HK1114338B - Stereoisomerically enriched 3-aminocarbonyl bicycloheptene pyrimidinediamine compounds and their uses - Google Patents

Description

3-aminocarbonyl bicycloheptene pyrimidinediamine compound rich in stereoisomer and application thereof

Cross Reference to Related Applications

This application claims priority from application No. 60/628,199 filed 11/15/2004 as 35u.s.c. § 119(e), the contents of which are incorporated herein by reference.

Technical Field

The present invention relates to a stereoisomerically-enriched composition of a 4N- (3-aminocarbonylbicyclo [2.2.1] hept-5-en-2-yl) -N2-substituted phenyl-2, 4-pyrimidinediamine compound that exhibits antiproliferative activity; prodrugs, intermediates of such compounds and synthetic methods for preparing such compounds and/or prodrugs; pharmaceutical compositions comprising these compounds and/or prodrugs; and the use of these compounds and/or prodrugs in a variety of contexts, including, for example, the treatment of proliferative diseases such as tumors and cancers.

Background

Cancer is a diverse group of diseases characterized by uncontrolled growth and spread of abnormal cells. Generally, all types of cancer have some abnormalities in the control of cell growth and division. Pathways that regulate cell division and/or cell communication are altered in cancer cells, and thus these regulatory mechanisms that control and limit cell growth are not functional or are bypassed. Through successive rounds of mutation and natural selection, a group of abnormal cells, usually derived from a single mutant cell, accumulate additional mutations that provide selective growth advantages over other cells, thereby evolving into the cell type that predominates in the cell population. The process of such mutation and natural selection is enhanced by the genetic instability exhibited by many types of cancer cells, which instability is obtained either from somatic mutations or through germline inheritance. The increased variability of cancer cells increases the likelihood that they will progress to form malignant cells. As cancer cells evolve further, some invade locally, then metastasize colonize (conize) in tissues other than the tissue source of the cancer cells. This property, along with the heterogeneity of tumor cell populations, makes cancer a particularly difficult disease to treat and eradicate.

Traditional cancer treatments take advantage of the strong proliferative capacity of cancer cells and their high sensitivity to DNA damage. Ionizing radiation (including gamma-and x-rays) and cytotoxic agents (e.g., bleomycin, cisplatin, vinblastine, cyclophosphamide, 5' -fluorouracil and methotrexate) rely on extensive damage to DNA and destabilization of the chromosomal structure, ultimately leading to destruction of cancer cells. These treatments are particularly effective for those types of cancer where the cell cycle checkpoint is deficient, which limits the ability of these cells to repair damaged DNA prior to cell division. However, the non-selective nature of these treatments often causes severe and debilitating adverse effects. Systemic application of these drugs can result in damage to normal healthy organs and tissues and compromise the long-term health of the patient.

Although a number of selective chemotherapeutic approaches have been developed based on the knowledge of how cancer cells develop, such as the antiestrogen compound tamoxifen, the effectiveness of all chemotherapeutic approaches is limited by the development of resistance. In particular, increased expression of cell membrane bound transporters such as MdrI has resulted in a multi-drug resistance phenotype characterized by increased efflux (efflux) of the drug from the cell. These types of cancer cell adaptation severely limit the effectiveness of certain classes of chemotherapeutic agents. Thus, the identification of other chemotherapeutic agents, particularly active stereoisomers and/or mixtures of stereoisomers, is critical to establishing a treatment that is effective against the heterogeneous nature of proliferative diseases and overcomes any resistance that may occur during treatment with other compounds. Moreover, the use of a combination of chemotherapeutic agents comprising different stereoisomers and/or mixtures of stereoisomers of a particular chemotherapeutic drug, which may have different properties and cellular targets, increases the effectiveness of chemotherapy and limits the development of resistance.

Disclosure of Invention

In one aspect, the present invention provides 4N- (3-aminocarbonylbicyclo [2.2.1] hept-5-en-2-yl) -2N-substituted phenyl-2, 4-pyrimidinediamine compounds that are enriched in specific diastereomers, which compounds exhibit antiproliferative activity against a variety of different types of tumor cells. In some embodiments, compounds according to structural formula (I) are provided:

including prodrugs, salts, hydrates, solvates and N-oxides thereof, which are enriched in the corresponding diastereomer of structural formula (Ia), referred to as the (1R, 2R, 3S, 4S) diastereomer:

wherein:

each R¹Are each independently selected from hydrogen, lower alkyl, - (CH)₂)_n-OH、-OR^a、-O(CH₂)_n-R^a、-O(CH₂)_n-R^b、-C(O)OR^aHalogen, -CF₃and-OCF₃；

Each R²Are each independently selected from hydrogen, lower alkyl, -OR^a、-O(CH₂)_n-R^a、-O(CH₂)_n-R^b、-NHC(O)R^aHalogen, -CF₃、-OCF₃、And

each R³Are each independently selected from hydrogen, lower alkyl, - (CH)₂)_n-OH、-OR^a、-O(CH₂)_n-R^a、-O(CH₂)_n-R^bHalogen, -CF₃、-OCF₃、And

each R⁴Are each independently selected from hydrogen, lower alkyl, arylalkyl, -OR^a、-NR^cR^c、-C(O)R^a、-C(O)OR^aand-C (O) NR^cR^c；

R⁵Is hydrogen, halogen, fluorine, -CN, -NO₂、-C(O)OR^aor-CF₃；

Each n is independently an integer from 1 to 3;

each R^aEach independently selected from hydrogen, lower alkyl and lower cycloalkyl;

each R^bAre all independently selected from-OR^a、-CF₃、-OCF₃、-NR^cR^c、-C(O)R^a、-C(O)OR^a、-C(O)NR^cR^cand-C (O) NR^aR^d；

Each R^cAre each independently selected from hydrogen and lower alkyl, or, two R^cThe substituents may be taken together with the nitrogen atom to which they are attached to form a 4-9 membered saturated ring, optionally including 1-2 additional heteroatom groups selected from O, NR^a、NR^a-C(O)R^a、NR^a-C(O)OR^aAnd NR^a-C(O)NR^a(ii) a And the number of the first and second electrodes,

each R^dEach independently is lower mono-hydroxyalkyl or lower di-hydroxyalkyl.

In some embodiments, the compound of structural formula (I) is a racemic mixture of the (2-exo-3-exo) cis isomer according to structural formula (IIa):

including prodrugs, salts, hydrates, solvates and N-oxides thereof, wherein R is¹、R²、R³And R⁵As defined above for formula (I).

In some embodiments, the compound is a stereoisomer-enriched diastereomer according to structural formula (Ia) above, including prodrugs, salts, hydrates, solvates, and N-oxides thereof, that is substantially free of its enantiomer as well as any other diastereomer thereof.

In another aspect, the invention provides prodrugs of the stereoisomerically enriched compounds. Such prodrugs may be active in their prodrug form or may be inactive until converted to the active drug form under physiological or other use conditions. In prodrugs, one or more functional groups of the stereoisomer-rich compound are contained in a precursor moiety (promoity) that is cleaved from the molecule under conditions of use, typically by hydrolysis, enzymatic cleavage, or some other cleavage mechanism, to yield the functional group. For example, the primary or secondary amino group can be contained in an amide precursor moiety that is cleaved under the conditions of use to form the primary or secondary amino group. Thus, prodrugs contain a special type of protecting group, called a "precursor group", which masks one or more functional groups of the compound, which is cleaved under the conditions of use to yield the active pharmaceutical compound. Functional groups in the stereoisomerically-rich compounds that can be masked with the precursor groups contained in the precursor moiety include, but are not limited to, amino (primary and secondary), hydroxyl, sulfanyl (thiol), carboxyl, carbonyl, and the like. A variety of precursor groups suitable for masking these functional groups to give precursor moieties that are cleavable under the desired conditions of use are well known in the art. All of these precursor groups may be contained in the prodrug either individually or in combination. Specific examples of primary or secondary amino-generating precursor moieties that may be included in a prodrug include, but are not limited to, amides, carbamates, imines, ureas, phenylphosphines, phosphoryls, and oxysulfides. Specific examples of sulfanyl-forming precursor moieties that can be included in a prodrug include, but are not limited to, thioethers, such as S-methyl derivatives (monothio, dithio, oxathio, aminothioacetals), silyl thioethers, thioesters, thiocarbonates, thiocarbamates, asymmetric disulfides, and the like. Specific examples of precursor moieties that can be included in a prodrug that are cleaved to form a hydroxyl group include, but are not limited to, sulfonates, esters, and carbonates. Specific examples of carboxyl-generating precursor moieties that may be included in prodrugs include, but are not limited to, esters (including silyl esters, oxamate and thioesters), amides and hydrazides.

In yet another aspect, the present invention provides compositions comprising one or more stereoisomer-enriched compounds. The compositions generally comprise one or more compounds, and/or prodrugs, salts, hydrates, solvates and/or N-oxides thereof, together with suitable carriers, excipients and/or diluents. The exact nature of the carrier, excipient and/or diluent will depend on the intended use of the composition, which may range from suitable for in vitro or in vitro use, to acceptable for veterinary or veterinary use, to acceptable for human or human use.

In vitro assays, the stereoisomer-enriched compounds described herein are potent inhibitors of proliferative abnormal cells, such as tumor cells. Thus, in a further aspect, the present invention provides a method of inhibiting the proliferation of abnormal cells, particularly tumor cells. The methods generally comprise contacting an abnormal cell, such as a tumor cell, with one or more of the stereoisomer-enriched compounds described herein and/or prodrugs, salts, hydrates, solvates, and/or N-oxides thereof, in an amount effective to inhibit cell proliferation. The cell may be contacted with the compound itself, or the compound may be formulated in a composition. The method can be performed under in vitro conditions, or as a therapeutic modality under in vivo conditions, for the treatment or prevention of proliferative diseases such as tumorigenic cancers.

In yet another aspect, the invention provides a method of treating a proliferative disease. The method can be carried out in animals in the veterinary field or in humans. The methods generally comprise administering one or more stereoisomer-enriched compounds described herein and/or prodrugs, salts, hydrates, solvates, and/or N-oxides thereof, to an animal or human subject in an amount effective to treat or prevent a proliferative disease. One or more compounds may be administered to the subject as such, or one or more compounds may be administered in the form of a composition. Proliferative diseases that can be treated according to this method include, but are not limited to, tumorigenic cancers.

The stereoisomerically enriched compounds described herein are potent inhibitors of Aurora kinases. Aurora kinases are a class of enzymes known as key regulators of cell division. Elevated levels of Aurora kinase have been found in several types of human cancer cells such as breast, colon, kidney, cervical, neuroblastoma, melanoma, lymphoma, pancreatic, prostate and other solid tumors (see, e.g., Bischott et al, 1998, EMBO J.17: 3052-. While not wishing to be bound by any particular theory of action, it is believed that the stereoisomer-enriched compounds described herein, and their active prodrugs, salts, hydrates, solvates and/or N-oxides, exert their antiproliferative activity by inhibiting one or more Aurora kinases.

Thus, in yet another aspect, the present invention provides a method of inhibiting Aurora kinase activity. The methods generally comprise contacting an Aurora kinase with one or more stereoisomer-enriched compounds described herein and/or active prodrugs, salts, hydrates, solvates, and/or N-oxides thereof in an amount effective to inhibit Aurora kinase activity. The methods can be performed under in vitro conditions using purified or partially purified Aurora kinases (e.g., using cell extracts expressing Aurora kinases), under in vitro conditions using intact cells expressing Aurora kinases, or under in vivo conditions, thereby inhibiting Aurora kinase-mediated processes (e.g., cellular mitosis) and/or as a therapeutic method for treating or preventing a disease or disorder mediated at least in part by Aurora kinase activity.

In yet another aspect, the present invention provides a method of treating or preventing an Aurora kinase-mediated disease or disorder. The methods generally comprise administering to an animal or human subject one or more stereoisomer-enriched compounds described herein and/or active prodrugs, salts, hydrates, solvates and/or N-oxides thereof, in an amount effective to treat or prevent an Aurora kinase-mediated disease or disorder. Aurora kinase-mediated diseases and dysfunctions include any disease, dysfunction or other undesirable condition in which enzymes of the Aurora kinase family play an important role. Specific examples of such Aurora kinase-mediated diseases or dysfunctions include, but are not limited to, melanoma; leukemia; and solid tumors such as colon cancer, breast cancer, stomach cancer, ovarian cancer, cervical cancer, melanoma, renal cancer, prostate cancer, lymphoma, neuroblastoma, pancreatic cancer, and bladder cancer.

Other aspects include, but are not limited to, intermediates, and methods for synthesizing stereoisomer-enriched compounds and prodrugs, which are described in detail below.

Drawings

FIGS. 1-4 illustrate the inhibitory effect of (1R, 2R, 3S, 4S) -N4- (3-aminocarbonylbicyclo [2.2.1] hept-5-en-2-yl) -5-fluoro-N2- [ (3-methyl-4- (4-methylpiperazin-1-yl) ] phenyl-2, 4-pyrimidinediamine dihydrochloride (compound 60 a.2HCl) on the growth of a number of different types of tumors in a standard xenograft treatment and regression model (regression model).

Detailed Description

Definition of

The following terms used herein have the following meanings:

"alkyl" by itself or as part of another substituent means a saturated or unsaturated, branched, straight chain or cyclic monovalent hydrocarbon radical having the indicated number of carbon atoms, either by removal of one hydrogen atom from a single carbon atom of a parent alkane, alkene or alkyne (i.e., C1-C6 represents 1 to 6 carbon atoms). Typical hydrocarbyl groups include, but are not limited to, methyl; ethyl groups such as ethyl, vinyl, ethynyl; propyl groups such as prop-1-yl, prop-2-yl, prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl, prop-1-en-1-yl; cyclopropyl-2-en-1-yl, prop-1-yn-1-yl, prop-2-yn-1-yl, and the like; butyl radicals, for example but-1-yl, but-2-yl, 2-methyl-prop-1-yl, 2-methyl-prop-2-yl, cyclobut-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-2-yl, but-1, 3-dien-1-yl, but-1, 3-dien-2-yl, cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobut-1, 3-dien-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, and the like; and the like. In the case where a specific degree of saturation is specified, the terms "alkyl", "alkenyl" and/or "alkynyl" are used as defined below. "lower alkyl" refers to a hydrocarbon group containing 1 to 6 carbon atoms.

"alkyl (alkanyl)" by itself or as part of another substituent refers to a saturated branched, straight-chain or cyclic hydrocarbon radical obtained by the removal of one hydrogen atom from a single carbon atom of a parent alkane. Typical alkyl groups include, but are not limited to, methyl; an ethyl group; propyl groups such as prop-1-yl, prop-2-yl (isopropyl), and cyclopropyl-1-yl; butyl groups such as but-1-yl, but-2-yl (sec-butyl), 2-methyl-propan-1-yl (isobutyl), 2-methyl-propan-2-yl (tert-butyl), cyclobut-1-yl, and the like; and the like.

"alkenyl" as itself or as part of another substituent means an unsaturated branched, straight chain or cyclic hydrocarbon radical having at least one carbon-carbon double bond derived by the removal of one hydrogen atom from a single carbon atom of a parent alkene. The double bond or bonds of the group may be in either the cis or trans configuration. Typical alkenyl groups include, but are not limited to, vinyl; acryl groups such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl, prop-2-en-2-yl, prop-1-en-1-yl, prop-2-en-1-yl; butenyls such as but-1-en-1-yl, but-1-en-2-yl, 2-methylprop-1-en-1-yl, but-2-en-2-yl, but-1, 3-dien-1-yl, but-1, 3-dien-2-yl, cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobut-1, 3-dien-1-yl and the like; and the like.

"alkynyl", by itself or as part of another substituent, refers to an unsaturated branched, straight chain or cyclic hydrocarbon radical having at least one carbon-carbon triple bond derived by the removal of one hydrogen atom from a single carbon atom of a parent alkyne. Typical alkynyl groups include, but are not limited to, ethynyl; propynyl groups such as prop-1-yn-1-yl group, prop-2-yn-1-yl group and the like; butynyl groups such as but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl and the like; and the like.

