Title of Invention

STEREOISOMERICALLY ENRICHED 3-AMINOCARBONYL BICYCLOHEPTENE PYRIMIDINEDIAMINE COMPOUNDS AND THEIR USES

Abstract The present invention provides stereoisomers and stereoisomeric mixtures of 3-aminocarbonyl-bicycloheptene-2,4-pyrimidinediamine compounds having antiprollferative activity, compositions comprising the compounds and methods of using the compounds to inhibit cellular proliferation and to treat proliferate diseases such as tumorigenic cancers.
Full Text STEREOISOMERICALLY ENRICHED 3-AMBNfOCARBONYL BICYCLOHEPTENE
PY R1MIIHNEDIAMINE COMPOUNDS AND THEIR USES
1. CROSS-REFERENCE TO RELATED APPLICATION
This application claims benefit under 35 U.S.C. § 119(e) to application Serial No.
60/628,199 filed November 15,2004, the contents of which are incorporated herein by reference.
2. FIELD
The present disclosure relates to stereoisomerically enriched compositions of 4N-(3-
aminocarbony lbicyclo[2.2.1 ]hept-5-en-2-y l)-N2~substituted pheny I-2,4-pyrimidinediamine
compounds that exhibit antiproliferative activity, prodrugs of the compounds, intermediates and
methods of synthesis for making the compounds and/or prodrugs, pharmaceutical compositions
comprising the compounds and/or prodrugs and the use of the compounds and/or prodrugs in a
variety of contexts, including, for example, the treatment of proliferative disorders, such as
tumors and cancers.
3. BACKGROUND
Cancer is a group of varied diseases characterized by uncontrolled growth and spread of
abnormal cells. Generally, all types of cancers involve some abnormality in the control of cell
growth and division. The pathways regulating cell division and/or cellular communication
become altered in cancer cells such mat the effects of these regulatory mechanisms hi controlling
and limiting cell growth fails or is bypassed. Through successive rounds of mutation and natural
selection, a group of abnormal celb, generally originating from a single mutant cell, accumulates
additional mutations that provide selective growth advantage over other cells, and thus evolves
into a cell type that predominates in the cell mass. This process of mutation and natural selection
is enhanced by genetic instability displayed by many types of cancer cells, an instability which is
gained either from somatic mutations or by inheritance from the germ line. The enhanced
mutability of cancerous cells increases the probability of their progression towards formation of
malignant cells. As the cancer cells further evolve, some become locally invasive and then
mestasize to colonize tissues other than the cancer cell's tissue of origin. This property along
with the heterogeneity of the tumor cell population makes cancer a particularly difficult disease to
treat and eradicate.
Traditional cancer treatments take advantage of the higher proliferative capacity of cancer
cells and their increased sensitivity to DNA damage. Ionizing radiation, including y-rays and
x-rays, and cytotoxic agents, such as bleomycin, cis-platin, vinblastine, cyclophosphamide,
5'-fluorouracil, and methotrexate rely upon a generalized damage to DNA and destabilization of
chromosomal structure which eventually lead to destruction of cancer cells. These treatments are
particularly effective for those types of cancers that have defects in cell cycle checkpoint, which
limits the ability of these cells to repair damaged DNA before undergoing cell division. The
non-selective nature of these treatments, however, often results in severe and debilitating side
effects. The systemic use of these drugs may result in damage to normally healthy organs and
tissues, and compromise the long-term health of the patient.
Although more selective chemotherapeutic treatments have been developed based on
knowledge of how cancer cells develop, for example, the anti-estrogen compound tamoxifen, the
effectiveness of all chemotherapeutic treatments are subject to development of resistance to the
drugs. In particular, the increased expression of cell membrane bound transporters, such as Mdrl,
produces a multidrug resistance phenotype characterized by increased efflux of drugs from the
cell. These types of adaptation by cancer cells severely limit the effectiveness of certain classes
of chemotherapeutic agents. Consequently, identification of other chemotherapeutic agents,
particularly active stereoisomers and/or stereoisomeric mixtures is critical for establishing
therapies effective for attacking the heterogeneous nature of proliferative disease and for
overcoming any resistance that may develop over the course of therapy with other compounds.
Moreover, use of combinations of chemotherapeutic agents, including different stereoisomers
and/or stereoisomeric mixtures of a particular chemotherapeutic agent, which may have differing
properties and cellular targets, increases the effectiveness of chemotherapy and limits the
generation of drug resistance.
4. SUMMARY
In one aspect, 4N-(3-ammocarbonylbicyclo[2.2.1]hept-5-en-2-yl)-2N-substituted phenyl-
2,4-pyrimidinediamine compounds enriched in specified diastereomers are provided mat 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, that are enriched in the
corresponding diastereomer of structural formula (la), designated the (1R,2R,3S,4S)
diastereomer
(Figure Removed)
wherein:
each R1 is independently selected from the group consisting of hydrogen, lower
alkyl, -(CH2),,-OH, -OR1, -O(CHa)B-R2, -O(CH2VRb, -C(OPRa, halo, -CF3 and -OCF3;
each R1 is independently selected from the group consisting of hydrogen, lower
5 -I-N 0
aikyl, -OR*, -O(CH2)n-R", -CKCH2VRb, -NHC(O)R', halo, -CF3, -OCF3, \ — ' and
(Figure Removed)
each R3 is independently selected from the group consisting of hydrogen, lower
s /~A
-f-N 0
alkyl, -(CH2)B-OH,-OR',-0(CH2VRa,-0(CH2VRb, halo, -CF3,-OCF3,
(Figure Removed)
each R4 is independently selected from the group consisting of hydrogen, lower
alkyl, arylalkyl, -OR*, -NRX -C(O)Ra, -C(O)OR and -C(O)NR°RC;
R5 is hydrogen, halo, fluoro, -CN, -NOz, -C(O)OR" or -CF3-
each n is independently an integer from 1 to 3;
each R* is independently selected from the group consisting of hydrogen, lower
alkyl and lower cycioalkyl;
each Rb is independently selected from the group consisting of -ORa, -Cf^,
-OCF3s -NRTl1; -C(0)R*, -OPPRa. -QOJNR'R6 and -C(O)NRtR(S;
each Rc is independently selected from the group consisting of hydrogen and
lower alkyl, or, alternatively, two Rc substituents may be taken together with the nitrogen atom to
which they are bonded to form a 4-9 membered saturated ring which optionally includes 1-2
additional heteroatomic groups selected from O, NR", NRB-C(O)R, NRa-C(OXRa and
NR-C(O)NRa;and
each Rd is independently lower monohydroxyalkyl or lower di-hydroxyalkyl.
In some embodiments, the compound of structural formula (I) is a racemic mixture of (2-
exo-3-exo) cis isomers according to structural formula (Ha):
including prodrugs, salts, hydrates, solvates and N-oxides thereof, wherein R1, R2, R3 and
R5 are as defined for structural formula (I), supra,
In some embodiments, the compound is a stereoisomerically enriched diastereomer
according to structural formula (la), supra, including prodrugs, salts, hydrates, solvates and Noxides
thereof, that is substantially free of its enantiomer and any other diastereomer thereof.
In still another aspect, prodrugs of the stereoisomerically enriched compounds are
provided. Such prodrugs may be active in their prodrug form, or may be inactive until converted
under physiological or other conditions of use to an active drug form. In the prodrugs, one or
more functional groups of the stereoisomerically enriched compounds are included in promoieties
that cleave from the molecule under the conditions of use, typically by way of hydrolysis,
enzymatic cleavage or some other cleavage mechanism, to yield the functional groups. For
example, primary or secondary amino groups may be included in an amide promoiety that cleaves
under conditions of use to generate the primary or secondary amino group. Thus, the prodrugs
include special types of protecting groups, termed "progroups," masking one or more functional
groups of the compounds that cleave under the conditions of use to yield an active drug
compound. Functional groups within the stereoisomerically enriched compounds that may be
masked with progroups for inclusion in a promoiety include, but are not limited to, amines
(primary and secondary), hydroxyls, sulfanyls (thiols), carboxyls, carbonyls, etc. Myriad
progroups suitable for masking such functional groups to yield promoieties that are cleavable
under the desired conditions of use are known in the art. All of these progroups, alone or in
combination, may be included in the prodrugs. Specific examples of promoieties that yield
primary or secondary amine groups mat can be included in the prodrugs include, but are not
limited to amides, carbatnates, imines, ureas, phosphenyls, phosphoryls and sulfenyls. Specific
examples of promoieties that yield sulfanyl groups mat can be included in the prodrugs include,
but are not limited to, thioethers, for example S-methyl derivatives (monothio, dithio, oxythio,
aminothio acetals), silyl thioethers, thioesters, thiocarbonates, thiocarbamates, asymmetrical
disulfides, etc. Specific examples of promoieties that cleave to yield hydroxyl groups that can be
included in the prodrugs include, but are not limited to, sulfonates, esters and carbonates.
Specific examples of promoieties that yield carboxyl groups that can be included in the prodrugs
include, but are not limited to, esters (including silyl esters, oxainic acid esters and thioesters),
amides and hydrazides.
In still another aspect, compositions comprising one or more stereoisomerically enriched
compounds are provided. The compositions generally comprise the compound(s), and/or
prodrugs, salts, hydrates, solvates and/or N-oxides thereof, and an appropriate carrier, excipient
and/or diluent. The exact nature of the carrier, excipient and/or diluent will depend upon the
desired use for the composition, and may range from being suitable or acceptable for /« vitro uses,
to being suitable or acceptable for veterinary uses, to being suitable or acceptable for use in
humans.
The stereoisomerically enriched compounds described herein are potent inhibitors of
proliferation abnormal cells, such as tumor cells, in in vitro assays. Thus, in still another aspect,
methods of inhibiting proliferation of abnormal cells, and in particular tumor cells, are provided.
The methods generally involve contacting an abnormal cell, such as a tumor cell, with an amount
of one or more stereoisomerically enriched compounds described herein, and/or prodrugs, salts,
hydrates, solvates and/or N-oxides thereof, effective to inhibit proliferation of the cell. The cells
can be contacted with the compound per se, or the compound can be formulated into a
composition. The methods may be practiced in in vitro contexts, or in in vivo contexts as a
therapeutic approach towards the treatment or prevention of prolifeiative disorders, such as
tumorigenic cancers.
In still another aspect, methods of treating proliferative disorders are provided. The
methods may be practiced in annuals in veterinary contexts or in humans. The methods generally
involve administering to an animal or human subject an amount of one or more stereoisomerically
enriched compounds described herein, and/or prodrugs, sans, hydrates, solvates and/or N-oxides
thereof, effective to treat or prevent the proliferative disorder. The compound(s) per se can be
administered to the subject, or the compound(s) can be administered in the form of a composition.
Proliferative disorders that can be treated according to the methods include, but are not limited to,
tumorigenic cancers.
The stereoisomerically enriched compounds described herein are potent inhibitors of
Aurora kinases. Aurora kinases are a family of enzymes known to be key regulators of cell
division. Elevated levels of Aurora kinases have been found in several types of human cancer
cells, such as breast, colon, renal, cervical, neuroblastomer, melanoma, lymphoma, pancreatic,
prostate and other solid tumors (see, e.g., Bischott et al., 1998, EMBO J. 17:3052-3065; Geopfert
& Briokley, 2000, Curr. Top. Dev. Biol. 49:331-342; Sakakura et aL, 2001, Br. J. Cancer 84:824-
831), and ova-expression of Aurora kinases has been shown to result in cell transformation, a
process by which normal cells become cancers. Although not intending to be bound by any
particular theory of operation, it is believed that the stereoisomerically enriched compounds
described herein, as well as the active prodrugs, salts, hydrates, solvates and/or N-oxides thereof,
exert their antiproliferative activity by inhibiting one or more Aurora kinases.
Thus, in yet another aspect, methods of inhibiting an activity of an Aurora kinase are
provided. Hie methods generally involve contacting an Aurora kinase with an amount of one or
more stereoisomerically enriched compounds described herein, and/or active prodrugs, salts,
hydrates, solvates and/or N-oxides thereof, effective to inhibit its activity. The methods can be
practiced in in vitro contexts with purified or partially purified Aurora kinase enzymes (e.g., with
extracts of cells expressing an Aurora kinase), in in vitro contexts with intact cells expressing an
Aurora kinase, or in in vivo contexts to inhibit an Aurora kinase-mediated process (for example
cellular mitotis) and/or as a therapeutic approach towards the treatment or prevention of diseases
or disorders that are mediated, at least in part, by Aurora kinase activity.
hi still another aspect, methods of treating or preventing Aurora kinase-mediated diseases
or disorders are provided. The methods generally involve administering to an animal or human
subject an amount of one or more stereoisomerically enriched compounds described herein,
and/or active prodrugs, salts, hydrates, solvates and/or N-oxides thereof, effective to treat or
prevent the Aurora kinase-mediated disease or disorder. Aurora kinase-mediated diseases and
disorders include any disease, disorder, or other deletarions condition in which a member of the
Aurora kinase family of enzymes plays a role. Specific examples of such Aurora kinase-mediated
diseases or disorders include, but are not limited to, melanoma, leukemia, and solid tumor
cancers, such as, for example, colon, breast, gastric, ovarian, cervical, melanoma, renal, prostate,
lymphoma, neuroblastoma, pancreatic and bladder cancers.
Other aspects include, but are not limited to, intermediates and methods useful for
synthesizing the stereoisomerically enriched compounds and prodrugs, as will be described in
more detail herein below.
5. BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1-4 illustrate the inhibitory effect of (lR,2R,3S,4S)N4-(3-
aminocarbonylbicyclo[2.2.1]hept-5-ene-2-yl)-5-fluoro-N2-[3-me1iiyl-4-(4-methylpipera2an-lyl)
phenyl]-2,4-pyrimidinediamine bis hydogen chloride salt (compound 60a-2HCl) on the growth
ox various different types of tumors in standard xenograft treatment and regression models.
6. DETAILED DESCRIPTION
6.1 Definitions
As used herein, the following terms are intended to have the following meanings:
"AJkyj" by itself or as part of another substituent refers to a saturated or unsaturated
branched, straight-chain or cyclic monovalent hydrocarbon radical having the stated number of
carbon atoms (i.e., CI-C6 means one to six carbon atoms) that is derived by the removal of one
hydrogen atom from a single carbon atom of a parent alkane, alkene or alkyne. Typical alkyl
groups include, but are not limited to, methyl; ethyls such as ethanyl, ethenyl, ethyny 1; propyls
such as propan-1-yl, propan-2-yl, cyclopropan-1-yl, prop-1-en-l-yl, prop-l-en-2-yl,
prop-2-en-l-yl, cycloprop-1-en-l-yl; cycloprop-2-en-l-yl, prop-1-yn-l-yl, prop-2-yn-l-yl, etc.;
butyls such as butan-1-yl, butan-2-yl, 2-methyl-propan-l-yl, 2-methyl-propan-2-yl>
cyclobutan-l-yl, but-1-en-l-yi, but-l-en-2-yl, 2-methyl-prop-l-en-l-yI, but-2-en-l-yl,
but-2-en-2-yl, buta-l,3-dien-l-yl, buta-l,3-dien-2-yl, cyclobut-l-en-l-yl, cyclobut-l-en-3-yl,
cyclobuta-l,3-dien-l-yl, but-1-yn-l-yl, but-l-yn-3-yl, but-3-yn-l-yl, etc.; and the like. Where
specific levels of saturation are intended, the nomenclature "alkanyl," "alkenyF and/or "alkynyl"
is used, as defined below. "Lower alky!" refers to an alkyl group containing from I to 6 carbon
atoms.
"AlkanyJ" by itself or as part of another substituent refers to a saturated branched,
straight-chain or cyclic alkyl derived by the removal of one hydrogen atom from a single carbon
atom of a parent alkane. Typical alkanyl groups include, but are not limited to, methanyl;
ethanyl; propanyls such as propan-1-yl, propan-2-yl (isopropyl), cyclopropan-l-yl, etc.; butanyls
such as butan-1-yl, butan-2-yl (sec-butyl), 2-methyl-propan-l-yl (isobutyl), 2-methyl-propan-2-yl
(t-butyl), cyclobutan-l-yl, etc.; and the like.
"Alkeavl" by itself or as part of another substituent refers to an unsaturated branched,
straight-chain or cyclic alkyl 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 group may be
in either the cis or trans conformation about the double bond(s). Typical alkenyl groups include,
but are not limited to, ethenyl; propenyls such as prop-1-en-l-yl, prop-l-en-2-yl, prop-2-en-l-yl,
prop-2-en-2-yl, cycloprop-1-en-l-yl; cycloprop-2-en-l-yl; butenyls such as but-1-en-l-yl,
but-l-en-2-yl, 2-methyl-prop-l-en-l-yl, but-2-en-l-yl, but-2-en-2-yl, buta-l,3-dien-l-yl,
buta-l,3-dien-2-yl, cyclobut-l-en-l-yl, cyclobut-l-en-3-yl, cyclobuta-13-dien-l-yl, etc.; and the
like.
"Alkynyl" by itself or as part of another substituent refers to an unsaturated branched,
straight-chain or cyclic aikyl 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; propynyls such as prop-1-yn-l-yl, prop-2-yn-l-yl, etc.;
butynyls such as but-1-yn-l-yl, but-l-yn-3-yl, but-3-yn-l-yl, etc.; and the like.
"Alkyldiyl" by itself or as part of another substituent refers to a saturated or unsaturated,
branched, straight-chain or cyclic divalent hydrocarbon group having the stated number of carbon
atoms (i.e., C1-C6 means from one to six carbon atoms) derived 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.
The two monovalent radical centers or each valency of the divalent radical center can form bonds
with the same or different atoms. Typical alkyldiyl groups include, but are not limited to,
methandiyl; ethyldiyls such as ethan-l,l-diyl, ethan-l,2-diyl, ethen-l,l-diyl, ethen-l,2-diyl;
propyldiyls such as propan-l,l-diyl, propan-l,2-diyl, propan-2,2-diyl, propan-l,3-diyl,
cyclopropan-l,l-diyl, cyclopropan-l,2-diyl, prop-l-en-l,l-diyl, prop-l-en-l,2-diyl,
prop-2-en-l,2-diyl., prop-l-en-l,3-diyl, cycloprop-l-en-l,2-diyl, cycloprop-2-en-l,2-diyl,
cycIoprop-2-en-l,l-diyl, prop-l-yn-l,3-diyl, etc.; butyldiyls such as, butan-l,l-diyl,
butan-l-diyl, butan-l,3-diyl, butan-l,4-diyl, butan-2,2-diyl, 2-methyl-propan-l,l-diyl,
2-methyl-propan-l,2-diyl, cyclobutan-l,l-diyl; cyclobutan-l,2-diyi, cyclobutan-l,3-diyl,
but-l-en-l,l-diyl, but-l-en-l,2-diyl, but-l-en-l,3-diyl, but-l-en-l,4-diyl,
2-methyl-prop~l-en-l,l-diyl, 2-methanylidene-propan-l,l-diyl, buta-l,3-dien-l,l-diyl,
buta-l,3-dien-l,2-diyl, buta-l,3-dien-l,3-diyl, buta-l,3-dien-l,4-diyl, cyclobut-l-en-l,2-diyl,
cyclobut-1 -en-1,3-diyl, cyclobut-2-en-1,2-diyl, cyclobuta-1,3-dien-1,2-diy 1,
cyclobuta-l,3-dien-l,3-diyl, but-l-yn-l,3-diyl, but-l-yn-l,4-diyl, buta-l,3-diyn-l,4-diyl, etc.; and
the like. Where specific levels of saturation are intended, the nomenclature alkanyldiyl,
alkenyldiyl and/or alkynyldiyl is used. Where it is specifically intended that the two valencies be
