Discovery of Novel Pyruvate Dehydrogenase Kinase 4 Inhibitors for

Article Cite This: J. Med. Chem. XXXX, XXX, XXX−XXX

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Discovery of Novel Pyruvate Dehydrogenase Kinase 4 Inhibitors for Potential Oral Treatment of Metabolic Diseases Dahye Lee,† Haushabhau S. Pagire,† Suvarna H. Pagire,† Eun Jung Bae,† Mahesh Dighe,† Minhee Kim,† Kyu Myung Lee,‡ Yoon Kyung Jang,‡ Ashok Kumar Jaladi,‡ Kwan-Young Jung,‡ Eun Kyung Yoo,§ Hee Eon Gim,§ Seungmi Lee,§ Won-Il Choi,§ Young-In Chi,§ Jin Sook Song,‡ Myung Ae Bae,‡ Yong Hyun Jeon,§,∥ Ga-Hyun Lee,⊥ Kwang-Hyeon Liu,⊥ Taeho Lee,⊥ Sungmi Park,§ Jae-Han Jeon,§,¶ In-Kyu Lee,*,§,¶ and Jin Hee Ahn*,† Downloaded via IOWA STATE UNIV on January 9, 2019 at 22:30:53 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

Department of Chemistry, Gwangju Institute of Science and Technology, Gwangju 61005, Republic of Korea Bio & Drug Discovery Division, Korea Research Institute of Chemical Technology, Daejeon 34114, Republic of Korea § Leading-edge Research Center for Drug Discovery and Development for Diabetes and Metabolic Disease, Kyungpook National University Hospital, Daegu 41404, Republic of Korea ∥ Laboratory Animal Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu 41061, Republic of Korea ⊥ College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu 41566, Republic of Korea ¶ Department of Internal Medicine, School of Medicine, Kyungpook National University, Kyungpook National University Hospital, Daegu 41944, Republic of Korea ‡

S Supporting Information *

ABSTRACT: Pyruvate dehydrogenase kinase 4 (PDK4) activation is associated with metabolic diseases including hyperglycemia, insulin resistance, allergies, and cancer. Structural modifications of hit anthraquinone led to the identification of a new series of allosteric PDK4 inhibitors. Among this series, compound 8c showed promising in vitro activity with an IC50 value of 84 nM. Good metabolic stability, pharmacokinetic profiles, and possible metabolites were suggested. Compound 8c improved glucose tolerance in diet-induced obese mice and ameliorated allergic reactions in a passive cutaneous anaphylaxis mouse model. Additionally, compound 8c exhibited anticancer activity by controlling cell proliferation, transformation, and apoptosis. From the molecular docking studies, compound 8c displayed optimal fitting in the lipoamide binding site (allosteric) with a full fitness, providing a new scaffold for drug development toward PDK4 inhibitors.

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INTRODUCTION Glycolysis, the initial stage of glucose metabolism, yields two adenosine 5′-triphosphates (ATPs), two reduced nicotinamide adenine dinucleotides (NADHs), and two molecules of threecarbon compound pyruvate from glucose. Subsequently, pyruvate generated from glycolysis is converted into acetylcoenzyme A (Acetyl CoA) by the pyruvate dehydrogenase complex (PDC).1−3 Acetyl CoA enters the tricarboxylic acid cycle and finally produces ATP, which is an energy source for the body. This cascade is coupled to the synthesis of 36 molecules of ATP by oxidative phosphorylation.4 The enzymatic activity of PDC is inhibited by pyruvate dehydrogenase kinases (PDK1-4) by phosphorylating three serine residues on its E1α subunit; Ser232, Ser293, and Ser300 with different specificities for each sites. Many previous studies have demonstrated that dysregulation of the PDH/PDK system is implicated in the onset of various diseases2,5,6 such © XXXX American Chemical Society

as cancer, metabolic diseases, and inflammation, suggesting that PDKs are a potent therapeutic target for these diseases. In diabetic mammals, among the four isotypes (PDK1-4), PDK4 is mainly and drastically increased in the liver, skeletal muscle, and adipose tissue.1,5,6 It has been reported that PDK4 knock-out mice show decreased blood glucose levels compared with wild-type (WT) mice.6 Moreover, high-fat diet-fed PDK4knockout mice were shown to have improved blood glucose levels as well as improved insulin sensitivity compared with WT mice.7 Mechanistically, PDK4 deficiency increases pyruvate oxidation in the muscle thereby limiting alanine, lactate, and pyruvate levels required for gluconeogenesis in the liver.7 Recently, it has been reported that the level of PDK4 is elevated in patients with type 2 diabetes.8 These findings Received: July 26, 2018

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DOI: 10.1021/acs.jmedchem.8b01168 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Diverse marketed drugs with an anthraquinone scaffold have been reported as having a wide range of pharmacological activities including laxative, anticancer, anti-inflammatory, and anti-arthritic effects (Figure 2).32 From the HTS results and biological activities associated with the anthraquinone scaffold, we attempted to achieve a lead compound via structural modifications of the hit moiety. The present study reports on the synthesis and biological evaluation of new anthraquinone derivatives as PDK4 inhibitors.

