Lipid accumulation inhibitory activities of novel isoxazole-based chenodeoxycholic acids: Design, synthesis and preliminary mechanism study

Article abstract of DOI:10.1016/j.bmcl.2018.07.026

In continuation of our drug discovery program on hyperlipidemia, a series of novel isoxazole-chenodeoxycholic acid hybrids were designed, synthesized and evaluated for their lipid-lowering effects. Preliminary screening of all the synthesized compounds was done by using a 3T3-L1 adipocyte model, in which the most active compound 16b could significantly reduce the lipid accumulation up to 30.5% at a nontoxic concentration 10 μM. Further mechanism studies revealed that 16b blocked lipid accumulation via activating FXR-SHP signaling pathway, efficiently down-regulated the expression of key lipogenesis regulator SREBP-1c.

Full text of DOI:10.1016/j.bmcl.2018.07.026

Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Lipid accumulation inhibitory activities of novel isoxazole-based
chenodeoxycholic acids: Design, synthesis and preliminary mechanism
study
Rongmao Qiua,b, Guoshun Luoa,b, Xinyu Lia,b, Fan Zhengb, Haolin Lib, Jin Zhangb, Qidong Youa,b
,
⁎
Hua Xianga,b,
a
State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing 210009, China
b
Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Hyperlipidemia
Hybrids
FXR
SREBP-1c
In continuation of our drug discovery program on hyperlipidemia, a series of novel isoxazole-chenodeoxycholic
acid hybrids were designed, synthesized and evaluated for their lipid-lowering effects. Preliminary screening of
all the synthesized compounds was done by using a 3T3-L1 adipocyte model, in which the most active compound
16b could significantly reduce the lipid accumulation up to 30.5% at a nontoxic concentration 10 μM. Further
mechanism studies revealed that 16b blocked lipid accumulation via activating FXR-SHP signaling pathway,
efficiently down-regulated the expression of key lipogenesis regulator SREBP-1c.
3T3-L1 adipocytes
Despite significant advances in the therapeutic management of
cardiovascular disease (CVD), it still remains the number one killer in
the developed countries. Hyperlipidemia, an abnormality in lipid
homeostasis, is a major risk factor for progression of atherosclerotic
disease which may account for up to 55% of CVD risk after correction
for age and gender.1 It is one of the most prevalent diseases, leading to
considerable financial and social burden worldwide.2 Present treatment
of hyperlipidemia comprises of various classes of drugs which chiefly
include statins, fibrates, niacin, cholesterol absorption inhibitors, bile
acid sequestrants and proprotein convertase subtilisin/kexin type 9
(PCSK9) inhibitors.3 Although there are many hypolipidemic drugs
used to reduce cholesterol as well as triglycerides in the blood, many
patients still exist residual cardiovascular risk or experience serious
treatment side effects.4 Therefore, it is still imperative to search for
more active and less toxic agents.
Bile acids (BAs) are a class of cholesterol derived, amphipathic
molecules that have early been recognized to aid in the transport, di-
gestion and absorption of nutrients, fat and vitamins. Long before the
era of modern medicine, traditional Chinese medicine recognized the
therapeutic value of animal biles whose main component are BAs.5 In
1999, the breakthrough discovery of BAs as the endogenous ligand of
farnesoid X receptor (FXR) brought a new life to BAs.6–8 FXR alters the
transcription of several genes involved in fatty acid, triglyceride and
lipoprotein metabolism, and administration of FXR agonists to rats re-
duces plasma triglyceride level.9 The main mechanism of inhibition of
triglyceride synthesis by FXR is inhibition of expression of the tran-
scription factor sterol-regulatory-element-binding protein-1c (SREBP-
1c) mediated by a signaling cascade that involves small heterodimer
partner (SHP).10 Chenodeoxycholic acid (CDCA), the most potent FXR
endogenous ligand, used for the treatment of patients with cholesterol
gallstone disease decreased plasma triglyceride level and was therefore
suggested as a drug for the treatment of hypertriglyceridemia.11–13
Isoxazole, a five-membered ring containing oxygen and nitrogen
atoms, have been reported to have many biological activities including
antimicrobial,14 antiviral,15 anticancer,16 anti-inflammatory,17 im-
munomodulatory,18 anticonvulsant,19 antihyperlipidemic20 and anti-
diabetic21 properties. A series of non-steroidal FXR agonists like
GW4064 and LY2562175 which contain isoxazole moiety possessed
robust lipid modulating properties.22,23 Furthermore, due to its easy
synthesis and low cytotoxicity, isoxazole is a very popular fragment for
the development of new agents with variable biological activities.24
In the design of new drugs, the development of hybrid molecules
through the combination of different pharmacophores in one frame
may possess interesting biological activities.25,26 And combination en-
dogenous metabolic products may provide a new molecule with less
toxicity and better bioavailability. Inspired by this approach, we herein
⁎
Corresponding author at: State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University,
Nanjing 210009, China.
