PHARMACEUTICAL COMPOSITIONS COMPRISING SAME

BRIEF DESCRIPTION OF INVENTION

The present invention relates to novel nontoxic diterpene compounds and their derivatives for use as medicaments, especially for the treatment of cancer, as well as dietary and pharmaceutical composition containing such diterpenes and derivates and their uses.

BACKGROUND OF THE INVENTION

Compounds extracted from plants have been used for centuries for medicinal purposes, and their exploration continues in modern times. Identification and characterization of newly extracted compounds and their therapeutic properties is vital in the on-going search for therapeutic agents to improve detrimental health conditions, such as cancer.

Cancer is a group of diseases (acute lymphoblastic leukemia (adult), acute aymphoblastic leukemia (childhood), acute myeloid leukemia (adult), acute myeloid leukemia (childhood), adrenocortical carcinoma, adrenocortical carcinoma (childhood), AIDS-related cancers, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytoma (childhood, cerebellar), astrocytoma (childhood cerebral), basal cell carcinoma (nonmelanoma), bile duct cancer (extrahepatic), bladder cancer, bladder cancer (childhood), bone cancer (osteosarcoma and malignant fibrous histiocytoma), brain stem glioma (childhood), brain tumor (adult), central nervous system embryonal tumors (childhood), cerebellar astrocytoma (childhood), cerebral astrocytoma/malignant glioma (childhood), ependymoblastoma (childhood), ependymoma (childhood), medulloblastoma (childhood), medulloepithelioma (childhood), pineal parenchymal tumors of intermediate differentiation (childhood), supratentorial primitive neuroectodermal tumors and pineoblastoma (childhood), visual pathway and hypothalamic glioma (childhood), brain and spinal cord tumors (childhood), breast cancer, breast cancer and pregnancy, breast cancer (childhood), breast cancer (male), bronchial tumors (childhood), Burkitt lymphoma, carcinoid tumor (childhood), carcinoid tumor (gastrointestinal), carcinoma of head and neck, central nervous system embryonal tumors (childhood), central nervous system lymphoma (primary), cerebellar astrocytoma (childhood), cerebral astrocytoma/malignant glioma (childhood), cervical cancer, cervical cancer (childhood), childhood cancers, chordoma (childhood), chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colorectal cancer, cutaneous T- cell lymphoma, embryonal tumors (central nervous system, childhood), endometrial cancer, ependymoblastoma (childhood), ependymoma (childhood), esophageal cancer, esophageal cancer (childhood), Ewing family of tumors, extracranial germ cell tumor (childhood), extragonadal germ cell tumor, extrahepatic bile duct cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric (stomach) cancer, gastric (stomach) cancer (childhood), gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), gastrointestinal stromal cell tumor (childhood), germ cell tumor (extracranial, childhood), germ cell tumor (extragonadal), germ cell tumor (ovarian), gestational trophoblastic tumor, glioma (adult), glioma (childhood) brain stem glioma, childhood cerebral astrocytoma, childhood visual pathway and hypothalamic, hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer (adult, primary), hepatocellular (liver) cancer (childhood, primary), Hodgkin lymphoma (adult), hodgkin lymphoma (childhood), hypopharyngeal cancer, hypothalamic and visual pathway glioma (childhood), intraocular melanoma, islet cell tumors (endocrine pancreas), Kaposi sarcoma, kidney (renal cell) cancer, kidney cancer, laryngeal cancer, laryngeal cancer (childhood), leukemia (acute lymphoblastic, adult), leukemia (acute lymphoblastic, childhood), leukemia (acute myeloid, adult), leukemia (acute myeloid, childhood), leukemia (chronic lymphocytic), leukemia (chronic myelogenous), hairy cell, lip and oral cavity cancer, non-small cell lung cancer, small cell lung cancer, non- Hodgkin adult lymphoma, non-Hodgkin childhood lymphoma, macroglobulinemia (Waldenstrom), melanoma, Merkel cell carcinoma, mesothelioma (adult malignant), mesothelioma (childhood), metastatic squamous neck cancer with occult primary, mouth cancer, multiple endocrine neoplasia syndrome (childhood), multiple myeloma/plasma cell neoplasm, mycosis fungoides, myelodysplastic syndromes, myelodysplastic / myeloproliferative diseases, myelogenous leukemia, chronic myeloid leukemia (adult acute), myeloid leukemia (childhood acute), myeloma (multiple), myeloproliferative disorders (chronic), nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, nasopharyngeal cancer (childhood), neuroblastoma, oral cancer (childhood), oral cavity cancer, lip and oropharyngeal cancer, ovarian cancer (childhood), ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, pancreatic cancer (childhood), papillomatosis (childhood), parathyroid Cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumors (childhood), pituitary tumor, pleuropulmonary blastoma, prostate Cancer, rectal cancer, renal pelvis and ureter transitional cell cancer, respiratory tract carcinoma involving the NUT gene on chromosome 15, rhabdomyosarcoma (childhood), salivary gland cancer, salivary gland cancer (childhood), soft tissue sarcoma (adult), soft tissue sarcoma (childhood), uterine sarcoma, small intestine cancer, squamous neck cancer with occult primary (metastatic), supratentorial primitive neuroectodermal tumors (childhood), testicular cancer, throat cancer, thymoma and thymic carcinoma, thymoma and thymic carcinoma (childhood), thyroid cancer, thyroid cancer (childhood), trophoblastic tumor (gestational, unknown primary site), carcinoma of adult (unknown primary site), cancer of childhood, unusual cancers of childhood, urethral cancer, uterine cancer (endometrial), uterine sarcoma, vaginal cancer, vaginal cancer (childhood), vulvar cancer, Wilms tumor, women's cancers) characterized by uncontrolled growth and spread of abnormal cells. The loss of differentiation, increased rate of growth, invasion of surrounding tissue and metastasis can result in death. Cancer is caused by both external factors (tobacco, chemicals, radiation, and infectious organisms) and internal factors (inherited mutations, hormones, immune conditions, and mutations that occur from metabolism). Cancer is treated with surgery, radiation, chemotherapy, hormone therapy, biological therapy, and targeted therapy.

