PHARMACOLOGICALLY ACTIVE COMPOUNDS

Abstract

The present invention relates to compounds of formula I shown below:

##STR00001##

wherein Q is as defined herein. The compounds of formula I act as selective positive allosteric modulators of strychnine-sensitive alpha 1-glycine receptors. The present invention further relates to the use of these compounds as therapeutic agents for the treatment and/or prevention of diseases or conditions in which strychnine-sensitive alpha 1-glycine receptor activity is implicated (such as, for example, chronic pain. The present invention also relates to processes for the preparation of these compounds and to pharmaceutical compositions comprising them.

Claims

1. A compound of formula (I) shown below: ##STR00032## wherein: Q is: wherein: indicates the point of attachment to the C(O) moiety of the compound of formula I; R.sub.1, R.sub.2a, R.sub.2b and R.sub.3 are each independently selected from hydrogen, halo, methyl, hydroxymethyl, CF.sub.3 and OCF.sub.3; or R.sub.2a and R.sub.2b are linked such that together they form a 4, 5 or 6-membered carbocyclic or heterocyclic ring; or a pharmaceutically acceptable salt thereof.

2. A compound according to claim 1, wherein R.sub.1, R.sub.2a, R.sub.2b and R.sub.3 are each independently selected from hydrogen, fluoro or methyl; or R.sub.2a and R.sub.2b are linked such that together they form a 4 or 5 membered carbocyclic or heterocyclic ring.

3. A compound according to claim 1, wherein R.sub.1, R.sub.2a, R.sub.2b and R.sub.3 are all hydrogen; or R.sub.2a and R.sub.2b are linked such that together they form a 4 or 5 membered heterocyclic ring.

4. A compound according to claim 1, wherein one or two of R.sub.1, R.sub.2a, R.sub.2b and R.sub.3 is a substituent other than hydrogen.

5. A compound according to claim 1, wherein R.sub.1 and R.sub.3 are hydrogen and one of R.sub.2a and R.sub.2b is fluoro.

6-18. (canceled)

19. A compound according to claim 1, which is selected from any one of the following: (3-fluoroazetidin-1-yl)(4-hydroxy-3,5-diisopropylphenyl) methanone; and (4-Hydroxy-3,5-diisopropylphenyl)(2-oxa-6-azaspiro[3.3]heptan-6-yl)methanone; or a pharmaceutically acceptable salt or solvate thereof.

20. A pharmaceutical composition comprising a compound according to claim 1, or a pharmaceutically acceptable salt thereof, in admixture with a pharmaceutically acceptable diluent or carrier.

21-23. (canceled)

24. A method of treating chronic pain or inducing anaesthesia in a patient in need of such treatment, said method comprising administering to said patient a therapeutically effective amount of a compound according to claim 1.

25. A method of synthesising a compound of claim 1, or a pharmaceutically acceptable salt or solvate thereof, the method comprising: a) reacting a compound of formula A ##STR00033## wherein X is a reactive group, for example chloro; and the hydroxyl group is optionally protected; with a compound of formula B:
H-Q wherein the H atom is attached to a nitrogen atom of the Q group.

26. A pharmaceutical composition comprising a compound according to claim 19, or a pharmaceutically acceptable salt thereof, in admixture with a pharmaceutically acceptable diluent or carrier.

27. A method of treating chronic pain or inducing anaesthesia in a patient in need of such treatment, said method comprising administering to said patient a therapeutically effective amount of a compound according to claim 19.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0192] The invention is described further in reference to the accompanying Figures, in which:

[0193] FIG. 1 shows the fractional potentiation (y-axis) against concentration (nM; x-axis) for the compound LT-01-25 in the expression and electrophysiology study described in Example 7 [Y axis: fractional potentiatione.g. 0.75 mean that if a response to glycine alone was 1 A then the response to glycine in the present of drug was 1.75 A];

[0194] FIGS. 2A and 2B show the effect of the compound LT-01-25 on paw withdrawal thresholds to (FIG. 2A) mechanical pressure in neuropathic rats and (FIG. 2B) cold (10 C.) stimulus in neuropathic rats compared with lamotrigine (see Example 8(i)); FIG. 3A shows the pharmacokinetic profile (plasma concentration (y-axis) versus time (x-axis)) for the LT-01-25 compound dosed at 10 mg/kg in 10% DMSO/10% Solutol/80% Saline in fasted and non-fasted rats;

[0195] FIG. 3B shows the pharmacokinetic profile (plasma concentration (y-axis) versus time (x-axis)) for the LT-01-25 compound dosed at 10 mg/kg in SSV in fasted and non-fasted rats;

[0196] FIG. 3C shows the pharmacokinetic profile (plasma concentration (y-axis) versus time (x-axis)) for the LT-01-25 and LT-01-89 (comparator) compounds dosed at 10 mg/kg in SSV in non-fasted rats.

[0197] FIGS. 4A and 4B show the comparison in effect of LT-01-25 (Example 1; 10 mg/kg p.o.) and LT-01-5 89 (30 mg/kg p.o.) on (FIG. 4A) ipsilateral paw withdrawal thresholds to mechanical pressure in neuropathic rats and (FIG. 4B) ipsilateral withdrawal latencies to a cold (10 C.) stimulus in neuropathic rats. Male, SD rats. n=6/group. Vehicle: 10% DMSO/10% Solutol HS15/80% saline.

[0198] FIG. 5 shows the effect of LT-01-26 (example 6) on percentage reversal to cold (10 C.) stimulus. In all cases fasted, male, Wistar rats were used (n=6/group). Vehicle: 10% DMSO/10% Solutol HS15/80% saline. 10 ml/kg p.o. One-way ANOVA, comparison with time-matched vehicle group using Tukey's HSD test * p<0.05, ** p<0.01, *** p<0.001.

[0199] FIGS. 6A and 6B show the efficacy of (FIG. 6A) LT-01-25 and (FIG. 6B) Gabapentin-on mechanical allodynia in Streptozocin (STZ) induced neuropathic rats. In all cases Wistar rats were used (n=2-6). Vehicle: 10% DMSO+1% Solutol+80% Saline, p.o. 10 mg/kg. The following administrative doses were used; Gabapentin: 30, 60 mg/kg in DSS, p.o. 10 ml/kg; and LT-01-25: 3, 10, 30 and 100 mg/kg in DSS, p.o. 10 ml/kg.

