ABSTRACT
PRMT5, a type 2 arginine methyltransferase, has a critical role in regulating cell growth and survival in cancer. With the aim of developing MTA-cooperative PRMT5 inhibitors suitable for MTAP-deficient cancers, herein we report our efforts to develop novel "MTA-cooperative" compounds identified through a high-throughput biochemical screening approach. Optimization of hits was achieved through structure-based design with a focus on improvement of oral drug-like properties. Bioisosteric replacement of the original thiazole guanidine headgroup, spirocyclization of the isoindolinone amide scaffold to both configurationally and conformationally lock the bioactive form, and fine-tuning of the potency, MTA cooperativity, and DMPK properties through specific substitutions of the azaindole headgroup were conducted. We have identified an orally available in vivo lead compound, 28 ("AZ-PRMT5i-1"), which shows sub-10 nM PRMT5 cell potency, >50-fold MTA cooperativity, suitable DMPK properties for oral dosing, and significant PRMT5-driven in vivo efficacy in several MTAP-deficient preclinical cancer models.
Subject(s)
Enzyme Inhibitors , Protein-Arginine N-Methyltransferases , Protein-Arginine N-Methyltransferases/antagonists & inhibitors , Protein-Arginine N-Methyltransferases/metabolism , Humans , Animals , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/chemical synthesis , Structure-Activity Relationship , Mice , Drug Discovery , Cell Line, Tumor , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/chemical synthesisABSTRACT
Homochiral α-amino acids are widely used in pharmaceutical design as key subunits in chiral catalyst synthesis or as building blocks in synthetic biology. Many synthetic methods have been developed to access rare or unnatural variants by controlling the installation of the α-stereocentre. By contrast, and despite their importance, α-amino acids possessing ß-stereocentres are much harder to synthesize. Here we demonstrate an iridium-catalysed protocol that allows the direct upconversion of simple alkenes and glycine derivatives to give ß-substituted α-amino acids with exceptional levels of regio- and stereocontrol. Our method exploits the native directing ability of a glycine-derived N-H unit to facilitate Ir-catalysed enolization of the adjacent carbonyl. The resulting stereodefined enolate cross-couples with a styrene or α-olefin to install two contiguous stereocentres. The process offers very high levels of regio- and stereocontrol and occurs with complete atom economy. In broader terms, our reaction design offers a unique directing-group-controlled strategy for the direct stereocontrolled α-alkylation of carbonyl compounds, and provides a powerful approach for the synthesis of challenging contiguous stereocentres.
Subject(s)
Alkenes , Amino Acids , Iridium , Stereoisomerism , Amino Acids/chemistry , Catalysis , Iridium/chemistry , Alkenes/chemistry , Glycine/chemistry , Molecular StructureABSTRACT
The preparation of bridged benzo[1,5]oxazocines and benzo[1,4]diazepines is demonstrated from simple aniline and aldehyde starting materials. A one-pot condensation/6π electrocyclization is followed by an intramolecular trapping of the 2,3-dihydroquinoline intermediate by nitrogen or oxygen nucleophiles to give bridged seven- and eight-membered products. Using 3-hydroxypyridinecarboxaldehydes results in a stable zwitterionic structure that can undergo a diastereoselective reduction under hydrogenative conditions. A similar cyclization/hydrogenation pathway with excellent diastereoselectivity is also demonstrated from 2-pyridyl-substituted 1,2,3,4-tetrahydroquinolines.
ABSTRACT
The glycine to cysteine mutation at codon 12 of Kirsten rat sarcoma (KRAS) represents an Achilles heel that has now rendered this important GTPase druggable. Herein, we report our structure-based drug design approach that led to the identification of 14, AZD4747, a clinical development candidate for the treatment of KRASG12C-positive tumors, including the treatment of central nervous system (CNS) metastases. Building on our earlier discovery of C5-tethered quinazoline AZD4625, excision of a usually critical pyrimidine ring yielded a weak but brain-penetrant start point which was optimized for potency and DMPK. Key design principles and measured parameters that give high confidence in CNS exposure are discussed. During optimization, divergence between rodent and non-rodent species was observed in CNS exposure, with primate PET studies ultimately giving high confidence in the expected translation to patients. AZD4747 is a highly potent and selective inhibitor of KRASG12C with an anticipated low clearance and high oral bioavailability profile in humans.
