Pre-clinical evaluation of the MDM2-p53 antagonist RG7388 alone and in combination with chemotherapy in neuroblastoma.

Neuroblastoma is a predominantly p53 wild-type (wt) tumour and MDM2-p53 antagonists offer a novel therapeutic strategy for neuroblastoma patients. RG7388 (Roche) is currently undergoing early phase clinical evaluation in adults. This study assessed the efficacy of RG7388 as a single-agent and in combination with chemotherapies currently used to treat neuroblastoma in a panel of neuroblastoma cell lines. RG7388 GI50 concentrations were determined in 21 p53-wt and mutant neuroblastoma cell lines of varying MYCN, MDM2 and p14(ARF) status, together with MYCN-regulatable Tet21N cells. The primary determinant of response was the presence of wt p53, and overall there was a >200-fold difference in RG7388 GI50 concentrations for p53-wt versus mutant cell lines. Tet21N MYCN+ cells were significantly more sensitive to RG7388 compared with MYCN- cells. Using median-effect analysis in 5 p53-wt neuroblastoma cell lines, selected combinations of RG7388 with cisplatin, doxorubicin, topotecan, temozolomide and busulfan were synergistic. Furthermore, combination treatments led to increased apoptosis, as evident by higher caspase-3/7 activity compared to either agent alone. These data show that RG7388 is highly potent against p53-wt neuroblastoma cells, and strongly supports its further evaluation as a novel therapy for patients with high-risk neuroblastoma and wt p53 to potentially improve survival and/or reduce toxicity.

A selective small molecule NOP (ORL-1 receptor) partial agonist for the treatment of anxiety.

Small molecule (1) has been identified as a selective partial agonist of Opioid Receptor Like-1 (ORL-1) with potential utility for the treatment of anxiety and other disorders. Nociceptin (orphanin FQ) is an endogenous peptide ligand that binds to ORL-1, however it does not bind the classical δ, µ and κ opioid receptors with high affinity. The synthesis of 1 involved using a molecular diversity approach, to rapidly advance a library of compounds for biological testing. A lead selective potent partial agonist (35-fold ORL-1/Mu) progressed to ORL-1 (NOP or OP4) proof of concept testing in advanced studies. The synthetic approach and biological data for the related chemical series will be presented.

Effect of the MDM2 antagonist RG7112 on the P53 pathway in patients with MDM2-amplified, well-differentiated or dedifferentiated liposarcoma: an exploratory proof-of-mechanism study.

BACKGROUND: We report a proof-of-mechanism study of RG7112, a small-molecule MDM2 antagonist, in patients with chemotherapy-naive primary or relapsed well-differentiated or dedifferentiated MDM2-amplified liposarcoma who were eligible for resection. METHODS: Patients with well-differentiated or dedifferentiated liposarcoma were enrolled at four centres in France. Patients received up to three 28-day neoadjuvant treatment cycles of RG7112 1440 mg/m(2) per day for 10 days. If a patient progressed at any point after the first cycle, the lesion was resected or, if unresectable, an end-of-study biopsy was done. The primary endpoint was to assess markers of RG7112-dependent MDM2 inhibition and P53 pathway activation (P53, P21, MDM2, Ki-67, macrophage inhibitory cytokine-1 [MIC-1], and apoptosis). All analyses were per protocol. This trial is registered with EudraCT, number 2009-015522-10. RESULTS: Between June 3, and Dec 14, 2010, 20 patients were enrolled and completed pretreatment and day 8 biopsies. 18 of 20 patients had TP53 wild-type tumours and two carried missense TP53 mutations. 14 of 17 assessed patients had MDM2 gene amplification. Compared with baseline, P53 and P21 concentrations, assessed by immunohistochemistry, had increased by a median of 4·86 times (IQR 4·38-7·97; p=0·0001) and 3·48 times (2·05-4·09; p=0·0001), respectively, at day 8 (give or take 2 days). At the same timepoint, relative MDM2 mRNA expression had increased by a median of 3·03 times (1·23-4·93; p=0·003) that at baseline. The median change from baseline for Ki-67-positive tumour cells was -5·05% (IQR -12·55 to 0·05; p=0·01). Drug exposure correlated with blood concentrations of MIC-1 (p<0·0001) and haematological toxicity. One patient had a confirmed partial response and 14 had stable disease. All patients experienced at least one adverse event, mostly nausea (14 patients), vomiting (11 patients), asthenia (nine patients), diarrhoea (nine patients), and thrombocytopenia (eight patients). There were 12 serious adverse events in eight patients, the most common of which were neutropenia (six patients) and thrombocytopenia (three patients). DISCUSSION: MDM2 inhibition activates the P53 pathway and decreases cell proliferation in MDM2-amplified liposarcoma. This study suggests that it is feasible to undertake neoadjuvant biopsy-driven biomarker studies in liposarcoma. FUNDING: F Hoffmann-La Roche.