on the same carbon atom, the nomenclature "alkylidene" is used. A "lower alkyldiyl" is an
alkyldiyl group containing 1 to 6 carbon atoms. In some embodiments the alkyldiyl groups are
saturated acyclic alkanyldiyl groups in which the radical centers are at the terminal carbons, e.g.,
methandiyl (methano); ethan-l,2-diyl (ethano); propan-l,3-diyl (propano); butan-l,4-diyl
(butano); and the like (also referred to as alkylenes, defined infra).
"Alkylcne" by itself or as part of another substituent refers to a straight-chain saturated or
unsaturated alkyldiyl group having two terminal monovalent radical centers derived by the
removal of one hydrogen atom from each of the two terminal carbon atoms of straight-chain
parent alkane, alkene or alkyne. The locant of a double bond or triple bond, if present, in a
particular alkylene is indicated in square brackets. Typical alkylene groups include, but are not
limited to, methylene (methano); ethylenes such as ethano, etheno, ethyno; propylenes such as
propano, prop[l]eno, propa[l,2]dieno, prop[l]yno, etc.; butylenes such as butano, but[l]eno,
but[2]eno, buta[l,3]dieno, but[l]yno, but[2]yno, buta[l,3]diyno, etc.; and the like. Where
specific levels of saturation are intended, the nomenclature alkano, alkeno and/or alkyno is used.
fa some embodiments, the alkylene group is (C1-C6) or (C1-C3) alkylene. In some
embodiments, the alkylene group is a straight-chain saturated alkano group, e.g., methano, ethano,
propane, butano, and the like.
"Cycloalkyl" by itself or as part of another substituent refers to a cyclic version of an
"alkyl" group. Typical cycloalkyl groups include, but are not limited to, cyclopropyl; cyclobutyls
such as cyclobutanyl and cyclobutenyl; cyclopentyls such as cyclopentanyl and cyclopentenyl;
cyclohexyls such as cyclohexanyl and cyclohexenyl; and the like.
"Parent Aromatic Ring gystero" refers to an unsaturated cyclic or polycyclic ring system
having a conjugated K electron system. Specifically included within the definition of "parent
aromatic ring system" are fused ring systems in which one or more of the rings are aromatic and
one or more of the rings are saturated or unsaturated, such as, for example, fluorene, indane,
indene, phenalene, tetrahydronaphthalene, etc. Typical parent aromatic ring systems include, but
are not limited to, aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene,
benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene, indacene,
s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene,
pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene,
pyranthrene, rubicene, tetrahydronaphthalene, triphenylene, trinaphthalene, and the like.
"Aryl" by itself or as part of another substituent refers to a monovalent aromatic
hydrocarbon group having the stated number of carbon atoms (i.e., C5-C15 means from 5 to 15
carbon atoms) derived by the removal of one hydrogen atom from a single carbon atom of a
parent aromatic ring system. Typical aryl groups include, but are not limited to, groups derived
from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene,
coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene, ov-indacene, s-indacene,
indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene,
pentalene, pentaphene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene,
pyranthrene, rubicene, triphenylene, trinaphtfaalene, and the like, as well as the various hydro
isomers thereof. In some embodiments, the aryl group is (C5-C15) aryl, with (C5-C10) being
more typical. Specific examples are phenyl and naphthyl.
"Halogen" or "Halo" by themselves or as part of another substituent, unless otherwise
stated, refer to fluoro, chloro, bromo and iodo.
"Haloalkyr by itself or as part of another substituent refers to an alkyl group in which
one or more of the hydrogen atoms are replaced with a halogen. Thus, the term "haloalkyl" is
meant to include monohaloalkyls, dihaloalkyls, trihaloalkyls, etc. up to perhaloalkyls. For
example, the expression "(C1-C2) haloalkyl" includes fluoromethyl, difluoromethyl,
trifluoromethyl, 1 -fluoroemyl, 1,1-difluoroethyI, 1,2-difluoroethyl, 1,1,1-trifluoroethyl,
perfluoroethyl, etc.
"Hydroxy alkyl" by itself or as part of another substituent refers to an alkyl group in which
one or more of the hydrogen atoms are replaced with a hydroxyl substituent. Thus, the term
"hydroxyalkyl" is meant to include ntonohydroxyalkyls, dihydroxyalkyls, trihydroxyalkyls, etc.
The above-defined groups may include prefixes and/or suffixes that are commonly used
in the art to create additional well-recognized substituent groups. As examples, "alkyloxy" or
"alkoxy" refers to a group of the formula -OR, "alkylamine" refers to a group of the formula
-NHR and "dialkylamine" refers to a group of the formula -NRR, where each R is independently
an alkyl. As another example, "haloalkoxy" or "haloalkyloxy" refers to a group of the formula
-OR', where R' is a haloalkyl,
"Prodrug" refers to a derivative of an active compound (drug) that may require a
transformation under the conditions of use, such as within the body, to release the active drug.
Prodrugs are frequently, but not necessarily, pharmacologically inactive until converted into the
active drug, Prodrugs are typically obtained by masking a functional group in the drug compound
believed to be in part required for activity with a progroup (defined below) to form a pi;omoiefy
which undergoes a transformation, such as cleavage, under the specified conditions of use to
release the functional group, and hence the active drug. The cleavage of the promoiety may
proceed spontaneously, such as by way of a hydrolysis reaction, or it may be catalyzed or Induced
by another agent, such as by an enzyme, by light, by acid or base, or by a change of or exposure to
a physical or environmental parameter, such as a change of temperature. The agent may be
endogenous to the conditions of use, such as an enzyme present in the cells to which the prodrug
is administered or the acidic conditions of the stomach, or it may be supplied exogenously.
A wide variety of pro groups, as well as the resultant promoieties, suitable for masking
functional groups in the active stereoisomerically enriched compounds described herein to yield
prodrugs are well-known in the art For example, a hydroxyl functional group may be masked as
a aulfbnate, ester or carbonate promoiety, which may be hydrolyzed in vivo to provide the
hydroxyl group. An amino functional group may be masked as an amide, carbamate, imine, urea,
phosphenyl, phosphoryl or sulfenyl promoiety, which may be hydrolyzed in vivo to provide the
amino group. A car boxy 1 group may be masked as an ester (including silyl esters and thioesters),
amide or hydrazide promoiety, which may be hydrolyzed in vivo to provide the carboxyl group.
Other specific examples of suitable progroups and their respective promoieties will be apparent to
those of skill in the art.
"Progroup" refers to a type of protecting group that, when used to mask a functional
group within an active stereoisomerically enriched drug compound to form a promoiety, converts
the drug into a prodrug. Progroups are typically attached to the functional group of the drug via
bonds that are cieavable under specified conditions of use. Thus, a progroup is that portion of a
promoiery that cleaves to release the functional group under the specified conditions of use. As a
specific example, an amide promoiery of the formula -NH-C(O)CH3 comprises the progroup
-C(COCH3.
"Proliferative disorder" refers to a disease or disorder characterized by aberrant cell
proliferation, for example, where cells divide more than their counterpart normal cells. The
aberrant proliferation may be caused by any mechanism of action or combination of mechanisms
of action. For example, the cell cycle of one or more cells may be affected such that cell(s) divide
more frequently than their counterpart normal cells, or as another example, one or more cells may
bypass inhibitory signals, which would normally limit their number of divisions. Proliferative
diseases include, but are not limited to, slow or fast growing tumors and cancers.
"Antiproliferative compound" refers to a compound that inhibits the proliferation of a cell
as compared to an untreated control cell of a similar type. The inhibition can be brought about by
any mechanism or combination of mechanisms, and may operate to inhibit proliferation
cytostatically or cytotoxically. As a specific example, inhibition as used herein includes, but is
not limited to, arrest of cell division, a reduction in the rate of cell division, proliferation and/or
growth and/or induction of cell death, by any mechanism of action, including, for example
apoptosis.
"Aurora kmase" refers to a member of the family of serine/threonine protein kinases that
are generally referred to as "Aurora" kinases. The Aurora family of serine/threonine protein
kinases are essential 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). Presently, there are
three known mammalian family members: Aurora-A ("2"), Aurora-B ("1") and Aurora-C ("3")
(see, e.g., Giet & Prigent, 1999,1 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
mammalian family members, but also later-discovered mammalian family members and
homologous proteins from other species and organisms (for non-limiting examples of
homologous members of the Aurora kinase family from other species and organisms see
Schumacher et al., 1998, J. Cell Biol. 143:1635-1646; Kimura et al., 1997, J. BioL Chem.
272:13766-13771).
"Aurora kjf "fff-'Wfiiflted process" or "Aurora kinase-mediated disease or disprder" refers
to a cellular process, disease or disorder hi which an Aurora kinase plays a role. The Aurora
kinases are believed to play a key role in protein phosphorylation events that regulate the mitotic
phase of the cell cycle. The human Aurora kinases display distinct subcellular locations during
mitosis. For example, Aurora-A is upregulated during the M phase of the cell cycle and localizes
to the spindle pole during mitosis, suggesting involvement in centrosomal functions. While
Aurora-A activity is maximized during prophase, Aurora-B is believed to play an important role
during chromatid separation and formation of the cleavage furrow in anaphase and telophase. The
role of Aurora-C is less clear, but it has been shown to localize to centrosomes during mitosis
from anaphase to cytokinesis. Moreover, inhibition of Aurora kinase activity in mammalian cells
leads to abnormal cell growth and polyploidy (Terada et al., 1998, EMBO J. 17:667-676). Thus,
Aurora kinases are thought to regulate ceil division, chromosome segregation, mitotic spindle
formation, and cytokinesis. As used herein, all of these various processes are within the scope of
"Aurora kinases-mcdiated process."
Moreover, since its discovery in 1997, the mammalian Aurora kinase family has been
closely linked to tumorigenesis. The most compelling evidence for this is that over-expression of
Aurora-A transforms rodent fibroblasts (Bischoff et al., 1998, EMBO J. 17:3052-3065). Cells
with elevated levels of this kinase contain multiple centrosomes and multipolar spindles, and
rapidly become aneuploid. The oncogenic activity of Aurora kinases is likely to be linked to the
generation of such genetic instability. Indeed, a correlation between amplification of the aurora-A
locus and chromosomal instability in mammary and gastric tumors has been observed (Miyoshi et
al., 2001, Int. J, Cancer 92:370-373; Sakakura et al., 2001, Brit. J. Cancer 84:824-831).
The Aurora kinases have been reported to be over-expressed in a wide range of human
tumors. Elevated expression of Aurora-A has been detected in over 50% of colorectal (Bischoff et
al., 1998, EMBO J. 17:3052-3065; Takahashi etal., 2000, Jpn. J. Cancer Res. 91:1007-1014),
ovarian (Gritsko et al., 2003, Clinical Cancer Research 9:1420-1426, and gastric tumors
(Sakakura, 2001, Brit. J. Cancer 84:824-831, and in 94% of invasive duct adenocarcinomas of the
breast (Tanaka, 1999, Cancer Research. 59:2041-2044). High levels of Aurora-A have also been
reported 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, Nature Genetics 20:189-193; Li et al., 2003, Clin Cancer Res.
9(3):991-7). Amplification/overexpression of Aurora-A is observed in human bladder cancers
and amplification of Aurora-A is associated with aneuploidy and aggressive clinical behavior
(Sen et al, 2002, J Natl Cancer List. 94(17): 1320-9. Moreover, amplification of the aurora-A locus
(20ql3) correlates with poor prognosis for patients with node-negative breast cancer (Isola et al.,
1995, American Journal of Pathology 147:905-911). Aurora-B is highly expressed hi multiple
human tumor cell lines, including leukemic cells (Katayama et al., 1998, Gene 244:1-7). Levels
of this enzyme increase as a function of Duke's stage in primary colorectal cancers (Katayama et
al., 1999, J. Nat'I Cancer Inst. 91:1160-1162). Aurora-C, which is normally only found in germ
cells, is also over-expressed in a high percentage of primary colorectal cancers and in a variety of
tumor cell lines including cervical adenocarcinoma and breast carcinoma cells (Kimura et al.,
1999, J. Biol. Chem. 274:7334-7340; Takahashi et al., 2000, Jpn. J. Cancer Res. 91:1007-1014).
In contrast, the Aurora family is expressed at a low level in the majority of normal tissues,
the exceptions being tissues with a high proportion of dividing cells, such as the thymus and testis
(Bischoffet al., 1998, EMBO J., 17:3052-3065).
For a further review of the role(s) Aurora kinases play in proliferative disorders, see
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 and Dutertre et al., 2002, Oncogene 21:6175-6183.
Although over-expression of proteins by cancer cells is not always indicative that
inhibition of the protein activity will yield anti-tumor effect, it has been confirmed in functional
assays that at least the following types of tumor cells are sensitive to inhibition of Aurora kinase
activity: prostate (DU145), cervical (Hela), pancreatic (Mia-Paca2, BX-PC3), histological
leukemia (U937), lung adenocarinoma, lung epidermoid, small lung cell carcinoma, breast, renal
carcinoma, Mo!T3 (all) and Molt4 (all).
Based on the established role of Aurora kinases in a variety of cancers, examples of
"Aurora kinases-mediated diseases and disorders" include, but are not limited to, melanoma,
leukemia, and solid tumor cancers, such as, for example, colon, breast, gastric, ovarian, cervical,
melanoma, renal, prostate, lymphoma, neuroblastoma, pancreatic and bladder cancers.
"Therapeutically effective amount" refers to an amount of a compound sufficient to treat
a specified disorder, or disease or one or more of its symptoms. In reference to tumorigenic
proliferative disorders, a therapeutically effective amount comprises an amount sufficient to,
among other things, cause the tumor to shrink, or to decrease the growth rate of the tumor.
In many situations, standard treatments for tumorigenic proliferative disorder involves
surgical interaction to remove the tumor(s), either alone or in combination with drug (chemo)
and/or radiation therapies. As used herein, a "therapeutically effect amount" of a compound is
intended to include an amount of compound that either prevents the recurrence of tumors in
subjects that have had tumor(s) surgically removed, or slows the rate of recurrance of tumor(s) in
such subjects.
Accordingly, as used herein, amounts of compounds that provide therapeutic benefit
adjunctive to another type of therapy, such as surgical intervention and/or treatment with other
antiproliferative agents, including, for example, 5-fluorouracil, vinorelbine, taxol, vinblastine,
cisplatin, topotecan, etc.), are included within the meaning of "therapeutically effective amount."
"ProphvlacticaUv effective amounf " refers to an amount of a compound sufficient to
prevent a subject from developing a specified disorder or disease. Typically, subjects in which
prophylaxis is practiced are not suffering from the specified disorder or disease, but are
recognized as being at an elevated risk for developing this disease or disorder based factors such
as, but not limited to, diagnostic markers and family history.
6.2 Stereoisomerically Enriched and Stereoisomerically Pure Compounds
It has been recently discovered that certain N4-(3-aminocarbonylbicyclo[2.2.1]hept-5-
ene-2-yl)-N2-substituted phenyl-2s4-pyrimidinediamine compounds, represented by structural
formula (I), below, are potent inhibitors of Aurora kinase activity and tumor cell proliferation in
in vitro assays (see, e.g., application Serial No, 11/133,419 filed May 18,2005, copending
application Serial No. ___ , entitled "StereoisomerieaUy Enriched p-Lactams Using
Candida Antarctica," filed concurrently herewith (identified by attorney docket no. 375462-
030US), and international application No. PCT/US05/17470 filed May 18,2005 and the priority
applications referenced therein):
Skilled artisans will appreciate that in structural formula (I), the stereochemistry at
carbons 1,2,3 and 4 is unspecified, such mat the compounds according to structural formula (I)
include eight diastereomers, illustrated by structural formulae (laXIh), below:
(Figure Removed)
Hie compounds of structural formula (I) also include two cis racemates, represented by
structural formulae (Ila) and (nb), and two trans racemates, represented by structural formulae
(ma) and (nib), below:
(Figure Removed)
The cis racemate of structural formula (fla) can be referred to as the 2-exo-3-exo
racemate, and includes the (1R,2R,3S,4S) and (1S,2S,3R,4R) diastereomers of structural formulae
(la) and (0)), respectively The cis racemate of structural formula (nb) can be refened to as the 2-
endo-3-endo racemate, and includes the (1R, 2S, 3R, 4S) and (1S, 2R, 3S, 4R) diastereomers of
structural formulae (Ic) and (Id), respectively. As described in more detail in the Examples
section, for compounds in which R5 is fluoro, R1 is hydrogen, R2 is 4-methylpiperazin-l-yl and R3
is methyl, these two cis racemates exhibit antiproliferative activity against a variety of different
tumor cell lines in in vitro antiproliferation assays. However, this 2-exo-3-exo racemate
(racemate rl) is approximately twenty-fold more potent than the corresponding 2-endo-3-endo
racemate (racemate r2) in all cell lines tested with both racemates. Moreover, it has been
discovered that the (1 R,2R,3S,4S) diastereomer of racemate rl is largely responsible for the
potency of the racemate rl. When tested as isolated stereoisomers, this (IR,2R,3S,4S)
diastereomer (designated the "a" diastereomer) generally exhibited ICSO's in the nanomolar
range, whereas the (1 S,2S,3R,4R) diastereomer (designated the "b" enantiomer) generally
exhibited ICSO's in the micromolar range against the same cell lines. Thus, in general, the
(1R,2R,3S,4S) diastereomer of this compound is generally 1000-fold more potent man its
corresponding (1 S,2S,3R,4R) enantiomer. It is also approximately 20-50 times more potent than
the corresponding 2-endo-3-endo r2 racemate in the cell lines tested. The (1R,2R3S,4S)
diastereomer exhibited similarly superior results compared to its (1S,2S,3R,4R) enantiomer in
cell-based inhibition assays against Aurora kinase B. Based on the observed potency of this
(1R,2R,3S,4S) diastereomer, it is expected that the full range of (1R^2R,3S,4S) diastereomers
according to structural formula (la) will exhibit similarly superior potencies as compared to their
corresponding (1 8,28,3 R,4R) enantiomers, 2-exo-3-exo racetnates, 2-endo-3-endo racemates and
other corresponding diastereomers.
Accordingly, provided herein are compounds that are enriched in this particularly potent
