Synthesis of new ent-labdane diterpene derivatives from andrographolide and evaluation of their anti-inflammatory activities

Accepted Manuscript Synthesis of new ent-labdane diterpene derivatives from andrographolide and evaluation of their anti-inflammatory activities Wang Wang, Yanli Wu, Xinxin Chen, Peng Zhang, Hua Li, Lixia Chen PII:

S0223-5234(18)30950-4

DOI:

https://doi.org/10.1016/j.ejmech.2018.11.002

Reference:

EJMECH 10859

To appear in:

European Journal of Medicinal Chemistry

Received Date: 20 September 2018 Revised Date:

30 October 2018

Accepted Date: 1 November 2018

Please cite this article as: W. Wang, Y. Wu, X. Chen, P. Zhang, H. Li, L. Chen, Synthesis of new entlabdane diterpene derivatives from andrographolide and evaluation of their anti-inflammatory activities, European Journal of Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.ejmech.2018.11.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Synthesis of new ent-labdane diterpene derivatives from andrographolide and evaluation of their anti-inflammatory

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activities

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Wang Wang,1,a Yanli Wu,1,a Xinxin Chen,a Peng Zhang,a Hua Li,*,a,b Lixia Chen*,a

Wuya College of Innovation, Key Laboratory of Structure-Based Drug Design &

110016, China b

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Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation,

School of Pharmacy, Tongji Medical College, Huazhong University of Science and

These authors contributed equally to this work.

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Technology, Wuhan 430030, China

*Corresponding author: Lixia Chen & Hua Li E-mail: [email protected] (Lixia Chen) [email protected] (Hua Li)

ACCEPTED MANUSCRIPT ABSTRACT: Two series of andrographolide derivatives with nitrogen-containing heterocycles, phenols and aromatic acids as bioisostere moiety of lactone ring were synthesized. 8 from 18 tested compounds showed stronger inhibitory effect on LPS-induced NO production in RAW264.7 macrophage than hydrocortisone. Among

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them, compound 8m exhibited the most potent inhibition with IC50 of 3.38 ± 1.03 µM. The structure-activity relationships (SARs) suggested that the replacement of lactone ring with small-molecule phenols could improve the anti-inflammatory efficacy. Furthermore, compound 8m significantly reduced the levels of pro-inflammatory

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cytokine IL-1β and IL-6 with no influence on cell survival, decreased the expression of iNOS and COX-2, and down-regulated the level and phosphorylation of IκBα, as

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well as the expression of NF-κB. Also it blocked the nuclear translocation of NF-κB in LPS-induced macrophage. Therefore, the anti-inflammation mechanism of compound 8m was related to the inhibition of COX-2, iNOS and NF-κB signal pathway.

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effect.

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Keywords: andrographolide, bioisostere, structure modification, anti-inflammatory

ACCEPTED MANUSCRIPT 1. Introduction In recent years, great research efforts have been focused on the development of anti-inflammatory drugs. Nonsteroidal anti-inflammatory drugs (NSAIDs) and

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anti-cytokine biologics are available for inflammation treatment, but their side effects or excessive costs make the development of safer and more effective anti-inflammatory agents remain a big challenge [1, 2]. While, natural-occurring

anti-inflammation pharmacotherapy [3].

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products and plant extracts have been considered as a rich source for

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Andrographolide is one of the main ingredients of Andrographis paniculata, a traditional Chinese medicine using for inflammatory treatment [4]. Many studies have shown that andrographolide possesses a wide spectrum of bioactivities such as [5],

anti-inflammatory

[6],

antimicrobial

[7],

antimalarial

[8],

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anticancer

immunomodulation [9], antithrombotic [10], and anti-influenza virus [11] properties. Its anti-inflammatory mechanisms have been reported that andrographolide can

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prevent LPS-induced inflammation-related proteins iNOS, COX-2 and NF-κB

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activation and expression [12-14]. Andrographolide and its derivatives, such as Chuanhuning, Xiyanping, Yanhuning and Lianbizhi (Fig. 1), have been used clinically for treating inflammation in China for decades [15]. But these derivatives have shown some side effects, such as pyrexia, allergy, syncope, even renal failure [16, 17]. Therefore, it is highly desirable to develop more derivatives of andrographolide with improved potency and safety profile.

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Fig.1. Structures of andrographolide and related derivatives.

Recently, the anti-cancer activity of andrographolide has gotten great attention, and

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many series of its derivatives have been designed and synthesized [18-22]. While, the researches on its anti-inflammatory derivatives are relatively few [23]. Previous studies of the structure-activity relationships (SARs) of andrographolide have

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revealed that the α-alkylidene-γ-butyrolactone moiety of andrographolide plays a

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crucial role in its activity profile [24, 25]. In recent years, bioisostere replacement has been used widely to decrease toxicity and side effects, and improve the bioactivity in the modification and optimization of lead compounds [26, 27]. So, we resort to this feasible strategy to synthesize a series of novel andrographolide derivatives in this study. In order to further investigate the influence of lactone ring moiety on the anti-inflammatory efficacy of andrographolide derivatives, and to find more potent

ACCEPTED MANUSCRIPT anti-inflammatory drugs with low toxicity, herein, we design and synthesize two series of andrographolide derivatives with nitrogen-containing heterocycles, phenols, and aromatic acids as bioisostere moiety of lactone ring. The inhibitory activity on

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mechanism of these derivatives were investigated as well.

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LPS-induced NO production in RAW264.7 macrophage and anti-inflammatory

Fig.2. Illustration of the design strategy for target compounds.

2.1. Chemistry

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2. Results and discussion

The synthetic route of series 1 and 2 was shown in Scheme 1. The intermediate

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compounds 2-6 were prepared and identified according to the literatures [18, 25,

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28]. The hydroxyl groups at C-3 and C-19 of andrographolide were first protected by reacting with 2,2-dimethoxypropane at the presence of catalytic pyridinium 4-toluenesulfonate (PPTS). Then the hydroxyl group at C-14 was removed by stirring with 4-dimethylamino-pyridin (DMAP) and acetic anhydride, and further the double bond between C-12 and C-13 was oxidized with KMnO4 to yield compound 4 [25]. The aldehyde group at C-12 of compound 4 was reduced with NaBH4 to form the key intermediate 5. In order to increase the reactivity of the

ACCEPTED MANUSCRIPT hydroxyl group, compound 5 was reacted with methanesulfonyl chloride (MsCl) to obtain methanesulfonate ester 6 [28]. The key intermediate 6 obtained above was converted into a number of derivatives through reacting with bioisosteresis

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(heterocyclic potassium salts or phenols) to afford compounds 7a-l, and then the protective groups at C-3 and C-19 were removed with HCl to yield the final

products of series 1. To improve the anti-inflammatory activity of target

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compounds, the well-known anti-inflammatory natural compounds with coumarin

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or flavonoid skeletons were also used as phenol bioisosteresis. On the other hand, Mitsunobu reaction was used to yield compounds 8m and 8n.

To investigate the influence of different types of bioisosteresis on the anti-inflammatory activity, several aromatic acids were chosen as bioisosteresis in

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series 2. Esterification of compound 5 with aromatic acids was carried out with dicyclohexylcarbodiimide (DCC) and DMAP to furnish compounds 9a-d, followed by removing the protective groups on C-3 and C-19 with HCl to yield the final

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products 10a-d.

