U.S. patent application number 11/717223 was filed with the patent office on 2008-01-24 for compounds, compositions, and methods.
This patent application is currently assigned to Cytokinetics, Inc.. Invention is credited to Gustave Bergnes, Andrew McDonald, David J. JR. Morgans, Whitney W. Smith, Bing Yao.
Application Number | 20080021050 11/717223 |
Document ID | / |
Family ID | 30771199 |
Filed Date | 2008-01-24 |
United States Patent
Application |
20080021050 |
Kind Code |
A1 |
Bergnes; Gustave ; et
al. |
January 24, 2008 |
Compounds, compositions, and methods
Abstract
Compounds, compositions and methods useful for treating cellular
proliferative diseases and disorders, for example, by modulating
the activity of KSP, are disclosed.
Inventors: |
Bergnes; Gustave; (Pacifica,
CA) ; Smith; Whitney W.; (El Cerrito, CA) ;
Yao; Bing; (Hayward, CA) ; Morgans; David J. JR.;
(Los Altos, CA) ; McDonald; Andrew; (Mill Valley,
CA) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER;LLP
901 NEW YORK AVENUE, NW
WASHINGTON
DC
20001-4413
US
|
Assignee: |
Cytokinetics, Inc.
|
Family ID: |
30771199 |
Appl. No.: |
11/717223 |
Filed: |
March 12, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10626012 |
Jul 23, 2003 |
7211580 |
|
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11717223 |
Mar 12, 2007 |
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60398224 |
Jul 23, 2002 |
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Current U.S.
Class: |
514/266.2 ;
544/287 |
Current CPC
Class: |
A61P 9/00 20180101; A61P
35/00 20180101; C07D 471/04 20130101; C07D 475/02 20130101; C07D
239/91 20130101; C07D 401/04 20130101; C07D 239/88 20130101; C07D
401/12 20130101; C07D 487/04 20130101; A61P 29/00 20180101; C07D
239/70 20130101; A61P 43/00 20180101; C07D 403/04 20130101; C07D
491/04 20130101; A61P 37/00 20180101; C07D 403/06 20130101; C07D
401/06 20130101 |
Class at
Publication: |
514/266.2 ;
544/287 |
International
Class: |
A61K 31/517 20060101
A61K031/517; C07D 239/72 20060101 C07D239/72; C07D 401/02 20060101
C07D401/02 |
Claims
1. A compound selected from the group represented by Formula I:
##STR16## where: V is a covalent bond, R.sup.1, R.sup.2, R.sup.3
and R.sup.4 are independently hydrogen, hydroxy, optionally
substituted alkyl, optionally substituted alkoxy, halogen or cyano;
R.sup.5 is hydrogen, optionally substituted alkyl, optionally
substituted aryl, optionally substituted aralkyl, optionally
substituted heteroaryl, or optionally substituted heteroaralkyl;
R.sup.6 to R.sup.9 are independently hydrogen, hydroxy, optionally
substituted alkyl, optionally substituted alkoxy, optionally
substituted aryl or optionally substituted alkylamino, and R.sup.10
is hydrogen, optionally substituted alkyl, optionally substituted
aryl, optionally substituted aralkyl, optionally substituted
heteroaryl, or optionally substituted heteroaralkyl, or a
pharmaceutically acceptable salt or thereof.
2. The compound of claim 1 wherein R.sup.1, R.sup.2, R.sup.3 and
R.sup.4 are independently hydrogen, halo, lower alkyl, substituted
lower alkyl, lower alkoxy or cyano.
3. The compound of claim 2 wherein R.sup.1, R.sup.2, R.sup.3 and
R.sup.4 are independently hydrogen, chloro, fluoro, methyl,
methoxy, or cyano.
4. The compound of claim 2 where R.sup.1, R.sup.2, R.sup.3 and
R.sup.4 are hydrogen, or three of R.sup.1, R.sup.2, R.sup.3 and
R.sup.4 are hydrogen and the fourth is halo, methoxy, methyl or
cyano.
5-10. (canceled)
11. A pharmaceutical formulation comprising a pharmaceutically
acceptable excipient and an effective amount of a compound of claim
1.
12-15. (canceled)
16. A compound of the group represented by Formula II: ##STR17##
where: T is a covalent bond or optionally substituted lower
alkylene; V is a covalent bond; W, X, Y and Z are independently N,
C, O, S or absent, provided that: no more than one of W, X, Y or Z
is absent, no more than two of W, X, Y and Z are --N.dbd., and W,
X, Y or Z can be O or S only when one of W, X, Y or Z is absent;
R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are independently hydrogen,
hydroxy, optionally substituted alkyl, optionally substituted
alkoxy, halogen or cyano, provided that R.sup.1, R.sup.2, R.sup.3
or R.sup.4 is absent where W, X, Y or Z, respectively, is --N.dbd.,
O, S or is absent; R.sup.5 is hydrogen, optionally substituted
alkyl, optionally substituted aryl, optionally substituted aralkyl,
optionally substituted heteroaryl, and optionally substituted
heteroaralkyl; R.sup.6 to R.sup.9 are independently hydrogen,
hydroxy, optionally substituted alkyl, optionally substituted
alkoxy, optionally substituted aryl or optionally substituted
alkylamino, and R.sup.10 is hydrogen, optionally substituted alkyl,
optionally substituted aryl, optionally substituted aralkyl,
optionally substituted heteroaryl, or optionally substituted
heteroaralkyl, or a pharmaceutically acceptable salt thereof.
17. The compound of claim 16 wherein T is optionally substituted
lower alkylene.
18. The compound of claim 17 wherein T is methylene.
19-23. (canceled)
24. The compound of claim 1 wherein R.sup.5 is aralkyl or
substituted aralkyl.
25. The compound of claim 24 wherein R.sup.5 is benzyl or
substituted benzyl.
26. The compound of claim 1 wherein R.sup.6 to R.sup.9 are
independently hydrogen or optionally substituted lower alkyl.
27. The compound of claim 26 wherein R.sup.6 to R.sup.9 are
hydrogen.
28. The compound of claim 1 wherein R.sup.10 is optionally
substituted benzyl or optionally substituted phenyl.
29. The compound of claim 28 wherein R.sup.10 is benzyl or
p-methyl-benzyl.
30. The compound of claim 16 wherein R.sup.1, R.sup.2, R.sup.3 and
R.sup.4 are independently selected from hydrogen, halo, lower
alkyl, substituted lower alkyl, lower alkoxy, and cyano.
31. The compound of claim 30 wherein R.sup.1, R.sup.2, R.sup.3 and
R.sup.4 are independently selected from hydrogen, chloro, fluoro,
methyl, methoxy, and cyano.
32. The compound of claim 30 wherein R.sup.1, R.sup.2, R.sup.3 and
R.sup.4 are hydrogen, or three of R.sup.1, R.sup.2, R.sup.3 and
R.sup.4 are hydrogen and the fourth is halo, methoxy, methyl or
cyano.
33. The compound of claim 16 wherein R.sup.5 is aralkyl or
substituted aralkyl.
34. The compound of claim 33 wherein R.sup.5 is benzyl or
substituted benzyl.
35. The compound of claim 16 wherein R.sup.6 to R.sup.9 are
independently hydrogen or optionally substituted lower alkyl.
36. The compound of claim 35 wherein R.sup.6 to R.sup.9 are
hydrogen.
37. The compound of claim 16 wherein R.sup.10 is optionally
substituted benzyl or optionally substituted phenyl.
38. The compound of claim 37 wherein R.sup.10 is benzyl or
p-methyl-benzyl.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of co-pending
provisional U.S. Application Ser. No. 60/398,224, filed Jul. 23,
2002 incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates to quinazolinone-like derivatives
that are inhibitors of the mitotic kinesin KSP and are useful in
the treatment of cellular proliferative diseases, for example
cancer, hyperplasias, restenosis, cardiac hypertrophy, immune
disorders and inflammation.
BACKGROUND OF THE INVENTION
[0003] The mitotic spindle is responsible for distribution of
replicate copies of the genome to each of the two daughter cells
that result from cell division. Disruption of the mitotic spindle
can inhibit cell division, and induce cell death. Microtubules are
the primary structural element of the mitotic spindle; they are the
site of action of certain existing therapeutic agents used to treat
cancer, such as taxanes and vinca alkaloids. Microtubules, however,
exist as elements in other types of cellular structures (including
tracks for intracellular transport in nerve processes). The
therapeutic targeting of microtubules can, therefore, modulate
processes in addition to cellular proliferation, leading to side
effects that limit the usefulness of such drugs.
[0004] Improvement in the specificity of agents used to treat
cancer is of considerable interest because of the therapeutic
benefits that would be realized if the side effects associated with
the administration of these agents could be reduced. Dramatic
improvements in the treatment of cancer have been associated with
identification of therapeutic agents acting through novel
mechanisms. Examples of this include not only the taxanes, but also
the camptothecin class of topoisomerase I inhibitors.
[0005] One novel anti-proliferative mechanism entails selective
inhibition of mitotic kinesins, enzymes that are essential for
assembly and function of the mitotic spindle, but are not generally
part of other microtubule structures, such as in nerve processes.
See, e.g., Guidebook to the Cytoskeletal and Motor Proteins, Kreis
and Vale, Eds., pp. 389-394 (Oxford University Press 1999). Mitotic
kinesins play essential roles during all phases of mitosis. These
enzymes are "molecular motors" that transform energy released by
hydrolysis of ATP into mechanical force that drives the directional
movement of cellular cargoes along microtubules. The catalytic
domain sufficient for this task is a compact structure of
approximately 340 amino acids. During mitosis, kinesins organize
microtubules into the bipolar structure that is the mitotic
spindle. Kinesins mediate movement of chromosomes along spindle
microtubules, as well as structural changes in the mitotic spindle
associated with specific phases of mitosis. Experimental
perturbation of mitotic kinesin function causes malformation or
dysfunction of the mitotic spindle, frequently resulting in cell
cycle arrest and cell death. Mitotic kinesins are attractive
targets for the discovery and development of novel anti-mitotic
chemotherapeutics.
[0006] Among the mitotic kinesins that have been identified is KSP.
KSP belongs to an evolutionarily conserved kinesin subfamily of
plus end-directed microtubule motors that assemble into bipolar
homotetramers consisting of antiparallel homodimers. During
mitosis, KSP associates with microtubules of the mitotic spindle.
Microinjection of antibodies directed against KSP into human cells
prevents spindle pole separation during prometaphase, giving rise
to monopolar spindles and causing mitotic arrest and induction of
programmed cell death. KSP and related kinesins in other,
non-human, organisms, bundle antiparallel microtubules and slide
them relative to one another, thus forcing the two spindle poles
apart. KSP may also mediate in anaphase B spindle elongation and
focussing of microtubules at the spindle pole.
[0007] Human KSP (also termed HsEg5) has been described [Blangy, et
al., Cell, 83:1159-69 (1995); Whitehead, et al., Arthritis Rheum.,
39:1635-42 (1996); Galgio et al., J. Cell Biol., 135:339-414
(1996); Blangy, et al., J Biol. Chem., 272:19418-24 (1997); Blangy,
et al., Cell Motil. Cytoskeleton, 40:174-82 (1998); Whitehead and
Rattner, J. Cell Sci., 111:2551-61 (1998); Kaiser, et al., JBC
274:18925-31 (1999); GenBank accession numbers: X85137, NM004523
and U37426], and a fragment of the KSP gene (TRIP5) has been
described [Lee, et al., Mol. Endocrinol., 9:243-54 (1995); GenBank
accession number L40372]. Xenopus KSP homologs (Eg5), as well as
Drosophila KLP61 F/KRP1 30 have been reported.
[0008] Recently, certain substituted quinazolinones have been
described as inhibitors of mitotic kinesins for the treatment of
cellular proliferative diseases (WO 01/30768 and WO 01/98278). It
is an object of the present invention to provide novel inhibitors
of mitotic kinesins such as KSP (particularly human KSP).
SUMMARY OF THE INVENTION
[0009] The present invention provides compounds, compositions and
methods useful in the inhibition of mitotic kinesins, particularly
KSP (more particularly human KSP). The compounds can be used to
treat cellular proliferative diseases and include certain
pyrrolidin-2-one, piperidin-2-one and tetrahydro-pyrimidin-2-one
quinazolinone derivatives.
[0010] In one aspect, the invention relates to one or more
compounds selected from the group represented by Formula I:
##STR1## where: [0011] V is chosen from a covalent bond, CR'R'' and
NR''', [0012] R' and R'' being independently chosen from hydrogen,
hydroxy, optionally substituted aryl, optionally substituted
heteroaryl, optionally substituted amino, optionally substituted
alkyl and optionally substituted alkoxy, and [0013] R''' being
chosen from hydrogen, optionally substituted alkyl, optionally
substituted aryl, optionally substituted aralkyl, optionally
substituted heteroaryl, and optionally substituted heteroaralkyl;
[0014] R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are independently
chosen from hydrogen, hydroxy, optionally substituted alkyl,
optionally substituted alkoxy, halogen, nitro, optionally
substituted amino, alkylsulfonyl, alkylsulfonamido, alkylsulfanyl,
alkoxycarbonyl, aminocarbonyl, optionally substituted aryl,
optionally substituted heteroaryl, and cyano; [0015] R.sup.5 is
chosen from hydrogen, optionally substituted alkyl, optionally
substituted aryl, optionally substituted aralkyl, optionally
substituted heteroaryl, and optionally substituted heteroaralkyl;
[0016] R.sup.6 to R.sup.9 are independently chosen from hydrogen,
hydroxy, optionally substituted alkyl, optionally substituted
alkoxy, optionally substituted aryl, and optionally substituted
alkylamino, provided that neither R.sup.8 nor R.sup.9 is hydroxy or
alkoxy when V is NR'''; and [0017] R.sup.10 is chosen from
hydrogen, optionally substituted alkyl, optionally substituted
aryl, optionally substituted aralkyl, optionally substituted
heteroaryl, and optionally substituted heteroaralkyl; including
single stereoisomers, mixtures of stereoisomers, and
pharmaceutically acceptable salts, solvates, and solvates of
pharmaceutically acceptable salts thereof. Compounds of Formula I
and pharmaceutically acceptable salts and solvates thereof are
useful as active agents in the practice of the methods of treatment
and in manufacture of compositions including the pharmaceutical
formulations of the invention, and may also be useful as
intermediates in the synthesis of such active agents.
[0018] In another aspect, the invention relates to one or more
compounds selected from the group represented by Formula II:
##STR2## where: [0019] T is a covalent bond or optionally
substituted lower alkylene; [0020] W, X, Y and Z are independently
N, C, O, S or absent, provided that: [0021] no more than one of W,
X, Y or Z is absent, [0022] no more than two of W, X, Y and Z are
--N.dbd., and [0023] W, X, Y or Z can be O or S only when one of W,
X, Y or Z is absent; and [0024] R.sup.1 to R.sup.10 and V are as
defined with regard to Formula I, provided that R.sup.1, R.sup.2,
R.sup.3 or R.sup.4 is absent where W, X, Y or Z, respectively, is
--N.dbd., O, S or absent; including single stereoisomers, mixtures
of stereoisomers, and pharmaceutically acceptable salts, solvates,
and solvates of pharmaceutically acceptable salts thereof. The
compounds encompassed by Formula II and pharmaceutically acceptable
salts and solvates thereof will be seen to include those of Formula
I; they are likewise useful as active agents in the practice of the
methods of treatment and in manufacture of compositions including
the pharmaceutical formulations of the invention, and may also be
useful as intermediates in the synthesis of such active agents.
[0025] In one of its particular aspects the present invention
pertains to a compound represented by Formula I or II, having a
substituent selected from one or more of the following for R.sup.1
to R.sup.4; R.sup.5; R.sup.6 to R.sup.9; R.sup.10; T; V; or W, X, Y
and Z: [0026] R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are
independently chosen from hydrogen, halo (particularly chloro and
fluoro), lower alkyl (particularly methyl), substituted lower
alkyl, lower alkoxy (particularly methoxy), and cyano; [0027]
R.sup.5 is aralkyl or substituted aralkyl (particularly benzyl or
substituted benzyl; most particularly benzyl); [0028] R.sup.6 to
R.sup.9 are hydrogen; [0029] R.sup.10 is optionally substituted
benzyl or optionally substituted phenyl (particularly tolylmethyl);
[0030] T is a covalent bond; [0031] V is CH.sub.2, N(H) or
N(optionally substituted alkyl); and [0032] W, X, Y and Z are
--C.dbd.. Other particular aspects of the invention pertain to
methods and to pharmaceutical formulations employing such a
compound.
[0033] In one aspect, the invention relates to methods for treating
cellular proliferative diseases, for disorders that can be treated
by modulating KSP kinesin activity, and for inhibiting KSP kinesin
by the administration of a therapeutically effective amount of a
compound of Formula I or II, or a pharmaceutically acceptable salt
or solvate of such compounds. Diseases and disorders that respond
to therapy with compounds of the invention include cancer,
hyperplasia, restenosis, cardiac hypertrophy, immune disorders and
inflammation.
[0034] In another aspect, the invention relates to a pharmaceutical
composition containing a therapeutically effective amount of a
compound of Formula I or II or a pharmaceutically acceptable salt
or solvate thereof admixed with at least one pharmaceutically
acceptable excipient.
[0035] Yet another aspect of the invention relates to a kit having
a compound, pharmaceutically acceptable salt or solvate of Formula
I or II and a package insert or other labeling including directions
for treating a cellular proliferative disease by administering an
effective amount of the compound, salt or solvate. In one
particular such aspect, the compound, pharmaceutically acceptable
salt or solvate of Formula I or II is provided as a pharmaceutical
composition.
[0036] In an additional aspect, the present invention provides
methods of screening for compounds that will bind to a KSP kinesin,
for example compounds that will displace or compete with the
binding of the compounds of the invention. The methods entail
combining a labeled compound of the invention, a KSP kinesin, and
at least one candidate agent and determining the binding of the
candidate agent to the KSP kinesin.
[0037] In a further aspect, the invention provides methods of
screening for modulators of KSP kinesin activity. The methods
entail combining a compound of the invention, a KSP kinesin, and at
least one candidate agent and determining the effect of the
candidate agent on the KSP kinesin activity.
DETAILED DESCRIPTION OF THE INVENTION
[0038] The present invention provides compounds, compositions and
methods useful in the inhibition of mitotic kinesins, particularly
KSP (more particularly human KSP). The compounds can be used to
treat cellular proliferative diseases and include certain
pyrrolidin-2-one, piperidin-2-one and tetrahydro-pyrimidin-2-one
quinazolinone derivatives. The invention further relates to
pharmaceutical formulations comprising compounds of the invention,
and methods of treatment employing such compounds or
compositions.