EXAMPLES

Example 1Synthesis of (4-(hydroxy)-3,5-diisopropylphenyl)(morpholino)methanone (LT-01-25)

Step 1Synthesis of 4-Hydroxy-3-5-diisopropylbenzaldehyde

[0200] ##STR00013##

[0201] Hexamethylenetetramine (15.8 g, 56 mmol) was added to a solution of 2,6-Diisopropylphenol (10.4 mL) in glacial acetic acid (50 mL) and H.sub.2O (10 mL). The resulting mixture was heated to reflux for 5 minutes and then short path distillation head was introduced and 10 ml of distillate was collected. The solution was allowed to continue refluxing for 6 hrs and the reaction was monitored by TLC. Upon completion of the reaction the solution was cooled to 00 C. and the resulting orange precipitation was isolated and washed with H.sub.2O (350 mL) to afford product as a pale orange solid (10.3 g, 89% yield). .sup.1H NMR (400 MHz, CDCl.sub.3) 9.86 (s, 1H), 7.63 (s, 2H), 5.49 (s, 1H), 3.21 (m, 2H), 1.31 (d, J=6.9 Hz, 12H). MS: C.sub.13H.sub.18O.sub.2 requires 206.3, found 206.3.

Step 2Synthesis of 4-(benzyloxy)-3,5-diisopropylbenzaldehyde

[0202] ##STR00014##

[0203] To a solution of 4-Hydroxy-3-5-diisopropylbenzaldehyde (4.18 g, 20.3 mmol) in acetone (50 mL) was added benzyl bromide (2.6 mL, 22.4 mmol) and potassium carbonate (5.6 g, 40.6 mmol). The resulting mixture was allowed to stir at room temperature for 18 hrs and the reaction was monitored by TLC. Upon completion the mixture was filtered through celite and the solvent was removed under vacuum. The product was purified by column chromatography (EtOAc/n-Hexane) to afford the product as an off white solid (5.3 g, 88% yield). .sup.1H NMR (400 MHz, CDCl.sub.3) 9.96 (s, 1H), 7.69 (s, 5H), 7.55-7.30 (m, 2H), 4.85 (s, 2H), 3.64-2.96 (m, 2H), 1.27 (d, J=6.9 Hz, 12H). MS: C.sub.20H.sub.24O.sub.2 requires: 319.1679, found: 319.1674

Step 3Synthesis of 4-(benzyloxy)-3,5-diisopropylbenzoic Acid

[0204] ##STR00015##

[0205] 4-(benzyloxy)-3,5-diisopropylbenzaldehyde (1.74 g, 5.87 mmol) was dissolved in THF (5 mL) under a blanket of N.sub.2. Selenium dioxide (325 mg, 2.94 mmol) was added to the solution along with Hydrogen peroxide (1.5 mL, 27 wt %) and the mixture was heated to reflux for 18 hrs. Upon completion Pd/C (10 mg) was added and the reaction mixture was allowed to stir for 10 mins. The mixture was filtered through Celite and the solvent was removed under vacuum. The product was purified by column chromatography (EtOAc/n-Hexane) to afford the product as a white solid (1.6 g, 85% yield). .sup.1H NMR (400 MHz, CDCl.sub.3) 7.92 (s, 2H), 7.57-7.27 (m, 5H), 4.85 (s, 2H), 3.49-3.24 (m, 2H), 1.27 (d, J=6.9 Hz, 12H). MS [M+Na].sup.+: C.sub.20H.sub.24O.sub.3 requires: 335.1625, found, 335.1623.

Step 4Synthesis of 4-(benzyloxy)-3,5-diisopropylbenzoyl Chloride

[0206] ##STR00016##

[0207] 4-(benzyloxy)-3,5-diisopropylbenzoic acid (200 mg, 0.6 mmol) was dissolved in DCM (5 mL) under a blanket of N.sub.2. Oxalyl chloride (0.12 mL, 0.72 mmol) was added along with DMF (1 drop from a pasture pipette). The reaction mixture was allowed to stir at room temperature for 2 hrs. The reaction was monitored by TLC and upon completion the solvent was removed under vacuum. The product was not isolated and was taken through crude.

Step 5Synthesis of (4-(benzyloxy)-3,5-diisopropylphenyl)(morpholino)methanone

[0208] ##STR00017##

[0209] Morpholine (67 L, 0.77 mmol) was added to a stirred solution of 4-(benzyloxy)-3,5-diisopropylbenzoyl chloride (200 mg, 0.64 mmol) in DCM (5 mL). Et.sub.3N (133 l, 9.6 mmol) was added and the resulting solution was allowed to stir at room temperature for 1.5 hours. The reaction was monitored by TLC and upon completion the reaction mixture was quenched with H.sub.2O (50 mL) and extracted with EtOAc (350 mL). The combined organic extracts were washed with brine, dried over MgSO.sub.4 and the solvent was removed under vacuum. The crude product was purified by column chromatography (EtOAc/n-Hexane) to afford the product as a white solid (192.9 mg 79% yield). .sup.1H NMR (400 MHz, CDCl.sub.3) 7.54-7.33 (m, 5H), 7.18 (s, 2H), 4.80 (s, 2H), 3.73 (s, 8H), 3.45-3.27 (m, 2H), 1.24 (d, J=6.9 Hz, 12H). MS [M+Na]+:C.sub.24H.sub.31NO.sub.3 requires: 404.2202, found: 404.2196.

Step 6Synthesis of (4-(benzyloxy)-3,5-diisopropylphenyl)(morpholino)methanone (LT-01-25)

[0210] ##STR00018##

[0211] (4-(benzyloxy)-3,5-diisopropylphenyl)(morpholino)methanone (520 mg, 1.4 mmol) was dissolved in MeOH (10 mL) under a blanket of H.sub.2. Pd/C (32 mg, 0.27 mmol) was added and the reaction mixture was allowed to stir for 18 hrs. Upon completion the reaction mixture was filtered through Celite and the solvent was removed under vacuum. The crude product was purified by column chromatography (EtOAc/n-Hexane) to afford the product as a white solid (346 mg, 85% yield). .sup.1H NMR (400 MHz, CDCl.sub.3) 7.14 (s, 2H), 5.06 (s, 1H), 3.71 (s, 8H), 3.15 (m, 2H), 1.26 (d, J=6.9 Hz, 12H). .sup.13C NMR (101 MHz, CDCl.sub.3) 171.74, 151.89, 134.12, 127.40, 123.58, 67.30, 27.47, 23.02. MS [M+Na]+:C.sub.17H.sub.25NO.sub.3 requires: 314.1732, found: 314.1725 CHN requires C: 70.07%, H: 8.65%, N: 4.81%, found C: 69.05%, H: 8.54%, N: 4.71%.

Example 2Synthesis of (R)-(4-(hydroxy)-3,5-diisopropylphenyl)(2-methylmorpholino)methanone

Synthesis of 4-(benzyloxy)-3,5-diisopropylbenzoyl Chloride

[0212] ##STR00019##

[0213] 4-(benzyloxy)-3,5-diisopropylbenzoic acid (200 mg, 0.6 mmol) was dissolved in DCM (5 mL) under a blanket of N.sub.2. Oxalyl chloride (0.12 mL, 0.72 mmol) was added along with DMF (1 drop from a pasture pipette). The reaction mixture was allowed to stir at room temperature for 2 hrs. The reaction was monitored by TLC and upon completion the solvent was removed under vacuum. The product was not isolated and was taken through crude.

