as a vital neurotransmitter , 
 acetylcholine ( ach ) activates ion 
 channels and g protein coupled receptors ( gpcrs ) through its interactions 
 with nicotinic and muscarinic ( machr ) acetylcholine receptors , respectively . among the five machrs , subtypes 1 , 4 , and 5 ( m1 , m4 , and m5 ) are most strongly associated with normal 
 central nervous system ( cns ) functioning .
the m2 and m3 subtypes are more broadly expressed 
 in the periphery on smooth muscle and glandular tissues such that aberrant overactivation of these receptors 
 leads to the adverse effects associated with nonselective muscarinic 
 agonists .
designing orthosteric small - molecule muscarinic ligands 
 with sufficient selectivity over the other four machrs has long been 
 a problem due to the highly conserved environment of the ach binding 
 site ( the orthosteric site ) . a prudent response to instances such 
 as this
has been to abandon orthosteric - acting molecules in favor 
 of ligands that interact at allosteric sites ( sites that are topographically 
 and structurally distinct from the endogenous agonist binding site ) .
we have employed this approach to identify a range of high quality 
 muscarinic ligands with positive allosteric modulation ( pam ) or negative 
 allosteric modulation ( nam ) at many of the cns - important machrs : m1 pams , m4 pams , m5 pams , and a novel m5 nam .
currently , m5 is the least characterized of the machrs 
 because of its low expression level and 
 until recently an absence of selective activators and inhibitors . 

nevertheless , phenotypic observations of m5 knockout ( ko ) 
 mice , m5 receptor localization 
 studies , and experiments utilizing nonselective , orthosteric muscarinic 
 ligands highlight this receptor s therapeutic potential
. m5 ko mice display decreased prepulse 
 inhibition ( a model of psychosis ) and 
 cognitive deficits associated with cns neuronal and cerebrovascular 
 abnormalities .
the loss of m5 machrs in ko mice prevents their cns vasculature from dilating in 
 response to ach , which could have implications 
 for cerebral hypoperfusion as related to alzheimer s disease , schizophrenia , ischemic stroke , and migraine . 

collectively , these data support the role of a m5 pam in 
 the treatment of numerous cns diseases . here
we report the development 
 of the first cns penetrant m5 pam , which is structurally 
 distinct from our previously reported isatin - containing m5 pams .
our initial foray into m5 pams began with 
 the identification of a nonselective m1 , m3 , 
 m5 pam as a confirmed hit from 
 an m1-focused high throughput screening ( hts ) campaign .
although very high levels of m5 selectivity 
 were engendered through a strategically placed substituent on the 
 isatin core , we were unable to detect these m5 pams in 
 rodent cns .
as such , we performed a high throughput screen directly 
 interrogating m5 functional activity in conjunction with 
 the scripps research institute molecular screening center ( srimsc ) . 
 for this campaign
, we used a triple addition protocol ( compound addition 
 followed by low and high concentrations of orthosteric agonist ( ach ) ) 
 to screen the mlpcn collection of 360  000 
 compounds .
this screening strategy allows for the identification of 
 activators ( agonists and pams ) while also surveying for inhibitors 
 ( nams and antagonists ) .
single concentration - point screening experiments 
 in chinese hamster ovary ( cho ) cells stably expressing the human m1 , m4 , and m5 receptors identified 3920 
 m5 hits ( 1.07% hit rate ) .
hits were triaged based on activity 
 in untransfected cells , structural tractability , and the elimination 
 of frequent hitters .
the most attractive 
 m5 activators were purchased from commercial sources and 
 reconfirmed using 10-point concentration  response curves ( crc ) . 
 these  triple - add  crc experiments resulted in the identification 
 of nine confirmed m5 pams , nine m5 antagonists , and zero m5 agonists .
structurally , the most promising of the m5 pam hits , 1 ( figure 1 ) , represented a novel chemotype for an m5 pam and offered 
 a wide range of straightforward modifications due to its highly modular 
 appearance .
the broad range of planned modifications to 1 , shown in figure 1 , could readily be accomplished 
 by employing the synthesis route shown in scheme 1 .
starting from the n - boc protected carboxylic 
 acid core 2 , peptide coupling with an amine introduced 
 the first alkyl group on amide 3 .
deprotonation of this 
 amide was followed by the addition of an alkylating agent ( e.g. , ethyl 
 iodide ) and a crown ether , necessary to facilitate the introduction 
 of the second alkyl group , providing 4 .
