Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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BRADYKININ ANTAGONISTS
CROSS-REFERENCE TO RELATED Al~~,, IONS~
This application claims the benefit of U.S. Patent Application Serial No.
60/088,466, filed June 8, 1998; U.S. Patent Application Serial No. 60/092,938,
filed July 15, 1998; and U.S. Patent Application Serial No. 60/125,751, filed
March
23, 1999; the disclosures of which are incorporated herein by reference in
their
entirety.
BACKGROUND OF THE INVENTION
Field of the Invention
This invention relates to novel multibinding compounds (agents) that are
non-peptidic bradykinin antagonists, pharmaceutical compositions comprising
such
5 compounds, and methods of preparing these compounds. Accordingly, the
multibinding compounds and pharmaceutical compositions of this invention are
useful in the treatment and prevention of diseases mediated by bradykinin such
as
cancer, pain, shock, asthma, rhinitis, arthritis, inflammatory.bowel disease,
and the
like.
10
$~~rences
The following publications are cited in this application as superscript
numbers:
' Burch et al., "Bradykinin Receptor Antagonists" J. Med.
15 Chem.,30:237-269 (1990).
Clark, W. G. Handbook of Expt. Pharmacol., Vol. XXV:
Bradykinin, kallidin, and kallidrein. Erdo, E. G, 311-322.
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Barnes, "Inflammatory Mediator Receptors and Asthma'' Am. Rev.
Respir. Dis., 135:526-31 (1987).
Fuller et al, "Bradykinin-induced Bronchoconstriction in Humans",
5 Am. Rev. Respir. Dis., 135:176-180 (1987).
Jin et al, "Inhibition of Bradykinin-induced Bronchoconstriction in
the Guinea-pig a Synthetic B2 Receptor Antagonist", Br. J.
Pharmacol., 9.7:598-602 (1989).
10
Polosa et al, "Contribution of Histamine and Prostanoids to
Bronchoconstriction Provoked by Inhaled Bradykinin in Atopic
Asthma", Allergy, 45:174-182 (1990).
15 ' Baumgarten et al, "Concentrations of Grandular Kallikrein in Human
Nasal Secretions Increase During Experimentally Induce Allergic
Rhinitis", J. Immunology, 137:1323-1328 (1986).
20 All of the above publications are herein incorporated by reference in their
entirety to the same extent as if each individual publication was specifically
and
individually indicated to be incorporated by reference in its entirety.
State of the Art
25 Bradykinin (BK) is one of the most important kinins. It is derived by
cleavage of precursor plasma proteins, through the kallikrein/kinin system. It
is a
potent inflammatory peptide whose generation in tissues and body fluids
elicits
many physiological responses including vasodilation, smooth muscle spasm,
edema, as well as pain and hyperalgesia. There is increasing evidence that BK
and
30 related kinins contribute to the inflammatory response in acute and chronic
diseases
including allergic reactions, arthritis, asthma, sepsis, viral rhinitis, and
inflammatory
bowel disease. For example, bradykinin receptors have been localized to
nociceptive peripheral nerve pathways and bradykinin has been demonstrated to
stimulate central fibers mediating pain sensation.''2 Numerous studies have
also
35 shown that bradykinin receptors are present in the lung and that bradykinin
can
cause bronchoconstriction in both animals and man and furthermore that
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bronchoconstriction can be inhibited by treatment with bradykinin
antagonists.'~3-a
Bradykinin has also been implicated in the production of symptoms in both
allergic
and viral rhinitis' and in the pathogenesis of human lung cancer. Therefore,
the
design and synthesis of specific, potent and stable bradykinin antagonists has
long
5 been considered a desirable goal in medicinal chemistry.
Stewart and Vavrek in "Chemistry of Peptide Bradykinin Antagonists",
Bradykinin Antagonists: Basic and Chemical Research, R. M. Burch (Ed.), pages
51-96, 1991 discuss peptide bradykinin antagonists and their possible use
against
effects of bradykinin. Regoli, D. et al Eur. J. ofPharmaco~~gy, 348, 1-10,
1998
10 disclose several peptidic and non-peptidic bradykinin antagonists and their
potential
use against effects of bradykinin. PCT Application No. 97/09347 discloses
bradykinin antagonists dimers composed of peptidic bradykinin antagonists
attached with a linking group for the treatment of cancer. PCT Application No.
94/11021 discloses heterodimers comprising a peptidic bradykinin antagonists
15 covalently linked to a peptide or a non-peptide pharmacophore which is not
a
bradykinin antagonist via a linking group for the treatment of pain and
inflammation. The major problems with presently available bradykinin
antagonists
are their low levels of potency and short duration of activity.
Accordingly, there is a need for bradykinin antagonists that are increased
20 potency and /or duration of action.
SUMMARY OF THE INVENTION
This invention is directed to novel multibinding compounds (agents) that are
non-peptidic bradykinin antagonists and are useful in the treatment and
prevention
25 of diseases such as cancer, pain, shock, asthma, rhinitis, arthritis,
inflammatory
bowel disease, and the like. In addition, it is contemplated that the non-
peptidic
multibinding compounds of the present invention will exhibit longer duration
of
activity vis-a-vis peptidic antagonists.
Accordingly, in one aspect, this invention provides a multibinding
30 compound comprising of from 2 to 10 ligands covalently attached to one or
more
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linkers, wherein each of said ligands comprises, independently of each other,
a non-
peptidic bradykinin antagonist, and pharmaceutically acceptable salts.
In a second aspect, this invention provides a multibinding compound of
Formula (I):
(L)v(X)q
(I)
wherein:
each L is, independently of each other, a non-peptidic bradykinin antagonist;
10 each X is independently a linker;
p is an integer of from 2 to I 0; and
q is an integer of from 1 to 20, and pharmaceutically acceptable salts
thereof.
Preferably, q is less than p in the multibinding compounds of this invention.
15 Preferably, each ligand, L, that is a non-peptidic bradykinin antagonist in
the
multibinding compound of Formula (I), is independently selected from the group
consisting of
(i) a compound of formula (a):
C B~A
Ry~ R2
~~R3
(a)
wherein:
20 A is selected from the group consisting of alkylene and substituted
alkylene;
B is selected from the group consisting of -O-, -NH-, and -S(O)~' (where n'
is an integer of from 0 to 2);
C is selected from the group consisting of a compound of formula ( 1 ) and
(2):
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O
X3X2 / N Rs
or
i N:X
(1) (2)
where:
X' is -N- or -CR4 where R4 is alkyl;
Xz is -N- or -CRS where RS is hydrogen or alkyl;
X3 is -N- or -CR6 where R6 is selected from the group consisting of
5 hydrogen, alkyl, alkoxy, halo, amino, aryl, carboxy, alkoxycarbonyl,
substituted
alkyl, substituted alkoxy, substituted amino, -CONHR (where R is hydrogen or
alkyl), cycloalkyloxy, and N-containing heterocycl-N-yl group optionally
substituted with alkyl;
R4 and RS are as defined above;
10 R' is selected from the group consisting of hydrogen, alkyl, substituted
alkyl, alkoxy, substituted alkoxy, and halo, or R' is a covalent bond linking
the
ligand to a linker;
Rz is selected from the group consisting of alkyl, substituted alkyl, alkoxy,
substituted alkoxy, and halo, or Rz is a covalent bond linking the ligand to a
linker;
15 R3 is selected from the group consisting of hydroxy, vitro, alkoxy,
substituted alkoxy, piperazinyl optionally substituted with one or two groups
selected from acylalkyl, oxo, and -NR'R8 [wherein R' is hydrogen, alkyl, or a
covalent bond linking the ligand to a linker, and R$ is hydrogen, -COORS
(where R9
is aryl), -COR'° (where R'° is aryl, heteroaryl, or
heterocyclyl)], or a group of
20 formula:
-(AA)-(CO-Q-R")~ or -(AA)-R'z
where:
nis0orl;
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AA is a amino acid residue wherein the terminal nitrogen atom of the amino
acid residue optionally links the ligand to a linker when n is 0;
Q is selected from the group consisting of alkylene, alkenylene, and a bond;
R" selected from the group consisting of aryl, heteroaryl, heterocyclyl, and
5 -X'Re (where X4 is -N-, -O-, or -S- and Ra is aryl, heteroaryl, or
heterocyclyl each of
which optionally links the ligand to a linker); and
R'z is selected from the group consisting of hydrogen and acylbiphenyl
which optionally link the ligand to a linker;
(ii) a compound of formula (b):
A~-O-R' 3
Rya R~s
Rts
(b)
10 wherein:
A' is selected from the group consisting of alkylene and substituted
alkylene;
R'3 is selected from the group consisting of quinolyl, quinazolinyl,
quinoxalinyl, benzimidazolyl, benzofuryl, benzoxazolyl, and imidazopyridyl,
each
15 of which is optionally substituted with one or more substituent(s) selected
from the
group consisting of halo, alkyl, substituted alkyl, alkoxy, substituted
alkoxy, amino,
substituted amino, heteroaryl, and heterocyclyl;
R'4 is selected from the group consisting of hydrogen, halo, alkyl, and
substituted alkyl, or R'4 is a covalent bond linking the ligand to a linker;
20 R'S is selected from the group consisting of halo and alkyl, or R'S is a
covalent bond linking the ligand to a linker; and
R'6 is carboxy or a group of formula:
n
-Q'-AZ-R~~ or --CO-N N-R~8
~J
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where:
Q' is alkylene or is a group of formula:
R~s
-N~ R~9 ~ /R~s l~~S Rzo Rz~
-N- o~ - NCO-
where:
R'9 is hydrogen or halo;
5 R'-° is selected from the group consisting of hydrog ~ ~ and alkyl,
or R'-° is a
covalent bond linking the ligand to a linker, or R'-° and R'S together
form alkylene;
and
Rz' is selected from the group consisting of hydrogen, alkyl, and aralkyl, or
RZ' is a covalent bond linking the ligand to a linker; provided that AZ is
alkylene
10 when RZ° is hydrogen;
AZ is selected from the group consisting of alkylene and a bond;
R" is selected from the group consisting of amino which optionally links the
ligand to a linker, aminoacyl, cyano, hydroxy, and acyl; and
R'g is selected from the group consisting of hydrogen and acyl; or
15 (iii) a compound of formula (c):
Rzz I ~ Rzs
Rz400C. rnnaz5
Rzy As
'N'
O N
~N_Rzs
(c)
wherein:
RZ-' and Rz3 are, independently of each other, halo or optionally link the
ligand to a linker;
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A3 is selected from the group consisting of a bond, alkylene, -CO-, -O-, and
-S(O)S (where n is an integer of 0 to 2);
R24 and R25 are, independently of each other, alkyl or optionally link the
ligand to a linker;
5 Rzb is selected from the group consisting of hydrogen, alkyl optionally
substituted with one or two substituents selected from hydroxy, amino,
substituted
amino, pyridyl, carbamoyl, pyrrolidinocarbonyl, propylaminocarbonyl,
piperidinocarbonyl or morpholinocarbonyl; piperidinyl optionally substituted
on the
nitrogen atom with alkyl or alkoxycarbonyl; cycloalkyl optionally substituted
with
10 one or two substituents selected from oxo, hydroxy, amino, alkylamino.
dialkylamino, methoxybenzamido, or morpholino; C,_,4 azacyclo, azabicyclo or
azatricycloalkyl in which the nitrogen atom is optionally substituted with a
substituent selected from alkyl, benzyl optionally substituted with one or two
substituents selected from halo, trihaloalkyl, acyl or -COOR'-8 (where R'-$ is
alkyl
15 optionally substituted with one or two halogen atoms); heteroaryl;
heterocyclyl;
cycloalkenyl; and phenyl fused to cycloalkyl; or R26 optionally links the
ligand to a
linker; and pharmaceutically acceptable salts thereof.
Preferably, each linker, X, in the multibinding compound of Formula {I) is a
non-peptidic linker.
20 More preferably, each linker, X, in the multibinding compound of Formula
(I) independently has the formula:
_Xa_Z_(ya_Z}m-Xa-
wherein:
25 m is an integer of from 0 to 20;
Xe at each separate occurrence is selected from the group consisting of
-O-, -S-, -NR-, -C(O)-, -C(O)O-, -C(O)NR-, -C(S), -C(S)O-, -C(S)NR- or a
covalent
bond where R is as defined below;
Z at each separate occurrence is selected from the group consisting of
30 alkylene, substituted alkylene, cycloalkylene, substituted cylcoalkylene,
alkenylene,
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substituted alkenylene, alkynylene, substituted alkynylene, cycloalkenylene,
substituted cycloalkenylene, arylene, heteroarylene, heterocyclene, or a
covalent
bond;
each Ya at each separate occurrence is selected from the group consisting of
5 -O-, -C(O)-, -OC(O)-, -C(O)O-, -NR-, -S(O)n-, -C(O)NR'-, -NR' C(O)-, -NR'
C(O)NR'-, -NR' C(S)NR'-, -C(=NR')-NR'-, -NR'-C(=NR')-, -OC(O)-NR'-, -NR'-
C(O)-O-, -N=C(Xa)-NR'-, -NR'-C(X8)=N-,-P(O)(OR')-O-, -O-P(O)(OR')-, -
S(O)~CR' R"-, -S(O)~-NR'-, -NR'-S(O)~-, -S-S-, and a covalent bond; where n is
0,
1 or 2; and R, R' and R" at each separate occurrence are selected from the
group
10 consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted
cycloalkyl,
alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl,
substituted alkynyl, aryl, heteroaryl and heterocyclic provided that at least
one of
Xa, Z, and Ya is not a covalent bond.
In a third aspect, this invention provides a pharmaceutical composition
15 comprising a pharmaceutically acceptable carrier and an effective amount of
a
multibinding compound comprising from 2 to 10 ligands covalently attached to
one
or more linkers, wherein each of said ligands comprises, independently of each
other, a non-peptidic bradykinin receptor antagonist and pharmaceutically
acceptable salts thereof.
20 In a fourth aspect, this invention provides a method of treating diseases
mediated by bradykinin in a mammal, said method comprising administering to
said
mammal a therapeutically effective amount of a pharmaceutical composition
comprising a pharmaceutically acceptable carrier and a multibinding compound
comprising from 2 to 10 ligands covalently attached to one or more linkers,
wherein
25 each of said ligands, comprises, independently of each other, a non-
peptidic
bradykinin antagonist, and pharmaceutically acceptable salts thereof.
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In a fifth aspect, this invention is directed to general synthetic methods for
generating large libraries of diverse multimeric compounds which multimeric
compounds are candidates for possessing multibinding properties for bradykinin
receptor. The diverse multimeric compound libraries provided by this invention
are
5 synthesized by combining a linker or linkers with a ligand or ligands to
provide for
a library of multimeric compounds wherein the linker and ligand each have
complementary functional groups permitting covalent linkage. The library of
linkers is preferably selected to have diverse properties such as valency,
linker
length, linker geometry and rigidity, hydrophilicity or hydrophobicity,
10 amphiphilicity, acidity, basicity and polarization and/or polarizability.
The library
of ligands is preferably selected to have diverse attachment points on the
same
ligand, different functional groups at the same site of otherwise the same
ligand, and
the like.
In a sixth aspect, this invention is directed to libraries of diverse
multimeric
15 compounds which multimeric compounds are candidates for possessing
multibinding properties for bradykinin receptor. These libraries are prepared
via the
methods described above and permit the rapid and efficient evaluation of what
molecular constraints impart multibinding properties to a ligand or a class of
ligands
targeting bradykinin receptor.
20 Accordingly, in one of its method aspects, this invention is directed to a
method for identifying multimeric ligand compounds possessing multibinding
properties for bradykinin receptor which method comprises:
(a) identifying a ligand or a mixture of ligands wherein each ligand
contains at least one reactive functionality;
25 (b) identifying a library of linkers wherein each linker in said library
comprises at least two functional groups having complementary reactivity to at
least
one of the reactive functional groups of the ligand;
(c) preparing a multimeric ligand compound library by combining at
least two stoichiometric equivalents of the ligand or mixture of ligands
identified in
30 (a) with the library of linkers identified in (b) under conditions wherein
the
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complementary functional groups react to form a covalent linkage between said
linker and at least two of said ligands; and
(d) assaying the multimeric ligand compounds produced in (c) above to
identify multimeric ligand compounds possessing multibinding properties for
5 bradykinin receptor.
In another of its method aspects, this invention is directed to a method
for identifying multimeric ligand compounds possessing multibinding properties
for
bradykinin receptor which method comprises:
(a) identifying a library of ligands wherein each ligand contains at least
10 one reactive functionality;
(b) identifying a linker or mixture of linkers wherein each linker
comprises at least two functional groups having complementary reactivity to at
least
one of the reactive functional groups of the ligand;
(c) preparing a multimeric ligand compound library by combining at
15 least two stoichiometric equivalents of the library of ligands identified
in (a) with
the linker or mixture of linkers identified in (b) under conditions wherein
the
complementary functional groups react to form a covalent linkage between said
linker and at least two of said ligands; and
(d) assaying the multimeric ligand compounds produced in (c) above to
20 identify multimeric ligand compounds possessing multibinding properties for
bradykinin receptor.
The preparation of the multimeric ligand compound library is achieved by
either the sequential or concurrent combination of the two or more
stoichiometric
equivalents of the ligands identified in (a) with the linkers identified in
(b).
25 Sequential addition is preferred when a mixture of different ligands is
employed to
ensure heterodimeric or multimeric compounds are prepared. Concurrent addition
of the ligands occurs when at least a portion of the multimer compounds
prepared
are homomultimeric compounds.
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The assay protocols recited in (d) can be conducted on the multimeric ligand
compound library produced in (c) above, or preferably, each member of the
library
is isolated by preparative liquid chromatography mass spectrometry (LCMS).
In one of its composition aspects, this invention is directed to a library of
S multimeric ligand compounds which may possess multivalent properties for
bradykinin receptor which library is prepared by the method comprising:
(a) identifying a ligand or a mixture of ligands wherein each ligand
contains at least one reactive functionality;
(b) identifying a library of linkers wherein each linker in said library
10 comprises at least two functional groups having complementary reactivity to
at least
one of the reactive functional groups of the ligand; and
(c) preparing a multimeric ligand compound library by combining at
least two stoichiometric equivalents of the ligand or mixture of ligands
identified in
(a) with the library of linkers identified in (b) under conditions wherein the
15 complementary functional groups react to form a covalent linkage between
said
linker and at least two of said ligands.
In another of its composition aspects, this invention is directed to a library
of
multimeric ligand compounds which may possess multivalent properties for
bradykinin receptor which library is prepared by the method comprising:
20 (a) identifying a library of ligands wherein each ligand contains at least
one reactive functionality;
(b) identifying a linker or mixture of linkers wherein each linker
comprises at least two functional groups having complementary reactivity to at
least
one of the reactive functional groups of the ligand; and
25 (c) preparing a multimeric ligand compound library by combining at
least two stoichiometric equivalents of the library of ligands identified in
(a) with
the linker or mixture of linkers identified in (b) under conditions wherein
the
complementary functional groups react to form a covalent linkage between said
linker and at least two of said ligands.
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In a preferred embodiment, the library of linkers employed in either the
methods or the library aspects of this invention is selected from the group
comprising flexible linkers, rigid linkers, hydrophobic linkers, hydrophilic
linkers,
linkers of different geometry, acidic linkers, basic linkers, linkers of
different
5 polarization and/or polarizability, and amphiphilic linkers. For example, in
one
embodiment, each of the linkers in the linker library may comprise linkers of
different chain length and/or having different complementary reactive groups.
Such
linker lengths can preferably range from about 2 to I OOA.
In another preferred embodiment, the ligand or mixture of ligands is selected
IO to have reactive functionality at different sites on said ligands in order
to provide for
a range of orientations of said ligand on said multimeric ligand compounds.
Such
reactive functionality includes, by way of example, carboxylic acids,
carboxylic
acid halides, carboxyl esters, amines, halides, pseudohalides, isocyanates,
vinyl
unsaturation, ketones, aldehydes, thiols, alcohols, anhydrides, boronates, and
15 precursors thereof. It is understood, of course, that the reactive
functionality on the
ligand is selected to be complementary to at least one of the reactive groups
on the
linker so that a covalent linkage can be formed between the linker and the
ligand.
In other embodiments, the multimeric ligand compound is homomeric (i.e.,
each of the ligands is the same, although it may be attached at different
points) or
20 heteromeric (i.e., at least one of the ligands is different from the other
ligands).
In addition to the combinatorial methods described herein, this invention
provides for an iterative process for rationally evaluating what molecular
constraints
impart multibinding properties to a class of multimeric compounds or ligands
targeting a receptor. Specifically, this method aspect is directed to a method
for
25 identifying multimeric ligand compounds possessing multibinding properties
for
bradykinin receptor which method comprises:
(a) preparing a first collection or iteration of multimeric compounds
which is prepared by contacting at least two stoichiometric equivalents of the
Iigand
or mixture of ligands which target a receptor with a linker or mixture of
linkers
30 wherein said ligand or mixture of ligands comprises at least one reactive
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functionality and said linker or mixture of linkers comprises at least two
functional
groups having complementary reactivity to at least one of the reactive
functional
groups of the ligand wherein said contacting is conducted under conditions
wherein
the complementary functional groups react to form a covalent linkage between
said
5 linker and at least two of said ligands;
(b) assaying said first collection or iteration of multimeric compounds to
assess which if any of said multimeric compounds possess multibinding
properties;
for bradykinin receptor
(c) repeating the process of (a) and (b) above until at least one
10 multimeric compound is found to possess multibinding properties for
bradykinin
receptor;
(d) evaluating what molecular constraints imparted multibinding
properties to the multimeric compound or compounds found in the first
iteration
recited in (a)- (c) above;
15 (e) creating a second collection or iteration of multimeric compounds
which elaborates upon the particular molecular constraints imparting
multibinding
properties to the multimeric compound or compounds found in said first
iteration;
(fJ evaluating what molecular constraints imparted enhanced
multibinding properties to the multimeric compound or compounds found in the
20 second collection or iteration recited in (e) above;
(g) optionally repeating steps (e) and (f) to further elaborate upon said
molecular constraints.
Preferably, steps (e) and (f) are repeated at least two times, more preferably
from 2-50 times, even more preferably from 3 to 50 times, and still more
preferably
25 at least 5-50 times.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates examples of multibinding compounds comprising 2
ligands attached in different formats to a linker.
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FIG. 2 illustrates examples of multibinding compounds comprising 3
ligands attached in different formats to a linker.
FIG. 3 illustrates examples of muItibinding compounds comprising 4
ligands attached in different formats to a linker.
5 FIG. 4 illustrates examples of multibinding compounds comprising >4
ligands attached in different formats to a linker.
FIG. 5 illustrates a synthesis of a compound of formula (b).
FIGS. 6-15 illustrate syntheses of bivalent multibinding compounds of
Formula (I).
10
DETAILED DESCRIPTION OF THE INVENTION
Definitions
This invention is directed to multibinding compounds which are bradykinin
receptor antagonists, pharmaceutical compositions containing such compounds
and
15 methods for treating diseases mediated by a bradykinin receptor in mammals.
When discussing such compounds, compositions or methods, the following terms
have the following meanings unless otherwise indicated. Any undefined terms
have
their art recognized meanings.
The term "alkyl" refers to a monoradical branched or unbranched saturated
20 hydrocarbon chain preferably having from 1 to 40 carbon atoms, more
preferably I
to 10 carbon atoms, and even more preferably 1 to 6 carbon atoms. This term is
exemplified by groups such as methyl, ethyl, n-propyl, iso-propyl, n-butyl,
iso-
butyl, n-hexyl, n-decyl, tetradecyl, and the like.
