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Title:
CYLODIMER OF DEHYDROSALICORTIN AND DERIVATIVES THEREOF ISOLATED FROM PLANT OF THE GENUS SALIX FOR USE IN CANCER THERAPY
Document Type and Number:
WIPO Patent Application WO/2020/157494
Kind Code:
A1
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.

Inventors:
BEALE MICHAEL HENRY (GB)
WARD JANE LOUISE (GB)
WU YANQI (GB)
MICHAELIS MARTIN (GB)
Application Number:
PCT/GB2020/050203
Publication Date:
August 06, 2020
Filing Date:
January 29, 2020
Export Citation:
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Assignee:
ROTHAMSTED RES LTD (GB)
UNIV OF KENT (GB)
International Classes:
C07H15/203; A61K31/7034; A61P35/00
Foreign References:
EP1374882A12004-01-02
Other References:
BONATERRA G A ET AL: "In vitro anti-proliferative effects of the willow bark extract STW 33-I", ARZNEIMITTEL FORSCHUNG. DRUG RESEARCH, ECV EDITIO CANTOR VERLAG, AULENDORF, DE, vol. 60, no. 6, 1 January 2010 (2010-01-01), pages 330 - 335, XP001526240, ISSN: 0004-4172, DOI: 10.1055/S-0031-1296296
GAGNEPAIN J ET AL: "Regio- and stereoselectivities in Diels-Alder cyclodimerizations of orthoquinonoid cyclohexa-2,4-dienones", TETRAHEDRON, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 63, no. 28, 9 July 2007 (2007-07-09), pages 6493 - 6505, XP025320916, ISSN: 0040-4020, [retrieved on 20070530], DOI: 10.1016/J.TET.2007.03.035
FELIX FEISTEL ET AL: "Idesia polycarpa (Salicaceae) leaf constituents and their toxic effect on Cerura vinula and Lymantria dispar (Lepidoptera) larvae", PHYTOCHEMISTRY, vol. 143, 1 November 2017 (2017-11-01), AMSTERDAM, NL, pages 170 - 179, XP055690997, ISSN: 0031-9422, DOI: 10.1016/j.phytochem.2017.08.008
JANE L. WARD ET AL: "Miyabeacin: A new cyclodimer presents a potential role for willow in cancer therapy", SCIENTIFIC REPORTS, vol. 10, no. 1, 15 April 2020 (2020-04-15), XP055690986, DOI: 10.1038/s41598-020-63349-1
KOTCHETKOV RDRIEVER PHCINATL JMICHAELIS MKARASKOVA JBLAHETA RSQUIRE JAVON DEIMLING AMOOG JCINATL J JR: "Increased malignant behavior in neuroblastoma cells with acquired multi-drug resistance does not depend on P-gp expression", INT J ONCOL., vol. 27, no. 4, October 2005 (2005-10-01), pages 1029 - 37
MICHAELIS MROTHWEILER FBARTH SCINATL JVAN RIKXOORT MLOSCHMANN NVOGES YBREITLING RVON DEIMLING ARODEL F: "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., vol. 2, 15 December 2011 (2011-12-15), pages e243
MICHAELIS MAGHA BROTHWEILER FLOSCHMANN NVOGES YMITTELBRONN MSTARZETZ THARTER PNABHARI BAFULDA S: "Identification of flubendazole as potential anti-neuroblastoma compound in a large cell line screen", SCI REP., vol. 3, no. 5, February 2015 (2015-02-01), pages 8202
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, vol. 376, 2010, pages 1741 - 50, XP027597942, DOI: 10.1016/S0140-6736(10)61543-7
Attorney, Agent or Firm:
SWEETINBURGH, Mark Roger et al. (GB)
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Claims:
CLAIMS

1. A compound comprising a dimer of dehydrosalicortin or a derivative, homologue, stereoisomer, prodrug or pharmaceutical salt thereof, preferably wherein the dimer is a result of a Diels- Alder reaction.

2. A compound of Formula I, or a derivative, homologue, stereoisomer, prodrug or pharmaceutical salt thereof,

R1— L— R2

Formula I wherein L is a linking member and, R1 and R2 are each independently selected from Formula III,

Formula III wherein R3, R4, R5 and R6 are each independently selected from (i) H, (ii) acetyl, (iii) benzoyl, (iv) ortho- or para-coumaroyl, (v) cinnamoyl, preferably wherein R3, R4, R5 and R6 are each independently selected from (i) H, and (ii) acetyl, preferably wherein R3, R4, R5 and R6 are each H.

3. A compound according to claim 2, wherein

(a) R1 is Formula IILA-

wherein R3, R4, R5 and R6 are each independently selected from (i) H, (ii) acetyl, (iii) benzoyl, (iv) ortho- or para-coumaroyl, (v) cinnamoyl, preferably wherein R3, R4, R5 and R6 are each independently selected from (i) H, and (ii) acetyl, preferably wherein R3, R4, R5 and R6 are each H, or

(b) R1 is Formula IIIB:-

wherein R3, R4, R5 and R6 are each independently selected from (i) H, (ii) acetyl, (iii) benzoyl, (iv) ortho- or para-coumaroyl, (v) cinnamoyl, preferably wherein R3, R4, R5 and R6 are each independently selected from (i) H, and (ii) acetyl, preferably wherein R3, R4, R5 and R6 are each H.

4. A compound according to claim 2 or 3, wherein

(a) R2 is Formula IIIC:-

Formula IIIC wherein R7, R8, R9 and R10 are each independently selected from (i) H, (ii) acetyl, (iii) benzoyl, (iv) ortho- or para-coumaroyl, (v) cinnamoyl, preferably wherein R7, R8, R9 and R10 are each independently selected from (i) H, and (ii) acetyl, preferably wherein R7, R8, R9 and R10 are each H, or

(b) R2 is Formula IIID:-

Formula HID wherein R7, R8, R9 and R10 are each independently selected from (i) H, (ii) acetyl, (iii) benzoyl, (iv) ortho- or para-coumaroyl, (v) cinnamoyl, preferably wherein R7, R8, R9 and R10 are each independently selected from (i) H, and (ii) acetyl, preferably wherein R7, R8, R9 and R10 are each H.

5. A compound according to any of claims 2 to 4, wherein L comprises a ring structure, preferably wherein L is the result of a Diels-Alder reaction and/or wherein L comprises a tricyclododecadiene derivative.

