Cyclic triazo and diazo sodium channel blockers

Abstract

A method of treating a disorder. The method includes administering to a subject in need thereof a compound of general formula (I): ##STR00001##
Each of A, N*, X, Y, R1, and R2 is defined herein. Also disclosed are compounds of the formula and a pharmaceutical composition containing such a compound.

Claims

1. A method of treating a disorder comprising administering to a subject in need thereof a compound of general formula (I), or a salt, solvate or tautomer thereof, ##STR00050## in which: X and Y are each N; A is selected from ditrifluoromethylphenyl, methoxyphenyl, ethoxyphenyl, dimethoxyphenyl, diethoxyphenyl, trimethoxyphenyl, triethoxyphenyl, fluoromethoxyphenyl, fluoroethoxyphenyl, difluoromethoxyphenyl, difluoroethoxyphenyl, trifluoromethoxyphenyl, trifluoroethoxyphenyl, and tetrafluoroethoxyphenyl; R1 is hydrogen or a substituent group selected from C.sub.1-10 alkyl, C.sub.2-10 alkenyl, C.sub.1-3 alkyl-aryl, C.sub.1-3 alkyl-heterocyclyl and C.sub.3-10 cycloalkyl, optionally substituted by hydroxy, halogen, carboxamido, halo C.sub.1-6 alkyl, C.sub.1-6 alkyl or C.sub.1-6 alkoxy, or the Y nitrogen is unsubstituted; R2 is amino or a substituent group selected from C.sub.1-6 alkyl groups and phenyl groups, in which the substituent group is optionally substituted by hydroxy, halogen, halo-C.sub.1-6 alkyl, C.sub.1-6 alkyl or C.sub.1-6 alkoxy; and N* is a group NRaRb where Ra and Rb are independently H or a C.sub.1-6 alkyl group; or N* is a piperazinyl ring optionally substituted by hydroxy, halogen, halo-C.sub.1-6 alkyl, C.sub.1-6 alkyl or C.sub.1-6 alkoxy; wherein the disorder is selected from epilepsy, multiple sclerosis, neuropathic pain, and motor neurone disease.

2. The method according to claim 1, wherein A is selected from 3,5-ditrifluoromethylphenyl, 4,5 dimethoxyphenyl, 3,4,5 trimethoxyphenyl, 2-fluoromethoxyphenyl, 2-fluoroethoxyphenyl, 4-fluoromethoxyphenyl, 4-fluoroethoxyphenyl, 2,4-difluoromethoxyphenyl and 2,4-difluoroethoxyphenyl.

3. The method according to claim 1, wherein the compound of formula (I) is selected from the group consisting of: 3,5-Diamino-6-(3,4,5 trimethoxyphenyl)-1,2,4-triazine [CEN-095]; 3,5-Diamino-6-(3,5-bistrifluoromethylphenyl)-1,2,4-triazine [CEN-092]; 3,5-Diamino-6-(3,4-dimethoxyphenyl)-1,2,4-triazine [CEN-115]; 3,5-Diamino-6-(2-trifluoromethoxyphenyl)-1,2,4-triazine [CEN-056]; 3,5-Diamino-6-[3-(1,1,2,2-tetrafluoroethoxy)phenyl]-1,2,4-triazine [CEN-108]; 3,5-Diamino-6-[2-(1,1,2,2-tetrafluoroethoxy)phenyl]-1,2,4-triazine [CEN-137]; 3,5-Diamino-6-[2,5-bis(2,2,2-trifluoroethoxy)phenyl]-1,2,4-triazine [CEN-140]; 3,5-Diamino-6-[2-difluoromethoxy)phenyl]-1,2,4-triazine [CEN-142]; 3,5-Diamino-6-(3,5-dimethoxyphenyl)-1,2,4-triazine [CEN-192]; 3,5-Diamino-6-[3,5-bis(2,2,2-trifluoroethoxy)phenyl]-1,2,4-triazine [CEN-193]; 3,5-Diamino-6-[2,5-bis(trifluoromethyl)phenyl]-1,2,4-triazine [CEN-198]; and salts, solvates and tautomers thereof.

