Anilino-monoindolylmaleimides as potent and selective JAK3 inhibitors

We designed a series of anilino-indoylmaleimides based on structural elements from literature JAK3 inhibitors 3 and 4, and our lead 5. These new compounds were tested as inhibitors of JAKs 1, 2 and 3 and TYK2 for therapeutic intervention in rheumatoid arthritis (RA). Our requirements, based on current scientific rationale for optimum efficacy against RA with reduced side effects, was for potent, mixed JAK1 and 3 inhibition, and selectivity over JAK2. Our efforts yielded a potent JAK3 inhibitor 11d and its eutomer 11e. These compounds were highly selective for inhibition of JAK3 over JAK2 and TYK. The compounds displayed only modest JAK1 inhibition.     

Pyrrolo[1,2-f]triazines as JAK2 inhibitors: achieving potency and selectivity for JAK2 over JAK3

SAR studies of pyrrolo[1,2-f]triazines as JAK2 inhibitors is presented. Achieving JAK2 inhibition selectively over JAK3 is discussed.     

Discovery of potent and efficacious pyrrolopyridazines as dual JAK1/3 inhibitors

A series of potent dual JAK1/3 inhibitors have been developed from a moderately selective JAK3 inhibitor. Substitution at the C6 position of the pyrrolopyridazine core with aryl groups provided exceptional biochemical potency against JAK1 and JAK3 while maintaining good selectivity against JAK2 and Tyk2. Translation to in vivo efficacy was observed in a murine model of chronic inflammation. X-ray co-crystal structure determination confirmed the presumed inhibitor binding orientation in JAK3. Efforts to reduce hERG channel inhibition will be described.     

Pharmacophore based 3D-QSAR modeling and free energy analysis of VEGFR-2 inhibitors

VEGFR-2, a transmembrane tyrosine kinase receptor is responsible for angiogenesis and has been an attractive target in treating cancers. The inhibition mechanism of structurally diverse urea derivatives, reported as VEGFR-2 inhibitors, was explored by pharmacophore modeling, QSAR, and molecular dynamics based free energy analysis.The pharmacophore hypothesis AADRR, resulted in a highly significant atom based 3D-QSAR model (r² = 0.94 and q² = 0.84). Binding free energy analysis of the docked complexes of highly active and inactive compounds, after 7 ns MD simulation, revealed the importance of van der Waals interaction in VEGFR-2 inhibition. The decomposition of binding free energy on a per residue basis disclosed that the residues in hinge region and hydrophobic pocket play a role in discriminating the active and inactive inhibitors. Thus, the present study proposes a pharmacophore hypothesis representing the identified interactions pattern and its further application as a template in screening databases to identify novel VEGFR-2 inhibitor scaffolds.     

Discovery of highly potent,selective, covalent inhibitors of JAK3

A useful and novel set of tool molecules have been identified which bind irreversibly to the JAK3 active site cysteine residue. The design was based on crystal structure information and a comparative study of several electrophilic warheads.     

Virtual screening and synthesis of quinazolines as novel JAK2 inhibitors

JAK2 is an important target in multiple processes associated with tumor growth. In this study, virtual screening was employed for hit compound identification with chemical libraries using SurflexDock. Subsequently, hit optimization for potent and selective candidate JAK2 inhibitors was performed through synthesis of diverse C-1 substituted quinazoline derivatives. A novel compound 5p, (6,7-dimethoxyquinazolin-4-yl)naphthalen-1-ylamine, was thus obtained. JAK2 inhibitory activity of 5p was 43% at 20 ??M and this was comparable to AG490, a representative JAK2 inhibitor. Moreover, 5p showed a positive correlation between JAK2 inhibition and cytotoxicity; 5p treatment in HT-29 cells strongly inhibited JAK2 activation and subsequent STAT3 phosphorylation, reduced anti-apoptotic protein levels, and finally induced apoptosis. This suggests that compound 5p is a candidate inhibitor of JAK2 and its downstream STAT3 signaling pathway for antitumor therapy. In the docking model, the quinazoline template of 5k, the lead compound, occupied a hydrophobic region such as Leu856, Leu855, Ala880, Leu932 and Gly935, and the highly conserved hydrogen bond was created by 6-OMe of the ring template, which binds to the NH of Arg980. Moreover, hydrophobic interactions were identified between morpholine moiety and the hydrophobic region formed by Leu855, Ala880, Tyr931, Val911 and Met929. Also, compound 5k more strongly inhibited JAK2 phosphorylation in mouse embryonic stem cells than AG490. Our study shows the successful application of virtual screening for lead discovery and we propose that the novel compound 5p can be an effective JAK2 inhibitor candidate for further antitumor agent research.     

