down syndrome ( ds ) 
 is one of the most frequent congenital disorders 
 in humans . many of them develop symptoms 
 of alzheimer s disease ( ad ) at a relatively young age , and 
 a high proportion of ds individuals develop dementia at a later stage . ds is caused by a trisomy of chromosome 21 as a consequence of nondisjunction 
 during meiotic cell division . although the mechanisms 
 leading from trisomy 21 to the clinical disease pattern are not yet 
 understood , the 30 genes located in the so - called down syndrome critical 
 region ( dscr ; 21q22.122.3 ) of chromosome 21 are highly likely 
 to be associated with the disease . there is mounting evidence 
 that overexpression of dyrk1a ( dual - specificity tyrosine phosphorylation - regulated 
 kinase 1a ) , a protein kinase encoded by a gene located within dscr , 
 contributes to mental retardation in ds . since dyrk1a is also connected 
 to neurological disorders such as ad , the early onset 
 of ad in ds individuals has been related to dyrk1a overexpression . two histopathological features are found in the 
 brains of ad patients : extracellular -amyloid plaques and intracellular 
 tangles consisting of hyperphosphorylated tau protein . hyperphosphorylation of tau by dyrk1a diminishes 
 its microtubule - stabilizing effects and increases aggregation . the neurological impairment of mice with modified 
 dyrk1a expression has also been attributed to deregulated splicing , 
 leading to an imbalance of 3r - tau and 4r - tau isoforms . the increased 3r - tau concentration ( by a factor of up to 4 ) observed 
 in ds has been associated with changes of the neuronal cytoskeleton 
 and neurofibrillary degeneration . dyrk1a - overexpressing 
 mice also exhibit increased production of a peptide , which 
 has been attributed to phosphorylation of the amyloid precursor protein 
 ( app ) and presenilin 1 by dyrk1a . dyrk1a 
 is a member of the dyrk protein kinases ( dyrk1a , dyrk1b , 
 dyrk2 , dyrk3 , dyrk4 ) , which are part of the cmgc superfamily and share 
 structural similarity of the catalytic domain and a small sequence 
 nearby , the so - called dyrk homology box ( dh - box ) . although dyrks are serine / threonine kinases , autophosphorylation 
 occurs at a conserved tyrosine residue of the activation loop . this 
 autophosphorylation is constitutive and seems to be not related to 
 regulation . in the light of its involvement 
 in ds and other neurodegenerative disorders , the inhibition of dyrk1a 
 with small chemical inhibitors has been suggested as a therapeutic 
 strategy . selective dyrk1a inhibitors would also be 
 valuable tools for the investigation of the role of dyrk1a in physiological 
 and pathobiochemical processes . to date , most protein kinase inhibitors 
 exert their activity by competing with atp in the binding pocket of 
 the kinase . since atp binding sites of protein kinases share a common 
 structure with subtle differences , selectivity with regard to closely 
 related kinases is not easily achieved . when selective 
 dyrk1a inhibitors are to be developed , special attention should therefore 
