Amino- or ammonium-containing sulfonic acid, phosphonic acid and carboxylic acid derivatives and their medical use

Abstract

The present invention relates to amino- or ammonium-containing sulfonic acid, phosphonic acid and carboxylic acid derivatives, in particular the compounds of formula 1, 2, 3, 4, 5 or 6, and their medical use, including their use in the treatment, prevention or amelioration of an inflammatory, autoimmune and/or allergic disorder. ##STR00001##

Claims

1. A method of treating or ameliorating an inflammatory, autoimmune and/or allergic disorder, the method comprising the administration of a compound of formula 1 ##STR00016## wherein: R.sup.1 is a C.sub.10-20 hydrocarbon group; R.sup.2 is a C.sub.1-4 alkyl group, and R.sup.3 is —H, a C.sub.1-4 alkyl group or R.sup.3 is absent; or R.sup.2 and R.sup.3 are mutually linked to form a pyrrolidine ring, a piperidine ring or an azepane ring together with the nitrogen atom X to which they are attached, wherein said pyrrolidine ring, said piperidine ring or said azepane ring is optionally substituted with one or more groups independently selected from —OH, —O(C.sub.1-3 alkyl), —O—C(O)—(C.sub.1-3 alkyl), C.sub.1-3 alkyl, —C(O)—(C.sub.1-3 alkyl), —C(O)—NH.sub.2, —C(O)—NH(C.sub.1-3 alkyl), —C(O)—N(C.sub.1-3 alkyl)(C.sub.1-3 alkyl), —NH.sub.2, —NH(C.sub.1-3 alkyl), —N(C.sub.1-3 alkyl)(C.sub.1-3 alkyl), —NH—C(O)—(C.sub.1-3 alkyl), —N(C.sub.1-3 alkyl)-C(O)—(C.sub.1-3 alkyl), —NH—C(O)—O(C.sub.1-3 alkyl), or —N(C.sub.1-3 alkyl)-C(O)—O(C.sub.1-3 alkyl); R.sup.4 is a C.sub.1-6 alkylene group; R.sup.5 is —SO.sub.3.sup.−, —SO.sub.3H, —PO.sub.3H.sup.−, —PO.sub.3.sup.2−, —PO.sub.3H.sub.2, —PO.sub.2(OC.sub.1-3 alkyl).sup.−, —PO.sub.2H(OC.sub.1-3 alkyl), —PO(OC.sub.1-3 alkyl).sub.2, —CO.sub.2.sup.−, or —CO.sub.2(C.sub.1-3 alkyl); and X is N.sup.+ or, if R.sup.3 is absent, X is N; or a pharmaceutically acceptable salt, solvate or prodrug thereof to a subject in need of such a treatment or amelioration, wherein the compound of formula 1 treats or ameliorates the inflammatory, autoimmune and/or allergic disorder, and wherein said inflammatory, autoimmune and/or allergic disorder is selected from: psoriasis, atopic dermatitis (atopic eczema), contact dermatitis, xerotic eczema, seborrheic dermatitis, neurodermitis, dyshidrosis, discoid eczema, venous eczema, dermatitis herpetiformis (Duhring's Disease), autoeczematization, dermatomyositis, hyper-IgE (Buckley) syndrome, Wiskott-Aldrich syndrome, anaphylaxis, food allergy, allergic reactions to venomous stings, acute urticarias, chronic urticarias, physical urticarias, aquagenic urticaria, cholinergic urticaria, cold urticaria (chronic cold urticaria), delayed pressure urticaria, dermatographic urticaria, heat urticaria, solar urticaria, vibration urticaria, adrenergic urticaria, urticaria angioedema, inflammatory bowel disease, Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, diversion colitis (diverticulitis), Behcet's syndrome, indeterminate colitis, celiac disease, irritable bowel syndrome, post-operative ileus, eosinophilic gastroenteropathy, gastritis, chronic allergic rhinitis, seasonal allergic rhinitis (hay-fever), allergic conjunctivitis, chemical conjunctivitis, neonatal conjunctivitis, Sjögren syndrome, open-angle glaucoma, dry eye disease, diabetic macular edema, chronic obstructive pulmonary disease (COPD), allergic asthma, allergic bronchopulmonary aspergillosis, hypersensitivity pneumonitis, lung fibrosis, rheumatoid arthritis, juvenile rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus (SLE), scleroderma, reactive arthritis, polymyalgia rheumatica, Guillain-Barre syndrome, Hashimoto's thyroiditis, Grave's disease, temporal arteritis, liver disease, primary biliary cirrhosis, sclerosing cholangitis, autoimmune hepatitis multiple sclerosis, or alopecia areata.

2. The method of claim 1, wherein said inflammatory, autoimmune and/or allergic disorder is selected from psoriasis, atopic dermatitis (atopic eczema), contact dermatitis, xerotic eczema, seborrheic dermatitis, neurodermitis, dyshidrosis, discoid eczema, venous eczema, dermatitis herpetiformis (Duhring's Disease), autoeczematization, dermatomyositis, hyper-IgE (Buckley) syndrome, Wiskott-Aldrich syndrome, anaphylaxis, food allergy, or allergic reactions to venomous stings.

3. The method of claim 1, wherein said inflammatory, autoimmune and/or allergic disorder is selected from acute urticarias, chronic urticarias, physical urticarias, aquagenic urticaria, cholinergic urticaria, cold urticaria (chronic cold urticaria), delayed pressure urticaria, dermatographic urticaria, heat urticaria, solar urticaria, vibration urticaria, adrenergic urticaria, or urticaria angioedema.

4. The method of claim 1, wherein said inflammatory, autoimmune and/or allergic disorder is selected from inflammatory bowel disease, Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, diversion colitis (diverticulitis), Behcet's syndrome, indeterminate colitis, celiac disease, irritable bowel syndrome, post-operative ileus, eosinophilic gastroenteropathy, or gastritis.

5. The method of claim 1, wherein said inflammatory, autoimmune and/or allergic disorder is selected from chronic allergic rhinitis, seasonal allergic rhinitis (hay-fever), allergic conjunctivitis, chemical conjunctivitis, neonatal conjunctivitis, Sjögren syndrome, open-angle glaucoma, dry eye disease, or diabetic macular edema.

6. The method of claim 1, wherein said inflammatory, autoimmune and/or allergic disorder is selected from chronic obstructive pulmonary disease (COPD), allergic asthma, allergic bronchopulmonary aspergillosis, hypersensitivity pneumonitis, or lung fibrosis.

7. The method of claim 1, wherein said inflammatory, autoimmune and/or allergic disorder is selected from rheumatoid arthritis, juvenile rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus (SLE), scleroderma, reactive arthritis, or polymyalgia rheumatica.

8. The method of claim 1, wherein said inflammatory, autoimmune and/or allergic disorder is selected from Guillain-Barre syndrome, Hashimoto's thyroiditis, Grave's disease, temporal arteritis, primary biliary cirrhosis, sclerosing cholangitis, autoimmune hepatitis, or alopecia areata.

9. The method of claim 1, whereby said compound is administered in combination with one or more immunomodulatory drugs and/or one or more anti-inflammatory drugs.

10. A method of treating or ameliorating an inflammatory, autoimmune and/or allergic disorder, the method comprising the administration of a compound of any of the following formulae to a subject in need of such a treatment or amelioration: ##STR00017## ##STR00018## ##STR00019## or a pharmaceutically acceptable salt, solvate or prodrug thereof, wherein the compound treats or ameliorates the inflammatory, autoimmune and/or allergic disorder.

11. The method of claim 10 whereby said compound is formulated for administration by any one of: an oral route; topical route, transdermal, intranasal, ocular, buccal, or sublingual route; parenteral route using injection techniques or infusion techniques, by subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, intrasternal, intraventricular, intraurethral, or intracranial route; pulmonary route, by inhalation or insufflation therapy; gastrointestinal route; intrauterine route; intraocular route; subcutaneous route; ophthalmic route, intravitreal, or intracameral route; rectal route; or vaginal route.

12. The method of claim 10, wherein said subject is a human.

13. The method of claim 10, wherein said subject is a non-human mammal.

14. The method of claim 1 whereby said compound is formulated for administration by any one of: an oral route; topical route, transdermal, intranasal, ocular, buccal, or sublingual route; parenteral route using injection techniques or infusion techniques, by subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, intrasternal, intraventricular, intraurethral, or intracranial route; pulmonary route, by inhalation or insufflation therapy; gastrointestinal route; intrauterine route; intraocular route; subcutaneous route; ophthalmic route, intravitreal, or intracameral route; rectal route; or vaginal route.

15. The method of claim 1, wherein said subject is a human.

16. The method of claim 1, wherein said subject is a non-human mammal.

17. The method of claim 11, whereby said compound is administered in combination with one or more immunomodulatory drugs and/or one or more anti-inflammatory drugs.

