Brief Article pubs.acs.org/jmc
Potent Antitumor Activities and Structure Basis of the Chiral β‑Lactam Bridged Analogue of Combretastatin A‑4 Binding to Tubulin Pengfei Zhou,†,§ Yan Liu,‡,§ Lu Zhou,†,§ Kongkai Zhu,‡ Kechang Feng,† Hao Zhang,‡ Yuru Liang,† Hualiang Jiang,‡ Cheng Luo,*,‡ Mingming Liu,*,† and Yang Wang*,† †
School of Pharmacy, Fudan University, Shanghai 201203, China Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
‡
S Supporting Information *
ABSTRACT: A series of chiral β-lactam bridged analogues (3-substituted 1,4-diaryl-2-azetidinones) of combretastatin A-4 (CA-4) were synthesized asymmetrically, and their antitumor activities were evaluated in vitro and in vivo. The cocrystal structure of tubulin in complex with compound 9 was determined by X-ray crystallography, which showed that 9 binds to the same site as colchicine with similar binding mode, and the absolute configuration of its C-4 was first identified and demonstrated to be critically important for their antiproliferative activities.
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INTRODUCTION Microtubule/tubulin is one of the most important and promising targets in cancer therapy. The heterodimer formed by α-subunit and β-subunit polymerizes to construct microtubules. Microtubule accomplishes its physiologic function by the dynamic equilibrium of the polymerization and depolymerization of α,β-heterodimer. This process plays an important role in cell mitosis. Either the promotion or inhibition of the polymerization can disturb the mitosis and lead to cell death.1 Paclitaxel, widely used for the cancer chemotherapy, binds to the inner surface of β-subunit and then stabilizes the tubulin, resulting in the stagnation of mitosis. Colchicine (Figure 1), the first discovered compound that can destabilize the tubulin, binds to the interface of α-subunit and β-subunit, resulting in the depolymerization of α,β-heterodimer.2 Combretastatin A-4 (CA-4), isolated from the South African tree Combretum caff rum, shows extremely strong effects on suppression of tubulin assembly through interaction with the colchicine binding site of tubulin, resulting in extensive inhibition of cell growth and angiogenesis,3 but the chemical instability and poor water solubility limit its further application in clinic. To optimize the stability and potency of CA-4, a large number of conformationally restricted analogues have been designed and discovered through replacement of cis-CC bond by a rigid carbocyclic or heterocyclic ring system.2,4 Among them, β-lactam bridged © 2016 American Chemical Society
Figure 1. Representative microtubule targeting agents.
analogues of CA-4, 1,4-diaryl-2-azetidinones, showed significant tubulin binding and depolymerizing effects, but the molecular mechanism stays obscure and the absolute configurations of these β-lactam analogues still remain unknown.5 To clarify the impact of stereochemistry on bioactivity and to investigate the binding mode with tubulin, a novel panel of optically active β-lactam bridged analogues of CA-4 were designed and synthesized asymmetrically and their in vitro and in vivo antitumor activity was evaluated in the present work. Here we report the results of the relationship between the absolute configuration of chiral center at β-lactam backbone Received: August 24, 2016 Published: November 2, 2016 10329
DOI: 10.1021/acs.jmedchem.6b01268 J. Med. Chem. 2016, 59, 10329−10334
Journal of Medicinal Chemistry
Brief Article
Scheme 1. Synthesis of 1,4-Diaryl-2-azetidinones
Table 1. Inhibition of Tubulin Polymerization and Growth of Four Human Cancer Cell Lines by 6, 8, 9 and Reference Compound CA-4 IC50 ± SD (μM)a
a
compd
A2780
MDA-MB-231
SK-OV-3
HeLa
tubulin assembly
6 8 9 CA-4
4.6 ± 1.9 0.115 ± 0.027 0.031 ± 0.004 0.006 ± 0.001
>10 0.308 ± 0.074 0.056 ± 0.016 0.008 ± 0.002
3.5 ± 1.6 0.114 ± 0.014 0.057 ± 0.009 0.012 ± 0.003
5.5 ± 2.3 0.243 ± 0.057 0.063 ± 0.015 0.010 ± 0.003
>100 6.6 ± 1.0 3.5 ± 0.1 1.32 ± 0.15b
IC50 is presented as the mean ± SD from at least two independent experiments. bThe data were reported in a previous literature.5b
To evaluate 3-methylene substituted analogues and to further clarify the impact of stereochemistry on the activity, the newly synthesized chiral β-lactam-bridged CA-4 analogues 6 and 8 were first screened for antiproliferative activities against human cancer cell lines and for inhibitory effects on tubulin polymerization using isolated pig brain tubulin (Table 1). The cytotoxicity and anti-tubulin activities of 6 were much weaker than that of its enantiomer 8, indicating the critical role of C-4 configuration for the activities. Therefore, the further hydrogenation catalyzed by Pd/C was only carried out with 8, providing (3R,4R)-3-methyl substituted target product 9 exclusively due to the induction of the neighboring chiral center, even under the condition of chiral hydrogenated catalysts. Preliminary stability study of 9 was carried out under both acidic and neutral conditions (pH 3.0 and 7.4) and demonstrated improved chemical stability as compared with CA-4, which was reported to have about 60% percentage recovery at pH 3.0 for 5 h.7 The aqueous solubility of 9 was also determined to be 115 μg/mL at pH 7.4, greatly improved compared with 1.04 μg/mL of CA-4.8 3-Methyl substituted analogue 9 was assessed for inhibition of cell growth and tubulin assembly and exhibited improved effects (Table 1). It showed that 9 disrupted in vitro and cellular tubulin polymerization (Figure 2A and Figure 2B) and induced G2/M cell cycle arrest (Figure S1 in Supporting Information) in a dose-dependent manner. Next, we evaluated the in vivo antitumor effects of 9 using human ovarian cancer xenograft mice model, which is established by subcutaneous
and the activities, which was strongly supported by the X-ray complex structure.
