NOVEL POTASSIUM CHANNEL DRUGS AND THEIR USES

MEDICAMENTS A CANAL POTASSIUM ET LEUR UTILISATION

English Abstract

This invention relates to novel multibinding compounds that bind to potassium
(K+) channels and modulate their activity. The compounds of this invention
comprise 2-10 K+ channel ligands covalently connected by a linker or linkers,
wherein the ligands in their monovalent (i.e., unlinked) state bind to one or
more types of K+ channel. The manner of linking the ligands together is such
that the multibinding agents thus formed demonstrate an increased biologic
and/or therapeutic effect as compared to the same number of unlinked ligands
made available for binding to the K+ channel. The invention also relates to
methods of using such compounds and to methods of preparing them. The
compounds of this invention are particularly useful for treating diseases and
conditions of mammals that are mediated by K+ channels. Accordingly, this
invention also relates to pharmaceutical compositions comprising a
pharmaceutically acceptable excipient and an effective amount of a compound of
this invention.

French Abstract

La présente invention concerne des composés à liaisons multiples se liant aux canaux potassium (canaux K?+¿) dont ils modulent l'activité. Les composés de l'invention comprennent de 2 à 10 ligands à canal potassium reliés par covalence au moyen d'au moins un lieur. A l'état monovalent, c'est-à-dire non liés, ces ligands se lient à au moins un type de canal potassium. La façon dont ces ligands sont liés fait que les agents à liaisons multiples ainsi constitués font preuve d'une efficacité biologique et/ou thérapeutique accrue par comparaison à ce que donnerait une même quantité de ligands non liés rendus disponibles pour une liaison avec le canal potassium. L'invention concerne également des procédés permettant de préparer et d'utiliser de tels composés. Les composés de l'invention conviennent particulièrement pour le traitement, chez les mammifères, d'états et d'affections à médiation des canaux potassium. L'invention concerne enfin des compositions pharmaceutiques comprenant un excipient pharmaceutiquement admis et une quantité suffisante d'un composé entrant par ailleurs dans le cadre de cette invention.

Claims

WHAT IS CLAIMED IS:
1. A multibinding compound 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, each of said ligands comprising a ligand domain capable of binding
to a K+ channel.
2. The multibinding compound of Claim 1 wherein said ligand is selected from
the group consisting of quinidine, glibenclamide, procaine, tetraethyl
ammonium, clofilium,
melperone, pinacidil, WAY-123,398, cromakalim, propoful, thiopentone,
risotilide,
almokalant, bretylium, N-acetylprocainamide, tacrine, UK66,914, RP58866,
4-aminopyridine, RP49356, afinidine, chromanol 293B, L-768,673 and its
analogs, bethanidine,
disopyramide, desethylamiodarone, NE-10064, artilide, dofetilide, E-4031,
sematilide,
ambasilide, azimilide, tedisamil, dronedarone, ibutilide, sotalol,
benzodiazepine analogs and
amiodarone.
3. The multibinding compound of Claim 1 which has 2 ligands.
4. A multibinding compound represented by Formula I:
(L)p(X)q I
where each L is a ligand that may be the same or different at each occurrence;
X is a linker that may be the same or different at each occurrence;
p is an integer of from 2 to 10; and
q is an integer of from 1 to 20;
wherein each of said ligands comprises a ligand domain capable of binding to a
K+ channel.
5. The multibinding compound of Claim 4, wherein q is less than p.
6. The multibinding compound of Claim 4 wherein said ligand is selected from
the group consisting of quinidine, glibenclamide, procaine, tetraethyl
ammonium, clofilium,
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melperone, pinacidil, WAY-123,398, cromakalim, propoful, thiopentone,
risotilide,
almokalant, bretylium, N-acetylprocainamide, tacrne, UK66,914, RP58866,
4-aminopyridine, RP49356, afinidine, chromanol 293B, L-768,673 and its
analogs, bethanidine,
disopyramide, desethylamiodarone, NE-10064, artilide, dofetilide, E-4031,
sematilide,
ambasilide, azimilide, tedisamil, dronedarone, ibutilide, sotalol,
benzodiazepine analogs and
amiodarone.
7. The multibinding compound of Claim 4 wherein p is 2 and q is 1.
8. A pharmaceutical composition comprising a pharmaceutically acceptable
excipient and a therapeutically effective amount of one or more multibinding
compounds, or
pharmaceutically acceptable salts 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, each of said ligands comprising a ligand domain capable of binding
to a K+ channel
of a cell mediating mammalian diseases or conditions, thereby modulating the
diseases or
conditions.
9. The pharmaceutical composition of Claim 8 wherein said ligand is selected
from the group consisting of quinidine, glibenclamide, procaine, tetraethyl
ammonium,
clofilium, melperone, pinacidil, WAY-123,398, cromakalim, propoful,
thiopentone, risotilide,
almokalant, bretylium, N-acetylprocainamide, tacrine, UK66,914, RP58866,
4-aminopyridine, RP49356, afinidine, chromanol 293B, L-768,673 and its
analogs, bethanidine,
disopyramide, desethylamiodarone, NE-10064, artilide, dofetilide, E-4031,
sematilide,
ambasilide, azimilide, tedisamil, dronedarone, ibutilide, sotalol,
benzodiazepine analogs and
amiodarone.
10. The pharmaceutical composition of Claim 8 which has 2 ligands.
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11. A pharmaceutical composition comprising a pharmaceutically acceptable
excipient and a therapeutically effective amount of one or more multibinding
compounds
represented by Formula I:
(L)p(X)q I
and pharmaceutically acceptable salts thereof,
where each L is a ligand that may be the same or different at each occurrence;
X is a linker that may be the same or different at each occurrence;
p is an integer of from 2 to 10; and
q is an integer of from 1 to 20;
wherein each of said ligands comprises a ligand domain capable of binding to a
K+ channel of
a cell mediating mammalian diseases or conditions, thereby modulating the
diseases or
conditions.
12. The pharmaceutical composition of Claim 11 wherein said ligand is selected
from the group consisting of quinidine, glibenclamide, procaine, tetraethyl
ammonium,
clofilium, melperone, pinacidil, WAY-123,398, cromakalim, propoful,
thiopentone, risotilide;
almokalant, bretylium, N-acetylprocainamide, tacrine, UK66,914, RP58866,
4-aminopyridine, RP49356, afinidine, chromanol 293B, L-768,673 and its
analogs, bethanidine,
disopyramide, desethylamiodarone, NE-10064, artilide, dofetilide, E-4031,
sematilide,
ambasilide, azimilide, tedisamil, dronedarone, ibutilide, sotalol,
benzodiazepine analogs and
amiodarone.
13. The pharmaceutical composition of Claim 11 which has 2 ligands.
14. A method for modulating the activity of a K+ channel in a biologic tissue,
which method comprises contacting a tissue having a K+ channel with a
multibinding
compound, or a pharmaceutically acceptable salt thereof, under conditions
sufficient to
produce a change in the activity of the channel in said tissue, wherein the
multibinding
compound comprises 2 to 10 ligands which may be the same or different and
which are
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covalently attached to a linker or linkers, which may be the same or
different, each of said
ligands comprising a ligand domain capable of binding to a K+ channel.
15. The method of Claim 14 wherein said ligand is selected from the group
consisting of quinidine, glibenclamide, procaine, tetraethyl ammonium,
clofilium,
melperone, pinacidil, WAY-123,398, cromakalim, propoful, thiopentone,
risotilide,
almokalant, bretylium, N-acetylprocainamide, tacrine, UK66,914, RP58866,
4-aminopyridine, RP49356, afinidine, chromanol 293B, L-768,673 and its
analogs, bethanidine,
disopyramide, desethylamiodarone, NE-10064, artilide, dofetilide, E-4031,
sematilide,
ambasilide, azimilide, tedisamil, dronedarone, ibutilide, sotalol,
benzodiazepine analogs and
amiodarone.
16. The method of Claim 14 wherein the multibinding compound has 2 ligands.
17. A method for treating a disease or condition in a mammal resulting from an
activity of a K+ channel, which method comprises administering to said mammal
a
therapeutically effective amount of a pharmaceutical composition comprising a
pharmaceutically acceptable excipient and one or more multibinding compounds,
or
pharmaceutically acceptable salts 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, each of said ligands comprising a ligand domain capable of
binding to a K+
channel of a cell mediating mammalian diseases or conditions.
18. The method of Claim 17 wherein said ligand is selected from the group
consisting of quinidine, glibenclamide, procaine, tetraethyl ammonium,
clofilium,
melperone, pinacidil, WAY-123,398, cromakalim, propoful, thiopentone,
risotilide,
almokalant, bretylium, N-acetylprocainamide, tacrine, UK66,914, RP58866,
4-aminopyridine, RP49356, afinidine, chromanol 293B, L-768,673 and its
analogs, bethanidine,
disopyramide, desethylamiodarone, NE-10064, artilide, dofetilide, E-4031,
sematilide,

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ambasilide, azimilide, tedisamil, dronedarone, ibutilide, sotalol,
benzodiazepine analogs and
amiodarone.
19. The method of Claim 17 wherein the multibinding compound has 2 ligands.
20. A method for treating a disease or condition in a mammal resulting from an
activity of a K+ channel, which method comprises administering to said mammal
a
therapeutically effective amount of a pharmaceutical composition comprising a
pharmaceutically acceptable excipient and one or more multibinding compounds
represented
by Formula I:
(L)p(X)q I
and pharmaceutically acceptable salts thereof,
where each L is a ligand that may be the same or different at each occurrence;
X is a linker that may be the same or different at each occurrence;
p is an integer of from 2 to 10; and
q is an integer of from 1 to 20;
wherein each of said ligands comprises a ligand domain capable of binding to a
K+ channel of
a cell mediating mammalian diseases or conditions.
21. The method of Claim 20 wherein said ligand is selected from the group
consisting of quinidine, glibenclamide, procaine, tetraethyl ammonium,
clofilium,
melperone, pinacidil, WAY-123,398, cromakalim, propoful, thiopentone,
risotilide,
almokalant, bretylium, N-acetylprocainamide, tacrine, UK66,914, RP58866,
4-aminopyridine, RP49356, afinidine, chromanol 293B, L-768,673 and its
analogs, bethanidine,
disopyramide, desethylamiodarone, NE-10064, artilide, dofetilide, E-4031,
sematilide,
ambasilide, azimilide, tedisamil, dronedarone, ibutilide, sotalol,
benzodiazepine analogs and
amiodarone.
22. The method of Claim 20 wherein the multibinding compound has 2 ligands.
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23. A method for identifying multimeric ligand compounds possessing
multibinding properties for potassium channels, which method comprises:
(a) identifying a ligand or a mixture of ligands wherein each ligand contains
at
least one reactive functionality;
(b) identifying a library of linkers wherein each linker in said library
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 said linker and at least two
of said ligands;
and
(d) assaying the multimeric ligand compounds produced in the library prepared
in (c) above to identify multimeric ligand compounds possessing multibinding
properties.
24. A method for identifying multimeric ligand compounds possessing
multibinding properties for potassium channels, which method comprises:
(a) identifying a library of ligands wherein each ligand contains at least one
reactive functionality;
(b) identifying a linker or mixture of linkers wherein each linker comprises
at
least two functional groups having complementary reactivity to at least one of
the reactive
functional groups of the ligand;
(c) preparing a multimeric ligand compound library by combining at least two
stoichiometric equivalents of the library of ligands identified in (a) with
the linker or
mixture of linkers identified in (b) under conditions wherein the
complementary functional
groups react to form a covalent linkage between said linker and at least two
of said ligands;
and
(d) assaying the multimeric ligand compounds produced in the library prepared
in (c) above to identify multimeric ligand compounds possessing multibinding
properties.

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25. The method according to Claim 23 or 24 wherein 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).
26. The method according to Claim 25 wherein the multimeric ligand
compounds comprising the multimeric ligand compound library are dimeric.
27. The method according to Claim 26 wherein the dimeric ligand compounds
comprising the dimeric ligand compound library are heterodimeric.
28. The method according to Claim 27 wherein the heterodimeric ligand
compound library is prepared by sequential addition of a first and second
ligand.
29. The method according to Claim 23 or 24 wherein, prior to procedure (d),
each member of the multimeric ligand compound library is isolated from the
library.
30. The method according to Claim 29 wherein each member of the library is
isolated by preparative liquid chromatography mass spectrometry (LCMS).
31. The method according to Claim 23 or 24 wherein the linker or linkers
employed are selected from the group comprising flexible linkers, rigid
linkers,
hydrophobic linkers, hydrophilic linkers, linkers of different geometry,
acidic linkers, basic
linkers, linkers of different polarization and amphiphilic linkers.
32. The method according to Claim 31 wherein the linkers comprise linkers of
different chain length and/or having different complementary reactive groups.
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33. The method according to Claim 32 wherein the linkers are selected to have
different linker lengths ranging from about 2 to 100.ANG..
34. The method according to Claim 23 or 24 wherein the ligand or mixture of
ligands is selected to have reactive functionality at different sites on said
ligands.
35. The method according to Claim 34 wherein said reactive functionality is
selected from the group consisting of carboxylic acids, carboxylic acid
halides, carboxyl
esters, amines, halides, pseudohalides, isocyanates, vinyl unsaturation,
ketones, aldehydes,
thiols, alcohols, anhydrides, boronates, and precursors thereof wherein 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.
36. The method according to Claim 23 or Claim 24 wherein the multimeric
ligand compound library comprises homomeric ligand compounds.
37. The method according to Claim 23 or Claim 24 wherein the multimeric
ligand compound library comprises heteromeric ligand compounds.
38. A library of multimeric ligand compounds which may possess multivalent
properties for potassium channels, which library is prepared by the method
comprising:
(a) identifying a ligand or a mixture of ligands wherein each ligand contains
at
least one reactive functionality;
(b) identifying a library of linkers wherein each linker in said library
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
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library of linkers identified in (b) under conditions .wherein the
complementary functional
groups react to form a covalent linkage between said linker and at least two
of said ligands.
39. A library of multimeric ligand compounds which may possess multivalent

properties for potassium channels, which library is prepared by the method
comprising;
(a) identifying a library of ligands wherein each ligand contains at least one
reactive functionality;
(b) identifying a linker or mixture of linkers wherein each linker comprises
at
least two functional groups having complementary reactivity to at least one of
the reactive
functional groups of the ligand; and
(c) preparing a multimeric ligand compound library by combining at least two
stoichiometric equivalents of the library of ligands identified in (a) with
the linker or
mixture of linkers identified in (b) under conditions wherein the
complementary functional
groups react to form a covalent linkage between said linker and at least two
of said ligands.
40. The library according to Claim 38 or Claim 39 wherein the linker or
linkers
employed are selected from the group comprising flexible linkers, rigid
linkers,
hydrophobic linkers, hydrophilic linkers, linkers of different geometry,
acidic linkers, basic
linkers, linkers of different polarization and amphiphilic linkers.
41. The library according to Claim 40 wherein the linkers comprise linkers of
different chain length and/or having different complementary reactive groups.~
42. The library according to Claim 41 wherein the linkers are selected to have
different linker lengths ranging from about 2 to 100.ANG..
43. The library according to Claim 38 or 39 wherein the ligand or mixture of
ligands is selected to have reactive functionality at different sites on said
ligands.~


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44. The library according to Claim 43 wherein said reactive functionality is
selected from the group consisting of carboxylic acids, carboxylic acid
halides, carbonyl
esters, amines, halides, pseudohalides, isocyanates; vinyl unsaturation,
ketones, aldehydes,
thiols, alcohols, anhydrides, boronates, and precursors thereof wherein 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.
45. The library according to Claim 38 or Claim 39 wherein the multimeric
ligand compound library comprises homomeric ligand compounds.
46. The library according to Claim 38 or Claim 39 wherein the multimeric
ligand compound library comprises heteromeric ligand compounds.
47. An iterative method for identifying multimeric ligand compounds possessing
multibinding properties for potassium channels, 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 a receptor with a linker or mixture of linkers wherein
said ligand or
mixture of ligands comprises at least one reactive functionality and said
linker or mixture of
linkers comprises at least two functional groups having complementary
reactivity to at least
one of the reactive functional groups of the ligand wherein said contacting is
conducted
under conditions wherein the complementary functional groups react to form a
covalent
linkage between said linker and at least two of said ligands;
(b) assaying said first collection or iteration of multimeric compounds to
assess
which if any of said multimeric compounds possess multibinding properties;
(c) repeating the process of (a) and (b) above until at least one multimeric
compound is found to possess multibinding properties;

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(d) evaluating what molecular constraints imparted or are consistent with
imparting 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 said first iteration;
(f) evaluating what molecular constraints imparted or are consistent with
imparting 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 said
molecular
constraints.
48. The method according to Claim 47 wherein steps (e) and (f) are repeated
from 2-50 times.
49. The method according to Claim 47 wherein steps (e) and (f) are repeated
from 5-50 times.
-146-

Description

CA 02318745 2000-07-24
WO 99/64050 PCTNS99/1Z777
NOVEL POTASSIUM CHANNEL DRUGS AND THEIR USES
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Applications Serial Nos. 60/088,465,
filed June
8, 1998; 60/093,068, filed July 16, 1998; and 60/113,864, filed December 24,
1998, the entire
contents of which are incorporated herein by reference.
BACKGROUND
This invention relates to novel multibinding compounds that bind to potassium
(K~
channels and modulate their activity. The compounds of this invention comprise
2-10 K+
channel ligands covalently connected by a linker or linkers, wherein the
ligands in their
monovalent (i.e., unlinked) state bind to one or more types of K' channel. The
manner of
linking the ligands together is such that the multibinding agents thus formed
demonstrate an
increased biologic and/or therapeutic effect as compared to the same number of
unlinked
ligands made available for binding to the K+ channel. The invention also
relates to methods
of using such compounds and to methods of preparing them.
The compounds of this invention are particularly useful for treating diseases
and
conditions of mammals that are mediated by K+ channels. Accordingly, this
invention also
relates to pharmaceutical compositions comprising a pharmaceutically
acceptable excipient
and an effective amount of a compound of this invention.
The following publications are cited in this application as superscript
numbers:
-1-


CA 02318745 2000-07-24
WO 99/64050 PCT/US99/12777
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-2-


CA 02318745 2000-07-24
WO 99/b4050 PCT/US99/12???
'°Bmwa, A.M., "Cardiac Potassium Channels in Health and Disease",
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-3-


CA 02318745 2000-07-24
WO 99/64050 PCTNS99/12777
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-4-


