tumor - associated cell cycle defects , manifesting in unscheduled proliferation , and the associated genomic and chromosomal instabilities are mediated by misregulation of cyclin - dependent kinases ( cdks ) .
because of the main role in the division cycle , cdks have been recognized as targets for anticancer therapy .
many small - molecule organic compounds , which have been identified as cdk modulators , are currently in preclinical or clinical development . however , no cdk inhibitors have gained marketing approval , despite 20 years of scientific investigation .
several studies have shown synergism when cdk inhibitors were combined with organic ( e.g. , doxorubicin , paclitaxel ) and inorganic ( e.g. , cisplatin , carboplatin ) cytotoxic drugs .
the first publications have appeared recently and include fe , cu , and pt complexes with cdk inhibitors derived from 6-benzylaminopurine , metal - based indolo-[3,2-d]benzazepines ( paullones ; ga , cu , ru , and os ) , and indolo-[3,2-c]quinolines ( ru , os ) . another class of compounds potentially suitable for targeted metal - based chemotherapy is that of 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines .
these have been documented recently as potent cdk1 inhibitors with antiproliferative activity in hela ( cervical carcinoma ) , hct116 ( colon carcinoma ) , and a375 ( melanoma ) human cancer cell lines .
comparison of cdk1 inhibitory activity with the inhibiting activity in four other protein kinases ( vegf - r2 , her2 , aurora - a , and ret ) revealed selectivity for cdk1 . structural modifications consisting of a replacement of both bicyclic rings in 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines by monocycles while retaining the imidazolyl pyrazole core have been proposed in order to obtain inhibitors with improved pharmacokinetic and solubility properties .
the most promising were suggested to be 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines ( see chart 1 ) .
the inspection of substitution patterns on the benzimidazole moiety and structure  activity relationships revealed that a methoxymethyl group in position 7b ( 4b ) is favorable for cdk1 inhibiting potency .
the role of the pyrazole nh group is also of note , since its methylation led to a significant reduction of cdk1 activity .
the effect of various heteroaryl groups in position 5a was also remarkable for the development of more - effective cdk inhibitors and antiproliferative agents .
our previous experience with metal - based indolo-[3,2-d]benzazepines prompted the use of the half - sandwich metal - arene moiety as a suitable scaffold to which 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines may be attached .
organometallic compounds [ m(-arene)(yz)x ] ( where m = ru , os ) exhibit promising anticancer activity and are the focus of attention for several groups .
these compounds have shown activity toward classic ( dna ) and nonclassic ( e.g. , cdks ) targets in anticancer chemotherapy .
herein , we report ( i ) the modified synthetic approach to 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines ( recall chart 1 : l1 , x = h , y = h ; l2 , x = br ; y = h ; l3 , x = br ; y = ch2och3 ) ; ( ii ) the synthesis and characterization of a new family of organoruthenium(ii ) ( 11a , 12a , 13a ) and osmium(ii ) ( 11b , 12b , 13b ) complexes of the general formula [ mcl(-p - cymene)l]cl , where l = 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines ( l1l3 ) ( chart 1 ) ; ( iii ) stabilization of the 7b tautomer of methoxymethyl - substituted l3 by metal coordination as well as ( iv ) nmr spectroscopic characterization of two tautomers 7b - l3 and 4b-l3 in a metal - free state ; and ( v ) cell cycle effects , as well as the antiproliferative and cdk inhibitory activities of both metal - free ligands and organometallic complexes .
1h - pyrazolo[3,4-b]pyridine-3-carboxylic acid ( 1 ) was obtained via the oxidation of 3-methyl-1h - pyrazolo[3,4-b]pyridine by kmno4 in the presence of a base , followed by acidification with 37% hcl .
solvents [ toluene , ethanol ( etoh ) , tetrahydrofuran ( thf ) , diethyl ether ( et2o ) ] were dried using standard procedures .
3-methyl-1h - pyrazolo[3,4-b]pyridine ( 10.9 g , 0.08 mol ) and anhydrous sodium acetate ( 10.21 g , 0.13 mol ) were suspended in glacial acetic acid ( 42 ml ) .
bromine ( 20.42 g , 0.13 mol ) was added , and the resulting mixture was stirred at room temperature for 22.5 h and then at 110115 c for 2.53 h. afterward , water ( 300350 ml ) was added and the mixture was stirred at room temperature .
the formed light yellow precipitate was filtered off and dried in vacuo at 4050 c .
yield : 17 g. the raw product was used without further purification in the next step .
purification by column chromatography afforded a white powder ( sio2 , etoac , rf = 0.79 ; 12.5 g , 72.6% yield ) .
esi - ms in meoh ( positive ) : m / z 213 [ m+h ] , 235 [ m+na ] , 253 [ m+k ] ; ( negative ) : m / z 211 [ m  h ] .
h nmr ( 500.32 mhz , meoh - d4 ) :  8.55 ( d , 1h , j = 2.16 hz , ch ) , 8.43 ( d , 1h , j = 2.15 hz , ch ) , 2.56 ( s , 3h , ch3 ) ppm .
h nmr ( 500.32 mhz , dmso - d6 ) :  13.42 ( brs , 1h , nh ) , 8.54 ( d , 1h , j = 2.19 hz , ch ) , 8.53 ( d , 1h , j = 2.18 hz , ch ) , 2.49 ( s , 3h , ch3 ) ppm .
colorless crystals of 20.5h2o suitable for x - ray diffraction ( xrd ) study were grown in etoac ( see figure s1 in the supporting information ) . to sodium hydroxide ( 9.54 g , 0.24 mol ) in water ( 150 ml ) was added the raw product 2 ( 7.1 g , 0.03 mol ) . after a dropwise addition of kmno4 ( 16.98 g , 0.11 mol ) in water ( 300 ml ) at 100 c over 2 h , the reaction mixture was further heated for 1 h. mno2 was filtered off from the hot reaction mixture and washed with hot water .
400 ml , and the yellow solution was acidified to ph 2 , using concentrated hcl .
yield : 2.28 g. the crude hydrochloride of 3 was used without further purification in the next step .
esi - ms in meoh ( positive ) : m / z 243 [ m+h ] , 265 [ m+na ] , 287 [ m+2na  h ] ; ( negative ) : m / z 241 [ m  h ] .
h nmr ( 500.32 mhz , meoh - d4 ) :  8.69 ( d , 1h , j = 2.22 hz , ch ) , 8.68 ( d , 1h , j = 2.22 hz , ch ) ppm .
h nmr ( 500.32 mhz , dmso - d6 ) :  14.65 ( s , 1h , nh ) , 13.45 ( brs , 1h , nh ) , 8.72 ( d , 1h , j = 2.23 hz , ch ) , 8.58 ( d , 1h , j = 2.25 hz , ch ) ppm .
nah ( 1.75 g , 0.07 mol ) was suspended in dry thf ( 200 ml ) . a solution of 2-amino-3-nitrobenzyl alcohol ( 5.23 g , 0.03 mol ) in dry thf ( 100 ml )
was added dropwise at 0 c and the mixture was stirred at the same temperature for 15 min . after a dropwise addition of mei ( 11.5 g , 0.08 mol ) ,
stirring was continued at room temperature for 3 h. a saturated aqueous solution of nahco3 ( 300 ml ) and meoh ( 300 ml ) then were added .
the organic phase was dried over na2so4 , filtered , and evaporated to yield a red oil ( 4.85 g ) .
the raw product was purified by column chromatography ( sio2 , etoac , or etoac / hexane 1/1 , first fraction , a red - orange oil crystallized to form a red solid at 4 c ; yield : 3.68 g , 65% ) .
h nmr ( 500.32 mhz , dmso - d6 ) :  8.00 ( d , 1h , j = 8.71 hz ,
c6h3 ) , 7.09 ( br , 2h , nh2 ) , 6.67 ( t , 1h , j = 8.45 hz , c6h3 ) , 4.48 ( s , 2h , ch2 ) , 3.31 ( s , 3h , ch3 ) ppm .
a mixture of 4 ( 1.4 g , 0.008 mol ) and 10% pd / c ( 0.18 g ) in dry etoh ( 55 ml ) was stirred under hydrogen atmosphere at room temperature for 1824 h. the catalyst was removed by filtration through gf-3-filter under argon and washed with dry etoh ( 5070 ml ) .
the filtrate was evaporated in vacuo to give a light - orange solid ( 1.17 g , 100% yield ) , which was used immediately in the next step .
h nmr ( 500.32 mhz , dmso - d6 ) :  6.52 ( dd , 1h , j = 1.87 hz , j = 7.25 , c6h3 ) , 6.426.36 ( m , 2h , c6h3 ) , 4.48 ( br , 2h , nh2 ) , 4.31 ( br , 4h , nh2+ch2 ) , 3.24 ( s , 3h , ch3 ) ppm .
n , n-carbonyldiimidazole ( cdi , 4.16 g , 0.026 mol ) was added in small portions to 1 ( 2.34 g ) in dry dmf ( 12 ml ) .
the mixture was stirred at room temperature for 2024 h. water ( 5 ml ) then was added and the suspension was stirred until all co2 was ceased .
the white precipitate was filtered off , washed with water ( 35 ml ) , and dried in a sublimator in vacuo at 60 c , to remove the imidazole as a contaminant .
esi - ms in methanol ( positive ) : m / z 214 [ m+h ] , 237 [ m+na ] ; ( negative ) : m / z 212 [ m  h ] .
h nmr ( 500.32 mhz , dmso - d6 ) :  14.95 ( brs , 1h , nh ) , 8.91 ( s , 1h , chim ) , 8.74 ( dd , 1h , j = 1.61 hz , j = 4.51 hz , chpy ) , 8.63 ( dd , 1h , j = 1.59 hz , j = 8.12 hz , chpy ) , 8.14 ( t , 1h , j = 1.23 hz , chim ) , 7.51 ( dd , 1h , j = 4.46 hz , j = 8.06 hz , chpy ) , 7.20 ( s , 1h , chim ) ppm .