18. The method of claim 11, wherein said inflammatory, autoimmune and/or allergic disorder is selected from: psoriasis, atopic dermatitis (atopic eczema), contact dermatitis, xerotic eczema, seborrheic dermatitis, neurodermitis, dyshidrosis, discoid eczema, venous eczema, dermatitis herpetiformis (Duhring's Disease), autoeczematization, dermatomyositis, hyper-IgE (Buckley) syndrome, Wiskott-Aldrich syndrome, anaphylaxis, food allergy, allergic reactions to venomous stings, acute urticarias, chronic urticarias, physical urticarias aquagenic urticaria, cholinergic urticaria, cold urticaria (chronic cold urticaria), delayed pressure urticaria, dermatographic urticaria, heat urticaria, solar urticaria, vibration urticaria, adrenergic urticaria, urticaria angioedema, inflammatory bowel disease, Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, diversion colitis (diverticulitis), Behcet's syndrome, indeterminate colitis, celiac disease, irritable bowel syndrome, post-operative ileus, eosinophilic gastroenteropathy, gastritis, chronic allergic rhinitis, seasonal allergic rhinitis (hay-fever), allergic conjunctivitis, chemical conjunctivitis, neonatal conjunctivitis, Sjögren syndrome, open-angle glaucoma, dry eye disease, diabetic macular edema, chronic obstructive pulmonary disease (COPD), allergic asthma, allergic bronchopulmonary aspergillosis, hypersensitivity pneumonitis, lung fibrosis, rheumatoid arthritis, juvenile rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus (SLE), scleroderma, reactive arthritis, polymyalgia rheumatica, Guillain-Barre syndrome, Hashimoto's thyroiditis, Grave's disease, temporal arteritis, liver disease, primary biliary cirrhosis, sclerosing cholangitis, autoimmune hepatitis multiple sclerosis, or alopecia areata.

Description

(1) The invention is also described by the following illustrative figures. The appended figures show:

(2) FIG. 1: Inhibition of mast cell degranulation by compounds 1a (FIG. 1A), 1b (FIG. 1B), 1c (FIG. 1C), 1d (FIG. 1D), 1e (FIG. 1E), 1f (FIG. 1F), 1g (FIG. 1G), 1h (FIG. 1H), 1i (FIG. 1I), 1j (FIG. 1J), 1k (FIG. 1K), 1n (FIG. 1L), 1q (FIG. 1M), 1r (FIG. 1N), 1s (FIG. 1O), 1t (FIG. 1P), 1u (FIG. 1Q), 1v (FIG. 1R), 1w (FIG. 1S), 1x (FIG. 1T), 2a (FIG. 1U), 2b (FIG. 1V) and miltefosine (FIG. 1W). Dose-response curves for inhibition of β-hexosaminidase release from RBL-2H3 cells stimulated with antigen-specific IgE and triggered with antigen are shown (means±standard error of the mean).

(3) FIG. 2: Inhibition of Akt phosphorylation on Ser473 by compounds 1a (FIG. 2A) and 1c (FIG. 2B). Percentage of total Akt phosphorylated on Ser473 is expressed as a percentage of control untreated cells induced with IgE and antigen for 15 min (shown are means±standard deviation).

(4) FIG. 3: Effect of compound 1a and dexamethasone on mouse ear swelling in the DTH response in mice (data are means±standard deviations of 8 mice; *p<0.01 vs. vehicle control (Dunnett's post hoc test)).

(5) FIG. 4: Effect of compound 1a on mouse ear swelling in the allergic contact dermatitis model in mice. FIG. 4A shows the inhibitory activity of compound 1a at different administration times before antigen challenge (data are means±SEM of 7 mice; *p<0.05 vs. vehicle control (Dunnett's post hoc test)). FIG. 4B shows the inhibitory activity of compound 1a after topical application (data are means±SEM of 7 mice; ***p<0.001 vs. vehicle control (Dunnett's post hoc test)).

(6) The invention will now be described by reference to the following examples which are merely illustrative and are not to be construed as a limitation of the scope of the present invention.

EXAMPLES

Example 1: 3-(N,N-Dimethylmyristylammonio)-propanesulfonate 1a

(7) Compound 1a is commercially available (Sigma-Aldrich Chemie GmbH, Munich, Germany, product number T7763).

Example 2: Preparation of 3-(N-methyltetradecylammonio)propanesulfonate 1b

(8) N-Methyltetradecylamine (454 mg, 2 mmol) and 1,3-propanesultone (280 mg, 2.3 mmol) are stirred in ethyl acetate (10 mL) for 24 h. The volatiles are removed and the residue is flash-chromatographed on silica using dichloromethane/methanol (4:1). Rotary evaporation and drying in high vacuum yields 363 mg (52%) of 1b as a white solid.

(9) .sup.1H-NMR (300 MHz, CDCl.sub.3): δ=0.81 (t, J=6.9, 3H), 1.1-1.35 (m, 22H), 1.25 (m, 2H), 2.22 (m, 2H), 2.81 (s, 3H), 2.98 (m, 4H), 3.25 (m, 2H).

(10) MS (ESI): 350.3 (M+H.sup.+), 372.6 (M+Na.sup.+), 699.6 (2M+H.sup.+), 721.6 (2M+Na.sup.+).

Example 3: Preparation of 3-N,N-dimethylmyristylammoniopropylphosphonic acid 1c

(11) N,N-Dimethyltetradecylamine is alkylated with commercial diethyl 3-bromopropyl-phosphonate to yield the corresponding ethyl ester, which is purified by crystallization. Treatment with trimethylsilylbromide in the presence of allyltrimethylsilane (Hammerschmidt 1991, Yan 2007) followed by hydrolysis of the resulting silylphosphonate yields compound 1c as hydrobromide salt (1c-HBr).

(12) The side products of the ester cleavage are volatile and can be removed in vacuum. 1c-HBr is subsequently purified by crystallization. The amount of water used should be kept to a minimum because 1c and its hydrobromide tend to intense foaming during rotary evaporation. Attempted aqueous workup of the abovementioned ethyl ester, 1c-HBr or 1c results in a stable emulsion. The hydrobromide is treated with exactly one equivalent of NaOH, desalted by passing through a RP-18 column and the resulting betaine 1c is purified by recrystallization.

(13) .sup.1H-NMR (600 MHz, CDCl.sub.3/CD.sub.3OD 8:2): δ=0.90 (t, 3H), [1.22-1.43 (m), 1.39 (br s), Σ=22H)], 1.66 (d/t, J=17.3/7.0, 2H), 1.75 (br m, 2H), 2.02 (m, 2H), 3.09 (s, 6H), 3.25 (m, 2H), 3.47 (m, 2H).

Example 4: Preparation of 2-(N,N-dimethyltetradecylammonio)ethanesulfonate 1d

(14) Sodium-2-bromoethansulfonate (411 mg, 1.95 mmol), N-methyltetradecylamine (342 mg, 1.50 mmol) and K.sub.2CO.sub.3 (269 mg, 1.95 mmol) are suspended in dimethylformamide (DMF) (3 mL) and stirred at reflux overnight. The volatiles are removed and the residue is purified by preparative HPLC to give, after drying at high vacuum, 324 mg of 2-(N-methyltetradecylammonio)-ethanesulfonate as a white solid.

(15) 2-(N-Methyltetradecylammonio)-ethanesulfonate (113 mg, 0.25 mmol), methyl iodide (284 mg, 2.0 mmol) and K.sub.2CO.sub.3 (103 mg, 0.75 mmol) are suspended in a mixture of acetone (3 mL) and dichloromethane (1 mL). The mixture is stirred at room temperature overnight, the solvent is removed and the residue purified by preparative HPLC to give 50 mg (57%) of 1d.

(16) .sup.1H-NMR (300 MHz, CDCl.sub.3/CD.sub.3OD 8:2): δ=0.80 (t, J=6.9, 3H), 1.1-1.45 (m, 22H), 1.68 (m, 2H), 3.02 (s, 6H), 3.19 (m, 4H), 3.61 (m, 2H).

(17) MS (ESI): 350.3 (M+H.sup.+), 699.6 (2M+H.sup.+).

Example 5: Preparation of 4-(N,N-dimethylmyristylammonio)butanesulfonate 1e

(18) 1,4-Butane sultone (681 mg, 5 mmol) and N,N-dimethyltetradecylamin (966 mg, 4 mmol) are dissolved in DMF (10 mL) and stirred under argon atmosphere at 130° C. bath temperature for 2 d. The volatiles are removed and the residue is purified by preparative HPLC to yield 831 mg (55%) of 1e as white solid.

(19) .sup.1H-NMR (300 MHz, CDCl.sub.3): δ=0.81 (t, J=6.9, 3H), 1.1-1.35 (m, 22H), 1.61 (m, 2H), 1.83 (m, 4H), 2.84 (m, 2H), 3.08 (s, 6H), 3.18 (m, 2H), 3.44 (m, 2H).

(20) MS (ESI): 378.3 (M+H.sup.+), 755.6 (2M+H.sup.+).

Example 6: Preparation of 2-((dimethyl(tetradecyl)ammonio)methyl)butane-1-sulfonate 1f

(21) Ethylmalonic acid diethylester (1.86 g, 9.9 mMol) and N-methyltetradecyl amine (1.5 g, 6.6 mmol) are dissolved in DMF (20 mL) and stirred at 130° C. bath temperature under argon atmosphere for 24h. The mixture is partitioned between water and EtOAc. The aqueous layer is extracted with EtOAc twice and the combined EtOAc layers are washed with sat. NaCl, dried over Na.sub.2SO.sub.4 and the solvent is removed. The residue is passed through a short plug of silica using dichloromethane/methanol 4:1, the solvent removed under reduced pressure and the residue is dried in high vacuum to provide 2.1 g (86%) of ethyl 2-(methyl(tetradecyl)carbamoyl)butanoate as a white material.