 be devoted to other members of the cmgc superfamily , consisting of 
 cdks , mapkinases ( such as erks ) , gsk-3 , and cdc - like kinases . the structures and properties 
 of dyrk1a inhibitors have been summarized 
 in recent reviews . a well established dyrk1a inhibitor 
 is the -carboline alkaloid harmine ( 1 , chart 1 ) , which however also shows high affinity for serotonine 
 and tryptamine receptor binding sites , acts as monoamine oxidase a ( mao a ) inhibitor , and also inhibits clks and 
 therefore is inappropriate for use as a cellular chemical probe . epigallocatechin 
 3-gallate ( egcg ) is a polyphenolic constituent of green tea reported 
 to inhibit dyrk1a in a non - atp - competitive manner , but it is chemically reactive and also interacts with numerous intracellular 
 signaling pathways by other mechanisms . the benzothiazole derivative indy showed potent inhibition of dyrk1a 
 and related kinases ( dyrk1b , dyrk2 , dyrk3 , clk1 , clk4 , ck1 , and pim1 ) . a prodrug of this compound , proindy ( 2 ) , has been shown 
 to protect xenopus tadpoles that overexpress dyrk1a 
 against head malformation during development . leucettine l41 ( 3 ) , derived from the marine natural 
 product leucettamine b , is an atp - competitive inhibitor of dyrks and 
 clks that also interacts with gsk-3 and ck2 to a lower extent . it 
 modulates pre - mrna splicing , protects ht22 hippocampal cells from 
 glutamate - induced cell death , induces autophagy , and inhibits phosphorylation 
 of tau on thr212 . similar to the agents mentioned before , the recently 
 reported chemical dyrk1a inhibitors comprising meridianines , indirubin 5-carboxylates , thiazolo[5,4-f]quinazolines , pyrido[2,3-d]pyrimidines , 3,5-diaryl-7-azaindoles ( dandys ) , kh - cb19 , 2,4-bisubstituted 
 thiophenes , and hydroxybenzothiophenes either show limited selectivity against structurally 
 closely related dyrk and clk kinase isoforms or were not tested on 
 these enzymes . in this regard , a dyrk1a inhibitor with high selectivity 
 over other cmgc kinases would be useful for biochemical and cellular 
 studies and as lead motif for the development of new pharmaceuticals 
 targeting neurodegenerative diseases . to identify novel hit matter , 
 we tested a small diverse in - house compound library against dyrk1a 
 and the following cmgc protein kinases : cdk1/cyclin b , cdk2/cylin 
 a , cdk5/p25 , ck1 , gsk-3 , and erk2 . the only compound showing a moderate 
 selective dyrk1a inhibition ( ic50 = 2.6 m ) was the 
 11h - indolo[3,2-c]quinoline-6-carboxylic 
 acid 5a ( table 1 ) . experimental dyrk1a inhibitors 
 described so far have suffered from 
 poor selectivity toward other closely related protein kinases , namely , 
 other dyrks and members of the clk family . we have developed two 10-iodo-11h - indolo[3,2-c]quinoline-6-carboxylic acids 5j and 5o as the first selective inhibitors of 
 dyrk1a . docking studies and x - ray structure analyses revealed 
 a nonclassical binding mode in which inhibitors were oriented via 
 the 10-halogen substituent toward the hinge region of the kinase . although the 10-substituent obviously was of central importance for 