Cancer is expected to overtake heart disease as the number one killer of people around the world by the year 2010. By 2030, the number of new cancer cases is expected to rise to 27 million, with 17 million cancer deaths.

Although many anticancer drugs are in research process and some of them are used in clinical practices (mechlorethamine, cyclophosphamide, busulfan, nitrosoureas, procarbazine, melphalan, methotrexate, 6-mercatopurine, fludarabine, capecitabine, 6-thioguanine, cytosine arabinoside, 5-fluorouracil, gemcitabine, dactinomycin, daunorubicin, doxorubicin, epirubicin, bleomycin, mitomycin C, etoposide, irinotecan, topotecan, vincristine, vinblastine, paclitaxel, docetaxel, hydroxyurea, cisplatin, carboplatin, asparaginase, mitoxantrone, amsacrine, imatinib, erlotinib, sorafenib, estrogens, tamoxifen, prednisone, flutamide, leuprolide, goserelin), there is no cure for cancer yet. Moreover, the cure rates of drugs for the treatment of cancers are unacceptably low, and the side effects of these drugs are severe. Therapeutic regimes with two cytotoxic drugs give better result compared to the use of only one drug, but the application of combination chemotherapy is usually followed by increase in toxicity. The toxic effects of the drugs on the cancer tissues only marginally exceed their toxic effects on normal, healthy cells of the body that should be protected from the effects of the drugs. Chemotherapy research in cancer treatment has been largely devoted to the search for drugs providing toxin specificity to destroy neoplastic tissue in the body without exceeding toxic exposure levels injurious to healthy tissues. For most cancer types, success has been quite limited.

Another obstacle in the cancer treatment is the acquirement of resistance after first-line therapy, which prevents the second-line chemotherapy to extend the life of patients. Some cancer types possess inherent resistance to the large scale of chemotherapeutics.

The discovery of new anti-cancer drugs that have more than one of the following characteristics is an imperative in our modern times: (1) ability to inhibit the growth of cancer cells, (2) ability to prevent cancer cell invasion to other tissues, (3) acceptable levels of toxicity to healthy cells, (4) effectiveness against cancer cells that are resistant to other drugs, and (5) a mechanism that differs from the action of conventional drugs, so the chance of the cancer cells to develop cross-resistance is reduced when the new drug is applied in combination with an existing drug.

The present invention relates to compounds having anti-cancer activity. The invention also relates to the use of combination of those compounds in non-toxic concentrations.

Up to now, not many diterpenes acting as anticancer agent were known. One of them was extracted from Taxus brevifolia, named Taxol or Docetaxel (references 2-23).Others have used agents from plant of the Euphorbiaceae family which are useful in the treatment and prophylaxis of prostate cancer in mammalian and in particular human subjects (United States Patent 7378445).

The Euphorbiaceae family of plants covers a wide variety of plants including weeds of Euphorbia species. There have been a variety of inconclusive reports on the potential effects of the sap of these plants on a variety of conditions as well as promoting tumorigenesis and causing skin and ocular irritation. A recent report describes selective cytotoxicity of a number of tiglilane diterpene esters from the latex of Euphorbia poisonii , a highly toxic plant found in Northern Nigeria, which is used as a garden pesticide. One of these compounds has a selective cytotoxicity for the human kidney carcinoma cell line A-498 more than 10,000 times greater than that of adriamycin (reference 28). Euphorbia hirta plants and extracts thereof 010 000004

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have been considered for a variety of purposes, including tumor therapy (EP 0 330 094), AIDS-related complex and AIDS (HU-208790) and increasing immunity and as an anti- fungoid agent for treatment of open wounds (DE-4102054).

Thus, while there are isolated reports of anti-cancer activity of various Euphorbia preparations (reference 28), not only are the compounds present in at least one Euphorbia species reported to be carcinogenic (references 30-32), but at least one species has a skin-irritant and tumor- promoting effect (reference 33) and another species reduces EBV-specific cellular immunity in Burkitt's lymphoma (reference 34).

On the other hand, till now, several types of diterpenes were isolated from Euphorbia dendroides plant, having the effect of aiding antineoplastic activities of certain cytostatic drugs (references 24-27). So for, no substances isolated from Euphorbia dendroides were known to work as an anticancer agent.

REFERENCES:

1. Mathers, Colin D, Cynthia Boschi-Pintb, Alan D Lopez and Christopher JL Murray (2001). Cancer incidence, mortality and survival by site for 14 regions of the world. Global Programme on Evidence for Health Policy Discussion Paper No. 13 (World Health Organization)

2. Lyseng- Williamson KA, Fenton C. Docetaxel: a review of its use in metastatic breast cancer. Drugs 2005;65(17):2513-31.