(22) Under argon atmosphere, LiAlH.sub.4 (608 mg, 16.0 mmol) is suspended in 10 mL of tetrahydrofuran (THF) and heated to reflux. A solution of ethyl 2-(methyl(tetradecyl)-carbamoyl)butanoate (3.0 g, 8.0 mmol) in 15 mL of THF is added dropwise with caution and the resulting mixture is heated to reflux overnight. Methanol is added dropwise with caution until the evolution of hydrogen ceases. 15 mL of water are added, whereupon the colour changes from grey to white. The mixture is diluted with water and ethyl acetate. The solids are filtered off using a pad of celite with EtOAc-washing. The aqueous layer is separated and extracted three times with ethyl acetate. The combined organic layers are washed with sat. NaCl (1×), dried over Na.sub.2SO.sub.4 and the solvent is removed under reduced pressure. The residue is chromatographed on silica using dichloromethane/methanol 10:1 to yield 550 mg (22%) of 2-((methyl(tetradecyl)amino)methyl)butan-1-ol as a white solid.

(23) 2-((Methyl(tetradecyl)amino)methyl)butan-1-ol (390 mg, 1.24 mmol) is dissolved in dichloromethane (8 mL) under argon atmosphere. N,N-Diisopropylethylamine (DIEA) (163 mg, 1.30 mmol) and 4-dimethylaminopyridine (DMAP) (15 mg, 0.12 mmol) are added. To this mixture is added dropwise methanesulfonyl chloride (148 mg, 1.30 mmol). The mixture is stirred at ambient temperature overnight. The mixture is quenched with methanol and the solvent is removed under reduced pressure. Chromatography on silica using dichloromethane/methanol 10:1 yields 208 mg (43%) of 2-((methyl(tetradecyl)amino)-methyl)butyl methanesulfonate.

(24) 2-((Methyl(tetradecyl)amino)-methyl)butyl methanesulfonate (200 mg, 0.5 mmol) is dissolved in ethanol (2 mL). A solution of Na.sub.2SO.sub.3 (315 mg, 2.5 mmol) in 1 mL of water is added and the mixture is stirred at 100° C. bath temperature for 3h. After removal of the solvent under reduced pressure, the residue is chromatographed on silica using dichloromethane/methanol 20:1 to yield 120 mg (64%) of 2-((methyl(tetradecyl)ammonio)methyl)butane-1-sulfonate.

(25) 2-((Methyl(tetradecyl)ammonio)methyl)butane-1-sulfonate (90 mg, 0.23 mmol) is dissolved in 2 mL of dichloromethane under argon atmosphere. K.sub.2CO.sub.3 (97 mg, 0.70 mmol) and methyl iodide (255 mg, 1.80 mmol) are added and the mixture is stirred at room temperature overnight. After removing the solvent under reduced pressure, the residue is purified by preparative HPLC to yield 78 mg (87%) of 1f as a white solid.

(26) .sup.1H-NMR (300 MHz, CDCl.sub.3): δ=0.81 (t, J=6.9, 3H), 0.92 (t, J=7.3, 3H), 1.1-1.35 (m, 22H), 1.50 (m, 2H), 1.66 (m, 2H), 2.40 (m, 1H), 2.75 (m, 1H), 2.91 (d/m, J=12.5, 1H), 3.10 (m, 1H), 3.12/3.16 (2s, 3H), 3.26 (m, 2H), 4.06 (d/m, J=12.9, 1H).

(27) MS (ESI): 392.4 (M+H.sup.+), 783.7 (2M+H.sup.+).

Examples 7, 8 and 9: Preparation of 3-(N,N-dimethyloctadecylammonio)propanesulfonate 1g, 3-(N,N-dimethylpalmitylammonio)propansulfonate 1h and 3-(N,N-dimethyldodecyl-ammonio)propanesulfonate 1i

(28) Compounds 1g, 1h and 1i are prepared in a similar way as described for compound 1b using N-methyloctadecylamine (for 1g), N-methylhexadecylamine (for 1h) or N-methyldodecylamine (for 1i) instead of N-methyltetradecylamine followed by quarternization of the nitrogen using methyl iodide as described in the final preparation step for compound 1d.

(29) Alternatively, compounds 1g, 1h and 1i are commercially available from Sigma-Aldrich GmbH, Munich, Germany (1g: product number 41570; 1h: product number H6883; 1i: product number D0431).

Example 10: N-Tetradecyl-N,N-dimethylglycine 1j

(30) Compound 1j is commercially available (Affymetrix, Santa Clara, Calif. 95051, USA, product number T305).

Example 11: Preparation of 3-(dimethyl(tetradecyl)ammonio)propanoate 1k

(31) Under argon atmosphere, β-propiolactone (216 mg, 3.0 mmol) is dissolved in a mixture of 4 mL of ether and 2 mL of acetonitrile. N,N-dimethyltetradecylamine (724 mg, 3.0 mmol) is added dropwise over a period of 2 h. The mixture is stirred for another 30 min and the product, a white precipitate, is collected by filtration, washed with several portions of ether and dried in vacuo, yielding 470 mg (50%) of 1k as a white powder. The product is stored below −15° C.

(32) .sup.1H-NMR (300 MHz, CDCl.sub.3): δ=0.81 (t, J=6.4, 3H), 1.1-1.35 (m, 22H), 1.65 (m, 2H), 2.55 (t, J=7.8, 2H), 3.13 (s, 6H), 3.20 (m, 2H), 3.69 (t, J=7.5, 2H).

(33) MS (ESI): 314.3 (M+H.sup.+), 627.5 (2M+H.sup.+).

Example 12: Preparation of 3-(dimethyl(dodecyl)ammonio)propanoate 1m

(34) Under argon atmosphere, β-propiolactone (216 mg, 3.0 mmol) is dissolved in a mixture of 4 mL of ether and 2 mL of acetonitrile. N,N-dimethyldodecylamine (639 mg, 3.0 mmol) is added dropwise over a period of 2 h. The mixture is stirred overnight and the product, a white precipitate, is collected by filtration, washed with several portions of ether and dried in vacuo, yielding 653 mg (76%) of 1 m as a white powder. The product is stored below −15° C.

(35) .sup.1H-NMR (300 MHz, CDCl.sub.3): δ=0.81 (t, J=6.4, 3H), 1.1-1.35 (m, 22H), 1.65 (m, 2H), 2.55 (t, J=7.8, 2H), 3.13 (s, 6H), 3.20 (m, 2H), 3.69 (t, J=7.5, 2H).

(36) MS (ESI): 314.3 (M+H.sup.+), 627.5 (2M+H.sup.+).

Example 13: Preparation of 4-(methyl(tetradecyl)ammonio)butanoate 1n

(37) To a solution of 681 mg (3.0 mmol) of N-methyl tetradecylamine in absolute dimethylformamide (DMF) (6 mL), are added 834 mg (6.0 mmol) of powdered K.sub.2CO.sub.3 and 1.34 g (6.0 mmol) of 4-bromobutyric acid. The mixture is stirred under argon atmosphere at 130° C. bath temperature for 3 d. The volatiles are removed on a rotary evaporator and the residue purified by preparative HPLC to yield 594 mg (41%) of 4-(methyl(tetradecyl)ammonio)butyric acid tert-butyl ester as a TFA salt.

(38) ESI-MS (pos.): 370.3 (M+H.sup.+).

(39) 4-(methyl(tetradecyl)ammonio)butyric acid tert-butyl ester (241 mg, 0.5 mmol) is suspended in 3 mL of TFA/H.sub.2O (95:5). The mixture is stirred for 1 h at room temperature, the volatiles removed on a rotary evaporator and the residue purified by preparative HPLC to yield 114 mg (36%) of 1n as a white solid.

(40) .sup.1H-NMR (300 MHz, CDCl.sub.3/CD.sub.3OD 8:2): δ=0.73 (t, J=6.9, 3H), 1.11 (m), 1.18 (m, Σ=22H), 1.56 (m, 2H), 1.85 (m, 2H), 2.29 (t, J=6.7, 2H), 2.66 (s, 3H), 2.89 (m), 2.97 (m, Σ=4H), 4.05 (br. s, 3H).

(41) MS (ESI): 314.2 (M+H.sup.+), 627.4 (2M+H.sup.+). Neg.: 312.0 (M−H.sup.−).

