Calanquinone A (1) was isolated from an EtOAc-soluble extract of Calanthe arisanensis through bioassay-guided fractionation. Its structure was identified by spectroscopic methods. Compound 1 showed potent cytotoxicity (EC50 < 0.5 µg/mL) against lung (A549), prostate (PC-3 and DU145), colon (HCT-8), breast (MCF7), nasopharyngeal (KB), and vincristine-resistant nasopharyngeal (KB-VIN) cancer cell lines, and interestingly, showed an improved drug resistance profile compared to paclitaxel. The total synthesis of 1 was also achieved and reported herein

Bastow, Kenneth F.

Chang, Fang Rong

Lee, Chia Lin

Lee, Kuo Hsiung

Liu, Yi Nan

Morris-Natschke, Susan L.

Nakagawa-Goto, Kyoko

Wu, Yang Chang

Yu, Donglei

PubMed

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Cytotoxic Calanquinone A from Calanthe arisanensis and Its FirstTotal SynthesisChia-Lin Leea,b, Kyoko Nakagawa-Gotoa, Donglei Yua, Yi-Nan Liua, Kenneth F. Bastowc,Susan L. Morris-Natschkea, Fang-Rong Changb, Yang-Chang Wub,d*, and Kuo-HsiungLeea*aNatural Products Research Laboratories, School of Pharmacy, University of North Carolina at Chapel Hill,NC 27599, USAbGraduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, TaiwancDivision of Medicinal Chemistry and Natural Products, School of Pharmacy, University of North Carolina,Chapel Hill, North Carolina 27599, USAdCenter of Excellence for Environmental Medicine, Kaohsiung Medical University, Kaohsiung 807, TaiwanAbstractCalanquinone A (1) was isolated from an EtOAc-soluble extract of Calanthe arisanensis throughbioassay-guided fractionation. Its structure was identified by spectroscopic methods. Compound 1showed potent cytotoxicity (EC50 < 0.5 µg/mL) against lung (A549), prostate (PC-3 and DU145),colon (HCT-8), breast (MCF7), nasopharyngeal (KB), and vincristine-resistant nasopharyngeal (KB-VIN) cancer cell lines, and interestingly, showed an improved drug resistance profile compared topaclitaxel. The total synthesis of 1 was also achieved and reported herein.The Calanthe genus in the Orchidaceae family contains terrestrial perennial herbs that arewidely distributed from tropical Africa and Madagascar to tropical and subtropical Asia, China,Japan, southward through Malaysia and Indonesia to the Pacific islands and Australia. Thisgenus includes more than 150 species, but only nineteen are found in Taiwan. Among themCalanthe arisanensis Hayata is endemic to Taiwan and grows in forests from 1000 to 2000 mthroughout the island.1A phytochemical study of this plant has not been reported to date. In cytotoxicity screening ofextracts of Formosan plants, an EtOAc extract of C. arisanensis was found to be active againstvarious human cancer cell lines with IC50< 20 µg/mL. Bioassay-directed chromatographicfractionation of this extract produced a new phenanthraquinone calanquinone A (1). Compound1 showed significant in vitro cytotoxic activicity against seven human cancer cell lines, asdescribed below.The active MeOH extract (225 g) of dry roots of C. arisanensis (5.42 kg) was partitionedbetween EtOAc and water (1:1, v/v). Further fractionation of the active EtOAc extract (32.7g) by repeated liquid chromatography on silica gel gave calanquinone A (1). HRESIMS of 1*Corresponding authors. K.H.L. Tel: 919-962-0066; fax: 919-966-3893; e-mail: khlee@unc.edu, Y.C.W. 886-7-312-1101 ext. 2197.Fax: 886-7-311-4773; e-mail: yachwu@kmu.edu.tw.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customerswe are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resultingproof before it is published in its final citable form. Please note that during the production process errors may be discovered which couldaffect the content, and all legal disclaimers that apply to the journal pertain.NIH Public AccessAuthor ManuscriptBioorg Med Chem Lett. Author manuscript; available in PMC 2009 August 1.Published in final edited form as:Bioorg Med Chem Lett. 2008 August 1; 18(15): 4275–4277. doi:10.1016/j.bmcl.2008.06.099.