3. Clarke SJ, Rivory LP. Clinical pharmacokinetics of docetaxel. Clin Pharmacokinet 1999;36(2):99-114

4. Anonymous. Oncology Tools: Approved Claims for microtubule inhibitors. US Food and Drug Administration. (17 Sep 2006). Last modified 22 Jun 1998.

5. Anonymous. Taxotere.com for Healthcare Professionals: About. Sanofi-aventis U.S.

LLC. (17 Sep 2006). Last modified Jul 2005.

6. Anonymous. Taxotere Docetaxel concentrate for infusion. Medsafe. (25 Sep 2006).

Last modified 6 Feb 2006.

7. Snyder JP, Nettles JH, Cornett B, Downing KH, Nogales E. The binding conformation of Taxol in b-tubulin: A model based on electron crystallographic density. PNAS. 2001;98(9):5312-16. Rang HP, Dale MM, Ritter JM, Moore PK. Pharmacology. 5th ed. London: Churchill Livingstone; 2003. p. 100-1

Anonymous. Drugdex Evaluations: Docetaxel. Thomson MICROMEDEX. (26 Sep 2006). Last modified 2006.

Anonymous. Taxotere.com for Healthcare Professionals: Efficacy and Safety. Sanofi- aventis U.S. LLC. (24 Sep 2006). Last modified Jul 2005.

Anonymous. New Zealand Pharmaceutical Schedule. Wellington: PHARMAC; 2006. p. 133.

Urien S, Barre J, Morin C, Paccaly A, Montay G, Tillement JP. Docetaxel serum protein binding with high affinity to alpha 1-acid glycoprotein. Invest New Drugs. 1996;14(2):147-51.

Baker SD, Zhao M, Lee CKK, Verweij J, Zabelina Y, Brahmer JR, et al. Comparative pharmacokinetics of weekly and every-three-weeks docetaxel. Clin Cancer Res. 2004;10(6):1976-83.

Anonymous. Taxotere.com for Healthcare Professionals: Pharmacokinetics. Sanofi- aventis U.S. LLC. (23 Sep 2006). Last modified Jul 2005.

Guitton J, Cohen S, Tranchand B, Vignal B, Droz JP, Guillaumont M, et al. Quantification of docetaxel and its main metabolites in human plasma by liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom. 2005;19(17):2419-26.

Yvon AC, Wadsworth P, Jordan MA. Taxol Suppresses Dynamics of Individual Microtubules in Living Human Tumor Cells. The American Society for Cell Biology. 1999;10:947-959.

Anonymous. Docetaxel: Clinical Pharmacology. RxList. (24 Sep 2006). Last modified 29 Jun 2006.

Eisenhauer EA, Vermorken JB. The taxoids: Comparative clinical pharmacology and therapeutic potential. Drugs. 1998;55(l):5-30.

Tannock IF, de Wit R, Berry WR, Horti J, Pluzanska A, Chi KN, et al. Docetaxel plus prednisone or mitoxantrone plus prednisone for advanced prostate cancer. N Engl J Med. 2004;351(15):1502-12.

Goyle S, Maraveyas A. Chemotherapy for colorectal cancer. Dig Surg. 2005;22(6):401-14.

Rang HP, Dale MM, Ritter JM, Moore PK. Pharmacology. 5th ed. London: Churchill Livingstone; 2003. p. 694-8. Pierre Potier, chemist, 1998 CNRS Gold Medalist

Vogel CL, Bellet RE, inventors; Aventis Pharma S.A., assignee. Use of docetaxel for treating cancers. United States patent US20016333348. 2001 Dec 25.

Corea, G.; Di Pietro, A.; Dumontet, C; Fattorusso, E.; Lanzotti, V Jatrophane diterpenes from Euphorbia spp. as modulators of multidrug resistance in cancer therapy. Phytochemistry Reviews (2009), 8(2), 431-447.

Barile, Elisa; Corea, Gabriella; Lanzotti, Virginia Diterpenes from Euphorbia as potential leads for drug design. Natural Product Communications (2008), 3(6), 1003- 1020.

Corea, Gabriella; Fattorusso, Ernesto; Lanzotti, Virginia; Taglialatela-Scafati, Orazio; Appendino, Giovanni; Ballero, Mauro; Simon, Pierre-Noel; Dumontet, Charles; Di Pietro, Attilio. Modified jatrophane diterpenes as modulators of multidrug resistance from Euphorbia dendroides L Bioorganic & Medicinal Chemistry (2003), 11(23), 5221-5227.

Corea, Gabriella; Fattorusso, Ernesto; Lanzotti, Virginia; Taglialatela-Scafati, Orazio; Appendino, Giovanni; Ballero, Mauro; Simon, Pierre-Noeel; Dumontet, Charles; Di Pietro, Attilio Jatrophane Diterpenes as P-Glycoprotein Inhibitors. First Insights of Structure-Activity Relationships and Discovery of a New, Powerful Lead Journal of Medicinal Chemistry (2003), 46(15), 3395-3402.

Fatope MO, Zeng L, Ohayaga JE, Shi G, McLaughlin JL 1996. Selectively cytotoxic diterpenes from Euphorbia poisonii. J Med Chem 39: 1005-1008.

