Germacrane Sesquiterpenoids as a New Type of Anticardiac Fibrosis

Subscriber access provided by Nottingham Trent University

Article

Germacrane Sesquiterpenoids as a New Type of Anticardiac Fibrosis Agents Targeting Transforming Growth Factor # Type I Receptor (T#RI) Lan-Lan Lou, Fu-Qiang Ni, Lin Chen, Sharpkate Shaker, Wei Li, Rong Wang, Gui-Hua Tang, and Sheng Yin J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00708 • Publication Date (Web): 13 Aug 2019 Downloaded from pubs.acs.org on August 13, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

Germacrane Sesquiterpenoids as a New Type of Anticardiac Fibrosis Agents Targeting Transforming Growth Factor  Type I Receptor (TRI) Lan-Lan Lou,† Fu-Qiang Ni,† Lin Chen, Sharpkate Shaker, Wei Li, Rong Wang, Gui-Hua Tang, Sheng Yin School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, 510006, People’s Republic of China

1

ACS Paragon Plus Environment

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT: A germacrane sesquiterpenoid library containing 30 compounds (231) was constructed by structural modification of a major component aristolactone (1) from the traditional Chinese medicine Aristolochia yunnanensis. Compound 11 was identified as a promising anticardiac fibrosis agent by systematic screening of this library. 11 could inhibit the expression of fibronectin (FN), -smooth muscle actin (-SMA), and collagens in transforming growth factor  1 (TGF)-stimulated cardiac fibroblasts at a micromolar level and ameliorate myocardial fibrosis and heart function in abdominal aortic constriction (AAC) rats at 5 mg/kg dose. Mechanistic study revealed that 11 inhibited the TGFsmall mother against decapentaplegic (Smad) signaling pathway by targeting TGF type I receptor (IC50  14.9  1.6 nM). The structureactivity relationships (SARs) study indicated that the unsaturated -lactone ring and oxidation of C-1 was important to the activity. These findings may provide a new type of structural motif for future anticardiac fibrosis drug development.

2


Page 2 of 54

Page 3 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION Cardiac fibrosis is a common pathological consequence of various cardiovascular diseases, such as hypertensive heart disease and myocardial infarction.13 It is characterized by an excessive synthesis and pathological accumulation of extracellular matrix (ECM) proteins in myocardial tissue, and plays a key role in ventricular remodeling, causing myocardial stiffness and leading to diastolic heart failure.4,5 Plenty of evidence has shown that cardiac fibrotic alterations may be reversible,6 suggesting that rationally designed antifibrotic therapies are likely to be invaluable in curbing the cardiovascular diseases. However, there is no effective therapy for fibrotic diseases in general, largely because the underlying basis of fibrosis is too elusive. An important event in cardiac fibrosis is the transformation of fibroblasts into the more active, smooth muscle-like contractile cells, termed the myofibroblasts (MFs).79 MFs could secrete the highly contractile protein -smooth muscle actin (-SMA) forming contractile microfilaments and produce the major portion of ECM proteins, such as fibronectin (FN) and collagens.4,5 In recent years, the molecular pathways that promote myofibroblast transformation have been intensively studied, uncovering a complex signaling network involving several hormones and cytokines, such as transforming growth factor  (TGF), endothelin-1, angiotensin II (Ang II), connective tissue growth factor (CTGF), and platelet-derived growth factor (PDGF).10 Among them, the small mother against decapentaplegic (Smad)-dependent TGF signaling is considered as a governing regulator in myofibroblast transformation and cardiac fibrosis.11 TGF exists as a secreted latent form in the normal heart, and once activated could phosphorylate TGF type II receptor (TRII), which in turn phosphorylates 3



TGF type I receptor (TRI, also known as ALK5). The phosphorylated TRI could further phosphorylate Smad2/3 to produce phosphorylated Smad2/3, which then forms the complex with Smad4 to translocate into the nucleus, finally activating the down-stream gene transcription of ECM.1214 Therefore, agents that block the TGF/Smad signaling are considered as promising therapies in cardiac fibrosis diseases. TGF/Smad signaling inhibitor FT01115 and TRI inhibitors SM1616 and GW78838817 have been proven previously to be effective anticardiac fibrosis agents in animal models. However, the associated adverse cardiac effects hampered their further progression into clinical studies.18 Thus, the discovery of novel TGF/Smad signaling inhibitors with clear mechanisms of action and less side effects continues unabated. Traditional Chinese medicine (TCM) is a valuable source of antifibrotic agents.19 Compounds isolated from TCMs, such as tanshinone IIA,20 astragaloside IV,21 3-[(Z)-Pentadec-8-enyl] catechol,22 and oxymatrine (OMT)23 were widely reported to relieve the fibrosis in different organs via comprehensive mechanisms. In our previous study, a crude extract of a traditional Chinese medicine Aristolochia yunnanensis was found to possess therapeutic effects on myocardial fibrosis.24 Subsequent chemical investigation led to the isolation of a group of sesquiterpenoids.25 These sesquiterpenoids exhibited certain anticardiac fibrosis effects on TGF1-stimulated cardiac fibroblasts, and the mechanistic study revealed their inhibition on the TGF/Smad signaling pathway.26 However, the potential target, the structureactivity relationships (SARs), and the in vivo efficacy of these compounds are still unclear. In the current study, a germacrane sesquiterpenoid library (Figure 1) containing 30 compounds was constructed based on the structural modification of the major 4


Page 4 of 54

Page 5 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


component aristolactone (1) from this plant. Systematic screening of this library led to the identification of 11 as a promising anticardiac fibrosis agent with clear mechanism and pronounced efficacy in vivo. Herein, we report the construction, the anticardiac fibrosis evaluation, and SARs of this germacrane library, and also address the mechanism of the active compound 11. 1

10

14

3 5 15

7

R

O

13

12

1

O

O O

2

R

O

R=

13

R=

10 R = 28 R = O

9(10) R = Br  R = Cl 9(10) 9(10) R=F 

O

OR

O

OH N3

8 R = Et

15

R=

O

O

17

R=

O O

O

CF3 CF3

CF3

R

O

O

N

O

N

26 R =

N

24 R = OCHO 27 R =

N

21 R = OMe

O

22 R = OEt 23 R = O-iPr

19 R = N O

O 25 R =

20 R = OH

O O 11

NO2

O

9 18 R =

S

O O

R

O

O

O O

R=

10(14) R=F 

OH

N

O

16

29 R = ONO2 30 R = OMe 31 R = OEt

7 R = Me

Cl

O

14 R = OAc

O 3 4 5 6

12

11

O

O

O

9

O

O

Figure 1. Structures of the compounds (131) in germacrane library.

RESULTS Isolation and Structural Modification. The stems of A. yunnanensis (5 kg) was extracted with ethanol at room temperature (rt) to give a crude extract, which was suspended in water and then partitioned with EtOAc. Subsequent purification of the EtOAc fraction afforded 5 g of 1.

5



The synthetic routes from 1 to the structurally diverse germacranes were shown in Scheme 1. Briefly, the modifications mainly occurred at C-1, C-9, and the unsaturated

-lactone. The oxidation of 1 with meta-chloroperoxybenzoic acid (m-CPBA) generated the 1,10-epoxide derivative 2. The electrophilic halogenation reactions of the 1,10 in 1 with different halogenating reagents gave the C-1 halogenated derivatives 36 with the formation of 9 or 10,14. The treatment of 1 with sodium alkoxide (NaOMe or NaOEt) yielded the lactone ring opening products 7 and 8. The treatment of 2 in aqueous hydrochloric acid solution generated the epoxy ring opening product 10 and a by-product 9 with opposite stereochemistry at C-1 and 9. The subsequent acylation of the 1-OH in 10 with different acyl chlorides yielded corresponding esters 1217, while the oxidation of 1-OH with 2-iodoxybenzoic acid (IBX) afforded 11. The addition of 1-ketocarbonyl in 11 with hydroxylamine, then followed by acylation with different acyl chlorides produced the acylated oximes 18 and 19. The nucleophilic substitution reaction of allyl bromide 3 with different nucleophiles such as H2O, alcohols, formic acid, secondary amines, and sodium azide generated the corresponding alcohol 20, ethers 2123, ester 24, tertiary amines 2527, and azide 28, respectively. The nucleophilic substitution of bromine atom in 3 with AgNO3 afforded the nitrate ester 29, which reacted with methanol or ethanol to generate ethers 30 and 31, respectively. The relative configurations for the newly generated chiral centers in these compounds were assigned by NOESY experiments (Figure S1, Supporting Information), and the absolute configuration of all these compounds were correlated with 11 possessing a single-crystal X-ray structure [Cu Kα radiation, flack parameter 0.01 (7)] (Scheme 1).

6


Page 6 of 54

Page 7 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Scheme 1. Synthesis of Germacrane Derivativesa 1

(a) O

OR

14 3

5

O O

7, 8

13

(i)

(b)

11

O 1

O 2

12

O

O

O

O

10 (k)

(j)

O

R

Br

O

O

9

(c)

(d)

R

15

7

OH

OH

O

9

10

O

R

(e)

O

O O

4-6

O

O

3

O 20-27

O

O

12-17

(g)

(f) N3

(l)

O 11

R

OR

ONO2

O

(h)

O

O 28

aReagents

O

O 29

O

O

O

30, 31

O 18,19

ORTEP of 11

and reaction conditions: (a) NaOMe, CH3OH, rt, 30 min, 85% for 7; NaOEt, EtOH, rt, 1

h, 40% for 8; (b) m-CPBA, CH2Cl2, rt, 30 min, 92%; (c) N-bromosuccinimide (NBS), CH2Cl2, rt, 30 min, 82%; (d) N-fluorobenzenesulfonimid (NFSI), NaH, CH2Cl2, 025 C, 3 h, 35% for 5 and 32% for 6; N-chlorosuccinimide (NCS), CH2Cl2, rt, 1 h, 56% for 4; (e) dimethylsulfoxide (DMSO)/H2O  100:1 (v/v), 60 C, 14% for 20; MeOH, 50 C, 1 h, 75% for 21; EtOH, 60 C, 2 h, 71% for 22; NaH, IPA, 050 C, 3 h, 32% for 23; HCOOH, N, N-dimethylformamide (DMF), 80 C, 1 h, 15% for 24; piperidine/pyrrolidine/morpholine, DMF, 50 C, 1 h, 15% for 25, 54% for 26, 46% for 27; (f) NaN3, DMF, 60 C, 1 h, 14%; (g) AgNO3, EtOH, rt, 10 min, 40%; (h) MeOH, 40 C, 30 min, 68% for 30; EtOH, 50 C, 1 h, 52% for 31; (i) 1% HCl in MeOH (m/v), 50 C, 3 h, 12% for 9 and 68% for 10; (j) 2-chloronicotinyl/2-thiophenecarbonyl/acetyl/4-nitrobenzoyl/2-trifluoromethylbenzoyl/ 3,5-bis(trifluoromethyl)benzoyl chlorides, Pyr, 80 C, overnight, 52% for 12, 62% for 13, 93% for 14, 42% for 15, 50% for 16, 47% for 17; (k) IBX, DMSO, 60 C, 2 h, 92%; (l) NH2OH·HCl, NaOAc, MeOH, rt, 30 min, then acetyl/2-furoyl chlorides, Et3N, CH2Cl2, rt, 1 h, 43% for 18 and 37% for 19.

Preliminary Screening of 131 on the Cardiac Fibrotic Biomarker FN. FN is one of the major forms of ECM proteins, which plays an important role in fibroblast adhesion, growth, migration, and differentiation.2729 Overexpression of FN is a

7



pathological feature in myocardial fibrosis response.30,31 Thus, compounds 131 were first evaluated for FN inhibition in TGF1-stimulated cardiac fibroblasts by using Western blot assay. As shown in Figure 2, the FN levels were dramatically elevated by the stimulation of TGF1, and the treatments of most of these germacranes at 50 M significantly decreased the FN levels by 50%. Among them, compounds 3, 4, 11, 12, 15, 17, and 20 still kept the efficacy at the concentration of 10 M. Compound 11 was identified as the most active compound, decreasing the FN level to 26.1  4.1% at 10

M (Table S1). The cytotoxicity of 11 was also evaluated (Figure S2, Supporting Information), which showed no obvious inhibition on the viability of cardiac fibroblasts at the concentration of 25 M.

Figure 2. The relative contents of FN under the treatments of 131 in TGF1-stimulated cardiac fibroblasts. Cardiac fibroblasts were treated with 50 M or 10 M each of 131 and 10 ng/mL TGF1 for 12 h. Proteins from total lysates were subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis, followed by Western blot with indicated antibodies. GAPDH was used as a loading control. The blots were then quantified by densitometry. UND: untreated cells; Ctrl: cells treated with 10 ng/mL TGF1; Nif: nifedipine; SB: SB431542. N = 3 independent experiments.

StructureActivity Relationships. In general, the structural diversities mainly occurred at C-1, C-9, and the -lactone of 1, and nearly 25 compounds exhibited better 8


Page 8 of 54

Page 9 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


activity than the initial compound. The presence of the unsaturated lactone ring was essential for the activity, as the lactone ring opening products 7 and 8 exhibited a dramatic decrease of the activity. The presence of the halogen atoms at C-1 was beneficial to the activity, as shown by the increasing activity of 36 vs 1. Among them, the bromide 3 exhibited the best activity, Br  Cl  F. The oxidation of C-1 generally increased the activity, as shown by 2, 917, and 2931 vs 1. Among them, ketone 11 was the most active compound, while the activity of others were ranked as 1-O-acyl series (1217)  epoxide (2)  hydroxides (9 and 10)  nitrate ester (29) and ethers (30 and 31). In the 1-O-acyl series, the electro-withdrawing substituents was favorable to the activity as shown by 15  12  17  13  16  14. The presence of the acylated oximes at C-1 dramatically decreased the activity as shown by 18 and 19, while the presence of nitrine group increased the activity as shown by 28. The modification of C-9 generally had little influence on the activity as shown by the oxidated derivatives 2124 and aminated derivatives 2527. The 9-OH derivative 20 with an improved activity stood as an exceptional case in this series, while the further etherification or esterification of 9-OH led to a decrease of activity. 11 Inhibited the Expression of Fibrotic Biomarkers FN, -SMA, and Collagen Ι and ΙΙΙ in Cardiac Fibroblasts. The increased expressions of ECM (FN, collagen , and ) and contractile protein -SMA in cardiac fibroblasts were characterized as the onset of fibrosis.4,5 As 11 exhibited significant inhibition on FN production, its inhibitory effects on other biomarkers and the dose-dependent relationships were further investigated in TGF1-stimulated cardiac fibroblasts. As shown in Figure 3A, 11 could dose-dependently decrease the protein levels of FN, -SMA, and collagen I and III at 9



2.5, 5, and 10 M. Accordingly, the mRNA levels of these biomarkers were also downregulated (Figure S3, Supporting Information). This inhibition was further compared with the natural antifibrotic agent OMT (2 mM)23, clinic drug nifedipine (20

M)32, and the TGF signaling inhibitor SB431542 (10 M)33. As shown in Figure 3B, 11 was more potent than OMT and nifedipine, but weaker than SB431542. These results suggested that 11 was a potential antifibrotic agent in cardiac fibroblasts.

