 94% yield).

Step 2 Synthesis of tert-butyl N- (2-cyclopropyl-2-oxo-ethyl) carbamate

To N- [2- (methoxy (methyl) amino) -2-oxo-ethyl at-30 ℃ over a period of 60 min via an addition funnel]To a solution of tert-butyl carbamate (2,455.3 g, 11.25 mol) in THF (9.0L) was added 2.0M isopropyl magnesium chloride/THF (5.34L, 10.69 mol) so that the internal temperature did not exceed 0 ℃. The mixture was then slowly warmed to 10 ℃ and 0.5M cyclopropylbromide was added via addition funnel over a period of 1 hourMagnesium oxide/THF (27.0L, 13.50 mol). The mixture was stirred at room temperature for 24 hours. The mixture was cooled to 0 ℃ and quenched with 1.0M HCl to pH 5-6, then warmed to room temperature and extracted with EtOAc (12L and 10L). The combined organic phases were washed successively with water (10L) and brine (8L), over Na₂SO₄Dried and filtered. The filtrate was evaporated under reduced pressure to obtain 2.24 kg (100% yield) of the title compound as a pale yellow oil, which was used directly in the next step.

Step 3N- [ (4-cyclopropyl-2, 5-dioxo-imidazolidin-4-yl) methyl group]Synthesis of tert-butyl carbamate

The compounds tert-butyl N- (2-cyclopropyl-2-oxo-ethyl) carbamate (4204 g, ca. 21.10 mol), KCN (1786 g, 27.43 mol) and (NH) were stirred at 65 ℃₄)₂CO₃(4866 g, 50.64 mol) in methanol (16.0L) and deionized water (19.5L) for 72 hours. The mixture was concentrated under reduced pressure to remove most of the methanol, then extracted with EtOAc (5 × 20L). The organic phase was washed with brine (8.0L) and Na₂SO₄Dried and filtered. The combined filtrates were divided into two equal portions. Each was concentrated to a volume of 15L and allowed to stand overnight at room temperature. The precipitate was filtered and washed with EtOAc (3 × 1.0L). The resulting white solids were combined and dried under vacuum at 45 ℃ for 3 days to obtain the title compound (3605 g, 64.3% yield).

Step 4N- [ [ (4R) -4-cyclopropyl-2, 5-dioxo-imidazolidin-4-yl]Methyl radical]Process for preparation of tert-butyl carbamate Separation of

Enantiomers of tert-butyl N- [ (4-cyclopropyl-2, 5-dioxo-imidazolidin-4-yl) methyl ] carbamate can be separated using a chiral column. Column: 11x33 cm Chiralpak AD of 20 microns, the flow rate/detection is 800 mL/min/230 nm, and the mobile phase is methanol.

Step 5 Synthesis of (5R) -5- (aminomethyl) -5-cyclopropyl-imidazolidine-2, 4-dione hydrochloride

Tert-butyl N- [ [ (4R) -4-cyclopropyl-2, 5-dioxo-imidazolidin-4-yl ] methyl ] carbamate (310g, 1151 mmol) was dissolved in MeOH (3.1L) at 6 ℃. 4M HCl/dioxane (310 mL) was added and the mixture was warmed to 25 ℃. After stirring for 22 hours, a second portion of 4M HCl/dioxane (110 mL) was added and stirring continued for another 16 hours. The reaction was then allowed to stand without stirring for 2 days. The mixture was then diluted with toluene (6L). The title compound (180 g) was collected as a white solid by filtering the mixture.

Example 1

(2R) -N- [ [ (4R) -4-cyclopropyl-2, 5-dioxo-imidazolidin-4-yl]Methyl radical]-2-methyl-3- [4- (trifluoro) Methyl) phenyl]Propionamide

Formula Ic

The synthesis essentially described in example 2 can be used to prepare the above compounds by using propionyl chloride instead of butyryl chloride.