Synthesis of (R)-(4-(Benzyloxy)-3,5-diisopropylphenyl)(2-methylmorpholino)methanone

[0214] ##STR00020##

[0215] (R)-2-Mehtylmorpholine hydrochloride (164 mg, 1.19 mmol) was added to a stirred solution of 4-(benzyloxy)-3,5-diisopropylbenzoyl chloride (264 mg, 0.80 mmol) in DCM (5 mL). Et.sub.3N (332 l, 2.4 mmol) was added and the resulting solution was allowed to stir at room temperature for 1.5 hours. The reaction was monitored by TLC and upon completion the reaction mixture was quenched with H.sub.2O (50 mL) and extracted with EtOAc (350 mL). The combined organic extracts were washed with brine, dried over MgSO.sub.4 and the solvent was removed under vacuum. The crude product was purified by column chromatography (EtOAc/n-Hexane) to afford the product as a white solid (151.7 mg 48% yield). .sup.1H NMR (400 MHz, CDCl.sub.3) 7.42 (m, 5H), 7.18 (s, 2H), 4.81 (s, 2H), 4.07-3.48 (m, 4H), 3.39 (m, 2H), 2.96 (m, 2H), 1.24 (d, J=6.9 Hz, 12H), 1.14 (s, 3H).

Synthesis of (R)-(4-(hydroxy)-3,5-diisopropylphenyl)(2-methylmorpholino)methanone

[0216] ##STR00021##

[0217] (4-(benzyloxy)-3,5-diisopropylphenyl)(morpholino)methanone (150 mg, 0.38 mmol) was dissolved in MeOH (10 mL) under a blanket of H.sub.2. Pd/C (32 mg, 0.27 mmol) was added and the reaction mixture was allowed to stir for 18 hrs. Upon completion the reaction mixture was filtered through Celite and the solvent was removed under vacuum. The crude product was purified by column chromatography (EtOAc/n-Hexane) to afford the product as an off white solid (111 mg, 96% yield). .sup.1H NMR (400 MHz, CDCl.sub.3) 7.13 (s, 2H), 5.17 (s, 1H), 3.74 (d, J=127.9 Hz, 4H), 3.22-3.07 (m, 2H), 2.74 (s, 1H), 1.26 (d, J=6.8 Hz, 12H), 1.18 (s, 3H). .sup.13C NMR (101 MHz, CDCl.sub.3) 171.43, 152.11, 134.14, 126.52, 123.21, 72.14, 66.68, 26.88, 22.68, 18.61. MS [M+Na].sup.+:C.sub.18H.sub.27NO.sub.3 requires: 328.1899, found: 328.1889. CHN requires C: 70.79%, H: 8.91%, N: 4.59%, found C: 70.56%, H: 8.55%, N: 4.61%

Example 3Synthesis of (3-Fluoroazetidin-1-yl)(4-hydroxy-3,5-diisopropylphenyl)methanone

Synthesis of 4-(benzyloxy)-3,5-diisopropylphenyl)(3-fluoroazetidin-1-yl)methanone

[0218] ##STR00022##

[0219] 3-Fluoroazetidine hydrochloride (2500 mg, 2.25 mmol) was added to a stirred solution of 4-(benzyloxy)-3,5-diisopropylbenzoyl chloride (prepared as described in Example 1; 500 mg, 1.5 mmol) in DCM (5 mL). Et.sub.3N (597 l, 4.5 mmol) was added and the resulting solution was allowed to stir at room temperature for 1.5 hours. The reaction was monitored by TLC and upon completion the reaction mixture was quenched with H.sub.2O (50 mL) and extracted with EtOAc (350 mL). The combined organic extracts were washed with brine, dried over MgSO.sub.4 and the solvent was removed under vacuum. The crude product was purified by column chromatography (EtOAc/n-Hexane) to afford the product as a white solid (387.4 mg 70% yield). .sup.1H NMR (400 MHz, CDCl.sub.3) 7.53-7.34 (m, 6H), 5.36 (dddd, J=56.8, 9.6, 6.2, 3.5 Hz, 1H), 4.58-4.46 (m, 2H), 4.45-4.30 (m, 2H), 3.44-3.33 (m, 2H), 1.24 (d, J=6.9 Hz, 12H).

Synthesis of (3-Fluoroazetidin-1-yl)(4-hydroxy-3,5-diisopropylphenyl)methanone

[0220] ##STR00023##

[0221] 4-(benzyloxy)-3,5-diisopropylphenyl)(3-fluoroazetidin-1-yl)methanone (387 mg, 1.05 mmol) was dissolved in MeOH (10 mL) under a blanket of H.sub.2. Pd/C (32 mg, 0.27 mmol) was added and the reaction mixture was allowed to stir for 18 hrs. Upon completion the reaction mixture was filtered through Celite and the solvent was removed under vacuum. The crude product was purified by column chromatography (EtOAc/n-Hexane) to afford the product as a white solid (223 mg, 76% yield).sup.1H NMR (400 MHz, CDCl.sub.3) 7.36 (s, 2H), 5.66 (s, 1H), 5.36 (dddd, J=56.8, 9.6, 6.2, 3.5 Hz, 1H), 4.58-4.26 (m, 4H), 3.26-3.12 (m, 2H), 1.25 (d, J=6.9 Hz, 12H). MS [M+H].sup.+:C.sub.16H.sub.22FNO.sub.3 requires: 280.1711, found: 280.1707.

Example 4Synthesis of (4-(Benzyloxy)-3,5-diisopropylphenyl)(piperidin-1-yl)methanone

Synthesis of 4-(benzyloxy)-3,5-diisopropylbenzoyl Chloride

[0222] ##STR00024##

[0223] 4-(benzyloxy)-3,5-diisopropylbenzoic acid (200 mg, 0.6 mmol) was dissolved in DCM (5 mL) under a blanket of N.sub.2. Oxalyl chloride (0.12 mL, 0.72 mmol) was added along with DMF (1 drop from a pasture pipette). The reaction mixture was allowed to stir at room temperature for 2 hrs. The reaction was monitored by TLC and upon completion the solvent was removed under vacuum. The product was not isolated and was taken through crude.

