Inder Chaudhary

SKI-606, a Src/Abl Inhibitor with In vivo Activity in Colon Tumor Xenograft Models

Src up-regulation is a common event in human cancers. In colorectal cancer, increased Src levels are an indicator of poor prognosis, and progression to metastatic disease is associated with substantial increases in Src activity. Therefore, we examined the activity of SKI-606, a potent inhibitor of Src and Abl kinases, against colon tumor lines in vitro and in s.c. tumor xenograft models. SKI-606 inhibited Src autophosphorylation with an IC(50) of approximately 0.25 micromol/L in HT29 cells. Phosphorylation of Tyr(925) of focal adhesion kinase, a Src substrate, was reduced by similar concentrations of inhibitor. Antiproliferative activity on plastic did not correlate with Src inhibition in either HT29 or Colo205 cells (IC(50)s, 1.5 and 2.5 micromol/L, respectively), although submicromolar concentrations of SKI-606 inhibited HT29 cell colony formation in soft agar. SKI-606 also caused loosely aggregated Colo205 spheroids to condense into compact spheroids. On oral administration to nude mice at the lowest efficacious dose, peak plasma concentrations of approximately 3 micromol/L, an oral bioavailability of 18%, and a t(1/2) of 8.6 hours were observed. SKI-606 was orally active in s.c. colon tumor xenograft models and caused substantial reductions in Src autophosphorylation on Tyr(418) in HT29 and Colo205 tumors. SKI-606 inhibited HT29 tumor growth on once daily administration, whereas twice daily administration was necessary to inhibit Colo205, HCT116, and DLD1 tumor growth. These results support development of SKI-606 as a therapeutic agent for treatment of colorectal cancer.

Bis(morpholino-1,3,5-triazine) derivatives: potent adenosine 5'-triphosphate competitive phosphatidylinositol-3-kinase/mammalian target of rapamycin inhibitors: discovery of compound 26 (PKI-587), a highly efficacious dual inhibitor.

The PI3K/Akt signaling pathway is a key pathway in cell proliferation, growth, survival, protein synthesis, and glucose metabolism. It has been recognized recently that inhibiting this pathway might provide a viable therapy for cancer. A series of bis(morpholino-1,3,5-triazine) derivatives were prepared and optimized to provide the highly efficacious PI3K/mTOR inhibitor 1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea 26 (PKI-587). Compound 26 has shown excellent activity in vitro and in vivo, with antitumor efficacy in both subcutaneous and orthotopic xenograft tumor models when administered intravenously. The structure-activity relationships and the in vitro and in vivo activity of analogues in this series are described.

Antitumor Efficacy of PKI-587, a Highly Potent Dual PI3K/mTOR Kinase Inhibitor

Purpose: The aim of this study was to show preclinical efficacy and clinical development potential of PKI-587, a dual phosphoinositide 3-kinase (PI3K)/mTOR inhibitor. Experimental Design: In vitro class 1 PI3K enzyme and human tumor cell growth inhibition assays and in vivo five tumor xenograft models were used to show efficacy. Results: In vitro, PKI-587 potently inhibited class I PI3Ks (IC50 vs. PI3K-α = 0.4 nmol/L), PI3K-α mutants, and mTOR. PKI-587 inhibited growth of 50 diverse human tumor cell lines at IC50 values of less than 100 nmol/L. PKI-587 suppressed phosphorylation of PI3K/mTOR effectors (e.g., Akt), and induced apoptosis in human tumor cell lines with elevated PI3K/mTOR signaling. MDA-MB-361 [breast; HER2+, PIK3CA mutant (E545K)] was particularly sensitive to this effect, with cleaved PARP, an apoptosis marker, induced by 30 nmol/L PKI-587 at 4 hours. In vivo, PKI-587 inhibited tumor growth in breast (MDA-MB-361, BT474), colon (HCT116), lung (H1975), and glioma (U87MG) xenograft models. In MDA-MB-361 tumors, PKI-587 (25 mg/kg, single dose i.v.) suppressed Akt phosphorylation [at threonine(T)308 and serine(S)473] for up to 36 hours, with cleaved PARP (cPARP) evident up to 18 hours. PKI-587 at 25 mg/kg (once weekly) shrank large (∼1,000 mm3) MDA-MB-361 tumors and suppressed tumor regrowth. Tumor regression correlated with suppression of phosphorylated Akt in the MDA-MB-361 model. PKI-587 also caused regression in other tumor models, and efficacy was enhanced when given in combination with PD0325901 (MEK 1/2 inhibitor), irinotecan (topoisomerase I inhibitor), or HKI-272 (neratinib, HER2 inhibitor). Conclusion: Significant antitumor efficacy and a favorable pharmacokinetic/safety profile justified phase 1 clinical evaluation of PKI-587. Clin Cancer Res; 17(10); 3193–203. ©2011 AACR.

