Structural Modification of Natural Product Ganomycin i Leading to Discovery of a α-Glucosidase and HMG-CoA Reductase Dual Inhibitor Improving Obesity and Metabolic Dysfunction in Vivo

Article abstract of DOI:10.1021/acs.jmedchem.8b00107

It is a great challenge to develop drugs for treatment of metabolic syndrome. With ganomycin I as a leading compound, 14 meroterpene derivatives were synthesized and screened for their α-glucosidase and HMG-CoA reductase inhibitory activities. As a result, a α-glucosidase and HMG-CoA reductase dual inhibitor ((R,E)-5-(4-(tert-butyl)phenyl)-3-(4,8-dimethylnona-3,7-dien-1-yl)furan-2(5H)-one, 7d) with improved chemical stability and long-term safety was obtained. Compound 7d showed multiple and strong in vivo efficacies in reducing weight gain, lowering HbAlc level, and improving insulin resistance and lipid dysfunction in both ob/ob and diet-induced obesity (DIO) mice models. Compound 7d was also found to reduce hepatic steatosis in ob/ob model. 16S rRNA gene sequencing, SCFA, and intestinal mucosal barrier function analysis indicated that gut microbiota plays a central and causative role in mediating the multiple efficacies of 7d. Our results demonstrate that 7d is a promising drug candidate for metabolic syndrome.

Full text of DOI:10.1021/acs.jmedchem.8b00107

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Structural modification of natural product ganomycin I leading
to discovery of a #-glucosidase and HMG-CoA reductase dual
inhibitor improving obesity and metabolic dysfunction in vivo
Kai Wang, Li Bao, Nan Zhou, Jinjin Zhang, mingfang Liao, Zhongyong Zheng, Yujing Wang,
Chang Liu, Jun Wang, Lifeng Wang, Wenzhao Wang, Shuang-Jiang Liu, and Hongwei Liu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00107 • Publication Date (Web): 10 Apr 2018
Downloaded from http://pubs.acs.org on April 10, 2018
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Structural modification of natural product ganomycin I leading to discovery of a
αꢀglucosidase and HMGꢀCoA reductase dual inhibitor improving obesity and
metabolic dysfunction in vivo
a,b,┴
a, b, ┴
c
a,b
a,b
Kai Wang,
Li Bao,
Nan Zhou, Jinjin Zhang, Mingfang Liao,
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Zhongyong Zheng , Yujing Wang , Chang Liu , Jun Wang , Lifeng Wang,
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Wenzhao Wang, ShuangJiang Liu , Hongwei Liu
*
a
State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of
Sciences, No.1 Beichenxi Road, Chaoyang District, Beijing 100101, P. R. China
b
University of Chinese Academy of Sciences, Beijing, 100049, P. R. China
c
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese
Academy of Sciences, No.1 Beichenxi Road, Chaoyang District, Beijing 100101, P. R.
China
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CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of
Microbiology, Chinese Academy of Sciences, No.1 Beichenxi Road, Chaoyang District,
Beijing 100101, P. R. China
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Beijing Kangyuan Pharmaceutical Co., Ltd., No. 3 Changliu Road, Changping
District, 102200, Beijing P. R. China
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State Key Laboratory of Mycology, Institute of Microbiology
b
University of Chinese Academy of Sciences.
c
State Key Laboratory of Microbial Resources, Institute of Microbiology
d
CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of
Microbiology
e
Beijing Kangyuan Pharmaceutical Co., Ltd.
┴
K. Wang, L. Bao contributed equally to this article.
*
Corresponding Author Tel: +86 10 64806074; Eꢀmail: liuhw@im.ac.cn (HꢀW, Liu)
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Abstract: It is a great challenge to develop drugs for treatment of metabolic
syndrome. With ganomycin I as a leading compound, fourteen meroterpene
derivatives were synthesized and screened for their αꢀglucosidase and HMGꢀCoA
reductase inhibitory activities. As a result, a αꢀglucosidase and HMGꢀCoA reductase
dual inhibitor ((R,E)ꢀ5ꢀ(4ꢀ(tertꢀbutyl)phenyl)ꢀ3ꢀ (4,8ꢀdimethylnonaꢀ3, 7ꢀdienꢀ1ꢀyl)
furanꢀ2(5H)ꢀone， 7d) with improved chemical stability and longꢀterm safety was
obtained. Compound 7d showed multiple and strong in vivo efficacies in reducing
weight gain, lowering HbAlc level, and improving insulin resistance and lipid
dysfunction in both ob/ob and dietꢀinduced obesity (DIO) mice models. Compound
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7d was also found to reduce hepatic steatosis in ob/ob model. 16S rRNA gene
sequencing, SCFA and intestinal mucosal barrier function analysis indicated that gut
microbiota plays a central and causative role in mediating of the multiple efficacies of
7d. Our results demonstrate that 7d is a promising drug candidate for metabolic
syndrome.
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Introduction
Metabolic syndrome (MS) has become an important public health issue worldwide.
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Take China as an example, it was estimated that 454 million adults were suffering
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from MS diseases in 2010. MS is a polygenetic, multiꢀorgan disease characterized by
insulin resistance, central obesity, elevated blood pressure, dyslipidemia, and
nonalcoholic steatohepatitis (NASH). The polypharmacology strategy by a multitude
of drugs for treating multifactorial diseases is unfavorable due to the complex and
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problematic drugꢀdrug interactions and additional side effects in clinics. An ideal
antiꢀMS drug should address the multitude of the desired therapeutic targets and
exhibit multiple effects in lowering blood glucose and plasma lipid levels, reducing
body weight, and improving cardiovascular health. Natural products with diverse
bioactivityꢀrelated scaffolds and pharmacophore patterns are often recognized with
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, 5
multiꢀeffects.
Recently, withaferin A, a steroidal lactone from Withania somnifera,
has been identified as potential candidate with multiple metabolic benefits of the
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bodyꢀweight loss, the reduced hepatic steatosis, and the antidiabetic action.
The enzyme 3ꢀhydroxyꢀ3ꢀmethylglutaryl coꢀenzyme A reductase (HMGꢀCoA
reductase) catalyzes the conversion of HMGꢀCoA to mevalonate, a key precursor of
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cholesterol biosynthesis. Statins, which decrease the level of plasma cholesterol
through inhibiting HMGꢀCoA reductase, represent the major class of hypolipidemic
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drugs in clinics. The enzyme αꢀglucosidase in the brush border of the intestinal
epithelial cells is a wellꢀknown target for the development of antiꢀdiabetic drugs.
αꢀGlucosidase inhibitors (AGIs: acarbose, miglitol, voglibose) can retard the digestion
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and absorption of carbohydrates in the small intestine, and thus reducing postprandial
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hyperglycemia and delaying the development of type 2 diabetes in patients. The
involvement of the gut microbiota in host health and pathogenesis of metabolic
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diseases have been demonstrated.
A ‘microbiotaꢀtoꢀhost paradigm’ has been
recommended as a new strategy for the development of new drugs or therapeutic
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approaches for the treatment metabolic diseases.
by AGIs accumulates more undigested carbohydrates in the lower parts of the gut,
which alteres gut microbial fermentation and further modulates the composition and
The inhibition of αꢀglucosidase
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function of gut microbiota.
Medicines (metformin, berberine,
or diet supplemented with plant extracts or plantꢀderived
have shown cures and/or prevention of metabolic diseases through
acarbose,
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and obeticholic acid
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components
modulating of gut microbiota. In light of the hypolipidemic efficacy of HMGꢀCoA
reductase inhibitor and the hypoglycemic and gut microbiota modulation effect of
αꢀglucosidase inhibitor, a dual inhibitor of HMGCR and αꢀglucosidase could be a
promising drug candidate to treat metabolic syndrome.
Ganoderma species (Ganodermataceae, Polyporales) are wellꢀknown as elixir in
China, and being widely used as functional foods and traditional medicine in Asian
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countries.
Ganomycin I, a meroterpeneꢀtype and dual inhibitor of both
αꢀglucosidase and HMGꢀCoA reductase from Ganoderma spp., was found to possess
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hypoglycemic, hypolipidemic, and insulinꢀsensitizing effects on KKꢀA mice in our
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early work.
However, the paraꢀdihydroxylbenzene moiety in its structure
disadvantages the further drug development due to its chemical instability. Herein, we
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synthesized fourteen ganomycin I derivatives and screened for their in vitro
αꢀglucosidase and HMGꢀCoA reductase inhibitory acitivities. As a result, a new dual
inhibitor (R,E)ꢀ5ꢀ(4ꢀ(tertꢀbutyl)phenyl)ꢀ3ꢀ(4,8ꢀdimethylnonaꢀ3,7ꢀdienꢀ1ꢀyl) furanꢀ 2
(5H)ꢀone (7d) with good stability and high potency was obtained. Compound 7d
exhibited multiple and potent antiꢀMS efficacies by a combination of the
hypoglycemic, hypolipidemic, and weight control activities in both ob/ob and DIO
mice. In addition, gut microbiota was confirmed to play a central role in mediating the
therapeutic effects of 7d.