Example 2

(2R) -N- [ [ (4R) -4-cyclopropyl-2, 5-dioxo-imidazolidin-4-yl]Methyl radical]-2- [ [4- (trifluoromethyl) Phenyl radical]Methyl radical]Butylamide

Formula Ie

Step 1 (4S) -4-benzyl-3-butyryl-oxazolidine-2-ketone synthesis

To a 3-neck RBF (round bottom flask) were added dichloromethane (2.4L), (S) -4-benzyl-2-oxazolidinone (200g, 1.13 mol) and N, N-dimethyl-4-aminopyridine, (13.6 g; 111.32 mmol). The flask was cooled in an ice-water bath and triethylamine (472 mL; 3.39 mol) was added dropwise at 0 ℃. Butyryl chloride (152.2 mL; 1.46 mol) was then added dropwise to the resulting solution over 3 hours while maintaining the temperature below 5 ℃. The reaction was then filtered, washed with 1M HCl (aq.) (500 ml x1) and saturated NaHCO₃The filtrate was washed (500 ml x 1). Through Na₂SO₄The organic phase was dried, filtered, and concentrated to obtain the title compound (275 g, MS: [ M + H ]]+= 248.1 m/z)。

Step 2 (4S) -4-benzyl-3- [ (2R) -2- [ [4- (trifluoromethyl) phenyl ] methyl ester]Methyl radical]Butyryl radical]Oxazolidine-2- Synthesis of ketones

In N₂Next, to 3 necks of 5L RBF were added THF (2L) and (4S) -4-benzyl-3-butyryl-oxazolidin-2-one (270 g 1.09 mol). The resulting solution was cooled to-68 ℃ in a dry ice acetone bath. To this cold solution was added sodium bis (trimethylsilyl) amide (1320 mL of a 1M solution in THF; 1.32 mol) dropwise over 1.5 hours while maintaining the internal temperature at-68 to-60 ℃. After the addition was complete, the reaction was stirred at-68 ℃ for 30 min. Then pass throughTo this cold solution was added 4-trifluoromethylbenzyl bromide (278 g; 1.16 mol)/THF (1L) at-68 ℃ for 30 min. After 1.5 h, the reaction was poured into 1M HCl. The mixture was extracted with ethyl acetate (3Lx 1). The extracts were combined and washed with NaHCO₃Aqueous (2Lx1) and brine (2Lx1) washes. Through Na₂SO₄The organic phase was dried, filtered and concentrated. The solid was triturated with EtOH (600mL) at 15 ℃. Filtration afforded the title compound (270 g, MS: [ M + H ] as a solid]+ = 406 m/z)。

Step 3 (2R) -2- [ [4- (trifluoromethyl) phenyl]Methyl radical]Synthesis of butyric acid

To a 3-neck RBF was added tetrahydrofuran (4.2L) and water (0.8L). (4S) -4-benzyl-3- (2- (benzyloxy) -3- (4- (trifluoromethyl) phenyl) propanoyl) oxazolidin-2-one (250 g; 616.65 mmol) was added and the solution cooled to 0 ℃. Hydrogen peroxide (4.93 mol; 500.30 mL) was added dropwise over 45 min. LiOH (1.08 mol; 45.28 g) in 1.2L of water was added dropwise over 1 hour. The resulting mixture was then stirred at 2 ℃ for 1 hour. Sodium sulfate (2.47 mol;310.90 g) was dissolved in 2L of water and the resulting solution was added dropwise to the reaction mixture over 1 h. After the addition was complete, the mixture was washed with DCM (2X 2L; 1 LX 1). The aqueous phase was then acidified with concentrated HCl (100 ml) to pH = 1. The resulting suspension was extracted with EA (2Lx 2). The organic extracts were combined and washed with Na₂SO₃The solution (2Lx1) and brine I (2Lx1) were washed with Na₂SO₄Dried and filtered to obtain the title compound (140 g, MS: [ M + H ]]+ = 247 m/z)。

Step 4 (2R) -N- [ [ (4R) -4-cyclopropyl-2, 5-dioxo-imidazolidin-4-yl]Methyl radical]-2- [ [4- (tris) Fluoromethyl) phenyl]Methyl radical]Synthesis of butanamide (example 2)

