Oxygenated and amino- or ammonium-containing phosphonic acid derivatives and their medical use

Abstract

The present invention relates to oxygenated amino and 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, or a proliferative, neoplastic or dysplastic disease or disorder. ##STR00001##

Claims

1. A compound of formula 1 or a pharmaceutically acceptable salt, solvate or prodrug thereof, ##STR00012## wherein: R.sup.1 is a C.sub.10-20 hydrocarbon group; 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), OC(O)(C.sub.1-3 alkyl), C.sub.1-3 alkyl, NH.sub.2, NH(C.sub.1-3 alkyl) or N(C.sub.1-3 alkyl)(C.sub.1-3 alkyl); R.sup.4 is a C.sub.3-6 alkylene group which is substituted with one or more groups independently selected from OH, O(C.sub.1-3 alkyl), OC(O)(C.sub.1-3 alkyl), OC(O)O(C.sub.1-3 alkyl), OC(O)NH.sub.2, OC(O)NH(C.sub.1-3 alkyl), OC(O)N(C.sub.1-3 alkyl)(C.sub.1-3 alkyl), O(CH.sub.2).sub.2OH or O(CH.sub.2).sub.3OH; R.sup.5 is PO.sub.3H.sup., PO.sub.3.sup.2, PO.sub.3H.sub.2, PO.sub.2(OC.sub.1-3 alkyl), PO.sub.2H(OC.sub.1-3 alkyl), or PO(OC.sub.1-3 alkyl).sub.2; and X is N.sup.+.

2. The compound according to claim 1, wherein R.sup.5 is PO.sub.3.sup.2, PO.sub.3H.sup., or PO.sub.3H.sub.2.

3. The compound according to claim 1, wherein R.sup.4 is CH.sub.2CH(R.sup.41)CH.sub.2 and R.sup.41 is selected from OH, O(C.sub.1-3 alkyl), OC(O)(C.sub.1-3 alkyl), OC(O)O(C.sub.1-3 alkyl), OC(O)NH.sub.2, OC(O)NH(C.sub.1-3 alkyl), OC(O)N(C.sub.1-3 alkyl)(C.sub.1-3 alkyl), O(CH.sub.2).sub.2OH, or O(CH.sub.2).sub.3OH.

4. The compound according to claim 1, wherein R.sup.1 is a linear C.sub.10-20 alkyl group, a linear C.sub.10-20 alkenyl group, or a linear C.sub.10-20 alkynyl group.

5. The compound according to claim 1, wherein R.sup.1 is (CH.sub.2).sub.11CH.sub.3, (CH.sub.2).sub.13CH.sub.3, or (CH.sub.2).sub.15CH.sub.3.

6. The compound of claim 1, wherein the compound is a compound of formula 2 ##STR00013## wherein: R.sup.1 is a C.sub.10-20 hydrocarbon group; R.sup.4 is a C.sub.3-6 alkylene group which is substituted with one or more groups independently selected from OH, O(C.sub.1-3 alkyl), OC(O)(C.sub.1-3 alkyl), OC(O)O(C.sub.1-3 alkyl), OC(O)NH.sub.2, OC(O)NH(C.sub.1-3 alkyl), OC(O)N(C.sub.1-3 alkyl)(C.sub.1-3 alkyl), O(CH.sub.2).sub.2OH or O(CH.sub.2).sub.3OH; R.sup.5 is PO.sub.3H.sup., PO.sub.3.sup.2, PO.sub.3H.sub.2, PO.sub.2(OC.sub.1-3 alkyl), PO.sub.2H(OC.sub.1-3 alkyl), or PO(OC.sub.1-3 alkyl).sub.2; n is 1 and m is 0, 1, 2, or 3; and each R.sup.6 is independently selected from OH, O(C.sub.1-3 alkyl), OC(O)(C.sub.1-3 alkyl), C.sub.1-3 alkyl, NH.sub.2, NH(C.sub.1-3 alkyl) or N(C.sub.1-3 alkyl)(C.sub.1-3 alkyl).

7. The compound of claim 1, wherein the compound is a compound of the following formula 2 ##STR00014## wherein: R.sup.1 is a C.sub.10-20 hydrocarbon group; R.sup.4 is a C.sub.3-6 alkylene group which is substituted with one or more groups independently selected from OH, O(C.sub.1-3 alkyl), OC(O)(C.sub.1-3 alkyl), OC(O)O(C.sub.1-3 alkyl), OC(O)NH.sub.2, OC(O)NH(C.sub.1-3 alkyl), OC(O)N(C.sub.1-3 alkyl)(C.sub.1-3 alkyl), O(CH.sub.2).sub.2OH or O(CH.sub.2).sub.3OH; R.sup.5 is PO.sub.3H.sup., PO.sub.3.sup.2, PO.sub.3H.sub.2, PO.sub.2(OC.sub.1-3 alkyl), PO.sub.2H(OC.sub.1-3 alkyl), or PO(OC.sub.1-3 alkyl).sub.2; n is 2 and m is 0, 1, 2, or 3 and each R.sup.6 is independently selected from OH, O(C.sub.1-3 alkyl), OC(O)(C.sub.1-3 alkyl), C.sub.1-3 alkyl, NH.sub.2, NH(C.sub.1-3 alkyl) or N(C.sub.1-3 alkyl)(C.sub.1-3 alkyl).

8. The compound of claim 1, wherein the compound is a compound of the following formula 2 ##STR00015## wherein: R.sup.1 is a C.sub.10-20 hydrocarbon group; R.sup.4 is a C.sub.3-6 alkylene group which is substituted with one or more groups independently selected from OH, O(C.sub.1-3 alkyl), OC(O)(C.sub.1-3 alkyl), OC(O)O(C.sub.1-3 alkyl), OC(O)NH.sub.2, OC(O)NH(C.sub.1-3 alkyl), OC(O)N(C.sub.1-3 alkyl)(C.sub.1-3 alkyl), O(CH.sub.2).sub.2OH or O(CH.sub.2).sub.3OH; R.sup.5 is PO.sub.3H.sup., PO.sub.3.sup.2, PO.sub.3H.sub.2, PO.sub.2(OC.sub.1-3 alkyl), PO.sub.2H(OC.sub.1-3 alkyl), or PO(OC.sub.1-3 alkyl).sub.2; n is 3 and m is 0, 1, 2, or 3; and each R.sup.6 is independently selected from OH, O(C.sub.1-3 alkyl), OC(O)(C.sub.1-3 alkyl), C.sub.1-3 alkyl, NH.sub.2, NH(C.sub.1-3 alkyl) or N(C.sub.1-3 alkyl)(C.sub.1-3 alkyl).