CA 02318745 2000-07-24
WO 99/64050 PGTNS99/12777
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CA 02318745 2000-07-24
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PCT/US99/12'7~7
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PCTNS99/12777
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'~EEuropean Patent 0 471 609 B 1.
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{aminoalkyloxy-4 dialkyl-3,5 benzoyl)-3 benzofurannes et alkyl-2
{aminoalkyloxy-4
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benzoylr3 benzofurannes; a~iangineux potentiels", Eur. J. Med. Chem., 9(1):19-
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Related
Compounds Possessing Both Class II and Class III Antiarrhyihmic Activity", J.
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[(Methylsulfonyl)amino]benza-
mides as Class III Antiarrhythmic Agents", J. Med. Cherri., 30{5):755 758 (May
1987).
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CA 02318745 2000-07-24
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1°~Lynch, J.J., Jr., et al., "Comparison of binding to rapidly
activating delayed
rectifier K+ channel, IKr, and effects on myocardial refractoriness for class
III
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1998).
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CA 02318745 2000-07-24
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1'lRoden, D.M., "Ibutilide and the Treatment of Atrial Arrhythmias. A New Drug
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118Yeola, ,S.W., et al., "Molecular Analysis of a Binding Site for Quinidine
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Human Cardiac Delayed Rectifier K* Channel. Role of S6 in Antiarrhythmic Drug
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The disclosure of each of the above publications is incorporated herein by
reference in
its entirety to the same extent as if each individual publication was
specifically and
m~~duallY indicated to be incorporated by reference in its entirety.
Voltage-regulated potassium channels mediate the flux of I~ out of cells in
response
to changes in membrane potential 2a Voltage-gated. K' channels in the open
state typically
transition to an inactivated state, and must reacquire the ability to respond
to an external
stimulus during a recovery period. An inward rectifying voltage-regulated
potassium channel
in cardiac muscle is also activated by acetylcholine (i.e., it is gated by
more than one type of
stimulus).'' A calcium-activated K* channel has been described.l6 Potassium
channels serve
a variety of important cellular functions, including excitability, setting and
maintaining the
resting potential, repolarizing action potentials, transmembrane transport,
volume regulation,
signal traasduction, and so on.~ They are implicated in a variety of
pathophysiological
disorders, including hypertension, cardiac arrhythmogenesis, insulin dependent
diabetes, non-
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insulin dependent diabetes mellitus, diabetic neuropathy, seizures,
tachycardia, ischemic heart
disease, cardiac failure, angina, myocardial infarction; transplant rejection,
autoimmune
disease, sickle cell anemia, muscular dystrophy, gastrointestinal distase,
mental disorder,
sleep disorder, anxiety disorder, neurosis, alcoholism, inflammation,
~brovascular
ischemia, CNS diseases, epilepsy, Parkinson's disease, asthma, incontinence,
urinazy
dysfunction , micturition disorder, irritable bowel syndrome, restenosis,
subarachnoid -
hemorrhage, Alzheimer disease, and they mediate the transmission of pain
impulses by
P~Pheral nerves:'s
Figure 1 illustrates in cross-sectional view the tran,~membrane domain/subunit
organization of various transporter molecules, as it is presently understood
by those working
in the field of transport physiology. It should be understood that, for
purposes of
simplification, other subunits that may be involved in or required for
transporter activity have
been omitted from the diagram.
Referring to Figure 1, voltage-gated ion channels and related proteins are
tetrameric
structures formed by the noncovalent association of individual subunits
(1),(2), or by the
interaction of homologous domains of a monomeric protein (3). The channels
differ as well
in the number of ttansmembrane segments per subunit or per domain. Inward-
rectifier type
K+ channels and P~ pminergic channels have two transmembrane-segments in each
subunit,
Shaker-type K" channels have six transmembrane segments per subunit and Na*
and Ca~'""
channels have six transmembrane segments per domain. Neurotransmitter-gated
ion channels
such as those shown in (4) are organized as pentamers, with each of the
subunits having four
t<ansmembrane segments/domains. The activation gate for potassium channels has
not been
identified, although a trap door mechanism has been proposed.a',m
Potassium channels are structurally similar to, but smaller and simpler than,
sodium
and calcium ion channels,9a with the K+ channel tetrameric structure being
formed by four
polypeptides' However, potassium channels represent a diverse class of ion
chamiels.'"
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CA 02318745 2000-07-24
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I~omotetramers can form, but there is evidence that heterotetramers may be
fuactionally
relevant in vivo.'° The x-ray structure of a bacterial K+ channel
(which is homologous to
mammalian K+ channels) has been disclosed.2' A prokaryotic IC~ channel was
found to have
the same structure as a eukaryotic K' channel.'°' The channel has an
inverted teepee struct~ne
with a large hydrophobic cavity. The cavity (1 OA) is centered in the channel
on the
cytoplasmic side, and appears to get larger upon channel opening i',~' IO,"'
Voltage-
dependent cardiac potassium channel genes have been cloned as cDNAs.'o,'~',"6
Variability
in the potassium channel genes may relate to disease
conditions.'4.a'~°a°
Drug binding sites for tetraethylammonium, quinidine and 4-aminopyridine are
found
in the inner vestibule of the K+ channel, and the amino acid side chains
involved are localized
in the S6 helix. Binding studies using mutagenesis show similarity to local
anesthetic (I,A)
binding to sodium channels, although sodium channel inhibitors bind more
deeply in the
cavity, r~.~=
There appear to be two types of potassium channel inactivation, N-type and C-
type,
which can occur simultaneously in Shaker potassium channels. Both are
partially coupled to
activation and are usually voltage insensitive once activation is complete. N-
type inactivation
in Shaker B channels depends on a group of amino acids at the N-terminal that
bind to the
activated channel and occlude the intracellular mouth of the channel. No
sequence similarity
has been found among the N-termini of the N-type inactivating channels. N-type
inactivation
is voltage insensitive at positive potentials and competes with drug binding
at the intracellular
face of the channel. C-type inactivation, which is less understood, occurs by
occlusion of the
external mouth of the channel during sustained depolarization. C-type
inactivation is voltage
insensitive at potentials where activation is complete, but recovery from C-
type inactivation is
voltage sensitive. Both C- and N-type inactivation are coupled or partially
coupled to
activation, and both require similar degrees of activation to
proceed.'°
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CA 02318745 2000-07-24
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Not surprisingly, potassium channels are recognized as important targets for
chug
therapy. For example, potassium channels are targeted by certain antidiabetic,
antihypertensive and antiarryhthmic drugs.
Potassium channel antagonists are used for treatment of arrhythmia
Antiarrhythmic
agents are classified into four classes under the Vaughan Williams
classification scheme:
Class I (sodium channel blockers); Class II (beta blockers); Class III
(potassium channel
biockers); and Class IV (calcium channel blockers). As shown in Table 1, an
antiarrhythmic
agent may have activity in several channels and/or with several receptors
~~'°' Newer drugs
are more selective to specific K+ channels, as shown in Table 2. Properties of
some known
K" channel blockers are given in Table 3. Table 5 sets forth the principal K+
currents and
some drugs that block them.4s The majority of drugs in developm~t are I~
blockers.w'°~~"2
Some agents appear to be cationic open-channel blockers."3~"~"'
Combination therapy with two separate agents, e.g., a potassium channel opener
with
little or no effect on cardiac action potential and a Class III antiarrhythmic
compound has
been disclosed.'s
The clinical shortcomings of drugs in current usage are considerable. Their
most
common adverse side effects include headache, hypotension, nausea, vomiting,
dizziness, and
the like. Other side effects may include photo sensitivity, corneal
microdeposits, neuropathy,
fatigue, 35,39,41,36,61 pn~onitis, hepatotoxicity, proarrhythmic effects, ~
thyroid abnormalities
and bradycardia. Reverse use dependence, which may lead to torsades de pointer
(an induced
arrhythmia), is a major problem of most or all known Class III agents
4~"'47.s6.117 F
there may be no survival benefit associated with the use of these agents 6'
With few
exceptions, the currently used drugs have a short duration of action and must
be administered
frequently for sustained effects.
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CA 02318745 2000-07-24
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Thus, there continues to exist a need for novel compounds with greater tissue
selectivity, increased e~cacy, reduced side effects and a more favorable
duration of action.
SI1MMARY Oh' TSE INVENTION
This invention is directed to novel multibinding compounds that bind to K+
channels
in mammalian tissues and can be used to treat diseases and conditions mediated
by such
channels.
This invention is also directed to general synthetic methods for generating
large
libraries of diverse multimeric compounds which multimeric compounds are
candidates for
possessing multi'binding properties for potassium channels. The diverse
multimeric
compound libraries provided by this invention are synthesized by combining a
linker or
linkers with a ligand or liga~s 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,
amphiphilicity, acidity, basicity and 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 multi'binding properties.
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 a potassium channel.
Accordingly, in one of its composition aspects, this invention is directed to
a
multibinding compound and salts thereof comprising 2 to 10 ligands which may
be the same
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or different and which are covalently attached to a linker or linkers, which
may be the same or
different, each of said ligsnds comprising a ligand domain capable of binding
to a K" channel.
The muitibinding compounds of this invention are prefeiably repres~ted by
Formula
I:
(I,~(~q I
where each L is a ligand that may be the same or different at each occurrence;
X is a linker
that may be the same or different at each occurrence; p is an integer of from
2 to 10; aad
q is an integer of from 1 to 20; wherein each of said ligands comprises a
ligand domain
capable of binding to a K+ channel. Preferably q is less than p.
Preferably, the binding of the multibinding compound to a K' channel or
channels in a
mammal modulates diseases and conditions mediated by the I~ channel or
channels.
In another of its composition aspects, this invention is directed to a
pharmaceutical
composition comprising a pharmaceutically acceptable excipient and a
therapeutically
effective amount of one or more multibinding compounds (or pharmaceutically
acceptable
salts 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, each of said
ligands comprising a ligand domain capable of binding to a K+ channel of a
cell mediating
mammalian diseases or conditions, thereby modulating the diseases or
conditions.
In still another of its composition aspects, this invention is directed to a
pharmaceutical composition comprising a pharmaceutically acceptable excipient
and a
therapeutically effective amount of one or more multibinding compounds
represented by
Formula I:
~~~9
or pharmaceutically acceptable salts thereof, where each L is a ligand that
may be the same or
different at each occurrence; X is a linker that may be the same or different
at each
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occurrence; p is an integer of from 2 to 10; and q is an integer of from 1 to
20; wherein each
of said ligands comprises a ligaad domain capable of binding to a K' channel
of a cell
mediating mammalian diseases or conditions, thereby modulating the diseases or
conditions.
Preferably q is less than p.
In one of its method aspects, this invention is directed to a method for
modulating the
acfiivity of a K' channel in a biologic tissue, which method comprises
contacting a tissue
having a K" channel with a multibinding compound (or pharmaceutically
acceptable salts
thereof) under conditions sufficient to produce a change in the activity of
the channel in said
tissue, wherein the multibinding compound comprises 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, each of said ligands comprising a ligand domain capable of binding
to a K' channel.
In another of its method aspects, this invention is directed to a method for
treating a
disease or condition in a mammal resulting from an activity of a K'' channel,
which method
comprises administering to said mammal a therapeutically effective amount of a
pharmaceutical composition comprising a pharmaceutically acceptable excipient
and one or
more multibinding compounds (or pharmaceutically acceptable salts 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, each of said ligands
comprising a ligaad
domain capable of binding to a K+ channel of a cell mediating mammalian
diseases or
conditions. .
In yet another of its method aspects, this invention is directed to a method
for treating
a disease or condition in a mammal resulting from an activity of a K" channel,
which method
comprises administering to said mammal a therapeutically effective amount of a
pharmaceutical composition comprising a pharmaceutically acceptable excipient
and one or
more multibinding compounds represented by Formula I:
(I.y(3~9
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and pharmaceutically acceptable salts thereof, where each L is a ligand that
may be the same
or different at each occurrence; X is a linker that may be the same or
different at each
occurrence; p is an integer of from 2 to 10; and q is an integer of from 1 to
20; wherein each
of said ligaads comprises a ligand domain capable of binding to a K'' channel
of a cell
mediating mammalian diseases or conditions. Preferably q is less than p.
la a further aspect, this invention provides processes for preparing the
multibinding
agents of Formula I. This can be accomplished by combining p appropriately
functionalized
ligands with q complementary functionalized linkers under conditions where
covalent bond
formulation between the ligands and linkers occurs; alternatively, linking
portions ofp
appropriately functionalized ligands to q complementary fimctionalized linkers
and then
completing the synthesis of the ligands in a subsequent step may be performed
to prepare
these compounds. Another method which may be used involves linking p
appropriately
funetionalized ligands to portions of the linkers) and then completing the
synthesis of the
linkers) in a subsequent step. Coupling one or more of an appropriately
functionalized
ligand to a complementary functionalized linker, and subsequently coupling one
or more
additional ligaads to said linker or linkers may be done to prepare the
claimed compounds.
Coupling as above wherein coupling of different appropriately functionalized
linkers occurs
simulataneously may also be used.
In one of its method aspects, this invention is directed to a method for
identifying
multimeric ligand compounds possessing multibinding properties for potassium
channels,
which method comprises:
(a) identifying a ligand or a mixture of ligands wherein each ligand contains
at
least one reactive functionality;
(b) identifying a library of linkers wherein each linker in said library
comprises
at least two fiuietional groups having complementary reactivity to at least
one of the
reactive functional groups of the ligand;
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pCTNS99112777
(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 said linker and at least two
of said ligands;
and
(d) assaying the multimeric ligand compounds produced in (c) above to identify
multimeric ligand compounds possessing multibinding Properties.
In another of its method aspects, this invention is directed to a method
for identifying multimeric ligand compounds possessing multz-binding
properties for
potassium channels, which method comprises:
(a) identifying a library of ligands wherein each ligand contains at least one
reactive functionality;
(b) identifying a linker or mixture of linkers wherein each linker comprises
at
least two functional groups having complementary reactivity to at least one of
the reactive
ftmctional groups of the ligand;
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 coiyditions wherein the
complementary functional
groups react to form a covalent linkage between said linker and at least two
of said ligands;
and
(d) assaying the multimeric ligand compounds produced in (c) above to 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 ligamis is employed to enswre heterodimeric or
multimeric
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compounds are prepared. Concurrent addition of the ligands occurs when at
least a portion
of the multimer comounds prepared are homomultimeric compOUn~ds.
The assay protocols recited in (d) can be conducted on the multimeric ligand
compound library produced in (c) above, or preferably, each member of the
library is
isolated by preparative liquid chromatography mass spectrometry (LCMS).
In one of its composition aspects, this invention is directed to a library of
multimeric
ligand compounds which may possess multivalent properties for potassium
channels, which
library is prepared by the method comprising:
(a) identifying a ligand or a mixture of ligands wherein each ligand contains
at
least one reactive functionality;
(b) identifying a library of linkers wherein each linker in said library
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 fimetional
groups react to form a covalent linkage between said linker and at least two
of said ligands.
In another of its composition aspects, this invention is directed to a library
of
multimeric ligand compounds which may possess multivalent properties for
potassium
channels, which library is prepared by the method comprising:
(a) identifying a library of ligands wherein each ligand contains at least one
reactive functionality;
(b) identifying a linker or mixture of linkers wherein each linker comprises
at
least two functional groups having complementary reactivity to at least one of
the reactive
functional groups of the ligand; and
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(c) Preparing a multimeric ligand compound library by combining at least two
stoichiometric equivalents of the library of ligands identified in (a) with
the linker or
mixture of linkers identified in (b) under conditions wherein the
complementary functional
groups react to form a covalent linkage between said linker and at least two
of said ligands.
In a preferred embodiment, the fbrary of linkers employed in either the
methods or
the library aspects of this invention is selected from the group comprising
flexible linkers,
rigid iinke:~, hydrophobic linkers, hydrophilic Linkers, Linkers of different
geometry, acidic
linkers, basic linkers, linkers of different 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 100A.
In another preferred embodiment, the potassium chapel ligand or mixture of
ligands is selected to have reactive functionality at different sites on said
ligands in order to
provide for a range of orientations of said ligand on said multimeric ligand
compounds.
Such reactive functionality includes, by way of example, carboxylic acids,
carboxylic acid
halides, carboxyl esters, amines, halides, isocyanates, vinyl unsaturation,
ketones,
aldehydes, thiols, alcohols, anhydrides, 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 Iigand.
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
heterodimeric
(i.e., at least one of the ligands is different from the other ligands).
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In addition to the combinatorial methods described herein, this invention
provides
for an iterative process for rationally evaluating what molecular constraints
impart
multibinding properties to a class of multimeric compounds or ligands
targeting a receptor.
Specifically, this method aspect is directed to a method for identifying
multimeric ligand
compounds possessing multibinding properties for potassium channels 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 a receptor with a linker or mixture of linkers wherein
said ligand or
mixture of ligands comprises at least one reactive functionality and said
linker or mixture of
linkers comprises at least two functional groups having complementary
reactivity to at least
one of the reactive functional groups of the ligand wherein said contacting is
conducted
under conditions wherein the complementary functional groups react to form a
covalent
linkage between said linker and at least two of said ligands;
(b) assaying said first collection or iteration of multimeric compounds to
assess
which if any of said multimeric compounds possess multibinding properties;
(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 said first iteration;
(f j evaluating what molecular constraints imparted enhanced muitibinding
properties to the multimeric compound or compounds found in the second
collection or
iteration recited in (e) above;
(g) optionally repeating steps (e) and (fj to further elaborate upon said
molecular
constraints.
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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
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a highly schematic illushation of the transmembrane organization
of
various cell membrane transporters.
Figure 2 illustrates a method for optimizing the linker geometry for
presentation of
ligands (filled circles) in bivalent compounds:
A. phenyldiacetylene core structure
B. cyclohexane dicarboxylic acid core structure
Figure 3 shows exemplary linker "core" structures.
Figure 4 illustrates examples of mufti-binding compounds comprising (A) 2
ligands,
(B) 3 ligands, (C) 4 ligands, and (D) >4 ligands attached in different formats
to a linker.
Figure 5 illustrates the Iigand amiodarone, Which may be used in preparing
multi-
binding compounds. Potentially modifiable positions are indicated by arrows.
Figure 6 illustrates numerous reactive functional groups and the resulting
bonds
formed by reaction therebetween.
Figures 7 to 21 illustrate convenient methods for preparing the multibinding
compounds of this invention. In each of these figures, the filled circles -
represent linkers,
referred to in the written Examples as "Link".
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DETAILED DESCRIPTION OF THE I1~1VENTION
Biological systems in general are controlled by molecular interactions between
bioactive ligands. and their receptors, in which the receptor "recognizes" a
molecule or a
portion thereof (i.e., a ligand domain) to produce a biological effect. The K+
channels are
considered to be pharmacological receptors: they possess specific binding
sites for ligands
having agonist and antagonist activities; the binding of ligands to such sites
modulates K" flux
through the channel; the channel properties (i.e., gating and ion selectivity)
are regulaxable.
Accordingly, diseases or conditions that involve, or are mediated by, K~
channels can be
treated with pharmacologically active ligaads that interact with such channels
to initiate,
modulaxe or abrogate transporter activity.
The interaction of a K" channel and a K'' channel-binding ligand may be
described in
terms of "affinity" and "specificity". The "affinity" and "specificity" of any
given ligand-K"
I S channel interaction is dependent upon the complementarity of molecular
binding surfaces and
the energetic costs of complexation (i.e., the net difference in free energy
between bound and
free states). Affinity may be quantified by the equilibrium constant of
complex formation, the
ratio of on/off rate constants, and/or by the free energy of complex
formation. Specificity
relates to the difference in binding affinity of a ligand for different
receptors.
The net free energy of interaction of such ligand with a K' channel is the
difference
between energetic gains (enthalpy gained through molecular complementarity and
entropy
gained through the hydrophobic effect) and energetic costs (enthalpy lost
through decreased
solvation and entropy lost through reduced transiational, rotational and
conformational
degrees of freedom).
The compounds of this invention comprise 2 to 10 K'' channel-binding ligaads
covalentiy linked together and capable of acting as multibinding agents.
Without wishing to
be bound by theory, the enhanced activity of these compounds is believed to
arise at least in
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part from their ability to bind in a multivalent manner with multiple ligand
binding sites on a
K'' channel or channels, which gives rise to a more favorable net free energy
of binding.
Multivalent interactions differ from collections of individual monovaient
(univalent)
interactions by being capable of providing enhanced biologic andlor thera~utic
effect
Multivalent binding can amplify binding affinities and differences in binding
affinities,
resulting in enhanced binding specificity as well as affinity.
As used herein:
The term "alkyl" refers to a monoradical branched or unbranched safi~rated
hydrocarbon chain, preferably having from 1 to 40 carbon atoms, preferably 1-
10 carbon
atoms, more preferably 1-6 carbon atoms, such as methyl, ethyl, n propyl,
isopropyl,
