Total Synthesis-Enabled Systematic StructureâActivity Relationship

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Total Synthesis Enabled Systematic SAR Study for Development of a Bioactive Alkyne-Tagged Derivative of Neolaxiflorin L Mengxun Zhang, Magnolia Muk-Lan Lee, Weijian Ye, Wing-Yan Wong, Brandon Dow Chan, Sibao Chen, Lizhi Zhu, William Chi-Shing Tai, and Chi-Sing Lee J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b00748 • Publication Date (Web): 14 May 2019 Downloaded from http://pubs.acs.org on May 20, 2019

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The Journal of Organic Chemistry

Total Synthesis Enabled Systematic SAR Study for Development of a Bioactive Alkyne-Tagged Derivative of Neolaxiflorin L Mengxun Zhang,† Magnolia Muk-Lan Lee,ǁ Weijian Ye,† Wing-Yan Wong,ǁ Brandon Dow Chan,ǁ Sibao Chen,*,‡ Lizhi Zhu,*,†,⁋ William Chi-Shing Tai*,ǁ,‡ and Chi-Sing Lee*,†,§ †Laboratory

of Chemical Genomics, Peking University Shenzhen Graduate School,

Xili, Shenzhen 518055, China ǁDepartment

of Applied Biology and Chemical Technology, Hong Kong Polytechnic

University, Hung Hom, Hong Kong ‡State

Key Laboratory of Chinese Medicine and Molecular Pharmacology

(Incubation), Shenzhen Research Institute of The Hong Kong Polytechnic University, Shenzhen 518057, China ⁋Institute

of Translational Medicine, Shenzhen Second People’s Hospital, The First

Affiliated Hospital of Shenzhen University, Health Science Centre, Shenzhen 518035, China §Department

of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong

Kong

*E-mail: [email protected] (S. Chen), [email protected] (L. Zhu), [email protected] (W. C.-S. Tai), [email protected] (C.-S. Lee)

ACS Paragon Plus Environment

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

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Table of Content/Abstract Graphics O

C

OB

A

H

O

D

D

O OH

A

O

total synthesis enabled SAR

O O

O

B

H

OH

Library 1: analogs with different funcationalities

C

O OH

Library 2: analogs with skeleton changes

H

O OH

OH

(±)-NL-A2

Abstract Neolaxiflorin L (NL) is a low-abundance Isodon 7,20-epoxy-ent-kuarenoid and was found to be a promising anti-cancer drug candidate in our previous study. In order to study its structure-activity relationship (SAR), a diversity-orientated synthetic route towards two libraries of (±)-NL analogs, including analogs containing different functionalities in the same 7,20-epoxy-ent-kuarene skeleton and analogs with skeletal changes, has been developed. The results of this total synthesis enabled SAR successfully led to a bioactive alkyne-tagged NL derivative, which could be a useful probe for proteomics studies.


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Introduction Despite the dominant role of natural products in cancer chemotherapy, the adverse effects caused by their non-specific interactions with multiple biological targets has limited their use in cancer treatment.1 Therefore, studies of the multiple modes of action for natural product drug candidates are very important for improving their safety profiles. Chemical proteomics2 is a promising strategy for identifying biological targets of natural products and it requires a bioactive probe tagged with a chemically-activatable functional group. The most common method for preparation of probes for chemical proteomics is direct derivatization of the parent natural product. This method employed the natural product as the starting material and derivatize the most reactive functional group of the molecule via a simple operation. However, due to the limited position for functionalization, this method could lead to a natural product derivative with very low bioactivity, which is not useful for proteomics studies. An alternative is to prepare natural product derivatives via total synthesis. This strategy could enable a systematic structure-activity relationship (SAR) study that could lead to a biotically active probe for identification of cellular targets in chemical proteomics studies. 7,20-Epoxy-ent-kaurenoids are a special sub-class of Isodon diterpenoids and some of the most abundant members, such as oridonin (Or) and eriocalyxin B (EB) (Figure 1), have been reported efficacious for several cancer cell lines with low toxicity.3 Mechanistic studies have revealed that NF-kB,4 STAT35 and VEGFR-26 are possible cellular targets of EB, but its modes of action remain unclear. Recently, our



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group has developed a scalable synthesis towards this sub-class of natural products, including (±)-eriocalyxin B (EB), (±)-neolaxiflorin L (NL) and (±)-xerophilusin I (XI) (Figure 1), and found that (±)-NL shows remarkable efficacy in mice xenograft tumor models with no observable toxicity.7 However, the major problem for further investigation of NL is its extremely low isolation yield (3 mg from 10 kg dried leaves of I. eriocalyx var. laxiflora).3c Based on our previous reported scalable synthesis of this sub-class of Isodon diterpenoids, we herein reported the development of a bioactive alkyne-tagged derivative of NL based on a total synthesis enabled systemic SAR study.

11

20 2 3

18

1

10 5

4 19

9 6

12

8

13 14

16

OH

20

17

15

O

O

7

7

H

H

ent-Kaurene

6

OH

H

O

H

O OH

OH

Eriocalyxin B (EB)

OH O OH

OH Oridonin (Or)

7,20-epoxy-ent-Kaurene

O O

H

OH O H

O

O OH

H

OH

Neolaxiflorin L (NL)

O OH

OH

Xerophilusin I (XI)

Figure 1. Representative members of 7,20-epoxy-ent-kaurenoids

Results and Discussion 1. Synthesis of alkyne-tagged NL analog via direct derivation Direct derivatization has been a popular strategy for preparation of Or and EB analogs and some of these analogs exhibited good anti-cancer activities.3c,8 Encouraged by these results, we prepared an alkyne-tagged NL derivative via direct esterification of (±)-NL. As shown in Scheme 1, esterification of (±)-NL at the


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C6-hydroxyl was found to be difficult due to its hindered environment and intramolecular hydrogen bonding with the C15-carbonyl. After a survey of the esterification conditions, (±)-NL-A1 was obtained using excessive hex-5-ynoic acid with DCC and DMAP in moderate yields along with some recovered (±)-NL. With (±)-NL-A1 in hand, its in vitro cytotoxicity towards SW480 were investigated. Unfortunately, (±)-NL-A1 was found to be non-cytotoxic towards SW480 (IC50 > 40 μM). This result indicated that modification of the C6-hydroxyl could lead to complete loss of the in vitro cytotoxicity. Scheme 1. Synthesis of (±)-NL-A1 and its bioactivity

O O 6

H

15

O OH

hex-5-ynoic acid DCC, DMAP CH2Cl2

IC50 > 40 μM O O

30%

6

H

OH

(±)-NL

15

O OH

O

(±)-NL-A1 O

2. De novo designed NL analogs To develop a bioactive alkyne-tagged NL analog for proteomics studies, rational design based on a reliable SAR study is crucial. Thus, we have designed two libraries of NL analogs for a systematic SAR study. As shown in Figure 2, the design of the NL analogs in Library 1 was guided by the single variable principle for evaluating the importance of a particular functional group including the C1-ketone, C6-hydroxyl and enone of the D ring. Library 2 was designed for investigating the influence of skeletal changes, including the position of the oxygen-bridge, relative configurations of the AB ring junction and the position of the D ring. De novo synthesis of these NL



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analogs could be achieved via a diversity-oriented synthesis9 starting from the common intermediate (±)-4, which can be obtained from simple substrates 1 – 3 in gram-scales using our previously reported synthetic strategy.7 OPMB OH 1

O

O MeO P MeO O

O

O

TBSO 6 steps, 31% TBSO (gram-scales)

2

ref. 6

H

H

OTIPS (±)-4

diversity-oriented synthesis

3

O

C

OB

A

H

O

D

D

O OH

A

Library 1: analogs with different funcationalities

O

B

H

OH

C

OH

Library 2: analogs with skeleton changes

Figure 2. Libraries of NL analogs for a systematic SAR study

3. Synthesis of NL analogs in Library 1 The NL analogs in Library 1 contain the skeleton of 7,20-epoxy-ent-kaurene with different functionalities and they were prepared from a common intermediate (±)-5, which was prepared from (±)-4 in 6 steps with 24% overall yield.7 As shown in Scheme 2, reduction of (±)-5 by LiAlH4 in high diluted condition provides analog (±)-6 (15-epi-enmelol) as a single diastereomer.10 The high diaselectivity of this reduction could be rationalized by addition of the hydride at the less hindered α-face of (±)-5. Selective oxidation of the allylic alcohol of (±)-5 using IBX in THF/DMSO led to analog (±)-7. Subsequent reduction of (±)-7 with NaBH4 inverted the configuration of the allylic alcohol and provided analog (±)-8 as a single diastereomer. The relative configurations of (±)-8 is determined by 2D NMR


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experiments. For synthesis of analog (±)-11, oxidation of (±)-5 with excessive Dess-Martin periodinane (DMP) gave trione (±)-9. Saegusa oxidation11 of (±)-9 followed by removal of silyl groups provided analog (±)-10. Finally, Michael addition of methanol to (±)-10 under acetic acid afforded analog (±)-11, which was characterized by 2D NMR experiments. Scheme 2. Synthesis of NL analogs (±)-6 – 11 in Library 1 OH