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Results and Discussion
Chemistry. It was observed that ganomycin I slowly decomposed after
twoꢀweeks exposure at room temperature due to its hydroquinone structural feature.
To search for new meroterpene derivatives with good stability and strong bioactivity,
fourteen ganomycin I analogues with different aromatic groups were synthesized. The
synthetic routes were developed by modifications of the reported synthetic method for
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ganomycin I (Scheme 1).
In brief, the key vinylbenzyl alcohols (2aꢀ2l) were
synthesized from commercially available benzaldehyde analogues (1aꢀ1l) by the
grignard reaction, and then transformed into 3aꢀ3l by manganese dioxide and
CoreyꢀBakshiꢀShibata (CBS) reduction. Compound 6 was prepared from geraniol (4)
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as previously reported. Compounds 3aꢀ3l and 6 were respectively connected, and
further converted into analogues by the same method as described for ganomycin I.
Compound 7m was prepared from 7l by hydrolysis. Compound 7n was synthesized in
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a similar manner as described above with naphthaldehyde 1n as starting material.
(Scheme S1).
Bioactivity Screening. The activities of analogues 7aꢀ7n against both
HMGꢀCoA reductase from pig liver microsomes and αꢀglucosidase from rat small
intestinal mucosa were shown in Table 1. Ganomycin I, atorvastatin, and acarbose
were used as reference compounds. Compounds 7aꢀ7n inhibited HMGꢀCoA reductase
in a concentration dependent manner with IC50 in the range between 5.82 and 112.12
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ꢁM. As to the inhibitory activity against αꢀglucosidase , compounds 7b, 7c, 7d and 7f
showed much stronger activity than ganomycin I. The structure of 7e is quite similar
to that of 7d, but it showed much weaker activities in both HMGꢀCoA reductase and
αꢀglucosidase assays than 7d. To further verify the biossay results, the αꢀglucosidase
inhibitory activity of 7c, 7d, 7e, and 7f was determined by another detection method
using high performance liquid chromatrography. As a result, the IC50 values of 7c, 7d,
7e, and 7f were determined to be 0.23, 0.04, 28.66, 0.16 ꢁM, respectively, similar to
those obtained by colorimetry (Figure S1). Therefore, a deep structureꢀactivity
relationship analysis needs further investigation. (R,E)ꢀ5ꢀ(4ꢀ(tertꢀbutyl)phenyl) ꢀ3ꢀ
(4,8ꢀdimethylnonaꢀ3,7ꢀdienꢀ1ꢀyl)furanꢀ2(5H)ꢀone (7d) displaying the strongest
activities against both αꢀglucosidase and HMGꢀCoA reductase (with a 6.3ꢀfold and
2.1ꢀfold stronger than ganomycin I respectively) was selected for in vivo study.
Stability and toxicity of 7d. In chemical stability assay, compound 7d showed a
better chemical stability than Ganomycin I when exposed under room temperature
(Figure S2). Compound 7d was also confirmed to be safe by a longꢀterm safety assay.
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Daily oral administration of 30.0 mg/kg of 7d for 90 days showed no negative effects
on behavior, body weight, blood glucose, liver function, organ coefficients, or
survival (Figure S3).
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In vivo PK Profile of 7d. Compound 7d was further investigated for the
pharmacokinetic (PK) properties (via p.o. route, with 2.0 mg/kg in SD rat). The mean
plasma concentrationꢀtime curve of 7d after intragastric administration in rats is
shown in Figure S4. The relevant pharmacokinetic parameters are listed in Table 2.
Compound 7d showed a halfꢀlife of 1.8 h, an AUC of 280.2 ng/mLꢂ× h, and a Cmax of
49.3 ng/mL.
In vivo efficacy. We used ob/ob diabetic and DIO (C57BL/6J) mice to evaluate the
hypoglycemic and hypolipidemic effects for 7d. Acarbose, the αꢀglucosidase inhibitor
used in clinics, was used as positive controls in ob/ob mice assay. Compound 7d (0.3
mg/kg, 1.0 mg/kg and 3.0 mg/kg) was daily administered by gavage method.
The ob/ob mice treated with 3.0 mg/kg of 7d showed a 68% reduction in weight
gain, as compared to that of the vehicleꢀtreated ob/ob group (2.0 g per mouse versus
6.5 g per mouse, respectively; Figure 1A). Moreover, the cumulative food intake for
25 days in the compound 7dꢀtreated group (3.0 mg/kg) was decreased to 78% of the
vehicleꢀtreated ob/ob group (Figure 1B). The LEE index and absolute fat mass in
compound 7dꢀtreated mice were also reduced substantially (Figures 1C, D), whereas
the lean mass was changed slightly (Figure 1E). In a separate experiment with DIO
mice, compound 7d treatment led to a 25.4% reduction in body weight (Figures S5),
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as compared with that of the vehicleꢀtreated group. The positive control, acarbose,
showed weak effects on body weight gain, cumulative food intake, and LEE index.
In addition, ob/ob mice treated with compound 7d (at 0.3, 1.0 and 3.0 mg/kg)
showed doseꢀdependent decreases in fasting blood glucose, free diet blood glucose,
and glycated hemoglobin A1c (HbA1c) levels (Figures 2AꢀC). Compound 7d
treatment also improved glucose tolerance and insulin resistance, and significantly
enhanced the insulin sensitivity index (ISI; Figure 2D). The mean area under curves
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(AUC) during the oral glucose tolerance test (OGTT) and the insulin tolerance test
(ITT) of 7dꢀtreated ob/ob mice was much lower than that of the vehicleꢀtreated mice
(Figures 2EꢀH). DIO mice treated with 7d produced similar effects on glucose
tolerance and insulin resistance as those observed in ob/ob mice (Figures S6).
Treating the ob/ob mice with 7d also decreased levels of serum total cholesterol
(TC), free fatty acids (FFAs), and triglycerides (TG) in a dose dependent manner
(Figures 3AꢀC). Particularly, the serum TG level in compound 7dꢀtreated mice (3.0
mg/kg) was decreased to 47% of the vehicleꢀtreated ob/ob mice (Figure 3B).
Compound 7d treatment at the 1.0 mg/kg and 3.0 mg/kg doses also caused reductions
of 43% and 51% in the LDLꢀC levels of ob/ob mice, respectively, as compared with
that of the vehicleꢀtreated group (Figure 3A). In recent studies, a group of
lowꢀdensityꢀlipoproteinꢀcholesterol (LDLꢀC)ꢀreducing medicines have been proved to
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be effective at treating dyslipidemia in clinical trials.
Examination of
hematoxylinꢀeosin (H&E)ꢀstained sections of white adipose tissue (WAT) indicated
the presence of macrovesicular steatosis in ob/ob mice. Compound 7d (3.0 mg/kg)
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effectively reduced the size of the adipocytes in WAT (Figures 3D, E). DIO mice
exhibited similar improvements on lipid metabolism as ob/ob mice under the oral
treatment of 7d (Figures S7).
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Steatosis and nonalcoholic steatohepatitis (NASH) commonly accompany with
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obesity and the obesityꢀrelated metabolic disorders. We found that compound 7d
reduced levels of FFAs, TG, TC, and LDLꢀC in the livers of ob/ob mice (Figures
4AꢀC) and decreased the activities of serum alanine aminotransferase (ALT) and
aspartate transaminase (AST; Figures 4D, E). In addition, compound 7d administered
at 3.0 mg/kg apparently reduced macrosteatosis, hepatocyte ballooning, and fat
deposition in the livers of ob/ob mice, as indicated by liver sections staining and
microscopy observation (Figure 4H). Compound 7d (3.0 mg/kg) also decreased
hepatic hydroxyproline levels, TGFꢀβ1 (a proꢀfibrogenic protein) expression, and
liver fibrosis (Figures 4FꢀH). These results support the effectiveness of 7d in reducing
hepatic steatosis and liver fibrosis.
Overall, compound 7d has been demonstrated so far to be a potential drug
candidate with multiple therapeutic effects including the reduction of weight gain, the
hypoglycemic and hypolipidemic actions, and the improvement of hepatic steatosis
and fibrosis. Compound 7d exhibited more potency than ganomycin I in lowering
body weight gain, and similar efficacies with that of ganomycin I in improving
hyperglycemia and hyperlipidemia (Figure S5ꢀS7).
