U.S. patent application number 12/145658 was filed with the patent office on 2008-10-30 for beta2-adrenergic receptor agonists.
Invention is credited to Seok-Ki Choi, Edmund J. Moran.
Application Number | 20080269344 12/145658 |
Document ID | / |
Family ID | 23817453 |
Filed Date | 2008-10-30 |
United States Patent
Application |
20080269344 |
Kind Code |
A1 |
Moran; Edmund J. ; et
al. |
October 30, 2008 |
Beta2-Adrenergic Receptor Agonists
Abstract
Disclosed are multibinding compounds which are .beta.2
adrenergic receptor agonists and are useful in the treatment and
prevention of respiratory diseases such as asthma, bronchitis. They
are also useful in the treatment of nervous system injury and
premature labor.
Inventors: |
Moran; Edmund J.; (San
Francisco, CA) ; Choi; Seok-Ki; (Palo Alto,
CA) |
Correspondence
Address: |
THERAVANCE, INC.
901 GATEWAY BOULEVARD
SOUTH SAN FRANCISCO
CA
94080
US
|
Family ID: |
23817453 |
Appl. No.: |
12/145658 |
Filed: |
June 25, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11784148 |
Apr 5, 2007 |
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12145658 |
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11049447 |
Feb 2, 2005 |
7217738 |
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11784148 |
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10108945 |
Mar 28, 2002 |
6916961 |
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11049447 |
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09637899 |
Aug 14, 2000 |
6576793 |
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10108945 |
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09457618 |
Dec 8, 1999 |
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09637899 |
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Current U.S.
Class: |
514/629 |
Current CPC
Class: |
A61P 11/08 20180101;
A61P 43/00 20180101; A61K 45/06 20130101; A61P 3/04 20180101; A61P
3/10 20180101; A61P 11/06 20180101; A61P 1/00 20180101; C40B 40/00
20130101; A61P 11/00 20180101; C07C 233/43 20130101; A61P 25/00
20180101; A61K 31/137 20130101; C07C 215/60 20130101; A61K 31/403
20130101; C07C 215/68 20130101; A61K 31/167 20130101; A61K 31/4164
20130101; A61P 15/06 20180101; A61K 31/4706 20130101; A61K 31/404
20130101 |
Class at
Publication: |
514/629 |
International
Class: |
A61K 31/16 20060101
A61K031/16; A61P 11/06 20060101 A61P011/06; A61P 11/00 20060101
A61P011/00 |
Claims
1-53. (canceled)
54. A method of treating asthma or chronic obstructive pulmonary
disease in a mammal, the method comprising administering to the
mammal a pharmaceutical composition comprising a
pharmaceutically-acceptable carrier and a
pharmaceutically-acceptable salt of a compound of the formula:
##STR00094##
55. The method of claim 54 wherein the method comprises
administering the pharmaceutical composition to the mammal by
inhalation.
56. The method of claim 54 wherein the method further comprises
administering a steroidal anti-inflammatory agent to the
mammal.
57. The method of claim 54 wherein the mammal is a human.
58. A method of treating asthma or chronic obstructive pulmonary
disease in a mammal, the method comprising administering to the
mammal a pharmaceutical composition comprising a
pharmaceutically-acceptable carrier and a compound of the formula:
##STR00095##
59. The method of claim 58 wherein the method comprises
administering the pharmaceutical composition to the mammal by
inhalation.
60. The method of claim 58 wherein the method further comprises
administering a steroidal anti-inflammatory agent to the
mammal.
61. The method of claim 58 wherein the mammal is a human.
62. A method of treating asthma or chronic obstructive pulmonary
disease in a mammal, the method comprising administering to the
mammal a pharmaceutically-acceptable salt of a compound of the
formula: ##STR00096##
63. The method of claim 62 wherein the method comprises
administering the pharmaceutically-acceptable salt of the compound
to the mammal by inhalation.
64. The method of claim 62 wherein the method further comprises
administering a steroidal anti-inflammatory agent to the
mammal.
65. The method of claim 62 wherein the mammal is a human.
66. A method of agonizing a .beta..sub.2 adrenergic receptor in a
mammal, the method comprising administering to the mammal a
compound of the formula: ##STR00097##
67. The method of claim 66 wherein the mammal is a human.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 09/323,943, filed on Jun. 2, 1999 which claims
the benefit of U.S. Patent Application Ser. No. 60/088,466, filed
Jun. 8, 1998; and U.S. patent application Ser. No. 60/092,938,
filed Jul. 15, 1998; the disclosures of which are incorporated
herein by reference in their entirety.
BACKGROUND OF THE INVENTION
Field of the Invention
[0002] This invention relates to novel multibinding compounds
(agents) that are adrenergic receptor agonists, partial agonists
and pharmaceutical compositions comprising such compounds.
Accordingly, the multibinding compounds and pharmaceutical
compositions of this invention are useful in the treatment and
prevention of respiratory diseases such as asthma, chronic
obstructive pulmonary disease and chronic bronchitis. They are also
useful in the treatment of nervous system injury and premature
labor.
