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Synthesis, biological activities, and docking studies of d -pantolactone derivatives as novel FAS inhibitors
Bioorganic & Medicinal Chemistry 27 (2019) 115069
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Synthesis, biological activities, and docking studies of D-pantolactone derivatives as novel FAS inhibitors
T
Hua Fanga,b,c, Jianlin Hea,c, Tan Rana,c, Hui Chena,b,c, Wenhui Jina,b,c, Bowen Tangd, ⁎ ⁎ Zhuan Honga,b,c, , Meijuan Fangd, a
Third Institute of Oceanography, Ministry of Natural Resources, Xiamen 361005, China Xiamen Ocean Vocational College, Xiamen 361005, China c Fujian Collaborative Innovation Center for Exploitation and Utilization of Marine Biological Resources, Xiamen 361005, China d School of Pharmaceutical Sciences, Xiamen University, Xiamen, Fujian 361005, China b
ARTICLE INFO
ABSTRACT
Keywords: D-pantolactone derivatives Fatty acid synthase (FAS) Lipid accumulation Molecular docking
A novel series of fatty acid synthase (FAS) inhibitors with D-(−)-pantolactone moiety and potential utility for the treatment of obesity were designed, synthesized and characterized, in which the structure of compound 3k was further confirmed by single X-ray diffraction. The mouse FAS inhibitory activity of synthesized compounds was evaluated. Major synthesized compounds (except 3g, 3i, 3k, 3l, and 3n) exhibited moderate FAS inhibitory properties with IC50 values in the range of 13.68 ± 1.52–33.19 ± 1.39 μM, reference inhibitor C75 has IC50 value of 13.86 ± 2.79 μM. Eight compounds (3c, 3d, 3e, 3f, 3j, 3m, 3q and 3r) also displayed inhibitory effect on lipid accumulation in human HepG2 cells. Additionally, the molecular docking study revealed that compound 3m having good inhibition activity against FAS and lipid accumulation also showed promising binding affinities with hFAS, while its binding model with hFAS (PDB ID: 4PIV) was different from that of reference compound C75.
1. Introduction Obesity and overweight, resulting from excessive adiposity is a serious public health problem worldwide with imminent clinical complications and economic burden. Resulting from a chronic imbalance between energy intake and energy expenditure, obesity is frequently associated with other diseases, such as diabetes and hypertension.1–3 On the other hand, Fatty acid synthase (E.C. 2.3.1.85; FAS) is a multifunctional enzyme system that catalyzes the formation of fatty acids from acetyl-CoA, malonyl-CoA, and NADPH, and plays a central role in lipid biosynthesis. FAS is a 270 kD cytosolic enzyme responsible for the conversion of dietary carbohydrates to triacylglycerols (TG) when energy is surfeit. Studies have recently associated FAS with a variety of human diseases and adverse health conditions including obesity, inflammation, cardiovascular disease, and especially cancer.4–8 Thus, FAS has been thought to be a potential target not only for cancer therapy but also for treating the obesity and diabetes. FAS is an attractive emerging target for anti-obesity drug discovery and pharmacological treatment may be required for the control of obesity in many patients. Currently, most of the identified lipid synthesis inhibitors are the inhibitors of fatty acid synthase. Cerulenin, a ⁎
natural metabolite product of the fungus Cephalosporium ceruleans, is the first identified FAS inhibitor.9 The clinical relevance of cerulenin, however, is limited due to the chemical instability.10 C75, a ceruleninderived semi-synthetic FAS inhibitor which binds irreversibly to KS (βketoacyl synthase), ER (enoyl reductase), and TE (thioesterase) as a competitive inhibitor, has been used as a leading compound for discovering the role of FAS in obesity and cancer. It can reduce food consumption and increased fatty acid oxidation in diet-induced obese mice.11,12 D-(−)-pantolactone, a similar five-membered lactone structure with C75 was also suggested to inhibit fatty acid synthase (Fig. 1). Owing to widespread interest in FAS and its relevance in a variety of diseases, it remains a strong need for potent and specific inhibitors of FAS with good develop ability properties that are suitable for the clinical treatment of obesity. We postulated that five-membered lactone of D-(−)-pantolactone would be an acceptable core for the KR (β-ketoacyl reductase) domain of FAS.13 Working from this hypothesis we have discovered a new series of D-pantolactone derivatives as FAS inhibitors. In this study, we first introduced aliphatic hydrocarbon or aromatic hydrocarbon on hydroxyl group at C-3 position of D(−)-pantolactone in order to obtain some advanced novel inhibitors of FAS. Then, the FAS inhibitory activity and the inhibition lipid
Corresponding authors. E-mail addresses: [email protected] (H. Fang), [email protected] (Z. Hong), [email protected] (M. Fang).
https://doi.org/10.1016/j.bmc.2019.115069 Received 26 July 2019; Received in revised form 22 August 2019; Accepted 22 August 2019 Available online 24 August 2019 0968-0896/ © 2019 Published by Elsevier Ltd.
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Table 1 Crystal Data and Structure Refinement for compound 3k. CCDC number Empirical formula Formula weight Crystal size (mm) Crystal system space group Unit cell dimensions a (Å) b (Å) c (Å) α/° β/° γ/° Volume (Å3) Z D (g/cm3) μ/mm−1 F(0 0 0) radiation 2θ range for data collection/° index ranges reflections collected independent reflections data/restraints/parameters goodness-of-fit on F2 final R indexes [I ≥ 2σ (I)] final R indexes [all data] largest diff. peak/hole/e Å−3
Fig. 1. Structures of Cerulenin, C75 and D-(−)-Pantolactone. O
OH
O +
O
Cl
O
R
DMAP, Et3N CH2Cl2, 0-5
2
1
O
oC
O
R O
3a-3s
3a
3f
3k
3p
3b
3g
3l
3q
3c
3h
3m
3r
3d
3i
3n
3s
3e
3j
3o
Scheme 1. General synthetic procedure of compounds 3a-3s.
accumulation of those synthetic D-(−)-pantolactone were evaluated in vitro bioassays. Finally, molecular docking study in the KR domain of human FAS (hFAS) was performed to determine the binding mode for the most potent compound (3m) and study the key interactions between 3m and hFAS.14–16
1909232 C13H13BrO4 313.14 0.15 × 0.12 × 0.11 Orthorhombic P 21 21 21 6.0168(2) 8.1332(3) 26.4618(10) 90.0 90.0 90.0 1294.93(9) 4 1.165 4.384 632 Cu Kα (λ = 1.54184) 6.68–123.92 −6 ≤ h ≤ 6, −9 ≤ k ≤ 8, −18 ≤ l ≤ 30 2761 1723 1723/0/164 1.046 R1 = 0.0612, wR2 = 0.1586 R1 = 0.0614, wR2 = 0.1589 1.89/-1.35
2. Results and discussion 2.1. Chemistry The synthetic procedures employed to obtain the target compounds fatty acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3a-3g) and substituted-benzoic acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3h-3s) are depicted in Scheme 1. The starting material D-(−)-pantolactone (1) reacted with corresponding fatty acid chloride acyl/substituted-benzoyl chloride in the presence of DMAP and Et3N to give fatty acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3a-3g)/substituted-benzoic acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3h-3s) with good yields. All of the synthetic compounds gave satisfactory analytical and spectroscopic data including 1H NMR, 13C NMR and HRMS, which were in accordance with their depicted structures. Among these compounds, 3c, 3g, 3i, 3l, 3n, 3p, 3q, 3r and 3s were reported for the first time. The structure of compound 3k was further confirmed by single X-ray diffraction. Their solubility was also surveyed, these synthesized compounds could be dissolved in DMF, DMSO, CH2Cl2 and CH3OH, but their solubility in water is < 1 mg/mL
Fig. 2. ORTEP drawing of the molecular structure of 3k. Displacement ellipsoids are at 50% probability level.
2.3. FAS inhibition activity of D-pantolactone derivatives The inhibitory activity of D-pantolactone derivatives (3a-3s) against FAS was investigated by usual procedure and compared with the reference compound C75.20 The IC50 values of all compounds are summarized in Table 2. D-(−)-pantolactone had no FAS inhibitory activity, while major synthesized D-pantolactone derivatives (except 3g, 3i, 3k, 3l and 3n) exhibited potent inhibitory effect on FAS with IC50 values in the range of 13.68 ± 1.52–33.19 ± 1.39 μM. So we attempted to establish the structure-activity relationship (SAR) among them based on data collected in Table 2. Among the compounds (3a-3g) with aliphatic hydrocarbon, the increase of the number of carbon atoms in group R led to an increase in activity when the number of carbon atoms was less 12, compound 3m exhibited more inhibitory activity with IC50 value of 13.68 ± 1.52 μM than other tested compounds with n-alkyl group. However, the introduction of an n-alkyl group that had more than 14 carbon atoms to the pharmaceutical core was unfavourable for FAS inhibitory activity, compound 3g carrying 18 carbon atoms of its side chains led to complete loss of inhibitory activity on FAS. We then turned our attention toward exploring the SAR profile of compound 3h-3r which contained aryl groups at the carbonyl position. It was found that compound 3h with phenyl group had good FAS inhibitory activity with IC50 value of 23.02 ± 1.20 μM. So, the impact of
2.2. Crystal structure of compound 3k Single crystals of C13H13BrO4 (3k) were grown from ethyl acetate. A suitable crystal was selected and mounted on an Ultima IV X-ray diffractometer (Rigaku, Japan). The crystal was kept at 100 (2) K during data. The structures were solved by direct methods yielding the positions of all non-hydrogen atoms, and refined with a full-matrix least squares procedure based on F2 using the Olex2 program system.17–19 The crystal data and the final refinement details of compound 3k are given in Table 1. Fig. 2 shows a perspective view of compound 3k with the atomic labeling system. The crystal structure is stabilized by weak intermolecular hydrogen bonds. Crystallographic data (excluding structure factors) for the structure have been deposited in the Cambridge Crystallographic Data Center as supplementary publication No. CCDC 1909232. 2
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Table 2 In vitro FAS inhibitory activity of the synthesized compounds (3a-3s).
O O O
R O
Compound ID
R
IC50 (μM)a
Compound ID
D-pantolactone
/
N.E.b
3k
N.E.
3a
33.19 ± 1.39
3l
N.E.
