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  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
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• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
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• • A—HUMAN NECESSITIES
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  • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
  • C07D239/70—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings condensed with carbocyclic rings or ring systems

Definitions

• the present invention is concerned with the finding that some of the macrocyclic quinazoline derivatives described in PCT publication WO2004/105765 are useful as inhibitors of VEGFR3 mediated biological activities, especially those activities which are mediated by VEGFR3 ligands VEGF-C and/or VEGF-D.
• Cancer is still a major cause of death in the world at the beginning of the 21st century and remains a major focus for ongoing research and development.
• Metastasis to regional lymph nodes via lymphatic vessels is a common step in the progression of cancer. Metastasis is an important prognostic factor in many types of cancer and forms the basis for surgical and radiation treatment of local lymph nodes.
• the process of tumor metastasis is a multistage event involving local invasion and destruction of intercellular matrix, intravasation into blood vessels, lymphatic or other channels of transport, survival in the circulation, extravasation out of the vessels in the secondary site and growth in the new location (Idler, et al., Adv. Cancer Res. 28,149-250 (1978), Liotta, et al., Cancer Treatment Res. 40,223-238 (1988), Nicolson, Biochim. Biophy. Acta 948, 175-224(1988) and Zetter, N. Eng. J. Med. 322,605-612 (1990)). Success in establishing metastatic deposits requires tumor cells to be able to accomplish these steps sequentially.
• lymphangiogenesis the formation of lymphatic vessels, promotes lymphatic metastasis (Stacker et al., Nature Med. 7(2), 186-191 (2001); Skobe et al., Nature Med. 7(2), 192-8 (2001); Makinen et al., Nature Med. 7(2), 199-205 (2001)).
• the control of lymphangiogenesis may provide a new strategy for preventing lymph node metastasis in cancer therapy.
• VEGF-C vascular endothelial growth factor family
• VEGFR3 vascular endothelial growth factor 3
• VEGF-C has also been shown to promote lymphatic-mediated metastasis via induction of tumor-associated lymphangiogenesis in numerous solid cancers, such as gastric cancer, prostatic cancer, human colorectal cancer, invasive cervical cancer, breast cancer metastases and human melanoma metastases.
• VEGFR3 a transmembrane tyrosine kinase receptor is expressed broadly in endothelial cells during early embryogenesis (Pajusola K., et al, Cancer Res. 53(16):3845 (1992)). During later stages of development, the expression of
• VEGFR-3 becomes restricted to developing lymphatic vessels [Kaipainen, A., et al., Proc. Natl. Acad. Sci. USA, 92: 3566-3570 (1995)].
• the lymphatic endothelium and some high endothelial venules express VEGFR-3, and increased expression occurs in lymphatic sinuses in metastatic lymph nodes and in lymphangioma.
• VEGFR-3 is also expressed in a subset of CD34+ hematopoietic cells which may mediate the myelopoietic activity of VEGF-C demonstrated by overexpression studies.
• VEGFR-3 Targeted disruption of the VEGFR-3 gene in mouse embryos leads to failure of the remodeling of the primary vascular network, and death after embryonic day 9.5 [Dumont et al., Science, 282: 946-949 (1998)]. These studies suggest an essential role for VEGFR-3 in the development of the embryonic vasculature, and also during lymphangiogenesis.
• inhibitors of VEGFR3 have tremendous potential as therapeutics, and new agents of this type represent a continuing need in the art for inhibiting growth and/or spread of a variety of neoplastic disorders or cell proliferative disorders.
• Inhibition of VEGFR3 activity is useful in the treatment of mammalian subjects that have been diagnosed with a disease characterized by proliferation of endothelial cells that express VEGFR-3.
• many tumors are characterized by blood vessel or lymphatic vessel neovascularization, wherein the neovascularization comprises endothelial cells that express VEGFR-3.
• the cancer cells themselves express VEGFR3, and represent the target cells.
• the cancer cells express a VEGFR-3 ligand selected from VEGF-C and VEGF-D.
• the ligand is believed to be involved in recruiting endothelial cells and stimulating their growth, thereby facilitating nourishment and/or spread of the cancer.
