Note: Descriptions are shown in the official language in which they were submitted.
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/1Z768
-1-
MULTIVALENT AGONISTS, PARTIAL AGONISTS, INVERSE
AGONISTS AND ANTAGONISTS OF THE SHT3 RECEPTORS
This application claims the benefit of U.S. Provisional Application Serial
5 Numbers 60/088,466, filed June 8, 1999; 60/092,938, filed July 16, 1998; and
60/120,282, filed February 16, 1999, all of which are herein incorporated by
reference in their entirety.
BACKGROUND OF THE INVENTION
F~'eld of the Invention
10 This invention relates to novel therapeutic agents which bind to mammalian
receptors and modulate their activity. More particularly, the invention
relates to
novel therapeutic agents that bind to and modulate the in vivo activity of 5-
HT3
receptors in mammals by acting as multi-binding compounds. The therapeutic
agents or mufti-binding compounds described herein comprise at least two
ligands
15 connected by a linker or linkers, wherein the ligands in their monovalent
state
bind to and/or are capable of modulating the activity of the 5-HT3 receptor.
The
linking moiety is chosen such that the mufti-binding compounds so constructed
demonstrate increased biological activity as compared to individual units of
the
ligand. The invention also relates to methods of using such compounds, to
20 methods of preparing such compounds and to pharmaceutical compositions
containing them.
These mufti-binding compounds are particularly useful in treating
mammalian conditions that are mediated by the 5-HT3 receptors targeted by the
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-2-
ligands, such as chemotherapy induced and radiation induced emesis, anxiety,
schizophrenia, drug withdrawal and cognitive disorders. Accordingly, this
invention also relates to pharmaceutical compositions comprising a
pharmaceutically acceptable excipient and an effective amount of a mufti-
binding
compound of this invention.
Additionally, the mufti-binding compounds are useful as affinity resins for
affinity chromatography. When so employed, the compounds of the invention
may be used as a tool in immunoprecipitation. The compounds may be used to
identify a receptor in vitro for example in microscopy, electrophoresis and
chromatography.
References
The following publications are cited in this application as superscript
numbers:
1. J. March, Advanced Organic Chemistry, 4'~' Edition, Wiley-
Interscience New York (1992);
2. Remington's Pharmaceutical Sciences, Mace Publishing Company,
Philadelphia, PA, 17th ed. (1985);
3. Green, Protective Groups in Organic Synthesis, 2"d Edition, John
Wiley & Sons, New York, New York (1991);
4. Gregory, R. E. et al., "5-HT3 Receptor Antagonists for the
Prevention of Chemotherapy-Induced Nausea and Vomiting" .
Drugs (1998) 55(2): 173-189;
5. Emesis in Anti-cancer Therapy Mechanisms and Treatment,
Andrews, P.L.R. and Sanger, G.J. eds., Chapman & Hall Medical
(1993).
b. Boess, ~.G. et al., "Molecular Biology of 5-HT Receptors",
Neuropharm. (1994) x(3/4):275-317;
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-3-
7. Gralla, R.J. "Anti-emesis with Cancer Chemotherapy", Europ. J.
of Cancer (1997) x(4):563-567;
8. Cunningham, R.S., "5-HT3-Receptor Antagonists: A Review of
5 Pharmacology and Clinical Efficacy", Oncology Nursing Forum,
(1997) ~(7, supp.):33-39;
9. Wilde et al., "Ondansetron A Review of its Pharmacology and
Preliminary Clinical Findings in Novel Applications", Drugs (1996)
10 5,"x(5):773-794;
10. S-Hydroxytryptamine in Peripheral Reactions, De Clerck et al.
eds., Raven Press, New York (1982) pp 1-35;
15 11. Oxford et al., "Ondansetron and Related 5-HT3 Antagonists: Recent
Advances", Progress in Med. Chem. (1992) x:239-270;
12. Chevallier, B. "The Control of Acute Cisplatin-induced Emesis - a
Comparative Study of Granisetron and a Combination Regimen of
20 High-dose Metoclopramide and Dexamethasone", Br. J. Cancer
(1993) x$:176-180;
13. Cubeddu, L.X., "Serotonin Mechanisms in Chemotherapy-Induced
Emesis in Cancer Patients", Oncology (1996) 5,~{suppl 1):18-35;
25
14. Rodriguez, M.L. et al., "Comparative Receptor Mapping of
Serotoninergic 5-HT3 and 5-HT4 Binding Sites", Journal of
Computer-Aided Molecular Design, (1997) x:589-599;
30 15. van Wijngaarden, Ineke et al., "Development of High-Affinity 5-
HT3 Receptor Antagonists. Structure-Affinity Relationships of
Novel 1,7-Annelated Indole Derivatives. 1", J. Med. Chem. (1993),
xø:3693-3699;
35 16. Parker, Rachel M.C. et al., "Allosteric Modulation of 5-HT3
Receptors: Focus on Alcohols and Anaesthetic Agents", TiPS
(March-1996) j~:95-99;
17. Fletcher, Stephanie et al., "Desperately Seeking Subunits: Are
40 Native 5-HT3 Receptors Really Homomeric Complexes?", TIPS
(June-1998) ~Q:212-215;
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
18. Yamada, Megumi et al., "A New 5-HT3 Receptor Ligand II
Structure-Activity Analysis of 5-HT3 Receptor Agonist Action in
the Gut", Chem. Pharm. Bull., (1998) 4(3):445-451;
5 19. Hirokawa, Yoshimi et al., "A Novel Series of N (Hexahydro-1,4-
Diazepin-6-YL) and N (Hexahydroazepin-3-YL) Benzamides with
High Affinity for 5-HT3 and Dopamine DZ Receptors", Bioorganic
& Medicinal Chemistry Letters, (1998) $:610-624;
10 20. Orjales, Aurelio et al., "New 2-Piperazinylbenzimidazole
Derivatives as 5-HT3 Antagonists. Synthesis and Pharmacological
Evaluation", J. Med. Chem. (1997), 4Q:586-593;
21. Ohta, Mitsuaki et al., "Novel 5-Hydroxytryptamine (5-HT3)
15 Receptor Antagonists. III. Pharmacological Evaluations and
Molecular Modeling Studies of Optically Active 4,5,6,7
Tetrahydro-1H-benzimidazole Derivatives", Chem. Pharm. Bull.
(1996), x(9):1707-1716;
20 22. Ohta, Mitsuaki et al., "Novel 5-Hydroxytryptamine (5-HT3)
Receptor Antagonists. I. Synthesis and Structure-Activity
Relationships of Conformationally Restricted Fused Imidazole
Derivatives", Chem. Pharm. Bull. (1996), x(5):991-999; and
25 23. Pinder, R.M., "Designing a New Generation of Antidepressant
Drugs", Acta Psychiatr Scand. (1997), Q~(suppl 391):7-13.
24. Hibert, et al. , J. Med. Chem. , x:1594 ( 1990)
30 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.
35 A receptor is a biological structure with one or more binding domains that
reversibly complexes with one or more ligands, where that complexation has
biological consequences.
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-5-
Receptors can exist entirely outside the cell (extracellular receptors),
within
the cell membrane (but presenting sections of the receptor to the
extracellular
milieu and cytosol), or entirely within the cell (intracellular receptors).
They may
also function independently of a cell (~.g., clot formation). Receptors within
the
cell membrane allow a cell to communicate with the space outside of its
boundaries (i.e., signaling) as well as to function in the transport of
molecules and
ions into and out of the cell.
A ligand is a binding partner for a specific receptor or family of receptors.
A ligand may be the endogenous ligand for the receptor or alternatively may be
a
synthetic ligand for the receptor such as a drug, a drug candidate or a
pharmacological tool.
The super family of seven transmembrane proteins (7-TMs), also called
G-protein coupled receptors (GPCRs), represents one of the most significant
classes of membrane bound receptors that communicates changes that occur
outside of the cell's boundaries to its interior, triggering a cellular
response when
appropriate. The G-proteins, when activated, affect a wide range of downstream
effector systems both positively and negatively (e.g., ion channels, protein
kinase
cascades, transcription, transmigration of adhesion proteins, and the like).
The ligands that bind to G-protein cellular receptors may be specifically
classified as follows:
1. Full agonists - ligands that when bound trigger the maximum activity
seen by natural ligands;
2. Partial agonists- ligands that when bound trigger sub-maximal activity;
3. Antagonist- ligands that when bound inhibit or prevent the activity
arising from a natural ligand binding to the receptor. Antagonists may be of
the
surmountable class (results in the parallel displacement of the dose-response
curve
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-6-
of the agonist to the right in a dose dependent fashion without reducing the
maximal response for the agonist) or insurmountable class (results in
depression of
the maximal response for a given agonist with or without the parallel shift);
4. Inverse antagonist-ligands that when bound decrease the basal activity of
the unbound receptor (if any).
There are four fundamental measurable properties that pertain to the
interaction of a ligand with its receptor including G-protein cellular
receptors:
1) the affinity of the ligand for the receptor, which relates to the
energetics
of the binding;
2) the efficacy of the ligand for the receptor, which relates to the
functional
downstream activity of the Iigand;
3) the kinetics of binding and function of the ligand at the receptor, which
defines the onset of action and the duration of action; and
4) the desensitization of the receptor for the ligand.
With regard to the iigand, it is the combination of these properties that
provides the foundation for defining the nature of the functional response.
Thus,
an activating ligand (or agonist) has affinity for the receptor and downstream
efficacy. In contrast, an inhibiting ligand (antagonist) has affinity for the
receptor,
and their efficacy lies in their ability to effectively block agonism of the
receptor.
Selectivity defines the ratios of affinities or the ratios of efficacies of a
given ligand compared across two receptors. It is the selectivity of a
specific drug
that provides the required biological profile. For example, in certain
therapeutic
settings, it is currently thought that a highly selective drug may be
preferred (e.g.,
Losartan (Cozaar), an antihypertensive, is a highly selective antagonist for
the
AT1 receptor). In contrast, it is considered that a drug with a broad spectrum
of
receptor activity may be preferred in other therapeutic settings.
CA 02321191 2000-08-15
WO 99/64046 PCTNS99/I2768
Current drugs (ligands) targeting receptors, including G-protein receptors,
have clinical shortcomings identified by one or more of low efficacy, low
affinity,
poor safety profile, lack of selectivity or overselectivity for the intended
receptor,
and suboptimal duration of action and onset of action. Accordingly, it would
be
beneficial to develop ligands that have improved affinity, efficacy,
selectivity, .
onset of action and duration of action.
Affinit~of Band for target rece~ntor
An increase in ligand affinity to the target receptor may contribute to
reducing the dose of ligand required to induce the desired therapeutic effect.
A
reduction in ligand affinity will remove activity and may contribute to the
selectivity profile for a ligand.
Efficacy of ligand at a targ -r rP~Pptor (funcr;~nal effect)
An increased ligand efficacy at a target receptor can lead to a reduction in
the dose required to mediate the desired therapeutic effect. This increase in
efficacy may arise from an improved positive functional response of the ligand
or
a change from a partial to full agonist profile. Reduced efficacy of a full
agonist
to a partial agonist may provide clinical benefit by modulating the biological
response. Further, antagonists have efficacy at inhibiting the agonism of the
receptor.
Selectivity of ligand compared across recept ~ yp~
An increase in the selectivity of the ligand across receptor subtypes
requires that the affinity or efficacy of the ligand at other receptors is
reduced
relative to the desired receptor.
A decrease in the selectivity of the ligand may also be desired. For
example, the angiotensin II endogenous ligand activates both the AT1 and AT2
receptor subtypes.
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
_g_
More rapid onset of action of the ligand to effect a biological response is
often preferred.
An increased duration of action of the ligand to effect a biological response
may be preferred. For example X32 adrenergic agonists such as albuterol have a
relatively short duration of action of approximately 3-4 hours and an increase
in
duration of action would simplify the dosing regimen required to administer
this
drug (ligand).
Desensitization of t_h_e recP~ptor for the lig'
Desensitization is best defined as the variety of processes by which the
functional interaction of the receptor with its G-protein are influenced.
These
processes lead ultimately to a reduction in cellular response to the
activating
agonist. Such phenomena are most often observed during prolonged stimulation
of
the receptor. The two main pathways for receptor desensitization are reduction
in
receptor density or changes in receptor structure by phosphorylation
mechanisms.
Receptor density is altered by receptor sequestration. This is a reversible
process that is observable within minutes and is a dynamic sorting of
receptors
with receptors being cycled to and from the membrane. On the other hand,
receptor down-regulation is generally slower, in the order of hours, and is
25 irreversible, involving destruction of the receptor. Finally, receptor
density may
be affected by an alteration in the rate of synthesis. For example, the rates
of Vii,
mRNA synthesis and degradation are controlled by levels of c-AMP within the
cell.
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-9-
Alternatively, receptor desensitization may occur through changes in
receptor structure, such as receptor phosphorylation. For example, agonist
induced activation of the (32-adrenergic receptor, which is positively coupled
to
adenylate cyclase through Gs, results in an elevation in an increase in the
levels of
c-AMP and an increase in the activity of protein kinase A. This kinase can
readily
phosphorylate the consensus site in the third intracellular loop. The
phosphorylated ~i~-adrenergic receptor exhibits significantly reduced coupling
to
Gs. Besides PKA, the G-protein coupled receptor kinases (GRK) are also
involved in the desensitization of GPCRs. For the X32-adrenergic receptor,
there
are two of these kinases bARKI and bARK2. These GRKs are more specific and
will only phosphorylate an agonist activated receptor. Furthermore this GRK
desensitization requires an arrestin protein.
Receptor oligomerization also plays a role in receptor function. This is
best exemplified in the area of growth receptors that are known to act
functionally
and structurally as dimers, e.g., EGF-R and interferon receptor. It is also
known
that dimerization is involved in the functioning of the steroid receptor.
Preliminary evidence is beginning to appear on the importance of
oligomerization
in G-protein coupling and signaling. It is proposed that receptor
oligomerization
may play a role in different receptor functions such as mediating coupling of
the
G-protein or receptor internalization.
One important class of GPCRs are the 5-HT receptors including the 5-HT3
(serotonin) receptor which plays a role in the mechanisms of nausea and emesis
in
mammals. 5-HT3 receptors are ligand-gated ion channels which are located on
neurons in many systems in the periphery and in the brain.b The 5-HT3 receptor
is
unique in that it belongs to the superfamily of ion-channel receptors'°
and is a
ligand-gated ion channel that is permeant to Na+, K+, Ca2+ and other cations.b
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-10-
Another family of receptors is the ligand-gated ion channel receptors.
Here, the binding of ligand to specific ligand binding sites on these
receptors
results in modulation of ion flux into or out of the cell. These membrane
bound
cell surface receptors are composed of multiple subunits, typically 5
subunits,
which may be the same or different.
These ligand gated ion channel receptors have important roles in the central
nervous system and the peripheral nervous system,. Examples of the receptors
include the 5 HT 3 receptor. Ligands which bind to this receptor and
therapeutic
indications include:
Disorder Ligands
Emesis, anxiety disorder,Ondansetron, lurosetron, azasetron,
cognitive disorders,itasetron, fabesetron, alosetron,
ramosetron,
dementia, depression,dolasetron, mirtazapine, palonosetron,
schizophrenia, gastriccilansetron, Granisetron, renzapride,
R-
motility disorder, zacopride, zatosetron, Tropisetron,
nausea,
irritable bowel syndrome,lerisetron, ricasetron, YM-114,
CP-93318,
heart arrhythmia, N-3256, WAY-100289, SC-50410, KF-
drug
dependence 18259, RS-56812, SC-52491, E-3620,
SC-
52246, SDZ-ICM-567, UCM-30593,
GR-
65630, DAT-582, ADR-851, N-3389,
RS-
33800, RS-56532, KB-6806, KGA-0941,
S-
21007, LY-278584, DAU-6285, R-093777,
GYKI-46903,
ML-1035, L-683877, KB-6933, RG-12915,
SC-52150, GK-128
Vomiting (emesis) results from an intricate series of physiological events
mediated by humoral factors and afferent fibers, and both inhibition and
excitation
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-11-
of somatic visceral musculature that are ultimately coordinated by the
vomiting
center in the brain." The emetic (vomiting) center is a nucleus of cells
located in
the medulla and is the motor center responsible for the coordination of
emesis.4
5 In the case of chemotherapy induced emesis, chemotherapy and radiation
therapy stimulate the enterochromaffln cells in the gut to release 5-hydroxy-
tryptamine (5-HT or serotonin). The binding of serotonin to the 5-HT3
receptor,
located in the gastrointestinal tract, emetic center in the medulla and
elsewhere in
the body, is a potent stimulant for nausea and vomiting.4 5-HT3 receptor
10 antagonists do not prevent the release of serotonin but instead create a
blockade
that prevents serotonin from binding to the 5-HT3 receptors. The binding of
the
antagonist to the receptor thereby prevents transmission of impulses that
initiate
nausea and vomiting.$ Other receptors associated with nausea and vomiting
include histamine (H1), dopamine (D2), muscarinic (M1), acetylcholine,
15 noradrenaline and endorphin.8
Currently, a variety of 5-HT3 receptor antagonists are used to treat
chemotherapy induced emesis. Examples of these antagonists include ondansetron
(Zofran~, Glaxo Wellcome), granisetron (Kytril~, SmithKline), dolasetron
20 (Anzemet°, Hoechst Marion Roussel), tropisetron (Sandoz), itasetron
(currently in
Phase III testing, Boehringer Ingelheim) and mirtazipine. These can be used
either alone as a single agent, in combination, or in a combination cocktail
with
dexamethasone, a corticosteroid.' The compounds can also be used in
combination with NK1 receptor antagonists. Currently, the use of these
25 compounds does not eiiminate chemotherapy induced emesis in all patients.
Accordingly, novel ligands having desired potency and therapeutic effect
for the 5-HT3 receptor. would be particularly desirable in order to further
inhibit
emesis, especially chemotherapy induced emesis, in mammalian patients. Such
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-12-
novel ligands would preferably achieve the desired potency and therapeutic
effect
by modulating one or more of the ligand's properties as to efficacy, affinity,
safety
profile, selectivity, duration of action and/or onset of action.
SUMMARY OF THE INVENTION
This invention is directed, in part, to novel multi-binding compounds that
bind SHT3 receptors and consequently these compounds can be used to treat
conditions mediated by SHT3 receptors such as chemotherapy induced and
radiation induced emesis, anxiety, schizophrenia, drug withdrawal and
cognitive
10 disorders.
Accordingly, in one of its composition aspects, this invention is directed to
a multi-binding compound and salts thereof comprising 2 to 10 ligands, which
may be the same or different and which are covalently attached to a linker or
15 linkers which may be the same of different, at least one of the ligands
comprising
a ligand domain capable of binding to a 5-HT3 receptor. Preferably, at least
two
and more preferably each of the ligands comprises a ligand domain capable of
binding to a SHT3 receptor. Since the 5-HT3 receptor is a pentameric
construct,
the most preferred compounds include between 2 and S ligands. More
20 preferably, each of the ligands independently comprises an agonist, partial
agonist,
inverse agonist or antagonist of the SHT3 receptors.
The mufti-binding compounds of this invention are preferably represented
by formula I:
25
~L~P~X~y I
wherein each L is independently selected from ligands comprising a ligand
domain
30 capable of binding to a 5-HT3 receptor; X is independently a linker; p is
an integer
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-13-
of from 2 to 10; g is an integer of from 1 to 20; and pharmaceutically
acceptable
salts thereof. Preferably, g is less than p.
Preferably, the ligands comprise a ligand domain capable of binding to one
5 or more 5-HT3 receptors which modulate chemotherapy induced and radiation
induced emesis, anxiety, schizophrenia, drug withdrawal and cognitive
disorders
in mammals. More preferably, the ligands are selected from the group
consisting
of ondansetron, granisetron, tropisetron, dolasetron, mirtazapine and
itasetron.
However, in one embodiment, at least one of the ligands in the mufti-binding
10 compound is a corticosteroid.
In still another of its composition aspects, this invention provides a
multibinding compound of formula II:
15 L'- X'- L' II
wherein each L' is independently a ligand comprising a ligand binding site
for the the SHT3 receptors, and is preferably an agonist, partial agonist,
inverse
agonist or antagonist of the SHT3 receptors and X' is a linker; and
20 pharmaceutically-acceptable salts thereof.
Preferably, in the multibinding compound of formula II, each Iigand, L', is
independently selected from the group consisting of ondansetron, granisetron,
tropisetron, dolasetron, mirtazapine and itasetron; X' is a linker; and
25 pharmaceutically-acceptable salts thereof.
Preferably, in the above embodiments, each linker (i.e., X or X')
independently has the formula:
CA 02321191 2000-08-15
WO 99/64046 , PCT/US99/12768
-14-
-Xa-Z_(ya-Z)m yb-Z_Xa_
wherein
m is an integer of from 0 to 20;
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 is at each separate occurrence is selected from the group consisting of
alkylene, substituted alkylene, cycloalkylene, substituted cylcoalkylene,
alkenylene, substituted alkenylene, alkynylene, substituted alkynylene,
cycloalkenylene, substituted cycloalkenylene, arylene, heteroarylene,
heterocyclene, or a covalent bond;
Ya and Yb at each separate occurrence are selected from the group
consisting of -C(O)NR'-, -NR'C{O)-, -NR'C(O)NR'-, -C(=NR')-NR'-,
-NR'-C(=NR')-, -NR'-C(O)-O-, -N=C(Xa)-NR'-, -P(O)(OR')-O-,
-S(O)"CR'R"-, -S(O)S NR'-, -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,
alkenyl,
substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl,
substituted
alkynyl, aryl, heteroaryl and heterocyclic.
