Honokiol, from magnolia bark, shuts down cancer cells in lab
Compound in magnolia may combat head and neck cancers
Honokiol, from magnolia bark, shuts down cancer cells in lab
Magnolias are prized for their large, colorful, fragrant flowers. Does the attractive, showy tree also harbor a potent cancer fighter?
Yes, according to a growing number of studies, including one from VA and the University of Alabama at Birmingham that is now online in the journal Oncotarget.
The study focused on squamous cell head and neck cancers, a scourge among those who use tobacco and alcohol. According to the National Cancer Institute, at least 3 in 4 head and neck cancers are caused by the use of tobacco and alcohol. The cancers have only a 50 percent survival rate, killing some 20,000 Americans each year.
Enter honokiol–chemical formula C18H18O2. As one of the major active compounds in magnolia extract, the phytochemical has been used for centuries in traditional Chinese and Japanese medicine to treat anxiety and other conditions. More recently, scientists have been discovering that the compound, found in magnolia bark, is a wily and versatile adversary of cancer. It seems to exploit many biochemical pathways to shrink tumors of various types, or to keep them from growing in the first place.
The Alabama scientists have now shown how it works against head and neck cancers: It blocks a protein called epidermal growth factor receptor, or EGFR. Prior research has found that almost all head and neck cancer cells display an over-abundance of the protein, and it had been suggested in the literature as a potential target.
The VA-UAB team says, based on its lab studies, that honokiol binds more strongly with EGFR than does the drug gefitinib (sold as Iressa), which is commonly used to treat head and neck cancers.
The researchers tested honokiol on cell lines derived from human cancers of the oral cavity, larynx, tongue, and pharynx. In all cases, the botanical shut down the aberrant cells. The team also tested it against tumors implanted into mice, with similar results.
Senior author Dr. Santosh K. Katiyar and his colleagues wrote, “Conclusively, honokiol appears to be an attractive bioactive small molecule phytochemical for the management of head and neck cancer which can be used either alone or in combination with other available therapeutic drugs.”
Katiyar has published extensively in the past on other natural substances that work against tumors, especially skin cancer. Some of his recent work has focused on compounds in green tea, for example, and grape seed proanthocyanidins.
There are several methods for purifying and isolating honokiol. In nature, honokiol exists with its structural isomer magnolol, which differs from honokiol only by the position of onehydroxyl group. Because of the very similar properties of magnolol and honokiol, purification has often been limited to a HPLC or electromigration. However, methods developed in 2006 by workers in the lab of Jack L. Arbiser, took advantage of the proximity of the phenolic hydroxyl groups in magnolol, which form a protectable diol, to generate amagnolol acetonide (Figure 1), with a subsequent simple purification via flash chromatography over silica.
Magnolol and Honokiol are normally inseparable. Honokiol is easily separable from the protected magnolol acetonide
Additionally a rapid separation approach was published in the Journal of Chromatography A in 2007. The process uses high-capacity high-speed countercurrent chromatography(high-capacity HSCCC). Through this method honokiol can be separated and purified to above 98% purity with a high yield in under an hour.
Honokiol is a lignan isolated from the bark, seed cones, and leaves of trees belonging to the genus Magnolia. It has been identified as one the chemical compounds in some traditional eastern herbal medicines along with magnolol, 4-O-methylhonokiol, andobovatol.
Extracts from the bark or seed cones of the Magnolia tree have been widely used in traditional medicine in China, Korea, and Japan.
Houpu has traditionally been used in Eastern medicine as analgesic and to treat anxiety and mood disorders. However, it has been shown to treat a number of other conditions. In China, magnolia bark is called Houpu and is most commonly taken from the Magnolia obovata and the Magnolia officinalis species. Some Chinese traditional formulas containing Houpu include Banxia Houpu Tang (半夏厚朴丸), Xiao Zhengai Tang, Ping Wei San(平胃散) and Shenmi Tang. Japanese Kampo formulas include, Hange-koboku-to (半夏厚朴湯) and Sai-boku-to (柴朴湯).
In the late 1990s, honokiol saw a revival in interest as a potent and highly tolerable antitumorigenic and neurotrophiccompound.
