one of the major risk factors for chd are elevated serum cholesterol concentrations . lowering the level of serum cholesterol is an established clinical practice for chd treatment , intervention , and prevention .
pharmacologically , circulating cholesterol concentrations are reduced by statins , which are 3-hydroxy-3-methylglutaryl coenzyme a ( hmg - coa ) reductase inhibitors , affecting biosynthesis of endogenous cholesterol .
another approach is to block the absorption of dietary cholesterol , which is the other major contributor to serum cholesterol concentrations in the small intestine .
ezetimibe 1 was originally discovered without a known molecular target through in  vivo screening of cholesterol - fed hamsters . in 2004 , researchers from the schering - plough research institute identified niemann pick c1-like1 ( npc1l1 ) protein as a molecular target of ezetimibe 1 
 .
ezetimibe 1 acts by blocking the internalization of npc1l1 , thereby preventing cholesterol from entering the cytoplasm of enterocytes .
a thorough structure  activity relationship ( sar ) study   of the -lactam cholesterol absorption inhibitors in cholesterol - fed hamsters revealed that the 2-azetidinone backbone as well as the aryl group at the n-1 and c-4 position of the -lactam ring are required for activity .
the aryl group at the c-4 position of the -lactam ring is optimally substituted with alkoxy or hydroxy groups at the para - position .
the side chain at the c-3 position of the -lactam ring with three linking atoms bearing a pendent aryl group is optimal .
preferred configuration at the c-4 chiral center of the -lactam ring is s and the c-3 atom tolerates s or r configurations .
introduction of a heteroatom at the 1-position of the c-3 side chain can also contribute to the activity , whereas isosteric groups at the 3-position of the side chain decrease the activity .
in continuation of our research in the field of -lactam chemistry [ 1115 ] and taking into consideration the requirements determined by sar studies , we synthesized bioisosteres 5 and 6 ( fig . 
2 ) of ezetimibe 1 bearing a nh group at the c-3 position of the -lactam ring .
bioisosterism is a useful approach for lead compound modification that can result in improved pharmacological activity , decreased toxicity , and optimized pharmacokinetics . with the classical bioisosteric exchange of the ch2 with a nh group we aimed at investigating whether the change in polarity of the side chain , the ability of additional h - bond , and ammonium salt formations would affect their cholesterol absorption inhibition and cytotoxicity .
here we show that our new ezetimibe analogs 5 , 6 and their diastereoisomeric mixture 5/6 ( 70:30 ) are potent novel cholesterol absorption inhibitors .
we synthesized two novel ezetimibe analogs 5 and 6 ( fig .  2 ) and their diastereoisomeric mixtures 5/6 ( 70:30 ) and 6/5 ( 85:15 ) from enantiomerically pure trans-(3r,4r)-amino--lactam 2 ( fig . 
3 ) and determined their in  vitro and in  vivo activities as cholesterol absorption inhibitors .
we extensively studied the stereoselectivity of the side chain keto group reduction with cbs - catalyst in proximity of the nh group at the c-3 position of 2-azetidinone .
enantiomeric purity of 2 ( > 99% ee ) was determined by the mosher 's mtpa method   on f nmr ( 67.79  ppm , s , 3f , cf3 and -116.22  ppm , s , 1f , n 
there are two possible approaches for the synthesis of ezetimibe analogs 5 and 6 and their diasteroisomeric mixtures 5/6 and 6/5 from trans - amino--lactam 2 : ( i ) n - alkylation of trans - amino--lactam 2 with commercially available 2-bromo-1-(4-fluorophenyl)ethan-1-one 3 followed by stereoselective reduction of the side chain keto group at the c-3 of the -lactam ring with cbs - catalyst or ( ii ) stereoselective reduction of the keto group of 3 preceding n - alkylation reaction . in the present study we applied both approaches.(i)n - alkylation of trans - amino--lactam
n - alkylation of trans - amino--lactam 2 was performed in mild conditions using nai for in situ generation of 2-iodo - derivative of 2-bromo-1-(4-fluorophenyl)ethan-1-one 3 in the presence of et3n at room temperature and provided 4 in a moderate yield ( 46% ) ( scheme 1a ) .
a mixture of thf and dmf in ratio 9:1 was found optimal for the reaction .
the c-3 side chain hydroxy group was obtained by stereoselective reduction of the keto group with cbs catalyst ( scheme 1b ) [ 1921 ] .
however , addition of cbs - catalyst ( 0.1 eq . ) and bh3me2s ( 1 eq . ) provided the diastereoisomeric mixture 5/6 ( 50:50 ) , determined by h nmr .
ratio ) had no effect on the stereoselectivity , providing mixture 5/6 ( 50:50 ) .
the absence of stereoselectivity in the reduction of amino--lactam ketone 4 was probably due to nitrogen proximity to the keto group and the ability of borane to form a complex with amine , which allowed a direct hydrogen delivery to the keto group without participation of a chiral catalyst .
addition of bh3me2s ( 2 eq . ) to cbs - catalyst ( 0.1 eq . ) did not result in improvement of stereoselectivity in keto group reduction of 4 either .
improvement of stereoselectivity was accomplished with the complex formation between cbs - catalyst and bh3me2s , ( 1:1 eq .
reaction with ( r)-cbs - catalyst provided a diastereoisomeric mixture of amino alcohols 5/6 ( 70:30 ) at  20  c , whereas ( s)-cbs - catalyst afforded 6/5 ( 85:15 ) ( fig .  4 ) .
lowering the temperature to  80  c did not improve the stereoselectivity of the reduction .
recrystallization of 6/5 ( 85:15 ) provided pure amino alcohol 6.(ii)stereoselective reduction of ketone 3 followed by n - alkylation .
n - alkylation of trans - amino--lactam 2 with ketone 3 and subsequent reduction of the side chain keto group .
n - alkylation of trans - amino--lactam 2 was performed in mild conditions using nai for in situ generation of 2-iodo - derivative of 2-bromo-1-(4-fluorophenyl)ethan-1-one 3 in the presence of et3n at room temperature and provided 4 in a moderate yield ( 46% ) ( scheme 1a ) .
a mixture of thf and dmf in ratio 9:1 was found optimal for the reaction .
the c-3 side chain hydroxy group was obtained by stereoselective reduction of the keto group with cbs catalyst ( scheme 1b ) [ 1921 ] .
however , addition of cbs - catalyst ( 0.1 eq . ) and bh3me2s ( 1 eq . ) provided the diastereoisomeric mixture 5/6 ( 50:50 ) , determined by h nmr .
ratio ) had no effect on the stereoselectivity , providing mixture 5/6 ( 50:50 ) .