In another of its composition aspects, this invention is directed to
a pharmaceutical composition comprising a pharmaceutically acceptable
excipient
and an effective amount of a mufti-binding compound, or a pharmaceutically
acceptable salt thereof, comprising 2 to 10 ligands which may be the same or
different and which are covalently attached to a linker or linkers which may
be the
same or different, at least one of the ligands comprising a ligand domain
capable
of binding to one or more 5-HT3 receptors. Preferably, the compound is a
compound of formula I or II.
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-15-
In one of its method aspects, this invention is directed to a method for
treating or preventing disorders mediated by 5-HT3 receptors, such as
chemotherapy induced and radiation induced emesis, anxiety, and schizophrenia
and cognitive disorders or ameliorating the effects of drug withdrawal in a .
mammal which method comprises administering to said mammal an effective
amount of a pharmaceutical composition comprising a pharmaceutically
acceptable
excipient and a mufti-binding compound, or a pharmaceutically acceptable salt
thereof, comprising 2 to 10 ligands which may be the same or different and
which
are covalently attached to a linker or linkers which may be the same or
different,
at least two of said ligands comprising a ligand domain capable of binding to
a 5-
HT3 receptor. Preferably, the pharmaceutical composition comprises a
pharmaceutically acceptable excipient and a mufti-binding compound represented
by formulas I or II as described above.
When the compounds are used in the methods described above, preferably,
q is less than p, and more preferably, the ligand is selected from the group
consisting of ondansetron, granisetron, tropisetron, dolasetron, mirtazapine
and
itasetron. However, in one embodiment, at least one of the ligands in said
multi-
binding compound is a corticosteroid.
This invention is also directed to general synthetic methods for generating
large libraries of diverse multimeric compounds which multimeric compounds are
candidates for possessing multibinding properties with respect to the SHT3
receptors. The diverse multimeric compound libraries provided by this
invention
are 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,
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-16-
amphiphilicity, acidity, basicity and polarizability and/or polarization. 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.
This invention is also directed to libraries of diverse multimeric compounds
which multimeric compounds are candidates for possessing multibinding
properties with respect to the SHT3 receptors. 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 the SHT3 receptors.
Accordingly, in one of its method aspects, this invention is directed to a
method for identifying multimeric ligand compounds possessing multibinding
properties with respect to the SHT3 receptors which method comprises:
(a) identifying a ligand or a mixture of ligands which bind to the SHT3
receptors wherein each ligand contains at least one reactive functionality;
(b) identifying a library of linkers wherein each linker in the 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 (a) with the library of linkers identified in (b) under conditions wherein
the
complementary functional groups react to form a covalent linkage between the
linker and at least two of the ligands; and
(d) assaying the multimeric ligand compounds produced in (c) above to
identify multimeric ligand compounds possessing multibinding properties.
In another of its method aspects, this invention is directed to a method
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-17-
for identifying multimeric ligand compounds possessing multibinding properties
which method comprises:
(a) identifying a library of ligands which bind to SHT3 receptors
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;
(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 the
linker and at least two of the ligands; and
(d) assaying the multimeric ligand compounds produced in (c) above to
identify multimeric ligand compounds possessing multibinding properties.
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).
Sequential addition is preferred when a mixture of different ligands is
employed to
ensure heteromeric or multimeric compounds are prepared. Concurrent addition
of the ligands occurs when at least a portion of the multimer comounds
prepared
are homomultimeric compounds.
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).
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-18-
In one of its composition aspects, this invention is directed to a library of
multimeric ligand compounds which may possess multivalent properties which
library is prepared by the method comprising:
(a) identifying a ligand or a mixture of ligands which bind to the SHT3
receptors wherein each ligand contains at least one reactive functionality;
(b) identifying a library of linkers wherein each linker in the library
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
complementary functional groups react to form a covalent linkage between the
linker and at least two of the ligands.
In another of its composition aspects, this invention is directed to a library
of multimeric ligand compounds which bind to the SHT3 receptors which may
possess multivalent properties which library is prepared by the method
comprising:
(a) identifying a library of ligands which bind to the SHT3 receptors
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
(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 the
linker and at least two of the ligands.
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-19-
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 polarizability and/or polarization 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 100.
In another preferred embodiment, the ligand or mixture of ligands is
selected to have reactive functionality at different sites on the ligands in
order to
provide for a range of orientations of the ligand on the 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 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
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 the SHT3 receptors. Specifically, this method aspect is
directed
CA 02321191 2000-08-15
WO 99/64046 PCTNS99/12768
-20-
to a method for identifying multimeric ligand compounds possessing
multibinding
properties with respect to the SHT3 receptors 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
ligand or mixture of ligands which target the SHT3 receptors with a linker or
mixture of linkers wherein the ligand or mixture of ligands comprises at least
one
reactive functionality and the 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 the contacting is conducted under
conditions wherein the complementary functional groups react to form a
covalent
linkage between the linker and at least two of the ligands;
(b) assaying the first collection or iteration of multimeric compounds to
assess which if any of the multimeric compounds possess multibinding
properties;
(c) repeating the process of (a) and (b) above until at least one
multimeric compound is found to possess multibinding properties;
(d) evaluating what molecular constraints imparted multibinding
properties to the multimeric compound or compounds found in the first
iteration
recited in (a)- (c) above;
(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 the first
iteration;
(f) evaluating what molecular constraints imparted enhanced
multibinding properties to the multimeric compound or compounds found in the
second collection or iteration recited in (e) above;
(g) optionally repeating steps (e) and (f) to further elaborate upon the
molecular constraints.
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-21-
Preferably, steps (e) and (f) are repeated at least two times, more
preferably at from 2-50 times, even more preferably from 3 to 50 times, and
still
more preferably at least 5-50 times.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1-4 illustrate reaction schemes for preparing mufti-binding
compounds of this invention.
Figure 5 illustrates examples of multibinding compounds comprising 2
ligands attached in different forms to a linker.
Figure 6 illustrates examples of multibinding compounds comprising 3
ligands attached in different forms to a linker.
Figure 7 illustrates examples of multibinding compounds comprising 4
ligands attached in different forms to a linker.
Figure 8 illustrates examples of multibinding compounds comprising 5-10
ligands attached in different forms to a linker.
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-22-
DETAILED DESCRIPTION OF THE INVENTION
Ligand (drug) interactions with cellular receptors are controlled by
molecular interaction/recognition between the ligand and the receptor. In
turn,
such interaction can result in modulation or disruption of the biological .
5 processes/functions of these receptors and, in some cases, leads to cell
death.
Accordingly, when cellular receptors mediate mammalian pathologic conditions,
interactions of the ligand with the cellular receptor can be used to treat
these
conditions. Of particular interest are mammalian 5-HT3 receptors which are
known to modulate emetic conditions in mammals; particularly chemotherapy
10 induced emetic conditions. As noted above, this invention is directed, in
part, to
multi-binding compounds that bind 5-HT3 receptors.
The "affinity" and "specificity" of the 5-HT3 receptor and a ligand thereto
are dependent upon the complementarity of molecular binding surfaces and the
15 energetic costs of complexation. "Affinity" is sometimes quantified by the
equilibrium constant of complex formation. Specificity relates to the
difference in
affinity between the same ligand binding to different ligand binding sites on
the
cellular receptor.
20 The mufti-binding compounds of this invention are capable of acting as
mufti-binding agents and the surprising activity of these compounds arises at
least
in part from their ability to bind in a multivalent manner with mammalian 5-
HT3
receptors.
25 Preferably, the ligands in the multibinding compounds comprise agonists,
partial agonists, inverse agonists or antagonists of the SHTz receptors.
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-23-
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
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. 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,
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,
15 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, vitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl,
-SO-heteroaryl, -SOZ-alkyl, -SOZ-substituted alkyl, -SO,-aryl and -SOZ-
heteroaryl.
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.
This term is exemplified by groups such as methylene (-CH,-), ethylene
(-CHZCHz-), the propylene isomers (e.g., -CH,CHZCH,- and -CH(CH3)CHZ-) and
the like. .
CA 02321191 2000-08-15
PCT/US99/12~68
WO 99/64046
-24-
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
from the group consisting of alkoxy, substituted alkoxy, cycloalkyl,
substituted
cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy,
.
S 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, -SO-substituted alkyl, -SO-aryl, -
SO-heteroaryl, -SOZ-alkyl, -SO~-substituted alkyl, -SOZ aryl and -SO,-
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, 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" refers to the groups -alkylene-aryl and -substituted
alkylene-aryl where alkylene, substituted alkylene and aryl are defined
herein.
Such alkaryl groups are exemplified by benzyl, phenethyl and the like.
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,
tert-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
CA 02321191 2000-08-15
WO 99/64046 PCTNS99/12768
-25-
substituted alkynyl-O- where substituted alkyl, substituted aIkenyl,
substituted
cycloalkyl, substituted cycloalkenyl and substituted alkynyl are as defined
herein.
The term "alkylalkoxy" refers to the groups -alkylene-O-alkyl,
S alkylene-O-substituted alkyl, substituted alkylene-O-alkyl and substituted
alkylene-
O-substituted alkyl wherein alkyl, substituted alkyl, alkylene and substituted
alkylene are as defined herein. Preferred alkylalkoxy groups are
alkylene-O-alkyl and include, by way of example, methylenemethoxy
(-CH~OCH3), ethylenemethoxy (-CH,CHZOCH3), n-propylene-iso-propoxy
(-CHZCH,CHZOCH(CH3)Z), methylene-t-butoxy (-CHz-O-C(CH3)3) and the like.
The term "alkylthioalkoxy" refers to the group -alkylene-S-alkyl,
alkylene-S-substituted alkyl, substituted alkylene-S-alkyl and substituted
alkylene-
S-substituted alkyl wherein alkyl, substituted alkyl, alkylene and substituted
alkylene are as defined herein. Preferred alkylthioalkoxy groups are alkylene-
S-
alkyl and include, by way of example, methylenethiomethoxy (-CHZSCH3),
ethylenethiomethoxy (-CHZCHZSCH3), n-propylene-iso-thiopropoxy
(-CHZCHZCHZSCH(CH3)2), methylene-t-thiobutoxy (-CHZSC(CH3)3) and the like.
The term "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=CHZ), n-propenyl
(-CHzCH=CHZ), iso-propenyl (-C(CH3)=CHz), and the like.
The term "substituted alkenyl" refers to an alkenyl 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
CA 02321191 2000-08-15
WO 99/64046
- PCT/US99/12768
-26-
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, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl,
-SO-heteroaryl, -SOZ-alkyl, -SO,-substituted alkyl, -SOz-aryl and -SOZ-
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 exemplified by groups such as ethenylene (-CH=CH-), the
propenylene isomers (e.g., -CHzCH=CH- and -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,
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, thioheterocyclooxy, thiol, thioalkoxy,
substituted
thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic,
heterocyclooxy,
hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -
SO-heteroaryl, -SO~-alkyl, -SO~-substituted alkyl, -SOZ-aryl and -SOZ-
heteroaryl.
Additionally, such substituted alkenylene groups include those where 2
substituents on the alkenylene group are fused to form one or more cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,
heterocyclic or
heteroaryl groups fused to the alkenylene group.
CA 02321191 2000-08-15
WO 99/64046
' PCTNS99/12768
-27-
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 from 1-6 sites of acetylene (triple bond) unsaturation. Preferred
alkynyl groups include ethynyl (-C---CH), propargyl (-CHIC--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, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy,
amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano,
halogen, hydroxyl, keto, thioketo, carboxyl, carbaxylalkyl, 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, -SO~-alkyl, -SOZ-substituted alkyl, -SO,-aryl and -SOZ-
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 from 1-6 sites of acetylene (triple bond) unsaturation. Preferred
alkynylene groups include ethynylene (-C---C-), propargylene (-CH,C---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 alkoxy, cycloalkyl,
substituted
cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy,
amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano,
CA 02321191 2000-08-15
WO 99/64046
- PCT/US99/12768
-28-
halogen, hydroxyl, keto, thioketo, carboxyl, carboxylaikyl, thioarylvxy,
thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted
thioalkoxy,
aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy,
hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -
SO-heteroaryl, -SOZ-alkyl, -SO,-substituted alkyl, -SOZ-aryl and -SO~-
heteroaryl
The term "acyl" refers to the groups HC(O)-, alkyl-C(O)-, substituted
alkyl-C(O)-, cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-, cycloalkenyl-C(O)-
,
substituted cycloalkenyl-C(O)-, aryl-C(O)-, heteroaryl-C(O)- and heterocyclic-
10 C(O)- where alkyl, substituted alkyl, 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,
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.
20 The term "aminoacyl" refers to the group -NRC(O)R where each R is
independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or
heterocyclic
wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as
defined
herein.
25 The term "aminoacyloxy" or "alkoxycarbonyiamino" refers to the group
-NRC(O)OR where each R is independently hydrogen, alkyl, substituted alkyl,
aryl, heteroaryl, or heterocyclic wherein alkyl, substituted alkyl, aryl,
heteroaryl
and heterocyclic are as defined herein.
CA 02321191 2000-08-15
WO 99/64046
- PCT/US99/12768
-29-
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,
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 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,
15 acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
substituted alkyl,
substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted
cycloalkyl,
substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino,
alkaryl,
aryl, aryloxy, azido, carboxyl, carboxyialkyl, cyano, halo, vitro, heteroaryl,
heteroaryloxy, heterocyclic, heterocyclooxy, aminoacyloxy, oxyacylamino,
20 thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-
alkyl, -SO-
substituted alkyl, -SO-aryl, -SO-heteroaryl, -SOZ-alkyl, -SOZ-substituted
alkyl, -
SOZ-aryl, -SOZ-heteroaryl and trihalomethyl. Preferred aryl substituents
include
alkyl, alkoxy, halo, cyano, vitro, trihalomethyl, and thioalkoxy.
25 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.
CA 02321191 2000-08-15
WO 99/64046
- PCTNS99/12768
-30-
The term "aryiene" 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 -NHS.
The term "substituted amino refers to the group -NRR where each R is
independently selected from the group consisting of hydrogen, alkyl,
substituted
alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl,
cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl,
heteroaryl and heterocyclic provided that both R's are not hydrogen.
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 alkenyl",
"-C(O)O-alkynyl" and "-C(O)O-substituted alkynyl" where alkyl, substituted
alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl,
alkynyl and
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 adamantanyl, and the like.
The term "substituted cycloalkyl" refers to cycloalkyl groups having from
CA 02321191 2000-08-15
WO 99/64046
' PCT/US99/12768
-31-
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, carboxyialkyl, 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, -SO~-alkyl, -SOZ-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
unsaturation. Examples of suitable cycloalkenyl groups include, for instance,
cyclobut-2-enyi, 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 alkoxy, substituted alkoxy, cycloalkyl, substituted
cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino,
20 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, -SO-substituted alkyl, -SO-aryl,
-SO-heteroaryl, -SOZ-alkyl, -SOZ-substituted alkyl, -SO~-aryl and -SOz-
heteroaryl.
The term "halo". or "halogen" refers to fluoro, chloro, bromo and iodo.
CA 02321191 2000-08-15
WO 99/64046
' PCT/US99/12768
-32-
The term "heteroaryl" refers to an aromatic group of from 1 to 15 carbon
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 S
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, 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, -SOZ-substituted alkyl, -SOZ-aryl, -SO,-heteroaryl and
trihalomethyl. Preferred aryl substituents include alkyl, alkoxy, halo, cyano,
vitro, trihalomethyl, and thioalkoxy. Such heteroaryl groups can have a single
ring (e.g., pyridyl or furyl) or multiple condensed rings (e.g., indolizinyl
or
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, 1,4-benzofuranylene, 2,5-pyridnylene, 2,5-indolenyl and the
like.
The term "heterocycle" or "heterocyclic" refers to a monoradical saturated
or unsaturated group having a single ring or multiple condensed rings, from 1
to
CA 02321191 2000-08-15
WO 99/64046
' PCTNS99/12768
-33-
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.
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, 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, alkoxyamino, nitro, -SO-alkyl, -SO-
substituted alkyl, -SO-aryl, -SO-heteroaryl, -SOZ-alkyl, -SOZ-substituted
alkyl, -
SOZ-aryl and -SOZ-heteroaryl. Such heterocyclic groups can have a single ring
or
multiple condensed rings. Preferred heterocyclics include morpholino,
piperidinyl, and the like.
Examples of nitrogen heterocycles and heteroaryls include, but are not
limited to, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine,
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, piperidine, piperazine, indoline, morpholino,
piperidinyl, tetrahydrofuranyl, and the like as well as N-alkoxy-nitrogen
containing heterocycles.
A preferred class of heterocyclics include "crown compounds" which
refers to a specific class of heterocyclic compounds having one or more
repeating
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-34-
units of the formula [-(CHZ-)mY-] where m is z 2, and Y at each separate
occurrence can be O, N, S or P. Examples of crown compounds include, by way
of example only, [-(CHZ)3-NH-]3, [-((CHZ),-O)4-((CHZ)~-NH)2] and the like.
Typically such crown compounds can have from 4 to 10 heteroatoms and 8 to 40
carbon atoms.
The term "heterocyclooxy" refers to the group heterocyclic-O-.
The term "thioheterocyclooxy" refers to the group heterocyclic-S-.
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, 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
attached to another ring via one carbon atom common to both rings.
The term "thiol" refers to the group -SH.
The term "thioalkoxy" refers to the group -S-alkyl.
The term "substituted thioalkoxy" refers to the group -S-substituted alkyl.
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-35-
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
heteroaryl group is as defined above including optionally substituted aryl
groups
as also 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.
The ligands and linkers which comprise the multibinding agents of the
invention and the multibinding compounds themselves may have various
stereoisomeric forms, including enantiomers and diastereomers. It is to be
understood that the invention contemplates all possible stereoisomeric forms
of
multibinding compounds, and mixtures thereof.
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
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
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-36-
salts. Salts derived from organic bases include, but are not limited to, salts
of
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,
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,
diheterocyclic amines, triheterocyclic amines; mixed di- and tri-amines where
at
least two of the substituents on the amine are different and are selected from
the
group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
aryl,
heteroaryl, heterocyclic, and the like. Also included are amines where the two
or
three 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, 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 carboxamides, dialkyl carboxamides, and the like.
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-37-
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,
5 glycolic 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
pharmaceutically-acceptable salt.
The term "protecting group" or "blocking group" refers to any group
which when bound to one or more hydroxyl, thiol, amino or carboxyl groups of
15 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. The particular removable blocking group employed is not critical and
preferred removable hydroxyl blocking groups include conventional substituents
20 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
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.
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-38-
Preferred removable amino blocking groups include conventional
substituents such as t-butyoxycarbonyl (t-BOC), benzyloxycarbonyl (CBZ),
fluorenylmethoxycarbonyl (FMOC), allyloxycarbonyl (ALOC), and the like which
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
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 the event or circumstance occurs and instances where
it
does not.
The term "ligand" as used herein denotes a compound that is a binding
partner for the SHT3 receptors and is bound thereto by complementarity. The
ligand is preferably an agonist, partial agonist, inverse agonist or
antagonist of the
SHT3 receptors. The specific region or regions of the ligand that is (are)
20 recognized by the receptor is designated as the "ligand domain". A ligand
may be
either capable of binding to a receptor by itself, or may require the presence
of
one or more non-ligand components for binding (e.g., Ca+2, Mg+~ or a water
molecule is required for the binding of a ligand to various ligand binding
sites).
The ligands and linkers which comprise the multibinding agents of the
invention
25 and the multibinding compounds themselves may have various stereoisomeric
forms, including enantiomers and diastereomers. It is to be understood that
the
invention contemplates all possible stereoisomeric forms of multibinding
compounds and mixtures thereof.
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-39-
Examples of ligands useful in this invention are described herein, and
specifically include mirtazapine, granisetron, ondansetron, paroxetine (binds
all 5-
HT receptor subtypes), tropisetron, dolasetron, and itasetron and analogs
thereof.
Those skilled in the art will appreciate that portions of the ligand structure
that are
5 not essential for specific molecular recognition and binding activity may be
varied
substantially, replaced or substituted with unrelated structures (for example,
with
ancillary groups 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
10 intended to be limited to compounds known to be useful in binding to SHT3
receptors. (e.g., known drugs). Those skilled in the art will understand that
the
term ligand can equally apply to a molecule that is not normally associated
with
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
15 as multivalent compounds because of the benefits conferred by multivalency.
Other ligands useful in this invention are ligands directed to.other receptors
which modulate chemotherapy induced emesis such as the dopamine (D2),
histamine (H1) and muscarinic (M1). D2 antagonists useful in this invention
20 include phenothiazines, such as chlorpromazine, perphenazine,
prochlorperazine,
promethazine, thiethylperazine, triflupromazine; benzimidazole derivatives
such as
domperidon; and butyrophenones such as haloperidol and droperidol. Substituted
benzamides, such as metoclopramide, trimethobenzamide and metopimazine are
both D2 and 5-HT3 antagonists. Combinations of ligands to the 5-HT3 and the D2
25 receptors may also be made as mufti-binding compounds. Antagonists to H1
(histamine) receptor, currently thought to be minimally effective in reducing
chemotherapy induced emesis, such as diphenhydramine and meclizine, rnay also
be used in this invention.
