Synthesis, biological activities, and docking studies of D-pantolactone derivatives as novel FAS inhibitors

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Synthesis, biological activities, and docking studies of d-pantolactone deriva-

tives as novel FAS inhibitors

Hua Fang, Jianlin He, Tan Ran, Hui Chen, Wenhui Jin, Bowen Tang, Zhuan

Hong, Meijua Fang

PII:

S0968-0896(19)31215-5

https://doi.org/10.1016/j.bmc.2019.115069

BMC 115069

DOI:

Reference:

Bioorganic & Medicinal Chemistry

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Accepted Date:

26 July 2019

22 August 2019

Please cite this article as: Fang, H., He, J., Ran, T., Chen, H., Jin, W., Tang, B., Hong, Z., Fang, M., Synthesis,

biological activities, and docking studies of d-pantolactone derivatives as novel FAS inhibitors, Bioorganic &

Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.115069

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Synthesis, Biological Activities, and Docking Studies of D-Pantolactone Derivatives

as Novel FAS Inhibitors

Hua Fang ^a,b,c, Jianlin He ^a,c, Tan Ran ^a,c, Hui Chen ^a,b,c, Wenhui Jin ^a,b,c, Bowen

Tang ^d, Zhuan Hong ^a,b,c*, Meijua Fang ^d*

^aThird Institute of Oceanography, Ministry of Natural Resources, Xiamen, 361005,

China

^bXiamen Ocean Vocational College, Xiamen, 361005, China

^cFujian 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

E-mail: fangmj@xmu.edu.cn; hfang@tio.org.cn

Abstract

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 IC₅₀values in the range of

13.68 ± 1.52 to 33.19 ± 1.39 μM, reference inhibitor C75 has IC₅₀value 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.

Keywords: D-Pantolactone Derivatives; Fatty Acid Synthase (FAS); Lipid

Accumulation; Molecular Docking

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.^4-8Thus, 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.⁹The clinical relevance of cerulenin,

however, is limited due to the chemical instability.¹⁰C75, a cerulenin-derived semi-

synthetic FAS inhibitor which binds irreversibly to KS (-ketoacyl 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 (Figure 1).

Insert Figure 1 here.

Figure 1. Structures of Cerulenin, C75 and D-(-)-Pantolactone

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 (-ketoacyl reductase) domain of FAS.¹³Working 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 position of D-(-)-pantolactone in order to obtain some advanced novel

inhibitors of FAS. Then, the FAS inhibitory activity and the inhibition lipid

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

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, CH₂Cl₂and CH₃OH, but their solubility in water is less

than 1 mg/mL

Insert Scheme 1 here.

Scheme 1. General synthetic procedure of compounds 3a-3s

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 collection. 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 F²using the Olex2

program system.^17-19The crystal data and the final refinement details of compound 3k

are given in Table 1. Figure 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.

Insert Table 1 here.

Table 1. Crystal Data and Structure Refinement for compound 3k

Insert Figure 2 here.

Figure 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 IC₅₀values 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 to 33.19 ± 1.39 M. So we attempted to establish

the structure-activity relationship (SAR) among them based on data collected in Table

2.

Insert Table 2 here.

Table 2. In vitro FAS inhibitory activity of the synthesized compounds (3a-3s)

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 M 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 IC₅₀value of 23.02 ±

1.20 M. So, the impact of the substituent at the C-4 position of phenyl ring was

investigated by introducing halogen (-F, -Cl, -Br, or -I), alkoxy (-OCH₃or -

OCH₂CH₂CH₃), alkyl (-CH₃, or -CH₂CH₂CH₂CH₃), 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 (4-

methoxyl-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 M and 3p (4-(n-butyl)-phenyl) had lower IC₅₀values 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 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.

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 IC₅₀less than 25.0 M (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 M 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 Figure 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.

Insert Figure 3 here.

Figure 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.

2.5 Molecular Docking.

Compound 3m showed the best FAS inhibitory activity and inhibition of lipid

accumulation in HepG2 cells. Therefore, molecular docking of 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 (Figure 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.

Insert Figure 4 here.

Figure 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 3m 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.

3. Conclusion

In this study, a series of novel D-pantolactone derivatives were designed and

synthesized. All of the synthetic compounds gave satisfactory analytical and

13

spectroscopic data including ¹H NMR, C 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M). In particular,

compound 3m exhibited greater FAS inhibitory activity with IC₅₀value of 13.68 ±

1.52 M 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.

4. Experimental section

4.1 Chemical reagents and instruments

All NMR spectra were recorded on a Bruker 400 MHz NMR spectrometer,

1

13

operating at 400 MHz for H, and 100 MHz for C, TMS was used as an internal

1

reference for H and ¹³C chemical shifts, and DMSO-d₆were 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 puri ed 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.