suggest that PDK4 activation is associated with metabolic diseases such as diabetes and insulin resistance.8 Furthermore, PDK4 inhibition has been shown to be effective in nonalcoholic steatohepatitis,9 cisplatin-induced nephrotoxicity,10 vascular calcification,11 and even diabetic cardiomyopathy.12 Mast cells (MCs) are classically considered to be effectors that play a critical role in allergic reactions.13 MC activation is represented by the release of preformed mediators including histamine.14,15 Aberrant activation of MC, in turn, causes various types of allergic diseases such as anaphylaxis, asthma, rhinitis, and atopic dermatitis.16−18 Recent findings by Phong et al.19 have shown that dichloroacetic acid (DCA) as a PDK inhibitor can effectively reduce the degranulation and inflammation cytokine levels in IgE/Ag-stimulated bone marrow-derived MCs. The authors showed enhanced aerobic glycolysis upon mast cell activation by showing changes of mitochondrial function such as oxygen consumption rate and extracellular acidification rate.19 Therefore, it is plausible that metabolic intervention by PDK modulation is a promising therapeutic approach for MC-mediated allergic diseases. Tumor metabolism is represented by aerobic glycolysis, so called Warburg effect. In other words, even in the presence of oxygen, cancer cells rely on cytoplasmic aerobic glycolysis.20 This metabolic switch is beneficial for providing ample glycolytic intermediates, which are utilized for macromolecule synthesis and eventually enhance chances for rapid proliferation of tumor cells.21−23 In this regard, inhibition of metabolic pathway of cancer cells by enforcing oxidative phosphorylation might act as an attractive strategy against cancer.24,25 PDK4 increases cancer cell proliferation,26 which implies that the inhibition of PDK4 might be effective in cancer treatment. Recent evidence has shown that PDK4 can upregulate mutant KRAS activity via post-translational regulation.27 Genetic PDK4 depletion or pharmacological PDK4 inhibition reduces both glucose and fatty acid oxidation, thereby inhibiting tumor growth of KRAS-mutant colorectal and lung cancer (Figure 1).27

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RESULTS AND DISCUSSION Chemistry. A series of anthraquinone derivatives were synthesized according to synthetic Schemes 1−3. Commercially available 1-bromoanthraquinone 3 was coupled with (4(methoxycarbonyl)phenyl)boronic acid via the Suzuki reaction followed by hydrolysis afforded compound 5a. The Suzuki reaction of 1-bromoanthraquinone 3 with pyridin-3-ylboronic acid and 4-hydroxyphenyl boronic acid in the presence of palladium catalyst afforded coupled products 4b and 4d with good yields, respectively. Additionally, 1-bromoanthraquinone 3 underwent Suzuki reaction with tert-butyl 4-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine1(2H)-carboxylate to produce compound 4c. Next, compound 4c was used for Boc group deprotection that resulted in compound 5b, which was further treated with benzyl chloroformate, methanesulfonyl chloride, and phenyl isocyanate to produce compounds 6a, 6b, and 6c, respectively (Scheme 1). The Suzuki reaction of 1-bromoanthraquinone 3 with pyrazole-THP boronate, pyrazole propionate boronate, and pyrazole-Boc piperidine boronate yielded compounds 7a−c with good yields, respectively. Compounds 8a and 8c were synthesized under acidic deprotection of the THP group and Boc group of compounds 7a and 7c, respectively. Compound 8b was synthesized under basic hydrolysis of 7b (Scheme 2). Trifluoromethylation of 1-bromoanthraquinone with CF3TMS in the presence of CsF yielded compound 9, and subsequent Suzuki reactions with corresponding boronate esters furnished compounds 10a, 10b, and 10c. The deprotection of Boc group and basic hydrolysis of ester group produced compounds 11a, 11b, and 11c. Compound 11a reacted with benzyl chloroformate to produce compound 12 with good yield (Scheme 3). Biological Activity. Thus, the synthesized novel series of anthraquinone derivatives was screened for PDK4 inhibition. PS10 was used as a reference compound.28 From the biological assay at 10 μM concentration, the hit anthraquinone 1 showed better in vitro activity than the reference compound PS10. Next, bromoanthraquinone 3 was evaluated and was found to have in vitro potential similar to that of anthraquinone 1 (Table 1). Further modification of compound 3 with sixmembered aryl (4a and 5a) or heteroaryl (4b) substitutions revealed decreased activities. Tetrahydropyridyl carbamate derivative 4c improved the potency with an IC50 value of 600 nM, which has a higher potency than the standard substrate PS10 (760 nM). Compound 5b, an amine hydrochloride, showed lower activity than carbamate 4c. Therefore, N-substituted tetrahydropyridyl anthraquinone derivatives such as benzyl carbamate, sulfonamide, and urea analogues (6a, 6b, and 6c) were synthesized and evaluated (Table 2). Compound 6a showed higher % inhibition than PS10 and lower potency than compound 4c (Table 2). As shown in

Figure 1. Mode of action of PDK and PDK4 inhibitor.

For this reason, we investigated small-molecule PDK4 inhibitors for the treatment of various diseases such as diabetes, allergies, and cancer. Several PDK4 inhibitors28−31 have been reported in patents and in the literature with limited structural diversity, which prompted us to identify novel PDK4 inhibitors. During the search for PDK4 inhibitors via high throughput screening (HTS) using the Korean Chemical Bank, anthraquinone (1) was discovered as a hit against PDK4. B



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Figure 2. Chemical structures of marketed drugs with an anthraquinone scaffold.