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptshowed a [M-H]− ion at m/z 313.0705 (C17H14O6 -H), indicating 11 degrees of unsaturation.The IR spectrum showed absorptions for hydroxyl (3348 cm−1), carbonyl (1642 cm−1), andaromatic ring (1626, 1514, 1460, 1411, and 844 cm−1) functional groups. UV absorptions at242, 308, and 426 nm also indicated an aromatic system. Seventeen carbon signals, includingthree methoxy, four methine, and ten quaternary carbons, were observed in the NMR spectraof 1 (Table 1). Among the ten quaternary carbons, two were identified as carbonyl carbons onthe basis of chemical shifts δC 184.7 and 186.2. Therefore, the data supported the presence oftwo carbonyls, six olefins, and three ring moieties to fulfill the 11 degrees of unsaturation, and1 was postulated to be an phenanthrenedione or anthrenedione.2 1D NMR and HSQC dataindicated the presence of three methoxy groups at δH 3.96, 4.01, and 4.02 (δC 57.1, 56.2, and61.0), one pair of ortho-coupled aromatic protons at δH 8.05 (d, J=8.7 Hz, δC 137.1) and 8.10(d, J=8.7 Hz, δC 122.0), and two olefinic protons at δH 6.15 (s, δC 107.4) and 6.86 (s, δC 101.4).In the HMBC spectra, the olefinic proton at δH 6.86 exhibited 2J interactions with a carbon atδC 155.2 (C-7), as well as 3J interactions with carbons at δC 118.7 (C-4b), 140.4 (C-6), and137.1 (C-9). The other olefinic proton at δH 6.15 exhibited 2J interactions with a carbon atδC 161.7 (C-3), as well as 3J interactions with carbons at δC 186.2 (C-4) and 133.0 (C-10a).Locations of methoxy groups at C-3, C-6, and C-7 were confirmed by the following NOESYcorrelations: δH 6.15 (H-2)/3.96 (3-OMe), δH 6.86 (H-8)/4.01 (7-OMe) and 8.05 (H-9), δH 4.01(7-OMe)/4.02 (6-OMe), and δH 8.05 (H-9)/8.10 (H-10). Thus, compound 1 was identified as5-hydroxy-3,6,7-trimethoxy-1,4-phenanthrenequinone and has been named as calanquinoneA (1).Compound 1 is related in structure to other naturally occurring phenanthrenequinones,including the des-oxy analog sphenone (lacking the C-5 OH group),3 cymbinodin A (lackingthe two methoxy groups at C-6 and C-7),4 and annoquinone A (lacking any substituents onring C).5,6 In prior studies, sphenone and annoquinone A showed cytotoxic activity againstthe KB cell line (reported EC50 2.73 and 1.65 µg/mL, respectively).Compound 1 exhibited potent cytotoxicity (EC50 0.03–0.45 µg/mL) against human lung(A549), prostate (PC-3 and DU145), colon (HCT-8), breast (MCF7), nasopharyngeal (KB),and vincristine-resistant nasopharyngeal (KB-VIN) cancer cell lines. Paclitaxel was used as apositive control (data shown in Table 2). Interestingly, 1 exhibited comparble potency againstboth KB and its drug-resistant KB-VIN subline, and thus, showed an improved drug resistanceprofile compared to paclitaxel. The cytotoxic values demonstrate the strong potential of 1 asa promising lead compound and C. arisanensis as a promising plant source of new agents forcancer chemotherapy.In order to make sufficient quantities of 1 for extensive biological evaluation, we modified thesynthetic procedure of Kraus and co-workers7,8 (Scheme 1) to synthesize 1. As shown inScheme 2, we prepared 2-methoxy-5-carboxylic acid methyl ester-1,4-quinone (4)9 by AgOoxidation of the methyl ester (3) of commercially available 2,4,5-trimethoxybenzoic acid (2).Compound 4 was coupled with 3,4,5-trimethoxytoluene in the presence of 1 equiv. oftrifluoroacetic acid to produce quinone 5, although Kraus reported the production ofhydroquinone 10 under these reaction conditions (Scheme 1). The quinone skeleton of 5 wasconfirmed from 13C-NMR data, which showed two carbonyl groups at 180.6 and 183.6 ppm.In addition, reaction of 5 with Me2SO4 did not give a methoxylated product (i.e., 11 in Scheme1). We attempted to reduce 5 with Na2S2O4 to obtain the corresponding hydroquinone, whichcould be transformed to the Kraus type of intermediate 11 (R = OMe) after methylation. Despiteextending the reaction time and adding more reducing agent, only starting material wasrecovered.Therefore, 5 was reduced with LAH (THF, reflux, 1 h) to alcohol, which was oxidizedselectively to aldehyde 6 with activated MnO2 (toluene, 110 °C, overnight) (Scheme 2). AfterLee et al. Page 2Bioorg Med Chem Lett. Author manuscript; available in PMC 2009 August 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptan unsuccessful attempt to obtain the phenanthraquinone 9 directly from 6 using P4-tBu,quinone 6 was first reduced to the hydroquinone using aq. Na2S2O4 (CH2Cl2, rt., overnight),10 and then methylated with Me2SO4 in the presence