Discovery of Potent Small-Molecule Inhibitors of Ubiquitin

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Discovery of Potent Small-Molecule Inhibitors of UbiquitinConjugating Enzyme UbcH5c from #-Santonin Derivatives Hao Chen, Guozhen Wu, Shuang Gao, Ruihua Guo, Zeng Zhao, Hu Yuan, Shanxiang Liu, Jian Wu, Xiaolong Lu, Xing Yuan, Zongmin Yu, Xianpeng Zu, Ning Xie, Niao Yang, Zhenlin Hu, Qingyan Sun, and Weidong Zhang J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01829 • Publication Date (Web): 11 Jul 2017 Downloaded from http://pubs.acs.org on July 11, 2017

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Journal of Medicinal Chemistry

Discovery of Potent Small-Molecule Inhibitors of Ubiquitin-Conjugating Enzyme UbcH5c from α-Santonin Derivatives Hao Chena,†, Guozhen Wua,†, Shuang Gaoa,†, Ruihua Guoa, Zeng Zhaoa, Hu Yuanb, Shanxiang Liua, Jian Wue, Xiaolong Luf, Xing Yuana, Zongmin Yua, Xianpeng Zua, Ning Xied, Niao Yanga, Zhenlin Hua,*, Qingyan Sunb,*, Weidong Zhanga,b,c,* a

School of Pharmacy, Second Military Medical University, Shanghai 200433, China.

b

c

Shanghai Institute of Pharmaceutical Industry, Shanghai 200040, China.

Institute of Interdisciplinary Research Complex, Shanghai University of Traditional

Chinese Medicine, Shanghai 201210, China. d

State Key Laboratory of Innovative Natural Medicine and TCM Injections, Jiangxi

Qingfeng Pharmaceutical Co., Ltd. Ganzhou 341000, Jiangxi, China. e

f

Progenra, Inc., 227 Great Valley Parkway, Malvern, PA 19355, USA

Lifesensors, Inc., 227 Great Valley Parkway, Malvern, PA 19355, USA

ACS Paragon Plus Environment


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ABSTRACT As a therapeutic target for anti-tumor necrosis factor (TNF)-α interventions, UbcH5c is one of the key ubiquitin-conjugating enzymes catalyzing ubiquitination during TNF-α-triggered nuclear factor kappa B (NF-κB) activation. In the present study, three series of analogs were designed and synthesized from α-santonin, and their UbcH5c inhibitory activities were screened by western blotting and NF-κB luciferase assay. Further BIAcore, in-gel fluorescence imaging and immunoprecipitation, assays demonstrated that compound 6d exhibited robust and specific inhibition of UbcH5c, exceeding that of the positive compound 1 (IJ-5). Mechanistic investigations revealed that compound 6d preferentially bound to and inactivated UbcH5c by forming a covalent adduct with its active site Cys85. Furthermore, compound 6d exhibited potent anti-inflammatory activity against complete Freund’s adjuvant-induced adjuvant arthritis in vivo. These findings suggest that the novel α-santonin-derived UbcH5c inhibitor 6d is a promising lead compound for the development of new anti-rheumatoid arthritis (RA) agent.

Keywords: α-Santonin; Derivatives; α-Methylene-γ-lactone; E2 UbcH5c; Selective inhibitor; NF-κB; Anti-RA


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INTRODUCTION Rheumatoid arthritis (RA) is a chronic and autoimmune disease of the synovial joints with progressive destruction of cartilage and underlying bone.1 RA affects approximately 1% of the population worldwide.2 However, available drugs have varying levels of defects and side effects. Conventional disease-modifying anti-rheumatic drug methotrexate (MTX) is the first-line therapy for RA. However, many patients fail to respond, and only 55% of patients remain on the drug after 2 years.3 In addition, several anti-tumor necrosis factor (TNF) agents are used to treat RA, including infliximab, adalimumab, and golimumab. These drugs inhibit the effects of TNF-α by preventing it from binding to its receptor. However, anti-TNF biologics are costly and have a high rate of non-response (approximately 30–40% of patients).4,5 Other biologics targeting different pathways are now available, including rituximab, which targets B cells, and tocilizumab, which targets the interleukin-6 (IL-6) pathway. Although considerable progress has been made in the treatment of RA over the past 10 years, there is still a critical need for novel agents with different mechanisms of action for patients unresponsive to current therapies.6 Inflammatory cytokines, such as interleukin-1 (IL-1), IL-6 and TNF-α, are abundant in the synovial tissue and synovial fluid of patients with RA.7 Nuclear factor kappa B (NF-κB) is activated in the chronic inflammation of RA.8-10 NF-κB is one of the

master

regulators

of

inflammatory

cytokines

produced

by

RA.11-15

TNF-α-stimulated NF-κB activation can induce the production of other inflammatory cytokines, induce collagenase release in synovial cells, and promote cartilage degradation and joint destruction. In addition, NF-κB can regulate the transcription of multiple genes in the pathogenesis of RA.16 Thus, NF-κB is regarded as an ideal target for anti-RA.



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NF-κB is a protein complex that controls the transcription of DNA, cytokine production and cell survival. Incorrect regulation of NF-κB has been linked to cancer, inflammation, autoimmune diseases, septic shock, viral infection, and improper immune development. Among most cell types, NF-κB is sequestered in the cytoplasm in an inactive form associated with inhibitors of NF-κB (IκB).17 The activation of NF-κB pathway is dependent on protein ubiquitination.18 Therefore, inhibitors of ubiquitination may suppress the activation of the NF-κB signaling pathway. Ubiquitination is an enzymatic post-translational modification in which a ubiquitin protein is attached to a substrate protein.19 In the protein ubiquitination cascade, ubiquitin (Ub) conjugation to a lysine residue on the target protein is catalyzed by three enzymes: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin ligase (E3).20 In recent years, a variety of small-molecule inhibitors for ubiquitination have been discovered (Chart 1).21-25 Among them, 4[4-(5-nitro-furan-2-ylmethylene)-3,5-dioxo-pyrazolidin-1-yl]-benzoic

acid

ethyl

ester (PYR-41)26 is an inhibitor of E1 ubiquitin ligases, which can inhibit the E1-Ub complex.22 The E2 inhibitor Leucettamol A,27 which was isolated from the marine sponge Leucetta aff. microrhaphis, inhibits ubiquitination mediated by the Ubc13-Uev1A

complex.23

Compound

2-nitro-5-(phenylsulfonyl)furan

(NSC697923),28 which is also an inhibitor of the E2 complex Ubc13-Uev1A, inhibits NF-κB activation in diffuse large B-cell lymphoma (DLBCL) cells.24 Manadosterols A and B inhibit ubiquitination by inhibiting the interaction of Ubc13 and Uev1A.25 Other

compounds,

such

10-(3-chloro-phenyl)-7-nitro-10H-pyrimido[4,5-b]quinoline-2,4-dione

as (HLI98C),

10-(4-chloro-phenyl)-7-nitro-10H-pyrimido[4,5-b]quinoline-2,4-dione (HLI98D) and 10-(4-methylphenyl)-7-nitro-10H-pyrimido[4,5-b]quinoline-2,4-dione


(HLI98E),29

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inhibit recognition of the E3 enzyme and substrate.21 However, no report has described how these small molecules act on ubiquitin enzymes. The concrete mechanism requires further investigation. The successful development of new anti-RA drugs depends, to a large extent, on the discovery of new drug targets, which not only reveals the mechanisms of action of drugs but is also important for the establishment of drug structure screening models and the discovery of lead compounds. In our previous study, an herb-derived sesquiterpene lactone (SL) 1 (IJ-5) (Chart 1),30 was identified based on its ability to inactivate the E2 enzyme UbcH5c, thereby attenuating TNF-α-triggered NF-κB activation and exerting anti-RA effects in vitro and in vivo.31 The mechanisms of action of SL 1 and UbcH5c have been clarified. Further research revealed that the α-methylene-γ-lactone moiety of 1 covalently reacts with the free thiol groups of Cys85 of UbcH5c via Michael addition, suggesting that the α-methylene-γ-lactone moiety may be a pharmacophore for inhibiting UbcH5c activity and that UbcH5c may be a key target in developing novel and effective therapeutic alternatives for RA. These findings provided critical information for the discovery of new anti-RA agents targeting UbcH5c. However, further investigation of 1 was limited due to its low content in natural medicinal plants. Furthermore, the complete synthesis of 1 is difficult given its multiple chiral centers and the β-hydroxy at the C1 position. Therefore, it is useful to search for new lead compounds with anti-RA effects as alternatives to 1. SLs are a large group of secondary metabolites in many medicinal plants.32 SLs exert their anti-inflammatory effects by inhibiting NF-κB activation as the α-methylene-γ-lactone moiety of sesquiterpene lactones can covalently bind to cysteine thiol groups via Michael-type additions.33 Most importantly, the



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α-methylene-γ-lactone moiety may be a pharmacophore for inhibiting UbcH5c, which is a potential therapeutic target for the development of new anti-RA agents. Thus, we sought to determine whether other types of SLs with α-methylene-γ-lactone have similar pharmacological activity to that exhibited by 1. α-Santonins, another type of compound with α-methylene-γ-lactone, were chosen as the starting material for library synthesis due to their favorable arrangement of functional groups and abundant availability at comparably low costs.34-37 To the best of our knowledge, there are no existing reports focused on screening for small molecules with inhibitory activity toward UbcH5c from sesquiterpene lactones and their analogs. In this study, three series of sesquiterpene lactones containing the α-methylene-γ-lactone moiety were successfully constructed by acid rearrangement, photochemical rearrangement and a series of functional group modifications. Subsequently, the effects of these semi-synthetic compounds against UbcH5c protein were evaluated, and the structure-activity relationships (SARs) were investigated. Among the compounds studied, compound 6d had a similar mechanism of action to 1 but exhibited better inhibitory activity toward UbcH5c than that of 1. Derivative 6d suppressed the activation of the NF-κB signaling pathway by selectively covalently binding to the Cys85 residue of UbcH5c. In addition, the anti-RA effect of 6d by oral administration in rats was studied in a preliminary pharmacological experimental study. Herein, we describe the design, synthesis, biological evaluation, preliminary structure-activity relationship analysis, and interactive mechanisms with UbcH5c and pharmacokinetics (PK) properties in rats, stability in vitro, anti-RA activity in vivo for this class of compounds. CHEMISTRY A convenient method for synthesizing guaiane-type sesquiterpene lactone 6 is


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presented in Scheme 1. In this route, the important intermediate compound 2 was first synthesized based on our previous study.38 Compound 2 was then treated with t-butyldimethylsilyl triflate (TBSOTf) to protect the 2-hydroxyl group and obtain compound 3. To form compound 5, a two-step procedure of selective dehydrogenation of the α-methyl-γ-lactone moiety was used that included the formation of phenylseleno-substituted 4 followed by treatment with hydrogen peroxide (H2O2). Finally,

after

deprotection

of

the

TBS

group

of

compound

5

using

tetrabutylammonium fluoride (TBAF), compound 6 was synthesized with excellent yield. As depicted in Scheme 2, compound 10 was also synthesized using α-santonin as a starting material. Compound 8 was first obtained using the building block of the α-methylene-γ-lactone moiety. Then, compound 9 with an aromatic ring was generated by acid rearrangement. The final target compound 10 was obtained from compound 9 under conditions of ammonium hydroxide in methanol. Alternatively, reduction of compound 8 with DIBAL-H in anhydrous tetrahydrofuran produced a separable mixture of the desired α-alcohol 11 and its β-isomer 12 in a ratio of 4:1 with 89% yield.39 The single-crystal X-ray structure of compound 12 (CCDC: 1014105) was obtained (Figure S1). In Scheme 3, compound 13 was synthesized from α-santonin by catalytic hydrogenation under the conditions of Pd/C and H2 at 3.5 atm in ethanol.40 Subsequently, reduction of carbonyl of compound 13 with NaBH4 yielded compounds 14, 15 and 16. Among them, compound 14 was isolated via silica gel column chromatography (PE/Acetone = 50:1), and compound 15 was purified by simple recrystallization (in CH2Cl2/MeOH = 10:1). Compound 16 was converted into compounds 23 and 24, which could be separated by silica gel column chromatography.



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The single-crystal X-ray structures of compounds 23 (CCDC: 1014107) and 24 (CCDC: 1014109) were obtained (Figures S2 and S3). Then, compounds 20, 21, 25 and 26 were synthesized following the approach used for compound 6. RESULTS AND DISCUSSION UbcH5c activity screening and preliminary SAR According to a limited screening of α-santonin-derived SLs for their inhibition activities toward UbcH5c by western blotting, compounds 6, 10 and 20 were identified as possessing weak UbcH5c inhibitory effects at 10 µM and could be used as precursors for further modification (Figure 1). To screen for easily available compounds with activity greater than 1, compounds 6, 10 and 20 were further modified, and 3 types, and a total of 73 derivatives were acquired. Their effects on the dimer formation of UbcH5c-Ub were measured by western blotting. The next step was to investigate the correlation between the structures of the derivatives and their activities with respect to inhibiting UbcH5c activities. Analogs 1a‒1f and 2a‒2f were derivatives of compound 6 (Scheme 4). According to a preliminary screen at a concentration of 10 µM, most of the derivatives had stronger activities than precursor 6 in inhibiting UbcH5c, except for compounds 1c, 1d, 2a and 2d. These results indicate that the introduction of benzoic acid and cinnamic acid side chains improves the activities (Figure 2A). Further screening at 5 µM (Figure 2A) revealed that compounds 1a, 1b, 1e and 2f still possess better activities. Comparing compounds 1a, 1b and 1e with 2a, 2b and 2e, the latter compounds had longer conjugated side chains, whereas the latter compounds were weaker than former compounds in the inhibition of UbcH5c activities. Therefore, the longer conjugated side chain structure may not enhance the effects of inhibiting UbcH5c.


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For derivatives 3a‒3q, which were benzoic acid derivatives of compound 10 (Scheme 5), these compounds exhibited no inhibitory effects toward UbcH5c at 5 µM (Figure 2B). For derivatives 4a‒4h, cinnamic acid side chains were introduced into

the R4 position (Scheme 5), whereas all of other compounds had no inhibitory activities except 4c (Figure 2B). Among compounds 5a‒5g, heterocyclics, such as furan, thiophene, and pyridine, were introduced into the derivatives (Scheme 5). Compounds 5a, 5f and 5g exhibited significant effects against UbcH5c (Figure 2B). Analysis of the structures of compounds 5a and 5g revealed that electron-withdrawing of the meta-nitrogen atom of pyridine may be responsible for enhancing the activities. Compounds 6a‒6i were benzyl ether derivatives of compound 10 (Scheme 5). Among these compounds, 6d, 6g and 6i, which contain o-bromine, o-trifluoromethyl and p-trifluoromethoxy, respectively, displayed significant effects in inhibiting UbcH5c activities (Figure 2B). For derivatives (7a‒7f, 8a‒8j, 9a‒9g) of compound 20 (Scheme 6), all of the aliphatic acid derivatives (7a‒7f) exhibited no inhibitory effects toward UbcH5c at 1 µM (Figure 2C). Simultaneously, almost all the other derivatives (8a‒8j, 9a‒9g) expressed no activities, except for compounds 8a, 8b and 9a, which contain m-iodin, o-iodin and a furan ring, respectively (Figure 2C). According to the results of screening derivatives by the inhibition of UbcH5c, 14 compounds 1a, 1b, 1e, 2f, 4c, 5a, 5g, 5f, 6i, 6d, 6g, 8a, 8b and 9a were identified to have inhibitory effects toward UbcH5c at 5 µM. Given that UbcH5c plays a key role in TNF-α-mediated NF-κB activation, the inhibitory effects of the above 14 compounds on NF-κB activation were measured by NF-κB luciferase reporter gene assay to reduce the subsequent workload. The pTA-NF-κB luciferase reporter gene vector contains four duplicated NF-κB cis-acting



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elements in the mini-promoter TATA box, which is located upstream of the luciferase gene-encoding region. When this vector is transfected into cells and NF-κB is activated, it translocates into the nucleus and combines with the NF-κB cis-acting element, accordingly promoting luciferase gene expression. Given that cell viability will affect luciferase activity, the optimal dosage of these compounds was first investigated with a Cell Counting Kit-8 (CCK-8) assay to eliminate the interference caused by cytotoxicity. The results showed that the inhibition rates of compounds on 293T cells were less than 20% at 5 µM, indicating that 5 µM could be used as the experimental dosage in the NF-κB luciferase assay. At a 5 µM concentration, the inhibition rates of 14 compounds on NF-κB activation were as follows: 1a (49.7%), 1b (45.0%), 1e (47.0%), 2f (32.8%), 4c (34.7%), 5a (63.8%), 5f (36.5%), 5g (69.2%), 6d (63.4%), 6g (31.4%), 6i (36.9%), 8a (62.1%), 8b (63.1%) and 9a (71.8%). To further confirm whether the above compounds possess inhibitory effects on TNF-α-induced NF-κB activation, we selected the compounds with inhibition rates greater than 40% for further screening by western blot assay (Figure 3A). The results showed that compounds 1a, 1b, 5g, 6d, 8a and 8b could inhibit the phosphorylation and degradation of IκB-α in b.End3 cells. Using b.End3 cells as a model, the 6 target compounds were further investigated to study their effects on IκB-α, p65 and the phosphorylation level of both (Figure 3B). The results showed that these 6 compounds could dose-dependently inhibit the phosphorylation and degradation of IκB-α and the phosphorylation of p65 in the range of 1‒10 µM. The 6 compounds could suppress the activation of the NF-κB signaling pathway. Another important E2 enzyme, Ubc13, could catalyze the formation of the K63 polyubiquitin chain, which can induce the activation of IKK and is involved in NF-κB


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activation to a certain extent. Therefore, it was necessary to investigate whether the target compounds could exert their inhibitory effect on NF-κB activation via inactivating Ubc13. Western blot assay demonstrated that compounds 1a, 1b, 5g, 6d, 8a, and 8b could dose-dependently inactivate UbcH5c in the range of 2.5‒10 µM, whereas 5g could not. Except for compound 5g, the other 5 compounds exhibited no inhibitory effects on Ubc13 (Figure 4A). This result not only confirmed the inhibitory effects of the active compounds on UbcH5c but also preliminary revealed that these 5 compounds may act on UbcH5c. To determine the adhesive strength between the above compounds and UbcH5c, the interaction kinetics of the interactions between the candidate compounds and UbcH5c were tested by surface plasmon resonance using a BIAcore T200 (Biacore AB, Uppsala, Sweden) system to identify the target compounds possessing the strongest affinity with UbcH5c for subsequent research. The results revealed that all compounds (except 1a) exhibited dose-dependent increases in response to the signal, indicating that those candidate compounds could bind to UbcH5c directly. The sensorgrams provided KD values for 1b, 6d, 8a, and 8b of 3.64 µM, 0.283 µM, 6.40 µM and 0.353 µM, respectively (Figure 5). Among these compounds, 6d exhibited the strongest affinity to UbcH5c, with a KD value of 0.283 µM. Compared with 1 (KD = 2.58 µM), compound 6d also had a strong affinity and interaction with UbcH5c. In addition, it is easier and cheaper to obtain 6d via semi-synthesis from α-santonin. These results indicated that compound 6d might have the potential to be a selective inhibitor of the ubiquitin-conjugating enzyme UbcH5c. Mechanistic study of compound 6d To further delineate the 6d mechanism of action, we tested its activity on the loading of Ub to other representative E2 enzymes in vitro Ub-loading assays. The



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results demonstrated that 6d (2.5–10 µM) dose-dependently inhibited Ub loading to UbcH5c (Figure 4A). In Figure 4B, 6d also inhibited Ub loading to the other two members of the UbcH5 family, UbcH5a and UbcH5b, but UbcH5a is slightly inhibited. Meanwhile, 6d exhibited an obviously weaker inhibitory effect against Ub loading to UbcH7 and Ubc10. Furthermore, it did not affect the Ub loading to Ubc13, E2-25K and UbcH2 (Figure 4A and 4B). To identify the proteomic selectivity of 6d, we conducted an in-gel fluorescence imaging assay according to the previous literature.41 We synthesized a probe 6d-1 with a terminal alkyne moiety at the 3´ position of 6d as a “clickable” tag (Figure 6A and Scheme S1). Total lysis of HeLa cells was treated with the small-molecule probe at different concentrations. After CuAAC (Cu(I)-catalyzed [3+2] azide-alkyne cycloaddtion) with Azide-fluor 488 and SDS-PAGE separation, in-gel fluorescence scanning revealed one major fluorescent band of ~15 kDa (Figure 6C). To clarify whether the cellular target labelled by probe was UbcH5c, we transferred the proteins to a poly-vinylidene fluoride (PVDF) membrane and conducted western blot assay. Immunoblot analysis using an UbcH5c antibody confirmed the protein labelled by probe 6d-1 was UbcH5c (Figure 6D). To sum, these data suggest that alkyne probe 6d-1 can potently and selectively engage UbcH5c in the human proteome. Combined with the above related experimental results, compound 6d can potently and selectively react with UbcH5c target protein. SL compounds with an α-methylene-γ-lactone moiety bind covalently to free cysteine thiol groups of proteins via a Michael addition.42,43 UbcH5c contains two free cysteine residues at positions 85 and 111.31 Cys85 is the E2 active site residue that accepts activated Ub from E1. In our previous study, SL 1 was shown to form a covalent adduct with Cys85 via a Michael addition, thereby inactivating UbcH5c.


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Thus, we predicted that compound 6d might have the same mechanism as 1. To test this prediction, we incubated synthetic peptide (IYHPNINSNGSICLDILR, purity ≥ 98%, peptide 73‒90 of UbcH5c) and recombinant UbcH5c with 6d separately. The former was used as a positive control, and the latter was digested with trypsin. Both

were

analyzed

using

online

liquid

chromatography

(LC)-Orbitrap

collision-induced dissociation (CID) tandem mass spectrometry (MS/MS). As shown in Figure S4A, a molecular mass of 819.38 was found for the synthetic peptide with modification by 6d (C111H165N26O30SBr) (R = 47000, Z = 3). Figures S4C and S4A show that the same peptide was modified by 6d, derived from the digestion of UbcH5c protein that was directly measured by high-resolution MS with high confidence. Thus, this peptide fragment should contain Cys85 of UbcH5c, and the mass shift suggested that UbcH5c was modified by 6d at Cys85. CID fragmentation MS/MS was also used to localize the modification site with single amino acid resolution. Detection of precursor ions at high resolution and an almost complete series of fragmentation ions from CID allowed the accurate sequencing and assignment of the modification site to Cys85 (Figures S4B, S4D and S4E). Interestingly, no mass shift was observed on the peptide with a molecular mass of 1353.73, which contains another free cysteine, Cys111, suggesting that the covalent binding is sterically selective for Cys85. Based on the results of mass spectrometry experiments, the terminal carbon atom with an α-methylene moiety of 6d and the sulfur atom of Cys85 were specified as the ligand reactive group and the receptor bond, respectively. To further investigate and determine the binding mode of 6d and UbcH5c, we performed a covalent docking simulation using Maestro software (Schrödinger, version 9.0). From the proposed binding mode, the stereo conformation of 6d fits well with the binding site around the



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reactive cysteine residue Cys85, and the hydroxyl and carbonyl groups of 6d can form additional hydrogen bonds with the amino groups of Gln92 and Arg90 of UbcH5c, respectively. These hydrogen bonds may help 6d conjugate to Cys85 but not other Cys proteins (Figure 7). Given that UbcH5c together with ligase E3 LUBAC can catalyze the formation of linear polyubiquitin chains of NEMO, ubiquitinated NEMO can be recognized and bind to another NEMO. Thus, it aids in IKKβ dimerization and ultimately leads to the trans-autophosphorylation of IKK, which is indispensable for TNF-α-mediated NF-κB activation. The effect of 6d on the linear ubiquitination of NEMO in HeLa cells was analyzed by immunoprecipitation to further explore the relationship between compound 6d and UbcH5c (Figure 8). The results demonstrated that 6d (2.5‒10 µM) could dose-dependently inhibit the TNF-α-induced linear polyubiquitination of NEMO, indicating that 6d could inhibit UbcH5c activity in cells and exert an inhibitory effect on the NF-κB pathway through suppressing the linear polyubiquitination of NEMO. NF-κB activation is an important factor contributing to inflammation in chronic diseases. NF-κB is highly activated in the synovium of patients with RA and can induce the transcription of proinflammatory cytokines, chemokines and matrix metalloproteinase, which are involved in the pathologic processes of RA.15 The precise etiology of RA is not known; synovial hyperplasia is the main pathogenic character of RA, and hyperplasia of the synovial membrane can induce the invasion of gristle and bone and destroy the structure of the joint. Fibroblast-like synoviocytes (FLSs) play a critical role in synovial hyperplasia given that they can produce cytokines that perpetuate inflammation and proteases that contribute to cartilage destruction.44

These

cytokines

include

matrix

metalloproteinases


(MMPs),

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pro-inflammatory cytokines, and chemokines. MMP-3 is the most important protease involved in cartilage degradation. Once activated, MMP-3 can not only directly degrade cartilage and bone but also activate other MMPs and degrade multiple proteins, such as cartilage link protein, fibronectin, and collagen types.45 In RA, IL-1 and MCP-1, which can be detected in synovial cells as well as in synovial fluid, could exert local effects on cartilage and bone matrix metabolism and enhance joint destruction indirectly.46,47 Synovial fibroblasts can be isolated from synovial tissue and grown in culture for a period of time. In addition, these cells secrete diverse cytokines. We used TNF-α as a stimulant to elevate mRNA levels of MMP-3, MCP-1 and IL-1 and test the inhibition effect of 6d on synovial fibroblasts (Figure 9). Quantitative real-time PCR (qPCR) results demonstrated that 6d could dose-dependently inhibit the mRNA levels of MMP-3, MCP-1 and IL-1 in the range of 1‒5 µM, indicating that 6d may have potential therapeutic value for rheumatoid arthritis. PK properties of compound 6d in rats and stability in vitro. The PK properties of 6d were evaluated in rats following intravenous and oral administration (Table 1). 6d was administered at a dose of 1 mg/kg in saline mixture (DMSO:castor-oil:saline = 3:3:94) (iv) or 5 mg/kg in 0.5% methlcellulose (po), respectively. Its bioavailability was determined to be 16.8%, and the half-life (t1/2) of 6d was 1.76 h (iv) and 2.86 h (po). It had a volume distribution (6.26 L/kg) and clearance at 2.48 L/h/kg. Next we examined the stability of 6d in vitro by measuring the half-life for compound loss upon incubation with rat liver microsomes, plasma and whole blood. Results are summarized in Figure S6 and Table 2, 6d shows good stability to microsomal and plasma degradation. However, in rat whole blood, the half-life of 6d



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is only 39.8 min, with relatively low stability. In this experiment process we found no lactone ring-opening metabolites hydrolyzed by the ester hydrolase. In terms of structure, α-methylene-γ-lactone of 6d is a Michael acceptor, and it is easy to covalently bind to cysteine thiol groups of protein via Michael-type additions.33 Therefore, a reasonable guess is that 6d can covalently bind to substance containing thiol group in blood cells, which leads to be lower stability in rat whole blood, but this was not investigated. So the above problems need for further study in the future. In vivo anti-inflammatory activity of compound 6d To evaluate the therapeutic value of 6d for the treatment of inflammatory diseases, we tested its in vivo anti-inflammatory effects in the complete Freund’s adjuvant (CFA)-induced adjuvant arthritis (AA) rat model.48 The AA model is a model of immune inflammation, and its histological features include synovial hyperplasia, mononuclear cell infiltration, articular cartilage and bone destruction. The model is similar to human rheumatoid arthritis and serves as a general model to study drugs in the treatment of rheumatoid arthritis. The AA model is generally divided into two phases: primary and secondary reaction stages. The primary reaction stage mainly involves an acute inflammation reaction, which mainly involves the ankles and can incorporate the foot pad and even the full foot. The secondary reaction stage is mainly manifested as a symptom of immunity-related inflammation caused by immune cell function disorder, which appears 10 to 16 days after inflammation and mainly involves non-inflammatory foot swelling, "arthritis" in ears and tail, significant weight loss and other chronic whole-body inflammation characterized by polyarthritis, all of which are signs of immune inflammation caused by immune dysfunction. In the AA rat model, daily oral administration of compound 6d (5 and 20 mg/kg per day) resulted in appreciable decreases in the arthritis index score and hind paw


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swelling rate compared with those of the vehicle-treated AA model control group (Figure 10A, 10B and 10C), indicating 6d has a potent in vivo anti-inflammatory activity. However, as shown in Figure 10D, compound 6d (5 mg/kg and 20 mg/kg) had no effect on weight loss in arthritic rats. We conjecture that the causes of this poor effect might be the relatively low stability in rat whole blood and an overly short duration of administration. However, this is a problem to be further studied and discussed. CONCLUSIONS The post-translational modification of proteins with ubiquitin (Ub) controls a number of fundamental processes within the cell, including protein degradation by the Ub-proteasome system (UPS).49 In the protein ubiquitination cascade, Ub conjugation to lysine residue on target proteins is catalyzed by the enzymes E1, E2 and E3. UbcH5 is an E2 enzyme family consisting of three homologs: UbcH5a, UbcH5b, and UbcH5c. Among them, UbcH5c is a key ubiquitin-conjugating enzyme catalyzing linear polyubiquitin chain formation and therefore plays a critical role in TNF-α-triggered NF-κB activation.50-52 More importantly, we reason that UbcH5c is a key target for the development of novel and effective therapeutic alternatives for RA. To discover novel potent small molecules which anti-RA by targeting UbcH5c, three libraries of 73 analogs in total were successfully designed and synthesized from α-santonin. These analogs all have an α-methylene-γ-lactone moiety as a pharmacophore. Structural optimization and SAR studies led to the discovery of sub-micromolar UbcH5c inhibitors. Their UbcH5c inhibitory activities were evaluated by western blotting and NF-κB luciferase assay. Among them, 14 compounds exhibited inhibitory effects against UbcH5c at 5 µM. Further mechanistic studies revealed that compounds 1a, 1b, 5g, 6d, 8a and 8b could suppress



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TNF-α-mediated NF-κB activation. Subsequently, we demonstrated that except for compound 5g, compounds 1a, 1b, 6d, 8a, and 8b dose-dependently inactivated UbcH5c in the range of 2.5‒10 µM but exhibited no inhibitory effects on Ubc13 by western blotting assay. Then, the interaction kinetics between each of the above five compounds and UbcH5c were tested using the BIAcore essay. Compound 6d has the most robust inhibition against UbcH5c, with a KD value of 0.283 µM, even exceeding that of 1. Next we conducted an in-gel fluorescence imaging assay, which proved 6d could potently and selectively engage UbcH5c in the human proteome. Furthermore, the mass spectrometry experiment and covalent docking simulation confirmed that compound 6d inactivated UbcH5c by covalently binding to the Cys85 residue. The immunoprecipitation results revealed that 6d could inhibit UbcH5c activity and exert an inhibitory effect on the NF-κB pathway by suppressing the linear polyubiquitination of NEMO. Subsequently, qRT-PCR results demonstrated that 6d dose-dependently decreased the mRNA levels of MMP-3, MCP-1 and IL-1 in the range of 1‒5 µM, indicating that 6d had a therapeutic effect on rheumatoid arthritis. Furthermore, 6d exhibited significant in vivo effects on the AA model of rats in a preliminary pharmacological experiment. These results highlighted the potential value of compound 6d as a novel anti-rheumatoid arthritis lead scaffold. However, 6d has a relatively low stability in rat whole blood, which points out future research directions. In conclusion, our current findings strongly demonstrate that 6d is a selective small-molecule inhibitor of ubiquitin-conjugating enzyme UbcH5c and deserves further investigation as a lead compound and may ultimately become an anti-RA agent.