The term "substituted alkyl" refers to an alkyl group as defined above,
25 having from 1 to 5 substituents, and preferably 1 to 3 substituents,
selected from the
group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted
cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino,
substituted
amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl,
keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,
30 thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl,
aryloxy,
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heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino,
alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-
heteroaryl,
-SOZ- -alkyl, -SO~-substituted alkyl, -SO~-aryl and -SO,-heteroaryl. This term
is
exemplified by groups such as hydroxymethyl, hydroxyethyl, hydroxypropyl, 2-
5 aminoethyl, 3-aminopropyl, 2-methylaminoethyl, 3-dimethylaminopropyl, 2-
sulfonamidoethyl, 2-carboxyethyl, and the like.
The term "alkylene" refers to a diradical of a branched or unbranched
saturated hydrocarbon chain, preferably having from 1 to 40 carbon atoms, more
preferably 1 to 10 carbon atoms and even more preferably 1 to 6 carbon atoms.
10 This term is exemplified by groups such as methylene (-CHI-), ethylene
(-CHZCH,- _ -), the propylene isomers (e.g., -CH,CH~CH~- and -CH(CH3)CH,-) and
the like.
The term "substituted alkylene" refers to an alkylene group, as defined
above, having from 1 to 5 substituents, and preferably 1 to 3 substituents,
selected
15 from the group consisting of alkoxy, substituted alkoxy, cycloalkyl,
substituted
cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy,
amino,
substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano,
halogen,
hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy,
thioheteroaryloxy,
thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy,
20 heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino,
alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-
heteroaryl,
-SO,-alkyl, -SOZ-substituted alkyl, -SOz-aryl and -SOZ-heteroaryl.
Additionally,
such substituted alkylene groups include those where 2 substituents on the
alkylene
group are fused to form one or more cycloalkyl, substituted cycloalkyl,
25 cycloalkenyl, substituted cycloalkenyl, aryl, heterocyclic or heteroaryl
groups fused
to the alkylene group. Preferably such fused groups contain from 1 to 3 fused
ring
structures.
The term "alkaryl" or "aralkyl" refers to the groups -alkylene-aryl and
-substituted alkylene-aryl where alkylene, substituted alkylene and aryl are
defined
30 herein. Such alkaryl groups are exemplified by benzyl, phenethyl and the
like.
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The term "alkoxy" refers to the groups alkyl-O-, alkenyl-O-, cycloalkyl-O-,
cycloalkenyl-O-, and alkynyl-O-, where alkyl, alkenyl, cycloalkyl,
cycloalkenyl,
and alkynyl are as defined herein. Preferred alkoxy groups are alkyl-O- and
include, by way of example, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy,
5 tent-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the
like.
The term "substituted alkoxy" refers to the groups substituted alkyl-O-,
substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-
, and
substituted alkynyl-O- where substituted alkyl, substituted alkenyl,
substituted
cycloalkyl, substituted cycloalkenyl and substituted alkynyl are as defined
herein.
10 The teen "alkenyl" refers to a monoradical of a branched or unbranched
unsaturated hydrocarbon group preferably having from 2 to 40 carbon atoms,
more
preferably 2 to 10 carbon atoms and even more preferably 2 to 6 carbon atoms
and
having at least 1 and preferably from 1-6 sites of vinyl unsaturation.
Preferred
alkenyl groups include ethenyl (-CH=CHI), n-propenyl (-CH,CH=CH,), iso-
15 propenyl (-C(CH3)=CH,), and the like.
The term "substituted alkenyl" refers to an alkenyl group as defined above
having from 1 to S substituents, and preferably 1 to 3 substituents, selected
from the
group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted
cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino,
substituted
20 amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen,
hydroxyl,
keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,
thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy,
heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino,
alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-
heteroaryl,
25 - _SO,-alkyl, -SO,-substituted alkyl, -SO~=aryl and -SO,-heteroaryl.
The term "alkenylene" refers to a diradical of a branched or unbranched
unsaturated hydrocarbon group preferably having from 2 to 40 carbon atoms,
more
preferably 2 to 10 carbon atoms and even more preferably 2 to 6 carbon atoms
and
having at least 1 and preferably from 1-6 sites of vinyl unsaturation. This
term is
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exemplified by groups such as ethenylene (-CH=CH-), the propenylene isomers
(e.g., -CHZCH=CH-, -C(CH3)=CH-, and the Like.
The term "substituted alkenylene" refers to an alkenylene group as defined
above having from 1 to 5 substituents, and preferably from 1 to 3
substituents,
5 selected from the group consisting of aIkoxy, substituted alkoxy,
cycloalkyl,
substituted cycloalkyl, cycloalkenyi, substituted cycloalkenyl, acyl,
acylamino,
acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl,
azido,
cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl,
thioaryloxy,
thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted
thioalkoxy,
10 aryl, aryloxy, heteroaryI, heteroaryloxy, heterocyclic, heterocyclooxy,
hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl,
-SO-heteroaryl, -SOz-alkyl, -SO,-substituted alkyl, -SO~-aryl and -SO,-
heteroaryl.
Additionally, such substituted alkenylene groups include those where 2
substituents
on the alkenylene group are fused to form one or more cycloalkyl, substituted
15 cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, heterocyclic or
heteroaryl
groups fused to the alkenylene group.
The term "alkynyl" refers to a monoradical of an unsaturated hydrocarbon
preferably having from 2 to 40 carbon atoms, more preferably 2 to 20 carbon
atoms
and even more preferably 2 to 6 carbon atoms and having at least 1 and
preferably
20 from 1-6 sites of acetylene (triple bond) unsaturation. Preferred alkynyl
groups
include ethynyl (-C=CH), propargyl (-CH,C---CH) and the like.
The term "substituted alkynyl" refers to an alkynyl group as defined above
having from 1 to 5 substituents, and preferably 1 to 3 substituents, selected
from the
group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted
cycloalkyl,
25 cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino,
substituted
amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl,
keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,
thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy,
heteroaryh heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino,
30 alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-
heteroaryl,
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-SO,-alkyl, -SO,-substituted alkyl, -SO~-aryl, and -SO,-heteroaryl.
The term "alkynylene" refers to a diradical of an unsaturated hydrocarbon
preferably having from 2 to 40 carbon atoms, more preferably 2 to 10 carbon
atoms
and even more preferably 2 to 6 carbon atoms and having at least 1 and
preferably
5 from 1-6 sites of acetylene (triple bond) unsaturation. Preferred alkynylene
groups
include ethynylene (-C---C-), propargylene (-CHIC=C-) and the like.
The term "substituted alkynylene" refers to an alkynylene group as defined
above having from 1 to 5 substituents, and preferably 1 to 3 substituents,
selected
from the group consisting of alkoxy, substituted aIkoxy, cy.,ioalkyl,
substituted
10 cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino,
acyloxy, amino,
substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano,
halogen,
hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy,
thioheteroaryloxy,
thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy,
heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino,
15 alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-
heteroaryl,
-SO~-alkyl, -SOZ-substituted alkyl, -SO,-aryl and -SOZ-heteroaryl.
The term "acyl" refers to the groups HC(O)-, alkyl-C(O)-, substituted alkyl-
C(O)-, alkenyl-C(O)-, substituted alkenyl-C(O)-, cycloalkyl-C(O)-, substituted
cycloalkyl-C(O)-, cycloalkenyl-C(O)-, substituted cycloalkenyl-C(O)-, aryl-
C(O)-,
20 heteroaryl-C(O)- and heterocyclic-C(O)- where alkyl, substituted alkyl,
alkenyl,
substituted alkenyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,
substituted
cycloalkenyl, aryl, heteroaryl and heterocyclic are as defined herein.
The term "acylamino" or "aminocarbonyl" refers to the group -C(O)NRR
where each R is independently hydrogen, alkyl, substituted alkyl, aryl,
heteroaryl,
25 heterocyclic or where both R groups are joined to form a heterocyclic group
(e.g.,
morpholino) wherein alkyl, substituted alkyl, aryl, heteroaryl and
heterocyclic are as
defined herein.
The term "aminoacyl" refers to the group -NRC(O)R where each R is
independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted
alkenyl,
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substituted amino, aryl, heteroaryl, or heterocyclic wherein alkyl,
substituted alkyl,
alkenyl, substituted alkenyl, aryl, heteroaryl and heterocyclic are as defined
herein.
The term "aminoacyloxy" or "alkoxycarbonylamino" refers to the group
-NRC(O)OR where each R is independently hydrogen, alkyl, substituted alkyl,
aryl,
5 heteroaryl, or heterocyclic wherein alkyl, substituted alkyl, aryl,
heteroaryl and
heterocyclic are as defined herein.
The term "acyloxy" refers to the groups alkyl-C(O)O-, substituted alkyl-
C(O)O-, cycloalkyl-C(O)O-, substituted cycloalkyl-C(O)O-, aryl-C(O)O-,
heteroaryl-C(O)O-, and heterocyclic-C(O)O- wherein alkyl, substituted alkyl,
10 cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, and heterocyclic are
as defined
herein.
The term "aryl" refers to an unsaturated aromatic carbocyclic group of from
6 to 20 carbon atoms having a single ring (e.g., phenyl) or multiple condensed
(fused) rings (e.g., naphthyl or anthryl). Preferred aryls include phenyl,
naphthyl
15 and the like. Unless otherwise constrained by the definition for the aryl
substituent,
such aryl groups can optionally be substituted with from 1 to 5 substituents,
preferably 1 to 3 substituents, selected from the group consisting of acyloxy,
hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl,
substituted alkyl, substituted alkoxy, substituted alkenyl, substituted
alkynyl,
20 substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino,
aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl,
cyano,
halo, nitro, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy,
aminoacyloxy,
oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy,
thioheteroaryloxy,
-SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO,-alkyl, -SO,-
25 substituted alkyl, -SO~-aryl, -SOZ-heteroaryl and trihalomethyl. Preferred
aryl
substituents include alkyl, alkoxy, halo, cyano, nitro, trihalomethyl, and
thioalkoxy.
The term "aryloxy" refers to the group aryl-O- wherein the aryl group is as
defined above including optionally substituted aryl groups as also defined
above.
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The term "arylene" refers to the diradical derived from aryl (including
substituted aryl) as defined above and is exemplified by 1,2-phenylene, 1,3-
phenylene, 1,4-phenylene, 1,2-naphthylene and the like.
The term "amino" refers to the group -NH,.
5 The term "substituted amino refers to the group -NRR where each R is
independently selected from the group consisting of hydrogen, alkyl,
substituted
alkyl, acyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl,
cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl,
heteroaryl
and heterocyclic provided that both R's are not hydrogen.
10 The term "carboxyalkyl" or "alkoxycarbonyl" refers to the groups
"-C(O)O-alkyl", "-C(O)O-substituted alkyl", "-C(O)O-cycloalkyl", "-C(O)O-
substituted cycloalkyl", "-C(O)O-alkenyl", "-C(O)O-substituted alkenyf',
"-C(O)O-alkynyl" and "-C(O)O-substituted alkynyl" where alkyl, substituted
alkyl,
cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, alkynyl and
15 substituted alkynyl alkynyl are as defined herein.
The term "cycloalkyl" refers to cyclic alkyl groups of from 3 to 20 carbon
atoms having a single cyclic ring or multiple condensed rings. Such cycloalkyl
groups include, by way of example, single ring structures such as cyclopropyl,
cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures
such as
20 adamantanyl, and the like.
The term "substituted cycloalkyl" refers to cycloalkyl groups having from
1 to 5 substituents, and preferably 1 to 3 substituents, selected from the
group
consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy,
substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl,
substituted
25 cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino,
aminoacyl,
aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo,
carboxyl, carboxylalkyl, .thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,
thiol,
thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy,
heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl,
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-SO- -substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO~-alkyl, -SO,-
substituted alkyl,
-SO,-aryl and -SO~-heteroaryl.
The term "cycloalkenyl" refers to cyclic alkenyl groups of from 4 to 20
carbon atoms having a single cyclic ring and at least one point of internal
5 unsaturation. Examples of suitable cycloalkenyl groups include, for
instance,
cyclobut-2-enyl, cyclopent-3-enyl, cyclooct-3-enyl and the like.
The term "substituted cycloalkenyl" refers to cycloalkenyl groups having
from 1 to 5 substituents, and preferably 1 to 3 substituents, selected from
the group
consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy,
10 substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl,
substituted
cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl,
aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo,
carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,
thiol,
thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy,
15 heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, vitro, -SO-alkyl, -
SO-
substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO,-alkyl, -SO~-substituted
alkyl, -
SOz-aryl and -SO~-heteroaryl.
The term "halo" or "halogen" refers to fluoro, chloro, bromo and iodo.
The term "heteroaryl" refers to an aromatic group of from 1 to 15 carbon
20 atoms and 1 to 4 heteroatoms selected from oxygen, nitrogen and sulfur
within at
least one ring (if there is more than one ring). Unless otherwise constrained
by the
definition for the heteroaryl substituent, such heteroaryl groups can be
optionally
substituted with 1 to 5 substituents, preferably 1 to 3 substituents, selected
from the
group consisting of acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl,
alkynyl,
25 cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy,
substituted alkenyl,
substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino,
substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido,
carboxyl,
carboxylalkyl, cyano, halo, vitro, heteroaryl, heteroaryloxy, heterocyclic,
heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted
thioalkoxy,
30 thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl,
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-SO-heteroaryl, -SOZ-alkyl, -SO,-substituted alkyl, -SOZ-aryl, -SO~-heteroaryl
and
trihalomethyl. Preferred heteroaryl substituents include alkyl, alkoxy, halo,
cyano,
nitro, trihalomethyl, and thioalkoxy. Such heteroaryl groups can have a single
ring
(;,.g., pyridyl or furyl) or multiple condensed rings (e.g., indolizinyl or
5 benzothienyl). Preferred heteroaryls include pyridyl, pyrrolyl and furyl.
The term "heteroaryloxy" refers to the group heteroaryl-O-.
The term "heteroarylene" refers to the diradical group derived from
heteroaryl (including substituted heteroaryl), as defined above, and is
exemplified
by the groups 2,6-pyridylene, 2,4-pyridiylene, 1,2-quinolinylene, 1,8-
quinolinylene,
10 1,4-benzofuranylene, 2,5-pyridnylene, 2,5-indolenyl, and the like.
The term "heterocycle" or "heterocyclyl" refers to a monoradical saturated
unsaturated group having a single ring or multiple condensed rings, from 1 to
40
carbon atoms and from 1 to 10 hetero atoms, preferably 1 to 4 heteroatoms,
selected
from nitrogen, sulfur, phosphorus, and/or oxygen within the ring and further
15 wherein one, two, or three of the ring carbon atoms may optionally be
replaced with
a carbonyl group (i.e., a keto group). Unless otherwise constrained by the
definition
for the heterocyclic substituent, such heterocyclic groups can be optionally
substituted with 1 to 5, and preferably 1 to 3 substituents, selected from the
group
consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl,
20 cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino,
substituted
amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl,
keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,
thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy,
heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino,
25 alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl,
-SO-heteroaryl, -SO~-alkyl, -SO,-substituted alkyl, -SO~-aryl and -SO,-
heteroaryl.
Such heterocyclic groups can have a single ring or multiple condensed rings.
Preferred heterocyclics include morpholino, piperidinyl, and the like.
Examples of heteroaryls and heterocycles include, but are not limited to,
30 pyrrole, thiophene, furan, imidazole, pyrazole, pyridine, pyrazine,
pyrimidine,
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pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine,
isoquinoline,
quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline,
pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline,
isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine,
imidazoline, pyrrolidine, piperidine, piperazine, indoline, morpholine,
tetrahydrofuranyl, tetrahydrothiophene, and the like as well as N-alkoxy-
nitrogen
containing heterocycles.
The term "heterocyclooxy" refers to the group heterocyclic-O-.
The term "thioheterocyclooxy" refers to the group heterocyclic-S-.
10 The term "heterocyclene" refers to the diradical group formed from a
heterocycle, as defined herein, and is exemplified by the groups 2,6-
morpholino,
2,5-morpholino and the like.
The term "oxyacylamino" or "aminocarbonyloxy" refers to the group
-OC(O}NRR where each R is independently hydrogen, alkyl, substituted alkyl,
aryl,
15 heteroaryl, or heterocyclic wherein alkyl, substituted alkyl, aryl,
heteroaryl and
heterocyclic are as defined herein.
The term "spiro-attached cycloalkyl group" refers to a cycloalkyl group
joined to another ring via one carbon atom common to both rings.
The term "thiol" refers to the group -SH.
20 The term "thioalkoxy" or "alkylthio" refers to the group -S-alkyl.
The term "substituted thioalkoxy" refers to the group -S-substituted alkyl.
The term "thioaryloxy" refers to the group aryl-S- wherein the aryl group is
as defined above including optionally substituted aryl groups also defined
above.
The term "thioheteroaryloxy" refers to the group heteroaryl-S- wherein the
25 heteroaryl group is as defined above including optionally substituted aryl
groups as
also defined above.
The term " amino acid residue " refers to compounds having both carboxylic
acid and amino functional groups and include both natural (L-amino acids) and
unnatural amino acids (D-amino acids). Natural amino acids, include by way of
30 examples, glycine, alanine, valine, serine, glutamic acid, aspartic acid,
lysine, and
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the like. Unnatural amino acids, include by way of examples, D-amino acids of
naturally occurring L-amino acids, sarcosine, 1-napthylalanine, and the like.
The term "acylbiphenyl " refers to a biphenyl ring substituted with an acyl
group as defined above.
As to any of the above groups which contain one or more substituents, it is
understood, of course, that such groups do not contain any substitution or
substitution patterns which are sterically impractical and/or synthetically
non-
feasible. In addition, the compounds of this invention include all
stereochemical
isomers arising from the substitution of these compounds.
10 The term "pharmaceutically-acceptable salt" refers to salts which retain
the
biological effectiveness and properties of the multibinding compounds of this
invention and which are not biologically or otherwise undesirable. In many
cases,
the multibinding compounds of this invention are capable of forming acid
and/or
base salts by virtue of the presence of amino and/or carboxyl groups or groups
15 similar thereto.
Pharmaceutically-acceptable base addition salts can be prepared from
inorganic and organic bases. Salts derived from inorganic bases, include by
way of
example only, sodium, potassium, lithium, ammonium, calcium and magnesium
salts. Salts derived from organic bases include, but are not limited to, salts
of
20 primary, secondary and tertiary amines, such as alkyl amines, dialkyl
amines,
trialkyl amines, substituted alkyl amines, di(substituted alkyl) amines,
tri(substituted alkyl) amines, alkenyl amines, dialkenyl amines, trialkenyl
amines,
substituted alkenyl amines, di(substituted alkenyl) amines, tri(substituted
alkenyl)
amines, cycloalkyl amines, di(cycloalkyl) amines, tri(cycloalkyl) amines,
25 substituted cycloalkyl amines, disubstituted cycloalkyl amine,
trisubstituted
cycloalkyl amines, cycloalkenyl amines, di(cycloalkenyl) amines,
tri(cycloalkenyl)
amines, substituted cycloalkenyl amines, disubstituted cycloalkenyl amine,
trisubstituted cycloalkenyl amines, aryl amines, diaryl amines, triaryl
amines,
heteroaryl amines; diheteroaryl amines, triheteroaryl amines, heterocyclic
amines,
30 diheterocyclic amines, triheterocyclic amines, mixed di- and tri-amines
where at
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least two of the substituents on the amine are different and are selected from
the
group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyh
cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,
heteroaryl,
heterocyclic, and the like. Also included are amines where the two or three
5 substituents, together with the amino nitrogen, form a heterocyclic or
heteroaryl
group.
Examples of suitable amines include, by way of example only,
isopropylamine, trimethyl amine, diethyl amine, tri(iso-propyl) amine, tri(n-
propyl)
amine, ethanolamine, 2-dimethylaminoethanol, tromethamine, lysine, arginine,
10 histidine, caffeine, procaine, hydrabamine, choline, betaine,
ethylenediamine,
glucosamine, N-alkylglucamines, theobromine, purines, piperazine, piperidine,
morpholine, N-ethylpiperidine, and the like. It should also be understood that
other
carboxylic acid derivatives would be useful in the practice of this invention,
for
example, carboxylic acid amides, including carboxamides, lower alkyl
15 carboxamides, dialkyl carboxamides, and the like.
Pharmaceutically acceptable acid addition salts may be prepared from .
inorganic and organic acids. Salts derived from inorganic acids include
hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric
acid, and
the like. Salts derived from organic acids include acetic acid, propionic
acid,
20 glycoiic acid, pyruvic acid, oxalic acid, malic acid, malonic acid,
succinic acid,
malefic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic
acid,
mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic
acid,
salicylic acid, and the like.
The term "pharmaceutically-acceptable cation" refers to the cation of a
25 pharmaceutically-acceptable salt.
The term "library" refers to at least 3, preferably from 1 OZ to I 09 and more
preferably from 10z to 104 multimeric compounds. Preferably, these compounds
are
prepared as a multiplicity of compounds in a single solution or reaction
mixture
which permits facile synthesis thereof. In one embodiment. the library of
30 multimeric compounds can be directly assayed for multibinding properties.
In
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another embodiment, each member of the library of multimeric compounds is
first
isolated and, optionally, characterized. This member is then assayed for
multibinding properties.
The term "collection" refers to a set of multimeric compounds which are
5 prepared either sequentially or concurrently (e.g., combinatorially). The
collection
comprises at least 2 members; preferably from 2 to 109 members and still more
preferably from 10 to 104 members.
The term "multimeric compound" refers to compounds comprising from 2 to
10 ligands covalently connected through at least one linker which compounds
may
10 or may not possess multibinding properties (as defined herein).
The term "pseudohalide" refers to functional groups which react in
displacement reactions in a manner similar to a halogen. Such functional
groups
include, by way of example, mesyl, tosyl, azido and cyano groups.
The term "protecting group" or "blocking group" refers to any group which
15 when bound to one or more hydroxyl, thiol, amino or carboxyl groups of the
compounds (including intermediates thereof) prevents reactions from occurring
at
these groups and which protecting group can be removed by conventional
chemical
or enzymatic steps to reestablish the hydroxyl, thiol, amino or carboxyl group
(See.,
T.W. Greene "Protective Groups in Organic Synthesis", 2"d Ed.).
20 The particular removable blocking group employed is not critical and
preferred removable hydroxyl blocking groups include conventional substituents
such as allyl, benzyl, acetyl, chloroacetyl, thiobenzyl, benzylidine,
phenacyl, t-
butyl-diphenylsilyl and any other group that can be introduced chemically onto
a
hydroxyl functionality and later selectively removed either by chemical or
25 enzymatic methods in mild conditions compatible with the nature of the
product.
Preferred removable thiol blocking groups include disulfide groups, acyl
groups, benzyl groups, and the like.
Preferred removable amino blocking groups include conventional
substituents such as t-butyoxycarbonyl (t-BOC), benzyloxycarbonyl (CBZ),
30 fluorenylmethoxy-carbonyl (FMOC), allyloxycarbonyl (ALOC), and the like
which
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can be removed by conventional conditions compatible with the nature of the
product.
Preferred carboxyl protecting groups include esters such as methyl, ethyl,
propyl, t-butyl etc. which can be removed by mild conditions compatible with
the
5 nature of the product.
The term "optional" or "optionally" means that the subsequently described
event, circumstance or substituent may or may not occur, and that the
description
includes instances where said event or circumstance occurs and instances where
it
does not.
10 The term "ligand" or " ligands" as used herein denotes a compound that is a
bradykinin receptor antagonist. The specific region or regions of the ligand
that is
(are) recognized by the receptor is designated as the "ligand domain". A
ligand may
be either capable of binding to the receptor by itself, or may require the
presence of
one or more non-ligand components for binding (e.g., Caa'-, Mg''-or a water
15 molecule is required for the binding of a ligand to various ligand binding
sites).
