Synthesis, Biological Evaluation and Molecular Docking Studies of Piperidinylpiperidines and Spirochromanones Possessing Quinoline Moieties as Acetyl-CoA Carboxylase Inhibitors

Acetyl-coenzyme A carboxylases (ACCs) play critical roles in the regulation of fatty acid metabolism and have been targeted for the development of drugs against obesity, diabetes and other metabolic diseases. Two series of compounds possessing quinoline moieties were designed, synthesized and evaluated for their potential to inhibit acetyl-CoA carboxylases. Most compounds showed moderate to good ACC inhibitory activities and compound 7a possessed the most potent biological activities against ACC1 and ACC2, with IC50 values of 189 nM and 172 nM, respectively, comparable to the positive control. Docking simulation was performed to position compound 7a into the active site of ACC to determine a probable binding model.


Introduction
Obesity is a chronic disease related to many other metabolic diseases such as type 2 diabetes mellitus (T2DM) and dyslipidemia [1,2]. Acetyl-coenzyme A carboxylases (ACCs) are key enzymes that catalyze the formation of malonyl coenzyme A (CoA), which contributes to the regulation of fatty acid biosynthesis and oxidation in humans and most other living organisms [3,4]. There are two isoforms of ACC in mammals identified as ACC1 and ACC2. ACC1 is a cytosolic enzyme and primarily expressed in lipogenic tissues responsible for the committed step in fatty acid biosynthesis. ACC2 is a mitochondrial enzyme that is highly expressed in oxidative tissues associated with mitochondrial fatty acid oxidation [2]. Thus, inhibition of the ACCs gives the potential for managing both long chain fatty acid biosynthesis and fatty acid oxidation, which has been regarded as a novel approach to treat obesity type 2 diabetes and other diseases associated with metabolic syndrome [5].
Wakil and coworkers [2] have reported that the ACC2-deficient mice exhibited increased fatty acid oxidation and reduced fat accumulation in skeletal muscle and heart tissue when placed on a high fat/high calorie diet. In contrast, liver ACC1-deficient mice showed a significant reduction in fatty acid synthesis as compared to wild type mice [6]. In the past few years, several classes of small molecules that inhibit both isoforms of mammalian ACC have been reported, such as spirochromanones [7], piperidinylpiperidines [8] and benzthiazolylamides [9], examples of which are shown in Figure 1. Among them is compound CP-640186 (I, Figure 1) which is the first well documented example from Pfizer's drug discovery program targeting ACCs with IC50 values of about 50 nM against ACC1 and ACC2 from mammalia [8,10]. In a subsequent publication, Pfizer described a series of spirochromanone ACC inhibitors (II, Figure 1) and discussed efforts to improve the properties [7]. Further work by Takeda focused on improving the compound of CP-640186 by replacing the anthracene ring and morpholine amide resulting in a benzthiazolylamides compound (III, Figure 1) [9]. Moreover, Pfizer is currently pursuing the clinical development of their clinical candidate PF-05175157 (structure not disclosed) targeting ACC in phase 2 [11]. It has been demonstrated that a spirochromanone and CP-640186 bound to the same active site at the interface of a dimer of ACC carboxyl transfer (CT) domain [12][13][14]. The shared binding pattern reveals that these two classes of inhibitors both interact with conserved residues Gly-2162 (yeast Gly-1958) and Glu-2230 (yeast Glu-2026). The crystal structure of the complex of ACC CT with CP-640186 indicates that the side chains of Glu-2230, Glu-2232, Ala-1964 and Lys-1967 are located near the anthracene ring ( Figure 2) [14]. We propose that the conversion of the anthracene ring into a heterocyclic ring may enhance the interaction of ligand and the binding site, resulting in increased ACC inhibitory activities. Designs based on the anthracene regions and piperidinylpiperidine core are shown in Figure 3. To increase the structural diversity and discover more potent inhibitors of ACC, we describe herein the synthesis of novel piperidinylpiperidines and spirochromanones bearing a quinoline moiety and evaluated their enzyme inhibitory activities against ACCs. Additionally, molecular docking was carried out to investigate the possible binding mode of representative compounds using the Sybyl software.

Scheme 2. Synthesis of spirochromanones.
As shown in Scheme 2, the spirochromanones 12a-12g were synthesized via a three-step procedure.
The structures of all the synthesized piperidinylpiperidines 7 and spirochromanones 12 have been confirmed by NMR, IR and MS, and all the corresponding data can be found in the Experimental section.