4. The method according to claim 3, wherein the compound of formula (I) is 3,5-Diamino-6-(3,5-bistrifluoromethylphenyl)-1,2,4-triazine [CEN-092], or a salt, solvate or tautomer thereof.

5. The method according to claim 1, wherein the disorder is epilepsy.

6. The method according to claim 3, wherein the disorder is epilepsy, multiple sclerosis, motor neurone disease, or neuropathic pain.

7. The method according to claim 4, wherein the disorder is epilepsy, multiple sclerosis, motorneurone disease, or neuropathic pain.

8. The method according to claim 1, in which the subject is a human.

9. A compound of general formula (I), or a salt, solvate or tautomer thereof, ##STR00051## in which: X and Y are each N; A is selected from ditrifluoromethylphenyl, methoxyphenyl, ethoxyphenyl, dimethoxyphenyl, diethoxyphenyl, trimethoxyphenyl, triethoxyphenyl, fluoromethoxyphenyl, fluoroethoxyphenyl, difluoromethoxyphenyl, difluoroethoxyphenyl, trifluoromethoxyphenyl, trifluoroethoxyphenyl, and tetrafluoroethoxyphenyl; R1 is hydrogen or a substituent group selected from C.sub.1-10 alkyl, C.sub.2-10 alkenyl, C.sub.1-3 alkyl-aryl, C.sub.1-3 alkyl-heterocyclyl and C.sub.3-10 cycloalkyl, optionally substituted by hydroxy, halogen, carboxamido, halo C.sub.1-6 alkyl, C.sub.1-6 alkyl or C.sub.1-6 alkoxy, or the Y nitrogen is unsubstituted; R2 is amino or a substituent group selected from C.sub.1-6 alkyl groups and phenyl groups, in which the substituent group is optionally substituted by hydroxy, halogen, halo-C.sub.1-6 alkyl, C.sub.1-6 alkyl or C.sub.1-6 alkoxy; and N* is a group NRaRb where Ra and Rb are independently H or a C.sub.1-6 alkyl group; or N* is a piperazinyl ring optionally substituted by hydroxy, halogen, halo-C.sub.1-6 alkyl, C.sub.1-6 alkyl or C.sub.1-6 alkoxy.

10. The compound according to claim 9, wherein A is selected from 3,5-ditrifluoromethylphenyl, 4,5 dimethoxyphenyl, 3,4,5 trimethoxyphenyl, 2-fluoromethoxyphenyl, 2-fluoroethoxyphenyl, 4-fluoromethoxyphenyl, 4-fluoroethoxyphenyl, 2,4-difluoromethoxyphenyl and 2,4-difluoroethoxyphenyl.

11. The compound according to claim 9, selected from the group consisting of: 3,5-Diamino-6-(3,4,5 trimethoxyphenyl)-1,2,4-triazine [CEN-095]; 3,5-Diamino-6-(3,5-bistrifluoromethylphenyl)-1,2,4-triazine [CEN-092]; 3,5-Diamino-6-(3,4-dimethoxyphenyl)-1,2,4-triazine [CEN-115]; 3,5-Diamino-6-(2-trifluoromethoxyphenyl)-1,2,4-triazine [CEN-056]; 3,5-Diamino-6-[3-(1,1,2,2-tetrafluoroethoxy)phenyl]-1,2,4-triazine [CEN-108]; 3,5-Diamino-6-[2-(1,1,2,2-tetrafluoroethoxy)phenyl]-1,2,4-triazine [CEN-137]; 3,5-Diamino-6-[2,5-bis(2,2,2-trifluoroethoxy)phenyl]-1,2,4-triazine [CEN-140]; 3,5-Diamino-6-[2-difluoromethoxy)phenyl]-1,2,4-triazine [CEN-142]; 3,5-Diamino-6-(3,5-dimethoxyphenyl)-1,2,4-triazine [CEN-192]; 3,5-Diamino-6-[3,5-bis(2,2,2-trifluoroethoxy)phenyl]-1,2,4-triazine [CEN-193]; 3,5-Diamino-6-[2,5-bis(trifluoromethyl)phenyl]-1,2,4-triazine [CEN-198]; and salts, solvates and tautomers thereof.