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EXPERIMENTAL SECTION Chemistry materials and methods Unless otherwise noted, all materials were obtained from commercial suppliers and used as obtained. Anhydrous organic solvents were purchased from J&K under N2 in Sure/Seal bottles and used directly. All solvents and reagents were of analytically pure grade, and no further purification was needed. Petroleum ether describes a mixture of hexanes in the bp range of 60 to 90 °C. Analytical thin-layer chromatography was performed on Yantai Huanghai HSGF254 silica gel plates and visualized by fluorescence quenching under UV light. Flash column chromatography was performed using silica gel (200‒300 mesh). Nuclear magnetic resonance (NMR) spectra were recorded using TMS as the internal standard in CDCl3 with a Bruker BioSpin GmbH spectrometer at 300 MHz or 500 MHz. When peak multiplicities are reported, the following abbreviations are used: s = singlet, d = doublet, t = triplet, m = multiplet, and dd = doublet of doublets. Melting points were determined using an X-4B micro-melting point apparatus. HRMS was recorded on an Agilent-6520 Q-TOF mass spectrometer (Agilent Technologies, Palo Alto, CA, USA). All the final compounds were tested by HPLC and the purity in every case was ≥ 95%. The reverse phase HPLC was conducted on Agilent Technologies 1260 Inﬁnity, which was equipped with C18 column (Aglilent Eclipse XDB-C18, 5 µm, 4.6 mm × 250 mm). The mobile phase A was 0.1% formic acid in water and mobile phase B was acetonitrile. The gradient of 20 ‒ 95% B was run at a flow rate of 1.0 mL/min over 30 min. (3S,3aS,6R,8S,9bS)-8-((Tert-butyldimethylsilyl)oxy)-3,6,9-trimethyl-2-oxo-2,3,3a,4,5, 6,6a,7,8,9b-decahydroazuleno[4,5-b]furan-6-yl acetate (3) To a solution of compound 2 (1.20 g, 3.9 mmol, 1.0 eq.) and N,N-diisopropylethylamine



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(DIPEA) (2.62 g, 20.3 mmol, 5.2 eq.）in dry CH2Cl2 (20 mL) being coolen to 0 °C was added dropwise tert-butyldimrthylsilyl trifluoromethanesulfonate (TBSOTf) (1.96 g, 7.4 mmol, 1.9 eq.). The reaction solution was stirred at 0 ºC for 30 min and then at room temperature for another 70 min. The reaction was complete detected by TLC and then quenched by the addition of the saturated aqueous NaHCO3. The mixture was extracted with CH2Cl2 three times (3 × 20 mL) and washed with brine. The combined organic layers were dried by anhydrous Na2SO4 and evaporated under vacuum to give a residue that was purified by silica-gel column chromatography (PE/EtOAc=20:1 – 15:1 – 10:1), resulting in a white solid product 3 (1.55 g, 94%). 1H NMR (500 MHz, CDCl3) δ 4.65 (dd, J = 10.9, 1.7 Hz, 1H), 4.49 (t, J = 6.7 Hz, 1H), 3.72 (s, 1H), 2.43 (td, J = 13.4, 4.3 Hz, 1H), 2.29 – 2.08 (m, 3H), 1.98 (s, 3H), 1.92 (dd, J = 6.9, 3.0 Hz, 1H), 1.81 (s, 3H), 1.59 (s, 1H), 1.53 (ddd, J = 13.5, 6.7, 5.9 Hz, 1H), 1.43 – 1.30 (m, 1H), 1.24 – 1.10 (m, 6H), 0.94 – 0.88 (m, 9H), 0.08 (dd, J = 6.5, 3.0 Hz, 6H); 13C NMR (125 MHz, CDCl3) δ 178.2, 170.4, 145.1, 129.7, 86.8, 81.7, 77.8, 51.2, 49.0, 41.5, 38.0, 35.2, 25.8 (3C), 25.2, 22.4, 20.2, 18.1, 12.7, 12.4, -4.5, -4.8. HRMS (ESI): m/z calcd for C23H38O5SiNa [M+Na]+: 445.2386, found 445.2390. (3R,3aR,6R,8S,9bS)-8-((Tert-butyldimethylsilyl)oxy)-3,6,9-trimethyl-2-oxo-3-(pheny lselanyl)-2,3,3a,4,5,6,6a,7,8,9b-decahydroazuleno[4,5-b]furan-6-yl acetate (4) To a stirred -78 °C solution of compound 3 (2.6 g, 6.2 mmol, 1.0 eq.) in dry THF (30 mL) under argon was added dropwise LDA (2.0 M, 3.7 mL, 1.2 eq.) via syringe over 5 min. After 30 min, a solution of PhSeCl (1.3 g, 6.8 mmol, 1.1 eq.) in dry THF (10 mL) was added dropwise via syringe to the above mixture while keeping internal temperature between -55 ̶ 78 °C. The reaction solution was stirred at -78 °C for 60 min and then at


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room temperature for overnight. The reaction was complete detected by TLC and then quenched by the addition of the saturated aqueous NH4Cl. The mixture was extracted with EtOAc three times (3 × 40 mL) and washed with brine. The combined organic layers were dried by anhydrous Na2SO4 and evaporated under vacuum to give a residue that was purified by silica-gel column chromatography (PE/EtOAc=25:1 – 20:1 – 15:1), resulting in a white solid product 4 (2.70 g, 80%). 1H NMR (500 MHz, CDCl3) δ7.64 (d, J = 7.3 Hz, 1H), 7.64 (d, J = 7.3 Hz, 1H), 7.43 (t, J = 7.3 Hz, 1H), 7.37 (t, J = 7.3 Hz, 1H), 7.37 (t, J = 7.3 Hz, 1H), 5.05 (d, J = 7.3 Hz, 1H), 4.42 (dd, J = 5.9, 6.6 Hz, 1H), 3.58 (dd, J = 3.8, 8.9 Hz, 1H), 2.67 (dd, J = 6.0, 13.9 Hz, 1H), 2.46 (dt, J = 7.3, 11.5 Hz, 1H), 2.19 (ddd, J = 3.8, 6.6, 14.4 Hz, 1H), 2.01 (ddd, J = 6.0, 11.5, 14.0 Hz, 1H), 1.96 (s, 3H), 1.84 (dd, J = 12.3, 13.9 Hz, 1H), 1.77 (s, 3 H), 1.66 (ddd, J = 11.5, 12.3, 14.0 Hz, 1H), 1.57 (s, 3H), 1.57 (ddd, J = 5.9, 8.9, 14.4 Hz, 1H), 1.09 (s, 3H), 0.89 (s, 9H), 0.08 (s, 6H);

13

C

NMR (125 MHz, CDCl3) δ175.8, 171.5, 142.4, 138.3, 138.3, 131.3, 129.7, 128.9, 128.9, 124.5, 87.4, 78.8, 76.8, 54.3, 53.0, 52.4, 38.9, 35.7, 25.9, 25.9, 25.9, 22.5, 21.8, 21.8, 19.8, 18.2, 12.4, -4.7, -4.7. HRMS (ESI): m/z calcd for C29H42O5SeSiNa [M+Na]+: 601.1864, found 601.1866. (3aS,6R,8S,9bS)-8-((Tert-butyldimethylsilyl)oxy)-6,9-dimethyl-3-methylene-2-oxo-2, 3,3a,4,5,6,6a,7,8,9b-decahydroazuleno[4,5-b]furan-6-yl acetate (5) To a solution of compound 4 (1.35 g, 2.34 mmol, 1.0 eq.) and acetic acid (HAc) (0.42g, 7.01 mmol, 3.0 eq.）in THF (15 mL) being coolen to 0 °C was added dropwise 30% H2O2 (1.72 mL, 15.2 mmol, 6.5 eq.). The reaction solution was stirred at 0 °C for 20 min and then at 20 °C for 10 min. The reaction was complete detected by TLC and then quenched by the addition of the saturated aqueous NaHCO3. The mixture was extracted with EtOAc



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three times (3 × 20 mL) and washed with brine. The combined organic layers were dried by anhydrous Na2SO4 and evaporated under vacuum to give a residue that was purified by silica-gel column chromatography (PE/EtOAc = 15:1 – 10:1 – 8:1), resulting in a white solid product 5 (0.94 g, 94%). 1H NMR (500 MHz, CDCl3) δ 6.23 (d, J = 3.4 Hz, 1H), 5.49 (d, J = 3.1 Hz, 1H), 4.66 (dd, J = 10.9, 1.8 Hz, 1H), 4.51 (t, J = 6.8 Hz, 1H), 3.76 (d, J = 1.8 Hz, 1H), 2.87 (dd, J = 11.4, 10.1 Hz, 1H), 2.57 – 2.43 (m, 1H), 2.31 – 2.10 (m, 3H), 1.99 (s, 3H), 1.86 (s, 3H), 1.60 – 1.48 (m, 2H), 1.20 (s, 3H), 0.91 (s, 9H), 0.10 (s, 3H), 0.08 (s, 3H);

13

C NMR (125 MHz, CDCl3) δ 170.4, 169.8, 145.2, 138.8,

129.2, 119.4, 86.7, 82.1, 77.8, 51.1, 45.2, 37.6, 35.4, 25.9 (3C), 24.2, 22.4, 20.2, 18.1, 12.7, -4.5, -4.8. HRMS (ESI): m/z calcd for C23H36O5SiNa [M+Na]+: 443.2224, found 443.2225. (3aS,6R,8S,9bS)-8-Hydroxy-6,9-dimethyl-3-methylene-2-oxo-2,3,3a,4,5,6,6a,7,8,9b-d ecahydroazuleno[4,5-b]furan-6-yl acetate (6) To a solution of compound 5 (1.20 g, 2.86 mmol, 1.0 eq.) in THF (20 mL) being coolen to 0 °C was added TBAF (1.72 mL, 15.2 mmol, 6.5 eq.) in portions. The reaction solution was stirred at 0 ºC for 30 min. Then the reaction was complete detected by TLC and then quenched by the addition of the saturated aqueous NaHCO3. The mixture was extracted with EtOAc three times (3 × 25 mL) and washed with brine. The combined organic layers were dried by anhydrous Na2SO4 and evaporated under vacuum to give a residue that was purified by silica-gel column chromatography (PE/EtOAc = 10:1 – 5:1 – 2:1), resulting in a white solid product 6 (0.84 g, 96%), mp 100.7–101.2 ºC. 1H NMR (500 MHz, CDCl3) δ 6.25 – 6.18 (m, 1H), 5.50 (d, J = 2.9 Hz, 1H), 4.66 (d, J = 10.8 Hz, 1H), 4.56 (s, 1H), 3.79 (s, 1H), 2.88 (t, J = 10.8 Hz, 1H), 2.55 – 2.44 (m, 1H), 2.40 (dt, J = 13.8, 8.0 Hz,


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1H), 2.19 (t, J = 15.8 Hz, 2H), 1.97 (d, J = 1.0 Hz, 3H), 1.92 (s, 3H), 1.62 – 1.53 (m, 1H), 1.41 (dt, J = 11.2, 7.1 Hz, 1H), 1.26 (s, 1H), 1.19 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 170.4, 169.7, 144.1, 138.6, 130.6, 119.6, 86.4, 82, 77.7, 51.2, 45.1, 37.5, 34.8, 24.2, 22.4, 20.1, 12.5. HPLC purity: 95.8%, retention time = 7.31 min. HRMS (ESI): m/z calcd for C17H22O5Na [M+Na]+: 329.1359, found 329.1365. (3R,3aR,5aS,9bS)-3,5a,9-Trimethyl-3-(phenylselanyl)-3a,5,5a,9b-tetrahydronaphtho [1,2-b]furan-2,8(3H,4H)-dione (7) According to the synthetic method of compound 4. 1H NMR (500 MHz, CDCl3) δ 7.65 (d, J = 7.3 Hz, 2H), 7.44 (d, J = 7.4 Hz, 1H), 7.35 (t, J = 7.5 Hz, 2H), 6.68 (d, J = 9.9 Hz, 1H), 6.25 (d, J = 9.9 Hz, 1H), 5.22 (d, J = 9.6 Hz, 1H), 2.14 (s, 3H), 2.02 – 1.95 (m, 3H), 1.60 (s, 3H), 1.53 (dd, J = 13.4, 7.6 Hz, 1H), 1.33 (s, 3H), 1.25 (s, 1H); 13C NMR (125 MHz, CDCl3) δ 186.2, 174.7, 154.7, 150.9, 138.2 (2C), 130.0, 129.3 (3C), 126.0, 123.9, 79.3, 57.6, 48.8, 41.3, 37.5, 25.1, 22.3, 20.6, 11.0. The spectroscopic data are consistent with the values reported in the literature.53 (3aS,5aS,9bS)-5a,9-Dimethyl-3-methylene-3a,5,5a,9b-tetrahydronaphtho[1,2-b]fura n-2,8(3H,4H)-dione (8) According to the synthetic method of compound 5. Mp 144.1–145.7 ºC. 1H NMR (500 MHz, CDCl3) δ 6.71 (td, J = 3.1, 9.9 Hz, 1H), 6.28 (d, J = 9.9 Hz, 1H), 6.14 (dd, J = 3.1, 3.3 Hz, 1H), 5.50 (dd, J = 3.1, 3.3 Hz, 1H), 4.78 (d, J = 11.4 Hz, 1H), 2.80 (ttd, J = 3.3, 7.2, 11.4 Hz, 1H), 2.17 (s, 3H), 2.18 (tdd, J = 6.9, 7.2, 14.1 Hz, 1H), 1.88 (dtd, J = 3.1, 6.9, 13.8 Hz, 1H), 1.85 (tdd, J = 6.9, 7.2, 14.1 Hz, 1H), 1.68 (dtd, J = 3.1, 6.9, 13.8 Hz, 1H), 1.32 (s, 3H);

13

C NMR (125 MHz, CDCl3) δ 185.9, 168.8, 155.4, 154.4, 138.0,

132.5, 126.1, 119.5, 81.3, 45.7, 40.2, 36.8, 25.0, 23.6, 11.3. HPLC purity: 95.7%,



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retention time = 6.95 min. The spectroscopic data are consistent with the values reported in the literature.53 (3aS,9bR)-6,9-Dimethyl-3-methylene-2-oxo-2,3,3a,4,5,9b-hexahydronaphtho[1,2-b]f uran-8-yl acetate (9) To a solution of compound 8 (1.00 g, 4.10 mmol, 1.0 eq.) in acetic anhydride (Ac2O) (5 mL) being coolen to 0 °C was added dropwise conc. H2SO4 (0.1 mL). After the reaction complete detected by TLC, the mixture was poured into glacial water (100 mL) and extracted with CH2Cl2 three times (3 × 30 mL) and washed with sat. NaHCO3 and brine. The combined organic layers were dried by anhydrous Na2SO4 and evaporated under vacuum to give crude product that was purified by silica-gel column chromatography (PE/EtOAc = 5:1 – 3:1), resulting in a white solid product 9 (0.82 g, 70%), mp 93.5–94.6 ºC. 1H NMR (300 MHz, CDCl3) δ 6.86 (s, 1H), 6.30 (d, J = 1.8 Hz, 1H), 5.72 (d, J = 1.8 Hz, 1H), 5.56 (d, J = 6.7 Hz, 1H), 3.23 – 3.38 (m, 1H), 2.69 – 2.76 (m, 1H), 2.44 – 2.59 (m, 1H), 2.33 (s, 3H), 2.22 (s, 6H), 1.93 – 2.01 (m, 1H), 1.77 – 1.87 (m, 1H); 13C NMR (125 MHz, CDCl3) δ 170.0, 169.6, 147.4, 147.0, 139.7, 134.4, 131.0, 129.0, 124.0, 121.6, 74.8, 39.2, 25.8, 23.9, 20.8. 19.5, 12.0. HPLC purity: 98.5%, retention time = 12.15 min. HRMS (ESI): m/z calcd for C17H18O4Na [M+Na]+: 309.1097, found 309.1097. (3aS,9bR)-8-Hydroxy-6,9-dimethyl-3-methylene-3a,4,5,9b-tetrahydronaphtho[1,2-b] furan-2(3H)-one (10) To a solution of compound 9 (1.0 g, 3.50 mmol, 1.0 eq.) in methanol (10 mL) being coolen to 0 °C was added dropwise ammonium hydroxide (NH3·H2O) (10 mL). After the reaction complete detected by TLC, the mixture evaporated under vacuum to give crude product, which was purified by silica-gel column chromatography (PE/EtOAc=5:1 – 3:1),


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resulting in a white solid product 10 (0.70 g, 82%), mp 200.6–201.4 ºC. 1H NMR (500 MHz, CDCl3) δ 6.68 (s, 1H), 6.31 (d, J = 1.9 Hz, 1H), 5.71 (d, J = 1.9 Hz, 1H), 5.59 (d, J = 6.7 Hz, 1H), 3.25 – 3.35 (m, 1H), 2.67 – 2.72 (m, 1H), 2.46 – 2.52 (m, 1H), 2.30 (s, 3H), 2.18 (s, 3H), 1.92 – 2.01 (m, 1H), 1.74 – 1.85 (m, 1H); 13C NMR (125 MHz, CDCl3) δ 170.4, 151.9, 140.0, 134.3, 130.5, 128.5, 122.4, 121.4, 117.7, 75.0, 39.4, 26.0, 23.4, 19.4, 11.1. HPLC purity: 99.5%, retention time = 9.58 min. HRMS (ESI): m/z calcd for C15H16O3Na [M+Na]+: 267.0992, found 267.0995. (3aS,5aS,8S,9bS)-8-Hydroxy-5a,9-dimethyl-3-methylene-3a,4,5,5a,8,9b-hexahydrona phtho[1,2-b]furan-2(3H)-one

(11)

and

(3aS,5aS,8R,9bS)-8-Hydroxy-5a,9-dimethyl-3-methylene-3a,4,5,5a,8,9b-hexahydron aphtho[1,2-b]furan-2(3H)-one (12) To a stirred -78 °C solution of compound 10 (0.16 g, 0.66 mmol, 1.0 eq.) in dry THF/Tol (1:1) (6 mL) under argon was added dropwise DIBAL-H (1.2 M, 0.81 mL, 1.5 eq.) via syringe over 5 min. The reaction solution was stirred at -78 °C for 2 h. Then the reaction was complete detected by TLC and quenched by the addition of the saturated aqueous NH4Cl. The mixture was extracted with EtOAc three times (3 × 15 mL) and washed with brine. The combined organic layers were dried by anhydrous Na2SO4 and evaporated under vacuum to give crude product, which was purified by silica-gel column chromatography (PE/EtOAc = 10:1 – 8:1 – 4:1), affording white solid compounds 11 (0.72 g, 71%) and 12 (0.18 g, 18%). 11: mp 120.1–121.8 ºC. 1H NMR (300 MHz, CDCl3) δ 6.16 (d, J = 3.1 Hz, 1H), 5.75 (dd, J = 10.0, 3.1 Hz, 1H), 5.61 (dd, J = 10.0, 1.2 Hz, 1H,), 5.47 (d, J = 3.1 Hz, 1H,), 4.69 (d, J = 11.3 Hz, 1H), 4.32 (d, J = 8.3 Hz, 1H), 2.53 – 2.69 (m, 1H), 2.03 – 2.18 (m, 1H), 2.08



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(s, 3H), 1.65 – 1.76 (m, 2H), 1.49 – 1.59 (m, 1H), 1.13 (s, 3H);

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13

C NMR (125 MHz,

CDCl3) δ 170.0, 138.5, 137.4, 132.2, 126.3, 123.9, 118.5, 82.3, 67.8, 50.1, 38.8, 38.5, 26.7, 22.4, 15.9. HPLC purity: 95.4%, retention time = 6.95 min. HRMS (ESI): m/z calcd for C15H18O3Na [M+Na]+: 269.1148, found 269.1159. 12: mp 118.7–119.2 ºC. 1H NMR (300 MHz, CDCl3) δ 6.17 (d, J = 3.1 Hz, 1H), 5.80 (dd, J = 9.9, 4.0 Hz, 1H), 5.65 (d, J = 9.9 Hz, 1H), 5.48 (d, J = 3.1 Hz, 1H), 4.64 (d, J = 11.1 Hz, 1H), 4.28 (dd, J = 9.0, 3.9 Hz, 1H), 2.58 – 2.66 (m, 1H), 2.03 – 2.17 (m, 1H), 1.60 – 1.78 (m, 2H), 1.57 (s, 3H), 1.39 – 1.52 (m, 2H), 1.20 (s, 3H);

13

C NMR (125 MHz,

CDCl3) δ 170.0, 138.8, 138.7, 133.2, 126.1, 123.6, 118.5, 82.3, 68.3, 50.3, 38.9, 38.7, 26.6, 22.5, 16.0. HPLC purity: 95.7%, retention time = 6.90 min. HRMS (ESI): m/z calcd for C15H18O3Na [M+Na]+: 269.1148, found 269.1159. (3S,3aS,5aS,9bS)-3,5a,9-Trimethyloctahydronaphtho[1,2-b]furan-2,8(3H,4H)-dione (13) A mixture of α-santonin (15.0 g, 61.0 mmol) and 10% Pd/C (1.5 g) in ethanol (1.5 L) was stirred under 3 atms at room temperature for 5 hours. The reaction was complete detected by TLC. Then the mixture was filtered through a Celite pad, and the filtrate was concentrated to give crude product, which was purified by silica-gel column chromatography (PE/Acetone = 20:1 – 15:1 – 12:1), affording white solid compounds 13 (14.0 g, 92%). 1H NMR (300 MHz, CDCl3) δ 3.99 (dd, J = 11.2, 10.2 Hz, 1H), 2.71 – 2.84 (m, 1H), 2.60 – 2.67 (m, 1H), 2.33 – 2.43 (m, 2H), 1.99 (dd, J = 11.4, 6.6 Hz, 1H), 1.85 – 1.89 (m, 1H), 1.74 – 1.80 (m, 1H), 1.66 – 1.71 (m, 1H), 1.52 – 1.63 (m, 4H), 1.24 (d, J = 7.5 Hz, 3H), 1.23 (d, J = 6.5 Hz, 3H), 1.19 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 213.7, 179.1, 79.5, 52.9, 49.1, 43.8, 42.2, 41.4, 40.0, 35.7, 35.0, 23.3, 20.1, 13.7, 12.5.