Definitions
[0039] As used in the present specification, the following words
and phrases are generally intended to have the meanings as set
forth below, except to the extent that the context in which they
are used indicates otherwise. The following abbreviations and terms
have the indicated meanings throughout:
[0040] Ac=acetyl
[0041] Boc=t-butyloxy carbonyl
[0042] Bu=butyl
[0043] c-=cyclo
[0044] CBZ=carbobenzoxy=benzyloxycarbonyl
[0045] DCM=dichloromethane=methylene chloride=CH.sub.2Cl.sub.2
[0046] DIEA=N,N-diisopropylethylamine
[0047] DMF=N,N-dimethylformamide
[0048] DMSO=dimethyl sulfoxide
[0049] Et=ethyl
[0050] Me=methyl
[0051] rt=room temperature
[0052] s-=secondary
[0053] t-=tertiary
[0054] TFA=trifluoroacetic acid
[0055] THF=tetrahydrofuran
[0056] The substituents identified as V, W and Y are intended to
have the meanings set forth in the Summary, this Detailed
Description and the Claims; they are not intended to designate the
atomic elements Vanadium, Tungsten and Yttrium.
[0057] The term "optional" or "optionally" means that the
subsequently described event or circumstance may or may not occur,
and that the description includes instances where said event or
circumstance occurs and instances in which it does not. For
example, "optionally substituted alkyl" includes "alkyl" and
"substituted alkyl," as defined below. It will be understood by
those skilled in the art with respect to any group containing one
or more substituents that such groups are not intended to introduce
any substitution or substitution patterns that are sterically
impractical, synthetically non-feasible and/or inherently
unstable.
[0058] "Alkyl" is intended to include linear, branched, or cyclic
aliphatic hydrocarbon structures and combinations thereof, which
structures may be saturated or unsaturated (particularly having up
to 20 carbon atoms, more particularly up to C.sub.13.). Lower alkyl
refers to alkyl groups of from 1 to 5 (particularly 1 to 4) carbon
atoms. Examples of lower alkyl groups include methyl, ethyl,
propyl, isopropyl, butyl, s- and t-butyl and the like. Cycloalkyl
is a subset of alkyl and includes cyclic aliphatic hydrocarbon
groups of from 3 to 13 carbon atoms. Examples of cycloalkyl groups
include c-propyl, c-butyl, c-pentyl, norbornyl, adamantyl and the
like. In this application, alkyl refers to alkanyl, alkenyl and
alkynyl residues; it is intended to include cyclohexylmethyl,
vinyl, allyl, isoprenyl and the like. Alkylene, alkenylene and
alkynylene are other subsets of alkyl, referring to the same
residues as alkyl, but having two points of attachment. Examples of
alkylene include ethylene (--CH.sub.2CH.sub.2--), ethenylene
(--CH.dbd.CH--), propylene (--CH.sub.2CH.sub.2CH.sub.2--),
dimethylpropylene (--CH.sub.2C(CH.sub.3).sub.2CH.sub.2--) and
cyclohexylpropylene (--CH.sub.2CH.sub.2CH(C.sub.6H.sub.13)--). When
an alkyl residue having a specific number of carbons is named, all
geometric isomers of that residue having the specified number of
carbons are meant to be included; thus, for example, "butyl" is
meant to include n-butyl, sec-butyl, isobutyl and t-butyl; "propyl"
includes n-propyl and isopropyl.
[0059] The term "alkoxy" or "alkoxyl" refers to the group
--O-alkyl, particularly including from 1 to 8 carbon atoms in a
straight, branched or cyclic configuration, or combinations
thereof, attached to the parent structure through an oxygen.
Examples include methoxy, ethoxy, propoxy, isopropoxy,
cyclopropyloxy, cyclohexyloxy and the like. Lower-alkoxy refers to
groups containing one to five carbons.
[0060] The term "substituted alkoxy" refers to the group
--O-(substituted alkyl). One particular substituted alkoxy group is
"polyalkoxy" or --O-(optionally substituted alkylene)-(optionally
substituted alkoxy), and includes groups such as
--OCH.sub.2CH.sub.2OCH.sub.3, and glycol ethers such as
polyethyleneglycol and --O(CH.sub.2CH.sub.2O).sub.xCH.sub.3, where
x is an integer of about 2-20, particularly about 2-10, and more
particularly about 2-5. Another particular substituted alkoxy group
is hydroxyalkoxy or --OCH.sub.2(CH.sub.2).sub.yOH, where y is an
integer of about 1-10, particularly about 1-4.
[0061] "Acyl" refers to groups of from 1 to 8 carbon atoms in a
straight, branched or cyclic configuration, or combinations
thereof, or to a hydrogen atom attached to the parent structure
through a carbonyl functionality. Such groups may be saturated or
unsaturated, and aliphatic or aromatic. One or more carbons in the
acyl residue may be replaced by nitrogen, oxygen or sulfur as long
as the point of attachment to the parent remains at the carbonyl.
Examples include formyl, acetyl, benzoyl, propionyl, isobutyryl,
t-butoxycarbonyl, benzyloxycarbonyl, aminocarbonyl, and the like.
Lower-acyl refers to an acyl group containing one to five carbons.
"Substituted acyl" refers to an acyl group where one or more of the
hydrogens otherwise attached to a carbon, nitrogen or sulfur atom
is substituted, the point of attachment to the parent moiety
remaining at the carbonyl.
[0062] The term "acyloxy" refers to the group --O-acyl.
"Substituted acyloxy" refers to the group --O-substituted acyl.
[0063] The term "amidino" refers to the group
--C(.dbd.NH)--NH.sub.2. The term "substituted amidino" refers to
the formula --C(.dbd.NR)--NRR in which each R is independently
selected from the group: hydrogen, optionally substituted alkyl,
optionally substituted alkoxy, optionally substituted
aminocarbonyl, optionally substituted aryl, optionally substituted
heteroaryl, optionally substituted heterocyclyl, acyl,
alkoxycarbonyl, sulfanyl, sulfinyl and sulfonyl, provided that at
least one R is not hydrogen.
[0064] The term "amino" refers to the group --NH.sub.2. The term
"substituted amino" refers to the group --NHR or --NRR where each R
is independently selected from the group: optionally substituted
acyl, optionally substituted alkyl, optionally substituted alkoxy,
optionally substituted amino, optionally substituted aryl,
optionally substituted heteroaryl, optionally substituted
heterocyclyl, sulfinyl and sulfonyl, e.g., methylamino,
dimethylamino, diethylamino, methylsulfonylamino,
furanyl-oxy-sulfonamino, guanidino.
[0065] "Aryl" and "heteroaryl" mean a 5- or 6-membered aromatic
ring or heteroaromatic ring containing 1-4 heteroatoms selected
from O, N, or S; a bicyclic 9- or 10-membered aromatic ring system
or heteroaromatic ring system containing 1-4 (or more) heteroatoms
selected from O, N, or S; or a tricyclic 13- or 14-membered
aromatic ring system or heteroaromatic ring system containing 1-4
(or more) heteroatoms selected from O, N, or S. The aromatic 6- to
14-membered carbocyclic rings include, e.g., benzene, naphthalene,
indane, tetralin, and fluorene and the 5- to 10-membered aromatic
heterocyclic rings include, e.g., imidazole, pyridine, indole,
thiophene, benzopyranone, thiazole, furan, benzimidazole,
quinoline, isoquinoline, quinoxaline, pyrimidine, pyrazine,
tetrazole and pyrazole; particularly imidazole and imidazoline.
[0066] "Aralkyl" refers to a residue in which an aryl moiety is
attached to the parent structure via an alkyl residue. Examples
include benzyl, phenethyl, phenylvinyl, phenylallyl and the like.
"Heteroaralkyl" refers to a residue in which a heteroaryl moiety is
attached to the parent structure via an alkyl residue. Examples
include furanylmethyl, pyridinylmethyl, pyrimidinylethyl and the
like.
[0067] The term "aryloxy" refers to the group --O-aryl. Similarly,
"aralkoxy" and "heteroaralkoxy" refer, respectively, to an aryl or
heteroaryl moiety attached to the parent structure via an alkoxy
residue.
[0068] "Halogen" or "halo" refers to fluorine, chlorine, bromine or
iodine (particularly fluorine, chlorine and bromine). Dihaloaryl,
dihaloalkyl, trihaloaryl etc. refer to aryl and alkyl substituted
with a plurality of halogens, but not necessarily a plurality of
the same halogen; thus 4-chloro-3-fluorophenyl is within the scope
of dihaloaryl.
[0069] "Heterocycle" or "heterocyclyl" means a cycloalkyl or aryl
residue in which one to four of the carbons is replaced by a
heteroatom such as oxygen, nitrogen or sulfur (i.e., encompassing
heterocycloalkyl and heteroaryl). Examples of heterocyclyl residues
that fall within the scope of the invention include imidazolyl,
imidazolinyl, pyrrolidinyl, pyrazolyl, pyrrolyl, indolyl,
quinolinyl, isoquinolinyl, tetrahydroisoquinolinyl, benzofuranyl,
benzodioxanyl, benzodioxolyl (commonly referred to as
methylenedioxyphenyl, when occurring as a substituent), tetrazolyl,
morpholinyl, thiazolyl, pyridinyl, pyridazinyl, pyrimidinyl,
thiophenyl, furanyl, oxazolyl, oxazolinyl, isoxazolyl, dioxanyl,
tetrahydrofuranyl and the like. "N-heterocyclyl" refers to a
nitrogen-containing heterocycle as a substituent residue. Examples
of N-heterocyclyl residues include 4-morpholinyl,
4-thiomorpholinyl, 1-piperidinyl, 1-pyrrolidinyl, 3-thiazolidinyl,
piperazinyl and 4-(3,4-dihydrobenzoxazinyl). Examples of
substituted heterocyclyl include 4-methyl-1-piperazinyl and
4-benzyl-1-piperidinyl.
[0070] The terms "heteroaryloxy" and "heterocyclooxy" refer,
respectively to the groups --O-heteroaryl and
---O-heterocyclyl.
[0071] The term "solvate" refers to a compound (e.g., a compound of
Formula I or II or a pharmaceutically acceptable salt thereof) in
physical association with one or more molecules of a
pharmaceutically acceptable solvent. It will be understood that
phrases such as "a compound of Formula I or II or a
pharmaceutically acceptable salt or solvate thereof" are intended
to encompass the compound of Formula I or II, a pharmaceutically
acceptable salt of the compound, a solvate of the compound, and a
solvate of a pharmaceutically acceptable salt of the compound.
[0072] The term "substituted" as used with regard to alkyl, aryl,
aralkyl, heteroaryl and heterocyclyl refers to an alkyl, aryl,
aralkyl, heteroaryl or heterocyclyl moiety wherein one or more (up
to about 5, particularly up to about 3) hydrogen atoms are replaced
by a substituent independently selected from the group: optionally
substituted acyl (e.g., aminocarbonyl and alkoxycarbonyl or
"esters"), optionally substituted acyloxy (e.g., acid esters,
carbamic acid esters, carbonic acid esters, and thiocarbonic acid
esters), optionally substituted alkyl (e.g., fluoroalkyl),
optionally substituted alkoxy (e.g., methoxy and methoxymethoxy),
alkylenedioxy (e.g. methylenedioxy), optionally substituted amino
(e.g., alkylamino, dialkylamino, carbonylamino,
benzyloxycarbonylamino or "CBZ-amino", and carboxamido), optionally
substituted amidino, optionally substituted aryl (e.g., phenyl and
4-methyl-phenyl or "tolyl"), optionally substituted aralkyl (e.g.,
benzyl), optionally substituted aryloxy (e.g., phenoxy), optionally
substituted aralkoxy (e.g., benzyloxy), optionally substituted
heteroaryl, optionally substituted heteroaralkyl, optionally
substituted heteroaryloxy, optionally substituted heteroaralkoxy,
carboxy (--COOH), cyano, halogen, hydroxy, nitro, sulfanyl,
sulfinyl, sulfonyl and thio. In the compounds of Formula II where T
is substituted alkylene, the term "substituted" also refers to
alkylene groups where one or more (up to about 3, particularly 1)
carbon atoms are replaced by a heteroatom independently selected
from O, N or S, such as --CH.sub.2--S--CH.sub.2--.
[0073] The term "sulfanyl" refers to the groups: --S-(optionally
substituted alkyl), --S-(optionally substituted aryl),
--S-(optionally substituted heteroaryl), and --S-(optionally
substituted heterocyclyl).
[0074] The term "sulfinyl" refers to the groups: --S(O)--H,
--S(O)-(optionally substituted alkyl), --S(O)-(optionally
substituted amino), --S(O)-(optionally substituted aryl),
--S(O)-(optionally substituted heteroaryl), and --S(O)-(optionally
substituted heterocyclyl).
[0075] The term "sulfonyl" refers to the groups: --S(O)--H,
--S(O.sub.2)-(optionally substituted alkyl),
--S(O.sub.2)-(optionally substituted amino),
--S(O.sub.2)-(optionally substituted aryl),
--S(O.sub.2)-(optionally substituted heteroaryl),
--S(O.sub.2)-(optionally substituted heterocyclyl),
--S(O.sub.2)-(optionally substituted alkoxy),
--S(O.sub.2)-optionally substituted aryloxy),
--S(O.sub.2)-(optionally substituted heteroaryloxy), and
--S(O.sub.2)-(optionally substituted heterocyclyloxy).
[0076] "Isomers" are different compounds that have the same
molecular formula. "Stereoisomers" are isomers that differ only in
the way the atoms are arranged in space. "Enantiomers" are a pair
of stereoisomers that are non-superimposable mirror images of each
other. A 1:1 mixture of a pair of enantiomers is a "racemic"
mixture. The term "(..+-..)" is used to designate a racemic mixture
where appropriate. "Diastereoisomers" are stereoisomers that have
at least two asymmetric atoms, but which are not mirror-images of
each other. The absolute stereochemistry is specified according to
the Cahn-Ingold-Prelog R-S system. When a compound is a pure
enantiomer the stereochemistry at each chiral carbon may be
specified by either R or S. Resolved compounds whose absolute
configuration is unknown are designated (+) or (-) depending on the
direction (dextro- or levorotatory) which they rotate plane
polarized light at the wavelength of the sodium D line. The term
"substantially pure" means having at least about 95% chemical
purity with no single impurity greater than about 1%. The term
"substantially optically pure" or "substantially enantiomerically
pure" means having at least about 95% enantiomeric excess. The
invention contemplates the use of pure enantiomers and mixtures of
enantiomers, including racemic mixtures, although the use of a
substantially optically pure enantiomer will generally be most
suitable.
[0077] "Mitotic spindle formation" refers to the organization of
microtubules into bipolar structures by mitotic kinesins. "Mitotic
spindle dysfunction" refers to mitotic arrest, monopolar spindle
formation or mitotic spindle malformation, in which context
"malformation" encompasses the splaying of mitotic spindle poles,
or otherwise causing morphological perturbation of the mitotic
spindle. The term "inhibit" as used with reference to mitotic
spindle formation, means altering mitotic spindle formation,
including decreasing spindle formation, and increasing or
decreasing spindle pole separation. "Anti-mitotic" means inhibiting
or having the potential to inhibit mitosis, for example, as
described above.
[0078] The term "pharmaceutically acceptable salts" is meant to
include both acid and base addition salts. A "pharmaceutically
acceptable acid addition salt" refers to those salts that retain
the biological effectiveness of the free bases and that are not
biologically or otherwise undesirable, formed with inorganic acids
such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric
acid, phosphoric acid and the like, or organic acids such as acetic
acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,
maleic acid, malonic acid, succinic acid, fumaric acid, tartaric
acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,
methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,
salicylic acid and the like. "Pharmaceutically acceptable base
addition salts" include those derived from inorganic bases such as
sodium, potassium, lithium, ammonium, calcium, magnesium, iron,
zinc, copper, manganese, aluminum salts and the like. Particularly
suitable are the ammonium, potassium, sodium, calcium, and
magnesium salts. Salts derived from pharmaceutically acceptable
organic non-toxic bases include salts of primary, secondary, and
tertiary amines, substituted amines including naturally occurring
substituted amines, cyclic amines and basic ion exchange resins,
such as isopropylamine, trimethylamine, diethylamine,
triethylamine, tripropylamine, and ethanolamine.
[0079] The term "therapeutically effective amount" or "effective
amount" refers to that amount of a compound of Formula I or II that
is sufficient to effect treatment, as defined below, when
administered to a patient in need of such treatment. The effective
amount will vary depending upon the patient and disease condition
being treated, the weight and age of the patient, the severity of
the disease condition, the particular compound, pharmaceutically
acceptable salt or solvate of Formula I or II chosen, the dosing
regimen to be followed, timing of administration, the manner of
administration and the like, all of which can readily be determined
by one of ordinary skill in the art. In a particular aspect of the
invention, the effective amount will be an amount sufficient to
inhibit KSP kinesin activity in cells involved with the disease
being treated.
[0080] The term "treatment" or "treating" means any treatment of a
disease in a patient, including: [0081] a) preventing the disease,
that is, causing the clinical symptoms of the disease not to
develop; [0082] b) inhibiting the disease, that is, slowing or
arresting the development of clinical symptoms; and/or [0083] c)
relieving the disease, that is, causing the regression of clinical
symptoms.
[0084] A "patient" for the purposes of the present invention
includes humans and other animals, particularly mammals, and other
organisms. Thus the methods are applicable to both human therapy
and veterinary applications. In a particular embodiment the patient
is a mammal, most particularly the patient is human.
COMPOUNDS OF THE PRESENT INVENTION
[0085] The present invention provides certain quinazolinone
derivatives. The compounds are inhibitors of one or more mitotic
kinesins. The present invention capitalizes on the finding that
perturbation of mitotic kinesin function causes malformation or
dysfunction of mitotic spindles, frequently resulting in cell cycle
arrest and cell death.
[0086] Accordingly, the present invention relates to one or more
compounds selected from the group represented by Formula I:
##STR3## where: [0087] V is chosen from a covalent bond, CR'R'' and
NR''', [0088] R' and R'' being independently chosen from hydrogen,
hydroxy, amino, optionally substituted aryl, optionally substituted
alkylamino, optionally substituted alkyl and optionally substituted
alkoxy, and [0089] R''' being chosen from hydrogen, optionally
substituted alkyl, optionally substituted aryl, optionally
substituted aralkyl, optionally substituted heteroaryl, and
optionally substituted heteroaralkyl; [0090] R.sup.1, R.sup.2,
R.sup.3 and R.sup.4 are independently chosen from hydrogen,
hydroxy, optionally substituted alkyl, optionally substituted
alkoxy, halogen and cyano; [0091] R.sup.5 is chosen from hydrogen,
optionally substituted alkyl, optionally substituted aryl,
optionally substituted aralkyl, optionally substituted heteroaryl,
and optionally substituted heteroaralkyl; [0092] R.sup.6 to R.sup.9
are independently chosen from hydrogen, hydroxy, optionally
substituted alkyl, optionally substituted alkoxy, optionally
substituted aryl, and optionally substituted alkylamino, provided
that neither R.sup.8 nor R.sup.9 is hydroxy or alkoxy when V is
NR'''; and [0093] R.sup.10 is chosen from hydrogen, optionally
substituted alkyl, optionally substituted aryl, optionally
substituted aralkyl, optionally substituted heteroaryl, and
optionally substituted heteroaralkyl, and the pharmaceutically
acceptable salts and solvates thereof.