removal of the 
 boc protecting group under standard anhydrous hcl conditions provided 
 the salt 5 .
the amine of 5 was then sulfonylated 
 to provide the hts hit 1 . alternatively
, this secondary 
 amine could be functionalized through reactions with a wide range 
 of electrophiles ( e.g. , acid chlorides , isocyanates , alkyl halides , 
 etc . ) .
although the synthesis route was quite flexible , when applied 
 to many of the modifications proposed in figure 1 , the sar was disappointingly rigid .
a litany of modifications were 
 not tolerated and resulted in a complete loss of m5 activity : 
 ( 1 ) removal of n - benzyl or n - ethyl 
 moiety to provide a secondary amide , ( 2 ) cyclizing the ethyl group 
 back to the piperidine , ( 3 ) relocating the carbonyl to produce the 
 acetamide or benzamide analogs , ( 4 ) replacing the amide with a sulfonamide , 
 ( 5 ) replacing the piperidine with an azetidine or [ 1.3.0 ] bicycle , 
 ( 6 ) replacing the sulfonamide with an amide , urea , or carbamate , and 
 ( 7 ) any alkylsulfonamide in place of an arylsulfonamide .
the failure 
 of these global modifications suggested that an improvement in potency 
 would require a better understanding of the m5 pam sar 
 brought about by more modest modifications .
reagents and conditions : ( a ) 
 benzylamine , hatu , dcm , dipea , 95% ; ( b ) naotbu , et - i , 
 15-crown-5 , thf , 88% ; ( c ) hcl , dioxane , 99% ; ( d ) 1,4-benzodioxan-6-sulfonyl 
 chloride , dcm , dipea , 70% . initial improvements 
 in pam potency were provided by modifications 
 to the arylsulfonamide ( table 1 ) .
the unadorned 
 phenylsulfonamide 6a showed slightly improved potency 
 over the hts hit ; however , potency could be further improved through 
 the introduction of methoxy groups at the para and meta positions . 

interestingly , the 3,4-dimethoxy analog 6e displayed 
 slightly reduced efficacy ( achmax ) relative to the hts 
 hit and may speak to the preference for sulfonamides with more planar 
 aryl groups at this location .
substitution at the ortho position was 
 clearly disfavored as indicated by 6d and the sulfonamide 
 regioisomer of the hts hit 6f .
a sampling of alternative 
 monocyclic heteroaryl groups ( 6g  m ) did not provide improved activity .
building on the importance of 
 substituents at the 3 and 4 positions , we explored a range of bicyclic 
 heteroarylsulfonamides with annulated rings spanning these two positions . 

in particular , the benzofuranyl ( 6o ) and indazolylsulfonamides 
 ( 6p and 6q ) provided hm5 pams 
 with ec50 of 1.62.4 m , representing an approximately 
 3-fold improvement over the naked phenylsulfonamide .
hm5 pec50 and 
 ach max data reported as averages  sem from our calcium mobilization 
 assay ; n = 34 determinations ;  , not 
 determined .
hm5 pec50 and 
 ach max data reported as averages  sem from our functional calcium 
 mobilization assay ; n = 34 determinations ; 
  , not determined . employing sulfonamides structurally related to those appearing 
 in table 1 , we explored changes to the western 
 region of 1 and were gratified that introduction of a 
 methyl group at the benzylic position improved potency while demonstrating 
 enantiospecific activity ( table 2 ) . while the 
 ( s)-enantiomer 7a was inactive at hm5 , the ( r)-methyl enantiomer 7b demonstrated a 3-fold improvement in potency relative to its des - methyl 
 analog 6b .
although slight , improvements in hm5 pam potency were realized with the incorporation of 2,3-dihydroindenyl 
 and the 6- and 5-indazolylsulfonamides ( 7e , 7f , and 7 g , respectively ) .
pec50 and 
 ach max data reported as averages  sem from our calcium mobilization 
 assay ; n = 35 determinations ;  , not 
 determined .
simultaneously , 
 we were exploring modifications to the western 
 aryl ring in the context of the indazole sulfonamides and identified 
 a number of productive alterations depicted in table 3 . systematically moving
a fluorine around the phenyl ring 
 revealed that substitution at the 2 and 3 positions was favored , with 
 a trend in preference for the 3-fluoro 8b .
most notably , the 3-methyl 
 analog 8f yielded a submicromolar potency ( hm5 ec50 = 0.87 m ) .