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RESULTS AND DISCUSSION The synthesis of optically active β-lactam derivatives was inspired by recent work of Ding and colleagues6 on the palladium catalyzed enantioselective allylic amination of racemic MBH adducts with aromatic amines in the presence of spiroketal-based bisphosphine ligands (SKPs). This chemistry has provided an efficient approach to access various optically active β-arylamino acid esters which can be readily transformed into their corresponding β-lactam derivatives without loss of enantioselectivity. On the basis of this methodology, several chiral 1,4-diaryl-3-methylene-2-azetidinones were synthesized asymmetrically as illustrated in Scheme 1. Racemic 1 was submitted to asymmetric allylic amination using aromatic amine 2 as the nucleophile under the catalysis of Pd2(dba)3 and corresponding chiral ligands, affording optically active amination products 3 (with (S,S,S)-Ph-SKP as ligand) and 4 (with (R,R,R)-Ph-SKP as ligand) in high yields (89% and 86% ,respectively) and enantioselectivities (98% ee both). The optically active allylic amination products 3 and 4 can be readily cyclized into their corresponding β-lactam derivatives 5 and 7 in good yields without loss of enantioselectivity by reaction with Sn[N(TMS)2]2 in refluxing toluene. Then the tert-butyldimethylsilyl protective group was removed by TBAF to provide the target 1,4-diaryl-3methylene-2-azetidinones 6 and 8. 10330
DOI: 10.1021/acs.jmedchem.6b01268 J. Med. Chem. 2016, 59, 10329−10334
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Figure 2. (A) Inhibition of in vitro tubulin polymerization by 9. (B) Immunofluorescence assay of SKOV-3 cells shows the effect of 9 on the organizations of cellular microtubule network. (C−E) Effects of 9 on xenografts tumor growth in nude mice. Shown are the average tumor volumes (C), weights (E), and the isolated tumors (E). (∗) p < 0.05, (∗∗∗) p < 0.001.
Figure 3. X-ray analysis of the tubulin−9 complex. (A) Chemical structure of 9. (B) Overall view of the complex formed between α,β-tubulin and 9. α- and β-tubulin are shown as cartoon, and 9 is shown in yellow sphere representation (PDB code 5GON). (C) Fo − Fc electron density (blue) of 9 is contoured at 3.0σ level. (D) Compound 9 binds to the colchicine site of β-tubulin (surface). (E) Close-up views of the hydrogen bonds observed between 9 (yellow sticks) and tubulin (white). Interacting residues of tubulin are shown in stick representation. Hydrogen bonds are shown by dashed lines. (F) Superimposition of 9 (yellow stick) and colchicine (PDB code 1SA0; cyan stick) and a CA-4P analogue TUB092 (PDB code 5JVD; white stick).
