Compounds for use in the treatment of inflammatory bowel disease

Abstract

Described are compounds of the structural formula (I): Also provided are pharmacologically acceptable isomers and salts of the compound of (I). The compounds are useful in the treatment of inflammatory bowel disease.

Claims

1. A compound of formula ##STR00019## and pharmaceutically acceptable salts thereof.

2. A N-Methyl-(D)-Glucamine salt compound of formula ##STR00020##

3. The pharmaceutical composition comprising an effective amount of a compound as claimed in claim 1 and a pharmaceutically acceptable carrier.

4. The pharmaceutical composition comprising an effective amount of a compound as claimed in claim 2 and a pharmaceutically acceptable carrier.

5. A method for the treatment of inflammatory bowel disease, comprising administering to a subject an effective amount of a compound as claimed in claim 1.

6. A method for the treatment of inflammatory bowel disease, comprising administering to a subject an effective amount of a compound as claimed in claim 2.

7. A method for the treatment of ulcerative colitis, comprising administering to a subject an effective amount of a compound as claimed in claim 1.

8. A method for the treatment of ulcerative colitis, comprising administering to a subject an effective amount of a compound as claimed in claim 2.

9. A method for the treatment of Crohn's disease, comprising administering to a subject an effective amount of a compound as claimed in claim 1.

10. A method for the treatment of Crohn's disease, comprising administering to a subject an effective amount of a compound as claimed in claim 2.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The invention will be more clearly understood from the following description thereof given by way of example only, in which:

(2) FIG. 1 is the X-ray crystal structure showing the absolute stereochemistry for the enantiomer compound 4 (R)-(+)-methylbenzylamine salt (compound 9);

(3) FIG. 2 is the X-ray crystal structure showing the absolute stereochemistry for the enantiomer compound 2 (S)-()-methylbenzylamine salt (compound 8);

(4) FIG. 2A is a view of a molecule of compound 8 from the crystal structure showing the numbering scheme employed. Anisotropic atomic displacement ellipsoids for the non-hydrogen atoms are shown at the 50% probability level. Hydrogen atoms are displayed with an arbitrarily small radius. Only the major disorder component is shown;

(5) FIG. 3 is a graph of the effect of compounds 2, 3, 4 and 5 at 30 mg/kg on disease activity index (DAI) over 7 days in 5% DSS colitis;

(6) FIG. 4 is a bar chart of the effect of compounds 2, 3, 4 and 5 at 30 mg/kg on disease activity index (DAI) at day 7 in 5% DSS colitis;

(7) FIG. 5 is a graph of the effect of compounds 5, 7, 2 and 6 at 10 mg/kg on disease activity index (DAI) over 7 days in 5% DSS colitis;

(8) FIG. 6 is a bar chart of the effect of compounds 5, 7, 2 and 6 at 10 mg/kg on disease activity index (DAI) at day 7 in 5% DSS colitis. Asterisks indicate a significant (P<0.05) difference (1 way ANOVA) from the vehicle control group.

(9) FIG. 7 Is a graph showing the effect of compound 6 on weight loss in 5% DSS-treated mice. Data are MeanSEM from 6-7 mice per group;

(10) FIG. 8 Is a graph showing the effect of compound 6 on DAI in 5% DSS-treated mice. Data are MeanSEM from 6-7 mice per group;

(11) FIG. 9 Is a bar chart showing the effect of compound 6 on DAI in 5% DSS-treated mice on day 7. Data are MeanSEM. Asterisks indicate a significant (P<0.05) difference (1 way ANOVA) from the vehicle control group;

(12) FIG. 10 Is a bar chart showing the effect of compound 6 on Colon length of 5% DSS-treated mice on day 7. Asterisks indicate a significant (P<0.05) difference (1 way ANOVA) from the vehicle control group;

(13) FIG. 11 Shows representative haematoxylin and eosin-stained sections from distal colons of mice. Higher magnifications (10) are shown;

(14) FIG. 12 Is a bar chart showing the effect of compound 6 on histology scores of colons from DSS-treated mice. Data are MeanSEM from 5-6 mice. Asterisks indicate a significant (P<0.05) difference (1 way ANOVA) from the vehicle control group. Note, maximum score 10;

(15) FIG. 13 Is a bar chart showing myeloperoxidase (MPO) activity in the colons of untreated or vehicle, prednisolone and compound 6 treated mice exposed to 5% DSS. Data are MeanSEM from 5-6 mice. Asterisks indicate a significant (P<0.05) difference (1 way ANOVA) from the vehicle control group;

(16) FIG. 14(A) to (C) are bar charts showing the effect of compound 6 on levels of cytokines (IL1, TNF and IL6) in mice treated with DSS. Data are MeanSEM from 5-6 mice. Asterisks indicate a significant (P<0.05) difference (1 way ANOVA) from the vehicle control group;

(17) FIG. 15 Is a grph showing weight loss in IL10.sup.--/--mice treated with vehicle or compound 6. Mice were administered compound 6 (300 mg/kg/week) or vehicle orally on a Monday/Wednesday/Friday (MWF) dosing schedule. Mice were 4 weeks of age at start of experiment and were treated for 9 weeks. Mice were weighed weekly and data are presented as MeanSEM from 9-12 mice per group. Mice were monitored for overt disease, rectal prolapse, and moribund animals were humanely killed;

(18) FIG. 16 Is a scatter graph representing Serum Amyloid A (SAA) levels of individual mice, and Mean (bar), from surviving animals at week 9 (11 and 9 mice in compound 6 or vehicle-treated groups, respectively). Student's t-test was used to test for statistical differences between groups;

(19) FIG. 17 Are representative hematoxylin and eosin-stained sections from distal colons from IL10.sup.--/-- mice treated for 9 weeks with vehicle or compound 6; and

(20) FIG. 18 Is a scatter graph showing histology scores of distal colons of IL10.sup.-/-- mice treated with vehicle or compound 6. Scatter graph representing histology score of individual mice, and Mean (bar), from surviving animals at week 9 (11 and 9 mice in compound 6 or vehicle-treated groups, respectively). Student's t-test was used to test for statistical differences between groups.

DETAILED DESCRIPTION OF THE INVENTION

(21) Compound 1 represents a pair of diastereoisomers that result from the reduction and demethylation of the ketone compound A which has a chiral centre at C-2, and is, as a result, a pair of enantiomers.

(22) ##STR00004##

(23) Reduction of this compound with LiAlH.sub.4 yields a compound of the formula

(24) ##STR00005##

(25) This compound comprises two diastereoisomers:

(26) ##STR00006##

(27) Hydrolysis of Diastereoisomer B gives rise to compounds 2 and 3

(28) ##STR00007##

(29) Hydrolysis of Diastereoisomer C gives rise to compounds 4 and 5.

(30) ##STR00008##

(31) The diastereoisomers can be resolved chemically or chromatographically into their constituent enantiomers.

(32) The absolute stereochemistry of compound 4 has been established by single crystal X-ray of compound 4 (R)-(+)-methylbenzylamine salt (compound 9) (FIG. 1).

(33) The absolute stereochemistry of compound 2 was confirmed by single crystal X-ray of compound 2 (S)-()-methylbenzylamine salt (compound 8) (FIGS. 2 and 2A).

(34) General Reaction Procedures

(35) General synthetic procedures for the coupling of enantiomeric mixtures as exemplified below are described in WO9720806A, the entire contents of which are herein incorporated by reference.

(36) General Preparation of Acid Derivative Compound A

(37) ##STR00009##

(38) To a stirred solution of the coupled product (4 mmol, 1.00 g) in tert-butanol (5 mL) and diethyl ether (30 mL) under nitrogen was added methyl (4-bromomethyl)benzoate (6 mmol, 1.41 g). To this was added a solution of potassium tert-butoxide in tert-butanol (30 mL) and diethyl ether (5 mL), slowly drop wise. With each drop, the mixture turned a yellow colour and it then reverted to its original grey colour. The mixture was stirred for a further 3 hours until the TLC (80:20, hexane:ethyl acetate) showed no more starting material. The reaction was quenched by the addition of sat. NH.sub.4Cl. The layers were separated and the aqueous layer extracted with diethyl ether (2120 mL). The combined organic layers were washed with water, brine, dried over MgSO.sub.4 and evaporated. The solid product precipitated from the crude on removal of most of the solvent. This was filtered off and washed with cold diethyl ether to give 0.98 g (62%) of a cream solid.