6. A compound according to any of claims 2 to 5, wherein L is selected from

(a) Formula II A: -

wherein R12 and R13 are each independently selected from H and OH, preferably wherein R12 and R13 are OH, or

(b) Formula IIB:-

Formula IIB wherein R14 and R15 are each independently selected from H and OH, preferably wherein R12 and R13 are OH.

7. A compound of Formula IIC, or a derivative, homologue, stereoisomer, prodrug or pharmaceutical salt thereof: -

wherein R16 and R17 are each independently selected from H, OH and Formula III, with the proviso that at least one of R16 and R17 is selected from Formula III, preferably wherein R16 and R17 are each independently selected from Formula III, and

wherein R12 and R13 are each independently selected from H and OH.

8. A compound of Formula IID, or a derivative, homologue, stereoisomer, prodrug or pharmaceutical salt thereof:-

Formula IID wherein R18 and R19 are each independently selected from H, OH and Formula III, with the proviso that at least one of R18 and R19 is selected from Formula III, preferably wherein R18 and R19 are each independently selected from Formula III, and

wherein R14 and R15 are each independently selected from H and OH.

9. A compound selected from

(a) Formula VII or a derivative, homologue, stereoisomer, prodrug or pharmaceutical salt thereof,

Formula VII wherein R3, R4, R5, R6, R7, R8, R9 and R10 are each independently selected from (i) H, (ii) acetyl, (iii) benzoyl, (iv) ortho or para-coumaroyl, (v) cinnamoyl, and

wherein R12 and R13 are each independently selected from H and OH, preferably wherein R14 and R15 are each OH, or

(b) Formula VIII or a derivative, homologue, stereoisomer, prodrug or pharmaceutical salt thereof, wherein R3, R4, R5, R6, R7, R8, R9 and R10 are each independently selected from (i) H, (ii) acetyl, (iii) benzoyl, (iv) ortho- or para-coumaroyl, (v) cinnamoyl, and

wherein R14 and R15 are each independently selected from H and OH, preferably wherein R14 and R15 are each OH.

10. A compound according to claim 9, wherein R3, R4, R5, R6, R7, R8, R9 and R10 are each independently selected from (i) H, and (ii) acetyl, preferably wherein R3, R4, R5, R6, R7, R8, R9 and R10 are each H.

11. A compound of Formula IX, or a derivative, homologue, stereoisomer, prodrug or pharmaceutical salt thereof, Formula IX wherein R7, R8, R9 and R10 are each independently selected from (i) H, (ii) acetyl, (iii) benzoyl, (iv) ortho- or para-coumaroyl, (v) cinnamoyl,

wherein R11 is selected from (i) H, (ii) OH, and (iii) Formula III.

wherein R13 is selected from H and OH.

12. A compound according to claim 11, wherein R7, R8, R9 and R10 are each independently selected from (i) H, and (ii) acetyl, preferably wherein R7, R8, R9 and R10 are each H.

13. A compound of Formula X, XI, XII, XIII, XIV, XV, XVI, XVII or XVIII, or a derivative, homologue, stereoisomer, prodrug or pharmaceutical salt thereof.

14. A composition comprising a compound according to any preceding claim.

15. A composition according to claim 14, wherein the composition comprises a compound of Formula VII, VIII or IX, most preferably a compound of Formula VII, or a derivative, homologue, stereoisomer, prodrug or pharmaceutical salt thereof.

16. A composition according to claim 14, wherein the composition comprises a compound of Formula X, XI or XTT

17. A composition according to claim 14, wherein the composition comprises a compound of Formula X, or a derivative, homologue, stereoisomer, prodrug or pharmaceutical salt thereof,

Formula X.

18. A composition according to any of claims 14 to 17 for use in therapy.

19. Use of a composition according to any of claims 14 to 17 for treating a disease.

20. Use of a composition according to any of claims 14 to 17 in the manufacture of a medicament for treating a disease.

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 any of claims 14 to 17.

22. A composition of claim 18 wherein therapy comprises treating a disease, a use of claim 19 or 20, or a method of claim 21, wherein the disease is cancer, preferably wherein the cancer is primary or secondary (metastatic) cancer and/or wherein the cancer is a drug-resistant cancer.

23. A composition according to claim 22, wherein the cancer is selected from neuroblastoma, breast cancer, oesophageal cancer or ovarian cancer.

24. A method for producing a compound according to any of claims 1 to 23, wherein the method comprises extracting the compound from a plant of the genus Salix, preferably wherein the method comprises extracting the compound from leaf or stem tissue of a plant of the genus Salix, preferably wherein Salix is selected from (i) Salix miyabeana, Salix dasyclados, Salix gilgiana, Salix gmelinii, Salix repens, Salix capsica or Salix adhenophylla, or (ii) a hybrid of Salix miyabeana, Salix dasyclados, Salix gilgiana, Salix gmelinii, Salix repens, Salix capsica or Salix adhenophylla.

25. A method according to claim 24, wherein Salix is Salix miyabeana or a hybrid thereof.

Description:
CYLODIMER OF DEHYDROSALICORTIN AND DERIVATIVES THEREOF

ISOLATED FROM PLANT OF THE GENUS SALIX FOR USE IN CANCER THERAPY

The present invention relates to novel compounds and their use in therapy, in particular for the treatment of cancer.

Cancer is a disease which effects millions of people around the world each year. Whilst many effective therapies exist for treating cancer, there are still a huge number of cancers for which there is either no treatment or for which current treatments remain largely ineffective. This, combined with the large number of different types of cancer now known, means that there is a great need for new therapies.

It is, therefore, an object of the present invention to seek to alleviate the above identified problem.

SUMMARY OF THE INVENTION

According to one aspect of the present invention, there is provided a compound comprising a dimer of dehydrosalicortin or a derivative, homologue, stereoisomer, prodrug or pharmaceutical salt thereof.

Preferably, the dimer is a result of a Diels- Alder reaction.

According to another aspect of the present invention, there is provided a compound of Formula I, or a derivative, homologue, stereoisomer, prodrug or pharmaceutical salt thereof.

Within this specification, reference to a compound of Formula I means a compound having the following structure:-

R 1 — L— R 2

Formula I wherein L is a linking member, R 1 and R 2 are each independently selected from Formula III. Within this specification, reference to Formula III means:-

Formula III wherein R 3 , R 4 , R 5 and R 6 are each independently selected from (i) H, (ii) acetyl, (iii) benzoyl, (iv) ortho- or para-coumaroyl, (v) cinnamoyl.