n , n-carbonyldiimidazole ( cdi , 16.2 g , 0.1 mol ) was added in small portions to crude 3 ( 11.58 g ) in dry dmf ( 70 ml ) .
the mixture was stirred at room temperature for 2024 h. water ( 10 ml ) then was added and the suspension was stirred until all co2 was ceased .
the white precipitate was filtered off , washed with water ( 1015 ml ) , and dried in a sublimator in vacuo at 60 c , to remove the imidazole as a contaminant .
esi - ms in methanol ( positive ) : m / z 315 [ m+na ] ; ( negative ) : m / z 291 [ m  h ] .
h nmr ( 500.32 mhz , dmso - d6 ) :  14.95 ( brs , 1h , nh ) , 8.89 ( s , 1h , chim ) , 8.83 ( d , 1h , j = 2.12 hz , chpy ) , 8.75 ( d , 1h , j = 2.07 hz , chpy ) , 8.13 ( s , 1h , chim ) , 7.20 ( s , 1h , chim ) ppm .
the solution of 1,2-diaminobenzene ( 0.5 g , 4.67 mmol ) in dry dmf ( 2 ml ) was added to the suspension of 6 ( 0.94 g , 4.41 mmol ) in dry dmf ( 13 ml ) , and this reaction mixture was heated under argon at 85 c for 5 h. dmf then was evaporated in vacuo at 50 c and water ( 1012 ml ) was added .
the light - yellow precipitate was filtered off and dried in vacuo at 50 c .
yield : 0.86 g. the raw product was used without purification in the next step .
esi - ms in methanol ( positive ) : m / z 255 [ m+h ] , 277 [ m+na ] ; ( negative ) : m / z 253 [ m  h ] .
h nmr ( 500.32 mhz , dmso - d6 ) :  14.30 ( brs , 1h , nhpz ) , 9.73 ( brs , 1h , conh ) , 8.64 ( dd , 1h , j = 1.59 hz , j = 4.44 hz , chpy ) , 8.56 ( dd , 1h , j = 1.53 hz , j = 8.02 hz , chpy ) , 7.397.36 ( m , 2h , chpy+chbz ) , 6.97 ( td , 1h , j = 1.31 hz , j = 7.73 hz , chbz ) , 6.82 ( dd , 1h , j = 1.2 hz , j = 7.89 hz , chbz ) , 6.64 ( td , 1h , j = 1.2 hz , j = 7.67 hz , chbz ) , 4.93 ( s , 2h , nh2 ) ppm .
( 10 ml ) was added to the suspension of 7 ( 2.28 g , 7.8 mmol ) in dry dmf ( 10 ml ) , and this mixture was heated under argon at 85 c for 7 h. dmf then was evaporated in vacuo at 50 c and water ( 20 ml ) was added .
yield : 2.2 g. the raw product was used without purification in the next step .
esi - ms in methanol ( positive ) : m / z 333 [ m+h ] , 355 [ m+na ] ; ( negative ) : m / z 331 [ m  h ] .
h nmr ( 500.32 mhz , dmso - d6 ) :  14.54 ( brs , 1h , nhpz ) , 9.80 ( brs , 1h , conh ) , 8.73 ( d , 1h , j = 2.26 hz , chpy ) , 8.69 ( d , 1h , j = 2.22 hz , chpy ) , 7.34 ( dd , 1h , j = 0.98 hz , j = 7.88 hz , chbz ) , 6.99 ( td , 1h , j = 1.43 hz , j = 8.03 hz , chbz ) , 6.82 ( dd , 1h , j = 1.22 hz , j = 7.96 hz , chbz ) , 6.64 ( td , 1h , j = 1.19 hz , j = 7.73 hz , chbz ) , 4.94 ( s , 2h , nh2 ) ppm .
a mixture of 5 ( 1.17 g , 7.69 mmol ) and 7 ( 2.1 g , 7.2 mmol ) in dry dmf ( 45 ml ) was heated under argon at 85 c for 20 h. dmf then was evaporated in vacuo at 50 c and water ( 20 ml ) was added .
yield : 2.3 g. the product was used without further purification in the next step .
esi - ms in methanol ( positive ) : m / z 399 [ m+na ] ; ( negative ) : m / z 375 [ m  h ] .
h nmr ( 500.32 mhz , dmso - d6 ) :  14.54 ( brs , 1h , nhpz ) , 9.85 ( brs , 1h , conh ) , 8.73 ( d , 1h , j = 2.13 hz , chpy ) , 8.68 ( d , 1h , j = 2.05 hz , chpy ) , 7.29 ( d , 1h , j = 7.56 hz , chbz ) , 7.03 ( d , 1h , j = 7.29 hz , chbz ) , 6.65 ( t , 1h , j = 7.79 hz , chbz ) , 4.78 ( brs , 2h , nh2 ) , 4.42 ( s , 2h , ch2 ) , 3.29 ( s , 3h , ch3 ) ppm . the raw product 8 ( 0.86 g ) was heated in a glacial acetic acid ( 10 ml ) at 125 c for 2.5 h. the solvent was evaporated under reduced pressure and the residue was dried in vacuo at 50 c , then resuspended in ch2cl2/meoh ( 4/1 , 25 ml ) , filtered off , and dried in vacuo to give l1 as a white powder ( 0.4 g ) .
the filtrate was evaporated and the residue was purified by column chromatography ( sio2 , etoac , rf = 0.58 ) to give an additional amount of l1 ( 0.22 g ) .
calcd for 110.15h2o0.1etoac ( mr = 246.76 g / mol ) : c , 65.22 ; h , 4.13 ; n , 28.38 . found : c , 65.56 ; h , 3.90 ; n , 28.39 .
esi - ms in methanol ( positive ) : m / z 237 [ m+h ] , 259 [ m+na ] ; ( negative ) : m / z 235 [ m  h ] .
vis ( methanol ) , max , nm (  , m cm ) : 232 ( 25618 ) , 275 ( 15198 ) , 324 ( 22333 ) .
h nmr ( 500.32 mhz , dmso - d6 ) :  14.19 ( brs , 1h , h1a ) , 13.11 ( brs , 1h , h1b ) , 8.85 ( dd , 1h , j = 1.5 hz ,
j = 8.1 hz , h4a ) , 8.66 ( dd , 1h , j = 1.5 hz , j = 4.5 hz , h6a ) , 7.75 ( d , 1h , j
= 7.3 hz , h4b or h7b ) , 7.54 ( d , 1h , j = 7.7 hz , h4b or h7b ) , 7.39 ( dd , 1h , j = 4.5 hz , j = 8.0 hz , h5a ) , 7.24 ( m , 2h , h5b+h6b ) ppm .
c nmr ( 125.81 mhz , dmso - d6 ) :  153.13 ( c8a ) , 150.22 ( c6a ) , 146.99 ( c2b ) , 144.34 ( c8b or c9b ) , 136.06 ( c3a ) , 134.68 ( c8b or c9b ) , 131.82 ( c4a ) , 123.35 ( c5b or c6b ) , 122.11 ( c5b or c6b ) , 119.44 ( c4b or c7b ) , 118.65 ( c5a ) , 113.33 ( c9a ) , 112.01 ( c4b or c7b ) ppm . n nmr ( 50.70 mhz , dmso - d6 ) :  166.2 ( n1a ) , 121.3 ( n1b ) ppm . the raw product 9 ( 2.2 g ) was heated in a glacial acetic acid ( 30 ml ) at 125 c for 2 h. the solvent was evaporated under reduced pressure , and the residue was dried in vacuo at 50 c , then resuspended in ch2cl2/meoh ( 7/1 , 50 ml ) , filtered off and dried in vacuo to give l2 as a white powder ( 1.15 g ) .
the filtrate was evaporated and the residue was purified by column chromatography ( sio2 , etoac / hexane 2/1 , rf = 0.6 ) to give an additional amount of the product ( 0.27 g ) . yield : 1.42 g , 58% based on 7 .
calcd for 120.25h2o0.04etoac ( mr = 322.17 g / mol ) : c , 49.06 ; h , 2.76 ; n , 21.74 .
esi - ms in methanol ( positive ) : m / z 315 [ m+h ] , 337 [ m+na ] ; ( negative ) : m / z 313 [ m  h ] .
vis ( methanol ) , max , nm (  , m cm ) : 234 ( 28154 ) , 282 ( 17628 ) , 335 ( 17198 ) .
h nmr ( 500.32 mhz , dmso - d6 ) :  14.43 ( brs , 1h , h1a ) , 13.19 ( brs , 1h , h1b ) , 8.99 ( d , 1h , j = 2.2 hz , h4a ) , 8.75 ( d , 1h , j = 2.3 hz , h6a ) , 7.77 ( d , 1h , j = 7.9 hz , h4b or h7b ) , 7.54 ( d , 1h , j = 7.9 hz , h4b or h7b ) , 7.26 ( m , 2h , h5b+h6b ) ppm .
c nmr ( 125.81 mhz , dmso - d6 ) :  151.49 ( c8a ) , 150.66 ( c6a ) , 146.42 ( c2b ) , 144.24 ( c8b or c9b ) , 135.61 ( c3a ) , 134.67 ( c8b or c9b ) , 133.35 ( c4a ) , 123.54 ( c5b or c6b ) , 122.27 ( c5b or c6b ) , 119.55 ( c4b or c7b ) , 114.87 ( c5a or c9a ) , 113.49 ( c5a or c9a ) , 112.09 ( c4b or c7b ) ppm . n nmr ( 50.70 mhz , dmso - d6 ) :  167.5 ( n1a ) , 121.3 ( n1b ) ppm .