Figure 3. The inhibitory effects of 11 on fibrotic biomarkers FN, -SMA, and collagen Ι and ΙΙΙ in TGF1-stimulated cardiac fibroblasts. Cardiac fibroblasts were treated with indicated concentrations of compounds and 10 ng/mL TGF1 for 12 h. Protein levels were determined by Western blot. The blots were then quantified by densitometry. (A) The inhibitory effects of 11 on the protein levels of FN, -SMA, and collagen Ι and ΙΙΙ at the concentrations of 2.5, 5, and 10 M. (B) Comparisons of the antifibrotic effects of 11 (10 M) with OMT (2 mM), nifedipine (20 M), and SB431542 (10

10


Page 10 of 54

Page 11 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


M). **P  0.01 and ***P  0.001 vs. UND group; #P  0.05, ##P  0.01, and

###P

 0.001 vs.

TGF1-treated group. N  3 independent experiments.

11 Inhibited the Phosphorylation and Nuclear Translocation of Smad2/3 in TGF Signaling Pathway. In fibroblasts, Smad2 and Smad3 are two major downstream signaling mediators in the TGF signaling pathway, which could be phosphorylated by the activated TRI.79 To explore the underlying mechanism of 11 regarding this signaling, the phosphorylation levels of Smad2 and Smad3 were studied. As shown in Figure 4A, the phosphorylation levels of Smad2 and Smad3 in cardiac fibroblasts were significantly elevated under the stimulation of 10 ng/mL TGF1, whereas the treatment of 11 at 10 M effectively decreased the levels of phosphorylated Smad2 and Smad3 at different times (5, 10, and 15 min). During this process, no change was observed in total Smad2 and Smad3 levels (Figure S4, Supporting Information). Upon phosphorylation, Smad2/3 form complexes with Smad4 and then translocate into the nucleus, activating the gene expression of ECM proteins.1214 As 11 could inhibit the phosphorylation of Smad2/3, the distribution of Smad2 and Smad3 in nucleus and cytoplasm was further examined to confirm its regulation on the downstream cascade. As shown in Figure 4B and 4C, under the stimulation of TGF1, the contents of Smad2 and Smad3 were increased in nucleus, while the treatment of 11 at 10 M significantly decreased these contents in nucleus and correspondingly increased these contents in cytoplasm. These results indicated that 11 inhibited the phosphorylation and nuclear translocation of Smad2/3 in TGF signaling pathway and the regulation may occur in the upstream of Smad2/3 cascade.

11



Figure 4. The inhibitory effects of 11 on the phosphorylation and nuclear translocation of Smad2/3 in TGF signaling pathway. (A) The protein levels of phosphorylated Smad2 and Smad3 in TGF1-stimulated cardiac fibroblasts. Cardiac fibroblasts were treated with 11 (10 M) and 10 ng/mL TGF1 for indicated time. The protein levels were determined by Western blot and then quantified by densitometry. (B and C) The protein levels of Smad2 and Smad3 in nucleus and cytoplasm of cardiac fibroblasts. Cardiac fibroblasts were treated with 10 M of 11 and 10 ng/mL TGF1 for 5 min. Subcellular cytosolic and nuclear fractions of Smad2 and Smad3 were analyzed by Western blot (B) and immunofluorescence (C). Red fluorescence signals represent Smad2 or Smad3 proteins. Blue signals represent nuclear, Scale bar: 20 m. ***P  0.001 vs. UND group; #P  0.05, ##P  0.01, and

###P

 0.001 vs. TGF1-treated group. N  3 independent experiments.

11 Inhibited the Kinase Activity of TRI. TGF signaling is initiated when the extracellular TGF ligand binds to its heteromeric receptor complex formed by TRI and TRII. Then, the activated TRI exert its kinase-like activity to further trigger the 12


Page 12 of 54

Page 13 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


downstream Smad2/3-dependent pathway.1214 As 11 could inhibit the phosphorylation of Smad2/3, its inhibitory effects on the upstream regulators TRI and TRII were further investigated. The protein levels of TRI and TRII were firstly detected under the treatment of 11 at different concentrations, which showed no influence on the protein levels (Figure S5, Supporting Information), suggesting that the inhibition of Smad2/3 signaling might be related to the kinases activity of TRI and TRII. Thus, kinase assays were performed using the human recombinant TRI and TRII kinases produced in Sf9 insect cells (Figure S6, Supporting Information). 11 exhibited potent inhibition on the TRI kinase with an IC50 value at 14.9  1.6 nM, being stronger than the TRI inhibitor SB431542 (IC50  67.1  13.1 nM), while had no obvious inhibition on TRII kinase up to 100 M. These results indicated that 11 blocked the TGF/Smad signaling via inhibition of TRI. 11 Ameliorated the Cardiac Fibrosis in AAC Rats. To assess the in vivo antifibrotic efficacy of 11, an abdominal aortic constriction (AAC) rat model was set up by surgery method. 11 was administrated once daily intraperitoneally (ip) at 5 mg/kg dose to the rats for 4 weeks and nifedipine was used as the positive control (ip, 10 mg/kg). The histological sections of the rat hearts were stained to detect the expressions of fibrotic biomarkers. As shown in Figure 5AC, the depositions of collagens, -SMA, and FN in AAC group increased significantly compared with sham group, while the treatment with 11 or nifedipine decreased the depositions of these biomarkers. 11 exhibited better efficacy than nifedipine with the reduction of FN, collagens, and

-SMA by 90.7%, 65.6%, and 88.8%, respectively. Accordingly, the protein levels of collagens, -SMA, and FN in heart tissues of AAC rats were downregulated by the 13



treatment of 11 (Figure 5D). These results suggested that 11 could ameliorate cardiac fibrosis in AAC rats.

Figure 5. The inhibitory effects of 11 on the expressions of FN, collagens, and -SMA in AAC rats. (AC) The depositions of FN (A), collagens (B), and -SMA (C) in the histological sections of heart tissues. FN and -SMA were stained by immunohistochemistry staining; Collagens was stained by Masson’s trichrome staining. Masson’s trichrome staining and immunohistochemistry areas were

14


Page 14 of 54

Page 15 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


determined with ImageJ software. The sum of 6 fields per rat (n  8 rats) was used for statistical analysis to determine significant difference. Scale bar: 100 m (A), 200 m (B), 100 m (C). (D) The protein levels of FN, -SMA, and collagen  and  in heart tissues. Protein levels were determined by Western blot. The blots were then quantified by densitometry. **P  0.01 and ***P  0.001 vs. Sham group; #P  0.05, ##P  0.01, and ###P  0.001 vs. AAC group.

11 Inhibited the TGF/Smad Signaling Pathway in AAC Rats. Consistent with the results from the in vitro experiments, the levels of the phosphorylated Smad2 and Smad3 were increased significantly in the heart tissues of the AAC rats, and were remarkably suppressed by the administration of 11 (Figure 6A and 6B). Besides, the elevated levels of Smad3 and Smad2 in the nucleus of AAC rat hearts were significantly decreased by the treatment of 11 (Figure 6C). These results suggested that 11 could inhibit the TGF/Smad signaling pathway in vivo.

15



Figure 6. The inhibitory effects of 11 on the phosphorylation and nuclear translocation of Smad2/3 in AAC rats. (A) The protein levels of phosphorylated Smad2/3 in heart tissue of AAC rats were determined by Western blot. (B) The protein levels of phosphorylated Smad2/3 in heart tissue of AAC rats were determined by immunohistochemistry staining of the heart histological section. Scale bar: 100 m. Immunohistochemistry areas were determined with ImageJ software. The sum of 6 fields per rat (n  8 rats) was used for statistical analysis to determine significant difference. (C) The contents of Smad2/3 in nucleus and cytoplasm of heart tissue were determined by Western blot. **P   and ***P  0.001 vs. Sham group; #P  

##P

 0.01, and ###P  0.001 vs. AAC group.

11 Improved the Heart Function of AAC Rats. Five weeks after AAC surgery, a significant enlargement of the heart volume (Figure 7A and 7B) and fibroblasts hypertrophy (Figure 7C) were observed in AAC rats. Accordingly, the ratios of heart weight to body weight (HW/BW), left ventricle weight to body weight (LVW/BW), and heart weight to tibia length (HW/TL) were increased in AAC group (Figure 7D). Echocardiography also revealed that the left ventricular posterior walls of AAC rats were increased, the internal dimensions in diastole and systole were decreased, and the ejection fraction (EF) and fractional shortening (FS) were increased significantly (Table 1). These results indicated that the left ventricular hypertrophy and impaired cardiac systolic function was successfully induced by AAC. The treatment of 11 at the 5 mg/kg in AAC rats ameliorated the cardiac hypertrophy, increased the left ventricle dimension, and ameliorated the deterioration of left ventricular performance, with the similar efficacy being comparable to the clinical drug nifedipine (10 mg/kg).

16


Page 16 of 54

Page 17 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 7. The therapeutic effects of 11 on the cardiac hypertrophy and cardiac function in AAC rats. (A) The gross morphologic examination of the rat hearts. (B and C) The hematoxylin-eosin (HE)-stained cross sections of rat hearts. Scale bar: 2 mm (B), 50 m (C). (D) the ratios of HW/TL, HW/BW, and LVW/BW in each group of rats. (E) Representative transthoracic M-mode echocardiograms of the rats in each group. *P  0.05 and **P  0.01 vs. Sham group; #P  0.05 and ##P

 0.01 vs. AAC group.

Table 1. Analysis of Cardiac Function of AAC Rats by Echocardiography (5 Weeks After AAC Surgery) Group LVAWd (mm) LVAWs (mm) LVIDd (mm) LVIDs (mm) LVPWd (mm) LVPWs (mm) EF (%) FS (%)

Sham

AAC

1.84  0.24 2.97  0.22 8.61  0.30 5.00  0.40 1.85  0.11 2.90  0.25 70.75  4.15 41.94  3.41

2.48  3.92  0.42** 7.60  0.63** 3.99  0.41** 2.33  0.31** 3.62  0.26** 79.59  3.13** 48.17  4.69* 0.42**

17

AAC  Nif 2.02  3.23  0.12## 8.32  0.43# 4.69  0.40# 1.97  0.22# 3.19  0.22## 72.60  5.37# 42.79  4.13


0.17##

AAC  11 2.11  0.11# 3.46  0.25# 8.30 0.53# 4.74  0.77# 2.00  0.23# 3.23  0.33# 71.38  6.94## 42.54  5.67


Abbreviations: LVAW, left ventricular anterior wall thickness; LVID, left ventricular internal diameter; LVPW, left ventricular posterior wall thickness; -d, diastolic; -s, systolic; EF, ejection fraction; FS, fractional shortening. All values are presented as means  standard deviation (SD). n  8, *P  0.05, and **P  0.01 vs. Sham group; #P  0.05 and ##P  0.01 vs. AAC group.

DISCUSSION AND CONCLUSIONS Sesquiterpenoids from the TCM A. yunnanensis were previously reported to possess certain anticardiac fibrosis effects on TGF1-stimulated cardiac fibroblasts.26 However, the potential target, the SARs, and the in vivo efficacy of these compounds are still unclear. In the current study, a germacrane sesquiterpenoid library containing 30 compounds was constructed based on the structural modification of the major component aristolactone from this plant. Systematic screening of this library led to the identification of 11 as a promising anticardiac fibrosis agent with clear mechanism and pronounced efficacy in vivo. 11 could inhibit the accumulations of ECM (FN and collagens) and -SMA in TGF1-stimulated cardiac fibroblasts at a micromolar level and ameliorate myocardial fibrosis and heart function in AAC rats at 5 mg/kg dose. Mechanistic study revealed that 11 blocked the TGF/Smad signaling pathway via inhibition of TRI (IC50  14.9  1.6 nM). The SARs study revealed that the unsaturated -lactone ring and oxidation of C-1 was important to the activity. It is worth noting that 11 exhibited a better enzymatic activity but a poorer cellular activity than that of SB431542, suggesting that the efforts towards the improvement of the cell

18


Page 18 of 54

Page 19 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


membrane permeability of 11 are still required in future modification. These findings may shed a light on future drug development for cardiac fibrosis and related diseases.