E-mail address: xianghua@cpu.edu.cn (H. Xiang).
https://doi.org/10.1016/j.bmcl.2018.07.026
Received 11 April 2018; Received in revised form 29 June 2018; Accepted 16 July 2018
0960-894X/©2018ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:Qiu,R.,Bioorganic&MedicinalChemistryLetters(2018),https://doi.org/10.1016/j.bmcl.2018.07.026

R. Qiu et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Fig. 1. Chemical structure of some potent lipid-lowering agents comprising isoxazole or BAs and general structure of our designed hybrids. (See above mentioned
references for further information.)
PPh3 to provide 9. Isoxazole fragment 13 was prepared as follows: ethyl
acetoacetate 10 was reacted with pyrrole to give 11; cyclization reac-
tion of 11 with nitroethane under the catalysis of phosphorus oxy-
chloride, followed by ester hydrolysis afforded intermediate 13. Amide
14 was obtained from 9 and 13 via condensation using DCC. After
hydrolysis of 14 to the corresponding 15 with LiOH, target compounds
16a–16h were obtained from 15 by reaction with EDCI and amines.
The structures of all the target compounds were confirmed by 1H NMR,
13C NMR and HRMS. Details of the synthetic procedures and structural
characterizations were described in the supplementary material.
It’s well known that 3T3-L1 cell-based method has been shown to be
an efficacious tool for screening beneficial compounds with lipid-low-
ering effects.29 To avoid the impact of cytotoxicity on the lipid accu-
mulation experiment using Oil Red O staining, our compounds were
first tested for cell viability in 3T3-L1 cells. 3T3-L1 cells were treated
with various concentrations (5, 10, 20, and 40 μM) of target compounds
and CDCA for 48 h. Then, the cell viability was measured using MTT
assays. As shown in Fig. 2, all of the tested compounds did not show
significant cytotoxicity to the 3T3-L1 cells even at 40 μM, except
compound 16g. Due to all of the compounds did not obviously affect
cell viability at 10 μM, the nontoxic concentrations of compounds se-
lected for further biological investigation were at 10 μM.
3T3-L1 preadipocytes were differentiated for 6 days into mature
adipocytes in accordance with the procedure which has been previously
reported by our group.30 To assess the effect of target compounds on
lipid accumulation in 3T3-L1 adipocytes, Oil Red O staining and its
subsequent quantification were performed to observe the intracellular
lipid accumulation. As shown in Fig. 3A, 3T3-L1 preadipocyte have a
fibroblast-like morphology. In the microscopic observation of Oil Red O
staining in 3T3-L1 adipocytes, we found that a large number of lipid
droplets were stained red (Fig. 3B). The biological evaluation results
were depicted in Fig. 3C. All of the 10 hybrids decreased lipid accu-
mulation and exhibited equal or greater activities than CDCA in 3T3-L1
adipocytes at 10 μΜ. Bulk amide group appeared to be unfavorable to
inhibitory on lipid accumulation. Compound 16b with N-methyl amide
group showed the highest level of reduction in lipid accumulation,
which reduced 30.5% lipid accumulation at 10 μΜ (*p < 0.05).
