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Progress in the Discovery and Development of Heat Shock Protein 90 (Hsp90) Inhibitors Miniperspective Rohit Bhat, Sreedhar R. Tummalapalli, and David P. Rotella* Department of Chemistry and Biochemistry, Sokol Institute for Pharmaceutical Life Sciences, Montclair State University, Montclair, New Jersey 07043, United States ABSTRACT: The discovery and clinical development of heat shock protein 90 (Hsp90) inhibitors continue to progress. A number of Hsp90 inhibitors are in clinical trials, and preclinical discoveries of new chemotypes that bind to distinct regions in the protein as well as isoform selective compounds are active areas of research. This review will highlight progress in the field since 2010.

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INTRODUCTION This review will summarize selected new information on the biology, medicinal chemistry, and clinical development of heat shock protein 90 (Hsp90) inhibitors since 2010. Previous reviews nicely summarized research in the field prior to this date.1−4 As the most abundant intracellular protein in mammalian cells, Hsp90 is essential for a wide range of protein assembly, trafficking, folding, and degradation processes.5 It has long been recognized as a potential target for cancer and more recently for neurodegenerative disease.6,7 The N- and C-termini as well as an internal region in Hsp90 contain distinct binding sites for ATP, and each is associated with a specific functional activity. ATP binding at the N-terminus provides energy to modulate protein folding and trafficking, whereas nucleotide binding at the C-terminus is associated with allosteric regulation of ATP binding to the N-terminal site. The C-terminal fragment controls Hsp90 dimerization, a key aspect of the functional properties of the protein. The internal domain of Hsp90 regulates client protein interactions and contains a site where the γ-phosphate of ATP binds and Hsp90 inhibitors interact with each of these domains. The majority of inhibitors bind in the N-terminal region; a smaller subset interacts with the Cterminus, and the depsipeptide sansalvamide A (1, Figure 1) is reported to bind to the internal region to allosterically modulate interaction with client proteins.8 Increasing attention is being focused on isoform-specific inhibition of Hsp90 to understand the pharmacology and cellular pathways associated with pan-inhibition that affects the function of over 200 client proteins. Hsp90α (inducible) and -β (constitutively active) are found in the cytosol. Trap1 is associated with mitochondria, and Grp94 is found in the endoplasmic reticulum; each of these is associated with a subset of client proteins.9 Strategies to identify isoform selective inhibitors are now beginning to emerge that attempt to use structural and conformation variations outside the nucleotide © XXXX American Chemical Society

binding sites rather than the homologous nucleotide binding domains. This topic will be addressed in the medicinal chemistry section. The remainder of the Introduction will summarize selected new results associated with biology, biochemistry, and functional activity associated with Hsp90. Molecular details for the mechanism by which the tumor suppressor p53 gene regulates the Wnt signaling pathway are not well established. Activation of the Wnt pathway plays an important role in colon cancer; consequently there is a strong impetus to dissect these details not only to understand cell transformation but also to illuminate potential drug targets. By use of wild type and p53 knockout mice, it was shown that levels of Hsp90 client proteins such as Akt and GSK3β as well as Hsp90 ATPase activity and Aha-1 were up-regulated in the colon of p53 KO animals.10 Increased Aha-1 expression led to increased Hsp90 ATPase activity and subsequently resulted in enhanced Wnt target gene expression associated with cellular transformation. Inhibition of Hsp90 activity using both 17allylamino geldanamycin (2)3,4 and the alkylamino pyrimdine 31 (Figure 1) decreased expression of key proteins in colon cancer cell lines. Fierro-Monti and co-workers investigated the quantitation and kinetics of Hsp90 inhibition on client proteins using T-cells as a model system.11 By use of SILAC technology (stable isotope labeling by amino acids in cell culture), Jurkat Tlymphocytes were treated with the isoform nonselective Hsp90 inhibitor geldanamycin (4) to study protein synthesis and degradation. A net reduction in protein synthesis was observed rather than an increase in protein degradation. Interestingly, selected protein families were affected more than others. For example, kinase enzymes, telomere maintenance, and nucleoporin proteins were measurably depressed while proteins Received: May 30, 2014

A

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Figure 1. Hsp90 inhibitors.