4.2 General Procedure for the Synthesis of Compounds 3a-3s

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 ^oC, 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 MgSO₄and 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% (t_R= 3.18 min), ¹H NMR (400 MHz, DMSO-

d₆): 5.53 (s, 1H, CH), 4.11 (d, J = 4.4 Hz, 1H_a, OCH_aH_b), 4.02 (d, J = 4.4 Hz, 1H_b,

13

OCH_aH_b), 2.13 (s, 3H, CH₃), 1.09 (s, 3H, CH₃), 0.99 (s, 3H, CH₃); C NMR (100

MHz, DMSO-d₆): 173.17, 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% (t_R= 3.45 min), ¹H NMR (400 MHz, DMSO-

d₆): 5.55 (s, 1H, CH), 4.12 (d, J = 4.4 Hz, 1H_a, OCH_aH_b), 4.03 (d, J = 4.4 Hz, 1H_b,

OCH_aH_b), 2.42-2.38 (t, J = 5.6 Hz, 2H, CH₂), 1.60-1.55 (m, 2H, CH₂), 1.09 (s, 3H,

13

CH₃), 0.97 (s, 3H, CH₃), 0.92-0.88 (t, J = 7.2 Hz, 3H, CH₃); C 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% (t_R= 3.87 min), ¹H NMR (400 MHz, DMSO-

d₆): 5.60 (s, 1H, CH), 4.17 (d, J = 4.2 Hz, 1H_a, OCH_aH_b), 4.08 (d, J = 4.2 Hz, 1H_b,

OCH_aH_b), 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% (t_R= 4.64 min), ¹H NMR (400 MHz, DMSO-

d₆): 5.60 (s, 1H, CH), 4.17 (d, J = 4.2 Hz, 1H_a, OCH_aH_b), 4.08 (d, J = 4.2 Hz, 1H_b,

OCH_aH_b), 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

1

solid, m.p. 53.7-54.4, yield: 74.69%, H NMR (400 MHz, DMSO-d₆): 5.60 (s, 1H,

CH), 4.17 (d, J = 4.2 Hz, 1H_a, OCH_aH_b), 4.08 (d, J = 4.2 Hz, 1H_b, OCH_aH_b), 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),

13

1.13 (s, 3H, CH₃), 1.02 (s, 3H, CH₃), 0.90-0.87 (t, J = 6.8 Hz, 3H, CH₃); C 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

1

solid, m.p. 56.0-57.9, yield: 92.66%, H NMR (400 MHz, DMSO-d₆): 5.55 (s, 1H,

CH), 4.12 (d, J = 4.2 Hz, 1H_a, OCH_aH_b), 4.05 (d, J = 4.2 Hz, 1H_b, OCH_aH_b), 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),

13

1.09 (s, 3H, CH₃), 0.97 (s, 3H, CH₃), 0.84 (t, J = 7.2 Hz, 3H, CH₃); C 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 (TOF-MS): Calcd for [C₂₂H₄₀O₄+H]⁺

369.2999, Found 369.2999.

(R)-Octadecanoic acid 4,4-dimethyl-2-oxo-tetrahydro-furan-3-yl ester (3g), white

1

solid, m.p. 45.6-46.3, yield: 91.94%, H NMR (400 MHz, DMSO-d₆): 5.55 (s, 1H,

CH), 4.12 (d, J = 4.2 Hz, 1H_a, OCH_aH_b), 4.04 (d, J = 4.2 Hz, 1H_b, OCH_aH_b), 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),

13

1.09 (s, 3H, CH₃), 0.97 (s, 3H, CH₃), 0.85-0.82 (t, J = 6.8 Hz, 3H, CH₃); C 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,

1

m.p. 42.3-44.1, yield: 90.59%, HPLC purity: 98.1% (t_R= 4.20 min), H 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, 1H_a, OCH_aH_b), 4.14 (d, J = 4.4 Hz, 1H_b,

13

OCH_aH_b), 1.19 (s, 3H, CH₃), 1.14 (s, 3H, CH₃); C 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),

1

white solid, m.p. 72.0-73.1, yield: 86.51%, HPLC purity: 97.6% (t_R= 4.15 min), H

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, 1H_a, OCH_aH_b), 4.16 (d, J = 4.4 Hz, 1H_b,