Examples of ligands useful in this invention are described herein. Those
skilled in
the art will appreciate that portions of the ligand structure that are not
essential for
specific molecular recognition and binding activity may be varied
substantially,
replaced or substituted with unrelated structures {for example, with ancillary
groups
20 as defined below) and, in some cases, omitted entirely without affecting
the binding
interaction. The primary requirement for a ligand is that it has a ligand
domain as
defined above. It is understood that the term ligand is not intended to be
limited to
compounds known to be useful in binding to bradykinin receptor (e.g., known
drugs). Those skilled in the art will understand that the term ligand can
equally
25 apply to a molecule that is not normally associated with bradykinin
receptor binding
properties. In addition, it should be noted that ligands that exhibit marginal
activity
or lack useful activity as monomers can be highly active as multivalent
compounds
because of the benefits conferred by multivalency.
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The term "ligand" or " ligands" as used herein is intended to include the
racemic forms of the ligands as well as individual enantiomers and
diasteromers and
non-racemic mixtures thereof.
The term "multibinding compound or agent" refers to a compound that is
5 capable of multivalency, as defined below, and which has 2-10 ligands
covalently
bound to one or more linkers. In all cases, each ligand and linker in the
multibinding compound is independently selected such that the multibinding
compound includes both symmetric compounds (i.e., where each ligand as well as
each linker is identical) and asymmetric compounds ( (i.e., where at least one
of the
10 ligands is different from the other ligand(s) and/or at least one linker is
different
from the other linker(s)). Additionally, the term is intended to include the
racemic
forms of the multibinding compound as well as individual enantiomers and
diasteromers and non-racemic mixtures thereof. Multibinding compounds provide
a
biological and/or therapeutic effect greater than the aggregate of unlinked
ligands
15 equivalent thereto which are made available for binding. That is to say
that the
biological and/or therapeutic effect of the ligands attached to the
multibinding
compound is greater than that achieved by the same amount of unlinked ligands
made available for binding to the ligand binding sites (receptors).
The phrase "increased biological or therapeutic effect" includes, for
20 example: increased affinity, increased selectivity for target, increased
specificity for
target, increased potency, increased efficacy, decreased toxicity, improved
duration
of activity or action, increased ability to kill cells such as fungal
pathogens, cancer
cells, etc., decreased side effects, increased therapeutic index, improved
bioavailibity, improved pharmacokinetics, improved activity spectrum, and the
like.
25 The multibinding compounds of this invention will exhibit at least one and
preferably more than one of the above-mentioned affects.
The term "univalency" as used herein refers to a single binding interaction
between one ligand as defined herein with one ligand binding site as defined
herein.
It should be noted that a compound having multiple copies of a ligand (or
ligands)
30 exhibit univalency when only one ligand is interacting with
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a ligand binding site. Examples of univalent interactions are depicted below.
~TJ
The term "multivalency" as used herein refers to the concurrent binding of
from 2 to 10 linked ligands (which may be the same or different) and two or
more
corresponding receptors (ligand binding sites) on one or more enzymes which
may
be the same or different.
For example, two ligands connected through a linker that bind concurrently
to two Iigand binding sites would be considered as bivalency; three ligands
thus
connected would be an example of trivalency. An example of trivalent binding,
illustrating a multibinding compound bearing three ligands versus a monovalent
binding interaction, is shown below:
univalent interaction
m
~~~ _ ~~
trivalent interaction
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It should be understood that not all compounds that contain multiple copies
of a ligand attached to a linker or to linkers necessarily exhibit the
phenomena of
multivalency, i.e., that the biological and/or therapeutic effect of the
multibinding
agent is greater than the sum of the aggregate of unlinked ligands made
available
for binding to the ligand binding site (receptor). For multivalency to occur,
the
ligands that are connected by a linker or linkers have to be presented to
their ligand
binding sites by the linkers) in a specific manner in order to bring about the
desired
ligand-orienting result, and thus produce a multibinding event.
The term "potency" refers to the minimum concentration at which a ligand is
10 able to achieve a desirable biological or therapeutic effect. The potency
of a ligand
is typically proportional to its affinity for its ligand binding site. In some
cases, the
potency may be non-linearly correlated with its affinity. In comparing the
potency
of two drugs, e.g., a multibinding agent and the aggregate of its unlinked
ligand, the
dose-response curve of each is determined under identical test conditions
(e.g., in an
15 in vitro or in vivo assay, in an appropriate animal model). The finding
that the
multibinding agent produces an equivalent biological or therapeutic effect at
a lower
concentration than the aggregate unlinked ligand is indicative of enhanced
potency.
The term "selectivity" or "specificity" is a measure of the binding
preferences of a ligand for different ligand binding sites (receptors). The
selectivity
20 of a ligand with respect to its target ligand binding site relative to
another ligand
binding site is given by the ratio of the respective values of Kd (i.e., the
dissociation
constants for each ligand-receptor complex) or, in cases where a biological
effect is
observed below the Kd , the ratio of the respective ECSO s (i.e., the
concentrations
that produce 50% of the maximum response for the ligand interacting with the
two
25 distinct ligand binding sites (receptors)).
The term "ligand binding site" denotes the site on the bradykinin receptor
that recognizes a ligand domain and provides a binding partner for the ligand.
The
ligand binding site may be defined by monomeric or multimeric structures. This
interaction may be capable of producing a unique biological effect, for
example,
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agonism, antagonism, modulatory effects, may maintain an ongoing biological
event, and the like.
It should be recognized that the ligand binding sites of the receptor that
participate in biological multivalent binding interactions are constrained to
varying
5 degrees by their infra- and inter-molecular associations. For example,
ligand
binding sites may be covalently joined to a single structure, noncovalently
associated in a multimeric structure, embedded in a membrane or polymeric
matrix,
and so on and therefore have less translational and rotational freedom than if
the
same structures were present as monomers in solution.
10 The term "antagonism" is well known in the art. The term "modulatory
effect" refers to the ability of the ligand to change the activity of an
agonist or
antagonist through binding to a ligand binding site.
The term "inert organic solvent" or "inert organic solvent" means a solvent
which is inert under the conditions of the reaction being described in
conjunction
15 therewith including, by way of example only, benzene, toluene,
acetonitrile,
tetrahydrofuran, dimethylformamide, chloroform, methylene chloride, diethyl
ether,
ethyl acetate, acetone, methylethyl ketone, methanol, ethanol, propanol,
isopropanol, t-butanol, dioxane, pyridine, and the like. Unless specified to
the
contrary, the solvents used in the reactions described herein are inert
solvents.
20 The term "treatment" refers to any treatment of a pathologic condition in a
mammal, particularly a human, and includes:
(i) preventing the pathologic condition from occurring in a subject
which may be predisposed to the condition but has not yet been diagnosed with
the
condition and, accordingly, the treatment constitutes prophylactic treatment
for the
25 disease condition;
(ii) inhibiting the pathologic condition, i.e., arresting its development;
(iii) relieving the pathologic condition, i.e., causing regression of the
pathologic condition; or
(iv) relieving the conditions mediated by the pathologic condition.
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The term "pathologic condition which is modulated by treatment with a
ligand" covers all disease states (i.e., pathologic conditions) which are
generally
acknowledged in the art to be usefully treated with a ligand for the
bradykinin
receptors in general, and those disease states which have been found to be
usefully
5 treated by a specific multibinding compound of our invention. Such disease
states
include, by way of example only, the treatment of a mammal afflicted with
cancer,
pain, shock, asthma, rhinitis, arthritis, inflammatory bowel disease, and the
like.
The term "therapeutically effective amount" refers to that amount of
multibinding compound which is sufficient to effect treatment, as defined
above,
10 when administered to a mammal in need of such treatment. The
therapeutically
effective amount will vary depending upon the subject and disease condition
being
treated, the weight and age of the subject, the severity of the disease
condition, the
manner of administration and the like, which can readily be determined by one
of
ordinary skill in the art.
15 The term "linker", identified where appropriate by the symbol 'X' refers to
a
group or groups that covalently attaches from 2 to 10 ligands (as identified
above)
in a manner that provides for a compound capable of multivalency. The term is
intended to include the racemic forms of the linker as well as individual
enantiomers and diasteromers and non-racemic mixtures thereof. Among other
20 features, the linker is a ligand-orienting entity that permits attachment
of multiple
copies of a ligand (which may be the same or different) thereto. In some
cases, the
linker may itself be biologically active. The term "linker" does not, however,
extend to cover solid inert supports such as beads, glass particles, fibers,
and the
like. But it is understood that the multibinding compounds of this invention
can be
25 attached to a solid support if desired. For example, such attachment to
solid
supports can be made for use in separation and purification processes and
similar
applications.
The extent to which multivalent binding is realized depends upon the
efficiency with which the linker or linkers that joins the ligands presents
these
30 ligands to the array of available ligand binding sites. Beyond presenting
these
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ligands for multivalent interactions with ligand binding sites, the linker or
linkers
spatially constrains these interactions to occur within dimensions defined by
the
linker or linkers. Thus, the structural features of the linker (valency,
geometry,
orientation, size, flexibility, chemical composition, etc.) are features of
multibinding
5 agents that play an important role in determining their activities.
The linkers used in this invention are selected to allow multivalent binding
of ligands to the ligand binding sites of a bradykinin receptor, whether such
sites are
located interiorly, both interiorly and on the periphery of the enzyme
structure, or at
any intermediate position thereof.
10
PREFERRED EMBODIMENTS
While the broadest definition of this invention is set forth in the Summary of
the Invention, certain compounds of Formula (I) are preferred.
(A) A preferred group is a multibinding compound of Formula (I) wherein:
15 p is 2 or 3, preferably 2; and
q is 1 or 2, preferably 1.
Within this group (A), a more preferred group of compounds is that wherein
the ligands are a compound of formula (b) as defined in the Summary of the
Invention.
20 Within compounds of formula (b), preferred compounds are:
(i) a compound of formula (II):
R13
O
O
N-C~ NH~
attachment
(II)
wherein:
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R'3 is quinolyl, quinoxalinyl, benzimidazolyl, or imidazopyridyl each of
which is substituted with one or more substituent(s) selected from halo, alkyl
or
alkoxy, preferably
R33 CH3
N
N
\ N~CH3 ~ , ~ I i , ~ ~ ~~OCH3
\N/ \CH3 ~ 'N CH3 H
wherein R33 is chloro, bromo or iodo;
R'4 and R'S are, independently of each other, hydro~ w, alkyl, or halo,
preferably hydrogen, methyl or chloro, most preferably methyl or chloro;
Rz' is hydrogen or methyl, preferably methyl; and
the terminal nitrogen atom attaches the ligand to a linker; or
(ii) a compound of formula (III):
R~3
O
R~s
R~4 w Ra
O i\
~H \ W
N N \
R21 O
(III)
wherein:
R'3 is quinolyl, quinoxalinyl, benzimidazolyl, or imidazopyridyl each of
which is substituted with one or more substituent(s) selected from halo, alkyl
or
alkoxy, preferably
R33 CH3
N
\ N T'CH3 ~ , ~ I i ~ , ( , N~OCH3
i H , ~N~ ~CH3 , ~N CH3 ~H
wherein R33 is chloro, bromo or iodo;
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R'° and R'S are, independently of each other, hydrogen, alkyl,
halo, or a
covalent bond linking the ligand to a linker, preferably hydrogen, methyl or
chloro,
or a covalent bond linking the ligand to a linker, most preferably methyl or
chloro;
R'-' is hydrogen, alkyl, or a covalent bond linking the ligand to a linker;
5 preferably hydrogen, methyl or a covalent bond linking the ligand to a
linker; and
Ra is -COOH, -NH2, -CONRZBR'-9 (wherein R'-g is hydrogen or alkyl and R'-9
is hydrogen, alkyl, or heteroaryl), -NR'°COR" (where R3° is
hydrogen or alkyl and
R3' is alkyl), -NR3°CONHR32 (where R'° is hydrogen or alkyl, and
R3'- is alkyl),
heteroaralkyl, heterocyclyl or a covalent bond linking the ligand to a linker,
10 preferably -CONHCH3, -CON(CH3)z, -NHCOCH3, -N(CH3)COCH3,
-NHCONHCH3,
H
~ \ \ N II ~ O
O~ ~ ' I ~ O
I N N N
or a covalent bond linking the ligand to a linker;
W is -CH- or -N-; or
(iii) a compound of formula (IV):
OR~3
R~4 R~5
i
N
NH2
attachment
15
(IV)
wherein:
R'3, R'4 and R'S are as defined in preferred embodiment (i) above; and
the terminal nitrogen atom attaches the ligand to a linker; or
(iv) a compound of formula (V):
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ORIs
R14 R1s
I ~ Ra
N
w ~ W
O
(V)
wherein:
R", R'4, R'S, W, and Ra are as defined in the preferred embodiments (ii)
above; and
S pharmaceutically acceptable salts thereof.
In one embodiment, the multibinding compound comprises of identical
ligands.
10 In one embodiment, the multibinding compound comprises of non-identical
ligands.
Within these preferred and more preferred groups, even more preferred
compounds are those shown below:
15 R13 R19
O O
R14 R15 R15 14
~ O H H O i I R
N~N X N~N w
R21 R21
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R13
I
O
R15
14
R I W O H ~- Ra
N~N \ \ W
O
O
N~N / ~ W
R14 I ~ Rt5 O H \ I Ra
O
R13
R13
O
R15
14
R \ O ~ Ra
N~N \ \ w R1s
O
O
R15 R14
O i
W / / N ~ N ~~~
R21
O
OR13 8130
14 R15
R I \ R15 R14
I
N ~ \ W W / N \
\/ ~ \/
O
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R13
i R13
O ,
O
R14 R1s
~R15 Ra
N Ir ~ / ~ H ~ I
W N \ W
N \
O Rz1
O
R13 R13
i
O O
R15 R15
14 14
R I i ~N / I X ~ \ / N~ \ I R
~W W / N
N
Rz1 O O R21
wherein R", R'4, R'S, Re, X, and W are as defined in preferred embodiments
above.
5 Within the above preferred groups, particularly preferred group of
compounds is that wherein the linker, X, in the bivalent multibinding compound
of
Formula (I) independently has the formula:
_X8_Z-(Ya_Z)m Xa_
10 wherein
m is an integer of from 0 to 20, preferably 0 to 5;
Xa at each separate occurrence is selected from the group consisting of
-O-, -S-, -NR-, -C(O)-, -C(O)O-, -C(O)NR-, -C{S), -C(S)O-, -C(S)NR- or a
covalent
bond where R is as defined below;
15 Z at each separate occurrence is selected from the group consisting of
alkylene, substituted alkylene, cycloalkylene, substituted cylcoalkylene,
alkenylene,
substituted alkenylene, alkynylene, substituted alkynylene, cycloalkenylene,
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substituted cycloalkenylene, arylene, heteroarylene, heterocyclene, or a
covalent
bond, preferably alkylene;
each Ya at each separate occurrence is selected from the group consisting of
-O-, -C(O)-, -OC(O)-, -C(O)O-, -NR-, -S(O)n-, -C(O)NR'-, -NR' C(O)-, -NR'
5 C(O)NR'-, -NR' C(S)NR'-, -C(=NR')-NR'-, -NR'-C(=NR')-, -OC(O)-NR'-, -NR'-
C(O)-O-, -N=C(Xa)-NR'-, -NR'-C(Xa)=N-,-P(O)(OR')-O-, -O-P(O)(OR')-,
S(O)~CR' R"-, -S(O)~-NR'-, -NR'-S(O)~-, -S-S-, and a covalent bond; where n is
0,
1 or 2; and R, R' and R" at each separate occurrence are selected from the
group
consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted
cycloalkyl,
10 alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyh
alkynyl,
substituted alkynyl, aryl, heteroaryl and heterocyclic provided that at least
one of
Xe, Z, and Ya is not a covalent bond, preferably -O-.
GENERAL SYNTHETIC SCHEME
15 Compounds of this invention can be made by the methods depicted in the
reaction schemes shown below.
The starting materials and reagents used in preparing these compounds are
either available from commercial suppliers such as Aldrich Chemical Co.,
(Milwaukee, Wisconsin, USA), Bachem (Torrance, California, USA),
20 Emka-Chemie, or Sigma (St. Louis, Missouri, USA) or are prepared by methods
known to those skilled in the art following procedures set forth in references
such as
Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-15 (John Wiley
and
Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and
Supplementals (Elsevier Science Publishers, 1989), Organic Reactions, Volumes
25 1-40 (John Wiley and Sons, 1991), March's Advanced Organic Chemistry, {John
Wiley and Sons, 4th Edition), and Larock's Comprehensive Organic
Transformations (VCH Publishers Inc., 1989).
The starting materials and the intermediates of the reaction may be isolated
and purified if desired using conventional techniques, including but not
limited to
30 filtration, distillation, crystallization, chromatography, and the like.
Such materials
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may be characterized using conventional means, including physical constants
and
spectral data.
Furthermore, it will be appreciated that where typical or preferred process
conditions (i.e., reaction temperatures, times, mole ratios of reactants,
solvents,
5 pressures, etc.) are given, other process conditions can also be used unless
otherwise
stated. Optimum reaction conditions may vary with the particular reactants or
solvent used, but such conditions can be determined by one skilled in the art
by
routine optimization procedures.
Additionally, as will be apparent to those skilled in the art, conventional
10 protecting groups may be necessary to prevent certain functional groups
from
undergoing undesired reactions. The choice of a suitable protecting group for
a
particular functional group as well as suitable conditions for protection and
deprotection are well known in the art. For example, numerous protecting
groups,
and their introduction and removal, are described in T. W. Greene and G. M.
Wuts,
15 Protecting Groups in Organic Synthesis, Second Edition, Wiley, New York,
1991,
and references cited therein.
These schemes are merely illustrative of some methods by which the
compounds of this invention can be synthesized, and various modifications to
these
schemes can be made and will be suggested to one skilled in the art having
referred
20 to this disclosure.
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Preparation of a multibinding compound of Formula fIl
In general, a bivalent multibinding compound of Formula (I) can be
prepared by covalently attaching the ligands, L, wherein at least one of the
ligand is
selected from a compound of formula (a) as defined in the Summary of the
S Invention, to a linker, X, as shown in Scheme A below.
S em A
M a
2 L~FG~
FGZ X -FGZ ---~- L X L
eth b
~. FG ~
FG2- X -FG2PG ----
L~ X -FGZPG
[intermediate]
(II)
deprotect ~ FGA
--.~ L~ X _FGZ + L~ ~ L~ X
10
In method (a), a bivalent multibinding compound of Formula (I) is prepared
in one step, by covalently attaching the ligands, L, to a linker, X, where FG'
and
FG'- represent a functional group such as halo, amino, hydroxy, thio,
aldehyde,
ketone, carboxy, carboxy derivatives such as acid halide, ester, amido, and
the like.
15 This method is preferred for preparing compounds of Formula (I) where the
ligands
are the same.
In method (b), the compounds of Formula (I) are prepared in a stepwise
manner by covalently attaching one equivalent of a ligand, L,, with a ligand X
where where FG' and FG'- repiesent a functional group as defined above, and
20 FGZPG is a protected functional group to give an intermediate of formula
(II).
Deprotection of the second functional group on the ligand, followed by
reaction
with a ligand LZ, which may be same or different than ligand L,, then provides
a
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compound of Formula (I). This method is suitable for preparing compounds of
Formula (I) where the ligands are the non-identical.
The ligands are covalently attached to the linker using conventional
chemical techniques providing for covalent linkage of the ligand to the
linker.
5 Reaction chemistries resulting in such linkages are well known in the art
and
involve the use of complementary functional groups on the linker and ligand as
shown in Table I below.
Table I
Represent ative Complementaryg Ci mistries
Bindin
10 First Reactive GroupSecond Reactive Link a
Group
carboxyl amine amide
sulfonyl halide amine sulfonamide
hydroxyl alkyl/aryl halide ether
hydroxyl isocyanate urethane
15 amine epoxide ~3-hydroxyamine
amine alkyl/aryl halide alkylamine
hydroxyl carboxyl ester
Reaction between a carboxylic acid of either the linker or the iigand and a
20 primary or secondary amine of the ligand or the linker in the presence of
suitable,
well-known activating agents such as dicyclohexylcarbodiimide, results in
formation of an amide bond covalently linking the ligand to the linker;
reaction
between an amine group of either the linker or the ligand and a sulfonyl
halide of
the ligand or the linker, in the presence of a base such as triethylamine,
pyridine, an
25 the like results in formation of a sulfonamide bond covalently linking the
ligand to
the linker; and reaction between an alcohol or phenol group of either the
linker or
the ligand and an alkyl or aryl halide of the ligand or the linker in the
presence of a
base such as triethylamine, pyridine, and the like, results in formation of an
ether
bond covalently linking the ligand to the linker.
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Any compound which inhibits bradykinin receptor can be used as a ligand in
this invention. As discussed in further detail below, numerous antagonists are
known in the art and any of these known compounds or derivatives thereof may
be
employed as ligands in this invention. Typically, a compound selected for use
as a
5 ligand will have at least one functional group, such as an amino, hydroxyl,
thiol or
carboxyl group and the like, which allows the compound to be readily coupled
to
the linker. Compounds having such functionality are either known in the art or
can
be prepared by routine modification of known compounds using conventional
reagents and procedures. The patents and publications set forth below provide
10 numerous examples of suitably functionalized bradykinin receptor antagonist
and
intermediates thereof which may be used as Iigands in this invention.
The compounds of formula (a) can be prepared as described in PCT
Application NO. 96/13485. The compounds of formula (b) can be prepared by the
methods described in PCT Application NO. 97/11069 and Y. Abe, et.al., J. Med
15 Chem., 41, pages 564, 4053, 4062, and 4587, (1998). The compounds of
formula
(c) can be prepared by the methods described in PCT Application NO. 96/06082.
Some of the methods for preparing compounds of formulae (a) and (b) of the
present invention are illustrated and described in Schemes B-F below.
20
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Svnt~sis of compounds of formula (al
Ligands of formula (a) can be prepared as described in Scheme B below.
Scheme B
Method i
X.A C_B~A
R~ ~ R2 + C-BH -.. ~ ~ R2
R ~~J
Ra Rs
1 ? (a)
Method ii
C B ~A
C B ~A
R~ ~ R2 + R"-Q-COOH
or acid derivative R~ ~ 2
N-AAH 4 ~~J R
R7 N-AA-CO-Q-R ~ ~
_3 R~
(a)
5 In general, compounds of formula (a) can be prepared as shown in methods
(i) or (ii) above.
In method (i), a compound of formula (a) is prepared by reacting a
compound of formula ~ where X is a leaving group under nucleophilic
displacement
reaction conditions [such as tosylate, mesylate, or halo (such as chloro,
bromo, or
10 iodo)] with an amine or alcohol of formula 2_ (B is -NH- or -O-). The
reaction is
typically carned out in the presence of a base such as triethylamine, and the
like.
In method (ii), a compound of formula (a) is prepared by reacting a
compound of formula ~, (where AAH is an amino acid residue) with an acid of
formula 4_ or its reactive derivative such as acid chloride, ester and the
like. The
15 reaction conditions used depend on the nature of compound 4_. If 4 is an
acid, the
reaction is carried out in the presence of a coupling agent such as
dicyclohexycarbodiimide. If 4_ is an acid derivative such as acid chloride,
then the
reaction is carried out in the presence of a suitable base such as
triethylamine, and
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the like. Detailed descriptions of the synthesis of compounds of formula (a)
via
these methods are given in PCT Application NO. 96/13485.
,~vnthesis of compounds of formula fbl
5 Compounds of formula (b) can be prepared as illustrated and described in
Schemes C-F below.