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HRMS (ESI): m/z calcd for C15H22O3Na [M+Na]+: 273.1461, found 273.1469. (3S,3aS,5aS,8S,9R,9bS)-8-Hydroxy-3,5a,9-trimethyldecahydronaphtho[1,2-b]furan2(3H)-one

(14)

and

(3S,3aS,5aS,8S,9S,9bS)-8-Hydroxy-3,5a,9-trimethyldecahydronaphtho[1,2-b]furan-2 (3H)-one (15) To a solution of compound 13 (15.0 g, 60 mmol, 1.0 eq.) in dry methanol (100 mL) being coolen to 0 °C was added NaBH4 (3.4 g, 90 mmol, 1.5 eq.) in portions. The reaction solution was stirred at 20 °C for 2.5 hours. The reaction was complete detected by TLC and then quenched by the addition of the saturated aqueous NH4Cl. The mixture was extracted with CH2Cl2 three times (3 × 120 mL) and washed with brine. The combined organic layers were dried by anhydrous Na2SO4 and evaporated under vacuum to give crude

product,

which

was

purified

by

silica-gel

column

chromatography

(PE/Acetone=50:1), affording white solid compound 14 (9.07 g, 60%), and 15 was afforded by recrystallized from CH2Cl2/CH3OH (10:1) (3.02 g, 20%). 14: 1H NMR (500 MHz, CDCl3) δ 3.97 (dd, J = 11.2, 10.2 Hz, 1H), 3.69 – 3.79 (m, 1H), 2.37 – 2.46 (m, 1H), 2.31 – 2.36 (m, 1H), 1.75 – 1.84 (m, 1H), 1.22 (d, J = 6.2 Hz, 3H), 1.02 (s, 3H), 0.97 (d, J = 7.5 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 179.6, 80.0, 73.0, 53.5, 49.6, 43.2, 41.8, 40.0, 35.8, 34.3, 25.8, 23.4, 21.0, 12.5, 8.6; HRMS (ESI): m/z calcd for C15H24O3Na [M+Na]+: 275.1618, found 275.1621. 15: 1H NMR (500 MHz, CDCl3) δ 4.40 (dd, J = 11.4, 4.4 Hz, 1H), 3.74 – 3.77 (m, 1H), 2.18 – 2.22 (m, 1H), 2.10 (dd, J = 11.4, 4.4 Hz, 1H), 1.76 – 1.95 (m, 5H), 1.64 – 1.74 (m, 2H), 1.44 – 1.51 (m, 2H), 1.22 (d, J = 7.0 Hz, 3H), 1.13 – 1.17 (m, 1H), 1.08 (s, 3H), 1.06 (d, J = 7.0 Hz, 3H);

13

C NMR (125 MHz, CDCl3) δ 179.0, 82.6, 72.8, 44.0, 43.2, 41.8,



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35.6, 34.2, 32.9, 30.1, 28.44, 28.37, 24.7, 18.1, 12.5. HRMS (ESI): m/z calcd for C15H24O3Na [M+Na]+: 275.1618, found 275.1621. (3S,3aS,5aS,8S,9R,9bS)-8-((Tert-butyldimethylsilyl)oxy)-3,5a,9-trimethyl-decahydro naphtho[1,2-b]furan-2(3H)-one (17) To a solution of compound 14 (9.0 g, 35.7 mmol, 1.0 eq.) in dry DMF (200 mL) was added TBSCl (26.9 g, 178.6 mmol, 5.0 eq.) and imidazole (24.3 g, 357.1 mmol, 10.0 eq.). The mixture was stirred at room temperature for 30 min and poured into water (200 mL). Then the mixture was extracted with CH2Cl2 three times (3 × 150 mL) and washed with brine. The combined organic layers were dried by anhydrous Na2SO4 and evaporated under vacuum to give crude product, which was purified by silica-gel column chromatography (PE/Acetone = 50:1), affording white solid compound 17 (10.4 g, 80%). 1

H NMR (500 MHz, CDCl3) δ 3.97 (dd, J = 11.2, 10.2 Hz, 1H), 3.65 – 3.69 (m, 1H), 2.27

– 2.36 (m, 2H), 1.69 – 1.80 (m, 2H), 1.41 – 1.53 (m, 6H), 1.16 – 1.26 (m, 2H), 1.21 (d, J = 7.0 Hz, 3H), 1.02 (s, 3H), 0.95 (d, J = 7.4 Hz, 3H), 0.88 (s, 9H), 0.04 (s, 3H), 0.03 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 179.8, 80.3, 73.5, 53.6, 49.7, 43.3, 41.8, 40.2, 35.8, 35.0, 26.6, 25.8 (3C), 23.4, 21.0, 12.5, 18.1, 9.0. HRMS (ESI): m/z calcd for C15H24O3Na [M+Na]+: 275.1618, found 275.1621. (3S,3aR,5aS,8S,9R,9bR)-8-((Tert-butyldimethylsilyl)oxy)-3,5a,9-trimethyl-3-(phenyl selanyl)decahydronaphtho[1,2-b]furan-2(3H)-one (18) According to the synthetic method of compound 4. No further purification for next step. (3aS,5aS,8S,9R,9bS)-8-((Tert-butyldimethylsilyl)oxy)-5a,9-dimethyl-3-methylene -decahydronaphtho[1,2-b]furan-2(3H)-one (19) According to the synthetic method of compound 5. 1H NMR (500MHz, CDCl3) δ 6.05 (d,


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J = 3.1 Hz, 1H), 5.38 (d, J = 3.1 Hz, 1H), 3.95 (t, J = 11.1 Hz, 1H), 3.64 – 3.78 (m, 1H), 2.42 – 2.55 (m, 1H), 2.30 – 2.40 (m, 1H), 2.00 – 1.95 (m, 1H), 1.67 – 1.78 (m, 1H), 1.53 – 1.65 (m, 3H), 1.45 – 1.53 (m, 2H), 1.19 – 1.32 (m, 2H), 1.01 (s, 3H), 0.96 (d, J = 7.4 Hz, 3H), 0.88 (s, 9H), 0.042 (s, 3H), 0.039 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 171.0, 140.0, 116.5, 80.5, 73.4, 51.0, 50.3, 43.0, 40.1, 35.9, 34.9, 26.7 (3C), 25.8, 21.8, 21.0, 18.1, 9.1. HRMS (ESI): m/z calcd for C21H36O3SiNa [M+Na]+: 387.2326, found 387.2325. (3aS,5aS,8S,9R,9bS)-8-Hydroxy-5a,9-dimethyl-3-methylenedecahydronaphtho[1,2-b ]furan-2(3H)-one (20) According to the synthetic method of compound 6. Mp 138.7–139.2 ºC. 1H NMR (500 MHz, CDCl3) δ 6.05 (d, J = 1.8 Hz, 1H), 5.36 (d, J = 1.8 Hz, 1H), 3.95 (t, J = 6.6 Hz, 1H), 3.74 – 3.78 (m, 1H), 2.43 – 2.49 (m, 2H), 1.96 – 2.03 (m, 1H), 1.62 – 1.70 (m, 4H), 1.46 – 1.60 (m, 3H), 1.22 – 1.33 (m, 2H), 1.01 (s, 3H), 0.99 (d, J = 4.5 Hz, 3H);

13

C NMR

(125 MHz, CDCl3) δ 171.0, 139.9, 116.7, 80.3, 72.9, 50.9, 50.1, 42.9, 40.0, 35.9, 34.2, 25.9, 21.8, 21.0, 8.7. HPLC purity: 96.4%, retention time = 11.26 min. HRMS (ESI): m/z calcd for C15H22O3Na [M+Na]+: 273.1461, found 273.1474. (3aS,5aS,8S,9S,9bS)-8-Hydroxy-5a,9-dimethyl-3-methylenedecahydronaphtho[1,2-b] furan-2(3H)-one (21) According to the synthetic method of compound 20. Mp 108.9–109.5 ºC. 1H NMR (300 MHz, CDCl3) δ 6.12 (d, J = 3.1 Hz, 1H), 5.43 (d, J = 3.1 Hz, 1H), 4.39 (dd, J = 11.7, 4.1 Hz, 1H), 3.78 (s, 1H), 2.70 – 2.78 (m, 1H), 2.12 – 2.17 (m, 1H), 2.02 – 2.07 (m, 1H), 1.77 – 1.94 (m, 3H), 1.69 – 1.72 (m, 1H), 1.51 – 1.60 (m, 1H), 1.25 – 1.30 (m, 2H), 1.15 – 1.19 (m, 1H), 1.10 (d, J = 6.8 Hz, 3H), 1.07 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 170.5,



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140.1, 118.1, 82.8, 72.6, 43.5, 40.9, 35.7, 34.1, 32.7, 29.9, 28.5, 28.4, 23.0, 18.0. HPLC purity: 97.5%, retention time = 11.45 min. HRMS (ESI): m/z calcd for C15H22O3Na [M+Na]+: 273.1461, found 273.1475. (3S,3aS,5aS,8R,9bS)-8-((Tert-butyldimethylsilyl)oxy)-3,5a,9-trimethyldecahy-drona phtho[1,2-b]furan-2(3H)-one (22) To a solution of compound 16 (1.6 g, 6.35 mmol, 1.0 eq.) in dry DMF (50 mL) was added TBSCl (4.8 g, 31.8 mmol, 5.0 eq.) and imidazole (4.3 g, 63.5 mmol, 10.0 eq.). The mixture was stirred at room temperature for 30 min and poured into water (150 mL). Then the mixture was extracted with CH2Cl2 three times (3 × 80 mL) and washed with brine. The combined organic layers were dried by anhydrous Na2SO4 and evaporated under vacuum to give crude product 22 without further purification for next step. (3S,3aR,5aS,8R,9S,9bR)-8-((Tert-butyldimethylsilyl)oxy)-3,5a,9-trimethyl-3-(phenyl selanyl)decahydronaphtho[1,2-b]furan-2(3H)-one

(23)

and

(3S,3aR,5aS,8R,9R,9bR)-8-((Tert-butyldimethylsilyl)oxy)-3,5a,9-trimethyl-3-(phenyl selanyl)decahydronaphtho[1,2-b]furan-2(3H)-one (24) According to the synthetic method of compound 4. 23: 1H NMR (300 MHz, CDCl3) δ 7.62 (d, J = 7.5 Hz, 2H), 7.39 (d, J = 7.5 Hz, 1H), 7.33 (d, J = 7.5 Hz, 2H), 4.78 (dd, J = 11.8, 6.2 Hz, 1H), 3.56 – 3.62 (m, 1H), 2.48 – 2.58 (m, 1H), 2.16 – 2.22 (m, 1H), 2.11 – 2.13 (m, 1H), 1.75 – 1.82 (m, 2H), 1.62 – 1.68 (m, 1H), 1.55 (s, 3H), 1.46 – 1.50 (m, 1H), 1.23 – 1.33 (m, 3H), 1.05 (d, J = 5.1 Hz, 3H), 1.04 (s, 3H), 0.87 (m, 9H), 0.04 (s, 3H), 0.02 (s, 3H);

13

C NMR (125 MHz, CDCl3) δ 176.7,

138.2 (2C), 129.5, 129.0 (2C), 124.4, 79.9, 74.2, 50.0, 49.9, 44.1, 40.3, 35.6, 34.5, 34.1, 30.2, 26.0, 25.8 (3C), 22.8, 21.5, 18.1, 12.7. ESI MS: m/z 523.1 [M+H]+.


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24: 1H NMR (500 MHz, CDCl3) δ 7.66 (2H, d, J = 7.5 Hz), 7.39 (1H, d, J = 7.5 Hz), 7.32 (2H, d, J = 7.5 Hz), 4.27 (1H, t, J = 10.9 Hz), 3.72 (1H, m), 1.99 – 2.03 (1H, m), 1.96 (1H, dd, J = 11.7, 4.9 Hz), 1.81 – 1.90 (1H, m), 1.73 – 1.82 (3H, m), 1.62 – 1.73 (1H, m), 1.55 (3H, s), 1.41 – 1.44 (1H, m), 1.22 – 1.37 (2H, m), 1.11 – 1.14 (1H, m), 0.99 (3H, s), 0.89 (3H, d, J = 7.5 Hz), 0.87 (9H, s), 0.02 (3H, s), 0.01 (3H, s);

13

C NMR (125 MHz,

CDCl3) δ 177.4, 138.0 (2C), 129.5, 129.1 (2C), 124.9, 78.2, 71.5, 58.2, 53.4, 50.5, 44.4, 43.2, 36.1, 36.0, 34.7, 25.9, 25.8 (3C), 24.8, 22.4, 21.5, 20.5, 0.05 (2C). ESI MS: m/z 523.2 [M+H]+. (3aS,5aS,8R,9S,9bS)-8-Hydroxy-5a,9-dimethyl-3-methylenedecahydronaphtho[1,2-b ]furan-2(3H)-one

(25)

and

(3aS,5aS,8R,9R,9bS)-8-Hydroxy-5a,9-dimethyl-3-methylenedecahydronaphtho[1,2-b ]furan-2(3H)-one (26) According to the synthetic method of compound 6. 25: mp 140.7–142.1 ºC. 1H NMR (500 MHz, CDCl3) δ 6.07 (d, J = 3.1 Hz, 1H), 5.38 (d, J = 3.1 Hz, 1H), 4.25 (dd, J = 12.2, 6.1 Hz, 1H), 3.70 – 3.74 (m, 1H), 3.21 – 3.25 (m, 1H), 2.34 – 2.38 (m, 1H), 2.11 – 2.22 (m, 1H), 2.01 – 2.06 (m, 1H), 1.78 – 1.84 (m, 1H), 1.55 – 1.73 (m, 4H), 1.46 – 1.55 (m, 2H), 1.14 (d, J = 7.3 Hz, 3H), 1.01 (s, 3H);

13

C NMR

(125 MHz, CDCl3) δ 171.1, 140.0, 117.0, 82.3, 73.4, 44.2, 43.4, 40.1, 35.1, 34.6, 34.2, 30.2, 25.2, 22.1, 12.2. HPLC purity: 98.6%, retention time = 10.83 min. HRMS (ESI): m/z calcd for C15H22O3Na [M+Na]+: 273.1461, found 273.1470. 26: mp 129.3–130.1 ºC. 1H NMR (300 MHz, CDCl3) δ 6.03 (d, J = 3.0 Hz, 1H), 5.36 (d, J = 3.0 Hz, 1H), 3.88 (m, 2H), 2.45 – 2.59 (m, 1H), 1.94 – 2.02 (m, 2H), 1.83 – 1.90 (m, 1H), 1.72 – 1.76 (m, 2H), 1.56 – 1.69 (m, 3H), 1.35 – 1.41 (m, 2H), 1.19 – 1.23 (m, 2H),



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1.00 (s, 3H), 0.98 (d, J = 6.0 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 171.1, 140.1, 116.5, 80.5, 71.1, 51.0, 44.7, 43.0, 36.2, 35.8, 34.6, 24.1, 21.9, 20.4, 14.0. HPLC purity: 97.1%, retention time = 13.15 min. HRMS (ESI): m/z calcd for C15H22O3Na [M+Na]+: 273.1461, found 273.1469. Preparation of derivatives General procedure of (1a‒1f, 2a‒2f, 3a‒3n, 4a‒4h, 5a‒5g, 7a‒7f, 8a‒8j and 9a‒9g): To a solution of compounds 6, 10 or 20 (1.0 eq.) and different substituted carboxylic acids (1.0 eq.) in CH2Cl2 was added DMAP (1.0 eq.). The mixture was stirred at ambient temperature. After 10 min, EDCI (2.0 eq.) was added to the reaction mixture in one portion. The mixture was left standing overnight and poured into water. Then the mixture was extracted with CH2Cl2 three times and washed with brine. The combined organic layers were dried by anhydrous Na2SO4 and evaporated under vacuum to give crude product, which was purified by silica-gel column chromatography (PE/EtOAc) to give the derivatives 1a‒1f, 2a‒2f, 3a‒3n, 4a‒4h, 5a‒5g, 7a‒7f, 8a‒8j and 9a‒9g. General procedure of (6a‒6i) To a solution of compound 10 (1.0 eq.) and different benzyl bromides (2.0 eq.) in acetone was added K2CO3 (1.1 eq.). The reaction suspension was stirred at ambient temperature overnight. Then the mixture was filtered through a celit pad, and the filtrate was concentrated to give the crude product, which was purified by silica-gel column chromatography (PE/EtOAc) to give the derivatives 6a‒6i. (3aS,6R,8S,9bS)-6-Acetoxy-6,9-dimethyl-3-methylene-2-oxo-2,3,3a,4,5,6,6a,7,8,9b-de cahydroazuleno[4,5-b]furan-8-yl benzoate (1a) Colorless solid (yield 76%), mp 148.7–149.6 ºC. 1H NMR (500 MHz, CDCl3) δ 8.08 –


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8.04 (m, 2H), 7.58 (t, J = 7.4 Hz, 1H), 7.46 (t, J = 7.8 Hz, 2H), 6.26 (d, J = 3.3 Hz, 1H), 5.78 (t, J = 5.7 Hz, 1H), 5.53 (d, J = 3.0 Hz, 1H), 4.70 (dd, J = 10.9, 1.6 Hz, 1H), 3.93 (s, 1H), 2.95 (t, J = 10.3 Hz, 1H), 2.51 (td, J = 13.4, 4.2 Hz, 2H), 1.96 (s, 3H), 1.96 (s, 3H), 1.40 (s, 1H), 1.25 (s, 3H), 1.24 (s, 3H);

13

C NMR (125 MHz, CDCl3) δ 170.3, 169.6,

166.2, 141.1, 138.4, 133.3, 133.1, 130.2, 129.6 (2C), 128.4 (2C), 119.8, 86.2, 81.6, 80.6, 52.2, 45.1, 37.4, 32.2, 24.2, 22.4, 20.0, 12.9. HPLC purity: 96.5%, retention time = 20.59 min. HRMS (ESI): m/z calcd for C24H27O6 [M+H]+: 411.1802, found 411.1808. (3aR,6R,8S,9bS)-6-Acetoxy-6,9-dimethyl-3-methylene-2-oxo-2,3,3a,4,5,6,6a,7,8,9b-de cahydroazuleno[4,5-b]furan-8-yl 4-chlorobenzoate (1b) Colorless solid (yield 90%), mp 191.2–192.5 ºC. 1H NMR (500 MHz, CDCl3) δ 7.98 (d, J = 8.6 Hz, 2H), 7.42 (d, J = 8.6 Hz, 2H), 6.26 (d, J = 3.3 Hz, 1H), 5.76 (t, J = 6.2 Hz, 1H), 5.53 (d, J = 3.0 Hz, 1H), 4.69 (dd, J = 10.9, 1.6 Hz, 1H), 3.93 (s, 1H), 2.95 (t, J = 10.3 Hz, 1H), 2.65 (dt, J = 14.6, 8.2 Hz, 1H), 2.55 – 2.46 (m, 1H), 2.29 – 2.19 (m, 2H), 1.96 (s, 3H), 1.94 (s, 3H), 1.79 – 1.69 (m, 1H), 1.45 (ddd, J = 14.1, 12.2, 7.0 Hz, 1H), 1.23 (s, 3H);

13

C NMR (125 MHz, CDCl3) δ 170.2, 169.5, 165.4, 140.8, 139.6, 138.3, 133.5,

131.0 (2C), 128.8 (2C), 128.7, 119.9, 86.1, 81.5, 80.9, 52.1, 45.1, 37.4, 32.2, 24.2, 22.4, 20.0, 12.9. HPLC purity: 97.7%, retention time = 23.87 min. HRMS (ESI): m/z calcd for C24H26ClO6 [M+H]+: 445.1412, found 445.1411. (3aS,6R,8S,9bS)-6-Acetoxy-6,9-dimethyl-3-methylene-2-oxo-2,3,3a,4,5,6,6a,7,8,9b-de cahydroazuleno[4,5-b]furan-8-yl 4-bromobenzoate (1c) Colorless solid (yield 92%), mp 180.3–181.4 ºC. 1H NMR (500 MHz, CDCl3) δ 7.91 (d, J = 8.5 Hz, 2H), 7.59 (d, J = 8.6 Hz, 2H), 6.26 (d, J = 3.3 Hz, 1H), 5.76 (s, 1H), 5.53 (d, J = 3.0 Hz, 1H), 4.66 (ddd, J = 32.9, 10.9, 1.5 Hz, 1H), 3.93 (s, 1H), 2.95 (t, J = 10.3 Hz,



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1H), 2.65 (dt, J = 14.7, 8.2 Hz, 1H), 2.51 (td, J = 13.3, 4.2 Hz, 1H), 2.30 – 2.18 (m, 2H), 1.96 (s, 3H), 1.94 (s, 3H), 1.79 – 1.71 (m, 1H), 1.44 (ddd, J = 14.2, 12.0, 7.1 Hz, 1H), 1.23 (s, 3H);

13

C NMR (125 MHz, CDCl3) δ 170.2, 169.5, 165.5, 140.8, 138.3, 133.5,

131.8 (2C), 131.1 (2C), 129.1, 128.2, 119.9, 86.1, 81.5, 80.9, 52.1, 45.1, 37.4, 32.2, 24.2, 22.4, 20.0, 12.9. HPLC purity: 97.1%, retention time = 21.23 min. HRMS (ESI): m/z calcd for C24H26BrO6 [M+H]+: 489.0907, found 489.0897. (3aS,6R,8S,9bS)-6-Acetoxy-6,9-dimethyl-3-methylene-2-oxo-2,3,3a,4,5,6,6a,7,8,9b-de cahydroazuleno[4,5-b]furan-8-yl 4-cyanobenzoate (1d) Colorless solid (yield 83%), mp 184.5–185.2 ºC. 1H NMR (500 MHz, CDCl3) δ 8.15 (d, J = 8.6 Hz, 2H), 7.76 (d, J = 8.6 Hz, 2H), 6.27 (d, J = 3.3 Hz, 1H), 5.79 (t, J = 6.6 Hz, 1H), 5.54 (d, J = 3.1 Hz, 1H), 4.70 (dd, J = 10.9, 1.8 Hz, 1H), 3.95 (s, 1H), 2.95 (dd, J = 11.7, 9.3 Hz, 1H), 2.67 (dt, J = 14.6, 8.2 Hz, 1H), 2.56 – 2.48 (m, 1H), 2.29 – 2.21 (m, 2H), 1.97 (s, 3H), 1.95 (s, 3H), 1.79 – 1.72 (m, 1H), 1.45 (t, J = 11.1 Hz, 1H), 1.23 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 170.2, 169.4, 164.6, 140.3, 138.2, 134.0, 134.0 (d, J = 10.4 Hz), 132.3 (2C), 130.1 (2C), 120.0, 117.9, 116.5, 86.0, 81.6, 81.4, 52.1, 45.1, 37.4, 32.1, 24.2, 22.4, 20.0, 12.9. HPLC purity: 96.9%, retention time = 24.27 min. HRMS (ESI): m/z calcd for C25H25NO6Na [M+Na]+: 458.1574, found 458.1578. (3aS,6R,8S,9bS)-6-Acetoxy-6,9-dimethyl-3-methylene-2-oxo-2,3,3a,4,5,6,6a,7,8,9b-de cahydroazuleno[4,5-b]furan-8-yl 4-methylbenzoate (1e) Colorless solid (yield 78%), mp 140.5–141.6 ºC. 1H NMR (500 MHz, CDCl3) δ 7.93 (d, J = 7.3 Hz, 1H), 7.44 – 7.38 (m, 1H), 7.27 (s, 1 H), 7.25 (s, 1H), 6.26 (d, J = 3.4 Hz, 1H), 5.76 (t, J = 6.1 Hz, 1H), 5.53 (d, J = 3.1 Hz, 1H), 4.69 (dd, J = 10.9, 1.7 Hz, 1H), 3.93 (s, 1H), 2.95 (dd, J = 10.9, 9.5 Hz, 1H), 2.71 – 2.64 (m, 1H), 2.63 (s, 3H), 2.51 (td, J = 13.4,


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4.2 Hz, 1H), 2.29 – 2.22 (m, 2H), 1.97 (s, 3H), 1.96 (s, 3H), 1.43 (dd, J = 21.1, 10.7 Hz, 2H), 1.23 (s, 3H);

13

C NMR (125 MHz, CDCl3) δ 170.3, 169.6, 167.1, 141.1, 140.4,

138.4, 133.2, 132.1, 131.8, 130.6, 129.5, 125.7, 119.8, 86.2, 81.6, 80.4, 52.2, 45.1, 37.4, 32.2, 24.2, 22.4, 21.9, 20.0, 13.0. HPLC purity: 98.7%, retention time = 22.75 min. HRMS (ESI): m/z calcd for C25H29O6 [M+H]+: 411.1958, found 411.1952. (3aS,6R,8S,9bS)-6-Acetoxy-6,9-dimethyl-3-methylene-2-oxo-2,3,3a,4,5,6,6a,7,8,9b-de cahydroazuleno[4,5-b]furan-8-yl 4-methoxybenzoate (1f) Colorless solid (yield 88%), mp 135.9–147.1 ºC. 1H NMR (500 MHz, CDCl3) δ 8.00 (t, J = 5.7 Hz, 2H), 6.92 (t, J = 8.3 Hz, 2H), 6.27 (d, J = 3.3 Hz, 1H), 5.75 (s, 1H), 5.53 (d, J = 3.0 Hz, 1H), 4.70 (dd, J = 10.9, 1.5 Hz, 1H), 3.92 (s, 1H), 3.87 (s, 3H), 2.95 (t, J = 10.3 Hz, 1H), 2.64 (dt, J = 14.7, 8.2 Hz, 1H), 2.50 (td, J = 13.3, 4.3 Hz, 1H), 2.31 – 2.21 (m, 2H), 1.96 (s, 3H), 1.95 (s, 3H), 1.75 (dd, J = 8.9, 5.7 Hz, 1H), 1.45 (dd, J = 10.9, 3.0 Hz, 1H), 1.24 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 170.3, 169.6 163.5, 141.3, 138.5, 133.1, 131.6 (2C), 122.7, 119.8, 113.7 (2C), 86.2, 81.6, 80.3, 55.5, 52.2, 45.1, 37.5, 32.3, 29.7, 24.2, 22.4, 20.0, 12.9. HPLC purity: 95.5%, retention time = 21.29 min. HRMS (ESI): m/z calcd for C25H29O7 [M+H]+: 441.1908, found 441.1910. (3aS,6R,8S,9bS)-6-Acetoxy-6,9-dimethyl-3-methylene-2-oxo-2,3,3a,4,5,6,6a,7,8,9b-de cahydroazuleno[4,5-b]furan-8-yl cinnamate (2a) Colorless solid (yield 78%), mp 121.1–122.6 ºC.1H NMR (500 MHz, CDCl3) δ 7.72 (d, J = 16.0 Hz, 1H), 7.56 – 7.51 (m, 2H), 7.42 – 7.37 (m, 3H), 6.47 (d, J = 16.0 Hz, 1H), 6.26 (d, J = 3.3 Hz, 1H), 5.75 – 5.64 (m, 1H), 5.53 (d, J = 3.1 Hz, 1H), 4.71 – 4.60 (m, 1H), 3.90 (s, 1H), 2.94 (dd, J = 11.8, 10.3 Hz, 1H), 2.61 (dt, J = 14.6, 8.2 Hz, 1H), 2.28 – 2.21 (m, 2H), 1.98 (s, 3H), 1.95 (s, 1H), 1.92 (s, 2H), 1.87 (s, 1H), 1.71 (dd, J = 8.7, 5.9 Hz,



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1H), 1.64 – 1.57 (m, 1H), 1.23 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 170.3 (2C), 166.7, 145.2, 141.1, 138.4, 134.3, 133.1, 130.4, 128.9 (2C), 128.1 (2C), 119.8, 118.0, 86.2, 81.6, 80.1, 52.1, 45.1, 37.5, 29.7, 24.2, 22.4, 20.0, 12.9. HPLC purity: 97.1%, retention time = 21.13 min. HRMS (ESI): m/z calcd for C26H29O6 [M+H]+: 437.1958, found 437.1945. (3aS,6R,8S,9bS)-6-Acetoxy-6,9-dimethyl-3-methylene-2-oxo-2,3,3a,4,5,6,6a,7,8,9b-de cahydroazuleno[4,5-b]furan-8-yl (E)-3-(4-chlorophenyl)acrylate (2b) Colorless solid (yield 75%), mp 145.6–146.8 ºC. 1H NMR (500 MHz, CDCl3) δ 7.66 (d, J = 16.0 Hz, 1H), 7.49 – 7.44 (m, 2H), 7.39 – 7.35 (m, 2H), 6.44 (d, J = 16.0 Hz, 1H), 6.26 (d, J = 3.3 Hz, 1H), 5.74 – 5.64 (m, 1H), 5.53 (t, J = 2.5 Hz, 1H), 4.65 (ddd, J = 30.8, 11.0, 1.7 Hz, 1H), 3.89 (d, J = 6.1 Hz, 1H), 2.98 – 2.90 (m, 1H), 2.60 (dt, J = 14.6, 8.2 Hz, 1H), 2.50 (td, J = 13.3, 4.3 Hz, 1H), 2.29 – 2.18 (m, 2H), 1.97 (s, 3H), 1.95 (s, 1H), 1.92 (s, 2H), 1.70 (dd, J = 8.6, 5.9 Hz, 1H), 1.49 – 1.40 (m, 1H), 1.23 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 170.3, 169.5, 166.4, 143.7, 141.0, 138.4, 136.3, 133.2, 132.8, 129.2 (4C), 119.8, 118.6, 86.2, 81.6, 80.2, 52.1, 45.1, 37.4, 32.2, 24.2, 22.4, 20.0, 12.8. HPLC purity: 96.1%, retention time = 23.23 min. HRMS (ESI): m/z calcd for C26H37ClO6Na [M+Na]+: 493.1388, found 493.1383. (3aS,6R,8S,9bS)-6-Acetoxy-6,9-dimethyl-3-methylene-2-oxo-2,3,3a,4,5,6,6a,7,8,9b-de cahydroazuleno[4,5-b]furan-8-yl (E)-3-(4-bromophenyl)acrylate (2c) Colorless solid (yield 94%), mp 140.3–141.2 ºC. 1H NMR (500 MHz, CDCl3) δ 7.64 (d, J = 16.0 Hz, 1H), 7.52 (dd, J = 8.8, 2.2 Hz, 2H), 7.42 – 7.37 (m, 2H), 6.48 – 6.42 (m, 1H), 6.28 – 6.24 (m, 1H), 5.73 – 5.64 (m, 1H), 5.52 (dd, J = 5.7, 3.0 Hz, 2H), 4.70 – 4.65 (m, 1H), 3.93 – 3.82 (m, 1H), 2.93 (t, J = 10.5 Hz, 1H), 2.64 – 2.56 (m, 1H), 2.21 (ddd, J = 10.3, 7.1, 4.1 Hz, 2H), 1.97 (s, 3H), 1.94 (s, 1H), 1.91 (s, 2H), 1.73 – 1.67 (m, 1H), 1.64 –


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1.58 (m, 1H), 1.22 (s, 3H);

