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A copper-mediated synthesis of diaryl sulfides utilizing Cu(I)-thiophene-2-carboxylate (CuTC) is described. We demonstrate the use of CuTC as a soluble, non-basic catalyst in the coupling of aryl iodides and aryl thiols in the synthesis of synthetically advanced diaryl sulfides. This method allows for the successful coupling of challenging substrates including ortho-substituted and heteroaryl iodides and thiols. Additionally, most of the aryl iodide substrates used here contain the privileged piperazine scaffold bound to a pyrimidine, pyridine, or phenyl ring and thus this method allows for the elaboration of complex piperazine scaffolds into molecules of biological interest. The method described here enables the incorporation of late-stage structural diversity into diaryl sulfides containing the piperazine ring, thus enhancing the number and nature of derivatives available for SAR investigation.

Heat shock protein 70 (Hsp70) is a family of proteins with key roles in regulating malignancy. Cancer cells rely on Hsp70 to inhibit apoptosis, regulate senescence and autophagy, and maintain the stability of numerous onco-proteins. Despite these important biological functions in cancer, robust chemical tools that enable the analysis of the Hsp70-regulated proteome in a tumor-by-tumor manner are yet unavailable. Here we take advantage of a recently reported Hsp70 ligand to design and develop an affinity purification chemical toolset for potential use in the investigation of the endogenous Hsp70-interacting proteome in cancer. We demonstrate that these tools lock Hsp70 in complex with onco-client proteins and effectively isolate Hsp70 complexes for identification through biochemical techniques. Using these tools we provide proof-of-concept analyses that glimpse into the complex roles played by Hsp70 in maintaining a multitude of cell-specific malignancy-driving proteins.

Heat shock protein 70 (Hsp70) is an important emerging cancer target whose inhibition may affect multiple cancer-associated signaling pathways and, moreover, result in significant cancer cell apoptosis. Despite considerable interest from both academia and pharmaceutical companies in the discovery and development of druglike Hsp70 inhibitors, little success has been reported so far. Here we describe structure-activity relationship studies in the first rationally designed Hsp70 inhibitor class that binds to a novel allosteric pocket located in the N-terminal domain of the protein. These 2,5'-thiodipyrimidine and 5-(phenylthio)pyrimidine acrylamides take advantage of an active cysteine embedded in the allosteric pocket to act as covalent protein modifiers upon binding. The study identifies derivatives 17a and 20a, which selectively bind to Hsp70 in cancer cells. Addition of high nanomolar to low micromolar concentrations of these inhibitors to cancer cells leads to a reduction in the steady-state levels of Hsp70-sheltered oncoproteins, an effect associated with inhibition of cancer cell growth and apoptosis. In summary, the described scaffolds represent a viable starting point for the development of druglike Hsp70 inhibitors as novel anticancer therapeutics.

SummaryHsp70s are important cancer chaperones that act upstream of Hsp90 and exhibit independent anti-apoptotic activities. To develop chemical tools for the study of human Hsp70, we developed a homology model that unveils a previously unknown allosteric site located in the nucleotide binding domain of Hsp70. Combining structure-based design and phenotypic testing, we discovered a previously unknown inhibitor of this site, YK5. In cancer cells, this compound is a potent and selective binder of the cytosolic but not the organellar human Hsp70s and has biological activity partly by interfering with the formation of active oncogenic Hsp70/Hsp90/client protein complexes. YK5 is a small molecule inhibitor rationally designed to interact with an allosteric pocket of Hsp70 and represents a previously unknown chemical tool to investigate cellular mechanisms associated with Hsp70.

The use of simple calix[4]arenes 1a,b for NO2/N2O4 sensing and conversion is demonstrated, both in solution and in the solid state. Upon reacting with these gases, compounds 1a,b encapsulate reactive NO+ cations within their cavities with the formation of deeply colored (λmax∼570 nm) charge-transfer complexes 2a,b . Further functionalization of the calix[4]arene platform is reported for attachment to solid supports. Polymer-supported calixarene material 3 was prepared, which reversibly traps NO2/N2O4 with the formation of nitrosonium storing polymer 4 . Material 4 was effectively used for nitrosation of amides.

