Methods and compositions utilizing quinazolinones

Description of the invention

The invention relates to quinazoline compounds. Another object of the invention is the use of these compounds. The invention further relates to pharmaceutical compositions containing these compounds.

Interest in the medical use of quinazoline derivatives began in the early 1950s with the elucidation of the structure of the quinazoline alkaloid, 3-e-keto-gamma- (3-hydroxy-2-piperidyl) propyl] -4-quinazolone, derived from an Asian plant with antimalarial properties. As a result of the search for other antimalarial agents, various substituted quinazolines have been synthesized. The synthesis of the 2-methyl-3-o-tolyl-4- (3H) -quinazolone derivative was of particular importance. This compound, known as methaqualone, has been shown to be effective in hypnotics despite having no effect on protozoa.

Since then, the pharmacological activity of quinazolones and their derivatives has been studied. It is now known that they have a hypnotic, sedative, analgesic, anticonvulsant, antitussive and anti-inflammatory effect.

Specific applications for quinazoline derivatives are described in US Patent Nos. 5,147,875, which discloses the activity of 2- (substituted phenyl) -4-oxoquinazoline as a bronchodilator; No. 3,723,432; No. 3,740,442 and No. 3,925,548 which describe a group of 1-substituent-4-aryl-2 (1H) -quinazolone derivatives used as anti-inflammatory agents. Other examples are given in European patents, namely: EP 0 056 637 B1 - 4 (3H) -quinazolone derivative group for the treatment of hypertension; EP 0 884 319 A1 compositions of 4-quinazolone for the treatment of disorders of the central and peripheral nervous system, which are degenerative in nature and caused by psychotropic agents, drugs and alcohol.

Quinazolones are part of a growing group of agents for the treatment of diseases related to cell proliferation, including cancer. For example, PCT Application No. WO 96/06616 describes a pharmaceutical composition containing a quinazoline derivative for inhibiting the proliferation of soft vascular cells, and PCT Application No. WO 96/19224 describes mesangial cells. U.S. Patent Nos. 4,981,856; No. 5,081,124 and No. 5,280,027 disclose quinazoline derivatives acting as inhibitors of thymidylate synthase, ie the enzyme catalyzing the methylation of deoxyuridine monophosphate, leading to the production of thymidine monophosphate needed for DNA synthesis. US Patent Nos. 5,747,498 and 5,773,476 disclose such derivatives for the treatment of tumors characterized by overactive or inadequate activity of tyrosine kinase receptors. US Patent No. 5,037,829 discloses (1H-1-azolylmethyl) substituted quinazoline compositions for treating cancer in epithelial cells; in PCT application under No. WO 98/34613 - derivatives for enhancing re-vascularization and treating malignancy; and in US Patent No. 5,187,167, 4-quinazolone derivatives with antitumor activity.

Other cancer treatments are taxane and vinca alkaloids. They act on microchannels located in the structure of cells. Microchannels are the basic building block of the mitotic spindle, which is responsible for delivering replicated copies of the genome to each of the two daughter cells that result from cell division. It is believed that damage to the mitotic spindle caused by these drugs inhibits cancer cell division and triggers their death. Since microchannels also form other types of cellular structures, such as pathways for intracellular transport in nervous processes, and the effects of these agents are not limited to mitotic spindles, there are side effects that reduce the usefulness of drugs.

Improving the specificity of action of agents used in the treatment of cancer is very important because of the therapeutic benefits that can be obtained while reducing the side effects of their administration. Traditionally, significant improvements in the treatment of cancer have been achieved through the identification of therapeutic agents with a novel mechanism of action. This applies not only to taxanes, but also to the camptothecin group of topoisomerase I inhibitors. For both these reasons, mitotic kinesins are interesting targets for new anti-cancer agents.

Mitotic kinesins are essential enzymes for the structure and function of the mitotic spindle, but are not generally part of other microchannel structures, such as in nervous processes. They play an essential role in all phases of mitosis as molecular motors

It converts the energy released by adenosine triphosphate hydrolysis into mechanical force that causes the directed movement of molecular charges along the microchannels. A catalytic domain suitable for this task is a compact structure of about 340 amino acids. During mitosis, kinesins organize the microchannels into a bipolar structure that is the mitotic spindle. Kinesins mediate the movement of chromosomes in the spindle microchannels as well as structural changes in the spindle during certain phases of mitosis. Experimental disruption of mitotic kinesins causes inappropriate formation or dysfunction of the mitotic spindle, sometimes leading to cell cycle arrest and cell death.

One of the identified mitotic kinesins is KSP. KSP belongs to the evolutionarily conserved subfamily of kinesins with microchannel drives directed to the positive pole, which form bipolar homotetramers composed of antiparallel homodimers. During mitosis, KSP binds to the microchannels of the mitotic spindle.

Injection of antibodies to KSP into human cells prevents detachment of the pole during prometaphase, increasing the growth of unipolar spindles and inhibiting mitosis and inducing cell death. In non-human organisms, KSP and other kinesins cause antiparallel microchannels to stick together and shift relative to each other, leading to a separation of the spindle poles. KSP may also participate in the elongation of the spindle during anaphase B and the focusing of microchannels on the spindle pole.

Human KSP (also called HsEg5) has been described in the literature [Blangy et al., Cell, 83: 1159-69 (1995); Whitehead et al., Arthritis Rheum., 39: 1635-42 (1996); Galgio et al., J. Cell. Biol., 135: 339-414 (1996); Blangy et al., J. Biol. Chem., 272: 19418-24 (1997); Blangy et al., Cell Motil Cytoskeleton, 40: 174-82 (1998); Whitehead and Rattner, J. Cell. Sci, 111: 2551-61 (1998); Kaiser et al., JBC 274: 18925-31 (1999); GenBank accession numbers: X85137, NM004523 and U37426] and the KSP gene fragment (TRIP5) is described in [Lee et al., Mol Endocrinol., 9: 243-54 (1995); GenBank accession number L40372]. The homologues of Xenopus KSP (Eg5) and Drosophila KLP61 F / KRP1 30 have also been reported.

Mitotic kinesins are attractive targets for the discovery and development of new mitotic chemotherapeutic agents. Accordingly, an object of the invention is to obtain new compounds useful for the inhibition of the mitotic kinesin KSP.

This aim is achieved in the solution according to the invention by obtaining and testing the action of quinazoline compounds of the formula:

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or a pharmaceutically acceptable salt thereof, in which the isopropyl group is attached in the R configuration; R1 is benzyl, chlobenzyl, methylbenzyl, methoxybenzyl, cyanobenzyl or hydroxybenzyl; R2 is halogen or cyano; and R3 is a phenyl group substituted with methyl and / or halogen.

Particularly useful are the above compounds or salts as substantially pure optical R isomers.

In a preferred embodiment of the invention, R1 is benzyl.

In another preferred embodiment, R2 is chlorine.

In a further preferred embodiment, R1 is benzyl, R2 is chlorine and R3 is 4-methylphenyl, where it is particularly preferred to act as substantially pure optical R isomers.

Another object of the invention that achieves the set aim is the use of quinazoline compounds of formula:

PL 204 525 B1

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or a pharmaceutically acceptable salt thereof, in which the isopropyl group is attached in the R configuration; R1 is benzyl, chlobenzyl, methylbenzyl, methoxybenzyl, cyanobenzyl or hydroxybenzyl; R2 is halogen or cyano; and R3 is a phenyl group substituted with methyl and / or halogen; preferably in the form of substantially pure optical R isomers; as pharmaceuticals.

This use particularly relates to pharmaceuticals for the treatment of diseases associated with cell proliferation.

In a preferred use, the pharmaceutical compositions are for the treatment of cancer, hyperplasia, restenosis, cardiac hypertrophy, immune system disease or inflammation.

The aim of the invention is also realized by pharmaceutical compositions, characterized in that they contain quinazoline compounds of the formula:

<img file="PL204525B1_D0003.tif" />

or a pharmaceutically acceptable salt thereof, in which the isopropyl group is attached in the R configuration; R1 is benzyl, chlobenzyl, methylbenzyl, methoxybenzyl, cyanobenzyl or hydroxybenzyl; R2 is halogen or cyano; and R3 is a phenyl group substituted with methyl and / or halogen; preferably in the form of substantially pure optical R isomers; and carriers.

