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 .
 the protein kinase dyrk1a has been 
 suggested to act as one of the 
 intracellular regulators contributing to neurological alterations 
 found in individuals with down syndrome . for an assessment of the 
 role of dyrk1a , selective synthetic inhibitors are valuable pharmacological 
 tools . 
 however , the dyrk1a inhibitors described in the literature 
 so far either are not sufficiently selective or have not been tested 
 against closely related kinases from the dyrk and the clk protein 
 kinase families . 
 the aim of this study was the identification of dyrk1a 
 inhibitors exhibiting selectivity versus the structurally and functionally 
 closely related dyrk and clk isoforms . 
 structure modification of the 
 screening hit 11h - indolo[3,2-c]quinoline-6-carboxylic 
 acid revealed structure  activity relationships for kinase inhibition 
 and enabled the design of 10-iodo - substituted derivatives as very 
 potent dyrk1a inhibitors with considerable selectivity against clks . 
 
 x - ray structure determination of three 11h - indolo[3,2-c]quinoline-6-carboxylic acids cocrystallized with dyrk1a 
 confirmed the predicted binding mode within the atp binding site . 
Introduction
Conclusion
Experimental Procedures