Design,synthesis, and biological characterization of novel PEG-linked dimeric modulators for CXCR4

CXCR4 dimerization has been widely demonstrated both biologically and structurally. This paper mainly focused on the development of structure-based dimeric ligands that target CXCL12–CXCR4 interaction and signaling. This study presents the design and synthesis of a series of [PEG]_n linked dimeric ligands of CXCR4 based on the knowledge of the homodimeric crystal structure of CXCR4 and our well established platform of chemistry and bioassays for CXCR4. These new ligands include [PEG]_n linked homodimeric or heterodimeric peptides consisting of either two DV3-derived moieties (where DV3 is an all-d-amino acid analog of N-terminal modules of 1–10 (V3) residues of vMIP-II) or hybrids of DV3 moieties and CXCL12_1–8. Among a total of 24 peptide ligands, four antagonists and three agonists showed good CXCR4 binding affinity, with IC₅₀ values of <50 nM and <800 nM, respectively. Chemotaxis and calcium mobilization assays with SUP-T1 cells further identified two promising lead modulators of CXCR4: ligand 4, a [PEG₃]₂ linked homodimeric DV3, was an effective CXCR4 antagonist (IC₅₀ = 22 nM); and ligand 21, a [PEG₃]₂ linked heterodimeric DV3–CXCL12_1–8, was an effective CXCR4 agonist (IC₅₀ = 407 nM). These dimeric CXCR4 modulators represent new molecular probes and therapeutics that effectively modulate CXCL12–CXCR4 interaction and function.     

UCLA1 aptamer inhibition of human immunodeficiency virus type 1 subtype C primary isolates in macrophages and selection of resistance

We have previously shown that the aptamer, UCLA1, is able to inhibit HIV-1 replication in peripheral blood mononuclear cells (PBMCs) by binding to residues in gp120. In this study we examined whether UCLA1 was effective against HIV-1 subtype C isolates in monocyte-derived macrophages (MDMs). Of 4 macrophage-tropic isolates tested, 3 were inhibited by UCLA1 in the low nanomolar range (IC₈₀<29 nM). One isolate that showed reduced susceptibility (<50 nM) to UCLA1 contained mutations in the α5 helix next to the CD4 and co-receptor (CoR) binding complex. To further evaluate aptamer resistance, two primary viruses were subjected to increasing concentrations of UCLA1 over a period of 84 days in PBMCs. One isolate showed a 7-fold increase in IC₈₀ (351 nM) associated with genetic changes, some of which were previously implicated in resistance. This included F223Y in the C2 region and P369L within the CD4 and CoR binding complex. A second isolate showed a 3-fold increase in IC₈₀ (118 nM) but failed to show any genetic changes. Collectively, these data show that UCLA1 can efficiently block HIV-1 infection in MDMs and PBMCs with escape mutations arising in some isolates after prolonged exposure to the aptamer. This supports the further development of the UCLA1 aptamer as a HIV-1 entry inhibitor.     

Synthesis of dammarane-type triterpene derivatives and their ability to inhibit HIV and HCV proteases

We synthesized dammarane-type triterpene derivatives and evaluated their ability to inhibit HIV-1 and HCV proteases to understand their structure–activity relationships. All of the mono- and di-succinyl derivatives (5a–5f) were powerful inhibitors of HIV-1 protease (IC₅₀ < 10 μM). However, only di-succinyl (5e) and 2,3-seco-2,3-dioic acid (3b) derivatives similarly inhibited HCV protease (IC₅₀ < 10 μM). A-nor dammarane-type triterpenes (4a and 4b, IC₅₀ 10.0 and 29.9 μM, respectively) inhibited HIV-1 protease moderately or strongly, but were inactive against HCV protease. All compounds that powerfully inhibited HIV-1 or HCV protease did not appreciably inhibit the general human proteases, renin and trypsin (IC₅₀ > 1000 μM). These findings indicated that the mono-succinyl dammarane type derivatives (5a–5d) selectively inhibited HIV-1 protease and that the di-succinyl (5e, 5f) as well as 2,3-seco-2,3-dioic acid (3b) derivatives preferably inhibited both viral proteases.     