Preferably, R 3 , R 4 , R 5 and R 6 are each independently selected from (i) H, and (ii) acetyl. Preferably, R 3 , R 4 , R 5 and R 6 are each H.

For the sake of convenience, Table 1 provides structures for some of the groups referred to herein. Table 1

Preferably, R 1 is Formula IIIA:-

wherein R 3 , R 4 , R 5 and R 6 are each independently selected from (i) H, (ii) acetyl, (iii) benzoyl, (iv) ortho- or para-coumaroyl, (v) cinnamoyl.

Preferably, R 3 , R 4 , R 5 and R 6 are each independently selected from (i) H, and (ii) acetyl. Preferably, R 3 , R 4 , R 5 and R 6 are each H.

Preferably, R 1 is Formula IIIB:-

Formula MB wherein R 3 , R 4 , R 5 and R 6 are each independently selected from (i) H, (ii) acetyl, (iii) benzoyl, (iv) ortho- or para-coumaroyl, (v) cinnamoyl. Preferably, R 3 , R 4 , R 5 and R 6 are each independently selected from (i) H, and (ii) acetyl. Preferably, R 3 , R 4 , R 5 and R 6 are each H.

Preferably, R 2 is Formula IIIC:-

Formula IIIC

wherein R 7 , R 8 , R 9 and R 10 are each independently selected from (i) H, (ii) acetyl, (iii) benzoyl, (iv) ortho- or para-coumaroyl, (v) cinnamoyl.

Preferably, R 7 , R 8 , R 9 and R 10 are each independently selected from (i) H, and (ii) acetyl. Preferably, R 7 , R 8 , R 9 and R 10 are each H.

Formula HID wherein R 7 , R 8 , R 9 and R 10 are each independently selected from (i) H, (ii) acetyl, (iii) benzoyl, (iv) ortho- or para-coumaroyl, (v) cinnamoyl.

Preferably, R 7 , R 8 , R 9 and R 10 are each independently selected from (i) H, and (ii) acetyl.

Preferably, R 7 , R 8 , R 9 and R 10 are each H.

Preferably, L comprises a ring structure.

Preferably, L is the result of a Diels- Alder reaction.

Preferably, L comprises a Diels- Alder reaction produced core element.

Preferably, L comprises a tricyclododecadiene derivative, preferably a substituted derivative, preferably wherein the cycloalkene is substituted by at least one group selected from OH, carbonyl,. Preferably, L is selected from Formula IIA or Formula IIB. Within this specification, reference to Formula IIA means:-

wherein R 12 and R 13 are each independently selected from H and OH. Preferably, R 12 and R 13 are each OH.

Within this specification, reference to Formula IIB means:-

Formula IIB

wherein R 14 and R 15 are each independently selected from H and OH.

Preferably, R 14 and R 15 are each OH. According to another aspect of the present invention, there is provided a compound of Formula IIC, or a derivative, homologue, stereoisomer, prodrug or pharmaceutical salt thereof.

Within this specification, reference to a compound of Formula IIC means a compound having the following structure:-

wherein R 16 and R 17 are each independently selected from H, OH and Formula III, with the proviso that at least one of R 16 and R 17 is selected from Formula III, and

wherein R 12 and R 13 are each independently selected from H and OH.

Preferably, R 16 and R 17 are selected from Formula III.

Preferably, either R 16 or R 17 is selected from Formula III.

Preferably, R 16 is H and R 17 is selected from Formula III.

Preferably, R 12 and R 13 are each OH.

According to another aspect of the present invention, there is provided a compound of Formula IID, or a derivative, homologue, stereoisomer, prodrug or pharmaceutical salt thereof. Within this specification, reference to a compound of Formula IID means a compound having the following structure:-

wherein R 18 and R 19 are each independently selected from H, OH and Formula III, with the proviso that at least one of R 18 and R 19 is selected from Formula III, and

wherein R 14 and R 15 are each independently selected from H and OH.

Preferably, R 18 and R 19 are selected from Formula III.

Preferably, either R 18 or R 19 is selected from Formula III.

Preferably, R 14 and R 15 are each OH.

Preferably, the invention relates to a compound of Formula VII or a derivative, homologue, stereoisomer, prodrug or pharmaceutical salt thereof.

Within this specification, reference to Formula VII means:-

Formula VII wherein R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 and R 10 are each independently selected from (i) H, (ii) acetyl, (iii) benzoyl, (iv) ortho or para-coumaroyl, (v) cinnamoyl, and

wherein R 12 and R 13 are each independently selected from H and OH.

Preferably, R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 and R 10 are each independently selected from (i) H, and (ii) acetyl.

Preferably, R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 and R 10 are each H.

Preferably, R 12 and R 13 are each OH.

Preferably, the invention relates to a compound of Formula VIII or a derivative, homologue, stereoisomer, prodrug or pharmaceutical salt thereof. Within this specification, reference to Formula VIII means:-

wherein R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 and R 10 are each independently selected from (i) H, (ii) acetyl, (iii) benzoyl, (iv) ortho- or para-coumaroyl, (v) cinnamoyl, and

wherein R 14 and R 15 are each independently selected from H and OH.

Preferably, R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 and R 10 are each independently selected from (i) H, and (ii) acetyl.

Preferably, R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 and R 10 are each H.

Preferably, R 14 and R 15 are each OH.

Preferably, the invention relates to a compound of Formula IX, or a derivative, homologue, stereoisomer, prodrug or pharmaceutical salt thereof. Within this specification, reference to Formula IX means:-

Formula IX wherein R 7 , R 8 , R 9 and R 10 are each independently selected from (i) H, (ii) acetyl, (iii) benzoyl, (iv) ortho- or para-coumaroyl, (v) cinnamoyl,

wherein R 1 1 is selected from (i) H, (ii) OH, and (iii) Formula III.

wherein R 13 is selected from H and OH.

Preferably, R 7 , R 8 , R 9 and R 10 are each independently selected from (i) H, and (ii) acetyl. Preferably, R 7 , R 8 , R 9 and R 10 are each H. Preferably, R 13 is H.

Preferably R 1 1 is H or OH. Preferably R 1 1 is OH.

Preferably, the invention relates to a compound of Formula X or a derivative, homologue, stereoisomer, prodrug or pharmaceutical salt thereof. Within this specification, reference to Formula X means:-

Formula X

Preferably, the invention relates to a compound of Formula XI, or a derivative, homologue, stereoisomer, prodrug or pharmaceutical salt thereof.