To further understand the mechanism of these compounds, the X-ray complex structure of tubulin and 9 was determined by soaking the compound into the well-established T2R-TTL complex and refined to 2.48 Å resolution (PDB code 5GON), which is the first complex structure of tubulin with β-lactam backbone inhibitor.9 Statistics of data collection and structural refinement are summarized in Table S2. As expected, 9 occupies the colchicine binding site at the intradimer interface with nice electron density (Figure 3B−D). The main binding driving force was thought to be shape matching with
inoculation of A2780 cells in the female Balb/C nude mice. As shown in Figure 2C−E, 9 significantly suppressed the tumor growth in a xenograft mice model at 12.5 mg/kg, without inducing significant body weight loss and detectable abnormalities of main organs (Figure S2). In vivo pharmacokinetic studies of 9 exhibited good PK characteristics as shown in its high peak concentration and AUC value (Table S1), suggesting promising potential for further clinical development. These results encouraged us to further discover the molecular mechanism of these β-lactam analogues binding to tubulin. 10331
DOI: 10.1021/acs.jmedchem.6b01268 J. Med. Chem. 2016, 59, 10329−10334
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hydrophobic interaction between 9 and β-tubulin (Figure 3E, Figure S3A). The C-4 aryl substituent inserts into a long narrow valley, which explained why the absolute configuration at this position is critical for the activities. As shown in Figure 3E, 9 also formed two hydrogen bonds with Thr179 of α-tubulin and Ala250 of β-tubulin. To further understand the detailed binding mode of 9, we superimposed the structure of tubulin−9 on T2R-TTL structure (rmsd of 0.463 over 1927 Cα atoms) and found that the binding of 9 did not affect the global conformation of tubulin. The major conformational changes involve two loops near the 9 binding site, including the flip-out of βH7 and the conformational change of αT5 (Figure S3B). We then superimposed the structure with two tubulin complex structures including colchicine and a newly reported chalcone analogue.10 Similar conformational changes were found in these complex structures,11 indicating that the inhibitory mechanism of 9 on tubulin may be the same as that of colchicine. The hydroxyl group on ring B was found to be conserved in these structures, implying that the hydrogen bond with Thr179 of α-tubulin is very important (Figure 3F). Moreover, ring A of 9 inserts into the pocket of β-tubulin much deeper than the other two small molecules, providing us a new possible modification site because 9 showed higher binding affinity than colchicine against tubulin (Figure 2A). As a class of druglike skeleton, β-lactam widely exists in the structures of drugs. Previous studies revealed that as β-lactam bridged CA-4 analogues, 1,4-diaryl-2-azetidinones exhibited potent antiproliferative activities through blocking of the polymerization of tubulin.5 However, to our knowledge, single isomer of 1,4-diaryl-2-azetidinones with determined absolute configuration was not obtained up until now. Hence, in this study, the role of the chiral configuration of aryl substituent at C-4 of the β-lactam ring on the antiproliferative activity was explored and well explained. A pair of enantiomers (6 and 8) were obtained with determined absolute configuration through asymmetric MBH reaction.6 The orientation of C-4 aryl substituent toward page plane below (shown in Scheme 1) is essential for the inhibitory activities of tubulin polymerization and cellular proliferation. In addition, 3-methylene analogue (8) and 3-methyl analogue (9) exhibited satisfactory antitubulin and antiproliferative effects, which may imply the configuration of C-3 is less crucial than that of C-4 for the activities. Besides, the carbonyl of β-lactam was also found to be of benefit for the activities, as the azetidine analogue is much less effective than the 2-azetidinone one. Moreover, 3-methyl substituted product 9 with cis-(3R,4R) configuration was obtained and exhibited more potent anticancer effect in vitro and in vivo than its 3-methylene precursor 8 through further structural modifications. The cocrystal structure revealed the binding mode which is similar to colchicine, and we found a new possible modification site on ring A which was considered as a conserved site before.12 The deeper binding of 9 increased binding affinity in vitro and showed greatly reduced toxicity comparing with colchicine (LD50 of mice of more than 500 mg/kg vs 4 mg/kg 13).
inhibitor. Compound 9 retained potency with acceptable therapeutic window, indicating a promising candidate for further clinical development.