13

C NMR (125 MHz, CDCl3) δ 170.3, 1696, 166.4, 143.8,

1410, 138.4, 133.2, 132.2 (2C), 129.5 (3C), 124.7, 119.8, 118.7, 86.2, 81.6, 80.2, 52.1, 45.1, 37.5, 32.2, 24.2, 22.4, 20.0, 12.8. HPLC purity: 97.5%, retention time = 20.54 min. HRMS (ESI): m/z calcd for C26H38BrO6 [M+H]+: 515.1064, found 515.1058. (3aS,6R,8S,9bS)-6-Acetoxy-6,9-dimethyl-3-methylene-2-oxo-2,3,3a,4,5,6,6a,7,8,9b-de cahydroazuleno[4,5-b]furan-8-yl (E)-3-(4-cyanophenyl)acrylate (2d) Colorless solid (yield 80%), mp 142.8–144.3 ºC. 1H NMR (500 MHz, CDCl3) δ 7.68 (dd, J = 12.1, 3.8 Hz, 3H), 7.62 (d, J = 8.3 Hz, 2H), 6.54 (d, J = 16.1 Hz, 1H), 6.26 (d, J = 3.3 Hz, 1H), 5.74 – 5.64 (m, 1H), 5.53 (d, J = 3.0 Hz, 1H), 4.68 (dd, J = 10.9, 1.6 Hz, 1H), 3.90 (s, 1H), 2.60 (dt, J = 14.6, 8.2 Hz, 1H), 2.50 (td, J = 13.4, 4.2 Hz, 1H), 2.29 – 2.17 (m, 2H), 1.97 (s, 3H), 1.95 – 1.82 (m, 4H), 1.73 – 1.66 (m, 1H), 1.44 (qd, J = 14.0, 3.3 Hz, 1H), 1.22 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 170.3, 169.5, 165.8, 142.7, 140.7, 138.6, 138.3, 133.5, 132.7 (2C), 128.4 (2C), 121.6, 119.9, 118.3, 113.5, 86.1, 81.5, 80.6, 52.1, 45.1, 37.4, 32.2, 24.2, 22.4, 20.0, 12.8. HPLC purity: 98.7%, retention time = 25.19 min. HRMS (ESI): m/z calcd for C27H27NO6Na [M+Na]+: 484.1731, found 484.1734. (3aS,6R,8S,9bS)-6-Acetoxy-6,9-dimethyl-3-methylene-2-oxo-2,3,3a,4,5,6,6a,7,8,9b-de cahydroazuleno[4,5-b]furan-8-yl (E)-3-(p-tolyl)acrylate (2e) Colorless solid (yield 86%), mp 129.7–131.5 ºC. 1H NMR (500 MHz, CDCl3) δ 7.69 (d, J = 15.9 Hz, 1H), 7.43 (t, J = 6.6 Hz, 2H), 7.20 (d, J = 8.0 Hz, 2H), 6.42 (d, J = 16.1 Hz, 1H), 6.26 (dd, J = 6.7, 3.3 Hz, 1H), 5.67 (t, J = 6.3 Hz, 1H), 5.52 (dd, J = 6.0, 3.0 Hz, 2H), 4.67 (ddd, J = 18.5, 9.1, 3.7 Hz, 1H), 2.61 (dd, J = 15.6, 7.3 Hz, 1H), 2.50 (dd, J = 12.4, 7.6 Hz, 1H), 2.38 (d, J = 2.5 Hz, 3H), 2.25 – 2.18 (m, 2H), 1.97 (s, 3H), 1.95 (s, 1H), 1.92 (s, 2H), 1.87 (s, 1H), 1.75 – 1.68 (m, 1H), 1.65 – 1.56 (m, 1H), 1.23 (s, 3H);


13

C NMR


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(125 MHz, CDCl3) δ 170.3, 169.6, 166.9, 145.2, 141.2, 140.9, 138.5, 133.0, 131.6, 129.6 (2C), 128.1 (2C), 119.8, 116.9, 86.2, 81.6, 80.0, 52.1, 45.1, 37.5, 32.2, 29.7, 24.2, 22.4, 21.5, 20.0, 12.9. HPLC purity: 97.2%, retention time = 22.73 min. HRMS (ESI): m/z calcd for C27H31O6 [M+H]+: 451.2115, found 437.2112. (3aS,6R,8S,9bS)-6-Acetoxy-6,9-dimethyl-3-methylene-2-oxo-2,3,3a,4,5,6,6a,7,8,9b-de cahydroazuleno[4,5-b]furan-8-yl (E)-3-(4-propoxyphenyl)acrylate (2f) Colorless solid (yield 87%), mp 116.2 – 117.7 ºC. 1H NMR (500 MHz, CDCl3) δ 7.66 (d, J = 15.9 Hz, 1H), 7.47 (d, J = 8.7 Hz, 2H), 6.91 – 6.88 (m, 2H), 6.32 (d, J = 15.9 Hz, 1H), 6.26 (d, J = 3.3 Hz, 1H), 5.66 (t, J = 6.7 Hz, 1H), 5.52 (d, J = 3.1 Hz, 1H), 4.68 (dd, J = 10.9, 1.8 Hz, 1H), 3.95 (t, J = 6.6 Hz, 2H), 3.89 (s, 1H), 2.94 (dd, J = 10.9, 9.6 Hz, 1H), 2.50 (dd, J = 13.5, 9.0 Hz, 1H), 2.27 – 2.17 (m, 2H), 1.97 (s, 3H), 1.95 (s, 1H), 1.92 (s, 3H), 1.82 (dd, J = 14.0, 6.7 Hz, 2H), 1.73 – 1.67 (m, 1H), 1.47 – 1.40 (m, 1H), 1.23 (s, 3H), 1.04 (dd, J = 8.1, 6.7 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 170.3, 169.6, 167.1, 161.1, 144.9, 141.3, 138.5, 132.9, 129.8 (2C), 126.8, 119.8, 115.2, 114.8 (2C), 86.2, 81.6, 79.8, 69.6, 52.1, 45.1, 37.5, 32.3, 29.7, 24.2, 22.5, 20.0, 12.9, 10.5. HPLC purity: 95.9%, retention time = 26.11 min. HRMS (ESI): m/z calcd for C29H35O7 [M+H]+: 495.2377, found 495.2372. (3aS,9bR)-6,9-Dimethyl-3-methylene-2-oxo-2,3,3a,4,5,9b-hexahydronaphtho[1,2-b]f uran-8-yl benzoate (3a) White solid (yield: 95%), mp 145.1–145.8 °C. 1H NMR (500 MHz, CDCl3) δ 8.23 (dd, J = 8.4, 1.2 Hz, 2H), 7.68 – 7.63 (m, 1H), 7.53 (t, J = 7.8 Hz, 2H), 7.00 (s, 1H), 6.33 (d, J = 2.1 Hz, 1H), 5.74 (d, J = 1.8 Hz, 1H), 5.61 (d, J = 6.7 Hz, 1H), 3.37 – 3.31 (m, 1H), 2.78 (ddd, J = 16.7, 6.2, 4.4 Hz, 1H), 2.57 (ddd, J = 16.7, 9.5, 4.5 Hz, 1H), 2.29 (s, 3H), 2.26


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(s, 3H), 2.01 (ddt, J = 13.7, 6.3, 4.7 Hz, 1H), 1.86 (dtd, J = 13.9, 9.6, 4.5 Hz, 1H);

13

C

NMR (125 MHz, CDCl3) δ 170.0, 165.2, 147.6, 139.7, 134.8, 134.4, 133.6, 131.0, 130.2 (2C), 129.3, 129.2, 128.6 (2C), 124.2, 121.6, 74.8, 39.3, 25.8, 24.0, 19.6, 12.1. HPLC purity: 97.5%, retention time = 20.74 min. HRMS (ESI): m/z calcd for C22H20O4Na [M+Na]+: 371.1254, found 371.1265. (3aS,9bR)-6,9-Dimethyl-3-methylene-2-oxo-2,3,3a,4,5,9b-hexahydronaphtho[1,2-b]f uran-8-yl 4-chlorobenzoate (3b) White solid (yield: 80%), mp 190.2–191.7 °C. 1H NMR (500 MHz, CDCl3) δ 8.17 – 8.14 (m, 2H), 7.50 (d, J = 8.6 Hz, 2H), 6.99 (s, 1H), 6.32 (d, J = 2.1 Hz, 1H), 5.74 (d, J = 1.8 Hz, 1H), 5.60 (d, J = 6.7 Hz, 1H), 3.34 (dtd, J = 9.6, 4.9, 2.6 Hz, 1H), 2.78 (ddd, J = 16.7, 6.2, 4.5 Hz, 1H), 2.57 (ddd, J = 16.7, 9.5, 4.5 Hz, 1H), 2.27 (s, 3H), 2.26 (s, 3H), 2.01 (dq, J = 13.7, 5.1 Hz, 1H), 1.86 (ddt, J = 14.1, 9.5, 4.8 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 170.0, 164.3, 147.4, 140.2, 139.6, 134.8, 134.6, 131.5 (2C), 131.1, 129.1 (2C), 129.0, 127.8, 124.0, 121.6, 74.7, 39.2, 25.8, 24.0, 19.6, 12.1. HPLC purity: 95.8%, retention time = 23.80 min. HRMS (ESI): m/z calcd for C22H19ClO4Na [M+Na]+: 405.0864, found 405.0873. (3aS,9bR)-6,9-Dimethyl-3-methylene-2-oxo-2,3,3a,4,5,9b-hexahydronaphtho[1,2-b]f uran-8-yl 4-bromobenzoate (3c) White solid (yield: 94%), mp 192.3–192.5 °C. 1H NMR (500 MHz, CDCl3) δ 8.10 – 8.06 (m, 2H), 7.69 – 7.65 (m, 2H), 6.99 (s, 1H), 6.33 (d, J = 2.1 Hz, 1H), 5.74 (d, J = 1.8 Hz, 1H), 5.60 (d, J = 6.7 Hz, 1H), 3.37 – 3.31 (m, 1H), 2.78 (ddd, J = 16.7, 6.2, 4.4 Hz, 1H), 2.57 (ddd, J = 16.7, 9.5, 4.5 Hz, 1H), 2.27 (s, 3H), 2.26 (s, 3H), 2.01 (ddt, J = 13.6, 6.2, 4.7 Hz, 1H), 1.86 (dtd, J = 13.9, 9.6, 4.4 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 170.0,



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164.4, 147.4, 139.6, 134.8, 134.6, 132.0 (2C), 131.6 (2C), 131.1, 129.1, 128.9, 128.2, 124.0, 121.6, 74.7, 39.2, 25.8, 24.0, 19.6, 12.0. HPLC purity: 98.3%, retention time = 24.45 min. HRMS (ESI): m/z calcd for C22H19BrO4Na [M+Na]+: 449.0359, found 449.0356. (3aS,9bR)-6,9-Dimethyl-3-methylene-2-oxo-2,3,3a,4,5,9b-hexahydronaphtho[1,2-b]f uran-8-yl 4-iodobenzoate (3d) White solid (yield: 75%), mp 175.4–176.1 °C. 1H NMR (500 MHz, CDCl3) δ 7.91 (q, J = 8.6 Hz, 4H), 6.98 (s, 1H), 6.32 (d, J = 2.0 Hz, 1H), 5.73 (d, J = 1.8 Hz, 1H), 5.60 (d, J = 6.7 Hz, 1H), 3.34 (ddd, J = 9.6, 7.0, 4.9 Hz, 1H), 2.77 (ddd, J = 16.7, 6.2, 4.4 Hz, 1H), 2.57 (ddd, J = 16.7, 9.5, 4.5 Hz, 1H), 2.27 (s, 3H), 2.25 (s, 3H), 2.01 (dq, J = 13.7, 5.0 Hz, 1H), 1.86 (dp, J = 14.1, 4.8 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 170.0, 164.7, 147.4, 139.6, 138.0 (2C), 134.8, 134.6, 131.5 (2C), 131.1, 129.1, 128.8, 124.0, 121.6, 101.7, 74.7, 39.2, 25.8, 24.0, 19.6, 12.1. HPLC purity: 99.5%, retention time = 25.10 min. HRMS (ESI): m/z calcd for C22H19O4Na [M+Na]+: 497.0220, found 497.0222. (3aS,9bR)-6,9-Dimethyl-3-methylene-2-oxo-2,3,3a,4,5,9b-hexahydronaphtho[1,2-b]f uran-8-yl 2,4-difluorobenzoate (3e) White solid (yield: 83%), mp 125.1–125.9 °C. 1H NMR (500 MHz, CDCl3) δ 8.16 (td, J = 8.5, 6.4 Hz, 1H), 7.01 (d, J = 7.0 Hz, 2H), 6.96 (ddd, J = 11.0, 8.7, 2.5 Hz, 1H), 6.32 (d, J = 2.1 Hz, 1H), 5.74 (d, J = 1.9 Hz, 1H), 5.60 (d, J = 6.7 Hz, 1H), 3.34 (dtd, J = 9.6, 4.9, 2.5 Hz, 1H), 2.78 (ddd, J = 16.7, 6.2, 4.4 Hz, 1H), 2.57 (ddd, J = 16.7, 9.5, 4.5 Hz, 1H), 2.30 (s, 3H), 2.26 (s, 3H), 2.05 – 1.98 (m, 1H), 1.85 (dtd, J = 13.9, 9.6, 4.5 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 170.0, 164.2, 161.9, 147.2, 139.6, 134.8, 134.6, 134.44, 134.36, 131.1, 129.1, 124.0, 121.6, 112.0, 112.0, 105.5, 74.7, 39.2, 25.8, 24.0, 19.6, 12.1.


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HPLC purity: 98.7%, retention time = 20.43 min. HRMS (ESI): m/z calcd for C22H18F4O4Na [M+Na]+: 407.1065, found 407.1066. (3aS,9bR)-6,9-Dimethyl-3-methylene-2-oxo-2,3,3a,4,5,9b-hexahydronaphtho[1,2-b]f uran-8-yl 3-(trifluoromethyl)benzoate (3f) White solid (yield: 79%), mp 70.9–70.4 °C. 1H NMR (500 MHz, CDCl3) δ 8.48 (s, 1H), 8.41 (d, J = 7.8 Hz, 1H), 7.91 (d, J = 7.8 Hz, 1H), 7.69 (t, J = 7.8 Hz, 1H), 7.00 (s, 1H), 6.33 (d, J = 2.0 Hz, 1H), 5.74 (d, J = 1.8 Hz, 1H), 5.61 (d, J = 6.7 Hz, 1H), 3.35 (dddd, J = 9.0, 6.9, 4.7, 2.2 Hz, 1H), 2.79 (dt, J = 16.7, 5.3 Hz, 1H), 2.58 (ddd, J = 16.7, 9.5, 4.5 Hz, 1H), 2.28 (d, J = 11.2 Hz, 6H), 2.09 – 1.99 (m, 1H), 1.87 (dp, J = 14.1, 4.8 Hz, 1H); 13

C NMR (125 MHz, CDCl3) δ 170.0, 163.9, 147.3, 139.6, 135.0, 134.8, 133.3, 131.2,

130.23, 130.17, 130.1, 129.4, 129.1, 127.1, 127.0, 123.9, 121.7, 74.7, 39.2, 25.8, 24.0, 19.6, 12.1. HPLC purity: 99.7%, retention time = 23.41 min. HRMS (ESI): m/z calcd for C23H19F3O4Na [M+Na]+: 439.1128, found 439.1124. (3aS,9bR)-6,9-Dimethyl-3-methylene-2-oxo-2,3,3a,4,5,9b-hexahydronaphtho[1,2-b]f uran-8-yl 4-(trifluoromethyl)benzoate (3g) White solid (yield: 84%), mp 123.5–124.5 °C. 1H NMR (500 MHz, CDCl3) δ 8.34 (d, J = 8.1 Hz, 2H), 7.80 (d, J = 8.2 Hz, 2H), 7.01 (s, 1H), 6.33 (d, J = 2.0 Hz, 1H), 5.74 (d, J = 1.8 Hz, 1H), 5.61 (d, J = 6.7 Hz, 1H), 3.35 (dtd, J = 9.6, 4.9, 2.5 Hz, 1H), 2.79 (ddd, J = 16.8, 6.2, 4.5 Hz, 1H), 2.58 (ddd, J = 16.7, 9.5, 4.5 Hz, 1H), 2.28 (d, J = 8.2 Hz, 6H), 2.07 – 1.99 (m, 1H), 1.86 (dtd, J = 13.9, 9.6, 4.4 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 170.0, 164.0, 147.3, 139.6, 135.2, 134.98, 134.95, 134.8, 132.6, 130.6 (2C), 129.0, 125.69, 125.66, 125.63, 123.8, 121.6, 74.6, 39.2, 25.8, 24.0, 19.6, 12.0. HPLC purity: 99.5%, retention time = 23.76 min. HRMS (ESI): m/z calcd for C23H19F3O4Na [M+Na]+:



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439.1128, found 439.1128. (3aS,9bR)-6,9-Dimethyl-3-methylene-2-oxo-2,3,3a,4,5,9b-hexahydronaphtho[1,2-b]f uran-8-yl 4-(trifluoromethoxy)benzoate (3h) White solid (yield: 54%), mp 90.6–91.8 °C. 1H NMR (500 MHz, CDCl3) δ 8.28 (d, J = 8.8 Hz, 2H), 7.36 (d, J = 8.3 Hz, 2H), 6.99 (s, 1H), 6.33 (d, J = 2.1 Hz, 1H), 5.74 (d, J = 1.8 Hz, 1H), 5.61 (d, J = 6.7 Hz, 1H), 3.34 (dtd, J = 9.6, 4.9, 2.5 Hz, 1H), 2.78 (ddd, J = 16.7, 6.2, 4.5 Hz, 1H), 2.57 (ddd, J = 16.7, 9.5, 4.5 Hz, 1H), 2.27 (d, J = 10.2 Hz, 6H), 2.07 – 1.98 (m, 1H), 1.87 (ddd, J = 13.8, 9.3, 4.4 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 170.0, 164.0, 153.2 , 147.4, 139.6, 134.9, 134.6, 132.2 (2C), 131.2, 129.1, 127.7, 124.0, 121.7, 120.5 (2C), 74.7, 39.2, 25.8, 23.9, 19.6, 12.0. HPLC purity: 99.1%, retention time = 24.36 min. HRMS (ESI): m/z calcd for C23H119F3O5Na [M+Na]+: 455.1077, found 455.1078. (3aS,9bR)-6,9-Dimethyl-3-methylene-2-oxo-2,3,3a,4,5,9b-hexahydronaphtho[1,2-b]f uran-8-yl 2-methylbenzoate (3i) White solid (yield: 63%), mp 107.8–108.7 °C. 1H NMR (500 MHz, CDCl3) δ 8.24 – 8.19 (m, 1H), 7.50 (td, J = 7.5, 1.5 Hz, 1H), 7.37 – 7.31 (m, 2H), 6.99 (s, 1H), 6.33 (d, J = 2.1 Hz, 1H), 5.74 (d, J = 1.9 Hz, 1H), 5.62 (d, J = 6.7 Hz, 1H), 3.34 (dtd, J = 9.6, 4.9, 2.5 Hz, 1H), 2.78 (ddd, J = 16.7, 6.1, 4.4 Hz, 1H), 2.68 (s, 3H), 2.57 (ddd, J = 16.7, 9.5, 4.5 Hz, 1H), 2.30 (s, 3H), 2.26 (s, 3H), 2.05 – 1.98 (m, 1H), 1.86 (ddt, J = 14.1, 9.5, 4.8 Hz, 1H); 13

C NMR (125 MHz, CDCl3) δ 170.0, 165.8, 147.6, 141.4, 139.7, 134.8, 134.4, 132.8,

132.0, 131.1, 131.0, 129.3, 128.3, 126.0, 124.2, 121.6, 74.8, 39.3, 25.8, 23.9, 21.9, 19.6, 12.2. HPLC purity: 95.8%, retention time = 22.89 min. HRMS (ESI): m/z calcd for C23H22O4Na [M+Na]+: 385.1410, found 385.1416.


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(3aS,9bR)-6,9-Dimethyl-3-methylene-2-oxo-2,3,3a,4,5,9b-hexahydronaphtho[1,2-b]f uran-8-yl 3-methylbenzoate (3j) White solid (yield: 47%), mp 115.6–115.8 °C. 1H NMR (500 MHz, CDCl3) δ 8.03 (d, J = 1.5 Hz, 2H), 7.49 – 7.38 (m, 2H), 6.99 (s, 1H), 6.32 (d, J = 2.0 Hz, 1H), 5.73 (d, J = 1.8 Hz, 1H), 5.61 (d, J = 6.7 Hz, 1H), 3.34 (dtd, J = 9.6, 4.9, 2.5 Hz, 1H), 2.78 (ddd, J = 16.7, 6.2, 4.5 Hz, 1H), 2.62 – 2.54 (m, 1H), 2.46 (s, 3H), 2.29 (s, 3H), 2.26 (s, 3H), 2.01 (ddt, J = 13.6, 6.3, 4.7 Hz, 1H), 1.87 (ddd, J = 13.8, 9.3, 4.4 Hz, 1H);

13

C NMR (125 MHz,

CDCl3) δ 170.0, 165.4, 147.6, 140.0, 138.5, 134.7, 134.41, 134.37, 131.0, 130.7, 129.3, 129.2, 128.5, 127.3, 124.2, 121.6, 74.8, 39.3, 25.8, 24.0, 21.3, 19.6, 12.1. HPLC purity: 99.0%, retention time = 22.97 min. HRMS (ESI): m/z calcd for C23H22O4Na [M+Na]+: 385.1410, found 385.1414. (3aS,9bR)-6,9-Dimethyl-3-methylene-2-oxo-2,3,3a,4,5,9b-hexahydronaphtho[1,2-b]f uran-8-yl 4-methylbenzoate (3k) White solid (yield: 86%), mp 100.4–101.2 °C. 1H NMR (300 MHz, CDCl3) δ 8.11 (2H, d, J = 8.0 Hz), 7.32 (2H, d, J = 8.0 Hz), 6.99 (1H, s), 6.32 (1H, s), 5.73 (1H, s), 5.60 (1H, d, J = 6.6 Hz), 3.30–3.36 (1H, m), 2.73–2.82 (1H, m), 2.65–2.49 (1H, m), 2.46 (3H, s), 2.28 (3H, s), 2.25 (3H, s), 2.09–1.93 (1H, m), 1.81–1.89 (1H, m); 13C NMR (125 MHz, CDCl3) δ 170.0, 165.2, 147.6, 144.5, 139.7, 134.7, 134.3, 131.0, 130.2 (2C), 129.3 (2C), 129.33 (2C), 129.27, 126.6, 124.2, 121.6, 74.8, 39.3, 25.8, 24.0, 21.7, 19.6, 12.06. HPLC purity: 98.5%, retention time = 22.92 min. HRMS (ESI): m/z calcd for C23H22O4Na [M+Na]+: 385.1410, found 385.1414. (3aS,9bR)-6,9-Dimethyl-3-methylene-2-oxo-2,3,3a,4,5,9b-hexahydronaphtho[1,2-b]f uran-8-yl 2-methoxybenzoate (3l)



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White solid (yield: 79%), mp 95.2–96.5 °C. 1H NMR (500 MHz, CDCl3) δ 8.03 (dd, J = 7.7, 1.8 Hz, 1H), 7.59 – 7.53 (m, 1H), 7.09 – 7.03 (m, 2H), 7.02 (s, 1H), 6.32 (d, J = 2.1 Hz, 1H), 5.73 (d, J = 1.8 Hz, 1H), 5.61 (d, J = 6.7 Hz, 1H), 3.94 (s, 3H), 3.33 (dtd, J = 9.6, 4.9, 2.5 Hz, 1H), 2.77 (ddd, J = 16.6, 6.1, 4.4 Hz, 1H), 2.56 (ddd, J = 16.6, 9.6, 4.5 Hz, 1H), 2.34 (s, 3H), 2.25 (s, 3H), 2.01 (ddt, J = 13.6, 6.2, 4.7 Hz, 1H), 1.85 (ddt, J = 14.2, 9.5, 4.8 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 170.0, 164.7, 159.8, 147.6, 139.8, 134.6, 134.2, 132.25, 134.19, 130.9, 129.3, 124.4, 121.5, 120.2, 119.2, 112.2, 74.8, 56.0, 39.2, 25.8, 23.9, 19.6, 12.1. HPLC purity: 99.1%, retention time = 18.76 min. HRMS (ESI): m/z calcd for C23H22O5Na [M+Na]+: 401.1359, found 401.1366. (3aS,9bR)-6,9-Dimethyl-3-methylene-2-oxo-2,3,3a,4,5,9b-hexahydronaphtho[1,2-b]f uran-8-yl 3-methoxybenzoate (3m) White solid (yield: 92%), mp 120.1–120.8 °C. 1H NMR (500 MHz, CDCl3) δ 7.83 (dt, J = 7.7, 1.2 Hz, 1H), 7.75 – 7.70 (m, 1H), 7.43 (t, J = 8.0 Hz, 1H), 7.19 (dd, J = 8.3, 2.7 Hz, 1H), 7.00 (s, 1H), 6.33 (d, J = 2.1 Hz, 1H), 5.74 (d, J = 1.8 Hz, 1H), 5.61 (d, J = 6.7 Hz, 1H), 3.89 (s, 3H), 3.34 (dtd, J = 9.6, 4.9, 2.5 Hz, 1H), 2.78 (ddd, J = 16.7, 6.2, 4.4 Hz, 1H), 2.57 (ddd, J = 16.7, 9.5, 4.5 Hz, 1H), 2.29 (s, 3H), 2.26 (s, 3H), 2.06 – 1.99 (m, 1H), 1.86 (dtd, J = 13.9, 9.6, 4.5 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 170.0, 165.0, 159.7, 147.6, 140.0, 134.8, 134.4, 131.0, 130.6, 130.0, 129.2, 124.1, 122.6, 121.6, 120.2, 114.5, 74.8, 55.5, 39.3, 25.8, 23.9, 19.6, 12.1. HPLC purity: 99.5%, retention time = 21.42 min. HRMS (ESI): m/z calcd for C23H22O5Na [M+Na]+: 401.1359, found 401.1357. (3aS,9bR)-6,9-Dimethyl-3-methylene-2-oxo-2,3,3a,4,5,9b-hexahydronaphtho[1,2-b]f uran-8-yl 4-methoxybenzoate (3n) White solid (yield: 91%), mp 170.5–171.1 °C. 1H NMR (500 MHz, CDCl3) δ 8.18 (d, J =


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8.9 Hz, 2H), 7.01 (s, 1H), 6.99 (d, J = 1.7 Hz, 2H), 6.32 (d, J = 2.1 Hz, 1H), 5.73 (d, J = 1.8 Hz, 1H), 5.61 (d, J = 6.7 Hz, 1H), 3.91 (s, 3H), 3.33 (dtd, J = 9.6, 4.9, 2.5 Hz, 1H), 2.77 (ddd, J = 16.7, 6.1, 4.4 Hz, 1H), 2.57 (ddd, J = 16.7, 9.5, 4.5 Hz, 1H), 2.28 (s, 3H), 2.25 (s, 3H), 2.04 – 1.98 (m, 1H), 1.86 (ddd, J = 13.8, 9.3, 4.4 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 170.0, 164.9, 163.9, 147.7, 139.7, 134.7, 134.2, 132.3 (2C), 130.9, 129.3, 124.3, 121.6, 121.5, 113.9 (2C), 74.8, 55.5, 39.3, 25.8, 24.0, 19.6, 12.1. HPLC purity: 99.2%, retention time = 20.97 min. HRMS (ESI): m/z calcd for C23H22O5Na [M+Na]+: 401.1359, found 401.1366. (3aS,9bR)-6,9-Dimethyl-3-methylene-2-oxo-2,3,3a,4,5,9b-hexahydronaphtho[1,2-b]f uran-8-yl (E)-3-(4-fluorophenyl)acrylate (4a) White solid (yield: 79%), mp 158.0–158.7 °C. 1H NMR (500 MHz, CDCl3) δ 7.85 (d, J = 16.0 Hz, 1H), 7.60 (dd, J = 8.7, 5.4 Hz, 2H), 7.12 (t, J = 8.6 Hz, 2H), 6.95 (s, 1H), 6.60 (d, J = 16.0 Hz, 1H), 6.32 (d, J = 2.1 Hz, 1H), 5.73 (d, J = 1.8 Hz, 1H), 5.60 (d, J = 6.7 Hz, 1H), 3.33 (dtd, J = 9.6, 4.9, 2.6 Hz, 1H), 2.77 (ddd, J = 16.7, 6.1, 4.5 Hz, 1H), 2.56 (ddd, J = 16.7, 9.6, 4.5 Hz, 1H), 2.27 (s, 3H), 2.25 (s, 3H), 2.01 (ddt, J = 13.6, 6.2, 4.7 Hz, 1H), 1.85 (ddt, J = 14.1, 9.5, 4.8 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 170.0, 165.3, 147.4, 145.4, 139.8, 137.0, 134.7, 134.4, 131.0, 130.3, 130.2, 129.2, 124.0, 121.6, 116.73, 116.72, 116.3, 116.1, 74.8, 39.2, 25.8, 24.0, 19.6, 12.0. HPLC purity: 98.5%, retention time = 22.31 min. HRMS (ESI): m/z calcd for C22H21FO4Na [M+Na]+: 415.1316, found 415.1319. (3aS,9bR)-6,9-Dimethyl-3-methylene-2-oxo-2,3,3a,4,5,9b-hexahydronaphtho[1,2-b]f uran-8-yl (E)-3-(4-chlorophenyl)acrylate (4b) White solid (yield: 34%), mp 135.4–136.7 °C. 1H NMR (500 MHz, CDCl3) δ 7.84 (d, J =