residues 210 and 380. This sequence includes residues known to be involved in microtubule binding. By use of a unique isotope labeling method for methyl groups in isoleucine, analysis of NMR spectra in the presence and absence of tau suggested that tau binds to N-terminal and middle regions of Hsp90, based on shifts in isoleucine signals at Ile20, -74, -369, and -440. Using small interfering RNA (siRNA) technology, Alani and co-workers demonstrated that silencing Hsp90 activity in PC12 cells protected against 6-hydroxydopamine (6OHDA) induced toxicity in culture.16 6OHDA is widely used in experimental models of Parkinson’s disease to induce oxidative stress associated with formation of reactive oxygen species. In response to stress, the transcription factor HSF-1 dissociates from Hsp90 and induces formation of cytoprotective Hsp70 that protects against apoptosis and reactive oxygen species. In culture, elevated levels of Hsp70 were measured following silencing Hsp90 expression with siRNA. Retinitis pigmentosa (RP) is most commonly associated with mutations in rhodopsin leading to a misfolded protein that does not function. Previous work with 2 with the P23H mutant rhodopsin in culture showed that the Hsp90 inhibitor decreased aggregation of the misfolded protein and resulted in increased cell viability.17 However, 2 does not effectively cross the blood−retina barrier, making it difficult to explore the potential of Hsp90 inhibitors for the treatment of RP in animal models. Aguilá and co-workers showed that NVP-99018 (5) was effective in a P23H transgenic rat model where the Hsp90 inhibitor reduced rhodopsin aggregation at a dose of 20 mg/kg orally.19 This effect was associated with induction of heat shock proteins such as Hsp70. Using a different hyperphosphorylated rhodopsin mutant, R135L, this group demonstrated in culture that 2 reduced accumulation of the protein, an effect that was dependent on decreased expression of the Hsp90-dependent rhodopsin kinase GRK1. These data suggest that Hsp90 inhibition may play a beneficial role in the treatment of RP. However, for this approach to be useful, it will be essential to establish formulations and dosage compatible with ophthalmic use that limit systemic exposure to avoid potential adverse events.

associated with the endoplasmic reticulum lumen and unfolded proteins were increased. Hsp90 inhibitors continue to be investigated for use in neurodegenerative disorders such as Alzheimer’s, Huntington’s, and Parkinson’s disease. The molecular basis of this approach to these complex disorders remains a primary objective. Inhibition of Hsp90 function is known to induce expression of protective chaperones Hsp70 and Hsp40.12 There are reports that prototypical Hsp90 inhibitors related to 4 such as 2 and related compounds demonstrate neuroprotective effects in a range of cell culture models; these molecules are cytotoxic in culture and in vivo.13 NXD30001 (structure not defined; reported to be a radicicol oxime derivative14) demonstrated neuroprotection comparable to geldanamycin with less cytotoxicity in an ALS1 cell culture model.14 Like 4, NXD30001 up-regulated expression of Hsp70 and Hsp40 in motor neurons from dissociated murine spinal cord tissue. This was associated with reduced cell death and mitochondrial fragmentation. When administered to mice by intraperitoneal injection, NXD30001 led to increased Hsp70 expression in skeletal and cardiac muscle but not in the spinal cord or brain in spite of the presence of the compound in the CNS of treated animals following single and multiple doses of the Hsp90 inhibitor. The authors suggest that these results indicate a complex relationship between Hsp90 inhibition and upregulation of protective heat shock proteins. This is an area where isoform-selective inhibitors may provide advantages to understand the proteins and pathways most relevant to cell survival in contrast to cell death. Using NMR and small-angle X-ray scattering, Karagöz and co-workers obtained a structure for the complex formed by Hsp90 and Tau protein.15 Tau and hyperphosporylated tau are often invoked in the neurotoxicity associated with both Parkinson’s and Alzheimer’s disease. In its native (unphosphorylated) form tau exists in an unfolded (random) state and is known to be an Hsp90 client. Hsp90 has moderate affinity for tau (KD ≈ 5 μM), regardless of the presence or absence of ATP. Analysis of the HSQC NMR spectrum of 15N labeled tau in the presence and absence of unlabeled Hsp90 enabled identification of a region in tau where Hsp90 binds between B

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Figure 2. Hsp90 inhibitors in clinical development.