Currently there are a large number of supplements containing honokiol on the market, and its use has been widely well received among practitioners of new age, homeopathic, and holistic medicine
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|Mature Magnolia fruit just starting to open, with a few seeds visible
Magnolia seeds and fruit on a tree in northern Argentina
The root and stem bark of Magnolia has been used as a traditional Chinese medicine for the treatment of thrombotic stroke, gastrointestinal complaints, and anxiety. Honokiol (HNK), a substituted biphenyl and an active component isolated and purified from Magnolia, has anti-oxidant, antithrombosis, antibacterial, neurotrophic, xanthine oxidase inhibitory, and anxiolytic effects (Taira et al., Free Radic Res Commun. 1993;19 Suppl l:S71-77; Teng et al. Thromb Res. 1988;50:757-765; Clark et al., J. Pharm. Sci. 1981;70:951-952; Chang et al., Anticancer Res. 1994;14:501-506; Kuribara et al., J. Pharm Pharmacol. 1998;50:819-826; Esumi et al., Bioorg & Medicinal Chem Let 2004, 14: 2621-25).
In the early 1990s, reports of HNK’s anticancer effects were published. In 1994, Hirano et al (Life Sci. 1994;55(13): 1061-9) examined the anti leukemic-cell efficacy of 28 naturally occurring and synthetic flavonoids and 11 naturally occurring ligands on human promyelocytic leukemic cell line HL-60, and cytotoxicity of these compounds was compared with four clinical anti-cancer agents. HNK was identified as one of the most potent compounds in this screen, with an IC50 value less than 100 ng/ml. In 1998, Hibasami et al. demonstrated that HNK induced apoptosis in human lymphoid leukemia Molt 4B cells (Hibasami et al., Int. J. MoI. Med. 1998).
HNK has also been found to induce apoptosis in human squamous cell lung cancer CH27 cells (Yang SE, et al Biochem Pharmacol. 2002;63:1641-1651) and in human colorectal RKO cells (Wang et al World J Gastroenterol. 2004; 10:2205-2208). In 2004, Chen et al. (World J Gastroenterol. 2004; 10: 3459-3463) reported that HNK was effective in an in vivo animal model of human colon cancer by inhibiting tumor growth and prolonging the lifespan of tumor bearing mice.
Honokiol is an inhibitor of angiogenesis and antitumor activity in vivo. HNK can cause apoptosis in tumor cells and inhibit angiogenesis through blocking phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2), the major mitogenic and chemoattractant endothelial growth factor (Bai et al. (2003) J. Biol. Chem. 278, 35501- 35507). Honokiol also exhibits direct antitumor activity through induction of apoptosis through tumor necrosis factor apoptosis-inducing ligand (TRAIL/ Apo2L) signaling and has been found to be highly effective against angiosarcoma in nude mice in vivo (Bai et al. (2003) J. Biol. Chem. 278, 35501-35507).
Esumi et al. (Biorganic & Medicinal Chemistry Letters (2004) 14: 2621-2625) describe a synthesis method to produce HNK. This report also evaluates the structure activity relationship of O-methylated and/or its hydrogenated analogs of HNK in an in vitro neurotrophic assay. Esumi et al. conclude that the 5-allyl and 4′-hydroxyl groups are essential for the neurotrophic activity of HNK.
PCT Publication No. WO 02/076393 and U.S. Publication No. 2004/0105906 to Emory University describe pharmaceutical compositions and methods of treating conditions such angiogenic-, neoplastic-, and cancer-related conditions and skin conditions by administration of honokiol-type and/or magnolol-type compounds, as shown in Figures 1-4. For example, such compositions comprise at least one compound of formula Al :
AI wherein R1, R2, R3, R4, R5, R1, R2, R3, R4, and R’5 can be independently selected from groups that include, but are not limited to, hydrogen, hydroxyl groups, amides, amines, hydrocarbons, halogenated hydrocarbons, cyclic hydrocarbons, cyclic heterocarbons, halogenated cyclic heterocarbons, benzyl, halogenated benzyl, organo selenium compounds, sulfides, carbonyl, thiol, ether, dinitrogen ring compounds, thiophenes, pyridines, pyrroles, imidazoles, and pyrimidines. Honokiol-type and magnolol-type compounds are shown to inhibit SVR cell proliferation.