the raw product 10 ( 2.3 g ) was heated in a glacial acetic acid ( 40 ml ) at 125 c for 2 h. the solvent was evaporated under reduced pressure , and the residue dried in vacuo at 50 c . after washing with ch2cl2 ( 30 ml ) , ch2cl2/meoh ( 2/1 , 57 ml ) the gray product was purified by column chromatography ( sio2 , etoac , rf = 0.68 ) to give a white powder ( 0.9 g ) . the filtrates were evaporated and the remaining solid was purified by column chromatography to give an additional amount of the product ( 0.3 g ) .
calcd for 13 : c , 50.29 ; h , 3.38 ; n , 19.55 . found : c , 50.03 ; h , 3.19 ; n , 19.19 .
esi - ms in methanol ( positive ) : m / z 381 [ m+na ] ; ( negative ) : m / z 357 [ m  h ] .
vis ( methanol ) , max , nm (  , m cm ) : 235 ( 29776 ) , 282 ( 17544 ) , 337 ( 16958 ) .
nmr characterization of 7b - l3 and 4b-l3 tautomers ( 1/1.3 ) in dmso - d6 : h nmr ( 500.32 mhz , dmso - d6 ) , 7b - l3 :  14.47 ( brs , 1h , h1a ) , 13.25 ( brs , 1h , h1b ) , 8.99 or 8.98 ( d+d , ( 1 + 1.3)h , j = 2.3 hz , h4a+h4a ) , 8.75 ( d , ( 1 + 1.3)h , j = 2.3 hz , h6a+h6a ) , 7.72 ( dd , 1h , j = 1.8 hz ,
j = 6.8 hz , h4b ) , 7.287.21 ( m , ( 2 + 2.6)h , h5a , h6a+h5a , h6a ) , 4.80 ( s , 2h , h10b ) , 3.37 ( s , 3h , h11b ) ppm .
h nmr ( 500.32 mhz , dmso - d6 ) , 4b-l3 :  14.43 ( brs , 1.3h , h1a ) , 13.22 ( brs , 1.3h , h1b ) , 8.99 or 8.98 ( d+d , ( 1 + 1.3)h , j = 2.3 hz , h4a+h4a ) , 8.75 ( d , ( 1 + 1.3)h , j = 2.3 hz , h6a+h6a ) , 7.47 ( dd , 1.3h , j = 1.2 hz ,
j = 7.7 hz , h7b ) , 7.287.21 ( m , ( 2 + 2.6)h , h5a , h6a+h5a , h6a ) , 4.96 ( s , 2.6h , h10b ) , 3.44 ( s , 3.9h , h11b ) ppm .
c nmr ( 125.81 mhz , dmso - d6 ) , 7b - l3 :  151.51 ( c8a+c8a ) , 150.69 ( c6a or c6a ) , 150.65 ( c6a or c6a ) , 146.70 ( c2b ) , 144.46 ( c9b ) , 135.62 ( c3a+c3a ) , 133.45 ( c4a or c4a ) , 133.42 ( c4a or c4a ) , 133.29 ( c8b ) , 123.38 ( c6b ; c5b or c6b ) , 123.36 ( c6b ; c5b or c6b ) , 122.95 ( c7b ) , 122.16 ( c5b or c6b ) , 118.97 ( c4b ) , 115.04 ( c5a or c9a or c5a or c9a ) , 114.92 ( c5a or c9a or c5a or c9a ) , 113.49 ( c5a or c9a or c5a or c9a ) , 70.49 ( c10b ) , 57.95 ( c11b ) ppm .
c nmr ( 125.81 mhz , dmso - d6 ) , 4b-l3 :  151.51 ( c8a+c8a ) , 150.69 ( c6a or c6a ) , 150.65 ( c6a or c6a ) , 146.13 ( c2b ) , 142.50 ( c9b ) , 135.62 ( c3a+c3a ) , 134.42 ( c8b ) , 133.45 ( c4a or c4a ) , 133.42 ( c4a or c4a ) , 129.38 ( c4b ) , 123.38 ( c6b ; c5b or c6b ) , 123.36 ( c6b ; c5b or c6b ) , 122.16 ( c5b or c6b ) , 120.81 ( c5b ) , 115.04 ( c5a or c9a or c5a or c9a ) , 114.92 ( c5a or c9a or c5a or c9a ) , 113.49 ( c5a or c9a or c5a or c9a ) , 111.17 ( c7b ) , 69.90 ( c10b ) , 58.35 ( c11b ) ppm .
n nmr ( 50.70 mhz , dmso - d6 ) :  167.6 ( n1a+n1a ) , 121.4 ( n1b+n1b ) ppm .
a mixture of l1 ( 54.7 mg , 0.23 mmol ) and [ rucl2(-p - cymene)]2 ( 70 mg , 0.11 mmol ) in dry ethanol ( 25 ml ) was stirred at room temperature for 1 h. ethanol then was evaporated up to ca .
calcd for 11ah2o ( mr = 559.45 g / mol ) : c , 49.38 ; h , 4.50 ; n , 12.52 ; cl , 12.67 .
found : c , 49.68 ; h , 4.25 ; n , 12.11 ; cl , 12.26 .
esi - ms in methanol ( positive ) : m / z 470 [ m  hcl  cl ] , 507 [ m  cl ] , 528 [ m  hcl+na ] ; ( negative ) : m / z 468 [ m2hcl  h ] , 505 [ m  hcl  h ] .
vis ( methanol ) , max , nm (  , m cm ) : 251 ( 16335 ) , 299 ( 26663 ) , sh 347 ( 16012 ) .
vis ( h2o ) , max , nm (  , m cm ) : sh 245 ( 10728 ) , 294 ( 18597 ) , 360 ( 9666 ) .
h nmr ( 500.32 mhz , dmso - d6 ) :  14.91 ( brs , 1h , h1b ) , 9.17 ( d , 1h , j = 7.7 hz , h4a ) , 8.82 ( d , 1h , j = 3.8 hz , h6a ) , 8.11 ( d , 1h , j = 7.1 hz , h4b ) , 7.84 ( d , 1h , j = 8.5 hz , h7b ) , 7.58 ( m , 3h , h5a+h5b+h6b ) , 6.43 ( m , 2h , h2c+h6c ) , 6.31 ( d , 1h , j = 5.3 hz , h3c or h5c ) , 6.12 ( d , 1h , j = 5.5 hz , h3c or h5c ) , 2.54 ( sep , 1h , h7c , under dmso - d6 peak ) , 2.21 ( s , 3h , h10c ) , 0.94 ( d , 3h , j = 6.6 hz , h8c or h9c ) , 0.91 ( d , 3h , j = 6.6 hz , h8c or h9c ) ppm .
c nmr ( 125.81 mhz , dmso - d6 ) :  153.97 ( c8a ) , 150.45 ( c6a ) , 146.52 ( c2b ) , 141.37 ( c9b ) , 134.70 ( c8b ) , 134.64 ( c3a ) , 131.48 ( c4a ) , 125.39 ( c5b or c6b ) , 124.92 ( c5b or c6b ) , 119.56 ( c5a ) , 117.85 ( c4b ) , 113.96 ( c7b ) , 111.54 ( c9a ) , 103.96 ( c4c ) , 103.74 ( c1c ) , 84.44 ( c2c or c6c ) , 83.73 ( c2c or c6c ) , 82.15 ( c3c or c5c ) , 80.84 ( c3c or c5c ) , 31.12 ( c7c ) , 22.19 ( c8c+c9c ) , 19.17 ( c10c ) ppm .
n nmr ( 50.70 mhz , dmso - d6 ) :  128.7 ( n1b ) ppm .
a mixture of l1 ( 42.5 mg , 0.18 mmol ) and [ oscl2(-p - cymene)]2 ( 70 mg , 0.09 mmol ) in dry ethanol ( 25 ml ) was stirred at room temperature for 2 h. ethanol then was removed under reduced pressure up to ca .
calcd for 11bh2o ( mr = 648.61 g / mol ) : c , 42.59 ; h , 3.88 ; n , 10.79 . found : c , 42.56 ; h , 3.57 ; n , 10.97 .
esi - ms in methanol ( positive ) : m / z [ m  hcl  cl ] , 596 [ m  cl ] , 618 [ m  hcl+na ] ; ( negative ) : m / z 595 [ m  hcl  h ] .
vis ( methanol ) , max , nm (  , m cm ) : sh 252 ( 17048 ) , 299 ( 20228 ) , 343 ( 19879 ) .
h nmr ( 500.32 mhz , meoh - d4 ) :  8.99 ( dd , 1h , j = 1.3 hz ,
j = 8.1 hz , h4a ) , 8.77 ( dd , 1h , j = 1.4 hz ,
j = 4.8 hz , h6a ) , 7.96 ( dd , 1h , j = 2.0 hz , j = 6.4 hz , h4b ) , 7.80 ( dd , 1h , j
= 1.9 hz , j = 6.1 hz , h7b ) , 7.647.59 ( m , 3h , h5a+h5b+h6b ) , 6.66 ( d , 1h , j = 5.6 hz , h2c or h6c ) , 6.59 ( d , 1h , j = 5.7 hz , h2c or h6c ) , 6.43 ( d , 1h , j = 5.6 hz , h3c or h5c ) , 6.21 ( d , 1h , j = 5.7 hz , h3c or h5c ) , 2.43 ( sep , 1h , j = 6.9 hz , h7c ) , 2.38 ( s , 3h , h10c ) , 0.96 ( d , 3h , j = 6.9 hz , h8c or h9c ) , 0.92 ( d , 3h , j = 6.9 hz , h8c or h9c ) ppm .
c nmr ( 125.81 mhz , meoh - d4 ) :  153.25 ( c8a ) , 148.91 ( c2b ) , 147.42 ( c6a ) , 140.49 ( c9b ) , 135.15 ( c3a ) , 134.25 ( c8b ) , 132.07 ( c4a ) , 125.47 ( c5b or c6b ) , 124.81 ( c5b or c6b ) , 118.48 ( c5a ) , 116.89 ( c4b ) , 112.98 ( c7b ) , 112.92 ( c9a ) , 97.71 ( c4c ) , 94.88 ( c1c ) , 76.44 ( c2c or c6c ) , 74.99 ( c2c or c6c ) , 71.93 ( c3c or c5c ) , 70.44 ( c2c or c6c ) , 31.37 ( c7c ) , 21.35 ( c8c or c9c ) , 21.09 ( c8c or c9c ) , 17.84 ( c10c ) ppm .