Example 14: Preparation of 4-(dimethyl(tetradecyl)ammonio)butanoate 1o

(42) Under argon atmosphere, 4-(methyl(tetradecyl)ammonio)butyric acid tert-butyl ester, TFA salt (described for 1n) (338 mg, 0.70 mmol) is dissolved in acetone (5 ml). Dry powdered K.sub.2CO.sub.3 (486 mg, 3.5 mmol) and methyl iodide (497 mg, 3.5 mmol) are added and the mixture stirred overnight. The product is purified by preparative HPLC and dried in high vacuum, yielding 266 mg (53%) of 4-(dimethyl(tetradecyl)ammonio)butyric acid tert-butyl ester, TFA salt, 100% pure by HPLC. This material is stirred with 3 ml of TFA/H.sub.2O (95:5) for 1 h. HPLC indicated complete conversion. The volatiles are removed on a rotary evaporator and the residue purified by preparative HPLC and dried in high vacuum to yield 105 mg (60%) of 1o.

(43) .sup.1H-NMR (300 MHz, CDCl.sub.3): δ=0.86 (t, J=6.4, 3H), 1.15-1.35 (m, 22H), 1.69 (m, 2H), 1.98 (m, 2H), 2.41 (t, J=6.1, 2H), 3.06 (s, 6H), 3.17 (m, 2H), 3.34 (m, 2H).

(44) MS (ESI): 328.3 (M+H.sup.+), 655.6 (2M+H.sup.+).

Example 15: N-Dodecyl-N,N-dimethylglycine 1p

(45) Compound 1p is commercially available (Affymetrix, Santa Clara, Calif. 95051, USA, product number D350).

Example 16: Preparation of 2-(N-methyldodecylammonio)ethanesulfonate 1q

(46) Sodium-2-bromoethane sulfonate (411 mg, 1.95 mmol) and powdered K.sub.2CO.sub.3 (269 mg, 1.95 mmol) are suspended in 3 mL of dry DMF under argon atmosphere. N-methyldodecylamine (298 mg, 1.50 mmol) is added and the mixture stirred at 130° C. overnight. The volatiles are removed on a rotary evaporator and the residue purified by preparative HPLC and dried in high vacuum to yield 342 mg (74%) of 1q as a white solid.

(47) .sup.1H-NMR (300 MHz, CDCl.sub.3): δ=0.81 (t, J=6.4, 3H), 1.10-1.35 (m, 18H), 1.70 (m, 2H), 2.88 (s), 2.89 (s, Σ=3H), 3.0-3.45 (m, 5H), 3.55 (m, 1H), 8.46 (br. s, 1H), 8.89 (br. s, 1H).

(48) MS (ESI): 308.2 (M+H.sup.+), 325.3 (M+NH.sub.4.sup.+), 615.4 (2M+H.sup.+).

Example 17: Preparation of 2-(N,N-dimethyldodecylammonio)ethanesulfonate 1r

(49) A mixture of 2-(N-methyldodecylammonio)ethanesulfonate 1q (225 mg, 0.73 mmol), methyl iodide (568 mg, 4.0 mmol), potassium carbonate (207 mg, 1.5 mmol) and acetone (5 mL) is stirred under argon atmosphere for 2.5 d. The volatiles are removed and the residue is purified by preparative HPLC and dried in high vacuum to yield 161 mg (68%) of product 1r.

(50) .sup.1H-NMR (300 MHz, CDCl.sub.3/CD.sub.3OD 8:2): δ=0.80 (t, J=6.5, 3H), 1.18/1.29 (2m, Σ=18H), 1.68 (m, 2H), 3.02 (s, 6H), 3.20 (m, 4H), 3.61 (m, 2H).

(51) MS (ESI): 322.2 (M+H.sup.+), 643.5 (2M+H.sup.+), 660.5 (2M+NH.sub.4.sup.+, 665.5 (2M+Na.sup.+).

Example 18: Preparation of 2-(N-methyltetradecylammonio)ethanesulfonate 1s

(52) A mixture of sodium-2-bromoethanesulfonate (411 mg, 1.95 mmol), N-methyltetradecylamine (342 mg, 1.50 mmol), K.sub.2CO.sub.3 (269 mg, 1.95 mmol) and DMF (3 mL) is stirred at 135° C. bath temperature under argon atmosphere overnight. The volatiles are removed on a rotary evaporator and the residue purified by preparative HPLC to yield 324 mg (64%) of 1s.

(53) .sup.1H-NMR (300 MHz, CDCl.sub.3): δ=0.90 (t, J=6.4, 3H), 1.15-1.45 (m, 22H), 1.78 (m, 2H), 2.97/2.99 (2s, Σ=3H), 3.05-3.55 (m, 5H), 3.64 (m, 1H), 7.36 (br. s, 1H).

(54) MS (ESI): 336.3 (M+H.sup.+), 671.5 (2M+H.sup.+), 693.5 (2M+Na.sup.+), 709.5 (2M+K.sup.+).

Example 19: Preparation of 3-(N,N-dimethyldodecylammonio)propylphosphonic acid 1t

(55) Under argon atmosphere, diethyl(3-bromopropyl)phosphonate (1.50 g, 5.75 mmol) is dissolved in 5 mL of absolute ether. N,N-dimethyldodecylamine (1.07 g, 5.00 mmol) is added and the mixture stirred overnight. The volatiles are removed and the residue dried in vacuo, resulting in solidification. The hygroscopic solid is broken up and triturated with ether and the ether removed by suction filtration. The residue is dried in high vacuum to yield 1.46 g (62%) of 3-(N,N-dimethyldodecylammonio)propylphosphonic acid diethylester (bromide salt) as a white, hygroscopic solid.

(56) .sup.1H-NMR (300 MHz, CDCl.sub.3): δ=0.81 (t, J=6.4, 3H), 1.19 (m), 1.27 (t, J=7.05, Σ=24H), 1.67 (m, 2H), 1.81 (d/t, J=18.1/6.8, 2H), 2.00 (m, 2H), 3.36 (s, 6H), 3.42 (m, 2H), 3.73 (m, 2H), 4.04 (m, 4H).

(57) MS (ESI): 392.4 (M+H.sup.+).

(58) 3-(N,N-dimethyldodecylammonio)propylphosphonic acid diethylester, bromide salt (175 mg, 0.37 mmol) is placed under argon atmosphere. Absolute dichloromethane (3 mL), bromotrimethylsilane (233 μL, 1.8 mmol) and allyltrimethylsilane (143 μL, 0.9 mmol) are added and the mixture stirred at room temperature for 2.5 d. The volatiles are removed on a rotary evaporator and the residue purified by preparative HPLC to yield 106 mg (85%) of 1t.

(59) .sup.1H-NMR (300 MHz, CDCl.sub.3/CD.sub.3OD 8:2): δ=0.78 (t, J=6.3, 3H), 1.1-1.3 (m, 18H), 1.62 (m, 4H), 2.10 (m, 2H), 2.97 (s, 6H), 3.13 (m, 2H), 3.27 (m, 2H).

(60) MS (ESI): 336.2 (M+H.sup.+), 671.5 (2M+H.sup.+).

Example 20: Preparation of 3-(N-methyl-N-hexadecylamino)propylphosphonic acid diethylester hydrobromide 1w

(61) Under argon atmosphere, N-methylhexadecylamine (510 mg, 2.0 mmol) is partially dissolved in absolute ether (4 mL). Diethyl(3-bromopropyl)phosphonate (647 mg, 2.50 mmol) and diisopropylethylamine (436 μL, 2.5 mmol) are added and the mixture stirred overnight at room temperature. The volatiles are removed and the residue purified by flash chromatography on silica using dichloromethane/methanol 10:1 to yield 216 mg (21%) of 1w.

(62) .sup.1H-NMR (300 MHz, CDCl.sub.3): δ=0.81 (t, J=6.3, 3H), 1.19 (m), 1.25 T, J=6.9, Σ=32H), 1.40 (m, 2H), 1.72 (m, 4H), 2.18 (s, 3H), 2.31 (m, 2H), 2.39 (br. t, J=6.5, 2H), 4.02 (m.sub.c, 4H).

(63) MS (ESI): 434.4 (M+H.sup.+), 889.7 (2M+Na.sup.+).

Example 21: Preparation of 3-(N-methyl-N-hexadecylamino)propylphosphonic acid 1v

(64) Under argon atmosphere, a mixture of 3-(N-methyl-N-hexadecylamino)propylphosphonic acid diethylester hydrobromide (1w) (130 mg, 0.25 mmol), absolute dichloromethane (2 mL), bromotrimethylsilane (184 mg, 1.20 mmol) and allyltrimethylsilane (68 mg, 0.60 mmol) is stirred overnight at room temperature. The volatiles are removed and the residue purified by preparative HPLC to yield 52 mg (55%) of 1v.

(65) .sup.1H-NMR (300 MHz, CDCl.sub.3/CD.sub.3OD 8:2): δ=0.89 (t, J=6.5, 3H), 1.2-1.4 (m, 26H), 1.65-1.87 (m, 4H), 2.06 (m, 2H), 2.80 (s, 3H), 2.85-3.25 (br. m, 4H).

(66) MS (ESI): 378.3 (M+H.sup.+), 755.6 (2M+H.sup.+).

Example 22: Preparation of 3-(N-methyl-N-tetradecylamino)propylphosphonic acid diethylester hydrobromide 1x

(67) N-methyltetradecylamine (454 mg, 2.0 mmol) is dissolved in absolute ether (3 mL) under argon atmosphere. Diispropylethylamine (322 mg, 2.5 mmol) and diethyl(3-bromopropyl)phosphonate (647 mg, 2.5 mmol) are added and the mixture stirred for 2.5 d at room temperature. A precipitate is removed and the supernatant concentrated on a rotary evaporator and purified by flash chromatography on silica using dichloromethane/methanol (10:1) to yield 356 mg (26%) of 1x a white solid.