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Hsp90 INHIBITORS IN CLINICAL DEVELOPMENT Hsp90 inhibitors have been in clinical development since 1999,1 and to date none have progressed to NDA status. Efficacy was observed with 2 and other analogs of 4 in phase I and/or II trials in melanoma, non-small-cell lung cancer, myeloma, acute myeloid leukemia, and refractory prostate cancer and in HER2 positive metastatic breast cancer in combination with trastuzumab.20 Clinical advancement of these derivatives was limited by efficacy and/or adverse events in more advanced trials, as well as pharmaceutical property and commercialization issues.20 In trials reported in peer reviewed literature since 2010, 2 was studied as a single agent in a phase II relapsed lymphoma trial that included 22 patients.21 Seven of 18 evaluable patients (39%) showed reduction in tumor volume. Hsp90 expression in lymphoid tissue did not change following treatment as measured by phosphorylated AKT or cyclin D1 levels. The most significant and severe adverse events included pain, dyspnea, and thrombocytopenia. In another phase II trial, 2 was evaluated as a single agent for treatment of metastatic melanoma.22 The primary end point in this trial was to determine the rate of disease stabilization at 24 weeks after initiation of therapy. If progression free survival rate was equal to or greater than 25%, the result would justify phase III evaluation. A total of 11 patients received the drug (450 mg/m2 weekly) and were evaluated. In this group the median survival was 232 days and 3 of the 11 achieved stable disease. The trial

was closed prematurely because it became impractical to balance patient accrual with drug related toxicity. A phase II study of 2 at doses of 50, 175, and 340 mg/m2 in combination with bortezomib in 22 patients with multiple myeloma reported responses (ranging from minimal to complete) 12 patients.23 Gastrointestinal, musculoskeletal, and connective tissue disorders were the most common adverse events resulting in study discontinuation. AUY922 (6, Figure 2) was discovered using a structure-based approach by scientists from Vernalis and the Cancer Research Center for Cancer Therapeutics and was licensed to Novartis.24 The molecule is currently in phase I and II clinical trials for patients with non-small-cell lung cancer (NSCLC), advanced solid tumors, and lymphoma. Additional phase II studies are underway in metastatic pancreatic cancer patients resistant to first line chemotherapy and in phase I for advanced HERpositive breast cancer. The compound was dosed at 70 mg/m2 once weekly up to 18 weeks in a phase II trial of 121 patients with either anaplastic lymphoma kinase (ALK) or EGFR mutated advanced NSCLC, some of whom received previous treatment with crizotinib.25 A clinical response was observed in 20−29% of patients with one of these two risk factors. AT13387 (7) was discovered in a fragment-based approach by Astex Pharmaceuticals and has an impressive profile against a panel of gastrointestinal stromal tumor cell lines (GIST) at low nanomolar concentrations.26 7 is also being tested for prostate cancer, NSCLC alone and combination with crizotinib C

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(13)36 is a tropane based Hsp90 inhibitor discovered by Exelixis in phase I alone or in combination with vemurafenib for the patients with unresectable BRAF mutated stage III/IV melanoma.