the absence of stereoselectivity in the reduction of amino--lactam ketone 4 was probably due to nitrogen proximity to the keto group and the ability of borane to form a complex with amine , which allowed a direct hydrogen delivery to the keto group without participation of a chiral catalyst .
addition of bh3me2s ( 2 eq . ) to cbs - catalyst ( 0.1 eq . ) did not result in improvement of stereoselectivity in keto group reduction of 4 either .
improvement of stereoselectivity was accomplished with the complex formation between cbs - catalyst and bh3me2s , ( 1:1 eq .
reaction with ( r)-cbs - catalyst provided a diastereoisomeric mixture of amino alcohols 5/6 ( 70:30 ) at  20  c , whereas ( s)-cbs - catalyst afforded 6/5 ( 85:15 ) ( fig .  4 ) .
lowering the temperature to  80  c did not improve the stereoselectivity of the reduction .
commercially available ketone 3 was reduced following the original protocol for cbs - reduction developed by corey et  al . and yielded alcohols 7a and 7b in > 99% ee ( scheme 2 ) .
protection of the oh group in 7a , b with tbdmscl was carried out in dmf in the presence of imidazole   to afford otbdms bromo derivatives 8a and 8b .
exchange of bromine in 8a , b with iodine ( in the presence of nai in acetone at 55  c ) yielded otbdms iodo derivatives 9a and 9b .
iodo - bromo exchange proceeded very slowly , providing the mixtures of 9a or 9b and unreacted 8a or 8b , respectively , in ratio 93:7 and 96% yield after 4 days .
this mixture was used in the n - alkylation reaction of amino--lactam 2 without further purification .
the reaction proceeded for 7 days in ch3cn to afford silyl intermediates 10a and 10b with a moderate yield ( 20% ) .
silyl intermediates 10a , b were further deprotected with 3% hcl in ethanol to produce ezetimibe bioisosteres 5 and 6 ( scheme 2 ) . the crystal structure of ( 3r,4r)-3-[(2s)-2-(4-fluorophenyl)-2-hydroxyethylamino]-1-(4-fluorophenyl)-4-(4-hydroxyphenyl)azetidin-2-one ( 2s,3r,4r)-6 was determined to establish unambiguously both absolute and relative configurations at the stereogenic centres c17   and n2 .
two symmetry independent molecules of 6 , related by a pseudo - twofold rotation axis , are present in the crystal structure designated as 6a and 6b ( fig . 
5 ) ; they are homochiral and of similar conformations ( fig .  6 ) .
configuration at the stereogenic centre c17 was assigned to be s in both 6a and 6b , according to the known r - configurations at the stereogenic centres c2 and c3 .
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 c hydrogen bonds into c2-symmetric dimers ( fig .  7 ,
each molecule has two hydroxy groups , which act as proton donors toward symmetry - equivalent molecules ; one toward a carbonyl and one toward another hydroxy group , giving a total of four o 
ho hydrogen bonds ( table  1 ) that link the dimers into layers parallel to ( 011 ) .
we measured an increase of the absorbance intensity at  247  nm by the addition of naoh in the ph range 612 ( fig . 
pka values calculated from the obtained sigmoidal curves for amino alcohol 5 ( fig . 
we got the same pka value for amino alcohol 6 ( data not shown ) , indicating that both compounds are present in the form of nh4 in the blood and small intestine .
cytotoxicity of amino alcohols 5 , 6 and their diastereoisomeric mixture 5/6 was analyzed using mtt cell proliferation assay and the lc50 values were determined in mdckii wild type , hnpc1l1/mdckii , and hepg2 cells ( table  2 ) .
mdckii cells stably expressing human niemann - pick c1-like protein 1 ( hnpc1l1/mdckii ) ( figure  s1 ) are a pharmacologically validated system for investigating npc1l1-mediated cholesterol uptake .
the lc50 values were higher than 100  m and considered nontoxic in all three cell lines .
lc50 values for ezetimibe 1 were 62.29  m in hnpc1l1/mdckii and 69.74  m in hepg2 cells .
in mdckii wild type cells , ezetimibe 1 showed no toxicity ( table  2 ) .
in addition , we tested the in  vitro cytotoxicity of ezetimibe 1 and compounds 5 , 6 , and 5/6 ( 70:30 ) in combination with micelles and found all lc50 values to be above 100  m ( figure  s2 ) .
first we tested ezetimibe analogs 5 and 6 and their diastereoisomeric mixtures 5/6 ( 70:30 ) and 6/5 ( 85:15 ) for their ability to inhibit cholesterol uptake .
in mdckii wildtype cells , ezetimibe 1 had no effect , but inhibited cholesterol uptake in hnpc1l1/mdckii cells ( figure  s3 ) . when hnpc1l1/mdckii were treated with 5 ( fig . 
10d ) , we observed 5055% inhibition of cholesterol uptake , reaching its maximum at 120  m concentration without a significant difference between the compounds .
these results show that the novel ezetimibe bioisosteres 5 , 6 and their diastereoisomeric mixtures 5/6 and 6/5 are potent inhibitors of cholesterol uptake in  vitro . in  vivo , we first determined the consequences of ezetimibe 1 and amino alcohols 5 and 6 on cholesterol absorption .
we therefore gavaged mice with 2  ci [ h]cholesterol and 10  mg / kg / day of each compound or vehicle , and radioactivity was measured in plasma , liver , and three equal parts of the intestine ( duodenum , jejunum , ileum ) .
both 5 and 6 showed significant inhibition of cholesterol absorption ( table  3 ) .
treatment of mice with either 5 and 6 resulted in reductions of [ h]cholesterol in plasma by 50% and 32% , respectively .
radioactivities in the liver were decreased by 44% and 47% , respectively , and were comparable to ezetimibe 1 .
amino alcohol 6 markedly lowered [ h]cholesterol in the small intestine with highest inhibition in the ileum by 5860% , whereas amino alcohol 5 reduced radioactivity in the small intestine by 22% without reaching statistical significance in either part of the intestine .
we therefore increased the dose to 20  mg / kg / day and determined inhibition of cholesterol absorption .
the inhibition in duodenum and jejunum was similar to the lower dose , whereas in ileum it raised from 21% to 41% ( table  3 ) . decreased cholesterol absorption of 5 in the small intestine compared to ezetimibe 1
might be explained by the fact that 2-azetidinones are moderate acyl - coenzyme a : cholesterol acyltransferase inhibitors and their level of activity is highly structure - dependent .
it might therefore be speculated that acyl - coenzyme a : cholesterol acyltransferase activity of amino alcohols 5 and 6 and their diastereoisomeric mixtures is altered compared to ezetimibe 1 .