Scheme 1. Synthesis of Anthraquinone with Phenyl, Pyridyl, and Tetrahydropyridne Derivatives (Compounds 4a−d, 5a,b, and 6a−c)a

a

Reagents and conditions: (a) (4-(methoxycarbonyl)phenyl)boronic acid or pyridin-3-ylboronic acid or tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate or 4-hydroxyphenyl boronic acid, 2 M K2CO3, Pd(PPh3)4, 1,4-dioxane, reflux. (b) NaOH, THF, water, rt. (c) 4 M HCl in dioxane, EtOAc, rt. (d) Benzyl chloroformate, TEA, THF, rt. (e) Methanesulfonyl chloride, DIPEA, THF, rt. (f) Phenyl isocyanate, DIPEA, THF, rt.

Table 3, five-membered pyrazole derivatives (7a, 8a, 7b, 8b, 7c, and 8c) were synthesized and evaluated for their ability to inhibit PDK4. Compound 8c showed good in vitro potency with an IC50 value of 84 nM, whereas the remaining derivatives displayed only moderate potency toward PDK4 inhibition. Next, the trifluoromethyl group was introduced into the anthraquinone scaffold, and its potency was screened for PDK4 inhibition. As shown in Table 4, compounds 10a, 10b, 11a, and 12 resulted in a loss of potency (compared to their respective oxo derivatives as displayed in Tables 2 and 3), except for compound 11c that showed improved potency.

Further, compound 11c exhibited enhanced potency with an IC50 value of 143 nM (Table 4). From in vitro data, we chose compound 8c as the prototype for further evaluation. To evaluate the in vitro cellular activity, compound 8c was treated in HEK293T human embryonic kidney cells. 8c successfully inhibited phosphorylation of Ser232, Ser293, and Ser300 of PDHE1α, as shown in Figure 3. The potency of compound 8c was greater than that of wellknown PDK2/4 inhibitor PS10. Next, compound 8c was evaluated for its liver microsomal stability in rat and human liver microsomes (HLMs). When C



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Scheme 2. Synthesis of Anthraquinone with Pyrazole Derivatives (Compounds 7a−c and 8a−c)a

a

Reagents and conditions: (a) pyrazole-THP boronate or pyrazole propionate boronate or pyrazole-Boc piperidine boronate, 2 M K2CO3, Pd(PPh3)4, 1,4-dioxane, reflux. (b) NaOH, THF, water, rt. (c) 4 M HCl in dioxane, EtOAc, rt. (d) Aqueous solution of HCl, EtOH, rt.

Scheme 3. Synthesis of 10-Hydroxy-10-(trifluoromethyl)anthracen-9(10H)-one Derivatives (Compounds 10a−c, 11a−c, and 12)a

Reagents and conditions: (a) K2CO3, CF3-TMS, CsF, DMF, 0 °C to rt. (b) tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6dihydropyridine-1(2H)-carboxylate or pyrazole propionate boronate or pyrazole-Boc piperidine boronate, 2 M K2CO3, Pd(PPh3)4, 1,4-dioxane, reflux. (c) NaOH, THF, water, rt. (d) 4 M HCl in dioxane, EtOAc, rt. (e) Benzyl chloroformate, TEA, THF, rt. a

incubated with HLMs together with reduced nicotinamide adenine dinucleotide phosphate (NADPH) and uridine 5’diphospho-glucuronic acid (UDPGA), in vitro half-life values

for 8c were >60 min. In vitro metabolic stability studies with 8c in HLMs revealed that phase I metabolism and glucuronidation were negligible. Following the incubation of D



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Table 1. SAR Data of Anthraquinone Derivatives

8c with HLMs together with NADPH and UDPGA, one minor phase I metabolite (M1) was profiled and identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Further analysis of 8c and M1 using LC-MS/MS produced an informative product ion for structural identification (Figure S1A). The MS/MS spectrum of 8c, which has a protonated molecular ion [M + H]+ at m/z 358.2, showed fragment ions at m/z 275.1 (loss of piperidine ring) and 84.1 (deprotonated piperidine moiety). M1 was tentatively identified as hydroxyl8c (Figure S1B). The mass spectrum of M1 contained a protonated molecular ion peak [M + H]+ at m/z 374.2, suggesting that one oxygen atom was added in compound 8c. The MS/MS spectrum of M1 showed fragmentation patterns like 8c (Figure S1). M1 showed fragment ions at m/z 275.1 (loss of hydroxyl-piperidine ring), 100.1 (deprotonated hydroxy-piperidine moiety), and 82.1 (loss of water in deprotonated hydroxy-piperidine moiety). However, the

exact hydroxylation site in the piperidine group of M1 could not be determined. Compound 8c showed moderate stability in the rat liver microsome (68% remained after 30 min incubation). Cytochrome P450 (P450) and uridine 5′-diphosphoglucuronyltransferase (UGT) enzymes have emerged as key determinants in drug interactions. Therefore, we examined the inhibitory effect of 8c against the activities of five human P450s and four UGTs in HLMs.33 The inhibitory potential of 8c against P450s and UGTs was determined in the absence and presence of 8c up to 50 μM final concentration using pooled HLMs (Table 5). 8c showed a slight inhibition (>10 μM) against CYP1A2, CYP2C19, and CYP3A isoforms and a negligible inhibition against CYP2C9, CYP2D6, and four UGT isoforms. These findings suggest that significant drug interactions between 8c and substrates of four UGT and five P450 enzymes would not be expected. Compound 8c showed E