Highly expressed in hepatocyte and adipose tissue, SREBP-1c sti-
mulates the expression of several lipogenic proteins, including acetyl-
CoA carboxylase (ACC), fatty acid synthase (FAS), and stearoyl-CoA
desaturase 1 (SCD-1).31 FXR activation suppresses the activity of
SREBP-1c through FXR-SHP-SREBP-1c signaling pathway.10 To uncover
the molecular mechanisms by which our compounds inhibited the lipid
accumulation, we examined whether the FXR signaling pathway was
influenced by 16b in HepG2 cells.32 We chose GW4064 as positive
control which has been successfully utilized as a research tool for
FXR.22 At first, GW4064 and 16b were tested for cell viability in HepG2
cells. HepG2 cells were treated with various concentrations (5, 10, 20,
Table 1
Structures of the designed compounds.
Compounds
R
Compounds
R
14
16d
15
16e
16f
16g
16a
16b
16c
16h
designed and synthesized a series of novel hybrids by a combination of
isoxazole and CDCA, and evaluated them for their lipid-lowering ac-
tivity (Fig. 1). The isoxazole pharmacophore was linked to the C-3
position of CDCA scaffold by amide bond which is widely existed in
compounds used to treat the metabolic syndrome such as Aramchol.27
In order to explore further the chemical space, we manipulated the side
chain at C-24 carboxylic acid moiety, producing the small library re-
ported in Table 1.
The preparation of designed compounds (Table 1) was depicted in
Scheme 1. As described in patent US9206220, CDCA (5) was obtained
by methyl esterification, acetylation, oxidation and Wolff-Kishner-
Huang reaction from CA (1).28 Esterification of CDCA (5) with me-
thanol, followed by sulfonylation with 4-tosyl chloride gave the sulfo-
nate 7. Treatment of 7 with NaN3 gave 8 which was further reduced by
2

R. Qiu et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Scheme 1. Synthesis of 14, 15 and 16a–16h. Reagents and conditions: (a) Conc HCl, CH3OH, reflux; (b) Ac2O, DMAP, Et3N, rt; (c) NaClO, AcOH, EA, 40 °C; (d)
N2H4·H2O, KOH, triethyleneglycol, 120 °C, 2 h; then, 210 °C, 3 h; (e) Conc HCl, CH3OH, reflux; (f) p-TsCl, Py, rt; (g) NaN3, DMF, 60 °C; (h) PPh3, THF, H2O, reflux; (i)
Pyrrole, PhCH3, reflux; (j) C2H5NO2, POCl3, Et3N, CHCl3, rt; (k) NaOH, H2O, CH3OH, rt; (l) DCC, HOBT, THF, rt, N2; (m) LiOH, H2O, CH3OH, rt; (n) RNH2, EDCI,
HOBT, DMF or ClCO2Et, Et3N, then RNH2, rt.
40 and 80 μM) of corresponding compounds for 48 h. Then, the cell
viability was measured using MTT assays. As shown in Fig. 4A, it was
very obvious that GW4064 and 16b did not show cytotoxicity to the
HepG2 cells at 10 μM. Therefore, the concentration of the compounds
selected for real-time PCR experiment was 10 μM. Similar to GW4064,
16b treatment markedly increased the expression of FXR and SHP,
while down-regulated the expression of SREBP-1c at 10 μM (Fig. 4B).
These results indicated that 16b inhibited lipid accumulation in 3T3-L1
adipocytes through FXR-SHP-SREBP-1c signaling pathway.
used to determine the orientation of compounds bound in the active site
of FXR (PDB ID: 3DCT) and the results of these studies were listed in
Table 2. Results showed that the compounds 16b bound effectively into
the active site of FXR with minimal CDOCKER_ENERGY (ΔG) 2.62 kcal/
mol as with reference to CDCA (ΔG) 9.54 kcal/mol (Table 2), but was
much larger than GW4064 (ΔG) −61.08 kcal/mol. Compounds with
bulk amide group like 16 g (ΔG = 48.89 kcal/mol) appeared to be un-
favorable to FXR binding compared to 16b (ΔG = 2.62 kcal/mol).