Within this specification, reference to Formula XI means:-

Formula XI Preferably, the invention relates to a compound of Formula XII, or a derivative, homologue, stereoisomer, prodrug or pharmaceutical salt thereof.

Within this specification, reference to Formula XII means:-

Formula XII

Preferably, the invention relates to a compound of Formula XIII or a derivative, homologue, stereoisomer, prodrug or pharmaceutical salt thereof.

Within this specification, reference to Formula XIII means:-

Preferably, the invention relates to a compound of Formula XIV or a derivative, homologue, stereoisomer, prodrug or pharmaceutical salt thereof.

Within this specification, reference to Formula XIV means:-

Formula XIV Preferably, the invention relates to a compound of Formula XV or a derivative, homologue, stereoisomer, prodrug or pharmaceutical salt thereof.

Within this specification, reference to Formula XV means:-

Formula XV

Preferably, the invention relates to a compound of Formula XVI or a derivative, homologue, stereoisomer, prodrug or pharmaceutical salt thereof.

Within this specification, reference to Formula XVI means:-

Formula XVI

Preferably, the invention relates to a compound of Formula XVII or a derivative, homologue, stereoisomer, prodrug or pharmaceutical salt thereof.

Within this specification, reference to Formula XVII means:-

Formula XVII

Preferably, the invention relates to a compound of Formula XVIII or a derivative, homologue, stereoisomer, prodrug or pharmaceutical salt thereof.

Within this specification, reference to Formula XVIII means:-

According to another aspect of the present invention, there is provided a compound of Formula XIX or a derivative, homologue, stereoisomer, prodrug or pharmaceutical salt thereof.

Within this specification, reference to Formula XIX means:-

wherein R 1 is selected from (i) H, (ii) acetyl, (iii) benzoyl, (iv) ortho- or para- coumaroyl, (v) cinnamoyl.

Preferably, the invention relates to a compound of Formula XX or a derivative, homologue, stereoisomer, prodrug or pharmaceutical salt thereof.

Within this specification, reference to Formula XX means:-

Formula XX Preferably, the derivative, homologue, stereoisomer, prodrug or pharmaceutical salt thereof is a therapeutically effective derivative, homologue, stereoisomer, prodrug or pharmaceutical salt thereof.

According to another aspect of the present invention, there is provided a composition comprising a compound as described herein, or a derivative, homologue, stereoisomer, prodrug or pharmaceutical salt thereof.

Preferably, the composition comprises a compound of Formula VII, VIII or IX, most preferably a compound of Formula VII, or a derivative, homologue, stereoisomer, prodrug or pharmaceutical salt thereof.

Preferably, the composition comprises a compound of Formula X, XI or XII, most preferably a compound of Formula X, or a derivative, homologue, stereoisomer, prodrug or pharmaceutical salt thereof.

Preferably, the composition is a pharmaceutical composition.

Preferably, the composition is a therapeutic composition.

Preferably, the composition comprises one or more pharmaceutically acceptable carriers, diluents or excipients.

According to another aspect of the present invention, there is provided a composition as described herein for use in therapy.

According to a further aspect of the present invention, there is provided use of a composition as described herein for treating a disease.

According to another aspect of the present invention, there is provided use of a composition as described herein in the manufacture of a medicament for treating a disease. According to a further aspect of the present invention, there is provided 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 as described herein.

Preferably, therapy comprises treating a disease.

Preferably, treating a disease comprises administering a therapeutically effective amount of a composition of the present invention to a patient suffering from a disease.

Preferably, the disease is cancer.

Preferably, the cancer is primary or secondary (metastatic) cancer.

Preferably, the cancer is a drug-resistant cancer. In this respect, it will be appreciated that reference to“drug resistant cancer” means a cancer which has previously shown resistance to treatment with another therapeutic composition, for example a cancer which has been unsuccessfully treated with another therapeutic composition.

Preferably, the cancer is resistant to an anti -microtubule agent, preferably an anti -microtubule alkaloid agent.

Preferably, the cancer is resistant to a vinca alkaloid.

Preferably, the cancer is resistant to vincristine.

Preferably, the cancer is selected from neuroblastoma, breast cancer, oesophageal cancer or ovarian cancer.

Preferably, the neuroblastoma is metastatic neuroblastoma in the bone marrow.

Preferably, the neuroblastoma is vincristine-resistant metastatic neuroblastoma in the bone marrow. Preferably, the breast cancer is invasive ductal carcinoma.

Preferably, the oesophageal cancer is oesophageal squamous cell carcinoma.

Preferably, the ovarian cancer is high grade ovarian serous adenocarcinoma or ovarian cystadenocarcinoma.

Preferably, the cancer is metastatic cancer.

Preferably, the subject is a mammal.

Preferably, the subject is a human.

Preferably, the compositions of the present invention comprise one or more additional active compounds. Preferably, the one or more additional active compounds are therapeutically active compounds, for example in the form of an additional therapeutic compound for co-delivery with the compositions described herein.

According to another aspect of the present invention, there is provided a method for producing a compound as described herein, wherein the method comprises extracting the compound from a plant of the genus Salix.

Preferably, the method comprises extracting the compound from leaf or stem tissue of a plant of the genus Salix.

Preferably, Salix is selected from (i) Salix miyabeana, Salix dasyclados, Salix gilgiana, Salix gmelinii, Salix repens, Salix capsica, Salix rehderiana, S. rossica, S. glaucophyloides or Salix adhenophylla, or (ii) a hybrid of Salix miyabeana, Salix dasyclados, Salix gilgiana, Salix gmelinii, Salix repens, Salix capsica, Salix rehderiana, S. rossica, S. glaucophyloides or Salix adhenophylla. Preferably, Salix is selected from (i) Salix miyabeana, Salix dasyclados, Salix gilgiana, Salix gmelinii, Salix repens, Salix capsica or Salix adhenophylla, or (ii) a hybrid of Salix miyabeana, Salix dasyclados, Salix gilgiana, Salix gmelinii, Salix repens, Salix capsica or Salix adhenophylla.

Preferably, Salix is Salix miyabeana or a hybrid of Salix miyabeana.

Preferably, Salix is S. miyabeana Seemen or a hybrid of S. miyabeana Seemen.

Preferably, Salix is S. miyabeana Purpurescens or a hybrid of S. miyabeana Purpurescens. Preferably, Salix is Salix dasyclados or a hybrid of Salix dasyclados.