Phase I study of RO4929097, a gamma secretase inhibitor of Notch signaling, in patients with refractory metastatic or locally advanced solid tumors.

PURPOSE: To determine the maximum-tolerated dose (MTD) and assess safety, pharmacokinetics, pharmacodynamics, and evidence of antitumor activity of RO4929097, a gamma secretase inhibitor of Notch signaling in patients with advanced solid malignancies. PATIENTS AND METHODS: Patients received escalating doses of RO4929097 orally on two schedules: (A) 3 consecutive days per week for 2 weeks every 3 weeks; (B) 7 consecutive days every 3 weeks. To assess reversible CYP3A4 autoinduction, the expanded part of the study tested three dosing schedules: (B) as above; modified A, 3 consecutive d/wk for 3 weeks; and (C) continuous daily dosing. Positron emission tomography scans with [(18)F]fluorodeoxyglucose (FDG-PET) were used to assess tumor metabolic effects. RESULTS: Patients on schedule A (n = 58), B (n = 47), and C (n = 5; expanded cohort) received 302 cycles of RO4929097. Common grade 1 to 2 toxicities were fatigue, thrombocytopenia, fever, rash, chills, and anorexia. Transient grade 3 hypophosphatemia (dose-limiting toxicity, one patient) and grade 3 pruritus (two patients) were observed at 27 mg and 60 mg, respectively; transient grade 3 asthenia was observed on schedule A at 80 mg (one patient). Tumor responses included one partial response in a patient with colorectal adenocarcinoma with neuroendocrine features, one mixed response (stable disease) in a patient with sarcoma, and one nearly complete FDG-PET response in a patient with melanoma. Effect on CYP3A4 induction was observed. CONCLUSION: RO4929097 was well tolerated at 270 mg on schedule A and at 135 mg on schedule B; the safety of schedule C has not been fully evaluated. Further studies are warranted on the basis of a favorable safety profile and preliminary evidence of clinical antitumor activity.

Synthesis and evaluation of 2,7-diamino-thiazolo[4,5-d] pyrimidine analogues as anti-tumor epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors.

2,7-Diamino-thiazolo[4,5-d]pyrimidine analogues were synthesized as novel epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors. Representative compounds showed potent and selective EGFR inhibitory activities and inhibited in vitro cellular proliferation in EGFR-overexpressing human tumor cells. The synthesis and preliminary biological, physical, and pharmacokinetic evaluation of these thiazolopyrimidine compounds are reported.

Synthesis, in vitro activities of (2-cyclopropoxyphenyl)piperidine derivatives for alpha 1a and alpha 1d adrenergic receptor inhibitors.

An alpha 1a- and alpha 1d-adrenergic receptor (AR) selective antagonist may be a more efficacious treatment for BPH/LUTS patients and may have fewer side effects than the existing pharmaceuticals. A facile synthesis for a series of (2-cyclopropoxyphenyl)piperidine derivatives has been developed, in which aryl vinyl ether formation and subsequent cyclopropyl formation provide efficient access to key intermediate N-Boc-4-(2-cyclopropoxyphenyl)piperidine. The synthesized (2-cyclopropoxyphenyl)piperidine derivatives display high affinity and selectivity for alpha1a-AR and alpha1d-AR compared to alpha1b-AR and D2 receptor, Ki values for alpha1a-AR are 0.91 nM to 79.0 nM and alpha1d-AR are 2.0 nM to 57 nM; Ki values for alpha1b-AR are 107 nM to 839.8 nM and D2 receptor are 66.2 nM to 187.1 nM. The selectivity ratios of Ki(alpha1b)/Ki(alpha1a) are 11 to 155 fold, Ki(alpha1b)/Ki(alpha1d) are 6 to 171 fold, Ki(D2)/Ki(alpha1a) are 2 to 158 fold, and Ki(D2)/Ki(alpha1d) are 1.2 to 89 fold. Compound 17a shows improved stability in human liver microsome test (t1/2 = 18 minutes).

7-[1H-Indol-2-yl]-2,3-dihydro-isoindol-1-ones as dual Aurora-A/VEGF-R2 kinase inhibitors: design, synthesis, and biological activity.

A novel series of 7-[1H-indol-2-yl]-2,3-dihydro-isoindol-1-ones designed to be inhibitors of VEGF-R2 kinase was synthesized and found to potently inhibit VEGF-R2 and Aurora-A kinases. The structure-based design, synthesis, and initial SAR of the series are discussed.

