Regio- and Stereospecific Synthesis of Oridonin D-Ring Aziridinated

Subscriber access provided by UNIV OF DURHAM

Regio- and Stereospecific Synthesis of Oridonin D-ring Aziridinated Analogues for the Treatment of Triple-Negative Breast Cancer via Mediated Irreversible Covalent Warheads Ye Ding, Dengfeng Li, Chunyong Ding, Pingyuan Wang, Zhiqing Liu, Eric A. Wold, Na Ye, Haiying Chen, Mark Andrew White, Qiang Shen, and Jia Zhou J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01514 • Publication Date (Web): 12 Mar 2018 Downloaded from http://pubs.acs.org on March 13, 2018

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

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

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

Journal of Medicinal Chemistry

Regio- and Stereospecific Synthesis of Oridonin D-ring Aziridinated Analogues for the Treatment of Triple-Negative Breast Cancer via Mediated Irreversible Covalent Warheads

Ye Ding,†,§ Dengfeng Li,‡,ξ,§ Chunyong Ding,† Pingyuan Wang,† Zhiqing Liu,† Eric A. Wold,† Na Ye,† Haiying Chen,† Mark A. White,∥ Qiang Shen,*,‡ Jia Zhou*,† †

Chemical Biology Program, Department of Pharmacology and Toxicology, ∥Sealy Center for

Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, Texas 77555, United States ‡

Department of Clinical Cancer Prevention, Division of Cancer Prevention and Population

Sciences, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, United States ξ

Department of Thyroid and Breast, Division of General Surgery, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai 200072, China

ACS Paragon Plus Environment

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

ABSTRACT Covalent drug discovery has undergone a resurgence in recent years due to comprehensive optimization of structure-activity relationship (SAR) and structure-reactivity relationship (SRR) for covalent drug candidates. The natural product oridonin maintains an impressive pharmacological profile through its covalent enone warhead on the D-ring and has attracted substantial SAR studies to characterize its potential in the development of new molecular entities for the treatment of various human cancers and inflammation. Herein, for the first time, we report the excessive reactivity of this covalent warhead and mediation of the covalent binding capability through a Rh2(esp)2-catalyzed mild and concise regio- and stereospecific aziridination approach. Importantly, aziridonin 44 (YD0514), with a more drug-like irreversible covalent warhead, has been identified to significantly induce apoptosis and inhibit colony formation against triple-negative breast cancer with enhanced antitumor effects in vitro and in vivo, while displaying lower toxicity to normal human mammary epithelial cells in comparison with oridonin.

Keywords: Natural product, Oridonin, Covalent warhead, Aziridination, Anticancer agent


Page 2 of 54

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


INTRODUCTION Covalent drugs can possess exceptionally high potency, ligand efficiency, and long-lasting effects given that they directly react with targets to form covalent bonds, and thereby generate corresponding bioactivities. Throughout the history of modern medicine, covalent drugs have been profoundly successful therapies for a wide array of human diseases. For example, from 1982–2009, thirty-nine covalent drugs had been approved by the FDA, with the majority having been approved before 2000.1 However, due to potential toxicities and safety risks, electrophiles have been considered un-druglike and were virtually nonexistent in modern target-specific drug discovery and development for many years. Recently, the notion of exploring new generations of covalent drugs has resurged with the advent of targeted covalent inhibitors, promising a positive benefit to risk ratio.1-3 The electrophilic Michael acceptor enamides and ynamides (Figure 1, 1-5) are frequently utilized as covalent warheads in the design of novel synthetic kinase inhibitors to enhance the biological efficacy, extend the duration of action, and overcome drug resistance.4-8 The successful comeback of covalent drugs is partially ascribed to the optimal mix of structure-activity relationship (SAR) of entire molecules and structure-reactivity relationship (SRR) of attached electrophiles, aiming to acquire the most suitable candidates.2 An additional support for covalent drug discovery is the broadly applied, simple and efficient methodology for determining the SRR by the assessment of the reaction rate between covalent warheads and a biologically relevant substrate glutathione (GSH) in vitro.9-13 An optimal balance of warhead reactivity is necessary, since excess reactivity of covalent warheads may hinder the overall selectivity and safety of drugs, while reduced reactivity may lead to failed covalent binding to the target protein to produce the corresponding effect.2



Figure 1. (A) Representative molecules bearing covalent warheads enamide and ynamide (highlighted in red). (B) Representative molecules with covalent warheads epoxide and aziridine (highlighted in red).

In addition to the above mentioned covalent synthetic drugs, covalent natural products from aspirin found in 1899 to the sesquiterpenoid artemisinin, have revolutionized medicine and


Page 4 of 54

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


have been an invaluable inspiration for the development of various therapeutic agents.1, 14, 15 In the kingdom of covalent natural products and derivatives, the enone, ethylene oxide and aziridine groups are three representative classes of warheads (Figure 1, 6-12), all of which can react with nucleophilic groups of target proteins, such as the thiol of cysteine residues, and share a similar reaction mechanism to form covalent bonds.16-18 Besides these examples depicted in Figure 1, another covalent natural product, the kaurane-type diterpenoid oridonin (13 in Table 1), has attracted increased attention in recent years, due to its high natural abundance, historic application in traditional herbal medicine (available over the counter as “Donglingcao Pian”), and impressive anti-inflammation and anticancer pharmacological activities.19, 20 Thus, oridonin as a single drug ingredient is currently undergoing

a

clinical

observational

study

in

China

(ChiCTR-OOB-16007883;

http://www.chictr.org.cn/enIndex.aspx). Over the past decade, several independent research groups, including our team, have utilized oridonin as a chemical lead for the design and synthesis of novel oridonin derivatives, with these studies resulting in pivotal SAR information.21 Briefly, modifications on the A-ring, the C-14 position (Table 1, entry 1, highlighted in blue), appear to be tolerable for improving the biological efficacy and drug-like profiles of oridonin. The enone system in D-ring (Table 1, entry 1, highlighted in red), as a classical covalent warhead, was identified to be a requisite Michael acceptor for biological activities,22 and removal of this enone system generally resulted in a dramatic loss of activity.21, 23 Importantly, among the bioactive oridonin derivatives depicted in Table 1, 14 was advanced into a Phase I human clinical trial for the treatment of leukemia in 2015 (CTR20150246; www.chinadrugtrials.org.cn), while all the others in Table 1 are currently in preclinical development.



Page 6 of 54

Table 1. Summary of the Preclinical and Clinical Development of Oridonin-like Compounds Entry

1

Compounds

Molecular targets regulated

Nrf2, NF-κB, ROS, p53, p21 and mitochondrial apoptosis pathways

Biological responds and

clinical indications

Ref.

Antioxidant, cytoprotection and antiinflammation at low concentrations (e.g. 1.4 μM); Anti-proliferation and pro-apoptosis at relative higher concentrations (e.g. 14 μM) in vitro Inhibition of tumor growth in t(8;21) leukemia murine model (7.5 and 15 mg/kg)

24-27

Oridonin-containing herb “Donglingcao” is an over-the-counter (OTC) anti-inflammatory medicine in China 2

3

4

5

NF-κB

An alanine ester prodrug of oridonin as Phase I clinical candidate for the treatment of leukemia (80-320 mg/d, iv)

p53, p21 and caspase-3

Antiproliferation against LX-2 cell line with IC50 value 0.49 μM, 10-fold more potent than oridonin in vitro

p21and p16

Significant antiproliferation against LX-2 cell line with 20-fold more potent activity than oridonin in vitro

NF-κB, Bcl-2/Bax and PARP

Antiproliferation against both ERpositive breast cancer MCF-7 and triple-negative MDA-MB-231 cell lines with IC50 values 0.98 and 5.6 μM in vitro, respectively Growth suppression of MDA-MB-231 xenograft tumors in vivo (5 mg/kg)


28, 29

30

31, 32

31

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


NF-κB, Bcl-2/Bax, PARP, DR5 and XBP1

6

Antiproliferation against various cancer cell lines with the IC50 values range from 0.2 ~ 2.0 μM in vitro 33-35

Growth suppression of MDA-MB-231 and HCC1806 xenograft tumors in vivo (5 mg/kg)

Inspired by recent advances of both synthetic and natural product covalent drugs, our approach reported here was guided by the idea that the exploring the SRR information of the covalent enone warhead in the D-ring is additionally beneficial to the further optimization for the biological activities, safety and other drug-like properties of oridonin. To our knowledge, this is the first attempt to measure the reactivity of the electrophile enone in D-ring with GSH. Based upon our SRR, favorable and drug-like covalent groups have been designed and incorporated to replace the overly reactive enone in the D-ring through a mild and concise homogeneous Rh2(esp)2-catalyzed direct aziridination in a regio- and stereospecific manner. As a continuation of our previous effort in the identification of novel oridonin analogues with the bioactive functionalities diversely installed in the A-ring, herein we describe the discovery of a new analog (2R,3'S,3a'R,3a1'R,6a'R,7'S,11'S,11a'S)-1-acryloyl-7',11'-dihydroxy-5',5',8',8'tetramethyldecahydro-2'H-spiro[aziridine-2,15'[6a,11a](epoxymethano)[3,3a1]ethanophenanthro[1,10-de][1,3]dioxin]-14'-one (Aziridonin 44, YD0514) with a more drug-like irreversible covalent warhead capable of significantly inducing apoptosis and inhibiting colony formation against triple-negative breast cancer with comparable to enhanced antitumor effects in vitro and in vivo, while displaying lower toxicity to normal human mammary epithelial cells in comparison with oridonin.

RESULTS AND DISCUSSION



Page 8 of 54

Activity and Reactivity of the Covalent Enone Warhead in the D-Ring. GSH is viewed as a major reducing small molecule participating in cellular redox reactions with reactive oxygen/nitrogen species (ROS/RNS), and maintaining intracellular redox balance. Accumulating evidence indicates that treatment with oridonin could decrease the intracellular GSH concentration, resulting in the increase of ROS and ROS-induced apoptosis in cancer cell lines.25, 36-39

The enone fragment in the D-ring of oridonin has been identified as a classical covalent

warhead Michael acceptor, which reacts with various thiol groups/residues of small molecules (e.g. GSH) and/or target proteins (e.g. thioredoxin reductase) to afford irreversible inhibition.25 A similar mechanism was revealed in the studies of oridonin-like kaurane diterpenoids.22,

40

Interestingly, the reduction of this enone by Pd/C-catalyzed hydrogenation significantly decreases anti-proliferation effects.41 Thus, the efficacy of oridonin is believed to largely depend on this D-ring enone pharmacophore by Michael addition with GSH and other protein targets. Given that systematic studies on the reactivities of various covalent groups with GSH and the corresponding SRR are useful for the development of novel covalent kinase inhibitors, we utilized nuclear magnetic resonance (NMR) spectroscopy to determine the experimental GSH reaction rates of oridonin, and its C-14 position derivatives 14 (Table 1) and 20 (Table 3). The oridonin-like substrates were incubated at 37 ˚C with 1.5 ~ 2 equivalents of GSH in 0.5 mL of DMSO-d6 and 0.05 mL of sodium phosphate deuterium oxide buffer (pH 7.4). The samples were measured by 1H NMR spectrum at time points of 1st, 5th and 10th min, respectively, for quantitatively describing the reactivities of the enones presented in these substrates with GSH. The reaction half-life (t1/2) of oridonin with GSH was found to be markedly less than 1 minute (Figure 2). The resultant product was further validated with high-resolution mass spectrometry (HRMS), indicating that the transformation of oridonin to its adduct 19 was complete (see the


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


supporting information Figure S1). Additionally, the alanine ester at the C-14 position (14) may enhance the enone’s reactivity (see the supporting information Figure S2). Introducing a sterically hindered acetonide group at the nearby C-7/C-14 positions (20) slightly weakens the reactivity of the enone; however, the t1/2 of the drug substrate is still less than 1 minute (see the supporting information Figure S3). Previous studies on the development of covalent inhibitors for epidermal growth factor receptor (EGFR) suggest that the suitable reactivity window of a covalent drug, in terms of t1/2, is from 25 minutes to 400 minutes.11 The half-life of the covalent drug dimethyl fumarate (Tecfidera®), an anti-multiple sclerosis (MS) blockbuster approved by the FDA in 2013, was identified to be approximately 50 ~ 60 minutes with 2 equivalents of GSH at pH 7.4.42,

43

Additionally, the binding of the natural covalent drug Bardoxolone Methyl (6) with GSH was confirmed to be mild and reversible.16, 42, 44 In comparison with the reactivity window of these reference compounds, oridonin displays a notably short half-life to form adducts and possesses highly irreversible binding with GSH, thereby leading to potential off-target effects and safety concerns for preclinical and clinical development. In particular, we found that the efficacious dose of oridonin-like compounds was between 5 and 15 mg/kg (Table 1) in vivo, while the median lethal dose (LD50) of oridonin was 37.5 mg/kg.45 A prodrug strategy, as utilized in human Phase I clinical trial candidate 14, could decrease the toxicity of oridonin and remedy the safety concerns.29 However, for continued development and safety we believe structural optimization of the enone remains imperative. From a chemistry standpoint, excessive reactivity, to a large degree, is ascribed to the exocyclic double bond of enone in a densely functionalized chemical microenvironment nearby the D-ring. Hence, we envisioned that the replacement of the enone with a more drug-like covalent warhead could maintain the original anticancer efficacy,



while decreasing the covalent binding reactivity to a suitable half-life value. As shown in Figure 1, epoxide and aziridine groups are two feasible covalent warheads for the replacement of the enone. Furthermore, aziridines are milder than epoxides in chemical reactions and have better aqueous solubility. Inspired by the approved, aziridinated drug 10 (Figure 1), the aziridine group was selected as a mild covalent warhead for our investigation on oridonin derivatives.

