1H NMR (CDCl3, 500 MHz) 9.80 (1H, s, CHO), 7.45 (1H, dd, = 8.5 Hz, = 2.0 Hz, ArH ), 7.35 (1H, d, = 2.0 Hz, ArH), 6.93 (1H, d, = 8.5 Hz, ArH), 3.87 (3H, s, OCH3), 0.99 (9H, s, C(CH3)3), 0.16 (6H, s, Si(CH3)2); 13C NMR (CDCl3, 125 MHz) 190.2, 156.2, 145.2, 130.0, 126.0, 119.4, 110.9, 55.1, 25.3, 18.0, ?5.0. 3-= 0.40 (hexanes-EtOAc 70:30)] as a yellow oil, which was taken to the next step without further purification. mouse model bearing an orthotopic PC-3 (prostate) tumor as imaged by color Doppler ultrasound. The combination of these results provides evidence that the indole-based phosphate prodrug 33 (OXi8007) functions as a vascular disrupting agent (VDA) that may prove useful for the treatment of cancer. The vast majority of natural products and synthetic compounds that bind to the tubulin-microtubule protein system and subsequently interfere with the dynamic assembly/disassembly inherent to the ,-tubulin/microtubule system do so by many variations of this overall mechanism of action. The most thoroughly studied are interactions at the colchicine, vinca alkaloid, and taxoid sites.1,2 A significant number of antiproliferative, anticancer agents function through this basic mechanism of action. The combretastatin family of natural products consists of a variety of Kuntze (Combretaceae).1 Combretastatin A-4 (CA4) is among the most potent antimitotic agents from this family of compounds and binds to the colchicine site on tubulin (Figure 1, compound 2).3C5 As with many natural products, the challenge of water solubility led to the development of a disodium phosphate prodrug 3, combretastatin A-4P (CA4P).6 The discovery of CA4 has led to a diverse library of anti-tubulin agents designed to mimic the simple stilbenoid structure.1,7 Open in a separate window Figure 1 Representative small-molecule inhibitors of tubulin assembly. The indole structure is prevalent as a core molecular component in a variety of inhibitors of tubulin assembly.8 As an early example, the vinca alkaloid natural products vinblastine and vincristine, originally isolated from the madagascar periwinkle plant (L.) G.Don (ApoCynaceae), both incorporate two key indole ring systems.9,10 A significant number of compounds that bind to the colchicine site and inhibit microtubule formation are also indole-based (see Figure 2 for representative molecules). To the best of our knowledge, the first examples of such colchicine site interactive, indole-based compounds were reported by von Angerer et al.11 (Figure 2, compound 6) and separately by Pinney et al.12,13 (Figure 2, Chlorhexidine compounds 7C8) in the middle-to-late 1990s. Our (Pinney and co-workers) 2-aryl-3-aroyl-indole analogues12C14 were originally inspired, in part, by the combretastatin collection of natural products (pioneered by George R. Pettit)1,15 and the non-steroidal, selective estrogen receptor modulator (SERM) work of Eli Lilly, Inc., featuring benzo[ 3 independent determinations. bFor additional data, see Ref.40 cSee Ref.41 dnd = not determined in this study. Although the reason is not known with certainty, several factors may contribute. For example, the stoichiometry that exists between the concentration of compound and the concentration of tubulin needed to achieve an IC50 value in the Chlorhexidine micromolar range in the pure protein (cell-free) assay may be drastically different from the requisite stoichiometry that exists when inhibiting tubulin in a cell-based assay leading to a measured GI50 value for cytotoxicity in the nanomolar range. It should be emphasized that the pure protein assay incorporates no cellular components other than tubulin. Importantly, tubulin disassembly (in cells) may release factors that are involved in intramolecular signal transduction leading to a major amplification. In addition, the amount of tubulin needed in the pure protein assay in order to accurately measure inhibition of microtubule formation (by monitoring absorbance at 350 nm spectrophotometrically) is large enough that the practical lower limit for this assay is around 0.5 to 1 1 micromolar (IC50 value) for even the most active inhibitors of tubulin polymerization. As a preliminary test of vascular disrupting activity in vivo, color Doppler ultrasound was applied to a PC-3 human tumor xenograft growing in a SCID mouse. Images were acquired over a period of 80 min starting immediately after administration of indole prodrug 33 (OXi8007). The tumor was clearly visible (Figure 3) with extensive bidirectional blood flow displayed in red and blue. Blood flow was also observed in vessels outside the tumor. Blood flow decreased progressively and by Mouse monoclonal to CD45RA.TB100 reacts with the 220 kDa isoform A of CD45. This is clustered as CD45RA, and is expressed on naive/resting T cells and on medullart thymocytes. In comparison, CD45RO is expressed on memory/activated T cells and cortical thymocytes. CD45RA and CD45RO are useful for discriminating between naive and memory T cells in the study of the immune system 80 min experienced essentially ceased in the core of the tumor. The extratumoral vessels remained undisrupted throughout. The differential activity in the tumor and animal tissue provides initial evidence of selective vascular disruption in vivo. Ultrasound offers emerged as a valuable imaging technique for VDAs42 and has recently been validated like a corollary imaging strategy to bioluminescence