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16.0 Hz, 1H), 7.54 (d, J = 8.5 Hz, 2H), 7.41 (d, J = 8.5 Hz, 2H), 6.94 (s, 1H), 6.65 (d, J = 16.0 Hz, 1H), 6.32 (d, J = 2.0 Hz, 1H), 5.73 (d, J = 1.8 Hz, 1H), 5.59 (d, J = 6.6 Hz, 1H), 3.33 (dtd, J = 9.5, 4.9, 2.6 Hz, 1H), 2.77 (ddd, J = 16.7, 6.1, 4.4 Hz, 1H), 2.56 (ddd, J = 16.7, 9.5, 4.5 Hz, 1H), 2.27 (s, 3H), 2.25 (s, 3H), 2.00 (ddt, J = 13.6, 6.2, 4.7 Hz, 1H), 1.85 (ddd, J = 13.8, 9.1, 4.4 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 170.0, 165.2, 147.4, 145.2, 139.7, 136.7, 134.7, 132.6, 131.0, 199.52, 129.46 (2C), 129.3 (2C), 129.2, 124.0, 121.6, 117.6, 74.8, 39.2, 25.8, 24.0, 19.6, 12.1. HPLC purity: 99.4%, retention time = 24.55 min. HRMS (ESI): m/z calcd for C22H21ClO4Na [M+Na]+: 431.1021, found 431.1026. (3aS,9bR)-6,9-Dimethyl-3-methylene-2-oxo-2,3,3a,4,5,9b-hexahydronaphtho[1,2-b]f uran-8-yl (E)-3-(4-(trifluoromethoxy)phenyl)acrylate (4c) White solid (yield: 82%), mp 101.5–102.3 °C. 1H NMR (500 MHz, CDCl3) δ 7.86 (d, J = 16.0 Hz, 1H), 7.64 (d, J = 8.7 Hz, 2H), 7.27 (dd, J = 8.9, 1.1 Hz, 2H), 6.95 (s, 1H), 6.65 (d, J = 16.0 Hz, 1H), 6.32 (d, J = 2.1 Hz, 1H), 5.73 (d, J = 1.8 Hz, 1H), 5.60 (d, J = 6.7 Hz, 1H), 3.33 (tdt, J = 7.0, 4.9, 2.0 Hz, 1H), 2.77 (ddd, J = 16.7, 6.1, 4.4 Hz, 1H), 2.56 (ddd, J = 16.7, 9.6, 4.5 Hz, 1H), 2.27 (s, 3H), 2.25 (s, 3H), 2.04 – 1.97 (m, 1H), 1.89 – 1.81 (m, 1H);

13

C NMR (125 MHz, CDCl3) δ 170.0, 165.1, 150.7, 147.4, 144.8, 139.6,

134.8, 134.4, 132.6, 131.0, 129.8 (2C), 129.1, 124.0, 121.6, 121.2 (2C), 117.9, 74.8, 39.2, 25.8, 24.0, 19.6, 12.0. HPLC purity: 99.0%, retention time = 24.75 min. HRMS (ESI): m/z calcd for C25H21F3O5Na [M+Na]+: 481.1233, found 481.1239. (3aS,9bR)-6,9-Dimethyl-3-methylene-2-oxo-2,3,3a,4,5,9b-hexahydronaphtho[1,2-b]f uran-8-yl (E)-3-(2,4-difluorophenyl)acrylate (4d) White solid (yield: 79%), mp 147.6–148.2 °C. 1H NMR (500 MHz, CDCl3) δ 7.94 (d, J =


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16.2 Hz, 1H), 7.61 (td, J = 8.5, 6.3 Hz, 1H), 6.99 – 6.95 (m, 1H), 6.95 (s, 1H), 6.90 (ddd, J = 11.0, 8.7, 2.5 Hz, 1H), 6.72 (dd, J = 16.1, 0.5 Hz, 1H), 6.32 (d, J = 2.1 Hz, 1H), 5.73 (d, J = 1.8 Hz, 1H), 5.59 (d, J = 6.6 Hz, 1H), 3.32 (dtd, J = 9.7, 4.9, 2.0 Hz, 1H), 2.76 (ddd, J = 16.7, 6.1, 4.5 Hz, 1H), 2.56 (ddd, J = 16.7, 9.6, 4.5 Hz, 1H), 2.27 (s, 3H), 2.25 (s, 3H), 2.04 – 1.97 (m, 1H), 1.85 (ddd, J = 13.8, 9.1, 4.4 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 170.0, 165.2, 147.4, 139.6, 138.2, 134.7, 134.4, 131.0, 130.6, 129.2, 124.0, 121.6, 119.2, 112.2, 104.8, 74.8, 39.2, 25.8, 24.0, 19.6, 12.0. HPLC purity: 98.9%, retention time = 23.27 min. HRMS (ESI): m/z calcd for C24H20F2O4Na [M+Na]+: 433.1222, found 433.1234. (3aS,9bR)-6,9-Dimethyl-3-methylene-2-oxo-2,3,3a,4,5,9b-hexahydronaphtho[1,2-b]f uran-8-yl (E)-3-(4-(trifluoromethyl)phenyl)acrylate (4e) White solid (yield: 78%), mp 114.3–115.7 °C. 1H NMR (500 MHz, CDCl3) δ 7.90 (d, J = 16.1 Hz, 1H), 7.70 (d, J = 3.2 Hz, 4H), 6.95 (s, 1H), 6.75 (d, J = 16.0 Hz, 1H), 6.33 (d, J = 2.1 Hz, 1H), 5.74 (d, J = 1.8 Hz, 1H), 5.60 (d, J = 6.6 Hz, 1H), 3.33 (dtd, J = 9.7, 4.8, 2.6 Hz, 1H), 2.77 (ddd, J = 16.7, 6.1, 4.4 Hz, 1H), 2.59 (d, J = 4.5 Hz, 1H), 2.28 (s, 3H), 2.25 (s, 3H), 2.05 – 1.98 (m, 1H), 1.85 (dt, J = 13.8, 4.7 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 170.7, 164.8, 147.3, 144.8, 139.6, 137.4, 134.8, 134.6, 131.0, 129.1, 128.4 (2C), 126.0, 125.98, 125.95, 125.91, 123.9, 121.6, 119.6, 74.8, 39.2, 25.8, 24.0, 19.6, 12.0. HPLC purity: 98.8%, retention time = 24.20 min. HRMS (ESI): m/z calcd for C25H21F3O4Na [M+Na]+: 465.1284, found 465.1284. (3aS,9bR)-6,9-Dimethyl-3-methylene-2-oxo-2,3,3a,4,5,9b-hexahydronaphtho[1,2-b]f uran-8-yl (E)-3-(4-cyanophenyl)acrylate (4f) White solid (yield: 84%), mp 186.3–187.8 °C. 1H NMR (500 MHz, CDCl3) δ 7.87 (d, J =



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16.0 Hz, 1H), 7.77 – 7.66 (m, 4H), 6.94 (s, 1H), 6.76 (d, J = 16.0 Hz, 1H), 6.33 (d, J = 2.1 Hz, 1H), 5.74 (d, J = 1.8 Hz, 1H), 5.60 (d, J = 6.7 Hz, 1H), 3.33 (dt, J = 6.8, 2.1 Hz, 1H), 2.77 (ddd, J = 16.7, 6.2, 4.4 Hz, 1H), 2.56 (ddd, J = 16.7, 9.5, 4.5 Hz, 1H), 2.26 (d, J = 8.7 Hz, 6H), 2.01 (ddt, J = 9.5, 6.1, 4.6 Hz, 1H), 1.85 (dt, J = 13.8, 4.8 Hz, 1H);

13

C

NMR (125 MHz, CDCl3) δ 170.0, 164.6, 147.2, 144.2, 139.6, 138.3, 134.8, 134.6, 132.7 (2C), 131.1, 129.0, 128.6 (2C), 123.8, 121.7, 120.6, 118.2, 113.8, 74.7, 39.2, 25.8, 24.0, 19.6, 12.0. HPLC purity: 98.5%, retention time = 18.72 min. HRMS (ESI): m/z calcd for C25H21NO4Na [M+Na]+: 422.1363, found 422.1365. (3aS,9bR)-6,9-Dimethyl-3-methylene-2-oxo-2,3,3a,4,5,9b-hexahydronaphtho[1,2-b]f uran-8-yl (E)-3-(p-tolyl)acrylate (4g) White solid (yield: 81%), mp 190.0–191.2 °C. 1H NMR (500 MHz, CDCl3) δ 7.87 (d, J = 16.0 Hz, 1H), 7.50 (d, J = 8.1 Hz, 2H), 7.24 (d, J = 7.9 Hz, 2H), 6.95 (s, 1H), 6.62 (d, J = 16.0 Hz, 1H), 6.32 (d, J = 2.1 Hz, 1H), 5.73 (d, J = 1.8 Hz, 1H), 5.60 (d, J = 6.7 Hz, 1H), 3.32 (ddd, J = 9.6, 6.8, 4.8 Hz, 1H), 2.82 – 2.73 (m, 1H), 2.56 (ddd, J = 16.6, 9.5, 4.5 Hz, 1H), 2.40 (s, 3H), 2.28 (s, 3H), 2.25 (s, 3H), 2.04 – 1.97 (m, 1H), 1.85 (dt, J = 13.8, 4.8 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 170.0, 165.6, 147.5, 146.7, 141.2, 139.7, 134.6, 134.2, 131.4, 130.9, 129.7 (2C), 129.2, 128.3 (2C), 124.1, 121.5, 115.8, 74.8, 39.2, 25.8, 24.0, 21.5, 19.6, 12.0. HPLC purity: 98.2%, retention time = 19.58 min. HRMS (ESI): m/z calcd for C23H23F3O2Na [M+Na]+: 411.1542, found 411.1565. (3aS,9bR)-6,9-Dimethyl-3-methylene-2-oxo-2,3,3a,4,5,9b-hexahydronaphtho[1,2-b]f uran-8-yl (E)-3-(4-methoxyphenyl)acrylate (4h) White solid (yield: 89%), mp 186.5–187.9 °C. 1H NMR (500 MHz, CDCl3) δ 7.85 (d, J = 15.9 Hz, 1H), 7.56 (d, J = 8.7 Hz, 2H), 6.98 – 6.92 (m, 3H), 6.54 (d, J = 15.9 Hz, 1H),


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6.32 (d, J = 2.1 Hz, 1H), 5.73 (d, J = 1.8 Hz, 1H), 5.60 (d, J = 6.7 Hz, 1H), 3.86 (s, 3H), 3.32 (dtd, J = 9.6, 5.0, 2.6 Hz, 1H), 2.76 (ddd, J = 16.8, 6.1, 4.4 Hz, 1H), 2.55 (ddd, J = 16.6, 9.6, 4.5 Hz, 1H), 2.27 (s, 3H), 2.24 (s, 3H), 2.02 – 1.98 (m, 1H), 1.85 (dt, J = 13.8, 4.8 Hz, 1H);

13

C NMR (125 MHz, CDCl3) δ 169.9, 165.8, 161.7, 147.6, 146.4, 139.8,

134.7, 134.2, 130.9, 130.0 (2C), 129.2, 126.9, 124.2, 121.5, 114.4 (2C), 114.3, 74.8, 55.4, 39.3, 25.8, 24.0, 19.6, 12.0. HPLC purity: 97.5%, retention time = 18.59 min. HRMS (ESI): m/z calcd for C25H24O5Na [M+Na]+: 427.1516, found 427.1522. (3aS,9bR)-6,9-Dimethyl-3-methylene-2-oxo-2,3,3a,4,5,9b-hexahydronaphtho[1,2-b]f uran-8-yl nicotinate (5a) White solid (yield: 34%), mp 119.1–120.5 °C. 1H NMR (500 MHz, CDCl3) δ 9.43 (d, J = 2.2 Hz, 1H), 8.89 (dd, J = 5.0, 1.7 Hz, 1H), 8.55 – 8.45 (m, 1H), 7.51 (dd, J = 8.0, 4.9 Hz, 1H), 7.01 (s, 1H), 6.32 (d, J = 2.1 Hz, 1H), 5.74 (d, J = 1.8 Hz, 1H), 5.60 (d, J = 6.7 Hz, 1H), 3.34 (dtd, J = 9.5, 4.7, 2.4 Hz, 1H), 2.78 (ddd, J = 16.7, 6.2, 4.5 Hz, 1H), 2.64 – 2.53 (m, 1H), 2.29 (s, 3H), 2.26 (s, 3H), 2.02 (ddt, J = 13.7, 6.4, 4.8 Hz, 1H), 1.86 (ddd, J = 13.8, 9.2, 4.4 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 167.0, 163.7, 154.0, 151.3, 147.2, 140.0, 137.9, 135.0, 134.8, 131.2, 129.0, 125.6, 123.9, 123.7, 121.7, 74.7, 39.2, 25.8, 24.0, 19.6, 12.1. HPLC purity: 97.5%, retention time = 13.68 min. HRMS (ESI): m/z calcd for C21H19NO4Na [M+Na]+: 350.1387, found 350.1397. (3aS,9bR)-6,9-Dimethyl-3-methylene-2-oxo-2,3,3a,4,5,9b-hexahydronaphtho[1,2-b]f uran-8-yl picolinate (5b) White solid (yield: 52%), mp 97.5–98.6 °C. 1H NMR (500 MHz, CDCl3) δ 8.86 (ddd, J = 4.7, 1.8, 0.9 Hz, 1H), 8.29 (dt, J = 7.8, 1.1 Hz, 1H), 7.93 (td, J = 7.7, 1.8 Hz, 1H), 7.57 (ddd, J = 7.6, 4.8, 1.2 Hz, 1H), 7.03 (s, 1H), 6.31 (d, J = 2.1 Hz, 1H), 5.73 (d, J = 1.8 Hz,



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1H), 5.59 (d, J = 6.6 Hz, 1H), 3.32 (dddt, J = 8.8, 6.8, 3.7, 1.9 Hz, 1H), 2.78 (ddd, J = 16.7, 6.0, 4.5 Hz, 1H), 2.57 (ddd, J = 16.7, 9.7, 4.5 Hz, 1H), 2.30 (s, 3H), 2.25 (s, 3H), 2.04 – 1.98 (m, 1H), 1.88 – 1.81 (m, 1H); 13C NMR (125 MHz, CDCl3) δ 170.0, 163.8, 150.2, 147.6, 147.3, 139.7, 137.2, 134.8, 134.7, 131.1, 129.2, 12.4, 125.8, 124.0, 121.6, 74.8, 39.3, 25.8, 24.0, 19.6, 12.1. HPLC purity: 95.6%, retention time = 12.02 min. HRMS (ESI): m/z calcd for C21H19NO4Na [M+Na]+: 350.1387, found 350.1385. (3aS,9bR)-6,9-Dimethyl-3-methylene-2-oxo-2,3,3a,4,5,9b-hexahydronaphtho[1,2-b]f uran-8-yl isonicotinate (5c) White solid (yield: 54%), mp 148.2–149.7 °C. 1H NMR (500 MHz, CDCl3) δ 8.93 – 8.85 (m, 2H), 8.07 – 7.99 (m, 2H), 7.00 (s, 1H), 6.33 (d, J = 2.1 Hz, 1H), 5.74 (d, J = 1.8 Hz, 1H), 5.60 (d, J = 6.7 Hz, 1H), 3.35 (dtt, J = 6.8, 4.9, 2.7 Hz, 1H), 2.78 (ddd, J = 16.8, 6.2, 4.4 Hz, 1H), 2.58 (ddd, J = 16.7, 9.5, 4.5 Hz, 1H), 2.27 (d, J = 6.5 Hz, 6H), 2.02 (ddt, J = 13.6, 6.2, 4.7 Hz, 1H), 1.91 – 1.80 (m, 1H); 13C NMR (125 MHz, CDCl3) δ 170.0, 163.7, 150.9 (2C), 147.2, 139.5, 136.7, 135.03, 134.95, 131.3, 128.9, 123.7, 123.3 (2C), 121.8, 74.6, 32, 25.7, 23.9, 19.6, 12.1. HPLC purity: 96.4%, retention time = 14.23 min. HRMS (ESI): m/z calcd for C21H19NO4Na [M+Na]+: 350.1387, found 350.1389. (3aS,9bR)-6,9-Dimethyl-3-methylene-2-oxo-2,3,3a,4,5,9b-hexahydronaphtho[1,2-b]f uran-8-yl 2-chloronicotinate (5d) White solid (yield: 72%), mp 94.7–95.2 °C. 1H NMR (500 MHz, CDCl3) δ 8.61 (dd, J = 4.8, 2.0 Hz, 1H), 8.40 (dd, J = 7.7, 2.0 Hz, 1H), 7.43 (dd, J = 7.7, 4.8 Hz, 1H), 7.02 (s, 1H), 6.33 (d, J = 2.0 Hz, 1H), 5.74 (d, J = 1.8 Hz, 1H), 5.61 (d, J = 6.8 Hz, 1H), 3.35 (dtt, J = 6.9, 4.8, 2.8 Hz, 1H), 2.78 (ddd, J = 16.7, 6.2, 4.4 Hz, 1H), 2.63 – 2.53 (m, 1H), 2.32 (s, 3H), 2.27 (s, 3H), 2.05 – 1.99 (m, 1H), 1.91 – 1.82 (m, 1H);


13

C NMR (125 MHz,

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CDCl3) δ 170.0, 163.0, 152.5, 150.6, 14.1, 140.7, 139.5, 135.1, 135.0, 131.3, 129.0, 126.1, 123.7, 122.2, 121.8, 74.7, 39.2, 25.7, 24.0, 19.6, 12.2. HPLC purity: 99.6%, retention time = 15.68 min. HRMS (ESI): m/z calcd for C21H18ClNO4Na [M+Na]+: 406.0817, found 406.0818. (3aS,9bR)-6,9-Dimethyl-3-methylene-2-oxo-2,3,3a,4,5,9b-hexahydronaphtho[1,2-b]f uran-8-yl (E)-3-(furan-2-yl)acrylate (5e) White solid (yield: 81%), mp 170.2–171.4 °C. 1H NMR (500 MHz, CDCl3) δ 7.62 (d, J = 15.7 Hz, 1H), 7.54 (d, J = 1.7 Hz, 1H), 6.94 (s, 1H), 6.70 (d, J = 3.4 Hz, 1H), 6.54 (d, J = 15.7 Hz, 1H), 6.51 (dd, J = 3.4, 1.8 Hz, 1H), 6.31 (d, J = 2.1 Hz, 1H), 5.72 (d, J = 1.8 Hz, 1H), 5.59 (d, J = 6.7 Hz, 1H), 3.35 – 3.28 (m, 1H), 2.76 (ddd, J = 16.7, 6.1, 4.4 Hz, 1H), 2.55 (ddd, J = 16.7, 9.6, 4.5 Hz, 1H), 2.26 (s, 3H), 2.24 (s, 3H), 2.02 – 1.96 (m, 1H), 1.84 (ddt, J = 13.8, 9.5, 4.8 Hz, 1H);

13

C NMR (125 MHz, CDCl3) δ 170.0, 165.5, 150.7,

147.4, 145.2, 139.7, 134.6, 134.3, 132.8, 130.9, 129.2, 124.1, 121.5, 115.8, 114.4, 112.4, 74.8, 39.2, 25.8, 24.0, 19.6, 12.0. HPLC purity: 96.6%, retention time = 20.34 min. HRMS (ESI): m/z calcd for C22H20O5Na [M+Na]+: 387.1203, found 387.1206. (3aS,9bR)-6,9-Dimethyl-3-methylene-2-oxo-2,3,3a,4,5,9b-hexahydronaphtho[1,2-b]f uran-8-yl (E)-3-(thiophen-2-yl)acrylate (5f) White solid (yield: 47%), mp 174.5–175.6 °C. 1H NMR (500 MHz, CDCl3) δ 7.98 (d, J = 15.7 Hz, 1H), 7.45 (d, J = 5.1 Hz, 1H), 7.34 (d, J = 3.6 Hz, 1H), 7.10 (dd, J = 5.1, 3.6 Hz, 1H), 6.94 (s, 1H), 6.46 (d, J = 15.7 Hz, 1H), 6.32 (d, J = 2.0 Hz, 1H), 5.73 (d, J = 1.8 Hz, 1H), 5.59 (d, J = 6.7 Hz, 1H), 3.32 (dddd, J = 9.7, 6.9, 4.7, 2.3 Hz, 1H), 2.76 (ddd, J = 16.7, 6.1, 4.5 Hz, 1H), 2.55 (ddd, J = 16.6, 9.6, 4.5 Hz, 1H), 2.27 (s, 3H), 2.24 (s, 3H), 2.00 (ddd, J = 13.6, 10.8, 4.8 Hz, 1H), 1.84 (dtd, J = 14.0, 9.7, 4.4 Hz, 1H);


13

C NMR


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(125 MHz, CDCl3) δ 170.0, 165.3, 147.4, 139.7, 139.2, 139.0, 134.6, 134.4, 131.6, 131.0, 129.23, 129.19, 128.2, 124.1, 121.6, 115.6, 74.8, 39.2, 25.8, 24.0, 19.6, 12.0. HPLC purity: 96.3%, retention time = 21.66 min. HRMS (ESI): m/z calcd for C22H20O4SNa [M+Na]+: 403.0975, found 403.0975. (3aS,9bR)-6,9-Dimethyl-3-methylene-2-oxo-2,3,3a,4,5,9b-hexahydronaphtho[1,2-b]f uran-8-yl (E)-3-(pyridin-3-yl)acrylate (5g) White solid (yield: 72%), mp 165.3–165.7 °C. 1H NMR (500 MHz, CDCl3) δ 8.84 (s, 1H), 8.68 (d, J = 3.9 Hz, 1H), 7.94 (dt, J = 8.0, 1.7 Hz, 1H), 7.88 (d, J = 16.1 Hz, 1H), 7.40 (dd, J = 7.9, 4.9 Hz, 1H), 6.95 (s, 1H), 6.76 (d, J = 16.1 Hz, 1H), 6.32 (d, J = 2.0 Hz, 1H), 5.73 (d, J = 1.8 Hz, 1H), 5.60 (d, J = 6.7 Hz, 1H), 3.33 (ddd, J = 9.6, 6.8, 4.9 Hz, 1H), 2.77 (dt, J = 16.7, 5.3 Hz, 1H), 2.62 – 2.53 (m, 1H), 2.28 (s, 3H), 2.25 (s, 3H), 2.01 (ddd, J = 13.5, 10.9, 4.8 Hz, 1H), 1.85 (ddt, J = 13.8, 9.5, 4.8 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 170.0, 164.7, 151.3, 149.8, 147.3, 142.8, 139.6, 134.8, 134.64, 134.56, 133.4, 131.1, 130.1, 129.1, 123.9, 121.6, 119.4, 74.8, 39.2, 25.8, 24.0, 19.6, 12.0. HPLC purity: 99.6%, retention time = 14.30 min. HRMS (ESI): m/z calcd for C23H21NO4Na [M+Na]+: 376.1543, found 376.1549. (3aS,9bR)-6,9-Dimethyl-8-((3-methylbenzyl)oxy)-3-methylene-3a,4,5,9b-tetrahydron aphtho[1,2-b]furan-2(3H)-one (6a) White solid (yield: 82%), mp 130.6–131.5 °C. 1H NMR (500 MHz, CDCl3) δ 7.28 (d, J = 7.4 Hz, 1H), 7.24 (s, 1H), 7.14 (d, J = 7.3 Hz, 1H), 6.81 (s, 1H), 6.30 (d, J = 2.0 Hz, 1H), 5.71 (d, J = 1.7 Hz, 1H), 5.63 (d, J = 6.7 Hz, 1H), 5.02 (s, 2H), 3.31 (ddd, J = 4.9, 6.9, 9.5 Hz, 1H), 2.75 – 2.67 (m, 1H), 2.53 (dd, J = 4.5, 9.6 Hz, 1H), 2.39 (s, 3H), 2.37 (s, 3H), 2.23 (s, 3H), 1.97 (dt, J = 6.1, 8.7 Hz, 1H), 1.83 (ddd, J = 4.3, 9.1, 13.6 Hz, 1H);


13

C

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NMR (125 MHz, CDCl3) δ 170.3, 155.1, 140.0, 138.2, 137.4, 133.9, 130.5, 128.5, 128.4, 127.9, 126.2, 124.2, 121.3, 115.1, 75.1, 70.6, 53.4, 39.4, 26.2, 23.6, 21.4, 20.0, 11.6. HPLC purity: 97.4%, retention time = 25.56 min. HRMS (ESI): m/z calcd for C23H24O3Na [M+Na]+: 371.1618, found 371.1618. (3aS,9bR)-8-((3,4-Dimethoxybenzyl)oxy)-6,9-dimethyl-3-methylene-3a,4,5,9b-tetrah ydronaphtho[1,2-b]furan-2(3H)-one (6b) White solid (yield: 97%), mp 155.7–156.9 °C. 1H NMR (500 MHz, CDCl3) δ 7.10 (s, 2H), 6.91 (dd, J = 2.4, 7.2 Hz, 1H), 6.87 (s, 1H), 6.30 (d, J = 1.9 Hz, 1H), 5.70 (d, J = 1.6 Hz, 1H), 5.62 (d, J = 6.7 Hz, 1H), 5.09 (s, 2H), 3.89 (d, J = 5.4 Hz, 6H), 3.30 (ddd, J = 5.0, 6.9, 9.6 Hz, 1H), 2.72 (dt, J = 4.7, 16.3 Hz, 1H), 2.56 – 2.48 (m, 1H), 2.35 (s, 3H), 2.24 (s, 3H), 1.96 (ddd, J = 4.7, 10.7, 13.5 Hz, 1H), 1.82 (dtt, J = 4.6, 9.6, 13.8 Hz, 1H);

13

C

NMR (125 MHz, CDCl3) δ 170.3, 155.0, 152.5, 146.8, 140.1, 134.0, 130.4, 128.4, 126.0, 124.2, 121.3, 120.7, 114.9, 112.0, 77.2, 75.1, 65.6, 61.0, 55.8, 39.5, 26.2, 23.6, 19.9, 11.6. HPLC purity: 97.2%, retention time = 22.32 min. HRMS (ESI): m/z calcd for C26H24O5Na [M+Na]+: 417.1672, found 417.1678. (3aS,9bR)-8-((2-Chlorobenzyl)oxy)-6,9-dimethyl-3-methylene-3a,4,5,9b-tetrahydron aphtho[1,2-b]furan-2(3H)-one (6c) White solid (yield: 93%), mp 146.7–148.1 °C. 1H NMR (500 MHz, CDCl3) δ 7.60 (d, J = 7.2 Hz, 1H), 7.43 – 7.38 (m, 1H), 7.35 – 7.27 (m, 2H), 6.81 (s, 1H), 6.31 (d, J = 2.0 Hz, 1H), 5.71 (d, J = 1.7 Hz, 1H), 5.63 (d, J = 6.7 Hz, 1H), 5.15 (s, 2H), 3.32 (ddd, J = 4.9, 6.9, 9.5 Hz, 1H), 2.72 (dt, J = 5.2, 16.4 Hz, 1H), 2.57 – 2.48 (m, 1H), 2.39 (s, 3H), 2.24 (s, 3H), 1.97 (ddd, J = 4.8, 10.9, 13.5 Hz, 1H), 1.84 (ddd, J = 4.3, 9.2, 13.7 Hz, 1H);