(68) .sup.1H-NMR (300 MHz, CDCl.sub.3): δ=0.81 (t, J=6.9, 3H), 1.19 (m), 1.25 (t, J=7.1, Σ=28H), 1.40 (m, 2H), 1.71 (m, 4H), 2.18 (s, 3H), 2.31 (m, 2H), 2.39 (br. t, J=7.2, 2H), 2.95 (br. s, 1H), 4.02 (m.sub.c, 4H).

(69) MS (ESI): 406.4 (M+H.sup.+), 833.6 (2M+Na.sup.+).

Example 23: Preparation of 3-(N-methyl-N-tetradecylamino)propylphosphonic acid (betaine) 1u

(70) 3-(N-methyl-N-tetradecylamino)propylphosphonic acid diethylester hydrobromide 1x (162 mg, 0.33 mmol) is dissolved in 2 mL of absolute dichloromethane under argon atmosphere. Trimethylsilyl bromide (245 mg, 1.6 mmol) and allyltrimethylsilane (91 mg, 0.8 mmol) are added and the mixture stirred overnight at room temperature. The volatiles are removed on a rotary evaporator and the residue dissolved in ethanol and purified by preparative HPLC to yield 105 mg (91%) of 1u as a white solid.

(71) .sup.1H-NMR (300 MHz, CDCl.sub.3): δ=0.81 (t, J=6.9, 3H), 1.1-1.3 (m, 20H), 1.61 (br.s, 2H), 1.75 (m, 2H), 2.00 (m, 2H), 2.73 (s, 3H), 2.86 (m), 3.02 (m), 3.14 (m, Σ=4H).

(72) MS (ESI): 350.3 (M+H.sup.+), 699.6 (2M+H.sup.+). Neg.: 697.3 (2M−H.sup.+).

Examples 24 and 25: Preparation of N-hexadecyl-N-(3-sulfonatopropyl)piperidinium 2a and 1-hexadecyl-1-(3-sulfonatopropyl)-4-hydroxypiperidinium 2b

(73) Piperidine (1.32 g, 15.5 mmol) and 1-iodohexadecane (1.82 g, 5.17 mmol) are dissolved in 6-8 mL of ethanol and stirred overnight while shielded from light. The mixture is partitioned between plenty of ether and dilute NaOH, washed with sat. NaCl once, dried over Na.sub.2SO.sub.4, rotavapped down to an oil and dried in vacuo. Yield: 1.45 g (90% of brownish oil). The product is used in the next step without further purification.

(74) In 4 mL of EtOAc, N-hexadecylpiperidine (300 mg, 0.97 mmol) is mixed with 1,3-propanesultone (142 mg, 1.63 mmol). The mixture is stirred first at room temperature (2 d), then at 50° C. overnight. After addition of another 142 mg of 1,3-propanesultone, the mixture is stirred at reflux for 2 days, at which point analytical HPLC indicates complete conversion. The volatiles are removed and the residue is purified by preparative HPLC to yield 298 mg (71%) of pure 2a as a white solid.

(75) .sup.1H-NMR (300 MHz, CDCl.sub.3): δ=0.81 (t, J=7.8, 3 H), 1.1-1.4 (m, 26H), 1.5-1.8 (m, 6H), 1.91 (m, 2H), 2.14 (m, 2H), 2.91 (t, J=6.2, 2H), 3.16 (m, 2H), 3.29 (m, 2H), 3.45 (M, 2H), 3.64 (m, 2H).

(76) MS (ESI): 432.4 (M+H.sup.+), 863.8 (2M+H.sup.+).

(77) Compound 2b is prepared in a similar way using 4-hydroxypiperidine instead of piperidine.

Example 26: Preparation of trans-N-Tetradecyl-N-(3-sulfonatopropyl)-3-diethylaminocarbonyl-piperidinium 2c

(78) A mixture of N,N-diethyl-3-piperidinecarboxamide (809 mg, 4.4 mmol), ethanol (3 mL) and tetradecyl iodide (620 mg, 1.9 mmol) is stirred overnight at room temperature. HPLC shows complete conversion. The mixture is diluted with ether (200 mL) and extracted with 1N NaOH (1×100 mL) and saturated NaCl (1×100 mL) and the organic layer dried over Na.sub.2SO.sub.4 and filtered. The volatiles are removed and the residue purified by flash chromatography on silica using dichloromethane/methanol (10:1). The product is dried in vacuo to yield 682 mg (94%) of 1-tetradecyl-3-diethylaminocarbonylpiperidine. The product contains residual N,N-diethyl-3-piperidinecarboxamide, which is removed in the next step.

(79) MS (ESI): 381.4 (M+H.sup.+).

(80) A mixture of 1-tetradecyl-3-diethylaminocarbonylpiperidine (380 mg, 1.0 mmol), ethyl acetate (3 mL) and 1,3-propanesultone (488 mg, 4.0 mmol) is stirred under argon atmosphere under reflux for 6 d. HPLC shows conversion is incomplete and the cis- and trans-stereoisomers give rise to two closely eluting peaks. The volatiles are removed and the residue purified by preparative HPLC. The two resulting fractions are purified again by preparative HPLC, yielding the racemic cis- and trans-stereoisomer in 95% purity, with a yield of 71 mg (14%) of trans-stereoisomer 2c and 54 mg (11%) of the cis-stereoisomer.

(81) .sup.1H-NMR (125 MHz, CDCl.sub.3): δ=0.81 (t, J=6.4, 3H), 1.03 (t, J=7.1, 3H), 1.10-1.35 (m, 25H), 1.50 (m, 1H), 1.60-1.90 (m, 3H), 1.90-2.20 (m, 3H), 2.20-2.45 (m, 1H), 2.85-3.15 (m, 5H), 3.15-3.55 (m, 7H), 3.60 (br. d, J=12.2, 1H), 3.75-3.95 (m, 3H).

(82) .sup.13C-NMR and DEPT (125.7 MHz, CDCl.sub.3): 12.73 (CH.sub.3), 14.09 (CH.sub.3), 14.74 (CH.sub.3), 18.15 (CH.sub.2), 18.88 (CH.sub.2), 21.60 (CH.sub.2), 22.66 (CH.sub.2), 25.91 (CH.sub.2), 26.37 (CH.sub.2), 28.99 (CH.sub.2), 29.32 (CH.sub.2), 29.38 (CH.sub.2), 29.52 (CH.sub.2), 29.60 (CH.sub.2), 29.62 (CH.sub.2), 31.88 (CH.sub.2), 32.92 (CH), 40.51 (CH.sub.2), 40.51 (CH.sub.2), 42.15 (CH.sub.2), 47.09 (CH.sub.2), 53.80 (CH.sub.2), 59.20 (CH.sub.2), 60.64 (CH.sub.2), 65.23 (CH.sub.2), 170.09 (CO).

(83) MS (ESI): 503.5 (M+H.sup.+).

Example 27: Inhibition of Mast Cell Degranulation

(84) Introduction

(85) Mast cells are key effector cells involved in allergic and inflammatory diseases, and the Rat Basophilic Leukemia clone 2H3 (RBL-2H3) cell line is a commonly used model of allergen dependent immune modulator release (degranulation) in mast cells. On their surface, they express the high affinity receptor for IgE (FcεRI). Upon binding of antigen-specific IgE to the receptor, cells become sensitized to the IgE specific antigen (allergen). When IgE-sensitized cells then encounter multivalent antigen, the antigen clusters IgE-FcεRI complexes and initiates a signal transduction cascade that leads to degranulation, that is, the release of inflammatory mediators, such as cytokines, eicosanoids, histamine and enzymes. The assay can be used as a screening method to identify immune-modulating compounds, in particular compounds useful in the medical management of allergic and inflammatory diseases and asthma. β-hexosaminidase was previously shown to be released with the same kinetics as histamine (Schwartz et al., J Immunology; 123:1445-1450 (1979)), thus offering a simple means to monitor degranulation. The RBL-2H3 cell line has been successfully used to identify compounds with anti-allergic activity (Choo et al. Planta Med., 69:518-522 (2003)).

(86) Materials and Methods

(87) Materials

(88) Chemicals: Rat anti-DNP IgE monoclonal antibody was acquired from Biozol (BZL06936), dinitrophenyl-conjugated human serum albumin (A6661) and Triton X-100 (T9284) were from Sigma-Aldrich, 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide (474502), Phorbol-12-myristate-13-acetate (524400) and thapsigargin (586005) from Calbiochem. Ionomycin (ALX-450-006) was purchased from Alexis Biochemicals. DMSO was from Merck (1.02950.0500) or Sigma-Aldrich (D2650). Cell culture media and supplements, Minimum Essential Medium (21090-022), Minimum Essential Medium without Phenol Red (51200-046), RPMI 1640 Medium (31870-025), L-Glutamine (25030-024) and 0.05% Trypsin-EDTA (25300-054), were obtained from Invitrogen. Fetal bovine serum (A15-151) was from PAA Laboratories. Other reagents were standard laboratory grade or better.

