Novel benzofuran-3-one indole inhibitors of PI3 kinase-α and the mammalian target of rapamycin: Hit to lead studies

A series of benzofuran-3-one indole phosphatidylinositol-3-kinases (PI3K) inhibitors identified via HTS has been prepared. The optimized inhibitors possess single digit nanomolar activity against p110α (PI3K-α), good pharmaceutical properties, selectivity versus p110γ (PI3K-γ), and tunable selectivity versus the mammalian target of rapamycin (mTOR). Modeling of compounds 9 and 32 in homology models of PI3K-α and mTOR supports the proposed rationale for selectivity. Compounds show activity in multiple cellular proliferation assays with signaling through the PI3K pathway confirmed via phospho-Akt inhibition in PC-3 cells.     

3-Amino-pyrazolo[3,4-d]pyrimidines as p38α kinase inhibitors: design and development to a highly selective lead

Learnings from previous Roche p38-selective inhibitors were applied to a new fragment hit, which was optimized to a potent, exquisitely selective preclinical lead with a good pharmacokinetic profile.     

Synthesis and structure–activity relationship study of pyrazolo[3,4-d]pyrimidines as tyrosine kinase RET inhibitors

Three series of pyrazolo[3,4-d]pyrimidine derivatives were synthesized and evaluated as RET kinase inhibitors. Compounds 23a and 23c were identified to show significant activity both in the biochemical and the BaF3/CCDC6-RET cell assays. Compound 23c was found to significantly inhibit RET phosphorylation and down-stream signaling in BaF3/CCDC6-RET cells, confirming its potent cellular RET-targeting profile. Different from other RET inhibitors with equal potency against KDR that associated with severe toxicity, 23c did not show significant KDR-inhibition even at the concentration of 1 μM. These results demonstrated that 23c is a potent and selective RET inhibitor.     

Microwave-assisted synthesis and structure–activity relationships of neuroactive pyrazolo[3,4-b]pyrrolo[3,4-d]pyridine derivatives

We described herein the optimization of the synthetic methodology exploited to obtain the pyrazolo[3,4-b]pyrrolo[3,4-d]pyridine sedative prototype 1a and novel analogues designed by successive molecular simplifications. By applying microwave irradiation during the hetero Diels-Alder key-step to obtain the heterotricyclic scaffold, under solvent-free conditions, we were able to obtain the desired compounds in drastically shorter times and better yields. Additionally, in vivo evaluation of the sedative effects of these heterocyclic derivatives showed that 1a and the novel structurally-related analogue 1e were the most efficient compounds to impair the locomotor activity in mice at the dose of 10 μmol/kg.     

Novel pyrazolo[1,5-a]pyridines as p110α-selective PI3 kinase inhibitors: Exploring the benzenesulfonohydrazide SAR

Structure-activity relationship studies of the pyrazolo[1,5-a]pyridine class of PI3 kinase inhibitors show that substitution off the hydrazone nitrogen and replacement of the sulfonyl both gave a loss of p110α selectivity, with the exception of an N-hydroxyethyl analogue. Limited substitutions were tolerated around the phenyl ring; in particular the 2,5-substitution pattern was important for PI3 kinase activity. The N-hydroxyethyl compound also showed good inhibition of cell proliferation and inhibition of phosphorylation of Akt/PKB, a downstream marker of PI3 kinase activity. It had suitable pharmacokinetics for evaluation in vivo, and showed tumour growth inhibition in two human tumour cell lines in xenograft studies. This work has provided suggestions for the design of more soluble analogues.     

Discovery of pyrazolo[1,5-a]pyridines as p110α-selective PI3 kinase inhibitors

We have made a novel series of pyrazolo[1,5-a]pyridines as PI3 kinase inhibitors, and demonstrated their selectivity for the p110α isoform over the other Class Ia PI3 kinases. We investigated the SAR around the pyrazolo[1,5-a]pyridine ring system, and found compound 5x to be a particularly potent example (p110α IC(50) 0.9nM). This compound inhibits cell proliferation and phosphorylation of Akt/PKB, a downstream marker of PI3 kinase activity, and showed in vivo activity in an HCT-116 human xenograft model.     