Sevil Oksiiza, b, Faliha Giireka, Long-ze Line, Roberto R. Gilc, John M. Pezzutoc and Geoffrey A. Cordell, Aleppicatines A and B from Euphorbia aleppica, Phytochemistry Volume 42, Issue 2, May 1996, Pages 473-478

A. Evans, M. A. Osman, Carcinogenicity of bracken and shikimic acid, Nature 250, 348 - 349 (26 July 1974)

Starvic and Stolz, Food Cosmet. Toxicol. 14: 141, 1976

Hecker "Cocarcinogens from Euphorbiaceae and Thymeleaceae" in " Symposium on Pharmacognosy and Phytochemistry", 147-165, (Wagner et al., eds., Springer Verlag, 1970).

Gundidza and Kufa, Centr. Afr. J. Med. 38: 444-447, 1992.

Imai, S., M. Sugiura, et al. (1994). African Burkitt's lymphoma: A plant, Euphorbia tirucalli, reduces Epstein-Barr virus-specific cellular immunity. Anticancer Research 000004

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14(3a): 933-936. Dep. Virol., Cancer Inst., Hokkaido Univ. Sch. Med., Sapporo 060, N15 W7, Japan

35. M. Pesic, J. Z. Markovic, D. Jankovic, S. Kanazir, I. D. Markovic, L. Rakic and S.

Ruzdijic, Induced Resistance in the Human Non Small Cell Lung Carcinoma (NCI- H460) Cell Line In Vitro by Anticancer Drugs, Journal of Chemotherapy, Vol. 18 (2006) - pp. 66-73

36. Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahon, J., Vistica, D., Warren, J. T., Bokesch, H., Kenney, S., and Boyd, M. R. (1990) New colorimetric cytotoxicity assay for anticancer-drug screening. J. Natl. Cancer Inst. 82, 1107-1112.

DISCLOSURE OF THE INVENTION

Antineoplastic agents are ususally highly toxic, and there is a strong need to find one with high antineoplastic activity and low toxicity. A pharmaceutical composition of very low toxicity containing diterpene compounds with antineoplastic activities was found in the Euphorbiaceae family of plants, including weeds of Euphorbia species, and particularly in Euphorbia dendroides, and is preferred in the practice of the present invention. Reference herein to Euphorbia dendroides includes various varieties, strains, lines, hybrids or derivatives of this plant as well as its botanical or horticultural relatives. Furthermore, the present invention may be practiced using a whole Euphorbiaceae plant or parts thereof including sap or seeds or other reproductive material. Generally, for seeds or reproductive material to be used, a plant or plantlet is first required to be propagated.

Reference herein to an Euphorbiaceae plant, a Euphorbia species or Euphorbia dendroides further encompasses genetically modified plants. Genetically modified plants include transgenic plants or plants in which a trait has been removed or where an endogenous gene sequence has been down-regulated, up-regulated, mutated or otherwise altered including the alteration or introduction of genetic material which exhibits a regulatory effect on a particular gene. Consequently, a plant which exhibits a character not naturally present in an

Euphorbiaceae plant or a species of Euphorbia or in Euphorbia dendroides is nevertheless encompassed by the present invention and is included within the scope of the above- mentioned terms. Furthermore, the present invention contemplates hybrid plant cells or plants comprising hybrid plant cells formed by the fusion of two or more plant cells from different strains, species or genera and optionally regenerating a plant therefrom. Such hybrid plant cells are proposed to generate novel secondary metabolites having useful therapeutic properties.

The diterpenes are generally in extracts of the Euphorbiaceae plants. An extract may comprise, therefore, sap or liquid or semi-liquid material exuded from, or present in, leaves, stem, flowers, seeds and bark or between the bark and the stem. Most preferably, the extract is from sap. Furthermore, the extract may comprise liquid or semi-liquid material located in fractions extracted from sap, leaves, stems, flowers, bark or other plant material of the Euphorbiaceae plant. For example, plant material may be subject to physical manipulation to disrupt plant fibres and extracellular matrix material and inter- and intra-tissue extracted into a solvent including an aqueous environment. The fractions may include aqueous or alcohol extracts. Other extraction media are also contemplated including fractions prepared by BPLC or other fractionation systems. All such sources of the d iterpenes are encompassed by the present invention including diterpenes obtained by synthetic routes.

The chemical agents of the present invention may be in purified or isolated form meaning that the preparation is substantially devoid of other compounds or contaminating agents other than diluent, solvent or carrier or isofbrms of the agents. Furthermore, the term "chemical agent" includes preparations of two or more compounds either admixed together or co-purified from a particular source. The chemical agent may also be a chemical fraction, extract or other preparation including sap from the Euphorbiaceae plant. The chemical agents or extracts or fractions of the present invention may also be referred to as "drugs" or "actives" or "active ingredients". The term "agent" is not to imply a synthetic compound and may include a fraction obtainable from the sap of the Euphorbiaceae plant. The term "obtainable" also includes "obtained".

Consequently, reference herein to a "chemical agent" includes a purified form of one or more compounds or a chemical fraction or extract such as from the sap of an Euphorbiaceae plant and in particular a species of Euphorbia, and most preferably from Euphorbia dendroides or botanical or horticultural relatives or variants thereof.