(39) Reduction of Methyl Benzoate Compound

(40) ##STR00010##

(41) To a stirred solution of the methyl benzoate compound (1.27 mmol; 0.50 g) in THF (15 mL) was added lithium tri-tert-butoxyaluminohydride (1.9 mmol, 0.48 g), slowly portion wise. The reaction was monitored by TLC (80:20, hexane:ethyl acetate) and after 3 h, all of the starting material had been consumed.

(42) The reaction was quenched by pouring onto ice and the crude product extracted into ethyl acetate by stirring the aqueous mixture for 10-15 min with ethyl acetate then pouring into a separatory funnel and then allowing it to separate. The combined organic layers were washed with water, brine, dried over MgSO.sub.4 and evaporated to give 0.34 g (68%) of a cream-tan solid. The product was isolated as a mixture of two diastereoisomers in an approximately 2:1 ratio.

(43) Analytical Results for the Mixture of Two Diastereoisomers

(44) Purity (HPLC): 94.9% (as a 2:1 ratio of diastereoisomers)

(45) .sub.H(300 MHz, CDCl.sub.3): 2.77-3.60 (6H, m, 3CH.sub.2) 3.85 (3H, s, CH.sub.3), [5.02 (1H, s, COH)] 5.18 (1H, s, COH), [6.23 (1H, s, CC] 6.43 (1H, s, CC), 6.90-6.98 (2H, m, ArH), 7.11-7.21 (1H, m, ArH), 7.22-7.31 (5H, m, ArH), 7.36-7.42 (2H, m, ArH), 7.78-7.84 (2H, m, ArH).

(46) Where possible, the value for the minor diastereoisomer is given in brackets.

(47) .sub.C(75.5 MHz, CDCl.sub.3): 38.3 (CH.sub.2), 38.4 (CH.sub.2), 38.6 (CH.sub.2), 39.9 (CH.sub.2), 40.3 (CH.sub.2), 43.4 (CH.sub.2), 51.9 (COOCH.sub.3), 52.0 (COOCH.sub.3), 55.9 (quat. C), 56.3 (quat. C), 82.0 (CHOH), 82.8 (CHOH), 120.5 (tert. C), 120.7 (tert. C), 123.5 (tert. C), 123.6 (tert. C), 124.0 (tert. C), 124.2 (tert. C), 124.5 (tert. C), 124.6 (tert. C), 124.8 (tert. C), 124.9 (tert. C), 125.1 (tert. C), 125.2 (tert: C), 126.1 (tert. C), 126.4 (tert. C), 127.0 (quat. C), 127.1 (quat. C), 128.0 (tert. C), 128.2 (tert. C), 128.5 (tert. C), 128.8 (tert. C), 129.0 (tert. C), 129.2 (tert. C), 129.5 (tert. C), 2130.0 (2tert. C), 2130.2 (2tert. C), 130.7 (tert. C), 140.4 (quat. C), 141.5 (quat. C), 142.8 (quat. C), 143.2 (quat. C), 143.5 (quat. C), 143.6 (quat. C), 143.7 (quat. C), 144.2 (quat. C), 144.3 (quat. C), 144.5 (quat. C), 150.4 (quat. C), 152.6 (quat. C), 167.0 (CO), 167.2 (CO).

(48) Hydrolysis of Methyl Benzoate Moiety

(49) ##STR00011##
The ester was placed in a round-bottomed flask and 10% aq. NaOH (1 mL) was added to it followed by sufficient methanol to form a solution (6 mL). The solution was heated at 40 C. and monitored by TLC (80:20, hexane:ethyl acetate). After ca. 4 h, no further ester was seen.

(50) The mixture was cooled and sat. NH.sub.4Cl added (solution at pH 12). Dilute HCl was added to acidic pH (pH 2). The product was extracted from the cloudy solution into ethyl acetate (310 mL). The combined extracts were dried over MgSO.sub.4 and evaporated in vacuo to give 0.15 g (quantitative) of a cream solid. The product was isolated as a mixture of two diastereoisomers in an approximately 2:1 ratio.

(51) Analytical Results for the Mixture of Two Diastereoisomers

(52) Purity (HPLC): 95.2% (as a 2:1 ratio of diastereoisomers)

(53) .sub.H(400 MHz, CDCl.sub.3): 2.81-3.59 (6H, m, 3CH.sub.2), [5.05 (1H, s, COH)], 5.23 (1H, s, COH), 6.46 (1H, s, CC), [6.66 (1H, s, CC)], 6.95-7.03 (2H, m, ArH), 7.12-7.17 (1H, m, ArH), 7.21-7.29 (5H, m, ArH), 7.37-7.43 (2H, m, ArH), 7.85-7.91 (2H, m, ArH).

(54) Where possible, the value for the minor diastereoisomer is given in brackets.

(55) .sub.C(100 MHz, CDCl.sub.3): 37.9 (CH.sub.2), 38.1 (CH.sub.2), 38.2 (CH.sub.2), 39.5 (CH.sub.2), 39.9 (CH.sub.2), 43.1 (CH.sub.2), 55.5 (quat. C), 55.9 (quat. C), 81.6 (CHOH), 82.4 (CHOH), 120.2 (tert. C), 120.3 (tert. C), 123.1 (tert. C), 123.2 (tert. C), 123.5 (tert. C), 123.9 (tert. C), 124.1 (tert. C), 124.4 (tert. C), 124.5 (tert. C), 124.7 (tert. C), 125.9 (tert. C), 126.0 (tert. C), 126.5 (tert. C), 2126.7 (quat. C & tert. C), 126.9 (quat. C), 128.1 (tert. C), 128.2 (tert. C), 128.4 (tert. C), 2129.2 (2tert. C), 2129.4 (2tert. C), 2129.8 (2tert. C), 2129.9 (2tert. C), 130.4 (tert. C), 140.0 (quat. C), 141.0 (quat. C), 142.3 (quat. C), 142.7 (quat. C), 143.0 (quat. C), 143.2 (quat. C), 143.8 (quat. C), 144.0 (quat. C), 144.1 (quat. C), 144.7 (quat. C), 150.0 (quat. C), 152.0 (quat. C), 170.8 (CO), 171.1 (CO).

(56) Chemical Separation of Enantiomers

(57) Preparation of NBOC D-phenylalanine derivative of methyl benzoate diastereoisomer and/or separation of subsequent diastereoisomers 1 and 2 (or 1 and 2)

(58) ##STR00012##

(59) Note: procedure applicable to both diastereoisomers but the example given is for the first diastereoisomer.

(60) Diastereoisomer A (2.5 mmol, 1.0 g) and NBOC D-phenylalanine (3.1 mmol, 0.8 g) were placed in a round bottom flask fitted with a condenser and suspended in CH.sub.3CN (25 mL) under nitrogen. To this suspension was added pyridine (3.1 mmol, 0.3 mL) followed by a solution of DCC (3.1 mmol, 0.7 g) and DMAP (10% mol, 0.25 mmol, 0.05 g) in CH.sub.3CN (2 mL). The mixture was stirred for 20 h at 50 C., and then allowed to reach room temperature.

(61) The white solid was filtered off and the solvent removed in vacuo. Ethyl acetate was added and the solution obtained was washed with 10% H.sub.2SO.sub.4, sat. NaHCO.sub.3, dried over MgSO.sub.4 and evaporated to give 2.1 g of a yellow oil (83% pure by HPLC, yield: quantitative).

(62) The diastereoisomers 1 and 2 were separated by flash chromatography (90 g of silica/g of product) using hexane/MTBE 90:10. From 4.17 g of mixture, 1.3 g of 2, derivative was obtained (as well as 1.71 g of the 1 derivative and 0.3 g as a mixture of both).

(63) Hydrolysis of NBOC D-phenylalanine Derivative of Methyl Benzoate Compound (1, 2, 1 or 2)

(64) ##STR00013##

(65) The diastereoisomer 2 (2.3 mmol, 1.45 g) was dissolved in methanol (25 mL) and NaOH (11.5 mmol, 0.45 g) was added and the mixture stirred at reflux temperature and monitored by TLC. After 20 h, the starting material was consumed.

