Abstract
4-Amino-2H-benzo[h]chromen-2-one (ABO) and 4-amino-7,8,9,10-tetrahydro-2H-benzo[h]chromen-2-one (ATBO) analogs were found to be significant in vitro anticancer agents in our previous research. Our continuing study has now discovered a new simplified (monocyclic rather than tricyclic) class of cytotoxic agents, 4-amino-2H-pyran-2-one (APO) analogs. By incorporating various substituents on the pyranone ring, we have established preliminary structure-activity relationships (SAR). Analogs 19, 20, 23, and 26–30 displayed significant tumor cell growth inhibitory activity in vitro. The most active compound 27 exhibited ED50 values of 0.059–0.090 μM.
Keywords: 4-Amino-2H-pyran-2-one (APO) analogs, Neo-tanshinlactone, Cytotoxicity
In 2004, our group first isolated and synthesized neo-tanshinlactone (1).1 Compound 1 was 10-fold more potent and 20-fold more selective as compared with tamoxifen citrate against the ER+ human breast cancer cell lines MCF-7 and ZR-75-1. Further structural optimization led to its 4-ethyl analog 2, which displayed significant and selective anti-breast cancer activity both in vitro and in vivo.2.3 Moreover, 2 was selective for a subset of breast cancer-derived cell lines and significantly less active against normal breast-derived tissue. In order to explore the effect of individual rings on the anticancer activity, identify new lead compounds, and discover new chemical entities, we designed and reported five classes of new anticancer agents, including 2-(furan-2-yl)naphthalen-1-ol (FNO),4 6-phenyl-4H-furo[3,2-c]pyran-4-one (AFPO),5 tetrahydronaphthalene-1-ol (TNO),6 4-amino-2H-benzo[h]chromen-2-one (ABO, 3, Figure 1),7 and 4-amino-7,8,9,10-tetrahydro-2H-benzo[h]chromen-2-one (ATBO, 4, Figure 1)8 analogs. Interestingly, the neo-tanshinlactone-inspired synthesis of a breast cancer selective ABO series was reported independently by others.9
Figure 1.
Structures of neo-tanshinlactone (1), 4-ethyl neo-tanshinlactone (2), previously reported ABO (3) and ATBO (4) scaffolds, and newly designed APO scaffold (5)
Importantly, ABO and ATBO compounds displayed much higher potency than 1-analogs, which encouraged us to further investigate these scaffolds. Structure-activity relationship (SAR) studies on 3 and 4 indicated that (1) a secondary amine (R2 or R3 = H) is preferred over tertiary amine (R2 and R3 ≠ H), (2) bulky groups are favored at the R2/R3 position, (3) a 3′-bromophenyl group can cause a dramatic loss of potency, and (4) a non-aromatic ring-A can increase potency and cancer cell line selectivity for certain analogs. Our studies also indicated that the lactone ring-C is critical to the cytotoxic activity.3,7,8 Therefore, we designed a structurally simplified monocyclic scaffold (5, Figure 1) and incorporated various substituents at the R and R′ positions to explore the contributions of ring-A and -B, develop new chemical entities and new leads, and establish the SAR. Herein, we report the design, synthesis, and biological activity of 4-amino-2H-pyran-2-one (APO) analogs.
As a first step in the current work, we designed two model compounds 12 and 13 by eliminating the fused ring-A/B system and incorporating pendant phenyl and styryl groups, respectively, on the remaining pyranone C-ring. 6-Substituted 4-hydroxy-2H-pyran-2-one 10 was synthesized according to the method reported by Bach et al.10 The appropriate aldehyde (6) and an acetoacetate equivalent (7) underwent a vinylogous Mukaiyama aldol addition to give 8, which was oxidized to 9 using the Dess–Martin method. A thermal cyclization of 9 yielded 4-hydroxy-2H-pyranone 10. Chlorination followed by amination of 10 provided model compounds 12 and 13.7
Analogs 12 and 13 were tested for in vitro cytotoxic activity against a panel of human tumor cell lines according to previously published methods (Table 1).3 Cell lines included A549 (non small cell lung cancer), DU145 (prostate cancer cell line), KB (nasopharyngeal carcinoma), and KB-VIN (vincristine-resistant MDR KB subline), SK-BR-3 (estrogen receptor negative, HER2 over-expressing breast cancer). Importantly, 13 showed significant inhibition of all human cancer cell lines tested with ED50 values from 1.23–2.02 μM, while 12 displayed moderate activity.
