Patent Application: US-44672199-A

Abstract:
the invention provides methods for enhancing cognitive activity and stimulating memory capacity , comprising the step of administering an effective amount of a compound with gaba c receptor antagonist activity to an animal in need of such treatment . preferably the compound has selective gaba c receptor antagonist activity , and more preferably comprises a phosphinic acid group . the invention also provides novel compounds and compositions . the methods of the invention are useful in the treatment of dementias and conditions involving cognitive deficit , or memory impairment .

Description:
the invention is described in detail by way of reference only to the following non - limiting general methods and experimental examples , and to the figures . ( 2r , 1 ′ s )-( 3 - n -[ 1 ′( 3 , 4 - dichlorophenyl ) ethyl ]) amino - 2 - hydroxypropyl ) benzylphosphinic acid ( cgp55845a ) were synthesised as described previously by froestl et al , ( 1992 ; 1995a ; 1995b ). caca and taca were prepared as previously described ( johnston et al , 1975 ). gaba was purchased from sigma chemical co ( st louis , mo ., usa ). human r 1 cdna in pcdna ( invitrogen , san diego , calif ., usa ) was obtained from dr . george uhl ( national institute for drug abuse , baltimore , usa ). the plasmid was linearized with xbai and crna made using the “ message machine ” kit from ambion inc . ( austin , tex ., usa ). 50 ng of crna was injected into defolliculated stage v xenopus oocytes . two to seven days later , receptor activity was measured by two - electrode voltage clamp recording , using a geneclamp 500 amplifier ( axon instruments inc ., foster city , calif ., usa ) and a maclab 2e recorder ( adinstruments , sydney , nsw , australia ). oocytes were voltage clamped at − 60 mv and continuously superfused with nd96 buffer ( 96 mm nacl , 2 mm kcl , 1 . 8 mm cacl 2 , 1 mm mgcl 2 and 5 mm hepes , ph 7 . 5 ). for receptor activation measurements , the indicated concentrations of agonist and antagonist were added to nd96 . current ( i ) as a function of agonist concentration ([ a ]) was fitted by least squares to i = i max [ a ] nh /( ec 50 nh +[ a ] nh ), where i max is the maximal current , the ec 50 is the effective dose that activates 50 % of the maximal current and n h is the hill coefficient . ec 50 values are expressed as mean ± s . e . m . ( n = 3 - 6 ) and were determined by fitting data from individual oocytes using kaleidagraph 2 . 1 ( 1990 ). current ( i ) as a function of antagonist concentration ([ ant ]) was fitted by least squares to i = i max —{ i max [ ant ] nh /( ic 50 nh +[ ant ] nh )}, where the ic 50 is the inhibition dose that blocks 50 % of the current generated by 1 μm gaba and n h is the hill coefficient . ic 50 values are expressed as mean ± s . e . m . ( n = 3 - 6 ). k b values are the apparent dissociation constants for the antagonists , and were determined using schild plot analysis ( arunlakshana and schild , 1959 ). - logk b values were determined using the following equation : log {( a )/( a *)− 1 }= m . log [ ant ]- logk b , where a is the ec 50 of gaba in the presence of a known antagonist concentration , a * is the ec 50 of gaba in the absence of an antagonist , [ ant ] is the concentration of the antagonist , and ‘ m ’ is the slope of the curve . for simple competitive antagonism , ‘ m ’ is 1 . - logk b values were determined by fitting data to the above function using kaleidagraph 2 . 