Regio- and Stereospecific Synthesis of Oridonin D

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Mar 12, 2018 - mediation of the covalent binding capability through a Rh2(esp)2- catalyzed ...... dimer; Rh2(TPA)4, rhodium(II) triphenylacetate dimer; Rh2(R-.

Article Cite This: J. Med. Chem. 2018, 61, 2737−2752

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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 A. White,‡ Qiang Shen,*,§ and Jia Zhou*,† †

Chemical Biology Program, Department of Pharmacology and Toxicology and ‡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 ∥ Department of Thyroid and Breast, Division of General Surgery, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai 200072, China S Supporting Information *

ABSTRACT: Covalent drug discovery has undergone a resurgence in recent years due to comprehensive optimization of the structure−activity relationship (SAR) and the 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)2catalyzed mild and concise regio- and stereospecific aziridination approach. Importantly, aziridonin 44 (YD0514), with a moredruglike 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 to oridonin.



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, 39 covalent drugs had been approved by the FDA, with the majority having been approved before the year 2000.1 However, due to potential toxicities and safety risks, electrophiles have been considered undruglike 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 © 2018 American Chemical Society

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 because the 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 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 have been an invaluable inspiration for the development of various therapeutic agents.1,14,15 In the kingdom of covalent Received: October 11, 2017 Published: March 12, 2018 2737

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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).

tolerable for improving the biological efficacy and druglike 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 of the others in Table 1 are currently in preclinical development. 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 on the covalent enone warhead in the D-ring is additionally beneficial to the further optimization for the biological activities, safety, and other druglike 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 on our SRR, favorable and druglike covalent groups have been designed and incorporated to

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 2738

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

druglike irreversible covalent warhead capable of significantly inducing apoptosis and inhibiting colony formation against triple-negative breast cancer with comparable enhanced antitumor effects in vitro and in vivo while displaying lower toxicity to normal human mammary epithelial cells in comparison to oridonin.

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[aziridine2,15′-[6a,11a](epoxymethano)[3,3a1]ethanophenanthro[1,10de][1,3]dioxin]-14′-one (aziridonin 44, YD0514) with a more-



RESULTS AND DISCUSSION Activity and Reactivity of the Covalent Enone Warhead in the D-Ring. GSH is viewed as a major reducing small 2739

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

positions (20) slightly weakens the reactivity of the enone; however, the t1/2 of the drug substrate is still less than 1 min (see 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 to 400 min.11 The half-life of the covalent drug dimethyl fumarate (Tecfidera), an antimultiple sclerosis (MS) blockbuster approved by the FDA in 2013, was identified to be approximately 50−60 min with 2 equiv 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 to 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 near the Dring. Hence, we envisioned that the replacement of the enone with a more-druglike 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.

molecule participating in cellular redox reactions with reactive oxygen and 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 ROSinduced 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 and residues of small molecules (e.g., GSH) and 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 antiproliferation effects.41 Thus, the efficacy of oridonin is believed to largely depend on this Dring 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 equiv 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 min (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 Figure S1). Additionally, the alanine ester at the C-14 position (14) may enhance the enone’s reactivity (see Figure S2). Introducing a sterically hindered acetonide group at the nearby C-7/C-14 2740

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Table 2. Exploration and Optimization of Oridonin-Based Aziridinationa

entries

DPH amount (equiv)

catalystb

recovery of 21c

yield of productsc

1 2 3 4 5 6 7 8

2.2 2.2 2.2 2.2 2.2 1.2 1.5 4.4

Rh2(esp)2 Rh2(OAc)4 Rh2(TPA)4 Rh2(R-DOSP)4 Rh2(S-DOSP)4 Rh2(esp)2 Rh2(esp)2 Rh2(esp)2

rt./12 h rt./12 h rt./12 h rt./12 h rt./12 h rt./12 h rt./12 h rt./24 h

trace trace >80% >80% >80% 18% 5% NA

Rh2(esp)2

50 °C/24 h

NA

Rh2(esp)2

reflux/48 h

NA

22 (69%) 22 (58%) 22 (trace) 22 (NA)d 22 (NA) 22 (54%) 22 (66%) 22 (71%) 23 (NA) 22 (66%) 23 (NA) 22 (56%) 23 (NA)

9

4.4

10

4.4

temp/time

a

All reaction conditions in this table were performed, utilizing 21 as the substrate. bThe equivalent of catalyst is 5%. cThe data were based on isolated yields. dNA: not available.

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 or basic media,49 and therefore, our ideal aziridination should be performed in a mild and near-neutral condition. Recently, Jat et al. published a moderate and scalable aziridination of olefins, utilizing O-(2,4dinitrophenyl)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. However, 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 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 and 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 no 1050339; see 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). 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). 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). 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 2741

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Table 3. Practical Extension of Rh-Catalyzed Aziridination

a

The data were based on isolated yields. bNR: no reaction.

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 druglike

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 reactivation 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. 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% 2742

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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, D) The comparison of two different substrates (C) 33 vs (D) 21, assisted by the neighboring oxygen group participation in the Rh2(esp)2-promoted regio- and stereospecific aziridination.

Scheme 1. Synthesis of the Aziridinated Oridonin Analoguesa

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%. a

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 noncovalent propionyl moiety leading to 45 for 2743

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

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

2744

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replacement of the covalent warhead enamide of 44 with a noncovalent 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-MB231, SK-BR3, and 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 MDA-MB-231, SK-BR3, MDAMB-468, and MCF-7 breast cancer cells in a dose-dependent manner.

