after decades of neglect , the search for 
 novel , effective antimalarials has been revived primarily due to emergence 
 of drug resistance .
widespread resistance 
 has been reported not only for 8-aminoquinolines but recent reports 
 indicate the appearance of increased parasite tolerance to artemisinin 
 analogues , potentially predicating clinical resistance .
the absence of highly efficacious malaria vaccines highlights 
 the importance of developing small - molecule drugs . with support from the wellcome trust and medicines for 
 malaria venture ( mmv ) , the ngbs consortium ( the novartis institute 
 for tropical diseases ( nitd ) , the genomics institute of the novartis 
 research foundation ( gnf ) , the biomedical primate research centre 
 ( bprc ) , and the swiss tropical and public health institute ( stphi ) ) 
 was formed and set up to discover new antimalarials which are effective 
 against multidrug resistant parasite strains
here we report our continued 
 effort to optimize a series of imidazolopiperazine compounds in search 
 of a preclinical candidate .
as described previously , an 
 extensive sar study on the three peripheral parts had yielded decent 
 in vitro and in vivo antimalarial activities as well as favorable 
 physiochemical and pharmacokinetic properties .
our efforts toward 
 the first - generation compounds led to candidates ( like compound 1 ) with double - digit nanomolar potency and moderate oral exposure 
 in mice . however , most potent compounds with an unsubstituted piperazine 
 had poor oral exposure which resulted in inferior in vivo efficacy 
 when administered orally , for example , compounds 28 and 31 ( scheme 1 ) in the previous communication 
 ( figure 1 ) .
we 
 were intrigued by the possibility of further refining the core structure 
 of the molecule to further improve the properties .
our hypothesis 
 was further reinforced by chemical stability and mouse in vitro metabolic 
 stability results which led to the identification of two biologically 
 inactive metabolites ( 1a and 1b ) arising 
 from the oxidation of piperazine ring .
our plan was to substitute 
 three possible positions ( positions 5 , 6 , and 8) to evaluate the corresponding 
 effects on potency as well as a means to improve oral exposure .
central piperazine ring oxidation is a major metabolic and chemical instability site in compound 1 .
on the basis of the designed molecules , it 
 was clear that previous synthetic routes were not amenable for the 
 desired compounds because we could not utilize the 3 + 2 cyclization .
distinct syntheses were designed and executed 
 to obtain substitution at different positions of a piperazine ring . 

the synthesis started with an alkylation 
 reaction between cbz - protected glycine derivative 2 and 
 bromide 3 .
adduct 4 was then refluxed with 
 nh4oac in toluene to give imidazole 5 with 
 removal of water using a dean  stark apparatus .
regioselective n - alkylation with ethyl 
 2-bromoacetate followed by a one - pot deprotection / cyclization sequence 
 furnished the lactam core , which was reduced by borane to furnish 
 amine 8 .
the quaternary -carbon did not pose a 
 significant amount of steric hindrance for the hatu mediated amidation 
 reaction with n - boc - glycine .
amide 9 was obtained at room temperature in decent yield and further was 
 brominated to provide intermediate 10 . then a standard 
 buchwald 
hartwig amination reaction with substituted aniline 
 followed by deprotection finished the synthesis of compound 12 with the desired alkyl modification at position 8 .
reagents and conditions : 
 ( a ) k2co3 , dmf , rt , 84% ; ( b ) nh4oac , 
 toluene , reflux , 88% ( c ) ethyl 2-bromoacetate , cs2co3 , dmf , rt , 83% ; ( d ) pd / c , h2 ( balloon ) , meoh , rt , 
 91% ; ( e ) bh3thf , thf , reflux , 95% ; ( f ) hatu , diea , 
 ch2cl2 , rt , 70% ; ( g ) br2 , acoh , ch2cl2 , rt , 100% ; ( h ) arnh2 , pd2(dba)3 , xantphos , cs2co3 , dioxane , 
 150 c , 89% ( for example 22 , ar = 4f - ph and r1 = r2 = me ) ; ( i ) tfa , ch2cl2 , rt , 52% ( for example 22 , ar = 4f - ph and r1 = r2 = me ) . scheme 3 describes the synthesis of 6,6-dimethyl analogues .
analogous 
 chemistry to compound 12 was used for the first two steps 
 to provide imidazole 5a .
the compound was regioselectively n - alkylated with methallyl chloride to obtain the key intermediate 
 for the piperazine cyclization step .
the intramolecular hydroamination 
 reaction proved to be difficult , and after extensive screening , treating 
 the substrate 5a with a 5:1 mixture of acetic acid and 
 methanesulfonic acid at 210 c produced the cyclized core with 
 simultaneous loss of the carbamate protecting group . despite the harsh 
 reaction condition ,
an acceptable yield of the product ( 39% ) was obtained 
 and the regiochemistry was confirmed by 2d nmr of the intermediate . 

the cyclized core was subjected to amide coupling with n - boc glycine to yield intermediate 13 .
the final steps 
 in the sequence are analogous to scheme 2 to 
 furnish analogue 14 .
the sequence involved six steps 
 from intermediate 5a with an overall yield of 3.7% .
the 
 synthesis of the 5,5-dimethyl modified analogues diverged from their 
 8,8-dimethyl modified counterparts at intermediate 7a .
n - pmb protection was necessary for lactam 7a before the nah - mediated bis - methylation at the 5-position 
 ( scheme 4 ) .
the bicyclic intermediate 
 was then deprotected and amine was subjected to a hatu mediated amidation 
 to obtain compound 7c .
the final sequence involved bromination 
 followed by palladium - catalyzed aniline amination and boc - deprotection 
 to furnish 5,5-dimethyl modified compound 26 .
the sequence 
 involved eight steps from intermediate 7a with an overall 
 yield of 10.9% .
reagents and conditions : 
 ( a ) methallyl chloride , k2co3 , ki , dmf , rt , 
 55% ; ( b ) acoh / msoh ( 6:1 ) , 210 c , 39% ; ( c ) n - boc - glycine , hatu , diea , dmf , rt ; 57% ; ( d ) br2 , acoh , 
 ch2cl2 , rt ; 55% ; ( e ) arnh2 , pd2(dba)3 , xantphos , cs2co3 , 
 dioxane , 150 c ; ( f ) tfa , ch2cl2 , rt , 55% 
 over 2 steps ( for example 25 , ar = 4f - ph ) . reagents and conditions : 
 ( a ) pmbcl , koh , dmf , 0 c to rt , 63% ; ( b ) mei , nah , dmf , rt , 
 86% ; ( c ) bh3thf , thf , reflux , 100% ; ( d ) tfa , 70 c , 
 quantitative ; ( e ) n - boc - glycine , hatu , tea , dmf , 
 rt , 49% ; ( f ) br2 , acoh , ch2cl2 , 90% ; 
 ( g ) arnh2 , pd2(dba)3 , xantphos , cs2co3 , dioxane , 150 c ; ( h ) tfa , ch2cl2 , rt , 46% over 2 steps for example 26 where 
 ar = 4f - ph .
we began exploring 
 the sar on the piperazine ring by substituting at the 8-position first , 
 and we decided to use 4-f - aniline and 3-f-4-cl aniline based on our 
 previous work at the r6 position ( table 1 ) . adding a methyl group to the 
 8-position led to loss of potency by 10-fold ( cmpd 17 ) , but the compound was still equipotent to compound 15 ( scheme 5 ) .
moreover , there was no profound 
 effect on the potency by changing the absolute configuration of the 
 methyl group . increasing the size of substituent to isopropyl group 
 led to dramatic loss in potency ( examples 19 and 20 ) .
a dimethyl group on the 8-postion not only led to more 
 favorable compound in terms of completely blocking the benzylic carbon 
 as indicated by our metabolite studies but also removing the stereogenic 
 carbon .
surprisingly , substitution of 3cl-4f aniline in the 8-disubstitution 
 case provided a less potent compound when compared to other anilines 
 ( 21 vs 22 , 23 , or 24 ) .
emboldened by the increase in the potency based 
 on the substitution on the 8-position , we decided to study the effect 
 of dime group on the ethylene carbons of the piperazine ring . on the 
 basis of examples 25 and 26 , it was abundantly 
 clear that the dimethyl group on the 8-position had the most significant 
 positive effect on potency .
the substitution on the other positions 
 of the piperazine ring only provided modest potencies when compared 
 to 8-substitution , with activities in the double - digit nanomolar range . 

as the 5-substituted compounds and the 6-substituted compounds were 
 less potent , we decided to focus our attention on the effect of the 
 amide substituent of the piperazine nitrogen with the 8,8-dimethyl 
 group constant .
our previous efforts demonstrated that dimethyl glycine 
 had slightly inferior potency but offered superior oral exposure . 
 in the case of the substituted piperazines , installation of  

methylalanine amide provided equipotent compounds ( 15 vs 27 , 22 vs 28 , and 23 vs 29 ) .
we proceeded to further understand 
 the overall effect of the dimethylpiperazine on the minimal pharmacophore .
after studying the effects of various substitutions on the piperazine 
 ring and obtaining a significant improvement in activity , we decided 
 to reconfirm the minimal pharmacophore needed for antiplasmodial activity . 

on the basis of the earlier results , we were able to achieve antiplasmodial 
 activity with ec50 in 160400 nm range for 
 the core devoid of the amino acid substituent on the piperazine nitrogen 
 ( compound 31 , table 2 ) .
modification 
 of the piperazine ring into the corresponding lactam led to a 7-fold 
 decrease in activity in the micromolar range ( 32 ) .
however , 
 the antiplasmodial activity is regained by introduction of dimethyl 
 substituent on the 8-position with a 7-fold improvement ( 33 ) .
the most optimal compound from this lactam subseries was a compound 
 with 3cl-4f aniline substitution with an activity around 50100 
 nm ( 34 ) .
the dimethyl substitution on the reduced piperazine 
 core led to 10-fold improvement in potency ( 31 vs 35 ) with compounds to 10 nm range .
these 
 results led us to conclude that the amino acid substitution had minimal 
 to no effect on the potency of the compound with substitution on the 
 8-position of the imidazolopiperazine core and that the basicity of 
 the piperazine nitrogen could be modulated without complete loss in 
 antimalarial activity .
in parallel to 
 the sar studies to optimize the potency of the compounds series , we 
 evaluated the in vitro adme and in vivo pharmacokinetic properties 
 of key analogues .
in general , this series of compounds had very good 
 lipinski 's rule - of - five compliance and most of the analogues were 
 highly soluble ( > 175 m at ph 6.8 ) , with the neutral lactams 
 being least soluble among all the compounds .
table 3 summarizes the in vitro adme profiles of the important compounds . 
 typically , a range of values in the permeability assays was observed . 
 in the caco-2 assays , the core piperazine compounds ( both lactams 
 as well as nonamide piperazine compounds ) exhibited high apical to 
 basal values ( 31 , 32 , 34 , and 35 ) .
the permeability of the compounds was reduced once the 
 side chain amino acid was installed , but metabolic stability is improved 
 ( 28 vs 35 ) .
however , the % absorbed was lower 
 in the pampa assay for compounds with poor permeability in the caco-2 
 assay . upon comparing the calculated log p values 
 with pampa as well as caco-2 values
unsurprisingly , log p of 4.5 or higher was needed for having excellent percentage 
 absorbed values in pampa assay ( see supporting 
 information ) . removing the basicity of the piperazine ring 
 by introduction of carbonyl group improved the mouse and human microsome 
 stability of the compound , presumably by blocking the ethylene bridge 
 ( 31 vs 32 ) but is not evident in rat microsomes . 

introduction of the dimethyl group on the 8-position of the piperazine 
 ring does not improve overall metabolic stability of the compound 
 ( 15 vs 28 or 31 vs 35 ) despite the metabolite identification results presented earlier . 

this may be due to the ethylene bridge in the piperazine ring representing 
 a metabolic soft spot and enabling metabolic switching .
in general , 
 this scaffold exhibited poor rat metabolic stability when compared 
 to other species .
one interesting finding with the substituted piperazine 
 analogues was the improvement of activity against the herg channel 
 as determined in the automated patch clamp assay .
compared to the 
 unsubstituted piperazine analogue 27 , we were able to 
 decrease inhibition of the herg channel by at least 23-fold , 
 with most analogues ( such as 22 ) suggesting a lower potential 
 for qt interval prolongation in compounds with a substituted piperazine .
compounds series found to 
 have favorable potency and in vitro adme properties were tested in 
 snapshot pk , an abbreviated pk study shown to accurately bin compounds 
 into low , moderate , or high oral exposure .
compounds predicted to have reasonable oral exposure would be tested 
 in mice using both po and iv routes of administration ( table 4 ) .
the in vivo clearance generally was low to moderate 
 and significantly improved when compared to the corresponding unsubstituted 
 analogues ( 15 , 22 ) .
this resulted in higher 
 maximum concentration as well as improvement in auc values in the 
 oral arms as well .
lactam compound 34 exhibited highest 
 oral auc than all other analogues , and it also had lower intrinsic 
 clearance with moderate volume of distribution . to test
whether red 
 cell partitioning was occurring to explain the high volume of distribution 
 for the basic compounds , we carried out an in vitro experiment in 
 which compound 22 was incubated in nave rat whole 
 blood as well as plasma for 30 min .
after centrifuging the blood and 
 quantifying the amount of compound in the red blood cells , we determined 
 that the concentration of the compound was 3.47 times higher in whole 
 blood than in plasma alone , indicating that the compound is either 
 adhering to the rbc s or getting distributed inside the cells .
po dosing ; dose ( mg / kg ) , 20 ; 
 species / strain , mouse , male balb / c ; formulation , 2.5 mg / ml in peg300/d5w , 
 3:1 , solution ; vd = volume of distribution ; 
 cl = clearance ; cmax = maximal concentration ; tmax = time at maximal concentration ; auc = area 
 under the curve ; f = oral bioavailability . because we had examples of compounds 
 with different modifications with acceptable bioavailability , we decided 
 to carry on the efficacy experiment in mice
. the in vivo antimalarial 
 activities of the optimized compounds were evaluated using a plasmodium berghei mouse survival model . in this test , groups of three p. berghei infected mice were treated orally with 
 compound one day after infection .
the key readouts were the percentage 
 of parasitemia reduction on day 3 postinfection compared to the untreated 
 mice and the prolongation of survival of the infected mice .
table 5 summarizes the in vivo efficacy test 
 results of compound in comparison with standard antimalarials chloroquine 
 ( cq ) and artesunate ( as ) .
it was observed that compounds 22 , 24 , and 29 exhibited > 99% parasitemia 
 reduction at a low dose of 30 mg / kg with a modest survival of 15 days 
 or higher .
increasing the dose to 100 mg / kg or dosing the compound 
 3  30 mg / kg did not lead to increase in survival .
currently , 
 we are investigating the possibility of recrudescence of parasites 
 or development of resistance for the compound in vivo to better explain 
 the lack of curative activity in this model .
we decided to determine 
 the ed99 representative examples for the substituted and 
 nonsubstituted piperazine cores and found the substituted piperazine 
 core was 10-fold more efficacious ( compound 22 ed99 = 2.2 mg / kg ) than the unsubstituted core ( compound 1 ed99 = 26.5 mg / kg ) .
compounds 34 and 36 clearly proved inferior both in terms of parasitemia 
 reduction as well as survival in mice , indicating the need for the 
 amino acid side chain in providing better mouse efficacy .
activity = average parasitemia 
 reduction ; survival = average lifespan after infection ( 67 
 days for untreated control mice ) ; compounds were formulated in 75% 
 peg300/25% d5w ( 5% dextrose in water ) ; cq , chloroquine ; as , artesunate .
10% ethanol , 30% peg400 , 6% 
 vitamin etpgs . on the basis of the efficacy results
rat was chosen 
 as the second animal species for oral exposure evaluation as it may 
 serve as the in vivo preclinical toxicity species . taking into account 
 the nonoptimal metabolic stability data for rats for this scaffold , 
 we decided to study the oral exposure of the compound in rat for a 
 period of 5 h in the oral arm only to get an estimation of the exposure 
 ( table 6 ) .
-methylalanine subclass of compounds like 29 did not exhibit any in vivo exposure and were not further pursued . 
 in line with the better rat metabolic stability ( er = 0.68 ) ,
nonbasic 
 lactam compound 34 demonstrated the best rat exposure 
 despite lower mouse efficacy .
po dosing ; dose ( mg / kg ) , 20 ; species / strain , 
 rat , wistar ; formulation , 2.5 mg / ml in peg300/d5w , 3:1 , solution ; 
 blq , below level of quantification ; na = could not be determined . as the oral exposure of compounds 22 was 
 low in rats but demonstrated superior mouse efficacy , we decided to 
 dose compound 22 at higher doses to make sure that sufficient 
 multiples of exposure of the mouse efficacious dose could be achieved 
 ( table 7 ) .
we observe that when the dose was 
 increased to 30 mg / kg from 10 mg / kg , there was overproportional 7-fold 
 increase in auc as well as 2-fold increase in bioavailability .
increasing 
 the dose further to 100 mg / kg dose with suspension formulation led 
 to 55-fold increase in auc with a 3-fold increase in oral bioavailability . 

