Paralog Specificity Determines Subcellular Distribution, Action

Article pubs.acs.org/jmc

Paralog Specificity Determines Subcellular Distribution, Action Mechanism, and Anticancer Activity of TRAP1 Inhibitors Hye-Kyung Park,†,∥ Hanbin Jeong,†,∥ Eunhwa Ko,‡ Geumwoo Lee,‡ Ji-Eun Lee,† Sang Kwang Lee,‡ An-Jung Lee,† Jin Young Im,† Sung Hu,† Seong Heon Kim,‡ Ji Hoon Lee,‡ Changwook Lee,*,† Soosung Kang,*,‡,§ and Byoung Heon Kang*,† †

Department of Biological Sciences, Ulsan National Institutes of Science and Technology (UNIST), Ulsan 44919, South Korea New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation (DGMIF), Daegu 41061, South Korea § College of Pharmacy and Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul 03760, South Korea ‡

S Supporting Information *

ABSTRACT: Although Hsp90 inhibitors can inhibit multiple tumorigenic pathways in cancer cells, their anticancer activity has been disappointingly modest. However, by forcing Hsp90 inhibitors into the mitochondria with mitochondrial delivery vehicles, they were converted into potent drugs targeting the mitochondrial Hsp90 paralog TRAP1. Here, to improve mitochondrial drug accumulation without using the mitochondrial delivery vehicle, we increased freely available drug concentrations in the cytoplasm by reducing the binding of the drugs to the abundant cytoplasmic Hsp90. After analyzing X-ray cocrystal structures, the purine ring of the Hsp90 inhibitor 2 (BIIB021) was modified to pyrazolopyrimidine scaffolds. One pyrazolopyrimidine, 12b (DN401), bound better to TRAP1 than to Hsp90, inactivated the mitochondrial TRAP1 in vivo, and it exhibited potent anticancer activity. Therefore, the rationale and feasible guidelines for developing 12b can potentially be exploited to design a potent TRAP1 inhibitor.

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INTRODUCTION TRAP1 is a mitochondrial Hsp90 paralog that controls cytoprotection, metabolic reprogramming, and drug resistance in cancer cells.1−3 TRAP1 possesses a structure similar to that of the cytosolic Hsp90, with increased structural similarity to the ATP-binding pockets at the N-terminal domains (NTDs).4,5 Various Hsp90 inhibitors targeting Hsp90 NTDs have been developed as potential anticancer drugs (Figure 1),6−14 and many of them have exhibited inhibitory activity against TRAP1 in vitro.15,16 However, we have previously reported that current Hsp90 inhibitors do not inactivate the mitochondrial TRAP1 because of an insufficient mitochondrial drug accumulation,4,17 which is closely related to their modest anticancer activity in vivo.16,18 Because the forced mitochondrial delivery of Hsp90 inhibitors increases their anticancer activity via a novel mode of action, improving mitochondrial drug accumulation is urgently required for transforming moderate drugs into potent ones.4,17−20 We hypothesized that an insufficient mitochondrial accumulation of Hsp90 inhibitors reflects a strong binding to abundant (∼2% of total cellular proteins) cytoplasmic Hsp90 proteins21−23 before entry into the mitochondria. Indeed, the concentration of the freely available portion of the inhibitor may possibly be too low to reach the mitochondria. Therefore, we decided to develop inhibitors with strong TRAP1-binding but weak Hsp90-binding, activities based on the structural analyses of Hsp90 inhibitors bound to TRAP1 and Hsp90.4,6 © 2017 American Chemical Society

On comparing the structures of TRAP1 and Hsp90 complexed with inhibitors, we identified that inhibitor−target protein interactions have very similar configurations. The pyrimidine of the purine ring and bromopiperonyl in 1 (PU-H71)6 or the pyridinyl of 2 (BIIB021)7 were well-occupied by the active sites of both the chaperones, and the imidazole ring of purine was relatively exposed to the solvent (Supporting Information, Figure S1).4 However, a part of the active site lid (Leu172− Phe201) structure was disordered (Figure 2A) and its flanking conserved residues, Asn171 and Gly202, had different configurations in TRAP1 compared with those in Hsp90 (Figure 2B,C). Thus, to generate TRAP1-selective interactions, we modified the imidazole ring of the purine scaffold, which is relatively close to the flanking regions of the disordered TRAP1 active site lid in the crystal structures.

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CHEMISTRY Pyrazolopyrimidin derivatives 12a and 12b were prepared from various benzyl halides in two steps: a benzylhydrazine formation and a cyclization for pyrazole ring formation. The benzylhydrazine formation was performed via addition of an excess amount of hydrated hydrazine into benzyl bromide solution in MeOH. Pyrazole ring formation was performed via mixing an equivalent amount of 2-amino-4,6-dichloropyrimiReceived: July 3, 2017 Published: August 17, 2017 7569

DOI: 10.1021/acs.jmedchem.7b00978 J. Med. Chem. 2017, 60, 7569−7578

Journal of Medicinal Chemistry

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Figure 1. Representative Hsp90 inhibitors. 1,6 2,7 3,8 4,9 5,10 6,11 7,12 8,13 and 9.14

dine-5-carbaldehyde with a benzylhydrazine under TEA basic conditions (Scheme 1). Scheme 1. Experimental Procedure for the Synthesis of 12a− ba

Reagents and conditions: (a) NH2NH2·ΗCl, MeOH, 0 to 60 °C, 3 h; (b) HCl, CH2Cl2, −2 °C, 13 h; (c) 2-amino-4,6-dichloropyrimidine-5carbaldehyde, TEA, CH2Cl2, −2 °C, 13 h. a

The synthesis of 16 is illustrated in Scheme 2. Tandem condensation of ethyl bromopyruvate with dibenzylamine and aminoguanidine bicarbonate provided the known intermediate 3-amino-6-((dibenzylamino)methyl)-1,2,4-triazin-5(4H)-one (14).24 Debenzylation via Pd/C catalyzed hydrogenation at hydrogen atmosphere, followed by amide bond formation with the activated ester 2,5-dioxopyrrolidin-1-yl 2-(benzo[d][1,3]dioxol-5-yl)acetate gave amide intermediate 15 in moderate yield. Cyclization of 15 under POCl3 condition and reflux with extra POCl3 afforded chlorinated product 16. Compound 20 was prepared from glycine in three steps (Scheme 3). Reductive amination of glycine with benzo[d][1,3]dioxole-5-carbaldehyde gave compound 18. Cyclization of 18 with 2-amino-4,6-dichloropyrimidine-5-carbaldehyde, followed by hydrolysis under aqueous LiOH conditions, afforded compound 20 in good yield.