Discovery of 2-arylthieno[3,2-d]pyrimidines containing 8-oxa-3-azabi-cyclo[3.2.1]octane in the 4-position as potent inhibitors of mTOR with selectivity over PI3K

2-Aryl-4-morpholinothieno[3,2-d]pyrimidines are known PI3K inhibitors. This class of compounds also potently inhibited the homologous enzyme mTOR. Replacement of the morpholine group in these compounds with an 8-oxa-3-azabicyclo[3.2.1]octane group led to mTOR inhibitors with selectivity over PI3K. Optimization of the 2-aryl substituent led to the discovery of 2-(4-ureidophenyl)-thienopyrimidines as highly potent (IC₅₀ <1 nM) mTOR inhibitors with excellent selectivity (up to >1000-fold) over PI3K and good potency in a cellular proliferation assay (IC₅₀ <50 nM).     

Synthesis and structure–activity relationships of 4-fluorophenyl-imidazole p38α MAPK,CK1δ and JAK2 kinase inhibitors

The synthesis and structure–activity relationships of novel 4-(4′-fluorophenyl)imidazoles as selective p38α MAPK, CK1δ and JAK2 inhibitors with improved water solubility are described. Microwave-assisted multicomponent reactions afforded 4-fluorophenyl-2,5-disubstituted imidazoles. Carboxylate and phosphonate groups were introduced via ‘click’ reactions. The kinase selectivity was influenced by the heteroaryl group at imidazole C-5 and the position of a carboxylic acid or tetrazole at imidazole C-2. For example, pyrimidines 15 and 34 inhibited p38α MAPK with IC₅₀ = 250 nM and 96 nM, respectively. Pyridine 3 gave CK1δ inhibition with IC₅₀ = 89 nM and pyridin-2-one 31 gave JAK2 inhibition with IC₅₀ = 62 nM.     

Potent BRAF kinase inhibitors based on 2,4,5-trisubstituted imidazole with naphthyl and benzothiophene 4-substituents

The RAS–RAF–MEK–ERK pathway is hyperactivated in 30% of human cancers. BRAF is a serine–threonine kinase, belonging to this pathway that is mutated with high frequency in human melanoma and other cancers thus BRAF is an important therapeutic target in melanoma. We have designed inhibitors of BRAF based on 2,4,5-trisubstituted imidazoles with naphthyl and benzothiophene-4-substituents. Two compounds were discovered to be potent BRAF inhibitors: 1-(6-{2-[4-(2-dimethylamino-ethoxy)phenyl]-5-(pyridin-4-yl)-1H-imidazol-4-yl} benzo[b]thiophen-3-yl)-2,2,2-trifluoroethanol (1i) with BRAF IC₅₀ = 190 nM and with cellular GI₅₀ = 2100 nM, and 6-{2-[4-(2-dimethylamino-ethoxy)-phenyl]-5-pyridin-4-yl-3H-imidazol-4-yl}-naphthalen-1-ol (1q) with IC₅₀ = 9 nM and GI₅₀ = 220 nM.     

Identification of 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-derived ureas as potent inhibitors of human nicotinamide phosphoribosyltransferase (NAMPT)

Potent nicotinamide phosphoribosyltransferase (NAMPT) inhibitors containing 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-derived ureas were identified using structure-based design techniques. The new compounds displayed improved aqueous solubilities, determined using a high-throughput solubility assessment, relative to previously disclosed urea and amide-containing NAMPT inhibitors. An optimized 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-derived compound exhibited potent anti-NAMPT activity (18; BC NAMPT IC₅₀ = 11 nM; PC-3 antiproliferative IC₅₀ = 36 nM), satisfactory mouse PK properties, and was efficacious in a PC-3 mouse xenograft model. The crystal structure of another optimized compound (29; NAMPT IC₅₀ = 10 nM; A2780 antiproliferative IC₅₀ = 7 nM) in complex with the NAMPT protein was also determined.     

