BRPI0718061A2 - VEGFR3 Inhibitors - Google Patents

Description

ι Patent Descriptive Report for "VEGFR3 INHIBITORS". FIELD OF INVENTION

The present invention relates to the finding that some of the macrocyclic quinazoline derivatives described in PCT publication W02004 / 105765 are useful as inhibitors of VEGFR3-mediated biological activities, especially those activities that are mediated by VEGFR3 VEGF-C and / or VEGF-D. BACKGROUND OF THE INVENTION Cancer is still a leading cause of death worldwide in the world.

early 21st century and remains a major focus for continuous research and development.

In recent years, a promising approach to cancer therapeutic intervention has focused on anti-angiogenesis therapies. This approach to intervening in cancer progression has the advantage of the idea that inhibiting or otherwise limiting tumor blood supply will deplete the tumor of oxygen and nutrients and cause disruption of tumor cell proliferation and growth. This approach has been found to be effective and there are now more than 20 antiangiogenic drugs undergoing various stages of evaluation in phase I1 Il or Ill clinical trials and numerous others in preclinical development.

While there are many different forms of cancer exhibiting a wide range of properties, one factor that many cancers share is that, to be fatal, they must metastasize. Until such time when metastasis occurs, a tumor, although it may be malignant, is limited to one area of the body. This can cause discomfort and / or pain, or even lead to more serious problems, although it can be localized before metastasis, the cancer can be administered or even removed by surgical intervention. As long as residual cancer cells are kept in check, such a discrete cancer if controlled without significant problems. However, metastasis will cause cancer cells to invade the body and while surgical resection may remove the primary tumor, metastatic expansion of the cancer to discrepant sites is very difficult to control.

Metastasis to regional lymph nodes through lymphatic vessels is a common step in cancer progression. Metastasis is an important prognostic factor in many cancers and forms the basis for surgical and radiation treatment of local lymph nodes. The tumor metastasis process is a multistage event involving local invasion and destruction of the intercellular matrix, intravasation into blood vessels, lymphatics or other transport channels, survival in circulation, extravasation of vessels in the secondary site, and growth in the site. New (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 that tumor cells be able to perform these steps sequentially. Recently, several lines of evidence have indicated that Iinfan-

genesis, lymphatic vessel formation, 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)). Control of lymphangiogenesis may provide a new strategy to prevent lymph node metastasis in cancer therapy.

Recent studies have shown that a member of the vascular endothelial growth factor (VEGF) family, VEGF-C, stimulates lymphangiogenesis and lymphatic endothelial cell growth and migration by binding to its receptor, VEGFR3 (Karkkainen MJ, et al. ., Semin Cell Dev Biol. 13: 9-18 (2002)). VEGF-C has likewise been shown to promote mediated lymphatic metastasis by inducing tumor-associated lymphangiogenesis in numerous solid cancers such as gastric cancer, prostate cancer, human colorectal cancer, invasive cervical cancer, breast cancer metastasis. and metastasis of human melanoma. In support of these are preclinical data demonstrating that overexpression of VEGF-C in cancer cells significantly increases tumor-associated lymphangiogenesis, resulting in increased metastasis in regional lymph nodes (Stacker SA., Et al., FASEB J 16: 922 -34 (2002)). In addition, VEGF-C / D-mediated signaling blockade has been shown to suppress tumor lymphangiogenesis and lymph node metastasis in mice (He Y., et al., J Natl Cancer Inst. 94: 819-25 (2002) ).

VEGFR3, a transmembrane tyrosine kinase receptor is

widely expressed in endothelial cells during early embryogenesis (Pajusola K., et al., Cancer Res. 53 (16): 3845 (1992)). During later stages of development, VEGFR-3 expression becomes restricted to developing lymphatic vessels [Kaipainen, A., et al.s., Proc. Natl. Cad. Know. USA, 92: 3566-3570 (1995)]. In adults, 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 similarly expressed in a subset of CD34 + hematopoietic cells that can mediate the myelopoietic activity of VEGF-C demonstrated by overexpression studies. Targeted disruption of the VEGFR-3 gene in mouse embryos leads to failure of primary vascular network remodeling, 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 embryonic vasculature, and also during lymphangiogenesis.

From the foregoing, it will be apparent that VEGFR3 inhibitors have tremendous potential as therapeutics, and new agents of this type represent a continuing need in the art to inhibit growth and / or dispersion of a variety of neoplastic disorders or cell proliferative disorders. Inhibition of VEGFR3 activity, including inhibition of VEGFR3 ligand is useful in the treatment of mammalian individuals who have been diagnosed with a disease characterized by proliferation of VEGFR-3-expressing endothelial cells. For example, many tumors are characterized by blood vessel or lymphatic vessel neovascularization, wherein the neovascularization comprises endothelial cells expressing VEGFR-3. In some cancers, the cancer cells themselves express VEGFR3, and represent the target cells. In another, possibly cancer overlapping set, cancer cells express a VEGFR-3 ligand selected from VEGF-C and VEGF-D. The Gypsy is believed to be involved in recruiting endothelial cells and stimulating their growth, thereby facilitating cancer nutrition and / or spread.

SUMMARY OF THE INVENTION

The invention is directed in part to methods of treating or preventing VEGFR3-mediated biological activities using certain compounds described in WO2004 / 105765, the disclosure of which is hereby incorporated by reference in its entirety.

In a related embodiment, 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 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 cancer a composition comprising a compound of the invention, in an amount effect to reduce cancer growth or neoplastic spread. It will be appreciated that any reduction in cancer growth rate or spread (which may prolong life and quality of life) is indicative of successful treatment. In preferred embodiments, cancer growth is completely arrested. In still more preferred embodiments, cancers diminish or are completely eradicated. Preferred subjects for treatment are human subjects, but other animals, especially murine, rat, canine, bovine, porcine, primate, etc. Model cancer treatment systems are considered.

In some cancers expressing VEGFR-3, direct inhibition of cancer growth or cancer death may be the mechanism. Treatment of all cancers expressing VEGFR-3 and all cancers characterized by angiogenesis or lymphangiogenesis and around a growing tumor is considered. For example, treatment is considered from 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, testis, ovary, skin, head and neck, esophagus, bone marrow and blood. In a particular embodiment of the present invention, treatment is considered from cancers of a tissue, organ or cell selected from the group consisting of Breast, Lung, Prostate and Colon.

In a variation of the above methods of treatment, the compounds are administered together with a second cancer therapeutic agent. The second agent may be any chemotherapeutic agent, radioactive agent, radiation, nucleic acid encoding a cancer therapeutic agent, antibody, protein, and / or other anti-lymphogenic agent or an antiangiogenic agent. The second agent may be administered before, after, or simultaneously with the compounds of the invention.

In one variation, the subject being treated has been diagnosed with an operable tumor, and the administration step is performed before, during, or after the tumor is resected from the individual. Compound treatment in combination with tumor resection is intended to reduce or eliminate the growth of unresected cancer cell tumors.

More generally stated, the invention provides a method of

treating a condition characterized by VEGFR-3 by binding to a natural ligand that binds to VEGFR-3, comprising the step of administering to a subject in need thereof a compound of the invention. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1: Effect of 4,6-ethanediylidenopyrimidate [4,5-b] [6,1,12]

zoxadiazacyclopentadecine, 17-bromo-8,9,10,1 1,12,13,14,19-octahydro-20-methoxy-13-methyl- (Compound 2) in VEGF-C induced VEGFR3 activity. Top structure provides the results of VEGFR-3 immunoprecipitates transferred on a nitrocellulose membrane, stained with phosphotyrosine antibodies. Lower structures provide the results of VEGFR-3 immunoprecipitates transferred on a nitrocellulose membrane stained with VEGFR-3 antibodies. Left lanes 1 and 2 provide the 10

15

20

negative and positive controls, ie DMSO Treatment in the absence and presence of VEGF-C.

Figure 2: VEGF and VEGF-C stimulation of Erk1 / 2 phosphorylation in HMVECd cells in the presence of 5uM of Compound 2. Top bands provide staining of phosphorylated Erk 1/2 (P-Erk 1/2) using a monoclonal antibody. Thr202 / Tir204 mouse as primary antibody and IRDye 800nm-conjugated goat anti-mouse as secondary antibody. Base strips provide total Erk 1/2 staining using Erk 1/2 specific rabbit polyclonal antibody as the primary antibody and Alexa 680 nm conjugated goat anti-rabbit as the secondary antibody. Left panel provides results for VEGF-induced phosphorylation of Erk-1/2 in HMVECd cells. Right pane provides the results for Erk 1/2 VEGF-C induced phosphorylation in HMVECd cells. DETAILED DESCRIPTION OF THE INVENTION

WO2004 / 105765 describes the preparation, formulation and pharmaceutical properties of a class of macrocyclic quinazoline derivatives of formula (I) as multi-targeted kinase inhibitors.