12. The compound according to claim 9, which is 3,5-Diamino-6-(3,5-bistrifluoromethylphenyl)-1,2,4-triazine [CEN-092], or a salt, solvate or tautomer thereof.

13. A pharmaceutical composition comprising a compound of claim 9 and a pharmaceutically acceptable carrier.

14. A pharmaceutical composition comprising a compound of claim 10 and a pharmaceutically acceptable carrier.

15. A pharmaceutical composition comprising a compound of claim 11 and a pharmaceutically acceptable carrier.

16. A pharmaceutical composition comprising a compound of claim 12 and a pharmaceutically acceptable carrier.

17. The method according to claim 1, wherein the disorder is multiple sclerosis.

18. The method according to claim 1, wherein the disorder is motor neurone disease.

19. The method according to claim 1, wherein the disorder is neuropathic pain.

20. The compound according to claim 9, wherein both R2 and N* are NH.sub.2.

Description

EXPERIMENTAL PROCEDURES

Preparation of Rat Forebrain Synaptosomes and Homogenates

(1) Experiments were performed using forebrain (whole brain less cerebellum/medulla) from Male Wistar rats weighing 175-250 g. All efforts were made to reduce the number of animals used and all experiments were carried out in accordance with the UK Animals (Scientific Procedures) Act, 1986 and the European Community Council Directive of 24 Nov. 1986 (86/609/EEC). Following killing of animals by stunning and decapitation, the forebrain (whole brain less cerebellum/medulla) was rapidly dissected and transferred to a weighed tube containing ice-cold 0.25M sucrose.

(2) Synaptosomes (heavy and light mitochondrial fraction containing synaptosomes) were prepared by transferring the forebrain (of known wet weight) to a glass Potter vessel to which 9 volumes ice-cold 0.25M sucrose had been added and homogenising, using a teflon pestle, by 8 up and down strokes of a Braun Potter S motor driven homogeniser set to 900 rpm. The resulting homogenate was centrifuged at 1036g at 4 for 10 min and the supernatant collected. The remaining pellet was resuspended, as above, in fresh ice-cold 0.25M sucrose and the centrifugation step repeated. The supernatant fractions were pooled and centrifuged at 40,000g (average) at 4 for 15 min and the resulting pellet resuspended in the appropriate assay buffer at a concentration of 20-25 mg wet weight per ml appropriate assay buffer.

(3) Homogenates were prepared by transferring the known weight of forebrain to a cooled tube containing 9 volumes of ice-cold 50 mM pH 7.4 HEPES buffer. The mixture was homogenised @ 4 by 35 sec bursts of an Ultra-Turrax homogeniser set at maximum speed. The resulting homogenate was centrifuged at 40,000g (average) at 4 for 15 min and the supernatant discarded. The resulting pellet was resuspended in 9 volumes of fresh ice-cold pH 7.4 buffer (as above), the centrifugation step was repeated and the resulting pellet resuspended in the [.sup.3H]BTX-B binding buffer at a concentration of 20-25 mg wet weight per ml assay buffer.

[14C] Guanidine Flux and Binding of [3H]BTX-B

(4) Both assays were carried out using 14 ml polypropylene test tubes to which a range of concentrations of the compounds under test were added. Test compounds were dissolved in DMSO and added to assays such that maximum concentration of DMSO did not exceed 2% v/v.

[14C]Guanidine Flux

(5) The [.sup.14C] guanidinine flux assay was measured using the method of Pauwels P J et al (1986) but carried out @ 30 for 2 min.

REFERENCE

(6) Pauwels P J, Leysen J E, Laduron P M. [3H]Batrachotoxinin A 20-alpha-benzoate binding to sodium channels in rat brain: characterization and pharmacological significance. Eur J Pharmacol. 1986 May 27; 124(3):291-8.

Binding of [3H]BTX-B

(7) [.sup.3H]BTX-B binding was carried out using the method described by Catterall et al (1981), except that both bovine serum albumin and TTX were omitted from the incubation medium.