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EXPERIMENTAL SECTION
General Methods. All reagents were commercially available and were used without further purification. Melting points were measured on a SGW X-4 apparatus and uncorrected. Optical rotations were measured at the sodium D line, using a Rudolph autopol IV automatic polarimeter and a 100 mm cell. 1H and 13C spectra were obtained by a Varian 400 MHz or a Bruker 600 MHz NMR spectrometer at 303 K, using tetramethylsilane as internal standard. MS was measured on Agilent 6120 quadrupole LC/MS. HRMS determinations for all new compounds were performed on AB SCIWX TRIPLETOF 5600+. Flash chromatography was carried out using standard silica gel 60 (300−400 mesh) and detected with UV light monitor at 254 nm. The chical HPLC analyses were performed on a JASCO LC-NetII/ ADC liquid chromatograph. The purities of all the activity tested compounds were confirmed to be ≥95% by this HPLC analysis. Preparation of (R)-Ethyl 2-((3-((tert-Butyldimethylsilyl)oxy)4-methoxyphenyl)((3,4,5-trimethoxyphenyl)amino)methyl)acrylate (3). A 50 mL round-bottom flask was charged with 10 mL of dichloromethane, and the solvent was deoxygenized with nitrogen bubbling for 15 min. Tris(dibenzylideneacetone)dipalladium (5 mg, 0.005 mmol) and (S,S,S)-Ph-SKP (8 mg, 0.012 mmol) were added into the flask. The resulting purple solution was stirred under nitrogen atmosphere for 30 min at room temperature. Then aniline 2 (0.13 g, 0.71 mmol), K2CO3 (1.0 M aq solution, 1.5 mL, 1.5 mmol), and 1 (0.2 g, 0.49 mmol, dissolved in 1 mL of oxygen free dichloromethane) were added under a steam of nitrogen. The solution was stirred for 5 h at room temperature. Water (10 mL) was added into the solution, and the mixture was extracted with dichloromethane (10 mL) 3 times. The organic layer was separated, washed with brine, dried over anhydrous Na2SO4, and filtered. The solvent was removed under reduced pressure and the residue was purified by flash column chromatography to give 0.23 g of the title compound 3 as colorless oil, yield 89%. [α]D20 −83.7 (c 0.56, CHCl3), 98% ee [determined by HPLC analysis using a Chiralcel AS-3 column; n-Hex/i-PrOH = 95:5, 1.0 mL/min, 254 nm; tR (minor) = 7.18 min; tR (major) = 9.28 min]. 1H NMR (400 MHz, CDCl3) δ 6.91 (dd, J = 8.3, 2.1 Hz, 1H), 6.83 (d, J = 2.1 Hz, 1H), 6.80 (d, J = 8.3 Hz, 1H), 6.34 (s, 1H), 5.88 (s, 1H), 5.81 (s, 2H), 5.28 (s, 1H), 4.21−4.11 (m, 2H), 4.07 (br s, 1H), 3.79 (s, 3H), 3.76 (s, 6H), 3.74 (s, 3H), 1.22 (t, J = 7.1 Hz, 3H), 0.97 (s, 9H), 0.12 (s, 6H). 13C NMR (150 MHz, CDCl3) δ 165.8, 153.2, 149.9, 144.4, 143.0, 140.3, 132.5, 129.6, 124.9, 120.1, 119.6, 111.5, 90.5, 60.5, 60.2, 58.1, 55.3, 54.9, 25.1, 17.8, 13.5, −5.3. ESI-MS (m/z): 532.1 (M + H+). ESI-HRMS (m/z): calcd for C28H42NO7Si [M + H]+, 532.2725; found, 532.2733. Preparation of (S)-Ethyl 2-((3-((tert-Butyldimethylsilyl)oxy)4-methoxyphenyl)((3,4,5-trimethoxyphenyl)amino)methyl)acrylate (4).6 A 250 mL round-bottom flask was charged with 100 mL of dichloromethane, and the solvent was deoxygenized with nitrogen bubbling for 15 min. Tris(dibenzylideneacetone)dipalladium (0.14 g, 0.15 mmol) and (R,R,R)-Ph-SKP (0.26 g, 0.39 mmol) were added into the flask. The resulting purple solution was stirred under nitrogen atmosphere for 30 min at room temperature. Then aniline 2 (4.4 g, 24.0 mmol), K2CO3 (1.0 M aq solution, 48 mL, 48 mmol), and 1 (6.5 g, 15.9 mmol, dissolved in 10 mL of oxygen free dichloromethane) were added under a steam of nitrogen. The solution was stirred for 5 h at room temperature. Water (50 mL) was added into the solution, and the mixture was extracted with dichloromethane (50 mL) 3 times. The organic layer was separated, washed with brine, dried over anhydrous Na2SO4, and filtered. The solvent was removed under reduced pressure and the residue was purified by flash column chromatography to give 7.3 g of the title compound 4 as colorless oil, yield 86%. [α]D20 +86.2 (c 0.41, CHCl3), 98% ee [determined by HPLC analysis using a Chiralcel AS-3 column; n-Hex/i-PrOH = 95:5, 1.0 mL/min, 254 nm; tR (major) = 7.47 min; tR (minor) = 9.72 min]. 1 H NMR (400 MHz, CDCl3): δ 6.91 (dd, J = 8.3, 2.1 Hz, 1H),