crystals of 11b4h2o suitable for xrd study have been obtained from a solution of 11b in ethanol .
a mixture of l2 ( 73.2 mg , 0.23 mmol ) and [ rucl2(-p - cymene)]2 ( 70 mg , 0.11 mmol ) in dry ethanol ( 25 ml ) was stirred at room temperature for 1 h. ethanol then was removed under reduced pressure up to ca .
calcd for 12ah2o ( mr = 638.35 g / mol ) : c , 43.28 ; h , 3.79 ; n , 10.97 .
esi - ms in methanol ( positive ) : m / z 548 [ m  hcl  cl ] , 608 [ m  hcl+na ] ; ( negative ) : m / z 549 [ m2hcl  h ] , 582 [ m  hcl  h ] .
vis ( methanol ) , max , nm (  , m cm ) : sh 254 ( 17778 ) , 303 ( 29953 ) , 351 ( 17387 ) .
h nmr ( 500.32 mhz , dmso - d6 ) :  14.34 ( brs , 1h , h1b ) , 9.21 ( s , 1h , h4a ) , 8.73 ( s , 1h , h6a ) , 8.06 ( d , 1h , j = 7.6 hz , h4b ) , 7.79 ( d , 1h , j = 8.5 hz , h7b ) , 7.52 ( m , 2h , h5b+h6b ) , 6.32 ( d , 2h , j = 5.8 hz , h2c+h6c ) , 6.22 ( d , 1h , j = 6.2 hz , h3c or h5c ) , 6.03 ( d , 1h , j = 6.1 hz , h3c or h5c ) , 2.52 ( sep , 1h , h7c , under dmso - d6 peak ) , 2.18 ( s , 3h , h10c ) , 0.94 ( d , 3h , j = 6.8 hz , h8c or h9c ) , 0.89 ( d , 3h , j = 6.9 hz , h8c or h9c ) ppm .
c nmr ( 125.81 mhz , dmso - d6 ) :  154.04 ( c8a ) , 151.24 ( c6a ) , 146.57 ( c2b ) , 141.37 ( c9b ) , 134.63 ( c8b ) , 133.43 ( c3a ) , 131.82 ( c4a ) , 125.33 ( c5b or c6b ) , 124.88 ( c5b or c6b ) , 117.78 ( c4b ) , 114.72 ( c5a or c9a ) , 113.85 ( c7b ) , 112.48 ( c5a or c9a ) , 103.96 ( c4c ) , 103.74 ( c1c ) , 84.44 ( c2c or c6c ) , 83.69 ( c2c or c6c ) , 82.16 ( c3c or c5c ) , 80.76 ( c3c or c5c ) , 31.09 ( c7c ) , 22.20 ( c8c or c9c ) , 22.17 ( c8c or c9c ) , 19.16 ( c10c ) ppm .
n nmr ( 50.70 mhz , dmso - d6 ) :  127.3 ( n1b ) ppm .
a mixture of l2 ( 56.4 mg , 0.18 mmol ) and [ oscl2(-p - cymene)]2 ( 70 mg , 0.09 mmol ) in dry ethanol ( 25 ml ) was stirred at room temperature for 2 h. ethanol then was removed under reduced pressure up to ca .
calcd for 12bh2o ( mr = 727.51 g / mol ) : c , 37.97 ; h , 3.33 ; n , 9.63 . found : c , 37.82 ; h , 3.02 ; n , 9.33 .
esi - ms in methanol ( positive ) : m / z 638 [ m  hcl  cl ] , 696 [ m  hcl+na ] ; ( negative ) : m / z 672 [ m  hcl  h ] .
vis ( methanol ) , max , nm (  , m cm ) : sh 253 ( 21638 ) , 302 ( 30505 ) , 357 ( 21813 ) .
h nmr ( 500.32 mhz , meoh - d4 ) :  9.08 ( d , 1h , j = 2.1 hz , h4a ) , 8.84 ( d , 1h , j = 2.1 hz , h6a ) , 7.96 ( dd , 1h , j = 2.1 hz ,
j = 6 hz , h4b ) , 7.80 ( dd , 1h , j = 2.1 hz ,
j = 6.1 hz , h7b ) , 7.63 ( m , 2h , h5b+h6b ) , 6.69 ( d , 1h , j = 5.7 hz , h2c or h6c ) , 6.63 ( d , 1h , j = 5.6 hz , h2c or h6c ) , 6.48 ( d , 1h , j = 5.6 hz , h3c or h5c ) , 6.22 ( d , 1h , j = 5.7 hz , h3c or h5c ) , 2.42 ( sep , 1h , j = 6.9 hz , h7c ) , 2.39 ( s , 3h , h10c ) , 0.95 ( d , 3h , j = 6.9 hz , h8c or h9c ) , 0.91 ( d , 3h , j = 6.9 hz , h8c or h9c ) ppm .
c nmr ( 125.81 mhz , meoh - d4 ) :  153.04 ( c8a ) , 152.49 ( c6a ) , 148.15 ( c2b ) , 140.47 ( c9b ) , 134.58 ( c3a ) , 134.19 ( c8b ) , 131.13 ( c4a ) , 125.79 ( c5b or c6b ) , 125.05 ( c5b or c6b ) , 116.99 ( c4b ) , 115.39 ( c5a or c9a ) , 113.09 ( c7b ) , 112.11 ( c5a or c9a ) , 98.54 ( c4c ) , 95.52 ( c1c ) , 75.73 ( c2c or c6c ) , 75.25 ( c2c or c6c ) , 71.87 ( c3c or c5c ) , 69.99 ( c3c or c5c ) , 31.41 ( c7c ) , 21.34 ( c8c or c9c ) , 21.16 ( c8c or c9c ) , 17.84 ( c10c ) ppm .
crystals of 12b and 12b2ch3oh2h2o suitable for xrd study have been obtained from a solution of 12b in methanol .
a mixture of l3 ( 64 mg , 0.18 mmol ) and [ rucl2(-p - cymene)]2 ( 50 mg , 0.08 mmol ) in dry ethanol ( 25 ml ) was stirred at room temperature for 1 h. ethanol then was removed under reduced pressure up to ca .
calcd for 13a1.5h2o ( mr = 691.41 g / mol ) : c , 43.43 ; h , 4.23 ; n , 10.13 ; cl , 10.26 .
found : c , 43.81 ; h , 4.24 ; n , 10.11 ; cl , 10.57 .
esi - ms in methanol ( positive ) : m / z 592 [ m  hcl  cl ] , 650 [ m  hcl+na ] ; ( negative ) : m / z 591 [ m2hcl  h ] , 628 [ m  hcl  h ] .
vis ( methanol ) , max , nm (  , mcm ) : sh 252 ( 18744 ) , 306 ( 28529 ) , 354 ( 16889 ) .
h nmr ( 500.32 mhz , dmso - d6 ) :  13.88 ( brs , 1h , h1b ) , 9.41 ( s , 1h , h4a ) , 8.68 ( s , 1h , h6a ) , 7.99 ( d , 1h , j = 7.7 hz , h4b ) , 7.49 ( m , 2h , h5b+h6b ) , 6.29 ( m , 2h , h2c+h6c ) , 6.20 ( d , 1h , j = 5.5 hz , h3c or h5c ) , 6.00 ( d , 1h , j = 5.9 hz , h3c or h5c ) , 4.88 ( s , 2h , h10b ) , 3.39 ( s , 3h , h11b ) , 2.48 ( sep , 1h , h7c , under dmso - d6 peak ) , 2.17 ( s , 3h , h10c ) , 0.93 ( d , 3h , j = 6.9 hz , h8c or h9c ) , 0.87 ( d , 3h , j = 6.8 hz , h8c or h9c ) ppm .
c nmr ( 125.81 mhz , dmso - d6 ) :  155.35 ( c8a ) , 150.27 ( c6a ) , 147.43 ( c2b ) , 141.64 ( c9b ) , 133.07 ( c8b ) , 132.68 ( c3a ) , 131.68 ( c4a ) , 124.87 ( c5b or c6b ) , 124.59 ( c5b or c6b ) , 124.53 ( c7b ) , 117.18 ( c4b ) , 114.23 ( c5a or c9a ) , 112.78 ( c5a or c9a ) , 103.74 ( c4c ) , 103.09 ( c1c ) , 85.38 ( c2c or c6c ) , 83.68 ( c2c or c6c ) , 82.13 ( c3c or c5c ) , 81.05 ( c3c or c5c ) , 70.19 ( c10b ) , 58.03 ( c11b ) , 31.04 ( c7c ) , 22.22 ( c8c or c9c ) , 22.09 ( c8c or c9c ) , 19.13 ( c10c ) ppm .
n nmr ( 50.70 mhz , dmso - d6 ) :  122.9 ( n1b ) ppm .
the red xrd - quality crystals of [ rucl(-p - cymene)(l3h ) ] ( 13c ) were obtained from etoh / et2o and an etoh solution of 13a ( long crystallization ) .
the yellow crystals of [ rucl(-p - cymene)(l3)]cl[rucl(-p - cymene)(l3h)]ch3oh ( 13dch3oh ) suitable for xrd study have been obtained from methanolic solution of 13a ( short crystallization ) .
