Christopher Thomas Brain

NOVEL PROCEDURE FOR THE SYNTHESIS OF 1,3,4-OXADIAZOLES FROM 1,2-DIACYLHYDRAZINES USING POLYMER-SUPPORTED BURGESS REAGENT UNDER MICROWAVE CONDITIONS

Abstract A novel and efficient means of effecting the cyclodehydration of 1,2-diacylhydrazines to provide 1,3,4-oxadiazoles is reported. Polymer supported Burgess reagent was utilised in combination with single-mode microwave heating.

An intramolecular palladium-catalysed aryl amination reaction to produce benzimidazoles

A novel synthesis of benzimidazoles by a palladium-catalysed intramolecular N-arylation reaction from (o-bromophenyl)amidine precursors is described.

Antihyperalgesic properties of the cannabinoid CT-3 in chronic neuropathic and inflammatory pain states in the rat.

&NA; CT‐3 (ajulemic acid) is a synthetic analogue of a metabolite of Δ9‐tetrahydrocannabinol that has reported analgesic efficacy in neuropathic pain states in man. Here we show that CT‐3 binds to human cannabinoid receptors in vitro, with high affinity at hCB1 (Ki 6 nM) and hCB2 (Ki 56 nM) receptors. In a functional GTP‐γ‐S assay CT‐3 was an agonist at both hCB1 and hCB2 receptors (EC50 11 and 13.4 nM, respectively). In behavioural models of chronic neuropathic and inflammatory pain in the rat, oral administration of CT‐3 (0.1–1 mg/kg) produced up to 60% reversal of mechanical hyperalgesia. In both models the antihyperalgesic activity was prevented by the CB1‐antagonist SR141716A but not the CB2‐antagonist SR144528. In the tetrad of tests for CNS activity, CT‐3 (1–10 mg/kg, po) produced dose‐related catalepsy, deficits in locomotor performance, hypothermia, and acute analgesia. Comparison of 50% maximal effects in the tetrad and chronic pain assays produced an approximate therapeutic index of 5–10. Pharmacokinetic analysis showed that CT‐3 exhibits significant but limited brain penetration, with a brain/plasma ratio of 0.4 measured following oral administration, compared to ratios of 1.0–1.9 measured following subcutaneous administration of WIN55,212‐2 or Δ9‐THC. These data show that CT‐3 is a cannabinoid receptor agonist and is efficacious in animal models of chronic pain by activation of the CB1 receptor. Whilst it shows significant cannabinoid‐like CNS activity, it exhibits a superior therapeutic index compared to other cannabinoid compounds, which may reflect a relatively reduced CNS penetration.

Abstract PR02: LEE011: An orally bioavailable, selective small molecule inhibitor of CDK4/6– Reactivating Rb in cancer.

The tumor suppressor Retinoblastoma protein (Rb) is often inactivated in cancer. In many tumors, the Rb protein itself is retained but functionally inactivated by increased CDK4/6 kinase activity. A number of key oncogenic aberrations can result in this increased activity, including inactivation of CDKN2A (p16), translocation or amplification of D-cyclins, amplification of CDK4/6 and mutations/deletions upstream of cyclin D, such as activating mutations of BRAF/PIK3CA and PTEN deletion. Abolishing CDK4/6 kinase activity and subsequent reactivation of Rb in these tumors has been demonstrated to inhibit tumor initiation and growth. Here we will describe LEE011- an orally bioavailable, selective small molecule inhibitor of CDK4/6 kinases. LEE011 inhibits CDK4/6 kinase activity with nM IC50 and is highly selective for these targets. In a number of preclinical tumor models, LEE011 demonstrates a dose dependent anti-tumor activity that tracks well with CDK4/6 inhibition. The primary mechanism for growth inhibitory effect appears to be G1 arrest in vitro , although, in some sensitive in vivo models, regressions are observed. Importantly, given the role of CDK4/6 downstream of other oncogenic driver mutations, LEE011 shows single agent activity in melanomas with activating mutations of BRAF or NRAS, and in breast cancers with intact estrogen receptor and/or activating aberrations of PIK3CA/Her-2. Combining LEE011 with LGX818, a V600E BRAF specific inhibitor, leads to robust anti-tumor activity in melanoma models that are both sensitive and, importantly, those that are resistant to LGX818. Furthermore, combining LEE011 with BYL719, a PIK3CA specific inhibitor, also leads to significant anti-tumor activity in breast cancer models both sensitive and resistant to BYL719. Several clinical studies evaluating LEE011 as single agent and in combinations are underway. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):PR02. Citation Format: Sunkyu Kim, Alice Loo, Rajiv Chopra, Giordano Caponigro, Alan Huang, Sadhna Vora, Sudha Parasuraman, Steve Howard, Nicholas Keen, William Sellers, Christopher Brain. LEE011: An orally bioavailable, selective small molecule inhibitor of CDK4/6– Reactivating Rb in cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr PR02.