the auc at the mouse efficacious dose ( 2 mg / kg ) was 780 hnm , 
 therefore a 45 exposure multiple is achieved at 100 
 mg / kg in the rat .
the multiple of exposure provides a sufficient window 
 to use the rat as a rodent toxicology species .
it is unclear at this 
 time if this overproportional exposure at higher doses is due to saturating 
 metabolism or an efflux transporter , but given the partial efflux 
 observed in caco-2 , this may explain this multiple dose exposure results .
compound 22 was subjected 
 to a series of assays to evaluate the toxicity profile .
this drug 
 candidate was inactive on a panel of human cytochromes p450 ( ic50s are greater than 6 m for cyp1a2 , cyp2c19 , cyp2c9 , 
 cyp2d6 , and cyp3a4 ) , and the observed cytotoxicity cc50 were all greater than 12 m on a panel of mammalian cell lines 
 ( 293 t , ba / f3 , cho , hep2 , hela , huh7 ) , which translates to an adequate 
 selectivity index ( si > 100 ) .
compound 20 was found 
 negative in the mini - ames and micronucleus tests , which indicated 
 a low mutagenic potential .
finally , considering the efficacious plasma cmax in the mouse at 2 mg / kg is low ( 75 
 nm ) , the risk of cardiotoxicity is considered to be minimal where 
 the herg channel inhibitory activity ic50 = 13.4 m , 
 providing a greater than 170-fold window .
imidazolopiperazine compounds had been proven 
 to be effective against multidrug resistant parasite strains with 
 minimum toxicity to host - cell lines . however , early lead compound 
 such as compound 15 and 27 still suffer 
 from moderate potencies , relatively poor rat pk , and potent herg inhibitory 
 activity
. directed by the identification of in vitro metabolites formed 
 by compound 1 , the piperazine core of 15 was systematically modified by a gem - dimethyl functionality 
 at all three possible places .
the 8,8-dimethyl analogues demonstrated 
 excellent potency as well as improved oral exposure in the mouse . 

this also allowed us to use the glycine amide that reduced our herg 
 risk when compared to 27 .
the first generation of compounds 
 exhibited efficacy that was comparable to the known antimalarials 
 in the p. berghei mouse model , however , 
 compound 22 was more efficacious than chloroquine and 
 artesunate especially when parasitemia reduction is compared in terms 
 of ed99 values . although compound 22 demonstrated poor 
 rat pk at low doses , with higher doses it was able to achieve the 
 necessary exposure multiples calculated based on the plasma concentration 
 found to be efficacious in the mouse model .
compound 22 is thus currently under preclinical safety assessment in rats as 
 a potential candidate for human trials .
plasmodium falciparum cultures were grown in o rbcs using culturing media : 
 rpmi 1640 media ( no phenol red ) with l - glutamine and containing 
 0.05 mg / ml gentamicin , 0.014 mg / ml hypoxanthine , 38.4 mm hepes , 0.20% 
 sodium bicarbonate , 0.20% glucose , 3.4 nm naoh , 5% human serum , and 
 1.25% albumax . using traditional p. falciparum protocols published previously , 25 ml cultures were maintained in 
 75 cm flasks ( fisher cat .
10 - 126 - 41 ) at 5% hematocrit 
 and parasitemia ranging between 1% and 10% .
once parasitemia reached 810% , cultures are diluted down 
 to 1% or higher depending on needs .
cultures were gassed for 
 approximately 30 s to 1 min using a blood gas mixture to maintain 
 a gas composition of 96% nitrogen , 3% carbon dioxide , and 1% oxygen 
 and incubated at 37 c . on day 1 of the assay ,
the smear was 
 fixed onto the slide by placing in methanol for 30 s , stained in 10% 
 geimsa stain , and the percent of parasitized erythrocytes vs uninfected 
 erythrocytes was determined by microscopy with a light microscope .
p. falciparum strains 
 to be used for screening were prepared with screening media ( culturing 
 media without human serum but supplemented with 0.5% albumax ii ) and 
 fresh erythrocytes .
twenty l of screening media were dispensed 
 via the microflo ( biotek ) into 384-well , black clear - bottom assay 
 plates ( clear gnf custom plates by griener bio - one ) .
then 50 
 nl of compounds were transferred using a platemate plus ( matrix ) or 
 the gnf systems pintool into the assay plates containing screening 
 media to a final maximum concentration of 10 m in a 12 point 
 dose  response ( 1/2 log serial dilution ) . on the basis of the 
 measured parasitemia levels ,
each parasite strain was diluted to yield 
 0.5% parasitemia , and 50% hematocrit blood ( uninfected erythrocytes ) 
 was added to a concentration of 4.17% .
this diluted culture was used 
 in dispensing the remaining 60% of the assay volume ( 30 l ) 
 into the prewarmed assay plates via the microflo liquid dispenser . 

the final parasitemia was 0.3% and final hematocrit was 2.5% in a 
 total volume of 50 l .
assay plates were incubated at 37 c 
 for 72 h in a gas chamber filled with low oxygen blood gas .
after 
 the 72 h incubation ( equivalent to 12 cycles during the blood 
 stage ) , a prepared mixture of the lysis buffer ( 5 mm edta , 1.6% triton 
 x-100 , 20 mm tris - hcl , 0.16% saponin ) in water and sybr green detection 
 ( 0.1% ) reagent was dispensed at 10 l per well using the microflo . 

cultures were incubated for an additional 24 h at 25 c before 
 measuring fluorescence intensity using the envision plate reader ( perkin - elmer ) 
 with a 505 dichroic mirror .
data were normalized based on maximum fluorescence 
 signal values for dmso treated wells ( no inhibition by compound ) and 
 the minimum fluorescence signal values for wells containing the highest 
 concentration of inhibitor control compounds , for example , pyrimethamine 
 at a final concentration of 10 m .
data were analyzed on a plate - by - plate 
 basis and compared to reference compounds that were always included 
 on every plate , typically artemisinin , mefloquine , and pyrimethamine . 

ec50 values were obtained using a custom curve fitting 
 model , and a standard logistic regression model was applied for curve 
 fitting .
the quality of the assay run was assessed by the performance 
 of the reference compounds where the ec50 must be within 
 3-fold of the standard reference values for the assay plate to pass 
 requisite data quality control needs .
additionally , all compounds 
 were typically assayed at a minimum in duplicate ( independent assay 
 plates ) and ec50 values ideally must not vary by more than 
 2-fold between plates .
the metabolic stability of drug 
 candidates was determined in human , mouse , and rat liver microsomes 
 using the compound depletion approach , quantified by lc / ms / ms .
the 
 assay measured the rate and extent of metabolism of chemical compounds 
 by measuring the disappearance of the parent compound .
the assay determined 
 the compound s in vitro half - life ( t1/2 ) and hepatic extraction ratios ( er ) and predicted metabolic clearance 
 in human , rat , and mouse species .
the compound concentration was 
 determined by uv absorption measured with a spectramax190 microplate 
 spectrophotometer ( molecular devices corporation , sunnyvale , ca , usa ) 
 at absorption wavelengths between 260 and 290 nm .
the caco-2 assay was carried out in a 
 96-well format , and compound concentrations in each chamber were measured 
 by lc / ms .
the assay offers apparent permeability data of test compounds 
 from the apical to the basolateral chambers [ papp ( a  b ) ] and from the basolateral to the apical chambers 
 [ papp ( b  a ) ] .
the results can be 
 used to predict the in vivo oral absorption ( using papp ( a  b ) ) and the transport mechanism of the test 
 compounds across gi membrane ( ratio of papp ( b  a):papp ( a  b ) and log p lipophilicity data ) .
the assay utilizes electrophysiology 
 measurement on the electric current passing through herg channel that 
 is heterologously expressed in a stable cho cell line .
channels were 
 open by a herg - specific voltage protocol , and the compound effects 
 were directly characterized by their block of the herg current .
all compounds 
 were tested by 4-point dose  response curves , and ic50 values ( between 0.2 and 30 m ) were measured .
in - life studies 
 were carried out under protocols approved by the animal care and use 
 committee ( iacuc ) of gnf .
all compounds were formulated at a concentration 
 of 2.5 mg / ml in 75% peg300 and 25% d5w ( 5% dextrose in distilled water ) 
 solution and were filtered using a 0.45 m syringe filter .
the 
 filtered solutions were dosed intravenously via the lateral tail vein 
 at 5 mg / kg with a dosing volume of 2 ml / kg to male balb / c mice ( n = 3 per group ) .
the filtered solutions were also administered 
 orally at 20 mg / kg ( with a dosing volume of 8 ml / kg ) to another group 
 of mice ( n = 3 per group ) .
six blood samples of 50 
 l each were collected serially from each animal via retro orbital 
 sinus up to 24 h after dosing .
blood samples were collected into heparin 
 microtainer and centrifuged and plasma separated and frozen until 
 analysis .
compounds were formulated at a concentration of 2.5 mg / ml 
 in 75% peg300 and 25% d5w solution . in rat snapshot pk , two rats ( wistar 
 rats ) were administrated orally at 10 mg / kg dose and four blood samples 
 ( 100 l ) were taken at 0.5 , 1 , 3 , and 5 h post dosing . before 
 the sample analysis , the plasma samples were pooled based on the sampling 
 time . in the rat full pk studies , the formulations were filtered before 
 the administration to rats .
three rats were dosed intravenously ( 3 
 mg / kg ) , and three rats were administrated orally ( 10 mg / kg ) .
six blood 
 samples ( 100 l per sample ) were collected serially from each 
 rat within 24 h post dosing .
plasma samples ( 20 l ) 
 were extracted with solution of acetonitrile : methanol ( 3:1 ) containing 
 internal standard .
the samples were vortexed and then centrifuged 
 with an eppendorf centrifuge 5810r ( eppendorf , hamburg , germany ) at 
 a setting of 4000 rpm for 5 min at 4 c .
the supernatant was 
 transferred to a clean 96-well analysis plate for lc / ms / ms analysis . 

an aliquot ( 10 l ) was injected onto a phenomenex c18 guard 
 column ( 4 mm  2 mm ) followed by a zorbax sb - c8 analytical column 
 ( 2.1 mm  30 mm , 3.5 m , agilent technologies inc . ,
separation was carried out using a gradient elution 
 method with a mobile phase consisting of 0.05% formic acid in water 
 ( solvent a ) and 0.05% formic acid in acetonitrile ( solvent b ) .
the hplc system , consisting of agilent 
 1100 series binary pump ( agilent technologies inc . ) , agilent 1100 
 series micro vacuum degasser ( agilent technologies inc . ) , htc pal 
 ctc analytics autosampler ( leap technologies , carborro , nc ) , and vici 
 two - position actuator ( valco int .
, houston , tx , usa ) was interfaced 
 to a mds sciex api-4000 triple quadruple mass spectrometer ( mds inc . , 

mass spectral analyses were carried out using electrospray 
 source in the positive ion mode and using multiple reaction monitoring 
 ( mrm ) for quantification .
mrm transition of compound 15 383.9/101.2 , compound 22 412.2/312.1 , compound 24 430.1/330.1 , compound 27 412.2/327.2 , compound 29 436.1/308.1 , compound 34 430.0/346.1 , and 
 compound 36 355.0/272.3 was used with its optimized ms 
 parameters , respectively .
the mean value from the three 
 animals at each time point was plotted against time to give plasma 
 concentration time profile .
pharmacokinetic parameters were determined 
 using watson lims , version 7.2 ( thermo electron corporation , pa , usa ) , 
 by noncompartmental analysis .
pharmacokinetic parameters for compounds 
 were calculated by noncompartmental regression analysis using an in - house 
 fitting program developed at gnf .
the oral bioavailability ( f ) was calculated as the ratio between the area under the 
 curve ( auc ) following oral administration and the auc following intravenous 
 administration corrected for dose ( f = aucpo  doseiv / auciv  dosepo ) .
all in vivo efficacy 
 studies were approved by the veterinary authorities of the canton 
 basel - stadt . in vivo
antimalarial activity was usually assessed for 
 groups of three female nmri mice ( 2022 g ) intravenously infected 
 on day zero with 2  10 erythrocytes parasitized 
 with p. berghei gfp anka malaria strain 
 ( pbgfpcon donation from a. p. waters and c. j. janse , leiden university ) .
chloroquine and artesunate were formulated in 10% 
 ethanol , 30% peg400 , and 6% vitamin e tpgs .
compounds were administered 
 orally in a volume of 10 ml / kg either as a single dose ( 24 h post 
 infection ) or as three consecutive daily doses ( 24 , 48 , and 72 h post 
 infection ) . with the single dose regimen , parasitemia was determined 
 ( 72 h post infection ) and for the triple dose regimen ( 96 h post infection ) 
 using standard flow cytometry techniques .
activity was calculated as the difference between the mean percent 
 parasitemia for the control and treated groups expressed as a percentage 
 of the control group .
the survival time in days was also recorded 
 up to 30 days after infection .
a compound was considered curative 
 if the animal survived to day 30 after infection with no detectable 
 parasites . unless otherwise noted , materials 
 were obtained from commercial suppliers and were used without purification . 