Table 1.
Cytotoxicity of 12–30 against Human Tumor Cell Line Panel
| Compd | ED50
(μM) a |
||||
|---|---|---|---|---|---|
| KB | KB-VIN | A549 | DU145 | SKBR-3 | |
| 3 | 0.11 | 0.13 | 0.17 | 0.11 | 0.13 |
| 12 | 9.69 | 9.15 | 16.02 | 7.47 | 10.91 |
| 13 | 1.25 | 1.24 | 2.02 | 1.23 | 1.53 |
| 14 | >30 | >30 | >30 | >30 | >30 |
| 15 | >30 | >30 | >30 | >30 | >30 |
| 16 | 4.42 | 4.84 | 10.06 | 5.94 | 4.93 |
| 17 | 5.42 | 4.98 | 8.38 | 5.42 | 4.73 |
| 18 | 4.62 | 4.39 | 8.32 | 8.61 | 5.29 |
| 19 | 0.093 | 0.098 | 0.16 | 0.12 | 0.079 |
| 20 | 0.11 | 0.10 | 0.15 | 0.11 | 0.083 |
| 21 | 1.62 | 1.30 | 2.42 | 1.60 | 1.34 |
| 22 | 1.91 | 1.46 | 2.66 | 1.95 | 1.76 |
| 23 | 0.15 | 0.13 | 0.18 | 0.15 | 0.13 |
| 24 | >30 | >30 | >30 | >30 | >30 |
| 25 | >30 | >30 | >30 | >30 | >30 |
| 26 | 0.12 | 0.15 | 0.13 | 0.098 | 0.13 |
| 27 | 0.067 | 0.059 | 0.064 | 0.073 | 0.090 |
| 28 | 0.31 | 0.24 | 0.32 | 0.34 | 0.40 |
| 29 | 0.44 | 0.49 | 0.35 | 0.37 | 0.54 |
| 30 | 0.63 | 0.55 | 0.49 | 0.66 | 0.74 |
mean from three or more independent tests.
Encouraged by these promising results, analogs 14–30 were designed to establish SAR correlations as well as to optimize structure and identify more active derivatives with the desired biological properties. Firstly, we installed various groups at the pyranone 6-position (R group) to explore the effect of group size, ring size, and aromaticity. Secondly, different substituents at the pyranone 4-position (R′ group) were investigated while retaining the best R group. As shown in Scheme 1, new analogs 14–30 were obtained through the five-step procedure similarly to 12 and 13, and then evaluated against five human tumor cell lines from different tissues.
Scheme 1.
Reagents and conditions: (a) TiCl4, CH2Cl2, -78 °C; (b) Dess-Martin reagent, rt; (c) toluene, reflux; (d) POCl3, Et3N, reflux, 1h; (e) aliphatic amines, EtOH, reflux, 2h; (f) aromatic amines, ethylene glycol, 160 °C, 1h.
Compounds 14 and 15 with methyl and isopropyl R groups were not active, and 16–18 with pentyl, cyclopentyl, and cyclohexyl groups showed moderate activity. These results suggested that long alkyl chains and cyclic alkyl groups were favored at the pyranone 6-position. Compounds 19–23, which contain different aromatic R groups, including phenyl, styryl, benzyl, furanyl, and naphthyl, displayed significant activity. Especially, 19 and 20 with phenyl and styryl groups, respectively, were the most potent analogs with ED50 values of 0.079–0.163 μM, and were equally or slightly more potent than ABO analog 3. The results indicated that an aromatic ring at the pyranone 6-position may be critical to the cytotoxic activity.