Compounds of formula (b) where R'3, R'~, R'S are as described in the
Summary of the Invention, A' is methylene, Q' is -NH-, A'- is a bond, and R"
is
acyl can be prepared as described below.
10 Scheme C
X ORls
R14 15 R13OH 14
~ R + ~ R I ~ R1s reduction
/ /
NOZ N02
5 6
OR13 ORIs
R14 \ R15 RbCOCI R14 \ R1s
/ /
NHZ NHCOR
7
Reaction of a 3-nitrophenyl compound of formula ~ where X is a leaving
group under nucleophilic displacement reaction conditions such as tosylate,
mesylate, or halo (such as chloro, bromo, or iodo), preferably mesylate, with
an
15 alcohol of formula R'30H where R'3 is as defined in the Summary of the
Invention
provides a compound of formula 6_. The reaction is carried out in the presence
of a
base such as sodium hydride in an aprotic solvent such as dimethylformamide.
Reduction of the nitro group with a suitable reducing agent such as hydrazine
monohydrate, iron (III) chloride hexahydrate and carbon provides an aniline of
20 formula Z which is then converted to a compound of formula (b) where Q' is -
NH-,
AZ is a bond, and R" is acyl by reaction 7 with an acylating agent such as as
acid
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chloride of formula RCOC1 where RCO- is an acyl group as defined in the
Summary of the invention.
Compounds of formula 5_ are either commercially available or they can be
prepared by methods well known in the art. For example, compound ~ where R'a
5 and R'S are chloro and X is -OMs can be prepared by the method described in
Abe,
Y. et al, J. Med. Chem., 41, 564, 1998. Compounds of formula R'30H can be
prepared by methods well known in the art. For example, 4-hydroxybenzimidazole
derivatives, 8-hydroxy-2-methylquinazoline, 2- and 3-methylquinoxaline
derivatives can be prepared by the methods described in Abe, Y. et al, J. Med.
10 Chem., 41, 4062, 1998.
Compounds of formula (b) where A'-, R'3, R'4, R'S are as described in the
Summary of the Invention, A' is methylene, Q' is -NR2'-, and R" is an
aminoacyl
(-NHCOR) group can be prepared as described below.
15 Scheme D
OR'3
O
~4
R'4 ~ ~s ~ R
R + ~ / ~N-AZ-COCI --
NHZ O
7
8
OR~3 OR~3
1. alkylation ~a R RCOCI R~4 \ R~s
(optional) R w 'S ~..
or acid derivatives
2. deprotection I / I / NH_Ca--A~-NHCOR
N-C4-~NHZ
R2' (b)
_10
1R= alkyl, aryl,
aralky, aralkenyl,
heteroaralkenyl, etcl
Treatment of a compound of formula 7 with an acid chloride of formula $
where Az is alkylene under the reaction conditions described in Abe, Y et al.,
J.
Med. Chem., 41, 564, 1998 provides N-phthaloyl derivatives of formula 9_ where
R'-'
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is hydrogen. Compound 9 where R'-' is hydrogen can optionally be converted to
a
corresponding compound of formula 9_ where R'-' is alkyl by treating it with
an
alkylating agent such as alkyl halide in the presence of a base such as sodium
hydride. Treatment of 9_ with hydrazine monohydrate provides a compound of
5 formula 10 which can be converted to a compound of formula (b) where R" is
aminoacyl by treating ~ with an acylating agent as described above. The
reaction
conditions employed depend on the nature of the acylationg agent. If the
acylating
agent agent is an acid chloride, the reaction reaction is carried out in the
presence of
a base such as triethylamine, pyridine or the like and in a suitable organic
solvent
10 such as dichloromethane, dimethylformamide, tetrahydrofuran, and the like.
If it is
an acid, then the reaction is carried out in the presence of a suitable
coupling agent
such as 1-ethoxy-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride in the
presence of 1-hydroxybenzotriazole as described in Abe, Y et al., J. Med.
Chem.,
41, 4053,1998.
15
Alternatively, compounds of formula (b) where A'-, R'3, R'4, R'' are as
described in the Summary of the invention, A' is methylene, Q' is -NR-'''-,
and R" is
an aminoacyl (-NHCOR) group can be prepared as described below.
scheme E
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OTBDPS O OTBDPS
1a R ~ R1a ~ R1s
R ~ 1s + I N-Az-COCI .~ O
NHz / O / NH--COIN
11
- 8 12 O
OMs OR13
14
i. alkylation (optR I ~ R1s O i R130H R14 \ R~s
ii. dep~ ~ N-CO-Az-N ~ \ iiii. deprotection ~ ,
iii. MsCI Rz1 ~ N-CO-Az-NHz
13 O Rz~
14
ORla
-~ R1a ~ R1s
N-CO-A? NHCOR
Rz~
(b)
Treatment of a compound of formula ~_l with an acid chloride of formula $
where A'- is alkylene under the reaction conditions described in Abe, Y et
al., J.
Med. Chem., 41, 564, 1998 provides N-phthaloyl derivatives of formula ~ where
R-'' is hydrogen. Removal of tert-butyldiphenylsilyl group with
tetrabutylammonium flouride followed by treatment with mesyl chloride provides
a
compound of formula ~ which can then be converted to a ligand of formula (b)
where R is arylalkenyl, heteroaralkenyl, alkyl, aralkyl, heteroaralkyl, etc as
described in Scheme D above.
10 Compounds of formula (b) where R'3, R'a, R'S are as described in the
Summary of the Invention, A' and A'- are methylene, R" is an aminoacy 1 (-
NHCOR
where R is aralkenyl or heteroaralkenyl) and Q' is a group of formula
/ ~,/
-- or
can be prepared as described in Scheme F below.
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ch m F
ORta
Rt4 \ Rts
CH2NH2
Ra \ ORt3
I
W / / Rt4 ~ Rt5 Ra
15a COOH I / v
or acid derivatives i t ~ W
ORts Q HN
Rta ~5 ~b~ 0
R
I , CHZNHz ~~~.
\ ~ w
N~ Q1 = -yN ~ or I
i
15b
A compound of formula (b) where Q' is pyrrole or phenyl can be prepared
by acylating a compound of formula 1 a or 15 under the reaction conditions
5 described above. Compounds ~5 and S]~ can be prepared by the methods
described in Abe, Y et al, J. Med. Chem., 41, 4587, (I998).
Synthesis of a compound of formula (b) where R'3 is as defined in the
Summary of the Invention, R'4 is hydroxy, R'S is chloro, R'6 is -Q'-Az-R"
where Q'
is -N(CH3)CO, AZ is -CHz-, and R" is -NHCOR (where R is aralkenyl or
10 heteroaralkenyl) is illustrated in Figure 5.
Syntheses of compounds of formula (b) where R'3, R'4, R'S, are as defined in
the Summary of the Invention, R'6 is -Q'-Az-R" where Q' is -N(CH3)CO, A'- is
-CHZ-, and R" is -NHCOR (where R is aralkenyl or heteroaralkenyl) are
illustrated
in Figures 6 and 7.
15 Syntheses of some representative compounds of Formula (I) are illustrated
in Figures 8-15 and described in detail in working examples 1-13 respectively.
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It will be apparent to one skilled in the art that the above chemistries ARE
not limited to preparing bivalent multibinding compounds of Formula (I) and
can be
used to prepare tri-, tetra-, etc., multibinding compounds of Formula (I).
The linker is attached to the ligand at a position such that it retains ligand
5 domain-ligand binding site interaction and specifically which permits the
ligand
domain of the ligand to orient itself to bind to the ligand binding site. Such
positions and synthetic protocols for linkage are well known in the art. The
term
linker embraces everything that is not considered to be part of the ligand.
The relative orientation in which the ligand domains are displayed derives
10 from the particular point or points of attachment of the ligands to the
linker, and on
the framework geometry. The determination of where acceptable substitutions
can
be made on a ligand is typically based on prior knowledge of structure-
activity
relationships (SAR) of the ligand and/or congeners and/or structural
information
about ligand-receptor complexes (e.g., X-ray crystallography, NMR, and the
like).
15 Such positions and the synthetic methods for covalent attachment are well
known in
the art. Following attachment to the selected linker (or attachment to a
significant
portion of the linker, for example 2-10 atoms of the linker), the univalent
linker-
ligand conjugate may be tested for retention of activity in the relevant
assay.
The linker, when covalently attached to multiple copies of the ligands,
20 provides a biocompatible, substantially non-immunogenic multibinding
compound.
The biological activity of the multibinding compound is highly sensitive to
the
valency, geometry, composition, size, flexibility or rigidity, etc. of the
linker and, in
turn, on the overall structure of the multibinding compound, as well as the
presence
or absence of anionic or cationic charge, the relative
hydrophobicity/hydrophilicity
25 of the linker, and the like on the linker. Accordingly, the linker is
preferably chosen
to maximize the biological activity of the multibinding compound. The linker
may
be chosen to enhance the biological activity of the molecule. In general, the
linker
may be chosen from any organic molecule construct that orients two or more
ligands to their ligand binding sites to permit multivalency. In this regard,
the
30 linker can be considered as a "framework" on which the ligands are arranged
in
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order to bring about the desired ligand-orienting result, and thus produce a
multibinding compound.
For example, different orientations can be achieved by including in the
framework groups containing mono- or polycyclic groups, including aryl and/or
5 heteroaryl groups, or structures incorporating one or more carbon-carbon
multiple
bonds (alkenyl, alkenylene, alkynyl or alkynylene groups). Other groups can
also
include oligomers and polymers which are branched- or straight-chain species.
In
preferred embodiments, rigidity is imparted by the presence of cyclic groups
(e.g.,
aryl, heteroaryI, cycloalkyl, heterocyclic, etc.). In other preferred
embodiments, the
10 ring is a six or ten membered ring. In still further preferred embodiments.
the ring
is an aromatic ring such as, for example, phenyl or naphthyl.
Different hydrophobic/hydrophilic characteristics of the linker as well as the
presence or absence of charged moieties can readily be controlled by the
skilled
artisan. For example, the hydrophobic nature of a linker derived from
15 hexamethylene diamine (HzN(CH~)6NH,) or related polyamines can be modified
to
be substantially more hydrophilic by replacing the alkylene group with a
poly(oxyalkylene) group such as found in the commercially available
"Jeffamines".
Different frameworks can be designed to provide preferred orientations of
the ligands. Such frameworks may be represented by using an array of dots (as
20 shown below) wherein each dot may potentially be an atom, such as C, O, N,
S, P,
H, F, Cl, Br, and F or the dot may alternatively indicate the absence of an
atom at
that position. To facilitate the understanding of the framework structure, the
framework is illustrated as a two dimensional array in the following diagram,
although clearly the framework is a three dimensional array in practice:
25
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g . . . . . . . . . .....
7 . . . . . . . . . .....
g . . . . . . . . . .....
5 . . . . . . . . . .....
. ._....
. . . . . . . . .....
. . . . . . . . .....
1 . . . . . . . . . .....
p . . . . . . . . . .....
p 1 2 3 4 5 6 7 8
Each dot is either an atom, chosen from carbon, hydrogen, oxygen, nitrogen,
sulfur, phosphorus, or halogen, or the dot represents a point in space {i.e.,
an
absence of an atom). As is apparent to the skilled artisan, only certain atoms
on the
grid have the ability to act as an attachment point for the ligands, namely,
C, O, N,
5 S and P.
Atoms can be connected to each other via bonds (single, double or triple
bonds with acceptable resonance and tautomeric forms), with regard to the
usual
constraints of chemical bonding. Ligands may be attached to the framework via
single, double or triple bonds (with chemically acceptable tautomeric and
resonance
10 forms). Multiple Iigand groups (2 to 10) can be attached to the framework
such that
the minimal, shortest path distance between adjacent ligand groups does not
exceed
100 atoms. Preferably, the linker connections to the ligand is selected such
that the
0
maximum spatial distance between two adjacent ligands is no more than 100A.
An example of a linker as presented by the grid is shown below for a
15 biphenyl construct.
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Q .
H H H
g . . . . . .
' ~ ~ H
H H
H CI
1
p . . ,
0 1 2 3 4 5 6 ~ 8 9 10
Nodes ( 1,2), (2,0), (4,4}, (5,2), (4,0), (6,2), (7,4), (9,4), ( 10,2), (9,0),
(7,0) all
represent carbon atoms. Node ( 10,0) represents a chlorine atom. All other
nodes
(or dots) are points in space (i.e., represent an absence of atoms).
Nodes (1,2) and (9,4) are attachment points. Hydrogen atoms are affixed to
nodes (2,4), (4,4), (4,0), (2,0), (7,4), (10,2) and (7,0). Nodes (5,2) and
(6,2) are
connected by a single bond.
The carbon atoms present are connected by either a single or double bonds,
taking into consideration the principle of resonance and/or tautomerism.
The intersection of the framework (linker) and the ligand group, and indeed,
the framework (linker) itself can have many different bonding patterns.
Examples
of acceptable patterns of three contiguous atom arrangements are shown in the
following diagram:
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CCC NCC OCC SCC pCC
CCN NCN OCN SCN pCN
CCO NCO OCO SCO PCO
CCS NCS OCS SCS PCS
CCP NCP OCP SCP PCP
CNC NNC ONC SNC PNC
S~ PNN
CNO NNO ON O
~ ~ P
CNP AR~ SN pNp
COC NOC OOC SOC POC
COO NO N 1~ SON pON
ft~ 0 ~ SOO
COP
CSC NSC
CSN NSN OSC SSC PSC
CSO NSO OSN SSN PSN
O S
CSP NSP OSS
OSP
CPC NPC
CPN NPN OPC SPC PPC
CPO NPO OPN SPN
CPS NPS OPO SPO lsp'O
CPP NPP app
SPP
One skilled in the art would be able to identify bonding patterns that would
produce multivalent compounds. Methods for producing these bonding
arrangements are described in March, "Advanced Organic Chemistry", 4th
Edition,
Wiley-Interscience, New York, New York (1992). These arrangements are
5 described in the grid of dots shown in the scheme above. All of the possible
arrangements for the five most preferred atoms are shown. Each atom has a
variety
of acceptable oxidation states. The bonding arrangements underlined are less
acceptable and are not preferred.
Examples of molecular structures in which the above bonding patterns could
10 be employed as components of the linker are shown below.
15
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O ~ ~ HN.C'Ci
wO.C'C~. ~
~O~O' ~N N ~O N
~C~N ~C~ ~
C C C. _C
O O O O
II II w .C II
~N~N~ wO~N~ wC~N~ C C wO
O
O O O
wC.S'Si
wS.S.N~ wS.S~N~ wC.s'C~ C C
O O O
C'S'C' \C~O~N~ ~ ~ ~ \C~O'C~
O N
O O~ O~ w .N.~ .-
., C C
wN~S.N~ O O \ ~N ~N~N
O N
w ~C' w .C' ,.
N O wN~N ~N~N
O
_ O O P N,N
w .N_N wC.PwC~ wN.PwC~ w0.0 C, N_N
N O O
The identification of an appropriate framework geometry and size for ligand
domain presentation are important steps in the construction of a multibinding
compound with enhanced activity. Systematic spatial searching strategies can
be
5 used to aid in the identification of preferred frameworks through an
iterative
process. Figure 1 illustrates a useful strategy for determining an optimal
framework
display orientation for ligand domains. Various other strategies are known to
those
skilled in the art of molecular design and can be used for preparing compounds
of
this invention.
10 As shown in Figure 1, display vectors around similar central core
structures
such as a phenyl structure (Panel A) and a cyclohexane structure (Panel B) can
be
varied, as can the spacing of the ligand domain from the core structure (i.e.,
the
length of the attaching moiety). It is to be noted that core structures other
than
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those shown here can be used for determining the optimal framework display
orientation of the ligands. The process may require the use of multiple copies
of the
same central core structure or combinations of different types of display
cores.
The above-described process can be extended to trimers (Figure ?) and
5 compound of higher valency. (Figures 3 & 4)
Assays of each of the individual compounds of a collection generated as
described above will lead to a subset of compounds with the desired enhanced
activities (e.g., potency, selectivity, etc.). The analysis of this subset
using a
technique such as Ensemble Molecular Dynamics will provide a framework
10 orientation that favors the properties desired. A wide diversity of linkers
is
commercially available (see, e.g., Available Chemical Directory (ACD)). Many
of
the linkers that are suitable for use in this invention fall into this categoy
. Other
can be readily synthesized by methods well known in the art and/or are
described
below.
15 Having selected a preferred framework geometry, the physical properties of
the linker can be optimized by varying the chemical composition thereof. The
composition of the linker can be varied in numerous ways to achieve the
desired
physical properties for the multibinding compound.
It can therefore be seen that there is a plethora of possibilities for the
20 composition of a linker. Examples of linkers include aliphatic moieties,
aromatic
moieties, steroidal moieties, peptides, and the like. Specific examples are
peptides
or polyamides, hydrocarbons, aromatic groups, ethers, lipids, cationic or
anionic
groups, or a combination thereof.
Examples are given below, but it should be understood that various changes
25 may be made and equivalents may be substituted without departing from the
true
spirit and scope of the invention. For example, properties of the linker can
be
modified by the addition or insertion of ancillary groups into or onto the
linker, for
example, to change the solubility of the multibinding compound (in water,
fats,
lipids, biological fluids, etc.), hydrophobicity, hydrophilicity, linker
flexibility,
30 antigenicity, stability, and the like. For example, the introduction of one
or more
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polyethylene glycol) (PEG) groups onto or into the linker enhances the
hydrophilicity and water solubility of the multibinding compound, increases
both
molecular weight and molecular size and, depending on the nature of the
unPEGylated linker, may increase the in vivo retention time. Further PEG may
5 decrease antigenicity and potentially enhances the overall rigidity of the
linker.
Ancillary groups which enhance the water solubility/hydrophilicity of the
linker and, accordingly, the resulting multibinding compounds are useful in
practicing this invention. Thus, it is within the scope of the present
invention to use
ancillary groups such as, for example, small repeating units of ethylene
glycols,
alcohols, polyols (e.g., glycerin, glycerol propoxylate, saccharides,
including mono-
oligosaccharides, etc.), carboxylates (e.g., small repeating units of glutamic
acid,
acrylic acid, etc.), amines (e.g., tetraethylenepentamine), and the like) to
enhance
the water solubility and/or hydrophilicity of the multibinding compounds of
this
invention. In preferred embodiments, the ancillary group used to improve water
solubility/hydrophilicity will be a polyether .
The incorporation of lipophilic ancillary groups within the structure of the
linker to enhance the lipophilicity and/or hydrophobicity of the multibinding
compounds described herein is also within the scope of this invention.
Lipophilic
groups useful with the linkers of this invention include, by way of example
only,
aryl and heteroaryl groups which, as above, may be either unsubstituted or
substituted with other groups, but are at least substituted with a group which
allows
their covalent attachment to the linker. Other lipophilic groups useful with
the
linkers of this invention include fatty acid derivatives which do not form
bilayers in
aqueous medium until higher concentrations are reached.
Also within the scope of this invention is the use of ancillary groups which
result in the multibinding compound being incorporated or anchored into a
vesicle
or other membranous structure such as a liposome or a micelle. The term
"lipid"
refers to any fatty acid derivative that is capable of forming a bilayer or a
micelle
such that a hydrophobic portion of the lipid material orients toward the
bilayer
while a hydrophilic portion orients toward the aqueous phase. Hydrophilic
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characteristics derive from the presence of phosphato, carboxylic, sulfato.
amino,
sulfhydryl, vitro and other like groups well known in the art. Hydrophobicity
could
be conferred by the inclusion of groups that include, but are not limited to,
long
chain saturated and unsaturated aliphatic hydrocarbon groups of up to 20
carbon
5 atoms and such groups substituted by one or more aryl, heteroaryl,
cycloalkyl,
and/or heterocyclic group(s). Preferred lipids are phosphglycerides and
sphingolipids, representative examples of which include phosphatidylcholine,
phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,
phosphatidic
acid, palmitoyleoyl phosphatidylcholine, lysophosphatidylcholine,
10 Iysophosphatidyl-ethanolamine, dipalmitoylphosphatidylcholine,
dioleoylphosphatidylcholine, distearoyl-phosphatidylcholine or
dilinoleoylphosphatidylcholine could be used. Other compounds lacking
phosphorus, such as sphingolipid and glycosphingolipid families are also
within the
group designated as lipid. Additionally, the amphipathic lipids described
above
15 may be mixed with other lipids including triglycerides and sterols.
The flexibility of the linker can be manipulated by the inclusion of ancillary
groups which are bulky and/or rigid. The presence of bulky or rigid groups can
hinder free rotation about bonds in the linker or bonds between the linker and
the
ancillary groups) or bonds between the linker and the functional groups. Rigid
20 groups can include, for example, those groups whose conformational lability
is
restrained by the presence of rings and/or multiple bonds within the group,
for
example, aryl, heteroaryl, cycloalkyl, cycloalkenyl, and heterocyclic groups.
Other
groups which can impart rigidity include polypeptide groups such as oligo- or
polyproline chains.
25 Rigidity can also be imparted electrostatically. Thus, if the ancillary
groups
are either positively or negatively charged, the similarly charged ancillary
groups
will force the presenter linker into a configuration affording the maximum
distance
between each of the like charges. The energetic cost of bringing the like-
charged
groups closer to each other will tend to hold the linker in a configuration
that
30 maintains the separation between the like-charged ancillary groups. Further
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ancillary groups bearing opposite charges will tend to be attracted to their
oppositely charged counterparts and potentially may enter into both inter- and
intramolecular ionic bonds. This non-covalent mechanism will tend to hold the
linker into a conformation which allows bonding between the oppositely charged
groups. The addition of ancillary groups which are charged, or alternatively,
bear a
latent charge when deprotected, following addition to the linker, include
deprotectation of a carboxyl, hydroxyl, thiol or amino group by a change in
pH,
oxidation, reduction or other mechanisms known to those skilled in the art
which
result in removal of the protecting group, is within the scope of this
invention.
Rigidity may also be imparted by internal hydrogen bonding or by
hydrophobic collapse.
Bulky groups can include, for example, large atoms, ions (e.g., iodine,
sulfur, metal ions, etc.) or groups containing large atoms, polycyclic groups,
including aromatic groups, non-aromatic groups and structures incorporating
one or
more carbon-carbon multiple bonds (i.e., alkenes and alkynes). Bulky groups
can
also include oligomers and polymers which are branched- or straight-chain
species.
Species that are branched are expected to increase the rigidity of the
structure more
per unit molecular weight gain than are straight-chain species.
In preferred embodiments, rigidity is imparted by the presence of cyclic
20 groups (e.g., aryl, heteroaryl, cycloalkyl, heterocyclic, etc.). In other
preferred
embodiments, the linker comprises one or more six-membered rings. In still
further
preferred embodiments, the ring is an aryl group such as, for example, phenyl
or
naphthyl.
In view of the above, it is apparent that the appropriate selection of a
linker
25 group providing suitable orientation, restricted/unrestricted rotation, the
desired
degree of hydrophobicity/hydrophilicity, etc. is well within the skill of the
art.
Eliminating or reducing antigenicity of the multibinding compounds described
herein is also within the scope of this invention. In certain cases, the
antigenicity of
a multibinding compound may be eliminated or reduced by use of groups such as,
30 for example, polyethylene glycol).
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As explained above, the multibinding compounds described herein comprise
2-10 ligands attached to a linker that attaches the ligands in such a manner
that they
are presented to the enzyme for multivalent interactions with ligand binding
sites
thereon/therein. The linker spatially constrains these interactions to occur
within
5 dimensions defined by the linker. This and other factors increases the
biological
activity of the multibinding compound as compared to the same number of
ligands
made available in monobinding form.