 the hinge  inhibitor attraction , the nature of this interaction 
 is not yet clear . the title compounds demonstrate that within the 
 cmgc family of protein kinases selective inhibition of dyrk1a is possible . 
 with the aim of developing compounds that are better suited for cellular 
 assays or animal disease models , further studies in this compound 
 class will be directed to increase solubility and cell permeability . starting materials were purchased 
 from acros organics ( geel , belgium ) or sigma - aldrich ( steinheim , germany ) . solvents were used as commercially available grades for synthesis , 
 with the exceptions of toluene , thf , ch2cl2 , 
 and diethyl ether , which were dried and purified by published methods.5b , 5c , and 5f were prepared as reported previously . synthetic procedures and structure characterization data 
 for the following compounds are available in the supporting information : 5a , 5d  e , 5g n , 5p  w , 6 , 7 , 10n , s , t . melting 
 points ( mp ) were determined on an electric variable heater ( electrothermal 
 ia 9100 , barnstedt international , southend - on - sea , u.k . ) in open glass 
 capillaries . ir spectra were recorded as kbr discs on a thermo nicolet 
 ft - ir 200 . h nmr spectra and c nmr spectra 
 were recorded on the following instruments : bruker avance drx-400 , 
 bruker avance iii-400 , and bruker avance ii-600 ( bruker , billerica , 
 ma , usa ) ; internal standard tetramethylsilane ; signals in ppm (  
 scale ) . signals in c spectra were assigned based on the 
 results of c dept135 experiments ( nmr laboratories of 
 the chemical institutes of the technische universitt braunschweig ) . elemental analyses were determined on a ce instruments flashea 1112 
 elemental analyzer ( thermo quest ) . mass spectra were recorded on a 
 finnigan - mat 95 ( thermo finnigan mat , bremen , germany ) . accurate measurements 
 were conducted according to the peak match method using perfluorokerosene 
 ( pfk ) as an internal mass reference . ( ei ) ms : ionization energy 70 
 ev ( department of mass spectrometry of the chemical institutes of 
 the technische universitt braunschweig ) . tlc : polygram sil 
 g / uv254 , macherey - nagel , 40 mm  80 mm , visualization 
 by uv illumination ( 254 and 366 nm ) . isocratic elution : elite 
 lachrom ( merck / hitachi ) , pump l-2130 , autosampler l-2200 , diode array 
 detector l-2450 , organizer box l-2000 ; column , merck lichrocart 1254 , 
 lichrosphere 100 , rp 18 , 5 m ; flow rate 1.000 ml / min ; volume 
 of injection , 10 l ; detection ( dad ) at 254 and 280 nm ; auc , 
 % method ; time of detection 15 min , net retention time ( tn ) , dead time ( tm ) related 
 to dmso . gradient elution : elite lachrom ( merck / hitachi ) , pump l-2130 , 
 autosampler l-2200 , uv detector l-2400 , organizer box l-2000 ; column , 
 merck lichrocart 125 - 4 , lichrosphere 100 , rp 18 , 5 m ; flow 
 rate 1.000 ml / min ; volume of injection , 10 l ; detection at 
 254 nm ; auc , % method ; net retention time ( tn ) , dead time ( tm ) related to dmso . 
 for all gradient runs , mixtures of acn and water or aqueous formic 
