CYLODIMER OF DEHYDROSALICORTIN AND DERIVATIVES THEREOF ISOLATED FROM PLANT OF THE GENUS SALIX FOR USE IN CANCER THERAPY
20220105113 · 2022-04-07
Assignee
Inventors
- Michael Henry BEALE (Harpenden, Hertfordshire, GB)
- Jane Louise WARD (Hertfordshire, GB)
- Yanqi WU (Harpenden, Hertfordshire, GB)
- Martin MICHAELIS (Canterbury Kent, GB)
Cpc classification
A61K31/704
HUMAN NECESSITIES
C07H15/203
CHEMISTRY; METALLURGY
International classification
Abstract
Described herein are compounds comprising a dimer of dehydrosalicortin or a derivative, homologue, stereoisomer, prodrug or pharmaceutical salt thereof. In particular embodiments, the dimer is a result of a Diels-Alder reaction. Also described are compositions comprising the compounds and their use in treating disease.
Claims
1. (canceled)
2. A compound of Formula I, or a derivative, homologue, stereoisomer, prodrug or pharmaceutical salt thereof,
R.sup.1-L-R.sup.2 Formula I wherein L is a linking member and, R.sup.1 and R.sup.2 are each independently selected from Formula III, ##STR00029## wherein R.sup.3, R.sup.4, R.sup.5 and R.sup.6 are each independently selected from (i) H, (ii) acetyl, (iii) benzoyl, (iv) ortho- or para-coumaroyl, (v) cinnamoyl, wherein L is selected from (a) Formula IIA:— ##STR00030## wherein R.sup.12 and R.sup.13 are each independently selected from H and OH, or (b) Formula IIB:— ##STR00031## wherein R.sup.14 and R.sup.15 are each independently selected from H and OH.
3. The compound according to claim 2, wherein (a) R.sup.1 is Formula IIIA:— ##STR00032## wherein R.sup.3, R.sup.4, R.sup.5 and R.sup.6 are each independently selected from (i) H, (ii) acetyl, (iii) benzoyl, (iv) ortho- or para-coumaroyl, (v) cinnamoyl, or (b) R.sup.1 is Formula IIIB:— ##STR00033## wherein R.sup.3, R.sup.4, R.sup.5 and R.sup.6 are each independently selected from (i) H, (ii) acetyl, (iii) benzoyl, (iv) ortho- or para-coumaroyl, (v) cinnamoyl.
4. The compound according to claim 2, wherein (a) R.sup.2 is Formula IIIC:— ##STR00034## wherein R.sup.7, R.sup.8, R.sup.9 and R.sup.10 are each independently selected from (i) H, (ii) acetyl, (iii) benzoyl, (iv) ortho- or para-coumaroyl, (v) cinnamoyl, or (b) R.sup.2 is Formula IIID:— ##STR00035## wherein R.sup.7, R.sup.8, R.sup.9 and R.sup.10 are each independently selected from (i) H, (ii) acetyl, (iii) benzoyl, (iv) ortho- or para-coumaroyl, (v) cinnamoyl.
5. (canceled)
6. (canceled)
7. (canceled)
8. (canceled)
9. The compound according to claim 1 selected from (a) Formula VII or a derivative, homologue, stereoisomer, prodrug or pharmaceutical salt thereof, ##STR00036## wherein R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9 and R.sup.10 are each independently selected from (i) H, (ii) acetyl, (iii) benzoyl, (iv) ortho or para-coumaroyl, (v) cinnamoyl, and wherein R.sup.12 and R.sup.13 are each independently selected from H and OH, or (b) Formula VIII or a derivative, homologue, stereoisomer, prodrug or pharmaceutical salt thereof, ##STR00037## wherein R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9 and R.sup.10 are each independently selected from (i) H, (ii) acetyl, (iii) benzoyl, (iv) ortho- or para-coumaroyl, (v) cinnamoyl, and wherein R.sup.14 and R.sup.15 are each independently selected from H and OH.
10. The compound according to claim 9, wherein R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9 and R.sup.10 are each independently selected from (i) H, and (ii) acetyl.
11. (canceled)
12. (canceled)
13. The compound according to claim 1 of Formula X or XI, or a derivative, homologue, stereoisomer, prodrug or pharmaceutical salt thereof.
14. A composition comprising a compound according to claim 1.
15. The composition according to claim 14, wherein the composition comprises a compound of Formula VII or VIII, or a derivative, homologue, stereoisomer, prodrug or pharmaceutical salt thereof.
16. The composition according to claim 14, wherein the composition comprises a compound of Formula X or XI.
17. The composition according to claim 14, wherein the composition comprises a compound of Formula X, or a derivative, homologue, stereoisomer, prodrug or pharmaceutical salt thereof, ##STR00038##
18. (canceled)
19. (canceled)
20. (canceled)
21. A method of treating a disease, wherein the method comprises administering to a patient suffering from a disease a therapeutically effective amount of a composition according to claim 14.
22. The method of claim 21, wherein the disease is cancer.
23. The method according to claim 22, wherein the cancer is selected from neuroblastoma, breast cancer, oesophageal cancer, or ovarian cancer.
24. (canceled)
25. (canceled)
26. A method of claim 22, wherein the cancer is primary or secondary (metastatic) cancer.
27. A method of claim 22, wherein the cancer is a drug-resistant cancer.
Description
DETAILED DESCRIPTION
[0142] Example embodiments of the present invention will now be described with reference to the accompanying Figures, in which:—
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[0163] The present invention relates to novel compounds and their use in therapy, in particular for the treatment of cancer.