Formula Ie

To a 3-neck RBF (2L) was added dichloromethane (996 mL), dimethylformamide (200 mL), (2R) -2- [ [4- (trifluoromethyl) phenyl ] under a nitrogen atmosphere at ambient temperature]Methyl radical]Butyric acid (53 g, 215 mmol) and O- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (215 mmol; 81.84 g). To this mixture was added N, N-dimethyl-ethylamine (1.08 mol; 116.80 mL) in one portion. The mixture was stirred for 30 min. To the resulting solution was added (5R) -5- (aminomethyl) -5-cyclopropyl-imidazolidine-2, 4-dione hydrochloride (237 mmol;49 g) in one portion. The resulting solution was stirred for 2.5 h. The stirring was then stopped and the mixture was left to stand open to the air for 16 hours. The reaction mixture was then diluted with EtOAc (200 mL), and washed with 2M HCl (aq.), (200mL x2), 5% NaHCO₃(aq.) (200mL x2) and saline (500 mL) washes. Through Na₂SO₄The organic phase was dried, filtered and concentrated to an oil. By CH₂Cl₂The oil was diluted (250 mL) resulting in the precipitation of a white solid. The solid was collected by filtration and washed with petroleum ether (100 mL x2) to provide the title compound (55 g; MS: [ M + H)]+ = 398 m/z)。

Example 3

(2S) -2-cyclopropyl-N- [ [ (4R) -4-cyclopropyl-2, 5-dioxo-imidazolidin-4-yl]Methyl radical]-3- [4- (tris) Fluoromethyl) phenyl]Propionamide

Formula Ik

The synthesis essentially described in example 2 can be used to prepare the above compounds by using 2-cyclopropylacetyl chloride instead of butyryl chloride.

Example 4

(2S) -N- [ [ (4R) -4-cyclopropyl-2, 5-dioxo-imidazolidin-4-yl]Methyl radical]-3-methyl-2- [ [4- (tris) Fluoromethyl) phenyl]Methyl radical]Butylamide

Formula Ii

The synthesis substantially described in example 2 can be used to prepare the above compound by using 3-methylbutanoyl chloride instead of butanoyl chloride.

Example 5

N- [ [ (4R) -4-cyclopropyl-2, 5-dioxoimidazolidin-4-yl]Methyl radical]-2, 2-dimethyl-3- [4- (trifluoromethyl) Radical) phenyl]Propionamide

Example Im

Step 1 (E) -2-methyl-3- [4- (trifluoromethyl) phenyl]Synthesis of prop-2-enoic acid esters

Ethyl 2-diethoxyphosphorylpropionate (5.24g, 22 mmol) and 4- (trifluoromethyl) benzaldehyde (3.48g, 20 mmol) were dissolved in dry THF (50 mL) under a nitrogen atmosphere. The resulting solution was cooled to 0 ℃. 60% wt NaH (960 mg, 24mmol) was carefully added. It was allowed to warm to ambient temperature and stirred for 12 hours. The reaction was concentrated. The residue was purified using flash chromatography (5% EtOAc/petroleum ether) to provide the title compound (4.18 g).

Step 2-2-methyl-3- [4- (trifluoromethyl) phenyl]Synthesis of ethyl propionate

Under nitrogen, (E) -2-methyl-3- [4- (trifluoromethyl) phenyl ] prop-2-enoic acid ethyl ester (2.58 g,10 mmol) was dissolved in a suspension of 10 wt% Pd-C (258 mg)/MeOH (20 mL) in RBF, as exposure of Pd-C to oxygen could lead to combustion. The flask was carefully purged with hydrogen and the resulting mixture was stirred under hydrogen (1 Atm) for 16 hours. The flask was purged with nitrogen and the solvent was degassed to remove all hydrogen before exposure to air. The suspension was filtered through a pad of celite. The filtrate was concentrated to give the title compound (2.39 g)

Step 3, 2-dimethyl-3- [4- (trifluoromethyl) phenyl]Synthesis of ethyl propionate

To a solution of LDA (2.9 mL 2M) in THF (30 mL) at-78 deg.C was added ethyl 2-methyl-3- [4- (trifluoromethyl) phenyl ] propionate (1 g, 3.85 mmol). Stir for 10 min, then add methyl iodide (1.48 g,10.4 mmol) and stir for another 15 min. The reaction was quenched with 10 mL of 1N HCl and allowed to warm to ambient temperature. Extract with EtOAc (50 mL). The extract was washed with brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified using flash chromatography (5% EtOAc/petroleum ether) to provide the title compound (475 mg).