h nmr ( 13c , 500.32 mhz , dmso - d6 ) :  13.43 ( s , 1h ) , 9.18 ( d , 1h , j = 2.1 hz ) , 8.48 ( d , 1h , j = 2.2 hz ) , 7.94 ( d , 1h , j = 8.2 hz ) , 7.45 ( t , 1h , j = 7.8 hz ) , 7.39 ( d , 1h , j = 7.2 hz ) , 6.16 ( d , 1h , j = 6.0 hz ) , 6.13 ( d , 1h , j = 6.1 hz ) , 6.07 ( d , 1h , j = 5.9 hz ) , 5.89 ( d , 1h , j = 6.4 hz ) , 4.85 ( s , 2h ) , 4.04 ( s , 3h ) , 2.14 ( s , 3h ) , 0.93 ( d , 3h , j = 6.8 hz ) , 0.86 ( d , 3h , j = 6.9
a mixture of l3 ( 59 mg , 0.17 mmol ) and [ oscl2(-p - cymene)]2 ( 60 mg , 0.08 mmol ) in dry ethanol ( 25 ml ) was stirred at room temperature for 3 h. ethanol was evaporated up to ca .
calcd for 13b : c , 39.85 ; h , 3.48 ; n , 9.29 . found : c , 39.60 ; h , 3.32 ; n , 9.20 .
esi - ms in methanol ( positive ) : m / z 682 [ m  hcl  cl ] , 704 [ m2hcl+na ] ; ( negative ) : m / z 680 [ m2hcl  h ] , 716 [ m  hcl  h ] .
vis ( methanol ) , max , nm (  , m cm ) : sh 256 ( 19825 ) , 303 ( 24634 ) , 357 ( 18850 ) .
h nmr ( 500.32 mhz , meoh - d4 ) :  9.33 ( d , 1h , j = 2.1 hz , h4a ) , 8.84 ( d , 1h , j = 2.2 hz , h6a ) , 7.91 ( dd , 1h , j = 1.2 hz ,
j = 7.8 hz , h4b ) , 7.647.58 ( m , 2h , h5b+h6b ) , 6.69 ( d , 1h , j = 5.6 hz , h2c or h6c ) , 6.62 ( d , 1h , j = 5.7 hz , h2c or h6c ) , 6.47 ( d , 1h , j = 5.5 hz , h3c or h5c ) , 6.21 ( d , 1h , j = 5.6 hz , h3c or h5c ) , 4.94 ( q , 2h , j = 12.4 hz , h10b ) , 3.49 ( s , 3h , h11b ) , 2.41 ( sep , 1h , j = 6.9 hz , h7c ) , 2.39 ( s , 3h , h10c ) , 0.94 ( d , 3h , j = 6.9 hz , h8c or h9c ) , 0.89 ( d , 3h , j = 6.9 hz , h8c or h9c ) ppm . c nmr ( 125.81 mhz , meoh - d4 ) :  153.18 ( c8a ) , 152.16 ( c6a ) , 148.36 ( c2b ) , 140.82 ( c9b ) , 134.35 ( c3a ) , 132.68 ( c8b ) , 131.68 ( c4a ) , 125.63 ( c5b or c6b ) , 124.85 ( c5b or c6b ) , 124.51 ( c7b ) , 116.72 ( c4b ) , 115.24 ( c5a or c9a ) , 112.26 ( c5a or c9a ) , 98.75 ( c4c ) , 95.50 ( c1c ) , 75.98 ( c2c or c6c ) , 75.34 ( c2c or c6c ) , 71.82 ( c3c or c5c ) , 70.29 ( c10b ) , 70.04 ( c3c or c5c ) , 57.17 ( c11b ) , 31.39 ( c7c ) , 21.34 ( c8c or c9c ) , 21.16 ( c8c or c9c ) , 17.85 ( c10c ) ppm .
the yellow crystals of [ oscl(-p - cymene)(l3)]cl[oscl(-p - cymene)(l3h)]0.75 ch3oh0.25h2o ( 13e0.75ch3oh0.25h2o ) suitable for xrd study have been obtained from a methanolic solution of 13b .
elemental analyses ( c , h , n , cl ) were performed by the microanalytical service of the institute of physical chemistry , university of vienna .
electrospray ionization mass spectrometry ( esi - ms ) was carried out with an esquire 3000 instrument ( bruker daltonics , bremen , germany ) , using solutions of compounds in methanol .
vis spectra were recorded on a lambda 20 uv  vis spectrophotometer ( perkin  elmer ) , using samples dissolved in methanol ( l1l3 , 11a , 12a , 13a , 11b , 12b , 13b ) and water ( 11a ) over 24 or 48 h , correspondingly . the one - dimensional ( h , c , n ) and two - dimensional spectra ( n , h hsqc , c , h hsqc , c , h hmbc , h , h cosy , h , h tocsy , h , h noesy ( l3 , 12a ) , h , h roesy ( l3 , 11a , 12a , 13a , 13b ) ) were recorded with a bruker model dpx500 ( ultrashield magnet ) system in dmso - d6 ( l1l3 , 11a , 12a , 13a ) , meoh - d4 ( 11b , 12b , 13b ) , d2o ( h nmr , 11a ) , and d2o / dmso - d6 ( h nmr , 12a ) , using standard pulse programs at 500.32 ( h ) , 125.81 ( c ) and 50.70 ( n ) mhz .
h signals are referenced relative to the solvent signals ( dmso - d6 at 2.51 , meoh - d4 at 3.33 ppm ) .
single crystals were positioned at distances of 35 , 40 , 40 , 40 , 40 , 40 , and 35 mm from the detector , and 1556 , 1131 , 518 , 1911 , 1176 , 1978 , and 1039 frames were measured , each for 30 , 60 , 30 , 20 , 70 , 60 , and 30 s over 1 scan width for 20.5h2o , 11b4h2o , 12b , 12b2ch3oh2h2o , 13c , 13dch3oh , and 13e0.75ch3oh0.25h2o , correspondingly .
crystal data , data collection parameters , and structure refinement details are given in table 1 .
the structures were solved by direct methods and refined by full - matrix least - squares techniques .
refinement of the structure of 12b revealed that the chloride counteranion occupies two statistically disordered positions with site occupation factor ( sof ) values of 0.57:0.43 , whereas , in 13c , the methoxymethylene group was found to be disordered over two positions with sof values of 0.80:0.20 .
similarly , the methoxymethylene group of one crystallographically independent complex in 13dch3oh or in both crystallographically independent complexes in 13e0.75ch3oh0.25h2o is disordered with sof values of 0.35:0.65 and 0.60:0.40 , 0.80:0.20 , correspondingly . the disorder was resolved with constrained anisotropic displacement parameters and restrained bond distances using eadp and sadi instructions of shelx97 , respectively .
structure solution was achieved with shelxs-97 and refinement with shelxl-97 , and graphics were produced with ortep-3 .
wr2 = { [w(fo  fc)]/[w(fo)]}. gof = { [w(fo  fc)]/(n  p ) } , where n is the number of reflections and p is the total number of parameters refined .
a549 ( non - small cell lung carcinoma , human ) and sw480 ( colon carcinoma , human ) cells were kindly provided by brigitte marian ( institute of cancer research , department of medicine i , medical university of vienna , austria ) .
ch1 cells ( ovarian cancer , human ) were a gift from lloyd r. kelland ( crc centre for cancer therapeutics , institute of cancer research , sutton , u.k . ) .
cells were grown in 75-cm culture flasks ( iwaki ) in a complete medium ( i.e. , minimum essential medium supplemented with 10% heat - inactivated fetal bovine serum , 1 mm sodium pyruvate , 4 mm l - glutamine , and 1% nonessential amino acids from 100 stock ) as adherent monolayer cultures .
cultures were grown at 37 c under a humidified atmosphere containing 5% co2 and 95% air .
antiproliferative activity in vitro was determined by the colorimetric mtt assay ( mtt = 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2h - tetrazolium bromide , fluka ) . for this purpose
, cells were harvested from culture flasks by the use of trypsin and seeded in complete medium ( 100 l / well ) into 96-well plates ( iwaki ) in densities of 4  10 ( a549 ) , 1.5  10 ( ch1 ) , and 2.5  10 ( sw480 ) viable cells per well .
test compounds were dissolved in dmso first , appropriately diluted in complete medium , and instantly added to the plates ( 100 l / well ) , where the dmso content did not exceed 0.4% and 1% for the ligands and complexes , respectively . after exposure for 96 h , the medium was replaced with 100 l / well rpmi 1640 medium ( supplemented with 10% heat - inactivated fetal bovine serum and 4 mm l - glutamine ) plus 20 l / well mtt solution in phosphate - buffered saline ( 5 mg / ml ) , followed by incubation for 4 h. subsequently , the medium / mtt mixture was removed , and the formazan product formed by viable cells was dissolved in dmso ( 150 l / well ) .
optical densities were measured with a microplate reader ( tecan spectra classic ) at 550 nm ( and a reference wavelength of 690 nm ) to yield relative quantities of viable cells as percentages of untreated controls , and 50% inhibitory concentrations ( ic50 ) were interpolated from concentration  effect curves .
calculations are based on at least three independent experiments with triplicates for each concentration level . to study the effects on the cell cycle of exponentially growing ch1 cells by flow cytometric analysis of their relative dna content
, cells were harvested from culture flasks , seeded in complete medium into 90-mm petri dishes ( 1  10 cells / dish ) and , after recovery for 24 h , exposed to various concentrations of the test compounds for 24 h. for this purpose , test compounds were diluted from dmso stocks with complete medium ( see above ) such that the effective dmso content did not exceed 0.5% . after exposure , treated and control cells were collected by scratching , washed with pbs , and stained with 5 g / ml propidium iodide overnight .
their fluorescence was measured with a facs calibur instrument ( becton dickinson ) , and the obtained histograms were analyzed with cell quest pro software ( becton dickinson ) .
at least two independent experiments were performed for each setting , and 2.5 or 3.0  10 cells were measured per sample .
the cdk - inhibitory capacities of test compounds were studied by a radiochemical assay using recombinant cdk1/cyclin b and cdk2/cyclin e isolated from sf21 insect cells and histone h1 as the substrate for phosphorylation , as described by marko et al .
