discoidin domain receptors ( i.e. , ddr1 
 and ddr2 ) are transmembrane 
 receptor tyrosine kinases ( rtks ) that specifically recognize fibrillar 
 collagens as extracellular ligands . ddr1 and ddr2 are highly 
 involved in fundamental cellular processes , including cell proliferation , 
 migration , adhesion , and matrix remodeling . the dysregulation of ddr1 has been linked to a variety of human 
 cancers and inflammatory conditions such as fibrotic disorders and 
 atherosclerosis . collective evidence indicates a critical link between ddr1 and pulmonary 
 fibrosis , a lethal disease with few therapeutic options . for instance , a ddr1 deletion has been reported to alleviate bleomycin 
 ( blm)-induced lung inflammation and pulmonary fibrosis by blocking 
 p38 mitogen - activated protein kinase ( p38 mapk ) activation . we and 
 others have identified several classes of ddr1 inhibitors 
 with different selectivity profiles and have demonstrated their therapeutic 
 potential for various human cancers ( figure 1 ) . however , these small molecules 
 still have relatively poor target specificity and there are limited 
 reports on the efficacy of the pharmacologic inhibition of ddr1 in 
 models of pulmonary fibrosis . in this 
 article , we report the structure - based design of tetrahydroisoquinoline 
 derivatives as new highly selective ddr1 inhibitors with promising 
 therapeutic effects in a blm - induced pulmonary fibrosis mouse model . selective 
 ddr1/ddr2 kinase inhibitors . ddr1 shares approximately 61% sequence 
 identity with abelson ( abl ) 
 kinase in its adenosine triphosphate ( atp ) binding domain , and most 
 reported selective ddr1 inhibitors are derivatized from abl antagonists . previous investigations revealed that a  
 stacking interaction between the chemical molecule and tyr of abl is critical for most of the reported abl inhibitors ( supporting 
 information ( si ) , figure s1a , b , c ) , but the corresponding interaction is unnecessary for ddr1 binding . 
 indeed , ddr1-in-1 achieved selective ddr1 inhibition because its hinge 
 binding moiety is oriented away from the p - loop , avoiding the potential 
  interactions with tyr in abl . however , the molecule could form an additional 
  interaction with phe in abl , 
 which contributed greatly to its relatively low selectivity between 
 ddr1 and abl ( si , figure s1d ) . diminishing this interaction a series of 1 , 2 , 3 , 4-tetrahydroisoquinoline 
 derivatives were designed as novel , selective ddr1 inhibitors in which 
 a pyrimidinyl group was utilized as the potential hinge binding moiety . a n-(3-((4-methylpiperazin-1-yl)methyl)-5-(trifluoromethyl ) 
 phenyl)carboxamide group was also introduced based on our previous 
 investigation ( figure 2a ) . ( b ) molecular 
 docking of 6a ( cyan ) into the ddr1-ponatinib ( yellow ) 
 costructure ( pdb i d : 3zos ) . ( c ) co - crystal structure of 6c with ddr1 ( pdb i d : 5fdp ) . ( e ) superposition of 6c with abl ( pdb i d : 3ik3 ) . our preliminary modeling suggested 
 that the initial lead , 6a , could fit nicely into the 
 ddr1 binding pocket and maintain 
 the key interactions with ddr1 . the pyrimidinyl moiety of 6a could form an essential hydrogen bond with the nh of met in the hinge region of ddr1 . two additional hydrogen bonds have 
 also formed between the linker amide and glu in the 
 c - helix and asp in the asp - phe - gly ( dfg ) motif , respectively 
 ( figure 2b ) . encouragingly , 
 compound 6a failed to dock into the abl binding pocket , 
 suggesting its potential selectivity among kinases . briefly , the substituted 
 methyl 4-(2-(2,2,2-trifluoroacetamido)ethyl)benzoate ( 8) was prepared by the trifluoroacetylation of methyl 4-(2-aminoethyl)benzoate 
 ( 7 ) and then underwent a classical pictet  spengler 
 reaction to yield the protected tetrahydroisoquinoline derivatives 
 ( 9 ) , which were deprotected and reacted with a hydrochloric 
 acid solution in meoh to form the key intermediate ( 10 ) . compound 10 was coupled with 5-bromopyrimidine , 3-bromopyridine , 
 or bromobenzene through a buchwald the final products were obtained 
 by treating intermediate 11 with different anilines under 
 basic conditions . reagents and conditions : ( a ) 
 trifluoroacetic anhydride , 0 c to rt , 5863% ; ( b ) ( hcho)n , conc h2so4 , 0 c 
 to rt , 5189% ; ( c ) ( i ) k2co3 , meoh / h2o ( 2:1 ) , rt , ( ii ) hclmeoh , meoh , rt , 8695% ( two 
 steps ) ; ( d ) 5-bromopyrimidine or 3-bromopyridine or bromobenzene , 
 pd(dba)2 , ruphos , cs2co3 , toluene , 
 80 c , 4289% ; ( e ) substituted aniline , t - buok , thf , 20 c to rt , 6986% . ddr1 inhibition of the compounds was determined using 
 a well - established 
 lance ultra kinase assay . the potential 