Subject(s)
Antineoplastic Agents , Lung Neoplasms , Neoplasms , Animals , Humans , Antineoplastic Agents/pharmacology , Proto-Oncogene Proteins p21(ras)/genetics , Neoplasms/drug therapy , Drug Design , Glycine/therapeutic use , Mutation , Lung Neoplasms/drug therapyABSTRACT
Spleen tyrosine kinase (SYK) is a non-receptor cytoplasmic kinase. Due to its pivotal role in B cell receptor and Fc-receptor signalling, inhibition of SYK has been a target of interest in a variety of diseases. Herein, we report the use of structure-based drug design to discover a series of potent macrocyclic inhibitors of SYK, with excellent kinome selectivity and in vitro metabolic stability. We were able to remove hERG inhibition through the optimization of physical properties, and utilized a pro-drug strategy to address permeability challenges.
Subject(s)
Protein-Tyrosine Kinases , Signal Transduction , Syk Kinase , Protein Kinase Inhibitors/pharmacologyABSTRACT
Condensation of 2-vinylanilines and conjugated aldehydes followed by an efficient light-mediated cyclisation selectively yields either substituted tetrahydroquinolines with typically high dr, or in the presence of an iridium photocatalyst the synthesis of quinoline derivatives is demonstrated. These atom economical processes require mild conditions, with the substrate scope demonstrating excellent site selectivity and functional group tolerance, including azaarene-bearing substrates. A thorough experimental mechanistic investigation explores multiple pathways and the key role that imine and iminium intermediates play in the absorption of visible light to generate reactive excited states. The synthetic utility of the reactions is demonstrated on gram scale quantities in both batch and flow, alongside further manipulation of the medicinally relevant products.
Subject(s)
Hydroxyquinolines , Quinolines , Cyclization , LightABSTRACT
Quaternary benzylic centers are accessed with high atom and step economy by Ir-catalyzed alkene hydroarylation. These studies provide unique examples of the use of non-polarized 1,1-disubstituted alkenes in branch selective Murai-type hydro(hetero)arylations. Detailed mechanistic studies have been undertaken, and these indicate that the first irreversible step is the demanding alkene carbometallation process. Structure-reactivity studies show that the efficiency of this is critically dependent on key structural features of the ligand. Computational studies have been undertaken to rationalize this experimental data, showing how more sterically demanding ligands reduce the reaction barrier via predistortion of the reacting intermediate. The key insight disclosed here will underpin the ongoing development of increasingly sophisticated branch selective Murai hydroarylations.
ABSTRACT
KRAS is an archetypal high-value intractable oncology drug target. The glycine to cysteine mutation at codon 12 represents an Achilles heel that has now rendered this important GTPase druggable. Herein, we report our structure-based drug design approach that led to the identification of 21, AZD4625, a clinical development candidate for the treatment of KRASG12C positive tumors. Highlights include a quinazoline tethering strategy to lock out a bio-relevant binding conformation and an optimization strategy focused on the reduction of extrahepatic clearance mechanisms seen in preclinical species. Crystallographic analysis was also key in helping to rationalize unusual structure-activity relationship in terms of ring size and enantio-preference. AZD4625 is a highly potent and selective inhibitor of KRASG12C with an anticipated low clearance and high oral bioavailability profile in humans.
Subject(s)
Antineoplastic Agents , Lung Neoplasms , Antineoplastic Agents/pharmacology , Drug Design , Humans , Lung Neoplasms/drug therapy , Mutation , Proto-Oncogene Proteins p21(ras)/genetics , Quinazolines/pharmacology , Structure-Activity RelationshipABSTRACT
Dual Bcl-2/Bcl-xL inhibitors are expected to deliver therapeutic benefit in many haematological and solid malignancies, however, their use is limited by tolerability issues. AZD4320, a potent dual Bcl-2/Bcl-xL inhibitor, has shown good efficacy however had dose limiting cardiovascular toxicity in preclinical species, coupled with challenging physicochemical properties, which prevented its clinical development. Here, we describe the design and development of AZD0466, a drug-dendrimer conjugate, where AZD4320 is chemically conjugated to a PEGylated poly-lysine dendrimer. Mathematical modelling was employed to determine the optimal release rate of the drug from the dendrimer for maximal therapeutic index in terms of preclinical anti-tumour efficacy and cardiovascular tolerability. The optimised candidate is shown to be efficacious and better tolerated in preclinical models compared with AZD4320 alone. The AZD4320-dendrimer conjugate (AZD0466) identified, through mathematical modelling, has resulted in an improved therapeutic index and thus enabled progression of this promising dual Bcl-2/Bcl-xL inhibitor into clinical development.