• the invention is directed in part to methods of treating or preventing VEGFR3 mediated biological activities utilizing certain compounds described in WO2004/105765, the disclosure of which is hereby incorporated by reference in its entirety.
• the invention provides a method of inhibiting metastatic spread of a cancer in a mammalian subject comprising administering to a mammalian subject suspected of having cancer a compound of the invention, in an amount effective to inhibit metastatic spread of the cancer; and a method for treating cancer comprising administering to a mammalian subject diagnosed with a cancer a composition comprising a compound of the invention, in an amount effect to reduce growth or neoplastic spread of the cancer.
• any reduction in the rate of cancer growth or spread is indicative of successful treatment.
• cancer growth is halted completely.
• cancers shrink or are eradicated entirely.
• Preferred subjects for treatment are human subjects, but other animals, especially murine, rat, canine, bovine, porcine, primate, and other model systems for cancer treatment, are contemplated.
• cancers that express VEGFR-3 direct inhibition of cancer growth or cancer killing may be the mechanism.
• Treatment of all cancers that express VEGFR-3 and all cancers characterized by arigiogenesis or lymphangiogenesis in and around a growing tumor is contemplated.
• treatment is contemplated of cancers of a tissue, organ, or cell selected from the group consisting of brain, lung, liver, spleen, kidney, lymph node, small intestine, blood cells, pancreas, colon, stomach, breast, endometrium, prostate, testicle, ovary, skin, head and neck, esophagus, bone marrow and blood.
• treatment is contemplated of cancers of a tissue, organ or cell selected from the group consisting of Breast, Lung, Prostate and Colon.
• the compounds are administered along with a second cancer therapeutic agent.
• the second agent can be any chemotherapeutic agent, radioactive agent, radiation, nucleic acid encoding a cancer therapeutic agent, antibody, protein, and/or other anti-lymphangio genie agent or an anti-angiogenic agent.
• the second agent may be administered before, after, or concurrently with the compounds of the invention.
• the subject to be treated has been diagnosed with an operable tumor, and the administering step is performed before, during, or after the tumor is resected from the subject.
• Compound treatment in conjunction with tumor resection is intended to reduce or eliminate regrowth of tumors from cancer cells that fail to be resected.
• the invention provides a method of treating a pathology characterized by VEGFR-3 binding to a natural ligand that binds VEGFR-3, comprising the step of administering to an individual in need thereof a compound of the invention.
• Top frame provides the results of V ⁇ GFR-3 immunoprecipitates transferred to a nitrocellulose membrane, stained with phospho-tyrosine antibodies.
• Lower frame provides the results of VEGFR- 3 immunoprecipitates transferred to a nitrocellulose membrane, stained with VEGFR-3 antibodies.
• Left lanes 1 and 2 provide the negative and positve controls, i.e. DMSO treatment in the absence and presence of VEGF-C.
• FIG. 2 VEGF and VEGF-C stimulation of Erkl/2 phosphorylation in HMVECd cells in the presence of 5uM of Compound 2.
• Top lane provides staining of phoshorylated Erk 1/2 (P-Erk 1/2) using Thr202/Tyr204 mouse monoclonal antibody as primary antibody and goat anti-mouse conjugated to IRDye 800nm as secondary antibody.
• Bottom lane provides staining of total Erk 1/2 using Erk 1/2 specific rabbit polyclonal antibody as primary antibody and goat anti-rabbit conjugated to Alexa 680 nm as secondary antibody.
• Left panel provides the results for VEGF induced phosphorylation of Erk- 1/2 in HMVECd cells.
• Right panel provides the results for VEGF-C induced phosphorylation of Erk 1/2 in HMVECd cells.
• WO-2004/105765 describes the preparation, formulation and pharmaceutical properties of a class of macrocyclic quinazoline derivatives of formula (I) as multi targeted kinase inhibitors.
• VEGFR3 inhibitory activity that makes them useful in the treatment or prevention of VEGFR3 mediated biological activities, in particular for the treatment or prevention of metastatic spread of a cancer in a mammalian subject.