25

It has now been found that a particular group of compounds within the aforementioned class have VEG-FR3 inhibitory activity, which 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 mammal individual.

Thus, in one aspect the present invention provides the use of the compounds of formula (I) wherein;

Z represents NH; Y represents -C3-9 alkyl-, -C1-Salkyl-NR13-Cl-Salkyl-, -C1. Salkyl-NR14-CO-Cl-Saalkyl-, -C1 -alkyl-NH-CO-Het20-, or -Het22-CH2 -CO-NH-C3-3 alkyl-;

X1 represents O, or -O-C1-4 alkyl;

X 2 represents a direct bond, -C 1-4 Alkyla, O, -O-C 1-4 Alkyla,

NR12 or NR12-C1-6 alkyl;

R1 represents hydrogen, cyano, halo or hydroxy, preferably

halo;

R 2 represents hydrogen, cyano, halo, or hydroxy; R3 represents hydrogen;

R4 represents C1-4 alkyloxy;

R12 represents hydrogen, or C1-4 alkyl-;

R13 represents hydrogen or C1-4 alkyl;

R14 represents hydrogen or C1-4 alkyl; Het 20 represents pyrrolidinyl, piperazinyl or piperidinyl; and

Het22 represents pyrrolidinyl, piperazinyl or piperidinyl;

pharmaceutically acceptable acid or base addition salts and stereochemically 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 dispersion of a cancer in a mammal individual.

Another aspect of the present invention is directed to a method for treating or preventing a VEGFR3 mediated biological activity, such as for example treating or preventing metastatic spread of a cancer in a mammalian subject comprising administering therapeutically effective amount. of a compound of formula I wherein; 7VAy2

8 nI

Z represents NH;

Y represents -C 3-9 alkyl-, -C 1-5 alkyl-NR 13 -C 15 alkyl-, -C 1. Salkyl-NR14-CO-Cl-Sakyla, -C1-Salkyl-NH-CO-Het20-, or -Het22-CH2-CO-NH-C1-Alkyl; X1 represents O, or -O-C1-4 alkyl;

X 2 represents a direct bond, -C 1-6 alkyl-, O, -O-C 1-4 alkyl-, NR 12 or NR 12 -C 1-4 alkyl;

R1 represents hydrogen, cyano, halo or hydroxy, preferably

halo;

R 2 represents hydrogen, cyano, halo, or hydroxy;

R3 represents hydrogen; R4 represents C1-4 alkyloxy; R12 represents hydrogen, or C1-4 alkyl-; R13 represents hydrogen or R14 represents hydrogen or C1-4 alkyl;

Het 20 represents pyrrolidinyl, piperazinyl or piperidinyl; Het22 represents pyrrolidinyl, piperazinyl or piperidinyl; and the pharmaceutically acceptable acid or base addition salts and stereochemically isomeric forms thereof; to a mammalian individual in need of such treatment.

Other VEGFR3-mediated biological activities are intended to include;

- metastatic spread of a cancer in a mammalian individual. Preferred subjects for treatment are human subjects, but other animals, especially murine, rat, canine, bovine, porcine, primate et al. Model cancer treatment systems are considered.

- Metastasis to regional lymph nodes through lymphatic vessels. In this manner it 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 by

example in gastric cancer, prostate cancer, human colorectal cancer, invasive cervical cancer, breast cancer, Kaposi's sarcoma and melanoma. It thus provides the use of the compounds according to the invention in the treatment or prevention of tumor-associated lymphangiogenesis in cancer, such as for example gastric cancer, prostate cancer, human colorectal cancer, invasive cervical cancer, breast cancer, Kaposi's sarcoma and melanoma;

- recruitment and proliferation of endothelial cells expressing VEGFR3 in neovascularization of cancer cells expressing

are a VEGFR3 Ligand selected from VEGF-C or VEGF-D. In this manner it 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.

In another embodiment, the present invention provides the use of a previously mentioned compound of formula (I) for the preparation of a pharmaceutical composition for treating cancers of a tissue, organ or cell selected from the group consisting of Breast, Lung (including treatment of both non-small cell and small cell lung cancer), colon and prostate. The present invention likewise relates to 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 a previously mentioned compound of formula (I) to said mammal. In yet another embodiment, the present invention provides the use of

of a previously mentioned compound of formula (I) for the preparation of a pharmaceutical composition for treating advanced breast cancer. The term "advanced breast cancer" is used herein to denote breast cancer that has not responded to, or resorted to following such treatment, and similarly breast cancer in patients who have metastatic disease at diagnosis.

The present invention likewise relates to a method of

of treating advanced breast cancer in a mammal, particularly a woman, comprising the step of administering a therapeutically effective amount of a previously mentioned compound of formula (I) to said mammal.

In particular, the present invention relates to 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 -

C1-Alkyl-NH-CO-Het20-;

X1 represents O;

X 2 represents -C 1-2 alkyl-, O, or NR 12 -C 12 alkyl-;

R1 represents hydrogen or halo; in particular R1 represents hydrogen or chlorine; more particularly R1 represents hydrogen;

R2 represents hydrogen or halo; in particular R2 represents

hydrogen, chlorine or bromine; more particularly R2 represents chloro or bromo

mo;

R3 represents hydrogen;

R4 represents C1-4 alkoxy; in particular R 4 represents methoxy; R12 represents C1-6 alkyl-; in particular R 12 represents methyl;

R14 represents hydrogen or C1-4 alkyl; in particular R14 represents hydrogen or methyl; more particularly R14 represents hydrogen;

Het 20 represents pyrrolidinyl, piperazinyl or piperidinyl; in particular Het20 represents piperidinyl.

Also of interest in the aforementioned uses are those compounds of formula (I) wherein R1 is in the 3 'position, R2 is in the 5' position, R4 is in the 7 position and X2 is in the 2 'position using - by numbering as given in Formula (I) above.

Most preferred compounds are those compounds selected from the group consisting of; 4,6-ethanedylidene-19H-pyrimido [4,5-6] [6,13,1] benzodioxa-azacyclopentadecine,

15-chloro-8,9,10,11,12,13-hexahydro-20-methoxy; 12H-4,6-ethanediylidene-13,17-methanopyrime [4,5-b] [6,1,10,16] benzoxatriazacyclononadecin-12-one,

21-chloro-8,9,10,11,13,14,15,16,18,23-decahydro-25-methoxy; 4,6-ethanediylidene-12H-pyrimido [4,5-b] [6,1,10,13] benzoxatriazacyclohexadecin-12-one,

18-chloro-8,9,10,11,13,14,15,20-octahydro-21-methoxy-13,14-

dimethyl-; and

4,6-ethanediylidene pyrimidated [4,5-b] [6,1,12] benzoxadiazacyclopentadecine,

17-bromo-8,9,10,11,12,13,14,19-octahydro-20-methoxy-13-methyl-; or a pharmaceutically acceptable acid addition salt thereof. The latter compound is especially preferred.

Most preferred compounds for use in accordance with the present invention are those selected from the group consisting of com- or a pharmaceutically acceptable acid addition salt thereof. The following compound is especially preferred:

or a pharmaceutically acceptable acid addition salt thereof.

As used in the foregoing definitions and below, - halo is generic to fluorine, chlorine, bromine and iodine;

C 1-2 alkyl defines methyl or ethyl;

C 1-3 alkyl defines straight or branched chain saturated hydrocarbon radicals having from 1 to 3 carbon atoms such as, for example, methyl, ethyl, propyl and the like;

- C1-4 alkyl defines saturated hydrocarbon radicals of

straight or branched chain having from 1 to 4 carbon atoms such as, for example, methyl, ethyl, propyl, butyl, 1-methylethyl, 2-methylpropyl, 2,2-dimethylethyl and the like;

- C1.5 alkyl defines saturated hydrocarbon radicals of

linear or branched chain having from 1 to 5 carbon atoms such as, for

for example methyl, ethyl, propyl, butyl, pentyl, 1-methylbutyl, 2,2-dimethylpropyl, 2,2-dimethylethyl and the like;

- C3.alkyl defines straight or branched chain saturated hydrocarbon radicals having from 3 to 9 carbon atoms such as propyl,

butyl, pentyl, hexyl, heptyl, octyl, nonyl and the like;

C1-4 alkyloxy defines straight or branched saturated hydrocarbon radicals such as methoxy, ethoxy, propyloxy, butyloxy, 1-methylethyloxy, 2-methylpropyloxy and the like;

- the term "CO" refers to a carbonyl group.