[0094] In another aspect, the invention relates to one or more
compounds selected from the group represented by Formula II:
##STR4## where: [0095] R.sup.1 to R.sup.10 and V are as defined
with regard to Formula I; [0096] T is a covalent bond or optionally
substituted lower alkylene; [0097] W, X, Y and Z are independently
N, C, O, S or absent, provided that: [0098] no more than one of W,
X, Y or Z is absent, [0099] no more than two of W, X, Y and Z are
--N.dbd., and [0100] W, X, Y or Z can be O or S only when one of W,
X, Y or Z is absent; and [0101] R.sup.1 to R.sup.10 and V are as
defined with regard to Formula I, provided that R.sup.1 , R.sup.2,
R.sup.3 or R.sup.4 is absent where W, X, Y or Z, respectively, is
--N.dbd., O, S or is absent, including single stereoisomers,
mixtures of stereoisomers, and the pharmaceutically acceptable
salts, solvates, and solvates of pharmaceutically acceptable salts
thereof. The compounds encompassed by Formula II will be seen to
include those of Formula I; they are likewise useful as active
agents in the practice of the methods of treatment and in
manufacture of compositions including the pharmaceutical
formulations of the invention, and may also be useful as
intermediates in the synthesis of such active agents. For the sake
of simplicity in the following description and claims, substituents
T, W, X, Y and Z will not be discussed in connection with certain
compounds falling within the scope of Formula I.
[0102] Many of the compounds described herein contain one or more
asymmetric centers and may thus give rise to enantiomers,
diastereomers, and other stereoisomeric forms that may be defined,
in terms of absolute stereochemistry, as (R)-- or (S)--. When the
compounds described herein contain olefinic double bonds or other
centers of geometric asymmetry, and unless specified otherwise, it
is intended that such compounds include both E and Z geometric
isomers. All tautomeric forms are also intended to be included. The
present invention is meant to include all such possible isomers,
including racemic mixtures, intermediate mixtures, optically pure
forms, substantially optically pure forms, enantiomerically pure
forms, and substantially enantiomerically pure forms.
Nomenclature
[0103] The compounds of Formula I and II can be named and numbered
(e.g., using AutoNom version 2.1 in ISIS-DRAW or ChemDraw) as
described below.
[0104] For example, the compound of Formula IA: ##STR5## i.e., the
compound according to Formula I where R.sup.1, R.sup.2 and R.sup.4
are H; R.sup.3 is chloro; R.sup.5 is benzyl; R.sup.6 to R.sup.9 are
H; R.sup.10 is 4-methyl-benzyl; and V is NH, can be named
3-benzyl-7-chloro-2-[3-(4-methyl-benzyl)-2-oxo-hexahydro-pyrimidin-4-yl]--
3H-quinazolin-4-one.
[0105] The compound of Formula IB: ##STR6## i.e., the compound
according to Formula I where R.sup.1, R.sup.2 and R.sup.4 are H;
R.sup.3 is chloro; R.sup.5 is benzyl; R.sup.6 to R.sup.9 are H;
R.sup.10 is benzyl; and V is CH.sub.2, can be named
3-benzyl-2-(1-benzyl-6-oxo-piperidin-2-yl)-7-chloro-3H-quinazolin-4-one.
[0106] The compound of Formula IIA: ##STR7## i.e., the compound
according to Formula II where R.sup.1, R.sup.2 and R.sup.4 are H;
R.sup.3 is chloro; R.sup.5 is benzyl; R.sup.6 to R.sup.9 are H;
R.sup.10 is 4-methyl-benzyl; T is isopropyl-methylene; V is NH; and
W, X, Y and Z are --C.dbd., can be named
3-benzyl-7-chloro-2-{2-methyl-1-[3-(4-methyl-benzyl)-2-oxo-hexahydro-pipe-
ridin-4-yl]-propyl}-3H-quinazolin-4-one.
[0107] The compound of Formula IIB: ##STR8## i.e., the compound
according to Formula II where R.sup.1, R.sup.3 and R.sup.4 are H;
R.sup.2 is di-methyl; R.sup.5 is benzyl; R.sup.6 to R.sup.9 are H;
R.sup.10is 4-methyl-benzyl; T is methylene; V is NR''' where R'''
is isopropyl; and W is CH.sub.2, X is C, and Y and Z are --C.dbd.,
can be named
3-benzyl-2-[1-isopropyl-3-(4-methyl-benzyl)-2-oxo-hexahydro-pyrimidin-4-y-
lmethyl]-6,6-dimethyl-5,6-dihydro-3H-quinazolin-4-one.
[0108] The compound of Formula IIC: ##STR9## i.e., the compound
according to Formula II where R.sup.1, R.sup.2 and R.sup.4 are H;
R.sup.3 is chloro; R.sup.5 is benzyl; R.sup.6 to R.sup.9 are H;
R.sup.10 is 4-methyl-benzyl; T is ethylene; V is CH.sub.2; W is
--N.dbd., and X, Y and Z are --C.dbd., can be named
3-benzyl-2-[2-(1-benzyl-6-oxo-piperidin-2-yl)-ethyl]-7-chloro-3H-pyrido[3-
,2-d]pyrimidin-4-one.
[0109] The compound of Formula IID: ##STR10## i.e., the compound
according to Formula II where R.sup.1 and R.sup.3 are H; R.sup.2
and R.sup.4 are absent; R.sup.5 is benzyl; R.sup.6 to R.sup.9 are
H; R.sup.10 is benzyl; T is oxo-ethylene; V is NH; and W and Y are
--C.dbd.; and X and Z are --N.dbd., can be named
3-benzyl-2[2-(1-benzyl-6-oxo-piperidin-2-yl)-2-oxo-ethyl]-3H-pyrimidino[4-
,5-d]pyrimidin-4-one.
[0110] The compound of Formula IIE: ##STR11## i.e., the compound
according to Formula II where R.sup.1 and R.sup.3 are H; R.sup.2
and R.sup.4 are absent; R.sup.5 is benzyl; R.sup.6 to R.sup.10 are
H; T is a covalent bond; V is NR''' where R''' is isopropyl; and W
and Y are C; X is O, and Z is absent, can be named
3-benzyl-2-(1-isopropyl-2-oxo-hexahydro-pyrimidin-4-yl)-5,7-dihydro-3H-fu-
ro[3,4-d]pyrimidin-4-one. Synthetic Reaction Parameters
[0111] The terms "solvent", "inert organic solvent" or "inert
solvent" mean a solvent inert under the conditions of the reaction
being described in conjunction therewith [including, for example,
benzene, toluene, acetonitrile, tetrahydrofuran ("THF"),
dimethylformamide ("DMF"), chloroform, methylene chloride (or
dichloromethane), diethyl ether, methanol, pyridine and the like].
Unless specified to the contrary, the solvents used in the
reactions of the present invention are inert organic solvents.
[0112] The term "q.s." means adding a quantity sufficient to
achieve a stated function, e.g., to bring a solution to the desired
volume (i.e., 100%).
[0113] Isolation and purification of the compounds and
intermediates described herein can be effected, if desired, by any
suitable separation or purification procedure such as, for example,
filtration, extraction, crystallization, column chromatography,
thin-layer chromatography or thick-layer chromatography, or a
combination of these procedures. Specific illustrations of suitable
separation and isolation procedures can be had by reference to the
examples hereinbelow. However, other equivalent separation or
isolation procedures can, of course, also be used.
[0114] When desired, the (R)- and (S)-isomers may be resolved by
methods known to those skilled in the art, for example by formation
of diastereoisomeric salts or complexes which may be separated, for
example, by crystallisation; via formation of diastereoisomeric
derivatives which may be separated, for example, by
crystallisation, gas-liquid or liquid chromatography; selective
reaction of one enantiomer with an enantiomer-specific reagent, for
example enzymatic oxidation or reduction, followed by separation of
the modified and unmodified enantiomers; or gas-liquid or liquid
chromatography in a chiral environment, for example on a chiral
support, such as silica with a bound chiral ligand or in the
presence of a chiral solvent. For example, a compound of Formula I
or II can be dissolved in a lower alkanol and placed on a Chiralpak
AD (205.times.20 mm) column (Chiral Technologies, Inc.) conditioned
for 60 min at 70% EtOAc in Hexane. It will be appreciated that
where the desired enantiomer is converted into another chemical
entity by one of the separation procedures described above, a
further step may be required to liberate the desired enantiomeric
form. Alternatively, specific enantiomer may be synthesized by
asymmetric synthesis using optically active reagents, substrates,
catalysts or solvents, or by converting one enantiomer to the other
by asymmetric transformation.
Synthesis of the Compounds of Formula I and II
[0115] Syntheses of the compounds of Formula I and II are described
below with reference to Reaction Schemes 1 to 4.
Brief Description Of Reaction Schemes
[0116] Reaction Scheme 1 illustrates a synthesis of the
N,N-optionally mono- or di-substituted pyrimidin-2-one compounds of
Formula I or II.
[0117] Reaction Scheme 2 illustrates a synthesis of intermediates
in preparation of the N-optionally substituted piperidin-2-one
compounds of Formula I or II.
[0118] Reaction Scheme 3 illustrates a synthesis of intermediates
in preparation of the N-optionally mono-substituted pyrimidin-2-one
compounds of Formula I or II (i.e., where the optional substituent
is at R.sup.10 and V is NH).
[0119] Reaction Scheme 4 illustrates a synthesis of the compounds
of Formula I or II from the intermediates prepared according to
Reaction Schemes 2 and 3.
[0120] It will be appreciated by those skilled in the art that one
or more of the reaction steps and/or conditions described with
reference to Reaction Schemes 1 to 4 may require adjustment to
accommodate non-hydrogen substituents at R', R'' and R.sup.1 to
R.sup.9.
Starting Materials
[0121] The N-protected 2,4-di-amino butyric acids of Formula 101
(e.g.,
4-tert-butoxycarbonylamino-2-(9H-fluoren-9-yl-methoxycarbonylamino)-butyr-
ic acid), the anthranilic acids of Formula 102 (e.g.,
4-chloro-anthranilic acid), aminoadipic acid hydrate,
di-aminobutyric acid and the like are commercially available, e.g.,
from Aldrich Chemical Company, Milwaukee, Wis. Other reactants are
likewise commercially available or may be readily prepared by those
skilled in the art using commonly employed synthetic methodology.
##STR12##
[0122] Preparation of Formula 103 Referring to Reaction Scheme 1,
Step 1, to an N-protected di-amino lower alkyl acid of Formula 101
(e.g., a protected 2,4-di-amino butyric acid, particularly
employing orthogonal amino-protecting groups "PG," such as Fmoc and
Boc, to facilitate selective de-protection) in solution with an
organic solvent (such as anhydrous THF) is added a slight molar
excess of N-methylmorpholine, at reduced temperature (e.g., in an
ice bath), followed by the addition of a slight molar excess of
isobutyl chloroformate, dropwise over 15 minutes. The mixture is
stirred at reduced temperature (e.g., 0.degree. C.) for an hour,
followed by the addition of a molar equivalent of an optionally
substituted o-amino allocyclic, heterocyclic or (hetero)aryl acid
of Formula 102 (such as anthranilic acid) with continued stirring
for another 2 hours at reduced temperature. The resulting protected
alkylamino-optionally substituted cyclic acid of Formula 103 can be
carried forward without isolation or purification.
[0123] Preparation of Formula 106 Referring to Reaction Scheme 1,
Step 2, to the intermediate of Formula 103 is added a slight molar
excess of N-methylmorpholine, and the mixture is allowed to warm to
room temperature with continued stirring over 16 hours. The mixture
is then cooled to 0.degree. C. and treated with a slight molar
excess of both N-methylmorpholine and isobutyl chloroformate,
followed by the room temperature addition of about 1.5 molar
equivalents of a primary amine of formula R.sup.5NH.sub.2 (such as
benzylamine) in several equal portions. Removal of the solvents,
partitioning between DCM and saturated sodium bicarbonate and
drying of the organic layer affords a mixture of Formula 104 and
Formula 105, which (in Step 3) is subsequently dried and treated
with a molar equivalent of lithium hydroxide monohydrate in a
solvent (e.g., 2/1 1,4-dioxane/ethylene glycol) at reflux for 5
hours. The reaction is quenched with water and the desired
substituted bi-cyclic product of Formula 106 (e.g., a
quinazolinone) is extracted with dichloromethane, dried, and
purified (e.g., by flash silica gel chromatography).
[0124] Preparation of Formula 107 Referring to Reaction Scheme 1,
Step 4, to a solution of a compound of Formula 106 (e.g., in
dichloromethane) is added a molar equivalent of a substituted
aldehyde (e.g., p-tolualdehyde or benzaldehyde). The mixture is
stirred for 1 hour after which a molar excess of sodium
triacetoxyborohydride is added with continued stirring for an
additional 3 hours. The corresponding 3-substituted amino compound
of Formula 107 is conventionally isolated and purified.
[0125] Preparation of Formula 108 Referring to Reaction Scheme 1,
Step 5, a compound of Formula 107 is deprotected (e.g., in the case
where the protecting group PG is t-BOC, by dissolution in aqueous
TFA) and stirred for 30 minutes, followed by evaporation of the
solvents, partitioning of the residue and drying of the organic
layer. The dried residue, and molar excesses of DIEA, sodium
triacetoxybyrohydride and an R'''-acyl compound (e.g., t-butyl
N-(2-oxoethyl) carbamate) are mixed in a solvent (e.g., DCM) and
stirred for 1 hour. The solution is washed, dried and evaporated to
give the corresponding N--R.sup.10,N--R'-disubstituted compound of
Formula 108. As will be appreciated by those skilled in the art,
this step can be omitted in the synthesis of compounds of Formula I
or II where R''' is hydrogen, proceeding directly to either of Step
6 with the compound of Formula 106 or 107.
[0126] Preparation of Formula I or II Referring to Reaction Scheme
1, Step 6, to a solution of Formula 106, 107 or 108 (e.g., in
dichloromethane) is added a molar excess of carbonyldiimidazole,
and the reaction is stirred for 1 hour. Evaporation of the solvent
and purification (e.g., by silica gel chromatography) gives the
corresponding compound of Formula I or II. Where the starting
material of Formula 108 is protected (e.g., where R' is
NHBoc-ethylene) the protecting group is removed (e.g., dissolving
the crude compound of Formula 1 in 95/5 TFA/H.sub.2O with stirring
for 1 hour) and the corresponding compound of Formula I or II is
conventionally isolated and purified. ##STR13##
[0127] Preparation of Formula 203 In Reaction Scheme 2, Step 1, to
a solution of an optionally substituted amino-dicarboxylic acid of
Formula 201 (e.g., aminoadipic acid hydrate dissolved in 2 molar
equivalents of 2 M NaOH) is added a molar equivalent of a solution
of an aldehyde of Formula 202 (e.g., dissolved in ethanol). After
10 minutes the mixture is cooled (e.g., to 0.degree. C.) and sodium
borohydride (0.3 molar equivalents) is added. Completion of the
reaction is monitored, e.g., by LCMS, followed by extraction and
isolation of a crude precipitate product, which is dissolved in
ethanol and boiled for 16 hours to afford the corresponding lactam
intermediate of Formula 203, which can be carried forward without
further purification. By substituting the compound of Formula 201
with an optionally substitued 2-amino-pentanedioic acid, the
corresponding intermediates of Formula 203 are obtained for the
synthesis of Formula I or II where V is a covalent bond.
[0128] Preparation of Formula 204 In Reaction Scheme 2, Step 2, the
lactam of Formula 203 and a molar equivalent of DIEA are dissolved
(e.g., in dichloromethane) and cooled (e.g., to 0.degree. C.). One
molar equivalent of isobutyl chloroformate is added and the mixture
stirred (e.g., for 20 minutes) followed by addition of 2 additional
molar equivalents of DIEA followed by a slight molar excess of an
optionally substituted o-amino-acid of Formula 102. The reaction
takes place, warming to room temperature, over 16 hours, to afford
the corresponding acid of Formula 204 (also a compound of Formula
401), which is washed, dried, evaporated, re-washed, cooled (e.g.,
to 0.degree. C.), acidified and then isolated for subsequent use
without further purification. ##STR14##
[0129] Preparation of Formula 302 In Reaction Scheme 3, Step 1, an
optionally substituted diamino lower alkyl acid of Formula 301 and
3 molar equivalents of sodium bicarbonate are dissolved in water,
to which one-half molar equivalent of copper sulfate dissolved in
water is added. A molar excess of di-(tert-butyl) pyrocarbonate
(dissolved, e.g., in acetone) is added followed by stirring for 24
hours, the addition of methanol and continued stirring for another
18 hours. The resulting intermediate mono-Boc-protected copper
complex is filtered, washed and dried, then suspended in water. Two
molar equivalents of quinolol are added to the suspension. After 5
hours, the suspension is filtered off and the liquid is evaporated.
The solid thus-obtained is dissolved (e.g., in 200 mL of 30%
methanol in benzene), and (trimethylsilyl)diazomethane is added,
dropwise to completion (indicated by color change and cessation of
bubbling), followed by stirring for 1 hour and the dropwise
addition of acetic acid to completion (indicated, e.g., by color
change and cessation of bubbling). The resulting material is
purified conventionally to provide the corresponding methyl ester
of Formula 302.
[0130] Preparation of Formula 303 In Reaction Scheme 3, Step 2, to
a solution of Formula 302 (e.g., in DCM) is added almost one molar
equivalent of an R.sup.10-aldehyde (e.g., p-tolualdehyde or
benzaldehyde) and the mixture stirred at room temperature for 1
hour. A slight molar excess of sodium triacetoxyborohydride is
added and the mixture is stirred for 16 hours. The protecting group
is removed (e.g., dissolving the R.sup.10-substituted amine in 2M
HCl in dioxane solution followed by stirring for 2 hours). The
solution is then washed, dried, isolated and purified
conventionally to give the corresponding optionally
substituted-aminomethyl ester compound of Formula 303.
[0131] Preparation of Formula 304 In Reaction Scheme 3, Step 3, to
a solution of Formula 303 and 2 molar equivalents of DIEA (e.g., in
DCM) is added a molar excess of carbonyldiimidazole. The reaction
mixture is stirred for 1 hour, after which the solvents are
evaporated. The residue is dissolved (e.g., in MeOH:H.sub.2O (2:1)
solution) to which solution is was added 2 molar equivalents of
LiOH. The reaction takes place with stirring over 3 hours after
which pH is adjusted to .about.7 (e.g., by adding Dowex-H+ resin).