of K2CO3(acetone, 60 °C, 1.5 h) to givethe desired compound 7 (Scheme 2). Cyclization of 7 with P4-t Bu (benzene, 100 °C, 63 h)gave 8, which was oxidized with AgO (6N HNO3, acetone, 50 °C, 2–3 min) tophenanthraquinone 9. Compound 9 was converted to calanquinone A (1) by selectivedemethylation with TMSI in CH2Cl2 at r.t. (Scheme 2).Synthesized 1 and intermediates 5–9 were screened in an in vitro cytotoxicity assay (data shownin Table 3). Compound 1 exhibited the highest potency (EC50 0.15–0.75 µg/mL) against allseven tested cancer cell lines. The remaining compounds showed no (6–8) or only weak (5 and9) activity. Clearly, the potency of 1 merits further study and our synthetic route can efficientlyproduce sufficient quantities of 1 for future extensive biological evaluation and SARinvestigation.Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.AcknowledgmentsThis work was supported by grants from National Cancer Institute, NIH, USA (CA 17625, K. H. Lee), National ScienceCouncil, Taiwan (Y.-C. Wu) and KMU-EM-97-2.1.b (Y.-C. Wu).References and notes1. Boufford, DE.; Hsieh, C-F.; Huang, T-C.; Kuoh, C-S.; Ohashi, H.; Su, H-J.; Yeh, H-Y.; Hsiao, J-L.;Hsiao, S-H. Flora of Taiwan. Vol. 5. Taipei, Taiwan: Editorial Committee of the Flora of Taiwan;2000. p. 776-777.p. 782-783.2. Fan C, Wang W, Wang Y, Qin G, Zhao W. Phytochemistry 2001;57:1255. [PubMed: 11454354]3. Wu TS, Huang SC, Chen MT, Jong TT. Phytochemistry 1989;28:1280.4. Barula AK, Ghosh BB, Ray S, Patra A. Phytochemistry 1990;29:3056.5. Wu TS, Jong TT, Tien HJ, Kuoh CS, Furukawa H, Lee KH. Phytochemistry 1987;26:1623.6. Krohn K, Loock U, Paavilainen K, Hausen B, Schmalle HW, Kiesele H. ARKIVOC 2001;2:88.7. Kraus GA, Hoover K, Zhang N. Tetrahedron Lett 2002;43:5319.8. Kraus GA, Zhang N. Tetrahedron Lett 2002;43:9597.9. Parker KA, Spero DM, Koziski KA. J. Org. Chem 1987;52:183.10. Hormi OEO, Moilanen AM. Tetrahedron 1998;54:1943.Lee et al. Page 3Bioorg Med Chem Lett. Author manuscript; available in PMC 2009 August 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 1.Structure of calanquinone A (1).Lee et al. Page 4Bioorg Med Chem Lett. Author manuscript; available in PMC 2009 August 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptScheme 1.Synthetic procedure of Kraus.Lee et al. Page 5Bioorg Med Chem Lett. Author manuscript; available in PMC 2009 August 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptScheme 2.Our total synthesis of calanquinone A (1).Lee et al. Page 6Bioorg Med Chem Lett. Author manuscript; available in PMC 2009 August 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptLee et al. Page 7Table 1NMR data of calanquinone A (1)aProton 13C (δC)1H (δH) HMBC (13C no) NOESY (1H no)1 184.72 107.4 6.15 s 3, 4, 10a C3-OCH33 161.74 186.24a 128.34b 118.75 148.36 140.47 155.28 101.4 6.86 s 4b, 6, 7, 9 9, C7-OCH38a 135.19 137.1 8.05 d (8.7) 4b, 8, 10a 8, 1010 122.0 8.10 d (8.7) 1, 4a, 8a 910a 133.0−OCH3 C3-OCH3 57.1 C3-OCH3 3.96 s 3 2C6-OCH3 61.0 C6-OCH3 4.02 s 6 C7-OCH3C7-OCH3 56.2 C7-OCH3 4.02 s 7 8, C6-OCH3C5-OH 10.73aMeasured in CDCl3 (300 and 500 MHz, δ in ppm, J in Hz).Bioorg Med Chem Lett. Author manuscript; available in PMC 2009 August 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptLee et al. Page 8Table 2Cytotoxicity of calanquinone A (1) isolated from C. arisanensis EC50 (µg/mL) / Cell lineCompoundA549PC-3DU145HCT-8MCF-7KBKB-VINCalanquinone A (1)0.190.160.340.200.030.320.45Paclitaxela<0.0050.0097<0.0050.210.0072<0.0052.16a positive controlBioorg Med Chem Lett. Author manuscript; available in PMC 2009 August 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptLee et al. Page 9Table 3Cytotoxicity of synthesized 1 and related intermediatesEC50 (µg/mL) / Cell lineCompoundA549PC3DU145HCT8MCF7KBKB-VIN10.310.750.480.290.150.300.2456.126.094.745.856.404.025.486NANANANANANANA7NANANANANANANA8NANANANANANANA97.067.814.4015.255.338.089.14NA: no activity up to 20 µg/mLBioorg Med Chem Lett. Author manuscript; available in PMC 2009 August 1.

Cytotoxic calanquinone A from Calanthe arisanensis and its first total synthesis

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Cytotoxic calanquinone A from Calanthe arisanensis and its first total synthesis

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