in a phase I/II trial. In a separate phase I trial, 7 is being studied in combination with dabrafenib and trametinib in patients with advanced melanoma. The combination of 7 and abiraterone offers a two-pronged approach at blocking androgen receptor mediated prostate cancer growth. In combination with imatinib, 7 is being examined for use in gastrointestinal stromal carcinomas. STA9090 (8, ganetespib), a triazole based analogue, is effective in modulating the cellular functions of Hsp90.27 8 is currently being studied in phase I and phase II for various malignancies including castration-resistant prostate cancer (CRPC) in patients who received prior docetaxel based therapy, relapsed or refractory small-cell lung cancer, unresectable stage III or stage IV melanoma, and metastatic ocular melanoma. In a phase II study in 98 ALK positive NSCLC patients who received 200 mg/m2 once weekly for 3 weeks with 1 week off, 4 of 8 patients with ALK gene rearrangements exhibited a partial response and 3 showed disease stabilization.28 The most common adverse events were diarrhea, fatigue, nausea, and anorexia. This suggests that more detailed investigation of the compound in ALK-positive patients is warranted. Pillai and Ramalingam summarized a number of abstract presentations reporting the activity of the drug candidate in NSCLC, noting that 8 is currently enrolling in a phase III study in combination with docetaxel in patients with lung adenocarcinomas.29 The nonansamycin derivative KW-2478 (9) is currently in phase I/II development by Kyowa Hakko for treatment of relapsed and/or refractory multiple myeloma in combination with bortezomib, a proteasome inhibitor.30 The purine based Hsp90 inhibitor BIIB028 (10) is an orally active prodrug initially developed by Conforma Therapeutics and then licensed to Biogen Idec that recently completed phase I clinical trials for the treatment of advanced solid tumors. The drug candidate was well-tolerated in 41 patients when administered twice a week in 21-day cycles with a maximum tolerated dose of 144 mg/m2.31 Another orally bioavailable Hsp90 inhibitor, Debio 0932 (11)32 entered clinical trials in 2010 for treatment of advanced solid tumors and lymphoma. On the basis of the promising results observed in phase I, the compound advanced to phase II in 2012. SNX-5422 (12) is in clinical studies for different cancer types. This aminobenzamide drug candidate is being developed by Esanex.33 Trials are ongoing for patients with lung adenocarcinoma, neuroendocrine tumors, HER2 positive cancers, and solid tumors. Results of a phase I trial in 25 patients with refractory hematologic cancers revealed that with an every other day schedule at doses ranging from 5.3 to 74 mg/m2, partial response was noted in a patient with transformed myeloma and disease stabilization occurred in a multiple myeloma patient.34 The most common toxicities included prolonged QTc interval, diarrhea, thrombocytopenia, pruritis, fatigue, and nausea. Severe thrombocytopenia occurred in five patients. While these results were considered encouraging, this trial was terminated prematurely because of ocular toxicity in patients enrolled in a separate study that used a different dosing regimen. Efficacy studies are under way for 3, a purine based Hsp90 inhibitor (Memorial Sloan Kettering) for solid tumors and lymphoma that have not responded to standard treatment.35 DS-2248 (structure not yet released) is an orally bioavailable Hsp90 inhibitor being developed by Daiichi, undergoing clinical trials for solid tumors and NSCLC treatment. XL888

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MEDICINAL CHEMISTRY UPDATE N-Terminal Inhibitors of Hsp90. The benzoquinone natural product 4 was among one of the first chemotypes to be

Figure 3. Templates for Hsp90 inhibitors.

Figure 4. Radicicol analogs.

Figure 5. Heterocyclic radicicol analogs.

identified as a potent N-terminal binding inhibitor of Hsp90.37 Chemical and metabolic instability stimulated the search for derivatives with improved pharmaceutical properties; however, all analogs in this series have been terminated in clinical trials for a variety of reasons.38 Recently Kitson and co-workers demonstrated that C-19 substitution with bulky groups not only reduces cellular toxicity but also induces a conformational change from a trans- to cis-amide conformation required for D

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Table 1. Isoform Selectivity of Hsp90 Inhibitors IC50 (nM) compd

Hsp90α

Hsp90β

Grp94

Trap-1

2 3 4 6 11 34

46 43 28 20 33 29

45 42 22 16 38 25

31 30 10 12 190 578

1496 205 661 38 1586 726

Figure 6. Purine-based Hsp90 inhibitors, part 1.

Figure 7. Purine-based Hsp90 inhibitors, part 2. Figure 11. Isoform-selective Hsp90 inhibitors.

Figure 8. Dihydroindolinone Hsp90 inhibitors. Figure 12. C-terminal Hsp90 inhibitors: epigallocatechin gallate and derivative.