CA 02321191 2000-08-15
WO 99/64046 PCTNS99/12768
-40-
The term "multibinding compound or agent" refers to a compound that is
capable of multivalency, as defined below, and which has 2-10 ligands which
comprise a ligand binding domain which is capable of binding to one or more
5HT3 receptors, and which is covalently bound to one or more linkers which may
be the same or different. Multibinding compounds provide a biological and/or
therapeutic effect greater than the aggregate of unlinked ligands 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, fvr example: increased affinity, increased
selectivity for target, increased specificity for target, increased potency,
increased
efficacy, decreased toxicity, improved duration of activity or action,
decreased
side effects, increased therapeutic index, improved bioavailibity, improved
pharmacokinetics, improved activity spectrum, and the like. The multibinding
compounds of this invention will exhibit at least one and preferably more than
one
of the above-mentioned effects.
"Emesis" is the act or instance of vomiting. An "emetic agent" is an agent
that induces vomiting. Emesis also includes nausea. In this invention, emetic
agents include compounds used in chemotherapy and radiation therapy.
"Chemotherapy induced emesis" refers to emetic episodes which are
induced by exposure to chemotherapy and/or anti-cancer treatments such as
treatment with, for example, cisplatin, adriamycin, apomorphine,
cyclohexamide,
cyclophosphamide, copper sulphate, ipecacuantla, mustine and radiation, among
others. Chemotherapy induced emetic episodes may be acute or may be delayed
up to several days. There are generally three types of chemotherapy induced
emesis seen clinically: acute, delayed and anticipatory (conditioned).' Acute
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-41-
chemotherapy induced nausea and vomiting generally is considered to be that
which occurs within the first 24 hours following drug administration.8 Delayed
emesis is generally defined as emetic episodes starting about 24 hours or more
following the last treatment.' This can be a serious complication for
patients, as it
5 can be protracted and severe and there are very few good treatment options
for its
prevention.4 Conditioned or anticipatory emesis results from poor control of
acute or delayed emesis. It is typically associated with anxiety prior to the
next
dose of chemotherapy, followed by nausea or vomiting before, during or
possibly
after the administration of chemotherapy.'
10
The term "potency" refers to the minimum concentration at which a ligand
is 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
15 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 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
20 indicative of enhanced potency.
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
25 ligands) exhibit univalency when only one ligand is interacting with
a.ligand
binding site. Examples of univalent interactions are depicted below.
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-42-
' Mi
10 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 receptors which
may be the same or different.
15 For example, two ligands connected through a linker that bind concurrently
to two ligand 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:
20
E--
_,r.- -
25 Univalent Interaction
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-43-
5
Trivalent Interaction
10
It should be understood that all compounds that contain multiple copies of a
ligand attached to a linker or to linkers do not 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
15 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.
20 The term "selectivity" or "specificity" is a measure of the binding
preferences of a ligand for different ligand binding sites (receptors). The
selectivity of a ligand with respect to its target ligand binding site
relative to
another Iigand 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
25 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 distinct ligand binding sites
(receptors)).
CA 02321191 2000-08-15
WO 99!64046 PCT/US99/12768
-44-
The term "ligand binding site" denotes the site on the SHT3 receptors 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,
agonism, antagonism, modulatory effects, may maintain an ongoing biological '
event, and the like. However, in one embodiment, the ligand(s) merely bind to
a
ligand binding site and do not have agonistic or antagonistic activity.
The terms "agonism" and "antagonism" are well known in the art.
Ligands which are full agonists are ligands which when bound trigger the
maximum activity seen by the natural ligands. Ligands which are partial
agonists
are ligands which when bound trigger sub-maximum activity. Ligands which are
antagonists are ligands that when bound, inhibit or prevent the activity
arising
from a natural ligand binding to the receptor. Antagonists may be of the
surmountable class (results in the parallel displacement of the dose-response
curve
of the agonist to the right in a dose dependent fashion without reducing the
maximal response for the agonist) or insurmountable class (results in
depression of
the maximal response for a given agonist with or without the parallel shift).
Ligands which are inverse agonists are ligands that, when bound, decrease the
basal activity of the unbound receptor or which provide an activity opposite
of the
natural agonist.
Ligands have measurable properties that relate to the interaction of the
ligand and the receptor. These include the affinity of the ligand for the
receptor,
which relates to the energetics of the binding, the efficacy of the ligand for
the
receptor, which relates to the functional downstream activity of the ligand,
the
kinetics of the ligand for the receptor, which defines the onset of action and
the
duration of action, and the desensitization of the receptor for the ligand.
Selectivity defines the ratio of the affinity and/or efficacy of a ligand
across two
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-45-
receptors. 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. It is a combination of these properties which provides the foundation
for
defining the nature of the functional response.
It should be recognized that the ligand binding sites of the receptor that
participate in biological multivalent binding interactions are constrained to
varying
degrees by their infra- and inter-molecular associations (e.g., such
macromolecular
structures 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.
The terms "inert organic solvent" or "inert solvent" means a solvent which
is inert under the conditions of the reaction being described in conjunction
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.
The "5-HT3 receptor" is a serotonin receptor and plays a role in the
mechanisms of nausea and emesis. 5-HT3 receptors are located on neurons in
many systems in the periphery and in the brain.6 The 5-HT3 receptor is unique
in
that it belongs to the superfamily of ion-channel receptors'4 and is a ligand-
gated
ion channel that is permeant to Na+, K+, Caz+ and other cations.b 5-HT;
receptor
antagonists are effective, in the control of chemotherapy induced emesis in
mammals. They are also promising in the control of central nervous system
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-46-
conditions such as anxiety, schizophrenia, drug withdrawal and cognitive
disorders.'4
It should be recognized that the 5-HT3 receptors that participate in
biological multivalent binding interactions are constrained to varying degrees
by
their intra- and intermolecular associations (e.g. cellular receptors may be
covalently joined in 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 cellular
receptors
were present as monomers in solution. The 5-HT~ receptors are generally
pentameric structures, formed by association of five identical subunits.
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
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.
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 SHT3
receptors
in general, and those disease states which have been found to be usefully
treated
by a specific multibinding compound of our invention. Such disease states
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-4.7-
include, by way of example only, chemotherapy induced and radiation induced
emesis, anxiety, schizophrenia, drug withdrawal and cognitive disorders.
The term "therapeutically effective amount" refers to that amount of
multibinding compound which is sufficient to effect treatment, as defined
above,
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.
The term "linker", identified where appropriate by the symbol X or X',
refers to a group or groups that covalently links from 2 to 10 ligands (as
identified
above) in a manner that provides for a compound capable of multivalency, for
example, when in the presence of at least one cellular receptor having 2 or
more
ligand binding sites. Among other features, the linker is a ligand-orienting
entity
that permits attachment of multiple copies of a Iigand (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 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 term "library" refers to at least 3, preferably from lOZ to 109 and more
preferably from 102 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
multimeric compounds can be directly assayed for multibinding properties. In
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-48-
another embodiment, each member of the library of multimeric compounds is
first
isolated and, optionally, characterized. This member is then assayed for
multibinding properties.
S The term "collection" refers to a set of multimeric compounds which are
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.
10 The term "multimeric compound" refers to compounds comprising from 2
to 10 ligands covalently connected through at least one linker which compounds
may or may not possess multibinding properties (as defined herein).
The term "pseudohalide" refers to functional groups which react in
15 displacement reactions in a manner similar to a halogen. Such functional
groups
include, by way of example, mesyl, tosyl, azido and cyano groups.
The extent to which multivalent binding is realized depends upon the
efficiency with which the linker or linkers that joins the ligands presents
these
20 ligands to the array of available ligand binding sites. Beyond presenting
these
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
25 multibinding 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 SHT3 receptors, wherever such sites
are
located on the receptor structure, whether such sites are located interiorly,
both
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-49-
interiorly and on the periphery of the molecule, or at any intermediate
position
thereof. In one embodiment, the distance between the nearest neighboring
ligand
domains is preferably in the range of about 2~ to about 100, more preferably
in
the range of about 101 to about 501. In another embodiment, the linker lengths
S are in the range of about 3l~ to about 10~.
The ligands are covalently attached to the linker or linkers using
conventional chemical techniques providing for covalent linkage of the ligand
to
the linker or linkers. Reaction chemistries resulting in such linkages are
well
10 known in the art and involve the use of complementary functional groups on
the
linker and ligand. Preferably, the complementary functional groups on the
linker
are selected relative to the functional groups available on the ligand for
bonding or
which can be introduced onto the ligand for bonding. Again, such complementary
functional groups are well known in the art. For example, reaction between a
15 carboxylic acid of either the linker or the ligand and a primary or
secondary amine
of the ligand or the linker in the presence of suitable, well-known activating
agents
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 results in formation of a sulfonamide bond
20 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 results in formation of an ether bond covalently linking
the
ligand to the linker.
25 Table I below illustrates numerous complementary reactive groups and the
resulting bonds formed by reaction there between.
Table I
I~nresentative Complement_a~v Binding Chemitrr;Ps
CA 02321191 2000-08-15
WO 99/64046 PCTNS99/12768
-50-
First Reactive Groun Second Reactive Gro Lid
~n
hydroxyl isocyanate urethane
amine epoxide ~3-hydroxyamine
sulfonyl halide amine sulfonamide
carboxyl amine amide
hydroxyl alkyl/aryl halide ether
aldehyde amine/NaCNBH4 amine
ketone amine/NaCNBH~ amine
amine isocyanate urea
The linker is attached to the ligand at a position that retains ligand 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 tinker
embraces everything that is not considered to be part of the ligand.
The relative orientation in which the ligand domains are displayed derives
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
20 can be made on a ligand is typically based on prior knowledge of structure-
activity
relationships (SAR) of the ligand andlor congeners and/or structural
information
about ligand-receptor complexes (e.g., X-ray crystallography, NMR, and the
like). 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
25 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.
Suitable linkers and ligands are discussed more fully below.
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-51-
At present, it is preferred that the multibinding agent is a bivalent
compound, e.g., two ligands which are covalently linked to linker X.
5 The linker, when covalently attached to multiple copies of the ligands,
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, position of
atachment, 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 of the linker, and the like
on the
linker. Accordingly, the linker is preferably chosen to maximize the
biological
activity of the multibinding compound. 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 linker can be
considered
as a "framework" on which the ligands are arranged in 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
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, heteroaryl, cycloalkyl, heterocyclic, etc.). In other preferred
embodiments,
the ring is a six, nine, ten, twelve, fifteen and eighteen member ring. In
still
further preferred embodiments, the ring is an aromatic ring such as, for
example,
phenyl or naphthyl.
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-52-
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
hexamethylene diamine (HZN(CHZ)6NH2) 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" .
By controlling the hydrophilicity/hydrophobicity, the ability of the compounds
to
cross the blood/brain barrier can be controlled. This can be important when
one
wishes to maximize or minimize CNS effects.
Examples of molecular structures in which the above bonding patterns
could be employed as components of the linker are shown below.
20
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-53-
O
w ~ ~ w w ~~ \O~C~C~ HN~C~ i
O O N N O N C
O
wC~C.C' ~C~O~C' wC~N.C'
O O O ~C~C~
II O
~N~N' \O N' ~C N' II
O O O ~ ~O~C'
ii ii ~ ~S~
wS~S.N' wS.o.N' wC.o ~C' C C w .S~ i
O C S
O ~~ O
~C~S~C' ~C'O~N' ~O~N~ ~C~,OS, ~C~ ~C~S~C~
O~ O~/,
~O.C.O'
wN.O N' wN~N w.N~N, wC.N.C~
S~'
~C~S~O~ wS.C~S..- wN.C~O~ \N~N~ S~~
~N,
~~ O O
wC.PwC' wN.P~C~ wO.,yC..-
~N N O 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 used to aid in the identification of preferred frameworks
through
an iterative process. Numerous strategies are known to those skilled in the
art of
molecular design and can be used for preparing compounds of this invention.
It is to be noted that core structures other than 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.
CA 02321191 2000-08-15
WO 99/64046
' PCT/US99/12768
-54-
The above-described process can be extended to trimers and compounds of
higher valency.
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
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
category.
Other can be readily synthesized by methods well known in the art and/or are
described below.
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
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 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
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-55-
water, fats, lipids, biological fluids, etc.), hydrophobicity, hydrophilicity,
linker
flexibility, antigenicity, stability, and the like. For example, the
introduction of
one or more 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
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.
CA 02321191 2000-08-15
WO 99/64046
PCT/US99/12768
-56-
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
characteristics derive from the presence of phosphato, carboxylic, sulfato,
amino,
sulfhydryl, nitro 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 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,
lysophosphatidyl-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 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 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
CA 02321191 2000-08-15
WO 99/64046
PCT/US99/12768
-57-
heterocyclic groups. Other groups which can impart rigidity include
polypeptide
groups such as oligo- or polyproline chains.
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
10 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.
15 In preferred embodiments, rigidity is imparted by the presence of cyclic
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.
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-
25 charged groups closer to each other will tend to hold the linker in a
configuration
that maintains the separation between the like-charged ancillary groups.
Further
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
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-58-
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,
5 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.
In view of the above, it is apparent that the appropriate selection of a
linker
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, for example, polyethylene glycol).
As explained above, the multibinding compounds described herein
comprise 2-10 ligands attached to a linker that links the ligands in such a
manner
that they are presented to the receptor for multivalent interactions with
ligand
binding sites thereon/therein. The linker spatially constrains these
interactions to
20 occur within 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
25 empirical formula (L)P(X)q where L, X, p and q are as defined above. This
is
intended to 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.
CA 02321191 2000-08-15
WO 99/64046 , PCT/US99/12768
-59-
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 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 multibindmg 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.
Examples of such bivalent compounds are provided in Figure 5, where each
10 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 central core, and thus represented as (L),X, where the linker X
could
15 include, for example, an aryl or cycloalkyl group. Illustrations of
trivalent and
tetravalent compounds of this invention are found in Figures 6 and 7
respectively
where, again, the shaded circles represent ligands. Tetravalent compounds can
be
represented in a linear array, e.g.,
20 L-X-L-X-L-X-L
in a branched array, e.g.,
25 L-X-L-X-L
L
(a branched construct analogous to the isomers of butane -- n-butyl, iso-
butyl, sec-
30 butyl, and t-butyl) or in a tetrahedral array, e.g.,
CA 02321191 2000-08-15
WO 99/64046
PCT/(JS99/12768
L~ ~L
X
.,,,
L/ ''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
core linker.
The same considerations apply to higher multibinding compounds of this
invention containing S-10 ligands as illustrated in Figure 8 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
10 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.
15 Certain of the above described compounds may alternatively be represented
as cyclic chains of the form:
L
X X
-L
and variants thereof.
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-61-
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
following formula:
_Xa_Z_(Ya_Z)m_Yb-Z_Xa_
in which:
m is an integer of from 0 to 20;
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 is at each separate occurrence is selected from the group consisting of
alkylene, substituted alkylene, cycloalkylene, substituted cylcoalkylene,
alkenylene, substituted alkenylene, alkynylene, substituted alkynylene,
cycloalkenylene, substituted cycloalkenylene, arylene, heteroarylene,
heterocyclene, or a covalent bond;
Ya and Yb at each separate occurrence are selected from the group
consisting of:
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-62-
O O O
N N' \ \
R'
R R. R.
R~
N iR
N
I1 N/ \ ~ O
N ~ -p-O-
O R'
R'
O Xa
\ ~ ~ \ ~ i S(O)n_CR'R"-
N O N N
-S(O)~_NR~_
R. R.
-S-S- or a covalent bond;
in which:
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,
alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl,
substituted alkynyl, aryl, heteroaryl and heterocyclic.
10 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.
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-63-
In one embodiment of this invention, the linker (i.e., X or X') is selected
those shown in Table II:
Table II
$epresentative Linkerc
Linker
-HN-(CHZ) 2-NH-C(O)-(CHZ)-C(O)-NH-(CHZ) ,-NH-
-HN-(CHZ) Z-NH-C(O)-(CHZ) ,-C(O)-NH-(CHI) ,-NH-
-HN-(CHZ)z-NH-C(O)-(CHZ)3-C(O)-NH-(CHz) ,-NH-
-HN-(CHZ) ~-NH-C(O)-(CH2)4-C{O)-NH-(CHI) ,-NH-
-HN-(CH~) z-NH-C(O)-(CHZ)5-C(O)-NH-(CH,) ,-NH-
-HN-(CHZ) 2-NH-C(O)-(CHZ)6-C(O)-NH-(CH,) ,-NH-
-HN-(CHz) 2-NH-C(O)-(CHZ),-C(O)-NH-(CHI) ~-NH-
-HN-(CHZ) ~-NH-C(O)-(CHZ)8-C(O)-NH-(CH,) ~-NH-
-HN-(CHZ) Z-NH-C(O)-(CHZ)9-C(O)-NH-(CH,) ,-NH-
-HN-(CHZ) ~-NH-C(O)-(CHZ),o-C(O)-NH-(CH,) ,-NH-
-HN-(CH2) 2-NH-C(O)-(CH2)"-C(O)-NH-(CH,) ,-NH-
-HN-(CHZ) 2-NH-C(O)-(CHZ),2-C(O)-NH-(CH,) ~-NH-
-HN-(CH2) Z-NH-C(O)-Z-C(O)-NH-(CHZ) ~-NH- where Z is
1,2-phenyl
-HN-(CHZ) Z-NH-C(O)-Z-C(O)-NH-(CHZ) 2-NH- where Z is
1,3-phenyl
-HN-{CHZ)2-NH-C(O)-Z-C(O)-NH-(CHZ),-NH- where Z is 1,4-phenyl
-HN-(CHZ) 2-NH-C(O)-Z-O-Z-C(O)-NH-(CHI) ~-NH- where Z
is 1,4-phenyl
-HN-(CHZ) ~-NH-C(O)-(CHZ) 2-CH(NH-C(O)-(CH~)g-CH3)-C(O)-NH-(CHZ),-
NH=
-HN-(CHZ) z-NH-C(O)-(CHZ)-O-(CHZ)-C(O)-NH-(CHz),-NH-
-HN-(CHZ) ~-NH-C(O~Z-C(O)-NH-(CHI) Z-NH-
where Z is 5-(n-octadecyloxy)-1,3-phenyl
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
Linker
-HN-(CHZ) 2-NH-C(O)-(CHz) Z-CH(NH-C(O)-Z)-C(O)-NH-(CHZ)
Z-NH-
where Z is 4-biphenyl
-HN-(CHZ) 2-NH-C(O)-Z-C(O)-NH-(CHZ)2-NH-
where Z is 5-(n-butyloxy)-1,3-phenyl
-HN-(CHZ) 2-NH-C(O)-(CH2)8-trans-(CH=CH)-C(O)-NH-(CHZ)
2-NH-
-HN-(CHZ) 2-NH-C(O)-(CH2) Z CH(NH-C(O)-(CHZ)12-CH3)-C(O)-NH-(CH2)z-
NH-
-HN-(CHZ)2-NH-C(O)-(CHZ) Z-CH(NH-C(O)-Z)-C(O)-NH-(CHZ)
2-NH-
where Z is 4-(n-octyl)-phenyl
-HN-(CHZ)-Z-O-(CHZ)6-O-Z-(CHz)-NH- where Z is 1,4-phenyl
-HN-(CHZ)2-NH-C(O)-(CH2)2-NH-C(O)-(CH~)3-C(O}-NH-(CHZ)z-C(O)-NH-
(CHZ)z-NH-
-HN-(CHZ) Z-NH-C(O)-(CHZ) Z CH(NH-C(O)-Ph)-C(O)-NH-(CHZ)
2-NH-
-HN-(CHz) ,-NH-C(O)-(CHZ)-N + ((CHZ)9-CH3)(CH,-C(O)-NH-(CHZ)
2-NHZ)-
(CH,)-C(O)-NH-(CHZ) Z-NH-
-HN-(CHZ) 2-NH-C(O)-(CHZ)-N((CHZ)9-CH3)-(CHZ)-C(O)-NH-(CHZ)
Z-NH-
-HN-(CHZ) 2-NH-C(O)-(CHZ) 2-NH-C(O)-(CHZ) ,-NH-C(O)-(CHZ)
3-C(O)-NH-
(CHZ) ~-C(O)-NH-(CHZ) 2-C(O)-NH-(CHZ)2-NH-
-HN-(CHZ) 2-NH-C(O)-Z-C(O)-NH-(CHZ) 2-NH-
where Z is 5-hydroxy-1,3-phenyl
In another embodiment of this invention, the linker (i.e., X or X') has the
formula
Ib Ib
Ra O-CH-CH O-Ra
wherein
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-65-
each Ra is independently selected from the group consisting of a covalent
bond, alkylene, substituted alkylene and arylene;
each Rb is independently selected from the group consisting of hydrogen,
alkyl and substituted alkyl; and
n ' is an integer ranging from 1 to about 20.
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.
Ligands
Any compound which is comprises a ligand domain capable of binding to a
SHT3 receptor, preferably, which is an agonist, partial agonist, inverse
agonist or
antagonist of the SHT3 receptors, and which can be covalently linked to a
linker
can be used as a ligand to prepare the compounds described herein. Such
ligands
are well known to those of skill in the art.