briefly , mops - buffered assay mixtures containing the test compound ( with a maximum of 1% dmso ) , the respective kinase / cyclin complex , histone h1 , and 0.4 ci ( -p)atp per sample were incubated for 10 min at 30 c .
aliquots of the solution were spotted onto phosphocellulose squares , which had been washed three times with 0.75% phosphoric acid , followed by acetone .
the dried squares were measured in scintillation vials by -counting ( perkin  elmer tri - carb 2800tr ; quanta smart software ) .
results were obtained in duplicate in at least two independent experiments , and ic50 values were calculated by interpolation .
several routes to 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines have been proposed by johnson & johnson pharmaceutical research & development l.l.c .
the first one was developed for 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines with an unsubstituted benzimidazole moiety and involved sulfur - induced benzimidazole ring formation via the treatment of 5-bromo-1h - pyrazolo[3,4-b]pyridine-3-carboxaldehyde with 1,2-diaminobenzene .
the poor reproducibility of this synthesis prompted the exploration of an alternative way , via 5-bromo-1h - pyrazolo[3,4-b]pyridine-3-carboxylic acid , which was used for the preparation of 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines with a substituted benzimidazole moiety .
the patented route to 5-bromo-1h - pyrazolo[3,4-b]pyridine-3-carboxylic acid ( 3 ) consists of four steps ( see scheme 1 , steps i  iv ) : oxidation of 3-methyl-1h - pyrazolo[3,4-b]pyridine by kmno4 in the presence of a base with subsequent acidification with h2so4 , esterification of 1h - pyrazolo[3,4-b]pyridine-3-carboxylic acid ( 1 ) in the presence of h2so4 in methanol , bromination of 1h - pyrazolo[3,4-b]pyridine-3-carboxylic acid methyl ester in an acoh / acona mixture , and hydrolysis of 5-bromo-1h - pyrazolo[3,4-b]pyridine-3-carboxylic acid methyl ester in the presence of naoh , followed by acidification with hcl .
reagents and conditions : ( i ) kmno4 , naoh , 3 h , 100 c , h2so4 ; ( ii ) methanol , h2so4 , reflux , 68 h , nahco3 , 42% ( i + ii ) ; ( iii ) br2 , acoh , acona , 115 c , overnight , chromatographic purification , 43% ; ( iv ) naoh , meoh , reflux , 4 h , hcl , 100% ; ( v ) br2 , acoh , acona , 115 c , 2.53 h ; and ( vi ) kmno4 , naoh , 3 h , 100 c , hcl , 2435% ( v + vi ) .
we performed the synthesis of 3 in two steps ( see scheme 1 , steps v and vi ) : bromination of 3-methyl-1h - pyrazolo[3,4-b]pyridine in acoh / acona mixture and oxidation of crude 5-bromo-3-methyl-1h - pyrazolo[3,4-b]pyridine ( 2 ) by kmno4 in a basic medium , followed by acidification with 37% hcl , with an overall yield of 2435% .
5-bromo-3-methyl-1h - pyrazolo[3,4-b]pyridine ( 2 ) is a known compound , the synthesis of which is well - documented . for the benzimidazole ring formation , 1,2-diaminobenzene and 1-methoxymethyl-2,3-diaminobenzene
the reported synthesis of 1-methoxymethyl-2,3-diaminobenzene ( 5 ) from 2,3-diaminobenzyl alcohol afforded the desired product in 34% yield ( see scheme 2 , step i ) .
however , the instability and low yield of diamines , as well as the necessity to purify the desired ether using column chromatography , stimulated the search for a more - convenient procedure that was subsequently proposed : etherification of 2-amino-3-nitrobenzyl alcohol , followed by the reduction of 1-methoxymethyl-2-amino-3-nitrobenzene ( 4 ) with 10% pd / c in ethanol under a hydrogen atmosphere afforded 5 in 6575% yield ( see scheme 2 , steps ii and iii ) .
reagents and conditions : ( i ) nah , mei , dry thf , 0 c , 30 min , room temperature , overnight , chromatographic purification , 34% yield ; ( ii ) nah , mei , dry thf , 0 c , 15 min , room temperature , 3 h , chromatographic purification , 6575% yield ; ( iii ) 10% pd / c , ethanol , h2 , room temperature , 1824 h , 100% yield .
patented benzimidazole ring formation by cyclization of the 3-carboxyl group of 5-bromo-1h - pyrazolo[3,4-b]pyridine-3-carboxylic acid ( 3 ) with substituted 1,2-diaminobenzenes was realized via amide formation using coupling reagents , followed by treatment with glacial acetic acid .
hatu ( n , n , n,n-tetramethyl - o-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate ) was used as a coupling reagent .
three 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines ( l1l3 ) ( where x = h , br ; and y = h , ch2och3 ) have been synthesized in this work ( see chart 1 ) : 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridine ( l1 ) is a new compound that we have prepared as a model ligand for coordination to metals ; 3-(1h - benzimidazol-2-yl)-5-bromo-1h - pyrazolo[3,4-b]pyridine ( l2 ) was previously known as boc - protected compound prepared via 5-bromo-1h - pyrazolo[3,4-b]pyridine-3-carboxaldehyde;5-bromo-3-(4-methoxymethyl-1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridine ( l3 ) was synthesized via 5-bromo-1h - pyrazolo[3,4-b]pyridine-3-carboxylic acid ( 3 ) and patented as a potential cdk inhibitor and antiproliferative agent . for the synthesis of l1l3 , we used n , n-carbonyldiimidazole ( cdi ) as an amide - coupling reagent , because it is relatively inexpensive and the side products , carbon dioxide and imidazole , could be easily removed from the reaction mixture .
cdi - mediated amidation of acids 1 and 3 was performed as shown in scheme 3 ( steps i and ii ) . in the first step ,
the acyl - imidazolides 6 and 7 were obtained in dry dmf at room temperature and isolated as white solids .
the yields based on 3-methyl-1h - pyrazolo[3,4-b]pyridine are : 2529% ( 6 ) and 1316% ( 7 ) . in the second step , monoacylation of phenylenediamines ( 1,2-diaminobenzene and 1-methoxymethyl-2,3-diaminobenzene ( 5 ) ) using acyl - imidazolides was performed in dmf at 8085 c .
reagents and conditions : ( i ) cdi , dry dmf , room temperature , 2024 h ; ( ii ) 1,2-diaminobenzene or 5 , dry dmf , 8085 c ; 520 h ; and ( iii ) glacial acoh , 120125 c , 2.54.5 h , chromatographic purification .
amides 810 were used without further purification in ring closure reactions in a glacial acetic acid at 120125 c and afforded the desired 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines : l1 ( 5560% ) , l2 ( 5861% ) , and l3 ( 4451% ) , based on 6 and 7 ( see scheme 3 , step iii ) .
the reported synthesis of l3 is a one - pot approach for amide formation using hatu with 57% yield , followed by cyclization under acidic conditions ( acetic acid ) with 87% yield .
thus , the ligands l1 , l2 , and l3 have been prepared in 7 , 8 , and 11 steps , correspondingly .
finally , the ligands l1l3 were reacted with [ mcl2(-p - cymene)]2 ( where m = ru , os ) in a 2:1 molar ratio in dry ethanol at room temperature to give [ mcl(-arene)(l)]cl complexes ( 11a , 11b , 12a , 12b , 13a , 13b ) in quantitative yields .
crystallization of [ rucl(-p - cymene)(l3)]cl ( 13a ) in etoh or etoh / et2o resulted in xrd - quality crystals of [ rucl(-p - cymene)(l3h ) ] ( 13c ) , while the crystallization of 13a in methanol afforded crystals of composition [ rucl(-p - cymene)(l3)]cl[rucl(-p - cymene)(l3h)]ch3oh ( 13dch3oh ) .
the osmium(ii ) analogue 13e0.75ch3oh0.25h2o was obtained via the crystallization of 13b in methanol . the full assignment of proton , carbon , and nitrogen resonances for l1l3 , 11a , 11b , 12a
, 12b , 13a , and 13b is quoted in tables s1s3 in the supporting information .
3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines with an unsubstituted benzimidazole moiety ( l1 , l2 ) display one set of signals .
l1 is characterized by three doublets of doublets for h4a , h5a , h6a ( 8.85 , 7.39 , 8.66 ppm , correspondingly ) of pyrazolopyridine moiety , two doublets for h4b , h7b ( 7.54 and 7.75 ppm ) , the overlapped h5b , h6b proton signals in one multiplet ( 7.24 ppm ) for a benzimidazole moiety and two singlet resonances for h1a and h1b ( for atom numbering , see chart 1 ) . the substitution of h5a by bromine ( l2 ) results in reduced multiplicity for the h4a and h6a signals ( two doublets at 8.99 and 8.75 ppm ) , whereas the benzimidazole moiety spectrum remains almost unchanged .
two singlets were attributed to nh protons and related to pyrazolopyridine ( h1a , at 14.19 ( l1 ) , 14.43 ( l2 ) ppm ) and benzimidazole ( h1b , at 13.11 ( l1 ) , 13.19 ( l2 ) ppm ) moieties .
nmr spectra for 11a , 11b , 12a , and 12b , where l1 and l2 coordinate as bidentate ligands via n2a and n3b with the formation of a stable five - membered chelate cycle , show one set of signals .
there was no evidence for monodentate or tridentate coordination of ligands with the formation of dinuclear or polynuclear complexes .
coordination of l1 and l2 to ruthenium(ii ) made it possible to assign the two doublets to proton resonances of h4b and h7b of the benzimidazole moiety . in the h ,
h roesy plots , one of them has cross - peaks with a ch cymene ring and the nearest to them is h4b ( e.g. , at 8.11 ( 11a ) , 8.06 ( 12a ) ppm ) .
a singlet at 14.91 ( 11a ) , 14.34 ( 12a ) ppm was assigned to nh proton and showed no couplings with other atoms .
