Development of the First Generation of Disulfide-Based Subtype

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Development of the First Generation of Disulfide-Based Subtype-Selective and Potent Covalent Pyruvate Dehydrogenase Kinase 1 (PDK1) Inhibitors Yifu Liu, Zuoquan Xie, Dan Zhao, Jin Zhu, Fei Mao, Shuai Tang, Hui Xu, Cheng Luo, Mei-Yu Geng, Min Huang, and Jian Li J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01245 • Publication Date (Web): 23 Feb 2017 Downloaded from http://pubs.acs.org on February 24, 2017

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Journal of Medicinal Chemistry

Development of the First Generation of Disulfide-Based Subtype-Selective and Potent Covalent Pyruvate Dehydrogenase Kinase 1 (PDK1) Inhibitors

Yifu Liua, †, Zuoquan Xieb, †, Dan Zhaoc, Jin Zhua, Fei Maoa, Shuai Tangb, Hui Xub, Cheng Luoc, Meiyu Gengb, Min Huangb,*, Jian Lia,*

a

Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University

of Science and Technology, Shanghai 200237, China b

Division of Antitumor Pharmacology, State Key Laboratory of Drug Research, Shanghai

Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China c

Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai

Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China

†

*

These authors contributed equally to this work. To

whom

correspondence

should

be

addressed:

[email protected]

ACS Paragon Plus Environment

[email protected]

or


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ABSTRACT

Pyruvate dehydrogenase kinases (PDKs) are overexpressed in most cancer cells and are responsible for aberrant glucose metabolism. We previously described Bis(4-morpholinyl thiocarbonyl)-disulfide (JX06, 16) as the first covalent inhibitor of PDK1. Here, based on the scaffold of 16, we identify two novel types of disulfide-based PDK1 inhibitors. The most potent analog, 3a, effectively inhibits PDK1 both at the molecular (kinact/Ki = 4.17×103M-1s-1) and the cellular level (down to 0.1 µM). In contrast to 16, 3a is a potent and subtype-selective inhibitor of PDK1 with >40-fold selectivity for PDK2–4. 3a also significantly alters glucose metabolic pathways in A549 cells by decreasing ECAR and increasing ROS. Moreover, in the xenograft models, 3a shows significant antitumor activity with no negative effect to the mice weight. Collectively, these data demonstrate that 3a may be an excellent lead compound for the treatment of cancer as a first-generation subtype-selective and covalent PDK1 inhibitor.


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INTRODUCTION As a major public health problem worldwide, cancer is a very complex set of diseases. Unlike their normal counterparts, most cancer cells have a preference for cytoplasm-based glycolysis rather than mitochondrial oxidative phosphorylation, even under normoxia. This unique reprograming of cellular metabolic pathways, first recognized by Otto Warburg in 1924, meets the survival and proliferation needs during the process of tumor progression.1 In recent years, a more intricate understanding of the metabolic alterations and adaptations of cancer cells has refocused efforts to target tumor metabolism as a selective anticancer strategy.2 The pyruvate dehydrogenase complex (PDC) is a gatekeeper of glucose oxidation, and by converting pyruvate to acetyl-CoA, it links glycolysis to the Krebs cycle.3 An emerging concept suggests that the Warburg effect may be partly due to the attenuation of mitochondrial function, which results from the inhibition of PDC via an increased expression of pyruvate dehydrogenase kinase.4 Four pyruvate dehydrogenase kinase isoenzymes (PDK1–4) have been identified in mammals with tissue-specific expression. They negatively regulate the activity of the PDC by reversible phosphorylation, which occurs independently on three different serines (Ser232, Ser293, and Ser300).5–7 PDK1 is the only isoform reported to phosphorylate all three serines and is closely associated with cancer malignancy.8–9 In cancer cells, the expression of the PDK1 gene is upregulated by the oncogenes c-Myc, hypoxia-inducible factor-1α (HIF-1α) and BRAFV-600E to control metabolic and malignant phenotypes.10–12 A recent study revealed that PDK1 is regulated by Wnt/β-catenin signaling



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in colon cancer cells.13 In addition, PDK1 activity is increased at the post-transcriptional level by diverse oncogenic tyrosine kinases. This enhancement of PDK1 activity results in a promotion of the Warburg effect and tumor growth.14 Furthermore, overexpression of PDK1 or PDK4 protects cells from anoikis, while depletion of the enzymes restores the susceptibility to anoikis and lowers the metastatic potential.15 These evidences suggest that the inhibition of PDK (especially PDK1) may offer a new therapeutic option for the treatment of cancer. Before our recent publications on two new classes of PDK inhibitors,16–17 three major classes of PDK inhibitors had been reported and distinguished by different binding sites (Figure 1). The well-known PDK inhibitor dichloroacetate (DCA, Figure 1), an analog of pyruvate, rapidly entered phase II clinical trials after its anticancer activity was first reported.18 However, the high effective dosage limits its further clinical application.18–19 Although there are several ingenious modifications of DCA, the results are far from satisfactory.20–22 Nov3r23 and AZD754524 (Figure 1) represent a class of PDK inhibitors that target the lipoyl-binding pocket of the enzymes; and recently CPI61325 (Figure 1), a lipoate derivative, demonstrates certain anticancer effects in an in vivo human xenograft tumor, and it has been investigated in phase I clinical trials for patients with relapsed or refractory hematological malignancies.23–25 Radicicol,26 M7797616 and its analogs, and PS1027 (Figure 1) belong to a class of ATP-competitive inhibitors that target the GHKL (gyrase, Hsp90, histidine kinase, MutL) domain in PDKs.16,

26–27

However, the high similarity of the

ATP-binding pocket of kinases may lead to off-target effects, resulting in side effects and


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toxicity issues. As the starting point of our efforts to discover a new type of PDK1 inhibitors, an in-house library of ~600 old drugs was screened using an enzymatic screening assay. Thiram17 (Figure 1), an existing pesticide with anticancer activity, was found to be capable of remarkably inhibiting PDK1 activity, and further chemical efforts led to the discovery of a more potent PDK1 inhibitor, 16 (JX06,17 Figure 1). Using 16 as a chemical probe, we found that the covalent inhibitory action mode of 16 and its analogs was through targeting a highly conserved cysteine residue (Cys240), and we identified the cancer subset responsive to PDK1 inhibition based on the ECAR (extracellular acidification rate)/OCR(oxygen consumption rate) ratio.17 Figure 1 Based on the therapeutic potential of 16, to obtain novel analogs with independent intellectual property rights and to explore the SAR (structure-activity relationship) of the 16 analogs, we designed two types of novel disulfide-based analogs, bis(thiazolyl)disulfides (1a–m) and bis(thiocarbonyl)disulfides (2a–s, 3a–j), derived from 16, under the premise minimizing the structural changes (Figure 2). Finally, a potent homomorpholinyl-derived PDK1 inhibitor with enhanced enzymatic, cellular inhibitory activities, and better performance in vivo, that is, 3a, was identified as a unique lead compound for the development of antitumor drugs through the selective and covalent inhibition of PDK1. Figure 2



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CHEMISTRY To further improve the pharmacologic activity of the lead compound 16 and to obtain novel analogs, chemical modifications were performed in three cycles (Figure 2). First, thirteen bis(thiazolyl)disulfides 1a–m (Table 1) were designed to determine whether the thiocarbamyl moiety of 16 was necessary for PDK1 inhibitory activity. Second, we substituted the morpholine with various steric and polar groups to design eighteen analogs (2a–f and 2h–s) (Table 2). Third, eleven ring contraction and ring expansion analogs (2g and 3a–f, Table 2) were prepared to determine whether the ring size affected PDK1 inhibitory activity. In this study, all the disulfides were obtained through oxidation of the corresponding thiols or dithiocarboxylic acids. The synthesis of analogs 1b–m is shown in Scheme 1, and the procedure generally followed the method reported by K. Ramadas.28 Treatment of commercially available thiols 4a–c with ammonium persulfate yielded target analogs 1b and 1d–e. Then, nitration of 1b afforded target analog 1c. Ring closure of α-bromo ketones 5a–g with ammonium dithiocarbamate gave rise to intermediates 4d–k (hydrolysis of 4i produced 4j), and the subsequent oxidation led to target analogs 1f–m. Scheme 1 Synthesis of target analogs 2a–g and 3a started from commercially available morpholine derivatives 6a–f and 7 and is shown in Scheme 2. The synthesis generally followed the method developed by Neelakantan.29 As an exception, target analog 2g used ammonium


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persulfate as the oxidant. Furthermore, acid 2c underwent esterification to provide target analog 2d. Scheme 2 Scheme 3 depicts the synthetic route for the preparation of target analogs 2h, 2j–k and 2s. O-ethylation of compound 8a30 formed 8b, and chlorination of 8a provided compound 9, which was further converted to 8c by alcoholysis. Debenzylation of 8a–c was performed by conventional catalytic hydrogenation using palladium. The resulting secondary amines were treated with CS2 without further purification, and the following oxidation by ammonium persulfate afforded target analogs 2h and 2j–k. The synthesis of 2s was used the same route of 2k, except for the different chiral material 8d. Scheme 3 Synthesis of 2i and 2l–r started from compound 10,31 as shown in Scheme 4. Nucleophilic substitution of compound 10 by sodium methoxide, phenols and water generated intermediates 11a, 11b–d and 11e, respectively. Reaction of 11e with iodoethane or acetyl chloride in the presence of base produced 11f and 11g. Replacement of the benzyl of 11e with t-butyloxy carbonyl led to 12, which was converted to 11h by acylation with dimethylcarbamic chloride. Deprotection of 11a–h yielded the corresponding secondary amines, which were used without further purification. Finally, target analogs 2i, 2l–m, and 2p–r were prepared in the same manner described in the preparation of 2a; however, target analogs 2n–o were obtained by oxidation of the dithiocarboxylic acids, as described in the



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preparation of 2h. Scheme 4 Target analogs 3b–j were prepared in parallel, as shown in Scheme 5. Nucleophilic substitution of compound 13 (Supporting Information) by sodium methoxide, phenols and water gave 14a, 14b–c, and 14d, respectively. The subsequent modification of 14d resulted in 14e–i. Deprotection of intermediates 14a–i led to the free secondary amines, which were used as nucleophiles in the reaction with CS2. Oxidation by NaNO2/HCl or ammonium persulfate yielded target analogs 3b–j. Scheme 5 RESULTS AND DISCUSSION Design and Synthesis of Disulfide-Based Analogs of 16 In total, thirty-two novel analogs of 16 (1a–m, 2a–s, and 3a–j) were designed and synthesized. Their chemical structures are shown in Tables 1–2. These analogs were synthesized through the routes outlined in Schemes 1–5, and the details of the synthetic procedures and structural characterization are described in the Experimental Section. All analogs were confirmed to be ≥95% purity (Table S1, Supporting Information). Tables 1–2 The IC50 Values of Analogs 1a–m, 2a–r, and 3a–j against PDK1 For the primary assay, the disulfide-based analogs were analyzed for their inhibitory activity against PDK1 using enzyme-linked immunosorbent assay (ELISA), and 16 was used