Structural diversity of nucleoside phosphonic acids as a key factor in the discovery of potent inhibitors of rat T-cell lymphoma thymidine phosphorylase

Structurally diverse, sugar-modified, thymine-containing nucleoside phosphonic acids were evaluated for their ability to inhibit thymidine phosphorylase (TP, EC 2.4.2.4) purified from spontaneous T-cell lymphomas of an inbred Sprague-Dawley rat strain. From a large set of tested compounds, among them a number of pyrrolidine-based derivatives, 10 nucleotide analogues with IC₅₀ values below 1 μM were selected. Out of them, four compounds strongly inhibited the enzyme with IC₅₀ values lying in a range of 11–45 nM. These most potent compounds might be bi-substrate analogues.     

Synthesis and CYP26A1 inhibitory activity of novel methyl 3-[4-(arylamino)phenyl]-3-(azole)-2,2-dimethylpropanoates

The role of all-trans-retinoic acid (ATRA) in the development and maintenance of many epithelial and neural tissues has raised great interest in the potential of ATRA and related compounds (retinoids) as pharmacological agents, particularly for the treatment of cancer, skin, neurodegenerative and autoimmune diseases. The use of ATRA or prodrugs as pharmacological agents is limited by a short half-life in vivo resulting from the activity of specific ATRA hydroxylases, CYP26 enzymes, induced by ATRA in liver and target tissues. For this reason retinoic acid metabolism blocking agents (RAMBAs) have been developed for treating cancer and a wide range of other diseases.The synthesis, CYP26A1 inhibitory activity and molecular modeling studies of novel methyl 3-[4-(arylamino)phenyl]-3-(azole)-2,2-dimethylpropanoates are presented. From this series of compounds clear SAR can be derived for 4-substitution of the phenyl ring with electron-donating groups more favourable for inhibitory activity. Both the methylenedioxyphenyl imidazole (17, IC₅₀ = 8 nM) and triazole (18, IC₅₀ = 6.7 nM) derivatives were potent inhibitors with additional binding interactions between the methylenedioxy moiety and the CYP26 active site likely to be the main factor. The 6-bromo-3-pyridine imidazole 15 (IC₅₀ = 5.7 nM) was the most active from this series compared with the standards liarozole (IC₅₀ = 540 nM) and R116010 (IC₅₀ = 10 nM).     

Synthesis and study of anti-HIV-1 RT activity of 5-benzoyl-4-methyl-1,3,4,5-tetrahydro-2H-1,5-benzodiazepin-2-one derivatives

In the present study, a series of fourteen 5-benzoyl-4-methyl-1,3,4,5-tetrahydro-2H-1,5-benzodiazepin-2-one derivatives were designed, synthesized and characterized by appropriate spectral analysis. Further, titled compounds were in-vitro screened against wild HIV-1 RT enzyme using ELISA based colorimetric assay, in which four compounds significantly inhibited the RT activity with IC₅₀ ≤ 25 µM. Moreover, two significantly active compounds of the series, A10 and A11 exhibited IC₅₀ values 8.62 and 6.87 µM respectively, during the in-vitro assay. Structure Activity Relationship (SAR) studies were performed for the synthesized compounds in order to estimate the effect of substitution pattern on the RT inhibitory potency. The cytotoxicity of the synthesized compounds was evaluated against T lymphocytes. Further, putative binding modes of the significantly active (A11) and the least active (A4) compounds with wild HIV-1 RT were also investigated using docking studies.     