A novel 5-[1,3,4-oxadiazol-2-yl]-N-aryl-4,6-pyrimidine diamine having dual EGFR/HER2 kinase activity: design, synthesis, and biological activity.

A novel 5-[1,3,4-oxadiazol-2-yl]-N-aryl-4,6-pyrimidine diamine was synthesized and found to have potent dual EGFR/HER2 kinase inhibitory activity. The structure-based drug design of this molecule as well as the kinase and cellular inhibition of HER2 kinase dependent cell lines will be discussed.

4-Amino-6-arylamino-pyrimidine-5-carbaldehyde hydrazones as potent ErbB-2/EGFR dual kinase inhibitors.

Members of a novel class of 4-amino-6-arylamino-pyrimidine-5-carbaldehyde hydrazones were identified as potent dual ErbB-2/EGFR kinase inhibitors using concept-guided design approach. These compounds inhibited the growth of ErbB-2 over-expressing human tumor cell lines (BT474, N87, and SK-BR-3) in vitro. Compound 15 emerged as a key lead and showed significant ability to inhibit growth factor-induced receptor phosphorylation in SK-BR-3 cells (IC(50)=54 nM) and cellular proliferation in vitro (IC(50)=14, 58, and 58 nM for BT474, N87, and SK-BR-3 respectively). The X-ray co-crystal structure of EGFR with a close analog (17) was determined and validated our design rationale.

Discovery of novel 4-amino-6-arylaminopyrimidine-5-carbaldehyde oximes as dual inhibitors of EGFR and ErbB-2 protein tyrosine kinases.

We herein disclose a novel series of 4-aminopyrimidine-5-carbaldehyde oximes that are potent and selective inhibitors of both EGFR and ErbB-2 tyrosine kinases, with IC(50) values in the nanomolar range. Structure-activity relationship (SAR) studies elucidated a critical role for the 4-amino and C-6 arylamino moieties. The X-ray co-crystal structure of EGFR with 37 was determined and validated our design rationale.

(Phenylpiperazinyl)cyclohexylureas: discovery of alpha1a/1d-selective adrenergic receptor antagonists for the treatment of benign prostatic hyperplasia/lower urinary tract symptoms (BPH/LUTS).

Benign prostatic hyperplasia/lower urinary tract symptoms (BPH/LUTS) can be effectively treated with alpha(1) adrenergic receptor antagonists. Unfortunately, currently marketed alpha(1) blockers produce CV-related side effects that are caused by the subtype non-selective nature of the drugs. To overcome this problem, it was postulated that an alpha(1a/1d) subtype-selective antagonist would bring more benefit for the treatment of BPH/LUTS. As a continuation of our effort to develop selective alpha(1a/1d) ligands, a series of (phenylpiperazinyl)cyclohexylureas was synthesized and evaluated for the ability to bind to three cloned human alpha(1)-adrenergic receptor subtypes. Several trans isomers were shown to have equal affinity for both alpha(1a), and alpha(1d) subtypes, with 14- to 47-fold selectivity versus the alpha(1b) subtype and >15-fold selectivity versus dopamine D(2).

Cellular and in vivo activity of JNJ-28871063, a nonquinazoline pan-ErbB kinase inhibitor that crosses the blood-brain barrier and displays efficacy against intracranial tumors.

JNJ-28871063 is a potent and highly selective pan-ErbB kinase inhibitor from a novel aminopyrimidine oxime structural class that blocks the proliferation of epidermal growth factor receptor (EGFR; ErbB1)- and ErbB2-overexpressing cells but does not affect the growth of non-ErbB-overexpressing cells. Treatment of human cancer cells with JNJ-28871063 inhibited phosphorylation of functionally important tyrosine residues in both EGFR and ErbB2 and blocked downstream signal transduction pathways responsible for proliferation and survival. A single dose of compound reduced phosphorylation of ErbB2 receptors in tumor-bearing mice, demonstrating target suppression in vivo. Tissue distribution studies show that JNJ-28871063 crosses the blood-brain barrier and penetrates into tumors, where it is able to accumulate to higher levels than those found in the plasma. JNJ-28871063 showed oral antitumor activity in human tumor xenograft models that overexpress EGFR and ErbB2. In an intracranial ErbB2-overexpressing tumor model, JNJ-28871063 extended survival relative to untreated animals. The brain is a primary site of metastasis for EGFR-overexpressing lung cancers and ErbB2-overexpressing breast cancers. Therefore, the ability to penetrate into the brain could be an advantage over existing therapies such as trastuzumab (Herceptin) and cetuximab (Erbitux), which are antibodies and do not cross the blood-brain barrier. These results show that JNJ-28871063 is orally bioavailable, has activity against EGFR and ErbB2-dependent tumor xenografts, and can penetrate into the brain and inhibit ErbB2-overexpressing tumor growth.