3b
28.17 ± 0.93
3m
13.68 ± 1.52
3c
23.33 ± 2.31
3n
N.E.
3d
20.81 ± 1.66
3o
17.20 ± 2.59
3e
15.16 ± 1.54
3p
27.66 ± 3.76
3f
19.56 ± 2.95
3q
21.46 ± 2.36
3g
N.E.
3r
13.92 ± 1.02
3h
23.02 ± 1.20
3s
28.19 ± 1.05
3i
N.E.
C75
3j
19.19 ± 2.83
a b
R
/
IC50 (μM)a
13.86 ± 2.79
IC50 values: the concentration of the inhibitor required to produce 50% inhibition of FAS, each value represents the mean ± SE of three experiments. N.E.: no inhibition in 80 μM.
the substituent at the C-4 position of phenyl ring was investigated by introducing halogen (eF, eCl, eBr, or eI), alkoxy (eOCH3 or eOCH2CH2CH3), alkyl (eCH3, or eCH2CH2CH2CH3), and cyano groups. Compound 3j carrying 4-chloro-phenyl group had better activity than 3h with phenyl group, while addition of fluorine, bromine, and iodine substituent at the C-4 position of phenyl ring (3i, 3k, and 3l) led to complete loss of activity. In addition, we observed that 3m (4methoxyl-phenyl) and 3o (4-methyl-phenyl) showed better FAS inhibitory activity than 3h (phenyl), while 3n (4-(n-propoxyl)-phenyl) exhibited no inhibition activity at the concentration of 80.0 μM and 3p (4-(n-butyl)-phenyl) had lower IC50 values than 3o and 3h. This indicated that the introduction of an n-alkyl or alkoxy group more than two carbon atoms at the C-4 position of phenyl ring was unhelpful for FAS inhibition activity. Introduction of canyon substituent into the phenyl ring (3q) led to a minor increase of activity. Addition of acetoxy groups at the C-3, C-4, and C-5 positions of phenyl ring (3r) led to almost a 2-fold improvement in potency, and exhibited almost equally potent activity with reference compound C75. Finally, substitution of aralkyl group at the carbonyl position gave lower potent compound 3s.
Fig. 3. The inhibition activity on lipid accumulation in HepG2 cells in vitro. Data are expressed as the mean ± SD of each group of cells from six independent experiments; *: 0.01 < p < 0.05, **: 0.001 < p < 0.01, compared with OA-treated cells.
2.4. D-pantolactone derivatives inhibited lipid accumulation in HepG2 cells The preliminary structure activity relationships of the synthesized compounds were achieved from the inhibitory activities on FAS from mouse liver. Then, ten compounds whose FAS inhibition of IC50 < 25.0 μM (3c, 3d, 3e, 3f, 3h, 3j, 3m, 3o, 3q, 3r) were selected for oil red staining test to further investigate their effect on suppressing lipid accumulation in HepG2 cells. In this study, we assessed the effect of the selected compounds at 10.0 μM concentration on oleic acid induced lipid accumulation in HepG2 cells in vitro. C75 and Fenofibrate (Feno) as positive controls were also tested their inhibition of lipid accumulation in the same manner. As shown in Fig. 3, comparison with the level of lipid in vehicle cells, the level of lipid in OA-treated cells were elevated by oleic acid, which was referred to as one hundred
percent. The experiment’s results revealed there was significant difference in the inhibition activity on lipid accumulation for eight of ten compounds (except 3h and 3o) in HepG2 cells in vitro. Especially, compounds 3e, 3f and 3m had almost the same inhibition activity on lipid accumulation in HepG2 cells compared to C75. 2.5. Molecular docking Compound 3m showed the best FAS inhibitory activity and inhibition of lipid accumulation in HepG2 cells. Therefore, molecular docking of 3
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H. Fang, et al.
Fig. 4. The interaction models of C75 and 3m with hFAS (PDB entry 4PIV). C75 (A) and 3m (B) docked into the ligand binding site of hFAS. C75 and 3 m are shown in stick with blue carbon and red oxygen. The binding site is presented with a coloured ribbon. Neighbouring amino acids were displayed in lines within a distance of 5 °A approximately to ligand. The detailed interaction diagram of C75 (C) and 3m (D) with hFAS was plotted using maestro 10.5 software. Hydrogen bonds formed by ligands with the side chain and backbone of protein are indicated with dashed arrows and solid arrows, respectively. Colour lines around ligands (C75 and 3m) stand for the binding pocket and the residues in colours nearby established the pocket. The green colour denotes the hydrophobic nature of amino acids, the red colour stands for the acid amino acids, the purple means the alkalinity of amino acids, the cyan denotes the polar amino acids, and the grey points of ligand atoms stands for the solvent accessibility.
C75 and 3m to the KR domain of hFAS (PDB code: 4PIV) was performed by using the Glide programme integrated in Maestro 10.5 to analyze the binding conformations of C75 and 3m with FAS. Molecular docking study indicated that both 3m and C75 exhibited promising binding affinities with hFAS with docking binding energies of −6.146 kcal/mol and −4.973 kcal/mol, respectively. Comparison of the binding poses of C75 and 3m in the KR domain of hFAS (Fig. 4) shows that both C75 and 3m form hydrogen bonds with THR2083 and ARG1462, but their binding features are apparently different. In C75-hFAS system, the oxygen atom of furan ring forms a hydrogen bonding interaction with the side chain of THR2083, the 3-carboxyl group on 5-oxotetrahydrofuran ring establishes two and one hydrogen bonds with the side chains of ARG1462 and SER2081, respectively. However, in 3m-hFAS system, the ester group on phenyl ring may form two hydrogen bonds with OH from the side chain of THR2083 and NH from the backbone of ALA2062, while another hydrogen bond may be formed between the 4-methoxy group on phenyl ring and NH from the side chain of ARG1462. Therefore, 4-methoxy-benzoate group are likely the important part of the pharmacophore for 3m, but 3-furancarboxylic acid for C75.
4. Experimental section 4.1. Chemical reagents and instruments All NMR spectra were recorded on a Bruker 400 MHz NMR spectrometer, operating at 400 MHz for 1H, and 100 MHz for 13C, TMS was used as an internal reference for 1H and 13C chemical shifts, and DMSOd6 were used as solvent. High-resolution mass spectra were determined on Micromass-LCT Premier Time of Flight (TOF) mass spectrometer (Waters, USA). The target compounds were purified by column chromatography. The purities of the target compounds were assessed by high performance liquid chromatography (HPLC) with a C18-MS-II column (250 mm × 4.6 mm i.d., 5 µm). The HPLC analysis was performed on an Agilent Technologies 1100 Series HPLC system. The mobile phase was methanol (A) and water (B), eluted as the following gradient program: A from 70% to 100% and B from 30% to 0% during 0–20 min. The flow rate was 5 µL min−1 and the detection wavelengths were 254 nm and 205 nm. All reagents used in this study were commercially available, analytically pure, and used without further purification.
3. Conclusion
4.2. General procedure for the synthesis of compounds 3a-3s
In this study, a series of novel D-pantolactone derivatives were designed and synthesized. All of the synthetic compounds gave satisfactory analytical and spectroscopic data including 1H NMR, 13C NMR and HRMS, especially the structure of compound 3k was further confirmed by single X-ray diffraction. The inhibition activity against fatty acid synthase for these compounds was determined. It showed that compounds 3e, 3j, 3m and 3r displayed similar inhibitory activities on FAS in vitro with the reference compound C75 (IC50 = 13.86 ± 2.79 μM). In particular, compound 3m exhibited greater FAS inhibitory activity with IC50 value of 13.68 ± 1.52 μM and inhibition activity on lipid accumulation in HepG2 cells than other tested compounds. The docking analysis predicted that 3m (docking binding energy: −6.146 kcal/mol) had better promising binding affinities with hFAS than C75 (docking binding energy: −4.973 kcal/mol), and formed three hydrogen bonds with hFAS. It indicated that compound 3m could well bind into the KR domain of human fatty acid synthase. Base on these results, compound 3m could be as a potential FAS inhibitor with high activity and could be considered as a promising lead compound for the treatment of obesity.