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Scheme 1. Synthetic route of series 1 and 2. Reagents and conditions: (a) DMP, PPTS, acetone, rt, 2 h; (b) Ac2O, DMAP, DCM, 40 °C, 8 h; (c) KMnO4, THF, 5-10 °C, 1 h; (d) NaBH4, MeOH, 0 °C, 1 h; (e) MsCl, TEA, DCM, 0 °C, 1 h; (f) heterocyclic potassium salts, K2CO3, NaI, DMF, 80 °C, 12 h (7a-e); phenols, K2CO3, CH3CN, reflux, 10 h (7f-l); (g) HCl/ethyl acetate, rt, 0.5 h; (h) phenols, P(Ph)3, DIAD, THF/N2, rt, 9 h; (i) aromatic acids, DCC, DMAP, DCM, rt, 2 h.

2.2. Biological evaluation

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2.2.1. Inhibitory activity on LPS-induced NO production and SAR study

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As an inflammatory mediator, high levels of NO are produced in response to inflammatory stimuli and mediation of inflammatory effects [29]. All the prepared compounds were tested for their inhibitory activity on LPS-induced NO production in RAW264.7

macrophage,

using

hydrocortisone

as

positive

control.

The

anti-inflammatory activity of all synthesized compounds was described as IC50 values of NO inhibition rates in Table 1. Among the tested compounds, series 1 displayed a better inhibitory activity than series 2, and compound 8m showed the most

ACCEPTED MANUSCRIPT remarkable inhibition against NO production (IC50 value: 3.38 ± 1.03 µM), which was more potent than andrographolide (Table 1, Fig. 3A). The preliminary SARs showed heterocycle and aromatic acids couldn't use as

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bioisosteresis to improve anti-inflammatory efficacy of andrographolide (i.e. compounds 8a-e, 10a-d). When phenols were used as the bioisosteresis, the derivatives exhibited better activity than hydrocortisone. But coumarin and flavonoid

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seemed useless to improve anti-inflammatory efficacy (i.e. compounds 8f-n). It could

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be concluded that small-molecule phenols could be used as bioisostere moiety of lactone ring for the improvement of anti-inflammation of andrographolide. Therefore, the above results would be helpful for the further structure modification of andrographolide.

Compounds

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Table 1. The IC50 values of the inhibition against NO production of series 1 and 2. a IC50 (µM)

Compounds

IC50 (µM)

8.81 ± 1.03

8j

>50

>100

8k

>100

>100

8l

>100

>50

8m

3.38 ± 1.03

>100

8n

40.25 ± 1.09

8e

>100

10a

>50

8f

44.68 ± 1.04

10b

>100

8g

12.78 ± 1.38

10c

35.24 ± 1.06

8h

35.77 ± 1.05

10d

37.33 ± 1.06

8i

47.74 ± 1.03

Hydrocortisone

58.79 ± 3.32

8a 8b 8c

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8d

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Andrographolide(1)

a

The results were showed as means ± SD of at least three independent experiments. .

2.2.2. Compound 8m inhibited IL-6 and IL-1β production in RAW264.7 cells Based on the initial screening results of these andrographolide derivatives, compound

ACCEPTED MANUSCRIPT 8m was chosen for further assessment of its inhibition of pro-inflammatory activities on LPS-induced macrophages. Compound 8m was first tested for its effect on RAW264.7 cell viability (Fig. 3B), showing no significant toxicity on cell survival at

were further used in subsequent experiment processes.

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the concentrations ranging from 3.125 to 100 µM. Thus, the non-toxic concentrations

LPS-activated macrophages produce a variety of inflammatory cytokines, including

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tumor necrosis factor (TNF-α), interleukins (ILs), and prostaglandins (PGs), which

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show a host defensive effect during inflammatory situations and also maintain normal cellular conditions [30]. As shown in Fig. 3C and 3D, the levels of the pro-inflammatory cytokine IL-1β and IL-6 were significantly elevated by LPS stimulation in the control group, on the contrary, obvious decrease was induced by

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compound 8m in a dose-dependent manner. These results indicated that compound 8m certainly attenuated an excessive immune reaction in RAW264.7 macrophages

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stimulated by LPS.

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Fig. 3. Inhibitory activity of compound 8m on LPS-induced NO and cytokine production. (A) The NO inhibition rates curve of compound 8m, with IC50 values of 3.38 ±1.03 µM. (B) The RAW264.7 cell viability after exposure to different concentrations of compound 8m for 24 h. (C, D) The production of IL-1β and IL-6 in the medium of RAW264.7 cells after treatment with compound 8m for 3 h and LPS for 24 h. ###, p<0.001, compared with control group that was untreated. **, p<0.01 and ***, p<0.001, compared with LPS-induced group.

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2.2.3. Compound 8m inhibited LPS-induced expression of COX-2 and iNOS in RAW264.7 cells

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Further, the possible anti-inflammatory mechanism of compound 8m was also investigated. During inflammation, pro-inflammatory stimuli induce the increased production of various cytokine and inflammatory mediators, such as NO, due to the increased production/activity of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) [31]. As expected, LPS stimulation could markedly increase COX-2 and iNOS protein expression, and compound 8m significantly decreased the expression of COX-2 and completely inhibited the high expression of

ACCEPTED MANUSCRIPT iNOS induced by LPS (Fig. 4). These results demonstrated that compound 8m could

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participate in signaling pathways activated by LPS in macrophages.

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Fig. 4. Inhibitory activity of compound 8m on LPS-induced protein expression of COX-2 and iNOS. (A) Western blot for COX-2. (B) Relative ratio of COX-2. (C) Western blot for iNOS. (D) Relative ratio of iNOS. RAW264.7 cells were pretreated with or without compound 8m for 3 h before the addition of LPS, and then the expression of iNOS was measured by western blot. ###, p<0.001, compared with control group that was untreated. ***, p<0.001, compared with LPS-induced group.

2.2.4. Compound 8m inhibits LPS-induced NF-κB signal pathway in RAW264.7 cells

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NF-κB is recognized as a crucial component of many immune responses, and for

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example, macrophages rely on NF-κB for the secretion of pro-inflammatory cytokines [32]. The phosphorylation of IκBα plays an important role in the process of the nuclear translocation and activation of NF-κB [33]. Therefore, Western blot and immunofluorescence analyses were used to examine the effects of compound 8m on LPS-induced transcriptional activity of NF-κB in RAW264.7 cells. As shown in Fig. 5A and 5B, compound 8m obviously decreased the phosphorylation of IκBα, at the same time, the protein expression of NF-κB and IκBα was also down-regulated,

ACCEPTED MANUSCRIPT compared with the LPS-induced group (Fig. 5A, 5C and 5D). Immunofluorescence assay demonstrated that LPS-stimulated translocations of NF-κB into the nucleus were significantly blocked by compound 8m at 10 µM (Fig. 5E). These results

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indicated that compound 8m exerts its anti-inflammatory activity by inhibiting the expression of NF-κB and IκBα, reducing the phosphorylation level of IκBα, and

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blocking the translocation of NF-κB into the nucleus.

Fig. 5. Inhibitory activity of compound 8m on NF-κB signal pathway. (A) Compound 8m

ACCEPTED MANUSCRIPT significantly downregulated the expression of NF-κB, IκBα and its phosphorylation. (B-D) Relative ratio of p-IκBα, IκBα and NF-κB. (E) Addition of compound 8m abolished LPS-induced nuclear translocation of NF-κB. RAW264.7 cells stained for NF-κB/p65 (green) and nuclei (DAPI, blue). #, p<0.05 and ###, p<0.001, compared with control group that was untreated. ***, p<0.001, compared with LPS-induced group.