Synthesis of (4-(Benzyloxy)-3,5-diisopropylphenyl)(piperidin-1-yl)methanone

[0224] ##STR00025##

[0225] Piperidine (230 L, 2.25 mmol) was added to a stirred solution of 4-(benzyloxy)-3,5-diisopropylbenzoyl chloride (500 mg, 1.5 mmol) in DCM (5 mL). Et.sub.3N (310 l, 2.25 mmol) was added and the resulting solution was allowed to stir at room temperature for 1.5 hours. The reaction was monitored by TLC and upon completion the reaction mixture was quenched with H.sub.2O (50 mL) and extracted with EtOAc (350 mL). The combined organic extracts were washed with brine, dried over MgSO.sub.4 and the solvent was removed under vacuum. The crude product was purified by column chromatography (EtOAc/n-Hexane) to afford the product as an off white solid (546 mg 95% yield). .sup.1H NMR (400 MHz, CDCl.sub.3) 7.52-7.32 (m, 5H), 7.16 (s, 2H), 4.80 (s, 1H), 3.71 (s, 4H), 3.35-3.45 (m, 2H), 1.64 (d, J=43.1 Hz, 6H), 1.23 (d, J=6.9 Hz, 12H). [M+H].sup.+].sup.+:C.sub.25H.sub.34NO.sub.2 requires: 380.2590, found: 380.2575

Synthesis of (4-hydroxy-3,5-diisopropylphenyl)(piperidine-1-yl)methanone

[0226] ##STR00026##

[0227] (4-hydroxy-3,5-diisopropylphenyl)(piperidine-1-yl)methanone (520 mg, 1.40 mmol) was dissolved in MeOH (10 mL) under a blanket of H.sub.2. Pd/C (32 mg, 0.27 mmol) was added and the reaction mixture was allowed to stir for 18 hrs. Upon completion the reaction mixture was filtered through Celite and the solvent was removed under vacuum. The crude product was purified by column chromatography (EtOAc/n-Hexane) to afford the product as an off white solid (323 mg, 80% yield). .sup.1H NMR (400 MHz, CDCl.sub.3) 7.12 (s, 2H), 4.97 (s, 1H), 3.50 (s, 4H), 3.20-3.08 (m, 2H), 1.64 (d, J=33.5 Hz, 1H), 1.26 (d, J=6.9 Hz, 1H). MS [M+Na].sup.+:C.sub.18H.sub.28NO.sub.2 requires: 290.2120, found: 290.2123.

Example 5Synthesis of (4-Hydroxy-3,5-diisopropylphenyl)(2-oxa-6-azaspiro[3.3]heptan-6-yl)methanone

Synthesis of 4-Hydroxy-3-5-diisopropylbenzoic Acid

[0228] ##STR00027##

[0229] NaClO.sub.2 (1.3 g, 14.4 mmol) was added to a solution of 4-Hydroxy-3-5-diisopropylbenzaldehyde (1.0 g, 4.8 mmol) NaH.sub.2PO.sub.4 (2.2 g, 14.4 mmol) and 2-methyl-2-butene (9.5 mL, 2M in THF) in BuOH/H.sub.2O (1:1, 15 mL). The reaction was allowed to stir at room temperature for 16 hours. Upon completion the reaction mixture was diluted with Na.sub.2CO.sub.3 (50 mL) and was washed with EtOAc (50 mL). The aqueous layer was acidified to pH 1 (20 mL HCl, 1 M) and extracted with EtOAc (330 mL). The organic extracts were collected and dried over MgSO.sub.4 and concentrated under vacuum to afford the product. The product was purified column chromatography (EtOAc/n-Hexane) to give the product as a white solid (739 mg, 68% yield). .sup.1H NMR (400 MHz, CDCl.sub.3) 7.85 (s, 2H), 5.30 (s, 1H), 3.21-3.11 (m, 2H), 1.30 (d, J=6.8 Hz, 12H). .sup.13C NMR (101 MHz, CDCl.sub.3) 172.25, 155.07, 133.42, 126.77, 121.39, 27.27, 22.45.MS: C13H18O3 [M+NH.sub.4].sup.+ requires 240.2, found 240.2.

Synthesis of 4-(benzyloxy)-3,5-diisopropylbenzoic Acid

[0230] ##STR00028##

[0231] 4-(benzyloxy)-3,5-diisopropylbenzaldehyde (1.74 g, 5.87 mmol) was dissolved in THF (5 mL) under a blanket of N.sub.2. Selenium dioxide (325 mg, 2.94 mmol) was added to the solution along with hydrogen peroxide (1.5 mL, 27 wt %) and the mixture was heated to reflux for 18 hours. Upon completion Pd/C (10 mg) was added and the reaction mixture was allowed to stir for 10 mins. The mixture was filtered through Celite and the solvent was removed under vacuum. The product was purified by column chromatography (EtOAc/n-Hexane) to afford the product as a white solid (1.6 g, 85% yield). .sup.1H NMR (400 MHz, CDCl.sub.3) 7.92 (s, 2H), 7.57-7.27 (m, 5H), 4.85 (s, 2H), 3.40 (hept, J=6.8 Hz, 2H), 1.27 (d, J=6.8 Hz, 12H). .sup.13C NMR (101 MHz, CDCl.sub.3) 171.53, 158.06, 142.57, 137.06, 128.65, 128.19, 127.43, 126.77, 125.49, 76.51, 26.76, 23.93. MS [M+Na].sup.+: C.sub.20H.sub.24O.sub.3 requires: 335.1625, found: 335.1623

Synthesis of (4-(Benzyloxy)-3,5-diisopropylphenyl)(2-oxa-6-azaspiro[3.3]heptan-6-yl)methanone

[0232] ##STR00029##

[0233] To a solution of 4-(benzyloxy)-3,5-diisopropylbenzoic acid (100 mg, 0.32 mmol) in DMF (5 mL) was added HATU (180, 0.48 mmol), K.sub.2CO.sub.3 (220 mg, 1.6 mmol) and 2-oxa-6-azaspiro[3.3]heptan-6-ium carboxyformate (101 mg, 0.35 mmol). The resulting solution was allowed to stir for 1 hour. Upon completion the reaction mixture was quenched with H.sub.2O (20 mL) and extracted with EtOAc (330 mL). The combined organic extracts were washed with H.sub.2O (330 mL), brine (20 mL), dried over MgSO.sub.4 and the solvent was removed under vacuum. The crude product was purified by column chromatography (EtOAc/n-Hexane) to afford the product as a white solid (62 mg, 50% yield). .sup.1H NMR (400 MHz, CDCl.sub.3) 7.54-7.31 (m, 7H), 4.83 (d, J=11.7 Hz, 6H), 4.40 (d, J=42.4 Hz, 4H), 3.39 (hept, J=6.8 Hz, 2H), 1.25 (d, J=6.8 Hz, 12H). .sup.13C NMR (101 MHz, CDCl.sub.3) 170.82, 157.83, 142.63, 128.66, 128.31, 127.37, 126.60, 125.44, 72.92, 60.89, 53.37, 26.39, 24.02. MS [M+Na]+:C.sub.25H.sub.31NO.sub.3 requires: 416.2202, found: 416.2186.