Methods for screening compounds that bind to the KSP kinesin, for example compounds that displace or compete with the binding of the compounds of the invention, include combining a labeled compound of the invention, a KSP kinesin, and at least one competitor, and then determining the binding of a bioactive candidate agent to KSP kinesins.

Screening for modulators of KSP kinesin activity is performed by combining the compositions of the invention, the KSP kinesin, and at least one competitor, and then determining the effect of a bioactive candidate on KSP kinesin activity.

The invention is further elucidated in the drawing, in which:

Figure 1 shows a general synthesis scheme for compositions according to the invention, Figure 2 is a route for the synthesis of related quinazoline KSP inhibitors, Figure 3 is a route for the synthesis of essentially pure single enantiomers, Figure 4 is a route for the synthesis of compounds of the invention.

The invention therefore relates to a group of novel compounds based on the basic quinazoline structure that are modulators of mitotic kinesins. By inhibiting or modulating mitotic kinesins, unrelated to other kinesins (e.g. transport kinesins), specific inhibition of cell proliferation is achieved. The invention is based on the discovery that disruption of mitotic kinesin function causes malformations or dysfunction of mitotic spindles, often leading to cell cycle arrest and cell death. The methods of inhibiting the human KSP kinesin include contacting the inhibitor according to the invention with KSP kinesin, especially human also with KSP fragments and variants. The inhibition may involve the activity of KSP kinesin in adenosine triphosphate hydrolysis and / or mitotic spindle formation, resulting in damage to the mitotic spindles. The meiotic spindles can also be damaged.

The main aim of the invention was to develop new inhibitors and modulators of mitotic kinesins, especially KSP, for the treatment of cell proliferative disorders. Typically, a significant improvement in the treatment of cancer, which is one of the disorders of cell proliferation, has been achieved through the identification of therapeutic agents with novel mechanisms of action. Examples are not only compounds from the group of taxanes, which influence the formation of microchannels, but also inhibitors of topoisomerase I from the group of camptothecin. The compositions and methods described may vary in selectivity and are preferably used to treat diseases associated with cell proliferation, including cancer, hyperplasia, restenosis, cardiac hypertrophy, immune disorders, and inflammation.

In the compounds described, halogen is fluorine, chlorine, bromine or iodine. It is preferably fluorine, chlorine and bromine.

These compounds contain an asymmetric center and can therefore form the (R) - or (S) - isomers. Optically active (R) isomers - can be prepared using chiral synthetics or chiral reagents, or resolved by conventional methods. The invention includes all tautomeric forms.

If desired, the R- and S- isomers can be separated by known methods, for example by formation of diastereoisomeric salts or complexes that can be separated, for example by crystallization; by formation of diastereomeric derivatives which may be separated, for example by crystallization, gas-liquid chromatography or liquid chromatography, by selective reaction of one of the enantiomers with a reagent specific for it, for example enzymatic oxidation or reduction, followed by separation of the modified or unmodified enantiomers; or gas-liquid or liquid chromatography in a chiral environment, for example on a chiral support such as silica with an attached chiral ligand or in the presence of a chiral solvent. It will be appreciated that when a desired enantiomer is converted into another chemical unit using one of the separation methods described, a next step will be required to release the desired enantiomeric form. Alternatively, a specific enantiomer may be synthesized by asymmetric synthesis with optically active reagents, substrates, catalysts or solvents, or by conversion to another form via asymmetric transformation. An example of a synthesis from optically active starting materials is shown in Figure 3.

The invention encompasses the use of pure enantiomers and mixtures of enantiomers, including racemic mixtures, although it is preferred to use an optically pure enantiomer, especially the R-enantiomer, in which the i-propyl group is attached to the first carbon atom in the side chain.

Compositions of the invention are synthesized as outlined below using known methods. For example, as described in Ager et al., J. of Med. Chem. 20: 379-386 (1977), quinazolones can be obtained by acid catalyzed condensation of N-acylanthranilic acids with primary aromatic amines. For other synthetic processes, see U.S. Patent Nos. 5,783,577, 5,922,866, and 5,187,167.

The compounds of the invention can be prepared as shown in Figures 1-4.

The compositions according to the invention find many uses. Mitosis can be influenced in many ways; that is, the activity of the component can be increased or decreased in the mitotic pathway. In other words, mitosis can be influenced (e.g., interrupted) by disturbing the equilibrium by inhibiting or activating certain components. Meiosis can be influenced in a similar way.

In a preferred embodiment, the compositions of the invention are used to modulate mitotic spindle formation, thereby prolonging cell cycle inhibition during mitosis. "Modulation is defined as a change in the formation of the mitotic spindle associated with its increase and decrease. The term mitotic spindle formation is understood as the arrangement of microchannels into bipolar structures by mitotic kinesins. "Mitotic spindle dysfunction" is defined as mitotic inhibition and unipolar spindle formation.

PL 204 525 B1

The compositions of the invention are useful for binding and / or modulating the activity of the mitotic kinesin, KSP. It is preferably human KSP, although KSP kinesins from other organisms can also be used. In this context, modulating means increasing or decreasing the separation of the spindle poles, causing inappropriate formation, i.e. distortion of the poles of the mitotic spindle, or its morphological disturbance. This also applies to variants and / or fragments of KSP. [see U.S. Patent No. 6,617,115 Methods of Screening for Modulators of Cell Proliferation and Methods of Diagnosing Cell Proliferation States; patent application October 22, 1999 (US Serial No. 09 / 428,156)]. Other mitotic kinesins may also be used in the uses of the invention, but the disclosed compositions are specific to KSP.

To determine the activity, KSP or a compound according to the invention is diffusion-inhibitingly bound to an insoluble support which has dedicated sample application sites (e.g., microtiter plate, matrix, etc.). The insoluble substrate can be made of any material to which the composition can be bonded. It can be easily separated from the soluble material and is compatible with the general screening method. The surface of such substrates may be solid or porous, and the shape may be any. Examples of suitable insoluble media are microtiter plates, matrices, membranes, and pellets. They are usually made of glass, plastics (e.g. polystyrene), polysaccharides, nylon or nitrocellulose, Teflon ™ etc. Microtiter plates and arrays are particularly convenient because they allow you to run a large number of tests simultaneously, using small amounts of reagents and samples. The particular method of binding the composition is not critical as long as it is compatible with the reagents and general methods of the invention, maintains the activity of the composition, and ensures the absence of diffusion. Preferred binding methods include the use of antibodies (which do not spatially block the ligand binding position or activation sequence when the protein is bound to a support), direct binding to sticky or ionic supports, chemical cross-linking, synthesis of the protein or agent on the support, etc. After binding the protein or agent, excess unbound material is removed by rinsing. The sample areas can then be blocked by incubation with bovine serum albumin (BSA), casein, or some other harmless protein, or some other molecule.

The antimitotic agents of the invention can be used to modulate the activity of a mitotic kinesin, especially KSP. In this embodiment, the agents of the invention are combined with KSP and the activity of KSP is determined. The activity of kinesins is known and consists of one or more activities. These include the possibility of influencing ATP hydrolysis, binding of microchannels, displacement and polymerization / depolymerization (effects on the dynamics of microchannels), binding to other proteins in the spindle, binding to proteins involved in the regulation of the cell cycle, acting as a substrate for other enzymes such as kinases or proteases, and kinesin specific cellular activities such as separating spindle poles.

Methods for carrying out mobility tests are well known [see, e.g., Hall et al., (1996), Biophys. J. 71: 3467-3476; Turner et al., 1996, Anal. Biochem. 242 (1): 20-5; Gittes et al., 1996, Biophys. J. 70 (1): 418-29; Shirakawa et al., 1995, J. Exp. Biol. 198: 1809-15; Winkelmann et al., 1995, Biophys. J. 68: 2444-53; Winkelmann et al., 1995, Biophys. J. 68: 72S].