the nitrogen resonance shift at 128.7 ( 11a ) , 127.3 ( 12a ) ppm is closer to the benzimidazole nh chemical shift in metal - free ligands ( 121.3 ( l1 , l2 ) ppm ) and , therefore , was assigned as n1b ( see table s3 in the supporting information ) .
the h1b resonance shows a significant shift by 1.8 and 1.15 ppm for 11a and 12a , respectively , upon ligand coordination ( l1 and l2 ) to the metal(ii)-arene moiety .
the nearest to the metal binding site pyrazolopyridine proton h1a was not detected for 11a , 11b , 12a , and 12b , and the proton resonance of h1b was also not seen for 11b or 12b .
l3 displays two sets of signals originated from 7b - l3 and 4b-l3 tautomers ( see chart 1 ) . the signals of pyrazolopyridine moieties are partially overlapped ( e.g. , h1a ( h1a ) at 14.47 and 14.43 ppm , h4a ( h4a ) at 8.99 and 8.98 ppm , h6a ( h6a ) at 8.75 ppm ) , whereas the signals of the benzimidazole moieties are better resolved ( see table s1 in the supporting information ) .
according to the h , h roesy plot , one of the ch2 groups at 4.80 ppm couples with the nh proton at 13.25 ppm , indicating their attribution to the 7b - l3 tautomer , whereas another nh proton at 13.22 ppm gives a cross - peak with h7b at 7.47 ppm and belongs to 4b-l3 tautomer ( figure 1 ) .
tautomers 7b - l3 and 4b-l3 are present in solution in 1:1.3 molar ratio and give , for the substituted benzimidazole moiety , two singlets ( h1b at 13.22 ppm , h1b at 13.25 ppm ) , two doublets of doublets ( h7b at 7.47 ppm , h4b at 7.72 ppm ) , one multiplet ( h5b , h6b , h5b , h6b at 7.287.21 ppm ) , two ch2 ( 4.80 ( h10b ) , 4.96 ( h10b ) ppm ) and two ch3 singlets at 3.37 ( h11b ) and 3.44 ( h11b ) ppm .
the absence of cross - peaks between h7b and h4b in the h , h cosy plot supports their relation to the two different molecules . the binding site in 4b-l3 tautomer is sterically shielded by a methoxymethyl group .
consistent with this nmr spectra display , only one set of signals is shown for 13a and 13b with the coordination of the 7b - l3 tautomer to ruthenium(ii ) and osmium(ii ) via n2a and n3b ( see scheme 4 ) .
the preference for coordination of the 7b - l3 tautomer is also confirmed by h , h roesy plots : h4b ( 7.99 ( 13a ) , 7.91 ( 13b ) ppm ) has couplings with ch protons of cymene ring .
the cross - peak between h10b at 4.88 ppm ( 13a ) and nh at 13.88 ppm ( 13a ) enabled the assignment of this singlet to a benzimidazole moiety ( h1b ) .
in addition , the chemical shifts for the c atoms of the benzimidazole moiety ( c4b , c7b , c5b , c6b ) of the coordinated ligand and metal - free 7b - l3 tautomer are very similar ( see table s2 in the supporting information ) .
the nitrogen resonance at 122.9 ppm ( 13a ) compares well to the benzimidazole nh chemical shift in metal - free l3 at 121.4 ppm . as for 11a , 11b , 12a , and
12b , the nearest to the coordination place pyrazolopyridine proton h1a resonance was not detected .
according to the h , h roesy plots of 11a , 12a , 13a , and 13b only ch cymene ring protons have couplings with the nearest h4b benzene ring proton , suggesting that the isopropyl or methyl groups are further away from the h4b proton .
the results of the xrd studies of [ oscl(-p - cymene)(l1)]cl4h2o ( 11b4h2o ) , [ oscl(-p - cymene)(l2)]cl ( 12b ) , [ oscl(-p - cymene)(l2)]cl2ch3oh2h2o ( 12b2ch3oh2h2o ) , [ rucl(-p - cymene)(l3h ) ] ( 13c ) , [ rucl(-p - cymene)(l3)]cl[rucl(-p - cymene)(l3h)]ch3oh ( 13dch3oh ) and [ oscl(-p - cymene)(l3)]cl[oscl(-p - cymene)(l3h)]0.75ch3oh0.25h2o ( 13e0.75ch3oh0.25h2o ) are shown in figures 2 , figure s2 in the supporting information , and figures 36 , respectively .
all complexes have a typical  three - legged piano - stool  geometry of ruthenium(ii ) and osmium(ii ) arene complexes , with an  -bound p - cymene ring forming the seat and three other donor atoms ( two nitrogens n1 and n5 of the corresponding 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridine and one chlorido ligand ) as the legs of the stool . selected bond distances and angles are quoted in the figure captions .
all complexes crystallize as racemates , because of the presence of the stereogenic metal center .
fragment of the crystal structure of 11b4h2o showing nitrogen atoms n3 and n4 , which act as proton donors in intermolecular hydrogen bonding interactions n3hcl2 [ n3cl2 3.067(7 )  , n3hcl2 177.3 ] and n4ho3 [ n4o3 2.671(9 )  , n4ho3 170.9 ] ; thermal ellipsoids have been drawn at 50% probability level .
selected bond lengths and angles : os  cl1 , 2.421(2 )  ; os 
c(arene)av , 2.198(32 )  ; c1n2 , 1.349(11 )  ; n2n1 , 1.366(10 )  ; n1c6 , 1.357(11 )  ; c6c7 , 1.428(12 )  ; c7n5 , 1.345(11 )  ; n5c13 , 1.391(11 )  ; n1os  n5 , 75.6(3) ; n1os  cl1 , 85.5(2) ; n5os  cl1 , 83.4(2). the bidentate 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines , which can have different substituents in positions 5a and 7b , reveal different acid  base properties .
it can act as a neutral organic ligand , with nitrogen atoms n2 and n4 as proton donors involved in intermolecular hydrogen bonding interactions n4ho1(x + 1 , y + 1 , z + 2 ) [ n4o1 , 2.730(4 )  ; n4ho1 , 176.1 ] and n2ho3(x + 2 , y + 1 , z + 1 [ n2o3 , 2.697(4 )  ; n4ho1 , 172.3 ] with one methanol and one water molecule , respectively , as is the case for 12b2ch3oh2h2o ( figure 3 ) or n2hcl2(cl2x ) and n4hcl1(x , y , z + 2 ) [ n4cl1 , 3.213(9 )  ; n4ho1 , 162.91 ] in 12b ( see figure s2 in the supporting information ) , correspondingly .
the ligand can be protonated at n3 ( n3hcl2 ) and deprotonated at n2 [ n2h  o4 ( x + 1 , y + 1 , z + 1 ) with n2o4 2.923(9 )  , n2h
 o4 138.7 ] ( overall charge zero ) with atom n4 as a proton donor to one of the four co - crystallized water molecules n4ho3 , as occurs in 11b4h2o ( see figure 2 ) .
ortep plot of the structure of the cation [ oscl(-p - cymene)(l2 ) ] in 12b2ch3oh2h2o .
thermal ellipsoids have been drawn at the 50% probability level . selected bond lengths and angles : os  cl1 , 2.4043(1 )  ; os 
c(arene)av , 2.197(26 )  ; c1n2 , 1.361(5 )  ; n2n1 , 1.344(4 )  ; n1c6 , 1.336(5 )  ; c6c7 , 1.447(6 )  ; c7n5 , 1.335(5 )  ; n5c13 , 1.380(5 )  ; n1os  n5 , 74.73(13) , n1os  cl1 , 84.29(10) ; and n5os  cl1 , 83.25(10). in 13c , the ligand was found to be deprotonated at n2 , acting as a proton acceptor in the intermolecular hydrogen bonding interaction n2h  n4 ( i denotes symmetry transformations x , y + 1.5 , z + 0.5 ) used to generate equivalent atoms ) [ n2n4 , 2.896(7 )  ; n2h  n4 , 155.6 ] ( see figure 4 ) . ortep plot of the complex [ rucl(-p - cymene)(l3h ) ] ( 13c ) .
thermal ellipsoids have been drawn at 30% probability level . selected bond lengths and angles : ru  cl , 2.399(2 )  ; ru  n1 ,
c(arene)av , 2.186(31 )  ; c1n2 , 1.366(8 )  ; n2n1 , 1.353(7 )  ; n1c6 , 1.362(7 )  ; c6c7 , 1.426(9 )  ; c7n5 , 1.335(8 )  ; n5c13 , 1.398(8 )  ; n1ru  n5 , 75.9(2) ; n1ru  cl , 87.35(17) ; and n5ru  cl , 85.73(17). complexes 13dch3oh and 13e0.75ch3oh0.25h2o crystallized both in the centrosymmetric triclinic space group p1. the asymmetric unit in both consists of a neutral complex [ mcl(-p - cymene)(l3h ) ] and a complex cation [ mcl(-p - cymene)(l3 ) ] ( m = ru or os ) , a chloride counteranion and co - crystallized solvent ( methanol or methanol / water )
. deprotonation of the organic ligand in [ mcl(-p - cymene)(l3h ) ] is corroborated by the presence of hydrogen bonding of the type n2ah  n2b ( x + 1 , y + 1 , z + 1 ) in the crystal structure of the ruthenium complex 13dch3oh ( figure 5 ) and a similar interaction n2bh  n2a ( x , y + 1 , z ) [ n2bn2a , 2.810(9 )  ; n2bh  n2a , 170.1 ] in the crystal of the osmium analogue 13e0.75ch3oh0.25h2o . in figure 6 the structure of [ oscl(-p - cymene)(l3h ) ]
is shown . in both structures , the atoms n4a and n4b act as proton donors in strong hydrogen bonding interactions to the chloride counteranion , namely , n4ah 
cl2 ( x + 1 , y + 2 , z + 1 ) [ n4cl2 , 3.275(6 )  ; n4ah  cl2 , 176.9 ] , n4bh 
cl2 [ n4bcl2 , 3.211(5 )  ; n4bh  cl2 , 176.0 ] ( 13dch3oh ) and n4ah  cl2 ( x + 1 , y + 1 , z + 1 ) [ n4acl2 , 3.273(8 )  ; n4ah  cl2 , 177.0 ] , n4bh 
cl2 [ n4bcl2 , 3.204(7 )  ; n4bh  cl2 , 177.0 ] ( 13e0.75ch3oh0.25h2o ) .