Mechanism study. The in vivo inhibitory effect of 7d on αꢀglucosidase was
confirmed by the oral sucrose tolerance test (OSTT) and oral maltose tolerance test
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(
OMTT) in both ob/ob mice and diet induced obesity (DIO) (Figures 5AꢀD and
Figures S8AꢀD). The administration of sucrose or maltose (4 g/kg of body weight,
p.o.) to the fasted mice resulted in a rapid increase in blood glucose concentrations
after 40 min, and then blood glucose was recovered to the pretreatment level at 120
min. Treatment of 7d (at 0.3, 1.0 and 3.0 mg/kg) and acarbose induced a significant
reduction of blood glucose levels at 40, 80 and 120 min when compared with that of
the model group (Figures 5AꢀD).
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As a potent αꢀglucosidase inhibitor, treatment with 7d at a dose of 3.0 mg/kg
accumulated more carbohydrates in the lower parts of the small intestine (Figure S8E)
in ob/ob mice, which may alter the composition and function of gut microbiota. To
evaluate the influence of 7d on gut microbiota, we investigated the composition and
abundance of gut microbiota by highꢀthroughput sequencing (Hiseq) of the V3ꢀV4
hypervariable region of the 16S rDNA genes from the caecum contents of mice and
performed comparative analysis between the vehicleꢀtreated and compound 7dꢀtreated
groups (at 1.0 and 3.0 mg/kg). The composition and abundance of gut microbiota
from the compound 7dꢀtreated ob/ob mice significantly differed from that of
vehicleꢀtreated ob/ob mice (NMDS plot, MRPP test, p = 0.001; Figures 6A, B). The
analysis with linear discriminant analysis (LDA) effect size (LEfSe) method revealed
a significant increase in Lachnospiraceae in compound 7dꢀtreated mice,
accompanying with the decrease of Alphaproteobacteria, Betaproteobacteria, and
Gammaproteobacteria (Figure 6C). Members of Lachnospiraceae were reported to
30
generate shortꢀchain fatty acids (SCFAs) from polysaccharides by fermentation.
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The increased level of gut butyrate in 7dꢀtreated mice (at a dose of 3.0 mg/kg) was
further confirmed by GCꢀMS analysis of SCFAs in mice feces (Figures 7A, B).
Metabolic endotoxemia (lowꢀgrade elevation in plasma LPS) has been regarded as
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31
the major cause for the initiation of obesity and obesityꢀrelated dysfunctions.
Increases in lipopolysaccharide (LPS)ꢀproducing gut bacteria or the occurrence of gut
31, 32
dysbiosis can trigger the state of metabolic endotoxemia.
It was found that the
abundance of LPSꢀproducing bacteria in the genus of Neisseri, Prevotella,
33, 34
Fusobacterium, and Selenomonas,
and the proꢀinflammatory bacteria in the
35ꢀ37
genus of Alistipes and Oscillibacter
was markedly reduced in the gut of
7dꢀtreated mice (Figure 6D). In accordance with above analysis, compound 7d
treatment at doses of 1.0 mg/kg and 3.0 mg/kg led to 29.8% and 40.9% reduction of
plasma LPS in ob/ob mice, respectively (Figure 7C), as compared with that in the
vehicleꢀtreated group. Meanwhile, treatment of compound 7d at a dose of 3.0 mg/kg
significantly reduced the level of TNFꢀα, an important inflammatory factor stimulated
by LPS (Figure 7D). Immunofluorescence analysis confirmed the reduced expression
of TNFꢀα in the liver of 7d treated mice (3.0 mg/kg; Figures 7E, F). RNAꢀseq
analysis of differentially expressed genes in liver also demonstrated reduced systemic
inflammation in the compound 7dꢀtreated mice (3.0 mg/kg). Geneꢀontology
(GO)ꢀbased functional annotation analysis of all downregulated and differentially
expressed genes (≥ 2ꢀfold) indicated that the top 10 enriched biological processes
correlated with inflammation and immunity (Figure S9A). A Kyoto Encyclopaedia of
Genes and Genomes (KEGG) analysis of these genes further indicated that 14 of the
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top 20 pathways were involved in the inflammatory response and autoimmunity (e.g.
Fcꢀγꢀreceptorꢀmediated phagocytosis, type 1 diabetes mellitus, leishmaniasis,
tuberculosis, asthma, inflammatory bowel disease (IBD), and rheumatoid arthritis;
Figure S9B).
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Butyrate produced by intestinal bacteria plays significant physiological roles in
stimulating mucin release, enhancing mRNA expression of mucosal integrity proteins
3
8, 39
40
41
and tight junction proteins,
and improving the energy metabolism. We
investigated the effects of 7d on intestinal inflammation and mucosal barrier function
in ob/ob mice. Results indicated that treatment with 7d at a dose of 3.0 mg/kg
decreased the mRNA expression of proꢀinflammatory cytokine TNFꢀα and increased
the mRNA expression of antiꢀinflammatory cytokine ILꢀ10, mucosal integrity proteins
(Mucꢀ1 and Mucꢀ5), and tight junction proteins (ZOꢀ1 and Occludin) in the ileum
4
2
(Figure 7G). Butyrate also participates in the maintenance of gut homeostasis. It
prevents dysbiotic expansion of Gramꢀnegative bacteria by limiting oxygen or nitrate
supply to these bacteria via butyrateꢀactivated PPARꢀγ signaling. The activation of
epithelial PPARꢀγ signaling by butyrate subsequently enhances βꢀoxidation of fatty
acids and reduces epithelial expression of Nos2. We found that treatment with 7d at a
dose of 3.0 mg/kg upregulated mRNA expression of Pparg and downregulated mRNA
expression of the inducible nitric oxide synthase gene Nos2 in epithelial colonocytes
(Figure 7H). We also detected decreased nitrate levels in the colonic lumen of
7dꢀtreated ob/ob mice (3.0 mg/kg; Figure 7I). Thus, the enhanced level of butyrate by
7d due to the increase of Lachnospiraceae in gut microbiota is conductive to
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improving gut barrier function and preventing the expansion of Gramꢀnegative
Proteobacteria.
Finally, we conducted experiments with pseudoꢀgermꢀfree ob/ob mice to further
confirm the role of gut microbiota in mediating the effects of 7d. Pseudoꢀgermꢀfree
mice were generated by administering a cocktail of broadꢀspectrum antibiotics
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(ampicillin, vancomycin, neomycin, gentamycin, and erythromycin) to ob/ob mice for
three weeks (Figure S10). Antibiotics pretreatment largely decreased the therapeutic
efficacies of 7d (at a dose of 3.0 mg/kg) on ameliorating endotoxemia, reducing
weight gain and food intake, and improving blood glucose and lipid metabolic
disorders in ob/ob mice (Figure S11). Interestingly, Abxꢀtreatment could not
completely abolish the hypolipidemic effects of 7d. Compound 7d treatment
significantly decreased the plasma TG level and the weight gain as compared to that
of the vehicleꢀtreated Abx group (Figure S11), which might be attributed to the
inhibitory activity of 7d against HMGꢀCoA reductase. Atorvastatin and rosuvastatin
have been reported to reduce body weight gain, improve diabetesꢀrelated metabolic
43
disorders, and alleviate inflammation.
Together, these data indicated that the
therapeutic effects of 7d are largely dependent on its modulation on gut microbiota.
Conclusion.
With natural product ganomycin I as a leading compound, fourteen meroterpene
derivatives (7aꢀ7n) were synthesized and screened for their in vitro HMGꢀCoA
reductase and αꢀglucosidase inhibitory activities. A potent dual inhibitor 7d with good
chemical stability, longꢀterm safety, and multiple in vivo beneficial effects was
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obtained. Compound 7d exhibited multiꢀeffects in reducing weight gain, lowering
HbAlc level, and improving insulin resistance, lipid dysfunction and hepatic steatosis.
It was demonstrated that 7d exerted its therapeutic effects through modulation of gut
microbiota. The changed gut microbiota enhanced the level of gut SCFAs (butyrate
and propionate) and restored intestinal mucosal barrier function, which consequently
reduced the metabolic endotoxemia. The worldwide epidemic of obesity and the
related metabolic diseases have become a serious public health problem. The current
work confirms that the new concept of “microbiota to host paradigm” for the
treatment of metabolic diseases is reasonable and promising.
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Experimental Section
Chemistry
Reagents and Methods. All commercial chemicals and solvents are reagent
grade and were used without further purification unless otherwise noted. All reactions
are stirred magnetically. Solvents used for extraction and chromatographic separation
were analytical grade. TLC was carried out on silica gel HSGF254, and the spots were
visualized by spraying with 10% H SO and heating. Silica gel (Qingdao Haiyang
2
4
Chemical Co., Ltd., People’s Republic of China). HPLC analysis was performed on
an Agilent 1200 HPLC system using the YMCꢀPack ODS column (C18, 250 × 4.6
mm, 5 ꢀm) with DAD detector at a flow rate of 1.0 mL/min. UV and IR spectral data
were acquired using a Thermo Genesysꢀ10S UV−vis and Nicolet IS5FTꢀIR
spectrophotometer, respectively. Specific rotations were recorded on a PerkinElmer
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41 polarimeter. NMR spectral data were obtained with a Bruker Avanceꢀ500
3 H C
spectrometer (CDCl , δ 7.26/δ 77.2). HRꢀTOFꢀMS data were measured using an
Agilent AccurateꢀMassꢀQꢀTOF LC/MS 6520 instrument. The purity of synthetic
compounds was determined by HPLC, and the compounds with purity of > 95% were
used for the following experiments.