Based on structural simplicity and chemical availability as well as the above results, we designed analogs 24–30, which have various substituents at the pyranone 4-position and a phenyl group fixed at the 6-position. Among them, 26–30 with substituted aniline groups displayed significant activity against all tested tumor cell lines compared with analogs containing cycloalkyl groups, including cyclohexylamine (12), cyclopropylamine (24), and piperidine (25). The latter two compounds totally lost cytotoxic activity. These data indicate that an aromatic amino R′ group is favored at the 4-position. In addition, the position and type of substituent on the aniline ring played an important role in the cytotoxic activity. The rank order of potency for all aromatic analogs against KB was 27 (4′-Me) > 19 (4′-OMe) > 26 (H) > 28 (4′-Br) ~ 29 (2′-OMe) ~ 30 (3′-OMe). 4-Methylaniline-substituted 27 was the most potent analog with ED50 values of 0.059–0.090 μM. It was about two-fold more potent than ABO analog 3.
Overall, 19 and 27 showed greater in vitro antitumor activity than other analogs, suggesting that the combination of a phenyl group at the 6-position and a 4′-methyl- or 4′-methoxy-aniline at the 4-position is favored for the APO analogs.
In summary, we designed and developed a new class (APO) of in vitro anticancer agents, through structural simplification and optimization. Lead compounds displayed potent antitumor activity with ED50 values in the low micromolar range. SAR studies indicated that: (1) an aromatic ring such as phenyl and styryl at the pyranone 6-position is critical to the antitumor activity, (2) a secondary amine at the 4-position is preferred over a tertiary amine, (3) aromatic amine groups at the 4-position are crucial, and (4) substituents on the aromatic amine group can increase potency. Compounds 19 and 27 were the most potent analogs (ED50 values of 0.059–0.163 μM) among all derivatives, and thus, are new lead compounds that are promising for further development of potential clinical trials candidates.
Acknowledgments
This work was supported by NIH grant CA-17625 from the National Cancer Institute, awarded to K.H. Lee.
Footnotes
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
References and notes
- 1.Wang X, Bastow KF, Sun CM, Lin YL, Yu HJ, Don MJ, Wu TS, Nakamura S, Lee KH. J Med Chem. 2004;47:5816. doi: 10.1021/jm040112r. [DOI] [PubMed] [Google Scholar]
- 2.Wang X, Nakagawa-Goto K, Bastow KF, Don MJ, Lin YL, Wu TS, Lee KH. J Med Chem. 2006;49:5631. doi: 10.1021/jm060184d. [DOI] [PubMed] [Google Scholar]