Molecular docking results concluded that interaction and environment
at the binding site of FXR were more favorable for 16b as compared to
other synthesized compounds, which were consistent with the results of
To help rationalize the effect of synthesized compounds on FXR-
SHP-SREBP-1c signaling pathway, Discovery Studio 3.0/CDOCKER was
Fig. 2. The effect of synthesized compounds on cell viability in 3T3-L1 preadipocytes. The cells were treated with various concentrations (5, 10, 20, and 40 μM) of
synthesized compounds and CDCA or control (1% DMSO) for 48 h, respectively. The results were expressed as the mean
SD of two independent experiments.
3

R. Qiu et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Fig. 3. (A) Microscopic images of 3T3-L1 preadipocyte. (B) Microscopic images of 3T3-L1 adipocytes stained with Oil Red O. (C) Screening of compounds inhibition
on lipid accumulation in 3T3-L1 adipocytes at 10 μM. The results were expressed as the mean
SD of two independent experiments (*p < 0.05 vs vehicle).
Fig. 4. (A) The effect of GW4064 and 16b on cell viability in HepG2 cells. (B) Real-time PCR analysis of mRNA expression of FXR-SHP-SREBP-1c in HepG2 cells
treated with compound 16b and GW4064 at 10 μM. The results were expressed as the mean
vs vehicle (0.1%DMSO)).
SEM (n = 3). (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
4

R. Qiu et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Table 2
Docking energy of FXR with compounds.
Compounds
R
CDOCKER_ENERGY
14
3.94
5.19
16.05
2.62
15
16a
16b
16c
16d
2.78
4.67
16e
16f
16g
38.17
27.65
48.89
Fig. 6. Partition coefficients of compound 16b and CDCA. The partition coef-
ficient represents P = Co/Cw of the compound between octanol (o) and water
(w). The results were expressed as the mean
SEM (n = 3).
which play important roles in the binding to FXR.34 These interactions
were postulated to be the primary reason for the 16b activity.
Oil-water partition coefficient (P) is an important standard to esti-
mate the druglike physical properties of compounds, which was mea-
sured by the distribution between octanol and water. The concentra-
tions of compounds in each phase were measured by HPLC and
partition coefficient was characterized by log P = log (Co/Cw).33As
shown in Fig. 6, compound 16b displayed satisfied physical property
(log P = 3.78), similar to the endogenous product CDCA (log P = 3.13).
In this study, we successfully designed and synthesized a series of
isoxazole-CDCA hybrids. Screening of all the synthesized compounds by
using a 3T3-L1 adipocyte model led to identify compound 16b as a
potent blocker of lipid accumulation in 3T3-L1 adipocytes at a nontoxic
concentration 10 μM. Further mechanism studies revealed that 16b
reduced lipid accumulation via activating FXR-SHP signaling pathway,
efficiently down-regulated the expression of key lipogenesis gene
SREBP-1c. Moreover, 16b displayed satisfied lipophilicity in a pre-
liminary study. In summary, our work revealed a new hybrid molecule
with therapeutic effects and pharmacological activities on hyperlipi-
demia.
16h
35.82
CDCA
GW4064
–
–
9.54
−61.10
lipid accumulation experiment.
The binding mode of 16b in the active site of FXR was shown in
Fig. 5. An excellent superimposition of compound 16b over the struc-
ture of GW4064 was observed in the binding pocket of FXR, suggested
that the designed compounds exhibited the similar binding patterns as
that of GW4064 (Fig. 5A). The docking model of 16b in Fig. 5B showed
that the isoxazole moiety appeared to make a hydrogen bond with
Thr288 and Pi-Pi interaction with Phe284. Moreover, the terminal
amide formed the essential hydrogen bonds interaction with Arg331
Fig. 5. (A) Superimposed poses of 16b (blue) with representative derivatives GW4064 (yellow) binding to FXR. (B) Detailed interaction of 16b with FXR. Green
dashed lines indicate potential hydrogen bonds to key residues and red dashed lines indicate Pi-Pi interaction.