Synthesis of (4-Hydroxy-3,5-diisopropylphenyl)(2-oxa-6-azaspiro[3.3]heptan-6-yl)methanone

[0234] ##STR00030##

[0235] (4-(Benzyloxy)-3,5-diisopropylphenyl)(2-oxa-6-azaspiro[3.3]heptan-6-yl)methanone (50 mg, 0.13 mmol) was reacted with Pd/C (15 mg, 0.12 mmol) according to the procedure described in paragraph [00163]. The crude product was purified by column chromatography (EtOAc/n-Hexane) to afford the product as a white solid (38 mg, 99% yield).sup.1H NMR (400 MHz, CDCl.sub.3) 7.35 (s, 2H), 5.43 (s, 1H), 4.82 (s, 4H), 4.39 (d, J=41.9 Hz, 4H), 3.16 (hept, J=6.8 Hz, 2H), 1.26 (d, J=6.8 Hz, 12H). .sup.13C NMR (101 MHz, CDCl.sub.3) 172.27, 154.00, 134.78, 125.74, 125.05, 82.10, 39.57, 28.22, 23.79. MS [M+H].sup.+:C.sub.18H.sub.26NO.sub.3 requires: 304.1907, found: 304.1906. CHN requires C: 71.26%, H: 8.31%, N: 4.62%, found C: 71.12%, H: 8.16%, N: 4.38%

Example 6Synthesis of (4-Hydroxy-3-5-diisopropylphenyl)(4-methylpiperazin-1-yl)methanone

[0236] ##STR00031##

[0237] 4-Hydroxy-3-5-diisopropylbenzoyl chloride (160 mg, 0.66 mmol) was reacted with N-methylpiperazine (0.088 mL, 0.80 mmol) according to general procedure 1 (below) to afford the product as a brown solid. The crude product was purified by trituration with EtOAc (109 mg, 55% yield). m.p.=154-156 C.: .sup.1H NMR (400 MHz, CDCl.sub.3) 7.13 (s, 2H), 5.18 (s, 1H), 3.74 (s, 4H), 3.16 (hept, J=6.8 Hz, 2H), 2.44 (s, 4H), 1.26 (d, J=6.8 Hz, 12H). .sup.13C NMR (101 MHz, CDCl.sub.3) 171.24, 151.59, 133.65, 127.18, 123.19, 65.87, 54.82, 45.78, 27.12, 22.62. MS: C.sub.18H.sub.28N.sub.2O.sub.2 requires: 305.2229, found: 305.2224. CHN requires C: 71.02%, H: 9.27%, N: 9.20%, found C: 68.44%, H: 9.03%, N: 8.39%.

General Procedure 1Amide Coupling

[0238] The appropriate morpholine derivative (1.2 eq) was added to a stirred solution of the acid chloride (1.0 eq) dissolved in DCM (10 mL/g). Et.sub.3N (1.5 eq) was added and the resulting solution was allowed to stir at room temperature for 1.5 hours. The reaction was monitored by TLC and upon completion the reaction mixture was quenched with H.sub.2O (50 mL) and extracted with EtOAc (350 mL). The combined organic extracts were washed with Na.sub.2CO.sub.3, dried over MgSO.sub.4 and the solvent was removed under vacuum.

Example 7Expression and Electrophysiological Characterization of Glycine and GABA Receptor Activity

[0239] Human embryonic kidney (HEK) 293 cells (ATCC CRL 1573) and/or Xenopus laevis oocytes were used for expressing the human al-3 glycine receptor subunit as well as human al, b3, g2 GABA receptor subunit cDNAs inserted into mammalian expression vectors. Patch- and voltage-clamp recording from receptor expressing cells was performed at a holding potential of 70 mV in normal Ringer solution and digitized for analyses. Dose-response curves of agonist induced peak currents (I) were normalized to the maximal current value obtained and fitted with the sigmoidal Hill equation using a Gauss Marquardt iteration, where EC50 represents the glycine/GABA concentration resulting in a half maximal response. Effects of the compounds tested on agonist induced currents were analysed after superfusing the cells with the respective compound for 5 seconds prior to and during agonist application and dose-response curves of modulated currents were determined in the presence of agonist concentrations eliciting a response corresponding to 20% of the maximal inducible current (EC20 value). Results represent meanss.d. and the significance of the data was evaluated using Student's paired t test and considered to be statistically significant at P<0.05.

[0240] The results are shown in FIG. 1 for the compound of Example 1 (LT-01-25).

[0241] The following EC.sub.50 data was obtained for the compounds of Examples 2 to 6:

TABLE-US-00001 Compound 1 glycine EC.sub.50 (nM) Example 2 0.06 +/ 0.01 Example 3 0.43 +/ 0.28 Example 5 0.00016 +/ 0.00004 Example 6 0.0012 +/ 0.0004

Example 8Biological Evaluation for LT-01-25 (Example 1)

(i) In Vivo Assessment of Paw Withdrawal Thresholds for LT-01-25 (Example 1)

Animals

[0242] All animal procedures were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986 and associated guidelines. Male Wistar rats (initial bodyweight 125-149 g; Harlan UK Ltd) were maintained in a controlled lighting environment, four to a cage and given food and water ad libitum.

Induction of Neuropathic Pain

[0243] Neuropathic pain was induced by partial ligation of the sciatic nerve. Briefly, the rats were anaesthetised (isoflurane/O.sub.2 inhalation), the left sciatic nerve exposed at mid-thigh level through a small incision and to of the nerve thickness tightly ligated within a 7.0 silk suture. The wound was closed with skin clips. Animals were allowed to recover and tested 12-15 days following surgery.

Behavioural Tests:

Mechanical Hyperalgesia

[0244] Mechanical hyperalgesia was examined in a model of neuropathic pain by measuring paw withdrawal thresholds (PWT) to increasing mechanical force applied to the dorsal surface of the rat paw using an Analgesymeter (Ugo-Basile, Milan) equipped with a wedge-shaped probe (area 1.75 mm.sup.2). Cut-off was set at 250 g and the end-point was taken as withdrawal of the hind paw. Both ipsilateral and contralateral paw withdrawal readings were taken.

Cold Sensitivity

[0245] Cold sensitivity was assessed using a commercially available cold-plate (Ugo Basile, Milan). The cold-plate was set according to pre-determined calibration data using a surface temperature probe to correlate set temperature to actual surface temperature over a wide temperature range (5 C. to 26 C.). The cold plate was allowed to stabilize for 5 minutes at the set temperature prior to testing. Paw withdrawal latencies were determined with the cold-plate set at 10 C. The animals were lightly restrained and each hind paw in turn placed onto the surface of the cold-plate. The end point was taken as the withdrawal of the paw and recorded as the withdrawal latency for the ipsilateral and the contralateral paw. A maximum cut-off of 30 seconds was used for each paw.

Testing Details and Data Handling

[0246] Withdrawal thresholds or latencies were measured on both the ipsilateral (ligated) and contralateral (non-ligated) paws. Treatment groups were randomised and blinded. Groups of 6 rats were used. Predose behavioural measurements were obtained by measuring paw withdrawals 14 days following nerve ligation; before the initiation of drug treatment. Following treatment, further readings were taken at 1, 2, 4, 6 and 24 hours after drug or vehicle administration.