the effect of the diastereoisomeric mixture 5/6 ( 70:30 ) on cholesterol absorption was comparable to ezetimibe 1 in plasma and ileum , lower in duodenum and jejunum , and higher in the liver ( table  3 ) .
this report demonstrates that we have successfully synthesized two novel ezetimibe bioisosteres 5 , 6 and their diastereoisomeric mixtures 5/6 ( 70:30 ) and 6/5 ( 85:15 ) from enantiomerically pure trans-(3r,4r)-amino--lactam 2 .
crystal structure of 6 was determined to establish unambiguously both absolute and relative configurations at the new stereogenic centre c17 and were assigned to be s. both diastereoisomeres 5 and 6 as well as their diastereoisomeric mixture 5/6 showed significant cholesterol absorption inhibitory activity both in  vitro and in  vivo . based on our data and the pka value for 5 and 6 being 9.35 , indicating that both compounds are present in the form of nh4 in the blood and small intestine , other diastereoisomeric mixtures ( e.g. 6/5 ( 85:15 ) ) may exhibit similar in  vivo results as 5/6 ( 70:30 ) .
results from this study implicate a therapeutic potential of these novel compounds to reduce cholesterol plasma concentrations and improve chd .
the ir spectra were recorded on a perkinelmer spectrum rx i ft - ir system spectrometer ( kbr pellets technique ) ( perkinelmer instruments , norwalk , ct , usa ) .
the h and c nmr spectra ( in cdcl3 and dmso - d6 at rt ) were measured on a bruker av 300 and/or av 600 spectrometer ( bruker biospin gmbh . ,
rheinstetten , germany ) ,  is given in ppm relative to tetramethylsilane as an internal reference .
microanalysis was performed on a pe 2400 series ii chns / o analyzer ( perkinelmer instruments , shelton , ct , usa ) .
optical rotations : optical activity automatic polarimeter aa-10 in 1  dm cell ; c in g/100  ml ( optical activity ltd . , ramsey , england ) .
column chromatography on silica gel , 70230  mesh , 60   ( sigma  aldrich , st .
louis , mo , usa or acros - organics , new jersey , usa ) was performed at rt .
thin layer chromatography was carried out on tlc aluminium sheets , 20    20  cm , silica gel 60 f254 ( merck , darmstadt , germany ) .
uv / vis measurements were performed on a t80 + spectrophotometer ( pg instruments limited , england ) .
samples for hr - ms analysis were resuspended in 5  l of thap / dac matrix and 1  l was spotted onto a maldi plate .
mass spectra were obtained on a matrix - assisted laser desorption / ionization - time - of - flight maldi - tof / tof mass spectrometer ( 4800 plus maldi - tof / tof analyzer , applied biosystems , foster city , ca , usa ) equipped with nd : yag laser operating at 355  nm with firing rate 200  hz in the positive ion reflector mode .
1600 shots per spectrum were taken covering mass range 1001000  da , focus mass 500  da and delay time 100  ns .
crystals of amino alcohol ( 2s,3r,4r)-6 suitable for data collection were grown from ethyl acetate and hexane by vapour diffusion .
the selected crystal was a needle with dimensions 0.10    0.03    0.02  mm .
single crystal measurement was performed on an oxford diffraction xcalibur nova r ( microfocus cu tube , oxford diffraction , u.k . ) at room temperature [ 293(2 ) k ] .
the model was refined using the full - matrix least squares refinement ; all non - hydrogen atoms were refined anisotropically .
hydrogen atoms bound to c and o were modelled as riding entities using the afix command .
hydrogen atoms bound to n2a and n2b could not be located from the electron density map due to the data poor quality .
however , it can be assumed that they are directed towards the nearest proton acceptor : the carbonyl oxygen of the neighbouring molecule .
therefore , they were generated at the appropriate positions and refined using appropriate restraints ( table  1 ) .
configuration of novel stereogenic centre c17 was established relative to the configurations of the known stereogenic centres c2 and c3 .
the structure comprises relatively large cavities filled by disordered water molecules . due to a very low electron density and low data quality ( due to small size of the crystal )
the disordered water was tentatively modelled as four isotropic oxygen atoms ( hydrogens could not have been located ) with occupancy of 0.5 .
molecular geometry calculations were performed by platon , and molecular graphics were prepared using ortep-3 , and ccdc - mercury .
crystallographic and refinement data for amino alcohol ( 2s,3r,4r)-6 reported in this paper are shown in table  4 .
supplementary crystallographic data for this paper can be obtained free of charge via 
 www.ccdc.cam.ac.uk/conts/retrieving.html ( or from the cambridge crystallographic data centre , 12 , union road , cambridge cb2 1ez , uk ; fax : ( + ) 44 1223 336033 ; or deposit@ccdc.cam.ac.uk ) .
amino--lactam 2 was synthesized following the procedure described by ojima et  al .   and habu et  al .   and purified by a silica gel column chromatography ( hexane / ethyl acetate 9:1 , gradually increasing the content of ethyl acetate in eluent to pure ethyl acetate ) and obtained as yellow oil ( 1.3  g , 43% ) .
[ ]d  =  26 ( c  =  1 , etoac ) .
ft - ir ( kbr ) cm : 2931 , 2858 , 1739 , 1609 , 1510 , 1390 , 1266 , 913 , 834 , 513 .
h nmr ( 300  mhz , cdcl3 ) : 0.19 ( s , 6h , si-(ch3)2 ) , 0.97 ( s , 9h , c-(ch3)3 ) , 1.81 ( bs , 2h , nh2 ) , 4.05 ( d , 1h , j  =  2.2  hz , c4 -lactam ) , 4.58 ( d , 1h , j  =  2.1  hz , c3 -lactam ) , 6.83 ( d , 2h , j  =  8.5  hz , ar 
h ) , 6.92 ( t , 2h , j1,2  =  8.7  hz , ar 
h ) , 7.18 ( d , 2h , j  =  8.5  hz , ar  h ) , 7.237.26 ( m , 2h , ar 
h ) ; c nmr ( 150  mhz , cdcl3 ) :  4.27 ( si-(ch3)2 ) , 18.32 ( c-(ch3)3 ) , 25.75 ( c-(ch3)3 ) , 66.69 ( c4 -lactam ) , 70.17 ( c3 -lactam ) , 115.95 ( d , j  =  22.8  hz , 4-f - c6h4 ) , 118.98 ( d , j  =  7.8  hz , 4-f - c6h4 ) , 120.86 ( 4-otbdms - c6h4 ) , 127.31 ( 4-otbdms - c6h4 ) , 129.18 ( 4-otbdms - c6h4 ) , 133.83 ( d , j  =  2.2  hz , 4-f - c6h4 ) , 156.25 ( 4-otbdms - c6h4 ) , 159.26 ( d , j  =  243.9  hz , 4-f - c6h4 ) , 168.02 ( co , -lactam ) . hrms for c21h27fn2o2si ( mr  =  386.53518 ) : calcd .
m / z [ m+h ] 387.1898 , found 387.1901 . to a solution of 2 ( 159  mg , 0.41  mmol ) in a mixture of anhydrous thf and dmf ( 9:1 , 7.8  ml ) , 2-bromo-1-(4-fluorophenyl)ethan-1-one 3 ( 98  mg , 0.45  mmol ) , sodium iodide ( 68  mg , 0.45  mmol ) and et3n ( 114  l , 0.82  mmol ) were added .
the reaction mixture was stirred at room temperature for 3  h after which solvent was evaporated to dryness .
distilled water ( 10  ml ) was added and the product extracted with ethyl acetate ( 3    20  ml ) .