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as the reference compound. The half-maximal inhibitory concentration (IC50) values of the bis(thiazolyl)disulfides (1a–m) are shown in Table 1. Based on the bioisosterism strategy, thiazoles with different substituents were induced to replace the thiocarbamyl moiety in 16. This set of heteroaryl disulfides displayed promising PDK1 inhibitory activity (IC50 < 0.5 µM), inheriting the activity of 16 (IC50 = 0.049 µM).17 Fusing aryl or cycloalkyl onto the thiazole can be tolerated (1a vs 1b–m). For the aryl analogs, introduction of a phenyl ring (1b, IC50 = 0.379 µM) and pyridine ring (1e, IC50 = 0.476 µM) produced activities similar to 1a (IC50 = 0.326 µM). An electron-withdrawing group or electron-donating group at the aryl ring improved the activity (1c–d vs 1b). For the cycloalkyl analogs, the substituent type on the cycloalkyl ring had little impact on the activity (1h vs 1i–l); however, the introduction of a large cycloalkyl ring was favorable. In the studied set of the cycloalkyl rings, the potency increased in the order cycloheptyl > cyclohexyl > cyclopentyl (1m > 1h > 1g). Notably, the inhibitory activity of analog 1m (IC50 = 0.039 µM) increased a lot compare to that of 1a, even more potent than that of 16. We also tested the inhibitory activities of two free thiols (the intermediates 4a and 4i) against PDK1, but no inhibition was observed at up to 10 µM, demonstrating that the disulfide bond is a critical pharmacophore for PDK1 inhibitory activity. Due to the difficulty of the structural modification of morpholinyl rings, we explored a limited range of substituent types. Analysis of the data in Table 2 revealed that the introduction of substituents at the 2 or 3 position of the morpholinyl ring of 16 substantially affected the activity (2a–s). Generally, fused ring substituents (2e–f vs 16) or bulky groups



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(2p–r vs 16) were not beneficial, and the terminal polar substituents (2g–i and 2o vs 16) led to a loss of activity. To our delight, small steric hydrophobic substituents on the morpholinyl ring maintained the potency (2a–b, 2d and 2i–m vs 16); especially, the inhibitory activity of analog 2k (IC50 = 0.024 µM) was greater than that of 16. Ring contraction (2g vs 16) of the morpholinyl ring was not beneficial, but ring expansion of the morpholinyl ring led to a homomorpholinyl analog (3a, IC50 = 0.026 µM) was favorable. Moreover, the effects of substituents on the homomorpholinyl ring were similar to the morpholinyl ring analogs. SAR of Disulfide-Based Analogs The SAR analysis of a set of thirty-one disulfide-based analogs provided important insight into the essential structural requirements for effective PDK1 inhibition. A preliminary analysis base on the IC50 data shown in Tables 1–2 reveals some noteworthy observations of the SAR for analogs 1a–m, 2a–r, and 3a–j: (1) the disulfide bond rather than the thiocarbamyl moiety of 16 is indispensable for PDK1 inhibitory activity; replacement of the thiocarbamyl moiety with a thiazolyl ring can be tolerated (16 vs 1a–m); (2) fusing a cycloalkyl (especially a larger ring) onto thiazole is favorable for high potency (1g, 1h, and 1m, Table 1), leading to identification of the most potent PDK1 inhibitor (1m) in the thiazole series; (3) among the different N,O-containing aliphatic rings, the potency increases in the order homomorpholinyl > morpholinyl > azetidinyl (3a > 16 > 2g); (4) the effects of substituents on the morpholinyl or homomorpholinyl ring are similar. Generally, fused ring substituents (2e–f), bulky groups (2p–r) and terminal polar substituents (2g–i and 2o) are not


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beneficial, but small steric hydrophobic substituents can maintain, even improve the potency (2k vs 16). Analysis of IC50 data has provided some interesting SARs. Considering the specialty of covalent inhibitors, IC50 value alone may be not sufficient for SAR discussion. In order to validate the SAR concluded above, we introduced the second-order rate constant of target inactivation (kinact/Ki) as the quantitative assessment of potency. Several representative compounds closely related to the above SAR were tested for their kinact/Ki and the resultant values were incorporated into a rectangular coordinate (Table 3, Figure 3). kinact/Ki of 16, 2k and 3a (1.94×103M-1s-1, 4.17×103M-1s-1 and 5.14×103M-1s-1, respectively), which filled in quadrant III, suggested the high affinity and moderate specific reactivity. In contrast, kinact/Ki of 2e, 2g, 2h and 2r (0.06×103M-1s-1, 0.07×103M-1s-1, 0.54×103M-1s-1, and 0.03×103M-1s-1, respectively), as located in quadrant IV, indicated weak affinity and resultant low inhibitory potencies. 1a and 1m (kinact/Ki = 1.53×103M-1s-1 and 3.93×103M-1s-1, respectively) were located in quadrant I, and their high activities likely stemmed from the strong specific reactivity whereas the affinity was responsible for the SAR. These results were overall in agreement with IC50 data indicated activities (Table 2) and the corresponding SAR. Moreover, the two isomers (2k and 2s) did not show difference in both IC50 and kinact/Ki values (Tables 2–3), indicating that the chirality of compounds had no influence on the enzymatic activities. Taken together, the SARs are obviously regular. A subtle interplay between N,O-containing aliphatic rings and small steric hydrophobic substituents is critical for potent



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PDK1 inhibitory activity. In our limited analogs, 2k and 3a represent the best combination of these factors, leading to ~2-fold potency improvement compared to the lead compound 16. Table 3 Figure 3

Cellular Activity of Representative Analogs Our chemical efforts based on 16 led to the discovery of two types of novel disulfide-based PDK1 inhibitors. Encouraged by the favorable PDK1 inhibitory activity in the biochemical kinase activity assay, we assessed the cellular activity using immunoblotting analysis, which examined the impact on PDHE1 phosphorylation downstream from PDK1. All analogs were subgrouped at a cutoff IC50 value of 0.3 µM, and the twenty-two best analogs (1c–d, 1f–m, 2a–d, 2f, 2i–k, 2m–n, 2p, and 3a) were selected and tested against A549 lung cancer cell lines. Firstly, we performed the immunoblotting analysis of the 22 analogs at 10 µM. DCA and 16 were used as positive controls. As shown in Figure 4A, among these samples, analogs 2a–b, 2d, 2i–k, 2m–n, and 3a displayed the strongest cellular potency. These nine analogs almost abolished PDHE phosphorylation at both S293 and S232, while the others showed marginal cellular activity or were only effective at one site (analogs 2f and 2p). For further screening, these nine potent analogs were investigated at much lower concentrations. As shown in Figure 4B, when the concentration was 1 µM, all the analogs, except for 2i, maintained similar strong inhibition of phosphorylation at both sites. We then


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adjusted the concentration to 0.3 µM, and the different cellular potency of the analogs could be easily distinguished. Among the nine analogs, only 2k and 3a maintained obvious activity comparable to that of 16. Considering the good performance in cell-based assays, together with 16, analogs 2k and 3a were selected for the next phase of investigation. For a more accurate comparison of the three analogs in the cell lines, we performed a concentration gradient assay (0.1 µM, 0.3 µM and 1 µM), and the semi-quantitative analysis was performed by Gray-Scale. As illustrated in Figure 4C, the cellular potency increased against a concentration gradient. Analog 3a exhibited the best inhibition at each concentration at both sites. Although analog 2k was slightly weaker than 3a, it exhibited stronger inhibition at S293 than that of 16. Figure 4 2k and 3a Inhibit PDK1 by Covalent Interaction Taking the similar chemical structures and comparable cellular activities into consideration, we believed that analogs 2k and 3a inhibited PDK1 through the same mechanism as 16, i.e., covalently binding to a conserved cysteine residue (C240).17 To confirm this hypothesis, we introduced the reducing thiol group to interrupt the inferential formation of disulfide bonds between PDK1 and the analogs by adding glutathione (GSH) to the system. As with 16,17 an increasing loss of activity was observed as the GSH concentration increased in the experiments with analogs 2k and 3a (Figure 5), while DCA was not affected.17 This result was consistent with our previous findings17 and suggested that 2k and 3a exerted strong inhibitions on the PDK1 activity through covalent interactions.



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Figure 5 2k and 3a Are Subtype-Selective and Potent PDK1 Inhibitors Due to the most significant role of PDK1 among the PDK subtypes in cancer therapy, we measured the kinact/Ki value of 16, 2k and 3a against all PDK isoforms using a biochemical enzymatic assay. As shown in Table 4, all three compounds exhibited more or less selectivity towards PDK1 over other PDK subtypes. Compounds 2k and 3a displayed better subtype-selectivity than 16. Among them, 3a exhibited ~40-fold selectivity towards PDK1 over PDK2 and 3, better than 2k and even 10-fold improvement than that of 16. All three compounds did not inhibit PDK4 at a concentration up to 10 µM. The control DCA had no subtype-selectivity for PDK1–4. To the best of our knowledge, these two analogs (2k and 3a) are the first generation of subtype-selective and potent PDK1 inhibitors. Table 4 Molecular Docking of 16, 2k and 3a with PDK1–4 To better understand the binding mode of 16 analogs with PDKs, we carried out molecular docking of 16, 2k and 3a with PDK1–4, respectively. Firstly, 16, 2k and 3a all suited well in the binding pocket near C240 of PDK1 (Figure 6A), which provided a good model to explain the improved activities of 2k and 3a towards PDK1. Apparently, the substituted morpholinyl of 2k and homomorpholinyl ring of 3a allowed stronger hydrophobic interactions with C240 surrounding residues, with more residues (A236, L239, N246, K282, M285, P351, L352, and A293) participating in 2k and 3a interactions compared with 16-PDK1 binding model. In the meanwhile, distance between the thiol of C240 and the


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thiocarbonyl of 2k (3.2 Å) and 3a (3.0 Å) was smaller than that of 16 (3.8 Å), suggesting a higher possibility to form a disulfide bond (Figure 6A). Secondly, the docking models may also explain the subtype-selectivity of 16 analogs for PDK1. The binding pockets of 16/2k/3a in PDK2 and PDK3 (Figures 6B–C) appeared less suitable (i.e. narrow) for compounds entry. For example, the binding pockets of 16/2k/3a in PDK3 showed rather small hole shapes, indicating that the compound need to overcome the solvent repulsion before binding and fitting into the enzyme pockets (Figure 6C). These findings were consistent with the measured Ki values of 16/2k/3a towards PDK2 and PDK3 versus PDK1 (Table 4). In the case of the irreversible binding, the kinact value reflects the reaction rate. In addition to the distances, many different factors contributed to kinact, including intrinsic chemical reactivity, reactant alignment, enforced local concentration, etc.32 As show in Figures 6A–B, the distances between the thiocarbonyl of these compounds and the thiol of C148 in PDK2 were much bigger than that of C240 in PDK1, consistent with the lower kinact of 16/2k/3a towards PDK2. Similarly, the distances of 16 and 2k to the cysteine in PDK1 and PDK3 were 3.8/3.5 Ǻ and 3.2/3.1 Ǻ, respectively, which were consistent with the higher kinact of 16/2k towards PDK3 over PDK1 (Table 4). One exception was 3a, whose distance to the cysteine in PDK1 was closer than that of PDK3 (3.0/3.2 Ǻ, respectively), but the kinact value towards PDK3 was obvious higher. We speculated that the binding pose of 3a in PDK3 may offer an advantageous trajectory of the thiocarbonyl to the cysteine. Altogether, these results suggested the lower kinact/Ki value of 16 analogs towards PDK2/3. Likewise, there were no obvious binding pockets for 16/2k/3a in PDK4 and appeared quite low possibility to form the