EXPERIMENTAL SECTION General. X-ray data were collected using an Angilent Xcalibur Nova X-ray diffractometer. Melting point was measured on a X-4 melting instrument and uncorrected. Optical rotations were measured on a Perkin-Elmer 341 polarimeter. UV spectra were recorded on a Shimadzu UV-2450 spectrophotometer. IR spectra were determined on a Bruker Tensor 37 infrared spectrophotometer with KBr disks. NMR spectra were measured on a Bruker AM-400 or AM-500 spectrometer at 25 C. HRESIMS were carried out on a Finnigan LCQ Deca instrument. A Shimadzu LC-20AT equipped with a SPD-M20A PDA detector was used for HPLC, and a YMC-pack ODS-A column (250  10 mm, S-5  m, 12 nm) was used for semipreparative HPLC separation. A chiral column (Phenomenex Lux, cellulose-2, 250  10 mm, 5  m) was used for chiral separation. Aluminium oxide neutral (100200 mesh, Sinopharm Chemical Reagent Co. Ltd.), Silica gel (300400 mesh, Qingdao Haiyang Chemical Co. Ltd.) and reversed-phase C18 (Rp-C18) silica gel (12 nm, S-50

 m, YMC Co. Ltd.) were used for column chromatography (CC). All commercial reagents (from Aladdin, JK Chemical Ltd, and Acros) were used without further purification. All solvents were of analytical grade (Guangzhou Chemical Reagents Company, Ltd.). The purity of the samples was determined by HPLC, conducted on a Shimadzu LC-20AT series system with a chiral column (Phenomenex Lux, cellulose-2, 250  10 mm, 5 m). The samples were eluted with a MeCNH2O mixture (90:10) at a 19



flow rate of 3 mLmin. The purity of all biologically evaluated compounds is greater than 95%. The pan assay interference compounds (PAINS) analysis on 11 was discussed (Supporting Information), which suggested that 11 was a genuine active compound. Plant Material. Stems of Aristolochia yunnanensis Franch. (synonym: Aristolochia griffithii) were collected in October 2010 from Yunnan Province, P. R. China, and were identified by Prof. You-Kai Xu of Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences. A voucher specimen (accession number: NMX201010) has been deposited at the School of Pharmaceutical Sciences, Sun Yat-sen University. Extraction and Isolation of 1. The air-dried powder of the stems of A. yunnanensis (5 kg) was extracted with 95% EtOH (4  5 L) at rt to give a crude extract (182 g). The extract was suspended in H2O (1 L) and then partitioned with EtOAc (4  1 L) to yield the corresponding portion. The EtOAc (95 g) was separated by column chromatography on C18 reversed-phase silica gel (RP-C18) eluted with a MeOH/H2O gradient (4:6  10:0) to give three fractions (Fr. IIII). Fr. II (50 g) was subjected to silica gel CC (petroleum ether (PE)/EtOAc, 15:1  1:1) to afford Fr. IIAIIB. Then Fr. IIA (34 g) was subjected to RP-C18 silica gel CC (MeOH/H2O, 7:3  10:0) to afford 1 (5 g). (1Z,4E,8S,9S)-10-oxabicyclo[7.2.1]dodeca-1(12),4-dien-11-one,5-methyl-8-(1-methyl ethenyl) (1). The spectroscopic data was identical to that reported.34 Preparation of 2 by Epoxidation of 1. To a stirred solution of 1 (100 mg, 0.43 mmol) in 5 mL of CH2Cl2 was added m-CPBA (81.9 mg, 0.51 mmol) at rt. After 30 min, excess saturated NaHCO3 solution was added to the mixture, while stirring was continuted for another 5 min. The resulting solution was washed with H2O and then 20


Page 20 of 54

Page 21 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


extracted with CH2Cl2. The concentrated residue was purified with silica gel flash column chromatography (PE:EtOAc  3:1) to yield 98.3 mg of 2 (92%). (1Z,4R,6S,9S,10S)-6-methyl-9-(1-methylethenyl)-5,11-dioxatricyclo[8.2.1.04,6]tridec1(13)-en-12-one (2). White powder; []25D 8.6 (c 0.2, MeOH); UV (CH2Cl2) max (log

) 228 (3.49) nm; IR (KBr) max 2954, 2922, 2852, 1749, 1641, 1459, 1376, 1261, 1097, 800, 751 cm1; 1H NMR (CDCl3, 400 MHz) H 7.07 (1H, s, H-5), 5.01 (1H, s, H-6), 4.85 (1H, s, H-12a), 4.75 (1H, m, H-12b), 2.69 (1H, ddd, J = 13.4, 7.2, 0.7 Hz, H-3a), 2.50 (1H, m, H-3b), 2.45 (1H, m, H-1), 2.41 (1H, m, H-7), 2.26 (1H, dd, J  14.6, 10.3 Hz, H-9a), 2.03 (1H, ddt, J  13.3, 7.5, 0.8 Hz, H-2a), 1.81 (3H, s, H3-13), 1.76 (1H, m, H-8a), 1.63 (1H, m, H-8b), 1.56 (1H, m, H-2b), 1.13 (3H, s, H3-14), 0.89 (1H, dd, J  14.6, 10.4 Hz, H-9b); 13C NMR (CDCl3, 100 MHz) C 173.5 (C-15), 151.5 (C-5), 149.3 (C-11), 132.5 (C-4), 111.3 (C-12), 82.5 (C-6), 68.6 (C-1), 60.9 (C-10), 52.9 (C-7), 38.1 (C-9), 23.4 (C-8), 21.6 (C-2), 21.3 (C-3), 20.6 (C-13), 17.3 (C-14); HRESIMS m/z 271.1305 [M + Na]+ (calcd for C15H20O3Na, 271.1306). Preparation of 3 and 4 by Halogenation of 1. To a solution of 1 (20 mg, 0.086 mmol) in 1 mL of CH2Cl2 was added NBS (22.9 mg, 0.13 mmol) or NCS (17.2 mg, 0.13 mmol). After being stirred at rt for 0.51 h, the resulting solution was concentrated via evaporation. The corresponding residues were purified on an aluminium oxide neutral flash column (PE:EtOAc  5:1) to obtain 21.9 mg of 3 (82%), and on a silica-gel column (PE:EtOAc  5:1) to obtain 15 mg of 4 (56%). (1Z,4R,5E,8S,9S)-10-Oxabicyclo[7.2.1]dodeca-5,12-dien-11-one,4-bromo-5-methyl8-(1-methylethenyl) (3). White powder; []25D 8.6 (c 0.3, CH2Cl2); UV (CH2Cl2) max (log ) 228 (3.42) nm; IR (KBr) max 2921, 2852, 1748, 1647, 1440, 1066, 899, 748 21



cm1; 1H NMR (CDCl3, 500 MHz) H 6.80 (1H, s, H-5), 5.07 (1H, s, H-6), 5.06 (1H, m, H-9), 5.00 (1H, s, H-12a), 4.90 (1H, s, H-12b), 4.76 (1H, dd, J  9.5, 2.3 Hz, H-1), 2.71 (1H, m, H-8a), 2.66 (2H, m, H2-3), 2.51 (1H, dd, J  11.8, 4.4 Hz, H-7), 2.19 (1H, dt, J  14.4, 2.1 Hz, H-8b), 2.10 (2H, m, H2-2), 1.86 (3H, s, H3-13), 1.76 (3H, s, H3-14); 13C NMR (CDCl3, 125 MHz) C 173.0 (C-15), 150.4 (C-5), 146.7 (C-11), 135.2 (C-10), 132.7 (C-9), 131.8 (C-4), 112.8 (C-12), 83.6 (C-6), 60.8 (C-1), 45.4 (C-7), 28.7 (C-2), 28.6 (C-8), 26.9 (C-3), 21.3 (C-13), 12.1 (C-14); HRESIMS m/z 333.0461 [M + Na]+ (calcd for C15H19O2BrNa, 333.0471). (1Z,4R,5E,8S,9S)-10-Oxabicyclo[7.2.1]dodeca-5,12-dien-11-one,4-chloro-5-methyl-8 -(1-methylethenyl) (4). White powder; []25D 9.4 (c 0.3, CH2Cl2); UV (CH2Cl2) max (log ) 220 (3.34) nm; IR (KBr) max 2954, 2921, 2852, 1749, 1647, 1459, 1376, 1261, 1096, 1020, 751 cm1; 1H NMR (CDCl3, 500 MHz) H 6.77 (1H, s, H-5), 5.07 (1H, s, H-6), 5.00 (1H, s, H-12a), 4.98 (1H, m, H-9), 4.90 (1H, t, J  1.9 Hz, H-12b), 4.58 (1H, dd, J  12.0, 2.2 Hz, H-1), 2.75 (1H, ddd, J  26.5, 11.8, 2.6 Hz, H-8a), 2.68 (2H, m, H2-3), 2.59 (1H, m, H-2a), 2.51 (1H, dd, J  11.9, 4.4 Hz, H-7), 2.19 (1H, dt, J  14.4, 1.8 Hz, H-8b), 1.93 (1H, m, H-2b), 1.89 (3H, s, H3-13), 1.71 (3H, s, H3-14); 13C NMR (CDCl3, 125 MHz) C 173.0 (C-15), 150.2 (C-5), 146.8 (C-11), 134.4 (C-10), 133.2 (C-9), 131.8 (C-4), 112.7 (C-12), 83.6 (C-6), 68.3 (C-1), 45.7 (C-7), 28.5 (C-8), 28.0 (C-2), 26.1 (C-3), 21.3 (C-13), 11.0 (C-14); HRESIMS m/z 289.0966 [M + Na]+ (calcd for C15H19O2ClNa, 289.0975). Preparation of 5 and 6 by Fluorination of 1. To a solution of 1 (50 mg, 0.21 mmol) in 2 mL of CH2Cl2 under N2 atmosphere was added 60% NaH (12.9 mg, 0.32 mmol) at 0 C. After being stirred for 30 min, NFSI (81.4 mg, 0.26 mmol) dissolved in CH2Cl2 22


Page 22 of 54

Page 23 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


was added dropwise to the solution. Then the mixture was stirred at rt for another 5 h, followed by the addition of 2 mL of H2O. The concentrated mixture was purified with semi-preparative HPLC (MeCN/H2O  70:30) to yield 5 (10.2 mg, 35%, tR 8.4 min), along with 10.5 mg of 6 (32%, tR 8.7 min). (1Z,4R,5E,8S,9S)-10-Oxabicyclo[7.2.1]dodeca-5,12-dien-11-one,4-fluoro-5-methyl-8 -(1-methylethenyl) (5). White powder; []25D 7.5 (c 0.2, MeOH); UV (CH2Cl2) max (log ) 227 (3.21) nm; IR (KBr) max 2955, 2921, 2852, 1745, 1646, 1459, 1376, 1261, 1095, 1021, 780, 751 cm1; 1H NMR (CDCl3, 500 MHz) H 6.73 (1H, s, H-5), 5.07 (1H, s, H-6), 5.00 (1H, s, H-12a), 4.94 (1H, m, H-9), 4.93 (0.5H, m, H-1), 4.90 (1H, s, H-12b), 4.84 (0.5H, dd, J  2.3, 1.2 Hz, H-1), 2.73 (1H, m, H-8a), 2.63 (2H, m, H2-3), 2.53 (1H, dd, J  11.9, 4.4 Hz, H-7), 2.37 (1H, ddd, J  25.8, 12.9, 4.7 Hz, H-2a), 2.20 (1H, t, J  11.9 Hz, H-8b), 1.89 (3H, s, H3-13), 1.84 (1H, m, H-2b), 1.63 (3H, s, H3-14); 13C

NMR (CDCl3, 125 MHz) C 172.9 (C-15), 150.1 (C-5), 146.8 (C-11), 133.8 (C-10),

133.7 (C-10), 133.1 (C-9), 133.0 (C-9), 131.4 (C-4), 112.7 (C-12), 98.8 (C-1), 97.4 (C-1), 83.5 (C-6), 46.0 (C-7), 27.9 (C-8), 23.5 (C-3), 23.3 (C-3), 23.2 (C-2), 23.1 (C-2), 21.3 (C-13), 10.2 (C-14); HRESIMS m/z 273.1261 [M + Na] (calcd for C15H19O2FNa, 273.1262). (1Z,4R,5E,8S,9S)-10-Oxabicyclo[7.2.1]dodeca-1(12)-en-11-one,4-fluoro-5-methylen e-8-(1-methylethenyl) (6). White powder; []25D 95.6 (c 0.3, MeOH); UV (CH2Cl2)

max (log ) 228 (3.50) nm; IR (KBr) max 2954, 2926, 2856, 1753, 1648, 1276, 1261, 1053, 902, 751 cm1; 1H NMR (CDCl3, 500 MHz) H 7.05 (1H, s, H-5), 5.50 (1H, d, J  1.3 Hz, H-14a), 5.17 (1H, d, J  1.3 Hz, H-14b), 5.03 (1H, s, H-6), 4.94 (1H, m, H-12a), 4.85 (1H, s, H-12b), 4.69 (0.5H, dd, J  5.2, 9.1 Hz, H-1), 4.60 (0.5H, dd, J  6.4, 2.7 23



Page 24 of 54

Hz, H-1), 2.56 (1H, m, H-3a), 2.51 (1H, dd, J  11.9, 3.1 Hz, H-7), 2.47 (1H, m, H-9a), 2.41 (1H, m, H-2a), 2.26 (1H, m, H-3b), 2.21 (1H, m, H-2b), 1.85 (3H, s, H3-13), 1.83 (1H, m, H-8a), 1.74 (1H, m, H-8b), 1.41 (1H, dd, J  17.3, 6.6 Hz, H-9b);

13C

NMR

(CDCl3, 125 MHz) C 174.2 (C-15), 149.0 (C-5), 147.3 (C-11), 146.3 (C-10), 146.2 (C-10), 133.3 (C-4), 115.2 (C-14), 115.1 (C-14), 112.3 (C-12), 94.5 (C-1), 93.1 (C-1), 82.8 (C-6), 52.1 (C-7), 34.1 (C-2), 33.9 (C-2), 31.1 (C-9), 31.0 (C-9), 24.2 (C-8), 21.4 (C-13), 19.0 (C-3), 18.9 (C-3); HRESIMS m/z 273.1261 [M + Na]+ (calcd for C15H19O2FNa, 273.1269). Preparation of 7 and 8 by Alcoholysis of 1. To a solution of 1 (20 mg, 0.086 mmol) in methanol or ethanol was added NaOMe or NaOEt, respectively. After the mixture was stirred at rt for 3050 min. The corresponding concentrated residue was purified with semi-preparative HPLC (MeCN/H2O  65:35) to afford 19.3 mg of 7 (85%, tR 12.6 min) or 9.6 mg of 8 (40%, tR 14.2 min), respectively. Methyl(1R,8S,4E)-5-methyl-9-oxo-8-(1-methylethenyl)cyclodec-4-ene-1-carboxylate (7). White powder; []25D 145 (c 0.3, CH2Cl2); UV (CH2Cl2) max (log ) 228 (3.27) nm; IR (KBr) max 2950, 2926, 2852, 1733, 1695, 1449, 1436, 1260, 1200, 1177, 1107, 898, 790 cm1; 1H NMR (CDCl3, 400 MHz) H 5.37 (1H, s, H-1), 4.80 (1H, s, H-12a), 4.77 (1H, s, H-12b), 3.09 (1H, m, H-7), 3.03 (1H, m, H-5a), 2.97 (1H, m, H-5b), 2.53 (1H, m, H-4), 2.45 (1H, m, H-2a), 2.40 (1H, m, H-8a), 2.05 (1H, m, H-3a), 2.04 (1H, m, H-9a), 1.93 (1H, m, H-9b), 1.83 (1H, m, H-3b), 1.83 (1H, m, H-2b), 1.62 (3H, s, H3-13), 1.54 (1H, m, H-8b), 1.47 (3H, s, H3-14), -COOCH3: 3.67 (3H, s); 13C NMR (CDCl3, 100 MHz) C 205.0 (C-6), 175.1 (C-15), 143.5 (C-11), 139.5 (C-10), 124.9 (C-1), 112.8