OH 20

20

LAH, THF

1 15

O

6

H

7

OH OH

1 6

H

OH (±)-15-epi-enmelol (6)

OH

O

HO

6 7

O

(±)-5

20

AlH3 H OH Me

OH OH

O

85% H

72%

1. TMSCl, NaI, HMDS 2. Pd(OAc)2, CH3CN 3. HCl (aq), THF/H2O

69%

O O

H

O

O OH

O

NaBH4, 0°C THF/H2O, 10 min

H

(±)-7 92%

OH

TsOH, MeOH

O OH

O (±)-10 80%

O O

H

O OH

O (±)-9

(±)-5

OH

1

Me

7

O

IBX, THF/DMSO 1h

15

15

O

89%

O

DMP, NaHCO3 CH2Cl2

O

OH OH (±)-8

OMe

H

O

O OH

O

(±)-11

4. Synthesis of NL analogs in Library 2 To study the effects of the oxygen bridge on the in vitro cytotoxicity of NL, the 3,20-epoxy analog (±)-12 and 6,20-epoxy analog (±)-14 were considered and they could be synthesized from the 7,20-epoxy framework via oxygen-bridge migration. As shown in Scheme 3, analog (±)-12 (Laxiflorin J) was obtained from (±)-EB via acid-catalyzed acetal ring-opening followed by oxa-Michael addition, which provide



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(±)-12 with 75% yield in one-pot. However, attempts of migrating the 7,20-epoxy bridge of (±)-5 to the 6,20-epoxy moiety using a variety of Brønsted and Lewis acids failed. These acidic conditions led to preliminary recovered starting materials or decomposition under high reaction temperature. Surprisingly, we finally found that (±)-5 can be converted to (±)-13 quantitatively after stirring in methanol at rt overnight. With the 6,20-epoxy moiety established, analog (±)-14 was obtained via Dess-Martin oxidation of (±)-13. Scheme 3. Synthesis of NL analogs (±)-12 – 14 in Library 2 O

O

H O

aq. HCl, THF

H

A

H BO 6

H

O

7

MeOH, overnight OH OH

(±)-5

100%

6

H

O

O

OH (±)-Laxiflorin J (LJ, 12)

OH (±)-EB

OH

O

86%

O OH

6

H

OH A

H

DMP, NaHCO3 CH2Cl2

B O7

H

6

O

OH

71%

OH (±)-13

O A

H B O7

H

6

O

O

OH (±)-14

An analog without the oxygen-bridge is also considered. However, attempts of acetal ring opening of (±)-5 failed. The 7,20-epoxy moiety of (±)-5 was found to be very stable under a variety of acid and basic conditions due to the trans-AB ring system. On the other hand, we found that the 7,20-epoxy moiety of (±)-16, which bears a cis-AB ring system, is much less stable. The oxygen-bridge of (±)-16 was opened successfully after allylic oxidation12 followed by Dess-Martin oxidation, which provided analog (±)-17 in good yields (Scheme 4). Compound (±)-16 was prepared by removal of silyl groups of (±)-15, which was obtained from (±)-4 in 65%.7


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Scheme 4. Synthesis of NL analog (±)-17 in Library 2 TBSO TBSO A

B

H

7

OTMS

6

1. SeO2, tBuOOH CH2Cl2 2. DMP, CH2Cl2

OH

TBAF THF

A

98%

O

B

H

(±)-15

7

6

O OH

57%

OH

A

B 6

H

(±)-16

7

O

O

(±)-17

Analog (±)-22 was designed for studying the effects of the enone moiety in a different C,D ring system. As shown in Scheme 5, synthesis of analog (±)-22 began with intramolecular Mukaiyama Michael reaction of (±)-4 using Me2AlCl. Selective deprotection of the primary TBS ether established the oxygen-bridge and gave (±)-19, which contains the suitable conformation for selective silyl enol ether formation at C11-12. Thus, ketal (±)-19 was converted to silyl enol ether followed by carbocyclization with ZnBr2,13 which gave (±)-20 as a single diastereomer. The structure of (±)-20 was determined by 2D NMR experiments. Finally, allylic and Dess-Martin oxidation of the cyclized product (±)-20 finished the synthesis of analog (±)-22. Scheme 5. Synthesis of NL analog (±)-22 in Library 2 TBSO TBSO

H

O 11

12

13

Me2AlCl CH2Cl2

OTIPS

O

TBSO TBSO

68%

11

(±)-18

O O OH (±)-22

DMP, NaHCO3, CH2Cl2

OH

11

OH SeO2, t-BuOOH CH2Cl2

O

82% H

OH

12

11

H

OH (±)-19 73% O 11

OH

13

O

90%

(±)-21

13

O

1. TMSCl, NaI, HMDS, CH3CN 2. ZnBr2, CH2Cl2 3. TBAF, THF

O

11

H

90%

O

O O

TBSO

13

TBAF, THF H

(±)-4

12

O

H

OH

(±)-20

The 6,7-seco analogs (±)-24 and (±)-25, contains the C,D ring system in a different skeleton, were synthesized from (±)-7 via reduction of the C6-ketone



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followed by oxidative cleavage14 of the intermediate diol (±)-23 that generated in situ (Scheme 6a). This synthetic sequence provided a 1:1 mixture of (±)-24 and (±)-25 with 86% yields. The structure of both (±)-24 and (±)-25 are determined by 2D NMR experiments and the comparison of the 3-dimentional conformations of (±)-24 and (±)-25 to (±)-NL was illustrated in Scheme 6b. Scheme 6. Synthesis of NL analog (±)-24 and (±)-25 (a) OH

NaBH4 EtOH

O H

(b)

O OH

O

H

O

HO

HO

H

OH OH

O

OH (±)-23

O HO

O

O (±)-24

H

O

H H

O

H

O (±)-25

1:1 OH H

O

H H

O

Me

O

O +

86%

H

Me

HO O

O

MnO2

O

0 °C

(±)-7

HO O

OH

Me O

O

O Me

(±)-24

(±)-NL

Me O

Me

(±)-25

5. In vitro cytotoxicity of NL analogs With the two libraries of (±)-NL analogs prepared, their in vitro cytotoxicity towards a panel of four human cancer cell lines, including colon adenocarcinoma (SW480), hepato-cellular carcinoma (SMMC7721), breast adenocarcinoma (MCF-7) and human alveolar basal epithelial adenocarcinoma (A549), were evaluated using MTT assay.15 In comparison of the IC50 values of (±)-EB, (±)-NL and (±)-XI, variation of oxidation states in the A ring showed only modest effects on the in vitro cytotoxicity (Figure 3).


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O

O

H O

A

H

OH

O OH

H

(±)-EB

OH

H O

A

OH

O OH

H

(±)-NL

H O

A

O OH OH (±)-XI

Figure 3. A comparison study on the A ring The effects of the C6-hydroxyl were examined by comparison of (±)-EB, (±)-NL and (±)-XI with their C6-keto analogs (±)-10, (±)-9 and (±)-7 respectively (Figure 4a – c). Among the three comparison groups, analog (±)-9 showed the most dramatic loss of in vitro cytotoxicity in all tested cell lines compare to (±)-NL (Figure 4b). These results indicated that the C6-hydroxyl of NL is important for its in vitro cytotoxicity and is consistent with the poor in vitro cytotoxicity of (±)-NL-A1. On the other hand, analog (±)-7 showed no significant loss in in vitro cytotoxicity compare to (±)-XI (Figure 4c). These differences could be rationalized by their different lipid-water partition coefficients and the intramolecular hydrogen bonding between the C6-hydroxyl and the enone of the D ring. (a)

(b) O

O O 6

H

OH

O OH (±)-EB

(c) O

O 6

H

O OH

O (±)-10

OH

O O 6

H

OH

O OH (±)-NL

O 6

H

O

O OH (±)-9

OH O 6

H

OH

Figure 4. Comparison studies on the C6-carbonyl


O OH (±)-XI

O

O OH

O

(±)-7

6

H


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The effects of the enone in D ring were examined by comparison of three groups of compounds including (±)-XI with (±)-6, (±)-7 with (±)-5 and (±)-6, and (±)-10 with (±)-11 (Figure 5). When the carbonyl of the enone was replaced by either an equatorial or axial hydroxyl, the in vitro cytotoxicity loss completely (Figure 5a, b). Analog (±)-11, without the enone in D ring, also showed no in vitro cytotoxicity (Figure 5c). These results indicated that the enone in D ring is essential for the in vitro cytotoxicity. (a)

(b) OH O H

OH

O OH (±)-XI

(c) OH

OH O H

OH OH OH (±)-6

H

OH O

O OH

O

(±)-7

H

O

OH O

OH OH

O

(±)-5

H

O

OH OH

O

(±)-8

OMe

O

H

O

O OH

O

(±)-10

H

O

O OH

O

(±)-11

Figure 5. Comparison studies on the enone in D ring Interestingly, the 3,20- and 6,7-epoxy analogs ((±)-12 and (±)-14 respectively) showed similar levels of in vitro cytotoxicity compare to (±)-NL (Figure 6a), suggesting that the 7,20-epoxy bridge may not be essential for the activities. Analog (±)-17, which contains a cis-AB ring system without the 7,20-epoxy bridge, exhibited good activities towards most of the tested cell lines. Change of the CD ring (analog (±)-22) led to dramatically decrease in in vitro cytotoxicity (Figure 6b). Moreover, the 6,7-seco derivatives (±)-24 and (±)-25 both showed good in vitro cytotoxicity (Figure 6c). These results are in good concurrent with other comparison groups, indicating


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that the CD ring is a more important moiety than the AB ring and the 7,20-epoxy moieties for the in vitro cytotoxicity of NL.