5

R. Qiu et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Acknowledgments
14. Padmaja A, Payani T, Reddy GD, Padmavathi V. Synthesis, antimicrobial and anti-
oxidant activities of substituted pyrazoles, isoxazoles, pyrimidine and thioxopyr-
imidine derivatives. Eur J Med Chem. 2009;44:4557–4566.
This work was supported by grants from the Natural Science
Foundation of China (NSFC, 81373279), the Twelfth Five-Year Plan
Major Project of Candidate Drugs (Ministry of National Science and
Technology, 2012ZX09103101048) and National Major Scientific and
Technological Special Project for “Significant New Drugs Development”
(Ministry of National Science and Technology, 2018ZX09301042-004).
15. Lee YS, Kim BH. Heterocyclic nucleoside analogues: design and synthesis of antiviral,
modified nucleosides containing isoxazole heterocycles. Bioorg Med Chem Lett.
2002;12:1395–1397.
16. Neckers L. Hsp90 inhibitors as novel cancer chemotherapeutic agents. Trends Mol
Med. 2002;8:S55–61.
17. Vitale P, Perrone MG, Malerba P, Lavecchia A, Scilimati A. Selective COX-1 inhibi-
tion as a target of theranostic novel diarylisoxazoles. Eur J Med Chem.
2014;74:606–618.
18. Maczyński M, Zimecki M, Ryng S. A new class of isoxazole derivatives: the M 1–9
series of compounds with immunotropic activity. Acta Pol Pharm. 2008;65:241–244.
19. Eddington ND, Cox DS, Roberts RR, et al. Synthesis and anticonvulsant activity of
enaminones. 4. investigations on isoxazole derivatives. Eur J Med Chem.
2002;37:635–648.
20. Mokale SN, Nevase MC, Sakle NS, et al. Synthesis and in-vivo hypolipidemic activity
of some novel substituted phenyl isoxazol phenoxy acetic acid derivatives. Bioorg
Med Chem Lett. 2014;24:2155–2158.
A. Supplementary data
Supplementary data (the 1H NMR, 13C NMR and HRMS spectra of
target compounds) associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.bmcl.2018.07.026.
21. Yang L, Zhang J, Si L, et al. Synthesis and biological evaluation of GPR40/FFAR1
agonists containing 3,5-dimethylisoxazole. Eur J Med Chem. 2016;116:46–58.
22. Maloney PR, Parks DJ, Haffner CD, et al. Identification of a chemical tool for the
orphan nuclear receptor FXR. J Med Chem. 2000;43:2971–2974.
23. Genin MJ, Bueno AB, Agejas Francisco J, et al. Discovery of 6-(4-{[5-cyclopropyl-3-
(2,6-dichlorophenyl)isoxazol-4-yl]methoxy}piperidin-1-yl)-1-methyl-1H-indole-3-
carboxylic acid: a novel FXR agonist for the treatment of dyslipidemia. J Med Chem.
2015;58:9768–9772.
References
1. Yusuf S, Hawken S, Ôunpuu S, et al. Effect of potentially modifiable risk factors
associated with myocardial infarction in 52 countries (the INTERHEART study): case-
control study. Orv Hetil. 2006;147:675–686.
2. Members WG, Benjamin EJ, Blaha MJ, et al. Heart disease and stroke statistics—2017
update: a report from the american heart association. Circulation.
2017;135:e146–e603.
24. Sysak A, Obminska-Mrukowicz B. Isoxazole ring as a useful scaffold in a search for
new therapeutic agents. Eur J Med Chem. 2017;137:292–309.
25. Muregi FW, Ishih A. Next-generation antimalarial drugs: hybrid molecules as a new
strategy in drug design. Drug Dev Res. 2010;71:20–32.
26. Sashidhara KV, Kumar A, Kumar M, et al. Synthesis and antihyperlipidemic activity
of novel coumarin bisindole derivatives. Bioorg Med Chem Lett. 2010;20:6504–6507.
