The orally active microtubule-disrupting agent (S)-1-ethyl-3-(2-methoxy-4-(5-methyl-4-((1-(pyridin-3-yl)butyl)amino)pyrimidin-2-yl)phenyl)urea (CYT997), reported previously by us (Bioorg Med Chem Lett 19:4639-4642, 2009; Mol Cancer Ther 8:3036-3045, 2009), is potently cytotoxic to a variety of cancer cell lines in vitro and shows antitumor activity in vivo. In addition to its cytotoxic activity, CYT997 possesses antivascular effects on tumor vasculature. To further characterize the vascular disrupting activity of CYT997 in terms of dose and temporal effects, we studied the activity of the compound on endothelial cells in vitro and on tumor blood flow in vivo by using a variety of techniques. In vitro, CYT997 is shown to potently inhibit the proliferation of vascular endothelial growth factor-stimulated human umbilical vein endothelial cells (IC(50) 3.7 ± 1.8 nM) and cause significant morphological changes at 100 nM, including membrane blebbing. Using the method of corrosion casting visualized with scanning electron microscopy, a single dose of CYT997 (7.5 mg/kg i.p.) in a metastatic cancer model was shown to cause destruction of tumor microvasculature in metastatic lesions. Furthermore, repeat dosing of CYT997 at 10 mg/kg and above (intraperitoneally, b.i.d.) was shown to effectively inhibit development of liver metastases. The time and dose dependence of the antivascular effects were studied in a DLD-1 colon adenocarcinoma xenograft model using the fluorescent dye Hoechst 33342. CYT997 demonstrated rapid and dose-dependent vascular shutdown, which persists for more than 24 h after a single oral dose. Together, the data demonstrate that CYT997 possesses potent antivascular activity and support continuing development of this promising compound.

CYT997 was discovered as a potent tubulin polymerization inhibitor possessing potent cytotoxic activity against a range of cancer cells. Details of SAR studies, pharmacokinetic investigations and synthesis of compounds leading to the discovery of CYT997 are reported.

AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO CYT997 is an orally available synthetic small molecule that is currently in phase II clinical studies within different oncology settings. CYT997 has been shown to exhibit potent vascular disrupting activity in vivo at doses well below its maximum tolerated dose. We have modeled this activity in vitro and found that CYT997 potently inhibited the proliferation of HUVEC primary endothelial cells at low nM concentrations. In order to evaluate the acute vascular effect of CYT997 on blood flow through tumour tissue in vivo , we have used Laser Doppler Flowmetry to measure blood flow in a mouse model of metastatic colorectal cancer. Oral administration of CYT997 led to a significant reduction in tumour blood flow 15 minutes post administration and this effect persisted for up to 96hrs post administration. Similarly, in athymic nude mice bearing subcutaneous DLD-1 human colon adenocarcinoma xenografts a single oral dose of CYT997 led to a time and concentration dependent shutdown of tumour vasculature. The observed vascular shutdown was rapid and was detected at 1-hour post administration of CYT997. Significant vascular shutdown was maintained in tumours at 24-hours post administration of CYT997 and subsequent histology demonstrated greater necrosis of the tumour core in mice treated with CYT997. In addition to vascular disrupting activity, CYT997 also causes G2/M cell cycle arrest and apoptosis of tumor cell lines. Time course analysis of the cell cycle in HCT-15 cells was performed and showed that CYT997 (250 nM) markedly and rapidly increased the fraction of cells in G2/M. As CYT997 is under clinical development as a therapy to be used in conjunction with current antitumor therapies we have assessed the combination of CYT997 and a number of clinically used anti-cancer agents with a range of different modes of action, on the proliferation of HCT-15 liver adenocarcinoma cells. CYT997 demonstrated marked synergy with 5FU (CI=0.55 at IC50), paclitaxel (CI=0.60 at IC50), and vincristine (CI=0.35 at IC50). In contrast, the kinase inhibitor sorafenib did not affect CYT997 activity on HCT-15 cell viability. Together, these findings clearly demonstrate that CYT997 is an orally available vascular disrupting agent that reduces tumour blood flow in a dose dependent manner in a two murine cancer models and raises the possibility that CYT997 may exhibit increased efficacy in combination with other common anticancer therapies. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr LB-211.

A series of phenylaminopyrimidines has been identified as inhibitors of Janus kinases (JAKs). Development of this initial series led to the potent JAK2/JAK1 inhibitor CYT387 (N-(cyanomethyl)-4-[2-[[4-(4-morpholinyl)phenyl]amino]-4-pyrimidinyl]-benzamide). Details of synthesis and SAR studies of these compounds are reported.

CYT997 is a wholly synthetic compound that possesses highly potent cytotoxic activity in vitro through inhibition of microtubule polymerization. CYT997 blocks the cell cycle at the G(2)-M boundary, and Western blot analysis indicates an increase in phosphorylated Bcl-2, along with increased expression of cyclin B1. Caspase-3 activation is also observed in cells treated with CYT997 along with the generation of poly(ADP-ribose) polymerase. The compound possesses favorable pharmacokinetic properties, is orally bioavailable, and is efficacious per os in a range of in vivo cancer models, including some refractory to paclitaxel treatment. CYT997 exhibits vascular disrupting activity as measured in vitro by effects on the permeability of human umbilical vein endothelial cell monolayers, and in vivo by effects on tumor blood flow. CYT997 possesses a useful combination of pharmacologic and pharmacokinetic properties and has considerable potential as a novel anticancer agent.

The non-natural enantiomeric forms of narciclasine and lycoricidine ((-)-1 and (-)-2, respectively), as well as congeners 3-6 are available through chemoenzymatic synthesis. Accordingly, they have now been tested for their cytotoxic effects in a 13-member human cancer cell-line panel and found to be only weakly active. In contrast, an authentic sample of the natural enantiomeric form of narciclasine ((+)-1) was found to be highly active in the same screens.

A series of 2-(alpha-methylbenzylamino) pyrazines have shown to be potent inhibitors of the FMS tyrosine receptor kinase. Details of SAR studies, modeling and synthesis of compounds within this series are reported.