Known methods for determining the hydrolytic activity of ATPase can also be used, preferably solution tests. For example, they are disclosed in US Patent No. 6,410,254 - Application No. 09 / 314,464, filed May 18, 1999. Alternatively, conventional methods are used. For example, you can mean the release of Pi from kinesin. In one preferred embodiment, 0.3 M PCA (perchloric acid) and a malachite green reagent (8.27 mM sodium molybdate II, 0.33 mM malachite green oxalate, and 0.8 mM Triton X-1- 00) are used for the ATPase hydrolytic activity assay. To perform the test, 10 μΐ of the reaction mixture is treated with 90 μΐ 0.3 M cold PCA. Phosphate standards are used so that the data can be converted to mM released inorganic phosphate. After all samples and standards have been treated with PCA, 100 µΐ of the malachite green reagent is added to the remaining wells of e.g. a microtiter plate. The mixture is left for 10-15 minutes and then the absorbance is read at 650 nm. If phosphate standards are used, absorbance readings can be converted to mM Pi and plotted against time. In addition, a luciferase assay can be used as an assay for ATPase.

An ATPase activity assay of kinesin domains can also be used to monitor the effects of modulating agents. In one embodiment, kinesin ATPase determinations are performed

PL 204 525 B1 in the absence of microchannels, in another - in their presence. Various types of modulating agents can be detected in the above tests. In a preferred embodiment, the effect of the modulating agent is independent of the microchannel content and ATP. In other embodiments, the effect of these agents on kinesin ATPase may be lowered or increased by increasing the concentrations of ATP, microchannels, or both.

Agents that modulate the biochemical activity of KSP in vitro can then be screened in vivo. In vivo methods for such agents include assays of cell cycle distribution, cell viability, or the presence, morphology, activity, distribution, or number of mitotic spindles. Methods for monitoring the distribution of the cell cycle in a cell population, for example by flow cytometry, are well known, as are methods for determining cell viability. See, for example, U.S. Patent No. 6,617,115 Methods of Screening for Modulators of Cell Proliferation and Methods of Diagnosing Cell Proliferation States; patent application October 22, 1999 (US Serial No. 09 / 428,156)].

Additionally, microscopic methods of monitoring mitotic spindle formation and deformation are also known [see, e.g., Whitehead and Rattner (1998), J. Cell. Sci. 111: 2551-61; Galgio et al. (1996) J. Cell. Biol. 135: 399-414].

The compositions of the invention inhibit the KSP kinesin. One measure of inhibition is the IC50, defined as the concentration of the composition at which the activity of KSP is reduced by fifty percent.

For the preferred compositions the IC50 values ​​are below 1 mM, more preferably below

100 μΜ, especially preferably - less than 10 μΜ, even more preferably - less than 1 μΜ, particularly preferably - less than 100 nM and most preferably - less than 10 nM. This value is determined using the ATPase test.

Another measure of inhibition is Ki. For compounds having an IC50 below 1 µ 1, Ki or Kd is defined as the dissociation rate constant of the interaction of quinazolone with KSP. For preferred compositions, Ki values ​​are below 100 µM, more preferably below 10 µM, more preferably below 1 µM, even more preferably below 100 nM and most preferably below 10 nM. The K value for a compound is determined from the IC value₅₀ based on three assumptions. First, there is only one compound molecule that binds to the enzyme, and there is no interaction. Second, the concentrations of the active enzyme and test compound are known (ie, there are no significant amounts of impurities or inactive forms in the formulations). Third, the enzyme rate of the enzyme-inhibitor complex is zero. The rate (i.e., the concentration of the compound) corresponds to the relationship:

^{V = V}max^E.0 (E0 +10 + Kd) -j (E0 + I0 + Kd)² -4E0I0

I2E0 where V is the observed rate, Vmax is the rate for the free enzyme, 10 is the inhibitor concentration, E0 - the enzyme concentration, Kd - the dissociation constant of the enzyme-inhibitor complex.

Another measure of inhibition is the GI50, defined as the concentration of the compound that reduces the rate of cell growth by 50 percent. Preferred compounds have a GI50 value below 1 mM. The degree of advantage of solutions is a function of their GI value₅₀: more preferably it is less than 20 µΜ, more preferably it is 10 µ jeszcze, even more preferably 1 µ, more preferably 100 nM and most preferably it is less than 10 nM. The GI50 determination is performed using a cell proliferation assay.

The compositions of the invention are used to treat diseases related to cell proliferation. Disease conditions that may be treated using the methods and compositions provided include, but are not limited to, cancer (discussed later in the description), autoimmune ailments, arthritis, transplant rejection, enteritis, hyperplasia as a result of medical procedures such as surgery, angioplasty, etc. In some cases, the cells may not be overgrown (abnormal) and require further treatment. For example, during wound healing, cells may proliferate normally, but augmentation may be required. Likewise, in the field of plant cultivation, cells may be normal, but modulation of growth may be required to increase yields by either directly promoting growth or by inhibiting the growth of a plant or organism that adversely affects fertility. Thus, in one embodiment of the invention, treatment is applied to cells or organisms afflicted with or at risk of one of the listed disease states.

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The compositions and methods of the invention are considered to be particularly useful for the treatment of neoplasms, including solid tumors such as skin, breast, brain, cervical, testicular cancer etc. In particular, without limiting the scope of the invention, these tumors include: cardiac-sarcoma (vascular sarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma; pulmonary - bronchial cancer (squamous cells, undifferentiated small cells, undifferentiated large cells, glandular), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondrosarcoma, mesothelioma; gastrointestinal: esophagus (squamous cell carcinoma, glandular, leiomyoma, lymphoma), stomach (cancer, lymphoma, leiomyoma), pancreas (ductal adenoma, islet cell adenoma, glucagon, gastrin-secreting adenoma, cancerous tumors), small intestine (adenoma, lymphoma, cancerous tumors, Karposi's myoma, leiomyoma, hemangioma, lipoma, fibro-neuroma, fibroma), large intestine (adenoma, tubular adenoma, papillary adenoma, hamartoma, leiomyoma); genitourinary tract: kidneys (adenoma, Wilms tumor [immature nephroma], lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, cancer of passage cells, adenoma), prostate (adenoma, sarcoma), testes (seminoma, teratoma, germ cell cancer, malignant teratoma, malignant chorionic epithelioma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, glandular tumors, lipoma); hepatic: hepatoma (liver cell carcinoma), biliary tract cancer, hepatoma, angiosarcoma; bones: bone sarcoma, fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma, multiple myeloma, malignant giant cell chord, benign chondrosarcoma, chondrosarcoma, osteosarcoma, osteosarcoma; nervous system: skull (osteoma, hemangioma, granuloma, jaundice, Paget's disease of the bone), meninges (meningioma, meningioma, glioblastoma), brain (astrocytoma, medulloblastoma, glioblastoma, ependymoma, glioblastoma, glioblastoma, neuroblastoma, retinal glioblastoma, congenital tumors), spinal cord (fibroadenoma, meningioma, glioma, sarcoma); gynecological: uterus (endometrial cancer), cervix (cervical cancer, cervical dysplasia), ovaries (ovarian cancer [serous cystadenoma, mucinous cystadenoma, cancer unspecified], granulosa cell carcinoma, Graaf cell tumors, Sertoli-Leydigoma cell tumors malignant), vulva (squamous cell carcinoma, endothelial cell carcinoma, adenoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, rhabdomyosarcoma), fallopian tube cancer; haematological: blood (myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloplastic diseases, multiple myeloma, myelodysplasia syndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignant lymphoma]; skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, lipoma, hemangioma, skin fibroma, keloids, psoriasis; and adrenal glands: neuroblastoma. Accordingly, the term cancer cell refers herein to cells afflicted with one of the above-mentioned conditions.

The compositions of the invention are incorporated into cells. By introducing is meant the administration of a therapeutically effective dose of the mitotic agents of the invention to a cell in culture or to a patient. A therapeutically effective dose is the dose that produces the desired results. The exact dose depends on the purpose of the treatment and can be determined by known methods. It may be necessary to consider: systemic or topical administration, age, body weight, general health, sex, diet, duration of administration, drug interactions, and the nature of the case. Almost all cells in which the course of mitosis or meiosis can be altered are defined as cells.