REFERENCES
[0003] The following publications are cited in this application as
superscript numbers: [0004] .sup.1 Hardman, J. G., et al. "The
Pharmacological Basis of Therapeutics", McGraw-Hill, New York,
(1996) [0005] .sup.2 Strosberg, A. D. "Structure, Function, and
Regulation of Adrenergic Receptors" Protein Sci 2, 1198-1209
(1993). [0006] .sup.3 Beck-Sickinger, A. G. "Structure
Characterization and Binding Sites of G-Protein-coupled Receptors"
DDT, 1, 502-513, (1996). [0007] .sup.4 Hein, L. & Kobilka, B.
K. "Adrenergic Receptor Signal Transduction and Regulation"
Neuropharmacol, 34, 357-366, (1995). [0008] .sup.5 Strosberg, A. D.
& Pietri-Rouxel, F. "Function, and Regulation of
.beta.3-Adrenoceptor" TiPS, 17, 373-381, (1996). [0009] 6 Barnes,
P. J. "Current Therapies for Asthma" CHEST, 111:17S-26S, (1997).
[0010] 7 Jack, D. A. "A way of Looking at Agonism and Antagonism:
Lessons from Salbutamol, Salmeterol and other .beta.-Adrenoceptor
Agonists" Br. J. Clin. Pharmac. 31, 501-514, (1991). [0011] 8
Kissei Pharmaceutical Co. Ltd.
"2-Amino-1-(4-hydroxy-2-methyl-phenyl)propanol derivatives"
JP-10152460 (Publication date Jun. 9, 1998).
[0012] All of the above publications are herein incorporated by
reference in their entirety to the same extent as if each
individual publication was specifically and individually indicated
to be incorporated by reference in its entirety.
STATE OF THE ART
[0013] A receptor is a biological structure with one or more
binding domains that reversibly complexes with one or more ligands,
where that complexation has biological consequences. Receptors can
exist entirely outside the cell (extracellular receptors), within
the cell membrane (but presenting sections of the receptor to the
extracellular milieu and cytosol), or entirely within the cell
(intracellular receptors). They may also function independently of
a cell (e.g., clot formation). Receptors within the cell membrane
allow a cell to communicate with the space outside of its
boundaries (i.e., signaling) as well as to function in the
transport of molecules and ions into and out of the cell.
[0014] A ligand is a binding partner for a specific receptor or
family of receptors. A ligand may be the endogenous ligand for the
receptor or alternatively may be a synthetic ligand for the
receptor such as a drug, a drug candidate or a pharmacological
tool.
[0015] The super family of seven transmembrane proteins (7-TMs),
also called G-protein coupled receptors (GPCRs), represents one of
the most significant classes of membrane bound receptors that
communicate changes that occur outside of the cell's boundaries to
its interior, triggering a cellular response when appropriate. The
G-proteins, when activated, affect a wide range of downstream
effector systems both positively and negatively (e.g., ion
channels, protein kinase cascades, transcription, transmigration of
adhesion proteins, and the like).
[0016] Adrenergic receptors (AR) are members of the G-protein
coupled receptors that are composed of a family of three receptor
sub-types: .alpha.1 (.sub.A, B, D) .alpha.2 (.sub.A, B, C), and
.beta.(.sub.1, 2, 3)..sup.1-5 These receptors are expressed in
tissues of various systems and organs of mammals and the
proportions of the .alpha. and the .beta. receptors are tissue
dependant. For example, tissues of bronchial smooth muscle express
largely .beta.2-AR while those of cutaneous blood vessels contain
exclusively .alpha.-AR subtypes.
[0017] It has been established that the .beta.2-AR sub-type is
involved in respiratory diseases such as such as asthma.sup.6,
chronic bronchitis, nervous system injury, and premature labor.
Currently, a number of drugs e.g., albuterol, formoterol,
isoprenolol, or salmeterol having .beta.2-AR agonist activities are
being used to treat asthma. However, these drugs have limited
utility as they are either non-selective thereby causing adverse
side effects such as muscle tremor, tachycardia, palpitations, and
restlesness.sup.6, or have short duration of action and/or slow
onset time of action..sup.7 Accordingly, there is a need for
.beta.2-selective AR agonists that are fast acting and have
increased potency and/or longer duration of action.
[0018] The multibinding compounds of the present invention fulfill
this need.
SUMMARY OF THE INVENTION
[0019] This invention is directed to novel multibinding compounds
(agents) that are agonists or partial agonists of .beta.2
adrenergic receptor and are therefore useful in the treatment and
prevention of respiratory diseases such as asthma, chronic
obstructive pulmonary disease, and chronic bronchitis. They are
also useful in the treatment of nervous system injury and premature
labor.