imaging (BLI)33 for the assessment of VDAs.43 Open in a separate window Number 3 Dynamics of vascular disruption caused by 33 (OXi8007) in human being prostate tumor PC-3 xenograft visualized by doppler ultrasound. Sequential transaxial images were acquired over a period of 80 min following injection of indole prodrug 33 (OXi8007) (350 mg/kg ip) inside a SCID mouse. (A) Immediately post injection, (B) 20 min, (C) 40 min, (D) 60 min, (E) 80 min. Each.1H NMR (CDCl3, 500 MHz) 8.11 (1H, br s, NH), 7.47 (1H, d, = 8.5 Hz, ArH), 7.16 (1H, dd, = 8.5 Hz, 2.0 Hz, ArH), 7.13 (1H, d, = 2.5 Hz, ArH), 6.90 (1H, d, = 8.5 Hz, ArH), 6.89 (1H, d, = 2.5 Hz, ArH), 6.79 (1H, dd, = 8.5 Hz, 2.5 Hz, ArH), 6.64 (1H, dd, = 2.0 Hz, 1.0 Hz, ArH), 3.86 (3H, s, OCH3), 3.84 (3H, s, OCH3), 1.04 (9H, s, C(CH3)3), 0.21 (6H, s, Si(CH3)2); 13C NMR (CDCl3, 125 MHz) 156.3, 150.5, 145.4, 137.4, 136.9, 125.8, 123.7, 120.9, 118.2, 117.8, 112.4, 109.9, 98.6, 94.5, 55.6, 55.4, 25.7, 18.5, ?4.6; HPLC: method B, found 384.1989 [M+H]+ (calcd for C22H30NO3Si, 384.1989). 2-(3-Hydroxy-4-methoxyphenyl)-6-methoxyindole (26a).46 To a solution of TBS-protected phenol 25 (0.10 g, 0.26 mmol) in THF (10 mL) at 0 C was added tetrabutylammonium fluoride (TBAF, 0.4 mL, 0.4 mmol, 1 M in THF) dropwise. these results provides evidence the indole-based phosphate prodrug 33 (OXi8007) functions like a vascular disrupting agent (VDA) that may Chlorhexidine demonstrate useful for the treatment of cancer. The vast majority of natural products and synthetic compounds that Chlorhexidine bind to the tubulin-microtubule protein system and subsequently interfere with the dynamic assembly/disassembly inherent to the ,-tubulin/microtubule system do this by many variations of this overall mechanism of action. The most thoroughly studied are relationships in the colchicine, vinca alkaloid, and taxoid sites.1,2 A significant quantity of antiproliferative, anticancer providers function through this fundamental mechanism of action. The combretastatin family of natural products consists of a variety of Kuntze (Combretaceae).1 Combretastatin A-4 (CA4) is among the most potent antimitotic providers from this family of compounds and binds to the colchicine site on tubulin (Number 1, compound 2).3C5 As with many natural products, the challenge of water solubility led to the development of a disodium phosphate prodrug 3, combretastatin A-4P (CA4P).6 The finding of CA4 offers led to a diverse library of anti-tubulin agents designed to mimic the simple stilbenoid structure.1,7 Open in a separate window Number 1 Representative small-molecule inhibitors of tubulin assembly. The indole structure is prevalent like a core molecular component in a variety of inhibitors of tubulin assembly.8 As an early example, the vinca alkaloid natural products vinblastine and vincristine, originally isolated from your madagascar periwinkle flower (L.) G.Don (ApoCynaceae), both incorporate two key indole ring systems.9,10 A significant quantity of compounds that bind to the colchicine site and inhibit microtubule formation will also be indole-based (observe Number 2 for representative molecules). To the best of our knowledge, the first examples of such colchicine site interactive, indole-based compounds were reported by von Angerer et al.11 (Number 2, compound 6) and separately by Pinney et al.12,13 (Number 2, compounds 7C8) in the middle-to-late 1990s. Our (Pinney and co-workers) 2-aryl-3-aroyl-indole analogues12C14 were originally inspired, in part, from the combretastatin collection of natural products (pioneered by George R. Pettit)1,15 and the non-steroidal, selective estrogen receptor modulator (SERM) work of Eli Lilly, Inc., featuring benzo[ 3 self-employed determinations. bFor additional data, observe Ref.40 cSee Ref.41 dnd = not determined with this study. Although the reason is not known with certainty, several factors may contribute. For example, the stoichiometry that is present between the concentration of compound and the concentration of tubulin needed to accomplish an IC50 value in the micromolar range in the pure protein (cell-free) assay may be drastically different from the requisite stoichiometry that is present when inhibiting tubulin inside a cell-based assay leading to a measured GI50 value for cytotoxicity in the nanomolar range. It should be emphasized the pure protein assay incorporates no cellular parts other than tubulin. Importantly, tubulin disassembly (in cells) may launch factors that are involved in intramolecular transmission transduction leading to a major amplification. In addition, the amount of tubulin needed in the genuine protein assay in order to accurately measure inhibition of microtubule formation (by monitoring absorbance at 350 nm spectrophotometrically) is definitely large enough the practical lower limit for this assay is around 0.5 to 1 1 micromolar (IC50 value) for even the most active inhibitors of tubulin polymerization. As a preliminary test of vascular disrupting activity in vivo, color Doppler ultrasound was applied to a Personal computer-3 human being tumor xenograft growing inside a SCID mouse. Images were Chlorhexidine acquired over a period of 80 min starting immediately after administration of indole prodrug 33 (OXi8007). The tumor was clearly visible (Number 3) with considerable bidirectional blood flow displayed in reddish and blue. Blood flow was also observed in vessels.