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disulfide bonds (Figure 6D), explaining the lack of activity against PDK4. Figure 6 2k and 3a Alter Glucose Metabolic Pathways Inhibition of PDK1 in cancer cells leads to the recovery of the impaired mitochondrial function, which switches the metabolism of pyruvate from cytoplasm-based lactate production to mitochondria-based oxidative phosphorylation. This metabolic pathway change is characterized by a decrease in ECAR and an increase in ROS. We tested the influence of 2k and 3a on the two indicators of metabolic pathway change and used 16 as the reference substance. As shown in Figure 7, ECAR was significantly decreased by analogs 2k and 3a in a dose-dependent manner, which was consistent with the expected reduction of lactate production in A549 cells. In line with the accelerated oxidative phosphorylation, ROS was increased by analogs 2k and 3a with increasing concentration. The regulative results of analogs 2k and 3a for ECAR and ROS were largely the same as lead compound 16. These data suggested that analogs 2k and 3a affected cell metabolism in the same way as 16. Figure 7 2k and 3a Exhibit Antiproliferative Effects in Cancer Cells but not in Normal Cells Encouraged by the above observations, we tested the antiproliferative effects of analogs 2k and 3a by the CCK8 assay. Two cancer cells, A549 (human lung cancer cells) and Kelly (human neural cancer cells), and two normal cells, GM00637 (human skin fibroblast cells) and LO2 (human normal liver cells), were employed. DCA was used as the positive control. All three compounds dose-dependently inhibited the proliferation of cancer cells and had no


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obvious influence on normal cells (IC50 > 10 µM, Table 5). As the best analog in the immunoblotting analysis (Figure 4), analog 3a sustained its advantage, and inhibited Kelly cell proliferation with a promising IC50 of 0.057 µM (Table 5), leading to ~5-fold potency improvement compared to the lead compound 16, whereas analog 2k possessed less than satisfactory potency (IC50 = 1.426 µM). In addition to Kelly cells, analog 3a effectively inhibited A549 cell proliferation (IC50 = 0.225 µM, Table 5), to a greater extent than both 16 (IC50 = 0.480 µM) and analog 2k (IC50 = 0.957 µM). Of note, compounds 2k and 3a barely inhibited the growth of two normal cell lines. Table 5 Compounds 2k and 3a Attenuates Tumor Growth in A549 Xenograft Models To further prove the therapeutic potential of compound 3a, we compared antitumor efficacy of 16, 2k and 3a in A549 subcutaneous xenograft mice models (Figure 8). Tumor-bearing mice were randomly subgrouped and intraperitoneally received vehicle control or 16 analogs at 40 and 80 mg/kg per day, respectively. A 21-day continuous treatment of 16 and 3a considerably reduced tumor volume at both dosages compared with the vehicle control (16, TGI = 27.7% at 40 mg/kg, TGI = 50.4% at 80 mg/kg; 3a, TGI = 26.2% at 40 mg/kg, TGI = 47.8% at 80 mg/kg; Figures 8A and 8C), but the effect of 2k was marginal (TGI = 11.0% at 40 mg/kg, TGI = 20.0% at 80 mg/kg; Figure 8B). Likewise, endpoint tumor weights in the 16 and 3a treated group were significantly less than those treated with vehicle control (Figure 8D). In this experiment, compound 3a showed similar antitumor efficacy as 16. The two-fold improvement in the enzymatic activity of 3a (Table 4)



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was not translated to in vivo efficacy in inhibiting tumor growth (Figure 8E). None of the results resulted in mice bodyweight loss even at higher dosage. CONCLUSIONS In summary, we have discovered two novel types of PDK1 inhibitors, i.e., bis(thiazolyl)disulfides (1a–m) and bis(thiocarbonyl)disulfides (2a–s, 3a–j). On the basis of the structure of the lead compound 16, thirty-one completely novel disulfide-based analogs have been synthesized and tested by a PDK1 enzymatic inhibitory assay. Twenty-two analogs (1c–d, 1f–m, 2a–d, 2f, 2i, 2j–k, 2m–n, 2p, and 3a) showed potent PDK1 inhibitory activity (IC50 < 0.3 µM). Preliminary SARs were obtained base on IC50 data and were further validated by kinact/Ki, showing that the disulfide bond and bulky N,O-containing aliphatic rings of 16 are indispensable for PDK1 inhibitory activity, and terminal small steric hydrophobic substituents can substantially improve the potency. The PDK1 inhibition assays at the cellular level further confirmed that nine analogs (2a– b, 2d, 2i–k, 2m–n, and 3a) were potent PDK inhibitors and effectively abolished PDHE phosphorylation at both S293 and S232. Among the nine analogs, analog 3a exhibited the best inhibition (down to 0.1 µM) at both S293 and S232 and the PDK-selective profile of 3a was observed. 3a was also identified to inhibit PDK1 by covalently binding to a conserved cysteine residue. Compound 3a demonstrated approximately 2 times higher potency in inhibiting PDK1 than that of 16 according to the kinact/Ki value. In the meanwhile, 3a had a ~40-fold selectivity for PDK1 over PDK2 and 3 and showed no inhibition of PDK4 at a


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concentration up to 10 µM. Subtype-selectivity of 3a was 10 times higher than that of 16. Further detailed analysis of molecular docking of 16, 2k and 3a with PDK1-4 gained a better understanding of the subtype-selectivity for PDK1-4 of these compounds. Compound 3a significantly altered glucose metabolic profile in a dose-dependent manner in A549 cells by decreasing ECAR and increasing ROS. Moreover, the in vitro antiproliferative assays showed that 3a had more potent inhibitory effects than that of 16 on the proliferation of two human cancer cell lines (A549: IC50 = 0.225 µM and Kelly: IC50 = 0.057 µM), without inhibition activity against normal cell lines GM00637 and LO2 (IC50 > 10 µM). Together with 16, compound 3a also showed significant antitumor activity in the in vivo

A549 xenograft models which was better than that of 2k. Altogether, these data demonstrate that 3a may be a promising starting point for the discovery of potential therapeutic drugs to specifically inhibit PDK1. Considering the small number of PDK1 inhibitors reported thus far, 3a could be regarded as the first generation of subtype-selective and covalent PDK1 inhibitors. Further characterization of 3a as a potential cancer therapeutic drug will be performed in the future. EXPERIMENTAL SECTION Chemistry. General Chemistry The synthetic starting materials, reagents and solvents were purchased from Alfa Aesar, Acros, Adamas-beta, Energy Chemical, J&K, Shanghai Chemical Reagent Co. and TCI at the highest commercial quality and were used without further purification. Analytical thin-layer chromatography (TLC) was performed on HSGF 254 (150−200 µm thickness; Yantai Huiyou



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Co., China), and the components were visualized by observation under UV light (254 nm and 365 nm). Melting points were determined on an SGW X-4 melting point apparatus without correction. Optical rotation of the chiral compounds was measured using a RUDOLPH (AUTOPOL V) automatic polarimeter. The products were purified by recrystallization or column chromatography on silica gel (200–300 mesh). The reaction yields were not optimized. Nuclear magnetic resonance (NMR) spectroscopy was performed on a Bruker AMX-400 NMR using deuterated chloroform (CDCl3), deuterated methanol (CD3OD), or deuterated dimethyl sulfoxide (DMSO-d6) as the solvent. The chemical shifts were reported in parts per million (ppm, δ) downfield from tetramethylsilane. The proton coupling patterns were described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), and broad (br). Low- and high-resolution mass spectra (LRMS and HRMS) were performed with electric and electrospray ionization (EI and ESI) with a Finnigan MAT-95 and an LCQ-DECA spectrometer. HPLC analysis of compounds 1a–m, 2a–s, and 3a–j was performed on an Agilent 1100 with a quaternary pump and a diode-array detector (DAD). The peak purity was checked by UV spectra. All analogs were confirmed to be ≥95% purity. Bis(thiazol-2-yl)disulfide (1a) This analog is commercially available from J&K Scientific Ltd. Bis(benzo[d]thiazol-2-yl)disulfide (1b) An aqueous solution of ammonium persulfate (342 mg, 1.5 mmol in 5 mL water) was added dropwise at room temperature to a stirred solution of benzo[d]thiazole-2-thiol (4a, 167 mg, 1 mmol) in THF (10 mL), and the reaction was monitored by TLC. After 0.5 h, the mixture was separated between EtOAc (50 mL) and water (50 mL). The organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced


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pressure. The residue was then purified by column chromatography on silica gel to afford analog 1b as a white solid (150 mg). Yield: 90%. M.p. 179–181 ºC. 1H-NMR (400 MHz, CDCl3): δ 7.94 (d, J = 8.2 Hz, 2H), 7.77 (d, J = 8.0 Hz, 2H), 7.49–7.45 (m, 2H), 7.38–7.34 (m, 2H); HRMS (EI) m/z calcd. C14H8N2S4 (M+) 331.9570, found 331.9576. Bis(6-nitrobenzo[d]thiazol-2-yl)disulfide (1c) Fuming nitric acid (0.016 mL, 0.35 mmol) was added to a stirred solution of 1b (100 mg, 0.3 mmol) in concentrated sulfuric acid (5 mL) maintained at 0 ºC. The solution was stirred at room temperature for 1 h and was then added to ice water (50 mL). The precipitated product was collected and crystallized from EtOAc to give analog 1c as a yellow solid (90 mg). Yield: 71%. M.p. 218–220 ºC; 1H-NMR (400 MHz, DMSO-d6): δ 9.13 (d, J = 2.3 Hz, 2H), 8.35 (dd, J = 9.0, 2.3 Hz, 2H); HRMS (EI) m/z calcd. C14H6N4O4S4 (M+) 421.9272, found 421.9277. Bis(6-ethoxybenzo[d]thiazol-2-yl)disulfide (1d) 1d was obtained in the same manner as described in the preparation of 1b from 6-ethoxybenzo[d]thiazole-2-thiol (4b) as a white solid. Yield: 85%. M.p. 132–135 ºC. 1

H-NMR (400 MHz, CDCl3): δ 7.83 (d, J = 9.0 Hz, 2H), 7.65 (d, J = 2.2 Hz, 2H), 7.10 (dd, J

= 9.0, 2.4 Hz, 2H), 4.06 (q, J = 6.9 Hz, 4H), 1.34 (t, J = 6.9 Hz, 6H); HRMS (EI) m/z calcd. C18H16N2O2S4 (M+) 420.0095, found 420.0096. Bis(thiazolo[5,4-b]pyridin-2-yl)disulfide (1e) 1e was obtained in the same manner as described in the preparation of 1b from thiazolo[5,4-b]pyridine-2-thiol (4c) as a white solid. Yield: 60%. M.p. 147–148 ºC. 1H-NMR (400 MHz, DMSO-d6): δ 8.61 (d, J = 4.6 Hz, 2H), 8.39 (d, J =8.3 Hz, 2H), 7.65–7.58 (m, 2H);