3. Conclusion

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To summarize, we designed and synthesized two series of new andrographolide derivatives with nitrogen-containing heterocycles, phenols and aromatic acids as

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bioisostere moiety of lactone ring. 8 from 18 tested compounds displayed inhibitory activity on LPS-induced NO production in RAW264.7 macrophage. Among them,

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compound 8m exhibited the most significant activity. The preliminary SARs illustrated that small-molecule phenols could be used as bioisostere moiety of lactone ring in andrographolide to improve the anti-inflammatory effect. The further study revealed that compound 8m could reduce the levels of IL-1β and

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IL-6, and its anti-inflammatory activity involved in the inhibition of COX-2 and iNOS, and the down-regulation of the activation of NF-κB signal pathway. These findings

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indicated that compound 8m could serve as an anti-inflammatory agent and deserve further study in vitro and in vivo.

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4. Experimental 4.1. Chemistry

All commercially available starting materials and solvents were reagent grade and used without further purification. Reactions were monitored by thin-layer chromatography (TLC) on 0.25 mm silica gel plates (GF254) and visualized under UV light or by heating after spraying with anisaldehyde-H2SO4 reagent. 1H NMR and 13C NMR spectra were recorded on a Bruker AV-600 spectrometer (Bruker Biospin,

ACCEPTED MANUSCRIPT Fallanden, Switzerland). Chemical shifts are stated relative to TMS and expressed in δ values (ppm), with coupling constants reported in Hz. High resolution mass spectra (HRMS) of all derivatives were recorded on a Waters Micromass Q-T of Micromass

4.2. Synthetic methods of all compounds 4.2.1. Synthesis of compound 2

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spectrometer by electrospray ionization (ESI).

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(4S,E)-4-hydroxy-3-(2-((4aR,6aS,7R,10bR)-3,3,6a,10b-tetramethyl-8-methylened

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ecahydro-1H-naphtho[2,1-d][1,3]dioxin-7-yl)ethylidene)dihydrofuran-2(3H)-one (compound 2). Andrographolide (1 g, 2.86 mmol) was dissolved in anhydrous acetone (25 mL). 2,2-dimethoxypropane (3.75 mL) and PPTS (catalytic amount) were added to the solution. The mixture was stirred for about 2 h at room

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temperature. Upon completion, acetone was evaporated to dryness. The crude product was redissolved in methylene chloride (DCM), washed with saturated sodium bicarbonate solution and brine, respectively, dried over anhydrous

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Na2SO4, filtered, evaporated and purified by silica gel column chromatography

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(CC) to obtain compound 2 as white solid (97% yield). HR-MS(ESI) m/z: 391.2476 [M+H]+ (Calcd. for C23H35O5, 391.2479); 1H-NMR (600 MHz, CDCl3):

δ 6.95 (1H, s), 5.04 (1H, s), 4.90 (1H, s), 4.62 (1H, s), 4.45 (1H, d, J = 4.2 Hz), 4.26 (1H, m), 3.95 (1H, d, J = 11.4 Hz), 3.49 (1H, d, J = 4.2 Hz), 3.17 (1H, d, J = 11.4 Hz), 2.57 (2H, s), 2.44 (2H, m), 1.98 (2H, m), 1.78 (5H, m), 1.41 (3H, s), 1.36 (3H, s), 1.20 (3H, s), 0.96 (3H, s). 4.2.2. Synthesis of compound 3

ACCEPTED MANUSCRIPT (E)-3-(2-((4aR,6aS,7R,10bR)-3,3,6a,10b-tetramethyl-8-methylenedecahydro-1Hnaphtho[2,1-d][1,3]dioxin-7-yl)ethylidene)furan-2(3H)-one

(compound

3).

Compound 2 (14 g, 35.8 mmol) was dissolved in anhydrous DCM (150 mL).

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DMAP (3.1 g, 25.1 mmol) and acetic anhydride (4.1 mL, 43.0 mmol) were added to the solution. The mixture was stirred for about 8 h at 40 °C. Upon completion,

the solution was washed with 20% citric acid solution, saturated sodium

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bicarbonate solution and brine, respectively, dried over anhydrous Na2SO4,

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filtered, evaporated and purified by silica gel CC to afford compound 3 as white solid (85% yield). HR-MS(ESI) m/z: 373.2372 [M+H]+ (Calcd. for C23H33O4, 373.2373); 1H-NMR (600 MHz, CDCl3): δ 7.00 (1H, s), 6.71 (1H, m), 6.19 (1H, s), 4.87 (1H, s), 4.43 (1H, s), 3.97 (1H, d, J = 11.4 Hz), 3.51 (1H, m), 3.19 (1H, d,

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J = 11.4 Hz), 2.51 (2H, m), 2.42 (2H, m), 1.99 (2H, m), 1.88 (2H, m), 1.77 (4H, m), 1.42 (3H, s), 1.37 (3H, s), 1.30 (6H, m), 1.21 (3H, s), 0.97 (3H, s). 4.2.3. Synthesis of compound 4

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2-((4aR,6aS,7R,10bR)-3,3,6a,10b-tetramethyl-8-methylenedecahydro-1H-

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naphtho[2,1-d][1,3]dioxin-7-yl)acetaldehyde (compound 4). Compound 3 (0.56 g, 1.51 mmol) was dissolved in THF (15 mL). The solution was put in an ice bath to cool down. Then KMnO4 (0.48 g, 3.04 mmol) was added to the solution. The mixture was stirred for 1 h at 5-10 °C. Upon completion, the soild was filtered off and the solvent was removed under reduced pressure. Then the residue was extracted with DCM, washed with brine, dried with anhydrous Na2SO4, filtered, evaporated and purified by silica gel CC to afford compound 4 as white solid (58%

ACCEPTED MANUSCRIPT yield). HR-MS(ESI) m/z: 307.2262 [M+H]+ (Calcd. for C19H31O3, 307.2268); 1H NMR (600 MHz, CDCl3): δ 9.65 (1H, s), 4.83 (1H, s), 4.41 (1H, s), 3.97 (1H, d, J = 11.6 Hz), 3.50 (1H, dd, J = 8.8, 4.1 Hz), 3.19 (1H, d, J = 11.6 Hz), 2.57 (1H, m),

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2.43 (2H, m), 2.43 (2H, m), 2.35 (1H, d, J = 10.9 Hz), 2.07 (1H, m), 2.00 (1H, m), 1.80 (1H, m), 1.73 (1H, m), 1.63 (1H, m), 1.40 (3H, s), 1.36 (3H, s), 1.31 (3H, m), 1.21 (3H, s), 0.92 (3H, s).