Conventional isolation and purification gives the corresponding
pyrimidine of Formula 304.
[0132] Preparation of Formula 305 In Reaction Scheme 3, Step 4, to
a solution of Formula 304 (e.g., in DMF) is added a slight molar
excess of anhydrous N-methylmorpholine. After cooling in an
ice-bath for 10 minutes, a slight molar excess of isobutyl
chloroformate is added dropwise while maintaining the temperature
below 5.degree. C. with stirring for 1 hour. A slight molar excess
of an optionally substituted o-amino allocyclic, heterocyclic or
(hetero)aryl acid of Formula 102 (such as anthranilic acid) of
Formula 102 (dissolved, e.g., in DMF) is added and the mixture
stirred for an additional 5 hours during which the temperature is
allowed to warm to room temperature to afford the corresponding
intermediate product of Formula 305 (also a compound of Formula
401), which can be carried on without isolation or purification.
##STR15##
[0133] Preparation of Formula 402 and Formula I or II In Reaction
Scheme 4, Step 1, to a solution of a piperidine compound of Formula
401 (e.g., in DMF) is added 3 molar equivalents of EDC followed by
stirring at room temperature for 1 hour. Three molar equivalents of
an R.sup.5-amine (e.g., benzylamine) are added followed by stirring
for an additional 3 hours. Conventional isolation and purification
provides the corresponding crude intermediate of Formula 402. In
Reaction Scheme 4, Step 2, the compound of Formula 402 is added to
a mixture of ethylene glycol with 1 molar equivalent of sodium
hydroxide, followed by stirring at 130.degree. C. for two days. The
mixture is then poured into water, extracted and purified to give
the corresponding pure product of Formula I or II.
[0134] Alternative Preparation of Formula 402 and Formula I or II
Alternatively, in Reaction Scheme 4, Step 1, two molar equivalents
of EDC are added to a solution of a pyrimidine compound of Formula
401 (e.g., in DMF) followed by stirring for 1 hour and then by the
addition of an R.sup.5-amine (e.g., benzylamine). The resulting
solution is stirred for 16 hours and the corresponding compound of
Formula 402 is isolated and purified conventionally. In Reaction
Scheme 4, Step 2, the compound of Formula 402 is dissolved (e.g.,
in ethylene glycol to which was added 2 molar equivalents of sodium
hydroxide). The mixture is stirred at 140.degree. C. for 20 hours.
Following consumption of starting material, the reaction mixture is
poured into 100 mL of water. After extraction with DCM, the crude
product is isolated and purified conventionally to give the
corresponding pure product of Formula I or II.
[0135] Compounds prepared by the above-described process of the
invention may be identified by the presence of a detectable amount
of Formula 402. While it is well known that pharmaceuticals must
meet pharmacopoeia standards before approval and/or marketing, and
that synthetic reagents (such as benzylamine, ethylene glycol or
NaOH) and precursors (such as Formula 402) should not exceed the
limits prescribed by pharmacopoeia standards, final compounds
prepared by a process of the present invention may have minor, but
detectable, amounts of such materials present, for example at
levels in the range of 95% purity with no single impurity greater
than 1%. These levels can be detected, e.g., by emission
spectroscopy. It is important to monitor the purity of
pharmaceutical compounds for the presence of such materials, which
presence is additionally disclosed as a method of detecting use of
a process of the invention.
Particular Optional Processes and Last Steps
[0136] A compound of Formula 402 is dissolved in an organic solvent
(e.g., ethylene glycol) to which was added about 2 molar
equivalents of sodium hydroxide.
[0137] A protected amino-substituted precursor to Formula I or II
(e.g. where R''' or R.sup.10 is NHBoc-protected amino ethyl) is
dissolved in TFA/H.sub.2O and stirred to afford the corresponding
de-protected compound of Formula I or II.
[0138] A racemic mixture of isomers of a compound of Formula I or
II is placed on a chromatography column and separated into (R)- and
(S)-enantiomers.
[0139] A compound of Formula I or II is contacted with a
pharmaceutically acceptable acid to form the corresponding acid
addition salt.
[0140] A pharmaceutically acceptable acid addition salt of Formula
I or II is contacted with a base to form the corresponding free
base of Formula I or II.
Particular Compounds
[0141] Particular embodiments of the invention include or employ
the compounds of Formula I and II having the following combinations
and permutations of substituent groups (indented/sub-grouped,
respectively, in increasing order of particularity). These are
presented in support of the appended claims as well as combinations
and permutations of substituent groups that may, for the sake of
brevity, not be specifically claimed but should be appreciated as
encompassed by the teachings of the present disclosure. In that
regard, the described subsets for each substituent are intended to
apply to that substituent alone or in combination with one,
several, or all of the described subsets for the other
substituents, for example, as illustrated with regard to the
compounds where V is CR'R'' or NR'''. [0142] W, X, Y and Z are
independently chosen from --C.dbd. and --N.dbd.; [0143] W, X, Y and
Z are --C.dbd.. [0144] R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are
independently chosen from hydrogen, halo (especially chloro and
fluoro), lower alkyl (especially methyl), substituted lower alkyl,
lower alkoxy (especially methoxy), and cyano. [0145] R.sup.1,
R.sup.2, R.sup.3 and R.sup.4 are independently hydrogen, chloro,
fluoro, methyl, methoxy or cyano. [0146] Where three or four of
R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are hydrogen. [0147] Where
four of R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are hydrogen or three
of R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are hydrogen and the
fourth is halo, methoxy, methyl, or cyano. [0148] Where halo is
chloro. [0149] Where R.sup.3 is hydrogen or chloro. [0150] Where
R.sup.3 is chloro. [0151] R.sup.5 is optionally substituted
aralkyl. [0152] R.sup.5 is benzyl or substituted benzyl. [0153]
R.sup.5 is benzyl. [0154] R.sup.6 is hydrogen or optionally
substituted lower alkyl. [0155] R.sup.6 is hydrogen. [0156] R.sup.7
is hydrogen or optionally substituted lower alkyl. [0157] R.sup.7
is hydrogen. [0158] R.sup.8 is hydrogen or optionally substituted
lower alkyl. [0159] R.sup.8 is hydrogen. [0160] R.sup.9 is hydrogen
or optionally substituted lower alkyl. [0161] R.sup.9 is hydrogen.
[0162] R.sup.10 is optionally substituted aryl or optionally
substituted aralkyl. [0163] R.sup.10 is optionally substituted
phenyl or optionally substituted benzyl. [0164] R.sup.10 is benzyl
or methyl-benzyl. [0165] T is optionally substituted C.sub.1 to
C.sub.4 alkylene or is absent. [0166] T is absent. [0167] Where T
is alkylene having a carbon substituted by a heteroatom, the
heteroatom is not bound directly to the bicyclic structure. [0168]
T is aminoalkylene or amidoalkylene. [0169] T is alkylene or
alkylene substituted with halo or oxo. [0170] V is CR'R'' or NR'''
[0171] V is CR'R'' (particularly where R' and/or R'' are hydrogen).
[0172] Where R' and R'' are hydrogen. [0173] Where W, X, Y and Z
are independently chosen from --C.dbd. and --N.dbd.. [0174] W, X, Y
and Z are --C.dbd.. [0175] Where R.sup.1, R.sup.2, R.sup.3 and
R.sup.4 are independently chosen from hydrogen, halo (particularly
chloro and fluoro), lower alkyl (particularly methyl), substituted
lower alkyl, lower alkoxy (particularly methoxy), and cyano. [0176]
R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are independently hydrogen,
chloro, fluoro, methyl, methoxy or cyano. [0177] Where three or
four of R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are hydrogen. [0178]
Where four of R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are hydrogen or
three of R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are hydrogen and the
fourth is halo, methoxy, methyl, or cyano. [0179] Where halo is
chloro. [0180] Where R.sup.3 is chloro. [0181] Where R' and R'' are
hydrogen. [0182] Where R.sup.5 is optionally substituted aralkyl.
[0183] Where R.sup.5 benzyl or substituted benzyl. [0184] Where
R.sup.5 is benzyl. [0185] Where R' and R'' are hydrogen. [0186]
Where R.sup.6, R.sup.7, R.sup.8 and R.sup.9 are independently
chosen from hydrogen or optionally substituted lower alkyl. [0187]
Where at least three of R.sup.6, R.sup.7, R.sup.8 and R.sup.9 are
hydrogen. [0188] Where R.sup.6, R.sup.7, R.sup.8 and R.sup.9 are
hydrogen. [0189] Where R' and R'' are hydrogen. [0190] Where
R.sup.10 is optionally substuted aryl or optionally substituted
aralkyl. [0191] R.sup.10 is optionally substituted phenyl or
optionally substituted benzyl. [0192] R.sup.10 is benzyl or
methyl-benzyl. [0193] Where R' and R'' are hydrogen. [0194] Where
W, X, Y and Z are chosen from --C.dbd. and --N.dbd.. [0195] W, X, Y
and Z are --C.dbd.. [0196] Where R.sup.1, R.sup.2, R.sup.3 and
R.sup.4 are independently chosen from hydrogen, halo (particularly
chloro and fluoro), lower alkyl (particularly methyl), substituted
lower alkyl, lower alkoxy (particularly methoxy), and cyano. [0197]
R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are independently hydrogen,
chloro, fluoro, methyl, methoxy or cyano. [0198] Where three or
four of R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are hydrogen. [0199]
Where four of R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are hydrogen or
three of R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are hydrogen and the
fourth is halo, methoxy, methyl, or cyano. [0200] Where halo is
chloro. [0201] Where R.sup.3 is chloro. [0202] Where R.sup.1,
R.sup.2, R.sup.3 and R.sup.4 are independently chosen from
hydrogen, halo (particularly chloro and fluoro), lower alkyl
(particularly methyl), substituted lower alkyl, lower alkoxy
(particularly methoxy), and cyano. [0203] R.sup.1, R.sup.2, R.sup.3
and R.sup.4 are independently hydrogen, chloro, fluoro, methyl,
methoxy or cyano. [0204] Where three or four of R.sup.1, R.sup.2,
R.sup.3 and R.sup.4 are hydrogen. [0205] Where four of R.sup.1,
R.sup.2, R.sup.3 and R.sup.4 are hydrogen or three of R.sup.1,
R.sup.2, R.sup.3 and R.sup.4 are hydrogen and the fourth is halo,
methoxy, methyl, or cyano. [0206] Where halo is chloro. [0207]
Where R.sup.3 is chloro. [0208] Where R.sup.5 is optionally
substituted aralkyl. [0209] Where R.sup.5 is benzyl or substituted
benzyl. [0210] Where R.sup.5is benzyl. [0211] Where R.sup.6,
R.sup.7, R.sup.8 and R.sup.9 are independently chosen from hydrogen
or optionally substituted lower alkyl. [0212] Where at least three
of R.sup.6, R.sup.7, R.sup.8 and R.sup.9 are hydrogen. [0213] Where
R.sup.6, R.sup.7, R.sup.8 and R.sup.9 are hydrogen. [0214] Where
R.sup.10 is optionally substituted aryl or optionally substituted
aralkyl. [0215] R.sup.10 is optionally substituted phenyl or
optionally substituted benzyl. [0216] R.sup.10 is benzyl or
methyl-benzyl. [0217] Where T is optionally substituted C.sub.1 to
C.sub.4 alkylene or is absent. [0218] T is absent. [0219] Where T
is alkylene having a carbon substituted by a heteroatom, the
heteroatom is not bound directly to the bicyclic structure. [0220]
T is aminoalkylene or amidoalkylene. [0221] T is alkylene or
alkylene substituted with halo or oxo. [0222] V is NR'''
(particularly where R''' is hydrogen or optionally substituted
alkyl). [0223] Where R'' is hydrogen or optionally substituted
amino-lower alkyl. [0224] Where W, X, Y and Z are chosen from
--C.dbd. and --N.dbd.. [0225] W, X, Y and Z are --C.dbd.. [0226]
Where R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are independently
chosen from hydrogen, halo (particularly chloro and fluoro), lower
alkyl (particularly methyl), substituted lower alkyl, lower alkoxy
(particularly methoxy), and cyano. [0227] R.sup.1, R.sup.2, R.sup.3
and R.sup.4 are independently hydrogen, chloro, fluoro, methyl,
methoxy or cyano. [0228] Where three or four of R.sup.1, R.sup.2,
R.sup.3 and R.sup.4 are hydrogen. [0229] Where four of R.sup.1,
R.sup.2, R.sup.3 and R.sup.4 are hydrogen or three of R.sup.1,
R.sup.2, R.sup.3 and R.sup.4 are hydrogen and the fourth is halo,
methoxy, methyl, or cyano. [0230] Where halo is chloro. [0231]
Where R.sup.3 is chloro. [0232] Where R''' is hydrogen or
amino-lower alkyl. [0233] Where R.sup.5 is optionally substituted
aralkyl. [0234] Where R.sup.5is benzyl or substituted benzyl.
[0235] Where R.sup.5 is benzyl. [0236] Where R''' is hydrogen or
optionally substituted amino-lower alkyl. [0237] Where R.sup.6,
R.sup.7, R.sup.8 and R.sup.9 are independently chosen from hydrogen
or optionally substituted lower alkyl. [0238] Where at least three
of R.sup.6, R.sup.7, R.sup.8 and R.sup.9 are hydrogen. [0239] Where
R.sup.6, R.sup.7, R.sup.8 and R.sup.9 are hydrogen. [0240] Where
R''' is hydrogen or amino-lower alkyl. [0241] Where R.sup.10 is
optionally substuted aryl or optionally substituted aralkyl. [0242]
R.sup.10 is optionally substituted phenyl or optionally substituted
benzyl. [0243] R.sup.10 is benzyl or methyl-benzyl. [0244] Where
R''' is hydrogen or amino-lower alkyl. [0245] Where W, X, Y and Z
are chosen from --C.dbd. and --N.dbd.. [0246] W, X, Y and Z are
--C.dbd.. [0247] Where R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are
independently chosen from hydrogen, halo (particularly chloro and
fluoro), lower alkyl (particularly methyl), substituted lower
alkyl, lower alkoxy (particularly methoxy), and cyano. [0248]
R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are independently hydrogen,
chloro, fluoro, methyl, methoxy or cyano. [0249] Where three or
four of R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are hydrogen. [0250]
Where four of R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are hydrogen or
three of R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are hydrogen and the
fourth is halo, methoxy, methyl, or cyano. [0251] Where halo is
chloro. [0252] Where R.sup.3is chloro. [0253] Where R.sup.1,
R.sup.2, R.sup.3 and R.sup.4 are independently chosen from
hydrogen, halo (particularly chloro and fluoro), lower alkyl
(particularly methyl), substituted lower alkyl, lower alkoxy
(particularly methoxy), and cyano. [0254] R.sup.1, R.sup.2, R.sup.3
and R.sup.4 are independently hydrogen, chloro, fluoro, methyl,
methoxy or cyano. [0255] Where three or four of R.sup.1, R.sup.2,
R.sup.3 and R.sup.4 are hydrogen. [0256] Where four of R.sup.1,
R.sup.2, R.sup.3 and R.sup.4 are hydrogen or three of R.sup.1,
R.sup.2, R.sup.3 and R.sup.4 are hydrogen and the fourth is halo,
methoxy, methyl, or cyano. [0257] Where halo is chloro. [0258]
Where R.sup.3is chloro. [0259] Where R.sup.5 is optionally
substituted aralkyl. [0260] Where R.sup.5 is benzyl or substituted
benzyl. [0261] Where R.sup.5 is benzyl. [0262] Where R.sup.6,
R.sup.7, R.sup.8 and R.sup.9 are independently chosen from hydrogen
or optionally substituted lower alkyl. [0263] Where at least three
of R.sup.6, R.sup.7, R.sup.8 and R.sup.9 are hydrogen. [0264] Where
R.sup.6, R.sup.7, R.sup.8 and R.sup.9 are hydrogen. [0265] Where
R.sup.10 is optionally substituted aryl or optionally substituted
aralkyl. [0266] R.sup.10 is optionally substituted phenyl or
optionally substituted benzyl. [0267] R.sup.10 is benzyl or
methyl-benzyl. [0268] Where T is optionally substituted C.sub.1 to
C.sub.4 alkylene or is absent. [0269] T is absent. [0270] Where T
is alkylene having a carbon substituted by a heteroatom, the
heteroatom is not bound directly to the bicyclic structure. [0271]
T is aminoalkylene or amidoalkylene. [0272] T is alkylene or
alkylene substituted with halo or oxo.
[0273] Compounds where V is CR'R'' or NR''', including those
illustrated by the above-described groupings and sub-groups of
substituents, individually and/or combined together, are
particularly suitable for practice of the present invention.
[0274] One group of compounds, pharmaceutically acceptable salts
and solvates thereof, compositions including pharmaceutical
formulations, and methods of manufacture and use of the present
invention are those wherein the compound of Formula I or II is
selected from: [0275]
3-benzyl-7-chloro-2-[3-benzyl-2-oxo-hexahydro-pyrimidin-4-yl]-3H-quinazol-
in-4-one; [0276]
3-benzyl-7-chloro-2-[3-(4-methyl-benzyl)-2-oxo-hexahydro-pyrimidin-4-yl]--
3H-quinazolin-4-one; [0277]
3-benzyl-2-(1-benzyl-6-oxo-piperidin-2-yl)-7-chloro-3H-quinazolin-4-one;
[0278]
3-benzyl-2-(1-(4-methyl-benzyl)-6-oxo-piperidin-2-yl)-7-chloro-3H-
-quinazolin-4-one; [0279]
2-[-1-(2-amino-ethyl)-3-(4-methyl-benzyl)-2-oxo-hexahydro-pyrimidin-4-yl]-
-3-benzyl-7-chloro-3H-quinazolin-4-one.
[0280] A particular group of compounds, pharmaceutically acceptable
salts and solvates thereof, compositions including pharmaceutical
formulations, and methods of manufacture and use of the present
invention are those wherein the compound of Formula I or II is
selected from: [0281]
3-benzyl-7-chloro-2-[3-(4-methyl-benzyl)-2-oxo-hexahydro-pyrimidin-4-yl]--
3H-quinazolin-4-one; [0282]
3-benzyl-2-(1-benzyl-6-oxo-piperidin-2-yl)-7-chloro-3H-quinazolin-4-one;
and [0283]
3-benzyl-2-(1-(4-methyl-benzyl)-6-oxo-piperidin-2-yl)-7-chloro-3H-quinazo-
lin-4-one.