n butyl, secondary butyl, tert-butyl, n-hexyl, n-octyl, n-decyl, n-dodecyl, 2-
ethyldodecyl,
tetradecyi, and the like, unless otherwise indicated.
The term "substituted alkyl" refers to an alkyl group as defined above having
from 1
to 5 substituents selected from the group consisting of alkoxy, substituted
alkoxy, cycloalkyl,
substituted cycloalkyl, cycloalkcnyl, substituted cycloalkenyl, acyl,
acylamino, acyloxy,
amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl,
keto,
thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,
thioheterocyclooxy, thiol,
thioalkoxy, substituted thioallcoxy, aryl, aryloxy, heteroaryl, heteroaryloxy,
heterocyclic,
heterocyciooxy, hydroxyamino, alkoxyamino, vitro, -SO-alkyl, -SO-aryl, -SO-
heteroaryl,
-S02-alkyl, -SO=-aryl, -SOZ-heteroaryl, and -NR'Rb, wherein R' and Rb may be
the same or
different and and are chosen from hydrogen, optionally substituted alkyl,
cycloalkyl, alkenyl,
cycloalkenyi, alkynyl, aryl, heteroaryl and heterocyclic.
The team "alkylene" refers to a diradical of a branched or unbranched
saturated
hydrocarbon chain, preferably having from 1 to 40 carbon atoms, preferably 1-
10 carbon
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atoms, more preferably 1-6 carbon atoms. This term is exemplified by groups
such as
methylene (-CH2-), ethylene (-CH~CH~-), the propylene isomers (e.g., -
CH2CH2CHz- and
-CH(CH3)CH2-) and the like.
The term "substituted alkylene" refers to: (1) An alkylene group as defined
above
having from 1 to 5 substituents selected from the group consisting of alkoxy,
substituted
alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, acyl,
aeylamino, aeyloxy, amino, aminoacyl; aminoacyloxy, oxyacylamino, azido;
cyano, halogen,
hydroxyl, keto, thioketo, carboxyl, carboxylallcyl, thiol, thioalkoxy,
substituted thioalkoxy,
aryl, aryloxy, thioaryloxy, heteroaryl, heteroaryloxy, thioheteroaryloxy,
heterocyclic,
heterocyclooxy, thioheterocyclooxy, vitro, and NR~Re, wherein R, and Re may be
the same or
different and are chosen from hydrogen, optionally substituted alkyl,
cycloalkyl, alkenyl,
cycloalkenyl, alkynyi, aryl, heteroaryl and heterocyclic. Additionally, such
substituted
alkylene gmups 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 gmup; (2) An alkylene
group as
defined above that is interrupted by 1-20 atoms independently chosen from
oxygen, sulfur and
NR,-, where R, is chosen from hydrogen, optionally substituted alkyl,
cycloalkyl, alkenyl,
cycloallcenyl, alkenyl, cycloalkenyl, alkynyi, aryl, heteroaryl and
heteiocyclic, or groups
selected from carbonyl, carboxyester, carboxyamide and sulfonyl; and (3) An
alkylene group
as'defined above that has both from 1 to 5 substituents as defined above and
is also
interrupted by 1-20 atoms as defined above. Examples of substituted alkylenes
are
chloromethylene (-CH(Cl)-), aminoethylene (-CH(NH~CHx-), 2-carboxypropylene
isomers
(-CH2CH(CO2H)CHz-),. ethoxyethyl (-CH2CHZ0-CH2CHZ-), ethylmethylaminoethyl
(-CHZCH2N(CH3)CH2CH2-), 1-ethoxy-2-(2-ethoxy-ethoxy)ethane (-CH2CH20-CH2CH2-
OCHZCH2- OCHZCH2-), and the like.
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The term "alkaryl" or "aralkyl" refers to the groups. -atkylene-aryl and -
substituted
alkylene-aryl in which alkylene and aryl are as defined herein. Such alkaryl
groups are
exemplified by benzyl, phcnethyl 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,
cycloallcenyl, and alkynyl
are as defined herein. Preferred allcoxy groups are alkyl-O- and include, by
way of example,
methoxy, ethoxy,.n-propoxy, iso-propoxy, n-butoxy, tart-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 cycloallcenyl-O-, and
substituted alkynyl-O-
where substituted alkyl, substituted alkenyl, substituted cycloalkyl,
substituted cycloalkenyl
and substituted alkynyl are as defined herein. .
The term "alkylalkoxy" refers to the groups -alkylene-O-alkyl, 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.
Examples of such
groups aie methylenemethoxy (-CH20CH3), ethylenemethoxy (-CHzCH20CH3), n-
propylene-
iso-propoxy (-CH2CHZCHiOCH(CH~, methylene-t-butoxy (-CHz-O-C(CH3)3) and the
like.
The term "alkylthioalkoxy" refers to the group -alkylene-S-allcyl, alkylene-S-
substituted alkyl, substituted alkylene-S-alkyl and substituted alkylene-S-
substituted alkyl
wherein alkyl, substituted alkyl, alkylene and substituted allcylene arc as
defined herein.
Preferred alkylthioalkoxy groups are alkylene-S-alkyl and include, by way of
example,
methylenethiomethoxy (-CH2SCH3), ethylenethiomethoxy (-CH2CH2SCH3), n-
propylene-iso-
thiopropoxy (-CH~CHZCHZSCH(CH3~, methylene-t-thiobutoxy (-CH2SC(CH3)3) and the
like.
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"Alkenyl" refers to a monoradical of a branched or unbraached unsaturated
hydrocarbon preferably having from 2 to 40 carbon atoms, preferably 2-10
carbon atoms,
more preferably 2-6 carbon atoms, and preferably having 1-6 double bonds. This
term is
further exemplified by such radicals as vinyl, prop-2-enyl, pent-3-enyl, hex-5-
enyl,
5-ethyldodec-3,6-dienyl, and the like.
The term "substituted alkenyl" refers to an alkenyl group as defined above
having
from 1 to 5 substituents selected from the group consisting of aikoxy,
substituted alkoxy,
aryl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido,
cyano,
halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thiol,
thioallcoxy, substituted
thioalkoxy, aryl, heteroaryl, heterocyclic, aryloxy, thioaryloxy,
heteroaryloxy,
thioheteroaryloxy, heterocyclooxy, thiohetemcyclooxy, vitro, -SO-alkyl, -SO-
substituted
alkyl, -SO-aryl, -SO-heteroaryl, -SOI-alkyl, -S02-substituted alkyl, -SO~-
aryl, -S02-
heteroaryl, and, NR"Rb, wherein R' and R" may be the same or different and are
chosen from
hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloatkenyl,
alkynyl, aryl,
heteroaryl and heterocyclic.
"Alkenylene" refers to a diradical of an unsaturated hydrocarbon, preferably
having
from 2 to 40 carbon atoms, preferably 2-10 carbon atoms, more preferably 2-6
carbon atoms,
and preferably having 1-6 double bonds. This term is further exemplified by
such radicals as
1,2=ethenyl, 1,3-prop-2-enyl, 1,5-pent-3-enyl,1,4-hex-5-enyl, 5-ethyl-1,12-
dodec-3,6-dienyl,
and the like.
The term "substituted alkenylene" refers to an alkenylene group as defined
above
having from 1 to 5 substituents, selected from the group consisting of alkoxy,
substituted
alkoxy, acyi, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy,
oxyacylamino, azido,
cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylallcyl, thiol,
thioalkoxy,
substituted thioalkoxy, aryl, aryloxy, thioaryloxy, heteroaryl, hetemaryloxy,
thioheteroaryloxy, heterocyclic, heterocyclooxy, thioheterocyclooxy, vitro,
and NRRb,
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wherein R' and Rb may be the same or different and are chosen from hydrogen;
optionally
substituted sikyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl,
heteroaryl and heterocyclic.
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.
"Alkynyl" refers to a monoradical of an unsaturated hydrocarbon, preferably
having
from 2 to 40 carbon atoms, preferably 2-10 carbon atoms, more preferably 2-6
carbon atoms,
and preferably having 1-6 triple bonds. This term is further exemplified by
such radicals as
acetylenyl, prop-2-ynyl, pent-3-ynyl, hex-5-ynyl, 5-ethyldodec-3,6-diynyl, and
the like. .
The term "substituted alkynyl" refers to an alkynyl group as defined above
having
from 1 to 5 substituents, selected from the group consisting of allcoxy,
substituted alkoxy,
aryl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyacylamino, azido,
cyano,
halogen, hydroxyl, keto, thioketo, carboxyl, carboxylallcyl, thiol,
thioalkoxy, substituted
thioalkoxy, aryl, aryloxy, thioaryloxy, heteroaryl, heteroaryloxy,
thioheteroaryloxy,
heterocyclic, heterocyclooxy, thioheterocycloxy, vitro, -SO-alkyl, -SO-
substituted alkyl,
-SO-aryl, -SO-heteroaryl, -SO~-alkyl, -SOZ-substituted alkyl, -S02-aryl, -S02-
heteroaryl, S02-
heterocyclic, NR'R°, wherein R' aad Rb may be the same or different and
are chosen from
hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloaikenyl,
alkynyi, aryl,
heteroaryl and heterocyclic.
"Alkynylene" refers to a diradical of an unsaturated hydrocarbon radical,
preferably
having from 2 to 40 carbon atoms, preferably 2-10 carbon atoms, more
preferably 2-6 carbon
atoms, and preferably having 1-6 triple bonds. This term is further
exemplified by such
radicals as 1,3-prop-2-ynyl, 1,5-pent-3-ynyl, 1,4-hex-5-ynyl, 5-ethyl-1,12-
dodec-3,6-diynyl,
and the like.
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The term "aryl" refers to the groups -CHO, alkyl-C(O)-, substituted alkyl-C(O)-
,
cycloatkyl-C(O)-, substituted cycioalkyl-C(O)-, cycioalkenyl-C(O)-,
substituted cycioalkenyl-
C(O)-, aryl-C(O~, heteroaryl-C(O)- and heterocyclic-C(O)- where alkyl,
substituted alkyl,
cycioaikyl, substituted cycloallcyl, cycloalkenyl, substituted cycloaikenyl,
aryl, heteroaryl and
heterocyclic are as defined herein.
The term "acylamino" refers to the group -C(O)NRR where each R is
independently
hydrogen, alkyl; substituted alkyl, aryl, heteroaryi, heterocyclic or where
both R groups are
joined to form a heterocyclic group (e.g., morpholine) wherein alkyl,
substituted alkyl, aryl,
heteroaryl and heterocyclic are as defined herein.
The term "aminoacyi" 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.
The term "aminoacyioxy" 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.
The term "acyloxy" refers to the groups alkyl-C(O)O-, substituted alkyl-C(O)O-
,
cycloalkyl-C(O)O-, substituted cycloalkyi-C(O)O-, aryl-C(O)O-, heteroaryl-
C(O)O-, and
heterocyciic-C(O)O- wherein alkyl, substituted alkyl, cycioalkyl, substituted
cycioalkyi, aryl,
heteroaryl, and heterocyciic 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 aathryl).
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Unless otherwise constrained by the definition for the aryl substituent, such
aryl
groups can optionally be substituted with from 1 to 5 substituents selected
from the group
consisting of acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl,
cycloalkyl,
cycloalkenyl, substituted alkyl, substituted alkoxy, substituted allcenyl,
substituted alkynyl,
substituted cyeloalkyl, substituted cycloalkenyl, amino, aminoacyl, acylamino,
alkaryl, aryl,
aryloxy, azido, carboxyl, carboxylalkyl, cyano, halo, vitro, heteroaryl,
heteroaryloxy,
heterocyclic, heterocyclooxy, aminoacyloxy, oxyacylamino, thioallcoxy,
substituted
thioalkoxy, thioaryloxy, thiohetemaryloxy, -SO-alkyl, -SO-substituted alkyl, -
SO-aryl,
-uv~-~vu°riViilyl, avs-"~~y, -- S3Z-sudstituted alkyl, -SO2-aryl, -S02-
heteroaryl, trihalomethyl,
NR'R", wherein R' and Rb may be the same or different and are chosen from
hydrogen,
optionally substituted alkyl, cycloallcyl, alkenyl, cycloalkenyl, alkynyl,
aryl, heteroaryl and
heteroeyclic. Preferred aryl substituents include alkyl, alkoxy, halo, cyano,
vitro,
trihalomcthyl, and thioalkoxy.
The term "aryloxy" refers to the group aryl-O- wherein the aryl group is as
defined
above including optionally substituted aryl groups as also defined above.
The term "arylene" refers to a diradical derived from aryl or 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 NH2
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, alkynyl, substituted alkynyl, aryl,
heteroaryl and heterocyclic
provided that both R's are not hydrogen.
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The term "carboxyallcyl" refers to the group "-C(O)O-alkyl", "-C(O)O-
substituted
alkyl", "-C(O)O-cycloallcyl", "-C(O)O-substituted cycloalkyl", "-C(O)O-
alkenyl", "-C(O)O-
substituted alkenyl", "C(O)O-atkynyl" and "-C(O)O-substituted alkynyl" where
alkyl,
substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted
alkenyl, alkynyl and
substituted alkynyl where 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 cycloallcyl groups having from
1 to 5 substituents selected from the group consisting of alkoxy, substituted
allcoxy,
cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, acylamino, acyloxy,
amino,
aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto,
thioketo,
carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,
thiol, thioalkoxy,
substituted thioaikoxy, aryl, aryloxy, heteroaryl, heteroaryloxy,
heterocyclic, heterocyclooxy,
hydroxyamino, alkoxyamino, vitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -
SO-
heteroaryl, -S02-alkyl, -SOZ-substituted alkyl, -SOZ-aryl, -S02-heteroaryl,
and NR'Rb, wherein
R' and Rb may be the same or different and are chosen from hydrogen,
optionally substituted
alkyl, cycloallcyl, alkenyl, cycloaikenyl, alkynyl, aryl, heteroaryl and
heterocyciic.
The term "cycloalkenyl" refers to cyclic allcenyl groups of from 4 to 20
carbon atoms
having a single cyclic ring or fused rings and at least one point of internal
unsaturation.
Examples of suitable cycloaikenyl groups include, for instance, cyclobut-2-
enyl, cyclopent-3-
enyl, cyclooct-3-enyl and the like.
The term "substituted cycloalkenyl" refers to cycloalkenyl groups having from
1 to 5
substituents selected from the group consisting of alkoxy, substituted alkoxy,
cycloalkyl,
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substituted cycloalkyl, cycloalkenyl, substituted cycioalkenyl, aryl,
acylamino, acyioxy,
amino, aminoacyl, aminoacyloxy, oxyaminoacyi, azido, cyano, halogen, hydroxyl,
keto,
thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,
thioheterocyclooxy, thioi,
thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryioxy,
heterocyclic,
heterocyclooxy, hydroxyamino, alkoxyamino, vitro, -SO-alkyl, -SO-substituted
allcyl, -SO-
aryl, -SO-heteroaryl, -S0I-alkyl, -S02-substituted alkyl, -S02-aryl, -
S02=heteroaryl, and
NR'R°, wherein R' and Rb may be the same or different and are chosen
from hydrogen,
optionally substituted alkyl, cycloalkyl, alkenyl, cycloalke~rl, alkynyl,
aryl, heteroaryl and
heterccyclic.
The term "halo" or "halogen" refers to fluoro, chioro, bromo and iodo.
"Haloalkyi" refers to alkyl as defined above substituted by 1-4 halo groups as
defined
above, which may be the same or different, such as 3-fluorododecyi, 12,12,12-
trifluorododecyi, 2-bromooctyl, -3-bromo-6-chloroheptyl, and the like.
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 5 substituents
selected from the
group consisting of acyloxy, hydroxy, thiol, acyi, alkyl, alkoxy, alkenyl,
alkynyl, cycloalkyl,
cycloalkenyl, substituted alkyl, substituted alkoxy, substituted allcenyl,
substituted alkynyi,
substituted cycloallcyi, substituted cycioallcenyl, amino, aminoacyl,
acyiamino, alkaryl, aryl,
aryloxy, azido, carboxyl, carboxylalkyl, cyano, halo, vitro, heteroaryl,
heteroaryioxy,
heterocyclic, heterocyciooxy, aminoacyloxy, oxyacylamino, thioallcoxy,
substituted
thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -
SO-aryl, -SO-
heteroaryi, -SOZ-allryl, -SOZ-substituted alkyl, -SOZ-aryl, -SOZ-heteroaryl,
trihalomethyl,
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mono-and di-alkylamino, mono- and NR'Rb, wherein R' and Rb may be the same or
different
and are chosen from hydrogen, optionally substituted alkyl, cycloaikyl,
alkenyl, cycloalkenyl,
alkynyl, aryl, heteroaryl and heterocyclic. Preferred heteroaryls include
pyridyl, pyrrolyl and
furyl.
The term "hcteroaryloxy" refers to the.group heteroaryl-O-.
The term "heteroarylene" refers to the diradical group derived from heteroaryl
or
substituted heteroaryl as defined above, and is exemplified by the groups 2,6-
pyridylene, 2,4-
pyridiylene, 1,2-quinoliaylene, 1,8-quinolinylene, 1,4-benzofuraaylene, 2,5-
pyridinylene, 1,3
morpholinylene, 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
40 carbon
atoms and firom 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 cycloallcenyl, acyl,
acylamino, acyloxy,
amino, aminoacyl,. aminoacyloxy, .oxyaminoacyl, cyano, halogen, hydroxyl,
keto, thioketo,
carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,
thiol, thioalkoxy,
- substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryioxy,
heterocyclic, heterocyclooxy,
~ hydroxyamino, alkoxyamino, vitro, -SO-alkyl, -SO-substituted alkyl, -SO-
aryl, -SO-
heteroaryl, -SO2-alkyl, -S02-substituted alkyl, -S02-aryl, -S02-heteroaryl,
and NR'Rb, wherein
R' and Rb may be the same or different and are chosen from hydrogen,
optionally substituted
alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and
heterocyclic. Such
heterocyclic groups can have a single ring or multiple condensed nags.
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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 units of
the formula
[-(CHx-)mY-] where m is equal to or greater than 2, and Y at each separate
occuaence can be
O, N, S or P. Examples of crown compounds include, by way of example only, [-
(CI-1~-
NH-]3, [-((CHx)x-O),-((CH~x-NH)xJ and the like. Typically such crown compounds
can have
from 4 to 10 heteroatoms and 8 to 40 carbon atoms.
The term "hetemcyclooxy" refers to the group heterocyclic-O-.
The term "thioheterocyclooxy" refers to the group heterocyclic-S-.
The term "heterocyclene" refers to the diradical group derived from'a
heterocycle as
defined herein, and is exemplified by the groups 2,6-morpholino, 2,5-
morpholino and the
like.
The term "oxyacylamino" refers to the group -OC(O)NRR where each R is
independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or
hetemcyclic wherein
alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined
herein.
The term "thiol" refers to the group -SH.
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The term "thioalkoxy" refers to the group -S-alkyl.
The term "substituted thioalkoxy" refers to the group -S-substituted alkyl.
'The term "thioaryloxy" refers to the group aryl-S- wherein the aryl group is
as defined
above including optionally substituted aryl groups also defined above.
The term "thioheteroaryloxy" refers to the group heteroaryl-S- wherein the
heteroaryl
group is as defined above including optionally substituted aryl groups as also
defined above.
As to suy 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 andlor synthetically non-feasible.
In addition, the
compounds of this invention include all stereochemical isomers arising from
the substitution
of these compounds.
"Alkyl optionally interrupted by 1-5 atoms chosen from O, S, or N" refers to
alkyl as
defined above in which the carbon chain is interrupted by O, S, or N. Within
the scope are
ethers, sulfides, and amines, for example 1-methoxydecyl, l-pentyloxynonane, 1-
(2-
isopropoxyethoxy~4-methylnonane, 1-(2-ethoxyethoxy)dodecyl, 2-(t-
butoxy)heptyl,
1-pentylsulfanylaonane, nonylpentylamine, and the like.
"Heteroarylalkyl" refers to hetcroaryl as defined above linked to alkyl as
defined
above, for example pyrid-2-ylmethyl, 8-quinolinylpropyl, and the like.
"Optional" or "optionally" means that the subsequently described event or
circumstance may or may not occur, and that the description includes instances
where said
event or circumstance occurs and instances ~in which it does not. For example,
optionally
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substituted alkyl means that alkyl may or may not be substituted by those
groups enumerated
in the definition of substituted alkyl.
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.
Phatmaceutically acceptable base addition salts can be prepared from inorganic
and
organic bases. Salts derived from inorganic bases, include by way of example
only, sodium,
potassium, lithium, ammonium, calcium and magnesium salts. Salts derived from
organic
bases include, but are not limited to, salts of 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,
eycloallcyl amines, di(cyeloalkyl) amines, tri(cycloalkyl) amines, substituted
cycloallcyl
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.
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Examples of suitable amines include, by way of example only, isopropylamine,
trimethyi amine, diethyl amine, tri(iso-propyl) amine, tri(n-pmpyl) amine,
ethanolamine,
2-dimethylaminoethanoi, tromethamine, lysine, arginine, histidine, caffeine,
procaine,
hydrabamine, choline, betaine, ethylenediamine, glucosamine, N-
alkylglucamines,
S 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.
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, 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 "protecting group" or "blocking group" refers to any group which when
bound to one or more hydroxyl, thiol, amino or carboxyl groups of the
compounds prevents
reactions from occiuring at these groups and which protecting group can be
removed by
conventional chemical or enzymatic steps to reestablish the hydroxyl, thiol,
amino or
carboxyl group. See, generally, T.W. Greene & P.G.M. Wuts, Protective Groups
in Organic
Synthesis, 2'~ Ed.,1991, John Wiley and Sons, N.Y.
The particular removable blocking group employed is not critical and preferred
removable hydroxyl blocking groups include conventional substituents such as
aliyl, benzyl,
acetyl, chloroacetyl, thiot~nzyl, benzylidine, phenacyl, t-butyl-diphenylsilyl
and any other
group that can be introduced chemically onto a hydroxyl functionality and
later selectively
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removed either by chemical or enzymatic methods in mild conditions compatible
with the
nature of the product.
Preferred removable amino blocking.groups include conventional substituents
such as
t-butyoxycarbonyl (t-BOC), benzyloxycarbonyl (CBS, fluorenyhnethoxycarbonyl
(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 hydrolysis conditions compatible
with the nature
of the product.
As used herein, the terms "inert organic solvent" or "inert solvent" mean a
solvent
inert under the conditions of the reaction being described in conjunction
therewith [including,
for example, benzene, toluene, acetonitrile, tetrahydrofuran ("T~'~,
dimethylformamide
("DMF'~, chloroform ("CHC13-), methylene chloride (or dichloromethane or
"CH2Ciz'~,
diethyl ether, ethyl acetate, acetone, methylethyl ketone, methanol, ethanol,
propanol,
isopropanol, tert-butanol, dioxane, pyridine, and the like]. Unless specified
to the contrary,
the solvents used in the reactions of the present invention are inert
solvents.
The term "K'' channel" refers to a structure comprised of integral membrane
proteins
that functions to allow K; to equilibrate across a membrane according to its
electrochemical
gradient and at rates that are diffusion limited.
"Ligaad" as used herein denotes a compound that is a binding partner for a K"
channel
receptor, and is bound thereto, for example, by complementarity. The specific
region or
regions of the ligand molecule that is recognized by the ligand binding site
of a K" channel
receptor is designated as the "ligand domain". A ligand may be either capable
of binding to a
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receptor by itself, or may require the presence of one or more non-ligand
components for
binding (e.g: ions; a lipid molecule, a solvent molecule, and the like).
Ligands useful in this invention comprise I~' channel modulators such as
quinidine,6~94
glibenclamide, procaine, tetraethyl ammonium ~° clofilium,l°x
melperone,a pinacidil, WAY-
123,398,9' cromakalim,xs propoful, thiopentone,'x risotilide, almokalant,'6
bretylium; a N_
acetylprocainamide, tacrine, IJK66,914, .RP58866, 4-aminopyridine, RP49356; s
afinidine,49
chromanol 293B,~' L-768,673 and its analogs,s' bethanidine,~' disopyramide; x
desethylamiodarone,l NE-10064,9~'4 artilide,", dofetilide,'9,'~,'4.~°w
E-4031,2°~'','°'
sematilide,lw'°' ambasilide,
aamilide,s~a°w.~''1°°.ioa tedisamil, dronedarone,"
ibutilide,'a~"'
sotalol,a' beazodiazepine analogs'6~" and amiodarone.ss,ss~ex.9s,~os See Table
4 for structures of .
various potassium channel ligands.
While it is contemplated that many potassium channel ligands that are
currently
known can be used is the preparation of multibinding compounds of this
invention (Table 2),
it should be understood that portions of the ligand structure that are not
essential for
molecular recognition and binding activity (i.e., that are not part of the
ligand domain) may be
varied substantially, replaced with unrelated structures and, in some cases,
omitted entirely
without affecting the binding interaction. Accordingly, it should be
understood that the term
"ligand" is not intended to be limited to compounds known to be useful as K+
channel
receptor-binding compounds (e.g., known drugs), in that ligaads that exhibit
marginal activity
or lack useful activity as monomers can be highly active as multibinding
compounds, because
of the biological benefit conferred by multivalency. The primary requirement
for a ligand as
defined herein is that it has a ligand domain, as defined above, which is
available for binding
to a recognition site on a K+ channel.
For purposes of the present invention, the term "ligand" or "ligands" is
intended to
include the racemic Iigands as well as the individual stereoisomers of the
ligands, including
pure enantiomers and non-raccmic mixtures thereof. The scope of the invention
as described
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WO 99/b4050 PCTNS99/12777
and claimed encompasses the racemic forms of the ligands as well as the
individual
enantiomers and non racemic mixtures thereof.
The term "ligand .binding site" as used herein denotes a site on a I~ channel
receptor
that recognizes a ligand domain and provides a binding partner for the ligand.
The ligaad
binding site may be defined by monomeric or multimeric structures. This
interaction may be
capable of producing a unique biological effect, fur example agonism,
antagonism,
modulation, or may maintain an ongoing biological event, aad the like.
It should be recognized that the ligand binding sites of K' channel receptors
that
participate in biological multivalent binding interactions are constrained to
varying degrees by
their infra- and intermolecular associations. For example, K+ channel ligand
binding sites
may be covalently joined in a single structure, noncovalently associated in
one or more
multimeric structures, embedded in a membrane or biopolymer matrix, and so on,
and
therefore have less translational and rotational freedom than if the same
sites were present as
monomers in solution.
The terms "agonism"and "antagonism" are well known in the art. As used herein,
the
term "agonist" refers to a ligand that when bound to a K+ channel stimulates
its activity. The
term "antagonist" refers to a ligand that when bound to a K+ channel inhibits