Accordingly, one aspect of the present invention contemplates a method for the treatment or prophylaxis of cancer or a related condition, said method comprising the administration to said subject of a symptom-ameliorating effective amount of a chemical agent obtainable from a plant of the Euphorbiaceae family or a derivative or chemical analog thereof which chemical agent is a diterpene selected from compounds extracted from an Euphorbiaceae plant of which chemical agent or derivative or chemical analog is represented by any one of the general formulae I, II and I I I,

Formula I Formula II Formula III wherein R represents hydrogen atom, hydroxy group, aliphatic alkoxy group with linear or branched, saturated or unsaturated, substituted or unsubstituted structure, or a radical represented by general formula RCOO-, wherein R denotes a linear or branched, saturated or unsaturated, substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic or heteroaromatic group. Supstituents R ² , R ³ , R ⁴ , R ⁵ , R ⁶ and R ⁷ may be same or different and represent hydrogen atoms, aliphatic groups with linear or branched, saturated or unsaturated, substituted or unsubstituted structure, or radicals represented by general formula RCO ^~ , wherein R denotes a linear or branched, saturated or unsaturated, substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic or heteroaromatic group.

R ¹ in the formulae I, II and III represents hydrogen atom, hydroxy group, aliphatic alkoxy group with linear or branched, saturated or unsaturated, substituted or unsubstituted structure, containing preferably from 1 to 30 carbon atoms, or a radical represented by general formula RCOO-. Supstituents R ² , R ³ , R ⁴ , R ⁵ , R ⁶ and R ⁷ in the formulae I, II and ΙΠ represent preferably linear or branched, saturated or unsaturated aliphatic groups containing preferably from 1 to 30 carbon atoms, or a radical represented by general formula RCO . The aliphatic group may be substituted with a halogen atom, a hydroxyl group, a carbonyl group, a carboxyl group, an amino group and an amide group.

Examples of carboxylic acids from which radicals RCOO- or RCO- are derived (wherein R represents an alkyl radical) include saturated aliphatic acids containing from 1 to 16 carbon atoms, such as acetic acid, propionoc acid, butiric acid, zso-butiric acid, 2,3-dimethyl butiric acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, and unsaturated aliphatic acids containing from 3 to 16 carbon atoms, such as 2,4-decadienoic acid. Examples of carboxylic acids from which RCOO- or RCO- is derived (wherein R represents an aromatic radical), include aromatic carboxylic acids, such as benzoic acid, phtalic acid, salicylic acid, and antranilic acid. Examples of carboxilic acid from which RCOO- or RCO- is derived (wherein R represents an heteroaromatic radical), include furanocarboxylic acid, thiophenecarboxilic acid, pyridinecarboxilic acid, such as nicotinic acid and wo-nicotinic acid. The aromatic and heteroaromatic radicals may be substituted with halogen atoms, hydroxyl radicals, carbonyl radicals, carboxyl radicals, amino radicals, and amide radicals.

A compound represented by the general formula I, wherein R ^! and R ⁷ are hydrogen atoms, R ² is propanoyl radical, R ³ and R ⁵ are acetyl radicals, R ⁴ is /so-butanoyl radical and R ⁶ is nicotinyl radical (compound 1), and a compound represented by the general formula I wherein R ^! and R ⁷ are hydrogen atoms, R ³ and R ⁵ are acetyl radicals, R ² and R ⁴ are iso- butanoyl radicals and R ⁶ is nicotinyl radical (compound 2), are prepared by extraction of lyophilized extract of Euphorbia dendroides with organic solvents such as hexane or methylene chloride at room temperature, followed by purifying the extracts according to known procedures.

A compound represented by the general formula Π, wherein R ¹ is acetoxy radical, R ² , R ³ , R ⁵ and R ⁷ are acetyl radicals, R ⁴ is /so-butanoyl radical, R ⁶ is nicotinyl radical (compound 6), is prepared by extraction of lyophilized extract of Euphorbia dendroides with organic solvents such as hexane or methylene chloride at room temperature, and then purifying the extracts. A compound represented by the general formula 111, wherein R ¹ is hydroxy radical, R ² hydrogen atom, R ³ benzoyl radical and R ⁴ z ^' so-butanoyl radical, (compound 7), is prepared extraction of lyophilized extract of Euphorbia dendroides with organic solvents such as hexane or methylene chloride at room temperature, and then purifying the extracts according to known procedures.

These isolated compounds may be used as starting materials for the preparation of other compounds represented by the general formulae I, II and ΙΠ, respectively. For example, compound 1 is hydrolyzed to give the compound of formula I wherein R ² is hydrogen atom, and then the obtained hydroxyl compound is converted to ether compound of the present invention in accordance with Williamson Synthesis described below, that is the action of sodium alkoxides and then alkylhalides R'X, wherein R' has the same meaning as R ² and X represents a halogen atom, (Fig. 1). Further, compound 1 after hydrolysis (formula I wherein R ² is a hydrogen atom) may react with acid anhydrides, i.e., (R"CO) ₂ 0 in the presence of anhidrous pyridine to give ester compounds of the present invention (Fig 2).