 target selectivity was additionally evaluated by monitoring their 
 inhibition against abl . compound 1 was included as a 
 positive control , which displayed similar ic50 values to 
 the previously reported data . it was 
 shown that 6a exhibited modest ddr1 inhibitory activity , 
 with an ic50 value of 442 nm , while its potency against 
 abl was strikingly inferior ( ic50 > 10.0 m ) . further 
 computational investigation suggested that a small hydrophobic 
 recess formed by val , ala , and met was available in the atp binding pocket of ddr1 ( figure 2b ) . a lipophilic 
 substituent at r1 may occupy this pocket to achieve improved 
 potency . the ( r)-methyl ( 6b ) and ( r)-ethyl derivatives ( 6d ) displayed a 20-fold 
 and 12-fold potency improvement , respectively . the ( s)-methyl compound ( 6c ) , in which the methyl moiety was 
 oriented away from the pocket , as confirmed by a 2.3  cocrystal 
 structure with ddr1 ( figure 2c ) , displayed a similar inhibitory potency to that of the 
 r1 unsubstituted 6a . a large group at the 
 r1 position was predicted to be detrimental to the binding 
 of ddr1 . as expected , r1-(r)-isopropyl 
 ( 6f ) caused a substantial potency loss for ddr1 . additionally , 
 compounds 6e and 6 g , which featured ( s)-ethyl and ( s)-isopropyl , respectively , 
 displayed almost no ddr1 inhibition . the x - ray crystal structure also 
 confirmed the presence of a strong hydrogen bonding network between 
 the new inhibitor and ddr1 ( figure 2c ) . the deletion of a hydrogen bond by eliminating 
 the n atoms in the pyrimidinyl group ( 6i ) totally abolished ddr1 inhibitory potency . not surprisingly , when 
 the pyrimidinyl group was replaced by a pyridinyl moiety , the resulting 
 compound , 6h , exhibited an almost identical ic50 value to that of 6a . it was also noteworthy that 
 all the new ddr1 inhibitors exhibited 
 excellent ddr1 selectivity over the structurally related abl kinase 
 ( table 1 ) . to rationalize 
 this target selectivity , the inhibitor 6c was superimposed 
 into the abl structure ( pdb i d : 3ik3 ) ( figure 2e ) . it was shown that the 1 , 2 , 3 , 4-tetrahydroisoquinoline 
 scaffold in 6c forced the pyrimidinyl moiety to adopt 
 a different dihedral angle that prevented the formation of critical 
 interactions with tyr and phe in abl . moreover , the distance between the n atom in the 
 pyrimidinyl group and met in abl was predicted to be 
 4.09  , which exceeds the limit to form a potential hydrogen 
 bond . ddr1 experiments 
 were performed 
 using the lance ultra kinase assay , according to the manufacturer s 
 instructions . abl1 activity 
 experiments were performed 
 using the fret - based z - lyte assay , according to the manufacturer s 
 instructions . further structural optimization 
 of the inhibitor 6b yielded 6j as a promising 
 candidate , with an ic50 value of 9.4 nm against ddr1 ( table 1 ) . the compound also 
 exhibited reasonable 
 pharmacokinetic ( pk ) properties , with an oral bioavailability of 66.8% 
 and a t1/2 value of 1.25 h at an oral 
 dose of 20 mg / kg ( table 2 ) in rats . the pk profile of compound 6j was also investigated 
 in icr mice , which showed that the compound had a similar oral bioavailability 
 to that of rats . however , the area under concentration  time 
 curve ( auc ) value of the compound in mice was obviously higher than 
 that in rats , suggesting its good absorption property in mice . icr mice 
 ( male , 24 animals per group ) 
 weighing 1830 g were used for the study . sd rats ( male , 3 animals per group ) 
 weighing 180220 g were used for the study . the ddr1 inhibition of 6j was further validated by 
 determining its binding affinity with the ddr1 protein ( conducted 
 by discoverx , san diego , ca ) . it was 
 shown that 6j bound tightly to ddr1 , with a binding constant 
 ( kd ) value of 4.7 nm . the target specificity 
 of 6j was also investigated by conducting a kinase selectivity 
 profiling study against a panel of 468 kinases ( including 403 nonmutated 
 kinases ) at 1.0 m , which is approximately 210-fold above its kd value against ddr1 using the discoverx screening 
 platform . it was shown that 6j displayed excellent target 
 selectivity , with s(10 ) and s(1 ) scores of 0.022 and 0.012 , respectively 
 ( table s5 ) . the potential  off - target  kinases tested included 
 cyclin - dependent kinase 11 ( cdk 11 ) , ddr2 , ephrin type - b receptor 
 8 ( ephb8 ) , muscle - specific receptor tyrosine kinase ( musk ) , nerve 
 growth factor receptor a ( trka ) , trkb , and trkc . however , the ic50 value of 6j against ddr2 was determined to 
 be 188 nm in our lance ultra kinase assay , indicating that 6j was 20-fold less potent against ddr2 . further determination of the 
 binding affinities ( kd values ) revealed 