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CONCLUSIONS In summary, the cocrystal structure of tubulin in complex with 1,4-diaryl-2-azetidinone 9 was obtained and the absolute configuration of C-4 of these β-lactams was first identified. The gained insight into the interaction of β-lactams with tubulin definitely provides guidance for further structural modification and optimization of this kind of tubulin polymerization 10332
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8.2, 2.0 Hz, 1H), 6.83 (d, J = 8.2 Hz, 1H), 6.59 (s, 2H), 5.81 (t, J = 1.6 Hz, 1H), 5.71 (s, 1H), 5.27 (s, 1H), 5.14 (s, 1H), 3.88 (s, 3H), 3.75 (s, 3H), 3.73 (s, 6H). 13C NMR (150 MHz, CDCl3): δ 160.3, 152.9, 149.2, 146.5, 145.6, 134.1, 133.2, 128.9, 118.1, 112.3, 110.3, 110.0, 94.31, 63.0, 60.3, 55.5, 55.4. ESI-MS (m/z): 372.1 (M + H+). ESI-HRMS (m/z): calcd for C20H22NO6 [M + H]+, 372.1442; found, 372.1441. Preparation of (3R,4R)-4-(3-Hydroxy-4-methoxyphenyl)-3methyl-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (9). A 250 mL round-bottom flask was charged with 8 (0.3 g, 0.80 mmol), 10% Pd/C (30 mg), and ethanol (10 mL). The solution was stirred for 12 h under H2 atmosphere (1 atm). Pd/C was filtrated, and then the solvent was removed under reduced pressure and the residue was purified by flash column chromatography (eluent EtOAc/hexane = 1:2, v/v) to give 0.28 g of the title compound 9 as white solid, yield 93%; mp 64− 66 °C; [α]D20 +138.9 (c 1.0, CHCl3). 1H NMR (400 MHz, CDCl3): δ 6.87−6.78 (m, 2H), 6.72 (d, J = 8.2 Hz, 1H), 6.56 (s, 2H), 5.71 (br s, 1H), 5.06 (d, J = 5.8 Hz, 1H), 3.89 (s, 3H), 3.77 (s, 3H), 3.72 (s, 6H), 3.67−3.58 (m, 1H), 0.91 (d, J = 7.6 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ 168.7, 153.7, 146.6, 146.0, 134.5, 134.2, 128.1, 118.9, 113.4, 110.8, 95.1, 61.2, 58.6, 56.3, 56.2 49.5, 29.9, 9.8. ESI-MS (m/z): 374.1 (M + H+). ESI-HRMS (m/z): calcd for C20H24NO6 [M + H+], 374.1589; found, 374.1597. Purity 97% [determined by HPLC analysis using a Chiralcel AD-H column; n-Hex/i-PrOH = 80:20, 1.0 mL/min, 254 nm; tR = 13.23 min].
6.83 (d, J = 2.1 Hz, 1H), 6.80 (d, J = 8.3 Hz, 1H), 6.34 (s, 1H), 5.88 (s, 1H), 5.81 (s, 2H), 5.28 (s, 1H), 4.15 (q, J = 7.1 Hz, 2H), 3.78 (s, 3H), 3.76 (s, 6H), 3.74 (s, 3H), 1.22 (t, J = 7.1 Hz, 3H), 0.96 (s, 9H), 0.11 (s, 6H). ESI-MS (m/z): 532.1 (M + H+). Preparation of (R)-4-(3-(tert-Butyldimethylsilyloxy)-4-methoxyphenyl)-3-methylene-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (5). To a 50 mL Schlenk flask equipped with a coldfinger were added appropriate allylic amination product 3 (0.13 g, 0.244 mmol), Sn[N(TMS)2]2 (0.14 g, 0.32 mmol), and dry toluene (5 mL). The mixture was heated to reflux for 3 h under nitrogen atmosphere. The solution was cooled and directly purified by flash chromatography to give 0.1 g of the title compound 5 as colorless oil, yield 86%. [α]D20 −38.0 (c 1.14, CHCl3). 1H NMR (400 MHz, CDCl3) δ 6.97 (dd, J = 8.3, 2.1 Hz, 1H), 6.86−6.83 (m, 2H), 6.59 (s, 2H), 5.82 (t, J = 1.7 Hz, 1H), 5.27 (s, 1H), 5.16−5.14 (m, 1H), 3.80 (s, 3H), 3.76 (s, 3H), 3.73 (s, 6H), 0.94 (s, 9H), 0.08 (d, J = 3.8 Hz, 6H). 