(66) The reaction was cooled to room temperature and quenched by addition of sat. NH.sub.4Cl. The methanol was removed in vacuo and the aqueous solution acidified to pH 1 with conc. HCl. The product was extracted with ethyl acetate, dried over MgSO.sub.4 and evaporated to give 1.6 g of a yellow gum, which was purified by a short silica column with hexane:MTBE 80:20 as eluent. 0.44 g of acid derivative compound 5 (50% yield) was obtained which was 97.2% pure by HPLC.

(67) Note: An alternative hydrolysis was also carried out using 10% aqueous NaOH in methanol at 40-50 C. This procedure took almost 5 days to go to completion.

(68) Analytical Results for Enantiomers 1, 2, 1, 2

(69) Enantiomer 1 from Diastereoisomer B-Compound 3

(70) ##STR00014##

(71) Description: Cream amorphous solid

(72) Melting point 195-196 C.

(73) [].sub.D: +98.51 (1.07%, MeOH)

(74) Purity: 99.0% .sub.H(400 MHz, CDCl.sub.3): 2.87 (1H, d, J=13.28 Hz, CH.sub.2), 3.00-3.09 (2H, m, CH.sub.2), 3.29 (1H, d, J=13.36 Hz, CH.sub.2), 3.43-3.61 (2H, m, CH.sub.2), 5.27 (1H, s, CHOH), 6.49 (1H, s, CHC), 7.00 (2H, d, J=7.88 Hz, ArH), 7.16-7.32 (6H, m, ArH), 7.44 (2H, d, J=7.24 Hz, ArH), 7.90 (2H, d, J=7.92 Hz, ArH).

(75) Enantiomer 2 from Diastereoisomer BCompound 2

(76) ##STR00015##

(77) Description: Cream amorphous solid

(78) Melting point 184-185 C.

(79) [].sub.D: 114.44 (0.18%, MeOH)

(80) Purity: 99.8% .sub.H(400 MHz, CDCl.sub.3): 2.87 (1H, d, J=13.32 Hz, CH.sub.2), 3.00-3.09 (2H, m, CH.sub.2), 3.29 (1H, d, J=13.28 Hz, CH.sub.2), 3.46 (1H, d, J=22.64 Hz, CH.sub.2), 3.58 (1H, d, J=22.56 Hz, CH.sub.2), 5.27 (1H, s, CHOH), 6.49 (1H, s, CHC), 7.00 (2H, d, J=8.04 Hz, ArH), 7.15-7.34 (6H, m, ArH), 7.44 (2H, d, J=7.20 Hz, ArH), 7.90 (2H, d, J=8.04 Hz, ArH).

(81) Enantiomer 1 from Diastereoisomer C-Compound 4

(82) ##STR00016##

(83) Description: Cream solid

(84) Melting point 136-140 C.

(85) [].sub.D: 39.3 (0.66%, MeOH)

(86) Purity: 94.0%

(87) .sub.H(400 MHz, CDCl.sub.3): 2.90-3.59 (6H, m, 3CH.sub.2), 5.08 (1H, s, CHOH), 6.70 (1H, s, CHC), 7.05 (2H, d, J=8.08 Hz, ArH), 7.19 (1H, t, J=7.34 Hz, ArH), 7.26-7.47 (7H, 2m, ArH), 7.93 (2H, d, J=8.08 Hz, ArH).

(88) Enantiomer 2 from Diastereoisomer C-Compound 5

(89) ##STR00017##

(90) Description: Cream amorphous solid

(91) Melting point 195-196 C.

(92) [].sub.D: +32.1 (1.18%, MeOH)

(93) Purity: 97.2%

(94) .sub.H(400 MHz, CDCl.sub.3): 2.94-3.59 (6H, m, 3CH.sub.2), 5.08 (1H, s, CHOH), 6.70 (1H, s, CHC), 7.05 (2H, d, J=8.12 Hz, ArH), 7.19 (1H, t, J=7.34 Hz, ArH), 7.26-7.47 (7H, 2m, ArH), 7.93 (2H, d, J=8.12 Hz, ArH).

(95) HPLC Method

(96) Achiral and Chiral HPLC methods were established for the qualitative and quantitative separation of enantiomers compounds 2, 3, 4, 5.

(97) HPLC Resolution of Enantiomers

(98) TABLE-US-00001 Reverse phase method Column Hypersil BDS C18, 5 , 250 4.6 mm Phenomenex Luna C18, 5, 250 4.6 mm, N: 32 Wavelength 210 nm Flow rate 1 mL/min (for ketone and esters) 0.6 mL/min (for acids and salts) Mobile phase 70:30 CH.sub.3CN:0.1% aq. Acetic acid Sample 1 mg/mL, made up in mobile phase (or CH.sub.3CN:dIW = 50:50 for acids/salts) Retention times Compound 1 - 20 min Diastereoisomers C (compounds 4/5) 9 min Diastereoisomers B (compounds 2/3) 10 min Chiral method Column ChiralPack IC, 5, 250 4.6 mm Wavelength 210 nm Temperature 25 C. Flow rate 0.35 mL/min Mobile phase n-Heptane/IPA/HOAc (or TFA) = 90/10/0.1 Sample 1 mg/mL, made up in mobile phase (or nHeptane/IPA/MeOH = 81/9/10 for salts) Retention times Compound A 54 min and >60 min Compound 4 - 30 min Compound 5 - 37 min Compound 3 - 18 min Compound 2 - 19 min
Salt Formation

(99) Salts were prepared by dissolving the free acid of compounds 2, 3, 4 and 5 in aqueous or aqueous organic solvent in the presence of the appropriate base and isolating the salt by evaporation of solvent.

(100) Compound 6: The N-Methyl-(D)-Glucamine salt (NMDG) of compound 2.

(101) ##STR00018##

(102) Compound 6 Physiochemical Properties:

(103) Appearance: Off-white solid

(104) Molecular Weight: 577 (free acid: 382)

(105) Molecular Formula: C.sub.33H.sub.39O.sub.8N (free acid: C.sub.26H.sub.22O.sub.3)

(106) Melting Point: 165-167 C.

(107) Compound 6: [].sub.D: 76.5 (sample concentration: 200 mg/10 ml in Water)

(108) Mass (Da): ES+ only [NMDG+Na] was visible

(109) Elemental analysis: Calc: C (68.61), H (6.80), N (2.42), O (22.16). Found: C (68.44), H (6.80), N (2.50), O (21.98).

(110) .sub.H(400 MHz, DMSO): 2.48 (3H, apparent s, NCH.sub.3), 2.65 (1H, d, J=13.56 Hz, HCH), 2.84-3.02 (4H, m), 3.16 (1H, d, J=13.60 Hz, HCH), 3.40-3.70 (7H, m), 3.85-3.92 (1H, in), 5.06 (1H, s, CHOH), 5.93 (1H, broad s, CHOH), 6.41 (1H, s, CHC), 6.80 (2H, d, J=7.92 Hz, ArH), 7.06-7.41 (8H, m, ArH), 7.64 (2H, d, J=7.80 Hz, ArH).

(111) .sub.C(100 MHz, DMSO): 33.8 (CH.sub.3), 37.9 (CH.sub.2), 38.2 (CH.sub.2), 39.5 (CH.sub.2), 51.6 (CH.sub.2N), 55.8 (quat. C), 63.5 (CH.sub.2O), 69.0 (CHO), 70.3 (CHO), 70.6 (CHO), 71.3 (CHO), 81.1 (CHOH), 120.1 (tert. C), 123.4 (tert. C), 123.7 (tert. C), 124.3 (tert. C), 124.4 (tert. C), 126.1 (tert. C), 126.3 (tert. C), 127.0 (tert. C), 127.5 (tert. C), 2128.5 (2tert. C), 2129.1 (2tert. C), 140.4 (quat. C), 141.1 (quat. C), 142.9 (quat. C), 144.5 (quat. C), 145.2 (quat. C), 154.3 (quat. C), 170.4 (CO).

(112) X-Ray Studies

(113) The absolute stereochemistry of compound 2 was established by single crystal X-ray analysis of its (S)-()-methylbenzylamine salt (compound 8). The results are given in Appendix 2. The results were in agreement with the stereochemistry shown in FIG. 2. The absolute stereochemistry of compounds 4 and 5 were established by conversion of the alcohols (compounds 2-5) to their ketenes and by correlation of their optical rotations.