(]R,2R,3S,4S) diastereomer. hi one embodiment, such stereoisomerically enriched compounds
include compounds according to structural formula (I):
(Figure Removed)
that are enriched in the corresponding diastereomer of structural formula (la):
(Figure Removed)
wherein:
each R1 is independently selected from the group consisting of hydrogen, lower
alkyl, -(CH2)B-OH, -OR', -O(CH2)B-Ra, -O(CHa)w-Rb. -CCOR, halo, -CF3 and -OCF3;
each R^ is independently selected from the group consisting of hydrogen, lower alkyl,
-OR", -(XCHjVR8, -O(CH2)n-Rb, -NHC(0)Ra, halo, -CF3, -OCF3, — / and
each R3 is independently selected from the group consisting of hydrogen, lower
-I-N 0
alkyl, -(CH2),,-OH, -OR4, -0(CH2)n-R',-0(CH2)n-Rk, halo, -CF3,-OCF3, V_7 ,
N and
each R4 is independently selected from the group consisting of hydrogen, lower
alkyl, arylalkyl, -OR8, -NRX -C(O)R", -C(O)ORand -C(O)NR°R°;
R5 is hydrogen, halo, fluoro, -CN, -NO2, -C(O)OR", or ~CF3;
each n is independently an integer from 1 to 3;
each R is independently selected from the group consisting of hydrogen, lower
alkyl and lower cycloalkyl;
each Rb is independently selected from the group consisting of-ORa, -CF3,
-OCF3, -NRCRC, ~C(0)Ra, -C(O)OR", -OfONR-R' and -C(O)NR*Rd;
each Rc is independently selected from the group consisting of hydrogen and
lower alkyl, or, alternatively, two R° substituents may be taken together with the nitrogen atom to
which they are bonded to form a 4-9 membered saturated ring which optionally includes 1 -2
additional heteroatomic groups selected from O, NR", NR*-C(O)Ra, NR'-C(O)ORa and
NR"-C(0)NR"; and
each Rd is independently lower mono-hydroxyalkyl or lower di-hydroxyalkyl.
In another embodiment, such stereoisomerically enriched compounds include 2-exo-3-exo
cis racemates according to structural formula (Ila), wherein R', R2, R3, R4 and R5 are as previously
defined for structural formula (I), that are enriched in the diastereomer of structural formula (fa),
supra.
As used herein, a compound is "enriched" in a particular diastereomer when that
diastereomer is present in excess over any other diastereomer present in the compound. The
actual percentage of the particular diastereomer comprising the compound will depend upon the
number of other diastereomers present. As a specific example, a racernic mixture is "enriched" in
a specified enantiomer when that enantiomer constitutes greater man 50% of the mixture.
Regardless of the number of diastereomers present, a compound that is enriched in a particular
diastereomer will typically comprise at least about 60%, 70%, 80%, 90%, or even more, of the
specified diastereomer. The amount of enrichment of a particular diastereomer can be confirmed
using conventional analytical methods routinely used by those of skill in the art, as will be
discussed in more detail, below.
In another embodiment, the stereoisomerically enriched compounds include compounds
according to structural formula (la), supra, wherein R1, R2, R3, R4 and R5 are as previously
defined for structural formula (I), that are substantially free of the corresponding enantiomer
and/or any other corresponding diastereomer. By "substantially free of is meant mat the
compound comprises less than about 10% of the undesired diastereomers and/or enantiomers as
established using conventional analytical methods routinely used by those of skill in the art
(discussed in more detail below). In some embodiments, the amount of undesired stereoisomers
may be less than 10%, for example, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or even less.
Stereoisomerically enriched compounds that contain about 95% or more of the desired
stereoisomer are referred to herein as "substantially pure" stereoisomers. Stereoisomerically
enriched compounds that contain about 99% or more of the desired stereoisomer are referred to
herein as "pure" stereoisomers. The purity of any stereoisomerically enriched compound
(diastereoisomeric purity; % de) can be confirmed using conventional analytical methods, as will
be described in more detail, below.
In some embodiments of the various stereoisomerically enriched compounds described
-f-N O -f-N N-R4 -f-N O
herein, R1 is hydrogen; R2 is \—' or V__Y ; ^d R3 jj oraer than ^—' ,
(Figure Removed)
N . hi other embodiments of Hie various stereoisomerically enriched
compounds described herein, R3 is hydrogen, methyl, methoxy, trifhioromethyl or chloro. In still
other embodiments, R4 is methyl, -C(O)CH3, -C(O)OCH3 or -C(O)OCH2CH3.
In still other embodiments of the various stereoisomerically enriched compounds
described herein, R1 is hydrogen, R1 is other than \ — / or \ — / and R3 is
In yet other embodiments, R2 is hydrogen, methyl,
methoxy, trifluoromethyl or chloro. Preferably, R4 is methyl, -C(O)CH3, -C(O)OCH3 or
-C(0)CH2CHj.
In still other embodiments of the various stereoisomerically enriched compounds
(Figure Removed)
described herein, R2 is other man \—' or \—' and R is other than \—' ,
In still other embodiments, R1 and R2 are each hydrogen and R3 is
-OCHaNHR*. In some other embodiments, R1, R2 and R3 are each, independently of one another
selected from the group consisting of hydrogen, methyl, methoxy, trifluoromethyl and chloro,
with the proviso that at least two of R1, R2 and R1 are other than hydrogen.
hi still other embodiments, R1 is hydrogen, R2 is selected from the group consisting of
R3 is selected from me group consisting of
-f-N 0 -|-N N-R4
hydrogen, lower alkyl, halo, -CF3) 'fa still other embodiments,
and R4 is methyl, -CORa or -CO(O)Ra where Ra is methyl or ethyl. In yet
R3 is selected from the group consisting of hydrogen, methyl, chloro, -CF3, \ — ' and
another embodiment, R2 is selected from the group consisting of hydrogen, NI — ' and
R3 is selected from the group consisting of hydrogen, lower alkyl, halo, -CF3)
In still other embodiments, R3 is selected from the group
(Figure Removed)
consisting of hydrogen, methyl, chloro, -CF3, \ — ' and ^ — / and R4 is methyl, -
-f-N N-R4
COR or -CO(O)R* wherein R* is methyl or ethyl. Preferably, R2 is \ — / ,R4is-€OR'
-f N N-R4
wherein R* is methyl; and R3 is hydrogen. In other embodiments, R2 is \ — / , R4 is-
CO(O)R" wherein R* is ethyl, and R3 is hydrogen. In still another embodiment, R2 is N| — '
and R3 is hydrogen.
to yet another embodiment, R is hydrogen; R is > — ' or \ — / ; and R4
-1-N N-R4
is methyl, -COR" or ~CO(O)Ra where R' is methyl or ethyl. Preferably, R2 is \ — f , R4
is methyl and R3 is selected from the group consisting of hydrogen, methyl, chloro and -CF3.
More preferably, R3 is methyl.
In still other embodiments of the stereoisomerically enriched compounds described
herein, R5 is fluoro.
In still other embodiments, the stereoisomerically enriched compound is substantially
stereoisomerically pure or stereoisomerically pure (lR,2R3S,4S)-N4-(3-
aminocarbonylbicyclo[2.2J]hept-5-ene-2-yl)-5-fluoro-N2-[3-methyl-4-(4-metiiylpiperazm-lyl)
phenyl]-2,4-pyrimidinediamine.
Additional exemplary embodiments of compounds according to structural formula (I) that
may be stereoisomerically enriched in the corresponding diastereomer of structural formula (la),
supra, substantially free of any enantiomets and/or diastereomer thereof, and/or substantially pure
or pure in the diastereomer of structural formula (la), supra, are illustrated in TABLE 1 , below:
(Figure Removed)
R5
When specific diastereomers and/or racemic mixtures of specific compounds described
herein, such as the compounds described in TABLEl, are intended, the compound number is
followed by a letter specifying the specific diastereomer or racemic mixture as follows:
Thus, as a specific example, the (1R,R,3S,4S) diastereomer of compound 60 is referred
to as compound 60s.
Those of ski]] in the art will appreciate that the stereoisomerically enriched compounds
described herein may include functional groups that can be masked with progroups to create
prodrugs. Such prodrugs are usually, but need not be, pharmacologically inactive until converted
into their active drug form. For example, ester groups commonly undergo acid-catalyzed
hydrolysis to yield the parent carboxylic acid when exposed to the acidic conditions of the
stomach, or base-catalyzed hydrolysis when exposed to the basic conditions of the intestine or
blood. Thus, when administered to a subject orally, stereoisomerically enriched compounds that
include ester moieties may be considered prodrugs of their corresponding carboxylic acid,
regardless of whether the ester form is pharmacologically active.
Included within the scope of the invention are prodrugs of the various stereoisomerically
enriched compounds described herein. In such prodrugs, any available functional moiety may be
masked with a progroup to yield a prodrug. Functional groups within the stereochemically
enriched compounds described herein that may be masked with progroups for inclusion in a
promoiety include, but are not limited to, amines (primary and secondary), hydroxyls, sulfanyls
(thiols), carboxyls, etc. Myriad progroups suitable for masking such functional groups to yield
promoieties that are cleavable under die desired conditions of use are known in the art. All of
these progroups, alone or in combinations, may be included in the stereoisomerically enriched
prodrugs of the invention.
In one illustrative embodiment, the stereoisomerically enriched prodrugs are compounds
according to structural formulae (I), supra, in which Ra Rb and Rc may be, in addition to their
previously-defined alternatives, a progroup, that are enriched in the corresponding diastereomer of
structural formula (la), supra.
Those of skill in the art will appreciate that many of the compounds and prodrugs
described herein, as well as the various compound species specifically described and/or illustrated
herein, may exhibit the phenomena of tautomerism and conformational isomerism. For example,
the compounds and prodrugs may exist in several tautomeric forms, including the enol form, the
keto form and mixtures thereof. As the various compound names, formulae and compound
drawings within the specification and claims can represent only one of the possible tautomeric or
conformational forms, it should be understood mat the invention encompasses any tautomers or
conformational isomers, of the compounds or prodrugs having one or more of the utilities
described herein, as well as mixtures of these various different isomeric forms. In cases of limited
rotation around the 2,4-pyrimidinediamine core structure, atrop isomers are also possible and are
also specifically included in the compounds and/or prodrugs of the invention.
Depending upon the nature of the various subsdtuents, the stereoisomerically enriched
compounds and prodrugs may be in the form of salts. Such salts include salts suitable for
pharmaceutical uses ("pharmaceutically-acceptable salts"), salts suitable for veterinary uses, etc.
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 those salts that retain substantially one or more of the
desired pharmacological activities of the parent compound and which are suitable for
administration to humans. Pharmaceutically acceptable salts include acid addition salts formed
with inorganic acids or organic acids. Inorganic acids suitable for forming pharmaceutically
acceptable acid addition salts include, by way of example and not limitation, hydrohalide acids
(e.g., hydrochloric acid, hydrobromic acid, hydriodic, etc.), sulfuric acid, nitric acid, phosphoric
acid, and the like. Organic acids suitable for forming pharmaceutically acceptable acid addition
salts include, by way of example and not limitation, 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,
alkylsulfonic acids (e.g., methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid,
2-hydroxyethanesulfbnic acid, etc.), arylsulfonic acids (e.g., benzenesulfonic acid,
4-chlorobenzenesulf onic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid,
camphorsulfonie acid, etc.), 4-methylbicyclo[2.2.2]-oct-2-ene-l-carboxylic acid, glucoheptonic
acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary 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 salts formed when an acidic proton present
in the parent compound is either replaced by a metal ion (e.g., an alkali metal ion, an alkaline
earth metal ion or an aluminum ion) or coordinates with an organic base (e.g., ethanolamine,
diethanoiamine, triethanolamine, N-memylglucamine, morpholine, piperidine, dimethylamine,
diethylamine, etc.).
The stereoisomerically enriched compounds and prodrugs, as well as the salts thereof,
may also be in the form of hydrates, solvates and/or N-oxides, as are well-known in the art.
Stereoisomeric enrichment and/or purity of compounds and prodrug described herein may
be established by conventional analytical methods well known to those of skill in the art. For
example, use of chiral NMR shift reagents, gas chromatographic analysis using chiral columns,
high pressure liquid chromatographic analysis using chiral columns, formation of diastereomeric
derivatives through reaction with chiral reagents and conventional analysis may be used to
establish the stereoisomeric enrichment and/or purity of a specific stereoisomer. Alternatively,
synthesis using starting materials of known stereoisomeric enrichment and/or purity may be used
to establish the stereoisomeric enrichment and/or purity of the compounds described herein.
Other analytical methods for demonstrating stereoisomeric homogeneity are well within the ambit
of the skilled artisan.
6.3 Methods of Synthesis
The stereoisomerically enriched compounds and prodrugs may be synthesized via a
variety of different synthetic routes using commercially available starting materials and/or starting
materials prepared by conventional synthetic methods. A variety of exemplary synthetic routes
that can be used to synthesize the stereoisomerically enriched compounds and prodrugs are
described in WO 03/063794 and US 2004/0029902, the disclosures of which are incorporated
herein by reference.
For purposes of illustration, an exemplary synthetic scheme that can be used to synthesize
the full range of compounds described herein is illustrated in Scheme (I), below:
Scheme (I)
(other hotogenaBng agants)
hi Scheme (1), R1, R2, R3 and R5 are as previously defined for structural formula (I),
supra, X is a halogen (e.g., F, Cl, Br or I), and each G is, independently of the other, selected
from O and S. It should be noted that an "*" in aminocarboxamide 6 indicates that the particular
stereocenter is not specified. Accordingly, those of skill in the art will appreciate that Scheme (1)
may be used to prepare racemic diastereomeric mixtures, diastereomerically enriched mixtures of
omDOunas according to structural formula (I), as well as stereoisomers of the compounds of
structural formula (I) that are substantially free of other specified diastereomers.
Referring to Scheme (I), uracil or thiouracil 2 is dihalogenated at the 2- and 4-positions
using the standard halogenating agent POX3 (or other halogenating agents) under standard
conditions to yield 2,4-bis-halo pyrimidine 4. The halide at the C4 position is more reactive
towards nucleophiles than the halide at the C2 position in pyrimidine 4. This differential
reactivity can be exploited to synthesize the compounds and prodrugs described herein by first
reacting 2,4-bis-halopyrimidine 4 with one equivalent of 2-aminobicyclo[2.2.1]hept-5-ene-3-
carboxamide 6, yielding 8, followed by reaction with aniline 10 to yield compounds according to
structural formula (I). Those of skill in the art will appreciate that the stereoisomeric
configuration and optical purity of aminocarboxamide 6 will, in most circumstances, determine
the stereoisomeric configuration and optical purity of the compounds of structural formula (I).
In most situations, the C4 halide is more reactive towards nucleophiles, as illustrated in
the Scheme. However, as will be recognized by skilled artisans, the identity of the R5 substituent
may alter this reactivity. For example, when Rs is trifluoromethyl, a 50:50 mixture of
4N-substituted-4-pyrimidinearnine 8 and the corresponding 2N-substituted-2-pyrimidineamine is
obtained. Regardless of the identity of the Rs substituent, the regioselectivity of the reaction can
be controlled by adjusting the solvent and other synthetic conditions (such as temperature), as is
well-known in the art.
The reactions depicted in Scheme (I) may proceed more quickly when the reaction
mixtures are heated via microwave. When heating in this fashion, the following conditions may
be used: heat to 17S°C in ethanol for 5-20 min. in a Smith Reactor (Personal Chemistry, Biotage
AB, Sweden) in a sealed tube (at 20 bar pressure).
The uracil or thiouracil 2 starting materials may be purchased from commercial sources 01
prepared using standard techniques of organic chemistry. Commercially available uracils and
thiouracils that can be used as starting materials in Scheme (I) include, by way of example and not
limitation, uracil (Aldrich #13,078-8; CAS Registry 66-22-8); 2-thio-uracil (Aldrich #11,558-4;
CAS Registry 141-90-2); 2,4-dithiouracil (Aldrich #15,846-1; CAS Registry 2001-93-6);
5-bromouracil (Aldrich #85,247-3; CAS Registry 51-20-7; 5-fluorouracil (Aldrich #85,847-1;
CAS Registry 51-21-8); 5-iodouracil (Aldrich #85,785-8; CAS Registry 696-07-1); 5-nitrouracil
(Aldrich #85,276-7; CAS Registry 611-08-5); 5-(trifluoromethyl)-uracil (Aldrich #22,327-1; CAS
Registry 54-20-6). Additional 5-suostituted uracils and/or thiouracils are available from General
Intermediates of Canada, Inc., Edmonton, CA (http://www.generalintermediates.com) and/or
Interchim, Cedex, France (http://www.interchim.com), or may be prepared using standard
techniques. Myriad textbook references teaching suitable synthetic methods are provided infra.
Anilines 10 may be purchased from commercial sources or, alternatively, may be
synthesized utilizing standard techniques. For example, suitable anilines may be synthesized
from nitro precursors using standard chemistry. Specific exemplary reactions are provided in the
Examples section. See also Vogel, 1989, Practical Organic Chemistry, Addison Wesley
Longman, Ltd. and John Wiley & Sons, be.
Skilled artisans will recognize that in some instances anilines 10 may include functional
groups that require protection during synthesis. The exact identity of any protecting group(s)
used will depend upon the identity of the functional group being protected, and will be apparent to
these of skill in the art. Guidance for selecting appropriate protecting groups, as well as synthetic
strategies for their attachment and removal, may be found, for example, in Greene & Wuts,
Protective Groups in Organic Synthesis, 3d Edition, John Wiley & Sons, Inc., New York (1999)
and the references cited therein (hereinafter "Greene & Wuts").
Prodrugs as described herein may be prepared by routine modification of the
above-described methods.
As skilled artisans will appreciate, the desired (3 R,2R,3S,4S) diastereomer corresponding
to structural formula (la), supra, can be isolated by chiral separation or other standard techniques.
Methods for chirally resolving 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 from 2-amino-3-carboxamide storting materials 6 having
specified stereochemistry, or with the aid of chiral auxiliaries.
In one exemplary embodiment, illustrated in Scheme (IT), below, the desired diastereomer
is resolved chemically using (R)-methyl-p-methoxybenzylamine 18 as a chiral auxiliary.
(Figure Removed)
to Scheme (II), 2-exo-3-exo racemic pMactam 14rl (prepared as described in Stajar et al.,