4 was purified by a silica gel column chromatography ( hexane / ethyl acetate 2:1 ) and obtained as light brown oil ( 99  mg , 46% ) .
[ ]d  =  + 20 ( c  =  1 , etoac ) .
ft - ir ( kbr ) cm : 3332 , 2931 , 1748 , 1693 , 1600 , 1506 , 1385 , 1260 , 1156 , 913 , 834 , 515 .
h nmr ( 600  mhz , cdcl3 ) : 0.18 ( s , 6h , si-(ch3)2 ) , 0.96 ( s , 9h , c-(ch3)3 ) , 1.60 ( bs , 1h , nh ) , 4.09 ( d , 1h , j  =  2.1  hz , c4 -lactam ) , 4.26 and 4.34 ( abq , 2h , jab  =  18.5  hz , c(=o)ch2nh ) , 4.80 ( d , 1h , j  =  1.9  hz , c3 -lactam ) , 6.80 ( d , 2h , j  =  8.5  hz , ar 
h ) , 6.93 ( t , 2h , j1,2  =  8.6  hz , ar  h ) , 7.127.16 ( m , 4h , ar 
h ) ; c nmr ( 75  mhz , cdcl3 ) :  4.28 ( si-(ch3)2 ) , 18.30 ( c-(ch3)3 ) , 25.74 ( c-(ch3)3 ) , 53.00 ( c(o)ch2nh ) , 63.79 ( c4 -lactam ) , 75.44 ( c3 -lactam ) , 115.98 ( d , j  =  22.6  hz , 4-f - c6h4 ) , 116.16 ( d , j  =  21.7  hz , 4-f - c6h4 ) , 119.06 ( d , j  =  7.9  hz , 4-f - c6h4 ) , 120.87 ( 4-otbdms - c6h4 ) , 127.30 ( 4-otbdms - c6h4 ) , 129.12 ( 4-otbdms - c6h4 ) , 130.62 ( d , j  =  9.4  hz , 4-f - c6h4 ) , 131.57 ( d , j  =  3.3  hz , 4-f - c6h4 ) , 133.68 ( d , j  =  2.8  hz , 4-f - c6h4 ) , 156.19 ( 4-otbdms - c6h4 ) , 159.31 ( d , j  =  243.9  hz , 4-f - c6h4 ) , 166.17 ( co , -lactam ) , 166.23 ( d , j  =  254.8  hz , 4-f - c6h4 ) , 195.58 ( co ) .
the reaction was carried out in dry conditions under argon atmosphere . to a solution of ( r)-(+)-2-methyl - cbs - oxazaborolidine catalyst ( 251  mg , 0.91  mmol ) in anhydrous thf
( 2  ml ) , 2 m thf solution of bh3me2s ( 454  l , 0.91  mmol ) was added and the mixture stirred at room temperature for 10  min .
the temperature was then lowered to  20  c and a solution of 4 ( 474  mg , 0.91  mmol ) in anhydrous thf ( 5  ml ) added dropwise .
after 24  h the reaction mixture was warmed to room temperature and quenched with methanol .
the solution was concentrated in vacuo , distilled water ( 15  ml ) added and the product extracted with ethyl acetate ( 3    40  ml ) . collected organic layers were dried over na2so4 and solvent evaporated to dryness .
crude product was dissolved in ethanol ( 22  ml ) and 1  m hcl ( 5  ml ) added .
reaction mixture was stirred at room temperature overnight , solvent evaporated to dryness and crude product purified by a silica gel column chromatography ( hexane / ethyl acetate 1:3 ) to afford diastereoisomeric mixture 5/6 ( 70:30 ) ( 283  mg , 76% ) .
ft - ir ( kbr ) cm : 3341 , 1736 , 1615 , 1601 , 1509 , 1390 , 1223 , 1154 , 1101 , 832 .
h nmr ( 600  mhz , dmso - d6 ) : 2.722.83 ( m , 2h , ch(oh)ch2nh , 5/6 ) , 2.93 ( bs , 1h , ch(oh)ch2nh , 5/6 ) , 3.97 ( s , 1h , c4 -lactam , 5/6 ) , 4.554.57 ( m , 1h , ch(oh)ch2nh , 6 ) , 4.594.62 ( m , 1h , ch(oh)ch2nh , 5 ) , 4.83 ( d , 1h , j  =  2.0  hz , c3 -lactam , 5/6 ) , 5.30 ( d , 1h , j  =  4.3  hz , ch(oh)ch2nh , 5 ) , 5.33 ( d , 1h , j  =  4.3  hz , ch(oh)ch2nh , 6 ) , 6.736.75 ( m , 2h , ar 
h , 5/6 ) , 9.43 ( s , 1h , ar  oh , 5/6 ) ; c nmr ( 150  mhz , dmso - d6 ) : 54.53 ( ch(oh)ch2nh , 5 ) , 54.78 ( ch(oh)ch2nh , 6 ) , 62.36 ( c4 -lactam , 5 ) , 62.58 ( c4 -lactam , 6 ) , 71.43 ( ch(oh)ch2nh , 5 ) , 71.61 ( ch(oh)ch2nh , 6 ) , 75.43 ( c3 -lactam , 5 ) , 75.63 ( c3 -lactam , 6 ) , 114.60 ( d , j  =  20.9  hz , 4-f - c6h4 , 5 ) , 114.62 ( d , j  =  21.2  hz , 4-f - c6h4 , 6 ) , 115.68 ( 4-oh - c6h4 , 5/6 ) , 115.79 ( d , j  =  22.7  hz , 4-f - c6h4 , 5/6 ) , 118.68 ( d , j  =  7.7  hz , 4-f - c6h4 , 5/6 ) , 127.40 ( 4-oh - c6h4 , 6 ) , 127.48 ( 4-oh - c6h4 , 5 ) , 127.52 ( 4-oh - c6h4 , 5 ) , 127.58 ( 4-oh - c6h4 , 6 ) , 127.85 ( d , j  =  7.8  hz , 4-f - c6h4 , 5/6 ) , 133.86 ( d , j  =  1.9  hz , 4-f - c6h4 , 5/6 ) , 140.33 ( d , j  =  2.7  hz , 4-f - c6h4 , 6 ) , 140.36 ( d , j  =  2.8  hz , 4-f - c6h4 , 5 ) , 157.34 ( 4-oh - c6h4 , 5/6 ) , 158.12 ( d , j  =  240.3  hz , 4-f - c6h4 , 5/6 ) , 161.19 ( d , j  =  242.4  hz , 4-f - c6h4 , 5/6 ) , 167.06 ( co , -lactam , 5 ) , 167.08 ( co , -lactam , 6 ) .