Preferably, Salix is Salix rehderiana or a hybrid of Salix rehderiana.

Preferably, Salix is RRes 710-27, RR09102 hybrid [NWC607 S. rehderiana x RR05337 (Aud x S. rossica)].

Preferably, Salix is S. miyabeana hybrid breeding line (RR10347) generated from a cross of NWC941 (S. miyabeana Purpurescens) with RR05326 (Resolution x S. rossica).

Preferably, Salix is willow breeding line RR10147.

RR10147 was 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 x 577“77056”) x RR07188 (944 S. glaucophyloides x 577“77056”)] as well as S. glaucophyloides (NWC 944).

Within this specification embodiments have been described in a way which enables a clear and concise specification to be written, but it is intended and will be appreciated that embodiments may be variously combined or separated without parting from the invention. For example, it will be appreciated that all preferred features described herein are applicable to all aspects of the invention.

DETAILED DESCRIPTION

Example embodiments of the present invention will now be described with reference to the accompanying Figures, in which:-

Figure 1 shows structures of isolated dimeric compounds from Salix miyabeana ;

Figure 2 shows reversed phase HPLC analysis of Salix miyabeana leaf extract indicating the peak of miyabeacin at 57.93 minutes;

Figure 3 shows Total Ion Chromatogram from LC-MS analysis (negative ion mode) of purified miyabeacin;

Figure 4 shows mass spectrum (negative ion mode) of miyabeacin at m/z 843.23529 with retention time 25.31 min;

Figure 5 shows MS-MS spectrum (negative ion mode) of m/z 843.23529 (25.31 min);

Figure 6 shows MS-MS spectrum (negative ion mode) of m/z 421.11404 (25.31 min);

Figure 7 shows 1 H-NMR spectrum of purified miyabeacin in CD30D . Expanded region shown between 57.50 - 53.49;

Figure 8 shows 1 H- 1 H COSY NMR spectrum of purified miyabeacin in CD 3 OD. Expanded region shown between 57.51 - 53.49;

Figure 9 shows 13 C-NMR spectrum of purified miyabeacin in CD 3 OD;

Figure 10 shows 13 C-DEPT135 spectrum of purified miyabeacin in CD 3 OD;

Figure 11 shows 1 H- 13 C-HMBC spectrum of purified miyabeacin in CD 3 OD;

Figure 12 shows reversed phase HPLC analysis of Salix miyabeana stem extract indicating the peak of miyabeacin B at 52.11 minutes;

Figure 13 shows Total Ion Chromatogram from LC-MS analysis (negative ion mode) of purified miyabeacin B;

Figure 14 shows mass spectrum (negative ion mode) of miyabeacin B at m/z 843.23474 with retention time 24.34 min;

Figure 15 shows Total Ion Chromatogram from LC-MS analysis (negative ion mode) of purified miyabeanol; Figure 16 shows mass spectrum (negative ion mode) of miyabeanol at m/z 531.15074 with retention time 20.11 min;

Figure 17 shows MSMS spectrum (negative ion mode) of miyabeanol at m/z 531.15074 with retention time 20.11 min;

Figure 18 shows 'H-NMR spectrum of purified miyabeanol in D20:CD30D (4: 1). Expanded region shown between 57.60 - 53.00;

Figure 19 shows ¾-¾ COSY NMR spectrum of miyabeanol in D20:CD30D (4: 1); and Figure 20 shows 13 C NMR spectrum of miyabeanol in D20:CD30D (4: 1).

The present invention relates to novel compounds and their use in therapy, in particular for the treatment of cancer.

The compounds described herein were extracted from plants of the genus Salix , in particular Salix miyabeana or Salix dasyclados.

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.

In relation to Salix miyabeana referred to herein, this species is believed to be native to Japan and Korea.

In relation to Salix dasyclados referred to herein, this species is believed to be native to Siberia.

In relation to Salix gilgiana referred to herein, this species is believed to be native to Japan and Korea.

In relation to Salix gmelinii referred to herein, this species is believed to be native to Kazakhstan.

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.

In relation to Salix capsica referred to herein, this species is believed to be native to Central Asia.

In relation to Salix adhenophylla referred to herein, this species is believed to be native to North America.

In relation to Salix rehderiana referred to herein, this species is believed to be native to China.

In relation to Salix rossica referred to herein, this species is believed to be native to Europe, Western Asia, and the Himalayas.

In relation to Salix glaucophyloides referred to herein, this species is believed to be native to North America.

Within this specification, the term“miyabeacin” means a compound of Formula X.

Within this specification, the term“miyabeacin B” means a compound of Formula XI.

Within this specification, the term“miyabeanol” means a compound of Formula XII.

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%.

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.

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.

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.

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.

Preferably, the composition comprises a therapeutically effective amount of at least one compound as described herein or a physiologically tolerated salt thereof.

Preferably, the composition comprises a physiologically tolerated carrier.

Within this specification, the term“prodrug” means to a compound that is biologically inactive, but is metabolized to produce an active therapeutic drug.

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.

Within this specification, the term“homologue” refers to a molecule having substantial structural similarities to the compounds described herein.

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.

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.

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.

The formulations may also contain other pharmacologically active agents, and conventional additives, such as stabilizers, wetting agents, emulsifiers, flavouring agents, buffers, and the like.

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.

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. 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.

Formulations intended for inhalation can be provided as an aerosol spray, for example in a pressurised container or dispenser which contains a suitable propellant.

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.

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.

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. 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.

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.

Pharmaceutical compositions containing the appropriately formulated compound can be included in a container, pack, or dispenser together with instructions for administration.

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. 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.

Freeze dried juvenile leaves of Salix miyabeana were used as the starting material for the initial isolation of Miyabeacin (Fig 1A) and Miyabeanol (Fig 1C). Freeze dried juvenile stems of Salix miyabeana were used as the starting material for the initial isolation of Miyabeacin B (Fig IB). Tissue was milled to a homogeneous powder prior to extraction.

Miyabeacin

For the initial isolation of Miyabeacin, lmL of watenmethanol (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 pL 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 pL 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 Cl 8 column (5um, 5 x 250mm (Supelco, UK). The operating solvents were: Solvent A: H20 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 57.93min (Fig 2). Equivalent fractions from 6 runs were combined and evaporated using a Speedvac concentrator (Genevac, Suffolk, UK) to yield 1.68 mg of purified Miyabeacin. It was also possible to recover the product from the following Salix species by the procedures described above: Salix dasyclados, Salix gilgiana, Salix gmelinii , Salix repens , Salix capsica and Salix adhenophylla and hybrids thereof. Table 2 Extraction and HPLC gradient conditions for the isolation of dimeric metabolites.