1984, Tetrahedron 40(12): 2385) is protected with a Boc group, yielding the corresponding
racemic Boc-protected p-lactam 16rl. Hoc-protected racemate 16rl is then reacted with
(/?)-memyl-/?ora-methoxybenzylamine 18, yielding a mixture of diastereomers 20a and 20b. This
diastereomeric mixture is treated with an acid such as TFA to cleave the Boc group, yielding a
mixture of diastereomers 22a and 22b, which can be reacted with 2,4-dihalopyrimidine 4 to afford
a racemic mixture of compounds 24a and 24b. At this stage, compounds 24a and 24b can be
separated from one another by crystallization and reacted with aniline 10 to afford isolated
diastereomers 25a and 25b. The chiral auxiliaries from isolated diasteromers 2Sa and 25b can
then be cleaved to yield isolated diastereomers according to structural formulae (la) and (Ib),
respectively.
For compounds 25a and 25b in which R1 is hydrogen, R2 is 4-methyl-piperazin-l-yl, R3 is
methyl and R5 is fluoro, cleavage of the chiral auxiliary proved difficult. For these and other
compounds where such cleavage proves difficult, the chiral auxiliary can be cleaved from
compounds 24a and 24b, and the resultant isolated compounds reacted with aniline 10 to yield
isolated diastereomers according to structural formulae (la) and (Ib). Specific examples of such
reactions are described in the Examples section.
Compounds that are stereoisomerically enriched, substantially stereoisomerically pure
and/or stereoisomerically pure in specified diastereomers can also be synthesized from
stereoisomerically enriched, substantially stereoisomerically pure, and/or stereoisomerically pure
p-lactams. Such stereoisomerically enriched and/or (substantially) stereoisomerically pure
P-lactams can be enzymatically resolved and isolated, m one exemplary embodiment,
(substantially) stereoisomerically pure P-lactams can be resolved and isolated from a racemic
mixture of 2-exo-3-exo P-lactam 14rl using an immobilized lipolase (available from Sigma
Chemical Co., catalog no. L4777) as described in Eniko et al., 2004, Tetrahedron Asymmetry
15:573-575. In another exemplary embodiment, (substantially) stereoisomerically pure P-lactams
can be resolved and isolated from 2-exo-3-exo Boc-protected racemic p-lactam 16rl using resin
bound, immobilized chirazyme L-2-type B, c.f. enzyme (Candida antarctica Type B, c-f,
available from Biocatalytics, Inc., Pasadena, CA) as described in application Serial
No. 60/628,401, filed November 15, 2004, co-pending application Serial No. 11/133,419 filed
May 18,2005, and international application No. PCT/US05/17470 filed May 18,2005, and
copending application Serial No. , entitled "Stereoisomerically Enriched PLactams
Using Candida Antarctica," filed concurrently herewith (identified by attorney docket
no. 375462-030US), the disclosures of which are incorporated herein by reference. A specific
example of the use of this enzyme to resolve specified diastereomers of p-lactams is described in
the Examples section, as is a method of synthesizing 2-exo-3-exo racemic p-lactam 16rl.
Examples of synthesizing specified diasteromers according to structural formula (la)
utilizing enzyme reactions are illustrated in Schemes (HI) and (IV), below. A specific example of
the use of Novozyme 435 enzyme as illustrated in Scheme (IV), which like the Chirazyme enyme
discussed supra and illustrated in Scheme (III), can be used to resolve enantiomers from racemic
p-lactams, is described in the Examples section.
(Figure Removed)
6.4 Activity of the Antiproliferative Compounds
Active stereoisomerically enriched compounds typically inhibit proliferation of desired
cells, such as tumor cells, with an IC5o in the range of about 20 μM or less, as measured in a
standard in vitro cellular proliferation assay. Of course, skilled artisans will appreciate that
compounds which exhibit lower ICsoS, for example on the order of 10 uM, 1 μM, 100 μM, 10 nM,
1 nM, or even lower, may be particularly useful in therapeutic applications. The antiproliferative
activity may be cytostatic or it may be cytotoxic. In instances where antiproliferative activity
specific to a particular cell type is desired, the compound may be assayed for activity with the
desired cell type and counter-screened for a lack of activity against other cell types. The desired
degree of "inactivity" in such counter screens, or the desired ratio of activity vs. inactivity may
vary for different situations, and may be selected by the user.
Active compounds also typically inhibit an activity of an Aurora kinase, with an IC50 in
the range of about 20 uM or less, typically in the range of about 10 uM, 1 uM, 100 nM, 10 mM,
1 mM, or even lower. The IC50 against an aurora kinase can be determined in a standard hi vitro
assay with an isolated aurora kinase, or in a functional cellular array. A suitable enzyme coupled
assay that can be used to determine the degree of Aurora kinase activity is described in Fox et al.,
1998, Protein Sci. 7:2249-2255. Kemptide peptide sequence LRRASLG (Bochern Ltd., UK) can
be used as a substrate for Aurora kinase-A Aurora kinase-B and/or Aurora kinase-C, and reactions
can be carried out at 30°C in a solution containing 100 mM HEPES (pH 7.5), 10 mM Mg C12,25
mM NaCl, 1 mM D1T. ICso values can be determined using computerized non-linear regression
with commercially-available software (e.g., Prism 3.0, GraphPed Software, San Diego, CA). A
suitable cell-based functional assay is described in the Examples section.
6.5 Uses of the Antiproliferative Compounds
The active stereoisomerically enriched compounds, including the various prodrugs, salts,
hydrates and/or N-oxide forms thereof, may be used to inhibit Aurora kinases, Aurora kinasemediated
processes, and/or cell proliferation in a variety of contexts. According to some
embodiments, a cell or population of cells is contacted with an amount of such a compound
effective to inhibit an activity of an Aurora kinase, an Aurora kinase-mediated process and/or
proliferation of the cell or cell population. When used to inhibit cellular proliferation, the
compound may act cytotoxically to kill the cell, or cytostatically to inhibit proliferation without
killing the cell.
In some embodiments, the methods may be practiced in vivo as a therapeutic approach
towards the treatment or prevention of Aurora kinase-mediated diseases or disorders, and in
particular proliferative disorders. Thus, in a specific embodiment, the stereoisomerically enriched
compounds described herein, (and the various forms described herein) may be used to treat or
prevent proliferative disorders in animal subjects, including humans. The method generally
zomprises administering to the subject an amount of a stereoisomerically enriched compound, or a
prodrug, salt, hydrate or N-oxide thereof, effective to treat or prevent the disorder. In one
embodiment, the subject is a mammal, including, but not Umited to, bovine, horse, feline, canine,
rodent, or primate. In another embodiment, the subject is a human.
A variety of cellular proliferative disorders may be treated or prevented with the
compounds described herein. In some embodiments, the compounds are used to treat various
cancers in afflicted subjects. Cancers are traditionally classified based on the tissue and cell type
from which the cancer cells originate. Carcinomas are considered cancers arising from epithelial
cells while sarcomas are considered cancers arising from connective tissues or muscle. Other
cancer types include leukemias, which arise from hematopoietic cells, and cancers of nervous
system cells, which arise from neural tissue. For non-invasive tumors, adenomas are considered
benign epithelial tumors with glandular organization while chondomas are benign tumor arising
from cartilage. In the present invention, the described compounds may be used to treat
proliferative disorders encompassed by carcinomas, sarcomas, leukemias, neural cell tumors, and
non-invasive tumors.
In a specific embodiment, the compounds are used to treat solid tumors arising from
various tissue types, including, but not limited to, cancers of the bone, breast, respiratory tract,
brain, reproductive organs, digestive tract, urinary tract, bladder, eye, liver, skin, head, neck,
thyroid, parathyroid, kidney, pancreas, blood, ovary, colon, germ/prostate, and mestastatic forms
thereof.
Specific proliferative disorders include the following: a) proliferative disorders of the
breast include, but are not limited to, invasive ductal carcinoma, invasive lobular carcinoma,
ductal carcinoma, lobular carcinoma in situ, and metastatic breast cancer; b) proliferative
disorders of the skin include, but are not limited to, basal cell carcinoma, squamous cell
carcinoma, malignant melanoma, and Karposi's sarcoma; c) proliferative disorders of the
respiratory tract include, but are not limited to, small cell and non-small cell lung carcinoma,
bronchial edema, pleuropulmonary blastema, and malignant mesothelioma; d) proliferative
disorders of the brain include, but are not limited to, brain stem and hyptothalamic glioma,
cerebellar and cerebral astrocytoma, medullablastoma, ependymal tumors, oligodendroglial,
meningiomas, and neuroectodermal and pineal tumors; e) proliferative disorders of the male
reproductive organs include, but are not limited to, prostate cancer, testicular cancer, and penile
cancer t) proliferative disorders of the female reproductive organs include, but are not limited to,
uterine cancer (endometrial), cervical, ovarian, vaginal, vulval cancers, uterine sarcoma, ovarian
germ cell tumor; g) proliferative disorders of the digestive tract include, but are not limited to,
anal, colon, colorectal, esopnageal, gallbladder, stomach (gastric), pancreatic cancer, pancreatic
cancer- Islet cell, rectal, small-intestine, and salivary gland cancers; h) proliferative disorders of
the liver include, but are not limited to, hepatocellular carcinoma, cholangiocarcinoma, mixed
hepatocellular cholangiocarcinoma, and primary liver cancer; i) proliferative disorders of the eye
include, but are not limited to, intraocular melanoma, retinoblastoma, and rhabdornyosarcoma; j)
proliferative disorders of the head and cancers include, but are not limited to, laryngeal,
hypopharyngeal, nasopharyngeal, oropharyngeal cancers, and lip and oral cancer, squamous neck
cancer, metastatic paranasal sinus cancer, k) proliferative disorders of the lymphomas include, but
are not limited to, various T cell and B cell lymphomas, non-Hodgkins lymphoma, cutaneous T
cell lymphoma, Hodgkins disease, and lymphoma of the central nervous system; 1) leukemias
include, but are not limited to, acute myeloid leukemia, acute lymphoblastic leukemia, chronic
lymphocytic leukemia, chronic myelogenous leukemia, and hair cell leukemia, m) proliferative
disorders of the thyroid include thyroid cancer, thymoma, and malignant thymoma; n) sarcomas
include, but are not limited to, sarcoma of the soft tissue, osteosarcoma, malignant fibrous
histiocvtoma, lymphosarcoma, and rhabdomyosarcoma.
It is to be understood that the descriptions of proliferative disorders is not limited to the
conditions described above, but encompasses other disorders characterized by uncontrolled
growth and malignancy. It is further understood that proliferative disorders include various
metastatic forms of the tumor and cancer types described herein. The compounds of the present
invention may be tested for effectiveness against the disorders described herein, and a
therapeutically effective regimen established. Effectiveness, as further described below, includes
reduction or remission of the tumor, decreases in the rate of cell proliferation, or cytostatic or
cytotoxic effect on cell growth.
6.6 Combination Therapies
The stereoisomerically enriched compounds described herein may be used alone, in
combination with one another, or as an adjunct to, or in conjunction with, other established
antiproliferative therapies. Thus, the compounds may be used with traditional cancer therapies,
such as ionization radiation in the form of y-rays and x-rays, delivered externally or internally by
implantation of radioactive compounds, and as a follow-up to surgical removal of tumors.
In another aspect, the compounds may be used with other chemotherapeutic agents useful
for the disorder or condition being treated. These compounds may be administered
simultaneously, sequentially, by the same route of administration, or by a different route.
In some embodiments, the present compounds are used with other anti-cancer or
cytotoxic agents. Various classes of anti-cancer and anti-neoplastic compounds include, but are
not limited to, alkylating agents, antimetabolites, vinca alkyloids, taxanes, antibiotics, enzymes,
cytokines, platinum coordination complexes, substituted ureas, tyrosine kinase inhibitors,
hormones and hormone antagonists. Exemplary alkylating agents include, by way of example
and not limitation, mechlorothamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil,
ethyleneimines, methylmelamines, alky I sulfonates (e.g., busulfan), and carmustine. Exemplary
antimetabolites include, by way of example and not limitation, folk acid analog methotrexate;
pyrimidine analog fluorouracil, cytosine arbinoside; purine analogs mecaptopurine, thioguanine,
and azathioprine. Exemplary vinca alkyloids include, by way of example and not limitation,
vinblastine, vincristine, paclitaxel, and colchicine. Exemplary antibiotics include, by way of
example and not limitation, actinomycin D, daunorubicin, and bleomycin. An exemplary enzyme
effective as anti-neoplastic agents include L-asparaginase. Exemplary coordination compounds
include, by way of example and not limitation, cisplatin and carboplatin. Exemplary hormones
and hormone related compounds include, by way of example and not limitation,
adrenocorticosteroids prednisone and dexamethasone; aromatase inhibitors amino glutethimide,
formestane, and anastrozole; progestin compounds hydroxyprogesteron caproate,
medroxyprogesterone; and anti-estrogen compound tamoxifen.
These and other useful anti-cancer compounds are described in Merck Index, 13th Ed.
(O"Neil M.J. et al., ed) Merck Publishing Group (2001) and Goodman and Gilmans The
Pharmacological Basis of Therapeutics, 10th Edition, Hardtnan, J.G. and Limbird, L.E. eds., pg.
1381-1287, McGraw Hill, (1996), both of which are incorporated by reference herein.
Additional anti-proliferative compounds useful in combination with the
stereoisomerically enriched compounds described herein include, by way of example and not
limitation, antibodies directed against growth factor receptors (e.g., anti-Her2); antibodies for
activating T cells (e.g., anti-CTLA-4 antibodies); and cytokines such as interferon-a and
interferon-y, interleukin-2 and GM-CSF.
6.7 Formulations and Administration
When used to treat or prevent such diseases, the active compounds and prodrugs may be
administered smgly, as mixtures of one or more active compounds, or in mixture or combination
with other agents useful for treating such diseases and/or the symptoms associated with such
diseases. The active compounds and prodrugs may also be administered in mixture or in
combination with agents useful to treat other disorders or maladies, such as steroids, membrane
stabilizers. The active compounds or prodrugs may be administered/jer^e, or as pharmaceutical
compositions comprising an active compound or prodrug.
Pharmaceutical compositions comprising the active compounds (or prodrugs thereof) may
be manufactured by means of conventional mixing, dissolving, granulating, dragee-making
levigating, emulsifying, encapsulating, entrapping or lyophilization 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, 15th
Ed., Hoover, J.E. ed.. Mack Publishing Co. (2003)
The active compound or prodrug may be formulated in the pharmaceutical compositions
per se, or in the form of a hydrate, solvate, N-oxide or pharmaceutically acceptable salt, as
previously described. Typically, such salts are more soluble in aqueous solutions than the
corresponding free acids and bases, but salts having lower solubility than the corresponding free
acids and bases may also be formed.
Pharmaceutical compositions may take a form suitable for virtually any mode of
administration, including, for example, topical, ocular, oral, buccal, systemic, nasal, injection,
transdermal, rectal, vaginal, etc., or a form suitable for administration by inhalation or
insufflation.
For topical administration, the active compound(s) or prodrug(s) may be formulated as
solutions, geis, ointments, creams, suspensions, etc. as are well-known in the art.
Systemic formulations include those designed for administration by injection, e.g.,
subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those
designed for transdermaJ, transmucosal oral or pulmonary administration.
Useful injectable preparations include sterile suspensions, solutions or emulsions of the
active compounds) in aqueous or oily vehicles. The compositions may also contain formulating
agents, such as suspending, stabilizing and/or dispersing agent. The formulations for injection
may be presented in unit dosage form, e.g., in ampoules or in multidose containers, and may
contain added preservatives.
Alternatively, the injectable formulation may be provided in powder form for
reconstitution with a suitable vehicle, including but not limited to sterile pyrogen free water,
buffer, dextrose solution, etc., before use. To this end, the active compound(s) may be dried by
any art-known technique, 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 known in the art.
For oral administration, the pharmaceutical compositions may take the form of, for
example, lozenges, tablets or capsules prepared by conventional means with pharmaceutically
acceptable excipients such as binding agents (e.g., pregelatinised maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline
cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium
lauryl sulfate, lecithin). The tablets may be coated by methods well known in the art with, for
example, sugars, films or enteric coatings.
Liquid preparations for oral administration may take the form of, for example, elixirs,
solutions, syrups or suspensions, or they may be presented as a dry product for constitution with
water or other suitable vehicle before use. Such liquid preparations may be prepared by
conventional means with 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 vehicles (e.g., almond oil, oily esters, ethyl alcohol, cremophore™ or
fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or
sorbic acid). The preparations may also contain buffer salts, preservatives, flavoring, coloring and
sweetening agents as appropriate.
Preparations for oral administration may be suitably formulated to give controlled release
of the active compound or prodrug, 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 routes of administration, the active compound(s) may be
formulated as solutions (for retention enemas) suppositories or ointments containing conventional
suppository bases such as cocoa butter or other glycerides.
For nasal administration or administration by inhalation or insufflation, the active
compound(s) or prodrug(s) can be conveniently delivered in the form of an aerosol spray from
pressurized packs or a 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 pressurized aerosol, the dosage unit may be
determined by providing a valve to deliver a metered amount Capsules and cartridges for use in
an inhaler or insufflator (for example capsules and cartridges comprised of gelatin) may be
formulated containing a powder mix of the compound and a suitable powder base such as lactose