Step 4: 2, 2-dimethyl-3- [4- (trifluoromethyl) phenyl]Synthesis of propionic acid

Ethyl 2, 2-dimethyl-3- [4- (trifluoromethyl) phenyl ] propionate (400 mg, 1.46 mmol) was dissolved in MeOH (2 mL) and aqueous NaOH (3 mL 3N) was added. Heat to 80 ℃ and stir for 3 hours. Cool to ambient temperature and acidify with 1N HCl until pH ≈ 4. Extracted with EtOAc. The organic extracts were combined, washed with brine, dried over sodium sulfate, filtered, and concentrated to provide the title compound (272 mg).

Step 5N- [ [ (4R) -4-cyclopropyl-2, 5-dioxo-imidazolidin-4-yl]Methyl radical]-2, 2-dimethyl-3- [4- (trifluoromethyl) phenyl group]Synthesis of propionamide

Example Im

To a solution of the amine (205 mg, 1mmol) in 15 mL dry acetonitrile was added N, N-diisopropylethylamine (387mg, 3 mmol). To the resulting solution was added 2, 2-dimethyl-3- [4- (trifluoromethyl) phenyl ] propionic acid (246 mg, 1mmol), EDCI (229 mg, 1.2 mmol), and HOAT (163 mg, 1.2 mmol). Stir at ambient temperature for 12 hours. Preparative HPLC was used to isolate the title compound (282 mg).

The following analytical protocols and results are provided for purposes of illustration to further demonstrate the use and effect of the compounds and/or methods of the present invention and are not intended to be limiting in any way. All ligands, radiolabels, solvents, and reagents used in the following assays were readily available from commercial sources or could be readily synthesized by one skilled in the art.

Aggrecanase binding assays were performed to demonstrate that the compounds included in the present invention exhibit affinity for aggrecanase. More specifically, preferred compounds of the present invention exhibit improved affinity for aggrecanase, as exemplified by their binding affinity in ADAMTS-4 and ADAMTS-5 AlphaScreen assays.

Matrix Metalloproteinases (MMPs) are known to be involved in several endo-stabilization processes including tissue remodeling, abscisic enzyme activity and endocytosis. The broad spectrum MMP inhibitors tested clinically are associated with cellulose proliferation and joint stiffness and an associated side effect collectively known as musculoskeletal syndrome (MSS). Thus, selectivity of the proteoglycan enzymes relative to MMPs is generally desired. Similarly, for the ADAMTS family, several members are associated with important functions other than the desired proteoglycan enzyme inhibition. The compounds of the present invention also exhibit efficacy in plasma (i.e., inhibition of ADAMTS-4 and ADAMTS-5) and/or increased selectivity of ADAMTS-4 and ADAMTS-5 over MMP-2 and MMP-14.

ADAMTS-4 and ADAMTS-5 AlphaScreen assays:

the compounds of the invention can be evaluated by using the aggrecanase ADAMTS-4 and ADAMTS-5 AlphaScreen assays (Miller J.A., et al., anal. biochem. 2003, 314, 260-: typically 3 or 4 nM ADAMTS-4 or 2.1 nM ADAMTS-5 was incubated with 80 nM 43-mer peptide substrate +/-inhibitor (1% final DMSO concentration) in white non-binding surface 96-well plates for 3 hours at room temperature (Corning 3990). Inhibitors were serially diluted 3-fold and tested at final starting concentrations up to about 100 μ M. The assay was then quenched with a mixture containing EDTA (62.5mM), 50 mM Tris (hydroxymethyl) aminomethane (Tris), (pH7.5), 10mM calcium chloride, 100 mM sodium chloride, 0.2% Brij 35 (the major component being polyoxyethylene (23) lauryl ether), 0.1% Bovine Serum Albumin (BSA), BC3 monoclonal antibody hybridoma supernatant (1:2000 final dilution), streptavidin-conjugated donor beads and anti-mouse IgG-conjugated acceptor beads (15. mu.g/mL final concentration of both beads). The plates were covered with aluminum foil tape and the combination was allowed to incubate overnight. Plates were then read on an AlphaScreen Fusion Alpha reader from Perkin Elmer. Data were analyzed using ActivityBase ­ software (IDBS Alameda, CA). A similar assay was used for the purified dog ADAMTS-4 enzyme. Data for representative compounds of the present invention are provided in table 1 below.