(114) Inflammatory Bowel Disease (IBD)

(115) Inflammatory Bowel Disease (IBD) consists of two idiopathic inflammatory diseases, Ulcerative Colitis (UC) and Crohn's Disease (CD). The greatest distinction between CD and UC is the range of inflamed bowel tissue. Inflammation in CD is discontinuously segmented, known as regional enteritis, while UC is superficial inflammation extending proximally and continuously from the rectum. At present the cause of IBD is unknown. The disease seems to be related to an exaggerated mucosal immune response to infection of the intestinal epithelium because of an imbalance of pro-inflammatory and immune-regulatory molecules. The inheritance of patterns of IBD, suggest a complex genetic component of pathogenesis that may consist of several combined genetic mutations. Currently no specific diagnosis exists for IBD, but as an understanding of pathogenesis improves so will testing methods. Treatment of IBD consists of inducing and maintaining remission. IBD patients may be maintained on remission by use of a 5-aminosalycilate. However, while the use of aminosalycilates in UC provides considerable benefit, both in inducing remission in mild to moderate disease and in preventing relapse, the usefulness of these drugs to maintain remission in CD is questionable and is no longer recommended. The mainstay of treatment of active disease is a corticosteroid, commonly used for limited periods to return both UC and CD patients to remission, though budesonide, designed for topical administration with limited systemic absorption, has no benefit in maintaining remission. Alternatives, such as the immunosuppressive drugs azathioprine and mercaptopurine, together with methotrexate and cyclosporine have limited efficacy and the capability of inducing grave adverse effects. Anti-TNF antibodies such as infliximab and adalimubab may be used in those patients unresponsive to standard immunosuppressive therapy. However, many patients fail to respond to anti-TNF therapy, either due to their particular phenotype or by the production of autoantibodies.

(116) Acute Murine DSS Colitis Model

(117) The dextran sodium sulphate (DSS) colitis model is an experimental mouse model that exhibits many of the symptoms observed in human UC, such as diarrhoea, bloody faeces, mucosal ulceration, shortening of the colon, weight loss and alterations in certain colon cytokines. The study is widely used as a model for studying the pathogenesis of UC and also for screening new therapeutic interventions for the treatment of UC.

(118) In these studies, an acute colitis model was used, with 5% DSS administered in the drinking water of BALB/c mice. This dosage regime induces severe acute colitis, by days 7-8 mice had overt rectal bleeding and marked weight loss; unless sacrificed beforehand, all mice would have died by days 10-12.

(119) Mice

(120) Specific Pathogen-Free BALB/c mice, 6-8 weeks of age, were obtained from a commercial supplier (Harlan UK). Mice were fed irradiated diet and housed in individually ventilated cages (Tecniplast UK) under positive pressure.

(121) DSS Treatment

(122) DSS (5%) was dissolved in drinking water. Compounds were administered orally at a dose of 10 mg/kg or 30 mg/kg on days 0-7, and mice were culled on day 8 or day 9, depending on the severity of the disease. The mice were checked each day for morbidity and the weight of individual mice was recorded. Induction of colitis was determined upon autopsy, length of colon and histology. Colons were recovered and stored at 20 C. for immunological analysis. All of the compounds and experimental groups are randomly alphabetically labelled. Throughout experiments all data recording was performed in a blind manner. The codes on boxes/groups were not broken until after the data was analysed i.e. boxes labelled A, B, C etc were identified as untreated, DSS-treated, or DSS+compound-treated.

(123) To quantify the extent of colitis, a disease activity index (DAI) was determined based on weight loss, faecal blood and stool consistency. A score was given for each parameter, with the sum of the scores used as the DAI. For each treatment group n=8.

(124) TABLE-US-00002 Description of DAI Score Weight loss % Stool consistency Faecal blood 0 None Normal None 1 1-3 2 3-6 Loose stool.sup.1 Visible in stool 3 6-9 4 >9 Diarrhea.sup.2 Gross bleeding.sup.3 Definitions: .sup.1Loose stool - stool not formed, but becomes a paste on handling. .sup.2Diarrhea - no stool formation, fur stained around the anus. .sup.3Gross bleeding - fresh blood on fur around the anus with excessive blood in the stool.
Administration of Compounds

(125) All compounds were prepared for oral gavage (0.1 mL per os (p.o.) per 10 g body weight) as a suspension in 0.5% carboxymethyl cellulose/2% Tween 80, at a dose of 3-30 mg/Kg. Compounds as free acid were initially dissolved in absolute alcohol and diluted with 14+1 with 0.5% carboxymethyl cellulose/2% Tween 80; this resulted in a fine precipitate in suspension while N-Methyl-(D)-Glucamine salts were soluble in the vehicle alone.

(126) Effect of Individual Enantiomers Compounds 2, 3, 4 and 5 in 5% DSS Murine Colitis

(127) BALB/c given 5% DSS in drinking water were administered compounds 2, 3, 4 and 5 at 30 mg/kg p.o. as a suspension in 0.5% carboxymethyl cellulose/2% Tween 80 daily for 7 days. DAI measures the extent of the disease in this model. Compound 4 was without activity on this variable, there not being any significant (P>0.05) difference in DAI at any time point (FIG. 3). At day 7, both compound 2 and compound 5 significantly (P<0.5) reduced DAI by a considerable margin, from 9.0+0.53 for vehicle controls to 3.20.73 for compound 5 and 2.50.71 for compound 2, there being no significant difference between the two (FIG. 4). In comparison, compound 3 reduced DAI to only 5.30.6. This was significantly (P>0.05) less potent than either compound 2 or compound 5. Further, while the DAI in compound 3-treated mice was statistically (P<0.05) less than vehicle controls at day 7 (FIG. 4), at day 6 there was no statistical (P>0.05) difference between compound 3 and vehicle (FIG. 3). In conclusion, of the four enantiomers, compounds 2, 3, 4 and 5 both compounds 2 and 5 are highly active in this model at 30 mg/kg. Compound 3 has minimal activity which is significantly (P<0.05) less than compound 2 and compound 5. Compound 4 is almost devoid of activity in this 5% DSS murine colitis model.

(128) Selection of a Salt of Compounds 2 and 5

(129) As a consequence of the limited aqueous solubility of the enantiomers compound 2 and compound 5, we attempted the synthesis of five salts of compound 5. The sodium salt, potassium salt, calcium salt, -methylbenzylamine salt and N-Methyl-(D)-Glucamine salt were synthesised. The sodium and calcium salt were unsuccessful. The three salts of compound 5, named potassium salt, -methylbenzylamine salt and N-Methyl-(D)-Glucamine salt were used for solubility and partition coefficient (log P) studies.

(130) The solubility of the four compounds was determined:

(131) TABLE-US-00003 Milli-RO H.sub.20 pH 4.0 Buffer pH 7.0 Buffer pH 9.0 Buffer Compound g/mL g/mL g/mL g/mL Compound 5 1.38 0.33 320.1 369.6 Compound 5 Potassium salt 217.0 0.15 54.71 340.3 Compound 5 Methyl- 413.9 0.20 227.4 311.0 benzylamine salt Compound 5 N-Methyl-D- >60,000* 0.14 >60,000* >60,000* Glucamine salt *Estimated value

(132) Compound 5 N-Methyl-(D)-Glucamine salt (compound 7) was determined, surprisingly, to be the most soluble compound from this group of analogous compounds by a considerable margin, with a solubility of >60,000 g/mL in Milli-RO water, 0.14 g/mL in pH 4 buffer, >60,000 g/mL in pH 7.0 and >3,000 g/mL in pH 9.0 buffer. Almost identical values were obtained with compound 2 N-Methyl-(D)-Glucamine (compound 6) with a solubility of >60,000 g/mL in Milli-RO water, 0.5 g/mL in pH4 buffer, >60,000 g/mL in pH 7.0 and >3,000 g/mL in pH 9.0 and buffer.

(133) The partition coefficient of compound 5 and related analogous compounds was investigated using the HPLC method (reverse phase C18 HPLC column) at neutral, acidic and alkaline pH.

(134) The partition coefficient of the four compounds was determined:

(135) TABLE-US-00004 Neutral Basic Acid Compound Log10 POW Log10 POW Log10 POW Compound 5 3.7 3.7 3.9 Compound 5 Potassium salt 3.7 3.7 3.9 Compound 5 Methyl- 3.6 3.6 3.9 benzylamine salt Compound 5 N-Methyl-D- 3.5 3.5 3.8 Glucamine salt

(136) The partition coefficient of each salt of compound 5 was found to be similar. It is suggested that this is happening because when the salt is in solution the compound dissociates into the parent compound 5 and the associated salt ion. As a result of this the measured partition coefficient was from the parent ion rather than the salt molecules.