Subject(s)
Antineoplastic Agents , Dendrimers , Neoplasms/drug therapy , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/therapeutic use , Dendrimers/chemical synthesis , Dendrimers/chemistry , Dendrimers/pharmacokinetics , Dendrimers/therapeutic use , Dogs , Female , Humans , Male , Mice , Mice, Inbred C57BL , Mice, SCID , Neoplasms/metabolism , Neoplasms/pathology , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Rats , Rats, Wistar , Therapeutic Index , Tumor Cells, Cultured , Xenograft Model Antitumor Assays , bcl-X Protein/antagonists & inhibitorsABSTRACT
Combined photochemical arylation, "nuisance effect" (SN Ar) reaction sequences have been employed in the design of small arrays for immediate deployment in medium-throughput X-ray protein-ligand structure determination. Reactions were deliberately allowed to run "out of control" in terms of selectivity; for example the ortho-arylation of 2-phenylpyridine gave five products resulting from mono- and bisarylations combined with SN Ar processes. As a result, a number of crystallographic hits against NUDT7, a key peroxisomal CoA ester hydrolase, have been identified.
Subject(s)
Benzene Derivatives/chemical synthesis , Enzyme Inhibitors/chemical synthesis , Small Molecule Libraries/chemical synthesis , Benzene Derivatives/metabolism , Catalysis , Chemistry Techniques, Synthetic/methods , Coordination Complexes/chemistry , Crystallography, X-Ray , Drug Design , Enzyme Inhibitors/metabolism , Feasibility Studies , Humans , Palladium/chemistry , Proof of Concept Study , Protein Binding , Pyridines/chemical synthesis , Pyridines/metabolism , Pyrophosphatases/metabolism , Pyrrolidinones/chemical synthesis , Pyrrolidinones/metabolism , Small Molecule Libraries/metabolism , Nudix HydrolasesABSTRACT
Spleen Tyrosine Kinase (SYK) is a well-studied enzyme with therapeutic applications in oncology and autoimmune diseases. We identified an azabenzimidazole (ABI) series of SYK inhibitors by mining activity data of 86,000 compounds from legacy biochemical assays with SYK and other homologous kinases as target enzymes. A structure-based design and hybridization approach was then used to improve the potency and kinase selectivity of the hits. Lead compound 23 from this novel ABI series has a SYK IC50 = 0.21 nM in a biochemical assay and inhibits growth of SUDHL-4 cells at a GI50 = 210 nM.
Subject(s)
Autoimmune Diseases/drug therapy , Aza Compounds/chemistry , Benzimidazoles/chemistry , Protein Kinase Inhibitors/chemistry , Syk Kinase/antagonists & inhibitors , Amino Acid Sequence , Aza Compounds/pharmacology , Benzimidazoles/pharmacology , Cell Line , Cell Proliferation/drug effects , Drug Design , Humans , Inhibitory Concentration 50 , Models, Molecular , Protein Binding , Protein Conformation , Protein Kinase Inhibitors/pharmacology , Structure-Activity Relationship , Substrate SpecificityABSTRACT
Spleen tyrosine kinase (SYK) is a non-receptor cytosolic kinase. Due to its pivotal role in B cell receptor and Fc-receptor signaling, inhibition of SYK has been targeted in a variety of disease areas. Herein, we report the optimization of a series of potent and selective SYK inhibitors, focusing on improving metabolic stability, pharmacokinetics and hERG inhibition. As a result, we identified 30, which exhibited no hERG activity but unfortunately was poorly absorbed in rats and mice. We also identified a SYK chemical probe, 17, which exhibits excellent potency at SYK, and an adequate rodent PK profile to support in vivo efficacy/PD studies.
Subject(s)
Indazoles/pharmacology , Protein Kinase Inhibitors/pharmacology , Syk Kinase/antagonists & inhibitors , Animals , Binding Sites , Caco-2 Cells , Crystallography, X-Ray , ERG1 Potassium Channel/antagonists & inhibitors , Humans , Indazoles/chemical synthesis , Indazoles/metabolism , Indazoles/pharmacokinetics , Mice , Microsomes, Liver/metabolism , Molecular Structure , Protein Binding , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/metabolism , Protein Kinase Inhibitors/pharmacokinetics , Rats, Wistar , Structure-Activity Relationship , Syk Kinase/chemistry , Syk Kinase/metabolismABSTRACT
Attempts to directly drug the important oncogene KRAS have met with limited success despite numerous efforts across industry and academia. The KRASG12C mutant represents an "Achilles heel" and has recently yielded to covalent targeting with small molecules that bind the mutant cysteine and create an allosteric pocket on GDP-bound RAS, locking it in an inactive state. A weak inhibitor at this site was optimized through conformational locking of a piperazine-quinazoline motif and linker modification. Subsequent introduction of a key methyl group to the piperazine resulted in enhancements in potency, permeability, clearance, and reactivity, leading to identification of a potent KRASG12C inhibitor with high selectivity and excellent cross-species pharmacokinetic parameters and in vivo efficacy.