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Table 2. SAR Data of Anthraquinone with Tetrahydropyridine Derivatives

no significant cytotoxicity in five representative cell lines (Table 5). The pharmacokinetic (PK) profiles of 8c were evaluated in rats and are summarized in Table 6. Compound 8c showed good bioavailability (64%), long half-life (>7 h), and moderate clearance (CL = 0.69) in rats. We next examined whether compound 8c could ameliorate insulin resistance. As shown in Figure 4, 10 μM of compound 8c significantly increased p-Akt in AML12 cells. To determine the antidiabetic efficacy of compound 8c, dietinduced obese mice were orally treated with 100 mg of compound 8c for 1 week, followed by undertaking the intraperitoneal glucose tolerance test. As shown in Figure 5, compound 8c significantly improved glucose tolerance, as quantified by the area under the curve (AUC). Anaphylaxis is initiated by cross-linking of IgE bound to FceRI which is evoked by specific allergen. This eventually causes production and immediate degranulation of various cytokines as well as chemokines. Upon the activation of MCs by IgE/antigen stimulation, the degranulation of MCs occurred rapidly and its level can be evaluated by the measurement of the granule component release. We investigated whether 8c inhibited the degranulation of bone marrow-derived mast cells (BMMCs), particularly β-hexosaminidase by IgE/antigen stimulation. As shown in Figure 6A, pre-incubation with 8c dose-dependently inhibited the release of β-hexosaminidase from IgE/antigen-activated BMMCs, showing that the absorbance values are 0.26 ± 0.025, 0.20 ± 0.015, and 0.126 ± 0.032 in IgE/Ag, 10 μM, and 20 μM 8ctreated BMMCs. When IgE/antigen-stimulated passive cutaneous anaphylaxis (PCA) reaction is induced in the ears of mice, excess extravasation occurs by increased permeability of vessels within the skin and the level of extravasation can be easily monitored by the administration of Evans blue solution to

mice during the PCA reaction.34,35 Thus, PCA can be used as a tool to study the sensitivity to allergens and compound efficacy in ameliorating these responses. Metabolism plays important roles in controlling cellular activation, differentiation, survival, and effector function.36−38 Especially, metabolic manipulation can induce the substantial alternations in the lymphocyte effector function as well as natural killer, B, and dendritic cells. Consistent with these findings, Phong et al. 38 have documented that FcεRI-mediated mast cell activation leads to alternation of their specific metabolism pathway including a rapid increase in glycolysis and mitochondrial respiration that is comparable to immediate reprogramming generated by lymphocyte activation,39,40 thereby resulting in early release of granules such as histamine and TNF-α and lipid mediators, and importantly its alternation can be restored by regulation of downstream metabolic pathways in MCs. Next, we examined the anti-allergic effects of the PDK4 inhibitors in mice using IgE/antigen-stimulated PCA. As shown in Figure 6, PDK4treated mice showed a marked reduction in extravasation by Evans blue solution. Levels of Evans blue exudation was 149.0 ± 16.9, and 49.9 ± 24.1 in vehicle, and 50 mg/kg 8c-treated mice. Even though detail mechanism is not addressed for 8cmediated anti-allergic effects, it could be postulated that metabolic reprogramming mediated by 8c-induced PDK4 inhibition effectively modulates the hyper-metabolic status in MCs so that marked reduction of β-hexosaminidase release is finally occurred in vitro and in vivo. These results indicate that our newly developed PDK4 inhibitors have great potential for anti-allergic effects in vitro and in vivo. Next, to evaluate the anticancer efficacy of compound 8c, we tested whether the treatment of compound 8c could regulate cancer cell proliferation, cellular transformation, and apoptosis. p53 is a transcription factor that restricts aberrant cell growth by regulation of cell death (apoptosis), cell cycle arrest, and F



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Table 3. SAR Data of Anthraquinone with Pyrazole Derivatives

significantly impeded the proliferation of human colon cancer cell lines, HCT116 and RKO (Figure 7B). The colony formation assay, which is considered as a key characteristic of cancer cells, is widely accepted in in vitro cancer research, also showing that the colony formation efficiency in HCT116 and RKO cells was significantly reduced after treatment of compound 8c compared with the DMSO-treated group. Intriguingly, compared with oxaliplatin alone, addition of 25 μM of the compound 8c or even compound 8c (25 μM) alone further decreased the colony formation (Figure 7C). These results suggest that compound 8c inhibits cellular proliferation and transformation. The above data suggest that compound 8c potently repressed cellular transformation and cellular proliferation (Figure 7). We further used flow cytometry to demonstrate whether compound 8c could cause apoptosis. Flow cytometry analysis of both HCT 116 and RKO cells treated with

cellular senescence in various pathologies such as neurodegeneration, ischemia cholestasis, atherosclerosis, and cancer.41−45 Therefore, we investigated whether compound 8c regulates the expression of genes important in the apoptosis such as p53, BAX, or BCLxL (Figure 7A). The western blot analysis showed that compound 8c-induced phosphorylation of p53 on serine 15 is a dose-dependent response in both HCT116 and RKO cells. Expression of BAX, which is downstream gene of p53 and induces apoptosis, was increased by compound 8c. Conversely, compound 8c decreased the expression of BCL-xL, which is an anti-apoptotic gene. Furthermore, cleavage of PARP1 and caspase 3, which is commonly used as an indicator of apoptosis, was increased by compound 8c (Figure 7A). These results indicate that compound 8c induces cell death (apoptosis) through inducing the phosphorylation of p53 (Ser15). In addition, CCK-8 assay showed that the treatment of 50 μM compound 8c G