or starch.
For ocular administration, the active compound(s) or prodrug(s) may be formulated as a
solution, emulsion, suspension, etc. suitable for administration to the eye. A variety of vehicles
suitable for administering compounds to the eye are known in the art Specific non-limiting
examples are described in 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. Patent No. 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 prolonged delivery, the active compound(s) or prodrug(s) can be formulated as a
depot preparation for administration by implantation or intramuscular injection. The active
ingredient may be formulated with suitable polymeric or hydrophobia materials (e.g., as an
emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, e.g., as a
sparingly soluble salt Alternatively, transdermal delivery systems manufactured as an adhesive
disc or patch which slowly releases the active compound(s) for percutaneous absorption may be
used. To this end, permeation enhancers may be used to facilitate transdermal penetration of die
active compound(s). Suitable transdermal patches are described in for example, U.S. Patent No.
5,407,713; U.S. Patent No. 5,352,456; U.S. Patent No. 5,332,213; U.S. Patent No. 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 pharmaceutical delivery systems may be employed. Liposomes and
emulsions are well-known examples of delivery vehicles that may be used to deliver active
compound(s) or prodrug(s). Certain organic solvents such as dimethylsulfoxide (DMSO) may
also be employed, although usually at the cost of greater toxicity.
The pharmaceutical compositions may, if desired, be presented in a pack or dispenser
device which may contain one or more unit dosage forms containing the active compound(s). The
pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for administration.
6.8 Effective Dosages
The active compound(s) or prodrug(s), or compositions thereof, will generally be used in
an amount effective to achieve the intended result, for example in an amount effective to treat or
prevent the particular disease being treated. The compound(s) may be administered
therapeutically to achieve therapeutic benefit. By therapeutic benefit is meant eradication or
amelioration of the underlying disorder being treated and/or eradication or amelioration of one or
more of the symptoms associated with the underlying disorder such that the patient reports an
improvement in feeling or condition, notwithstanding that the patient may still be afflicted with
the underlying disorder. Therapeutic benefit also includes halting or slowing the progression of
the disease, regardless of whether improvement is realized.
The amount of compound administered will depend upon 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, etc. Determination of an effective dosage is well within the capabilities of
those skilled in the art.
Effective dosages may be estimated initially from in vitro assays. For example, an initial
dosage for use in animals may be formulated to achieve a circulating blood or serum
concentration of active compound that is at or above an IC50 of the particular compound as
measured in an in vitro assay, such as the in vitro assays described in the Examples section.
Calculating dosages to achieve such circulating blood or serum concentrations taking into account
the bioavailability of the particular compound is well within the capabilities of skilled artisans.
For guidance, the reader is referred to Fingl & Woodbury, "General Principles," In: Goodman
and Oilman "s The Pharmaceutical Basis of Therapeutics, Chapter 1, pp. 1-46, latest edition,
Pergamon Press, and the references cited therein.
Initial dosages may also be estimated from in vivo data, such as animal models. Animal
models useful for testing the efficacy of compounds to treat or prevent the various diseases
described above are well-known in the art. Dosage amounts will typically be in the range of from
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 upon, among other factors, the activity of the compound, its bioavailability, the mode
of administration and various factors discussed above. Dosage amount and interval may be
adjusted individually to provide plasma levels of the compound(s) which are sufficient to
maintain therapeutic or prophylactic effect. For example, the compounds may be administered
once per week, several times per week (e.g., every other day), once per day or multiple times per
day, depending upon, among other things, the mode of administration, the specific indication
being treated and the judgment of the prescribing physician. In cases of local administration or
selective uptake, such as local topical administration, the effective local concentration of active
compound(s) may not be related to plasma concentration. Skilled artisans will be able to optimize
effective local dosages without undue experimentation.
Preferably, the compound(s) will provide therapeutic or prophylactic benefit without
causing substantial toxic ity. Toxicity of the compound(s) may be determined using standard
pharmaceutical procedures. The dose ratio between toxic and therapeutic (or prophylactic)
LD5o/EDSo effect is the therapeutic index (LDso is the dose lethal to 50% of the population and
EDso is the dose therapeutically effective in 50% of the population). Compounds(s) that exhibit
high therapeutic indices are preferred.
6.9 Kits
The compounds and/or prodrugs described herein may be assembled in the form of kits,
hi some embodiments, the kit provides the compound(s) and reagents to prepare a composition
for administration. The composition may be in a dry or lyophilized form, or in a solution,
particularly a sterile solution. When the composition is in a dry form, the reagent may comprise a
pharmaceutically acceptable diluent for preparing a liquid formulation. The kit may contain a
device for administration or for dispensing the compositions, including, but not limited to syringe,
pipette, transdermal patch, or inhalant
The kite may include other therapeutic compounds for use in conjunction with the
compounds described herein, hi some embodiments, the therapeutic agents are other anti-cancer
and anti-neoplastic compounds. These compounds may be provided in a separate form, or mixed
with the compounds of the present invention.
The kits will include appropriate instructions for preparation and administration of the
composition, side effects of the compositions, and any other relevant information. The
instructions may be in any suitable format, including, but not limited to, printed matter, videotape,
computer readable disk, or optical disc.
7. EXAMPLES
The inventions are further defined by reference to the following examples, which describe
the preparation of the various compounds described herein, methods for assaying their biological
activity, and methods for then- use. It will be apparent to the skilled artisan that many
modifications, both to the materials and methods may be practiced without departing from the
scope of the inventions.
7.1 Preparation of 4-(4-Metkylpiperazin-l-yl>-3-Methylinitrobenzene
(Figure Removed)
Procedure: A homogeneous mature of 4-fluoro-3-methylnitrobenzene 1 (20 g, 129
mmol) and N-methylpiperazine 3 (25.82 g, 258 mmol) in JV-methylpyrrolidone (NMP) (10 mL)
was refluxed (120 °C) under Nj for 24 hours. The reaction mixture upon cooling to room
temperature was poured over a saturated NaCl solution (100 mL). The resulting solid was
sonicated for approx. 30 seconds, filtered, washed with ice-cold water (2x10 mL) and dried
under high vacuum to obtain 4-(4-methylpiperazin-l-yl)-3-methyhitrobenzene 5 (28 g, 92%). ]H
NMR (CD3OD): 5 8.02 (m, 2H), 7.13 (d, 1H, J= 9.3 Hz), 3.08 (m, 4H), 2.66 (m, 4H), 2.38 (s,
6H); LCMS: purity: 99%, MS (m/e): 236 (MH*).
7.2 Preparation of 4-(4-Methylpiperazin-l-yl)-3-MethyIaniiine
Reaction:
(Figure Removed)
Procedure: A heterogeneous mixture of 4-(4-methylpiperazinyl)-3-methyhiitrobenzene 5
(20 g, 85 mmol), 10% Pd/C (1.3 g) in methanol (1.2 liter) was hydrogenated [H2] at 40 PSI for 3
hours. The palladium catalyst was filtered through a pad of celite, washed with methanol (3 x 50
mL) and the combined filtrate was concentrated to afford 4-(4-methylpiperazin-l-yl)-3-
methylaniline 7 (15 g, 86%). 1H NMR (CD3OD): 8 6.83 (d, 1H, J= 8.7 Hz), 6.59 (d, 1H, J= 2.7
Hz), 6.54 (dd, 1H, J= 8.4 and 2.7 Hz), 2.84 (t, 4H, J= 4.8 Hz), 2.60 (bm, 4H), 2.34 (s, 3H), 2.20 (s,
3H); LCMS: purity: 99.9%, MS (m/e): 206 (MH4).
7.3 Preparation of 3-Aza-4-oxo-tricyclo[4.2.1.0(2,5)]non-7-ene
Procedure: Parti: A solution of 2,5-norbornadiene 47 (25.0 mL, 0.246 mole) in
CH2C12 (110 ml,, fresh bottle) was cooled in an ice/NaCl bath (-10 °C). To this was added dropwise
a solution of CSI (21.4 mL, 0.246 mole) in CH2C12 (45 mL, fresh bottle) at a rate to maintain
the temperature below 5°C (the addition took approx. 1.25 hr.). Upon completion of the addition,
the reaction mixture was stirred for 1 hour at 0-5°C and then removed from the cooling bath and
allowed to warm to 20°C. The reaction mixture was quenched with water (60 mL) and vigorously
stirred for several minutes. The organic layer was separated, washed with brine, and dried with
NaaSCV Concentration gave light brown oil.
Part 2: A mixture of Na2SOs (24.5 g), water (70 mL), and CH2C12 (30 mL) was cooled in
an ice/NaCl bath. The oil from Part 1 was diluted to lOOmL with CH2C12 and added dropwise to
the above mixture at a rate to maintain the temperature below 15°C (the addition took approx.
1.75 hr). The pH of the reaction mixture was monitored with a pH meter and kept basic (pH 7-
10) by adjusting with 10% NaOH (w/v) (as needed). Upon completion of the addition, the
reaction mixture was stirred for 1 hour at 5-10°C (final pH was 8.5). The reaction mixture was
poured into a separatory funnel and the CH2C12 layer separated. This organic phase was a thick
and gelatinous solid suspension. It was diluted with water (approx. 400 mL) to make a more free
flowing solution. The aqueous layer was further extracted with CH2C12 (4 x 100 mL).
(Alternatively, the solids can be separated from the CH2C12 by centrifugation. The solids can then
be diluted with water (until almost all dissolved) and extracted with CH2C12). The aqueous layer
was further extracted with CH2C12 (10 X lOOmL). The CH2C12 extracts were monitored by TLC
for the presence of product. The combined organic extracts were washed with brine, dried with
MgSC4, and filtered through celite. Removal of solvent gave the desired product, racemic-2-exo-
3-endo 3-aza~4-oxo-tricyclo[4.2.1.0(2,5)]non-7-ene 14rl as white solid (20.5 g, 62%). !HNMR
(DMSO-/6): 8 8.01 (bs, 1H), 6.22 (dd, J= 3.3 and 5.4 Hz, 1H), 6.12 (dd, J= 3.3 and 5.4 Hz, 1H),
2.88 (dd, J- 1.5 and 3.3,1H), 2.79 (bs, 1H), 2.74 (bs, 1H), 1.58 (d, J= 9.3 Hz, 1H), and 1.47 (d, J-
9.3 Hz, 1H).
7.4 Preparation of 4-Oxo-3-tert-butoiycarbonylaza-tricyclo[4.2.1.0(2,5)]non-7-
(Figure Removed)
(racemic, 2-exo-3-«xo) (racemic, 2-exo-3-exo)
Procedure: A homogeneous mixture of 3-aza-4-oxo-tricyclo[4.2.1.0(2,5)]non-7-ene
(14rl; racemic-2-exo-3-exo; 10.0 g, 74 mmol), (BOQjO (16.1 g, 74 mmol) and DMAP (1.1 g) in
CH2C12 was stirred under N2 at room temperature for 24 hours. To this reaction mixture were
added EtOAc (100 mL) followed by H2O (100 mL) and stirred for additional 1 hour. The organic
layer was separated and washed with H^O (2 x 100 mL). The organic layer was dried over
anhydrous Na2SC>4 and solvent was removed under a reduced pressure to afford 4-oxo-3~tertbutoxycarbonylaza-
tricyclo[4.2.1.0(2,5)]non-7-ene (16rl; racemic-2-exo-3-exo) (16.5 g, 70%);
!HNMR(DMSO-6):56JZ9(dd,J=3.3and5.4Hz, lH),6.19(dd, J= 3.3 and5.4Hz, 1H), 3.77
(d, J- 4.5 Hz, 1H), 3,13 (bs, 1H), 3.08-3.04 (m, 1H), 2.93 (bs, 1H), 1.45 (s, 9H). LCMS: 95%.
7.5 Preparation of, aad Isolation of, Stereoisomerically Pare Diastereomers
From (±) Racemic (2-exo-3-exo)-N4-(3-aminocarbonyIbicyclo[2.2.1]hept-5- •
en-2-y!)-5-fluoro-N2-[3-methyl-4-(4-niethylpiperazin-l-yI)pheByll-2,4-
pyrimidinediamine
A racemic mixture of the title compound was prepared from the 2-exo-3-exo racemate of
2-aminobicylco[2,2.1]hept-5-ene-3-carboxamide as follows.
Reaction:
(Figure Removed)
Procedure: A round bottom flask equipped with a rubber septum and a magnetic stirring
bar was charged with racemic N-BOC-p-lactam 16rl (2.0 g) under a positive pressure of nitrogen.
To mis were added ethyl acetate (25 mL) followed by 30% ammonia in water (25 mL) and stirred
at room temperature for 3 hours. The ethyl acetate layer was separated and washed with 5%
aqueous solution of NaHCOj (20 mL), dried over anhydrous Na2SO4 and solvent was evaporated
to afford 1.10 gm of racemic N-BOC carboxyamide 28rl.
(Figure Removed)
Procedure: A round bottom flask equipped with N2 inlet and a magnetic stirring bar was
charged with racemic N-BOC lactam 28rl (2.00 g, 7.9 mmol) and then treated with 20% of TFA
in CH2C12 at room temperature for 2 hours. The resulting solution was concentrated under a
reduced pressure. The trace of TFA was removed under high vacuum for several hours to afford
the intermediate, TFA salt (30rl, racemic). The resulting racemic TFA salt 30rl was treated with
2,4-dichloro-5-fluoropyrimidme 10 (1.58 g, 9.51 mm) in MeOH:H2O (20:10 mL) in the presence
of NaHCO3 (1 .33 g, 15.84 mmol) at room temperature for 48 hours. The reaction mixture was
diluted with H2O (25 mL), satured with Nad and extracted with EtOAc (3 x 50 mL). Upon
drying over anhydrous Na2SCO4, the solvent was evaporated and the residue was chromatographed
(silica gel, CH2C12 then 2-4% 2N NHs/MeOH in CH2C12) to afford 1 .3 g of racemic mono-SNAr
product 36rl.
Reaction:
(Figure Removed)
Procedure: A sealed tube charged with racemic mono-SNAr product 36rl (1.1 g, 8
mmol), aniline 7 (0.90 g, 4.4 mmol), TFA (0.6 mL) and methanol (9 mL) was stirred at 100 °C for
24 hours. The resulting viscous homogeneous solution was concentrated and the residue was
chromatographed (silica gel, CH2C12 then 2-5% 2N NH3/MeOH in CH2C12) to afford the expected
2-exo-3-exo racemic 2,4-pyrimidinediamine derivative 60rl (1.12 g; purity: 95%);
Isolation of Enantionmers: The diastereomers were resolved and isokted from
racemate 60rl by chiral preparative HPLC chromatography Phenomenex Chkex 3020 250 x
at a flow rate of 6mL/min. The enantiomer eluting at 9.44 min. was designated the El enantiomer
and the enantiomer etuting at 12.74 min. was designated the E2 enantiomer.
7.6 Enzymatic Preparation of StereoisomericaUy Pure (lIpR^S,4S)-N4-(3-
Aminocarbonylbkyclo[2.2.1]hept-5-en-2-yl)-5-fl«oro-N2-[3-methvl-4-(4-
methylpiperazin-l-yl)phenyI]-2,4-pyrimidinediamine Using Chirazyme
7.6.1 Preparation of Stereochemically Pure N-Boc-p-Lactam
Reaction:
Chirazyme L-2, type B, c.f. ,/ x // x 2
diteopropyf ether. 60°C, 60 hr s ^ fSl-NBoc + s ( A . , .NHBoc
(racemic, 2-exo-3~exo) N-Boc carboxylic acid
Procedure: A dry sealed tube charged with 4-oxo-3~tert-butoxycarbonylazatricyclo[
4.2.1.0(2,5)]non-7-ene (16rl; racemic-2-exo-3-exo) (4.0 g, 17.02 mmol), resin
bound/immobilized chirazyme L-2, type B, c.f. (8.0 g, purchased from BioCatalytics Lie.,
Pasadena, CA) and diisopropyl ether (80 mL) was gently shaken in an incubator at 60 °C for 60
hours. (The enzymatic resolution of racemic N-BOC pMactam 16rl was followed by proton
NMR. The integration of tert-butyl group of enantiomerically pure N-BOC lactam 16a and NBOC
carboxylic acid 26b was seen in 1:1 ratio). The resulting reaction mixture was filtered and
the solid resin was washed with diisopropyl ether (2 x 40 mL). The filtrate was concentrated to
afford a mixture of enatiomerically pure N-BOC-pMactam 16a and N-BOC carboxylic acid 26b
(total mass: 4.0 gm).
Reaction:
(Figure Removed)
N-Boc carboxylic acid (remains in organic phase) (remains in aqueous solution)
Procedure: A round bottom equipped with a rubber septum and a magnetic stirring bar
was charged with a mixture of enantiomerically pure N-BOC-lactam 16a and N-BOC carboxylic
acid 26b (4.0 g) under a positive pressure of nitrogen. To this were added ethyl acetate (SO mL)
followed by 25% aqueous ammonium hydroxide (50 mL) and stirred at room temperature for 3
hours. The reaction progress was monitored by TLC. The ethyl acetate layer was separated and
washed with 5% aqueous solution of NaHCOj (40 mL), dried over anhydrous NazSO4 and solvent
was evaporated to afford 2.00 gm (7.93 mmol out of a theoretical 8.51 mmol; 93% yield) of the
desired enantiomerically pure N-BOC carboxamide 28a with greater than 99% enantiomeric
excess, as determined by chiral HPLC. The aqueous solution containing the N-BOC ammonium
carboxylate upon acidification with cold IN HC1 followed by extraction with CH2C12 regenerated
me N-BOC carboxylic acid 26b (1.8 g, 7.11mmol out of a theoretical S.Slmmol, 84% yield). 'H
NMR (DMSO-d6): 7.26 (bs, IH), 6.66 (bs, IH), 6.13 (m, 2H), 3.59 (t, IH, J= 6.9 Hz), 2.80 (s,
IH), 2.54 (s, IH), 2.31 (d, IH, J= 8.1 Hz), 2.00 (d, IH, J= 8.7 Hz), 1.36 (s, 9H), 1.30 (d, IH, J=
8.1 Hz); LCMS. MS (m/z): 254 (MH4); [o]D -76.78 ° (c 1.0, MeOH).
7.6.2 Preparation of Stereoisomerically Pure Mono SNAr Product
Reaction.
(Figure Removed)
Procedure: A round bottom flask equipped with N2 inlet and a magnetic stirring bar was
charged with enantiomerically pure N-BOC carboxyamide 28a (2.00 g, 7.93 mmol) and then
treated with 20% of TFA in CH2C12 at room temperature for 2 hours. The reaction progress was
monitored by TLC. The resulting solution was concentrated under a reduced pressure. The trace
of TFA was removed under high vacuum for several hours to afford the enantiomerically pure
intermediate, TFA salt 30a in quantitative yield. 'H NMR (DMSO-d6): 8.10(bs,2H),7.92(s,
IH), 7.25 (s, IH), 6.29 (m, IH), 6.18 (m, IH), 4.38 (bs, IH), 3.06 (d, IH, J= 7.2 Hz), 2.97 (s, IH),
2.87 (s, IH), 2.43 (d, IH, J= 7.5 Hz), 2.10 (d, IH, J= 6 Hz), 1.36 (d, IH, J= 8.7 Hz); LCMS: MS
(m/z):152(MH+).
The resulting TFA salt 30a was treated with 2,4-dichloro-5-fluoropyrimidine 34 (1.58 g,
9.51 mmol) in MeOH;H2O (20:10 mL) in the presence of NaHCO3 (1.33 g, 15.84 mmol) at room
temperature for 48 hours. The reaction mixture was diluted with H2O (25 mL), saturated with
NaCl and extracted with EtOAc (3 x 50 mL). Upon drying over anhydrous NaaSO4 the solvent
was evaporated and the residue was chromatographed (silica gel, CH2C12 men 2-4% 2N
NHs/MeOH in CH2C12) to afford 2.02 g (91%) of desired mono-SNAr product 36a 'H NMR
(DMSO-d6): 8.25 (d, IH, J= 7.2 Hz), 8.07 (d, IH, J=3.3 Hz), 7.71 (s, IH), 7.19 (s, IH), 6.29 (m,
2H), 3.99 (t, IH, J= 7.8 Hz), 2.85 (s, IH), 2.75 (s, IH), 2.49 (d, IH, J= 0.9 Hz), 2.11 (d, IH, J=
8.7 Hz), 1.39 (d, IH, J= 8,7 Hz); LCMS: purity: 95%, MS (rn/z): 283 (MH4). The enantiomeric
purity was greater than 99% as determined by chiral HPLC; [a]D + 61.10° (c 1.0, MeOH).
7.63 Preparation of Stereoisomericalry Pure (lR>2R^S,4S)-N4-(3-
Aminocarbonylbkyclo[2.2.1]hept-5-en-2-yI)-5-fluoro-N2-[3-methyl-
4-(4-methylpiperazin-l-yl)phenyl]-2,4-pyrimidinediamine
Reaction:
(Figure Removed)
Procedure: A dry reaction flask equipped with a stirring bar, relflux condenser and an N2
inliet was charged with enantiomerically pure mono-SNAr product 36a (2.25 g, 8 mmol), aniline
7 (1.80 g, 8.8 mmol), TFA (1.12 mL) and isopropanol (18 mL) and the resulting reaction mixture
was stirred at reflux temperature for 8-10 hours. After cooling the reaction mixture to room
temperature, ethyl acetate (20 mL) was added. The solid obtained was filtered and washed with
ethyl acetate (2x5 mL) to afford compound 60a in the form of acidic salt The resulting solid
was then taken into water and the aqueous mixture adjusted to pH 9 with aqueous NaHCO3,
which caused precipitation of a solid. The solid was filtered from the mixture, washed with water
