Synthesis of a Next-Generation Taxoid by Rapid Methylation Amenable for 11C-Labeling.

Next-generation taxoids, such as SB-T-1214, are highly potent cytotoxic agents that exhibit remarkable efficacy against drug-resistant tumors in vivo, including those that overexpress the P-glycoprotein (Pgp) efflux pump. As SB-T-1214 is not a substrate for Pgp-mediated efflux, it may exhibit a markedly different biodistribution and tumor-accumulation profile than paclitaxel or docetaxel, which are both Pgp substrates. To investigate the biodistribution and tumor-accumulation levels of SB-T-1214 using positron emission tomography (PET), a new synthetic route has been developed to allow the incorporation of 11C, a commonly employed positron-emitting radionucleide, via methyl iodide at the last step of chemical synthesis. This synthetic route features a highly stereoselective chiral ester enolate-imine cyclocondensation, regioselective hydrostannation of the resulting ß-lactam, and the Stille coupling of the novel vinylstannyl taxoid intermediate with methyl iodide. Conditions have been established to allow the rapid methylation and HPLC purification of the target compound in a time frame amenable to 11C-labeling for applications to PET studies.

Design and synthesis of tumor-targeting theranostic drug conjugates for SPECT and PET imaging studies.

Theranostics will play a significant role in the next-generation chemotherapy. Two novel tumor-targeting theranostic drug conjugates, bearing imaging arms, were designed and synthesized. These theranostic conjugates consist of biotin as the tumor-targeting moiety, a second generation taxoid, SB-T-1214, as a potent anticancer drug, and two different imaging arms for capturing 99mTc for SPECT (single photon emission computed tomography) and 64Cu for PET (positron emission tomography). To explore the best reaction conditions for capturing radionuclides and work out the chemistry directly applicable to "hot" nuclides, cold chemistry was investigated to capture 185Re(I) and 63Cu(II) species as surrogates for 99mTc and 64Cu, respectively.

Design, synthesis and biological evaluation of a highly-potent and cancer cell selective folate-taxoid conjugate.

The folate receptor (FR) has been widely recognized as an excellent target for the tumor-selective delivery of cytotoxic agents, and four folate-drug conjugates have entered clinical evaluations for the treatment of solid tumors to date. However, most of these conjugates required structural modification of the cytotoxic warheads in order to achieve efficient drug release from the linkers. We designed and constructed a novel folate conjugate of a highly-potent next-generation taxoid, SB-T-1214, by exploiting bioorthogonal Cu-free 'click' chemistry. The synthesis was highly convergent and required no HPLC purification to obtain the final folate-taxoid conjugate 1. Conjugate 1 demonstrated highly FR-specific potency (IC50 2.1-3.5 nM) against a panel of cancer cell lines, with a >1000-fold decrease in cytotoxicity against normal human cells (IC50>5000 nM). The remarkable potency and selectivity of conjugate 1 can be attributed to highly FR-specific receptor-mediated endocytosis as well as efficient release of the unmodified cytotoxic warhead using a mechanism-based self-immolative linker.

Design, synthesis, and biological evaluation of theranostic vitamin-linker-taxoid conjugates.

Novel tumor-targeting theranostic conjugates 1 and 2, bearing either a fluorine-labeled prosthetic as a potential (18)F-PET radiotracer (1) or a fluorescence probe (2) for internalization studies in vitro, were designed and synthesized. We confirmed efficient internalization of 2 in biotin-receptor positive (BR+) cancer cells via receptor-mediated endocytosis (RME) based on flow cytometry and confocal fluorescence microscopy (CFM) analyses, which exhibited very high specificity to BR+ cancer cells. The potency and cancer-cell selectivity of 1 were evaluated against MX-1, L1210FR and ID8 cancer cells (BR+) as well as L1210 cells and WI38 normal human lung fibroblast cells (biotin-receptor negative: BR-). In particular, we designed and performed an assay in the presence of glutathione ethyl ester (GSH-OEt) wherein only 1 molecules internalized into cells via RME in the first 24 h period exert cytotoxic effect. The observed selectivity of 1 was remarkable, with 2 orders of magnitude difference in IC50 values between BR+ cancer cells and WI38 cells, demonstrating a salient feature of this tumor-targeted drug delivery system.

Design, Synthesis and Application of Fluorine-Labeled Taxoids as 19F NMR Probes for the Metabolic Stability Assessment of Tumor-Targeted Drug Delivery Systems.