The compounds of this invention are preferably represented by the empirical
Formula (L)P(X)q where L, X, p and q are as defined above. This is intended to
10 include the several ways in which the ligands can be linked together in
order to
achieve the objective of multivalency, and a more detailed explanation is
described
below.
As noted previously, the linker may be considered as a framework to which
ligands are attached. Thus, it should be recognized that the ligands can be
attached
15 at any suitable position on this framework, for example, at the termini of
a linear
chain or at any intermediate position.
The simplest and most preferred multibinding compound is a bivalent
compound which can be represented as L-X-L, where each L is independently a
ligand which may be the same or different and each X is independently the
linker.
20 Examples of such bivalent compounds are provided in FIG. 2 where each
shaded
circle represents a ligand. A trivalent compound could also be represented in
a
linear fashion, i.e., as a sequence of repeated units L-X-L-X-L, in which L is
a
ligand and is the same or different at each occurrence, as can X. However, a
trimer
can also be a radial multibinding compound comprising three ligands attached
to a
25 central core, and thus represented as (L)3X, where the linker X could
include, for
example, an aryl or cycloalkyl group. Illustrations of trivalent and
tetravalent
compounds of this invention are found in figures 2 and 3 respectively where,
again.
the shaded circles represent ligands. Tetravalent compounds can be represented
in a
linear array, e.g.,
30
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L-X-L-X-L-X-L
in a branched array, e.g.,
5
L-X-L-X-L
L
10 (a branched construct analogous to the isomers of butane -- n-butyl, iso-
butyl, sec-
butyl, and t-butyl) or in a tetrahedral array, e.g.,
L~ ~L
X
,~~' L
where X and L are as defined herein. Alternatively, it could be represented as
an
alkyl, aryl or cycloalkyl derivative as above with four (4) ligands attached
to the
15 core linker.
The same considerations apply to higher multibinding compounds of this
invention containing 5-10 ligands as illustrated in FIG. 4 where, as before,
the
shaded circles represent ligands. However, for multibinding agents attached to
a
central linker such as aryl or cycloalkyl, there is a self evident constraint
that there
20 must be sufficient attachment sites on the linker to accommodate the number
of
ligands present; for example, a benzene ring could not directly accommodate
more
than 6 ligands, whereas a multi-ring linker (e.g., biphenyl) could accommodate
a
larger number of ligands.
Certain of the above described compounds may alternatively be represented
25 as cyclic chains of the form:
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L1
X X
-L
and variants thereof.
All of the above variations are intended to be within the scope of the
invention defined by the Formula (L)P(X)q
With the foregoing in mind, a preferred linker may be represented by the
5 following:
_Xa_Z_(ya_Z)m_Xa-
wherein
m is an integer of from 0 to 20;
10 Xa at each separate occurrence is selected from the group consisting of
-O-, -S-, -NR-, -C(O)-, -C(O)O-, -C(O)NR-, -C(S), -C(S)O-, -C(S)NR- or a
covalent
bond where R is as defined below;
Z at each separate occurrence is selected from the group consisting of
alkylene, substituted alkylene, cycloalkylene, substituted cylcoalkylene,
alkenylene,
15 substituted alkenylene, alkynylene, substituted alkynylene,
cycloalkenylene,
substituted cycloalkenylene, arylene, heteroarylene, heterocyclene, or a
covalent
bond;
each Ye at each separate occurrence is selected from the group consisting of
-O-, -C(O)-, -OC(O)-, -C(O)O-, -NR-, -S(O)n-, -C(O)NR'-, -NR' C(O)-, -NR'
20 C(O)NR'-, -NR' C( S)NR'-, -C(=NR')-NR'-, -NR'-C(=NR')-, -OC(O)-NR'-, -NR'
C(O)-O-, -N=C(Xa)-NR'-, -NR'-C(Xa)=N-,-P(O)(OR')-O-, -O-P(O)(OR')-,
S(O)~CR' R"-, -S(O)"-NR'-, -NR'-S(O)~ -, -S-S-, and a covalent bond; where n
is 0,
1 or 2; and R, R' and R" at each separate occurrence are selected from the
group
consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted
cycloalkyl,
25 alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl,
alkynyl,
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substituted alkynyl, aryl, heteroaryl and heterocyclic provided that at least
one of
Xa, Z, and Ya is not a covalent bond.
Additionally, the linker moiety can be optionally substituted at any atom
therein by one or more alkyl, substituted alkyl, cycloalkyl, substituted
cycloalkyl,
alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl,
substituted alkynyl, aryl, heteroaryl and heterocyclic group.
In one embodiment of this invention, the linker (i.e., X, X' or X") is
selected those shown in Table II:
Table II
Representative Linkers
Linker
-(CHZ)~- where n is an integer of from 2-10
-(CH,) ~-O-(CHZ)z-O-(CH,) ~-
-CH,-Z-CH,- where Z is 1,4-phenylene
-(CHCO,Et)-(CHZ)2-(CHCOZEt)-
-CH,-Z-CH,- where Z is 2,3-quinoxaline
-(CHZ) -(CH=CH)-(CHZ)-
-(CHZ)-C(O)-C(O)-(CH~)-
-(CHZ) -(C_C)-(CHZ)-
-CHZ-Z-CHz- where Z is 1,2-phenylene
-(CHZ)-C(O)-(CHZ)-
-(CHz)-CH(COOH)-(CHZ)-
-(CHI)-CH(COOEt)-(CHZ)-
-(CHZ)-CH(OH)-(CHZ)-
-CH(CH3)-(CHz)-
-CH(CN)-(CHZ)-
-CH,-Z-CH,- where Z is 2,6-pyridyl
-(CH,)-C(=CH,)-(CHZ)-
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Linker
-(CH,)-CH(OH)-CH(OH)-(CH,)-
-(CH,) ,-N(CH3)-(CH,)~-
-(CH,) ,-O-(CH,),-
-(CH~)-Z-(CH~)-
where Z is 4-biphenyl
-CHZ-Z-CH,- where Z is 1,2-phenylene
-CHI-Z-CH,- where Z is 1,3-phenylene
-CHI-O-C(O)-NH-Z-NH-C(O)-O-CH,- where Z is 1,3-phenylene
-CHI-O-C(O)-(CHz)g-C(O)-O-CH~-
-CH,-O-C(O)-(CH~),o-C(O)-O-CH~-
-CHz-O-C(O)-Z-C(O)-O-CH,- where Z is 1,3-phenylene
-CHI-O-C(O)-(CH,)6-C(O)-O-CH~-
-CH,-O-C(O)-Z-C(O)-O-CHz- where Z is 1,4-phenylene
-CH,-O-C(O)-Z-C(O)-O-CHZ- where Z is 2,3-(5-norbornene)
In view of the above description of the linker, it is understood that the term
"linker" when used in combination with the term "multibinding compound"
includes both a covalently contiguous single linker (e.g., L-X-L) and multiple
covalently non-contiguous linkers (L-X-L-X-L) within the multibinding
compound.
Combinatorial Libraries
The methods described above lend themselves to combinatorial approaches
for identifying multimeric compounds which possess multibinding properties.
Specifically, factors such as the proper juxtaposition of the individual
25 ligands of a multibinding compound with respect to the relevant array of
binding
sites on a target or targets is important in optimizing the interaction of the
multibinding compound with its targets) and to maximize the biological
advantage
through multivalency. One approach is to identify a library of candidate
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multibinding compounds with properties spanning the multibinding parameters
that
are relevant for a particular target. These parameters include: ( 1 ) the
identity of
ligand(s), (2} the orientation of ligands, (3) the valency of the construct,
(4) linker
length, (5) linker geometry, (6) linker physical properties, and (7) linker
chemical
5 functional groups.
Libraries of multimeric compounds potentially possessing multibinding
properties (i.e., candidate multibinding compounds) and comprising a
multiplicity -
of such variables are prepared and these libraries are then evaluated via
conventional assays corresponding to the ligand selected and the multibinding
10 parameters desired. Considerations relevant to each of these variables are
set forth
below:
selection of li;g~(sl:
A single Iigand or set of ligands is (are) selected for incorporation into the
libraries of candidate multibinding compounds which library is directed
against a
15 particular biological target or targets e.g., bradykinin receptor. The only
requirement for the Iigands chosen is that they are capable of interacting
with the
selected target(s). Thus, ligands may be known drugs, modified forms of known
drugs, substructures of known drugs or substrates of modified forms of known
drugs (which are competent to interact with the target), or other compounds.
20 Ligands are preferably chosen based on known favorable properties that may
be
projected to be carned over to or amplified in multibinding forms. Favorable
properties include demonstrated safety and efficacy in human patients,
appropriate
PK/ADME profiles, synthetic accessibility, and desirable physical properties
such
as solubility, log P, etc. However, it is crucial to note that ligands which
display an
25 unfavorable property from among the previous list may obtain a more
favorable
property through the process of multibinding compound formation; i.e., ligands
should not necessarily be excluded on such a basis. For example, a ligand that
is
not sufficiently potent at a particular target so as to be efficacious in a
human
patient may become highly potent and efficacious when presented in
multibinding
30 form. A ligand that is potent and efficacious but not of utility because of
a non-
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mechanism-related toxic side effect may have increased therapeutic index
(increased potency relative to toxicity) as a multibinding compound. Compounds
that exhibit short in vivo half lives may have extended half lives as
multibinding
compounds. Physical properties of ligands that limit their usefulness (e.g.
poor
5 bioavailability due to low solubility, hydrophobicity, hydrophilicity) may
be
rationally modulated in multibinding forms, providing compounds with physical
properties consistent with the desired utility.
Orientatiow selection of ligand attachment dints and linkin~~chemistrv:
Several points are chosen on each ligand at which to attach the ligand to the
linker. The selected points on the ligand/linker for attachment are
functionalized to
contain complementary reactive functional groups. This permits probing the
effects of presenting the ligands to their receptors) in multiple relative
orientations,
an important multibinding design parameter. The only requirement for choosing
attachment points is that attaching to at least one of these points does not
abrogate
15 activity of the ligand. Such points for attachment can be identified by
structural
information when available. For example, inspection of a co-crystal structure
of a
protease inhibitor bound to its target allows one to identify one or more
sites where
linker attachment will not preclude the enzyme:inhibitor interaction.
Alternatively,
evaluation of ligand/target binding by nuclear magnetic resonance will permit
the
identification of sites non-essential for ligand/target binding. See, for
example,
Fesik, et al., U.S. Patent No. 5,891,643. When such structural information is
not
available, utilization of structure-activity relationships (SAR) for ligands
will
suggest positions where substantial structural variations are and are not
allowed. In
the absence of both structural and SAR information, a library is merely
selected
25 with multiple points of attachment to allow presentation of the ligand in
multiple
distinct orientations. Subsequent evaluation of this library will indicate
what
positions are suitable for attachment.
It is important to emphasize that positions of attachment that do abrogate the
activity of the monomeric ligand may also be advantageously included in
candidate
30 multibinding compounds in the library provided that such compounds bear at
least
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one ligand attached in a manner which does not abrogate intrinsic activity.
This
selection derives from, for example, heterobivalent interactions within the
context
of a single target molecule. For example, consider a receptor antagonist
ligand
bound to its target receptor, and then consider modifying this ligand by
attaching to
5 it a second copy of the same ligand with a linker which allows the second
ligand to
interact with the same receptor molecule at sites proximal to the antagonist
binding
site, which include elements of the receptor that are not part of the formal
antagonist
binding site and/or elements of the matrix surrounding the receptor such as
the
membrane. Here, the most favorable orientation for interaction of the second
10 ligand molecule with the receptor/matrix may be achieved by attaching it to
the
linker at a position which abrogates activity of the ligand at the formal
antagonist
binding site. Another way to consider this is that the SAR of individual
ligands
within the context of a multibinding structure is often different from the SAR
of
those same ligands in momomeric form.
15 The foregoing discussion focused on bivalent interactions of dimeric
compounds bearing two copies of the same ligand joined to a single linker
through
different attachment points, one of which may abrogate the binding/activity of
the
monomeric Iigand. It should also be understood that bivalent advantage may
also
be attained with heterodimeric constructs bearing two different ligands that
bind to
20 common or different targets. For example, a SHT4 receptor antagonist and a
bladder-selective muscarinic M3 antagonist may be joined to a linker through
attachment points which do not abrogate the binding affinity of the monomeric
ligands for their respective receptor sites. The dimeric compound may achieve
enhanced affinity for both receptors due to favorable interactions between the
SHT~
25 ligand and elements of the M3 receptor proximal to the formal M3 antagonist
binding site and between the M3 ligand and elements of the SHT4 receptor
proximal
to the formal SHT4 antagonist binding site. Thus, the dimeric compound may be
more potent and selective antagonist of overactive bladder and a superior
therapy
for urinary urge incontinence.
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Once the ligand attachment points have been chosen, one identifies the types
of chemical linkages that are possible at those points. The most preferred
types of
chemical linkages are those that are compatible with the overall structure of
the
ligand (or protected forms of the ligand) readily and generally formed, stable
and
5 intrinsically inocuous under typical chemical and physiological conditions,
and
compatible with a large number of available linkers. Amide bonds, ethers,
amines,
carbamates, ureas, and sulfonamides are but a few examples of preferred
linkages.
Linkers: spanning relevant multibinding parameters through selection of
valency.
linker length. l~, inker geo, metrr~. rigidity .physical properties. and
chemical functional
10 groups
In the library of linkers employed to generate the library of candidate
multibinding compounds, the selection of linkers employed in this library of
linkers
takes into consideration the following factors:
Valence:
15 In most instances the library of linkers is initiated with divalent
linkers. The
choice of ligands and proper juxtaposition of two ligands relative to their
binding
sites permits such molecules to exhibit target binding affinities and
specificities
more than sufficient to confer biological advantage. Furthermore, divalent
linkers
or constructs are also typically of modest size such that they retain the
desirable
20 biodistribution properties of small molecules.
Linker length:
Linkers are chosen in a range of lengths to allow the spanning of a range of
inter-ligand distances that encompass the distance preferable for a given
divalent
interaction. In some instances the preferred distance can be estimated rather
25 precisely from high-resolution structural information of targets, typically
enzymes
and soluble receptor targets. In other instances where high-resolution
structural
information is not available (such as 7TM G-protein coupled receptors), one
can
make use of simple models to estimate the maximum distance between binding
sites
either on adjacent receptors or at different locations on the same receptor.
In
30 situations where two binding sites are present on the same target (or
target subunit
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for multisubunit targets), preferred linker distances are 2-20 A, with more
preferred
linker distances of 3-12 ~1. In situations where two binding sites reside on
separate
(e.g., protein) target sites, preferred linker distances are 20-100 A, with
more
a
preferred distances of 30-70 A.
Linker eeometr~~ and rieiditw
The combination of ligand attachment site, linker length, linker geometry,
and linker rigidity determine the possible ways in which the ligands of
candidate
multibinding compounds may be displayed in three dimensions and thereby
presented to their binding sites. Linker geometry and rigidity are nominally
determined by chemical composition and bonding pattern, which may be
controlled
and are systematically varied as another spanning function in a multibinding
array.
For example, linker geometry is varied by attaching two ligands to the ortho,
meta,
and para positions of a benzene ring, or in cis- or trans-arrangements at the
1,1- vs.
1,2- vs. 1,3- vs. 1,4- positions around a cyclohexane core or in cis- or trans-
arrangements at a point of ethylene unsaturation. Linker rigidity is varied by
controlling the number and relative energies of different conformational
states
possible for the linker. For example, a divalent compound bearing two ligands
joined by 1,8-octyl linker has many more degrees of freedom, and is therefore
less
rigid than a compound in which the two ligands are attached to the 4,4'
positions of
a biphenyl linker.
I i~k~r ohvsicai properties:
The physical properties of linkers are nominally determined by the chemical
constitution and bonding patterns of the linker, and linker physical
properties
impact the overall physical properties of the candidate multibinding compounds
in
which they are included. A range of linker compositions is typically selected
to
provide a range of physical properties (hydrophobicity, hydrophilicity,
amphiphilicity, polarization, acidity, and basicity) in the candidate
multibinding
compounds. The particular choice of linker physical properties is made within
the
context of the physical properties of the ligands they join and preferably the
goal is
to generate molecules with favorable PK/ADME properties. For example, linkers
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can be selected to avoid those that are too hydrophilic or too hydrophobic to
be
readily absorbed and/or distributed in vivo.
I.in_ker chemical functional groups:
Linker chemical functional groups are selected to be compatible with the
5 chemistry chosen to connect linkers to the ligands and to impart the range
of
physical properties sufficient to span initial examination of this parameter.
Combinatorial synthesis:
Having chosen a set of n ligands (n being determined by the sum of the
10 number of different attachment points for each Iigand chosen) and m linkers
by the
process outlined above, a library of (n!)m candidate divalent multibinding
compounds is prepared which spans the relevant multibinding design parameters
for
a particular target. For example, an array generated from two ligands, one
which
has two attachment points (AI, A2) and one which has three attachment points
(B1,
15 B2, B3) joined in all possible combinations provide for at least 15
possible
combinations of multibinding compounds:
AI-A1 A1-A2 A1-B1 A1-B2 AI-B3 A2-A2 A2-B1 A2-B2
A2-B3 B1-BI B1-B2 BI-B3 B2-B2 B2-B3 B3-B3
20
When each of these combinations is joined by 10 different linkers, a library
of 150 candidate multibinding compounds results.
Given the combinatorial nature of the library, common chemistries are
preferably used to join the reactive functionalies on the ligands with
complementary
25 reactive functionalities on the linkers. The library therefore lends itself
to efficient
parallel synthetic methods. The combinatorial library can employ solid phase
chemistries well known in the art wherein the ligand and/or linker is attached
to a
solid support. Alternatively and preferably, the combinatorial libary is
prepared in
the solution phase. After synthesis, candidate multibinding compounds are
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optionally purified before assaying for activity by, for example,
chromatographic
methods (e.g., HPLC).
AnalXsis of array by biochemical analytical pharmacological and computational
~nelhods:
5 Various methods are used to characterize the properties and activities of
the
candidate multibinding compounds in the library to determine which compounds
possess multibinding properties. Physical constants such as solubility under
various
solvent conditions and logD/clogD values can be determined. A combination of
NMR spectroscopy and computational methods is used to determine low-energy
10 conformations of the candidate multibinding compounds in fluid media. The
ability
of the members of the library to bind to the desired target and other targets
is
determined by various standard methods, which include radioligand displacement
assays for receptor and ion channel targets, and kinetic inhibition analysis
for many
enzyme targets. Ira vitro efficacy, such as for receptor agonists and
antagonists, ion
15 channel blockers, and antimicrobial activity, can also. be determined.
Pharmacological data, including oral absorption, everted gut penetration,
other
pharmacokinetic parameters and efficacy data can be determined in appropriate
models. In this way, key structure-activity relationships are obtained for
multibinding design parameters which are then used to direct future work.
20 The members of the library which exhibit multibinding properties, as
defined herein, can be readily determined by conventional methods. First those
members which exhibit multibinding properties are identified by conventional
methods as described above including conventional assays (both in vitro and in
vivo).
25 Second, ascertaining the structure of those compounds which exhibit
multibinding properties can be accomplished via art recognized procedures. For
example, each member of the library can be encrypted or tagged with
appropriate
information allowing determination of the structure of relevant members at a
later
time. See, for example, Dower, et al., International Patent Application
Publication
30 No. WO 93/06121; Brenner, et al., Proc. Natl. Acad. Sci., USA, 89:5181
(1992);
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Gallop, et al., U.S. Patent No. 5,846,839; each of which are incorporated
herein by
reference in its entirety. Alternatively, the structure of relevant
multivalent
compounds can also be determined from soluble and untagged libaries of
candidate
multivalent compounds by methods known in the art such as those described by
Hindsgaul, et al., Canadian Patent Application No. 2,240,32 which was
published
on July 11, 1998. Such methods couple frontal affinity chromatography with
mass
spectroscopy to determine both the structure and relative binding affinities
of
candidate multibinding compounds to receptors.
The process set forth above for dimeric candidate multibinding compounds
can, of course, be extended to trimeric candidate compounds and higher analogs
thereof.
Follow up synthesis and analysis of additional arravlsl:
Based on the information obtained through analysis of the initial library, an
optional component of the process is to ascertain one or more promising
multibinding "lead" compounds as defined by particular relative ligand
orientations,
linker lengths, linker geometries, etc. Additional libraries can then be
generated
around these leads to provide for further information regarding structure to
activity
relationships. These arrays typically bear more focused variations in linker
structure in an effort to further optimize target affinity and/or activity at
the target
(antagonism, partial agonism, etc.), andlor alter physical properties. By
iterative
redesign/analysis using the novel principles of multibinding design along with
classical medicinal chemistry, biochemistry, and pharmacology approaches, one
is
able to prepare and identify optimal multibinding compounds that exhibit
biological
advantage towards their targets and as therapeutic agents.
To further elaborate upon this procedure, suitable divalent linkers include,
by way of example only, those derived from dicarboxylic acids,
disulfonylhalides,
dialdehydes, diketones, dihalides, diisocyanates,diamines, diols, mixtures of
carboxylic acids, sulfonylhalides, aldehydes, ketones, halides, isocyanates,
amines
and diols: In each case, the carboxylic acid, sulfonylhalide, aldehyde,
ketone,
halide, isocyanate, amine and diol functional group is reacted with a
complementary
CA 02319730 2000-08-02
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WO 99/64039
-74-
functionality on the ligand to form a covalent linkage. Such complementary
functionality is well known in the art as illustrated in the following table:
COMPLEMENTARY BINDING CHEMISTRIES
First Reactive Groun ~ec~nd Reactive GrounL' a
hydroxyl isocyanate urethane
amine epoxide ~i-hydroxyamine
hydroxyamine sulfonyl halide sulfonamide
carboxyl acid amine amide
hydroxyl alkyl/aryl halide ether
aldehyde amine/NaCNBH3 amine
ketone amine/NaCNBH3 amine
amine isocyanate urea
Exemplary linkers include the following linkers identified as X-1 through X-
418 as set forth below:
CA 02319730 2000-08-02
WO 99/64039 PCT/US99/12674 _
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o --___.-~-._-_. ....___ -__..... --... ~_..... ..
. _.-._._ .. .
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.
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p~.~C~ ~/W/~s' ~ y'i~o Np'\~'p np~'
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on nc ~" o
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w.
x.l x-I__._ _x.3._ _--_- x.'_- _ _.a~s.. x
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pr\/U\/~-~p o ~ a
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xas.e x.H x.If x~' _. .... x.'.
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p IC pip p ,_. ~ . w
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a
w w N,e ' ~~,--o ~'N~ ~L.
'~nv"~ ~ ' _~ ~~
e~ y
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e CN, ' /
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1 ee ve~/ No 1 1 np.._ a
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xsl X.aI x.~.-_ ~ x!!- xsf0 0 x-w
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ea; ~\-w o~
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f r o ~ o
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CA 02319730 2000-08-02
pC'f/US99/126~4
WO 99/64039 _~6_
ow " v/v a P
' "" o
.. ~. ~.:~-.~~i ~ tee.. - ~ e ~
, ~_ . ~ "
\s\.~". w~/ ../~on T ". ~...;
n.y i
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~ Ic-a ~_. x'~~__ .x.p_.... x.
___ ~ - . .
x-' -_ ... .
_ .
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xa ~._~_ -_ __-_ ~ o .,
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__ _ . .. ~ /~/t\/t\~'p N '
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, \ . ~N . ~ ~ 0 . .0
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ew en x~i x:w. X-os._.....x.w.
-_--' _ ___. _. .
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CA 02319730 2000-08-02
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WO 99/64039
-80-
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CA 02319730 2000-08-02
WO 99/64039 PCT/US99/12674
-81-
Representative ligands for use in this invention include, by way of example,
ligands of L-1 through L-3 wherein L-1 through L-3 are selected from the
compounds of formulae (a)-(c): LI= (a), L2= (b), and L3= (c).