[0284] Another particular group of compounds, pharmaceutically
acceptable salts and solvates thereof, compositions including
pharmaceutical formulations, and methods of manufacture and use of
the present invention are those wherein the compound of Formula I
or II is selected from: [0285]
3-benzyl-2-(1-benzyl-6-oxo-piperidin-2-yl)-7-chloro-3H-quinazolin-
-4-one; and [0286]
3-benzyl-2-(1-(4-methyl-benzyl)-6-oxo-piperidin-2-yl)-7-chloro-3H-quinazo-
lin-4-one.
Utility, Testing and Administration
[0286] General Utility
[0287] The compounds of the invention find use in a variety of
applications, including as therapeutic active agents, in the
practice of the methods of treatment, in compositions, particularly
pharmaceutical formulations and in methods for the manufacture of
pharmaceutical formulations, and as intermediates in the synthesis
of such therapeutic active agents.
[0288] As will be appreciated by those in the art, mitosis can be
altered in a variety of ways; that is, one can affect mitosis
either by increasing, decreasing or otherwise interfering with the
activity of a component in the mitotic pathway. Stated differently,
mitosis can be affected (e.g., disrupted) by disturbing
equilibrium, either by inhibiting or activating certain mitotic
components. Similar approaches can be used to alter meiosis.
[0289] The compounds of the invention can be used to inhibit
mitotic spindle formation. Such inhibition may take the form of
lessening a mitotic kinesin's organization of microtubules into
bipolar structures, increasing or decreasing spindle pole
separation, and/or inducing mitotic spindle dysfunction. In
particular, the compounds of the invention are useful to bind to
and/or inhibit the activity of a mitotic kinesin, KSP, especially
human KSP, although KSP kinesins from other organisms may also be
used. Also included within the definition of the term "KSP" for
these purposes are variants and/or fragments of KSP. See, U.S. Pat.
No. 6,437,115. While other mitotic kinesins may be used in the
present invention, the compounds of the invention have been shown
to have specificity for KSP. Contacting a compound of the invention
with a KSP kinesin, particularly human KSP kinesin, can lead to
diminished KSP-mediated ATP hydrolysis activity and/or diminished
KSP-mediated mitotic spindle formation activity. Meiotic spindles
can be similarly disrupted.
[0290] In another embodiment, the compounds of the invention can be
used to modulate one or more other human mitotic kinesins, in
addition to inhibiting KSP, including: HSET (see, U.S. Pat. No.
6,361,993); MCAK (see, U.S. Pat. No. 6,331,424); CENP-E (see, PCT
Publication No. WO 99/13061); Kif4 (see, U.S. Pat. No. 6,440,684);
MKLP1 (see, U.S. Pat. No. 6,448,025); Kif15 (see, U.S. Pat. No.
6,355,466); Kid (see, U.S. Pat. No. 6,387,644); Mpp1, CMKrp, KinI-3
(see, U.S. Pat. No. 6,461,855); Kip3a (see, PCT Publication No. WO
01/96593); Kip3d (see, U.S. Pat. No. 6,492,151); and RabK6.
[0291] Therapeutic uses facilitated by the mitotic
kinesin-inhibitory activity of the compounds of the present
invention include the treatment of disorders associated with cell
proliferation. Particular disease states that can be treated by the
methods, pharmaceutical formulations, and compounds provided herein
include, but are not limited to, cancer (further discussed below),
autoimmune disease, arthritis, graft rejection, inflammatory bowel
disease, proliferation induced after medical procedures, including,
but not limited to, surgery, angioplasty, and the like. In one
embodiment, the invention includes application to cells or
individuals afflicted or impending afflication with any one of
these disorders or states.
[0292] The compounds, pharmaceutical formulations and methods
provided herein are particularly deemed useful for the treatment of
cancer including solid tumors such as skin, breast, brain, cervical
carcinomas, testicular carcinomas, etc. More particularly, cancers
that can be treated include, but are not limited to: [0293]
Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma,
liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma;
[0294] Lung: bronchogenic carcinoma (squamous cell,
undifferentiated small cell, undifferentiated large cell,
adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial
adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma;
[0295] Gastrointestinal: esophagus (squamous cell carcinoma,
adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma,
lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma,
insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma),
small bowel (adenocarcinoma, lymphoma, carcinoid tumors, Karposi's
sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma),
large bowel (adenocarcinoma, tubular adenoma, villous adenoma,
hamartoma, leiomyoma); [0296] Genitourinary tract: kidney
(adenocarcinoma, Wilm's tumor [nephroblastoma], lymphoma,
leukemia), bladder and urethra (squamous cell carcinoma,
transitional cell carcinoma, adenocarcinoma), prostate
(adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal
carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial
cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma);
[0297] Liver: hepatoma (hepatocellular carcinoma),
cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular
adenoma, hemangioma; [0298] Bone: osteogenic sarcoma
(osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma,
chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell
sarcoma), multiple myeloma, malignant giant cell tumor chordoma,
osteochronfroma (osteocartilaginous exostoses), benign chondroma,
chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell
tumors; [0299] Nervous system: skull (osteoma, hemangioma,
granuloma, xanthoma, osteitis deformans), meninges (meningioma,
meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma,
glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform,
oligodendroglioma, schwannoma, retinoblastoma, congenital tumors),
spinal cord neurofibroma, meningioma, glioma, sarcoma); [0300]
Gynecological: uterus (endometrial carcinoma), cervix (cervical
carcinoma, pre-tumor cervical dysplasia), ovaries (ovarian
carcinoma [serous cystadenocarcinoma, mucinous cystadenocarcinoma,
unclassified carcinoma], granulosa-thecal cell tumors,
Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma),
vulva (squamous cell carcinoma, intraepithelial carcinoma,
adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell
carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal
rhabdomyosarcoma], fallopian tubes (carcinoma); [0301] Hematologic:
blood (myeloid leukemia [acute and chronic], acute lymphoblastic
leukemia, chronic lymphocytic leukemia, myeloproliferative
diseases, multiple myeloma, myelodysplastic syndrome), Hodgkin's
disease, non-Hodgkin's lymphoma [malignant lymphoma]; [0302] Skin:
malignant melanoma, basal cell carcinoma, squamous cell carcinoma,
Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma,
dermatofibroma, keloids, psoriasis; and [0303] Adrenal glands:
neuroblastoma. As used herein, treatment of cancer includes
treatment of cancerous cells, including cells afflicted by any one
of the above-identified conditions.
[0304] Another useful aspect of the invention is a kit having a
compound, salt or solvate of Formula I or II and a package insert
or other labeling including directions treating a cellular
proliferative disease by administering an effective amount of the
compound, salt or solvate. The compound, salt or solvate of Formula
I or II in the kits of the invention is particularly provided as
one or more doses for a course of treatment for a cellular
proliferative disease, each dose being a pharmaceutical formulation
including a pharmaceutically accepted excipient and a compound,
salt or solvate of Formula I or II.
Testing
[0305] To assay activity, generally, either KSP or a compound
according to the invention is non-diffusably bound to an insoluble
support having isolated sample receiving areas. The insoluble
support can be made of any material to which the compounds can be
bound, is readily separated from soluble material, and is otherwise
compatible with the overall method of screening. The surface of
such supports can be solid or porous and of any convenient shape.
Examples of suitable insoluble supports include microtiter plates,
arrays, membranes and beads. These are typically made of glass,
plastic (e.g., polystyrene), polysaccharides, nylon or
nitrocellulose, Teflon.TM., etc. Microtiter plates and arrays are
especially convenient because a large number of assays can be
carried out simultaneously, using small amounts of reagents and
samples. The particular manner of binding of the compound is not
crucial so long as it is compatible with the reagents and overall
methods of the invention, maintains the activity of the compound
and is nondiffusable. Particular methods of binding include the use
of antibodies (which do not sterically block either the ligand
binding site or activation sequence when the protein is bound to
the support), direct binding to "sticky" or ionic supports,
chemical crosslinking, the synthesis of the protein or agent on the
surface, etc. Following binding of the protein or agent, excess
unbound material is removed by washing. The sample receiving areas
can then be blocked through incubation with bovine serum albumin
(BSA), casein or other innocuous protein or other moiety.
[0306] The compounds of the invention can be used on their own to
modulate the activity of a mitotic kinesin, particularly KSP. In
this embodiment, a compound of the invention is combined with KSP
and the activity of KSP is assayed. Measurable kinesin activities
include the ability to affect ATP hydrolysis; microtubule binding;
gliding and polymerization/depolymerization (effects on microtubule
dynamics); binding to other proteins of the spindle; binding to
proteins involved in cell-cycle control; serving as a substrate to
other enzymes, such as kinases or proteases; and specific kinesin
cellular activities such as spindle pole separation.
[0307] Methods of performing motility assays are well known to
those of skill in the art. [See e.g., Hall, et al. (1996), Biophys.
J., 71: 3467-3476, Turner et al., 1996, Anal. Biochem. 242
(1):20-5; Gittes et al., 1996, Biophys. J. 70(I): 418-29; Shirakawa
et al., 1995, J. Exp. Biol. 198: 1809-15; Winkelmann et al., 1995,
Biophys. J. 68: 2444-53; Winkelmann et al., 1995, Biophys. J. 68:
72S.]
[0308] Methods known in the art for determining ATPase hydrolysis
activity also can be used. Solution based assays are particularly
suitable (see, U.S. Pat. No. 6,410,254); alternatively,
conventional methods are used. For example, P.sub.i release from
kinesin can be quantified. In one embodiment, the ATPase hydrolysis
activity assay utilizes 0.3 M PCA (perchloric acid) and malachite
green reagent (8.27 mM sodium molybdate II, 0.33 mM malachite green
oxalate, and 0.8 mM Triton X-100). To perform the assay, 10 .mu.L
of reaction is quenched in 90 .mu.L of cold 0.3 M PCA. Phosphate
standards are used so data can be converted to mM inorganic
phosphate released. When all reactions and standards have been
quenched in PCA, 100 .mu.L of malachite green reagent is added to
the relevant wells in e.g., a microtiter plate. The mixture is
developed for 10-15 minutes and the plate is read at an absorbance
of 650 nm. When phosphate standards are used, absorbance readings
can be converted to mM P.sub.i and plotted over time. Additionally,
ATPase assays known in the art include the luciferase assay.
[0309] ATPase activity of kinesin motor domains also can be used to
monitor the effects of modulating agents. In one embodiment ATPase
assays of kinesin are performed in the absence of microtubules. In
another embodiment, the ATPase assays are performed in the presence
of microtubules. Different types of modulating agents can be
detected in the above assays. In one particular embodiment, the
effect of a modulating agent is independent of the concentration of
microtubules and ATP. In another embodiment, the effect of the
agents on kinesin ATPase can be decreased by increasing the
concentrations of ATP, microtubules or both. In yet another
embodiment, the effect of the modulating agent is increased by
increasing concentrations of ATP, microtubules or both.
[0310] Agents that modulate the biochemical activity of KSP in
vitro may then be screened in vivo. Methods for testing such agents
in vivo include assays of cell cycle distribution, cell viability,
or the presence, morphology, activity, distribution or amount of
mitotic spindles. Methods for monitoring cell cycle distribution of
a cell population, for example, by flow cytometry, are well known
to those skilled in the art, as are methods for determining cell
viability. See, for example, WO 01/31335, entitled "Methods of
Screening for Modulators of Cell Proliferation and Methods of
Diagnosing Cell Proliferation States."
[0311] In addition to the assays described above, microscopic
methods for monitoring spindle formation and malformation are well
known to those of skill in the art (see, e.g., Whitehead and
Rattner (1998), J. Cell Sci. 111:2551-61; Galgio et al, (1996) J.
Cell Biol., 135:399-414).
[0312] The compounds of the invention inhibit KSP kinesin. One
measure of inhibition, IC.sub.50, is defined as the concentration
of the compound at which the activity of KSP is decreased by fifty
percent. Particularly suitable compounds have IC.sub.50's of less
than about 1 mM, with more particularly suitable compounds having
IC.sub.50's of less than about 100 .mu.M. IC.sub.50's of less than
about 10 nM can be attained by certain compounds of the invention,
and the pharmaceutically acceptable salts and solvates thereof, it
being appreciated that a smaller IC.sub.50 is generally considered
advantageous. Measurement of IC.sub.50 is done using an ATPase
assay.
[0313] Another measure of inhibition is K.sub.i. For compounds with
IC.sub.50's less than 1 .mu.M, the K.sub.i or Kis defined as the
dissociation rate constant for the interaction of the test compound
with KSP. Particularly suitable compounds have K.sub.i's of less
than about 100 .mu.M, more particularly suitable compounds having
K.sub.i's of less than about 10 .mu.M. K.sub.i's of less than about
10 nM can be attained by certain compounds of the invention, and
the pharmaceutically acceptable salts and solvates thereof, it
being appreciated that a smaller K.sub.i is generally considered
advantageous. The K.sub.i for a compound is determined from the
IC.sub.50 based on three assumptions. First, only one compound
molecule binds to the enzyme and there is no cooperativity. Second,
the concentrations of active enzyme and the compound tested are
known (i.e., there are no significant amounts of impurities or
inactive forms in the preparations). Third, the enzymatic rate of
the enzyme-inhibitor complex is zero. The rate (i.e., compound
concentration) data are fitted to the equation: V = V max .times. E
0 .function. [ I - ( E 0 + I 0 + Kd ) - ( E 0 + I 0 + Kd ) 2 - 4
.times. E 0 .times. I 0 2 .times. E 0 ] ##EQU1## Where V is the
observed rate, V.sub.max is the rate of the free enzyme, I.sub.0 is
the inhibitor concentration, E.sub.0 is the enzyme concentration,
and K.sub.d is the dissociation constant of the enzyme-inhibitor
complex.
[0314] Another measure of inhibition is GI.sub.50, defined as the
concentration of the compound that results in a decrease in the
rate of cell growth by fifty percent. Anti-proliferative compounds
that have been successfully applied in the clinic to treatment of
cancer (cancer chemotherapeutics) have GI.sub.50's that vary
greatly. For example, in A549 cells, paclitaxel GI.sub.50 is 4 nM,
doxorubicin is 63 nM, 5-fluorouracil is 1 .mu.M, and hydroxyurea is
500 .mu.M (data provided by National Cancer Institute,
Developmental Therapeutic Program, http://dtp.nci.nih.gov/).
Therefore, compounds that inhibit cellular proliferation at
virtually any concentration may be useful. Particularly suitable
compounds have GI.sub.50's of less than about 1 mM, with more
particularly suitable compounds having a GI.sub.50 of less than
about 10 .mu.M. GI.sub.50's of less than about 10 nM can be
attained by certain compounds of the invention, and the
pharmaceutically acceptable salts and solvates thereof, it being
appreciated that a smaller GI.sub.15 is generally considered
advantageous. Measurement of GI.sub.50 is done using a cell
proliferation assay.
[0315] Testing for growth inhibition using cell lines (such as
MCF-7/ADR-RES and HCT15) that express P-glycoprotein (also known as
Multi-drug Resistance, or MDR.sup.+), which conveys resistance to
other chemotherapeutic drugs, such as pacilitaxel, can identify
anti-mitotic agents that inhibit cell proliferation and are not
subject to resistance by overexpression of MDR.sup.+ by
drug-resistant tumor lines.
[0316] In vitro potency of small molecule inhibitors is determined
by assaying human ovarian cancer cells (SKOV3) for viability
following a 72-hour exposure to a 9-point dilution series of
compound. Cell viability is determined by measuring the absorbance
of formazon, a product formed by the bioreduction of MTS/PMS, a
commercially available reagent. Each point on the dose-response
curve is calculated as a percent of untreated control cells at 72
hours minus background absorption (complete cell kill).
[0317] To employ the compounds of the invention in a method of
screening for compounds that bind to KSP kinesin, the KSP is bound
to a support, and a compound or composition of the invention is
added to the assay. Alternatively, a composition of a compound of
the invention bound to a solid support can be made, and KSP added
to the assay. Classes of compounds among which novel binding agents
may be sought include specific antibodies, non-natural binding
agents identified in screens of chemical libraries, peptide
analogs, etc. Of particular interest are screening assays for
candidate agents that have a low toxicity for human cells. A wide
variety of assays may be used for this purpose, including labeled
in vitro protein-protein binding assays, electrophoretic mobility
shift assays, immunoassays for protein binding, functional assays
(phosphorylation assays, etc.) and the like.
[0318] The determination of the binding of the mitotic agent to KSP
may be done in a number of ways. In a particular embodiment, the
compound of the invention is labeled, for example, with a
fluorescent or radioactive moiety and binding determined directly.
For example, this may be done by attaching all or a portion of KSP
to a solid support, adding a labeled compound (for example a
compound of the invention in which at least one atom has been
replaced by a detectable isotope), washing off excess reagent, and
determining whether the amount of the label is that present on the
solid support. Various blocking and washing steps may be utilized
as is known in the art.
[0319] By "labeled" herein is meant that the compound is either
directly or indirectly labeled with a label which provides a
detectable signal, e.g., radioisotope, fluorescent tag, enzyme,
antibodies, particles such as magnetic particles, chemiluminescent
tag, or specific binding molecules, etc. Specific binding molecules
include pairs, such as biotin and streptavidin, digoxin and
antidigoxin etc. For the specific binding members, the
complementary member would normally be labeled with a molecule
which provides for detection, in accordance with known procedures,
as outlined above. The label can directly or indirectly provide a
detectable signal.
[0320] In some embodiments, only one of the components is labeled.
For example, the kinesin proteins may be labeled at tyrosine
positions using .sup.125I, or with fluorophores. Alternatively,
more than one component may be labeled with different labels; using
.sup.125I for the proteins, for example, and a fluorophor for the
anti-mitotic agents.
[0321] The compounds of the invention may also be used as
competitors to screen for additional drug candidates. "Candidate
agent" or "drug candidate" or grammatical equivalents as used
herein describe any molecule, e.g., protein, oligopeptide, small
organic molecule, polysaccharide, polynucleotide, etc., to be
tested for bioactivity. They may be capable of directly or
indirectly altering the cellular proliferation phenotype or the
expression of a cellular proliferation sequence, including both
nucleic acid sequences and protein sequences. In other cases,
alteration of cellular proliferation protein binding and/or
activity is screened. Screens of this sort may be performed either
in the presence or absence of microtubules. In the case where
protein binding or activity is screened, particular embodiments
exclude molecules already known to bind to that protein, for
example, polymer structures such as microtubules, and energy
sources such as ATP. Particular embodiments of assays herein
include candidate agents that do not bind the cellular
proliferation protein in its endogenous native state termed herein
as "exogenous" agents. In another particular embodiment, exogenous
agents further exclude antibodies to KSP.
[0322] Candidate agents can encompass numerous chemical classes,
though typically they are organic molecules, particularly small
organic compounds having a molecular weight of more than 100 and
less than about 2,500 daltons. Candidate agents comprise functional
groups necessary for structural interaction with proteins,
particularly hydrogen bonding and lipophilic binding, and typically
include at least an amine, carbonyl, hydroxyl, ether, or carboxyl
group, especially at least two of the functional chemical groups.