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HRMS (EI) m/z calcd. C12H6N4S4 (M+) 333.9475, found 333.9476. Bis(6,7-dihydro-4H-pyrano[4,3-d]thiazol-2-yl)disulfide (1f) Ammonium dithiocarbamate (110 mg, 1 mmol) was added to a solution of 3-bromooxan-4-one (5a, 179 mg, 1 mmol) in EtOH (20 mL), and the mixture was heated to reflux for 1 h and concentrated under reduced pressure. The residue was separated between EtOAc (50 mL) and water (50 mL), the organic layer was washed with brine and dried over anhydrous sodium sulfate, and the solvent was removed to give the crude product 6,7-dihydro-4H-pyrano[4,3-d]thiazole-2-thiol (4d, 180 mg), which was used in the next step without further purification. Analog 1f was obtained in the same manner as described in the preparation of 1b from the crude product 4d as a white solid. Yield: 52% over two steps. M.p. 125–127 ºC. 1H-NMR (400 MHz, DMSO-d6): δ 4.75 (s, 4H), 3.96–3.88 (m, 4H), 2.83–2.75 (m, 4H); HRMS (EI) m/z calcd. C12H12N2O2S4 (M+) 343.9782, found 343.9781. Bis(5,6-dihydro-4H-cyclopenta[d]thiazol-2-yl)disulfide (1g) 1g was obtained in the same manner as described in the preparation of 1f from 2-bromocyclopentanone (5b) as a white solid. Yield: 70% over two steps. M.p. 115–118 ºC. 1

H-NMR (400 MHz, DMSO-d6): δ 2.91 (t, J = 7.2 Hz, 4H), 2.77 (t, J = 7.4 Hz, 4H), 2.46–

2.37 (m, 4H); HRMS (EI) m/z calcd. C12H12N2S4 (M+) 311.9883, found 311.9884. Bis(4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl)disulfide (1h) 1h was obtained in the same manner as described in the preparation of 1f from 2-bromocyclohexanone (5c) as a white solid. Yield: 85% over two steps. M.p. 102–105 ºC.


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1

H-NMR (400 MHz, DMSO-d6): δ 2.78–2.71 (m, 4H), 2.71–2.63 (m, 4H), 1.84–1.72 (m, 4H);

HRMS (EI) m/z calcd. C14H16N2S4 (M+) 340.0196, found 340.0197. Bis(6-methyl-4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl)disulfide (1i) 1i was obtained in the same manner as described in the preparation of 1f from 2-bromo-4-methylcyclohexanone (5d) as a light yellow solid. Yield: 80% over two steps. M.p. 110–112 ºC. 1H-NMR (400 MHz, CDCl3): δ 2.93–2.79 (m, 4H), 2.79–2.68 (m, 2H), 2.40– 2.29 (m, 2H), 1.94–1.79 (m, 4H), 1.56–1.43 (m, 2H), 1.10 (s, 3H), 1.08 (s, 3H); HRMS (EI) m/z calcd. C16H20N2S4 (M+) 368.0509, found 368.0508. Bis(6,6-dimethyl-4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl)disulfide (1j) 1j was obtained in the same manner as described in the preparation of 1f from 2-bromo-4,4-dimethylcyclohexanone (5e) as a light yellow solid. Yield: 77% over two steps. M.p. 110–111 ºC. 1H-NMR (400 MHz, CDCl3): δ 2.83–2.75 (m, 4H), 2.53 (s, 4H), 1.67–1.58 (m, 4H), 1.03 (s, 12H); HRMS (EI) m/z calcd. C18H24N2S4 (M+) 396.0822, found 396.0829. Bis(6-ethoxycarbonyl-4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl)disulfide (1k) 1k was obtained in the same manner as described in the preparation of 1f from ethyl 3-bromo-4-oxocyclohexanecarboxylate (5f) as a white solid. Yield: 67% over two steps. M.p. 129–131 ºC. 1H-NMR (400 MHz, CDCl3): δ 4.18 (q, J = 7.0 Hz, 4H), 3.08–2.99 (m, 4H), 2.99–2.87 (m, 2H), 2.87–2.74 (m, 4H), 2.32–2.21 (m, 2H), 2.07–1.90 (m, 2H), 1.28 (t, J = 7.1 Hz, 6H); HRMS (ESI) m/z calcd. C20H25N2O4S4 [M+H]+ 485.0697, found 485.0700. Bis(6-carboxyl-4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl)disulfide (1l) A mixture of ethyl 2-mercapto-4,5,6,7-tetrahydrobenzo[d]thiazole-6-carboxylate (243



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mg, 1 mmol) and LiOH (100 mg, 4 mmol) was combined in 4 mL of THF and H2O solvent (VTHF:VH2O = 3:1) at room temperature for 0.5 h, and was acidified to pH 5.5. Then, the precipitate

was

collected,

washed

with

H2O,

and

dried

to

afford

2-mercapto-4,5,6,7-tetrahydrobenzo[d]thiazole-6-carboxylic acid (4j, 200 mg, 93%) as a white solid. 1l was obtained in the same manner as described in the preparation of 1f from 4j as a white solid. Yield: 95%. M.p. 222–223 ºC. 1H-NMR (400 MHz, CDCl3): δ 12.48 (brs, 2H), 3.07–2.97 (m, 2H), 2.95–2.83 (m, 2H), 2.83–2.68 (m, 6H), 2.19–2.06 (m, 2H), 1.93–1.79 (m, 2H); HRMS (EI) m/z calcd. C16H16N2O2S4 (M+) 427.9993, found 427.9991. Bis(5,6,7,8-tetrahydro-4H-cyclohepta[d]thiazol-2-yl)disulfide (1m) 1m was obtained in the same manner described in the preparation of 1f from 2-bromocycloheptanone (5g) as a white solid. Yield: 60% over two steps. M.p. 87–88 ºC. 1

H-NMR (400 MHz, CDCl3): δ 2.98–2.89 (m, 4H), 2.83–2.75 (m, 4H), 1.89–1.81 (m, 4H),

1.77–1.64 (m, 4H); HRMS (EI) m/z calcd. C16H20N2S4 (M+) 368.0509, found 368.0510. Bis(2-methylmorpholin-4-yl-thiocarbonyl)disulfide (2a) A mixture of 2-methylmorpholine (6a, 505 mg, 5 mmol), carbon disulfide (0.6 mL, 10 mmol), potassium hydroxide (616 mg, 11 mmol), and water (50 mL) was stirred at 50 ºC overnight. After cooling to room temperature, the solution was mixed with sodium nitrite (345 mg, 5 mmol) and MeOH (0.3 mL), and concentrated HCl (1 mL) was added dropwise at 0 ºC under vigorous stirring. After 0.5 h, the system was separated between EtOAc (200 mL) and water (100 mL), and the organic layer was washed with brine, dried over anhydrous


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sodium sulfate, and concentrated under reduced pressure. The residue was then purified by column chromatography on silica gel to afford analog 2a as a colorless oil (818 mg). Yield: 93%. 1H-NMR (400 MHz, CDCl3): δ 5.63–4.65 (m, 4H), 4.10–3.92(m, 2H), 3.88–3.65 (m, 4H), 3.62–3.36 (m, 2H), 3.32–2.94 (m, 2H), 1.27 (s, 3H), 1.26(s, 3H); HRMS (EI) m/z calcd. C12H20N2O2S4 (M+) 352.0408, found 352.0405. Bis((2R,6S)-2,6-dimethylmorpholin-4-yl-thiocarbonyl)disulfide (2b) 2b was obtained in the same manner as described in the preparation of 2a from (2S, 6R)-2,6-dimethylmorpholine (6b) as a white solid. Yield: 90%. M.p. 124–125 ºC. [α]D25 = -0.010 (c 1, methanol). 1H-NMR (400 MHz, CDCl3): δ 5.30 (br s, 2H), 4.82 (br s, 2H), 3.77 (br s, 4H), 3.09 (br s, 2H), 294 (br s, 2H); HRMS (EI) m/z calcd. C14H24N2O2S4 (M+) 380.0721, found 380.0723. Bis(2-carboxyl-morpholin-4-yl-thiocarbonyl)disulfide (2c) 2c was obtained in the same manner as described in the preparation of 2a from morpholine-2-carboxylic acid (6c) as a white solid. Yield: 90%. M.p. 112–113 ºC. 1H-NMR (400 MHz, DMSO-d6): δ 13.30 (brs, 2H), 5.14–4.90 (m, 1H), 4.79–4.17 (m, 5H), 4.16–3.73 (m, 6H), 3.73–3.63 (m, 2H); HRMS (ESI) m/z calcd. C12H16N2O6S4Na [M+Na]+ 434.9789, found 434.9784. Bis(2-methoxycarbonylmorpholin-4-yl-thiocarbonyl)disulfide (2d) Concentrated sulfuric acid (0.05 mL) was added to a stirred solution of 2c (412 mg, 1 mmol) in MeOH (50 mL), and the solution was stirred at room temperature for 10 h. The system was separated between EtOAc (200 mL) and water (100 mL), the organic layer was



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washed with water and brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was then purified by column chromatography on silica gel to afford analog 2d as a white solid (380 mg). Yield: 86%. M.p. 150–152 ºC. 1H-NMR (400 MHz, CDCl3): δ 5.50–5.00 (m, 2H), 4.96–4.72 (m, 2H), 4.34 (dd, J = 9.4, 2.9 Hz, 2H), 4.23– 4.11 (m, 2H), 3.93–3.72 (m, 12H); HRMS (EI) m/z calcd. C12H20N2O2S4 (M+) 440.0204, found 440.0205. Bis(3,4-dihydro-2H-benzo[b]morpholin-4-yl-thiocarbonyl)disulfide (2e) 2e was obtained in the same manner as described in the preparation of 2a from 3,4-dihydro-2H-benzo[b]morpholine (6d) as a colorless oil. Yield: 65%. 1H-NMR (400 MHz, DMSO-d6): δ 8.05 (d, J=8.2 Hz, 2H), 7.27 (t, J = 8.0 Hz, 2H), 7.16–7.04 (m, 4H), 4.28 (t, J = 4.8 Hz, 2H), 4.03 (t, J = 4.8 Hz, 2H); HRMS (EI) m/z calcd. C18H16N2O2S4 (M+) 420.0095, found 420.0093. Bis(trans-octahydro-2H-benzo[b][1,4]oxazin-4-yl-thiocarbonyl)disulfide (2f) 2f was obtained in the same manner as described in the preparation of 2a from trans-octahydro-2H-benzo[b][1,4]oxazine (6e) as a light yellow solid. Yield: 70%. M.p. 55– 56 ºC. 1H-NMR (400 MHz, CDCl3): δ 5.27–5.16 (m, 2H), 4.36–4.24 (m, 2H), 4.23–4.15 (m, 2H), 4.10–4.06 (m, 2H), 4.00–3.89 (m, 2H), 3.86–3.76 (m, 2H), 2.88–2.72 (m, 2H), 2.04– 1.98 (m, 2H), 1.88–1.78 (m, 4H), 1.27–1.25 (m, 8H); HRMS (EI) m/z calcd. C18H28N2O2S4 (M+) 432.1034, found 432.1036. Bis(3-hydroxyazetidin-1-yl-thiocarbonyl)disulfide (2g) Carbon disulfide (0.6 mL, 10 mmol) was added to a solution of azetidin-3-ol (6f, 730 mg,