Another natural product, radicicol (15) serves as a template for Hsp90 inhibitor discovery.40 Recently Moody and coworkers have reported a series of macrolactam ring substituted analogs of 15 with metabolic stability superior to that of radicicol and related lactone analogs.41 The N-benzylamide derivative (16, Figure 4) was metabolized to a lesser extent (47%) by human liver microsomes than radicicol (83%) and lactone 17 (90%). In addition 16 demonstrated 10-fold higher antiproliferative activity (IC50 = 600 nM) than the lactone analog (IC50 = 7600 nM) against human colon cancer cell lines in spite of a lower biochemical activity (IC50 = 200 nM) than the lactone (IC50 = 40 nM). Utilizing a fragment based drug discovery and lead optimization approach, Brasca et al. identified an isoxazole 3carboxamide (18) exhibiting high Hsp90 binding affinity (IC50 = 48 nM) and efficacy in human tumor model (A2780 IC50 =

Figure 9. Triazine-based Hsp90 inhibitors.

Hsp90 binding.39 When tested on human epithelial (ARPE-19) and endothelial cell (HUVEC) lines for cellular toxicity, the C19 phenyl substituted ansamycin (14, IC50 < 5 μM, Figure 3) was found to be less toxic than 17-AAG. Cellular evaluation showed that 14 causes depletion of Hsp90 client proteins in human cancer cell lines as well as up-regulation of Hsp70 and Hsp27 in human neuronal cell lines via Hsp90 inhibition.

Figure 10. Hsp90 isoform probe molecules. E

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Figure 13. Novologues.

Figure 14. Sansalvamide A derivatives.

80 nM).42 A secondary carboxamide was found to be essential for activity, as deletion of this group or a tertiary amide resulted in substantial loss of biochemical and cellular activity. The Xray crystal structure analysis of 18 revealed that the carboxamide moiety expands into the solvent exposed region and forms H-bonding interactions with amino acids Gly97 and Lys58. Subsequent optimization by adding bulkier hydrophilic residues to the amide resulted in identification of compound 19 with high Hsp90 binding affinity (IC50 = 10 nM) and improved antiproliferative activity (A2780 IC50 = 69 nM). Taddei and co-workers reported a triazole carboxamide derivative (20, Figure 5) that exhibited high Hsp90 binding affinity (IC50 < 5 nM) and cytotoxicity toward a non-small-cell lung carcinoma (NCI-H460, IC50 = 4 nM) cell line. A 1,5 arrangement of the phenyl groups on the triazole and a 4ethylcarboxamide were found to be optimum for activity.43 The O-acetylated derivative of this compound showed antiproliferative activity similar to the parent compound (NCI-H460, IC50 = 6.5 nM) albeit with much reduced Hsp90 binding affinity (>1000 nM), confirming the crucial role of both hydroxyl groups in Hsp90 binding.

Baruchello and co-workers investigated a library of 3,4isoxazole diamides for Hsp90 binding and cytotoxicity against a non-small-cell lung carcinoma cell line (NCI-H460).44 The structure−activity study concluded that an H-bond donor at the C-4 position on the isoxazole was essential for activity. Decreased Hsp90 binding affinity was observed when the amide was replaced with substituted amines. Compound 21 exhibited potent antitumor activity in vivo (48% tumor inhibition) at a dose of 60 mg/kg ip against human epidermoid carcinoma (A431) tumor xenograft model in spite of only moderate in vitro activity (200 nM) against a non-small-cell lung cancer cell line. The same group also reported a small series of tetrahydropyridoisoxazole compounds carrying a resorcinol moiety. The most active compound 22 showed strong affinity to Hsp90α (IC50 = 29 nM) but only moderate antiproliferative activity (IC50 = 450 nM) against NCI-H460 cell lines.45 Purine Inhibitors and Analogs. As noted earlier, Hsp90 has three distinct ATP binding sites.5 This observation provided the structural foundation to explore purine-based candidates as Hsp90 inhibitors. Compounds in this group F