The candidate agents often comprise cyclical carbon or heterocyclic
structures and/or aromatic or polyaromatic structures substituted
with one or more of the above functional groups. Candidate agents
are also found among biomolecules including peptides, saccharides,
fatty acids, steroids, purines, pyrimidines, derivatives,
structural analogs or combinations thereof.
[0323] Candidate agents are obtained from a wide variety of sources
including libraries of synthetic or natural compounds. For example,
numerous means are available for random and directed synthesis of a
wide variety of organic compounds and biomolecules, including
expression of randomized oligonucleotides. Alternatively, libraries
of natural compounds in the form of bacterial, fungal, plant and
animal extracts are available or readily produced. Additionally,
natural or synthetically produced libraries and compounds are
readily modified through conventional chemical, physical and
biochemical means. Known pharmacological agents may be subjected to
directed or random chemical modifications, such as acylation,
alkylation, esterification, amidification to produce structural
analogs.
[0324] Competitive screening assays can be done by combining KSP
and a drug candidate in a first sample. A second sample may be made
combining a compound of the invention, KSP and a drug candidate.
This may be performed in either the presence or absence of
microtubules. The binding of the drug candidate is determined for
both samples, and a change or difference in binding between the two
samples indicates the presence of an agent capable of binding to
KSP and potentially modulating its activity. That is, if the
binding of the drug candidate is different in the second sample
relative to the first sample, the drug candidate is capable of
binding to KSP.
[0325] In a particularly suitable embodiment, the binding of the
candidate agent is determined through the use of competitive
binding assays. In this embodiment, the competitor is a binding
moiety known to bind to KSP, such as an antibody, peptide, binding
partner, ligand, etc. Under certain circumstances, there may be
competitive binding as between the candidate agent and the binding
moiety, with the binding moiety displacing the candidate agent.
[0326] In one embodiment, the candidate agent is labeled. Either
the candidate agent, or the competitor, or both, is added first to
KSP for a time sufficient to allow binding, if present. Incubations
can be performed at any temperature that facilitates optimal
activity, typically between 4 and 40.degree. C. Incubation periods
are selected for optimum activity, but may also be optimized to
facilitate rapid high throughput screening. Typically between 0.1
and 1 hour will be sufficient. Excess reagent is generally removed
or washed away. The second component is then added, and the
presence or absence of the labeled component is followed, to
indicate binding.
[0327] In a particularly suitable embodiment, the competitor is
added first, followed by the candidate agent. Displacement of the
competitor is an indication the candidate agent is binding to KSP
and thus is capable of binding to, and potentially modulating, the
activity of KSP. In this embodiment, either component can be
labeled. Thus, for example, if the competitor is labeled, the
presence of label in the wash solution indicates displacement by
the agent. Alternatively, if the candidate agent is labeled, the
presence of the label on the support indicates displacement.
[0328] In an alternative embodiment, the candidate agent is added
first, with incubation and washing, followed by the competitor. The
absence of binding by the competitor may indicate the candidate
agent is bound to KSP with a higher affinity. Thus, if the
candidate agent is labeled, the presence of the label on the
support, coupled with a lack of competitor binding, may indicate
the candidate agent is capable of binding to KSP.
[0329] It may be of value to identify the binding site of KSP. This
can be done in a variety of ways. In one embodiment, once KSP has
been identified as binding to the compound, KSP is fragmented or
modified and the assays repeated to identify the necessary
components for binding.
[0330] Modulation is tested by screening for candidate agents
capable of modulating the activity of KSP comprising the steps of
combining a candidate agent with KSP, as above, and determining an
alteration in the biological activity of KSP. Thus, in this
embodiment, the candidate agent should both bind to KSP (although
this may not be necessary), and alter its biological or biochemical
activity as defined herein. The methods include both in vitro
screening methods and in vivo screening of cells for alterations in
cell cycle distribution, cell viability, or for the presence,
morpohology, activity, distribution, or amount of mitotic spindles,
as are generally outlined above.
[0331] Alternatively, differential screening may be used to
identify drug candidates that bind to the native KSP, but cannot
bind to modified KSP.
[0332] Positive controls and negative controls may be used in the
assays. Preferably all control and test samples are performed in at
least triplicate to obtain statistically significant results.
Incubation of all samples is for a time sufficient for the binding
of the agent to the protein. Following incubation, all samples are
washed free of non-specifically bound material and the amount of
bound, generally labeled agent determined. For example, where a
radiolabel is employed, the samples may be counted in a
scintillation counter to determine the amount of bound
compound.
[0333] A variety of other reagents can be included in the screening
assays. These include reagents like salts, neutral proteins, e.g.,
albumin, detergents, etc which may be used to facilitate optimal
protein-protein binding and/or reduce non-specific or background
interactions. Also reagents that otherwise improve the efficiency
of the assay, such as protease inhibitors, nuclease inhibitors,
anti-microbial agents, etc., may be used. The mixture of components
may be added in any order that provides for the requisite
binding.
Formulation and Administration
[0334] The compounds, pharmaceutically acceptable salts and
solvates of Formula I and II are administered at a therapeutically
effective dosage, e.g., a dosage sufficient to provide treatment
for the disease states previously described. Human dosage levels
are typically determined by escalating dose ranging studies
conducted in accordance with current Good Clinical Practice, FDA
and local guidelines. The amount of active compound administered
will, of course, be dependent on the subject and disease state
being treated, the severity of the affliction, the manner and
schedule of administration and the judgment of the prescribing
physician.
[0335] The administration of the compounds and pharmaceutical
formulations of the present invention can be done in a variety of
ways, including, but not limited to, orally, subcutaneously,
intravenously, intranasally, transdermally, intraperitoneally,
intramuscularly, intrapulmonary, vaginally, rectally, or
intraocularly. In some instances, for example, in the treatment of
wounds and inflammation, the compound or composition may be
directly applied as a solution or spray.
[0336] Pharmaceutical formulations include a compound of Formula I
or II or a pharmaceutically acceptable salt or solvate thereof, and
one or more pharmaceutically acceptable excipients. As is known in
the art, pharmaceutical excipients are secondary ingredients that
function to enable or enhance the delivery of a drug or medicine in
a variety of dosage forms (e.g.: oral forms such as tablets,
capsules, and liquids; topical forms such as dermal, opthalmic, and
otic forms; suppositories; injectables; respiratory forms and the
like). Pharmaceutical excipients include inert or inactive
ingredients, synergists or chemicals that substantively contribute
to the medicinal effects of the active ingredient. For example,
pharmaceutical excipients may function to improve flow
characteristics, product uniformity, stability, taste, or
appearance, to ease handling and administration of dose, for
convenience of use, or to control bioavailability. While
pharmaceutical excipients are commonly described as being inert or
inactive, it is appreciated in the art that there is a relationship
between the properties of the pharmaceutical excipients and the
dosage forms containing them.
[0337] Pharmaceutical excipients suitable for use as carriers or
diluents are well known in the art, and may be used in a variety of
formulations. See, e.g., Remington's Pharmaceutical Sciences, 18th
Edition, A. R. Gennaro, Editor, Mack Publishing Company (1990);
Remington: The Science and Practice of Pharmacy, 20th Edition, A.
R. Gennaro, Editor, Lippincott Williams & Wilkins (2000);
Handbook of Pharmaceutical Excipients, 3rd Edition, A. H. Kibbe,
Editor, American Pharmaceutical Association, and Pharmaceutical
Press (2000); and Handbook of Pharmaceutical Additives, compiled by
Michael and Irene Ash, Gower (1995). The concentration of a
therapeutically active agent in a formulation can vary widely, from
about 0.1 to 99.9 wt.%, depending on the nature of the
formulation.
[0338] Oral solid dosage forms such as tablets will typically
comprise one or more pharmaceutical excipients, which may for
example help impart satisfactory processing and compression
characteristics, or provide additional desirable physical
characteristics to the tablet. Such pharmaceutical excipients may
be selected from diluents, binders, glidants, lubricants,
disintegrants, colorants, flavorants, sweetening agents, polymers,
waxes or other solubility-modulating materials.
[0339] Dosage forms for parenteral administration will generally
comprise fluids, particularly intravenous fluids, i.e., sterile
solutions of simple chemicals such as sugars, amino acids or
electrolytes, which can be easily carried by the circulatory system
and assimilated. Such fluids are typically prepared with water for
injection USP. Fluids used commonly for intravenous (IV) use are
disclosed in Remington, The Science and Practice of Pharmacy [full
citation previously provided], and include: [0340] alcohol, e.g.,
5% alcohol (e.g., in dextrose and water ("D/W") or D/W in normal
saline solution ("NSS"), including in 5% dextrose and water
("D5/W"), or D5/W in NSS); [0341] synthetic amino acid such as
Aminosyn, FreAmine, Travasol, e.g., 3.5 or 7; 8.5; 3.5, 5.5 or 8.5%
respectively; [0342] ammonium chloride e.g., 2.14%; [0343] dextran
40, in NSS e.g., 10% or in D5/W e.g., 10%; [0344] dextran 70, in
NSS e.g., 6% or in D5/W e.g., 6%; [0345] dextrose (glucose, D5/W)
e.g., 2.5-50%; [0346] dextrose and sodium chloride e.g., 5-20%
dextrose and 0.22-0.9% NaCl; [0347] lactated Ringer's (Hartmann's)
e.g., NaCl 0.6%, KCl 0.03%, CaCl.sub.2 0.02%; [0348] lactate 0.3%;
[0349] mannitol e.g., 5%, optionally in combination with dextrose
e.g., 10% or NaCl e.g., 15 or 20%; [0350] multiple electrolyte
solutions with varying combinations of electrolytes, dextrose,
fructose, invert sugar Ringer's e.g., NaCl 0.86%, KCl 0.03%,
CaCl.sub.2 0.033%; [0351] sodium bicarbonate e.g., 5%; [0352]
sodium chloride e.g., 0.45, 0.9, 3, or 5%; [0353] sodium lactate
e.g., 1/6 M; and [0354] sterile water for injection The pH of such
IV fluids may vary, and will typically be from 3.5 to 8 as known in
the art.
[0355] The compounds, pharmaceutically acceptable salts and
solvates of the invention can be administered alone or in
combination with other treatments, i.e., radiation, or other
therapeutic agents, such as the taxane class of agents that appear
to act on microtubule formation or the camptothecin class of
topoisomerase I inhibitors. When so-used, other therapeutic agents
can be administered before, concurrently (whether in separate
dosage forms or in a combined dosage form), or after administration
of an active agent of the present invention.
[0356] The following examples serve to more fully describe the
manner of using the above-described invention, as well as to set
forth the best modes contemplated for carrying out various aspects
of the invention. It is understood that these examples in no way
serve to limit the true scope of this invention, but rather are
presented for illustrative purposes.
EXAMPLES
Example 1
N-(3-amino-propyl)-N-[1-(3-benzyl-4-oxo-3,4-dihydro-pyrido[2,3-d]pyrimidin-
-2-yl)-2-methyl-propyl]-4-methyl-benzamide
[0357] 1A. Formula 106 where R.sup.1, R.sup.2 R.sup.4, R.sup.6,
R.sup.7, R.sup.8 and R.sup.9 are H; R.sup.3 is Chloro; and R.sup.5
is Benzyl: To a solution of Formula 101,
4-tert-butoxycarbonylamino-2-(9H-fluoren-9-ylmethoxycarbonylamino)-butyri-
c acid (1.00 g, 2.27 mmol) in 20 mL of anhydrous THF was added
anhydrous N-methylmorpholine (274 .mu.L, 2.50 mmol). After the
mixture was stirred in an ice-bath for 1 minute, isobutyl
chloroformate (324 .mu.L, 2.50 mmol) was added dropwise over 15
minutes at 0.degree. C. The mixture was stirred in the ice-bath for
1 hour, after which 4-chloro-anthranilic acid (389 mg, 2.27 mmol)
in 2 mL of THF was added, affording the corresponding compound of
Formula 103,
2-[4-tert-butyxycarbonylamino-2-(9H-fluoren-9-ylmethoxy-carbonylamino)-bu-
tyrylamino]-4-chloro-benzoic acid, following 2 hours of additional
stirring at 0.degree. C. To the stirring compound of Formula 103,
anhydrous N-methylmorpholine (274 .mu.L, 2.50 mmol) was added and
the mixture stirred overnight while slowly warming to room
temperature. The mixture was then cooled to 0.degree. C. and
treated with anhydrous N-methylmorpholine (274 .mu.L, 2.50 mmol)
and isobutyl chloroformate (324 .mu.L, 2.50 mmol). This was
followed by addition of benzylamine (992 .mu.L, 3.92 mmol) in four
equal portions at room temperature. Once the starting material was
consumed, the solvents were evaporated and the residue partitioned
between DCM and saturated sodium bicarbonate. The organic layer was
dried over sodium sulfate and the solvent was evaporated, affording
a mixture of the corresponding compounds of Formula 104 and Formula
105,
[1-(2-benzylcarbamoyl-5-chloro-phenyl-carbamoyl)-3-tert-butoxycarbonylami-
no-propyl]-carbamic acid 9H-fluoren-9-ylmethyl ester and
[1-(3-benzyl-7-chloro-4-oxo-c,4-3,4-dihydro-quinazolin-2-yl)-3-tert-butox-
ycarbonyl-amino-propyl]-carbamic acid 9H-fluoren-9-ylmethyl ester,
respectively (about 6:1 by LCMS) which was dried under vacuum and
then treated with lithium hydroxide monohydrate (95 mg, 2.27 mmol)
in 60 mL of 1,4-dioxane/ethylene glycol (2/1) under reflux for 5
hours. The reaction solution was poured into 200 mL of water and
the product extracted with DCM. After drying the organic layers
over sodium sulfate, the solvent was evaporated and the crude
product purified by flash silica gel chromatography (stepwise
gradient 1:4, 1:2, 1:1, 2:1, 4:1 with ethyl acetate:hexanes as
eluent) to give the corresponding product of Formula 106,
[3-amino-3-(3-benzyl-7-chloro-4-oxo-3,4-dihydro-quinazolin-2-yl)-pro-
pyl]-carbamic acid tert-butyl ester (700 mg, 70%).
[0358] 1B. Formula 107 where R.sup.1, R.sup.2, R.sup.4, R.sup.6,
R.sup.7, R.sup.8 and R.sup.9 are H; R.sup.3 is Chloro; R.sup.5 is
Benzyl; and R.sup.10 is p-Methyl-benzyl: To a solution of
[3-amino-3-(3-benzyl-7-chloro-4-oxo-3,4-dihydro-quinazolin-2-yl)-propyl]--
carbamic acid tert-butyl ester (700 mg, 1.58 mmol) and DIEA (275
.mu.L, 2.37 mmol) in 50 mL of DCM was added p-tolualdehyde (188
[2L, 1.58 mmol). The mixture was stirred for 1 hour, after which
sodium triacetoxyborohydride (500 mg, 2.37 mmol) was added. After
stirring an additional 3 hours, the mixture was washed with
saturated sodium bicarbonate solution, dried over sodium sulfate.
The solvents were evaporated to dryness and the residue was
purified by flash silica gel chromatography (stepwise gradient 1:4,
1:2, 1:1, 2:1, 4:1 with ethyl acetate:hexanes as eluent) to give
the corresponding, pure compound of Formula 107,
[3-(3-benzyl-7-chloro-4-oxo-3,4-dihydro-quinazolin-2-yl)-3-(4-methyl-benz-
ylamino)-propyl]-carbamic acid tert-butyl ester (760 mg, 88%).
[0359] 1C. Formula 108 where R.sup.1, R.sup.2, R.sup.4, R.sup.6,
R.sup.7, R.sup.8 and R.sup.9 are H; R.sup.3 is Chloro; R.sup.5 is
Benzyl; R.sup.10 is p-Methyl-benzyl; and R''' is NHBoc-Ethyl:
[3-(3-Benzyl-7-chloro-4-oxo-3,4-dihydro-quinazolin-2-yl)-3-(4-methyl-benz-
ylamino)-propyl]-carbamic acid tert-butyl ester (360 mg, 0.658
mmol) was dissolved in 10 mL of TFA/H.sub.2O (95/5) solution and
stirred for 30 minutes. The solvents were evaporated and the
residue partitioned between DCM and saturated sodium bicarbonate
solution. The organic layer was dried over sodium sulfate and the
solvents evaporated to dryness. The resulting residue, DIEA (168
.mu.L, 0.966 mmol), tert-butyl N-(2-oxoethyl) carbamate (122 mg,
0.767 mmol) and sodium triacetoxyborohydride (318 mg, 0.966 mmol)
were mixed in 50 mL of DCM and stirred for 1 hour. The solution was
then washed with 100 mL of saturated sodium bicarbonate solution
and dried over sodium sulfate. The solvent was evaporated to
dryness to give the corresponding compound of Formula 108,
{2-[3-(3-benzyl-7-chloro-4-oxo-3,4-dihydro-quinazolin-2-yl)-3-(4-met-
hyl-benzylamino)-propylamino]-ethyl}-carbamic acid tert-butyl ester
(380 mg, 98%).
[0360] 1D. Formula I where R.sup.1, R.sup.2, R.sup.4, R.sup.6,
R.sup.7, R.sup.8 and R.sup.9 are H; R.sup.3 is Chloro; R.sup.5 is
Benzyl; R.sup.10 is p-Methyl-benzyl; and V is NR' where R''' is
Amino-ethyl: To a solution of crude
{2-[3-(3-benzyl-7-chloro-4-oxo-3,4-dihydro-quinazolin-2-yl)-3-(4-
-methyl-benzylamino)-propylamino]-ethyl}-carbamic acid tert-butyl
ester (380 mg, 0.644 mmol) and DIEA (167 .mu.L, 0.966 mmol) in 50
mL of DCM was added carbonyldiimidazole (157 mg, 0.966 mmol) and
the reaction mixture stirred for 1 hour. The solvent was evaporated
and the residue purified by flash silica gel chromatography
(stepwise gradient 1:4, 1:2, 1:1, 2:1, 4:1 with ethyl
acetate:hexanes as eluent) to give the corresponding, pure
R'''-Boc-protected precursor to Formula I (250 mg, 63%). This
product (250 mg, 0.405 mmol) was dissolved in 10 mL of TFA/H.sub.2O
(95/5) solution, stirred for 1 hour, and then evaporated to
dryness. The residue was partitioned between DCM and saturated
sodium bicarbonate, the organic layer was dried over sodium
sulfate, and the solvents evaporated to dryness. The residue was
dried under vacuum for a few hours to give the desired product of
Formula I,
2-[-1-(2-amino-ethyl)-3-(4-methyl-benzyl)-2-oxo-hexahydro-pyrimidin-4-yl]-
-3-benzyl-7-chloro-3H-quinazolin-4-one (188 mg, 90%).