[0020] Accordingly, in one of its composition aspects, this
invention provides a multibinding compound of Formula (I):
(L).sub.p(X).sub.q (I)
wherein:
[0021] p is an integer of from 2 to 10;
[0022] q is an integer of from 1 to 20;
[0023] X is a linker; and
[0024] L is a ligand wherein:
[0025] one of the ligands, L, is a compound of formula (a):
##STR00001##
wherein:
[0026] Ar.sup.1 and Ar.sup.2 are independently selected from the
group consisting of aryl, heteroaryl, cycloalkyl, substituted
cycloalkyl, and heterocyclyl wherein each of said Ar.sup.1 and
Ar.sup.2 substituent optionally links the ligand to a linker;
[0027] R.sup.1 is selected from the group consisting of hydrogen,
alkyl, and substituted alkyl, or R.sup.1 is a covalent bond linking
the ligand to a linker;
[0028] R.sup.2 is selected from the group consisting of hydrogen,
alkyl, aralkyl, acyl, substituted alkyl, cycloalkyl, and
substituted cycloalkyl, or R.sup.2 is a covalent bond linking the
ligand to a linker;
[0029] W is a covalent bond linking the --NR.sup.2-- group to
Ar.sup.2, alkylene or substituted alkylene wherein one or more of
the carbon atoms in said alkylene or substituted alkylene group is
optionally replaced by a substituent selected from the group
consisting of --NR.sup.a-- (where R.sup.a is hydrogen, alkyl, acyl,
or a covalent bond linking the ligand to a linker), --O--,
--S(O).sub.n (where n is an integer of from 0 to 2), --CO--,
--PR.sup.b-- (where R.sup.b is alkyl), --P(O).sub.2--, and
--O--P(O)O-- and further wherein said alkylene or substituted
alkylene group optionally links the ligand to a linker provided
that at least one of Ar.sup.1, Ar.sup.2, R.sup.1, R.sup.2, or W
links the ligand to a linker; and
[0030] the other ligands are independently of each other a compound
of formula (b):
-Q-Ar.sup.3 (b)
wherein:
[0031] Ar.sup.3 is selected from the group consisting of aryl,
heteroaryl, cycloalkyl, substituted cycloalkyl, and
heterocyclyl;
[0032] Q, which links the other ligand to the linker, is selected
from the group consisting of a covalent bond, alkylene, and
substituted alkylene wherein one or more of the carbon atoms in
said alkylene and substituted alkylene is optionally replaced by a
substituent selected from the group consisting of --NR-- (where
R.sup.a is hydrogen, alkyl, acyl, or a covalent bond linking the
ligand to a linker), --O--, --S(O).sub.n-- (where n is an integer
of from 0 to 2), --CO--, --PR.sup.b-- (where R.sup.b is alkyl),
--P(O).sub.2--, and --O--P(O)O--; and
individual isomers, mixtures of isomers and pharmaceutically
acceptable salts thereof provided that: (i) when the multibinding
compound of Formula (I) is a compound of formula:
##STR00002##
where Ar.sup.1 and Ar.sup.2 are aryl, then W and X both are not
alkylene or alkylene-O--; (ii) when the multibinding compound of
Formula (I) is a compound of formula:
##STR00003##
where Ar.sup.1 is 4-hydroxy-2-methylphenyl, Ar.sup.1 is aryl,
Ar.sup.1 is aryl or heterocyclyl, W is ethylene, Q is a covalent
bond, R.sup.1 is alkyl, then the linker X is not linked to the
Ar.sup.2 group through an oxygen atom; (iii) when the multibinding
compound of Formula (I) is a compound of formula:
##STR00004##
where Ar.sup.1 and Ar.sup.3 are aryl, W is alkylene, Ar.sup.2 is
aryl or cycloalkyl, Q is a covalent bond, then X is not
-alkylene-O--; and (iv) when the multibinding compound of Formula
(I) is a compound of formula:
##STR00005##
where Ar.sup.1 is 4-benzyloxy-3-formylamino, R.sup.2 is aralkyl, W
is --CH(CH.sub.3)CH.sub.2--, Ar.sup.2 and Ar.sup.3 are phenyl, Q is
a covalent bond, then the linker X is not linked to the Ar.sup.2
group through an oxygen atom.
[0033] More preferably, each linker, X, in the multibinding
compound of Formula (I) independently has the formula:
--X.sup.a-Z--(Y.sup.a-Z).sub.m-X.sup.a--
wherein
[0034] m is an integer of from 0 to 20;
[0035] X.sup.a at each separate occurrence is selected from the
group consisting of --O--, --S--, --NR--, --C(O)--, --C(O)O--,
--OC(O)--, --C(O)NR--, --NRC(O)--, C(S), --C(S)O--, --C(S)NR--,
--NRC(S)--, or a covalent bond where R is as defined below;
[0036] Z at each separate occurrence is selected from the group
consisting of alkylene, substituted alkylene, cycloalkylene,
substituted cycloalkylene, alkenylene, substituted alkenylene,
alkynylene, substituted alkynylene, cycloalkenylene, substituted
cycloalkenylene, arylene, heteroarylene, heterocyclene, or a
covalent bond;
[0037] each Y.sup.a at each separate occurrence is selected from
the group consisting of --O--, --C(O)--, --OC(O)--, --C(O)O--,
--NR--, --S(O).sub.n--, --C(O)NR'--, --NR'C(O)--, --NR'C(O)NR'--,
--NR'C(S)NR'--, --C(.dbd.NR')--NR'--, --NR'--C(.dbd.NR')--,
--OC(O)--NR'--, --NR'--C(O)--O--, --N.dbd.C(X.sup.a)--NR'--,
--NR'--C(X.sup.a).dbd.N--, --P(O)(OR')--O--, --O--P(O)(OR')--,
--S(O).sub.nCR'R''--, --S(O).sub.n--NR'--, --NR'--S(O).sub.n--,
--S--S--, and a covalent bond; where n is 0, 1 or 2; R, R' and R''
at each separate occurrence are selected from the group consisting
of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted
cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted
cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl and
heterocyclic, and X.sup.a is as defined above.