Compound Amount Tissue Extraction Number HPLC HPLC Amount

Extracted Volume of 100 pL Gradient Retention Isolated

(solvent: Injections [mobile time of

H 2 0:Me0H) made into phases Peak

HPLC water (A)

and

acetonitri

le (B),

both

contaimn

g 0.1%

formic

acid.l

X 50 mg Salix 1 mL 6 5% B (0- 57.93 1.68 miyabeana 10 min), min mg Seemen. Ill 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 (0- 52.11 0.67 miyabeana 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 (0- 44.87 1.05 miyabeana 10 min), min mg Seemen. Ill 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 (0- 20.9 0.9 mg miyabeana 20 min), min

Seemen. 40% B

“Purpurescens’ (20-25

leaf tissue. min) to 50

Line: NWC941 % B (25- XIII & 150 mg RRes 710-27, 2.4 mL (2 10 20% B (0 41.4 0.75

XIV (2 x 75 RR09102 1.2 mL) mm), 40% mg mg) hybrid B (0 - 45

[NWC607 S. min) to

rehderiana c 100 % B

RR05337 (45.0-50

(Aud x S. min)

rossica)] leaf

tissue

XV 150 mg RRes 710-27, 2.4 mL (2 10 20% B (0 45.5 0.25

(2 x 75 RR09102 1.2 mL) mm), 40% mg mg) hybrid B (0 - 45

[NWC607 S. min) to

rehderiana c 100 % B

RR05337 (45.0-50

(Aud x S. min)

rossica)] leaf

tissue

Further structural diversity was evident in the analysis of a S. miyabeana hybrid breeding line (RR10347) generated from a cross of NWC941 (S. miyabeana Purpurescens) with RR05326 (Resolution x S. rossica). Analysis of RR10347 by LC-MS indicated the presence of tremulacin (C27H28O11), a 2'-0-benzoylated derivative of salicortin which is well known in the Salicaceae. A new peak also appeared at 30.95 min with a mass of 947.2561 that corresponded to an ion with molecular formula C47H47O21. As in the case of the mass spectrum of miyabeacin, a smaller ion (m/z 421.1125) corresponded to that observed for salicortenone (C20H21O10). An additional small ion (m/z 525.1465) was also evident with a suggested formula of C27H25O11. MSMS of m/z 947.2561 yielded two strong ions at m/z 121 (C7H5O2) and m/z 123 (C7H7O2), the former corresponding to a benzoate moiety and the latter to a salicyl moiety. The data were suggestive of a compound of Formula XVI/XVII, novel mono-benzoylated derivative of miyabeacin. The peak was isolated by repeated injection into an HPLC system and the structure characterised by 1 H-NMR. This data, via comparison to the NMR spectrum of miyabeacin, confirmed the presence of a dimeric compound. Additional peaks at 58.06, 7.70 and 7.54 were characteristic of a benzoate group but a doubling of most peaks indicated a 1 : 1 mixture of isomers which could not be separated further. A set of multiplet peaks appearing at 55.25 confirmed the benzoyl substitution at the 2'-position of the glucoside in the first isomer and the 2"-position in the second isomer. This was further confirmed in the ¾-¾ -COSY spectrum. 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 x 577 “77056”) x RR07188 (944 S. glaucophyloides x 577“77056”)] as well as S. glaucophyloides (NWC 944). In the Total Ion Chromatogram of the negative ion mode LC-MS data salicortin, 2'-0-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''-0-acetylmiyabeacin (Formula XIII/XIV) appearing at 26.90 min and 2'/2''-0-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 C49H49O22 . Although there was insufficient for isolation, the MS was suggestive of the predicted miyabeacin analogue bearing both an acetyl and benzoyl substitution.

The structure of Miyabeacin was determined by various forms of spectroscopy. Table 3 shows the general measurement conditions for spectroscopic analyses.

Table 3 General Conditions and parameters for spectroscopic measurements.

Measurement Conditions

High resolution LC-MS

LC apparatus Ultimate 3000 RS uHPLC (Thermo)

Chromatography Column Ci 8 Hypersil gold column (1.9 pm, 30 x 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 10pL

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 ¾: 600.05, 13 C: 150.9

Solvent D 2 0:CD 3 0D (80:20)

Concentration 0.6 mg/mL

Internal Standard d 4 -TSP

Temperature 300K

Probe 5mm Selective Inverse

1 H 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 300K

Number of transients 32

2D HSQC Measurement

Pulse program hsqcetgpsi2

Observation width 7180, 30150 Hz Data points 2048, 1024

Temperature 300K

Number of transients 128 2D HMBC Measurement

Pulse program hmbcgpndqf

Observation width 7182, 33165 Hz

Data points 4096, 256

Temperature 300K

Number of transients 256

13 C NMR _

Apparatus Avance 400 (Bruker)

Observation Frequency 13 C: 100.61

Solvent D 2 0:CD 3 0D (80:20)

Concentration 0.6 mg/mL

Internal Standard d 4 -TSP

Temperature 300K

Probe 5mm Broadband BBO

13 C NMR Measurement

Pulse sequence deptl35

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 ¾-¾ COSY)

HSQC: Heteronuclear Single Quantum Coherence (A method of ¾- 13 C COSY)

HMBC: Heteronuclear Multiple Bond Correlation (A method of long-range ¾- 13 C COSY) Miyabeacin Spectroscopic Analyses

High Resolution LC-MS: LC-MS was carried out in negative ion mode using a Cl 8 column. Conditions of analysis are outlined in Table 3. Figure 3 shows a total ion chromatogram of purified miyabeacin which appeared as a single peak at 25.31 min. A high-resolution mass spectrum (Figure 4) was collected in negative ion mode and showed an m/z ion at 843.23529 (C40H43O20), corresponding to the [M-H]- of miyabeacin (molecular formula C40H44O20). Smaller ions also present in the mass spectrum were m/z 889.2396 (C41H45O22, formate adduct), 557.1300 (C27H25O13), 421.1140 (C20H21O10), 331.1034 (C14H19O9) and 217.0507 (C12H9O4). MS-MS of m/z 843.23529 (Figure 5) revealed a variety of low abundance fragments including m/z 123.04538 (C7H7O2), 201.05629 (C12H9O3), 227.03554 (C13H7O4), 245.04494 (C13H9O5) and 557.13739 (C27H25O13). MS-MS of m/z 421.11404 ion (Figure 6) gave fragments at m/z 297.06246 (C13H13O8), 153.02017 (C7H5O4), 135.00946 (C7H3O3), 123.04583 (C7H7O2), 109.03004 (C 6 H 5 0 2 ) and 81.03513 (C5H5O).

