Mitigation of reactive metabolite formation for a series of 3-amino-2-pyridone inhibitors of Bruton’s tyrosine kinase (BTK)

Reactive metabolites have been putatively linked to many adverse drug reactions including idiosyncratic toxicities for a number of drugs with black box warnings or withdrawn from the market. Therefore, it is desirable to minimize the risk of reactive metabolite formation for lead molecules in optimization, in particular for non-life threatening chronic disease, to maximize benefit to risk ratio. This article describes our effort in addressing reactive metabolite issues for a series of 3-amino-2-pyridone inhibitors of BTK, e.g. compound 1 has a value of 459 pmol/mg protein in the microsomal covalent binding assay. Parallel approaches were taken to successfully resolve the issues: establishment of a predictive screening assay with correlation association of covalent binding assay, identification of the origin of reactive metabolite formation using MS/MS analysis of HLM as well as isolation and characterization of GSH adducts. This ultimately led to the discovery of compound 7 (RN941) with significantly reduced covalent binding of 26 pmol/mg protein.     

Discovery and SAR of substituted 3-oxoisoindoline-4-carboxamides as potent inhibitors of poly(ADP-ribose) polymerase (PARP) for the treatment of cancer

Through conformational restriction of a benzamide by formation of a seven-membered hydrogen-bond with an oxindole carbonyl group, a series of PARP inhibitors was designed for appropriate orientation for binding to the PARP surface. This series of compounds with a 3-oxoisoindoline-4-carboxamide core structure, displayed modest to good activity against PARP-1 in both intrinsic and cellular assays. SAR studies at the lactam nitrogen of the pharmacophore have suggested that a secondary or tertiary amine is important for cellular potency. An X-ray structure of compound 1e bound to the protein confirmed the formation of a seven-membered intramolecular hydrogen bond. Though revealed previously in peptides, this type of seven-membered intramolecular hydrogen bond is rarely observed in small molecules. Largely due to the formation of the intramolecular hydrogen bond, the 3-oxoisoindoline-4-carboxamide core structure appears to be planar in the X-ray structure. An additional hydrogen bond interaction of the piperidine nitrogen to Gly-888 also contributes to the binding affinity of 1e to PARP-1.     

Anti-cancer activity of m-carborane-containing trimethoxyphenyl derivatives through tubulin polymerization inhibition

m-Carborane-containing compound 1a was identified as a cell growth inhibitor from a random screening of a boron compound library. As 1a is a mixture of diastereomers due to the presence of two chiral carbons, we designed achiral derivatives 2–4 and studied the structure-activity relationships of the methoxy groups on the benzene ring. 3,4,5-Trimethoxybenzyl derivative 2a and 3,4,5-trimethoxybenzoyl derivative 3a showed more potent anti-cancer activity against the human breast cancer cell line MDA-MB-453 than lead compound 1a. Compound 3a inhibited tubulin polymerization in a dose-dependent manner.     

Purine derivatives as potent Bruton’s tyrosine kinase (BTK) inhibitors for autoimmune diseases

Investigation of various heterocyclic core isosteres of imidazopyrazines 1 & 2 yielded purine derivatives 3 & 8 as potent and selective BTK inhibitors. Subsequent SAR studies of the purine series led to the discovery of 20 as a leading compound. Compound 20 is very selective when screened against a panel of 400 kinases and is a potent inhibitor in cellular assays of human B cell function including B-Cell proliferation and CD86 cell surface expression and exhibited in vivo efficacy in a mouse PCA model. Its X-ray co-crystal structure with BTK shows that the high selectivity is gained from filling a BTK specific lipophilic pocket. However, physical and ADME properties leading to low oral exposure hindered further development.     

Discovery and Biological evaluation of pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione derivatives as potent Bruton’s tyrosine kinase inhibitors

Aberrant activation of B cell receptor (BCR) signal transduction cascade contributes to the propagation and maintenance of B cell malignancies. The discovery of mall molecules with high potency and selectivity against Bruton’s tyrosine kinase (BTK), a key signaling molecule in this cascade, is particularly urgent in modern treatment regimens. Herein, a series of pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione derivatives were reported as potent BTK inhibitors. Compounds 17 and 18 displayed strong BTK inhibitory activities in the enzymatic inhibition assay, with the IC₅₀ values of 1.2 and 0.8 nM, respectively, which were comparable to that of ibrutinib (IC₅₀ = 0.6 nM). Additionally, compound 17 had a more selective profile over EGFR than ibrutinib. According to the putative binding poses, the molecular basis of this series of compounds with respect to potency against BTK and selectivity over EGFR was elucidated. In further experiments at cellular level, compounds 17 and 18 significantly inhibited the proliferation of Ramos and TMD8 cells. And they arrested 75.4% and 75.2% of TMD8 cells in G1 phase, respectively, at the concentration of 1 µM.     