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Synthesis of 3aꢀ3l, and 3n. The solution of benzaldehyde analogues (1aꢀ1l, and
1n) (10 mmol) in THF (10 mL) and 30 mL of allyl magnesium bromide (1.0 M in
THF) were stirred at ꢀ78 ºC for 1 h. The reaction was quenched by the addition of
NaHCO in saturated aqueous solution, followed by 15 min of agitation. The mixture
3
was then concentrated and filtered through a silica plug (~100 g) with hexane/EtOAc
(20:1, 1000 mL) as the eluant. The filtrate was concentrated in vacuo to give 2aꢀ2l,
and 2n
To a solution of alcohols (2aꢀ2l, and 2n) (7.8 mmol) in hexane (10 mL) was added
activated MnO (7.2 g, 81 mmol). The solution was stirred for 6 h at room
2
temperature. The resulting mixture was filtered through a pad of celite, and the filtrate
was concentrated. The solvent was removed in vacuo to afford crude aldehyde.
(
R)ꢀCBS catalyst (1.0 mol/L, 8.1 mL, 8.1 mmol) and BH·Sme
mmol) were added into 10 mL of anhydrous CH Cl at 20 °C. After stirring for 20
min at 20 °C, the crude aldhyde in anhydrous CH Cl (5 mL) was added dropwisely.
2
(10 mol/L, 8.1 mL, 81
2
2
2
2
After 3 h, the reaction was quenched by methanol (10 mL). The resulting mixture was
then concentrated and filtered through a silica plug (~100 g) with hexane/EtOAc (20:1,
1000 mL) as the eluant. The filtrate was concentrated in vacuo to give 3aꢀ3l, and 3n.
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1
ꢀphenylpropꢀ2ꢀenꢀ1ꢀol (2a): colourless oil; UV (methanol) λmax (log ε) 218 (3.20),
249 (0.59) nm; IR (neat) νmax 2810, 1640, 1428, 1423, 1325, 1223, 1018, and 625
ꢀ1
+
1
9
cm ; positive HRTOFMS m/z [M+H] 135.0810 (calcd. for C H11O, 135.0810). H
0
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NMR data are in accordance with literature data.
ꢀ(2ꢀnitrophenyl)propꢀ2ꢀenꢀ1ꢀol (2b): colourless oil; UV (methanol) λmax (log ε)
20 (3.25), 260 (0.50) nm; IR (neat) νmax 2824, 1645, 1425, 1348, 1225, 1029, 798,
1
2
ꢀ1
+
9 3
and 613 cm ; positive HRTOFMS m/z [M+H] 180.0655 (calcd. for C H10NO ,
1
45
1
80.0655). H NMR data are in accordance with literature data.
ꢀ(pꢀtolyl)propꢀ2ꢀenꢀ1ꢀol (2c): colourless oil; UV (methanol) λmax (log ε) 218
3.25), 237 (0.34) nm; IR (neat) νmax 2810, 1645, 1623, 1423, 1418, 1225, 1025, 798,
1
(
ꢀ1
+
723 cm ; positive HRTOFMS m/z [M+H] 149.0970 (calcd. for C10H13O, 149.0967).
1
46
H NMR data are in accordance with literature data.
1ꢀ(4ꢀ(tertꢀbutyl)phenyl)propꢀ2ꢀenꢀ1ꢀol (2d): colourless oil; UV (methanol) λmax
log ε) 220 (3.25), 260 (0.50) nm; IR (neat) νmax 3335, 2810, 2795, 1645, 1620, 1425,
(
ꢀ1
+
1
348, 1253, 1029, 799, 723, and 613 cm ; positive HRTOFMS m/z [M+H] 191.1432
1
(
calcd. for C13
H
19O, 191.1430). H NMR (500 MHz, CDCl
3
): δ 7.38 (d, J = 8.1 Hz,
2
1
1
H), 7.29 (d, J = 8.1 Hz, 2H), 6.04 (ddd, J = 16.6, 10.3, 10.3 Hz, 1H), 5.34 (d, J =
13
6.6 Hz, 1H), 5.17 (m, 1H), 1.31 (s, 9H); C NMR (125 MHz, CDCl
3
): δ 150.8,
40.3, 139.7, 126.1 (×2), 125.5 (×2), 114.9, 75.2, 34.6, 31.4 (×3).
1ꢀ(4ꢀisopropylphenyl)propꢀ2ꢀenꢀ1ꢀol (2e): colourless oil; UV (methanol) λmax (log
ε) 218 (3.18), 251 (0.89) nm; IR (neat) νmax 3318, 3152, 2823, 1629, 1442, 1425,
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ꢀ
1
+
1
289, 1225, 925, and 623 cm ; positive HRTOFMS m/z [M+H] 177.1280 (calcd. for
1
47
C
12
H
17O, 177.1279). H NMR data are in accordance with literature data.
ꢀ(4ꢀethylphenyl)propꢀ2ꢀenꢀ1ꢀol (2f): colourless oil; UV (methanol) λmax (log ε)
218 (2.85), 260 (0.52) nm; IR (neat) νmax 2823, 2772, 1634, 1608, 1425, 1225, 1025,
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ꢀ1
+
7
95, 723, and 613 cm ; positive HRTOFMS m/z [M+H] 163.1118 (calcd. for
1
48
C
11
H
15O, 163.1117). H NMR data are in accordance with literature data.
ꢀ(4ꢀmethoxyphenyl)propꢀ2ꢀenꢀ1ꢀol (2g): colourless oil; UV (methanol) λmax (log
ε) 220 (2.54), 268 (0.78) nm; IR (neat) νmax 3320, 2925, 1625, 1428, 1423, 1245,
1
ꢀ1
+
1
223, 725, and 625 cm ; positive HRTOFMS m/z [M+H] 165.0918 (calcd. for
1
46
C
10
H
13
O
2
, 165.0916). H NMR data are in accordance with literature data.
1ꢀ(4ꢀnitrophenyl)propꢀ2ꢀenꢀ1ꢀol (2h): colourless oil; UV (methanol) λmax (log ε)
20 (3.23), 237 (0.34) nm; IR (neat) νmax 2825, 1645, 1623, 1452,1423, 1223, 798,
2
ꢀ1
+
723, 623 cm ; positive HRTOFMS m/z [M+H] 149.0965 (calcd. for C10H13O,
1
49
1
49.0967). H NMR data are in accordance with literature data.
ꢀ(2,5ꢀdifluorophenyl)propꢀ2ꢀenꢀ1ꢀol (2i): colourless oil; UV (methanol) λmax (log
ε) 220 (3.25), 246 (0.58) nm; IR (neat) νmax 3309, 3128, 2825, 1625, 1445, 1423,
1
ꢀ1
+
1
245, 723, and 618 cm ; positive HRTOFMS m/z [M+H] 171.0621 (calcd. for
1
50
C
9
H
9
F
2
O, 171.0621). H NMR data are in accordance with literature data.
ꢀ(3,4,5ꢀtrifluorophenyl)propꢀ2ꢀenꢀ1ꢀol (2j): colourless oil; UV (methanol) λmax
(log ε) 215 (3.23), 268 (0.34) nm; IR (neat) νmax 2895, 2765, 1623, 1608, 1423, 1225,
1
ꢀ1
+
1
9 8 3
125, 798, 623 cm ; positive HRTOFMS m/z [M+H] 189.0444 (calcd. for C H F O,
1
189.0444). H NMR (500 MHz, CDCl
3
): δ 7.00 (d, J = 8.1 Hz, 2H), 5.92 (ddd, J =
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4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
7.2, 10.2, 6.5 Hz, 1H), 5.35 (d, J = 17.1 Hz, 1H), 5.25 (d, J = 17.1 Hz, 1H), 5.12 (d,
13
J = 6.5 Hz, 1H); C NMR (125 MHz, CDCl
10.2, 110.1, 74.0.
1ꢀ(2,5ꢀdimethoxyphenyl)propꢀ2ꢀenꢀ1ꢀol (2k): colourless oil; UV (methanol) λmax
log ε) 221 (1.25), 254 (2.59) nm; IR (neat) νmax 3316, 2825, 2421, 1640, 1445, 1428,
3
): δ 139.0 (×2), 116.8 (×2), 110.3, 110.3,
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
ꢀ1
+
1327, 1245, 1228, 928, and 625 cm ; positive HRTOFMS m/z [M+H] 195.1022
1
(calcd. for C11
H
15
O
3
, 195.1021). H NMR data are in accordance with literature
49
data.