(89) Buffers and solutions: Phosphate buffered saline (PBS) and 1 M HEPES were provided by the in-house service facility. Tyrode's buffer (TyB) consisted of Minimum Essential Medium without Phenol Red supplemented with 2 mM L-glutamine and 20 mM HEPES. Lysis buffer consisted of 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA and 0.1% (w/v) Triton) X-100. DNP-HSA was dissolved to 1 mg/ml in water. MUG substrate solution consisted of 2.5 mM 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide in 0.05 M citrate, pH 4.5; stop solution was 0.1 M NaHCO.sub.3/0.1 M Na.sub.2CO.sub.3, pH 10.

(90) Consumables and equipment: For small-volume liquid handling procedures, Rainin LTS electronic pipettes were routinely used (Mettler-Toledo). Costar-Corning 24-well plates (3337) were centrifuged in an Eppendorf 5804 R centrifuge. A Heraeus B15 table top incubator was used for incubations at 37° C. under non-sterile conditions. Fluorescence was measured in black Nunc 96-well plates (237105) using a microplate reader (Tecan Safire) or FlexStation 3 (Molecular Devices) multi-mode plate reader. Cells were maintained in Hera Cell 240 CO.sub.2 incubators (Thermo Scientific). Serological pipettes (4487, 4488 and 4489) and cell culture flasks (431080) were from Corning-Costar, 1.5 and 2 ml microcentrifuge tubes (0030 120.086 and 0030 120.094) were from Eppendorf.

(91) Cell Culture: RBL-2H3 cells obtained from the German Collection of Microorganisms and Cell Cultures (ACC312) (Braunschweig, Germany) were maintained in 70% Minimum Essential Medium with Earle's Salts, 20% RPM 1640 Medium, 10% FBS and 2 mM L-glutamine in 95% air/5% CO.sub.2 at 37° C. and routinely checked for mycoplasma contamination. Cells were passaged every 3-4 days; after washing cells once with 35 ml PBS cells were incubated 8 min with 5 ml 0.05% Trypsin-EDTA solution at 37° C. Cells were removed from the incubator, 15 ml culture medium was added and cells were resuspended by repeated pipetting.

(92) Cell seeding: cells were harvested with Trypsin-EDTA as described and 50-100 μl cell suspension seeded into Costar CellBind 24 well cluster plates (no. 3337). Plates were kept for 30 min at RT under the sterile hood before being transferred to the incubator. Cells were used within one or two days after seeding.

(93) Measurement of β-Hexosaminidase Release

(94) Experimental Procedures

(95) For sensitization, cells for immediate use were sensitized 6-12 h after plating; cells to be used the following day were sensitized 26-38 h after plating. Culture plates were removed from the incubator and checked for cell growth and contamination. The medium was discarded and cells were sensitized with anti-DNP IgE (0.4 μg/ml) in 0.4 ml culture medium overnight. Following overnight sensitization, cells were washed with 0.8 ml pre-warmed TyB and 0.38 ml test compound or vehicle control (supplemented or not with 1% FBS) were added to duplicate wells. Samples were adjusted to contain 1% vehicle for test compounds dissolved in organic solvents. Cells were incubated for 1 h at 37° C. At the end of the incubation period, cells were routinely stimulated with 20 μl DNP-HSA (2 μg/ml; final concentration 0.1 μg/ml) diluted in TyB and cells were incubated for 15 min at 37° C. Alternatively, cells were stimulated with 20 μl 5 μM ionomycin (final concentration 0.25 μM) or 20 μl 5 μM thapsigargin (final concentration 0.25 μM), both in the absence or presence of 20 nM PMA (final concentration).

(96) Plates were removed from the incubator and immediately centrifuged at 4° C. for 5 min at 250×g and transferred to an ice bath. Aliquots of supernatants, 25 μl, were transferred to 96-well plates. Remaining supernatant was aspirated from control wells and cells were lysed in 400 μl lysis buffer for 5 min at RT on an orbital shaker at 450 rpm under non-sterile conditions. After lysis, 25 μl aliquots of lysates were transferred to 96-well plates. MUG substrate solution, 100 μl, were added to supernatant and lysate samples and plates were incubated 30 min at 37° C. The reaction was terminated by addition of 150 μl stop solution. Fluorescence was measured at 365 nm excitation and 440 nm emission wavelengths.

(97) Test compound preparation: test compounds were prepared in 1.5 or 2 ml microcentrifuge tubes and incubated for 30 min at 37° C. in a Thermomixer Comfort with agitation (750 rpm). An electronic multichannel pipette was used for rapid transfer of compound dilutions from microcentrifuge tubes to the cells.

(98) Controls: controls used are defined as follows: negative control, supernatant of unstimulated cells was measured for unspecific β-hexosaminidase release; positive control, supernatant of DNP-HSA stimulated cells was measured for specific, antigen-stimulated β-hexosaminidase release; maximum control, lysate of unstimulated cells was measured for total β-hexosaminidase content.

(99) Assessment of Pharmacologic Effect

(100) Degranulation (β-hexosaminidase release): Degranulation was calculated as the percentage of β-hexosaminidase released with respect to maximum control (total β-hexosaminidase) after subtraction of negative control (unspecific release) using the formula;
% Degranulation=100×(test compound−negative control)/(maximum control−negative control).

(101) Inhibition of degranulation (inhibition of β-hexosaminidase release): Inhibition of degranulation was calculated as percent reduction of β-hexosaminidase release with respect to positive control (antigen-stimulated release) after subtraction of negative control (unspecific release) using the formula;
% Inhibition=100×(1−(test compound−negative control)/(positive control−negative control)).
Measurement of Maximum Tolerated Concentration

(102) The maximum tolerated concentration (MTC), i.e. the highest concentration of test compound that does not cause cytotoxicity, as determined by the release of lactate dehydrogenase, was measured over the tested concentration range. A commercially available cytotoxicity test was used (Promega Cytotox-One cat. #67891).

(103) The safety index (SI) of a test compound is the ratio between the maximum tolerated concentration and the IC50 and is used as a measure of the relative safety of the test compound.

(104) Results

(105) Concentration-dependent inhibition of degranulation was determined for all test compounds over a concentration range, as shown in FIG. 1, and IC50 values (concentration at which 50% of maximal inhibition is reached) were determined for each compound together with the MTC values over the same concentration range (Table 1). Results are taken from at least three independent experiments.

(106) TABLE-US-00001 TABLE 1 Inhibition of degranulation: IC50, MTC and SI values Compound IC50 (μM) MTC (μM) SI 1a 3.2 100 31.3 1b 4.3 100 23.3 1c 2.8 200 71.4 1d 3.9 100 25.6 1e 3.7 150 40.5 1f 4.0 75 18.8 1g 9.5 50 5.3 1h 3.7 50 13.5 1i 4.1 200 48.8 1j 5.1 75 14.7 1k 5.3 100 18.9 1m 73% 100 — inhibition at 25 μM 1n 8.0 100 12.5 1o 65% 100 — inhibition at 25 μM 1p 50% 100 — inhibition at 25 μM 1q 8.8 200 22.7 1r 4.2 200 47.6 1s 5.6 100 17.9 1t 6.1 200 32.8 1u 5.9 200 33.9 1v 8.8 200 22.7 1w 6.9 200 29.0 1x 5.5 100 18.2 2a 4.5 50 11.1 2b 4.3 50 11.6 2c 50% 100 — inhibition at 25 μM Miltefosine 4.2 25 6.0

(107) The MTC of the test compounds was 5-70 fold higher than their respective IC50s and hence, the inhibition of degranulation can be ascribed to a pharmacological effect and not to an effect secondary to cytotoxicity.

(108) All substances outlined in Table 1 show IC50 values in the low micromolar range combined with high MTC values when compared to Miltefosine. Thus, the compounds according to the invention and, in particular compounds 1a to 1x and compounds 2a to 2c, have an advantageously low cytotoxicity.

(109) Mast cell degranulation is a key cellular event in allergic and inflammatory reactions, in particular in pathological events involving the release of mediators such as histamine, leukotrienes and prostaglandins as well as proteases. As consequence, the inhibition of mast cell degranulation is a valuable strategy for prevention or treatment of pathological processes involving the aforementioned mediators. Furthermore, the mast cell degranulation assay provides an estimate of the activity of test compounds in other cells that play a key role in the inflammatory response, such as granulocytes, macrophages and thymocytes, which release proinflammatory cytokines and chemokines and tissue eroding proteases.

Example 28: Inhibition of Activation of Akt Kinase

(110) Introduction

(111) The mast cell degranulation assay using the RBL-2H3 cell line (see Example 27) was also used to determine the status of the PI3K/Akt axis. Activation of PI3K leads to production of PIP3 on the cytosolic side of the lipid bilayer. Akt is recruited to the PIP3 domain and subsequently activated by phosphorylation on residues Ser473 and Thr308. (Franke et al., Cell 81:727-736, (1995)). Once recruited to the membrane, it is phosphorylated and activated by other kinases (Hemmings, Science 275:628-630 (1997); Hemmings, Science 276:534 (1997); Downward, Science 279:673-674 (1998); Alessi et al., EMBO J. 15:6541-6551 (1996)). Western blotting of the phosphorylated Ser473 residue on Akt (phospho-Akt Ser473) is widely used to assess the level of activation of the PI3K/Akt axis.