removal of solvent under reduced pressure refers to distillation using 
 bchi rotary evaporator attached to a vacuum pump ( 3 
 mmhg ) .
products obtained as solids or high boiling oils were dried 
 under vacuum ( 1 mmhg ) .
purification of compounds by high pressure 
 liquid chromatography was achieved using a waters 2487 series with 
 ultra 120 5 m c18q column with a linear gradient from 10% solvent 
 a ( acetonitrile with 0.035% trifluoroacetic acid ) in solvent b ( water 
 with 0.05% trifluoroacetic acid ) to 90% a in 7.5 min , followed by 
 2.5 min elution with 90% a. h nmr spectra were recorded 
 on bruker xwin - nmr ( 400 or 600 mhz ) .
proton resonances are reported 
 in parts per million ( ppm ) downfield from tetramethylsilane ( tms ) .
h nmr data are reported as multiplicity ( s singlet , d doublet , 
 t triplet , q quartet , quint quinted , sept septed , dd doublet of doublets , 
 dt doublet of triplet , bs broad singlet ) , number of protons , and coupling 
 constant in hertz . for spectra obtained in cdcl3 , dmso - d6 , and cd3od , the residual protons 
 ( 7.27 , 2.50 , and 3.31 ppm , respectively ) were used as the reference .
analytical thin - layer chromatography ( tlc ) was performed on commercial 
 silica plates ( merck 60-f 254 , 0.25 mm thickness ) ; compounds were 
 visualized by uv light ( 254 nm ) .
100c , isco ) or using silica 
 gel ( merck kieselgel 60 , 230400 mesh ) .
the purity and quantitative 
 analysis were determined with a waters zq 2000 lc / ms system , which 
 employed an acquity uplc system ( binary pump , column compartment , 
 autosampler , and diode array uv detector ) .
the eluent was split between 
 the mass spectrometer and an antek chemiluminescent nitrogen detector 
 ( clnd ) .
the mobile phases used were ( a ) 95% h2o/5% meoh / ipa 
 ( 75/25 , v / v ) + 0.05% formic acid and
( b ) meoh / ipa ( 75/25 , v / v ) + 0.035% 
 formic acid . a gradient hplc method with flow of 0.4
ml / min started 
 at 2% b , with a hold of 1.0 min before a linear increase to 95% b 
 at 3.50 min , with a 30 s hold . from 4.04.25 min ,
the column used was a thermo syncronis c18 2.1 mm  30 mm , 
 3 m .
the mass spectrometer was operated in positive mode , with 
 a spray voltage of 3.2 kv and cone voltage of 30 v. the source and 
 desolvation temperatures were 130 and 400 c , respectively , with 
 600 l / h of nitrogen desolvation flow .
all the final compounds reported 
 in this communication had the purity of at least 95% .
elemental analyses 
 were carried out by midwest microlabs llc , indianapolis , in , usa . compound 1a was prepared from 1 by the following method : to a stirred 
 solution of 1 ( 12 mg , 0.03 mmol ) was added mno2 ( 52 mg , 0.60 mmol ) .
the reaction mixture was stirred at room temperature 
 ( rt ) for 2 h. lc / ms test showed that 1 was almost consumed 
 and the desired product ( [ m + 1 ] = 428 ) was detected as one of the 
 major peaks .
the 
 residue was subjected to a mass - triggered hplc purification to give 1a as well as 1b .
h nmr for 1a ( 400 mhz , cd3od )  7.597.63 ( m , 
 2h ) , 7.09 ( t , j = 6.4 hz , 2h ) , 6.957.02 ( m , 
 1h ) , 6.466.52 ( m , 1h ) , 6.346.38 ( m , 1h ) , 5.92 ( s , 
 1h ) , 4.33 ( dd , j = 4.4 , 14 hz , 1h ) , 3.96 ( dd , j = 4.8 , 13.2 hz , 1h ) , 3.703.78 ( m , 1h ) , 3.463.54 
 ( m , 1h ) , 1.73 ( s , 3h ) , 1.36 ( s , 3h ) .
cd3od )  7.757.78 ( m , 2h ) , 6.957.01 
 ( m , 3h ) , 6.446.50 ( m , 1h ) , 6.326.36 ( m , 1h ) , 4.19 
 ( dd , j = 1.6 , 6.8 hz , 2h ) , 4.00 ( dd , j = 1.6 , 6.8 hz , 2h ) , 1.47 ( s , 6h ) .
to a solution of 2-bromo-1-(4-fluorophenyl)ethanone ( 46.5 
 g , 214.29 mmol ) in dmf ( 400 ml ) was added 2-(benzyloxycarbonyl)-2-methylpropanoic 
 acid ( 55.8 g , 236.4 mmol ) and potassium carbonate ( 35.4 g , 256.5 mmol ) . 

the resulting solution was extracted 
 with ethyl acetate ( 2  800 ml ) , and the combined organic layer 
 was washed with water ( 2  800 ml ) and then brine ( 1  800 
 ml ) .
this 
 resulted in 67 g ( 84% ) of compound 4 as a white solid .
to a solution of compound 4 ( 70 g , 187.7 
 mmol ) in toluene ( 700 ml ) was added nh4oac ( 144.5 g , 1.88 
 mol ) .
the resulting solution was heated to reflux for 3 h in an oil 
 bath .
the resulting solution was extracted 
 with ethyl acetate ( 2  500 ml ) , and the combined organic layer 
 was washed with water ( 2  800 ml ) and brine ( 1  800 ml ) . 

this resulted 
 in 58 g ( 88% ) of compound 5 as a white solid .
h nmr ( 300 hz , dmso - d6 )  11.91 
 ( s , nh ) , 7.807.75 ( m , 2h ) , 7.547.12 ( m , 8h ) , 4.97 
 ( m , 2h ) , 1.6 ( s , 6h ) . to a solution of compound 5 ( 58 g , 164.3 
 mmol ) in dmf ( 400 ml ) was added cesium carbonate ( 134 g , 411.0 mmol ) . 

this was followed by the addition of ethyl 2-bromoacetate ( 33 g , 197.6 
 mmol ) dropwise with stirring at rt in 30 min .
the resulting solution was extracted with ethyl 
 acetate ( 2  700 ml ) , and the combined organic layer was washed 
 with water ( 2  800 ml ) and brine ( 1  800 ml ) . the resulting 
 mixture was dried concentrated under vacuum .
this resulted in 60 g 
 ( 83% ) of compound 6 as a yellow solid .
h 
 nmr ( 300 hz , dmso - d6 )  7.767.72 
 ( m , 2h ) , 7.327.28 ( m , 5h ) , 7.087.03 ( m , 3h ) , 5.08 
 ( m , 2h ) , 4.87 ( m , 2h ) , 4.23 ( q , j = 5.4 hz , 2h ) , 
 2.0 ( s , 6h ) , 1.28 ( t , j = 5.4 hz , 3h ) .
to a solution of compound 6 ( 70 g , 159.45 
 mmol , 1.00 equiv ) in methanol ( 800 ml ) was added palladium on carbon 
 ( 10 g ) .
this resulted in 7 ( 38 g , 145.56 mmol , 
 91% ) as a white solid .
lc - ms : ( es , m / z ) [ m + h ] calcd for c14h14fn3o , 260 ; found , 260 .
h nmr ( 300 hz , dmso - d6 )  7.797.74 ( m , 2h ) , 7.137.07 
 ( m , 3h ) , 6.35 ( s , 1h ) , 4.73 ( s , 2h ) , 1.79 ( s , 6h ) . to a stirred solution of compound 7 ( 5 g , 
 19.2 mmol , 1 equiv ) was dissolved in 50 ml of thf , 1 m borane / thf 
 complex ( 57 ml , 57 mmol , 3 equiv ) was added slowly and reaction was 
 refluxed overnight .
the crude product of 8 ( 4.5 g , 18.3 mmol , 
 95% ) was used in the next step .
lc - ms : ( es , m / z ) [ m + h ] calcd for c14h17fn3 246 , found 246 .
h nmr ( dmso , 300 hz )  
 7.757.70 ( m , 2h ) , 7.4 ( s , 1h ) , 7.14 ( t , j = 9.0 hz , 2h ) , 3.9 ( t , j = 5.4 hz , 2h ) , 2.51 ( t , j   = 5.4 hz , 2h ) , 1.41(s , 6h ) ; nh proton not observed .
to a stirred solution of compound 8 ( 2.9 
 g , 11.82 mmol , 1.1 equiv ) and 2-(tert - butoxycarbonylamino)acetic 
 acid ( 2.27 g , 13 mmol , 1.1 equiv ) in 15 ml of dichloromethane were 
 added diea ( 2.47 ml , 14.18 mmol , 1.2 equiv ) and hatu ( 5.39 g , 14.18 
 mmol , 1.2 equiv ) .
the reaction mixture was stirred at rt for 8 h. 
 hplc / ms analysis showed that desired product compound 9 was the major product .
the organic layer was washed with washed with water ( 1  
 30 ml ) , followed by saturated nahco3 ( 1  30 ml ) and 
 finally with brine ( 1  30 ml ) .
the resulting oil was purified using column chromatography with hexanes / ethyl 
 acetate ( 0100% linear gradient ) used as eluant .
the desired 
 product compound 9 was obtained as oil ( 3.3 g , 8.27 mmol , 
 70% ) .
h nmr ( 300 hz , dmso - d6 , )  7.777.72 ( m , 2h ) , 7.54 ( s , 1h ) , 7.207.14 
 ( m , 2h ) , 6.846.80 ( m , 1h ) , 4.07 ( s , 2h ) , 3.90 ( d , j = 3.0 hz , 2h ) , 3.70 ( s , 2h ) , 1.80 ( s , 6h ) , 1.40 ( s , 9h ) . 
 lc - ms : ( es , m / z ) [ m + h ] calcd for c21h28fn4o3 , 403 ; found , 403 . to a stirred solution of compound 9 ( 3.02 
 g , 7.51 mmol ) in dichloromethane ( 30 ml )
was added br2 ( 0.43 
 ml , 8.26 mmol ) in acetic acid ( 3 ml ) .
hplc / ms test showed that desired product ( ii ) was 
 the only peak .
the product was confirmed by 400 mhz proton nmr to be 
 the title compound 10 .
the product was used in the next 
 step without further purification and exhibited quantitative mass 
 recovery .
lc - ms : ( es , m / z ) [ m + 
 h ] calcd for c21h27brfn4o3 , 482 ; found , 482 .
h nmr ( meoh - d4 , 400 hz )  7.847.81 ( m , 2h ) , 7.14 ( t , j = 8.8 hz , 2h ) , 4.094.01 ( m , 4h ) , 3.81 ( t , j = 4.8 hz , 2h ) , 1.89 ( s , 6h ) , 1.46 ( s , 9h ) .
( s)-2-amino-1-(3-(4-chloro-3-fluorophenylamino)-2-(4-fluorophenyl)-8-methyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 17 was prepared from compound 10 ( r1 = me , r2 = h ) by a pd2(dba)3 mediated amination reaction with 4-chloro-3-fluoroaniline 
 followed by a tfa mediated deprotection in a protocol similar to compound 22 .
h nmr ( meoh - d4 , 400 mhz )  7.617.58 ( m , 2h ) , 7.167.12 ( m , 
 1h ) , 7.077.02 ( m , 2h ) , 6.476.42 ( m , 2h ) , 5.74 ( m , 
 1h ) , 4.073.68 ( m , 6h ) , 1.56 ( d , j = 6.8 hz , 
 3h ) .
( r)-2-amino-1-(3-(4-chloro-3-fluorophenylamino)-2-(4-fluorophenyl)-8-methyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 18 was prepared from compound 10 ( r1 = me , r2 = h ) by a pd2(dba)3 mediated amination reaction with 4-chloro-3-fluoroaniline 
 followed by a tfa mediated deprotection in a protocol similar to compound 22 .
( s)-2-amino-1-(3-(4-chloro-3-fluorophenylamino)-2-(4-fluorophenyl)-8-isopropyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 19 was prepared from compound 10 ( r1 = isopropyl , r2 = h ) by a pd2(dba)3 mediated amination reaction with 4-chloro-3-fluoroaniline 
 followed by a tfa mediated deprotection in a protocol similar to compound 22 .
h nmr ( meoh - d4 , 400 mhz ) 7.617.58 ( m , 2h ) , 7.267.20 ( m , 
 3h ) , 6.456.38 ( m , 2h ) , 5.5 ( d , j = 7.9 hz , 
 1h ) , 4.113.75 ( m , 6h ) , 2.362.31 ( m , 1h ) , 1.15 ( d , j = 6.7 hz , 3h ) , 0.95 ( d , j = 6.7 hz , 3h ) .
( r)-2-amino-1-(3-(4-chloro-3-fluorophenylamino)-2-(4-fluorophenyl)-8-isopropyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 20 was prepared from compound 10 ( r1 = isopropyl , r2 = h ) by a pd2(dba)3 mediated amination reaction with 4-chloro-3-fluoroaniline 
 followed by a tfa mediated deprotection in a protocol similar to compound 22 .
h nmr ( meoh - d4 , 400 mhz )  7.697.67 ( m , 2h ) , 7.277.20 ( m , 
 3h ) , 6.686.65 ( dd , j = 2.5 hz , j = 11.0 hz , 1h ) , 6.606.55 ( dd , j = 2.25 
 hz , j = 8.7 hz , 1h ) , 5.78 ( d , j = 
 7.7 hz , 1h ) , 4.424.02 ( m , 4h ) , 3.943.87 ( m , 2h ) , 2.55 
 ( m,1h ) , 1.24(d , j = 6.8 hz , 3h ) , 1.05 ( d , j = 6.8 hz , 3h ) .
m / z = 
 460.2 ( m + 1 ) . 2-amino-1-(3-(4-chloro-3-fluorophenylamino)-2-(4-fluorophenyl)-8,8-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 21 was prepared from compound 10 ( r1 = me , r2 = me ) by a pd2(dba)3 mediated amination reaction with 4-chloro-3-fluoroaniline 
 followed by a tfa mediated deprotection in a protocol similar to compound 22 .
h nmr ( meoh - d4 , 400 mhz )  7.597.55 ( m , 2h ) , 7.207.13 ( m , 
 3h ) , 6.60 ( dd , j = 11.2 , 10.8 hz ) , 1h ) , 6.53 ( dd , j = 2.4 , 0.8 hz , 1h ) , 4.0 ( m , 4h ) , 3.78 ( m , 2h ) , 2.00 ( s , 
 6h ) .
elemental 
 analysis ( compound + 2.0 hcl + 2.0 h2o ) : % c , 47.62 ; % h , 
 5.09 ; % n , 12.62 ; ( calc ) .
% c = 47.54/47.38 ; % n = 12.44/12.46 ; % h = 
 4.62/4.57 ( experimental ) .
2-amino-1-(2-(4-fluorophenyl)-3-(4-fluorophenylamino)-8,8-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : in a glass vial , cs2co3 , 4f - aniline 
 ( 0.462 g , 4.1 mmol , 2.0 equiv ) , pd2(dba)3 ( 0.095 
 g , 0.104 mmol , 0.05 equiv ) , xantphos ( 0.120 g , 0.208 mmol , 0.1 equiv ) , 
 and dioxane were stirred for 5 min at rt .
compound 10 ( 1 g , 2.08 mmol , 1.0 equiv ) was added to the reaction mixture , after 
 which the reaction mixture was degassed for 15 min and then stirred 
 at 120 c under n2 for 8 h. hplc / ms test showed that 
 the starting material compound 10 was consumed and desired 
 product was formed predominantly along with some 9 .
the reaction mixture was concentrated 
 and then purified by normal phase column chromatography ( silica gel 
 80 g ) using a gradient of 1000% to 0100% hexane : etoac . 