Figure 2. The reactivity of oridonin with GSH determined by 1H NMR analysis.

Chemistry for Aziridinated Oridonin Analogues. Aziridines, the smallest nitrogenous heterocycles, are important synthetic target structures and building blocks.46, 47 Although several methodologies are available for the synthesis of aziridines from olefins, the development of an efficient and stereospecific aziridination with conjugated olefins remains a challenge.48 Additionally, the oridonin skeleton is not stable for a relatively long time (hours) in strong acidic


Page 10 of 54

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


or basic media,49 and therefore, our ideal aziridination should be performed in a mild and nearneutral condition. Recently, Jawahar L. Jat et al. published a moderate and scalable aziridination of olefins, utilizing O-(2,4-dinitrophenyl)hydroxylamine (DPH) as the aminating agent and Du Bois’s catalyst Rh(esp)2 at room temperature.50 However, the practical application of their new methodology was not extended to the aziridination of enone substrates. On the other hand, some researchers report that the aziridination of enones could be promoted by specific organic amine catalysts under basic conditions.51-53 Given the chemical sensitivity of oridonin to strong acidic or basic conditions, our investigation began by subjecting mixtures of the representative oridonin-like substrates (15 and 21, Table 2) and DPH in the presence of 5% mol equivalent of various homogeneous rhodium catalysts at room temperature, referring to the method reported by Jawahar L. Jat et al. The direct aziridination of 15 failed to proceed in our initial screening of rhodium catalysts. However, the transformation of its C-7/C-14 acetonide–protected substrate 21 to the corresponding aziridine 22 as a sole regio-specific product could be efficiently promoted by Rh2(esp)2 or Rh2(OAc)4 under mild conditions (Table 2, entries 1-5). The equivalent amount of DPH was optimized from 2.2 to 1.5 for improving efficiency of the aminating agent and eliminating the serious dyeing effect (dark yellow) of DPH, further leading to a more scalable purification in a best yield of 66% (Table 2, entries 6-7). The structure of 22, obtained under the reaction condition illustrated in Table 2 (entry 7), was unambiguously determined by X-ray crystallographic analysis (CCDC number 1050339, see the supporting information Table S3), indicating that the carbon atom of 21 at the C-16 position was stereospecifically converted to the R stereo configuration. Interestingly, the olefin at the C1-C2 position of 21 or 22 could not be further aziridinated to compound 23, even utilizing more aminating agent, higher temperature or extended time (Table 2, entries 8-10).



Page 12 of 54

This selectivity of our aziridination may be owing to the steric hindrance of the oridonin skeleton, consequently inactivating the olefin at the A-ring. Hereafter, the deprotection of C1-C14 dihydroxyl of 22 with 5% HCl aqueous solution afforded compound 24 (Table 2).

Table 2. Exploration and Optimization of Oridonin-based Aziridinationa

Entries

DPH Amount (equiv.)

Catalystb

Temp. /Time

Recovery of 21c

Yield of Productsc

1

2.2

Rh2(esp)2

r.t. / 12 h

trace

22 (69%)

2

2.2

Rh2(OAc)4

r.t. / 12 h

trace

22 (58%)

3

2.2

Rh2(TPA)4

r.t. / 12 h

> 80%

22 (trace)

4

2.2

Rh2(R-DOSP)4

r.t. / 12 h

> 80%

22 (NAd)

5

2.2

Rh2(S-DOSP)4

r.t. / 12 h

> 80%

22 (NA)

6

1.2

Rh2(esp)2

r.t. / 12 h

18%

22 (54%)

7

1.5

Rh2(esp)2

r.t. / 12 h

5%

22 (66%)

8

4.4

Rh2(esp)2

r.t. / 24 h

NA

9

4.4

Rh2(esp)2

50 ˚C / 24 h

NA

10

4.4

Rh2(esp)2

reflux / 48 h

NA


22 (71%) 23 (NA) 22 (66%) 23 (NA) 22 (56%)

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


23 (NA) a

b

All reaction conditions in this table were performed utilizing 21 as the substrate. The equivalent of catalyst is 5%.

c

The data were based upon isolated yields. d NA: not available.

To further explore the practicality and scope of this aziridination reaction, several diverse and bioactive oridonin derivatives, identified from our previous efforts (e.g. 16, 20, 26 and 28), were selected as representative substrates and successfully converted to the corresponding oridonin-like aziridines (Table 3, entries 1-4). However, the thiazole analogue 31 failed to be transformed likely due to the catalyst poisoning by sulfur in the thiazole moiety. Interestingly, the small regular cyclic or exocyclic enones (e.g. 32 and 33) also failed in the examination of the aziridination reaction (Table 3, entries 6-7).

Table 3. Practical Extension of Rh-catalyzed Aziridination Entries

Substrates

Products

a

Yields

1

76%

2

82%

3

61%

4

71%



a

Page 14 of 54

5

NR

b

NR

6

NR

NR

7

NR

NR

The data were based upon isolated yields. b NR: no reaction.

In the structural analysis of the reactive fragment (Figure 3A, substructure of 21) and the similar inactive substrate (33), we turned our attention to the possible critical role of the neighboring substituted oxygen (Figure 3A, red) in the Rh2(esp)2-catalyzed regio- and stereospecific aziridination. The catalyst Rh2(esp)2, with large steric hindrance (Figure 3B), captures “:NH” from DPH to afford triplet-spin state nitrene intermediate 34.50 We assumed that the electron density of 33’s olefin is decreased by the α-site ketone, thereby inducing chemical inactivation, while the intermediate 34 charges the corresponding olefin (Figure 3C). The neighboring substituted oxygen donates electron density to the olefin, leading to the re-activation of the olefin for aziridination. Meanwhile, this oxygen group anchors the “:NH” group of nitrene 34 at one side of the olefin via an intermolecular hydrogen bond, consequently leading to the regio- and stereospecific aziridination. Otherwise, the regio- and stereospecific properties of the aziridination may be alternatively ascribed to the interactions between the unique steric microenvironment of oridonin’s D-ring and nitrene intermediate 34.


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


Figure 3. Plausible reaction mechanism for Rh2(esp)2 catalyzed regio- and stereospecific aziridination of oridonin-like substrates. (A) Analysis of the reactive substructure and substrate participating in the aziridination. (B) The structures of Rh2(esp)2. (C) and (D) The comparison of two different substrates 33 (C) vs. 21 (D), assisted by the neighboring oxygen group participation in the Rh2(esp)2-promoted regio- and stereospecific aziridination.

To pursue diverse aziridine derivatives and highlight the application potential of our chemistry, the follow-up synthesis commenced with the preparation of oridonin aziridinated analogs with selected substrates, as mentioned above. The deprotection of C1-C14 dihydroxyl of 25, 27, 29 or 30 with 5% HCl aqueous solution achieved the desired compounds 35-38, respectively (Scheme 1A). According to the previously established SAR, the fused thiazole moieties on A-ring could significantly enhance anticancer activities of oridonin (Table 1, entry 6). With the aim of further improving the anticancer profiles of oridonin aziridines, the representative target compound 43 was designed with an additional N-allyl substituted thiazole at



the A-ring, and synthesized from an aziridinated substrate 27 prior to the thiazole ring formation due to the aforementioned reason for aziridination failure of 31 directly (Table 3, entry 5). As depicted in Scheme 1B, protection of the secondary amine of aziridine 27 with Fmoc chloride in the presence of Na2CO3 provided 39 in 34% yield. Treatment of 39 with 5% aqueous HCl readily resulted in the deprotected derivative 40 in 75% yield. Subsequently, compound 40 was brominated by PyHBr3 in THF, followed by the Hantzsch reaction of 41 with N-allylthiourea yielding the thiazole 42. Finally, removal of the acetonide protection group with piperidine in DMF afforded the desired compound 43. Moreover, the secondary amine of aziridine provides an additional site for further structural modifications or the formation of salts to improve the bioactivities and drug-like properties such as aqueous solubility. As presented in Scheme 1C, we could attach various functional fragments such as an additional enone to the aziridine ring leading to compound 44, and a non-covalent propionyl moiety leading to 45 for comparison through different conventional mild reactions. Such mediated covalent warheads at the secondary amine may directly exert bioactivities independent of the aziridine ring, and alternatively, regulate the covalent bond formation reactivities of the aziridine ring indirectly.

Scheme 1. Synthesis of the Aziridinated Oridonin Analoguesa


Page 16 of 54

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


a

Reagents and conditions: (a) 5% HCl (aq), MeOH, CH2Cl2, 15 min, 28% ~ 62%; (b) Fmoc

chloride, Na2CO3, dioxane, 0 ˚C → rt, 12 h, 34%; (c) 5% HCl (aq), MeOH, CH2Cl2, 15 min, 75%; (d) PyHBr3, THF, 0 °C, 2 h, 82%; (e) N-allylthiourea, EtOH, reflux, 5 h, 44%; (f) piperidine, DMF, rt, 1 h, 82%; (g) acrylic acid, HBTU, DIPEA, CH2Cl2, 0 ˚C → rt, 12 h, 33%; (h) propionic acid, HBTU, DIPEA, CH2Cl2, 0 ˚C → rt, 12 h, 74%.

Covalent Bond Formation Reactivity of the New Warheads. From our initial investigation of reactions between aziridines and thiol groups from the SciFinder database, we found that the representative compound 2-aziridinecarboxylic acid, which is similar to the reactive fragments of our aziridinated oridonin analogs, could react with the thiol of cysteine in buffer at room temperature for 20 h in high yield (see the supporting information Figure S4).54



To determine the experimental GSH reaction rates of aziridinated oridonin analogs, compound 25 was incubated at 37 ˚C with 1.5 ~ 2 equivalents of GSH in 0.5 mL of d6-DMSO and 0.05 mL of sodium phosphate deuterium oxide buffer (pH 7.4). The sample was analyzed by 1H NMR at 11 time points (Figure 4) and indicated that the reactivity of the covalent warhead aziridine in the D-ring is much more moderate than the enone at the same position. Based upon the integrated area of 1H NMR peak d generated from the reaction, t1/2 was identified as approximately 4 ~ 6 hrs. Additionally, the adduct product was also validated by HRMS analysis of the reaction at 5 hrs, indicating the dominant component of the sample as the adduct 46. Meanwhile, the signals of starting materials GSH and aziridine 25 were obviously observed in such analysis, suggesting that aziridinated oridonin analogs are much more stable compared to oridonin with exposure to GSH (see the supporting information Figure S5). Moreover, compound 25 with the aziridination of the D-ring enone has higher microsomal and plasma stability compared to oridonin in vitro (see the supporting information Table S2 and Figure S9), indicating that the D-ring aziridine is a drug-like pharmacophore as well as an available site of modification for improving pharmacokinetics profiles.


Page 18 of 54

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


Figure 4. The reactivity of aziridinated oridonin analogue 25 with GSH determined by 1H NMR.

Under the same conditions, the acrylamide analogue 44 possesses a more reactive warhead in comparison to the aziridinated oridonin 25. Based upon the integrated area of 1H NMR peak a disappearing during the reaction, the half-life is identified from 30 to 60 min (Figure 5). Similarly, the adduct product 47 was validated by HRMS analysis after the reaction at 1 h (see the supporting information Figure S7). In conclusion, the aziridine or acrylamide aziridine attached to the D-ring of oridonin are milder covalent warheads with suitable reactivity in terms of the half-life values, which are in line with the reactivity window (25 min ~ 400 min)11 suggested for covalent drugs.