(112) Materials and Methods

(113) Materials

(114) All buffers and solutions used for the phosphor-Akt Ser473 assay were from Meso Scale Discovery. Tris Lysis Buffer consisted of 150 mM NaCl, 20 mM Tris, pH 7.5, 1 mM EDTA, 1 mM EGTA and 1% Triton-X-100. Complete Tris Lysis Buffer was prepared prior to use by addition of protease inhibitor, phosphatase inhibitors and PMSF. The 10× Tris Wash Buffer consisted of 500 mM Tris, pH 7.5, 1.5 M NaCl and 0.2% Tween-20. Blocker A was made up of bovine serum albumin in Tris Wash Buffer. Read Buffer T was used according to manufacturer's instructions. The Whole Cell Lysate Kits used were phospho-Akt Ser473 (K11100D, Lot K0011749) and total ERK1/2 (K11107D, Lot K0011698) as a loading control.

(115) Equipment

(116) 12-well multichannel pipettes (30-300 μl) from Eppendorf were used. Assay plates were agitated on a TiMix 5 control (Edmund Bühler). Electrochemiluminescence detection was performed on a SECTOR Imager 6000 (Meso Scale Discovery).

(117) Measurement of Phospho-Akt Ser473

(118) Experimental Procedures

(119) Protein assay: protein concentration was determined using the BCA (bicinchoninic acid) Protein Assay kit according to the manufacturer's instructions. Briefly, duplicate 10 μl samples of bovine serum albumin (BSA) standards, blank and lysates were incubated in a 96-well plate with 0.2 ml working reagent for 30 min at 37° C. Plates cooled to room temperature for 5 min and absorbance at 562 nm measured in a multi-mode plate reader. Protein concentrations were calculated using FlexStation 3 software (SoftMax Pro version 5.3). Protein concentration of lysates was determined from a standard curve (BSA) using a linear curve fit.

(120) Phosphoprotein assay: protein phosphorylation was determined using the MULTI-SPOT® Assay System (Meso Scale Discovery), providing simultaneous detection of phosphorylated and total proteins. Briefly, capture antibodies against phosphorylated and total protein are patterned on distinct spots in the same well of 96-well plates. Sandwich immunoassay and electrochemiluminescence detection technology are combined to measure intensity of the emitted light from phosphorylated and total protein spots. For analysis of phosphor-Akt Ser473 was performed according to the manufacturer's instructions. The optimal amount of protein was determined at 5 μg lysate per well for ERK1/2 and 10 μg/well for phospho-Akt Ser473. Plates were blocked with 25 μl/well Blocker A for 1 h at room temperature with gentle agitation. During this time, the lysates were thawed and diluted to the desired protein concentration in complete Tris Lysis Buffer. Plates were washed four times in Tris Wash Buffer and 25 μl lysate per well added. Plates were incubated for 1-3 h at room temperature with agitation according to the manufacturer's recommendations. Plates were washed four times with Tris Wash Buffer, followed by addition of 25 μl/well of the respective detection antibody and incubation for 1 h at room temperature, with agitation. After a final four washes with Tris Wash Buffer 150 μl/well, Read Buffer T was added. and plates read on a SECTOR Imager 6000 plate reader.

(121) Assessment of Effects of Phospho-Akt Ser473

(122) The mean background signal from each plate was subtracted from averaged raw data. The amount of total protein phosphorylated was expressed as % phosphoprotein according to the manufacturer's (Meso Scale Discovery) instructions.

(123) Results

(124) Levels of phospho-Akt Ser473 were determined in IgE sensitized and antigen stimulated cells after treatment without (positive control) or with 1, 5 and 25 μM test compound and normalized to levels of total Akt. Concentration-dependent inhibition of Akt phosphorylation on Ser473 was demonstrated, as shown in FIG. 2. Table 2 shows levels of normalized phospho-Akt Ser473 as a percentage of those in the positive control.

(125) TABLE-US-00002 TABLE 2 Inhibition of Akt phosphorylation on Ser473 by compounds 1a and 1c Level of phospho-Akt Ser473 (% positive contol) Compound 1 μM 5 μm 25 μM 1a 103.1 ± 17.4 57.0 ± 15.2 13.2 ± 4.2 1c  83.2 ± 17.3 11.7 ± 7.7   2.2 ± 1.4 Percentage of total Akt phosphorylated on Ser473 expressed as percentage of control untreated cells, after induction with IgE and antigen for 15 min.

(126) A dose-dependent decrease in levels of phospho-Akt Ser473 was observed after treatment with all compounds outlined in Table 2. Thus, the compounds according to the invention can be used to reduce levels of activated Akt and, accordingly, are useful in the medical intervention in indications in which hyperactivated Akt plays a pathogenic role, such as inflammatory and allergic diseases.

Example 29: Inhibition of the Delayed-Type Hypersensitivity (DTH) Reaction in Mice

(127) Introduction

(128) The anti-inflammatory and anti-allergic effects of compound 1a was assessed in a mouse model of skin delayed-type hypersensitivity (DTH) reactions and compared to a vehicle control and to the reference drug dexamethasone. DTH reactions are antigen-specific cell-mediated immune responses, driven primarily by T helper type 1 (Th1) cells, similar to the tuberculin immunization response. The immune reaction induced by an ovalbumin challenge to animals previously sensitized with ovalbumin in Complete Freund's Adjuvant, is characterized by swelling (edema) at the site of challenge, e.g. the mouse ear. Dexamethasone, an anti-inflammatory steroid, reduces cell-mediated immune responses and was employed to validate the responsiveness of the assay to pharmacological treatment.

(129) Materials and Methods

(130) Materials

(131) Ovalbumin (fraction V, lyophilized powder), complete Freud's adjuvant (CFA) and methylcellulose were obtained from Sigma-Aldrich, dexamethasone from Pharmaceutical Works Polfa (Pabianice, Poland).

(132) Animals

(133) Female BALB/cJW mice were bred at the University of Lodz, Lodz, Poland and housed in groups of 8 in makrolon cages with a 12 h light-dark cycle. Mice were given free access to food (Agropol S.j., Motycz, Poland) and water.

(134) Antigen Sensitization and Challenge

(135) Group size was n=8 mice unless otherwise stated. Test compound was freshly prepared before administration.

(136) Sensitization: The protein antigen, ovalbumin, was reconstituted in PBS at 4 mg/ml. An ovalbumin-CFA emulsion was prepared by mixing the protein solution with the CFA suspension at a ratio of 1:1, using two Luer-lock syringes. The emulsion was tested by putting a drop of emulsion onto PBS; if the emulsion remained as a tight droplet on the PBS, the emulsion was deemed ready. Mice were sensitized by subcutaneously injecting 25 μL of emulsion into each side of the tail (100 μg ovalbumin per mouse).

(137) Challenge: On the sixth day after sensitization, DTH was elicited by challenging animals subcutaneously (gauge 30 needle, B. Braun Melsungen, Melsungen, Germany) in the left ears with 10 μL of a 1% suspension of heat-aggregated ovalbumin (HOVA) (100 μg ovalbumin per mouse). The right ears were administered subcutaneously with PBS and served to determine the individual differences in ear thicknesses. HOVA was prepared by heating a 5% solution of ovalbumin in saline for 1 h at 80° C. with occasional swirling. After cooling to room temperature and centrifugation (400 g, 10 min at 4° C.), the pellet was washed twice with saline, resuspended at 2% in PBS and aliquots stored at −30° C. Before injection, HOVA was diluted with an equal volume of PBS and sonicated. Ear thickness was measured with a precise spring-loaded caliper (Arta No. 7309, Mitutoyo, Kawasaki, Japan) before challenge, and 24 h after challenge.

(138) Sensitization, challenge and ear thickness measurement were performed under anesthesia (ketamine 80 mg/kg plus xylazine 8 mg/kg, intraperitoneally).

(139) Compound Administration

(140) The anti-inflammatory effects of compound 1a were compared to a vehicle control (0.5% methyl cellulose solution) and to the reference drug, dexamethasone. Test compound was given orally by gavage (Art. No. 432093, Harvard Apparatus GmbH, March-Hugstetten, Germany) as follows: 16 h prior to ovalbumin sensitization, a loading dose of 100 mg/kg was administered; the first maintenance dose of 25 mg/kg was given 3 h before sensitization (day 0) and on each of the next five consecutive days (day 1 to 5) as well as on the day of antigen challenge (day 6) (a total of 8 administrations). Three hours after the last dose, the antigen challenge was performed on the ears as described above. Dexamethasone was given at 1 mg/kg orally by gavage 3 h before sensitization and once daily with the final dose given 3 h prior to antigen challenge (a total of 7 administrations). All administrations were given in a volume of 10 mL/kg.