1ꢀ(2ꢀ(methoxymethoxy)phenyl)propꢀ2ꢀenꢀ1ꢀol (2l): colourless oil; UV (methanol)
λ
max (log ε) 218 (1.25), 287 (2.59), 311 (1.32) nm; IR (neat) νmax 3321, 1655, 1442,
ꢀ1
+
1428, 1321, 1212, 723, and 625 cm ; positive HRTOFMS m/z [M+H] 195.1025
1
(calcd. for C11
H
15
O
3
, 195.1021). H NMR data are in accordance with literature data.
5
1
1ꢀ(naphthalenꢀ2ꢀyl)propꢀ2ꢀenꢀ1ꢀol (2n): yellow oil; UV (methanol) λmax (log ε)
218 (2.28), 272 (1.58), 321 (1.98) nm; IR (neat) νmax 3310, 3115, 2823, 1643, 1476,
ꢀ1
+
1445, 1425, 1423, 1245, 1223, 723, and 625 cm ; positive HRTOFMS m/z [M+H]
1
185.0966 (calcd. for C13
H
13O, 185.0966). H NMR data are in accordance with
5
1
literature data.
25
(R)ꢀ1ꢀphenylpropꢀ2ꢀenꢀ1ꢀol (3a): colourless oil; [α]
+16.0 (c 0.1 mg/mL,
D
1
13
MeOH); the IR and H and C NMR data of 3a are those of 2a; positive HRTOFMS
+
m/z [M+H] 135.0812 (calcd. for C
9
H11O, 135.0810).
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(
D
R)ꢀ1ꢀ(2ꢀnitrophenyl)propꢀ2ꢀenꢀ1ꢀol (3b): colourless oil; [α] +49.0 (c 0.1
1
13
mg/mL, MeOH); the IR and H and C NMR data of 3b are identical with those of
+
2
b; positive HRTOFMS m/z [M+H] 180.0655 (calcd. for C
9
H
10NO
3
, 180.0655).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
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4
4
4
4
4
5
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5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
25
(R)ꢀ1ꢀ(pꢀtolyl)propꢀ2ꢀenꢀ1ꢀol (3c): colourless oil; [α]
+25.0 (c 0.1 mg/mL,
D
1
13
MeOH); the IR and H and C NMR data of 3c are identical with those of 2c;
+
positive HRTOFMS m/z [M+H] 149.0966 (calcd. for C10
H
13O, 149.0967).
25
(
D
R)ꢀ1ꢀ(4ꢀ(tertꢀbutyl)phenyl)propꢀ2ꢀenꢀ1ꢀol (3d): colourless oil; [α] +54.2 (c
1
13
0
.1 mg/mL, MeOH); the IR and H and C NMR data of 3d are identical with those
+
of 2d; positive HRTOFMS m/z [M+H] 191.1432 (calcd. for C H O, 191.1430).
13
19
2
5
(
D
R)ꢀ1ꢀ(4ꢀisopropylphenyl)propꢀ2ꢀenꢀ1ꢀol (3e): colourless oil; [α] +89.0 (c 0.1
1
13
mg/mL, MeOH); the IR and H and C NMR data of 3e are identical with those of
+
2
e; positive HRTOFMS m/z [M+H] 177.1279 (calcd. for C12
H
17O, 177.1279).
2
5
(R)ꢀ1ꢀ(4ꢀethylphenyl)propꢀ2ꢀenꢀ1ꢀol (3f): colourless oil; [α]
D
+32.0 (c 0.1
1
13
mg/mL, MeOH); the IR and H and C NMR data of 3f are identical with those of 2f;
+
positive HRTOFMS m/z [M+H] 163.1118 (calcd. for C11
H
15O, 163.1117).
2
5
(
D
R)ꢀ1ꢀ(4ꢀmethoxyphenyl)propꢀ2ꢀenꢀ1ꢀol (3g): colourless oil; [α] +23.0 (c 0.1
1
13
mg/mL, MeOH); the IR and H and C NMR data of 3g are identical with those of
+
2
g; positive HRTOFMS m/z [M+H] 165.0916 (calcd. for C10
H
13
O
2
, 165.0916).
2
5
(
R)ꢀ1ꢀ(4ꢀnitrophenyl)propꢀ2ꢀenꢀ1ꢀol (3h): colourless oil; [α]
+9.0 (c 0.1
D
1
13
mg/mL, MeOH); the IR and H and C NMR data of 3h are identical with those of
+
2
h; positive HRTOFMS m/z [M+H] 180.0655 (calcd. for C
9
H
10NO
3
, 180.0655).
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2
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(
D
R)ꢀ1ꢀ(2,5ꢀdifluorophenyl)propꢀ2ꢀenꢀ1ꢀol (3i): colourless oil; [α] +25.0 (c 0.1
1
13
mg/mL, MeOH); the IR and H and C NMR data of 3i are identical with those of 2i;
+
positive HRTOFMS m/z [M+H] 171.0625 (calcd. for C
9
H
8
F
2
O, 171.0621).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
5
(R)ꢀ1ꢀ(3,4,5ꢀtrifluorophenyl)propꢀ2ꢀenꢀ1ꢀol (3j): colourless oil; [α]
+23.1 (c
D
1
13
0
.1 mg/mL, MeOH); the IR and H and C NMR data of 3j are identical with those of
+
2
j; positive HRTOFMS m/z [M+H] 189.0448 (calcd. for C
9
H
7
F O
3 3
, 189.0444).
25
(
D
R)ꢀ1ꢀ(2,5ꢀdimethoxyphenyl)propꢀ2ꢀenꢀ1ꢀol (3k): colourless oil; [α] +42.0 (c
1
13
0
.1 mg/mL, MeOH); the IR and H and C NMR data of 3k are identical with those
+
of 2l; positive HRTOFMS m/z [M+H] 195.1021 (calcd. for C H O , 195.1021).
11
15
3
25
(
R)ꢀ1ꢀ(2ꢀ(methoxymethoxy)phenyl)propꢀ2ꢀenꢀ1ꢀol (3l): colourless oil; [α]
D
1
13
+39.0 (c 0.1 mg/mL, MeOH); the IR and H and C NMR data of 3l are identical
+
with those of 2l; positive HRTOFMS m/z [M+H] 195.1020 (calcd. for C11
H
15
O
3
,
195.1021).
25
(
R)ꢀ1ꢀ(naphthalenꢀ2ꢀyl)propꢀ2ꢀenꢀ1ꢀol (3n): yellow oil; [α] +8.0 (c 0.1 mg/mL,
D
1
13
MeOH); the IR and H and C NMR data of 3n are identical with those of 2n;
+
positive HRTOFMS m/z [M+H] 185.0968 (calcd. for C13
H
13O, 185.0966).
Synthesis of (E)ꢀ7,11ꢀdimethylꢀ3ꢀmethylenedodecaꢀ6,10ꢀdienoic acid (6). With
geraniol as starting material, compound 6 (3.16 g, 85%) was obtained via 5 (6.07 g,
2
5
9
0% yield) as an intermediate according to the previously reported method.
1
Compound 6: colorless oil; H NMR (500 MHz, CDCl
3
): δ 6.29 (s, 1H), 5.65 (s,
1H), 5.09 (m, 2H), 2.35 (t, J = 7.8 Hz, 2H), 2.16 (t, J = 7.8, 2H), 2.06 (t, J = 7.8 Hz,
H), 1.98 (t, J = 7.8 Hz, 2H), 1.78 (s, 3H), 1.60 (s, 6H); IR (neat) νmax 3212, 2945,
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ꢀ
1
+
2
915, 1694, 1635, 1425, 1304, 942 cm ; positive HRTOFMS m/z [M+H] 223.1692
(calcd. for C14 , 223.1693).
23 2
H O
Synthesis of 7aꢀ7n. By the same method as described for ganomycin I, compounds
3aꢀ3m were connected, and further converted into analogues 7aꢀ7n.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
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4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
R,E)ꢀ3ꢀ(4,8ꢀdimethylnonaꢀ3,7ꢀdienꢀ1ꢀyl)ꢀ5ꢀphenylfuranꢀ2(5H)ꢀone
(7a):
25
Colorless oil; [α]
D
+ 16.0 (c 0.1 mg/mL, MeOH); IR (neat) νmax 3210, 2873, 1662,
ꢀ1
+
1
3
7
632, 1445, 1423, 1237, 942, 712, 623 cm ; positive HRTOFMS m/z [M+H]
1
11.2012 (calcd. for C21
H
27
O
2
, 311.2011). H NMR (500 MHz, CDCl
3
): δ 7.37 (d, J =
.1 Hz, 2H), 7.25 (m, 3H) 7.08 (s, 1H), 5.87 (s, 1H), 5.12 (d, J = 7.5 Hz, 1H), 5.07 (d,
J = 7.5 Hz, 1H), 2.41 (t, J= 7.8 Hz, 2H), 2.32 (t, J=7.8 Hz, 2H), 2.05 (t, J = 7.8 Hz,
13
3
2H), 1.98 (t, J = 7.8 Hz, 2H), 1.67 (s, 3H), 1.59 (s, 6H); C NMR (125 MHz, CDCl ):
δ 173.9, 147.9, 136.9, 135.2, 133.5, 131.5, 129.1, 128.9 (×2), 126.5 (×2), 124.1, 122.6,
82.3, 39.6, 26.6, 25.7 (×2), 25.4, 17.7, 16.2.