[0164] The compounds described herein were extracted from plants of the genus Salix, in particular Salix miyabeana or Salix dasyclados.
[0165] The genetic origin of Salix plants in general is unknown, although they are most abundant in cold and temperate regions of the Northern Hemisphere, including, for example Europe, Asia and North America.
[0166] In relation to Salix miyabeana referred to herein, this species is believed to be native to Japan and Korea.
[0167] In relation to Salix dasyclados referred to herein, this species is believed to be native to Siberia.
[0168] In relation to Salix gilgiana referred to herein, this species is believed to be native to Japan and Korea.
[0169] In relation to Salix gmelinii referred to herein, this species is believed to be native to Kazakhstan.
[0170] In relation to Salix repens referred to herein, this species is believed to be native to Austria, Baltic States, Belgium, Central European Russia, Czechoslovakia, Denmark, Finland, France, Germany, Great Britain, Ireland, Netherlands, North European Russia, Norway, Portugal, Spain, Sweden, Switzerland and Yugoslavia.
[0171] In relation to Salix capsica referred to herein, this species is believed to be native to Central Asia.
[0172] In relation to Salix adhenophylla referred to herein, this species is believed to be native to North America.
[0173] In relation to Salix rehderiana referred to herein, this species is believed to be native to China.
[0174] In relation to Salix rossica referred to herein, this species is believed to be native to Europe, Western Asia, and the Himalayas.
[0175] In relation to Salix glaucophyloides referred to herein, this species is believed to be native to North America.
[0176] Within this specification, the term “miyabeacin” means a compound of Formula X.
[0177] Within this specification, the term “miyabeacin B” means a compound of Formula XI.
[0178] Within this specification, the term “miyabeanol” means a compound of Formula XII.
[0179] Within this specification, the term “about” means plus or minus 20%, more preferably plus or minus 10%, even more preferably plus or minus 5%, most preferably plus or minus 2%.
[0180] As used herein, the term “therapeutically effective amount” means the amount of a composition which is required to reduce the severity of and/or ameliorate at least one condition or symptom which results from the disease in question.
[0181] Within this specification, the term “treatment” means treatment of an existing disease and/or prophylactic treatment in order to prevent incidence of a disease. As such, the methods and compositions of the invention can be used for the treatment, prevention, inhibition of progression or delay in the onset of disease.
[0182] Within this specification, reference to a “a compound as described herein” preferably means a compound of any of Formulas I to XX, or a derivative, homologue, stereoisomer, prodrug or pharmaceutical salt thereof.
[0183] Within this specification, reference to “a composition as described herein” means a composition comprising a compound as described herein. Preferably, the composition is a pharmaceutical composition.
[0184] Preferably, the composition comprises a therapeutically effective amount of at least one compound as described herein or a physiologically tolerated salt thereof.
[0185] Preferably, the composition comprises a physiologically tolerated carrier.
[0186] Within this specification, the term “prodrug” means to a compound that is biologically inactive, but is metabolized to produce an active therapeutic drug.
[0187] Within this specification, the term “derivative” means a molecule derived from the compounds described herein. Such derivatives may, for example, be synthetically altered derivatives of these compounds.
[0188] Within this specification, the term “homologue” refers to a molecule having substantial structural similarities to the compounds described herein.
[0189] Within this specification, the term “stereoisomer” means a molecule that has the same molecular formula and sequence of bonded atoms as another molecule, but which differs in the three-dimensional orientations of its atoms in space.
[0190] The compounds and compositions of the present invention can be formulated for clinical use into pharmaceutical formulations for administration by any suitable route. Examples include via oral, nasal, rectal, topical, sublingual, transdermal, intrathecal, transmucosal or parenteral (e.g. subcutaneous, intramuscular, intravenous and intradermal) administration.
[0191] Pharmaceutical formulations can be prepared by mixing the active substance, or a pharmaceutically acceptable salt thereof, with conventional pharmaceutically acceptable carriers, diluents or excipients. Examples of excipients include water, gelatin, gum arabicum, lactose, microcrystalline cellulose, starch, sodium starch glycolate, calcium hydrogen phosphate, magnesium stearate, talcum, colloidal silicon dioxide, and so on. Pharmaceutically acceptable carriers include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration, unless use thereof is incompatible with the active compound.
[0192] The formulations may also contain other pharmacologically active agents, and conventional additives, such as stabilizers, wetting agents, emulsifiers, flavouring agents, buffers, and the like.
[0193] The formulations may be prepared by conventional methods in dosage forms such as tablets, capsules, granules, powders, syrups, suspensions, suppositories or injections. Liquid formulations may be prepared by dissolving or suspending the active substance in water or other suitable vehicles. Tablets and granules may be coated in a conventional manner.
[0194] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
[0195] For oral administration, the compositions can be in the form of soft gelatin capsules or tablets and will usually include an inert diluent or an edible carrier. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavouring agent such as peppermint, methyl salicylate, or orange flavouring.
[0196] Formulations intended for inhalation can be provided as an aerosol spray, for example in a pressurised container or dispenser which contains a suitable propellant.