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4.2.4. Synthesis of compound 5

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2-((4aR,6aS,7R,10bR)-3,3,6a,10b-tetramethyl-8-methylenedecahydro-1Hnaphtho[2,1-d][1,3]dioxin-7-yl)ethanol (compound 5). Compound 4 (0.56 g, 1.82 mmol) was dissolved in THF (15 mL). The solution was put in an ice bath to cool down. Then NaBH4 (83 mg, 2.19 mmol) was added to the solution for several

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times. The mixture was stirred for 1 h at 0-5 °C. Upon completion, water (5 mL) was added to quench reaction, then the solvent was removed under reduced pressure, and the residue was extracted with DCM, washed with brine, dried with

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anhydrous Na2SO4, filtered, evaporated and purified by CC to afford compound 5

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as white solid (96% yield). HR-MS(ESI) m/z: 309.2420 [M+H]+ (Calcd. for C19H33O3, 309.2424); 1H-NMR (600 MHz, CDCl3): δ 4.86 (1H, s), 4.59 (1H, s), 3.97 (1H, d, J = 12.0 Hz), 3.74 (1H, m), 3.51 (2H, m), 3.17 (1H, d, J = 12.0 Hz), 2.40 (1H, dd, J = 13.2, 3.4 Hz), 1.97 (2H, m), 1.79 (2H, m), 1.72 (3H, m), 1.42 (3H, s), 1.37 (3H, s), 1.32 (1H, m), 1.27 (3H, m), 1.19 (3H, m), 0.92 (3H, s). 4.2.5. Synthesis of compound 6 2-((4aR,6aS,7R,10bR)-3,3,6a,10b-tetramethyl-8-methylenedecahydro-1H-

ACCEPTED MANUSCRIPT naphtho[2,1-d][1,3]dioxin-7-yl)ethyl

methanesulfonate

(compound

6).

Compound 5 (0.3 g, 0.97 mmol) was dissolved in anhydrous DMF (10 mL). The solution was put in an ice bath to cool down. Triethylamine (0.69 mL) was added

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to the solution. Then MsCl (0.25 mL, 3.13 mmol) was added to the mixture dropwise after 10 minutes. The mixture was stirred in an ice bath for another 1 h. Upon completion, water (5 mL) was added to quench reaction. The organic layer

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was washed with 20% citric acid solution, saturated sodium bicarbonate solution,

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water and brine, respectively, as well as dried over anhydrous Na2SO4, filtered, evaporated and purified by CC to afford compound 6 as light yellow solid (92% yield). HR-MS(ESI) m/z: 387.2195 [M+H]+ (Calcd. for C20H35O5S, 387.2200); 1

H-NMR (600 MHz, CDCl3): δ 4.90 (1H, s), 4.53 (1H, s), 4.33 (1H, m), 4.10 (1H,

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td, J = 9.0, 6.4 Hz), 3.94 (1H, d, J = 11.6 Hz), 3.50 (1H, dd, J = 8.2, 3.9 Hz), 3.16 (1H, d, J = 11.6 Hz), 2.97 (3H, s), 2.41 (1H, m), 1.97 (4H, m), 1.86 (1H, m), 1.78

(3H, s).

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(3H, m), 1.71 (2H, m), 1.40 (3H, s), 1.36 (3H, s), 1.29 (4H, m), 1.18 (3H, s), 0.92

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4.2.6. General procedure for the synthesis of compounds 7a-e and 8a-e Compound 6 (0.3 g, 0.78 mmol) was dissolved in anhydrous DMF (10 mL). Then K2CO3 (0.13 g, 0.94 mmol), heterocyclic potassium salt (1.56 mmol) and NaI (catalytic amount) were added to the solution. The mixture was stirred for 12 h at 80 °C. Upon completion, the solvent was removed under reduced pressure, and the residue was extracted with ethyl acetate, washed with water, brine, and dried with anhydrous Na2SO4, respectively, and then filtered, evaporated and purified by CC to

ACCEPTED MANUSCRIPT afford compounds 7a-e. Thus, compounds 7a-e were dissolved in MeOH and stirred with HCl for 2 h at room temperature to afford compounds 8a-e. 1-(2-((1R,5R,6R,8aS)-6-hydroxy-5-(hydroxymethyl)-5,8a-dimethyl-2-methylenedecahy

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dronaphthalen-1-yl)ethyl)pyrimidine-2,4(1H,3H)-dione (compound 8a). White solid, 76% yield; HR-MS(ESI) m/z: 385.2091 [M+Na]+ (Calcd. for C20H30N2NaO4, 385.2098); 1H-NMR (600 MHz, DMSO-d6): δ 11.18 (1H, s, N-H), 7.59 (1H, d, J =

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7.8 Hz, 1'-H), 5.51 (1H, dd, J = 7.8 Hz, 2.2Hz, 2'-H), 4.73 (1H, s, 13a-H), 4.48 (1H, s,

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13b-H), 3.81 (1H, d, J = 10.9 Hz, 15a-H), 3.74 (1H, m, 12a-H), 3.41 (1H, m, 12b-H), 3.23 (1H, d, J = 10.9 Hz, 15b-H), 3.21 (1H, m, 3-H), 1.07 (3H, s, 14-H), 0.59 (3H, s, 16-H);

13

C-NMR (150 MHz, DMSO-d6): δ 163.7, 150.9, 147.2, 145.8, 107.0, 100.7,

78.4, 62.5, 54.6, 53.0, 47.3, 42.2, 38.6, 37.7, 36.4, 27.8, 24.0, 23.2, 23.0, 14.7.

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2-amino-3-(2-((1R,5R,6R,8aS)-6-hydroxy-5-(hydroxymethyl)-5,8a-dimethyl-2-methyle nedecahydronaphthalen-1-yl)ethyl)pyrimidin-4(3H)-one (compound 8b). White solid, 73% yield; HR-MS(ESI) m/z: 362.2435 [M+H]+ (Calcd. for C20H32N3O3, 362.2438); H-NMR (600 MHz, DMSO-d6): δ 8.86 (1H, s, 4'-N-H), 7.99 (1H, d, J = 7.5 Hz, 3'-H),

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1

AC C

6.08 (1H, d, J = 7.5 Hz, 2'-H), 4.85 (1H, s, 13a-H), 4.72 (1H, s, 13b-H), 3.83 (1H, m, 12a-H), 3.81 (1H, d, J = 11.1 Hz, 15a-H), 3.54 (1H, m, 12b-H), 3.23 (1H, d, J = 10.8 Hz, 15b-H), 3.21 (1H, m, 3-H), 1.07 (3H, s, 14-H), 0.59 (3H, s, 16-H); 13C-NMR (150 MHz, DMSO-d6): δ 159.8, 149.8, 147.5, 147.2, 107.0, 93.1, 78.3, 62.5, 54.4, 52.9, 48.7, 42.2, 38.7, 37.7, 36.4, 27.8, 24.0, 23.0, 23.0, 14.7. (1R,2R,4aS,5R)-5-(2-(1H-imidazol-1-yl)ethyl)-1-(hydroxymethyl)-1,4a-dimethyl-6-met hylenedecahydronaphthalen-2-ol

(compound

8c).

White

solid,

84%

yield;

ACCEPTED MANUSCRIPT HR-MS(ESI) m/z: 319.2377 [M+H]+ (Calcd. for C19H31N2O2, 319.2380); 1H-NMR (600 MHz, DMSO-d6): δ 9.22 (1H, s, 1'-H), 7.83 (1H, t, J = 1.5 Hz, 3'-H), 7.68 (1H, t, J = 1.5 Hz, 2'-H), 4.86 (1H, s, 13H-a), 4.68 (1H, s, 13H-b), 4.19 (1H, m, 12H-a), 4.01

RI PT

(1H, m, 12H-b), 3.81 (1H, d, J = 11.0Hz, 15a-H), 3.23 (1H, d, J = 11.0 Hz, 15b-H), 3.18 (1H, m, 3-H), 1.06 (3H, s, 14-H), 0.61 (3H, s, 16-H);

13

C-NMR (150 MHz,

38.6, 37.6, 36.3, 27.7, 24.5, 24.0, 23.0, 14.7.