• the present invention provides the use of the compounds of formula (I) wherein;
• Z represents NH
• Y represents -C 3-9 alkyl-, -C 1-5 alkyl-NR 13 -C 1-5 alkyl-, -Ci -5 alkyl-NR 14 -CO-C 1-5 alkyl-,
• X 1 represents O, or -O-C 1-2 alkyl-;
• X 2 represents a direct bond, -Ci -2 alkyl-, O, -O-Ci -2 alkyl-, NR 12 or NR 12 -C 1-2 alkyl-;
• R 1 represents hydrogen, cyano, halo or hydroxy, preferably halo;
• R 2 represents hydrogen, cyano, halo, or hydroxy;
• R 3 represents hydrogen;
• R 4 represents C 1-4 alkyloxy-;
• R represents hydrogen, or C ⁇ alkyl-
• R 13 represents hydrogen or Ci -4 alkyl
• R 14 represents hydrogen or Q ⁇ alkyl
• Het 20 represents pyrrolidinyl, piperazinyl or piperidinyl
• Het 22 represents pyrrolidinyl, piperazinyl or piperidinyl; the pharmaceutically acceptable acid or base addition salts and the stereochemical ⁇ isomeric forms thereof, in the manufacture of a medicament for the treatment or prevention of VEGFR3 mediated biological activities, such as for example in the treatment or prevention of metastatic spread of a cancer in a mammalian subject.
• a further aspect of the present invention is directed to a method for the treatment or prevention of a VEGFR3 mediated biological activity, such as for example in the treatment or prevention of metastatic spread of a cancer in a mammalian subject, comprising administering therapeutically effective amount of a compound of formula I
• Z represents NH
• Y represents -C 3-9 alkyl-, -C 1-5 alkyl-NR 13 -Ci -5 alkyl-, -C 1-5 alkyl-NR 14 -CO-C 1-5 alkyl-, -C 1-3 alkyl-NH-CO-Het 20 -, or -Het 22 -CH 2 -CO-NH-C 1-3 alkyl-;
• X 1 represents O, or -O-C 1-2 alkyl-
• X 2 represents a direct bond, -C 1-2 alkyl-, O, -O-Ci -2 alkyl-, NR 12 or NR 12 -C 1-2 alkyl-;
• R 1 represents hydrogen, cyano, halo or hydroxy, preferably halo;
• R 2 represents hydrogen, cyano, halo, or hydroxy
• R 3 represents hydrogen
• R 4 represents C 1-4 alkyloxy-
• R 12 represents hydrogen, or C 1-4 alkyl-;
• R 13 represents hydrogen or
• R 14 represents hydrogen or Ci ⁇ alkyl
• Her 20 represents pyrrolidinyl, piperazinyl or piperidinyl
• Her 22 represents pyrrolidinyl, piperazinyl or piperidinyl; and the pharmaceutically acceptable acid or base addition salts and the stereochemically isomeric forms thereof; to a mammalian subject in need of such treatment.
• VEGFR3 mediated biological activities are meant to include; metastatic spread of a cancer in a mammalian subject.
• Preferred subjects for treatment are human subjects, but other animals, especially murine, rat, canine, bovine, porcine, primate and other model systems for cancer treatment are contemplated.
• Metastasis to regional lymph nodes via lymphatic vessels provides the use of the compounds according to the invention in the treatment or prevention of lymph node metastasis; tumor-associated lymphangiogenesis in cancers, such as for example in gastric cancer, prostatic cancer, human colorectal cancer, invasive cervical cancer, breast cancer, Kapsoi's sarcoma and melanoma.
• the compounds according to the invention in the treatment or prevention of tumor-associated lymphangiogenesis in cancers, such as for example in gastric cancer, prostatic cancer, human colorectal cancer, invasive cervical cancer, breast cancer, Kapsoi's sarcoma and melanoma; recruitment and proliferation of endothelial cells that express VEGFR3 in neovascularization of cancer cells that express a VEGFR3 ligand selected from VEGF-C or VEGF-D. It accordingly provides the use of the compounds according to the invention in the treatment of cancers characterized by angiogenesis or lymphangiogenesis in and around a growing tumor.