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10 mmol) in THF (50 mL), and the mixture was stirred at room temperature for 1 h before an aqueous solution of ammonium persulfate (2280 mg, 10 mmol in 10 mL water) was added. After 0.5 h, the mixture was separated between EtOAc (200 mL) and water (100 mL). The organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was then purified by column chromatography on silica gel to afford analog 2g as a colorless oil (800 mg). Yield: 54%. 1H-NMR (400 MHz, CDCl3): δ 4.86–4.80 (m, 2H), 4.79–4.71 (m, 2H), 4.64–4.56 (m, 2H), 4.39–4.31 (m, 2H), 4.25–4.18 (m, 2H); HRMS (ESI) m/z calcd. C8H12N2O2S4Na [M+Na]+ 318.9679, found 318.9678. Bis((R)-3- hydroxymethylmorpholin-4-yl-thiocarbonyl)disulfide (2h) (R)-(4-benzylmorpholin-3-yl)methanol (8a, 207 mg, 1 mmol) was dissolved in MeOH (50 mL) in a hydrogenation reactor. Then, 10% Pd/C (50 mg) was added, the reactor was filled with hydrogen gas to a pressure of 1 atm, and the reaction mixture was stirred overnight. The spent catalyst was filtered through Celite, and the filtrate was evaporated to dryness. The residue was stirred with CS2 (114 mg, 1.5 mmol) in THF (10 mL) for 1 h before an aqueous solution of ammonium persulfate (228 mg, 1 mmol in 1 mL water) was added. After 0.5 h, the mixture was separated between EtOAc (100 mL) and water (100 mL), and the organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was then purified by column chromatography on silica gel to afford analog 2h as a colorless oil (158 mg). Yield: 80% over two steps. [α]D25 = -0.201 (c 1, methanol). 1H-NMR (400 MHz, CDCl3): δ 5.69–4.62 (m, 4H), 4.11–3.93 (m, 10H), 3.80– 3.56 (m, 6H); HRMS (ESI) m/z calcd. C12H20N2O4S4Na [M+Na]+ 407.0204, found 407.0197.



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Bis(2-methanolylmorpholin-4-yl-thiocarbonyl)disulfide (2i) 4-Benzyl-2-chloromethylmorpholine (10, 2.25 g, 10 mmol), 10 mL of water and 50 mL of formamide were placed in a 250 mL round-bottomed flask with a condenser. The mixture was heated at 190 ºC overnight. After returning to room temperature, the medium was poured into 100 mL of ice-cold water, and the resulting mixture was basified with 10 M sodium hydroxide to pH=12. Extraction was conducted with 400 mL EtOAc, and the organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was then purified by column chromatography on silica gel to afford 4-benzyl-2-(hydroxymethyl)morpholine (11e) as an amber oil (1.8 g). Yield: 87%. 1H-NMR (400 MHz, CDCl3): δ 7.57–7.22 (m, 5H), 3.94–3.86 (m, 1H), 3.78–3.65 (m, 2H), 3.64–3.46 (m, 4H), 2.76–2.63 (m, 2H), 2.21 (td, J = 11.4, 3.3 Hz, 1H), 2.03 (t, J = 10.8 Hz, 1H). 11e (207 mg, 1 mmol) was dissolved in MeOH (50 mL) in a hydrogenation reactor. Then, 10% Pd/C (50 mg) was added, the reactor was filled with hydrogen gas to a pressure of 1 atm, and the reaction mixture was stirred overnight. The spent catalyst was filtered through Celite, and the filtrate was evaporated to dryness. The residue was stirred with carbon disulfide (0.06 mL, 1 mmol) and potassium hydroxide (61.6 mg, 1.1 mmol) in 10 mL of water at 50 ºC overnight. After cooling to room temperature, the solution was mixed with sodium nitrite (34.5 mg, 0.5 mmol) and MeOH (0.1 mL) before concentrated HCl (0.1 mL) was added dropwise at 0 ºC under vigorous stirring. After 0.5 h, the mixture was separated between EtOAc (100 mL) and water (100 mL), the organic layer was washed with brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure. The residue was


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then purified by column chromatography on silica gel to afford analog 2i as a colorless oil (140 mg). Yield: 73% over two steps. 1H-NMR (400 MHz, CDCl3): δ 5.67–4.42 (m, 4H), 4.10–4.00 (m, 2H), 3.84–3.62 (m, 8H), 3.61–3.30 (m, 4H); HRMS (ESI) m/z calcd. C12H20N2O4S4Na [M+Na]+ 407.0204, found 407.0205. Bis((R)-3-(ethoxymethyl)morpholin-4-yl-thiocarbonyl)disulfide (2j) 2j was obtained in the same manner as described in the preparation of 2h from (R)-4-benzyl-3-(ethoxymethyl)morpholine (8b) as a light yellow oil. Yield: 75%. [α]D25 = -0.185 (c 1, methanol). 1H-NMR (400 MHz, CDCl3): δ 5.74–4.50 (m, 4H), 4.15–4.09 (m, 2H), 4.04–3.94 (m, 2H), 3.93–3.31 (m, 14H), 1.21 (t, J = 6.8 Hz, 6H); HRMS (EI) m/z calcd. C16H28N2O4S4 (M+) 440.0932, found 440.0931. Bis((R)-3-(methoxymethyl)morpholin-4-yl-thiocarbonyl)disulfide (2k) 2k was obtained in the same manner as described in the preparation of 2h from (R)-4-benzyl-3-(methoxymethyl)morpholine (8c) as a light yellow solid. Yield: 75%. M.p. 100–102 ºC. [α]D25 = -0.094 (c 1, methanol). 1H-NMR (400 MHz, CDCl3): δ 5.74–4.50 (m, 4H), 4.17–4.06 (m, 2H), 4.02–4.01 (m, 2H), 3.89–3.52 (m, 10H), 3.43 (s, 6H); HRMS (EI) m/z calcd. C14H24N2O4S4 (M+) 412.0619, found 412.0621. Bis(2-(ethoxymethyl)morpholin-4-yl-thiocarbonyl)disulfide (2l) Sodium hydride (60%, 120 mg, 3 mmol) was added slowly to a stirred solution of (4-benzylmorpholin-2-yl)methanol (11e, 207 mg, 1 mmol) in DMF (10 mL). The resulting mixture was stirred for 20 minutes at room temperature. Subsequently, iodoethane (300 mg, 2 mmol) was added to the resulting suspension, followed by stirring at 80 ºC overnight. The



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mixture was separated between EtOAc (100 mL) and water (100 mL), and the organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was then purified by column chromatography on silica gel to afford 4-benzyl-2-(ethoxymethyl)morpholine (11f) as a yellow oil (141 mg). Yield: 60%. 1H-NMR (400 MHz, CDCl3): δ 7.40–7.29 (m, 5H), 3.92–3.84 (m, 1H), 3.83–3.66 (m, 2H), 3.58–3.35 (m, 6H), 2.82–2.63 (m, 2H), 2.28–2.15 (m, 1H), 2.08–1.93 (m, 1H), 1.19 (t, J = 7.2 Hz, 3H). 2l was obtained in the same manner as described in the preparation of 2i from 11f as a light yellow oil. Yield: 74%. 1H-NMR (400 MHz, CDCl3): δ 5.45–4.66 (m, 4H), 4.09–4.00 (m, 2H), 3.85–3.71 (m, 4H), 3.66–3.25 (m, 12H), 1.23 (t, J = 6.8 Hz, 6H); HRMS (EI) m/z calcd. C14H28N2O4S4 (M+) 440.0932, found 440.0936. Bis(2-(methoxymethyl)morpholin-4-yl-thiocarbonyl)disulfide (2m) 4-benzyl-2-(methoxymethyl)morpholine (11a) was obtained in the same manner as described in the preparation of 8c from 4-benzyl-2-(chloromethyl)morpholine (10). 2m was obtained in the same manner as described in the preparation of 2i from 11a as a light yellow oil. Yield: 68%. 1H-NMR (400 MHz, CDCl3): δ 5.61–4.50 (m, 4H), 4.12–4.01 (m, 2H), 3.85–3.67 (m, 4H), 3.60–3.44 (m, 6H), 3.41 (s, 6H), 3.39–3.14 (m, 2H); HRMS (EI) m/z calcd. C14H24N2O4S4 (M+) 412.0619, found 412.0618. Bis(2-(acetoxymethyl)morpholin-4-yl-thiocarbonyl)disulfide (2n) TEA

(300

mg,

3

mmol)

was

added

to

a

stirred

solution

of

(4-benzylmorpholin-2-yl)methanol (11e, 207 mg, 1 mmol) in DCM (10 mL). Subsequently, acetyl chloride (156 mg, 2 mmol) was added to the solution, and the solution was stirred at


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room temperature overnight. The mixture was separated between EtOAc (50 mL) and cold water (50 mL), and the organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was then purified by column chromatography on silica gel to afford 4-benzyl-2-(acetoxymethyl)morpholine (11g) as a yellow oil (181 mg). Yield: 73%. 1H-NMR (400 MHz, CDCl3): δ 7.37–7.22 (m, 5H), 4.09– 4.05 (m, 2H), 3.94–3.86 (m, 1H), 3.85–3.65 (m, 2H), 3.53 (s, 2H), 2.78–2.62 (m, 2H), 2.28– 2.16 (m, 1H), 2.08 (s, 3H), 2.03–1.93 (m, 1H). 2n was obtained in the same manner as described in the preparation of 2h from 11g as a yellow oil. Yield: 85%. 1H-NMR (400 MHz, CDCl3): δ 3.53–4.79 (m, 4H), 4.15–4.16 (m, 4H), 4.10–4.03 (m, 2H), 3.91–3.83 (m, 2H), 3.82–3.74 (m, 2H), 3.64–3.48 (m, 2H), 3.45– 3.25 (m, 2H), 2.12 (s, 6H); HRMS (EI) m/z calcd. C16H24N2O6S4 (M+) 468.0517, found 468.0519. Bis(2-((dimethylcarbamoyloxy)methyl)morpholin-4-yl-thiocarbonyl)disulfide (2o) 11e (207 mg, 1 mmol) was dissolved in MeOH (50 mL) in a hydrogenation reactor. Then, 10% Pd/C (50 mg) was added, the reactor was filled with hydrogen gas to a pressure of 1 atm, and the reaction mixture was stirred overnight. The spent catalyst was filtered through Celite, and the filtrate was evaporated to dryness. The residue was stirred with di-tert-butyl dicarbonate (240 mg, 1.1 mmol), sodium bicarbonate (250 mg, 3 mmol) in 20 mL of THF and 5 mL of water at room temperature overnight. The mixture was separated between EtOAc (100 mL) and water (50 mL), the organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was then purified by