SC

DMSO-d6): δ 146.8, 135.1, 121.9, 119.7, 107.2, 78.3, 62.5, 54.4, 52.7, 47.9, 42.2,

M AN U

(1R,2R,4aS,5R)-5-(2-(1H-1,2,4-triazol-1-yl)ethyl)-1-(hydroxymethyl)-1,4a-dimethyl-6 -methylenedecahydronaphthalen-2-ol (compound 8d). White solid, 81% yield; HR-MS(ESI) m/z: 386.2542 [M+H]+ (Calcd. for C21H32N5O2, 386.2551); 1H-NMR (600 MHz, DMSO-d6): δ 9.11 (1H, s, 1'-H), 8.40 (1H, s, 2'-H), 4.88 (1H, s, 13a-H),

TE D

4.65 (1H, s, 13b-H), 4.24 (1H, m, 12a-H), 4.08 (1H , m, 12b-H), 3.81 (1H, d, J = 10.9 Hz, 15a-H), 3.23 (1H, d, J = 10.9 Hz, 15b-H), 3.17 (1H, m, 3-H), 1.06 (3H, s, 14-H), 0.61 (3H, s, 16-H);

13

C-NMR (150 MHz, DMSO-d6): δ 148.2, 147.1 ,142.9, 106.9,

EP

78.3, 62.5, 54.4, 52.5, 48.8, 42.2, 38.6, 37.7, 36.3, 27.8, 24.0, 23.9, 23.0, 14.7.

AC C

(1R,2R,4aS,5R)-5-(2-(6-amino-9H-purin-9-yl)ethyl)-1-(hydroxymethyl)-1,4a-dimethyl -6-methylenedecahydronaphthalen-2-ol (compound 8e). White solid, 43% yield; HR-MS(ESI) m/z: 445.2261 [M+H]+ (Calcd. for C24H33N2O6, 445.2260); 1H-NMR (600 MHz, DMSO-d6): δ 8.16 (1H, s, 4'-H), 8.11 (1H, s, 1'-H), 7.16 (2H, s, 3'-N-H), 4.89 (1H, s, 13a-H), 4.82 (1H, s, 13b-H), 3.80 (1H, d, J = 10.9 Hz, 15a-H), 3.23 (1H, d, J = 10.9 Hz, 15b-H), 3.22 (1H, m, 3-H), 1.06 (3H, s, 14-H), 0.59 (3H, s, 16-H); 13

C-NMR (150 MHz, DMSO-d6): δ 155.9, 152.3, 149.5, 147.1, 140.7, 118.7, 107.1,

ACCEPTED MANUSCRIPT 78.3, 62.5, 54.5, 53.0, 42.3, 42.2, 38.6, 37.7, 36.4, 27.8, 24.4, 24.0, 22.9, 14.8. 4.2.7. General procedure for the synthesis of compounds 7f-l and 8f-l Compound 6 (1.0 g, 2.6 mmol) was dissolved in anhydrous acetonitrile (10 mL). Then

RI PT

phenol (3.1 mmol) and K2CO3 (0.65 g, 4.7 mmol) were added to the solution. The mixture was refluxed and stirred for 10 h. Upon completion, K2CO3 was filtered off, and the solvent was evaporated to dryness. The residue was extracted with ethyl

SC

acetate, washed with water, brine, and dried with anhydrous Na2SO4, respectively, and

M AN U

then filtered, evaporated and purified by silica gel CC to afford compounds 7f-l. Then compounds 7f-l were dissolved in MeOH and stirred with HCl for 0.5 h at room temperature to obtain compounds 8f-l.

(1R,2R,4aS,5R)-1-(hydroxymethyl)-1,4a-dimethyl-6-methylene-5-(2-(naphthalen-1-ylo

TE D

xy)ethyl)decahydronaphthalen-2-ol (compound 8f). White solid, 81% yield; HR-MS(ESI) m/z: 417.2395 [M+Na]+ (Calcd. for C26H34NaO3, 417.2400); 1H-NMR (600 MHz, CDCl3): δ 8.26 (1H, t, J = 5.4 Hz, 8′-H), 7.79 (1H, t, J = 4.2 Hz, 5′-H),

EP

7.48 (2H, m, 6′, 7′-H), 7.39 (1H, d, J = 8.4 Hz, 4′-H), 7.34 (1H, t, J = 7.2 Hz, 3′-H),

AC C

6.74 (1H, d, J = 7.8 Hz, 2′-H), 4.89 (1H, s, 13a-H), 4.64 (1H, s, 13b-H), 4.21 (2H, m, 12a, 15a-H), 4.00 (1H, m, 12b-H), 3.53 (1H, m, 3-H), 3.34 (1H, d, J = 10.8 Hz, 15b-H), 1.25 (3H, s, 16-H), 0.71 (3H, s, 14-H). 13C-NMR (150 MHz, CDCl3): δ 154.7, 147.4, 134.4, 127.4, 126.3, 125.9, 125.6, 125.0, 121.9, 119.9, 117.2, 114.6, 80.6, 67.2, 64.2, 55.2, 52.7, 42.8, 38.8, 38.0, 36.9, 28.2, 24.1, 23.9, 22.7, 15.2. (1R,2R,4aS,5R)-1-(hydroxymethyl)-1,4a-dimethyl-6-methylene-5-(2-(4-phenoxypheno xy)ethyl)decahydronaphthalen-2-ol (compound 8g). White solid, 85% yield;

ACCEPTED MANUSCRIPT HR-MS(ESI) m/z: 459.2500 [M+Na]+ (Calcd. for C28H36NaO4, 459.2506); 1H-NMR (600 MHz, CDCl3): δ 7.29 (2H, t, J = 7.8 Hz, 3′′, 5′′-H), 7.03 (1H, t, J = 7.2 Hz, 4′′-H), 6.94 (4H, m, 3′, 5′, 2′′, 6′′-H), 6.83 (2H, d, J = 9.0 Hz, 2′, 6′-H), 4.87 (1H, s,

RI PT

13a-H), 4.59 (1H, s, 13b-H), 4.21 (1H, d, J = 11.4 Hz, 15a-H), 4.02 (1H, m, 12a-H), 3.80 (1H, m, 12b-H), 3.51 (1H, m, 3-H), 3.34 (1H, d, J = 11.4 Hz, 15b-H), 1.26 (3H, s, 16-H), 0.68 (3H, s, 14-H). 13C-NMR (150 MHz, CDCl3): δ 158.5, 155.3, 149.9, 147.3,

SC

129.5, 129.5, 122.3, 120.7, 120.7, 117.5, 117.5, 115.4, 115.4,107.2, 80.6, 67.5, 64.1,

M AN U

55.2, 52.5, 42.8, 38.8, 38.0, 36.7, 28.2, 23.9, 23.8, 22.6, 15.1.