In November of 2004, Arbiser et al. reported that honokiol inhibited the growth of multuple myeloma cell lines via induction of Gl growth arrest, followed by apoptosis with IC50 values at 48h of 5 to 10 μg/mL. It was also reported that honokiol inhibited growth of doxorubin (Dox)-resistant (RPMI-Dox40), mephalan resistant (RPMI-LR5) and dexamethasone (Dex)-resistant (MM. IR) cell lines. It was suggested that the mechanism of honokiol triggered cytotoxicity is the honokiol induced increased expressin of Bax and Bad, down-regulated Mc-I protein expression, followed by caspase-8/9/3 cleavage, (Arbiser, J. et al. Poster at the American Society of Hematology Annual Meeting, 2004. Abstract published online November 4, 2004).
In July of 2005, Battle et al. reported that honokiol induces caspase-dependent apoptosis in B-cell chronic lymphocytic leukemia (B-CLL) cells (Blood. July 2005; 106:690- 697). Honokiol induced caspase-dependent cell death in all of the B-CLL cells examined, which were primary tumor cells derived from B-CLL patients, and was more toxic toward B- CLL cells than to normal mononuclear cells. The honokiol-induced apoptosis was characterized by the activation of caspase-3, -8, and -9 and cleavage of poly(adenosine diphosphate-ribose) polymerase (PARP). It was also reported that honokiol enhanced cytotoxicity induced by fludarabine, cladribine, or chlorambucil.
In September 2005, Ishitsuka et al reported that honokiol overcomes conventional drug resistance in human multiple myeloma by induction of caspase-dependent and – independent apoptosis (Blood, 1 September 2005, Vol. 106, No. 5, pp.1794-1800). HNK induced cytotoxicity in human multiple myeloma (MM) cell lines and tumor cells from patients with relapsed refractory MM through induction of apoptosis via both caspase- dependent and -independent pathways. HNK also enhanced MM cell cytotoxicity and apoptosis induced by bortezomib.
It is an object of the present invention to provide new compounds, compositions, methods and uses for the treatment of disorders associated with angiogenesis, cell proliferation, tumor growth, tumorogenesis, and myeloma.
the intermediates for the synthesis of honokiol are 3-allyl-4- hydroxybenzeneboronate 5 and 4-allyl-2-bromophenol 9. The boronate 5 can be prepared from 2-iodophenol 1 by bromination, followed by Suzuki coupling to introduce the allyl group, and boronation under Suzuki conditions. Compound 9 can be prepared from 4- iodophenol 6 by bromination and allylation (Suzuki coupling). The coupling of 5 and 9 under Suzuki conditions can yield honokiol from Suzuki coupling, not other allyl-oriented products from the Heck reaction, as it was shown that Suzuki coupling can succeed in the presence of C=C double bond (see Miyaura, N.; Suzuki, A. (1995), Chem. Rev. 95, 2457- 2483; and Suzuki, A. (1999), J. Organometal. Chem. 576, 147-168, and the references cited therein). Thus, honokiol and derivatives can be synthesized from commercially available starting materials in 6 steps (Scheme 1). Scheme 1
Treatment of honokiol with TMS-diazomethane in methanol results in mono- and di- methylated compounds I-III, and hydrogenation of honokiol with Wilkinson’s catalyst yields di- and tetrahydrohonokiols VI-VIII, as reported by Esumi, T. et al. (2004), Bioorg. Med. Chern. Lett. 14, 2621-2625. The amino and fluoro analogues (IV and V) can be constructed from iodoacetanilide under Suzuki coupling conditions. From 2-iodoacetanilide 10, after bromination, allylation, and boronation, the boronated intermediate 13 can be prepared. The other bromo intermediate 16 can be prepared from 4-iodoacetanilide 14 via bromination and allylation. The coupling of boronate 13 and bromide 16 under Suzuki conditions can afford, after deprotection, the compound IV. Diazotization followed by Schiemann reaction can convert the amino analogue TV to fluoro analogue V (Scheme 2).
The dimethoxy honokiol derivative, III, can also be prepared, for example, by the treatment of honokiol with potassium carbonate, iodomethane. (Scheme 2a). The hydrogenated honokiol analog can alternatively be prepared by the hydrogenation of honokiol with sodium borohydride and nickel(II) chloride to yields tetrahydrohonokiols VI- Vπi. (Scheme 2a). Scheme 2a
The preparation of the vinyl analogue IX is based on combining the Wittig reaction with Suzuki coupling. The intermediate aldehyde 18 can be prepared from 4-iodophenol 17 via the Reimer-Tiemann reaction, while 3-bromo-4-hydroxybenzenealdehyde 23 can be prepared from para-hydroxybenzoic ester 21 via bromination and reduction. The Wittig reaction of these two aldehydes can yield the corresponding vinyl substituted benzenes 19 and 24. Compound 19 can afford the boronate 20, which can be coupled with 24, to yield the compound IX (Scheme 3).