• the present invention provides the use of an aforementioned compound of formula (I) for the preparation of a pharmaceutical composition for treatment of cancers of a tissue, organ or cell selected from the group consisting of Breast, Lung (including the treatment of both small cell and non- small cell lung cancer), Colon and Prostate.
• the present invention also concerns a method of treating breast cancer, lung cancer, colon cancer and/or prostate cancer in a mammal, comprising the step of administering a therapeutically effective amount of an aforementioned compound of formula (I) to said mammal.
• the present invention provides the use of an aforementioned compound of formula (I) for the preparation of a pharmaceutical composition for treating advanced breast cancer.
• advanced breast cancer is used herein to denote breast cancer which has not responded to previous treatment, or which has recurred following such treatment, and also breast cancer in patients who present with metastatic disease at diagnosis.
• the present invention also concerns a method of treating advanced breast cancer in a mammal, particularly a woman, comprising the step of administering a therapeutically effective amount of an aforementioned compound of formula (I) to said mammal.
• the present invention is concerned with the use of those compounds of formula (I) wherein one or more of the following restrictions apply;
• Z represents NH;
• Y represents -C 3-9 alkyl-, -C 1-5 alkyl-NR 14 -CO-C 1-5 alkyl-, or -C 1-3 alkyl-NH-CO-Het 20 -;
• X 1 represents O;
• X 2 represents -C 1-2 alkyl-, O, or NR 12 -C 1-2 alkyl-;
• R represents hydrogen or halo; in particular R 1 represents hydrogen or chloro; more in particular R 1 represents hydrogen;
• R 2 represents hydrogen or halo; in particular R 2 represents hydrogen, chloro or bromo; more in particular R represents chloro or bromo;
• R 3 represents hydrogen;
• R 4 represents Ci -4 alkoxy; in particular R 4 represents methoxy; R 12 represents C 1-4 alkyl-; in particular R 12 represents methyl; R 14 represents hydrogen or Ci -4 alkyl; in particular R 14 represents hydrogen or methyl; more in particular R 14 represents hydrogen; Her 20 represents pyrrolidinyl, piperazinyl or piperidinyl; in particular Het represents piperidinyl.
• Most preferred compounds are those compounds selected from the group consisting of; 4,6-ethanediylidene- 19H-pyrimido[4,5-6] [6,13,1 Jbenzodioxaazacyclo-pentadecine,
• - halo is generic to fluoro, chloro, bromo and iodo
• - C ⁇ alkyl defines methyl or ethyl
• - C,_ 3 alkyl defines straight and branched chain saturated hydrocarbon radicals having from 1 to 3 carbon atoms such as, for example, methyl, ethyl, propyl and the like;
• - C j _ 4 alkyl defines straight and branched chain saturated hydrocarbon radicals having from 1 to 4 carbon atoms such as, for example, methyl, ethyl, propyl, butyl,
• - C j.5 alkyl defines straight and branched chain saturated hydrocarbon radicals having from 1 to 5 carbon atoms such as, for example, methyl, ethyl, propyl, butyl, pentyl, 1-methylbutyl, 2,2-dimethylpropyl, 2,2-dimethylethyl and the like;
• - C 3-9 alkyl defines straight and branched chain saturated hydrocarbon radicals having from 3 to 9 carbon atoms such as propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl and the like;
• - Cj -4 alkyloxy defines straight or branched saturated hydrocarbon radicals such as methoxy, ethoxy, propyloxy, butyloxy, 1-methylethyloxy, 2-methylpropyloxy and the like;
• the pharmaceutically acceptable acid or base addition salts as mentioned hereinabove are meant to comprise the therapeutically active non-toxic acid and nontoxic base addition salt forms which the compounds of formula (I) are able to form.
• the compounds of formula (I) which have basic properties can be converted in their pharmaceutically acceptable acid addition salts by treating said base form with an appropriate acid.
• Appropriate acids comprise, for example, inorganic acids such as hydrohalic acids, e.g. hydrochloric or hydrobromic acid; sulfuric; nitric; phosphoric and the like acids; or organic acids such as, for example, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic, malonic, succinic (i.e.