Novel tumor-targeting drug conjugates, BLT-F2 (1) and BLT-S-F6 (2), bearing a fluorotaxoid as the warhead, a mechanism-based self-immolative disulfide linker, and biotin as the tumor-targeting module, were designed and synthesized as 19F NMR probes. Fluorine atoms and CF3 groups were strategically incorporated into the conjugates to investigate the mechanism of linker cleavage and factors that influence their plasma and metabolic stability by real-time monitoring with 19F NMR. Time-resolved 19F NMR study on probe 1 disclosed a stepwise mechanism for release of a fluorotaxoid, which might not have been detected by other analytical methods. Probe 2 was designed to bear two CF3 groups in the taxoid moiety as "3-FAB" reporters for enhanced sensitivity and a polyethylene glycol oligomer insert to improve solubility. The clean analysis of the linker stability and reactivity of drug conjugates in blood plasma or cell culture media by HPLC and 1H NMR is troublesome, due to the overlap of key signals/peaks with background arising from highly complex ingredients in biological systems. Accordingly, the use of 19F NMR would provide a practical solution to this problem. In fact, our "3-FAB" probe 2 was proven to be highly useful to investigate the stability and reactivity of the self-immolative disulfide linker system in human blood plasma by 19F NMR. It has also been revealed that the use of polysorbate 80 as excipient for the formulation of probe 2 dramatically increases the stability of the disulfide linker system. This finding further indicates that the tumor-targeting drug conjugates with polysorbate 80/EtOH/saline formulation for in vivo studies would have high stability in blood plasma, while the drug release in cancer cells proceeds smoothly.

Design, synthesis, and biological evaluations of tumor-targeting dual-warhead conjugates for a taxoid-camptothecin combination chemotherapy.

Novel tumor-targeting dual-warhead conjugates, 2 (DW-1) and 3 (DW-2), which consist of a next-generation taxoid, 1 (SB-T-1214), and camptothecin as two warheads, self-immolative disulfide linkers for drug release, biotin as the tumor-targeting moiety, and 1,3,5-triazine as the tripod splitter module, were designed and synthesized. The potency of 2 was evaluated against MX-1, MCF-7, ID8, L1210FR (BR+, biotin receptor overexpressed) and WI38 (BR-, normal) cell lines in the absence and presence of glutathione (GSH), which is an endogenous thiol that triggers drug release inside the cancer cells. With the GSH and resuspension protocol, 2 exhibited IC50 values of 3.22-9.80 nM against all BR+ cancer cell lines, and 705 nM against WI38. Thus, there was a two orders of magnitude higher selectivity to cancer cells. Also, a clear cooperative effect was observed for the taxoid-camptothecin combination when two drugs were delivered to the cancer cells specifically in the form of a dual-warhead conjugate.

Drug discovery targeting cell division proteins, microtubules and FtsZ.

Eukaryotic cell division or cytokinesis has been a major target for anticancer drug discovery. After the huge success of paclitaxel and docetaxel, microtubule-stabilizing agents (MSAs) appear to have gained a premier status in the discovery of next-generation anticancer agents. However, the drug resistance caused by MDR, point mutations, and overexpression of tubulin subtypes, etc., is a serious issue associated with these agents. Accordingly, the discovery and development of new-generation MSAs that can obviate various drug resistances has a significant meaning. In sharp contrast, prokaryotic cell division has been largely unexploited for the discovery and development of antibacterial drugs. However, recent studies on the mechanism of bacterial cytokinesis revealed that the most abundant and highly conserved cell division protein, FtsZ, would be an excellent new target for the drug discovery of next-generation antibacterial agents that can circumvent drug-resistances to the commonly used drugs for tuberculosis, MRSA and other infections. This review describes an account of our research on these two fronts in drug discovery, targeting eukaryotic as well as prokaryotic cell division.

Fluorine-Containing Taxoid Anticancer Agents and Their Tumor-Targeted Drug Delivery.

A long-standing problem of conventional chemotherapy is the lack of tumor-specific treatments. Traditional chemotherapy relies on the premise that rapidly proliferating cancer cells are more likely to be killed by a cytotoxic agent. In reality, however, cytotoxic agents have very little or no specificity, which leads to systemic toxicity, causing undesirable severe side effects. Consequently, various "molecularly targeted cancer therapies" have been developed for use in specific cancers, including tumor-targeting drug delivery systems. In general, such a drug delivery system consists of a tumor recognition moiety and a cytotoxic "warhead" connected through a "smart" linker to form a conjugate. When a multi-functionalized nanomaterial is used as the vehicle, a "Trojan Horse" approach can be used for mass delivery of cytotoxic "warheads" to maximize the efficacy. Exploitation of the special properties of fluorine has proven successful in the development of new and effective biochemical tools as well as therapeutic agents. Fluorinated congeners can also serve as excellent probes for the investigation of biochemical mechanisms. 19F-NMR can provide unique and powerful tools for mechanistic investigations in chemical biology. This account presents our recent progress, in perspective, on the molecular approaches to the design and development of novel tumor-targeted drug delivery systems for new generation chemotherapy by exploiting the unique nature of fluorine.