9. A pharmaceutical composition comprising the compound of claim 1 and a pharmaceutically acceptable excipient.

10. A method of treating or ameliorating a disorder in a subject in need thereof, the method comprising administering the compound of claim 1 to the subject, wherein said disorder is selected from: psoriasis, atopic dermatitis, contact dermatitis, xerotic eczema, seborrheic dermatitis, neurodermitis, dyshidrosis, discoid eczema, venous eczema, dermatitis herpetiformis, autoeczematization, dermatomyositis, hyper-IgE syndrome, Wiskott-Aldrich syndrome, anaphylaxis, food allergy, allergic reactions to venomous stings, acute urticarias, chronic urticarias, physical urticarias including aquagenic urticaria, cholinergic urticaria, 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, Behget's syndrome, indeterminate colitis, celiac disease, irritable bowel syndrome, post-operative ileus, eosinophilic gastroenteropathy, gastritis, chronic allergic rhinitis, seasonal allergic rhinitis, allergic conjunctivitis, chemical conjunctivitis, neonatal conjunctivitis, Sjogren syndrome, open-angle glaucoma, dry eye disease, diabetic macular edema, chronic obstructive pulmonary disease, allergic asthma, allergic bronchopulmonary aspergillosis, hypersensitivity pneumonitis, lung fibrosis, rheumatoid arthritis, juvenile rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, scleroderma, reactive arthritis, polymyalgia rheumatica, Guillain-Barre syndrome, Hashimoto's thyroiditis, Grave's disease, temporal arteritis, primary biliary cirrhosis, sclerosing cholangitis, autoimmune hepatitis, alopecia areata, a graft-versus-host disease, a host-versus-graft disease, or a transplant rejection.

11. The method of claim 10, wherein said disorder is selected from psoriasis, atopic dermatitis, contact dermatitis, xerotic eczema, seborrheic dermatitis, neurodermitis, dyshidrosis, discoid eczema, venous eczema, dermatitis herpetiformis, autoeczematization, dermatomyositis, hyper-IgE syndrome, Wiskott-Aldrich syndrome, anaphylaxis, food allergy, or allergic reactions to venomous stings.

12. The method of claim 10, wherein said disorder is selected from acute urticarias, chronic urticarias, physical urticarias including aquagenic urticaria, cholinergic urticaria, cold urticaria, delayed pressure urticaria, dermatographic urticaria, heat urticaria, solar urticaria, vibration urticaria, adrenergic urticaria, or urticaria angioedema.

13. The method of claim 10, wherein said disorder is selected from inflammatory bowel disease, Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, diversion colitis, Behget's syndrome, indeterminate colitis, celiac disease, irritable bowel syndrome, post-operative ileus, eosinophilic gastroenteropathy, or gastritis.

14. The method of claim 10, wherein said disorder is selected from chronic allergic rhinitis, seasonal allergic rhinitis, allergic conjunctivitis, chemical conjunctivitis, neonatal conjunctivitis, Sjogren syndrome, open-angle glaucoma, dry eye disease, or diabetic macular edema.

15. The method of claim 10, wherein said disorder is selected from chronic obstructive pulmonary disease, allergic asthma, allergic bronchopulmonary aspergillosis, hypersensitivity pneumonitis, or lung fibrosis.

16. The method of claim 10, wherein said disorder is selected from rheumatoid arthritis, juvenile rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, scleroderma, reactive arthritis, or polymyalgia rheumatica.

17. The method of claim 10, wherein said 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.

18. The method of claim 10, wherein said disorder is selected from a graft-versus-host disease, a host-versus-graft disease or a transplant rejection.

19. A method of treating or ameliorating a disorder in a subject in need thereof, the method comprising administering the pharmaceutical composition of claim 10 to the subject, wherein said disorder is selected from: psoriasis, atopic dermatitis, contact dermatitis, xerotic eczema, seborrheic dermatitis, neurodermitis, dyshidrosis, discoid eczema, venous eczema, dermatitis herpetiformis, autoeczematization, dermatomyositis, hyper-IgE syndrome, Wiskott-Aldrich syndrome, anaphylaxis, food allergy, allergic reactions to venomous stings, acute urticarias, chronic urticarias, physical urticarias including aquagenic urticaria, cholinergic urticaria, 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, Behcee s syndrome, indeterminate colitis, celiac disease, irritable bowel syndrome, post-operative ileus, eosinophilic gastroenteropathy, gastritis, chronic allergic rhinitis, seasonal allergic rhinitis, allergic conjunctivitis, chemical conjunctivitis, neonatal conjunctivitis, Sjogren syndrome, open-angle glaucoma, dry eye disease, diabetic macular edema, chronic obstructive pulmonary disease, allergic asthma, allergic bronchopulmonary aspergillosis, hypersensitivity pneumonitis, lung fibrosis, rheumatoid arthritis, juvenile rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, scleroderma, reactive arthritis, polymyalgia rheumatica, Guillain-Barre syndrome, Hashimoto's thyroiditis, Grave's disease, temporal arteritis, primary biliary cirrhosis, sclerosing cholangitis, autoimmune hepatitis, alopecia areata, a graft-versus-host disease, a host-versus-graft disease, or a transplant rejection.

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) and miltefosine (FIG. 1F). Dose-response curves for inhibition of -hexosaminidase release from RBL-2H3 cells stimulated with antigen-specific IgE and triggered with antigen are shown (meansstandard 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 meansstandard deviation).

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

(5) FIG. 4: Effect of compounds 1a and 1c on mouse ear swelling in the allergic contact dermatitis model in mice. FIG. 4A shows a comparison of the inhibitory activity of compounds 1a and 1c and miltefosine at different administration times before antigen challenge (data are meansSEM of 7 mice; **p<0.01, ***p<0.001 vs. vehicle control (Dunnett's post hoc test), p<0.05 vs. vehicle control (t-test)). FIGS. 4B and 4C show a comparison of the inhibitory activity of compound 1a and corticosteroids after systemic (oral) application (FIG. 4B) or topical application (FIG. 4C) (data are meansSEM of 7 mice; **p<0.01, ***p<0.001 vs. vehicle control (Dunnett's post hoc test)). FIGS. 4D and 4E show the local lymph node reaction, comparing the effect of compound 1a and topical diflorasone on local lymph node weight (FIG. 4D) and cell number (FIG. 4E) (data are meansSEM of 7 mice; **p<0.01, ***p<0.001 vs. vehicle control (Dunnett's post hoc test)).

(6) FIG. 5: Effect of compound 1a on collagen type II-induced arthritis (CIA) in mice. FIG. 5A shows the effects on arthritis score during the course of type II CIA of compound 1a and dexamethasone using a prophylactic regimen (data are means of 10-11 mice; *p<0.02, **p<0.01, ***p<0.001 vs. vehicle control (t-test)). FIG. 5B shows the effects on body weight changes during the course of type II CIA of compound 1a and dexamethasone using a prophylactic regimen (data are means of 10-11 mice; *p<0.02, **p<0.01, ***p<0.001 vs. vehicle control (t-test)). FIG. 5C shows the effects on arthritis score during the course of type II CIA of compound 1a using a therapeutic regimen (data are means of 11 mice; *p<0.02 vs. vehicle control (t-test)). FIG. 5D shows the effects on spleen and thymus weight during the course of type II CIA of compound 1a and dexamethasone using a prophylactic regimen (data are means of 10-11 mice; ***p<0.001 vs. vehicle control (t-test)).

(7) FIG. 6: Mean plasma concentration-time profiles (semi-logarithmic) of five polymorphic forms of compound 1a in female Balb/c mice (n=3) following a single oral administration of 100 mg/kg in 0.5% methylcellulose solution.