"Hydrocarbdiyl" by itself or as part of another substituent means a saturated or unsaturated, branched, straight chain, or cyclic divalent hydrocarbon radical having the indicated number of carbon atoms, either by the removal of one hydrogen atom from each of two different carbon atoms of a parent alkane, alkene, or alkyne, or by the removal of two hydrogen atoms from a single carbon atom of a parent alkane, alkene, or alkyne (i.e., C1-C6 represents 1 to 6 carbon atoms). Each valence of two monovalent radical centers or a divalent radical center may be attached to the same or different atoms. Typical hydrocarbondiyl groups include, but are not limited to, methyldiyl; ethanediyl radicals, such as the ethane-1, 1-diyl, ethane-1, 2-diyl, ethene-1, 1-diyl, ethene-1, 2-yl; propanediyl radicals, such as the propane-1, 1-diyl, propane-1, 2-diyl, propane-2, 2-diyl, propane-1, 3-diyl, cyclopropane-1, 1-diyl, cyclopropane-1, 2-diyl and propane-1-ene-1, 1-diyl radical, prop-1-en-1, 2-diyl, prop-2-en-1, 2-diyl, prop-1-en-1, 3-diyl, prop-1-en-1, 2-diyl, prop-2-en-1, 1-diyl, prop-1-yn-1, 3-diyl, and the like; butandiyls, for example butan-1, 1-diyl, butan-1, 2-diyl, butan-1, 3-diyl, butan-1, 4-diyl, butan-2, 2-diyl, 2-methyl-propan-1, 1-diyl, 2-methyl-propan-1, 2-diyl, cyclobutan-1, 1-diyl, cyclobutan-1, 2-diyl, cyclobutan-1, 3-diyl, butan-1-en-1, 1-diyl, butan-1-en-1, 2-diyl, butan-1-en-1, 3-diyl, butan-1-en-1, 4-diyl, 2-methyl-propan-1-en-1-ene-1, 1-diyl, 2-methylene-prop-1, 1-diyl, but-1, 3-dien-1, 2-diyl, but-1, 3-dien-1, 3-diyl, but-1, 3-dien-1, 4-diyl, cyclobut-1-en-1, 2-diyl, cyclobut-1-en-1, 3-diyl, cyclobut-2-en-1, 2-yl, cyclobut-1, 3-dien-1, 2-diyl, cyclobut-1, 3-dien-1, 3-diyl, but-1-yn-1, 3-diyl, But-1-yne-1, 4-diyl, but-1, 3-diyne-1, 4-diyl, and the like; and the like. In the case of specifying a particular degree of saturation, the terms "alkanediyl", "alkenediyl" and/or "alkynediyl" are used. Wherein, when two valencies are specified on the same carbon atom, the term "alkylene (alkylene)" is used. "lower hydrocarbadiyl" is a hydrocarbadiyl group containing from 1 to 6 carbon atoms. In some embodiments, the hydrocarbadiyl group is a saturated non-cycloalkanediyl group, the radical center of which is on a terminal carbon atom, e.g., a methyldiyl (methylene); 1, 2-diyl (ethylene); prop-1, 3-diyl (propylene); but-1, 4-diyl (tetramethylene); and the like (also known as hydrocarbylene), which is defined below.

"hydrocarbylene" as it stands or as part of another substituent refers to a linear saturated or unsaturated hydrocarbadiyl group having two terminal monovalent radical centers derived by removing one hydrogen atom from each of the two terminal carbon atoms of a linear parent alkane, alkene, or alkyne. If a double or triple bond is present in a particular hydrocarbylene group, the position of that double or triple bond is indicated by brackets. Typical hydrocarbylenes include, but are not limited to, methylene (methylene); ethylenes such as ethylene, vinylene, ethynylene; propylenes such as propylene, propen 1 ylene, propadiene 1, 2-ene, propyne 1-ene, etc.; butyne groups such as butyne, butene [1] ene, butene [2] ene, butene [1, 3] diyl, butyne [1] ene, butyne [2] ene, and butyne [1, 3] ene; and the like. In the case of specifying a specific degree of saturation, the terms alkylene (alkano), alkenylene (alkeno) and/or alkynylene (alkyno) are used. In some embodiments, the hydrocarbylene is (C1-C6) or (C1-C3) hydrocarbylene. In some embodiments, the alkylene is a straight chain saturated alkylene, e.g., methylene, ethylene, propylene, butylene, and the like.

"cycloalkyl" as it is or as part of another substituent means a cyclic form of "alkyl". Typical cycloalkyl groups include, but are not limited to, cyclopropyl; cyclobutyl groups, such as cyclobutyl and cyclobutenyl: cyclopentyl groups, such as cyclopentyl and cyclopentenyl: cyclohexyl, such as cyclohexyl and cyclohexenyl; and the like.

"parent aromatic ring system" refers to an unsaturated or polycyclic ring system having a conjugated pi-electron system. In particular, the definition of "parent aromatic ring system" includes fused ring systems in which one or more rings are aromatic and one or more rings are saturated or unsaturated, such as fluorene, indane, indene, Phenalene (Phenalene), tetrahydronaphthalene, and the like. Typical parent aromatic ring systems include, but are not limited to, aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene (azulene), benzene, chrysene (chrysene), coronene (coronene), fluoranthene, fluorene, hexacene, hexalene, indacene, s-indacene, indane, indene, naphthalene, octacene, octaphene, aldrin (octalene), ovalene, penta-2, 4-diene, pentacene, pentalene, perylene, phenalene, phenanthrene, picene, hepta ene (pleiadene), pyrene, pyranthrene, rubicene, tetrahydronaphthalene, benzo [9, 10] phenanthrene, binaphthalene, and the like.

"aryl" by itself or as part of another substituent means a monovalent aromatic hydrocarbon radical having the indicated number of carbon atoms, obtained by removing one hydrogen atom from a single carbon atom of the parent aromatic ring system (i.e., C5-C15 represents 5 to 15 carbon atoms). Typical aryl groups include, but are not limited to, groups derived from: aceanthrylene, acenaphthene, acephenanthrene, anthracene, azulene (azulene), benzene, chrysene (chrysene), coronene (coronene), fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene, indane, indene, naphthalene, octacene, octaphene, aldrin (octalene), ovalene, penta-2, 4-diene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picene, hepta ene (pleiadene), pyrene, pyranthrene, rubicene, tetrahydronaphthalene, benzo [9, 10] phenanthrene, binaphthalene and the like, as well as various hydroisomers thereof. In some embodiments, the aryl group is a (C5-C15) aryl group, more typically a (C5-C10) aryl group. Specific examples are phenyl and naphthyl.

"halo" or "halo" as it is or as part of another substituent refers to fluoro, chloro, bromo, and iodo unless otherwise specified.

"haloalkyl" as employed by itself or as part of another substituent means a hydrocarbyl group in which one or more hydrogen atoms are replaced by a halogen. Thus, the term "halogenated hydrocarbon group" is meant to include monohalogenated hydrocarbon groups, dihalogenated hydrocarbon groups, trihalohydrocarbon groups, and the like, up to perhalogenated hydrocarbon groups. For example, the expression "(C1-C2) halohydrocarbyl" includes fluoromethyl, difluoromethyl, trifluoromethyl, 1-fluoroethyl, 1, 1-difluoroethyl, 1, 2-difluoroethyl, 1, 1, 1-trifluoroethyl, perfluoroethyl, and the like.

"Hydroxyhydrocarbyl" by itself or as part of another substituent refers to a hydrocarbyl group in which one or more hydrogen atoms are replaced by a hydroxy substituent. Thus, the term "hydrocarbyl" is meant to include monohydroxy hydrocarbyl, dihydroxy hydrocarbyl, trihydroxy hydrocarbyl and the like.

The above-defined groups may include prefixes and/or suffixes commonly used in the art to derive other well-known substituents. Examples are "hydrocarbyloxy" OR "hydrocarbyloxy" referring to a group of formula-OR, "hydrocarbylamine" referring to a group of formula-NHR, and "dihydrocarbylamine" referring to a group of formula-NRR, where each R is independently a hydrocarbyl group. Another example is "halohydrocarbyloxy" OR "halohydrocarbyloxy" refers to a group of formula-OR 'wherein R' is halohydrocarbyl.

"prodrug" refers to a derivative of an active compound (drug) that may need to be converted under conditions of use (e.g., in vivo) to release the active drug. Prodrugs are often, but not necessarily, pharmacologically inactive prior to conversion to the active drug. Prodrugs are typically obtained by: masking of functional groups (which are considered in part to be required for activity) in a drug compound with a precursor group (defined below) forms a precursor moiety which under the particular conditions of use is converted (e.g. cleaved) to release the functional group and thereby yield the active drug. The cleavage of the precursor portion may occur spontaneously, for example by means of a hydrolysis reaction; or it may be catalyzed or initiated by another agent, such as by an enzyme, light, acid or base, or to alter a physical or environmental parameter with which it is in contact, such as to change temperature. The agent may be endogenous to the conditions of use, such as the enzymes present in the cell to which the prodrug is administered or the acidic conditions of the stomach; or may be provided exogenously.

A number of precursor groups and resulting precursor moieties suitable for use in masking the functional groups in the stereoisomer-enriched active compounds described herein to produce prodrugs are well known in the art. For example, the hydroxyl functionality may be masked as a sulfonate, ester, or carbonate precursor moiety that can be hydrolyzed in vivo to yield a hydroxyl group. The amino functional group can be masked as an amide, carbamate, imine, urea, phenylphosphino, phosphoryl, or sulfenyl precursor moiety that can be hydrolyzed in vivo to yield an amino group. The carboxyl group can be masked as an ester (including silyl esters and thioesters), amide or hydrazide precursor moiety that can be hydrolyzed in vivo to produce a carboxyl group. Other specific examples of suitable precursor groups and their respective precursor moieties will be apparent to those skilled in the art.

"precursor group" refers to a type of protecting group: when used to mask a functional group to form a precursor moiety in a stereoisomer-enriched active drug compound, the drug is converted to a prodrug. The precursor group is typically linked to the functional group of the drug via a bond that can be cleaved under the particular conditions of use. Thus, a precursor group is part of a precursor moiety that cleaves under specific conditions of use to release a functional group. Specific examples thereof are those of the formula-NH-C (O) CH₃The amide precursor moiety of (a) includes a precursor group-C (O) CH₃。

"proliferative disease" refers to a disease or disorder characterized by abnormal cell proliferation (e.g., where cells divide more than their corresponding normal cells). Abnormal proliferation may be caused by a mechanism of action or a combination of mechanisms of action. For example, the cell cycle of one or more cells may be affected such that the cell(s) divide more frequently than their corresponding normal cells, or, as another example, one or more cells may bypass inhibitory signals that normally limit their number of divisions. Proliferative diseases include, but are not limited to, tumors and cancers that grow slowly or rapidly.

An "anti-proliferative compound" refers to a compound that inhibits cell proliferation as compared to an untreated control cell of similar type. Inhibition may be caused by any mechanism or combination of mechanisms, and may inhibit proliferation in a manner that inhibits cell growth or cytotoxicity. As specific examples, the inhibitory effect described herein includes, but is not limited to, stopping cell division, reducing the rate of cell division, proliferation and/or growth, and/or inducing cell death by any mechanism of action including, for example, apoptosis.

"Aurora kinase" refers to a member of the serine/threonine protein kinase family, which is commonly referred to as "Aurora" kinase. The Aurora family of serine/threonine protein kinases is important for Cell proliferation (see, e.g., Bischhoff & Plowman, 1999, Trends Cell biol.9: 454-459; Giet & Prigent, 1999, J.cell Science 112: 3591-3601; Nigg, 2001, nat. Rev. mol. Cell biol.2: 21-32; Adams et al, 2001, Trends Cell biol.11: 49-54). Currently, there are three known members of the mammalian family: Aurora-A ("2"), Aurora-B ("1"), and Aurora-C ("3") (see, e.g., Giet & Prigent, 1999, J.cell Sci.112: 3591-3601; Bischoff & Plowman, 1999, Trends Cell biol.9: 454-459). As used herein, "Aurora kinase" includes not only these three known members of the mammalian family, but also those later discovered members of the mammalian family as well as homologous proteins from other species and organisms (non-limiting examples of homologous members of the Aurora kinase family from other species and organisms are described in Schumacher et al, 1998, J.cell biol.143: 1635-.

By "Aurora kinase-mediated process" or "Aurora kinase-mediated disease or disorder" is meant a cellular process, disease or disorder in which Aurora kinase plays an important role. Aurora kinases are believed to have a key role in the protein phosphorylation events that regulate the mitotic phase of the cell cycle. Human Aurora kinase shows clear subcellular localization during mitosis. For example, Aurora-a is upregulated during the M phase of the cell cycle and is localized on the spindle during mitosis, suggesting involvement in the function of the centrosome. Aurora-a activity is maximized in the pre-mitotic phase and Aurora-B is believed to play an important role in the separation of chromatids and cleavage furrow formation during the post-and post-mitotic phase. The role of Aurora-C is less clear, but it has been shown to localize to the centrosome during mitosis from anaphase to cytokinesis. Furthermore, inhibition of Aurora kinase activity in mammalian cells causes abnormal cell growth and polyploidy (Terada et al, 1998, EMBO J.17: 667-. Thus, Aurora kinases are thought to regulate cell division, chromosome segregation, mitotic spindle formation, and cytokinesis. The various processes used herein are within the scope of "Aurora kinase mediated processes".

Moreover, the mammalian Aurora kinase family has been intimately linked to tumorigenesis since its discovery in 1997. In this regard, the most convincing evidence is that overexpression of Aurora-A transformed rodent fibroblasts (Bischoff et al, 1998, EMBO J.17: 3052-one 3065). Cells with higher levels of this kinase contain multiple centrosomes and multipolar spindles and rapidly become aneuploid. The tumorigenic activity of Aurora kinases may be associated with the development of this genetic instability. In fact, a correlation between the increase of the Aurora-A locus and chromosomal instability has been observed in breast and stomach tumors (Miyoshi et al, 2001, int.J. cancer 92: 370-831; Sakakura et al, 2001, Brit.J. cancer 84: 824-831).

Aurora kinase has been reported to be overexpressed in a large number of human tumors. Increased expression of Aurora-a has been detected in the following cases: more than 50% of colorectal (Bischoff et al, 1998, EMBO J.17: 3052 + 3065; Takahashi et al, 2000, Jpn. J. cancer Res.91: 1007 + 1014), ovarian (Gritsko et al, 2003, Clinical cancer research 9: 1420 + 1426) and gastric (Sakakura, 2001, Brit. J. cancer 84: 824 + 831); and 94% of invasive breast ductal adenomas (Tanaka, 1999, cancer research.59: 2041-2044). It has also been reported that high levels of Aurora-A exist in renal, cervical, neuroblastoma, melanoma, lymphoma, pancreatic and prostate tumor cell lines (Bischoff et al, 1998, EMBO J.17: 3052-3065; Kimura et al, 1999, J.biol.chem.274: 7334-7340; Zhou et al, 1998, Naturegenetics 20: 189-193; Li et al, 2003, Clin Cancer Res.9 (3): 991-7). Increased/over-expression of Aurora-A was observed in human bladder Cancer, and the increase in Aurora-A was correlated with aneuploidy and aggressive clinical behavior (Sen et al, 2002, J Natl Cancer Inst.94 (17): 1320-9). Furthermore, an increase in the Aurora-A locus (20q13) was associated with a poor prognosis in breast cancer patients who were node-negative (Isola et al, 1995, American Journal of Pathology 147: 905-911). Aurora-B is highly expressed in a variety of human tumor cell lines, including leukemia cells (Katayama et al, 1998, Gene 244: 1-7). Elevated levels of this enzyme became an indicator of the Duke's stage of primary colorectal Cancer (Katayama et al, 1999, J.nat' l Cancer Inst.91: 1160-1162). Aurora-C is usually found only in germ cells, and is also overexpressed in a high percentage of primary colorectal cancers and in a variety of tumor cell lines, including cervical adenocarcinoma and breast cancer cells (Kimura et al, 1999, j.biol.chem. 274: 7334-7340; Takahashi et al, 2000, jpn.j.cancer res.91: 1007-1014).