this potency was mirrored by the 
 analogous 2-methyl analog 8 g and prompted a further exploration 
 of substituents at the ortho position .
the 2-chloro ( 8i ) and 2-trifluoromethyl ( 8j ) groups provided further 
 improvements in potency , but interestingly the 3- and 4-trifluoromethyl 
 analogs ( 8k and 8l , respectively ) possessed 
 greatly reduced activity and illustrated the frequently steep nature 
 of allosteric sar .
the two most potent ortho - substituted analogs ( cl 
 and cf3 ) with the 6-indazolylsulfonamide were also 
 examined in the context of the 5-indazolylsulfonamide ( 8 m and 8n ) and found to be among the most potent 
 hm5 pams prepared to date .
specifically , 8n , displaying a hm5 pam ec50 = 0.19 m , 
 was 8-fold more potent than its des - cf3 congener ( 6p , table 1 ) .
disappointingly , 
 addition of a methyl group in the ( r ) configuration 
 to 8n , analogous to the compounds in table 2 , resulted in a 10-fold decrease in potency ( hm5 ec50 = 2.4 m , structure not shown ) .
hm5 pec50 and 
 ach max data reported as averages  sem from our calcium mobilization 
 assay ; n = 35 determinations ;  , not 
 determined . in vitro metabolite identification 
 experiments performed on 7
while we were simultaneously 
 exploring modification to this n - ethyl group with 
 variations at other locations , a concise but still representative 
 description of these efforts can be summarized in the context of the 
 highly optimized 8n and its analogs appearing in table 4 .
although not bearing the optimized 5-indazolylsulfonamide 
 shown in table 4 , early results indicated that 
 groups smaller than ethyl ( i.e. , hydrogen or methyl ) resulted in a 
 complete loss of , or greatly diminished , activity at hm5 ( respectively , structures not shown ) .
the ethyl group in 8n could be extended without incurring a loss in potency as demonstrated 
 by the n - propyl analog 9a .
however , 
 the other three - carbon isomers ( 9b  d ) all suffered a > 10-fold drop in activity .
attempts to mitigate 
 metabolism through the introduction of polarity on the terminus of 
 the ethyl group in 8n similarly engendered an unacceptable 
 decrease in activity upon introduction of a hydroxyl ( 9f ) or even a single fluorine atom ( 9 g ) . given the complete 
 absence of activity demonstrated by the cyclopropyl analog 9d , it was surprising to find that the cyclobutyl version ( 9e ) displayed mid - micromolar potency .
supporting the hypothesis that 
 alkyl branching is not well tolerated directly adjacent to the amide 
 nitrogen ( i.e. , 9c 
e ) but that larger 
 alkyl groups could be present more distally , the sec - butyl analog 9h was equipotent to its n - propyl conger ( 9a ) .
furthermore , even larger alkyl 
 groups at this location ( 9i  k ) displayed 
 consistently high levels of activity .
unfortunately , none of these 
 modifications could shift the primary route of metabolism away from 
 this region of these molecules while maintaining high levels of m5 pam activity , nor could they attenuate an inherently high 
 rate of in vitro metabolism ( i.e. , rat hepatic microsomal clint > 500 ml min kg ) .
however , 9d was able to reduce intrinsic microsomal clearance by an 
 order of magnitude , but this came at the expense of losing all hm5 activity . a subset 
 of m5 pams were further assessed for their ability to enhance 
 the potency 
 of ach at the hm5 receptor using a fluorescence based calcium 
 mobilization assay .
experimentally , a fixed concentration of pam ( 10 
 m ) or vehicle was added prior to the addition of a concentration 
 response curve ( crc ) of ach , and the left shift in potency of ach 
 was determined as the ratio ( fold shift ) of the potency in the absence 
 and presence of pam .
as shown in table 5 , the 
 hts hit 1 produced a fold shift of 2.3 , while the more 
 potency - optimized analogs showed at least twice that value .
four of 
 the most potent compounds from tables 2 and 3 ( 7b , 7 g , 8j , and 8n ) gave fold shift values in the 7- to 12-fold 
 range , similar to earlier m5 pams .
ach fold - shift 
 data , for compounds 
 at 10 m , reported as averages  sem from our calcium mobilization 
 assay and represent leftward shifts in ach potency ; n = 34 .
although 7 g and 8n displayed similar 
 fold shift values , 8n was superior to 7 g with respect to hm5 pam potency and by virtue of its 
 superior muscarinic subtype selectivity profile . the muscarinic subtype selectivity profile for 8n across the five human and rat receptor subtypes can be seen in figure 2 .