(8) 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: Preparation of 2-hydroxy-3-(N-methylhexadecylammonio)propane-sulfonate 1a, 2-hydroxy-3-(N-methyltetradecylammonio)propanesulfonate 1b and 2-hydroxy-3-(N-methyldodecylammonio)propanesulfonate 1c

(9) Sodium-3-chloro-2-hydroxy-1-propanesulfonate (3.83 g, 19.5 mmol), N-methyldodecylamine (3 g, 15 mmol) and N,N-diisopropylethylamine (DIEA) (2.5 g, 19.5 mmol) are suspended in 20 mL of dry dimethylformamide (DMF) and heated to 130 C. under argon atmosphere for 24 h. The volatiles are removed under reduced pressure and the residue is chromatographed on silica using dichloromethane/methanol using a stepwise increase of eluent strength from 9:1 to 4:1. Compound 1c is obtained as white material (4.61 g, 91%).

(10) MS (ESI): 338.2 (M+H.sup.+), 675.5 (2M+H.sup.+), 697.5 (2MNa.sup.+).

(11) .sup.1H-NNMR (300 MHz, CDCl.sub.3): =0.81 (t, J=6.9, 3H), 1.1-1.35 (m, 18H), 1.68 (m, 2H), 2.89 (d/d, J=4.5/13.1, 3H), 2.95-3.45 (m, 6H), 4.52 (m, 1H), 8.90 (br. s, 1H), 9.17 (br. s. 1H).

(12) Compounds 1a and 1b are prepared in a similar way using N-methylhexadecylanine (for 1a) or N-methyltetradecylamine (for 1b) instead of N-methyldodecylamine.

Examples 2 and 3: Preparation of 2-methoxy-3-(N,N-dimethyl-N-tetradecylammonio)-propane-1-sulfonate 1d and 7-acetoxy-3-(N,N-dimethyltetradecylammonio)propanesulfonate 1e

(13) Compound 1b (440 mg, 1.2 mmol), methyl iodide (1.36 g, 9.6 mMol) and K.sub.2CO.sub.3 (497 mg, 3.6 mmol) are suspended in a mixture of acetone (10 mL) and dichloromethane (2 mL). The mixture is stirred at room temperature overnight. The volatiles are removed under reduced pressure and the residue is purified by preparative HPLC to yield 287 mg of 2-hydroxy-3-(N,N-dimethyl-N-tetradecylammonio) propane-1-sulfonate as a white solid.

(14) For the preparation of compound 1d sodium hydride (12 mg, 0.52 mmol) is suspended in dry THF (2 mL) under argon atmosphere. The aforementioned 2-hydroxy-3-(N,N-dimethyl-N-tetradecylammonio) propane-1-sulfonate (65 mg, 0.17 mmol) is dissolved in a mixture of DMF (1 mL) and THF (1 mL) and added dropwise. Methyl iodide (111 mg, 0.78 mmol) is added and the mixture is stirred at room temperature for 4 d. The mixture is quenched with 0.5 mL of methanol, the solvent is removed under reduced pressure and the residue is purified by preparative HPLC to yield 57 mg (85%) of 1d as white material.

(15) MS (ESI): 394.4 (M+H.sup.+), 787.7 (2M+H.sup.+).

(16) .sup.1H-NMR (300 MHz, CDCl.sub.3): =0.81 (t, J=6.9, 3H), 1.1-1.35 (m, 22H), 1.70 (m, 2H), 2.80 (m, 1H), 3.13 (s, 6H), 3.2-3.45 (m, 4H), 3.33 (s, 3H), 4.18 (m, 2H).

(17) For the preparation of compound 1e the aforementioned 2-hydroxy-3-(N,N-dimethyl-N-tetradecylammonio)propane-1-sulfonate (140 mg, 0.37 mmol) and 4-dimethylaminopyridine (DMAP) (3.4 mg, 0.028 mmol) are dissolved in dichloromethane (5 mL) under argon atmosphere. Acetic anhydride (32 mg, 0.31 mmol) and DIEA (40 mg, 0.31 mmol) are added and the mixture is stirred at room temperature for 24 h. The volatiles are removed under reduced pressure and the residue is purified by preparative HPLC to give 123 mg (79%) of 1e as a solid material.

(18) MS (ESI): 422.3 (M+H.sup.+), 843.6 (2M+H.sup.+).

(19) .sup.1H-NMR (300 MHz, CDCl.sub.3): =0.81 (t, J=6.9, 3H), 1.1-1.35 (m, 22H), 1.69 (m, 2H), 2.02 (s, 3H), 3.14 (d, J=5.5, 6H), 3.0-3.3 (m, 4H), 3.69 (m, 1H), 4.30 (d/m, J=14.4, 1H), 5.64 (m, 1H).

Example 4: Large Scale Synthesis of Compound 1a

N-methyl hexadecanamide

(20) A suspension of 1775 g of palmitic acid (6.92 mol) and 12.5 L of toluene was stirred at 20-25 C. and 1245 g (1.5 eq., 10.38 mol) thionyl-chloride added. The reaction mixture was heated under reflux for 12 h, then cooled to 20-25 C. and evaporated to dryness on a rotary evaporator under vacuum (bath temperature, 70 C.). The yield of crude palmitoyl chloride was 2475 g.

(21) The crude palmitoyl chloride was dissolved in 5.0 L of dichloromethane (DCM), cooled to 0-5 C. and 3.5 L of methylamine (5.7 eq., 39.44 mol) in 5.8 L of dichloromethane was added dropwise over a period of 105 minutes (maintaining the temperature at 5-10 C.). The suspension was allowed to warm to 20-25 C. and stirred overnight and the mixture then evaporated to dryness on a rotary evaporator under vacuum (bath temperature, 40 C.). The residue was suspended in 3.1 L of deionized water and the product collected by filtration. The filter cake was washed with 3.0 L of deionized water and 0.5 L of methylcyclohexane and then dried to a constant weight in vacuo at 55 C. The yield was 96.0% (1790 g) of a beige powder; GC: 99.3 area %, water content: 0.07%.

Hexadecyl(methyl)amine

(22) To a mixture of 15.75 kg of tetrahydrofuran (THF) and 2.76 kg (1.3 eq., 8.64 mol) lithium aluminium hydride (solution in THF), was added 1.79 kg (6.64 mol) of the N-methyl hexadecanamide in portions, at 20-25 C. (gas evolution). The reaction mixture (a yellowish-brown suspension) was stirred at reflux for 3 h. The reaction mixture was cooled to 5-10 C. and quenched by dropwise addition of 360 ml of deionized water, 360 ml of 20% sodium hydroxide solution and 1.04 L of deionized water. Tonsil (Sd-Chemie), 370 g, was added to the reaction mixture and the resulting suspension filtered and the filter cake washed with 2.08 kg of THF. The filtrate was evaporated to dryness on a rotary evaporator under vacuum (bath temperature, 60 C.). The yield was 88.0% (1495 g) of an off-white powder; GC: 98.2 area %. The material was stored under nitrogen.