In contrast, in most normal tissues, the Aurora family is expressed at low levels, with the exception of tissues with a high proportion of dividing cells, such as the thymus and testis (Bischoff et al 1998, EMBO J., 17: 3052-3065).

To further review the role of Aurora kinases in proliferative diseases, please see bischoff & Plowman, 1999, Trends Cell biol.9: 454-459; giet & priment, 1999, j.cell Science 112: 3591-3601; nigg, 2001, nat. rev. mol. cell biol.2: 21-32; adams et al, 2001, Trends Cell biol.11: 49-54; and dutetrre et al, 2002, Oncogene 21: 6175-6183.

Although cancer cells overexpress proteins not always indicating that inhibition of protein activity would produce an anti-tumor effect, it has been demonstrated in functional assays that at least the following types of tumor cells are sensitive to inhibition of Aurora kinase activity: prostate cancer (DU145), cervical cancer (Hela), pancreatic cancer (Mia-Paca2, BX-PC3), histological leukemia (U937), lung adenocarcinoma, lung epidermoid carcinoma, small cell lung cancer, breast cancer, kidney cancer, MolT3 (all), and MolT4 (all).

Based on the discovered role of Aurora kinases in a variety of cancers, examples of "Aurora kinase-mediated diseases and disorders" include, but are not limited to, melanoma, leukemia, and solid tumor cancers, such as colon, breast, stomach, ovary, cervix, melanoma, kidney, prostate, lymphoma, neuroblastoma, pancreatic, and bladder.

"therapeutically effective amount" refers to an amount of a compound sufficient to treat a particular disorder or disease or one or more symptoms thereof. For neoplastic proliferative diseases, a therapeutically effective amount includes an amount sufficient to cause, among other things, shrinkage of the tumor, or decrease the rate of tumor growth.

In many cases, standard treatment for neoplastic proliferative diseases involves surgery to remove the tumor, either alone or in combination with drugs (chemotherapy) and/or radiation therapy. As used herein, a "therapeutically effective amount" of a compound means that the amount of the compound included is that which prevents tumor recurrence in subjects having tumor(s) removed by surgery, or delays the rate of tumor(s) recurrence in these subjects.

Thus, as used herein, the amount of a compound that provides adjunctive therapeutic benefit for other types of treatment (e.g., surgical intervention and/or treatment with other antiproliferative agents), including, for example, 5-fluorouracil, vinorelbine, paclitaxel, vinblastine, cisplatin, topotecan, and the like, is included within the meaning of "therapeutically effective amount".

By "prophylactically effective amount" is meant an amount of a compound sufficient to prevent the development of a particular disease or disorder in a subject. Typically, the subject undergoing prophylaxis does not suffer from a particular disorder or disease, but is considered to have a high risk of developing such a disease or disorder, such as, but not limited to, diagnostic markers and family history.

Stereoisomer-enriched compounds and pure stereoisomer compounds

In recent years, it has been found that certain N4- (3-aminocarbonylbicyclo [2.2.1] hept-5-en-2-yl) -N2-substituted phenyl-2, 4-pyrimidinediamine compounds, represented by the following structural formula (I), are potent inhibitors of Aurora kinase activity and tumor cell proliferation in vitro assays (see, for example, application 11/133,419 filed on 18.5.2005, co-pending application entitled "Stereooisomic enzymatic entity. beta. -Lactams Using Candidaantarctica" (application No. unknown, attorney docket No. 375462. sup. 030US), and International application PCT/US05/17470 and priority applications referenced thereto filed on 18.5.2005):

it will be appreciated by those skilled in the art that in formula (I), the stereochemistry at the 1,2, 3 and 4 carbon atoms is not specified, and therefore, the compounds according to formula (I) include 8 diastereomers represented by the following formulae (Ia) to (Ih):

the compound of structural formula (I) also includes two cis racemates represented by the following structural formulae (IIa) and (IIb), and two trans racemates represented by the following structural formulae (IIIa) and (IIIb):

(2-outer-3-outer) (2-inner-3-inner)

The cis-racemate of formula (IIa) may be referred to as 2-exo-3-exo-racemateRacemates and include the (1R, 2R, 3S, 4S) and (1S, 2S, 3R, 4R) diastereomers of formulae (Ia) and (Ib), respectively. The cis racemate of formula (IIb) may be referred to as the 2-endo-3-endo racemate and includes the (1R, 2S, 3R, 4S) and (1S, 2R, 3S, 4R) diastereomers of formulae (Ic) and (Id), respectively. As described in more detail in the examples section, for wherein R⁵Is fluorine, R¹Is hydrogen, R²Is 4-methylpiperazin-1-yl and R³Are methyl compounds, and the two cis racemates show antiproliferative activity in vitro antiproliferative assays against a variety of different tumor cell lines. However, the efficacy of this 2-rac-3-racemate (racemate r1) was approximately 20-fold higher than the corresponding 2-endo-3-racemate (racemate r2) in all cell lines tested with both racemates. Furthermore, it has been found that the (1R, 2R, 3S, 4S) diastereomer of racemate R1 plays a major role in the efficacy of racemate R1. When tested as isolated stereoisomers, such (1R, 2R, 3S, 4S) diastereomer (referred to as the "a" diastereomer) typically shows IC50 in the nanomolar range, while the (1S, 2S, 3R, 4R) diastereomer (referred to as the "b" enantiomer) typically shows IC50 in the micromolar range, for the same cell line. Thus, in general, the (1R, 2R, 3S, 4S) diastereomer of this compound is 1000 times more potent than its corresponding (1S, 2S, 3R, 4R) enantiomer. The efficacy was also approximately 20-50 fold higher in the cell lines tested than in the 2-endo-3-endo r2 racemate. The (1R, 2R, 3S, 4S) diastereomer also showed similar better results in cell-based inhibition assays for Aurora kinase B compared to its (1S, 2S, 3R, 4R) enantiomer. Based on the observation of the effect of such (1R, 2R, 3S, 4S) diastereomers, it is expected that all (1R, 2R, 3S, 4S) diastereomers according to formula (Ia) will exhibit similar advantageous effects as compared to their corresponding (1S, 2S, 3R, 4R) enantiomer, 2-outer-3-outer racemate, 2-inner-3-inner racemate, and other corresponding diastereomers.

Thus, compounds enriched in this particularly potent (1R, 2R, 3S, 4S) diastereomer are provided herein. In one embodiment, such stereoisomer-enriched compounds include compounds according to structural formula (I):

it is enriched in the corresponding diastereoisomer of structural formula (Ia):

wherein:

each R¹Are each independently selected from hydrogen, lower alkyl, - (CH)₂)_n-OH、-OR^a、-O(CH₂)_n-R^a、-O(CH₂)_n-R^b、-C(O)OR^aHalogen, -CF₃and-OCF₃；

Each R²Are each independently selected from hydrogen, lower alkyl, -OR^a、-O(CH₂)_n-R^a、-O(CH₂)_n-R^b、-NHC(O)R^aHalogen, -CF₃、-OCF₃、And

each R³Are each independently selected from hydrogen, lower alkyl, - (CH)₂)_n-OH、-OR^a、-O(CH₂)_n-R^a、-O(CH₂)_n-R^bHalogen, -CF₃、-OCF₃、And

each R⁴Are each independently selected from hydrogen, lower alkyl, arylalkyl, -OR^a、-NR^cR^c、-C(O)R^a、-C(O)OR^aand-C (O) NR^cR^c；

R⁵Is hydrogen, halogen, fluorine, -CN, -NO₂、-CO(O)R^aor-CF₃；

Each n is independently an integer from 1 to 3;

each R^aEach independently selected from hydrogen, lower alkyl and lower cycloalkyl;

each R^bAre all independently selected from-OR^a、-CF₃、-OCF₃、-NR^cR^c、-C(O)R^a、-C(O)OR^a、-C(O)NR^cR^cand-C (O) NR^aR^d；

Each R^cAre each independently selected from hydrogen and lower alkyl, or two R^cThe substituents may be taken together with the nitrogen atom to which they are attached to form a 4-9 membered saturated ring, which optionally includes 1-2 additional ring members selected from O, NR^a、NR^a-C(O)R^a、NR^a-C(O)OR^aAnd NR^a-C(O)NR^aA heteroatom group of (a); and is

Each R^dAre each independently a lower monohydroxyhydrocarbyl group or a lower dihydroxyhydrocarbyl group.

In another embodiment, such stereoisomer-enriched compounds include those according to the structure2-exo-3-exo cis racemate of formula (IIa), wherein R¹、R²、R³、R⁴And R⁵As defined above for formula (I) enriched in the diastereomer of formula (Ia).

As used herein, a compound is "enriched" in a particular diastereomer when that diastereomer is present in an amount that greatly exceeds any other diastereomer present in the compound. The actual percentage of a compound containing that particular diastereomer will depend on the number of other diastereomers present. As a specific example, a racemic mixture is "enriched" in a particular enantiomer when that enantiomer is more than 50% of the composition of the mixture. Regardless of the number of diastereomers present, a compound enriched in a particular diastereomer generally comprises at least about 60%, 70%, 80%, 90%, or even more of that particular diastereomer. The enrichment (enrichment) of this particular diastereomer can be confirmed using conventional analytical methods commonly used by those skilled in the art, as discussed in detail below.

In another embodiment, the stereoisomer-enriched compound comprises the aforementioned compound according to structural formula (Ia), wherein R¹、R²、R³、R⁴And R₅As previously stated for structural formula (I), the compounds are substantially free of the corresponding enantiomer and/or any other corresponding diastereomer. By "substantially free" it is meant that the compound comprises less than about 10% of undesired diastereomers and/or enantiomers (discussed in detail below) as determined using conventional analytical methods routinely employed by those skilled in the art. In some embodiments, the amount of undesired stereoisomer can be less than 10%, e.g., 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or even less. Stereoisomerically enriched compounds containing about 95% or more of the desired stereoisomer are referred to herein as "substantially pure" stereoisomers. Stereoisomerically enriched compositions containing about 99% or more of a desired stereoisomerThe compounds of the formula are referred to herein as "pure" stereoisomers. The purity of any stereoisomerically-enriched compound (diastereomeric purity;% de) can be confirmed using conventional analytical methods, which are discussed in detail below.

In some embodiments of the various stereoisomer-enriched compounds described herein, R¹Is hydrogen; r²Is thatOrAnd R is³Is not provided withOrIn other embodiments of the various stereoisomer-enriched compounds described herein, R³Is hydrogen, methyl, methoxy, trifluoromethyl or chlorine. In other embodiments, R⁴Is methyl, -C (O) CH₃、-C(O)OCH₃or-C (O) OCH₂CH₃。

In still other embodiments of the various stereoisomer-enriched compounds described herein, R¹Is hydrogen; r²Is not provided withOrAnd R is³Is thatOrIn still other embodiments, R²Is hydrogen, methyl, methoxy, trifluoromethyl or chlorine. Preferably R⁴Is methyl, -C (O) CH₃、-C(O)OCH₃or-C (O) CH₂CH₃。

In still further embodiments of the various stereoisomer-enriched compounds described herein, R²Is not provided withOrAnd R is³Is not provided withOrIn still other embodiments, R¹And R²Are both hydrogen, and R³is-OCH₂NHR^a. In some other embodiments, R¹、R²And R³Each independently of the others, selected from the group consisting of hydrogen, methyl, methoxy, trifluoromethyl and chloro, with the proviso that R is¹、R²And R³At least two of which are not hydrogen.

In still other embodiments, R¹Is hydrogen; r²Selected from hydrogen,Andand R is³Selected from hydrogen, lower alkyl, halogen, -CF₃、Andin still other embodiments, R³Selected from hydrogen, methyl, chloro, -CF₃、Andand R is⁴Is methyl, -COR^aor-CO (O) R^aWherein R is^aIs methyl or ethyl. In yet another embodiment, R²Selected from hydrogen,Andand R is³Selected from hydrogen, lower alkyl, halogen, -CF₃、Andin still other embodiments, R³Selected from hydrogen, methyl, chloro, -CF₃、 Andand R is⁴Is methyl, -COR^aor-CO (O) R³Wherein R is^aIs methyl or ethyl. Preferably R²Is thatR⁴is-COR^aWherein R is^aIs methyl; and R is³Is hydrogen. In other embodiments, R²Is thatR⁴is-CO (O) R^aWherein R is^aIs an ethyl group; and R is³Is hydrogen. In yet another embodiment, R²Is thatAnd R is³Is hydrogen.

In yet another embodiment, R²Is hydrogen; r³Is thatOrAnd R is⁴Is methyl, -COR^aor-CO (O) R^aWherein R is^aIs methyl or ethyl. Preferably R²Is thatR⁴Is methyl; and R is³Selected from hydrogen, methyl, chlorine and-CF₃. More preferably R³Is methyl.

In still other embodiments of the stereoisomerically-enriched compounds described herein, R⁵Is fluorine.

In still other embodiments, the stereoisomerically-enriched compound is (1R, 2R, 3S, 4S) -N4- (3-aminocarbonylbicyclo [2.2.1] hept-5-en-2-yl) -5-fluoro-N2- [ (3-methyl-4- (4-methylpiperazin-1-yl) ] phenyl-2, 4-pyrimidinediamine that is substantially pure stereoisomerically or pure stereoisomerically.

Further exemplary embodiments of the compounds according to formula (I) which may be stereoisomerically enriched with the corresponding diastereoisomer of the aforementioned formula (Ia), substantially free of any enantiomer and/or diastereoisomer thereof, and/or substantially pure or pure diastereoisomer of the aforementioned formula (Ia) are illustrated in table I below:

when referring to a particular diastereomer and/or racemic mixture of a particular compound described herein as a compound described in table I, the compound number is followed by a letter that designates the particular diastereomer or racemic mixture as follows:

a＝(1R，2R，3S，4S)

b＝(1S，2S，3R，4R)

c＝(1R，2S，3R，4S)

d＝(1S，2R，3S，4R)

e＝(1R，2R，3R，4S)

f＝(1S，2S，3S，4R)

g＝(1R，2S，3S，4S)

h＝(1S，2R，3R，4R)

r 1-rac-3-rac-racemic

r 2-endo-3-endo-cis racemate

r 3-rac-3-endo-trans racemate

r 4-endo-3-racemic form

Thus, a specific example is the (1R, 2R, 3S, 4S) diastereomer of compound 60, referred to as compound 60 a.

One skilled in the art will appreciate that the stereoisomerically-enriched compounds described herein may include functional groups that can be masked with a precursor group to form a prodrug. Such prodrugs are typically, but not necessarily, pharmacologically inactive until converted to their active pharmaceutical form. For example, ester groups are typically subjected to acid-catalyzed hydrolysis upon exposure to acidic conditions in the stomach to yield the parent carboxylic acid, or to base-catalyzed hydrolysis upon exposure to alkaline conditions in the intestine or blood. Thus, when a subject is administered orally, a compound comprising an ester moiety that is enriched in stereoisomers may be considered a prodrug of its corresponding carboxylic acid, regardless of whether the ester form is pharmacologically active.

Prodrugs of the various stereoisomer-enriched compounds described herein are included within the scope of the present invention. In these prodrugs, any functional moiety that may be used may be masked with a precursor group to form a prodrug. Functional groups within the stereoisomer-rich compounds described herein, which may be masked in the precursor moiety with precursor groups, include, but are not limited to, amines (primary and secondary), hydroxyls, thioalkyls (thiols), carboxyls, and the like. A variety of precursor groups suitable for masking these functional groups to produce precursor moieties that are cleavable under the desired conditions of use are well known in the art. All of these precursor groups, alone or in combination, can be included in the stereoisomer-enriched prodrugs of the invention.