ISOLATION

To the aerial parts of Euphorbia dendroides (1 kg) collected on the territory of west Balkans, 60% (v/v) of aqueous ethanol (3 L) was added and the mixture left for ten days at room temperature with stirring. The extract was lyophilized at 40° C to give the concentrated extract (lOOg). The concentrated extract was mixed with hexane (350 ml) and placed in an ultra sound bath for 45 minutes. The soluble part was decanted and the extraction repeated under same conditions. The combined extracts were concentrated under reduced pressure at 40° C to give the concentrated extract (4.65 g). The crude extract was subjected to dry flash chromatography on silica gel using toluene/ethyl acetate in different proportions as eluent. The fraction 1, eluated with 5% - 20 % ethyl acetate hadn't shown biological activity and consisted mostly of fatty acid esters, according to proton NMR spectrum. Fractions 2-6 were eluated with higher percent of ethyl acetate in toluene and exibited biological activity. These fractions were purified by preparative thin layer chromatography (TLC) on silica gel plates 20 cm x 20 cm, with the layer thicknes of 0.5 mm, 1 mm or 2 mm, the choice of the layer thickness depended on complexity of the separating mixture. Final purification was performed by HPLC to obtain diterpene compounds 1-7.

Fraction 2 (0.26 g, eluated with 30 % ethyl acetate in toluene) was subjected to preparative TLC on silica gel, layer thicknes 2 mm, developing system hexane-acetone = 7:3 (the plates were developed 3 times) to give subtraction 2-1 (77.7 mg). Final purification by HPLC ¹ afforded pure compounds 4 (4.5 mg), 5 (4.8 mg) and 7 (5 mg).

Fraction 3 (0.25 g, eluated with the second portion of 30 % ethyl acetate in toluene) after preparative TLC on silica gel, layer thickness 1 mm, developing system hexane-acetone = 7:3 (the plates were developed 5 times) gave two subfractions. Subtraction 3-1 (81 mg) was purified by HPLC1 to give 1 (8 mg) and 2 (16 mg). Subtraction 3-2 (63 mg) after purification by HPLC yelded additional amount of 2 ( 8.6 mg).

¹ Performed on Zorbax XDB-C18 column, with the mobile phase a mixture of 0.20 % formic acid in MilliQ water and acetonitrile (gradient mode). P T/RS2010/000004

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Preparative TLC of fraction ^ (0.16 g, eluated with 40 % ethyl acetate in toluene) on silica gel, layer thickness 2 mm, developing system hexane-acetone = 7:3 (the plates were developed 3 times) afforded two subfractions. Subtraction 4-1 (50 mg), after HPLC purification afforded 1 (17 mg) and 3 (2 mg). Subtraction 4-2 (51 mg) upon HPLC yelded aditional 1 (16 mg) and 2 (8 mg).

Fraction 5 (0.09 g, eluated with 50 %- 100 % ethyl acetate in toluene) was subjected to preparative TLC on silica gel, layer thickness 0.5 mm, developing system hexane-acetone = 65:35 (the plate was developed 4 times) to afford three subfractions. Subfractions 5-1 (12.4 mg) and 5-2 (23 mg), after purification by HPLC, both yielded compound 6 (2 mg and 4.4 mg, respectively). Purification of subfraction 5-3 (21 mg) by HPLC afforded additional amount of 1 (2.2 mg).

Preparative TLC of fraction 6 (0.12 g, eluated with 10 % methanol in ethyl acetate) on silica gel plate, layer thickness 0.5 mm, developing system hexane-acetone = 65:35 (the plate was developed 4 times) afforded subfraction 6-1 (30 mg), purified by HPLC, to give additional amount of compound 6 (2.5 mg).

The MS, UV and IR data for compounds 1-7 are given in Table 1.

Table 1

1H NMR and ¹³ C NMR data for compounds 1 -7 are given in Tables 2-4. 04

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Table 2

Table 3

Table 4

Accordingly, the structure of compounds 1 to 7 were identified from the data given above, and interpretation of two dimensional NMR spectroscopy (COSY, HSQC, HMBC, NOESY).

Table 5

Structures of the compounds 1 to 7

Formula I Formula II Formula III

Pr-propanoyl, Ac- acetyl, iBu- iso-butanoyl, Nic- nicotinyl. and Bz- benzoyl

THE PROCEDURE FOR DETERMINATION OF BIOLOGICAL ACTIVITY

Each compound was tested against four cultured, cancer cell lines of human origin, three of which (NCI-H460, DLD-1 and U-87 MG) were from ATCC (American Type Culture Collection). NCI-H460/R cell line with multidrug resistant phenotype was originally selected from NCI-H460 cells and cultured in a medium containing ΙΟΟηΜ doxorubicin (DOX) (reference 35). NCI-H460 (non-small cell lung carcinoma), NCI-H460/R (multidrug resistant non-small cell lung carcinoma) and DLD-1 (colorectal adenocarcinoma) cells were 10 000004

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maintained in RPMI 1640 supplemented with 10% FBS (Fetal Bovine Serum), 2mM L- glutamine, 4.5 g/1 glucose, 10.000 U/ml penicillin, 10 mg/ml streptomycin, 25 μ¾ ι 1 amphotericin B solution at 37° C in a humidified 5 % C0 ₂ atmosphere. U-87 MG (glioblastoma) cells were maintained in Eagle Minimum Essential Medium supplemented with 10% FBS, 2mM L-glutamine, 10.000 U/ml penicillin and 10 mg/ml streptomycin at 37° C in a humidified 5 % C0 ₂ atmosphere. All cell lines were sub cultured at 72 h intervals using 0.25 % trypsin/EDTA and seeded into a fresh medium at the following densities: 8.000 cells/cm ² for NCI-H460, 16.000 cells/cm ² for NCI-H460/R, 16.000 cells/cm ² for DLD-1 and 16.000 cells/cm ² for U-87 MG. Paclitaxel (PTX) is used as positive control for cross- resistance.