and dried to give 3.3 g (93%) of 2,4-pyrimidinediamine derivative 60s. 1H NMR (DMSO-d6):
8.85 (s, IH), 7.83 (d, IH, J= 2.7 Hz), 7.68 (s, IH), 7.47 (s, 2H), 7.36 (d, IH, J= 7.8 Hz), 7.18 (s,
IH), 6.89 (d, IH, J= 8.7 Hz), 6.32 (m, IH), 6.25 (m, IH), 4.11 (t, IH, J= 7.8 Hz), 3.32 (s, 3H),
2.86 (s, IH), 2.76 (m, 4H), 2.49 (m,4H), 2.46(m, 2H), 2.21 (s, 3H), 2.11 (d, IH, J= 8.4Hz), 1.39
(d, IH, J= 9Hz); LCMS: purity: 100 %, MS (m/z): 452 (M1); > 99 %ee as determined by chiral
HPLC; |a]D
RT +101.2° (c 1.0, MeOH). The chiral analytical data, JH NMR and LCMS were
found to be identical with the enantiomer designated El.
7.7 Enzymatic Preparation of Stereoisomerically Pure (lR^2R,3S,4S)-N4-(3-
AminocarbonylbicycIo[2.2.11hept-5-en-2-yl)-5-fluoro-N2-[3-methyl-4-(4-
methyipiperaziii~l~yl)phenyI]-2,4-pyrimidinediamuie Using Novazyme 435
Enzyme
7.7.1 Preparation of Stereoisomerically Pure pVLactam
Reaction:
lipolase
Procedure: Immobilized Lipolase (8.0 g, from Sigma, order number L4777), (Mactam
14rl (racemic: 2-exo-3-exo) (4.0 g, 7.4 mmol) and water (0.13 ml, 7.4 mmol) were added to 250
ml diisopropyl ether in a pressure flask. The mixture was degassed with nitrogen for 20 minutes
and the flask was sealed and incubated for 14 days at 70°C. The mixture was cooled to room
temperature, filtered over celite and washed with 300 ml diisopropyl ether. The combined filtrate
was concentrated to dryness and the residue was crystallized from diisopropyl ether to give the
enantiomerically pure EMactam 14a as colorless needles (1.22 g, 61%). The enantiomeric purity
was greater than 99% as determined by chiral HPLC.
7.7.2 Preparation of StereoisomericaUy Pure 2-N-Boc-amino-3-
aniiiH>earbonyl-bicyclo[2.2.1] hept-5-ene
Reaction:
(Figure Removed)
Procedure: A homogeneous mixture of enantiomerically pure 3-aza-4-oxotricyclo[
4.2.1.0(2,5)]non-7-ene 14a (1.1 g, 8.2 mmol), (BOC^O (2.76 g, 12.3 mmol) and DMAP
(100 mg) in CH2C12 was stirred under N2 at room temperature for 3 hours to give enantiomerically
pure N-BOC lactam 16a, which was used further without isolation. To this reaction mixture was
added 20 ml of 25% aqueous ammonium hydroxide and stirring was continued for another 4
hours. Water was added and the reaction mixture was extracted with dichloromethane (2 x 50ml).
The combined organic phase was washed with aqueous HC1 (5%), dried over sodium suifate and
reduced to dryness under reduced pressure to give enantiomerically pure N-BOC carboxyamide
28a (2.51 g) as a white solid, which was used in the next step without further purification.
7.7.3 Preparation of Stereoisomerically Pure Mono SNAr Product
(lRv2R,3S,4S)-N4-(3-AminocobonyIbicyclo[2^.1]hepl-5-en-2-yl)-2-
chloro-5-fluoro-4-aininopvriduie
Reaction:
(Figure Removed)
Procedure: The enatiomerically pure N-BOC carboxyamide 28a (2.51 g) was dissolved
in 10 ml dichloromethane and treated with 10 ml TFA. The mixture was stirred for 1 hour at room
temperature and concentrated to dryness under reduced pressure. The residue was suspended in
toluene and again concentrated to dryness. The resulting solid was dissolved in methanol;water
(30 ml:3 ml) and treated with 1.5 g sodium bicarbonate. The 5-fluoro-2,4-dichloropyrimidine 34
(3 g, 17.9 mmol) was added and the mixture was stirred for 2 days at room temperature. The
volatiles were removed under vacuum and the residue was suspended in brine. The precipitate
was filtered, dried and subjected to column chromatography (silica gel, dichloromethanemethanol,
20:1) to give the desired enantiomerically pure mono-SNAr product 36a as a white
solid (1.7 g, 74%).
7.7.4 Preparation of Stereoisomerically Pore (lR^R3S,4S)-N4-(3-
AmiBocarboaylbicyclo[2.2.1]hept-5-en-2-yl)-5-fluoro-N2-l3-methyl-
4-(4-methylpiperazin-l-yI)phenyl]-2,4-pyrimidinediamine
(Figure Removed)
Procedure: A homogeneous mixture of aniline 7 (400mg, 1.95 mmol), enantiomerically
pure mono-SNAr product 36* (400 mg, 1.41 mmol) and 0.2 ml TFA in 4 ml isopropanol in a
sealed tube was stirred at 100 °C for 20 hours. The mixture was cooled to room temperature,
diluted with 2 ml diethylether and the resulting precipitate was filtered and washed with
diethylether. The remaining solids were dissolved in water and treated with aqueous 25%
ammonium hydroxide solution. The resulting precipitate was filtered, washed with water and
dried to give 527 mg (83%) of desired product, 2,4-pyrimidindiamine derivative 60a as an offwhite
solid. Purity was determined by LCMS to be greater than 97% and the enantiomeric purity
was determined by chiral HPLC to be greater than 99%. The chiral analytical data, 'H NMR and
LCMS analyses were identical with the enantiomer that was designated El.
7.8 Preparatioa of Stereoisomerically Pure Compounds Using (R)-Methyl-/>-
Methoxybenzylamine as a Chiral Auxiliary
7.8.1 Preparation of 2-Eio-3-Exo Racemic Amines
(Figure Removed)
Procedure: A homogeneous mixture of 4-oxo-3-tert-butoxycarbonylazatricyclo[
4.2.1.0(2,S)]non-7-ene (16rl; racemic~2-exo-3-exo) (9.2 g, 40 mmol) and CR)-methyl-4-
methoxylbenzylamine 13 (18, 24 g, 48 mmol) in dry THF (75 mL) was stirred at room
temperature for 48 hours. The reaction mixture was concentrated, suspended in hexanes (5 mL),
sonicated and the solid was separated by filtration to give mixture of diasterisomers 20a and 20b
(12 mg). Alternatively, the purification can be done using column chromatography (silica gel,
hexanes then 5%, 10%, 20% and 50% EtOAc in hexanes).
7.8.2 Preparation of 2-Exo-3-Exo Racemic Mono SISi Ar Products Followed
By Separation of Isoraericalty Pare Compounds by Crystallization
Reaction:
(Figure Removed)
Procedure: A heterogeneous mixture of diasterisomers 20a and 20b (6.0 g g, 17 mmol),
TFA (20 mL) in CHzClj was stirred at room temperature for 2 hours. TLC was used to monitor
the progress of the reaction. The resulting reaction was concentrated to dryness and dried under a
high vacuum for several hours to afford a diasterisomeric mixture of intermediates 22a and 22 b.
This mixture was then reacted with 2,4-dichloro-S-fluoropyrimiduie 34 (3.4 g, 20 mmol) in the
presence of NaHCO3 (5.7 g, 68 mmol) in MeOH'-HjO (50 mL, each) at room temperature for 24
hours. The reaction mixture was then diluted with NaCl-saturated water (50 mL) and extracted
with CHiCl2. The extract upon drying over anhydrous NajSC^ followed by removal of solvent
under reduced pressure gave a residue, which was chromatographed (silica gel, CHjCfe then 2%
2N NHa/MeOH in CH^Ck). The chromatographic purification gave a mixture diasterisomers 38a
and 38b (4.0 g) (1:1 ratio can be seen with a clear separation on reverse phase LCMS). The
resulting 4.0 grams upon crystallization using EtOAc:hexanes (30:150 mL; v/v) afforded
crystalline material of intermediate 38a, which was confirmed by X-ray crystal structure;
chemical purity: 96% and % de: 96%. [a]D -36.7° (c, 0.18 MeOH). The mother liquor containing
the other isomer had poor % de (70-80%), which is assumed to be diastereoisomer 38b.
7.8.3 Preparation of Stereoisomerically Pure Product Including the Chiral
Auxiliary
(Figure Removed)
Procedure: A mixture of diastereoisomer 38a (1.42 g, 3.4 mmol), aniline 7 (0.834 g, 4.0
mmol) and TFA (700 mg) in MeOH (10 mL) was heated in a sealed tube at 100 °C for 24 hours.
The resulting residue was chromatographed (silica gel, CHjClj then 2% 2N NH3/MeOH in
CH2C12) to afford product 40a as colorless solid, chemical purity: 96%.
7.8.4 Cleavage of the Chiral Auxiliary
The cleavage of chiral auxiliary from 40a was found to be difficult, therefore the cleavage
of chiral auxiliary from intermediate compounds 38a and 38b followed by the second SNAr
reaction with aniline 7 was carried as follows.
7.8.5 Cleavage of the Chiral Auxiliary From Stereoisomerically Pure
Intermediate 38a and Preparation of Stereoisomerically Pure
(!R^Rr3S,4S)-N4-(3-Aminocarbonylbicyclo[2.2.1]hept-5-en-2-yl)-5-
fluoro-N2-[3-methyl-4-(4-methylpiDerazin-l-yl)phenyl]-2,4-
pyrimidwediamine
(Figure Removed)
Procedure: The mono-SNAr product with chiral auxiliary 38a was allowed to react
with DDQ (3 equivalents) in CH2Cl2:H2O at room temperature to obtain the desired mono-SNAr
product 36a. The mono-SNAr product was purified by column chromatography and found to be
same as compound 36a obtained via enzymatic route, which was confirmed by chiral analytical
HPLC, LCMS and 'H NMR. Further, the reaction of mono-SNAr product 36a with aniline 7 in
MeOH:TFA at 100 °C in a sealed tube for 24 h gave the desired product 60a. It was purified by
column chromatography and analyzed by 'HNMR, LCMS and chiral analytical HPLC. The chiral
analytical HPLC, LCMS and *H NMR analyses indicated that the data for the product 60a was
matching with the enantiomer designated El.
7.8.6 Cleavage of the Chiral Auxiliary From Intermediate 38b and
Preparation of Stereoiaomerically Pare (IS^S 3R,4R>-N4-(3-
AminocarbonyIbkycio[2.2.1]hept-S-eB-2.yI).5-fluoro-N2-[3-methyI-
4-(4-methylpJpentrin-l-vf)pfaeiiyl]-2,4-pyrijmfdinediamine
Reaction:
Procedare: The mono-SNAr product 38b was allowed to react with DDQ (3 equivalents)
in CHjCkHjO at room temperature to obtain the desired mono-SNAr product 36b (after the
cleavage of chiral auxiliary). The mono-SNAr product was purified by column chromatography
and found to be a different diastereoisomer than that-was obtained via enzymatic route, and this
was confirmed by chiral analytical HPLC. Further, the reaction of mono-SNAr product 36b with
aniline 7 in MeOH:TFA at 100 °C in a sealed tube for 24 h gave the desired product 60b. ft was
purified by column chromatography and analyzed by 'HNMR, LCMS and chiral analytical
HPLC. The chiral analytical HPLC, LCMS and JH NMR analyses radicated that the data for
product 60b was identical with the enantiomer designed E2. [a]D
RT -102.00° (c, 1.0 MeOH).
7.9 Preparation of HC1 Salts
HC1 salts of the racemate 60rl and stereoisomericalry pure 60a were prepared as
described below. .
7.9,1 Preparation of Racemic N4-(3-Amuiocarboaylbicyclo[2J.l]hept-5-
en-2-y!)-5-flnoro-N2-[3-inetliyl-4-(4-methylpipera2iii-l-yt)pheByl]-
2,4-pyTimidinediamine Hydrogen Chloride Salt
To a solution of 2-exo-3-exo racemic N4-(3-amfaocarbonylbicyclo[2,2.1]hept-5-ene-2-
yl)-5-fluoro-N2-{3-methyl-4- (0.140 g, 0.3 mmol) in MeOH (3 mL) at 0 °C was added HC1 (4M, dioxane, 0.170 mL, 0.681
mmol) dropwise and then stirred at 0 °C for Ih and room temperature for 15 minutes. The clear
homogeneous solution was filtered, concentrated and redissolved in EtOH. Ethyl acetate was
added to the ethanolic solution to precipitate the desired product, which was isolated to give 2-
exo-3-exo racemic N4-(3-aminocarbonylbicyclo[2.2.1 ]hept-5-ene-2-yl)-5-fluoro-N2-[3-methyl-4-
(4-methylpiperazm-1-yl)phenyl]-2,4-pyrimidinediamine bis hydrogen chloride salt (compound
60rl-2HCl). LCMS: purity: 98%; MS (m/e): 453 (MH+).
7.9.2 Preparation of Stereofeomericatty pure (lR^Ry3Sy4S)-N4-(3-
Aminocarbonylbicyclo[2.2.1]hept-5-en-2-yl)-5-fluoro-N2-[3-methyl-
4-(4-metbyIpiperazin-l-yl)phenyl]-2,4-pyrimidinediamine Hydrogen
Chloride Salt
In a like manner, supra, the interaction of 2 equivalents of HC1 (4M, dioxane) with
stereoisomericaily pure (1 R,2R,3S,4S)-N4-(3-aminocarbonylbicyclo[2.2. l]hept-5-ene-2-yl)-5-
fluoro-N2-[3-methyl-4^4-me1ivylpipera2Ui-l-yl)phenyl]-2,4-pyrimidinediamine (60a) gave
stereoisomericaily pure (!R,2R,3S,4S)-N4-(3-aminocarbonylbicyclo[2.2.1]hept-5-ene-2-yl)-5-
fluoro-N2~[3-inethy l-4-(4-methylpiperazin-l -yl)phenyl]-2,4-pyrimidinediamine bis hydogen
chloride salt (compound 60a-2HCI). LCMS: purity: 97%; MS (m/e): 453 (MH4); [a]D +46.3° (c,
0.04 MeOH).
7.10 Preparation of (1R^R,3S,4S) N4-(3-AminocarbonylbicycIo[2J.lJhept-5-ene-
2-yI)-5-fluon>-N2-[3-(lrJ-oxazol-2-yl)phenyl]-2,4-pyriniidinedianiine
H2NOC
(lR,2R,3S,4S)N4^3-aminocarbonylbicyclo[2.2.1]hept-5-ene-2-yl)-5-fluoro-N2-[3-(l,3-
oxazol-2-yl)phenyl]--2,4-pyrimidinediamine (Compound 90a) was prepared as described above.
'HNMRtDMSO-dC): 9.36 (s, 1H), 8.48 (s, 1H), 8.14 (s, 1H), 9.92 (d, 1H, J= 3 Hz), 7.79 (d, 1H,
J= 7.8 Hz), 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=
4.8 Hz), 2.86 (s, 1H), 2.77 (s, 1H), 2.55 (d, 1H, J= 8.1 Hz), 2.14 (d, 1H, J= 8.4 Hz), 1.39 (d, 1H,
J= 8.7 Hz); LCMS: purity: 98%, MS (m/e): 407 (MH4).
7.11 Inhibition of Cellular Proliferation la Vitro
Compounds 60rl, 60r2,60rl-2HCl, 60a, 60b and 60a-HCl were tested against a variety
of different types of tumor cells for their ability to inhibit proliferation using standard in vitro
antiproliferation assays. The various cells lines tested included: A549 (lung carcinoma); ASPC-1
(pancreatic adenocarcinoma); BXPC-3 (pabcreatic adenocarcinoma); CaOV-3 (ovarian
adenocarcinoma); COLO 205 (colorectal adenocarcinoma); DU145 (prostate carcinoma); ES-2
(ovarian clear cell carcinoma); H1299 (non-small cell lung carcinoma); H1155 (non-small cell
lung carcinoma); H460 (large cell lung carcinoma); HELA (cervical adenocarcinoma); HL160
(promyeloblast promyelocytic leukemia); K562 (bone marrow chronic myelogenous leukemia);
L1210 (mouse lymphocytic leukemia); MiaPaCa-2 (pancreatice carcinoma); MOLT4 (T
lymphoblast acute lyraphoblastic leukemia); OVCAR-3 (ovarian adenocarcinoma); MOLT3 (T
lymphoblast acute lymphoblastic leukemia); OVCAR-8 (ovarian carcinoma); PC3 (prostate
adenocarcinoma); SK-OV-3 (ovarian adenocarcinoma); SU86.86 (pancreatic carcinoma); SW620
(colorectal adenocarcinoma); THP-1 (monocyte acute monocytic leukemia); TOV-21G (ovarian
clear cell carcinoma); U2OS (bone osteosarcoma); and U937 (histiocytic lymphoma).
The ICso values obtained with the compounds are provided in TABLE 2, below. In
TABLE 2, a "+" indicates an IC50 value of "+++" indicates an IC50 value of 1 uM. A blank
indicates that the compound was not tested against the specific cell line.
7.12 Inhibition of Aurora Kinases in Functional Cellular 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 the assay,
A549 cells were seeded into the wells of a microtiter tray (5000 cells/well in 100 ul F12K media)
late in the afternoon on Day 1. The cells were grown overnight (37°C, 5% COj). On Day 2, 50 ul
nocodazole (1 uM in media) was added to each well, giving a final concentration of 333 nM.
Cells were grown for an additional 18 hrs under the same conditions.
On Day 3, 50 ul aligupts of varying concentrations of test compound were added to the
wells. Test compounds were prepared by 2-fold serial dilution of a 2mM stock (in DMSO). The
diluted compounds in DMSO were then further diluted 1:50 with media to yield a final solution
containing 4X test compound, 98% media, 2% DMSO. After incubation, the media/test
compound was washed and the cells fixed with 2% para-formaldehyde (in Dulbecco's phosphate
buffered saline "DPBS"; 25 ul per well; > 20 mm incubation). The fixed cells were washed once
with DPBS (200 ul/well), stained with phospho-Histone H3 (Cell Signaling Technology; 1:500 in
DPBS, 10% normal goat serum "NGS", 0.05% Triton X-100; 1-2 hrs at room temperature), and
washed twice with DPBS (200 ul/well). The cells were then stained with a secondary antibody
labeled with a fluorescent dye (secondary antibody donkey anti-mouse AlexFluor 488 (Invitrogen
Molecular Probes; 1:2000) arid DAPI (1:15,000 of Img/ml stock) for 1 hr at room temperature,
washed three times with DPBS (200 ul/well) and stored under DPBS (100 ul/well) at 4°C until
ready for analysis.
A Zeiss Axiovert SI00 inverted fluorescent microscope with a Plan-NEOFLUAR lOx
objective, a Hamamatsu Lightningcure 200 Mercury-Xenon light source and an Omega Optical
XF57 quad filter was used for all data collection. The system was equipped with a Ludl Mac2000
motorized stage with X/Y/Z control, a Ludl filter wheel, a Zymark Twister robot arm and a
Quantix digital camera from Roper Scientific. All hardware was controlled with ImagePro 4.5
with the ScopePro/StagePro 4.1 module (Media Cybernetics) on a PC running Win2000. Visual
Basic Scripts were written for ImagePro to automate hardware control and image collection.
Focusing was performed with a software auto-focus routine contained with StagePro that used the
maximum local contrast to determine the best plane of focus from a Z series captured once in each
well. Once proper focus was achieved images were captured in a 3x3 grid pattern of adjacent
images next to, but not including, the position of focusing. Images were captured and analyzed in
12-bit format using segmentation and morphological routines contained in the Image Pro software
package. Identified nuclei were counted and pixel data for each cell along with experimental
conditions was stored in a database using MySQL 4.0.14. Subsequent analysis of experimental
results and graph creation was done using Matlab 6.5.
For phospho-histone H3 analysis the data is converted to Facs files and analysed using
FlowJo. The percent Phospho-H3 cells are plotted at each compound concentration to determine
an EC50 for Aurora B inhibition.
Results. Compound 60a inhibited Aurora kinase-B with an ICso of about 7 nM in this
assay. By contrast, the ICso of its enantiomer, compound 60b, was 2.49 uM, approx. 350 times
greater.
7.13 Phannacokinetics of Compound El in Monkeys
ompound 60a was administered to monkeys intraveinously (1 mg/kg in saline) and orally
(5 mg/kg in saline) and the plasma concentrations monitored over time. When administered by
i.v., the plasma concentration of compound remained above the ICso of 7 nM for 11 hrs following
administration; when administered orally, a plasma concentration of compound above the ICso
was maintained for over 20 hrs.
7.14 Compound 60a Shrinks Tumors In Vivo
Compound 60a-2HCl, was tested for its ability to shrink A549 and Colo205 tumors in a
standard xenograft therapeutic model in SCED mice, and Colo205 and MiaPaCa tumors in a
standard xeuograph regression model in SCID mice. When palpable tumors appeared and were of
a preselected volume (approx. 100 mm3 for treatment model; >300 mm3 for regression model),
the mice were administered test compounds in the amounts and according to the dosing regimens
specified in TABLE 3 (treatment protocol) and TABLE 4 (regression protocol), below.
(Table Removed)
Results. The inhibitory effects of Compound 60a-2HCl on Colo205 tumor growth in the
treatment model are illustrated in FIGS. 1 and 2. The results of the daily dosing regimen are
illustrated in FIG. 1; the results of the pulsed dosing regimens in FIG. 2. Both dosing regimens
yielded significant (p all dosage levels tested. A 549 tumors were less responsive to treatment resulting in an
approximate 40% reduction hi mean tumor volume following a dosing regimen of S days on/2
days off and a dose level of 10 rag/kg qd (p>0.05).
The inhibitory effects of Compound 60a-2HCl on Colo205 tumor growth in the
regression model are illustrated in FIG. 3. The effects of Compound 60a-2HCI on MiaPaCa
tumors in the regression model are illustrated in FIG. 4. Significant reductions in tumor growth
rate were observed with both tumor lines. These reductions were independent of the mode of
administration. Moreover, the reductions observed in MiaPaCa tumors were similar to those
observed with taxol (see FIG. 4).
Although the foregoing inventions have been described in some detail to facilitate
understanding, it will be apparent that certain changes and modifications may be practiced within
the scope of the appended claims. Accordingly, the described embodiments are to be considered
as illustrative and not restrictive, and the invention is not to be limited to die details given herein,
but may be modified within the scope and equivalents of the appended claims.
All literature and patent references cited throughout the application are incorporated into
the application by reference for all purposes.