Subject(s)
Antineoplastic Agents/therapeutic use , Neoplasms/drug therapy , Piperazines/therapeutic use , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Quinazolines/therapeutic use , Quinolones/therapeutic use , Allosteric Regulation , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacokinetics , Caco-2 Cells , Cell Line, Tumor , Drug Design , Humans , Male , Mice, Nude , Molecular Conformation , Mutation , Piperazines/chemical synthesis , Piperazines/pharmacokinetics , Proto-Oncogene Proteins p21(ras)/genetics , Quinazolines/chemical synthesis , Quinazolines/pharmacokinetics , Quinolones/chemical synthesis , Quinolones/pharmacokinetics , Rats, Wistar , Structure-Activity Relationship , Xenograft Model Antitumor AssaysABSTRACT
The 4-π-photocyclization of a range of 1,2-dihydropyridazines is described, generating bicyclic 1,2-diazetidines in high yields on multigram scale. The key bicyclic 1,2-diazetidines are versatile synthetic intermediates and were easily converted into a range of novel derivatives, including functionalized 1,2-diazetidines, cyclobutenes, cyclobutanes, and 1,3-dienes.
ABSTRACT
B-cell lymphoma 6 (BCL6) inhibition is a promising mechanism for treating hematological cancers but high quality chemical probes are necessary to evaluate its therapeutic potential. Here we report potent BCL6 inhibitors that demonstrate cellular target engagement and exhibit exquisite selectivity for BCL6 based on mass spectrometry analyses following chemical proteomic pull down. Importantly, a proteolysis-targeting chimera (PROTAC) was also developed and shown to significantly degrade BCL6 in a number of diffuse large B-cell lymphoma (DLBCL) cell lines, but neither BCL6 inhibition nor degradation selectively induced marked phenotypic response. To investigate, we monitored PROTAC directed BCL6 degradation in DLBCL OCI-Ly1 cells by immunofluorescence and discovered a residual BCL6 population. Analysis of subcellular fractions also showed incomplete BCL6 degradation in all fractions despite having measurable PROTAC concentrations, together providing a rationale for the weak antiproliferative response seen with both BCL6 inhibitor and degrader. In summary, we have developed potent and selective BCL6 inhibitors and a BCL6 PROTAC that effectively degraded BCL6, but both modalities failed to induce a significant phenotypic response in DLBCL despite achieving cellular concentrations.
Subject(s)
Antineoplastic Agents/pharmacology , Proto-Oncogene Proteins c-bcl-6/antagonists & inhibitors , Quinolones/pharmacology , Thalidomide/analogs & derivatives , Thalidomide/pharmacology , Adaptor Proteins, Signal Transducing , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/metabolism , Cell Line, Tumor , HEK293 Cells , Humans , Ligands , Lymphoma, Large B-Cell, Diffuse/drug therapy , Peptide Hydrolases/metabolism , Protein Binding , Proteolysis , Proto-Oncogene Proteins c-bcl-6/chemistry , Proto-Oncogene Proteins c-bcl-6/metabolism , Quinolones/chemical synthesis , Quinolones/metabolism , Thalidomide/chemical synthesis , Thalidomide/metabolism , Ubiquitin-Protein Ligases/metabolismABSTRACT
Inhibition of the protein-protein interaction between B-cell lymphoma 6 (BCL6) and corepressors has been implicated as a therapeutic target in diffuse large B-cell lymphoma (DLBCL) cancers and profiling of potent and selective BCL6 inhibitors are critical to test this hypothesis. We identified a pyrazolo[1,5-a]pyrimidine series of BCL6 binders from a fragment screen in parallel with a virtual screen. Using structure-based drug design, binding affinity was increased 100000-fold. This involved displacing crystallographic water, forming new ligand-protein interactions and a macrocyclization to favor the bioactive conformation of the ligands. Optimization for slow off-rate constant kinetics was conducted as well as improving selectivity against an off-target kinase, CK2. Potency in a cellular BCL6 assay was further optimized to afford highly selective probe molecules. Only weak antiproliferative effects were observed across a number of DLBCL lines and a multiple myeloma cell line without a clear relationship to BCL6 potency. As a result, we conclude that the BCL6 hypothesis in DLBCL cancer remains unproven.