Figure 2. Structural comparison between TRAP1 and Hsp90. (A) Ribbon diagram of Hsp90 (cyan; PDB: 2FWZ) and TRAP1 (pink; PDB: 4Z1F) complexed with 1. The dotted line indicates structurally disordered region. (B) Close-up view. The structural difference between the interactions of Hsp90 and TRAP1 with 1 is highlighted. (C) Sequence alignment of TRAP1 and Hsp90. The structurally disordered region in the complex structure of TRAP1-1 and its flanking amino acid residues are indicated.

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Scheme 2. Experimental Procedure for the Synthesis of 16a

a

Reagents and conditions: (a) dibenzylamine, ethyl acetate, reflux, 2 h; (b) aminoguanidine bicarbonate, EtOH, reflux, 12 h; (c) Pd/C, H2, AcOH, H2O, 55 °C, 4 h; (d) 2,5-dioxopyrrolidin-1-yl 2-(benzo[d][1,3]dioxol-5-yl)acetate, TEA, CH3CN, 50 °C; (e) POCl3, CH3CN, reflux, 5 h.

Scheme 3. Experimental Procedure for the Synthesis of 20a

Reagents and conditions: (a) benzo[d][1,3]dioxole-5-carbaldehyde, NaBH4, TEA, CH2Cl2, 50 °C, 12 h; (b) 2-amino-4,6-dichloropyrimidine-5carbaldehyde, DBU, DMF, room temperature, 2 h; (c) LiOH, THF/H2O, 65 °C, 12 h.

a

Figure 3. Development of the mitochondrial TRAP1 inhibitor 12b. (A) Initially synthesized 2 mimic molecules. (B) Structures of 21 and 12b. (C) Inhibitor binding to TRAP1 and Hsp90. Fluorescence polarization was measured with purified recombinant proteins and 1−FITC3,25 as described in the Biological Experiment Section, compared with maximum and minimum millipolarization (mP) values, and presented as the mean ± SEM of two independent experiments.

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Table 1. Drug IC50 Valuesa

RESULTS AND DISCUSSION Among various purine mimic derivatives, guanine mimics with the piperonyl side chain derived from 2, such as 12a, 16, and 20, were discovered to have favorable inhibitory activity toward TRAP1 (Figure 3A). Additional syntheses of pyrazolopyrimidines provided us with 21 (Figure 3B), which exhibited stronger inhibitory activities against TRAP1 (IC50 = 138 nM) but comparable inhibitory activities against Hsp90 (IC50 = 97 nM) compared with those exhibited by 2 (Figure 3C). Subsequently, the pyridinyl of 21 was replaced with bromopiperonyl to yield 12b, which further improved the inhibitory activities against TRAP1 but reduced those against Hsp90 (Figure 3C). Compound 12b exhibited highly potent inhibition of TRAP1 (IC50 = 79 nM) but weak inhibition of Hsp90 (IC50 = 698 nM) compared with that exhibited by the purine scaffold Hsp90 inhibitors 1 and 2 (Table 1). To our knowledge, 12b is the most selective TRAP1 inhibitor (over Hsp90) identified until date.

Name 1 2 12a 12b 16 20 21

IC50 for TRAP1 (μM) 0.257 0.535 6.335 0.079 >10 8.434 0.138

± ± ± ±

0.001 0.001 0.541 0.001

± 0.641 ± 0.001

IC50 for Hsp90 (μM) 0.089 0.052 8.932 0.698 >10 >10 0.097

± ± ± ±

0.003 0.001 3.472 0.021

± 0.006

a

Inhibitory activities of the drugs against TRAP1 and Hsp90 in the fluorescence polarization experiments.

We determined the crystal structures of human TRAP1-NM (residues 60−561)4 in complex with 12b and 21. Structural analyses revealed that both pyrazolopyrimidines of 12b and 21 established additional π−π stacking interactions with the conserved Phe201 residue26 in TRAP1 (Figure 4A) compared with the structures of Hsp90 or TRAP1 complexed with the 7571



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Figure 4. Comparison of inhibitor-bound structures. (A) Determined X-ray cocrystal structures of 21 (left; PDB: 5Y3O) and 12b (right; PDB: 5Y3N) bound to human TRAP1. Dotted yellow lines and red spheres indicate intermolecular hydrogen bonds and water molecules, respectively. (B) Structural comparison of TRAP1−1 (left; PDB: 4Z1F) and TRAP1−12b (right). (C) Structural comparison between the crystal structure of Hsp90−2 (left; PDB: 3QDD) and the computational model structure of Hsp90-12b (right). Structures are presented in the same orientation. Hsp90 and TRAP1 are colored cyan and pink, respectively. Oxygen and nitrogen atoms are colored red and blue, respectively. The computational binding experiment was completed using AutoDock Vina.27 All structural figures were prepared using PyMOL (www.pymol.org).