(
R,E)ꢀ3ꢀ(4,8ꢀdimethylnonaꢀ3,7ꢀdienꢀ1ꢀyl)ꢀ5ꢀ(2ꢀnitrophenyl)furanꢀ2(5H)ꢀone
25
(
D
7b): Colorless oil; [α] + 38.0 (c 0.1 mg/mL, MeOH); IR (neat) νmax 2932, 1693,
ꢀ1
+
1
654, 1423, 1418, 1265, 1245, 942, 712 cm ; positive HRTOFMS m/z [M+H]
1
356.1856 (calcd. for C21
H26NO
4
, 356.1856). H NMR (500 MHz, CDCl
3
): δ 8.17 (d, J
=
8.1 Hz, 1H), 7.68 (t, J = 7.6 Hz) 7.53 (t, J = 8.1 Hz, 2H), 7.34 (s, 1H), 6.53 (s, 1H),
5
.06 (m, 2H), 2.38 (t, J = 7.8 Hz, 2H), 2.27 (t, J =7.8 Hz, 2H), 1.99 (t, J = 7.8 Hz, 2H),
13
1.94 (t, J = 7.8 Hz, 2H), 1.66 (s, 3H), 1.57 (s, 3H), 1.56 (s, 3H); C NMR (125 MHz,
CDCl ): δ 173.7, 148.1, 147.9, 137.0, 134.7, 133.6, 132.3, 131.5, 129.4, 126.8 125.3,
124.1, 122.3, 78.4, 39.6, 26.6, 25.7 (×2), 25.4, 17.7, 16.1.
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2
2
2
2
2
2
2
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(
R,E)ꢀ3ꢀ(4,8ꢀdimethylnonaꢀ3,7ꢀdienꢀ1ꢀyl)ꢀ5ꢀ(pꢀtolyl)furanꢀ2(5H)ꢀone
(7c):
25
Colorless oil; [α]
D
+ 21.0 (c 0.1 mg/mL, MeOH); IR (neat) νmax 2893, 2877, 1654,
ꢀ1
+
1
625, 1425, 1398, 1344, 1223, 945, 723 cm ; positive HRTOFMS m/z [M+H]
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
325.2164 (calcd. for C H O , 325.2162). H NMR (500 MHz, CDCl ): δ 7.18 (d, J =
22
29
2
3
8
1
2
.1 Hz, 2H), 7.13 (d, J = 8.1 Hz, 2H), 7.05 (s, 1H), 5.83 (s, 1H), 5.12 (t, J = 7.2 Hz,
H), 5.07 (t, J = 7.2 Hz, 1H), 2.40 (t, J = 7.8 Hz, 2H), 2.35 (s, 3H), 2.31 (t, J = 7.8 Hz,
13
H), 2.04 (t, J = 7.8 Hz, 2H), 1.99 (t, J =7.8 Hz, 2H), 1.67 (s, 3H), 1.59 (s, 6H); C
): δ 174.0, 147.9, 139.1, 136.8, 133.5, 132.1, 131.5, 129.8
×2), 126.6 (×2), 124.1, 122.6, 82.2, 39.6, 26.6, 25.7, 25.7, 25.4, 21.2, 17.7, 16.2.
R,E)ꢀ5ꢀ(4ꢀ(tertꢀbutyl)phenyl)ꢀ3ꢀ(4,8ꢀdimethylnonaꢀ3,7ꢀdienꢀ1ꢀyl)furanꢀ2(5H)ꢀo
NMR (125 MHz, CDCl
3
(
(
25
ne (7d): Colorless oil; [α]
D
+ 58.0 (c 0.1 mg/mL, MeOH); IR (neat) νmax 3212, 2923,
ꢀ1
2
907, 1694, 1631, 1423, 1418, 1304, 1245, 942, 725 cm ; positive HRTOFMS m/z
+
1
35 2 3
[M+H] 367.2632 (calcd. for C25H O , 367.2632). H NMR (500 MHz, CDCl ): δ
7
.40 (d, J = 8.1 Hz, 2H), 7.18 (d, J = 8.1 Hz, 2H), 7.06 (d, J = 11.9 Hz, 1H), 5.85 (s,
H), 5.12 (t, J = 6.5 Hz, 1H), 5.07 (t, J = 6.5 Hz, 1H), 2.40 (t, J = 7.8 Hz, 2H), 2.31 (t,
1
J = 7.8 Hz, 2H), 2.05 (t, J = 7.8 Hz, 2H), 1.98 (t, J = 7.8 Hz, 2H), 1.67 (s, 3H), 1.59 (s,
13
6
1
2
3
H), 1.31 (s, 9H); C NMR (125 MHz, CDCl ): δ 174.0, 152.3, 147.9, 136.8, 133.5,
32.1, 131.5, 126.4 (×2), 125.9 (×2), 124.1, 122.6, 82.2, 39.6, 34.7, 31.2 (×3), 26.6,
5.7, 25.7, 25.4, 17.7, 16.2.
(R,E)ꢀ3ꢀ(4,8ꢀdimethylnonaꢀ3,7ꢀdienꢀ1ꢀyl)ꢀ5ꢀ(4ꢀisopropylphenyl)furanꢀ2(5H)ꢀon
25
e (7e): Colorless oil; [α]
D
+ 48.0 (c 0.1 mg/mL, MeOH); IR (neat) νmax 3312, 2828,
ꢀ1
+
1645, 1623, 1465, 1425, 1371, 1225, 723, 623 cm ; positive HRTOFMS m/z [M+H]
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53.2481 (calcd. for C24
H
33
O
2
, 353.2481). H NMR (500 MHz, Acetoneꢀd
6
): δ 7.36 (s,
1H), 7.30 (d, J = 7.5 Hz, 2H), 7.24 (d, J = 7.5 Hz, 2H), 5.89 (s, 1H), 5.19(d, J = 7.5
Hz, 1H), 5.06 (d, J = 7.5 Hz, 1H), 2.92 (m, 1H), 2.36 (m, 4H), 2.09 (t, J = 7.8 Hz, 2H),
2.00 (t, J = 7.8 Hz, 2H), 1.65 (s, 3H), 1.62 (s, 3H), 1.59 (s, 3H), 1.24 (d, J = 7.5 Hz,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6H).
(
R,E)ꢀ3ꢀ(4,8ꢀdimethylnonaꢀ3,7ꢀdienꢀ1ꢀyl)ꢀ5ꢀ(4ꢀethylphenyl)furanꢀ2(5H)ꢀone
25
(
D
7f): Colorless oil; [α] + 54.0 (c 0.1 mg/mL, MeOH); IR (neat) νmax 2925, 2918,
ꢀ1
+
1
3
8
652, 1624, 1424, 1303, 1223, 923, 723 cm ; positive HRTOFMS m/z [M+H]
1
39.2320 (calcd. for C H O , 339.2319). H NMR (500 MHz, CDCl ): δ 7.20 (d, J =
23
31
2
3
.1 Hz, 2H), 7.16 (d, J = 8.1 Hz, 2H), 7.06 (s, 1H), 5.84 (s, 1H), 5.12 (t, J = 6.5 Hz,
1H), 5.07 (t, J= 6.5 Hz, 1H), 2.65 (q, J = 7.6 Hz, 2H), 2.41 (t, J = 7.8 Hz, 2H), 2.31 (t,
J = 7.8 Hz, 2H), 2.04 (t, J = 7.8 Hz, 2H), 1.98 (t, J = 7.8 Hz, 2H), 1.67 (s, 3H), 1.59 (s,
13
6H), 1.23 (t, J = 7.6 Hz, 3H); C NMR (125 MHz, CDCl
3
): δ 174.0, 147.9, 145.4,
36.8, 133.5, 132.4, 131.5, 128.4 (×2), 126.6 (×2), 124.1, 122.6, 82.3, 39.6, 28.6, 26.6,
5.7, 25.7, 25.4, 17.7, 16.2, 15.5.