Figure 5. The reactivity of aziridinated oridonin analogue 44 with GSH determined by 1H NMR.

In Vitro Biological Activities against Breast Cancer Cell Lines. The growth inhibitory effects of these newly synthesized oridonin aziridinated derivatives were initially evaluated in two breast cancer cell lines, MCF-7 (ER-positive) and MDA-MB-231 (ER-negative and triplenegative), using MTT assays as described in the in vitro screening protocol (Experimental Section). The ability of these new analogs to inhibit the growth of cancer cells was summarized in the supporting information Table S1 and compared with oridonin. Given that the original enone warhead in the D-ring is a critical pharmacophore, it is not surprising that the overall antiproliferative effects of these aziridinated oridonin molecules are overall lower than those lead compounds we previously reported.31, 33, 55, 56 However, several aziridinated oridonin derivatives


Page 20 of 54

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


such as 42 and 43 displayed moderate activities. Compound 44 exhibited significantly improved antiproliferative effects against highly invasive triple-negative breast cancer MDA-MB-231 cells and MCF-7 cells with IC50 values of 8.32 µM and 9.39 µM, respectively, in comparison with oridonin (IC50 values of 29.4 µM and 6.72 µM against corresponding cell lines). Interestingly, replacement of the covalent warhead enamide of 44 with a non-covalent propionyl moiety (compound 45) resulted in a complete loss of activity, indicating the important presence of the covalent warhead for biological activity. Next, aziridinated oridonin analogue 44 was selected for the colony formation assay and was found to significantly inhibit the colony formation of highly invasive triple-negative breast cancer cells MDA-MB-231, SK-BR3, MDA-MB-468 and also the ER-positive breast cancer cells MCF-7 at low micromolar concentrations of the compound (Figure 6A). As shown in Figure 6B, 44 also suppressed the proliferation of MDAMB-231, SK-BR3, MDA-MB-468 and MCF-7 breast cancer cells in a dose-dependent manner.



Figure 6. Growth inhibitory effects of 44 against MDA-MB-231, SK-BR3, MDA-MB-468 and MCF-7 breast cancer cells. (A) 44 significantly inhibits colony formation of MDA-MB-231, SKBR3, MDA-MB-468 and MCF-7 cells. (B) 44 suppresses the proliferation of MDA-MB-231, SK-BR3, MDA-MB-468 and MCF-7 cells. Data is presented as the mean ± SD of at least three independent experiments. Significance between different groups was determined by using one way ANOVA, p = 0.0003 (MDA-MB-231) and p = 0.0012 (MCF-7).

Based on its anti-proliferative effects shown in Figure 6, further preliminary mechanistic studies of 44 were explored to determine whether 44 can induce apoptosis of breast cancer cells. MDAMB-231 cells were treated with 44 at varying concentrations (1 μM, 5 μM, 10 μM, 15 μM, or 20 μM) for 48 h and stained with FITC-Annexin V and propidium iodide (PI). The percentages of apoptotic MDA-MB-231 cells were determined by flow cytometry. As shown in Figure 7A and 7B, 44 can induce apoptosis in breast cancer cells at 10 μM (18.2%), 15 μM (26.8%), and 20 μM


Page 22 of 54

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


(47.6%) concentrations at 48 h (early and late apoptosis together) dose-dependently compared to vehicle control. Apparently, 44 induces apoptosis of MDA-MB-231 cells, which at least in part contributes to its antiproliferative effects. Previous studies have demonstrated that oridonin induces apoptosis of cancer cells by regulating a series of transcription factors, protein kinases as well as pro- and/or anti-apoptotic proteins such as NF-κB37, 57, 58 and Bcl-259, 60. To elucidate the potential mechanisms contributing to apoptosis induction by 44, expression levels of several proteins related to apoptosis were determined by Western blotting. As shown in Figure 7C, treatment of MDA-MB-231 cells with 44 dose-dependently led to the down-regulation of NF-κB (p65) protein and up-regulation of cleaved PARP, with the latter as an apoptosis marker. In addition, 44 also reduced the ratio of Bcl-2/Bax, which are important apoptotic marker proteins. These results suggest that 44 is capable of inducing apoptosis to decrease cellular proliferation. The parental chemotype, oridonin, has been reported to inhibit tumor cell proliferation and induce cancer cell death through cell cycle arrest58, 59, 61, autophagy37, 57, 62, 63 and necrosis64. Therefore, more extensive mechanistic studies in terms of major drug targets and signaling pathways associated with 44 are under investigation, and the results will be reported in due course.



Figure 7. Induction of apoptosis in MDA-MB-231 breast cancer cells by 44. (A) Flow cytometry analysis of apoptotic MDA-MB-231 breast cancer cells induced by 44 at different concentrations. (B) Apoptotic ratio of different concentrations of 44 in MDA-MB-231 breast cancer cells. The values are means ± SD of at least three independent experiments. *represents p < 0.05, **represents p < 0.01, ***represents p < 0.001 to 0 M group (DMSO, vehicle control). (C) Western blot analysis of biomarkers for apoptosis induced by 44 in MDA-MB-231 breast cancer cells at different concentrations (48 h). In Vitro Growth Inhibition on Normal Mammary Epithelial Cells. Selective toxicity for cancer, but not normal cells, is essential in the development of targeted cancer experimental therapeutics. To investigate whether the improved antiproliferative effects of 44 against breast cancer cells were attributed to undesired cell toxicities, we further examined its inhibitory effects on the growth of normal mammary epithelial cells MCF-10A. As shown in Figure 8, 44 exhibited comparable (0 ~ 10 µM) or significantly lower (15 µM ~ 100 µM) growth inhibitory activity against MCF-10A cells at all tested concentrations, while displaying significantly enhanced anticancer activities against triple-negative MDA-MB-231 cancer cells when compared with oridonin. These results provide a proof of concept that designing novel covalent


Page 24 of 54

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


warheads in the D-ring of oridonin could be beneficial in lead optimization for improved biological activities, safety and other drug-like properties.

Figure 8. Effect of 44 and oridonin on normal mammary epithelial cells (MCF-10A) proliferation. MCF-10A cells were treated with varying concentrations of 44 and oridonin for 48 h. Values are mean ± SD of three independent experiments. Statistical significance was determined using Student’s t-test, in comparison with corresponding value of oridonin treatment at the same concentration. ns means not significant, * p < 0.05, *** p < 0.001, ****p < 0.0001.

Figure 9. In vivo efficacy of 44 in suppressing the xenograft tumor growth of triple-negative breast cancer. Breast cancer cell line MDA-MB-231 was used to generate xenograft tumors in nude mice for 10 mg/kg and 15 mg/kg doses via i.p., respectively. Data is presented as the mean ± SEM of tumor volume at each time point. Significance between different groups was determined by using one way ANOVA, p = 0.0023.



Growth Suppression of 44 on Triple-Negative Breast Cancer Xenografts in Mice. Compound 44 was further evaluated for its suppression of tumor growth in the triple-negative breast cancer MDA-MB-231 xenograft model. As shown in Figure 9, mice treated with 10 and 15 mg/kg of 44 (i.p.) showed a significant inhibitory effect on tumor growth compared to the mice treated with vehicle. The dose of 15 mg/kg group with the treatment of 44 showed a better inhibitory effect than the oridonin group (one way ANOVA, p = 0.0023), while both 44 and oridonin displayed a comparable efficacy at 10 mg/kg. These findings suggest that 44 is a potential anticancer drug candidate with effective antitumor activity and better druglike properties for further preclinical development.

CONCLUSIONS From incidental discovery to rational design, the SRR of warheads has been recognized as a critical index in the development of covalent drugs. For the first time, SRR studies on the natural product oridonin have been implemented by 1H NMR and HRMS analyses monitoring its reaction with GSH and determining its D-ring electrophile enone reactivity. To explore more drug-like oridonin derivatives with optimized covalent warheads, we utilized a mild and efficient Rh2(esp)2-catalyzed reaction to regio- and stereo-specifically construct new aziridinated oridonin analogues, replacing the overly reactive enone at the same site on D-ring. For the synthetic chemistry approach, this Rh2(esp)2 promoted reaction may undergo the neighboring oxygen group participation. Moreover, the reaction methodology was demonstrated to be feasible for the aziridination of various oridonin-like substrates. Furthermore, the reactivities of the representative aziridinated oridonin derivatives 25 and 44 have been successfully mediated to be much milder than oridonin. The initial GSH t1/2 values of oridonin, 25 and 44 are classified to be


Page 26 of 54

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


< 1 min (excessively active), 240 ~ 360 min (relatively inactive) and 30 ~ 60 min (drug-like), respectively, indicating new aziridinated analogues have more suitable druglike properties in line with the referenced reactivity window of covalent drugs. Accordingly, aziridonin 44 has demonstrated significantly enhanced apoptosis induction and colony formation inhibition against triple-negative breast cancer in vitro and in vivo with lower toxicity to normal human mammary epithelial cells in comparison with oridonin. Taken together, our findings support that optimization of diverse bioactive functionalities at the A-ring, while moderation of the covalent warhead reactivities at the D-ring, may be a viable approach to the discovery and development of effective and drug-like oridonin derivatives in the future.

EXPERIMENTAL SECTION General. All commercially available starting materials and solvents were reagent grade and used without further purification. Reactions were performed under a nitrogen atmosphere in dry glassware with magnetic stirring. Preparative column chromatography was performed using silica gel 60, particle size 0.063−0.200 mm (70−230 mesh, flash). Analytical TLC was carried out employing silica gel 60 F254 plates (Merck, Darmstadt). Visualization of the developed chromatograms was performed with detection by UV (254 nm). NMR spectra were recorded on a Bruker-600 (1H, 300 MHz;

13

C, 75 MHz) spectrometer. 1H and

13

C NMR spectra were

recorded with TMS as an internal reference. Chemical shifts downfield from TMS were expressed in ppm, and J values were given in Hz. High-resolution mass spectra (HRMS) were obtained from Thermo Fisher LTQ Orbitrap Elite mass spectrometer. Parameters include the following: nano ESI spray voltage was 1.8 kV, capillary temperature was 275 °C, and the resolution was 60000; ionization was achieved by positive mode. Melting points were measured



on a Thermo Scientific Electrothermal digital melting point apparatus and uncorrected. Purity of final compounds was determined by analytical HPLC, which was carried out on a Shimadzu HPLC system (model: CBM-20A LC-20AD SPD-20A UV/vis). HPLC analysis conditions: Waters μBondapak C18 (300 mm × 3.9 mm), flow rate 0.5 mL/min, UV detection at 270 and 254 nm, linear gradient from 10% acetonitrile in water (0.1% TFA) to 100% acetonitrile (0.1% TFA) in 20 min, followed by 30 min of the last-named solvent. All biologically evaluated compounds are >95% pure. (2R,3'S,3a'R,3a1'R,6a'R,7'S,11a'R)-7'-Hydroxy-5',5',8',8'-tetramethyl1',3',3a',7',7a',8',9',11b'-octahydro-2'H-spiro[aziridine-2,15'[6a,11a](epoxymethano)[3,3a1]ethanophenanthro[1,10-de][1,3]dioxin]-14'-one (22). To a suspension of 2155 (28.2 mg, 0.07 mmol) in CF3CH2OH (0.7 mL) was added Rh2(esp)2 (1.1 mg) and DPH (17.5 mg, 0.09 mmol) at room temperature. The resulting mixture was stirred at room temperature for 24 h. Then the solvent was removed under reduced pressure and the residue was dissolved in CH2Cl2 (20 mL). The organic layer was washed with water (20 mL) and saturated brine (20 mL), dried over anhydrous Na2SO4, filtered, and evaporated to give an oily residue. The residue was purified using silica gel column; elution with 50% EtOAc in hexanes afforded the desired product 22 (21.6 mg, 74%) as a yellowish amorphous solid; [α]25 = -72.4 (c 0.20, D MeOH); 1H NMR (300 MHz, CDCl3): δ 5.81 (m, 1H), 5.24 (m, 1H), 5.11 (d, J = 11.7 Hz, 1H), 4.97 (d, J = 1.8 Hz, 1H), 4.07 – 3.79 (m, 3H), 2.47 – 2.34 (m, 1H), 2.24 (d, J = 9.2 Hz, 2H), 2.04 – 1.72 (m, 6H), 1.69 (s, 3H), 1.64 (d, J = 6.2 Hz, 3H), 1.41 (s, 3H), 1.18 (s, 3H), 1.07 (s, 3H); 13C NMR (75 MHz, CDCl3): δ 216.8, 130.4, 124.1, 101.4, 95.2, 72.0, 70.9, 65.0, 58.1, 56.6, 48.9, 48.7, 41.1, 38.5, 38.0, 32.3, 31.9, 31.1, 30.3, 26.5, 25.4, 22.1, 17.2; HRMS (ESI) Calcd for C23H32NO5 [M+H]+ 402.2280; found 402.2268. The stereochemistry of 22 was secured by X-ray