the organic layer 
 was concentrated at reduced pressure to yield the boc derivative ( 950 
 mg , 89% ) yield .
lc - ms : ( es , m / z ) 
 [ m + h ] calcd for c27h32f2n5o3 , 512 ; found , 512 .
the boc compound 
 was treated with 20% tfa in ch2cl2 ( 50 ml ) and 
 was added to the mixture .
after the completion of this reaction ( monitored 
 by lcms ) , the resulting mixture was concentrated under reduced pressure . 

the resulting residue was purified by reverse phase hplc to yield 
 product as a tfa salt .
lc - ms : ( es , m / z ) [ m + h ] calcd for c22h23f2n5o , 412.2 ; found , 412.1 .
h nmr ( meoh - d4 , 400 hz )  7.617.57 ( m , 2h ) , 
 6.94 ( t , j = 8.8 hz , 2h ) , 6.81 ( t , j = 8.8 hz , 2h ) , 6.47 ( m , 2h ) , 3.72 ( m , 2h ) , 3.58 ( m , 2h ) , 3.42 ( m , 
 2h ) , 1.85 ( s , 6h )
. elemental analysis compound 22 with 
 0.65 equiv h2o : c , 62.44 ; n , 16.55 ; h , 5.79 ( calculated ) . 

c = 62.54/62.44 ; n = 16.35/16.29 ; h = 5.52/5.61 ( experimental ) .  
the regiochemistry of the dime groups was unambiguously assigned 
 using 2-d nmr techniques ( table 8) .
2-amino-1-(2-(4-fluorophenyl)-8,8-dimethyl-3-(p - tolylamino)-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 23 was prepared from compound 10 ( r1 = me , r2 = me ) by a pd2(dba)3 mediated amination reaction with 4-methylaniline 
 followed by a tfa mediated deprotection in a protocol similar to compound 22 .
h nmr ( meoh - d4 , 400 mhz )  7.797.69 ( m , 2h ) , 7.21 ( t , j = 8.4 hz , 2h ) , 7.04 ( d , j = 8.0 hz , 2h ) , 6.76 ( d , j = 8.2 hz , 2h ) , 4.11 ( s , 2h ) , 3.88 ( m , 4h ) , 2.22 ( s , 3h ) , 
 2.12 ( s , 6h ) .
lc / ms major mass : 408.1 ( m + h ) . 2-amino-1-(3-((3,4-difluorophenyl)amino)-2-(4-fluorophenyl)-8,8-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 24 was prepared from compound 10 ( r1= me , r2 = me ) by a pd2(dba)3 mediated amination reaction with 3,4-difluoroaniline 
 followed by a tfa mediated deprotection in a protocol similar to compound 22 .
h nmr ( meoh - d4 , 400 mhz )  7.737.69 ( m , 2h ) , 7.21 ( t , j = 8.8 hz , 2h ) , 7.067.03 ( m , 1h ) , 6.766.65 ( m , 1h ) , 
 6.536.51 ( m , 1h ) , 4.084.06 ( m , 4h ) , 3.863.83 
 ( m , 2h ) , 2.07 ( s , 6h ) .
elemental analysis ( compound + 2.9 hcl ) : % c , 
 49.37 ; % h , 4.69 ; % n , 13.09 ; ( calc ) .
% c = 49.43/49.06 ; % n = 12.89/12.77 ; 
 % h = 4.7/4.81 ( experimental ) .
13 was prepared from compound 5a by the following way : to a stirred solution of compound 5a ( 1.1 
 g , 3.38 mmol , 1.00 equiv ) in dmf ( 30 ml ) was added 3-chloro-2-methylprop-1-ene 
 ( 500 mg , 5.49 mmol , 1.50 equiv ) , potassium carbonate ( 560 mg , 4.06 
 mmol , 1.10 equiv ) , and potassium iodide ( 1.12 g , 6.75 mmol , 2.00 equiv ) 
 at rt .
the mixture 
 was washed with brine ( 3  10 ml ) , dried over sodium sulfate , 
 and concentrated under vacuum .
the residue was applied onto a silica 
 gel column with petroleum ether / etoac ( 5:1 ) to give 0.7 g ( 55% ) of 
 compound 5a-1 as a light - yellow solid .
m / z = 380 ( m + 1 ) . into a 30 ml sealed tube , was placed compound 5a-1 ( 2.0 g , 5.28 mmol , 1.00 equiv ) , acetic acid ( 12 ml ) , 
 and methanesulfonic acid ( 2 ml ) .
the reaction mixture was stirred 
 for 12 h at 260 c ( the temperature of the sand bath ) .
the aqueous layer was washed with ethyl acetate ( 3  10 ml ) . 

the aqueous 
 layer was extracted with ethyl acetate ( 3  10 ml ) .
the combined 
 organic layers were washed with brine ( 3  10 ml ) , dried over 
 sodium sulfate , and concentrated under vacuum .
the solid was collected 
 by filtration and washed with 5 ml of n - hexane to 
 give 500 mg ( 39% ) of compound 5a-2 as a 
 white solid .
h nmr ( 400 mhz , cdcl3 )  
 7.707.65 ( m , 2h ) , 7.046.9 ( m , 2h ) , 6.96 ( s , 1h ) , 4.12 
 ( s , 2h ) , 3.69 ( s , 2h ) , 1.23 ( s , 6h ) . to a stirred solution of compound 5a-2 ( 280 mg
, 1.14 mmol , 1.0 equiv ) in dmf ( 10 ml ) was added 
 2-(tert - butoxycarbonyl)acetic acid ( 600 mg , 3.43 
 mmol , 3.0 equiv ) , hatu ( 1.3 g , 3.42 mmol , 3.0 equiv ) , and diea ( 880 
 mg , 6.82 mmol , 6.0 equiv ) at rt .
the organic layer was washed with brine ( 3  10 ml ) , 
 dried over sodium sulfate , and concentrated under vacuum .
the residue 
 was applied onto a silica gel column with ch2cl2/meoh ( 10:1 ) to give 280 mg ( 57% ) of compound 13 as 
 a brown solid .
h nmr ( 400 mhz , cdcl3 )  
 7.727.68 ( m , 2h ) , 7.23 ( s , 1h ) , 7.137.07 ( m , 2h ) , 
 5.46 ( s , nh ) , 4.65 ( s , 2h ) , 4.054.04 ( m , 2h ) , 3.97 ( s , 2h ) , 
 1.551.39 ( m , 15h ) .  
the structure of 13 was verified by hmqc , cosy , hmbc , and roesy ( table 9 ) .
2-amino-1-(2-(4-fluorophenyl)-3-(4-fluorophenylamino)-6,6-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : to a stirred solution of compound 13 ( 386 
 g , 0.96 mmol ) in 6 ml of dichloromethane was added br2 ( 55 
 l , 1.06 mmol ) in acetic acid ( 2 ml ) .
after neutralization , the residue was subjected 
 to flash chromatography ( 40 g , 0100% ethyl acetate in hexane , 
 50 min , dry loading ) purification to give 256 mg ( 55% ) of the title 
 compound 13a as a colorless oil .
h nmr ( 400 
 mhz , cdcl3 )  7.877.83 ( m , 2h ) , 7.03 ( t , j = 8.8 hz , 2h ) , 5.46 ( s , 1h ) , 4.5 ( s , 2h ) , 3.97 ( d , j = 4.2 hz , 2h ) , 3.81 ( s , 2h ) , 1.45 ( s , 6h ) , 1.39 ( s , 9h ) .
example 25was obtained from 13a by a 
 pd2(dba)3 mediated amination reaction with 4-fluoroaniline 
 followed by a tfa mediated deprotection by analogy to compound 22 ( 55% yield over 2 steps ) .
h nmr ( 400 mhz , dmso - d6 ) :  8.06 ( m , 2h ) , 7.98 ( m , 1h ) , 7.80 
 ( m , 2h ) , 7.16 ( m , 2h ) , 6.98 ( m , 2h ) , 6.60 ( m , 2h ) , 4.64 ( m , 2h ) , 4.02 
 ( m , 2h ) , 3.80 ( m , 2h ) , 1.38 ( s , 6h ) .
2-(4-fluorophenyl)-7-(4-methoxybenzyl)-5,5-dimethyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine : compound 7b was prepared from compound 7a ( r1 = r2 = h ) by the following way : to a solution of compound 7a ( 231 mg , 1.0 
 mmol ) in dmf ( 10 ml ) were added koh ( 168 mg , 3.0 mmol ) and pmbcl ( 405 
 l , 3.0 mmol ) at 0 c .
the reaction mixture was stirred 
 at the same temperature for 2 h and at rt for 2 additional hours . 

the hplc 
 fraction were evaporated under reduced pressure and neutralized by 
 satd nahco3 .
the organic layer was concentrated 
 under reduced pressure to give compound 7a-1 ( 221 mg , 63% ) as a white solid .
h nmr ( 400 mhz , cdcl3 )  7.677.63 ( m , 2h ) , 7.287.25 ( m , 2h ) , 
 7.077.02 ( m , 2h ) , 6.896.86 ( m , 2h ) , 4.74 ( s , 2h ) , 
 4.70 ( m , 2h ) , 4.53 ( s , 2h ) , 3.79 ( s , 3h ) . to a solution of compound 7a-1 ( 253 mg , 0.72 mmol ) in dmf ( 15 ml ) were added 60% sodium hydride 
 ( 87 mg , 0.085 mmol ) and methyl iodide ( 0.45 ml , 7.2 mmol ) at rt .
the 
 reaction mixture was stirred at rt for 2 h. the reaction mixture was 
 quenched with methanol and directly subjected to mass - triggered hplc 
 purification to give 7a-2 as a white solid 
 after evaporation of acetonitrile  water in vacuo .
the tfa salt 
 was subsequently neutralized by saturated sodium bicarbonate solution 
 and extracted with dichloromethane to get the compound as a free base . 

the organic layer was dried over sodium sulfate and concentrated under 
 reduced pressure to yield the desired compound 7a-2 ( 234 mg , 86% ) .
the structure of 7a-2 was verified by 2-d nmr techniques ( table 10 ) . to a solution of compound 7a-2
( 170 mg , 0.45 mmol ) in thf ( 9 ml ) was added 1.0 n bh3thf ( 2.70 ml , 2.70 mmol ) at rt .
the reaction mixture was stirred 
 at reflux for 2 h. pd / c was added ( gas generated ) .
the reaction mixture 
 was stirred for 1 h. solid was filtered off , and solvent was removed . 

the product was assumed to be of 100% yield and used in the next 
 step without further purification .
h nmr ( 400 mhz , cd3od )  8.1 ( s , 1h ) , 7.747.70 ( m , 2h ) , 7.34 ( d , j = 8.6 hz , 2h ) , 7.27 ( m , 2h ) , 6.94 ( d , j = 8.6 hz , 2h ) , 3.91 ( s , 2h ) , 3.80 ( s , 3h ) , 3.79 ( s , 2h ) , 2.87 ( s , 
 2h ) , 1.61 ( s , 6h ) .
7-(4-methoxybenzyl)-2-(4-fluorophenyl)-5,5-dimethyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine ( 1.07 g , 2.93 mmol , 1.00 equiv ) ( 7b ) was added to trifluoroacetic acid ( 15 ml ) .
the resulting solution 
 was stirred for 2 h at 70 c in an oil bath .
the ph value of the solution was adjusted to 8 
 with aqueous sodium bicarbonate .
7b-1 was 
 extracted from the aqueous layer using 3  100 ml of ethyl acetate . 