• the compounds of formula (I) which have acidic properties may be converted in their pharmaceutically acceptable base addition salts by treating said acid form with a suitable organic or inorganic base.
• Appropriate base salt forms comprise, for example, the ammonium salts, the alkali and earth alkaline metal salts, e.g.
• the lithium, sodium, potassium, magnesium, calcium salts and the like salts with organic bases, e.g. the benzathine, iV-methyl-D-glucamine, hydrabamine salts, and salts with amino acids such as, for example, arginine, lysine and the like.
• acid or base addition salt also comprise the hydrates and the solvent addition forms which the compounds of formula (I) are able to form. Examples of such forms are e.g. hydrates, alcoholates and the like.
• stereochemically isomeric forms of compounds of formula (I), as used hereinbefore, defines all possible compounds made up of the same atoms bonded by the same sequence of bonds but having different three-dimensional structures which are not interchangeable, which the compounds of formula (I) may possess.
• chemical designation of a compound encompasses the mixture of all possible stereochemically isomeric forms which said compound may possess. Said mixture may contain all diastereomers and/or enantiomers of the basic molecular structure of said compound.
• All stereochemically isomeric forms of the compounds of formula (I) both in pure form or in admixture with each other are intended to be embraced within the scope of the present invention.
• the compounds according to the invention can be prepared and formulated into pharmaceutical compositions by methods known in the art and in particular according to the methods described in the published patent specification mentioned herein and incorporated by reference; for the compounds of formula (I) suitable examples can be found in PCT publication WO-2004/105765.
• a therapeutically effective amount of the particular compound, optionally in addition salt form, as the active ingredient is combined in intimate admixture with a pharmaceutically acceptable carrier, which may take a wide variety of forms depending on the form of preparation desired for administration.
• compositions are desirably in unitary dosage form suitable, preferably, for systemic administration such as oral, percutaneous, or parenteral administration; or topical administration such as via inhalation, a nose spray, eye drops or via a cream, gel, shampoo or the like.
• systemic administration such as oral, percutaneous, or parenteral administration
• topical administration such as via inhalation, a nose spray, eye drops or via a cream, gel, shampoo or the like.
• any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols and the like in the case of oral liquid preparations such as suspensions (including nanosuspensions), syrups, elixirs and solutions; or solid carriers such as starches, sugars, kaolin, lubricants, binders, disintegrating agents and the like in the case of powders, pills, capsules and tablets.
• tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed.
• the carrier will usually comprise sterile water, at least in large part, though other ingredients, for example, to aid solubility, may be included.
• injectable solutions for example, may be prepared in which the carrier comprises saline solution, glucose solution or a mixture of saline and glucose solution.
• injectable solutions containing compounds of formula (I) may be formulated in an oil for prolonged action.
• Appropriate oils for this purpose are, for example, peanut oil, sesame oil, cottonseed oil, corn oil, soy bean oil, synthetic glycerol esters of long chain fatty acids and mixtures of these and other oils.
• Injectable suspensions may also be prepared in which case appropriate liquid carriers, suspending agents and the like may be employed.
• the carrier optionally comprises a penetration enhancing agent and/or a suitable wettable agent, optionally combined with suitable additives of any nature in minor proportions, which additives do not cause any significant deleterious effects on the skin. Said additives may facilitate the administration to the skin and/or may be helpful for preparing the desired compositions.
• These compositions may be administered in various ways, e.g., as a transdermal patch, as a spot-on or as an ointment.
• compositions for topical application there may be cited all compositions usually employed for topically administering drugs e.g.
• compositions may be by aerosol, e.g. with a propellent such as nitrogen, carbon dioxide, a freon, or without a propellent such as a pump spray, drops, lotions, or a semisolid such as a thickened composition which can be applied by a swab.
• a propellent such as nitrogen, carbon dioxide, a freon
• a propellent such as a pump spray
• drops lotions
• a semisolid compositions such as salves, creams, gels, ointments and the like will conveniently be used.