In an illustrative embodiment, the stereoisomer-enriched prodrug is a compound according to the aforementioned structural formula (I), wherein R^a、R^bAnd R^cIn addition to the aforementioned options, it may also be a precursor group enriched in the corresponding diastereomer of the aforementioned formula (Ia).

It will be appreciated by those skilled in the art that many of the compounds and prodrugs described herein, as well as the various compound classes specifically described and/or illustrated herein, may exhibit tautomerism and conformational isomerism. For example, the compounds and prodrugs may exist in several tautomeric forms, including enolic forms, ketonic forms, and mixtures thereof. With respect to the various compound names, structural formulae and compound figures within the specification and claims can represent only one of the possible tautomeric or conformational forms, it being understood that the invention includes any tautomer or conformational isomer of a compound or prodrug having one or more of the uses described herein, as well as mixtures of these various isomeric forms. Atrop isomers are also possible with limited rotation around the 2, 4-pyrimidinediamine core structure, and are also specifically included in the compounds and/or prodrugs of the invention.

Depending on the nature of the various substituents, the stereoisomerically-enriched compounds and prodrugs may be in the form of salts. Such salts include those suitable for pharmaceutical use ("pharmaceutically acceptable salts"), veterinary use, and the like. Such salts may be derived from acids or bases, as is well known in the art.

In some embodiments, the salt is a pharmaceutically acceptable salt. Generally, pharmaceutically acceptable salts are salts that substantially retain one or more of the desired pharmacological activities of the parent compound and are suitable for administration to humans. Pharmaceutically acceptable salts include acid addition salts formed with inorganic or organic acids. Inorganic acids suitable for forming pharmaceutically acceptable acid addition salts include, by way of example and not limitation, hydrohalic acids (e.g., hydrochloric, hydrobromic, hydroiodic, and the like), sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids suitable for forming pharmaceutically acceptable acid addition salts include, for example, but are not limited to, acetic acid, trifluoroacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, oxalic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, palmitic acid, benzoic acid, 3- (4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, hydrocarbyl sulfonic acids (e.g., methanesulfonic acid, ethanesulfonic acid, 1, 2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, and the like), arylsulfonic acids (e.g., benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, and the like), 4-methylbicyclo [2.2.2] -oct-2-ene-1-carboxylic acid, heptonic acid, 3-phenylpropionic acid, heptonic acid, pivalic acid, t-butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like.

Pharmaceutically acceptable salts also include those formed when an acidic proton present in the parent compound is replaced with a metal ion (e.g., an alkali metal ion, alkaline earth metal ion, or aluminum ion), or is coordinated with an organic base (e.g., ethanolamine, diethanolamine, triethanolamine, N-methylglucamine, morpholine, piperidine, dimethylamine, diethylamine, and the like).

The stereomerically enriched compounds and prodrugs and salts thereof, which may also be in the form of hydrates, solvates and/or N-oxides, are well known in the art.

The degree of stereoisomeric enrichment (Stereoiso-polymeric enrichment) and/or purity of the compounds and prodrugs described herein can be determined by routine analytical methods well known to those skilled in the art. For example, the use of chiral NMR shifting reagents, gas chromatography using chiral columns, high pressure liquid chromatography using chiral columns, formation of diastereomeric derivatives by reaction with chiral reagents, and conventional analytical methods can all be used to determine the degree of stereoisomer enrichment and/or purity of a particular stereoisomer. Alternatively, starting material syntheses using known degrees of stereoisomer enrichment and/or purity can be used to determine the degree of stereoisomer enrichment and/or purity of the compounds described herein. Other analytical methods for determining the homogeneity of stereoisomers are also well known to the skilled person.

Synthesis method

The stereoisomerically enriched compounds and prodrugs can be synthesized via a variety of different synthetic routes using commercially available starting materials and/or starting materials prepared by conventional synthetic methods. Various exemplary synthetic routes that can be used to synthesize the stereoisomer-enriched compounds and prodrugs described herein are described in WO03/063794 and US2004/0029902, the disclosures of which are incorporated herein by reference.

For purposes of illustration, an exemplary synthetic scheme that can be used to synthesize all of the compounds described herein is set forth in scheme (I) below:

scheme (I)

In scheme (I), R¹、R²、R³And R⁵As previously defined for structural formula (I), X is halogen (e.g., F, Cl, Br, or I) and each G is independently selected from O and S. It should be noted that "+" in the aminocarboxamide 6 means that no specific stereocenter is specified. Thus, one skilled in the art will appreciate that scheme (I) may be used to prepare racemic diastereomeric mixtures, diastereomeric-enriched mixtures, and stereoisomers of compounds of formula (I) (substantially free of other specific diastereomers) according to formula (I).

For scheme (I), uracil or Thiouropyrimidine 2 uses the standard halogenating agent POX₃(or other halogenating agent) under standard conditions to double-halogenate at the 2-and 4-positions to give 2, 4-bis-halogenopyrimidine 4. The halide at the C4 position in pyrimidine 4 is more reactive towards nucleophiles than the halide at the C2 position. This difference in reactivity can be exploited to synthesize the compounds and prodrugs described herein in the following manner: first, 2, 4-bis-halopyrimidine 4 is reacted with 1 equivalent of 2-aminobicyclo [2.2.1]Hept-5-ene-3-carboxamide 6 to give 8; and then reacted with aniline 10 to give the compound according to formula (I). It will be appreciated by the person skilled in the art that the stereoisomeric conformation and the optical purity of the aminocarboxamide 6 will in most cases determine the stereoisomeric conformation and the optical purity of the compound of formula (I).

In most cases, as illustrated in this scheme, the C4 halide is more nucleophilicAnd (4) reactivity. However, one skilled in the art will recognize that R⁵The nature of the substituents may alter their reactivity. For example, when R is⁵Is trifluoromethyl, a 50 of 4N-substituted-4-pyrimidinamine 8 and the corresponding 2N-substituted-2-pyrimidinamine is obtained: 50 of the mixture. Whether or not R⁵How the nature of the substituents can be controlled by adjusting the solvent and other synthesis conditions (e.g., temperature) is well known in the art.

The reaction depicted in scheme (I) proceeds more rapidly when the reaction mixture is heated by microwaves. When heating in this manner, the following conditions were used: in a Smith reactor (Personal chemistry, Biotage AB, Sweden) in a sealed tube (at 20 bar pressure) heated to 175 ℃ in ethanol for 5-20 minutes.

Uracil or thiouracil 2 starting materials are commercially available or can be prepared using standard techniques of organic chemistry. Commercially available uracils and thiouracils that can be used as starting materials in scheme (I) include, for example, but are not limited to, uracil (Aldrich #13,078-8; CAS registry number 66-22-8); 2-Thiouropyrimidine (Aldrich #11,558-4; CAS registry number 141-90-2); 2, 4-dithiouracil (Aldrich #15,846-1; CAS registry number 2001-93-6); 5-bromouracil (Aldrich #85,247-3; CAS registry number 51-20-7); 5-Fluorouracil (Aldrich #85,847-1; CAS registry number 51-21-8); 5-iodouracil (Aldrich #85,785-8; CAS registry number 696-07-1); 5-Nitroiuracils (Aldrich #85,276-7; CAS registry number 611-08-5); 5- (trifluoromethyl) -uracil (Aldrich #22,327-1; CAS registry number 54-20-6). Other 5-substituted uracils and/or thiouracils are available from general intermediates of Canada, Inc., Edmonton, CA (http:// www.general-intermediates. com) and/or Interchim, Cedex, France (http:// www.inter-chim. com), or can be prepared using standard techniques. Numerous textbook references are provided below which teach suitable synthetic methods.

Aniline 10 can be purchased from commercial sources or can be synthesized using standard techniques. For example, a suitable aniline can be synthesized from the nitro precursor using standard chemical techniques. Specific exemplary reactions are provided in the examples section. See also Vogel, 1989, Practical organic chemistry, Addison Wesley Longman, Ltd., and John Wiley & Sons, Inc.

It will be understood by those skilled in the art that in some cases, the aniline 10 may include functional groups that need to be protected during synthesis. The exact nature of any protecting group(s) used will depend on the nature of the functional group being protected, as will be apparent to those skilled in the art. Guidance in the selection of suitable protecting Groups and synthetic strategies for their attachment and removal can be found, for example, in Greene & Wuts, Protective Groups in Organic Synthesis, third edition, John Wiley & Sons, Inc., New York (1999) and references cited therein (hereinafter "Greene & Wuts").

The prodrugs described herein may be prepared by conventional modifications of the methods described above.

It will be appreciated by those skilled in the art that the desired (1R, 2R, 3S, 4S) diastereomer corresponding to the aforementioned formula (Ia) may be separated by chiral separation or other standard techniques. Methods for chiral resolution of specific diastereomers are described in more detail in the examples section.

Stereoisomerically enriched compounds and/or substantially pure and/or pure diastereomers can also be synthesized starting from 2-amino-3-carboxamide starting material 6 with a specific stereochemistry, or with the aid of chiral auxiliaries.

In an exemplary embodiment, the desired diastereomer is chemically cleaved using (R) -methyl-p-methoxybenzylamine 18 as a chiral auxiliary, as described in scheme (II) below.

In scheme (II), the 2-rac-3-racemic β -lactam 14r1 (prepared as described in Stajar et al, 1984, Tetrahedron40 (12): 2385) was protected with a Boc group to give the corresponding racemic Boc-protected β -lactam 16r 1. The Boc-protected racemate 16R1 was then reacted with (R) -methyl-p-methoxybenzylamine 18 to give a mixture of diastereomers 20a and 20 b. This mixture of diastereomers is reacted with an acid such as TFA to cleave the Boc group to provide a mixture of diastereomers 22a and 22b, which can be reacted with 2, 4-dihalopyrimidine 4 to provide a racemic mixture of compounds 24a and 24 b. At this stage, compounds 24a and 24b can be separated from each other by crystallization and reacted with aniline 10 to give the separated diastereomers 25a and 25 b. The chiral auxiliary is then cleaved from the separated diastereomers 25a and 25b to give the separated diastereomers according to formulae (Ia) and (Ib), respectively.

For compounds 25a and 25b, where R is¹Is hydrogen, R²Is 4-methyl-piperazin-1-yl, R³Is methyl and R⁵Is fluorine, the cleavage of the chiral auxiliary proves to be difficult. For these and other compounds where such cleavage has proven difficult, the chiral auxiliary can be cleaved from compounds 24a and 24b and the resulting isolated compounds reacted with aniline 10 to give isolated diastereomers according to formulae (Ia) and (Ib). Specific examples of these reactions are described in the examples section.

Stereoisomerically enriched compounds, substantially stereoisomeric (substitantialy) specific diastereomers and/or pure stereoisomeric (substitantialy pure) specific diastereomers can also be synthesized from stereoisomerically enriched, substantially pure and/or pure stereoisomeric beta-lactams. Such stereoisomer-enriched and/or (substantially) pure stereoisomer beta-lactams can be enzymatically cleaved and isolated. In an exemplary embodiment, as described by Eniko et al, 2004, Tetrahedron Asymmetry 15: 573-575 the (essentially) pure stereoisomer of a beta-lactam can be decomposed and isolated from a racemic mixture of 2-exo-3-exo beta-lactams using immobilized lipase (from Sigma Chemical Co., catalog number L4777). In another exemplary embodiment, a (substantially) pure stereoisomer of a beta-lactam can be resolved and isolated from a 2-exo-3-exo Boc-protected racemic beta-lactam 16r1 using an immobilized chiralyme L-2 type B lipase (Candida antarctica type B, c-f, available from Biocatalytics, Inc., Pasadena, Calif.) bound to a resin, the disclosures of which are incorporated herein by reference, as described in application No. 60/628,401 filed on 15/11/2004, co-pending application No. 11/133,419 filed on 18/5/2005, and International application No. PCT/US05/17470 filed on 18/5/2005, and co-pending application entitled "Stereorganics engineered beta-membranes Using Candida Antarctica" (application No. unknown, attorney docket No. 375462-. Specific examples of the use of this enzyme to isolate specific diastereoisomers of β -lactam are described in the examples section, which is a method of synthesizing 2-rac-3-racemic β -lactam 16r 1.

Examples of the synthesis of specific diastereomers according to formula (Ia) using enzymatic reactions are described in schemes (III) and (IV) below. Specific examples of the use of the Novozyme435 enzyme as described in schemes (III) and (IV) are described in the examples section, which, like the chiralzyme enzyme discussed above and shown in scheme (III), can be used to dissociate enantiomers from racemic β -lactams.

Scheme (IV)

Activity of antiproliferative active Compounds

Rich in vitamin CStereoisomeric active compounds generally inhibit the proliferation of desired cells, such as tumor cells, the IC of which is measured in a standard in vitro cell proliferation assay₅₀In the range of about 20 μ M or less. Of course, one skilled in the art will appreciate that a lower IC is shown₅₀Compounds (e.g. in the order of 10. mu.M, 1. mu.M, 100nM, 10nM, 1nM or even lower) may be used in particular in therapeutic applications. The antiproliferative activity may be cytostatic or cytotoxic. Where anti-proliferative activity is desired to be specific for a particular cell type, the compounds can be assayed for activity using the desired cell type and screened against activities lacking in other cell types. In such a reverse screen, the desired degree of "inactivity", or desired ratio of activity to inactivity, may vary from case to case and may be selected by the user.

The active compounds also typically inhibit the activity of Aurora kinases, the IC thereof₅₀In the range of about 20. mu.M or less, typically in the range of about 10. mu.M, 1. mu.M, 100nM, 10mM, 1mM, or even less. IC against Aurora kinase₅₀Can be determined in standard in vitro assays using isolated Arurora kinase, or in functional cell arrays. Suitable enzyme-linked assays that can be used to determine the degree of Aurora kinase activity are described in Fox et al, 1998, Protein sci.7: 2249-2255. The Kentucky peptide sequence LRRASLG (Bochern Ltd., UK) can be used as a substrate for Aurora kinase-A, Aurora kinase-B and/or Aurora kinase-C, in the presence of 100mM HEPES (pH7.5), 10mM MgCl, at 30 deg.C₂25mM NaCl and 1mM DTT. IC (integrated circuit)₅₀Values can be determined using programmed nonlinear regression using commercially available Software (e.g., prism3.0, GraphPed Software, San Diego, CA). Suitable cell-based functional assays are described in the examples section.

Use of antiproliferative compounds

The stereomerically enriched active compounds, including various prodrug, salt, hydrate, and/or N-oxide forms thereof, can be used to inhibit Aurora kinase, Aurora kinase-mediated processes, and/or cell proliferation in a variety of contexts. According to some embodiments, the cell or population of cells is contacted with such a compound in an amount effective to inhibit Aurora kinase activity, an Aurora kinase-mediated process, and/or proliferation of the cell or population of cells. When used to inhibit cell proliferation, the compounds may exert cytotoxic killing of cells, or may exert cytostatic effects to inhibit proliferation without killing cells.

In some embodiments, these methods may be performed in vivo as therapeutic methods to treat or prevent Aurora kinase-mediated diseases or dysfunctions, particularly proliferative diseases. Thus, in one embodiment, the stereoisomerically enriched compounds described herein (and the various forms described herein) may be used for the treatment or prophylaxis of a proliferative disease in an animal subject, including a human. The methods generally comprise administering to the subject a stereoisomer-enriched compound, or a prodrug, salt, hydrate, or N-oxide thereof, in an amount effective for the treatment or prevention of the disease. In one embodiment, the subject is a mammal, including but not limited to a bovine, equine, feline, canine, rodent, or primate. In another embodiment, the subject is a human.

Various cell proliferative disorders may be treated or prevented using the compounds described herein. In some embodiments, these compounds are used to treat various cancers in an afflicted subject. Generally, cancers are classified according to the tissue and cell type from which the cancer cells are derived. Carcinoma (carcinoma) is considered to be a cancer derived from epithelial cells (cancer), and sarcoma is considered to be a cancer derived from connective tissue and muscle. Other cancer types include leukemias derived from hematopoietic cells and cancers derived from nervous system cells of neural tissue. For non-invasive tumors, adenomas are considered benign epithelial tumors with glandular structure, while chondroma are considered benign tumors derived from cartilage. Proliferative diseases that can be treated with the compounds of the present invention include carcinomas, sarcomas, leukemias, neuronal tumors, and non-invasive tumors.