REFERENCE

(8) Catterall W A, Morrow C S, Daly J W, Brown G B. Binding of batrachotoxinin A 20-alpha-benzoate to a receptor site associated with sodium channels in synaptic nerve ending particles. J Bio. Chem. 1981 Sep. 10; 256(17): 8922-7.

Binding of [3H]Methotrexate

(9) All steps were carried out at 4 (or on ice). Freshly dissected rat liver was dissected into 0.25M ice-cold Sucrose and subsequently homogenised (U-turrax) in 50 mM pH 6.0 phosphate buffer (10 ml/g tissue) containing 15 mM Dithiothreitol. The resulting homogenate was centrifuged @ 47,500g for 20 min and supernatant (filtered through cotton wool to remove fatty lumps) stored @80 before use (Rothenberg et al).

(10) Inhibition of the binding of [.sup.3H]methotrexate to rat liver homogenate supernatant fractions were carried out essentially as described by Arons et al, 1975. Results were calculated, either as IC.sub.50 values (see below) derived from concentration-effect curves or as percentage inhibition values determined by comparison with control and cold Methotrexate (10 M final concentration) binding values.

REFERENCE

(11) Elliot Arons, Sheldon P. Rothenberg, Maria da Costa, Craig Fischer and M. Perwaiz Iqbal; Cancer Research 35, Aug. 1, 1975, 2033-2038,

Computation of IC50 Values

(12) Data are presented as meansem of number of experiments indicated in brackets. IC.sub.50 values were obtained from radioligand displacement or guanidine flux inhibition curves by plotting log.sub.10 concentration vs bound ligand/guanidine uptake according the equation:
y=Rmin+Rsp/{1+exp[n(xC)]} where y=bound (dpm) x=log.sub.10 compound concentration Rmin=lower asymptote (i.e. 100% inhibition) Rsp=upper asymptoteRmin (i.e. specific binding) n=slope (log.sub.e) and C=IC.sub.50 (i.e. concentration required to inhibit 50% of specific binding

Hippocampal Slice Assay

(13) Neuroprotective efficacy was measured in 0.4 mm slices of rat hippocampus using the method described by Fowler and Li (1998).sup.1 except that lodoacetate (400 M).sup.2 was used as the metabolic insult. Compounds (usually 30 M) were always directly compared with tetrodotoxin (1 M).sup.3 for their ability to maintain slice concentrations of ATP following inhibition of glycolysis.

REFERENCES

(14) 1. Fowler J C, Li Y. Contributions of Na.sup.+ flux and the anoxic depolarization to adenosine 5-triphosphate levels in hypoxic/hypoglycemic rat hippocampal slices. Neuroscience 1998, 83, 717-722. 2. Reiner P B, Laycock A G, Doll C J. A pharmacological model of ischemia in the hippocampal slice. Neurosci Lett 1990; 119:175-8 3. Boening J A, Kass I S, Cottrell J E, Chambers G. The effect of blocking sodium influx on anoxic damage in the rat hippocampal slice. Neuroscience. 1989. vol 33 (2), 263-268.

Measurement of ATP and Protein

(15) Individual slices were disrupted by ultra-sonication and the resulting homogenates centrifuged @ 10000g for 5 min @ 4. The supernatant was decanted into a fresh tube and any remaining supernatant removed by vacuum aspiration. The pellet was resuspended in 0.5 ml 0.1M KOH by ultra-sonication and the resulting suspensions warmed with gentle agitation @ 37 for 30 minutes. Concentrations of ATP were measured in 6 l of supernatant by mixing with Luciferase reagent (ATPLite from Perkin Elmer) and measuring subsequent luminescence in a 96-well plate Counter.

(16) Protein concentration was measured using BCA protein assay (Pierce) with Bovine Serum albumin as reference standard.

(17) ATP concentrations were expressed as nmoles/mg protein and neuroprotective indices (% protection) calculated by direct comparison with the effect of 1 M TTX.

hERG

(18) Compounds were sent to MDS Pharma for measurement of their inhibition @ 10 M concentration of the binding of [.sup.3H]astemizole to HEK-293 cells expressing human recombinant hERG. Making the assumption that binding slopes would be 1.0 IC.sub.50 values could be calculated (see above) for compounds exhibiting between 5% and 95% inhibition of binding.