Figure 5. TRAP1 inhibition by 12b. (A) Measurement of mitochondrial membrane potential. HeLa cells were treated with 20 μM of FCCP, 6, and 12b for 4 h. The cells were stained with JC-1 and analyzed using flow cytometry. (B) Mitochondrial ROS production. HeLa cells were treated with drugs for 6 h, and the cells were stained with Mito-Sox and analyzed using flow cytometry. (C) Expression of Hsp70 and Hsp90 client proteins. HeLa cells were treated with 6 and 12b at 20 μM for 6 h. The expression of CHOP, Chk1, Akt, Hsp70, and β-actin was analyzed using Western blotting. (D) Cytochrome c release assay. HeLa cells were treated with drugs at 20 μM for 4 h, and the cytoplasmic fraction was analyzed using Western blotting. (E) Inhibition of TRAP1 ATPase activity. Data are presented as means ± SEM from experiments performed in triplicate.

purine derivatives 1 and 2 (Supporting Information, Figure S2 and S3).4,6 Furthermore, 12b exhibited TRAP1 inhibition that was superior to 1, 2, and 21, reflecting the importance of additional water-mediated hydrogen bonds between the pyrazole N-2 and the Asn171 amide (Figure 4A−C; Supporting Information, Figure S2). Thus, the TRAP1-specific binding of 12b was enhanced by establishing additional interactions with Asn171 and Phe201, located on the flanked residues of the disordered TRAP1 structure.

Compound 12b triggered the signature responses indicative of TRAP1 inactivation including the loss of mitochondrial membrane potential,18,28 overproduction of mitochondrial ROS,29−31 elevation of CHOP expression,32,33 and subsequent discharge of cytochrome c in HeLa cells within 6 h (Figure 5A− D).18,28 In contrast, these responses were not observed in the cancer cell (Figure 5A−D) after treatment with the Hsp90 inhibitor 2,11 which most potently inhibited purified recombinant TRAP1 in vitro (Figure 5E). 12b degraded Hsp90 client 7572



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Figure 6. Analysis of cytoplasmic Hsp90 inhibition. (A) Short exposure to inhibitors. Cytoplasmic Hsp90 inhibition was assessed by analyzing Hsp70 induction and client protein degradation. Prostate and cervical human cancer cell lines, PC3 and HeLa, were treated with drugs (10 μM) for 6 h as indicated and were analyzed by Western blotting. (B) Long exposure to inhibitors. HeLa cells were incubated with 6 and 12b for 36 h as indicated and were analyzed by Western blotting. Gamitrinib (22) is a mitochondria-targeted TRAP1 inhibitor.17 2, 5, and 6 are representative Hsp90 inhibitors with purine, geldanamycin, and resorcinol scaffolds, respectively.37 Akt, Chk1, and Cdk4 are Hsp90 client proteins.38−40

Figure 7. In vitro and in vivo anticancer activities of 12b. (A) Cytotoxic activities of 12b against several cancer cell lines. Cancer cells were incubated with the indicated drug concentrations for 24 h, and cell viability was analyzed using MTT assay. Data are presented as means ± SEM from two independent experiments performed in triplicate. (B) Cell death induction by 12b. HeLa and PC3 cells were incubated with 20 μM 6 or 12b for 24 h, labeled with FITC-DEVD-fmk and propidium iodide, and analyzed using flow cytometry. 12b elevated dead cell population (PI positive) with apoptotic phenotype (AnnexinV positive), whereas the Hsp90 inhibitor 6 did not trigger cell death. (C) Cytotoxic activity in normal cells. Mouse primary hepatocytes and human corneal cells were incubated with various drug concentrations for 24 h, and cell viability was measured using MTT assay. Data are presented as means ± SEM from two independent experiments performed in duplicate; *p < 0.05; **p < 0.003; ***p < 0.0001 (12b vs other drugs). (D) In vivo anticancer activity of 12b. PC3 cells (1 × 107) were subcutaneously implanted into nude mice. After the establishment of tumors, mice were intraperitoneally injected with vehicle or 30 mg/kg 12b daily, and tumor sizes were measured using calipers. (E) Mouse body weights. Vehicle (DMSO) and 12b-treated mouse body weights were measured at the end of the experiment and are presented in the bar graph; n.s., nonsignificant.

proteins less effectively compared with Hsp90 inhibitors and did not induce expression of the biomarker (Hsp70) for Hsp90 inhibition34 during both short-term (6 h) and long-term (36 h) drug treatment (Figures 5C and 6), likely due to reduced binding affinity toward Hsp90 (Figure 3C). Hsp70 suppresses cancer cell death, which is closely related to the moderate anticancer activity of and drug-resistance development against the Hsp90 inhibitors.35,36 Compound 12b completely avoided Hsp70 induction, the adverse effect of Hsp90 inhibitors. Compound 12b exhibited cytotoxic activity that was superior to Hsp90 inhibitors in several human cancer cells (Figure 7A,B). The cytotoxicity is largely dependent on TRAP1

inhibition of the drug, considering significant reduction in cancer cell viability at low drug concentrations (∼5 μM) (Figure 7A) without signs of cytoplasmic Hsp90 inhibition (Figure 6B). However, normal human corneal cells and mouse primary hepatocytes were only marginally affected by 12b treatment, whereas Hsp90 inhibitors caused considerably more cytotoxicity in these normal cells (Figure 7C). Taken together, these data suggested that 12b has superior cancer-specific cytotoxicity but reduced cytotoxic effects in normal cells compared with those of other Hsp90 inhibitors. Thus, to examine anticancer activities in vivo, 30 mg/kg 12b was intraperitoneally administered once daily to PC3 xenograft 7573