Pharmaceutically acceptable base or acid addition salts as mentioned hereinabove are intended to comprise the therapeutically active non-toxic acid and non-toxic base addition salt forms which the compounds of formula (I) are capable of forming. Compounds of formula (I) which have basic properties may be converted to their pharmaceutically acceptable acid addition salts by treating said base form with an appropriate acid. Suitable acids include, for example, inorganic acids such as hydroalic acids, for example hydrochloric or hydrobromic acid; sulfuric; nitric; phosphoric and similar acids; or organic acids such as, for example, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic, malonic, succinic (i.e. buttanoic acid), maleic, fumaric, malic, tartaric, citric, methanesulfonic, ethanesulfonic, benzenesulfonic, p -toluenesulfonic, cyclamic, salicylic, p-aminosalicylic, pamoic and similar acids.

The compounds of formula (I) which have acidic properties may be converted and their pharmaceutically acceptable base addition salts by treating said acid form with a suitable organic or inorganic base. Suitable base salt forms include, for example, ammonium salts, alkali metal and alkaline earth salts, for example lithium, sodium, potassium, magnesium, calcium salts and the like, salts with organic bases, for example. , benzathine, N-methyl-D-glycamine, hydrabamine salts, and amino acid salts such as, for example, arginine, lysine and the like.

The terms acid or base addition salt likewise include hydrates and solvent addition forms that the compounds of formula (I) may form. Examples of such forms are, for example, hydrates, alcoholates and the like.

The term stereochemically isomeric forms of compounds of formula (I) as used herein defines all possible compounds prepared from the same atoms bonded by the same sequence of bonds but having different non-exchangeable three-dimensional structures, wherein The compounds of formula (I) may have. Unless otherwise stated or indicated, the 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 fall within the scope of the present invention.

Some of the compounds of formula (I) may likewise exist in their tautomeric forms. Such forms although not explicitly indicated in the above formula are intended to be included within the scope of the present invention. Whenever used below, the term "compounds of the formula

(I) "or" compounds according to the invention "are intended to include in the same manner the pharmaceutically acceptable base or acid addition salts and all stereoisomeric forms.

The compounds according to the invention may 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; Suitable examples of the compounds of formula (I) can be found in PCT publication W0-2004 / 105765. To prepare the aforementioned pharmaceutical compositions, a therapeutically effective amount of the particular compound, optionally, moreover, salt form, as the active ingredient is combined in intimate admixture with a pharmaceutically acceptable carrier, which may take a wide variety. depending on the desired preparation form for administration. These pharmaceutical compositions are desirably in unit dosage form suitable, preferably for systemic administration such as oral, percutaneous, or parenteral administration; or topical administration such as by inhalation, a nasal spray, eye drops or by a cream, gel, shampoo or the like. For example, by preparing the compositions in oral dosage form, 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. Because of their ease in administration, tablets and capsules represent the advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. For parenteral compositions, the carrier will usually comprise sterile water, at least to a large extent, however other ingredients, for example to aid solubility, may be included. Injectable solutions, for example, may be prepared wherein 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. Suitable oils for this purpose are, for example, peanut oil, sesame oil, cottonseed oil, corn oil, soybean oil, synthetic long chain fatty acid glycerol esters and mixtures thereof. s oils. Injectable suspensions may likewise be prepared in which case appropriate liquid vehicles, suspending agents and the like may be employed. In compositions suitable for percutaneous administration, the carrier optionally comprises a penetration enhancing agent and / or a suitable wetting agent, optionally combined with suitable additives of any nature in minor proportions, the additives of which do not cause any significant deleterious effect on the composition. skin. Said additives may facilitate administration to the skin and / or may be useful for preparing the desired compositions. These compositions may be administered in various ways, for example, as a transdermal patch, as a blemish or as an ointment. Suitable compositions for topical application may include all compositions commonly employed for topically administering drugs, for example creams, gels, dressings, shampoos, tinctures, pastes, ointments, ointments, powders and the like. Application of said compositions may be aerosolized, for example, with a propellant such as nitrogen, carbon dioxide, a freon, or without a propellant such as a spray pump, drops, lotions, or a semi-solid such as a composition. thick that can be applied by a mop. In particular, semi-solid compositions such as ointments, creams, gels, ointments and the like will be conveniently used. It is especially advantageous to formulate pharmaceutical compositions

mentioned above in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used in the specification and claims herein refer to physically discrete units suitable as unitary dosages, each unit containing a predetermined amount of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. . Examples of such dosage unit forms are tablets (including marked or coated tablets), capsules, pills, powder packets, lozenges, suspensions or injectable solutions, full teaspoons, full tablespoons and the like, and segregated multiple of these.

Preferably, 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. On the basis of current data, it appears that a pharmaceutical composition comprising a compound of the present invention, and in particular 4,6-ethanediylidenopyrimidate [4,5-6] [6,1,12] benzoxadiazacyclopentadecine, 17- bromo- 8,9,10,11,12,13,14,19-octahydro-20-methoxy-13-methyl- as the active ingredient may be orally administered in an amount from 10 mg to several (1 to 5 ) grams daily as a single dose or subdivided into more than one dose, including for example two, three or even four times a day. 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 per day. It will be appreciated that the amount of a compound according to the present invention likewise refers herein as the active ingredient, which is required to obtain a therapeutic effect, of course, which will vary with the routine administration, the age and condition of the recipient, and the particular disorder or disease to be treated. Optimal dosage amounts and regimen can be readily determined by those skilled in the art using conventional methods and due to the information mentioned herein. This treatment may be produced continuously or intermittently, including, but not limited to, 3-4 week cycles with treatment determined for 1-21 days per cycle or other times shown to be effective and safe.

The above 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 into systemic therapy, or local instillation / administration (e.g. intrathecally), depending on efficacy / safety requirements.

In certain preferred embodiments, one or more other suitable cancer treatments in combination with the VEGFR3 inhibitors of the present invention, with respect to different tumor types, include, but are not limited to;

breast: herceptin, docetaxel, anthracycline, capecitabine prostate: docetaxel, mitoxantrone

colon: oxaliplatin, 5-FU, avastin, irinotecan, cetuximab lung: taxotere, carboplatin, gemicitabine

The VEGFR3 inhibitor of the invention and the other anticancer agent may be administered simultaneously (e.g., in separate or unitary compositions) or sequentially in any case. In the latter case, the two compounds will be administered within a period and in such amount and manner as is sufficient to ensure that an advantageous or synergistic effect is obtained. It will be appreciated that the preferred method and order of administration and the respective dosage amounts and regimens for each component of the combination will depend upon the particular VEGFR3 inhibitor et al., Anticancer agents to be administered, its administration routine, the tumor. particular to be treated and the particular host treated. The ideal method and order of administration and the dosage and regimen amounts can be readily determined by those skilled in the art using conventional methods and due to the information mentioned herein. PREPARATION OF COMPOUNDS AND FORMULATIONS: Preparation of Compound 95. 12H-4,6-ethanediylidene-13.17-methanopyrimidor4,5-b16.1,10,161benzoxatriazacyclononadecin-12-one. 21-chloro-8,9,10.11.13,14.15.16,18.23-decahydro-25-methoxy Step 1

Titanium, tetracis (2-propanolate) (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 CH 2 Cl 2 (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 saturated NaH-CO3. H2O) was added. The organic layer was separated, dried (K 2 CO 3), filtered and the solvent was evaporated and coevaporated with toluene to remove residual CH 2 Cl 2. 3-Piperidinecarboxylic acid, 1 - [(4-chloro

2-nitrophenyl) methyl] -, ethyl ester as crude residue was used as such in the next reaction step.