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. 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 Figure S4).54To determine the experimental GSH reaction rates of aziridinated oridonin analogs, compound 25 was incubated at 37 °C with 1.5−2 equiv 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 on the integrated area of 1H NMR peak d generated from the reaction, t1/2 was identified as approximately 4−6 h. Additionally, the adduct product was also validated by HRMS analysis of the reaction at 5 h, 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 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 Table S2 and Figure S9), indicating that the D-ring aziridine is a druglike pharmacophore as well as an available site of modification for improving pharmacokinetics profiles. Under the same conditions, the acrylamide analogue 44 possesses a more reactive warhead in comparison to the aziridinated oridonin 25. Based on the integrated area of a 1H NMR peak 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 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∼400 min)11 suggested for covalent drugs. 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 triple-negative) using MTT assays as described in the in vitro screening protocol (see the Experimental section). The ability of these new analogs to inhibit the growth of cancer cells was summarized in Table S1 and compared to 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 such as 42 and 43 displayed moderate activities. Compound 44 exhibited significantly improved antiproliferative effects against highly invasive triple-negative breast cancer MDA-MB231 cells and MCF-7 cells with IC50 values of 8.32 and 9.39 μM, respectively, in comparison with oridonin (IC50 values of 29.4 μM and 6.72 μM against corresponding cell lines). Interestingly,

Figure 6. Growth inhibitory effects of 44 against MDA-MB-231, SKBR3, MDA-MB-468, and MCF-7 breast cancer cells. (A) 44 significantly inhibits colony formation of MDA-MB-231, SK-BR3, 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 are 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), p = 0.0002 (MDA-MB-468), p < 0.0001 (SK-BR3), and p = 0.0012 (MCF-7).

Based on its antiproliferative effects shown in Figure 6, further preliminary mechanistic studies of 44 were explored to determine whether 44 can induce apoptosis of breast cancer cells. MDA-MB-231 cells were treated with 44 at varying concentrations (1, 5, 10, 15, 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,B, 44 can induce apoptosis in breast cancer cells at 10 μM (18.2%), 15 μM (26.8%), and 20 μM (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 2745

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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. A single asterisk represents p < 0.05, two asterisks represent p < 0.01, and three asterisks represent p < 0.001 to 0 μM 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).

cancer cells by regulating a series of transcription factors and protein kinases as well as pro- and anti-apoptotic proteins such as NF-κB37,57,58 and Bcl-2.59,60 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 upregulation of cleaved PARP, with the latter as an apoptosis marker. In addition, 44 also reduced the ratio of Bcl-2 to 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 arrest,58,59,61 autophagy,37,57,62,63 and necrosis.64 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. 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∼100 μM) growthinhibitory activity against MCF-10A cells at all tested concentrations while displaying significantly enhanced anticancer activities against triple-negative MDA-MB-231 cancer cells in comparison to oridonin. These results provide a proof of concept that designing novel covalent warheads in the D-ring of oridonin could be beneficial in lead optimization for improved biological activities, safety, and other druglike properties. 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 triplenegative breast cancer MDA-MB-231 xenograft model. As shown in Figure 9, mice treated with 10 and 15 mg/kg of 44

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 a Student t-test, in comparison to the corresponding value of oridonin treatment at the same concentration. ns indicates not significant, a single asterisk indicates p < 0.05, triple asterisks indicate p < 0.001, and quadruple asterisks indicate p < 0.0001.

Figure 9. In vivo efficacy of 44 in suppressing the xenograft tumor growth of triple-negative breast cancer. The breast cancer cell line MDAMB-231 was used to generate xenograft tumors in nude mice for 10 and 15 mg/kg doses via intraperitoneal injection, respectively. Data are 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.

(intraperitoneal) 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 2746

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Journal of Medicinal Chemistry

Article

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′-tetramethyl-1′,3′,3a′,7′,7a′,8′,9′,11b′-octahydro-2′H-spiro[aziridine2,15′-[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. Next, 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 D = −72.4 (c 0.20, 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); 13 C 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): [M + H]+ calcd for C23H32NO5, 402.2280; found, 402.2268. The stereochemistry of 22 was secured by X-ray crystallographic analysis. The data have been assigned at the Cambridge Crystallographic Data Centre to the deposition number CCDC 1050339. (2R,5′S,6′S,6a′R,9′S,11b′R,14′R)-5′,6′,14′-Trihydroxy-4′,4′-dimethyl-4′,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, 1 78%) as a white amorphous solid. [α]25 D = −102.5 (c 0.11, MeOH); H NMR (300 MHz, CDCl3, δ): 5.91−5.77 (m, 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). 13C 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 [M + H]+: calcd for C20H28NO5, 362.1967; found, 362.1958. (2R,3′S,3a′R,3a 1 ′R,6a′R,7′S,11′S,11a′S)-7′,11′-Dihydroxy5′,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 solid. [α]25 D = −102.8 (c 0.13, MeOH); 1H NMR (300 MHz, CDCl3, δ): 5.46 (d, J = 11.6 Hz, 1H), 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). 13C 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 [M + H]+: calcd for C23H34NO6, 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′H-spiro[aziridine-2,15′-[6a,11a](epoxymethano)[3,3a1]ethanophenanthro[1,10-de][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 1 yellowish amorphous solid. [α]25 D = −59.6 (c 0.18, MeOH); H NMR (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),

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-druglike oridonin derivatives with optimized covalent warheads, we utilized a mild and efficient Rh2(esp)2-catalyzed reaction to regio- and stereospecifically 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

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