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Trap-1. Selective modulation of these differentially expressed, regulated, and located proteins may result in distinct biological effects compared to pan-Hsp90 modulators.52 Taldone and coworkers developed a fluorescence polarization assay using a new purine based probe (33, Figure 10) to investigate the Hsp90 selectivity and affinity (Table 1) of different structural classes.53 The binding affinity of these inhibitors for cytosolic Hsp90 (α and β) was comparable. However, 17-AAG and 11 exhibited 40-fold lower affinity for Trap-1, whereas the trisubstituted benzamide 34 (an analog of 12) had a 20-fold lower preference for Grp94. Computational analysis of the cocrystal structure/docking of these inhibitors overlaid on the N-terminal binding region/ homology model of individual isoforms identified three binding regions: the ATP-binding region (pocket A), lipophilic region (pocket B), and an exit pocket C. It was proposed that the observed differences in the binding affinity are a result of differential interaction of these inhibitors to binding regions B and C. For example, the likely cause of diminished binding of 34 to Grp94 was attributed to polar group (−CF3) with suboptimal interactions with Leu163 and Phe195 in pocket B. Similarly, the authors hypothesized that significant drop in the affinity of 11 for Trap-1 could be a result of Lys126 and Phe201 in pocket C interfering in the interactions of the tert-butyl group of 11 with the solvent exposed region of Trap-1. In SkBr3 cell membranes Grp94 associates with HER2 thereby stabilizing the protein. The alkylpyrimidine derivative 35, a Grp94 preferring inhibitor (Grp94 IC50 = 220 nM; Hsp90α/Grp94 = 140), destabilized membrane bound HER2 in SKBr3 cells; however, it failed to induce Hsp70, an apparent demonstration of a specific biological effect in cells.54 Using structure based approach, Duerfeldt et al. have designed N-terminal inhibitors exhibiting preferential binding to Grp94 isoform.55 In the cellular assay, compound 36 (Figure 11) inhibited (IC50 = 32 nM) the trafficking of Toll-like receptors (TLRs) to the cell membrane of HEK293 cells expressing Grp94. However, when tested for cytotoxicity in MCF-7 and SkBr3 cell lines, 36 did not induce the degradation of Hsp90α/β dependent client proteins. These two specific cellular responses have been attributed to the preferential binding of compound 36 to Grp94. Ernst et al. identified a dihydroindole-4-one class of inhibitor (37) exhibiting selectivity for Hsp90α/β (Ki = 5 nM) over Grp94 and TRAP1 (Ki > 10 μM).56 In vitro compound 37 promoted the clearance of mutant Huntington protein (mHtt) in HEK cells (IC50 = 24 nM). Compound 37 was orally bioavailable (F = 34%) showing appreciable CNS penetration [B/P (T = 6 h) of 0.6] and was able to reduce the Huntington protein (Htt) by 50% when administered to rats orally (5 mg/ kg). C-Terminal Inhibitors. Epigallocatechin gallate (EGCG, 38, Figure 12) is a major flavonoid of green tea, and it has been shown to inhibit Hsp90 by binding to the C-terminal region of the protein and thereby inducing the degradation of its client proteins.57,58 Recently Khandewal and co-workers published a structure−activity study in which analogs of 38 were evaluated against breast cancer cell lines for antiproliferative activity.59 The study suggested that while A-ring hydroxyl groups were essential in this series, deletion of C-ring hydroxyl groups could be beneficial for Hsp90 inhibition. A des-hydroxy analog (39) of 38 showed 18-fold improvement in antiproliferative activity over EGCG in an MCF-7 breast cancer cell line (39 IC50 = 4 μM vs 38 IC50 = 74 μM). Client protein (Her2, Raf and pAkt)