NMR spectroscopy: Ή-NMEI data of miyabeacin was collected at 600 MHz in aqueous d 4 - methanol containing 0.01% w/v d4-TSP as internal standard. The spectrum showed peaks relating to 34 coupled protons (Fig 7 and Table 4).

Table 4. Ή-NMEI assignments for mivabeacin. Data collected at 600 MHz in

(4: IT referenced to cb-TSP (0.01% w/vY

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

7a 5.40 (d, 11.9)

7b 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

22b 5.38 (d, 12.1)

22a 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

r 5.09 (d, 1.5)15.01 (d, 7.8)

3.55-3.63 (m)

3’ 3.56-3.62 (m)

4’ 3.47-3.52 (m)

5’ 3.56-3.62 (m)

6’b 3.77 (dd, 12.4, 6.0)/3.73 (dd, 12.4,

6.0)

6’a 3.94 (dd, 12.4, 2.1)/3.92 (dd, 12.4,

2.1)

1 5.09 (d, 1.5)15.01 (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 , b 3.77 (dd, 12.4, 6.0)/3.73 (dd, 12.4,

6.0)

6”a 3.94 (dd, 12.4, 2.1)/3.92 (dd, 12.4,

2L}

Four signals were observed between d 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 : 55.40 and 55.19; pair 2: 55.38 and 55.16). Similarly, the molecule contained two separate glucoside moieties with characteristic doublet signals relating to the H-G anomeric protons being duplicated (55.09 and 55.07) as were those corresponding to the glucosyl 6'-methylenes. Four separate olefin signals were present between 56.60 and 5.85 each integrating for one proton, two appearing as double doublets and the others as simple triplets. ¾-¾ COSY analysis (Fig 8) demonstrated that the two double bonds were isolated from each other. Integration of the carbohydrate region (53.96 - 3.40) suggested a total of 16 coupled protons. Of these, 12 could be accounted for in two glucose units leaving 4 unaccounted for. 13 C NMR data (Fig. 9 and Table 5) confirmed the presence of 40 carbon atoms in the molecule including two ketone carbonyls at 5199.6 and 210.0 and two ester carbonyls at 5 173.6 and 173.2 while 13 C DEPT135 (Fig 10) identified four non-aromatic methine signals, in addition to those of glucose (x2) and two olefmic signals.

Table 5 13 C-NMR assignment for mivabeacin. Data collected at 100 61 MHz in

(4: 11. referenced to d4-TSP (0.01% w/vT

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

7a 67.3

7b 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

22b 66.7

22a 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

G 103.0/102.9

76.0 3’ 79.1

4’ 72.5/72.4

5’ 78.7/78.8

63.7

63.7

103.0/102.9

76.0

79.1

72.5/72.4

78.7/78.8

63.7

63.7

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 1 H- 13 C HMBC were observed around all the positions of the dimeric core structure (Fig 11) and between H-10 (5 3.60) and C-8 (d 173.6) confirming the attachment of a carboxyl group to the core structure at C-9. The correlation between H-7 (d 5.19 and 5.40) and C-8 (d 173.6) confirmed the linkage via the ester carbonyl, to a salicyl moiety. Similar correlations were observed between H-15 (d 3.50) to C-21 (d 173.2) and also between H-22 (d 5.16 and 5.38) and C-21 (d 173.2), suggesting a second carboxy-salicyl entity attached to the tricyclododecadiene core at C-20. Additional correlations between C-l (d 158.0) and H-7 (d 5.19 and 5.40) and H-G (d 5.09 / 5.07) and also between C-28 (d 157.7) to H-22 (d 5.16 and 5.38) and H-l " (d 5.07 /5.09) were consistent with placement of the O-glucosides at C-l and C-28.

Miyabeacin B

For the initial isolation of Miyabeacin B, 2.5 mL of watenmethanol (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 pL 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 pL 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 Cl 8 column (5um, 5 x 250mm (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), 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 (Fig 12). Equivalent fractions from multiple runs were combined and evaporated using a Speedvac concentrator (Genevac, Suffolk, UK) to yield 0.67 mg of purified Miyabeacin B. The structure of Miyabeacin B was determined by various forms of spectroscopy.

Miyabeacin B Spectroscopic Analyses

High Resolution LC-MS: LC-MS was carried out in negative ion mode using a Cl 8 column. Conditions of analysis are outlined in Table 3. Figure 13 shows a total ion chromatogram of purified miyabeacin B which appeared as a single peak at 24.34 min. A high-resolution mass spectrum (Figure 14) was collected in negative ion mode and showed an m/z ion at 843.23474 (C40H43O20), corresponding to the [M-H]- of miyabeacin B (molecular formula C40H44O20). A smaller ion at m/z 889.23895 (C41H45O22) corresponded to the formate adduct.

NMR spectroscopy: 1H-NMR spectroscopy in aqueous methanol showed a total of 17 signals which related to 34 separate protons (Table 6).

Table 6 1 H-NMR assignments for miyabeacin B Data collected at 600 MHz in D20:CD30D (4: 11. referenced to d4-TSP (0.01% w/yl.

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 7a 5.46 (d, 11.7)

7b 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

22b 5.46 (d, 11.7)

22a 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

V 5.07 (d, 7.8)

T 3.51 (dd, 9.4, 7.8)

3’ 3.58 (m)

4’ 3.45 (t, 9.4)

5’ 3.58 (m)

6’b 3.72 (dd, 12.4, 6.0)

6’a 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 , b 3.72 (dd, 12.4, 6.0)

6”a 3.99 (dd, 12.5, 2.2)

The presence of signals relating to benzyl and glucosyl moieties compared well with those observed in the 1 H-NMR spectrum of miyabeacin. Absence of the four olefin signals (55.91 to 6.59) previously observed in miyabeacin was accompanied by a movement upfield of the four bridgehead protons (53.43-3.63) to give a set of four signals at 52.76, 2.88, 2.99 and 3.12 each integrating for 2 protons. The 1 H-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 1 H-NMR spectrum for miyabeacin B relative to that observed for miyabeacin. [ 1 H- 1 H] correlation spectroscopy confirmed the linkages around the tricyclic core of the molecule. 13C data for miyabeacin B is given in Table 7.