Step 2

A mixture of 1 - [(4-chloro-2-nitrophenyl) methyl] -, ethyl ester

3-Piperidinecarboxylic acid (1.69 g, 0.005 mol) in ethanol (150 mL) was hydrogenated with 5% Pt / C (0.5 g) as a catalyst in the presence of a thiophene solution (1 mL). After uptake of H2 (3 equiv.), The catalyst was filtered and the filtrate was evaporated. The residue was redissolved in EtOAc and this solution was filtered over Extrelute. Heptane was added but no crystals appeared. The solvent was evaporated and the residue was redissolved in CH 3 OH and filtered on a paper filter to remove silica grease. The solvent was evaporated to yield 1.35 g of 3-piperidinecarboxylic acid ethyl ester, 1 - [(2-amino-4-chlorophenyl) methyl] - as a brown oil. Step 3

4-Chloro-7-methoxy-6-quinazolinol 6-acetate (0.273 g, 0.00108 mol) was added to a solution of 1 - [(2-amino-4-chlorophenyl) methyl] -, ethyl ester of 3-piperidinecarboxylic acid (0.0011) in 2-propanol (qs) and then the reaction mixture was stirred overnight at 80 ° C. Then the mixture was also stirred for 6.5 hours at 80 ° C and the solvent was evaporated. Yield: 1 - [[2 - [[6- (acetyloxy) -7-methoxy-4-quinazolinyl] amino] -4-chlorophenyl] methyl] -, 3-piperidinecarboxylic acid ethyl ester (crude; used as such in the next reaction step ). Step 4

7N NH3 / CH30H (approx. 10 ml) was added to 1 - [[2 - [[6- (acetyloxy) -7-methoxy-4-quinazolinyl] amino] -4-chlorophenyl] methyl] - ethyl ester. 3-piperidinecarboxylic acid (0.00114 mol) and the reaction mixture was stirred for 1 hour at 35 ° C and then the solvent was evaporated. Yield: 1 - [[4-chloro-2 - [(6-hydroxy-7-methoxy-4-quinazolinyl) amino] phenyl] methyl] - ethyl ester of 3-piperidinecarboxylic acid (used as such in the next reaction step). nal). Step 5

A mixture of 1 - [[4-chloro-2 - [(6-hydroxy-7-methoxy-4-quinazolinyl) amino] phenyl] methyl] -, ethyl 3-piperidinecarboxylic acid ester (0.00114 mol), 1 N, - (3-bromopropyl) carbamic acid 1-dimethylethyl ester (1 eq) and Cs 2 CO 3 (1.8582 g) was stirred overnight at room temperature and then the reaction mixture was stirred for 30 minutes at room temperature. 50 ° C. When needed further N- (3-bromopropyl) carbamic acid 1,1-dimethylethyl ester was added and the mixture was stirred for 4 hours at room temperature for another 15 minutes at 50 ° C. The solvent was evaporated (Genevac) and the residue was dissolved in CH 2 Cl 2. This solution was filtered over dicalite and the filtrate was evaporated. Yield: 1 - [[4-chloro-2 - [[6- [3 - [[(1,1-dimethylethyloxy) carbonyl] amino] propoxy] -7-methoxy-4-quinazolinyl] amino] phenyl] methyl] - 3-piperidinecarboxylic acid ethyl ester. Step 6

A solution of 1 - [[4-chloro-2 - [[6- [3 - [[(1,1-

dimethylethyloxy) carbonyl] amino] propoxy] -7-methoxy-4-quinazolinyl] amino] phenyl] methyl] -, 3-piperidinecarboxylic acid ethyl ester (residue; approx. 0.00114 mol) in TFA / CH2 Cl2 / TIS (90 (8.4 ml) was stirred for 7-8 hours, then the solvent was evaporated and the residue obtained was dried overnight in an oven. Yield: 3-piperidinecarboxylic acid 1 - [[2 - [[6- (3-aminopropoxy) -7-methoxy-4-quinazolinyl] amino] -4-chlorophenyl] methyl] -. CF3COOH. Step 7

A 1 - [[2 - [[6- (3-Aminopropoxy) -7-methoxy-4-quinazolinyl] amino] -4-chlorophenyl] methyl] - solution of 3-piperidinecarboxylic acid. CF3COOH (0.00114 mol) and DIPEA (0.00684 mol) was added to a solution of HBTU (0.00342 mol) and HOBt (0.00228 mol) in dry DMF (285 ml) and then the mixture. The reaction mixture was reacted for 1 hour. The solvent was evaporated and the dried residue was purified by reverse phase high performance liquid chromatography. Product fractions were collected, Na 2 CO 3 was added and the organic solvent was evaporated. CH 2 Cl 2 was added to the aqueous concentrate and the resulting mixture was extracted 3 times with CH 2 Cl 2, then the organic extract was dried and collected. Yield: 0.0007 mol of 12H-4,6-ethanediylidene-13,17-methanopyrime [4,5-b] [6,1,10,16] benzoxatriazacyclononadecin-12-one, 21-chloro-

8,9,10,11,13,14,15,16,18,23-decahydro-25-methoxy (62% yield). Preparation of Compound 96, 4,6-ethanediylidene-12H-pyrimidin4,5-

b1f6.1.10,131benzoxatriazacyclohexadecin-12-one, 18-chloro-

8.9.10.11,12,13,14.15.20-octahydro-21-methoxy-13.14-dimethyl (13S) - Step 1 A mixture of N-methyl-L-alanine methyl ester hydrochloride (1.4 g, 0.010 mol), 4-chloro-2-nitrobenzaldehyde (0.010 mol) and Titanium tetracis (2-propanolate) (0.010 mol) in CH 2 Cl 2 (qs) was stirred for 1 hour at room temperature. Then NaBH (OAc) 3 (0.022 mol) was added and the mixture was stirred for 3 hours at room temperature. More NaBH (OAc) 3 (0.011 mol) was added and the mixture was stirred overnight at room temperature. Then saturated (NaHCO3) was added while still basic and the mixture was filtered through a P3 filter. The organic layer was separated and the water layer was extracted 3 times with CH 2 Cl 2. The combined organic layers were dried (K2 CO3) and the solvent was evaporated to dryness. The crude residue, L-alanine, N / - [(4-chloro-2-nitrophenyl) methyl] -Î ”/ methyl-methyl ester was used as such in the next reaction step. Step 2

A mixture of L-alanine, N / - [(4-chloro-2-nitrophenyl) methyl] - / V-methyl-,

methyl ester (1.03 g, 0.0036 mol) in CH30H (50 ml) was hydrogenated with 5% Pt / C (0.5 g) as a catalyst in the presence of a 4% thiophene solution in DIPE (0 0.5 ml). After uptake of H2 (3 equiv.), The catalyst was filtered off and the filtrate was evaporated. The residue was purified on silica gel (with cartridge) (eluent: 4% Et 3 N / CH 2 Cl 2). The desired fractions were collected. Heptane was added to the desired fraction (2 phase form). The heptane layer was separated to remove silica grease. After solvent removal and drying 0.5674 g of L-alanine, N - - [(2-amino-4-chlorophenyl) methyl] -A / methylmethyl ester was obtained (22%; yellow oil ). The crude product was used as such in the next reaction step. Step 3

4-Chloro-7-methoxy-6-quinazolinol 6-acetate (0.00055 mol) was added to a solution of L-alanine, V - [(2-amino-4-chlorophenyl) methyl] -A / - methyl-, methyl ester (0.00055 mol) in 2-propanol (5 ml) and then the reaction mixture was stirred overnight at 80 ° C and the solvent was evaporated. Yield: L-alanine, [V - [[2 - [[6- (acetyloxy) -7-methoxy-4-quinazolinyl] amino] -4-chlorophenyl] methyl] -N-methyl methyl ester (crude, used as next reaction stage). Step 4

7N NH 3 / CH 30 H (10 mL) was added to L-alanine, β / - [[2 - [[6- (acetyloxy) -7-methoxy-4-quinazolinyl] amino] -4-chlorophenyl] methyl] -N -methyl- methyl ester (0.00055 mol) and the reaction mixture was stirred for 1-2 hours and then the solvent was evaporated. Yield: L-alanine, [V - [[4-chloro-2 - [(6-hydroxy-7-methoxy-4-quinazolinyl) amino] phenyl] methyl] [? / Methyl] methyl ester (used as such in the next reaction step). Step 5