In a particular embodiment, these compounds are used to treat solid tumors derived from a variety of tissue types, including, but not limited to, bone cancer, breast cancer, respiratory tract cancer, brain cancer, reproductive organ cancer, digestive tract cancer, urinary tract cancer, bladder cancer, eye cancer, liver cancer, skin cancer, head cancer, neck cancer, thyroid cancer, parathyroid cancer, kidney cancer, pancreatic cancer, blood cancer, ovarian cancer, colon cancer, male germ cell/prostate cancer, and metastases thereof.

Specific proliferative diseases include the following: a) proliferative diseases of the breast including, but not limited to, invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ, and metastatic breast cancer; b) proliferative diseases of the skin including, but not limited to, basal cell carcinoma, squamous cell carcinoma, malignant melanoma, and Karposi's sarcomas (Karposi's sarcomas); c) respiratory proliferative diseases including, but not limited to, small and non-small cell lung cancer, bronchial edema, pleuropulmonary blastoma, malignant mesothelioma; d) brain proliferative diseases including, but not limited to, hypothalamic glioma, cerebellar and brain astrocytoma, medulloblastoma, ependymoma, oligodendroglioma, meningioma, and neuroectodermal and pineal tumors; e) male reproductive organ proliferative disorders including, but not limited to, prostate cancer, testicular cancer, and penile cancer; f) proliferative diseases of female reproductive organs including, but not limited to, uterine cancer (endometrial cancer), cervical cancer, ovarian cancer, vaginal cancer, vulvar cancer, uterine sarcoma, ovarian germ cell tumors; g) proliferative diseases of the digestive tract including, but not limited to, anal, colon, colorectal, esophageal, gallbladder, gastric, pancreatic-islet cell, rectal, small intestine, and salivary gland cancers; h) liver proliferative diseases including, but not limited to hepatocellular carcinoma, cholangiocarcinoma, mixed hepatocellular cholangiocarcinoma, and primary liver cancer; i) ocular proliferative diseases including, but not limited to, intraocular melanoma, retinoblastoma, and rhabdomyosarcoma; j) proliferative diseases and cancers of the head including, but not limited to, laryngeal, hypopharyngeal, nasopharyngeal, oropharyngeal cancer, as well as lip and oral cancer, squamous carcinoma of the neck, metastatic sinus cancer; k) proliferative diseases of lymphoma, including but not limited to various T-cell and B-cell lymphomas, non-hodgkin's lymphoma, cutaneous T-cell lymphoma, hodgkin's disease, and lymphoma of central nervous system cells; l) leukemias, including but not limited to acute myelogenous leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell leukemia; m) proliferative disorders of the thyroid gland, including thyroid cancer, thymoma and malignant thymoma; n) sarcomas including, but not limited to, soft tissue tumors, osteosarcomas, malignant fibrous histiocytomas, lymphosarcomas, and rhabdomyosarcomas.

It is to be understood that the description of proliferative diseases is not limited to the various conditions described herein, but includes other diseases characterized by uncontrolled growth and malignant manifestations. It is also understood that proliferative diseases include various metastatic forms of the tumors and cancer types described herein. The compounds of the invention can detect efficacy against the diseases described herein and establish effective treatment regimens. As described further below, efficacy includes reduction or remission of tumors, a decrease in the rate of cell proliferation, or cytostatic or cytotoxic effects on cell growth.

Combination therapy

The stereoisomerically enriched compounds described herein may be used alone, in combination with another compound, or in addition to or in combination with other antiproliferative therapies. Thus, these compounds can be used with conventional cancer treatment methods, such as ionizing radiation in the form of gamma-rays and x-rays, external delivery or internal delivery by implantation of radioactive compounds, and continued treatment after surgical removal of the tumor.

In another aspect, these compounds may be used with other chemotherapeutic agents useful in the disease or condition being treated. These compounds may be administered simultaneously, sequentially by the same route of administration, or by different routes of administration.

In some embodiments, the compounds of the present invention may be used with other anti-cancer agents or cytotoxic agents. Various types of anticancer and antineoplastic compounds include, but are not limited to, alkylating agents, antimetabolites, vinca alkaloids (vinca alkyloids), taxanes, antibiotics, enzymes, cytokines, platinum coordination complexes, substituted ureas, tyrosine kinase inhibitors, hormones, and hormone antagonists. Exemplary alkylating agents include, but are not limited to, mechlorodiethylamine (mechlorothiamine), cyclophosphamide, ifosfamide, melphalan, chlorambucil, ethylenimine, methyl melamine, alkyl sulfonates (e.g., busulfan), and carmustine. Exemplary antimetabolites include, for example, but are not limited to, the folate analogue methotrexate; pyrimidine analogs fluorouracil, cytosine arabinoside; the purine analogs mercaptopurine, thioguanine, and azathioprine. Exemplary vinca alkaloids include, for example, but are not limited to, vinblastine, vincristine, paclitaxel, and colchicine. Exemplary antibiotics include, for example, but are not limited to, actinomycin D, daunorubicin, and bleomycin. Exemplary enzymes effective as antineoplastic agents include L-asparaginase. Exemplary coordination compounds include, for example, but are not limited to, cisplatin and carboplatin. Exemplary hormones and hormone-related compounds include, for example, but are not limited to, prednisones and dexamethasone, the adrenocorticoid steroids; aromatase inhibitors aminohypnotic (amiglutethimide), formestane (formestane) and anastrozole (anastrozole); progesterone compounds hydroxyprogesterone caproate, medroxyprogesterone; and the antiestrogenic compound tamoxifen.

These and other useful anti-cancer compounds are described in The Merck Index, thirteenth edition (O' NeilMJ. et al eds.) Merck Publishing Group (2001) and Goodman and Gilmans The pharmaceutical Basis of Therapeutics, tenth edition, Hardman, J.G. and Limbird, L.E. eds., 1381. and 1287 pages, McGraw Hill, (1996), both of which are incorporated herein by reference.

Other anti-proliferative compounds that may be used in combination with the stereoisomer-enriched compounds described herein include, for example, but are not limited to, antibodies directed against growth factor receptors (e.g., anti-Her 2); antibodies that activate T cells (e.g., anti-CTLA-4 antibodies); and cytokines such as interferon-alpha and interferon-gamma, interleukin-2 and GM-CSF.

Dosage forms and administration

When used to treat or prevent such diseases, the active compounds and prodrugs can be administered alone, as a mixture of one or more active compounds, or in combination with other agents that treat the diseases and/or symptoms associated with the diseases. The active compounds and prodrugs can also be administered in combination with other dysfunctional or disease agents such as steroids, mixtures or combinations of membrane stabilizers. The active compounds or prodrugs can be administered as such or as pharmaceutical compositions comprising the active compounds or prodrugs.

Pharmaceutical compositions comprising the active compounds (or prodrugs thereof) may be prepared by conventional mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, entrapping or lyophilizing processes. The compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically (see Remington's Pharmaceutical Sciences, fifteenth edition, Hoover, j.e. eds., Mack Publishing Co. (2003)).

The active compound or prodrug may be formulated as a pharmaceutical composition per se, or as a hydrate, solvate, N-oxide or pharmaceutically acceptable salt as hereinbefore described. In general, these salts have a higher solubility in aqueous solution than the corresponding free acids and bases, but salts having a lower solubility than the corresponding free acids and bases may also be formed.

The pharmaceutical compositions may take virtually any form suitable for administration, including, for example, by topical, ocular, oral, buccal, systemic, nasal, injection, transdermal, rectal, vaginal routes, or by administration suitable for inhalation or insufflation.

For topical administration, the active compound(s) or prodrug(s) can be formulated as solutions, gels, ointments, creams, suspensions, and the like, as is well known in the art.

Systemic formulations include those designed for administration by injection, such as subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal buccal or pulmonary administration.

Useful injectable formulations include sterile suspensions, solutions or emulsions of the active compound(s) in aqueous or oily vehicles. The compositions may also contain formulating agents, such as suspending, stabilizing and/or dispersing agents. Injectable formulations may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, and may contain an added preservative.

Alternatively, the injectable dosage forms may be provided in powder form for reconstitution with a suitable vehicle before use, including, but not limited to, sterile pyrogen-free water, buffers, dextrose solutions, and the like. To this end, the active compound(s) may be dried by any technique known in the art, such as lyophilization, and reconstituted prior to use.

For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are well known in the art.

For oral administration, the pharmaceutical compositions may be prepared in conventional manner, e.g., as lozenges, tablets or capsules using pharmaceutically acceptable excipients such as binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or dibasic calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silicon dioxide); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate, lecithin). Tablets may be coated by methods well known in the art, using, for example, sugar, film or enteric coatings.

Liquid preparations for oral administration may take the form of, for example, elixirs, solutions, syrups or suspensions, or may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared in a conventional manner using pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous carriers (e.g. almond oil, oily esters, ethanol, cremophore)^TMOr fractionated vegetable oils); and preservatives (e.g., methyl or propyl parabens or sorbic acid). The formulation may also contain buffer salts, preservatives, flavouring agents, colouring agents and appropriate sweetening agents.

Formulations for oral administration may be suitably configured to provide controlled release of the active compound or a prodrug thereof, as is well known in the art.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For rectal and vaginal administration the active compound(s) may be formulated as solutions (for retention enemas), or as suppositories or ointments containing conventional suppository bases such as cocoa butter or other glycerides.

For intranasal administration or administration by inhalation or insufflation, the active compound(s) or prodrug(s) may be conveniently delivered in the form of an aerosol spray from a compressed pack or nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, fluorocarbons, carbon dioxide or other suitable gas. In the case of a compressed aerosol, the dosage unit may be determined for metered delivery by providing a valve. Capsules and cartridges (e.g., of gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

For intraocular administration, the active compound(s) or prodrug(s) can be formulated as solutions, emulsions, suspensions, and the like, suitable for intraocular administration. A variety of carriers suitable for administering the compounds to the eye are well known in the art. Specific non-limiting ions such as U.S. patent No. 6,261,547; U.S. patent No. 6,197,934; U.S. patent No. 6,056,950; U.S. patent No. 5,800,807; U.S. Pat. nos. 5,776,445; U.S. patent No. 5,698,219; U.S. patent No. 5,521,222; U.S. patent No. 5,403,841; U.S. patent No. 5,077,033; U.S. patent No. 4,882,150; and U.S. patent No. 4,738,851.

For long-term drug delivery, the active compound(s) or prodrug(s) can be formulated in a sustained release (depot) formulation for administration by implantation or intramuscular injection. The active ingredient may be formulated using suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives such as a sparingly soluble salt. Alternatively, transdermal delivery systems made as adhesive discs or patches can be used that slowly release the active drug(s) for transdermal absorption. To this end, penetration enhancers may be used to facilitate transdermal penetration of the active compound(s). Suitable transdermal patches are described, for example, in U.S. patent nos. 5,407,713; U.S. patent No. 5,352,456; U.S. patent No. 5,332,213; U.S. Pat. nos. 5,336,168; U.S. patent No. 5,290,561; U.S. patent No. 5,254,346; U.S. patent No. 5,164,189; U.S. patent No. 5,163,899; U.S. patent No. 5,088,977; U.S. patent No. 5,087,240; U.S. patent No. 5,008,110; and U.S. patent No. 4,921,475.

Alternatively, other drug delivery systems may be employed. Liposomes and emulsions are well known ions of delivery vehicles that can be used to deliver the active compound(s) or prodrug(s). Certain organic solvents such as Dimethylsulfoxide (DMSO) may also be employed, although usually at the expense of higher toxicity.

If desired, the pharmaceutical compositions may be presented in a pack or dispenser device containing one or more unit dosage forms containing the active compound(s). The packaging, for example, may comprise a metal foil or a plastic film, such as a film wrapper. The packaging or dispensing device may be accompanied by instructions for administration.

Effective dose

The active compound(s) or prodrug(s), or combination thereof, is generally used in an amount effective to achieve the desired result, e.g., an amount effective to treat or prevent the particular disease being treated. The compound(s) may be administered therapeutically to achieve a therapeutic benefit. Therapeutic benefit refers to the elimination or amelioration of the underlying disease being treated and/or the elimination or amelioration of one or more symptoms associated with the underlying disease such that the patient reports an improvement in the sensation or condition, even though the patient may still be afflicted with the underlying disease. Therapeutic benefits also include halting or delaying progression of the disease, whether or not improvement is achieved.

The amount of compound administered will depend on a variety of factors including, for example, the particular indication being treated, the mode of administration, the severity of the indication being treated and the age and weight of the patient, the bioavailability of the particular active compound, and the like. Determination of an effective dose is included within the ability of those skilled in the art.

Initially, the effective dose can be estimated from in vitro testing. For example, an initial dose for an animal can be configured to achieve an IC for a particular compound as determined in an in vitro assay₅₀Or above, circulating blood or serum concentrations of the active compound, as described in the examples section. Calculation of the dose to achieve such circulating blood or serum concentrations is within the ability of the skilled artisan to take into account the bioavailability of the particular compound. For guidance, the reader is referred to Fingl&Woodbury, "General Principles," In: goodman and Gilman's The Pharmaceutical Basis of Therapeutics, Chapter I, pages 1-46, latest edition, Pergamon Press, and references cited therein.

Initial doses can also be estimated from in vivo data such as animal models. Animal models for testing the efficacy of a compound to treat or prevent the various diseases described above are well known in the art. The dosage will generally be in the range of about 0.0001 or 0.001 or 0.01 mg/kg/day to about 100 mg/kg/day, but may be higher or lower, depending on, among other factors, the activity of the compound, its bioavailability, mode of administration, and the various factors mentioned above. The dosage and interval may be adjusted individually to provide plasma levels of the compound(s) sufficient to maintain a therapeutic or prophylactic effect. For example, the compounds may be administered once a week, several times a week (e.g., every other day), once a day, or several times a day, depending on, among other factors, the mode of administration, the particular indication being treated, and the judgment of the attending physician. In the case of topical administration and selective uptake, such as localized topical administration, the effective local concentration of the compound(s) is not related to the plasma concentration. The skilled artisan will be able to optimize effective topical dosages without further experimentation.

Preferably, the compound(s) will provide a therapeutic or prophylactic benefit without substantially causing toxicity. Standard pharmaceutical procedures can be employed to determine the toxicity of the compound(s). Quantitative ratio LD between toxic and therapeutic (or prophylactic) effects₅₀/ED₅₀Is the therapeutic index (LD)₅₀Is half the lethal dose, ED₅₀Is half the therapeutically effective amount). Compound(s) exhibiting a high therapeutic index are preferred.

Reagent kit

The compounds and/or prodrugs described herein may be assembled in the form of a kit. In some embodiments, the kit provides the compound(s) and the reagents to prepare the composition for administration. The composition may be in dry or lyophilized form or in solution, especially sterile solution. When the composition is in dry form, the reagents may include pharmaceutically acceptable diluents to prepare a liquid dosage form. The kit may contain a device for administering or dispensing the composition, including but not limited to a syringe, dropper, transdermal patch, or inhaler.

Kits may include other therapeutic compounds for use in combination with the compounds described herein. In some embodiments, the therapeutic agent is an additional anti-cancer or anti-neoplastic compound. These compounds may be provided in individual form or mixed with the compounds of the present invention.

Kits will include appropriate instructions for preparation and administration of the compositions, side effects of the compositions, and other relevant information. These instructions may be in any suitable format, including but not limited to printed matter, videotape, computer-readable magnetic or optical disk.

Examples

The invention is further illustrated by reference to the following examples which describe the preparation of the various compounds described herein, methods for determining their biological activity, and methods of use thereof. Many modifications of the materials and methods of practice will be apparent to those skilled in the art without departing from the scope of the invention.