TABLE 1

Rat and dog plasma excursion AlphaScreen assay

This AlphaScreen assay was modified to include testing of inhibitors of ADAMTS-5 in the presence of plasma against 50% Lewis rat to determine the effect of plasma protein binding on inhibitor function. Calculating the IC of an inhibitor against ADAMTS-5 in 50% Lewis rat plasma₅₀IC compared to inhibitor in buffer₅₀And is described as the plasma drift of the inhibitor. The assay was performed in the same manner using 10 nM ADAMTS-5 instead of 2.1 nM. A similar assay was used for dog ADAMTS-4 in the presence of 25% dog plasma. Data for representative compounds of the present invention are provided in table 2 below.

TABLE 2

In vitro fluorescence assay for MMP-2 activity

A continuous assay was used in which the substrate was a synthetic peptide containing a fluorescent group (7-methoxycoumarin, Mca), which was quenched by energy and converted to 2, 4-dinitrophenyl. The substrate is peptide Mca-PQGL- (3- [2, 4-dinitrophenyl ] -L-2, 3-diaminopropionyl) -AR-OH. When the peptide is cleaved by MMP, a substantial increase in fluorescence is observed. The enzyme source used for this analysis was full-length recombinant human pro-MMP-2 expressed in Chinese Hamster Ovary (CHO) cells, which was subsequently activated by the organomercury preparation compound 4-aminophenylmercuric acetate (APMA). APMA was removed by desalting column (MMP-2 Calbiochem catalog No. PF 023). The assay buffer consisted of 100 mM Tris-HCl (pH7.5), 100 mM NaCl, 10mM CaCl2, and 10. mu.M human serum albumin. Each well of the 96-well plate consisted of 100 μ L of reaction mixture consisting of assay buffer MMP (final concentration 0.2 nM, prepared by dilution in assay buffer), and different concentrations of inhibitor (prepared by serial dilution of a given inhibitor in DMSO in 96-well polypropylene plates using either a 10-point or 11-point dilution protocol). The enzymatic reaction was initiated by adding substrate to a final concentration of 20. mu.M. The final DMSO concentration in the assay was 1.0%. The plates were incubated at room temperature for 2-4 hours and substrate cleavage was determined on LJL Analyst or Wallac Envision using a fluorescent plate reader (excitation filter 320 and emission filter 436) at room temperature.

Using the ActivinyBase software program vs. 5.3 to analyze the data using the 4 parameter fitting model equation 205, relative IC was generated therefrom₅₀. The maximum signal was calculated from wells that were not treated with inhibitor but with enzyme, substrate and 1.0% DMSO. The minimum signal was calculated from wells with buffer only (no enzyme), substrate and 1.0% DMSO.

In vitro fluorescence analysis of other MMP activity

Procedures essentially identical to the MMP-2 assay described above or as known in the art were used for the remaining MMP assays. For example, for MMP-14, the enzyme source is the MMP-14 (MT1-MMP) catalytic region produced by activation of a recombinant soluble form of the enzyme purified from the periplasm. It is made up by using amino acid residue Tyr of mature human MT1-MMP (Calbiochem catalog No. 475935)⁸⁹To Gly²⁶⁵And (4) forming. The final concentration in each well was 0.5 nM instead of 0.2 nM in the MMP-2 assay. Data for representative compounds of the present invention are provided in table 3 below.

TABLE 3

MIA-injected PD model in rats

The assay described in Swearingen et al, Osteoarthritis and Cartilige 18 (2010) 1159-1166 may be used. MIA (Sigma, Cat # I2512, sodium salt) was prepared fresh on the day of use at 3 mg in 50 ul sterile 0.9% saline. On day 0, 7-8-week old male Lewis rats were anesthetized and injected intra-articularly with MIA into the right knee (to induce endogenous aggrecanase activity and release aggrecan into the synovial fluid) and saline to the left knee (contralateral). The proteoglycanase inhibitor (3, 10 or 30 mg/kg) or vehicle [1% hydroxyethyl cellulose (HEC); 0.25% Tween 80; 0.05% antifoam ] was administered orally twice daily from day 3. A single dose of compound was administered on day 7, and the animals were sacrificed 4 hours later and the knee joints were irrigated with 200 ul saline. The aggrecan was analyzed for proteoglycan enzyme cleaved fragments by synovial lavage using a NITEGE sandwich enzyme-linked immunosorbent assay (NITEGESANDwich ELISA). The amount of aggrecan fragments present in synovial lavage was determined based on a standard curve generated from rat aggrecan digested with aggrecanase. Statistical analysis was performed using the Dunnett test.