 or trifluoroacetic acid were used as specified for particular compounds . preparation of h2o + ( et3nh)2so4 buffer ( ph 2.6 ) for isocratic hplc : triethylamine ( 20.0 ml ) 
 and sodium hydroxide ( 242 mg ) are dissolved in water to 1 l. the solution 
 is adjusted to ph 2.6 by addition of sulfuric acid . all compounds 
 that were biologically tested were of > 95% purity with the exception 
 of 5h ( hplc , 93.6% at 254 nm and 94.8% at 280 nm detection 
 wavelength ) and 5w ( hplc , 95.1% at 254 nm and 94.2% at 
 280 nm detection wavelength ) . absorption maxima ( max ) were extracted from the spectra recorded by the dad in the hplc 
 peak maxima in isocratic runs ( software , ez chrom elite client / server , 
 version 3.1.3 . ) . an appropriate substituted 3,4-dihydro-1h-1-benzazepine-2,5-dione 
 ( 1 equiv ) and an appropriately substituted phenylhydrazine hydrochloride 
 ( 1.21.5 equiv ) and sodium acetate ( 1.21.5 equiv ) are 
 stirred in acetic acid ( 10 ml ) at 70 c for the indicated time . sulfuric acid ( 0.1 ml ) is added , and stirring at 70 c is continued 
 for the indicated time . after cooling to room temperature , the mixture 
 is poured into 5% aqueous sodium acetate solution ( 20 ml ) and kept 
 at 8 c for 2 h. a precipitate is formed , which is filtered off 
 with suction , washed with water and petrol ether . the appropriate 
 paullone 4 ( 1 equiv ) , nhpi 
 ( 2 equiv ) , and cobalt(ii ) acetate tetrahydrate ( 0.25 equiv ) are dissolved 
 in the given volume of dmf . oxygen is bubbled through the stirred 
 mixture for the given time at the indicated temperature . if no precipitate appears , water ( 5 ml ) is added and the resulting 
 precipitate is separated and purified by centrifugation , decanting , 
 and washing with water . the resulting solid is washed with a small 
 amount of acetone . the predried product is finally dried at 130 c 
 in vacuo . ethanol ( 100 ml ) is added to a solution of an appropriate 11h - indolo[3,2-c]quinoline-6-carboxylic acid 5 in dmso ( 5 ml ) . hydrogen chloride gas is bubbled through 
 the solution for 30 min at room temperature . after removal of the 
 ethanol by evaporation , water ( 100 ml ) is added and the mixture is 
 kept at 8 c for 1 h. the resulting precipitate is filtered off , 
 washed with water , and dissolved in acetone ( 5 ml ) . after addition 
 of silica gel ( 2 g ) , the mixture is evaporated . the product is subsequently 
 eluted from the silica gel by flash chromatography using the indicated 
 eluent . synthesis was according 
 to general procedure a from 3,4-dihydro-1h-1-benzazepine-2,5-dione ( 88 mg , 0.50 mmol ) , 2-iodophenylhydrazine hydrochloride ( 203 mg , 0.750 mmol ) , and sodium acetate ( 62 
 mg , 0.75 mmol ) . reaction times were 1 h before and 4 h after addition 
 of sulfuric acid , respectively . formic acid 5:5:1 ) yielded a 
 dark yellow powder ( 51 mg , 28% ) ; mp ( dec ) starting at 288 c . ir ( kbr ) : 3194 cm ( nh ) , 1666 cm ( c = o ) . h nmr ( dmso - d6 , 400.4 mhz ) :  ( ppm ) = 3.47 ( s , 2h ) , 6.90 ( t , 1h , j = 7.6 hz ) , 7.227.31 ( m , 2h ) , 7.40 ( ddd , 1h , j = 7.9/7.4/1.6 hz ) , 7.59 ( dd , 1h , j = 