[0197] Transmucosal or transdermal delivery means can be used for systemic administration. Penetrants appropriate to the barrier in question can be used and are well known in the art. Examples include detergents, bile salts, and fusidic acid derivatives. Nasal sprays and suppositories can be used for transmucosal delivery. Creams, ointments, salves and gels can be used for transdermal delivery. In the case of rectal delivery, the formulations can also be provided as retention enemas.
[0198] Formulations intended for targeted delivery of the compositions and compounds described herein can also be provided, for example using targeting agents such as antibodies, antibody fragments, receptor binding agents, nanoparticles, nanocarriers or combinations thereof. In this respect, it is known that cancer cells exhibit cancer specific markers which means that agents specific for these markers can be used to direct the compounds and compositions described herein to cancer cells and tissues in a selective manner.
[0199] In one example, the compounds described herein can be bound to antibodies or fragments thereof specific for one or more cancer cell specific markers or conjugated to nanoparticles coupled to a targeting ligand specific for one or more cancer cell specific markers. Examples of nanoparticles include lipid cationic nanoparticles, gold nanoparticles, silica nanoparticles, PEGylated nanoparticles and amphiphilic polymeric nanoparticles. The compositions can comprise nanoparticles with multiple functional ligands which can include, for example, diagnostic and/or other therapeutic agents in addition to the compounds described herein.
[0200] Nanocarriers, such as liposomes and micelles, conjugated to targeting molecules, such as ligands, antibodies or antibody fragments, can be used to deliver unmodified compounds and compositions described herein to cancer cells and tissues.
[0201] The compounds and compositions may also be provided in formulations which prevent rapid elimination from the body. Examples include known modified release formulations such as implants and microencapsulated delivery systems.
[0202] Pharmaceutical compositions containing the appropriately formulated compound can be included in a container, pack, or dispenser together with instructions for administration.
[0203] The appropriate dosage form for the formulation will depend upon the intended route of administration, the required quantity of drug to be delivered and the potential toxicity of the compound. This can be determined in accordance with standard procedures known in the art.
[0204] For example, toxicity and therapeutic efficacy of compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals and evaluated by considering the LD50 (the dose lethal to 50% of the population), the ED50 (the dose therapeutically effective in 50% of the population) and the resultant therapeutic index (LD50/ED50). Appropriate dosage forms may also depend upon the potential side effects of particular routes of delivery and the amount of active compound required to effect sufficient delivery to the intended site of therapeutic need.
EXAMPLES
Isolation and Characterisation of Dimeric Compounds Miyabeacin, Miyabeacin B and Miyabeanol.
[0205] Freeze dried juvenile leaves of Salix miyabeana were used as the starting material for the initial isolation of Miyabeacin (
Miyabeacin
[0206] For the initial isolation of Miyabeacin, 1 mL of water:methanol (80:20) was added to Salix miyabeana leaf tissue (50 mg). The suspension was agitated for 5 minutes at room temperature and then heated to 50° C. for 10 minutes using a water bath. The resultant solution was centrifuged at 13,000 rpm for 5 minutes. 800 μL of the supernatant, removed to a clean tube, was heated at 90° C. for 2 minutes. The solution was cooled (5° C.) for 30 minutes and centrifuged at 13,000 rpm for 5 minutes. The supernatant, containing the target compound was subjected to purification using reversed-phase HPLC. 6 repeated injections of 100 μL each were made into an analytical HPLC using an Agilent 1100 HPLC system equipped with a quaternary pump, diode array detector, column oven and auto sampler. Peaks were separated using an Ascentis C18 column (Sum, 5×250 mm (Supelco, UK). The operating solvents were: Solvent A: H2O with 0.1% formic acid, solvent B: acetonitrile with 0.1% formic acid. The operating gradient for peak isolation was from 5% B (0-10 min), 22% B (10-50 min) to 37% B (60-70 min) at a constant flow of 1 mL/min and using a total chromatographic run of 72 min. Peaks were identified and monitored using a wavelength of 254 nm and were collected manually into glass tubes. Miyabeacin eluted at 57.93 min (