(a)

(b)

O

O O H

O OH

OH

(±)-NL

O

O H

O OH

O

O

O H

(±)-12 (LJ)

(c)

O OH

O

O

(±)-14

O H

OH

O OH (±)-NL

O

H

HO O

O

O

O O OH H

(±)-17

O

O

O

O H

OH H

(±)-22

H

OH

O OH (±)-NL

HO O O

O

O (±)-24

O O

(±)-25

(h) Figure 6. Comparison studies on skeletal changes

6. Synthesis of alkyne-tagged NL analog based on the SAR study Based on the results of the SAR study,16 the best position for introducing an alkyne-tag to the natural product could be the A ring. As shown in Scheme 7, silylation of (±)-NL led to the silyl enol ether on the A ring with TMS ethers formation at C6- and C7-hydroxyl. Upon treatment of m-CPBA followed by acid hydrolysis of the tris(TMS) intermediate provided (±)-26.17 Finally, selective esterification of the less hindered C2-hydroxyl afforded (±)-NL-A2 in good yields. To our delight, this alkyne-tagged NL derivative showed significant in vitro cytotoxicity towards SW480 with IC50 value equals 7.9 μM. Although (±)-NA-A2 showed modest in vitro cytotoxicity, it will be useful in proteomics studies due to its potential covalent binding ability towards its cellular targets via the enone in the D ring.



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Scheme 7. Synthesis of (±)-NL-A2 and its in vitro cytotoxicity 1. HMDS, NaI TMSCl, CH3CN 2. mCPBA, NaHCO3 3. HCl (aq.), THF

O O H

O HO

O OH

H

OH (±)-NL

IC50 = 7.9 μM

5-hexynoic acid, EDCI, DMAP

O

O OH

OH

(±)-26

53% from NL

O O

O O H

O OH

OH

(±)-NL-A2

Conclusion In summary, synthesis and bio-evaluated of alkyne-tagged NL derivatives, (±)-NL-A1 and (±)-NL-A2, have been accomplished. (±)-NL-A1 was prepared rapidly via direct derivatization from (±)-NL, which led to significantly loss of in vitro cytotoxicity. This problem was solved by a total synthesis enabled systematic structure-activity relationship study, which led to the development of (±)-NL-A2. This alkyne-tagged NL derivative exhibited significant in vitro cytotoxicity and could be a useful probe for proteomics studies. We are currently investigating the biological targets of NL and its mode of actions using (±)-NL-A2.

Experimental Section


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General information for synthesis Unless otherwise noted, all air and water sensitive reactions were carried out under a nitrogen atmosphere with dry solvents under anhydrous conditions. All the chemicals were purchased commercially and used without further purification. Anhydrous THF and toluene were distilled from sodium-benzophenone, and dichloromethane was distilled from calcium hydride. Yields were determined chromatographically, unless otherwise stated. Reactions were monitored by thin-layer chromatography (TLC) carried out on 0.25 mm silica gel plates (60F-254) that were analyzed by staining with KMnO4 solution (200 mL H2O of 1.5 g KMnO4, 10 g K2CO3 and 1.25 mL of 10% aqueous NaOH), fluorescence following 254 nm irradiation or by staining with anisaldehyde (450 mL of 95% EtOH, 25 mL of conc. H2SO4, 15 mL of AcOH, and 25 mL of anisaldehyde). Silica gel (60, particle size 0.040-0.063 mm) was used for flash chromatography. NMR spectra were recorded at 300 MHz (1H: 300 MHz, MHz), 400 MHz (1H: 400 MHz,

13C:

13C:

100 MHz) and 500 MHz (1H: 500 MHz,

75

13C:

125 MHz). The following abbreviations were for multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, b = broad. High-resolution mass spectra were obtained from a MALDI-TOF Mass Spectrometer. All the IR spectra were obtained using FT-IR Spectrometer.

General information for MTT assays Cell culture: Four human cancer cell lines, SW480 (colon adenocarcinoma), SMMC7721 (hepatocellular carcinoma), MCF-7 (breast adenocarcinoma) and A549



Page 16 of 38

(human alveolar basal epithelial adenocarcinoma) were purchased from the American Type Culture Collection (Manassas, USA) and maintained in DMEM or RPMI-1640 medium (Life Technologies, USA) supplemented with 10% heat-inactivated FBS and penicillin/ streptomycin (50 U/mL) at 37 °C, 5% CO2. Cell viability and cytotoxicity assays: The cell viability of different cancer cell lines under drug treatment was determined using the MTT assay. 5 × 104 HL60 or 3 – 6 × 103 SW480, SMMC7721, MCF-7 or A549 cells were seeded in each well of 96-well plates. After 24 h, cells were treated with different drugs at various concentrations for another

48

h.

Cells

were

then

treated

with

0.5

mg/mL

3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at 37 °C for 4 h. Media was removed after incubation and DMSO was added to each well to dissolve the formazan crystals. Absorbance was measured at 570 nm using a Multiskan™ FC Microplate Photometer (ThermoFisher Scientific, USA). The IC50 of the cell lines were calculated.

Experimental Details (±)-6-Hydroxy-4,4-dimethyl-8-methylene-1,7-dioxododecahydro-1H-6,11b-(epox ymethano)-6a,9-methanocyclohepta[a]naphthalen-5-yl hex-5-ynoate ((±)-NL-A1) To a stirred solution of (±)-NL (25 mg, 0.072 mmol) in CH2Cl2 (3.5 mL), hex-5-ynoic acid (0.08 mL, 0.72 mmol), DCC (148.6 mg, 0.72 mmol), DMAP (8.8 mg, 0.072 mmol) were added at rt. After stirring at rt for 10 h, the reaction was quenched by addition of water. The aqueous layer was extracted with ethyl acetate (15 mL × 4).


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The combined organic extracts were washed with brine, dried over MgSO4, filtered, and concentrated. Silica gel flash column chromatography (hexanes/ethyl acetate = 10:1) of the residue gave a colorless oil (9.5 mg, 0.02 mmol, 30%) as the product. (±)-NL-A1: 1H NMR (300 MHz, chloroform-d) δ 5.92 (s, 1H), 5.34 (s, 1H), 5.15 (d, J = 9.0 Hz, 1H), 4.36 (d, J = 9.9 Hz, 1H), 4.32-4.29 (m, 1H), 4.02 (d, J = 12.0 Hz, 1H), 3.00 (dd, J = 9.9, 4.5 Hz, 1H), 2.68 (q, J = 7.8 Hz, 2H), 2.54-2.44 (m, 2H), 2.36-2.27 (m, 4H), 2.09 (d, J = 12.3 Hz, 1H), 1.98-1.87 (m, 4H), 1.78-1.67 (m, 2H), 1.50-1.39 (m, 2H), 1.16 (dd, J = 13.5, 6.6 Hz, 1H), 1.01 (s, 3H), 0.99 (s, 3H). 13C{1H} NMR (125 MHz, chloroform-d) δ 211.7, 202.1, 176.6, 152.9, 116.3, 97.1, 83.2, 74.6, 69.1, 64.9, 57.4, 55.2, 49.0, 47.1, 37.8, 35.5, 33.6, 33.4, 32.8, 29.6, 29.4, 25.5, 23.5, 23.4, 18.5, 17.9. IR (KBr) 3368, 2852, 2786, 2022, 1720, 1114, 1036, 679 cm-1 HRMS (ESI/[M+Na]+) calcd. for C26H32O6Na: 463.2097. found 463.2105. (±)-15-epi-Enmelol ((±)-6) To a stirred solution of (±)-5 (1.5 g, 4.5 mmol) in THF (900 mL) was added LiAlH4 (0.57 g, 14.9 mmol) slowly at rt. After stirring at rt for 1.5 h, the reaction was quenched by addition of 4 N HCl aqueous solution (10 mL) at 0 ˚C. The aqueous layer was then extracted with ethyl acetate/THF = 2:1 (200 mL × 4). The combined organic extracts were washed with brine, dried over MgSO4, filtered, and concentrated. Silica gel flash column chromatography (hexanes/THF = 1:1) of the residue gave a white solid (1.4 g, 4.0 mmol, 89%) as the product. (±)-15-epi-enmelol ((±)-6): 1H NMR (500 MHz, pyridine-d5) δ 7.94 (s, 1H), 6.97 (d, J = 3.5 Hz, 1H), 6.31 (s, 1H), 5.73 (d, J = 3.0 Hz, 1H), 5.32 (s, 1H), 5.16 (s, 1H), 5.09 (s, 1H), 5.03 (s, 1H), 4.39 (s, 1H), 4.16 (s, 2H), 3.70 (s, 1H), 2.65-2.76 (m, 2H),