13

C

NMR (125 MHz, CDCl3) δ 170.2, 154.6, 140.0, 135.1, 134.1, 134.1, 132.4, 130.6, 129.3,



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128.8, 128.5, 127.0, 126.1, 121.3, 114.9, 75.1, 67.6, 39.4, 26.1, 23.6, 20.0, 11.6. HPLC purity: 99.1%, retention time = 26.21 min. HRMS (ESI): m/z calcd for C22H21ClO3Na [M+Na]+: 391.1071, found 391.1076. (3aS,9bR)-8-((2-Bromobenzyl)oxy)-6,9-dimethyl-3-methylene-3a,4,5,9b-tetrahydron aphtho[1,2-b]furan-2(3H)-one (6d) White solid (yield: 97%), mp 144.4–145.8 °C. 1H NMR (500 MHz, CDCl3) δ 7.59 (d, J = 8.1 Hz, 2H), 7.36 (t, J = 7.5 Hz, 1H), 7.19 (t, J = 7.7 Hz, 1H), 6.80 (s, 1H), 6.31 (d, J = 1.9 Hz, 1H), 5.71 (d, J = 1.7 Hz, 1H), 5.63 (d, J = 6.7 Hz, 1H), 5.10 (s, 2H), 3.31 (ddd, J = 5.0, 6.9, 9.5 Hz, 1H), 2.76 – 2.69 (m, 1H), 2.57 – 2.49 (m, 1H), 2.40 (s, 3H), 2.24 (s, 3H), 1.97 (ddd, J = 4.8, 10.8, 13.4 Hz, 1H), 1.82 (dtd, J = 4.4, 9.6, 13.8 Hz, 1H);

13

C

NMR (125 MHz, CDCl3) δ 170.3, 154.6, 140.0, 136.7, 134.1, 132.5, 130.6, 129.1, 128.8, 128.7, 127.6, 126.1, 122.1, 121.3, 114.9, 75.1, 69.8, 53.4, 39.4, 26.1, 23.6, 20.0, 11.6. HPLC purity: 99.5%, retention time = 26.76 min. HRMS (ESI): m/z calcd for C22H21BrO3Na [M+Na]+: 435.0566, found 435.0564. (3aS,9bR)-8-((4-Bromobenzyl)oxy)-6,9-dimethyl-3-methylene-3a,4,5,9b-tetrahydron aphtho[1,2-b]furan-2(3H)-one (6e) White solid (yield: 80%), mp 130.5–132.1 °C. 1H NMR (500 MHz, CDCl3) δ 7.51 (d, J = 8.2 Hz, 2H), 7.31 (d, J = 8.1 Hz, 2H), 6.76 (s, 1H), 6.30 (s, 1H), 5.71 (s, 1H), 5.62 (d, J = 6.7 Hz, 1H), 5.01 (s, 2H), 3.35 – 3.27 (m, 1H), 2.71 (dt, J = 5.1, 16.4 Hz, 1H), 2.51 (ddd, J = 4.4, 9.5, 16.1 Hz, 1H), 2.35 (s, 3H), 2.22 (s, 3H), 1.97 (dd, J = 5.7, 13.4 Hz, 1H), 1.83 (tt, J = 4.2, 8.8 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 170.2, 154.6, 139.9, 136.5, 134.0, 131.6, 130.7, 128.8, 128.8, 126.1, 121.6, 121.4, 115.0, 75.0, 69.8, 39.4, 26.1, 23.6, 20.0, 11.6. HPLC purity: 96.3%, retention time = 26.00 min. HRMS (ESI): m/z calcd for


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C22H21BrO3Na [M+Na]+: 435.0566, found 435.0561. (3aS,9bR)-8-((2-Iodobenzyl)oxy)-6,9-dimethyl-3-methylene-3a,4,5,9b-tetrahydronap htho[1,2-b]furan-2(3H)-one (6f) White solid (yield: 95%), mp 145.0–146.9 °C. 1H NMR (500 MHz, CDCl3) δ 7.87 (d, J = 7.8 Hz, 1H), 7.55 (d, J = 7.6 Hz, 1H), 7.39 (t, J = 7.5 Hz, 1H), 7.07 – 6.99 (m, 1H), 6.79 (s, 1H), 6.31 (d, J = 1.9 Hz, 1H), 5.72 (d, J = 1.6 Hz, 1H), 5.63 (d, J = 6.7 Hz, 1H), 5.01 (s, 2H), 3.36 – 3.28 (m, 1H), 2.72 (dt, J = 4.8, 16.4 Hz, 1H), 2.59 – 2.48 (m, 1H), 2.40 (s, 3H), 2.24 (s, 3H), 1.97 (ddd, J = 4.8, 10.8, 13.5 Hz, 1H), 1.83 (ddd, J = 4.3, 9.2, 13.7 Hz, 1H);

13

C NMR (125 MHz, CDCl3) δ 170.3, 154.5, 140.0, 139.5, 139.2, 134.1, 130.6,

129.4, 128.8, 128.5, 128.4, 126.1, 121.3, 114.9, 97.0, 75.1, 74.4, 39.4, 26.1, 23.6, 20.0, 11.7. HPLC purity: 97.0%, retention time = 21.16 min. HRMS (ESI): m/z calcd for C22H21IO3Na [M+Na]+: 483.0428, found 483.0429. (3aS,9bR)-6,9-Dimethyl-3-methylene-8-((2-(trifluoromethyl)benzyl)oxy)-3a,4,5,9b-te trahydronaphtho[1,2-b]furan-2(3H)-one (6g) White solid (yield: 92%), mp 125.5–126.7 °C. 1H NMR (500 MHz, CDCl3) δ 7.79 (d, J = 7.8 Hz, 1H), 7.70 (d, J = 7.8 Hz, 1H), 7.59 (t, J = 7.6 Hz, 1H), 7.43 (t, J = 7.6 Hz, 1H), 6.76 (s, 1H), 6.31 (d, J = 1.9 Hz, 1H), 5.72 (d, J = 1.7 Hz, 1H), 5.64 (d, J = 6.7 Hz, 1H), 5.26 (s, 2H), 3.32 (ddd, J = 5.0, 6.9, 9.5 Hz, 1H), 2.72 (dt, J = 4.7, 16.4 Hz, 1H), 2.57 – 2.47 (m, 1H), 2.40 (s, 3H), 2.22 (s, 3H), 1.97 (ddd, J = 4.8, 10.9, 13.5 Hz, 1H), 1.83 (ddd, J = 4.3, 9.1, 13.7 Hz, 1H);

13

C NMR (125 MHz, CDCl3) δ 170.2, 154.5, 140.0, 136.1,

134.1, 132.2, 130.7, 128.9, 128.4, 127.5, 127.2, 126.0, 125.8, 125.8, 121.4, 114.8, 75.1, 66.4, 39.4, 26.1, 23.6, 20.0, 11.6. HPLC purity: 99.6%, retention time = 25.73 min. HRMS (ESI): m/z calcd for C23H21F3O3Na [M+Na]+: 425.1335, found 425.1338.



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3-((((3aS,9bR)-6,9-Dimethyl-3-methylene-2-oxo-2,3,3a,4,5,9b-hexahydronaphtho[1,2 -b]furan-8-yl)oxy)methyl)benzonitrile (6h) White solid (yield: 67%), mp 128.6–129.7 °C. 1H NMR (500 MHz, CDCl3) δ 7.73 (s, 1H), 7.68 (d, J = 7.8 Hz, 1H), 7.62 (d, J = 7.7 Hz, 1H), 7.51 (t, J = 7.7 Hz, 1H), 6.75 (s, 1H), 6.31 (d, J = 2.0 Hz, 1H), 5.72 (d, J = 1.7 Hz, 1H), 5.63 (d, J = 6.7 Hz, 1H), 5.08 (s, 2H), 3.32 (ddd, J = 4.9, 7.0, 9.5 Hz, 1H), 2.76 – 2.68 (m, 1H), 2.58 – 2.48 (m, 1H), 2.37 (s, 3H), 2.23 (s, 3H), 2.02 – 1.94 (m, 1H), 1.88 – 1.79 (m, 1H); 13C NMR (125 MHz, CDCl3) δ 170.2, 154.4, 139.8, 134.1, 131.5, 131.3, 130.9, 130.4, 129.4, 129.2, 126.1, 121.4, 118.7, 114.9, 112.7, 75.0, 69.3, 53.4, 39.4, 26.1, 23.6, 20.0, 11.6. HPLC purity: 97.5%, retention time = 26.31 min. HRMS (ESI): m/z calcd for C23H21NO3Na [M+Na]+: 382.1414, found 382.1418. (3aS,9bR)-6,9-Dimethyl-3-methylene-8-((4-(trifluoromethoxy)benzyl)oxy)-3a,4,5,9btetrahydronaphtho[1,2-b]furan-2(3H)-one (6i) White solid (yield: 85%), mp 125.3–125.9 °C. 1H NMR (500 MHz, CDCl3) δ 7.47 (d, J = 8.6 Hz, 2H), 7.24 (d, J = 8.3 Hz, 2H), 6.78 (s, 1H), 6.30 (d, J = 2.0 Hz, 1H), 5.71 (d, J = 1.7 Hz, 1H), 5.62 (d, J = 6.7 Hz, 1H), 5.05 (s, 2H), 3.31 (ddd, J = 4.9, 6.9, 9.5 Hz, 1H), 2.71 (ddd, J = 4.6, 6.0, 16.4 Hz, 1H), 2.52 (ddd, J = 4.5, 9.5, 16.4 Hz, 1H), 2.36 (s, 3H), 2.23 (s, 3H), 2.00 – 1.93 (m, 1H), 1.83 (ddd, J = 4.3, 9.2, 13.7 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 170.2, 154.7, 148.7, 139.9, 136.1, 134.0, 130.7, 128.9, 128.4, 126.1, 121.4, 121.0, 115.0, 75.0, 69.6, 60.4, 39.4, 26.1, 23.6, 21.0, 19.9, 14.2, 11.5. HPLC purity: 99.2%, retention time = 25.59 min. HRMS (ESI): m/z calcd for C23H21F3O4Na [M+Na]+: 441.1284, found 441.1293. (3aS,5aS,8S,9R,9bS)-5a,9-Dimethyl-3-methylene-2-oxododecahydronaphtho[1,2-b]fu


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ran-8-yl acetate (7a) White solid (yield: 70%), mp 97.6–98.3 °C. 1H NMR (500 MHz, CDCl3) δ 6.05 (d, J = 3.2 Hz, 1H), 5.38 (d, J = 3.0 Hz, 1H), 4.83 (dt, J = 5.1, 12.0 Hz, 1H), 3.92 (t, J = 11.1 Hz, 1H), 2.61 – 2.54 (m, 1H), 2.47 (td, J = 3.3, 11.5 Hz, 1H), 2.04 (s, 3H), 1.78 (dd, J = 3.7, 12.2 Hz, 1H), 1.73 (dd, J = 4.2, 8.0 Hz, 1H), 1.59 – 1.48 (m, 3H), 1.36 (td, J = 3.6, 13.6 Hz, 1H), 1.32 – 1.24 (m, 3H), 1.04 (s, 3H), 0.98 (d, J = 7.4 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 170.3, 139.8, 116.7, 79.9, 74.9, 53.4, 50.8, 49.9, 42.8, 39.7, 35.9, 31.5, 22.5, 21.8, 21.2, 21.0, 9.4. HPLC purity: 97.6%, retention time = 17.06 min. HRMS (ESI): m/z calcd for C17H24O4Na [M+Na]+: 315.1567, found 315.1565. (3aS,5aS,8S,9R,9bS)-5a,9-Dimethyl-3-methylene-2-oxododecahydronaphtho[1,2-b]fu ran-8-yl propionate (7b) White solid (yield: 85%), mp 82.5–83.7 °C. 1H NMR (500 MHz, CDCl3) δ 6.06 (d, J = 3.2 Hz, 1H), 5.38 (d, J = 3.0 Hz, 1H), 4.85 (dt, J = 5.1, 11.9 Hz, 1H), 3.93 (t, J = 11.1 Hz, 1H), 2.61 – 2.43 (m, 2H), 2.32 (q, J = 7.6 Hz, 2H), 2.00 (dd, J = 2.9, 13.2 Hz, 1H), 1.78 (dd, J = 3.6, 12.3 Hz, 1H), 1.72 (dd, J = 4.6, 11.5 Hz, 1H), 1.60 (d, J = 3.5 Hz, 1H), 1.57 (dd, J = 3.5, 9.7 Hz, 1H), 1.52 – 1.48 (m, 1H), 1.37 (td, J = 3.7, 13.7 Hz, 2H), 1.32 – 1.27 (m, 2H), 1.14 (t, J = 7.6 Hz, 3H), 1.04 (s, 3H), 0.99 (d, J = 7.4 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 173.7, 170.7, 139.8, 116.7, 79.9, 74.6, 50.8, 49.9, 42.8, 39.7, 35.9, 31.5, 27.9, 22.5, 21.8, 21.0, 9.5, 9.2. HPLC purity: 97.6%, retention time = 20.00 min. HRMS (ESI): m/z calcd for C18H26O4Na [M+Na]+: 329.1723, found 329.1747. (3aS,5aS,8S,9R,9bS)-5a,9-Dimethyl-3-methylene-2-oxododecahydronaphtho[1,2-b]fu ran-8-yl propionate (7c) White solid (yield: 83%), mp 60.2–61.5 °C. 1H NMR (500 MHz, CDCl3) δ 6.02 (d, J =



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3.2 Hz, 1H), 5.34 (d, J = 3.1 Hz, 1H), 3.76 (t, J = 10.9 Hz, 1H), 3.73 – 3.69 (m, 1H), 2.54 (dd, J = 2.8, 11.2 Hz, 1H), 1.96 (dq, J = 2.6, 3.3, 9.4 Hz, 1H), 1.86 (t, J = 11.2 Hz, 1H), 1.75 – 1.66 (m, 3H), 1.63 – 1.60 (m, 1H), 1.57 (dt, J = 2.3, 8.7 Hz, 1H), 1.53 – 1.48 (m, 1H), 1.37 (td, J = 3.9, 13.4 Hz, 1H), 1.26 (d, J = 6.3 Hz, 1H), 1.08 (d, J = 6.7 Hz, 3H), 0.92 (s, 3H), 0.90 (s, 7H); 13C NMR (125 MHz, CDCl3) δ 171.2 (2C), 139.7, 116.1, 83.7, 72.9, 51.1, 47.7, 40.6, 36.9, 36.0, 35.7, 29.7, 25.9 (2C), 25.8, 21.6, 18.8, 18.1. HPLC purity: 95.8%, retention time = 22.37 min. HRMS (ESI): m/z calcd for C19H28O4Na [M+Na]+: 343.1880, found 343.1882. (3aS,5aS,8S,9R,9bS)-5a,9-Dimethyl-3-methylene-2-oxododecahydronaphtho[1,2-b]fu ran-8-yl pentanoate (7d) White solid (yield: 70%), mp 57.3–58.6 °C. 1H NMR (500 MHz, CDCl3) δ 6.05 (d, J = 3.2 Hz, 1H), 5.38 (d, J = 3.0 Hz, 1H), 4.87 – 4.80 (m, 1H), 2.61 – 2.44 (m, 2H), 2.29 (t, J = 7.5 Hz, 2H), 1.99 (dt, J = 2.9, 9.7 Hz, 1H), 1.77 (dd, J = 3.4, 12.6 Hz, 1H), 1.72 (dd, J = 4.5, 11.5 Hz, 1H), 1.69 – 1.64 (m, 2H), 1.59 (t, J = 7.7 Hz, 2H), 1.54 – 1.45 (m, 2H), 1.35 (dd, J = 5.7, 13.3 Hz, 3H), 1.27 (dd, J = 12.2, 16.0 Hz, 2H), 1.04 (s, 3H), 0.98 (d, J = 7.4 Hz, 3H), 0.91 (t, J = 7.4 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 173.0, 170.7, 139.8, 116.7, 79.9, 74.5, 50.8, 49.9, 42.8, 39.7, 35.9, 34.3, 31.5, 27.2, 22.5, 22.2, 21.8, 21.0, 13.7, 9.5. HPLC purity: 97.0%, retention time = 24.41 min. HRMS (ESI): m/z calcd for C20H30O4Na [M+Na]+: 357.2036, found 357.2041. (3aS,5aS,8S,9R,9bS)-5a,9-Dimethyl-3-methylene-2-oxododecahydronaphtho[1,2-b]fu ran-8-yl cyclopentanecarboxylate (7e) White solid (yield: 72%), mp 77.2–78.1 °C. 1H NMR (500 MHz, CDCl3) δ 6.05 (d, J = 3.2 Hz, 1H), 5.38 (d, J = 3.0 Hz, 1H), 4.83 (dt, J = 4.9, 12.0 Hz, 1H), 3.92 (t, J = 11.1 Hz,


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1H), 2.76 – 2.69 (m, 1H), 2.48 (tq, J = 3.2, 10.3 Hz, 1H), 1.99 (dq, J = 3.6, 13.2 Hz, 1H), 1.90 – 1.75 (m, 7H), 1.70 – 1.66 (m, 3H), 1.57 (tdd, J = 3.3, 6.6, 8.6 Hz, 4H), 1.49 (dt, J = 3.0, 13.5 Hz, 1H), 1.36 (td, J = 3.5, 13.6 Hz, 1H), 1.29 (dd, J = 3.7, 13.2 Hz, 1H), 1.04 (d, J = 3.5 Hz, 3H), 0.98 (d, J = 7.4 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 175.9, 170.7, 139.8, 116.7, 79.9, 74.4, 50.8, 49.9, 44.1, 42.8, 39.7, 35.9, 31.5, 30.2, 29.8, 25.8, 25.7, 22.5, 21.8, 21.0, 9.5. HPLC purity: 95.2%, retention time = 25.06 min. HRMS (ESI): m/z calcd for C21H30O4Na [M+Na]+: 369.2036, found 369.2033. (3aS,5aS,9R,9bS)-5a,9-Dimethyl-3-methylene-2-oxododecahydronaphtho[1,2-b]fura n-8-yl cyclohexanecarboxylate (7f) White solid (yield: 83%), mp 80.8–81.2 °C. 1H NMR (500 MHz, CDCl3) δ 6.05 (d, J = 3.2 Hz, 1H), 5.38 (d, J = 3.0 Hz, 1H), 4.83 (dt, J = 5.0, 11.9 Hz, 1H), 3.92 (t, J = 11.1 Hz, 1H), 2.61 – 2.43 (m, 2H), 2.28 (tt, J = 3.7, 11.2 Hz, 1H), 1.99 (dd, J = 2.9, 13.2 Hz, 1H), 1.88 (dd, J = 6.4, 9.8 Hz, 2H), 1.77 (dd, J = 3.7, 13.5 Hz, 1H), 1.75 – 1.71 (m, 2H), 1.69 – 1.59 (m, 3H), 1.60 – 1.52 (m, 2H), 1.52 – 1.46 (m, 1H), 1.43 (dd, J = 3.5, 7.7 Hz, 2H), 1.37 (dd, J = 3.6, 13.6 Hz, 1H), 1.31 – 1.23 (m, 4H), 1.04 (s, 3H), 0.98 (d, J = 7.4 Hz, 3H);

13

C NMR (125 MHz, CDCl3) δ 175.2, 170.7, 139.8, 116.7, 79.9, 74.2, 50.8, 49.9,

43.4, 42.8, 39.7, 35.9, 31.5, 30.9, 29.2, 28.9, 25.7, 25.4, 25.3, 22.5, 21.8, 21.0, 9.5. HPLC purity: 97.2%, retention time = 26.85 min. HRMS (ESI): m/z calcd for C22H32O4Na [M+Na]+: 383.2193, found 383.2198. (5aS,8S,9R)-5a,9-Dimethyl-3-methylene-2-oxododecahydronaphtho[1,2-b]furan-8-yl 3-iodobenzoate (8a) White solid (yield: 83%), mp 148.5–150.1 °C. 1H NMR (500 MHz, CDCl3) δ 8.34 (t, J = 1.4 Hz, 1H), 8.02 – 7.96 (m, 1H), 7.96 – 7.83 (m, 1H), 7.18 (t, J = 7.8 Hz, 1H), 6.06 (d, J



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= 3.2 Hz, 1H), 5.39 (d, J = 3.0 Hz, 1H), 5.08 (dt, J = 5.1, 11.7 Hz, 1H), 3.96 (t, J = 11.1 Hz, 1H), 2.78 – 2.66 (m, 1H), 2.50 (td, J = 3.2, 11.4 Hz, 1H), 2.07 – 1.98 (m, 1H), 1.91 (td, J = 3.5, 12.8 Hz, 1H), 1.80 (ddd, J = 4.1, 9.9, 15.9 Hz, 2H), 1.61 (dd, J = 3.1, 13.2 Hz, 2H), 1.53 (dt, J = 2.8, 13.5 Hz, 1H), 1.44 (td, J = 3.4, 13.6 Hz, 1H), 1.31 (td, J = 3.7, 13.1 Hz, 1H), 1.09 (d, J = 7.3 Hz, 6H);

13

C NMR (125 MHz, CDCl3) δ 170.6, 163.8, 141.7,

139.7, 138.4, 132.5, 130.0, 128.7, 116.8, 93.8, 79.8, 75.9, 50.8, 49.9, 42.8, 39.7, 35.9, 31.7, 22.6, 21.8, 21.0, 9.7. HPLC purity: 96.1%, retention time = 27.67 min. HRMS (ESI): m/z calcd for C22H25IO4Na [M+Na]+: 503.0690, found 503.0681. (3aS,5aS,8S,9R,9bS)-5a,9-Dimethyl-3-methylene-2-oxododecahydronaphtho[1,2-b]fu ran-8-yl 4-iodobenzoate (8b) White solid (yield: 85%), mp 148.7–149.7 °C. 1H NMR (500 MHz, CDCl3) δ 7.82 – 7.77 (m, 2H), 7.75 – 7.69 (m, 2H), 6.07 (d, J = 3.2 Hz, 1H), 5.39 (d, J = 3.0 Hz, 1H), 5.08 (dt, J = 5.0, 11.9 Hz, 1H), 3.96 (t, J = 11.1 Hz, 1H), 2.74 – 2.65 (m, 1H), 2.51 (td, J = 3.3, 11.4 Hz, 1H), 2.02 (dt, J = 3.2, 13.3 Hz, 1H), 1.97 – 1.87 (m, 1H), 1.83 (dd, J = 4.2, 8.8 Hz, 1H), 1.79 (dd, J = 4.5, 11.5 Hz, 1H), 1.62 (d, J = 3.4 Hz, 1H), 1.60 (dd, J = 3.5, 6.5 Hz, 1H), 1.53 (dt, J = 3.0, 13.5 Hz, 1H), 1.45 (dd, J = 3.7, 13.6 Hz, 1H), 1.35 – 1.28 (m, 1H), 1.11 – 1.08 (m, 6H); 13C NMR (125 MHz, CDCl3) δ 170.6, 165.2, 139.7, 137.7 (2C), 131.0, 130.1 (2C), 116.8, 100.6, 79.8, 75.7, 50.8, 49.9, 42.8, 39.7, 35.9, 31.7, 22.6, 21.8, 21.0, 9.7. HPLC purity: 99.8%, retention time = 27.83 min. HRMS (ESI): m/z calcd for C22H25IO4Na [M+Na]+: 503.0690, found 503.0691. (3aS,5aS,8S,9R,9bS)-5a,9-Dimethyl-3-methylene-2-oxododecahydronaphtho[1,2-b]fu ran-8-yl 3-fluorobenzoate (8c) White solid (yield: 81%), mp 120.2–121.3 °C. 1H NMR (500 MHz, CDCl3) δ 7.83 (dt, J


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= 1.2, 7.7 Hz, 1H), 7.70 (ddd, J = 1.5, 2.5, 9.3 Hz, 1H), 7.42 (td, J = 5.6, 8.0 Hz, 1H), 7.25 (tdd, J = 0.9, 2.7, 8.3 Hz, 1H), 6.07 (d, J = 3.2 Hz, 1H), 5.40 (d, J = 3.0 Hz, 1H), 5.09 (dt, J = 5.0, 11.9 Hz, 1H), 3.96 (t, J = 11.1 Hz, 1H), 2.75 – 2.68 (m, 1H), 2.51 (ddt, J = 3.3, 7.2, 11.4 Hz, 1H), 2.02 (dd, J = 2.8, 13.2 Hz, 1H), 1.93 (qd, J = 3.7, 13.3 Hz, 1H), 1.85 (dt, J = 2.2, 4.6 Hz, 1H), 1.80 (dd, J = 4.5, 11.5 Hz, 1H), 1.64 – 1.60 (m, 2H), 1.53 (dt, J = 3.0, 13.4 Hz, 1H), 1.45 (td, J = 3.6, 13.6 Hz, 1H), 1.32 (td, J = 3.7, 13.1 Hz, 1H), 1.13 – 1.08 (m, 6H);

13

C NMR (125 MHz, CDCl3) δ 170.6, 164.5, 163.5, 139.7, 130.0,

125.2, 120.0, 116.8, 116.5, 116.3, 79.8, 75.9, 50.8, 49.9, 42.8, 39.7, 35.9, 31.7, 22.5, 21.8, 21.0, 9.7. HPLC purity: 97.9%, retention time = 24.72 min. HRMS (ESI): m/z calcd for C22H25FO4Na [M+Na]+: 395.1629, found 395.1637. (3aS,5aS,8S,9R,9bS)-5a,9-Dimethyl-3-methylene-2-oxododecahydronaphtho[1,2-b]fu ran-8-yl 3-chlorobenzoate (8d) White solid (yield: 95%), mp 121.4–122.5 °C. 1H NMR (500 MHz, CDCl3) δ 8.00 – 7.98 (m, 1H), 7.91 (dt, J = 1.3, 7.8 Hz, 1H), 7.52 (ddd, J = 1.1, 2.1, 8.0 Hz, 1H), 7.38 (t, J = 7.9 Hz, 1H), 6.06 (d, J = 3.2 Hz, 1H), 5.39 (d, J = 3.0 Hz, 1H), 5.09 (dt, J = 5.0, 11.9 Hz, 1H), 3.96 (t, J = 11.1 Hz, 1H), 2.76 – 2.67 (m, 1H), 2.51 (tq, J = 3.2, 10.3 Hz, 1H), 2.01 (dq, J = 3.6, 13.1 Hz, 1H), 1.93 (qd, J = 3.7, 13.3 Hz, 1H), 1.84 (d, J = 3.7 Hz, 1H), 1.81 – 1.76 (m, 1H), 1.61 (dt, J = 3.0, 13.5 Hz, 2H), 1.53 (dt, J = 3.0, 13.5 Hz, 1H), 1.44 (td, J = 3.5, 13.6 Hz, 1H), 1.31 (td, J = 3.8, 13.1 Hz, 1H), 1.12 – 1.08 (m, 6H); 13C NMR (125 MHz, CDCl3) δ 170.6, 164.4, 139.7, 134.5, 132.9, 132.4, 129.7, 129.6, 127.7, 116.8, 79.8, 75.9, 50.8, 49.9, 42.8, 39.7, 35.9, 31.7, 22.6, 21.8, 21.0, 9.7. HPLC purity: 96.1%, retention time = 26.76 min. HRMS (ESI): m/z calcd for C22H25ClO4Na [M+Na]+: 411.1334, found 411.1338.