A mixture of L-alanine, N - - [[4-chloro-2 - [(6-hydroxy-7-methoxy-4

quinazolinyl) amino] phenyl] methyl] - [? / - methyl-, methyl ester (0.00055 mol), N- (3-bromopropyl) carbamic acid 1,1-dimethyl ester and Cs 2 CO 3 (0, 8965 g) was stirred overnight at room temperature and then the reaction mixture was stirred for 30 minutes at 50 ° C. When necessary, more CAS 1- (3-bromopropyl) carbamic acid 1,1-dimethylethyl ester was added and the mixture was stirred for 4 hours at room temperature and for another 15 minutes at 50 ° C. The solvent was evaporated (Gene-vac) and the residue was dissolved in CH 2 Cl 2. This solution was filtered over and the filtrate was evaporated. Yield: L-alanine, N - - [[4-chloro-2 - [[6- [3 - [[(1,1-dimethylethyloxy) carbonyl] amino] propoxy] -7-methoxy-4-quinazolinyl] amino] phenyl] methyl] -Î ”-methyl- methyl ester. Step 6

A solution of L-alanine, N - [[4-chloro-2 - [[6- [3 - [[(1,1-dimethylethyloxy) carbonyl] amino] propoxy] -7-methoxy-4-quinazolinyl] amino] phenyl] methyl] -N-methyl- methyl ester (crude residue; 0.00055 mol) in TFA / CH2 Cl2 / TIS (90/8/2) (5.5 ml) was stirred for 7-8 hours then The solvent was evaporated and the residue obtained was dried overnight in an oven. Yield: L-alanine, N - [[2 - [[6- (3-aminopropoxy) -7-methoxy-4-quinazolinyl] amino] -4-chlorophenyl] methyl] - V-methyl-. CF3COOH. Step 7

A solution of L-alanine, N - - [[2 - [[6- (3-aminopropoxy) -7-methoxy-4-quinazolinyl] amino] -4-chlorophenyl] methyl] -N-methyl-COF 3 COOH (0 , 00055 mol) and DIPEA (0.0033 mol) was added to a solution of an HBTU (0.00165 mol) and HOBt (0.0011 mol) in dry DMF (137 ml) and then a reaction mixture was reacted for 1 hour. The solvent was evaporated and the dried residue was purified by reverse phase high performance liquid chromatography. Product fractions were collected, Na 2 CO 3 was added and the organic solvent was evaporated. CH 2 Cl 2 was added to the aqueous concentrate and the resulting mixture was extracted 3 times with CH 2 Cl 2, then the organic extract was dried and collected. Yield: 0.049 g of 4,6-ethanediylidene-12 / - / - pyrimido [4,5-b] [6,1,10,13] benzoxatriazacyclohexadecine-12-one, 18-chloro-8.9 10,11,13,14,15,20-octahydro-21-methoxy-13,14-dimethyl- (13S) -.

When used in the examples, 'CH 3 OH' means methanol, 'Et 3 N' means triethylamine, 'CH 2 Cl 2' means dichloromethane, 'HBTU' means 1- [bis (dimethylamino) methylene] -1H-Benzotriazoliumhexafluorophosphate (1 -) 3-oxide 'DMF' means 'V' / dimethylformamide, 'NaBH (OAc) 3' means sodium triacetoxyborohydride, 'DIPEA' means N-ethyl-N- (1-methylethyl) -2-propanamine, 'HOBt' means 1-hydroxy-1H-benzotriazole, 'TFA' means trifluoroacetic acid, 'TIS' means tris (1-methylethyl) silane, 'K 2 CO 3' means potassium carbonate, 'C 2 CO 3' means cesium carbonate, ' Na 2 CO 3 'means carbonic acid disodium salt,' NaHCO 3 'means monosodium carbonic acid salt.

Preparation of Compound 22. MTK11

A suitable preparation of the preferred compound used in this invention, taken from WO-2004/105765, follows: Example A

a) Preparation of 1-pentanol. 5-fr (4-bromo-2-nitropheninmetinamino1- (intermediate 1)

A solution of 4-bromo-2-nitro-benzaldehyde (0.013 mol), 5-amino-1-pentanol (0.013 mol) and titanium tetracis (2-propanolate) (0.014 mol) in EtOH (15 ml) was Stirred at room temperature for 1 hour, then the reaction mixture was heated to 50 ° C and stirred for 30 minutes. The mixture was cooled to room temperature and NaBH4 (0.013 mol) was added portion by portion. The reaction mixture was stirred overnight and then poured into ice water (50 mL). The resulting mixture was stirred for 20 minutes, the precipitate formed was filtered (yielding Filtrate (I)), washed with H 2 O and stirred in DCM (to dissolve the product and remove from the Ti salt). The mixture was filtered and then the filtrate was dried (MgSO4) and filtered, finally the solvent was evaporated. Filtrate (I) was evaporated until EtOH was removed and the aqueous concentrate was extracted 2 times with DCM. The organic layer was separated, dried (MgSO 4), filtered and the solvent was evaporated, yielding 3.8 g (93%) of intermediate 1.

Example B

a) Preparation of 1-pentanol, 5-Î ± (4-bromo-2-nitropheninmethylmethylamino1- (intermediate 2)

A solution of intermediate 50 (0.0047 mol), formaldehyde (0.025 mol) and titanium, tetracis (2-propanolate) (0.0051 mol) in EtOH (150 ml) was heated to 50 ° C and stirred for 1 hour, then NaBH4 (0.026 mol) was added portion by portion at room temperature. The reaction mixture was stirred overnight and then quenched with water (100 mL). The resulting mixture was stirred for 1 hour; The precipitate formed was filtered off and washed. The organic filtrate was concentrated, then the aqueous concentrate was extracted with DCM and dried. The solvent was evaporated and the residue was filtered on silica gel (eluent: DCM / CH 3 OH from 98/2 to 95/5). The product fractions were collected and the solvent was evaporated, yielding 0.5 g of intermediate 2.

b) Preparation of 1-pentanol, 5- (4-bromo-2-nitrophenylmethylmethylamino) acetate (ester) (intermediate 3)

A solution of intermediate 2 (0.0015 mol) and pyridine (0.015 mol) in acetic anhydride (8 ml) was stirred overnight at room temperature, then the solvent was evaporated and coevaporated with toluene, yielding intermediate 3. c) Preparation of 1-pentanol. 5-fr (2-amino-4-bromopheninmethylmethylamino1-, acetate (ester) (intermediate 4)

A mixture of intermediate 3 (0.0015 mol) in THF (50 ml) was hydrogenated with 5% Pt / C (0.5 g) as a catalyst in the presence of thiophene solution (0.5 ml) [H179 After uptake of H2 (3 equiv.), The catalyst was filtered off and the filtrate was evaporated, yielding 0.5 g of intermediate 4.

d) Preparation of 6-quinazolinol. 4-Rf-2Î ± -5- (acetyloxy) pentinmethylamino1methyl1-5-bromophenyl1amino1-7-methoxy acetate (ester) (intermediate 5)

A mixture of intermediate 4 (0.0015 mol) and 4-chloro-7-methoxy-6-quinazolinol acetate (ester) (0.0015 mol) in 2-propanol (30 ml) was heated to 80 ° C and reaction mixture for 1 day. The solvent was evaporated under reduced pressure and the residue was used as such in the next reaction step, yielding 0.83 g of intermediate 5.

e) Preparation of 6-quinazolinol, 4- (N-5-bromo-2- [r (5-

hydroxypentyl) methylamino1metinfeninamino1-7-methoxy (intermediate 6)

A solution of intermediate 5 (0.0015 mol) in methanol (25 ml) was stirred at room temperature and a solution of K 2 CO 3 (0.003 mol) in H 2 O (2.5 ml) was added, then the reaction mixture was heated to 60 ° C and stirred for 18 hours. The solvent was evaporated and H 2 O (20 mL) was added, then the mixture was neutralized with acetic acid and the precipitate formed was filtered. The filtrate was concentrated under reduced pressure and the concentrate was extracted with DCM, filtered, then dried (MgSO 4) and the mixture was concentrated under reduced pressure yielding 0.5 g (70%) of intermediate 6. Example C

a) Preparation of 4,6-ethanediylidene pyrimidof4,5-iro 6,1,121benzoxadiazacyclopentadecine, 17-bromo-8,9,10,11.12.13.14,19-octahydro-20-methoxy-13-methyl- MTK11 compound)

A solution of intermediate 6 (0.0011 mol) in THF (50 ml) was stirred at room temperature and tributylphosphine (0.0016 mol) was added, then 1,1 '- (azodicarbonyl) bispiperidine (0 0.0016 mol) was added and the reaction mixture was stirred for 2 hours. The solvent was evaporated to 1/3 of the initial volume. The resulting precipitate was filtered off and washed. The filtrate was evaporated and the residue was purified by reverse phase high performance liquid chromatography. The product fractions were collected and the organic solvent was evaporated. The aqueous concentrate was extracted 2 times with DCM and the organic layer was dried (MgSO4) 1 then filtered. The solvent was evaporated and the residue was dried (vac.) At 50 ° C, yielding 0.004 g (0.8%) of compound MTKI1.