1 ( 1990 ). schild analyses were carried out for compounds that had ic 50 values of less than 30 μm . gaba c receptor antagonists block activation of chloride channels by gaba expression of human r 1 mrna in xenopus oocytes generated gaba c receptors which showed a dose - dependent gaba activated inward current when the cell was voltage clamped at − 60 mv . this could be blocked by compounds such as cgp44530 , cgp38593 , cgp70523 , cgp70522 and cgp36742 , as shown in fig1 . the structures of the compounds are shown in fig2 and fig3 . these compounds were first screened at 100 μm to determine agonist activity , by activation of cl − channels , or antagonist activity , by blocking the activation of the channels by 1 μm gaba . fig2 shows the active compounds that had some effect at 100 μm as agonists ( fig2 a ) or antagonists ( fig2 b ) at gaba c receptors , and fig3 shows the compounds that had no effect at 100 μm as agonists or antagonists at gaba c receptors . only the carboxylic acids , taca , gaba and caca activated the cl − channel . taca was more potent than gaba , with an ec 50 of 0 . 44 ± 0 . 02 μm , and was almost a full agonist , with a maximal taca dose generating 95 % of the maximal gaba activated current . gaba was found to have an ec 50 value of 0 . 82 ± 0 . 09 μm . caca was less potent than gaba , with an ec 50 value of 37 . 4 ± 6 . 1 μm , and was a partial agonist , with a maximal caca dose generating 75 % of the maximal gaba activated current . these results are summarised in table 1 . the hill coefficients ( n h ) as shown in tables 1 and 2 were greater or equal to 2 , which suggests that more than one molecule of the agonist is required to bind before the cl − channels can open . these findings are in agreement with those of woodward et al ( 1993 ). d k b is the binding constant for the antagonist . these were determined using schild plot analysis assuming competitive antagonism over the tested concentrations ( table 2 ). a ec 50 is the effective dose that activates 50 % of the maximal current when tested at r 1 receptors expressed in xenopus oocytes . ec 50 values are expressed as mean ± s . e . m . ( n = 3 - 6 ) and are determined by fitting data from individual oocytes using kaleidagraph 2 . 1 ( 1990 ). ec 50 values of gaba have shifted to the right in the presence of a known concentration of the antagonist . - log k b values were determined as described in materials and methods section . the k b values are shown in table 1 . b n h is the hill coefficient . these are greater than 1 indicating that more than 1 molecule of gaba is required for the channel to open . c slope of schild plot analysis indicating competitive antagonism over the tested concentrations . gp35024 , cgp27492 , cgp44530 , cgp38593 , cgp70523 and cgp70522 did not activate any current on their own ( fig1 ). they acted as gaba c receptor antagonists , inhibiting the current activated by 1 μm gaba ( fig1 ). ic 50 values were obtained for these compounds ( table 1 ) and schild analyses were carried out for the active compounds ( table 2 ). k b ( binding constant ) values for cgp27492 , cgp44530 , cgp35024 , and cgp38593 are shown in table 1 . the methylphosphinic analogue , cgp44530 , and phosphinic analogue , cgp38593 of taca were antagonists with ic 50 values of 5 . 5 ± 1 . 2 μm and 7 . 7 ± 0 . 7 μm respectively . these compounds had lower affinity for the gaba c receptor expressed in xenopus oocytes than that of the corresponding methylphosphinic analogue , cgp35024 , and phosphinic analogue , cgp27492 , of gaba . cgp35024 had an ic 50 of 0 . 75 ± 0 . 07 μm and cgp27492 had an ic 50 of 2 . 47 ± 0 . 04 μm . the methylphosphinic analogue , cgp70523 and phosphinic analogue , cgp70522 , of caca were antagonists , with ic 50 values of 38 . 9 ± 4 . 9 μm and & gt ; 100 μm respectively . these compounds had lower affinity for gaba c receptors than the methylphosphinic and phosphinic analogues of gaba and taca . the order of potency of the methyl phosphinic acids and phosphinic acids is cgp35024 & gt ; cgp27492 & gt ; cgp44530 & gt ; cgp38593 & gt ; cgp70523 & gt ;& gt ; cgp70522 . the new compounds cgp44530 , cgp38593 , cgp70523 and cgp70522 were weaker at the gaba c receptor than the existing phosphinic acid , cgp27492 and the methylphosphinic acid , cgp35024 . cgp35024 , cgp27492 , cgp44530 and cgp38593 were found to be competitive antagonists . the gradients of the schild regression plots were not significantly different from 1 over the concentrations tested , indicating that these compounds compete for the same site as gaba . cgp36742 was found to be an antagonist with moderate potency at the gaba c receptor , with an ic 50 value of 62 . 