(141) Quantification of Assay Results

(142) To account for individual variability, the increase in right ear thickness, before and 24 h after administration of PBS, was subtracted from the HOVA-induced increase in left ear thickness. The increase in ear thickness was calculated by the difference between ear thickness before and 24 h after antigen challenge. Percent inhibition of ear swelling was calculated according to the following formula:
% inhibition=100×(IET.sub.vehicle−IET.sub.compound)/IET.sub.vehicle where IET=(ET.sub.24 hrs pc−ET.sub.predose).sub.HOVA-treated ears−(ET.sub.24 hrs pc−(ET.sub.predose).sub.PBS-treated ears (IET, increase ear thickness; ET, ear thickness; pc, post challenge)
Statistical Evaluation

(143) Mean and standard deviation (SD) were calculated from individual ear edema values. Statistical evaluation was a one-way analysis of variance (ANOVA) with Dunnett's post hoc test or Student's t-test where appropriate.

(144) Results

(145) Suppression of mouse ear swelling by compound 1a and dexamethasone, compared to vehicle control is shown in FIG. 3. Table 3 summarizes the inhibition of DTH for compounds 1a.

(146) TABLE-US-00003 TABLE 3 Effect of compound 1a on ear swelling in the DTH response in mice. Inhibition of Compound mouse ear swelling 1a, 100 mg/kg 32* Dexamethasone, 1.0 mg/kg 49* *p < 0.01 vs. vehicle control (Dunnett's post hoc test)

(147) Dexamethasone administered orally at a dose of 1 mg/kg, once daily over the whole sensitization period resulted in as significantly reduced DTH with inhibition of 49%. Such high dosing (overdose) is, however, not suitable for treatment of humans due to severe side effects of the corticosteroid and was only used to validate the responsiveness of the model. In addition, in the course of the current study, administration of dexamethasone resulted in a significant loss in body weight of 4.4% (p<0.01 vs. vehicle control with the paired Student's t-test), a typical sign of corticosteroid toxicity. In contrast, no toxic side-effects of compound 1a were observed during the course of the study.

(148) Compound 1a, administered orally twice daily over the whole sensitization period at 20 mg/kg (loading dose 100 mg/kg), significantly reduced the DTH response by 32%. Hence, compound 1a was able to produce an inhibition equivalent to 65% that of a high dose of dexamethasone.

(149) The reduction of DTH response obtained by treatment with compound 1a demonstrates that the compounds according to the invention and, in particular compound 1a, are effective in the pharmaceutical intervention in allergic and inflammatory diseases involving antigen-specific cell-mediated immune responses.

Example 30: Inhibition of the Allergic Contact Dermatitis Inflammatory Response in Mice

(150) Introduction

(151) The anti-inflammatory and anti-allergic effects of compound 1a were assessed in a mouse model of allergic contact dermatitis, a response driven primarily by T helper type 2 (Th2) cells. It has been demonstrated that BALB/c mice are susceptible to the allergen toluene-2,4-diisocyanate (TDI), producing an inflammatory condition of the skin with similar aspects to that of human atopic dermatitis (Baumer et al., J Pharm Pharmacol, 55:1107-1114 (2003); Baumer et al., Br J Dermatol. 151:823-830 (2004); Ehinger et al., Eur J Pharmacol. 392:93-99 (2000)). In this model, an allergic dermatitis response is obtained by sensitizing mice to TDI and subsequently challenging them with antigen by topical administration onto the ears. A quantitative assessment of anti-inflammatory and anti-allergic effects of topically or orally administered test compounds is possible by measuring the resulting ear swelling.

(152) The advantages of the allergic contact dermatitis model (Zöliner et al., Bioessays 26:693-6 (2004)) are reproducibility and reliability (>90% of BALB/c mice respond to sensitization), a short induction protocol, quantitative assessment by measuring ear thickness, atopic dermatitis-like skin lesions can be induced, and clinically relevant pharmaceuticals, such as corticosteroids, calcineurin-inhibitors and PDE4-inhibitors, are effective in this model.

(153) Materials and Methods

(154) Animals

(155) Female BALB/c-mice were obtained from Charles River (Sulzfeld, Germany) at age 8 weeks. All animals were housed in groups of eight per cage at 22° C. with a 12 h light/dark-cycle. Water and a standard diet (Altromin, Lage/Lippe, Germany) were available ad libitum. All animals were acclimatized for one week before experimental procedures were commenced.

(156) TDI Sensitization, Allergen Challenge and Mouse Ear Swelling Test

(157) Experimental procedures for BALB/c mice housing, TDI sensitization and challenge, and measurement of ear thickness were performed as previously described (Baumer et al., J Pharm Pharmacol. 55:1107-1114 (2003)) with the following modifications. For active sensitization, 100 μL of 5% (w/v) TDI was administered to the shaved and stripped abdominal epidermis on day one, and for the next three consecutive days, 50 μL of 5% (w/v) TDI was applied. The allergic reaction was boosted 21 days later by application of 50 μL of 0.5% (w/v) TDI. For the examination of test compound effects, the left ears were used for the TDI challenge (20 μL of 0.5% in acetone) and ear thickness measured 3 h before and 24 h after challenge.

(158) Compound Administration for Systemic Treatment

(159) Group size was n=7 mice unless otherwise stated. Test compounds were freshly prepared before administration.

(160) Administration time: to determine optimal time for administration treatment groups were treated orally by gavage with 100 mg/kg of compound 1a (suspended in phosphate-buffered saline (PBS), 10 mL/kg) 4 or 16 h before topical TDI challenge. Vehicle treated mice received PBS (10 mL/kg) orally, 4 h before challenge.

(161) Dose-response: two groups of mice were treated orally with compound 1a at 20 mg/kg or 100 mg/kg suspended in PBS, 4 h before topical TDI challenge. Vehicle treated mice received PBS orally 4 h before challenge.

(162) Compound Administration for Topical Treatment

(163) Compound 1a was administered to two groups of mice topically in 20 μl of a 2% or 6% solution in acetone/water (1:1). The solution was applied, 2 h before topical TDI challenge by administration of 10 μl onto each of the inner and outer surfaces of the left ears. A vehicle group (n=7) was treated with acetone/water (1:1).

(164) Determination of Local Lymph Node Weight and Cell Count

(165) Directly after sacrifice, the ear draining lymph node (Ln. auricularis) was prepared and excised. Organ weight was determined by means of an analytical balance (Kern, Balingen, Germany). Single cell suspensions were prepared by means of a glass potter (VWR, Darmstadt, Germany) and cells were counted with a hemocytometer (Neubauer, VWR, Germany).

(166) Statistical Evaluation

(167) Mean and standard error of the mean (SEM) were calculated from individual ear edema values. Statistical evaluation was a one-way analysis of variance (ANOVA) (if the test for normal distribution was passed) or the Kruskal-Wallis one-way ANOVA on Ranks (if the normal distribution test failed). Both were followed by a post-hoc test (Dunnett's method or Dunn's test, respectively). A p<0.05 was considered to be significant.

(168) Results

(169) Suppression of mouse ear swelling by compound 1a after oral administration, compared to vehicle control is shown in FIG. 4A. Table 4 summarizes inhibition of the allergic contact dermatitis response by compound 1a.

(170) TABLE-US-00004 TABLE 4 Effect of orally administered compound 1a on ear swelling in the allergic contact dermatitis response in mice. Inhibition of Compound mouse ear swelling Administration time (oral) 1a, 100 mg/kg, 4 h 51.7* 1a, 100 mg/kg, 16 h 32.2 *p < 0.05 vs. vehicle control (Dunnett's post hoc test) compared to vehicle

(171) In the administration time study with oral administration, compound 1a reduced ear swelling significantly (52% of vehicle control) when administered 4 h before challenge, as also shown in FIG. 4A.

(172) Compound 1a had a significant impact on the TDI induced inflammatory reaction in a pilot study at 100 mg/kg. Thus, the compounds according to the invention and, in particular compound 1a, are particularly effective and thus useful for the oral pharmaceutical intervention in inflammatory diseases, in particular in atopic dermatitis.

(173) Suppression of mouse ear swelling by compound 1a after topical administration, compared to vehicle control is shown in FIG. 4B. Table 5 summarizes inhibition of the allergic contact dermatitis response by compound 1a.

(174) TABLE-US-00005 TABLE 5 Effect of topically administered compound 1a on ear swelling in the allergic contact dermatitis response in mice. Inhibition of Compound mouse ear swelling 1a, 2% 72.0*** 1a, 6% 86.0*** ***p < 0.001 vs. vehicle control (Dunnett's post hoc test) compared to vehicle

(175) Compound 1a topically administered as a solution at 2% or 6% reduced ear swelling highly significantly by 72 or 86%, respectively.

(176) One of the most undesirable side-effects of corticosteroid administration is immunosuppression, which leads to the inability to effectively address parasitic infection, wound healing and tumor growth. In the current study, the local lymph node reaction after TDI challenge (lymph node weight and cell number) was determined to assess the response of immune organs. Systemic treatment with compound 1a at 100 mg/kg or topical treatment at 2% or 6% did not have any impact on the local lymph node reaction.

(177) In view of the strong effect shown in the allergic contact dermatitis model, the compounds of the present invention and, including compound 1a, are particularly effective and thus useful for the topical pharmaceutical intervention in inflammatory diseases, in particular in atopic dermatitis.