this resulted in 7b-1 ( 718 mg , quantitative ) 
 as a yellow solid .
to a solution of 7b-1 ( 750 
 mg , 3.06 mmol , 1.00 equiv ) in n , n - dimethylformamide ( 30 ml ) were added 2-(tert - butoxycarbonyl)acetic 
 acid ( 850 mg , 4.86 mmol , 1.50 equiv ) , hatu ( 1.3 g , 3.42 mmol , 1.10 
 equiv ) , and triethylamine ( 650 mg , 6.44 mmol , 2.00 equiv ) . the resulting 
 solution was stirred overnight at rt .
the resulting mixture was washed with brine 
 ( 3  100 ml ) , dried over anhydrous sodium sulfate , and concentrated 
 under vacuum .
the residue was applied onto a silica gel column with 
 petroleum ether / ethyl acetate ( 4:1 ) .
this resulted in 600 mg ( 49% ) 
 of 7c as a pale - yellow solid .
h nmr ( 300 
 mhz , cdcl3 ) :  7.75 ( m , 2h ) , 7.22 ( s , 1h ) , 7.137.07 
 ( m , 2h ) , 4.994.87 ( m , 2h ) , 4.12 ( s , 2h ) , 3.91 3.65 ( m , 2h ) , 
 1.57 ( m , 6h ) , 1.43 ( s , 9h ) . to a stirred solution of compound 7b-2 ( 583 g , 1.45 mmol ) in dichloromethane ( 6 ml ) was added br2 ( 82 l , 1.60 mmol ) in acetic acid ( 2 ml ) .
after neutralization , 
 the residue was subjected to flash chromatography ( 40 g , 0100% 
 ethyl acetate in hexane , 50 min , dry loading ) purification to give 
 628 mg ( 90% ) of the title compound 7c as a yellow solid .
h nmr ( 400 mhz , cdcl3 )  7817.78 
 ( m , 2h ) , 7.07 ( t , j = 8.6 hz , 2h ) , 5.28 ( s , nh ) , 
 4.844.7 ( m , 2h ) , 4.073.58 ( m , 4h ) , 1.491.44 
 ( m , 6h ) , 1.28 ( s , 9h ) .
2-amino-1-(2-(4-fluorophenyl)-3-(4-fluorophenylamino)-5,5-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 26 was prepared from compound 7c by a pd2(dba)3 mediated amination 
 reaction with 4-fluoroaniline followed by a tfa mediated deprotection 
 in 46% yield over 2 steps .
h nmr ( 400 mhz , cd3od ) :  7.57 ( m , 2h ) , 7.02 ( m , 2h ) , 6.77 ( m , 2h ) , 6.52 ( m , 2h ) , 
 4.974.91 ( m , 2h ) , 4.093.74 ( m , 4h ) , 1.56 ( s , 3h ) , 
 1.51 ( s , 3h ) .
2-amino-1-(2-(4-fluorophenyl)-3-(4-fluorophenylamino)-8,8-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)-2-methylpropan-1-one : compound 28 was prepared from compound 8 ( r1 = r2 = me ) in the following fashion 
 using a slightly modified route to the one described in scheme 2 . to a stirred solution of compound 8 ( 1.48 
 g , 6.04 mmol ) and net3 ( 6.0 g , 59.4 mmol ) in dichloromethane 
 ( 20 ml ) was added 2-bromo-2-methylpropanoyl bromide ( 14 g , 60.9 mmol ) 
 dropwise at rt . after being stirred for 3 h at rt , the reaction was 
 then quenched by the addition of water ( 30 ml ) .
the combined organic 
 layers were washed with brine , dried over anhydrous sodium sulfate , 
 and concentrated under vacuum .
this resulted in crude product as a 
 dark solid , which was washed with etoac : petroleum ether ( 1:10 ) to 
 remove the impurities to produce compound 8 - 1 as a gray solid .
( 2.0 g , 5.08 mmol , 1.00 equiv ) in dmf ( 10 ml ) was nan3 ( 1.0 g , 15.38 mmol , 3.00 equiv ) at rt .
the resulting mixture was washed with 3  
 20 ml of brine , dried over anhydrous sodium sulfate , and concentrated 
 under vacuum .
the residue was applied onto a silica gel column with 
 petroleum ether / etoac ( 5:1 ) to give compound 8 - 2 as a white solid .
m / z 357 
 ( m + h ) . to a stirred solution of compound 8 - 2 ( 1.2 g
, 3.37 mmol , 1.00 equiv ) in methanol ( 20 ml ) was added 
 pd / c ( 80 mg , 0.75 mmol , 0.20 equiv ) at rt .
m / z 331 ( m + h ) . to a stirred solution of compound 8 - 3 ( 910 mg
, 2.76 mmol , 1.00 equiv ) in thf ( 50 ml ) was added 
 boc anhydride ( 3.2 g , 14.68 mmol , 5.00 equiv ) , followed by aqueous 
 naoh ( 1n , 6 ml , 2.0 equiv ) at rt .
the organic layer 
 was washed with 3  10 ml of brine , dried over na2so4 , and concentrated under vacuum .
the solid was collected 
 by filtration and washed with n - hexane ( 5 ml ) to 
 give compound 8 - 4 as a white solid .
h nmr ( 300 mhz , cdcl3 )  7.757.71 
 ( m , 2h ) ,
7.097.02 ( m , 3h ) , 4.84(s , nh ) , 4.094.0 ( m , 
 4h ) , 1.98 ( s , 6h ) , 1.55 ( s , 6h ) , 1.45 ( s , 9h ) . to a stirred solution of compound 8 - 4 ( 32 mg , 0.074 mmol ) in dichloromethane ( 3 ml ) was added 
 br2 ( 4.2 l , 0.082 mmol ) in acetic acid ( 1 ml ) .
after neutralization , 
 the residue was subjected to flash chromatography ( 4 g , 060% 
 ethyl acetate in hexane , 16 min ) purification to give the compound 8 - 5 as colorless oil , was collected by filtration , 
 and washed with n - hexane ( 5 ml ) to give compound 8 - 4 as a white solid .
h nmr ( 400 
 mhz , cdcl3 )  7.917.88 ( m , 2h ) , 7.107.04 
 ( m , 2h ) , 5.11(s , nh ) , 4.01 ( m , 2h ) , 3.96 ( s , 2h ) , 1.94 ( s , 6h ) , 1.51 
 ( s , 6h ) , 1.41 ( s , 9h ) .
compound 28 was prepared 
 from compound 8 - 5 by a pd2(dba)3 mediated amination reaction with 4-fluoroaniline followed 
 by a tfa mediated deprotection .
h nmr ( 400 mhz , meoh - d4 )  7.66 ( dd , j = 9.2 , 
 5.2 hz , 2h ) , 7.11 ( t , j = 8.8 hz , 2h ) , 6.81 ( t , j = 8.8 hz , 2h ) , 6.726.68 ( m , 2h ) , 4.043.99 
 ( m , 4h ) , 2.01 ( s , 6h ) , 1.66 ( s , 6h ) .
2-amino-1-(2-(4-fluorophenyl)-8,8-dimethyl-3-(p - tolylamino)-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)-2-methylpropan-1-one : compound 29 was prepared from 35 by the following way : to a stirred solution of 35 ( 21 mg , 0.06 
 mmol ) and et3n ( 83 l , 0.60 mmol ) in dry dichloromethane 
 ( 6 ml ) was added 2-bromo-2-methylpropanoyl bromide ( 71 l , 0.60 
 mmol ) .
the reaction mixture was stirred at rt for 5 h. the reaction 
 mixture was concentrated and subjected to mass - triggered lc / ms purification 
 directly .
the obtained solution was concentrated to give 22 mg ( 73% ) 
 of compound 35 - 1as yellow oil after neutralization .
h nmr ( 400 mhz , meoh - d4 )  
 7.717.66 ( m , 2h ) , 7.237.19 ( m , 2h ) , 7.02 ( d , j = 8.4 hz , 2h ) , 6.66 ( d , j = 8.4 hz , 2h ) , 
 4.31 ( m , 2h ) , 4.09 ( m , 2h ) , 2.21 ( s , 3h ) , 2.06 ( s , 6h ) , 2.00 ( s , 6h ) . to a solution of compound 35 - 1
( 22 mg , 0.044 mmol ) in dmf ( 3 ml ) was added nan3 ( 8.6 
 mg , 0.132 mmol ) at rt .
the reaction mixture was stirred at rt for 
 2 h. the reaction mixture was directly subjected to mass - triggered 
 hplc purification directly .
the obtained mecn / aqueous solution was 
 combined and concentrated to give 15 mg ( 75% ) of compound 35 - 2 as yellow oil after neutralization .
h 
 nmr ( 400 mhz , meoh - d4 )  7.70 7.66 
 ( m , 2h ) , 7.237.19 ( m , 2h ) , 7.04 ( d , j = 8.3 
 hz , 2h ) , 6.66 ( d , j = 8.4 hz , 2h ) , 4.26 ( m , 2h ) , 
 4.074.05 ( m , 2h ) , 2.24 ( s , 3h ) , 2.03 ( s , 6h ) , 1.58 ( s , 6h ) . to a solution of compound 35 - 2 ( 15 mg , 0.033 mmol ) in meoh ( 3 ml ) was added 10% pd / c ( 4 mg , 0.003 
 mmol ) at rt .
the reaction 
 mixture was stirred at rt for 2 h. solid was filtered off , and solvent 
 was removed .
the reaction mixture was directly subjected to mass - triggered 
 hplc purification to give 15 mg ( 100% ) of the title compound yellow 
 oil .
h nmr ( meoh - d4 , 400 hz ) 
  7.787.75 ( m , 2h ) , 7.066.97 ( m , 4h ) , 6.56 ( d , j = 8.4 hz , 2h ) , 5.16 ( s , nh ) , 4.33 ( t , j = 4.8 hz , 2h ) , 3.74 ( t , j = 4.8 hz , 2h ) , 2.29 ( s , 
 3h ) , 1.95 ( s , 6h ) , 1.45 ( s , 6h ) .
2-amino-1-(3-(3,4-difluorophenylamino)-2-(4-fluorophenyl)-8,8-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)-2-methylpropan-1-one : compound 30 was prepared from compound 8 - 5 by a pd2(dba)3 mediated 
 amination reaction with 3,4-difluoroaniline followed by a tfa mediated 
 deprotection.h nmr ( 400 mhz , meoh - d4 )  7.637.60 ( m , 2h ) , 7.14 ( t , j = 8.4 hz , 2h ) , 7.01 ( dd , j = 18.8 , 8.8 hz , 1h ) , 
 6.686.63 ( m , 1h ) , 6.5 ( m , 1h ) , 4.03 ( m , 2h ) , 4.01 ( m , 2h ) , 
 2.01 ( s , 6h ) , 1.66 ( s , 6h ) .
2-(4-fluorophenyl)-n-(p - tolyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazin-3-amine : the details of the synthesis involving an ugi reaction 
 between 4-fluorobenzaledehyde , 2-aminopyrazine , and 1-isocyano-4-methylbenzene 
 has been reported in the previous communication.h nmr ( 400 mhz , meoh - d4 )  7.73 ( d , j = 6.8 hz , 2h ) , 7.006.99 
 ( m , 4h ) , 6.49 ( d , j = 8.0 hz , 2h ) , 4.01 ( s , 2h ) , 
 3.72 ( m , 2h ) , 3.15 ( m , 2h ) , 2.19 ( s , 3h ) .
2-(4-fluorophenyl)-3-(p - tolylamino)-7,8-dihydroimidazo[1,2-a]pyrazin-6(5h)-one : compound 32 was synthesized from compound 7 ( r1 = r2 = h ) pd2(dba)3 mediated coupling reaction with p - toluidine .
h nmr ( 400 mhz , dmso - d6 )  
 8.55 ( s , 1h ) , 7.777.75 ( m , 2h ) , 7.267.14 ( m , 4h ) , 
 7.016.95 ( m , 2h ) , 6.53 ( m , 1h ) , 4.53 ( m , 2h ) , 4.22 ( m , 2h ) , 
 2.17 ( s , 3h ) .
2-(4-fluorophenyl)-8,8-dimethyl-3-(p - tolylamino)-7,8-dihydroimidazo[1,2-a]pyrazin-6(5h)-one : compound 33a was prepared from 7 by the following way : to a stirred solution of compound 7 ( 390 mg , 1.51 mmol , 1.00 equiv ) in dichloromethane ( 20 ml ) 
 was added nbs ( 0.28 g , 1.00 equiv ) .
the solid was filtered out , and the mixture was washed 
 with a saturated solution of na2s2o3 and dried over na2so4 .
the solids was purified by silica gel chromatography 
 ( petroleum ether / etoac = 1:2 ) to result in 432 mg ( 85% ) of the title 
 compound as a white solid .
h nmr ( 300 mhz , dmso - d6 )  8.76 ( s , 1h ) , 7.947.89 ( m , 
 2h ) , 7.317.25 ( m , 2h ) , 4.57 ( s , 2h ) , 1.56 ( s , 6h ) .
compound 33 was prepared from compound 33a by a pd2(dba)3 mediated amination reaction with p - toluidine .
h nmr ( meoh - d4 , 400 hz )  7.627.58 ( m , 2h ) , 7.05 ( t , j = 8.4 hz , 2h ) , 6.92 ( d , j = 8.4 hz , 2h ) , 
 6.52 ( d , j = 8.4 hz , 2h ) , 4.42 ( s , 2h ) , 2.12 ( s , 
 3h ) , 1.71 ( s , 6h ) .
3-(4-chloro-3-fluorophenylamino)-2-(4-fluorophenyl)-8,8-dimethyl-7,8-dihydro 
 imidazo[1,2-a]pyrazin-6(5h)-one : compound 34 was prepared from compound 7b by a pd2(dba)3 mediated amination 
 reaction with 4-chloro-3-fluoroaniline .
h nmr ( meoh - d4 , 400 hz )  7.747.70 ( m , 2h ) , 
 7.21 ( t , j = 8.4 hz , 1h ) , 7.08 ( m , 2h ) , 6.496.40 
 ( m , 2h ) , 4.43 ( s , 2h ) , 1.72 ( s , 6h ) .
2-(4-fluorophenyl)-8,8-dimethyl - n - p - tolyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazin-3-amine : compound 35 was prepared from compound 35 - 1 ( shown below and prepared by analogy to 
 compound 9 and using cbz - glycine ) by a pd2(dba)3 mediated amination reaction with p - toluidine followed by a 6n hcl mediated hydrolysis .
h nmr ( meoh - d4 , 400 hz )  7.787.75 
 ( m , 2h ) , 7.066.97 ( m , 4h ) , 6.56 ( d , j = 8.4 
 hz , 2h ) , 5.16 ( s , nh ) , 4.33 ( t , j = 4.8 hz , 2h ) , 
 3.74 ( t , j = 4.8 hz , 2h ) , 2.29 ( s , 3h ) , 1.95 ( s , 
 6h ) .
n,2-bis(4-fluorophenyl)-8,8-dimethyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazin-3-amine : compound 36 was prepared from compound 35 - 1 by a pd2(dba)3 mediated 
 amination reaction with 4-fluoroaniline followed by a 6n hcl mediated 
 hydrolysis .
h nmr ( 400 mhz , meoh - d4 )  7.627.58 ( m , 2h ) , 7.127.08 ( m , 2h ) , 
 6.686.63 ( m , 2h ) , 6.67 ( m , 2h ) , 4.16 ( m , 2h ) , 3.78 ( m , 2h ) , 
 1.92 ( s , 6h ) .
unless otherwise noted , materials 
 were obtained from commercial suppliers and were used without purification . 