13C NMR (150 MHz, CDCl3) δ 160.3, 152.9, 150.9, 149.3, 144.9, 134.0, 133.2, 128.1, 119.7, 118.7, 111.6, 110.0, 94.3, 63.0, 60.3, 55.4, 54.9, 25.0, 17.8, −5.3. ESI-MS (m/z): 486.1 (M + H+). ESI-HRMS (m/z): calcd for C26H36NO6Si [M + H]+, 486.2306; found, 486.2315. Preparation of (R)-4-(3-Hydroxy-4-methoxyphenyl)-3-methylene-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (6). A 50 mL round-bottom flask was charged with 5 (45 mg, 0.093 mmol) and THF (5 mL). The solution was cooled to 0 °C by an ice base, then TBAF (27 mg, 0.1 mmol) was added into the flask. After the mixture was stirred for 10 min at 0 °C, 30 mL of water was added into the solution. The mixture was extracted with ethyl acetate (10 mL) 3 times. The organic layer was separated, washed with brine, dried over anhydrous Na2SO4, and filtered. The solvent was removed under reduced pressure and the residue was purified by flash column chromatography to give 28 mg of the title compound 6 as colorless oil, yield 82%; mp 110−112 °C; [α]D20 −31.2 (c 0.64, CHCl3). 99% ee [determined by HPLC analysis using a Chiralcel AD-H column; n-Hex/i-PrOH = 80:20, 1.0 mL/min, 254 nm; tR (minor) = 13.83 min; tR (major) = 17.46 min]. 1H NMR (400 MHz, CDCl3): δ 6.92 (d, J = 1.6 Hz, 1H), 6.87 (dd, J = 8.2, 1.6 Hz, 1H), 6.80 (d, J = 8.2 Hz, 1H), 6.57 (s, 2H), 5.92 (s, 1H), 5.77 (s, 1H), 5.25 (s, 1H), 5.11 (s, 1H), 3.83 (s, 3H), 3.72 (s, 3H), 3.70 (s, 6H). 13C NMR (100 MHz, CDCl3): δ 160.9, 153.4, 149.6, 147.1, 146.2, 134.5, 133.7, 129.4, 118.7, 112.9, 110.8, 110.7, 94.8, 63.6, 60.9, 56.0, 55.9. ESI-MS (m/z): 372.1 (M + H+). ESI-HRMS (m/z): calcd for C20H22NO6 [M + H]+, 372.1442; found, 372.1451. Preparation of (S)-4-(3-(tert-Butyldimethylsilyloxy)-4-methoxyphenyl)-3-methylene-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (7).6 To a 50 mL Schlenk flask equipped with a coldfinger were added appropriate allylic amination product 4 (0.44 g, 0.83 mmol), Sn[N(TMS)2]2 (0.44 g, 1 mmol), and dry toluene (10 mL). The mixture was heated to reflux for 3 h under nitrogen atmosphere. The solution was cooled and directly purified by flash chromatography to give 0.37 g of the title compound 7 as colorless oil, yield 92%; [α]D20 +37.6 (c 1.00, CHCl3). 1H NMR (400 MHz, CDCl3) δ 6.95 (dd, J = 8.3, 2.1 Hz, 1H), 6.85−6.81 (m, 2H), 6.58 (s, 2H), 5.80 (s, 1H), 5.26 (s, 1H), 5.13 (s, 1H), 3.78 (s, 3H), 3.74 (s, 3H), 3.71 (s, 6H), 0.92 (s, 9H), 0.06 (d, J = 4.1 Hz, 6H). ESI-MS (m/z): 486.1 (M + H+). Preparation of (S)-4-(3-Hydroxy-4-methoxyphenyl)-3-methylene-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (8). A 50 mL round-bottom flask was charged with 7 (0.65 g, 1.34 mmol) and THF (10 mL). The solution was cooled to 0 °C by an ice base, and then TBAF (0.39 g, 1.5 mmol) was added into the flask. After the mixture was stirred for 10 min at 0 °C, 30 mL of water was added into the solution. The mixture was extracted with ethyl acetate (15 mL) 3 times. The organic layer was separated, washed with brine, dried over anhydrous Na2SO4, and filtered. The solvent was removed under reduced pressure and the residue was purified by flash column chromatography to give 0.42 g of the title compound 8 as white solid; yield 85%; mp 115− 117 °C; [α]D20 +33.6 (c 1.0, CHCl3). 99% ee [determined by HPLC analysis using a Chiralcel AD-H column; n-Hex/i-PrOH = 80:20, 1.0 mL/min, 254 nm; tR (major) = 13.76 min; tR (minor) = 17.72 min]. 1 H NMR (400 MHz, CDCl3): δ 6.95 (d, J = 2.0 Hz, 1H), 6.90 (dd, J =
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b01268. Experimental procedures and spectral data for compounds, biological evaluation assay, and crystallographic data (PDF) Molecular formula strings and some data (CSV) Accession Codes
The PDB code of the complex of tubulin and compound 9 is 5GON. The authors will release the atomic coordinates and experimental data upon article publication.