Antiproliferative Effects of CDK4/6 Inhibition in CDK4-Amplified Human Liposarcoma In Vitro and In Vivo

Well-differentiated/dedifferentiated liposarcomas (WD/DDLPS) are among the most common subtypes of soft tissue sarcomas. Conventional systemic chemotherapy has limited efficacy and novel therapeutic strategies are needed to achieve better outcomes for patients. The cyclin-dependent kinase 4 (CDK4) gene is highly amplified in more than 95% of WD/DDLPS. In this study, we explored the role of CDK4 and the effects of NVP-LEE011 (LEE011), a novel selective inhibitor of CDK4/CDK6, on a panel of human liposarcoma cell lines and primary tumor xenografts. We found that both CDK4 knockdown by siRNA and inhibition by LEE011 diminished retinoblastoma (RB) phosphorylation and dramatically decreased liposarcoma cell growth. Cell-cycle analysis demonstrated arrest at G0–G1. siRNA-mediated knockdown of RB rescued the inhibitory effects of LEE011, demonstrating that LEE011 decreased proliferation through RB. Oral administration of LEE011 to mice bearing human liposarcoma xenografts resulted in approximately 50% reduction in tumor 18F-fluorodeoxyglucose uptake with decreased tumor biomarkers, including RB phosphorylation and bromodeoxyuridine incorporation in vivo. Continued treatment inhibited tumor growth or induced regression without detrimental effects on mouse weight. After prolonged continuous dosing, reestablishment of RB phosphorylation and cell-cycle progression was noted. These findings validate the critical role of CDK4 in maintaining liposarcoma proliferation through its ability to inactivate RB function, and suggest its potential function in the regulation of survival and metabolism of liposarcoma, supporting the rationale for clinical development of LEE011 for the treatment of WD/DDLPS. Mol Cancer Ther; 13(9); 2184–93. ©2014 AACR.

4-(Pyrazol-4-yl)-pyrimidines as selective inhibitors of cyclin-dependent kinase 4/6.

Identification and structure-guided optimization of a series of 4-(pyrazol-4-yl)-pyrimidines as selective CDK4/6 inhibitors is reported herein. Several potency and selectivity determinants were established based on the X-ray crystallographic analysis of representative compounds bound to monomeric CDK6. Significant selectivity for CDK4/6 over CDK1 and CDK2 was demonstrated with several compounds in both enzymatic and cellular assays.

Fragment-Based Discovery of 7-Azabenzimidazoles as Potent, Highly Selective, and Orally Active CDK4/6 Inhibitors

Herein, we describe the discovery of potent and highly selective inhibitors of both CDK4 and CDK6 via structure-guided optimization of a fragment-based screening hit. CDK6 X-ray crystallography and pharmacokinetic data steered efforts in identifying compound 6, which showed >1000-fold selectivity for CDK4 over CDKs 1 and 2 in an enzymatic assay. Furthermore, 6 demonstrated in vivo inhibition of pRb-phosphorylation and oral efficacy in a Jeko-1 mouse xenograft model.

N-Thioacylation of β-Amino Alcohols by N-(Thioacyl)phthalimides: A Facile Synthesis of α-Amino Acid Thiazolines

Abstract β-amino alcohols are selectively N -thioacylated by N -(thioacyl)phthalimides under very mild conditions to provide N -(hydroxyethyl)thioamides in high yields. Cyclodehydration with Burgess reagent then provides α-amino acid thiazolines. This approach provides a convenient alternative to those based upon thionation of a preformed N -(hydroxyethyl)amide.

Sulfonamido-aryl ethers as bradykinin B1 receptor antagonists.

The synthesis and identification of sulfonamido-aryl ethers as potent bradykinin B1 receptor antagonists from a approximately 60,000 member encoded combinatorial library are reported. Two distinct series of compounds exhibiting different structure-activity relationships were identified in a bradykinin B1 whole-cell receptor-binding assay. Specific examples exhibit K(i) values of approximately 10nM.

Abstract A236: Human liposarcoma growth inhibition by novel CDK4/6 inhibitor LEE011.

Background: Well-differentiated and de-differentiated liposarcomas (WD/DDLPS) are characterized by high-level chromosomal amplification of 12q13–15, a region including the cyclin-dependent kinase 4 (CDK4) oncogene. In this study, we explored the effects of LEE011, a novel selective inhibitor of CDK4/CDK6 on human liposarcoma cell lines and primary tumor xenografts. Materials and Methods: We examined the expression of cell cycle regulatory proteins in human liposarcoma cell lines and human normal preadipocytes/adipocytes. CDK4 small interfering RNA (siRNA) or LEE011 was applied to liposarcoma cell lines and the biological consequences were determined by phospho-RB immunoblot, cell cycle assay and cell enumeration. In addition, liposarcoma cells were transfected with RB siRNA to determine the specificity of the effects of LEE011. Nude mice implanted with human liposarcoma cell lines or primary tumors were treated with LEE011 or vehicle by oral gavage. After 3 daily doses, in vivo BrdU incorporation assay, immunostains for phospho-RB, and 18F-fluorodeoxyglucose (FDG)-positron emission tomography (PET) scan were performed. Tumor size and animal weight were serially measured during 3 weeks of dosing. Results: CDK4 and p-Rb are highly expressed in human liposarcoma cell lines in comparison with normal preadipocytes/adipocytes. Both CDK4 siRNA and LEE011 inhibited RB phosphorylation at the CDK4/6-specific sites Ser780 and Ser807/811 in a dose-dependent manner and dramatically inhibited liposarcoma cell growth. Cell cycle analysis demonstrated arrest at G0/G1. siRNA-mediated knockdown of RB1 rescued the inhibitory effects of LEE011, suggesting that LEE011 inhibited proliferation as expected through the RB pathway. Oral administration of LEE011 to mice bearing human liposarcoma xenografts reduced tumor 18F-FDG uptakes by approximately 50% and dramatically decreased biomarkers including RB phosphorylation and BrdU incorporation in vivo. Continued LEE011 treatment inhibited growth of established cell line xenografts as well as primary human liposarcoma tumor xenografts without detrimental effects on mouse weight. Conclusions: LEE011 decreases cell cycle progression, hypermetabolism, and proliferation of human liposarcomas in an RB-dependent manner, consistent with inhibition of CDK4. Clinical trials of LEE011 in patients with liposarcoma are warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A236.