Discovery of novel selective Janus kinase 2 (JAK2) inhibitors bearing a 1H-pyrazolo[3,4-d]pyrimidin-4-amino scaffold

Janus kinases (JAKs) regulate various cancers and immune responses and are targets for the treatment of cancers and immune diseases. A new series of 1H-pyrazolo[3,4-d]pyrimidin-4-amino derivatives were synthesized and optimized by introducing a functional 3,5-disubstituted-1H-pyrazole moiety into the C-3 moiety of pyrazole template, and then were biologically evaluated as potent Janus kinase 2 (JAK2) inhibitors. Among these molecules, inhibitors 11f, 11g, 11h and 11k displayed strong activity and selectivity against the JAK2 kinase, with IC₅₀ values of 7.2?nM, 6.5?nM, 8.0?nM and 9.7?nM, respectively. In particular, the cellular inhibitory assay and western blot analysis further support the JAK2 selectivity of compound 11g also in cells. Furthermore, compound 11g also exhibited potent inhibitory activity in lymphocytes proliferation assay and delayed hypersensitivity assay. Taken together, the novel JAK2 selective inhibitors discovered in this study may be potential lead compounds for new drug discovery via further development of more potent and selective JAK2 inhibitors.     

The discovery of 2,5-isomers of triazole-pyrrolopyrimidine as selective Janus kinase 2 (JAK2) inhibitors versus JAK1 and JAK3

Members of the Janus kinase (JAK) family are potential therapeutic targets. Abnormal signaling by mutant JAK2 is related to hematological malignancy, such as myeloproliferative neoplasms (MPNs), and tyrosine kinase inhibitor (TKI)-resistance in non-small cell lung cancer (NSCLC). We discovered a potent and highly selective inhibitor of JAK2 over JAK1 and -3 based on the structure of 4-(2,5-triazole)-pyrrolopyrimidine. Among all triazole compounds tested, 2,5-triazole regioisomers more effectively inhibited JAK2 kinase activity than isomers with substitutions of various alkyl groups at the R₂ position, except for methyl-substituted 1,5-triazole, which was more potent than the corresponding 1,4- and 2,5-triazoles. None of the synthesized 1,4-isomers inhibited all three JAK family members. Compounds with phenyl or tolyl group substituents at the R₁ position were completely inactive compared with the corresponding analogues with a methyl substituted at the R₁ position. As a result of this structure–activity relationship, 54, which is substituted with a cyclopropylmethyl moiety, exhibited significant inhibitory activity and selectivity (IC₅₀ = 41.9 nM, fold selectivity JAK1/2 10.6 and JAK3/2 58.1). Compound 54 also exhibited an equivalent inhibition of wild type JAK2 and the V617F mutant. Moreover, 54 inhibited the proliferation of HEL 92.1.7 cells, which carry JAK2 V617F, and gefitinib-resistant HCC827 cells. Compound 54 also suppressed STAT3 phosphorylation at Y705.     

Role of JAK inhibitors and immune cells in transplantation

Immunosuppressive challenge after transplantation has dual objectives, namely, to efficiently inhibit immune populations involved in acute, chronic, humoral or cellular transplant rejection while minimizing the effect on immune integrity toward pathogens. The current immunosuppressive strategies show limited efficacy and remain associated with strong side effects, and thus, it is essential to develop new strategies. The use of Janus kinase (JAK) inhibitors is one of the new strategies focusing on cytokine pathways. Specifically, the first-generation JAK inhibitors (JAKis) showed low specificity toward the four known JAK molecules and did not exhibit better effects than calcineurin inhibitors, which constitute the standard treatment posttransplantation. However, because the new generation of JAKis present higher specificity, we are gaining further insights on the response of cells to these inhibitions. This review focuses on the impact of JAKis on different immune cell subsets, focusing on their role in transplantation.     

Thiophene carboxamide inhibitors of JAK2 as potential treatments for myleoproliferative neoplasms

A series of carboxamide-substituted thiophenes demonstrating inhibition of JAK2 is described. Development of this chemical series began with the bioisosteric replacement of a urea substituent by a pyridyl ring. Issues of chemical and metabolic stability were solved using the results of both in vitro and in vivo studies, ultimately delivering compounds such as 24 and 25 that performed well in an acute PK/PD model measuring p-STAT5 inhibition.     