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(3aS,5aS,8S,9R,9bS)-5a,9-Dimethyl-3-methylene-2-oxododecahydronaphtho[1,2-b]fu ran-8-yl 3-bromobenzoate (8e) White solid (yield: 84%), mp 130.5–130.8 °C. 1H NMR (500 MHz, CDCl3) δ 8.15 (t, J = 1.7 Hz, 1H), 7.96 (dt, J = 1.2, 7.8 Hz, 1H), 7.68 (ddd, J = 1.0, 2.0, 8.0 Hz, 1H), 7.32 (t, J = 7.9 Hz, 1H), 6.07 (d, J = 3.2 Hz, 1H), 5.40 (d, J = 3.0 Hz, 1H), 5.09 (dt, J = 5.0, 11.9 Hz, 1H), 3.96 (t, J = 11.1 Hz, 1H), 2.76 – 2.68 (m, 1H), 2.51 (td, J = 3.3, 11.4 Hz, 1H), 2.02 (dq, J = 3.5, 13.2 Hz, 1H), 1.94 (qd, J = 3.7, 13.3 Hz, 1H), 1.84 (q, J = 4.0, 4.5 Hz, 1H), 1.80 (dd, J = 4.5, 11.5 Hz, 1H), 1.63 (t, J = 3.0 Hz, 1H), 1.60 (d, J = 3.4 Hz, 1H), 1.53 (dt, J = 3.0, 13.4 Hz, 1H), 1.45 (td, J = 3.4, 13.6 Hz, 1H), 1.36 – 1.31 (m, 1H), 1.10 (d, J = 7.7 Hz, 6H);

13

C NMR (125 MHz, CDCl3) δ 170.6, 164.3, 139.7, 135.8, 132.6,

132.5, 129.9, 128.1, 122.4, 116.8, 79.8, 76.0, 50.8, 49.9, 42.8, 39.7, 35.9, 31.7, 22.6, 21.8, 21.0, 9.7. HPLC purity: 97.5%, retention time = 27.30 min. HRMS (ESI): m/z calcd for C22H25BrO4Na [M+Na]+: 455.0828, found 455.0826. (3aS,5aS,8S,9R,9bS)-5a,9-Dimethyl-3-methylene-2-oxododecahydronaphtho[1,2-b]fu ran-8-yl 3-(trifluoromethyl)benzoate (8f) White solid (yield: 79%), mp 110.3–111.3 °C. 1H NMR (500 MHz, CDCl3) δ 8.28 (s, 1H), 8.22 (d, J = 7.8 Hz, 1H), 7.82 (d, J = 7.8 Hz, 1H), 7.59 (t, J = 7.8 Hz, 1H), 6.08 (d, J = 3.2 Hz, 1H), 5.40 (d, J = 3.0 Hz, 1H), 5.13 (dt, J = 4.9, 12.0 Hz, 1H), 3.97 (t, J = 11.1 Hz, 1H), 2.78 – 2.71 (m, 1H), 2.52 (ddd, J = 3.3, 9.9, 14.3 Hz, 1H), 2.03 (ddd, J = 3.9, 6.4, 13.2 Hz, 1H), 1.95 (td, J = 3.7, 12.8, 13.3 Hz, 1H), 1.88 – 1.78 (m, 2H), 1.64 (ddd, J = 3.4, 7.6, 13.2 Hz, 2H), 1.54 (dt, J = 3.1, 13.4 Hz, 1H), 1.46 (td, J = 3.7, 13.7 Hz, 1H), 1.33 (td, J = 3.8, 13.4 Hz, 1H), 1.14 – 1.09 (m, 6H); 13C NMR (125 MHz, CDCl3) δ 170.6, 164.3, 139.7, 132.7, 131.5, 129.4, 129.4, 129.0, 126.4, 126.4, 116.8, 79.8, 76.2, 50.8, 49.9,


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42.8, 39.7, 35.9, 31.7, 22.6, 21.8, 21.0, 9.7. HPLC purity: 96.8%, retention time = 26.23 min. HRMS (ESI): m/z calcd for C23H25F3O4Na [M+Na]+: 440.2043, found 440.2047. (3aS,5aS,8S,9R,9bS)-5a,9-Dimethyl-3-methylene-2-oxododecahydronaphtho[1,2-b]fu ran-8-yl 3-(trifluoromethoxy)benzoate (8g) White solid (yield: 80%), mp 91.4–92.1 °C. 1H NMR (500 MHz, CDCl3) δ 7.97 (dt, J = 1.3, 7.7 Hz, 1H), 7.86 (s, 1H), 7.48 (td, J = 2.1, 7.9 Hz, 1H), 7.43 – 7.38 (m, 1H), 5.40 (d, J = 3.0 Hz, 1H), 5.10 (dt, J = 5.0, 11.9 Hz, 1H), 3.97 (t, J = 11.1 Hz, 1H), 2.71 (tt, J = 5.7, 11.2 Hz, 1H), 2.51 (tq, J = 3.2, 10.3 Hz, 1H), 2.06 – 2.00 (m, 1H), 1.99 – 1.89 (m, 1H), 1.88 – 1.77 (m, 2H), 1.65 – 1.59 (m, 2H), 1.53 (dt, J = 3.0, 13.4 Hz, 1H), 1.45 (td, J = 3.5, 13.6 Hz, 1H), 1.32 (td, J = 3.7, 13.1 Hz, 1H), 1.13 – 1.08 (m, 6H); 13C NMR (125 MHz, CDCl3) δ 170.6, 164.2, 149.2, 139.7, 132.7, 129.9 (2C), 127.8, 125.2, 122.0, 116.8, 79.8, 76.1, 50.8, 49.9, 42.8, 39.7, 35.9, 31.7, 22.5, 21.8, 21.0, 9.7. HPLC purity: 97.2%, retention time = 26.83 min. HRMS (ESI): m/z calcd for C23H25F3O5Na [M+Na]+: 461.1546, found 461.1544. (3aS,5aS,8S,9R,9bS)-5a,9-Dimethyl-3-methylene-2-oxododecahydronaphtho[1,2-b]fu ran-8-yl benzoate (8h) White solid (yield: 92%), mp 112.0–113.9 °C. 1H NMR (500 MHz, CDCl3) δ 8.06 – 8.02 (m, 2H), 7.55 (d, J = 7.4 Hz, 1H), 7.47 – 7.41 (m, 2H), 6.07 (d, J = 3.2 Hz, 1H), 5.40 (d, J = 3.0 Hz, 1H), 5.10 (dt, J = 5.1, 11.8 Hz, 1H), 3.97 (t, J = 11.1 Hz, 1H), 2.77 – 2.68 (m, 1H), 2.52 (ddt, J = 3.3, 7.1, 11.4 Hz, 1H), 2.02 (ddd, J = 3.3, 6.1, 13.2 Hz, 1H), 1.97 – 1.88 (m, 1H), 1.87 – 1.78 (m, 2H), 1.61 (dq, J = 3.5, 10.0 Hz, 2H), 1.53 (dt, J = 3.0, 13.5 Hz, 1H), 1.45 (td, J = 3.7, 13.6 Hz, 1H), 1.32 (td, J = 3.8, 13.2 Hz, 1H), 1.14 – 1.08 (m, 6H); 13C NMR (125 MHz, CDCl3) δ 170.6, 165.6, 139.8, 132.8, 130.6, 129.5 (2C), 128.3



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(2C), 116.8, 79.9, 75.4, 50.8, 50.0, 42.8, 39.8, 36.0, 31.8, 22.6, 21.8, 21.0, 9.7. HPLC purity: 98.6%, retention time = 24.13 min. HRMS (ESI): m/z calcd for C22H26O4Na [M+Na]+: 377.1723, found 377.1721. (3aS,5aS,8S,9R,9bS)-5a,9-Dimethyl-3-methylene-2-oxododecahydronaphtho[1,2-b]fu ran-8-yl 3-methylbenzoate (8i) White solid (yield: 38%), mp 170.2–171.5 °C. 1H NMR (500 MHz, CDCl3) δ 7.86 – 7.81 (m, 2H), 7.38 – 7.29 (m, 2H), 6.07 (d, J = 3.2 Hz, 1H), 5.40 (d, J = 3.0 Hz, 1H), 5.09 (dt, J = 5.0, 11.8 Hz, 1H), 3.97 (t, J = 11.1 Hz, 1H), 2.77 – 2.65 (m, 1H), 2.51 (ddt, J = 3.3, 7.1, 11.4 Hz, 1H), 2.41 (s, 3H), 2.05 – 1.99 (m, 1H), 1.98 – 1.89 (m, 1H), 1.84 (dd, J = 4.3, 8.9 Hz, 1H), 1.80 (dd, J = 4.5, 11.5 Hz, 1H), 1.61 (dq, J = 3.5, 9.6 Hz, 2H), 1.53 (dt, J = 3.1, 13.4 Hz, 1H), 1.45 (td, J = 3.6, 13.6 Hz, 1H), 1.32 (td, J = 3.8, 13.1 Hz, 1H), 1.14 – 1.08 (m, 6H); 13C NMR (125 MHz, CDCl3) δ 170.7, 165.8, 139.8, 138.1, 133.6, 130.6, 130.1, 128.2, 126.7, 116.7, 79.9, 75.3, 50.8, 50.0, 42.8, 39.8, 36.0, 31.8, 22.6, 21.8, 21.3, 21.0, 9.7. HPLC purity: 98.2%, retention time = 26.00 min. HRMS (ESI): m/z calcd for C23H28O4Na [M+Na]+: 391.1880, found 391.1889. (3aS,5aS,8S,9R,9bS)-5a,9-Dimethyl-3-methylene-2-oxododecahydronaphtho[1,2-b]fu ran-8-yl 3-methoxybenzoate (8j) White solid (yield: 68%), mp 110.8–111.5 °C. 1H NMR (500 MHz, CDCl3) δ 7.63 (dt, J = 1.2, 7.7 Hz, 1H), 7.56 (dd, J = 1.5, 2.5 Hz, 1H), 7.34 (t, J = 7.9 Hz, 1H), 7.10 (ddd, J = 0.9, 2.7, 8.2 Hz, 1H), 6.07 (d, J = 3.2 Hz, 1H), 5.40 (d, J = 3.0 Hz, 1H), 5.09 (dt, J = 5.1, 11.8 Hz, 1H), 3.96 (t, J = 11.1 Hz, 1H), 3.85 (s, 3H), 2.71 (d, J = 7.3 Hz, 1H), 2.51 (d, J = 2.9 Hz, 1H), 2.02 (dd, J = 2.8, 13.2 Hz, 1H), 1.97 – 1.87 (m, 1H), 1.87 – 1.82 (m, 1H), 1.80 (dd, J = 4.5, 11.5 Hz, 1H), 1.61 (dq, J = 3.0, 3.6, 13.4 Hz, 2H), 1.53 (dt, J = 3.0, 13.5


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Hz, 1H), 1.46 (dd, J = 3.8, 13.6 Hz, 1H), 1.36 – 1.28 (m, 1H), 1.12 – 1.08 (m, 6H); 13C NMR (125 MHz, CDCl3) δ 170.7, 165.5, 159.5, 139.8, 132.0, 129.3, 121.9, 119.1, 116.8, 114.2, 79.9, 75.5, 55.4, 50.8, 50.0, 42.8, 39.7, 35.9, 31.8, 22.6, 21.8, 21.0, 9.7. HPLC purity: 96.2%, retention time = 24.49 min. HRMS (ESI): m/z calcd for C23H28O5Na [M+Na]+: 407.1829, found 407.1839. (5aS,8S,9R)-5a,9-Dimethyl-3-methylene-2-oxododecahydronaphtho[1,2-b]furan-8-yl furan-2-carboxylate (9a) White solid (yield: 89%), mp 136.5–137.8 °C. 1H NMR (500 MHz, CDCl3) δ 7.57 (dd, J = 0.8, 1.7 Hz, 1H), 7.15 (dd, J = 0.8, 3.5 Hz, 1H), 6.50 (dd, J = 1.7, 3.5 Hz, 1H), 6.06 (d, J = 3.2 Hz, 1H), 5.39 (d, J = 3.0 Hz, 1H), 5.07 (dt, J = 4.9, 12.0 Hz, 1H), 3.95 (t, J = 11.1 Hz, 1H), 2.73 – 2.62 (m, 1H), 2.50 (ddt, J = 3.3, 7.1, 11.4 Hz, 1H), 2.01 (dq, J = 3.7, 13.2 Hz, 1H), 1.97 – 1.86 (m, 1H), 1.84 – 1.73 (m, 2H), 1.65 (dd, J = 3.9, 10.6 Hz, 1H), 1.62 – 1.58 (m, 1H), 1.52 (dt, J = 3.0, 13.4 Hz, 1H), 1.42 (td, J = 3.6, 13.6 Hz, 1H), 1.30 (td, J = 3.7, 13.1 Hz, 1H), 1.09 – 1.06 (m, 6H);

13

C NMR (125 MHz, CDCl3) δ 170.6, 157.9,

146.2, 144.9, 139.7, 117.7, 116.8, 111.7, 79.8, 75.5, 50.8, 49.9, 42.8, 39.7, 35.9, 31.8, 22.6, 21.8, 21.0, 9.5. HPLC purity: 99.6%, retention time = 19.10 min. HRMS (ESI): m/z calcd for C20H24O5Na [M+Na]+: 367.1516, found 367.1517. (5aS,8S,9R)-5a,9-Dimethyl-3-methylene-2-oxododecahydronaphtho[1,2-b]furan-8-yl thiophene-2-carboxylate (9b) White solid (yield: 95%), mp 130.3–131.2 °C. 1H NMR (500 MHz, CDCl3) δ 7.79 (dd, J = 1.3, 3.7 Hz, 1H), 7.54 (dd, J = 1.3, 5.0 Hz, 1H), 7.09 (dd, J = 3.7, 5.0 Hz, 1H), 6.06 (d, J = 3.2 Hz, 1H), 5.39 (d, J = 3.0 Hz, 1H), 5.05 (dt, J = 5.2, 11.8 Hz, 1H), 3.96 (t, J = 11.1 Hz, 1H), 2.73 – 2.66 (m, 1H), 2.50 (ddt, J = 3.3, 7.1, 11.4 Hz, 1H), 2.01 (dq, J = 3.7, 13.2



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Hz, 1H), 1.94 – 1.88 (m, 1H), 1.85 (ddt, J = 3.7, 8.4, 13.0 Hz, 1H), 1.78 (dd, J = 4.5, 11.5 Hz, 1H), 1.63 – 1.56 (m, 2H), 1.52 (dt, J = 3.1, 13.4 Hz, 1H), 1.43 (td, J = 3.8, 13.5 Hz, 1H), 1.31 (td, J = 3.6, 13.1 Hz, 1H), 1.11 – 1.07 (m, 6H); 13C NMR (125 MHz, CDCl3) δ 170.6, 161.3, 139.7, 134.2, 133.2, 132.2, 127.7, 116.8, 79.8, 75.7, 50.8, 49.9, 42.8, 39.7, 35.9, 31.8, 22.6, 21.8, 21.0, 9.6. HPLC purity: 99.6%, retention time = 22.28 min. HRMS (ESI): m/z calcd for C20H24SO4Na [M+Na]+: 383.1288, found 383.1298. (3aS,5aS,8S,9R,9bS)-5a,9-Dimethyl-3-methylene-2-oxododecahydronaphtho[1,2-b]fu ran-8-yl nicotinate (9c) White solid (yield: 84%), mp 134.3–134.7 °C. 1H NMR (500 MHz, CDCl3) δ 9.22 (d, J = 1.5 Hz, 1H), 8.77 (dd, J = 1.7, 4.8 Hz, 1H), 8.29 (dt, J = 1.9, 7.9 Hz, 1H), 7.39 (ddd, J = 0.8, 4.9, 7.9 Hz, 1H), 6.07 (d, J = 3.2 Hz, 1H), 5.40 (d, J = 3.0 Hz, 1H), 5.13 (d, J = 12.1 Hz, 1H), 3.96 (t, J = 11.1 Hz, 1H), 2.76 – 2.69 (m, 1H), 2.51 (ddd, J = 3.3, 10.0, 14.3 Hz, 1H), 2.02 (dq, J = 3.7, 13.2 Hz, 1H), 1.95 (dd, J = 3.7, 12.2 Hz, 1H), 1.88 – 1.83 (m, 1H), 1.80 (dd, J = 4.5, 11.5 Hz, 1H), 1.62 (dd, J = 3.1, 12.9 Hz, 2H), 1.54 (dt, J = 3.0, 13.3 Hz, 1H), 1.45 (td, J = 3.5, 13.5 Hz, 1H), 1.32 (td, J = 3.8, 13.1 Hz, 1H), 1.13 – 1.08 (m, 6H); 13

C NMR (125 MHz, CDCl3) δ 170.6, 164.4, 153.3, 150.8, 139.7, 137.1, 126.4, 123.3,

116.9, 79.8, 76.1, 50.8, 49.9, 42.7, 39.7, 35.9, 31.7, 22.6, 21.8, 21.0, 9.7. HPLC purity: 95.1%, retention time = 17.90 min. HRMS (ESI): m/z calcd for C21H25NO4Na [M+Na]+: 356.1856, found 356.1859. (3aS,5aS,8S,9R,9bS)-5a,9-Dimethyl-3-methylene-2-oxododecahydronaphtho[1,2-b]fu ran-8-yl isonicotinate (9d) White solid (yield: 87%), mp 142.1–142.9 °C. 1H NMR (500 MHz, CDCl3) δ 8.88 – 8.53 (m, 2H), 7.92 – 7.72 (m, 2H), 6.07 (d, J = 3.2 Hz, 1H), 5.40 (d, J = 3.0 Hz, 1H), 3.96 (t, J


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= 11.1 Hz, 1H), 2.75 – 2.69 (m, 1H), 2.51 (ddt, J = 3.3, 7.1, 11.4 Hz, 1H), 2.02 (dq, J = 3.6, 13.2 Hz, 1H), 1.93 (td, J = 3.7, 12.7, 13.3 Hz, 1H), 1.87 – 1.75 (m, 3H), 1.67 – 1.58 (m, 2H), 1.59 – 1.50 (m, 1H), 1.44 (td, J = 3.5, 13.5 Hz, 1H), 1.32 (td, J = 3.8, 13.1 Hz, 1H), 1.12 – 1.08 (m, 6H); 13C NMR (125 MHz, CDCl3) δ 170.2, 164.2, 150.6 (2C), 139.6, 137.6, 122.8 (2C), 116.9, 79.7, 76.5, 50.8, 49.9, 42.7, 39.6, 35.9, 31.7, 22.5, 21.8, 21.0, 9.6. HPLC purity: 98.7%, retention time = 18.44 min. HRMS (ESI): m/z calcd for C21H25NO4Na [M+Na]+: 356.1856, found 356.1856. (3aS,5aS,8S,9R,9bS)-5a,9-Dimethyl-3-methylene-2-oxododecahydronaphtho[1,2-b]fu ran-8-yl (E)-3-(furan-2-yl)acrylate (9e) White solid (yield: 70%), mp 130.7–131.5 °C. 1H NMR (500 MHz, CDCl3) δ 7.47 (d, J = 1.4 Hz, 1H), 7.41 (d, J = 15.7 Hz, 1H), 6.61 (d, J = 3.3 Hz, 1H), 6.46 (dd, J = 1.8, 3.4 Hz, 1H), 6.31 (d, J = 15.7 Hz, 1H), 6.06 (d, J = 3.2 Hz, 1H), 5.39 (d, J = 3.0 Hz, 1H), 4.95 (dt, J = 5.0, 11.9 Hz, 1H), 3.94 (t, J = 11.1 Hz, 1H), 2.68 – 2.60 (m, 1H), 2.49 (td, J = 3.3, 11.4 Hz, 1H), 2.00 (dt, J = 2.9, 9.6 Hz, 1H), 1.85 (qd, J = 3.7, 13.3 Hz, 1H), 1.76 (dd, J = 4.5, 11.5 Hz, 2H), 1.63 (s, 1H), 1.60 – 1.54 (m, 2H), 1.51 (dt, J = 3.0, 13.4 Hz, 1H), 1.40 (td, J = 3.6, 13.5 Hz, 1H), 1.31 (dd, J = 4.0, 13.3 Hz, 1H), 1.27 – 1.24 (m, 1H), 1.06 (s, 3H), 1.04 (d, J = 7.4 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 170.6, 166.1, 150.9, 144.7, 139.8, 131.0, 116.7, 116.1, 114.6, 112.2, 79.9, 74.9, 50.8, 49.9, 42.8, 39.8, 35.9, 31.6, 22.6, 21.8, 21.0, 9.5. HPLC purity: 95.2%, retention time = 23.80 min. HRMS (ESI): m/z calcd for C22H26O5Na [M+Na]+: 393.1672, found 393.1679. (3aS,5aS,8S,9R,9bS)-5a,9-Dimethyl-3-methylene-2-oxododecahydronaphtho[1,2-b]fu ran-8-yl (E)-3-(thiophen-2-yl)acrylate (9f) White solid (yield: 77%), mp 184.7–185.2 °C. 1H NMR (500 MHz, CDCl3) δ 7.76 (d, J =



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15.7 Hz, 1H), 7.37 (d, J = 5.1 Hz, 1H), 7.27 – 7.24 (m, 1H), 7.05 (dd, J = 3.6, 5.1 Hz, 1H), 6.23 (d, J = 15.7 Hz, 1H), 6.06 (d, J = 3.2 Hz, 1H), 5.39 (d, J = 3.0 Hz, 1H), 4.95 (dt, J = 5.0, 11.9 Hz, 1H), 3.94 (t, J = 11.1 Hz, 1H), 2.68 – 2.61 (m, 1H), 2.49 (ddt, J = 3.3, 7.2, 11.3 Hz, 1H), 2.00 (dq, J = 3.6, 13.2 Hz, 1H), 1.85 (qd, J = 3.7, 13.2 Hz, 1H), 1.78 – 1.73 (m, 2H), 1.58 (ddd, J = 3.4, 8.1, 13.6 Hz, 2H), 1.51 (dt, J = 3.0, 13.4 Hz, 1H), 1.41 (td, J = 3.7, 13.6 Hz, 1H), 1.29 (td, J = 3.8, 13.3 Hz, 1H), 1.08 – 1.03 (m, 6H); 13C NMR (125 MHz, CDCl3) δ 170.6, 165.6, 139.8, 139.6, 137.0, 130.8, 128.4, 128.0, 117.2, 116.7, 79.9, 75.0, 50.8, 49.9, 42.8, 39.8, 35.9, 31.6, 22.6, 21.8, 21.0, 9.6. HPLC purity: 96.5%, retention time = 24.84 min. HRMS (ESI): m/z calcd for C22H26SO4Na [M+Na]+: 409.1444, found 409.1447. (3aS,5aS,8S,9R,9bS)-5a,9-Dimethyl-3-methylene-2-oxododecahydronaphtho[1,2-b]fu ran-8-yl (E)-3-(pyridin-3-yl)acrylate (9g) White solid (yield: 53%), mp 150.5–151.2 °C. 1H NMR (500 MHz, CDCl3) δ 8.75 (s, 1H), 8.63 – 8.57 (m, 1H), 7.85 (dt, J = 1.8, 8.0 Hz, 1H), 7.65 (d, J = 16.1 Hz, 1H), 7.33 (dd, J = 4.8, 7.9 Hz, 1H), 6.50 (d, J = 16.1 Hz, 1H), 6.06 (d, J = 3.2 Hz, 1H), 5.39 (d, J = 3.0 Hz, 1H), 4.98 (dt, J = 5.0, 11.9 Hz, 1H), 3.95 (t, J = 11.1 Hz, 1H), 2.70 – 2.59 (m, 1H), 2.54 – 2.45 (m, 1H), 2.01 (dd, J = 2.9, 13.3 Hz, 1H), 1.87 (qd, J = 3.7, 13.2 Hz, 1H), 1.77 (dd, J = 4.5, 11.5 Hz, 2H), 1.62 – 1.56 (m, 2H), 1.52 (dt, J = 3.1, 13.5 Hz, 1H), 1.42 (td, J = 3.6, 13.6 Hz, 1H), 1.30 (td, J = 3.7, 13.2 Hz, 1H), 1.09 – 1.03 (m, 6H); 13C NMR (125 MHz, CDCl3) δ 170.7, 165.4, 150.8, 149.6, 140.8, 139.7, 134.3, 130.3, 123.8, 120.7, 116.8, 79.9, 75.4, 50.8, 49.9, 42.8, 39.7, 35.9, 31.6, 22.6, 21.8, 21.0, 9.6. HPLC purity: 96.3%, retention time = 23.57 min. HRMS (ESI): m/z calcd for C23H27NO4Na [M+Na]+: 382.2013, found 382.2012.


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Biology Cell lines and cell cultures Mouse brain microvascular endothelial cells (bEnd.3) and 293T cells were purchased from ATCC and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO) supplemented with 10% fetal bovine serum (FBS) (Sciencell) at 37 °C in a humidified atmosphere with 5% CO2. Synovial fibroblasts were isolated from synovial tissue specimens obtained from RA patients who had undergone total joint replacement surgery. Synovial fibroblasts were grown in DMEM supplemented with 10% FBS and 1% synoviocyte growth supplement (Sciencell). E2 ubiquitin-loading assays In total, 0.5 µM UbcH5c in assay buffer (25 mM Tris-HCl, 50 mM MgCl2, pH = 7.5) with different concentrations of compounds was incubated at room temperature for 15 min. Then, 2 mM ATP, 0.2 µM E1, and 10 µM Ub were added and incubated at 37 °C for 40 min. The reactions were terminated by the addition of the SDS-PAGE sample buffer without reducing agent and heating at 95 °C for 3 min. The samples were loaded on 12% SDS-PAGE and processed for immunoblot analysis with anti-UbcH5c antibody (Cell Signaling Technology) NF-κB luciferase assay The NF-κB luciferase reporter plasmid pNF-κB-luc (Beyotime) contains an ampicillin resistance gene to allow selection in E. coli. Transfected pNF-κB-luc into E. coli, and E. coli were grown in LB medium overnight at 250 rpm. Plasmids were extracted using plasmid DNA purification kits (Invitrogen) according to the manufacturer's instructions. Briefly, 293T cells were seeded onto 96-well plates at 37 °C. When the cells reached 80



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to 90% confluence, pNF-κB-luc plasmids were transfected into cells using LipofectamineTM 2000 (Invitrogen). After 48 h, the cells were treated or untreated with compounds (5 µM) for 2 h before stimulation with 20 ng/mL of TNF-α for 8 h. Cells were lysed, and luciferase assay substrate was added (Promega). The luciferase activity was quantified on a Synergy 4 instrument (Bio-tek) using Gene5 software (Bio-tek) and reported as relative luminescence units. Western blotting bEnd.3 cells were cultured in 6-well plates for 36 h. Then, the cells were treated or untreated with different concentrations of compounds (2.5, 5, 10 µM) for 2 h followed by stimulation with 20 ng/mL TNF-α for 15 min. Subsequently, the cells were washed three times with ice-cold PBS, and total cell extracts were extracted in 80 µL lysis buffer containing a protease and phosphatase inhibitor cocktail (Biotool). Cellular proteins were obtained via centrifugation at 13,000 rpm. The protein concentrations were determined with a BCA protein assay kit (Pierce), and equal amounts of protein were loaded per well on a 10% SDS-PAGE. Separated proteins were transferred to pure PVDF membrane (Bio-Rad) and blocked with 5% slim milk for 1 h. The blots were incubated with rabbit anti-phospho-IκBα, anti-IκBα, anti-phospho-p65, anti-p65 and anti-GAPDH primary antibodies (Cell Signaling Technology) overnight at 4 °C. Membranes were washed in TBST solution three times followed by incubation with IRDy 800 CW goat anti-rabbit secondary antibody (LI-COR Biotechnology). Immunodetection of proteins was performed with an Odyssey infrared imaging system (LI-COR Biotechnology). Quantitative real-time PCR (qPCR) Total RNA from synovial fibroblasts was extracted using an RNA Miniprep kit (Biomiga)


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according to the manufacturer's instructions. In total, 1000 ng of total RNA was reverse transcribed using a PrimeScript RT reagent Kit (Takara). The primers used were designed and synthesized by Takara. The sequences of the primers are indicated in Table 1. qPCR amplification was performed on an StepOne Plus real-time PCR system (Applied Biosystems, Foster City, CA) using the SYBR Green Master Mix (Applied Biosystems). All experiments were performed in triplicate, and the relative levels of assayed mRNAs were calculated with the delta–delta CT method using GAPDH expression as an endogenous control. The values were normalized to non-treated control. BIAcore Experiments were performed on a BIAcore T200 instrument (Biacore AB, Uppsala, Sweden). Recombinant human UbcH5c dissolved in 10 mM sodium acetate buffer (pH = 5.0) was covalently immobilized in the dextran matrix of a CM5 sensor chip with the Amine Coupling Kit using a standard primary amine coupling procedure. Gradient concentrations of compounds were injected into the flow cells in running buffer (PBS, 5% DMSO) at a flow rate of 30 µL/min for 90 s of association phase followed by a 120 s dissociation phase and a 30 s regeneration phase. The surface of the sensor chip was regenerated via the injection of 10 µL of the regeneration buffer (5 mM NaOH). The data were analyzed using Biacore T200 Evaluation Software 2.0.1 and fitted to the standard 1:1 interaction model to obtain results. The dissociation constant (KD) was calculated using the global fitting of the kinetic data from gradient concentrations. This work is supported by Lili Zhu and Lina Quan who are from East China University of Science and Technology. In-gel fluorescence imaging assay