Page 28 of 54

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


crystallographic analysis. The data have been assigned at the Cambridge Crystallographic Data Centre to the following deposition number CCDC 1050339. (2R,5'S,6'S,6a'R,9'S,11b'R,14'R)-5',6',14'-Trihydroxy-4',4'-dimethyl4',4a',5',6',9',10',11',11a'-octahydro-3'H,7'H-spiro[aziridine-2,8'[6,11b](epoxymethano)[6a,9]methanocyclohepta[a]naphthalen]-7'-one (24). To a solution of 22 (101.0 mg, 0.24 mmol) in MeOH (2 mL) and CH2Cl2 (0.5 mL) was added hydrochloric acid aqueous solution (1 N, 0.5 mL) at room temperature. The resulting mixture was stirred at room temperature for 15 min. The solvent was removed under reduced pressure and the residue was dissolved in CH2Cl2 (20 mL) and saturated NaHCO3 aqueous solution (10 mL). The organic layer was washed with water (20 mL) and saturated brine (20 mL), dried over anhydrous Na2SO4, filtered, and evaporated to give an oily residue. The residue was purified using silica gel column; elution with 2.5% MeOH in CH2Cl2 afforded the desired product 24 (71.0 mg, 78%) as a white amorphous solid. [α]25 = -102.5 (c 0.11, MeOH); 1H NMR (300 MHz, CDCl3): δ 5.91 – 5.77 (m, D 1H), 5.25 – 5.14 (m, 1H), 4.95 (s, 1H), 4.78 (d, J = 11.3 Hz, 1H), 4.06 – 3.66 (m, 6H), 2.54 – 2.22 (m, 2H), 2.02 – 1.46 (m, 6H), 1.41 (m, 1H), 1.32 – 1.23 (m, 1H), 1.18 (s, 3H), 1.06 (s, 3H), 0.99 – 0.80 (m, 1H).

13

C NMR (75 MHz, CDCl3): δ 216.0, 130.9, 124.1, 97.2, 74.6, 72.8, 65.2,

63.7, 58.4, 52.3, 49.7, 43.7, 41.1, 38.4, 32.4, 30.9, 22.2, 22.1, 21.8, 16.2. HRMS Calcd for C20H28NO5: [M+H]+ 362.1967; found 362.1958. (2R,3'S,3a'R,3a1'R,6a'R,7'S,11'S,11a'S)-7',11'-Dihydroxy-5',5',8',8'tetramethyldecahydro-2'H-spiro[aziridine-2,15'[6a,11a](epoxymethano)[3,3a1]ethanophenanthro[1,10-de][1,3]dioxin]-14'-one

(25).

Compound 25 (211.6 mg) was prepared from 2055 in 76% yield by a procedure similar to that used to prepare compound 22. The title compound 25 was obtained as a yellowish amorphous



Page 30 of 54

solid. [α]25 = -102.8 (c 0.13, MeOH); 1H NMR (300 MHz, CDCl3): δ 5.46 (d, J = 11.6 Hz, 1H), D 4.94 (d, J = 1.7 Hz, 1H), 4.28 (dd, J = 9.9, 1.5 Hz, 1H), 4.06 (dd, J = 9.9, 1.2 Hz, 1H), 3.90 (dd, J = 11.6, 7.6 Hz, 1H), 3.50 (m, 1H), 2.39 (m, 1H), 2.23 (m, 2H), 1.92 (m, 3H), 1.82 – 1.21 (m, 15H), 1.20 – 1.11 (m, 6H).

13

C NMR (75 MHz, CDCl3): δ 217.9, 101.1, 94.7, 74.5, 73.0, 70.9,

62.8, 59.0, 56.6, 50.5, 49.3, 40.7, 39.0, 38.2, 33.7, 33.3, 31.9, 30.4, 29.8, 26.8, 25.4, 22.6, 19.9. HRMS Calcd for C23H34NO6: [M+H]+ 420.2386; found 420.2371. (2R,3'S,3a'R,3a1'R,6a'R,7'S,11a'S)-7'-Hydroxy-5',5',8',8'-tetramethyloctahydro-2'Hspiro[aziridine-2,15'-[6a,11a](epoxymethano)[3,3a1]ethanophenanthro[1,10de][1,3]dioxine]-11',14'(7'H)-dione (27). Compound 27 (20.1 mg) was prepared from 2656 in 82% yield by a procedure similar to that used to prepare compound 22. The title compound 27 was obtained as obtained as a yellowish amorphous solid. [α]25 = -59.6 (c 0.18, MeOH); 1H NMR D (300 MHz, CDCl3): δ 4.97 (dd, J = 6.7, 5.0 Hz, 2H), 4.25 (dd, J = 10.2, 1.5 Hz, 1H), 4.06 (dd, J = 10.1, 1.6 Hz, 1H), 3.94 (dd, J = 11.7, 8.7 Hz, 1H), 2.43 (m, 3H), 2.25 (m 2H), 2.11 (m, 1H), 2.02 – 1.62 (m, 10H), 1.54 (m, 1H), 1.40 (s, 3H), 1.22 (s, 3H), 1.07 (s, 3H); 13C NMR (75 MHz, CDCl3): δ 217.1, 211.5, 101.5, 95.6, 71.8, 70.8, 64.7, 59.9, 56.0, 48.9, 48.1, 46.5, 38.4, 38.3, 35.8, 33.0, 32.1, 31.0, 30.2, 26.4, 25.3, 23.1, 18.6; HRMS Calcd for C23H32NO6: [M+H]+ 418.2230; found 418.2215. (2R,3'S,3a'R,3a1'R,6a'R,7'S,11a'R)-7'-hydroxy-5',5',8',8'-tetramethyl-1',3',3a',7a',8',11b'hexahydro-2'H-spiro[aziridine-2,15'[6a,11a](epoxymethano)[3,3a1]ethanophenanthro[1,10-de][1,3]dioxine]-9',14'(7'H)-dione (29). Compound 29 (29.1 mg) was prepared from 2831 in 61% yield by a procedure similar to that used to prepare compound 22. The title compound 29 was obtained as obtained as a yellowish amorphous solid. [α]25 = -95.4 (c 0.29, MeOH); 1H NMR (300 MHz, CDCl3): δ 6.33 (d, D


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


J = 10.2 Hz, 1H), 6.04 (d, J = 10.3 Hz, 1H), 5.23 (d, J = 11.8 Hz, 1H), 5.00 (d, J = 1.6 Hz, 1H), 4.11 (m, 3H), 2.44 (m, 1H), 2.27 (m, 2H), 2.06 (s, 1H), 2.02 – 1.53 (m, 8H), 1.50 – 1.07 (m, 10H);

13

C NMR (75 MHz, CDCl3): δ 216.6, 203.2, 142.4, 129.9, 101.7, 95.2, 70.8, 70.7, 64.3,

56.2, 56.1, 48.8, 48.0, 44.6, 38.7, 32.1, 30.2, 26.3, 25.4, 23.9, 22.5, 17.1, 14.2; HRMS Calcd for C23H30NO6: [M+H]+ 416.2073; found 416.2058. (2R,3'S,3a'R,3a1'R,6a'R,7'S,11a'S)-7'-hydroxy-5',5',8',8'-tetramethyl-1',3',3a',7a',8',11b'hexahydro-2'H-spiro[aziridine-2,15'[6a,11a](epoxymethano)[3,3a1]ethanophenanthro[1,10-de][1,3]dioxine]-11',14'(7'H)-dione (30). Compound 30 (11.0 mg) was prepared from 1631 in 71% yield by a procedure similar to that used to prepare compound 22. The title compound 30 was obtained as obtained as a yellowish amorphous solid. [α]25 = -111.6 (c 0.10, MeOH); 1H NMR (300 MHz, CDCl3): δ 6.81 D (d, J = 10.1 Hz, 1H), 5.87 (d, J = 10.1 Hz, 1H), 5.17 – 4.96 (m, 2H), 4.27 (dd, J = 9.9, 1.6 Hz, 1H), 4.16 – 4.03 (m, 2H), 2.45 – 2.32 (m, 1H), 2.29 – 2.18 (m, 2H), 2.04 (d, J = 7.3 Hz, 2H), 1.97 – 1.85 (m, 2H), 1.77 – 1.65 (m, 6H), 1.41 (d, J = 2.2 Hz, 6H), 1.27 (d, J = 2.2 Hz, 3H);

13

C

NMR (75 MHz, CDCl3): δ 217.3, 196.3, 162.1, 126.7, 101.5, 95.5, 71.6, 70.8, 65.2, 56.6, 56.2, 49.0, 47.1, 45.7, 38.2, 35.9, 32.1, 30.4, 30.2, 26.5, 25.3, 25.0, 19.1; HRMS Calcd for C23H30NO6: [M+H]+ 416.2073; found 416.2063. (6S,6aR,6a1R,9aR,10S,12bR)-2-(Cyclohexylamino)-6-hydroxy-5,5,8,8-tetramethyl-15methylene-4,5,5a,6,9a,10,12,12a-octahydro-11H-6a,12b-(epoxymethano)-6a1,10ethano[1,3]dioxino[4',5',6':8,9]phenanthro[4,3-d]thiazol-16-one (31). To a solution of 18a33 (190.8 mg, 0.38 mmol) in acetone (10 mL) was added 2,2-dimethoxypropane (1.5 mL) and pTsOH (10 mg) at room temperature. The resulting mixture was refluxed for 8 h. The solvent was removed under reduced pressure and the residue was dissolved in CH2Cl2 (20 mL) and saturated



NaHCO3 aqueous solution (10 mL). The organic layer was washed with water (20 mL) and saturated brine (20 mL), dried over anhydrous Na2SO4, filtered, and evaporated to give an oily residue. The residue was purified using silica gel column; elution with 25% EtOAc in hexane afforded the desired product 31 (168.1 mg, 82%) as a white amorphous solid. [α]25 = -68.0 (c D 0.10, MeOH); 1H NMR (300 MHz, CDCl3): δ 6.13 (d, J = 1.3 Hz, 1H), 5.54 – 5.47 (m, 2H), 4.92 (d, J = 1.7 Hz, 1H), 4.81 (d, J = 7.8 Hz, 1H), 4.33 (dd, J = 9.8, 1.5 Hz, 1H), 4.04 – 3.86 (m, 2H), 3.25 – 3.10 (m, 1H), 3.05 (dd, J = 8.9, 1.6 Hz, 1H), 2.57 – 2.44 (m, 2H), 2.29 (m, 1H), 2.10 – 1.94 (m, 4H), 1.92 – 1.83 (m, 1H), 1.78 – 1.69 (m, 3H), 1.64 – 1.55 (m, 2H), 1.36 (s, 4H), 1.27 (m, 6H), 1.19 (m, 3H), 1.01 (s, 3H), 0.88 (s, 1H); 13C NMR (75 MHz, CDCl3): δ 204.8, 165.1, 150.9, 142.9, 120.0, 118.5, 101.0, 95.4, 72.0, 70.2, 65.0, 58.5, 56.6, 54.7, 49.9, 40.4, 40.2, 39.0, 34.9, 33.2, 33.1, 30.9, 30.7, 30.2, 25.5, 25.4, 24.7, 24.7, 21.4, 20.1. HRMS Calcd for C30H41N2O5S: [M+H]+ 541.2736; found 541.2734. (1'S,2R,5'S,6'S,6a'R,9'S,11b'S,14'R)-1',5',6',14'-Tetrahydroxy-4',4'-dimethyldecahydro1'H,7'H-spiro[aziridine-2,8'[6,11b](epoxymethano)[6a,9]methanocyclohepta[a]naphthalen]-7'-one (35). Compound 35 (30.1 mg) was prepared from 25 in 62% yield by a procedure similar to that used to prepare compound 24. The title compound 35 was obtained as a white amorphous solid. [α]25 = -110.9 (c D 0.11, MeOH); 1H NMR (300 MHz, CDCl3): δ 5.18 (d, J = 11.1 Hz, 1H), 4.93 (d, J = 1.3 Hz, 1H), 4.27 – 4.09 (m, 2H), 3.89 – 3.76 (m, 2H), 3.68 (d, J = 12.1 Hz, 1H), 3.46 (dd, J = 11.0, 5.9 Hz, 1H), 2.49 – 2.26 (m, 2H), 2.22 – 1.98 (m, 1H), 1.80 – 1.38 (m, 7H), 1.37 – 1.20 (m, 2H), 1.14 (m, 8H), 0.95 – 0.80 (m, 1H). 13C NMR (75 MHz, CDCl3): δ 217.9, 96.6, 75.0, 73.7, 73.6, 67.0, 63.9, 62.9, 59.8, 54.4, 49.7, 43.2, 41.3, 38.7, 33.8, 32.8, 30.0, 22.3, 21.9, 18.4. HRMS Calcd for C20H30NO6: [M+H]+ 380.2073; found 380.2063.