Design,synthesis and biological evaluation of novel non-covalent piperidine-containing peptidyl proteasome inhibitors

A series of novel non-covalent piperidine-containing dipeptidyl derivatives were designed, synthesized and evaluated as proteasome inhibitors. All target compounds were tested for their proteasome chymotrypsin-like inhibitory activities, and selected derivatives were evaluated for the anti-proliferation activities against two multiple myeloma (MM) cell lines RPMI 8226 and MM-1S. Among all of these compounds, eight exhibited significant proteasome inhibitory activities with IC₅₀ less than 20 nM, and four are more potent than the positive control Carfilzomib. Compound 28 displayed the most potent proteasome inhibitory activity (IC₅₀: 1.4 ± 0.1 nM) and cytotoxicities with IC₅₀ values at 13.9 ± 1.8 nM and 9.5 ± 0.5 nM against RPMI 8226 and MM-1S, respectively. Additionally, the ex vivo blood cell proteasome inhibitory activities of compounds 24 and 27–29 demonstrated that the enzymatic metabolism in the whole blood could be well tolerated. All these experiments confirmed that the piperidine-containing non-covalent proteasome inhibitors are potential leads for exploring new anti-cancer drugs.     

Isoquinoline derivatives as potent CRTH2 antagonists: Design,synthesis and SAR

In this study, we describe the synthesis and structure–activity relationship (SAR) of a series of isoquinoline chemoattractant receptor–homologous molecule expressed on Th2 cells (CRTH2) antagonists. TASP0376377 (15-20), one of the most potent compounds, showed a potent binding affinity (IC₅₀ = 19 nM) in addition to the excellent functional antagonist activity (IC₅₀ = 13 nM). Moreover, the efficacy of this compound in a chemotaxis assay (IC₅₀ = 23 nM) was in good agreement with its potency as a CRTH2 antagonist. In addition, 15-20 exhibited greater selectivity in binding to CRTH2 than to the DP1 prostanoid receptor (IC₅₀ >1 μM) or the enzymes COX-1 and COX-2 (IC₅₀ >10 μM).     

C-5 substituted heteroaryl-3-pyridinecarbonitriles as PKCθ inhibitors: Part II

We previously reported that a 3-pyridinecarbonitrile analog with a furan substituent at C-5 and a 4-methylindol-5-ylamino substituent at C-4, 1, was a potent inhibitor of PKCθ (IC₅₀ = 4.5 nM). Replacement of the C-5 furan ring of 1 with bicyclic heteroaryl rings, led to compounds with significantly improved potency against PKCθ. Analog 6b with a 4-methylindol-5-ylamino group at C-4 and a 5-[(4-methylpiperazin-1-yl)methyl]-1-benzofuran-2-yl group at C-5 had an IC₅₀ value of 0.28 nM for the inhibition of PKCθ.     

Discovery of new [1,4]dioxino[2,3-f]quinazoline-based inhibitors of EGFR including the T790M/L858R mutant

A novel series of 2,3-dihydro-[1,4]dioxino[2,3-f]quinazoline derivatives were designed, synthesized and evaluated as reversible and noncovalent epidermal growth factor receptor (EGFR) inhibitors. Most of the compounds exhibited good potency against EGFR^wt and some showed moderate to excellent potency against EGFR^T790M/L858R mutant. The half-maximal inhibitory concentration (IC₅₀) values of twenty-one compounds against EGFR^wt were less than 50 nM, and those of six compounds were less than 10 nM. The IC₅₀ values of eleven compounds against EGFR^T790M/L858R were less than 100 nM. Among these, compound b1 displayed the most potent inhibitory activity against EGFR^wt (IC₅₀ = 2.0 nM) and EGFR^T790M/L858R (IC₅₀ = 6.9 nM). Compounds with excellent inhibitory activities against EGFR^wt and EGFR^T790M/L858R kinase inhibitory activities showed good antiproliferative activities against H358 and A549 cells. Docking study was performed to position compound b1 into the EGFR active pocket to determine the probable binding conformation.     