[0038] Preferably, q is less than p in the multibinding compounds
of this invention.
[0039] In still another of its composition aspects, this invention
provides a pharmaceutical composition comprising a pharmaceutically
acceptable carrier and an effective amount of a multibinding
compound of Formula (I):
(L).sub.p(X).sub.q (I)
wherein:
[0040] p is an integer of from 2 to 10;
[0041] q is an integer of from 1 to 20;
[0042] X is a linker; and
[0043] L is a ligand wherein:
[0044] one of the ligands, L, is a compound of formula (a):
##STR00006##
wherein:
[0045] Ar.sup.1 and Ar.sup.2 are independently selected from the
group consisting of aryl, heteroaryl, cycloalkyl, substituted
cycloalkyl, and heterocyclyl wherein each of said Ar.sup.1 and
Ar.sup.2 substituent optionally links the ligand to a linker;
[0046] R.sup.1 is selected from the group consisting of hydrogen,
alkyl, and substituted alkyl, or R.sup.1 is a covalent bond linking
the ligand to a linker;
[0047] R.sup.2 is selected from the group consisting of hydrogen,
alkyl, aralkyl, acyl, substituted alkyl, cycloalkyl, and
substituted cycloalkyl, or R.sup.2 is a covalent bond linking the
ligand to a linker,
[0048] W is a covalent bond linking the --N.sup.2-- group to
Ar.sup.2, alkylene or substituted alkylene wherein one or more of
the carbon atoms in said alkylene and substituted alkylene is
optionally replaced by a substituent selected from the group
consisting of --NR.sup.a-- (where R.sup.a is hydrogen, alkyl, acyl,
or a covalent bond linking the ligand to a linker), --O--,
--S(O).sub.n (where n is an integer of from 0 to 2), --CO--,
--PR.sup.b-- (where R.sup.b is alkyl), --P(O).sub.2--, and
--O--P(O)O-- and further wherein said alkylene or substituted
alkylene group optionally links the ligand to a linker provided
that at least one of Ar.sup.1, Ar.sup.2, R.sup.1, R.sup.2, or W
links the ligand to a linker, and
[0049] the other ligands are independently of each other a compound
of formula (b):
-Q-Ar.sup.3 (b)
wherein:
[0050] Ar.sup.3 is selected from the group consisting of aryl,
heteroaryl, cycloalkyl, substituted cycloalkyl, and
heterocyclyl;
[0051] Q, which links the other ligand to the linker, is selected
from the group consisting of a covalent bond, alkylene, or a
substituted alkylene group wherein one or more of the carbon atoms
in said alkylene or substituted alkylene group is optionally
replaced by a substituent selected from the group consisting of
--NR.sup.a-- (where R.sup.a is hydrogen, alkyl, acyl, or a covalent
bond linking the ligand to a linker), --O--, --S(O).sub.n-- (where
n is an integer of from 0 to 2), --CO--, --PR.sup.b-- (where
R.sup.b is alkyl), --P(O).sub.2--, and --O--P(O)O--; and
individual isomers, mixtures of isomers and pharmaceutically
acceptable salts thereof provided that: (i) when the multibinding
compound of Formula (I) is a compound of formula:
##STR00007##
where Ar.sup.1 and Ar.sup.3 are aryl, then W and X both are not
alkylene or alkylene-O--; (ii) when the multibinding compound of
Formula (I) is a compound of formula:
##STR00008##
where Ar.sup.1 is 4-hydroxy-2-methylphenyl, Ar.sup.2 is aryl,
Ar.sup.3 is aryl or heterocyclyl, W is ethylene, Q is a covalent
bond, R.sup.1 is alkyl, then the linker X is not linked to the
Ar.sup.2 group through an oxygen atom; (iii) when the multibinding
compound of Formula (I) is a compound of formula:
##STR00009##
where Ar.sup.1, Ar.sup.2, Ar.sup.3, R.sup.1, R.sup.2 are as defined
above, W is alkylene, and Q is a covalent bond, then X is not
-alkylene-O--; and (iv) when the multibinding compound of Formula
(I) is a compound of formula:
##STR00010##
where Ar.sup.1 is 4-benzyloxy-3-formylamino, R.sup.2 is aralkyl, W
is --CH(CH.sub.3)CH.sub.2--, Ar.sup.2 and Ar.sup.3 are phenyl, Q is
a covalent bond, then the linker X is not linked to the Ar.sup.2
group through an oxygen atom.