removal of solvent under reduced pressure refers to distillation using 
 bchi rotary evaporator attached to a vacuum pump ( 3 
 mmhg ) .
products obtained as solids or high boiling oils were dried 
 under vacuum ( 1 mmhg ) .
purification of compounds by high pressure 
 liquid chromatography was achieved using a waters 2487 series with 
 ultra 120 5 m c18q column with a linear gradient from 10% solvent 
 a ( acetonitrile with 0.035% trifluoroacetic acid ) in solvent b ( water 
 with 0.05% trifluoroacetic acid ) to 90% a in 7.5 min , followed by 
 2.5 min elution with 90% a. h nmr spectra were recorded 
 on bruker xwin - nmr ( 400 or 600 mhz ) .
proton resonances are reported 
 in parts per million ( ppm ) downfield from tetramethylsilane ( tms ) .
h nmr data are reported as multiplicity ( s singlet , d doublet , 
 t triplet , q quartet , quint quinted , sept septed , dd doublet of doublets , 
 dt doublet of triplet , bs broad singlet ) , number of protons , and coupling 
 constant in hertz . for spectra obtained in cdcl3 , dmso - d6 , and cd3od , the residual protons 
 ( 7.27 , 2.50 , and 3.31 ppm , respectively )
analytical thin - layer chromatography ( tlc ) was performed on commercial 
 silica plates ( merck 60-f 254 , 0.25 mm thickness ) ; compounds were 
 visualized by uv light ( 254 nm ) .
100c , isco ) or using silica 
 gel ( merck kieselgel 60 , 230400 mesh ) .
the purity and quantitative 
 analysis were determined with a waters zq 2000 lc / ms system , which 
 employed an acquity uplc system ( binary pump , column compartment , 
 autosampler , and diode array uv detector ) .
the eluent was split between 
 the mass spectrometer and an antek chemiluminescent nitrogen detector 
 ( clnd ) .
the mobile phases used were ( a ) 95% h2o/5% meoh / ipa 
 ( 75/25 , v / v ) + 0.05% formic acid and ( b ) meoh / ipa ( 75/25 , v / v ) + 0.035% 
 formic acid . a gradient hplc method with flow of 0.4
ml / min started 
 at 2% b , with a hold of 1.0 min before a linear increase to 95% b 
 at 3.50 min , with a 30 s hold . from 4.04.25 min ,
the column used was a thermo syncronis c18 2.1 mm  30 mm , 
 3 m .
the mass spectrometer was operated in positive mode , with 
 a spray voltage of 3.2 kv and cone voltage of 30 v. the source and 
 desolvation temperatures were 130 and 400 c , respectively , with 
 600 l / h of nitrogen desolvation flow .
all the final compounds reported 
 in this communication had the purity of at least 95% .
elemental analyses 
 were carried out by midwest microlabs llc , indianapolis , in , usa . compound 1a was prepared from 1 by the following method : to a stirred 
 solution of 1 ( 12 mg , 0.03 mmol ) was added mno2 ( 52 mg , 0.60 mmol ) .
the reaction mixture was stirred at room temperature 
 ( rt ) for 2 h. lc / ms test showed that 1 was almost consumed 
 and the desired product ( [ m + 1 ] = 428 ) was detected as one of the 
 major peaks .
the 
 residue was subjected to a mass - triggered hplc purification to give 1a as well as 1b .
h nmr for 1a ( 400 mhz , cd3od )  7.597.63 ( m , 
 2h ) , 7.09 ( t , j = 6.4 hz , 2h ) , 6.957.02 ( m , 
 1h ) , 6.466.52 ( m , 1h ) , 6.346.38 ( m , 1h ) , 5.92 ( s , 
 1h ) , 4.33 ( dd , j = 4.4 , 14 hz , 1h ) , 3.96 ( dd , j = 4.8 , 13.2 hz , 1h ) , 3.703.78 ( m , 1h ) , 3.463.54 
 ( m , 1h ) , 1.73 ( s , 3h ) , 1.36 ( s , 3h ) .
h nmr for 1b ( 400 mhz , 
 cd3od )  7.757.78 ( m , 2h ) , 6.957.01 
 ( m , 3h ) , 6.446.50 ( m , 1h ) , 6.326.36 ( m , 1h ) , 4.19 
 ( dd , j = 1.6 , 6.8 hz , 2h ) , 4.00 ( dd , j = 1.6 , 6.8 hz , 2h ) , 1.47 ( s , 6h ) .
to a solution of 2-bromo-1-(4-fluorophenyl)ethanone ( 46.5 
 g , 214.29 mmol ) in dmf ( 400 ml ) was added 2-(benzyloxycarbonyl)-2-methylpropanoic 
 acid ( 55.8 g , 236.4 mmol ) and potassium carbonate ( 35.4 g , 256.5 mmol ) . 

the resulting solution was extracted 
 with ethyl acetate ( 2  800 ml ) , and the combined organic layer 
 was washed with water ( 2  800 ml ) and then brine ( 1  800 
 ml ) .
this 
 resulted in 67 g ( 84% ) of compound 4 as a white solid .
to a solution of compound 4 ( 70 g , 187.7 
 mmol ) in toluene ( 700 ml ) was added nh4oac ( 144.5 g , 1.88 
 mol ) .
the resulting solution was heated to reflux for 3 h in an oil 
 bath .
the resulting solution was extracted 
 with ethyl acetate ( 2  500 ml ) , and the combined organic layer 
 was washed with water ( 2  800 ml ) and brine ( 1  800 ml ) . 

this resulted 
 in 58 g ( 88% ) of compound 5 as a white solid .
h nmr ( 300 hz , dmso - d6 )  11.91 
 ( s , nh ) , 7.807.75 ( m , 2h ) , 7.547.12 ( m , 8h ) , 4.97 
 ( m , 2h ) , 1.6 ( s , 6h ) . to a solution of compound 5 ( 58 g , 164.3 
 mmol ) in dmf ( 400 ml ) was added cesium carbonate ( 134 g , 411.0 mmol ) . 

this was followed by the addition of ethyl 2-bromoacetate ( 33 g , 197.6 
 mmol ) dropwise with stirring at rt in 30 min .
the resulting solution was extracted with ethyl 
 acetate ( 2  700 ml ) , and the combined organic layer was washed 
 with water ( 2  800 ml ) and brine ( 1  800 ml ) . the resulting 
 mixture was dried concentrated under vacuum .
this resulted in 60 g 
 ( 83% ) of compound 6 as a yellow solid .
h 
 nmr ( 300 hz , dmso - d6 )  7.767.72 
 ( m , 2h ) , 7.327.28 ( m , 5h ) , 7.087.03 ( m , 3h ) , 5.08 
 ( m , 2h ) , 4.87 ( m , 2h ) , 4.23 ( q , j = 5.4 hz , 2h ) , 
 2.0 ( s , 6h ) , 1.28 ( t , j = 5.4 hz , 3h ) .
to a solution of compound 6 ( 70 g , 159.45 
 mmol , 1.00 equiv ) in methanol ( 800 ml ) was added palladium on carbon 
 ( 10 g ) .
this resulted in 7 ( 38 g , 145.56 mmol , 
 91% ) as a white solid .
lc - ms : ( es , m / z ) [ m + h ] calcd for c14h14fn3o , 260 ; found , 260 .
h nmr ( 300 hz , dmso - d6 )  7.797.74 ( m , 2h ) , 7.137.07 
 ( m , 3h ) , 6.35 ( s , 1h ) , 4.73 ( s , 2h ) , 1.79 ( s , 6h ) . to a stirred solution of compound 7 ( 5 g , 
 19.2 mmol , 1 equiv ) was dissolved in 50 ml of thf , 1 m borane / thf 
 complex ( 57 ml , 57 mmol , 3 equiv ) was added slowly and reaction was 
 refluxed overnight .
the crude product of 8 ( 4.5 g , 18.3 mmol , 
 95% ) was used in the next step .
lc - ms : ( es , m / z ) [ m + h ] calcd for c14h17fn3 246 , found 246 .
h nmr ( dmso , 300 hz )  
 7.757.70 ( m , 2h ) , 7.4 ( s , 1h ) , 7.14 ( t , j = 9.0 hz , 2h ) , 3.9 ( t , j = 5.4 hz , 2h ) , 2.51 ( t , j   = 5.4 hz , 2h ) , 1.41(s , 6h ) ; nh proton not observed .
to a stirred solution of compound 8 ( 2.9 
 g , 11.82 mmol , 1.1 equiv ) and 2-(tert - butoxycarbonylamino)acetic 
 acid ( 2.27 g , 13 mmol , 1.1 equiv ) in 15 ml of dichloromethane were 
 added diea ( 2.47 ml , 14.18 mmol , 1.2 equiv ) and hatu ( 5.39 g , 14.18 
 mmol , 1.2 equiv ) .
the reaction mixture was stirred at rt for 8 h. 
 hplc / ms analysis showed that desired product compound 9 was the major product .
the organic layer was washed with washed with water ( 1  
 30 ml ) , followed by saturated nahco3 ( 1  30 ml ) and 
 finally with brine ( 1  30 ml ) .
the resulting oil was purified using column chromatography with hexanes / ethyl 
 acetate ( 0100% linear gradient ) used as eluant .
the desired 
 product compound 9 was obtained as oil ( 3.3 g , 8.27 mmol , 
 70% ) .
h nmr ( 300 hz , dmso - d6 , )  7.777.72 ( m , 2h ) , 7.54 ( s , 1h ) , 7.207.14 
 ( m , 2h ) , 6.846.80 ( m , 1h ) , 4.07 ( s , 2h ) , 3.90 ( d , j = 3.0 hz , 2h ) , 3.70 ( s , 2h ) , 1.80 ( s , 6h ) , 1.40 ( s , 9h ) . 

lc - ms : ( es , m / z ) [ m + h ] calcd for c21h28fn4o3 , 403 ; found , 403 .
to a stirred solution of compound 9 ( 3.02 
 g , 7.51 mmol ) in dichloromethane ( 30 ml ) was added br2 ( 0.43 
 ml , 8.26 mmol ) in acetic acid ( 3 ml ) .
hplc / ms test showed that desired product ( ii ) was 
 the only peak .
the product was confirmed by 400 mhz proton nmr to be 
 the title compound 10 .
the product was used in the next 
 step without further purification and exhibited quantitative mass 
 recovery .
lc - ms : ( es , m / z ) [ m + 
 h ] calcd for c21h27brfn4o3 , 482 ; found , 482 .
h nmr ( meoh - d4 , 400 hz )  7.847.81 ( m , 2h ) , 7.14 ( t , j = 8.8 hz , 2h ) , 4.094.01 ( m , 4h ) , 3.81 ( t , j = 4.8 hz , 2h ) , 1.89 ( s , 6h ) , 1.46 ( s , 9h ) .
( s)-2-amino-1-(3-(4-chloro-3-fluorophenylamino)-2-(4-fluorophenyl)-8-methyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 17 was prepared from compound 10 ( r1 = me , r2 = h ) by a pd2(dba)3 mediated amination reaction with 4-chloro-3-fluoroaniline 
 followed by a tfa mediated deprotection in a protocol similar to compound 22 .
h nmr ( meoh - d4 , 400 mhz )  7.617.58 ( m , 2h ) , 7.167.12 ( m , 
 1h ) , 7.077.02 ( m , 2h ) , 6.476.42 ( m , 2h ) , 5.74 ( m , 
 1h ) , 4.073.68 ( m , 6h ) , 1.56 ( d , j = 6.8 hz , 
 3h ) .
r2 = h ) by a pd2(dba)3 mediated amination reaction with 4-chloro-3-fluoroaniline 
 followed by a tfa mediated deprotection in a protocol similar to compound 22 .
( s)-2-amino-1-(3-(4-chloro-3-fluorophenylamino)-2-(4-fluorophenyl)-8-isopropyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 19 was prepared from compound 10 ( r1 = isopropyl , r2 = h ) by a pd2(dba)3 mediated amination reaction with 4-chloro-3-fluoroaniline 
 followed by a tfa mediated deprotection in a protocol similar to compound 22 .
h nmr ( meoh - d4 , 400 mhz ) 7.617.58 ( m , 2h ) , 7.267.20 ( m , 
 3h ) , 6.456.38 ( m , 2h ) , 5.5 ( d , j = 7.9 hz , 
 1h ) , 4.113.75 ( m , 6h ) , 2.362.31 ( m , 1h ) , 1.15 ( d , j = 6.7 hz , 3h ) , 0.95 ( d , j = 6.7 hz , 3h ) .
( r)-2-amino-1-(3-(4-chloro-3-fluorophenylamino)-2-(4-fluorophenyl)-8-isopropyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 20 was prepared from compound 10 ( r1 = isopropyl , r2 = h ) by a pd2(dba)3 mediated amination reaction with 4-chloro-3-fluoroaniline 
 followed by a tfa mediated deprotection in a protocol similar to compound 22 .
h nmr ( meoh - d4 , 400 mhz )  7.697.67 ( m , 2h ) , 7.277.20 ( m , 
 3h ) , 6.686.65 ( dd , j = 2.5 hz , j = 11.0 hz , 1h ) , 6.606.55 ( dd , j = 2.25 
 hz , j = 8.7 hz , 1h ) , 5.78 ( d , j = 
 7.7 hz , 1h ) , 4.424.02 ( m , 4h ) , 3.943.87 ( m , 2h ) , 2.55 
 ( m,1h ) , 1.24(d , j = 6.8 hz , 3h ) , 1.05 ( d , j = 6.8 hz , 3h ) .
2-amino-1-(3-(4-chloro-3-fluorophenylamino)-2-(4-fluorophenyl)-8,8-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 21 was prepared from compound 10 ( r1 = me , r2 = me ) by a pd2(dba)3 mediated amination reaction with 4-chloro-3-fluoroaniline 
 followed by a tfa mediated deprotection in a protocol similar to compound 22 .
h nmr ( meoh - d4 , 400 mhz )  7.597.55 ( m , 2h ) , 7.207.13 ( m , 
 3h ) , 6.60 ( dd , j = 11.2 , 10.8 hz ) , 1h ) , 6.53 ( dd , j = 2.4 , 0.8 hz , 1h ) , 4.0 ( m , 4h ) , 3.78 ( m , 2h ) , 2.00 ( s , 
 6h ) .
elemental 
 analysis ( compound + 2.0 hcl + 2.0 h2o ) : % c , 47.62 ; % h , 
 5.09 ; % n , 12.62 ; ( calc ) .
% c = 47.54/47.38 ; % n = 12.44/12.46 ; % h = 
 4.62/4.57 ( experimental ) .
2-amino-1-(2-(4-fluorophenyl)-3-(4-fluorophenylamino)-8,8-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : in a glass vial , cs2co3 , 4f - aniline 
 ( 0.462 g , 4.1 mmol , 2.0 equiv ) , pd2(dba)3 ( 0.095 
 g , 0.104 mmol , 0.05 equiv ) , xantphos ( 0.120 g , 0.208 mmol , 0.1 equiv ) , 
 and dioxane were stirred for 5 min at rt .
compound 10 ( 1 g , 2.08 mmol , 1.0 equiv ) was added to the reaction mixture , after 
 which the reaction mixture was degassed for 15 min and then stirred 
 at 120 c under n2 for 8 h. hplc / ms test showed that 
 the starting material compound 10 was consumed and desired 
 product was formed predominantly along with some 9 .
the reaction mixture was concentrated 
 and then purified by normal phase column chromatography ( silica gel 
 80 g ) using a gradient of 1000% to 0100% hexane : etoac . 