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Figure 8. In vivo activity of 12b. (A) Cell death induction in vivo. After 12b treatment of PC3-implanted nude mice, as in Figure 7D, tumor tissues from vehicle (DMSO)- and 12b-treated mice were analyzed by TUNEL staining and examined using a fluorescence microscope (left). Data are presented as means ± SEM; ***p < 0.0001. Dead cell (TUNEL+) population was markedly elevated in the 12b treated group. (B) Histological analyses of drug-treated mouse organs. Organs harvested from vehicle (DMSO)- or 12b-treated mice were fixed with formalin. Fixed tissues were stained with H&E and analyzed by dot slide microscope. No histologic abnormality was observed in 12b-treated mice. (C) In vivo mode of action of 12b. After 12b treatment of PC3 implanted nude mice, as shown in Figure 7D, tumor tissues from vehicle (DMSO)- and 12b-treated mice were analyzed by Western blotting (left). Quantitation of the band intensity (right). The data are the mean ± SEM; n.s. for p > 0.05; *p < 0.05; **p < 0.01. 12b treatment elevated CHOP expression, indicative of TRAP1 inhibition, without inducing Hsp70, a biomarker for Hsp90 inhibition. a supported copper redox catalyst. All reactions were performed under an atmosphere of dry argon. A Biotage Initiator microwave system was used for microwave-assisted reaction. Thin-layer chromatography was carried out on Merck precoated silica gel 60 F254 plates. A Teledyne ISCO flash purification system with various prepacked silica gel cartridges was used for flash column chromatography. 1H and 13C NMR spectra were recorded in the indicated solvent on a Bruker AVANCE III HD (400 and 100 MHz for 1H and 13C, respectively) spectrometer. Chemical shifts are reported as δ values in parts per million downfield from TMS (δ 0.0) as the internal standard in DMSO-d6 or CDCl3. MS was performed on a system consisting of an electrospray ionization (ESI) source in a Shimadzu LCMS-2020 liquid chromatography−mass spectrometer (column: Shim-pack GIS, 100 × 3.0 mm, 3 μm ODS). The purity of the compounds was evaluated on a Shimadzu reverse-phase analytical HPLC system (column: Ace C18, 150 × 4.6 mm, 3 μm). Purities of all compounds that were subjected to biological assay were >95%. Preparation of Hsp90 Inhibitors. 1, 2, 3, 4, 5, and 6 were purchased from Tocris, Toronto Research Chemicals, and SigmaAldrich. Piperonylhydrazine 11a and 11b. Hydrazine monohydrate (6.33 mL, 130 mmol) was added to a solution of 3-piperonylcloride (1a-b: 130 mmol) in MeOH (40 mL) at 0 °C, followed by stirring for 30 min at the same temperature. After stirring for 2.5 h at 60 °C, the reaction mixture was cooled to room temperature and quenched by addition of water (24 mL). The solvent was distilled to approximately 55 mL under reduced pressure. To the residue, 2 N NaOH (aq. 160 mL) was added; this was then extracted with CH2Cl2. The organic layer was dried over Na2SO4, filtered, and then concentrated under

nude mice. Compound 12b treatment reduced tumor growth (Figure 7D) and increased apoptotic cell death in tumor tissues (Figure 8A) and did not cause weight loss or histologic abnormalities (Figures 7E and 8B). Moreover, in agreement with in vitro results, tumors isolated from 12b-treated animals showed reduced expression of Hsp90 client proteins and induction of CHOP expression without elevated Hsp70 expression (Figure 8C).

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CONCLUSIONS In summary, for developing a TRAP1 inhibitor not requiring a mitochondrial delivery vehicle that occasionally causes nonspecific toxicity and poor ADME properties, we hypothesized that the binding affinity to the abundant cytoplasmic Hsp90 should be minimized to increase freely available drug concentrations around the mitochondria. Collectively, our findings indicated that the development of the pyrazolopyrimidine 12b for optimized TRAP1 and Hsp90 binding changed the subcellular drug distribution and subsequently generated a potent TRAP1 inhibitor with a novel mode of action.

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EXPERIMENTAL SECTION

Synthetic Methods and Molecular Characterization. Starting materials were purchased from Sigma-Aldrich, TCI, and Alfa-Aesar and were used without further purification. Solvents were purified by passage through a solvent column composed of activated alumina and 7574



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below 30 °C. Once the quench was complete (pH= 9), the suspension was stirred at room temperature for 20 min and then filtered to obtain the crude solid product. The solid was washed with water, and dried in vacuum. The dried solid (37 mg) was dissolved in POCl3 (3 mL) and heated to reflux for 10 h. The mixture was cooled, concentrated under vacuum, and poured into ice water. The pH of the mixture was adjusted to 8 using saturated aqueous NaHCO3. The organic material was extracted with EA, dried over MgSO4, and filtered. The filtrated was concentrated under reduced pressure and the residue was purified by column chromatography to give the target compound 16 (10 mg, 11% yield). 1H NMR (400 MHz, CDCl3) δ 7.75 (s, 1H), 7.04 (s, 2H), 6.83 (d, J = 1.5 Hz 1H), 6.80 (d, J = 7.9 Hz 1H), 6.68 (dd, J = 8.0, 1.6 Hz 1H), 5.91 (s, 2H), 4.17 (s, 2H): MS (ESI, m/z) calculated for C13H11ClN5O2 [M+1]+ 304.1, found 304.9. Methyl (Benzo[d][1,3]dioxol-5-ylmethyl)glycinate (18). To a solution of Glycine methyl ester hydrochloride (3 g, 23.9 mmol), piperonal (4.3 g, 28.7 mmol) in CH2Cl2 (5 mL) was added TEA (3.9 mL, 28.7 mmol). After stirring for 1 h a room temperature, NaBH4 (497 mg, 13.14 mmol) was added to the mixture at 0 °C. The reaction mixture was stirred for 12 h at room temperature, after which water (30 mL) and ethyl acetate (30 mL) were added to the mixture. The organic layer was partitioned, dried over MgSO4, filtered, and concentrated under reduced pressure. The crude residue was purified by flash column chromatography (EA/Hex) to give the titled compound 18 as colorless oil (3.5 g, 65.6%). 1H NMR (400 MHz, CDCl3) δ 6.83 (s, 1H), 6.74 (s, 2H), 5.93 (s, 2 H), 3.71 (s, 3 H), 3.69 (s, 2H), 3.39 (s, 2H); MS (ESI, m/z) calculated for C11H14NO4 [M +1]+ 224.1, found 224.0. Methyl 2-Amino-7-(benzo[d][1,3]dioxol-5-ylmethyl)-4chloro-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate (19). To a solution of 18 (590 mg, 2.64 mmol) in DMF was added TEA (0.57 mL, 3.2 mmol) and 2-amino-4,6-dichloropyrimidine-5-carbaldehyde (500 mg, 3.2 mmol). After stirring for 6 h at room temperature, DBU (0.45 mL, 3.0 mmol) was added. The mixture was stirred for a further 12 h at 60 °C, then diluted with EA and water. The organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure. The crude residue was purified by flash column chromatography (EA/ Hex) to give the title compound 19 as yellow solid (200 mg, 70% yield). 1H NMR (400 MHz, DMSO-d6) δ 7.23 (bs, 2H), 7.02−7.19 (m, 1H), 6.80 (d, J = 8.07 Hz, 1H), 6.74 (s, 1H), 6.56 (d, J = 8.07 Hz, 1H), 5.96 (s, 2H), 5.51 (s, 2H), 3.78 (s, 3H); MS (ESI, m/z) calculated for C16H14ClN4O4 [M+1]+ 361.1, found 361.5. 2-Amino-7-(benzo[d][1,3]dioxol-5-ylmethyl)-4-chloro-7Hpyrrolo[2,3-d]pyrimidine-6-carboxylic Acid (20). A mixture of 19 (150 mg, 0.42 mmol) and LiOH (15 mg, 0.64 mmol) in THF/water (2:1) was stirred for overnight at 65 °C. The reaction mixture was cooled to room temperature, acidified with 1 N HCl, and extracted with EA. The organic phase was dried over MgSO4, filtered, and concentrated under reduced pressure. The crude residue was purified by flash column chromatography to give 20 (126 mg, 84% yield). 1H NMR (400 MHz, DMSO-d6) δ 13.07 (bs, OH), 7.06 (s, 1H), 6.80 (d, J = 8.0 Hz, 1H), 6.73 (d, J = 1.7 Hz, 1H), 6.55 (dd, J = 8.0, 1.6 Hz, 1H), 5.95 (s, 2H), 5.53 (s, NH2); MS (ESI, m/z) calculated for C15H12ClN4O4 [M+1]+ 347.1, found 347.2. 4-Chloro-1-((4-methoxy-3,5-dimethylpyridin-2-yl)methyl)1H-pyrazolo[3,4-d]pyrimidin-6-amine (21, DN320). 21 was prepared using a published procedure:7 1H NMR (400 MHz, DMSO- d6) δ 8.09 (s, 1H), 7.91 (s, 1H), 5.53 (s, 2H), 3.79 (s, 3H), 2.26 (s, 6H); 13C NMR (100 MHz, DMSO-d6) δ 164.7, 161.8, 156.2, 154.7, 153.2, 148.3, 132.6, 126.3, 125.5, 106.8, 59.2, 49.2, 11.9, 9.6; MS (ESI, m/z) calcd for C14H15ClN6O [M+1]+ 318.8, found 320.0.