[0052] More preferably, each linker, X, in the multibinding
compound of Formula (I) independently has the formula:
--X.sup.a-Z-(Y.sup.a-Z).sub.m_X.sup.a--
wherein
[0053] m is an integer of from 0 to 20;
[0054] X.sup.a at each separate occurrence is selected from the
group consisting of --O--, --S--, --NR--, --C(O)--, --C(O)O--,
--OC(O)--, --C(O)NR--, --NRC(O)--, C(S), --C(S)O--, --C(S)NR--,
--NRC(S)--, or a covalent bond where R is as defined below;
[0055] Z at each separate occurrence is selected from the group
consisting of alkylene, substituted alkylene, cycloalkylene,
substituted cycloalkylene, alkenylene, substituted alkenylene,
alkynylene, substituted alkynylene, cycloalkenylene, substituted
cycloalkenylene, arylene, heteroarylene, heterocyclene, or a
covalent bond;
[0056] each Y.sup.a at each separate occurrence is selected from
the group consisting of --O--, --C(O)--, --OC(O)--, --C(O)O--,
--NR--, --S(O).sub.n--, --C(O)NR'--, --NR'C(O)--, --NR'C(O)NR'--,
--NR'C(S)NR'--, --C(.dbd.NR')--NR'--, --NR'--C(.dbd.NR')--,
--OC(O)--NR'--, --NR'--C(O)--O--, --N.dbd.C(X.sup.a)--NR'--,
--NR_C(X.sup.a).dbd.N--, --P(O)(OR')--O--, --O--P(O)(OR')--,
--S(O).sub.nCR'R''--, --S(O).sub.n--NR'--, --NR'--S(O).sub.n--,
--S--S--, and a covalent bond; where n is 0, 1 or 2; R, R' and R''
at each separate occurrence are selected from the group consisting
of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted
cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted
cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl and
heterocyclic, and X.sup.a is as defined above.
[0057] In still another aspect, this invention provides a method of
treating diseases mediated by a .beta.2 adrenergic receptor in a
mammal, said method comprising administering to said mammal a
therapeutically effective amount of a pharmaceutical composition
comprising a pharmaceutically acceptable carrier and a multibinding
compound of Formula (I):
(L).sub.p(X).sub.q (I)
wherein:
[0058] p is an integer of from .sup.2 to 10;
[0059] q is an integer of from 1 to 20;
[0060] X is a linker, and
[0061] L is a ligand wherein:
[0062] one of the ligands, L, is a compound of formula (a):
##STR00011##
wherein:
[0063] Ar.sup.1 and A.sup.2 are independently selected from the
group consisting of aryl, heteroaryl, cycloalkyl, substituted
cycloalkyl, and heterocyclyl wherein each of said Ar.sup.1 and
Ar.sup.2 substituent optionally links the ligand to a linker,
[0064] R.sup.1 is selected from the group consisting of hydrogen,
alkyl, and substituted alkyl, or R.sup.1 is a covalent bond linking
the ligand to a linker;
[0065] R.sup.2 is selected from the group consisting of hydrogen,
alkyl, aralkyl, acyl, substituted alkyl, cycloalkyl, and
substituted cycloalkyl, or R.sup.2 is a covalent bond linking the
ligand to a linker;
[0066] W is a covalent bond linking the --NR.sup.2-- group to
Ar.sup.2, alkylene or substituted alkylene wherein one or more of
the carbon atoms in said alkylene and substituted alkylene group is
optionally replaced by a substituent selected from the group
consisting of --NR.sup.a-- (where R.sup.a is hydrogen, alkyl, acyl,
or a covalent bond linking the ligand to a linker), --O--,
--S(O).sub.n (where n is an integer of from 0 to 2), --CO--,
--PR.sup.b-- (where R.sup.b is alkyl), --P(O).sub.2--, and
--O--P(O)O-- and further wherein said alkylene or substituted
alkylene group optionally links the ligand to a linker provided
that at least one of Ar.sup.1, Ar.sup.2, R.sup.1, R.sup.2, or W
links the ligand to a linker, and
[0067] the other ligands are independently of each other a compound
of formula (b):
-Q-Ar.sup.3 (b)
wherein:
[0068] Ar.sup.3 is selected from the group consisting of aryl,
heteroaryl, cycloalkyl, substituted cycloalkyl, and
heterocyclyl;
[0069] Q, which links the other ligand to the linker, is selected
from the group consisting of a covalent bond, alkylene, and
substituted alkylene wherein one or more of the carbon atoms in
said alkylene and substituted alkylene group is optionally replaced
by a substituent selected from the group consisting of --NR.sup.a--
(where R.sup.a is hydrogen, alkyl, acyl, or a covalent bond linking
the ligand to a linker), --O--, --S(O).sub.n-- (where n is an
integer of from 0 to 2), --CO--, --PR.sup.b-- (where R.sup.b is
alkyl), --P(O).sub.2--, and --O--P(O)O--; and
individual isomers, mixtures of isomers and pharmaceutically
acceptable salts thereof provided that: (i) when the multibinding
compound of Formula (I) is a compound of formula:
##STR00012##
where Ar.sup.1 and Ar.sup.3 are aryl, then W and X both are not
alkylene or alkylene-O--; (ii) when the multibinding compound of
Formula (I) is a compound of formula:
##STR00013##
where Ar.sup.1 is 4-hydroxy-2-methylphenyl, Ar.sup.2 is aryl,
Ar.sup.3 is aryl or heterocyclyl, W is ethylene, Q is a covalent
bond, R.sup.1 is alkyl, then the linker X is not linked to the
Ar.sup.2 group through an oxygen atom; (iii) when the multibinding
compound of Formula (I) is a compound of formula:
##STR00014##
where Ar.sup.1, Ar.sup.2, Ar.sup.3, R.sup.1, R.sup.2 are as defined
above, W is alkylene, and Q is a covalent bond, then X is not
-alkylene-O; and (iv) when the multibinding compound of Formula (I)
is a compound of formula:
##STR00015##
where Ar.sup.1 is 4-benzyloxy-3-formylamino, R.sup.2 is aralkyl, W
is --CH(CH.sub.3)CH.sub.2--, A.sup.2 and Ar.sup.3 are phenyl, Q is
a covalent bond, then the linker X is not linked to the A.sup.2
group through an oxygen atom.