Discovery of 3-morpholino-imidazole[1,5-a]pyrazine BTK inhibitors for rheumatoid arthritis

8-Amino-imidazo[1,5-a]pyrazine-based Bruton’s tyrosine kinase (BTK) inhibitors, such as 6, exhibited potent inhibition of BTK but required improvements in both kinase and hERG selectivity (Liu et al., 2016; Gao et al., 2017). In an effort to maintain the inhibitory activity of these analogs and improve their selectivity profiles, we carried out SAR exploration of groups at the 3-position of pyrazine compound 6. This effort led to the discovery of the morpholine group as an optimized pharmacophore. Compounds 13, 23 and 38 displayed excellent BTK potencies, kinase and hERG selectivities, and pharmacokinetic profiles.     

Design and synthesis of novel pyrimidine analogs as highly selective,non-covalent BTK inhibitors

BTK is a promising target for the treatment of multiple diseases such as B cell malignances, asthma, and rheumatoid arthritis. Here, we report the discovery of a series of novel pyrimidine analogs as potent, highly selective, non-covalent inhibitors of BTK. Compound 25d demonstrated higher affinity to an unactivated conformation of BTK that resulted in an excellent kinase selectivity. Compound 25d showed a good oral bioavailability in mice, and significantly inhibits the PCA reaction in mice.     

Design,synthesis and biological evaluation of novel 3-substituted pyrazolopyrimidine derivatives as potent Bruton’s tyrosine kinase (BTK) inhibitors

A series of 3-substituted pyrazolopyrimidine derivatives as BTK inhibitors were designed by structure-based drug design and they were synthesized, evaluated by enzyme-based assay and anti-proliferation against Ramos and Raji cells. Most of them displayed good inhibitory activities against both BTK and B-cell lymphoblastic leukemia lines in vitro. Among them, compound 8a exhibited excellent potency (IC₅₀?=?7.95?nM against BTK enzyme, 8.91?μM against Ramos cells and 1.80?μM against Raji cells), with a better hydrophilicity (ClogP?=?3.33). These explorations provided new clues to discover 3-substituted pyrazolopyrimidine derivatives as novel anti-tumor agents.     

Cyclic glycopeptidomimetics through a versatile sugar-based scaffold

Cyclic peptidomimetics are attracting structures to obtain a distinct, bioactive conformation. Even more attractive are sugar-containing cyclic peptidomimetics which present turn structures induced by the pyranose ring when incorporated in cyclic peptides. The use of a new and versatile saccharidic scaffold to achieve sugar-based peptidomimetics is here reported together with the successful synthesis of diastereomerically pure cyclic SAA peptidomimetics 15 and 16.     

Optimization of a 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione series of HIV capsid assembly inhibitors 1: Addressing configurational instability through scaffold modification

The optimization of a 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione series of inhibitors of HIV-1 capsid assembly that possess a labile stereocenter at C3 is described. Quaternization of the C3 position of compound 1 in order to prevent racemization gave compound 2, which was inactive in our capsid disassembly assay. A likely explanation for this finding was revealed by in silico analysis predicting a dramatic increase in energy of the bioactive conformation upon quaternization of the C3 position. Replacement of the C3 of the diazepine ring with a nitrogen atom to give the 1,5-dihydro-benzo[f][1,3,5]triazepine-2,4-dione analog 4 was well tolerated. Introduction of a rigid spirocyclic system at the C3 position gave configurationally stable 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione analog 5, which was able to access the bioactive conformation without a severe energetic penalty and inhibit capsid assembly. Preliminary structure–activity relationships (SAR) and X-ray crystallographic data show that knowledge from the 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione series of inhibitors of HIV-1 capsid assembly can be transferred to these new scaffolds.     

Conformation of the cyclic tetrapeptide dihydrochlamydocin. Iabu-L-Phe D-Pro-LX, and experimental values for 3 leads to 1 intramolecular hydrogen bonds by X-ray diffraction.

Chlamydocin, Iabu-L -Phe-D -Pro-L X, is a naturally occurring cyclic tetrapeptide that exhibits high cytostatic activity. The conformation of the peptide ring, as well as the stereo configuration in the vicinity of the epoxide ring, have been established by a single-crystal X-ray study of dihydrochlamydocin: C₂₈H₄₀N₄O₆·H₂O. It crystallizes in the monoclinic space group P2₁ with a = 12.616(6) Å, b = 12.355(6) Å, c = 9.442(5) Å, and β = 99.5(1)°. The structure was solved by the symbolic addition procedure for phase determination followed by the tangent formula method of phase refinement. This structure represents the first cyclic tetrapeptide in which all four peptide units have been found in the trans conformation; however, each peptide unit is significantly nonplanar with ω angles deviating by 14–24° from the ideal value of 180°. This molecule contains two intramolecular 3 → 1 hydrogen bonds and experimentally determined parameters for these seven-membered turns are presented.     