- 3.Dong Y, Shi Q, Pai HC, Peng CY, Pan SL, Teng CM, Nakagawa-Goto K, Yu D, Liu YN, Wu PC, Bastow KF, Morris-Natschke SL, Brossi A, Lang JY, Hsu JL, Hung MC, Lee EY, Lee KH. J Med Chem. 2010;53:2299. doi: 10.1021/jm1000858. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Dong Y, Shi Q, Liu YN, Wang X, Bastow KF, Lee KH. J Med Chem. 2009;52:3586. doi: 10.1021/jm9001567. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Dong Y, Shi Q, Nakagawa-Goto K, Wu PC, Morris-Natschke SL, Brossi A, Bastow KF, Lang JY, Hung MC, Lee KH. Bioorg Med Chem. 2010;8:803. doi: 10.1016/j.bmc.2009.11.049. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Dong Y, Shi Q, Nakagawa-Goto K, Wu PC, Bastow KF, Morris-Natschke SL, Lee KH. Bioorg Med Chem Lett. 2009;19:6289. doi: 10.1016/j.bmcl.2009.09.092. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Dong Y, Nakagawa-Goto K, Lai CY, Morris-Natschke SL, Bastow KF, Lee KH. Bioorg Med Chem Lett. 2010;20:4085. doi: 10.1016/j.bmcl.2010.05.079. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Dong Y, Nakagawa-Goto K, Lai CY, Morris-Natschke SL, Bastow KF, Lee KH. Bioorg Med Chem Lett. 2011;21:545. doi: 10.1016/j.bmcl.2010.10.074. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Sashidhara KV, Rosaiah JN, Kumar M, Gara RK, Nayak LV, Srivastava K, Bid HK, Konwar R. Bioorg Med Chem Lett. 2010;20:7127. doi: 10.1016/j.bmcl.2010.09.040. [DOI] [PubMed] [Google Scholar]
- 10.Bach T, Kirsch S. Synlett. 2001:1974–1976. [Google Scholar]
- 11.Spectroscopic data. 4-(Cyclohexylamino)-6-phenyl-2H-pyran-2-one (12). 1H NMR (400 MHz, DMSO-d6): δ 7.75 (m, 2H, Ar-H), 7.39 (m, 3H, Ar-H), 6.21 (d, 1H, J = 2.0 Hz,), 5.11 (d, 1H, J = 1.6 Hz, 3-H), 4.94 (d, 1H, J = 7.2 Hz, NH), 3.29 (m, 1H, NCH), 2.05 (m, 2H, NCHCH2), 1.78 (m, 2H, NCHCH2), 1.26 (m, 6H, cyclohexyl-H). MS m/z 270 (M++1). (E)-4-(Cyclohexylamino)-6-styryl-2H-pyran-2-one (13) 1H NMR (400 MHz, DMSO-d6): δ 7.47 (m, 3H, Ar & olefin-H), 7.32 (m, 3H, Ar-H), 6.53 (d, 1H, J = 13.6 Hz, olefin-H), 5.71 (s, 1H, 3-H), 5.08 (d, 1H, J = 1.6 Hz,), 4.73 (d, 1H, J = 7.2 Hz, NH), 3.27 (m, 1H, NCH), 2.05 (m, 2H, NCHCH2), 1.78 (m, 2H, NCHCH2), 1.26 (m, 6H, cyclohexyl-H). MS m/z 296 (M++1). 4-[(4-Methoxyphenyl)amino]-6-methyl-2H-pyran-2-one (14) 1H NMR (400 MHz, CD3OD): δ 7.14 (d, 2H, J = 8.8 Hz, Ar-H), 6.97 (d, 2H, J = 8.8 Hz, Ar-H), 5.97 (d, 1H, J = 2.0 Hz, 5-H), 5.14 (d, 1H, J = 1.6 Hz, 3-H), 3.82 (s, 1H, 4′-OCH3), 2.20 (d, 3H, J = 0.8 Hz, 6-CH3). MS m/z 230 (M+-1). 