5 ± 0 . 5 μm . this compound is orally active , showing cognitive enhancement effects . other related compounds , such as cgp35348 , cgp46381 , cgp51176 and cgp55845a ( fig3 ), are also orally active , but do not show cognitive enhancement effects . these were screened at 100 μm , and had no effect as either agonists or antagonists at gaba c receptors . these compounds show high selectivity as gaba b receptor antagonists . relative effects of compounds on gaba a , gaba b and gaba c receptors the development of many alkylphosphinic and phosphinic analogues of gaba has yielded novel gaba b receptor agonists and antagonists ( olpe et al , 1990 ; 1993 ; bittiger et al , 1992 ; 1993 ; froestl et al , 1992 ; 1995a ; 1995b ), including the methylphosphinic and phosphinic analogues of taca and caca , ie . cgp44530 , cgp38593 , cgp70522 and cgp70523 . in this study , we tested these compounds on gaba c receptors expressed in xenopus oocytes , and found them to be competitive antagonists . the antagonist potencies of cgp44530 , cgp38593 , cgp70522 and cgp70523 were found to be lower than that of the methylphosphinic and phosphinic analogues of gaba , cgp35024 and cgp27492 . the relative effects of the compounds at gaba a , gaba b and gaba c receptors are shown in table 3 . three compounds , cgp38593 , cg ? 70522 and cgp27492 , were moderately potent at gaba a receptors when tested using radioligand binding assays ( ic 50 = 6 . 8 μm ; ic 50 = 6 . 6 μm and ic 50 = 1 . 7 μm , respectively ) ( froestl et al , 1995a ). however , the compounds were more potent at gaba b receptors than at gaba a receptors using this assay . similarly , these compounds appear more potent at gaba b receptors than at gaba c receptors . d ic 50 values for the inhibition of the response of 1 μm gaba using human r 1 mrna expressed in xenopus oocytes as described in the materials and methods section . e ec 50 values ie . the effective dose that activates 50 % of the maximal current when tested at r 1 receptors expressed in xenopus oocytes as described herein . f ic 50 values for the inhibition of the total na - independent [ 3 h ] gaba binding using rat brain membranes ( johnston et al , 1978 ). g data from kerr and ong ( 1995 ) using guinea pig ileum , in the presence of bicuculline , against baclofen - depression of twitch contractions . h data from ragozzino et al ( 1996 ) using whole cell patch recordings from pyrimidal neurons in hippocampal slices in the presence of bicuculline ( 20 μm ). i data from murata et al ( 1996 ) using human r 1 mrna expressed in xenopus oocytes . j data from ragozzino et al ( 1996 ) using poly ( a ) + rna from rat cortex expressed in xenopus oocytes . k ec 50 values for gaba , cgp27492 , cgp35024 , taca , caca and isoguvacine using poly ( a ) + rna from rat cortex expressed in xenopus oocytes are 107 μm , 938 μm , inactive at 1 mm , 133 μm , inactive at 5 mm , and 305 μm respectively ( woodward et al , 1993 ). these values are different to the values obtained from radio - ligand binding assays . l data from measurement of the frequency of spontaneous discharges in rate neocortical slices using the grease - gap recording system . we have demonstrated that the benchmark gaba c antagonist , tpmpa , is an order of magnitude less potent at blocking human homo - oligomeric rho - 2 receptors than rho - 1 gaba c receptors . of particular interest is the dihydro derivative of tpmpa , piperidine - 4 -( methyl ) phosphinic acid ( pmpa ): ( piperid - 4 - yl ) methylphosphinate ( pmpa ) was synthesized by reduction of a precursor of tmpa and subsequent hydrolysis , as follows : platinum oxide ( pto 2 . h 2 o ) ( 50 mg ) was added to a solution of recrystallised troc - precursor ( isopropyl [ 1 -( 2 , 2 , 2 - trichloroethoxycarbonyl )- 1 , 2 , 5 , 6 - tetrahydropyridin - 4 - yl ] methylphosphinate , a ) ( 1 . 