Aminocyclohexylsulfonamides: discovery of metabolically stable alpha(1a/1d)-selective adrenergic receptor antagonists for the treatment of benign prostatic hyperplasia/lower urinary tract symptoms (BPH/LUTS).

Benign prostatic hyperplasia/lower urinary tract symptoms (BPH/LUTS) can be effectively treated by alpha(1) adrenergic receptor antagonists, but these drugs also produce side effects that are related to their subtype non-selective nature. To overcome this limitation, it was hypothesized that an alpha(1a/1d) subtype-selective antagonist would be efficacious while keeping side effects to a minimum. To discover alpha(1a/1d)-selective antagonists and improve metabolic stability of our previously reported compounds, we have designed and synthesized a series of (phenylpiperazinyl)- or (phenylpiperidinyl)-cyclohexylsulfonamides. By incorporating the information obtained from metabolism studies, we were able to discover several compounds that are both alpha(1a/1d) adrenoceptor subtype selective and show increased stability toward human liver microsomal metabolism. The selectivity profile of these compounds provides great improvement over the commercial drug tamsulosin, hence may pave the way to the development of new and efficacious therapeutic agents with reduced side effects.

Synthesis and evaluation of pyrazolo[3,4-b]pyridine CDK1 inhibitors as anti-tumor agents.

A series of 3,5-disubstituted pyrazolo[3,4-b]pyridine cyclin-dependent kinase (CDK) inhibitors was synthesized. These compounds showed potent and selective CDK inhibitory activities and inhibited in vitro cellular proliferation in cultured human tumor cells. Selected compounds were evaluated in an in vivo tumor xenograft model. The synthesis and biological evaluation of these pyrazolo[3,4-b]pyridines and related compounds are reported.

(Phenylpiperidinyl)cyclohexylsulfonamides: development of alpha1a/1d-selective adrenergic receptor antagonists for the treatment of benign prostatic hyperplasia/lower urinary tract symptoms (BPH/LUTS).

Although alpha(1) adrenergic receptor blockers can be very effective for the treatment of benign prostatic hyperplasia/lower urinary tract symptoms (BPH/LUTS), their usage is limited by CV-related side-effects that are caused by the subtype non-selective nature of the current drugs. To overcome this problem, it was hypothesized that a alpha(1a/1d) subtype selective antagonist would bring more benefit for the therapy of BPH/LUTS. In developing such selective alpha(1a/1d) ligands, a series of (phenylpiperidinyl)cyclohexylsulfonamides has been synthesized and evaluated for binding to three cloned human alpha(1)-adrenergic receptor subtypes. Many compounds showed equal affinity for both alpha(1a) and alpha(1d) subtypes with good selectivity versus the alpha(1b) subtype.

4-Aryl-5-cyano-2-aminopyrimidines as VEGF-R2 inhibitors: synthesis and biological evaluation.

A novel series of 4-aryl-5-cyano-2-aminopyrimidines were synthesized and found to have potent VEGF-R2 kinase inhibitory activity. Structure-activity relationships were investigated and compound 14a was shown to be efficacious in a mouse model of corneal neovascularization.

(Arylpiperazinyl)cyclohexylsufonamides: discovery of alpha(1a/1d)-selective adrenergic receptor antagonists for the treatment of Benign Prostatic Hyperplasia/Lower Urinary Tract Symptoms (BPH/LUTS).

Benign Prostatic Hyperplasia/Lower Urinary Tract Symptoms (BPH/LUTS) can be effectively treated by alpha(1)-adrenergic receptor antagonists. Unfortunately, all currently marketed alpha(1) blockers produced CV related side effects that are caused by the subtype non-selective nature of the drugs. To overcome this problem, it was postulated that a alpha(1a/1d) subtype selective antagonist would bring more benefit for the treatment of BPH/LUTS. In developing selective alpha(1a/1d) ligands, (arylpiperazinyl)cyclohexylsulfonamides were synthesized and their binding profiles against three cloned human alpha(1)-adrenergic receptor subtypes were evaluated. Many compounds show equal affinity for both alpha(1a) and alpha(1d) subtypes with good selectivity against the alpha(1b) subtype. They also overcome the problem of dopamine receptor affinity that previous analogues had exhibited.

Synthesis and biological study of 2-amino-4-aryl-5-chloropyrimidine analogues as inhibitors of VEGFR-2 and cyclin dependent kinase 1 (CDK1).

The series of 2-amino-4-aryl-5-chloropyrimidines was discovered to be potent for both VEGFR-2 and CDK1. Described here are the chemistry for analogue synthesis, SAR study, and its kinase selectivity prolifing. The full rat PK data and in vivo efficacy study are also included.