to a solution of ( s)-()-2-methyl - cbs - oxazaborolidine catalyst ( 275  mg , 0.99  mmol ) in anhydrous thf ( 2  ml ) , 2  m thf solution of bh3me2s ( 496  l , 0.99  mmol ) and 4 ( 519  mg , 0.99  mmol ) in anhydrous thf ( 5  ml ) were added .
diastereoisomeric mixture 6/5 ( 85:15 ) ( 278  mg , 68% ) was obtained .
amino alcohol 6 was obtained ( 111  mg , 25% ) by recrystallization of diastereoisomeric mixture 6/5 ( 85:15 ) in ch2cl2 .
[ ]d  =  + 9 ( c  =  1 , etoac ) .
ft - ir ( kbr ) cm : 3344 , 1723 , 1615 , 1510 , 1394 , 1233 , 1155 , 829 .
h nmr ( 300  mhz , dmso - d6 ) : 2.702.86 ( m , 2h , ch(oh)ch2nh ) , 3.08 ( bs , 1h , ch(oh)ch2nh ) , 3.97 ( d , 1h , j  =  1.9  hz , c4 -lactam ) , 4.544.59 ( m , 1h , ch(oh)ch2nh ) , 4.84 ( d , 1h , j  =  1.9  hz , c3 -lactam ) , 5.38 ( d , 1h , j  =  4.3  hz , ch(oh)ch2nh ) , 6.75 ( d , 2h , j  =  8.5  hz , ar 
oh ) ; c nmr ( 75  mhz , dmso - d6 ) : 54.76 ( ch(oh)ch2nh ) , 62.57 ( c4 -lactam ) , 71.56 ( ch(oh)ch2nh ) , 75.57 ( c3 -lactam ) , 114.58 ( d , j  =  21.1  hz , 4-f - c6h4 ) , 115.65 ( 4-oh - c6h4 ) , 115.74 ( d , j  =  22.6  hz , 4-f - c6h4 ) , 118.63 ( d , j  =  8.0  hz , 4-f - c6h4 ) , 127.34 ( 4-oh - c6h4 ) , 127.52 ( 4-oh - c6h4 ) , 127.78 ( d , j  =  8.1  hz , 4-f - c6h4 ) , 133.85 ( d , j  =  2.5  hz , 4-f - c6h4 ) , 140.25 ( d , j  =  2.8  hz , 4-f - c6h4 ) , 157.33 ( 4-oh - c6h4 ) , 158.08 ( d , j  =  240.7  hz , 4-f - c6h4 ) , 161.17 ( d , j  =  242.2  hz , 4-f - c6h4 ) , 167.02 ( co , -lactam ) .
for the synthesis of 7a , 2  m thf solution of bh3me2s ( 1.15  ml , 2.30  mmol ) was added to a solution of ( r)-(+)-2-methyl - cbs - oxazaborolidine catalyst ( 64  mg , 0.23  mmol ) in anhydrous thf ( 2  ml ) .
the mixture was stirred for 10  min  and a solution of 2-bromo-1-(4-fluorophenyl)ethan-1-one 3 ( 500  mg , 2.30  mmol ) in anhydrous thf ( 4  ml ) was added dropwise .
reaction mixture was quenched with methanol , solvent evaporated to dryness and distilled water ( 15  ml ) added .
the product was extracted with ch2cl2 ( 3    30  ml ) , collected organic layers dried over na2so4 and solvent evaporated to dryness .
7a was purified by a silica gel column chromatography ( hexane / ethyl acetate 6:1 ) and obtained as colourless oil ( 503  mg , 100% ) .
the ee>99% of 7a was determined by chiral hplc ( 200/4 nucleodex beta - pm column , macherey 
nagel , germany , method : 0.1% teaa in h2o : methanol 55 : 45 , 30  min , flow 0.7  ml / min ,   =  254  nm , retention time 19.5  min ) .
. 7b was synthesized following the same procedure using ( s)-()-2-methyl - cbs - oxazaborolidine catalyst .
[ ]d  =  + 28 ( c  =  1 , etoac ) .
ft - ir ( kbr ) cm : 3401 , 2963 , 2897 , 1896 , 1605 , 1513 , 1306 , 1224 , 1158 , 1067 , 992 , 838 , 779 , 645 , 556 , 523 .
h nmr ( 300  mhz , cdcl3 ) : 2.75 ( bs , 1h , ch(oh)ch2 ) , 3.463.61 ( m , 2h , ch(oh)ch2 ) , 4.89 ( dd , 1h , j1  =  8.7  hz , j2  =  3.5  hz , ch(oh)ch2 ) , 7.05 ( t , 2h , j1,2  =  8.6  hz , ar 
h ) ; c nmr ( 150  mhz , cdcl3 ) : 40.23 ( ch(oh)ch2 ) , 73.33 ( ch(oh)ch2 ) , 115.76 ( d , j  =  21.6  hz , 4-f - c6h4 ) , 127.88 ( d , j  =  8.2  hz , 4-f - c6h4 ) , 136.25 ( d , j  =  3.1  hz , 4-f - c6h4 ) , 162.86 ( d , j  =  247.0  hz , 4-f - c6h4 ) .
mr  =  219.05 ) : c , 43.86 ; h , 3.68 . found : c , 43.63 ; h 4.01 .
for the synthesis of 8a a solution of imidazole ( 254  mg , 3.74  mmol ) in dmf ( 0.5  ml ) was added to a solution of 7a ( 327  mg , 1.49  mmol ) in dmf ( 1  ml ) and stirred for 10  min , followed by dropwise addition of tbdmscl ( 293  mg , 1.94  mmol ) solution in dmf ( 1.3  ml ) .
solvent was evaporated to dryness , distilled water ( 15  ml ) added and the resulting mixture extracted with ethyl acetate ( 3    30  ml ) .