Combinations of ligands (L} and linkers (X) per this invention include, by
way example only, homo- and hetero-dimers wherein the first ligand is selected
from L-1 through L-3 above and the second ligand and linker is selected from
the
following:
L-1/X-1- L-1/X-2- L-1/X-3- L-1/X-4- L-I/X-5- L-lIX-6-
1 L-1/X-7- L-1/X-8- L-1/X-9- L-1/X-10- L-1/X-I1-L-1/X-12-
0
L-1/X-13-L-1/X-14- L-1/X-15- L-1/X-16- L-I/X-17-L-1/X-18-
L-1/X-19-L-I/X-20- L-1/X-21- L-1/X-22- L-1/X-23-L-1/X-24-
L-1 lX-25-L-1 /X-26-L-1 /X-27-L-1 /X-2 L- I /X-29-L-1 /X-30-
8-
L-1/X-31-L-I/X-32- L-1/X-33- L-1/X-34- L-I/X-35-L-I/X-36-
1 L-1/X-37-L-1/X-38- L-I/X-39- L-1/X-40- L-1/X-41-L-1/X-42-
5
L-1/X-43-L-1/X-44- L-1/X-45- L-1/X-46- L-I/X-47-L-1/X-48-
L-1/X-49-L-1/X-50- L-1/X-51- L-I/X-52- L-1/X-53-L-I/X-54-
L-1 /X-5 L-1 /X-56-L-1 /X-57-L-1 /X-5 L- I /X-59-L-1 /X-60-
5- 8-
L-1/X-61-L-1/X-62- L-I/X-63- L-1/X-64- L-1/X-65-L-1/X-66-
20 L-I/X-67-L-1/X-68- L-1/X-69- L-1/X-70- L-1/X-71-L-l/X-72-
L-1/X-73-L-1/X-74- L-1/X-75- L-1/X-76- L-1/X-77-L-1/X-78-
L-1/X-79-L-I/X-80- L-1/X-81- L-1/X-82- L-1/X-83-L-1/X-84-
L-1/X-85-L-1/X-86- L-I/X-87- L-1/X-88- L-1/X-89-L-1/X-90-
L-IlX-91-L-1/X-92- L-1/X-93- L-1/X-94- L-I/X-95-L-I/X-96-
25 L-1/X-97-L-i/X-98- L-1/X-99- L-1/X-100-L-1/X-101-L-1/X-102-
L-1/X-103-L-1/X-104-L-1/X-105-L-1/X-106-L-l/X-107-L-I/X-108-
L-I/X-109-L-l/X-110-L-I/X-111-L-1/X-112-L-1/X-113-L-1/X-114-
L-1/X-115-L-1/X-116-L-1/X-117-L-1/X-118-L-1/X-119-L-1/X-120-
L-1/X-121-L-I/X-122-L-1/X-123-L-1/X-124-L-I/X-125-L-1/X-126-
30 L-l/X-127-L-1/X-128-L-t/X-129-L-1/X-130-L-1/X-131-L-I/X-132-
L-1/X-133-L-1/X-134-L-1/X-135-L-lr'X-136-L-1/X-137-L-1/X-138-
L-1/X-139-L-1/X-140-L-1/X-141-L-1/X-142-L-1/X-143-L-1/X-144-
L-I/X-145-L-1/X-146-L-1/X-147-L-1/X-148-L-I/X-149-L-1/X-l50-
L-I/X-151-L-I/X-152-L-1/X-153-L-1/X-154-L-1/X-155-L-1/X-156-
CA 02319730 2000-08-02
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-82-
L-I/X-157-L-I/X-158-L-1/X-159-L-1/X-160-L-1/X-161-L-1/X-162-
L-1/X-I63-L-I/X-164-L-1/X-165-L-1/X-166-L-1/X-167-L-I/X-168-
L-1/X-169-L-1/X-170-L-i/X-171-L-1/X-172-
L-1/X-173-L-1/X-174-L-1/X-175-L-I/X-176-L-1/X-177-L-I/X-178-
5 L-1/X-179-L-1/X-180-L-1/X-181-L-1/X-182-L-1/X-183-L-1/X-184-
L-1/X-185-L-1/X-186-L-1/X-187-L-1/X-188-L-I/X-189-L-1/X-190-
L-1/X-191-L-1/X-192-L-1/X-193-L-1/X-194-L-1/X-195-L-1/X-196-
L-1/X-197-L-1/X-198-L-1/X-199-L-1/X-200-L-I/X-201-L-I/X-202-
L-1/X-203-L-1/X-204-L-1/X-205-L-1/X-206-L-1/X-207-L-1/X-208-
1 L-1/X-209-L-I/X-210-L-1/X-211-L-I/X-212-L-1/X-213-L-1/X-2l4-
0
L-1/X-215-L-1/X-216-L-1/X-217-L-t/X-218-L-1/X-219-L-1/X-220-
L-1/X-221-L-I/X-222-L-1/X-223-L-1/X-224-L-I/X-225-L-1/X-226-
L-1/X-227-L-1/X-228-L-1/X-229-L-1/X-230-L-1/X-231-L-I/X-232-
L-1/X-233-L-1/X-234-L-1/X-235-L-1/X-236-L-1/X-237-L-1/X-238-
1 L-1/X-239-L-1/X-240-L-l/X-241-L-1/X-242-L-1/X-243-L-1/X-244-
S
L-1/X-245-L-1/X-246-L-1/X-247-L-1/X-248-L-1/X-249-L-I/X-250-
L-1/X-251-L-1/X-252-L-I/X-253-L-I/X-254-L-I/X-255-L-1/X-256-
L-1/X-257-L-1/X-258-L-1/X-259-L-1/X-260-L-1/X-261-L-I/X-262-
L-1/X-263-L-l/X-264-L-1/X-265-L-I/X-266-L-1/X-267-L-11X-268-
20 L-I/X-269-L-i/X-270-L-1/X-271-L-1/X-272-L-1/X-273-L-1/X-274-
L-1/X-275-L-1/X-276-L-1/X-277-L-I/X-278-L-i/X-279-L-1/X-280-
L-1/X-281-L-I/X-282-L-1/X-283-L-1/X-284-L-I/X-285-L-1/X-286-
L-1/X-287-L-1/X-288-L-I/X-289-L-1/X-290-L-I/X-291-L-1/X-292-
L-1/X-293-L-1/X-294-L-1/X-295-L-1/X-296-L-1/X-297-L-1/X-298-
25 L-1/X-299-L-I/X-300-L-I/X-301-L-1/X-302-L-1/X-303-L-1/X-304-
L-I/X-305-L-1/X-306-L-1/X-307-L-1/X-308-L-1/X-309-L-1/X-310-
L-1/X-311-L-1/X-312-L-1/X-313-L-I/X-314-L-1/X-315-L-1/X-316-
L-1/X-317-L-1/X-3I8-L-I/X-319-L-I/X-320-L-I/X-321-L-I/X-322-
L-l/X-323-L-1/X-324-L-1/X-325-L-I/X-326-L-1/X-327-L-IlX-328-
30 L-1/X-329-L-1/X-330-L-1/X-331-L-1/X-332-L-1/X-333-L-1/X-334-
L-1/X-335-L-1/X-336-L-1/X-337-L-1/X-338-L-I/X-339-L-1/X-340-
L-1/X-341-L-1/X-342-L-l/X-343-L-1/X-344-L-1/X-345-L-I/X-346-
L-1/X-347-L-1/X-348-L-I/X-349-L-1/X-350-L-1/X-351-L-1/X- 352-
L-I/X-353-L-1/X-354-L-1/X-355-L-I/X-356-L-1/X-357-L-I/X-358-
3 L-1/X-359-L-1/X-360-L-l/X-361-L-1/X-362-L-1/X-363-L-1/X-364-
5
L-1/X-365-L-1/X-366-L-1/X-367-L-1/X-368-L-I/X-369-L-1/X-370-
CA 02319730 2000-08-02
WO 99/64039 PCT/US99/12674
-83-
L-1/X-371-L-1/X-372-L-1/X-373-L-1/X-374-L-1/X-375-L-1/X-376-
L-I/X-377-L-I/X-378-L-I/X-379-L-1/X-380-L-1/X-381-L-1/X-382-
L-I/X-383-L-I/X-384-L-1/X-385-L-1/X-386-L-1/X-387-L-1/X-388-
L-1/X-389-L-1/X-390-L-1/X-391-L-1/X-392-L-1/X-393-L-I/X-394-
L-1/X-395-L-1/X-396-L-1/X-397-L-I/X-398-L-1/X-399-L-1/X-400-
L-I/X-401-L-I/X-402-L-1/X-403-L-I/X-404-L-1/X-405-L-1/X-406-
L-t/X-407-L-1/X-408-L-1/X-409-L-1/X-410-L-1/X-411-L-I/X-412-
L-1/X-413-L-1/X-414-L-1/X-415-L-1/X-416-L-I/X-417-L-1/X-418-
1 L-2/X-1- L-2/X-2- L-2/X-3- L-2/X-4- L-2/X-5- L-2/X-6-
0
L-2/X-7- L-2/X-8- L-2/X-9- L-2/X-10- L-2/X-11- L-2/X-i2-
L-2/X-13- L-2/X-14-L-2/X-15- L-2/X-16- L-2/X-17- L-2/X-18-
L-2/X-i9- L-2/X-20-L-2/X-21- L-2/X-22- L-2/X-23- L-2/X-24-
L-2/X-25- L-2/X-26-L-2/X-27- L-2/X-28- L-2/X-29- L-2/X-30-
1 L-2/X-31- L-2/X-32-L-2/X-33- L-2/X-34- L-2/X-35- L-2/X-36-
5
L-2/X-37- L-2/X-38-L-2/X-39- L-2/X-40- L-2/X-41- L-2/X-42-
L-2/X-43- L-2/X-44-L-2/X-45- L-2/X-46- L-2/X-47- L-2/X-48-
L-2/X-49- L-2/X-50-L-2/X-51- L-2/X-52- L-2/X-53- L-2/X-54-
L-2/X-55- L-2/X-56-L-2/X-57- L-2/X-58- L-2/X-59- L-2/X-60-
20 L-2/X-61- L-2/X-62-L-2/X-63- L-2/X-64- L-2/X-65- L-2/X-66-
L-2/X-67- L-2/X-68-L-2/X-69- L-2/X-70- L-2/X-71- L-2/X-72-
L-2/X-73- L-2/X-74-L-2/X-75- L-2/X-76- L-2/X-?7- L-2/X-78-
L-2/X-79- L-2/X-80-L-2/X-81- L-2/X-82- L-2/X-83- L-2/X-84-
L-2/X-85- L-2/X-86-L-2/X-87- L-2/X-88- L-2/X-89- L-2/X-90-
25 L-2/X-91- L-2/X-92-L-2/X-93- L-2/X-94- L-2/X-95- L-2/X-96-
L-2/X-97- L-2/X-98-L-2/X-99- L-2/X-100-L-2/X-101-L-2/X-102-
L-2/X-103-L-2/X-104-L-2/X-105-L-2/X-106-L-2/X-107-L-2/X-108-
L-2/X-109-L-2/X-110-L-2/X-111-L-2/X-112-L-2/X-113-L-2/X-114-
L-2lX-115-L-2/X-116-L-2/X-117-L-2/X-118-L-2/X-119-L-2/X-120-
30 L-2/X-121-L-2/X-122-L-2/X-123-L-2/X-124-L-2/X-125-L-2/X-126-
L-2/X-127-L-2/X-128-L-2/X-129-L-2/X-130-L-2/X-131-L-2/X-132-
L-2/X-133-L-2/X-134-L-2/X-135-L-2/X-136-L-2/X-137-L-2/X-I38-
L-2/X-139-L-2/X-140-L-2/X-141-L-2/X-142-L-2/X-143-L-2/X-i44-
L-2/X-145-L-2/X-146-L-2/X-147-L-2/X-148-L-2/X-149-L-2/X-150-
3 L-2/X-151-L-2/X-152-L-2/X-153-L-2/X-154-L-2/X-155-L-2/X-156-
5
L-2/X-157-L-2/X-158-L-2/X-159-L-2/X-160-L-2/X-161-L-2/X-162-
CA 02319730 2000-08-02
WO 99/64039 PCT/US99/12674
-84-
L-2/X-163-L-2/X-164-L-2/X-165-L-2/X-166-L-2/X-167-L-2/X-168-
L-2/X-169-L-2/X-170-L-2/X-171-L-2/X-172-
L-2/X-173-L-2/X-174-L-2/X-175-L-2/X-176-L-2/X-177-L-2/X-178-
L-2/X-179-L-2/X-180-L-2/X-181-L-2/X-182-L-2/X-183-L-2/X-184-
L-2/X-185-L-2/X-186-L-2/X-187-L-2/X-188-L-2/X-189-L-2/X-190-
L-2/X-191-L-2/X-192-L-2/X-193-L-2/X-194-L-2/X-195-L-2/X-196-
L-2/X-197-L-2/X-198-L-2/X-199-L-2/X-200-L-2/X-201-L-2/X-202-
L-2/X-203-L-2/X-204-L-2/X-205-L-2/X-206-L-2/X-207-L-2!X-208-
L-2/X-209-L-2/X-210-L-2/X-211-L-2!X-212-L-2/X-213-L-2/X-214-
1 L-2/X-215-L-2/X-216-L-2/X-217-L-2/X-218-L-2/X-219-L-2/X-220-
0
L-2/X-221-L-2/X-222-L-2/X-223-L-2/X-224-L-2/X-225-L-2/X-226-
L-2/X-227-L-2/X-228-L-2/X-229-L-2/X-230-L-2/X-231-L-2/X-232-
L-2/X-233-L-2/X-234-L-2/X-235-L-2/X-236-L-2/X-237-L-2/X-238-
L-2/X-239-L-2/X-240-L-2/X-241-L-2/X-242-L-2/X-243-L-2/X-244-
L-2/X-245-L-2/X-246-L-2/X-247-L-2/X-248-L-2/X-249-L-2/X-250-
L-2/X-251-L-2/X-252-L-2/X-253-L-2/X-254-L-2/X-255-L-2/X-256-
L-2/X-257-L-2/X-258-L-2/X-259-L-2/X-260-L-2/X-261-L-2/X-262-
L-2/X-263-L-2/X-264-L-2/X-265-L-21X-266-L-2/X-267-L-2/X-268-
L-2/X-269-L-2/X-270-L-2/X-271-L-2/X-272-L-2/X-273-L-2/X-274-
L-2/X-275-L-2/X-276-L-2/X-277-L-2/X-278-L-2/X-279-L-2/X-280-
L-2/X-281-L-2/X-282-L-2/X-283-L-2/X-284-L-2/X-285-L-2/X-286-
L-2/X-287-L-2/X-288-L-2/X-289-L-2/X-290-L-2/X-291-L-2/X-292-
L-2/X-293-L-2/X-294-L-2/X-295-L-2/X-296-L-2/X-297-L-2/X-298-
L-2/X-299-L-2/X-300-L-2/X-301-L-2/X-302-L-2/X-303-L-2/X-304-
L-2/X-305-L-2/X-306-L-2/X-307-L-2/X-308-L-2/X-309-L-2/X-310-
L-2/X-311-L-2/X-312-L-2/X-313-L-2/X-314-L-2/X-315-L-2/X-316-
L-2/X-317-L-2/X-318-L-2/X-319-L-2/X-320-L-2/X-321-L-2/X-322-
L-2/X-323-L-2/X-324-L-2/X-325-L-2/X-326-L-2/X-327-L-2/X-328-
L-2/X-329-L-2/X-330-L-2/X-331-L-2/X-332-L-2/X-333-L-2/X-334-
L-2/X-335-L-2/X-336-L-2/X-337-L-2/X-338-L-2/X-339-L-2/X-340-
L-2/X-341-L-2/X-342-L-2/X-343-L-2/X-344-L-2/X-345-L-2/X-346-
L-2/X-347-L-2/X-348-L-2/X-349-L-2/X-350-L-2/X-351-L-2/X-352-
L-2/X-353-L-2/X-354-L-2/X-355-L-2/X-356-L-2/X-357-L-2/X-358-
L-2/X-359-L-2/X-360-L-2/X-361-L-2/X-362-L-2/X-363-L-2/X-364-
L-2/X-365-L-2/X-366-L-2/X-367-L-2/X-368-L-2/X-369-L-2/X-370-
L-2/X-371-L-2/X-372-L-2/X-373-L-2/X-374-L-2/X-375-L-2/X-376-
CA 02319730 2000-08-02
WO 99/64039 PCTNS99/12674
-85-
L-2/X-377-L-2/X-378-L-2/X-379-L-2/X-380-L-2/X-38i-L-2/X-382-
L-2/X-383-L-2/X-384-L-2/X-385-L-2/X-386-L-2/X-387-L-2/X-388-
L-2/X-389-L-2/X-390-L-2/X-391-L-2/X-392-L-2/X-393-L-2/X-394-
L-2/X-395-L-2/X-396-L-2/X-397-L-2/X-398-L-2/X-399-L-2/X-400-
5 L-2/X-401-L-2/X-402-L-2/X-403-L-2/X-404-L-2/X-405-L-2/X-406-
L-2/X-407-L-2/X-408-L-2/X-409-L-2/X-410-L-2/X-41 L-2/X-412-
1-
L-2/X-413-L-2/X-414-L-2/X-415-L-2/X-416-L-2/X-417-L-2/X-418-
L-3/X-1- L-3/X-2- L-3/X-3- L-3/X-4- L-3/X-5- L-3/X-6-
1 L-3/X-7- L-3/X-8- L-3/X-9- L-3/X-10- L-3/X-11-L-3/X-12-
0
L-3/X-13- L-3/X-14- L-3/X-15- L-3/X-16- L-3/X-17-L-3/X-t
8-
L-3/X-19- L-3/X-20- L-3/X-21- L-3/X-22- L-3/X-23-L-3/X-24-
L-3/X-25- L-3/X-26- L-3/X-27- L-3/X-28- L-3/X-29-L-3/X-30-
L-3/X-31- L-3/X-32- L-3/X-33- L-3/X-34- L-3/X-35-L-3/X-36-
1 L-3/X-37- L-3/X-38- L-3/X-39- L-3/X-40- L-3/X-41-L-3/X-42-
5
L-3/X-43- L-3/X-44- L-3/X-45- L-3/X-46- L-3/X-47-L-3/X-48-
L-3/X-49- L-3/X-50- L-3/X-51- L-3/X-52- L-3/X-53-L-3/X-54-
L-3/X-55- L-3/X-56- L-3lX-57- L-3/X-58- L-3/X-59-L-3/X-60-
L-3/X-61- L-3/X-62- L-3/X-63- L-3/X-64- L-3/X-65-L-3/X-66-
20 L-3/X-67- L-3lX-68- L-3/X-69- L-3/X-70- L-3/X-71-L-3/X-72-
L-3/X-73- L-3/X-74- L-3IX-75- L-3/X-76- L-3/X-77-L-3/X-78-
L-3/X-79- L-3/X-80- L-3/X-81- L-3/X-82- L-3/X-83-L-3/X-84-
L-3/X-85- L-3/X-86- L-3/X-87- L-3/X-88- L-3/X-89-L-3/X-90-
L-3/X-91- L-3/X-92- L-3/X-93- L-3/X-94- L-3/X-95-L-3/X-96-
25 L-3/X-97- L-31X-98- L-3/X-99- L-3/X-100-L-3/X-101-L-3/X-102-
L-3/X-103-L-3/X-104-L-3/X-105-L-3/X-106-L-3/X-107-L-3/X-108-
L-3/X-109-L-3IX-I10-L-3/X-111-L-3/X-112-L-3/X-113-L-3/X-114-
L-3/X-115-L-3/X-116-L-3/X-117-L-3/X-118-L-3/X-119-L-3IX-120-
L-3/X-121-L-3/X-122-L-3/X-123-L-3/X-124-L-3/X-125-L-3/X-126-
30 L-3!X-127-L-3/X-128-L-3lX-l29-L-3/X-130-L-3/X-131-L-3/X-132-
L-3/X-133-L-3/X-134-L-3/X-135-L-3/X-136-L-3IX-137-L-3/X-138-
L-3/X-139-L-3IX-140-L-3/X-141-L-3/X-142-L-3/X-143-L-3/X-144-
L-3/X-145-L-3/X-146-L-3/X-147-L-3/X-148-L-3/X-149-L-3/X-150-
L-31X-151-L-3/X-152-L-3/X-153-L-3/X-154-L-3/X-155-L-3/X-156-
3 L-3/X-157-L-3/X-158-L-3/X-159-L-3/X-160-L-3/X-161-L-3/X-162-
S
CA 02319730 2000-08-02
WO 99/64039 PCT/US99/12674
-86-
L-3/X-163-L-3/X-164-L-3/X-165-L-3/X-166-L-3/X-167-L-3/X-168-
L-3/X-169-L-3/X-170-L-3/X-171-L-31X-172-
L-3/X-173-L-3/X-174-L-3/X-175-L-31X-176-L-3/X-177-L-3/X-178-
L-3/X-179-L-3/X-180-L-3/X-181-L-3/X-182-L-3/X-183-L-3/X-184-
S L-3/X-185-L-3/X-186-L-3/X-187-L-3/X-188-L-3/X-189-L-3/X-190-
L-3/X-191-L-3/X-192-L-3/X-193-L-3/X-194-L-3/X-195-L-3/X-196-
L-3/X-197-L-3/X-198-L-3/X-199-L-3/X-200-L-3/X-201-L-3/X-202-
L-3/X-203-L-3/X-204-L-3/X-205-L-3/X-206-L-3/X-207-L-3/X-208-
L-3/X-209-L-3/X-210-L-3/X-211-L-3/X-212-L-3/X-213-L-3/X-2l4-
10 L-3/X-215-L-3/X-216-L-3/X-217-L-3/X-218-L-3/X-2l9-L-3/X-220-
L-3/X-221-L-3/X-222-L-3/X-223-L-3/X-224-L-3/X-225-L-3/X-226-
L-3/X-227-L-3/X-228-L-3/X-229-L-3!X-230-L-3/X-231-L-3/X-232-
L-3/X-233-L-3/X-234-L-3/X-235-L-3/X-236-L-3/X-237-L-3/X-238-
L-3/X-239-L-3/X-240-L-3/X-241-L-3/X-242-L-3/X-243-L-3/X-244-
1 L-3/X-245-L-3/X-246-L-3/X-247-L-3/X-248-L-3/X-249-L-3/X-250-
5
L-3/X-251-L-3/X-252-L-3/X-253-L-3/X-2S4-L-3/X-25S-L-3/X-2S6-
L-3/X-257-L-3/X-258-L-3/X-259-L-3/X-260-L-3/X-261-L-3/X-262-
L-3/X-263-L-3/X-264-L-3/X-265-L-3/X-266-L-3/X-267-L-3/X-268-
L-3/X-269-L-3/X-270-L-3/X-271-L-3/X-272-L-3/X-273-L-3/X-274-
20 L-3/X-275-L-3/X-276-L-3/X-277-L-3/X-278-L-3/X-279-L-3/X-280-
L-3/X-281-L-3/X-282-L-3/X-283-L-3/X-284-L-3/X-285-L-3/X-286-
L-3/X-287-L-3/X-288-L-3/X-289-L-3/X-290-L-3/X-291-L-3/X-292-
L-3/X-293-L-3/X-294-L-3/X-295-L-3/X-296-L-3/X-297-L-3/X-298-
L-3/X-299-L-3/X-300-L-3/X-301-L-3/X-302-L-3/X-303-L-3/X-304-
25 L-3/X-305-L-3/X-306-L-3/X-307-L-3/X-308-L-3/X-309-L-3/X-310-
L-3/X-311-L-3/X-312-L-3/X-313-L-3/X-314-L-3/X-315-L-3/X-316-
L-3/X-317-L-3/X-318-L-3/X-319-L-3/X-320-L-3/X-321-L-3/X-322-
L-3/X-323-L-3/X-324-L-3/X-325-L-3/X-326-L-3/X-327-L-3/X-328-
L-3/X-329-L-3/X-330-L-3/X-331-L-3/X-332-L-3/X-333-L-3/X-334-
30 L-3/X-335-L-3/X-336-L-3/X-337-L-3/X-338-L-3/X-339-L-3/X-340-
L-3/X-341-L-3/X-342-L-3/X-343-L-3/X-344-L-3/X-345-L-3/X-346-
L-3/X-347-L-3/X-348-L-3/X-349-L-3/X-350-L-3/X-351-L-3/X-352-
L-3/X-353-L-3/X-3S4-L-3/X-355-L-3/X-356-L-3/X-357-L-3/X-358-
L-3/X-359-L-3/X-360-L-3/X-361-L-3/X-362-L-3/X-363-L-3/X-364-
35 L-3/X-365-L-3/X-366-L-3/X-367-L-3/X-368-L-3/X-369-L-3/X-370-
L-3/X-371-L-3/X-372-L-3/X-373-L-3/X-3?4-L-3/X-375-L-3/X-376-
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L-3/X-377- L-3/X-378-L-3/X-379-L-3/X-380-L-3/X-381-L-3/X-382-
L-3/X-383- L-3/X-384-L-3/X-385-L-3/X-386-L-3/X-387-L-3/X-388-
L-3/X-389- L-3/X-390-L-3/X-391-L-3/X-392-L-3/X-393-L-3/X-394-
L-3/X-395- L-3/X-396-L-3/X-397-L-3/X-398-L-3/X-399-L-3/X-400-
S L-3/X-401-L-3/X-402-L-3/X-403-L-3/X-404-L-3/X-405-L-3/X-406-
L-3/X-407- L-3/X-408-L-3JX-409-L-3/X-410-L-3/X-411-L-3/X-412-
L-3/X-413- L-3/X-414-L-3/X-415-L-3/X-416-L-3/X-417-L-3/X-418-
10 Utility, Testing, and Administration
i 't
The multibinding compounds of this invention are bradykinin antagonists.