An approach toward visual detection and chemical utilization of NO(2)/N(2)O(4) is proposed, which employs simple calix[4]arenes. Exposure of tetra-O-alkylated calix[4]arenes 1 and 2, possessing either a cone or a 1,3-alternate conformation, to NO(2)/N(2)O(4), both in chloroform solution and in the solid state, results in deeply colored calixarene-nitrosonium (NO(+)) complexes. In the presence of a Lewis acid, such as SnCl(4), stable calixarene-NO(+) complexes 7 and 8 were isolated in a quantitative yield and characterized by UV-vis, FTIR, high-resolution (1)H NMR spectroscopy and elemental analysis. NO(+) is found encapsulated within the calixarene cavity, and stable charge-transfer complexes result with K(ass)10(6) M(-1) (CDCl(3)). The NO(+) encapsulation was also demonstrated in titration experiments with calixarenes 1, 2, and 5 and commercially available NO(+)SbF(6)(-) salt in chloroform. The complexation process is reversible, and the complexes dissociate upon addition of water and alcohol, recovering the parent calixarenes. Attachment of functionalized calix[4]arenes to silica gel was demonstrated, which afforded a solid material 15 capable of visual detection and entrapment of NO(2)/N(2)O(4). Calixarene-NO(+) complexes can be utilized for the NO(+) transfer processes and nitrosation reactions. The NO(+) guest transfer between two calixarene containers 2 and 5 was achieved and studied by UV-vis and (1)H NMR spectroscopy. Chemical fixation of NO(2)/N(2)O(4) was demonstrated through their quantitative transformation into the calixarene-NO(+) complex and its use as a nitrosonium transfer agent in the synthesis of N-nitrosoamides. These results may lead toward novel nitrogen oxides storing materials.

Molecular chaperone heat shock protein 90 (Hsp90) is an important target in cancer and neurodegenerative diseases, and has rapidly become the focus of several drug discovery efforts. Among small molecule Hsp90 inhibitors with clinical applicability are derivatives of 8-arylmethyl-9H-purin-6-amine class. Here we report the use of microwave-assisted chemistry for the successful one-pot delivery of 8-arylmethyl-9H-purin-6-amines. We discuss the applicability as well as the limitations of this method towards the creation of a large chemical diversity in the 8-arylmethyl-9H-purin-6-amine series.

The 70 kDa heat shock proteins (Hsp70) are molecular chaperones that assist in folding of newly synthesized polypeptides, refolding or denaturation of misfolded proteins, and translocation of proteins across biological membranes. In addition, Hsp70 play regulatory roles in signal transduction, cell cycle, and apoptosis. Here, we present a novel assay platform based on fluorescence polarization that is suitable for investigating the yet elusive molecular mechanics of human Hsp70 allosteric regulation.

PU-H71 is a purine-scaffold Hsp90 inhibitor currently undergoing late stage preclinical evaluation for the treatment of cancer. In this investigation, we present a simple method for the synthesis of d6-labeled PU-H71 for use as an internal standard to accurately quantitate the drug in biological matrices based on an LC-MS-MS method. PU-H71-d6 was synthesized in five steps using readily available 1,3-dibromopropane-d6 and is an important compound for the advancement of our clinical program. Copyright © 2009 John Wiley & Sons, Ltd.