8a was purified by a silica gel column chromatography ( hexane / ethyl acetate 6:1 ) and obtained as colourless oil ( 452  mg , 91% ) .
[ ]d  =  46 ( c  =  1 , etoac ) .
8b was synthesized following the same procedure starting from 7b ( 424  mg , 1.94  mmol ) and obtained as colourless oil ( 602  mg , 94% ) .
[ ]d  =  + 46 ( c  =  1 , etoac ) .
ft - ir ( kbr ) cm : 2957 , 2930 , 2887 , 2858 , 1606 , 1509 , 1463 , 1417 , 1362 , 1257 , 1226 , 1155 , 1113 , 1012 , 914 , 834 , 778 , 724 , 646 .
h nmr ( 300  mhz , cdcl3 ) :  0.09 ( s , 3h , si  ch3 ) , 0.10 ( s , 3h , si 
ch3 ) , 0.88 ( s , 9h , c-(ch3)3 ) , 3.363.48 ( m , 2h , ch(otbdms)-ch2 ) , 4.82 ( dd , 1h , j1  =  7.4  hz , j2  =  4.8  hz , ch(otbdms)-ch2 ) , 7.02 ( t , 2h , j1,2  =  8.7  hz , ar 
h ) , 7.287.33 ( m , 2h , ar  h ) ; c nmr ( 75  mhz , cdcl3 ) :  4.75 ( si  ch3 ) ,  4.60 ( si  ch3 ) , 18.35 ( c-(ch3)3 ) , 25.86 ( c-(ch3)3 ) , 39.48 ( ch(otbdms)-ch2 ) , 74.76 ( ch(otbdms)-ch2 ) , 115.39 ( d , j  =  21.6  hz , 4-f - c6h4 ) , 127.98 ( d , j  =  8.1  hz , 4-f - c6h4 ) , 138.20 ( d , j  =  3.2  hz , 4-f - c6h4 ) , 162.59 ( d , j  =  245.8  hz , 4-f - c6h4 ) . anal . calcd . for c14h22brfosi
( mr  =  333.31 ) : c , 50.45 ; h , 6.65 .
reaction was carried out in a reaction flask protected from light . to a solution of 8a ( 113  mg , 0.34  mmol ) in acetone ( 3  ml ) , nai ( 254  mg , 1.68  mmol ) was added .
the reaction proceeded at 55  c for 4 days after which distilled water ( 15  ml ) was added and product extracted with ethyl acetate ( 3    30  ml ) .
collected organic layers were dried over na2so4 and solvent evaporated to dryness . thus obtained crude product was purified by a silicagel column chromatography ( hexane ) .
compound 9a was obtained as a mixture of 9a and unreacted 8a ( 93:7 ) as light brown oil ( 124  mg , 96% ) .
9b was synthesized following the same procedure starting from 8b ( 75  mg , 0.22  mmol ) and nai ( 168  mg , 1.12  mmol ) to give after a silicagel column chromatography ( hexane ) a mixture of 9b and unreacted 8b ( 94:6 ) as light brown oil ( 39  mg , 46% ) .
ft - ir ( kbr ) cm : 3448 , 2956 , 2930 , 2887 , 2858 , 1605 , 1509 , 1463 , 1408 , 1362 , 1257 , 1224 , 1106 , 997 , 887 , 837 , 776 .
h nmr ( 300  mhz , cdcl3 ) :  0.13 ( s , 3h , si  ch3 ) , 0.10 ( s , 3h , si 
ch3 ) , 0.89 ( s , 9h , c-(ch3)3 ) , 3.283.30 ( m , 2h , ch(otbdms)-ch2 ) , 4.73 ( t , 1h , j1,2  =  6.0  hz , ch(otbdms)-ch2 ) , 7.00 ( t , 2h , j1,2  =  8.7  hz , ar 
4.68 ( si  ch3 ) ,  4.56 ( si  ch3 ) , 15.03 ( ch(otbdms)-ch2 ) , 18.36 ( c-(ch3)3 ) , 25.93 ( c-(ch3)3 ) , 74.65 ( ch(otbdms)-ch2 ) , 115.38 ( d , j  =  21.5  hz , 4-f - c6h4 ) , 127.80 ( d , j  =  8.2  hz , 4-f - c6h4 ) , 138.87 ( d , j  =  3.1  hz , 4-f - c6h4 ) , 162.51 ( d , j  =  246.0  hz , 4-f - c6h4 ) .
reaction was carried out in a reaction flask protected from light . to a solution of 2 ( 154  mg , 0.40  mmol ) in ch3cn ( 3  ml ) 9a ( 151  mg , 0.40  mmol ) was added .
the reaction proceeded at 80  c for 7 days after which the solvent was evaporated to dryness .
compound 10a was purified by a silica gel column chromatography ( hexane / ethyl acetate 6:1 ) and obtained as light brown oil ( 52  mg , 20% ) .
[ ]d  =  9 ( c  =  1 , etoac ) .
ft - ir ( kbr ) cm : 3480 , 2931 , 2858 , 1748 , 1607 , 1505 , 1464 , 1385 , 1259 , 1226 , 1101 , 1006 , 914 , 834 , 515 .
h nmr ( 300  mhz , cdcl3 ) :  0.15 ( s , 3h , si  ch3 ) , 0.02 ( s , 3h , si 
ch3 ) , 0.18 ( s , 6h , si-(ch3)2 ) , 0.86 ( s , 9h , c-(ch3)3 ) , 0.96 ( s , 9h , c-(ch3)3 ) , 2.01 ( bs , 1h , ch(otbdms)ch2nh ) , 2.782.99 ( m , 2h , ch(otbdms)-ch2-nh ) , 4.00 ( d , 1h , j  =  2.1  hz , c4 -lactam ) , 4.61 ( d , 1h , j  =  1.8  hz , c3 -lactam ) , 4.77 ( dd , 1h , j1  =  7.8  hz , j2  =  4.3  hz , ch(otbdms)ch2nh ) , 6.81 ( d , 2h , j  =  8.7  hz , ar 
h ) , 6.91 ( t , 2h , j1,2  =  8.8  hz , ar 
h ) , 6.99 ( t , 2h , j1,2  =  8.8  hz , ar 
h ) , 7.13 ( d , 2h , j  =  8.5  hz , ar  h ) , 7.217.30 ( m , 4h , ar 
4.75 ( si  ch3 ) ,  4.41 ( si  ch3 ) ,  4.28 ( si-(ch3)2 ) , 18.29 ( c-(ch3)3 ) , 25.73 ( c-(ch3)3 ) , 25.96 ( c-(ch3)3 ) , 55.69 ( ch(otbdms)-ch2-nh ) , 63.86 ( c4 -lactam ) , 74,18 ( ch(otbdms)ch2nh ) , 75.73 ( c3 -lactam ) , 115.24 ( d , j  =  21.3  hz , 4-f - c6h4 ) , 115.90 ( d , j  =  22.6  hz , 4-f - c6h4 ) , 118.92 ( d , j  =  7.9  hz , 4-f - c6h4 ) , 120.84 ( 4-otbdms - c6h4 ) , 127.23 ( 4-otbdms - c6h4 ) , 127.95 ( d , j  =  8.0  hz , 4-f - c6h4 ) , 129.40 ( 4-otbdms - c6h4 ) , 133.78 ( d , j  =  2.7  hz , 4-f - c6h4 ) , 138.86 ( d , j  =  3.0  hz , 4-f - c6h4 ) , 156.11 ( 4-otbdms - c6h4 ) , 159.20 ( d , j  =  243.6  hz , 4-f - c6h4 ) , 162.33 ( d , j  =  245.5  hz , 4-f - c6h4 ) , 166.38 ( co , -lactam ) .