Page 32 of 54

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


(2R,5'S,6'S,6a'R,9'S,11b'S,14'R)-5',6',14'-Trihydroxy-4',4'-dimethyldecahydro-1'H,7'Hspiro[aziridine-2,8'-[6,11b](epoxymethano)[6a,9]methanocyclohepta[a]naphthalene]-1',7'dione (36). Compound 36 (79.1 mg) was prepared from 27 in 28% yield by a procedure similar to that used to prepare compound 24. The title compound 36 was obtained as a white amorphous solid. [α]25 = -100.0 (c 0.14, MeOH); 1H NMR (300 MHz, CDCl3): δ 5.37 (d, J = 11.1 Hz, 1H), D 5.04 (s, 1H), 4.27 (dd, J = 10.4, 1.4 Hz, 1H), 4.03 (dd, J = 10.4, 1.4 Hz, 1H), 3.90 – 3.74 (m, 1H), 2.54 – 2.24 (m, 5H), 2.11 (m, 1H), 2.00 – 1.53 (m, 7H), 1.39 – 1.21 (m, 2H), 1.16 (s, 3H), 1.01 (s, 3H), 0.96 – 0.63 (m, 1H).

13

C NMR (75 MHz, CDCl3): δ 217.9, 211.7, 97.9, 73.2, 72.8, 64.9,

61.5, 60.1, 49.1, 49.1, 48.4, 40.5, 38.4, 35.7, 32.9, 31.5, 30.5, 26.0, 23.3, 18.6. HRMS Calcd for C20H28NO6: [M+H]+ 378.1917; found 378.1907. (2R,5'S,6'S,6a'R,9'S,11b'R,14'R)-5',6',14'-Trihydroxy-4',4'-dimethyl4',4a',5',6',9',10',11',11a'-octahydro-3'H,7'H-spiro[aziridine-2,8'[6,11b](epoxymethano)[6a,9]methanocyclohepta[a]naphthalene]-3',7'-dione (37). Compound 37 (16.6 mg) was prepared from 29 in 35% yield by a procedure similar to that used to prepare compound 24. The title compound 37 was obtained as a white amorphous solid. [α]25 = -55.0 (c D 0.12, MeOH); 1H NMR (300 MHz, CDCl3): δ 6.35 (d, J = 10.4 Hz, 1H), 6.05 (d, J = 10.4 Hz, 1H), 5.51 (s, 1H), 5.14 (s, 1H), 4.29 – 3.83 (m, 3H), 2.57 – 2.21 (m, 3H), 1.93 (m, 4H), 1.72 (m, 3H), 1.30 (m, 7H), 0.89 (m, 1H); 13C NMR (75 MHz, CDCl3): δ 217.7, 202.8, 142.6, 130.2, 97.5, 73.4, 72.0, 64.7, 61.8, 56.1, 50.7, 44.5, 40.5, 39.0, 31.6, 29.7, 26.0, 23.6, 22.1, 17.3; HRMS Calcd for C20H26NO6: [M+H]+ 376.1760; found 376.1750. (2R,5'S,6'S,6a'R,9'S,11b'S,14'R)-5',6',14'-Trihydroxy-4',4'-dimethyl4',4a',5',6',9',10',11',11a'-octahydro-1'H,7'H-spiro[aziridine-2,8'-



[6,11b](epoxymethano)[6a,9]methanocyclohepta[a]naphthalene]-1',7'-dione (38). Compound 38 (11.0 mg) was prepared from 30 in 61% yield by a procedure similar to that used to prepare compound 24. The title compound 38 was obtained as a white amorphous solid. [α]25 = -206.9 (c D 0.19, MeOH); 1H NMR (300 MHz, CDCl3): δ 6.80 (d, J = 10.1 Hz, 1H), 5.89 (d, J = 10.1 Hz, 1H), 5.51 (d, J = 11.5 Hz, 1H), 5.10 (d, J = 23.9 Hz, 2H), 4.31 (d, J = 10.2 Hz, 1H), 4.05 (dd, J = 19.5, 10.0 Hz, 2H), 3.51 (s, 1H), 2.28 (d, J = 11.7 Hz, 3H), 2.07 (d, J = 4.1 Hz, 2H), 1.90 (d, J = 9.0 Hz, 2H), 1.62 (d, J = 8.8 Hz, 3H), 1.37 (s, 2H), 1.27 (d, J = 5.5 Hz, 4H); 13C NMR (75 MHz, CDCl3): δ 218.3, 196.2, 161.3, 127.1, 97.8, 73.4, 72.6, 65.6, 62.0, 56.5, 49.8, 49.2, 46.2, 40.5, 35.8, 30.0, 29.7, 26.2, 24.7, 19.0; HRMS Calcd for C20H26NO6: [M+H]+ 376.1760; found 376.1750. (9H-Fluoren-9-yl)methyl

(2R,3'S,3a'R,3a1'R,6a'R,7'S,11a'S)-7'-hydroxy-5',5',8',8'-

tetramethyl-11',14'-dioxodecahydro-2'H-spiro[aziridine-2,15'[6a,11a](epoxymethano)[3,3a1]ethanophenanthro[1,10-de][1,3]dioxine]-1-carboxylate (39). To a solution of 27 (203.1 mg, 0.49 mmol) and Na2CO3 (154.7 mg, 1.46 mmol) in dioxane (20 mL) was added Fmoc chloride (151.0 mg, 0.58 mmol) at 0 ˚C. The resulting mixture was stirred at room temperature for 12 h. Then the solution was removed and the residue was dissolved with CH2Cl2 (50 mL). The organic layer was washed with saturated NaHCO3 solution (50 mL) and brine (50 mL), dried over anhydrous Na2SO4, filtered, and evaporated to give an oily residue. The residue was purified using silica gel column; elution with 33% EtOAc in hexane afforded the desired product 39 as a white solid (106.1 mg, 34%). [α]25 = -45.9 (c 0.19, MeOH); 1H NMR D (300 MHz, CDCl3): δ 7.83 – 7.73 (m, 2H), 7.65 (dd, J = 10.5, 7.4 Hz, 2H), 7.37 (dtt, J = 26.4, 7.6, 1.4 Hz, 4H), 4.99 (d, J = 1.5 Hz, 1H), 4.65 (d, J = 11.9 Hz, 1H), 4.61 – 4.49 (m, 1H), 4.36 – 4.19 (m, 3H), 4.08 (dd, J = 10.1, 1.5 Hz, 1H), 3.96 (dd, J = 11.9, 8.8 Hz, 1H), 2.75 (d, J = 1.1 Hz,


Page 34 of 54

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


1H), 2.56 – 2.32 (m, 5H), 2.15 – 2.07 (m, 1H), 1.99 – 1.54 (m, 9H), 1.44 (s, 3H), 1.22 (s, 3H), 1.08 (s, 3H).

13

C NMR (75 MHz, CDCl3): δ 211.9, 211.2, 159.4, 143.6, 143.6, 141.2, 141.2,

127.7, 127.7, 127.1, 127.1, 125.4, 125.4, 119.9, 119.9, 101.9, 95.5, 71.8, 70.4, 68.7, 64.7, 59.6, 56.2, 54.5, 48.2, 46.9, 46.8, 38.5, 38.2, 35.8, 35.4, 33.0, 31.0, 29.8, 26.5, 25.4, 23.0, 18.5. HRMS Calcd for C38H42NO8: [M+H]+ 640.2943; found 640.2897. (9H-Fluoren-9-yl)methyl

(2R,5'S,6'S,6a'R,9'S,11b'S,14'R)-5',6',14'-trihydroxy-4',4'-

dimethyl-1',7'-dioxodecahydro-1'H,7'H-spiro[aziridine-2,8'[6,11b](epoxymethano)[6a,9]methanocyclohepta[a]naphthalene]-1-carboxylate

(40).

Compound 40 (106.1 mg) was prepared in 75% yield by a procedure similar to that used to prepare compound 24. The title compound 40 was obtained as a white solid. [α]25 = -110.2 (c D 0.13, MeOH); 1H NMR (300 MHz, CDCl3 and CD3OD): δ 7.72 (d, J = 7.4 Hz, 2H), 7.54 (dd, J = 7.1, 2.3 Hz, 2H), 7.44 – 7.20 (m, 4H), 5.98 (d, J = 125.4 Hz, 1H), 5.05 (d, J = 11.6 Hz, 1H), 4.85 (s, 1H), 4.45 – 4.14 (m, 4H), 4.04 – 3.92 (m, 1H), 3.85 – 3.53 (m, 2H), 2.60 – 2.39 (m, 2H), 2.30 (ddt, J = 15.3, 11.5, 5.8 Hz, 2H), 2.12 – 1.98 (m, 2H), 1.97 – 1.53 (m, 5H), 1.24 (dt, J = 10.7, 5.4 Hz, 2H), 1.13 (s, 3H), 0.97 (s, 3H).

13

C NMR (75 MHz, CDCl3 and CD3OD): δ 214.2, 211.6,

154.8, 143.8, 143.7, 141.2, 141.2, 127.6, 127.6, 127.1, 127.1, 125.1, 125.1, 119.9, 119.9, 97.8, 73.3, 72.7, 67.2, 66.8, 64.7, 62.7, 59.8, 49.1, 48.2, 47.0, 44.5, 44.0, 38.2, 35.7, 32.8, 30.4, 23.1, 21.1, 17.1. HRMS Calcd for C35H38NO8: [M+H]+ 600.2597; found 600.2587. (9H-Fluoren-9-yl)methyl

(2R,6'S,7'S,7a'R,10'S,12b'R,15'R)-2'-(allylamino)-6',7',15'-

trihydroxy-5',5'-dimethyl-8'-oxo-5',5a',6',7',10',11',12',12a'-octahydro-4'H,8'Hspiro[aziridine-2,9'-[7,12b](epoxymethano)[7a,10]methanocyclohepta[7,8]naphtho[1,2d]thiazole]-1-carboxylate (42).



Page 36 of 54

To a solution of 40 (70.0 mg, 0.12 mmol) in THF (4 mL) was added PyHBr3 (41.6 mg, 0.13 mmol) at room temperature. The reaction mixture was stirred at room temperature for 4 hours and then poured into water and extracted with CH2Cl2 (20 mL × 3). The combined organic layer was washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo to give a crude oily product 41 (65.2 mg, 82%), which was directly used in the next reaction. To a solution of 41 (65.2 mg, 0.10 mmol) in ethanol (4 mL) was added N-Allylthiourea (13.4 mg, 0.12 mmol) at room temperature. The reaction mixture was heated under reflux for 6 h. After cooling and basifying with saturated NaHCO3 aqueous solution, the mixture was concentrated in vacuo to give an oily residue. The residue was purified by silica gel column; elution with 33% EtOAc in hexane afforded the desired product 42 (29.4 mg, 44%) as an amorphous gel. [α]25 = -102.5 (c D 0.08, MeOH); 1H NMR (300 MHz, CDCl3): δ 7.76 (d, J = 7.5 Hz, 2H), 7.59 (d, J = 7.4 Hz, 2H), 7.40 (t, J = 7.4 Hz, 2H), 7.33 (d, J = 7.4 Hz, 2H), 6.13 (s, 1H), 5.90 (ddt, J = 16.2, 10.7, 5.6 Hz, 1H), 5.49 (s, 1H), 5.34 – 5.12 (m, 4H), 5.07 (s, 1H), 4.53 (d, J = 10.2 Hz, 1H), 4.32 (ddd, J = 31.8, 13.6, 7.6 Hz, 3H), 3.96 (d, J = 10.2 Hz, 1H), 3.88 – 3.78 (m, 2H), 3.65 (d, J = 12.1 Hz, 1H), 2.63 – 2.46 (m, 2H), 2.45 – 2.28 (m, 2H), 2.05 – 1.88 (m, 2H), 1.84 – 1.74 (m, 1H), 1.61 (d, J = 9.4 Hz, 1H), 1.26 (d, J = 8.3 Hz, 6H), 0.98 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 214.6, 166.6, 154.7, 144.0, 143.9, 142.1, 141.3, 141.3, 133.6, 133.6, 127.6, 127.6, 127.1, 127.1, 125.2, 125.2, 119.9, 119.7, 117.3, 97.6, 73.5, 73.4, 67.7, 66.7, 65.3, 64.0, 57.7, 52.8, 48.3, 47.1, 44.7, 41.0, 38.8, 35.0, 30.4, 29.7, 21.7, 20.9, 18.6. HRMS Calcd for C39H42N3O7S: [M+H]+ 696.2743; found 696.2698. (2R,6'S,7'S,7a'R,10'S,12b'R,15'R)-2'-(Allylamino)-6',7',15'-trihydroxy-5',5'-dimethyl5',5a',6',7',10',11',12',12a'-octahydro-4'H,8'H-spiro[aziridine-2,9'[7,12b](epoxymethano)[7a,10]methanocyclohepta[7,8]naphtho[1,2-d]thiazol]-8'-one


(43).