8n shows no activity at hm2 or hm4 ( the natively gi / o coupled machrs ; 
 our assays employed cells co - transfected with chimeric gqi5 to facilitate m2/m4 coupling to ca mobilization ) and displays greater than 10-fold selectivity over 
 hm1 and hm3 ( the gq coupled machrs ) . 

the lower potencies , combined with lower efficacies , at hm1 ( hm1 pam ec50 = 5.4 m , achmax = 52% ) and hm3 ( hm3 pam ec50 = 
 2.1 m , achmax = 67% ) when compared to those for 8n at hm5 ( hm5 pam ec50 = 
 0.19 m , achmax = 96% ) may actually afford a greater 
 than 10-fold selectivity window . interestingly , when assessed at the 
 rat muscarinic receptors , the level of subtype selectivity diminished 
 and rm1 was now closest in potency to rm5 .
muscarinic 
 subtype selectivity profile of 8n : ( a ) 
 human selectivity ( hm5 ec50 = 0.19 m , 
 hm4 ec50 > 30 m , hm3 ec50 = 2.1 m , hm2 ec50 > 30 m , 
 hm1 ec50 = 5.4 m ) ; ( b ) rat selectivity 
 ( rm5 ec50 = 0.61 m , rm4 ec50 > 30 m , hm3 ec50 = 3.1 m , 
 hm2 ec50 > 30 m , hm1 ec50 = 2.0 m ) .
data represent the mean  sem from 
 at least three independent determinations employing highly expressing 
 cell lines with similarly high expression levels of muscarinic receptors .
8n has 
 no inhibitory effect on [ h]nms binding ( 97.1% max ) , while 
 the control ( atropine ) inhibits [ h]nms binding in a concentration 
 dependent manner ( ki = 1.47 nm , 1.8% max ) . 

( b ) acetylcholine affinity shift profile of 8n . increasing 
 fixed concentrations of 8n result in progressive left 
 shifts of the ach inhibition curve , with a maximal shift of approximately 
 15 .
the pharmacology of 8n was further profiled 
 in radioligand 
 binding experiments ( figure 3 ) .
increasing 
 concentrations of 8n or atropine ( control ) were incubated 
 with a fixed concentration of [ h]n - methylscopolamine 
 ( nms , 0.3 nm , an orthosteric antagonist ) and membranes expressing 
 the hm5 receptor . while atropine displaced [ h]nms binding in a concentration dependent manner , 8n had no effect on [ h]nms binding ( figure 3a ) , suggesting that 8n interacts with the hm5 receptor via an allosteric mechanism . to further characterize 
 the interaction of 8n with the hm5 receptor , 
 increasing fixed concentrations of 8n
were incubated 
 with a crc of ach in the presence of a fixed concentration of [ h]nms ( 0.4 nm ) to determine the effect of 8n on 
 the affinity of ach .
8n shifted the ach competition curve 
 leftward by 15-fold ( figure 3b ) , further 
 demonstrating its function as a pam , acting through modulation of 
 the potency and affinity of ach .
however , it has yet to be defined 
 what in vitro properties are required for a hm5 pam to 
 generate a specific in vivo outcome .
only now are we beginning to 
 amass the necessary tool compounds to explore this question .
interestingly , this novel class of hm5 pams showed a 
 clear preference for the gq coupled machrs over the gi / o coupled receptors , which is reminiscent of the nonselective 
 pan - gq pam hts hit that served 
 as the progenitor for three previous m5 pam probe molecules and an m1 selective pam .
this similarity points to the possibility of 
 a common allosteric binding site and the high probability that further 
 sar will reveal completely selective m5 pams from this 
 series . to more broadly explore this new scaffold s potential 
 for nonmuscarinic , off - target activity , 8n was submitted 
 to eurofin s pan labs lead profiling screen .
this battery of 
 radioligand binding assays consists of 68 common gpcrs , ion channels , 
 and transporters where the test compound ( 8n ) was present 
 at 10 m .
remarkably , 8n showed a significant response 
 ( > 49% radiologand displacement ; see supporting 
 information for complete results ) in just two assays : cannabinoid 
 cb1 receptor ( 50% displacement ) and 1 receptor ( 53% displacement ) .