its activity.
Cliannel block or activation may result from aliosteric effects of ligand
binding to the channel
rather than occupancy of the channel pore. These allosteric effects may
produce changes in
protein conformation that affect K" binding sites, Bating mechanisms and/or
the pore region
(i.e., ion permeation).
A potassium channel can exist in several modes: C (closed resting state); C*
(activated closed state); O (open state); and I (inactivated state) ~ The
probability that a
channel will exist in one of these four states changes with voltage. A given
ligand may have


CA 02318745 2000-07-24
PCT/US99/12777
different binding amities for different states, and be capable of producing
agonist or
antagonist activity.
The terns "modulatory effect" is intended to refer to the ability of a ligand
to chaage
the activity of a K'" channel through binding to the channel.
"Multibindi~ag agent" or "multibinding compound" refers herein to a compound
that
has from 2 to 10 K+ channel ligands as defined herein (which may be the same
or different)
covalently bound to one or more linkers (which may be the same or different),
and is capable
of multivalency, as defined below.
A multibinding compound provides an improved biologic and/or therapeutic
effect
compared to that of the same number of vmlinked ligands available for binding
to the ligand
binding sites on a K'' channel or channels. Examples of improved "biologic
and/or
therapeutic effect" include increased ligand-receptor binding interactions
(e.g., increased
affinity, increased ability to elicit a functional change in the target,
improved kinetics),
increased selectivity for the target, increased potency, increased efficacy,
decreased toxicity,
increased therapeutic index, improved duration of action, improved
bioavailability, 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.
"Univalency" as used herein refers to a single binding interaction between one
ligand
with one ligaad binding site as defined herein. It should be noted that a
compound having
multiple copies of a ligand (or ligands) exhibits univalency when only one
ligand of that
compound interacts with a ligand binding site. Examples of univalent
interactions are
depicted below.
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,T i
t1r11Va1Cnt lntCfaCtiOn
"Multivalency" as used herein refers to the conciurent binding of from 2 to 10
linked
ligands, which may be the same or different, and two or more corresponding
ligand binding
sites, which may be the same or different. An example of trivalent binding is
depicted below
for illustrative purposes.
v v v v v ..
trivalent interaction
It should be understood that not all compounds that contain multiple copies of
a ligand
attached to a linker necessarily exhibit the phenomena of multivalency, i.e.,
that the biologic
and/or therapeutic effect of the multibinding agent is greater than that of
the same numbeLof
unlinked ~ligands made available for binding to the ligand binding sites. For
multivalency to
occur, the ligand domains of the ligands that are linked together must be
presented to their
cognate ligand binding sites by the linker or linkers in a specific manner in
order to bring
about the desired ligand-orienting result, and thus produce a multi'binding
interaction.
The term "li'brary" refers to at least 3, preferably from l Oz to 109 and more
preferably
from 10z to 104 multimeric compounds. Rreferably, these compounds are prepared
as a
multiplicity of compounds in.a single solution or reaction mixtime which
permits facile
synthesis thereof. In one embodiment, the. library of multimcric compounds can
be directly
assayed for multi'binding properties. In another embodiment, each member of
the library of
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WO 99/64050 PCTNS99/12777
multimeric compounds is first isolated and, optionally, characterized. This
member is then
assayed for multibinding properties.
The term "collection" refers to a set of muitimeric compounds which are
prepared
either sequentially or concurrently (e.g., combinatorially). The collection
comprises at least 2
members; preferably from 2 to 10' members and still more preferably from 10 to
10°
members.
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
displacement
reactions in a manner similar to a halogen. Such functional groups include, by
way of
example, mesyl, tosyl, azido and cyano gmups.
The term "linker", identified where appropriate by the symbol X, refers to a
group or
groups that covalently links from 2 to 10 ligands (as defined above) in a
manner that provides
a compound capable of multivalency. The linker is a ligand-orienting entity
that permits
attachment of multiple copies of a ligand (which may be the same or different)
thereto.
The term "linker" includes everything that is not considered to be part of the
ligand,
e.g., ancillary groups such as solubilizing groups, lipophilic gmups, groups
that alter
phatmacodynamics or pharmacokinetics, groups that modify the diffusability of
the
multibinding compound, spacers that attach the ligand to the linker, groups
that aid the
ligand-orienting function of the linker, for example, by imparting flexibility
or rigidity to the
linker as a whole, or to a portion thereof, and so on. The term "linker" does
not, however,
cover solid inert supports such as beads, glass particles, rods, and the like,
but it is to be
understood that the multibinding compounds of this invention can be attached
to a solid
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vmderstood that the multibinding compounds of this invention can be attached
to a solid
support if desired, for example, for use in separation and purification
processes and for
similar applications.
The extent to which the previously discussed enhanced activity of multibinding
compounds is realized is this invention depends upon the e~ciency with which
the lirJker or
linkers that joins the ligands presents them to their array of ligand binding
sites. Beyond
presenting these ligands for multivalent interactions with ligand binding
sites, the linker
spatially constrains these interactions to occur within dimensions defined by
the linker.
The linkers used in this invention are selected to allow multivalent binding
of ligands
to any desired ligand binding sites of a K'' channel, whether such sites are
located within the
cell membrane, interiorly (e.g., within a channel/translocation pore), both
interiorly and on the
periphery of a channel, at the boundary region between the lipid bilayer and
the channel, or at
any intermediate position thereof. The preferred linker length will vary
depending on the
distance between adjacent ligand binding sites, and the geometry, flexibility
and composition
of the linker. The length of the linker will preferably be in the range of
about 2A to about
100A, more preferably from about 2A to about SOt~ and even more preferably
from about SA
to about 20A.
The ligands are covalently attached to the linker or linkers using
conventional
chemical techniques. The reaction chemistiries resulting in such linkage are
well known in the
art and involve the use of reactive functional groups present on the linker
and ligand.
Preferably, the reactive functional groups on the linker are selected relative
to the fimctional
groups available on the ligand for coupling,'or which can be introduced onto
the ligand for
this purpose. Again, such reactive functional groups are well known in the
art. For example,
reaction between a 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;
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PCTNS99/127~7
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 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 is formation
of an ether bond
covalently linking the lig~d to. the linker. The table below and Figure 6
illustrate numerous
reactive tonal groups and the resulting bonds formed by reaction therebetween.
Where
functional groups are lacking, they can be created by suitable chemistries
that are described in
standard organic chemistry texts ooh ~ J. March, Advanced Organic Chemistry,
4'" Ed.,
(W'iley-Interscience, N.Y.,1992).
Complementary Binding Chemistries
First Reactive Group Second Reactive Gronp Linkage
hydroxyl isooyWn~cthane
amine epoxide - l ~-hydroxyamine
sulfonyl halide
carboxyl amine amide
hydroxyl . ~ alkyl/aryl halide ~h~
amine ~ alkyl halide ~ substituted amine
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 linker embraces everything t~ is
not
considered to be part of the ligand.
The relative orientation in which the ligand domains are displayed depends
both on
the particular point or points ~of attachment of the ligands to the linker,
and on the framework
geometry. The determination of where acceptable substitutions can be made on a
ligand is
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PGTNS99/127~7
typically based on prior knowledge of structure-activity relationships of the
ligand and/or
congeners and/or structural information about ligand-receptor complexes (e.g.,
X-ray
crystallography, NMR, and the like). Such positions and synthetic protocols
for linkage are
well known in the art and can be determined by those with ordinary skill in
the art (see, e.g.,
METHODS OF PREPARATION, Examples 1-29 and Figures 7 to 21. Following
attachment of a ligand to the linker or linkers, or to a significant portion
thereof (e.g., 2-10
atoms of linker), the linker-ligand conjugate may be tested for retention of
activity in a
relevant assay system (see 3ltilitv and Testing below for representative
assays).
At present, it is preferred that the multibinding compound is a bivalent
compound in
which two ligands are covalently linked, or a trivalent compound, in which
three ligaads are
covalently linked. Linker design is further discussed under METHODS OF
PREPARATION.
"Potency" as used herein 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 receptor. In some cases, the
potency may be non-
linearly correlated with its affinity. In comparing the potency of two drugs,
e.g., a
multibinding agent and the aggregate of its unlinked ligand, the dose-response
curve of each
is determined under identical test conditions (e.g., in an in vitro or in vivo
assay, in an
appropriate animal model (such as a human patient)). The finding that the
multibinding agent
produces as equivalent biologic or therapeutic effect at a lower concentration
than the
aggregate unlinked ligand (e.g., on a per weight, per mole or per ligaad
basis) is indicative of
enhanced potency.
.
"Selectivity" or "specificity" is a measure of the binding preferences of a
ligand for
different receptors. The selectivity of a ligand with respect to its target
receptor relative to
another receptor is given by the ratio of the respective values of K,a (i.e.,
the dissociation
constants for each ligand-receptor complex) or, in cases where a biological
effect is observed
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below the Kd, the ratio of the respective EC~s or ICs (i.e., the
concentrations that produce
50% of the maximum response for the ligand interacting with the two distinct
receptflrs).
The term "treatment" refers to any treatment of a disease or condition in a
mammal,
particularly a human, and includes:
(i) preventing the disease or 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 treatanent constitutes prophylactic treatment for the
pathologic condition;
(ii) inhibiting the disease or condition, i.e., arresting its development;
(iii) relieving the disease or condition, i.e., causing regression of the
disease or
condition; or
(iv) relieving the symptoms resulting from the disease or condition without
addressing the underlying disease or condition, e.g., relieving symptoms of
angina pectoris
and other conditions of ischemia but not an underlying cause such as, for
example,
atheroscierotic disease or hypertension.
The phrase "disease or condition which is modulated by dent with a
multibinding
I~'' channel ligand" covers all disease states and/or conditions that are
generally
acknowledged in the art to be usefully treated with a ligand for a K" channel
in general, and
those disease states and/or conditions that have been found to be usefully
treated by a specific
multibinding compound of our invention, i.e., the compounds of Formula I. Such
disease
states include, by way of example only, hypertension, cerebral ischemia,
cardiac arrythmias
(particularly, anythmias resulting from potassium-related changes in membrane
potential and
conduction), cardiac hypertrophy due to systolic or diastolic overload,
congestive heart
failure, and the like.
The term "therapeutically effective amount" refers to that amount of
multibinding
compound that 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
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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 tenor "pharmaceutically acceptable eXCipient" is intended to include
vehicles and
carriers capable of being coadministered with a multibinding compound to.
facilitate the
performance of its intended function. The use of such media foi
pharmaceutically active
substances is well known in the art. Examples of such vehicles and carriers
include solutions,
solvents, dispersion media, delay agents, emulsions and the like. Any other
conventional
carrier suitable for use with the multibinding compounds also falls within the
scope of the
present invention.
METHODS OP' PREPARATION
I,ink~
The linker or linkers, when covalently attached to multiple copies of the
ligands,
provides a biocompatible, substantially non immunogenic multibinding compound.
The
biological activity of the multibinding I~ channel compound is highly
sensitive to the
geometry, composition, size, length, flexibility or rigidity, the presence or
absence of anionic
or cationic charge, the relative hydrophobicity/hydrophilicity, and similar
properties of the
linker. Accordingly, the linker is preferably chosen to maximize the
biological activity of the
compound. The linker may be biologically "neutral," i.e., not itself
contribute any additional
biological activity to the multibinding compound, or it may be chosen to
further enhance the
biological activity of the compound: In general, the linker may be chosen from
any organic
molecule construct that orients two or more ligands for binding to the
receptors 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.
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For example, different orientations of ligands can be achieved by varying the
geometry of the framework (linker) by use of mono- or polycyclic groups, such
as aryl and/or
heteroaryl groups, or structures incorporating one or more carbon-carbon
multiple bonds
(alkenyl, alkenylene, alkynyl or alkynylene groups). The optimal geometry and
composition
of frameworks (linkers) used in the multibinding compounds of this invention
are based upon
the properkies of their intended receptors. For example, it is preferred to
use rigid cyclic
groups (e.g., aryl, heteroaryl), or non-rigid cyclic groups.(e.g., cyeloalkyl
or cmwn groups) to
reduce conformational entropy when such may be necessary to achieve
energetically coupled
binding.
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
(H2N(CH2)6NH~ or
related polyamines can be modified to be substantially more hydrophilic by
replacing the
i 5 alkylene group with a poly(oxyalkylene) group such as found in the
commercially available
"Jeffamines" (class of surfactants).
Different fiameworks can be designed to provide preferred orientations of the
ligands.
The identification of an appropriate framework geometry for ligand domain
presentation is an
important first step in the construction of a multi binding agent with
enhanced activity.
Systematic spatial searching strategies can be used to aid in the
identification of preferred
frameworks through an iterative process. Figure 2 illustrates a useful
strategy for determining
an optimal framework display orientation for ligand domains and can be used
for preparing
the bivalent compounds of this invention. Various alternative strategies known
to those
skilled in the art of molecular design can be substituted for the one
described here.
As shown in Figure 2, the ligands (shown as filled circles) are attached to a
central
core structure such as phenyldiacetylene (Panel A) or cyclohexane dicarboxylic
acid (Panel
B). The ligands are spaced apart from the core by an attaching moiety of
variable lengths m
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WO 99/64050 PCTNS99/12777
and n. If the ligand possesses multiple attachment sites (see discussion
below), the
orientation of the ligand on the attaching moiety may be varied as well. The
positions of the
display vectors around the central core structures are varied, thereby
generating a collection of
compounds. Assay of each of the individual compounds of a collection generated
as
described will lead to a subset of compounds with the desired enhanced
activities (e.g.,
potency, selectivity). The analysis of this subset using a technique such as
Ensemble
Molecular Dynamics will suggest a framework orientation that favors the
properties desired.
The process may require the use of multiple copies of the same central core
structure
or combinations of different types of display cores. It is to be noted that
core structures other
than those shown here can be used for determining the optimal fiamework
display orientation
of the ligands. The above-described technique can be extended to trivalent
compounds and
compounds of higher-order valeacy.
A wide variety of linkers is commercially available (Chew Sources USA and Chew
Sources International; the ACD electronic database; and Chemical Abstracts).
Maay of the
linkers that are suitable for use in this inveation fall into this category.
Others can be readily
synthesized by methods known in the art, and as described below. Examples of
linkers
include aliphatic moieties, aromatic moieties, steroidal moieties, peptides,
and the like.
Specific examples ale peptides or polyamides, hydrocarbons, aromatics,
heterocyclics, ethers,
lipids, cationic or anionic groups, or a combination thereof.
Examples are given below and in Figure 3, 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 the linker, for example, to
change the solubility
of the multibinding compound (in 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 the linker
enhances the
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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 that enhance the water solubility/hydrophilicity of the
linker, and
accordingly, the resulting multibiading 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 andlor hydrophilicity of the
multibinding compounds
of this invention. In preferred embodiments, the ancillary group used to
improve water
solubility/hydrophilicity will be a polyether. In particularly preferred
embodiments, the
ancillary group will contain a small number of repeating ethylene oxide (-
CI~CH20-) units.
The incorporation of lipophilic ancillary groups within the structure of the
linker to
enhance the lipophilicity and/or hydrophobicity of the compounds of Formula I
is also within
the scope of this invention. Lipophilic groups useful with the linkers of this
invention
include, but are not limited to, lower alkyl, aromatic groups and polycyclic
aromatic groups.
The aromatic groups 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. As used
herein the term "aromatic groups" incorporates both aromatic hydrocarbons and
heterocyclic
aromatics. Other lipophilic groups useful with the linkers of this invention
include fatty acid
derivatives which may or may not form micelles in aqueous medium and other
specific
lipophilic groups which modulate interactions between the multibinding
compound and
biological membranes.
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Also within the scope of this invention is the use of ancillary groups which
result in
the compound of Formula I being incorporated into a vesicle, such as a
liposome, or a
micelle. The term "lipid" refers to aay fatty acid derivative that is capable
of forming a
bilayer or 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,
vitro and other like groups well known in the art. Hydrophobicity could be
conferred by the
inclusion of groups that include, but are not limited to, long chain saturated
and unsaturated
aliphatic hydrocarbon groups of up to 20 carbon atoms and such groups
substituted by one or
more aryl, heteroaryl, cycloalkyl, and/or hetemcyclic group(s). Preferred
lipids are
phosphoglycerides and sphingolipids, representative examples of which include
phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,
phosphatidylinositol,
phosphatidic acid, palmitoyleoyl phosphatidylcholine, iysophosphatidylcholine,
lysophosphatidyl-ethanolamine, dipalmitoylphosphatidylcholine,
dioleoylphosphatidyl-
choline, distearoyl-phosphatidyicholine and dilinoleoyiphosphatidylcholine.
Other
compounds lacking phosphorus, such as sphingolipid and glycosphingolipid
families, are also
withia the gmup designated as lipid. Additionally, the amphipathic lipids
described above
may be mixed with other lipids including triglycerides and sterols.
The flexibility ~of the linkei can be manipulated by the inclusion of
ancillary groups
which are bulky andlor 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
group(s), or bonds
between the linker and the functional groups. Rigid groups can include, for
example, those
groups whose conformational freedom is restizained by the presence of rings
and/or ~-bonds,
for example, aryl, heteroaryl and heterocyclic gmups. Other groups which can
impart rigidity
include polypeptide groups such as oligo- or polyproline chains.
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 linker
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into a configuration affording the maximum distance betvv~een each of the like
charges. The
energetic cost of bringing the like-charged groups closer to each other, which
is inversely
related to the square of the distance between the groups, 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 oppositeiy
charged counterparts and potentially may enter into both inter- and
intramolecular ionic
bonds. This non-covalent mechanism will tend to hold the linker in a
conformation which
allows bonding between the oppositely charged groups. The addition of
ancillary groups
which are charged, or alternatively, protected groups that bear a latent
charge which is
unmasked, following addition to the linker, by deprotection, a change in pH,
oxidation,
reduction or other mechanisms known to those skilled in the art, is within the
scope of this
invention.
Bulky groups can include, for example, large atoms, ions (e.g., iodine,
sulfur, metal
ions, etc.) or groups containing large atoms, polycyclic groups, including
aromatic groups,
non-aromatic groups and structures incorporating one or more carbon-carbon a-
bonds (i.e.,
alkenes and alkynes). Bulky groups can also include oligomers and polymers
which are
branched'- or straight-chain species. Species that are branched are expected
to increase the
rigidity of the structure more per unit molecular weight gain than are
straight-chain species.
In preferred embodiments, rigidity (entropic control) is, imparted by the
presence of
alicyclic (e.g., cycloalkyl), aromatic and heterocyclic groups. In other
preferred
embodiments, this comprises one or more six-membered rings. In still fiuther
preferred
embodiments, the ring is an aryl group such as, for example, phenyl or
naphthyl, or a
macrocyclic ring such as, for example, a crown compound
In view of the above, it is apparent that the appropriate selection of a
linker group
providing suitable orientation, entropy and physico-chemical properties is
well within the
skill of the art.
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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 multibiading
compound may be eliminated or reduced by use of groups such as, for example,
polyethylene
glycol).
As explained above, the multibiading compounds described herein comprise 2-10
ligands attached covalently to a linker that links the ligands in a maser that
allows their
multivalent binding to ligand binding sites of IC' channels. The linker
spatially constrains
these interactions to occur within dimensions defined by the linker. This and
other factors
increases the biologic and/or therapeutic effect of the multibinding compound
as compared to
the same number of ligands used in monobinding form.
The compounds of this invention are preferably represented by the empirical
formula
(L)p(3~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 provided below. .
As noted previously, the linker may be considered as a framework to which
ligands
are attached. Thus, it should be recognized that the ligands can be attached
at any suitable
position on this framework, for example, at the termini of a linear chain or
at any intermediate
position thereof.
The simplest and most preferred multibinding compound is a bivalent compound
which can be represented as L-X-L, where L is a ligand and is the same or
different and X is
the linker. 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 is X. However, a trivalent compound can also comprise
three ligaads
a~ to a central con, and thus be represented as (LAX, where the linker X could
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inclu~ie,for example, an aryl or cycloalkyl group. Tetravalent compounds can
be represented
in a linear stray:
L-X-L-X-L-X L,
or a branched array:
L-X-L-X-L,
L
i.e., a branched construct analogous to the isomers of butane (n-butyl, iso-
butyl, sec-butyl,
and t butyl). Alternatively, it could be represented as an aryl or cycloalkyl
derivative as
described above with four (4) ligands attached to the core linker.
The same considerations apply to higher multibinding compounds of this
invention
1 S containing from 5-10 ligands. However, for multibinding, agents attached
to a central linker
such as an aryl, cycloalkyl or heterocyclyl group, or a crown compound, there
is a self-evident
constraint that there must be sufficient attachment sites on the linker to
accommodate the
number of ligands present; for example, a benzene ring could not accommodate
more than 6
ligands, whereas a multi-ring linker (e.g., biphenyl) could accommodate a
larger number of
ligands.
The fOImtlla (L)P(X)q is also intended to represent a cyclic compound of
formula (-L-
X-)o ,where n is 2-10.