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AUTHOR INFORMATION
Corresponding Authors
*C.L.: phone, 86-21-5080-6600; e-mail,
[email protected]. *M.L.: phone, 86-21-5198-0121; e-mail,
[email protected]. *Y.W.: phone, 86-21-5198-0115; e-mail,
[email protected]. Author Contributions §
P.Z., Y.L., and L.Z. contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
We thank staff members of beamline BL19U at SSRF for assistance in data collection and Prof. Dr. Ao Zhang from Shanghai Institute of Materia Medica, Chinese Academy of Sciences for the valuable discussions and help. This work was supported by the National Natural Science Foundation of China (Grant 21472025 to Y.W., Grant 81302257 to M.L., Grants 21472208, 81625022, and 81430084 to C.L, and Grant 21210003 to H.J.), the National Basic Research Program (Grant 2015CB910304 to H.J.), and the Fund of State Key Laboratory of Toxicology and Medical Countermeasures, Academy of Military Medical Science (Grant TMC201505 to C.L.). 10333
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Brief Article
(9) (a) Prota, A. E.; Bargsten, K.; Zurwerra, D.; Field, J. J.; Diaz, J. F.; Altmann, K. H.; Steinmetz, M. O. Molecular mechanism of action of microtubule-stabilizing anticancer agents. Science 2013, 339, 587−590. (b) Prota, A. E.; Magiera, M. M.; Kuijpers, M.; Bargsten, K.; Frey, D.; Wieser, M.; Jaussi, R.; Hoogenraad, C. C.; Kammerer, R. A.; Janke, C.; Steinmetz, M. O. Structural basis of tubulin tyrosination by tubulin tyrosine ligase. J. Cell Biol. 2013, 200, 259−270. (10) Canela, M. D.; Noppen, S.; Bueno, O.; Prota, A. E.; Bargsten, K.; Saez-Calvo, G.; Jimeno, M. L.; Benkheil, M.; Ribatti, D.; Velazquez, S.; Camarasa, M. J.; Diaz, J. F.; Steinmetz, M. O.; Priego, E. M.; PerezPerez, M. J.; Liekens, S. Antivascular and antitumor properties of the tubulin-binding chalcone TUB091. Oncotarget DOI: 10.18632/ oncotarget.9527. Published online: May 20, 2016. (11) (a) Wei, W.; Ayad, N. G.; Wan, Y.; Zhang, G. J.; Kirschner, M. W.; Kaelin, W. G., Jr. Degradation of the SCF component Skp2 in cellcycle phase G1 by the anaphase-promoting complex. Nature 2004, 428, 194−198. (b) Dorleans, A.; Gigant, B.; Ravelli, R. B.; Mailliet, P.; Mikol, V.; Knossow, M. Variations in the colchicine-binding domain provide insight into the structural switch of tubulin. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 13775−13779. (12) Tron, G. C.; Pirali, T.; Sorba, G.; Pagliai, F.; Busacca, S.; Genazzani, A. A. Medicinal chemistry of combretastatin A4: present and future directions. J. Med. Chem. 2006, 49, 3033−3044. (13) Mehta, B. M.; Rosa, E.; Fissekis, J. D.; Bading, J. R.; Biedler, J. L.; Larson, S. M. In-vivo identification of tumor multidrug resistance with 3H-colchicine. J. Nucl. Med. 1992, 33, 1373−1377.