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Table 4. SAR Data of 10-Hydroxy-10-(trifluoromethyl)anthracen-9(10H)-one Derivatives

compound 8c showed that compound 8c inhibited cell proliferation and increased the number of cells undergoing apoptosis (Figure 8). Annexin V/propidium iodide (PI) staining showed that compound 8c dose-dependently increased apoptosis (gray bar in Figure 8B). In addition, combination treatment of both compound 8c and oxaliplatin markedly induced cell apoptosis compared with the treatment of oxaliplatin alone [14.2% or 7.05% in oxaliplatin (lane 5) alone versus 21.8% or 33.8% in oxaliplatin and 25μM of 8c combined (lane 6)] (Figure 8B).

Next, cells were treated with compound 8c and oxaliplatin for 24 h before harvest and analyzed for apoptosis by PI staining. The number of cells undergoing apoptosis (sub-G1 cell population) was increased by the combined compound 8c and oxaliplatin compared with oxaliplatin alone (25.1% or 12% in oxaliplatin alone vs 36 or 57.4% in oxaliplatin and compound 8c combined) (Figure 8). Taken together, this result indicated that compound 8c might be a potent anticancer drug via the induction of apoptosis. On the basis of the crystal structure of human PDK4 (Protein Data Bank access code 2ZKJ) and the compound’s H



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Figure 3. Cellular assay of PDK4 inhibitors. Left panel: HEK293T human embryonic kidney cells were treated with vehicle, 10 μM compound 8c, PS10, and 5 mM DCA for 24 h. Right panel: Relative band intensity of pSer232, pSer293, and pSer300. The experiments were performed in triplicate.

SwissDock web server.46 The predicted PDK4/inhibitor complex displayed an optimal fitting of the compound into the lipoamide binding site (an allosteric site away from the active site) with full fitness of −1972.78 kcal/mol and estimated ΔG of −8.14 kcal/mol for the most favorable interactions (Figure 9). The binding pocket remained virtually unaltered, and the inhibitor made the best fit into the pre-made cavity. Although the pyrazole branch group made an entrance into the pocket, the anthraquinone group remained at the gate by intercalating between Phe43 and Phe56 on the protein surface. The main protein-inhibitor interactions consisted of three hydrogen bonds with a side chain of Ser53 and the backbone carbonyl oxygen of Gln175 as well as van der Waals interactions with the surrounding hydrophobic residues in the pocket. On the protein surface, Phe43 and Phe56 residues at the gate provided additional pi (stacking interactions with the anthraquinone group of the compound).

Table 5. Liver Microsomal Stability and CYP Inhibition of Compound 8c assay

results

liver microsomal stability (human) T1/2 (half-life) > 60 min liver microsomal stability (rat)a 67.8% remained after 30 min incubation CYP inhibition CYP1A2 IC50: 16.3 μM CYP2C9 IC50: >50 μM CYP2C19 IC50: 42.4 μM CYP2D6 IC50: >50 μM CYP3A IC50: 36.6 μM UGT inhibition UGT1A1 IC50: >50 μM UGT1A4 IC50: >50 μM UGT1A9 IC50: >50 μM UGT2B7 IC50: >50 μM cytotoxicity VERO IC50: 36.42 μM HFL-1 IC50: 36.54 μM L929 IC50: 35.82 μM NIH 3T3 IC50: 39.64 μM CHO K1 IC50: 36.02 μM

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CONCLUSIONS From the HTS, we found that the anthraquinone scaffold has the potential to inhibit PDK4. A series of novel anthraquinone derivatives were synthesized and examined for their ability of PDK4 inhibition. Among them, compound 8c exhibited good in vitro activity with an IC50 value of 84 nM. Good metabolic stability, PK profiles, and possible metabolites were suggested. Compound 8c improved glucose tolerance in diet-induced obesity mice and ameliorated allergic reactions in a PCA mouse model. Additionally, compound 8c exhibited anticancer activity by controlling cell proliferation, transformation, and apoptosis. From the molecular docking studies, compound 8c was determined as an allosteric modulator of the human PDK4 isozyme. Consequently, this novel series of small-molecule PDK4 inhibitors (anthraquinone scaffold) have the potential for further drug development as the therapeutic agent in diabetes, cancer, and allergies.

a

Buspirone was used as a positive control [0.1% (rat) and 4% (human)] remained after 30 min incubation.

Table 6. PK Parameters of 8c in Male Rats parameters

I.V. (5 mg/kg)

P.O. (10 mg/kg)

Tmax (h) Cmax (μg/mL) T1/2 (h) AUCt (μg·h/mL) AUC∞ (μg·h/mL) CL (L/h/kg) Vss (L/kg) F (%)

NA NA 7.06 ± 1.61 7.02 ± 1.84 7.61 ± 2.02 0.685 ± 0.158 5.52 ± 1.66 NA

6±0 0.565 ± 0.208 21.6 ± 3.37 8.92 ± 3.79 18.46 ± 9.68 NA NA 63.6

atomic coordinates generated from its chemical structure, molecular docking calculations were conducted using the

Figure 4. PDK4 inhibitors attenuate insulin resistance in hepatocyte. AML12 cells challenged with palmitate were treated with vehicle, 10 μM compound 8c, 5 mM DCA for 24 h. Right panel: relative band intensity of p-Akt to t-Akt. *p < 0.05, ***p < 0.001 compared with untreated. I