Simultaneous activation of JAK1 and JAK2 confers IL-3 independent growth on Ba/F3 pro-B cells

JAK1 and JAK2 are tyrosine kinases involved in the regulation of cell proliferation, differentiation, and survival. These proteins may play a key role in mediating the effects of the cytokine IL-3 on hematopoietic cells. IL-3 induces tyrosine phosphorylation of both JAK1 and JAK2. However, it is not clear whether the activation of JAK1, JAK2, or both is sufficient to confer factor-independent growth in IL-3 dependent cells. To address this issue, fusion proteins CD16/CD7/JAK (CDJAK), comprised of a CD16 extracellular domain, a CD7 transmembrane domain, and a JAK cytoplasmic region (either a wild-type JAK or a dominant negative mutant of JAK) were constructed. We established several Ba/F3 derivatives that stably overexpress the conditionally active forms of either CDJAK1, CDJAK2, or both these fusion proteins. In this study, the autophosphorylation of CDJAK1 or CDJAK2 was induced by crosslinking with anti-CD16 antibody. We demonstrated that, like their wild-type counterparts, CDJAK1 and CDJAK2 were preassociated with the IL-3 receptor beta and alpha subunits, respectively. Furthermore, the simultaneous activation of both CDJAK1 and CDJAK2 fusion proteins, but not either one alone, led to the tyrosine phosphorylation of the IL-3 receptor beta subunit, the activation of downstream signaling molecules, including STAT5, Akt, and MAPK, and the conferring of factor-independent growth to IL-3-dependent Ba/F3 cells. Coexpression of dominant negative mutants CDJAK1KE or CDJAK2KE with wild type CDJAK2 or CDJAK1, respectively, inhibited these activation activities. These results suggest that JAK1 and JAK2 must work cooperatively and not independently and that their actions are dependent on having normal kinase activity to trigger downstream signals leading to IL-3 independent proliferation and survival of Ba/F3 cells.     

Discovery of pyrrolo[1,2-b]pyridazine-3-carboxamides as Janus kinase (JAK) inhibitors

A new class of Janus kinase (JAK) inhibitors was discovered using a rationally designed pyrrolo[1,2-b]pyridazine-3-carboxamide scaffold. Preliminary studies identified (R)-(2,2-dimethylcyclopentyl)amine as a preferred C4 substituent on the pyrrolopyridazine core (3b). Incorporation of amino group to 3-position of the cyclopentane ring resulted in a series of JAK3 inhibitors (4g–4j) that potently inhibited IFNγ production in an IL2-induced whole blood assay and displayed high functional selectivity for JAK3–JAK1 pathway relative to JAK2. Further modifications led to the discovery of an orally bioavailable (2-fluoro-2-methylcyclopentyl)amino analogue 5g which is a nanomolar inhibitor of both JAK3 and TYK2, functionally selective for the JAK3–JAK1 pathway versus JAK2, and active in a human whole blood assay.     

Pharmacophore feature prediction and molecular docking approach to identify novel anti‐HCV protease inhibitors

Discovering a potential drug for HCV treatment is a challenging task in the field of drug research. This study initiates with computational screening and modeling of promising ligand molecules. The foremost modeling method involves the identification of novel compound and its molecular interaction based on pharmacophore features. A total of 197 HCV compounds for NS3/4A protein target were screened for our study. The pharmacophore models were generated using PHASE module implemented in Schrodinger suite. The pharmacophore features include one hydrogen bond acceptor, one hydrogen bond donor, and three hydrophobic sites. As a result, based on mentioned hypothesis the model ADHHH.159 corresponds to the CID 59533233. Furthermore, docking was performed using maestro for all the 197 compounds. Among these, the CID 59533313 and 59533233 possess the best binding energy of ?11.75 and ?10.40 kcal/mol, respectively. The interactions studies indicated that the CID complexed with the NS3/4A protein possess better binding affinity with the other compounds. Further the compounds were subjected to calculate the ADME properties. Therefore, it can be concluded that these two compounds could be a potential alternative drug for the development of HCV.     

Pharmacophore modelling and atom-based 3D-QSAR studies on N-methyl pyrimidones as HIV-1 integrase inhibitors

Pharmacophore modelling and atom-based 3D-QSAR studies were carried out for a series of compounds belonging to N-methyl pyrimidones as HIV-1 integrase inhibitors. Based on the ligand-based pharmacophore model, we got 5-point pharmacophore model AADDR, with two hydrogen bond acceptors (A), two hydrogen bond donors (D) and one aromatic ring (R). The generated pharmacophore-based alignment was used to derive a predictive atom-based 3D-QSAR model for the training set (r²?=?0.92, SD?=?0.16, F?=?84.8, N?=?40) and for test set (Q²?=?0.71, RMSE?=?0.06, Pearson R?=?0.90, N?=?10). From these results, AADDR pharmacophore feature was selected as best common pharmacophore hypothesis, and atom-based 3D-QSAR results also support the outcome by means of favourable and unfavourable regions of hydrophobic and electron-withdrawing groups for the most potent compound 30. These results can be useful for further design of new and potent HIV-1 IN inhibitors.     