 7.5/0.9 hz ) , 7.71 ( d , 1h , j = 7.9 hz ) , 7.91 ( dd , 
 1h , j = 7.8/1.5 hz ) , 10.11 ( s , 1h , lactam nh ) , 11.29 
 ( s , 1h , indole nh ) . c nmr ( dmso - d6 , 100.7 mhz ) :  ( ppm ) = 31.6 ( ch2 ) ; 118.0 , 
 121.1 , 122.1 , 123.3 , 128.3 , 128.5 , 131.5 ( ch ) ; 76.7 , 109.4 , 122.4 , 
 127.1 , 133.6 , 135.6 , 139.4 ( c ) ; 171.7 ( c = o ) . c16h11in2o ( 374.18 ) . ms ( ei ) : m / z ( % ) = 374 [ m ] ( 100 ) , 373 
 [ m  h ] ( 35 ) , 345 [ m  cho ] ( 49 ) . 
 hrms ( ei ) : m / z [ m ] calcd 373.991  06 , found 373.990  84 . hplc ( isocr ) : 
 96.1% at 254 nm and 92.9% at 280 nm , tn = 4.7 min , tm = 1.0 min ( acn / h2o 50:50 ) ; max : 230 and 313 nm . synthesis was according 
 to general procedure a from 8-methoxy-3,4-dihydro-1h-1-benzazepine-2,5-dione ( 500 mg , 2.44 mmol ) , 
 2-iodophenylhydrazine hydrochloride ( 993 
 mg , 3.67 mmol ) , and sodium acetate ( 301 mg , 3.67 mmol ) . reaction times 
 were 3 h before and 16 h after addition of sulfuric acid , respectively . purification by column chromatography ( toluene  ethyl acetate  formic 
 acid 5:5:1 ) yielded a dark yellow powder ( 340 mg , 34% ) ; mp ( dec ) starting 
 at 290 c . ir ( kbr ) : 3205 cm ( nh ) , 1680 cm ( c = o ) . h nmr ( dmso - d6 , 600.1 mhz ) :  ( ppm ) = 3.47 ( s , 2h ) , 3.81 ( s , 
 3h ) , 6.82 ( d , 1h , j = 2.6 hz , ) , 6.88 ( t , 1h , j = 7.7 hz ) , 6.91 ( dd , 1h , j = 8.7/2.6 
 hz ) , 7.55 ( dd , 1h , j = 7.5/0.9 hz ) , 7.67 ( d , 1h , j = 7.8 hz ) , 7.85 ( d , 1h , j = 8.7 hz ) , 
 10.04 ( s , 1h , lactam nh ) , 11.19 ( s , 1h , indole nh ) . c 
 nmr ( dmso - d6 , 150.9 mhz ) :  ( ppm ) 
 = 55.2 ( ch3 ) , 31.6 ( ch2 ) , 106.6 , 109.9 , 117.6 , 
 121.0 , 129.6 , 130.9 ( ch ) , 76.5 ( c(11 ) ) , 107.6 , 115.3 , 127.1 , 133.8 , 
 136.9 , 139.0 , 159.2 ( c ) . c 50.51 , h 3.24 , n 6.93 , 
 found c 50.38 , h 3.29 , n 6.72 . ms ( ei ) : m / z ( % ) = 404 [ m ] ( 100 ) , 403 [ m  
 h ] ( 36 ) , 375 [ m  cho ] ( 42 ) . hrms ( ei ) : m / z [ m ] calcd 404.001  62 , 
 found 404.001  61 . hplc ( isocr ) : 96.0% at 254 nm and 95.2% at 
 280 nm , tn = 5.2 min , tm = 1.0 min ( acn / h2o 50:50 ) . synthesis was according to general 
 procedure b1 from 11-iodo-7,12-dihydroindolo[3,2-d]benzazepin-6(5h)-one ( 4j , 43 mg , 
 0.11 mmol ) , nhpi ( 36 mg , 0.22 mmol ) , and cobalt(ii ) acetate tetrahydrate 
 ( 7 mg , 0.03 mmol ) in dmf ( 2 ml ) . oxygen was bubbled through the reaction 
 mixture at room temperature for 4 h. a yellow powder was formed , which 
 was purified by washing with acetone ( 34 mg , 82% ) ; mp ( dec ) 358359 
 c . ir ( kbr ) : 3224 cm ( nh ) , 1659 cm ( c = o ) . h nmr ( dmso - d6 , 400.4 mhz ) :  ( ppm ) = 7.10 ( t , 1h , j = 7.7 hz ) , 7.357.42 ( m , 1h ) , 7.567.62 
 ( m , 2h ) , 7.82 ( dd , 1h , j = 7.7/1.0 hz ) , 8.30 ( dd , 
 1h , j = 8.0/1.0 hz ) , 8.38 ( d , 1h , j = 8.0 hz ) , 11.54 ( s , 1h , nh ) , 11.95 ( s , 1h , cooh ) . c nmr ( dmso - d6 , 100.7 mhz ) :  ( ppm ) 
 = 121.2 ( 2c ) , 123.4 , 124.4 , 129.2 , 131.1 , 134.8 ( ch ) ; 76.7 ( c(10 ) ) , 
 115.5 , 117.2 , 127.3 , 135.2 , 139.3 , 142.5 , 161.8 ( c ) ; 176.3 ( c = o ) . ms 