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


To a solution of 42 (28.0 mg, 0.04 mmol) in DMF (0.8 mL) was added piperidine (0.2 mL) at room temperature. The reaction mixture was stirred at room temperature for 1 hour and then poured into water and extracted with CH2Cl2 (10 mL × 3). The combined organic layer was washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo to give a crude oily residue. The residue was purified by silica gel column; elution with 5% MeOH in CH2Cl2 afforded the desired product 43 (15.6 mg, 82%) as an amorphous gel. [α]25 = -30.8 (c 0.13, D MeOH); 1H NMR (300 MHz, CDCl3): δ 5.90 (ddt, J = 17.2, 10.2, 5.6 Hz, 1H), 5.38 – 5.11 (m, 3H), 5.07 (d, J = 1.3 Hz, 1H), 4.86 (d, J = 11.6 Hz, 1H), 4.39 (dd, J = 10.1, 1.4 Hz, 1H), 4.00 (dd, J = 10.1, 1.4 Hz, 1H), 3.91 (dd, J = 11.3, 8.8 Hz, 1H), 3.82 (d, J = 3.9 Hz, 2H), 3.73 (d, J = 25.0 Hz, 2H), 3.50 (s, 1H), 2.94 (d, J = 22.3 Hz, 2H), 2.58 – 2.25 (m, 4H), 2.23 – 1.97 (m, 2H), 1.97 – 1.62 (m, 3H), 1.62 – 1.54 (m, 1H), 1.27 (d, J = 2.4 Hz, 5H), 1.00 (s, 3H).

13

C NMR (75 MHz,

CDCl3): δ 218.5, 165.7, 142.8, 133.8, 119.7, 117.2, 97.5, 73.3, 73.2, 65.6, 62.7, 58.2, 52.7, 49.2, 48.2, 40.8, 38.9, 35.0, 31.8, 30.5, 29.7, 26.5, 21.0, 19.9. HRMS Calcd for C24H32N3O5S: [M+H]+ 474.2063; found 474.2053. (2R,3'S,3a'R,3a1'R,6a'R,7'S,11'S,11a'S)-1-acryloyl-7',11'-dihydroxy-5',5',8',8'tetramethyldecahydro-2'H-spiro[aziridine-2,15'[6a,11a](epoxymethano)[3,3a1]ethanophenanthro[1,10-de][1,3]dioxin]-14'-one (44). To a solution of 25 (66.0 mg, 0.16 mmol), acrylic acid (12.5 mg, 0.17 mmol) and DIPEA (60.9 mg, 0.47 mmol) in CH2Cl2 (20 mL) was added HBTU (89.5 mg, 0.24 mmol) at 0 ˚C. The resulting mixture was stirred at room temperature for 12 h. Then the mixture was diluted with CH2Cl2 (30 mL). The organic layer was washed with saturated NaHCO3 solution (50 mL) and brine (50 mL), dried over anhydrous Na2SO4, filtered, and evaporated to give an oily residue. The residue was purified using silica gel column; elution with 33% EtOAc in hexane afforded the desired product



Page 38 of 54

44 as a white solid (48.1 mg, 65%). [α]25 = -25.4 (c 0.13, MeOH); 1H NMR (300 MHz, CDCl3): D δ 6.71 – 5.96 (m, 2H), 5.89 – 5.52 (m, 1H), 4.97 (m, 2H), 4.56 – 3.98 (m, 2H), 3.88 (m, 1H), 3.47 (m, 1H), 2.77 (m, 4H), 2.43 (m, 3H), 1.95 (m, 3H), 1.72 (m, 4H), 1.43 (m, 4H), 1.24 (m, 2H), 1.12 (m, 6H). 13C NMR (75 MHz, CDCl3): δ 212.1, 175.0, 131.0, 129.0, 101.6, 94.7, 74.5, 72.9, 70.7, 62.7, 58.2, 56.8, 55.1, 51.2, 41.0, 39.0, 38.1, 34.5, 33.6, 33.4, 29.9, 29.8, 27.5, 25.5, 22.7, 19.8. HRMS Calcd for C26H36NO7: [M+H]+ 474.2492; found 474.2478; C26H35NNaO7: [M+Na]+ 496.2311; found 496.2303. (2R,3'S,3a'R,3a1'R,6a'R,7'S,11'S,11a'S)-7',11'-Dihydroxy-5',5',8',8'-tetramethyl-1propionyldecahydro-2'H-spiro[aziridine-2,15'[6a,11a](epoxymethano)[3,3a1]ethanophenanthro[1,10-de][1,3]dioxin]-14'-one

(45).

Compound 45 (35.0 mg) was prepared in 74% yield by a procedure similar to that used to prepare compound 44. The title compound 45 was obtained as a white solid. [α]25 = -36.0 (c 0.10, D CHCl3); 1H NMR (300 MHz, CDCl3): δ 5.03 (d, J = 12.0 Hz, 1H), 4.99 – 4.92 (m, 1H), 4.28 (d, J = 9.9 Hz, 1H), 4.05 (d, J = 9.9 Hz, 1H), 3.91 (dd, J = 12.0, 8.1 Hz, 1H), 3.48 (dd, J = 11.4, 5.5 Hz, 1H), 2.69 (d, J = 1.0 Hz, 1H), 2.55 – 2.28 (m, 5H), 2.03 – 1.88 (m, 2H), 1.79 (m, 1H), 1.73 (s, 3H), 1.71 – 1.57 (m, 2H), 1.57 – 1.45 (m, 2H), 1.43 (s, 3H), 1.35 – 1.22 (m, 3H), 1.15 (s, 3H), 1.13 (s, 3H),1.08 (m, 3H).

13

C NMR (75 MHz, CDCl3): 213.5, 184.8, 101.6, 94.7, 74.4, 73.0,

70.8, 62.8, 58.3, 56.9, 54.9, 51.2, 40.9, 39.0, 38.2, 34.7, 33.6, 33.4, 30.3, 29.9, 29.7, 27.4, 25.5, 22.7, 19.8, 8.6. HRMS Calcd for C26H38NO7: [M+H]+ 476.2648; found 476.2632. Measuring Compound Reactivity with GSH. The oridonin or its derivatives (10.0 mg) were incubated with an excess of GSH (15 ~ 20 mg) in deuterated phosphate buffer at pH 7.4 (0.05 mL) and d6-DMSO (0.50 mL) at 37 °C. The reaction was monitored by the change of


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


representative hydrogen signals with 1H NMR analysis, and the adduct products were confirmed with the HRMS. The reactivities of the covalent warheads could be initially classified based upon the integration change of the representative proton NMR. In Vitro Determination of Newly Synthesized Compounds against Cancer Cell Proliferation. Breast cancer cells (MDA-MB-231 and MCF-7 lines) were seeded in 96-well plates at a density of 1 × 104 cells/well and treated with DMSO or testing compounds at 0.1 μM, 0.33 μM, 1.0 μM, 3.3 μM, and 10 μM concentrations for 72 h. Proliferation was measured by treating cells with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in a CellTiter 96t AQueous Non-Radioactive Cell Proliferation Assay kit (Promega,Madison, WI, USA). Absorbance of all wells was determined by measuring OD at 560 nm after 1 h incubation at 37 °C on a 96-well iMark™ Microplate absorbance Reader (BioRad,Hercules, CA). Each individual compound was tested in quadruplicate wells for each concentration. Colony Formation Assay. Breast cancer MDA-MB-231 cells were seeded in 6-well tissue culture plates with a density of 600 cells/per well and maintained in regular culture media. After 24 h, the cells were treated with 44 at different concentrations (1 μM, 5 μM, 10 μM, 15 μM, and 20 μM respectively) or DMSO as the vehicle. The culture media with the compounds were changed every 72 h. At the end of two weeks, the wells were washed twice with PBS buffer and 2 mL of 0.01% crystal violet staining buffer was added to each well and incubated for 10 min. The wells were then washed with PBS for 5 min for three times, and allowed to dry. Photographs were then taken. Cellular Apoptosis Assay. Breast cancer MDA-MB-231 cells were incubated in 6-well plates (2.5 × 105 cells/well). Cells were then treated with DMSO, oridonin or testing compounds at



different concentrations for 48 h, and then both adherent and floating cells were collected, washed once with PBS. Resuspended cells were incubated with 100 μL of PBS containing 1% BSA and 100 μL of Annexin V and dead cell detection reagent at room temperature for 20 min. Apoptosis was measured immediately using the Muse Cell Analyzer with the Muse™ Apoptosis Kit (Catalog No. MCH100105). Western Blot Analysis. Breast cancer MDA-MB-231 cells were treated with DMSO or 44 at different concentrations respectively. After 48 h of treatment, cells were harvested and lysed. Protein concentrations were quantified by the method of Bradford with bovine serum albumin as the standard. Equal amounts of total cellular protein extract (40 μg) was separated by electrophoresis on SDS-polyacrylamide gels and transferred to NC membranes. After blocking with 5% non-fat milk, the membrane was incubated with the desired primary antibody overnight at the following dilution: anti-Bcl-2 (1:200), anti-cleaved-PARP (1:500), anti-NF-κB (1:1000), and β-actin (1:2000), anti-Bax (1:1000). Subsequently, the membrane was incubated with appropriate secondary antibody. The immunoreactive bands were visualized by enhanced chemiluminescence as recommended by the manufacturer. In Vivo Antitumor Activity Determination. All procedures including mice and in vivo experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of UT M. D. Anderson Cancer Center (MDACC). A total of 31 female nude mice were obtained from MDACC and were used for orthotopic tumor studies at 6 to 8 weeks of age. The mice were maintained in a barrier unit with 12 h light-dark switch. Freshly harvested MDA-MB-231 cells (1.0 × 106 cells per mouse, resuspended in 100 μL PBS) were injected into the #3 mammary fat pad of each mice, and then randomly assigned into 4 groups. The mice were treated daily with 10 or 15 mg/kg of 44, oridonin or vehicle through intraperitoneal injection, when the tumor volume


Page 40 of 54

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


reached 80-100 mm3 in volume. All agents were dissolved in DMSO for in vivo administration. Body weights and tumors volume were measured daily and tumor volume was calculated according to the formula V = 0.5 × L × W2, where L = length (mm) and W = width (mm). Statistical Analysis. Statistical significance was determined using student’s t-test or one way ANOVA in vivo experiments. *represents a p value less than 0.05, **represents a p value less than 0.01, and ***represents a p value less than 0.001.

ASSOCIATED CONTENT Supporting Information The reactivity of oridonin derivatives with GSH determined by the 1H NMR and HRMS. Growth inhibitory effects of the oridonin derivatives on MCF-7 and MDA-MB-231 cell lines. 1H and 13C NMR spectra for the new compounds described in this paper, and X-ray CIF file for compound 22. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors *(J.Z.) Phone: (409) 772-9748; Fax: (409) 772-9818; E-mail: [email protected]. *(Q.S.) Phone: (713) 834-6357; Fax: (713) 834-6350; E-mail: [email protected]. Author Contributions §

Y.D. and D.L. contributed equally to this work.

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.