however , these binding results do not 
 guarantee functional activity and the mid - range values did not prompt 
 functional determinations
. encouraged 
 by these initial 
 results , 8n was characterized in a variety of dmpk assays 
 ( table 6 ) . from an in vitro standpoint
, 8n displayed minimal metabolic stability with a very high 
 hepatic microsomal intrinsic clearance in rat and human ( clint ; rat , 2600 ml min kg ; human , 
 540 ml min kg ) and a correspondingly 
 high predicted hepatic clearance in both species ( clhep ; rat , 68 ml min kg ; human , 
 20 ml min kg ) , on par with 
 hepatic blood flow . a low fraction unbound in plasma was observed 
 for rat and human ( fu , plasma ; rat , 0.014 ; 
 human , 0.015 ) and in rat brain homogenate ( fu , brain ; rat , 0.011 ) . in rodents ( male , sprague 
dawley 
 rats , 1 mg / kg iv , n = 3 ) , similar instability was 
 observed after intravenous dosing ; 8n displayed high 
 clearance ( 66 ml min kg ) , 
 a moderate volume of distribution ( 1.6 l kg ) , 
 and a short half - life ( t1/2 , 22 min ) . 

a modest total brain - plasma partition coefficient ( kp = 0.36 ) was also determined from these experiments ( 15 
 min after administration ) ; however , the unbound brain - plasma partition 
 coefficient ( kp , uu = 0.28 ) tempers the 
 attractiveness of 8n as a highly cns penetrant compound . 

still , the high permeability determined in caco-2 cells ( papp = 2.5  10 cm / s ) represents 
 an attractive starting point to further optimize cns exposure . determined using clint values in the 
 well - stirred model of organ clearance not corrected 
 for fraction unbound in plasma .
in summary , we have developed 8n ( also referred to 
 as ml380 or vu0481443 ) , which is among the most potent m5 pams reported to date ( hm5 ec50 = 190 nm , 
 rm5 ec50 = 610 nm ) and is m5 preferring 
 with some functional activity remaining at m1 and m3 .
this compound will be a useful tool to further investigate 
 the in vitro properties of the m5 receptor as more advanced 
 pams are identified .
this novel chemotype distinguishes itself from 
 our previously published isatin - containing m5 pams in its 
 ability to be detected within the cns even though its partition coefficients 
 ( kp and kp , uu ) are less than optimal .
however , the highly modular nature of this 
 ligand will allow for continued structural optimization to further 
 improve potency , selectivity , metabolic stability , and cns penetration . 

continuing efforts around this scaffold are in progress and will be 
 reported in due course .
the general chemistry , experimental information , 
 and syntheses of key compounds are supplied in the supporting information .
purity for all final compounds was 
 > 95% , and each showed a parent mass ion consistent with the desired 
 structure ( lcms ) . to a solution of 1-boc-4-piperidinecarboxylic 
 acid ( 2.00 g , 8.72 mmol , 1 equiv ) and dipea ( 4.48 ml , 26.2 mmol , 3 
 equiv ) in dcm ( 30 ml , 0.3 m ) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide 
 hcl ( 2.51 g , 13.1 mmol , 1.5 equiv ) , hydroxybenzotriazole ( 1.77 g , 
 13.1 mmol , 1.5 equiv ) , and ethylamine hcl ( 1.42 g , 17.5 mmol , 2.0 
 equiv ) .
the mixture was stirred for 2 h at room temperature before 
 being quenched with aqueous nahco3 .
the organic layer was 
 separated , and the aqueous layer was extracted with dcm .
the combined 
 organic layers were washed with brine , dried over mgso4 , filtered , and concentrated under reduced pressure .
the residue 
 was purified via silica gel column chromatography to give 1.89 g of 
 1-boc-4-(ethylcarbamoyl)piperidine ( 83% yield ) .
to a solution 
 of 1-boc-4-(ethylcarbamoyl)piperidine ( 50.0 mg , 0.195 mmol , 
 1 equiv ) and 15-crown-5 ( 77.4 l , 0.390 mmol , 2 equiv ) in thf 
 ( 2 ml , 0.1 m ) was added naobu ( 28.1 mg , 
 0.293 mmol , 1.5 equiv ) .
the mixture was stirred for 30 min at room 
 temperature before adding 2-(trifluoromethyl)benzyl bromide ( 59.4 
 l , 0.39 mmol , 2 equiv ) .
after 16 h , the mixture was concentrated 
 under reduced pressure and the residue partitioned between h2o and dcm . the organic layer was separated , and the aqueous layer 
 was extracted with dcm .
the combined organic layers were concentrated 
 under reduced pressure and the residue was purified via gilson preparative 
 lc ( mecn / water/0.1% tfa gradient as the mobile phase through a c-18 
 column ) to obtain 1-boc-4-(ethyl(2-(trifluoromethyl)benzyl)carbamoyl)piperidine . 