(137) The partition coefficient (Log 10 POW) of compound 2 N-Methyl-D-Glucamine salt (compound 6) was successfully determined in neutral, basic and acidic conditions as 3.5, 4.3 and 2.6 respectively.

(138) N-Methyl-(D)-Glucamine was chosen as the salt candidate for both compound 2 and compound 5.

(139) Effect of Enantiomers Compound 2 and Compound 5 and their N-Methyl-(D)-Glucamine Salts (Compounds 6 and 7) at 10 mg/kg in 5% DSS Murine Colitis

(140) Given that both compounds 2 and 5 show considerable activity in the 5% DSS model at 30 mg/kg, we then re-examined their activity, together with their N-Methyl-(D)-Glucamine salts at the lower dose of 10 mg/kg, given daily for 7 days as a suspension or solution in 0.5% carboxymethyl cellulose/2% Tween 80. No adjustment was made in the dosages of the salts to compensate for their increased molecular weight. Both compounds 5 and 7, at 10 mg/kg, had no significant (P>0.05) effect on DAI in the 5% DSS murine colitis model when compared to vehicle control (see FIG. 5). In contrast, at day 7, both compound 2 and compound 6, the N-Methyl-(D)-Glucamine salt, at 10 mg/kg significantly (P<0.05) and potently reduced DAI from 9.30.51 (vehicle) to 2.10.7 and 3.30.52 respectively (FIG. 6).

(141) In conclusion, compound 2 (and its N-Methyl-(D)-Glucamine salt, compound 6) is the most potent of the four enantiomers by a considerable margin, and the only enantiomer to retain activity at the lower dose level of 10 mg/kg.

(142) Effect of a Range of Doses of Compound 6 and a Comparison with Prednisolone on 5% DSS Murine Colitis

(143) Compound 6 was selected as the most favoured enantiomer. The activity of compound 6 in the 5% DSS murine model of colitis at varying dose levels was tested to ascertain if there was a dose/response relationship and to make a comparison with a potent oral steroid, Prednisolone, commonly used to return patients suffering from acute exacerbations of IBD to remission.

(144) Mice were administered compound 6 at dose levels 3, 10 and 30 mg/Kg (equivalent to 6.6-20 mg/Kg of the compound 2). A group of DSS-treated mice was also treated with prednisolone, 5 mg/Kg. Prednisolone is a corticosteroid in clinical use in the treatment of human IBD and the quantity used in this study is the optimal dose of prednisolone for this model. After 3 days of treatment of BALB/c mice with 5% DSS in the drinking water signs of colitis were apparent. This was manifested as weight loss (FIG. 7) and an increase in the disease DAI (FIG. 8). However, following oral administration daily for 7 days, compound 6 at three doses (3, 10 and 30 mg/Kg) caused no overt reactions in mice. Compound 6 ameliorated the severity of colitis following acute DSS treatment in multiple parameters of disease examined. The capacity of compound 6 to ameliorate disease in the DSS model was dose-dependent. Compound 6 at 30 mg/Kg was therapeutic in the DSS model at a comparable, or better, efficacy relative to prednisolone at 5 mg/Kg.

(145) The severity of these symptoms are progressive; by day 7 the DSS-treated mice have lost up to 15% of their body weight and all mice have perfuse rectal bleeding. The DAI values on the day of autopsy showed that mice treated with compound 6 3-30 mg/kg had at each dose level, a significantly (P<0.05P<0.01) lower DAI than vehicle controls. Prednisolone (5 mg/kg) also significantly (P<0.01; ANOVA; Dunnett Multiple Comparison Test), reduced DAI scores (FIG. 9).

(146) At autopsy on day 7, there was significant shortening of colon length (P<0.05P<0.01; ANOVA; Dunnett Multiple Comparison Test) in all DSS treated groups compared to colons from mice not treated with DSS (FIG. 10). The lowest dose of 3 mg/kg of compound 6 did not have a significant effect in inhibiting colon shortening when compared to vehicle controls whereas the 10 and 30 mg/kg groups and the Prednisolone group did have a significant effect. Compound 6 at 30 mg/kg was significantly better than Prednisolone (P<0.05; ANOVA; Dunnett Multiple Comparison Test) (FIG. 10).

(147) Following DSS treatment histology sections of the distal colon showed extensive crypt damage and cell infiltration (FIG. 11).

(148) The extent of colon damage was quantified using an arbitrary scoring system. Compound 6 at both 10 and 30 mg/Kg, caused a dose-dependent and highly statistically significant reduction (P<0.01; Kruksal-Wallis ANOVA; Dunnett Multiple Comparison Test) in colon pathology relative to the vehicle group. In contrast, there was no significant improvement in histology scores with the prednisolone (5 mg/Kg) treated group relative to vehicle-treated mice (FIG. 12).

(149) Consistent with the histology results showing inflammation in the colons of mice, there was a significant (P<0.001; Kruksal-Wallis ANOVA; Dunnett Multiple Comparison Test) elevation in colon myeloperoxidase (MPO) activity in DSS-treated mice administered vehicle only. Colonic myeloperoxidase activity (MPO), representing the level of inflammatory neutrophil cell infiltration into the gut wall which was increased by almost 8-fold by DSS treatment but was significantly (P<0.05) reduced by both compound 6 at 30 mg/kg and Prednisolone, at 63% and 54% respectively by day 7 (FIG. 13).

(150) Quantification of levels of colon cytokines showed that DSS-treatment induces elevated IL1 (FIG. 14(a)), TNF (FIG. 14(b)) and IL6 (FIG. 14(c)), to 0.7440.076 ng/mg, 1.4780.378 ng/mg and 1.0570.1784 ng/mg respectively. In each case, compound 6 caused a significant (P<0.05, 30 mg/kg) and dose-dependant reduction in these cytokine levels. Prednisolone (5 mg/kg) also reduced (p<0.05) these increases in cytokine levels; for each cytokine there was no significant difference between the effect of prednisolone 5 mg/kg and compound 6 at the higher dose level of 30 mg/kg at day 7

(151) In summary, following oral administration daily for 7 days, compound 6 at three doses (3, 10 and 30 mg/Kg) caused no overt reactions in mice. Compound 6 ameliorated the severity of colitis following acute 5% DSS treatment by multiple parameters of disease examined and the capacity to ameliorate the disease is dose-dependent. Further, compound 6 at 30 mg/Kg was therapeutic in the DSS model at a comparable or better efficacy, relative to prednisolone (5 mg/Kg).

(152) Chronic IL10.sup.--/-- Model

(153) Mice with a deletion in the IL10.sup.--/-- gene spontaneously develop chronic colitis, with the age of onset and the severity of the disease being dependent on background mouse strain and the conditions in which the animals are housed. The onset of colitis in IL10.sup.--/-- mice housed under the conditions used in this study was also strain dependent, with an earlier onset and greater severity, in terms of mortality, in BALB/c strain mice relative to C57BL/6 strain animals. In this experiment, animals received oral treatment on a MWF regime over 9 weeks. Initially, both groups of mice progressively gain weight (FIG. 15). Vehicle treated mice stopped gaining weight from week 5 of treatment, whereas compound 6-treated mice maintained weight gain until week 8. By week 9 animals had marked weight loss, with one moribund animal humanely killed on day 60 in each group. As other mice were losing weight and developing clinical symptoms of disease, both groups were culled at week 9 (day 63) and analysed. While there were greater mortalities in the vehicle-treated group (25%) relative to compound 6 treated mice (9.2%) by Kaplan-Meier analysis, there was no statistical difference in survival of IL10.sup.-/- mice over the 9 weeks. Serum was recovered from mice and Serum Amyloid A (SAA) and was analysed as a marker for severity of colitis. There were significantly (P<0.05; Student's t-test) reduced SAA levels in compound 6 treated mice relative to vehicle treated IL10.sup.--/-- mice (FIG. 16).

(154) Histology sections of colons from IL10.sup.--/-- mice treated with vehicle or compound 6 are shown in FIG. 17.

(155) Histology sections of colons from IL10.sup.--/-- mice treated with vehicle or compound 6 were scored. The extent of colon pathology was significantly reduced (P<0.05; Student's t-test) in IL10.sup.--/-- mice receiving compound relative to mice treated with vehicle (FIG. 18).