• Dosage unit form as used in the specification and claims herein refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
• dosage unit forms are tablets (including scored or coated tablets), capsules, pills, powder packets, wafers, injectable solutions or suspensions, teaspoonfuls, tablespoonfuls and the like, and segregated multiples thereof.
• a therapeutically effective amount of the pharmaceutical composition comprising a compound according to the invention is administered orally or parenterally.
• Said therapeutically effective amount is the amount that effectively prevents metastasis and/or growth or reduces the size of a variety of neoplastic disorders or cell proliferative disorders (supra) in patients.
• a pharmaceutical composition comprising a compound of the present invention, and in particular 4,6-ethanediylidenepyrimido [4,5-6][6,l,12]benzoxadiazacyclo-pentadecine, 17-bromo-8,9,10,l l,12,13,14,19- octahydro-20-methoxy- 13 -methyl- as the active ingredient can be administered orally in an amount of from 10 mg to several (1 to 5) grams daily, either as a single dose or subdivided into more than one dose, including, e.g. two, three or even four times daily. A preferred amount ranges from 500 to 4,000 mg daily.
• a particularly, preferred dosage for such a compound is in the range of 750 mg to 3,000 mg daily. It will be appreciated that the amount of a compound according to the present invention, also referred to here as the active ingredient, which is required to achieve a therapeutic effect will, of course, vary with, the route of administration, the age and condition of the recipient, and the particular disorder or disease being treated. The optimum dosage amounts and regimen can be readily determined by those skilled in the art using conventional methods and in view of the information set out herein.
• This treatment can be given either continuously or intermittently, including, e.g., but not limited to, cycles of 3-4 weeks with treatment given for 1-21 days per cycle or other schedules shown to be efficacious and safe.
• VEGFR3 inhibitors may be used in combination with one or more other cancer treatments. Such combinations could encompass any established antitumor therapy, such as, but not limited to, chemotherapies, irradiation, and target based therapies such as antibodies and small molecules. These therapies may be combined in systemic therapy, or local instillation/administration (e.g. intrathecally), depending on optimum efficacy/safety requirements.
• one or more other cancer treatments suitable in combination with the VEGFR3 inhibitors of the present invention, with respect to the different tumor types include, but are not limited to; breast: herceptin, docetaxel, anthracyclin, capecitabine prostate: docetaxel, mitoxantrone colon: oxaliplatin, 5-FU, avastin, irinotecan, cetuximab lung; taxotere, carboplatin, gemicitabine
• VEGFR3 inhibitor of the invention and the further anti-cancer agent may be administered simultaneously (e.g. in separate or unitary compositions) or sequentially in either order. In the latter case, the two compounds will be administered within a period and in an amount and manner that is sufficient to ensure that an advantageous or synergistic effect is achieved.
• the preferred method and order of administration and the respective dosage amounts and regimens for each component of the combination will depend on the particularVEGFR3 inhibitor and further anti-cancer agents being administered, their route of administration, the particular tumor being treated and the particular host being treated. The optimum method and order of administration and the dosage amounts and regimen can be readily determined by those skilled in the art using conventional methods and in view of the information set out herein.
• Step l Titanium, tetrakis(2-propanolato) (0.005 mol) was added to a solution of 3- piperidinecarboxylic acid ethyl ester (0.54 g, 0.005 mol) and 4-chloro-2- nitrobenzaldehyde (0.93 g, 0.005 mol) in CH2C12 (40 ml). The mixture was stirred for 1 hour at room temperature and NaBH(OAc)3 (0.0055 mol) was added. The reaction mixture was stirred for 1 hour at room temperature and extra NaBH(OAc)3 (0.00275 mol) was added and the mixture was stirred for 2 hours at room temperature and then NaHCC ⁇ (saturated in H2O) was added.
• Step 5 A mixture of 3-piperidinecarboxylic acid, 1 -[[4-chloro-2-[(6-hydroxy-7-methoxy-4- quinazolinyl)amino]phenyl]methyl]-, ethyl ester (0.00114 mol), N-(3- bromopropyl)carbamic acid 1,1-dimethylethyl ester (1 eq) and Cs2CO3 (1.8582 g) was stirred overnight at room temperature and then the reaction mixture was stirred for 30 minutes at 5O 0 C.