50 g , 3 . 96 mmol ) in methanol ( 25 ml ) and the mixture was shaken with h 2 ( 5 atm .) at room temperature for 24 h . the catalyst was filtered off through celite , and the residue concentrated under reduced pressure to afford a viscous colourless oil . a n . m . r . examination of the crude reduction product indicated complete reduction of the olefinic bond together with significant concomitant reduction and partial deprotection of the troc group . a mixture of the residue from above , 48 % aq . hbr ( 40 ml ) and glacial acetic acid ( 40 ml ) was refluxed for 60 h . the reaction mixture was concentrated under reduced pressure ( water pump ) and the final traces of hbr / acoh were removed by the sequential addition of h 2 o and concentration ( several cycles ). the final residue ( hbr salt ) was dissolved in a small volume of h 2 o and applied to a dowex ag 50 ( h + ) column . after initial elution with h 2 o until the eluant was neutral , the eluting agent was changed to 1m aq . pyridine . ninhydrin - positive factions were combined and concentrated under reduced pressure ( water pump ). final traces of pyridine were removed by the sequential addition of h 2 o and concentration under reduced pressure ( several cycles ) to afford a quantitative yield of crude ( piperid - 4 - yl ) methylphosphinic acid ( pmpa )( c ) as an off - white solid ( ca . 645 mg , air dried ) which was recrystallised from etoh / h 2 o ( 450 mg , 70 %): m . p . 289 - 291 °; 1 h nmr ( 300 mhz , d 2 o , ref : doh = δ 4 . 8 ) δ 1 . 19 ( 3h , d , j = 13 . 2 hz , pch 3 ), 1 . 56 - 1 . 82 ( 3h , 2 × overlapping m , 2 × nch 2 ch b and pch ), 2 . 00 - 2 . 08 ( 2h , m , 2 × nch 2 ch a ), 2 . 96 ( 2h , ( apparent ?) dt , j = 3 . 0 , 12 . 8 hz , 2 × nch b ch 2 ), 3 . 43 - 3 . 51 ( 2h , m , 2 × nch a ch 2 ); 13 c nmr ( d 2 o , 75 . 64 mhz , ref : ( internal ) dioxane = δ 67 . 4 ) δ 13 . 6 ( d , j = 91 . 5 hz ), 23 . 1 , 36 . 0 ( d , j = 96 hz ), 44 . 8 ( d , j = 14 . 2 hz ). the chemical synthesis is shown in fig5 . we have found that pmpa is a much more potent antagonist than tpmpa against rho - 2 receptors , and less potent than tpmpa against rho - 1 receptors , as indicated in table 4 . this procedure trains each chick on anthranilate - coated red beads , which have a bitter taste . in the test , 120 min . after the initial exposure to the red bead each chick is presented with a blue and a red bead , and normally will avoid pecking at the red bead ; the discrimination ratio measures how well it remembers to do this . chicks trained on 100 % anthranilate - coated beads produce a discrimination ratio better than 0 . 9 , and drug - induced memory deficits can be detected in this group . however , chicks trained on 20 % anthranilate - coated beads produce a discrimination ratio of around 0 . 6 , and this group can be used to detect drug - induced memory enhancement . drugs are delivered by two bilateral intracranial injections ( 10 μl each ). fig6 shows that tpmpa at a dose of 30 μm enhances memory with the group trained with 20 % anthranilate performing as well as the 100 % anthranilate group . fig7 shows the dose - response relationship for the effects of tpmpa on discrimination ratio , with an ec 50 between 1 and 10 μm . fig8 shows the dependence of this effect on time of injection , with an optimum effect produced by injecting in the 2 . 