(156) In summary, oral treatment with compound 6 (300 mg/kg/week) in IL10.sup.--/-- BALB/c strain mice, using a MWF regime over 9 weeks, delayed weight loss and reduced deaths from colitis relative to vehicle-treated mice. In this model of chronic colitis, compound 6 significantly reduced disease indices with respect to a serum marker of colon inflammation and the degree of inflammation and damage to the colon. This is particularly noteworthy in view of the fact that the plasma half-life (t.sub.1/2) for compound 6 is 3 hours in the rat. With the standard MWF dosing schedule, mice will have been unexposed to compound 6 for substantial periods during the experiment.

(157) The invention is not limited to the embodiments hereinbefore described which may be varied in detail.

APPENDIX 1

List of Abbreviations Used

(158) aq aqueous b.p. boiling point CDCl.sub.3 chloroform-d CH(OCH.sub.3).sub.3 trimethylsilyl orthoformate CO.sub.2 carbon dioxide DCM dichloromethane dIW distilled ionized water DMSO dimethyl sulphoxide Et.sub.2O ether EtOH ethanol H.sub.2O water HCl hydrochloric acid IR infra red IPA isopropyl alcohol KCl potassium chloride M molar min minutes microliters mM milli-molar m.p. melting point N.sub.2 nitrogen NaBH.sub.4 sodium borohydride NaOH sodium hydroxide Na.sub.2SO.sub.4 sodium sulphate NMR nuclear magnetic resonance O.sub.2 oxygen RT room temperature .sup.tBuOH tert butanol .sup.tBuOK potassium tert butoxide S.E.M. standard error of mean THF tetrahydrofuran TLC thin layer chromatography l microliters Triflic Acid trifluoromethanesulfonic acid TMS Triflate trimethyl silyl trifluoromethanesulfonate v/v volume per volume w/v weight per volume .sub.em emission wavelength .sub.exc excitation wavelength

APPENDIX 2

(159) X-Ray Studies

(160) A single crystal X-ray analysis was carried out on compound 2 (S)-()-methylbenzylamine salt (compound 8), using a SuperNova, Dual, Cu at zero, Atlas Diffractometer and the parameters outlined in Table 1.

(161) TABLE-US-00005 TABLE 1 Data collection and structure refinement for compound 8, the (S)-()- methylbenzylamine salt of compound 2. Diffractometer SuperNova, Dual, Cu at zero, Atlas Radiation source SuperNova (Cu) X-ray Source, Cu K Data collection method Omega scans Theta range for data collection 3.74 to 76.22 Index ranges 13 h 13, 11 k 12, 14 l 14 Reflections collected 12753 Independent reflections 5263 [R(int) = 0.0196] Coverage of independent 99.4% reflections Variation in check reflections N/A Absorption correction Semi-empirical from equivalents Max. and min. transmission 1.00000 and 0.90238 Structure solution technique direct Structure solution program Bruker SHELXTL Refinement technique Full-matrix least-squares on F.sup.2 Refinement program Bruker SHELXTL Function minimized w(F.sub.o.sup.2 F.sub.c.sup.2).sup.2 Data/restraints/parameters 5263/1/363 Goodness-of-fit on F.sup.2 1.007 /.sub.max 0.001 Final R indices 5161 data; I > 2(I) R1 = 0.0321, wR2 = 0.0857 all data R1 = 0.0327, wR2 = 0.0865 Weighting scheme w = 1/[.sup.2 (F.sub.o.sup.2) + (0.0600P).sup.2 + 0.2200P] where P = (F.sub.o.sup.2 + 2F.sub.c.sup.2)/3 Absolute structure parameter 0.04(14) Extinction coefficient 0.0035(5) Largest diff. peak and hole 0.214 and 0.154 e .sup.3
Refinement Summary:

(162) Ordered Non-H atoms, XYZ Freely refining

(163) Ordered Non-H atoms, U Anisotropic

(164) H atoms (on carbon), XYZ Idealized positions riding on attached atoms

(165) H atoms (on carbon), U Appropriate multiple of U(eq) for bonded atom

(166) H atoms (on heteroatoms), XYZ Freely refining

(167) H atoms (on heteroatoms), U Isotropic

(168) Disordered atoms, OCC Refined with a two part model constrained to a total of unity

(169) Disordered atoms, XYZ freely refining

(170) Disordered atoms, U freely refining

(171) The single crystal X-ray data establishes that the structure of compound 6 is monoclinic, space group P2.sub.1, with one molecule of compound 6 in the asymmetric unit (Table 2).

(172) TABLE-US-00006 TABLE 2 Sample and crystal data for compound 8 Crystallization solvents Diethyl ether, MeOH, THF Crystallization method Slow evaporation Empirical formula C.sub.34H.sub.33N.sub.1O.sub.3 Formula weight 503.61 Temperature 100(1) K Wavelength 1.54178 Crystal size 0.50 0.50 0.50 mm Crystal habit Colourless Block Crystal system Monoclinic Space group P2.sub.1 Unit cell dimensions a = 11.0344(2) = 90 b = 10.1727(2) = 93.682(2) c = 11.8532(2) = 90 Volume 1327.77(4) .sup.3 Z 2 Density (calculated) 1.260 Mg/m.sup.3 Absorption coefficient 0.627 mm.sup.1 F(000) 536

(173) The absolute stereochemistry was determined as S, S at C9 and C10 for compound 2 and S at C33 for the methylbenzylamine cation. The assignment was made from consideration of both the Flack parameter which was determined to be 0.04 (14) and from the a priori knowledge of the stereochemistry of the salt former.

(174) The absolute stereochemistry was also determined using Bayesian statistics on the Bijvoet pair differences which resulted in a probability of the stereochemistry at the chiral centers C9, C10 and C33 being S, S and S respectively as 1.000 and R, R and R as 0.000. This supports the assignment of S, S and S for C9, C10 and C33 respectively from the Flack parameter measurement.

(175) The calculated X-ray powder diffraction pattern from the single crystal X-ray structure was in agreement with the stereochemistry shown in FIG. 2 (or the following).

(176) TABLE-US-00007 TABLE 3 Atomic coordinates and equivalent isotropic, atomic displacement parameters, (.sup.2), for compound 8. U(eq) is defined as one third of the trace of the orthogonalised U.sub.ij tensor. x/a y/b z/c U(eq) O1 0.02763(10) 0.17316(11) 1.16556(8) 0.0228(2) O2 0.07430(9) 0.03465(10) 1.12294(7) 0.0194(2) O3 0.10561(8) 0.01057(10) 1.90142(8) 0.0184(2) C1 0.08315(12) 0.12167(14) 1.47373(12) 0.0198(3) C2 0.07248(13) 0.09752(14) 1.35802(12) 0.0192(3) C3 0.08014(11) 0.02912(13) 1.31666(11) 0.0158(3) C4 0.05975(11) 0.05851(14) 1.19195(11) 0.0164(3) C5 0.10196(12) 0.13219(14) 1.39262(11) 0.0184(3) C6 0.11261(13) 0.10790(14) 1.50817(11) 0.0197(3) C7 0.10101(11) 0.01884(14) 1.55106(10) 0.0164(3) C8 0.09988(12) 0.04205(14) 1.67717(10) 0.0177(3) C9 0.22568(11) 0.05199(14) 1.74191(10) 0.0160(3) C10 0.20981(12) 0.06390(14) 1.87231(10) 0.0173(3) C11 0.32285(12) 0.00001(14) 1.92450(11) 0.0183(3) C12 0.36695(13) 0.00323(15) 2.03747(11) 0.0217(3) C13 0.46523(13) 0.07703(16) 2.07062(12) 0.0263(3) C14 0.51796(13) 0.15733(16) 1.99312(13) 0.0271(3) C15 0.47368(13) 0.16061(15) 1.87974(13) 0.0237(3) C16 0.37476(12) 0.08173(14) 1.84684(11) 0.0188(3) C17 0.30303(12) 0.07486(14) 1.73362(11) 0.0189(3) C18 0.29536(12) 0.17122(14) 1.70380(10) 0.0170(3) C19 0.24493(13) 0.29849(15) 1.68674(11) 0.0224(3) C20 0.34284(13) 0.38466(15) 1.64945(10) 0.0202(3) C21 0.34340(15) 0.51740(16) 1.62093(12) 0.0279(3) C22 0.45250(18) 0.57426(17) 1.59308(13) 0.0363(8) C23 0.55837(16) 0.50075(18) 1.59165(13) 0.0317(4) C24 0.55735(14) 0.36697(17) 1.61785(12) 0.0269(3) C25 0.44911(13) 0.31016(15) 1.64729(11) 0.0212(3) C26 0.42215(14) 0.17370(16) 1.68241(12) 0.0238(3) C18A 0.29536(12) 0.17122(14) 1.70380(10) 0.0170(3) C19A 0.24493(13) 0.29849(15) 1.68674(11) 0.0224(3) C20A 0.34284(13) 0.38466(15) 1.64945(10) 0.0202(3) C21A 0.34340(15) 0.51740(16) 1.62093(12) 0.0279(3) C22A 0.45250(18) 0.57426(17) 1.59308(13) 0.0279(3) C23A 0.55837(16) 0.50075(18) 1.59165(13) 0.0317(4) C24A 0.55735(14) 0.36697(17) 1.61785(12) 0.0269(3) C25A 0.44911(13) 0.31016(15) 1.64729(11) 0.0212(3) C26A 0.42215(14) 0.17370(16) 1.68241(12) 0.0238(3) N1 0.09024(11) 0.21952(13) 1.02800(10) 0.0194(2) C27 0.18541(12) 0.06679(15) 0.92258(12) 0.0220(3) C28 0.19466(13) 0.15069(16) 0.82981(13) 0.0256(3) C29 0.23606(14) 0.10317(17) 0.72421(13) 0.0273(3) C30 0.26855(15) 0.02757(18) 0.71195(13) 0.0301(3) C31 0.26063(14) 0.11089(16) 0.80481(13) 0.0255(3) C32 0.21928(12) 0.06417(15) 0.91135(11) 0.0200(3) C33 0.21444(12) 0.15827(15) 1.01084(12) 0.0205(3) C34 0.24587(14) 0.09613(16) 1.12172(13) 0.0256(3)

