ABSTRACT
Design and synthesis of a series of 4-morpholino-6-aryl-1H-pyrazolo[3,4-d]pyrimidines as potent and selective inhibitors of the mammalian target of rapamycin (mTOR) are described. Optimization of the 6-aryl substituent led to the discovery of inhibitors carrying 6-ureidophenyl groups, the first reported active site inhibitors of mTOR with subnanomolar inhibitory concentrations. The data presented in this paper show that 6-arylureidophenyl substituents led to potent mixed inhibitors of mTOR and phosphatidylinositol 3-kinase alpha (PI3K-alpha), whereas 6-alkylureidophenyl appendages gave highly selective mTOR inhibitors. Combination of 6-alkylureidophenyl groups with 1-carbamoylpiperidine substitution resulted in compounds with subnanomolar IC(50) against mTOR and greater than 1000-fold selectivity over PI3K-alpha. In addition, structure based drug design resulted in the preparation of several 6-arylureidophenyl-1H-pyrazolo[3,4-d]pyrimidines, substituted in the 4-position of the arylureido moiety with water solubilizing groups. These compounds combined potent mTOR inhibition (IC(50) < 1 nM) with unprecedented activity in cellular proliferation assays (IC(50) < 1 nM).
Subject(s)
Adenosine Triphosphate/chemistry , Protein Kinase Inhibitors/pharmacology , Protein Kinases/chemistry , Pyrimidines/pharmacology , Adenosine Triphosphate/metabolism , Binding, Competitive , Cell Growth Processes/drug effects , Cell Line, Tumor , Humans , Male , Models, Molecular , Morpholines/chemical synthesis , Morpholines/chemistry , Morpholines/pharmacology , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/pathology , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Protein Kinases/metabolism , Pyrazoles/chemical synthesis , Pyrazoles/chemistry , Pyrazoles/pharmacology , Pyrimidines/chemical synthesis , Pyrimidines/chemistry , Structure-Activity Relationship , TOR Serine-Threonine KinasesABSTRACT
The mammalian target of rapamycin (mTOR) is a central regulator of cell growth, metabolism, and angiogenesis and an emerging target in cancer research. High throughput screening (HTS) of our compound collection led to the identification of 3-(4-morpholin-4-yl-1-piperidin-4-yl-1H-pyrazolo[3,4-d]pyrimidin-6-yl)phenol (5a), a modestly potent and nonselective inhibitor of mTOR and phosphoinositide 3-kinase (PI3K). Optimization of compound 5a, employing an mTOR homology model based on an X-ray crystal structure of closely related PI3Kgamma led to the discovery of 6-(1H-indol-5-yl)-4-morpholin-4-yl-1-[1-(pyridin-3-ylmethyl)piperidin-4-yl]-1H-pyrazolo[3,4-d]pyrimidine (5u), a potent and selective mTOR inhibitor (mTOR IC(50) = 9 nM; PI3Kalpha IC(50) = 1962 nM). Compound 5u selectively inhibited cellular biomarker of mTORC1 (P-S6K, P-4EBP1) and mTORC2 (P-AKT S473) over the biomarker of PI3K/PDK1 (P-AKT T308) and did not inhibit PI3K-related kinases (PIKKs) in cellular assays. These pyrazolopyrimidines represent an exciting new series of mTOR-selective inhibitors with potential for development for cancer therapy.
Subject(s)
Drug Discovery , Protein Kinase Inhibitors/pharmacology , Protein Kinases/metabolism , Pyrimidines/pharmacology , Binding, Competitive , Cell Line, Tumor , Humans , Inhibitory Concentration 50 , Models, Molecular , Molecular Conformation , Molecular Weight , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/metabolism , Protein Kinases/chemistry , Pyrimidines/chemical synthesis , Pyrimidines/chemistry , Pyrimidines/metabolism , Signal Transduction/drug effects , Substrate Specificity , TOR Serine-Threonine KinasesABSTRACT
The mammalian target of rapamycin (mTOR), a central regulator of growth, survival, and metabolism, is a validated target for cancer therapy. Rapamycin and its analogues, allosteric inhibitors of mTOR, only partially inhibit one mTOR protein complex. ATP-competitive, global inhibitors of mTOR that have the potential for enhanced anticancer efficacy are described. Structural features leading to potency and selectivity were identified and refined leading to compounds with in vivo efficacy in tumor xenograft models.
Subject(s)
Adenosine Triphosphate/physiology , Antineoplastic Agents/chemical synthesis , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyrazoles/chemical synthesis , Pyrimidines/chemical synthesis , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Binding Sites , Carbamates/chemical synthesis , Carbamates/chemistry , Carbamates/pharmacology , Cell Line, Tumor , Class Ib Phosphatidylinositol 3-Kinase , Crystallography, X-Ray , Drug Design , Intracellular Signaling Peptides and Proteins/chemistry , Isoenzymes/chemistry , Mice , Mice, Nude , Microsomes/metabolism , Models, Molecular , Phosphatidylinositol 3-Kinases/chemistry , Protein Serine-Threonine Kinases/chemistry , Pyrazoles/chemistry , Pyrazoles/pharmacology , Pyrimidines/chemistry , Pyrimidines/pharmacology , Structure-Activity Relationship , TOR Serine-Threonine Kinases , Urea/analogs & derivatives , Urea/chemical synthesis , Urea/chemistry , Urea/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
The mammalian target of rapamycin (mTOR) is centrally involved in cell growth, metabolism, and angiogenesis. While showing clinical efficacy in a subset of tumors, rapamycin and rapalogs are specific and allosteric inhibitors of mTOR complex 1 (mTORC1), but they do not directly inhibit mTOR complex 2 (mTORC2), an emerging player in cancer. Here, we report chemical structure and biological characterization of three pyrazolopyrimidine ATP-competitive mTOR inhibitors, WAY-600, WYE-687, and WYE-354 (IC(50), 5-9 nmol/L), with significant selectivity over phosphatidylinositol 3-kinase (PI3K) isofoms (>100-fold). Unlike the rapalogs, these inhibitors acutely blocked substrate phosphorylation by mTORC1 and mTORC2 in vitro and in cells in response to growth factor, amino acids, and hyperactive PI3K/AKT. Unlike the inhibitors of PI3K or dual-pan PI3K/mTOR, cellular inhibition of P-S6K1(T389) and P-AKT(S473) by the pyrazolopyrimidines occurred at significantly lower inhibitor concentrations than those of P-AKT(T308) (PI3K-PDK1 readout), showing mTOR selectivity in cellular setting. mTOR kinase inhibitors reduced AKT downstream function and inhibited proliferation of diverse cancer cell lines. These effects correlated with a strong G(1) cell cycle arrest in both the rapamycin-sensitive and rapamycin-resistant cells, selective induction of apoptosis, repression of global protein synthesis, and down-regulation of angiogenic factors. When injected into tumor-bearing mice, WYE-354 inhibited mTORC1 and mTORC2 and displayed robust antitumor activity in PTEN-null tumors. Together, our results highlight mechanistic differentiation between rapalogs and mTOR kinase inhibitors in targeting cancer cell growth and survival and provide support for clinical development of mTOR kinase inhibitors as new cancer therapy.