reduced pressure. The resulting residue was dissolved in CH2Cl2 (100 mL) and then cooled to 0 °C. Subsequently, 4 N HCl in dioxane (38 mL, 150 mmol) was added to the solution, and the mixture was stirred at 0 °C for 13 h. The resulting precipitate was collected via filtration, followed by washing with CH2Cl2, isopropyl ether, and CH2Cl2, and it was finally dried to obtain piperonylhydrazine hydrochloride (11a−b) as a white salt (78% for 11a, 82% for 11b). 11a: 1H NMR (400 MHz, CDCl3) δ 6.84 (s, 1H), 6.78 (bs, 2H), 5.96 (s, 2H), 3.83 (s, 2H), 2.98 (bs,3H); MS (ESI, m/z) calculated for C8H11N2O2 [M+1]+ 167.1, found 167.0. 11b: 1H NMR (400 MHz, DMSO-d6) δ 7.21 (s, 1H), 7.11 (s, 1H), 6.07 (s, 2H), 5.74 (br, 3H), 4.03 (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 148.3, 147.6, 128.2, 114.2, 112.9, 110.7, 102.6, 52.9; MS (ESI, m/z) calculated for C8H10BrN2O2 [M+1]+ 245.0, found 245.5. 1-(Benzo[d][1,3]dioxol-5-ylmethyl)-4-chloro-1H-pyrazolo[3,4-d]pyrimidin-6-aminepiperonyl hydrazine (12a) and 1-((6bromobenzo[d][1,3]dioxol-5-yl)methyl)-4-chloro-1H-pyrazolo[3,4-d]pyrimidin-6-amine (12b, DN401). A solution of triethylamine (1.2 mL, 8.4 mmol) in CH2Cl2 (3.0 mL) was added dropwise to piperonylhydrazine hydrochloride (11a or 11b) (2.5 mmol) and 2amino-4,6-dichloropyrimidien-5-carbaldehyde (0.4 g, 2.1 mmol) in CH2Cl2 (10 mL) at 0 °C for 15 min. After stirring for 1.5 h at 0 °C, the reaction mixture was poured into 0.2 N HCl (aq) and then extracted with CH2Cl2 (×3). The combined organic layer was washed with brine and dried over MgSO4. The resulting residue was purified by column chromatography (CH2Cl2 to 1:4 EtOAc/CH2Cl2) to obtain the target compound 12a (0.60 g, 95%) or 12b (0.79 g, 99%) as a white solid. 12a: 1H NMR (400 MHz, MeOD) δ 7.97 (s, 1H), 6.93 (s, 2H), 6.48 (s, 1H), 5.97 (bs, 3H), 5.44 (s,2H); MS (ESI, m/z) calculated for C13H11ClN5O2 [M+1]+ 304.1, found 304.0.; 12b: 1H NMR (400 MHz, DMSO-d6) δ 8.04 (s, 1H), 7.39 (s, 2H), 7.25 (s, 1H), 6.48 (s, 1H), 6.04 (s, 2H), 5.32 (s,2H); 13C NMR (100 MHz, DMSO-d6) δ 161.4, 155.9, 153.5, 147.7, 147.2, 132.8, 128.6, 112.5, 112.3, 108.9, 106.0, 102.0, 49.49; MS (ESI, m/z) calculated for C13H10BrClN5O2 [M+1]+ 382.0, found 382.0. 3-Amino-6-((dibenzylamino)methyl)-1,2,4-triazin-5(4H)-one (14). A solution of dibenzylamine (4.05 g, 20.5 mmol) and ethyl bromopyruvate (2.0 g, 10.3 mmol) in ethyl aetate (10 mL) was heated to reflux for 2 h. The reaction mixture was cooled to room temperature and filtered to remove precipitated solid. The filtrates were concentrated under reduced pressure. The residue and aminoguanidine bicarbonate (1.4 g, 10.3 mmol) in EtOH (10 mL) were heated to reflux for 12 h. After cooling to ambient temperature, the mixture was concentrated under reduced pressure. The crude residue was purified by flash column chromatography (EA/Hex) to afford the target compound 14 (487 mg, 14.9% yield). δ 1H NMR (400 MHz, DMSO-d6) δ 12.08 (s, 1H), 7.54−7.04 (m, 10H), 6.74 (s, 2H), 3.64 (s, 4H), 3.47 (s, 2H); MS (ESI, m/z) calculated for C18H20N5O [M+1]+ 224.1, found 224.5. N-((3-Amino-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl)methyl)-2(benzo[d][1,3]dioxol-5-yl) Acetamide (15). Under hydrogen atmosphere, a mixture of 14 (343 mg, 1.07 mmol) and 10% Pd/C (11 mg, 0.01 mmol) in AcOH (0.2 mL) and H2O (3 mL) was stirred for 4 h at 55 °C. After cooling to room temperature, the mixture was filtered through a plug of Celite, and the filtrate evaporated to dryness. The residue was dissolved in TEA (0.42 mL, 2.61 mmol) and CH3CN (2 mL) and heated to 50 °C. 2,5-dioxopyrrolidin-1-yl 2-(benzo[d][1,3]dioxol-5-yl)acetate (296 mg, 1.07 mmol) was added to the solution and the solution was stirred until no starting material was shown in TLC. After cooling to room temperature, the mixture was filtered, the residue washed with water/CAN, and the filtrate dried in vacuum to give the crude titled compound (151 mg, 46.6% yield). 1H NMR (400 MHz, DMSO-d6) δ 12.06 (s, 1H), 8.11 (s, 1H), 6.81 (m, 3H), 5.97 (s, 2H), 4.06 (d, J = 5.7 Hz, 2H), 3.72 (s, 2H); MS (ESI, m/ z) calculated for C13H14N5O4 [M+1]+ 304.1, found 304.9. 7-(Benzo[d][1,3]dioxol-5-ylmethyl)-4-chloroimidazo[5,1-f ][1,2,4]triazin-2-amine (16). To a suspension of 15 (150 mg, 0.5 mmol) in CH3CN (3 mL) was added POCl3 (97 uL) and heated to reflux for 5 h. After cooling to 0 °C, a saturated aqueous K2CO3 (5 mL) was added dropwise to the mixture at a rate to keep temperature