PWT-458, a novel pegylated-17-hydroxywortmannin, inhibits phosphatidylinositol 3-kinase signaling and suppresses growth of solid tumors.

Deregulated phosphatidylinositol 3-kinase (PI3K) signaling pathway is widely implicated in tumor growth and resistance to chemotherapy. While a strong rationale exists for pharmacological targeting of PI3K, only a few proof-of-principle in-vivo efficacy studies are currently available. PWT-458, pegylated-17-hydroxywortmannin, is a novel and highly potent inhibitor of PI3K in animal models. Upon in vivo cleavage of its poly(ethyleneglycol) (PEG), PWT-458 releases its active moiety 17-hydroxywortmannin (17-HWT), the most potent inhibitor in its class. Here we show that a single intravenous injection of PWT-458 rapidly inhibited PI3K signaling, as measured by a complete loss of AKT (Ser-473) phosphorylation in xenograft tumors grown in nude mice. Following a daily X5 dosing regimen, PWT-458 demonstrated single-agent antitumor activity in nude mouse xenograft models of U87MG glioma, non-small cell lung cancer (NSCLC) A549, and renal cell carcinoma (RCC) A498. Efficacious doses ranged from 0.5 mg/kg to 10 mg/kg, achieving a superior therapeutic index over 17-HWT. PWT-458 augmented anticancer efficacy of a suboptimal dose of paclitaxel against A549 and U87MG tumors. Combination treatment of PWT-458 and an mTOR inhibitor, Pegylated-Rapamycin (Peg-Rapa), resulted in an enhanced antitumor efficacy in U87MG. Finally, PWT-458 in combination with interferon-? (Intron-A) caused a dramatic regression of RCC A498, which was not achieved by either agent alone. These studies identify PWT-458 as an effective anticancer agent and provide strong proof-of-principle for targeting the PI3K pathway as novel anticancer therapy.

2,4-Diamino-quinazolines as inhibitors of β-catenin/Tcf-4 pathway: Potential treatment for colorectal cancer

The synthesis and SAR of a series of 2,4-diamino-quinazoline derivatives as beta-catenin/Tcf-4 inhibitors are described. This series was developed by modifying the initial lead 1, which was identified by screening of our compound library and found to inhibit the beta-catenin/Tcf-4 pathway. Replacement of the biphenyl moiety in compound 1 with the N-phenylpiperidine-4-carboxamide chain as in 2, resulted in a number of new analogues, which are potent inhibitors of the beta-catenin/Tcf-4 pathway. Compound such as 16k exhibited good cellular potency, solubility, metabolic stability and oral bioavailability.

Lead Optimization of N-3-Substituted 7-Morpholinotriazolopyrimidines as Dual Phosphoinositide 3-Kinase/Mammalian Target of Rapamycin Inhibitors: Discovery of PKI-402

Herein we describe the identification and lead optimization of triazolopyrimidines as a novel class of potent dual PI3K/mTOR inhibitors, resulting in the discovery of 3 (PKI-402). Compound 3 exhibits good physical properties and PK parameters, low nanomolar potency against PI3Kalpha and mTOR, and excellent inhibition of cell proliferation in several human cancer cell lines. Furthermore, in vitro and in vivo biomarker studies demonstrated the ability of 3 to shut down the PI3K/Akt pathway and induce apoptosis in cancer cells. In addition, 3 showed excellent in vivo efficacy in various human cancer xenografts, validating suppression of PI3K/mTOR signaling as a potential anticancer therapy.