The term patient includes both humans and animals, especially mammals, and other organisms. Thus, the described methods relate to human treatment and veterinary practice. Preferably the patient is a mammal, and most preferably a human.

Mitotic agents having the desired pharmacological activity can be administered in a physiologically acceptable carrier. Depending on the method of introduction, the compounds can be formulated in various ways as described below.

The concentration of the therapeutically active agent in the formulation may be from 0.1 to 100% by weight. The agents can be used alone or in combination with other therapies such as radiation or other chemotherapeutic agents.

In a preferred embodiment, the pharmaceutical compositions are in water-soluble form as pharmaceutically acceptable acid and base addition salts. The term pharmaceutically acceptable acid addition salts means salts which retain the biological effectiveness of the free acids and do not act in any undesirable way, formed from inorganic acids, such as hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, etc., or organic acids, such as acetic acid. , propionic, glycolic, pyruvic, oxalic, maleic, malonic, amber, fumaric, tartaric, lemon, benzoic, cinnamic, almond, methanesulfonic, ethanesulfonic, p-toluenesulfonic, salicylic and the like. The pharmaceutically acceptable base addition salts are derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, etc. salts, especially the ammonium, potassium, sodium, calcium and magnesium. Salts derived from pharmaceutically acceptable non-toxic organic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring, cyclic amines, and basic ion exchange resins such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.

The pharmaceutical compositions may be prepared in various forms, such as granules, tablets, pills, suppositories, capsules, suspensions, lotions, lotions, etc. To supplement the formulation, pharmaceutical grade organic and inorganic carriers and / or diluents suitable for oral or topical use are added. These include water centers, vegetable and animal oils and fats. As auxiliary ingredients, there are stabilizers, wetting and emulsifying agents, salts for modifying the osmotic pressure, buffers for adjusting the pH, and skin penetration enhancers. The compositions may also contain one or more of the following ingredients: protein carriers such as albumin serum; buffers; fillers such as microcrystalline cellulose, lactose, corn starch and other starches; binding agents; sweeteners and flavorings; dyes; and polyethylene glycol. The additives are known in the art and used in various formulations.

The administration of the mitotic agents of the invention can be carried out in various ways, particularly orally, subcutaneously, intravenously, intranasally, transdermally, intraperitoneally, intramuscularly, intrapleurally, vaginally, rectally or intraocularly. In some cases, for example in the treatment of wounds and inflammations, the antimitotic agents can be applied directly as a solution or spray.

To use the agents of the invention to screen for compounds that bind to the KSP kinesin, KSP is first bound to the substrate and an agent of the invention (which is a mitotic agent) is added to the assay. Alternatively, the agent of the invention binds to the substrate and adds KSP. Compounds from which to search for new binders include specific antibodies, non-naturally occurring binders identified when screening chemical libraries, peptide analogs, etc. Of particular interest are candidate screening assays with low toxicity to human cells. Various assays are used for this purpose, such as in vitro labeled protein binding assays, electrophoretic mobility assays, protein binding immunoassays, functional assays (phosphorylation assays etc.) etc.

Determination of the binding of a mitotic agent to KSP can be accomplished in various ways. In a preferred embodiment, the mitotic agent (a compound of the invention) is labeled, for example, with a fluorescent or radioactive molecule, and the binding is directly determined. This can be done by attaching all or part of the KSP to a solid support, adding a labeled mitotic agent (e.g. a compound of the invention in which at least one atom has been replaced with a detectable isotope), washing off excess reagent, and determining the amount of labeling agent on the solid support. Known blocking and washing steps are used.

A compound that is labeled directly or indirectly with a label that provides a detectable signal, e.g., a radioisotope, a fluorescent marker, an enzyme, an antibody, molecules such as magnetic, chemiluminescent markers, or specific binding molecules, etc. Specific binding molecules include pairs such as biotin and streptavidin, digoxin and antidigoxin etc. For specific binding molecules, the complementary molecule is usually designated by known methods. The label may directly or indirectly give a detectable signal.

In some embodiments, only one component is labeled. For example, kinesin proteins can be labeled at the tyrosine position using the iodine isotope¹²⁵l, or fluorophores. Alternatively, more components are labeled with different labels; using e.g. an isotope¹²⁵l for proteins, and the fluorophore for mitotic agents.

The agents of the invention may also be used as competitors in the screening of additional drug candidates. A candidate bioactive agent or drug candidate, or their grammatical equivalents, means any molecule, e.g. protein, oligopeptide,

A small organic molecule, polysaccharide, polynucleotide, etc., the bioactivity of which is being tested. They may be capable of directly or indirectly altering the cellular proliferation phenotype or the expression of cellular proliferation sequences, including nucleic acid sequences and protein sequences. In other instances, a change in binding and / or function of a cell proliferation protein will be screened. This type of screening is performed in the presence or absence of microchannels. In the case of screening for protein binding or activity, preferred embodiments exclude molecules already known to bind to the protein in question, for example polymeric structures such as microchannels, and energy sources such as ATP. Preferred assays contain candidate agents that do not bind cellular proliferation proteins in their native state, referred to as exogenous agents. In another preferred embodiment, the exogenous agents exclude antibodies to KSP.

Candidate agents can belong to many chemical groups, although they are typically organic molecules, especially small organic compounds with a molecular weight of 100 to about 2,500 daltons (100-2500 x 10^-24 g). These agents contain functional groups necessary for structural interaction with proteins, especially hydrogen and lipophilic bonding, and typically contain at least an amine, carbonyl, hydroxyl, ether or carboxyl, preferably at least two functional groups. They often include cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Agents of this type are often found among biomolecules such as peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs, and combinations thereof.

Peptides are especially preferred.

Candidate agents are obtained from a variety of sources, including libraries of synthetic or natural compounds. For example, there are many methods available for the random and directed synthesis of a wide variety of organic compounds and biomolecules, including the expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Moreover, natural and synthetic compounds can be readily modified by known chemical, physical and biochemical methods. Known pharmaceuticals can be subjected to controlled or random chemical modifications, such as acylation, alkylation, esterification, amidification, so as to obtain structural analogs.

Competitive screening tests can be performed by combining KSP and the candidate in the first sample. The second sample contains the mitotic agent, KSP, and a candidate compound. This is done in the absence of, or in the presence of, microchannels. Candidate binding is determined for both samples, and a change or difference between the two samples indicates the presence of an agent capable of binding KSP and potentially modulating its activity. That is, if the binding of the candidate in the second sample is different from the first sample, it is capable of binding to KSP.

In a preferred embodiment, the binding ability for a candidate is determined by a competitive binding test. In such an embodiment, the competitor is a molecule known to bind to KSP, such as an antibody, peptide, binding partner, ligand, etc. Under certain conditions, there may be a competitive binding, such as between a candidate and a binding molecule, in which the binding molecule excludes the candidate. .

The candidate agent can be marked. In that case, the candidate or competitor, or both, are first attached to the KSP for a period of time sufficient for binding, if any. Incubations are performed at any temperature that provides optimal activity, typically between 4 ° and 40 ° C.

The incubation period is optimal for activity, or so as to achieve high screening efficiency. Typically 0.1 to 1 hour is enough. Excess reagent is removed or rinsed. The second component is then added and the presence or absence of the labeled component indicates binding.

In a preferred embodiment, the competitor is added first, followed by the candidate agent. Eliminating a competitor indicates that the candidate has attached to, and therefore has the ability to, KSP, and may possibly also modulate the activity of KSP. In this solution, one of the ingredients is marked. Thus, for example, if a competitor is labeled, the presence of the label in the washing solution indicates that it has been displaced by the agent. Alternatively, if the candidate is identified, the presence of the marker on the substrate indicates displacement.

In an alternate embodiment, the candidate is introduced first, incubated and washed, and then the competitor is added. The lack of binding by the competitor may indicate that the candidate binds to KSP with higher affinity. Thus, if kanPL 204 525 B1 is labeled, the presence of the label on the substrate, coupled with the lack of binding by a competitor, may indicate the ability of the agent to bind KSP.