[0247] Data were expressed as withdrawal threshold (g) or withdrawal latencies (s) and percentage reversals calculated according to the following formula:

[00001]$\%.Math..Math.\mathrm{reversal}=\frac{\mathrm{ipsilateral}.Math..Math.\mathrm{threshold}.Math..Math.\mathrm{postdose}-\mathrm{ipsilateral}.Math..Math.\mathrm{threshold}.Math..Math.\mathrm{predose}}{\mathrm{contralateral}.Math..Math.\mathrm{threshold}.Math..Math.\mathrm{predose}-\mathrm{ipsilateral}.Math..Math.\mathrm{threshold}.Math..Math.\mathrm{predose}}100$

General Observations

[0248] In addition to behavioural pain readings, each rat was observed throughout the study for changes in general behaviour.

Drug Administration

[0249] The compounds were made up in a vehicle containing 10% DMSO/10% Solutol HS15/80% saline. Rats were fasted overnight prior to dosing. The compounds were administered by oral gavage at 10 ml/kg bodyweight. Control animals received vehicle alone.

[0250] Compound or vehicle solutions were coded and allocated randomly to the animals (coded A-F) at predose. Data was uncoded and sorted at the end of the experiment.

Statistical Analysis

[0251] Statistical analysis was carried out on withdrawal threshold readings using ANOVA with repeated measures followed by post-hoc analysis using Tukey's HSD test. The level for statistical significance was set as p<0.05.

Results

[0252] The results are shown in FIG. 2.

[0253] The LT-01-25 compound produced a marked and long-lasting reversal of both the cold (10 C. cold plate) and mechanical (paw pressure) parameters. All three doses of the LT-01-25 compound showed good and dose-related efficacy. Peak reversal of 90% with mechanical occurred at 3 h and 93% with cold occurred at 1 h. The positive control, lamotrigine, gave reversals of 65% and 72% in mechanical and cold respectively.

[0254] LT-01-25 therefore showed a significant increase in contralateral paw withdrawal thresholds to mechanical pressure. At 30 mg/kg, LT-01-25 produced increased contralateral paw withdrawal latencies to cold.

[0255] There were no apparent drug-induced behavioural side effects.

(ii) Pharmacokinetic Evaluation

[0256] CompoundsLT-01-25 (Example 1) and LT-01-89 [a comparator: (4-(hydroxy)-3,5-di-tertbutyl)(morpholino)methanone]
Dose10 mg/kg (5 mL/kg)

RouteORAL

Vehicles10% DMSO/10% Solutol and SSV

Time24 h

Procedure Number6

Vehicles

10% DMSO/10% Solutol/80% Saline

[0257] 1) Compound was weighed out into a clean vial. [0258] 2) DMSO was added into the vial; the vial was vortexed and sonicated it for 15 min. [0259] 3) Solutol HS 15 was heated at 50 C. until a liquid formed, and then was added into the vial. The vial was vortexed for 1-2 min. [0260] 4) 0.9% Saline solution was added to the vial. The vial was vortexed and then sonicated for 10 min.

Standard Suspension Vehicle (SSV)

[0261] 1) A solution of SSV was made using 0.5% Sodium carboxymethylcellulose, 0.5% Benzyl alcohol, 0.4% Tween80 and 98.6% saline (0.9%) [0262] 2) Compound was weighed out into a clean vial [0263] 3) SSV solution was added into the vial; the vial was vortexed and sonicated it for 15 min.

Fasted Rats

[0264] Fasted rats were fasted overnight and given access to food immediately after dosing.

Protocol

[0265] 1) Rats were manually restrained and given a 10 mg/kg oral dose of compound at 5 mL/kg. [0266] 2) At designated time points (0.25, 0.5, 1, 3, 5, 7 and 24 hours) the rats were anaesthetised using isoflurane. [0267] 3) Approximately 300 L of blood was taken from the tail vein using a 1.5 inch needle treated with heparin and collected into 1.5 mL eppendorf tubes. [0268] 4) After sampling, pressure was applied to the puncture until bleeding ceased. The animals were returned to their cages and given free access to food and water. [0269] 5) Blood samples were stored on ice and then centrifuged at 13,000 rpm for 15 min to separate the plasma. [0270] 6) 150 L of the plasma was treated with 300 L of ACN:MeOH (1:1) solution containing the internal standard. The samples were then centrifuged for a further 15 min at 13,000 rpm. [0271] 7) 200 L of the supernatant was collected and placed in LC-MS vials for analysis. All samples were stored at 80 C.

Results

[0272] The results for the LT-01-25 compound dosed at 10 mg/kg in 10% DMSO/10% Solutol/80% Saline in fasted and non-fasted rats are shown in FIG. 3A and in Table 1 shown below.

TABLE-US-00002 TABLE 1 PK Parameters Non-Fasted 10% DMSO Fasted 10% DMSO Half Life (hr) 1.4 1.4 Cmax (ng/mL) 1710.8 2415.5 Tmax (hr) 4 0.3 AUC (area) (ng-hr/mL) 20421.4 8532.8 Vd (area)/kg (mL/kg) 973.7 2300.6 CL (area)/kg (mL/hr/kg) 505.4 1171.9

[0273] The results for the LT-01-25 compound dosed at 10 mg/kg in SSV in fasted and non-fasted rats are shown in FIG. 3B and in Table 2 shown below.

TABLE-US-00003 TABLE 2 PK Parameters Non-Fasted SSV Fasted SSV Half Life (hr) 3.62 1.4 Cmax (ng/mL) 2124 2592.6 Tmax (hr) 0.25 1 AUC (area) (ng-hr/mL) 19032.8 18130 Vd (area)/kg (mL/kg) 2710.9 1142.6 CL (area)/kg (mL/hr/kg) 519.7 551.6

[0274] The results for the LT-01-25 and LT-01-89 (comparator) compounds dosed at 10 mg/kg in SSV in non-fasted rats are shown in FIG. 3C and in Table 3 shown below.