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Figure 5. Antidiabetic efficacy of compound 8c in high-fat diet-induced obesity mice. Eight-week old C57BL/6J mice were fed a high-fat diet composed of 60% kcal fat, 0.5% cholesterol, and 10% fructose diet for 4 weeks. On the fourth week of high-fat feeding, mice were treated with vehicle, 10 mg/kg sitagliptin, 100 mg/kg PS10, and 100 mg/kg 8c orally for 1 week (n = 6). Following 1 week of administration of the compounds, the intraperitoneal glucose tolerance test (1.5 g/kg) was performed. *p < 0.05, **p < 0.01 compared with vehicle. mmol) in 1,4-dioxane (20 mL) was refluxed for 3 h. The reaction mixture was cooled to room temperature and extracted with ethyl acetate. The extract was dried over anhydrous Na2SO4 and evaporated. The residue was purified by silica gel column chromatography to give tert-butyl 4-(4-(9,10-dioxo-9,10-dihydroanthracen-1-yl)-1H-pyrazol-1-yl) piperidine-1-carboxylate 7c (120 mg, 75%) of a solid. 1H NMR (400 MHz, CDCl3): δ 8.34 (dd, J = 7.6, 1.5 Hz, 1H), 8.30−8.25 (m, 1H) 8.21−8.15 (m, 1H), 7.80−7.65 (m, 6H), 4.36 (tt, J = 11.4, 4.0 Hz, 3H), 2.96 (d, J = 11.3 Hz, 2H), 2.26 (dd, J = 12.4, 2.6 Hz, 2H), 2.14−1.90 (m, 2H), 1.48 (d, J = 6.4 Hz, 9H); 13C NMR (101 MHz, DMSO-d6): δ 183.35, 182.66, 153.86, 138.96, 137.87, 134.71, 134.68, 134.52, 133.79, 133.29, 132.05, 130.07, 128.09, 126.91, 126.07, 125.81, 119.89, 78.85, 58.03, 41.98, 28.08, 24.96; HRMS (ESI) m/z calculated for C27H27N3O4 [M + H]+ 457.2002; found 457.1984; HPLC purity 97.85%. To a solution of compound 7c (120 mg, 0.262 mmol) in ethyl acetate (20 mL) was added hydrogen chloride, 4.0 M solution in 1,4dioxane (5 mL). Reaction mixture was stirred at ambient temperature for overnight. The product was collected by filtration and washed with ethyl acetate to give 1-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)anthracene-9,10-dione hydrochloride 8c (90 mg, 96%) of yellow solid. 1H NMR (400 MHz, DMSO-d6): δ 8.88 (s, 1H), 8.65 (s, 1H), 8.23−8.17 (m, 1H), 8.08 (d, J = 5.0 Hz, 1H), 7.96−7.80 (m, 3H), 7.81−7.75 (m, 1H), 7.71 (d, J = 1.4 Hz, 1H), 4.55 (t, J = 10.8 Hz, 1H), 3.43 (d, J = 12.4 Hz, 2H), 3.11 (q, J = 11.1 Hz, 2H), 2.44−1.84 (m, 5H). 13C NMR (100 MHz, DMSO-d6): δ 183.9, 183.2, 139.7, 138.5, 135.2 (2CH), 134.9 (2CH), 134.4, 133.9, 132.6, 130.7, 129.0, 127.5, 126.5, 120.6, 55.6, 42.4 (2CH2), 29.2 (2CH2). HRMS (ESI) m/z calculated for C22H19N3O2 [M + H]+ 357.1477; found 357.1480. HPLC purity, 98.1%. In Vitro Assay of PDK4 Inhibitors. HEK293T cells were cultured in Dulbecco’ modified Eagle’s medium (DMEM) high glucose supplemented with 10% fetal bovine serum (FBS) (Hyclone, AUS) for 24 h. All cells were incubated at 37 °C under 5% CO2 in a humidified incubator. After 24 h, the cultured media were changed with DMEM high glucose containing 10% FBS. Then, cells were stimulated with compounds 8c, PS10, DCA for 24 h and harvested with a lysis buffer (5 mmol/L ethylenediaminetetraacetic acid (EDTA), pH 8.0, 2 mmol/L Na3VO4, 20 mmol/L Tris, pH 7.4, 10 mmol/L Na4P2OH, 100 mmol/L, 0.1 mmol/L PMSF, and 1% NP40) containing protease and phosphatase inhibitor cocktail 3 (Sigma, P0044). Proteins (20 μg) were separated on Bolt gel and transferred to the PVDF membrane (Millipore, GVWP2932A). In vitro assay was performed using antibodies specific for phospho-PDHE1α ser232 (Calbiochem, AP1063), phospho-PDHE1α ser293 (Calbiochem, AP1062), phospho-PDHE1α ser300 (Calbiochem, AP1064), and PDHE1α (Cell signal, 2784S) for in vitro assay. In Vitro Metabolic Stability Study. To evaluate the metabolic stability of 8c, pooled HLMs (1.0 mg/mL, Xenotech) and 8c (1 μM) were preincubated (37 °C, 5 min). To initiate the reaction, NADPH and UDPGA were added to the samples and incubated for specific time points (10, 20, 40, and 60 min). Then, sample aliquots were

Figure 6. Effects of PDK4 inhibitors in vitro and in vivo. (A) IgElabeled BMMCs were pre-incubated with 10 and 20 μM of 8c for 30 min and then stimulated with human serum albumin (HSA). The supernatant was collected to determine levels of β-hexosaminidase release (at 15 min after stimulation). (B) Mice received anti-DNP IgE solution via intradermal injection. After 24 h, mice were intraperitoneally administrated with 50 mg/kg 8c followed by intravenous injection of 60 μg/200 μL DNP-HSA with 2% Evans blue in PBS 1 h later. Dye extravasation in ear tissues was measured by experimental sections. The data are presented as the mean ± SD (n = 5). *, p < 0.05, **, p < 0.01, ***, p < 0.001.