Preparation of 4- (4-methylpiperazin-1-yl) -3-methylnitrobenzene

Reaction:

the method comprises the following steps: a homogeneous mixture of 4-fluoro-3-methylnitrobenzene 1(20g, 129mmol) and N-methylpiperazine 3(25.82g, 258mmol) in N-methylpyrrolidone (NMP) (10mL) was N₂Reflux (120 ℃ C.) for 24 hours. The reaction mixture was cooled to room temperature and poured into a saturated NaCl solution (100 mL). The resulting solid was sonicated for approximately 30 seconds, filtered, washed with ice-cold water (2 × 10mL), and dried under high vacuum to give 4- (4-methylpiperazin-1-yl) -3-methylnitrobenzene 5(28g, 92%).¹H NMR(CD₃OD)：δ8.02(m，2H)，713(d, 1H, J ═ 9.3Hz), 3.08(m, 4H), 2.66(m, 4H), 2.38(s, 6H); LCMS: purity: 99%, MS (m/e): 236 (MH)⁺)。

Preparation of 4- (4-methylpiperazin-1-yl) -3-methylaniline

Reaction:

the method comprises the following steps: hydrogenation of a heterogeneous mixture of 4- (4-methylpiperazine) -3-methylnitrobenzene 5(20g, 85mmol), 10% Pd/C (1.3g) in methanol (1.2 liters) at 40PSI [ H₂]For 3 hours. The palladium catalyst was filtered through a pad of celite, washing with methanol (3 × 50mL), and the combined filtrates were concentrated to give 4- (4-methylpiperazin-1-yl) -3-methylaniline 7(15g, 86%).¹HNMR(CD₃OD): δ 6.83(d, 1H, J ═ 8.7Hz), 6.59(d, 1H, J ═ 2.7Hz), 6.54(dd, 1H, J ═ 8.4 and 2.7Hz), 2.84(t, 4H, J ═ 4.8Hz), 2.60(bm, 4H), 2.34(s, 3H), 2.20(s, 3H); LCMS: purity: 99.9%, MS (m/e): 206 (MH)⁺)。

Preparation of 3-aza-4-oxo-tricyclo [4.2.1.0(2, 5) ] non-7-ene

Reaction:

racemic, 2-exo-3-exo

The method comprises the following steps: a first part: 2, 5-norbornadiene 47(25.0mL, 0.246mol) in CH₂Cl₂The solution in (110mL, clean bottle) was cooled in an ice/NaCl bath (-10 ℃). Dropping CSI (21.4mL, 0.246mole)/CH at a rate that maintains the temperature below 5 deg.C₂Cl₂(45mL, clean bottle) solution (addition procedure approximately 1.25 hours). After the addition was complete, the reaction mixture was stirred at 0-5 ℃ for 1 hour and then removed from the cooling bath and allowed to warm to 20 ℃. The reaction mixture was cooled with water (60mL) and stirred vigorously for a few minutes. The organic layer was separated, washed with brine and Na₂SO₄And (5) drying. Concentration gave a light brown oil.

A second part: na (Na)₂SO₃(24.5g), water (70mL) and CH₂Cl₂The mixture (30mL) was cooled in an ice/NaCl bath. Using CH for oil from the first part₂Cl₂Diluted to 100mL and added dropwise to the above mixture at a rate that maintains the temperature below 15 ℃ (about 1.75 hours for the addition). The pH of the reaction mixture was monitored by a pH meter and maintained basic (pH7-10) as required with 10% NaOH (w/v). After the addition was complete, the reaction mixture was stirred at 0-5 ℃ for 1 hour (final pH 8.5). The reaction mixture was poured into a separatory funnel and CH was separated₂Cl₂And (3) a layer. The organic phase was a thick colloidal solid suspension. It was diluted with water (approximately 400mL) to prepare a more fluid solution. The aqueous layer is further treated with CH₂Cl₂(4(× 100mL) extraction (alternatively, solids may be separated from CH by centrifugation)₂Cl₂Is separated out. The solid can then be diluted with water (until almost completely dissolved) and treated with CH₂Cl₂Extraction). The aqueous layer is further treated with CH₂Cl₂Extraction (10X 100 mL). Monitoring CH by TLC₂Cl₂Presence of product in the extract. The combined organic extracts were washed with brine, over MgSO₄Dried and filtered through celite. Removal of the solvent afforded the desired product, rac-2-exo-3-endo 3-aza-4-oxo-tricyclo [4.2.1.0(2, 5) as a white solid]Non-7-ene 14r1 (20.5g, 62%).¹HNMR(DMSO-d₆): δ 8.01(bs, 1H), 6.22(dd, J ═ 3.3 and 5.4Hz, 1H), 6.12(dd, J ═ 3.3 and 5.4Hz, 1H), 2.88(dd, J ═ 1.5 and 3.3, IH), 2.79(bs, 1H), 2.74(bs, 1H), 1.58(d, J ═ 9.3Hz, 1H), and 1.47(d, J ═ 9.3Hz, 1H).

Preparation of 4-oxo-3-tert-butoxycarbonylaza-tricyclo [4.2.1.0(2, 5) ] non-7-ene

Reaction:

(rac, 2-rac-3-rac)

The method comprises the following steps: 3-aza-4-oxo-tricyclo [4.2.1.0(2, 5)]Non-7-ene (14r 1; rac-2-exo-3-exo; 10.0g, 74mmol), (BOC)₂O (16.1g, 74mmol) and DMAP (1.1g) in CH₂Cl₂In N₂And stirred at room temperature for 24 hours. To this reaction mixture was added EtOAc (100mL) followed by H₂O (100mL), and stirring was continued for 1 hour. Separating the organic layer and reacting with H₂O (2X 100 mL). Anhydrous Na for organic layer₂SO₄Drying and removal of the solvent under reduced pressure gave 4-oxo-3-tert-butoxycarbonylaza-tricyclo [4.2.1.0(2, 5) ]]Non-7-ene (16r 1; rac-2-rac-3-rac) (16.5g, 70%);¹H NMR(DMSO-d₆): δ 6.29(dd, J ═ 3.3 and 5.4Hz, 1H), 6.19(dd, J ═ 3.3 and 5.4Hz, 1H), 3.77(d, J ═ 4.5Hz, 1H), 3.13(bs, 1H), 3.08-3.04(m, 1H), 2.93(bs, 1H), 1.45(s, 9H). LCMS: 95 percent.

Preparation and isolation of a diastereomer pure in stereoisomeric terms from (±) racemic (2-exo-3-exo) -N4- (3-aminocarbonylbicyclo [2.2.1] hept-5-en-2-yl) -5-fluoro-N2- [ 3-methyl-4- (4-methylpiperazin-1-yl) phenyl ] -2, 4-pyrimidinediamine

From the (2-rac-3-rac) racemate of 2-aminobicyclo [2.2.1] hept-5-ene-3-carboxamide a racemic mixture of the title compound was prepared as follows.

Reaction:

(rac, 2-rac-3-rac)

The method comprises the following steps: racemic N-BOC-. beta. -lactam 16r1(2.0g) was charged under positive pressure nitrogen to a round bottom flask equipped with a rubber septum and a magnetic stir bar. To this was added ethyl acetate (25mL), followed by 30% ammonia/water (25mL), and stirred at room temperature for 3 hours. The ethyl acetate layer was separated and washed with 5% NaHCO₃(20mL) washed with aqueous Na₂SO₄Drying and evaporation of the solvent gave 1.10gm of racemic N-BOC carboxamide 28r 1.

Reaction:

the method comprises the following steps: racemic N-BOC lactam 28r1(2.00g, 7.9mmol) was charged with N₂Round-bottomed flask with inlet and magnetic stir bar, then with 20% TFA/CH₂Cl₂The treatment was carried out at room temperature for 2 hours. The resulting solution was concentrated under reduced pressure. Traces of TFA were removed under high vacuum for several hours to give the intermediate TFA salt (30r1, racemic). The resulting racemic TFA salt, 30r1, was treated with 2, 4-dichloro-5-fluoropyrimidine 10(1.58g, 9.51mm)/MeOH₂O (20:10mL) in NaHCO₃(1.33g, 15.84mmol) at room temperature for 48 hours. Reaction mixture with H₂O (25mL) was diluted, saturated with NaCl, and extracted with EtOAc (3X 50 mL). By using anhydrous Na₂SO₄Drying, evaporating the solvent and subjecting the residue to chromatography (silica gel, CH)₂Cl₂Then 2-4% 2N NH₃/MeOH/CH₂Cl₂) Work-up gave 1.3g of racemic mono-SNAr product 36r 1.

Reaction:

the method comprises the following steps: a sealed tube containing the racemic mono-SNAr product 36r1(1.1g, 8mmol), aniline 7(0.90g, 4.4mmol), TFA (0.6mL), and methanol (9mL) was stirred at 100 ℃ for 24 h. The resulting viscous homogeneous solution is concentrated and the residue is chromatographed (silica gel, CH)₂Cl₂Then 2-5% 2N NH₃/MeOH/CH₂Cl₂) Treatment to give the desired 2-rac-3-racemic 2, 4-pyrimidinediamine derivative 60r1(1.12 g; purity: 95 percent):

separation of enantiomers: diastereoisomers were resolved and separated from the racemate 60r1 by chiral preparative HPLC chromatography Phenomenex Chirex3020 (250X 10mm column), eluting with a 35: 63: 2 (vol: vol) mixture of hexane: dichloromethane: methanol at a flow rate of 6 mL/min. The enantiomer eluting at 9.44 minutes was designated as the E1 enantiomer and the enantiomer eluting at 12.74 minutes was designated as the E2 enantiomer.

Enzymatic preparation of pure stereoisomers of (1R, 2R, 3S, 4S) -N4- (3-aminocarbonylbicyclo [2.2.1] hept-5-en-2-yl) -5-fluoro-N2- [ 3-methyl-4- (4-methylpiperazin-l-yl) phenyl ] -2, 4-pyrimidinediamine using Chirazyme

Preparation of pure stereoisomeric N-Boc-beta-lactams

Reaction:

the method comprises the following steps: a dry sealed tube containing 4-oxo-3-tert-butoxycarbonylaza-tricyclo [4.2.1.0(2, 5) ] non-7-ene (16r 1; rac-2-exo-3-exo) (4.0g, 17.02mmol), conjugate resin/immobilized chiralzyme L-2, type B, c.f. (8.0g, from BioCatalytics Inc., Pasadena, Calif.) and diisopropyl ether (80mL) was gently shaken in an incubator at 60 ℃ for 60 hours. (proton NMR after enzymatic cleavage of racemic N-BOC. beta. -lactam 16r 1. it can be seen that the tert-butyl group of the pure enantiomeric N-BOC lactam 16a and the N-BOC carboxylic acid 26b are bound in a ratio of 1: 1). The resulting reaction mixture was filtered and the solid resin was washed with diisopropyl ether (2X 40 mL). The filtrate was concentrated to give a mixture of pure enantiomers of N-BOC-. beta. -lactam 16a and N-BOC carboxylic acid 26b (total mass: 4.0 gm).

Reaction:

the method comprises the following steps: a mixture of pure enantiomeric N-BOC-lactam 16a and N-BOC carboxylic acid 26b (4.0g) was charged under positive pressure nitrogen into a round bottom flask equipped with a rubber septum and a magnetic stir bar. To this was added ethyl acetate (50mL), followed by 25% aqueous ammonium hydroxide (50mL), and stirred at room temperature for 3 hours. The progress of the reaction was monitored by TLC. The ethyl acetate layer was separated and washed with 5% NaHCO₃Washed with aqueous solution (40mL) and anhydrous Na₂SO₄Drying and evaporation of the solvent gave 2.00gm (7.93mmol, deviation from theory 8.51 mmol; 93% yield) of the desired pure enantiomeric N-BOC carboxamide 28a, with enantiomeric excess of more than 99% as determined by chiral HPLC. The aqueous solution containing N-BOC ammonium hydroxide was acidified with cold 1N HCl and then with CH₂Cl₂The regenerated N-BOC carboxylic acid 26b (1.8g, 7.11mmol, deviation from theory 8.51mmol, 84% yield) was extracted.¹H NMR(DMSO-d₆)：7.26(bs，1H)，6.66(bs，1H)，6.13(m，2H)，3.59(t，1H，J＝6.9Hz)，2.80(s，1H)，2.54(s，1H)，2.31(d，1H，J＝8.1Hz)，2.00(d，1H，J＝8.7Hz)，1.36(s，9H)，1.30(d，1H，J＝8.1Hz)；LCMS：MS(m/z)：254(MH⁺)；[α]D-76.78°(c1.0，MeOH)。

Preparation of pure stereoisomeric Mono-SNAr products

Reaction:

the method comprises the following steps: the pure enantiomer of N-BOC carboxamide 28a (2.00g, 7.93mmol) was charged to the N-complex₂Round-bottomed flask with inlet and magnetic stir bar, then with 20% TFA/CH₂Cl₂The treatment was carried out at room temperature for 2 hours. The progress of the reaction was monitored by TLC. The resulting solution was concentrated under reduced pressure. Removal of traces of TFA under high vacuum for several hours provided a quantitative yield of the pure enantiomeric intermediate TFA salt 30 a.¹H NMR(DMSO-d₆)：8.10(bs，2H)，7.92(s，1H)，7.25(s，1H)，6.29(m，1H)，6.18(m，1H)，4.38(bs，1H)，3.06(d，1H，J＝7.2Hz)，2.97(s，1H)，2.87(s，1H)，2.43(d，1H，J＝7.5Hz)，2.10(d，1H，J＝6Hz)，1.36(d，1H，J＝8.7Hz)；LCMS：MS(m/z)：152(MH⁺)。

The resulting TFA salt 30a was treated with 2, 4-dichloro-5-fluoropyrimidine 34(1.58g, 9.51mmol) in MeOH H₂O (20:10mL) in NaHCO₃(1.33g, 15.84mmol) at room temperature for 48 hours. Reaction mixture with H₂O (25mL) was diluted, saturated with NaCl, and extracted with EtOAc (3X 50 mL). With anhydrous Na₂SO₄Drying, evaporating the solvent and subjecting the residue to chromatography (silica gel, CH)₂Cl₂Then 2-4% 2N NH₃/MeOH/CH₂Cl₂) Work-up gave 2.02g (91%) of the desired mono-SNAr product 36 a.¹H NMR(DMSO-d₆): 8.25(d, 1H, J ═ 7.2Hz), 8.07(d, 1H, J ═ 3.3Hz), 7.71(s, 1H), 7.19(s, 1H), 6.29(m, 2H), 3.99(t, 1H, J ═ 7.8Hz), 2.85(s, 1H), 2.75(s, 1H), 2.49(d, 1H, J ═ 0.9Hz), 2.11(d, 1H, J ═ 8.7Hz), 1.39(d, 1H, J ═ 8.7 Hz); LCMS: purity: 95%, MS (m/z): 283 (MH)⁺). Enantiomeric purity as determined by chiral HPLC was over 99%; [ alpha ] to]D+61.10°(c1.0，MeOH)。

Preparation of pure stereoisomer of (1R, 2R, 3S, 4S) -N4- (3-aminocarbonylbicyclo [2.2.1] hept-5-en-2-yl) -5-fluoro-N2- [ 3-methyl-4- (4-methylpiperazin-1-yl) phenyl ] -2, 4-pyrimidinediamine

Reaction:

the method comprises the following steps: the pure enantiomeric mono-SNAr product 36a (2.25g, 8mmol), aniline 7(1.80g, 8.8mmol), TFA (1.12mL), and isopropanol (18mL) were charged to a flask equipped with a stir bar, reflux condenser, and N₂In an inlet dry reaction flask, the resulting reaction mixture was stirred at reflux temperature for 8-10 hours. After the reaction mixture was cooled to room temperature, ethyl acetate (20mL) was added. The resulting solid was filtered and washed with ethyl acetate (2 × 5mL) to give compound 60a as the acid salt. The resulting solid was then poured into water and washed with NaHCO₃The aqueous solution adjusted the aqueous mixture to a pH of 9, which caused the solid to precipitate. The solid was filtered from the mixture, washed with water and dried to give 3.3g (93%) of the 2, 4-pyrimidinediamine derivative 60 a.¹H NMR(DMSO-d₆): 8.85(s, 1H), 7.83(d, 1H, J ═ 2.7Hz), 7.68(s, 1H), 7.47(s, 2H), 7.36(d, 1H, J ═ 7.8Hz), 7.18(s, 1H), 6.89(d, 1H, J ═ 8.7Hz), 6.32(m, 1H), 6.25(m, 1H), 4.11(t, 1H, J ═ 7.8Hz), 3.32(s, 3H), 2.86(s, 1H), 2.76(m, 4H), 2.49(m, 4H), 2.46(m, 2H), 2.21(s, 3H), 2.11(d, 1H, J ═ 8.4Hz), 1.39(d, 1H, J ═ 9 Hz); LCMS: purity: 100%, MS (m/z): 452 (M)⁺) (ii) a Determination by chiral HPLC>99％ee；[α]_D ^RT+101.2 ° (c1.0, MeOH). Chiral analysis data,¹H NMR and LCMS found the same enantiomer designated E1.