ACKNOWLEDGMENTS This work was supported by Breast Cancer Research Program (BCRP) Breakthrough Award (BC160038 and BC160038P1) from the Department of Defense (DoD) (to J.Z. and Q.S.), grants P30 DA028821 and R01 DA038446 from the National Institutes of Health (to J.Z.), Sanofi Innovation Awards (iAward) Program (to J.Z.), and Cancer Prevention Research Institute of Texas (CPRIT) award (to J.Z.), Cancer Center Support Grant P30 CA016672 from the United States National Institutes of Health (to The University of Texas MD Anderson Cancer Center), startup funds from MD Anderson Cancer Center (to Q.S.), and the Duncan Family Institute Seed Funding Research Program (to Q.S). We want to thank Drs. Lawrence C. Sowers at the Department of Pharmacology as well as Dr. Tianzhi Wang at the NMR core facility of UTMB for the NMR spectroscopy assistance, and Dr. Xuemei Luo at UTMB mass spectrometry core with funding support from UT system proteomics network for the HRMS analysis.

ABBREVIATION USED SAR, structure-activity relationship; SRR, structure-reactivity relationship; FDA, the Food and Drug Administration; GSH, glutathione; NRF2, nuclear factor erythroid-derived 2-like 2; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; ROS, reactive oxygen species; p53, tumor protein p53; p21, cyclin-dependent kinase inhibitor 1; OTC, over-the-counter; IC50 half maximal inhibitory concentration; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; PARP, Poly (ADP-ribose) polymerase; ER, estrogen receptor; DR5, death receptor 5; XBP1,

X-box

binding

protein

1;

Rh2(esp)2,

Bis[rhodium(α,α,α′,α′-tetramethyl-1,3-

benzenedipropionic acid)]; RNS, reactive nitrogen species; NMR, nuclear magnetic resonance; t1/2, half-life; HRMS, high-resolution mass spectrometry; EGFR, epidermal growth factor


Page 42 of 54

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


receptor;

MS,

multiple

sclerosis;

LD50,

median

lethal

dose;

DPH,

O-(2,4-

dinitrophenyl)hydroxylamine; Rh2(OAc)4, rhodium(II) acetate dimer; Rh2(TPA)4, Rhodium(II) triphenylacetate

dimer;

Rh2(R-DOSP)4,

pyrrolidinecarboxylate]dirhodium(II);

tetrakis[1-[[4-alkyl(C11-C13)phenyl]sulfonyl]-(2R)Rh2(S-DOSP)4,

tetrakis[1-[[4-alkyl(C11-

C13)phenyl]sulfonyl]-(2S)-pyrrolidinecarboxylate]dirhodium (II); PyHBr3, pyridinium tribromide; THF, Tetrahydrofuran; DMF, dimethylformamide; HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3tetramethyluronium hexaﬂuorophosphate; DIPEA, N,N-Diisopropylethylamine; MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;; PI, propidium iodide; DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline; BCA, bicinchoninic acid; BSA, bovine serum albumin; SDS, sodium dodecyl sulfate; NC, nitrocellulose.

REFERENCES

(1) Singh, J.; Petter, R. C.; Baillie, T. A.; Whitty, A. The resurgence of covalent drugs. Nat. Rev. Drug Discovery 2011, 10, 307-317. (2) Barf, T.; Kaptein, A. Irreversible protein kinase inhibitors: balancing the benefits and risks. J. Med. Chem. 2012, 55, 6243-6262. (3) Liu, Q.; Sabnis, Y.; Zhao, Z.; Zhang, T.; Buhrlage, S. J.; Jones, L. H.; Gray, N. S. Developing irreversible inhibitors of the protein kinase cysteinome. Chem. Biol. 2013, 20, 146159. (4) Finlay, M. R.; Anderton, M.; Ashton, S.; Ballard, P.; Bethel, P. A.; Box, M. R.; Bradbury, R. H.; Brown, S. J.; Butterworth, S.; Campbell, A.; Chorley, C.; Colclough, N.; Cross, D. A.; Currie, G. S.; Grist, M.; Hassall, L.; Hill, G. B.; James, D.; James, M.; Kemmitt, P.; Klinowska, T.;



Lamont, G.; Lamont, S. G.; Martin, N.; McFarland, H. L.; Mellor, M. J.; Orme, J. P.; Perkins, D.; Perkins, P.; Richmond, G.; Smith, P.; Ward, R. A.; Waring, M. J.; Whittaker, D.; Wells, S.; Wrigley, G. L. Discovery of a potent and selective EGFR inhibitor (AZD9291) of both sensitizing and T790M resistance mutations that spares the wild type form of the receptor. J. Med. Chem. 2014, 57, 8249-8267. (5) Engel, J.; Richters, A.; Getlik, M.; Tomassi, S.; Keul, M.; Termathe, M.; Lategahn, J.; Becker, C.; Mayer-Wrangowski, S.; Grütter, C.; Uhlenbrock, N.; Krüll, J.; Schaumann, N.; Eppmann, S.; Kibies, P.; Hoffgaard, F.; Heil, J.; Menninger, S.; Ortiz-Cuaran, S.; Heuckmann, J. M.; Tinnefeld, V.; Zahedi, R. P.; Sos, M. L.; Schultz-Fademrecht, C.; Thomas, R. K.; Kast, S. M.; Rauh, D. Targeting drug resistance in EGFR with covalent inhibitors: a structure-based design approach. J. Med. Chem. 2015, 58, 6844-6863. (6) Song, Z.; Ge, Y.; Wang, C.; Huang, S.; Shu, X.; Liu, K.; Zhou, Y.; Ma, X. Challenges and perspectives on the development of small-molecule EGFR inhibitors against T790M-mediated resistance in non-small-cell lung cancer. J. Med. Chem. 2016, 59, 6580-6594. (7) Schwartz, P. A.; Kuzmic, P.; Solowiej, J.; Bergqvist, S.; Bolanos, B.; Almaden, C.; Nagata, A.; Ryan, K.; Feng, J.; Dalvie, D.; Kath, J. C.; Xu, M.; Wani, R.; Murray, B. W. Covalent EGFR inhibitor analysis reveals importance of reversible interactions to potency and mechanisms of drug resistance. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 173-178. (8) Backus, K. M.; Correia, B. E.; Lum, K. M.; Forli, S.; Horning, B. D.; González-Páez, G. E.; Chatterjee, S.; Lanning, B. R.; Teijaro, J. R.; Olson, A. J.; Wolan, D. W.; Cravatt, B. F. Proteome-wide covalent ligand discovery in native biological systems. Nature 2016, 534, 570574.


Page 44 of 54

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


(9) Flanagan, M. E.; Abramite, J. A.; Anderson, D. P.; Aulabaugh, A.; Dahal, U. P.; Gilbert, A. M.; Li, C.; Montgomery, J.; Oppenheimer, S. R.; Ryder, T.; Schuff, B. P.; Uccello, D. P.; Walker, G. S.; Wu, Y.; Brown, M. F.; Chen, J. M.; Hayward, M. M.; Noe, M. C.; Obach, R. S.; Philippe, L.; Shanmugasundaram, V.; Shapiro, M. J.; Starr, J.; Stroh, J.; Che, Y. Chemical and computational methods for the characterization of covalent reactive groups for the prospective design of irreversible inhibitors. J. Med. Chem. 2014, 57, 10072-10079. (10) Cee, V. J.; Volak, L. P.; Chen, Y.; Bartberger, M. D.; Tegley, C.; Arvedson, T.; McCarter, J.; Tasker, A. S.; Fotsch, C. Systematic study of the glutathione (GSH) reactivity of Narylacrylamides: 1. effects of aryl substitution. J. Med. Chem. 2015, 58, 9171-9178. (11) Ward, R. A.; Anderton, M. J.; Ashton, S.; Bethel, P. A.; Box, M.; Butterworth, S.; Colclough, N.; Chorley, C. G.; Chuaqui, C.; Cross, D. A.; Dakin, L. A.; Debreczeni, J. É.; Eberlein, C.; Finlay, M. R.; Hill, G. B.; Grist, M.; Klinowska, T. C.; Lane, C.; Martin, S.; Orme, J. P.; Smith, P.; Wang, F.; Waring, M. J. Structure- and reactivity-based development of covalent inhibitors of the activating and gatekeeper mutant forms of the epidermal growth factor receptor (EGFR). J. Med. Chem. 2013, 56, 7025-7048. (12) Palkowitz, M. D.; Tan, B.; Hu, H.; Roth, K.; Bauer, R. A. Synthesis of diverse N-acryloyl azetidines and evaluation of their enhanced thiol reactivities. Org. Lett. 2017, 19, 2270-2273. (13) Kathman, S. G.; Xu, Z.; Statsyuk, A. V. A fragment-based method to discover irreversible covalent inhibitors of cysteine proteases. J. Med. Chem. 2014, 57, 4969-4974. (14) Wang, J.; Zhang, C. J.; Chia, W. N.; Loh, C. C.; Li, Z.; Lee, Y. M.; He, Y.; Yuan, L. X.; Lim, T. K.; Liu, M.; Liew, C. X.; Lee, Y. Q.; Zhang, J.; Lu, N.; Lim, C. T.; Hua, Z. C.; Liu, B.; Shen, H. M.; Tan, K. S.; Lin, Q. Haem-activated promiscuous targeting of artemisinin in plasmodium falciparum. Nat. Commun. 2015, 6, 10111.



(15) Baillie, T. A. Targeted covalent inhibitors for drug design. Angew. Chem. Int. Ed. Engl. 2016, 55, 2-17. (16) Wong, M. H.; Bryan, H. K.; Copple, I. M.; Jenkins, R. E.; Chiu, P. H.; Bibby, J.; Berry, N. G.; Kitteringham, N. R.; Goldring, C. E.; O'Neill, P. M.; Park, B. K. Design and synthesis of irreversible analogues of bardoxolone methyl for the identification of pharmacologically relevant targets and interaction sites. J. Med. Chem. 2016, 59, 2396-2409. (17) Federspiel, J. D.; Codreanu, S. G.; Goyal, S.; Albertolle, M. E.; Lowe, E.; Teague, J.; Wong, H. L.; Guengerich, F. P.; Liebler, D. C. Specificity of protein covalent modification by the electrophilic proteasome inhibitor carfilzomib in human cells. Mol. Cell. Proteomics 2016, 15, 3233-3242. (18) Bauer, R. A. Covalent inhibitors in drug discovery: from accidental discoveries to avoided liabilities and designed therapies. Drug Discovery Today 2015, 20, 1061-1073. (19) Li, C. Y.; Wang, E. Q.; Cheng, Y.; Bao, J. K. Oridonin: an active diterpenoid targeting cell cycle arrest, apoptotic and autophagic pathways for cancer therapeutics. Int. J. Biochem. Cell Biol. 2011, 43, 701-704. (20) Xu, J.; Wold, E. A.; Ding, Y.; Shen, Q.; Zhou, J. Therapeutic potential of oridonin and its analogs: from anticancer and antiinflammation to neuroprotection. Molecules 2018, 23, 474. (21) Ding, Y.; Ding, C.; Ye, N.; Liu, Z.; Wold, E. A.; Chen, H.; Wild, C.; Shen, Q.; Zhou, J. Discovery and development of natural product oridonin-inspired anticancer agents. Eur. J. Med. Chem. 2016, 122, 102-117. (22) Lin, Z.; Guo, Y.; Gao, Y.; Wang, S.; Wang, X.; Xie, Z.; Niu, H.; Chang, W.; Liu, L.; Yuan, H.; Lou, H. ent-Kaurane diterpenoids from Chinese liverworts and their antitumor activities


Page 46 of 54

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


through Michael addition as detected in situ by a fluorescence probe. J. Med. Chem. 2015, 58, 3944-3956. (23) Nan, F. J.; Zhang, Y. M.; Liu, H. N.; Ding, J.; Meng, L. H.; Xu, C. H. Synthesis and Derivatization of an ent-Kaurane Diterpenoid. Chinese Patent CN102584760A, 2011. (24) Du Y; Villeneuve, N. F.; Wang, X. J.; Sun, Z.; Chen, W.; Li, J.; Lou, H.; Wong, P. K.; Zhang, D. D. Oridonin confers protection against arsenic-induced toxicity through activation of the Nrf2-mediated defensive response. Environ. Health Persp. 2008, 116, 1154-1161. (25) Zhen, T.; Wu, C. F.; Liu, P.; Wu, H. Y.; Zhou, G. B.; Lu, Y.; Liu, J. X.; Liang, Y.; Li, K. K.; Wang, Y. Y.; Xie, Y. Y.; He, M. M.; Cao, H. M.; Zhang, W. N.; Chen, L. M.; Petrie, K.; Chen, S. J.; Chen, Z. Targeting of AML1-ETO in t(8;21) leukemia by oridonin generates a tumor suppressor-like protein. Sci. Transl. Med. 2012, 4, 127ra38. (26) Zhou, G. B.; Kang, H.; Wang, L.; Gao, L.; Liu, P.; Xie, J.; Zhang, F. X.; Weng, X. Q.; Shen, Z. X.; Chen, J.; Gu, L. J.; Yan, M.; Zhang, D. E.; Chen, S. J.; Wang, Z. Y.; Chen, Z. Oridonin, a diterpenoid extracted from medicinal herbs, targets AML1-ETO fusion protein and shows potent antitumor activity with low adverse effects on t(8;21) leukemia in vitro and in vivo. Blood 2007, 109, 3441-3450. (27) De Silva C; Stevens, R.; Jordan, K. M. Inflammatory aortitis controlled by the Chinese herbal remedy Donglingcao Pian. Rheumatology (Oxford) 2008, 47, 1257-1259. (28) Liu, Q. Q.; Wang, H. L.; Chen, K.; Wang, S. B.; Xu, Y.; Ye, Q.; Sun, Y. W. Oridonin derivative ameliorates experimental colitis by inhibiting activated T-cells and translocation of nuclear factor-kappa B. J. Dig. Dis. 2016, 17, 104-112. (29) Sun, P. Y.; Wu, G. L.; Qiu, Z. J.; Chen, Y. J. L-alanine-(14-oridonin) Ester Trifluoroacetate as well as Preparation Method and Application. Chinese Patent CN104017000A, 2014.