And (3) sandwich enzyme-linked immunosorbent assay analysis: for NITEGE ELISA, NITEGE monoclonal antibodies were immobilized on white high binding ELISA plates (Nunc) overnight at 4C. After blocking, rat synovial lavage samples were added to the plates and fragments with C-terminal NITEGE sequences were captured. The captured fragments were detected using HRP-conjugated HABR monoclonal antibody. ELISA signals were tested using Supersignal ELISA femto maximum sensitivity substrate (Pierce) and read on a Victor luminometer. The amount of aggrecan fragments present in the sample was determined based on a standard curve generated using rat chondrosarcoma aggrecan digested with aggrecanase (850 mg/ml stock solution diluted in antibody dilution buffer). The data are presented in table 4.

TABLE 4

In the bone jointStudy of plasma biomarker ARGN in inflamed dogs

The objective of this study was to determine the response of the plasma biomarker ARGN to a range of doses of the compound of the invention in Osteoarthritis (OA) dogs after daily oral administration for 21 days. Sixteen (16) adult laboratory Beagle dogs ≧ 8 years of age (radiographic evidence of OA in the hip joint) and 4 age-matched control Beagle dogs without OA were involved in this study.

Blood samples of baseline plasma ARGN concentrations were collected from all dogs on days 30, 28, and 26 prior to the start of dose administration. 16 dogs with OA were randomly batched by their mean baseline plasma ARGN concentration into 1 of 4 treatment groups: placebo, 0.1, 1, and 10 mg/kg of the compound of example 2. All 4 age-matched control dogs without OA were assigned to placebo treated groups. Starting on day 0, dogs received once daily oral gavage of the compound of example 2 in solution/suspension for 3 weeks, depending on their assigned treatment group. Blood samples of plasma ARGN and the compound concentration of example 2 were collected before the first dose administration and 3 times per week for an additional 4 weeks (day 28). Additional blood samples of plasma ARGN and the compound concentration of example 2 were collected at 1, 2, 6, 12 and 24 hours before and after the last dose administration (day 20). Plasma ARGN concentrations were determined by immunoassay using a sandwich enzyme-linked immunosorbent assay procedure and plasma concentrations of the compound of example 2 were determined using the LC-MS/MS method. Summary statistics of plasma ARGN and compound concentrations of example 2 were calculated and non-compartmentalized PK analyses of compound concentrations of example 2 were performed.

Plasma ARGN concentrations were inhibited following daily doses of 0.1, 1, and 10 mg/kg of the compound of example 2, respectively, in a manner that inhibited the drug response by 33.8%, 70.7%, and 80.3% on average day 21. ARGN inhibition was relatively low in normal and OA placebo-treated dogs, ranging from-1.30% to 13.4%. The ARGN concentrations did not differ substantially between normal and OA placebo dogs. Example 2 plasma concentrations of compound increase with dose in a sub-proportional (sub-proportional) manner and a steady-state trough concentration is rapidly achieved. It was demonstrated that increasing dose and systemic exposure of the compound of example 2 resulted in an increase in inhibition of plasma ARGN concentration. Thus, after once daily oral administration, the compound of example 2 inhibited its target proteoglycan enzyme in dogs that naturally suffer from OA.

The present invention is described by the following clauses.

1. A compound having the formula:

formula I

Wherein R is₁Selected from the group consisting of methyl, ethyl, propyl, dimethyl, and cyclopropyl.

2. A compound according to clause 1, or a pharmaceutically acceptable salt thereof, having the formula:

formula Ia, or

The formula Im.

3. A compound according to clause 1, or a pharmaceutically acceptable salt thereof, having the formula:

formula Ib.

4. A compound according to clause 3, or a pharmaceutically acceptable salt thereof, having the formula:

formula Ic.

5. A compound according to clause 1, or a pharmaceutically acceptable salt thereof, having the formula:

formula Id.

6. A compound according to clause 5, or a pharmaceutically acceptable salt thereof, having the formula:

formula Ie.

7. A compound according to clause 1, or a pharmaceutically acceptable salt thereof, having the formula:

formula If.

8. A compound according to clause 7, or a pharmaceutically acceptable salt thereof, having the formula:

formula Ig.

9. A compound according to clause 1, or a pharmaceutically acceptable salt thereof, having the formula:

formula Ih.