Additional cancer cell line against which compounds were tested in certain series was K-562 (chronic myelogenous leukemia). K-562 cells were maintained in RPMI 1640 with 10% FCS (Fetal Calf Serum).This cell line was sub cultured at 72 h intervals using 0.1 % trypsin/EDTA and seeded into a fresh medium at 20.000 cells/cm ² . Human normal lymphocytes were used in order to determine the level of toxicity on healthy cells. Cisplatin (CPt) is used as positive control for toxicity.

Cells grown in 25 cm ² tissue flasks were trypsinized, seeded into flat-bottomed 96-well tissue culture plates in 150 μΐ medium and incubated overnight. The working solution of ImM for each compound was in 25 % ethanol. Further dilutions were made in a cell culture medium. Tested concentrations in final volume of culture medium (200 μΐ) were 0.1 μΜ, 0.5 μΜ, 1 μΜ, 5 μΜ, 10 μΜ, 25 μΜ and 50 μΜ. The compounds were added to the cells in 50 μΐ of all samples under continuous culture for 72 hours. Inhibition of growth relative to untreated control was determined by the SRB (Sulforhodamine B) assay and MTT (3-(4,5- Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) assay. These assays test the relative ability of a compound to inhibit cell growth, but not survival of the cells. However, inhibition of growth may reflect cell death (necrosis or apoptosis) and/or cytostasis.

SRB assay (reference 36): The cellular proteins were stained with sulforhodamine B (SRB) assay that was performed with some changes. Briefly, the cells in 96-well plates were fixed in 50 % trichloroacetic acid (50 μΐ/well) for 1 h at 4° C, rinsed four times in tap water and stained with 0.4 % (w/v) sulforhodamine B in 1% acetic acid (50 μΐ/well) for 30 min at room temperature. The cells were then rinsed three times in 1% acetic acid (250 μΐ) to remove the unbound stain. The protein-bound stain was extracted with 200 μΐ 10 mM Tris base (pH 10.5) per well. The optical density was read at 540 nm, with correction at 670 nm (LKB 5060-006 Micro Plate Reader, Vienna, Austria). Values are average of quanta plicate determinations. Growth inhibition (I) was determined according to the following equitation:

I (%) = (1 - (A treated sample / untreated control) X 100.

IC ₃₀ and IC ₅₀ values were defined as the concentration of the drug that inhibited cell growth by 30% and 50%, respectively. The inhibitory concentrations for the compounds were calculated by linear regression analysis using Excel software.

RESULTS

Results of the in vitro cytotoxicity testing, specifically IC ₃₀ and IC ₅ o values for the various cancer cell lines, are given in Table 1. Cross-resistance profile of tested compounds compared with PTX is shown in Table 2.

Table 1. Growth inhibition effect of tested compounds on various cancer cell lines

Co NCI-H460 NCI-H460/R DLD-1 U-87 MG K-562 mp IC30 IC50 IC30 IC50 IC30 IC50 IC30 IC50 IC30 IC50 ou

nd

1 >50μΜ >50μΜ >50μ >50μ 6.1 Μ 46μΜ >50μΜ >50μΜ 1.4μΜ 5.1μΜ

2 2.5μΜ 5.6μΜ 25μΜ 46μΜ 1.9μΜ 18μΜ 5.6μΜ >50μΜ >50μΜ >50μΜ

3 4.8μΜ Ι Ι . ΙμΜ 12μΜ 23μΜ 12μΜ 25μΜ 35μΜ >50μΜ 4.5μ 13μΜ

4 Ι ΙμΜ 20μ 14μΜ 28μΜ 14μΜ 38μΜ 10μΜ 46μΜ 6.2μΜ 14μΜ

5 8.3μΜ 22μΜ 15μΜ 50μΜ 14μΜ 39μΜ 10μΜ >50μΜ 5.7μΜ 15μΜ

6 5μΜ 16μΜ 50μΜ >50μΜ 37μ >50μΜ 24μΜ >50μΜ 3.1μΜ 6.7μΜ

7 6.3μΜ 13μΜ 16μΜ 24μΜ 13μΜ 24μΜ 35μΜ >50μΜ 4.6μΜ Π μΜ

An IC50 value above 50 μΜ indicates that there was insufficient cytotoxicity of the compound to achieve a 50% inhibition of cell growth at 50 μΜ.

Table 2. Relative cross-resistance to tested compounds

Resistance

Compound Factor

Paclitaxel 185.7

Vinblastine 34,6

Etoposide 21,2

Epirubicin 8,9 Doxorubicin 54,5

2 8.2

3 2.1

4 1.4

5 2.3

7 1.8

Resistance factor is relation between ICso values of resistant and sensitive non-small cell carcinoma cell lines (Resistance factor = ICso ofNCI-H460/R/IC ₅₀ ofNCI-H460)

Figure 1. Effect of 1, 2, 3, 4, 5, 6, 7 and CPt on viability of normal human lymphocytes measured by MTT. The healthy lymphocytes were preserved after 72h treatment with 1, 2, 3, 4, 5, 6 and 7 (at tested concentrations the percent of viable lymphocytes was about 90-95%). CPt induced significant decrease in lymphocyte counts (57% of lymphocytes survived at 5 μΜ and only 35% at 50 μΜ). 04

22

1 is active against colorectal adenocarcinoma and chronic myelogenous leukemia (Table 1). 1 shows great potential for treatment of leukemia, but not for solid tumors. This cytotoxic effect is followed by low toxicity of 1 observed on healthy lymphocytes (Figure 1). IC ₅₀ for 1 was above 50 μΜ for sensitive and resistant non-small cell lung carcinoma cell lines, and therefore resistance factor was not shown in Table 2.