[0070] More preferably, each linker, X, in the multibinding
compound of Formula (I) independently has the formula:
--X.sup.a-Z-(Y.sup.a-Z).sub.m-X.sup.a--
wherein
[0071] m is an integer of from 0 to 20;
[0072] X.sup.a at each separate occurrence is selected from the
group consisting of --O--, --S--, --NR--, --C(O)--, --C(O)O--,
--OC(O)--, --C(O)NR--, --NRC(O)--, C(S), --C(S)O--, --C(S)NR--,
--NRC(S)--, or a covalent bond where k is as defined below;
[0073] Z at each separate occurrence is selected from the group
consisting of alkylene, substituted alkylene, cycloalkylene,
substituted cycloalkylene, alkenylene, substituted alkenylene,
alkynylene, substituted alkynylene, cycloalkenylene, substituted
cycloalkenylene, arylene, heteroarylene, heterocyclene, or a
covalent bond;
[0074] each Y.sup.a at each separate occurrence is selected from
the group consisting of -O--; --C(O)--, --OC(O)--, --C(O)O--,
--NR--, --S(O).sub.n--, --C(O)NR'--, --NR'C(O)--, --NR'C(O)NR'--,
--NR'C(S)NR'--, --C(.dbd.NR')--NR'--, --NR'--C(.dbd.NR')--,
--OC(O)--NR'--, --NR'--C(O)--O--, --N.dbd.C(X.sup.a)--NR'--,
--NR'--C(X.sup.a).dbd.N--, --P(O)(OR')--O--, --O--P(O)(OR')--,
--S(O).sub.nCR'R''--, --S(O).sub.n--NR'--, --NR'--S(O).sub.n--,
--S--S--, and a covalent bond; where n is 0, 1 or 2; R, R' and R''
at each separate occurrence are selected from the group consisting
of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted
cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted
cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl and
heterocyclic, and X.sup.a is as defined above.
[0075] Preferably p is less than q.
[0076] In still another aspect, this invention is directed to
general synthetic methods for generating large libraries of diverse
multimeric compounds which multimeric compounds are candidates for
possessing multibinding properties for .beta.2 adrenergic receptor.
The diverse multimeric compound libraries provided by this
invention are synthesized by combining a linker or linkers with a
ligand or ligands to provide for a library of multimeric compounds
wherein the linker and ligand each have complementary functional
groups permitting covalent linkage. The library of linkers is
preferably selected to have diverse properties such as valency,
linker length, linker geometry and rigidity, hydrophilicity or
hydrophobicity, 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.
[0077] This invention is also directed to libraries of diverse
multimeric compounds which multimeric compounds are candidates for
possessing multibinding properties for .beta.2 adrenergic receptor.
These libraries are prepared via the methods described above and
permit the rapid and efficient evaluation of what molecular
constraints impart multibinding properties to a ligand or a class
of ligands targeting a receptor.
[0078] Accordingly, in one of its method aspects, this invention is
directed to a method for identifying multimeric ligand compounds
possessing multibinding properties for .beta..sub.2 adrenergic
receptor which method comprises:
[0079] (a) identifying a ligand or a mixture of ligands wherein
each ligand contains at least one reactive functionality;
[0080] (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;
[0081] (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
[0082] (d) assaying the multimeric ligand compounds produced in (c)
above to identify multimeric ligand compounds possessing
multibinding properties for .beta.2 adrenergic receptor.
[0083] In another of its method aspects, this invention is directed
to a method for identifying multimeric ligand compounds possessing
multibinding properties for .beta.2 adrenergic receptor which
method comprises:
[0084] (a) identifying a library of ligands wherein each ligand
contains at least one reactive functionality;
[0085] (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;
[0086] (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
[0087] (d) assaying the multimeric ligand compounds produced in (c)
above to identify multimeric ligand compounds possessing
multibinding properties for .beta.2 adrenergic receptor.