A solution of dry dichloromethane (10.0 mL) containing the substituted acyl chloride 2 (12.0 mmol) was added dropwise to the solution of dichloromethane (20.0 mL) containing D-(−)-pantolactone (1, 10.0 mmol, 1.30 g), 4-dimethylaminopyridine (1.0 mmol, 0.12 g) and triethylamine (12.0 mmol, 0.95 g). The reaction mixture was stirred overnight at 0–5 °C, and the reaction process was monitored by TLC. After completion of the reaction, the solvent was then removed under reduced pressure to give a residue which was extracted with ethyl acetate (3 × 50 mL). The solution was dried over anhydrous MgSO4 and concentrated under vacuum to obtain a slurry residue, which was purified by silica gel column chromatography (petroleum ether/ethyl acetate 2:1) to give products 3a-3s in yields of 57.19–94.94% (Scheme 1). (R)-Acetic acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3a), pale yellow oil, yield: 94.94%, HPLC purity: 96.9% (tR = 3.18 min), 1H NMR (400 MHz, DMSO-d6): 5.53 (s, 1H, CH), 4.11 (d, J = 4.4 Hz, 1Ha, OCHaHb), 4.02 (d, J = 4.4 Hz, 1Hb, OCHaHb), 2.13 (s, 3H, CH3), 1.09 (s, 3H, CH3), 0.99 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): 173.17, 4
Bioorganic & Medicinal Chemistry 27 (2019) 115069
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170.0 (C]O), 75.8, 75.2, 40.2, 22.3, 20.7, 20.0. HRMS (TOF-MS): Calcd for [C8H12O4 + H]+ 173.0808, Found 173.0811. (R)-Butyric acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3b), pale yellow oil, yield: 87.50%, HPLC purity: 95.8% (tR = 3.45 min), 1H NMR (400 MHz, DMSO-d6): 5.55 (s, 1H, CH), 4.12 (d, J = 4.4 Hz, 1Ha, OCHaHb), 4.03 (d, J = 4.4 Hz, 1Hb, OCHaHb), 2.42–2.38 (t, J = 5.6 Hz, 2H, CH2), 1.60–1.55 (m, 2H, CH2), 1.09 (s, 3H, CH3), 0.97 (s, 3H, CH3), 0.92–0.88 (t, J = 7.2 Hz, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): 173.2, 172.3 (C]O), 75.8, 75.1, 40.2, 35.4, 22.4, 20.0, 18.4, 13.7. HRMS (TOF-MS): Calcd for [C10H16O4 + H]+ 201.1121, Found 201.1127. (R)-Hexanoic acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3c), pale yellow oil, yield: 88.38%, HPLC purity: 92.9% (tR = 3.87 min), 1H NMR (400 MHz, DMSO-d6): 5.60 (s, 1H, CH), 4.17 (d, J = 4.2 Hz, 1Ha, OCHaHb), 4.08 (d, J = 4.2 Hz, 1Hb, OCHaHb), 2.53–2.45 (t, J = 5.6 Hz, 2H, CH2), 1.63–1.59 (m, 2H, CH2), 1.34–1.30 (m, 4H, 2CH2), 1.14 (s, 3H, CH3), 1.02 (s, 3H, CH3), 0.93–0.88 (t, J = 6.8 Hz, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): 173.2, 172.5 (C]O), 75.8, 75.1, 40.2, 33.6, 31.2, 24.6, 22.4, 22.3, 22.2, 20.1, 14.2. HRMS (TOF-MS): Calcd for [C12H20O4 + H]+ 229.1434, Found 201. 229.1442. (R)-Octanoic acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3d), pale yellow oil, yield: 88.30%, HPLC purity: 93.1% (tR = 4.64 min), 1H NMR (400 MHz, DMSO-d6): 5.60 (s, 1H, CH), 4.17 (d, J = 4.2 Hz, 1Ha, OCHaHb), 4.08 (d, J = 4.2 Hz, 1Hb, OCHaHb), 2.53–2.45 (t, J = 5.6 Hz, 2H, CH2), 1.62–1.58 (m, 2H, CH2), 1.31–1.28 (m, 8H, 4CH2), 1.13 (s, 3H, CH3), 1.02 (s, 3H, CH3), 0.90–0.87 (t, J = 7.2 Hz, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): 173.2, 172.5 (C]O), 75.8, 75.1, 40.2, 33.6, 31.6, 28.7, 28.7, 24.9, 22.5, 20.1, 14.4. HRMS (TOF-MS): Calcd for [C14H24O4 + H]+ 257.1747, Found 257.1759. (R)-Dodecanoic acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3e), white solid, m.p. 53.7–54.4, yield: 74.69%, 1H NMR (400 MHz, DMSO-d6): 5.60 (s, 1H, CH), 4.17 (d, J = 4.2 Hz, 1Ha, OCHaHb), 4.08 (d, J = 4.2 Hz, 1Hb, OCHaHb), 2.53–2.44 (t, J = 5.6 Hz, 2H, CH2), 1.61–1.58 (m, 2H, CH2), 1.30–1.27 (m, 16H, 8CH2), 1.13 (s, 3H, CH3), 1.02 (s, 3H, CH3), 0.90–0.87 (t, J = 6.8 Hz, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): 173.1, 172.5 (C]O), 75.8, 75.1, 33.6, 40.2, 31.8, 29.4, 29.4, 29.3, 29.2, 29.1, 29.0, 28.7, 25.0, 22.6, 22.3, 20.1, 14.4. HRMS (TOF-MS): Calcd for [C18H32O4 + H]+ 313.2373, Found 313.2383. (R)-Hexadecanoic acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3f), white solid, m.p. 56.0–57.9, yield: 92.66%, 1H NMR (400 MHz, DMSO-d6): 5.55 (s, 1H, CH), 4.12 (d, J = 4.2 Hz, 1Ha, OCHaHb), 4.05 (d, J = 4.2 Hz, 1Hb, OCHaHb), 2.49–2.41 (t, J = 6.0 Hz, 2H, CH2), 1.61–1.58 (m, 2H, CH2), 1.30–1.27 (m, 24H, 12CH2), 1.09 (s, 3H, CH3), 0.97 (s, 3H, CH3), 0.84 (t, J = 7.2 Hz, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): 173.1, 172.5 (C]O), 75.8, 75.1, 40.3, 33.6, 31.7, 29.4, 29.3, 29.2, 29.0, 28.7, 24.9, 22.5, 22.3, 20.1, 14.4. HRMS (TOFMS): Calcd for [C22H40O4 + H]+ 369.2999, Found 369.2999. (R)-Octadecanoic acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3g), white solid, m.p. 45.6–46.3, yield: 91.94%, 1H NMR (400 MHz, DMSO-d6): 5.55 (s, 1H, CH), 4.12 (d, J = 4.2 Hz, 1Ha, OCHaHb), 4.04 (d, J = 4.2 Hz, 1Hb, OCHaHb), 2.49–2.41 (t, J = 5.6 Hz, 2H, CH2), 1.61–1.48 (m, 2H, CH2), 1.30–1.21 (m, 28H, 14CH2), 1.09 (s, 3H, CH3), 0.97 (s, 3H, CH3), 0.85–0.82 (t, J = 6.8 Hz, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): 173.1, 172.5 (C]O), 75.8, 75.1, 40.2, 33.6, 31.7, 29.5, 29.4, 29.3, 29.2, 29.0, 28.7, 24.9, 22.5, 22.3, 20.1, 14.4. HRMS (TOF-MS): Calcd for [C24H44O4 + H]+ 397.3312, Found 397.3321. (R)-Benzoic acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3h), white solid, m.p. 42.3–44.1, yield: 90.59%, HPLC purity: 98.1% (tR = 4.20 min), 1H NMR (400 MHz, DMSO-d6): 8.04–8.02 (m, 2H, Ar), 7.73–7.71 (m, 1H, Ar), 7.61–7.57 (m, 2H, Ar), 5.85 (s, 1H, CH), 4.25 (d, J = 4.4 Hz, 1Ha, OCHaHb), 4.14 (d, J = 4.4 Hz, 1Hb, OCHaHb), 1.19 (s, 3H, CH3), 1.14 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): 173.1,
165.2 (C]O), 134.5, 130.0, 129.5, 129.0, 76.0, 40.4, 22.4, 20.2. HRMS (TOF-MS): Calcd for [C13H14O4 + H]+ 235.0965, Found 235.0960. (R)-4-Fluoro-benzoic acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3i), white solid, m.p. 72.0–73.1, yield: 86.51%, HPLC purity: 97.6% (tR = 4.15 min), 1H NMR (400 MHz, DMSO-d6): 8.12 (d, J = 6.8 Hz, 2H, Ar), 7.44 (d, J = 6.8 Hz, 2H, Ar), 5.86 (s, 1H, CH), 4.26 (d, J = 4.4 Hz, 1Ha, OCHaHb), 4.16 (d, J = 4.4 Hz, 1Hb, OCHaHb), 1.21 (s, 3H, CH3), 1.16 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): 173.0, 167.2 (C]O), 164.7, 133.0, 132.9, 125.5, 116.6, 76.1, 76.0, 40.5, 22.3, 20.2. HRMS (TOF-MS): Calcd for [C13H13FO4 + H]+ 253.0871, Found 253.0866. (R)-4-Chloro-benzoic acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3j), white solid, m.p. 66.5–68.3, yield: 76.86%, HPLC purity: 99.5% (tR = 5.15 min), 1H NMR (400 MHz, DMSO-d6): 8.01 (d, J = 6.8 Hz, 2H, Ar), 7.64 (d, J = 6.8 Hz, 2H, Ar), 5.82 (s, 1H, CH), 4.22 (d, J = 4.4 Hz, 1Ha, OCHaHb), 4.12 (d, J = 4.4 Hz, 1Hb, OCHaHb), 1.17 (s, 3H, CH3), 1.12 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): 173.0, 164.4 (C]O), 139.5, 131.8, 129.7, 127.8, 76.2, 76.0, 40.6, 22.3, 20.2. HRMS (TOF-MS): Calcd for [C13H13ClO4 + H]+ 269.0575, Found 269.0572. (R)-4-Bromo-benzoic acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3k), white solid, m.p. 84.3–85.2, yield: 90.70%, HPLC purity: 99.7% (tR = 5.20 min), 1H NMR (400 MHz, DMSO-d6): 7.93 (d, J = 6.8 Hz, 2H, Ar), 7.78 (d, J = 6.8 Hz, 2H, Ar), 5.82 (s, 1H, CH), 4.21 (d, J = 4.4 Hz, 1Ha, OCHaHb), 4.12 (d, J = 4.4 Hz, 1Hb, OCHaHb), 1.17 (s, 3H, CH3), 1.11 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): 173.0, 164.6 (C]O), 132.6, 131.9, 128.7, 128.2, 76.2, 76.0, 40.6, 22.3, 20.2. HRMS (TOF-MS): Calcd for [C13H13BrO4 + H]+ 313.0070, Found 313.0065. (R)-4-Iodo-benzoic acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3l), white solid, m.p. 81.4–82.3, yield: 89.12%, HPLC purity: 97.3% (tR = 9.88 min), 1H NMR (400 MHz, DMSO-d6): 7.96 (d, J = 6.8 Hz, 2H, Ar), 7.75 (d, J = 6.8 Hz, 2H, Ar), 5.81 (s, 1H, CH), 4.21 (d, J = 4.4 Hz, 1Ha, OCHaHb), 4.12 (d, J = 4.4 Hz, 1Hb, OCHaHb), 1.16 (s, 3H, CH3), 1.10 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): 173.0, 164.9 (C]O), 138.5, 131.5, 128.4, 103.2, 76.1, 76.0, 40.6, 22.3, 20.2. HRMS (TOF-MS): Calcd for [C13H13IO4 + H]+ 360.9931, Found 360.9926. (R)-4-Methoxy-benzoic acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3yl ester (3m), pale yellow oil, yield: 63.28%, HPLC purity: 96.4% (tR = 6.38 min), 1H NMR (400 MHz, DMSO-d6): 7.99–7.97 (m, 2H, Ar), 7.11–7.09 (m, 2H, Ar), 5.80 (s, 1H, CH), 4.23 (d, J = 4.4 Hz, 1Ha, OCHaHb), 4.13 (d, J = 4.4 Hz, 1Hb, OCHaHb), 3.86 (s, 3H, OCH3), 1.17 (s, 3H, CH3), 1.12 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): 173.3, 164.8 (C]O), 121.1, 114.7, 75.9, 56.1, 40.4, 22.4, 20.2. HRMS (TOFMS): Calcd for [C14H16O5 + H]+ 265.1071, Found 265.1067. (R)-4-Ethoxy-benzoic acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3n), white solid, m.p. 84.1–85.4, yield: 57.19%, HPLC purity: 98.7% (tR = 3.48 min), 1H NMR (400 MHz, DMSO-d6): 7.95 (d, J = 6.8 Hz, 2H, Ar), 7.07 (d, J = 6.8 Hz, 2H, Ar), 5.79 (s, 1H, CH), 4.49 (d, J = 4.4 Hz, 1Ha, OCHaHb), 4.24–4.4.02 (m, 3H, OCHaHb, OCH2), 1.38 (t, J = 8.0 Hz, 3H, CH3), 1.28 (s, 3H, CH3), 1.12 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): 173.3, 164.8 (C]O), 163.5, 132.2, 120.9, 115.1, 76.0, 75.6, 40.6, 32.4, 20.2, 14.9. HRMS (TOF-MS): Calcd for [C15H18O5 + H]+ 279.1227, Found 279.1223. (R)-4-Methyl-benzoic acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3o), white solid, m.p. 54.0–56.3, yield: 63.21%, HPLC purity: 96.8% (tR = 10.30 min), 1H NMR (400 MHz, DMSO-d6): 7.92 (d, J = 8 Hz, 2H, Ar), 7.38 (d, J = 8 Hz, 2H, Ar), 5.82 (s, 1H, CH), 4.23 (d, J = 4.2 Hz, 1Ha, OCHaHb), 4.13 (d, J = 4.2 Hz, 1Hb, OCHaHb), 2.41 (s, 3H, CH3), 1.18 (s, 3H, CH3), 1.13 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): 173.2, 165.2 (C]O), 145.0, 130.0, 129.8, 129.6, 126.3, 76.0, 75.8, 40.6, 22.4, 21.7, 20.2. HRMS (TOF-MS): Calcd for [C14H16O4 + H]+ 249.1121, Found 249.1110. (R)-4-Butyl-benzoic acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3p), pale yellow oil, yield: 74.83%, HPLC purity: 96.3% 5
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(tR = 4.75 min), 1H NMR (400 MHz, DMSO-d6): 7.93 (d, J = 8 Hz, 2H, Ar), 7.40 (d, J = 8 Hz, 2H, Ar), 5.82 (s, 1H, CH), 4.23 (d, J = 4.4 Hz, 1Ha, OCHaHb), 4.13 (d, J = 4.4 Hz, 1Hb, OCHaHb), 2.70–2.66 (m, 2H, CH2), 1.60–1.54 (m, 2H, CH2), 1.33–1.28 (m, 2H, CH2), 1.20 (s, 3H, CH3), 1.16 (s, 3H, CH3), 0.92–0.88 (m, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): 173.2, 165.2 (C]O), 149.7, 130.1, 129.4, 126.5, 76.0, 75.8, 40.6, 35.3, 33.2, 22.4, 22.2, 20.2, 14.2. HRMS (TOF-MS): Calcd for [C17H22O4 + H]+ 291.1591, Found 291.1586. (R)-4-Cyano-benzoic acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3q), white solid, m.p. 110.6–111.9, yield: 88.03%, HPLC purity: 97.9% (tR = 8.55 min), 1H NMR (400 MHz, DMSO-d6): 8.18 (d, J = 6.8 Hz, 2H, Ar), 8.05 (d, J = 6.8 Hz, 2H, Ar), 5.88 (s, 1H, CH), 4.24 (d, J = 4.4 Hz, 1Ha, OCHaHb), 4.15 (d, J = 4.4 Hz, 1Hb, OCHaHb), 1.20 (s, 3H, CH3), 1.15 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): 172.8, 164.1 (C]O), 133.5, 132.9, 130.6, 118.4, 116.6 (CN), 76.6, 76.1, 40.6, 22.3, 20.2. HRMS (TOF-MS): Calcd for [C14H13NO4 + H]+ 260.0917, Found 260.0886. (R)-3,4,5-Triacetoxy-benzoic acid 4,4-dimethyl-2-oxo-tetrahydrofuran-3-yl ester (3r), white solid, m.p. 107.6–108.3, yield: 80.39%, HPLC purity: 97.8% (tR = 3.70 min), 1H NMR (400 MHz, DMSO-d6): 7.86 (s, 2H, Ar), 5.86 (s, 1H, CH), 4.22 (d, J = 4.4 Hz, 1Ha, OCHaHb), 4.14 (d, J = 4.4 Hz, 1Hb, OCHaHb), 2.50 (s, 3H, COCH3), 2.32 (s, 6H, 2COCH3), 1.18 (s, 3H, CH3), 1.16 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): 172.8, 168.4, 167.3, 163.5 (C]O), 144.0, 139.8, 127.0, 122.8, 76.6, 76.0, 40.6, 22.3, 20.9, 20.3, 20.2. HRMS (TOF-MS): Calcd for [C19H20O10 + H]+ 409.1129, Found 409.1133. (R)-2-(4-Isobutyl-phenyl)-propionic acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3s), pale yellow oil, yield: 88.99%, HPLC purity: 97.3% (tR = 9.98 min), 1H NMR (400 MHz, DMSO-d6): 7.28–7.24 (m, 2H, Ar), 7.17–7.14 (m, 2H, Ar), 5.57 (d, J = 10.8 Hz, 1H, CH), 4.16–4.04 (m, 1Ha, OCHaHb), 4.00–3.93 (m, 2H, OCHaHb, CH), 2.53–2.44 (m, 2H, CH2), 1.84–1.82 (m, 1H, CH), 1.50–1.44 (m, 3H, CH3), 0.94–0.85 (m, 12H, 4CH3); 13C NMR (100 MHz, DMSO-d6): 173.7, 172.8 (C]O), 140.4, 138.2, 137.5, 129.5, 127.6, 75.7, 75.1, 44.6, 40.5, 30.1, 22.6, 22.3, 19.9, 19.4, 18.7, 18.4. HRMS (TOF-MS): Calcd for [C19H26O4 + H]+ 319.1904, Found 319.1899.
a humidified atmosphere of 95% air-5% carbon dioxide. Culture medium was changed every 2 days, and the number of viable cells was determined using the MTT method and approximately 2.5 × 104 cells were plated in each well. Control cells received only with the solvent (DMSO) and the OA cells treated only with 5 mM oleic acid (OA-treated cells). In parallel, the experiment cells were treated with different target compounds (10 μM) dissolved in DMSO and oleic acid.22 A single experiment was repeated three times to calculate the standard deviation. 4.5. Oil red O staining For quantification, The HepG2 cells were fixed with 10% neutral formalin for 1 h at room temperature, washed with phosphate-buffered saline and then stained for 1 h with 0.5% oil red O in 60% isopropanol. After washing with distilled water, the stained cells were observed under a microscope. The lipid droplets in cells were stained with oil red O for 30 min and then washed with 70% ethanol for quantification by measuring its absorbance at 358 nm.23,24 4.6. X-ray diffraction Single crystals of C13H13BrO4 (3k) were grown from ethyl acetate. A suitable crystal was selected and mounted on a SuperNova, Dual, Cu at zero, AtlasS2 diffractometer (Rigaku Oxford Diffraction, Tokyo, Japan). The crystal was kept at 290 (2) K during data collection. Using Olex 2, the structure was solved with the ShelXT31 structure solution program using Direct Methods and refined with the ShelXL32 refinement package using Least Squares minimization. 4.7. Molecular modeling Molecular docking was performed using the Glide programme integrated in Maestro 10.5 (Schrödinger Release 2016-1: Maestro, Schrödinger, LLC, New York, NY, 2016). The protein structure of hFAS (PDB code: 4PIV) was prepared with Protein Preparation Wizard panel. The 3D structure of GSK2194069, C75 and 3m were optimized using LigPrep panel with OPLS2005 force field. The binding pocket of the native ligand GSK2194069 in KR domain of hFAS (4PIV) was chosen for docking site. Then based on this docking grid, GSK2194069, C75 and 3m were docked flexibly by Glide 7.0 in the extra precision (XP) mode. Default values were used for all the parameters during docking. The redocking run for GSK2194069 to KR domain of hFAS showed that Glide XP mode could be successful in reproducing the binding mode of native ligand GSK2194069 with a RMSD value of 1.2° A.
4.3. Fatty acid synthase activity assay To study the inhibitory effect of synthetic D-pantolactone derivatives on FAS, the crude FAS activity of 196.2 U/mg was incubated with Dpantolactone derivatives at a ratio of 1:1 (v/v) to obtain the suitable concentration.21 The FAS inhibitory activity of compounds was measured enzymatically with commercial assay kits. The FAS, D-pantolactone derivative, NADPH and acetyl-CoA were incubated in K2HPO4 buffer (pH 7.0) at 37 °C for 30 min. then malonyl-CoA was added. The reaction was assayed for an additional 3 min to determine FAS-dependent oxidation of NADPH and the absorbance was monitored at 340 nm in a heated chamber UH5300 spectrophotometer at 37 °C. The assay solution without D-pantolactone derivatives was run in the same manner which was served as control. FAS inhibitory activity was determined and expressed as the sample concentration that gave a 50% inhibition in the enzyme activity (IC50). The percent of inhibition of FAS was calculated as follows.
Percent inhibition (%) = [(B
4.8. Statistical analysis All data are presented as mean ± SD. Differences between variants were analyzed by a Student’s t-test (Graph Pad Prism 5) for unpaired data. Data of cell experiments were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s (SPSS 19.0). Values of p < 0.05 were considered statistically significant. Declaration of Competing Interest
S)/B] × 100
None.
where B: FAS activity of control; S: FAS activity in the presence of Dpantolactone derivatives. Each sample was assayed in triplicate and at least five different concentrations. The data were expressed as mean ± SE of three experiments.
Acknowledgments We appreciate financial support from the Scientific Research Foundation of Third Institute of Oceanography, State Oceanic Administration (2016042), Marine Economy Innovation Development Area Demonstration Project of Beihai (Bhsfs009), the Natural Science Foundation of Fujian Province of China (No. 2018 J01132), and the Fundamental Research Funds for the Central Universities (No. 20720180051) to M. F.