the organic layer 
 was concentrated at reduced pressure to yield the boc derivative ( 950 
 mg , 89% ) yield .
lc - ms : ( es , m / z ) 
 [ m + h ] calcd for c27h32f2n5o3 , 512 ; found , 512 .
the boc compound 
 was treated with 20% tfa in ch2cl2 ( 50 ml ) and 
 was added to the mixture .
after the completion of this reaction ( monitored 
 by lcms ) , the resulting mixture was concentrated under reduced pressure . 

the resulting residue was purified by reverse phase hplc to yield 
 product as a tfa salt .
lc - ms : ( es , m / z ) [ m + h ] calcd for c22h23f2n5o , 412.2 ; found , 412.1 .
h nmr ( meoh - d4 , 400 hz )  7.617.57 ( m , 2h ) , 
 6.94 ( t , j = 8.8 hz , 2h ) , 6.81 ( t , j = 8.8 hz , 2h ) , 6.47 ( m , 2h ) , 3.72 ( m , 2h ) , 3.58 ( m , 2h ) , 3.42 ( m , 
 2h ) , 1.85 ( s , 6h )
. elemental analysis compound 22 with 
 0.65 equiv h2o : c , 62.44 ; n , 16.55 ; h , 5.79 ( calculated ) . 

c = 62.54/62.44 ; n = 16.35/16.29 ; h = 5.52/5.61 ( experimental ) .  
the regiochemistry of the dime groups was unambiguously assigned 
 using 2-d nmr techniques ( table 8) .
2-amino-1-(2-(4-fluorophenyl)-8,8-dimethyl-3-(p - tolylamino)-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 23 was prepared from compound 10 ( r1 = me , r2 = me ) by a pd2(dba)3 mediated amination reaction with 4-methylaniline 
 followed by a tfa mediated deprotection in a protocol similar to compound 22 .
h nmr ( meoh - d4 , 400 mhz )  7.797.69 ( m , 2h ) , 7.21 ( t , j = 8.4 hz , 2h ) , 7.04 ( d , j = 8.0 hz , 2h ) , 6.76 ( d , j = 8.2 hz , 2h ) , 4.11 ( s , 2h ) , 3.88 ( m , 4h ) , 2.22 ( s , 3h ) , 
 2.12 ( s , 6h ) .
lc / ms major mass : 408.1 ( m + h ) . 2-amino-1-(3-((3,4-difluorophenyl)amino)-2-(4-fluorophenyl)-8,8-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 24 was prepared from compound 10 ( r1= me , r2 = me ) by a pd2(dba)3 mediated amination reaction with 3,4-difluoroaniline 
 followed by a tfa mediated deprotection in a protocol similar to compound 22 .
h nmr ( meoh - d4 , 400 mhz )  7.737.69 ( m , 2h ) , 7.21 ( t , j = 8.8 hz , 2h ) , 7.067.03 ( m , 1h ) , 6.766.65 ( m , 1h ) , 
 6.536.51 ( m , 1h ) , 4.084.06 ( m , 4h ) , 3.863.83 
 ( m , 2h ) , 2.07 ( s , 6h ) .
elemental analysis ( compound + 2.9 hcl ) : % c , 
 49.37 ; % h , 4.69 ; % n , 13.09 ; ( calc ) .
% c = 49.43/49.06 ; % n = 12.89/12.77 ; 
 % h = 4.7/4.81 ( experimental ) .
13 was prepared from compound 5a by the following way : to a stirred solution of compound 5a ( 1.1 
 g , 3.38 mmol , 1.00 equiv ) in dmf ( 30 ml ) was added 3-chloro-2-methylprop-1-ene 
 ( 500 mg , 5.49 mmol , 1.50 equiv ) , potassium carbonate ( 560 mg , 4.06 
 mmol , 1.10 equiv ) , and potassium iodide ( 1.12 g , 6.75 mmol , 2.00 equiv ) 
 at rt .
the mixture 
 was washed with brine ( 3  10 ml ) , dried over sodium sulfate , 
 and concentrated under vacuum .
the residue was applied onto a silica 
 gel column with petroleum ether / etoac ( 5:1 ) to give 0.7 g ( 55% ) of 
 compound 5a-1 as a light - yellow solid .
m / z = 380 ( m + 1 ) . into a 30 ml sealed tube , was placed compound 5a-1 ( 2.0 g , 5.28 mmol , 1.00 equiv ) , acetic acid ( 12 ml ) , 
 and methanesulfonic acid ( 2 ml ) .
the reaction mixture was stirred 
 for 12 h at 260 c ( the temperature of the sand bath ) .
the aqueous layer was washed with ethyl acetate ( 3  10 ml ) . 

the aqueous 
 layer was extracted with ethyl acetate ( 3  10 ml ) .
the combined 
 organic layers were washed with brine ( 3  10 ml ) , dried over 
 sodium sulfate , and concentrated under vacuum .
the solid was collected 
 by filtration and washed with 5 ml of n - hexane to 
 give 500 mg ( 39% ) of compound 5a-2 as a 
 white solid .
h nmr ( 400 mhz , cdcl3 )  
 7.707.65 ( m , 2h ) , 7.046.9 ( m , 2h ) , 6.96 ( s , 1h ) , 4.12 
 ( s , 2h ) , 3.69 ( s , 2h ) , 1.23 ( s , 6h ) . to a stirred solution of compound 5a-2 ( 280 mg
, 1.14 mmol , 1.0 equiv ) in dmf ( 10 ml ) was added 
 2-(tert - butoxycarbonyl)acetic acid ( 600 mg , 3.43 
 mmol , 3.0 equiv ) , hatu ( 1.3 g , 3.42 mmol , 3.0 equiv ) , and diea ( 880 
 mg , 6.82 mmol , 6.0 equiv ) at rt .
the organic layer was washed with brine ( 3  10 ml ) , 
 dried over sodium sulfate , and concentrated under vacuum .
the residue 
 was applied onto a silica gel column with ch2cl2/meoh ( 10:1 ) to give 280 mg ( 57% ) of compound 13 as 
 a brown solid .
h nmr ( 400 mhz , cdcl3 )  
 7.727.68 ( m , 2h ) , 7.23 ( s , 1h ) , 7.137.07 ( m , 2h ) , 
 5.46 ( s , nh ) , 4.65 ( s , 2h ) , 4.054.04 ( m , 2h ) , 3.97 ( s , 2h ) , 
 1.551.39 ( m , 15h ) .  
the structure of 13 was verified by hmqc , cosy , hmbc , and roesy ( table 9 ) .
2-amino-1-(2-(4-fluorophenyl)-3-(4-fluorophenylamino)-6,6-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : to a stirred solution of compound 13 ( 386 
 g , 0.96 mmol ) in 6 ml of dichloromethane was added br2 ( 55 
 l , 1.06 mmol ) in acetic acid ( 2 ml ) .
after neutralization , the residue was subjected 
 to flash chromatography ( 40 g , 0100% ethyl acetate in hexane , 
 50 min , dry loading ) purification to give 256 mg ( 55% ) of the title 
 compound 13a as a colorless oil .
h nmr ( 400 
 mhz , cdcl3 )  7.877.83 ( m , 2h ) , 7.03 ( t , j = 8.8 hz , 2h ) , 5.46 ( s , 1h ) , 4.5 ( s , 2h ) , 3.97 ( d , j = 4.2 hz , 2h ) , 3.81 ( s , 2h ) , 1.45 ( s , 6h ) , 1.39 ( s , 9h ) .
example 25was obtained from 13a by a 
 pd2(dba)3 mediated amination reaction with 4-fluoroaniline 
 followed by a tfa mediated deprotection by analogy to compound 22 ( 55% yield over 2 steps ) .
h nmr ( 400 mhz , dmso - d6 ) :  8.06 ( m , 2h ) , 7.98 ( m , 1h ) , 7.80 
 ( m , 2h ) , 7.16 ( m , 2h ) , 6.98 ( m , 2h ) , 6.60 ( m , 2h ) , 4.64 ( m , 2h ) , 4.02 
 ( m , 2h ) , 3.80 ( m , 2h ) , 1.38 ( s , 6h ) .
2-(4-fluorophenyl)-7-(4-methoxybenzyl)-5,5-dimethyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine : compound 7b was prepared from compound 7a ( r1 = r2 = h ) by the following way : to a solution of compound 7a ( 231 mg , 1.0 
 mmol ) in dmf ( 10 ml ) were added koh ( 168 mg , 3.0 mmol ) and pmbcl ( 405 
 l , 3.0 mmol ) at 0 c .
the reaction mixture was stirred 
 at the same temperature for 2 h and at rt for 2 additional hours . 

the hplc 
 fraction were evaporated under reduced pressure and neutralized by 
 satd nahco3 .
the organic layer was concentrated 
 under reduced pressure to give compound 7a-1 ( 221 mg , 63% ) as a white solid .
h nmr ( 400 mhz , cdcl3 )  7.677.63 ( m , 2h ) , 7.287.25 ( m , 2h ) , 
 7.077.02 ( m , 2h ) , 6.896.86 ( m , 2h ) , 4.74 ( s , 2h ) , 
 4.70 ( m , 2h ) , 4.53 ( s , 2h ) , 3.79 ( s , 3h ) . to a solution of compound 7a-1 ( 253 mg , 0.72 mmol ) in dmf ( 15 ml ) were added 60% sodium hydride 
 ( 87 mg , 0.085 mmol ) and methyl iodide ( 0.45 ml , 7.2 mmol ) at rt .
the 
 reaction mixture was stirred at rt for 2 h. the reaction mixture was 
 quenched with methanol and directly subjected to mass - triggered hplc 
 purification to give 7a-2 as a white solid 
 after evaporation of acetonitrile  water in vacuo .
the tfa salt 
 was subsequently neutralized by saturated sodium bicarbonate solution 
 and extracted with dichloromethane to get the compound as a free base . 

the organic layer was dried over sodium sulfate and concentrated under 
 reduced pressure to yield the desired compound 7a-2 ( 234 mg , 86% ) .
the structure of 7a-2 was verified by 2-d nmr techniques ( table 10 ) . to a solution of compound 7a-2
( 170 mg , 0.45 mmol ) in thf ( 9 ml ) was added 1.0 n bh3thf ( 2.70 ml , 2.70 mmol ) at rt .
the reaction mixture was stirred 
 at reflux for 2 h. pd / c was added ( gas generated ) .
the reaction mixture 
 was stirred for 1 h. solid was filtered off , and solvent was removed . 

the product was assumed to be of 100% yield and used in the next 
 step without further purification .
h nmr ( 400 mhz , cd3od )  8.1 ( s , 1h ) , 7.747.70 ( m , 2h ) , 7.34 ( d , j = 8.6 hz , 2h ) , 7.27 ( m , 2h ) , 6.94 ( d , j = 8.6 hz , 2h ) , 3.91 ( s , 2h ) , 3.80 ( s , 3h ) , 3.79 ( s , 2h ) , 2.87 ( s , 
 2h ) , 1.61 ( s , 6h ) .
7-(4-methoxybenzyl)-2-(4-fluorophenyl)-5,5-dimethyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine ( 1.07 g , 2.93 mmol , 1.00 equiv ) ( 7b ) was added to trifluoroacetic acid ( 15 ml ) .
the resulting solution 
 was stirred for 2 h at 70 c in an oil bath .
the ph value of the solution was adjusted to 8 
 with aqueous sodium bicarbonate .
7b-1 was 
 extracted from the aqueous layer using 3  100 ml of ethyl acetate . 