[0088] The preparation of the multimeric ligand compound library is
achieved by either the sequential or concurrent combination of the
two or more stoichiometric equivalents of the ligands identified in
(a) with the linkers identified in (b). Sequential addition is
preferred when a mixture of different ligands is employed to ensure
heterodimeric or multimeric compounds are prepared. Concurrent
addition of the ligands occurs when at least a portion of the
multimer compounds prepared are homomultimeric compounds.
[0089] 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).
[0090] In one of its composition aspects, this invention is
directed to a library of multimeric ligand compounds which may
possess multivalent properties for .beta.2 adrenergic receptor
which library is prepared by the method comprising:
[0091] (a) identifying a ligand or a mixture of ligands wherein
each ligand contains at least one reactive functionality;
[0092] (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
[0093] (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.
[0094] In another of its composition aspects, this invention is
directed to a library of multimeric ligand compounds which may
possess multivalent properties for .beta.2 adrenergic receptor
which library is prepared by the method comprising:
[0095] (a) identifying a library of ligands wherein each ligand
contains at least one reactive functionality;
[0096] (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
[0097] (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.
[0098] In a preferred embodiment, the library of linkers employed
in either the methods or the library aspects of this invention is
selected from the group comprising flexible linkers, rigid linkers,
hydrophobic linkers, hydrophilic linkers, linkers of different
geometry, acidic linkers, basic linkers, linkers of different
polarization and amphiphilic linkers. For example, in one
embodiment, each of the linkers in the linker library may comprise
linkers of different chain length and/or having different
complementary reactive groups. Such linker lengths can preferably
range from about 2 to 100 .ANG..
[0099] In another preferred embodiment, the 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 ligand.
[0100] 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).
[0101] In addition to the combinatorial methods described herein,
this invention provides for an interactive 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 .beta.2 adrenergic receptor which
method comprises:
[0102] (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;
[0103] (b) assaying said first collection or iteration of
multimeric compounds to assess which if any of said multimeric
compounds possess multibinding properties for .beta.2 adrenergic
receptor;
[0104] (c) repeating the process of (a) and (b) above until at
least one multimeric compound is found to possess multibinding
properties for .beta.2 adrenergic receptor;
[0105] (d) evaluating what molecular constraints imparted
multibinding properties for .beta.2 adrenergic receptor to the
multimeric compound or compounds found in the first iteration
recited in (a)-(c) above;
[0106] (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;
[0107] (f) evaluating what molecular constraints imparted enhanced
multibinding properties to the multimeric compound or compounds
found in the second collection or iteration recited in (e)
above;
[0108] (g) optionally repeating steps (e) and (f) to further
elaborate upon said molecular constraints.
[0109] Preferably, steps (e) and (f) are repeated at least two
times, more preferably at from 2-50 times, even more preferably
from 3 to 50 times, and still more preferably at least 5-50
times.
BRIEF DESCRIPTION OF THE DRAWINGS
[0110] FIG. 1 illustrates examples of multibinding compounds
comprising 2 ligands attached in different formats to a linker.
[0111] FIG. 2 illustrates examples of multibinding compounds
comprising 3 ligands attached in different formats to a linker.
[0112] FIG. 3 illustrates examples of multibinding compounds
comprising 4 ligands attached in different formats to a linker.
[0113] FIG. 4 illustrates examples of multibinding compounds
comprising >4 ligands attached in different formats to a
linker.
[0114] FIGS. 5-15 illustrate synthesis of compounds of Formula
(I).
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0115] This invention is directed to multibinding compounds which
are .beta.2 adrenergic receptor agonists pharmaceutical
compositions containing such compounds and methods for treating
diseases mediated by .beta.2 adrenergic receptor in mammals. When
discussing such compounds, compositions or methods, the following
terms have the following meanings unless otherwise indicated. Any
undefined terms have their art recognized meanings.
[0116] The term "alkyl" refers to a monoradical branched or
unbranched saturated hydrocarbon chain preferably having from 1 to
40 carbon atoms, more preferably 1 to 10 carbon atoms, and even
more preferably 1 to 6 carbon atoms. This term is exemplified by
groups such as methyl, ethyl, n-propyl, iso-propyl, n-butyl,
iso-butyl, n-hexyl, n-decyl, tetradecyl, and the like.
[0117] The term "substituted alkyl" refers to an alkyl group as
defined above, having from 1 to 5 substituents, and preferably 1 to
3 substituents, selected from the group consisting of alkoxy,
substituted alkoxy, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy,
amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl,
azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl,
carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,
thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy,
heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy,
hydroxyamino, alkoxyamino, nitro, --SO-alkyl, --SO-substituted
alkyl, --SO-aryl, --SO-heteroaryl, --SO.sub.2-alkyl,
--SO.sub.2-substituted alkyl, --SO.sub.2-aryl and
--SO.sub.2-heteroaryl. This term is exemplified by groups such as
hydroxymethyl, hydroxyethyl, hydroxypropyl, 2-aminoethyl,
3-aminopropyl, 2-methylaminoethyl, 3-dimethylaminopropyl,
2-sulfonamidoethyl, 2-carboxyethyl, and the like.