24


Page 25 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(C-12), 61.5 (C-7), 42.8 (C-5), 40.5 (C-9), 39.0 (C-4), 32.1 (C-8), 25.6 (C-3), 24.4 (C-2), 19.9 (C-13), 15.9 (C-14), -COOCH3: 51.7; HRESIMS m/z 287.1618 [M + Na]+ (calcd for C16H24O3Na, 287.1616). Ethyl(1R,8S,4E)-5-methyl-9-oxo-8-(1-methylethenyl)cyclodec-4-ene-1-carboxylate (8). White powder; []25D 150.6 (c 0.3, CH2Cl2); UV (CH2Cl2) max (log ) 228 (3.21) nm; IR (KBr) max 2926, 2853, 1748, 1702, 1418, 1259, 1093, 1074, 749, 735 cm1; 1H NMR (CDCl3, 400 MHz) H 5.16 (1H, d, J  9.9 Hz, H-1), 4.82 (1H, s, H-12a), 4.76 (1H, s, H-12b), 3.11 (1H, d, J  11.4 Hz, H-7), 2.86 (1H, m, H-5a), 2.81 (1H, m, H-4), 2.75 (1H, dd, J  16.5, 8.7 Hz, H-5b), 2.46 (1H, ddd, J  25.0, 13.2, 3.2 Hz, H-8a), 2.19 (2H, m, H2-2), 2.11 (1H, m, H-3a), 2.08 (1H, m, H-9a), 1.92 (1H, ddd, J  16.3, 12.7, 3.5 Hz, H-9b), 1.65 (3H, s, H3-13), 1.57 (1H, m, H-3b), 1.52 (1H, m, H-8b), 1.45 (3H, s, H3-14), -COOCH2CH3: 4.10 (2H, q, J  7.0 Hz), 1.23 (3H, m); 13C NMR (CDCl3, 100 MHz) C 205.0 (C-6), 176.3 (C-15), 143.2 (C-11), 139.2 (C-10), 125.6 (C-1), 113.1 (C-12), 62.9 (C-7), 45.9 (C-5), 40.8 (C-9), 40.2 (C-4), 32.7 (C-3), 32.3 (C-8), 27.5 (C-2), 19.8 (C-13), 15.8 (C-14), -COOCH2CH3: 60.7, 14.2; HRESIMS m/z 301.1774 [M + Na]+ (calcd for C17H26O3Na, 301.1772). Preparation of 9 and 10 by Hydrolysis of 2. To a stirred solution of 2 (100 mg, 0.43 mmol) in 5 mL of MeOH was added excess 1% HCl solution at rt. The temperature was allowed to maintain at 50 C for 3 h. And then, the resultant concentrated mixture was purified with silica gel flash column chromatography (PE:EtOAc  4:1  3:1) to afford 12 mg of 9 (12%), along with 68 mg of 10 (68%). (1Z,4S,5Z,8S,9S)-10-Oxabicyclo[7.2.1]dodeca-5,12-dien-11-one,4-hydroxy-5-methyl8-(1-methylethenyl) (9). White powder; []25D 29.5 (c 0.2, MeOH); UV (CH2Cl2) max 25



(log ) 227 (3.16) nm; IR (KBr) max 3291, 2955, 2922, 2852, 1741, 1461, 1275, 1262, 751 cm1; 1H NMR (CDCl3, 500 MHz) δH 6.77 (1H, s, H-5), 5.23 (1H, dd, J  11.7, 3.9 Hz, H-9), 5.13 (1H, s, H-6), 4.90 (2H, s, H2-12), 4.41 (1H, dd, J  10.3, 5.2 Hz, H-1), 2.63 (1H, m, H-3a), 2.49 (1H, d, J  11.3 Hz, H-7), 2.41 (1H, dd, J  11.7, 2.3 Hz, H-8a), 2.25 (1H, m, H-3b), 2.19 (1H, m, H-2a), 1.84 (3H, s, H3-13), 1.80 (1H, d, J  14.4 Hz, H-8b), 1.72 (3H, s, H3-14), 1.62 (1H, m, H-2b); 13C NMR (CDCl3, 125 MHz)

C 174.0 (C-15), 153.0 (C-5), 146.0 (C-11), 136.3 (C-10), 129.4 (C-9), 127.2 (C-4), 112.9 (C-12), 83.3 (C-6), 66.8 (C-1), 50.3 (C-7), 25.5 (C-8), 23.9 (C-2), 21.6 (C-13), 20.7 (C-3), 17.0 (C-14); HRESIMS m/z 271.1305 [M + Na]+ (calcd for C15H20O3Na, 271.1294). (1Z,4R,5E,8S,9S)-10-Oxabicyclo[7.2.1]dodeca-5,12-dien-11-one,4-hydroxy-5-methyl -8-(1-methylethenyl) (10). The spectroscopic data was identical to that reported.35 Preparation of 11 by Oxidation of 10. To a solution of 10 (43 mg, 0.17 mmol) in 3 mL of DMSO at rt was added 73.2 mg of IBX (0.26 mmol). After being stirred at 60 C for 2 h, the mixture was washed with H2O and extracted with EtOAc. The organic layer was dried over anhydrous Na2SO4 and then concentrated via evaporation. The resulting residue was purified with silica gel flash column chromatography (PE:EtOAc  4:1  3:1) to afford 39 mg of 11 (92%). (1Z,5E,8S,9S)-10-Oxabicyclo[7.2.1]dodeca-5,12-dien-4,11-dione,5-methyl-8-(1-meth ylethenyl) (11). Colorless crystals; mp 131.4132.8 C; []25D 189.5 (c 0.3, MeOH); UV (CH2Cl2) max (log ) 243 (3.58), 221 (3.39) nm; IR (KBr) νmax 2973, 2923, 1744, 1642, 1444, 1281, 1081, 885, 769, 561 cm1; 1H NMR (CD3OD, 400 MHz) H 7.39 (1H, s, H-5), 6.22 (1H, dd, J  11.2, 1.2 Hz, H-9), 5.29 (1H, s, H-6), 5.09 (1H, s, 26


Page 26 of 54

Page 27 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


H-12a), 4.97 (1H, dd, J  4.3, 2.9 Hz, H-12b), 3.32 (1H, m, H-2a), 3.04 (1H, dd, J  11.5, 5.4 Hz, H-7), 2.80 (1H, m, H-3a), 2.75 (1H, m, H-8a), 2.60 (1H, ddd, J  12.2, 3.9, 3.9 Hz, H-2b), 2.49 (1H, m, H-8b), 2.36 (1H, m, H-3b), 1.95 (3H, s, H3-13), 1.65 (3H, s, H3-14); 13C NMR (CD3OD, 100 MHz) C 204.3 (C-1), 175.7 (C-15), 152.4 (C-5), 150.7 (C-9), 148.5 (C-11), 136.7 (C-4), 136.5 (C-10), 113.0 (C-12), 85.6 (C-6), 48.4 (C-7), 41.9 (C-2), 31.5 (C-8), 23.0 (C-3), 21.3 (C-13), 11.7 (C-14); HRESIMS m/z 269.1148 [M + Na]+ (calcd for C15H18O3Na, 269.1147). Preparation of 1217 by Acylation of 10. To a solution of 10 (20 mg, 0.08 mmol) in freshly distilled pyridine (2 mL) was added excess acyl chlorides (100 L). The resulting solution was stirred at 80 C overnight and then quenched by adding 2 mL of ethanol. After removal of the solvent under vacuum, the residues were purified by silica gel flash column chromatography (PE:EtOAc  20:1  5:1) to afford 16.4 mg of 12 (52%), 17.9 mg of 13 (62%), 21.8 mg of 14 (93%), 13.4 mg of 15 (42%), 16.9 mg of 16 (50%), and 18.5 mg of 17 (47%), respectively. (1Z,4R,5E,8S,9S)-5-methyl-11-oxo-8-(1-methylethenyl)-10-oxabicyclo[7.2.1]dodeca1(12),5-dien-4-yl 2-chloronicotinate (12). White powder; []25D 8.6 (c 0.4, CH2Cl2); UV (CH2Cl2) max (log ) 228 (3.43), 268 (3.05) nm; IR (KBr) max 2953, 2922, 2852, 1744, 1579, 1451, 1403, 1298, 1263, 1145, 1063, 1054, 940, 794, 766 cm1; 1H NMR (CDCl3, 400 MHz) H 6.83 (1H, s, H-5), 5.52 (1H, s, H-1), 5.13 (1H, m, H-9), 5.11 (1H, s, H-6), 5.02 (1H, s, H-12a), 4.91 (1H, s, H-12b), 2.78 (1H, m, H-8a), 2.73 (2H, m, H2-3), 2.56 (1H, dd, J  11.6, 3.3 Hz, H-7), 2.47 (1H, ddd, J  17.0, 13.2, 4.7 Hz, H-2a), 2.22 (1H, d, J  13.9 Hz, H-8b), 1.90 (3H, s, H3-13), 1.80 (1H, d, J = 14.3 Hz, H-2b), 1.69 (3H, s, H3-14), 1-O-2-chloronicotinyl: 8.51 (1H, d, J = 4.7, 1.9 Hz), 8.12 (1H, dd, J 27



 7.7, 1.9 Hz), 7.32 (1H, dd, J  7.7, 4.8 Hz);

13C

NMR (CDCl3, 100 MHz) C 173.1

(C-15), 150.2 (C-5), 146.3 (C-11), 134.6 (C-9), 131.8 (C-10), 131.7 (C-4), 112.7 (C-12), 83.6 (C-6), 82.6 (C-1), 45.8 (C-7), 28.1 (C-8), 24.0 (C-3), 22.9 (C-2), 21.3 (C-13), 11.1 (C-14), 1-O-2-chloronicotinyl: 163.5, 151.9, 150.2, 140.3, 127.0, 122.0; HRESIMS m/z 388.1310 [M + H]+ (calcd for C21H23NO4Cl, 388.1309). (1Z,4R,5E,8S,9S)-5-methyl-11-oxo-8-(1-methylethenyl)-10-oxabicyclo[7.2.1]dodeca1(12),5-dien-4-yl thiophene-2-carboxylate (13). White powder; []25D 50.2 (c 0.2, MeOH); UV (CH2Cl2) max (log ) 250 (3.86), 221 (3.80) nm; IR (KBr) max 2926, 1748, 1702, 1418, 1260, 1075, 963, 942, 862, 789, 747 cm1; 1H NMR (CDCl3, 400 MHz) H 6.82 (1H, s, H-5), 5.45 (1H, dd, J  11.7, 1.9 Hz, H-1), 5.10 (1H, s, H-6), 5.08 (1H, m, H-9), 5.01 (1H, s, H-12a), 4.90 (1H, s, H-12b), 2.78 (1H, m, H-3a), 2.72 (1H, m, H-8a), 2.67 (1H, m, H-3b), 2.54 (1H, dd, J  11.8, 4.2 Hz, H-7), 2.44 (1H, ddd, J  25.8, 12.5, 4.8 Hz, H-2a), 2.20 (1H, d, J  14.5 Hz, H-8b), 1.89 (3H, s, H3-13), 1.77 (1H, m, H-2b), 1.68 (3H, s, H3-14), 1-O-thiophene-2-carbonyl: 7.77 (1H, d, J  3.6 Hz), 7.54 (1H, d, J  4.9 Hz), 7.08 (1H, t, J  4.3 Hz); 13C NMR (CDCl3, 100 MHz) C 173.2 (C-15), 150.2 (C-5), 146.9 (C-11), 134.0 (C-10), 133.9 (C-9), 131.8 (C-4), 112.6 (C-12), 83.6 (C-6), 81.2 (C-1), 45.8 (C-7), 28.1 (C-8), 24.0 (C-3), 23.0 (C-2), 21.3 (C-13), 10.9 (C-14), 1-O-thiophene-2-carbonyl: 161.1, 133.4, 132.4, 132.4, 127.7; HRESIMS m/z 381.1131 [M + Na]+ (calcd for C20H22O4SNa, 381.1142). (1Z,4R,5E,8S,9S)-5-methyl-11-oxo-8-(1-methylethenyl)-10-oxabicyclo[7.2.1]dodeca1(12),5-dien-4-yl acetate (14). White powder; []25D 16.5 (c 0.4, CH2Cl2); UV (CH2Cl2) max (log ) 221 (3.49) nm; IR (KBr) νmax 2955, 2921, 2852, 1737, 1459, 1376, 1260, 1096, 1019, 801, 763, 751 cm1; 1H NMR (CDCl3, 400 MHz) H 6.77 (1H, 28


Page 28 of 54

Page 29 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


s, H-5), 5.24 (1H, dd, J  11.6, 1.9 Hz, H-1), 5.07 (1H, s, H-6), 5.01 (1H, d, J  10.5 Hz, H-9), 5.00 (1H, s, H-12a), 4.89 (1H, s, H-12b), 2.73 (1H, m, H-8a), 2.67 (2H, m, H2-3), 2.51 (1H, dd, J  11.9, 4.2 Hz, H-7), 2.31 (1H, m, H-2a), 2.16 (1H, m, H-8b), 1.88 (3H, s, H3-13), 1.62 (1H, m, H-2b), 1.59 (3H, s, H3-14), 1-OAc: 2.02 (3H, s);

13C

NMR

(CDCl3, 100 MHz) C 173.1 (C-15), 150.1 (C-5), 146.9 (C-11), 133.8 (C-9), 132.4 (C-10), 131.8 (C-4), 112.6 (C-12), 83.6 (C-6), 80.5 (C-1), 45.8 (C-7), 28.1 (C-8), 24.0 (C-3), 23.0 (C-2), 21.3 (C-13), 10.8 (C-14), 1-OAc: 170.1, 21.2; HRESIMS m/z 313.1410 [M + Na]+ (calcd for C17H22O4Na, 313.1418). (1Z,4R,5E,8S,9S)-5-methyl-11-oxo-8-(1-methylethenyl)-10-oxabicyclo[7.2.1]dodeca1(12),5-dien-4-yl 4-nitrobenzoate (15). White powder; []25D 26.7 (c 0.3, CH2Cl2); UV (CH2Cl2) max (log ) 263 (3.76), 226 (3.61) nm; IR (KBr) νmax 2923, 2853, 1738, 1717, 1525, 1352, 1299, 1277, 1118, 1104, 956, 871, 785, 716 cm1; 1H NMR (CDCl3, 400 MHz) H 6.84 (1H, s, H-5), 5.53 (1H, dd, J  11.8, 2.3 Hz, H-1), 5.15 (1H, d, J  11.5 Hz, H-9), 5.12 (1H, s, H-6), 5.03 (1H, s, H-12a), 4.92 (1H, m, H-12b), 2.77 (2H, m, H2-3), 2.73 (1H, m, H-8a), 2.56 (1H, dd, J  11.7, 4.3 Hz, H-7), 2.49 (1H, m, H-2a), 2.22 (1H, m, H-8b), 1.90 (3H, s, H3-13), 1.79 (1H, m, H-2b), 1.71 (3H, s, H3-14), 1-O-4-nitrobenzoyl: 8.28 (2H, d, J  8.9 Hz), 8.17 (2H, m);