Novel pyrazolo[1,5-a]pyridines with improved aqueous solubility as p110α-selective PI3 kinase inhibitors

As part of our investigation into pyrazolo[1,5-a]pyridines as novel p110α selective PI3 kinase inhibitors, we report a range of analogues with improved aqueous solubility by the addition of a basic amine. The compounds demonstrated comparable p110α potency and selectivity to earlier compounds but with up to 1000× greater aqueous solubility, as the hydrochloride salts. The compounds also displayed good activity in a cellular assay of PI3 kinase activity.     

Design,synthesis and structure-activity relationship study of piperazinone-containing thieno[3,2-d]pyrimidine derivatives as new PI3Kδ inhibitors

Two classes of piperazinone-containing thieno[3,2-d]pyrimidines were designed and synthesized as new PI3Kδ inhibitors in this study. Detailed SAR study with respect to the piperazinone substituents at the 6-position of thieno[3,2-d]pyrimidine core demonstrated that piperazinone-containing thieno[3,2-d]pyrimidines would be more potent and selective for PI3Kδ than their piperazine counterparts, which led to the discovery of several potent PI3Kδ inhibitors with comparable or better antiproliferative activity against a panel of non-Hodgkin lymphoma (NHL) cell lines as compared with idelalisib. Our study will promote the development of new PI3Kδ inhibitors based on piperazinone-containing thieno[3,2-d]pyrimidine scaffold.     

5,6,7,8-Tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine derivatives as inhibitors of full-length RORγt

Retinoid-related orphan receptor gamma-t (RORγt) belongs to the nuclear receptor superfamily that takes vital roles in the development and maturation of T-helper 17 cell (Th17) and lymph-node genesis. Because Th17 cells have been proved to be major effectors in human autoimmune and inflammatory diseases, the agonists and antagonists of RORγt have been discovered as promising leads for the therapeutics of these diseases. Most of the current studies of RORγt inhibitors have been focused on ligand binding domain (LBD) of RORγt because the structure and binding pockets of LBD have been elucidated and studied in detail. Recent research elucidated that the hinge domain (HD) of RORγt was significantly involved in the SUMOylation of RORγt and thus specifically affecting T cell development but not lymph-node genesis. These discoveries highlighted the potential of HD of RORγt as the target of RORγt inhibitors that could specifically inhibit Th17-related activities without affecting lymph-node genesis. In this study, we utilized a screening system with full-length RORγt including DBD, HD and LBD to evaluate the activities of a synthesized library of tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine derivatives. We identified a potent lead compound (28) that effectively inhibited Th17 cell differentiation. Docking and structure–activity relationship (SAR) studies showed that compound 28 may not bind in the binding pocket as most of the known inhibitors, but may bind in the pocket closed to Gln223 and Leu244 in HD. Our studies showed evidence that the HD of RORγt could afford a binding pocket for Th17 specific inhibitors and this domain should be further studied to discover potent and specific RORγt inhibitors.     

The conversion of 4-hydroxypyrazolo-[3,4-d]pyrimidine (allopurinol) into 4,6-dihydroxypyrazolo[3,4-d]pyrimidine (oxipurinol) in vivo in the absence of xanthine–oxygen oxidoreductase

1. A patient with congenital deficiency of xanthine oxidase (EC 1.2.3.2) (xanthinuria) excreted the xanthine isomer 4,6-dihydroxypyrazolo[3,4-d]pyrimidine (oxipurinol) in his urine when the hypoxanthine isomer 4-hydroxypyrazolo[3,4-d]pyrimidine (allopurinol) was given by mouth. 2. The identity of the oxipurinol that the patient excreted was established by mass spectrometry. 3. The mass spectra and infrared spectra of allopurinol, oxipurinol, hypoxanthine and xanthine are compared. 4. A mechanism for the fragmentation of these compounds that occurs during their mass-spectrometric investigation is proposed. 5. A possible metabolic pathway for the oxidation of allopurinol to oxipurinol in the absence of xanthine oxidase is discussed.     

Synthesis and structure–activity relationships of PI3K/mTOR dual inhibitors from a series of 2-amino-4-methylpyrido[2,3-d]pyrimidine derivatives

Inhibition of the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway by PI3K/mTOR dual inhibitors provides a promising new approach to the treatment of cancers. In this Letter, we identified structurally novel and potent PI3K/mTOR dual inhibitors from a series of 2-amino-4-methylpyrido[2,3-d]pyrimidine derivatives. Their synthesis and structure–activity relationships are reported.     