CLAIMS
What is Claimed Is
1. A compound according to structural formula (I):
including prodrugs, sate, hydrates, solvates and N-oxides thereof, that is enriched in the
corresponding diastereomer of structural formula (la):
(Figure Removed)
wherein:
each R1 is independently selected from the group consisting of hydrogen, lower
alkyl, -(CH2)B-OH, -OR, -O(CH2)B-R", -O(CH2)n-Rb, -C(O)OR", halo, -CF3 and -OCF3;
each R2 is independently selected from the group consisting of hydrogen, lower
alkyl,-ORa,-0(CH2)B-Ra,-CKCHj)b,-NHC(O)R", halo,-CF3,-OCF3, N—/ and
(Figure Removed)
each R3 is independently selected from the group consisting of hydrogen, lower
alkyl, -(CH2)n-OH, -OR8, -O(CH2)n-R8,0(CH2)n-Rb, halo, -CF3,-OCF3, ^—* ,
N and
each R4 is independently selected from the group consisting of hydrogen, lower
alkyl, arylalkyl, -OR*, -NRX -C(O)R", -C(OX)Ra and -C(O)NR°RC;
R5 is hydrogen, halo, fluoro, -CN, -NO2, CO2R, or -CF3;
each n is independently an integer from 1 to 3;
each Ra is independently selected from the group consisting of hydrogen, lower
alkyl and lower cycloalkyl;
each Rb is independently selected from the group consisting of -OR", -CF3)
-OCF3 -NRCR°, -C(0)Ra, -C(O)OR", -C(O)NRCRC and -C(O)NR1IR1;
each RQ is independently selected from the group consisting of hydrogen and
lower alkyl, or, alternatively, two Rc substituents may be taken together with the nitrogen atom to
which they are bonded to form a 5-7 membered saturated ring which optionally includes
additional heteroatomic groups selected from O, NR1, NR-C(O)Ra, NRa-C(O)ORa and
NR'-C(O)NR"; and
each Rd is independently lower mono-hydroxyalkyl or lower di-hydroxyalkyl.
2. The compound of Claim 1 in which the compound according to structural
formula (I) is a (2-exo-3-exo) cis racemate.
3. The compound of Claim 1 which contains about 60% or more of the diastereomer
of structural formula (la).
4. The compound of Claim 1 which contains about 90% or more of the diastereomer
of structural formula (la).
5. The compound of Claim 1 which contains about 99% or more of the diastereomer
of structural formula (la).
6. The compound of any one of Claims 1-5 in which Rs is fluoro.
-f-N O
7. The compound of Claim 6 m which R1 is hydrogen; R2 is \ — /
(Figure Removed)
8 . The compound of Claim 7 in which R3 is hydrogen, methyl, methoxy,
trifluoromethyl or chloro.
9. The compounds of Claim 7 in which R4 is methyl, -C(O)CH3, -C(O)OCH3 or
(Figure Removed)
10. The compound of Claim 6 in which R1 is hydrogen; R2 is other than \—'
(Figure Removed)
11. The compound of Claim 10 in which R2 is hydrogen, methyl, methoxy,
trifluoromethyl or chloro.
12. The compound of Claim 10 in which R4 is methyl, -C(O)CH3, -C(O)OCH3 or
-C(O)CH2CH3.
13. The compound of Claim 6 in which R2 is other than —/
(Figure Removed)
.
14. The compound of Claim 13 in which R1 and R2 are each hydrogen and R3 is
-OCH2NHR",
15. The compound of Claim 13 in which R1, R2 and R3 are each, independently
selected from the group consisting of hydrogen, methyl, methoxy, trifluoromethyl and chloro,
with the proviso that at least two of R!, R2 and R3 are other than hydrogen.
16. The compound of Claim 6 in which R1 is hydrogen; R2 is selected from the group
-f-N O -jj-N N-R4
consisting of hydrogen, \—' and \—/ ; and R3 is selected from the group
(Figure Removed)
consisting of hydrogen, lower alkyl, halo, -CFs, VN—' and ^^—/
17. The compound of Claim 16 in which R3 is selected from the group consisting of
(Figure Removed)
4 hydrogen, methyl, chloro, -CF3, N'—' and ^—' ; and R is methyl, -COR" or
-CO(O)Ra where R" is methyl or ethyl.
1 8. The compound of Claim 16 in which R2 is selected from the group consisting of
and R3 is selected from the group consisting of
-j-N O -|-N N-R4
hydrogen, lower alkyl, halo, -CF3, \ — ' and \ — /
19. The compound of Claim 18 in which R3 is selected from the group consisting of
(Figure Removed)
20. The compound of Claim 19 in which R2 is \—f ; R4 is-COR* wherein
R* is methyl; and R3 is hydrogen.
-1-N N-R4
21. The compound of Claim 19 hi which R2 is \—f ; R4 is -CO(O)R'
wherein Ra is ethyl; and R3 is hydrogen.
22. The compound of Claim 19 in which R2 is \—' and R3 is hydrogen
-|-N O
23. The compound of Claim 19 in which R2 is hydrogen; R3 is x*—' or
-I-N N-R4
(Figure Removed)
24. The compound of Claim 19 in which R2 is \—f ; R4 is metfryl; and R3
is selected from the group consisting of hydrogen, methyl, chloro and -CF3.
25. The compound of Claim 24 in which R3 is methyl.
26. The compound of Claim 1 which is substantially pure (lR,2R,3S,4S)-N4-(3-
aminocarbonylbicyclo[2.2.1 ]hept-5-ene-2-yl)-5-fluoro-N2-[(3-methyl-4-(4-methylpiperazin-l -
yl)]phenyl~2,4-pyrimidinediamme.
27. The compound of Claim 1 which is pure (lR,2R,3S,4S)-N4-(3-
aminocarbonylbicyclo[2.2.1]hept-5-ene-2-yl)-5-fluoro-N2-t(3-methyl-4-(4-methylpiperazin-lyl)]
phenyl~2,4-pyrimidinediamine.
28. A composition comprising a compound according to Claim 1 and a carrier,
excipient and/or diluent.
29. The composition of Claim 28 in which the carrier, excipient and/or diluent is
acceptable for pharmaceutical uses.
30. A method of inhibiting proliferation of a cell comprising contacting the cell with
an amount of a compound according to Claim 1 effective to inhibit its proliferation.
31. The method of Claim 30 in which the cell is a tumor cell.
32. The method of Claim 31 in which the tumor cell is a lung, colon, breast, gastric,
ovarian, cervical, melanoma., renal, prostrate, lymphoma, neuroblastoma, pancreatic, bladder or
hepatic tumor cell.
33. A method of inhibiting an activity of an Aurora kinase comprising contacting the
Aurora kinase with an amount of a compound according to Claim 1 effective to inhibit its activity.
34. The method of Claim 33 which is carried out in vitro with an isolated or partially
isolated Aurora kinase.
35. The method of Claim 33 which is carried out in vitro with a cell expressing an
Aurora kinas.
36. A method of inhibiting an Aurora kinase-mediated process comprising contacting
a cell expressing an Aurora kinase with an amount of a compound according to Claim 1 effective
to inhibit the Aurora kinase-mediated process.
3 7. The method of Claim 3 6 in which the Aurora kinase-mediated process inhibited
is mitosis.
3 8. The method of Claim 36 in which the cell is a tumor cell.
39. The method of Claim 36 in which the cell is contacted with a concentration of the
compound that is equal to or greater than its IC50 as measured in an in vitro assay.
40. A method of treating a Aurora kinase-mediated disease, comprising administering
to a subject in need thereof an amount of a compound according to Claim 1 effective to treat the
disease.
4 i. The method of Claim 40 in which the Aurora kinase-mediated disease is a
proliferative disease.
42. The method of Claim 41 in which the proliferative disease is cancer.
43. The method of Claim 42 in which the cancer is a metastatic tumor.
44. The method of Claim 43 in which the cancer is selected from lung cancer, breast
cancer, gastric cancer, ovarian cancer, cervical cancer, melanoma, renal cancer, prostrate cancer,
lymphoma, neuroblastoma, pancreatic cancer, bladder cancer, and liver cancer.
45. The method of Claim 40 in which the compound is administered in the form of a
pharmaceutical composition.
46. The method of Claim 40 in which the compound is administered orally.
47. The method of Claim 40 in which the compound is administered intravenously.
48. The method of Claim 40 in which the subject is a human.
49. The method of Claim 40 in which the compound is administered in an amount
effective to achieve a serum concentration that is at or above the IC50 of the compound, as
measured in an in vitro assay.