(1R,2R,4aS,5R)-5-(2-(4-(tert-butyl)phenoxy)ethyl)-1-(hydroxymethyl)-1,4a-dimethyl-6 -methylenedecahydronaphthalen-2-ol (compound 8h). White solid, 76% yield; HR-MS(ESI) m/z: 423.2862 [M+Na]+ (Calcd. for C26H40NaO3, 423.2870); 1H-NMR

TE D

(600 MHz, CDCl3): δ 7.27 (2H, d, J = 8.4 Hz, 3′, 5′-H), 6.79 (2H, d, J = 9.0 Hz, 2′, 6′-H), 4.85 (1H, s, 13a-H), 4.58 (1H, s, 13b-H), 4.20 (1H, d, J = 11.4 Hz, 15a-H), 4.02 (1H, m, 12a-H), 3.79 (1H, m, 12b-H), 3.50 (1H, m, 3-H), 3.30 (1H, d, J = 10.2 Hz,

EP

15b-H), 1.29 (9H, s, t-Bu-H), 1.25 (3H, s, 16-H), 0.67 (3H, s, 14-H). 13C-NMR (150

AC C

MHz, CDCl3): δ 156.7, 147.3, 143.1, 126.1, 126.1, 113.8, 113.8, 107.2, 80.6, 67.0, 64.1, 55.2, 52.5, 42.8, 38.9, 38.0, 36.7, 34.0, 31.5, 31.5, 31.5, 28.2, 24.0, 23.9, 22.6, 15.1.

(1R,2R,4aS,5R)-1-(hydroxymethyl)-5-(2-(2-hydroxyphenoxy)ethyl)-1,4a-dimethyl-6-m ethylenedecahydronaphthalen-2-ol

(compound

8i).

White solid,

80%

yield;

HR-MS(ESI) m/z: 383.2190 [M+Na]+ (Calcd. for C22H32NaO4, 383.2193); 1H-NMR (600 MHz, CDCl3): δ 6.92 (1H, dd, J = 8.4, 1.2 Hz, 3′-H), 6.85 (1H, td, J = 7.2, 1.8

ACCEPTED MANUSCRIPT Hz, 4′-H), 6.79 (2H, m, 5′, 6′-H), 4.90 (1H, s, 13a-H), 4.61 (1H, s, 13b-H), 4.20 (1H, d, J = 11.4 Hz, 15a-H), 4.09 (1H, m, 12a-H), 3.95 (1H, m, 12b-H), 3.50 (1H, m, 3-H), 3.32 (1H, d, J = 10.8 Hz, 15b-H), 1.25 (3H, s, 16-H), 0.67 (3H, s, 14-H).

13

C-NMR

RI PT

(150 MHz, CDCl3): δ 147.5, 145.8, 145.7, 121.3, 120.0, 114.4, 111.6, 107.3, 80.5, 68.2, 64.1, 55.2, 52.9, 42.8, 38.8, 38.0, 36.8, 28.2, 23.9, 23.8, 22.6, 15.1.

7-(2-((1R,5R,6R,8aS)-6-hydroxy-5-(hydroxymethyl)-5,8a-dimethyl-2-methylenedecahy

SC

dronaphthalen-1-yl)ethoxy)-2H-chromen-2-one (compound 8j). Yellow solid, 78%

1

M AN U

yield; HR-MS(ESI) m/z: 435.2140 [M+Na]+ (Calcd. for C25H32NaO5, 435.2142); H-NMR (600 MHz, CDCl3): δ 7.63 (1H, d, J = 9.0 Hz, 4′-H), 7.35 (1H, d, J = 8.4

Hz, 5′-H), 6.81 (1H, dd, J = 8.4, 1.8 Hz, 6′-H), 6.76 (1H, d, J = 2.4 Hz, 8′-H), 6.24 (1H, d, J = 9.6 Hz, 3′-H), 4.90 (1H, s, 13a-H), 4.58 (1H, s, 13b-H), 4.20 (1H, d, J =

TE D

10.8 Hz, 15a-H), 4.11 (1H, m, 12a-H), 3.89 (1H, m, 12b-H), 3.53 (1H, m, 3-H), 3.33 (1H, d, J = 11.4 Hz, 15b-H), 1.26 (3H, s, 16-H), 0.68 (3H, s, 14-H).

13

C-NMR (150

MHz, CDCl3): δ 162.2, 161.2, 155.8, 147.1, 143.3, 128.6, 112.9, 112.8, 112.4, 107.3,

EP

101.4, 80.5, 67.7, 64.1, 55.2, 52.4, 42.9, 38.8, 38.0, 36.8, 28.2, 23.9, 23.7, 22.6, 15.1.

AC C

2-(4-(2-((1R,5R,6R,8aS)-6-hydroxy-5-(hydroxymethyl)-5,8a-dimethyl-2-methylenedec ahydronaphthalen-1-yl)ethoxy)phenyl)-6-methyl-4H-chromen-4-one (compound 8k). Yellow solid, 83% yield; HR-MS(ESI) m/z: 525.2608 [M+Na]+ (Calcd. for C32H38NaO5, 525.2611); 1H-NMR (600 MHz, CDCl3): δ 8.03 (1H, s, 5′-H), 7.91 (2H, d, J = 9.0 Hz, 2′′, 6′′-H), 7.56 (1H, dd, J = 9.0, 2.4 Hz, 7′-H), 7.51 (1H, d, J = 9.0 Hz, 8′-H), 7.09 (1H, s, 3′-H), 6.99 (2H, d, J = 9.0 Hz, 3′′, 5′′-H), 4.90 (1H, s, 13a-H), 4.60 (1H, s, 13b-H), 4.20 (1H, d, J = 10.8 Hz, 15a-H), 4.14 (1H, m, 12a-H), 3.92 (1H, m,

ACCEPTED MANUSCRIPT 12b-H), 3.52 (1H, m, 3-H), 3.33 (1H, d, J = 10.8 Hz, 15b-H), 2.49 (3H, s, 6′-CH3), 1.27 (3H, s, 16-H), 0.69 (3H, s, 14-H). 13C-NMR (150 MHz, CDCl3): δ 178.3, 164.8, 162.4, 154.5, 147.2, 135.7, 135.6, 128.4, 128.4, 124.9, 123.2, 122.3, 117.7, 115.0,

RI PT

115.0, 107.2, 104.9, 80.5, 67.5, 64.1, 55.2, 52.4, 42.9, 38.8, 38.0, 36.8, 28.2, 23.9, 23.8, 22.7, 21.0, 15.1.

6-(2-((1R,5R,6R,8aS)-6-hydroxy-5-(hydroxymethyl)-5,8a-dimethyl-2-methylenedecahy

SC

dronaphthalen-1-yl)ethoxy)-2-phenyl-4H-chromen-4-one (compound 8l). Yellow solid,

1

M AN U

82% yield; HR-MS(ESI) m/z: 511.2450 [M+Na]+ (Calcd. for C31H36NaO5, 511.2455); H-NMR (600 MHz, CDCl3): δ 7.95 (2H, dd, J = 7.8, 1.2 Hz, 2′′, 6′′-H), 7.54 (5H, m,

5′, 8′, 3′′, 4′′, 5′′-H), 7.32 (1H, dd, J = 9.0, 3.0 Hz, 7′-H), 7.03 (1H, s, 3′-H), 4.89 (1H, s, 13a-H), 4.60 (1H, s, 13b-H), 4.21 (1H, d, J = 11.4 Hz, 15a-H), 4.16 (1H, m, 12a-H),

TE D

3.93 (1H, m, 12b-H), 3.53 (1H, m, 3-H), 3.34 (1H, d, J = 11.4 Hz, 15b-H), 1.27 (3H, s, 16-H), 0.69 (3H, s, 14-H). 13C-NMR (150 MHz, CDCl3): δ 178.3, 163.8, 156.6, 151.0, 147.0, 131.8, 131.6, 129.0, 129.0, 126.4, 126.4, 124.4, 124.0, 119.4, 107.3, 106.3,

EP

105.5, 80.5, 68.0, 64.1, 55.2, 52.5, 42.9, 38.8, 38.0, 36.8, 28.2, 23.9, 23.8, 22.6, 15.2.