Example 2
Other Compounds of Formulae I and II
[0361] 2A. Formula I where R.sup.1, R.sup.2, R.sup.4, R.sup.6,
R.sup.8 and R.sup.9 are H; R.sup.3 is Chloro; R.sup.5 is Benzyl;
R.sup.7 is i-Propyl; R.sup.10 is p-Methyl-benzyl; and V is NR'
where R''' is Amino-ethyl: By following the procedure described in
Example 1 and substituting
4-tert-butoxycarbonylamino-2-(9H-fluoren-9-ylmethoxycarbonylamino)-butyri-
c acid with
3-(tert-butoxycarbonylamino-methyl)-2-(9H-fluoren-9-ylmethoxycarbonylamin-
o)-4-methyl-pentanoic acid, there is obtained
2-[-1-(2-amino-ethyl)-5-isopropyl-3-(4-methyl-benzyl)-2-oxo-hexahydro-pyr-
imidin-4-yl]-3-benzyl-7-chloro-3H-quinazolin-4-one.
[0362] 2B. Formula I where R.sup.1, R.sup.4, R.sup.6, R.sup.7,
R.sup.8 and R.sup.9 are H; R.sup.2 and R.sup.3 are Methoxy; R.sup.5
is Benzyl; R.sup.10 is p-Methyl-benzyl; and V is NR' where R''' is
Amino-ethyl: By following the procedure described in Example 1 and
substituting 4-chloro-anthranilic acid with 2-amino-4,5-dimethoxy
benzoic acid, there is obtained
2-[-1-(2-amino-ethyl)-3-(4-methyl-benzyl)-2-oxo-hexahydro-pyrimidin-4-yl]-
-3-benzyl-6,7-dimethoxy-3H-quinazolin-4-one.
[0363] 2C. Formula I where R.sup.1, R.sup.2, R.sup.4, R.sup.6,
R.sup.7, R.sup.8 and R.sup.9 are H; R.sup.3 is Methoxy; R.sup.5 is
Benzyl; R.sup.10 is p-Methyl-benzyl; and V is NR''' where R''' is
Isopropyl: By following the procedure described in Example 1 and
substituting tert-butyl N-(2-oxoethyl) carbamate with
2-methylpriopionaldehyde, there is obtained
2-[-1-isopropyl-3-(4-methyl-benzyl)-2-oxo-hexahydro-pyrimidin-4-yl]-3-ben-
zyl-7-chloro-3H-quinazolin-4-one.
[0364] 2D. Formula II where T is Methylene; W, X, Y and Z are
--C.dbd.; R.sup.1, R.sup.2, R.sup.4, R.sup.6, R.sup.8 and R.sup.9
are H; R.sup.3 is Chloro; R.sup.5 is Benzyl; R.sup.7 is i-Propyl;
R.sup.10 is p-Methyl-benzyl; and V is NR' where R''' is
Amino-ethyl: By following the procedure described in Example 1 and
substituting
4-tert-butoxycarbonylamino-2-(9H-fluoren-9-ylmethoxycarbonylamino)-butyri-
c acid with
4-(tert-butoxycarbonylamino-methyl)-3-[(9H-fluoren-9-yl)-methoxycarbonyl--
amino]-5-methyl-hexanoic acid, there is obtained
2-[-1-(2-amino-ethyl)-5-isopropyl-3-(4-methyl-benzyl)-2-oxo-hexahydro-pyr-
imidin-4-ylmethyl]-3-benzyl-7-chloro-3H-quinazolin-4-one.
[0365] 2E. Formula II where T is Carboxyethylene; W, X, Y and Z are
--C.dbd.; R.sup.1, R.sup.2, R.sup.4, R.sup.6, R.sup.8 and R.sup.9
are H; R.sup.3 is Chloro; R.sup.5 is Benzyl; R.sup.10 is
p-Methyl-benzyl; and V is NR' where R''' is Isopropyl: By following
the procedure described in Example 1, substituting in
4-tert-butoxycarbonylamino-2-(9H-fluoren-9-ylmethoxycarbonylamino)-butyri-
c acid with
6-(tert-butoxycarbonylamino)-4-[(9H-fluoren-9-yl)-methoxycarbonyl-amino]--
3-oxo-hexanoic acid in Example 1A, and substituting tert-butyl
N-(2-oxoethyl) carbamate with 2-methyl-propionaldehyde in Example
1C, there is obtained
3-benzyl-7-chloro-2-{2-[1-isopropyl-3-(4-methyl-benzyl)-2-oxo-hexahydro-p-
yrimidin-4-yl]-2-oxo-ethyl}-3H-quinazolin-4-one.
[0366] 2F. Formula II where T is Carboxyethylene; W and Y are
--C.dbd.; X and Z are --N.dbd.; R.sup.1, R.sup.2, R.sup.4, R.sup.6,
R.sup.8 and R.sup.9 are H; R.sup.3 is Chloro; R.sup.5 is Benzyl;
R.sup.10 is p-Methyl-benzyl; and V is NR' where R''' is Isopropyl:
By following the procedure described in Example 2E and additionally
substituting 4-chloro-anthranilic acid with
4-amino-pyridine-5-carboxylic acid in Example 1A, there is obtained
3-benzyl-2-{2-[1-isopropyl-3-(4-methyl-benzyl)-2-oxo-hexahydro-pyrimidin--
4-yl]-2-oxo-ethyl}-3H-pyrimido[4,5-d]pyrimidin-4-one.
Example 3
3-Benzyl-2-(1-benzyl-6-oxo-piperidin-2-yl)-7-chloro-3H-quinazolin-4-one
[0367] 3A. Formula 203 where R', R'', R.sup.6, R.sup.7, R.sup.8 and
R.sup.9 are H; and R.sup.10 is Benzyl;
[0368] DL-2-Aminoadipic acid hydrate (2 g, 11 mmol) was dissolved
in 2 M NaOH (11 mL, 22 mmol). Benzaldehyde (1.4 mL, 11 mmol) was
dissolved in 3.0 mL of ethanol and this solution was added to the
first solution. After 10 minutes the mixture was cooled to
0.degree. C. and sodium borohydride (0.13 g, 3.3 mmol) was added.
After 1 hour, LCMS analysis showed the reaction to be complete. The
solution was extracted 3 times with 20 mL portions of ether, cooled
to 0.degree. C., acidified to pH 2 with conc. HCl, and the
resulting precipitate was filtered to afford a damp white solid.
The solid was washed once with a minimum amount of acetonitrile
(.about.1 mL) and three times with ether. The crude solid was
dissolved in 55 mL ethanol and the solution was boiled overnight.
The solution was evaporated to provide 1.22 g (47% yield for 2
steps) of the lactam product of Formula 203,
1-benzyl-6-oxo-piperidine-2-carboxylic acid, which was carried on
without further purification.
[0369] 3B. Formula 204 where R', R'', R.sup.1 , R.sup.2, R.sup.4,
R.sup.6, R.sup.7, R.sup.8 and R.sup.9 are H; R.sup.3 is Chloro; and
R.sup.10 is Benzyl: 1-Benzyl-6-oxo-piperidine-2-carboxylic acid (1
g, 4.3 mmol) and DIEA (0.75 mL, 4.3 mmol) were dissolved in 20 mL
of dichloromethane and cooled to 0.degree. C. Isobutylchloroformate
(0.59 mL, 4.3 mmol) was added and the mixture was stirred for 20
minutes. More DIEA (1.5 mL, 8.6 mmol) was added, followed by
4-chloroanthranilic acid (0.77 g, 4.5 mmol), and the mixture was
allowed to warm to room temperature overnight. The mixture was
washed with 1 M HCl and brine, dried over MgSO.sub.4, and
evaporated to provide a yellow oily solid. This material was
dissolved in 3 mL of 2 M NaOH, washed twice with ether, cooled to
0.degree. C., and acidified with conc. HCl. A pale yellow oil
formed, which solidified upon standing. This material was filtered
and dried under vacuum to provide 0.57 g (34% yield) of the benzoic
acid of Formula 204,
2-[(1-benzyl-6-oxo-piperidine-2-carbonyl)-amino]-4-chloro-benzoic
acid, which was carried on without further purification.
[0370] 3C. Formula I where R.sup.1, R.sup.2, R.sup.4, R.sup.6,
R.sup.7, R.sup.8 and R.sup.9 are H; R.sup.3 is Chloro; R.sup.5 is
Benzyl, R.sup.10 is Benzyl; and V is CR'R''' where R' and R'' are
H: To a solution of
2-[(1-benzyl-6-oxo-piperidine-2-carbonyl)-amino]-4-chloro-benzoic
acid (165 mg, 0.427 mmol) in 5 mL of DMF was added EDC (245 mg,
1.28 mmol) and the mixture stirred at room temperature. After 1
hour, benzylamine (140 .mu.L, 1.28 mmol) was added, and the
reaction mixture was stirred at room temperature for an additional
3 hours. After evaporating the solvents, the residue was dissolved
in 100 mL of DCM, and washed with saturated sodium bicarbonate and
brine. The organic layer was dried over sodium sulfate and
filtered. The solvent was removed to afford crude
1-benzyl-6-oxo-piperidine-2-carboxylic acid
(2-benzylcarbamoyl-5-chloro-phenyl)-amide, a compound of Formula
402. This was dried under vacuum and then added to a mixture of 10
mL of ethylene glycol and sodium hydroxide (17 mg, 0.425 mmol),
followed by stirring at 130.degree. C. for two days with monitoring
by LC-MS. The mixture was then poured into 100 mL of water. After
extraction with DCM, the resulting crude product was purified over
silica gel (stepwise gradient 1:4, 1:2, 1:1, 2:1, 4:1 with ethyl
acetate:hexanes as eluent) to give the desired pure product of
Formula I,
3-benzyl-2-(1-benzyl-6-oxo-piperidin-2-yl)-7-chloro-3H-quinazolin-4-one
(20 mg,10%).
Example 4
Other Compounds of Formulae I and II
[0371] 4A. Varying T, V and R.sup.6 to R.sup.9 By following the
procedure described in Example 3 and substituting DL-2-aminoadipic
acid hydrate with the following: [0372] 2-amino-pentanedioic acid;
[0373] 4-amino-2,2-dimethyl-pentanedioic acid; [0374]
2-amino-3-methyl-hexanedioic acid; [0375]
2-amino-4-isopropyl-hexanedioic acid; [0376]
2-amino-3,4-dihydroxy-hexanedioic acid; [0377]
5-amino-2,3-diethoxy-hexanedioic acid; [0378] 4-amino-octanedioic
acid; and [0379] 5-amino-6-carboxymethoxy-hexanoic acid, there are
obtained the following respective compounds: [0380]
3-benzyl-2-(1-benzyl-5-oxo-pyrrolidin-2-yl)-7-chloro-3H-quinazolin-4-one;
[0381]
3-benzyl-2-(1-benzyl-4,4-dimethyl-5-oxo-pyrrolidin-2-yl)-7-chlor-
o-3H-quinazolin-4-one; [0382]
3-benzyl-2-(1-benzyl-3-methyl-6-oxo-piperidin-2-yl)-7-chloro-3H-quinazoli-
n-4-one; [0383]
3-benzyl-2-(1-benzyl-4-isopropyl-6-oxo-piperidin-2-yl)-7-chloro-3H-quinaz-
olin-4-one; [0384]
3-benzyl-2-(1-benzyl-3,4-dihydroxy-6-oxo-piperidin-2-yl)-7-chloro-3H-quin-
azolin-4-one; [0385]
3-benzyl-2-(1-benzyl-4,5-diethoxy-6-oxo-piperidin-2-yl)-7-chloro-3H-quina-
zolin-4-one; [0386]
3-benzyl-2-[3-(1-benzyl-5-oxo-pyrrolidin-2-yl)-propyl]-7-chloro-3H-quinaz-
olin-4-one [0387]
3-benzyl-2-(1-benzyl-6-oxo-piperidin-2-ylmethoxymethyl)-7-chloro-3H-quina-
zolin-4-one.
[0388] 4B. Varying R.sup.10 By following the procedure described in
Examples 3 and 4A, and substituting benzaldeyhde with
4-methyl-benzaldehyde, there are obtained: [0389]
3-benzyl-2-[1-(4-methyl-benzyl)-5-oxo-pyrrolidin-2-yl]-7-chloro-3H-quinaz-
olin-4-one; [0390]
3-benzyl-2-[4,4-dimethyl-1-(4-methyl-benzyl)-5-oxo-pyrrolidin-2-yl]-7-chl-
oro-3H-quinazolin-4-one; [0391]
3-benzyl-2-[3-methyl-1-(4-methyl-benzyl)-6-oxo-piperidin-2-yl]-7-chloro-3-
H-quinazolin-4-one; [0392]
3-benzyl-2-[4-isopropyl-1-(4-methyl-benzyl)-6-oxo-piperidin-2-yl]-7-chlor-
o-3H-quinazolin-4-one; [0393]
3-benzyl-2-[3,4-dihydroxy-1-(4-methyl-benzyl)-6-oxo-piperidin-2-yl]-7-chl-
oro-3H-quinazolin-4-one; [0394]
3-benzyl-2-[4,5-diethoxy-1-(4-methyl-benzyl)-6-oxo-piperidin-2-yl]-7-chlo-
ro-3H-quinazolin-4-one; [0395]
3-benzyl-2-{3-[1-(4-methyl-benzyl)-5-oxo-pyrrolidin-2-yl]-propyl}-7-chlor-
o-3H-quinazolin-4-one; and [0396]
3-benzyl-2-[1-(4-methyl-benzyl)-6-oxo-piperidin-2-ylmethoxymethyl]-7-chlo-
ro-3H-quinazolin-4-one;
[0397] 4C. Varying T, V, and R' or R.sup.9 By following the
procedure described in Example 3, substituting DL-2-aminoadipic
acid hydrate with the following: [0398]
2-amino-4-(2-tert-butoxycarbonylamino-ethyl)-pentanedioic acid;
[0399] 2-amino-5-(2-tert-butoxycarbonylamino-ethyl)-hexanedioic
acid; and [0400]
5-amino-2-(2-tert-butoxycarbonylamino-ethyl)-6-(tert-butoxycarbon-
yl-carboxymethyl-amino)-hexanoic acid, and de-protecting the
product thus-obtained (for example, as described in the second part
of Example 1D), there are obtained the following respective
compounds: [0401]
3-benzyl-2-[4-(2-amino-ethyl)-1-benzyl-5-oxo-pyrrolidin-2-yl]-7-chloro-3H-
-quinazolin-4-one; [0402]
3-benzyl-2-[5-(2-amino-ethyl)-1-benzyl-6-oxo-piperidin-2-yl]-7-chloro-3H--
quinazolin-4one; and [0403]
2-({[5-(2-amino-ethyl)-1-benzyl-6-oxo-piperidin-2-ylmethyl]-amino}-methyl-
)-3-benzyl-7-chloro-3H-quinazolin-4-one.
[0404] 4D. Varying T, V, R', R.sup.9 and R.sup.10 By following the
procedure described in Example 4C and substituting benzaldeyhde
with (2-oxo-ethyl)-carbamic acid tert-butyl ester, there are
obtained: [0405]
3-benzyl-2-[1,4-bis-(2-amino-ethyl)-5-oxo-pyrrolidin-2-yl]-7-chlo-
ro-3H-quinazolin-4-one; [0406]
3-benzyl-2-[1,5-bis-(2-amino-ethyl)-6-oxo-piperidin-2-yl]-7-chloro-3H-qui-
nazolin-4-one; and [0407]
3-benzyl-2-({[1,5-bis-(2-amino-ethyl)-6-oxo-piperidin-2-ylmethyl]-amino}--
methyl)-7-chloro-3H-quinazolin-4-one.
[0408] 4E. Varying T, V, R.sup.1to R.sup.4, R.sup.6 to R.sup.9 and
W, X, Y and Z By following the procedure as described with
2-amino-pentanedioic acid, 4-amino-2,2-dimethyl-pentanedioic acid
and 2-amino-3-methyl-hexanedioic acid in Example 4A, and
additionally substituting 4-chloroanthranilic acid (from Example
3B) with the following: [0409] 3-amino-pyrazine-2-carboxylic acid;
[0410] 3-amino-1,4-dihydro-pyridine-2-carboxylic acid; [0411]
2-amino-cyclopent-1-enecarboxylic acid; [0412]
4-amino-2,5-dihydro-furan-3-carboxylic acid; and [0413]
3-amino-1H-pyrrole-2-carboxylic acid, there are obtained the
following respective compounds: [0414]
3-benzyl-2-(1-benzyl-5-oxo-pyrrolidin-2-yl)-3H-pteridin-4-one;
[0415]
3-benzyl-2-(1-benzyl-5-oxo-pyrrolidin-2-yl)-5,8-dihydro-3H-pyrido[3,2-d]p-
yrimidin-4-one; [0416]
3-benzyl-2-(1-benzyl-5-oxo-pyrrolidin-2-yl)-3,5,6,7-tetrahydro-cyclopenta-
pyrimidin-4-one; [0417]
3-benzyl-2-(1-benzyl-5-oxo-pyrrolidin-2-yl]-5,7-dihydro-3H-furo[3,4-d]pyr-
imidin-4-one; [0418]
3-benzyl-2-(1-benzyl-5-oxo-pyrrolidin-2-yl]-3,7-dihydro-pyrrolo[3,2-d]pyr-
imidin-4-one; [0419]
3-benzyl-2-(1-benzyl-4,4-dimethyl-5-oxo-pyrrolidin-2-yl)-3H-pteridin-4-on-
e; [0420]
3-benzyl-2-(1-benzyl-4,4-dimethyl-5-oxo-pyrrolidin-2-yl)-5,8-dihydro-3H-p-
yrido[3,2-d]pyrimidin-4-one; [0421]
3-benzyl-2-(1-benzyl-4,4-dimethyl-5-oxo-pyrrolidin-2-yl)-3,5,6,7-tetrahyd-
ro-cyclopentapyrimidin-4-one; [0422]
3-benzyl-2-(1-benzyl-4,4-dimethyl-5-oxo-pyrrolidin-2-yl]-5,7-dihydro-3H-f-
uro[3,4-d]pyrimidin-4-one; [0423]
3-benzyl-2-(1-benzyl-4,4-dimethyl-5-oxo-pyrrolidin-2-yl]-3,7-dihydro-pyrr-
olo[3,2-d]pyrimidin-4-one; [0424]
3-benzyl-2-(1-benzyl-3-methyl-6-oxo-piperidin-2-yl)-3H-pteridin-4-one;
[0425]
3-benzyl-2-(1-benzyl-3-methyl-6-oxo-piperidin-2-yl)-5,8-dihydro-3-
H-pyrido[3,2-d]pyrimidin-4-one; [0426]
3-benzyl-2-(1-benzyl-3-methyl-6-oxo-piperidin-2-yl)-3,5,6,7-tetrahydro-cy-
clopentapyrimidin-4-one; [0427]
3-benzyl-2-(1-benzyl-3-methyl-6-oxo-piperidin-2-yl]-5,7-dihydro-3H-furo[3-
,4-d]pyrimidin-4-one; and [0428]
3-benzyl-2-(1-benzyl-3-methyl-6-oxo-piperidin-2-yl]-3,7-dihydro-pyrrolo[3-
,2-d]pyrimidin-4-one.