10. A compound according to clause 9, or a pharmaceutically acceptable salt thereof, having the formula:

formula Ii.

11. A compound according to clause 1, or a pharmaceutically acceptable salt thereof, having the formula:

formula Ij.

12. A compound according to clause 11, or a pharmaceutically acceptable salt thereof, having the formula:

formula Ik.

13. A compound according to clause 1, or a pharmaceutically acceptable salt thereof, having the formula:

formula II.

14. A compound according to clause 13, or a pharmaceutically acceptable salt thereof, having the formula:

the formula Im.

15. A pharmaceutical composition comprising a compound according to any one of clauses 1 to 14 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier, excipient or diluent.

16. The pharmaceutical composition of clause 15, wherein the composition comprises at least one additional active agent.

17. The pharmaceutical composition of clause 15 or 16, wherein the composition is a human pharmaceutical composition.

18. The pharmaceutical composition of clauses 15 or 16, wherein the composition is a veterinary composition.

19. The pharmaceutical composition of any of clauses 15 to 18, wherein the pharmaceutical composition is suitable for oral administration.

20. The pharmaceutical composition of any of clauses 15 to 19, wherein the pharmaceutical composition is in the form of a tablet, capsule, solution, or suspension.

21. A method of treating arthritis in a patient in need thereof, the method comprising administering to the patient an effective amount of a compound according to any one of clauses 1 to 14 or a pharmaceutically acceptable salt thereof.

22. The method of clause 21, wherein the patient is administered at least one additional active agent.

23. The method of clause 21 or 22, wherein the patient is a human.

24. The method of clause 21 or 22, wherein the patient is a dog.

25. The method of any of clauses 21 to 24, wherein the administration is oral administration.

26. The method of any of clauses 21 to 25, wherein the administration is carried out using the compound in the form of a tablet, capsule, solution, or suspension.

27. A method of inhibiting cartilage erosion in a patient in need thereof, the method comprising administering to the patient an effective amount of a compound according to any one of clauses 1 to 14 or a pharmaceutically acceptable salt thereof.

28. The method of clause 27, wherein the patient is administered at least one additional active agent.

29. The method of clause 27 or 28, wherein the patient is a human.

30. The method of clause 27 or 28, wherein the patient is a dog.

31. The method of any one of clauses 27 to 30, wherein the administration is oral administration.

32. The method of any of clauses 27 to 31, wherein the administration is carried out using the compound in the form of a tablet, capsule, solution, or suspension.

33. A compound according to any one of clauses 1 to 14, or a pharmaceutically acceptable salt thereof, for use in therapy.

34. A compound according to any one of clauses 1 to 14, or a pharmaceutically acceptable salt thereof, for use in the treatment of arthritis.

35. A compound according to any one of clauses 1 to 14, or a pharmaceutically acceptable salt thereof, for use in inhibiting cartilage erosion.

36. Use of a compound according to any one of clauses 1 to 14, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament.

37. The use of clause 36, wherein the medicament is for treating arthritis.

38. The use of clause 36 or 37, wherein the medicament is for inhibiting cartilage erosion.

39. The use of any of clauses 36 to 38, wherein the pharmaceutical composition is suitable for oral administration.

40. The use of any of clauses 36 to 39, wherein the medicament is in the form of a tablet, capsule, solution, or suspension.

Claims (8)

1. A compound having the formula:

formula I

Wherein R is₁Selected from the group consisting of methyl, ethyl, propyl, dimethyl, and cyclopropyl.

2. A compound having the formula:

formula Ia

Wherein R is₁Selected from the group consisting of methyl, ethyl, propyl, dimethyl, and cyclopropyl.

3. A compound according to claim 1, or a pharmaceutically acceptable salt thereof, having the formula:

formula Ib;

formula Id;

formula If;

formula Ih;

formula Ij; or

Formula II.

4. A compound according to claim 2, or a pharmaceutically acceptable salt thereof, having the formula:

formula Ic;

formula Ig;

formula Ii;

formula Ik; or

The formula Im.

5. A compound according to claim 2, or a pharmaceutically acceptable salt thereof, having the formula:

formula Ie.

6. A pharmaceutical composition comprising a compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier, excipient or diluent.

7. Use of a compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for treating arthritis in a patient in need thereof.

8. Use of a compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for inhibiting cartilage erosion in a patient in need thereof.