13C

NMR (CDCl3, 100

MHz) C 173.1 (C-15), 150.2 (C-5), 146.8 (C-11), 134.5 (C-9), 131.9 (C-10), 131.8 (C-4), 112.7 (C-12), 83.6 (C-6), 82.2 (C-1), 45.8 (C-7), 28.2 (C-8), 24.0 (C-3), 23.0 (C-2), 21.3 (C-13), 11.0 (C-14), 1-O-4-nitrobenzoyl: 163.5, 150.6, 135.8, 130.7  2, 123.6  2; HRESIMS m/z 420.1418 [M + Na]+ (calcd for C22H23NO4Na, 420.1415). (1Z,4R,5E,8S,9S)-5-methyl-11-oxo-8-(1-methylethenyl)-10-oxabicyclo[7.2.1]dodeca1(12),5-dien-4-yl 4-(trifluoromethyl)benzoate (16). Yellow powder; []25D 95.6 (c 0.3, 29



MeOH); UV (CH2Cl2) max (log ) 228 (3.50) nm; IR (KBr) νmax 2924, 2853, 1744, 1713, 1324, 1273, 1166, 1123, 1101, 1066, 1017, 940, 774, 557, 497 cm1; 1H NMR (CDCl3, 400 MHz) H 6.84 (1H, s, H-5), 5.52 (1H, dd, J  11.8, 2.3 Hz, H-1), 5.13 (1H, d, J  12.3 Hz, H-9), 5.11 (1H, s, H-6), 5.02 (1H, s, H-12a), 4.91 (1H, s, H-12b), 2.78 (1H, m, H-8a), 2.75 (2H, m, H2-3), 2.55 (1H, m, H-7), 2.48 (1H, ddd, J  17.0, 8.5, 3.2 Hz, H-2a), 2.21 (1H, d, J  14.2 Hz, H-8b), 1.90 (3H, s, H3-13), 1.78 (1H, ddd, J  13.7, 6.5, 3.9 Hz, H-2b), 1.70 (3H, s, H3-14), 1-O-trifluoromethylbenzoyl: 8.11 (2H, d, J  8.1 Hz), 7.69 (2H, d, J  8.2 Hz); 13C NMR (CDCl3, 100 MHz) C 173.1 (C-15), 150.2 (C-5), 146.8 (C-11), 134.3 (C-9), 132.1 (C-10), 131.8 (C-4), 112.7 (C-12), 83.6 (C-6), 81.7 (C-1), 45.9 (C-7), 28.2 (C-8) 24.1 (C-3), 23.0 (C-2), 21.3 (C-13), 11.1 (C-14), 1-O-trifluoromethylbenzoyl: 164.2, 134.3, 133.7, 130.0  2, 125.4  2, 122.3; HRESIMS m/z 443.1441 [M + Na]+ (calcd for C23H23O4F3Na, 443.1447). (1Z,4R,5E,8S,9S)-5-methyl-11-oxo-8-(1-methylethenyl)-10-oxabicyclo[7.2.1]dodeca1(12),5-dien-4-yl 3,5-bis(trifluoromethyl)benzoate (17). Yellow powder; []25D 26.5 (c 0.3, CH2Cl2); UV (CH2Cl2) max (log ) 228 (3.81) nm; IR (KBr) max 2928, 2854, 1752, 1728, 1277, 1251, 1177, 1134, 963, 942, 911, 769, 701, 682 cm1; 1H NMR (CDCl3, 400 MHz) H 6.85 (1H, s, H-5), 5.54 (1H, dd, J  11.8, 2.3 Hz, H-1), 5.15 (1H, d, J  11.2 Hz, H-9), 5.11 (1H, s, H-6), 5.02 (1H, s, H-12a), 4.91 (1H, s, H-12b), 2.80 (1H, m, H-3a), 2.76 (1H, m, H-8a), 2.73 (1H, m, H-3b), 2.57 (1H, dd, J  8.3, 5.5 Hz, H-7), 2.52 (1H, m, H-2a), 2.21 (1H, dt, J  14.2, 1.9 Hz, H-8b), 1.90 (3H, s, H3-13), 1.79 (1H, m, H-2b), 1.70 (3H, s, H3-14), 1-O-3,5-bis(trifluoromethyl)benzoyl: 8.42 (2H, s), 8.05 (1H, s);

13C

NMR (CDCl3, 100 MHz) C 173.1 (C-15), 150.2 (C-5), 146.7 (C-11), 134.7

(C-9), 131.7 (C-4), 131.7 (C-10), 112.7 (C-12), 83.6 (C-6), 82.5 (C-1), 45.8 (C-7), 28.1 30


Page 30 of 54

Page 31 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(C-8),

24.0

(C-3),

23.0

(C-2),

21.3

(C-13),

11.0

(C-14),

1-O-3,5-bis(trifluoromethyl)benzoyl: 162.8, 132.6, 132.3, 132.01, 129.6  2, 126.3, 124.2, 121.5; HRESIMS m/z 511.1315 [M + Na]+ (calcd for C24H22O4F6Na, 511.1317). Preparation of 18 and 19 by Oximation and Subsequent Acylation of 11. To a stirred solution of 11 (15 mg, 0.06 mmol) in methanol (2 mL) was added NaOAc (15 mg, 0.18 mmol) and NH2OH·HCl (6.3 mg, 0.09 mmol). The resulting solution was stirred at rt for 30 min, and then washed with H2O. The mixture was extracted with EtOAc, and evaporated to generate a crude product. The crude product dissolved in CH2Cl2 (2 mL) was added 100 L of Et3N, followed by the treatment of acetyl or 2-furoyl chloride. After being stirred at rt for 1 h, the corresponding residue was purified with semi-preparative HPLC (MeCN/H2O  70:30) to afford 8 mg of 18 (43%, tR 8.5 min) or 7.7 mg of 19 (37%, tR 9.4 min). (1Z,5E,8S,9S)-10-oxabicyclo[7.2.1]dodeca-5,12-dien-11-one,4-(acetoxyimino)-5-met hyl-8-(1-methylethenyl) (18). White powder; []25D 68 (c 0.3, MeOH); UV (CH2Cl2)

max (log ) 246 (3.89), 228 (3.78) nm; IR (KBr) max 2954, 2922, 2852, 1752, 1642, 1460, 1276, 1262, 750 cm1; 1H NMR (CD3OD, 500 MHz) H 7.29 (1H, s, H-5), 5.54 (1H, d, J  11.1 Hz, H-9), 5.22 (1H, s, H-6), 5.06 (1H, s, H-12a), 4.94 (1H, s, 12b), 3.25 (1H, m, H-2a), 2.89 (1H, d, J  11.6 Hz, H-7), 2.81 (1H, m, H-3a), 2.80 (1H, m, H-2b), 2.78 (1H, m, H-8a), 2.44 (1H, m, H-3b), 2.36 (1H, m, H-8b), 2.22 (3H, s, H3-13), 1.78 (3H, s, H3-14), -C=NOCOCH3: 1.92 (3H, s);

13C

NMR (CD3OD, 125 MHz) C 175.7

(C-15), 169.6 (C-1), 153.1 (C-5), 148.7 (C-12), 142.5 (C-9), 134.6 (C-4), 130.7 (C-10), 112.9 (C-12), 85.6 (C-6), 47.0 (C-7), 30.7 (C-8), 25.3 (C-2), 23.8 (C-3), 21.4 (C-13),

31



14.3 (C-14), -C=NOCOCH3: 171.2, 19.6; HRESIMS m/z 326.1363 [M + Na]+ (calcd for C17H21NO4Na, 326.1348). (1Z,5E,8S,9S)-10-oxabicyclo[7.2.1]dodeca-5,12-dien-11-one,4-(((furan-2-carbonyl)o xy)imino)-5-methyl-8-(1-methylethenyl) (19). White powder; []25D 48.0 (c 0.3, MeOH); UV (CH2Cl2) max (log ) 227 (3.69), 251 (3.61) nm; IR (KBr) max 2951, 2919, 2850, 1747, 1713, 1666, 1652, 1470, 1438, 1361, 1309, 1209, 1163, 1093, 768 cm1; 1H NMR (CD3OD, 400 MHz) H 7.31 (1H, s, H-5), 5.59 (1H, dd, J  10.5 Hz, H-9), 5.23 (1H, s, H-6), 5.07 (1H, s, H-12a), 4.95 (1H, s, H-12b), 3.34 (1H, m, H-2a), 2.93 (1H, dd, J  9.7, 3.9 Hz, H-7), 2.89 (1H, m, H-2b), 2.83 (1H, m, H-3a), 2.77 (1H, m, H-8a), 2.54 (1H, m, H-3b), 2.39 (1H, m, H-8b), 1.93 (3H, s, H3-13), 1.84 (3H, s, H3-14), for furoyl moiety: 7.85 (1H, s), 7.42 (1H, d, J  3.5 Hz), 6.69 (1H, dd, J  3.5, 1.7 Hz);

13C

NMR (CD3OD, 100 MHz) C 175.7 (C-15), 171.1 (C-1), 153.3 (C-5),

148.7 (C-11), 142.9 (C-9), 134.4 (C-10), 130.8 (C-4), 113.0 (C-12), 85.6 (C-6), 47.0 (C-7), 30.7 (C-8), 25.4 (C-2), 24.0 (C-3), 21.4 (C-13), 11.0 (C-14), for furoyl moiety: 158.1, 149.0, 144.3, 120.3, 113.3; HRESIMS m/z 378.1312 [M + Na]+ (calcd for C20H21NO5Na, 378.1313). Preparation of 20 by Hydroxylation of 3. To a stirred solution of 3 (20 mg, 0.06 mmol) in DMSO (2 mL) was added 20 L of H2O. The mixture was maintained at 50 C for 3 h, and then the solution was diluted with H2O and extracted with EtOAc. After removal of the solvent, the remaining residue was purified with silica gel flash column chromatography (PE:EtOAc  4:1  2:1) to yield 2.2 mg of 20 (14%). (1Z,4Z,6R,8S,9S)-10-Oxabicyclo[7.2.1]dodeca,1(12),4-dien-11-one,6-hydroxy-5-met hyl-8-(1-methylethenyl) (20). Yellow oil; []25D 47.4 (c 0.3, MeOH); UV (CH2Cl2) 32


Page 32 of 54

Page 33 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


max (log ) 227 (3.38) nm; IR (KBr) max 3447, 2926, 2855, 1747, 1648, 1444, 1275, 993, 751 cm1; 1H NMR (CDCl3, 500 MHz) H 6.59 (1H, s, H-5), 5.21 (1H, s, H-1), 5.03 (1H, s, H-6), 4.91 (1H, s, H-12a), 4.82 (1H, s, H-12b), 3.93 (1H, dd, J  10.7, 6.0 Hz, H-9), 2.75 (1H, d, J  12.6 Hz, H-3a), 2.56 (1H, dd, J  12.3, 5.2 Hz, H-7), 2.24 (1H, m, H-2a), 2.09 (1H, m, H-8a), 2.08 (1H, m, H-2b), 1.92 (1H, dt, J  9.2, 4.6 Hz, H-3b), 1.87 (3H, s, H3-13), 1.78 (3H, s, H3-14), 1.67 (1H, m, H-8b); 13C NMR (CDCl3, 125 MHz) C 174.6 (C-15), 152.4 (C-5), 147.9 (C-11), 140.4 (C-10), 128.4 (C-4), 125.6 (C-1), 112.3 (C-12), 82.8 (C-6), 65.5 (C-9), 47.5 (C-7), 34.5 (C-8), 24.8 (C-2), 24.5 (C-3), 20.1 (C-13), 17.1 (C-14); HRESIMS m/z 271.1305 [M + Na]+ (calcd for C15H20O3Na, 271.1307). Preparation of 2123 by Etherification of 3. To a stirred solution of 1 mL of methanol or 1 mL of ethanol was added 20 mg of 3. The mixture was reacted at 50 C for 12 h. The corresponding concentrated residue was purified with silica gel flash column chromatography (PE:EtOAc  6:1) to afford 12.6 mg of 21 (75%) or 12.6 mg of 22 (71%), respectively. To a stirred solution of 3 (20 mg, 0.06 mmol) in 1 mL of isopropanol was added 60% NaH (7.7 mg, 0.19 mmol) under N2 atmosphere at rt. The solution was heated to 50 C and stirred for another 3 h, followed by the addition of excess H2O. The resultant mixture was then extracted with EtOAc and concentrated via evaporation. The obtained crude product was purified with silica gel flash column chromatography (PE:EtOAc  6:1) to afford 6 mg of 23 (32%). (1Z,4Z,6R,8S,9S)-10-Oxabicyclo[7.2.1]dodeca,1(12),4-dien-11-one,6-methoxy-5-met hyl-8-(1-methylethenyl) (21). White powder; []25D 45.8 (c 0.4, CH2Cl2); UV (CH2Cl2) 33



max (log ) 228 (3.42) nm; IR (KBr) max 3291, 2955, 2922, 2852, 1741, 1461, 1275, 1262, 751 cm1; 1H NMR (CDCl3, 400 MHz) H 6.60 (1H, s, H-5), 5.40 (1H, m, H-1), 5.02 (1H, s, H-6), 4.90 (1H, m, H-12a), 4.81 (1H, m, H-12b), 3.41 (1H, dd, J  10.1, 6.2 Hz, H-9), 2.74 (1H, m, H-3a), 2.55 (1H, dd, J  12.4, 5.0 Hz, H-7), 2.26 (1H, m, H-3b), 2.11 (1H, m, H-8a), 2.09 (1H, m, H-2a), 1.93 (1H, ddd, J  13.3, 12.7, 4.2 Hz, H-2b), 1.86 (3H, s, H3-13), 1.69 (3H, s, H3-14), 1.62 (1H, m, H-8b), 9-OMe: 3.04 (3H, s); 13C NMR (CDCl3, 100 MHz) C 174.4 (C-15), 152.5 (C-5), 147.9 (C-11), 136.8 (C-10), 128.1 (C-4), 127.4 (C-1), 112.0 (C-12), 82.6 (C-6), 73.8 (C-9), 47.3 (C-7), 32.7 (C-8), 24.58 (C-2), 24.55 (C-3), 19.9 (C-13); 17.2 (C-14), 9-OMe: 54.8; HRESIMS m/z 285.1461 [M + Na]+ (calcd for C16H22O3Na, 285.1448). (1Z,4Z,6R,8S,9S)-10-Oxabicyclo[7.2.1]dodeca,1(12),4-dien-11-one,6-ethoxy-5-methy l-8-(1-methylethenyl) (22). White powder; []25D 47.0 (c 0.3, CH2Cl2); UV (CH2Cl2)

max (log ) 228 (3.39) nm; IR (KBr) max 2953, 2925, 2852, 1750, 1640, 1461, 1276, 1262, 750, 596 cm1; 1H NMR (CDCl3, 400 MHz) H 6.60 (1H, s, H-5), 5.34 (1H, m, H-1), 5.02 (1H, s, H-6), 4.89 (1H, s, H-12a), 4.80 (1H, t, J  1.2 Hz, H-12b), 3.51 (1H, dd, J  10.7, 6.1 Hz, H-9), 2.73 (1H, d, J = 12.3 Hz, H-3a), 2.54 (1H, dd, J  12.4, 5.0 Hz, H-7), 2.25 (1H, m, H-2a), 2.13 (1H, m, H-2b), 2.08 (1H, m, H-8a), 1.94 (1H, dd, J  12.6, 4.1 Hz, H-3b), 1.86 (3H, s, H3-13), 1.70 (3H, s, H3-14), 1.66 (1H, m, H-8b), 9-OEt: 3.26 (1H, dq, J  8.8, 7.0 Hz), 3.08 (1H, dq, J  9.0, 7.0 Hz), 1.07 (3H, t, J  7.0 Hz);