L-Type Calcium Channels

(19) Compounds were sent to MDS Pharma for measurement of their inhibition @ 10 M concentration of the binding of [.sup.3H]nitrendipine to rat cerebral cortex membranes. Making the assumption that binding slopes would be 1.0 IC.sub.50 values could be calculated (see above) for compounds exhibiting between 5% and 95% inhibition of binding.

Rat Microsome Stability

(20) Compounds were sent to BioFocus for measurement of their stability @ 1 M concentration following incubation with rat liver microsomes for 40 minutes @ 37.

Results

(21) Data from the various testing procedures is set out in the Table below:

(22) TABLE-US-00001 Microsome [.sup.14C]guanidine [.sup.3H]mtx binding hERG L-type Ca.sup.2+ stability flux IC.sub.50 (M) hERG IC.sub.50 (M) L-type Ca.sup.2+ IC.sub.50 (M) % metabolised CEN Mean (% inhibition % inhibition (extrapolated from % inhibition (extrapolated from (40 min nr IC.sub.50 (M) @ 125 M) @ 10 M 10 M inh'n) @ 10 M 10 M inh'n) incubation 37) 1 (Ltg) 219.2 631(17 2(4)) 1 989 17 48.8 0.5 11** 68*.sup.(***.sup.) 41 60.3 76 3 >200 42 616.6 32 43 631.0 20 47 13.2 22 35 48 5.8 18 46 1 >>190 57 >2000 44 61 676.1 87 62 141.3 46 13 >200 64 >2000 52 67 4.3 11 15 57 16 >>190 0.5 68 794.3 101 69 776.2 66 70 1513.6 66 71 512.9 54 72 131.8 101 73 81.2 5 3 >200 8 114.9 62.5 74 295.1 99 75 49.0 5 78 3 76 77.6 6 74 4 77 14.5 1 36 18 78 102.3 9 24 32 79 208.9 1 3 >200 17 48.8 0.0 80 123 0 1 14 61 81 251.1 2 17 49 82 >1000 8 83 40.8 5 16 52 84 43.7 4 9 101 7 132.8 33.0 85 3.6 5 39 16 15 >>190 86 14.1 5 1 >200 9 101.1 87 288.4 5 3 >200 7 132.8 88 190.5 82 89 724.4 101 90 97.7 53 91 371.5 94 92 144.5 15 4 >200 3 >190 0.0 93 63.1 4 35 19 94 398.1 2 95 >>1000 6 96 109.6 105 97 363.1 101 98 8.9 41 18 46 18 >>190 6.0 99 134.9 1 23 34 0.0 100 77.6 10 30 23 101 >>1000 65 102 58.9 25 103 >>1000 47 104 >>1000 23 105 100.0 86 35 19 106 26.9 43 107 1174.9 48 108 812.8 81 109 >>1000 80 110 134.9 23 12 >200 61.5 111 912.0 97 112 1000.0 84 113 1995.3 65 114 173.8 46 115 >>1000 61 116 955.0 70 117 >>1000 79 118 1148.2 7 119 1949.8 52 120 195.0 18 6 157 121 >>1000 92 122 >>1000 86 123 41.7 86 124 871.0 90 125 7585.8 6 126 58.9 12 79 3 127 85.1 24 2 >200 128 251.2 0 16 53 129 190.5 3 7 133 130 20.4 2 2 >200 131 288.4 94 132 69.2 4 133 >>2000 17 134 4.9 6 135 43.7 89 136 229.1 27 137 >>2000 23 138 >>2000 67 139 288.4 46 140 229.1 3 141 288.0 30 142 >>2000 12 143 >>2000 27 144 57.5 96 145 9.6 39 146 871.0 3 147 60.3 4 148 20.4 38 149 23.4 4 150 16.2 27 151 67.6 76 152 478.6 1 153 50.1 2 154 162.2 13 155 34 6 22 35 156 246 32 157 204 1 6 157 158 >1000 1 159 44 97 160 295 11 0 >200 161 16 7 88 1 162 12 57 163 8 13 11 81 164 692 0 165 372 4 23 34 166 1175 88 167 >>2000 2 168 1000 95 169 347 95 170 263 29 171 2 91 172 234 100 173 159 34 174 589 4 9 101 175 >>2000 4 176 309 55 177 14 16 22 35 178 28 27 179 >>2000 1 180 >>2000 2 181 74 101 182 214 6 10 90 183 39 106 184 34 104 185 3 90 186 >>2000 83 187 447 99 188 2570 76 189 468 2 10 90 190 813 6 191 1950 31 192 >>2000 91*.sup.(***.sup.) 193 135 99*.sup.(***.sup.) 194 9 6*.sup.(***.sup.) 195 4 2*.sup.(***.sup.) 196 912 41*.sup.(***.sup.) 197 91 95*.sup.(***.sup.) *99 M **198 M ***uses fresh batch of supernatant Inhibition of binding of [3H]batrachotoxinin binding to rat (wistar) brain Data are presented as % inhibition @ 10 M and extrapolated IC.sub.50's (which assumes hill slope = 1). Compounds which give <5% inhibition are ascribed IC5.sub.0's of >200 M Compounds which give >95% inhibition are ascribed IC.sub.50's of <0.5 M