1.99-2.34 (m, 6H), 1.72-1.85 (m, 3H), 1.52-1.62 (m, 1H), 1.34 (s, 3H), 1.20-1.30 (m, 1H), 1.24 (s, 3H). 13C{1H} NMR (125 MHz, pyridine-d5) δ 163.2, 109.6, 98.3, 82.0, 74.6, 65.2, 65.1, 52.8, 50.8, 44.9, 41.0, 36.0, 34.3, 33.8, 33.5, 32.6, 27.8, 27.5, 23.2, 15.2. IR (KBr) 3213, 2965, 2927, 1859, 2855, 1709, 1114, 1069, 1039 cm-1 HRMS (ESI/[M+Na]+) calcd. for C20H30O5Na: 373.1991. found 373.1998. Melting point: 296-297 ℃ (±)-1,6-Dihydroxy-4,4-dimethyl-8-methyleneoctahydro-1H-6,11b-(epoxymethano)6a,9-methanocyclohepta[a]naphthalene-5,7(6H,8H)-dione ((±)-7) To a stirred solution of (±)-5 (45.0 mg, 0.13 mmol) in DMSO (5 mL) and THF (8 mL) was added IBX (44.8 mg, 0.16 mmol) at 0 ˚C and the mixture was stirred at 0 ˚C for 4 h. The reaction was quenched by saturated Na2S2O3 (aq.) at 0 ˚C, the aqueous layer was then extracted with ethyl acetate/THF = 2:1 (20 mL × 4). The combined organic extracts were washed with water, saturated NaHCO3 aqueous solution, brine, dried over MgSO4, filtered, and concentrated. Silica gel flash column chromatography (hexanes/ethyl acetate = 2:1) of the residue gave a white solid (32.4 mg, 0.09 mmol, 72%) as the product. (±)-7: 1H NMR (300 MHz, methanol-d4) δ 5.80 (s,1H), 5.40 (s, 1H), 4.60 (s, 1H), 4.11 (d, J = 10.2 Hz, 1H), 4.00 (d, J = 10.2 Hz, 1H), 3.54 (s, 1H), 3,12-3.06 (m, 1H), 2.44-2.32(m, 2H), 2.24 (d, J = 12.3, 1H), 2.19 (m, 2H), 2.06-1.97 (m, 1H), 1.94-1.83 (m, 4H), 1.75-1.70 (m, 1H), 1.58-1.43 (m, 2H), 1.41 (s, 3H), 1.24-1.13(m, 1H), 1.04 (s, 3H).

13C{1H}

NMR (125 MHz, methanol-d4) δ 205.2,

204.2, 152.9, 114.9, 92.4, 65.1, 63.9, 57.4, 54.9, 44.7, 43.2, 34.6, 34.1, 33.8, 33.3, 29.5, 25.7, 23.7, 21.2, 15.6. IR (KBr) 3318, 2986, 2956, 2876, 1752, 1718, 1073,


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1115, 913 cm-1 HRMS (ESI/[M+Na]+) calcd. for C20H26O5Na: 369.1678. found 369.1680. Melting point: 197-198 ℃ (±)-1,6,7-Trihydroxy-4,4-dimethyl-8-methylenedecahydro-1H-6,11b-(epoxymeth ano)-6a,9-methanocyclohepta[a]naphthalen-5(6H)-one ((±)-8) To a stirred solution of (±)-7 (52.0 mg, 0.15 mmol) in H2O (7.5 mL) and THF (7.5 mL) was added NaBH4 (5.7 mg, 0.15 mmol) at 0 ˚C. The resulting solution was stirred at 0 ˚C for 15 min. The aqueous layer was then extracted with ethyl acetate/THF = 2:1 (20 mL × 4). The combined organic extracts were washed with brine, dried over MgSO4, filtered, and concentrated. Silica gel flash column chromatography (hexanes/THF = 2:1) of the residue gave a white solid (47.9 mg, 0.14 mmol, 92%) as the product. (±)-8: 1H NMR (400 MHz, DMSO-d6) δ 5.99 (s, 1H), 5.14 (d, J = 6.0 Hz, 1H), 4.88 (s, 1H), 4.80 (s, 1H), 4.38 (d, J = 4 Hz, 1H), 3.92-3.83 (m, 2H), 3.72 (d, J = 11.2 Hz, 1H), 2.50-2.43 (m, 2H), 2.24 (s, 1H), 2.13-2.01 (m, 2H), 1.74-1.67 (m, 1H), 1.59-1.45 (m, 4H), 1.39-1.29 (m, 2H), 1.23 (s, 3H), 0.97 (d, J = 13.2 Hz, 1H), 0.88 (s, 3H).

13C{1H}

NMR (100 MHz, DMSO-d6) δ 206.7, 161.9, 107.3, 93.7, 74.0, 64.7, 63.8, 56.5, 48.7, 42.2, 36.1, 35.9, 34.7, 34.4, 34.0, 32.8, 26.3, 24.9, 22.7, 14.3. IR (KBr) 3221, 2988, 2870, 2769, 1763, 1734, 1096. 1088 cm-1 HRMS (ESI/[M+Na]+) calcd. for C20H28O5Na: 371.1843. found 371.1850. Melting point: 257-258 ℃ (±)-6-Hydroxy-4,4-dimethyl-8-methyleneoctahydro-1H-6,11b-(epoxymethano)-6a,9 -methanocyclohepta[a]naphthalene-1,5,7(6H,8H)-trione

((±)-9)

To

a

stirred

solution of (±)-5 (45.0 mg, 0.13 mmol) and NaHCO3 (163.8 mg, 1.95 mmol) in CH2Cl2 (13 mL) was added DMP (276.9 mg, 0.65 mmol) at 0 ˚C. After stirring at rt



Page 20 of 38

for 1 h, the reaction was quenched by addition of saturated Na2S2O3 aqueous solution (4 mL). The aqueous layer was extracted with ethyl acetate/THF = 2:1 (20 mL × 4). The combined organic extracts were washed with saturated NaHCO3 aqueous solution, brine, dried over MgSO4, filtered, and concentrated. Silica gel flash column chromatography (hexanes/EA = 5:1) of the residue gave a white solid (36.6 mg, 0.11 mmol, 85%) as the product. (±)-9: 1H NMR (500 MHz, chloroform-d) δ 5.91 (s, 1H), 5.41 (s, 1H), 4.36 (d, J = 10.0 Hz, 1H), 4.14 (d, J = 10.0 Hz, 1H), 3.11 (dd, J = 10.0, 4.5 Hz, 1H), 2.70 (s, 1H), 2.48-2.41 (m, 2H), 2.39-2.30 (m, 1H), 2.28 (d, J = 12.5 Hz, 1H), 2.12 (dd, J = 12.5, 4.5 Hz, 1H), 2.01-1.93 (m, 2H), 1.89-1.83 (m, 2H), 1.78-1.72 (m, 1H), 1.50 (s, 3H), 1.48-1.43 (m, 1H), 1.20 (s, 3H).

13C{1H}

NMR (75 MHz,

chloroform-d) δ 208.8, 203.9, 201.3, 151.4, 117.2, 91.9, 65.1, 62.6, 54.7, 51.6, 45.5, 40.1, 37.0, 34.0, 32.4, 29.3, 23.8, 23.7, 18.4. IR (KBr): 3356, 2965, 2922, 2863, 1747, 1734, 1701, 1697, 1263, 1048, 799. HRMS (ESI/[M+Na]+) calcd. for C20H24O5Na: 367.1521. found 367.1529. Melting point: 182-183 ℃ (±)-6-Hydroxy-4,4-dimethyl-8-methylene-4,4a,9,10,11,11a-hexahydro-1H-6,11b-( epoxymethano)-6a,9-methanocyclohepta[a]naphthalene-1,5,7(6H,8H)-trione ((±)-10) To a stirred mixture of (±)-9 (156 mg, 0.45 mmol) in CH3CN (2 mL), NaI (800 mg, 5.33 mmol) and HMDS (1.8 mL, 8.63 mmol) were added at rt, TMSCl (0.8 mL, 6.31 mmol) was added slowly at 0 ˚C. After stirring at rt for 12 h, the reaction was diluted with hexanes (30 mL) and quenched by addition of water (5 mL). The aqueous phase was extracted with hexanes (20 mL × 3). The combined organic extracts were washed with Na2S2O3 aqueous solution until clear, and then washed


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with brine, dried over MgSO4, filtered, and concentrated. To a stirred solution of the residue in CH3CN (5 mL) was added Pd(OAc)2 (200 mg, 0.90 mmol) at room temperature. After stirring at rt for 6 h, the reaction was quenched by addition of a 0.5 N HCl aqueous solution (5 mL). The aqueous phase was extracted with ethyl acetate (20 mL × 3). The combined organic extracts were washed with brine, dried over MgSO4, filtered, and concentrated. Silica gel flash column chromatography (hexanes/ethyl acetate = 4:1) of the residue gave a white solid (106 mg, 0.31 mmol, 69%) as the product. (±)-10: 1H NMR (500 MHz, methanol-d4) δ 6.74 (d, J = 10.0 Hz, 1H), 5.85-5.79 (m, 2H), 5.42 (s, 1H), 4.37 (d, J = 10.0 Hz, 1H), 4.01 (d, J = 10.0 Hz, 1H), 3.16-3.08 (m, 1H), 2.95 (s, 1H), 2.38 (d, J = 12.0 Hz, 2H), 2.28-2.18 (m, 1H), 2.11-2.03 (m, 2H), 1.95-1.86 (m, 1H), 1.61 (s, 3H), 1.45-1.38 (m, 1H), 1.34-1.28 (m, 1H), 1.25 (s, 3H).