fragment of the crystal structure of [ rucl(-p - cymene)(l3)]cl[rucl(-p - cymene)(l3h)]ch3oh ( 13dch3oh ) showing the intermolecular hydrogen bonding n2a  hn2b [ n2an2b ( x + 1 , y + 1 , z + 1 ) , 2.814(7 )  ; n2a  hn2b , 163.3 ] . selected bond lengths and angles : ru1a  cl1a , 2.3952(17 )  ; ru1a 
c(arene)av , 2.194(33 )  ; c1a  n2a , 1.358(8 )  ; n2a  n1a , 1.354(7 )  ; n1a  c6a , 1.350(8 )  ; c6a  c7a , 1.441(9 )  ; c7a  n5a , 1.335(8 )  ; n5a  c13a , 1.381(8 )  ; n1a  ru1a  n5a , 75.6(2) ; n1a 
ru1a  cl1a , 84.45(15) ; and n5a  ru1a  cl1a , 85.05(15). ortep plot of [ oscl(-p - cymene)(l3h ) ] in [ oscl(-p - cymene)(l3)]cl[oscl(-p - cymene)(l3h)]0.75ch3oh0.25h2o ( 13e0.75ch3oh0.25h2o ) .
selected bond lengths and angles : os1a  cl1a , 2.402(2 )  ; os1a  n1a , 2.068(7 )  ; os1a  n5a , 2.086(7 )  ; os1a  c(arene)av , 2.192(25 )  ; c1a  n2a , 1.349(10 )  ; n2a  n1a 1.369(9 )  ; n1a  c6a , 1.361(10 )  ; c6a  c7a , 1.431(11 )  ; c7a  n5a , 1.339(10 )  ; n5a  c13a , 1.398(11 )  ; n1a  os1a  n5a , 75.1(3) ; n1a 
os1a  cl1a , 83.56(18) ; and n5a  os1a  cl1a , 84.2(2). ligands l1l3 , as well as the corresponding ruthenium(ii ) ( 11a13a ) and osmium(ii ) ( 11b13b ) arene complexes , were studied with regard to their capacity of inhibiting cell growth in vitro in the human cancer cell lines ch1 ( ovarian carcinoma ) , sw480 ( colon carcinoma ) , and a549 ( non - small cell lung cancer ) , yielding the ic50 values listed in table 2 .
all compounds show the strongest effect in the generally chemosensitive ch1 cells , whereas the more chemoresistant a549 cells are also less affected by the compounds investigated here .
> l2 > l1 , indicating that bromination ( l2 , l3 ) and , even more so , the double substitution ( bromination and an additional replacement of h by a methoxymethyl group ( l3 ) ) are advantageous , with regard to activity .
these structure  activity relationships are also reflected in the rank orders of the corresponding ruthenium and osmium complexes for the ruthenium complexes , 13a > 12a > 11a , and for the osmium complexes , 13b > 12b > 11b .
however , the ic50 values are shifted to higher concentrations ( see figure 7 ) .
the differences between the ruthenium and osmium analogues are mostly small ( ic50 values differ only up to a factor of 2.4 ) , with the osmium complexes being at least as active as their ruthenium counterparts .
ch1 denotes ovarian cancer , human ; sw480 denotes colon carcinoma , human ; and a549 denotes non - small - cell lung carcinoma , human .
50% inhibitory concentrations ( mean value  standard deviation from at least three independent experiments ) obtained by the mtt assay ( 96-h exposure ) . concentration
 effect curves of each ligand , compared to the corresponding ruthenium and osmium complexes , in ch1 ovarian cancer cells ( mtt assay , 96 h exposure ) : ( a ) l1 , 11a , 11b ; ( b ) l2 , 12a , 12b ; and ( c ) l3 , 13a , 13b . both the presence of substituents in the ligands and complexation result in a marked shift of antiproliferative activity . since 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines have been reported to be potent cdk inhibitors , we expected the investigated ligands and complexes to induce cell cycle perturbations . to confirm this assumption in a sensitive cell line ,
exponentially growing ch1 cells were treated with the compounds for 24 h , stained with propidium iodide , and analyzed for their dna content by fluorescence - activated cell sorting ( facs ) .
surprisingly , the metal - free ligands l1l3 turned out to exert only modest effects on cell cycle distribution , with a slight increase of the g2/m fraction from 32% in the untreated control to 53% by 20 m l2 as the strongest effect observed in this setting ( higher concentrations of this compound led to disintegration of cells already after 24 h ) .
however , the less cytotoxic ruthenium complexes 12a and 13a , both bearing substituted ligands ( l2 and l3 correspondingly ) , cause a more pronounced g2/m phase arrest , as reflected by an increase of this cell fraction to 65% at 80 m and 59% at 40 m , respectively , and a concomitant decrease of the g0/g1 fraction to 21% and 24% ( compared to 42% in controls ) , whereas ruthenium complex 11a bearing an unsubstituted ligand l1 is devoid of activity on the cell cycle . on the other hand
, the osmium complexes do not generally show stronger effects on the cell cycle than the metal - free ligands , perhaps with the exception of 13b , which induces an increase of the g2/m fraction up to 53% at 80 m , accompanied by a decline of the g0/g1 fraction to 31% ( see figure 8) .
concentration - dependent effects of metal - free ligands ( top ) , and the corresponding ruthenium ( middle ) and osmium complexes ( bottom ) , on the cell cycle distribution of ch1 cells ( (  ) g0/g1 , (  ) s , and (  ) g2/m phase ) .
although the lack of generally pronounced cell cycle effects does not argue for a strong role of cdk inhibition in the mechanism of action of the investigated compounds , inhibitory potencies were studied in cell - free experiments with two recombinant cdk / cyclin complexes , by means of the histone h1 kinase assay .
results reveal that all compounds are capable of inhibiting kinase activities in a concentration - dependent manner , being more effective on cdk2/cyclin e than on cdk1/cyclin b ( see figure 9 ) .
in contrast to the observed cell cycle effects , but in accordance with antiproliferative activities , cdk - inhibitory potency of the metal - free ligands is consistently higher than that of the metal complexes . in particular , only l1l3 effectively inhibit cdk2/cyclin e in low concentrations ( 1 m or 10 m ) , whereas all complexes require higher concentrations to exert > 50% inhibitory effects . as in the mtt assay ,
differences between the effects of corresponding ruthenium and osmium complexes are minor , compared to the differences from those of the metal - free ligands .
concentration - dependent effects of metal - free ligands ( l1l3 ) as well as corresponding ruthenium ( 11a13a ) and osmium complexes ( 11b13b ) , on kinase activities of recombinant cdk1/cyclin b ( top ) and cdk2/cyclin e ( bottom ) .
the known multistep synthesis of 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines , which we have modified , essentially afforded three organic compounds ( l1l3 ) that possess cdk - inhibiting properties .
these were used as bidentate ligands , and six novel organometallic complexes of the general formula [ mcl(-p - cymene)(l)]cl , where m = ru ( 11a , 12a , 13a ) or os ( 11b , 12b , 13b ) and l = l1l3 , have been synthesized and comprehensively characterized using spectroscopic and x - ray diffraction methods .
complexation of l1l3 with ruthenium or osmium yielded compounds with increased solubility in the biological medium , yet lowered antiproliferative activity in human cancer cell lines .
modulation of biological activities by substitution at the ligands can be accomplished in the metal - free molecules and their metal complexes in a similar way .
the known cdk - inhibitory activity of the ligands could be confirmed in cell - free experiments , in particular in cdk2/cyclin e , and their stronger effects on cdk activity parallel their higher capacity of inhibiting cancer cell growth in vitro , compared to their metal complexes .
nevertheless , the lack of pronounced effects on the cell cycle of chemosensitive ovarian cancer cells argues against a major role for inhibition of cell growth , at least in this setting .
 six organometallic complexes of the general formula [ miicl(6-p - cymene)(l)]cl , where m = ru ( 11a , 12a , 13a ) or os ( 11b , 12b , 13b ) and l = 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines ( l1l3 ) have been synthesized . 
 the latter are known as potential cyclin - dependent kinase ( cdk ) inhibitors . 
 all compounds have been comprehensively characterized by elemental analysis , one- and two - dimensional nmr spectroscopy , uv  vis spectroscopy , esi mass spectrometry , and x - ray crystallography ( 11b and 12b ) . 
 the multistep synthesis of 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines ( l1l3 ) , which was reported by other researchers , has been modified by us essentially ( e.g. , the synthesis of 5-bromo-1h - pyrazolo[3,4-b]pyridine-3-carboxylic acid ( 3 ) via 5-bromo-3-methyl-1h - pyrazolo[3,4-b]pyridine ( 2 ) ; the synthesis of 1-methoxymethyl-2,3-diaminobenzene ( 5 ) by avoiding the use of unstable 2,3-diaminobenzyl alcohol ; and the activation of 1h - pyrazolo[3,4-b]pyridine-3-carboxylic acids ( 1 , 3 ) through the use of an inexpensive coupling reagent , n , n-carbonyldiimidazole ( cdi ) ) . 
 stabilization of the 7b tautomer of methoxymethyl - substituted l3 by coordination to a metal(ii ) center , as well as the nmr spectroscopic characterization of two tautomers 7b - l3 and 4b-l3 in a metal - free state are described . 
 structure  
 activity relationships with regard to cytotoxicity and cell cycle effects in human cancer cells , as well as cdk inhibitory activity , are also reported . 
Introduction
Experimental Section
Results and Discussion
Conclusion