20 Examples of SHT3 ligands which can be used as ligands to prepare the
compounds of the present invention include mirtazapine, granisetron,
ondansetron,
paroxetine (binds all 5-HT receptor subtypes), tropisetron, dolasetron, and
itasetron. and analogs thereof. Analogs (for these and other known compounds
which bind to the SHT3 receptors) which can be used include alkylated,
acylated,
25 carboxylated, amidated, sulfonated, phosphorylated, aminated, hydroxylated,
thiolated, and halogenated derivatives. Examples of some of these ligands are
shown below:
CA 02321191 2000-08-15
WO 99/64046
PCT/US99/12768
-66-
N
\ ~N
CH3 (Ondansetron)
N
N,CH3
\ N
H (Granisetron)
,N
N
CH3
O
/ \ "O
y
N N~ (Tropisetron)
H CH3
O
O
(Dolasetron)
N N
H O
CA 02321191 2000-08-15
WO 99/64046 PCTNS99/I27G8
-67-
Ligands which bind to SHT3 receptors (and the precursors and analogs
thereof) are well-known in the art and can be readily prepared using art-
recognized starting materials, reagents and reaction conditions. By way of
illustration, the following patents and publications disclose compounds,
intermediates and procedures useful in the preparation of ligands which bind
to
SHT3 receptors:
Ahlman and Dahlstrom, "Storage and Release of S-Hydroxytryptamine in
Enterochromaffin Cells of the Small Intestine," in S- vdroxvtrvn a",;"P ;.,
Perir~heral Reaction , ed. De Clerk and Vanhoutte, Raven Press, NY, (1982)
Andrews, "5-HT3 receptor antagonists and antiemesis,", pp. 255-317, in S-
Hydroxytryptamine-3-receptor antagonists, King, Jones and Sanger, eds., Boca
Raton, CRC Press, (1994)
Boess and Martin, Neurouharmacoloev 33(3/4):275-317 (1994)
Chevallier, Br. J. Cancer. 68:1760180 (1993)
Cubeddu, On~ø,~ 53(suppl. 1):18-25 (1996)
Cunningham, ONF. 24(7):33-40 (1997)
Fletcher and Barnes, TIPS. 19:212-215 (1998)
Gralla, Eur. J. Cancer 33(suppl 4):563-567 (1997)
Gregory and Ettinger, 55(2):173-189 (1998)
Hirokawa et al., Biooreanic and Medicinal hem .err 8:619-6234
(1998)
Lovinger, J. Neuro~hvcinino~ 66:1329-1337 (1991)
Marr et al., Pharmacolo~~ 48:283-292 (1994)
Milano et al., J Pharmacol And xp Thera~T_ 274:951-961 (1995)
Naylor and Rudd, OncolQg~ 53:8-17 (1996)
Ohta et al., Chem. Pharrn Rmn 44(5):991-999 (1996)
Ohta et al., Chem. Pharm R nt 44(9):1707-1716 (1996)
Orjales et al., J. Med. Chem._ 40:586-593 (1997)
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-68-
Oxford et al., Proe. Med. hem 29:239-271 (1992)
Parker et al., TIPS. 17:95-99 (1996)
Pinder, Acta Psy~hiatr Scand 96(suppl. 391):7-13 (1997)
Rodriguez et al., J. CompurPr_A;~P.~ ~r"m,o~ 11:589-599 (1997)
Roila and DelFavero, Eur. J. Cancer_ 33:1364-1370 (1997)
Wilde and Markham, ,~,~g,~, 52(5):73-794 (1996)
van Wijngaarden et al., J. Med. _hPt" 36:3693-3699 (1993)
Yamada et al., Chem. Pharm Rmtt 46(3):445-451 (1998)
Each of these patents and publications is incorporated herein by reference
in its entirety to the same extent as if each individual patent or publication
was
specifically and individually indicated to be incorporated by reference in its
entirety.
Preparation of Mult~h~" ine Com
The multibinding compounds of this invention can be prepared from
readily available starting materials using the following general methods and
procedures. It will be appreciated that where typical or preferred process
conditions (i.e., reaction temperatures, times, mole ratios of reactants,
solvents,
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
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
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-69-
groups, and their introduction and removal, are described in T. W. Greene and
G.
M. Wuts, Protecting Groups in Organic Synthesis, Second Edition, Wiley, New
York, 1991, and references cited therein.
Any compound which is an agonist, partial agonist, inverse agonist or
antagonist of the SHT3 receptors, preferably antagonists of the SHT3
receptors,
can be used as a ligand in this invention. As discussed above, numerous such
receptor agonists, partial agonists, inverse agonists and 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 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 above provide
numerous examples of suitably functionalized agonists, partial agonists and
antagonists of the SHT3 receptors, and intermediates thereof, which may be
used
as ligands in this invention.
The ligands can be covalently attached to the linker through any available
position on the ligands, provided that when the ligands are attached to the
linker,
at least one of the ligands retains its ability to bind to the SHT3 receptors.
Certain
sites of attachment of the linker to the ligand are preferred based on known
structure-activity relationships. Preferably, the linker is attached to a site
on the
ligand where structure-activity studies show that a wide variety of
substituents are
tolerated without loss of receptor activity.
It will be understood by those skilled in the art that the following methods
may be used to prepare other multibinding compounds of this invention. Ligand
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-70-
precursors, for example, ligands containing a leaving group or a nucleophilic
group, can be covalently linked to a linker precursor containing a
nucleophilic
group or a leaving group, using conventional reagents and conditions.
Other methods are well known to those of skill in the art for coupling
molecules such as the ligands described herein with the linker molecules
described
herein. For example, two equivalents of ligand precursor with a halide,
tosylate,
or other leaving group, can be readily coupled to a linker precursor
containing two
nucleophilic groups, for example, amine groups, to form a dimer. The leaving
group employed in this reaction may be any conventional leaving group
including,
by way of example, a halogen such as chloro, bromo or iodo, or a sulfonate
group
such as tosyl, mesyl and the like. When the nucleophilic group is a phenol,
any
base which effectively deprotonates the phenolic hydroxyl group may be used,
including, by way of illustration, sodium carbonate, potassium carbonate,
cesium
carbonate, sodium hydride, sodium hydroxide, potassium hydroxide, sodium
ethoxide, triethylamine, diisopropylethylamine and the like. Nucleophilic
substition reactions are typically conducted in an inert diluent, such as
tetrahydrofuran, N,IV dimethylformamide, N,N dimethylacetamide, acetone, 2-
butanone, 1-methyl-2-pyrrolidinone and the like. After the reaction is
complete,
the dimer is typically isolated using conventional procedures, such as
extraction,
filtration, chromatography and the like.
By way of further illustration, dimers with a hydrophilic linker can be
formed using a ligand precursor containing nucleophilic groups and a a
polyoxyethylene containing leaving groups, for example, poly(oxyethylene)
dibromide (where the number of oxyethylene units is typically an integer from
1 to
about 20). In this reaction, two molar equivalents of the ligand precursor are
reacted with one molar equivalent of the poly(oxyethylene) dibromide in the
presence of excess potassium carbonate to afford a dimer. This reaction is
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-71-
typically conducted in N,N dimethylformamide at a temperature ranging from
about 25°C to about 100°C for about 6 to about 48 hours.
Alternatively, the linker connecting the Iigands may be prepared in several
steps. Specifically, a ligand precursor can first be coupled to an "adapter",
i.e., a
bifunctional group having a leaving group at one end and another functional
group
at the other end which allows the adapter to be coupled to a intermediate
linker
group. In some cases, the functional group used to couple to the intermediate
linker is temporarily masked with a protecting group ("PG"). Representative
examples of adapters include, by way of illustration, tert-butyl bromoacetate,
1-
Fmoc-2-bromoethylamine, 1-trityl-2-bromoethanethiol, 4-iodobenzyl bromide,
propargyl bromide and the like. After the ligand precursor is coupled to the
adapter and the protecting group is removed from the adapter's functional
group
(if a protecting group is present) to form an intermediate, two molar
equivalents of
the intermediate are then coupled with an intermediate linker to form a dimer.
Ligand precursors can be coupled with adapters which include both leaving
groups and protecting groups to form protected intermediates. The leaving
group
employed in this reaction may be any conventional leaving group including, by
20 way of example, a halogen such as chloro, bromo or iodo, or a sulfonate
group
such as tosyl, mesyl and the like. Similarly, any conventional protecting
group
may be employed including, by way of example, esters such as the methyl, tert-
butyl, benzyl ("Bn") and 9-fluorenylmethyl ("Fm") esters.
Protected intermediates can then be deprotected using conventional
procedures and reagents to afford deprotected intermediates. For example, tert-
butyl esters are readily .hydrolyzed with 95 % trifluoroacetic acid in
dichloromethane; methyl ester can be hydrolyzed with lithium hydroxide in
tetrahydrofuran/water; benzyl esters can be removed by hydrogenolysis in the
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-72-
presence of a catalyst, such as palladium on carbon; and 9-fluorenylmethyl
esters
are readily cleaved using 20% piperidine in DMF. If desired, other well-known
protecting groups and deprotecting procedures may be employed in these
reactions
to form deprotected intermediates.
Similarly, ligand precursors having an adapter with an amine functional
group can be prepared. Ligand precursors can be coupled with adapters which
include leaving groups and protected amine groups to afford protected
intermediates. The leaving group employed in this reaction may be any
conventional leaving group. Similarly, any conventional amine protecting group
may be employed including, by way of example, trityl, tert-butoxycarbonyl
("Boc"), benzyloxycarbonyl ("CBZ") and 9-fluorenylmethoxy-carbonyl
("Fmoc"). After coupling the adapter to the ligand precursor, the resulting
protected intermediate is deprotected to afford a ligand precursor including
an
15 amine group using conventional procedures and reagents. For example, a
trityl
group is readily removed using hydrogen chloride in acetone; a Boc group is
removed using 95 % trifluoroacetic acid in dichloromethane; a CBZ group can be
removed by hydrogenolysis in the presence of a catalyst, such as palladium on
carbon; and a 9-fluorenylmethoxycarbonyl group is readily cleaved using 20%
piperidine in DMF to afford the deblocked amine. Other well-known amine
protecting groups and deprotecting procedures may be employed in these
reactions
to form amine-containing intermediates and related compounds.
Ligand precursors having an adapter, for example, one including a free
carboxylic acid group or a free amine group, can be readily coupled to
intermediate linkers having complementary functional groups to form
multibinding
compounds as described herein. For example, when one component includes a
carboxylic acid group, and the other includes an amine group, the coupling
reaction typically employs a conventional peptide coupling reagent and is
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/t2768
-73-
conducted under conventional coupling reaction conditions, typically in the
presence of a trialkylamine, such as ethyldiisopropylamine. Suitable coupling
reagents for use in this reaction include, by way of example, carbodiimides,
such
as ethyl-3-(3-dimethylamino)propylcarboiimide (EDC), dicyclohexylcarbodiimide
(DCC), diisopropylcarbodiimide (DIC) and the like, and other well-known
coupling reagents, such as N,N'-carbonyldiimidazole, 2-ethoxy-1-ethoxycarbonyl-
1,2-dihydroquinoline (EEDQ), benzotriazol-1-yloxy-
tris(dimethylamino)phosphonium hexafluorophosphate (BOP), O-(7-
azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate
(HATU) and the like. Optionally, well-known coupling promoters, such N
hydroxysuccinimide, 1-hydroxybenzotriazole (HOBT), 1-hydroxy-7-
azabenzotriazole (HOAT), N,N-dimethylaminopyridine (DMAP) and the like, may
be employed in this reaction. Typically, this coupling reaction is conducted
at a
temperature ranging from about 0°C to about 60°C for about 1 to
about 72 hours
in an inert diluent, such as THF, to afford the dimer.
The multibinding compounds described herein can also be prepared using a
wide variety of other synthetic reactions and reagents. For example, ligand
precursors having aryliodide, carboxylic acid, amine and boronic acid
functional
groups can be prepared. Hydroxymethyl pyrrole can be readily coupled under
Mitsunobu reaction conditions to various phenols to provide, after
deprotection,
functionalized intermediates. The Mitsunobu reaction is typically conducted by
reacting hydroxymethyl pyrrole and the appropriate phenol using diethyl
azodicarboxylate (DEAD) and triphenylphosphine at ambient temperature for
25 about 48 hours. Deprotection, if necessary, using conventional procedures
and
reagents then affords the functionalized intermediates.
The functionalized intermediates can be employed in the synthesis of
multibinding compounds. For example, aryliodide intermediates can be coupled
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-74-
with bis-boronic acid linkers to provide dimers. Typically, this reaction is
conducted by contacting two molar equivalents of the aryliodide and one molar
equivalent of the bis-boronic acid in the presence of
tetrakis(triphenylphosphine)palladium(0), sodium carbonate and water in
refluxing
toluene.
Aryliodide intermediates can also be coupled with acrylate intermediates or
alkyne intermediate to afford dimers. These reactions are typically conducted
by
contacting two molar equivalents of aryliodide intermediates with one molar
equivalent of either acrylates or alkynes in the presence of
dichlorobis(triphenylphosphine)palladium (II), copper (I) iodide and
diisopropylethylamine in N,N dimethylformamide to afford the respective
dimers.
More specifically, and in addition to the above, recent attempts have been
made to rationalize the activity of several 5-HT; antagonists. Hibert, et
a1.24
assessed the low-energy conformations to define a pharmacophore and receptor
map that may account for the activity of ondansetron, metoclopramide and some
tropane-based esters and amides. The basic pharmacophore consists essentially
of
a carbonyl group coplanar to an aromatic ring and a basic center with the
relative
positions and dimensions illustrated below:
linking aryl functional group - can be
substituted by other hydrogen bond
aooeptor analogous to a carbonyl
group
3.6 + 0.1
O~~ 0.2
N
7.5 ~ 0.2
aromatic ring
basic nitrogen atom
CA 02321191 2000-08-15
WO 99/64046 PCTNS99/12768
-75-
Based on the basic pharmacophore for 5-HT3 receptor ligands and the
current available materials, several classes of bivalent 5-HT3 ligands
(compounds)
can be prepared. Typically, such compounds selected for use as a ligand will
have
S at least one functional group, such as an amino, thiol, hydroxyl or carboxyl
group
and the like, which allows the compound to be readily coupled to another
ligand
via a suitable 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 ligand can be covalently attached to
the
10 linker through any available position on the ligand, provided that when the
ligand
is attached to the linker, the ligand retains its ability to inhibit 5-HT3.
A first group of preferred bivalent ligands for use in this invention are
prepared via attachment through the basic nitrogen atom of the indole ring to
15 provide for N-N linked compounds. The preparation of such N-N bivalent
ligands
is illustrated in FIG. 1. Specifically, in FIG. 1, a para-substituted phenyl
hydrazine, compound I, is combined with at least a stoichiometric equivalent
and
preferably a slight excess of 1,3-cyclohexanedione wherein, in compound I, R
is
selected from the group consisting of hydrogen, alkyl, substituted alkyl,
alkenyl,
20 substituted alkenyl, alkoxy, substituted alkoxy, acyl, aryl, cycloalkyl,
substituted
cycloalkyl, heteroaryl, aryloxy, heteroaryloxy and the like. Generally, this
reaction is conducted in an inert diluent, such as methanol, ethanol,
isopropanol
and mixtures thereof, at a temperature of about 25°C to about
100°C until
reaction completion as evidenced by, for example, tlc. The resulting N-phenyl,
25 N'-cyclohex-2-en-I-on-3-yl hydrazine, compound II, is readily recovered by
stripping the solvent. Purification, if desired, can be achieved by
conventional
methods such as chromatography, crystallization, filtration and the like.
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-76-
N-phenyl, N'-cyclohex-2-en-1-on-3-yl hydrazine, compound II, is then
cyclized under conventional conditions to provide for tricyclic compound III.
Generally, this reaction is conducted in a diluent comprising acetic acid and
concentrated HCl and the reaction is preferably conducted at reflux until
reaction
completion as evidenced by, for example, tlc. The resulting tricyclic compound
III is readily recovered by stripping the solvent. Purification, if desired,
can be
achieved by conventional methods such as chromatography, crystallization,
filtration and the like.
Tricyclic compound III is then coupled via a linking arm utilizing the basic
amine group of the indole portion of the molecule to form compound IV. The
linking arm selected for linkage is illustrated by an a,c.>-dibromoalkylene
compound wherein n is preferably an integer from 2 to 12. It is understood,
however, that other linking groups can be employed in this reaction.
Generally, this reaction is conducted by contacting compound III with
about one half of an equivalent of a a,c.~-dibromoalkylene in an inert diluent
such
as toluene, benzene, tetrahydrofuran, and the like. A stoichiometric excess of
a
base such as potassium carbonate, sodium bicarbonate, sodium hydroxide, etc.
is
employed to scavenge the acid generated by the reaction. The reaction is
preferably conducted at a temperature of from about 0°C to about
50°C and is
continued until reaction completion as evidenced by, for example, tlc. The
resulting compound IV is readily recovered by extraction, filtration,
stripping, etc.
Purification, if desired, can be achieved by conventional methods such as
chromatography, crystallization, filtration and the like to provide for
dimeric
compound IV.
Compound IV is then alkylated at each of the methylene groups located a
to the carbonyl of the cyclohexenone group to provide for compound V.
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-77-
Specifically, alkylation provides for substitution at this position with an
N,N-
dialkylaminomethylene group (illustrated by N,N-dimethylaminomethylene).
Generally, this reaction is conducted by contacting compound IV in acetic acid
in
the presence of at least 2 equivalents of paraformaldehyde, and dimethylamine
5 hydrochloride (or other dialkylamine hydrochloride). The reaction is
preferably
conducted at a temperature of from about 60 ° C to about 120 ° C
and is continued
until reaction completion as evidenced by, for example, tlc. The resulting
compound V is readily recovered by extraction, filtration, stripping, etc.
Purification, if desired, can be achieved by conventional methods such as
10 chromatography, crystallization, filtration and the like to provide for
dimeric
compound V.
In the last step of the synthetic protocol, displacement of the
dimethylamino group by an imidazole compound provides for compound VI
15 wherein each of RZ, R3 and R4 are independently selected from the group
consisting of hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy,
acyl,
aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, aryloxy, heteroaryloxy
and the
like.
20 Generally, this reaction is conducted by contacting at least two
equivalents
of the imidazole compound with compound V in an inert diluent. The reaction is
preferably conducted at a temperature of from about 60°C to about
120°C and is
continued until reaction completion as evidenced by, for example, tlc. The
resulting compound VI is readily recovered by extraction, filtration,
stripping, etc.
25 Purification, if desired, can be achieved by conventional methods such as
chromatography, crystallization, filtration and the like to provide for
dimeric
compound VI.
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
_78_
A second group of preferred bivalent ligands for use in this invention are
prepared via attachment through the saturated methylene group a to the
carbonyl
of the cyclohexenone group to provide for C-C linked compounds. The
preparation of such C-C bivalent ligands is illustrated in FIG. 2 and FIG. 3.
S Specifically, FIG. 2 illustrates a reaction scheme resulting in bivalent 5-
HT3
ligands coupled via a suitable linking group (illustrated as the -NH(CHZ)~NH-
group wherein n is preferably from 2 to 40, more preferably, from 2 to 12,
although longer alkyl chains are contemplated). Specifically, in FIG. 2,
compound VII is contacted with at least a stoichiometric amount of a
10 diaminoalkylene compound to provide for compound VIII wherein R' and n are
as
defined above and RZ is selected from the group consisting of hydrogen, alkyl,
substituted alkyl, aryl, aryl, cycloalkyl, substituted cycloalkyl, heteroaryl,
and the
like. It being understood that when RZ is hydrogen, then a suitable protecting
group is employed to protect the basic aromatic nitrogen during the reaction.
Generally, this reaction is conducted by contacting compound VII with a
half of an equivalent of the alkylene diamine linking agent in an inert
diluent.
The reaction is preferably conducted at a temperature of from about
60°C to about
120°C and is continued until reaction completion as evidenced by, for
example,
tlc. The resulting compound VI is readily recovered by extraction, filtration,
stripping, etc. Purification, if desired, can be achieved by conventional
methods
such as chromatography, crystallization, filtration and the like to provide
for
dimeric compound VI.
Compound VII, in turn, is prepared in a manner consistent with that
illustrated in FIG. 1 with the exception that the alkylation reaction is
conducted on
the monomeric tricyclic compound rather than on the dimer.
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-79-
FIG. 3 illustrates the preparation of compounds similar to those depicted in
FIG. 2 having a different linking agent showing versatility in the linker arm.
Specifically, in FIG. 3, the displacement reaction employs an amino acid
which, if
necessary, is optionally protected at the carboxyl group to provide for an N-
5 substituted amino acid identified as compound IX. Subsequent reaction of the
amino acid group with the alkylene diamine provides for bivalent compound X:
As is apparent, additional amino acids can be incorporated into the linker
group by
conventional peptide chemistry prior to reaction with the alkylene diamine and
typically 1 to 10 amino acid groups are incorporated into compound IX.
10
Generally, this reaction is conducted by contacting compound VII with an
at least an equivalent of an amino acid (R' is hydrogen, alkyl, substituted
alkyl,
aryl, heteroaryl, cycloalkyl, heterocyclic and the like) in an inert diluent.
The
reaction is preferably conducted at a temperature of from about 60°C to
about
15 120°C and is continued until reaction completion as evidenced by,
for example,
tlc. The resulting compound IX is readily recovered by extraction, filtration,
stripping, etc. Purification, if desired, can be achieved by conventional
methods
such as chromatography, crystallization, filtration and the like to provide
for
compound IX.
20
The reaction coupling two copies of compound IX via the alkylene diamine
is achieved through conventional acylation reactions well known in the art.
Such
acylation reactions are typically conducted using conventional coupling
reagents
and procedures optionally using well known coupling reagents such as
25 carbodiimides with or without the use of well known additives such as N-
hydroxysuccinimide, 1-hydroxybenzotriazole, etc. can be used to facilitate
coupling. The reaction is conventionally conducted in an inert aprotic polar
diluent such as dimethylformamide, dichloromethane, chloroform, acetonitrile,
tetrahydrofuran and the like.