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HeLa cells were cultured in 10 cm dishes for 24 h. The growth media was removed and the cells were washed twice with ice-cold PBS. The cells were harvested and the pellet was resuspended in 80 µL of NP40 lysis buffer (50 mM HEPES, pH 7.4, 1% NP-40, 150 mM NaCl) with protease inhibitor cocktail (Beyotime). The lysate was incubated on ice for 20 min and fractionated by centrifugation at 12,000 rpm for 10 min at 4 °C. The protein concentrations were measured from each of the supernatant sample by BCA assay (Beyotime) and adjusted to 1 mg/mL. Lysates were incubated with various concentrations of the probe for 40 min at 37 °C. CuACC was performed at a final concentration of 25 µΜ Azide-fluor 488 (Click Chemistry Tools, Sigma-Aldrich), 1 mM Tris(2-carboxyethyl) phosphine (TCEP, TCI), 100 µΜ Tris[(1-benzyl-1H-1,2,3-triazol-4-yl) methyl] amine (TBTA, Sigma-Aldrich), and 1 mM CuSO4 (Sigma-Aldrich) in a total volume of 100 µL. Click reactions were set up in the dark for 1 h before termination by addition of 25 µL of 5 × Laemmli sample buffer (Bio-Rad) and boiled for 5 min. Then, 30 µL of the samples were loaded in a 10-wells gel and separated by a 15% SDS-PAGE before visualization on a G:BOX Imager (Syngene). Fluorescence images were displayed as gray scale, and the fluorescent band of Azide-fluor 488 was displayed as internal reference. After visualization, the proteins in gel were transferred to a PVDF membrane at 250 mA for 40 min. Then, the membrane was blocked in 5% nonfat milk TBST buffer for 30 min and incubated with antibody for UbcH5c (Cell Signaling Technology) overnight at 4°C. After washing with TBST buffer for three times, the membranes were incubated with IRDy 800 CW goat anti-rabbit secondary antibody for 1 h at room temperature. Fluorescence image was obtained with the Odessey Infrared Imaging System. Immunoprecipitation (IP)


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HeLa cells were cultured in 6-well plates for 24 h. Then, cells were treated or untreated with different concentrations of compounds (2.5, 5, 10 µM) for 2 h followed by stimulation with 1 µg/mL TNF-α for 5 min. Subsequently, the cells were harvested and lysed in lysis buffer containing protease inhibitor. For detection of the ubiquitinated NEMO, cell lysates were incubated at 90 °C for 15 min in 1% SDS lysis buffer to eliminate noncovalent binding. The lysate was diluted to 0.1% SDS with lysis buffer, and samples were incubated at 4 °C for 1 h with rotation. NEMO was immunoprecipitated using anti-NEMO antibody (Abcam) at 4 °C overnight with rotation followed by incubation with protein A/G-PLUS agarose beads (Santa) for 4 h at room temperature. Immunoprecipitates were resolved on 10% SDS-PAGE and processed for immunoblot analysis with anti-linear polyubiquitin antibody (Millipore). Determination of 6d binding site on UbcH5c by LC/MS/MS analysis Recombinant UbcH5c was incubated for 1 h at 37 °C with 6d, which was digested with trypsin directly. Positive control experiments were conducted in the same manner using synthetic peptide (IYHPNINSNGSICLDILR, purity ≥ 98%, peptide 73‒90 of UbcH5c) instead of UbcH5c without trypsin digestion. The two samples were subjected to liquid chromatography performed on a nano-Acquity UPLC system (Waters Corporation) connected to an LTQ Orbitrap XL mass spectrometer (Thermo Scientific) equipped with an online nanoelectrospray ion source (Michrom Bioresources). Briefly, 10 µL peptide solution was loaded onto the Captrap Peptide column (2 mm × 0.5 mm, Michrom Bioresources) at a 20 µL/min flow rate of solvent A (5% acetonitrile, 0.1% formic acid in water) for 5 min and then separated on a Magic C18AQ reverse-phase column (100 µM id × 15 cm, Michrom Bioresources) with a three-step linear gradient of solvent A and



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solvent B (90% acetonitrile, 0.1% formic acid in water). The following conditions were employed: starting from 5% B to 45% B for 40 min, increased to 80% B for 3 min, and then to 5% B for 2 min. The column was re-equilibrated at initial conditions for 15 min. The column flow rate was maintained at 500 nL/min, and the column temperature was maintained at 35 °C. The electrospray voltage of 1.8 kV versus the inlet of the mass spectrometer was used. An LTQ Orbitrap XL mass spectrometer was operated in the data-dependent mode to switch automatically between MS and MS/MS acquisition. The spectrums were recorded with Xcalibur (version 2.0.7) software. Covalent molecular docking After the derived structure of UbcH5c (PDB ID: 1X23) was prepared using the protein preparation wizard Maestro software package (Schrödinger Inc., version 9.0) and 6d was prepared using LigPrep module with default parameters, the terminal carbon atom of α-methylene moiety of 6d and the sulfur atom of Cys85 were specified as the ligand reactive group and the receptor bond, respectively. The covalent ligand docking of 6d on UbcH5c was performed with the conformation of the residues within 10 Å around Cys85 being sampled, while other residues were fixed. Finally, the top 20 docked poses of 6d remained for further visual investigation. This work is supported by Junsheng Zhu who is from East China University of Science and Technology. In vivo PK experimental Male Sprague-Dawley rats (about 200‒220 g) were used and randomly divided into two groups (n = 6 in each group). Rats were fasted overnight before dosing. Compound 6d was administered by intravenous injection at a dose of 1 mg/kg in saline mixture (DMSO: castor-oil: saline = 3:3:94). Blood each timepoints (0, 3, 8, 15, 30, 60, 90, 120, 150, 180,


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240 and 300 min) were collected into heparin tubes via the orbital artery. 6d was formulated as a suspension in 0.5% methlcellulose and administered orally by gavage at a dose of 5 mg/kg. Blood of each timepoints (0.083, 0.25, 0.5, 1, 2, 3, 5, 7, 9, 11, 22 and 24 h) were collected into heparin tubes via the orbital artery. Plasma Sample were analyzed via LC/MS/MS utilizing electrospray ionization (ESI) and MRM transition(s) specific to the analyte on a Waters Xevo™ UPLC-TQ-S (Waters Corporation, UAS), which was equipped with ACQUITY UPLC® BEH C18 (1.7 µm, 50 × 2.1 mm). All data were analyzed using UNIFI 1.7.1 (Waters Corporation, UAS). PK parameters were estimated by noncompartmental analysis using the DAS 2.0 software package (Wannan Medical College, Wuhu, China).54 Stability of 6d in vitro Microsome Metabolism. The incubation mixture, with a total volume of 200 µL, consisted of 100 mM potassium phosphate buffer (pH 7.4), an NADPH-generating system (4 mM MgCl2, 1 mM NADP+, 10 mM Glucose 6-phosphate, and 1 unit/mL glucose-6-phosphate dehydrogenase) and 0.5 mg/mL rat liver microsomes (BD Gentest Woburn, MA). In the experiment, 6d (25 mM in DMSO) was serially diluted to the required concentrations (final sample volume 500 µL, final 6d concentration 5 µM). After preincubation at 37 °C for 3 min, the reaction was initiated by addition of the NADPH-generating system and further incubated at 37 °C in a shaking water bath for 0, 15, 30, 45, 60 and 90 min. The reaction was terminated by the addition of ice-cold acetonitrile (100 µL). The mixture was kept on ice until it was centrifuged at 13000 rpm for 10 min at 4 °C. Aliquots of supernatants for each time point were stored at -20 °C until analysis.



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Plasma and whole blood stability. 6d (25 mM in DMSO) were incubated with rat plasma or whole blood (final sample volume 500 µL, final 6d concentration 5 µM) for 0, 15, 30, 45, 60, 90 min and 0, 5, 10, 30, 60, 90 min at 37 °C, respectively. The incubation was terminated by the addition of 100 µL ice-cold acetonitrile. The mixture was kept on ice until it was centrifuged at 13000 rpm for 10 min at 4 °C. Aliquots of supernatants for each time point were stored at -20 °C until analysis. Adjuvant-induced arthritis (AA) model AA was induced in rats as previously described.48 In brief, arthritis was induced by an intradermal injection of 0.1 mL of CFA into the base of the left hind paw. The day of CFA injection was designated day 0. To investigate the therapeutic effects of 6d, the CFA injection was administered following therapeutic protocols. The 2 groups (10 rats/group) were orally administered with 6d (5 and 20 mg/kg) daily starting on day 10 after CFA immunization. The rats in normal control and AA model groups were administered an equal volume of 0.3% sodium carboxymethylcellulose simultaneously with the 6d administration. To evaluate the severity of arthritis, the volume of left and right hind paw was measured with a volume meter (YLS-7B, Ji Nan, China). The arthritis index was assessed by 2 independent observers under blinded conditions using a scoring system of 0‒4, where 0 = no swelling, 1 = swelling of finger joints, 2 = mild swelling of ankle or wrist joints, 3 = swelling of the paw below the ankle, 4 = swelling of the entire paw, the including ankle. Each limb was graded with a maximum possible score of 16 per animal. ASSOCIATED CONTENT Supporting Information Single crystal X-ray diffraction information of compounds 12, 23 and 24. Determination


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of 6d binding site on UbcH5c by LC/MS/MS analysis. Drug concentration-time curve of 6d. Stability of compound 6d in vitro. Synthesis of probe 6d-1. The 1H NMR, 13C NMR and HRMS of representative compounds 1a, 1b, 5g, 6d, 8a, 8b and 6d-1. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * W.-D.Z. E-mail: [email protected]. Phone/Fax: +86-21-81871244. Q.-Y.S. E-mail: [email protected]. Phone/Fax: +86-21-20572000-2028. Z.-L.H. E-mail: [email protected]. Phone/Fax: +86-21-81871332. Author Contributions †

H.C., G.-Z.W. and S.G. contributed equally to this work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENT The work was supported by Professor of Chang Jiang Scholars Program, NSFC (81230090, 81520108030, 81573318, 81373301, 1302658), Shanghai Engineering Research Center for the Preparation of Bioactive Natural Products (16DZ2280200), the Scientific Foundation of Shanghai China (12401900801, 13401900103, 13401900101), National Major Project of China (2011ZX09307-002-03) and the National Key Technology R&D Program of China (2012BAI29B06), State Key Laboratory of Innovative Natural Medicine and TCM Injections.



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ABBREVIATIONS AA, Adjuvant arthritis; CCK-8, Cell Counting Kit-8; eq., Equivalent; IKK, IκB kinase; IL-1, Interleukin-1; IκB-α, Inhibitor of NF-κB; LUBAC, Linear ubiquitin assembly complex; MCP, Monocyte chemoattractant protein; MMP, Matrix matalloproteinase; mRNA, Messenger RNA; NEMO, NF-κB Essential Modulator; NF-κB, Nuclear factor-κB; NMR, Nuclear magnetic resonance; PCR, Polymerase chain reaction; RA, Rheumatoid arthritis; TNF-α, Tumor necrosis factor-α; Ub, Ubiquitin.

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guaiane-type sesquiterpene lactone derivatives with respect to inhibiting NO production in lipopolysaccharide-induced RAW 264.7 macrophages. Eur. J. Med. Chem. 2014, 83, 307‒316. (39). Miyashita, M.; Suzuki, T.; Yoshikoshi, A. Synthesis of Dehydroisoerivanin, Isoerivanin,

Ludovicin

C,

and

1α,3α-Dihydroxyarbusculin

B

utilizing

the

organoselenium-mediated reduction of epoxy ketone. Chem. Lett. 1987, 16, 2387‒2390. (40). García-Granados, A.; Parra, A.; Rivas, F.; Segovia, A. J. Seven-membered cyclic sulfite eudesmane derivatives: partial synthesis, structural determination, and enzymatic resolution. J. Org. Chem. 2007, 72, 643‒646. (41). Chen Y. C.; Backus K. M.; Merkulova M.; Yang C.; Brown D.; Cravatt B. F.; Zhang C. Covalent modulators of the vacuolar ATPase. J. Am. Chem. Soc. 2017, 139, 639‒642. (42). Zhang, S.; Won, Y. K.; Ong, C. N.; Shen, H. M. Anti-cancer potential of sesquiterpene lactones: bioactivity and molecular mechanisms. Curr. Med. Chem. Anti-Cancer Agents 2005, 5, 239‒249. (43). Merfort, I. Perspectives on sesquiterpene lactones in inflammation and cancer. Curr. Drug Targets 2011, 12, 1560‒1573. (44). Bartok, B.; Firestein, G. S. Fibroblast-like synoviocytes: key effector cells in rheumatoid arthritis. Immunol. Rev. 2010, 233, 233‒255. (45). Mamehara, A.; Sugimoto, T.; Sugiyama, D.; Morinobu, S.; Tsuji, G.; Kawano, S.; Morinobu, A.; Kumagai, S. Serum matrix metalloproteinase-3 as predictor of joint destruction in rheumatoid arthritis, treated with non-biological disease modifying


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anti-rheumatic drugs. Kobe J. Med. Sci. 2010, 56, E98‒107. (46). Szekanecz, Z.; Koch, A. E.; Kunkel, S. L.; Strieter, R. M. Cytokines in rheumatoid arthritis. Potential targets for pharmacological intervention. Drugs Aging 1998, 12, 377‒ 390. (47). Klimiuk, P. A.; Sierakowski, S.; Latosiewicz, R.; Skowronski, J.; Cylwik, J. P.; Cylwik, B.; Chwiecko, J. Histological patterns of synovitis and serum chemokines in patients with rheumatoid arthritis. J. Rheumatol. 2005, 32, 1666‒1672. (48). Kim, K. S.; Choi, Y. H.; Kim, K. H.; Lee, Y. C.; Kim, C. H.; Moon, S. H.; Kang, S. G.; Park, Y. G. Protective and anti-arthritic effects of deer antler aqua-acupuncture (DAA), inhibiting dihydroorotate dehydrogenase, on phosphate ions-mediated chondrocyte apoptosis and rat collagen-induced arthritis. Int. Immunopharmacol. 2004, 4, 963‒973. (49). Silva, S. R. D.; Paiva, S. L.; Lukkarila, J. L.; Gunning, P. T. Exploring a new frontier in cancer treatment: targeting the ubiquitin and ubiquitin-like activating enzymes. J. Med. Chem. 2013, 56, 2165‒2177. (50). Kirisako, T.; Kamei, K.; Murata, S.; Kato, M.; Fukumoto, H.; Kanie, M.; Sano, S.; Tokunaga, F.; Tanaka, K.; Iwai, K. A ubiquitin ligase complex assembles linear polyubiquitin chains. EMBO J. 2006, 25, 4877‒4887. (51). Smit, J. J.; Monteferrario, D.; Noordermeer, S. M.; van Dijk, W. J.; van der Reijden, B. A.; Sixma, T. K. The E3 ligase HOIP specifies linear ubiquitin chain assembly through its RING-IBR-RING domain and the unique LDD extension. EMBO J. 2012, 31, 3833‒ 3844.



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(52). Stieglitz, B.; Morris-Davies, A. C.; Koliopoulos, M. G.; Christodoulou, E.; Rittinger, K. LUBAC synthesizes linear ubiquitin chains via a thioester intermediate. EMBO Rep. 2012, 13, 840‒846. (53). Arantes, F. F. P.; Barbosa, L. C. A.; Alvarenga, E. S.; Demuner, A. J.; Bezerra, D. P.; Ferreira, J. R. O.; Costa-Lotufo, L. V.; Pessoa, C.; Moraes, M. O. Synthesis and cytotoxic activity of α-santonin derivatives. Eur. J. Med. Chem. 2009, 44, 3739‒3745. (54). Hu, N.; Xie, S.; Liu, L.; Wang, X.; Pan, X.; Chen, G.; Zhang, L.; Liu, H.; Liu, X.; Liu, X. Opposite effect of diabetes mellitus induced by streptozotocin on oral and intravenous pharmacokinetics of verapamil in rats. Drug Metab. Dispos. 2011, 39, 419‒ 425.


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Legends of Chart, Schemes and Figures Chart 1. Small-molecule inhibitors for ubiquitination Scheme 1. Synthesis of guaiane-type sesquiterpene lactone 6 Scheme 2. Synthesis of compound 10 Scheme 3. Synthesis of compound 20 Scheme 4. Synthesis of derivatives (1a‒1f, 2a‒2f) of compound 6 Scheme 5. Synthesis of derivatives (3a‒3n, 4a‒4h, 5a‒5g, 6a‒6i) of compound 10 Scheme 6. Synthesis of derivatives (7a‒7f, 8a‒8j, 9a‒9g) of compound 20 Figure 1. The effects of α-santonin derivatives on UbcH5c Figure 2. The effects of derivatives of compounds 6, 10 and 20 on UbcH5c Figure 3. Effects of derivatives on TNF-α induced activation of the NF-κB pathway Figure 4. Effects of α-santonin derivatives on the activity of representative E2 enzymes Figure 5. SPR analysis of the interaction between active compounds and UbcH5c Figure 6. Potent, specific and rapid labeling of a cellular protein by the electrophilic probe 6d-1 in HeLa cells Figure 7. The proposed binding mode of compound 6d and UbcH5c Figure 8. Compound 6d inhibits TNF-α-induced linear polyubiquitination of NEMO Figure 9. Compound 6d suppresses TNF-α-induced expression of inflammatory cytokines in synovial fibroblasts Figure 10. The effect of compound 6d treatment in adjuvant arthritis (AA) rats Table 1. PK properties of compound 6d in rats. Table 2. Stability of compound 6d in vitro



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Chart 1. Small molecule inhibitors for ubiquitination

OAc a

OAc b

O

OAc c

HO

TBSO

O O

O

α-santonion O

O 2

O OAc

d

O 3

O OAc

OAc e

TBSO

f

TBSO

O

HO

SePh O 4

O O

5

O O

6

O

Scheme 1. Synthesis of guaiane-type sesquiterpene lactone 6a a

Reagents and conditions: (a) hυ (Hg-AP/RB, 500 W), anhydrous AcOH, 12 h, 30%; (b)

NaBH4, MeOH, 0 °C, 92%; (c) TBSOTf/DIPEA, CH2Cl2, 0 °C, 94%; (d) LDA/PhSeCl, THF, -78 °C ̶ -20 °C, 80%; (e) 30% H2O2/AcOH, THF, 94%; (f) TBAF, THF, 96%.


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Scheme 2. Synthesis of compound 10a a

Reagents and conditions: (a) PhSeCl, LDA, THF, -78 °C, 83%; (b) H2O2, AcOH, THF,

0 °C, 97%; (c) Con. H2SO4, Ac2O, 0 °C; (d) NH3·H2O, MeOH, 0 °C; (e) DIBAL-H, THF, -78 °C.

Scheme 3. Synthesis of compound 20a a

Reagents and conditions: (a) Pd/C, H2, EtOH, 92%; (b) NaBH4, dry MeOH; (c) TBSCl,

Imidazole, DMF, rt, 79% ̶ 82%; (d) PhSeCl, LDA, THF, -78 °C, 76% ̶ 86%; (e) 30%



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H2O2, AcOH, THF, 0 °C, 50%‒65%; (f) TBAF, THF, rt, 96%.

Scheme 4. Synthesis of derivatives (1a‒1f, 2a‒2f) of compound 6a a

Reagents and conditions: (i) EDCI, DMAP, CH2Cl2, rt, overnight, 80% ̶ 98%.

Scheme 5. Synthesis of derivatives (3a‒3n, 4a‒4h, 5a‒5g, 6a‒6i) of compound 10a a

Reagents and conditions: (i) EDCI, DMAP, CH2Cl2, rt, overnight, 80%‒98%; (ii) K2CO3,

acetone, rt, overnight.


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O

O R6

Hetero

O

O O O

R6

Hetero

O

Hetero= i

R6 = 8a: m-I, 8b: p-I, 8c: m-F, 8d: m-Cl, 8e: m-Br, 8f: m-CF3 , 8g: m-OCF 3, 8h: -H, 8i: m-Me, 8j: m-OMe

i

9a;

9b:

O

HO

alkyl

S

O

Hetero

O i

Hetero

O O

O

O

9e-9g

O

Hetero=

alkyl= H3C

N

OH

O

7a-7f

9d: N

O

O

OH i

O

7d: H 3C

9c:

O

20

7a:

O

9a-9d

OH

8a-8j

alkyl

O

O

OH

7b: H 3C

7c: H3C

7e:

7f:

9e;

9g:

9f: O

S

N

Scheme 6. Synthesis of derivatives (7a‒7f, 8a‒8j, 9a‒9g) of compound 20a a

Reagents and conditions: (i) EDCI, DMAP, CH2Cl2, rt, overnight, 80%‒98%.

Figure 1. The effects of α-santonin derivatives on UbcH5c UbcH5c was incubated with E1, Ub and ATP in the absence or presence of α-santonin derivatives at 10 µM concentration for 40 min. The reaction was terminated by SDS-PAGE sample without DTT, then detected by western blotting with anti-UbcH5c antibody.



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Figure 2. The effects of derivatives of compounds 6, 10 and 20 on UbcH5c UbcH5c was incubated with E1, Ub and ATP in the absence or presence of derivatives at 5 or 10 µM concentrations for 40 min. The reaction was terminated by SDS-PAGE sample without DTT, then detected by western blotting with anti-UbcH5c antibody. (A) The effects of derivatives of compound 6 on UbcH5c. (B) The effects of derivatives of compound 10 on UbcH5c. (C) The effects of derivatives of compound 20 on UbcH5c.


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Figure 3. Effects of derivatives on TNF-α induced activation of NF-κB pathway B.End3 cells were treated with target compounds for 2 h at indicated concentrations, followed by stimulated with 20 ng/mL TNF-α for 20 min. After treatment, the total proteins were extracted. (A) The levels of IκB-α were evaluated by western blotting. (B) The levels of IκB-α, p65, p-IκB-α and p-p65 were evaluated by western blotting.



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Figure 4. Effects of α-santonin derivatives on the activity of representative E2 enzymes (A) and (B): E2 enzymes were pretreated with target compounds for 20 min at indicated concentration, then add E1, Ub and ATP to incubate for 40 min in this reaction system. The reaction was detected by western blotting with anti-E2 antibody.


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Figure 5. SPR analysis of the interaction between active compounds and UbcH5c Kinetics of target compounds binding to UbcH5c were determined by Biacore. The purified UbcH5c was covalently immobilized onto the CM5 sensor chip. Different concentrations of compound were injected at a flow rate of 30 µL/min. The surface was regenerated with 5 mM NaOH. The kinetic parameters KD were measured by Biacore evaluation software.



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Figure 6. Potent, specific and rapid labeling of a cellular protein by the electrophilic probe 6d-1 in HeLa cells (A) Structures of 6d and its clickable analog (6d-1). (B) Internal reference control of Azide-fluor 488. (C) After CuAAC (Cu(I)-catalyzed [3+2] azide-alkyne cycloaddtion) with Azide-fluor 488 and SDS-PAGE separation, in-gel fluorescence scanning revealed one major fluorescent band of ~15 kDa. (D) Immunoblot analysis using an UbcH5c antibody confirmed the protein labelled by probe 6d-1 was UbcH5c.

Figure 7. The proposed binding mode of compound 6d and UbcH5c


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UbcH5c (Protein Data Bank ID code 1X23) is shown as the surface (light blue) and 6d (green), and the key residues (cyan) are represented as sticks. Hydrogen bonds were identified according to rational bond angles and distances, which are indicated by red dashed lines. Oxygen, nitrogen, and sulfur atoms are shown in red, blue, and orange, respectively.

Figure 8. Compound 6d inhibit TNF-α induced linear polyubiquitination of NEMO Hela cells were pretreated with 6d for 2 h at indicated concentration, followed by stimulated with 1 µg/mL TNF-α for 5 min. Cell lysates were immunoprecipitated with anti-NEMO antibody and detected with anti-linear polyubiquitin antibody by western blotting.



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Figure 9. Compound 6d suppresses TNF-α-induced expression of inflammatory cytokines in synovial fibroblasts The synovial fibroblasts were pretreated with 6d for 2 h at indicated concentrations, then stimulated with 20 ng/mL TNF-α for 3h. The mRNA levels of IL-1, MCP-1 and MMP-3 were measured by qPCR. Data were obtained from three independent experiments, values are means ± SEM. # p < 0.05 compared to medium control, *p < 0.05 compared toTNF-α only.

Figure 10. Effect of compound 6d treatment in adjuvant arthritis (AA) rats Wistar rats were immunized with CFA and boosted 10 days later to induce AA. Rats were oral administration with vehicle, 6d at 5 mg/kg and 20 mg/kg once daily between day 10 and day 20 postimmunization. The severity of AA in control and 6d-treated rats was evaluated every 2 or 3 days by 2 independent observers under blinded conditions (n = 10).


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(A) The severity of AA in control and 6d-treated rats. (B) and (C) The inhibitory effect of 6d-treated on the primary and secondary foot lesion in rats with AA. (D) Effect of 6d-treated on the body weight of rats with AA. Data are mean ± S.E.M. of 10 rats per group. *p < 0.05 and **p < 0.01 versus AA control.

Table 1. PK properties of compound 6d in rats.

(A) Intravenous Administration Dose (mg/kg)

CL (L·h-1 kg-1)

Vss (L/kg)

t1/2 (h)

AUC(0-∞) (ng·h/mL)

1

2.48 ± 0.269

6.26 ± 2.07

1.76 ± 0.55

408 ± 48.9

(B) Oral Administration AUC(0-∞) Dose (mg/kg)

tmax (h)

Cmax (ng/mL)

t1/2 (h)

F (%) (ng·h/mL)

5

3.83 ± 2.56

42.2 ± 24.8

2.86 ± 1.05

314 ± 197

16.8%

Data represent the mean ± SD of six rats.

Table 2. Stability of compound 6d in vitro

t1/2 (min)

Rat liver microsomes

Rat plasma

Rat whole blood

126.5

85.6

39.8



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Table of Contents Graphic



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Small-molecule inhibitors for ubiquitination 128x101mm (300 x 300 DPI)


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Synthesis of guaiane-type sesquiterpene lactone 6 174x63mm (300 x 300 DPI)



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Synthesis of compound 10 177x69mm (300 x 300 DPI)


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Synthesis of compound 20 177x101mm (300 x 300 DPI)



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Synthesis of derivatives (1a‒1f, 2a‒2f) of compound 6 177x96mm (300 x 300 DPI)


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Synthesis of derivatives (3a‒3n, 4a‒4h, 5a‒5g, 6a‒6i) of compound 10 171x101mm (300 x 300 DPI)



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Synthesis of derivatives (7a‒7f, 8a‒8j, 9a‒9g) of compound 20 177x84mm (300 x 300 DPI)


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The effects of α-santonin derivatives on UbcH5c 61x45mm (300 x 300 DPI)



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The effects of derivatives of compounds 6, 10 and 20 on UbcH5c. 119x83mm (300 x 300 DPI)


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Effects of derivatives on TNF-α induced activation of the NF-κB pathway. 123x90mm (300 x 300 DPI)



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Effects of α-santonin derivatives on the activity of representative E2 enzymes 124x105mm (300 x 300 DPI)


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SPR analysis of the interaction between active compounds and UbcH5c 73x63mm (300 x 300 DPI)



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Potent, specific and rapid labeling of a cellular protein by the electrophilic probe 6d-1 in HeLa cells 83x54mm (300 x 300 DPI)


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The proposed binding mode of compound 6d and UbcH5c 69x58mm (300 x 300 DPI)



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Compound 6d inhibits TNF-α-induced linear polyubiquitination of NEMO 59x42mm (300 x 300 DPI)


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Compound 6d suppresses TNF-α-induced expression of inflammatory cytokines in synovial fibroblasts 62x47mm (300 x 300 DPI)



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The effect of compound 6d treatment in adjuvant arthritis (AA) rats 177x127mm (300 x 300 DPI)


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Table of Contents Graphic 210x55mm (300 x 300 DPI)


Discovery of Potent Small-Molecule Inhibitors of Ubiquitin

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