TABLE-US-00002 TABLE 2 Extraction and HPLC gradient conditions for the isolation of dimeric metabolites. HPLC Gradient [mobile phases water (A) and acetonitrile (B), Number both Extraction of 100 μL containing HPLC Volume Injections 0.1% Retention Amount (solvent: made into formic time of Amount Compound Extracted Tissue H.sub.2O:MeOH) HPLC acid.] Peak Isolated X 50 mg Salix 1 mL 6 5% B 57.93 1.68 miyabeana (10-0 min), min mg Seemen. III 22% B leaf tissue. (10-50 Line: min) to 37% NWC837 B (60- 70 min). XI 200 mg Salix 2.5 mL >10 5% B 52.11 0.67 miyabeana (0-10 min), min mg Seemen. 29% B “Purpurescens” (10-60 stem tissue. min) to 29% Line: NWC941 B (60- 70 min) XII 150 mg Salix 2 mL 8 5% B 44.87 1.05 miyabeana (0-10 min), min mg Seemen. III 22% B leaf tissue. (10-50 Line: min) to 37% NWC837 B (60- 70 min) XIX 450 mg Salix 4.5 mL 44 20% B 20.9 0.9 miyabeana (0-20 min), min mg Seemen. 40% B “Purpurescens” (20-25 leaf tissue. min) to 50% Line: NWC941 B (25- 35 min) XIII & 150 mg RRes 710-27, 2.4 mL (2 10 20% B 41.4 0.75 XIV (2 × 75 RR09102 1.2 mL) (0 min), 40% mg mg) hybrid B (0-45 [NWC607 S. min) to rehderiana × 100% B RR05337 (45.0-50 (Aud × S. min) rossica)] leaf tissue XV 150 mg RRes 710-27, 2.4 mL (2 10 20% B 45.5 0.25 (2 × 75 RR09102 1.2 mL) (0 min), 40% mg mg) hybrid B (0-45 [NWC607 S. min) to rehderiana × 100% B RR05337 (45.0-50 (Aud × S. min) rossica)] leaf tissue
[0207] A further example, extending the range of substituted dimeric compounds was seen in the LC-MS analysis of a willow breeding line (RR10147) developed as part of a biomass improvement programme at Rothamsted Research. This hybrid line included S. dasyclados (NWC577) in both parents [RR07187 (944 S. glaucophyloides×577 “77056”)×RR07188 (944 S. glaucophyloides×577 “77056”)] as well as S. glaucophyloides (NWC 944). In the Total Ion Chromatogram of the negative ion mode LC-MS data salicortin, 2′-O-acetylsalicortin and tremulacin appeared as major peaks. Given that this cross has generated a hybrid capable of producing both acetylated and benzoylated salicinoids alongside salicortin it followed that associated dimeric analogues would also be expected to be formed via a matrix of cross-over reactions involving the three corresponding dienones. This was indeed the case with miyabeacin appearing at 25.03 min, 2′/2″-O-acetylmiyabeacin (Formula XIII/XIV) appearing at 26.90 min and 2′/2″-O-benzoylmiyabeacin (Formula XVI/XVII) appearing at 30.95 min. A further intriguing peak was observed at 32.48 min which showed an ion at m z 989.2617, corresponding to a formula of C.sub.49H.sub.49O.sub.22. Although there was insufficient for isolation, the MS was suggestive of the predicted miyabeacin analogue bearing both an acetyl and benzoyl substitution.
[0208] The structure of Miyabeacin was determined by various forms of spectroscopy. Table 3 shows the general measurement conditions for spectroscopic analyses.
TABLE-US-00003 TABLE 3 General Conditions and parameters for spectroscopic measurements. Measurement Conditions High resolution LC-MS LC apparatus Ultimate 3000 RS uHPLC (Thermo) Chromatography Column C.sub.18 Hypersil gold column (1.9 μm, 30 × 2.1 mm i.d.) Column Temperature 35° C. Solvents Water/0.1% formic acid (A) and acetonitrile/0.1% formic acid (B) Solvent Gradient 0 min, 0% B; 27 min, 70% B; 28 min, 100% B. Flow rate 0.3 mL/min Run time 30 min Injection volume 10 μL MS Apparatus LTQ-Orbitrap Elite (Thermo) Source Heated ESI source Ionisation mode Negative Resolution 120,000 Capillary temperature 350° C. Source heater temperature 350° C. Source voltage 2500 V Source current 100 uA Sheath gas flow 35 Auxiliary gas 10 R.F. Lens 50% Scan range m/z 50-1500 MS-MS fragmentation Automatic on top 3 ions Ion isolation width for MSMS m/z 2 Fragmentation mode HCD Normalised collision energy 65 Activation time 0.1 ms NMR Apparatus Avance 600 (Bruker) Observation Frequency .sup.1H: 600.05, .sup.13C: 150.9 Solvent D.sub.2O:CD.sub.3OD (80:20) Concentration 0.6 mg/mL Internal Standard d.sub.4-TSP Temperature 300 K Probe 5 mm Selective Inverse .sup.1H NMR Measurement Pulse sequence zgpr Sweep width 7183 Hz Spectrum offset 2879.40 Hz Data points 32,768 Pulse angle 90° Delay 5 s Number of scans 64 2D COSY 45 Measurement Pulse program cosyqf45 Observation width 2973, 2973 Hz Data points 1024, 1024 Temperature 300 K Number of transients 32 2D HSQC Measurement Pulse program hsqcetgpsi2 Observation width 7180, 30150 Hz Data points 2048, 1024 Temperature 300 K Number of transients 128 2D HMBC Measurement Pulse program hmbcgpndqf Observation width 7182, 33165 Hz Data points 4096, 256 Temperature 300 K Number of transients 256 .sup.13C NMR Apparatus Avance 400 (Bruker) Observation Frequency .sup.13C: 100.61 Solvent D.sub.2O:CD.sub.3OD (80:20) Concentration 0.6 mg/mL Internal Standard d.sub.4-TSP Temperature 300 K Probe 5 mm Broadband BBO .sup.13C NMR Measurement Pulse sequence dept135 Sweep width 23,980 Hz Spectrum offset 10363 Hz Data points 32768 Pulse angle 30° Delay 0.7 s Number of scans 46,191 DEPT Measurement Observation width 23980 Hz Data points 65536 Pulse repetition time 2 Number of scans 4096 Abbreviations DEPT: Distortionless Enhancement by Polarization Transfer (A method for determining a carbon type (distinguishing among CH3, CH2, CH, and C)) COSY: Correlation SpectroscopY (A method of .sup.1H-.sup.1H COSY) HSQC: Heteronuclear Single Quantum Coherence (A method of .sup.1H-.sup.13C COSY) HMBC: Heteronuclear Multiple Bond Correlation (A method of long-range .sup.1H-.sup.13C COSY)
Miyabeacin Spectroscopic Analyses
[0209] High Resolution LC-MS: LC-MS was carried out in negative ion mode using a C18 column. Conditions of analysis are outlined in Table 3.