4.4. Isolation and culture of HepG2 cell The HepG2 cells were from ATCC and grown Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% FBS and antibiotics (100 μg/L penicillin, 100 μg/L streptomycin) and maintained at 37 °C in 6
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Synthesis, biological activities, and docking studies of D-pantolactone derivatives as novel FAS inhibitors
T
Hua Fanga,b,c, Jianlin Hea,c, Tan Rana,c, Hui Chena,b,c, Wenhui Jina,b,c, Bowen Tangd, ⁎ ⁎ Zhuan Honga,b,c, , Meijuan Fangd, a
Third Institute of Oceanography, Ministry of Natural Resources, Xiamen 361005, China Xiamen Ocean Vocational College, Xiamen 361005, China c Fujian Collaborative Innovation Center for Exploitation and Utilization of Marine Biological Resources, Xiamen 361005, China d School of Pharmaceutical Sciences, Xiamen University, Xiamen, Fujian 361005, China b
ARTICLE INFO
ABSTRACT
Keywords: D-pantolactone derivatives Fatty acid synthase (FAS) Lipid accumulation Molecular docking
A novel series of fatty acid synthase (FAS) inhibitors with D-(−)-pantolactone moiety and potential utility for the treatment of obesity were designed, synthesized and characterized, in which the structure of compound 3k was further confirmed by single X-ray diffraction. The mouse FAS inhibitory activity of synthesized compounds was evaluated. Major synthesized compounds (except 3g, 3i, 3k, 3l, and 3n) exhibited moderate FAS inhibitory properties with IC50 values in the range of 13.68 ± 1.52–33.19 ± 1.39 μM, reference inhibitor C75 has IC50 value of 13.86 ± 2.79 μM. Eight compounds (3c, 3d, 3e, 3f, 3j, 3m, 3q and 3r) also displayed inhibitory effect on lipid accumulation in human HepG2 cells. Additionally, the molecular docking study revealed that compound 3m having good inhibition activity against FAS and lipid accumulation also showed promising binding affinities with hFAS, while its binding model with hFAS (PDB ID: 4PIV) was different from that of reference compound C75.
1. Introduction Obesity and overweight, resulting from excessive adiposity is a serious public health problem worldwide with imminent clinical complications and economic burden. Resulting from a chronic imbalance between energy intake and energy expenditure, obesity is frequently associated with other diseases, such as diabetes and hypertension.1–3 On the other hand, Fatty acid synthase (E.C. 2.3.1.85; FAS) is a multifunctional enzyme system that catalyzes the formation of fatty acids from acetyl-CoA, malonyl-CoA, and NADPH, and plays a central role in lipid biosynthesis. FAS is a 270 kD cytosolic enzyme responsible for the conversion of dietary carbohydrates to triacylglycerols (TG) when energy is surfeit. Studies have recently associated FAS with a variety of human diseases and adverse health conditions including obesity, inflammation, cardiovascular disease, and especially cancer.4–8 Thus, FAS has been thought to be a potential target not only for cancer therapy but also for treating the obesity and diabetes. FAS is an attractive emerging target for anti-obesity drug discovery and pharmacological treatment may be required for the control of obesity in many patients. Currently, most of the identified lipid synthesis inhibitors are the inhibitors of fatty acid synthase. Cerulenin, a ⁎
natural metabolite product of the fungus Cephalosporium ceruleans, is the first identified FAS inhibitor.9 The clinical relevance of cerulenin, however, is limited due to the chemical instability.10 C75, a ceruleninderived semi-synthetic FAS inhibitor which binds irreversibly to KS (βketoacyl synthase), ER (enoyl reductase), and TE (thioesterase) as a competitive inhibitor, has been used as a leading compound for discovering the role of FAS in obesity and cancer. It can reduce food consumption and increased fatty acid oxidation in diet-induced obese mice.11,12 D-(−)-pantolactone, a similar five-membered lactone structure with C75 was also suggested to inhibit fatty acid synthase (Fig. 1). Owing to widespread interest in FAS and its relevance in a variety of diseases, it remains a strong need for potent and specific inhibitors of FAS with good develop ability properties that are suitable for the clinical treatment of obesity. We postulated that five-membered lactone of D-(−)-pantolactone would be an acceptable core for the KR (β-ketoacyl reductase) domain of FAS.13 Working from this hypothesis we have discovered a new series of D-pantolactone derivatives as FAS inhibitors. In this study, we first introduced aliphatic hydrocarbon or aromatic hydrocarbon on hydroxyl group at C-3 position of D(−)-pantolactone in order to obtain some advanced novel inhibitors of FAS. Then, the FAS inhibitory activity and the inhibition lipid
Corresponding authors. E-mail addresses: [email protected] (H. Fang), [email protected] (Z. Hong), [email protected] (M. Fang).
https://doi.org/10.1016/j.bmc.2019.115069 Received 26 July 2019; Received in revised form 22 August 2019; Accepted 22 August 2019 Available online 24 August 2019 0968-0896/ © 2019 Published by Elsevier Ltd.
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Table 1 Crystal Data and Structure Refinement for compound 3k. CCDC number Empirical formula Formula weight Crystal size (mm) Crystal system space group Unit cell dimensions a (Å) b (Å) c (Å) α/° β/° γ/° Volume (Å3) Z D (g/cm3) μ/mm−1 F(0 0 0) radiation 2θ range for data collection/° index ranges reflections collected independent reflections data/restraints/parameters goodness-of-fit on F2 final R indexes [I ≥ 2σ (I)] final R indexes [all data] largest diff. peak/hole/e Å−3
Fig. 1. Structures of Cerulenin, C75 and D-(−)-Pantolactone. O
OH
O +
O
Cl
O
R
DMAP, Et3N CH2Cl2, 0-5
2
1
O
oC
O
R O
3a-3s
3a
3f
3k
3p
3b
3g
3l
3q
3c
3h
3m
3r
3d
3i
3n
3s
3e
3j
3o
Scheme 1. General synthetic procedure of compounds 3a-3s.
accumulation of those synthetic D-(−)-pantolactone were evaluated in vitro bioassays. Finally, molecular docking study in the KR domain of human FAS (hFAS) was performed to determine the binding mode for the most potent compound (3m) and study the key interactions between 3m and hFAS.14–16
1909232 C13H13BrO4 313.14 0.15 × 0.12 × 0.11 Orthorhombic P 21 21 21 6.0168(2) 8.1332(3) 26.4618(10) 90.0 90.0 90.0 1294.93(9) 4 1.165 4.384 632 Cu Kα (λ = 1.54184) 6.68–123.92 −6 ≤ h ≤ 6, −9 ≤ k ≤ 8, −18 ≤ l ≤ 30 2761 1723 1723/0/164 1.046 R1 = 0.0612, wR2 = 0.1586 R1 = 0.0614, wR2 = 0.1589 1.89/-1.35
2. Results and discussion 2.1. Chemistry The synthetic procedures employed to obtain the target compounds fatty acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3a-3g) and substituted-benzoic acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3h-3s) are depicted in Scheme 1. The starting material D-(−)-pantolactone (1) reacted with corresponding fatty acid chloride acyl/substituted-benzoyl chloride in the presence of DMAP and Et3N to give fatty acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3a-3g)/substituted-benzoic acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3h-3s) with good yields. All of the synthetic compounds gave satisfactory analytical and spectroscopic data including 1H NMR, 13C NMR and HRMS, which were in accordance with their depicted structures. Among these compounds, 3c, 3g, 3i, 3l, 3n, 3p, 3q, 3r and 3s were reported for the first time. The structure of compound 3k was further confirmed by single X-ray diffraction. Their solubility was also surveyed, these synthesized compounds could be dissolved in DMF, DMSO, CH2Cl2 and CH3OH, but their solubility in water is < 1 mg/mL
Fig. 2. ORTEP drawing of the molecular structure of 3k. Displacement ellipsoids are at 50% probability level.
2.3. FAS inhibition activity of D-pantolactone derivatives The inhibitory activity of D-pantolactone derivatives (3a-3s) against FAS was investigated by usual procedure and compared with the reference compound C75.20 The IC50 values of all compounds are summarized in Table 2. D-(−)-pantolactone had no FAS inhibitory activity, while major synthesized D-pantolactone derivatives (except 3g, 3i, 3k, 3l and 3n) exhibited potent inhibitory effect on FAS with IC50 values in the range of 13.68 ± 1.52–33.19 ± 1.39 μM. So we attempted to establish the structure-activity relationship (SAR) among them based on data collected in Table 2. Among the compounds (3a-3g) with aliphatic hydrocarbon, the increase of the number of carbon atoms in group R led to an increase in activity when the number of carbon atoms was less 12, compound 3m exhibited more inhibitory activity with IC50 value of 13.68 ± 1.52 μM than other tested compounds with n-alkyl group. However, the introduction of an n-alkyl group that had more than 14 carbon atoms to the pharmaceutical core was unfavourable for FAS inhibitory activity, compound 3g carrying 18 carbon atoms of its side chains led to complete loss of inhibitory activity on FAS. We then turned our attention toward exploring the SAR profile of compound 3h-3r which contained aryl groups at the carbonyl position. It was found that compound 3h with phenyl group had good FAS inhibitory activity with IC50 value of 23.02 ± 1.20 μM. So, the impact of
2.2. Crystal structure of compound 3k Single crystals of C13H13BrO4 (3k) were grown from ethyl acetate. A suitable crystal was selected and mounted on an Ultima IV X-ray diffractometer (Rigaku, Japan). The crystal was kept at 100 (2) K during data. The structures were solved by direct methods yielding the positions of all non-hydrogen atoms, and refined with a full-matrix least squares procedure based on F2 using the Olex2 program system.17–19 The crystal data and the final refinement details of compound 3k are given in Table 1. Fig. 2 shows a perspective view of compound 3k with the atomic labeling system. The crystal structure is stabilized by weak intermolecular hydrogen bonds. Crystallographic data (excluding structure factors) for the structure have been deposited in the Cambridge Crystallographic Data Center as supplementary publication No. CCDC 1909232. 2
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Table 2 In vitro FAS inhibitory activity of the synthesized compounds (3a-3s).
O O O
R O
Compound ID
R
IC50 (μM)a
Compound ID
D-pantolactone
/
N.E.b
3k
N.E.
3a
33.19 ± 1.39
3l
N.E.
3b
28.17 ± 0.93
3m
13.68 ± 1.52
3c
23.33 ± 2.31
3n
N.E.