Discovery of novel BTK inhibitors with carboxylic acids

We report the design and synthesis of a series of novel Bruton’s Tyrosine Kinase (BTK) inhibitors with a carboxylic acid moiety in the ribose pocket. This series of compounds has demonstrated much improved off-target selectivities including adenosine uptake (AdU) inhibition compared to the piperidine amide series. Optimization of the initial lead compound 4 based on BTK enzyme inhibition, and human peripheral blood mononuclear cell (hPBMC) and human whole blood (hWB) activity led to the discovery of compound 40, with potent BTK inhibition, reduced off target activities, as well as favorable pharmacokinetic profile in both rat and dog.     

A bicyclic and hsst2 selective somatostatin analogue: design, synthesis, conformational analysis and binding

A backbone bridged and disulfide bridged bicyclic somatostatin analogue, compound 1 (PTR-3205), was designed and synthesized by solid-phase methodology. The binding of compound 1 to the five different somatostatin receptors, expressed in CHO or COS-7 cells, indicate a high degree of selectivity towards hsstr2. The three-dimensional structure of this compound has been determined in DMSO-d₆ and in water by ¹H NMR and by molecular dynamics simulations. Similar backbone conformations were observed in both solvents. We have established direct evidence that the backbone of this bicyclic somatostatin analogue assumes a ‘folded’ conformation in solution, where the lactam ring extends roughly in the plane of the β-turn. The pharmacophoric region Phe-(d)-Trp-Lys-Thr of compound 1 is in accord with that of both the Veber compound L-363,301 (Merck) and sandostatin. We believe that the enhanced selectivity towards the hsst2 receptor, in comparison with other analogues, is due to its large hydrophobic region, composed of the lactam ring and the Phe side chains at positions 1 and 8.     

Photo-induced synthesis of Axinastatin 3 analogs,the secondary structures and their in vitro antitumor activities

Cyclic peptides combine several favorable properties such as good binding affinity, target selectivity and low toxicity that make them an attractive modality for drug development. In an effort to identify what conformation could be accounting for the bioactive disparity of natural and synthetic cyclic peptides, some structurally-constrained analogs of cyclopeptide Axinastatin 3 were prepared by photo-induced single electron transfer (SET) reaction. Detailed stereochemistry study was performed by experimental electronic circular dichroism combined with theoretical calculations. Our study suggested that the cyclopeptide 1 with βI-turn presented stronger antitumor activity comparing with those without such secondary structures. Moreover, a rare ‘π helix unit’ (compound 3) was realized because of the constrained cyclic structure, which could be considered an important research object for future study of unique helix secondary structures.     

Novel amino acid-substituted diphenylpyrimidine derivatives as potent BTK inhibitors against B cell lymphoma cell lines

A new family of diphenylpyrimidine derivatives bearing an amino acid substituent were identified as potent BTK inhibitors. Among them, compound 7b, which features an l-proline substituent, was identified as the strongest BTK inhibitor, with an IC₅₀ of 8.7?nM. Compound 7b also displayed similar activity against B-cell lymphoma cell lines as ibrutinib. Moreover, 7b exhibited low cytotoxic activity against normal PBMC cells. In addition, the acridine orange/ethidium bromide (AO/EB) staining assay, Western blot analysis and flow cytometry analysis also showed its effectiveness in interfering with B-cell lymphoma cell growth. The molecular simulation performance showed that 7b forms additional strong hydrogen bonds with the BTK protein. All these findings provided new clues about the pyrimidine scaffold as an effective BTK inhibitor for the treatment of B-cell lymphoma.     

Anti-liver fibrotic lignans from the fruits of Schisandra arisanensis and Schisandra sphenanthera

Three new polyoxygenated C₁₈-dibenzocyclooctadiene lignans, arisanschinins M and N (1 and 2) and schisphenin A (3), together with eight related metabolites (4–11), were isolated from the fruits of Schisandra arisanensis and Schisandra sphenanthera, respectively. The structures of 1–3 were elucidated on the basis of extensive spectroscopic and 2D NMR (HSQC, HMBC, and NOESY) analyses. The configuration of the biphenyl moiety in the octadiene ring was determined by circular dichroism (CD). Compound 1 possessed an unprecedented 3-(1-hydroxypropan-2-yl)-3-methyl-1,4-dioxo-2-one lactonide ring system attaching at C-6/C-14. Pharmacological studies revealed that compounds 3, 4, 6, 7, and 10 exhibited significant anti-hepatic fibrosis activity, while 9 and 11 showed cytotoxicity against HSC-T6 cells. The biogenetic pathway for compound 1 was also proposed.     