2-hydroxy-3-(N-methylhexadecylammonio)propane-sulfonate, sodium/potassium salt (Compound 1a)

(23) Batch 1: Hexadecyl(methyl)amine (814 g, 3.18 mol) was suspended in 10.7 L of dimethylformamide (DMF). The off-white suspension was stirred at 20-25 C. and 847.5 g (1.3 eq., 4.13 mol) of sodium 3-chloro-2-hydroxypropane sulfonate hemihydrate, 573 g (1.3 eq., 4.13 mol) of potassium carbonate and 99.4 g (0.2 eq., 0.63 mol) of sodium iodide were added. The mixture was stirred at 50-60 C. for 69 h under nitrogen. HPLC showed 25.1 area % of product in the reaction mixture. DMF was removed by vacuum distillation (bath temperature, 80 C.) and the residue suspended in 4.95 L of THF and evaporated to 1.3 kg of celite. Soxhlet extraction with 20.0 L of THF:methanol (9:1) was carried out for 4.5 days. The extract was evaporated to dryness to leave a residue, which was confirmed by HPLC to be mostly side-products and which was discarded. The celite was removed from the Soxhlet extractor and dried to constant weight at 60 C. in vacuo. (3220 g). The celite was suspended in 32.0 L of methanol and heated to reflux. The mixture was filtered hot and the filtrate crystallized at 0-5 C., yielding 1016 g of yellowish powder (HPLC: 96.6 area %) of compound 1a as the sodium/potassium salt.

(24) Batch 2: Hexadecyl(methyl)amine (480.8 g, 1.88 mol) was suspended in 6.25 L of DMF. The off-white suspension was stirred at 20-25 C. and 500 g (1.3 eq., 2.44 mol) of sodium 3-chloro-2-hydroxypropane sulfonate hemihydrate, 338.4 g (1.3 eq., 2.44 mol) of potassium carbonate and 58.6 g (0.2 eq., 0.37 m lol) of sodium iodide were added. The mixture was stirred at 50-60 C. for 82 h under nitrogen. HPLC showed 30.5 area % of product in the reaction mixture. DMF was removed by vacuum distillation (bath temperature, 80 C.) and the residue crystallized from 12.35 L of boiling methanol. The mixture was filtered hot (insoluble part: 226.0 g) and the filtrate was crystallized at 0-5 C., yielding 562.0 g of yellowish powder, (HPLC: 97.6 area %) of compound 1a as the sodium/potassium salt.

2-hydroxy-3-(N-methylhexadecylammonio)propane-sulfonate (Compound 1a)

(25) The sodium/potassium salt, 1100 g, obtained as described above, were suspended in 11 L of chloroform:methanol (4:1). The pH was adjusted to 5 with saturated HCl solution in 2-propanol (the amount of HCl was determined by titration of a sample). Filtration and evaporation to dryness yielded 990 g of brownish-yellow, amorphous solid (HPLC: 96.8 area %). Crystallization of this solid, as described above, from 8.0 L of methanol yields 715.0 g of compound 1a (HPLC: 98.6 area %).

(26) Material of this grade was further purified by repeated crystallization from boiling methanol, with removal of insolubles by hot filtration if required. Recrystallization from isopropanol:water (4:1) may also be used. In a typical experiment, 805 g of compound 1a were dissolved in 2.4 L of isopropanol:water (4:1) at reflux, slowly cooled to 0-5 C., filtered and dried to yield 730 g (90% recovery) of compound 1a as an off-white powder (HPLC: 98.8 area %). In order to obtain a uniform polymorph, recrystallization from methanol under anhydrous conditions is preferred for the final crystallization step.

(27) Purification is continued until the following criteria are met: Purity by HPLC: >99 area % Residue on ignition: <0.8% Conductivity of 1% aqueous suspension: <50 S/cm NMR, MS: Conform with structure. Elemental analysis: consistent with composition.

Example 5: Polymorphism of Compound 1a

(28) Compound 1a can be crystallized from a variety of solvents. From a practical standpoint (ease of filtration, low toxicity of solvent), methanol, ethanol and isopropanol are preferred. Addition of 5-25% water to water-miscible solvents increases solubility and leads to a steeper temperature coefficient of solubility, reducing the amount of solvent required. Less preferred solvents for recrystallization are 1-propanol, n-butanol, acetone, acetonitrile, THF and ethyl acetate. Recrystallization from water is hard to control and frequently leads to difficulties in filtration.

(29) Starting from dry crude material and solvent, compound 1a is obtained as an anhydrate. Pure monohydrate can be obtained by crystallization from water. In a typical procedure, 150 g of compound 1a (anhydrate) was dissolved in 2 L of deionized boiling water, then seeded with compound 1a hydrate and the mixture was allowed to slowly cool to room temperature (RT). The precipitate was collected by filtration and dried at 35 C., 15 mbar, yielding 134.6 g (86% recovery) of an off-white solid. Karl Fischer titration and elementary analysis indicated the presence of a monohydrate.

(30) NMR indicates that compound 1a monohydrate remains associated with water even in solution, leading to an unexpectedly complex pattern of signals. Reversible coalescence is observed upon heating. This water-associated form is likely to be present under physiological conditions. The anhydrate shows the expected, simpler NMR spectrum.

(31) Compound 1a monohydrate (batch 2338-CF/30): 1H-NMR (630 MHz, CDCl3): =0.87 (t, 3H), [1.15-1.35 (m), 1.31 (br, s), =26H], 1.74 (br. s, 2H), [2.93 (d, J=4.5), 2.98 (d, J=4.7), 2.95-3.35 (m), 3.44 (br. d, J=12.7), =12H], 4.58 (hr. s. 1H), 9.21 (br. s, 0.5H), 9.48 (br. s, 0.5H).

(32) Compound 1a anhydrate (batch 2208-CF/1): 1H-NMR (500 MHz, CDCl3): =0.86 (t, 3H), [1.15-1.35 (m), 1.30 (br. s), =26H], 1.73 (br. s, 2H), 2.94 (s, 3H), [3.05-3.25 (m), 3.12 (d, J=5.7), =5H], 3.39 (br. d, J=12.1, 1H), 4.58 (br. d, J=6.1, 1H). (>5 ppm: no signals.)

(33) Both the monohydrate from water and the anhydrate from methanol form thin, micrometer-sized platelets of irregular shape. X-ray powder diffraction (XRPD) indicates a layered structure with a base period of 27 for both forms. Both forms can be easily interconverted. According to differential scanning calorimetry (DSC) analysis, the monohydrate loses its water between 60 C. and 90 C. The anhydrate can be converted to monohydrate by exposure to air of 100% relative humidity for 4 days. Only one equivalent of water is taken up and the consistency of the material remains unchanged.

(34) Crystallization from mixtures of water and water-miscible solvents yielded the anhydrate in most cases. In one experiment with methanol containing 10-25% water, formation of monohydrate was observed, but was poorly reproducible. Due to the more predictable outcome, anhydrous conditions are preferred for the final steps of purification.

(35) A pseudo-polymorph can be characterized by crystallizing compound 1a from methanol and analyzing the wet material by XRPD. In the methanol-wet sample, a layered structure is observed in which the base period is 28.5 . During drying at ambient temperature, both 27 (anhydrate) and 28.5 are observed together. Upon complete drying at ambient temperature, only the diffraction pattern of the anhydrate is observed. Thus, this crystallization proceeds through the intermediate stage of an instable methanol solvate in which methanol is weakly intercalated between the layers of the anhydrate structure.