entered clinical trials as noted above. A recent addition to this group (no longer in active development) is MPC-3100 (23, Figure 6) that was derived from a known PU class of inhibitors, e.g. 24.46,47 Efforts to modify the N-alkyl region of 24 by substitution of the propargyl group with phenyl and subsequently with 4-aminopiperidine led to the identification of N-formyl-4-piperidine analog (25) with acceptable Hsp90 affinity and cellular activity (IC50 = 150 nM; HCT116 IC50 = 430 nM) and oral bioavailability (F = 35%). The SAR study indicated that a two-carbon linker between N-9 and piperidine was optimum for activity. Deletion of the acyl group or substitution with an alkyl moiety was found to be detrimental for Hsp90 affinity. One of the most potent analogs, 23 (Hsp90 IC50 = 140 nM; HCT116 IC50 = 540 nM) had reasonable plasma stability (42% drug remaining after 40 min) and demonstrated a favorable pharmacokinetic profile (t1/2 = 1.4 h; F = 38%) when administered intravenously (2.5 mg/kg) to Swiss nude mice as well as orally (10 mg/kg). Recently, Davies et. al. identified a cyanopyrrolo[2,3d]pyrimidine 26 (Figure 7) by structure based optimization of the purine nucleus exhibiting modest Hsp90 affinity (IC50 = 200 nM).48 X-ray crystallography and molecular modeling study suggested that the cyano group makes an H-bond interaction with Asn51 by displacing a conserved water molecule. Further optimization of 26 at the 4-aryl and 2pyrrolopyrimidine position resulted in the development of potent analogs 27 (Hsp90, IC50 < 1 nM) and 28 (Hsp90, IC50 < 1 nM) demonstrating potent antiproliferative activity (BT474 GI50, 72 and 34 nM, respectively). The study revealed that the S-acetamide series of compounds (exemplified by 28; hERG IC50 > 30 μM) have enhanced safety profile compared with the S-alkylamines (27, hERG IC50 = 1.4 μM). Upon oral administration to mice, 28 was found to be orally bioavailable with moderate plasma exposure and half-life (t1/2 = 1.4 h). A dihydroindole-4-one class of Hsp90 inhibitors exemplified by 29 (Figure 8) was identified by focused screening of purine, and pyrrolocyclohexanone-based scaffolds showed modest antiproliferative activity (AU565 IC50 = 10 μM).49 Activity was improved by relocating the methyl group from C-2 to C-3 on the pyrrolocyclohexanone core and introducing a methyl group at C-4 of the quinazoline ring to block metabolism. This led to analog 30 that showed appreciable Hsp90 affinity (IC50 = 98 nM) and potent cellular activity (AU565 IC50 = 4 nM). This quinazoline also showed antiproliferative activity in a panel of cancer cell lines (e.g., MCF, IC50 = 7 nM; HT29 IC50 = 5 nM). Suda et al. identified a 1,3,5-triazine derivative (31, Figure 9) showing high binding affinity (Kd = 3.4 nM) for Hsp90 and antiproliferative activity against human cancer cell lines (HCT116, IC50 = 460 nM; NCI-N87 IC50 = 570 nM).50 However, poor solubility and low oral bioavailability in murine tumor models limited its clinical potential. These properties of 31 were improved by replacing the S-methyl moiety with butyramide (32), a derivative with enhanced Hsp90 binding affinity (IC50 = 0.48 nM), solubility, and bioavailability (F = 44%). The compound also exhibited potent antiproliferative activity against mammalian cancer cell lines (HCT 116, IC50 = 98 nM; NCI-N87 IC50 = 66 nM). An X-ray crystal structure revealed that the methylene groups in the butyramide form hydrophobic interactions with Met98 and Ile96 while the amide group forms H-bonding interactions with the protein via water molecules contributing to improved Hsp90 affinity.51 Isoform-Selective Hsp90 Inhibitors. As noted earlier, Hsp90 exists in four isoforms: Hsp90α, Hsp90β, Grp94, and G

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degradation was observed in MCF-7 cell lysate, confirming Hsp90 inhibition by 39. Novobiocin and Analogs. The antibiotic novobiocin manifests Hsp90 inhibition by occupying the C-terminal ATP binding site. The poor affinity of novobiocin (40, Figure 13) for Hsp90 (SKBr3, IC50 ≈ 700 μM) was greatly improved by making systematic structural modifications to the benzamide chain, the coumarin core, and the noviose sugar regions.60 In order to gain further insights into the structure−activity of the noviose fragment, Blagg and co-workers examined several analogs with simplified sugar surrogates. SAR suggested that it was possible to improve the antiproliferative activity by replacing noviose with azasugars, cyclic (41, SkBr3 IC50 = 800 nM) and acyclic amines (42, SkBr3 IC50 = 400 nM). In these derivatives, a three-carbon linker between the ether and the amino group was found to be optimum for activity.61−63 Compounds lacking a coumarin core showed antiproliferative activity (43, MCF IC50 = 880 nM65; 44, MCF-7 IC50 = 490 nM64) comparable to that of novobiocin analogs with a coumarin ring.64 These studies confirmed previous observations that the coumarin lactone is not essential for Hsp90 inhibition.65 A series of biaryl analogs were tested for neuroprotective efficacy. This revealed that compounds with an acetamide side chain and an electronegative substituent on the m-position of the B-ring (exemplified by 45) show significant protection against glucose induced neurotoxicity (cell viability of 95%) as compared to reference compound 46 (cell viability of 86%).66 Allosteric Modulation of Hsp90. The marine natural product sansalvamide A and its amide derivative San a-A-amide were shown to have cytotoxic activity against a panel of cancer cell lines. Vasko and co-workers reported that sansalvamide Aamide (47, Figure 14) inhibits the C-terminal binding of Hsp90 client proteins by allosteric modulation of Hsp90.8 San A-amide derivatives, 48 and 49, have shown promising antitumor activity against human colon cancer (HCT-116; IC50 of 7.4 and 5.0 μM, respectively) and pancreatic carcinoma (MiaPaCa-2; IC50 of 7.8 and 5.5 μM, respectively) cell lines. However, unlike the N-terminal Hsp90 inhibitors, sansalvamide A-amide analogs show cytotoxicity without eliciting a heat shock response.67,68