13C

NMR (CDCl3, 100 MHz) C 174.4 (C-15), 152.5 (C-5), 148.0 (C-11), 137.6

(C-10), 128.1 (C-4), 126.8 (C-1), 111.9 (C-12), 82.7 (C-6), 72.0 (C-9), 47.2 (C-7), 33.0 (C-8), 24.6 (C-3), 24.6 (C-2), 20.0 (C-13), 17.4 (C-14), 9-OEt: 62.3, 15.4; HRESIMS m/z 299.1618 [M + Na]+ (calcd for C17H24O3Na, 299.1619). 34


Page 34 of 54

Page 35 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(1Z,4Z,6R,8S,9S)-10-Oxabicyclo[7.2.1]dodeca,1(12),4-dien-11-one,6-isopropoxy-5methyl-8-(1-methylethenyl) (23). White powder; []25D  (c 0.2, CH2Cl2); UV (CH2Cl2) max (log ) 228 (3.25) nm; IR (KBr) max 2955, 2921, 2852, 1749, 1646, 1460, 1377, 1260, 1095, 1020, 799 cm1; 1H NMR (CDCl3, 400 MHz) H 6.61 (1H, s, H-5), 5.30 (1H, m, H-1), 5.02 (1H, s, H-6), 4.89 (1H, s, H-12a), 4.80 (1H, s, H-12b), 3.58 (1H, dd, J  10.7, 6.1 Hz, H-9), 2.74 (1H, d, J = 11.9 Hz, H-2a), 2.51 (1H, dd, J  12.3, 4.8 Hz, H-7), 2.30 (1H, m, H-3a), 2.09 (1H, m, H-3b), 2.02 (1H, m, H-8a), 1.95 (1H, dd, J  12.8, 3.9 Hz, H-2b), 1.86 (3H, s, H3-13), 1.72 (3H, s, H3-14), 1.66 (1H, m, H-8b), 9-O-iPr: 3.31 (1H, dt, J  12.2, 6.0 Hz), 1.02 (3H, d, J  2.6 Hz), 1.01 (3H, d, J  2.5 Hz);

13C

NMR (CDCl3, 100 MHz) C 174.5 (C-15), 152.6 (C-5), 148.0 (C-11),

138.6 (C-10), 128.2 (C-4), 126.2 (C-1), 111.9 (C-12), 82.6 (C-6), 69.0 (C-9), 47.2 (C-7), 33.5 (C-8), 24.8 (C-2), 24.7 (C-3), 20.0 (C-13), 17.5 (C-14), 9-O-iPr: 66.9, 23.4, 21.7; HRESIMS m/z 313.1774 [M + Na]+ (calcd for C18H26O3Na, 313.1776). Preparation of 24 by Esterification of 3. To a solution of 3 (20 mg, 0.06 mmol) in 2 mL of DMF was added formic acid (10 L, 0.12 mmol). The solution was heated to 80 C and stirred for another 2 h. After the reaction was completed, the solution was washed with H2O and extracted with EtOAc. The concentrated residue was purified with semi-preparative HPLC (MeCN/H2O  70:30) to yield 2.5 mg of 24 (15%, tR 9.5 min). (1Z,4Z,6R,8S,9S)-5-methyl-11-oxo-8-(1-methylethenyl)-10-Oxabicyclo[7.2.1]dodeca1(12),4-dien-6yl formate (24). White powder; []25D 26.2 (c 0.3, CH2Cl2); UV (CH2Cl2) max (log ) 228 (3.03) nm; IR (KBr) max 2954, 2921, 2852, 1749, 1460, 1377, 1261, 1095, 1019, 801, 750 cm1; 1H NMR (CDCl3, 500 MHz) H 6.59 (1H, s, 35



H-5), 5.30 (1H, t, J  8.3 Hz, H-1), 5.08 (1H, m, H-9), 5.03 (1H, s, H-6), 4.93 (1H, m, H-12a), 4.85 (1H, s, H-12b), 2.81 (1H, d, J  12.3 Hz, H-3a), 2.60 (1H, dd, J  12.3, 3.9 Hz, H-7), 2.43 (1H, dd, J  23.7, 12.5 Hz, H-2a), 2.25 (1H, m, H-2b), 2.09 (1H, ddd, J  14.5, 10.3, 6.2 Hz, H-8a), 1.95 (1H, t, J  13.1 Hz, H-3b), 1.87 (3H, s, H3-13), 1.84 (1H, m, H-8b), 1.80 (3H, s, H3-14); 9-OCHO: 7.86 (1H, s); 13C NMR (CDCl3, 125 MHz) C 173.8 (C-15), 152.0 (C-5), 147.3 (C-11), 135.5 (C-10), 129.0 (C-1), 128.8 (C-4), 112.6 (C-12), 82.1 (C-6), 68.4 (C-9), 47.2 (C-7), 31.5 (C-8), 24.9 (C-2), 24.4 (C-3), 19.9 (C-13), 17.6 (C-14), 9-OCHO: 159.2; HRESIMS m/z 299.1254 [M + Na]+ (calcd for C16H20O4Na, 299.1247). Preparation of 2527 by Amination of 3. To a solution of 3 (20 mg, 0.06 mmol) in 2 mL of DMF was respectively added piperidine, pyrrolidine, or morpholine (0.19 mmol). After being stirred at 50 C for 2 h, each solution was diluted with H2O and extracted with EtOAc. The concentrated products were purified with preparative TLC (PE:EtOAc  4:1) to afford 3 mg of 25 (15%), 11 mg of 26 (54%), and 9.3 mg of 27 (46%), respectively. (1Z,4Z,6R,8S,9S)-10-Oxabicyclo[7.2.1]dodeca,1(12),4-dien-11-one,6-(piperidin-1-yl) -5-methyl-8-(1-methylethenyl) (25). Yellow powder; []25D 145 (c 0.3, CH2Cl2); UV (CH2Cl2) max (log ) 228 (3.50) nm; IR (KBr) max 2930, 2853, 1750, 1441, 1275, 895, 751 cm1; 1H NMR (CDCl3, 400 MHz) H 6.62 (1H, s, H-5), 5.27 (1H, t, J  8.0 Hz, H-1), 5.00 (1H, s, H-6), 4.87 (1H, s, H-12a), 4.78 (1H, s, H-12b), 2.70 (1H, dt, J  12.5, 3.8 Hz, H-3a), 2.51 (1H, dd, J  12.2, 4.2 Hz, H-7), 2.44 (1H, dd, J  11.1, 5.8 Hz, H-9), 2.18 (2H, m, H2-2), 2.13 (1H, m, H-8a), 1.94 (1H, ddd, J  13.2, 12.4, 5.4 Hz, H-3b), 1.86 (3H, s, H3-13) , 1.72 (3H, s, H3-14), 1.55 (1H, m, H-8b), for piperidine 36


Page 36 of 54

Page 37 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


moiety: 2.33 (4H, t, J  5.3 Hz), 1.45 (4H, m), 1.34 (2H, m);

13C

NMR (CDCl3, 100

MHz) C 174.6 (C-15), 152.8 (C-5), 148.9 (C-11), 138.5 (C-10), 127.6 (C-1), 127.5 (C-4), 111.6 (C-12), 82.5 (C-6), 59.9 (C-9), 47.4 (C-7), 31.9 (C-8), 25.2 (C-2), 24.6 (C-3), 20.1 (C-13), 19.5 (C-14), for piperidine moiety: 51.8  2, 26.5  2, 24.5; HRESIMS m/z 316.2271 [M + H]+ (calcd for C20H30NO2, 316.2265). (1Z,4Z,6R,8S,9S)-10-Oxabicyclo[7.2.1]dodeca,1(12),4-dien-11-one,6-(pyrrolidin-1-y l)-5-methyl-8-(1-methylethenyl) (26). Yellow powder; []25D 87.3 (c 0.4, MeOH); UV (CH2Cl2) max (log ) 228 (3.59) nm; IR (KBr) max 2960, 2926, 1754, 1457, 1276, 898, 751 cm1;

1H

NMR (CD3OD, 500 MHz) H 6.92 (1H, s, H-5), 5.32 (1H, t, J  8.5 Hz,

H-1), 5.11 (1H, s, H-6), 4.95 (1H, s, H-12a), 4.85 (1H, s, H-12b), 2.70 (1H, dd, J  12.2, 4.1 Hz, H-7), 2.63 (1H, d, J  12.4 Hz, H-3a), 2.51 (1H, m, H-9), 2.19 (2H, m, H2-2), 2.08 (1H, m, H-8a), 2.03 (1H, m, H-3b), 1.89 (3H, s, H3-13), 1.80 (3H, s, H3-14), 1.66 (1H, m, H-8b), for pyrrolidine moiety: 2.53 (2H, m), 2.40 (2H, m), 1.72 (4H, m);

13C

NMR (CD3OD, 125 MHz) C 177.1 (C-15), 155.7 (C-5), 150.2 (C-11), 140.0 (C-10), 128.6 (C-1), 128.3 (C-4), 112.2 (C-12), 84.5 (C-6), 61.9 (C-9), 47.8 (C-7), 34.3 (C-8), 26.2 (C-2), 25.2 (C-3), 20.4 (C-13), 19.1 (C-14), for pyrrolidine moiety: 53.2  2, 23.8  ; HRESIMS m/z 302.2115 [M + H]+ (calcd for C19H28NO2, 302.2117). (1Z,4Z,6R,8S,9S)-10-Oxabicyclo[7.2.1]dodeca,1(12),4-dien-11-one,6-morpholino-5methyl-8-(1-methylethenyl) (27). Yellow powder; []25D 20.4 (c 0.3, MeOH); UV (CH2Cl2) max (log ) 228 (3.52) nm; IR (KBr) max 2954, 2921, 2852, 1751, 1645, 1459, 1377, 1262, 1019, 800, 751 cm1; 1H NMR (CD3OD, 400 MHz) H 6.92 (1H, s, H-5), 5.40 (1H, m, H-1), 5.10 (1H, s, H-6), 4.94 (1H, m, H-12a), 4.84 (1H, d, J  1.5 Hz, H-12b), 2.70 (1H, dd, J  12.3, 4.3 Hz, H-7), 2.62 (1H, m, H-3a), 2.46 (1H, dd, J 

37



Page 38 of 54

11.1, 5.9 Hz, H-9), 2.17 (2H, m, H2-2), 2.09 (1H, m, H-8a), 2.03 (1H, m, H-3b), 1.88 (3H, s, H3-13), 1.75 (3H, s, H3-14), 1.54 (1H, ddd, J  13.8, 11.1, 4.3 Hz, H-8b), for morpholine moiety: 3.59 (4H, m), 2.40 (4H, m);

13C

NMR (CD3OD, 100 MHz) C

177.0 (C-15), 155.8 (C-5), 150.9 (C-11), 138.5 (C-10), 130.1 (C-1), 128.3 (C-4), 112.2 (C-12), 84.5 (C-6), 61.3 (C-9), 48.1 (C-7), 32.6 (C-8), 26.2 (C-2), 25.1 (C-3), 20.3 (C-13), 19.2 (C-14), for morpholine moiety: 68.1  2, 52.5  2; HRESIMS m/z 318.2064 [M + H]+ (calcd for C19H28NO3, 318.2079). Preparation of 28 by Azidation of 3. To a solution of 3 (50 mg, 0.16 mmol) in 4 mL of DMF was added NaN3 (31 mg, 0.48 mmol). After being stirred at 50 C for 4 h, the resulting solution was subsequently diluted with H2O and extracted with EtOAc. The concentrated residue was purified with semi-preparative HPLC (MeCN/H2O  70:30), yielding

6.2

mg

of

28

(14%).

(1Z,4R,5E,8S,9S)-10-Oxabicyclo[7.2.1]dodeca-5,12-dien-11-one,4-azido-5-methyl-8-(1 -methylethenyl) (28). White powder; []25D 28.3 (c 0.2, CH2Cl2); UV (CH2Cl2) max (log ) 228 (3.60), 261 (3.14) nm; IR (KBr) max 2921, 2850, 2092, 1739, 1649, 1274, 1261, 751 cm1; 1H NMR (CDCl3, 500 MHz) H 6.78 (1H, s, H-5), 5.08 (1H, s, H-6), 5.01 (1H, s, H-12a), 4.97 (1H, d, J  11.3 Hz, H-9), 4.90 (1H, m, H-12b), 4.14 (1H, dd, J  12.0, 2.2 Hz, H-1), 2.77 (1H, ddd, J  23.4, 11.6, 11.6 Hz, H-8a), 2.64 (2H, m, H2-3), 2.52 (1H, dd, J  11.9, 4.5 Hz, H-7), 2.23 (1H, m, H-2a), 2.18 (1H, m, H-8b), 1.89 (3H, s, H3-13), 1.62 (3H, s, H3-14), 1.54 (1H, m, H-2b);

13C

NMR (CDCl3, 125

MHz) C 173.1 (C-15), 150.2 (C-5), 146.8 (C-11), 134.9 (C-9), 132.3 (C-10), 131.8 (C-4), 112.7 (C-12), 83.6 (C-6), 71.2 (C-1), 45.7 (C-7), 28.4 (C-8), 24.9 (C-3), 22.9 (C-2), 21.3 (C-13), 11.1 (C-14); HRESIMS m/z 296.1369 [M + Na]+ (calcd for C15H19N3O2Na, 296.1375).