Biological Activities
The in vitro inhibitory activities of compounds 7a-7i and 12a-12g against both ACC1 and ACC2 are listed in Table 1. Among the tested compounds, the selected potent compounds (ACC IC50 < 1000 nM) were further evaluated for their cytotoxicity against human embryonic lung fibroblast (HELF) cells using the 3-[4,5-dimethylthylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) method [15]. For the convenience of structure-activity relationship analysis, compounds 7a-7i and 12a-12g were defined as piperidinylpiperidine derivatives and spirochromanone derivatives, respectively. As shown in Table 1, we can conclude that most of the compounds showed moderate to good activities against ACC1 and ACC2 and low toxic effects against normal human embryonic lung fibroblasts. For example, 7a-7g, 12a, 12b and 12d showed promising ACC2 inhibitory activities with IC50 values ranging from 172 nM to 940 nM and low cytotoxic activities (>100 μM). 7a-7e, 7g, 12a and 12b showed promising ACC1 inhibitory activity with IC50 values below 1000 nM. Moreover, 7a-7g and 12d displayed more potent ACC2 inhibitory activity compared to their anti-ACC1 activity and exhibited a relative selectivity. It is worth mentioning that the most active compound, the piperidinylpiperidine derivative 7a displayed comparable inhibitory activity against ACC1/2 as the parent compound CP-640186. The octanol/water partition coefficients (miLogP) and drug-likeness model scores were also computed for all the compounds using the online molinspiration LogP calculation program and molsoft software, respectively. All the synthesized compounds showed moderate to good drug-likeness score ranging from 0.41 to 1.61, which is higher than CP-640186 (0.27). The logP values of most of the test compounds range from 3.18 to 5.0 within the acceptable criteria.

Molecular Docking Study
The ACCase is a multienzyme complex composed of three function domains: biotin carboxylase domain (BC), biotin carboxyl carrier protein domain (BCCP) and carboxyltransferase domain (CT) [18].
The CT domain catalyzes the transfer of an activated carboxyl group to acetyl CoA to form malonyl CoA. The crystal structures of the CT domain in complex with CP-640186 [12], coenzyme A [19], and pinoxaden [20] have been reported, respectively. The cocrystal structures of CP-640186 reveal that the active site of the CT domain resides at the dimeric interface of the N and C domains. The binding pattern suggests that the anthracenyl moiety is sandwiched between helix α6 in the N domain and helix α6′ in the C domain near the dimer interface; the carbonyl oxygen atom adjacent to the anthracene is hydrogen bonded to Glu-2026 and the carbonyl oxygen atom adjacent to the morpholine is hydrogen bonded to Gly-1958. In order to investigate the interactions of these two series of compounds with ACC, a molecular docking study was performed by docking of several representative compounds into the active site of CT domain, using the Sybyl 7.1 program package (Tripos International, St. Louis, MO, USA). The three-dimensional structure of CT domain of human ACC was taken from the Protein Data Bank (PDB ID: 3FF6) [14]. The synthesized compounds were energy minimized and docked into the active site, which formed by residues Ala-1964, Lys-1967, Val-1968, Leu-1965, Gly-2162, Ser-2161, Phe-2160, Arg-2158, Glu-2230, Glu-2232, Glu-2236 and Ile-2237. The clustering of docked compounds 7a, 7g, 7h, 7i, 12a, 12e and CP-640186 is shown in Figure 4. These compounds exhibited similar conformations and binding modes as CP-640186. The best inhibitor 7a with a higher docking score (8.01) in comparison to CP-640186 (7.18) and binds at the same site as the ligand. Two hydrogen bonds were observed between the carbonyl groups with residues Glu-2230 and Gly-2162 ( Figure 4, right), respectively. The quinoline moiety is surrounded by the side chain of Ala-1964, Lys-1967, Val-1968, Gly-2233, Glu-2236 and Ile-2237. More importantly, the oxygen atom associated with 2-phenylquinoline moiety interacts with the residue Lys-1967 by an additional hydrogen bond. We speculate that the enhanced activity of 7a is partly due to the additional hydrogen bond with Lys-1967.