Preparation of Compound 2. 4.6-ethanediylidene-19 / - / - pyrimidin4,5-b1f6,13,11benzodioxa-azacyclopentadecine, 15-chloro-8,9,10,11,12,13-hexahydro 20-methoxy; Example D

Preparation of 4,6-ethanediylidene-19H-pyrimidof4,5-6,16,13,1benzodioxazacyclopentadecine. 15-chloro-8.9,10.11.12.13-hexahydro-20-methoxy (compound 2)

A solution of intermediate 9 (0.0024 mol) and triphenylphosphine (0.0036 mol) in dry THF (100 ml) was stirred at room temperature and then a solution of bis (1-methylethyl) diazenedicarboxylate (0.0036 mol). ) in THF (10 ml) was added dropwise. The reaction mixture was stirred for 6 hours and extra bis (1-methylethyl) diazenedicarboxylate (0.35 mL) in THF (10 mL) was added. The mixture was stirred overnight and concentrated. The residue was purified by column chromatography over silica gel (eluent: DCM / CH 3 OH / THF 90/5/5). Product fractions were collected and also purified by reverse phase high performance liquid chromatography. The product fractions were collected and concentrated. The aqueous concentrate was filtered, and the retained solid washed and dried (vac.) At 65 ° C, yielding 0.065 g of compound 2, mp 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 1 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 minutes. 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)

A mixture of intermediate 6 (0.046 mol) and ethanimate (2 g) in

THF (ml) was hydrogenated with 5% Pt / C (3 g) as a catalyst in the presence of DIPE (2 ml). After uptake of H2 (3 equiv.), The reaction mixture was filtered in small buffer from Dicalite and the filtrate was concentrated, yielding intermediate 7. c) Preparation of hexanoic acid, 6-r2-f [6 - (acetyloxy) -7-methoxy-4-quinazolinylaminamin-6-chlorophenoxy- methyl ester (intermediate 8)

A mixture of 4-chloro-6-methylcarbonyloxy-7-methoxyquinazoline (0.022 mol) and intermediate 7 (0.022 mol) in 2-propanol (170 ml) was stirred and heated at 80 ° C for 2 hours, concentrated and the residue was concentrated. chromatographed on silica gel (eluent: DCM / CH 3 OH 97/3). The product fractions were collected and the solvent was evaporated, yielding 5.1 g of intermediate 8 (used as such in the next reaction step).

d) Preparation of 6-quinazolinol, 4-fr3-chloro-2- (6-hydroxyhexyl) oxyphenylamino1-7-methoxy (intermediate 9) A mixture of LAH (0.0246 mol) in THF (40 ml) it was hectic

at room temperature. A solution of intermediate 8 (0.006 mol) in THF (60 ml) was added dropwise. The reaction mixture was stirred for 1 day, then extra LAH (0.0123 mol) was added dropwise. The mixture was also stirred over the weekend then H 2 O (2 mL) was added dropwise, followed by the dropwise addition of a 15% NaOH solution (2 mL) and H 2 O (6 mL). This mixture was stirred for 15 minutes filtered and the filtrate was concentrated. The residue was stirred in boiling CH 3 CN, filtered and the retained solid was dried (vac.) At 60 ° C. The solids were redissolved in CH 3 OH / DCM (10/90) and this mixture was neutralized with HCl (1 Ν). The organic layer was separated, dried (MgSO4), filtered and concentrated to yield 1 g of intermediate 9.

An example of illustrative formulation for the most preferred compound, MYKI1, is as follows: Example F: Formulation:

MTKI1 may be prepared as an oral solution at 10-mg / mL, pH 2. Contains an excipient, Captisol® (chemical name: sulfo-butyl ether-p-cyclodextrin, SBE-CD), citric acid, Tween® 20, HCl and NaOH in purified water. The formulation may be stored refrigerated (2-8 ° C (36-46 ° F)) and allowed to warm to room temperature for up to 1 hour prior to dose preparation.

The MTKI1 product may likewise be prepared as 50-mg, 100-mg and 300-mg oral immediate release capsules containing the active chemical entity MTKI1, Iactose monohydrate (200 mesh), sodium lauryl sulfate and Magnesium stearate in hard gelatin capsules, sizes 3, 4 and 00, respectively. The capsules may likewise contain any or all of the following ingredients: gelatin, red iron oxide and titanium oxide. EXPERIMENTAL DATA Inhibition of VEGFR3

Iymphode involvement is a poor prognostic factor in many types of cancer. Recent studies have shown that lymphatic vessel formation plays an important role in tumor progression and CeD vascular endothelial growth factors (VEGF) have been identified as specific lymphangiogenic factors acting through activation of the VEGFR3 cognate receptor. 4,6-ethanediylidenopyrimidate [4,5-δ] [6,1,12] benzoxadiazacyclopentadecine, 17-bromo-8,9,10,11,12,13,14,19-octahydro Methoxy-13-methyl-, hereinafter also referred to as Compound 2, is a multi-targeted kinase inhibitor that has been shown to have a unique kinase inhibition profile (pan Her and Src family) as well as a distribution profile. of favorable tissue resulting in potent antitumor activity in various experimental models.

In in vitro kinase assays showed that the compounds of the present

This invention has potent inhibition of EGFR1 family activity Her2, Her4 and Src kinases (Src, Fyn, Lck, Yes, Lyn). In vitro kinase inhibition was validated here in cell-based assays using human vascular endothelial cells (HM-VECd), where Compound 2 prevented Erkl / 2 VEGFR3-dependent VEGF-C stimulation as it did not affect signaling. of VEGFRI-mediated VEGF. Compound 2 was likewise shown herein to inhibit VEGF-C-induced phosphorylation of VEGFR3 using a human VEGFR3 overexpression directed cell line.

These results have also been validated here in a newly developed Xenopus tadpole model where Compound 2 was found to phenocopy the knockout effects of VEGF-C. (Ny et al., Nat. Med. 2005 Sep; 11: 998-1004). Methods

In vitro kinase assays

The VEGFR3 kinase reaction was performed at 30 ° C for 10 minutes in a 96-well microtiter plate. The 25 μΙ reaction volume contained 8 mM MOPS pH 7, 200 μΜ EDTA, 10 mM MgAc, unlabeled 10 μΜ ATP, 0.5 mCi AT33P, 500 μΜ JAK3-tide (AcGGEEEEYFELVKKKK- NH 2), 25 ng VEGFR 3 and 2% compound in 100% DMSO.

The reaction was stopped by adding 5 μΙ of a

3% phosphoric acid. 10 μΙ of the reaction mixture was then stained on a Filtermat P30 (WaIIac) filter and washed 3 times for 5 minutes in 0.75% phosphoric acid and 1 time for 2 minutes in methanol before transferring into a sealable plastic bag containing 4 ml of scintillation liquid and read on a scintillation counter. VEGFR3 Cellular Assays

VEGF-C-induced inhibition of VEGFR3 activity

For this assay, porcine aortic endothelial cells (PAE) stably expressing human VEGFR-3 were grown in a 10 cm-dish. The cells were starved overnight and the medium replaced with fresh serum free media. Predetermined amounts of Compound 2 or control vehicle were added to the cells, and then incubated at 37 ° C for 15 minutes. This was followed by the addition of VEGF-C to a final concentration of 100 ng / ml and the cells incubated for 10 minutes at 37 ° C, after which they were washed with ice cold PBS and then lysed. Cell lysates containing protease inhibitors were immunoprecipitated with a VEGFR-3 specific antibody and the immunoprecipitates separated by SDS-PAGE. Proteins were transferred on a nitrocellulose membrane, and then first tested with a phosphotyrosine antibody and then a VEGFR-3 antibody. VEGF-C-Induced Erk Activation Inhibition in Human Micro-Vessel Endothelial Dermal Cells (HMVEC-d)