Antagonists of 5-HT₆ receptors. Substituted 3-(phenylsulfonyl)pyrazolo[1,5-a]pyrido[3,4-e]pyrimidines and 3-(phenylsulfonyl)pyrazolo[1,5-a]pyrido[4,3-d]pyrimidines-Synthesis and 'structure-activity' relationship

Synthesis and biological evaluation of a new series of structurally unrestricted and intramolecular hydrogen bond restricted derivatives of 3-(phenylsulfonyl)pyrazolo[1,5-a]pyrido[3,4-e]pyrimidines (angular tricyclics) and 3-(phenylsulfonyl)pyrazolo[1,5-a]pyrido[4,3-d]pyrimidines (linear tricyclics) are described. Structurally restricted derivatives are highly potent and selective blockers of 5-HT(6) receptors with little difference between angular or linear shape of the tricyclic core, the angular species being only slightly more potent. The angular representative of 3-(phenylsulfonyl)pyrazolo[1,5-a]pyrido[3,4-e]pyrimidines, 5, can be considered as more favorable candidate for further development as it shows only weak 5-HT(2B) blocking activity (IC(50)=6.16 μM as compared with IC(50)=1.8 nM for 5-HT(6) receptors) and very low hERG potassium channel blocking potency (IC(50)=54.2 μM). The linear analog, 11, is less favorable as while showing no binding to the 5-HT(2B) receptor at concentrations of up to 10 μM, it exhibits quite a high potency to block the hERG channel (IC(50)=0.5 μM).     

Synthesis and Biological Activity of the Novel Adenosine Analogs; 3-Amino-6-(β-D-ribofuranosyl)pyrazolo[3,4-c]pyrazole and 3-Amino-1-methyl-6-(β-D-ribofuranosyl)pyrazolo[3,4-c]pyrazole

Abstract

Chemical modification of the 4-nitrile group in 5-amino-1-(2,3,5-tri-O-benzyl-β-D-ribofuranosyl)pyrazole-4-carbonitrile (1) afforded 5-amino-4-(5-methyl-1,2,4-oxadiazol-3-yl)-1-(2,3,5-tri-O-benzyl-β-D-ribofuran osyl)pyrazole (3). The methylation of 3, via a three step procedure, gave 5-methylamino-4-(5-methyl-1,2,4-oxadiazol-3-yl)-1-(2,3,5-tri-O-benzyl-β-D-ribofuranosyl)pyrazole (3a). The mononuclear heterocyclic rearrangement (m.h.r) of 3 and 3a, provided a convenient route to the novel azapentalene adenosine analogs 3-amino-6-(β-D-ribofuranosyl)pyrazolo[3,4-c]pyrazole (6) and 3-amino-1-methyl-6-(β-D-ribofuranosyl)pyrazolo[3,4-c]pyrazole (6a), respectively. Compound 6 exhibited no cytotoxicity when screened in vitro against either mouse L1210 leukemic cells or human foreskin fibroblasts. Nor was it active against human cytomegalovirus. Compound 6a was designed and prepared to investigate the possibility that the lack of biological activity of 6 might be due to annular tautomerization limiting the ability of 6 to serve as a substrate for the activating enzyme adenosine kinase. This hypothesis was neither supported nor disproved by the results, as compound 6a was also inactive in both the antiproliferative and antiviral test systems.     

Synthesis and antiviral evaluation of thieno[3,4-d]pyrimidine C-nucleoside analogues of 2′,3′-dideoxy- and 2′,3′-dideoxy-2′,3′-didehydro-adenosine and -inosine

Several thieno[3,4-d]pyrimidine derivatives, including four hitherto unknown 2′,3′-dideoxy- and 2′,3′-dideoxy-2′,3′-didehydro-C-nucleoside analogues of adenosine and inosine have been synthesized. When evaluated in cell culture experiments against human immunodeficiency virus, none of the tested compounds exhibited any significant antiviral effect, while two of them showed some cytotoxicity.     

Benzo[d]imidazole inhibitors of Coactivator Associated Arginine Methyltransferase 1 (CARM1)—Hit to Lead studies

Hit to Lead optimization and SAR development led to the identification of the potent and selective benzo[d]imidazole inhibitor (17b) of Co-activator Associated Arginine Methyltransferase (CARM1).     