As molecularly targeted agents assume a more prominent role in anticancer therapy there is a growing need to determine in a noninvasive manner whether the target is being engaged and to what extent such drug-target binding results in desirable effects. We address this need in the context of Hsp90, a target of significant value and one in critical need for such assessment tools, by combining a novel chemical tool selective for tumor Hsp90 with PET imaging and mathematical modeling. The chemical tool is [124I]-PU-H71, the iodine-124 radiolabeled analog of the potent Hsp90 inhibitor PU-H71, which can be administered in tracer quantities for PET imaging. The resulting diagnostic, PU-PET, has been optimized and validated preclinically in mouse models of cancer and then translated to the clinic. The exquisite design of this assay is based on three essential concepts as it relates to the target (Hsp90) as well as to the PET tracer (124I-PU-H71). First, the target is “oncogenic” Hsp90 and has been shown by numerous biochemical and pharmacokinetic studies to have a strong affinity for inhibitors and a very low koff resulting in selective and prolonged retention in tumor. Secondly, the tracer incorporates a 124I in place of the naturally occurring 127I in the structure of PU-H71 and therefore there is no change in the chemical structure. This feature in a PET tracer intended as a companion diagnostic is unprecedented and ensures that the PK properties are identical to the therapeutic agent (PU-H71). Finally, the radionuclide 124I has a four-day half-life and thus is well-suited to monitor the extended tumor retention profile observed for Hsp90 inhibitors. We here demonstrate that this PET assay informs on Hsp90 targeting in individual tumors in real time and provides accurate tumor drug concentrations for at least four chemically distinct Hsp90 drugs. In contrast, we find that plasma pharmacokinetics is not predictive of intratumor parameters and therefore provides limited value in estimating target engagement. Using PU-PET we demonstrate that at least one Hsp90 inhibitor exhibits tumor targeting and retention in humans, delivering and retaining therapeutic, micromolar, concentrations at safe doses. PU-PET is currently being evaluated in Phase 0/1 (NCT01269593) clinical trials as a noninvasive companion diagnostic to determine intratumoral concentration as well as to identify those patients who would best benefit from Hsp90 inhibitor therapy. This diagnostic assay is intended to be incorporated into future Phase 2 clinical trials in order to preselect those patients who would most likely benefit from Hsp90 inhibitor treatment. Citation Format: Tony Taldone, Nagavarakishore Pillarsetty, Mark PS Dunphy, John F. Gerecitano, Eloisi Caldas-Lopes, Brad Beattie, Radu I. Peter, Yanlong Kang, Anna Rodina, Pengrong Yan, Erica M. DaGama Gomes, Alexander Bolaender, Christina Pressl, Blesida Punzalan, Anson Ku, Thomas Ku, Smit Shah, Mohammad Uddin, Mei H. Chen, Elmer Santos, Jacek Koziorowski, Adriana Corben, Shanu Modi, Komal Jhaveri, Oscar Lin, Efsevia Vakiani, Yelena Janjigian, Pat Zanzonico, Clifford Hudis, Steven M. Larson, Jason S. Lewis, Gabriela Chiosis. Development of a noninvasive assay to determine drug concentration in tumor during hsp90 inhibitor therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5444. doi:10.1158/1538-7445.AM2015-5444

The synthesis of a red-shifted cy3B-GM ligand and its evaluation as a fluorescence polarization probe for Hsp90 is presented.

Abstract 2493 Prognosis for acute myelogenous leukemia (AML) with traditional chemotherapeutic agents remains poor, most patients relapse and die of their disease. Studies have shown that AML can be originated and maintained by leukemia stem cells (LSCs) and that a high percentage of phenotypically-defined LSCs correlate to especially poor prognosis. Thus, novel therapies that can eliminate LSCs are needed. Upregulation of HSP70 has been described in human cancer cells and is often associated with chemoresistance and poor prognosis. Therefore, HSP70 represents a novel target for cancer therapy. Due to this, we tested a novel inhibitor of tumor-HSP70, YK5, and found that it can induce potent cell death in AML cells including progenitor and stem cell populations with minimal effects in normal hematopoietic cells. Fifteen primary AML patient samples were treated with YK5 in varying concentrations for 48 hours to determine the LD50 of YK5. The viability of these samples was determined by multiparameter flow cytometry using cell surface markers to distinguish, lymphocytes, blasts, progenitor and stem cell populations in conjunction with Annexin V and 7AAD. Mean LD50 for all samples tested was 4.5μM (CI 95% 2.239–6.853). Three of these samples had very low LD50s below 2.5μM and only one sample demonstrated relative resistance with a LD50 that was greater than 15μM. Notably, progenitor and stem cell populations were also targeted while normal remaining lymphocytes were spared. Furthermore, we found that LSCs in a subset of patient samples treated with YK5 exhibited higher sensitivity to the compound when compared to blast cells. Importantly, treatment of primary AML samples with 5μM YK5 resulted in a 63% decrease in colony forming ability when compared to untreated control (N=5), suggesting that YK5 can impair malignant progenitors. In contrast, normal CD34+ cord blood cells after treatment with 5μM YK5 for 48hrs exhibited a viability of 57% (N=3). Xenotransplant assays are currently ongoing to determine the ability of YK5 to inhibit LSC function. Treatment with YK5 resulted in a decrease in phosphorylation of STAT5, determined by flow cytometry. Taken together, HSP70 inhibition is a promising potential treatment for AML and has the potential to eradicate leukemic stem and progenitor cells while sparing normal hematopoietic cells. Thus, targeting HSP70 in AML appears to hold great promise. *JFR and KKS contributed equally to this project Disclosures: Roboz: EpiCept: Consultancy; ChemGenex: Consultancy; Celgene: Consultancy; Boehringer Ingelheim: Consultancy.