Reagents and conditions: (a) CHCl3, aq. NaOH, 70 °C; (b) Ph3PCH3Br1 n-BuLi, THF; (c) PdC!2(dppf), dppf, KOAc, dioxane, bis(pinacoato)diboron, 80 °C; (d) DIBALH, -70 °C; (e) PdCI2(dppf), dppf, K3PO4, dioxane, reflux.
For the synthesis of honokiol analogues with changed positions of the allyl or hydroxyl groups, the boronate 5, and the bromophenols 4 and 9 can be used as intermediates. Suzuki coupling of one of these intermediates with an appropriate halide or boronate can provide the compounds X-XVII. Compounds X-XII and XTV-XV can be prepared by Suzuki coupling of boronate 5 with an appropriate halide. Halide 25, needed for compound X, can be prepared from 2-bromo-6-iodophenol 2 via allylation, while the intermediate, 5-allyl-2- bromophenol 29 for compound XI, can be furnished from 3-iodophenol 26 via bromination and allylation. The preparation of halide 5-allyl-3-bromophenol 33, an intermediate for the synthesis of compound XIV, requires an organothallium reagent. The thallation of 3- bromophenol 30 followed by treatment with iodide can yield 3-bromo-5-iodophenol 32. After allylation, the allyl-substituted intermediate 33 can be prepared. The synthesis of compound XII can begin with 2-iodoacetanilide 10, via sulfonation, nitration, and reduction to obtain the intermediate 36. Aniline 36, after diazotization, followed by acid and base treatments, will afford 2-amino-3-iodophenol 37. Diazotization, Sandmeyer reaction, and allylation of compound 37 will yield halide 39. By a coupling reaction of these halides (25, 29, 33, and 39) with boronate 5, these compounds (X-XII, and XTV) can be prepared. Compound XV can be synthesized by Suzuki coupling of halide 4 with boronate 5 (Scheme 4).
Alternatively, compounds X, XV, and XVII can be synthesized by an allylation- Claisen pathway. Biphenol compounds can be reacted first with potassium carbonate and allyl bromide, followed by reaction with BCl3 to yield honokiol-like compounds, for example, X, XV, and XVII. (Scheme 4a). To a cooled solution (O0C with an ice bath) of diallyl starting material (1 eq.) in dry diehloromethane (Concentration of the solution : 0.1 mol.L“1) was added dropwise a solution Of BCl3 (IM in diehloromethane; 1.5 eq. = 0.75 eq for each allyl group). The reaction is then stirred at O0C until disappearance of the starting material on TLC (If after 15 minutes, the reaction is not complete, 1 more equivalent of BCl3 can be added). After hydrolysis with water (about same volume than diehloromethane), the two layers are separated. The organic layer is washed again with water, dried under MgSO4 then evaporated under vacuum. The residue is finally purified by column chromatography to give the di- hydroxy derivative . Scheme 4a
Bromide 9 is also a useful intermediate for coupling with some boronates. For example, Suzuki coupling of bromide 9 with boronate 42, which is prepared from 4-bromo-3- iodophenol 40 via allylation and boronation, can yield the compound XIII. Similarly, the coupling between bromide 9 and boronate 43 can afford the compound XVT. The compound XVII can be prepared from 4-allyl-2-bromophenol 9 via boronation followed by Suzuki coupling with 2-allyl-6-bromophenol 25 (Scheme 5).
The compounds XVIII and XIX can be synthesized from commercially available bisphenol 45 and the dihydroxynaphthalene-disulfuric acid salt 47. Thus, the bisphenol 45, through the Williamson reaction and Claisen rearrangement, can be converted to compound XVπi. Similarly, desulfonation of dihydroxynaphthalene-disulfuric acid salt .47, followed by the Williamson reaction and Claisen rearrangement, can produce the compound XIX (Scheme 6).Scheme 6
Dioxolane compounds can be prepared from magnoliol by reaction of magnoliol with 2,2′-dimethoxypropane and p-toluenesulfonic acid. (Scheme 7). This synthesis also provides a method of separating mixtures of honokiol and magnoliol. Scheme 7
The following examples are offered by way of illustration and not by way of limitation.