Synthesis of novel spiro[pyrazolo[4,3-d]pyrimidinones and spiro[benzo[4,5]thieno[2,3-d]pyrimidine-2,3′-indoline]-2′,4(3H)-diones and their evaluation for anticancer activity

An efficient and novel method for the preparation of spiro[pyrazolo[4,3-d]pyrimidin]-7′(1′H)-ones by the condensation of 4-amino-1-methyl-3-propylpyrazole-5-carboxamide with ketones under mild conditions using catalytic InCl₃ was reported. This method has been extended for the synthesis of novel spiro[benzo[4,5]thieno[2,3-d]pyrimidine-2,3′-indoline]-2′,4(3H)-dione which are having potential applications in medicinal chemistry. All the synthesized compounds were evaluated for their anti-proliferative properties in vitro against cancer cell lines and several compounds were found to be active. Further in vitro studies revealed that inhibition of sirtuins could be the possible mechanism of action of these molecules.     

Optimization of 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidines to generate a highly selective PI3Kδ inhibitor

Chemical optimization of the 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine (THPP) scaffold was conducted with a focus on cellular potency while maintaining high selectivity against PI3K isoforms. Compound 11f was identified as a potent, highly selective and orally available PI3Kδ inhibitor. In addition, 11f exhibited efficacy in an in vivo antibody production model. The desirable drug-like properties and in vivo efficacy of 11f suggest its potential as a drug candidate for the treatment of autoimmune diseases and leukocyte malignancies.     

Alternating Dependency of Cytokinin Activity on the Number of Methylene Units in ω-Phenylalkyl Derivatives of Some Purine Cytokinins and 4-Substituted Pyrido[3,4-d]pyrimidine

ω-Phenylalkyl derivatives of N⁶-substituted adenine, N⁶-substituted adenosine and 6-alkoxy-purine with odd numbers of methylenes had relatively high activity, while the corresponding 2-methyl-4-substituted-aminopyrido[3,4-d]pyrimidines with even numbers of methylenes had high activity in an Amaranthus betacyanin test and lettuce seed germination test. These results suggest that the ω-phenyl groups of the cytokinins play a specific role for cytokinin-receptor binding and that the pyrido[3,4-d]pyrimidines interact with a receptor in a different binding mode.     

Synthesis and biological evaluation of 3-tetrazolo steroidal analogs: Novel class of 5α-reductase inhibitors

In the present study, a series of steroidal tetrazole derivatives of androstane and pregnane have been prepared in which the tetrazole moiety was appended at C-3 and 17a-aza locations. 3-Tetrazolo-3,5-androstadien-17-one (6), 3-tetrazolo-19-nor-3,5-androstadien-17-one (10), 3-tetrazolo-3,5-pregnadien-20-one (14), 17a-substituted 3-tetrazolo-17a-aza-d-homo-3,5-androstadien-17-one (26–31) and 3-(2-acetyltetrazolo)-17a-aza-d-homo-3,5-androstadien-17-one (32) were synthesized from dehydroepiandrosterone acetate (1) through multiple synthetic steps. Some of the synthesized compounds were evaluated for their in vitro 5α-reductase (5AR) inhibitory activity by measuring the conversion of [³H] androstenedione in human embryonic kidney (HEK) cells. In vivo 5α-reductase inhibitory activity also showed a significant reduction (p <0.05) in rat prostate weight. The most potent compound 14 showed 5AR-2 inhibition with IC₅₀ being 15.6 nM as compared to clinically used drug finasteride (40 nM). There was also a significant inhibition of 5AR-1 with IC₅₀ 547 nM compared to finasteride (453 nM).     

5-Ureidobenzofuranone indoles as potent and efficacious inhibitors of PI3 kinase-α and mTOR for the treatment of breast cancer

A series of 5-ureidobenzofuran-3-one indoles as potent inhibitors of PI3Kα and mTOR has been developed. The best potency in cells was obtained when the urea group was extended to a 4-[2-(dimethylamino)ethyl]methylamino amidophenyl group. A 7-fluoro group on the indole ring also enhanced cellular potency. Compound 18i, incorporating the optimal functional groups, showed high potency in cellular lines and was further studied in vivo. It was able to inhibit the biomarker phosphorylation for 8 h when dosed at 25 mg/kg iv. In the MDA-MB-361 breast cancer model, it shrank the tumor size remarkably when dosed at 25 mg/kg iv on days 1, 5, and 9.