AC C

4.2.8. General procedure for the synthesis of compounds 7m-n and 8m-n PPh3 (0.84 g, 3.2 mmol) and DIAD (0.63 mL, 3.2 mmol) were dissolved in anhydrous THF (5 mL) with the protection of nitrogen. After 30 minutes, the solution of phenol (1.9 mmol) in anhydrous THF (5 mL) was added dropwise. Then the solution of compound 5 (0.5 g, 1.6 mmol) in anhydrous THF (5 mL) was added to the mixture after stirring for another 30 minutes. The mixture was stirred at room temperature for 8 h. Upon completion, the solvent was removed under reduced pressure, then the

ACCEPTED MANUSCRIPT residue was extracted with ethyl acetate, washed with water, brine, and dried with anhydrous Na2SO4, respectively, and then filtered, evaporated and purified by CC to afford compounds 7m-n. Then compounds 7m-n were dissolved in MeOH and stirred

RI PT

with HCl for 0.5 h at room temperature to obtain compounds 8m-n. (1R,2R,4aS,5R)-1-(hydroxymethyl)-1,4a-dimethyl-6-methylene-5-(2-(4-nitrophenoxy)e thyl)decahydronaphthalen-2-ol (compound 8m). White solid, 96% yield; HR-MS(ESI)

SC

m/z: 412.2091 [M+Na]+ (Calcd. for C22H31NNaO5, 412.2094); 1H-NMR (600 MHz,

M AN U

CDCl3): δ 8.18 (2H, d, J = 9.6 Hz, 3′, 5′-H), 6.91 (2H, d, J = 9.6 Hz, 2′, 6′-H), 4.89 (1H, s, 13a-H), 4.58 (1H, s, 13b-H), 4.20 (1H, d, J = 11.4 Hz, 15a-H), 4.13 (1H, m, 12a-H), 3.92 (1H, m, 12b-H), 3.51 (1H, m, 3-H), 3.34 (1H, d, J = 11.4 Hz, 15b-H), 1.26 (3H, s, 16-H), 0.68 (3H, s, 14-H). 13C-NMR (150 MHz, CDCl3): δ 164.0, 147.1,

TE D

141.3, 125.8, 125.8, 114.4, 114.4, 107.2, 80.5, 68.0, 64.0, 55.2, 52.4, 42.8, 38.8, 38.0, 36.8, 28.2, 23.9, 23.7, 22.6, 15.1.

(1R,2R,4aS,5R)-5-(2-(4-bromophenoxy)ethyl)-1-(hydroxymethyl)-1,4a-dimethyl-6-met

EP

hylenedecahydronaphthalen-2-ol

(compound

8n).

White

solid,

65%

yield;

1

AC C

HR-MS(ESI) m/z: 445.1347 [M+Na]+ (Calcd. for C22H31BrNaO3, 445.1349); H-NMR (600 MHz, CDCl3): δ 7.34 (2H, d, J = 9.0 Hz, 3′, 5′-H), 6.73 (2H, d, J = 9.0

Hz, 2′, 6′-H), 4.87 (1H, s, 13a-H), 4.57 (1H, s, 13b-H), 4.20 (1H, d, J = 11.4 Hz, 15a-H), 4.00 (1H, m, 12a-H), 3.77 (1H, m, 12b-H), 3.51 (1H, m, 3-H), 3.33 (1H, d, J = 10.8 Hz, 15b-H), 1.25 (3H, s, 16-H), 0.67 (3H, s, 14-H).

13

C-NMR (150 MHz,

CDCl3): δ 158.0, 147.2, 132.1, 132.1, 116.2, 116.2, 112.5, 107.2, 80.6, 67.3, 64.1, 55.2, 52.4, 42.8, 38.8, 38.0, 36.7, 29.6, 28.2, 23.9, 22.6, 15.1.

ACCEPTED MANUSCRIPT 4.2.9. General procedure for the synthesis of compounds 9a-d and 10a-d DCC (72 mg, 0.34 mmol) and acid molecules (0.40 mmol) were dissolved in DCM (5 mL). After 20 minutes, compound 5 (100 mg, 0.32 mmol) and DMAP

RI PT

(catalytic amount) were added to the mixture. The mixture was stirred for about 2 h at 25 °C, then the soild was filtered off. The organic layer was washed with 20% citric acid solution, and saturated sodium bicarbonate solution and brine,

SC

respectively, and then dried over anhydrous Na2SO4, filtered, evaporated and

M AN U

purified by CC to afford compounds 9a-d. Then compounds 9a-d were dissolved in MeOH and stirred with HCl for 0.5 h at room temperature to obtain compounds 10a-d.

2-((1R,5R,6R,8aS)-6-hydroxy-5-(hydroxymethyl)-5,8a-dimethyl-2-methylenedecahydr

TE D

onaphthalen-1-yl)ethyl benzoate (compound 10a). White solid, 82% yield; HR-MS(ESI) m/z: 374.2320 [M+H]+ (Calcd. for C22H32NO4, 374.2326); 1H-NMR (600 MHz, DMSO-d6): δ 9.03 (1H, d, J = 1.5 Hz, 2′-H), 8.82 (1H, dd, J = 1.6, 4.8 Hz,

EP

4′-H), 8.30 (1H, dt, J = 8.0, 1.9 Hz, 6′-H), 7.58 (1H, m, 5′-H), 4.88 (1H, s, 13a-H),

AC C

4.57 (1H, s, 13b-H), 4.24 (2H, m, 12-H), 3.85 (1H, d, J = 10.9 Hz, 15a-H), 3.28 (1H, d, J = 10.9 Hz, 15b-H), 3.20 (1H, m, 3-H), 1.09 (3H, s, 14-H), 0.92 (3H, s, 16-H). 13

C-NMR (150 MHz, DMSO-d6): δ 164.7, 153.6, 150.0, 136.8, 134.8, 128.7, 125.7,

123.9, 78.3, 64.2, 62.7, 51.2, 42.1, 37.9, 34.5, 33.8, 27.7, 26.9, 22.9, 20.1, 19.4, 19.0. 2-((1R,5R,6R,8aS)-6-hydroxy-5-(hydroxymethyl)-5,8a-dimethyl-2-methylenedecahydr onaphthalen-1-yl)ethyl nicotinate (compound 10b). White solid, 78% yield; HR-MS(ESI) m/z: 395.2190 [M+Na]+ (Calcd. for C23H32NaO6, 395.2193); 1H-NMR

ACCEPTED MANUSCRIPT (600 MHz, DMSO-d6): δ 7.98 (2H, dd, J = 8.1, 0.9 Hz, 2′, 6′-H), 7.66 (1H, t, J = 7.4 Hz, 4′-H), 7.53 (2H, t, J = 7.8 Hz 3′, 5′-H), 4.85 (1H, s, 13a-H), 4.58 (1H, s, 13b-H), 4.21 (2H, m, 12-H), 3.86 (1H, d, J = 11.1 Hz, 15a-H), 3.28 (1H, d, J = 10.9 Hz, 13

C-NMR (150

RI PT

15b-H), 3.19 (1H, m, 3-H), 1.10 (3H, s, 14-H), 0.92 (3H, s, 16-H).

MHz, DMSO-d6): δ 165.7, 134.9, 133.2, 129.8, 129.1, 129.0, 128.7, 78.4, 63.9, 62.7, 51.2, 42.1, 37.9, 34.5, 33.8, 27.7, 27.0, 22.9, 20.1, 19.4, 19.0.