13C{1H}

NMR (125 MHz, methanol-d4) δ 204.8, 201.5, 195.5,

159.4, 152.7, 125.3, 115.2, 92.8, 65.1, 59.8, 55.2, 46.7, 35.8, 34.3, 30.3, 29.5, 23.5, 22.2, 19.1. IR (KBr): 3363, 2989, 2931, 2865, 1754, 1703, 1680, 1057, 699. HRMS (ESI/[M+Na]+) calcd. for C20H22O5Na: 365.1365. found 365.1371. Melting point: 185-186 ℃ (±)-6-Hydroxy-8-(methoxymethyl)-4,4-dimethyl-4,4a,9,10,11,11a-hexahydro-1H6,11b-(epoxymethano)-6a,9-methanocyclohepta[a]naphthalene-1,5,7(6H,8H)-trio ne ((±)-11) To a stirred solution of (±)-10 (30.0 mg, 0.09 mmol) in MeOH (9 mL) was added TsOH (0.4 mg, 0.02 mmol) at rt. After stirring at rt for 4 h, the aqueous layer was extracted with ethyl acetate/THF = 2:1 (20 mL × 4). The combined organic extracts were washed with saturated NaHCO3 aqueous solution, brine, dried over



MgSO4, filtered, and concentrated. Silica gel flash column chromatography (hexanes/ethyl acetate = 4:1) of the residue gave a white solid (27.0 mg, 0.07 mmol, 80%) as the product. (±)-11: 1H NMR (500 MHz, chloroform-d) δ 6.65 (d, J = 10.0 Hz, 1H), 5.84 (d, J = 10.0 Hz, 1H), 4.48 (s, 1H), 4.34 (d, J = 10.0 Hz, 1H), 3.98 (d, J = 12.0 Hz, 1H), 3.61 (dd, J = 10.0, 4.5 Hz, 1H), 3.55-3.48 (m, 1H), 3.32 (s, 3H), 2.86 (s, 1H), 2.81-2.77 (m, 1H), 2.68-2.64 (m, 1H), 2.39 (d, J = 13.0 Hz, 1H), 2.18-2.11 (s, 2H), 2.03-1.98 (s, 1H), 1.86-1.79 (m, 2H), 1.62 (s, 3H), 1.51-1.45 (s, 1H), 1.24 (s, 3H). 13C{1H}

NMR (125 MHz, chloroform-d) δ 215.9, 201.2, 194.8, 159.3, 125.9, 92.1,

68.5, 65.6, 59.9, 58.9, 55.9, 55.2, 47.9, 47.1, 36.2, 31.4, 29.3, 25.8, 23.2, 19.8, 18.9. IR (KBr): 3362, 2987, 2928, 2865, 2859, 1751, 1700, 1680, 1112, 696. HRMS (ESI/[M+Na]+) calcd. for C21H26O6Na: 397.1627. found 397.1630. Melting point: 182-184 ℃ (±)-Laxiflorin J ((±)-12) To a stirred solution of (±)-EB (45.0 mg, 0.13 mmol) in THF (8 mL) was added 0.5 N HCl aqueous solution (5 mL). After stirring at rt for 48 h, the solution was extracted with ethyl acetate/THF = 2:1 (20 mL × 4). The combined organic extracts were washed with saturated NaHCO3 aqueous solution, brine, dried over MgSO4, filtered, and concentrated. Silica gel flash column chromatography (hexanes/ethyl acetate = 2:1) of the residue gave a white solid (38.7 mg, 0.11 mmol, 86%) as the product. (±)-laxiflorin J ((±)-12): 1H NMR (500 MHz, pyridine-d5) δ 7.94 (s, 1H), 6.97 (d, J = 3.5 Hz, 1H), 6.31 (s, 1H), 5.73 (d, J = 3.0 Hz, 1H), 5.32 (s, 1H), 5.16 (s, 1H), 5.09 (s, 1H), 5.03 (s, 1H),

4.39 (s, 1H), 4.16 (s, 2H), 3.70 (s, 1H),

2.65-2.76 (m, 2H), 1.99-2.34 (m, 6H), 1.72-1.85 (m, 3H), 1.52-1.62 (m, 1H), 1.34 (s,


Page 22 of 38

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3H), 1.20-1.30 (m, 1H), 1.24 (s, 3H). 13C{1H} NMR (125 MHz, pyridine-d5) δ 163.2, 109.6, 98.3, 82.0, 74.6, 65.2, 65.1, 52.8, 50.8, 44.9, 41.0, 36.0, 34.3, 33.8, 33.5, 32.6, 27.8, 27.5, 23.2, 15.2. IR(KBr) 3428, 2935, 1741, 1699,1641, 1452, 1203, 1127, 938. HRMS (ESI/[M+Na]+) calcd. for C20H24O5Na: 367.1521 found 367.1531. Melting point: 173-175℃ (±)-1,5,7-Trihydroxy-4,4-dimethyl-8-methylenedodecahydro-6H-5,11b-(epoxyme thano)-6a,9-methanocyclohepta[a]naphthalen-6-one ((±)-13) A solution of (±)-5 (480 mg, 1.38 mmol) in CH3OH (300 mL) was stirred at rt for 12 h. Concentration and silica gel flash column chromatography (hexanes/THF = 2:1) of the residue gave a white solid (480 mg, 1.38 mmol, 100%) as the product. (±)- 13: 1H NMR (400 MHz, methanol-d4) δ 5.24 (s, 1H), 5.17 (s, 1H), 4.44 (d, J = 12.0 Hz, 1H), 4.36 (s, 1H), 4.22 (d, J = 12.0 Hz, 1H), 3.47 (t, J = 3.2 Hz, 1H), 3.31 (p, J = 1.6 Hz, 4H), 2.70 (t, J = 6.8 Hz, 1H), 2.30 (s, 1H), 2.20-2.11 (m, 1H), 2.04-1.82 (m, 3H), 1.73-1.60 (m, 2H), 1.54-1.46 (m, 1H), 1.44-1.31 (m, 3H), 1.21 (s, 3H), 1.06 (s, 3H). 13C{1H} NMR (100 MHz, methanol-d4) δ 175.7, 159.5, 110.8, 82.0, 80.5, 70.4, 63.9, 51.3, 50.7, 45.5, 41.5, 36.3, 34.9, 33.1, 32.2, 31.7, 26.9, 25.5, 20.7, 15.9. IR(KBr) 3410, 2948, 2876, 1734, 1191, 1136, 1060, 1001, 909. HRMS (ESI/[M+Na]+) calcd. for C20H28O5Na: 371.1834 found 371.1839. Melting point: 225-226 ℃ (±)-5-Hydroxy-4,4-dimethyl-8-methyleneoctahydro-1H-5,11b-(epoxymethano)-6a,9 -methanocyclohepta[a]naphthalene-1,6,7(2H,8H)-trione ((±)-14) To a stirred solution of (±)-13 (40.0 mg, 0.11 mmol) and NaHCO3 (138.6 mg, 1.65 mmol) in CH2Cl2 (5 mL) was added DMP (243.3 mg, 0.55 mmol) was at 0 ˚C. After stirring at



rt for 14 h, the reaction was quenched by addition of a saturated Na2S2O3 aqueous solution (4 mL). The aqueous layer was extracted with ethyl acetate/THF = 2:1 (20 mL × 4). The combined organic extracts were washed with NaHCO3 aqueous solution, brine, dried over MgSO4, filtered, and concentrated. Silica gel flash column chromatography (hexanes/ethyl acetate = 3:1) of the residue gave a white solid (26.9 mg, 0.08 mmol, 71%) as the product. (±)-14: 1H NMR (500 MHz, chloroform-d) δ 6.01 (s, 1H), 5.45 (s, 1H), 4.63 (d, J = 12.5 Hz, 1H), 4.34 (d, J = 12.5 Hz, 1H), 3.70 (s, 1H), 3.02 (dd, J = 9.0, 4.5 Hz, 1H), 2.84 (s, 1H), 2.51-2.45 (m, 2H), 2.33-2.29 (m, 1H), 2.20 (dd, J = 13.5, 5.0 Hz, 1H), 1.95-1.85 (m, 2H), 1.84-1.71 (m, 4H), 1.56-1.45 (m, 2H), 1.37 (s, 3H), 1.28 (s, 3H). 13C{1H} NMR (125 MHz, chloroform-d) δ 208.9, 203.0, 171.9, 151.5, 118.3, 78.8, 68.4, 57.3, 54.7, 54.3, 44.6, 39.7, 36.6, 34.2, 32.5, 31.8, 29.7, 24.7, 23.4, 19.1. IR(KBr) 2981, 2956, 2867, 1747, 1709, 1153, 1111, 913. HRMS (ESI/[M+Na]+) calcd. for C20H24O5Na: 367.1521 found 367.1532. Melting point: 187-189℃ (±)-4,4-Dimethyl-8-methylenedodecahydro-6H-6,11b-(epoxymethano)-6a,9-meth anocyclohepta[a]naphthalene-1,6-diol ((±)-16) To a stirred solution of (±)-15 (420.0 mg, 0.68 mmol) in THF (7.0 mL) was added TBAF (2.0 mL, 1.0 M in THF) at rt. After stirring at rt for 14 h, the solution was quenched by addition of water (10 mL) and then the aqueous layer was extracted with ethyl acetate/THF = 2:1 (20 mL × 4). The combined organic extracts were washed with brine, dried over MgSO4, filtered, and concentrated. Silica gel flash column chromatography (hexanes/ethyl acetate = 3:1) of the residue gave a white solid (211.9 mg, 0.67 mmol, 98%) as the product.