(36) In total, four different polymorphs could be identified by XRPD (Table 1). Upon heating, clear solutions could be obtained with water, methanol, ethanol, isopropanol and the solvent:water (9:1) mixtures. Polymorphs from other solvents were obtained by equilibration of suspensions of form A in the respective solvents.

(37) TABLE-US-00001 TABLE 1 Polymorphs of compound 1a obtained from various solvents. Water of Batch Crystal Solvent system crystallization number form* Water; methanol; acetonitrile: n/a A water (1:1); acetone:water (1:1); ethanol:water (1:1) Solvent:water (9:1); solvents n/a A tested were methanol, ethanol, isopropanol, THF Water Monohydrate KP-0722.11 A Methanol Anhydrate KP-0726.11 A Methanol; partial sodium salt Anhydrate TN-0382.11 A (alkaline) Methanol:water (1:1) Monohydrate 2338-CF/30 A Methanol, traces of sodium salt Anhydrate 2208-CF/1 A Ethanol; n-butanol; ethylacetate; n/a B THF Acetonitrile n/a C Isopropanol n/a D *Arbitrary nomenclature

(38) Crystal forms A-C are relatively similar and share the same base period of 27 . While form A shows two broad reflexions at about 4.0 and 4.4 , form B shows a single sharp reflexion at about 4.5 . Form C shows the same sharp 4.5 reflexion as form B, plus a complex pattern of four more medium broad reflexions between 3.6 and 4.4 .

(39) Surprisingly, crystallization from neat isopropanol yields a highly crystalline polymorph which is very different from all other samples in Table 1. In form D, the base period is reduced to 22.8 and it does not share any major reflexion with form A. Unlike form A from methanol, this form does not change during drying. Crystallizing from isopropanol containing 10% of water is enough to yield form A instead.

(40) This demonstrates that methanol is to be preferred for the final steps of crystallization, because it yields a uniform lattice type (form A), even in the presence of small amounts of water. Ease of removal and ease of filtration are also best for methanol. Of form A, both the anhydrate and the monohydrate (which can be prepared by hydration of anhydrate) are suitable for pharmaceutical use. The monohydrate of form A is advantageous in that it will not take up further water during stress-testing in stability tests (such as, e.g., open incubation at 40C./80% humidity for 3 to 6 months). Accordingly, the use of the monohydrate of form A may be preferred in terms of storage stability.

(41) The present invention embraces all polymorphs of the compounds disclosed herein, including the above-described polymorphs of compound 1a.

Example 6: Inhibition of Mast Cell Degranulation

(42) Introduction

(43) 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 (FcRI). 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-FcRI 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, -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)).

Materials and Methods

Materials

(44) 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--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.

(45) 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--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.

(46) 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.

(47) 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% RPMI 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.

(48) Cell seeding: cells were harvested with Trypsin-EDTA as described and 50-100 l 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.

Measurement of -Hexosaminidase Release

Experimental Procedures

(49) 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 g/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 l DNP-HSA (2 g/ml; final concentration 0.1 g/ml) diluted in TyB and cells were incubated for 15 min at 37 C. Alternatively, cells were stimulated with 20 l 5 M ionomycin (final concentration 0.25 M) or 20 l 5 M thapsigargin (final concentration 0.25 M), both in the absence or presence of 20 nM PMA (final concentration).

(50) Plates were removed from the incubator and immediately centrifuged at 4 C. for 5 min at 250g and transferred to an ice bath. Aliquots of supernatants, 25 l, were transferred to 96-well plates. Remaining supernatant was aspirated from control wells and cells were lysed in 400 l lysis buffer for 5 min at RT on an orbital shaker at 450 rpm under non-sterile conditions. After lysis, 25 l aliquots of lysates were transferred to 96-well plates.

(51) MUG substrate solution, 100 l, were added to supernatant and lysate samples and plates were incubated 30 min at 37 C. The reaction was terminated by addition of 150 l stop solution. Fluorescence was measured at 365 nm excitation and 440 nm emission wavelengths.

(52) 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.

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

(54) Assessment of Pharmacologic Effect

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

(56) Inhibition of degranulation (inhibition of -hexosaminidase release): inhibition of degranulation was calculated as percent reduction of -hexosaminidase release with respect to positive control (antigen-stimulated release) after subtraction of negative control (unspecific release) using the formula;
% Inhibition=100(1(test compoundnegative control)/(positive controlnegative control)).

Measurement of Maximum Tolerated Concentration

(57) 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).

(58) 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.

Results

(59) 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 2). Results are taken from at least three independent experiments.

(60) TABLE-US-00002 TABLE 2 Inhibition of degranulation: IC50, MTC and SI values Compound IC50 (M) MTC (M) SI 1a 6.9 75 10.9 1b 5.1 100 19.6 1c 4.1 200 48.8 1d 3.3 75 22.7 1e 3.9 100 25.6 Miltefosine 4.2 25 6.0

(61) The MTC of the test compounds was 11-50 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.

(62) All substances outlined in Table 2 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 1e, have an advantageously low cytotoxicity.

(63) 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 7: Inhibition of Activation of Akt Kinase

(64) Introduction

(65) The mast cell degranulation assay using the RBL-2H3 cell line (see example 6) 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.

Materials and Methods

Materials

(66) 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.

(67) Equipment

(68) 12-well multichannel pipettes (30-300 l) from Eppendorf were used. Assay plates were agitated on a TiMix 5 control (Edmund Bhler). Electrochemiluminescence detection was performed on a SECTOR Imager 6000 (Meso Scale Discovery).

Measurement of Phospho-Akt Ser473

Experimental Procedures

(69) Protein assay: protein concentration was determined using the BCA (bicinchoninic acid) Protein Assay kit according to the manufacturer's instructions. Briefly, duplicate 10 l 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 were cooled to room temperature for 5 min and absorbance at 562 nm was 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.

(70) 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. Analysis of phosphor-Akt Ser473 was performed according to the manufacturer's instructions. The optimal amount of protein was determined at 5 g lysate per well for ERK1/2 and 10 g/well for phospho-Akt Ser473. Plates were blocked with 25 l/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 l 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 l/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 l/well, Read Buffer T was added and plates read on a SECTOR Imager 6000 plate reader.

Assessment of Effects of Phospho-Akt Ser473

(71) 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.

Results

(72) 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 M 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 3 shows levels of normalized phospho-Akt Ser473 as a percentage of those in the positive control.

(73) TABLE-US-00003 TABLE 3 Inhibition of Akt phosphorylation on Ser473 by compounds 1a and 1c Level of phospho-Akt Ser473 (% positive control) Compound 1 M 5 M 25 M 1a 80.8 17.9 62.4 16.1 9.7 4.9 1c 119.7 35.7 67.9 17.3 17.4 6.4 Percentage of total Akt phosphorylated on Ser473 expressed as percentage of control untreated cells, after induction with IgE and antigen for 15 min.

(74) A dose-dependent decrease in levels of phospho-Akt Ser473 was observed after treatment with all compounds outlined in Table 3. 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, hyperproliferative diseases and other indications.

Example 8: Inhibition of the Delayed-Type Hypersensitivity (DTH) Reaction in Mice

(75) Introduction

(76) The anti-inflammatory and anti-allergic effects of compounds 1a and 1c were 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 (T1) 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.

Materials and Methods

Materials

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

(78) Animals

(79) 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.