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EXPERIMENTAL SECTION

General. All reported yields are isolated yields after column chromatography or crystallization. All solvents and chemicals were used as purchased without further purification. 1H NMR spectra and 13 C spectra were recorded on a JEOL JNM-ECS400 and JEOL JNMECX400 spectrometers at 400 MHz for 1H NMR and 100 MHZ for 13 C NMR, respectively. Chemical shifts (δ) are expressed in parts per million downfield from TMS as an internal standard. The letters are s, d, t, q, and multiplet. HRMS data were obtained by the JMS 700 (Jeol, Japan). Purity of all tested compounds was ≥95%, as estimated by high-performance liquid chromatography (HPLC) analysis. Samples were analyzed on a Waters Agilent HPLC system equipped with a PDA detector and a Waters SB-C18 column (2.1 × 50 mm). The mobile phase consisted of buffer A (ultrapure H2O containing 0.1% trifluoroacetyl) and buffer B (chromatographic grade CH3CN) was applied at a flow rate of 0.4 mL min−1. All experiments were approved by the Institutional Animal Care and Use Committee of Daegu-Gyeongbuk Medical Innovation Foundation (DGMIF1751101-00). Synthesis of 1-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)anthracene9,10-dione Hydrochloride (8c). A mixture of 1-bromoanthraquinone 3 (100 mg, 0.348 mmol), tert-butyl 4-(4-(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (145 mg, 0.383 mmol), 2 M aqueous potassium carbonate (0.348 mL, 0.697 mmol), tetrakis(triphenylphosphine) palladium(0) (20 mg, 0.017 J



Article

Figure 7. Compound 8c inhibits cellular proliferation and transformation. (A) Western blot analysis. HCT116 and RKO cells were treated with various dose of compound 8c for 24 h. (B) CCK-8 assay of cell proliferation. HCT116 and RKO cells were treated with compound 8c or/and oxaliplatin, grown for 0−72 h, and analyzed for viability by CCK-8 using colorimetry at 450 nm. *p < 0.05. (C) Colony formation assays HCT116 and RKO cells were seeded onto 6-well plates and treated with 10−50 mM/mL compound 8c or 5 mM/mL oxaliplatin for 7 days. After fixation, the cells were stained with crystal violet.

Figure 8. Compound 8c induces cellular apoptosis. (A) Annexin V cell surface binding in HCT116 and RKO cells after treatment with compound 8c or/and oxaliplatin for 24 h. Cell cycle analysis after treatment of compound 8c or/and oxaliplatin for 24 h before PI staining and DNA content analysis by flow cytometry. (B) Bar graph indicating ratio of the cell population in different phases after staining with annexin V/PI or PI. mM) system for 2 h with agitation. The reaction was terminated by

taken and terminated with acetonitrile. After centrifugation, supernatants were analyzed using LC-MS/MS. In Vitro Metabolism Study. To evaluate the microsomal metabolism of 8c, pooled HLMs (1.0 mg/mL, Xenotech) and alamethicin (25 μg/mg protein) were mixed and placed on ice for 15 min. After pre-incubation at 37 °C for 5 min with 8c (100 μM), the reaction was initiated by the NADPH (1.3 mM) and UDPGA (5

adding ice-cold acetonitrile. After termination of the reaction, the mixture was vortexed and centrifuged at 18 000 × g for 5 min. Finally, the supernatant was injected into the LC-MS/MS system. The mass transition used for analysis of 8c was 358.2 → 84.1 (collision energy 22 eV). K



Article

β-hexosaminidase release from BMMCs, they were added 1 h before the addition of DNP-HSA. IgE-Mediated Passive Cutaneous Anaphylaxis. Anti-DNP IgE (80 ng/20 μL, Sigma) was intradermally injected into the ears of sixweek old male mice. After 24 h, mice were intraperitoneally administrated with a single dose of 50 mg/kg 8c. One hour later, the mice were intravenously challenged with 60 μg/200 μL DNPHSA with 2% Evans blue (Sigma) in PBS (200 μL). The mice were euthanized 1 h after treatment with DNP-HSA, and their ears removed and dissolved in 500 μL of dimethylformamide (JUNSEI, Japan) at 37 °C overnight. The amount of dye extravasation was determined at 650 nm using a microplate reader (BMG Labtech). In Silico Modeling of PDK4-Inhibitor Interactions. The threedimensional structure of human PDK4 was obtained from the Protein Data Bank (PDB access code 2ZKJ), and the compound’s atomic coordinates were generated from its chemical structure using the online SIMLES translator.46 The inhibitor compound was docked into PDK4 using the SwissDock web service (http://swissdock.vitalit.ch/) based on the CHARMM force field implemented in the docking software EADock DSS.47 The docking was performed using the “accurate” parameter at otherwise default parameters, with no region of interest defined (blind docking approach). The results were downloaded and viewed in UCSF Chimera package.48

Figure 9. Predicted binding mode of PDK4-inhibitor. The protein is shown as a ribbon diagram and a partially transparent surface representation, whereas the docked compound is shown as a ball-andstick model. The inset shows a zoomed-in view of the main interactions highlighted by a network of hydrogen bonds within the pocket and the pi stacking interactions at the gate. Figure was prepared with PyMOL.