 ( ei ) : m / z ( % ) = 388 [ m ] ( 37 ) , 360 [ m  co ] ( 100 ) . hrms ( ei ) : m / z [ m ] calcd 387.970  32 , 
 found 387.968  99 . hplc ( isocr ) : 96.7% at 254 nm and 98.6% at 
 280 nm , tn = 2.8 min , tm = 1.0 min ( acn / h2o + ( et3n)2so4 50:50 ) . hplc ( grad ) : 97.4% at 254 nm , tn = 10.0 min , tm = 1.1 min ( acn / h2o ; 0 min , 10/90  13 min , 90/10 ( linear ) ; 20 min , 90/10 ) . synthesis 
 was according to general procedure b1 from 11-iodo-3-methoxy-7,12-dihydroindolo[3,2-d]benzazepin-6(5h)-one ( 4o , 40 mg , 0.10 mmol ) , nhpi ( 33 mg , 0.20 mmol ) , and cobalt(ii ) acetate 
 tetrahydrate ( 7 mg , 0.03 mmol ) in dmf ( 2 ml ) . oxygen was bubbled through 
 the reaction mixture for 1 h. for the purification by column chromatography , 
 impurities were removed initially by elution with toluene  ethyl 
 acetate  diethylamine ( 1:1:1 ) prior to elution by toluene  ethyl 
 acetate yellow powder ( 20 mg , 48% ) ; mp ( dec ) 
 346348 c . ir ( kbr ) : 3200 cm ( nh ) , 
 1669 cm ( c = o ) . h nmr ( dmso - d6 , 600.1 mhz ) :  ( ppm ) = 3.86 ( s , 3h ) , 
 7.01 ( dd , 1h , j = 9.0/2.6 hz ) , 7.07 ( t , 1h , j = 7.7 hz ) , 7.17 ( d , 1h , j = 2.5 hz ) , 
 7.78 ( dd , 1h , j = 7.5/0.9 hz ) , 8.31 ( dd , 1h , j = 7.9/0.7 hz ) , 8.36 ( d , 1h , j = 9.0 hz ) , 
 11.44 ( s , 1h , nh ) , 11.76 ( s , 1h , cooh ) . c nmr ( dmso - d6 , 150.9 mhz ) :  ( ppm ) = 55.5 ( ch3 ) ; 104.9 , 110.6 , 121.1 , 124.2 , 130.8 , 134.4 ( ch2 ) ; 76.3 , 110.3 , 114.2 , 127.5 , 137.0 , 139.1 , 143.0 , 161.1 , 161.5 ( c ) ; 
 175.5 ( c = o ) . ms ( ei ) : m / z ( % ) 
 = 418 [ m ] ( 47 ) , 390 [ m  co ] ( 100 ) , 375 [ m  43 ] ( 24 ) , 347 [ m  71 ] ( 22 ) . hrms ( ei ) : m / z [ m ] calcd 417.980  89 , found 417.981  26 . hplc ( isocr ) : 96.3% at 254 nm and 97.7% at 280 nm , tn = 3.2 min , tm = 1.0 min 
 ( acn / h2o + ( et3n)2so4 50:50 ) . hplc ( grad ) : 99.4% at 
 254 nm , tn = 10.8 min , tm = 1.2 min ( gradient , acn / h2o ; 0 min , 10/90 
  13 min , 90/10 ( linear ) ; 20 min , 90/10 ) . synthesis 
 was according to general procedure c from 10-iodo-3-methoxy-11h - indolo[3,2-c]quinoline-6-carboxylic acid 
 ( 5o , 120 mg , 0.300 mmol ) . diethylamine 2:2:1 ) yielded pale 
 yellow crystals ( 100 mg , 75% ) ; mp 214217 c . ir ( kbr ) : 
 3384 ( nh ) , 1726 cm ( c = o ) . h 
 nmr ( dmso - d6 , 400.4 mhz ) :  ( ppm ) 
 = 1.45 ( t , 3h , j = 7.1 hz ) , 3.99 ( s , 3h ) , 4.59 ( q , 
 2h , j = 7.1 hz ) , 7.14 ( t , 1h , j = 
 7.8 hz ) , 7.46 ( dd , 1h , j = 9.1/2.6 hz ) , 7.63 ( d , 
 1h , j = 2.6 hz ) , 7.93 ( dd , 1h , j = 7.6/1.0 hz ) , 8.33 ( dd , 1h , j = 8.1/0.9 hz ) , 9.05 
 ( d , 1h , j = 9.1 hz ) , 12.34 ( s , 1h , nh ) . c nmr ( dmso - d6 , 100.7 mhz ) :  ( ppm ) 
 = 14.2 ( ch2ch3 ) ; 55.5 ( och3 ) ; 61.7 ( ch2 ) ; 108.7 , 119.1 , 122.0 , 122.5 , 124.7 , 135.1 ( ch ) ; 76.8 , 111.2 , 111.8 , 
 121.4 , 141.1 , 142.0 , 145.1 146.0 , 160.1 ( c ) ; 166.7 ( c = o ) . c19h15in2o3 ( 446.24 ) ; calcd 
 c 51.14 , h 3.39 , n 6.28 , found c 51.21 , h 3.41 , n 6.01 . ms ( ei ) : m / z ( % ) = 446 [ m ] ( 22 ) , 
 374 [ m  c3h4o2 ] ( 100 ) . hplc ( isocr ) : 99.7% at 254 nm and 99.8% at 280 nm , tn = 7.6 min , tm = 