All of the above variations are intended to be within the scope of the
invention
defined by the formula (L~(X~q. Examples of bivalent and higher-order valency
compounds
of this invention are provided in Figures 4A to 4D.
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With the foregoing in mind, a preferred linker may be represented by the
following
formula:
X'=Z-(Y'-Z~-Y"-Z X'-
in which: m is an integer of from 0 to 20; X' at each separate occurrence is -
O-, -S-, -S(O~, -
S(O~-, NR-, N' R R'-, -C(O~, -C(O)O-, -C(O)NH-, -C(S), -C(S)O-, -C(S)NH- or a
covalent
bond, where R and R' at each separate occurrence are as defined below for R'
and R"; Z is at
each separate occuaence selected from alkylene, substituted alkylene,
alkylalkoxy,
cycloalkylene, substituted cycloalkylene, alkenylene, substituted alkenylene,
alkynylene,
substituted alkynylene, cycloalkenylene, substituted alkenylene, arylene,
substituted arylene,
heteroarylene, heterocyclene, substituted heterocyclene, crown compounds, or a
covalent
bond; Y' and Y" at each separate occurrence are selected from the group
consisting of
0 0 0
N~ \N 'N N/
,. ~ . .
R
R'
R~N N~
N/ ~N -pUyIWR'~'C
R'
o x
25~
~N/ N~ -S(O~,-CRW"-, -S(~~; NR~-,
p
R. ~ R.
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-S-S- or a covalent bond; in which: n is.0,1 or 2; and R' and R" at each
separate occw~ace
re selected from hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted
cycloallcyl, alkenyl,
substituted aikenyl, alkynyl, substituted alkynyl, aryl, hetemaryl or
heterocyclic.
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, alkynyl, substituted alkynyl, aryl, heteroaryl and heterocyclic
group.
As indicated above, the simplest (and preferred) construct is a bivalent
compound
which can be represented as L-X-L, where L is a K' channel ligand that is the
same or
different at each occurrence, and X is the linker. Accordingly, examples of
the preparation of
a bivalent ligand are given below as an illustration of the manner in which
multibinding
compounds of Formula I are obtained.
The reaction schemes that follow illustrate preferred linking strategies for
linking
phenylmethane sulfonamide (dofetilide, ibutilide, sematilide, sotalol, and E-
4031 ) and
benzofuran (amiodarone, desethylamiodarone, NE-10064) classes of potassium
channel
modulators. These strategies are intended to apply as well to any K' channel
ligand that
includes, or can be functionalized with groups compatible with the chosen
linker (e.g.,
azirnilide and tedisamil).
As was previously discussed, the linker or linkers can be attached to
different
positions on the ligand molecule to achieve different orientations of the
ligand domains and
thereby facilitate multivalency. For example, the positions that are
potentially available for
linking a benzofiwan such as amiodarone are indicated by arrows in the
structure shown in
Figure 5.
Preferred positions of attachment suggested by known SAR are illustrated in
the
reaction schemes of Figures 7 to 21. Examples of ligands are shown in Table 4.
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Certain K'' chazmel ligands may be chiral and exhibit stereoselectivity. The
most
active enantiomers are preferably used as ligaads in the multibinding
compounds of this
invention. The chiral resolution of enantiomers is accomplished by well known
procedures
that result in the formation of diastereomeric derivatives or salts, followed
by conventional
separation by chromatographic procedures or by fractional crystallization
(see, e.g., Bossert,
et al., Angew. Che~n. Int. Ed, 20:762-769 (1981) and U.S. Patent No. 5,571,827
and
references cited therein). They may also be obtained by asymmetric synthesis.
The ligaads are covalently attached to the linker using conventional chemical
techniques. The reaction chemistries resulting in such linkage are well known
in the art and
involve the coupling of reactive functional groups present on the linker and
ligand. In some
cases, it may be necessary to protect portions of the ligand that are not
involved in linking
reactions. Protecting groups for this purpose are well known in the art and
are indicated
generally in the reaction schemes by the symbols PG and PG'.
Preferably, the reactive functional groups on the linker are selected relative
to the
functional groups on the ligand that are available for coupling, or can be
introduced onto the
ligand for this purpose. In some embodiments, the linker is coupled to ligand
precursors,
with the completion of ligand synthesis being carried out in a subsequent
step. Whore
functional groups are lacking, they can be created by suitable chemistries
thax arc described in
standard organic chemistry texts such as J. March, Advanced Organic Chemistry,
4'~ Ed.
(W'~ley- Interscience, N.Y.,1992). Examples of the chemistry for connecting
ligands by a
linker are shown in Figure 6, where R' and R2 represent a ligand and/or the
linking group.
One skilled in the art will appreciate that synthetically equivalent coupling
reactions can be
substituted for the reactions illustrated herein.
The linker to which the ligaads or ligand precursors are attached comprises a
'.'core"
molecule having two or more functional groups with reactivity thax is
complementary to that
of the,functional groups on the ligand. Figure 3. illustrates the diversity of
"cores" that are


CA 02318745 2000-07-24
WO 99/64050 PCT/US99/1Z777
useful for varying the linker sire, shape, length, orientation, rigidity,
acidity/basiciiy,
hydmphobicity/hydrophilicity, hydrogen bonding characteristics and number of
ligands
connected. This pictorial representation is intended only to illustrate the
invention, and not to
limit its scope to the structures shown. In the Figures and reaction schemes
that follow, a
solid circle is used to generically represent a core molecule, referred to as
"Link" in the
Examples. The solid circle is equivalent to a linker as defined above after
reaction.
The preferred compounds of Formula I are bivalent. Accordingly, and for the
purpose
of simplicity, most of the figures and reaction schemes below illus~ate the
synthesis of
bivalent K+ channel modulators. It should be noted, however, that the same
techniques can be
used to generate higher order multibinding compounds, i.e., the compounds of
the invention
where p is 3-10. (See, e.g., Figure 15 and 20.)
Reactions performed under standard amide coupling conditions are carried out
in an
inert polar solvent (e.g., DMF, DMA) in the presence of a hindered base {e.g.,
TEA, DIPEA)
and standard amide coupling reagents {e.g., DPPA, PyBOP, HATU, DCC).
Several methods for preparing bivalent benzofuran (BF) compounds, as
exemplified
here for amiodarone and structurally analogous molecules, are illustrated in
the reaction
schemes for amiodarone and dronedarone shown in Figure 7. These are described
in detail in
Examples 1-3.
Several methods for preparing bivalent phenylmethane sulfonamide (PMS)
compounds, as exemplified by dofetilide, ibutilide, sematilide and sotalol,
and structurally
analogous molecules are illustrated in the reaction schemes shown in Figures 8
-11. These
are described in detail in Examples 4-11.
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Several methods for preparing bivalent aamilide and tedisamil compounds are
illustirated in the reaction schemes shown in Figures 12 -13. These are
described in detail in
Examples 12 - I4.
The strategies for preparing compounds of Formula I discussed above involve
coupling the ligand directly to a homobifunetional core. Another strategy that
can be used
with all ligands, and for the preparation of both bivalent and higher order
multibinding
compounds, is to introduce a 'spacer' before coupling to a central core. Such
a spacer can
itself be selected from the same set as the possible core compounds. Examples
of this
linking strategy using starting materials prepared as described above, are
shown in Figure 14,
where the spacer is~represented by a black circle. As defined herein, the
linker comprises the
spacer + core. These are described in detail in Examples I S-17.
Compounds of Formula I of higher order valency, i.e., p>2, can be prepared by
simple
extension of the above strategies. As shown in Figure 15, compounds are
prepared by
coupling ligaads to a central core bearing multiple functional groups. The
reaction conditions
are the same as described above for the preparation of bivalent compounds,
with appropriate
adjustments made in the molar quantities of ligand and reagents. These are
described in
detail in Examples 18-21.
Figures 16 and 17 show ligaads coupled to a polypeptide core with a sidechain
spacer.
Solid phase peptide synthesis can be used to produce a wide variety of
peptidic core
molecules. Techniques well-known to those skilled in the art (including
combinatorial
methods) are used to vary the distance between ligaad a#schment sites on the
core molecule,
the number of attachment sites available for coupling, and the chemical
properties of the core
molecule. Orthogonal protecting groups are used to selectively protect
functional groups on
the core molecule, thus allowing ancillary groups to be inserted into the
linker of the
multibinding compound and/or the preparation of "heterovalomers" (i.e.,
multibinding
- compounds with nonidentical ligands).
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All of the synthetic strategies described above employ a step in which the
ligand,
attached to spacers or not, is symmetrically linked to functionally equivalent
positions on a
central core. Compounds of Formula I can also be synthesized using an
asymmetric linear
approach. This strategy is preferred when linking two or more ligands at
different points of
connectivity (see, e.g., Figure 18) or when preparing heterovalomers (see,
e.g., Figure 19).
These are described in detail in Examples 22-25.
Isolation and purification of the compounds and intermediates described herein
can be
effected, if desired, by any suitable separation or purification such as, for
example, filtration,
extraction, crystallization, column chromatography, thin-layer chromatography,
thick-layer
chromatography, preparative low or high pressure liquid chromatography or a
combination of
these procedures. Characterization is preferably by NMR and mass spectroscopy.
~jtihp~and Testing
The multibinding compounds of this invention can be used to modulate potassium
channels in various tissues including heart, muscle, and neurons. They will
typically be used
for the treatment of diseases and conditions in mammals that involve or are
mediated by Ki
channels, such as hypertension, cardiac arrythmias, cerebral ischemia,
congestive heart
failure, and the like.
The multibinding compounds of this invention are tested in well-known and
reliable
assays and their activities are compared with those of the corresponding
unlinked (i.e.,
monovalent) ligands.
$~LnCllIlg~,ni~,r~,n ~,~~taasi ,m ch nnelc
The binding affinity is determined by a radioligand competitive inhibition
assay ~
The ability of the present compounds to compete with ~H]dofetilide or a
similar radioactive
ligand in binding to high- and low-affinity binding sites of guinea pig
ventricular myocytes is
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measured in vitro. The binding affinity, calculated from competition curves,
is compared
with that of the monovaient ligand andlor monovalent linker-ligand conjugate.
Antiarrhythmic effect of compounds of this invention may be determined in vivo
in
dogs with induced myocardial infarction and reproducibly inducible ventricular
tachycardia or
ventricular fibrillation.' Suppression of inducible arrhythmias is measured.
The antifibrillatory and antiarrhythmic effects of the compounds of this
invention may
be determined in vivo using a canine model of sudden death.' Reduction of the
incidence of
programmed electrical stimulation (PES) induced ventricular tachycardia and
protection
against ischemia induced ventricular fibrillation ate measured.
The antiarrhythmic effect of the compounds of this invention may be determined
in
vivo using the mouse chloroform model.' The percentage of animals showing
normal sinus
rhythm is measured.
The antiarrhythmic effect of the compounds of this invention may be determined
in
vivo using the rat coronary ligation model ° Ventricular extrasystoles
occurring during the 30
minutes following the procedure are counted.
The antiarrhythmic effect of the compounds of this invention may be determined
in
vitro or in vivo using rat coronary artery ligation/reperfusion models
° In the in vitro model,
excised rat hearts are retrogradely perfused with a solution of the compound
to be tested, then
the coronary artery is ligated, followed by reperfusion. In the in vivo
evaluation, the
compound is administered i.p., then the coronary artery is ligated, followed
by reperfusion. In
both models the incidence and time to onset during reperfusion of ventricular
extrasystole,
tachyarrhythmia and fibrillation ate measured.
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The antiarrhythmic effect of the compounds of this invention may be determined
in
vivo using an anesthetized rat model of ventricular arrhythmias 9 The time to
onset of
ventricular extrasystoles is measured.
The antiarrhythmic effect of the compounds of this invention may be~
determined in
vivo using a canine myocardial infarction model where compound is administered
24 hours
after ligation of the left anterior descending coronary artery" Right
ventricular effective
refractory period, monophasic action potential duration and reduction of PES
induced
ventricular tachycardia and ventricular fibrillation are measured.
The ability of the compounds of this invention to prolong the action potential
(achieve
a slower onset of active state block) and recover faster from block may be
determined in vitro
using rabbit ventricular myocytes!'~° Development of block during a
long depolarizing
clamp and recovery from block are measured.
The ability of the compounds of this invention to suppress repolarization
arrhythmias
may be determined in vitro using canine epicardium nudmyocardium and
endocardium and
canine cardiac Purkinje fibers and,in vivo using anesthetized
rabbits.'°w
The ability of the compounds of this invention to suppress arrhythmias may be
determined in vivo using the feline coronary occlusion and left stellate
ganglion stimulation
model, the conscious canine model of transient ischemia during exercise in the
presence of a
healed MI and the conscious canine model of complete occlusion after recent MI
~'
The ability of the compounds of this invention to pmlong action potential
duration
may be determined in vivo and i~ vitro using guinea pig hearts;' and in vitro
using calf
cardiac Purkinje fibers.
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The ability of the compounds of this invention to prevent atrial fibrillation
(A~ may
be determined using a canine model of sustained vagotonic AF 6' Prevention of
AF induction
is measured. Reverse use dependence may also be determined.
. $ffect on t~hyca~'~llia
The effect of compounds of this invention on tachycardia may be determined in
vitro
using rabbit right atrial preparations i Micro-electrode techniques are used
to measure the
ability to prolong the refractory period and thus prevent initiation of
tachycardia.
The effect of the compounds of this invention on tachyarrhythmias may be
determined
in vitro using guinea pig right ventricular papillary muscle r The action
potential duration at
different extracellular potassium concentrations is measured. .
The effect of compounds of this invention on the repolarization cuwents IK and
Lro
may be determined in vitro using whole cell recordings in cat ventricular
myocytes and
papillary muscles from the hearts of oophorectomized rabbits.4~'
The effect of compounds of this invention on various specific potassium
current niay
be determined in vitro using guinea pig ventricular myocytes and sinoatrial
node cells, human
atiial myocytes, canine ventricular muscle and Purldnje fibers, guinea pig
papillary muscle,
single voltage clamped guinea pig ventricular myocytes and human ventricular
endomyocardium ~ml'~~~~3.s~,sl.~.~al
The ability of compounds of this invention to inhibit potassium currents in a
non
cardiac preparation may be determined using rat taste receptor cells ~°
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The ability of compounds of this invention to modulate the KATP channel may be
determined using a °~Rb efflux assay.'fw Thus, this is a potency assay.
The selectivity and/or specificity of the compounds of this invention may be
determined using CfIO cell lines expressing specific recombinant potassium
channel
X34
The selectivity of compounds of this invention for various potassium channel
currents
may be determined in vitro using cloned K channels expressed in cells or
ventricular
myocytes.'~"
The selectivity of compounds of this invention for various receptors may be
determined in vitro using rat synaptosomal membrane. (Pong, et al. "Binding
profile of NE-
10064, a novel Class III anti-arrhythmic agent to rat brain receptors", Faseb
J., 7:A474
(1993)).
Antivasoconstrictor activity is determined as described in Brittain, et al.,
Physiologist,
28:325 (1985) as the concentration of a compound required to produce 50'/o
vasoreiaxation in
KCl-contracted rabbit thoracic aorta strips in the presence of calcium.
Alternatively, the
concentration of a compound required to inhibit coronary vasoconstriction
induced by a
th=omboxane mimetic (U-46619, i.e., 9,11-methanoepoxy-PGH~ in guinea pig
Langendorff
heart preparation is measured as described in Eltze, et ai., Chirality, 2:233-
240 (1990).
Antihypertensive activity is determined in male spontaneously hypertensive
rats by
measurement of mean arterial blood pressure (Rovnyak, et al., J. Med Chem.,
35:3254-3263
(1992)).
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Selectivity for vascular smooth muscle as compared with cardiac muscle can be
assessed by comparing the concentration of a multibinding compound that
produces a 50%
increase in coronary blood flow in an isolated guinea pig heart with that
required to inhibit
myocardial contractility. See, e.g , Osterrieder, W. and Holck, M., J.
Cardiovasc. Pharm.,
13:754-9 (1989); and Cremate, et al., J. Cardiovasc. Pharm., 29:692-696
(1997).
The methods described above lend themselves to combinatorial approaches for
identifying multimeric compounds which possess multibinding properties for
potassium
channels.
Specifically, factors such as the proper juxtaposition of the individual
ligands of a
multl'binding 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 ligand(s), (2) the orientation of ligands, (3) the valency
of the construct,
(4) linker length, (~ linker geometry, (6) linker physical properties, and ('n
linker chemical
functional groups.
Libraries of multimeric compounds potentially possessing multibinding
properties
(i. e. , candidate multibinding compounds] and comprising a multiplicity of
such variables
are prepared and these libraries are then evaluated via conventional assays
corresponding to
the ligand selected and the multibinding parameters desired. Considerations
relevant to
each of these variables are set forth below:
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A single Iigand or set of ligands is (are) selected for incorporation into the
libraries
bf candidate multibinding compounds which library is directed against a
particular
biological target or targets. 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
PKlADME
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 effcacious in a human patient may become highly potent and efficacious when
presented
in multibinding form. A ligand that is potent and efRcacious 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 Iigands 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.
Several points are chosen on each ligand at which to attach the ligand to the
linker.
The selected points on the ligand/linker for attacbment are functionalized to
contain
complementary reactive functional groups. This permits probing the effects of
presenting
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WO 99/64050 PCT/US99/12777
the ligands to their receptors) in multiple relative orientations, an
important multibinding
design parameter. The only~requireme~ for choosing attachment poi~s 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 protease inlu'bitor bound to its
target allows one to
identify one or more sites where linker attachment will not preclude the
enzyme:inlu'bitor
interaction. Alternatively, evaluation of ligandltarget binding by mtclear
magnetic
resonance will permit the identification of sites non-essential for
ligand/target binding. See,
for example, Fesik, et al., U.S. Patent No. 5,891,643. When such structural
information is
not available, utilization of structure-activity relationships (SAR) for
ligands will suggest
positions where substantial structural variations are and are not allowed. In
the absence of
both structural and SAR information, a library is merely selected with
multiple points of
attachment to allow presentation of the ligand in multiple distinct
orientations. Subsequent
evaluation of this library will indicate what positions are suitable for
attachment.
It is important to emphasize that positions of attachment that do abrogate the
activity
of the monomeric ligand may also be advantageously inchtded 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, heterobivaient interactions within the content of a single target
molecule. For
example, consider a receptor antagonist ligand bound to its target receptor,
and then
consider modifying this ligand by attaching to it a second copy of the same
ligand with a
linker which allows the second ligand to, interact with the same receptor
molecule at sites
proximal to the antagonist binding site, which include elements of the
receptor that are not
part of the formal antagonist binding site andlor elements of the matrix
surrounding the
receptor such as the membrane. Here, the most favorable orientation for
interaction of the
second ligand molecule with the receptor/matrix may be achieved by attaching
it to the
linker at a position which abrogates activity of the ligand at the formal
antagonist binding
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PGTNS99/12777
site. Another way to consider this is that the SAR of individual ligands
within the content
of a multibinding structure is often different from the SAR of those same
ligands in
momomeric form.
The foregoing discussion focused on bivalent interactions of dinuric compounds
bearing two copies of the same ligand joined to a single linker through
different attachment
points, one of which may.abrogate the binding/activity of the monomeric
ligand. It should
also be understood that bivale~ advantage may also be attained with
heterodimeric
constructs bearing two different ligands that bind to common or different
targets. For -
example, a SHT4 receptor antagonist and a bladder-selective muscarinic N13
antagonist may
be joined to a linker through attachment points which do not abrogate the
binding amity of
the monomeric ligands for their respective receptor sites. The dimeric
compound may
achieve enhanced affinity for both receptors due to favorable interactions
between the SHT,
ligand and elements of the N13 receptor proximal to the formal N13 antagonist
binding site
and between the Ni3 ligand and elements of the SHT,, receptor proximal to the
formal 5HT4
antagonist binding site: Thus, the dimeric compound may be more potent and
selective
antagonist of overactive bladder and a superior therapy for urinary urge
incontinence.
Once the ligand attachment points have been chosen, one. ide~ifies the types
of
chemical linkages that are possible at those points. The most preferred types
of chemical
linkages are those that are compatible with the overall structure of the.
ligand (or protected
forms of the ligand)~ readily and generally formed, stable and intrinsically
inocuous under
typical chemical and physiological conditions, and compatible with a large
number of
available linkers. Amide bonds, ethers, amines, carbamates, areas, and
s~on~des are
but a few examples of preferred linkages.
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In the library of linkers employed to generate the library of candidate
multi'bindin~g
compounds, the selection of linkers employed in this library of linkers takes
into
consideration the following factors:
y~~,, In most instances the library of linkers is initiated with divalent
linkers.
The choice of ligands and proper juxtaposition of two ligands relative to
their binding sites
permits such molecules to exhibit target binding affinities and specificities
more than
sufficient to confer biological advantage. Furthermore, divalent linkers or
constructs are
also typically of modest size such that they retain the desirable
biodistribution properties of
small molecules.
j'n~~th. Linkers are chosen in a range of lengths to allow the spanning of a
range of inter-ligand distances that encompass the distance preferable for a
given divalent
interaction. In some instances the preferred distance can be estimated rather
precisely from
high-resolution structural information of targets, typically enzymes and
soluble receptor
targets. In other instances where high-resolution structural information is
not available
(such as 7TM G-protein coupled receptors), one can make use of simple models
to estimate
the maximum distance between binding sites either on adjacent receptors or at
different
locations on the same receptor. In situations where two binding sites are
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 A. In situations where two
binding sites
reside on separate (e.g., protein) target sites, preferred linker distances
are 20-100 A, with
more preferred distances of 30-70 A.
~~ om and ~ riai~ The combination of ligand attachment site, linker
leagth, linker geometry, and linker rigidity determine the possible ways. in
which the
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CA 02318745 2000-07-24
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ligands of candidate multi'binding compounds may be displayed in three
dimensions and
thereby prese~d to their binding sites. Linker geometry and rigidity are
nominally
determined by chemical composition and bonding pattern, which may be
controlled and are
systematically varied as another spanning function in a multibinding array.