5 minutes after training . in this assay mice are trained by placing them at the end of the open arm of the plus maze and allowing them to find the shelter of the closed arms . the time taken is measured as the ‘ latency ’. immediately after the trial the mice are injected with the test drug or with a saline control . two and ten days later the test is repeated . an agent which enhances memory consolidation will significantly reduce the latency time relative to the although the possibility that pmpa might have activity as a competitive antagonist of gaba c receptors is mentioned in u . s . pat . no . 5 , 627 , 169 : “ selective antagonists for gaba rho receptor ” and in a paper by woodward et al ( 1993 ), it appears that neither this compound nor its analogues was actually synthesised and tested consequently these prior disclosures are merely speculative paper examples . cgp36742 was shown to be a moderately potent antagonist at gaba b receptors using a [ 3 h ]- cgp27492 binding assay ( ic 50 = 35 μm ) ( bittiger et al , 1992 ; olpe et al , 1993 ; froestl et al , 1995a ). it had weak effects at gaba a receptors ( ic 50 = 500 μm ) ( bittiger et al , 1992 ), and had no effect at other receptor types , including nmda , benzodiazepine , quisqualate , kainate , muscarinic cholinergic , adrenergic , serotoninergic and histaminergic receptors ( 1 mm ) ( bittiger et al , 1992 ; froestl et al , 1995b ). however , we have now found that cgp36742 showed moderate antagonist activity at gaba c receptors ( ic 50 = 62 μm ), and that its apparent selectivity for gaba b compared to gaba c receptors was approximately 2 - fold . this compound has shown promising therapeutic potential in the treatment of cognitive deficits , petit mal epilepsy and depression ( bittiger et al , 1992 ). therefore it is possible that antagonism of gaba c receptors contributes to the cognitive enhancement potentiation by cgp36742 , such enhancement is not shown by other orally - active gaba b receptor antagonists ( froestl et al , 1995b ). tpmpa was recently synthesised and tested at gaba a , gaba b , and gaba c receptors ( murata et al , 1996 ). it is a conformationally - restricted analogue of cgp44530 , and is the methylphosphinic analogue of isoguvacine . it was found to be more than 100 - fold more selective as an antagonist for gaba c receptors than for gaba b receptors , and is 500 - fold more selective at gaba c receptors than at gaba a receptors ( murata et al , 1996 ; ragozzino et al , 1996 ). saline controls . the results are summarized in fig9 and show that tpmpa at 200 mg / kg , but not at 50 mg / kg , significantly reduces the latency in the 14 day test . this is consistent with enhancement of memory consolidation . when we repeated the experiment , this time testing only after 14 days and using swiss mice from a different source , we found that tpmpa at 50 mg / kg but not 200 mg / kg significantly reduced latency . the experiments overall are therefore positive but inconclusive , since the different origin of the mice may be a contributing factor . it will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding , various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification . references cited herein are listed on the following pages , and are incorporated herein by this reference . arunlakshana , o . and schild , h . o . br . j . pharmacol ., 1959 14 48 - 58 bittiger , h ., bernasconi , r ., froestl , w ., hall , r ., jaekel , j ., klebs , k ., krueger , l ., mickel , s . j ., mondadori , c ., olpe , h . r ., pfannkuch , f ., pozza , m ., probst , a ., van riezen , h ., schmutz , m ., schuetz , h ., steinmann , m . w ., vassout , a ., waldmeier , p . pharmacol . com ., 1992 2 70 - 74 bittiger , h ., froestl , r . w ., mickel , s . j ., olpe , h . r ., 1993 trends pharmacol . sci ., 1993 14 391 - 393 cutting , g . r ., lu , l ., o &# 39 ; 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