Accordingly, the multibinding compounds and pharmaceutical compositions of
this
invention are useful in the treatment and prevention of diseases mediated by
15 bradydinin such as cancer, pain, shock, asthma, rhinitis, arthritis,
inflammatory
bowel disease, and the like.
Te i
The in vitro bradykinin antagonist activity of the compounds of Formula (I)
20 may be tested by the assay described in Example 24. The effectiveness of
the
compounds of Formula (I} in inhibiting bradykinin-induced brochoconstriction
can
be tested using an asthma model as described in Example 25. The effectiveness
of
the compounds of Formula (I) in inhibiting bradykinin-induced inflammation can
be
tested using the carrageenin-induced paw edema model as described in Example
26
25 and bradykinin-induced pancreatitis can be tested using Caerulein-induced
pancreatitis model as described in Example 27.
Pharmaceutical Formulations
When employed as pharmaceuticals, the compounds of this invention are
30 usually administered in the form of pharmaceutical compositions. These
compounds can be administered by a variety of routes including oral, rectal,
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transdermal, subcutaneous, intravenous, intramuscular, and intranasal. These
compounds are effective as both injectable and oral compositions. Such
compositions are prepared in a manner well known in the pharmaceutical art and
comprise at least one active compound.
This invention also includes pharmaceutical compositions which contain, as
the active ingredient, one or more of the compounds described herein
associated
with pharmaceutically acceptable carriers. In making the compositions of this
invention, the active ingredient is usually mixed with an excipient, diluted
by an
excipient or enclosed within such a carrier which can be in the form of a
capsule,
sachet, paper or other container. When the excipient serves as a diluent, it
can be a
solid, semi-solid, or liquid material, which acts as a vehicle, carrier or
medium for
the active ingredient. Thus, the compositions can be in the form of tablets,
pills,
powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions,
solutions;
syrups, aerosols (as a solid or in a liquid medium), ointments containing, for
15 example, up to 10% by weight of the active compound, soft and hard gelatin
capsules, suppositories, sterile injectable solutions, and sterile packaged
powders.
In preparing a formulation, it may be necessary to mill the active
compound to provide the appropriate particle size prior to combining with the
other
ingredients. If the active compound is substantially insoluble, it ordinarily
is milled
20 to a particle size of less than 200 mesh. If the active compound is
substantially
water soluble, the particle size is normally adjusted by milling to provide a
substantially uniform distribution in the formulation, e.g. about 40 mesh.
Some examples of suitable excipients include lactose, dextrose, sucrose,
sorbitol; mannitol, starches, gum acacia, calcium phosphate, alginates,
tragacanth,
25 gelatin, calcium silicate, microcrystalline cellulose,
polyvinylpyrrolidone, cellulose,
sterile water, syrup, and methyl cellulose. The formulations can additionally
include: lubricating agents such as talc, magnesium stearate, and mineral oil;
wetting agents; emulsifying and suspending agents; preserving agents such as
methyl- and propylhydroxy-benzoates; sweetening agents; and flavoring agents.
30 The compositions of the invention can be formulated so as to provide quick,
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sustained or delayed release of the active ingredient after administration to
the
patient by employing procedures known in the art.
The compositions are preferably formulated in a unit dosage form, each
dosage containing from about 0.001 to about 1 g, more usually about 1 to about
30
5 mg, of the active ingredient. The term "unit dosage forms" refers to
physically
discrete units suitable as unitary dosages for human subjects and other
mammals,
each unit containing a predetermined quantity of active material calculated to
produce the desired therapeutic effect, in association with a suitable
pharmaceutical
excipient. Preferably, the compound of Formula (I) above is employed at no
more
10 than about 20 weight percent of the pharmaceutical composition, more
preferably
no more than about 15 weight percent, with the balance being pharmaceutically
inert carrier(s).
The active compound is effective over a wide dosage range and is generally
administered in a pharmaceutically effective amount. It, will be understood,
15 however, that the amount of the compound actually administered will be
determined
by a physician, in the light of the relevant circumstances, including the
condition to
be treated, the chosen route of administration, the actual compound
administered
and its relative activity, the age, weight, and response of the individual
patient, the
severity of the patient's symptoms, and the like.
20 For preparing solid compositions such as tablets, the principal active
ingredient is mixed with a pharmaceutical excipient to form a solid
preformulation
composition containing a homogeneous mixture of a compound of the present
invention. When referring to these preformulation compositions as homogeneous,
it
is meant that the active ingredient is dispersed evenly throughout the
composition
25 so that the composition may be readily subdivided into equally effective
unit dosage
forms such as tablets, pills and capsules. This solid preformulation is then
subdivided into unit dosage forms of the type described above containing from,
for
example, 0.1 to about 500 mg of the active ingredient of the present
invention.
The tablets or pills of the present invention may be coated or otherwise
30 compounded to provide a dosage form affording the advantage of prolonged
action.
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For example, the tablet or pill can comprise an inner dosage and an outer
dosage
component, the latter being in the form of an envelope over the former. The
two
components can be separated by an enteric layer which serves to resist
disintegration in the stomach and permit the inner component to pass intact
into the
5 duodenum or to be delayed in release. A variety of materials can be used for
such
enteric layers or coatings, such materials including a number of polymeric
acids and
mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and
cellulose acetate.
The liquid forms in which the novel compositions of the present invention
10 may be incorporated for administration orally or by injection include
aqueous
solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored
emulsions with edible oils such as corn oil, cottonseed oil, sesame oil,
coconut oil,
or peanut oil, as well as elixirs and similar pharmaceutical vehicles.
Compositions for inhalation or insufflation include solutions and
15 suspensions in pharmaceutically acceptable, aqueous or organic solvents, or
mixtures thereof, and powders. The liquid or solid compositions may contain
suitable pharmaceutically acceptable excipients as described supra. Preferably
the
compositions are administered by the oral or nasal respiratory route for local
or
systemic effect. Compositions in preferably pharmaceutically acceptable
solvents
20 may be nebulized by use of inert gases. Nebulized solutions may be inhaled
directly from the nebulizing device or the nebulizing device may be attached
to a
face mask tent, or intermittent positive pressure breathing machine. Solution,
suspension, or powder compositions may be administered, preferably orally or
nasally, from devices which deliver the formulation in an appropriate manner.
25
EXAMPLES
The following preparations and examples are given to enable those skilled in
the art to more clearly understand and to practice the present invention. They
should not be considered as limiting the scope of the invention, but merely as
being
30 illustrative and representative thereof.
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In the examples below, the following abbreviations have the following
meanings. Unless otherwise stated, all temperatures are in degrees Celsius. If
an
abbreviation is not defined, it has its generally accepted meaning.
5 DMF - N,N dimethylformamide
g - gram
HPLC - high performance liquid chromatography
mg - milligram
ml - milliliter
10 mmol - millimol
N - normal
THF - tetrahydrofuran
~g - micrograms
Na,C03 - sodium carbonate
15 Na~S04 - sodium sulfate
NaOH - sodium hydroxide
KZC03 - sodium carbonate
NaH - sodium hydride
TLC - thin layer chromatography
20 EtOH - ethanol
NaHC03 - sodium bicarbonate
Pd/C - palladium on carbon
MeCN - acetonitrile
HCI - hydrochloric acid
25
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Synthetic Exam I~e~
Example 1
(Following Fig. 8)
Preparation of a comnou_nd of Formula I wherein p is 2 ands is 1 and the
ligands
5 L, are a com>'ound of formula fIII (where R''' is 2-meth~quinolin-8 yl and
R'~ and
R'S are chlorol which are li ked via the terminal amine nitrogen
I ~ ~ i i
N
N
O O
CI I ~ CIO H OC! , CI
H
i N~N~-- ~
N~N W
CH3 CH3
A solution of compound (II) (where R" is 2-methylquinolin-8-yl and R'~
and R'S are chloro) (2 mmols), triethylamine (2 mmols), and a dibromo linker
molecule (1 mmol) in DMF (5 ml) is warmed. The course of the reaction is
10 followed by thin layer chromatography. After reaction occurs, the reaction
solution
is quenched in water and the aqueous mixture is extracted with methylene
chloride.
The organic layer is dried (Na,SO,), filtered, and concentrated under reduced
pressure to give the crude product. The desired compound is obtained by
purification of the crude product by use of HPLC.
15
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Ex 2
(Following Fig. 8)
P_~gnaration of a compound of Formula I wherein n i 2 and a is 1 and the
ligands_
L are a compound of formula~IIl lwhere R''' is 2-bromo-3-methylimidazo(1 2
5 ~)py it din-8-yl and R'4 and R'S are chloro) which are linked via the
terminal amine
nitrogen
Br Br
,N~ ~N
i N N w
O O
CI ~ Cf O OCI , CI
N~N ~ ~ NH~N ~
n
CH3 O O CH3
A solution of compound (II) (where R'~ is 2-bromo-3-methylimidazo( 1,2-
a)pyridin-8-yl and R'~ and R'S are Cl) (2 mmols) and benzene-1,4-bisacetic
acid (1
mmol) in methylene chloride (5 ml) is prepared under argon in a flask equipped
10 with magnetic stirrer and a drying tube. To this solution is added
dicyclohexylcarbodiimide (solid, 2.1 mmols). The course of the reaction is
followed by thin layer chromatography. After reaction occurs, the reaction
solution
is quenched in water and the aqueous mixture is extracted with methylene
chloride.
The organic layer is washed with aqueous Na,C03, with water, and is dried
15 (Na,S04), filtered and concentrated under reduced pressure to give the
crude
product. The desired compound is obtained by purification of the crude product
by
use of HPLC.
20
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(Following Fig 8)
Preparation of a compound of Formula I wherein his 2 and a is 1 and the
ligands.
js, are a compound of formula l~) (where R'3 is 2-bromo-3-methvlimidazo
jl 2-alvvridin-8-xl and R'4 and R'S are chlorol which are linked via
the terminal aminP~nitrogen
Br Br
I
O O
CI ~ CIO OCI , CI
NH~N ~
CH3 O ~ O CH3
A solution of the benzene-1,4-bisacetyl chloride ( 1 mmol) in methylene
chloride is added slowly to a solution of compound (II) where (R'3 is 2-bromo-
3-
10 methylimidazo(1,2-a)pyridin-8-yl and R'4 and R'S are Cl) (2 mmols) in
methyiene
chloride (5 ml) and pyridine (0.5 ml) in a flask equipped with a magnetic
stirrer and
a drying tube and which is cooled in an ice-water bath. The course of the
reaction is
followed by thin layer chromatography. After reaction occurs, the reaction
solution
is quenched in water and the aqueous mixture is extracted with ethyl acetate.
The
organic layer is washed with aqueous Na~C03, with water, and is dried
(Na.=SO,),
filtered and concentrated under reduced pressure to give the crude product.
The
desired compound is obtained by purification of the crude product by use of
HPLC.
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Exa pie 4
(Following Fig. 8)
Preparation of a compound of Formula I wherein n is 2 and a is 1 and the
liQands.
L are a compound of formula (I_Il (where R'3 is ~-meth~lauinoxalin-8-Yl_ and
R'4
~d R'S are chloro~which are linked via the terminal amine nitrogen
w N~ I N~ i
w
~N N
O O
CI ~ CIO OCI , CI
I
I , N~NH~1CH2 NH N w
CH3 CH3
A solution of compound (II) (where R'3 is 2-methylquinoxal-8-yl and R'~
and R'S are Cl) (2 mmols) in methanol (4 ml) is acidified with acetic acid to
pH 6.6
(pH meter) under a nitrogen atmosphere. 1,6-Hexanedial (1 mmol) is added neat
followed by sodium cyanoborohydride (1.1 mmol). The course of the reaction is
followed by thin layer chromatography. After reaction occurs, the reaction
solution
is quenched in water and the pH of the aqueous mixture is adjusted to greater
than
10 with aqueous NaOH. The mixture is extracted with ether, the organic
extracts
are washed with half saturated saline, dried (Na.=SOa), filtered and
concentrated
under reduced pressure to give the crude product. The desired compound is
obtained by purification of the crude product by use of HPLC.
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Exam le
(Following Fig. 9)
Preparation of a Compound of Formula I wherein ids 2-a is 1 and the ligands L
are a of formula (IIII where R'' is 2-met~ylq inoli - -vl R'~ and R'S are
chloro R,'-'
i-s methyl. and W is -CH-
w
O O O N O
O
CI I ~ CIO H ~ ~ H~ ~H ~ \ OCI , CI
N~N ~ ~ N
N
CH3 O O CH3
A solution of compound (III) (where R'3 is 2-methyiquinolin-8-yl, R'a and
10 R'S are chloro, RZ' is methyl, and W is -CH-) (2 mmols} and 1,5-bisamino-3-
oxapentane ( 1 mmol) in methylene chloride (20 ml) is prepared under argon in
a
flask equipped with magnetic stirrer and drying tube. To this solution is
added
dicyclohexylcarbodiimide (solid, 2.1 mmols). The course of the reaction is
followed by thin layer chromatography while stirring at room temperature.
After
15 reaction occurs, the reaction solution is diluted with ethyl acetate and
washed with
water and with aqueous Na,C03. The organic layer is dried (Na,S04), filtered
and
concentrated under reduced pressure to give the crude product. The desired
compound is obtained by purification of the crude product by use of HPLC.
20
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Example 6
(Following Fig. 9)
Preparation of a Compound of Formula I wherein ~ is 2 q is 1 and the ligandS L
are a compound of formula IIII) where R'3 is 2-methyl~uinolin-8-yl R" and R'S
are
chloro. R'-' is methyl. and W is -CH-
I
~N w
O H O
N O
CI ~ CIO ~ \ ~ ~ H
H O / ~ CI , CI
1
I i N~N H O
N~N w
CH3 O O CH3
A solution of compound (III) (where R" is 2-methylquinolin-8-yl, R'a and
R'S are chloro, R'-' is methyl, and W is -CH-) (2 mmols) and benzene-1,4-
bisacetic
acid (1 mmol) in methylene chloride (20 ml) is prepared under argon in a flask
equipped with magnetic stirrer and drying tube. To this solution is added
dicyclohexylcarbodiimide (solid, 2.1 mmols) while stirring at room
temperature.
The course of the reaction is followed by thin layer chromatography. After
reaction
occurs, the reaction solution is diluted with ethyl acetate and washed with
water and
with aqueous Na,C03. The organic layer is dried (Na,SOa), filtered and
concentrated under reduced pressure to give the cntde product. The desired
compound is obtained by purification of the crude product by use of HPLC.
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x m le 7
(Following Fig. 9)
Preparation of a Compound of Formula I wherein p is 2. q is 1. and the
ligands. L.
are a compound of formula (III) where R'' is 2-methylimidazo( 1.2-a)
S pyridin-8-yl, R'S is chloro, R'-' is meth,. and W is -CH-
i
H3CHNOC CONHCH3
N N \
~ N
O H3C O
A solution of compound (III) (where R" is 2-methylimidazo(1,2-a)pyridin-
8-yl, R'4 is hydroxy, R'S is chloro, R'-' is methyl, and W is -CH-) (2 mmols)
and
1,4-bisiodomethylbenzene (1 mmol) in acetone (5 ml) containing K~CO; is
stirred
10 and heated at reflux temperature under an inert atmosphere. The course of
the
reaction is followed by thin layer chromatography. After reaction occurs, the
reaction solution is diluted with ethyl acetate and washed with water and with
aqueous Na2C03. The organic layer is dried (Na.=S04), filtered and
concentrated
under reduced pressure to give the crude product. The desired compound is
15 obtained by purification of the crude product by use of HPLC.
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Example 88
(Following Fig. 10)
Preparation of a Compound f Formula I wherein p is 2. a, is 1. and the
ligands. L,
are a compound of formula (IIII where R'3 i~2-meth, ly~uinolin-8-.~
R'4 and R'S are chloro, RZ' is methyl. and W is -CH-
i i
I ~
N' \ ~ \ (
0 0 .N.
H O
CI ~ CI O ~ \ HN
H ~ I I ~ ~ O CI / CI
I \ O ~j
N~N / N~N w
CH3 O
O CHa
St
A solution of compound (III) (where R'3 is 2-methylquinolin-8-yl,
R'° and
R'S are chloro, RZ' is methyl, and W is -CH-) (1 mmol) and an aminoacid,
methyl
ester linker molecule (1 mmol) in methylene chloride (20 ml) is stirred under
argon
in a flask equipped with magnetic stirrer and drying tube. To this solution is
added
dicyclohexylcarbodiimide (solid, 1.1 mmol). The course of the reaction is
followed
by thin layer chromatography while stirring at room temperature. After
reaction
occurs, the reaction solution is diluted with ethyl acetate and washed with
water and
with aqueous Na,C03. The organic layer is dried (Na2S0,), filtered and
concentrated under reduced pressure to give the crude product. The desired
compound is obtained by purification of the crude product by use of HPLC.
Step 2
A solution of the product from Step 1 above, and lithium hydroxide (10
mmols) in methanol (6 ml) and water (2 ml) is stirred at room temperature. The
reaction is followed by thin layer chromatography. After reaction occurs, the
pH of
the solution is adjusted to 7 by the addition of dilute aq. hydrochloric acid.
The
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solvent is removed by lyophilization and the dry, crude product is used
directly in
the next reaction.
Step
A solution containing the crude product from Step 2 above, and a compound
5 of formula (III) (where R" is 2-methylquinolin-8-yl, R'a and R'S are chloro,
and W
is -CH-) (1 mmol) in methylene chloride (20 ml) is prepared under argon in a
flask
equipped with magnetic stirrer and drying tube. To this solution is added
dicyclohexylcarbodiimide (solid, 1.1 mmols). The course of the reaction is
followed by thin layer chromatography while stirring at room temperature.
After
10 reaction occurs, the reaction solution is diluted with ethyl acetate and
washed with
water and with aqueous Na,C03. The organic layer is dried (Na.=SO~), filtered
and
concentrated under reduced pressure to give the crude product. The desired
compound is obtained by purification of the crude product by use of HPLC.
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Example 99
(Following Fig. l l )
Preparation of a ComrJOUnd of Formula I wherein p is 2 q is 1 and one of the
ligands. L,, is a compound of formula III wherein R'' is 7 methylauinoIin 8
R'4 and R'S are chloro and Re is -NHCO H3 and the other li ag nd.
L_2 is a compound of formula~IIII wherein R,'3 is 2 methyl
auinolin-8-vl R'~ and R'' are chloro and R-'' is methyl
i
N
O
CI ~ CIO ~ NHCOCH3
i N~N ~ w
HN O
CI , CI
O O
i / N
N
O CH3
to 1
10 A mixture of NaH ( 1.1 mmol) and DMF ( 1 ml) is prepared under an inert
atmosphere in a flask equipped with a stirring bar and a drying tube. To this
is
added a solution of phthaloyl derivative _9 (where R'3 is 2-methylquinolin-8-
yl and
R'4 and R'S are Cl) (1 mmol) and an N-Cbz-bromomethyl linker molecule in dry
DMF (5 ml). The resulting mixture is stirred and the course of the reaction is
15 followed by thin layer chromatography. After reaction occurs, the reaction
is
quenched with cold dilute aq. Na~C03 and extracted with methylene chloride.
The
organic layer is dried (Na,SO,), filtered and concentrated under reduced
pressure to
give the crude product. The desired compound ~ is obtained by purification of
the
crude product by use of HPLC.
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to
Product 1~( obtained in Step 1 above, is suspended in EtOH (S ml) and
hydrazine hydrate (2 mmols) is added. The reaction mixture is warmed and the
course of the reaction followed by TLC. After reaction occurs, the reaction
solution
is filtered to remove solids and the filtrate is quenched with cold dilute aq.
Na,C03
and extracted with methylene chloride. The organic layer is dried (Na,SO,),
filtered
and concentrated under reduced pressure to give the crude product. The desired
compound is obtained by purification of the crude product by use of HPLC.
Step 3
The product obtained from Step 2 above, is carefully dried and placed in a
solution in dry DMF (5 ml) with the carboxylic acid ]$ (where W is -CH- and Ra
is
-NHAc) (1 mmol) and 1-hydroxybenzotriazole (1.4 mmols) under an inert
atmosphere. The solution is stirred, cooled in an ice-water bath and protected
from
the atmosphere with a drying tube. To the stirred solution is added 1-ethoxy-3-
[3-
(dimethylamino)propyl]carbodiimide hydrochloride (1.1 mmol). The course of the
reaction is followed by tlc. The cooling bath is removed and after reaction
occurs,
the reaction mixture is partitioned between methylene chloride and saturated
aqueous NaHC03. The organic layer is washed with water and brine, dried and
concentrated under reduced pressure. The desired product ~ 7 is obtained by
20 purification of the crude product by use of HPLC.
Ste~4
Ammonium formate (96 mg, 1.5 mmol) and 10% Pd/C (50 mg) are added to a
solution of compound 17 in methanol (2 ml) and THF (1 ml). The mixture is
stirred
at room temperature. The reaction is monitored by tlc and after reaction
occurs, the
mixture is filtered through Celite and rinsed with ethyl acetate. The filtrate
is
washed successively with aq. NaHC03 and with half saturated brine, then
filtered
and concentrated under reduced pressure to give the crude product. The desired
compound is obtained by purification of the crude product 19 by use of HPLC.
Ste~S .