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BIOLOGICAL EXPERIMENTAL SECTION

Chemicals, Plasmids, and Antibodies. All chemicals were purchased from Sigma. Anti-CHOP and anti-Cdk4 antibodies were purchased from Cell Signaling; anti-Akt and anti-Chk1 were purchased from Santa Cruz Biotechnology; anti-β-actin was obtained from MP Biomedicals; anticytochrome c and anti-Hsp70 were purchased from BD Biosciences.

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Cells and Culture Conditions. Human cells from cervix (HeLa), liver (SK-HEP-1), brain (T98G), lung (H460), and prostate (PC3) cancers were purchased from the American Type Culture Collection (ATCC) and were maintained as recommended by the supplier. Cells were cultured in DMEM or RPMI medium (GIBCO) containing 10% fetal bovine serum (FBS; GIBCO) and 1% penicillin/streptomycin (GIBCO) at 37 °C in a humidified atmosphere of 5% CO2. Human corneal cells were purchased from ATCC and were cultured in ATCC corneal medium (ATCC) according to the manufacturer’s instructions. Primary hepatocytes were isolated from 8-week-old BALB/c mice as previously described4 and were cultured in M199/EBSS medium (Hyclone) containing 10% FBS at 37 °C in a humidified atmosphere of 5% CO2. Analyses of Cell Viability and Apoptosis Induction. Cell viability was determined using 3-(4,5-dimethyl-thyzoyl-2-yl)-2,5diphenyltetrazolium bromide (MTT) assays and was quantified by measuring absorbance of the tetrazolium dye at 595 nm using an Infinity M200 microplate reader (TECAN). Absorbance values were normalized to the DMSO control and data were expressed as percent viability. To measure apoptosis induction, DNA contents (propidium iodide) and externalized phosphatidylserine (Annexin V) using apoptosis kit (Molecular Probe). Labeled cells were quantified using the FACS Calibur system (BD Biosciences). Data were processed using FlowJo software (TreeStar). Cytochrome c release was determined by Western blotting after mitochondrial fractionation as described previously.18 Apoptosis in the tumor sections was assessed using in situ apoptosis detection kit (Millipore) according to the manufacturer’s protocols and analyzed using an FV1000 laser confocal scanning microscope (Olympus). Recombinant Protein Preparation and Fluorescence Polarization Assays. Recombinant TRAP1 and Hsp90α were prepared (Supporting Information, Figure S4) as described previously.4 For fluorescence polarization experiments, the fluorescence probe 1FITC3 was synthesized as described previously,21 and 10 nM 1-FITC3 and 100 nM protein were incubated for 24 h at 4 °C with various concentrations of inhibitors in FP buffer containing 135 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, 1 mM DTT, 2 mM MgCl2, 0.1 mg/mL BSA, and 0.05% NP40 (pH 7.3). Fluorescence polarization was finally measured using a SYNERGY NEO microplate reader (BioTek Instruments, Inc.). ATPase Activity Assay. ATPase activity was measured with the PiColorLock Gold Phosphate Detection Kit (Innova Biosciences) as described previously.4 Briefly, 200 nM TRAP1 was preincubated with various concentrations of inhibitors in assay buffer (100 mM Tris, 20 mM KCl, and 6 mM MgCl2, pH 7.0) for 30 min at 37 °C. Then, the mixture was incubated with 0.2 mM ATP for 3 h and the 20 μL mixture of Pi Color Lock Gold reagent and the accelerator (100:1) was added to the 80 μL ATP hydrolysate sample. After 5 min incubation at 25 °C, color development was stopped by the addition of 10 μL stop solution, and absorbance was measured at 620 nm with an Infinity M200 microplate reader (Tecan). Analysis of Mitochondrial Membrane Potential and ROS Production. To examine mitochondrial membrane potential or superoxide production, HeLa cells were incubated with 5 μg/mL JC-1 (Molecular Probes) or 200 nM Mito-SOX Red (Invitrogen) for 20 min at 37 °C. Subsequently, the cells were washed with PBS, trypsinized, resuspended in 500 μL of PBS, and analyzed using the FACS Calibur system (BD Bioscience). Crystallization and Structure Determination. Protein production for human TRAP1 lacking the C terminal domain (residues 60− 561, hTRAP1-NM) was performed as described previously.4,41 To crystallize hTRAP1-NM and inhibitor complexes, 50 μL of 20 mg/mL purified hTRAP1 NM domain was mixed with 3.7 μL of 10 mM inhibitors in DMSO for 1 h on ice and adjusted to a final volume of 90 μL in buffer containing 25 mM Tris-Cl (pH 7.5), 150 mM NaCl, and 5 mM DTT to reduce the DMSO concentration to 4%. Complexes were then crystallized using the hanging drop diffusion method at room temperature by mixing 1 μL of protein solution with 1 μL of reservoir buffer. Crystals of the TRAP1−21 complex grew in reservoir buffers comprising 100 mM sodium cacodylate (pH 6.76), 14%−16% 8K