It may be important to identify the KSP binding position. This is done in different ways. In one embodiment, once KSP is bound to a mitotic agent, KSP is fragmented or modified and the assays repeated to identify components necessary for binding.

Modulation is tested by screening for agents capable of modulating the activity of KSP. It comprises the step of combining the candidate with KSP, as above, and determining the change in biological activity of KSP. In this embodiment, the candidate should both bind to KSP (although not necessarily) and alter its biological or biochemical activity. The methods include in vitro and in vivo screening of cells for changes in cell cycle distribution, cell viability, or the presence, morphology, activity, distribution, or number of mitotic spindles.

Alternatively, differential screening can be used to identify candidates that bind to native KSP but are unable to bind to the modified KSP.

You can use positive and negative tests in the tests. Preferably, all standard and test samples are tested in triplicate to obtain statistically significant results. All samples are incubated for a period of time sufficient for the agent to bind to the protein. After incubation, all samples are washed until nonspecifically bound material is washed away and the amount of bound, usually labeled agent is determined. For example, if a radioactive label is used, samples are counted in a scintillation counter to determine the amount of bound agent.

Many different reagents are used for screening tests. These are salts, neutral proteins, e.g. albumin, detergents etc. which are used to obtain optimal protein binding and / or to reduce non-specific or background interactions. Reagents can also be used to improve the performance of the assay, such as protease inhibitors, nuclease inhibitors, antimicrobial agents, etc. The mixture of ingredients may be added in any order required to ensure binding.

The following examples are intended to provide a more complete description of the manner of implementing the described invention.

Examples

Abbreviations and definitions

The following abbreviations and terms have the meanings indicated:

Ac

BNB

Boc

Bu cCBZ

DBU

DCM

DCE

DEAD

DIC

DIEA

DMAP

DMF

DMSO

DVB

EEDQ

Et

Fmoc

GC

HATU

HMDS

HO Ac

HOBt

Me acetyl 4-bromomethyl-3-nitrobenzoic acid t-butyloxycarbonyl butyl cyclo carbobenzoxy = benzyloxycarbonyl diazobicyclo [5.4.0] undecane-7 dichloromethane = methylene chloride = CH2Cl2 dichloroethylene diethylazodicarboxylate diisopropylcarbodiimide

N, N-diisopropylethylamine

4-N, N-dimethylaminopyridine

N, N-dimethylformamide dimethylsulfoxide

1,4-divinylbenzene

2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline ethyl

9-fluorenylmethoxycarbonyl gas chromatography hexafluorophosphate 0- (7-azobenzotnazolyl-1) -1,1,3,3-tetramethyluronium hexamethyldisilazane acetic acid hydroxybenzotriazole methyl

Mesyl

MTBE

NMO

PEG

Ph

PhOH

PfP

PPTS

Py

PyBroP rt sat stTBDMS

TES

TFA

THF

TMOF

TMS tosyl

Trt methanesulfonyl methyl t-butyl ether N-methylmorpholine oxide polyethylene glycol phenyl phenol pentafluorophenol p-toluenesulfonate pyridinium pyridine hexafluorophosphate bromo-tris-pyrrolidine-phosphonium room temperature d = tetraethyl-tetra-methyl-tetra-methyl tetra-methyl tetra-saturated secondary tetra-methyl tetra-tetraethyl-saturated pentafluorophenol p-toluenesulfonate pyridinium phosphonium; amethylsilyl p-toluenesulfonyl triphenylmethyl

Example 1

Synthesis of compounds

A general scheme of the synthesis is shown in Fig. 1 and Fig. 2.

Step 1: N-butyrylanthranilic acid

Anthranilic acid (1) (0.5 mol, 68.5 g) and dimethylformamide (250 ml) were charged to a 500 mL three-necked flask with a thermometer, dropping funnel, and efficient stirrer. To this solution was added butyryl chloride (0.55 mol, 57.1 ml) dropwise at such a rate that the temperature of the mixture did not rise above 40 ° C. The suspension was stirred vigorously at room temperature for at least an additional 3 hours. It was then poured into water (2000 ml) and stirred for another hour. The precipitated product was filtered off, washed with cold water and dried in vacuo over P2O5, yielding compound 2 (67.3 g, 65%).

Step 2: 2-propyl-3,1- [4H] benzoxazinone-4

Compound 2 (51.8 g, 0.25 mol) was dissolved in acetic anhydride (180 mL) in a 500 mL round bottom flask with stirrer, Claissen distillation head (with vacuum inlet), and thermometer. The flask was placed in an oil bath and slowly heated to 170-180 ° C with vigorous stirring. The resulting acetic acid was slowly distilled off at atmospheric pressure. To keep the transformation progress, the temperature of the distillation head was monitored. The reaction mixture was then cooled to 60 ° C and the excess acetic anhydride was removed by distillation under reduced pressure (ca. 2.666 kPa = 20 mm Hg). The residue was cooled and the product was recrystallized. Trituration with n-hexane (75 ml) and filtration provided 2-propyl-3,1- [4H] benzoxazinone-4 (3) (29.3 g, 62%). The above procedure gave compound 3 sufficiently pure to be used directly in the next step.

Step 3: 2-propyl-3-benzylquinazolone-4

Compound 3 (28.4 g, 0.15 mol) and benzylamine (17.5 mL, 0.16 mol) were recycle distilled in chloroform (50 mL) in a 250 mL single neck round bottom flask for 6 hours. After compound 3 was completely absorbed, chloroform was evaporated under reduced pressure. Ethylene glycol (100 ml) and NaOH tablets (0.60 g) were added to the residue and a Claissen distillation head and a magnetic stirrer were placed in the flask. The flask was immersed in an oil bath, heated to a bath temperature of 130-140 ° C (with vigorous stirring) which was held for 5 hours, removing the water formed by distillation. After completion of the reaction, the clear solution was cooled to room temperature and the product was allowed to precipitate overnight. The pH of the slurry was adjusted to 7-8 by adding a 3% aqueous solution of HCl. The crystals were filtered off, washed with cold water and recrystallized from isopropanol (acetone may also be present). Compound 2-propyl-3-benzylquinazolone-4 (compound 4) (28.0 g, 67%) was obtained.

PL 204 525 B1

Step 4: 2- (1'-bromopropyl) -3-benzylquinazolone-4

To a 250 mL three-neck round bottom flask with a thermometer, dropping funnel, and stirrer, was charged compound 4 (27.8 g, 0.10 mol), anhydrous sodium acetate (10.0 g), and glacial acetic acid (130 mL). Bromine (16.0 g, 0.10 mol) dissolved in acetic acid (10 ml) was added dropwise to the above solution at 40 ° C for 1-2 hours. The precipitated product, 2- (1'-bromopropyl) -3-benzylquinazolone-4 (5), was filtered off, washed with warm water until traces of acetic acid were removed, and rinsed with a little isopropanol. After drying, compound 5 was obtained (33.0 g, 92%).

Step 5: 2- [1 '- (N, N-dimethylethylenediamino) propyl] -3-benzylquinazolone-4

Compound 5 (10.7 g, 0.03 mol) and N, N-dimethylethylenediamine (6.6 ml, 0.06 mol) were dissolved in dry alcohol (60 ml) and refluxed for 6 hours. After completion of the reaction, the solvent was evaporated under reduced pressure. The residue was dissolved in dichloromethane (150 ml) and washed with a 3% aqueous NaOH solution (ca. 10-20 ml). The organic layer was dried over MgSO4 and evaporated to dryness under reduced pressure. The remaining oily product was purified by flash chromatography on silica gel, eluting with CHCl3 - MeOH - aqueous NH3 solution 90: 10: 0.1, yielding the expected compound (5) in the form of 2- [1 '- (N, N-dimethylethylenediamine) propyl] -3-benzylquinazolone-4 (6.0 g, 55%).