10b was synthesized following the procedure for 10a and obtained as light brown oil ( 9  mg , 19% ) .
[ ]d  =  + 16 ( c  =  1 , etoac ) .
ft - ir ( kbr ) cm : 3439 , 2929 , 2857 , 1749 , 1608 , 1506 , 1472 , 1385 , 1259 , 1101 , 1012 , 913 , 833 , 516 .
h nmr ( 300  mhz , cdcl3 ) :  0.14 ( s , 3h , si  ch3 ) , 0.04 ( s , 3h , si  ch3 ) , 0.18 ( s , 6h , si-(ch3)2 ) , 0.86 ( s , 9h , c-(ch3)3 ) , 0.96 ( s , 9h , c-(ch3)3 ) , 1.80 ( bs , 1h , ch(otbdms)ch2nh ) , 2.802.96 ( m , 2h , ch(otbdms)-ch2-nh ) , 4.02 ( d , 1h , j  =  2.0  hz , c4 -lactam ) , 4.62 ( d , 1h , j  =  2.0  hz , c3 -lactam ) , 4.81 ( dd , 1h , j1  =  7.6  hz , j2  =  4.3  hz , ch(otbdms)ch2nh ) , 6.81 ( d , 2h , j  =  8.6  hz , ar 
h ) , 6.90 ( t , 2h , j1,2  =  8.8  hz , ar 
h ) , 6.99 ( t , 2h , j1,2  =  8.7  hz , ar 
h ) , 7.12 ( d , 2h , j  =  8.5  hz , ar  h ) , 7.207.31 ( m , 4h , ar 
4.72 ( si  ch3 ) ,  4.39 ( si  ch3 ) ,  4.27 ( si-(ch3)2 ) , 18.30 ( c-(ch3)3 ) , 25.74 ( c-(ch3)3 ) , 25.97 ( c-(ch3)3 ) , 55.68 ( ch(otbdms)-ch2-nh ) , 63.35 ( c4 -lactam ) , 74,25 ( ch(otbdms)ch2nh ) , 75.82 ( c3 -lactam ) , 115.25 ( d , j  =  21.5  hz , 4-f - c6h4 ) , 115.91 ( d , j  =  22.8  hz , 4-f - c6h4 ) , 118.94 ( d , j  =  7.9  hz , 4-f - c6h4 ) , 120.84 ( 4-otbdms - c6h4 ) , 127.25 ( 4-otbdms - c6h4 ) , 127.96 ( d , j  =  8.0  hz , 4-f - c6h4 ) , 129.35 ( 4-otbdms - c6h4 ) , 133.77 ( d , j  =  2.8  hz , 4-f - c6h4 ) , 138.89 ( d , j  =  3.0  hz , 4-f - c6h4 ) , 156.13 ( 4-otbdms - c6h4 ) , 159.22 ( d , j  =  243.0  hz , 4-f - c6h4 ) , 162.34 ( d , j  =  245.4  hz , 4-f - c6h4 ) , 166.36 ( co , -lactam ) .
compound 10a ( 24  mg , 0.04  mmol ) was dissolved in 3% hcl in ethanol ( 1.59  ml ) and the mixture was stirred at room temperature overnight .
solvent was evaporated to dryness and crude product purified by a silica gel column chromatography ( hexane / ethyl acetate 1:3 , 5% meoh ) .
product 5 was obtained as light yellow powder ( 13  mg , 84% ) .
[ ]d  =  18 ( c  =  1 , etoac ) .
ft - ir ( kbr ) cm : 3448 , 1735 , 1617 , 1509 , 1389 , 1225 , 1102 , 833 .
h nmr ( 300  mhz , dmso - d6 ) : 2.752.77 ( m , 2h , ch(oh)ch2nh ) , 2.95 ( bs , 1h , ch(oh)ch2nh ) , 3.97 ( bs , 1h , c4 -lactam ) 4.584.63 ( m , 1h , ch(oh)ch2nh ) , 4.84 ( d , 1h , j  =  2.0  hz , c3 -lactam ) , 5.33 ( d , 1h , j  =  4.4  hz , ch(oh)ch2nh ) , 6.74 ( d , 2h , j  =  8.5  hz , ar 
oh ) ; c nmr ( 75  mhz , dmso - d6 ) : 54.55 ( ch(oh)ch2nh ) , 62.34 ( c4 -lactam ) , 71.45 ( ch(oh)ch2nh ) , 75.47 ( c3 -lactam ) , 114.60 ( d , j  =  21.0  hz , 4-f - c6h4 ) , 115.66 ( 4-oh - c6h4 ) , 115.80 ( d , j  =  21.7  hz , 4-f - c6h4 ) , 118.65 ( d , j  =  8.0  hz , 4-f - c6h4 ) , 127.49 ( 4-oh - c6h4 ) , 127.52 ( 4-oh - c6h4 ) , 127.84 ( d , j  =  8.1  hz , 4-f - c6h4 ) , 133.87 ( d , j  =  2.3  hz , 4-f - c6h4 ) , 140.40 ( d , j  =  2.7  hz , 4-f - c6h4 ) , 157.31 ( 4-oh - c6h4 ) , 158.08 ( d , j  =  240.7  hz , 4-f - c6h4 ) , 161.17 ( d , j  =  241.6  hz , 4-f - c6h4 ) , 167.10 ( co , -lactam ) .
stock solution of amino alcohols 5 and 6 ( 160  l , 2  mg / ml methanol ) was added to nacl solution in water ( 20  ml , 0.1  mol / l ) , thus resulting in a solution of each amino alcohol 5 and 6 in concentration of 4    10 
furthermore , solutions of naoh ( 0.1  mol / l , 1  mol / l and 2 
mol / l ) were prepared in nacl solution in water ( 100  ml , 0.1  mol / l ) .