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Product ~ is placed in a dry DMF solution (3 ml) with formula (III)
compound (where R'3 is 2-methylquinolin-8-yl, R'4 and R'S are Cl, W is -CH-,
and
Q' is -N(CH),C(O)-) (0.8 mmols) under argon in a flask equipped with magnetic
stirrer and drying tube. To this solution is added dicyclohexylcarbodiimide
(solid,
5 1. l mmols). The course of the reaction is followed by thin layer
chromatography
while stirring at room temperature. After reaction occurs, the reaction
solution is
diluted with ethyl acetate and washed with water and with aqueous Na,CO;. The
organic layer is dried (Na.=SO,), filtered and concentrated under reduced
pressure to
give the crude product. The desired compound (I) is obtained by purification
of the
crude product by use of HPLC.
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exam l,~e 10
(Following Fig. I2)
Preparation of a compound of Formula I wherein ~ is 2 and q is I and the
ligands.
L, are a compound of formula ~III~where R'3 is 2-methXlauinolin- =,vl. R'~ and
R'S
~e chloro, and Ra is -NHCOCH3,1 which is linked via the anilide nitrog-en,
i
N
O
CI ~ CIO ~ NHAc
~N~N W w
O
(I Hz)s O
N /
~N
CI I ~ CIO H I ~ NHAc
O
N\
i
Step _l
To a stirred mixture of NaH (2.1 mmol) in dry DMF (3 ml) under argon is
added phthaloyl derivative Q (where R'3 is 2-methylquinolin-8-yl and R'a and
R'S
are chloro) (2 mmol). After stirring for 20 min, 1,5-dibromopentane (neat, I
mmol)
is added and stirring is continued. The course of the reaction is followed by
thin
layer chromatography and the reaction mixture is warmed to complete the
reaction.
After reaction occurs, the reaction solution is poured into water and the
aqueous
mixture extracted with methylene chloride. The organic extracts are washed
with
water and with brine, dried (Na,SO,), filtered and concentrated under reduced
pressure to give the crude product. The desired compound ~Q is obtained by
purification of the crude product by use of HPLC. The pure product is used in
the
following reaction.
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a 2
To a solution of compound ~ in 95% EtOH (S ml) is added hydrazine hydrate
(2 mmols). The stirred mixture is warmed and the course of the reaction
followed
by tlc. After reaction occurs, the reaction solution is filtered to remove
solids and
5 the filtrate is quenched with cold dilute aq. Na,C03 and extracted with
methylene
chloride. The organic layer is dried (Na2S04), filtered and concentrated under
reduced pressure to give the crude product. The desired compound 21 is
obtained
by purification of the crude product by use of HPLC.
Ste~3
IO The product 2_~ from Step 2 above is carefully dried and placed in a
solution
in dry DMF (5 ml) with the carboxylic acid ~$ (where W is -CH- and Ra is -
NHAc)
(1 mmol) and 1-hydroxybenzotriazole (1.4 mmols) under an inert atmosphere. The
solution is stirred, cooled in an ice-water bath and protected from the
atmosphere
with a drying tube. To the stirred solution is added 1-ethoxy-3-[3-
(dimethylamino)-
15 propyl]carbodiimide hydrochloride (l . l mmol). The course of the reaction
is
followed by tlc. The cooling bath is removed and after reaction occurs, the
reaction
mixture is partitioned between methylene chloride and saturated aqueous
NaHC03.
The organic layer is washed with water and brine, dried and concentrated under
reduced pressure. The desired compound of Formula I, is obtained by
purification
20 of the crude product by use of HPLC.
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Exam In a 1 I
(Following Fig. 13)
~'renaration of a compound of Formula I wherein n is 2 ~ is 1 and one of the
ligands"~,, is a comnound~f forr,~la jI_Ij~ wherein R" is ~3 bromo 2
S methvllimidazo[I 2-a],p ridin-8-vl and R" and R'S are methyl and Ra is
-NHCOCH3 and the other ligand, Lz. is a compound of formula ,(III) where,.j~
R" is (3-bromo-2-meth~)imidazo~~]~pvridin 8 yl and R'a is chloro
Br
~N~
~N
O
O H ~ I N HAc
i N~N ~ w
I O
O
O
p H ~ I ~NHCH3
O ~ N~N
N ,
N CI CH3 O
Br
Step 1
A mixture of NaH ( 1.1 mmol) and DMF ( 1 ml} is prepared under an inert
10 atmosphere in a flask equipped with a stirring bar and a drying tube. To
this is
added a solution of phthaloyl derivative ~ [where R'' is 8-(3-bromo-2-methyl)-
8-
imidazo[1,2-a]pyridyl and R'~ and R'S are Me] (1 mmol) and a tert-
butyldimethylsilyl-protected hydroxymethyl-bromomethyl linker molecule (1
mmol) in dry DMF (5 ml). The resulting mixture is stirred and the course of
the
15 reaction is followed by thin layer chromatography. After reaction occurs,
the
reaction is quenched with cold dilute aq. Na~CO; and extracted with methylene
chloride. The organic layer is dried (Na,SO~), filtered and concentrated under
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reduced pressure to give the crude product. The desired compound ~ is obtained
by purification of the crude product by use of HPLC.
Step 2
To a solution of the product ~ in 95% EtOH (S ml) is added hydrazine
5 hydrate (2 mmols). The stirred mixture is warmed and the course of the
reaction
followed by TLC. After reaction occurs, the reaction solution is filtered to
remove
solids and the filtrate is quenched, with cold dilute aq. Na,C03 and extracted
with
methylene chloride. The organic layer is dried (Na.'SOa}, filtered and
concentrated
under reduced pressure to give the crude product. The desired compound is
obtained by purification of the crude product by use of HPLC.
Step
The product obtained in Step 2 is carefully dried and placed in a solution in
dry DMF (5 ml) with the carboxylic acid ~$ (where W is -CH= and Ra is -NHAc)
(1
mmol) and 1-hydroxybenzotriazole (1.4 mmols) under an inert atmosphere. The
15 solution is stirred, cooled in an ice-water bath and protected from the
atmosphere
with a drying tube. To the stirred solution is added 1-ethoxy-3-[3-
(dimethylamino)-
propyl]carbodiimide hydrochloride (1.1 mmol). The course of the reaction is
followed by tlc. The cooling bath is removed and after reaction occurs, the
reaction
mixture is partitioned between methylene chloride and saturated aqueous
NaHCO;.
The organic layer is washed with water and brine, dried and concentrated under
reduced pressure. The desired product ~3 is obtained by purification of the
crude
product by use of HPLC.
Step 4
A solution compound ~, obtained from Step 3 above and Et;N-(HF)3 in
25 MeCN (5 ml) is stirred at room temperature. After reaction occurs as
detected by
tlc, the solution is diluted with EtOAc and then washed with water-brine. The
organic layer is dried (Na,S04), filtered and concentrated under reduced
pressure to
give the crude product. The desired hydroxy compound is obtained by
purification
of the crude product with the use of HPLC.
Ste~S
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A solution of the hydroxy compound obtained in Step 4 above in methylene
chloride (5 ml) and triethyl amine (S drops) is stirred under argon and cooled
in an
ice-water bath. Methanesulfonyl chloride ( 1.2 mmol) in methylene chloride
(0.5
ml) is added and the solution is stirred and allowed to warm to room
temperature.
S The progress of the reaction is followed by TLC and after reaction occurs,
the
solution is poured into aqueous Na,C03. The mixture is extracted with
methylene
chloride and the organic layer is washed with water and with brine, dried
(Na=SOa),
filtered and concentrated under reduced pressure to give the crude product.
The
desired mesylated compound is obtained by purification of the crude product by
use
10 of HPLC.
Ste~6
A mixture of the mesylated compound obtained in Step 5 above, KI ( 10
mmol), and 18-crown-6 (1 mmol) in DMF (2 ml) is stirred and warmed. The
progress of the reaction is followed by tlc and after reaction occurs, the
mixture is
15 poured into water and extracted with ethyl acetate. The combined organic
extract
solution is washed with water, with brine, dried (Na,S04), filtered and
concentrated
under reduced pressure to give the crude product. The desired compound 24 is
obtained by purification of the crude product by use of HPLC.
t 7
20 A solution of the compound 24 and compound (III) (where R" is 8-(2-
methyl)imidazo(1,2-a)pyridinyl and W is -CH-) in acetone (5 ml) containing
K~C03 is stirred and heated at reflux temperature under an inert atmosphere.
The
course of the reaction is followed by thin layer chromatography. After
reaction
occurs, the reaction solution is diluted with ethyl acetate and washed with
water and
25 with aqueous Na~C03. The organic layer is dried (Na.:S04), filtered and
concentrated under reduced pressure to give the crude product. The desired
compound (I) is obtained by purification of the crude product by use of HPLC.
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Ex~ In a 12
(Following Fig 14)
Preparation of a comn~nd of Formula I wherein p is 2 q is 1 and one of the
ands L,~comnound of formula (IIj) wherein R'3 is 2 methyl~uinolin8 vl
R''' and R'S are chloro and RZ' i metl~vl and the other li~,~,. is a
compound of formula IIII) wherein R'3 is 2-met~vlimidazo[1 ~ a)
p ri in-8-yl, R'4 is chloro and RZ' is methyl
N O
O O 10 H ~ I NHCH3
CI ~ CI O H , N ~ N \ \
I i N~N \ \ I H CHs 0
CH3 O
S~
10 A solution of compound (III) (where R'3 is 2-methylquinolin-8-yl, R'4 and
R'S are Cl, W is -CH-, and R'-' is methyl) (1 mmol) and a tert-
butyldimethylsilyl-
protected hydroxymethyl amine linker molecule (lmmol) in methylene chloride
(20 ml) is prepared under argon in a flask equipped with magnetic stirrer and
drying
tube. To this solution is added dicyclohexylcarbodiimide (solid, 1.1 mmol).
The
15 course of the reaction is followed by thin layer chromatography while
stirring at
room temperature. After reaction occurs, the reaction solution is diluted with
ethyl
acetate and washed with water and with aqueous Na~C03. The organic layer is
dried (Na.:S04), filtered and concentrated under reduced pressure to give the
crude
product. The desired compound ~5_ is obtained by purification of the crude
product
20 by use of HPLC and is then converted to a compound of formula ~ as
described in
Example 12, Steps 2-4 above.
Stelz2_
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A solution of the compound 26 and of formula (III) compound (where R'3 is
8-(2-methyl)imidazo(1,2-a)pyridinyl and W is -CH-) in acetone (5 ml)
containing
K,C03 is stirred and heated at reflux temperature under an inert atmosphere.
The
course of the reaction is followed by thin layer chromatography. After
reaction
S occurs, the reaction solution is diluted with ethyl acetate and washed with
water and
with aqueous Na2C03. The organic layer is dried (Na,SO,), filtered and
concentrated under reduced pressure to give the crude product. The desired
compound (I) is obtained by purification of the crude product by use of HPLC.
Example 13
(Following Fig. 15)
Preparation of a compound of Formula I wherein p is 2j q is I and one of the
~g~nds. ,, is a com~und of formula (III~wherein R" is 2-methyl uinolin-8-vl
~'~ and R'S are chloro and R'-' is meths and the other li ag nd. Lz is a
compound of
formula~IIIZwherein R'3 is (,~ methyllimidazof I 2-a~pyridin-8-yl_ R'~ is
chloro R-''
ie methyl and Ra is -CONHCH;
i o
O CI ,., H ~ ~ NHCH3
H ~N
CI I ~ CIO , N N
~N~N \ \ ~ CH3 O
CH3 O
Step 1
A solution of compound (III) (where R'3 is 2-methylquinolin-8-yl, R" and
R'S are Cl, W is -CH-, and RZ' is methyl) (1 mmol) and a t-butyldimethylsilyl-
protected hydroxymethyl carboxylic acid linker molecule ( 1 mmol) in methylene
chloride (20 ml) is prepared under argon in a flask equipped with magnetic
stirrer
and drying tube. To this solution is added dicyclohexylcarbodiimide (solid,
1.1
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mmol). The course of the reaction is followed by thin layer chromatography
while
stirring at room temperature. After reaction occurs, the reaction solution is
diluted
with ethyl acetate and washed with water and with aqueous Na~C03. The organic
layer is dried (Na,SO,), filtered and concentrated under reduced pressure to
give the
crude product. The desired compound 27 is obtained by purification of the
crude
product by use of HPLC which is then converted to a compound of formula ~$ as
described in Example 12, Steps 2-4 above.
Ste
A solution of the compound 2~ and a compound (III) (where A is 8-(2-
methyl)imidazo(1,2-a)pyridinyl and W is -CH-) in acetone (5 ml) containing
K,C03 is stirred and heated at reflux temperature under an inert atmosphere.
The
course of the reaction is followed by thin layer chromatography. After
reaction
occurs, the reaction solution is diluted with ethyl acetate and washed with
water and
with aqueous NazC03. The organic layer is dried (Na,SO,), filtered and
concentrated under reduced pressure to give the crude product. The desired
compound (I) is obtained by purification of the crude product by use of HPLC.
Exam In a 14
Hard gelatin capsules containing the following ingredients are prepared:
Quantity
redie (m T/ca sp ulel_
Active Ingredient 30.0
Starch 305.0
Magnesium stearate 5.0
The above ingredients are mixed and filled into hard gelatin capsules in 340
mg quantities.
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Example 15
A tablet Formula is prepared using the ingredients below:
Quantity
Ingredient /t blet
5
Active Ingredient 25.0
Cellulose, microcrystalline 200.0
Colloidal silicon dioxide 10.0
Stearic acid 5.0
10
The components are blended and compressed to form tablets, each weighing
240 mg.
Exam lp a 16
15 A dry powder inhaler formulation is prepared containing the following
components:
Wei hit
Active Ingredient 5
20 Lactose 95
The active ingredient is mixed with the lactose and the mixture is added to a
dry powder inhaling appliance.
25 Exam 1p a 17
Tablets, each containing 30 mg of active , are prepared
ingredient as follows:
Quantity
In r '
~g/tabletl
30 Active Ingredient 30.0 mg
Starch 45.0 mg
Microcrystalline cellulose 35.0 mg
Polyvinylpyrrolidone
(as 10% solution in sterile water) 4.0 mg
35 Sodium carboxymethyl starch 4.5 mg
Magnesium stearate 0.5 mg
Talc 1~
Total 120 mg
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The active ingredient, starch and cellulose are passed through a Nu. 20 mesh
U.S. sieve and mixed thoroughly. The solution of polyvinylpyrrolidone is mixed
with the resultant powders, which are then passed through a 16 mesh U.S.
sieve.
5 The granules so produced are dried at 50 ° to 60 °C and passed
through a 16 mesh
U.S. sieve. Tlie sodium carboxymethyl starch, magnesium stearate, and talc,
previously passed through a No. 30 mesh U.S. sieve, are then added to the
granules
which, after mixing, are compressed on a tablet machine to yield tablets each
weighing 120 mg.
Ex~ple 18
Capsules, each containing 40 mg of medicament are made as follows:
Quantity
re ' (mg/ca sulel
Active Ingredient 40.0 mg
Starch 109.0 mg
Magnesium stearate 1.
Total 150.0 mg
20 The active ingredient, starch, and magnesium stearate are blended, passed
through a No. 20 mesh U.S. sieve, and filled into hard gelatin capsules in 150
mg
quantities.
Suppositories, each containing 25 mg of active ingredient are made as
follows:
I r i ou
Active Ingredient 25 mg
Saturated fatty acid glycerides to 2,000 mg
The active ingredient is passed through a No. 60 mesh U.S. sieve and
suspended in the saturated fatty acid glycerides previously melted using the
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minimum heat necessary. The mixture is then poured into a suppository mold of
nominal 2.0 g capacity and allowed to cool.
Example 20
Suspensions, each containing 50 mg
of medicament per 5.0 mL dose are
made as follows:
r i m nt
Active Ingredient 50.0 mg
Xanthan gum 4.0 mg
Sodium carboxymethyl cellulose ( 11
%)
Microcrystalline cellulose (89%) 50.0 mg
Sucrose 1.75 g
Sodium benzoate 10.0 mg
Flavor and Color q.v,
Purified water to 5.0 mL
The active ingredient, sucrose and xanthan gum are blended, passed through
a No. 10 mesh U.S. sieve, and then mixed with a previously made solution of
the
microcrystalline cellulose and sodium carboxymethyl cellulose in water. The
20 sodium benzoate, flavor, and color are diluted with some of the water and
added
with stirring. Sufficient water is then added to produce the required volume.
Exam In a 21
A formulation may be prepared as follows:
I r ' n Quantity
Active Ingredient 1 5.o sulel
g
Starch 407.0 mg
30 Magnesium stearate 3.0 me
Total 425.0 mg
The active ingredient, starch, and magnesium stearate are blended, passed
through a No. 20 mesh U.S. sieve, and filled into hard gelatin capsules in
425.0 mg
quantities.
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Example 22
A formulation may be prepared as follows:
Ing ~el dient S~tantitv
Active Ingredient 5.0 mg
5 Corn Oil 1.0 mL
Exam la a 23
A topical formulation may be prepared as follows:
10 Ingredient Quantity
Active Ingredient 1-10 g
Emulsifying Wax 30 g
Liquid Paraffin 20 g
White Soft Paraffin to 100 g
15
The white soft paraffin is heated until molten. The liquid paraffin and
emulsifying wax are incorporated and stirred until dissolved. The active
ingredient
is added and stirring is continued until dispersed. The mixture is then cooled
until
solid.
20 Another preferred formulation employed in the methods of the present
invention employs transdermal delivery devices ("patches"). Such transdermal
patches may be used to provide continuous or discontinuous infusion of the
compounds of the present invention in controlled amounts. The construction and
use of transdermal patches for the delivery of pharmaceutical agents is well
known
25 in the art. See, e.g., U.S. Patent 5,023,252, issued June 11, 1991, herein
incorporated by reference in its entirety. Such patches may be constructed for
continuous, pulsatile, or on demand delivery of pharmaceutical agents.
Other suitable formulations for use in the present invention can be found in
Remington's Pharmaceutical Sciences, edited by E. W. Martin (Mack Publishing
30 Company, 18th ed.,1990).
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Biological ExamRles
Exam In a 24
Bradykinin Antagonist Activit~...ln vitro assay
The bradykinin antagonistic activity of the compounds of the invention can
5 be tested as described below.
Tissue Preparation:
The specif c binding of [3H]BK (a high affinity B, ligand) is assayed as
follows. Male Hartley guinea pigs are killed by exsanguination under
anesthesia.
The ilea are removed and homogenized in ice-cold buffer (~~mM sodium
10 (trimethylamino)ethanesulfonate (TES) and 1mM l, 10-phenanthroline, pH6.8)
with a Polytron homogenizer (PT-10, Brinkmann Instruments, Inc., Westbury,
NY).
Cellular debris is removed by centrifuging at 1000g at 4°C for 20
minutes. The
supernatant is centrifuged at 100000g at 4°C for 60 minutes. The pellet
is then
resuspended in ice-cold assay buffer containing SOmM TES, 1 mM 1,10-
15 phenanthroline, 140pg/ml bacitracin, 1mM dithiothreiol, lp.M captopril, and
0.1%
bovine serum albumin (BSA), pH 6.8. The amount of protein is determined by the
method of Lowry et al. using a kit (Catalog # P5656, Sigma Chemical Co., St.
Louis, MO). The pellet is stored at -80 °C until use.
Receptor binding:
20 The bradykinin antagonist activity of the compounds of the invention is
tested as follows. 0.2mg/ml of the receptor is incubated with 0.06 nM [3H]
bradykinin and varying concentrations of either a test compound or unlabeled
BK at
room temperature for 60 minutes. Receptor bound [3H] bradykinin is harvested
by
filtration through Whatman glass fiber filters (Catalog # GFB, Whatman, Inc.,
25 Clifton, NJ) under reduced pressure, and the filter is washed five times
with 300 ml
of ice-cold buffer (SOmM Tris HCl). The radioactivity retained on the filter
is
measured with a scintillation counter. Specific binding is calculated by
subtracting
the nonspecific binding from total binding.
30
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Inhibition of bradykinin-induced broncho onstriction in vivo asthma model
The ability of the compounds of the invention to inhibit bradykinin induced
bronchoconstriction can be tested using an asthma model as described below.
Male
Hartley guinea pigs, obtained from Charles River, weighing 470-750 g are used.
The guinea pigs are fasted overnight and anesthetized by intraperitoneal
injection of
sodium pentobarbital (30 mg/kg). The trachea, jugular vein, and esophagus are
cannulated. The animals are ventilated at a tidal volume of 10 mg/kg with a
frequency of 60 breathes/min through the tracheal cannula. To suppress
spontaneous respiration, alcuronium chloride (0.5 mg/kg) is administered
intravenously through the jugular vein cannula. Then, propanolol ( 10 mg/kg is
administered subcutaneously. After 10 min., S ,ug/kg bradykinin is dissolved
in
saline with 0.1% BSA arid admininstered intravenously via the jugular vein
cannula. Bronchoconstriction is measured as the peak increase of pulmonary
insufflation pressure. Each dose of the test compound or control compound is
suspended in 0.5% methylcellulose solution and administered through the
esophageal cannula after the first bradykinin-induced bronchoconstriction. The
bradykinin is administered again at 30 min. and bronchoconstriction is
measured. A
0% response is determined as peak increase of pulmonary insufflation pressure
before administration of bradykinin and the 100% response is determined as the
first bradykinin-induced bronchaconstriction before drug administration. The
percent response was calculated from the following formula: % response =
change
in peak increase of pulmonary insufflation after drug/peak increase in
pulmonary
insufflation before drug) X 100.
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Exam Ip a 26
Inhibition of inflammation...in vivQ Carraeeenan-Induced ~w edema model
Carrageenin induced paw edema in rats is a standard model of inflammation.
In this model, male Sprague Dawley rats are deprived of food overnight and
then
5 various concentrations of test compounds, dissolved in 0.05 N HCI, are
administered orally. After 1 S minutes, 0.1 ml of carrageenan ( 1 % in saline)
is
injected into the right hind paw intraplantar. Paw volume is measured using a
water
plethysmometer (Catalog # Ugo Basile 57140, Stoelting Co., Wood Dale, IL)
before, and 1, 2, 3, and 4 hours after injection of carrageenin. The ability
of the test
10 compounds to inhibit bradykinin induced inflammation is measured as
duration of
action.
Exam In a 27
Inhibition of bradykinin-induced Pancreatitis...ln vivo Caerulein-Induced
15 Pancreatitis model
Female Sprague Dawley rats (9-l Oweeks old) are deprived of food for
l8hours and caerulein (20pg/ml) is injected intraperitoneally 4 times at
hourly
intervals. Test compounds, dissolved in O.OSN HCl or vehicle, are administered
orally 30 minutes before the first caerulein injection. Three hours after the
last
20 caerulein injection, a blood sample is taken from the abdominal artery with
heparin
under anesthesia (diethyl ether inhalation), and the animal is killed by
exsanguination. Serum amylase and lipase levels are determined using a Vet
Test
8000 chemistry analyzer model (see, Gukovskaya et al., "Pancreatic Acinar
Cells
Produce, Release, and Respond to Tumor Necrosis Factor-a: Role in Regulating
25 Cell Death and Pancreatitis" J. Clin. Invest., 100(7), 1853-1862. (1997)).
The foregoing invention has been described in some detail by way of
illustration
and example, for purposes of clarity and understanding. It will be obvious to
one of
skill in the art that changes and modificationsmay be practiced within the
scope of the
30 appended claims. Therefore, it is to be understood that the above
description is
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intended to be illustrative and not restrictive. The scope of the invention
should,
therefore, be determined not with reference to the above description, but
should instead
be determined with reference to the following appended claims, along with the
full
scope of equivalents to which such claims are entitled.
5 All patents, patent applications and publications cited in this application
are
hereby incorporated by reference in their entirety for all purposes to the
same extent
as if each individual patent, patent application or publication were so
individually
denoted.
10
15