(177) TABLE-US-00008 TABLE 4 Selected bond lengths, (), for compound 8 O1C4 1.2528(18) O2C4 1.2688(17) O3C10 1.4373(16) O3H3A 0.88(2) C1C2 1.3909(19) C1C7 1.3964(19) C2C3 1.383(2) C3C5 1.3929(19) C3C4 1.5108(17) C5C6 1.3893(18) C6C7 1.395(2) C7C8 1.5141(16) C8C9 1.5457(17) C9C18 1.5203(19) C9C17 1.5537(19) C9C10 1.5713(16) C10C11 1.5040(18) C11C16 1.391(2) C11C12 1.3953(17) C12C13 1.394(2) C13C14 1.385(2) C14C15 1.401(2) C15C16 1.390(2) C16C17 1.5152(18) C18C19 1.418(2) C18C26 1.4380(19) C19C20 1.481(2) C20C21 1.392(2) C20C25 1.398(2) C21C22 1.394(2) C22C23 1.388(3) C23C24 1.396(2) C24C25 1.391(2) C25C26 1.485(2) N1C33 1.5073(18) N1H1B 0.91(2) N1H1C 0.93(2) N1H1D 0.90(2) C27C32 1.388(2) C27C28 1.390(2) C28C29 1.391(2) C29C30 1.383(3) C30C31 1.387(2) C31C32 1.398(2) C32C33 1.5172(19) C33C34 1.518(2)

(178) TABLE-US-00009 TABLE 5 Selected bond angles, (), for compound 8 C10O3H3A 107.0(15) C2C1C7 120.96(13) C3C2C1 120.72(13) C2C3C5 118.95(12) C2C3C4 121.50(12) C5C3C4 119.48(12) O1C4O2 125.44(12) O1C4C3 116.78(12) O2C4C3 117.77(12) C6C5C3 120.23(13) C5C6C7 121.32(13) C6C7C1 117.75(12) C6C7C8 120.71(12) C1C7C8 121.43(13) C7C8C9 115.87(10) C18C9C8 111.09(11) C18C9C17 110.70(11) C8C9C17 113.19(11) C18C9C10 108.74(10) C8C9C10 109.89(10) C17C9C10 102.86(10) O3C10C11 109.18(11) O3C10C9 109.69(10) C11C10C9 103.26(10) C16C11C12 121.07(13) C16C11C10 110.61(11) C12C11C10 127.84(13) C13C12C11 118.29(14) C14C13C12 120.72(13) C13C14C15 120.99(14) C16C15C14 118.34(14) C15C16C11 120.58(13) C15C16C17 129.14(13) C11C16C17 110.16(12) C16C17C9 103.90(11) C19C18C26 109.64(13) C19C18C9 124.70(12) C26C18C9 125.65(13) C18C19C20 107.21(12) C21C20C25 120.31(14) C21C20C19 131.41(14) C25C20C19 108.27(13) C20C21C22 118.51(15) C23C22C21 121.31(15) C22C23C24 120.24(15) C25C24C23 118.68(15) C24C25C20 120.94(14) C24C25C26 130.48(14) C20C25C26 108.57(13) C18C26C25 106.29(13) C33N1H1B 108.3(13) C33N1H1C 112.0(13) H1BN1H1C 107.4(18) C33N1H1D 111.6(13) H1BN1H1D 112.5(18) H1CN1H1D 105.0(17) C32C27C28 120.51(14) C27C28C29 120.09(15) C30C29C28 119.78(14) C29C30C31 120.10(14) C30C31C32 120.61(15) C27C32C31 118.89(14) C27C32C33 122.36(13) C31C32C33 118.74(13) N1C33C32 110.61(11) N1C33C34 108.16(11) C32C33C34 114.30(12)

(179) TABLE-US-00010 TABLE 6 Selected torsion angles, (), for compound 8 C7C1C2C3 0.4(2) C1C2C3C5 1.7(2) C1C2C3C4 175.50(12) C2C3C4O1 156.41(13) C5C3C4O1 20.75(18) C2C3C4O2 22.38(18) C5C3C4O2 160.46(12) C2C3C5C6 1.7(2) C4C3C5C6 175.57(12) C3C5C6C7 0.5(2) C5C6C7C1 2.5(2) C5C6C7C8 173.65(12) C2C1C7C6 2.52(19) C2C1C7C8 173.64(12) C6C7C8C9 83.92(16) C1C7C8C9 100.03(15) C7C8C9C18 64.43(16) C7C8C9C17 60.83(15) C7C8C9C10 175.19(12) C18C9C10O3 155.49(11) C8C9C10O3 33.70(15) C17C9C10O3 87.10(12) C18C9C10C11 88.22(13) C8C9C10C11 149.99(11) C17C9C10C11 29.19(13) O3C10C11C16 96.36(13) C9C10C11C16 20.29(15) O3C10C11C12 75.67(18) C9C10C11C12 167.68(14) C16C11C12C13 0.5(2) C10C11C12C13 171.75(13) C11C12C13C14 0.3(2) C12C13C14C15 0.3(2) C13C14C15C16 0.4(2) C14C15C16C11 1.2(2) C14C15C16C17 174.32(14) C12C11C16C15 1.2(2) C10C11C16C15 173.88(13) C12C11C16C17 175.05(12) C10C11C16C17 2.40(16) C15C16C17C9 167.34(14) C11C16C17C9 16.79(15) C18C9C17C16 88.09(12) C8C9C17C16 146.44(11) C10C9C17C16 27.92(13) C8C9C18C19 44.46(16) C17C9C18C19 171.10(11) C10C9C18C19 76.60(15) C8C9C18C26 137.25(13) C17C9C18C26 10.60(17) C10C9C18C26 101.70(15) C26C18C19C20 1.81(14) C9C18C19C20 179.67(11) C18C19C20C21 179.77(14) C18C19C20C25 1.34(15) C25C20C21C22 1.5(2) C19C20C21C22 177.24(14) C20C21C22C23 1.1(2) C21C22C23C24 0.3(2) C22C23C24C25 1.2(2) C23C24C25C20 0.7(2) C23C24C25C26 177.73(14) C21C20C25C24 0.6(2) C19C20C25C24 178.39(12) C21C20C25C26 179.41(12) C19C20C25C26 0.38(15) C19C18C26C25 1.57(15) C9C18C26C25 179.92(11) C24C25C26C18 179.32(14) C20C25C26C18 0.71(15) C32C27C28C29 1.2(2) C27C28C29C30 0.3(2) C28C29C30C31 0.4(2) C29C30C31C32 0.3(2) C28C27C32C31 1.2(2) C28C27C32C33 178.07(13) C30C31C32C27 0.5(2) C30C31C32C33 178.85(14) C27C32C33N1 86.99(16) C31C32C33N1 93.72(15) C27C32C33C34 35.36(18) C31C32C33C34 143.93(14)