• 'CH 3 OH' means methanol
• 'Et 3 N' means triethylamine
• 'CH 2 Cl 2 ' means dichloromethane
• 'HBTU' means 1- [bis(dimethylamino)methylene]-lH-Benzotriazoliumhexafluorophosphate(l-)3- oxide'DMF' means N, JV-dimethylformamide
• 'NaBH(OAc) 3 ' means sodium triacetoxyborohydride
• 'DIPEA' means iV-ethyl-iV-(l -methyl ethyl)- 2-propanamine
• 'HOBt' means 1 -hydroxy- lH-benzotriazole
• 'TFA' means trifluoroacetic acid
• 'TIS' means tris(l-methylethyl)silane
• 'K 2 CO 3 ' means potassium carbonate
• 'Cs 2 CO 3 ' means cesium
• reaction mixture was stirred for 6 hours and extra bis(l - methylethyl)diazenedicarboxylate (0.35 ml) in T ⁇ F (10 ml) was added. The mixture was stirred overnight and concentrated. The residue was purified by column chromatography over silica gel (eluent: DCM/C ⁇ 3 O ⁇ /T ⁇ F 90/5/5). The product fractions were collected and further purified by RP high-performance liquid chromatography. The product fractions were collected and concentrated. The aqueous concentrate was filtered , and the solid retained washed and dried (vac.) at 65 0 C, yielding 0.065 g of compound 2, melting point 255.5-260.2°C.
• Example E a) Preparation of hexanoic acid, 6-(2-chloro-6-nitrophenoxy)-, methyl ester (intermediate 6) A solution of 2-chloro-6-nitro- phenol (0.046 mol) in N,N-dimethylformamide (150 ml) was heated to 50 °C, then K 2 CO 3 (0.069 mol) was added and the reaction mixture was stirred for 15 min. 6-Bromo-,methyl ester hexanoic acid (0.069 mol) was added and the mixture was stirred overnight . The reaction mixture was filtered and the filtrate was concentrated and the residue was used as such in the next step, yielding 13.88 g of intermediate 6. b) Preparation of hexanoic acid, 6-(2-amino-6-chlorophenoxy)-, methyl ester (intermediate 7)
• the product MTKIl can be prepared as a 10-mg/mL oral solution, pH 2. It contains an excipient, Captisol® (chemical name: sulfobutyl ether- ⁇ -cyclodextrin, SBE- ⁇ -CD), citric acid, Tween® 20, HCl, and NaOH in purified water.
• Captisol® chemical name: sulfobutyl ether- ⁇ -cyclodextrin, SBE- ⁇ -CD
• citric acid citric acid
• Tween® 20 HCl
• NaOH NaOH
• the product MTKIl can also be prepared as 50-mg, 100-mg and 300-mg oral immediate release capsules, containing the active chemical entity MTKIl , lactose monohydrate (200 mesh), sodium lauryl sulphate and magnesium stearate in hard gelatin capsules, sizes 3, 4 and 00, respectively.
• the capsules may also contain any or all of the following ingredients: gelatin, red iron oxide and titanium oxide.
• lymphatic vessel formation plays an important role in tumor progression and vascular endothelial growth factor (VEGF) C and D have been identified as specific lymphangiogenic factors that act via activation of the cognate receptor VEGFR3.
• VEGF vascular endothelial growth factor
• Compound 2 4,6-ethanediylidenepyrimido [4,5- 6][6,l,12]benzoxadiazacyclo-pentadecine, 17-bromo-8,9,10,l l,12,13,14,19- octahydro-20-methoxy-13-methyl- , hereinafter also referred to as Compound 2, is a multitargeted kinase inhibitor that has been shown to have a unique kinase inhibition profile (pan Her and Src family) as well as a favourable tissue distribution profile resulting in potent anti tumor activity in a number of experimental models.
• HMVECd human vascular endothelial cells
• the VEGFR3 kinase reaction was performed at 30°C for 10 minutes in a 96-well microtiterplate.