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-80-
A third group of preferred multiligand compounds per this invention are
prepared by attachment of two or more different ligands to the linkers wherein
at
least one of the ligands comprises a Iigand domain capable of binding to one
or
more S-HT3 receptors. In one embodiment, at least one of the other Iigands is
a
ligand which binds 5-HT3 receptors. In another embodiment, at least one of the
other ligands is does not bind 5-HT3 receptors and includes, for example,
corticosteriods, ligands binding DZ receptors, and the like. Examples of
corticosteroid are cortisone, desoximetasone, dexamethazone, hydrocortisone,
betamethasone, fluorinated derivatives, and the like. Examples of Iigands
which
bind to DZ receptors include, for instance, metopimazine, e.g.,
O
HZN
N
N I ~ S02CH3
S
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/1Z768
-$1-
and trunetgibebzanude, e.g.,
O
CH3
NH ~ OCH
NCO I / I /
CH30/ OCH
OCH3
FIG. 4 illustrates a synthetic method for preparing such heterologous
bivalent multibinding compounds. Specifically, in FIG. 4, compound VII,
described as above, is coupled with compound XI under standard coupling
conditions, also described above, to provide for a heterologous bivalent
multibinding compound of this invention, i.e., compound XII.
As will be readily apparent to those of ordinary skill in the art, the
synthetic procedures described herein or those known in the art may be readily
modified to afford a wide variety of compounds within the scope of this
invention.
The methods described herein lend themselves to combinatorial approaches
for identifying multimeric compounds which possess multibinding properties.
Specifically, factors such as the proper juxtaposition of the individual
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
multibinding compounds with properties spanning the multibinding parameters
that
are relevant for a particular target. These parameters include: (1) the
identity of
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-82-
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
functional groups.
Libraries of multimeric compounds potentially possessing muitibinding
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
parameters desired. Considerations relevant to each of these variables are set
forth below:
Selection of Ligandl,~
A single ligand or set of ligands is (are) selected for incorporation into the
libraries of candidate multibinding compounds which library is directed
against a
particular biological target or targets, i.e., binding of SHT3 receptors. The
only
requirement for the ligands 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.
Ligands are preferably chosen based on known favorable properties that may be
projected to be carried 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, loge, etc. However, it is crucial to note that ligands which
display
an 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
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-83-
form. A ligand that is potent and efficacious but not of utility because of a
non-
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
bioavailability due to low solubility, hydrophobicity, hydrophilicity) may be
rationally modulated in multibinding forms, providing compounds with physical
properties consistent with the desired utility.
Orientation' Selection of igand A tac'hmP~,t pnint~ amd imL~iiir~,1 i1C-
mlSiiV
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 target binding sites)
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 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 ligand bound to its target allows
one to
identify one or more sites where linker attachment will not preclude the
ligand/target 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,
the disclosure of which is incorporated herein by reference in its entirety.
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 with multiple points of attachment
to
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-84-
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
5 the activity of the monomeric ligand may also be advantageously included in
candidate multibinding compounds in the library provided that such compounds
bear at least 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 ligand
10 bound to its target, and then consider modifying this ligand by attaching
to it a
second copy of the same ligand with a linker which allows the second ligand to
interact with the same target at sites proximal to the first binding site,
which
include elements of the target that are not part of the formal ligand binding
site
and/or elements of the matrix surrounding the formal binding site, such as the
15 membrane. Here, the most favorable orientation for interaction of the
second
ligand molecule may be achieved by attaching it to the linker at a position
which
abrogates activity of the ligand at the first 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
20 form.
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
25 monomeric ligand. It should also be understood that bivalent advantage may
also
be attained with heteromeric constructs bearing two different ligands that
bind to
common or different targets.
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-85-
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 Iigand) readily and generally formed,
5 stable and intrinsically innocuous 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.
Linker Selection
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:
15 V~ encv: 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 aff
nities 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 biodistribution properties of small molecules.
Linker Length: Linkers are chosen in a range of lengths to allow the
spanning of a range of inter-Iigand distances that encompass the distance
preferable for a given divalent interaction. In some instances the preferred
25 distance can be estimated rather precisely from high-resolution structural
information of targets. In other instances where high-resolution structural
information is not available, 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 situations where two binding
sites are
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-86-
present on the same target (or target subunit for multisubunit targets),
preferred
linker distances are 2-20 ~, with more preferred linker distances of 3-12 ~.
In
situations where two binding sites reside on separate target sites, preferred
linker
distances are 20-100 h, with more preferred distances of 30-70 A.
5
Linker Geometry and Rigiditw 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
10 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 traps-arrangements at the 1,1- vs. 1,2- vs. 1,3- vs. 1,4- positions
around
15 a cyclohexane core or in cis- or traps-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
20 ligands are attached to the 4,4' positions of a biphenyl linker.
i ker P sical 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
25 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
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-87-
join and preferably the goal is to generate molecules with favorable PK/ADME
properties. For example, linkers can be selected to avoid those that are too
hydrophilic or too hydrophobic to be readily absorbed and/or distributed in
vivo.
5 Linker Chemical Functio a1 rrn, p~ Linker chemical functional groups
are selected to be compatible with the chemistry chosen to connect linkers to
the
ligands and to impart the range of physical properties sufficient to span
initial
examination of this parameter.
10
Combinatorial Synthesis
Having chosen a set of n ligands (n being determined by the sum of the
number of different attachment points for each ligand chosen) and m linkers by
the
15 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 (A1, A2) and one which has three attachment
points (B1, B2, B3) joined in all possible combinations provide for at least
15
20 possible combinations of multibinding compounds:
A1-A1 A1-A2 A1-B1 A1-B2 A1-B3 A2-A2 A2-B1 A2-B2
A2-B3 B1-B1 B1-B2 B1-B3 B2-B2 B2-B3 B3-B3
25 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
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
_88_
complementary 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
5 combinatorial libary is prepared in the solution phase. After synthesis,
candidate
multibinding compounds are optionally purified before assaying for activity
by,
for example, chromatographic methods (e.g., HPLC).
Analysis of the Library
10 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
15 determine low-energy 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. In vitro efficacy, such as for
receptor
20 agonists and antagonists, ion 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
25 work.
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
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-89-
methods as described above including conventional assays (both in vitro and in
vivo) .
Second, ascertaining the structure of those compounds which exhibit
5 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 No. WO 93/06121; Brenner, et al., Proc. Natl. Acad. Sci.iUSA;
89:5181 (1992); 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.
2,240,325
15 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 S,~thesis and Analysis of Additional Libraries
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 iigand
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
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-90-
variations in linker structure in an effort to further optimize target
affinity and/or
activity at the target (antagonism, partial agonism, etc.), and/or 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 functionality on the ligand to form a covalent linkage.
Such
complementary functionality is well known in the art as illustrated in the
following
table:
Reyresentative Complementarv Binding hemic~ aP~
First Reactive ~rounSecond Reactive GrounLinkage
hydroxyl isocyanate urethane
amine epoxide ~3-hydroxyamine
sulfonyl halide amine sulfonamide
carboxyl acid amine amide
hydroxyl alkyl/aryl halide ether
aldehyde amine( + reducing amine
agent)
ketone amine(+ reducing agent)amine
amine isocyanate urea
Exemplary linkers include the following linkers identified as X-1 through
X-418 as set forth below:
CA 02321191 2000-08-15
WO 99/64046 _91 _ PCTNS99/12768
Diacids
_.-__ - --
_- - ~ ~ ~ p
p a 0
~ ~
Ip~'\~-~ OM./~ON NO~F ~O NOMO M~O
ON
ON ,5C
NO~O CN, O
~,
CN,
xI x.: x7 X.I x-s x
__ O ON 0 ON ON NO O ON
p ON ~~~~/lO ~ ~Y.ON 0~0 ~O
NpM/\/\/\~O N~ ~ ~
O ~.- NO CN, '-~N,
Mp
ND-(v ~/
x7_ X.tO X.9 X.10- , x-1
x-II.
O - O ON O O O ON ~ ON
/ ON
/
0
p ~ ~.~~/'. ~.~ NO /\ O~~O ~~
/\_/~O ) G~O
II
ON NO N,C ~
CN ~ ~
,
N,C
x-a x.u x-IS x-is x17 x.u
o NO O oNNo OH N o o O
~ pi"' No~''oN OfON
.~ /~~
~a a_/
O~
ON
O \/ NO _ \/
5~ N
X.19 X.M'. %ilO X-23 X.pO X-71
e~. c"" o O O N0~0
D
N
No~sfs ~.. /-.~ Nod. No~oN O~cNn
ON ~~ oN r~
~, ~
a~~ O
O
X-35 xZ6a X1T x7t_. xM %
0 0 oN
_ O ~, oN
a, ~~ a a ~~ ~ y
No~~ s ~.." a~~.,~., No No No
~. .
;,
oN ~ v N
Am C-.~
,
CN,
xal.~~ x.7:~~ x.57_-- _ x-T _x.ss_ , x-a
- - - -
0 - r o~~ ~cN, ,~.w o
~ .~.
O y
~
~ ~/' s \ ON ~..0 ~ ON
~H./~~ o ~
w ~ f ..~. ~
a np ... CN, V'i pN
X.77 x-7t X.79 x"M X-il. %~l
'i
-..
O OH N0 Np~O 0 .. 0
G i\ .. .s:
~
~ ~ ~~
NO~\/~MO ,.- O NOv 1
p~ /\/-\ ~~
. N~/
ON O
_
~ ~
x.17 x-11. x-IS x.u~u xr7~ xrt
"
-
~ r ~~ ~o" ~ ~. ~~ o~N
.. ." o
N r
~
e F No--. . >--/ oN
F-F oN ~
~J ~-l o
r No o No No
w-n
x.,9' ' x-so. xal x-s: x.ss. x-
"'
a ~ ~ ~
~ o~ '
p
~y -; > ~ No~/~~ ~~'-~ N
~a ~ <
ow ~
~
N ~
< p '~
X.55~ i\.~\/' x36p X.S7 X-St X-1!i X
N~ , o" -
0 M 0 ~" N C CN N,C\ O~N
N O O ~ O M
N ~
~
W~ D\~/"ON / ~ ON Ki N
V /\ C N "
p~/\/'V ' '
F
oN N~~ No a t No-~ ~aN ~/~/
~./ ~o F9 o
o No
0
x.al xs: x.a7 x.w x.as- x
0 0.. o Ro ~~ YoN'.. c
' 's a
Ip~/~~'\/\M/\/e ~./~N -i )
~~~/~./~~ '~ i-~F N'-,l~-
~~
c ~..o o i: a
\,
x.61 X.ta xfi9~ ~ X-m X.11- x-77
~--
f F t t F F -_ O ON N~.O r% l NO
F F .p...~,~ '
' ' ~ 0 ~
~
NO~~,~/~ai~\i NO /J ~
ON e~ 0 OO ~~~ ~~ ' -
NO~ F f F F ON
/ F F f
"~,~'-" ~ ~/
~
~0
~0v. NO
x-n xa1_'. X'7s xa6 x.m. x.7a
..- -
-_-
_- i~ ,!\ d., r
o N
-~.. -., i 'Y'. .~oN
...~.~....~.~.~ N,!_..o. ~.nl~v" No''\i'>o
.N _. /~,
.. ,
cN, o _w. a No
" r.,~e oN a
~~aN y No~o
o ' "oN .a'~
.J~w
X7y x.an x.al x-is x.a5 x.u
CA 02321191 2000-08-15
WO 99/64046 _92_ PCT/US99/12768
ON N wp'~ 0 W
~/v/r/v/'~/~/'-~W~' ~~ ~ O
/te
, p -: 0~. ~ 0 /
a '~'~ ~
N
"-~~v"~W~~ NOJ~ ..I~n ON i %
N
O a
\/~O" N ON
x-If_" _ __ X.16 Xd1 X-SI X-19 X
-- o _ O ON 0 0 0 ./\
~ v/\/~.// o
o ~
/ " ~ \ . IIO~~BVS~-O n N~N 'EMON
e-~
p,.\~"'w~.'~'~i~~/ ON s , ,d\~\~o
N v o o ~ ON ~ N
~.i~
O
- x.91d, oN x-v: x-a7 x-sn x.~r x
IAI IxI OyeO ~ I N'~ ~ NO ~ 7~ON
O ! I I I ON' t0
I r ON
N NO
~
- ..
N0~\/~~~/ ~MOII ~,~/\ ,
N~.~
~r ~
I 0 \/ \. 0 O O Ii,
11 I I I I NO ~(v OON 0
I I Ip
~0
x.97_ x-w- x.99: x.lno x-lol x.197
- -..
-
-
fF FF FF CF O ON e a
~o i"~y
Ny-.v~/N ~w NO~/sv/s\/~O I--~f~ W
\ ~
N f ~
~~ r vN~ N
ON o" a
O F F f f .l~\/~O o \~n '~o .Mp
F V
F O
X-I1'o~'' x.IM x.i9f X106.~o x.lOTw X.IM
0 oN p ,." 0 0
... ~ "e~ MaN
NO/W/\./'\/'r~\/Q NO y~W
a tl ON w. Ve' \/N~./~CN .~...\/~"
,~~p e~'e o ~!i
x.lo9 x.uo x.m"~ x.117 x.117, x.u
W/
n ." /a "~' o oN No o'~ o ON
ON
0T~ 0~
O /vf~ NO'~N~O O 'O ~~
l p
-O ' fo '~ -
_O~i- 1-ON 6 ON ON
~--
~.
N
t W,/ a O MP
.
e
N0 ON NO ON
x-Its x-us x.ln x-lu: x.119,_ x.l
._... _.__.
O ON --.- ,o.,.,p 0 O ON
~ NO
~
Noes~s~ ~ o~ .c No oho
N0 V F
y" r' w~ ON ON e0N
1<N .~~0
a ~1
0 o s n~'
x.171 x.177 x.177 x~i x.l7s x.1
- .
__...-- o oN
N n~
~1 ~~v. ,~o~ ~:W~-p" ~ .5~/~
.: '\.~.
oN
v..\~v'V~.I\- .~~~~o ~ ./~oN
no
.e'~
x-I=7 x-In x.179 x.un:- x.ul x.177
Disultonvl
Halides
0 0 0 - , .~. ? I. .o ~ /\
i_s-v ;~'\ - =s--~"_... ~i s'I ,s'' G o o,,
/.r's-n ' /,~
- .o
wN N~ o o a o -._' ;s~~s s s
a I '
' ~
'
~G~i o 'a/\ u i
~ o o
'G a o
o
e.'\/rcN,
o
%-I77' X171 x.Ufc", %-176 XI77 %IN
-- -
F .o o ,G o, ,~. Q.s.o "'G~aS o,.i
r a ,o a
s~ vN~~o ~ .-'-r, . o,~-'~ , - ,swfs? G
s..
I
\% O \/ J o, p\ . ,a ~~
cl 'Nio p l
W%\/~: G ~ G a
o
x.179__ _%L10 X.111~ %.112' x.117 X.IN
'SC/a/
r
a o p
a ye I O. CI CN, ~ ' a ' a
. ~ ~O ! 0=%/0
v 1
\ s~s G' ~' ~ 1'
p
v /\/N~ ~ 'e/~N~. CI O . O ~9' ~ ~G
~G
~N~ N ~ \~ a ~(; /~C~ ~ Np~ ~/
1
.
x113a / x.116"' x.117 x.116 x.119' x.l
'''~J ~\ O . ~ \~
O
Clvs/~%~./~%'\B'u C1~61~./W
:~v6.C1
'
~~ \\O O O O O
O
x.ul x-IS7
Dialdehvdes
_ ....___ _..._._ ._--..__
. _. ~ _ ~_ _. .. ~\
.__ , -~i ..-. .vl. ~ O
pc\ ~...p~ .._. . .. ~O.~.J O ~N~'
,v~. . .0
w i
\,.--et'
\
,
. , q o'
x.ls7 X-Iy1 X.Ifs x-ix X.ur x.IS1
CA 02321191 2000-08-15
WO 99/64046 _93_ PC"T/US99/12768
'° .Q\r0, -:: \ ~ _.. _ ~ O . O ° y\ O
_ .'~\..~ _c . . ~'>~.'
a\ o ... ~o/\.-~o~ .. _ w~'i
. °..'.' ° o~ '"o ~ a oA
, . o \~o o .
°
wF 'CH, X~I!9 X.166 X.IbI _ _ X.161 _~_X.161 _
i'.o~ __--. ..... _ ~.-_ ~~~~.° _-...__ .....__. _.._. .._~- _.
_.__~.o. ~1~/p __-.___. ... _° ___._~_. - ___.y.0
'. ~\
\y~: '. ,_.\
°Q/~/\r°
o ~~0 0 0
x.16! X.166 X.167 X.l6t x.169
_- __ . ._..._._._~._._~.._.__ -..N...~.__..... _.._.__ .._ O_-_~.~~..-
~_.___~___ _____----_ .-_.-_-__. -
\' O O O.y~f~0
\/
\.I-'V'~~t~:
O° ~._
V/'o-
w.c x~171 X.177 x~171 X.17~
G -.____._.._._.- ~ -- ._ ~1 1 r' p~\%\i1r
_ i~' \'"-Ir
. I
, ~ ._. G\/~N/\/G 0H ON
W
G~ ,
_ w.
- 0
x17!_ _ X-176 x177__ X.176 X.179
- _.
_ cl % .., ~ _ er'f6r G/\~G
W .~./'~. ,.o.,~o..,~ ~_.~'i~-.
~'N 'a I
O .~\/\~% Wlt
i"B/
X.111 X.111 X.111_ X-IH__ x11!_
.. ...._ ____ ~ _.__ __.
. . _
._ -_
__ . ...__... ~ er 1' 4~'.. Iv.i1/I
N\/\. ~.i . 1
..1/\: N..._ Ow ~ ~ '
O"\.' ..- . er.
~ _ v \%
... G ~l~"~..-
0
x.117._.- X./11_-.. X.119 x.1911_ _ X-191._..
. ._ -__.-___..____-_.__.. ~-...._. _-.._
__ _._._ ___.-.
_____._.
N\,.~v.~ /v _ N'' ~ Glw ~...-\.~.0" '
."..!\''~p - v
.
_ .. o- /~ /~rc/ u__:
w , v
',
we
a 1..:
X.191 x.l9J x.193 X196__ X.191___
_ . _~. ~.._..~- ~- _ .._-
--.
Ir\%\y'1 /y _ ~c ~ 4 e _./G
-._.- _/y/~_.''. N O ~~'.OM
r
O ~6
V' - O. y-' 1r _ _~
V 'Ir
Ir CI
x.lw_ x.7m x.1I x-71n x-1m
.- - -
~-
__ _ b~.~0~,.1~ HG'O' p ~ Nyl.
~\/W /~'iliN ~Ir
N~Ir
0 . G\~~
/G
N ~ M ,
X.tyf_ X.7u6 X-167 X.pw X-7a9
_._.__ __- -.__.__ _--..____
try~,-\. -\~er sr.". ~ cl
~_i\::i:
cwl o
G\/'~I~'
w
O
X-711 x.711 X-717 X.116
___...-...__.....__.~_..-._-o__.__..-._..._...._. __________.__ -_..-_'.___-
__-
._......._.. _..-_~._ o>.rzw
0 0
,
..\~J\%' w~ p ' .. , - r
O wf
.'v.n '/
O \'e...vd.% y' w w ~ n 1~_ Ou'w . .' .xi
a
-
. ,
X.TI! x.116 X.117 X.711 x.119
.. ..._... ..._. __....__.....__.___......~ _ ~.__
. .. _. .______ ___._- __ __ ..._~.
__ _-
. _ r
_..._._.__ ._....._.-.._.
~._... _
O., O ~ . _ ._ ~~"N ' ej . ~ -0
-...
'"~.
cw ... .._ '
...
sr
'
o "ca cly . v.
~w ~' 'w4;
;: Y
X.711 ..\'772 x.777 _ _ X.77a X.12!
_. __.~_.' -_ -_,
... __.___-_ __ .__. ___.
__._....... .._
.
..._. . ...... ._.. . ./v N ~
... ._._... ..._.._.- ~ w
_.. . \;
..
i ~.~~\.w ow' . . .. cl,\s'y,~ ,
',~~
' o '_. ~.: 1: ~ w.w.% a ,,' q._e.
-. w
o
X.117 X.711 \.179 x.7N1 x.pl
CA 02321191 2000-08-15
WO 99/64046 94_ PCTNS99/12768
p, p .p N~N .... 9 p
.~ \\N~~N/ ~ %v .
N\ y '
" l/ '~/~ ~ N~~ \ % p ~
~ '. M,
O
11~C , ,
p GM~CN ~y/N~~ ,~~, hC~G",
0
X.!).7 X.E7a x.375"F eN, X.177 X.D
- ..__ ~-- X.1J6 -.' -
____ _--
- p /NWN/
~=v ~_~%' ~ .~nr"~ ~N O
N_' v.--1 N.
~ "
O , ~
r y b/ O
J'1, _ X-779 _ X.7J11 X.211_ X.7a7 N'c ~~
~~ ~ X11)
- q ~/ o o .~-. b o
~ o G
\ . -
/\M ~ \,
y
\/~/\/~/ N N~N ~. .'
'CS N ~/~ ~ v. /r
N ,.,
C~
~N~ \~'~N GI~ n p ~
/~ ' N
p N,c '-
"Fr 'Scr o 0
xau x.7fs x.=a6x.:u x.7af
Diamins
/~ "~.N~.N N~ '~.. NNi\/~Ni'\/.'.NllzNxNMNN~ \Jv~.~
N
p N n CN, ON
/~/\NN ~pr\i"v/19'~ N~/
~. N
\~'
~!
x7as x.7sn x-alx-fs7 x-of t x.7u
"~~-
~YS/\rpv/\/\/W~p~~NSr~/N ~ y.hN~ %'~ N~N~y,~, N~NfNN~
\~ / ":N~ r~
~ ~~~z
' ~
N
_ X753 _ _ X-23i X-!f7_ X131 X119 X
_ NzNV/\/Ov./\./~z N,N~NIIZ ANN, NzNyf/\/WI~
"FWW /\/\/\y/\/~CN,
\~-\% ~f/'~/
\
N
z / ~/
X-261' %-76! X367X-f6a XMf _ _
~ - _. ~ -~~ X-
-__ __ ~ ~ qlp N~11~/N r'\ p /~ N CvN h11\/\/~S
/~.nly CN \/\
M~y/V' YOr \ / ~ NN7
\~ M A CN
~ /"1
w y Np N NN, D
M N~N
J ~
Np
X.:67 xasf Xa69_ x.77n _ x.:71 x.:n
_
.