(180) TABLE-US-00011 TABLE 7 Anisotropic atomic displacement parameters, (.sup.2), for compound 8 The anistropic atomic displacement factor exponent takes the form: 2.sup.2 [h.sup.2a*.sup.2 U.sub.11 + . . . + 2hka* b* U.sub.12 U.sub.11 U.sub.22 U.sub.33 U.sub.23 U.sub.13 U.sub.12 O1 0.0325(5) 0.0206(5) 0.0153(4) 0.0025(4) 0.0015(4) 0.0044(4) O2 0.0255(5) 0.0206(5) 0.0123(4) 0.0017(4) 0.0024(3) 0.0014(4) O3 0.0205(4) 0.0232(5) 0.0116(4) 0.0002(4) 0.0027(3) 0.0016(4) C1 0.0257(7) 0.0179(7) 0.0159(6) 0.0017(5) 0.0018(5) 0.0028(5) C2 0.0267(7) 0.0171(7) 0.0139(6) 0.0035(5) 0.0024(5) 0.0019(5) C3 0.0160(6) 0.0187(7) 0.0128(6) 0.0004(5) 0.0017(4) 0.0011(5) C4 0.0166(5) 0.0193(7) 0.0134(6) 0.0000(5) 0.0014(4) 0.0018(5) C5 0.0234(6) 0.0155(7) 0.0159(6) 0.0001(5) 0.0011(5) 0.0020(5) C6 0.0251(6) 0.0175(7) 0.0158(6) 0.0030(5) 0.0024(5) 0.0028(5) C7 0.0150(5) 0.0213(7) 0.0129(6) 0.0000(5) 0.0009(4) 0.0028(5) C8 0.0188(6) 0.0217(7) 0.0124(6) 0.0007(5) 0.0000(4) 0.0018(5) C9 0.0186(6) 0.0177(7) 0.0117(5) 0.0004(5) 0.0007(4) 0.0002(5) C10 0.0206(6) 0.0190(7) 0.0121(6) 0.0000(5) 0.0005(4) 0.0022(5) C11 0.0201(6) 0.0185(7) 0.0163(6) 0.0030(5) 0.0004(5) 0.0033(5) C12 0.0234(6) 0.0249(8) 0.0166(6) 0.0018(5) 0.0015(5) 0.0056(5) C13 0.0237(7) 0.0322(9) 0.0216(7) 0.0074(6) 0.0074(5) 0.0074(6) C14 0.0196(7) 0.0284(8) 0.0324(8) 0.0099(6) 0.0049(6) 0.0015(6) C15 0.0199(6) 0.0229(7) 0.0282(7) 0.0035(6) 0.0008(5) 0.0012(6) C16 0.0186(6) 0.0198(7) 0.0178(6) 0.0023(5) 0.0007(5) 0.0028(5) C17 0.0213(6) 0.0203(7) 0.0151(6) 0.0004(5) 0.0018(5) 0.0008(5) C18 0.0200(6) 0.0206(7) 0.0101(5) 0.0011(5) 0.0009(4) 0.0004(5) C19 0.0245(7) 0.0249(8) 0.0176(6) 0.0024(5) 0.0008(5) 0.0029(5) C20 0.0256(7) 0.0227(7) 0.0124(6) 0.0001(5) 0.0015(5) 0.0027(5) C21 0.0392(8) 0.0237(8) 0.0215(6) 0.0032(6) 0.0059(6) 0.0017(7) C22 0.063(2) 0.0236(16) 0.0226(13) 0.0024(11) 0.0090(13) 0.0165(15) C23 0.0359(8) 0.0356(9) 0.0240(7) 0.0034(6) 0.0049(6) 0.0140(7) C24 0.0253(7) 0.0331(9) 0.0225(7) 0.0050(6) 0.0034(5) 0.0047(6) C25 0.0253(7) 0.0253(8) 0.0129(5) 0.0003(5) 0.0016(5) 0.0035(5) C26 0.0277(7) 0.0248(8) 0.0197(6) 0.0005(6) 0.0069(5) 0.0012(6) C18A 0.0200(6) 0.0206(7) 0.0101(5) 0.0011(5) 0.0009(4) 0.0004(5) C19A 0.0245(7) 0.0249(8) 0.0176(6) 0.0024(5) 0.0008(5) 0.0029(5) C20A 0.0256(7) 0.0227(7) 0.0124(6) 0.0001(5) 0.0015(5) 0.0027(5) C21A 0.0392(8) 0.0237(8) 0.0215(6) 0.0032(6) 0.0059(6) 0.0017(7) C22A 0.0392(8) 0.0237(8) 0.0215(6) 0.0032(6) 0.0059(6) 0.0017(7) C23A 0.0359(8) 0.0356(9) 0.0240(7) 0.0034(6) 0.0049(6) 0.0140(7) C24A 0.0253(7) 0.0331(9) 0.0225(7) 0.0050(6) 0.0034(5) 0.0047(6) C25A 0.0253(7) 0.0253(8) 0.0129(5) 0.0003(5) 0.0016(5) 0.0035(5) C26A 0.0277(7) 0.0248(8) 0.0197(6) 0.0005(6) 0.0069(5) 0.0012(6) N1 0.0248(6) 0.0191(6) 0.0143(5) 0.0013(5) 0.0005(4) 0.0007(5) C27 0.0216(6) 0.0233(7) 0.0213(7) 0.0001(5) 0.0017(5) 0.0030(5) C28 0.0250(7) 0.0228(8) 0.0293(7) 0.0035(6) 0.0038(6) 0.0021(6) C29 0.0265(7) 0.0298(9) 0.0254(7) 0.0087(6) 0.0001(5) 0.0017(6) C30 0.0326(8) 0.0357(9) 0.0214(7) 0.0019(6) 0.0041(6) 0.0050(7) C31 0.0286(7) 0.0238(8) 0.0234(7) 0.0001(6) 0.0034(6) 0.0053(6) C32 0.0169(6) 0.0233(7) 0.0198(6) 0.0024(5) 0.0007(5) 0.0024(5) C33 0.0196(6) 0.0205(7) 0.0212(6) 0.0023(5) 0.0001(5) 0.0031(5) C34 0.0280(7) 0.0264(8) 0.0232(7) 0.0029(6) 0.0065(6) 0.0024(6)

APPENDIX 3

(181) Compound 1 4-((1-hydroxy-2,3-dihydro-1H,1H-2,2-biinden-2-yl)methyl)benzoic acid Compound 2 4-(((1S,2S)-1-hydroxy-2,3-dihydro-1H,1H-2,2-biinden-2-yl)methyl)benzoic acid Compound 3 4-(((1R,2R)-1-hydroxy-2,3-dihydro-1H,1H-2,2-biinden-2-yl)methyl)benzoic acid Compound 4 4-(((1R,2S)-1-hydroxy-2,3-dihydro-1H,1H-2,2-biinden-2-yl)methyl)benzoic acid Compound 5 4-(((1S,2R)-1-hydroxy-2,3-dihydro-1H,1H-2,2-biinden-2-yl)methyl)benzoic acid Compound 6 6-(Methylamino)hexane-1,2,3,4,5-pentanol 4-(((1S,2S)-1-hydroxy-2,3-dihydro-1H,1H-2,2-biinden-2-yl)methyl)benzoate Compound 7 6-(Methylamino)hexane-1,2,3,4,5-pentanol 4-(((1S,2R)-1-hydroxy-2,3-dihydro-1H,1H-2,2-biinden-2-yl)methyl)benzoate Compound 8 (S)-1-Phenylethylammonium 4-(((1S,2S)-1-hydroxy-2,3-dihydro-1H,-1H-2,2-biinden-2-yl)methyl)benzoate Compound 9 (R)-1-Phenylethylammonium 4-(((1R,2S)-1-hydroxy-2,3-dihydro-1H,1H-2,2-biinden-2-yl)methyl)benzoate