HMVEC-d cells (Cambrex) were cultured at 37 ° C in 5% CO 2 in medium containing appropriate serum (Cambrex) in fibronectin-coated 12-well plates (BD Biocoat, Becton Dickinson) for 4 days. The cells were then deprived of food for 16 hours in the same medium as above but requiring serum and containing Sl-TE + 3 (Invitrogen) in place. The cells were then preincubated with 0.2% DMSO (the solvent) or 5uM Compound 2 for 60 minutes and then either 10 ng / ml VEGF or 100 ng / ml VEGF-C ( R&D) were added to the cells. Prior to, or 10 and 30 minutes after challenge, the medium was removed, cells were rinsed with cold PBS containing 0.1 mM sodium orvanadate and lysed in M-PER (PIERCE) buffer containing 0% SDS. , 1%, and 1x phosphatase and protease inhibitors (HALT, Pl-ERCE). Cells were scraped from the plates and incubated in lysis buffer for 30 minutes on ice and then centrifuged at 4 ° C for 10 minutes at 16000xg. Lysates were quantified using BCA reagent according to the manufacturer's instructions (PIERCE), and 5 mg of total protein was loaded onto SDS-polyacrylamide gels (NUPAGE, Invitrogen). Electrophoresis and staining on PVDF membranes were all performed using NUPAGE system reagents according to the manufacturer's instructions. Spots were blocked for 1 hour with Odyssey Blocking Buffer (LICOR) and then primary antibodies (see below) were diluted 1: 1000 in a 1: 1 mixture of Odyssey Blocking Buffer and PBS containing Tween-20 at 0 ° C. , 1%, and applied overnight at 4 ° C: P-Erkl / 2 (Thr202 / Tir204, clone E10, mouse monoclonal No. 9106, Erk1 / 2 (polyclonal rabbit No. 9102) (all from Cell Signaling Technologies, The spots were washed three times in PBS containing 0.1% Tween for 5 minutes and then incubated for 1 hour with secondary antibodies (diluted 1: 1000 in PBS containing 0.1% Tween-20): anti - 800nm IRDye-conjugated goat mouse (Rockland, Inc) or Alexa 680nm-conjugated goat anti-rabbit (Molecular Probes, Inc).

The spots were washed three times in PBS containing 0.1% Tween for 5 minutes and once in PBS and evaluated using the 700nm and 800nm LICOR system using intensity 5 and all appropriate settings for the sweep. membranes. VEGFR3 In vivo Xenopus Assay

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 middle of the Xenopus tadpoles beginning at stage 26-28, that is, prior to the formation of equally blood and lymphatic vessels. Compound effects were monitored by "live analysis", ie live embryos were examined 4 days after evidence of lymph vascular defects by trained observers. As an additional means of confirming the results, in a limited number of experiments, tadpoles were fixed at stage 35-36 and in situ hybridizations to Proxl were performed to evaluate posterior cardinal vein (PCV) lymphatic endothelial cell (ECF) migration to their dorsal end point, this reflecting an essential step in lymphangiogenesis. Under normal circumstances, ECFs arise from the accumulated PCV1 region, and during the study period they migrate dorsally to "defined areas" (Area 1 to Area 2 and Area 3), where they then also migrate rostrally and caudally to form the lymphatic vasculature in the tail and trunk. Defects in ECF migration from PCV were quantified by determining the maximum cell migration, and counting the number of cells in these different tail regions. Results

In vitro kinase assays

The following table provides the p150 values obtained for the compounds of the present invention using the VEGFR3 kinase assay mentioned above.

Structure Name Pic50 ι — ι Cl 4,6-ethanediylidene-19 H-pyrimido [4,5-δ] [6,13,1] benzodioxazacyclopentadecine, 15-chloro-8,9,10,11, 12,13-hexahydro-20-methoxy-4,6-ethanediylidopyrimidate [4,5-b] [6,1,12] benzoxadiazacyclopenta decine , 17-bromo-8,9,10,1 1,12,13,14,19-octahydro-20- methoxy-13-methyl-7,74 OVN 12H-4,6-ethanediylidene-13, 17-methanopyrime [4,5-b] [6,1,10,16] benzoxatriazacyclone nadecin-12-one, 21-chloro-8,9,10,11,13,14,15,16,18,23- decahydro-25-methoxy; 6 H CYsOCX \ HN ^^ Cl \ J, A 4,6-ethanediylidene-12H-pyrimido [4,5-b] [6,1,10,13] benzoxatriazacyclohexadecin-12-one, 18-chloro - 8,9,10,11,13,14,15,20-octahydro-21-methoxy-13,14-dimethyl-6

VEGF-C-Induced VEGFR-3 Phosphorylation Inhibition Induced in VEGFR-3 Expressing Porcine Aortic Endothelial Cells

The effect of 4,6-ethanediylidenopyrime [4,5-b] [6,1,12] benzoxadiazacyclopentadecine, 17-bromo-8,9,10,11,12,13,14,19-octa -hydro-20-methoxy-13-methyl- (Compound 2) in VEGF-C-induced VEGFR3 activity is evident from the dose-dependent reduction in VEGFR-3 phosphorylation (Top Structure of Figure 1). Presence of VEGFR-3 at different concentration is confirmed under Western blotting with VEGFR-3 antibodies (Base Structure of Figure 1). Erk phosphorylation assays in HMVEC-d cells As illustrated in Figure 2, Compound 2 at 5 μΜ, phosphorylation

prevented Erk1 / 2 by VEGF-C but did not interfere with Erk1 / 2 VEGF stimulation. These data are consistent with selective inhibition of VEGFR-3 by Compound 2. Xenopus Study Results The effects of Compound 2 were evaluated using screening.

In vivo, concentrations of Compound 2 of> 50 µM induced bleeding in 30% of the tadpoles. At 20 µM, 10% of tadpoles developed edema, which was not seen in vehicle-treated tadpoles alone. Compound 2 dose dependent inhibits Lymphatic Endothelial Cell (ECF) Migration in tadpoles (Table 1). Stage 35-36 ECF migration was particularly decreased, even at the lowest concentrations of the test compound (0.31 µM), and this was more than observed with vehicle alone. These data support the conclusion that this compound interferes with normal lymphangiogenesis. Table 1

Quantification of lymphatic endothelial cell migration (detected by Proxl in situ hybridization) from the posterior cardinal vein to the dorsum after exposure of tadpoles to stages 35-36 for compounds (n = 10-15 embryos per condition) Exp. 08.08.06 Concentration of Compound 2 in μΜ 80 20 5 1.25 0.3125 DMSO% embryos with 80 60 40 63 57 21 reduced migration% Relative Area 56 ± 15 19 ± 8 123 ± 32 20 ± 8 37 ± 20% Bad Migration - 67 ± 21 35 ± 1 121 ± 3 47 ± 5 73 ± 22 maximum Reduced

Note: The above assessments of ECL migration from Proxl positive cells were first performed microscopically in a semi-quantitative manner by a trained observer ('% of reduced migrating embryos'). Quantitative computer generated analysis was then performed. "Relative Area 2-3" is the number of ECFs that migrated to control relative area 2-3 (vehicle only). "Relative maximum migration" is the most distant ECF migration from PCV under treatment relative to the maximum observed in vehicle-only controls.

While the foregoing specification teaches the principles of the present invention, with examples provided for purposes of illustration, it will be understood that the practice of the invention encompasses all customary variations, adaptations and / or modifications as they fall within the scope of the present invention. following claims and their equivalents.

Claims (20)