 that 6j exhibited an approximately 21120-fold 
 less potency against the majority of the other  off target kinases , with the exception of trkb and trkc , which displayed kd values of 22 and 18 nm , respectively ( figure 3 ) . compound 6j was profiled at a concentration of 1.0 m 
 against a diverse panel of 468 kinases by discoverx . ( b ) binding constants 
 ( kd values ) of compound 6j against the top hits . further investigation revealed that the activation of ddr1 
 as well 
 as its downstream signaling intermediate , p38 , were both dose - dependently suppressed by 6j in primary human lung fibroblasts ( figure 4 ) , suggesting the efficacy of this compound 
 against ddr1-induced signaling . in light of the critical role of ddr1 
 in blm - induced pulmonary fibrosis , we 
 treated mice with compound 6j after the onset of a blm 
 challenge . inhibitor 6j was orally administered at 10 
 and 50 mg / kg twice daily ( bid ) for 2 weeks based on its pk properties 
 ( table 2 ) . unlike those 
 from the phosphate buffered saline ( pbs ) treated mice , which had large 
 alveolar spaces and were weakly stained by masson s trichrome , 
 the lungs from blm - challenged animals exhibited a reduction in alveolar 
 spaces and were stained blue by masson s trichrome , demonstrating 
 typical fibrotic features . the compound prevented these blm - induced 
 pathological changes in a dose - dependent manner ( figure 5a ) . these results agreed with 
 the expression levels of fibrotic markers in lung tissue lysates , 
 including fibronectin and -smooth muscle actin ( sma ) ( figure 5b ) . further analyses also revealed that the administration 
 of compound 6j caused a dose - dependent suppression in 
 the content of hydroxyproline ( figure 5c ) , a unique amino acid found in collagen . the above data collectively indicate the promising 
 therapeutic potential of 6j against the blm - induced pulmonary 
 fibrosis . 6j inhibited ddr1-mediated signaling 
 in a concentration - dependent manner in primary human lung fibroblasts 
 ( 24 h treatment ) . fourteen 
 days after the onset of blm injury , c57bl/6 mice ( five animals each 
 group ) received an oral gavage of 6j twice daily at the 
 indicated dosages , and the lungs were collected on day 28 . the upper 
 panels in ( a ) display the hematoxylin and eosin ( h&e ) staining 
 images of the dissected lungs . the images in ( b ) show the results of the immunoblotting 
 with the indicated antibodies . the histogram in ( c ) shows the determined 
 hydroxyproline content . * * * p < 0.001 . in summary , a series 
 of 1 , 2 , 3 , 4-tetrahydroisoquinoline derivatives 
 were designed as novel highly selective ddr1 inhibitors . compound 6j strongly suppressed ddr1 , with a single digital nm ic50 value , but it is significantly less potent in a panel of 
 400 nonmutated kinases . thus , to the best of our knowledge , this compound 
 represents one of the most selective ddr1 inhibitors to date . the 
 compound also demonstrated reasonable pk properties and a promising 
 oral therapeutic effect in a blm - induced mouse pulmonary fibrosis 
 model . its strong ddr1 inhibitory potency and extraordinary target 
 specificity make compound 6j not only a promising lead 
 compound for new drug discovery but also a valuable research probe 
 for further biological investigation of its target . reagents and solvents 
 were obtained 
 from commercial suppliers and used without further purification . h and c nmr spectra were recorded on a bruker 
 av-400 spectrometer at 400 mhz and bruker av-500 spectrometer at 125 
 mhz . the low or high resolution of esi - ms was recorded on an agilent 
 1200 hplc - msd mass spectrometer or applied biosystems q - star elite 
 esi - lc - ms / ms mass spectrometer , respectively . the purity of compounds 
 was determined to be over 95% ( > 95% ) by reverse - phase high performance 
 liquid chromatography ( hplc ) analysis . hplc instrument : dionex summit 
 hplc ( column , diamonsil c18 , 5.0 m , 4.6 mm  250 mm ( dikma 
 technologies ) ; detector , pda-100 photodiode array ; injector , asi-100 
 autoinjector ; pump , p-680a ) . elution : 85% meoh in water with 0.1% 
 modifier ( ammonia , v / v ) ; flow rate , 1.0 ml / min .
the structure - based design of 1 , 
 2 , 3 , 4-tetrahydroisoquinoline 
 derivatives as selective ddr1 inhibitors is reported . 
 one of the representative 
 compounds , 6j , binds to ddr1 with a kd value of 4.7 nm and suppresses its kinase activity with 
 an ic50 value of 9.4 nm , but it is significantly less potent 
 for a panel of 400 nonmutated kinases . 
 6j also demonstrated 
 reasonable pharmacokinetic properties and a promising oral therapeutic 
 effect in a bleomycin - induced mouse pulmonary fibrosis model .