38


Page 39 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Preparation of 29 From 3. To a stirred solution of 3 (20 mg, 0.06 mmol) in ethanol (2 mL) was treated with AgNO3 (43.87 mg, 0.25 mmol) at rt. After 30 min, the concentrated mixture was purified with preparative TLC (PE:EtOAc  5:1) to afford 18.8 mg of 29 (40%). (1Z,4R,5E,8S,9S)-5-methyl-11-oxo-8-(1-methylethenyl)-10-oxabicyclo[7.2.1]dodeca1(12),5-dien-4-yl nitrate (29). White powder; []25D 8.8 (c 0.3, CH2Cl2); UV (CH2Cl2)

max (log ) 228 (3.21) nm; IR (KBr) max 2926, 2854, 1749, 1626, 1442, 1274, 1179, 1098, 1017, 859, 794 cm1; 1H NMR (CDCl3, 400 MHz) H 6.82 (1H, s, H-5), 5.32 (1H, m, H-1), 5.13 (1H, d, J  12.5 Hz, H-9), 5.10 (1H, s, H-6), 5.01 (1H, s, H-12a), 4.92 (1H, m, H-12b), 2.75 (2H, m, H2-3), 2.71 (1H, m, H-8a), 2.55 (1H, dd, J  11.8, 4.1 Hz, H-7), 2.35 (1H, ddd, J  25.6, 25.6, 12.1 Hz, H-2a), 2.23 (1H, m, H-8b), 1.89 (3H, s, H3-13), 1.75 (1H, m, H-2b), 1.61 (3H, s, H3-14); 13C NMR (CDCl3, 100 MHz) C 172.8 (C-15), 150.3 (C-5), 146.5 (C-11), 136.3 (C-9), 131.4 (C-4), 130.4 (C-10), 112.9 (C-12), 89.7 (C-1), 83.6 (C-6), 45.7 (C-7), 28.1 (C-8), 23.9 (C-3), 21.3 (C-13), 20.6 (C-2), 10.7 (C-14); HRESIMS m/z 316.1155 [M + Na]+ (calcd for C15H19NO5Na, 316.1161). Preparation of 30 and 31 by Etherification of 3. 10 mg of 29 (0.04 mmol) was dissolved in 1 mL of methanol or 1 mL of ethanol, and then was stirred at 50 C for 0.51 h. The corresponding mixture was concentrated and was then purified with semi-preparative HPLC (MeCN/H2O  70:30) to afford 6 mg of 30 (68%, tR = 10.3 min) or 4.9 mg of 31 (52%, tR = 10.8 min), respectively. (1Z,4R,5E,8S,9S)-10-Oxabicyclo[7.2.1]dodeca-5,12-dien-11-one,4-methoxy-5-methyl -8-(1-methylethenyl) (30). White powder; []25D 3.6 (c 0.1, MeOH); UV (CH2Cl2) max (log ) 228 (3.24) nm; IR (KBr) max 2925, 2853, 1746, 1644, 1458, 1260, 1099, 1014, 39



793, 767, 750 cm1; 1H NMR (CDCl3, 400 MHz) H 6.74 (1H, s, H-5), 5.07 (1H, s, H-6), 5.01 (1H, s, H-12a), 4.93 (1H, d, J  11.4 Hz, H-9), 4.90 (1H, m, H-12b), 3.68 (1H, dd, J  11.3, 2.2 Hz, H-1), 2.78 (1H, dt, J  14.2, 11.7 Hz, H-8a), 2.63 (2H, m, H2-3), 2.51 (1H, dd, J  11.9, 4.3 Hz, H-7), 2.20 (1H, m, H-2a), 2.17 (1H, m, H-8b), 1.90 (3H, s, H3-13), 1.65 (1H, m, H-2b), 1.52 (3H, s, H3-14), 1-OCH3: 3.11 (3H, s); 13C NMR (CDCl3, 100 MHz) C 173.2 (C-15), 149.8 (C-5), 147.1 (C-11), 134.7 (C-10), 133.9 (C-9), 132.0 (C-4), 112.5 (C-12), 88.1 (C-1), 83.5 (C-6), 45.9 (C-7), 28.2 (C-8), 24.4 (C-3), 23.7 (C-2), 21.4 (C-13), 10.0 (C-14), 1-OCH3: 55.0; HRESIMS m/z 285.1461 [M + Na]+ (calcd for C16H22O3Na, 285.1464). (1Z,4R,5E,8S,9S)-10-Oxabicyclo[7.2.1]dodeca-5,12-dien-11-one,4-ethoxy-5-methyl8-(1-methylethenyl) (31). White powder; []25D 3.8 (c 0.3, CH2Cl2); UV (CH2Cl2) max (log ) 227 (3.01) nm; IR (KBr) max 2959, 2924, 2854, 1750, 1275, 1262, 1103, 1076, 751 cm1; 1H NMR (CDCl3, 400 MHz) H 6.72 (1H, s, H-5), 5.06 (1H, s, H-6), 5.00 (1H, s, H-12a), 4.91 (1H, s, H-9), 4.89 (1H, s, H-12b), 3.79 (1H, dd, J  11.2, 2.1 Hz, H-1), 2.77 (1H, dt, J  14.1, 11.7 Hz, H-8a), 2.62 (2H, dd, J  11.6, 4.1 Hz, H2-3), 2.50 (1H, dd, J  11.8, 4.3 Hz, H-7), 2.19 (1H, m, H-2a), 2.15 (1H, m, H-8b), 1.89 (3H, s, H3-13), 1.65 (1H, m, H-2b), 1.52 (3H, s, H3-14), 1-OEt: 3.28 (1H, dq, J  9.1, 7.1 Hz), 3.17 (1H, dq, J  9.3, 7.0 Hz), 1.14 (3H, s);

13C

NMR (CDCl3, 100 MHz) C 173.2

(C-15), 149.6 (C-5), 147.1 (C-11), 135.3 (C-10), 133.3 (C-9), 132.2 (C-4), 112.5 (C-12), 86.3 (C-1), 83.5 (C-6), 46.0 (C-7), 28.2 (C-8), 24.5 (C-3), 23.9 (C-2), 21.3 (C-13), 10.2 (C-14), 1-OEt: 62.3, 15.2; HRESIMS m/z 299.1618 [M + Na]+ (calcd for C17H24O3Na, 299.1609).

40


Page 40 of 54

Page 41 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


X-ray Crystal Structure Analysis. Single crystals of 11 was collected on an Xcalibur, Onyx, Nova diffractometer equipped with Cu K radiation (  1.54184 Å). The structure was determined using direct methods and refined using olex 2. All non-hydrogen atoms were refined using anisotropic thermal parameters. Hydrogen atoms were located by geometrical calculations. The absolute configurations of 11 was confirmed by refinement of the Flack parameters. Crystallographic data have been deposited at the Cambridge Crystallographic Data Centre [deposition numbers: CCDC 1905680]. These data are available free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk. Crystal Data for 11. C15H18O3 (M  246.29 g/mol): monoclinic, space group P21 (no. 4), a = 6.56770 (10) Å, b = 12.1333 (2) Å, c  8.18530 (10) Å,   99.2150 (10), V  643.851 (17) Å3, Z  2, T  100.00 (10) K,  (CuK)  0.706 mm-1, Dcalc  1.270 g/cm3, 12541 reflections measured (10.95  2  152.712), 2583 unique (Rint  0.0326, Rsigma  0.0244) which were used in all calculations. The final R1 was 0.0278 (I  2 (I)) and wR2 was 0.0708 (all data). Flack parameter  0.01 (7). Rat Cardiac Fibroblasts Isolation and Culture. Animal handling and the experimental procedures were conformed to the Guidelines of Animal Experiments from Ethical Committee for Animal Research of Sun Yat-sen University. Primary cultured adult rat cardiac fibroblasts were prepared as described in our previous study.26 Briefly, male Sprague Dawley rats (180220 g) were anesthetized by intraperitoneal injection of sodium pentobarbital (30 mg/kg). The hearts from rats were minced and digested in 0.1% collagenase II (Gibco, USA) for three 30 min, 0.25% trypsin (SigmaAldrich, USA) for three 5 min periods. Cells were collected and suspended in 41



DMEM (Gibco, USA) containing 10 fetal bovine serum (Gibco, USA). After 1 hour incubation, the debris and non-adherent cells were removed, and the attached cells were cultured in a humidified atmosphere containing 5 CO2 at 37 C. Cardiac fibroblasts were cultured for 34 days and passaged further. The second to fourth passages of the cardiac fibroblasts were used in the current study. Cytotoxicity Assay. Cell cytotoxicity was measured using the MTT assay. Briefly, cells (5000 per well) were seeded into each well of a 96-well plate and treated with 11 at indicated concentrations for 48 h. After then, the cells were incubated with 5 mg/mL MTT (Millipore, USA) for another 4 h. The suspension was discarded and the formazans were dissolved in DMSO (Millipore, USA). The absorbance was measured at 570 nm with a multifunction microplate reader (Biotech, USA). Cells were plated in triplicates and experiments were repeated three times. Immunofluorescence Assay. Cardiac fibroblasts were cultured on confocal dishes (Axygen, USA), treated with 11 (10 M) and TGF1 (Millipore, USA) (10 ng/mL) for 5 min. The cells were successively washed (three times with phosphate buffer saline), fixed with 4% paraformaldehyde (Beyotime, China) for 10 min, permeabilized with 0.1% Triton X‐100 (Beyotime, China) for 30 min at rt, blocked for 30 min with normal goat serum (Beyotime, China), and then followed by incubation with specific primary antibodies Smad2 and Smad3 (Santa, sc-101154, sc-101153, 1:800) overnight at 4 C. After a wash step, cells were incubated with Alexa 594-labeled anti-mouse IgG (red) (Thermo, #A-11005, 1:1,000 dilution) for 1 h at rt and the nucleus was stained by DAPI (Beyotime, China) for another 10 min in the dark at rt. The images were acquired using a LSM 510 laser confocal microscope (Leica Microsystems, Zeiss, Germany). 42


Page 42 of 54

Page 43 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


TRI and TRII Kinases Assay. Kinases assays were performed by using TRI and TRII kinase kits (promega, USA) according to the manufacturer’s instructions. In brief, the solution of 11 with different concentrations was prepared in DMSO. Each solution was mixed with reaction buffer including purified TRI or TRII protein, 50

M ATP, and 0.2 g/L casein (for TRI) or 0.1 g/L MBP (for TRII). The mixtures were incubated for 2 h at room temperature. Then, the ADP generated was measured, which is indicative of kinase activities. The IC50 values of 11 and SB431542 were calculated from dose-response curves generated using Graphpad prism software.

AAC Model and Drug Treatment. Male Sprague Dawley rats (n  32) weighing 200250 g were purchased from the Animal Center of Sun Yat-Sen University (Guangzhou, China) and were divided randomly into 4 groups: (1) the sham-operated group (sham  vehicle, n  8), (2) the AAC group (AAC  vehicle, n  8), (3) the AAC  nifedipine group (n  8), and (4) the AAC  11 group (n  8). Rats were housed in temperature-controlled (2025 C) and humidity-controlled (4070%) rooms with 12 h light/12 h dark. Animal handling and the experimental procedures were conforming to the Guidelines of Animal Experiments from Ethical Committee for Animal Research of Sun Yat-sen University (approval number: SYSU-IACUC-2018-000112). AAC was induced. Briefly, rats were anesthetized intraperitoneal injection of 30 mg/kg sodium pentobarbital. The abdominal aorta proximal to the renal arteries was exposed and a 40 silk suture was tied securely around a blunt 21-G probe placed along the aorta above both renal arteries. The probe was removed promptly to create constriction. Saline (1 mL) was added to the peritoneal cavity to replace incidental fluid 43



loss and the abdominal wall and skin were sutured. A similar surgical procedure was performed without AAC in the sham group. AAC  11 group was treated with 11 (5 mg/kg) and AAC  nifedipine (MedChemExpress, USA) group was treated with nifedipine (10 mg/kg). The AAC and sham groups were treated with an equivalent volume of normal saline (including 0.5% DMSO, 5% Kolliphor HS 15). Drugs or vehicle were administered every day for 4 weeks. After treatments in the last day, two-dimensional-guided M-mode echocardiography was performed by a Technos MPX ultrasound system (ESAOTE, SpAESAOTESpA, Italy) equipped with a 8.5-MHz imaging transducer to assess cardiac function. Basic hemodynamic parameters, such as EF, FS, LVPWd, LVPWs, LVIDd, LVIDs, LVAWd, and LVAWs were measured. Histological Examination. After 4 weeks of treatment, the SD rats were sacrificed and their heart tissues were quickly removed out. Heart tissues of the rats were fixed in 10% buffered formalin (Beyotime, China). 2 mm thick slices were prepared from the ventricle, embedded in paraffin, and then cut into 4 m sections. The transverse myocardial sections were stained with phosphorylated Smad2/3, -SMA (Abcam, ab124964, 1:200), and FN (Proteintech, 15613-1-AP, 1:200). Western Blot Analysis. Cardiac fibroblasts (3  105 per well) were seeded into a six-well plate (Corning, USA). The cells were treated with indicated concentrations of 11, OMT (MedChemExpress, USA), nifedipine, SB431542 (MedChemExpress, USA), and 10 ng/mL TGFβ1 for different times. The cells were successively washed (three times with PBS). After treatments, cardiac fibroblasts were lysed in cell lysis buffer 44


Page 44 of 54

Page 45 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(Beyotime, China) containing 1% NP-40, 20 mM Tris-HCl (pH 7.6), 0.15 M NaCl, 3 mM EDTA, 3 mM EGTA, 1 mM phenyl methyl sulfonyl fluoride (Beyotime, China), 20 mg/mL aprotinin, and 5 mg/mL leupeptin. Nuclear and cytoplasmic protein fractions were obtained by a commercially available separation kit according to the manufacturer's protocol (Active Motif, USA). To determine the cardiac protein expression of FN, collagen  (Zenbio, 250064, 1:800) and  (Proteintech, 14695-1-Ap, 1:800), and -SMA, the hearts were homogenized in lysis buffer containing protease and phosphatase inhibitors. Lysates were centrifuged at 12,000 rpm for 30 min at 4 °C, and the supernatants were saved. Nuclear and cytoplasmic protein fractions were obtained by a commercially available separation kit according to the manufacturer's protocol. The protein concentrations were determined by a bicinchoninic acid (BCA) (Beyotime, China) protein assay kit. Equal amount of protein (30 g) were run on 10% sodium dodecylsulfate polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore, USA). Nonspecific binding sites on the membranes were blocked by 5% nonfat milk (Yuanye, China) for 2 h at rt, followed by incubated with the primary antibodies at 1:1,000 dilution over night at 4 °C and then incubated with specific secondary antibodies at 1:5,000 dilution for 1 hour at rt. The bands were analyzed by ImageJ software. Quantitative Real Time Reverse Transcription Polymerase Chain Reaction (qRT-PCR). Cardiac fibroblasts (3  105 per well) were seeded into a six well plate. The cells were treated with indicated concentrations of 11 and 10 ng/mL TGF1 for 12 h. The cells were successively washed three times with PBS. RNAiso Plus (Yeasen, China) was added to each well for extracting total RNA. Complementary DNA was 45



synthesized with equal amount of total RNA (1 g) according to the instructions of the Prime Scrip™RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara, Japan). The GAPDH was utilized as a reference to normalize the amount of transcript. Amplification of the cDNA was performed using PCR. Briefly, cDNA template and PCR master mix were mixed, which complied to the instructions of the SYBR Premix Ex Taq™ (Tli RNaseH Plus) kit. The PCR amplifications were carried out in 96 well plates. Quantification of transcript levels of FN, collagen  and , and ‐SMA genes were conducted according to the 2−ΔΔCt method. The expression data of these proteins were denoted as means  SD. The PCR primers used in this study were shown in Table S2 in Supporting Information. Statistical Analysis. All statistical analyses were performed using GraphPad Prism version 5.0 software (GraphPad Software San Diego; CA, USA). Independent experiments involved triplicate analyses for each sample conditions. The data were presented as means ± SD. Differences were considered to be statistically significant when P values were less than 0.05.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Figures and tables related to the article, 1D and 2D NMR spectra and the purity analyses by HPLC of 131, and IR, HRESIMS spectra of new compounds (PDF) Molecular formula strings for compounds 131 (CSV) 46


Page 46 of 54

Page 47 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


AUTHOR INFORMATION Corresponding Author *(S. Yin) Tel and fax: +86-20-39943090. E-mail: [email protected]. Author Contributions †L.-L.