Page 24 of 38

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(±)-16: 1H NMR (300 MHz, pyridine-d5) δ 7.93 (s, 1H), 5.98 (s, 1H), 5.15 (d, J = 9.0 Hz, 1H), 5.07 (s, 1H), 4.91 (s, 1H), 3.81 (d, J = 9.0 Hz, 1H), 3.70-3.63 (m, 1H), 3.60 (d, J = 11.7 Hz, 1H), 3.14 (d, J = 16.2 Hz, 1H), 2.69 (d, J = 6.6 Hz, 1H), 2.57-2.45 (m, 1H), 2.29- 2.16 (m, 3H), 2.09 (s, 2H), 2.02 (dd, J = 13.2, 7.8 Hz, 1H), 1.96-1.81 (m, 1H), 1.81-1.67 (m, 2H), 1.54-1.43 (m, 2H), 1.40-1.23 (m, 2H), 1.11 (s, 3H), 0.81 (s, 3H).

13C{1H}

NMR (75 MHz, pyridine-d5) δ 139.1, 125.5, 103.7, 78.3, 73.2, 50.7,

48.2, 43.0, 41.9, 40.5, 39.6, 34.6, 34.1, 33.4, 32.0, 31.4, 28.8, 22.9, 21.0, 18.9. IR (KBr) 3402, 3381, 2956, 2930, 1469, 1258, 1115, 1077, 887, 833, 744. HRMS (ESI/[M+Na]+) calcd. for C20H30O3Na: 341.2093 found 341.2084. Melting point: 251-252 ℃ (±)-1-Hydroxy-4,4-dimethyl-8-methylene-6,7-dioxododecahydro-6a,9-methanocy clohepta[a]naphthalene-11b(1H)-carbaldehyde ((±)-17) To a stirred solution of (±)-16 (50.0 mg, 0.16 mmol) in CH2Cl2 (8 mL) was added t-butyl peroxide (0.07 mL, 5.5 M in heptane) and SeO2 (5.3 mg, 0.05 mmol) at rt. The resulting solution was stirred at rt for 24 h. Upon addition of hexanes, a cloudy solution formed. The precipitate was filtered off through a plug of silica gel and then concentrated. To a stirred of the residue in CH2Cl2 (8 mL) was added DMP (150 mg, 0.35 mmol) at rt After stirring at rt for 4 h, the reaction was quenched by addition of a saturated Na2S2O3 aqueous solution (4 mL). The aqueous layer was extracted with ethyl acetate (20 mL × 4). The combined organic extracts were washed with NaHCO3 aqueous solution, brine, dried over MgSO4, filtered, and concentrated. Silica gel flash column chromatography (hexanes/ethyl acetate = 3:1) of the residue gave a white solid



product (30.0 mg, 0.09 mmol, 57%). (±)-17: 1H NMR (500 MHz, pyridine-d5) δ 10.44 (s, 1H), 6.04 (s, 1H), 5.25 (s, 1H), 3.52 (d, J = 7.5 Hz, 1H), 3.01 (dd, J = 14.0, 3.5 Hz, 1H), 2.93 (s, 1H), 2.69-2.56 (m, 2H), 2.55-2.48 (m, 2H), 2.31 (dd, J = 12.5, 5.0 Hz, 1H), 2.00 (dd, J = 16.0, 5.5 Hz, 1H), 1.91-1.81 (m, 2H), 1.70-1.56 (m, 2H), 1.55-1.43 (m, 3H), 1.28 (s, 1H), 0.93 (s, 3H), 0.72 (s, 3H). 13C{1H} NMR (75 MHz, pyridine-d5) δ 210.0, 207.9, 202.7, 147.9, 116.1, 66.5, 61.7, 43.9, 40.8, 38.7, 37.4, 36.5, 34.9, 33.8, 32.7, 30.2, 29.0, 23.4, 19.5. IR: IR (KBr) 3398, 2855, 2778, 1830, 1403, 1255, 1112, 1071, 991, 835, 747. HRMS (ESI/[M+Na]+) calcd. for C20H26O4Na: 353.1729, found 353.1737. Melting point: 165-166 ℃ (±)-5-((tert-Butyldimethylsilyl)oxy)-4b-(((tert-butyldimethylsilyl)oxy)methyl)-8,8 -dimethyl-10a-(prop-2-yn-1-yl)decahydrophenanthrene-3,10(2H,4H)-dione ((±)-18) To a stirred solution of (±)-4 (60.0 mg, 0.08 mmol) in CH2Cl2 (8 mL) was added Me2AlCl (0.1 mL, 1.0 M in hexane ) slowly at rt. After stirring at rt for 1 h, the reaction was quenched by addition of a saturated sodium potassium tartrate aqueous solution (8 mL). The aqueous layer was extracted with ethyl acetate (20 mL × 4). The combined organic extracts were washed with brine, dried over MgSO4, filtered, and concentrated. Silica gel flash column chromatography (hexanes/ethyl acetate = 75:1) of the residue gave a brown oil product (30.5 mg, 0.05 mmol, 68%). (±)-18: 1H NMR (500 MHz, chloroform-d) δ 5.16 (s, 1H), 4.98 (s, 1H), 3.91 (d, J = 10.0 Hz, 1H), 3.84 (dd, J = 11.5, 3.5 Hz, 1H), 3.73 (d, J = 10.0 Hz, 1H), 3.28 (s, 1H), 3.19 (dt, J = 17.0, 3.0 Hz, 1H), 2.85 (q, J = 5.5 Hz, 3H), 2.62 (dd, J = 18.0, 8.0 Hz, 1H), 2.51 (dd, J = 18.0, 2.5 Hz, 1H), 2.39 (d, J = 16.5 Hz, 1H), 2.13 (s, 2H), 2.06 (dd, J = 8.0, 2.5 Hz,


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1H), 1.80-1.69 (m, 1H), 1.57 (s, 2H), 1.46 (dt, J = 13.5, 3.5 Hz, 1H), 1.36-1.26 (s, 1H), 1.05 (s, 2H), 0.91 (m, 25H), 0.09-0.03 (m, 12H).

13C{1H}

NMR (125 MHz,

chloroform-d) δ 213.6, 211.1, 80.6, 74.9, 71.8, 61.5, 48.8, 47.5, 43.7, 41.4, 39.9, 38.9, 36.1, 36.1, 34.1, 33.4, 32.9, 27.4, 26.0, 25.9, 25.3, 24.9, 18.1, 18.0, -4.6, -4.7, -5.0, -5.6. IR(KBr) 2956, 2860, 1770, 1754, 1701, 1258, 1052, 837, 825, 748. HRMS (ESI/[M+Na]+) calcd. for C32H56O4Si2Na: 583.3615, found 583.3622. (±)-4-((tert-Butyldimethylsilyl)oxy)-9-hydroxy-1,1-dimethyl-8a-(prop-2-yn-1-yl)d odecahydro-6H-9,4a-(epoxymethano)phenanthren-6-one ((±)-19) To a stirred solution of (±)-18 (220 mg, 0.39 mmol) in THF (4 mL) was added TBAF (0.5 mL, 1.0 M in THF) at rt. The resulting mixture was stirred at rt for 14 h. The solution was quenched by addition of water (5 mL) and the aqueous layer was then extracted with ethyl acetate (20 mL × 4). The combined organic extracts were washed with brine, dried over MgSO4, filtered, and concentrated. Silica gel flash column chromatography (hexanes/ethyl acetate = 30:1) of the residue gave a colorless oil (156.5 mg, 0.35 mmol, 90%) as the product. (±)-19: 1H NMR (300 MHz, chloroform-d) δ 4.07 (d, J = 12.3 Hz, 1H), 3.73 (s, 1H), 3.49 (d, J = 12.3 Hz, 1H), 3.12 (s, 1H), 2.85 (d, J = 14.7 Hz, 1H), 2.76 (d, J = 5.4 Hz, 1H), 2.62-2.42 (m, 4H), 2.07-1.99 (m, 2H), 1.94-1.82 (m, 3H), 1.74-1.66 (m, 4H), 1.42 (d, J = 13.8 Hz, 2H), 0.98 (s, 3H), 0.89 (s, 10H), 0.84 (s, 3H), 0.10 (d, J = 5.1 Hz, 6H). 13C{1H} NMR (75 MHz, chloroform-d) δ 215.4, 95.5, 70.9, 68.0, 47.6, 44.2, 41.6, 40.6, 34.4, 34.4, 32.6, 32.2, 31.9, 28.5, 27.41, 26.2, 25.6, 23.6, 17.9, -4.5, -4.7. IR(KBr) 3419, 2956, 2935, 2859, 1705, 1262, 1056, 837, 748. HRMS (ESI/[M+Na]+) calcd. for C26H42O4Si1Na: 469.2750, found 469.2761.