Step 6: 2- [1 '- (N-4-fluorobenzoyl) - (N, N-dimethylethylenediamino) propyl] -3-benzylquinazolone-4 Prepared: Stock compound 5 (1.822 g, 5.0 mmol) in CHCl3 (0.5 mL ) HPLC grade; stock solution of p-fluorobenzole chloride (160.2 mg, 1 mmol) in 1,2-dichloroethane (2.0 mL), HPLC grade, in a 2.0 mL volumetric flask; and a solution of triethylamine (2.0 ml, 0.5 mol) in the same reagent. 100 µl aliquots of each solution were pipetted into a glass reaction vessel using a Beckman Biomet 2000 automatic liquid dispenser. The reaction mixture was shaken on a mechanical shaker, sonicated in a water rinse and then incubated overnight at room temperature. The mixture was diluted

WANT₃ (300 μΐ), washed with 5% aqueous NaHCO solution₃ and water. The solvent was removed in vacuo to afford compound 6 in 65% yield. The purity of the product was determined by liquid chromatography using the eluent CH2Cl2 - ethanol - concentrated aqueous NH3 solution in the ratio 100: 10: 1.

Examples 2 and 3

Synthesis of compounds with the structure of amines

<img file="PL204525B1_D0004.tif" />

Most reagents were purchased from Aldrich Chemical Company including - all non-aqueous solvents in SureSeal® containers. The abbreviations mean: DCM = dichloromethane; DIEA = N, N14

PL 204 525 B1

-diisopropylethylamine, DMF = N, N-dimethylformamide; TES = triethylsilane; TFA = trifluoroacetic acid. The template synthesis was carried out in 15 x 75 mm round screw cap glass vials placed in 4x6 aluminum synthesis blocks sealed with a Teflon lined rubber membrane. Reagent addition and aqueous extractions were performed using single or multi-channel pipetting kits. Filtration was performed using Whatman / Polyfiltronics 24 10 ml filter blocks. A Labconco Vortex-Evaporator or a nitrogen blast was used to evaporate the volatile materials.

Example 2 (single compound synthesis in solid phase)

Step 1 / 1,3-diaminopropanetrityl resin (Novabiochem, 1.2 mmol / g) (0.20 g, 0.24 mmol) was weighed into a screw cap vial and 3 ml of a 1: 1 mixture of DMF and chloroform was added. Then DIEA (0.130 ml, 0.72 mmol) and 2- (1'-bromopropyl) -3-benzylquinazolone-4) (from example 1) (0.188 g, 0.48 mmol) were added. The vial was capped, heated to 70 ° C and shaken overnight. The resin was filtered off, washed (3 x DCM, 2 x MeOH, 1 x DCM, 2 x ether) and dried in vacuo. An aliquot of 27 mg of the resin was taken and treated with TFA: TES: DCM 5: 5: 90. After 15 minutes, it was filtered and evaporated to give 8 mg (64% yield) of the quinazolinone diamine intermediate. Analysis by LCMS (Liquid Chromatography Mass Spectrometry) showed a purity&gt; 80%.

Step 2 / The resin from step 1 was swelled with 3 ml of DCM. DIEA (0.130 ml, 0.72 mmol) and 4-bromobenzyl bromide (0.12 g, 0.48 mmol) were added. The vial was capped and shaken overnight. LCMS analysis of the decomposed aliquot showed that the mixture ratio of starting material to product was approximately 1: 1. Equal amounts of DIEA and 4-benzyl bromide were added again, followed by shaking at 70 ° C for 8 hours. The resin was filtered off, washed as above and dried in vacuo.

Step 3 / The resin from step 2 was shaken twice for 30 minutes with a mixture of TFA: TFS: DCM in a ratio of 5: 5: 90. The filtrates were combined and evaporated to give 140 mg of an orange oil. The material was purified using preparative reverse-phase HPLC (acetonitrile-water gradient) to afford 27 mg (17% yield for 3 steps) of the mono TFA salt.

Example 3 (combined synthesis of multiple compounds)

Step 1 / 1,2-diaminoethanetrityl resins (Novabiochem, 0.95 mmol / g) (200 g, 1.9 mmol) and 1,3-diaminopropanetrityl (Novabiochem, 1.14 mmol.g) (2.0 g, 2.28 mmol) were placed separately in 10 mL polypropylene vials (Bio-Rad). To each was added 4 ml of DMF, 4 ml of chloroform, 3 equivalents of DIEA (1.0 ml and 1.2 ml respectively) and 2 equivalents of 2- (1'-bromopropyl) -3-benzylquinazolinone-4 (from Example 1) (1.5 g and 1.8 g). The mixtures were shaken overnight at 70 ° C. Each was washed (3 x DCM, 2 x MeOH, 1 x DCM, 2 x ether) and dried in vacuo. Distribution analysis showed the presence of the corresponding quinazolone diamine with a purity of&gt; 90%.

Step 2 / Quinazoline ethyl diamine resin (105 mg, 0.10 mmol) was placed in each vial in the first 2 rows of the matrix and quinazoline propyl diamine resin (88 mg, 0.10 mmol) in the last 2 rows of the matrix. DIEA (0.131 mL, 0.75 mmol) was added to each vial. A different amine was added to each vial in the first two rows, and these additions were repeated for the last two rows. The reaction block was shaken overnight at 70 ° C. The liquid from each vial was removed using a multichannel pipetting kit, then the resins were washed (2 x DCM, 1 x MeOH, 1 x DCM) and dried in vacuo.

Step 3 / To each vial in the matrix was added 2 ml of a 10: 5: 85 TFA: TES: DCM solution. The reaction block was shaken for 45 minutes after which the mixtures were transferred to a filter block, filtered and washed twice with 0.75 ml of DCM. The solutions were evaporated to give yellow to orange oils. These thick oils were triturated twice with ether, dissolved in DCM, and treated with a 4M solution of HCl in dioxane to yield the hydrochloric acid salts (unknown number of salts per compound) as light brown to white powdery or amorphous solids. LCMS analysis showed&gt; 75% purity.

Examples 4 - 6

The six racemic quinazolones were separated into their enantiomers using chiral chromatography. The chiral chromatography of three of these compounds is described below:

PL 204 525 B1

Example 4:

<img file="PL204525B1_D0005.tif" />

Column - Chiralpak AD, 250 x 4.6 mm (Diacel Inc.). Sample - 0.5 mg / ml in ethanol. Condition 15 minutes in 60% ethanol in hexane, enantiomer 1 elutes 4.5 minutes, enantiomer 2 4.9 minutes.

Example 5:

<img file="PL204525B1_D0006.tif" />

Chiralcel OJ column, 250 x 4.6 mm (Diacel Inc.). Sample - 0.5 mg / ml in ethanol. Condition 15 minutes in 10% ethanol in hexane, (R) enantiomer eluted after 8.4 minutes, (S) enantiomer eluted after 9.6 minutes.

Example 6:

<img file="PL204525B1_D0007.tif" />

Column - Chiralpak AD, 250 x 4.6 mm (Diacel Inc.). Sample - 0.5 mg / ml in ethanol. Conditions 15 minutes in 70% ethanol in hexane, enantiomer 1 elutes 6.5 minutes, enantiomer 2 8.8 minutes.

The table below summarizes the IC50 activities of the racemate and the enantiomers of the three other compounds separated as above. In all three cases, one enantiomer had much greater potential than the other. Independent chiral synthesis showed that the R-enantiomer was more active.

PL 204 525 B1

<img file="PL204525B1_D0008.tif" />

The following two compounds were synthesized as single enantiomers as shown in Figure 3. The data indicate that the R-enantiomer is more active.

<td></td><td>Κι (μΜ)</td><td>Ki (PM)</td>

<td></td><td>The S enantiomer</td><td>The R enantiomer</td>

<td>oQ what Sr</td><td> 2</td><td> &lt; 0.1</td>

<td> .9 ^θ^)^^ΝΗίBr</td><td> &gt;0.5</td><td> &lt;0.05</td>

PL 204 525 B1

Example 9

Chiral separation by recrystallization with tartaric acid

Intermediate A as obtained in Example 1 can be processed into Intermediate B which, after separation, is an alternative to the first five steps shown in Fig. 3. The process is illustrated in the following diagram:

<img file="PL204525B1_D0009.tif" />

The R-enantiomer of compound B can be selectively crystallized by heating mixture B with 1.1 equivalents of D-tartaric acid in a mixture of isopropanol and methanol and then allowing the mixture to reach room temperature.

Example 9: X = Cl, R = H.