For example,
linker geometry is varied by attaching two ligands to the ortho, meta, and
para positions of
a benzena ring, or in cis- or traps-arrangements at the 1,1- vs. I,2- vs. 1,3-
vs. 1,4-
positions around a cyclohexane core or in cis- or traps-arrangements at a
point of ethylene
unsaturation. Linker rigidity is varied by controlling the ~oumber and
relative energies of
different conformational states possible for the linker. For example, a
divalent compound
bearing two ligands joined by 1,8-octyl linker has many more degrees of
freedom, and is
therefore less rigid than a compound in which the two ligands are attached to
the 4,4'
positions of a biphenyl linker.
t .'ynk~r pbyci 1 rone~, The physical properties of linkers are nominally
determined by the chemical constitution and bonding patterns of the linker,
and linker
physical properties impact the overall physical properties of the candidate
multibinding
compounds in which they are included. A range of linker compositions is
typically selected
to provide a range of physical properties (hydrophobicity, hydrophilicity,
amphiphilicity,
polarization, acidity, and basicity) in the candidate multibinding compounds.
The
particular choice of linker physical properties is made within the content of
the physical
properties of the ligands they join and preferably the goal is to generate
molecules with
favorable PK/ADME properties. For example, linkers can be selected to avoid
those that
are too hydrophilic or too hydrophobic to be readily absorbed and/or
distributed in vivo.
L' ~ , ~ ~j~' 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.
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~inat~aL~hs~i~
~iaving chosen a set of n ligaads (n being determined by the sum of the number
of
different attachment points for each Iigand chosen) and m linkers by the
process outlined
above, a library of (n!)m candidate divalent multibinding compounds is
prepared which
spans the relevant multibinding design parameters for a particular target. For
example, an
array generated from two ligands, one which has two attachment points (Al, A2)
and one
which has three attachment points (B1, B2, B3) joined in all possible
combinations provide
for at least 15 possible combinations of multibinding coa~OUnds:
Al-A1 Al-AZ A1 Bl Al-B2 Al-B3 A2-A2 A2 B1 A2-B2
A2 B3 Bl B1 Bl-B2 Bl-B3 B2 B2 B2 B3 B3-B3
When each of these combinations is joined by 10 different linkers, a library
of 150
candidate multi'binding compounds results.
Given the combinatorial nature of the library, common chemistries are
preferably
used to join the reactive functionalies on the ligands with 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 andlor linker is attached to a solid support.
Alternatively and
preferably; the combinatorial libary is prepared in the solution phase. After
synthesis,
candidate multibinding compounds are optionally purified before assaying for
activity by,
for example, chromatographic methods (e.g., HPLC).
Various methods are used to characterize the properties and activities of the
candidate multibinding compounds in the library to deternnine which compounds
possess
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CA 02318745 2000-07-24
WO ~/~~ PCT/US99/12777
multz'binding properties. Physical constants such as solubility under various
solvent
conditions and logDlclogD values can be determined. A combination of NMR
spectroscopy and computational methods is used to determine low-energy
conformations of
the candidate multibinding compounds in fluid aardia. 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 agonists and antagonists. ion channel blockers, and a~imicrobial
activity, can
also be determined. Pharnoacological data, including oral absorption, evened
gut -
penetration, other pharmacokiiietic parameters and efficacy data can be
determined in
appropriate models. In this way, key structure-activity relationships are
obtained for
multi'binding design parameters which are then used to direct future 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 methods as
described above
including conventional assays (both in vitro and in vivo).
Second, ascertaining the st~~ of those compounds which exhibit multibinding
properties can be accomplished via art recognized Procedures. For example,
each member
of the library can be encrypted or tagged ~~ apPi'opriate 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;
Bremner, et
al., Proc. Nail. Acad. Sci., USA, 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.
-79-


CA 02318745 2000-07-24
WO 99/64050
PCT/US99/I2777
2,240,325 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 muldbinding 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.
Based on the information obtained through analysis ~of the initial library,.an
optional
component of the process is to ascertain one or more promising multibinding
"lead"
compounds as defined by particular relative ligand orientations, linker
lengths, linker
geometries, etc. Additional libraries can then be generated around these leads
to provide
for further information regarding structure to activity relationships. These
arrays typically
bear more focused variations in linker structure in an effort to further
optimize target
affinity and/or activity at the target (antagonism, partial agonism, etc.),
and/or alter
physical properties. By iterative redesignlanalysis using the novel principles
of
multibinding design along with classical medicinal chemistry, biochemistry,
and
pharmacology approaches, one is able to prepare and identify optimal
multibmdmg
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,diaznines, diols, mixtures of carboxylic
acids,
sulfonylhalides, aldehydes, keton,es, halides, isocyanates, amines and diols.
In each case,
the carboxylic acid, sulfonylhalide, aldehyde, ketone, halide, isocyanate,
amore and diol
functional graup 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:
-80-


CA 02318745 2000-07-24
WO 99/64050 . PCTNS99/12777
COMP1LEMENTARY BINDING CHEMISTRIES
F31'~I. eaetive ~g . $e: o~d ~aMive Can
hydroxyl isocyanate iu~thane
amine . epoxide p-hydroxyamine
sulfonyl halide amibe . sulfonamide
carboxyl acid amine amide
hydroxyl alkyl/aryl halide ' ether
aldehyde amine/NaCNBH~ canine
ketone amine/NaCNBH4 amine
isocyanate carbamate
Facemplary .linkers inchide the following linkers identified as X-1 through
X1.18 as
set forth below:
20
-81-


CA 02318745 2000-07-24
WO 99/64050 PCT/US99/12777
_
x t xa xr xa
__ _
~S
x_ xs xte x.n x.ts
~~o tn' " ' H° °
-o
r
xlf X-H 7Flf 74ia 7G1 x-11
ND ND p N
_..
x.b X-I1 Xat xif
_
w
xs~ xa~ xa, xas
_
o w
xat xaf xas xar xaf xaa
a -- ~ .
0
o d~~~o
o"b
X.17 xa1 x. ~ X.11 X.11 7611
0
w
X.11 x~11 x.11 XAi X-17 X.41
x.N ' X. x.71 X.7i X-!f X.fs
0
0
0
X.u x.fa xa1 xf1 xso
a ~ ~ .
X~1 X.lt X.if xi1 Xi7 X.i6
~ _ .
i ei
xn xa x.H xx xr xn
o ~ ~s~o H° ~°~ , 01
v w Ip
X.n x-x xr xx xar xn
-82-


CA 02318745 2000-07-24
WO 99/64050 PCT/US99/12777
... _. ..
__ __ _ o
x. X.11 ~ X.11 X.t7 X.11
_ _
X.If 741f X~fl 7411 X-11
-__ QQ1111
I~~o Na off er
I11 ae
X-~ X.11 X,lf ~ ~ X.li
r r r~ ow oS
o'
x,~ x.w xor xlao x.lol ~ x.wn
t 1 t t a
ow ~
t w '
X,yp X~111 X.IIf X~IIf X.iN X.111
0
a
xle1 X.uo x.lu x-lu x.lu x~In
o~ a~
x.us r x.u1 x.ln x.lu X.no x.lm
a ~ __
- ~ o~0 0
t~I~o 0
~' ~°'
X~11! X~l7t a X.lif X.131 X~Il3 X-0S
_ .
eo
X.It1 X~111 x-Im Xdlo X-111 X.lll
Disulfoayl Halide
oa . o,,~s'o
o'I~a d ~a o' ~a d t
xl» x.lx x.l» xlss x.lr xl»
a
a
o a ~ ~ o o~la os a a
1
x.l» xla x.lu ~ X.la x-In x.lw
0.~ o w ~~sp
o I a
0
x.lu d 'r x.lr1 ~ x.la X~Iw x.u1 x.lso
_ - 0.
Gj ~G a~ \~
X.IfI X~ISl
-83-


CA 02318745 2000-07-24
WO 99/61050 PCT/US99/12777
Dialdehydee
lw a
x-m xix x.m wx x-m x.ix
0
at xm xm xui xia x.ia x.ia
~ ._
xia x.~a xm x.iw x.ia x.no
ono .
xrn xin xm ~m
Dihalides
°~°~a W
a
a '~
x.~» xtx x.~n x~~» xt» x.~w
x.ui x.~a xaa xvN xm x.us
rr
~a
x.m ~ xaa ~ ~ x.m xa~o x.~m x.us
,.
xt~ xn4 ~ x.m xax xa~ xiw
ear ~/'~/~/~1 110 Gt ON
Y et
x.~» xaeo xaoi iwm xaa xaw
r~r r r h/~.~r
Ko-o a
r
xms 'xao~ xxo~ xaoe xmr xa~
a a r~
xau xm x~u xau
DiixOCyanata '
-84-


CA 02318745 2000-07-24
WO 99/6~t050 PCTNS99/12777
a
e~w~~°
xu! xau xat~ x as xau xsno
_ a _~~o ~~
0
~II~ ~ °4
x-at x-us xaa xau x-rn xas
__°
e~"~ o o 'o
V
xa>h xm xar xvs xut ~ xm
°
0
7Gb7 x->W 7C~! X~171 ~ Xalf
oo
xa>» xaa xaa xaa w= w xan
o~o ~o
0
)G>w x,11! xalf X,7lt
Dimness .
x.u, xa>e xmt xaa x.s!! ~ xa>,
xass xrn xan xaso
w~.."wv."~a.~ ~w0..n:"S I w~.~.,~ ~»5 .
xae xaa xao xaa xx! x-us
xu! x.>a xae xate x-rn xm
xrn xa~~ xa>s :-s:>s x-m
w
x a» x-no xut 1~-T'1-~"' xaa xaa x-u~i
X.u! X-u' X~u7 ~ X~u1 x-IH X-790
~gs~


CA 02318745 2000-07-24
WO 99/64050 PCTNS99/12777
-~w°.~~ ~~ ~~,~
I,
xan xas: xan xas, xan x-zx
oMO
x xs» xah x-m xam
W~~./w~"4 ~o~°~ ~~ Wow,
xaaa xaer xaes xao~ xaa~r xae,
WWW~r~ ~ , ~
xa" xal xau xa xau xaN
. iy ry . . . . W~
I HP
xais xau xan xau xa~s xaso
ww"~w~,~''oS w~aw.~w ~~~~ ~.~~°4 ~~
xasi xasa xap xau xaa
Diols
Nae ~o~Maw
r w ~ off
I 0N~ off
0
X-7N xri! X-a7° Xaal
,yo ~ °o o"
x~~ xaas A~ ~~ x-»~ xaas xass
wi'~~o~i°v~o~ HoMWa~
r o"
r
x.~, x-a» xao xaa xaa xaa
°" ~a~ ~ °' ro~~aH
Ho a" n°
bH
xsu xxs xa~ xau x
~v°a~..°iv°vyvo" ~owa, °" w r r No oN
~~I''~~//~
_ xaso ~ xan xasa xass xas4 xass
aH
I
ON
QI
i I ~ xa xa>t xa» xsso x-x~
xaa xaa xas~ xau xaa xas~
-86-


CA 02318745 2000-07-24
WO 99/6~t050 PCT/US99/12777
~ ~


xas xan xsn xan



xan xax x xaw
' _.


w~i~--~.-i~ w~~~ w~~a~~~ NoN~a~ . won


p )tall ' Xal1 ~ Xa0 XaM ~ X.ltf



w~ ~/~~ . w~/'~~
p~
w



xa ~ xaa xaso x.t


N~w
~4


x-~ xaa xa~e xan


N~~o . ww~



xa~ xa~ x~oo xa~ xps x.as


w~~~ W /W~~ ~



x.w s xao c . x.~ x
w, o~~
w


xm o Xau xau x.~u xan xau


w / ~ a
. w\~
9


a .
w ,


x.4t s xm x.~ u




CA 02318745 2000-07-24
PCTNS99/12777
Represernative ligands for use in this invention inchide, by way of example, L-
1
through L-4. L-1 ligands are benzofuran compounds (e.g.; 7A-1, 7B-1 or 7C-1 of
Examples 1-3). Phenylmethane sulfonamide structures are designated L-2 ligands
(e.g.,
8A-1, 8B-1, 8C-1, 9A 1, 9B-1, l0A 1, lOB-1, or 11-1 of Fxamples 4-11). L-3
ligands are
azimilide compounds (e.g., 12-1, 12-3 of Examples 12-13). L-4 ligands are
tedisamil
compounds (e.g., 13-1 of Exana~ple 14).
Combinations of ligands {L) and linkers (X) Per this invention include, by way
example only, home- a~ hetero-dimers wherein a first ligand is selected from L-
1 through
L-4. above and the second ligand and linker is selected from the following:
L-1lX-1- L-1IX-2- L-1/X-3- L-1/X-4- L-1!X-5- L-1IX-6-


L-1/X-7- L-1/X-8- L-1IX 9- L-1IX-10- L-1/X-11- L-1/X-12-


L-11X-13- L-1/X-14- L-1/X-15- L-1IX-16- L-1/X-17- L-11X-1&


L-1/X-19- L-1/X-20- L-11X-21- L-1/X-22- L-1/X 23- L-1/X 24-


L-1lX-25- L-1IX-26- L-1/X-27- L-1IX-28- L-1/X-29- L-11X-30-
.


L-1IX-31- L-1lX-32- L-1/X-33- L-1/X-34- L-1IX-35- L-1/X-36-


L-1/X-37- L-1/X-38- L-1/X-39- L-1/X-40- L-1lX-41- L-1/X-42-


L-1/X-43- L-1/X-44- L-1/X-45- L-1/X-46- L-1lX-47- L-1!X-48-


L-1IX-49- L-1IX-50- L-11X-51- , L-i/X-52-L-1lX-53- L-1JX-54-
.


L-1IX-55- L-1!X-56- L-1IX-57- L-1/X-58- L-ilX-59- L-1/X-60-


L-1/X-61- L-11X-62- L-1/X-63- L-1/X-64-~L-1/X-65- L-11X-66-


L-1/X-67- L-1IX-68- L-1IX-69- L-1/X-70- L-1/X-71- L-1IX-72-


L-1IX-73- L-1/X-74- L-1IX 75- L-11X-76- L-1IX 77- L-11X-78-


L-1/X-79- L-1/X-80- L-1IX-81- L-1/X-82- L-1IX-83- L-1/X-84-


L-1/X-85- L-1/X-86- L-1/X-87- L-1/X-88- L-1IX-89- L-1IX-90-
.


L-1/X-91- L-1IX-92- L-1/X-93- ~L-1/X-94-L-1/X-95- L-1IX-96-


L-1/X-97- L-1/X-98- L-1/X-99- L-1IX-100-L-1/X-101-L-1/X-102-


-88-


CA 02318745 2000-07-24
WO 99/b4050 PCTNS99/12777
L-1lX-103- L-1/X-104-L-1/X-105-L-1/X-106-L-1/X-107-L-1/X-108-


L-1/X 109- L-1lX-110-L-1IX-111-L-1/X-112-L-1/X-113-L-1/X-114-


L-1IX-115- L-1/X-llfrL-1lX 117-L-1/X-118-L-1/X-119-L-1/X-120-


L-1IX-121- L-1/X-122-L-1/X-123-L-1/X-124-L-1IX-125-L-I/X-126-


L-I/X-127- L-1/X-128-L-1/X-129-L-1/X-130-L-1/X-131-L-1/X-132-


L-1JX-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-1/X-143-L-1/X-144-
-


L-1/X-145- L-1IX-146-L-1/X-147-L-1/X-148-L-1/X-149-L-1/X-150-


L-1/X-151- L-1/X-I52-L-1/X-153=L-1/X-154-L-1/X-ISS-L-1/X-156-


L-.1/X-1S7-L-1/X-158-L-1/X-159-L-1lX-160-L-1/X-161-L-1/X-162-


L-1/X-163- L-1IX-164-L-1IX-165-L-1/X-166-L-1/X-167-L-1/X-168=


L-1/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-1IX-180-


L-1IX-181- L-1/X-182-L-1/X-183-.L-1/X-184-L-1/X-185-L-1IX-186-


L-1/X-187- L-1IX-188-L-1IX-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-1lX-197-L-1IX-198-


L-1/X-199- L-1/X-200-L-1lX-201-L-1/X-202-L-1/X-203-L-1/X-204-


L-1lX-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-1IX-217- L-1/X-218-L-1/X 219-L-1/X-ZZO-L-1/X-221-L-1/X-222-


L-1/X 223- L-1IX-224-L-1/X-225-L-1/X-226-L-1/X-227-L-1IX-228-
.


.L-1/X-229-L-1/X-230-L-1/X-231-L-1/X-232-L-1/X-233-L-1/X-234j


L-1IX-235- L-1/X-236-L-1/X-237-L-1IX-238-L-1/X-239-L-1IX-240-


L-1/X-241- L-1/X-242-L-1/X-243-L-1/X-244-L-1/X-245-L-1IX-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-1IX-253- L-1/X-254-L-1/X-255-L-1IX-256-L-1/X-257-L-1/X-258-


L-1/X-259- L-1/X-260-L-1/X-261-L-11X-262-L-I/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-


-89-


CA 02318745 2000-07-24
WO 99164050 PCTNS99/12777
L-1/X-271- L-1/X-272-L-1IX 273-L-1/X-274- L-1/X-275-L-1/X-276-


L-1/X-277- L-1/X-278-L-1lX-279-L-1/X-280- L-1/X-281-L-1/X-282-


L-1/X 283- L-1/X-284-L-1IX-285-L-1/X-286- L-1/X-287-L-1/X-288-


L-1/X-289- L-1IX-290-L-1/X 291-L-1/X-292- L-1IX-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-1IX-300-


L-1/X-301- L-1/X-302-L-1/X-303-L-1/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-
w


L-1/X-313- L-1/X-314-L-1IX-315-L-1/X-316- I~1/X-317-L-1/X-318-
.


L-1/X-319- L-1/X-320-L-IlX-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-1IX-337- L-1/X-338-L-IIX-339-L-1/X-340- L-1IX-341-L-1/X-342-


L-1/X-343- L-1/X-344-L-11X-345-L-1/X-346- L-1/X-347-L-1/X-348-


L-1/X-349- L-1/X-350-L-1lX-351-L-1/X-352- L-1/X-353-L-1/X-354-


L-1/X-355- L-1/X-356-L-1IX-357-L-1/X-358- L-11X-359-L-1/X-360-


L-1/X-361- L-1/X-362-L-1/X-3fi3-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-1IX-371-L-1IX-372-


L-1/X-373- L-1/X-374-L-1/X-375-L-1/X-376- L-1/X-377-L-1/X-378-


L-1/X-379- L-1/X-380-L-1IX-381-L-1/X-382- L-1IX-383-L-1/X-384-


L-1/X-385- L-1/X-386-L-1lX-387-L-1/X-388- L-1/X-389-L-1/X-390-


L-1~IX-391-L-1/X-392-L-11X-393-L-1/X-394- L-1/X-395-L-1lX-396-


L-1/X-397- . L-1/X-398-L-1/X-399-L-1/X-400- L-1/X-401-L-1/X-4.02-


L-1/X-403- L-1/X-404-L-1/X-405-L-1/X-406- L-1lX-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-2IX-2-L-2/X-3- L-2/X-4- L-2IX-5- L 2/X-6-


L-2/X-7- L-2/X-8- L-2/X-9- L-2/X-10- L-2IX-11- L-2/X-12-


-90-


CA 02318745 2000-07-24
PCTNS99/12777
WO 99/64050
L-2/X-13- L-2/X-14- L-2/X-15- L-2IX-16- L-2IX-17- L-2IX-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-2IX-25- L-2/X-26- L-Z/X-27- L-2/X-28- L-2/X-29- L-2IX-30-


L-2IX-31- L-2/X-32- L-2/X-33- L-2/X-34- L-ZIX-35- L-2lX-36-
.


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-2IX-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-2IX-60-


L-2/X-61- L-2/X-62- L-2IX-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-2IX-75- L-2/X-76- L-2/X-77- L-2/X-78-


L-2/X-79- L-2/X-80- Ir2/X-81- L-2IX-82- L-2IX-83- L-2IX-84-


L-2lX-85- L-2/X-86- L-2/X-87- , L-2IX-88-L-21X-89- L-2IX-90-


L-2/X-91- L-2/X-92- L-2/X-93- L-2/X-94- L-2/X-95- L-2lX-96-


L-2/X-97- L-2/X-98- L-2/X-99- L-2/X-100- L-2/X-101-L-2lX-102-


L-2/X-103- L-2IX-104-L-2IX-105-L-2/X-106- L-2/X-107-L-2/X-108-


L-2IX-109- L-2IX-110-L-2/X-111-L-2/X-112- L-2/X-113-L-2IX-114-


L-2/X-115- L-2/X-116-L-2/X-117-L-2/X-118- L-2/X-119-L-2IX-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- Lr2/X-128-ir2lX-129=L-2/X-130- L-2/X-131-L-2/X-132-


L-2/X-133- L-2lX-134-L-2/X-135-Ir2lX-136- L-2IX-137-L-2lX-I38-


L-2/X-139- L-2/X-140-L-2lX-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-2IX-149-L-2lX-150-


L-2/X-151- L-2/X-152-L-21X-153-L-2/X-154- L-2/X-155-L-2IX-.156-


L-2/X-157- L-2/X-158-L 2!X-159-L-2/X-160- L-2/X-161-L-2IX-162-


L-2/X-163 L-2/X-164 L-2JX-165 L-2/X-166 L-2/X-167 L-2!X-168


L-2lX-169 L-2lX-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-2IX-180-


-91-


CA 02318745 2000-07-24
WO 99/64050 PCT/US99/12777
L-2/X-181- L-2/X-182-L-2/X-183-L-2/X-184-L-2IX 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-2IX-192-


L-2/X-193- L-2/X-194-L-2/X-195-L-2IX-196-L-2/X-197-L-2/X-198-


L-2/X-199- L-2IX-200-L-2/X-201-L-2IX-202-L-2/X 203-L-2/X-204-
. ~


L-2/X-205- L-Z/X-206-L-2/X-207-L-2/X-208-L-2/X-209-L-2/X-210-


L-2/X-211- L-2/X-212-L-2/X-213-L-2lX-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-21X-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-2IX 242-L-2/X 243-L-2/X-244-L 2IX 245-L-2/X-246-


L-2/X-247- L-2/X-248-L-2/X-249-L-2lX-250-L-2/X-251-L-2lX-252-


L-2IX-253- L-2/X-254-L-2/X-255-L-2IX-256-L-2/X-257-L-2IX 258-


L-2/X-259- L-2/X-260-L-Z/X-261-L-2/X 262-L-2lX 263-L-2IX 264-


L-2/X-265- L-21X-266-L-2/X-267-L-2/X-268-L-2/X-269-L-2/X-270-


L-2IX-271y L-2IX-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-2IX 287-L-2/X-288-


L-2/X-289- L-2/X-290-L-2/X-291-L-2/X-292-L-21X-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-2IX 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-2IX-307- L-2/X-308-L-2lX-309-L-2/X-310-L-2/X-311-L-2/X-312-


Il2/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-2IX-320-L-21X-321-L-2/X-322-~ L-2/X-323-L-2IX-324-


L-2/X-325- L-2/X-326-L-2/X-327-L-2/X-328-L-2/X-329-L-2IX-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-2IX-340-L-2lX-34I-L-2IX-342-


L-2IX-343- L-2/X-344-L-21X-345-L-2/X-346-L-2/X-347-L 2/X-348-


-92-


CA 02318745 2000-07-24
WO 99/64050 PCT/US99/12777
L-2IX-349-L-2/X-350- L-2/X-35I-L-2IX-352- L-2IX-353-L-2IX-354-


L-2/X-355-L-2!X-356- L-2/X-357-L-2IX-358- L-2/X-359-L-2/X-360-


L-2/X-361-L-2/X-362- L-2/X-363-L-21X-364- L-2/X-365-i.-2IX-366-


L-2/X-367-L-2/X-368- L-2/X-369-L-2IX-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-2IX-377-L-2/X-378-


L-2/X-379-L-2/X-380- L-2/X-381-L-2IX-382- L-2/X-383-L-2lX-384-


L-2/X-385-L-2IX-386- L-2/X-387-L-2/X-388- L-2lX-389-L-2/X-390-
w


L-2lX-391-L-2/X-392- L-2IX-393-L-2IX-394- L-2/X-395-L-2/X-396-


L-2IX-397-.L-2/X-398-L-2/X-399-L-2IX-400- L-2/X-401-L-2IX-402-


L-2IX-403-L-2/X-404- L-2/X-405-L-2IX-406- L-2/X-407-L-2/X-408-


L-21X-409-L-2/X-410- L-2/X-411-L-21X-412- L-2IX-413-L-2/X-414-


L-2/X-415-L-21X16- L-2/X-417-L-2/X-418-


L-3IX-1- L-3/X-2- L-3/X-3- L-3IX-4- L-3/X-5- . L-3/X-6-
'


L-3/X-7- L-3/X-8- L-3/X-9- L-3IX-10- L-3/X-11- L-3/X-12-


L-3/X-13- L-3/X-14- L-3/X-15-L-3IX-16- L-3/X-17- L-3/X-18-


L-3IX-19- L-3IX-20- L-3/X-21-L-3IX-22- L-3/X-23- L-3IX-24-


L-3lX-25- L-3/X-26- L-3/X-27-L-3/X-28- L-3/X-29- L-3/X-30-


L-31X-31- L-3/X-32- L-3/X-33-L-3IX-34- L-3/X-35- L-3/X-36-


24 L-3/X-37- L-3/X-38- L-3/X-39-L-3IX-40- L-3/X-41- L-3IX~42-


L-3~/X-43-L-3/X-44- L-3/X-45-L-3IX-46- L-3/X-47- L-31X-48-


L-3IX~9- L-3/X-50- L-3/X-51-L-3IX-52- L-3IX-53- L-3/X-54-


L-3/X-55- L-3/X-56- L-3/X-57-L-3IX-58- L-3/X-59- L-3/X-60-


L-3/X-61- L-3/X-62- L-3/X-63-L-3IX-64- L-3/X-65- L-3/X-66-


L-3IX-67- . Lr3/X-68-L-3/X-69-L-3IX-70- L-3/X-71- L-3IX-72-


L-3IX-73- L-3/X-74- L-3/X-75-L-3/X-76- L-3IX-77- L-3lX-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-3IX-88- ~ L-3/X-89-L-3IX-90-


-93-


CA 02318745 2000-07-24
PCT/US991127~7
L-31X-91- L-3/X-92- L-31X-93= L-3/X-94- L-3IX-95- L-31X-96-
.


L-3IX-97- L-3IX-98- L-3/X-99- L-3/X-100-L-3/X-101-L-3lX-102-


L-3/X-103- L-3/X-104-L-3/X-105-L-3/X-106-L-3/X-107-L-3/X-108-
.


L-3IX-109- L-31X-110-L-3/X-111-L-31X-112-L-3/X-113-L-3/X-114-


L-3/X-115- L-3/X-116-L-3IX-117-L-3/X-118-L-3/X-119-L-3IX-120-


L-3/X-121- L-3/X-122-L-3/X-123-L-3/X-124-L-3/X-125-L-3/X-126-
. .


L-3/X-127.-L-3/X-128-L-3lX-129-L-3/X-130-L-3IX-131-L-3/X-132-
w


L-3/X-133- L-3/X-134-L-3/X-135-L-3/X-136-L-3IX-137-L-3/X-138-


L-3/X-139- L-3/X-140-L-3/X-141-L-3/X-142-L-3IX-143-L-3/X-144-


L-3/X-145- L-3IX-146-L-3/X-147-L-3/X-148-L-3IX-149-L-3/X-150-


L-31X-151- L-3/X- 152-L-3/X-153-L-3JX-154-L-31X-155-L-3IX-156-


L-3/X-157- L-3IX-158-L-3/X-159-L-3IX-160-L-3/X-161-L-3IX-162-


L-3/X-163- L-3/X-164-L-3/X-165-L-3/X-166-L-3IX-167-L-3/X-168-


L-3/X-169- L-3IX-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-3lX-1?9-L-3lX-180-


L-3/X-181- I:3/X-182-L-3IX-183-L-3IX-184-L-3IX-185-L-3/X-186-


L-3/X-187- L-3/X-188-L-31X-189-L-3IX-190-L-3IX-191-L-3/X-192-


L-3/X-193- L-3/X-194-L-3/X-195-L-3/X-196-L-3/X-197-L-3/X-198-
~


L-3/X-199- L-3/X-200-L-3/X-201-L-3/X-202-L-3lX-203-L-3/X-204-
~


L-3/X-205- L-3IX-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-3IX-213-L-3/X-214-L-3IX-215-L-3/X-216-


L-3IX-217- L-3/X-218-L-3/X 219-L-3/X-220-L-3IX-221-L-3IX-222-


L-3/X-223- L-3/X-224-L-3lX-225-L-3/X-226-L-3/X-227-L-3lX-228-


L-3IX-229- L-3/X-230-L-3IX-231-L-3/X-232-L-3/X-233-L-3/X-234-


L-3JX-235- L-31X-236-L-3/X-237-L-3/X-238-L-3/X-239-L-31X-240-


L-3IX-241- L-3IX-242-L-3IX-243-L-3/X-244-L-3/X-245-L-3/X 246-


L-3/X-247- Ir3/X-248-L-3/X-249-L-3/X-250-L-3/X-251-L-31X 252-


L-3IX-253- L-3/X-254-L-3IX-255-L-3/X-256-L-3/X-257-L-3IX-258-.


-94-


CA 02318745 2000-07-24
WO 99/64050 PCT/US99/12777
L-3/X-2S9- L-3/X-260-L-3IX-261-L-3/X-262-L-3/X-263-L-3IX-264-


L-31X-26S- L-3/X-266-L-3IX-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-27S-L-3/X-276-


L-3/X-277- L-3/X-278-L-3/X 279-L-3lX-280-L-3IX-281-L-3/X-282-
.


S L-3/X-283- L-3/X-284-L-3/X-28S-L-3/X-286-L-3IX-287-L-3IX-288-


L-3JX-289- L-3/X-290-L-3IX-291-L-31X-292-L-3/X-293-L-3lX-294-
.


L-3IX-29S- L-3/X-296-L-3IX-297-L-3/X-298-L-3/X-299-L-3/X-300-
~-


L-31X-301- L-3/X-302-L-3IX-303-L-3/X-304-L-3/X-30S-L-3IX-306-


L-3IX-307- L-3/X-308-L-3/X-309-L-3/X-310-L-3/X-311-L-3/X-312-
.


L 3lX-313- L-3/X-314-L-3IX-31S-L-3IX-316-L-3/X-317-L-3/X-318-


L-31X-319- L-3/X-320-L-3IX-321-L-3/X-322-L-31X-323-L-3/X-324-


L-3lX-32S- L-3/X-326-L-3/X-327-L-3IX-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-3IX-33S-L-3/X-336-


L-3lX-337- L-3/X-338-L-3/X-339-L-3/X-340-L-3/X-341-L-3IX-342-


1S L-31X-343- L-3/X-344-L-31X-34S-L-3/X-346-L-3/X-347-L-3/X-348-
.


L-3lX-349- L-3/X-3S0-L-31X-3S1-L-3/X-3S2-L-3/X-3S3-L-3/X-3S4-


L-31X-3SS- . L-3/X-3S6-L-3/X-3S7-L-3/X-358-L-3/X-3S9-L-3/X-360-


L-3/X-361- L-3/X-362-L-3IX-363-L-3/X-364-L-31X-36S-L-3/X-366-


L-31X-367- L-3/X-368-L-3/X-369-L-3/X-370-L-3IX-371-L-3IX-372-


L-3/X-373- L-3/X-374-L-3/X-37S-L-3/X-376-L-3IX-377-L-31X-378-


L-3~IX-379-L-3/X-380-L-3/X-381-L-3/X-382-L-3/X-383-L-3IX-384-


L-3/X-38S- 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-39S-L-3/X-396-


L-31X-397- L-3IX-398-L-3/X-399-L-3/X-400-L-3/X-401-L-3/X-402-


2S L-3IX-403- L-3/X-404-L-3/X-40S-L-3/X-406-L-3/X-407-L-31X-408-


L-31X-4.09-L-3/X-410-L-31X-411-L-3/X-412-L-3/X-413-L-3/X-414-


L-3/X-4.15-L-3lX-416-L-3/X-417-L-3IX-418-


-95-


CA 02318745 2000-07-24
WO 99/64050 PCT/US99/12777
L-4/X-1- L-4lX-2- L-4/X-3- L-4/X-4- L-4IX-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-4lX-12-


L-4/X-13- L-4/X-14- L-4/X-15- L-4/X-16- L.4/X-17-L-4/X-I8-


L-4/X-19- L-4./X L-4IX-21- L-4/X-22- L-4/X L-4/X-24-
20- 23-


L-4/X-25- L-4IX-26- L-4/X-27- L-4/X-28- L-4/X L-4IX-30-
29-


L-4/X-31- L~/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.IX~0- L-4/X-41-L-4/X-42-
.~


L-4/X-43- L-4./X-44-L-4IX-45- L-4./X-46- L-4/X-47-L-4.IX-48-


L-4IX-49- L-4./X-50-.L-4./X-51-L-4/X-52- L-4.IX-53-L-4/X-54-


L-4IX-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-4IX-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 L-4lX-72-
71-


L-4/X-73- L-4/X-74- L-4IX-75- L~/X-76- L-4/X-77-L-4IX-78-


L-4/X-79- L~L/X-80- L-4./X L-4/X-82- L-4/X-83-L-4IX-84-
81-


L-4/X-85- L-4/X-86- L-4.IX-87-L-4/X-88- L-4/X-89-L-4IX-90-


L-4/X-9I- L-4/X-92- L-4IX-93- L-4IX-94- L-4/X-95-L-4./X-96-


L-4/X-97- L-4/X-98- L-4lX-99- L-4/X-100- L-4/X-101-L-4/X-102-


L-4/X-103- L-4/X-104-L-4/X-105-L~4/X-106- L-4lX-107-L-41X-108-


L-4/X-109- L-4/X-110-~L-4/X-111-L~/X-112- L-4/X-113-L-41X-114-


L-4/X-115- L-4/X-116-L-4/X-117-L-4/X-118- L-4IX-119-L-4IX-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-4IX-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-4IX-136- L-~/X-137-L-4/X-138-


L-4/X-139- L-4./X-140-L-4/X-141-L-4./X-142-L-4IX-143-L-4./X-144-


L-4.I X-145=L-4/X-146-L-4IX-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.IX-156-


L-4lX-157- L-4/X-158-L-4./X-159-L-4/X-160- L-4IX-161-L-4/X-162-


-96-


CA 02318745 2000-07-24
WO 99/64050 PCTNS99/12777
L-.4IX-163 L-4/X-164 L-4/X-165 L-4IX-166 L-4/X-167 L-4/X-168


L-4/X-169 L-4./X-170L-41X-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-4IX-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-4IX-201-L-4/X-202-L-4/X-203-L-4/X-204-
w


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-4fX-212-L-4/X-213-L-4/X-214-L-4/X-215-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-225-L-4/X-226-L-4/X-22?-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-235- L-4/X-236-L-4/X-237-L-4/X-238-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-245-L-4/X-246-


L-4/X-247- L-4/X-248-L-4/X-249-L-4/X-250-L-4/X-251-L-4/X-252-


L-4/X-253- L-4/X-254-L-4/X-255-L-4/X-256-L-4/X-257-L-4/X-258-


L-4/X-259- L-4/X-260-L-4/X-261-L-4/X-262-L-4/X-263-L-4/X-264-


L-4/X-265- 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-275-L-4/X-276-


L-4/X-277- L-4/X-278-L-4/X-279-L-4IX-280-L-4/X-281-L-4/X-282-


L-4/X-283- L-4/X 284-L-4/X-285-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-295- L-4/X-296-L-4/X-297-L-4/X-298-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-305-L-4/X 306-


L-4/X-307- L-4/X-308-L-4/X-309-L-4/X-310-L-4/X-31.1-L-4/X-312-


L-4/X-313- L-4/X-314-L-4/X-315-L-4/X-3-l L-4/X-317-L-4/X-318-
b-


L-4/X-319- L-4/X-320-L-4/X-321-L-4IX-322-L-4/X-323-L-4/X-324-


L-4/X-325- 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-335-L-4/X-336-


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CA 02318745 2000-07-24
WO 99/64050 PGT/US99/12777
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-3.45-L-4./X-346-L-4/X-347-L-4/X-348-


L-4/X-349- .L-4/X-350-L-4/X-351-L-4lX-352- L-4/X-353-L-4!X
354-


L-4/X-355- L-4/X-356-L-4/X-357-L-4/X-358- L-4/X-359-L-4/X-360-


L-4/X-361- L-4/X-362-L-4/X-363-L-4IX-364- L-4IX-365-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-375-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-4IX-382- L-4/X-383-L-4/X-384-


L-4/X-385- L-4!X-386-L-4/X-387-L-4/X-388- . 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-4JX-399-L-4/X-400- L-4/X-40I-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-4.18-


E~u~s
When employed as pharmaceuticals, the compounds of Formula I are usually
administered in the form of pharmaceutical compositions. This invention