A method for treating or preventing a VEGFR3-mediated biological activity in a mammalian subject comprising administering a therapeutically effective amount of a compound of formula (I). pharmaceutically acceptable or base addition salts and stereochemically isomeric forms thereof, wherein Z represents NH; Y represents -C3-9 alkyl-, -C1-5 alkyl-NR13 -C1-5 alkyl-, -C1-5 alkyl-NR14-CO-C1.5 alkyl-, -C4 alkyl-NH-CO-Het20-, or -Het22-CH2 -CO- NH-C1-3 alkyl-; X1 represents O, or -O-C1-2 alkyl-; X 2 represents a direct bond, -C 1-2 alkyl-, O, -0-C 1-2 alkyl-, NR 12 or NR 12 -C 12 alkyl-; R1 represents hydrogen, cyano, halo or hydroxy, preferably halo; R 2 represents hydrogen, cyano, halo, or hydroxy; R3 represents hydrogen; R4 represents C1-4 alkyloxy; R 12 represents hydrogen, or C 1-4 alkyl-; R13 represents hydrogen or C1-4 alkyl; R14 represents hydrogen or C1-4 alkyl; Het 20 represents pyrrolidinyl, piperazinyl or piperidinyl; and Het22 represents pyrrolidinyl, piperazinyl or piperidinyl in a mammalian subject in need of such treatment. A method according to claim 1, wherein in the compound of formula (I); Z represents NH; Y represents -C 3-9 alkyl-, -C 1-5 alkyl-NR 14 -CO-C 1-5 alkyl-, or -C 1-4 alkyl-NH-CO-Het 2 O-; X1 represents O; X 2 represents -C 1-6 alkyl, O, or NR 12 -C 12 alkyl-; R1 represents hydrogen or halo; R2 represents hydrogen or halo; R3 represents hydrogen; R4 represents C1-4 alkoxy; R 12 represents C 1-4 alkyl-; R14 represents hydrogen or C1-4 alkyl; and Het 20 represents pyrrolidinyl, piperazinyl or piperidinyl. A method according to claim 1, wherein the compound of formula (I) is selected from the group consisting of 4,6-ethanediylidene-19 / - / - pyrimido [4,5-ib] [6,13,1] benzodioxa-azacyclopentadecine, 15-chloro-8,9,10,11,12,13-hexahydro-20-methoxy; 12H-4,6-ethanediylidene-13,17-methanopyrime [4,5-b] [6,1,10,16] benzoxatriazacyclononadecin-12-one, 21-chloro-8,9,10,11,13 , 14,15,16,18,23-decahydro-25-methoxy; 4,6-ethanediylidene-12H-pyrimido [4,5-b] [6,1,10,13] benzoxatriazacyclohexadecin-12-one, 18-chloro-8,9,10,11,13,14,15 20-octahydro-21-methoxy-13,14-dimethyl-; and 4,6-ethanediylidenopyrimidate [4,5-b] [6,1,12] benzoxadiazacyclopentadecin, 17-bromo-8,9,10,11,12,13,14,19-octa-hydrodiazide 20-methoxy-13-methyl; or a pharmaceutically acceptable acid addition salt thereof. The method according to claim 3, wherein the compound is 4,6-ethanediylidene pyrimidated [4,5-b] [6,1,12] benzoxadiazacyclopentadecine, 17-bromo-8,9,10 11,12,13,14,19-octahydro-20-methoxy-13-methyl-; or a pharmaceutically acceptable acid addition salt thereof. The method of claim 1, wherein a therapeutically effective amount of the compound is administered orally or parenterally. A method according to claim 1, wherein the compound of formula (I) is administered in combination with another anticancer agent. A method according to claim 6, wherein the other anticancer agent is selected from the group consisting of herceptin, docetaxel, anthracycline and capecitabine in the case of breast cancer; docetaxel and mitoxantrone in the case of prostate cancer; oxaliplatin, 5-FU, avastin, irinotecan and cetuximab in the case of colon cancer; and taxotere, carboplatin and gemicitabine in the case of lung cancer. A method according to claim 1, wherein the VEGFR3 mediated biological activity is selected from the group consisting of metastatic spread of a cancer in a mammalian subject; metastasize to regional lymph nodes through lymphatic vessels; tumor-associated lymphangiogenesis in cancers; recruitment and proliferation of VEGFR3-expressing endothelial cells in the neovascularization of cancer-expressing cells expressing a VEGFR3 ligand; and combinations thereof. The method of claim 8, wherein said VEGFR3 Binder is VEGF-C, VEGF-D, or a combination thereof. A method according to claim 1 wherein the VEGFR3-mediated biological activity is advanced breast cancer in a mammal comprising the steps of administering a therapeutically effective amount of a compound as defined in any one of the above. claims 1 to 4 to said mammal. Use of a compound of formula (I) is the pharmaceutically acceptable acid or base addition salts and stereochemically isomeric forms thereof, wherein Z represents NH; Y represents -C3-9 alkyl-, -C1-5 alkyl-NR13 -C1.5 alkyl-, -C1.5 alkyl-NR14 -CO-C1-5 alkyl-, -C4 alkyl-NH-CO-Het20-, or -Het22- CH2 -CO-NH-C1-3 alkyl-; X 1 represents O, or -O-C 1 -C 2 alkyl-; X 2 represents a direct bond, -C 1-2 alkyl-, O, -O-C 1-2 alkyl-, NR 12 or NR 12 -C 1-4 alkyl; R1 represents hydrogen, cyano, halo or hydroxy, preferably halo; R 2 represents hydrogen, cyano, halo, or hydroxy; R3 represents hydrogen; R4 represents C1-4 alkyloxy; R12 represents hydrogen, or C1-4 alkyl; R13 represents hydrogen or C1-4 alkyl; R14 represents hydrogen or C1-4 alkyl; Het 20 represents pyrrolidinyl, piperazinyl or piperidinyl; and Het22 represents pyrrolidinyl, piperazinyl or piperidinyl; and in the manufacture of a medicament for the treatment of VEGFR3-mediated biological activities. Use according to claim 10, wherein in the compound of formula (I); Z represents NH; Y represents -C 3-9 alkyl-, -Cl-Salkyl-NR 14 -CO-C 1 -C 7 -alkyl-, or -C 1 -Salkyl-NH-CO-Het 2 O-; X1 represents O; X 2 represents -C 1-6 alkyl, O, or NR 12 -C 1-2 alkyl-; R1 represents hydrogen or halo; in particular R1 represents hydrogen or chlorine; more particularly R1 represents hydrogen; R2 represents hydrogen or halo; in particular R2 represents hydrogen, chlorine or bromine; more particularly R2 represents chloro or bromo; R3 represents hydrogen; R4 represents C1-4 alkoxy; in particular R 4 represents methoxy; R12 represents C1-4 alkyl-; in particular R 12 represents methyl; R14 represents hydrogen or C1-4 alkyl; in particular R14 represents hydrogen or methyl; more particularly R14 represents hydrogen; Het 20 represents pyrrolidinyl, piperazinyl or piperidinyl; in particular Het20 represents piperidinyl; in particular Het20 represents piperidinyl. Use according to claim 11, wherein the compound of formula (I) is selected from the group consisting of; 4,6-ethanediylidene-19H-pyrimido [4,5-b] [6,13,1] benzodioxazacyclo-pentadecine, 15-chloro-8,9,10,11,12,13-hexahydro Methoxy; 12H- 4,6-ethanediylidene-13,17-methanopyrime [4,5-b] [6,1,10,16] benzoxatriazacyclononadecin-12-one, 21-chloro-8,9,10,11,13 , 14,15,16,18,23-decahydro-25-methoxy; 4,6-ethanediylidene-12H-pyrimido [4,5-b] [6,1,10,13] benzoxatriazacyclohexadecin-12-one, 18-chloro-8,9,10,11,13,14 , 15,20-octahydro-21-methoxy-13,14-dimethyl-; and 4,6-ethanediylidenopyrime [4,5-b] [6,1,12] benzoxadiazacyclopentadecin, 17-bromo-8,9,10,11,12,13,14,19-octahydro-20- methoxy-13-methyl-; or a pharmaceutically acceptable acid addition salt thereof. Use according to claim 11, wherein the compound of formula (I) is 4,6-ethanediylidenopyrimidated [4,5- /]] [6,1,12] benzoxadiazacyclopentadecine, 17-bromo -8,9,10,11,12,13,14,19-octahydro-20-methoxy-13-methyl; or a pharmaceutically acceptable acid addition salt thereof. Use according to any one of claims 11 to 14, wherein the medicament is for oral or parenteral administration. Use according to claim 15, wherein the compound of formula (I) is administered in combination with another anticancer agent. Use according to claim 16, wherein the other anticancer agent is selected from the group consisting of herceptin, docetaxel, anthracycline and capecitabine in the case of breast cancer; docetaxel and mitoxantrone in the case of prostate cancer; oxaliplatin, 5-FU, avastin, irinotecan and cetuximab in the case of colon cancer; and taxotere, carboplatin and gemicitabine in the case of lung cancer. Use according to any one of claims 11 to 14, wherein the VEGFR3-mediated biological activity is selected from the group consisting of: - metastatic spread of a cancer in a mammalian subject; - regional lymph node metastasis by lymphatic vessels; - tumor-associated lymphangiogenesis in cancers, such as for example in gastric cancer, prostate cancer, human colorectal cancer, invasive cervical cancer, breast cancer, Kaposi's sarcoma and melanoma; and recruitment and proliferation of endothelial cells expressing VEGFR3 in neovascularization of cancer cells expressing a VEGFR3 ligand. Use according to claim 8, wherein said VEGF3 ligand is VEGF-C, VEGF-D or a combination thereof. Use according to any one of claims 1 to 4, wherein the VEGFR3-mediated biological activity is advanced breast cancer.