Hybrid Inhibitors of Phosphatidylinositol 3-Kinase (PI3K) and the Mammalian Target of Rapamycin (mTOR): Design, Synthesis, and Superior Antitumor Activity of Novel Wortmannin−Rapamycin Conjugates

Hyperactivation of the PI3K/AKT/mTOR signaling pathway is common in cancer, and PI3K and mTOR act synergistically in promoting tumor growth, survival, and resistance to chemotherapy. Thus, combined targeting of PI3K and mTOR presents an opportunity for robust and synergistic anticancer efficacy. 17-Hydroxywortmannin (2a) analogues conjugated to rapamycin (3a) analogues via a prodrug linker are uniquely positioned for this approach. Our efforts led to the discovery of diester-linked conjugates that, upon in vivo hydrolysis, released two highly potent inhibitors. Conjugate 7c provided enhanced solubility relative to 3a and to an equivalent mixture of 3a and 9a and demonstrated profound activity in U87MG mouse xenografts, achieving an MED of 1.5 mg/kg, following weekly intravenous dosing. At 15 mg/kg, 7c completely inhibited the growth of HT29 tumors, whereas an equivalent mixture of the inhibitors was poorly tolerated. In the A498 renal tumor model, 7c exhibited superior efficacy over 3a or 9a when administered as a single agent or in combination with bevacizumab. Thus, we have uncovered a novel approach to target both PI3K and mTOR via hybrid inhibitors, leading to a broader and more robust anticancer efficacy.

PKI-179: An orally efficacious dual phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibitor

A series of mono-morpholino 1,3,5-triazine derivatives (8a-8q) bearing a 3-oxa-8-azabicyclo[3.2.1]octane were prepared and evaluated for PI3-kinase/mTOR activity. Replacement of one of the bis-morpholines in lead compound 1 (PKI-587) with 3-oxa-8-azabicyclo[3.2.1]octane and reduction of the molecular weight yielded 8m (PKI-179), an orally efficacious dual PI3-kinase/mTOR inhibitor. The in vitro activity, in vivo efficacy, and PK properties of 8m are discussed.

Design and Synthesis of Novel Diaminoquinazolines with in Vivo Efficacy for β-Catenin/T-Cell Transcriptional Factor 4 Pathway Inhibition

We are introducing a novel series of 2,4-diaminoquinazolines as beta-catenin/Tcf4 inhibitors which were identified by ligand-based design. Here we elucidate the SAR of this series and explain how we were able to improve key molecular properties such as solubility and cLogP leading to compound 9. Analogue 9 exhibited better biological activity and improved physical and pharmacological properties relative to the HTS hit 49. Furthermore, 9 demonstrated good cell growth inhibition against several human colorectal cancer lines such as LoVo and HT29. In addition, treatment with compound 9 led to gene expression changes that overlapped significantly with the transcriptional profile resulting from the pathway inhibition by siRNA knockdown of beta-catenin or Tcf4. Subsequently, 9 was tested for efficacy in a beta-catenin/RKE-mouse xenograft, where it led to more then 50% decrease in tumor volume.

Application of Quantitative NMR in Pharmacological Evaluation of Biologically Generated Metabolites: Implications in Drug Discovery

It is important to gain an understanding of the pharmacological activities of metabolite(s) of compounds in development, especially if they are found in systemic circulation in humans. Pharmacological evaluation of metabolites is normally conducted with synthetic standards, which become available during various stages of drug development. However, the synthesis of metabolite standards may be protracted, taking anywhere from several weeks to months to be completed. This often slows down early pharmacological evaluation of metabolites. Once a metabolite(s) is found to possess comparable (or greater) pharmacological activity than the parent compound, additional studies are performed to better understand the implications of circulating pharmacologically active metabolite(s). To conduct some of these studies as early as possible without slowing the progression of a compound in development is important, especially if critical go or no-go decisions impinge on the outcomes from these studies. Early pharmacological evaluation of significant metabolites is hereby proposed to be conducted in the drug discovery stage so that all pertinent studies and information can be gathered in a timely manner for decision-making. It is suggested that these major metabolites be isolated, either from biological or chemical sources, and quantified appropriately. For biologically generated metabolites, NMR is proposed as the tool of choice to quantitate these metabolites before their evaluation in pharmacological assays. For metabolites that have the same UV characteristics as the parent compound, quantitation can be conducted using UV spectroscopy instead of NMR. In this article, we propose a strategy that could be used to determine the pharmacological activities of metabolites isolated in submilligram quantities.