R,E)ꢀ3ꢀ(4,8ꢀdimethylnonaꢀ3,7ꢀdienꢀ1ꢀyl)ꢀ5ꢀ(4ꢀmethoxyphenyl)furanꢀ2(5H)ꢀone
1
2
(
2
5
(
D
7g): Colorless oil; [α] + 78.0 (c 0.1 mg/mL, MeOH); IR (neat) νmax 3310, 2723,
ꢀ1
+
1
665, 1446, 1422, 1225, 823, 723, 625 cm ; positive HRTOFMS m/z [M+H]
1
3
41.2120 (calcd. for C H O , 341.2117). H NMR (500 MHz, CDCl ): δ 7.13 (t, J =
2
2
28
3
3
7.7 Hz, 2H), 7.07(d, J = 7.5 Hz, 2H), 6.81 (d, J = 7.7 Hz, 1H), 6.15(d, J = 7.5 Hz, 1H),
.03(d, J = 7.5 Hz, 1H), 4.99(d, J = 7.5 Hz, 1H), 3.42 (s, 3H), 2.38 (t, J = 7.8 Hz, 2H),
2.28 (t, J = 7.8 Hz, 2H), 2.02 (t, J = 7.8 Hz, 2H), 1.96 (t, J = 7.8 Hz, 2H), 1.67 (s, 3H),
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1
3
1
.59 (s, 3H), 1.58 (s, 3H); C NMR (125 MHz, CDCl
3
): δ 174.2, 153.1, 147.8, 136.9,
133.2, 131.6, 129.9, 126.6, 124.2, 122.7, 122.2, 121.2, 115.8, 78.1, 51.0, 39.6, 26.6,
2
5.7 (×2), 25.4, 17.7, 16.1.
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1
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4
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(R,E)ꢀ3ꢀ(4,8ꢀdimethylnonaꢀ3,7ꢀdienꢀ1ꢀyl)ꢀ5ꢀ(4ꢀnitrophenyl)furanꢀ2(5H)ꢀone
25
D
(7h): Colorless oil; [α]
+ 92.0 (c 0.1 mg/mL, MeOH); IR (neat) νmax 2932, 1692,
ꢀ1
+
1
648, 1425, 1418, 1248, 1225, 845, 725, 623 cm ; positive HRTOFMS m/z [M+H]
1
356.1856 (calcd. for C21
H
26NO
4
, 356.1856). H NMR (500 MHz, CDCl
3
): δ 8.07 (d, J
=
7.4 Hz, 2H), 7.48 (d, J = 7.4 Hz, 2H) 7.29 (m, 2H), 7.34 (s, 1H), 5.01 (t, J = 7.4 Hz,
1
H), 4.98 (t, J = 7.4 Hz, 1H), 2.34 (t, J = 7.8 Hz, 2H), 2.19 (t, J = 7.8 Hz, 2H), 1.92 (t,
J = 7.8 Hz, 2H), 1.88 (t, J = 7.8 Hz, 2H), 1.67 (s, 3H), 1.57 (s, 6H).
(R,E)ꢀ5ꢀ(2,5ꢀdifluorophenyl)ꢀ3ꢀ(4,8ꢀdimethylnonaꢀ3,7ꢀdienꢀ1ꢀyl)furanꢀ2(5H)ꢀon
25
e (7i): Colorless oil; [α]
D
+ 89.0 (c 0.1 mg/mL, MeOH); IR (neat) νmax 2893, 1623,
ꢀ1
+
1448, 1422, 1252, 1224, 842, 723, 625 cm ; positive HRTOFMS m/z [M+H]
1
3
1
47.1823 (calcd. for C H F O , 347.1823). H NMR (500 MHz, CDCl ): δ 7.33 (m,
21
24
2
2
3
H), 7.07 (s, 1H), 6.90 (t, J = 7.4 Hz, 2H), 6.22 (s, 1H), 5.13 (t, J = 7.4 Hz, 1H), 5.07
(
t, J = 7.4 Hz, 1H), 2.42 (t, J = 7.8 Hz, 2H), 2.32 (t, J =7.8 Hz, 2H), 2.05 (t, J = 7.8 Hz,
H), 1.99 (t, J = 7.8 Hz, 2H), 1.66 (s, 3H), 1.61 (s, 3H), 1.59 (s, 3H).
R,E)ꢀ3ꢀ(4,8ꢀdimethylnonaꢀ3,7ꢀdienꢀ1ꢀyl)ꢀ5ꢀ(3,4,5ꢀtrifluorophenyl)furanꢀ2(5H)ꢀ
2
(
2
5
one (7j): Colorless oil; [α]
+ 21.0 (c 0.1 mg/mL, MeOH); IR (neat) νmax 2923,
D
ꢀ1 1
2907, 1694, 1625, 1423, 926, 723 cm ; H NMR (500 MHz, CDCl
.1 Hz, 1H), 6.91 (t, J = 8.1 Hz, 2H), 5.77 (s, 1H), 5.08 (m, 2H), 2.41 (t, J = 7.8 Hz,
2H), 2.31 (t, J = 7.8 Hz, 2H), 2.03 (t, J = 7.8 Hz, 2H), 1.98 (t, J = 7.8 Hz, 2H), 1.67 (s,
3
): δ 7.02 (d, J =
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3
H), 1.59 (s, 6H); C NMR (125 MHz, CDCl ): δ 172.9, 146.4, 137.2, 134.6, 131.6,
124.0 (×2), 122.3 (×2), 110.9, 110.8, 110.7, 110.7, 80.2, 39.6, 26.6, 25.7, 25.6, 25.4,
+
1
7.7, 16.2; positive HRTOFMS m/z [M+H] 365.1726 (calcd. for C21
H
24
F
3
O
2
,
1
1
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4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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4
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6
7
8
9
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2
3
4
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7
8
9
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365.1723).
(
R,E)ꢀ5ꢀ(2,5ꢀdimethoxyphenyl)ꢀ3ꢀ(4,8ꢀdimethylnonaꢀ3,7ꢀdienꢀ1ꢀyl)furanꢀ2(5H)ꢀ
2
5
one (7k): Colorless oil; [α]
D
+ 44.2 (c 0.1 mg/mL, MeOH); IR (neat) νmax 3303,
ꢀ1
2
901, 1723, 1623, 1444, 1421, 1225, 825, 729, 627 cm ; positive HRTOFMS m/z
+
1
[
M+H] 371.2193 (calcd. for C23
H
31
O
4
, 371.2196). H NMR (500 MHz, CDCl
3
): δ
7
.19 (s), 6.83 (m, 2H) 6.76 (m, 1H), 6.19 (m, 1H), 5.11 (d, J = 7.5 Hz, 1H), 5.07 (d, J
=
7.5 Hz, 1H), 3.84 (s, 3H), 3.74 (s, 3H), 2.35 (t, J = 7.8 Hz, 2H), 2.27 (t, J = 7.8 Hz,
2H), 2.01 (t, J = 7.8 Hz, 2H), 1.95 (t, J = 7.8 Hz, 2H), 1.67 (s, 3H), 1.58 (s, 6H);
(
R,E)ꢀ3ꢀ(4,8ꢀdimethylnonaꢀ3,7ꢀdienꢀ1ꢀyl)ꢀ5ꢀ(2ꢀ(methoxymethoxy)phenyl)furanꢀ
2
5
2(5H)ꢀone (7l): Colorless oil; [α]
D
+ 71.0 (c 0.1 mg/mL, MeOH); IR (neat) νmax
ꢀ1
3
310, 2912, 1701, 1645, 1452, 1445, 1423, 1225, 823, 734, 625 cm ; positive
+
1
HRTOFMS m/z [M+H] 371.2196 (calcd. for C23
MHz, CDCl ): δ 7.28 (dd, J = 7.1, 2.5 Hz, 1H), 7.18 (m, 1H) 7.08 (m, 2H), 7.01 (m,
H), 6.26 (d, J = 7.5 Hz, 1H), 5.30 (s, 2H), 5.11 (d, J = 7.5 Hz, 1H), 5.07 (d, J = 7.5
31 4
H O , 371.2196). H NMR (500
3
1
Hz, 1H), 3.51 (s, 2H), 2.37 (t, J = 7.8 Hz, 2H), 2.28 (t, J = 7.8 Hz, 2H), 2.02 (t, J = 7.8
Hz, 2H), 1.96 (t, J = 7.8 Hz, 2H), 1.67 (s, 3H), 1.58 (s, 6H);
(R,E)ꢀ3ꢀ(4,8ꢀdimethylnonaꢀ3,7ꢀdienꢀ1ꢀyl)ꢀ5ꢀ(2ꢀhydroxyphenyl)furanꢀ2(5H)ꢀone
25
D
(
7m): Colorless oil; [α]
+ 54.0 (c 0.1 mg/mL, MeOH); IR (neat) νmax 3325, 3310,
ꢀ1
+
2923, 1628, 1455, 1423, 1223, 725, 623 cm ; positive HRTOFMS m/z [M+H]
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1
3
27.1883 (calcd. for C21
H
27
O
3
, 327.1882). H NMR (500 MHz, CDCl
3
): δ 7.21 (t, J =
7.7, 2H), 7.15 (d, J = 7.5, 1H), 6.93 (t, J = 7.5, 1H), 6.81 (d, J = 7.7, 1H), 6.23 (d, J =
.5 Hz, 1H), 5.11 (d, J = 7.5 Hz, 1H), 5.06(d, J = 7.5 Hz, 1H), 2.38 (t, J = 7.8 Hz, 2H),
7
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
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1
2
3
4
5
6
7
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0
2.29 (t, J = 7.8 Hz, 2H), 2.02 (t, J = 7.8 Hz, 2H), 1.96 (t, J =7.8 Hz, 2H), 1.67 (s, 3H),
13
1
.59 (s, 3H), 1.58 (s, 3H); C NMR (125 MHz, CDCl
3
): δ 174.1, 153.0, 147.7, 136.8,
1
33.1, 131.5, 129.8, 126.6, 124.2, 122.6, 122.1, 121.3, 115.8, 78.0, 39.6, 26.6, 25.7
(×2), 25.4, 17.7, 16.1.