6-Isopropyl-4-[(4-methoxyphenyl)amino]-2H-pyran-2-one (15) 1H NMR (400 MHz, CD3OD): δ 7.15 (d, 2H, J = 8.8 Hz, Ar-H), 6.97 (d, 2H, J = 8.8 Hz, Ar-H), 5.97 (d, 1H, J = 2.0 Hz, 5-H), 5.15 (d, 1H, J = 1.6 Hz, 3-H), 3.81 (d, 3H, J = 1.2 Hz, 4′-OCH3), 2.72 (h, 1H, J = 6.8 Hz, isopropyl-H), 1.24 (d, 6H, J = 6.8 Hz, isopropyl-H). MS m/z 258 (M+-1). 4-[(4-Methoxyphenyl)amino]-6-pentyl-2H-pyran-2-one (16) 1H NMR (400 MHz, CD3OD): δ 7.15 (d, 2H, J = 9.2 Hz, Ar-H), 6.97 (d, 2H, J = 8.8 Hz, Ar-H), 5.97 (d, 1H, J = 2.4 Hz, 5-H), 5.15 (d, 1H, J = 2.4 Hz, 3-H), 3.81 (s, 3H, 4′-OCH3), 2.46 (t, 2H, J = 7.6 Hz, pentyl-H), 1.66 (p, 2H, J = 7.6 Hz, pentyl-H), 1.34–1.38 (m, 4H, pentyl-H), 0.93 (t, 3H, J = 7.2 Hz, pentyl-H). MS m/z 286 (M+-1). 6-Cyclopentyl-4-[(4-methoxyphenyl)amino]-2H-pyran-2-one (17) 1H NMR (400 MHz, CD3OD): δ 7.14 (d, 2H, J = 8.8 Hz, Ar-H), 6.97 (d, 2H, J = 8.8 Hz, Ar-H), 5.99 (d, 1H, J = 2.4 Hz, 5-H), 5.14 (d, 1H, J = 2.0 Hz, 3-H), 3.81 (s, 3H, 4′-OCH3), 2.40 (m, 1H, cyclopentyl-H), 2.89 (p, 1H, J = 8.0 Hz, cyclopentyl-H), 1.96–2.01 (m, 2H, cyclopentyl-H), 1.66–1.82 (m, 6H, cyclopentyl-H). MS m/z 284 (M+-1). 6-Cyclohexyl-4-[(4-methoxyphenyl)amino]-2H-pyran-2-one (18) 1H NMR (400 MHz, CD3OD): δ 7.14 (d, 2H, J = 8.8 Hz, Ar-H), 6.97 (d, 2H, J = 8.8 Hz, Ar-H), 5.95 (s, 1H, 5-H), 5.15 (s, 1H, 3-H), 3.81 (s, 1H, 4′-OCH3), 2.40 (m, 1H, cyclohexyl-H), 1.72–1.94 (m, 5H, cyclohexyl-H), 1.25–1.47 (m, 5H, cyclohexyl-H). MS m/z 298 (M+-1). 4-[(4-Methoxyphenyl)amino]-6-phenyl-2H-pyran-2-one (19) 1H NMR (400 MHz, CDCl3): δ 7.82–7.85 (m, 2H, Ar-H), 7.48–7.51 (m, 3H, Ar-H), 7.20 (d, 2H, J = 8.8 Hz, Ar-H), 7.00 (d, 2H, J = 8.8 Hz, Ar-H), 6.66 (d, 1H, J = 2.0 Hz,5-H), 5.29 (d, 1H, J = 2.0 Hz, 3-H), 3.83 (s, 3H, 4′-OCH3). MS m/z 292 (M+-1). (E)-4-[(4-Methoxyphenyl)amino]-6-styryl-2H-pyran-2-one (20) 1H NMR (400 MHz, CD3OD): δ 7.60 (d, 2H, J = 7.2 Hz, Ar-H), 7.33–7.44 (m, 4H, Ar & olefin-H), 7.17–7.20 (m, 2H, Ar-H), 6.98–7.00 (m, 2H, Ar-H), 6.85 (d, 1H, J = 16.0 Hz, olefin-H), 6.20 (d, 1H, J = 2.0 Hz, 5-H), 5.25 (d, 1H, J = 1.6 Hz, 3-H), 3.82 (s, 1H, 4′-OCH3). MS m/z 318 (M+-1). 6-Benzyl-4-[(4-methoxyphenyl)amino]-2H-pyran-2-one (21) 1H NMR (400 MHz, CD3OD): δ 7.30–7.36 (m, 5H, Ar-H), 7.11 (d, 2H, J = 8.8 Hz, Ar-H), 6.95 (d, 2H, J = 9.2 Hz, Ar-H), 5.89 (s, 1H, 5-H), 5.14 (d, 1H, J = 1.6 Hz, 3-H), 3.80 (s, 3H, 4′-OCH3), 3.79 (s, 2H, 6-CH2). MS m/z 306 (M+-1). 