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column

chromatography

on

silica

gel

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to

afford

tert-butyl

2-(hydroxymethyl)morpholine-4-carboxylate (12) as a colorless oil (173 mg). Yield: 80% over two steps. 1H-NMR (400 MHz, CDCl3): δ 4.02–3.78 (m, 3H), 3.70–3.66 (m, 1H), 3.61– 3.49 (m, 3H), 3.02–2.86 (m, 1H), 2.84–2.68 (m, 1H), 1.47 (s, 9H). TEA (300 mg, 3 mmol) was added to a stirred solution of 12 (217 mg, 1 mmol) in DCM (10 mL). Subsequently, dimethylcarbamic chloride (214 mg, 2 mmol) was added to the solution, and the solution was stirred at room temperature overnight. The mixture was separated between EtOAc (50 mL) and cold water (50 mL), and the organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was then purified by column chromatography on silica gel to afford tert-butyl 2-((dimethylcarbamoyloxy)methyl)morpholin-4-carboxylate (11h) as a white solid (175 mg). Yield: 61%. 1H-NMR (400 MHz, CDCl3): δ 4.19–4.03 (m, 2H), 4.00–3.75 (m, 3H), 3.68– 3.47 (m, 2H), 3.04–2.97 (m, 1H), 2.92 (s, 6H), 2.79–2.63 (m, 1H), 1.47 (s, 9H). 11h (288 mg, 1 mmol) was treated with HCl in dioxane (10 mL, 4 mol/L) for 1 h at room temperature and was then evaporated to dryness. The residue was stirred with CS2 (114 mg, 1.5 mmol) in THF (10 mL) for 1 h before an aqueous solution of ammonium persulfate (228 mg, 1 mmol in 1 mL water) was added. After 0.5 h, the mixture was separated between EtOAc (100 mL) and water (100 mL), and the organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was then purified by column chromatography on silica gel to afford analog 2o as a colorless oil (118 mg). Yield: 45% over two steps. 1H-NMR (400 MHz, CDCl3): δ 5.64–4.60 (m, 4H),


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4.26–4.02 (m, 6H), 3.92–3.82 (m, 2H), 3.79–3.73 (m, 2H), 3.63–3.15 (m, 4H), 2.94 (s, 12H); HRMS (ESI) m/z calcd. C18H30N4O6S4Na [M+Na]+ 549.0946, found 549.0943. Bis(2-(phenoxymethyl)morpholin-4-yl-thiocarbonyl)disulfide (2p) Sodium hydride (60%, 120 mg, 3 mmol) was added slowly to a stirred solution of phenol (188 mg, 2 mmol) in DMF (10 mL). The resulting mixture was stirred for 20 minutes at room temperature. Subsequently, 10 (225 mg, 1 mmol) was added to the resulting suspension, followed by stirring at 120 ºC overnight. The mixture was separated between EtOAc (100 mL) and water (100 mL), and the organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was then

purified

by

column

chromatography

on

silica

gel

to

afford

4-benzyl-2-(phenoxymethyl)morpholine (11b) as a white solid (136 mg). Yield: 48%. 1

H-NMR (400 MHz, CDCl3): δ 7.40–7.28 (m, 7H), 6.97–6.86 (m, 3H), 4.05–3.87 (m, 4H),

3.84–3.69 (m, 1H), 3.58 (s, 2H), 2.98–2.66 (m, 2H), 2.34–2.09 (m, 2H). 2p was obtained in the same manner as described in the preparation of 2i from 11b as a light yellow oil. Yield: 76%. 1H-NMR (400 MHz, CDCl3): δ 7.34–7.26 (m, 5H), 7.00–6.89 (m, 5H), 5.92–4.57 (m, 4H), 4.33–3.22 (m, 14H); HRMS (ESI) m/z calcd. C24H28N2O4S4Na [M+Na]+ 559.0830, found 559.0823. Bis(2-((p-tolyloxy)methyl)morpholin-4-yl-thiocarbonyl)disulfide (2q) 2q was obtained in the same manner as described in the preparation of 2p from 4-benzyl-2-((p-tolyloxy)methyl)morpholine (11c) as a light yellow oil. Yield: 70%. 1H-NMR (400 MHz, CDCl3): δ 7.09–7.07 (m, 4H), 6.84–6.82 (m, 4H), 5.63–4.60 (m, 4H), 4.09–3.92



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(m, 8H) , 3.87–3.74 (m, 2H) , 3.67–3.36 (m, 4H) , 3.29 (s, 6H); HRMS (ESI) m/z calcd. C26H32N2O4S4Na [M+Na]+ 587.1143, found 587.1147. Bis(2-((naphth-1-yloxy)methyl)morpholin-4-yl-thiocarbonyl)disulfide (2r) 2r was obtained in the same manner as described in the preparation of 2p from 4-benzyl-2-((naphth-1-yloxy)methyl)morpholine (11d) as a light yellow solid. Yield: 70%. M.p. 69–70 ºC. 1H-NMR (400 MHz, CDCl3): δ 8.33–8.18 (m, 2H), 7.85–7.75 (m, 2H), 7.57– 7.42 (m, 6H), 7.41–7.32 (m, 2H), 6.89–6.68 (m, 2H), 5.88–4.42 (m, 4H), 4.37–4.11 (m, 8H), 3.89–3.39 (m, 6H); HRMS (ESI) m/z calcd. C32H32N2O4S4Na [M+Na]+ 659.1143, found 659.1141. Bis((S)-3-(methoxymethyl)morpholin-4-yl-thiocarbonyl)disulfide (2s) 2s was obtained in the same manner as described in the preparation of 2h from (S)-4-benzyl-3-(methoxymethyl)morpholine (8c) as a white solid. Yield: 80%. M.p. 100–102 ºC. [α]D25 = +0.117 (c 1, methanol). 1H-NMR (400 MHz, CDCl3): δ 5.74–4.50 (m, 4H), 4.17– 4.06 (m, 2H), 4.02–4.01 (m, 2H), 3.89–3.52 (m, 10H), 3.43 (s, 6H); HRMS (EI) m/z calcd. C14H24N2O4S4 (M+) 412.0619, found 412.0621. Bis(1,4-oxazepan-4-yl-thiocarbonyl)disulfide (3a) 3a was obtained in the same manner as described in the preparation of 2a from 1,4-oxazepane (7) as a white solid. Yield: 87%. M.p. 100–102 ºC. 1H-NMR (400 MHz, CDCl3): δ 4.42–4.26 (m, 7H), 4.15–3.96 (m, 2H), 3.92–3.70 (m, 7H), 2.31–2.21 (m, 2H), 2.18–2.06 (m, 2H); HRMS (EI) m/z calcd. C12H20N2O2S4 (M+) 352.0408, found 352.0405. Bis(2-hydroxymethyl-1,4-oxazepan-4-yl-thiocarbonyl)disulfide (3b)


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(4-benzyl-1,4-oxazepan-2-yl)methanol (14d) was obtained in the same manner as described in the preparation of 11e from 1-benzyl-3-(chloromethyl)azepane (13). 3b was obtained in the same manner as described in the preparation of 2h from 14d as a light yellow oil. Yield: 65%. 1H-NMR (400 MHz, CDCl3): δ 5.08–4.97 (m, 1H), 4.87–4.60 (m, 2H), 4.25–4.12 (m, 3H), 4.07–3.41 (m, 12H) , 2.50–1.90 (m, 4H); HRMS (ESI) m/z calcd. C14H24N2O4S4Na [M+Na]+ 435.0517, found 435.0518. Bis(2-methoxymethyl-1,4-oxazepan-4-yl-thiocarbonyl)disulfide (3c) 4-benzyl-2-(methoxymethyl)-1,4-oxazepane (14a) was obtained in the same manner as described in the preparation of 8c from 13. 3c was obtained in the same manner as described in the preparation of 2i from 14a as a light yellow oil. Yield: 84%. 1H-NMR (400 MHz, CDCl3): δ 5.21–5.10 (m, 1H), 4.92–4.68 (m, 2H), 4.27–4.08 (m, 3H), 3.99–3.45 (m, 12H) , 3.42 (s, 3H) , 3.39 (s, 3H) , 2.51–1.98 (m, 4H); HRMS (EI) m/z calcd. C16H28N2O4S4 (M+) 440.0932, found 440.0930. Bis(2-ethoxymethyl-1,4-oxazepan-4-yl-thiocarbonyl)disulfide (3d) 3d was obtained in the same manner as described in the preparation of 2j from 14e as a light yellow oil. Yield: 83%. 1H-NMR (400 MHz, CDCl3): δ 5.21–5.11 (m, 1H), 4.91–4.67 (m, 2H), 4.27–4.10 (m, 3H), 3.99–3.45 (m, 16H), 2.51–2.03 (m, 4H), 1.26–1.17 (m, 6H); HRMS (EI) m/z calcd. C18H32N2O4S4 (M+) 468.1245, found 468.1246. Bis(2-(n-butoxymethyl)-1,4-oxazepan-4-yl-thiocarbonyl)disulfide (3e) 3e was obtained in the same manner as described in the preparation of 2i from 14f as a light yellow oil. Yield: 83%. 1H-NMR (400 MHz, CDCl3): δ 5.21–5.11 (m, 1H), 4.91–4.68



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(m, 2H), 4.27–4.05 (m, 3H), 4.00–3.74 (m, 3H), 3.66–3.40 (m, 12H), 2.51–1.96 (m, 4H), 1.63–1.56 (m, 4H), 1.42–1.32 (m, 4H), 0.97–0.87 (m, 6H); HRMS (ESI) m/z calcd. C22H40N2O4S4 Na [M+Na]+ 547.1769, found 547.1774. Bis(2-decyloxymethyl-1,4-oxazepan-4-yl-thiocarbonyl)disulfide (3f) 3f was obtained in the same manner as described in the preparation of 2i from 14g as a light yellow oil. Yield: 90%. 1H-NMR (400 MHz, CDCl3): δ 5.21–5.11 (m, 1H), 4.91–4.68 (m, 2H), 4.29–4.03 (m, 3H), 4.02–3.74 (m, 4H), 3.73–3.37 (m, 12H), 2.50–1.96 (m, 4H), 1.65–1.51 (m, 4H), 1.41–1.19 (m, 28H), 0.88 (t, J = 6.5 Hz, 6H); HRMS (ESI) m/z calcd. C34H64N2O4S4Na [M+Na]+ 715.3647, found 715.3645. Bis(2-phenoxymethyl-1,4-oxazepan-4-yl-thiocarbonyl)disulfide (3g) 3g was obtained in the same manner as described in the preparation of 2p from 14b as a yellow solid. Yield: 55%. 1H-NMR (400 MHz, CDCl3): δ 7.33–7.21 (m, 4H), 7.04–6.86 (m, 6H), 5.32–5.21 (m, 1H), 5.06–4.70 (m, 2H), 4.40–4.14 (m, 5H), 4.14–4.12 (m, 4H), 4.02– 3.83 (m, 3H), 3.77–3.54 (m, 3H), 2.54–2.03 (m, 4H); HRMS (ESI) m/z calcd. C26H32N2O4S4Na [M+Na]+ 587.1143, found 587.1135. Bis(2-(p-tolyloxymethyl)-1,4-oxazepan-4-yl-thiocarbonyl)disulfide (3h) 3h was obtained in the same manner as described in the preparation of 2q from 14c as a light yellow oil. Yield: 61%. 1H-NMR (400 MHz, CDCl3): δ 7.15–7.01 (m, 4H), 6.90–6.77 (m, 4H), 5.29–5.20 (m, 1H), 5.05–4.68 (m, 2H), 4.38–4.11 (m, 5H), 4.11–3.90 (m, 5H), 3.90– 3.50 (m, 5H), 2.53–2.00 (m, 4H); HRMS (ESI) m/z calcd. C28H36N2O4S4Na [M+Na]+ 615.1456, found 615.1456.