(
R,E)ꢀ3ꢀ(4,8ꢀdimethylnonaꢀ3,7ꢀdienꢀ1ꢀyl)ꢀ5ꢀ(naphthalenꢀ2ꢀyl)furanꢀ2(5H)ꢀone
25
(7n): Colorless oil; [α]
+ 17.0 (c 0.1 mg/mL, MeOH); IR (neat) νmax 3121, 1643,
D
ꢀ1
+
1
624, 1465, 1445,1425, 1227, 825, 725, 623 cm ; positive HRTOFMS m/z [M+H]
1
361.2171 (calcd. for C25
H
28
O
2
, 361.2168). H NMR (500 MHz, CDCl
3
): δ 8.08 (d, J =
8
.4 Hz, 1H), 7.91 (d, J = 8.4 Hz, 1H), 7.86 (d, J = 8.4 Hz, 1H), 7.60 (t, J = 7.4 Hz,
1H), 7.56 (t, J = 7.4 Hz, 1H), 7.44 (m, 1H), 7.37 (m, 2H), 6.66 (s, 1H), 5.13 (t, J = 7.4
Hz, 1H), 5.06 (t, J = 7.4 Hz, 1H), 2.43 (t, J= 7.8 Hz, 2H), 2.33 (t, J=7.8 Hz, 2H), 2.02
(
t, J = 7.8 Hz, 2H), 1.97 (t, J =7.8 Hz, 2H), 1.66 (s, 3H), 1.59 (s, 3H), 1.58 (s, 3H);
1
3
3
C NMR (125 MHz, CDCl ): δ 173.8, 147.4, 136.9, 134.0, 133.8, 131.5, 131.3, 130.7,
1
29.6, 129.1, 126.9, 126.1, 125.4, 124.1, 123.5, 122.6, 122.5, 79.3, 39.6, 26.6, 25.7
(×2), 25.5, 17.7, 16.2.
Inhibition Assay against αꢀGlucosidase and HMGꢀCoA Reductase. The
preperation of αꢀglucosidase from rat small intestinal mucosa and HMGꢀCoA
24
reductase from pig liver was performed as previously described. The bioassay was
conducted using a 96ꢀwell plate, and the absorbance was determined at 405 nm using
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a Spectra Max 190 microplate reader (Molecular Devices Inc.). The enzyme assay
were performed in triplicate with different concentrations of individual compounds.
Each concentration was repeated three times in parallel. The control was prepared by
adding a phosphate buffer instead of tested compounds. The blank was prepared by
adding phosphate buffer instead of the αꢀglucosidase or HMGꢀCoA reductase. The
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inhibition rates (%) = [(OD control ꢀ OD control blank)ꢀ(OD testꢀOD test blank)]/ (OD control
OD control blank) × 100%.
ꢀ
In an another assay against αꢀglucosidase, the enzyme reaction product
4ꢀnitrophenol) was immediately dectected after incubation by a HPLC method. Each
(
concentration was repeated 3 times in parallel. The gradient elution started with 20%
acetonitrile in water (with 0.1 ‰ trifluoroacetic acid) and linearly increased to 100%
acetonitrile in 10 min, and followed by 5 min of reꢀequilibration. The inhibition rates
(%) =[(Area control ꢀ Area control blank)ꢀArea test]/ (Area control ꢀ Area control blank) × 100%.
IC50 values were determined by the inhibition rates of different concentration
gradients and calculated by Graphpad 6.0.
Animal care and experiments. All procedures were performed in accordance with
recommendations in the Guide for the Care and Use of Laboratory Animals of the
Institute of Microbiology, Chinese Academy of Sciences (IMCAS) Ethics Committee.
The protocol was approved by the Committee on the Ethics of Animal Experiments of
IMCAS (permit no. APIMCAS2017023). C57BL/6J and ob/ob mice were purchased
from the Experimental Animal Center, Chinese Academy of Medical Sciences. For
Abx mice study, male ob/ob mice were fed with ampicillin (1 g/L), vancomycin (0.5
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g/L), neomycin (0.5 g/L), gentamycin (100 mg/L), and erythromycin (10 mg/L) in
5
2
drinking water beginning at 7ꢀ8 weeks of age through 10ꢀ11 weeks of age. Cecum
observation and anaerobic cultivation of faecal samples confirmed successful
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53
depletion of gut microbiota after the antibiotic treatment. In the study with DIO
mice, the C57BL/6J mice were fed with a highꢀfat diet (60 kcal% fat, 20 kcal%
proteins and 20 kcal% carboꢀhydrates, Cat. D12492i, Research Diet, New Brunꢀswick,
NJ, USA) at the age of 8 weeks and maintained on the same diet for 8 weeks.
Eightꢀweekꢀold male ob/ob mice were kept on normal chow diet (NCD, 13.5 %
calories from fat) that was purchased from Vital River Laboratory Animal
Technology Co., Ltd. (Beijing, China). Mice were housed in a 12ꢀh darkꢀlight cycle,
with the dark cycle encompassing 7 p.m. to 7 a.m., and had free access to food and
water.
For the longꢀterm safety assay, 20 male and 20 female C57BL/6J mice were sorted
into four groups (n = 10 each, two male and two female groups) based on gender,
blood glucose level, and body weight. The treatment groups (one male, one female)
were given 30.0 mg/kg of 7d daily by gavage. The vehicle groups (one male, one
female) were given oral gavage of an equivalent volume of soybean (Pharmaceutical
excipients, Maya Regent, Zhejiang, China). Daily treatment continued for three
months. Behavior, survival, body weight, blood glucose, liver function, and organ
coefficients were recorded.
In assays with 7d on male ob/ob mice, animals were sorted into six groups (n = 10
each) based on their blood glucose levels and body weight. 7dꢀtreated groups were
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given with 7d (3.0 mg/kg, 1.0 mg/kg, and 0.3 mg/kg) daily by gavage.
Acarboseꢀtreated group was given with acarbose (10.0 mg/kg). The vehicle group was
treated with an oral gavage of an equivalent volume of soybean. Treatments continued
for 31 days. Body composition was assessed by using 7.5 MHz time domainꢀnuclear
magnetic resonance (TDꢀNMR; LF50 Minispec, Bruker, Rheinstetten, Germany).
In assays with 7d on the DIO mice, male C57BL/6J mice fed with highꢀfat diet
were sorted into five groups (n = 8 each) based on their blood glucose levels and body
weight. Compound 7d treated groups were daily administrated with 7d (3.0 mg/kg,
1
1
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1
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1
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1.0 mg/kg, and 0.3 mg/kg), Ganomycin Iꢀtreated group was given with ganomycin I
(3.0 mg/kg) daily. Vehicle groups (normal diet and highꢀfat diet) were treated with an
oral gavage of an equivalent volume of soybean. Treatment was continued for 5
weeks.
In the Abx experiments, sixteen male ob/ob mice and sixteen Abx male ob/ob mice
were sorted into five groups (n = 8 each) based on their blood glucose levels and body
weight. Treatment group of ob/ob mice was given 3.0 mg/kg/day 7d by mouth. The
vehicle group of ob/ob mice was given an equivalent volume of soybean by gavage.
The treatment group of Abx ob/ob mice was given 3.0 mg/kg/day of 7d, and the
vehicle group of Abx ob/ob mice was given an equivalent volume of soybean. All
treatments continued for 3 weeks.
Tissue sampling. After treatment, animals were anesthetized with diethyl ether
(Beijing Chemical Works, Beijing, China) and blood was sampled from the portal and
cava veins. After exsanguination, mice were euthanized by cervical dislocation.
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