A change of hydrophilic 1-(4-methyl) piperazinylmethyl moiety from R2 to R3 position (7t) obviously improved the DDR1 inhibitory activity with an IC50 value of 19.9 nM, but the selectivity over DDR2 and Bcr-Abl was significantly decreased. were first utilized for the structureCactivity relationship (SAR) exploration. Inhibitory Activities of Compounds 7aC7j against DDR1, DRR2, Bcl-Abl, and c-Kita Open in a separate window Open in a separate window aDDR1 and DDR2 experiments were performed using LANCE ULTRA kinase assay according to the manufacturers instructions. Bcr-Abl and c-Kit activity experiments were performed using the f?rster resonance energy transfer (FRET)-based Z-Lyte assay according to the manufacturers instructions. All the data are mean values from at least three independent experiments. Our previous investigation suggested that a hydrogen bond between the pyrimidinyl moiety of 7a or 7b with the NH of Met704 in the hinge region of DDR1 was critical for the compounds to exhibit strong DDR1 inhibition.20 Not surprisingly, replacement of the hinge binding 5-pyrimidinyl group (7a) by a 4-pyrimidinyl (7d) or a 5-pyrimidinylmethyl moiety (7e) caused total loss of the DDR1 inhibitory activity, which likely results from unfavorable orientations of the heterocyclic heads that prevent the formation of critical hydrogen bonds. Our previous study also revealed that the (Inhibitory Activities of Compounds 7kC7af against DDR1, DRR2, Bcl-Abl, and c-Kita Open in a separate window Open in a separate window aDDR1 and DDR2 experiments were performed using LANCE ULTRA kinase assay according to the manufacturers instructions. Bcr-Abl and c-Kit activity experiments were performed using the f?rster resonance energy transfer (FRET)-based Z-Lyte assay according to the manufacturers instructions. All NVS-PAK1-1 the data are mean values from at least three independent experiments. Further investigation demonstrated that a hydrophilic group at R2 also contributed greatly to the DDR1 kinase inhibition. When the 1-(4-methyl) piperazinylmethyl moiety was removed, the resulting compound 7r exhibited an IC50 value of 191 nM against DDR1, which was approximately 5-fold less potent than the original compound 7c. When 1-(4-methyl)piperazinylmethyl moiety was moved at R5 position, the resulting compound 7s totally abolished the kinase activity. A change of hydrophilic 1-(4-methyl) piperazinylmethyl moiety from R2 to R3 position (7t) obviously improved the DDR1 inhibitory activity with an IC50 value of 19.9 nM, but the selectivity over DDR2 and Bcr-Abl was significantly decreased. It was also found that the 1-(4-methyl)piperazinyl group at R2 position could be replaced by a 1-(4-methyl)piperazinyl (7u), 1-(4-methyl)piperazinylethyl (7v), 1-(4-ethyl)piperazinylmethyl (7w), or 1-(4-cyclohexyl)piperazinylmethyl (7x) to maintain strong DDR1 inhibitory activities with IC50 values ranging from 71.1 to 132 nM. However, when the 1-(4-methyl)piperazinylmethyl group was replaced by a morpholinomethyl (7y), thiomorpholinomethyl (7z), piperidin-1-ylmethyl (7aa), pyrrolidin-1-ylmethyl (7ab), or dimethylaminomethyl (7ac) substituent, the resulting compounds were 4.4C6.6-fold less potent than 7c. This might be rationalized by the fact that all of the new molecules lacked a solvent-exposing anti-inflammatory activity. Open in a separate window Figure 4 Compound 7ae inhibited LPS-induced IL-6 and TNF- release in a dose-dependent manner in MPMs. Each bar represents mean SE of 3C5 independent experiments. Statistical significance relative to LPS is indicated: * 0.05, ** 0.01. The therapeutic potential of 7ae was further studied in a LPS-induced ALI model.28 Compound 7ae was orally administered at 20 or 40 mg/kg twice daily (BID) based on its pharmacokinetics (PK) parameters (Table S3) for 7 days prior to the administration of LPS (20 L, 5 mg/kg). It was evident that pretreatment with compound 7ae markedly reduced the LPS-induced pulmonary edema as determined by lung wet/dry (W/D) ratio (Figure ?Figure55A). Meanwhile, the total cell number and total protein concentration in bronchial alveolar lavage fluid (BALF) were increased amazingly after LPS administration compared to the control group (Number ?Number55B,C). Administration of 7ae dose-dependently inhibited the LPS-induced increase in total cell number and total protein concentration in BALF (Number ?Number55B,C). LPS treatment also resulted in significant pulmonary congestion, thickening of alveolar wall, and interstitial edema (Number ?Number55D). These pathological changes were also markedly reduced from NVS-PAK1-1 the administration of 7ae (Number ?Number55D). Open in a separate window Number 5 Compound 7ae attenuated LPS induced ALI in mice. (A) Lung W/D percentage. (B) Total amount of cells in BALF. (C) Protein concentration in BALF. (D) Hematoxylin and eosin staining. Statistical significance relative to LPS group was indicated: * 0.05, ** 0.01. In summary, an extensive SAR investigation.Inhibitory Activities of Compounds 7aC7j against DDR1, DRR2, Bcl-Abl, NVS-PAK1-1 and c-Kita Open in a separate window Open in a separate window aDDR1 and DDR2 experiments were performed using LANCE ULTRA kinase assay according to the manufacturers instructions. the manufacturers instructions. Bcr-Abl and c-Kit activity experiments were performed using the f?rster resonance energy transfer (FRET)-based Z-Lyte assay according to the manufacturers instructions. All the data are imply ideals from at least three self-employed experiments. Our earlier investigation suggested that a hydrogen relationship between the pyrimidinyl moiety of 7a or 7b with the NH of Met704 in the hinge region of DDR1 was critical for the compounds to exhibit strong DDR1 inhibition.20 Not surprisingly, replacement of the hinge binding 5-pyrimidinyl group (7a) by a 4-pyrimidinyl (7d) or a 5-pyrimidinylmethyl moiety (7e) caused total loss of the DDR1 inhibitory activity, which likely effects from unfavorable orientations of the heterocyclic mind that prevent the formation of critical hydrogen bonds. Our earlier study also exposed the (Inhibitory Activities of Compounds 7kC7af against DDR1, DRR2, Bcl-Abl, and c-Kita Open in a separate window Open in a separate windows aDDR1 and DDR2 experiments were performed using LANCE ULTRA kinase assay according to the manufacturers instructions. Bcr-Abl and c-Kit activity experiments were performed using the f?rster resonance energy transfer (FRET)-based Z-Lyte assay according to the manufacturers instructions. All the data are imply ideals from at least three self-employed experiments. Further investigation demonstrated that a hydrophilic group at R2 also contributed greatly to the DDR1 kinase inhibition. When the 1-(4-methyl) piperazinylmethyl moiety was eliminated, the producing compound 7r exhibited an IC50 value of 191 nM against DDR1, which was approximately 5-fold less potent than the initial compound 7c. When 1-(4-methyl)piperazinylmethyl moiety was relocated at R5 position, the producing compound 7s totally abolished the kinase activity. A change of hydrophilic 1-(4-methyl) piperazinylmethyl moiety from R2 to R3 position (7t) obviously improved the DDR1 inhibitory activity with an IC50 value of 19.9 nM, but the selectivity over DDR2 and Bcr-Abl was significantly decreased. It was also found that the 1-(4-methyl)piperazinyl group at R2 position could be replaced by a 1-(4-methyl)piperazinyl (7u), 1-(4-methyl)piperazinylethyl (7v), 1-(4-ethyl)piperazinylmethyl (7w), or 1-(4-cyclohexyl)piperazinylmethyl (7x) to keep up strong DDR1 inhibitory activities with IC50 ideals ranging from 71.1 to 132 nM. However, when the 1-(4-methyl)piperazinylmethyl group was replaced by a morpholinomethyl (7y), thiomorpholinomethyl (7z), piperidin-1-ylmethyl (7aa), pyrrolidin-1-ylmethyl (7ab), or dimethylaminomethyl (7ac) substituent, the producing compounds were 4.4C6.6-fold less potent than 7c. This might become rationalized by the fact that all of the new molecules lacked a solvent-exposing anti-inflammatory activity. Open in a separate window Number 4 Compound 7ae inhibited LPS-induced IL-6 and TNF- launch inside a dose-dependent manner in MPMs. Each pub represents imply SE of 3C5 self-employed experiments. Statistical significance relative to LPS is definitely indicated: * 0.05, ** 0.01. The restorative potential of 7ae was further studied inside a LPS-induced Rgs4 ALI model.28 Compound 7ae was orally administered at 20 or 40 mg/kg twice daily (BID) based on its pharmacokinetics (PK) parameters (Table S3) for 7 days prior to the administration of LPS (20 L, 5 mg/kg). It was obvious that pretreatment with compound 7ae markedly reduced the LPS-induced pulmonary edema as determined by lung damp/dry (W/D) percentage (Number ?Number55A). Meanwhile, the total cell number and total protein concentration in bronchial alveolar lavage fluid (BALF) were improved amazingly after LPS administration compared to the control group (Number ?Number55B,C). Administration of 7ae dose-dependently inhibited the LPS-induced increase in total cell number and total protein concentration in BALF (Number ?Number55B,C). LPS treatment also resulted in significant pulmonary congestion, thickening of alveolar wall, and interstitial edema (Number ?Number55D). These pathological changes were also markedly reduced from the administration of 7ae (Number ?Number55D). Open in a separate window Number 5 Compound 7ae attenuated LPS induced ALI in mice. (A) Lung W/D percentage. (B) Total amount of cells in BALF. (C) Protein concentration in BALF. (D) Hematoxylin and eosin staining. Statistical significance relative to LPS group was indicated: * 0.05, ** 0.01. In summary, an extensive SAR investigation was carried out based on our recently disclosed tetrahydroisoquinoline-7-carboxamide centered DDR1 inhibitors. The effort yielded a.