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LC-MS/MS Analysis of 8c and Its Metabolite. Separation and identification of 8c and its metabolite were carried out using a ThermoFisher UltiMate HPLC system with a Thermo Q Exactive Focus Quadrupole-Orbitrap mass spectrometer (ThermoFisher Scientific, San Jose, CA, USA). Electrospray ionization was used for positive ion modes. Compound separation was performed on a Capcell core ADME column (100 × 2.1 mm, 2.7 μm; Shiseido). The mobile phase consisted (A) of water with 0.1% formic acid and (B) of acetonitrile with 0.1% formic acid. To achieve chromatographic separation, a gradient elution program was optimized as follows: 0− 2.0 min, 20% B; 2.0−6.0 min, 20−90% B; 6.1−12 min, 20% B. The flow rate was 0.2 mL/min, and the injection volume was 1 μL. The retention times for 8c and M1 were 6.99 and 7.18 min, respectively. Cytochrome P450 (P450) and UGT Inhibition Assay. The inhibitory potency of 8c was determined with five P450 and four UGT assays in the absence and presence of 8c (final concentrations of 0−50 μM) using pooled HLMs (Xenotech H0630). All experiments were performed in duplicate. Phenacetin O-deethylation, tolbutamide 4-hydroxylation, omeprazole hydroxylation, dextromethorphan Odemethylation, midazolam 1′-hydroxylation, SN-38 glucuronidation, trifluoperazine N-glucuronidation, mycophenolic acid glucuronidation, and naloxone 3-glucuronidation were determined as probe activities for CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A, UGT1A1, UGT1A, UGT1A9, and UGT2B7 respectively, using cocktail incubation and tandem mass spectrometry, as described previously.31 In Vivo Antidiabetic Efficacy. Briefly, eight-week old C57BL/6J mice were fed with a high-fat diet (60% kcal fat + 0.5% cholesterol + 10% fructose diet) for 4 weeks. On the fourth week of high-fat diet feeding, mice were orally treated with vehicle, 10 mg/kg sitagliptin, 100 mg/kg PS10, and 100 mg/kg compound 8c for 1 week (n = 6 per group). Following 1 week administration of the compounds, an intraperitoneal glucose tolerance test (1.5 g/kg of glucose) was performed and area-under the curve analyzed. Degranulation Assays. BMMCs (106 cells/mL) were sensitized overnight with 500 ng/mL mouse anti-DNP IgE (Sigma). Sensitized cells were pretreated 10 and 20 μM 8c for 30 min and then stimulated for appropriate periods with 100 ng/mL DNP−HSA (Sigma-Aldrich). Either supernatant or total cell lysate was placed into 96-well plates together with 50 μL of 2.5 mM 4-nitrophenyl-N-acetyl β-D glucosaminide (Sigma) solubilized in 0.04 M citrate buffer adjusted with disodium phosphate to pH 4.5. After incubation at 37 °C for 60 min, the reaction was terminated by the addition of 50 μL of 0.2 M glycine adjusted with sodium hydroxide to pH 10.7. The colored product was measured at 405 nm using a microplate reader (BMG Labtech, Offenburg, Germany). When effects of 8c were examined on

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.8b01168. Synthetic procedure, 1H NMR, 13C NMR, HRMS spectra, and HPLC assessment of purity for all of the final compounds, HPLC assessment of purity for target compounds, product ion scan mass spectra, and MS/MS fragmentation schemes (PDF) Molecular formula strings and in vitro data (CSV)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +82-62-715-4621. *E-mail: [email protected]. Phone: +82-53-420-5564. ORCID

Minhee Kim: 0000-0001-5384-7342 Yoon Kyung Jang: 0000-0002-9227-8995 Yong Hyun Jeon: 0000-0003-3161-7698 Jin Hee Ahn: 0000-0002-6957-6062 Author Contributions

D.L., H.S.P., and S.H.P. contributed equally. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from Korea Drug Development Fund (KDDF-201601-03), the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare (HI16C1501, HI12C1006), the Ministry of Science, ICT & Future Planning(MSIP)/National Research Foundation of Korea (NRF) (2016M3A9B6902868), and GIST Research Institute (GRI) grant funded by GIST. L



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ABBREVIATIONS PDK4, pyruvate dehydrogenase kinase 4; THF, tetrahydrofuran; K2CO3, potassium carbonate; EtOAc, ethyl acetate; HCl, hydrochloric acid; DMF, dimethylformamide; Et3N, trimethylamine; NaOH, sodium hydroxide; DIPEA, N,N-diisopropylethylamine; Pd (PPh 3 ) 4 , tetrakis(triphenylphosphine)palladium(0); EtOH, ethanol; CsF, caesium fluoride; DCA, dichloroacetic acid; CF3-TMS, trifluoromethyltrimethylsilane

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Discovery of Novel Pyruvate Dehydrogenase Kinase 4 Inhibitors for

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