Inhibition of Binding of [3H]BTX-B

(23) TABLE-US-00002 % inhibition Extrapolated Compound (@ 10 M) IC50 (M) CEN-1 28 >200 CEN-198 23 34 CEN-199 29 25 CEN-200 14 61 CEN-201 3 >200 CEN-202 90 1 CEN-203 102 <0.5 CEN-204 52 9 CEN-205 79 3 CEN-206 24 32 CEN-207 30 23 CEN-208 31 22 CEN-209 36 18 CEN-210 43 13 CEN-211 106 <0.5 CEN-212 0 >200 CEN-213 2 >200 CEN-214 10 90 CEN-215 22 35

Summary of [3H]Batrachotoxinin Binding Method279510 Sodium Channel, Site 2

(24) Source: Wistar Rat brain Ligand: 5 nM [.H] Batrachotoxin Vehicle: 1% DMSO Incubation Time/Temp: 60 minutes @ 37.0 Incubation Buffer: 50 mM HEPES, 50 mM Tris-HCl, pH7.4, 130 mM Choline Chloride, 5.4 mM KCl, 0.8 mM MgCl., 5.5 mM Glucose, 40 g/ml LqTx K.sub.D 0.052 M* Non-Specific Ligand: 100 M Veratridine Bmax: 0.7 pmole/mg Protein * Specific binding: 77% Quantitation Method: Radioligand Binding Significance Criteria: >/=50% of max stimulation or Inhibition

Hippocampal Slice Data

(25) TABLE-US-00003 % protection Conc'n (v 1 M TTX) (M) (mean sem) Standard Compound TTX 1 100 Lamotrigine 30 41 5 (3) [CEN-001] Sipatrigine 30 58 6 (7) DPH 30 48 Compound CEN-47 30 98 CEN-67 30 3 CEN-86 30 98 CEN-92 30 32 CEN-98 30 11 CEN-130 30 39 CEN-140 30 11 CEN-152 30 10 CEN-160 30 0 CEN-163 30 56 (no. of expt's)

(26) The screening data obtained in respect of representative compounds of the invention points to the suitability of compounds of general formula (I)) for treatment of disorders in mammals that are susceptible to sodium channel blockers and antifolates, and particularly disorders such epilepsy, multiple sclerosis, glaucoma and uveitis, cerebral traumas and cerebral ischaemias, stroke, head injury, spinal cord injury, surgical trauma, neurodegenerative disorders, motorneurone disease, Alzheimer's disease, Parkinson's disease, chronic inflammatory pain, neuropathic pain, migraine, bipolar disorder, mood, anxiety and cognitive disorders, schizophrenia and trigeminal autonomic cephalalgias; for treatment of mammalian cancers; and for treatment of malaria.