• the 25 ⁇ l reaction volume contained 8 mM MOPS pH 7, 200 ⁇ M EDTA, 10 mM MgAc, 10 ⁇ M unlabeled ATP, 0.5 mCi AT33P, 500 ⁇ M JAK3-tide (Ac-GGEEEEYFELVKKKK-NH2) , 25 ng VEGFR3 and 2 % compound in 100 % DMSO.
• the reaction was stopped by adding 5 ⁇ l of a 3% phosphoric acid solution. 10 ⁇ l of the reaction mixture was then spotted onto a Filtermat P30 filter (Wallac) and washed 3 times for 5 min. in 0.75 % phosphoric acid and 1 time for 2 min. in methanol prior to transferring to a sealable plastic bag containing 4 ml of scintillation liquid and reading in a scintillation counter.
• porcine aortic endothelial (PAE) cells stably expressing human VEGFR-3 were grown to confluency on 10 cm dishes. The cells were serum starved overnight and the media replaced with fresh serum-free media. Pre-determined amounts of Compound 2 or control vehicle was added to the cells, and then incubated at 37 0 C for 15 min. This was followed by the addition of VEGF-C to a final concentration of 100 ng/ml and the cells incubated for 10 min at 37 °C, after which they were washed with ice-cold PBS and then lysed.
• PAE porcine aortic endothelial
• Cell lysates containing protease inhibitors were subjected to immunoprecipitation with an antibody specific for VEGFR-3 and the immunoprecipitates separated by SDS-PAGE. Proteins were transferred to a nitrocellulose membrane, and then probed first with a phospho- tyrosine antibody and then a VEGFR-3 antibody.
• HMVEC-d Human Micro Vessel Endothelial Cells-dermal
• HMVEC-d cells were cultured at 37C in 5% CO2 in appropriate serum containing medium (Cambrex) on fibronectin coated 12-well plates (BD Biocoat, Becton Dickinson) for 4 days. Cells were then starved for 16 hours in the same medium as before, but lacking serum and containing SITE+3 (Invitrogen) instead. Cells were then preincubated with DMSO at 0.2% (the solvent) or 5uM Compound 2 for 60 minutes and then either 10 ng/ml VEGF or 100 ng/ml VEGF-C (R&D systems) was added to the cells.
• Electrophoresis and blotting onto PVDF membranes was all performed using reagents from the NUPAGE system, according to the manufacturer's instructions. Blots were blocked for 1 hour with Odyssey blocking buffer (LICOR) and then primary antibodies (see below) were diluted 1 : 1000 in a 1 : 1 mix of Odyssey blocking buffer and PBS containing 0.1% Tween-20, and applied overnight at 4°C: P-Erkl/2 (Thr202/Tyr204, clone ElO, mouse monoclonal #9106, Erkl/2 (rabbit polyclonal #9102) (all from Cell Signaling Technologies, Inc).
• LICOR Odyssey blocking buffer
• primary antibodies see below
• Blots were washed three times in PBS containing 0.1% Tween for 5 min and then incubated for 1 hour with secondary antibodies (diluted 1 :1000 in PBS containing 0.1% Tween-20):goat anti-mouse conjugated to IRDye 800nm (Rockland, Inc) or goat anti-rabbit conjugated to Alexa 680nm (Molecular Probes, Inc). Blots were washed three times in PBS containing 0.1% Tween for 5 min and once in PBS and scanned using the LICOR system at 700nm and 800nm using intensity 5 and all settings appropriate for the scanning of membranes.
• Xenopus tadpoles were used as a model (Ny et al Nature Medicine 11 : 998-1004 (2005)) to analyze the effects of Compound 2 on lymphangiogenesis.
• the compound was administered daily in a dose dependent manner, with vehicle controls, to the medium of the Xenopus tadpoles starting at stage 26-28, i.e., before the formation of both blood and lymphatic vessels.
• the effects of the compound was monitored by "live analysis", i.e. live embryos were examined 4 days later for evidence of lymph- vascular defects by trained observers.
• LECs lymphatic endothelial cells
• PCV posterior cardinal vein
• the following table provides the pIC50 values obtained for the compounds of the present invention using the above-mentioned VEGFR3 kinase assay.

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