polyethylene glycol (PEG), and 100 mM calcium acetate. The bestdiffracted crystals of the TRAP1−21 complex were obtained in reservoir buffer containing 14%−16% 20K PEG instead of 8K PEG. Crystals were then cryoprotected in a crystallization solution supplemented with 30% glycerol and were flash frozen in liquid nitrogen. X-ray diffraction data were collected in the 5C beamline of the Pohang Accelerator laboratory (PAL), and data were processed using HKL2000 software.42 Both crystals had almost the same space group (P41212) and unit cell (a = b = 69.5 Å, c = 253.0 Å) as previously reported for the TRAP1−1 complex crystal.4 Electron densities of inhibitors were calculated using difference Fourier methods with the unliganded TRAP1 structure from TRAP1−1 (PDB 4Z1F),4 and were subsequently built into residual electron densities using Coot.43 Final models for TRAP1−12b (PDB 5Y3N) and TRAP1−21 (PDB 5Y3O) were refined to 2.4 and 2.7 Å resolution, respectively, using Phenix.44 Crystallography statistics are summarized in Supporting Information Table S1. All structural figures were generated using PyMOL (http://www.pymol.com). Tumor Xenograft Experiments. All experiments involving animals were approved by UNIST (IACUC-12-003-A). Cancer cells (1 × 107 PC3) were suspended in 200 μL of PBS and were injected subcutaneously into both flanks of 6-week-old BALB/c nu/nu male mice (Charles River Laboratories). Tumors are then allowed to grow to an average volume of approximately 100 mm3 and animals were randomly grouped (two tumors/mouse, five mice/group). Subsequently, DMSO (vehicle) and 12b dissolved in DMSO were each mixed with propylene glycol at a 1:1 ratio (final propylene glycol concentration, 50%) and was administered intraperitoneally (30 mg/ kg) every day. Tumors were measured using calipers, and tumor volumes were calculated using the following formula: V = 1/2 × (width)2 × length. At the end of experiment, animals were euthanized, and organs including brain, heart, kidney, liver, lung, spleen, stomach, and tumor were collected for histology and Western blotting. Harvested organ specimens were also fixed in 10% formalin and embedded in paraffin for histological analyses. Briefly, sections (5 μm) were placed on high-adhesive slides, were stained with H&E, and were scanned using the Dotslide system (Olympus) with 20× magnification. For Western blot analyses, tissue samples were homogenized in RIPA buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, 1% NP-40, and 0.25% N-deoxycholate, and protease inhibitor and phosphatase inhibitor cocktails (Calbiochem), and soluble factions were used for analysis as described previously.32 The band intensity was quantified using ImageJ software (National Institute of Health, USA). Statistical Analyses. MTT experiments were duplicated and repeated independently at least three times. Statistical analyses were performed using the software program Prism 7.0 (GraphPad). Differences were identified using unpaired t-tests and were considered significant when p < 0.05.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00978. Additional structural comparison; TRAP1 SDS-PAGE analysis; and crystallography parameters and refinements (PDF) Molecular formula strings (CSV) Accession Codes

The atomic coordinates and crystallographic structure factors can be accessed using PDB code 5Y3O (21) and 5Y3N (12b) in the Protein Data Bank (www.rcsb.org).

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AUTHOR INFORMATION

Corresponding Authors

*Changwook Lee, Ph.D. Phone: +82-52-217-2534, E-mail: [email protected]. 7576



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*Soosung Kang, Ph.D. Phone: +82-2-3277-6619, E-mail: [email protected]. *Byoung Heon Kang, Ph.D. Phone: +82-52-217-2521, E-mail: [email protected].