Pyrido[2,3-b]pyrazines,discovery of TRPV1 antagonists with reduced potential for the formation of reactive metabolites

The transient receptor potential cation channel, subfamily V, member 1 (TRPV1) is a non-selective cation channel that can be activated by a wide range of noxious stimuli, including capsaicin, acid, and heat. Blockade of TRPV1 activation by selective antagonists is under investigation in an attempt to identify novel agents for pain treatment. During pre-clinical development, the 1,8-naphthyridine 2 demonstrated unacceptably high levels of irreversible covalent binding. Replacement of the 1,8-naphthyridine core by a pyrido[2,3-b]pyrazine led to the discovery of compound 26 which was shown to have significantly lower potential for the formation of reactive metabolites. Compound 26 was characterized as an orally bioavailable TRPV1 antagonist with moderate brain penetration. In vivo, 26 significantly attenuated carrageenan-induced thermal hyperalgesia (CITH) and dose-dependently reduced complete Freund’s adjuvant (CFA)-induced chronic inflammatory pain after oral administration.     

Kristall- und molekülstruktur des mono-O-isopropylidenderivatives der dimeren 1,6-anhydro-β-D-arabino-hexopyranos-3-ulose

Acetonation of dimeric 1,6-anhydro-β-D-arabino-hexopyranos-3-ulose yields, besides a monomeric di-O-isopropylidene compound, the dimer 2, which crystallizes in space group P2₁2₁2₁ with a  1.3680 (9), b  1.0686 (7), and c  1.0319 (7) nm, Z  4. The crystal and molecular structure of 2 have been determined by X-ray analysis with direct methods and was refined to a final R_w of 5.55% for 2468 reflections. Compound 2 has not the same dimeric structure as the parent compound with a central 1,4-dioxane ring, but contains instead a central 1,3-dioxolane ring. The pyranose ring bearing the isopropylidene group adopts an almost ideal sofa conformation, with a nearly planar arrangement of C-1, C-2, C-3, C-4, and C-5. By analogy, it was concluded that the dimeric mono-O-isopropylidene derivative 7 of 1,6-anhydro-β-D-xylo-hexopyranos-3-ulose has the same asymmetric structure. The 360-MHz ¹H-n.m.r. spectra of both compounds are in full agreement with the proposed structures.     

Design,synthesis and biological evaluation of novel dithiocarbamate-substituted diphenylaminopyrimidine derivatives as BTK inhibitors

Bruton’s tyrosine kinase (BTK) has emerged as an attractive target related to B-lymphocytes dysfunctions, especially hematologic malignancies and autoimmune diseases. In our study, a series of diphenylaminopyrimidine derivatives bearing dithiocarbamate moieties were designed and synthesized as novel BTK inhibitors for treatment of B-cell lymphoma. Among all these compounds, 30ab (IC₅₀ = 1.15 ± 0.19 nM) displays similar or more potent inhibitory activity against BTK than spebrutinib (IC₅₀ = 2.12 ± 0.32 nM) and FDA approved drug ibrutinib (IC₅₀ = 3.89 ± 0.57 nM), which is attributed to close binding of 30ab with BTK predicted by molecular docking. In particular, 30ab exhibits enhanced anti-proliferative activity against B-lymphoma cell lines at the IC₅₀ concentration of 0.357 ± 0.02 μM (Ramos) and 0.706 ± 0.05 μM (Raji), respectively, almost 10-fold better than ibrutinib and spebrutinib. In addition, 30ab displays stronger selectivity on B-cell lymphoma over other cancer cell lines than spebrutinib. Furthermore, 30ab efficiently blocks BTK downstream pathways and results in apoptosis of cancer cells. In vivo xenograft model evaluation demonstrates the significant efficacy and broad safety margin of 30ab in treatment of B-cell lymphoma. We propose that compound 30ab is a candidate for further study and development based on our current findings.     

Catenulisporidins A and B, 16-membered macrolides of the hygrolidin family produced by the chemically underexplored actinobacterium Catenulispora species

Two new macrolide metabolites of the hygrolidin family, catenulisporidins A and B (1 and 2), together with a known compound hygrolidin (3), were isolated from the culture broth of the rare actinobacterium Catenulispora sp. KCB13F192. Their structures were elucidated on the basis of HRESIMS spectrometric and NMR spectroscopic analyses. Catenulisporidins A and B are the first example of natural hygrolidin and bafilomycin derivatives featuring a modified macrolide ring, and catenulisporidin A possesses a tetrahydrofuran ring through an ether linkage between C-7 and C-10. In cell-based fluorescent imaging and immunoblot assays, the three compounds were shown to inhibit autophagic flux in HeLa cells.