TABLE-US-00004 TABLE 3 PK Parameters L1-01-89 LT-01-25 Half Life (hr) 20.4 3.6 Cmax (ng/mL) 471.2 2124.0 Tmax (hr) 1.2 0.3 AUC (area) (ng-hr/mL) 5931.7 19032.8 Vd (area)/kg (mL/kg) 32729.9 2710.9 CL (area)/kg (mL/hr/kg) 1076.8 519.7
(iii) In Vivo Model of Neuropathic Pain: Reversal of Tactile Allodynia

[0275] A comparison of LT-01-25 (Example 1) and L1-01-89 was conducted using the following protocol:

Summary

[0276] 1) In rats a peripheral neuropathy was induced by partial ligation of the sciatic nerve in one hind limb. [0277] 2) Two weeks (12-15 days) after induction of peripheral neuropathy stable mechanical (tactile) allodynia was induced in the hind paw of the affected limb. [0278] 3) Five treatment groups of male SD rats (n=8) were used: vehicle control (drug formulation), 3 drug doses, and a positive control, lamotrigine (30 mg/kg). Animals were randomized between groups and the experiment was carried out using blinded conditions. The study was split with n=4/group in each experiment. [0279] 4) Baseline behavioural measurements were obtained prior to surgery and at intervals post-surgery. Predose behavioural measurements were obtained by measuring paw withdrawal thresholds 12-15 days following nerve ligation. [0280] 5) Compound efficacy was determined by measuring paw withdrawal thresholds at specified intervals following vehicle/compound treatment.

Methods

Animals

[0281] All animal procedures were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986 and associated guidelines. Animals were maintained in a controlled lighting environment and given food and water ad libitum. Male Sprague Dawley rats (120-140 g at time of surgery) were used.

Drug Administration

[0282] Rats were fasted overnight with free access to water and fed 4 hours post-dose.

[0283] Tests compounds as well as Lamotrigine (30 mg/kg, volume: 10 ml/kg)) were prepared in the designated formulation (10% DMSO, Solutol HS 15/80% and 0.9% saline for p.o. administration) and administered via the chosen route.

Induction of Neuropathic Tactile Allodynia

[0284] Allodynia was examined in the model of neuropathic pain induced by partial ligation of the sciatic nerve as described by Seltzer et al (1990). Rats were anaesthetised (isoflurane/O2 inhalation), the left sciatic nerve was exposed at mid-thigh level through a small incision and to of the nerve thickness tightly ligated within a 7.0 silk suture. The wound was closed with skin clips. Animals were allowed to recover and compounds administered 12-15 days following surgery.

Behavioural Tests

[0285] Tactile allodynia was assessed by measuring withdrawal thresholds to calibrated von Frey hairs. As a force higher than 15 g can lift the paw as well as eliciting a response, 15 g represented the cut-off point. Animals were placed into a perspex chamber with metal grid floor giving access to the underside of their paws and allowed to acclimatise prior to the start of the experiment. Tactile allodynia was tested by touching the plantar surface of the hind paw with von Frey hairs in ascending order of force for up to 6 seconds. A positive response was noted if the paw is sharply withdrawn or there was flinching upon removal of the hair. Once a positive withdrawal response had been established, the paw was re-tested, starting with the next descending von Frey hair until no response occurred. The lowest amount of force required to elicit a response was recorded as the paw withdrawal threshold (in grams).

[0286] Data were also expressed as percentage of the maximum possible effect (% MPE) defined as:

[0287] Allodynia was measured on both the ipsilateral (ligated) and contralateral (non-ligated) paw prior to (pre-dose) and at a set time point following compound or vehicle administration (post-dose). Treatment groups were randomised and blinded. Groups of eight rats were used.

[0288] Predose behavioural measurements were obtained by measuring paw withdrawal thresholds 12-15 days following nerve ligation; before the initiation of drug treatment.

[0289] Compound/vehicle were administered at specified doses. Following treatment, further readings were taken; 1, 3, 6 and 24 hour after p.o. administration.

Statistical Analysis

[0290] Raw data were analysed using parametric statistical tests, including one-way analysis of variance (ANOVA) followed by Tukey's post hoc test repeated measures of ANOVA.

[0291] P<0:05 was set as the level of statistical significance.

[0292] Reference: Seltzer Z, Dubner R, Shir Y. A novel behavioural model of neuropathic pain disorders produced in rats by partial sciatic nerve injury. Pain 1990; 43:205218.

Results

[0293] The results are shown in FIG. 4 and Table 4 below:

TABLE-US-00005 TABLE 4 comparison of LT-01-25 and L1-01-89 LT-01-25 (10 mg/kg) LT-01-89 (30 mg/kg) Mechanical pressure 1 hr - - - 80% 1 hr - - - 70% Percentage reversal of predose 3 hr - - - 90% 3 hr - - - 50% hyperalgesia 6 hr - - - 40% 6 hr - - - 20% Cold (10 C.) Stimulus 1 hr - - - 85% 1 hr - - - 70% Percentage reversal of predose 3 hr - - - 35% 3 hr - - - 65% Hyperalgesia after 1 hr 6 hr - - - 35% 6 hr - - - 10%

(iv) Cerebrospinal Fluid (CSF) and Plasma Levels

[0294]

TABLE-US-00006 Test system SD rat, 243-272 g, male, N = 9, purchased from SLAC Laboratory Animal Co. LTD Food status Fasted overnight, free access to water and fed 4 hr post dose. Administration PO: 3 mg/kg (10 mL/kg) via oral gavage (N = 9) Blood collection The animal was restrained manually at the designated time points, approximately 150 L of blood sample was collected via cardioac puncture into EDTA-2K tubes. The blood samples were maintained in wet ice first and centrifuged to obtain plasma (2000 g, 4 C., 10 min) within 30 minutes post sampling. CSF collection 1. The animal was euthanized under pure CO.sub.2. 2. The CSF was collected by direct puncture of butterfly needle into the cisterna magna, using the occipital bone and the wings of the atlas as landmarks. 3. A piece of white paper was used as a background to monitor color change in the sample just above the needle during collection. 4. Upon observation of color change, the PE tubing was quickly clamped off above the color change and cut just above the clamped site. The clear sample is drawn into the syringe. Sample storage and Plasma samples will be stored in dry ice temporarily and transferred into- disposition 80 C. freezer for long term preservation. The backup samples will be discarded after 3 weeks unless specified. The unused dosing solutions will be stored at 4 C. fridge and discarded within 1 week after completion of the study.

Results

[0295] The results are shown in Table 5 below.

TABLE-US-00007 TABLE 5 CSF data for LT-01-25 and L1-01-89 (comparator) LT-01-25 LT-01-89 CSF levels (p.o. dose 3 95 ng/mL 27 ng/mL mg/kg after 2 hr) (900 EC.sub.50) (180 EC.sub.50)

(v) Microsomal Stability Data

[0296] Rat and human microsomal stability data was obtained using well established protocols known to those skilled in the art.

[0297] The human microsomal stability protocol is detailed below.

Human Microsomal Data

Incubation Methods

[0298] The metabolic stability assay was performed by incubating each test compound (1 M) with human liver microsomes in duplicate at 37 C. and 0.4 mg/mL protein concentration. The metabolic reaction was initiated by the addition of a NADPH-regenerating system (i.e. NADPH is the cofactor required for CYP450-mediated metabolism) and quenched at various time points over the 60 minute incubation period by the addition of acetonitrile. Control samples (containing no NADPH) were included (and quenched at 2, 30 and 60 minutes) to monitor for potential degradation in the absence of cofactor. Samples were analysed by UPLC-MS (Waters/Micromass Xevo G2 QTOF) under positive electrospray ionisation and MS spectral data acquired in a mass range of 80 to 1200 Daltons.