ABBREVIATIONS USED MBH, Morita−Baylis−Hillman reaction; SKP, spiroketal-based bisphosphine ligand; rmsd, root-mean-square deviation; TBAF, tetrabutylammonium fluoride; THF, tetrahydrofuran
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REFERENCES
(1) Jordan, A.; Hadfield, J. A.; Lawrence, N. J.; McGown, A. T. Tubulin as a target for anticancer drugs: agents which interact with the mitotic spindle. Med. Res. Rev. 1998, 18, 259−296. (2) Lu, Y.; Chen, J.; Xiao, M.; Li, W.; Miller, D. D. An overview of tubulin inhibitors that interact with the colchicine binding site. Pharm. Res. 2012, 29, 2943−2971. (3) Mikstacka, R.; Stefanski, T.; Rozanski, J. Tubulin-interactive stilbene derivatives as anticancer agents. Cell. Mol. Biol. Lett. 2013, 18, 368−397. (4) For reviews, see (a) and (b). (a) Kaur, R.; Kaur, G.; Gill, R. K.; Soni, R.; Bariwal, J. Recent developments in tubulin polymerization inhibitors: an overview. Eur. J. Med. Chem. 2014, 87, 89−124. (b) Zheng, S.; Zhong, Q.; Mottamal, M.; Zhang, Q.; Zhang, C.; Lemelle, E.; McFerrin, H.; Wang, G. Design, synthesis, and biological evaluation of novel pyridine-bridged analogues of combretastatin-A4 as anticancer agents. J. Med. Chem. 2014, 57, 3369−3381. (c) Chaudhary, V.; Venghateri, J. B.; Dhaked, H. P.; Bhoyar, A. S.; Guchhait, S. K.; Panda, D. Novel combretastatin-2-aminoimidazole analogues as potent tubulin assembly inhibitors: exploration of unique pharmacophoric impact of bridging skeleton and aryl moiety. J. Med. Chem. 2016, 59, 3439−3451. (5) (a) O’Boyle, N. M.; Carr, M.; Greene, L. M.; Bergin, O.; Nathwani, S. M.; McCabe, T.; Lloyd, D. G.; Zisterer, D. M.; Meegan, M. J. Synthesis and evaluation of azetidinone analogues of combretastatin A-4 as tubulin targeting agents. J. Med. Chem. 2010, 53, 8569−8584. (b) Carr, M.; Greene, L. M.; Knox, A. J.; Lloyd, D. G.; Zisterer, D. M.; Meegan, M. J. Lead identification of conformationally restricted β-lactam type combretastatin analogues: synthesis, antiproliferative activity and tubulin targeting effects. Eur. J. Med. Chem. 2010, 45, 5752−5766. (c) Tripodi, F.; Pagliarin, R.; Fumagalli, G.; Bigi, A.; Fusi, P.; Orsini, F.; Frattini, M.; Coccetti, P. Synthesis and biological evaluation of 1,4-diaryl-2-azetidinones as specific anticancer agents: activation of adenosine monophosphate activated protein kinase and induction of apoptosis. J. Med. Chem. 2012, 55, 2112−2124. (d) Greene, T. F.; Wang, S.; Greene, L. M.; Nathwani, S. M.; Pollock, J. K.; Malebari, A. M.; McCabe, T.; Twamley, B.; O’Boyle, N. M.; Zisterer, D. M.; Meegan, M. J. Synthesis and biochemical evaluation of 3-phenoxy-1,4-diarylazetidin-2-ones as tubulin-targeting antitumor agents. J. Med. Chem. 2016, 59, 90−113. (e) Perez-Perez, M. J.; Priego, E. M.; Bueno, O.; Martins, M. S.; Canela, M. D.; Liekens, S. Blocking blood flow to solid tumors by destabilizing tubulin: an approach to targeting tumor growth. J. Med. Chem. 2016, 59, 8685− 8711. (6) (a) Wang, X.; Meng, F.; Wang, Y.; Han, Z.; Chen, Y. J.; Liu, L.; Wang, Z.; Ding, K. Aromatic spiroketal bisphosphine ligands: palladium-catalyzed asymmetric allylic amination of racemic MoritaBaylis-Hillman adducts. Angew. Chem., Int. Ed. 2012, 51, 9276−9282. (b) Wang, X.; Guo, P.; Han, Z.; Wang, X.; Wang, Z.; Ding, K. Spiroketal-based diphosphine ligands in Pd-catalyzed asymmetric allylic amination of Morita-Baylis-Hillman adducts: exceptionally high efficiency and new mechanism. J. Am. Chem. Soc. 2014, 136, 405−411. (7) Greene, L. M.; Wang, S.; O’Boyle, N. M.; Bright, S. A.; Reid, J. E.; Kelly, P.; Meegan, M. J.; Zisterer, D. M. Combretazet-3 a novel synthetic cis-stable combretastatin A-4-azetidinone hybrid with enhanced stability and therapeutic efficacy in colon cancer. Oncol. Rep. 2013, 29, 2451−2458. (8) Chen, J.; Wang, Z.; Li, C.-M.; Lu, Y.; Vaddady, P. K.; Meibohm, B.; Dalton, J. T.; Miller, D. D.; Li, W. Discovery of novel 2-aryl-4benzoyl-imidazoles targeting the colchicines binding site in tubulin as potential anticancer agents. J. Med. Chem. 2010, 53, 7414−7427. 10334
DOI: 10.1021/acs.jmedchem.6b01268 J. Med. Chem. 2016, 59, 10329−10334