(±)-4,9-Dihydroxy-1,1-dimethyl-14-methylenedecahydro-2H,6H-9,4a-(epoxymeth ano)-5,8a-ethanophenanthren-6-one ((±)-20) To a stirred mixture of (±)-19 (578 mg, 1.29 mmol), NaI (967.5 mg, 6.45 mmol) and HMDS (1.6 mL, 7.77 mmol) in CH3CN (2.6 mL) was added TMSCl (0.97 mL, 7.77 mmol) slowly at 0 ˚C. After stirring at rt for 12 h, the reaction was diluted with hexanes (30 mL) and quenched by addition of water (5 mL). The aqueous phase was extracted with hexanes (20 mL × 3). The combined organic extracts were washed with saturated Na2S2O3 aqueous solution until clear, and then washed with brine, dried over MgSO4, filtered, and concentrated. To a stirred solution of the residue in CH2Cl2 (13 mL) was added ZnBr2 (575.3 mg, 2.58 mmol) at rt. After stirring at rt for 2 h, the reaction was filtered through a plug of silica gel to remove ZnBr2, and then concentrated. To a stirred solution of the residue in THF (6.5 mL) was added TBAF (3.8 ml, 1.0 M) at rt. After stirring at rt for 14 h, the reaction was quenched by addition of water and the aqueous layer was then extracted with ethyl acetate (20 mL × 4). The combined organic extracts were washed with brine, dried over MgSO4, filtered, and concentrated. Silica gel flash column chromatography (hexanes/ethyl acetate = 2:1) of the residue gave a white solid (315.5 mg, 0.95 mmol, 73%) as the product. (±)-20: 1H NMR (400 MHz, pyridine-d5) δ 6.58 (s, 1H), 5.37 (s, 1H), 5.06 (s, 1H), 4.31 (d, J = 12.0 Hz, 2H), 4.19 (d, J = 12.0 Hz, 1H), 3.57 (d, J = 4.8 Hz, 1H), 3.19 (d, J = 4.8 Hz, 1H), 3.10 (dd, J = 16.0, 14.0 Hz, 1H), 2.72 (dd, J = 16.0, 2.4 Hz, 1H), 2.61 (dd, J = 16.8, 3.2 Hz, 2H), 2.54-2.42 (m, 1H), 2.33-2.22 (m, 2H), 2.17 (dd, J = 14.0, 7.6 Hz, 1H), 2.11-1.87 (m, 3H), 1.77 (dd, J = 13.6, 8.0 Hz, 1H), 1.13-1.07 (m, 1H), 1.05 (s, 3H), 1.01 (s, 3H). 13C{1H} NMR (100


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MHz, pyridine-d5) δ 216.4, 150.4, 110.3, 98.4, 73.7, 69.4, 54.3, 49.9, 45.8, 45.4, 43.2, 41.4, 40.7, 33.4, 33.2, 32.7, 32.6, 31.9, 31.1, 28.1. IR (KBr) 3398, 2956, 2905, 2854, 1721, 1654, 1461, 1254, 1163, 1069. HRMS (ESI/[M+Na]+) calcd. for C20H28O4Na: 355.1885, found 355.1878. Melting point: 221-223 ℃. (±)-4,9,13-Trihydroxy-1,1-dimethyl-14-methylenedecahydro-2H,6H-9,4a-(epoxy methano)-8a,5-ethanophenanthren-6-one ((±)-21) To a stirred solution of (±)-20 (800 mg, 2.44 mmol) in CH2Cl2 (24 mL) was added t-butyl peroxide (1.11 mL, 5.5 M in heptane) and SeO2 (81 mg, 0.73 mmol) at rt. The mixture was stirred at rt for 24 h. A plug of silica gel was added to the solution and then concentrated. Silica gel flash column chromatography (hexanes/ethyl acetate = 1:1) of the residue gave a white solid (764.2 mg, 2.20 mmol, 90%) as the product. (±)-21: 1H NMR (400 MHz, pyridine-d5) δ 5.62 (s, 2H), 4.88 (s, 1H), 4.53 (s, 2H), 4.40-4.18 (m, 2H), 3.38-3.16 (m, 2H), 2.60 (d, J = 14.0 Hz, 1H), 2.47-2.31 (m, 1H), 2.32-2.08 (m, 3H), 1.98 (dt, J = 18.9, 11.3 Hz, 4H), 1.23-1.08 (m, 1H), 1.01 (d, J = 18.8 Hz, 6H).13C{1H} NMR (100 MHz, pyridine-d5) δ 214.8, 154.0, 112.9, 97.0, 77.8, 73.9, 67.2, 57.2, 47.4, 45.2, 43.4, 39.7, 38.3, 31.8, 31.7, 31.5, 31.1, 29.4, 28.3, 26.7. IR (KBr) 3398, 3318, 2989, 2955, 2901, 2833, 1707, 1651, 1456, 1159, 1063. HRMS (ESI/[M+Na]+) calcd. for C20H28O5Na: 371.1834, found 371.1828. Melting point: 241-243 ℃. (±)-9-Hydroxy-1,1-dimethyl-14-methylenedecahydro-4H,6H-9,4a-(epoxymethano)5,8a-ethanophenanthrene-4,6,13-trione ((±)-22) To a stirred solution of (±)-21 (79.0 mg, 0.24 mmol) in CH2Cl2 (2.4 mL) was added NaHCO3 (151 mg, 1.80 mmol) and then cool to 0 ˚C. DMP (255 mg, 0.60 mmol) was added at 0 ˚C. After stirring at



rt for 2 h, the reaction was quenched by addition of a saturated Na2S2O3 aqueous solution (4 mL). The aqueous layer was extracted with ethyl acetate (20 mL × 4). The combined organic extracts were washed with NaHCO3 aqueous solution, brine, dried over MgSO4, filtered, and concentrated. Silica gel flash column chromatography (hexanes/ethyl acetate = 2:1) of the residue gave a white solid (67.7 mg, 0.20 mmol, 82%) as the product. (±)-22: 1H NMR (300 MHz, pyridine-d5) δ 6.26 (s, 1H), 5.64 (s, 1H), 4.73 (d, J = 13.5 Hz, 1H), 4.56 (d, J = 13.5 Hz, 1H), 3.76 (d, J = 4.5 Hz, 1H), 3.55 (d, J = 4.5 Hz, 1H), 2.86 (dd, J = 18.6, 7.5 Hz, 1H), 2.75-2.61 (m, 1H), 2.52-2.36 (m, 3H), 2.30-2.20 (m, 4H), 1.98-1.81 (m, 1H), 1.29 (s, 2H), 0.90 (s, 3H), 0.69 (s, 3H). 13C{1H}

NMR (75 MHz, pyridine-d5) δ 212.7, 205.4, 196.7, 146.2, 118.9, 96.5, 72.9,

57.8, 49.7, 47.8, 45.9, 40.5, 40.3, 35.6, 33.8, 32.6, 30.9, 30.6, 29.8, 21.1. IR (KBr) 3318, 2955, 2833, 2786, 2759, 1714, 1661, 1463, 1201, 1069. HRMS (ESI/[M+Na]+) calcd. for C20H24O5Na: 367.1521, found 367.1523. Melting point: 172-174 ℃ (±)-8-Hydroxy-4,4-dimethyl-10-methyleneoctahydro-1H,8H-8a,11-methanocyclo hepta[c]furo[3,4-e]chromene-3,9(3aH,10H)-dione ((±)-24 and (±)-25) To a stirred solution of (±)-7 (126 mg, 0.36 mmol) in EtOH (36 mL) was added NaBH4 (34.2 mg, 0.90 mmol) slowly at 0 ˚C. After stirring at 0 ˚C for 2 h, the reaction was quenched by addition of water (15 mL). The aqueous layer was extracted with ethyl acetate/THF = 2:1 (40 mL × 4). The combined organic extracts were washed with brine, dried over MgSO4, filtered by silica gel flash column and concentrated. To a stirred solution of the residue in acetone (24 mL) was added MnO2 (313 mg, 3.6 mmol) slowly at rt. After stirring at rt for 14 h, the solution was filtered through a plug of silica gel to


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remove MnO2, and then the filtrate was concentrated. Silica gel flash column chromatography (hexanes/EA = 3:1) of the residue gave two white solids ((±)-24: 53.5 mg, 0.15 mmol, 43%; (±)-25: 54.0 mg, 0.15 mmol, 43%) as the products. (±)-24: 1H

NMR (400 MHz, pyridine-d5) δ 5.89 (s, 1H), 5.40 (s, 1H), 5.05 (s, 1H), 4.06 (d, J