Example 5
3-Benzyl-7-chloro-2-[3-(4-methyl-benzyl)-2-oxo-hexahydro-pyrimidin-4-yl]-3-
H-quinazolin-4-one
[0429] 5A. Formula 302 where R.sup.6, R.sup.7, R.sup.8 and R.sup.9
are H: DL-2,4-Diaminobutyric acid (4.9 g, 26 mmol) and sodium
bicarbonate (6.5 g, 77 mmol) was dissolved in 25 mL of water.
Copper sulfate (3.2 g, 13 mmol) was dissolved in 25 mL of water and
this solution was added to the first solution. To this mixture was
added a solution of di-(tert-butyl) pyrocarbonate (7.3 g, 33.5
mmol) dissolved in 32 mL of acetone. After stirring for 24 hours,
methanol was added to the mixture and stirring was continued for
another 18 hours. The resulting light blue precipitate was
filtered, washed twice with water, and dried under vacuum to
provide 3.3 g (51%) of the mono-Boc-protected copper complex of
diaminobutyric acid. This copper complex (3.3 g, 6.7 mmol) was
suspended in 300 mL of water and quinolinol (2.5 g, 17 mmol) was
added. After 5 hours, the suspension was filtered off and the
liquid was evaporated to provide approximately 3.5 g of a wet
yellow solid. This material was dissolved in 200 mL of 30% methanol
in benzene. To this solution was added dropwise
(trimethylsilyl)diazomethane (2.0 M in hexanes, approximately 12
mL, approximately 24 mmol) until a deep yellow color persisted and
bubbling ceased. This solution was stirred for 1 hour. Acetic acid
was added dropwise until the deep yellow color was discharged and
bubbling ceased, and then the mixture was evaporated. The material
was purified twice by flash chromatography (EtOAc to 5% MeOH in
EtOAc) to provide the methyl ester of Formula 302,
2-amino-4-tert-butoxycarbonylamino-butyric acid methyl ester, as a
pale yellow wax (1.4 g, 58%).
[0430] 5B. Formula 303 where R.sup.6, R.sup.7, R.sup.8 and R.sup.9
are H; and R.sup.10 is p-Methyl-benzyl:
[0431] To a solution of 2-amino-4-tert-butoxycarbonylamino-butyric
acid methyl ester (0.60 g, 2.58 mmol) in 100 mL of DCM was added
p-tolualdehyde (282 .mu.L, 2.39 mmol) and the mixture stirred at
room temperature for 1 hour. Sodium triacetoxyborohydride (820 mg,
3.87 mmol) was added and the mixture stirred overnight. The
solution was then washed with water and dried over magnesium
sulfate. The solvents were evaporated and the residue dissolved in
100 mL of 2M HCl in dioxane solution and stirred for 2 hours. The
solvent was evaporated and the residue dried under vacuum to give
the desired compound of Formula 303,
4-amino-2-(4-methyl-benzylamino)-butyric acid methyl ester as its
HCl salt (475 mg, 62%). This was taken on without further
purification.
[0432] 5C. Formula 304 where R.sup.6 , R.sup.7, R.sup.8 and R.sup.9
are H; and R.sup.10 is p-Methyl-benzyl:
[0433] To a solution of 4-amino-2-(4-methyl-benzylamino)-butyric
acid methyl ester HCl salt (923 mg, 3.13 mmol) and DIEA (1.08 mL,
6.26 mmol) in 100 mL of DCM was added carbonyldiimidazole (760 mg,
4.69 mmol). The reaction mixture was stirred for 1 hour, after
which the solvents were evaporated. The residue was dissolved in 20
mL of MeOH:H.sub.2O (2:1) solution to which was added LiOH (150 mg,
6.26 mmol). The mixture was stirred at room temperature for 3 hours
and then adjusted to pH .about.7 by adding Dowex-H+ resin. The
solution was filtered, the solvent was evaporated, and the residue
dried under vacuum to give the desired compound of Formula 304,
3-(4-methyl-benzyl)-2-oxo-hexahydro-pyrimidine-4-carboxylic acid
(520 mg, 67%), which was taken on without further purification.
[0434] 5D. Formula 402 where R.sup.1, R.sup.2, R.sup.4, R.sup.6,
R.sup.7, R.sup.8 and R.sup.9 are H; R.sup.5 is Benzyl; R.sup.10 is
p-Methyl-benzyl; and V is N--R''' where R''' is H: To a solution of
3-(4-methyl-benzyl)-2-oxo-hexahydro-pyrimidine-4-carboxylic acid
(400 mg, 1.61 mmol) in 20 mL of DMF was added anhydrous
N-methylmorpholine (212 .mu.L, 1.93 mmol). After cooling in an
ice-bath for 10 min, isobutyl chloroformate (251 .mu.L, 1.93 mmol)
was added dropwise at while maintaining the temperature below
5.degree. C. The mixture was stirred in the ice-bath for 1 hour,
and 4-chloroanthranilic acid (331 mg, 1.93 mmol) in 1 mL of DMF was
added. The mixture was stirred an additional 5 h during which the
temperature was allowed to warm to room temperature to afford the
corresponding intermediate product of Formula 305, which was
carried on without isolation or purification. EDC (618 mg, 3.22
mmol) was then added into the reaction, the mixture was stirred for
1 hour, and benzylamine (528 .mu.L, 4.83 mmol) was added. The
resulting solution was stirred overnight, after which the solvents
were evaporated and the residue purified by flash silica gel
chromatography (stepwise gradient 1:4, 1:2, 1:1, 2:1, 4:1 with
ethyl acetate:hexanes as eluent) to give the pure bis-amide product
of Formula 402,
3-(4-methyl-benzyl)-2-oxo-hexahydro-pyrimidine-4-carboxylic acid
(2-benzylcarbamoyl-5-chloro-phenyl)-amide (430 mg, 54%).
[0435] 5E. Formula I where R.sup.1, R.sup.2, R.sup.4, R.sup.6,
R.sup.7, R.sup.8 and R.sup.9 are H; R.sup.3 is Chloro; R.sup.5 is
Benzyl; R.sup.10 is p-Methyl-benzyl; and V is N--R''' where R''' is
H: 3-(4-Methyl-benzyl)-2-oxo-hexahydro-pyrimidine-4-carboxylic acid
(2-benzylcarbamoyl-5-chloro-phenyl)-amide (350 mg, 0.713 mmol) was
dissolved in 60 mL of ethylene glycol to which was added sodium
hydroxide (60 mg, 1.5 mmol). The mixture was stirred at 140.degree.
C. for 20 hours and monitored by LC-MS. Following consumption of
starting material, the reaction mixture was poured into 100 mL of
water. After extraction with DCM, the crude product was purified by
flash silica gel chromatography (stepwise gradient 1:4, 1:2, 1:1,
2:1, 4:1 with ethyl acetate:hexanes as eluent) to give the desired
product of Formula I,
3-benzyl-7-chloro-2-[3-(4-methyl-benzyl)-2-oxo-hexahydro-pyrimidin-4-yl]--
3H-quinazolin-4-one (180 mg, 53%).
Example 6
Compounds of Formula II where R.sup.6 to R.sup.9 are H; R.sup.5 is
Benzyl; R.sup.10 is p-Methyl-benzyl; T is Methylene and V is
N--R''' where R''' is H, Varying R.sup.1 to R.sup.4 and W, X, Y and
Z
[0436] By following the procedure as described in Example 5 and
substituting 4-chloroanthranilic acid with the following: [0437]
3-amino-pyrazine-2-carboxylic acid; [0438]
3-amino-1,4-dihydro-pyridine-2-carboxylic acid; [0439]
2-amino-cyclopent-1-enecarboxylic acid; [0440]
4-amino-2,5-dihydro-furan-3-carboxylic acid; and [0441]
3-amino-1H-pyrrole-2-carboxylic acid, there are obtained the
following respective compounds: [0442]
3-benzyl-2-[3-(4-methyl-benzyl)-2-oxo-hexahydro-pyrimidin-4-yl]-3H-pterid-
in-4-one; [0443]
3-benzyl-2-[3-(4-methyl-benzyl)-2-oxo-hexahydro-pyrimidin-4-yl]-5,8-dihyd-
ro-3H-pyrido[3,2-d]pyrimidin-4-one; [0444]
3-benzyl-2-[3-(4-methyl-benzyl)-2-oxo-hexahydro-pyrimidin-4-yl]-3,5,6,7-t-
etrahydro-cyclopentapyrimidin-4-one; [0445]
3-benzyl-2-[3-(4-methyl-benzyl)-2-oxo-hexahydro-pyrimidin-4-yl]-5,7-dihyd-
ro-3H-furo[3,4-d]pyrimidin-4-one; and [0446]
3-benzyl-2-[3-(4-methyl-benzyl)-2-oxo-hexahydro-pyrimidin-4-yl]-3,7-dihyd-
ro-pyrrolo[3,2-d]pyrimidin-4-one.
Example 7
Induction of Mitotic Arrest in Cell Populations Treated with a KSP
Inhibitor
[0447] FACS analysis to determine cell cycle stage by measuring DNA
content is performed as follows. Skov-3 cells (human ovarian
cancer) are split 1:10 for plating in 10 cm dishes and grown to
subconfluence with RPMI 1640 medium containing 5% fetal bovine
serum (FBS). The cells are then treated with either 10 nM
paclitaxel, 400 nM test compound, 200 nM test compound, or 0.25%
DMSO (vehicle for compounds) for 24 hours. A well known
anti-mitotic agent, such as placitaxel, is used as a positive
control. Cells are then rinsed off the plates with PBS containing 5
mM EDTA, pelleted, washed once in PBS containing 1% FCS, and then
fixed overnight in 85% ethanol at 4.degree. C. Before analysis, the
cells are pelleted, washed once, and stained in a solution of 10
.mu.g propidium iodide and 250 .mu.g of ribonuclease (RNAse) A per
milliliter at 37.degree. C. for half an hour. Flow cytometry
analysis is performed on a Becton-Dickinson FACScan, and data from
10,000 cells per sample is analyzed with Modfit software.
Monopolar Spindle Formation Following Application of a
Quinazolinone KSP Inhibitor
[0448] To determine the nature of G2/M accumulation, human tumor
cell lines Skov-3 (ovarian), HeLa (cervical), and A549 (lung) are
plated in 96-well plates at densities of 4,000 cells per well
(SKOV-3 & HeLa) or 8,000 cells per well (A549), allowed to
adhere for 24 hours, and treated with various concentrations of the
test compounds for 24 hours. Cells are fixed in 4% formaldehyde and
stained with antitubulin antibodies (subsequently recognized using
fluorescently-labeled secondary antibody) and Hoechst dye (which
stains DNA). The cells can be visually inspected to assess the
effects of the test compounds. For example, microinjection of
anti-KSP antibodies causes mitotic arrest with arrested cells
displaying monopolar spindles.
Example 8
Inhibition of Cellular Proliferation in Tumor Cell Lines Treated
with KSP Inhibitors
[0449] Cells are plated in 96-well plates at densities from
1000-2500 cells/well (depending on the cell line) and allowed to
adhere/grow for 24 hours. They are then treated with various
concentrations of test compound for 48 hours. The time at which
compounds are added is considered T.sub.0. A tetrazolium-based
assay using the reagent
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
-2H-tetrazolium (MTS) (U.S. Pat. No. 5,185,450) (see Promega
product catalog #G3580, CellTiter 96.RTM. AQ.sub.ueous One Solution
Cell Proliferation Assay) is used to determine the number of viable
cells at T.sub.0 and the number of cells remaining after 48 hours
compound exposure. The number of cells remaining after 48 hours is
compared to the number of viable cells at the time of test compound
addition, allowing for calculation of growth inhibition. The growth
over 48 hours of cells in control wells treated with vehicle only
(0.25% DMSO) is considered 100% growth and the growth of cells in
wells with compounds is compared to this. Active KSP inhibitors
inhibit cell proliferation in one or more human tumor cell lines of
the following tumor types: lung (NCI-H460, A549), breast
(MDA-MB-231, MCF-7, MCF-7/ADR-RES), colon (HT29, HCT15), ovarian
(SKOV-3, OVCAR-3), leukemia (HL-60(TB), K-562), central nervous
system (SF-268), renal (A498), osteosarcoma (U2-OS), and cervical
(HeLa), and mouse tumor line (B16, melanoma).
[0450] Calculation Of GI.sub.50: A GI.sub.50 is calculated by
plotting the concentration of compound in .mu.M vs the percentage
of cell growth of cell growth in treated wells. The GI.sub.50
calculated for the compounds is the estimated concentration at
which growth is inhibited by 50% compared to control, i.e., the
concentration at which:
100.times.[(Treated.sub.48-T.sub.0)/(Control.sub.48-T.sub.0)]=50.
All concentrations of compounds are tested in duplicate and
controls are averaged over 12 wells. A very similar 96-well plate
layout and GI.sub.50 calculation scheme is used by the National
Cancer Institute (see Monks, et al., J. Natl. Cancer Inst.
83:757-766 (1991)). However, the method by which the National
Cancer Institute quantitates cell number does not use MTS, but
instead employs alternative methods.
[0451] Calculation Of IC.sub.50: Measurement of a compound's
IC.sub.50 for KSP activity uses an ATPase assay. The following
solutions are used: Solution 1 consists of 3 mM phosphoenolpyruvate
potassium salt (Sigma P-7127), 2 mM ATP (Sigma A-3377), 1 mM IDTT
(Sigma D-9779), 5 .mu.M paclitaxel (Sigma T-7402), 10 ppm antifoam
289 (Sigma A-8436), 25 mM Pipes/KOH pH 6.8 (Sigma P6757), 2 mM
MgC12 (VWR JT400301), and 1 mM EGTA (Sigma E3889). Solution 2
consists of 1 mM NADH (Sigma N8129), 0.2 mg/ml BSA (Sigma A7906),
pyruvate kinase 7U/ml, L-lactate dehydrogenase 10 U/ml (Sigma
P0294), 100 nM KSP motor domain, 50 .mu.g/ml microtubules, 1 mM DTT
(Sigma D9779), 5 .mu.M paclitaxel (Sigma T-7402), 10 ppm antifoam
289 (Sigma A-8436), 25 mM Pipes/KOH pH 6.8 (Sigma P6757), 2 mM
MgC12 (VWR JT4003-01), and 1 mM EGTA (Sigma E3889). Serial
dilutions (8-12 two-fold dilutions) of the composition are made in
a 96-well microtiter plate (Corning Costar 3695) using Solution 1.
Following serial dilution each well has 50 .mu.l of Solution 1. The
reaction is started by adding 50 .mu.l of Solution 2 to each well.
This can be done with a multichannel pipettor either manually or
with automated liquid handling devices. The microtiter plate is
then transferred to a microplate absorbance reader and multiple
absorbance readings at 340 nm are taken for each well in a kinetic
mode. The observed rate of change, which is proportional to the
ATPase rate, is then plotted as a function of the compound
concentration. For a standard IC.sub.50 determination the data
acquired is fit by the following four parameter equation using a
nonlinear fitting program (e.g., Grafit 4): y = Range 1 + ( x IC 50
) s + Background ##EQU2## where y is the observed rate and x the
compound concentration.
Example 9
Inhibition of Cellular Viability in Tumor Cell Lines Treated with
KSP Inhibitors
[0452] Materials and Solutions: [0453] Cells: SKOV3, Ovarian Cancer
(human). [0454] Media: Phenol Red Free RPMI+5% Fetal Bovine Serum+2
mM L-glutamine. [0455] Colorimetric Agent for Determining Cell
Viability: Promega MTS tetrazolium compound. [0456] Control
Compound for max cell kill: Topotecan, 1 .mu.M.
[0457] Procedure: Day 1--Cell Plating: Adherent SKOV3 cells are
washed with 10 mLs of PBS followed by the addition of 2 mLs of
0.25% trypsin and incubation for 5 minutes at 37.degree. C. The
cells are rinsed from the flask using 8 mL of media (phenol
red-free RPMI+5% FBS) and transferred to fresh flask. Cell
concentration is determined using a Coulter counter and the
appropriate volume of cells to achieve 1000 cells/100.mu.L is
calculated. 100 .mu.L of media cell suspension (adjusted to 1000
cells/100 .mu.L) is added to all wells of 96-well plates, followed
by incubation for 18 to 24 hours at 37.degree. C., 100% humidity,
and 5% CO.sub.2, allowing the cells to adhere to the plates.
[0458] Procedure: Day 2--Compound Addition: To one column of the
wells of an autoclaved assay block are added an initial 2.5 .mu.L
of test compound(s) at 400.times. the highest desired
concentration. 1.25 .mu.L of 400.times. (400 .mu.M) Topotecan is
added to other wells (ODs from these wells are used to subtract out
for background absorbance of dead cells and vehicle). 500 .mu.L of
media without DMSO are added to the wells containing test compound,
and 250 .mu.L to the Topotecan wells. 250 .mu.L of media+0.5% DMSO
is added to all remaining wells, into which the test compound(s)
are serially diluted. By row, compound-containing media is replica
plated (in duplicate) from the assay block to the corresponding
cell plates. The cell plates are incubated for 72hours at
37.degree. C., 100% humidity, and 5% CO.sub.2.
[0459] Procedure: Day 4--MTS Addition and OD Reading: The plates
are removed from the incubator and 40 .mu.l MTS/PMS is added to
each well. Plates are then incubated for 120 minutes at 37.degree.
C., 100% humidity, 5% CO.sub.2, followed by reading the ODs at 490
nm after a 5 second shaking cycle in a ninety-six well
spectrophotometer.
[0460] Data Analysis The normalized % of control
(absorbance-background) is calculated and an XLfit is used to
generate a dose-response curve from which the concentration of
compound required to inhibit viability by 50% is determined.
[0461] The compounds of the present invention show activity when
tested in one or more of the methods described in Examples 7, 8 and
9.
[0462] While the present invention has been described with
reference to the specific embodiments thereof, it should be
understood by those skilled in the art that various changes may be
made and equivalents may be substituted without departing from the
true spirit and scope of the invention. In addition, many
modifications may be made to adapt a particular situation,
material, composition of matter, process, process step or steps, to
the objective, spirit and scope of the present invention. All such
modifications are intended to be within the scope of the claims
appended hereto. All patents and publications cited above are
hereby incorporated by reference.
* * * * *
References