3d
20.81 ± 1.66
3o
17.20 ± 2.59
3e
15.16 ± 1.54
3p
27.66 ± 3.76
3f
19.56 ± 2.95
3q
21.46 ± 2.36
3g
N.E.
3r
13.92 ± 1.02
3h
23.02 ± 1.20
3s
28.19 ± 1.05
3i
N.E.
C75
3j
19.19 ± 2.83
a b
R
/
IC50 (μM)a
13.86 ± 2.79
IC50 values: the concentration of the inhibitor required to produce 50% inhibition of FAS, each value represents the mean ± SE of three experiments. N.E.: no inhibition in 80 μM.
the substituent at the C-4 position of phenyl ring was investigated by introducing halogen (eF, eCl, eBr, or eI), alkoxy (eOCH3 or eOCH2CH2CH3), alkyl (eCH3, or eCH2CH2CH2CH3), and cyano groups. Compound 3j carrying 4-chloro-phenyl group had better activity than 3h with phenyl group, while addition of fluorine, bromine, and iodine substituent at the C-4 position of phenyl ring (3i, 3k, and 3l) led to complete loss of activity. In addition, we observed that 3m (4methoxyl-phenyl) and 3o (4-methyl-phenyl) showed better FAS inhibitory activity than 3h (phenyl), while 3n (4-(n-propoxyl)-phenyl) exhibited no inhibition activity at the concentration of 80.0 μM and 3p (4-(n-butyl)-phenyl) had lower IC50 values than 3o and 3h. This indicated that the introduction of an n-alkyl or alkoxy group more than two carbon atoms at the C-4 position of phenyl ring was unhelpful for FAS inhibition activity. Introduction of canyon substituent into the phenyl ring (3q) led to a minor increase of activity. Addition of acetoxy groups at the C-3, C-4, and C-5 positions of phenyl ring (3r) led to almost a 2-fold improvement in potency, and exhibited almost equally potent activity with reference compound C75. Finally, substitution of aralkyl group at the carbonyl position gave lower potent compound 3s.
Fig. 3. The inhibition activity on lipid accumulation in HepG2 cells in vitro. Data are expressed as the mean ± SD of each group of cells from six independent experiments; *: 0.01 < p < 0.05, **: 0.001 < p < 0.01, compared with OA-treated cells.
2.4. D-pantolactone derivatives inhibited lipid accumulation in HepG2 cells The preliminary structure activity relationships of the synthesized compounds were achieved from the inhibitory activities on FAS from mouse liver. Then, ten compounds whose FAS inhibition of IC50 < 25.0 μM (3c, 3d, 3e, 3f, 3h, 3j, 3m, 3o, 3q, 3r) were selected for oil red staining test to further investigate their effect on suppressing lipid accumulation in HepG2 cells. In this study, we assessed the effect of the selected compounds at 10.0 μM concentration on oleic acid induced lipid accumulation in HepG2 cells in vitro. C75 and Fenofibrate (Feno) as positive controls were also tested their inhibition of lipid accumulation in the same manner. As shown in Fig. 3, comparison with the level of lipid in vehicle cells, the level of lipid in OA-treated cells were elevated by oleic acid, which was referred to as one hundred
percent. The experiment’s results revealed there was significant difference in the inhibition activity on lipid accumulation for eight of ten compounds (except 3h and 3o) in HepG2 cells in vitro. Especially, compounds 3e, 3f and 3m had almost the same inhibition activity on lipid accumulation in HepG2 cells compared to C75. 2.5. Molecular docking Compound 3m showed the best FAS inhibitory activity and inhibition of lipid accumulation in HepG2 cells. Therefore, molecular docking of 3
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Fig. 4. The interaction models of C75 and 3m with hFAS (PDB entry 4PIV). C75 (A) and 3m (B) docked into the ligand binding site of hFAS. C75 and 3 m are shown in stick with blue carbon and red oxygen. The binding site is presented with a coloured ribbon. Neighbouring amino acids were displayed in lines within a distance of 5 °A approximately to ligand. The detailed interaction diagram of C75 (C) and 3m (D) with hFAS was plotted using maestro 10.5 software. Hydrogen bonds formed by ligands with the side chain and backbone of protein are indicated with dashed arrows and solid arrows, respectively. Colour lines around ligands (C75 and 3m) stand for the binding pocket and the residues in colours nearby established the pocket. The green colour denotes the hydrophobic nature of amino acids, the red colour stands for the acid amino acids, the purple means the alkalinity of amino acids, the cyan denotes the polar amino acids, and the grey points of ligand atoms stands for the solvent accessibility.
C75 and 3m to the KR domain of hFAS (PDB code: 4PIV) was performed by using the Glide programme integrated in Maestro 10.5 to analyze the binding conformations of C75 and 3m with FAS. Molecular docking study indicated that both 3m and C75 exhibited promising binding affinities with hFAS with docking binding energies of −6.146 kcal/mol and −4.973 kcal/mol, respectively. Comparison of the binding poses of C75 and 3m in the KR domain of hFAS (Fig. 4) shows that both C75 and 3m form hydrogen bonds with THR2083 and ARG1462, but their binding features are apparently different. In C75-hFAS system, the oxygen atom of furan ring forms a hydrogen bonding interaction with the side chain of THR2083, the 3-carboxyl group on 5-oxotetrahydrofuran ring establishes two and one hydrogen bonds with the side chains of ARG1462 and SER2081, respectively. However, in 3m-hFAS system, the ester group on phenyl ring may form two hydrogen bonds with OH from the side chain of THR2083 and NH from the backbone of ALA2062, while another hydrogen bond may be formed between the 4-methoxy group on phenyl ring and NH from the side chain of ARG1462. Therefore, 4-methoxy-benzoate group are likely the important part of the pharmacophore for 3m, but 3-furancarboxylic acid for C75.
4. Experimental section 4.1. Chemical reagents and instruments All NMR spectra were recorded on a Bruker 400 MHz NMR spectrometer, operating at 400 MHz for 1H, and 100 MHz for 13C, TMS was used as an internal reference for 1H and 13C chemical shifts, and DMSOd6 were used as solvent. High-resolution mass spectra were determined on Micromass-LCT Premier Time of Flight (TOF) mass spectrometer (Waters, USA). The target compounds were purified by column chromatography. The purities of the target compounds were assessed by high performance liquid chromatography (HPLC) with a C18-MS-II column (250 mm × 4.6 mm i.d., 5 µm). The HPLC analysis was performed on an Agilent Technologies 1100 Series HPLC system. The mobile phase was methanol (A) and water (B), eluted as the following gradient program: A from 70% to 100% and B from 30% to 0% during 0–20 min. The flow rate was 5 µL min−1 and the detection wavelengths were 254 nm and 205 nm. All reagents used in this study were commercially available, analytically pure, and used without further purification.
3. Conclusion
4.2. General procedure for the synthesis of compounds 3a-3s
In this study, a series of novel D-pantolactone derivatives were designed and synthesized. All of the synthetic compounds gave satisfactory analytical and spectroscopic data including 1H NMR, 13C NMR and HRMS, especially the structure of compound 3k was further confirmed by single X-ray diffraction. The inhibition activity against fatty acid synthase for these compounds was determined. It showed that compounds 3e, 3j, 3m and 3r displayed similar inhibitory activities on FAS in vitro with the reference compound C75 (IC50 = 13.86 ± 2.79 μM). In particular, compound 3m exhibited greater FAS inhibitory activity with IC50 value of 13.68 ± 1.52 μM and inhibition activity on lipid accumulation in HepG2 cells than other tested compounds. The docking analysis predicted that 3m (docking binding energy: −6.146 kcal/mol) had better promising binding affinities with hFAS than C75 (docking binding energy: −4.973 kcal/mol), and formed three hydrogen bonds with hFAS. It indicated that compound 3m could well bind into the KR domain of human fatty acid synthase. Base on these results, compound 3m could be as a potential FAS inhibitor with high activity and could be considered as a promising lead compound for the treatment of obesity.