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Bis(2-((dimethylcarbamoyloxy)methyl)-1,4-oxazepan-4-yl-thiocarbonyl)disulfide (3i) 3i was obtained in the same manner as described in the preparation of 2o from 13 as a light yellow oil. Yield: 66%. 1H-NMR (400 MHz, CDCl3): δ 5.26–5.18 (m, 1H), 4.93–4.65 (m, 2H), 4.34–4.00 (m, 8H), 4.00–3.71 (m, 3H), 3.69–3.39 (m, 4H), 3.01–2.85 (m, 12H), 2.49–1.98 (m, 4H); HRMS (ESI) m/z calcd. C20H34N4O6S4Na [M+Na]+ 577.1259, found 577.1262. Bis(2-((2-hydroxyethoxy)methyl)-1,4-oxazepane-4-yl-thiocarbonyl)disulfide (3j) 3j was obtained in the same manner as described in the preparation of 2h from 14h as a light yellow oil. Yield: 78%. 1H-NMR (400 MHz, CDCl3): δ 5.22–5.11 (m, 1H), 4.90–4.65 (m, 2H), 4.26–4.08 (m, 3H), 4.01–3.40 (m, 22H), 2.51–2.00 (m, 4H); HRMS (ESI) m/z calcd. C18H32N2O6S4Na [M+Na]+ 523.1041, found 523.1041. Biological Procedures. Chemicals and Antibodies DCA was purchased from Sigma-Aldrich. The following antibodies were used: pyruvate dehydrogenase E1-alpha subunit [pSer293] antibody (Novus Biologicals); PhosphoDetect™ anti-PDH-E1α (pSer²³²) rabbit pAb (Calbiochem); GAPDH (Cell Signaling); horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies (Kang Chen Bio-tech, China). Cell Lines A549 cells were obtained from American Type Culture Collection (ATCC). Kelly cells were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). GM00637 cells were gifted by Dr. Yves Pommier (NCI, Bethesda, MD). LO2 cells were



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obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). The cells were authenticated by analyzing the DNA profile of eight short tandem repeat (STR) loci plus amelogenin (Genesky Biotechnologies Inc.). The cells were maintained in appropriate culture media suggested by the supplier. ELISA-based kinase activity assay The 6xHis-tagged full-length coding sequences of PDK1 and PDHA1 were expressed in E. coli and purified with Ni-NTA column following the manufacturer's instructions (Qiagen). The enzymatic reaction was performed with enzyme PDK1 (0.25 µg/well), substrate PDHA1 (0.5 µg/well), and pre-incubated with compound or with GSH (2, 10, 50 µM) for 30 min in 50 µL of reaction buffer (50 mM HEPES, 10 mM MgCl2 and 1 mM EGTA), then adding 50 µL of reaction buffer containing 10 µM of ATP to initiate the reaction and placed in 37°C incubator for 1 hr. After the reaction, the plate was washed with Tween-PBS, followed by incubation with primary antibody (p-PDHE1, S293; Abgent) for 1 h and horseradish peroxidase-conjugated secondary antibody (Calbiochem) for 1 h. The plate was washed and visualized with citrate buffer containing 0.1% H2O2 and 2 mg/mL o-phenylenediamine. The absorbance was measured at 490 nm after termination by H2SO4 (2 M; 50 µL/well). For enzymatic assay of other PDK isoforms, the procedure was similar to PDK1 except using of different amount of enzymes, as PDK2 (0.5 µg), PDK3 (0.2 µg) and PDK4 (0.3 µg). kinact and Ki assay The method for measuring kinact and Ki is according to the classical enzymatic assay described by Singh et al.33 The procedure is similar to the PDKs enzymatic activity assay as


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described above except using various pre-incubation time points (45, 35, 25, 15, 5, 0 min) at each concentration of compounds, then adding ATP to initiate the reaction and placed in 37 °C incubator for 15 min. After the reaction, the plate was washed with Tween-PBS, followed by incubation with primary antibody (p-PDHE1, S293; Abgent) for 1 h and horseradish peroxidase-conjugated secondary antibody (Calbiochem) for 1 h. The plate was washed and visualized with citrate buffer containing 0.1% H2O2 and 2 mg/mL o-phenylenediamine. The absorbance was measured at 490 nm after termination by H2SO4 (2 M; 50 µL/well). Following least-squares regression analysis to determine the negative slope of the logarithm of the % remaining activity (corrected for negative control) versus pre-incubation time at each concentration of compound, non-linear regression analysis of the negative slopes against compound concentration enables kinact and Ki to be calculated. Immunoblotting Analysis Cells were lysed using preheated 2% SDS by vortexing vigorously for 2–3 sec followed by boiling for 30 min. The protein content was measured using a BCA protein assay kit (Beyotime). Blocking was performed for 1 h with 5% non-fat milk in Tris-buffered saline Tween 20 (TBST) (25 mM Tris, 150 mM NaCl, 2 mM KCl, pH 7.4, supplemented with 0.1% Tween 20), and blotting was performed with primary antibodies (1:2000) at 4 °C overnight. After washing the membranes with TBST three times for 10 min, horseradish peroxidase-conjugated anti-rabbit IgG (1:2000) or anti-mouse IgG (1:2000) antibodies were incubated at room temperature for 1 hr. The membranes were washed for 10 min with TBST for three times before visualization using an enhanced chemiluminescence assay (Thermo



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Scientific) or femtochemiluminescence assay (Thermo Scientific). Extracellular Acidification Rate (ECAR) Analysis A Seahorse XF96 extracellular flux analyzer (Seahorse Bioscience, North Billerica, MA) was used to assess the extracellular acidification rate (ECAR). Cells were plated, and each XF96 assay well was equipped with a disposable sensor cartridge and embedded with 96 pairs of fluorescent biosensors (pH) coupled to fiber-optic waveguides. The ECAR measurement was expressed in mpH/min. Reactive Oxygen Species (ROS) Measurement After treatment for 24 hr, cells were harvested and washed with 1 × PBS, followed by incubation with 3 mM dihydroethidium (Invitrogen) in the dark for at 37 °C for 20 min. The cells were then washed in 1 × PBS and resuspended in regular DMEM. Intracellular ROS was measured by FACS analysis (FACSCalibur flow cytometer; BD Biosciences). Cell Proliferation Assay Cells were seeded into 96-well plates, and cell proliferation was assessed using the CCK8 assay (Life Technologies) after incubation for 72 hr. The absorbance (optical density, OD) was read at 450 nm on an ELISA plate reader. The IC50 values were calculated by concentration-response curve fitting using the four-parameter method. Animal studies All procedures were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and European Union directives and guidelines and were approved by the local ethics committees. Four to six weeks-old female


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BALB/c-nude mice were inoculated subcutaneously with 5×106 A549 cells which mixed with equal volumes of Matrigel Matrix (Cat: 356237, Corning) in right forearm and treatment was started when tumor volume reached around 100 mm3. Mice grouped into six mice per group were intraperitoneally treated with saline or 16, 2k, 3a at a dose of 80 or 40 mg/kg daily for 21 days. Tumor growth was monitored by measurement of tumor size slide caliper twice a week. Mice were sacrificed and analyzed after last dosing. Percent tumor growth inhibition (% TGI) was calculated based on the ratio of the change in average tumor volumes for inhibitors vs vehicle treatment: TGI = 100(1 - [{Vday21 - Vday0}dose/{Vday21- Vday0}vehicle]). Molecular docking Missing residues of PDK1 (PDB entry: 2Q8F,34 residue 68-71, 168-169, 204-214, 415-416), PDK2 (PDB entry: 2BTZ,35 residue 165-172, 300-313), PDK3(PDB entry: 1Y8N,36 residue 317-319, 322-323) and PDK4 (PDB entry: 2E0A,26 residue 46-48, 318-323) were complemented using “Build Homology Models” Module of Discovery Studio 2.5 (Accelrys Software Inc., San Diego, CA). Then Autodock 4.0 software was used for molecular docking. The binding sites were defined as a sphere with a radius of 10 Å around the sulphur atom of PDK1 Cys240, PDK2 Cys148, PDK3 Cys208, and PDK4 Cys215 respectively. The grid parameter files (gpf) and the docking parameter files (dpf) were generated by AutoDock Tools (ADT). The Lamarckian genetic algorithm (LGA) was employed to explore the optimal chemical space in the binding site.37 Docking parameters were kept the default values. ASSOCIATED CONTENT



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Supporting Information The HPLC reports for the purity check of analogs 1a–m, 2a–s, and 3a–j, and the source and chemical data of intermediates 4a–k, 5a–g, 6a–f, 7, 8a–c, 9–10, 11a, 11c–d, 13, 14a–i, and 15. This material is available free of charge via the Internet at http://pubs.acs.org. . AUTHOR INFORMATION Corresponding Author * For J.L.: phone, +86-21-64252584; fax, +86-21-64252584; E-mail, [email protected]; For M.H.: phone, +86-21-50806600-2431; E-mail, [email protected]. Author Contributions †

These authors contributed equally to this work.

Notes The authors declare no competing financial interest. ACKNOWLEDGMENT Financial support of this research provided by the National Natural Science Foundation of China (Grants 21672064, 21372001, 81573464), the “Shu Guang” project supported by the Shanghai Municipal Education Commission and the Shanghai Education Development Foundation (Grant 14SG28), and the Fundamental Research Funds for the Central Universities is gratefully acknowledged. ABBREVIATIONS


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PDC, pyruvate dehydrogenase complex; PDK, pyruvate dehydrogenase kinase; HIF-1α, hypoxia-inducible factor-1α; DCA, dichloroacetate; GHKL, gyrase, Hsp90, histidine kinase, MutL; ECAR, extracellular acidification rate; OCR, oxygen consumption rate; SAR, structure-activity

relationship;

ELISA,

enzyme-linked

immunosorbent

assay;

IC50,

half-maximal inhibitory concentration; GSH, glutathione; A549, human lung cancer cells; Kelly, human neural cancer cells; GM00637, human skin fibroblast cells; LO2, human normal live cells; TGI, tumor growth inhibition; TLC, thin-layer chromatography; HPLC, high-performance liquid chromatography; NMR, nuclear magnetic resonance; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; EtOH, ethanol; EtOAc, ethyl acetate; MeOH, methanol; THF, tetrahydrofuran; DCM, dichloromethane; TEA, triethylamine.