 1.0 min ( acn / h2o 80:20 ) . hplc ( grad ) : 99.7% at 254 nm , tn = 14.1 min , tm = 1.1 min ( gradient , 
 acn / h2o ; 0 min , 10/90  13 min , 90/10 ( linear ) ; 
 20 min , 90/10 ) . crystals for x - ray structure analysis were prepared 
 by crystallization from ethanol ( 96% ) . crystal data for 10oh2o at 100 k : orthorhombic , p212121 , a = 6.36362(12 ) , b = 15.0401(3 ) , c = 18.3209(4 )  , v = 1753.48  , z = 4 . a yellow irregular crystal 0.3 mm  
 0.2 mm  0.15 mm was used to record 76097 intensities , 5096 independent 
 ( rint = 0.036 ) on an oxford diffraction 
 xcalibur e diffractometer using mo k radiation (  = 0.710  73 
  , 2max = 60 ) . the structure was refined 
 anisotropically on f using the program 
 shelxl-97 to wr2 = 0.049 , r1 = 0.020 for 249 parameters ; s = 1.12 , max . crystallographic data have been deposited with the cambridge crystallographic 
 data centre as supplementary publication no . buffer a consisted of 10 mm mgcl2 , 1 mm egta , 1 mm dtt , 
 25 mm tris - hcl , ph 7.5 , 50 g heparin / ml . buffer c consisted 
 of 60 mm -glycerophosphate , 15 mm p - nitrophenylphosphate , 
 25 mm mops ( ph 7.2 ) , 15 mm egta , 15 mm mgcl2 , 1 mm dtt , 
 1 mm sodium vanadate , 1 mm phenylphosphate . kinase activities 
 were assayed in buffer a or c at 30 c at a final atp concentration 
 of 15 mol / l . blank values were subtracted , and activities were 
 expressed in percent of the maximal activity , i.e. , in the absence 
 of inhibitors . the gs-1 , cks , cdk7/9 tide , and rs peptide substrates were 
 obtained from proteogenix ( oberhausbergen , france ) . cdk1/cyclin 
 b ( m phase starfish oocytes , native ) , cdk2/cyclin a , 
 and cdk5/p25 ( human , recombinant ) were prepared as previously described . their kinase activity was assayed in buffer 
 a , with 1 mg of histone h1/ml , in the presence of 15 mol / l 
 [ -33p ] atp ( 3000 ci / mmol ; 10 mci / ml ) in a final volume of 30 
 l . after a 30 min incubation at 30 c , the reaction was 
 stopped by harvesting onto p81 phosphocellulose supernatant ( whatman ) 
 using a filtermate harvester ( packard ) and washing in 1% phosphoric 
 acid . cdk9/cyclin t ( human , recombinant , expressed 
 in insect cells ) was 
 assayed as described for cdk1/cyclin b but using cdk7/9 tide ( ysptspsysptspsysptspskkkk ) 
 ( 8.1 g / assay ) as a substrate . gsk-3 ( porcine brain , native , 
 affinity purified on axin1-sepharose 
 beads ) was assayed , as described for cdk1 , with 0.5 mg of bsa / ml + 
 1 mm dtt and using a gsk-3 specific substrate ( gs-1 , yrraavppspslsrhssphqspedeee ) 
 ( ps stands for phosphorylated serine ) . ck1/ ( porcine brain , native , affinity purified 
 on 
 axin2-sepharose beads ) was assayed as described for cdk1 but in buffer 
 c and using 25 m cks peptide ( rrkhaaigpsaysita ) , a ck1-specific 
 substrate . clk1 , -2 , -3 , and -4 
 ( mouse , recombinant , expressed in e. coli as gst 
 fusion proteins ) were assayed as described 
 for cdk1/cyclin b with 0.5 mg bsa / ml + 1 mm dtt and rs peptide ( grsrsrsrsrsr ) 
 ( 1 g / assay ) as a substrate . dyrk1a , -1b , -2 , -3 ( human , 
 recombinant , expressed in e. 