Calculations

[0299] Test compound concentration versus time data were fitted to an exponential decay function to determine the first-order rate constant for substrate depletion. In cases where clear deviation from first-order kinetics was evident, only the initial linear portion of the profile was utilised to determine the degradation rate constant (k). Each substrate depletion rate constant was then used to calculate: [1] a degradation half-life, [2] an in vitro intrinsic clearance value (CLint, in vitro); [3] a predicted in vivo hepatic intrinsic clearance value (CLint); [4] a predicted in vivo blood clearance value (CLblood); and [5] a predicted in vivo hepatic extraction ratio (EH).

TABLE-US-00008 [1] [00002]$t_{1/2}=\frac{\ln \left(2\right)}{k}$ [2] [00003]${\mathrm{CL}}_{\mathrm{int},.Math.\mathrm{in}.Math..Math.\mathrm{vitro}}=\frac{k}{\mathrm{microsomal}.Math..Math.\mathrm{protein}.Math..Math.\mathrm{content}.Math..Math.\left(0.4.Math..Math.\mathrm{mg}.Math..Math.\mathrm{protein}.Math.\text{/}.Math.\mathrm{mL}\right)}$ [3].sup.* [00004]${\mathrm{CL}}_{\mathrm{int}}={\mathrm{CL}}_{\mathrm{int},.Math.\mathrm{in}.Math..Math.\mathrm{vitro}}\frac{\mathrm{liver}.Math..Math.\mathrm{mass}.Math..Math.\left(g\right)}{\mathrm{body}.Math..Math.\mathrm{weight}.Math..Math.\left(\mathrm{kg}\right)}\frac{\mathrm{mg}.Math..Math.\mathrm{microsomal}.Math..Math.\mathrm{protein}}{g.Math..Math.\mathrm{liver}.Math..Math.\mathrm{mass}}$ [4].sup.* [00005]${\mathrm{CL}}_{\mathrm{blood}}=\frac{Q{\mathrm{CL}}_{\mathrm{int}}}{Q+{\mathrm{CL}}_{\mathrm{int}}}$ [5].sup.* [00006]$E_H=\frac{{\mathrm{CL}}_{\mathrm{blood}}}{Q}=\frac{{\mathrm{CL}}_{\mathrm{int}}}{Q+{\mathrm{CL}}_{\mathrm{int}}}$ .sup.*The following scaling parameters.sup.a were assumed in the above calculations: Liver Mass Microsomal Hepatic blood flow (Q) (g liver/kg protein (mL/min/kg Species body weight) (mg/g liver mass) body weight) Human 25.7 32 20.7 .sup.aRing et al. (2011) Journal of Pharmaceutical Sciences, 100:4090-4110. NOTE: Calculations of intrinsic clearance are based on the in vitro T.sub.1/2 method (Obach, 1999, Drug Metab. Dispos. 27: 1350-1359), which assumes: 1) The substrate concentration employed is well below the apparent K.sub.M for substrate turnover; and, 2) There is no significant product inhibition, nor is there any mechanism-based inactivation of enzyme. The use of hepatic microsomes in the prediction of the in vivo hepatic extraction ratio has two further inherent assumptions (Obach, 1999) which: 1) NADPH-dependent oxidative metabolism predominates over other metabolic routes (i.e. direct conjugative metabolism, reduction, hydrolysis, etc.); and, 2) Rates of metabolism and enzyme activities in vitro are truly reflective of those that exist in vivo. Data should be considered within these terms of reference.

[0300] The limit of sensitivity of this assay corresponds to 15% loss of compound over the assay duration. For compounds showing <15% loss over 60 minutes (i.e. degradation half-life >247 min), metabolic stability parameters based on 0.4 mg/mL microsomal protein concentration are reported as below:

TABLE-US-00009 In vitro CL.sub.int Microsome-Predicted Species (L/min/mg protein) E.sub.H Human <7 <0.22 Rat <7 <0.15 Mouse <7 <0.13

Results

[0301] The results for the rat and human microsomal studies are shown in Table 6 below:

TABLE-US-00010 TABLE 6 Rat and human microsomal data for LT-01-25 and L1-01-89 (comparator) Microsome used LT-01-25 L1-01-89 Rat microsomes 741 min, 121 min (1 M) (t.sub.1/2), CI 3.35 mL/min/kg 20.38 mL/min/kg 1155 min (t.sub.1/2)* 165 min (t.sub.1/2)* 1.07 mL/min/kg (CI)* 5.26 mL/min/kg (CI)* human microsomes 495 min (t.sub.1/2)* 138.6 min (t.sub.1/2)* (1 M) (t.sub.1/2), CI 2.7 mL/min/kg (CI)* 6.2 mL/min/kg (CI)*

Example 9Biological Evaluation of LT-01-26 (Example 6)

[0302] The effect of LT-01-26 on percentage reversal to cold (10 C.) stimulus was investigated using the protocol described in Example 8, section (iii) above. In all cases fasted, male, Wistar rats were used (n=6/group). The vehicle was 10% DMSO/10% Solutol HS15/80% saline. 10 ml/kg p.o. was administered. The results were subject to one-way ANOVA, comparison with time-matched vehicle group using Tukey's HSD test * p<0.05, ** p<0.01, *** p<0.001.

Results

[0303] The results are shown in FIG. 5.

Example 10Streptozocin-Induced Diabetic Neuropathic Pain Model

[0304] The efficacy of LT-01-25 on mechanical allodynia in Streptozocin (STZ) induced neuropathic rats was measured using the methodology described below. Comparative data for the efficacy of Gabapentin (Gbp) was also generated.

Method

[0305] Mechanical allodynia was measured using hindpaw withdrawal threshold (PWT) using von Frey hair test. In all cases Wistar rats (200 g, SLAC) were used. Streptozocin, obtained from Sigma, was injected intraperitoneally (IP injection) at time zero (0 days). An effect amount of 60 mg/kg of Streptozocin was injected. PWT and blood glucose tests were then measured after 7 days (blank (BL)) before the vehicle or compound was administered and further PWT measurements were taken at either 1, 3, 4 and 6 hours, 1, 3 and 6 hours or 3, 6, 9 and 12 hours after vehicle or compound administration.

Compounds Used:

[0306] Vehicle II (DSS): 10% DMSO+1% Solutol+80% Saline, p.o. 10 mg/kg
Gabapentin: 30, 60 mg/kg in DSS, p.o. 10 ml/kg
LT-01-25: 3, 10, 30 and 100 mg/kg in DSS, p.o. 10 ml/kg

Results

[0307] The results are shown in FIGS. 6(a) and 6(b).