(80) Antigen Sensitization and Challenge

(81) Group size was n=8 mice unless otherwise stated. Test compounds were freshly prepared before administration.

(82) 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 L of emulsion into each side of the tail (100 g ovalbumin per mouse).

(83) 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 L of a 1% suspension of heat-aggregated ovalbumin (HOVA) (100 g 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 (Art. No. 7309, Mitutoyo, Kawasaki, Japan) before challenge, and 24 h after challenge.

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

(85) Compound Administration

(86) The anti-inflammatory effects of compounds 1a and 1c were compared to a vehicle control (0.5% methyl cellulose solution) and to the reference drug, dexamethasone. Test compounds were given at 25 or 100 mg/kg orally by gavage (Art. No. 432093, Harvard Apparatus GmbH, March-Hugstetten, Germany) 16 h and 3 h before sensitization and then twice daily with the final dose given 3 h prior to ear challenge (a total of 14 administrations). Dexamethasone was given at 0.1 or 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.

(87) Quantification of Assay Results

(88) 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.vehicleIET.sub.compound)/IET.sub.vehicle
where IET=(ET.sub.24 hrs pcET.sub.predose).sub.HOVA-treated ears(ET.sub.24 hrs pcET.sub.predose).sub.PBS-treated ears (IET, increase ear thickness; ET, ear thickness; pc, post challenge)
Statistical Evaluation

(89) 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.

Results

(90) Suppression of mouse ear swelling by compounds 1a and 1c as well as dexamethasone, compared to vehicle control is shown in FIG. 3. Table 4 summarizes the inhibition of DTH for compounds 1a and 1c.

(91) TABLE-US-00004 TABLE 4 Inhibition of mouse ear swelling by compounds 1a and 1c in the DTH response in mice. Inhibition of Compound mouse ear swelling 1a, 25 mg/kg 49* 1a, 100 mg/kg 52* 1c, 100 mg/kg 41* Dexamethasone, 0.1 mg/kg 14 Dexamethasone, 1.0 mg/kg 53* *p < 0.01 vs. vehicle control (Dunnett's post hoc test)

(92) Dexamethasone administered orally at a dose of 1 mg/kg, once daily over the whole sensitization period resulted in a significantly reduced DTH response, with inhibition of 53%. 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, a significant loss in body weight of 9% (p<0.01 vs. vehicle Control with the paired Student's t-test) was seen in the high dose dexamethasone group. A more clinically representative dose of dexamethasone in the mouse is 0.1 mg/kg, but at this dose inhibition was very low (14%) and did not reach significance indicating that only steroid doses which result in significant body weight loss upon repeated administration are active in this model.

(93) Compound 1a, administered orally twice daily over the whole sensitization period at two dosing regimens, 25 mg/kg or 100 mg/kg, reduced the DTH response by 49% and 52%, respectively. The higher dose administration of compound 1c reduced DTH response by 41%. Hence, these compounds were able to produce an inhibition almost equivalent to that of the high dose of dexamethasone (up to 98% for 1a and 77% for 1c). In contrast to dexamethasone, no significant toxic side-effects of compounds 1a or 1c were observed during the course of the study.

(94) The reduction of DTH response obtained by treatment with compounds 1a and 1c demonstrates that the compounds according to the invention and, in particular compounds 1a and 1c, are effective in the pharmaceutical intervention in allergic and inflammatory diseases involving antigen-specific cell-mediated immune responses. Even at the low dose, compound 1a provided for the same high inhibition of the DTH response as obtained by an overdose of dexamethasone, and thus represents a particularly preferred compound of the present invention.

Example 9: Inhibition of the Allergic Contact Dermatitis Inflammatory Response in Mice

(95) Introduction

(96) The anti-inflammatory and anti-allergic effects of compounds 1a and 1c 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. The advantages of the allergic contact dermatitis model (Zllner 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.

Materials and Methods

Materials

(97) Dexamethasone dihydrogenphosphate (Dexa-Inject) was obtained from Mibe GmbH, Jena, Germany and diflorasone diacetate from Basotherm, Biberach an der Riss, Germany.

(98) Animals

(99) 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.

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

(101) 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 L 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 L of 5% (w/v) TDI was applied. The allergic reaction was boosted 21 days later by application of 50 L of 0.5% (w/v) TDI. For the examination of test compound effects, the left ears were used for the TDI challenge (20 L of 0.5% in acetone) and ear thickness measured 3 h before and 24 h after challenge.

(102) Compound Administration for Systemic Treatment

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

(104) Administration time: to determine optimal time for administration treatment groups were treated orally by gavage with 100 mg/kg of compound 1a or 1c (suspended in 0.5% tylose, 10 mL/kg) 1, 4 or 16 h before topical TDI challenge. One group was treated with 100 mg/kg miltefosine orally, 16 h before challenge (based on available data for optimal administration time for miltefosine) and vehicle treated mice received tylose (10 mL/kg) orally, 4 h before challenge.

(105) Dose-response: two groups of mice were treated orally with compound 1a at 25 mg/kg or 100 mg/kg suspended in 0.5% tylose, 4 h before topical TDI challenge. Vehicle treated mice received 0.5% tylose orally 4 h before challenge. As a positive control, dexamethasone was administered in saline solution at 1 mg/kg or 3 mg/kg, 2 h and 30 min before challenge and 1 h after challenge. The dose and dosing scheme for dexamethasone was based on previous experience showing a maximal effect in this model.

(106) Compound Administration for Topical Treatment

(107) Compound 1a was administered to two groups of mice topically in 20 l of a 2% or 6% solution in propyleneglycol. The suspension was heated to 60 C. and mixed using a thermomixer (Eppendorf) until it became clear. The solution was applied, 2 h before topical TDI challenge by administration of 10 l onto each of the inner and outer surfaces of the left ears. A vehicle group (n=5) was treated with propyleneglycol. As a positive control, diflorasone, diacetate was administered at 0.01% (low dose) and 0.05% (high dose) in 20 l acetone, 2 h before challenge. A basal control group was left untreated.

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

(109) 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).

(110) Statistical Evaluation

(111) 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.

Results

(112) Suppression of mouse ear swelling by compounds 1a and 1c after oral administration, compared to vehicle control is shown in FIG. 4A. Table 5 summarizes inhibition of the allergic contact dermatitis response by compounds 1a and 1c.

(113) TABLE-US-00005 TABLE 5 Effect of orally administered compounds 1a and 1c on ear swelling in the allergic contact dermatitis response in mice. Inhibition of Compound mouse ear swelling Administration time (oral) 1a, 100 mg/kg, 1 h 72.6*** 1a, 100 mg/kg, 4 h 73.4*** 1a, 100 mg/kg, 16 h 29.0 1c, 100 mg/kg, 1 h 58.1** 1c, 100 mg/kg, 4 h 68.5*** 1c, 100 mg/kg, 16 h 42.7.sup. Miltefosine, 100 mg/kg, 16 h 47.3.sup. Dose-response (oral) 1a, 25 mg/kg 44.9*** 1a, 100 mg/kg 44.4*** Dexamethasone, 1 mg/kg 78.6*** Dexamethasone, 3 mg/kg 87.1*** **p < 0.01, ***p < 0.001 vs. vehicle control (Dunnett's post hoc test) compared to vehicle, .sup.p < 0.05 vs. vehicle control (t-test)

(114) In the administration time study with oral administration, compounds 1a and 1c reduced ear swelling significantly (up to 73% of vehicle control) when administered 1 h or 4 h before challenge, as also shown in FIG. 4A. Miltefosine has previously been shown to be maximally effective after oral administration when given 16 h before challenge and also in this study significantly reduced ear swelling (47% of vehicle control). However, miltefosine was not as maximally effectively as compounds 1a and 1c at their optimal administration time of 4 h, reaching only 64% of the inhibitory efficacy of 1a and 69% of that of compound 1c.