Enzymatic preparation of pure stereoisomer of (1R, 2R, 3S, 4S) -N4- (3-aminocarbonylbicyclo [2.2.1] hept-5-en-2-yl) -5-fluoro-N2- [ 3-methyl-4- (4-methylpiperazin-1-yl) phenyl ] -2, 4-pyrimidinediamine using Novazyme435 enzyme

Preparation of pure stereoisomeric beta-lactams

Reaction:

the method comprises the following steps: to a pressure-resistant flask containing 250ml of diisopropyl ether were added immobilized Lipolase (8.0g, purchased from Sigma, order number L4777), β -lactam 14r1 (rac: 2-rac-3-rac) (4.0g, 7.4mmol) and water (0.13ml, 7.4 mmol). The mixture was degassed with nitrogen for 20 minutes, the flask was sealed and incubated at 70 ℃ for 14 days. The mixture was cooled to room temperature, filtered through celite and washed with 300ml of diisopropyl ether. The combined filtrates were concentrated to dryness and the residue was crystallized from diisopropyl ether to give the pure enantiomeric β -lactam 14a (1.22g, 61%) as colorless needles. Enantiomeric purity by chiral HPLC was over 99%.

Preparation of pure stereoisomer 2-N-Boc-amino-3-aminocarbonyl-bicyclo [2.2.1] hept-5-ene

Reaction:

the method comprises the following steps: the pure enantiomer of 3-aza-4-oxo-tricyclo [4.2.1.0(2, 5) ]]Non-7-ene 14a (1.1g, 8.2mmol), (BOC)₂O (2.76g, 12.3mmol) and DMAP (100mg)/CH₂Cl₂In N₂And stirred at room temperature for 3 hours to give the pure enantiomer of N-BOC lactam 16a, which was used further without isolation. To this reaction mixture was added 20ml of 25% aqueous ammonium hydroxide solution and stirring was continued for an additional 4 hours. Water was added and the reaction mixture was extracted with dichloromethane (2X 50 ml). The combined organic phases were washed with aqueous HCl (5%), dried over sodium sulfate and concentrated to dryness under reduced pressure to give the pure enantiomeric N-BOC carboxamide 28a (2.51g) as a white solid which was used without further treatmentOne step purification was used for the next step.

Preparation of pure stereoisomer of single SNAr product (1R, 2R, 3S, 4S) -N4- (3-aminocarbonylbicyclo [2.2.1] hept-5-en-2-yl) -2-chloro-5-fluoro-4-aminopyridine

Reaction:

the method comprises the following steps: the pure enantiomer of N-BOC carboxamide 28a (2.51g) was dissolved in 10ml of dichloromethane and treated with 10ml of TFA. The mixture was stirred at room temperature for 1 hour and concentrated to dryness under reduced pressure. The residue was suspended in toluene and concentrated again to dryness. The resulting solid was dissolved in methanol: water (30ml:3ml) and treated with 1.5g of sodium bicarbonate. 5-fluoro-2, 4-dichloropyrimidine 34(3g, 17.9mmol) was added, and the mixture was stirred at room temperature for 2 days. Volatiles were removed in vacuo and the residue was suspended in brine. The precipitate was filtered, dried and treated with column chromatography (silica gel, dichloromethane-methanol, 20:1) to afford the desired pure enantiomeric mono-SNAr product 36a (1.7g, 74%) as a white solid.

Preparation of pure stereoisomer of (1R, 2R, 3S, 4S) -N4- (3-aminocarbonylbicyclo [2.2.1] hept-5-en-2-yl) -5-fluoro-N2- [ 3-methyl-4- (4-methylpiperazin-1-yl) phenyl ] -2, 4-pyrimidinediamine

Reaction:

the method comprises the following steps: a homogeneous mixture of aniline 7(400mg, 1.95mmol), the pure enantiomeric mono-SNAr product 36a (400mg, 1.41mmol) and TFA/isopropanol (0.2ml/4ml) in a sealed tube was stirred at 100 ℃ for 20 h. The mixture is cooled to room temperature, diluted with 2ml of diethyl ether, the precipitate obtained is filtered off and taken up with diethyl etherAnd (4) washing with ether. The remaining solid was dissolved in water and treated with 25% aqueous ammonium hydroxide. The resulting precipitate was filtered, washed with water and dried to give 527mg (83%) of the desired product, 2, 4-pyrimidinediamine derivative 60a as an off-white solid. Purity by LCMS was greater than 97% and enantiomeric purity by chiral HPLC was greater than 99%. Chiral analysis data,¹H NMR and LCMS analysis were identical to the enantiomer designated E1.

Preparation of pure stereoisomeric compounds using (R) -methyl-p-methoxybenzylamine as chiral auxiliary

Preparation of 2-exo-3-exo racemic amines

Reaction:

the method comprises the following steps: a homogeneous mixture of 4-oxo-3-tert-butoxycarbonylaza-tricyclo [4.2.1.0(2, 5) ] non-7-ene (16R 1; rac-2-exo-3-exo) (9.2g, 40mmol) and (R) -methyl-4-methoxybenzylamine 13(18, 24g, 48mmol) in anhydrous THF (75mL) was stirred at room temperature for 48 h. The reaction mixture was concentrated, suspended in hexane (5mL), sonicated, and the solid was isolated by filtration to give a mixture of diastereomers 20a and 20b (12 mg). Alternatively, purification can be performed using column chromatography (silica gel, hexanes, then 5%, 10%, 20%, and 50% EtOAc/hexanes).

Preparation of 2-rac-3-rac-mono SNAr product followed by isolation of the pure isomeric compound by crystallization

Reaction:

the method comprises the following steps: diastereoisomers 20a and 20b (6)0g, 17mmol), TFA (20mL) in CH₂Cl₂The homogeneous mixture of (a) was stirred at room temperature for 2 hours. The progress of the reaction was monitored by TLC. The resulting reaction was concentrated to dryness and dried under high vacuum for several hours to give a mixture of diastereomers of intermediates 22a and 22 b. The mixture was then reacted with 2, 4-dichloro-5-fluoropyrimidine 34(3.4g, 20mmol) in NaHCO₃(5.7g，68mmol)/MeOH:H₂O (50mL each) was reacted at room temperature for 24 hours. The reaction mixture was then diluted with water saturated with NaCl (50mL) and with CH₂Cl₂And (4) extracting. Extracting with anhydrous Na₂SO₄Drying, and removing the solvent under reduced pressure to give a residue, which is chromatographed (silica gel, CH)₂Cl₂Then 2% 2N NH₃/MeOH/CH₂Cl₂). Purification by chromatography gave a mixture of diastereomers 38a and 38b (4.0g) (clear separation in the 1:1 ratio was seen on reverse phase LCMS). The resulting 4.0 grams of compound was crystallized from EtOAc: hexane (30:150 mL; v/v) to afford the crystalline material of intermediate 38a, which was confirmed by the X-ray crystal structure; chemical purity: 96% and% de: 96 percent. [ alpha ] to]D-36.7 ° (c, 0.18 MeOH). The mother liquor containing the other isomer had a poor% de (70-80%), which was assumed to be diastereomer 38 b.

Preparation of pure stereoisomeric products comprising chiral auxiliary Agents

Reaction:

the method comprises the following steps: a mixture of diastereomer 38a (1.42g, 3.4mmol), aniline 7(0.834g, 4.0mmol) and TFA (700mg) in MeOH (10mL) was heated in a sealed tube at 100 deg.C for 24 h. The residue obtained is chromatographed (silica gel, CH)₂Cl₂Then 2% 2N NH₃/MeOH/CH₂Cl₂) Work-up to give product 40a as a colourless solid, chemical purity: 96 percent.

Splitting of chiral auxiliary

Cleavage of the chiral auxiliary from 40a has been found to be difficult, and therefore the chiral auxiliary is cleaved from intermediate compounds 38a and 38b, followed by a second SNAr reaction with aniline 7, as follows.

The chiral auxiliary is cleaved from the pure stereoisomer intermediate 38a and the pure stereoisomer of (1R, 2R, 3S, 4S) -N4- (3-aminocarbonylbicyclo [2.2.1] hept-5-en-2-yl) -5-fluoro-N2- [ 3-methyl-4- (4-methylpiperazin-1-yl) phenyl ] -2, 4-pyrimidinediamine is prepared

Reaction:

the method comprises the following steps: is a mono-SNAr product with chiral auxiliary 38a and DDQ (3 equivalents)/CH₂Cl₂:H₂O is reacted at room temperature to yield the desired mono-SNAr product 36 a. Purification of the mono-SNAr product by column chromatography found to be identical to compound 36a obtained via the enzymatic route by chiral analytical HPLC, LCMS and¹h NMR confirmed. Further, the mono-SNAr product 36a was reacted with aniline 7 in a sealed tube in MeOH: TFA at 100 deg.C for 24 hours to afford the desired product 60 a. Purifying by column chromatography, and purifying with¹HNMR, LCMS and chiral analytical HPLC analysis. Chiral analytical HPLC, LCMS and¹h NMR analysis showed that the data for product 60a matched the enantiomer designated E1.

Cleavage of the chiral auxiliary from intermediate 38b and preparation of the pure stereoisomer of (1S, 2S, 3R, 4R) -N4- (3-aminocarbonylbicyclo [2.2.1] hept-5-en-2-yl) -5-fluoro-N2- [ 3-methyl-4- (4-methylpiperazin-1-yl) phenyl ] -2, 4-pyrimidinediamine

Reaction:

the method comprises the following steps: mono-SNAr product 38b with DDQ (3 equivalents)/CH₂Cl₂:H₂The O is reacted at room temperature to give the desired mono-SNAr product 36b (after cleavage of the chiral auxiliary). The mono-SNAr product was purified by column chromatography and found to be a different diastereomer than the compound obtained via the enzymatic route, which could be confirmed by chiral analytical HPLC. Further, the mono-SNAr product 36b was reacted with aniline 7 in a sealed tube in MeOH: TFA at 100 deg.C for 24 hours to afford the desired product 60 b. Purifying by column chromatography, and purifying with¹HNMR, LCMS and chiral analytical HPLC analysis. Chiral analytical HPLC, LCMS and¹h NMR analysis showed that the data for product 60b was the same as the enantiomer designated E2. [ alpha ] to]_D ^RT-102.00°(c，1.0MeOH)。

Preparation of HCl salt

The HCl salt of rac 60r1 and the pure stereoisomer of 60a were prepared as described below.

Preparation of racemic N4- (3-aminocarbonylbicyclo [2.2.1] hept-5-en-2-yl) -5-fluoro-N2- [ 3-methyl-4- (4-methylpiperazin-1-yl) phenyl ] -2, 4-pyrimidinediamine hydrochloride

To 2-exo-3-exo racemic N4- (3-aminocarbonylbicyclo [2.2.1] at 0 DEG C]Hept-5-en-2-yl) -5-fluoro-N2- [ 3-methyl-4- (4-methylpiperazin-1-yl) phenyl]A solution of-2, 4-pyrimidinediamine (60r1) (0.140g, 0.3mmol) in MeOH (3mL) was added HCl (4M, dioxane, 0.170mL, 0.681mmol) dropwise, followed by stirring at 0 ℃ for 1 hour and reaction at room temperature for 15 minutes. The clear homogeneous solution was filtered, concentrated and redissolved in EtOH. Adding ethyl acetate into ethanol solution to precipitate desired product, and separating to obtain 2-rac-3-rac-N4- (3-aminocarbonylbicyclo [ 2.2.1%]Hept-5-en-2-yl) -5-fluoro-N2- [ 3-methyl-4- (4-methylpiperazin-1-yl) phenyl]2, 4-pyrimidinediamine dihydrochloride (Compound 60r1-2 HCl). LCMS: purity: 98 percent; MS (m/e): 453 (MH)⁺)。

Preparation of pure stereoisomer of (1R, 2R, 3S, 4S) -N4- (3-aminocarbonylbicyclo [2.2.1] hept-5-en-2-yl) -5-fluoro-N2- [ 3-methyl-4- (4-methylpiperazin-1-yl) phenyl ] -2, 4-pyrimidinediamine hydrochloride

In a similar manner to that described above, 2 equivalents of HCl (4M, dioxane) were reacted with the pure stereoisomer of (1R, 2R, 3S, 4S) -N4- (3-aminocarbonylbicyclo [2.2.1]Hept-5-en-2-yl) -5-fluoro-N2- [ 3-methyl-4- (4-methylpiperazin-1-yl) phenyl]Interaction between (2, 4-pyrimidinediamine (60a) to give (1R, 2R, 3S, 4S) -N4- (3-aminocarbonylbicyclo [ 2.2.1) pure stereoisomer]Hept-5-en-2-yl) -5-fluoro-N2- [ 3-methyl-4- (4-methylpiperazin-1-yl) phenyl]2, 4-pyrimidinediamine dihydrochloride (compound 60 a.2HCl). LCMS: purity: 97 percent; MS (m/e): 453 (MH)⁺)；[α]_D+46.3°(c，0.04MeOH)。

Preparation of (1R, 2R, 3S, 4S) -N4- (3-aminocarbonylbicyclo [2.2.1] hept-5-en-2-yl) -5-fluoro-N2- [3- (1, 3-oxazol-2-yl) phenyl ] -2, 4-pyrimidinediamine

Preparation of (1R, 2R, 3S, 4S) -N4- (3-aminocarbonylbicyclo [2.2.1] as described above]Hept-5-en-2-yl) -5-fluoro-N2- [3- (1, 3-oxazol-2-yl) phenyl]2, 4-pyrimidinediamine (Compound 90 a).¹HNMR(DMSO-d₆): 9.36(s, 1H), 8.48(s, 1H), 8.14(s, 1H), 9.92(d, 1H, J ═ 3Hz), 7.79(d, 1H, J ═ 7.8Hz), 7.68(s, 1H), 7.42(m, 4H), 7.18(s, 1H), 6.29(m, 1H), 6.13(m, 1H), 4.21(t, 1H, J ═ 4.8Hz), 2.86(s, 1H), 2.77(s, 1H), 2.55(d, 1H, J ═ 8.1Hz), 2.14(d, 1H, J ═ 8.4Hz), 1.39(d, 1H, J ═ 8.7 Hz); LCMS: purity: 98%, MS (m/e): 407 (MH)⁺)。

Inhibition of cell proliferation in vitro

The proliferation inhibitory potency of compounds 60r1, 60r2, 60r 1.2HCl, 60a, 60b and 60 a.HCl on a variety of different types of tumor cells was tested using standard in vitro antiproliferation assay methods. Various cell lines tested included: a549 (lung cancer); ASPC-1 (pancreatic cancer); BXPC-3 (pancreatic cancer); CaOV-3 (ovarian adenocarcinoma); COLO205 (colorectal adenocarcinoma); DU145 (prostate cancer); ES-2 (clear cell carcinoma of the ovary); h1299 (non-small cell lung cancer); h1155 (non-small cell lung cancer); h460 (large cell lung cancer); HELA (cervical adenocarcinoma); HL160 (promyelocytic leukemia); k562 (myeloid chronic myelogenous leukemia); l1210 (mouse lymphocytic leukemia); MiaPaCa-2 (pancreatic cancer); MOLT4(T lymphocyte acute lymphoblastic leukemia); OVCAR-3 (ovarian adenocarcinoma); MOLT3(T lymphocyte acute lymphoblastic leukemia); OVCAR-8 (ovarian cancer); PC3 (prostatic adenocarcinoma); SK-OV-3 (ovarian adenocarcinoma); SU86.86 (pancreatic cancer); SW620 (colorectal adenocarcinoma); THP-1 (monocytic acute monocytic leukemia); TOV-21G (clear cell carcinoma of ovary); u2OS (osteosarcoma); and U937 (histiocytic lymphoma).