Subject(s)
Protein Interaction Maps/drug effects , Proto-Oncogene Proteins c-bcl-6/metabolism , Pyrazoles/chemistry , Pyrazoles/pharmacology , Pyridines/chemistry , Pyridines/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Design , Humans , Lymphoma, Large B-Cell, Diffuse/drug therapy , Molecular Docking Simulation , Proto-Oncogene Proteins c-bcl-6/antagonists & inhibitorsABSTRACT
Irinotecan is used clinically for the treatment of colorectal cancer; however, its utility is limited by its narrow therapeutic index. We describe the use of a generation 5 l-lysine dendrimer that has been part-modified with a polyoxazoline as a drug delivery vehicle for improving the therapeutic index of SN-38, the active metabolite of irinotecan. By conjugating SN-38 to the dendrimer via different linker technologies we sought to vary the release rate of the drug to generate diverse pharmacokinetic profiles. Three conjugates with plasma release half-lives of 2.5h, 21h, and 72h were tested for efficacy and toxicity using a mouse SW620 xenograft model. In this model, the linker with a plasma release half-life of 21h achieved sustained SN-38 exposure in blood, above the target concentration. Control over the release rate of the drug from the linker, combined with prolonged circulation of the dendrimer, enabled administration of an efficacious dose of SN-38, achieving significant regression of the SW620 tumours. The conjugates with 2.5 and 72h release half-lives did not achieve an anti-tumour effect. Intraperitoneal dosing of the clinically used prodrug irinotecan produces high initial and local concentrations of SN-38, which are associated with gastrointestinal toxicity. Administration of the 21h release dendrimer conjugate did not produce a high initial Cmax of SN-38. Consequently, a marked reduction in gastrointestinal toxicity was observed relative to irinotecan treatment. Additional studies investigating the dose concentrations and dose scheduling showed that a weekly dosing schedule of 4mg SN-38/kg was the most efficacious regimen. After 4 doses at weekly intervals, the survival period of the mice extended beyond 70 days following the final dose. These extensive studies have allowed us to identify a linker, dose and dosing regimen for SN-38 conjugated to polyoxazoline-modified dendrimer that maximised efficacy and minimised adverse side effects.
Subject(s)
Antineoplastic Agents, Phytogenic/administration & dosage , Camptothecin/analogs & derivatives , Colorectal Neoplasms/drug therapy , Delayed-Action Preparations/chemistry , Dendrimers/chemistry , Animals , Antineoplastic Agents, Phytogenic/adverse effects , Antineoplastic Agents, Phytogenic/pharmacokinetics , Antineoplastic Agents, Phytogenic/therapeutic use , Camptothecin/administration & dosage , Camptothecin/adverse effects , Camptothecin/pharmacokinetics , Camptothecin/therapeutic use , Colon/drug effects , Colon/pathology , Colorectal Neoplasms/pathology , Female , Irinotecan , Mice , Mice, Nude , Oxazoles/chemistry , Rats, Wistar , Rectum/drug effects , Rectum/pathologyABSTRACT
TC AC 28, 6-(1H-Indol-4-yl)-8-methoxy-1-methyl-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepine-4-acetic acid methyl ester, has been synthesized on a near-gram scale in seven steps with notable improvements in the reported poor-yielding last two steps enabling this key chemical probe compound to be available for researchers.
ABSTRACT
5-Phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-ones react under palladium- and visible light photoredox catalysis, in refluxing methanol, with aryldiazonium salts to afford the respective 5-(2-arylphenyl) analogues. With 2- or 4-fluorobenzenediazonium derivatives, both fluoroaryl- and methoxyaryl- products were obtained, the latter resulting from a SNAr on the fluorobenzenediazonium salt ("nuisance effect"). A computational DFT analysis of the palladium-catalysed and the palladium/ruthenium-photocalysed mechanism for the functionalization of benzodiazepines indicated that, in the presence of the photocatalyst, the reaction proceeds via a low-energy SET pathway avoiding the high-energy oxidative addition step in the palladium-only catalysed reaction pathway.