to a solution of 1-boc-4-(ethyl(2-(trifluoromethyl)benzyl)carbamoyl)piperidine 
 in dcm was added mp - tsoh ( 5 equiv ) .
the mixture was filtered , 
 and the resin was rinsed with meoh before washing with nh3/meoh to elute product .
solvent was removed under reduced pressure 
 to give 43 mg of pure 4-(ethyl(2-(trifluoromethyl)benzyl)carbamoyl)piperidine 
 ( 70% yield , two steps ) . to a solution of 4-(ethyl(2-(trifluoromethyl)benzyl)carbamoyl)piperidine 
 ( 20 mg ,
0.063 mmol , 1 equiv ) and dipea ( 33 l , 0.20 mmol , 3 
 equiv ) in dcm was added 1h - indazole-5-sulfonyl chloride 
 ( 21 mg , 0.095 mmol , 1.5 equiv ) .
the mixture was allowed to stir for 
 2 h at room temperature and was then quenched with meoh and concentrated 
 under reduced pressure .
the residue was purified via gilson preparative 
 lc ( mecn / water/0.1% tfa gradient as the mobile phase through a c-18 
 column ) to obtain 4.2 mg of 8n ( 15% yield ) .
hrms ( tof , 
 es+ ) c23h26n4o3f3s [ m + h ] calcd mass 495.1678 , found 495.1679 .
h nmr ( 1:1.25 rotamer ratio ,  denotes minor rotamer , 400.1 
 mhz , cdcl3 )  ( ppm ) : 8.31 ( s , 1h ) ; 8.25 ( m , 1h ) ; 
 7.78 , 7.72 * ( d , j = 8.8 hz , 1h ) ; 7.687.57 
 ( m , 2h ) ; 7.52 * , 7.46 ( t , j = 7.6 hz , 1h ) ; 7.38 * , 
 7.32 ( t , j = 7.6 hz , 1h ) ; 7.217.13 ( m , 1h ) ; 
 4.76 , 4.64 * ( s , 2h ) ; 3.953.86 , 3.853.76 * ( m , 2h ) ; 
 3.41 * , 3.22 ( q , j = 7.2 hz , 2h ) ; 2.602.46 
 ( m , 2h ) ; 2.372.26 ( m , 1h ) ; 2.111.92 ( m , 2h ) ; 1.911.81 , 
 1.741.65 * ( m , 2h ) ; 1.161.05 ( m , 3h ) .
c nmr ( 1:1.35 rotamer ratio ,  denotes minor rotamer , 100.6 
 mhz , cdcl3 )  ( ppm ) : 174.70 ; 141.40 , 141.34 * ; 136.29 , 
 135.77 * ; 135.95 ; 132.67 * , 132.31 ; 129.31 * , 129.19 ; 127.94 , 127.86 * ; 
 127.90 ( q , j = 30.3 hz ) ; 126.65 ( q , j = 5.3 ) ; 126.47 ; 126.26 ( q , jcf = 245 hz ) ; 126.04 ( q , j = 5.6 hz ) ; 122.81 , 
 122.73 * ; 122.66 ; 110.92 , 110.89 * ; 46.94 * , 44.23 ; 45.64 , 45.46 * ; 42.12 , 
 41.87 * ; 37.96 * , 37.68 ; 28.48 , 28.27 * ; 14.44 , 12.67*.
 a functional 
 high throughput screen identified a novel chemotype 
 for the positive allosteric modulation ( pam ) of the muscarinic acetylcholine 
 receptor ( machr ) subtype 5 ( m5 ) . 
 application of rapid analog , 
 iterative parallel synthesis efficiently optimized m5 potency 
 to arrive at the most potent m5 pams prepared to date and 
 provided tool compound 8n ( ml380 ) demonstrating modest 
 cns penetration ( human m5 ec50 = 190 nm , rat 
 m5 ec50 = 610 nm , brain to plasma ratio ( kp ) of 0.36 ) . 
Introduction
Results and Discussion
Conclusion
Experimental Section