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FIGURE Figure 1. Structures of the PDK1 inhibitors reported in the literature. reported by other groups: O

O

O S

Cl

N

OH

Cl

OH N

O

(R)

N

N

N H

CF3 O

O

DCA, Phase II

Cl

O

CF3

(R)

OH

AZD7545

Nov3r

O O OH O

OH S

S

O

Cl CPI613, Phase I HO O N S O

HO

O H

HO

HO H

N NH OH

O Radicicol

M77976

OH

OH PS10 reported by our group: S N

S S

N

S S

Thiram

N O

O S

S

N S

16 (JX06)


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Figure 2. Scaffold hopping (1) and three chemical modification strategies (2–3) of lead compound 16.



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Figure 3. Representative inhibitors characterization based on kinetic properties with PDK1: quadrant I, low affinity and high reactivity; quadrant II, high affinity and high reactivity; quandrant III, high affinity and moderate reactivity; quadrant IV, weak affinity and moderate to low reactivity.


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Figure 4. Cellular potencies of representative analogs on PDK1 inhibition evaluated by downstream PDHE1 phosphorylation in A549 cells. (A) Screening of analogs at 10 µM. (B) Screening of analogs at 1 µM and 0.3 µM. (C) Analogs 2k, 3a and 16 inhibited PDHE1 phosphorylation in a dose-dependent manner, and the comparison was determined by semi-quantitative analysis of the Gray-Scale. All cells treated for 24 h with the analogs at the indicated concentrations were lysed and subjected to immunoblotting analysis. Error bars represent the average ± S.D. of two independent experiments.



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Figure 5. Inhibition curves of analogs 2k (A) and 3a (B) toward PDK1 at different concentrations of GSH.


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Figure 6. Molecular docking of 16, 2k and 3a with PDK1–4. (A–C) the binding pocket of 3a (white sticks) in PDK1–3 (green surface) and the detailed interactions between inhibitors and their surrounding residues (green sticks) in PDK1–3. (D) No obvious binding pockets for 16, 2k, 3a in PDK4. A

B

C

D



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Figure 7. Metabolic alteration caused by 2k, 3a and 16 on A549 cells. (A) Effect on the extracellular acidification rate (ECAR) in a dose-dependent manner. (B) Effect on the reactive oxygen species (ROS) in a dose-dependent manner. Cells were treated with compounds at the indicated concentrations for 24 hr. Error bars represent the average ± S.D. of two independent experiments.


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Figure 8. 2k, 3a and 16 inhibit tumor growth in vivo. A549 xenograft mouse models were treated with inhibitors or vehicle control daily at indicated dosages for 21 consecutive days. (A)–(C) the tumor growth inhibition values (TGI) were measured on the final day of the study; (D) tumor weight after last dosing; (E) body weight change of 16 and 3a. One-way ANOVA analysis, *, P < 0.05.



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SCHEMES Scheme 1. Syntheses of Analogs 1b–ma

a

Reagents and conditions: (a) (NH4)2S2O8, H2O, THF, rt; (b) HNO3, H2SO4, 0 ºC to rt; (c)

ammonium dithiocarbamate, EtOH, reflux, 1 h; (d) LiOH, H2O, THF, rt, 1 h.


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Scheme 2. Syntheses of Analogs 2a–g and 3aa

a

Reagents and conditions: (a) (1) CS2, KOH, H2O, 50 ºC, overnight; (2) NaNO2, H2O, MeOH,

HCl, 0 ºC; (b) (1) CS2, THF, rt, 1 h; (2) (NH4)2S2O8, H2O, THF, rt, 0.5 h; (c) H2SO4, MeOH, rt, overnight.



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Scheme 3. Syntheses of Analogs 2h, 2j, 2k and 2sa

a

Reagents and conditions: (a) iodoethane, NaH, DMF, 80 ºC, overnight; (b) SOCl2, DCM, rt,

2 h; (c) MeONa, NaI, MeOH, reflux, overnight; (d) H2, Pd/C, MeOH, rt, overnight; (e) (1) CS2, THF, rt, 1 h; (2) (NH4)2S2O8, H2O, THF, rt, 0.5 h.


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Scheme 4. Syntheses of Analogs 2i and 2l–ra R4 O

O a

Bn N

b Cl

O 10

4

11e, R =H

O 12

4

11f, R =Et d

4

11g, R =Ac e

j OH

S

N

O

R4

S

2p, R4=Ph

2q, R4=p-MePh

2r, R4=naphth-1-yl

2i, R4=H

2l, R4=Et

2n, R4=Ac

Boc N

Boc N

S

f, g (11g: f, h)

4

N

2m, R4=Me 4

f, i

a

R4

11b, R =Ph 11c, R =p-MePh 11d R =naphth-1-yl

c

O O

11a, R4=Me 4

O

S

Bn N

k, h O

O

N

O

2o, R4=

N

O 11h

Reagents and conditions: (a) MeONa, NaI, MeOH, reflux overnight; (b) R4OH, NaH, DMF,

120 ºC, overnight; (c) formamide, H2O, 190 ºC, overnight; (d) iodoethane, NaH, DMF, 80 ºC, overnight; (e) acetyl chloride, TEA, DCM, rt, overnight; (f) H2, Pd/C, MeOH, rt, overnight; (g) (1) CS2, KOH, H2O, 50 ºC, overnight; (2) NaNO2, H2O, MeOH, HCl, 0 ºC; (h) (1) CS2, THF, rt, 1 h; (2) (NH4)2S2O8, H2O, THF, rt, 0.5 h; (i) (Boc)2O, NaHCO3, THF, H2O, rt, overnight; (j) dimethylcarbamic chloride, TEA, DCM, rt, overnight; (k) HCl, dioxane, rt, 1 h.



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Scheme 5. Syntheses of Analogs 3b–ja

a

Reagents and conditions: (a) MeONa, NaI, MeOH, reflux overnight; (b) R5OH, NaH, DMF,

120 ºC, overnight; (c) formamide, H2O, 190 ºC, overnight; (d) R5-I, NaH, DMF, 80 ºC, overnight; (e) H2, Pd/C, MeOH, rt, overnight; (f) (1) CS2, KOH, H2O, 50 ºC, overnight; (2) NaNO2, H2O, MeOH, HCl, 0 ºC; (g) (1) CS2, THF, rt, 1 h; (2) (NH4)2S2O8, H2O, THF, rt, 0.5 h; (h) (Boc)2O, NaHCO3,THF, H2O, rt, overnight; (i) dimethylcarbamic chloride, TEA, DCM, rt, overnight; (j) HCl, dioxane, rt, 1 h.


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TABLES Table 1. Chemical structures of analogs 1a–m and their PDK1 inhibitory activities in the ELISA assay a

PDK1

PDK1 Compd.

Compd.

Core A

Core A

IC50 (µM)

a

IC50 (µM)

1a

0.326±0.140

1h

0.090±0.026

1b

0.379±0.279

1i

0.119±0.02

1c

0.161±0.051

1j

0.218±0.026

1d

0.109±0.014

1k

0.109±0.036

1e

0.476±0.103

1l

0.130±0.059

1f

0.169±0.053

1m

0.039±0.012

1g

0.139±0.023

16

0.049±0.003

The IC50 was calculated from two independent experimental measurements. The values are reported as the average ± S.D.



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Table 2. Chemical structures of analogs 2a–s and 3a–j and their PDK1 inhibitory activities in the ELISA assay a S R

S

S

R S

PDK1 Compd.

R Group

PDK1 Compd.

IC50 (µM)

R Group IC50 (µM)

2a

0.096±0.021

2p

0.247±0.047

2b

0.032±0.001

2q

2.414±0.777

2c

0.126±0.015

2r

3.875±0.483

2d

0.085±0.017

2s

0.030±0.007


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2e

1.048±0.349

3a

0.026±0.001

2f

0.289±0.011

3b

2.854±0.379

2g

0.395±0.105

3c

0.350±0.117

2h

2.437±0.032

3d

0.622±0.071

2i

0.702±0.192

3e

0.588±0.146

2j

0.072±0.006

3f

>10

2k

0.024±0.003

3g

0.609±0.202

2l

0.101±0.033

3h

>10



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

a

Page 66 of 70

2m

0.090±0.022

3i

1.459±0.668

2n

0.113±0.035

3j

6.832±1.190

2o

1.701±0.479

The IC50 was calculated from two independent experimental measurements. The values are reported as the average ± S.D.


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Table 3. kinact/Ki values for representative analogs a

a

Compd.

kinact(10-4s-1)

Ki(10-7M)

kinact/Ki(103M-1s-1)

1a

56.47±3.47

37.31±3.30

1.53±0.21

1m

69.88±5.46

18.03±1.65

3.93±0.63

2e

2.10±0.12

33.45±1.15

0.06±0.01

2g

4.74±0.32

8.72±0.67

0.54±0.01

2h

3.24±0.26

40.28±6.94

0.07±0.01

2k

4.43±0.23

1.10±0.20

4.17±0.94

2r

6.90±0.10

225.73±25.04

0.03±0.01

2s

4.95±0.21

1.2±0.29

4.06±0.90

3a

5.47±0.41

1.09±0.11

5.14±0.68

16

8.75±0.21

4.62±0.50

1.94±0.27

The experiments were carried out in triplicates. The values are reported as the average ±

S.D.



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Page 68 of 70

Table 4. PDK1–4 inhibitory activities of analogs 2k, 3a, 16 and DCA a PDK1 Compd.

PDK3

PDK4

kinact

Ki

kinact/Ki

kinact

Ki

kinact/Ki

kinact

Ki

kinact/Ki

kinact

Ki

kinact/Ki

(10-4s-1)

(10-7M)

(103M-1s-1)

(10-4s-1)

(10-7M)

(103M-1s-1)

(10-4s-1)

(10-7M)

(103M-1s-1)

(10-4s-1)

(10-7M)

(103M-1s-1)

2k

4.43±0.23

1.10±0.20

4.17±0.94

2.07±0.7

7.02±0.05

0.30±0.01

17.64±1.26

29.87±0.93

0.59±0.03

No inhibition at 10 µM

3a

5.47±0.41

1.09±0.11

5.14±0.68

1.56±0.06

12.78±2.06

0.12±0.02

8.58±0.58

77.62±3.50

0.11±0.02


16

8.75±0.21

4.62±0.50

1.94±0.27

0.99±0.14

1.76±0.02

0.56±0.07

12.80±1.12

28.51±1.61

0.45±0.06


DCA a

PDK2

IC50=1025±19 µM

IC50=504±205 µM

IC50=5560±540 µM

The data was calculated from two independent experimental measurements. The values are reported as the average ± S.D.


IC50=727±251 µM

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Table 5. The antiproliferative effects of analogs 2k, 3a, and 16 on different cell lines a IC50 (µM) Cell

a

16

2k

3a

DCA

A549

0.480±0.014

0.957±0.009

0.225±0.045

14827±5333

Kelly

0.289±0.017

1.426±0.010

0.057±0.002

11095±28

GM00637

>10

>10

>10

13812±4587

LO2

>10

>10

>10

>15000

The experiments were carried out in triplicates by CCK8 assay after incubation for 72 hours. The IC50 values are reported as the average ± S.D.



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Table of Contents graphic 206x64mm (220 x 220 DPI)


Page 70 of 70

Development of the First Generation of Disulfide-Based Subtype

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