Table 7 13 C-NMR assignment for miyabeacin B Data collected at 100 61 MHz in

DTQ (4: 11 referenced to d4-TSP (0.01% w/vY

Position

1 158.1

2 117.9

3 133.9

4 125.9

5 134.3

6 126.4

7a 67.1

7b 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

22b 67.1

22a 67.1

23 126.4

24 134.3

25 125.9

26 133.9

27 117.9

28 158.1

G 102.9

t 76.0 3’ 79.0

4’ 72.8

5’ 79.0

63.9

63.9

102.9

76.0

79.0

72.8

79.0

63.9

63.9

Miyabeanol

For the initial isolation of Miyabeanol, 2mL of watenmethanol (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 pL 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 pL 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 Cl 8 column (5um, 5 x 250mm (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. Miyabeanol eluted at 44.87min (Fig 1). Equivalent fractions from 8 runs were combined and evaporated using a Speedvac concentrator (Genevac, Suffolk, UK) to yield 1.05mg of purified Miyabeanol. Miyabeanol Spectroscopic Analyses

High Resolution LC-MS: LC-MS was carried out in negative ion mode using a Cl 8 column. Conditions of analysis are outlined in Table 3. Figure 15 shows a total ion chromatogram of purified miyabeacin B which appeared as a single peak at 20.11 min. A high-resolution mass spectrum (Figure 16) was collected in negative ion mode and showed an m/z ion at 531.15074(C 26 H 27 Oi 2 ), corresponding to the [M-H]- of miyabeanol (molecular formula C26H28O12). Additional ions at 421.11421 (C20H21O10) and 467.11943 (C21H23O12) corresponded to the product of a reverse Diels Alder reaction (salicortenone) and its corresponding formate adduct. MSMS analysis of m/z 531.15074 (Fig. 17) gave rise to fragment ions at 245.04634 (C13H9O5), 217.05150 (C12H9O4) and 123.04579 (C7H7O2).

NMR spectroscopy: The ¾ NMR spectrum of miyabeanol (Fig 18 and Table 8) suggested an analogous structure to the cyclodimer miyabeacin, although certain regions of the spectrum, including those relating to the benzyl and glucosyl groups, were no longer duplicated suggesting that one of each of these units had been lost.

Table 8 ¾-NMK assignments for miyabeanol. Data collected at 600 MHz in D70:CD70D

(4: 11. referenced to d 4 -TSP (0.01% w/vT

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

7a 5.39 (d, 11.9)

7b 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

22b

22a

23

24

25

26

27

28

r 5.06 (d, 7.3)

t 3.49-3.59 (m)

3’ 3.54-3.61 (m)

4’ 3.45-3.53 (m)

5’ 3.54-3.61 (m)

6’b 3.76 (dd, 12.5, 5.9)

6’a 3.92 (dd, 12.4, 2.2)

1

2

3”

4”

5”

6 , b

6”a

¾ signals at d 6.63 and d 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 d 6.27 and d 5.94 were also present. This data and additional 'H-'H COSY correlations (Fig 19) of these signals to 4 additional methine protons confirmed that the molecule retained the Diels- Alder“core”. 13C NMR (Fig 20 and Table 9) showed 26 separate carbon signals including 2 ketone signals at d 213.29 and d 199.1. Table 9 13 C-NMR assignments for mivabeanol. Data collected at 100 61 MHz in DTQ (4: 11. referenced to cU-TSP (0.01% w/vY

Position [7] [7]

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

7a 67.2 67.0

7b 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

22b

22a

23

24

25

26

27

28

r 103.0 102.7

T 76.0 75.8

3’ 79.1 78.7

4’ 72.4 72.2

5’ 78.8 78.5

6’b 63.6 63.5

6’a 63.6 63.5

1

2

3” 4”

5”

6”p

6”a

The position of side-chain loss and decarboxylation was confirmed via extensive analysis of COSY, HSQC and HMBC correlation spectroscopy. Key 1 H- 13 C 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 (d 81.4) were all present.

Bioassay Data of Miyabeacin

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 10. Bioactivitv data of miyabeacin in six cancer cell lines

Miyabeacin IC50 cancer type cell line (ug/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

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'VCR (Rothwell et al., 2010) (adapted to grow in the presence of vincristine lOng/mL). At a concentration of 20 pg/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-S'VCR 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 100pL 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 2 in the absence or presence of varying compound concentrations for 120h. Then, 25 pL of MTT solution (2pg/mL dissolved in PBS) were added for 4h. This was followed by the addition of 100 pL of 20% sodium dodecyl sulphate (50:50 purified water/ DMF) solution adjusted to pH 4.7 for an additional 4h in order to lyse cells and dissolve formazan precipitates. Plates were then read at 600nm. 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-68ON and COLO-704) and ranged from 17.15 pM to 40.18 mM (Table 11).

Table 11 Replicated IC% determination in three cancer cell lines

miyabeacm IC50 concentration IC50

(ug/mL) (mIUI)

cancer type cell line _ expt 1 expt 2 expt 3 mean S.D. mean oesophageal COLO- cancer 680N 5.08 15.08 51.46 23.87 19.93 28.28 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

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. 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

Kotchetkov R, Driever PH, Cinatl J, Michaelis M, Karaskova J, Blaheta R, Squire JA, 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 Oct;27(4): 1029-37.

Michaelis M, Rothweiler F, Barth S, Cinatl J, van Rikxoort M, Loschmann N, Voges Y, Breitling R, von Deimling A, Rodel F, Weber K, Fehse B, Mack E, Stiewe T, Doerr HW, 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.

Michaelis M, Agha B, Rothweiler F, Loschmann N, Voges Y, Mittelbronn M, Starzetz T, Harter PN, Abhari BA, Fulda S, Westermann F, Riecken K, Spek S, Langer K, Wiese M, Dirks WG, Zehner R, Cinatl J, Wass MN, Cinatl J Jr. Identification of flubendazole as potential anti-neuroblastoma compound in a large cell line screen. Sci Rep. 2015a Feb 3;5:8202.

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).

The content of all references cited herein are incorporated herein by reference in their entirety.