therefore provides
pharmaceutical compositions which contain, as the active ingredient, one or
more of the
compounds of Formula I above or a pharmaceutically acceptable salt thereof and
one or more
pharmaceutically acceptable excipients, carriers, diluents, permeation,
enhancers, solubilizers
and adjuvants. The compounds may be administered alone or in combination with
other
therapcutic agents (e.g., other antihypertensive drugs, diuretics and the
like). Such
compositions are prepared in a manner well known in the pharmaceutical art
(see, e.g.,
Remington's Pharm. Sci., Mack Publishing Co., Philadelphia, PA, 17'~ Ed.
(1985) and
"Modern Pharm. ", Mattel Dekker, Inc., 3'~ Ed. (G.S. Banker & C.T. Rhodes,
Eds.).
The compounds of Formula I may be administered by any of the accepted modes of
administration of agents having similar utilities, for example, by oral,
parenteraZ, rectal,
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WO 99/f4050 PCT/US99/12777
buccal, intranasai or transderma~l routes. The most suitable route will depend
on the nature
and severity of the condition being treated. Oral administration is a
preferred route for the
compounds of this. invention. In making the compositions of this invention,
the active
ingredient is usually diluted by an excipient or enclosed within such a
carrier which can be in
the form of a capsule, sachet, paper or other container. When the excipient
serves as a
diiuent, it can be a solid, semi-solid, or liquid material, which acts as a
vehicle, carrier or
medium for the active ingreclient. Thus, the compositions can be in the form
of tablets, pills,
powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions,
sohrtions, syrups,
aerosols (as a solid or in a liquid medium), ointments containing, for
example, up to 10% by
weight of the active compound, soft and hard gelatin capsules, suppositories,
sterile injectable
solutions, and sterile packaged powders. Pharmaceutically acceptable salts of
the active
agents may be prepared using standard procedures known to those skilled in the
art of
synthetic organic chcmist<y and described, e.g., by J. March, Advanced Organic
Chem.
Reactions, Mechanisms and Structure, 4"' Ed. (N.Y.: Whey-Interscience, 1992).
Some examples of suitable excipients include lactose, dextrose, sucrose,
sorbitol,
mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth,
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 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. Controlled release drug delivery systems for oral
administration
include osmotic pump systems and dissolutional systems containing polymer-
coated
reservoirs or drug-polymer matrix foanulations. Examples of controlled release
systems are
given in U.S. Patent Nos. 3,845,770; 4,326,525; 4,902514; and 5,616,345.
Another preferred
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CA 02318745 2000-07-24
WO 99164050 PCTNS99/12777
formulation for use in the methods of the present invention employs
traasdcrmal 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. PatentNos. 5,023,252; 4,992,4.45 and
5,001,139. Such
patches may be constructed for continuous, pulsatile, or on demand delivery of
.
pharmaceutical agents.
The compositions are preferably formulated in a unit dosage form. 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 pmduce the desired therapeutic erect, in association with a
suitable
pharmaceutical excipient (e.g., a tablet, capsule, ampoule). The active
compound is effective
over a wide dosage range and is generally administered in a pharmaceutically
effective
amount. Preferably, for oral administration, each dosage unit contains from 1-
250 mg of a
compound of Formula I, and for parenteral administration, preferably from 0.1
to 60 mg of a
compound of Formula I or a pharmaceutically acceptable salt thereof. 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 be treated, the
chosen ioute of administration, the actual compound administered and its
relative activity, the
age, weight, and response of the individual patient, the seventy of the
patient's symptoms, and
the like.
For preparing solid compositions such as tablets, the principal active
ingredient is
mixed with a pharmaceutical excipient to form a solid preformulation
composition containing
a homogeneous mixture of a compound of the present invention. When referring
to these
preformulation compositions as homogeneous, it is meant that the active
ingredient is
dispersed evenly throughout the composition so that the composition may be
readily
subdivided into equally effective unit dosage forms such as tablets, pills and
capsules.
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WO 99/64050 PCT/US99/12777
The tablets or pills of the present invention may be coated or otherwise
compounded
to pmvide 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 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.
i0
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, or peanut oil, as well as
elixirs and similar
pharmaceutical vehicles.
Compositions for inhalation or insuftlation 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
acceptable excipients
as described supra. Preferably the compositions are administered by the oral
or nasal
respiratory mute 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 nebuiizing device or the nebulizing device may be
attached to a face
mask tent, or inten~rittent 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
. compositions of the present invention.
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WO 99/64050 PCfNS99/12777
Hard gelatin capsules containing the following ingredients are prepared:
. Q~antitY
In~i~nt SU~l
Active Ingredient 30.0
Starch 305.0
Magnesium stearate 5.0
~ The above ingredients are mixed and filled into hard gelatin capsules in 340
mg
quantities.
En~xam~l~~
A tablet formula is prepared using the ingredients below:
Quantity


Active Ingredieat 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.
F~rmul~tiQn.Ex~mgl~
A dry powder inhaler formulation is prepared containing the following
components:
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WO 99/64050 PGT/US99/12'f77
Active Ingredient
S The active ingredient is mixed with the lactose and the mixture is added to
a dry
powder inhaling appliance.
~y~,]~~pile 4
Tablets, each containing 30 mg of active ingredient, are prepared as follows:
Quantity
Lmgl~l~
Active Ingredient 30.0
Starch 45.0
Microcrystalline cellulose 35.0
Polyvinylpyrrolidone (as 10% solution in sterile water) 4.0
Sodium carboxymethyl starch 4.5
0.5
Talc 1.0
Total 120.0
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°C 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:
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WO 99/bA050 PCTNS99/12777
Capsules, each containing 40 mg of medicament are made as follows:
Q~t3'


Intent


Active Ingredient ~ 40.0


Starch 109.0


Magnesium steaxate 1.0


Total 150.0


The active ingredient, starch, and magnesium stearate are blended, passed
thmugh a
No. 20 mesh U.S. sieve, and filled into hard gelatin capsules in 150 mg
quantities.
E~laam~l~.~
Suppositories, each containing 25 mg of active ingredient are made as follows:
Active Ingredient 25.0 mg
Saturated fatty acid glycerides to 2,000.0 mg
The active ingredient is passed through a No. 60 mesh U.S. sieve and suspended
in the
saxurated 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.
E~rmui~~o,~xam»1~Z
Suspensions, each containing 50 mg of medicament per 5.0 mL dose are made as
follows:
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WO 99/64050 PCTNS99/12777
Active Ingredient 50.0 mg


~~ g~ 4.0 mg


Sodium carboxymethyl cellulose (11%)


Micmcrystalline cellulose (89%) 50.0 mg


Sucrose 1.75 g


Sodium benzoate . 10.0 mg


Flavor and Color qv.


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.
Q~t3'
~g,/c~p~s~el
Active Ingredient 15.0 mg
Starch 407.0 mg
3.0 mg
Total 425.0 mg
The active ingredient, starch, and magnesium stearate are blended, passed
through a
No. 20 mesh U.S. sieve, and filled into hard gelatin capsules in 425.0 mg
quantities.
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WO 99/6A050 PCTNS99/12777
A subcutaneous formulation may be prepared as follows:
Active Ingredient 5.0 mg
~rn pd 1.0 mL
Frequently, it will be desirable or necessary to introduce the pharmaceutical
composition to the brain, either directly or indirectly. Direct techniques
usually involve
placement of a drug delivery catheter into the host's ventricular system to
bypass the
blood-brain barrier. One such implantable delivery system used for the
transport of biological
factors to specific anatomical regions of the body is described in U.S. Patent
5,011,472 which
is herein incorporaxed by reference.
Indirect techniques, which are genaally preferred, usually involve formulating
the
compositions to provide for drug latentiation by the conversion of hydrophilic
drugs into
lipid-soluble drugs. l..atentiation is generally achieved through blocking of
the hydroxy,
carbonyl, sulfate, and primary amine groups present on the drug to render the
drug more lipid
soluble and amenable to transportation across the blood-brain barrier.
Alternatively, the
delivery of hydrophilic dnigs may be enhanced by infra arterial infusion of
hypertonic
solutions which can transiently open the blood-brain barrier.
Example 1. (Figure 7A)
Preparation of N,N'-dimethyl-N,N'-di-[2-[4-[2-butyl-3-benzofaranylcarbonyl]-
2,6-
diiodophenory]ethyl]hezane, (7A-2), in which n~l, and Link is (CH~6.
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WO 99/6~t050 PCT/US99/127~7
A. A solution of 3-[(2-bromoethoacy)-3,5-diodobenzoyl]-2-butylbenzofuiaa (7A
1), prepared as described in Eia: J. Med Chem., 1974, 19-25, and in Belgian
Patent 900138,
(2 mmol), diisopropylethylamine (5 mmol) and 1,6-di-{methylamino~exane (1
mmol) in
acetonitrile (25mL) is maintained at room temperature, and the reaction is
monitored by thin
layer chromatography (tlc). When it is complete, the solution is added to
water and extracted
with CH2ClZ. The extract is dried and evaporated, and the residue is
chromatographed to
afford the title compound 7A-2.
B. In a similar manner, by employing different diami~es in place of 1,6-di-
(aiethylamino)hexane, as described herein, in A above, different compounds of
Formula 7A-2
are obtained.
C. In similar manner, by employing different bromo compounds of Formula. 7A-
1, as described herein, in A above, different compounds of Formula 7A-2 are
obtained
Example 2. {Figure 7B) '
Preparation of 1,8-di-[2-[4-[2-butylbenzofuran-3-yicarbonyl]-2,6-
diiodophenory]ethyl]methylamino]-3,5-diozaoctane, 7B-2, in which n is 1, and
Link is
(~~z(~(~x~s~
A. A solution of N-methyl 2-[4-[2-butylbenzofuran 3-ylcarbonyl]-2,6-
diiodophenoxy]-ethylamine (7B-1), prepared according to procedures described
in Eur: J.
Med Chem., 1974,19-25, (5 mmol),1,8-dibromo-3,5-dioxaoctane (2.5 mmol) and
diisopropylethylamine (2mL) in EtOH (25mL) is maintained at room temperature.
The
~ progress of the reaction is followed by tlc. When it is complete, the
mixture is poured into
water and extracted with CH2CIz. The extract is dried and evaporated, and the
residue is
chromatographed to afford the title compound 7B-2, in which n is 1 and Link is
(CH~s(O(CH~.
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CA 02318745 2000-07-24
wo ~is~ooso rcrms~nz~~~
B. ~ In a similar meaner, by employing diffaeat compounds 7B-1, as described
herein, different linked compounds of Formula 7B-2 are obi.
C. in a similar memner, by employing different linking compounds, as descxibed
herein, in place of 1,8-dibromo-3,5-dioxaoctane, different linked compo~mds of
Formula 7B-
2 are obtained.
Example 3. (Figure 7C)
Preparation of 1,10-di-[2-butyl-[3-[4-(3-
dibuh'laminoProPo=3')benzoyljbenzofaran-5-
yl]aminosulfonyl]decane, 7C-2, in which Link is ((CH~1~.
A. 5 Amino-2 butyl-3-[4-(3-dibutylaminopropoxy)ben~yl]beu~ofuran (7C-1),
prepared as described in EP 0471609, (1 mmol) and 1,10-di(chlorosulfonyle (0.5
mmol) are heated at reflex in CHiCII (20mL). The progress of the reaction is
followed by tlc.
1 S ~Vhea it is complete, the solution is added to dilute NazC03 : The organic
phase is sepa~cated,
dried and evaporated, and the residue is chromatographed to a$ord the title
compound 7C 2.
B. In a similar manner, by employing diffemat di-(chlorosulfonyl) linking
~po~, as described heroin, different linked compounds of Formula 7C-2 are
obtained
Example 4. (Figure 8A)
Preparation of 1,6-di[4-[2-[2-['t-(met6~'laulfonylamino)phenosy]-
ethylmethylamino]ethyl]phanylamioosalfonyl]hezane SA 2, in which Link is
(C>8~,.
A. Z-[4-(MethylsulfonYlamino~nOxyl~hYl 21-1, (10 mmol) and N-
methyl 2-(4-nitrophen3rl)ethylamine, Zl-2. (10 mmol) both prepped as described
in J. Med
Cham.,1990,1151, art heated at reflex in MeCN (100mL) containing ICzCO~ (3g)
and KI
(0.2g). The reaction is monitored by tlc. Whm it is complete, the elution is
added to water
and with EtOAc. Tlie eacttact is dried and evaporated and the residue is
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CA 02318745 2000-07-24
WO 99/64050 PCTNS99/12777
chromatogiaphed to afford N-methyl N-(4-nitrophenylethyl) 2-[4-
(methylsulfonylamino)phenoxy]ethylamine (21-3).
B. The above compound (3mmol) is dissolved is EtOH (50mL) and ltaney nickel
(1 g) is added. The miUChwe is stirred in a hydrogen atmosphere. The progress
of the reaction
is monitored by tic. When it is complete, the solution is filtered and then
evaporated. The
residue is chromatographed to afford N-methyl N-(4-aminophenylethyl) 2-[4-
(methylsulfonylamino~henoxy]ethylamine, 8A 1.
C. A solution of hexane-1,6-disulfonyl chloride (1 mmol),
diisopropylethylamine
(1mL) and 8A-1 (0.5 mmol) in dry CI~CIz (25mL) is maintained at room
temperature. The
progress of the reaction is monitored by tlc. When it is complete, the solvent
is removed
under reduced pressure and the residue is chromatographed to afford the title
compound 8A-
2, in which Link is (CH~~.
D. In a similar manner, by employing different di(chlorosulfonyl) linking
compounds, as described herein, in C above, different linked compounds SA-2
are prepared.
Facample 5. (Figure 8B)
Preparation of 1,4-di[-4-[2-methyl-2-[4-
(methylsnlfonylamino)phenyl]ethylamino]-
ethoryJphenyl]aminosulfonylmethyl]benzene, 8B-2, where Link is p-CHzC6H,CH=.
A. 2-(4-Nitrophenoxy)ethyl bmmide, 21-4, (10 mmol) and 2-{4-
methylsulfonylaminophenyl) N-methylethylamine 21-5 (10 mmol) are heated at
reflex in
MeCN (50mL) containing K,1C03 (2g). The progress of the reaction is monitored
by tlc.
When it is complete, the solution is added to watei and eactracted with EtOAc.
The extract is
dried and evaporated and the residue is chromatographed to afford N-methyl N-
(4-
(methylsulfonyl-amino~henylethyl) 2-(4-aminophenoxy)ethylamine 8B-1.
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CA 02318745 2000-07-24
WO 99/64050 PC"T/US99/12777
B. The above compound 8B-1, (I mmol) and 1,4-di-
(chlorosulfonylmcthyl)benzene (0.5 mmol) are dissolved in CI~C12 (50 mL). The
progress of
the reaction is monitored by tlc. QVhen it is complete,.the solution is washed
with dilute
NazC03, then dried and evaporated. The residue is chromatographed to afford 8B-
2, is which
Link is p-CHzC6H,,CH2.
C. In a similar ~aaner, by employing different di(sulfonyl chloride) linking
compounds, as described herein, in B above, different linked compounds 8B-2
are prepared
~ Example 6. (Figure 8C)
Preparation of 1,10-di [2-(4-(methylsulfonylaminophenory)ethyl] 2-[4-
[methylsulfonylaminophenyljethyljaminojdecane, 8C-2, in which Link is (CH~1~.
A. N-Benzyl 2-[4- (methylsulfonylaminophenyl)]ethylamine, (21-7 prepared as
described in EP 338793), {10 mmol), 2-[4-(methylsulfonylaminophenoxy)ethyl
bromide 21-8
(10 mmol) and ICzC03 (1 g) are heated at reflux in MeCN {50 mL). The progress
of the
reaction is monitored by tlc. When it is complete, the solution is added to
water and extracted
with EtOAc. The extract is dried and evaporated, and the residue is
chromatographed to
afford N-benzyl N-2-(4-methylsulfonylaminophenoxy)ethyl 2-(4-
methylsulfonylaminophenyl)ethylamine, 21-9..
B. The compound 21-9 (1 mmol) is dissolved in EtOH (20 mL) and ammonium
formats (100 mg) and 10% Pd/C (50 mg) are added. The progress of the reaction
is
monitored by tlc. When it is complete, the solution is filtered then added to
water and
extracted with EtOAc. The extract is dried and evaporated, and the residue is
chromatographed to afford N-[2-(4-methylsulfonylaminophenoxy)ethyl] 2-(4-
methylsulfonylaminophenyl)-ethylamine, 8C-1.
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CA 02318745 2000-07-24
WO 99164050 PGTNS99/12777
C. The above compound (lmmol), 1,10-dibromodecane (0.5 mmol), K2C03 (lg)
and KI (0.05g) are heated at reflex in MeCN. The progress of the reaction is
monitored by
tlc. When it is complete, the solution is addod to water and extracted with
EtOAc. The
extract is dried and evaporated. The residue is chmmatographed to afford the
title compound
8C-2, in which Link is (CH~,a,
D. ' In a similar manner, by employing different dialkylating agents, as
described
herein, in place of 1,10-dibmmodecane, in C above, different compounds of
Formula 8C-2
are obtained.
Example 7. (Figure 9A)
Preparation of 1,8-di-[4-[4-(ethylheptylamino)-1-hydrorybntyl]phenylamino-
snlfonyl]octane, 9A-2, in which Link is (CH~6.
A. 4-Nitrophenyl-4-oxobutanoic acid, 21-10, prepared as described in Gaza
Chim. Ital., 1967, 97, 654, and U.S. Patent 5,405,851 (10 mmol),1-
hydroxybenztciazole (10
mmol) and dicyclohexylcarbodiimide (10 mmol) are dissolved in DMF (50mL). To
the
solution is added ethylheptylamine (10 mmol). The progress of the reaction is
monitored by
tlc. When it is complete, the solution is added to water and extr~a~cted with
EtOAc. The
extract is dried and evaporated, and the residue is chromatographed to afford
N-ethyl N-
heptyl 4-{4-nitrophenyl)-4-oxobutanamide, 21-11.
B. The above prepared compound (2 mmol) is dissolved in dry diglyme (20mL)
and MeOH {1mL). Lithium borohydride (25 mmol) is added and the solution is
heated to
reflex. The progress of the reaction is monitored by tlc. When it is complete,
the solution'is
added to water and extracted with EtOAc. The e~ctract is dried and evaporated
and the residue
is chromatographed to afford 4-[1-hydroxy-4{ethylheptylamino)butyl]aniline 9A-
1.
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C. The compound 9A-1 (1 mmol) and octane-1,8-disuZfonyl chloride (0.5 mmol)
are dissolved in CHiCiz (25mL). The progress of the reaction is monitored by
dc. When it is
complete, the solution is added to dilute Na2C03, and then extracted with
EtOAc. The extract
is dried and evaporated, and the residue is chromatographed to afford the
title compound 9A-
2.,_ in which Link is (CH~6.
D. In a similar manner, by employing different disulfonyl chlorides, as
described
herein, in C above, different compounds of Formula 9A-2 are obtained.
Example 8. (Figure 9B)
Preparation of 1,6-di-[4-[4-hydrory-4-(methylsulfonylaminophenynbniylJ-
ethylamino]he=ave 9B-2, in which Link is (CH~6.
A. 4-(4-Aminophenyl)-4-oxobutanoic acid 21-12, (5 mmol) is added to a solution
of dicyclohexylcarbodiimide (5 mmol) and ethylamine (5 mmol) in THF (SOmL).
After 12
hours, the mixtine is added to crater and extracted with EtOAc. The extract is
washed with
dilute NaOH, then dried and evaporated. The residue is chromatographed to
afford N-ethyl 4-
(4-aminophenyl)-4-oxobutanamide, 21-13.
B. The product from A above '(3 maiol) is dissolved in THF (25mL) and to the
solution is added diisopropylethylamine (5 mmol) and inethanesulfonyl chloride
(3 mmol).
The reaction is monitored by tlc. When it is complete, the solution is addod
to water and
extracted with EtOAc. The extract is dried and evaporated and the residue is
chromatographed to afford N-ethyl 4-(4-methylsulfonylaminophenyl)-4-
oxobvrtanaanide, 21-
14.
C. The above-described compound (1 mmol) is dissolved in ether (SOmL); the
solution is cooled to 0°C, and to it is added lithium aluminum hydride
(5 mmol). The
reaction is monitored by tlc. When it is complete, excess hydride is
decomposed by addition
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of aqueous potassium sodium tarGrate. The organic phase is separated, dried
and evaporated,
and the residue is chromatographed to afford N-ethyl 4-(4-
methylsulfonylaminophenyl)-4-
hydmxybutylamine, 9B-1:
S . D. The compound 9B-1 (lmmol), diisopropylethylamine (2 mmol) and 1,6-
dibromohexane (0.5 mmol) are dissolved in MeCN (2S mL). The progress of the
reaction is
monitored by tlc. When it is complete, the mixture is added to dilute NazC03,
and extracted
with CHZCIz. The extract is dried and evaporated, and the residue is
ehromatographed to
afford the title compouad 9B-2, in which Link is (CH~6.
E. In a similar manner, by employing different dialkylating agents, as
described
herein, in place of 1,6-dibromohexane, different compounds of Formula 9B-2 are
obtained.
Example 9. (Figure l0A)
1S Preparation of 1,8-di-[4-[2-[diethylaminoethyi]aminocarbonyl]phenylamino-
sulfonyl]octane, l0A-2, in which Link is (CH~6.
A. Procaine amide (l0A-1) (10 mmol) is dissolved in MeCN (SO mL) and 1;8-di-
(chlorosulfonyl)octane (S mmol) is added. The progress of the reaction is
monitored by tlc.
When it is complete, the solution is added to dilute NalC03 and extracted with
CH2Ch. The
extract is dried and evaporated to afford the title compound, l0A-2, in which
Link is (CH2)s.
B. In a similar manner, by employing different disulfonyl chlorides, as
described
herein, different compounds of Formula l0A-2 are obtained.
2S
Example 10. (Figure 10B)
Preparation of 1,8-df-[N-ethyl N'-[2-[4-
[methylsulfonylamino]benzoylaminoethyl]-
amino] 3,5-diozaoctane,10B-2, in which Link is (CH~(O(CH~=.
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A. N-Ethyl N'-(4-methylsulfonylaminobenzoyl~thylenediamine, (lOB-1),
prepared as described in J. Med Chem.,1987, 30, 755, (5 mmol)
diisopropylethyiamine (5
mmol) and 1,8-dibromo-3,5-dioxaoctane (2.5 mmol) are dissolved in MeCN (25
mL): The
progress of the reaction is monitored by tlc. When it is complete, the
solution is added to
dilute NazC03 and exh~acted with CHZCh. The solution is dried and evaporated,
and the
residue is chromatographed to afford the title compound 108-2, in which Link
is
(CH~(O(CH~-
B. In a similar manaar, by employing different dialkylating agents, as
described
herein, different compounds of Formula lOB-2 are obtained.
Example 11. (Figure 11)
Preparation of 1,10-di-[2-hydro~cy-2-[4-methylsnlfonylaminophenyl]ethylamino]-
decane,
11-2, in which Link is {CHyo.
A. 1,10-Dibromodecane (Smmol), 2-hydroxy-2-(4-methylsulfonylaminophenyl)-
ethylamine (11-1) (10 mmol), prepared as described in European Patent 338?93,
potassium
iodide (O.lg) and K.~C03 {lg) are stirred in MeCN (SOmL). The reaction is
monitored by tlc.
When it is complete, the mixtiue is added to water and extracted with CH2C1~,
The extract is
dried and evaporated, and the residue is chromatographed to afford the title
compound 11-2,
in which Link is (CI~,o.
B. In a similar manner, by employing different dialkylating agents, as
described
herein, different compounds of Formula 11-2 are obtained:
Example 12. (Figure 12)
Preparation of l,b-di[4-[1-[[5-(4-chlorophenyl)-2-furanylmethylene]amino]-
imidazolidine-2,4-dion 3-yl]bntylmethylamino]hezane,12-2, where Link is (CH~6.
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A. 1-Benzylamino-3-(4-iodobutyl)imidazolidine-2,4-dione (12-S~, prepared as
described in W093/04061, (5 mmol) is added to a solution of methylamine (2g)
in MeOH
(40mL). The progress of the reaction is monitored by tlc. When it is complete,
the mixture is
added to water and e~cttacted with CHZC12. The extract is dried and evaporated
and the
residue is chromaxographed to afford 1-benzylamino-3-(4-
methylaminobutyl)imidazolin: 2,4-
dione,12-6.
B. The latter compound 12-6, (2 mmol) is added to EtOH (25mL) containing i 0%
Pd/C (54mg) and ammonium formats (O.Sg). The progress of the reaction is
followed by tlc.
When it is complete, the solution is filtered and added to water. The aqueous
solution is
extracted with EtOAc. The extract is dried and evaporated. The residue is
chromatographed
to afford 1-amino-3-(4-methylaminobutyl)imidazolidine-2,4-dione,12-7.
C. The above-described compound (1 mmol) is dissolved in EtOH (20mL). To
the solution is added 5-(4-chloropheayl)furan 2-carboxaldehyde (12-$), (1
mmol) and p-
toluenesulfonic acid (1 Omg). The progress of the reaction is followed by tlc.
When it is
complete, the mixture is added to water and extracted with CHzCl2. The extract
is dried and
evaporated to afford 1-[5-(4-chlorophenyl)-2-furanylmethyleneaminoj-3-[4-
(methylamino)butyljimidazolidine-2,4-dione,12-1.
D. A solution of 12-1 (1 mmol),1,6-di-(p-toluenesulfonyloxy)hexane (0.5 mmol)
and diisopropylethylamine (3 mmol) in CI~C12 (50 mL) is heated at reflux. The
progress of
the reaction is monitored by tlc. When it is complete, the solution is cooled
and added to
water. The aqueous solution is extracted with CHsCIZ, and the extract is dried
and
evaporated. The residue is chromatographed to afford the title compound 12 2,
in which Link
is .(CH~6.
E. In a similar manner, by employing other dialkylating agents, as described
herein, different compounds of Formula 12-2 are obtained.
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Example 13. (Figure 12)
Preparation of 1,4-di[4-[1-[[5-(4-chlorophenyl)-2-faranylmethylene]amino]-
imidazolidine-2,4-dion-3-yl]4-bntyl(piperazin-1 yn]batane,12-4, where Link is
(CHI,.
A. I-Benzylamino-3-(4-iodobutyl)imidazolidine-2,4-dione (12-S~, prepared as
described in W093/04061, (5 mmol), diisopropylethylamine (10 mmol) and N-
benzyloxycarbonylpipaaziile (5 mmol) are dissolved in MeCN (50 mL). The
progress of the
reaction is monitored by tlc. When it is complete, the mixture is added to
water and extracted
with CHZCl2. The extract is dried and evaporated, and the residue is
chromatographed to
afford 1-benzylamino-3-[4-(4-benzyloXycarbonylpiperazinyl)butyl]-imidazoline-
2,4-dione,
12-9.
B. The above compound, (2 mmol) is dissolved in EtOH (25 mL), and to the
solution are added 5% Pd/C (100mg) and ammonium formate (250mg). The progress
of the
reaction is monitored by tlc. When it is complete, the mixture is filtered and
added to water,
then extracted with EtOAc. The extract is dried and evaporated and the residue
is
chmmatographed to afford 1-amino-3-[4-(piperazia-1-yl)butyl]imidazoline-2,4-
dione,12-10.