Structural insights of JAK2 inhibitors: pharmacophore modeling and ligand-based 3D-QSAR studies of pyrido-indole derivatives

Abstract

In this study we have performed pharmacophore modeling and built a 3D QSAR model for pyrido-indole derivatives as Janus Kinase 2 inhibitors. An efficient pharmacophore has been identified from a data set of 51 molecules and the identified pharmacophore hypothesis consisted of one hydrogen bond acceptor, two hydrogen bond donors and three aromatic rings, i.e. ADDRRR. A powerful 3D-QSAR model has also been constructed by employing Partial Least Square regression analysis with a regression coefficient of 0.97 (R²) and Q² of 0.95, and Pearson-R of 0.98.     

A panoramic review of IL-6: Structure,pathophysiological roles and inhibitors

Interleukin-6 (IL-6) is a pleiotropic pro-inflammatory cytokine. Its deregulation is associated with chronic inflammation, and multifactorial auto-immune disorders. It mediates its biological roles through a hexameric complex composed of IL-6 itself, its receptor IL-6R, and glycoprotein 130 (IL-6/IL-6R/gp130). This complex, in turn, activates different signaling mechanisms (classical and trans-signaling) to execute various biochemical functions. The trans-signaling mechanism activates various pathological routes, like JAK/STAT3, Ras/MAPK, PI3K–PKB/Akt, and regulation of CD4+ T cells and VEGF levels, which cause cancer, multiple sclerosis, rheumatoid arthritis, anemia, inflammatory bowel disease, Crohn’s disease, and Alzheimer’s disease. Involvement of IL-6 in pathophysiology of these complex diseases makes it an important target for the treatment of these diseases. Though some anti-IL-6 monoclonal antibodies are being used clinically, but their high cost, only parenteral administration, and possibility of immunogenicity have limited their use, and warranted the development of novel small non-peptide molecules as IL-6 inhibitors. In the present report, all molecules reported in literature as IL-6 inhibitors have been classified as IL-6 production, IL-6R, and IL-6 signaling inhibitors. Reports available till date are critically studied to identify important and salient structural features common in these molecules. These analyses would assist medicinal chemists to design novel and potent IL-6 production and signaling inhibitors, through knowledge- and/or computer-based approaches, for the treatment of complex multifactorial diseases.     

Discovery of a series of novel 5H-pyrrolo[2,3-b]pyrazine-2-phenyl ethers,as potent JAK3 kinase inhibitors

We report the discovery of a novel series of ATP-competitive Janus kinase 3 (JAK3) inhibitors based on the 5H-pyrrolo[2,3-b]pyrazine scaffold. The initial leads in this series, compounds 1a and 1h, showed promising potencies, but a lack of selectivity against other isoforms in the JAK family. Computational and crystallographic analysis suggested that the phenyl ether moiety possessed a favorable vector to achieve selectivity. Exploration of this vector resulted in the identification of 12b and 12d, as potent JAK3 inhibitors, demonstrating improved JAK family and kinase selectivity.     

Combination treatment for myeloproliferative neoplasms using JAK and pan‐class I PI3K inhibitors

Current JAK2 inhibitors used for myeloproliferative neoplasms (MPN) treatment are not specific enough to selectively suppress aberrant JAK2 signalling and preserve physiological JAK2 signalling. We tested whether combining a JAK2 inhibitor with a series of serine threonine kinase inhibitors, targeting nine signalling pathways and already used in clinical trials, synergized in inhibiting growth of haematopoietic cells expressing mutant and wild‐type forms of JAK2 (V617F) or thrombopoietin receptor (W515L). Out of 15 kinase inhibitors, the ZSTK474 phosphatydylinositol‐3′‐kinase (PI3K) inhibitor molecule showed strong synergic inhibition by Chou and Talalay analysis with JAK2 and JAK2/JAK1 inhibitors. Other pan‐class I, but not gamma or delta specific PI3K inhibitors, also synergized with JAK2 inhibitors. Synergy was not observed in Bcr‐Abl transformed cells. The best JAK2/JAK1 and PI3K inhibitor combination pair (ruxolitinib and GDC0941) reduces spleen weight in nude mice inoculated with Ba/F3 cells expressing TpoR and JAK2 V617F. It also exerted strong inhibitory effects on erythropoietin‐independent erythroid colonies from MPN patients and JAK2 V617F knock‐in mice, where at certain doses, a preferential inhibition of JAK2 V617F mutated progenitors was detected. Our data support the use of a combination of JAK2 and pan‐class I PI3K inhibitors in the treatment of MPNs.     

Pyrrole-3-carboxamides as potent and selective JAK2 inhibitors

We report herein the discovery, structure guided design, synthesis and biological evaluation of a novel class of JAK2 inhibitors. Optimization of the series led to the identification of the potent and orally bioavailable JAK2 inhibitor 28 (NMS-P953). Compound 28 displayed significant tumour growth inhibition in SET-2 xenograft tumour model, with a mechanism of action confirmed in vivo by typical modulation of known biomarkers, and with a favourable pharmacokinetic and safety profile.