[0210] NMR spectroscopy: .sup.1H-NMR data of miyabeacin was collected at 600 MHz in aqueous d.sub.4-methanol containing 0.01% w/v d.sub.4-TSP as internal standard. The spectrum showed peaks relating to 34 coupled protons (
TABLE-US-00004 TABLE 4 .sup.1H-NMR assignments for miyabeacin. Data collected at 600 MHz in D.sub.2O:CD.sub.3OD (4:1), referenced to d.sub.4-TSP (0.01% w/v). Position 1 — 2 7.19 (d, 8.3) 3 7.41 (ddd, 8.0, 7.5, 2.0) 4 7.12 (t, 7.5)/7.11 (t, 7.5) 5 7.32 (dd, 7.6, 1.5)/7.34 (dd, 7.6.1.5) 6 — .sup. 7α 5.40 (d, 11.9) 7β 5.19 (d, 11.9) 8 — 9 — 10 3.59-3.63 (m) 11 3.58-3.55 (m) 12 6.59 (dd, 10.2, 4.1) 13 6.02 (dd, 10.2, 1.5) 14 — 15 3.50-3.53 (m) 16 6.19 (t, 7.9) 17 5.91 (ddd, 7.9, 6.5, 1.4) 18 3.43 (m) 19 — 20 — 21 — 22β 5.38 (d, 12.1) .sup. 22α 5.16 (d, 12.1) 23 — 24 7.32 (dd, 7.6, 1.5)/7.34 (dd, 7.6, 1.5) 25 7.12 (t, 7.5)/7.11 (t, 7.5) 26 7.41 (ddd, 8.0, 7.5, 2.0) 27 7.20 (d, 8.3) 28 — .sup. 1′ 5.09 (d, 7.5)/5.07 (d, 7.8) .sup. 2′ 3.55-3.63 (m) .sup. 3′ 3.56-3.62 (m) .sup. 4′ 3.47-3.52 (m) .sup. 5′ 3.56-3.62 (m) 6′β 3.77 (dd, 12.4, 6.0)/3.73 (dd, 12.4, 6.0) .sup. 6′α 3.94 (dd, 12.4, 2.1)/3.92 (dd, 12.4, 2-1) 1″ 5.09 (d, 7.5)/5.07 (d, 7.8) 2″ 3.55-3.63 (m) 3″ 3.56-3.62 (m) 4″ 3.47-3.52 (m) 5″ 3.56-3.62 (m) 6″β 3.77 (dd, 12.4, 6.0)/3.73 (dd, 12.4, 6.0) 6″α 3.94 (dd, 12.4, 2.l)/3.92 (dd, 12.4, 2.1)
[0211] Four signals were observed between δ 7.34-7.10 and were consistent with those obtained in salicyl containing compounds. Integration of these aromatic peaks corresponded to 8 protons suggestive of two such salicyl rings. This was confirmed by the presence of two pairs of J=12 Hz doublet signals relating to the distinctive salicyl hydroxymethylene group (pair 1: δ5.40 and δ5.19; pair 2: δ5.38 and δ5.16). Similarly, the molecule contained two separate glucoside moieties with characteristic doublet signals relating to the H-1′ anomeric protons being duplicated (δ5.09 and δ5.07) as were those corresponding to the glucosyl 6′-methylenes. Four separate olefin signals were present between δ6.60 and 5.85 each integrating for one proton, two appearing as double doublets and the others as simple triplets. .sup.1H-.sup.1H COSY analysis (
TABLE-US-00005 TABLE 5 .sup.13C-NMR assignment for mivabeacin. Data collected at 100.61 MHz in D.sub.2O:CD.sub.3OD (4:1), referenced to d.sub.4-TSP (0.01% w/v). Position 1 158.0 2 117.9 3 133.5/133.4 4 125.6/125.7 5 133.7/133.6 6 126.9 .sup. 7α 67.3 7β 67.3 8 173.6 9 82.2 10 40.3 11 43.5 12 152.5 13 130.9 14 198.6 15 45.1 16 135.5 17 132.8 18 54.2 19 210.0 20 80.0 21 173.2 22β 66.7 .sup. 22α 66.7 23 126.6 24 133.7/133.6 25 125.6/125.7 26 133.5/133.4 27 117.7 28 157.7 .sup. 1′ 103.0/102.9 .sup. 2′ 76.0 .sup. 3′ 79.1 .sup. 4′ 72.5/72.4 .sup. 5′ 78.7/78.8 6′β 63.7 .sup. 6′α 63.7 1″ 103.0/102.9 2″ 76.0 3″ 79.1 4″ 72.5/72.4 5″ 78.7/78.8 6″β 63.7 6″α 63.7
[0212] Given the molecular formula from accurate mass, the similarity in fragmentation pattern of the smaller m/z 421 fragment to that of the known molecule salicortin, and the duplication of benzyl and glycosyl related NMR signals we postulated that miyabeacin was an unsymmetrical dimeric structure formed via conjugation of two molecules of a dehydro analogue of salicortin. The structure has a tricyclododecadiene core. Key correlations in the .sup.1H-.sup.13C HMBC were observed around all the positions of the dimeric core structure (
Miyabeacin B
[0213] For the initial isolation of Miyabeacin B, 2.5 mL of water:methanol (80:20) was added to Salix miyabeana stem tissue (200 mg). The suspension was agitated for 5 minutes at room temperature and then heated to 50° C. for 10 minutes using a water bath. The resultant solution was centrifuged at 13,000 rpm for 5 minutes. 800 μL of the supernatant, removed to a clean tube, was heated at 90° C. for 2 minutes. The solution was cooled (5° C.) for 30 minutes and centrifuged at 13,000 rpm for 5 minutes. The supernatant, containing the target compound was subjected to purification using reversed-phase HPLC. Injections of 100 μL each were made into an analytical HPLC using an Agilent 1100 HPLC system equipped with a quaternary pump, diode array detector, column oven and auto sampler. Peaks were separated using an Ascentis C18 column (5 um, 5×250 mm (Supelco, UK). The operating solvents were: Solvent A: H.sub.2O with 0.1% formic acid, solvent B: acetonitrile with 0.1% formic acid. The operating gradient for peak isolation was from 5% B (0-10 min), 29% B (10-60 min) to 29% B (60-70 min) at a constant flow of 1 mL/min and using a total chromatographic run of 70 min. Peaks were identified and monitored using a wavelength of 254 nm and were collected manually into glass tubes. Miyabeacin B eluted at 52.11 min (
Miyabeacin B Spectroscopic Analyses
[0214] High Resolution LC-MS: LC-MS was carried out in negative ion mode using a C18 column. Conditions of analysis are outlined in Table 3.