Racemic intermediate B (1.5 g), dissolved in 100 ml of boiling isopropanol, was mixed with 0.8 g of D-tartaric acid in 100 ml of boiling methanol. The mixture was allowed to slowly reach room temperature. After standing overnight, the precipitate was separated by filtration, rinsed with ethyl acetate and hexane, and air dried. The dry solid (0.8 g) was dissolved in a boiling mixture of 50 ml of isopropanol and 50 ml of methanol and allowed to cool slowly to room temperature. After standing overnight, the precipitate was separated by filtration, rinsed with ethyl acetate and hexane, and air dried. The dry material was stirred with saturated sodium bicarbonate for 30 minutes then extracted with ethyl acetate. The organic portion was dried (MgSO4), filtered and evaporated to dryness. The clear oil obtained weighed 345 mg. The chiral purity was assessed&gt; 95% by converting a portion to the S-Mosher amide and testing the product by¹HNMR. The enantiomerically pure compounds below were prepared according to the rest of the steps in Figure 3, from material prepared according to the described procedure using variants D - and L - tartaric acid.

<td></td><td>The racemate ICsofaM)</td><td>The R isomer IC50 (μΜ)</td><td>Isomer S IC₅₀(mm)</td>

<td>° 9 ΝΗ₂ch₃</td><td> &lt;0.05</td><td> &lt;0.05</td><td> &gt;0.5</td>

Example 10

Induction of mitotic inhibition in a population of cells treated with the quinazoline inhibitor of KSP

FACS (fluorescent cell sorter method) analysis was performed as follows to determine the stage of the cell cycle by measuring the DNA content. Skov-3 cells (cancer

Of human ovary) was split 1:10 for seeding in 10 cm plates and grown to sub-confluence with RPMI 1640 medium containing 5% fetal bovine serum (FBS). Cells were then treated with 10 nM paclitaxel, 400 nM quinazolinone 1, 200 nM quinazolinone 2, or 0.25% DMSO (vehicle for compounds) for 24 hours. Cells were then washed from the plates with PBS containing 5 mM EDTA, pelleted, washed once with PBS containing 1% FCS and placed in 85% ethanol at 4 ° C overnight. Prior to analysis, cells were plasticized, washed once and stained in a solution of 10 µg propidium iodide and 250 µg ribonuclease (RNAse) A per milliliter at 37 ° C for half an hour. Flow cytometric analysis was performed on a Becton-Dickinson FACScan instrument, and data from 10,000 cells per sample were analyzed using Modfit software.

Quinazoline compounds as well as the known antimitotic agent paclitaxel shift the cell population from the G0 / G1 cell cycle stage (2n DNA content) to the G2 / M stage (4n DNA content). Other compounds of the same class have been found to have a similar effect.

Formation of a unipolar mitotic spindle after the introduction of a quinazoline inhibitor of KSP.

To determine the nature of G2 / M accumulation, human Skov-3 (ovarian), HeLa (cervical) and A549 (pulmonary) tumor cells were seeded in 96-well plates at densities of 4,000 cells per well (SKOV-3 and HeLa) or 8,000 cells per well. a well (A549) allowed to settle for 24 hours, then quinazoline compounds were administered at various concentrations over 24 hours. Cells were suspended in 4% formaldehyde and stained with anti-tubulin antibodies (which were then detected with fluorescently labeled secondary antibodies) and Hoechst dye (which stains DNA).

Visual inspection showed that the quinazoline compounds cause a cell cycle inhibition in the mitotic prometaphase stage. The DNA thickened and mitotic spindle formation began, but the inhibited cells uniformly showed spindle polarity, indicating there was an inhibition of spindle pole separation. Microinjection of antibodies to KSP also inhibited mitosis and the arrested cells showed unipolar spindles.

Inhibition of cell proliferation in cancer cells caused by quinazoline inhibitors of KSP

Cells were plated in 96-well plates at densities of 1,000 to 2,500 cells per well (depending on cell type) and allowed to grow for 24 hours. They were then treated with drugs at various concentrations for 48 hours. The time of compound addition is reported as T0. A tetrazolium-based assay was used to quantify the number of viable cells after T0 and the remaining cells after 48 hours of exposure, using the compound 3- (4,5-dimethylthiazolyl-2) -5- (3-carboxymethoxyphenyl) -2- (4- sulfophenyl) -2H-tetrazolium (MTS) (IS&gt; Patent No. 5,185,450) (see Promega Catalog No. G3580, CellTiter 96® AQueousOne Solution Cell Proliferation Assay). The number of cells remaining after 48 hours was compared with the number of cells alive at the time of drug administration to calculate growth inhibition.

Cell growth after 48 hours in vehicle control wells (0.25% DMSO) was determined as 100% growth and compared to the growth of cells treated with compounds. Quinazoline KSP inhibitors inhibited cell proliferation of the following human cancers: lung (NCI-H460, A549), breast (MDA-MB-231, MCF-7, MCF-7 / ADR-RES), colon (HT29, HCT15), ovary ( SKOV-3, OVCAR-3), leukemia (HL-60 (TB), K-562), central nervous system (SF-268), kidney (A498), osteosarcoma (U2-OS) and cervical (HeLa). In addition, mice tumors (B16, melanoma) were also found to be inhibited by quinazoline compounds.

GI was calculated₅₀ by plotting the compound concentration in μΜ and the percent cell growth in the compound added wells. The GI50 calculated for a compound is the concentration at which growth is inhibited 50% of the reference, i.e. the concentration at which:

100 x [(treated ₄₈ - T.₀) / (control₄₈ - T.₀)] = 50

All compound concentrations were tested in duplicate and references averaged across 12 wells. A very similar 96-well plate scheme with GI50 calculation is used by the National Cancer Institute (see Monks et al., Natl. Cancer. Inst. 83: 757-766 (1991)). The difference is that the NCI uses a different method of cell counting, without using the MTS.

PL 204 525 B1

Calculation of the IC50

The ATPase assay is used to measure the IC50 for the activity of the composition against KSP. The following solutions are used: Solution 1 consisting of 3 mM phosphoenolpyruvate potassium (Sigma P-7127), 2 mM ATP (Sigma A-3377), 1 mM IDTT (Sigma D-9779), 5 paclitaxel (Sigma T-7402) , 10 ppm of 289 skimmer (Sigma A-8436), 25 mM Pipes / KOH pH 6.8 (Sigma P6757), 2 mM MgCl2 (VWR JT400301), and 1 mM EGTA (Sigma E3889); Solution 2 containing 1 mM NADH (Sigma N8129), 0.2 mg / ml BSA (Sigma A7906), pyruvic kinase 7U / ml, L-lactate dehydrogenase 10 U / ml (Sigma P0294), 100 nM KSP drive domain, 50 μg / ml microchannels, 1 mM DTT (Sigma D9779), 5 μM paclitaxel (Sigma T-7402), 10 ppm 289 skimmer (Sigma A-8436), 25 mM Pipes / KOH pH 6.8 (Sigma P6757), 2 mM MgCl2 (VWR JT4003- 01), and 1 mM EGTA (Sigma E3889). Serial dilutions (8-12 double dilutions) of the composition were made in a 96-well microtiter plate (Corning Costar 3695) with Solution 1. After dilution, there was 50 μl of solution 1 in each well. The reaction was initiated by adding 50 μl of solution 2 to each well. using a multi-channel pipetting kit. The plate was transferred to an absorbance reader and several absorbance readings were taken for each well at 340 nm (kinetic mode). The observed rate of change, which is proportional to the ATPase rate, is plotted against compound concentration. The following equation with four parameters, solved using a non-linear fit program (e.g., Graphite 4), was used to derive a standard IC50 value:

y range = ----------------------------- + background + (x / IC50)^s where y is the observed rate and x - compound concentration.

Quinazoline compounds inhibit growth in various groups of cells, including (MCF-7 / ADRRES, HCT1 5) developing P-glycoprotein (also known as Multidrug Resistance or MDR⁺), which transfers resistance to other chemotherapeutic agents such as paclitaxel. Thus, quinazolinones are antimitotics that inhibit cell proliferation and are not susceptible to immunity due to MDR overexpression.⁺ in drug-resistant tumor cells.