preliminary results in treatment of retinitis pigmentosa and treatment of glucotoxicity in animal models.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 973-655-7204. E-mail: [email protected]. Notes

The authors declare no competing financial interest. Biographies Rohit Bhat received his Master’s degree in 2006 from Manipal University in India, where he studied pharmaceutical chemistry. He earned his Ph.D. (Medicinal Chemistry) in 2012 at the University of Mississippi (Dr. Christopher R. McCurdy). He is currently a postdoctoral fellow with Dr. David P. Rotella (Montclair State University, NJ). His research interest includes discovery of small molecule inhibitors targeting Hsp90 and parasitic protein kinases. Sreedhar R. Tummalapalli received his Bachelor of Science (B.Sc., Biology and Chemistry) in India. He earned a JRF (Junior Research Fellowship) from the CSIR (Council of Scientific and Industrial Research) affiliated with Government of India. Sreedhar earned an M.S. in Chemistry from the University of Minnesota, Duluth, and continued his Ph. D. studies in Medicinal Chemistry in the College of Pharmacy under the supervision of Professor Gunda Georg. Sreedhar began his current appointment at Montclair State University, NJ, under Dr. David Rotella in 2012. He has worked on the synthesis of kinase inhibitors and more recently is engaged in a protease inhibitor project. David P. Rotella is Sokol Professor of Medicinal Chemistry at Montclair State University, NJ. His research interests include discovery of protein kinase and Hsp90 inhibitors and novel scaffolds for medicinal chemistry. David earned his Ph.D. in Medicinal Chemistry at The Ohio State University under Donald T. Witiak and did postdoctoral research at Pennsylvania State University with Ken S. Feldman. Prior to Montclair State University, he was a medicinal chemist in the pharmaceutical industry where he worked on neurodegeneration, psychiatry, cancer, and cardiovascular and metabolic disease projects. He authored over 35 papers and is an inventor on eight issued U.S. patents. Dr. Rotella coedited the 7th edition of Burger’s Medicinal Chemistry with Donald J. Abraham and is a Senior Editor for the Royal Society of Chemistry’s drug discovery book series.

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SUMMARY AND OUTLOOK The biology, pharmacology, and medicinal chemistry of Hsp90 remain very active fields in drug discovery and clinical development. First generation compounds in clinical trials are being investigated in combinations where available biomarkers help predict efficacy; however, these molecules are not isoform selective, which may contribute to cardiotoxicity and gastrointestinal side effects that limit dosing and efficacy. As understanding of the complex cellular pathways regulated by Hsp90 grows, the ability to manipulate them in a beneficial manner will increase and therapeutic opportunities associated with Hsp90 function will grow. Medicinal chemists are now beginning to address the issue of Hsp90 isoform selectivity. This goal will be a significant challenge given the close structural homology between Hsp90s in mammalian cells, and it is likely that binding sites outside the respective ATP pockets will need to be identified to succeed. In spite of these challenges, tool compounds and analytical methods to assess selectivity are beginning to appear. These collective achievements will create opportunities to apply molecules that affect Hsp90 function in diseases beyond cancer, as evidenced by the

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REFERENCES

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