this resulted in 7b-1 ( 718 mg , quantitative ) 
 as a yellow solid .
to a solution of 7b-1 ( 750 
 mg , 3.06 mmol , 1.00 equiv ) in n , n - dimethylformamide ( 30 ml ) were added 2-(tert - butoxycarbonyl)acetic 
 acid ( 850 mg , 4.86 mmol , 1.50 equiv ) , hatu ( 1.3 g , 3.42 mmol , 1.10 
 equiv ) , and triethylamine ( 650 mg , 6.44 mmol , 2.00 equiv ) . the resulting 
 solution was stirred overnight at rt .
the resulting mixture was washed with brine 
 ( 3  100 ml ) , dried over anhydrous sodium sulfate , and concentrated 
 under vacuum .
the residue was applied onto a silica gel column with 
 petroleum ether / ethyl acetate ( 4:1 ) .
this resulted in 600 mg ( 49% ) 
 of 7c as a pale - yellow solid .
h nmr ( 300 
 mhz , cdcl3 ) :  7.75 ( m , 2h ) , 7.22 ( s , 1h ) , 7.137.07 
 ( m , 2h ) , 4.994.87 ( m , 2h ) , 4.12 ( s , 2h ) , 3.91 3.65 ( m , 2h ) , 
 1.57 ( m , 6h ) , 1.43 ( s , 9h ) . to a stirred solution of compound 7b-2 ( 583 g , 1.45 mmol ) in dichloromethane ( 6 ml ) was added br2 ( 82 l , 1.60 mmol ) in acetic acid ( 2 ml ) .
after neutralization , 
 the residue was subjected to flash chromatography ( 40 g , 0100% 
 ethyl acetate in hexane , 50 min , dry loading ) purification to give 
 628 mg ( 90% ) of the title compound 7c as a yellow solid .
h nmr ( 400 mhz , cdcl3 )  7817.78 
 ( m , 2h ) , 7.07 ( t , j = 8.6 hz , 2h ) , 5.28 ( s , nh ) , 
 4.844.7 ( m , 2h ) , 4.073.58 ( m , 4h ) , 1.491.44 
 ( m , 6h ) , 1.28 ( s , 9h ) .
2-amino-1-(2-(4-fluorophenyl)-3-(4-fluorophenylamino)-5,5-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 26 was prepared from compound 7c by a pd2(dba)3 mediated amination 
 reaction with 4-fluoroaniline followed by a tfa mediated deprotection 
 in 46% yield over 2 steps .
h nmr ( 400 mhz , cd3od ) :  7.57 ( m , 2h ) , 7.02 ( m , 2h ) , 6.77 ( m , 2h ) , 6.52 ( m , 2h ) , 
 4.974.91 ( m , 2h ) , 4.093.74 ( m , 4h ) , 1.56 ( s , 3h ) , 
 1.51 ( s , 3h ) .
2-amino-1-(2-(4-fluorophenyl)-3-(4-fluorophenylamino)-8,8-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)-2-methylpropan-1-one : compound 28 was prepared from compound 8 ( r1 = r2 = me ) in the following fashion 
 using a slightly modified route to the one described in scheme 2 . to a stirred solution of compound 8 ( 1.48 
 g , 6.04 mmol ) and net3 ( 6.0 g , 59.4 mmol ) in dichloromethane 
 ( 20 ml ) was added 2-bromo-2-methylpropanoyl bromide ( 14 g , 60.9 mmol ) 
 dropwise at rt . after being stirred for 3 h at rt , the reaction was 
 then quenched by the addition of water ( 30 ml ) .
the combined organic 
 layers were washed with brine , dried over anhydrous sodium sulfate , 
 and concentrated under vacuum .
this resulted in crude product as a 
 dark solid , which was washed with etoac : petroleum ether ( 1:10 ) to 
 remove the impurities to produce compound 8 - 1 as a gray solid .
( 2.0 g , 5.08 mmol , 1.00 equiv ) in dmf ( 10 ml ) was nan3 ( 1.0 g , 15.38 mmol , 3.00 equiv ) at rt .
the resulting mixture was washed with 3  
 20 ml of brine , dried over anhydrous sodium sulfate , and concentrated 
 under vacuum .
the residue was applied onto a silica gel column with 
 petroleum ether / etoac ( 5:1 ) to give compound 8 - 2 as a white solid .
m / z 357 
 ( m + h ) . to a stirred solution of compound 8 - 2 ( 1.2 g
, 3.37 mmol , 1.00 equiv ) in methanol ( 20 ml ) was added 
 pd / c ( 80 mg , 0.75 mmol , 0.20 equiv ) at rt .
m / z 331 ( m + h ) . to a stirred solution of compound 8 - 3 ( 910 mg
, 2.76 mmol , 1.00 equiv ) in thf ( 50 ml ) was added 
 boc anhydride ( 3.2 g , 14.68 mmol , 5.00 equiv ) , followed by aqueous 
 naoh ( 1n , 6 ml , 2.0 equiv ) at rt .
the organic layer 
 was washed with 3  10 ml of brine , dried over na2so4 , and concentrated under vacuum .
the solid was collected 
 by filtration and washed with n - hexane ( 5 ml ) to 
 give compound 8 - 4 as a white solid .
h nmr ( 300 mhz , cdcl3 )  7.757.71 
 ( m , 2h ) ,
7.097.02 ( m , 3h ) , 4.84(s , nh ) , 4.094.0 ( m , 
 4h ) , 1.98 ( s , 6h ) , 1.55 ( s , 6h ) , 1.45 ( s , 9h ) . to a stirred solution of compound 8 - 4 ( 32 mg , 0.074 mmol ) in dichloromethane ( 3 ml ) was added 
 br2 ( 4.2 l , 0.082 mmol ) in acetic acid ( 1 ml ) .
after neutralization , 
 the residue was subjected to flash chromatography ( 4 g , 060% 
 ethyl acetate in hexane , 16 min ) purification to give the compound 8 - 5 as colorless oil , was collected by filtration , 
 and washed with n - hexane ( 5 ml ) to give compound 8 - 4 as a white solid .
h nmr ( 400 
 mhz , cdcl3 )  7.917.88 ( m , 2h ) , 7.107.04 
 ( m , 2h ) , 5.11(s , nh ) , 4.01 ( m , 2h ) , 3.96 ( s , 2h ) , 1.94 ( s , 6h ) , 1.51 
 ( s , 6h ) , 1.41 ( s , 9h ) .
compound 28 was prepared 
 from compound 8 - 5 by a pd2(dba)3 mediated amination reaction with 4-fluoroaniline followed 
 by a tfa mediated deprotection .
h nmr ( 400 mhz , meoh - d4 )  7.66 ( dd , j = 9.2 , 
 5.2 hz , 2h ) , 7.11 ( t , j = 8.8 hz , 2h ) , 6.81 ( t , j = 8.8 hz , 2h ) , 6.726.68 ( m , 2h ) , 4.043.99 
 ( m , 4h ) , 2.01 ( s , 6h ) , 1.66 ( s , 6h ) .
2-amino-1-(2-(4-fluorophenyl)-8,8-dimethyl-3-(p - tolylamino)-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)-2-methylpropan-1-one : compound 29 was prepared from 35 by the following way : to a stirred solution of 35 ( 21 mg , 0.06 
 mmol ) and et3n ( 83 l , 0.60 mmol ) in dry dichloromethane 
 ( 6 ml ) was added 2-bromo-2-methylpropanoyl bromide ( 71 l , 0.60 
 mmol ) .
the reaction mixture was stirred at rt for 5 h. the reaction 
 mixture was concentrated and subjected to mass - triggered lc / ms purification 
 directly .
the obtained solution was concentrated to give 22 mg ( 73% ) 
 of compound 35 - 1as yellow oil after neutralization .
h nmr ( 400 mhz , meoh - d4 )  
 7.717.66 ( m , 2h ) , 7.237.19 ( m , 2h ) , 7.02 ( d , j = 8.4 hz , 2h ) , 6.66 ( d , j = 8.4 hz , 2h ) , 
 4.31 ( m , 2h ) , 4.09 ( m , 2h ) , 2.21 ( s , 3h ) , 2.06 ( s , 6h ) , 2.00 ( s , 6h ) . to a solution of compound 35 - 1
( 22 mg , 0.044 mmol ) in dmf ( 3 ml ) was added nan3 ( 8.6 
 mg , 0.132 mmol ) at rt .
the reaction mixture was stirred at rt for 
 2 h. the reaction mixture was directly subjected to mass - triggered 
 hplc purification directly .
the obtained mecn / aqueous solution was 
 combined and concentrated to give 15 mg ( 75% ) of compound 35 - 2 as yellow oil after neutralization .
h 
 nmr ( 400 mhz , meoh - d4 )  7.70 7.66 
 ( m , 2h ) , 7.237.19 ( m , 2h ) , 7.04 ( d , j = 8.3 
 hz , 2h ) , 6.66 ( d , j = 8.4 hz , 2h ) , 4.26 ( m , 2h ) , 
 4.074.05 ( m , 2h ) , 2.24 ( s , 3h ) , 2.03 ( s , 6h ) , 1.58 ( s , 6h ) . to a solution of compound 35 - 2 ( 15 mg , 0.033 mmol ) in meoh ( 3 ml ) was added 10% pd / c ( 4 mg , 0.003 
 mmol ) at rt .
the reaction 
 mixture was stirred at rt for 2 h. solid was filtered off , and solvent 
 was removed .
the reaction mixture was directly subjected to mass - triggered 
 hplc purification to give 15 mg ( 100% ) of the title compound yellow 
 oil .
h nmr ( meoh - d4 , 400 hz ) 
  7.787.75 ( m , 2h ) , 7.066.97 ( m , 4h ) , 6.56 ( d , j = 8.4 hz , 2h ) , 5.16 ( s , nh ) , 4.33 ( t , j = 4.8 hz , 2h ) , 3.74 ( t , j = 4.8 hz , 2h ) , 2.29 ( s , 
 3h ) , 1.95 ( s , 6h ) , 1.45 ( s , 6h ) .
2-amino-1-(3-(3,4-difluorophenylamino)-2-(4-fluorophenyl)-8,8-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)-2-methylpropan-1-one : compound 30 was prepared from compound 8 - 5 by a pd2(dba)3 mediated 
 amination reaction with 3,4-difluoroaniline followed by a tfa mediated 
 deprotection.h nmr ( 400 mhz , meoh - d4 )  7.637.60 ( m , 2h ) , 7.14 ( t , j = 8.4 hz , 2h ) , 7.01 ( dd , j = 18.8 , 8.8 hz , 1h ) , 
 6.686.63 ( m , 1h ) , 6.5 ( m , 1h ) , 4.03 ( m , 2h ) , 4.01 ( m , 2h ) , 
 2.01 ( s , 6h ) , 1.66 ( s , 6h ) .
2-(4-fluorophenyl)-n-(p - tolyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazin-3-amine : the details of the synthesis involving an ugi reaction 
 between 4-fluorobenzaledehyde , 2-aminopyrazine , and 1-isocyano-4-methylbenzene 
 has been reported in the previous communication.h nmr ( 400 mhz , meoh - d4 )  7.73 ( d , j = 6.8 hz , 2h ) , 7.006.99 
 ( m , 4h ) , 6.49 ( d , j = 8.0 hz , 2h ) , 4.01 ( s , 2h ) , 
 3.72 ( m , 2h ) , 3.15 ( m , 2h ) , 2.19 ( s , 3h ) .
2-(4-fluorophenyl)-3-(p - tolylamino)-7,8-dihydroimidazo[1,2-a]pyrazin-6(5h)-one : compound 32 was synthesized from compound 7 ( r1 = r2 = h ) pd2(dba)3 mediated coupling reaction with p - toluidine .
h nmr ( 400 mhz , dmso - d6 )  
 8.55 ( s , 1h ) , 7.777.75 ( m , 2h ) , 7.267.14 ( m , 4h ) , 
 7.016.95 ( m , 2h ) , 6.53 ( m , 1h ) , 4.53 ( m , 2h ) , 4.22 ( m , 2h ) , 
 2.17 ( s , 3h ) .
2-(4-fluorophenyl)-8,8-dimethyl-3-(p - tolylamino)-7,8-dihydroimidazo[1,2-a]pyrazin-6(5h)-one : compound 33a was prepared from 7 by the following way : to a stirred solution of compound 7 ( 390 mg , 1.51 mmol , 1.00 equiv ) in dichloromethane ( 20 ml ) 
 was added nbs ( 0.28 g , 1.00 equiv ) .
the solid was filtered out , and the mixture was washed 
 with a saturated solution of na2s2o3 and dried over na2so4 .
the solids was purified by silica gel chromatography 
 ( petroleum ether / etoac = 1:2 ) to result in 432 mg ( 85% ) of the title 
 compound as a white solid .
h nmr ( 300 mhz , dmso - d6 )  8.76 ( s , 1h ) , 7.947.89 ( m , 
 2h ) , 7.317.25 ( m , 2h ) , 4.57 ( s , 2h ) , 1.56 ( s , 6h ) .
compound 33 was prepared from compound 33a by a pd2(dba)3 mediated amination reaction with p - toluidine .
h nmr ( meoh - d4 , 400 hz )  7.627.58 ( m , 2h ) , 7.05 ( t , j = 8.4 hz , 2h ) , 6.92 ( d , j = 8.4 hz , 2h ) , 
 6.52 ( d , j = 8.4 hz , 2h ) , 4.42 ( s , 2h ) , 2.12 ( s , 
 3h ) , 1.71 ( s , 6h ) .
3-(4-chloro-3-fluorophenylamino)-2-(4-fluorophenyl)-8,8-dimethyl-7,8-dihydro 
 imidazo[1,2-a]pyrazin-6(5h)-one : compound 34 was prepared from compound 7b by a pd2(dba)3 mediated amination 
 reaction with 4-chloro-3-fluoroaniline .
h nmr ( meoh - d4 , 400 hz )  7.747.70 ( m , 2h ) , 
 7.21 ( t , j = 8.4 hz , 1h ) , 7.08 ( m , 2h ) , 6.496.40 
 ( m , 2h ) , 4.43 ( s , 2h ) , 1.72 ( s , 6h ) .
2-(4-fluorophenyl)-8,8-dimethyl - n - p - tolyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazin-3-amine : compound 35 was prepared from compound 35 - 1 ( shown below and prepared by analogy to 
 compound 9 and using cbz - glycine ) by a pd2(dba)3 mediated amination reaction with p - toluidine followed by a 6n hcl mediated hydrolysis .
h nmr ( meoh - d4 , 400 hz )  7.787.75 
 ( m , 2h ) , 7.066.97 ( m , 4h ) , 6.56 ( d , j = 8.4 
 hz , 2h ) , 5.16 ( s , nh ) , 4.33 ( t , j = 4.8 hz , 2h ) , 
 3.74 ( t , j = 4.8 hz , 2h ) , 2.29 ( s , 3h ) , 1.95 ( s , 
 6h ) .
n,2-bis(4-fluorophenyl)-8,8-dimethyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazin-3-amine : compound 36 was prepared from compound 35 - 1 by a pd2(dba)3 mediated 
 amination reaction with 4-fluoroaniline followed by a 6n hcl mediated 
 hydrolysis .
h nmr ( 400 mhz , meoh - d4 )  7.627.58 ( m , 2h ) , 7.127.08 ( m , 2h ) , 
 6.686.63 ( m , 2h ) , 6.67 ( m , 2h ) , 4.16 ( m , 2h ) , 3.78 ( m , 2h ) , 
 1.92 ( s , 6h ) .
 on the basis of the initial success of optimization of 
 a novel series of imidazolopiperazines , a second generation of compounds 
 involving changes in the core piperazine ring was synthesized to improve 
 antimalarial properties . 
 these changes were carried out to further 
 improve the potency and metabolic stability of the compounds by leveraging 
 the outcome of a set of in vitro metabolic identification studies . 
 
 the optimized 8,8-dimethyl imidazolopiperazine analogues exhibited 
 improved potency , in vitro metabolic stability profile and , as a result , 
 enhanced oral exposure in vivo in mice . 
 the optimized compounds were 
 found to be more efficacious than the current antimalarials in a malaria 
 mouse model . 
 they exhibit moderate oral exposure in rat pharmacokinetic 
 studies to achieve sufficient multiples of the oral exposure at the 
 efficacious dose in toxicology studies . 
Introduction
Chemistry
Results and Discussion
Summary
Experimental Section
Experimental Protocols (Chemistry)