Inhibition of cell proliferation has also been found for other compounds of this class, although GI50 values ​​vary. GI50 values ​​for the quinazoline compounds tested ranged from 200 nM to greater than the highest concentrations tested. This means that although most of the compounds biochemically inhibiting the activity of KSP inhibit cell proliferation, in some cases at the highest concentration tested (about 20 µM), growth was inhibited below 50%. Many of these compounds have GI values₅₀ lower than 10 µM, and for some they are less than 1 µM. Anti-proliferative compounds that have been successfully used in clinical practice for the treatment of cancer (cancer chemotherapeutic agents) have widely varying Gl50 values. For example, for cells A549 - GI₅₀ is 4 nM for paclitaxel, 63 nM for doxorubicin, 1 μM for 5-fluorouracil, and 500 μM for hydroxycarbamide (data provided by the National Cancer Institute, Developmental Therapeutic Program, http://dtp.nci.nih.gov/). Thus, the cell proliferation inhibiting compounds can be used at any concentration. However, it is preferable to use compounds with GI values₅₀ they are less than 1 nM, more preferably - from 20 µM, and even more preferably - from 10 µM. It is advisable to lower these values ​​further down to the order of 1 µM. For some quinazoline compounds of the invention, GI50 values ​​are from less than 200 nM to less than 10 nM.

Example 11

Female mice weighing about 20 g were subcutaneously implanted with a trocar with human cancerous tumor fragments grown subcutaneously in a mouse host. After a tumor size of approximately 77 mg was obtained, animals were paired to create treatment and control groups. There were 8 tumor bearing mice in each group, and each was labeled on the ear and observed individually throughout the experiment. Initial doses (10 ml / kg 66 mM citrate buffer pH 5.0 / 0.9% saline / 10% Tween 80 of each test compound formulation at a maximum concentration of 5 mg / ml) were administered on the first day after pairing, in the amounts and aliquots listed.

The mice were weighed twice a week, and the size of the tumors was also measured frequently with calipers, starting on day 1. From these tumor size measurements, their weight was calculated according to the known formula W² x L / 2. The experiment was terminated when an average tumor weight of about 1 g in the control group was achieved. Upon completion, the mice were weighed, sacrificed and the tumors were excised. The tumors were weighed and then calibrated20

The average weight of the treated tumor for the group was calculated. In this model, the ratio of the mean treated tumor weight change to the mean tumor weight change for the control x 100% (∆Ϊ / ∆ ^ was subtracted from 100% to determine tumor growth inhibition (TGI) for each group.

The following compounds (1-5) were tested according to the method described and the results are given in Tables AD. Other compounds of the invention when tested by this method showed comparable activities.

Relationship 1

<img file="PL204525B1_D0010.tif" />

Relationship 2

<img file="PL204525B1_D0011.tif" />

Relationship 3

<img file="PL204525B1_D0012.tif" />

Relationship 4

<img file="PL204525B1_D0013.tif" />

Compound 5

<img file="PL204525B1_D0014.tif" />

PL 204 525 B1

<td colspan="4">Table A SKOV3 tumor xenograft</td>

<td></td><td>Vehicle</td><td>Relationship 1</td><td>Taxol</td>

<td>Dose and Administration</td><td>5 times a day</td><td>80 mg / kg every 3 days x 4</td><td>20 mg / kg daily x 5</td>

<td>Method of administration</td><td>intravenously</td><td>intravenously</td><td>intraperitoneally</td>

<td>Number of mice in the beginning</td><td> 8</td><td> 8</td><td> 8</td>

<td>Final tumor mass (mean + standard error)</td><td> 904 ±126.5</td><td> 554.3 ± 76.8</td><td> 90.4 ± 36.0</td>

<td>Tumor growth inhibition</td><td> -</td><td> 41.5%</td><td> 91.7%</td>

<td>Mice with partial tumor contraction</td><td> 0</td><td> 0</td><td> 4</td>

<td>Mean% of tumor contraction</td><td> -</td><td> -</td><td> 27.9%</td>

<td>Maximum weight loss</td><td>lack</td><td>lack</td><td> 16.5%</td>

<td>Mortality</td><td> 0</td><td> 1</td><td> 0</td>

<td colspan="5">Table B SKOV3 tumor xenograft</td>

<td></td><td>Vehicle</td><td>Relationship 2</td><td>Relationship 3</td><td>Taxol</td>

<td>Dose and Administration</td><td>5 times a day</td><td>50 mg / kg co 3rd day x 4</td><td>60 mg / kg daily x 5</td><td>20 mg / kg daily x 5</td>

<td>Method of administration</td><td>intravenously</td><td>intravenously</td><td>intravenously</td><td>intraperitoneally</td>

<td>Number of mice in the beginning</td><td> 8</td><td> 8</td><td> 8</td><td> 8</td>

<td>Final tumor mass (mean ± standard error)</td><td> 1506.3 ±227.1</td><td> 340.8 ± 93.0</td><td> 806.1 ± 163.8</td><td> 55.9 + 29.4</td>

<td>Tumor growth inhibition</td><td> -</td><td> 81.5%</td><td> 48.3%</td><td> 99.7%</td>

<td>Mice with partial tumor contraction</td><td> 0</td><td> 0</td><td> 0</td><td> 7</td>

<td>Mean% of tumor contraction</td><td> -</td><td> -</td><td> -</td><td> 43.5%</td>

<td>Maximum weight loss</td><td>lack</td><td>lack</td><td> 2.76%</td><td> 7.49%</td>

<td>Mortality</td><td> 0</td><td> 0</td><td> 1</td><td> 0</td>

<td colspan="4">Table C SKOV3 tumor xenograft</td>

<td></td><td>Vehicle</td><td>Relationship 4</td><td>Taxol</td>

<td> 1</td><td> 2</td><td> 3</td><td> 4</td>

<td>Dose and Administration</td><td>5 times a day</td><td>4mg / kg weekly x 3</td><td>20 mg / kg daily x 5</td>

<td>Method of administration</td><td>intravenously</td><td>intravenously</td><td>intraperitoneally</td>

<td>Number of mice in the beginning</td><td> 8</td><td> 8</td><td> 8</td>

PL 204 525 B1 cont. table C

<td> 1</td><td> 2</td><td> 3</td><td> 4</td>

<td>Final tumor mass (mean ± standard error)</td><td> 1191.1 ±239.6</td><td> 726.9 ±147.2</td><td> 90.6 ± 34.5</td>

<td>Tumor growth inhibition</td><td> -</td><td> 40.1%</td><td> 87.1%</td>

<td>Mice with partial tumor contraction</td><td> 0</td><td> 0</td><td> 4</td>

<td>Mean% of tumor contraction</td><td> -</td><td> -</td><td> 44.4%</td>

<td>Maximum weight loss</td><td>lack</td><td> 0.02%</td><td> 12.26%</td>

<td>Mortality</td><td> 1</td><td> 1</td><td> 1</td>

<td colspan="4">Table D SKOV3 tumor xenograft</td>

<td></td><td>Vehicle</td><td>Compound 5</td><td>Taxol</td>

<td>Dose and Administration</td><td>5 times a day</td><td>25 mg / kg daily x5</td><td>20 mg / kg daily x 5</td>

<td>Method of administration</td><td>intravenously</td><td>intravenously</td><td>intraperitoneally</td>

<td>Number of mice in the beginning</td><td> 8</td><td> 8</td><td> 8</td>

<td>Final tumor mass (mean ± standard error)</td><td> 1230.4 ± 227.3</td><td> 405.6 ± 124.8</td><td> 379.0 ± 154.0</td>

<td>Tumor growth inhibition</td><td> -</td><td> 71.0%</td><td> 73.0%</td>

<td>Mice with partial tumor contraction</td><td> 0</td><td> 1</td><td> 0</td>

<td>Mean% of tumor contraction</td><td> -</td><td> 56.3%</td><td> -</td>

<td>Maximum weight loss</td><td>lack</td><td>lack</td><td> 8.77%</td>

<td>Mortality</td><td> 0</td><td> 0</td><td> 0</td>

Patent claims