[0215] NMR spectroscopy: 1H-NMR spectroscopy in aqueous methanol showed a total of 17 signals which related to 34 separate protons (Table 6).
TABLE-US-00006 TABLE 6 .sup.1H-NMR assignments for miyabeacin B. Data collected at 600 MHz in D.sub.2O:CD.sub.3OD (4:1), referenced to d.sub.4-TSP (0.01% w/v). Position 1 — 2 7.20 (d, 8.2) 3 7.43 (ddd, 8.5, 7.5, 1.5) 4 7.12 (ddd, 7.5, 7.4, 0.9) 5 7.35 (dd, 7.5, 1.5) 6 — .sup. 7α 5.46 (d, 11.7) 7β 5.13 (d, 11.7) 8 — 9 — 10 2.76 (dd, 4.4, 2.1) 11 2.99 (m) 12 2.88 (m) 13 3.12 (dd, 7.6, 4.0) 14 — 15 2.76 (dd, 4.4, 2.1) 16 2.99 (m) 17 2.88 (m) 18 3.12 (dd, 7.6, 4.0) 19 — 20 — 21 — 22β 5.46 (d, 11.7) .sup. 22α 5.13 (d, 11.7) 23 24 7.35 (dd, 7.5, 1.5) 25 7.12 (ddd, 7.5, 7.4, 0.9) 26 7.43 (ddd, 8.5, 7.5, 1.5) 27 7.20 (d, 8.2) 28 .sup. 1′ 5.07 (d, 7.8) .sup. 2′ 3.51 (dd, 9.4, 7.8) .sup. 3′ 3.58 (m) .sup. 4′ 3.45 (t, 9.4) .sup. 5′ 3.58 (m) 6′β 3.72 (dd, 12.4, 6.0) .sup. 6′α 3.99 (dd, 12.5, 2.2) 1″ 5.07 (d, 7.8) 2″ 3.51 (dd, 9.4, 7.8) 3″ 3.58 (m) 4″ 3.45 (t, 9.4) 5″ 3.58 (m) 6″β 3.72 (dd, 12.4, 6.0) 6″α 3.99 (dd, 12.5, 2.2)
[0216] The presence of signals relating to benzyl and glucosyl moieties compared well with those observed in the .sup.1H-NMR spectrum of miyabeacin. Absence of the four olefin signals (δ5.91 to 6.59) previously observed in miyabeacin was accompanied by a movement upfield of the four bridgehead protons (δ3.43-3.63) to give a set of four signals at 62.76, 2.88, 2.99 and 3.12 each integrating for 2 protons. The .sup.1H-NMR data suggested a further [2+2] intramolecular cyclization of the olefin units in miyabeacin to give a “caged” structure which we have named miyabeacin B. The cycloaddition of the double bonds in now confers a 2-fold axis of symmetry resulting in a significant simplification of the .sup.1H-NMR spectrum for miyabeacin B relative to that observed for miyabeacin. [.sup.1H-.sup.1H] correlation spectroscopy confirmed the linkages around the tricyclic core of the molecule. 13C data for miyabeacin B is given in Table 7.