C. The above-described compound (1 mmol) is dissolved in EtOH (20mL). To
the solution is added 5-(4-chlorophenyl)furan 2-carboxaldehyde (12-8), (1
irimol) and p-
toluenesulfonic acid (1 Omg). The progress of the reaction is followed by tlc.
When it is
complete, the mixt<ne is added to water and extracted wlth~CH2C12. The extract
is dried and
evaporated to afford 1-[5-(4-chlorophenyl~2-furaaylmethyleneamino]-3-[4-
(piperazin-1-
yl)butyl]imidazolidine-2,4-dione,12-3.
D. A solution of 1,4-dibromobutaae, (0.5 mmol) and 12-3 (1 m:hol) in EtOH is
maintained at room temperat<n~e, while the progress of the reaction is
monitored by tlc. When
it is complete, the mixture is evaporated to dryness under reduced pressure,
and the residue is
chromatographed to afford 12-4, in which Link is (CHI,.
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E. In a similar manner, by employing different dialkylating agents, as
described
herein, in part D atmve, different compounds of Formula 12-4 are obtained.
Example 14. (Figurel3)
Preparation of 1,10-di-(3-cyclopropylmethyl-9,9-tetramethylene-3,7-
diazabicyclo-
(33.lJnon-7-yndecane,13-2, in which Link is (CH~IO.
3-Benzy!-7-cyclopropylmethyl-9,9-tetramethylene-3,7-
diazabicyclo[3.3.1]nonane, (20-1, in which Rl is benzyl), the preparation of
which is
i0 described in European Patent 461574, (Table i, compound 22), (S mmol) is
dissolved in
MeOH (20 mL) containing 5% Pd/C (SOmg) and formic acid (0.5 mL). The progress
of the
reaction is monitored by tlc. When it is complete, the solution is filtered
end the solvent is
removed under reduced pressure. The residue is chromatographed to afford 3-
cyclopropyhnethyl-9,9-tetramcthylene-3,7-diazabicyclo[3.3.1]nonane, (20-1, in
which R, is
H).
B. 1,10-Dibmmodecane (0.5 mmol) is added to a solution of the compound 20-1
in which R, is H, prepared as described above, (1 mmol) and
diisopropylethylamine (0.5 mL)
in~ DMF (10 mL). The progress of the reaction is monitored by tlc. When it is
complete, the
solution is added to dilute NazC03 and extracted with EtOAc. The extract is
dried and
evaporated and the residue is chromatographed to afford the title compound 13
2, in which
Link is (CHyo.
C. In a similar manner, by employing different dialkylating agents, as
described
herein, different compounds of structure 13-2 are obtained.
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Example 15. (Figure 14)
Preparation of 1,4-di-[Z-[Z-[4-{Z-butylbenzofuran-3-yl)carbonyl-2,6-
diiodophenoryethyl]methylamino]acetylamino]batane,14-Z, in which n is 1 and
Link is
(
A. The compound 7B-1, prepared according to procedures described in Ew. J.
Med Chem.,1974,19-25, (5 mmol), diisoprogylethyiamine (5 mmol) and ethyl
bromoacetate
(5 mmol) are dissolved in CH=C12. The reaction is monitored by tlc. When it
is~ complete, the
mixture is added to water and extracted with EtOAc. The extract is dried and
evaporated; the
residue is chromatographed to afford ethyl N-methyl 2-[4-[2-butylbenzofuran-3-
ylcarbonyl]-
2,6-diiodophenoxy]ethylaminoacetate,14-1, in which R is ethyl.
B. The above compound (1 mmol) is dissolved in THF (10 mL) and water (3
mL), and lithium hydroxide monohydrate (1.25 mmol) is added. The reaction is
monitored by
tlc. When it is complete, acetic acid (2 mmol) is added, and the solvents are
removed under
reduced pressure. The residue is cluomatographed to afford N-methyl 2-[4-[2-
butylbenzofuran-3-ylcarbonyl]-2,6-diiodophenoxy]ethylaminoacetic acid,14-1, in
which R is
H.
~ C. The above-prepared compound (1 mmol) is dissolved in DMF (20 mL) and
dicyclohexylcarbodiimide (1 mmol) and 1,4-diaminobutane (0.5 mmol) are added.
The
reaction is monitored by tlc. When it is complete, the mixhire is added to
water and extracted
with EtOAc. The extract is dried and evaporated; the residue is
chromatographed to afford
the title compound 14-Z, in which n is 1 and Link is (CH2),,.
D. In a similar manner, by employing different diaminGg, as described herein,
different compounds of Formula 14-2 are obtained.
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Example 16. (Figure 14)
Preparation of 1,4-di-[3-[4-[Z-[4-(methylaulfonylamino)phenory]ethyl]-
aminoethyi]phenylaminoaulfonyl]propybnethylamino]batane,14-7, in which Link is
(~4~
A. N-methyl N-(4=aminophenylethyl) 2-[4-
(methylsulfonylamino~henoxy]ethylamine,14-3, prepared as described in Examples
3A and
3B, (5 mmol) is dissolved in CHZC12 (25 mL) and 3-azidopropylsulfonylchloride
(5 mmol) is
added. The reaction is monitored by tlc. When it is complete, the mixture is
added to water
and extracted with EtOAc. The extract is dried and evaporated; the residue is
chromatographed to afford N-methyl N-[4-(3-
aadopropylsulfonyl)aminophenylethyl] 2-[4-
(methylsulfonylamino~henoxy]-ethylamine,14-5.
B. The above-prepared compound (I mmol) is dissolved in MeOH (20 mL) and
5% Pd/C (50 mg) is added. The mixtin~e is stirred in a hydrogen atmosphere.
The progress of
the reaction is followed by tlc. When it is complete, the solution is filtered
and the solvent is
removed under reduced pressure. 'The residue is redissoived in MeOH (20 mL)
and
paraformaldehyde (1 mmol) and sodium cyanoborohydride (1 mmol) are added. The
reaction
is monitored by tlc. When it is complete, the mixture is added to water and
extracted with
' EtOAc. The extract is dried and avapoiated~and the residue is
chroaiatographed to afford N-
methyl N-[4-(3-methylaminopropylsulfonyl)-aminophenylethyl]-2-j4-
(methylsulfonylamino)phenoxy]ethylamine,14-6.
C. 1,4-Dibromobutane (0.5 mmol) is dissolved in MeCN, and KzC03 (0.5 g) and
the compound 14-6 (0.25 mmol) are added. The reaction is monitored by tlc.
When it is
complete, the mixture is added to water and extracted with EtOAc. The extract
is dried and
evaporated and the residue is chromatographed to afford the title compound 14-
7, in 'vrrhich
Link is (CHI,,.
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D. In a similar manner, by employing different dialkylating agents, as
described
herein, in step C above, different compounds of Formula 14-7 are obtained.
Example 17. (Figure 14)
Preparation of 1,8-di-[[N-[2-(4-methylsnlfonylaminophenory)ethyl] N-2-(4-
methylsulfonylaminophenyl)ethyl] Z-aminoethory]octane,14-10, in which Link is
(fir
A. N-2-(4-Methylsulfonylam~inophenoxykthyl2-(4-methylsulfonylaminophenyl~
ethylamine, (14-8), the preparation of which is described above in Examples 6A
and 6B, (1
mmol) is dissolved is EtOH (20 mL) and to the solution is added 2-bromoethanol
(1 mmol)
and diisopmpylethylamine (5 mmol). The reaction is monitored by tlc. When it
is complete,
the mixture is added to water and extracted with EtOAc. The extract is dried
and evaporated
and the residue is chromatographed to afford N-(2-hydroxyethyl) N-[2-(4-
methyisulfonylaminophenoxy)ethyl] 2-(4-methylsulfonylaminophenyl)ethylamine,14-
9.
B. The compouad 14-9, (1 mmol) is dissolved in DMSO (10 mL) and KOH (10
mmol) and 1,8-dibromooctane (0.5 mmol) are added. The progress of the reaction
is
monitored by tlc. When it is complete, the mixture is added to water and
extracted with
EtOAc. The extract is dried and evaporated and the residue is chromatographed
to afford the
title compound,14-10, in which Link is (CH~a.
C. In a similar meaner, by employing different dihalo compounds, in place of
1,8-
dibromooctane, different compounds of Formula 14-10 can be obtained.
Example 18. (Figure 15)
Preparation of 1,4,8,12-tetra-[2-(4-methylsulfonylaminophenory)ethyl]-1,4,8,12-

tetraazacyclohezadecane,15=3, in which X is O.
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A. 2-(4-Methyisulfonylamino~henoxyethyi bromide (15-1, in which X is O), the
preparation of which is described in J. Med Chem., 1990, 1551, (4 mmol) and
1,4,8,12-
tetraazacyclohexade~e (15-2) (1 mmol) are dissolved in MeCN (25 mL). The
progress of
the reaction is monitored by tlc. When it is complete, the mixture is added to
water and
extracted with EtOAc. The extract is dried and evaporated aad the residue is
chromatographed to afford the title compound,15-3, in which X is O.
B. In a similar manner, by employing 2-(4-
methylsulfonylaminophenyl~thylamine, (15-1, in which X is a direct bond, the
preparation of
which is described in J. Mec~ Chem., 1990, 1551), there is obtained 1,4,8;12-
tetra [2-(4-
methylsulfonylaminophenyl)ethyl]-1,4,8,12-te;traazacyciohexadecane,15-3, in
which X is a
direct bond.
Example 19. (Figure 15)
Preparation of 1,3,5-tri-[N-methyl 2-[2,6-diiodo-4-[2-butyl-3-benzofuran-
ylcarbonyl]phenory]ethy!]aminometbylJbenzene,15-6.
N-Methyl 2-[4-[2-butylbenzofuran-3-ylcarbonyl]-2,6-diiodophenoxy]ethylamine
(15-
4), prepared according to procedures described in Eur. J. Med Chem., 1974,19-
25, (3 mmol)
is dissolved in MeCN (30 W L), and 1,3,5-tri(bromomethyl)benzene (1 mmol) and
K2C03
(O.Sg) are added. The progress of the reaction is monitored by flc. When it is
complete, the
mixture is added to water and extracted with EtOAc. The extract is dried and
evaporated and
the residue is chromatographed to afford the title compound 15-6.
Example 20. (Figure 15)
Preparation of the trimeric amide 15-9.
A. N-Methyl 2-{4-[2-butylbenzofuran 3-ylcarbonyl]-2,6-
diiodophenoxy]ethylamine (15-4), prepared according to procedures described in
Ew. J.
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Med Chem., 1974,19-25, (5 mmol) is dissolved in EtOH (25 mL) and ethyl
bmmoacetate (5
mmol) and diisopropylethylamine (10 mmol) are added. The progress of the
reaction is
followed by tlc. When it is complete, the reaction is added to water and
extracted with
EtOAc. The extract is washed with dilute HCI, the dried and the solvent is
evaporated under
reduced pressure. The residue is dissolved is THF ( 15 mL), and LiOH, ISO (S
mmol) is
added. The .pmgof the reaction is followed by tlc. When it is complete, the pH
is .
adjusted to 7 by addition ofdilute Hcl. The solvents are removed under reduced
pressure and
the residue is chroraatographed.to afford N-methyl N-[2-[4-[2-butylbenzofuran-
3-ylcarbonyl]-
2,6-diodophenoxy]ethyl]glycine,15-7.
B. The compound 15-7 (3 mmol) is dissolved in DMF (25 mL) and
dicyclohexylcarbodiimide (3 mmol) aad tris(2-aminoethyl)amine (1 mmol) are
added. The
progress of the reaction is monitored by tlc. When it is complete, the mixhare
is added to
water and extracted with EtOAc. The extract is dried and evaporated and the
residue is
chromatographed to afford the triamide product 15-9.
Example 21. (Figure 15)
Preparation of 1,4..di-[N-methyl 2-(4-
methylsulfonylaminophenory~ethyIamino]butane,
15-12, in which X is O, R is methyl and Link is (CHI,,
A. 2-(4-Methylsulfonylaminophcnoxy)ethyl bromide,15-10, in which X is O, (2
mmol) and 1,4-di(methylamino)butane,15-11, (1 mmol) are dissolved in MeCN (20
mL)
containing KzC03 (O.Sg). The progress of the reaction is followed by tlc. When
it is
complete, the reaction is added to water and extiracted with EtOAc. The
extract is dried and
evaporated, and the residue is chromatographed to afford the title compound 15-
12.
B. In a similar manner, by employing 2-(4-methylsulfonylaminophenyl)ethyl
bmmide in A above, there is obtained the corresponding product 1,4-di-[N-
methyl 2-(4-
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methylsulfonylaminophenyl)-ethylamino]butane,15-12, in which X is a direct
bond, R is
methyl and Link is (CH~4~
C. In a similar manner, by employing different diamines 15-11, as described
herein, in A and B above, there are obtained the corresponding diamine
products 15-12.
Example 22. (Figure 18)
Preparation of the asymmetricavy linked aminosulfonamide 18-4, in which Link
is
(CH~=.
A. N-Methyl N-(4-aminophenylethyl) 2-[4-(methylsulfonylaanino)phenoxy]-
ethylamine,l8-1, the preparation of which is described in Examples 4A and 4B
above, (2
mmol) is dissolved in dry CH2Ciz (25 mL); diisopropylethylamine (10 mmol) and
3-
bromopropanesulfonyl chloride (2 mmol) are added. The progress of the reaction
is followed
by tlc. When it is complete, the reaction is added to water and extracted with
EtOAc. The
extract is washed aad dried and the solvent is evaporated under reduced
pressure. The residue
is chromatographed to afford 1-bromo-3-[4-[N-methyl 2-[2-[4-
methylsulfonylamino]phenoxy]ethyiamino]phenylaminosulfonyl]propane,18-2, in
which
Link is (CH~2.
B. N 2-(4-aminophenyl)ethyl 2-[4-methylsuifonylaminophenoxy]ethylamine,18-
3, prepared using methods described in EP 338793, (1 mmol) and the compound 18-
2, (1
mmol) are dissolved in CH2Cli (25 mL) and to the solution is added
diisopropylethylamine
(I O mmol). The progress of the reaction is monitored by tlc. When it is
complete, the
mixture is added to water and extracted with EtOAc. The extract is dried and
evaporated and
the residue is chmmatographed to afford the title compound I8-4, in which Link
is (CHI.
C. In a similar manner, by employing different bromosulfonyl chlorides, as
described herein, the corresponding compounds of Formula 18-4 are obtainod.
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Example 23. (Figure 19)
Preparation of the dimeric heterovalomer 19-4, in which Link is {CHs.
A. Using the procedure described in Example 22A, but using 6-
bromohexanesulfonyl chloride instead of 3-bromopropanesulfonyl chloride;1-
bromo-6-[4-
[N-methyl 2-[2-[4-
methylsulfonylamino]phenoxy]ethylamino]phenylaminosulfonyl]hexane,
19-2, in which Link is (CH~3, is prepared.
B. The above compound (2 mmol) and N-ethyl 2-[4-[2 butylbenzofuran-3-
ylcarbonyl]-2,6-diiodophenoxy]ethylamine (i9-3), prepared acxording to
procedures
described in Eur. J. Med Chem., 1974, 19-25, are dissolved in MeCN (25 mL).
The progress
of the reaction is monitored by tlc. When it is complete, the mixture is added
to dilute
NaHC03 and extracted with EtOAc. The extract is dried and evaporated and the
residue is
chromatographed to afford the title compound 19-4, in which Link is (CHI,.
C. In a similar manner, by employing different bromo compounds 19-2, the
corresponding heterovalomers 19-4 are obtained.
Example 24. (Figure 19)
Preparation of the dimeric heterovalomer 19-8,, in which Link is (CH~~.
A. N-[4-[[2-(6-methyl-2-pyridinylxthyl]-4-piperidinyl]carbonyl]phenyl
methanesulfonamide, (E-4031, Table 4) prepared as described in J. Med Chem.,
1990, 903,
(10 mmol) is dissolved is 48% HBr in AcOH (SO mL). The solution is heated to
60°C and
the progress of the reaction is monitored by tlc. When it is complete, the
mixture is cooled
and the solvent is removed under reduced pressure. The residue is taken up in
water and the
solution is basified with aqueous NaOH. The aqueous solution is extracted with
CHzCl2, and
the extract is dried and evaporated. The residue is chmmatographed to afford 6-
[2-[4-[4-
aminobenzoyl-1-piperidyl]ethyl]-2-methylpyridine,19-5.
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CA 02318745 2000-07-24
WO 99/64050 PCTNS99/12777
B. The above compound 19-5 (2 mmol) is dissolved in CH=Clz (35 mL) and to
the solution are added diisopropylethylamine (5 mmol) and 6-
bromohexanesulfonyl chloride
(2 mmol). The progress of the reaction is monitored by tlc. When it is
complete, the mixture
is added to dilute NaHC03 and extrac,~ted with EtOAc. The extract is dried and
evaporated
and the residue is chromatographed to afford 19-6, in which Link is (CHs.
C. To a solution of the above compound (1 mmol) in CH2Clz (20 mL) is added N-
[2-(4-methylsulfonylaminophenoxy)ethyl] 2-(4-methylsulfonylaminophenyl)-
ethylamine,
19-7, the preparation of which is described in Example 6A and 6B, (1 mmol).
The progress
of the reaction is monitored by tlc. When it is complete, the mixture is added
to dilute
NaHC03 and extracted with EtOAc. The extract is dried and evaporated and the
residue is
chromatographed to afford the dimeric product 19-8, in which Lick is (CHs.
D. In a similar manner, by employing different bromoalkyl sulfonyl chlorides,
as
i 5 described herein, the corresponding products of Formula 19-8 are obtained.
Example 25. (Figure 19)
Preparation of the dimeric heterovalomer 19-11, in which Link is (CH~3.
A. Using the procedure of Example 24A, except that 4-bromobutanesulfonyl
chloride is employed instead of 6-bromohexanesulfonyl chloride, there is
prepared the
compound 19-9, in which Link is (CH~3.
B. N-Methyl N-(4-aminophenylethyl) 2-[4-
(methylsulfonylamino)phenoxy]ethylamine, (8A-1; the preparation of which is
described in
Examples 4A and 4B) (2 mmol) is dissolved in MeCN (25 mL) and to the solution
is added
3-bromopropanesulfonyl chloride (2 mmol). After 6 hours, 10% methanolic
methylamine (1
mL) is added. The progress of the reaction is followed by tlc. When it is
complete, the
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CA 02318745 2000-07-24
WO 99/64050 PCT/US99/12777
mixture is added to water and extracted with CH2Cl2. The extract is dried and
evaporated,
and the residue is chromatographed to afford N-methyl N-[4-(3-
mcthylaminopropylsulfonyl~
~aminophenylethyl]-2-[4-(methylsulfonylamino~henoxy)ethylamine,19-10.
C. The product from A above (1 mmol) and the product from H above (1 mmol)
are dissolved in EtOH (20 mL). To the mixture is added diisopropylethylamine
(20 mmol).
The progress of the reaction-is monitored by tlc. When it is complete, the
mixture is added to
water and extracted with CHzCl2. The extract is dried and evaporated, and the
residue is
chromatographed to afford the title compound 19-11, in which Link is (CH~3.
D. In a similar manner, by employing different bromoalkyl sulfonyl chlorides,
as
described herein, in step A above, there are obtained the corresponding
products of Formula
19-11.
Example 26. (Figure 20)
Preparation of 3-cyclupropylmethyl 7-[2-[4-methyLsalfonylaminophenory)ethyl]-
9,9-
tetramethylene-3,7-diazabicyclo[3.3.I)nonane, 20-3, in which X is O.
A. 3-Cyclopropylmethyl-9,9-tetramethylene-3,7-diazabicyclo[3.3.1)nonane, (20-
1, in which R is H, the preparation of which is described in Example 14A), (2
mmol) is
dissolved in MeCN (20 mL). To the solution is added 2-[4- .
methylsulfonylaminophenoxy)ethyl bromide (20-2, in which R is O) (2 mmol) and
KZC03
(O.Sg). The progress of the reaction is monitored by tlc. When it is complete,
the mixture is
added to dilute NaHC03 and extracted with EtOAc. The extract is dried and
evaporated and
the residue is chromatogtaphed to afford the title compound 20-3.
B. In a similar manner, by employing 2-[4-methylsulfonylaminophenyl)ethyl
bromide 20-2, in which R is a direct bond, in A above, there is obtained the
corresponding
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CA 02318745 2000-07-24
WO 99/64050 PCT/US99/12777
product 3-cyclopropylmethyl 7-[2-[4-methylsulfonylaminophenyl]ethyl]-9,9-
tetramethylene_
3,7-diazabicyclo[3.3.1Jnonane, 20-3, in which X is a direct bond.
Example 27. (Figure 20)
Preparation of 3,8-di-[2-[4-methylsulfonylaminophenory)ethyl)-9,9-
tetramethylene-3,7-
diazabicyclo[3.3:1)nonane, 20-5, in which X is O.
A. 3,$-Dibenzyl-9,9-tetramethylene-3,7-diazabicyclo[3.3.1]nonane, 20-4, in
which R, and RZ are benzyl, the preparation of which is described in European
Patent 461574,
(5 mmol) is dissolved in EtOH (25 mL). 10% Pd/C (50 mg) and ammonium fomnate
(200
mg) are added. The progress of the reaction is followed by tlc. When it is
complete, the
solution is filtered and the solvent is removed under vacuum to afford 9,9-
tetramethylene-3,7-
diazabicyclo[3.3.1)nonane, 20-4, in which R, and R2 are H.
B. The above compound (1 mmol) is dissolved in MeCN, and to the solution is
added diisopropylethylamine (5 mmol) and 2-[4-methylsulfonylaminophenoxy]ethyl
bromide
(0.5 mmol). The progress of the reaction is monitored by tlc. When it is
complete, the
mixture is added to water and extracted with EtOAc. The extract is dried and
evaporated and
the residue is chromatographed to afford the title compound 20-5, in which X
is O.
C. In a similar manner, by employing 2-[4-methylsulfonylaminophenyl]ethyl
bromide in B above, there is obtained the corresponding product 3,$-di-[2-[4-
methylsulfonylaminophenyl]ethyl]-9,9-tetramethylene-3,7-
diazabicyclo[3.3.1)nonane 20-5, in
which X is a direct bond.
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CA 02318745 2000-07-24
WO 99/64050 PCT/US99/12777
Example 28. (Figure 20)
Preparation of 3-cyclopropylmethyl-7-[4-[2-[4-methylaalfonylaminophenory]-
ethylmethylamino]butyl]-9,9-tetramethylene-3,7-diazabicyclo[33.1]nonane, 20-5,
in
which X is O.
A. 3-Cyclopropylmethyl-9,9-tetramethylene-3,7-diazabicyclo[3:3.1]nonane, (20-
1, in which R is H), (5 mmol) and 1,4-dibromobutane (5 mmol) are dissolved in
EtOH (30
mL). The progress of the reaction is followed by tlc. When it is complete, N-
methyl 2-(4-
methylsulfonylaminopheaoxy~thylamine (20-6, in which X is O), (5 mmol) is
added. The
progress of the reaction is followed by tlc. When it is complete, the mixture
is added to dilute
NaHC03 and extracted with EtOAc. The extract is dried and evaporated and the
residue is
chromatographed to afford the title compound 20-5, in which X is O.
'B. In a similar manner, by employing 2-[4-methylsulfonylaminophenyl]ethyl
bromide in A above, there is obtained the corresponding product 3-
cyclopropylmethyl-7-[4-
[2-[4-methylsulfonylaminophenyl]-ethylmethylamino]butyl]-9,9-tetranaethylene-
3,7-
diazabicyclo[3.3.1]nonane, 20-5, in which X is a direct bond.
C. In a similar meaner, by employing different dibromo compounds in place of
1,4-diliromobutane, there are obtained the con~esponding products similar to
20-7.
Example 29. (Figure 20)
Preparation of I,3,5-tri-[2-[4-(methylsnlfonylamino)phenory]ethylmethylamino-
methyl]benzene, 20-9, in which X is O.
A. N-Methyl 2-[(4=methylsulfonylamino)phenoxy]ethylamine (20-6, in which X
is O), (3 mmol) and 1,3,5-tri-(bromomethyl) benzene (20-8), (1 mmol) are
dissolved in .
MeCN (25 mL) containing KZC03 (O.Sg). The progress of the reaction is
monitored by tlc.
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CA 02318745 2000-07-24
WO 99/64050 PCT/US99/12777
When it is complete, the mixture is added to water and attracted with EtOAc.
The extract is
dried and evaporated and the residue is chromatographed to afford the tide
compound, 20-9,
in which X is O.
B. ~ In a similar manner, by employing N-methyl 2-[(4-
methylsulfonylamino~henyl]-ethylamine, (20-6, in which X is a direct bond)
there is
obtained the cornesponding product 1,3,5-tri-[2-[4-
(methylsulfonylamino)phenyl]ethylmethylamino-methyl]benzene, 20.9, in which X
is a
direct bond.
While the present invention has been described with reference to the specific
embodiments thereof, it should be understood by those skilled in the art that
various changes
may be made and equivalents may be substituted without departing from the true
spiiit and
scope of the invention. In addition, many modifications may be made to adapt a
particular
situation, material, composition of matter, process, process step or steps; to
the objective
spirit and scope of the present invention. All such modifications are intended
to be within the
scope of the claims appended hereto.
All of the publications, patent applications and patents cited in this
application are
herein incorporated by reference in their entirety to the same extent as if
each individual
publication, patent application or patent was specifically and individually
indicated to be
incorporated by reference in its entirety.
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CA 02318745 2000-07-24
WO 99/64050 PCT/US99/12777
Table 1: Multiple Activities of Antiarrhythmic Agent
Drag Channels Receptors


Na


Fast Med. Slow Ca K a ~i M=


Class I


Lidocaine


Mexiletine ~


Tocainide ~


Moricizine '


Procainamide


Disopyramide ~


Quinidine a a a


Propafenone


Flecainide


Encainide


Class IV


Bepridil o


Verapamil ~


. Diltiazem '


Class III


Bretylitun


Sotalol


Amiodarone ~ ~


. Afinidine


Potency of block: ~ Low ~ Moderate ~ High * Biphasic Action
-130-


CA 02318745 2000-07-24
WO 99/6d050 PC1'/US99/12777
Table 2: Activity of Potassium Channel Ligands
Drug DevelopmentI,~ Ix, I,o Na,,",Use


Stage ~ Dependence


AmiodaroneApproved ~ ~ None


DofetiiidePhase 3 ~ Reverse


S~~~ Phase 3 a Reverse


E-4031 Phase 2 ~ ' Reverse


Aamilide Phase 3 o a ~ Reverse


AmbasilideTerminated ~ ~ None


Ibutilide Approved ~ t None


Tedisamil Phase 3 ~ ~ Reverse


-I31-


CA 02318745 2000-07-24
WO 99/64050 PGTIUS99/1Z777
0
Z
. .~ .~
0o c
~~ en
w y ~ ~ ~ o ono
g
a~
H.
s.
N
d' N
G C N O V O
e~ . N cn car e~ N ~ ~ 'O~
w
0
o ~ a
i '~ .r~ .~ ~' a,
W v~~a~,v°~ ~w~a
a
v
H ~=
a a b
a 'd a
a b ~ ~ b
x ~ b
..
A '" °~. ~ a c°
H A
0
132


CA 02318745 2000-07-24
PCTNS99/i2777
WO 99/64050
Table 4: Potuasinm Channel Ligands
~,.. '
U1~914 ~ a ~ t
~ta~
' Amledaress
fha) .
V
TaatM ~ ~nd IK1~ '
Dnoduea~
T
~~ (he1
i
43~ ~ ~ Def~lild~
. ~11 N
~b,r~daarav th~~''11 ~ Seeaist
0
4
RP4f33e l~lt[A'T~ ~P~ ~ ~ p-fC31 ~
o
S~aaditdy,
4 . ,
'C2H5 ~
CH3-~C-NH ~ H -NH~CHZ-CHZ-N ' H .
C2 5
N-Acetyl Procoinomide (NAPA)
. ~H5
CI-~~-CHZ-CHZ-C1~-CHZ"~5 Hi3
Ciotilium
-133-


CA 02318745 2000-07-24
WO 991!14050
Table 4: Potaaaium Channel Ligsada (COQ
Effect of Subsdtustaa oa Iw Poe~meF
z - 7ss.s~3
and analogs
PCT/US99/12777
ht, lur ICw CC8r8
(aD~' m ICm


~ IGr 9G fa~u ~aa~' mp'C
(ate at o~nea
(a3~


E " ~lsCHa Z7.4 a000 Z t 0.3


S'4 R Ms o m ~ i~ ' lal
t Lt


0 _ ~ 2991 at '1000 224-223'
6~rl ~ 1000


R My p ~ 1996 at 179-Id0
~ 1000.
,[


4 R ~, 1 ,~ soot sly at looo>looo ul-uz
~


a R My p s~-slo


ee~uvi~ar~ 140 loo-los


~ at loco >looo u9
9a


u~ slat at >looo .
~o ~-


s R n~. 2 ~ ~ lsoo . >iooo irt-lsa
o Cs-~s-s


to R au . 5 DBOO ~ '1000 a4-a6
I aoo~
~
'


11 R Ms o iHs_~ _ '1000 lTb-I9a
a
CF~


_ . '10000. '1000 1S8-141
~
~t


R ~ Z 4"~, r 1~ '1000 ~
p


1~ 193


15 R$ H Z P~ d20t 100 >I0o0 L98-201
Z ~ .


1s RS MvsNCBsC~ . ~ i~ 41


iT R t-Pt S 140-1


18 R . 0 S.3 6
i-Pt l ~ ~ 1~ 140-141


1 S.diehl ips '1000 .
s 90-98
43
0


20 R i Pr 0 , 11~ 2aS~r at 14
9~Y1 100 -1
100
9


Zl R F~CCH~ ~ 90 4000 9
~' -


22 R F~CCHa 1 ~.P 143-143
~4


23 R F~CCRs 1 Z.i.dliehlorop~80~ 35Si at '1000
1 4-bb(bitlnasomethyl~P~7~ 1000 2134
?


S4 S F~CCHa 1 , ~ ~ '1000 13
4~idtsiNuoromsthyllpha~t
2


1 R FaCCHs . ~ ....


~134-


CA 02318745 2000-07-24
WO 99/(t4050 Pcrnrs99nZ~~~
Table 5: Principal Cardiac K'' Currents snd Some Drags that Block Them
CurrentDrugs that Block CurrentReference


Ix tJKb6,914, dofetilide, Argentieri,1992;
Cmmeliet,


senlatilide, d solatol 1985; Gwilt, et al.,
1990,


1991


IK1 RP58866, RP62719 Frscande, et al.,1992;


Imoto, et al., 1987


L,.o~ Tedisamil Dukes, et al., 1990


Ix~,rr,P~Glibenclamide, 5- K~ntor, et al., 1990;
Notsu,


hydroxydecanoate et al.,1989;1992


-135-