General Information
All solvents were dried according to standard methods before use. Melting points were measured on a Buchi B-545 melting point apparatus and are uncorrected. IR spectra were determined using KBr disks with a PE-983 infrared spectrometer and only major absorptions are listed. NMR spectra were recorded on a Mercury-Plus 400 spectrometer in CDCl3 using TMS as an internal reference. LC-MS data were obtained on an API 2000 liquid chromatography-tandem mass spectrometer. Unless otherwise noted, all materials were commercially available and were used directly without further purification. To a solution of piperidin-4-one hydrochloride (2, 3.24 g, 24 mmol) and triethylamine (4.86 g, 48 mmol) in DMF (40 mL) was added substituted quinoline-4-carboxylic acid (1, 20 mmol) followed by HATU (9.12 g, 24 mmol). The reaction mixture was stirred at room temperature for 6 h and then poured into ice-cold water (50 mL). The precipitate was filtered and dried to give the corresponding substituted 1-(quinoline-4-carbonyl)piperidin-4-one 3. This product was used directly in the next reaction step without further purification.

Molecular Docking
The molecular docking was performed and analyzed with the Sybyl 7.1 program package [21]. The X-ray structure of CT domain bounded with CP-640186 was taken from the Protein Data Bank (PDB ID: 3FF6). All water molecules and ligands were removed from the original enzyme structure. For docked ligands, non-polar hydrogens were added and Gasteiger-Huckl charges were assigned. To validate our docking procedure, the co-crystallized ligand was redocked into the active site of the hACC2 and the reasonable RMSD value of 1.9620 Å was obtained. Dock scores were evaluated by Consensus Score (CScore), which integrates a number of popular scoring functions for ranking the affinity of ligands bound to the active site of a receptor.

ACC1 and ACC2 in Vitro Assay
To investigate the biological activities of the synthesized compounds, the in vitro inhibitory activities of these compounds against both rat ACC1 and ACC2 were evaluated according to the previous experimental method with some changes [8]. The ACC1 and ACC2 enzymes were supplied by Huawei Pharmaceutical Co. Ltd (Shanghai, China). The compound CP-640186 was supplied by Selleck Chemicals Co. Ltd. (Houston, TX, USA) Briefly, the test compounds were diluted in 10% DMSO at 10 μM. Then, the solution was diluted with buffer solution until it reached the required concentration (1 nM to 10 μM) and 5μL of the dilution was added to a 50 μL reaction mixture containing 30 mM HEPES, pH 7.4, 2 mM MgCl2, 2 mM sodium citrate, 1 mM DTT, 25 μM ATP, 16 mM NaHCO3 and 15 μM acetyl-CoA. All of the enzymatic reactions were conducted at 30 °C for 60 min. The assay was performed using Kinase-Glo Plus luminescence kinase assay kit (Beijing, China), which measures kinase activity by quantifying the amount of ATP remaining in solution following a kinase reaction. The luminescent signal from the assay is correlated with the amount of ATP present and is correlated with the amount of kinase activity. The same concentrations of DMSO were tested in parallel and used as background control. The inhibition percentage was expressed as the average value from three independent experiments. CP-640186, an ACC1/ACC2 dual inhibitor developed by Pfizer, was used as a control.

In Vitro Cytotoxic Activities
In the cytotoxicity experiment, tetrazolium-based colorimetric assay (MTT test) was used to measure the cytotoxic effect of these selected potent compounds on normal cells [15,22]. Human embryonic lung fibroblast cell line (HELF, WI-38) was purchased from the Cell Resource Center, Chinese Academy of Medical Sciences (Beijing, China). Briefly, cells (5000 per well) were collected and seeded in 96-well plates. After incubation for 24 h, cells were exposed to fresh medium containing various concentrations of test compound at 37 °C. After incubation for 48 h, 20 μL of MTT tetrazolium salt dissolved in phosphate buffered saline (PBS) at a concentration of 5 mg/mL was added to each well and incubated in a CO2 incubator for 4 h. The medium was then aspirated from each well and 150 μL of DMSO was added to dissolve formazan crystals. The absorbance of each well was measured by ELx808 Microplate Reader (BioTek, Winooski, VT, USA) at a test wavelength of 490 nm. TC50, the drug concentration which reduces the cell viability by 50% was calculated by the Logit method.

Conclusions
In conclusion, we have designed and synthesized two series of novel ACC inhibitors bearing a quinoline moiety. The molecular docking study revealed that compounds from these two series possessed similar conformations and binding modes as CP-640186 in the CT domain of human ACC. The preliminary in vitro assay indicated that most of the synthesized compounds possessed considerable ACC1/2 inhibitory activities. Moreover, compound 7a displayed comparable inhibitory activity against ACC2 as the parent compound CP-640186, which may partly due to the additional hydrogen bond with the side chain amide of Lys-1967. Further studies in animal models are in progress in our laboratory.