27. Gonen A, Shaish A, Leikin-Frenkel A, Gilat T, Harats D. Fatty acid bile acid con-
jugates inhibit atherosclerosis in the C57BL/6 mouse model, Pathobiology.
2002–2003; 70(4):215–218.
28. Ferrari M, Zinetti F. Process for preparing high purity ursodeoxycholic acid.
US9206220, 2015.
29. Zeng XY, Zhou X, Xu J, et al. Screening for the efficacy on lipid accumulation in 3T3-
L1 cells is an effective tool for the identification of new anti-diabetic compounds.
Biochem Pharmacol. 2012;84:830–837.
30. Xiang H, Zhao W, Xiao H, et al. Synthesis and biological evaluation of isoflavone
fatty acid esters with potential weight loss and hypolipidemic activities. Bioorg Med
Chem. 2010;18:3036–3042.
3. Ewang-Emukowhate M, Wierzbicki AS. Lipid-lowering agents. J Cardiovasc
Pharmacol Ther. 2013;18:401–411.
4. Santos RD, Waters DD, Tarasenko L, et al. A comparison of non-HDL and LDL cho-
lesterol goal attainment in a large, multinational patient population: the lipid
treatment assessment project 2. Atherosclerosis. 2012;224:150–153.
5. Qiao X, Ye M, Pan DL, et al. Differentiation of various traditional Chinese medicines
derived from animal bile and gallstone: simultaneous determination of bile acids by
liquid chromatography coupled with triple quadrupole mass spectrometry. J
Chromatogr A. 2011;1218:107–117.
6. Parks DJ, Blanchard SG, Bledsoe RK, et al. Bile acids: natural ligands for an orphan
nuclear receptor. Science. 1999;284:1365–1368.
7. Makishima M, Okamoto AY, Repa JJ, et al. Identification of a nuclear receptor for
bile acids. Science. 1999;284:1362–1365.
8. Wang H, Chen J, Hollister K, et al. Endogenous bile acids are ligands for the nuclear
receptor FXR/BAR. Mol Cell. 1999;3:543–553.
9. Cariou B, Staels B. FXR: a promising target for the metabolic syndrome? Trends
Pharmacol Sci. 2007;28:236–243.
10. Watanabe M, Houten SM, Wang L, et al. Bile acids lower triglyceride levels via a
pathway involving FXR, SHP, and SREBP-1c. J Clin Invest. 2004;113:1408–1418.
11. Angelin B, Einarsson K, Hellström K, et al. Effects of cholestyramine and cheno-
deoxycholic acid on the metabolism of endogenous triglyceride in hyperlipoprotei-
nemia. J Lipid Res. 1978;19:1017–1024.
12. Bell GD, Lewis B, Petrie A, et al. Serum lipids in cholelithiasis: effect of cheno-
deoxycholic acid therapy. Br Med J. 1973;3:520–523.
13. Bateson MC, Maclean D, Evans JR, et al. Chenodeoxycholic acid therapy for hyper-
triglyceridaemia in men. Br J Clin Pharmacol. 1978;5:249–254.
31. Ferre P, Foufelle F. SREBP-1c transcription factor and lipid homeostasis: clinical
perspective. Horm Res. 2007;68:72–82.
32. Gu M, Zhang S, Zhao Y, et al. Cycloastragenol improves hepatic steatosis by acti-
vating farnesoid X receptor signalling. Pharmacol Res. 2017;121:22–32.
33. Yuan X, Xia Y, Lu P, et al. Synthesis and evaluation of 1H-pyrrole-2,5-dione deri-
vatives as cholesterol absorption inhibitors for suppressing the formation of foam
cells and inflammatory response. Bioorg Med Chem. 2017;26:1435–1447.
34. Feng S, Yang M, Zhang Z, et al. Identification of an N-oxide pyridine GW4064 analog
as a potent FXR agonist. Bioorg Med Chem Lett. 2009;19 2595–1598.
6