~" C ~N\/~ NzN ~ t , ~.- /'
y
~~ / N~ NBC NBC y NzNy~/NNz
C" GN, ,
~
. N C ~ \/
F :% '.
!
~ n
x-:n x-77a: xfn~~<-776 x-nn x-7n
'Yv/\/\/\/\/\/'~"",W /\ /\/N~./"- ul~ Nn, o ,-
N ON . ~' "vp~\y
~ ~ ~'a ~ N~N~\/\ ~
N . \
N
~../ N~ /
NN>
~/
p h ~~f.yN,
x-779 x-7el x-nlx.79: x.:~s _ x-:u
p ~
~
ISNWOV~p~\/OWNMZN,CfNr\/\/N~/\CN,NzN\/\.r\/~z 0 O MN NrCNz
., z \/\ N~M~~"~S
MzN~f
%
%
\
\
N11,
x.ns x-n6 xaax.m x-m. x.
~SNV/\/O\/\/~p~/~~z~\/\ ~ NiN\/\/\/~NNZ ~. ~ n //\/\/\\
N
Nr\
~ //\/6~ N,N\/\/\/"".\ /
r
N~~/ \~N~S
N14
NIh
x791 _ _ X397___ X-297X.79J X.793: X.796
O 0 NfC\/N\.r\.N\/CNZ NzN\/\rr\/\/NNi .:L~'\\ NzN' NNZ
NzN~/~.r~z
.~\/~p~~p/\/~ ~~y ~~ \%
a'\' ./a
~/ \%\~z KN~e/ \f~Ny,
x-J97 x.i9,-_ _ _ _x._=99x-7ao x.7ol. x-7o:
~--
KN /\/\ Nzll~-~Or\NN.~ N~~ ~ NN
~' ~_r .. rOyW Ch
' W
'
...,. W"
1-0; M
_
_._ p ~~~- ..
N,N , ~r "G",
/\%
_ x.7a7 x.3lu__ _ X.7NSx-7a6 x.7m xam
. _.__.__.- ~_-_ _-__~_-- -
_~__ _ -
-._._.
__.
y- 1 ~ . __ p:_ ~ N'Ny~~ z M ..- ~
. O " 1 .CND
C~ ~ .- . ~
N_! _, ~__. '
.,-NN
"
~ O .__. . N,C~
, eN.. _Cw .\~
.__ H,, .a~ 0 - GNZ '
CN,~a-
\/~ \
, W
NN,
X.7m x-sIN X.711x7a xau. x.7la
CA 02321191 2000-08-15
WO 99/b4046 PCT/US99/12768
-95-
N.\,M/\/\/\w ..... ~ /, y\ NIN
-..... W, .. N
/' ' NF.N ,
"' /\ /.y N111
~~
." .. i\./\/'\:: NIt
Nt i-. :N~i.
: . N
N N
NBC ~~~CN~ ~-~ _/ ~
~CN,
xals ___ __ xale xals__ xau___-_._Xaxa
__ .- 19
__ _ y/\/\:~v/\/~NN~ N~t~h ~\/NNvtN~ NIC~N/~
~St\.~N\/1/\/'~-N/~M /\NitNl
.__~
"1N\/~'
O ~
~\/'IN7
X~?n -_- x.7n_ xan_~- xau__-_ xav
~_
Diols ._ - .
_ _.- ~~ 0 1 ~ ~~~i..a,
_. N0~/N\/\N/'v//~, ~/ /\"y.-\Oh
N0 \ \%\O~/O" 0~ O ~ ,CN/
W O/\/ _
~ '
1. ' .77s oN xan xa71_ xass xa7tx.s71
__
Np/\/\/\/\M/\/\~0 O O ON ~OW\ON
CN
M~C~O'~'\0"~CN~ ~~ N~C~M ~~~0~ '~N\''~0N
ON\0N nW O/~ CN, ON
CND
x.77: _ xa77 xa7a xaa xa7sxar
9 O \ i1/\f!'v/\.ON /~ CNI dl 0 ON
' ~e'~' wc~' ~ s r DN
~d
N/t~ oN
/ ~ON "1t~0'~t"
r "O~CN~ CND ~/
1 I CNI
I CMn
x.7 I x.7ao x.m__ x-7f7x.yl7
xa
No'
_ . O 0 ON
Np/\/\/\/\/\/\/\/"~ f ,~ ~ NOMON
.-'\/f\/\
/\i~
"O\~~~ N~ ~'. GN
ON Nt~/\/
I
ON
x.7u. ___ x.NS x.7~_s__- x.7m-.__ x_N!__ xa
_
NO/\/O\/\O/\/O\/\O/\i0-_- !~.'/0\~ NO~/\%0" NO/\~~~/10N I I
NO/\/W../\ON
ON iON
N\:\O/~~~i \:/ r _~I \//
F. .f
f
'
xaso __ _ xasl x-n7 x-7s7NO xas.,____ x-us
--_-.~_
_ /\./\~%\.WO" NO/\/\/\/\O" oN ~ i V -tN
/\/O~' ~O\~
~ ~ N0
~ Nt/\/'\/'~O" ~ ~ON \/\/
~ON
W.~'~/ 7s, x-7s7 xasl xa x.7wxul
....
~M/\M/1/" NO/\/\!\/~G1 ~\/~ Np/\/\/N M ON
ON
N\/\N/s\/~ MO\~\/ N~t~/O~C1S
ON
NO.~/ \~tN~
xas7 xau xas~ x-71s xatax.7e
-
'
No.~/t\/\t~/\/\tN.=\ Ntn/\~/\/\oN oN a. ~ ,G\
~
.. /O~/ I Nlt - / \ ,.~/~/
NO 'tNl
NO f-O N W
ON
x-7s1 xas9 xa7o xa71_ xa7rxars
_ NO/'\/\/\/\/O" 't"~N NOON /GI
"o
ON ~ p/\/N\/\ON
CND y v'tNl
' ~ '
"It Y
v
110 .
-'o-. xa7, xa7s__ X._7n_ t"1 x.7m x.7wxa
-_-
o _. o _ /\/f\\ ~.-\/\/\p/\/N N~/\/\..~'~N Nor~tN s / a
~-,..~
W M/~
I t
I
-_-- -. X71 -._~..____._...x-711___._ -_ X-713__ ~_ X-717~_-_.._-
X'711x.7tf
_._.
Dithiols
NI\/.\~~w.-~.1" Nf'./~.. -~~ -. -_._.-.._~_._.___..-__.-. -- ___. "f
ffN
-- - _- f ~~
_.__. NS~/
\~.6N ~.__t /i\
Q"~ :_:
_ Nf ~2/
rN
x.716 x.717 x.711 x.719 x.7911Xa91
CA 02321191 2000-08-15
WO 99/64046 _96_ PCT/US99/12768
o ",~~~.~~'w..s. N6~ ~ ~~ sN eN, sN !N
NL~/~..Oi~..'Of6N 7N ~ //~
O N!~
"O~%~~
fN
X-797 X-797 X.>9J X.79! X-)91-_ _ X-M
--_- .-_.-~ .---__ _
_ -- :_,-__~ Ns "L~n/t"
Ns~o~o~sN o
- 1
~
~/ ~CN~ -,
Nf 011 Ny~~fN -
O -~'iN
Nip
x-79i xa99 x-Nn_ ~!H xaoi x.,t7. xrw
_ _ _ Nt .-. N6.~/y'~/dHN8~~~/sN Ili~lN
"!-~.~/~/~/~fN~ G O tN
~
NS~~N~~~1N ~i~
x-~m x.~o~ x-aw x-m x.,ai x
_- _~
~
._ ON Nt~O-~~1NNip r-tN N ON
ON "!~~ ~ O~
O ~
~/ill v- ~-.O N /
O
O" aH NO ~ ON 1M
x-iW X-~n x-W7 x-IU X-tl~ X-1u
off ~-- ,~~ NsH T.
~aH ~s~i "'W ~iN
No
xaie Xan x.~u
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-97-
Representative ligands for use in this invention include, by way of
example, L-1 through L-6, where ondansetron (L-I), granisetron (L-2),
tropisetron (L-3), dolasetron (L-4), mirtazapine (L-5) and itasetron (L-6).
Combinations of ligands (L) and linkers (X) per this invention include, by
way example only, homo- and hetero-dimers wherein a first ligand is selected
from L-1 through L-6 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-1/X-5- L-1/X-6-
L-1/X-7- L-1/X-8- L-1/X-9- L-1/X-10- L-1/X-11- L-1/X-12-
L-1/X-13- L-1/X-14- L-1/X-15- L-1/X-16- L-1/X-17- L-1/X-18-
L-1/X-19- L-I/X-20- L-1/X-21- L-I/X-22- L-1/X-23- L-1/X-24-
L-I/X-25- L-1/X-26- L-1/X-27- L-1/X-28- L-I/X-29- L-1/X-30-
L-1/X-31- L-1/X-32- L-1/X-33- L-1/X-34- L-1/X-35- L-1/X-36-
L-1/X-37- L-1/X-38- L-1/X-39- L-1/X-40- L-1/X-41- L-1/X-42-
L-1/X-43- L-1/X-44- L-1/X-45- L-1/X-46- L-1/X-47- L-1/X-48-
L-1/X-49- L-1/X-SO- L-1/X-51- L-1/X-52- L-1/X-53- L-I/X-54-
L-1/X-55- L-1/X-56- L-1/X-57- L-1/X-58- L-1/X-59- L-1/X-60-
L-1/X-61- L-1/X-62- L-1/X-63- L-1/X-64- L-1/X-65- L-1/X-66-
L-1/X-67- L-1/X-68- L-I/X-69- L-1/X-?0- L-1/X-71- L-1/X-72-
L-1/X-73- L-I/X-74- L-l/X-75- L-1/X-76- L-1/X-77- L-1/X-78-
L-1/X-79- L-1/X-80- L-1/X-81- L-I/X-82- L-1/X-83- L-1/X-84-
L-1/X-85- L-1/X-86- L-1/X-87- L-I/X-88- L-1/X-89- L-1/X-90-
L-1/X-91- L-1/X-92- L-1/X-93- L-1/X-94- L-1/X-95- L-1/X-96-
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-I/X-106-L-1/X-107-L-1/X-108-
L-1/X-109-L-1/X-110-L-1/X-111-L-I/X-112-L-1/X-113-L-1/X-114-
L-I/X-115-L-1/X-116-L-1/X-117-L-1/X-118-L-1/X-119-L-I/X-120-
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-98-
L-1/X-121-L-1/X-122-L-1/X-123-L-1/X-124-L-1/X-125- L-1/X-126-
L-1/X-127-L-1/X-128-L-1/X-129-L-1/X-130-L-1/X-131- L-1/X-132-
L-I/X-133-L-1/X-134-L-1/X-135-L-1/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-I/X-143- L-1/X-144-
L-1/X-145-L-1/X-146-L-1/X-147-L-1/X-148-L-1/X-149- L-1/X-150-
L-1/X-151-L-1/X-152-L-1/X-153-L-1/X-154-L-1/X-155- L-1/X-156-
L-I/X-157-L-1/X-158-L-1/X-159-L-1/X-160-L-I/X-161- L-I/X-162-
L-1/X-163-L-1/X-164-L-1/X-165-L-1/X-166-L-1/X-167- L-1/X-168-
L-l/X-169-L-1/X-170-L-1/X-171-L-1/X-172-
L-1/X-173-L-1/X-174-L-1/X-175-L-1/X-176-L-1/X-177- L-1/X-178-
L-1/X-179-L-1/X-180-L-1/X-181-L-1/X-I82-L-1/X-183- L-1/X-184-
L-1/X-185-L-1/X-186-L-1/X-187-L-I/X-188-L-1/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-1/X-201- L-1/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-
L-1/X-209-L-1/X-210-L-1/X-211-L-1/X-212-L-1/X-213- L-1/X-214-
L-1/X-215-L-1/X-216-L-1/X-217-L-1/X-218-L-1/X-219- L-1/X-220-
L-1/X-221-L-1/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-1/X-232-
L-1/X-233-I; 1/X-234-L-1/X-235-L-1/X-236-L-1/X-237- L-1/X-238-
L-1/X-239-L-l/X-240-L-1/X-241-L-1/X-242-L-1/X- 243- L-1lX-244-
L-1/X-245-L-1/X-246-L-1/X-247-L-1/X-248-L-1/X-249- L-1/X-250-
L-1/X-251-L-1/X-252-L-1/X-253-L-1/X-254-L-1/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-1/X-262-
L-1/X-263-L-1/X-264-L-1/X-265-L-1/X-266-L-1/X-267- L-1/X-268-
L-1/X-269-L-1/X-270-L-1/X-271-L-1/X-272-L-1/X-273- L-I/X-274-
L-I/X-275-L-1/X-2.76-L-1/X-277-L-1/X-278-L-I/X-279- L-1/X-280-
L-1/X-281-L-1/X-282-L-1/X-283-L-1/X-284-L-1/X-285- L-1/X-286-
L-I/X-287-L-1/X-288-L-1/X-289-L-1/X-290-L-1/X-291- L-1/X-292-
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-99-
L-1/X-293-L-1/X-294-L-1/X-295-L-1/X-296-L-1/X-297-L-1/X-298-
L-1/X-299-L-1/X-300-L-1/X-301-L-1/X-302-L-1/X-303-L-l/X-304-
L-1/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-1/X-314-L-1/X-315-L-1/X-316-
L-1/X-317-L-1/X-318-L-1/X-319-L-1/X-320-L-1/X-321-L-1/X-322-
L-1/X-323-L-1/X-324-L-1/X-325-L-1/X-326-L-1/X-327-L-1/X-328-
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-I/X-338-L-1/X-339-L-1/X-340-
L-1/X-341-L-1/X-342-L-1/X-343-L-1/X-344-L-1/X-345-L-1/X-346-
L-1/X-347-L-1/X-348-L-1/X-349-L-1/X-350-L-1/X-351-L-1/X-352-
L-1/X-353-L-1/X-354-L-1/X-355-L-1/X-356-L-1/X-357-L-1/X-358-
L-1/X-359-L-1/X-360-L-1/X-361-L-1/X-362-L-1/X-363-L-1/X-364-
L-1/X-365-L-1/X-366-L-1/X-367-L-1/X-368-L-1/X-369-L-1/X-370-
L-1/X-371-L-1/X-372-L-1/X-373-L-I/X-374-L-1/X-375-L-1/X-376-
IS L-1/X-377-L-1/X-378-L-1/X-379-L-1/X-380-L-1/X-381-L-1/X-382-
L-I/X-383-L-1/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-1/X-394-
L-1/X-395-L-1/X-396-L-1/X-397-L-1/X-398-L-1/X-399-L-1/X-400-
L-1/X-401-L-1/X-402-L-1/X-403-L-1/X-404-L-1/X-4.05-L-1/X-406-
L-1 /X-407-L-1 /X-408-L-1 /X-409-L-1 /X-410-L-1 /X-411-L-1 /X-412-
L-1/X-413-L-1/X-414-L-1/X-415-L-1/X-416-L-1/X-417-L-1/X-418-
L-2/X-1- L-2/X-2- L-2/X-3- L-2/X-4- L-2/X-5- L-2/X-6-
L-2/X-7- L-2/X-8- L-2/X-9- L-2/X-10- L-2/X-11- L-2/X-12-
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-19- 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-
L-2/X-31- L-2/X-32-L-2/X-33- L-21X-34- L-2/X-35- L-2/X-36-
CA 02321191 2000-08-15
WO 99/64046 PCTNS99/I2768
-100-
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-2IX-57- L-2/X-58- L-2/X-59- L-2/X-60-
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-77- 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-
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-2/X-115- L-2/X-116-L-2/X-117-L-2/X-118-L-2/X-119-L-2/X-120-
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-138-
L-2/X-139- L-2/X-140-L-2/X-141-L-2/X-142-L-2/X-143-L-2/X-144-
L-2/X-145- L-2/X-146-L-2/X-147-L-2/X-148-L-2/X-149-L-2/X-150-
L-2/X-151- L-2/X-152-L-2/X-153-L-2/X-154-L-2/X-155-L-2/X-156-
L-2/X-157- L-2/X-158-L-2/X-159-L-2/X-160-L-2/X-161-L-2/X-162-
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-1.92-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-
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-101-
L-2/X-209-L-2/X-210-L-2/X-211-L-2/X-212-L-2/X-213-L-2/X-214-
L-2/X-215-L-2/X-216-L-2/X-217-L-2/X-218-L-2/X-219-L-2/X-220-
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-2/X-26b-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-2IX-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-
L-2/X-377-L-2/X-378-L-2/X-379-L-2/X-380-L-2/X-381-L-2/X-382-
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-102-
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-
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-411-L-2/X-412-
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-
L-3/X-7- L-3/X-8- L-3/X-9- L-3/X-10- L-3/X-11- L-3/X-I2-
L-3/X-13- L-3/X-14- L-3/X-15- L-3/X-lb- L-3/X-17- L-3/X-18-
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-
L-3/X-37- L-3/X-38- L-3/X-39- L-3/X-40- L-3/X-41- L-3/X-42-
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-3IX-56- L-3/X-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-
L-3/X-67- L-3/X-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-3/X-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-
L-3/X-97- L-3/X-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-31X-105-L-3/X-106-L-3/X-107-L-3/X-108-
L-3/X-109-L-3/X-1 L-3/X-111-L-3/X-112-L-3/X-113-L-3/X-114-
i0-
L-3/X-115-L-3/X-116-L-3/X-1 L-3/X-i L-3/X-119-L-3/X-120-
i7- 18-
L-3/X-121-L-3/X-122-L-3/X-123-L-3/X-124-L-3/X-125-L-3/X-126-
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-103-
L-3/X-127- L-3/X-128-L-3/X-129-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-3/X-137-L-3/X-138-
L-3/X-139- L-3/X-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-3lX-148-L-3/X-149-L-3/X-150-
L-3/X-151- L-3/X-152-L-3/X-153-L-3/X-154-L-3/X-155-L-3/X-156-
L-3/X-157- L-3/X-158-L-3/X-159-L-3/X-160-L-3/X-161-L-3/X-162-
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-3lX-170-L-3/X-171-L-3/X-172-
L-3/X-173- L-3/X-174-L-3/X-175-L-3/X-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-
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-I97- 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-2I3-L-3/X-214-
L-3/X-215- L-3/X-216-L-3/X-217-L-3/X-218-L-3/X-219-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-
L-3/X-245- L-3/X-246-L-3/X-247-L-3/X-248-L-3/X-249-L-3/X-250-
L-3/X-251- L-3/X-252-L-3/X-253-L-3/X-254-L-3/X-255-L-3/X-256-
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-
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-
CA 02321191 2000-08-15
WO 99164046 PCT/US99/12768
-104-
L-3/X-299-L-3/X-300-L-3/X-301-L-3/X-302-L-3/X-303-L-3/X-304-
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-
S 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-
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-3IX-352-
L-3/X-353-L-3/X-354-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-
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-374-L-3/X-375-L-3/X-376-
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-
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-3/X-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-
L-4/X-1- L-4/X-2- L-4/X-3- L-4/X-4- L-4/X-5- L-4/X-6-
L-4/X-7- L-4/X-8- L-4/X-9- L-4/X-10- L-4/X-11- L-4/X-12-
L-4/X-13- L-4/X-14- L-4/X-15- L-4/X-16- L-4/X-17- L-4/X-18-
L-4/X-19- L-4/X-20- L-4/X-21- L-4/X-22- L-4/X-23- L-4/X-24-
L-4/X-25- L-4/X-26- L-4/X-27- L-4/X-28- L-4/X-29- L-4/X-30-
L-4/X-31- L-4/X-32- L-4/X-33- L-4/X-34- L-4/X-35- L-4/X-36-
L-4/X-37- L-4/X-38- L-4/X-39- L-4/X-40- L-4/X-41- L-4/X-42-
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-i05-
L-4/X-43- L-4/X-44- L-4/X-45- L-4/X-46- L-4/X-47- L-4/X-48-
L-4/X-49- L-4./X-50-L-4/X-51- L-4/X-52- L-4/X-53- L-4/X-54-
L-4/X-55- L-4/X-56- L-4/X-57- L-4/X-58- L-4/X-59- L-4/X-60-