The IC's obtained with these compounds are provided in Table 2 below₅₀The value is obtained. In Table 2, "+" indicates IC₅₀The value ≦ 1 μ M, "+" indicates IC₅₀A value of ≦ 20nM, "+ + +" indicates IC₅₀A value of ≦ 10nM, and "-" means IC₅₀Value of>1 μ M. Blanks indicate that the compound was not detected on the indicated cell lines.

Inhibition of Aurora kinase in functional cell assays

Compounds 60a and 60B were tested for their ability to inhibit Aurora kinase-B in a functional cellular assay involving phosphorylation of its substrate histone H3. For measuringSpecifically, a549 cells were seeded in wells of a microtiter plate (5000 cells/well in 100 μ l F12K medium) later in the afternoon of the first day. Cells were grown overnight (37 ℃, 5% CO)₂). The following day, 50. mu.l nocodazole (nocodazole) (1. mu.M in medium) was added to each well to give a final concentration of 333 nM. Under the same conditions, the cells were grown for an additional 18 hours.

On day three, 50 μ l aliquots of test compounds at different concentrations were added to the wells. Test compounds were prepared by serial 2-fold dilution with 2mM stock solution (in DMSO). Then, the diluted compounds in DMSO were further diluted 1:50 with medium to give a final solution containing 4 × test compound, 98% medium, 2% DMSO. After incubation, the medium/test compound was washed and the cells were fixed with 2% p-formaldehyde (in Dulbecco's phosphate buffered saline "DPBS"; 25. mu.l per well; incubation >20 mm). The fixed cells were washed once with DPBS (200. mu.l/well), stained with phospho-histone H3 (Cell Signaling Technology; 1:500, 10% normal sheep serum "NGS" in DPBS, 0.05% Triton X-100; room temperature 1-2 hours), and washed twice with DPBS (200. mu.l/well). Then, the cells were stained with fluorescent dye-labeled secondary antibodies (secondary antibodies donkey anti-murine AlexFluor488(Invitrogen molecular probes; 1:2000) and DAPI (1:15,0001mg/ml stock) for 1 hour at room temperature, washed three times with DPBS (200. mu.l/well), and stored in DPBS (100. mu.l/well) at 4 ℃ for assay use.

A Zeiss Axiovert S100 inverted fluorescence microscope with a Plan-NEOFLUAR10 Xobjective, Hamamatsu lightning 200 mercury-xenon illuminant, and Omega Optical XF57 quad filter (quad filter) was used for all data collection. The system was equipped with a Ludl Mac2000 motorized stage with X/Y/Z controls, a Ludl Filter wheel, a Zymark Twister robot, and a Quantix digital camera from Roper Scientific. All hardware was controlled on a PC running Win2000 with ImagePro4.5 with ScopePro/StagePro4.1 module (Media Cybernetics). Visual Basic script (Visual Basic script) is written for ImagePro for automatic hardware control and image acquisition. The focusing was performed using autofocus software containing stageprol which determined the maximum plane of focus from the Z series captured once per well using the maximum local contrast. Once proper focusing is completed, the image is captured within the 3 x3 grid lines of the next adjacent image, but not including the location of the focus. Images were captured and analyzed in a 12-bit format using segmentation and morphology programs contained in the Image Pro software package. The identified nuclei were counted and the pixel data for each cell and assay conditions were stored in a database using mysql 4.0.14. Analysis of the test results and tracing of the graphs was then done using matlab6.5.

For phospho-histone H3 analysis, the data were converted to a Facs file and analyzed using FlowJo. The concentration of each compound was plotted against the percentage of phospho-H3 cells to determine the EC50 for Aurora B inhibition.

As a result, in this assay, Compound 60a inhibits the IC of Aurora kinase-B₅₀Is about 7 nM. In contrast, IC of its enantiomeric compound 60b₅₀2.49 μ M, approximately 350 times higher.

Pharmacokinetics of compound E1 in monkeys

Compound 60a was administered to monkeys both intravenously (1 mg/kg in saline) and orally (5 mg/kg in saline) and plasma concentrations were monitored over time. When administered intravenously, the plasma concentrations of the compounds were still higher than an IC of 7nM at 11 hours after administration₅₀(ii) a When administered orally, the plasma concentrations of the compounds were still above IC over 20 hours₅₀。

Compound 60a tumor reduction in vivo

Compound 60 a.2hcl was tested to test its ability to reduce a549 and Colo205 tumors in a standard xenograft therapy model in SCID mice, and to reduce Colo205 and MiaPaCa tumors in a standard xenograft regression model in SCID mice. When present, is accessible and has a preselected volume (the treatment model is about 100 mm)³(ii) a Regression model>300mm³) Is administered to the mouse an amount of the test compound, andaccording to the dosing regimen specified in table 3 below (treatment regimen) and table 4 (regression regimen).

As a result: the inhibitory effect of compound 60 a.2hci on Colo205 tumor growth in the treatment model is shown in fig. 1 and 2. The results of the daily dosing regimen are shown in figure 1; the results of the pulsatile dosing regimen are shown in figure 2. For all dose levels tested, both regimens significantly reduced the tumor growth rate compared to the vehicle control group (p < 0.050). 549 tumors responded poorly to treatment, with an average tumor volume approximately 40% reduction (p >0.05) at a dosing schedule of 5 days dosing/2 days off and a dose level of 10mg/kg four times a day.

The inhibitory effect of compound 60 a.2hci on Colo205 tumor growth in the regression model is shown in figure 3. The effect of 60 a.2hci on MiaPaCa tumors in a regressive model is shown in figure 4. For both tumor lines, a significant reduction in tumor growth rate was observed. These reductions are independent of the mode of administration. Moreover, the decrease observed in MiaPaCa tumors was similar to that observed for paclitaxel (see fig. 4).

Although the foregoing invention has been described in some detail for purposes of clarity of understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, and the invention is not to be limited to the details given herein, but may be modified within the scope and equivalents of the appended claims.

All documents and patent documents cited in this application are incorporated by reference into this application for all purposes.

Claims (54)

1. A compound according to structural formula (I):

or a salt thereof, which is enriched in the corresponding diastereomer of structural formula (Ia):

wherein:

R¹selected from hydrogen, C1-C6 hydrocarbyl radicals, - (CH)₂)_n-OH、-OR^a、-O(CH₂)_n-R^a、-O(CH₂)_n-R^b、-C(O)OR^aHalogen, -CF₃and-OCF₃；

Each R²Are each independently selected from hydrogen, C1-C6 hydrocarbyl, -OR^a、-O(CH₂)_n-R^a、-O(CH₂)_n-R^b、-NHC(O)R^aHalogen, -CF₃、-OCF₃、

Each R³Are each independently selected from hydrogen, C1-C6 hydrocarbyl, - (CH)₂)_n-OH、-OR^a、-O(CH₂)_n-R^a、-O(CH₂)_n-R^bHalogen, -CF₃、-OCF₃、

Each R⁴Are each independently selected from hydrogen, C1-C6 hydrocarbyl, C5-C15 arylalkyl, -OR^a、-NR^cR^c、-C(O)R^a、-C(O)OR^aand-C (O) NR^cR^c；

R⁵Is hydrogen, halogen, fluorine, -CN, -NO₂、CO₂R^aor-CF₃；

Each n is independently an integer from 1 to 3;

each R^aAre each independently selected from hydrogen, C1-C6 hydrocarbyl;

each R^bAre all independently selected from-OR^a、-CF₃、-OCF₃、-NR^cR^c、-C(O)R^a、-C(O)OR^a、-C(O)NR^cR^cand-C (O) NR^aR^d；

Each R^cAre each independently selected from hydrogen and C1-C6 hydrocarbyl, or two R^cThe substituents may be taken together with the nitrogen atom to which they are attached to form a 5-7 membered saturated ring, optionally including 1-2 additional substituents selected from O, NR^a、NR^a-C(O)R^a、NR^a-C(O)OR^aAnd NR^a-C(O)NR^aA heteroatom group of (a); and is

Each R^dAre independently C1-C6 monohydroxy hydrocarbyl or C1-C6 dihydroxy hydrocarbyl.

2. The compound of claim 1, comprising only the (2-exo-3-exo) stereoisomer.

3. The compound of claim 1, which contains 60% or more of the diastereomer of structural formula (Ia).

4. The compound of claim 1, which contains 90% or more of the diastereomer of structural formula (Ia).

5. The compound of claim 1, which contains 99% or more of the diastereomer of structural formula (Ia).

6. The compound of claim 1, wherein R⁵Is fluorine.

7. The compound of claim 6, wherein R¹Is hydrogen; r²Is thatR³Is not provided with

8. The compound of claim 7, wherein R³Is hydrogen, methyl, methoxy, trifluoromethyl or chlorine.

9. The compound of claim 7, wherein R⁴Is methyl, -C (O) CH₃、-C(O)OCH₃or-C (O) OCH₂CH₃。

10. The compound of claim 6, wherein R¹Is hydrogen; r²Is not provided withR³Is that

11. The compound of claim 10, wherein R²Is hydrogen, methyl, methoxy, trifluoromethyl or chlorine.

12. The compound of claim 10, wherein R⁴Is methyl, -C (O) CH₃、-C(O)OCH₃or-C (O) CH₂CH₃。

13. The compound of claim 6, wherein R²Is not provided withR³Is not provided with

14. The compound of claim 13, wherein R¹And R²Each is hydrogen, R³is-OCH₂NHR^a。

15. The compound of claim 13, wherein R¹、R²And R³Each independently selected from hydrogen, methyl, methoxy, trifluoromethyl and chloro, with the proviso that R is¹、R²And R³At least two of which are not hydrogen.

16. The compound of claim 6, wherein R¹Is hydrogen; r²Selected from hydrogen,R³Selected from hydrogen, C1-C6 hydrocarbyl, halogen, -CF₃、

17. The compound of claim 16, wherein R³Selected from hydrogen, methyl, chloro, -CF₃、R⁴Is methyl, -COR^aor-CO (O) R^aWherein R is^aIs methyl or ethyl.

18. The compound of claim 16, wherein R²Selected from hydrogen,R³Selected from hydrogen, C1-C6 hydrocarbyl, halogen, -CF₃、

19. The compound of claim 18, wherein R³Selected from hydrogen, methyl, chloro, -CF₃、R⁴Is methyl, -COR^aor-CO (O) R^aWherein R is^aIs methyl or ethyl.

20. The compound of claim 19, wherein R²Is thatR⁴is-COR^aWherein R is^aIs methyl; r³Is hydrogen.

21. The compound of claim 19, wherein R²Is thatR⁴is-CO (O) R^aWherein R is^aIs an ethyl group; r³Is hydrogen.

22. The compound of claim 19, wherein R²Is thatR³Is hydrogen.

23. The compound of claim 19, wherein R²Is hydrogen; r³Is thatOrR⁴Is methyl, -COR^aor-CO (O) R^aWherein R is^aIs methyl or ethyl.

24. The compound of claim 19, wherein R²Is thatR⁴Is methyl; r³Selected from hydrogen, methyl, chlorine and-CF₃。

25. A compound which is N4- (3-aminocarbonylbicyclo [2.2.1] hept-5-en-2-yl) -5-fluoro-N2- [ 3-methyl-4- (4-methylpiperazin-1-yl) phenyl-2, 4-pyrimidinediamine, wherein the compound is enriched in the (1R, 2R, 3S, 4S) diastereoisomer.

26. The compound of claim 25, containing 95% or more of the (1R, 2R, 3S, 4S) diastereomer.

27. The compound of claim 25 or 26, which is in the form of a pharmaceutically acceptable salt.

28. The compound of claim 27, which is a salt of an organic acid selected from the group consisting of: acetic acid, trifluoroacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, oxalic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, palmitic acid, benzoic acid, 3- (4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1, 2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo [2.2.2] -oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, and muconic acid.

29. The compound of claim 28, which is a benzoate salt.

30. A pharmaceutical composition comprising a compound according to any one of claims 1, 25, 26, 27, 28 or 29 and a carrier and/or excipient.

31. The pharmaceutical composition of claim 30, wherein the carrier and/or excipient is pharmaceutically acceptable.

32. A composition for inhibiting cell proliferation comprising a compound of any one of claims 1-29.

33. The composition of claim 32, wherein the cell is a tumor cell.

34. The composition of claim 33, wherein said tumor cell is a lung, colon, breast, stomach, ovarian, cervical, melanoma, kidney, prostate, leukemia, lymphoma, neuroblastoma, pancreatic, bladder or liver tumor cell.

35. A method of inhibiting Aurora kinase activity in vitro, comprising contacting Aurora kinase with an amount of a compound according to any one of claims 1-29.

36. The method of claim 35 which is carried out in vitro, wherein the Aurora kinase is isolated or partially isolated.

37. The method of claim 35, which is performed in vitro using cells expressing Aurora kinase.

38. A method of inhibiting an Aurora kinase-mediated process comprising contacting a cell expressing an Aurora kinase with the compound of claim 1 in vitro in an amount effective to inhibit an Aurora kinase-mediated process.

39. The method of claim 38, wherein the inhibited Aurora kinase-mediated process is mitosis.

40. The method of claim 38, wherein said cell is a tumor cell.

41. The method of claim 38, wherein the cell is contacted with a compound at a concentration equal to or greater than its IC as determined in an in vitro assay₅₀。

42. A composition for the treatment of an Aurora kinase-mediated disease comprising a compound according to any one of claims 1-28.

43. The composition according to claim 42, wherein the Aurora kinase-mediated disease is a proliferative disease.

44. The composition of claim 43, wherein said proliferative disease is cancer.

45. The composition of claim 44, wherein the cancer is a metastatic tumor.

46. The composition of claim 42, for oral administration.

47. The composition of claim 42, for intravenous administration.

48. The composition of any one of claims 30-34 and 42-47, wherein said compound is (1R, 2R, 3S, 4S) -N4- (3-aminocarbonylbicyclo [2.2.1] hept-5-en-2-yl) -5-fluoro-N2- [ 3-methyl-4- (4-methylpiperazin-1-yl) phenyl-2, 4-pyrimidinediamine.

49. The use of a compound according to any one of claims 1 to 29 in the manufacture of a medicament for the inhibition of Aurora kinase mediated diseases.

50. The use according to claim 49 wherein the Aurora kinase-mediated disease is a proliferative disease.

51. The use according to claim 50, wherein the proliferative disease is cancer.

52. The use according to claim 51, wherein the cancer is a metastatic tumor.

53. The use according to claim 51, wherein the cancer is selected from the group consisting of lung cancer, breast cancer, stomach cancer, ovarian cancer, cervical cancer, melanoma, renal cancer, prostate cancer, leukemia, lymphoma, neuroblastoma, pancreatic cancer, bladder cancer, and liver cancer.

54. The use according to any one of claims 49 to 53, wherein the compound is (1R, 2R, 3S, 4S) -N4- (3-aminocarbonylbicyclo [2.2.1] hept-5-en-2-yl) -5-fluoro-N2- [ 3-methyl-4- (4-methylpiperazin-1-yl) phenyl-2, 4-pyrimidinediamine.