(115) In the dose-response study, compound 1a administered 4 h before challenge reduced ear swelling significantly (45%) at 25 or 100 mg/kg, as shown in FIG. 4B. In comparison, dexamethasone administered orally at doses of 1 and 3 mg/kg significantly inhibited ear swelling (78 and 87% respectively). As discussed in example 8 for the DTH response, such high doses (overdose) of dexamethasone are unsuitable for treatment of humans due to severe side effects of the corticosteroid and were used to validate the responsiveness of the model. Nevertheless, compound 1a was able to effect an inhibition equal to 52% of the highest dose of dexamethasone at doses of 1a, which showed no toxicity.

(116) Compounds 1a (25 mg/kg and 100 mg/kg) and 1c (100 mg/kg) had a significant impact on the TDI induced inflammatory reaction. Thus, the compounds according to the invention and, in particular compound 1a and 1c, are particularly effective and thus useful for the oral pharmaceutical intervention in inflammatory diseases, in particular in atopic dermatitis.

(117) Suppression of mouse ear swelling by compounds 1a and 1c after topical administration, compared to vehicle control is shown in FIG. 4C. Table 6 summarizes inhibition of the allergic contact dermatitis response by compounds 1a and 1c.

(118) TABLE-US-00006 TABLE 6 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% 67.9** 1a, 6% 63.1** Diflorasone, 0.1% 101.1*** Diflorasone, 0.5% 110.8*** **p < 0.01, ***p < 0.001 vs. vehicle control (Dunnett's post hoc test) compared to vehicle. Diflorasone treatment reduced ear thickness below that of untreated mice.

(119) Compound 1a topically administered as a solution at 2% or 6% significantly reduced ear swelling up to 68%, compared to vehicle control. The positive control, diflorasone, completely eliminated ear swelling and even reduced ear thickness to below the level of untreated mice. This indicates that the doses of diflorasone used here are not representative of a clinical benchmark, but were used to validate the responsiveness of the model. It must also be stressed, that diflorasone is one of the strongest dermal corticosteroids and is taken for severe eczema.

(120) 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. Diflorasone produced a highly significant reduction in the local lymph node reaction at both 0.1% and 0.5%, by completely inhibiting the increase in lymph node weight and cell number (FIGS. 4D and 4E), even reducing this to levels below untreated animals. In contrast, topical treatment with compound 1a at 2% or 6% did not have any impact on the local lymph node reaction (FIGS. 4D and 4E).

(121) 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. In addition, the compounds of the present invention, including compound 1a, do not show adverse effects typical of topically administered corticosteroids, such as inhibition of the lymph node reaction and loss in body weight.

Example 10: Inhibition of Collagen Type II-Induced Arthritis (CIA) in the Mouse

(122) Introduction

(123) The inhibitory effect of compound 1a was assessed for anti-inflammatory and anti-arthritic activity in the type II collagen-induced arthritis (CIA) model in the mouse. CIA has been proposed as a pertinent animal model of rheumatoid arthritis in humans. In this model, a peripheral arthritis is elicited by intradermal injection of homologous or heterologous (e.g. bovine, chicken) type II collagen (CII) in complete Freund's adjuvant (CFA) into rats or mice (Stuart et al., Ann Rev Immunol. 2:199-218 (1984); Marty et al., J Clin Invest. 107:631-640 (2001); Boissier et al., Eur J Immunol. 25:1184-90 (1995)). The central role played by T cells in the development of type II CIA is demonstrated by the T cell proliferative response to mouse CII in immunized mice, the successful adoptive transfer of the disease with immune cells from the spleen, and the resistance of athymic nude mice to the induction of the pathology (Stuart et al., Ann Rev Immunol. 2:199-218 (1984); Marty et al., J Clin Invest. 107:631-640 (2001)). An advantage of this model of arthritis as compared to others is the development of an arthritogenic response toward a well defined antigen (CII), which also permits the study of antigen-induced immunological phenomena and their selective modification by immunopharmacological intervention.

Materials and Methods

Materials

(124) Bovine type II collagen (Chondrex, Redmond WA, USA) was dissolved at 2 mg/ml in 0.05 M acetic acid by gentle stirring overnight at 4 C. CFA was prepared by adding Mycobacterium tuberculosis H37Ra (Difco, Detroit, MI)) at 2 mg/ml to IFA (incomplete Freund's adjuvant, Sigma Aldrich, Milano, Italy). Before injection, CII was emulsified with an equal volume of CFA.

(125) Animals

(126) Eight to 9 week old male DBA/1j mice were purchased from Harlan Laboratories srl (San Pietro al Natisone, Udine, Italy) and kept under standard laboratory conditions with free access to food and water. Mice were allowed to adapt one week to their environment before starting the study.

(127) Induction of Collagen Induced Arthritis (CIA)

(128) Group size was n=11 mice unless otherwise stated. Test compounds were freshly prepared before administration. Mice were injected intradermally at the base of the tail with 100 L of an emulsion containing 100 g of CII, IFA and 100 g of Mycobacterium tuberculosis. On day 21, a boost of CII in IFA was administered.

(129) Prophylactic Treatment

(130) Five groups of mice were treated under a prophylactic regimen from day 0 to 47 and an additional group of sham treated mice was treated only with the CII vehicle, on days 0 and 21. Compound 1a was administered as a suspension in 0.5% carboxymethylcellulose (10 mL/kg) and, as a positive control, dexamethasone was administered at 0.3 mg/kg as detailed below: Group 1: compound 1a at 25 mg/kg, orally by gavage, twice daily Group 2: compound 1a at 100 mg/kg, orally by gavage, twice daily Group 3: dexamethasone 0.3 mg/kg, intraperitoneally, once daily Group 4: vehicle (carboxymethylcellulose), orally by gavage Group 5: sham treated mice

(131) Animals were sacrificed on day 47 after immunization.

(132) Therapeutic Treatment

(133) Groups of mice were treated with compound 1a from the onset of arthritic symptoms, defined as first day on which a disease score of 1 or higher was observed and mice expressing the respective disease score were randomly assigned to each experimental group. Treatment was continued for 20 consecutive days. Group 6: compound 1a at 100 my/kg, orally by gavage, twice daily

(134) As the treatment was based on the individual expression of arthritis symptoms, the mice were synchronized to the first day of treatment for evaluation of the disease progression. No separate vehicle group was included for the therapeutic treatment regimen hence vehicle group 4 was reanalyzed after synchronization to the first day on which a disease score of 1 or higher was observed. Animals were sacrificed after 20 days of treatment.