SC

2-((1R,5R,6R,8aS)-6-hydroxy-5-(hydroxymethyl)-5,8a-dimethyl-2-methylenedecahydr

M AN U

onaphthalen-1-yl)ethyl 2-nitrobenzoate (compound 10c). White solid, 86% yield; HR-MS(ESI) m/z: 440.2041 [M+Na]+ (Calcd. for C23H31NNaO6, 440.2044); 1H-NMR (600 MHz, DMSO-d6): δ 8.06 (1H, m, 3'-H), 7.91 (3H, m, 4',5',6'-H), 4.87 (1H, s, 13a-H), 4.57 (1H, s, 13b-H), 4.19 (2H, m, 12-H), 3.85 (1H, d, J = 10.8 Hz, 15a-H), 13

C-NMR (150 MHz,

TE D

3.21 (1H, m, 3-H), 1.09 (3H, s, 14-H), 0.91 (3H, s, 16-H).

DMSO-d6) δ 164.6, 147.8, 134.5, 133.6, 132.8, 129.8, 128.9, 126.4, 124.1, 78.3, 65.0, 62.7, 51.2, 42.1, 37.9, 34.5, 33.8, 27.6, 26.4, 22.9, 20.1, 19.3, 18.9.

EP

2-((1R,5R,6R,8aS)-6-hydroxy-5-(hydroxymethyl)-5,8a-dimethyl-2-methylenedecahydr

AC C

onaphthalen-1-yl)ethyl 4-nitrobenzoate (compound 10d). White solid, 85% yield; HR-MS(ESI) m/z: 440.2040 [M+Na]+ (Calcd. for C23H31NNaO6, 440.2044); 1H-NMR (600 MHz, DMSO-d6): δ 8.36 (2H, d, J = 8.8 Hz, 3′, 5′-H), 8.18 (2H, d, J = 8.8 Hz, 2′, 6′-H), 4.86 (1H, s, 13a-H), 4.66 (1H, s, 13b-H), 4.12 (2H, m, 12-H), 3.83 (1H, dd, J = 10.9, 2.7 Hz, 15a-H), 3.24 (2H, m, 15b, 3-H), 1.08 (3H, s, 14-H), 0.63 (3H, s, 16-H). 13

C-NMR (150 MHz, DMSO-d6): δ 164.3, 150.2, 147.5, 135.2, 130.6, 123.9, 106.8,

78.4, 65.1, 62.6, 54.4, 52.1, 42.2, 38.5, 37.7, 36.4, 27.8, 24.0, 23.0, 22.8, 14.7.

ACCEPTED MANUSCRIPT 4.3. Biology 4.3.1. Cell culture and cell viability The RAW264.7 murine macrophage cell line was purchased from ATCC. The cells

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were cultured at 37 °C in 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM; Gibco Inc., NY, USA) supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin and 10% fetal bovine serum (FBS; Gibco Inc., NY, USA). To evaluate

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the effect of compounds on the cell viability, the cells were seeded into 96-well plates

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at a density of 5×104 cells/well and incubated with serum-free medium in the presence of different concentrations of compounds. After incubation for 24 h, the cell viability was determined by Cell-Counting Kit-8 (CCK8) methods. DMSO was also treated as a vehicle control. All of the experiments were performed in triplicate.

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4.3.2 Identification of nitric oxide (NO)

The RAW264.7 cells were seeded in 96-well plates and treated with different concentrations (0-100 µM) of compounds for 3 h, and then incubated with LPS (1

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µg/mL) for 24 h. DMSO with or without LPS were treated as a vehicle control or a

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model control. Nitrite accumulation in the culture medium was measured using Griess reagent at 540 nm with a microplate reader. The inhibition rates (%) of compounds treated groups were calculated and the IC50 value was determined to evaluate the NO inhibitory activity.

4.3.3 Enzyme Linked Immunosorbent Assay (ELISA) RAW264.7 cells in 96-well plates were treated with DMSO or compound 8m at 5, 10 and 20 µM for 3 h, and then stimulated with LPS, 1µg/mL, for 24 h. The production

ACCEPTED MANUSCRIPT of inflammatory cytokines IL-1β and IL-6 were determined by a commercial ELISA kit according to the manufacturer’s instructions. The optical density (OD) was measured at 540 nm.

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4.3.4 Western blot analysis The cells in 6-well plates were treated as described above and then incubated in the presence of LPS (1 µg/mL) for 24 h. The cells were collected and suspended in an

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extraction lysis buffer (Sigma-Aldrich) containing protease inhibitors. The protein

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concentration was determined using a protein assay reagent (Bio-Rad Laboratories) according to the manufacturer’s instructions. Equal amounts of total cellular protein (30 µg) were resolved by 10% SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membrane was incubated with blocking

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solution (5% skim milk), followed by an overnight incubation at 4 °C with the appropriate primary antibody. The following primary antibodies and dilutions were used: anti-β-actin (1:2000 dilution; Cell Signaling, MA, USA); anti-iNOS (1:200

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dilution; Cell Signaling); anti-COX-2 (1:2000 dilution; Cell Signaling); anti-NF-κB

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(1:2000 dilution; Cell Signaling); anti-IκBα (1:2000 dilution; Cell Signaling); anti-pIκBα (1:2000 dilution; Cell Signaling). The blots were washed three times with Tris-buffered saline containing Tween 20 (TBST), and then incubated with a 1:5000 dilution of a horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. The blots were again washed three times with TBST, and then developed using an enhanced chemiluminescence (ECL) kit (Thermo, CA, USA). 4.3.5 NF-κB/p65 nuclear translocation immunofluorescence assay

ACCEPTED MANUSCRIPT RAW264.7 cells were grown in a glass chamber and pretreated with DMSO or compound 8m for 2 h and then stimulated by 1 µg/mL LPS for 12 h. Cell treatment was terminated by washing with PBS, followed by fixation in freshly prepared 4%

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paraformaldehyde in PBS for 15 min. The fixed cells were washed three times with PBS and then permeabilized in 0.25% Triton X-100. After incubating with P65 antibody and secondary AlexaFluor488 antibody, DAPI was used to stain the nuclei at

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37 °C for 30 min in dark. Microscopy was performed using a Nikon eclipse.

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4.4 Statistical Analysis

Data were presented as the mean ± standard deviation of at least three independent experiments. Statistical analysis was performed using the GraphPad Prism software, version 4.00 (GraphPad Software Inc., San Diego, CA, USA) and a one-way analysis

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of variance (ANOVA) followed by Tukey’s multiple comparison tests. P<0.05 was considered statistically significant.

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Acknowledgements

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This study was financially supported by the National Natural Science Foundation of China (Nos. 81202426, 81473254, and 81773637), and Training Program Foundation for the Distinguished Young Scholars of University in Liaoning Province (No. LJQ2014104).

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Highlights 1. The derivatives of andrographolide with nitrogen-containing heterocycles, phenols and aromatic acids as bioisostere of lactone ring

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were synthesized. 2. Small-molecule phenols as bioisostere of lactone ring could improve the anti-inflammation efficacy of andrographolide.

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production in RAW264.7 macrophage.

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3. Compound 8m showed the most potent inhibition on LPS-induced NO

4. Compound 8m suppressed LPS-induced NO, IL-1β and IL-6 production.

5. The anti-inflammatory action mechanism of 8m was related to the

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inhibition of COX-2, iNOS and NF-κB signal pathway.