TABLE-US-00007 TABLE 7 .sup.13C-NMR assignment for miyabeacin B. Data collected at 100.61 MHz in D.sub.2O:CD.sub.3OD (4:1), referenced to d.sub.4-TSP (0.01% w/v). Position 1 158.1 2 117.9 3 133.9 4 125.9 5 134.3 6 126.4 .sup. 7α 67.1 7β 67.1 8 174.0 9 80.6 10 43.1 11 41.0 12 37.7 13 48.1 14 210.6 15 43.1 16 41.0 17 37.7 18 48.1 19 210.6 20 80.6 21 174.0 22β 67.1 .sup. 22α 67.1 23 126.4 24 134.3 25 125.9 26 133.9 27 117.9 28 158.1 .sup. 1′ 102.9 .sup. 2′ 76.0 .sup. 3′ 79.0 .sup. 4′ 72.8 .sup. 5′ 79.0 6′β 63.9 .sup. 6′α 63.9 1″ 102.9 2″ 76.0 3″ 79.0 4″ 72.8 5″ 79.0 6″β 63.9 6″α 63.9
Miyabeanol
[0217] For the initial isolation of Miyabeanol, 2 mL of water:methanol (80:20) was added to Salix miyabeana leaf tissue (150 mg). The suspension was agitated for 5 minutes at room temperature and then heated to 50° C. for 10 minutes using a water bath. The resultant solution was centrifuged at 13,000 rpm for 5 minutes. 800 μL of the supernatant, removed to a clean tube, was heated at 90° C. for 2 minutes. The solution was cooled (5° C.) for 30 minutes and centrifuged at 13,000 rpm for 5 minutes. The supernatant, containing the target compound was subjected to purification using reversed-phase HPLC. 8 repeated injections of 100 μL each were made into an analytical HPLC using an Agilent 1100 HPLC system equipped with a quaternary pump, diode array detector, column oven and auto sampler. Peaks were separated using an Ascentis C18 column (Sum, 5×250 mm (Supelco, UK). The operating solvents were: Solvent A: H.sub.2O with 0.1% formic acid, solvent B: acetonitrile with 0.1% formic acid. The operating gradient for peak isolation was from 5% B (0-10 min), 22% B (10-50 min) to 37% B (60-70 min) at a constant flow of 1 mL/min and using a total chromatographic run of 72 min. Peaks were identified and monitored using a wavelength of 254 nm and were collected manually into glass tubes. Miyabeanol eluted at 44.87 min (
Miyabeanol Spectroscopic Analyses
[0218] High Resolution LC-MS: LC-MS was carried out in negative ion mode using a C18 column. Conditions of analysis are outlined in Table 3.
[0219] NMR spectroscopy: The .sup.1H NMR spectrum of miyabeanol (
TABLE-US-00008 TABLE 8 .sup.1H-NMR assignments for miyabeanol. Data collected at 600 MHz in D.sub.2O:CD.sub.3OD (4:1), referenced to d.sub.4-TSP (0.01% w/v). Position 1 2 7.19 (d, 8.0) 3 7.40 (m) 4 7.12 (td, 7.5, 0.9) 5 7.31 (dd, 7.6, 1.5) 6 — .sup. 7α 5.39 (d, 11.9) 7β 5.18 (d, 11.9) 8 — 9 — 10 3.57-3.61 (m) 11 3.48-3.53 (m) 12 6.63 (dd, 10.2, 4.1) 13 6.02 (dd, 10.1, 1.7) 14 — 15 3.28-3.33 (m) 16 6.27 ( ddd, 7.9, 6.9, 17 5.94 (1H, ddd, 7.9, 18 3.36 (1H, ddd, 6.0, 19 — 20 — 21 — 22β — .sup. 22α 23 24 25 26 27 28 .sup. 1′ 5.06 (d, 7.3) .sup. 2′ 3.49-3.59 (m) .sup. 3′ 3.54-3.61 (m) .sup. 4′ 3.45-3.53 (m) .sup. 5′ 3.54-3.61 (m) 6′β 3.76 (dd, 12.5, 5.9) .sup. 6′α 3.92 (dd, 12.4, 2.2) 1″ 2″ 3″ 4″ 5″ 6″β 6″α
[0220] .sup.1H signals at δ 6.63 and δ 6.02 corresponded to those observed in miyabeacin and related to the enone protons, H-12 and H-13. Signals corresponding to the isolated olefin protons at δ 6.27 and δ 5.94 were also present. This data and additional .sup.1H-.sup.1H COSY correlations (
TABLE-US-00009 TABLE 9 .sup.13C-NMR assignments for miyabeanol. Data collected at 100.61 MHz in D.sub.2O:CD.sub.3OD (4:1), referenced to d.sub.4-TSP (0.01% w/v). [7] [7] Position (D2O:CD.sub.3OD) (D2O) 1 158.0 157.8 2 117.7 117.3 3 133.7 133.2 4 125.7 125.3 5 133.7 133.2 6 126.6 126.6 .sup. 7α 67.2 67.0 7β 67.2 67.0 8 173.7 173.6 9 82.5 82.4 10 40.6 40.4 11 43.9 43.7 12 152.8 152.7 13 130.8 130.6 14 199.0 199.1 15 45.7 45.5 16 136.0 135.6 17 132.2 132.2 18 54.6 54.5 19 213.3 213.4 20 missing 81.4 21 — — 22β — — .sup. 22α — — 23 — — 24 — — 25 — — 26 — — 27 — — 28 — — .sup. 1′ 103.0 102.7 .sup. 2′ 76.0 75.8 .sup. 3′ 79.1 78.7 .sup. 4′ 72.4 72.2 .sup. 5′ 78.8 78.5 6′β 63.6 63.5 .sup. 6′α 63.6 63.5 1″ — — 2″ — — 3″ — — 4″ — — 5″ — — 6″β — — 6″α — —
[0221] The position of side-chain loss and decarboxylation was confirmed via extensive analysis of COSY, HSQC and HMBC correlation spectroscopy. Key .sup.1H-.sup.13C correlations were identified between H-10 and C-8, H-10 to C-14 and H-13 to C-9. This allowed placement of the carboxy-salicylglycoside moiety at C-9. Correlations from H-15 and H-18 to the carbonyl at C-19 and from H-16, H-18 and H-10 to C-20 (δ81.4) were all present.