A solution of dry dichloromethane (10.0 mL) containing the substituted acyl chloride 2 (12.0 mmol) was added dropwise to the solution of dichloromethane (20.0 mL) containing D-(−)-pantolactone (1, 10.0 mmol, 1.30 g), 4-dimethylaminopyridine (1.0 mmol, 0.12 g) and triethylamine (12.0 mmol, 0.95 g). The reaction mixture was stirred overnight at 0–5 °C, and the reaction process was monitored by TLC. After completion of the reaction, the solvent was then removed under reduced pressure to give a residue which was extracted with ethyl acetate (3 × 50 mL). The solution was dried over anhydrous MgSO4 and concentrated under vacuum to obtain a slurry residue, which was purified by silica gel column chromatography (petroleum ether/ethyl acetate 2:1) to give products 3a-3s in yields of 57.19–94.94% (Scheme 1). (R)-Acetic acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3a), pale yellow oil, yield: 94.94%, HPLC purity: 96.9% (tR = 3.18 min), 1H NMR (400 MHz, DMSO-d6): 5.53 (s, 1H, CH), 4.11 (d, J = 4.4 Hz, 1Ha, OCHaHb), 4.02 (d, J = 4.4 Hz, 1Hb, OCHaHb), 2.13 (s, 3H, CH3), 1.09 (s, 3H, CH3), 0.99 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): 173.17, 4
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170.0 (C]O), 75.8, 75.2, 40.2, 22.3, 20.7, 20.0. HRMS (TOF-MS): Calcd for [C8H12O4 + H]+ 173.0808, Found 173.0811. (R)-Butyric acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3b), pale yellow oil, yield: 87.50%, HPLC purity: 95.8% (tR = 3.45 min), 1H NMR (400 MHz, DMSO-d6): 5.55 (s, 1H, CH), 4.12 (d, J = 4.4 Hz, 1Ha, OCHaHb), 4.03 (d, J = 4.4 Hz, 1Hb, OCHaHb), 2.42–2.38 (t, J = 5.6 Hz, 2H, CH2), 1.60–1.55 (m, 2H, CH2), 1.09 (s, 3H, CH3), 0.97 (s, 3H, CH3), 0.92–0.88 (t, J = 7.2 Hz, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): 173.2, 172.3 (C]O), 75.8, 75.1, 40.2, 35.4, 22.4, 20.0, 18.4, 13.7. HRMS (TOF-MS): Calcd for [C10H16O4 + H]+ 201.1121, Found 201.1127. (R)-Hexanoic acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3c), pale yellow oil, yield: 88.38%, HPLC purity: 92.9% (tR = 3.87 min), 1H NMR (400 MHz, DMSO-d6): 5.60 (s, 1H, CH), 4.17 (d, J = 4.2 Hz, 1Ha, OCHaHb), 4.08 (d, J = 4.2 Hz, 1Hb, OCHaHb), 2.53–2.45 (t, J = 5.6 Hz, 2H, CH2), 1.63–1.59 (m, 2H, CH2), 1.34–1.30 (m, 4H, 2CH2), 1.14 (s, 3H, CH3), 1.02 (s, 3H, CH3), 0.93–0.88 (t, J = 6.8 Hz, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): 173.2, 172.5 (C]O), 75.8, 75.1, 40.2, 33.6, 31.2, 24.6, 22.4, 22.3, 22.2, 20.1, 14.2. HRMS (TOF-MS): Calcd for [C12H20O4 + H]+ 229.1434, Found 201. 229.1442. (R)-Octanoic acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3d), pale yellow oil, yield: 88.30%, HPLC purity: 93.1% (tR = 4.64 min), 1H NMR (400 MHz, DMSO-d6): 5.60 (s, 1H, CH), 4.17 (d, J = 4.2 Hz, 1Ha, OCHaHb), 4.08 (d, J = 4.2 Hz, 1Hb, OCHaHb), 2.53–2.45 (t, J = 5.6 Hz, 2H, CH2), 1.62–1.58 (m, 2H, CH2), 1.31–1.28 (m, 8H, 4CH2), 1.13 (s, 3H, CH3), 1.02 (s, 3H, CH3), 0.90–0.87 (t, J = 7.2 Hz, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): 173.2, 172.5 (C]O), 75.8, 75.1, 40.2, 33.6, 31.6, 28.7, 28.7, 24.9, 22.5, 20.1, 14.4. HRMS (TOF-MS): Calcd for [C14H24O4 + H]+ 257.1747, Found 257.1759. (R)-Dodecanoic acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3e), white solid, m.p. 53.7–54.4, yield: 74.69%, 1H NMR (400 MHz, DMSO-d6): 5.60 (s, 1H, CH), 4.17 (d, J = 4.2 Hz, 1Ha, OCHaHb), 4.08 (d, J = 4.2 Hz, 1Hb, OCHaHb), 2.53–2.44 (t, J = 5.6 Hz, 2H, CH2), 1.61–1.58 (m, 2H, CH2), 1.30–1.27 (m, 16H, 8CH2), 1.13 (s, 3H, CH3), 1.02 (s, 3H, CH3), 0.90–0.87 (t, J = 6.8 Hz, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): 173.1, 172.5 (C]O), 75.8, 75.1, 33.6, 40.2, 31.8, 29.4, 29.4, 29.3, 29.2, 29.1, 29.0, 28.7, 25.0, 22.6, 22.3, 20.1, 14.4. HRMS (TOF-MS): Calcd for [C18H32O4 + H]+ 313.2373, Found 313.2383. (R)-Hexadecanoic acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3f), white solid, m.p. 56.0–57.9, yield: 92.66%, 1H NMR (400 MHz, DMSO-d6): 5.55 (s, 1H, CH), 4.12 (d, J = 4.2 Hz, 1Ha, OCHaHb), 4.05 (d, J = 4.2 Hz, 1Hb, OCHaHb), 2.49–2.41 (t, J = 6.0 Hz, 2H, CH2), 1.61–1.58 (m, 2H, CH2), 1.30–1.27 (m, 24H, 12CH2), 1.09 (s, 3H, CH3), 0.97 (s, 3H, CH3), 0.84 (t, J = 7.2 Hz, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): 173.1, 172.5 (C]O), 75.8, 75.1, 40.3, 33.6, 31.7, 29.4, 29.3, 29.2, 29.0, 28.7, 24.9, 22.5, 22.3, 20.1, 14.4. HRMS (TOFMS): Calcd for [C22H40O4 + H]+ 369.2999, Found 369.2999. (R)-Octadecanoic acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3g), white solid, m.p. 45.6–46.3, yield: 91.94%, 1H NMR (400 MHz, DMSO-d6): 5.55 (s, 1H, CH), 4.12 (d, J = 4.2 Hz, 1Ha, OCHaHb), 4.04 (d, J = 4.2 Hz, 1Hb, OCHaHb), 2.49–2.41 (t, J = 5.6 Hz, 2H, CH2), 1.61–1.48 (m, 2H, CH2), 1.30–1.21 (m, 28H, 14CH2), 1.09 (s, 3H, CH3), 0.97 (s, 3H, CH3), 0.85–0.82 (t, J = 6.8 Hz, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): 173.1, 172.5 (C]O), 75.8, 75.1, 40.2, 33.6, 31.7, 29.5, 29.4, 29.3, 29.2, 29.0, 28.7, 24.9, 22.5, 22.3, 20.1, 14.4. HRMS (TOF-MS): Calcd for [C24H44O4 + H]+ 397.3312, Found 397.3321. (R)-Benzoic acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3h), white solid, m.p. 42.3–44.1, yield: 90.59%, HPLC purity: 98.1% (tR = 4.20 min), 1H NMR (400 MHz, DMSO-d6): 8.04–8.02 (m, 2H, Ar), 7.73–7.71 (m, 1H, Ar), 7.61–7.57 (m, 2H, Ar), 5.85 (s, 1H, CH), 4.25 (d, J = 4.4 Hz, 1Ha, OCHaHb), 4.14 (d, J = 4.4 Hz, 1Hb, OCHaHb), 1.19 (s, 3H, CH3), 1.14 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): 173.1,
165.2 (C]O), 134.5, 130.0, 129.5, 129.0, 76.0, 40.4, 22.4, 20.2. HRMS (TOF-MS): Calcd for [C13H14O4 + H]+ 235.0965, Found 235.0960. (R)-4-Fluoro-benzoic acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3i), white solid, m.p. 72.0–73.1, yield: 86.51%, HPLC purity: 97.6% (tR = 4.15 min), 1H NMR (400 MHz, DMSO-d6): 8.12 (d, J = 6.8 Hz, 2H, Ar), 7.44 (d, J = 6.8 Hz, 2H, Ar), 5.86 (s, 1H, CH), 4.26 (d, J = 4.4 Hz, 1Ha, OCHaHb), 4.16 (d, J = 4.4 Hz, 1Hb, OCHaHb), 1.21 (s, 3H, CH3), 1.16 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): 173.0, 167.2 (C]O), 164.7, 133.0, 132.9, 125.5, 116.6, 76.1, 76.0, 40.5, 22.3, 20.2. HRMS (TOF-MS): Calcd for [C13H13FO4 + H]+ 253.0871, Found 253.0866. (R)-4-Chloro-benzoic acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3j), white solid, m.p. 66.5–68.3, yield: 76.86%, HPLC purity: 99.5% (tR = 5.15 min), 1H NMR (400 MHz, DMSO-d6): 8.01 (d, J = 6.8 Hz, 2H, Ar), 7.64 (d, J = 6.8 Hz, 2H, Ar), 5.82 (s, 1H, CH), 4.22 (d, J = 4.4 Hz, 1Ha, OCHaHb), 4.12 (d, J = 4.4 Hz, 1Hb, OCHaHb), 1.17 (s, 3H, CH3), 1.12 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): 173.0, 164.4 (C]O), 139.5, 131.8, 129.7, 127.8, 76.2, 76.0, 40.6, 22.3, 20.2. HRMS (TOF-MS): Calcd for [C13H13ClO4 + H]+ 269.0575, Found 269.0572. (R)-4-Bromo-benzoic acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3k), white solid, m.p. 84.3–85.2, yield: 90.70%, HPLC purity: 99.7% (tR = 5.20 min), 1H NMR (400 MHz, DMSO-d6): 7.93 (d, J = 6.8 Hz, 2H, Ar), 7.78 (d, J = 6.8 Hz, 2H, Ar), 5.82 (s, 1H, CH), 4.21 (d, J = 4.4 Hz, 1Ha, OCHaHb), 4.12 (d, J = 4.4 Hz, 1Hb, OCHaHb), 1.17 (s, 3H, CH3), 1.11 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): 173.0, 164.6 (C]O), 132.6, 131.9, 128.7, 128.2, 76.2, 76.0, 40.6, 22.3, 20.2. HRMS (TOF-MS): Calcd for [C13H13BrO4 + H]+ 313.0070, Found 313.0065. (R)-4-Iodo-benzoic acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3l), white solid, m.p. 81.4–82.3, yield: 89.12%, HPLC purity: 97.3% (tR = 9.88 min), 1H NMR (400 MHz, DMSO-d6): 7.96 (d, J = 6.8 Hz, 2H, Ar), 7.75 (d, J = 6.8 Hz, 2H, Ar), 5.81 (s, 1H, CH), 4.21 (d, J = 4.4 Hz, 1Ha, OCHaHb), 4.12 (d, J = 4.4 Hz, 1Hb, OCHaHb), 1.16 (s, 3H, CH3), 1.10 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): 173.0, 164.9 (C]O), 138.5, 131.5, 128.4, 103.2, 76.1, 76.0, 40.6, 22.3, 20.2. HRMS (TOF-MS): Calcd for [C13H13IO4 + H]+ 360.9931, Found 360.9926. (R)-4-Methoxy-benzoic acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3yl ester (3m), pale yellow oil, yield: 63.28%, HPLC purity: 96.4% (tR = 6.38 min), 1H NMR (400 MHz, DMSO-d6): 7.99–7.97 (m, 2H, Ar), 7.11–7.09 (m, 2H, Ar), 5.80 (s, 1H, CH), 4.23 (d, J = 4.4 Hz, 1Ha, OCHaHb), 4.13 (d, J = 4.4 Hz, 1Hb, OCHaHb), 3.86 (s, 3H, OCH3), 1.17 (s, 3H, CH3), 1.12 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): 173.3, 164.8 (C]O), 121.1, 114.7, 75.9, 56.1, 40.4, 22.4, 20.2. HRMS (TOFMS): Calcd for [C14H16O5 + H]+ 265.1071, Found 265.1067. (R)-4-Ethoxy-benzoic acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3n), white solid, m.p. 84.1–85.4, yield: 57.19%, HPLC purity: 98.7% (tR = 3.48 min), 1H NMR (400 MHz, DMSO-d6): 7.95 (d, J = 6.8 Hz, 2H, Ar), 7.07 (d, J = 6.8 Hz, 2H, Ar), 5.79 (s, 1H, CH), 4.49 (d, J = 4.4 Hz, 1Ha, OCHaHb), 4.24–4.4.02 (m, 3H, OCHaHb, OCH2), 1.38 (t, J = 8.0 Hz, 3H, CH3), 1.28 (s, 3H, CH3), 1.12 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): 173.3, 164.8 (C]O), 163.5, 132.2, 120.9, 115.1, 76.0, 75.6, 40.6, 32.4, 20.2, 14.9. HRMS (TOF-MS): Calcd for [C15H18O5 + H]+ 279.1227, Found 279.1223. (R)-4-Methyl-benzoic acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3o), white solid, m.p. 54.0–56.3, yield: 63.21%, HPLC purity: 96.8% (tR = 10.30 min), 1H NMR (400 MHz, DMSO-d6): 7.92 (d, J = 8 Hz, 2H, Ar), 7.38 (d, J = 8 Hz, 2H, Ar), 5.82 (s, 1H, CH), 4.23 (d, J = 4.2 Hz, 1Ha, OCHaHb), 4.13 (d, J = 4.2 Hz, 1Hb, OCHaHb), 2.41 (s, 3H, CH3), 1.18 (s, 3H, CH3), 1.13 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): 173.2, 165.2 (C]O), 145.0, 130.0, 129.8, 129.6, 126.3, 76.0, 75.8, 40.6, 22.4, 21.7, 20.2. HRMS (TOF-MS): Calcd for [C14H16O4 + H]+ 249.1121, Found 249.1110. (R)-4-Butyl-benzoic acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3p), pale yellow oil, yield: 74.83%, HPLC purity: 96.3% 5
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(tR = 4.75 min), 1H NMR (400 MHz, DMSO-d6): 7.93 (d, J = 8 Hz, 2H, Ar), 7.40 (d, J = 8 Hz, 2H, Ar), 5.82 (s, 1H, CH), 4.23 (d, J = 4.4 Hz, 1Ha, OCHaHb), 4.13 (d, J = 4.4 Hz, 1Hb, OCHaHb), 2.70–2.66 (m, 2H, CH2), 1.60–1.54 (m, 2H, CH2), 1.33–1.28 (m, 2H, CH2), 1.20 (s, 3H, CH3), 1.16 (s, 3H, CH3), 0.92–0.88 (m, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): 173.2, 165.2 (C]O), 149.7, 130.1, 129.4, 126.5, 76.0, 75.8, 40.6, 35.3, 33.2, 22.4, 22.2, 20.2, 14.2. HRMS (TOF-MS): Calcd for [C17H22O4 + H]+ 291.1591, Found 291.1586. (R)-4-Cyano-benzoic acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3q), white solid, m.p. 110.6–111.9, yield: 88.03%, HPLC purity: 97.9% (tR = 8.55 min), 1H NMR (400 MHz, DMSO-d6): 8.18 (d, J = 6.8 Hz, 2H, Ar), 8.05 (d, J = 6.8 Hz, 2H, Ar), 5.88 (s, 1H, CH), 4.24 (d, J = 4.4 Hz, 1Ha, OCHaHb), 4.15 (d, J = 4.4 Hz, 1Hb, OCHaHb), 1.20 (s, 3H, CH3), 1.15 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): 172.8, 164.1 (C]O), 133.5, 132.9, 130.6, 118.4, 116.6 (CN), 76.6, 76.1, 40.6, 22.3, 20.2. HRMS (TOF-MS): Calcd for [C14H13NO4 + H]+ 260.0917, Found 260.0886. (R)-3,4,5-Triacetoxy-benzoic acid 4,4-dimethyl-2-oxo-tetrahydrofuran-3-yl ester (3r), white solid, m.p. 107.6–108.3, yield: 80.39%, HPLC purity: 97.8% (tR = 3.70 min), 1H NMR (400 MHz, DMSO-d6): 7.86 (s, 2H, Ar), 5.86 (s, 1H, CH), 4.22 (d, J = 4.4 Hz, 1Ha, OCHaHb), 4.14 (d, J = 4.4 Hz, 1Hb, OCHaHb), 2.50 (s, 3H, COCH3), 2.32 (s, 6H, 2COCH3), 1.18 (s, 3H, CH3), 1.16 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): 172.8, 168.4, 167.3, 163.5 (C]O), 144.0, 139.8, 127.0, 122.8, 76.6, 76.0, 40.6, 22.3, 20.9, 20.3, 20.2. HRMS (TOF-MS): Calcd for [C19H20O10 + H]+ 409.1129, Found 409.1133. (R)-2-(4-Isobutyl-phenyl)-propionic acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3s), pale yellow oil, yield: 88.99%, HPLC purity: 97.3% (tR = 9.98 min), 1H NMR (400 MHz, DMSO-d6): 7.28–7.24 (m, 2H, Ar), 7.17–7.14 (m, 2H, Ar), 5.57 (d, J = 10.8 Hz, 1H, CH), 4.16–4.04 (m, 1Ha, OCHaHb), 4.00–3.93 (m, 2H, OCHaHb, CH), 2.53–2.44 (m, 2H, CH2), 1.84–1.82 (m, 1H, CH), 1.50–1.44 (m, 3H, CH3), 0.94–0.85 (m, 12H, 4CH3); 13C NMR (100 MHz, DMSO-d6): 173.7, 172.8 (C]O), 140.4, 138.2, 137.5, 129.5, 127.6, 75.7, 75.1, 44.6, 40.5, 30.1, 22.6, 22.3, 19.9, 19.4, 18.7, 18.4. HRMS (TOF-MS): Calcd for [C19H26O4 + H]+ 319.1904, Found 319.1899.
a humidified atmosphere of 95% air-5% carbon dioxide. Culture medium was changed every 2 days, and the number of viable cells was determined using the MTT method and approximately 2.5 × 104 cells were plated in each well. Control cells received only with the solvent (DMSO) and the OA cells treated only with 5 mM oleic acid (OA-treated cells). In parallel, the experiment cells were treated with different target compounds (10 μM) dissolved in DMSO and oleic acid.22 A single experiment was repeated three times to calculate the standard deviation. 4.5. Oil red O staining For quantification, The HepG2 cells were fixed with 10% neutral formalin for 1 h at room temperature, washed with phosphate-buffered saline and then stained for 1 h with 0.5% oil red O in 60% isopropanol. After washing with distilled water, the stained cells were observed under a microscope. The lipid droplets in cells were stained with oil red O for 30 min and then washed with 70% ethanol for quantification by measuring its absorbance at 358 nm.23,24 4.6. X-ray diffraction Single crystals of C13H13BrO4 (3k) were grown from ethyl acetate. A suitable crystal was selected and mounted on a SuperNova, Dual, Cu at zero, AtlasS2 diffractometer (Rigaku Oxford Diffraction, Tokyo, Japan). The crystal was kept at 290 (2) K during data collection. Using Olex 2, the structure was solved with the ShelXT31 structure solution program using Direct Methods and refined with the ShelXL32 refinement package using Least Squares minimization. 4.7. Molecular modeling Molecular docking was performed using the Glide programme integrated in Maestro 10.5 (Schrödinger Release 2016-1: Maestro, Schrödinger, LLC, New York, NY, 2016). The protein structure of hFAS (PDB code: 4PIV) was prepared with Protein Preparation Wizard panel. The 3D structure of GSK2194069, C75 and 3m were optimized using LigPrep panel with OPLS2005 force field. The binding pocket of the native ligand GSK2194069 in KR domain of hFAS (4PIV) was chosen for docking site. Then based on this docking grid, GSK2194069, C75 and 3m were docked flexibly by Glide 7.0 in the extra precision (XP) mode. Default values were used for all the parameters during docking. The redocking run for GSK2194069 to KR domain of hFAS showed that Glide XP mode could be successful in reproducing the binding mode of native ligand GSK2194069 with a RMSD value of 1.2° A.
4.3. Fatty acid synthase activity assay To study the inhibitory effect of synthetic D-pantolactone derivatives on FAS, the crude FAS activity of 196.2 U/mg was incubated with Dpantolactone derivatives at a ratio of 1:1 (v/v) to obtain the suitable concentration.21 The FAS inhibitory activity of compounds was measured enzymatically with commercial assay kits. The FAS, D-pantolactone derivative, NADPH and acetyl-CoA were incubated in K2HPO4 buffer (pH 7.0) at 37 °C for 30 min. then malonyl-CoA was added. The reaction was assayed for an additional 3 min to determine FAS-dependent oxidation of NADPH and the absorbance was monitored at 340 nm in a heated chamber UH5300 spectrophotometer at 37 °C. The assay solution without D-pantolactone derivatives was run in the same manner which was served as control. FAS inhibitory activity was determined and expressed as the sample concentration that gave a 50% inhibition in the enzyme activity (IC50). The percent of inhibition of FAS was calculated as follows.
Percent inhibition (%) = [(B
4.8. Statistical analysis All data are presented as mean ± SD. Differences between variants were analyzed by a Student’s t-test (Graph Pad Prism 5) for unpaired data. Data of cell experiments were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s (SPSS 19.0). Values of p < 0.05 were considered statistically significant. Declaration of Competing Interest
S)/B] × 100
None.
where B: FAS activity of control; S: FAS activity in the presence of Dpantolactone derivatives. Each sample was assayed in triplicate and at least five different concentrations. The data were expressed as mean ± SE of three experiments.
Acknowledgments We appreciate financial support from the Scientific Research Foundation of Third Institute of Oceanography, State Oceanic Administration (2016042), Marine Economy Innovation Development Area Demonstration Project of Beihai (Bhsfs009), the Natural Science Foundation of Fujian Province of China (No. 2018 J01132), and the Fundamental Research Funds for the Central Universities (No. 20720180051) to M. F.
4.4. Isolation and culture of HepG2 cell The HepG2 cells were from ATCC and grown Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% FBS and antibiotics (100 μg/L penicillin, 100 μg/L streptomycin) and maintained at 37 °C in 6
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