naoh solutions ( 1.520  l ) were added to each solution of amino alcohol 5 or 6 , ph values measured and uv spectra (  200350  nm ) recorded at ph 612 . the titrations were performed at rt ( 20  c ) and inflection points of sigmoid curves at  247  nm were calculated as pka values for both amino alcohols .
darby canine kidney ii ( mdckii ) cells , mdckii cells stably expressing human npc1l1 ( hnpc1l1/mdckii ) , and human hepg2 liver cells were maintained in dmem ( dulbecco 's modified eagle medium , invitrogen , usa ) containing 100 units / ml penicillin and 100  g / ml streptomycin supplemented with 10% fcs ( sigma  aldrich , germany ) .
stable transfection of mdckii cells with human npc1l1 protein was performed using lipofectamine ltx ( invitrogen , usa ) according to the manufacturer 's protocol .
selection with 1  mg / ml g418 ( sigma  aldrich , germany ) was started 2 days after transfection .
the concentration of g418 was decreased to 500  g / ml after 14 days .
stably transfected cells were maintained in dmem containing 100 units / ml penicillin and 100  g / ml streptomycin supplemented with 10% fcs and 500  g / ml g418 .
mdckii , hnpc1l1/mdckii , and hepg2 cells were seeded in 96-well microtiter plates on day 0 at densities of 3000 cells / well . on day 1 ,
tested compounds ( 73  mm stock solution in dmso ) were added in five consecutive 10-fold dilutions ( 10 to 10  mol / l ) and incubated for 72  h. cytotoxicity of the compounds was assessed on day 4 by the mtt cell proliferation assay ( sigma  aldrich , germany ) which detects mitochondrial dehydrogenase activity in viable cells .
the results are expressed as lc50 and the values for each compound were calculated from dose  response curves using linear regression analysis .
toxicity of the compounds in combination with micelles was also tested using the same method .
mdckii and hnpc1l1/mdckii were seeded in 96-well microtiter plates ( 150  l medium / well ) at a density of 2    10  cells / ml medium 24  h before the experiment .
micelles were prepared according to the modified method reported by field et  al . .
in brief , 0.25  mm oleic acid , 50  m cholesterol , 10  m compactin , 50  m mevalonate , 5  mm na - taurocholate ( sigma  aldrich , germany ) in dmem/10% fcs were mixed and sonicated .
following the micellar formation , compounds 1 , 5 , 6 , and 5/6 ( 70:30 ) ( 73  mm stock solution in dmso ) were added to the medium and vortexed to obtain the final concentrations of the compounds ( 25 , 50 , 100 , 150 , 200  m ) and applied to the cells for 1  h. thereafter , cytotoxicity was determined using the mtt assay .
medium a contained dmem plus 100 units / ml penicillin and 100  g / ml streptomycin supplemented with 5% lipoprotein - deficient serum ( density  >  1.21  g / ml , prepared from fcs by ultracentrifugation ) .
cholesterol replenishing medium ( medium b ) was prepared according to the modified method reported by field et  al . .
in brief , 0.25  mm oleic acid , 50  m free cholesterol , 10  m compactin , 50  m mevalonate , 5  mm na - taurocholate ( sigma  aldrich , germany ) , and [ h]cholesterol ( 0.18  ci / ml medium , [ 1,23h(n ) ] cholesterol , 1  mci / ml ( arc inc . , usa ) ) in medium a were mixed and sonicated . following the micellar formation , compounds 1 , 5 , 6 , 5/6 ( 70:30 ) , and 6/5 ( 85:15 ) ( 73  mm stock solution in dmso ) were added to medium b and vortexed to obtain the final concentrations ( 10 , 30 , 60 , 90 , 120 , 150 , 200  m ) .
mdckii wildtype and hnpc1l1/mdckii cells were seeded in 24-well plates at a density of 5    10 cells / ml medium ( 500  l medium / well ) in medium a 24  h before the treatment .
cells were washed once with pbs and incubated in medium b ( 500  l medium / well ) containing micelles and tested compounds in indicated concentrations for 1  h. following the incubation , the medium was removed and cells were washed three times with ice - cold 0.2% free fatty acid - free bsa .
cells were lysed and radioactivity in the lysate ( 100  l ) was determined by liquid scintillation counting .
protein concentrations of the lysates were determined with the dc protein assay ( bio - rad , usa ) .
the results were calculated as cpm / mg protein and expressed as percentage of inhibition compared to untreated cells .
male c57bl/6 mice aged 2528 weeks were maintained under a 12  h light/12  h dark cycle in a temperature - controlled environment with free access to chow diet ( ssniff , germany ) and water . for 2 days , mice ( n  =  35 ) were fasted for 4  h and gavaged with corn oil ( 100  l ) ( vehicle ) containing ezetimibe 1 ( 10  mg / kg / day ) or compounds 5 , 6 , and 5/6 ( 70:30 ) ( 10 or 20  mg / kg / day ) . on day 2 , 90  min post vehicle or compound treatment , mice were gavaged with corn oil ( 200  l ) containing [ h]cholesterol ( 2  ci ) ( [ 1,23h(n ) ] cholesterol , 1  mci / ml , arc inc . , st .
plasma , liver , and small intestines were collected 4  h post - gavage .
the results are presented as mean    s.d . or mean    s.e.m .
statistical analyses were performed using unpaired two - tailed t - test or one - way analysis of variance ( anova ) followed by dunnett 's test for multiple comparisons using graphpad prism 5.0 software ( san diego , ca , usa ) .
 two new trans-(3r,4r)-amino--lactam derivatives and their diastereoisomeric mixtures were synthesized as ezetimibe bioisosteres and tested in in  vitro and in  vivo experiments as novel -lactam cholesterol absorption inhibitors . both compounds exhibited low cytotoxicity in mdckii , hnpc1l1/mdckii , and hepg2 cell lines and potent inhibitory effect in hnpc1l1/mdckii cells . in addition , these compounds markedly reduced cholesterol absorption in mice , resulting in reduced cholesterol concentrations in plasma , liver , and intestine . 
 we determined the crystal structure of one amino--lactam derivative to establish unambiguously both the absolute and relative configuration at the new stereogenic centre c17 , which was assigned to be s. the pka values for both compounds are 9.35 , implying that the amino--lactam derivatives and their diastereoisomeric mixtures are in form of ammonium salt in blood and the intestine . 
 the ic50 value for the diastereoisomeric mixture is 60  m . in  vivo 
 , it efficiently inhibited cholesterol absorption comparable to ezetimibe . 
Introduction
Results and discussion
Conclusion
Experimental protocols