Design,synthesis and biological evaluation of novel substituted benzoylguanidine derivatives as potent Na+/H+ exchanger inhibitors

A novel series of substituted benzoylguanidine derivatives were designed and synthesized as potent NHE1 inhibitors. Most compounds can significantly inhibit NHE1-mediated platelet swelling in a concentration-dependent manner, among which compound 5f (IC₅₀ = 3.60 nM) and 5l (IC₅₀ = 4.48 nM) are 18 and 14 times respectively more potent than cariporide (IC₅₀ = 65.0 nM). Furthermore, when tested in vivo and in vitro, compound 5f showed superior cardioprotective effects against SD rat myocardial ischemic-reperfusion injury over cariporide, representing a promising lead compound for further exploration.     

Highly potent tyrosinase inhibitor,neorauflavane from Campylotropis hirtella and inhibitory mechanism with molecular docking

Tyrosinase inhibition may be a means to alleviate not only skin hyperpigmentation but also neurodegeneration associated with Parkinson’s disease. In the course of metabolite analysis from tyrosinase inhibitory methanol extract (80% inhibition at 20 μg/ml) of Campylotropis hirtella, we isolated fourteen phenolic compounds, among which neorauflavane 3 emerged as a lead structure for tyrosinase inhibition. Neorauflavane 3 inhibited tyrosinase monophenolase activity with an IC₅₀ of 30 nM. Thus this compound is 400-fold more active than kojic acid. It also inhibited diphenolase (IC₅₀ = 500 nM), significantly. Another potent inhibitor 1 (IC₅₀ = 2.9 μM) was found to be the most abundant metabolite in C. hirtella. In kinetic studies, compounds 3 showed competitive inhibitory behavior against both monophenolase and diphenolase. It manifested simple reversible slow-binding inhibition against monophenolase with the following kinetic parameters: K_i^app = 1.48 nM, k₃ = 0.0033 nM⁻¹ min⁻¹ and k₄ = 0.0049 min⁻¹. Neorauflavane 3 efficiently reduced melanin content in B16 melanoma cells with 12.95 μM of IC₅₀. To develop a pharmacophore model, we explored the binding mode of neuroflavane 3 in the active site of tyrosinase. Docking results show that resorcinol motif of B-ring and methoxy group in A-ring play crucial roles in the binding the enzyme.     

Structure activity relationships of fused bicyclic and urea derivatives of spirocyclic compounds as potent CCR1 antagonists

A series of fused bicyclic and urea derivatives of spirocyclic compounds were designed, synthesised and evaluated in vitro as potent CCR1 antagonists. In particular, 4 (7 nM), 44 (1.3 nM), 48 (0.89 nM) and 50 (0.63 nM) were the most potent hCCR1 antagonists in this series of compounds. Moreover, some of these substances demonstrated good rodent cross-over, especially 46 which exhibited very high rat CCR1 binding affinity with an IC₅₀ value of 16 nM.     

5-(1,3-Benzothiazol-6-yl)-4-(4-methyl-1,3-thiazol-2-yl)-1H-imidazole derivatives as potent and selective transforming growth factor-β type I receptor inhibitors

A series of 5-(1,3-benzothiazol-6-yl)-4-(4-methyl-1,3-thiazol-2-yl)-1H-imidazole derivatives was synthesized as transforming growth factor-β (TGF-β) type I receptor (also known as activin-like kinase 5 or ALK5) inhibitors. These compounds were evaluated for their ALK5 inhibitory activity in an enzyme assay and for their TGF-β-induced Smad2/3 phosphorylation inhibitory activity in a cell-based assay. As a representative compound, 16i was a potent and selective ALK5 inhibitor, exhibiting a good enzyme inhibitory activity (IC₅₀ = 5.5 nM) as well as inhibitory activity against TGF-β-induced Smad2/3 phosphorylation at a cellular level (IC₅₀ = 36 nM). Furthermore, the topical application of 3% 16i lotion significantly inhibited Smad2 phosphorylation in Mouse skin (90% inhibition compared with vehicle-treated animals).     

Discovery of a novel series of CXCR3 antagonists

The discovery of a novel series of CXCR3 antagonists is described. Starting from an HTS positive, iterative optimization gave potent compounds (IC₅₀ 15 nM in a chemotaxis assay). The strategy employed to improve the metabolic stability of these derivatives is described.