2 is active against non-small cell lung carcinoma, colorectal adenocarcinoma and glioblastoma (Table 1). 2 shows great potential for treatment of non-small cell lung carcinoma and other solid tumors, but not for leukemia. This cytotoxic effect is followed by low toxicity of 2 observed on healthy lymphocytes (Figure 1). 2 is also active against resistant non-small cell lung carcinoma, with cross-resistance below the one obtained with PTX (Table 2).

4 is active against non-small cell lung carcinoma, colorectal adenocarcinoma, glioblastoma and leukemia (Table 1). 4 is also active against resistant non-small cell lung carcinoma, with very low cross-resistance (Table 2).

7 is active against non-small cell lung carcinoma, colorectal adenocarcinoma and leukemia (Table 1). 7 is also active against resistant non-small cell lung carcinoma, with very low cross-resistance (Table 2).

5 is active against non-small cell lung carcinoma, colorectal adenocarcinoma, glioblastoma and leukemia (Table 1). 5 is also active against resistant non-small cell lung carcinoma, with very low cross-resistance (Table 2).

6 is active against non-small cell lung carcinoma and chronic myelogenous leukemia (Table 1). 6 shows great potential for treatment of leukemia, but not for solid tumors. This cytotoxic effect is followed by low toxicity of 6 observed on healthy lymphocytes (Figure 1). IC50 for 6 was above 50 μΜ for resistant non-small cell lung carcinoma cell line, and therefore resistance factor was not shown in Table 2.

3 is active against against non-small cell lung carcinoma, both nonresistant and resistant cell lines, colorectal adenocarcinoma as well as chronic myelogenous leukemia (Table 1). Resistance factor was shown in Table 2. The best effect on non-small cell lung carcinoma (NCI-H460) is achieved by 2, 7, 6 and 3 (Table 1).

The best effect on colorectal adenocarcinoma (DLD-1) is achieved by 2, 7, 3 and 1 (Table 1). The best effect on glioblastoma (U-87 MG) is achieved by 4, 2 and 5 (Table 1). The best effect on chronic myelogenous leukemia (K-562) is achieved by 1, 6 and 3 (Table 1). The lowest cross-resistance is obtained with 4, 7, 5, and 3 (Table 2).

2 and 3 demonstrate great potential for treatment of solid tumors, while 1, 6 and 3 show strong anti-cancer effect toward lymphoid malignances. All seven compounds do not exert toxic effect on healthy human cells. CPt, which is already in use as first line chemotherapeutic, significantly lowers the number of healthy human lymphocytes at 5 μΜ (the concentration effective against cancerous cells). 2, 4, 7, 5 and 3 are prospective drugs for treatment of resistant cancer cells. The cross-resistance to these compounds is enormously lower than the one obtained with PTX, which is also first line chemotherapeutic.

Raising the dose of paclitaxel, vinblastine, etoposide, epirubicin or doxorubicin, as shown in Table 2 by any amount increases their toxicity by the same amount, and therefore increases negative side effects to the human organism. In order to eliminate resistant cancer cells in vitro or in human organism, substances 2, 4, 7, 5 and 3 have in vitro shown results under 50 μΜ while, at the same time, it showed no toxic effects on healthy human cells.

BEST MODE FOR CARRYING OUT THE INVENTION

The novel substances may be used as dietary supplements or pharmaceutical compositions. The dietary composition may be in form of food such as dairy products (yoghurts), in form of fortified food such as cereal bars and bakery items such as cakes and cookies, in form of dietary supplements such as tablets, pills, granules, dragees, capsules, and effervescent formulations, in form of non-alcoholic drinks such as soft drinks, sport drinks, fruit juices, lemonades, near-water drinks, teas and milk based drinks, in form of liquid food such as soups and dairy products (muesli drinks), in form of alcoholic drinks such as brandy, wme or beer, or in any other beverage.

The pharmaceutical compositions may be administered in various forms, such as tablets, powders, granules, capsules, injections, suppositories, ointments and cataplasms. The pharmaceutical composition containing the active compounds of the present invention may be formulated by using conventional carriers and additives such as vehicles and resolvents, bases, diluents, fillers, adjuvants, such as solvent adjuvants, emulsifying agents, dispersers, disintegrants, solubilizers, viscosity-increasing agents, and lubricants, and additives such as antioxidans, preservatives, flavoring agents and sweetening agents.

INDUSTRIAL APPLICABILITY

Euphorbia dendroides can be planted industrially, it gives lots of juice from which the basic diterpenes may be extracted. It is a long living plant, and only the aerial parts of it are used, so it need not be destroyed when taking parts of it for the process needed. The chemical processes needed to extract the diterpenes which are presented by this invention are standard, whereas the processes needed to transform them are tested during the preparation of this application, and are fully feasible in ammounts needed for mass production of the compounds.