= 10.0 Hz, 1H), 3.81 (d, J = 10.0 Hz, 1H), 3.36 (s, 1H), 3.07 (s, 1H), 2.95 (s, 1H), 2.41 (dd, J = 12.4, 4.4 Hz, 1H), 2.25 (d, J = 12.4 Hz, 2H), 1.97 (dd, J = 12.8, 10.0 Hz, 2H), 1.86-1.76 (m, 2H), 1.71-1.62 (m, 2H), 1.47-1.41 (m, 1H), 1.19 (s, 3H), 1.06 (d, J = 12.8 Hz, 1H), 0.92 (s, 3H). 13C{1H} NMR (100 MHz, pyridine-d5) δ 206.9, 178.1, 114.1, 95.8, 75.7, 71.4, 56.2, 50.7, 48.7, 45.0, 37.7, 32.5, 32.2, 31.5, 27.1, 24.5, 24.5, 19.2. IR: 3382, 2935, 2836, 2812, 1747, 1695, 1642, 1203 780. HRMS (ESI/[M+Na]+) calcd. for C20H26O5Na: 369.1678. found 369.1673. Melting point: 284-287 ℃. (±)-25: 1H

NMR (500 MHz, pyridine-d5) δ 6.03 (s, 1H), 5.29 (s, 1H), 5.22 (s, 1H), 4.39 (t, J =

2.5 Hz, 1H), 4.19 (d, J = 10.0 Hz, 1H), 4.00 (s, 1H), 3.18 (s, 1H), 3.08-3.00 (m, 1H), 2.64 (d, J = 7.5 Hz, 1H), 2.54 (d, J = 12.5 Hz, 1H), 2.13 (tdd, J = 13.5, 5.0, 2.5 Hz, 1H), 2.02-1.89 (m, 3H), 1.80-1.73 (m, 1H), 1.72-1.64 (m, 2H), 1.64-1.56 (m, 2H), 1.36 (s, 3H), 1.22 (d, J = 10.5 Hz, 1H), 1.06 (s, 3H).

13C{1H}

NMR (125 MHz,

pyridine-d5) δ 208.06, 178.32, 115.28, 94.77, 76.59, 63.34, 53.81, 51.27, 45.39, 43.01, 37.86, 33.07, 32.90, 32.48, 32.31, 31.42, 24.58, 24.20, 19.48. IR: 3410, 2938, 2833, 2815, 1751, 1695, 1642, 1403, 1114, 780. HRMS (ESI/[M+Na]+) calcd. for C20H26O5Na: 369.1678. found 369.1663. Melting point: 284-287 ℃ (±)-2,5,6-Trihydroxy-4,4-dimethyl-8-methylenedecahydro-1H-6,11b-(epoxymeth ano)-6a,9-methanocyclohepta[a]naphthalene-1,7(8H)-dione ((±)-26)：To a stirred



mixture of (±)-NL (47 mg, 0.14 mmol) in CH3CN (1.4 mL) were added NaI (183.6 mg, 1.22 mmol) and HMDS (0.43 mL, 2.04 mmol) at rt and then TMSCl (0.15 mL, 1.22 mmol) was added slowly at 0 ˚C. After stirring at rt for 12 h, the reaction was diluted with hexanes (30 mL) and quenched by addition of water (5 mL). The aqueous layer was extracted with hexanes (20 mL × 3). The combined organic extracts were washed with a saturated Na2S2O3 aqueous solution until clear, and then washed with brine, dried over MgSO4, filtered, and concentrated. To a stirred solution of the residue in CH2Cl2 (5.6 mL) was added mCPBA (46.9 mg, 0.27 mmol) at rt. After stirring at rt for 1 h, the reaction was quenched by addition of Na2SO3 aqueous solution (5 mL). The aqueous layer was extracted with ethyl acetate (20 mL × 3). The combined organic extracts were washed with brine, dried over MgSO4, filtered, and concentrated to provide crude product, which was dissolved in THF (5.6 mL) was added 1.0 N HCl aqueous solution (0.7 mL) at rt. After stirring at rt for 6 h, the reaction was quenched by addition of a saturated NaHCO3 aqueous solution (5 mL). The aqueous layer was extracted with ethyl acetate (20 mL × 3). The combined organic extracts were washed with brine, dried over MgSO4, filtered, and concentrated. Silica gel flash column chromatography (hexanes/ethyl acetate = 2:1) of the residue gave a white solid (33.0 mg, 0.09 mmol, 65%) as the product. 1H NMR (300 MHz, pyridine-d5) δ 6.76 (d, J = 10.5 Hz, 1H), 6.01 (s, 1H), 5.31 (s, 1H), 4.65 (t, J = 7.2 Hz, 1H), 4.46 (s, 2H), 4.37 (t, J = 7.2 Hz, 1H), 2.86-2.90 (m, 1H), 2.60 – 2.38 (m, 4H), 2.31-2.27 (m, 3H), 2.13-2.10 (m, 1H), 2.04 – 1.89 (m, 2H), 1.30 (s, 4H), 1.23 (s, 4H).

13C

NMR (75 MHz, pyridine-d5) δ13C{1H} NMR (75 MHz, Pyr) δ 213.0,


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210.7, 154.7, 118.7, 97.7, 75.3, 73.6, 66.3, 63.9, 61.0, 52.8, 51.8, 48.3, 35.9, 35.2, 33.3, 31.2, 27.3, 25.4, 19.7. IR: 3857, 3752, 3238, 2934, 1738, 1709, 1136, 1064. HRMS (ESI/[M+Na]+) calcd. for C20H26O6Na: 385.1627. found 385.1627. Melting point: 318-319 ℃. (±)-5,6-Dihydroxy-4,4-dimethyl-8-methylene-1,7-dioxododecahydro-1H-6,11b-(e poxymethano)-6a,9-methanocyclohepta[a]naphthalen-2-yl

hex-5-ynoate

((±)-NL-A2) To a stirred solution of (±)-26 (33.0 mg, 0.09 mmol) in CH2Cl2 (2 mL) was added hex-5-ynoic acid (12.4 mg, 0.11 mmol), EDCI (21.0 mg, 0.11 mmol), DMAP (1.4 mg, 0.01 mmol) at rt. After stirring at rt for 10 h, the reaction was quenched by addition of water (5 mL). The aqueous layer was extracted with ethyl acetate (20 mL × 3). The combined organic extracts were washed with brine, dried over MgSO4, filtered, and concentrated Silica gel flash column chromatography (hexanes/ethyl acetate = 4:1) of the residue gave an amorphous solid (33 mg, 0.07mmol, 82%) as the product. (±)-NL-A2: 1H NMR (300 MHz, chloroform-d) δ 6.20 (d, J = 11.7 Hz, 1H), 6.02 (s, 1H), 5.50 (s, 1H), 5.01 (dd, J = 11.7, 6.0 Hz, 1H), 4.30 (d, J = 9.9 Hz, 1H), 4.19 (d, J = 9.9 Hz, 1H), 3.94 (dd, J = 11.7, 7.8 Hz, 1H), 3.83 (s, 1H), 3.07 (dd, J = 9.6, 4.5 Hz, 1H), 2.51 (t, J = 7.2 Hz, 2H), 2.30-2.25 (m, 4H), 2.18 – 2.06 (m, 4H), 1.98 (t, J = 2.7 Hz, 1H), 1.91-1.81 (m, 4H), 1.53 – 1.42 (m, 2H), 1.26 (d, J = 1.5 Hz, 6H). 13C{1H} NMR (75 MHz, CDCl3) δ 208.3, 204.0, 171.9, 152.2, 118.7, 95.2, 82.9, 73.6, 72.8, 69.1, 64.9, 61.3, 58.9, 51.0, 46.6, 46.1, 34.2, 34.0, 32.3, 31.8, 24.8, 23.4, 18.1, 17.6. HRMS (ESI/[M+Na]+) calcd. for C26H32O7Na: 479.2046. found 479.2058.



Page 34 of 38

Acknowledgements This work is financially supported by the National Natural Science Foundation of China (Grant No. 21502002, 21572006, 21871017 and 81872769), the Natural Science Foundation of Guangdong Province, China (Grant No. 2017A030313042), the Hong Kong Research Grants Council General Research Fund (Project No. 12103515), the Health and Medical Research Fund (Project No. 15161401), the Shenzhen

Science

and

JCYJ20151030164022389,

Technology

Innovation

JCYJ20160229173844278,

Committee

(Grant

No.

JCYJ20160428153421635

and JCYJ20160330095659560), Peking University Shenzhen Graduate School, Hong Kong Polytechnic University and Hong Kong Baptist University.

Supporting Information 1H

NMR and

13C

NMR spectra for all the new compounds. COSY, HSQC and

NOESY NMR spectra for (±)-8, (±)-11, (±)-13, (±)-20, (±)-24 and (±)-25. This material is available free of charge via the Internet at http://pubs.acs.org.

References 1. (a) Sharp, M.; Dohme, W. P. Combination Chemotherapy with Bleomycin, Cyclophosphamide and Dactinomycin for the Treatment of Osteogenic Sarcoma. Cancer 1977, 40, 2779. (b) Boyette-Davis, J. A.; Walters, E. T.; Dougherty, P. M. Mechanisms Involved in the Development of Chemotherapy-induced Neuropathy.


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