6-(Furan-2-yl)-4-[(4-methoxyphenyl)amino]-2H-pyran-2-one (22) 1H NMR (400 MHz, CDCl3): δ 7.68 (s, 1H, Ar-H), 7.18 (d, 2H, J = 8.8 Hz, Ar-H), 6.98–7.00 (m, 3H, Ar-H), 6.61–6.63 (m, 1H, Ar-H), 6.49 (d, 1H, J = 1.6 Hz, 5-H), 5.22 (d, 1H, J = 2.0 Hz, 3-H), 3.82 (s, 3H, 4′-OCH3). MS m/z 282 (M+-1). 4-[(4-Methoxyphenyl)amino]-6-(naphthalen-2-yl)-2H-pyran-2-one (23) 1H NMR (400 MHz, CD3OD): δ 8.42 (s, 1H, Ar-H), 7.86–7.99 (m, 4H, Ar-H), 7.57–5.59 (m, 2H, Ar-H), 7.23 (d, 2H, J = 8.8 Hz, Ar-H), 7.02 (d, 2H, J = 9.2 Hz, Ar-H), 6.81 (d, 1H, J = 2.0 Hz,5-H), 5.33 (d, 1H, J = 2.0 Hz, 3-H), 3.84 (s, 3H, 4′-OCH3). MS m/z 342 (M+-1). 4-(Cyclopropylamino)-6-phenyl-2H-pyran-2-one (24) 1H NMR (400 MHz, CD3OD): δ 7.80 (m, 2H, Ar-H), 7.46 (m, 3H, Ar-H), 6.46 (s, 1H, 5-H), 5.41 (s, 1H, 3-H), 2.52 (s, 1H, 1′-H), 0.85 (d, 2H, J = 6.4 Hz,2′ & 3′-H), 0.58 (m, 2H, 2′ & 3′-H). MS m/z 226 (M+-1). 6-Phenyl-4-(piperidin-1-yl)-2H-pyran-2-one (25) 1H NMR (400 MHz, CD3OD): δ 7.89–7.92 (m, 2H, Ar-H), 7.47–7.49 (m, 3H, Ar-H), 6.92 (d, 1H, J = 1.6 Hz,5-H), 5.28 (d, 1H, J = 2.0 Hz, 3-H), 3.57 (t, 4H, J = 4.8 Hz, 2′ & 6′-H), 1.68–1.75 (m, 6H, 3′, 4′, & 5′-H). MS m/z (M+-1). 6-Phenyl-4-(phenylamino)-2H-pyran-2-one (26) 1H NMR (400 MHz, DMSO-d6): δ 9.42 (s, 1H, NH), 7.76–7.78 (m, 2H, Ar-H), 7.53–7.56 (m, 3H, Ar-H), 7.44 (t, 2H, J = 7.6 Hz, Ar-H), 7.28 (d, 2H, J = 8.4 Hz, Ar-H), 7.21 (t, 1H, J = 7.2 Hz, Ar-H), 6.70 (d, 1H, J = 1.6 Hz,5-H), 5.34 (t, 1H, J = 1.2 Hz, 3-H). MS m/z 262 (M+-1). 6-Phenyl-4-(p-tolylamino)-2H-pyran-2-one (27) 1H NMR (400 MHz, CD3OD): δ 7.83–7.86 (m, 2H, Ar-H), 7.50–7.52 (m, 3H, Ar-H), 7.26 (d, 2H, J = 8.4 Hz, Ar-H), 7.17 (d, 2H, J = 8.4 Hz, Ar-H), 6.69 (d, 1H, J = 2.0 Hz,5-H), 5.41 (d, 1H, J = 2.0 Hz, 3-H), 2.37 (s, 3H, CH3). MS m/z 276 (M+-1). 4-[(4-Bromophenyl)amino]-6-phenyl-2H-pyran-2-one (28) 1H NMR (400 MHz, CD3OD): δ 7.84–7.87 (m, 2H, Ar-H), 7.59 (d, 1H, J = 8.4 Hz, Ar-H), 7.50–7.52 (m, 3H, Ar-H), 7.23 (d, 1H, J = 8.8 Hz, Ar-H), 6.70 (d, 1H, J = 2.0 Hz,5-H), 5.50 (d, 1H, J = 2.0 Hz, 3-H). MS m/z 340 (M+-1). 4-[(2-Methoxyphenyl)amino]-6-phenyl-2H-pyran-2-one (29) 1H NMR (400 MHz, CD3OD): δ 7.83–7.86 (m, 2H, Ar-H), 7.50–7.52 (m, 3H, Ar-H), 7.34 (t, 1H, J = 8.0 Hz, Ar-H), 6.81–6.89 (m, 3H, Ar-H), 6.71 (d, 1H, J = 2.0 Hz,5-H), 5.53 (d, 1H, J = 2.0 Hz, 3-H), 3.82 (s, 3H, 4′-OCH3). MS m/z 292 (M+-1). 4-[(3-Methoxyphenyl)amino]-6-phenyl-2H-pyran-2-one (30) 1H NMR (400 MHz, CD3OD): δ 7.83–7.86 (m, 2H, Ar-H), 7.50–7.52 (m, 3H, Ar-H), 7.34 (t, 1H, J = 8.0 Hz, Ar-H), 6.81–6.89 (m, 3H, Ar-H), 6.71 (d, 1H, J = 2.0 Hz,5-H), 5.53 (d, 1H, J = 2.0 Hz, 3-H), 3.82 (s, 3H, 4′-OCH3). MS m/z 292 (M+-1).