(10) Jez, J. M.; Chen, J. C.; Rastelli, G.; Stroud, R. M.; Santi, D. V. Crystal structure and molecular modeling of 17-DMAG in complex with human Hsp90. Chem. Biol. 2003, 10, 361−368. (11) Brough, P. A.; Aherne, W.; Barril, X.; Borgognoni, J.; Boxall, K.; Cansfield, J. E.; Cheung, K. M.; Collins, I.; Davies, N. G.; Drysdale, M. J.; Dymock, B.; Eccles, S. A.; Finch, H.; Fink, A.; Hayes, A.; Howes, R.; Hubbard, R. E.; James, K.; Jordan, A. M.; Lockie, A.; Martins, V.; Massey, A.; Matthews, T. P.; McDonald, E.; Northfield, C. J.; Pearl, L. H.; Prodromou, C.; Ray, S.; Raynaud, F. I.; Roughley, S. D.; Sharp, S. Y.; Surgenor, A.; Walmsley, D. L.; Webb, P.; Wood, M.; Workman, P.; Wright, L. 4,5-diarylisoxazole Hsp90 chaperone inhibitors: potential therapeutic agents for the treatment of cancer. J. Med. Chem. 2008, 51, 196−218. (12) Lin, T. Y.; Bear, M.; Du, Z.; Foley, K. P.; Ying, W.; Barsoum, J.; London, C. The novel HSP90 inhibitor STA-9090 exhibits activity against Kit-dependent and -independent malignant mast cell tumors. Exp. Hematol. 2008, 36, 1266−1277. (13) Woodhead, A. J.; Angove, H.; Carr, M. G.; Chessari, G.; Congreve, M.; Coyle, J. E.; Cosme, J.; Graham, B.; Day, P. J.; Downham, R.; Fazal, L.; Feltell, R.; Figueroa, E.; Frederickson, M.; Lewis, J.; McMenamin, R.; Murray, C. W.; O’Brien, M. A.; Parra, L.; Patel, S.; Phillips, T.; Rees, D. C.; Rich, S.; Smith, D. M.; Trewartha, G.; Vinkovic, M.; Williams, B.; Woolford, A. J. Discovery of (2,4dihydroxy-5-isopropylphenyl)-[5-(4-methylpiperazin-1-ylmethyl)-1,3dihydrois oindol-2-yl]methanone (AT13387), a novel inhibitor of the molecular chaperone Hsp90 by fragment based drug design. J. Med. Chem. 2010, 53, 5956−5969. (14) Nakashima, T.; Ishii, T.; Tagaya, H.; Seike, T.; Nakagawa, H.; Kanda, Y.; Akinaga, S.; Soga, S.; Shiotsu, Y. New molecular and biological mechanism of antitumor activities of KW-2478, a novel nonasnamycin heat shock protein 90 inhibitor in multiple myeloma cells. Clin. Cancer Res. 2010, 16, 2792−2802. (15) Patel, P. D.; Yan, P.; Seidler, P. M.; Patel, H. J.; Sun, W.; Yang, C.; Que, N. S.; Taldone, T.; Finotti, P.; Stephani, R. A.; Gewirth, D. T.; Chiosis, G. Paralog-selective Hsp90 inhibitors define tumor-specific regulation of HER2. Nat. Chem. Biol. 2013, 9, 677−684. (16) Kang, B. H.; Altieri, D. C. Compartmentalized cancer drug discovery targeting mitochondrial Hsp90 chaperones. Oncogene 2009, 28, 3681−3688. (17) Kang, B. H. TRAP1 regulation of mitochondrial life or death decision in cancer cells and mitochondria-targeted TRAP1 inhibitors. BMB Rep. 2012, 45, 1−6. (18) Kang, B. H.; Plescia, J.; Song, H. Y.; Meli, M.; Colombo, G.; Beebe, K.; Scroggins, B.; Neckers, L.; Altieri, D. C. Combinatorial drug design targeting multiple cancer signaling networks controlled by mitochondrial Hsp90. J. Clin. Invest. 2009, 119, 454−464. (19) Butler, L. M.; Ferraldeschi, R.; Armstrong, H. K.; Centenera, M. M.; Workman, P. Maximizing the therapeutic potential of HSP90 inhibitors. Mol. Cancer Res. 2015, 13, 1445−1451. (20) Kang, B. H.; Siegelin, M. D.; Plescia, J.; Raskett, C. M.; Garlick, D. S.; Dohi, T.; Lian, J. B.; Stein, G. S.; Languino, L. R.; Altieri, D. C. Preclinical characterization of mitochondria-targeted small molecule hsp90 inhibitors, gamitrinibs, in advanced prostate cancer. Clin. Cancer Res. 2010, 16, 4779−4788. (21) Taldone, T.; Patel, P. D.; Patel, M.; Patel, H. J.; Evans, C. E.; Rodina, A.; Ochiana, S.; Shah, S. K.; Uddin, M.; Gewirth, D.; Chiosis, G. Experimental and structural testing module to analyze paraloguespecificity and affinity in the Hsp90 inhibitors series. J. Med. Chem. 2013, 56, 6803−6818. (22) Moulick, K.; Ahn, J. H.; Zong, H.; Rodina, A.; Cerchietti, L.; Gomes DaGama, E. M.; Caldas-Lopes, E.; Beebe, K.; Perna, F.; Hatzi, K.; Vu, L. P.; Zhao, X.; Zatorska, D.; Taldone, T.; Smith-Jones, P.; Alpaugh, M.; Gross, S. S.; Pillarsetty, N.; Ku, T.; Lewis, J. S.; Larson, S. M.; Levine, R.; Erdjument-Bromage, H.; Guzman, M. L.; Nimer, S. D.; Melnick, A.; Neckers, L.; Chiosis, G. Affinity-based proteomics reveal cancer-specific networks coordinated by Hsp90. Nat. Chem. Biol. 2011, 7, 818−826. (23) Whitesell, L.; Lindquist, S. L. HSP90 and the chaperoning of cancer. Nat. Rev. Cancer 2005, 5, 761−772.

ORCID

Soosung Kang: 0000-0001-7016-2417 Byoung Heon Kang: 0000-0001-5902-0549 Author Contributions ∥

H.-K.P. and H.J. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the staff of the 5C beamline of the PAL for assistance with synchrotron facilities and the staff of the UNIST-Olympus Biomedical Imaging Center for technical support. This work was supported by the 2015 UNIST Research Fund (1.150098.01), the National Research Foundation of Korea (NRF) grants funded by MEST (2015R1D1A1A01058016; 2010-0028684; 2016R1A2B2012624), and the Korea Drug Development Fund funded by MSIP, MOTIE, and MOHW (KDDF-201512-02).

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ABBREVIATIONS USED CypD, cyclophilin D; DMAG, 17-(dimethylaminoethylamino)17-demethoxygeldanamycin; Hsp90, 90 kDa heat shock protein; MTT, 3-(4,5-dimethyl-thyzoyl-2-yl)-2,5-diphenyltetrazolium bromide; NTD, N-terminal domain; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TPP, triphenylphosphonium; TRAP1, tumor necrosis factor receptor-associated protein 1; TRAP1-NM, C-terminal deleted TRAP1

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REFERENCES

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Paralog Specificity Determines Subcellular Distribution, Action

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