Lou and F.-Q. Ni contributed equally.

Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS The authors thank the National Natural Science Foundation of China (Nos. 81573302 and 81722042), the Science and Technology Planning Project of Guangdong Province, China (2015A020211007), and Engineering and Technology Research Center for New drug

Druggability

Evaluation

(Seed

Program

of

Guangdong

Province,

2017B090903004) for providing financial support to this work. ABBREVIATIONS USED TGF1, transforming growth factor  1; AAC, abdominal aortic constriction; -SMA,

-smooth muscle actin; FN, fibronectin; ECM, extracellular matrix; Smad, small mother against decapentaplegic; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; SARs,

structureactivity

relationships;

Nif,

nifedipine;

4-[4-(1,3-benzodioxol-5-yl)-5-pyridin-2-yl-1H-imidazol-2-yl]benzamide;

SB431542, OMT,

oxymatrine; TCM, Traditional Chinese medicine; TRI, TGF type I receptor; TRII, TGF type II receptor; qRT-PCR, quantitative real time reverse transcription polymerase chain reaction; SD, standard deviation; IBX, 2-iodoxybenzoic acid; 47



m-CPBA,

meta-chloroperoxybenzoic

acid;

NBS,

Page 48 of 54

N-bromosuccinimide;

NCS,

N-chlorosuccinimide; NFSI, N-fluorobenzenesulfonimide; PE, petroleum ether; rt, room temperature; DMSO, dimethylsulfoxide; DMF, N, N-dimethylformamide.

REFERENCES (1) Kong, P.; Christia, P.; Frangogiannis, N. G. The Pathogenesis of Cardiac Fibrosis. Cell. Mol. Life. Sci. 2014, 71, 549–574. (2) Hattab, D. A.; Czubryt, M. P. A Primer on Current Progress in Cardiac Fibrosis. Can. J. Physiol. Pharmacol. 2017, 95, 1091–1099. (3) Zhang, N.; Wei, W. Y.; Li, L. L.; Hu, C.; Tang, Q. Z. Therapeutic Potential of Polyphenols in Cardiac Fibrosis. Front. Pharmacol. 2018, 9, 122. (4) Marcin, D.; Quesada, C. G.; Frangogiannis, N. G. The Extracellular Matrix as a Modulator of the Inflammatory and Reparative Response Following Myocardial Infarction. J. Mol. Cell. Cardiol. 2010, 48, 504511. (5) Fan, D.; Takawale, A.; Lee, J.; Kassiri, Z. Cardiac Fibroblasts, Fibrosis and Extracellular Matrix Remodeling in Heart Disease. Fibrogenesis Tissue Repair 2012, 5, 15. (6) Lim, D. S.; Lutucuta, S.; Bachireddy, P.; Youker, K.; Evans, A.; Entman, M.; Roberts, R.; Marian, A. J. Angiotensin II Blockade Reverses Myocardial Fibrosis in a Transgenic Mouse Model of Human Hypertrophic Cardiomyopathy. Circulation 2001, 13, 789−791. 48


Page 49 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(7) Krenning, G.; Zeisberg, E. M.; Kalluri, R. The Origin of Fibroblasts and Mechanism of Cardiac Fibrosis. J. Cell. Physiol. 2010, 225, 631637. (8) Baum, J.; Duffy, H. S. Fibroblasts and Myofibroblasts: What are We Talking About? J. Cardiovasc. Pharmacol. 2011, 57, 376379. (9) Travers, J. G.; Kamal, F. A.; Robbins, K. J.; Yutzey, K. E.; Blaxall, B. C. Cardiac Fibrosis: The Fibroblast Awakens. Circ. Res. 2016, 118, 10211040. (10) Leask, A. Potential Therapeutic Targets for Cardiac Fibrosis: TGF, Angiotensin, Endothelin, CCN2, and PDGF, Partners in Fibroblast Activation. Circ. Res. 2010, 106, 1675−1680. (11) Frangogiannis, N. G. The Inflammatory Response in Myocardial Injury, Repair, and Remodeling. Nat. Rev. Cardiol. 2014, 11, 255–265. (12) Derynck, R.; Zhang, Y. E. Smad-Dependent and Smad-Independent Pathways in TGF Family Signalling. Nature 2003, 425, 577–584. (13) Leask, A. TGFβ, Cardiac Fibroblasts, and the Fibrotic Response. Cardiovasc. Res. 2007, 74, 207–217. (14) Worthington, J. J.; Klementowicz, J. E.; Travis, M. A. TGF: A Sleeping Giant Awoken by Integrins. Trends Biochem. Sci. 2011, 36, 47–54. (15) Zhang, Y.; Edgley, A. J.; Cox, A. J.; Powell, A. K.; Wang, B.; Kompa, A. R.; Stapleton, D. I.; Zammit, S. C.; Williams, S. J.; Krum, H.; Gilbert, R. E.; Kelly, D. J. FT011, A New Anti-Fibrotic Drug, Attenuates Fibrosis and Chronic Heart Failure in Experimental Diabetic Cardiomyopathy. Eur. J. Heart Fail. 2012, 14, 549562. (16) Engebretsen, K. V. T.; Skårdal, K.; Bjørnstad, S.; Marstein, H. S.; Skrbic, B.; Sjaastad, I.; Christensen, G.; Bjørnstad, J. L.; Tønnessen, T. Attenuated Development of 49



Cardiac Fibrosis in Left Ventricular Pressure Overload by SM16, an Orally Active Inhibitor of ALK5. J. Mol. Cell. Cardiol. 2014, 76, 148157. (17) Derangeon, M.; Montnach, J.; Cerpa, C. O.; Jagu, B.; Patin, J.; Toumaniantz, G.; Girardeau, A.; Huang, C. L. H.; Colledge, W. H.; Grace, A. A.; Baró, I.; Charpentier, F. Transforming Growth Factor  Receptor Inhibition Prevents Ventricular Fibrosis in a Mouse Model of Progressive Cardiac Conduction Disease. Cardiovasc. Res. 2017, 113, 464–474. (18) Park, S.; Nguyen, N. B.; Pezhouman, A.; Ardehali, R. Cardiac Fibrosis: Potential Therapeutic Targets. Transl. Res. 2019, DOI: 10.1016/j.trsl.2019.03.001. (19) Li, X. Y.; Zhu, L. X.; Wang, B. B.; Yuan, M. F.; Zhu, R. X. Drugs and Targets in Fibrosis. Front. Pharmacol. 2017, 8, 855. (20) Huang, L.; Zhu, J.; Zheng, M.; Zou, R.; Zhou, Y.; Zhu, M. Tanshinone IIA Protects Against Subclinical Lipopolysaccharide Induced Cardiac Fibrosis in Mice through Inhibition of NADPH Oxidase. Int. Immunopharmacol. 2018, 60, 59–63. (21) Li, X.; Wang, X.; Han, C.; Wang, X.; Xing, G.; Zhou, L.; Li, G.; Niu, Y. Astragaloside IV Suppresses Collagen Production of Activated Hepatic Stellate Cells via Oxidative Stress-Mediated P38 MAPK Pathway. Free Radic. Biol. Med. 2013, 60, 168–176. (22) Ai, J.; Nie, J.; He, J.; Guo, Q.; Li, M.; Lei, Y.; Liu, Y.; Zhou, Z.; Zhu, F.; Liang, M.; Cheng, Y.; Hou, F. F. GQ5 Hinders Renal Fibrosis in Obstructive Nephropathy by Selectively Inhibiting TGF-Induced Smad3 Phosphorylation. J. Am. Soc. Nephrol. 2015, 26, 1827–1838.

50


Page 50 of 54

Page 51 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(23) Wu, X. L.; Zeng, W. Z.; Jiang, M. D.; Qin, J. P.; Xu, H. Effect of Oxymatrine on the TGF-Smad Signaling Pathway in Rats with CCl4-Induced Hepatic Fibrosis. World J. Gastroenterol. 2008, 14, 2100–2105. (24) Shao, W. W.; Li, D.; Peng, J.; Chen, S. R.; Zhou, C.; Cheng, Z. B.; Yu, Y.; Li, H.; Li, C. X.; You, Y.; Ma, Y. Z.; Liu, P. Q.; Yin, S.; Shen, X. Y. Inhibitory Effect of Ethyl Acetate Extract of Aristolochia yunnanensis on Cardiac Fibrosis through Extracellular Signal-Regulated Kinases 1/2 and Transforming Growth Factor /Small Mother Against Decapentaplegic Signaling Pathways. Transl. Res. 2014, 163, 160–170. (25) Cheng, Z. B.; Shao, W. W.; Liu, Y. N.; Liao, Q.; Lin, T. T.; Shen, X. Y.; Yin, S. Extracellular

Signal-Regulated

Kinases

(ERK)

Inhibitors

from

Aristolochia

yunnanensis. J. Nat. Prod. 2013, 76, 664671. (26) Lou, L. L.; Li, W.; Zhou, B. H.; Chen, L.; Weng, H. Z.; Zou, Y. H.; Tang, G. H.; Bu, X. Z.; Yin, S. (+)-Isobicyclogermacrenal and Spathulenol from Aristolochia yunnanensis Alleviate Cardiac Fibrosis by Inhibiting Transforming Growth Factor β/Small Mother Against Decapentaplegic Signaling Pathway. Phytother. Res. 2019, 33, 214223. (27) Ruoslahti, E. Fibronectin in Cell Adhesion and Invasion. Cancer Metastasis Rev. 1984, 3, 43–51. (28) Sottile, J.; Hocking, D. C. Fibronectin Polymerization Regulates the Composition and Stability of Extracellular Matrix Fibrils and Cell-Matrix Adhesions. Mol. Biol. Cell 2002, 13, 3546–3559. (29) van Dijk, A. V.; Niessen, H. W. M.; Ursem, W.; Twisk, J. W. R.; Visser, F. C.; van Milligen, F. J. Accumulation of Fibronectin in the Heart after Myocardial Infarction: a 51



Putative Stimulator of Adhesion and Proliferation of Adipose-Derived Stem Cells. Cell Tissue Res. 2008, 332, 289–298. (30) Heling, A.; Zimmermann, R.; Kostin, S.; Maeno, Y.; Hein, S.; Devaux, B.; Bauer, E.; Klövekorn, W. P.; Schlepper, M.; Schaper, W.; Schaper, J. Increased Expression of Cytoskeletal, Linkage, and Extracellular Proteins in Failing Human Myocardium. Circ. Res. 2000, 86, 846–853. (31) Valiente-Alandi, I.; Potter, S. J.; Salvador, A. M.; Schafer, A. E.; Schips, T.; Carrillo-Salinas, F.; Gibson, A. M.; Nieman, M. L.; Perkins, C.; Sargent, M. A.; Huo, J.; Lorenz, J. N.; DeFalco, T.; Molkentin, J. D.; Alcaide, P.; Blaxall, B. C. Inhibiting Fibronectin Attenuates Fibrosis and Improves Cardiac Function in a Model of Heart Failure. Circulation 2018, 138, 12361252. (32) Yamashita, C.; Hayashi, T.; Mori, T.; Matsumoto, C.; Kitada, K.; Miyamura, M.; Sohmiya, K.; Ukimura, A.; Okada, Y.; Yoshioka, T.; Kitaura, Y.; Matsumura, Y. Efficacy of Olmesartan and Nifedipine on Recurrent Hypoxia-Induced Left Ventricular Remodeling in Diabetic Mice. Life Sci. 2010, 27, 322330. (33) Davies, M. R.; Liu, X. H.; Lee, L.; Laron, D.; Ning, A. Y.; Kim, H. T.; Freeley, B. T. TGF Small Molecule Inhibitor SB431542 Reduces Rotator Cuff Muscle Fibrosis and Fatty Infiltration by Promoting Fibro/Adipogenic Progenitor Apoptosis. PLoS One 2016, 11 (5), e0155486. (34) Wu, T. S.; Chan, Y. Y.; Leu, Y. L. The Constituents of the Root and Stem of Aristolochia heterophylla Hemsl. Chem. Pharm. Bull. 2000, 48, 357−361.

52


Page 52 of 54

Page 53 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(35) Zhang, J.; He, L. X. Determation of the Structures of Versicolatone B and Versicolatone C in the Root of Aristolochia versicolar S. M. Hwang. Acta Pharm. Sin. 1986, 21, 273−278.

53



Table of Contents graphic:

54


Page 54 of 54

Germacrane Sesquiterpenoids as a New Type of Anticardiac Fibrosis

Recommend Documents