(30) Bohanon, F. J.; Wang, X.; Graham, B. M.; Ding, C.; Ding, Y.; Radhakrishnan, G. L.; Rastellini, C.; Zhou, J.; Radhakrishnan, R. S. Enhanced effects of novel oridonin analog CYD0682 for hepatic fibrosis. J. Surg. Res. 2015, 199, 441-449. (31) Ding, C.; Zhang, Y.; Chen, H.; Yang, Z.; Wild, C.; Ye, N.; Ester, C. D.; Xiong, A.; White, M. A.; Shen, Q.; Zhou, J. Oridonin ring A-based diverse constructions of enone functionality: identification of novel dienone analogues effective for highly aggressive breast cancer by inducing apoptosis. J. Med. Chem. 2013, 56, 8814-8825. (32) Wang, X.; Bohanon, F.; Kandathiparampil, A.; Rastellini, C.; Ding, C.; Ding, Y.; Zhou, J.; Radhakrishnan, R. Suppressive effects of novel oridonin derivative CYD0625 on activated hepatic stellate cells. FASEB J. 2015, 29, 611.5. (33) Ding, C.; Zhang, Y.; Chen, H.; Yang, Z.; Wild, C.; Chu, L.; Liu, H.; Shen, Q.; Zhou, J. Novel nitrogen-enriched oridonin analogues with thiazole-fused A-ring: protecting group-free synthesis, enhanced anticancer profile, and improved aqueous solubility. J. Med. Chem. 2013, 56, 5048-5058. (34) Chen, W.; Zhou, J.; Wu, K.; Huang, J.; Ding, Y.; Yun, E. J.; Wang, B.; Ding, C.; Hernandez, E.; Santoyo, J.; Chen, H.; Lin, H.; Sagalowsky, A.; He, D.; Zhou, J.; Hsieh, J. T. Targeting XBP1-mediated β-catenin expression associated with bladder cancer with newly synthetic oridonin analogues. Oncotarget 2016, 7, 56842-56854. (35) Wu, J.; Ding, Y.; Chen, C. H.; Zhou, Z.; Ding, C.; Chen, H.; Zhou, J.; Chen, C. A new oridonin analog suppresses triple-negative breast cancer cells and tumor growth via the induction of death receptor 5. Cancer Lett. 2016, 380, 393-402. (36) Zang, L.; He, H.; Xu, Q.; Yu, Y.; Zheng, N.; Liu, W.; Hayashi, T.; Tashiro, S.; Onodera, S.; Ikejima, T. Reactive oxygen species H2O2 and •OH, but not O2•(-) promote oridonin-induced


Page 48 of 54

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


phagocytosis of apoptotic cells by human histocytic lymphoma U937 cells.

Int.

Immunopharmacol. 2013, 15, 414-423. (37) Cheng, Y.; Qiu, F.; Ye, Y. C.; Guo, Z. M.; Tashiro, S.; Onodera, S.; Ikejima, T. Autophagy inhibits reactive oxygen species-mediated apoptosis via activating p38-nuclear factor-kappa B survival pathways in oridonin-treated murine fibrosarcoma L929 cells. FEBS J. 2009, 276, 12911306. (38) Kuo, L. M.; Kuo, C. Y.; Lin, C. Y.; Hung, M. F.; Shen, J. J.; Hwang, T. L. Intracellular glutathione depletion by oridonin leads to apoptosis in hepatic stellate cells. Molecules 2014, 19, 3327-3344. (39) Xu, Z. Z.; Fu, W. B.; Jin, Z.; Guo, P.; Wang, W. F.; Li, J. M. Reactive oxygen species mediate oridonin-induced apoptosis through DNA damage response and activation of JNK pathway in diffuse large B cell lymphoma. Leuk. Lymphoma 2016, 57, 888-898. (40) Kubo, I.; Taniguchi, M.; Satomura, Y.; Kubota, T. Antibacterial activity and chemical structure of diterpenoids. Agric. Biol. Chem. 1974, 38, 1261-1262. (41) Xu, S.; Luo, S.; Yao, H.; Cai, H.; Miao, X.; Wu, F.; Yang, D. H.; Wu, X.; Xie, W.; Yao, H.; Chen, Z. S.; Xu, J. Probing the anticancer action of oridonin with fluorescent analogues: visualizing subcellular localization to mitochondria. J. Med. Chem. 2016, 59, 5022-5034. (42) Schmidt, T. J.; Ak, M.; Mrowietz, U. Reactivity of dimethyl fumarate and methylhydrogen fumarate towards glutathione and N-acetyl-L-cysteine-preparation of S-substituted thiosuccinic acid esters. Bioorg. Med. Chem. 2007, 15, 333-342. (43) Wilson, A. J.; Kerns, J. K.; Callahan, J. F.; Moody, C. J. Keap calm, and carry on covalently. J. Med. Chem. 2013, 56, 7463-7476.



(44) Couch, R. D.; Browning, R. G.; Honda, T.; Gribble, G. W.; Wright, D. L.; Sporn, M. B.; Anderson, A. C. Studies on the reactivity of CDDO, a promising new chemopreventive and chemotherapeutic agent: implications for a molecular mechanism of action. Bioorg. Med. Chem. Lett. 2005, 15, 2215-2219. (45) Fujita, E.; Nagao, Y.; Kaneko, K.; Nakazawa, S.; Kuroda, H. The antitumor and antibacterial activity of the Isodon diterpenoids. Chem. Pharm. Bull. 1976, 24, 2118-2127. (46) Zhu, Y.; Wang, Q.; Cornwall, R. G.; Shi, Y. Organocatalytic asymmetric epoxidation and aziridination of olefins and their synthetic applications. Chem. Rev. 2014, 114, 8199-8256. (47) Degennaro, L.; Trinchera, P.; Luisi, R. Recent advances in the stereoselective synthesis of aziridines. Chem. Rev. 2014, 114, 7881-7929. (48) Smith, D. T.; Njardarson, J. T. A scalable rhodium-catalyzed intermolecular aziridination reaction. Angew. Chem. Int. Ed. Engl. 2014, 53, 4278-4280. (49) Xu, J.; Zhao, J.; Wang, J.; Feng, N.; Tan, R.; Liu, Y. Study on stability of oridonin solution. Zhongguo Zhong Yao Za Zhi 2009, 34, 47-49. (50) Jat, J. L.; Paudyal, M. P.; Gao, H.; Xu, Q. L.; Yousufuddin, M.; Devarajan, D.; Ess, D. H.; Kürti, L.; Falck, J. R. Direct stereospecific synthesis of unprotected N-H and N-Me aziridines from olefins. Science 2014, 343, 61-65. (51) Armstrong, A.; Baxter, C. A.; Lamont, S. G.; Pape, A. R.; Wincewicz, R. Amine-promoted, organocatalytic aziridination of enones. Org. Lett. 2007, 9, 351-353. (52) Shen, Y. M.; Zhao, M. X.; Xu, J.; Shi, Y. An amine-promoted aziridination of chalcones. Angew. Chem. Int. Ed. Engl. 2006, 45, 8005-8008.


Page 50 of 54

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


(53) Pesciaioli, F.; De Vincentiis F; Galzerano, P.; Bencivenni, G.; Bartoli, G.; Mazzanti, A.; Melchiorre, P. Organocatalytic asymmetric aziridination of enones. Angew. Chem. Int. Ed. Engl. 2008, 47, 8703-8706. (54) Hata, Y.; Watanabe, M. Reaction of aziridinecarboxylic acids with thiols in aqueous solution. the formation of β-amino acid. Tetrahedron 1987, 43, 3881-3888. (55) Ding, C.; Zhang, Y.; Chen, H.; Wild, C.; Wang, T.; White, M. A.; Shen, Q.; Zhou, J. Overcoming synthetic challenges of oridonin A-ring structural diversification: regio- and stereoselective installation of azides and 1,2,3-triazoles at the C-1, C-2, or C-3 position. Org. Lett. 2013, 15, 3718-3721. (56) Ding, C.; Wang, L.; Chen, H.; Wild, C.; Ye, N.; Ding, Y.; Wang, T.; White, M. A.; Shen, Q.; Zhou, J. ent-Kaurane-based regio- and stereoselective inverse electron demand hetero-DielsAlder reactions: synthesis of dihydropyran-fused diterpenoids. Org. Biomol. Chem. 2014, 12, 8442-8452. (57) Zhang, Y.; Wu, Y.; Wu, D.; Tashiro, S.; Onodera, S.; Ikejima, T. NF-kappab facilitates oridonin-induced apoptosis and autophagy in HT1080 cells through a p53-mediated pathway. Arch. Biochem. Biophys. 2009, 489, 25-33. (58) Hsieh, T. C.; Wijeratne, E. K.; Liang, J. Y.; Gunatilaka, A. L.; Wu, J. M. Differential control of growth, cell cycle progression, and expression of NF-kappaB in human breast cancer cells MCF-7, MCF-10A, and MDA-MB-231 by ponicidin and oridonin, diterpenoids from the Chinese herb rabdosia rubescens. Biochem. Biophys. Res. Commun. 2005, 337, 224-231. (59) Chen, S.; Gao, J.; Halicka, H. D.; Huang, X.; Traganos, F.; Darzynkiewicz, Z. The cytostatic and cytotoxic effects of oridonin (rubescenin), a diterpenoid from rabdosia rubescens, on tumor cells of different lineage. Int. J. Oncol. 2005, 26, 579-588.



(60) Zhang, C. L.; Wu, L. J.; Zuo, H. J.; Tashiro, S.; Onodera, S.; Ikejima, T. Cytochrome c release from oridonin-treated apoptotic A375-S2 cells is dependent on p53 and extracellular signal-regulated kinase activation. J. Pharmacol. Sci. 2004, 96, 155-163. (61) Cheng, Y.; Qiu, F.; Ye, Y. C.; Tashiro, S.; Onodera, S.; Ikejima, T. Oridonin induces G2/M arrest and apoptosis via activating ERK-p53 apoptotic pathway and inhibiting PTK-Ras-RafJNK survival pathway in murine fibrosarcoma L929 cells. Arch. Biochem. Biophys. 2009, 490, 70-75. (62) Cui, Q.; Tashiro, S.; Onodera, S.; Ikejima, T. Augmentation of oridonin-induced apoptosis observed with reduced autophagy. J. Pharmacol. Sci. 2006, 101, 230-239. (63) Cui, Q.; Tashiro, S.; Onodera, S.; Minami, M.; Ikejima, T. Autophagy preceded apoptosis in oridonin-treated human breast cancer MCF-7 cells. Biol. Pharm. Bull. 2007, 30, 859-864. (64) Zhang, C. L.; Wu, L. J.; Tashiro, S.; Onodera, S.; Ikejima, T. Oridonin induces a caspaseindependent but mitochondria- and MAPK-dependent cell death in the murine fibrosarcoma cell line L929. Biol. Pharm. Bull. 2004, 27, 1527-1531.


Page 52 of 54

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


Table of Contents Graphic:



105x84mm (220 x 220 DPI)


Page 54 of 54

Regio- and Stereospecific Synthesis of Oridonin D-Ring Aziridinated

Recommend Documents