Bioassay Data of Miyabeacin
[0222] The activity of miyabeacin was tested against a range of cancer cell lines including those in neuroblastoma and breast, oesophageal and ovarian cancers (Table 10).
TABLE-US-00010 TABLE 10 Bioactivity data of miyabeacin in six cancer cell lines Miyabeacin IC50 cancer type cell line (μg/mL) breast BT-474 27.04 oesophageal COLO-680N 5.08 ovarian COLO-704 20.18 ovarian EFO-21 12.69 breast MCF-7 2.19 neuroblastoma UKF-NB-3 7.12
[0223] The MYCN-amplified neuroblastoma cell line UKF-NB-3 was established from a stage 4 neuroblastoma patient (Kotchetkov et al., 2005). Also tested was the vincristine-resistant UKF-NB-3 sub-line UKF-NB-3.sup.rVCR (Rothwell et al., 2010) (adapted to grow in the presence of vincristine 10 ng/mL). At a concentration of 20 μg/mL of miyabeacin, the cell viability, relative to non-treated cells, after 120 hours was 0% for UKF-NB-3 and 4.22±2.89% for the vincristine resistant UKF-NB-3.sup.rVCR line. The oesophageal cancer cell line COLO-680N was obtained from ATCC (Manassas, Va., USA) and the ovarian cancer cell line COLO-704 from DSMZ (Braunschweig, Germany). All cell lines were propagated in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% FCS, 100 IU/ml penicillin and 100 mg/ml streptomycin at 37° C. Cells were routinely tested for mycoplasma contamination and authenticated by short tandem repeat profiling. Cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye reduction assay after 120 h incubation as described previously (Michaelis et al., 2015). Briefly, 5000 cells (suspended in 100 μL IMDM supplemented with 10% FCS, 100 IU/ml penicillin and 100 mg/ml streptomycin) were incubated in 96-well plates at 37° C. and 5% CO.sub.2 in the absence or presence of varying compound concentrations for 120 h. Then, 25 μL of MTT solution (2 μg/mL dissolved in PBS) were added for 4 h. This was followed by the addition of 100 Lp of 20% sodium dodecyl sulphate (50:50 purified water/DMF) solution adjusted to pH 4.7 for an additional 4 h in order to lyse cells and dissolve formazan precipitates. Plates were then read at 600 nm. The relative viability was determined as the relative reduction of the optical density relative to an untreated cell control (=100%). Replicated IC50 values for miyabeacin activity were determined on three selected lines (UKF-NB-3, COLO-680N and COLO-704) and ranged from 17.15 μM to 40.18 μM (Table 11).
TABLE-US-00011 TABLE 11 Replicated IC.sub.50 determination in three cancer cell lines miyabeacin IC50 concentration IC50 (μg/mL) (μM) cancer type cell line expt 1 expt 2 expt 3 mean S.D. mean oesophageal COLO- 5.08 15.08 51.46 23.87 19.93 28.28 cancer 680N ovarian cancer COLO-704 20.18 32.38 50.79 34.45 12.58 40.18 neuroblastoma UKF-NB-3 7.12 20.97 15.33 14.47 5.69 17.15
[0224] Whilst the results are important for all of the cells lines shown above, of particular note is the activity against neuroblastoma cell lines. Overall survival rates are below 50% and it represents the most frequent extracranial solid childhood tumour. With resistance acquisition being a significant issue in neuroblastoma, new compounds effective against neuroblastoma are in great need.
[0225] It should be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present invention and without diminishing its attendant advantages. It is therefore intended that such changes and modifications are covered by the appended claims.
REFERENCES
[0226] Kotchetkov R, Driever P H, Cinatl J, Michaelis M, Karaskova J, Blaheta R, Squire J A, Von Deimling A, Moog J, Cinatl J Jr. Increased malignant behavior in neuroblastoma cells with acquired multi-drug resistance does not depend on P-gp expression. Int J Oncol. 2005 October; 27(4):1029-37. [0227] Michaelis M, Rothweiler F, Barth S, Cinatl J, van Rikxoort M, Loschmann N, Voges Y, Breitling R, von Deimling A, Rödel F, Weber K, Fehse B, Mack E, Stiewe T, Doerr H W, Speidel D, Cinatl J Jr. Adaptation of cancer cells from different entities to the MDM2 inhibitor nutlin-3 results in the emergence of p53-mutated multi-drug-resistant cancer cells. Cell Death Dis. 2011 Dec. 15; 2:e243. [0228] Michaelis M, Agha B, Rothweiler F, Loschmann N, Voges Y, Mittelbronn M, Starzetz T, Harter P N, Abhari B A, Fulda S, Westermann F, Riecken K, Spek S, Langer K, Wiese M, Dirks W G, Zehner R, Cinatl J, Wass M N, Cinatl J Jr. Identification of flubendazole as potential anti-neuroblastoma compound in a large cell line screen. Sci Rep. 2015a Feb. 3; 5:8202. [0229] Rothwell, P. M., et al. Long-term effect of aspirin on colorectal cancer incidence and mortality: 20-year follow-up of five randomised trials. Lancet 376:1741-50 (2010).
[0230] The content of all references cited herein are incorporated herein by reference in their entirety.