L-4/X-61- L-4/X-62- L-4/X-63- L-4./X-64-L-4/X-65- L-4/X-66-
L-4/X-67- L-4/X-68- L-4/X-69- L-4/X-70- L-4/X-71- L-4/X-72-
L-4/X-73- L-4/X-74- L-4/X-75- L-4/X-76- L-4/X-77- L-4/X-78-
L-4/X-79- L-4/X-80- L-4/X-81- L-4/X-82- L-4/X-83- L-4/X-84-
L-4/X-85- L-4/X-86- L-4/X-87- L-4/X-88- L-4/X-89- L-4/X-90-
L-4/X-91- L-4/X-92- L-4/X-93- L-4./X-94-L-4/X-95- L-4/X-96-
L-4/X-97- L-4/X-98- L-4/X-99- L-4/X-100-L-4/X-101-Ir4/X-102-
L-4/X-103-L-4/X-104-L-4/X-105-L-4/X-106-L-4/X-107-L-4/X-108-
L-4/X-109-L-4/X-110-L-4/X-111-L-4/X-112-L-4/X-113-L-4/X-114-
L-4/X-115-L-4/X-116-L-4/X-117-L-4/X-118-L-4/X-119-L-4/X-120-
L-4/X-121-L-4/X-122-L-4/X-123-L-4/X-124-L-4/X-125-L-4/X-126-
L-4/X-127-L-4/X-128-L-4/X-129-L-4/X-130-L-4/X-131-L-4/X-132-
L-4/X-133-L-4/X-134-L-4/X-135-L-4/X-136-L-4/X-137-L-4/X-138-
L-4/X-139-L-4/X-140-L-4/X-141-L-4/X-142-L-4/X-143-L-4/X-144-
L-4/X-145-L-4/X-146-L-4/X-147-L-4/X-148-L-4/X-149-L-4/X-150-
L-4/X-151-L-4/X-152-L-4/X-153-L-4/X-154-L-4/X-155-L-4/X-156-
L-4/X-157-L-4/X-158-L-4/X-159-L-4/X-160-L-4/X-161-L-4/X-162-
L-4/X-163-L-4/X-164-L-4/X-165-L-4/X-166-L-4/X-167-L-4/X-168-
L-4/X-169-L-4/X-170-L-4/X-171-L-4/X-172-
L-4/X-173-L-4/X-174-L-4/X-175-L-4/X-176-L-4/X-177-L-4/X-178-
L-4/X-179-L-4/X-180-L-4/X-181-L-4/X-182-L-4/X-183-L-4/X-184-
L-4/X-185-L-4/X-186-L-4/X-187-L-4/X-188-L-4/X-189-L-4/X-190-
L-4/X-191-L-4/X-192-L-4/X-193-L-4/X-194-L-4/X-195-L-4/X-196-
L-4/X-197-L-4/X-198-L-4/X-199-L-4/X-200-L-4/X-201-L-4/X-202-
L-4/X-203-L-4./X-204-. L-4/X-205-L-4/X-206-L-4/X-207-L-4/X-208-
L-4/X-209-L-4/X-210-L-4/X-211-L-4/X-212-L-4/X-213-L-4/X-214-
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-106-
L-4/X-21S- L-4/X-216-L-4/X-217-L-4/X-218-L-4/X-219-L-4/X-220-
L-4/X-221- L-4/X-222-L-4/X-223-L-4/X-224-L-4/X-22S-L-4/X-226-
L-4/X-227- L-4/X-228-L-4/X-229-L-4/X-230-L-4/X-231-L-4/X-232-
,
L-4/X-233- L-4/X-234-L-4/X-23S-L-4/X-236-L-4/X-237-L-4/X-238-
S L-4/X-239- L-4/X-240-L-4/X-241-L-4/X-242-L-4/X-243-L-4/X-244-
L-4/X-24S- L-4/X-246-L-4/X-247-L-4/X-248-L-4/X-249-L-4/X-2S0-
L-4/X-2S1- L-4/X-2S2-L-4/X-2S3-L-4/X-2S4-L-4/X-2SS-L-4/X-2S6-
L-4/X-2S7- L-4/X-2S8-L-4/X-2S9-L-4/X-260-L-4/X-261-L-4/X-262-
L-4/X-263- L-4/X-264-L-4/X-26S-L-4/X-266-L-4./X-267-L-4/X-268-
L-4/X-269- L-4/X-270-L-4/X-271-L-4/X-272-L-4/X-273-L-4/X-274-
L-4/X-27S- L-4/X-276-L-4/X-277-L-4/X-278-L-4/X-279-L-4/X-280-
L-4/X-281- L-4/X-282-L-4/X-283-L-4/X-284-L-4/X-28S-L-4/X-286-
L-4/X-287- L-4/X-288-L-4/X-289-L-4/X-290-L-4/X-291-L-4/X-292-
L-4/X-293- L-4/X-294-L-4/X-29S-L-4/X-296-L-4/X-297-L-4/X-298-
1S L-4./X-299-L-4/X-300-L-4/X-301-L-4/X-302-L-4/X-303-L-4/X-304-
L-4/X-30S- L-4/X-306-L-4/X-307-L-4/X-308-L-4/X-309-L-4/X-310-
L-4/X-311- L-4/X-312-L-4/X-313-L-4/X-314-L-4/X-31S-L-4/X-316-
L-4/X-317- L-4/X-318-L-4/X-319-L-4/X-320-L-4/X-321-L-4/X-322-
L-4/X-323- L-4/X-324-L-4/X-32S-L-4/X-326-L-4/X-327-L-4/X-328-
L-4/X-329- L-4/X-330-L-4/X-331-L-4/X-332-L-4/X-333-L-4./X-334-
L-4/X-33S- L-4/X-336-L-4/X-337-L-4/X-338-L-4./X-339-L-4/X-340-
L-4/X-341- L-4/X-342-L-4/X-343-L-4/X-344-L-4/X-34S-L-4/X-346-
L-4/X-347- L-4/X-348-L-4/X-349-L-4/X-3S0-L-4/X-3S1-L-4/X-3S2-
L-4/X-3S3- L-4/X-3S4-L-4/X-3SS-L-4/X-3S6-L-4/X-3S7-L-4/X-3S8-
2S L-4/X-3S9- L-4/X-360-L-4/X-361-L-4/X-362-L-4/X-363-L-4./X-364-
L-4/X-36S- L-4./X-366-L-4/X-367-L-4/X-368-L-4/X-369-L-4/X-370-
L-4/X-371- L-4/X-372-L-4/X-373-L-4/X-374-L-4/X-37S-L-4/X-376-
L-4/X-377- L-4/X-378-L-4/X-379-L-4/X-380-L-4./X-381-L-4/X-382-
L-4/X-383- L-4/X-384-L-4/X-38S-L-4/X-386-L-4/X-387-L-4/X-388-
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-107-
L-4/X-389- L-4/X-390-L-4/X-391-L-4/X-392-L-4/X-393-L-4/X-394-
L-4/X-395- L-4/X-396-L-4/X-397-L-4/X-398-L-4/X-399-L-4/X-400-
L-4/X-401- L-4/X-402-L-4/X-403-L-4/X-404-L-4/X-405-L-4/X-406-
L-4/X-407- L-4/X-408-L-4/X-409-L-4/X-410-L-4/X-411-L-4/X-412-
L-4/X-413- L-4/X-414-L-4/X-415-L-4/X-416-L-4/X-417-L-4/X-418-
L-SIX-1- L-5/X-2- L-5/X-3- L-5/X-4- L-5/X-5- L-SIX-6-
L-5/X-7- L-5/X-8- L-5/X-9- L-S/X-10- L-5/X-11- L-5/X-12-
L-5/X-13- L-5/X-14- L-5/X-15- L-5/X-16- L-5/X-17- L-5/X-18-
L-5/X-19- L-5/X-20- L-5/X-21- L-5/X-22- L-5/X-23- L-5/X-24-
L-5/X-25- L-5/X-26- L-5/X-27- L-5/X-28- L-5/X-29- L-5/X-30-
L-5/X-31- L-5/X-32- L-5/X-33- L-5/X-34- L-S/X-35- L-5/X-36-
L-5/X-37- L-5/X-38- L-5/X-39- L-5/X-40- L-5/X-41- L-5/X-42-
L-5/X-43- L-5/X-44- L-5/X-45- L-5/X-46- L-5/X-47- L-5/X-48-
L-5/X-49- L-5/X-50- L-5/X-51- L-5/X-52- L-5/X-53- L-5/X-54-
L-5/X-55- L-SIX-56- L-5/X-57- L-SIX-58- L-S/X-59- L-5/X-60-
L-5/X-61- L-5/X-62- L-5/X-63- L-5/X-b4- L-S/X-65- L-S/X-66-
L-5/X-67- L-5/X-68- L-5/X-69- L-5/X-70- L-S/X-71- L-5/X-72-
L-5/X-73- L-S/X-74- L-5/X-75- L-5/X-7b- L-5/X-77- L-5/X-78-
L-SIX-79- L-5/X-80- L-5/X-81- L-5/X-82- L-5/X-83- L-5/X-84-
L-5/X-85- L-5/X-86- L-5/X-87- L-5/X-88- L-SIX-89- L-5/X-90-
L-5/X-91- L-SIX-92- L-SlX-93- L-5/X-94- L-5/X-95- L-SIX-96-
L-5/X-97- L-5/X-98- L-5/X-99- L-SlX-100-L-5/X-101-L-SIX-102-
L-5/X-103-L-SIX-104-L-SIX-105-L-5/X-106-L-5/X-107-L-5/X-108-
L-5/X-109-L-SIX-110-L-5/X-111-L-5/X-112-L-5/X-113-L-5/X-114-
L-5/X-115-L-SIX-116-L-5/X-117-L-5/X-118-L-5/X-119-L-5/X-120-
L-5/X-121-L-5/X-122-L-SIX-123-L-SIX-124-L-5/X-125-L-5/X-126-
L-5/X-127-L-5/X-128-L-SIX-129-L-5/X-130-L-SIX-131-L-5/X-132-
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-108-
L-5/X-133- L-5/X-134-L-5/X-135-L-5/X-13b-L-5/X-137-L-5/X-138-
L-5/X-139- L-5/X-140-L-5/X-141-L-5/X-142-L-5/X-143-L-5/X-144-
L-5/X-145- L-5/X-146-L-5/X-147-L-5/X-148-L-5/X-149-L-5/X-150-
L-5/X-151- L-5/X-152-L-5/X-153-L-5/X-154-L-5/X-155-L-5/X-156-
L-5/X-157- L-5/X-158-L-5/X-159-L-5/X-160-L-5/X-161-L-5/X-162-
L-5/X-163- L-5/X-164-L-5/X-165-L-5/X-166-L-5/X-167-L-5/X-168-
L-5/X-169- L-5/X-170-L-5/X-171-L-5/X-172-
L-5/X-173- L-5/X-174-L-5/X-175-L-5/X-176-L-5/X-177-L-5/X-178-
L-5/X-179- L-5/X-180-L-5/X-181-L-5/X-182-L-5/X-183-L-5/X-184-
L-5/X-185- L-5/X-186-L-5/X-187-L-5/X-188-L-5/X-189-L-5/X-190-
L-5/X-191- L-5/X-192-L-5/X-193-L-5/X-194-L-5/X-195-L-5/X-196-
L-5/X-197- L-5/X-198-L-5/X-199-L-SIX-200-L-5/X-201-L-5/X-202-
L-5/X-203- L-5/X-204-L-5/X-205-L-5/X-206-L-5/X-207-L-5/X-208-
L-5/X-209- L-5/X-210-L-5/X-211-L-5/X-212-L-5/X-213-L-5/X-214-
. L-5/X-215- L-5/X-216-L-5/X-217-L-5/X-218-L-5/X-219-L-5/X-220-
L-5/X-221- L-5/X-222-L-5/X-223-L-5/X-224-L-5/X-225-L-5/X-226-
L-SIX-227- L-5/X-228-L-5/X-229-L-5/X-230-L-5/X-231-L-5/X-232-
L-5/X-233- L-5/X-234-L-5/X-235-L-5/X-236-L-5/X-237-L-5/X-238-
L-5/X-239- L-5/X-240-L-5/X-241-L-5/X-242-L-5/X-243-L-5/X-244-
L-5/X-245- L-5/X-246-L-5/X-247-L-5/X-248-L-5/X-249-L-5/X-250-
L-5/X-251- L-5/X-252-L-5/X-253-L-5/X-254-L-5/X-255-L-5/X-256-
L-5/X-257- L-5/X-258-L-5/X-259-L-5/X-260-L-5/X-261-L-5/X-262-
L-5/X-263- L-5/X-264-L-5/X-265-L-5/X-266-L-5/X-267-L-5/X-268-
L-5/X-269- L-5/X-270-L-5/X-271-L-5/X-272-L-5/X-273-L-5/X-274-
L-5/X-275- L-5/X-276-L-5/X-277-L-5/X-278-L-5/X-279-L-5/X-280-
L-5/X-281- L-5/X-282-L-5/X-283-L-5/X-284-L-5/X-285-L-5/X-286-
L-5/X-287- L-5/X-288-L-5/X-289-L-5/X-290-L-5/X-291-L-5/X-292-
L-5/X-293- L-SIX-294-L-5/X-295-L-5/X-296-L-5/X-297-L-5/X-298-
L-5/X-299- L-5/X-300-L-5/X-301-L-5/X-302-L-5/X-303-L-5/X-304-
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-109-
L-5/X-305- L-5/X-306-L-S/X-307-L-S/X-308-L-5/X-309-L-SIX-310-
L-5/X-311- L-5/X-312-L-5/X-3I3-L-5/X-314-L-5/X-315-L-5/X-316-
L-5/X-317- L-5/X-318-L-5/X-319-L-5/X-320-L-5/X-321-L-5/X-322-
L-5/X-323- L-5/X-324-L-5/X-325-L-5/X-326-L-5/X-327-L-5/X-328-
L-5/X-329- L-5/X-330-L-5/X-331-L-S/X-332-L-S/X-333-L-5/X-334-
L-5!X-335- L-5/X-336-L-5/X-337-L-5/X-338-L-SIX-339-L-5/X-340-
L-5/X-341- L-5/X-342-L-5/X-343-L-5/X-344-L-SIX-345-L-5/X-346-
L-5/X-347- L-5/X-348-L-SIX-349-L-5/X-350-L-5/X-351-L-5/X-352-
L-5/X-353- L-5/X-354-L-S/X-355-L-5/X-356-L-5/X-357-L-5/X-358-
L-5/X-359- L-5/X-360-L-SIX-361-L-5/X-362-L-5/X-363-L-SIX-3b4-
L-5/X-365- L-5/X-366-L-S/X-367-L-5/X-368-L-5/X-369-L-5/X-370-
L-5/X-371- L-5/X-372-L-5/X-373-L-5/X-374-L-5/X-375-L-5/X-376-
L-S/X-377- L-5/X-378-L-5/X-379-L-SIX-380-L-5/X-381-L-SIX-382-
L-5/X-383- L-5/X-384-L-5/X-385-L-5/X-386-L-5/X-387-L-SIX-388-
L-5/X-389- L-5/X-390-L-5/X-391-L-5/X-392-L-S/X-393-L-5/X-394-
L-5/X-395- L-5/X-396-L-5/X-397-L-5/X-398-L-5/X-399-L-5/X-400-
L-5/X-401- L-5/X-402-L-5/X-403-L-SIX-404-L-S/X-405-L-5/X-406-
L-5/X-407- L-5/X-408-L-5/X-409-L-5/X-410-L-5/X-411-L-5/X-412-
L-5/X-413- L-S/X-414-L-5/X-415-L-5/X-416-L-S/X-417-L-5/X-418-
L-6/X-1- L-6/X-2- L-6/X-3- L-6/X-4- L-6/X-5- L-6/X-6-
L-6/X-7- L-6/X-8- L-6/X-9- L-6/X-10- L-6/X-11- L-6/X-12-
L-6/X-13- L-6/X-14- L-6/X-15- L-6/X-16- L-6/X-17- L-6/X-18-
L-6/X-19- L-6/X-20- L-6/X-21- L-6/X-22- L-6/X-23- L-6/X-24-
L-6/X-25- L-6/X-26- L-6/X-27- L-6/X-28- L-6/X-29- L-6/X-30-
L-6/X-31- L-6/X-32- L-6/X-33- L-6/X-34- L-6/X-35- L-6/X-36-
L-6/X-37- L-6/X-38- L-6/X-39- L-6/X-40- L-6/X-41- L-6/X-42-
L-6/X-43- L-6/X-44- L-6/X-45- L-6/X-46- L-6/X-47- L-6/X-48-
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-110-
L-6/X-49- L-6/X-50- L-6/X-51- L-6/X-52- L-6/X-53- L-6/X-54-
L-6/X-55- L-6/X-56- L-6/X-57- L-6/X-58- L-6/X-59- L-6/X-60-
L-6/X-61- L-6/X-62- L-6/X-63- L-6/X-64- L-6/X-65- L-6/X-66-
L-6/X-67- L-6/X-68- L-6/X-69- L-6/X-70- L-6/X-71- L-6/X-72-
L-6/X-73- L-6/X-74- L-6/X-75- L-6/X-76- L-6/X-77- L-6/X-78-
L-6/X-79- L-6/X-80- L-6/X-81- L-6/X-82- L-6/X-83- L-6/X-84-
L-6/X-85- L-6/X-86- L-6/X-87- L-6/X-88- L-6/X-89- L-6/X-90-
L-6/X-91- L-6/X-92- L-6/X-93- L-6/X-94- L-6/X-95- L-6/X-96-
L-6/X-97- L-6/X-98- L-6/X-99- L-6/X-100-L-6/X-101-L-6/X-102-
L-6/X-103-L-6/X-104-L-6/X-105-L-6/X-106-L-6/X-107-L-6/X-108-
L-6/X-109-L-6/X-110-L-6/X-111-L-6/X-112-L-6/X-113-L-6/X-114-
L-6/X-115-L-6/X-116-L-6/X-117-L-6/X-118-L-6/X-119-L-6/X-120-
L-6/X-121-L-6/X-122-L-6/X-123-L-6/X-124-L-6/X-125-L-6/X-126-
L-6/X-127-L-6/X-128-L-6/X-129-L-6/X-130-L-6/X-131-L-6/X-132-
L-6/X-133-L-6/X-134-L-6/X-135-L-6/X-136-L-6/X-137-L-6/X-138-
L-6/X-139-L-6/X-140-L-6/X-141-L-6/X-142-L-6/X-143-L-6/X-144-
L-6/X-145-L-6/X-146-L-6/X-147-L-6/X-148-L-6/X-149-L-6/X-150-
L-6/X-151-L-6/X-152-L-6/X-153-L-6/X-154-L-6/X-155-L-6/X-156-
L-6/X-157-L-6/X-158-L-6/X-159-L-6/X-160-L-6/X-161-L-6/X-162-
L-6/X-163-L-6/X-164-L-6/X-165-L-6/X-166-L-6/X-167-L-6/X-168-
L-6/X-169-L-6/X-170-L-6/X-171-L-6/X-172-
L-6/X-173-L-6/X-174-L-6/X-175-L-6/X-176-L-6/X-177-L-6/X-178-
L-6/X-179-L-6/X-180-L-6/X-181-L-6/X-182-L-6/X-183-L-6/X-184-
L-6/X-185-L-6/X-186-L-6/X-187-L-6/X-188-L-6/X-189-L-6/X-190-
L-6/X-191-L-6/X-192-L-6/X-193-L-6/X-194-L-6/X-195-L-6/X-196-
L-6/X-197-L-6/X-198-L-6/X-199-L-6/X-200-L-6/X-201-L-6/X-202-
L-6/X-203-L-6/X-204-L-6/X-205-L-6/X-206-L-6/X-207-L-6/X-208-
L-6/X-209-L-6/X-210-L-6/X-211-L-6/X-212-L-6/X-213-L-6/X-214-
L-6/X-215-L-6/X-216-L-6/X-217-L-6/X-218-L-6/X-219-L-6/X-220-
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-111-
L-6/X-221- L-6/X-222-L-6/X-223-L-6/X-224- L-6/X-22S-L-6/X-226-
L-6/X-227- L-6/X-228-L-6/X-229-L-6/X-230- L-6/X-231-L-6/X-232-
L-6/X-233- L-6/X-234-L-6/X-23S-L-6/X-236- L-6/X-237-L-6/X-238-
L-6/X-239- L-6/X-240-L-6/X-241-L-6/X-242- L-6/X-243-L-6/X-244-
S L-6/X-24S- L-6/X-246-L-6/X-247-L-6/X-248- L-6/X-249-L-6/X-2S0-
L-6/X-2S1- L-6/X-2S2-L-6/X-2S3-L-6/X-2S4- L-6/X-2SS-L-6/X-2S6-
L-6/X-2S7- L-6/X-2S8-L-6/X-2S9-L-6/X-260- L-6/X-261-L-6/X-262-
L-6/X-263- L-6/X-264-L-6/X-26S-L-6/X-266- L-6/X-267-L-6/X-268-
L-6/X-269- L-6/X-270-L-6/X-271-L-6/X-272- L-6/X-273-L-6/X-274-
L-6/X-27S- L-6/X-276-L-6/X-277-L-6/X-278- L-6/X-279-L-6/X-280-
L-6/X-281- L-6/X-282-L-6/X-283-L-6/X-284- L-6/X-28S-L-6/X-286-
L-6/X-287- L-6/X-288-L-6/X-289-L-6lX-290- L-6/X-291-L-6/X-292-
L-6/X-293- L-6/X-294-L-6/X-29S-L-6/X-296- L-6/X-297-L-6/X-298-
L-6/X-299- L-6/X-300-L-6/X-301-L-6/X-302- L-6/X-303-L-6/X-304-
1S L-6/X-305- L-6/X-306-L-6/X-307-L-6/X-308- L-6/X-309-L-6/X-310-
L-6/X-311- L-6/X-312-L-6/X-313-L-6/X-314- L-6/X-31S-L-6/X-316-
L-6/X-317- L-6/X-318-L-6/X-319-L-6/X-320- L-6/X-321-L-6/X-322-
L-6/X-323- L-6/X-324-L-6/X-32S-L-6/X-326- L-6/X-327-L-6/X-328-
L-6/X-329- L-6/X-330-L-6/X-331-L-6/X-332- L-6/X-333-L-6/X-334-
L-6/X-33S-. L-6/X-336-L-6/X-337-L-6/X-338- L-6/X-339-L-6/X-340-
L-6/X-341- L-6/X-342-L-6/X-343-L-6/X-344- L-6/X-34S-L-6/X-346-
L-6/X-347- L-6/X-348-L-6/X-349-L-6/X-3S0- L-6/X-3S1-L-6/X-3S2-
L-6/X-3S3- L-6/X-3S4-L-6/X-3SS-L-6/X-3S6- L-6/X-3S7-L-6/X-3S8-
L-6/X-3S9- L-6/X-360-L-6/X-361-L-6/X-362- L-6/X-363-L-6/X-364-
2S L-6/X-36S- L-6/X-366-L-6/X-367-L-6/X-368- L-6/X-369-L-6/X-370-
L-6/X-371- L-6/X-372-L-6/X-373-L-6/X-374- L-6/X-37S-L-6/X-376-
L-6/X-377- L-6/X-378-L-6/X-379-L-6/X-380- L-6/X-381-L-6/X-382-
L-6/X-383- L-6/X-384-L-6/X-38S-L-6/X-386- L-6/X-387-L-6/X-388-
L-6/X-389- L-6/X-390-L-6/X-391-L-6/X-392- L-6/X-393-L-6/X-394-
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-112-
L-6/X-395-L-6/X-396- L-6/X-397-L-6/X-398-L-6/X-399-L-6/X-400-
L-6/X-401-L-6/X-402- L-6/X-403-L-6/X-404-L-6/X-405-L-6/X-406-
L-6/X-407-L-6/X-408- L-6/X-409-L-6/X-410-L-6/X-411-L-b/X-412-
L-6/X-413-L-6/X-414- L-6/X-415-L-6/X-416-L-6/X-417-L-6/X-418-
Pharmaceutical Formulations
When employed as pharmaceuticals, the compounds of this invention are
usually administered in the form of pharmaceutical compositions. These
compounds
can be administered by a variety of routes including oral, rectal,
transdermal,
10 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.