Documents:

2632-delnp-2007-abstract.pdf

2632-delnp-2007-Assignment-(05-12-2013).pdf

2632-delnp-2007-Claims-(05-12-2013).pdf

2632-delnp-2007-claims.pdf

2632-delnp-2007-Correspondance Others-(30-04-2013).pdf

2632-delnp-2007-correspondece-others.pdf

2632-delnp-2007-Correspondence Others-(05-12-2013).pdf

2632-delnp-2007-description (complete).pdf

2632-delnp-2007-drawings.pdf

2632-delnp-2007-form-1.pdf

2632-delnp-2007-form-2.pdf

2632-delnp-2007-form-3.pdf

2632-delnp-2007-form-5.pdf


Patent Number 258447
Indian Patent Application Number 2632/DELNP/2007
PG Journal Number 02/2014
Publication Date 10-Jan-2014
Grant Date 09-Jan-2014
Date of Filing 09-Apr-2007
Name of Patentee RIGEL PHARMACEUTICALS, INC.
Applicant Address 1180, VETERANS BLVD., SOUTH SAN FRANCISCO, CA 94080, USA
Inventors:
# Inventor's Name Inventor's Address
1 HUI LI 2576 AMETHYST DR. SANTA CLARA, CALIFORNIA 95051, USA
2 ANKUSH ARGADE 639-1 CATAMARAN STREET, FOSTER CITY, CALIFORNIA 94404, USA
3 RAJINDER SINGH 1832, HILLMAN AVENUE, BELMONT, CALIFORNIA 94002, USA
PCT International Classification Number A61K 31/506
PCT International Application Number PCT/US2005/041359
PCT International Filing date 2005-11-15
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/628,199 2004-11-15 U.S.A.