(135) Clinical Assessment

(136) Mice were evaluated for arthritis daily by an observer unaware of the treatment regimens according to a macroscopic scoring system: 0=no signs of arthritis; 1=swelling and/or redness of the paw or one digit; 2=involvement of 2 joints; 3=involvement of more than 2 joints; 4=severe arthritis of the entire paw and digits. An arthritis index was calculated for each mouse by summing the scores for individual paws. Clinical severity was also determined by evaluation of paw thickness of both front and hind-paws using a thickness gauge. An index was calculated for each mouse by summing the thickness for individual paws. Body weights were also recorded daily.

(137) Statistical Evaluation

(138) Mean and standard deviation (SD) were calculated from individual score values.

(139) For the arthritis score two different statistical calculations were performed. For each treatment day the arthritis scores of each group were compared to the vehicle control group using the student's-t test and a p<0.05 was considered significant.

(140) Additionally, a cumulative arthritis score was calculated for each treatment group by summing all arthritis scores throughout the study period. The cumulative arthritis scores were compared using the student's-t test and a p<0.05 was considered significant. The cumulative arthritis score requires that all animals are evaluated for the same length of time; in order to determine the cumulative arthritis score of animals which had died during the study, the missing values were substituted with the group mean for the day of the missing value. The substitution of missing values was only performed for the cumulative arthritis score and not used for other calculations.

Results

(141) Effects of the Prophylactic Treatment of Test Compounds on Arthritic Score

(142) As expected, starting 5-6 days after the CII boost, clinical signs of arthritis became observable in vehicle treated control mice, consisting of progressively augmenting arthritic scores, accompanied by increased paw thickness. Significant loss in body weight of vehicle treated animals compared to the sham-treated group was observed after the CII boost.

(143) Compound 1a reduced the cumulative arthritis score and paw thickness at both 25 and 100 mg/kg compared to vehicle treated mice, as shown in FIG. 5A. Dexamethasone nearly completely suppressed the clinical signs of arthritis, but also induced a significant reduction in body weight from day 8 until the end of the study, when compared to vehicle treated mice (FIG. 5B). However, no effects on body weight were observed in mice treated with compound 1a at both low and high doses (FIG. 5B).

(144) Effects of the Therapeutic Treatment of Test Compounds on Arthritic Score

(145) Compound 1a at 100 mg/kg significantly reduced the arthritis score and the cumulative arthritis score compared to vehicle-treated mice from day 11 to 13 and on day 21, as also shown in FIG. 5C. Furthermore, a trend to a reduction in the duration of disease compared to vehicle-treated mice was observed.

(146) Immune Organ Weights

(147) At sacrifice, thymuses and spleens were collected and weighed to assess the effect on these important immune organs. Compared to the sham-treated mice, animals treated with the vehicle exhibited a significant increase in spleen weight, due to the proliferation of lymphocytes in answer to elicitation by CII injection. As expected, treatment with the positive control drug, dexamethasone, markedly reduced the weights of both spleens and thymuses compared to both vehicle treated and sham groups demonstrating the known immunosuppressive effect of corticosteroids (FIG. 5D). In contrast, treatment with compound 1a did not reduce thymus or spleen weights compared to vehicle treated mice (FIG. 5D).

(148) The data demonstrate that compound 1a ameliorated the clinical course of type II CIA when administered prophylactically, reducing the arthritis score and paw thickness. When administered under the therapeutic regimen, 1a a significant reduction in the cumulative disease score was evidenced. Compound 1a did not show any toxic effects, whereas dexamethasone caused a significant loss in body weight and spleen and thymus weights. These results suggest that the compounds of the present invention, including compound 1a, are particularly effective and thus useful for the medical intervention in rheumatoid arthritis.

Example 11: Pharmacokinetic Study of Five Different Batches of Compound 1a After a Single Oral Administration to Mice

(149) Introduction

(150) The aim of the study was to evaluate pharmacokinetic (PK) properties of five different batches including anydrates and monohydrates of the crystal form A of compound 1a (cf. Table 1 in Example 5) after a single oral administration to mice. Three of the forms were anhydrous and two were monohydrates.

Materials and Methods

(151) Compound 1a was administered as a suspension of 0.5% methyl cellulose (Sigma-Aldrich) in water at a dose of 100 mg/kg (10 ml/kg) by oral gavage to non-fasted female Balb/c mice (n=3). This dose had previously been shown to be well tolerated. No overt toxicity was shown regardless of the polymorphic crystal form.

(152) Blood was collected by decapitation at 0.5, 1, 2, 4, 8, 12, 24, and 48 h after administration. Blood plasma was isolated by centrifugation at 4 C. (20,000g for 10 min), transferred to microtubes containing lithium-heparin and frozen in aliquots at 70 C. until assayed by mass-spectrometry. Mean plasma concentration-time profiles (semi-logarithmic) of the five polymorphic crystal forms of compound 1a are shown in FIG. 6.

(153) Pharmacokinetic Parameters

(154) For PK evaluation, summary statistics and plotting of concentration/time curves, all values below the lower limit of quantification (LLOQ) were set to zero. PK parameters for the five forms of compound 1a are listed in Table 7.

(155) TABLE-US-00007 TABLE 7 Pharmacokinetic parameters of polymorphic forms of compound 1a in female Balb/c mice (n = 3) following a single oral administration of 100 mg/kg in 0.5% methylcellulose solution. Polymorphic form PK parameter 2338-CF/30 2208-CF/1 KP-0722.11 KP-0726.11 TN-0382.11 c.sub.max (ng/ml) 1063 1003 571 759 627 t.sub.max (h) 1.0 1.0 2.0 1.0 1.0 c.sub.last (ng/ml) 1.8 1.1 3.8 1.2 3.4 t.sub.last (h) 48.0 48.0 24.0 48.0 24.0 AUC.sub.0-t (ng*h/ml) 3118 4400 3162 3744 3535 AUC.sub.0-inf (ng*h/ml) 3144 4412 3179 3758 3547 MRT (h) 5.5 5.0 4.8 5.5 5.9 t.sub.1/2 (h) 9.9 7.3 n.d. 8.2 n.d. c.sub.max Observed maximum plasma concentration t.sub.max Time of occurrence of c.sub.max c.sub.last Concentration at last sampling time point t.sub.last Time of last sampling time point AUC.sub.0-t Area under the plasma concentration versus time curve from time zero to t.sub.last, calculated by the trapezoidal rule AUC.sub.0-inf Area under the plasma concentration versus time curve from time zero to infinity with extrapolation of the terminal phase MRT Mean residence time calculated using trapezoid area calculations extrapolated to infinity t.sub.1/2 Terminal half-life

(156) The PK behaviour was comparable for all polymorphic forms of compound 1a. The compound was rapidly absorbed from the gastro-intestinal (GI)-tract, reaching a maximum plasma concentration after 1-2 h. The overall exposure was highest with form 2208-CF/1. A second plasma peak was observed at 8 h after administration of forms KP-0726.11 and TN-0382.11, indicating a second absorption window that might be caused by improved solubility of these polymorphs in the lower part of the GI-tract. The mean residence time was in the same range for all polymorphic forms tested and the PK curves and terminal half-lives were similar between 8-24 h, as also shown in FIG. 6. No terminal half-lives were determined for KP-0722.11 or TN-0382.11, due to the lack of 48 h values for these forms.