15 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
20 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
example, up to
25 10% by weight of the active compound, soft and hard gelatin capsules,
suppositories,
sterile injectable solutions, and sterile packaged powders.
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-113-
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
to a particle size of less than 200 mesh. If the active compound is
substantially
5 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,
10 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. The
15 compositions of the invention can be formulated so as to provide quick,
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
20 dosage containing from about 0.001 to about 1 g, more usually about 1 to
about 30
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
25 excipient. Preferably, the compound of formula I above is employed at no
more
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).
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/IZ768
-114-
The active compound is effective over a wide dosage range and is generally
administered in a pharmaceutically effective amount. It, will be understood,
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
S 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.
For preparing solid compositions such as tablets, the principal active
10 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 so
that the composition may be readily subdivided into equally effective unit
dosage
15 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
20 compounded to provide a dosage form affording the advantage of prolonged
action.
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 duodenum
or to
25 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.
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-115-
The liquid forms in which the novel compositions of the present invention
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,
S or peanut oil, as well as elixirs and similar pharmaceutical vehicles.
Compositions for inhalation or insufflation include solutions and suspensions
in pharmaceutically acceptable, aqueous or organic solvents, or mixtures
thereof, and
powders. The liquid or solid compositions may contain suitable
pharmaceutically
10 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 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
15 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.
The following formulation examples illustrate representative pharmaceutical
20 compositions of the present invention.
Formulation ~xamnle 1
Hard gelatin capsules containing the following ingredients are prepared:
Quantity
25 Ingredient (mg/calas~le)
Active Ingredient 30.0
Starch
305.0
Magnesium stearate 5.0
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-116-
The above ingredients are mixed and filled into hard gelatin capsules in 340
mg quantities.
Formulation Example 2
A tablet formula is prepared
using the ingredients below:
Quantity
Ingredient m / 1
Active Ingredient 25.0
Cellulose, microcrystalline 200.0
Colloidal silicon dioxide 10.0
Stearic acid 5.0
The components are blended and compressed to form tablets, each weighing
240 mg.
Formulation Example ~
A dry powder inhaler formulation is prepared containing the following
components:
~edient Weir
Active Ingredient
Lactose 95
The active ingredient is mixed with the lactose and the mixture is added to a
dry powder inhaling appliance.
Formulation xamnle 4
Tablets, each containing 30 mg of active ingredient, are prepared as follows:
Quantity
Img/tablet)
Active Ingredient 30.0 mg
Starch 45.0 mg
Microcrystalline cellulose 35.0 mg
CA 02321191 2000-08-15
WO 99/64046 PCT/US99I12768
-117-
Polyvinylpyrrolidone
(as 10 % solution in sterile water) 4.0 mg
Sodium carboxymethyl starch 4.5 mg
Magnesium stearate 0.5 mg
Talc 1.
Total 120 mg
The active ingredient, starch and cellulose are passed through a No. 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.
The granules so produced are dried at 50° to 60°C and passed
through a 16 mesh
U.S. sieve. The 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.
Formulation Example 5
Capsules, each containing 40 mg of medicament are made as follows:
Quantity
Ingredient (mg/c~psule)
Active Ingredient 40.0 mg
Starch 109.0 mg
Magnesium stearate 1,
Total 150.0 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 150
mg
quantities.
Formulation Exampj~ø
Suppositories, each containing 25 mg of active ingredient are made as follows:
a i Amount
Active Ingredient 25 mg
Saturated fatty acid glycerides to 2,000 mg
CA 02321191 2000-08-15
WO 99/64046 PCTNS99/12768
-11$-
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
minimum
heat necessary. The mixture is then poured into a suppository mold of nominal
2.0 g
capacity and allowed to cool.
Formulation .xamp~?
Suspensions, each containing 50 mg of medicament per 5.0 mL dose are made
as follows:
n redient
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
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.
Formulation Example 8
A formulation may be prepared as follows:
Quantity
Ingredient lmg/capsule)
Active Ingredient 15.0 mg
Starch 407.0 mg
Magnesium stearate 3,
Total 425.0 mg
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-119-
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.
A formulation may be prepared as follows:
I~redie~
Active Ingredient S.~~itv
Corn Oil g
1.0 mL
Formulation Example 10
A topical formulation may be prepared as follows:
,~ngc'edient want tw
Active Ingredient 1-10 g
Emulsifying Wax 30 g
Liquid Paraffin 20 g
White Soft Paraffin to 100 g
The white soft paraffin is heated until molten. The liquid paraffin and
20 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.
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
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.
CA 02321191 2000-08-15
WO 99/64046 PCTNS99/12768
-120-
Other suitable formulations for use in the present invention can be found in
Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia,
PA,
17th ed. (19$5).
LTtilitv
The multibinding compounds of this invention are agonists, partial agonists,
inverse agonists or antagonists of the SHT3 receptors, which are known to
mediate
numerous disorders. Accordingly, the multibinding compounds and pharmaceutical
compositions of this invention are useful in the treatment and prevention of
various
disorders, in particular, chemotherapy induced and radiation induced emesis,
anxiety, schizophrenia, drug withdrawal and cognitive disorders.
When used in treating or ameliorating such conditions, the compounds of this
invention are typically delivered to a patient in need of such treatment by a
pharmaceutical composition comprising a pharmaceutically acceptable diluent
and an
effective amount of at least one compound of this invention. The amount of
compound administered to the patient will vary depending upon what compound
and/or composition is being administered, the purpose of the administration,
such as
prophylaxis or therapy, the state of the patient, the manner of
administration, and the
like.
In therapeutic applications, compositions are administered to a patient
already
suffering from, for example, emesis, in an amount sufficient to at least
partially
reduce the symptoms. Amounts effective for this use will depend on the
judgment of
25 the attending clinician depending upon factors such as the degree or
severity of the
disorder in the patient, the age, weight and general condition of the patient,
and the
like. The pharmaceutical compositions of this invention may contain more than
one
compound of the present invention.
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-121-
As noted above, the compounds administered to a patient are in the form of
pharmaceutical compositions described above which can be administered by a
variety
of routes including oral, rectal, transdermal, subcutaneous, intravenous,
intramuscular, etc. These compounds are effective as both injectable and oral
deliverable pharmaceutical compositions. Such compositions are prepared in a
manner well known in the pharmaceutical art and comprise at least one active
compound.
The multibinding compounds of this invention can also be administered in the
form of pro-drugs, i.e., as derivatives which are converted into a
biologically active
compound in vivo. Such pro-drugs will typically include compounds in which,
for
example, a carboxylic acid group, a hydroxyl group or a thiol group is
converted to
a biologically liable group, such as an ester, lactone or thioester group
which will
hydrolyze in vivo to reinstate the respective group.
Additionally, the compounds of the invention may be bound to affinity resins
for affinity chromatography. The compounds of the invention may be used as a
tool
in immunoprecipitation. The compounds may be used to identify a receptor in
vitro
for example in microscopy, electrophoresis and chromatography.
Combinatorial Chemistry
The compounds can be assayed to identify which of the multimeric ligand
compounds possess multibinding properties. First, one identifies a ligand or
mixture
of ligands which each contain at least one reactive functionality and a
library of
linkers which each include at least two functional groups having complementary
reactivity to at least one of the reactive functional groups of the ligand.
Next one
prepares a multimeric ligand compound library by combining at least two
stoichiometric equivalents of the ligand or mixture of ligands with the
library of
linkers under conditions wherein the complementary functional groups react to
form
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-122-
a covalent linkage between the linker and at least two of the ligands. The
multimeric
ligand compounds produced in the library can be assayed to identify multimeric
ligand compounds which possess multibinding properties.
The method can also be performed using a library of ligands and a linker or
mixture
of linkers.
The preparation of the multimeric ligand compound library can be achieved by
either the sequential or concurrent combination of the two or more
stoichiometric
equivalents of the ligands with the linkers. The multimeric ligand compounds
can be
dimeric, for example, homomeric or heteromeric. A heteromeric ligand compound
library can be prepared by sequentially adding a first and second ligand.
Each member of the multimeric ligand compound library can be isolated from
the library, for example, by preparative liquid chromatography mass
spectrometry
15 (LCMS). The linker or linkers can be flexible linkers, rigid linkers,
hydrophobic
linkers, hydrophilic linkers, linkers of different geometry, acidic linkers,
basic
linkers, linkers of different polarization and/or polarizability or
amphiphilic linkers.
The linkers can include linkers of different chain lengths and/or which have
different
complementary reactive groups. In one embodiment, the linkers are selected to
have
20 different linker lengths ranging from about 2 to 100. The ligand or mixture
of
ligands can have reactive functionality at different sites on the ligands. The
reactive
functionality can be, for example, carboxylic acids, carboxylic acid halides,
carboxyl
esters, amines, halides, pseudohalides, isocyanates, vinyl unsaturation,
ketones,
aldehydes, thiols, alcohols, anhydrides, boronates, and precursors thereof, as
long as
25 the reactive functionality on the ligand is 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.
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-123-
A library of multimeric ligand compounds can thus be formed which possesses
multivalent properties.
Multimeric ligand compounds possessing multibinding properties can be
identified in an iterative method by preparing a first collection or iteration
of
multimeric compounds by contacting at least two stoichiometric equivalents of
the
ligand or mixture of ligands which target the SHT, receptors with a linker or
mixture
of linkers, where the ligand or mixture of ligands includes at least one
reactive
functionality and the linker or mixture of linkers includes at least two
functional
groups having complementary reactivity to at least one of the reactive
functional
groups of the ligand. The ligand(s) and linkers) are reacted under conditions
which
form a covalent linkage between the linker and at least two of the ligands.
The first
collection or iteration of multimeric compounds can be assayed to assess which
if
any of the compounds possess multibinding properties. The process can be
repeated
15 until at least one multimeric compound is found to possess multibinding
properties.
By evaluating the particular molecular constraints which are imparted or are
consistent with imparting multibinding properties to the multimeric compound
or
compounds in the first iteration, a second collection or iteration of
multimeric
compounds which elaborates upon the particular molecular constraints can be
20 assayed, and the steps optionally repeated to further elaborate upon the
molecular
constraints. For example, the steps can be repeated from between 2 and 50
times,
more preferably, between 5 and 50 times.
The following synthetic and biological examples are offered to illustrate this
25 invention and are not to be construed in any way as limiting the scope of
this
invention. Unless otherwise stated, all temperatures are in degrees Celsius.
EXAMPLES
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-124-
In the examples below, the following abbreviations have the following
meanings. If an abbreviation is not defined, it has its generally accepted
meaning.
- Angstroms
cm - centimeter
DCC - dicyclohexyl carbodiimide
DMF - N, N dimethylformamide
DMSO - dimethylsulfoxide
EDTA - ethylenediaminetetraacetic acid
10 g - gram
HPLC - high performance liquid chromatography
MEM - minimal essential medium
mg - milligram
MIC - minimum inhibitory concentration
15 min - minute
mL - milliliter
- millimeter
mmol - millimol
- normal
20 THF - tetrahydrofuran
uL - microliters
~m - microns
CA 02321191 2000-08-15
WO 99/64046 PCTNS99/12768
-125-
Example 1: In vitro Radioligand binding studies.
Receptor binding assays for the SHT~ receptors are performed on rat
5 membrane preparations. In general, after rapid decapitation and dissection
of the
appropriate brain tissue, crude membrane pellets are prepared by
homogenization
and centrifugation. Aliquots of membrane homogenate are added to test tubes
containing buffer, radioligand, and various concentrations of inhibitor.
Nonspecific
binding is determined using an excess of the specified inhibitor for the
receptor of
10 interest. Following the appropriate incubation conditions, the reactions
are
terminated by separating the membrane bound from free radioligand by rapid
filtration over Whatman GF/C filters. Bound radioligand is quantified by
scintillation spectroscopy. The percent inhibition of radiotigand binding at
each test
concentration is calculated, and an ICS is determined by log-probit analysis.
Example 2: Determination of the Efficacy of the Compounds
The following assays are used to evaluate the multi-binding compounds of this
invention.
In vitro binding assay
Using the 5-HT3 receptor binding assay described by Ohta et al.,z' the pKi for
5-HT3 receptor binding is determined for the multi-binding compounds of this
invention.
Ex vivo model
Using the isolated guinea pig colon assay described by Ohta et a1.,2'
contractile
responses to the mufti-binding compounds of this invention are measured.
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-126-
In vivo model
Using the von Bezold-Jarisch reflex test described by Ohta et al., '-' multi-
binding compounds of this invention are tested.
Cisplatin-induced emesis in Ferrets
Using the cisplatin-induced emesis ferret model described by Ohta et a1.,2'
mufti-binding compounds of this invention are tested for their ability to
reduce the
emetic effects of cisplatin in ferrets. Male ferrets weighing 1-1.5 kg are
used.
Thirty minutes after i.v. injection of the test compound into the instep of a
foot,
10 cisplatin (10 mg/kg) is injected into a vein of another foot. Emesis is
recorded for
four hours after injection of cisplatin and defined as a rhythmic abdominal
contraction, either with vomiting or without (retching) associated expulsion
of solid
or liquid material. To evaluate p.o. effects, the test drug is orally
administered one
hour prior to intra-peritoneal injection of cisplatin (10 mg/kg).
S3rntheses of Bivalent 5-HT, Recep or An monists
Basic pharmacophore for 5-HT3 receptor antagonists
Recently, attempts have been made to rationalize the activity of several 5-HT3
antagonists of diverse structural type employing computer-modelling
techniques.
Hibert et al. [J. Med. Chem. 33: 1594 (1990)] computed low-energy
conformations
to define a pharmacophore and receptor map that may account for the activity
of
ondansetron, metoclopramide and some tropane-based esters and amides. The
basic
pharmacophore consists essentially of a carbonyl group coplanar to an aromatic
ring
and a basic centre with. the relative positions and dimensions illustrated
above.
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-127-
Based on the basic pharmacophore for 5-HT3 receptor antagonists and the
current available materials, several classes of bivalent 5-HT3 receptor
antagonists are
designed, the syntheses are described in the following four examples.
Example 1: Indole-linked [N-N] Compounds
Compounds of Formula VI (n = 2-12, R1 = H, alkyl, alkenyl, Ph,
phenylalkyl; 1 of R2-R4 = H, alkyl, alkenyl, phenylalkyl, the others = H,
alkyl)
linked via their indole nitrogen positions, may be prepared as shown in FIGURE
1.
Example 2: Preparation of a Compound of Formula VI In Which
Rl=R3=R4=H, R2=Me, n=2
A mixture of phenylhydrazine (1.08 g, 10 mmol, 1.0 equiv) and 1,3-
cyclohexanedione (1.18 g, 10.5 mmol, 1.05 equiv) in absolute ethanol (15 mL)
is
boiled for 1 h. The reaction mixture is then evaporated to dryness, and the
residue
is dissolved in methanol. Ethyl acetate is added, and the mixture is left to
crystallize
overnight at 0 °C. After vacuum filtration, the hydrazone II is
obtained as a solid.
The hydrazone II from last step is mixed with acetic acid (20 mL) and
concentrated hydrochloric acid (3.2 mL), refluxed for 1 h, and evaporated to
dryness. The residue is taken up in CHZC12 and washed with water. The organic
layer is concentrated, and the residue is purified by silica gel
chromatography using
ethyl acetate as eluant to afford the tetrahydrocarbazolone III.
To a mixture of the tetrahydrocarbazolone III (1.5 g, 8.1 mmol, 2.1 equiv),
K,C03 (2.8 g, 20 mmol, 5.0 equiv) and toluene (50 mL) is added 1,2-
dibromoethane
(0.72 g, 3.8 mmol, 1.0 equiv). After stirring several hours at rt, the
reaction
mixture is washed with water and sat. NH4Cl, organic layer is concentrated in
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/t2768
-128-
vacuo. The residue is purified by silica gel chromatography to give the
dimeric
product IV.
A mixture of IV (1.0 g, 2.5 mmol, 1.0 equiv), paraformaldehyde (0.15 g, 5.0
mmol, 2.0 equiv), dimethylamine hydrochloride (0.45 g, 5.5 mmol, 2.2 equiv),
and
acetic acid (7.5 mL), is stirred for 3 h at 100 °C. After evaporating
to dryness the
residue is treated with 2 N NaOH and CHzCIz. The organic layer is washed with
water and concentrated. The residue is purified by silica gel chromatography
using
methanol containing 3% Et3N as eluant. The desired,fractions are collected and
concentrated, the residue is dissolved in a mixture of ethanol (2.5 mL) and
concentrated HCl (0.2 mL,), giving V as its hydrochloric salt.
The compound V (1.2 g, 2.0 mmol, 1.0 equiv) is mixed with 2-
methylimidazole (0.5 g, 6.0 mmol, 3.0 equiv) and water (6 mL). The mixture is
stirred for 20 h at 100 °C. After cooling at 0 °C, the product
is isolated by
filtration, dried, and purified by silica gel chromatography with CH,Ch
containing
5 % methanol as eluant to afford the compound VI.
L inking at t_h_e tetrahvdrocarbaznlnnP cide-rha;..
Compounds of Formula VIII (n = 2-12, R1 = R2 = H, alkyl, alkenyl, Ph,
phenylalkyl) linked from side-chain of the tetrahydrocarbazolones, may be
prepared
as shown in FIGURE 2, and the synthesis of Formula VII could use similar
chemistries described in FIGURE 1
Example 3: Preparation of a Compound of Formula VIII in which
Rl=H, R2=Me, n=2
The compound VII (R1=H, R2=Me, 1.0 g, 3.4 mmol, 2.1 equiv) is mixed
with ethylenediamine (0.1 g, 1.6 mmol, 1.0 equiv) and water (6 mL). The
mixture
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-129-
is stirred for 20 h at 100 °C. After cooling at 0 °C, the
product is isolated by
filtration, dried, and purified by silica gel chromatography with CHzCIz
containing
% methanol as eluant to afford the compound VIII.
5 Bivalent compound using amino acids ac handle
Compounds of Formula X (n = 2-12, R1 = R2 = H, alkyl, alkenyl, Ph,
phenylalkyl, R3 = available amino acids), may be prepared as shown in FIGURE 3
by standard amide bond formation chemistry, and the precursor IX could be
synthesized from the common intermediate VII by similar chemistries described
earlier.
Example 4: Preparation of a Compound of Formula X
In Which Rl=H, R2=R3=Me, n=2
To a solution of compound of Formula IX (R1=H, R2=R3=Me, 0.6 g, 2
mmol, 2.0 equiv), ethylenediamine (0.06 g, 1.0 mrnol, 1.0 equiv) and DMF (10.0
mL) is added a solution of PyBOP (1.2 g, 2.4 mmol, 2.4 equiv), HOBT (0.3 g,
2.4
mmol, 2.4 equiv), followed by diisopropylethylamine (0.65 g, 5.0 mmol, 5.0
equiv)
and is stirred at room temperature for several hours. The reaction mixture is
diluted
with 200 mL of diethyl ether to give a white precipitate which is collected by
vacuum filtration. The solid is purified by reverse-phase HPLC using a mixed
acetonitrile/water solvent system to afford the final compound IX.
Heterodimer of 5-HT~, rece for antagonist and D2 receptor ants onic
Compounds of Formula XII (R1 = R2 = H, alkyl, alkenyl, Ph, phenylalkyl),
may be prepared as shown in FIGURE 4 by similar chemistries described earlier.
The precursor XI, described by Sindelar et al., Czech. Collect. Chem. Commun.
CA 02321191 2000-08-15
WO 99/64046 PCT/US99/12768
-130-
55(6):1586-601 (1990), is the key structure unit of metopimazine-a D~ receptor
antagonist.
While the above examples are described with respect to particular SHT3
5 receptor antagonists, the chemistry is applicable to all of the ligands
described
herein.