 coli as gst fusion proteins ) and clk1 , -2 , -3 , and -4 ( mouse , 
 recombinant , expressed in e. coli as gst fusion proteins ) 
 were assayed in buffer a ( supplemented extemporaneously with 0.15 
 mg of bsa / ml + 1 mm dtt ) with 1 g of rs peptide ( grsrsrsrsrsr ) 
 as a substrate . response 
 curves 
 were recorded in triplicate . typically , the standard deviation of 
 single data points was below 10% . the assay for 
 inhibition of sf3b1 phosphorylation was performed as described previously . for the assay of tau phosphorylation , we used 
 a hek293 subclone with regulatable expression of gfp - dyrk1a and constitutive 
 expression of gfp - tau ( hek293-tau - dyrk1a ) that was kindly provided 
 by dr . matthias engel ( department of pharmaceutical and medicinal 
 chemistry , saarland university , saarbrcken , germany ) . cells were grown overnight in six - well plates 
 before expression of gfp - dyrk1a was induced with 2 g / ml doxycyclin . the inhibitors were then added from stock solutions in dmso to the 
 desired final concentration and cells were further incubated for 20 
 h. cells were lysed in sds lysis buffer ( 20 mm tris - hcl , ph 7.4 , 1% 
 sds ) . samples were sonicated and cleared by centrifugation before 
 sds  page and immunoblotting with a goat antibody for gfp ( no . 
 600 - 101 - 215 , rockland immunochemicals , gilbertsville , pa , usa ) and 
 a phosphorylation state specific antibody directed against pthr212 
 in the tau protein ( no . immunoreactivities 
 were detected by enhanced chemiluminescence using hrp - coupled secondary 
 antibodies and quantified using the aida image analyzer 5.0 program 
 ( raytest , straubenhardt , germany ) . relative tau phosphorylation was 
 calculated by normalization to total tau expression , as determined 
 from gfp immunoreactivity . to calculate relative dyrk1a activity , the basal pt212 signal in control cells not treated with doxycyclin 
 was subtracted from all values , and the phosphorylation in dyrk1a 
 expressing cells not treated with inhibitors was set to 100% . curve 
 fitting for ic50 determination was done with the help of 
 the graphpad prism 5.0 program ( graphpad software , la jolla , ca , usa ) . recombinant dyrk1a was purified 
 as previously described and was treated 
 with tev protease to remove the n - terminal his6 tag . the 
 kinase at 1315 mg / ml in 50 mm hepes , ph 7.5 , 500 mm 
 nacl and 5 mm dtt was incubated with the inhibitors at 1 mm prior 
 to crystallization . crystals were obtained using the sitting drop 
 vapor diffusion method at 4 c using either 2 m ammonium sulfate , 
 0.2 m na / k tartrate , 0.1 m citrate , ph 5.6 ( for 5s and 5 t ) , or 37% peg 400 , 0.2 m lithium sulfate , 0.1 m tris , ph 
 8.8 ( for 5j ) , as the reservoir solutions . diffraction 
 data collected at diamond light source , beamline i04 - 1 , were processed 
 with xds or mosflm and subsequently scaled with scala from ccp4 suite . structures were determined by molecular replacement 
 method using phaser and the coordinates 
 of dyrk1a structure as a search model . iterative cycles of manual model building in coot alternated with refinement using refmac , and a tls model calculated from tlsmd server was performed . dyrk1a - ligand complexes ( pdb i d ) : dyrk1a-5j ( 4ylj ) ; dyrk1a-5s ( 4ylk ) ; dyrk1a-5 t ( 4yll ) . molecular 
 docking was performed using gold , version 
 4.0 , running under linux ubuntu dapper 
 drake . the poses obtained with gold 4.0 were later reproduced using 
 gold , version 5.1 . the pdb file 2wo6 was downloaded from the protein data 
 bank . only the b chain present in the structure hydrogen atoms were added , and the protonation / tautomerization 
 status of amino acid side chains within the atp binding site was checked 
 and adjusted manually , if necessary . a zone of 10  around the original 
 ligand was defined as relevant for binding . the options flip 
 side chains of asn / gln  and  alter tautomers of his  
 were switched off . ligands were constructed with moe , energy minimized , and saved as mol2 files . the option redocking of the originally bound ligand djm2005 into chain 
 b of the protein reproduced the original pose of the inhibitor , albeit 
 not as highest ranked pose .