13

OCH_aH_b), 1.21 (s, 3H, CH₃), 1.16 (s, 3H, CH₃); C 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),

1

white solid, m.p. 66.5-68.3, yield: 76.86%, HPLC purity: 99.5% (t_R= 5.15 min), H

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, 1H_a, OCH_aH_b), 4.12 (d, J = 4.4 Hz, 1H_b,

13

OCH_aH_b), 1.17 (s, 3H, CH₃), 1.12 (s, 3H, CH₃); C 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),

1

white solid, m.p. 84.3-85.2, yield: 90.70%, HPLC purity: 99.7% (t_R= 5.20 min), H

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, 1H_a, OCH_aH_b), 4.12 (d, J = 4.4 Hz, 1H_b,

13

OCH_aH_b), 1.17 (s, 3H, CH₃), 1.11 (s, 3H, CH₃); C 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

1

solid, m.p. 81.4-82.3, yield: 89.12%, HPLC purity: 97.3% (t_R= 9.88 min), H 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, 1H_a, OCH_aH_b), 4.12 (d, J = 4.4 Hz, 1H_b, OCH_aH_b),

13

1.16 (s, 3H, CH₃), 1.10 (s, 3H, CH₃); C 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-3-yl ester (3m),

1

pale yellow oil, yield: 63.28%, HPLC purity: 96.4% (t_R= 6.38 min), H 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, 1H_a, OCH_aH_b), 4.13 (d, J = 4.4 Hz, 1H_b, OCH_aH_b), 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 (TOF-MS): 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),

1

white solid, m.p. 84.1-85.4, yield: 57.19%, HPLC purity: 98.7% (t_R= 3.48 min), H

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, 1H_a, OCH_aH_b), 4.24-4.4.02 (m, 3H,

OCH_aH_b，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% (t_R= 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, 1H_a, OCH_aH_b), 4.13 (d, J = 4.2 Hz, 1H_b,

13

OCH_aH_b), 2.41 (s, 3H, CH₃), 1.18 (s, 3H, CH₃), 1.13 (s, 3H, CH₃); C 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% (t_R= 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, 1H_a, OCH_aH_b), 4.13 (d, J = 4.4 Hz, 1H_b, OCH_aH_b), 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,

13

3H, CH₃), 0.92-0.88 (m, 3H, CH₃); C 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% (t_R= 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, 1H_a, OCH_aH_b), 4.15 (d, J = 4.4 Hz, 1H_b,

13

OCH_aH_b), 1.20 (s, 3H, CH₃), 1.15 (s, 3H, CH₃); C 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-tetrahydro-furan-3-yl ester

(3r), white solid, m.p. 107.6-108.3, yield: 80.39%, HPLC purity: 97.8% (t_R= 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, 1H_a, OCH_aH_b), 4.14 (d, J = 4.4 Hz, 1H_b, OCH_aH_b), 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

1

ester (3s), pale yellow oil, yield: 88.99%, HPLC purity: 97.3% (t_R= 9.98 min), H

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, 1H_a, OCH_aH_b), 4.00-3.93 ( m, 2H, OCH_aH_b,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.

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 D-pantolactone derivatives

at a ratio of 1:1 (v/v) to obtain the suitable concentration.²¹The 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

o

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

o

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-S)/B] × 100

Where B: FAS activity of control; S: FAS activity in the presence of D-

pantolactone derivatives. Each sample was assayed in triplicate and at least five

different concentrations. The data were expressed as mean ± SE of three experiments.

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 g/L penicillin,100 g/L

streptomycin) and maintained at 37°C in 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× 10⁴cells 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 ) dissolved in DMSO and

oleic acid.²²A 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 minutes 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 re-docking 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.

4.8 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.

Acknowledgments

We appreciate financial support from the Scientific Research Foundation of Third

Institute of Oceanography, SOA (2016042), Marine Economy Innovation

Development Area Demonstration Project of Beihai (Bhsfs009), the Natural Science

Foundation of Fujian Province of China (No. 2018J01132), and the Fundamental

Research Funds for the Central Universities (No. 20720180051) to M. F.

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OH

O

HOOC

O

NH

O

2

O

D-(-)-Pantolactone

C75

Cerulenin

Figure 1. Structures of Cerulenin, C75 and D-(-)-Pantolactone

Figure 2. ORTEP drawing of the molecular structure of 3k. Displacement ellipsoids

are at 50% probability level.

100

90

*

80

**

70

60

0

5

3f

3j

3c

3e

3r

3d

3q

3m

icle

OA

C7

h

Feno

Ve

Compounds (10 )