[0118] The term "alkylene" refers to a diradical of a branched or
unbranched saturated hydrocarbon chain, preferably having from 1 to
40 carbon atoms, more preferably 1 to 10 carbon atoms and even more
preferably 1 to 6 carbon atoms. This term is exemplified by groups
such as methylene (--CH.sub.2--), ethylene (--CH.sub.2CH.sub.2--),
the propylene isomers (e.g., --CH.sub.2CH.sub.2CH.sub.2-- and
--CH(CH.sub.3)CH.sub.2--) and the like.
[0119] The term "substituted alkylene" refers to an alkylene group,
as defined above, having from 1 to 5 substituents, and preferably 1
to 3 substituents, selected from the group consisting of alkoxy,
substituted alkoxy, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy,
amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl,
azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl,
carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,
thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy,
heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy,
hydroxyamino, alkoxyamino, nitro, --SO-alkyl, --SO-substituted
alkyl, --SO-aryl, --SO-heteroaryl, --SO.sub.2-alkyl,
--SO.sub.2-substituted alkyl, --SO.sub.2-aryl and
--SO.sub.2-heteroaryl. Additionally, such substituted alkylene
groups include those where 2 substituents on the alkylene group are
fused to form one or more cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, aryl, heterocyclic or
heteroaryl groups fused to the alkylene group. Preferably such
fused groups contain from 1 to 3 fused ring structures.
[0120] The term "alkaryl" or "aralkyl" refers to the groups
-alkylene-aryl and -substituted alkylene-aryl where alkylene,
substituted alkylene and aryl are defined herein. Such alkaryl
groups are exemplified by benzyl, phenethyl and the like.
[0121] The term "heteroaralkyl" refers to the groups
-alkylene-heteroaryl and -substituted alkylene-heteroaryl where
alkylene, substituted alkylene and heteroaryl are defined herein.
Such heteroaralkyl groups are exemplified by pyridin-3-lmethyl,
pyridin-3-ylmethyloxy, and the like.
[0122] The term "alkoxy" refers to the groups alkyl-O--,
alkenyl-O--, cycloalkyl-O--, cycloalkenyl-O--, and alkynyl-O--,
where alkyl, alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as
defined herein. Preferred alkoxy groups are alkyl-O-- and include,
by way of example, methoxy, ethoxy, n-propoxy, iso-propoxy,
n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy,
1,2-dimethylbutoxy, and the like.
[0123] The term "substituted alkoxy" refers to the groups
substituted alkyl-O--, substituted alkenyl-O--, substituted
cycloalkyl-O--, substituted cycloalkenyl-O--, and substituted
alkynyl-O-- where substituted alkyl, substituted alkenyl,
substituted cycloalkyl, substituted cycloalkenyl and substituted
alkynyl are as defined herein.
[0124] The term "alkenyl" refers to a monoradical of a branched or
unbranched unsaturated hydrocarbon group preferably having from 2
to 40 carbon atoms, more preferably 2 to 10 carbon atoms and even
more preferably 2 to 6 carbon atoms and having at least 1 and
preferably from 1-6 sites of vinyl unsaturation. Preferred alkenyl
groups include ethenyl (--CH.dbd.CH.sub.2), n-propenyl
(--CH.sub.2CH.dbd.CH.sub.2), iso-propenyl
(--C(CH.sub.3).dbd.CH.sub.1), and the like.
[0125] The term "substituted alkenyl" refers to an alkenyl group as
defined above having from 1 to 5 substituents, and preferably 1 to
3 substituents, selected from the group consisting of alkoxy,
substituted alkoxy, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy,
amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl,
azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl,
carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,
thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy,
heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy,
hydroxyamino, alkoxyamino, nitro, --SO-alkyl, --SO-substituted
alkyl, --SO-aryl, --SO-heteroaryl, --SO.sub.2-alkyl,
--SO.sub.2-substituted alkyl, --SO.sub.2-aryl and
--SO.sub.2-heteroaryl.
[0126] The term "alkenylene" refers to a diradical of a branched or
unbranched unsaturated hydrocarbon group preferably having from 2
to 40 carbon atoms, more preferably 2 to 10 carbon atoms and even
more preferably 2 to 6 carbon atoms and having at least 1 and
preferably from 1-6 sites of vinyl unsaturation. This term is
exemplified by groups such as ethenylene (--CH.dbd.CH--), the
propenylene isomers (e.g., --CH.sub.2CH.dbd.CH--,
--C(CH.sub.3).dbd.CH--, and the like.
[0127] The term "substituted alkenylene" refers to an alkenylene
group as defined above having from 1 to 5 substituents, and
preferably from 1 to 3 substituents, selected from the group
consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted
cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl,
acylamino, acyloxy, amino, substituted amino, aminoacyl,
aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto,
thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,
thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy,
aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic,
heterocyclooxy, hydroxyamino, alkoxyamino, nitro, --SO-alkyl,
--SO-substituted alkyl, --SO-aryl, --SO-heteroaryl,
--SO.sub.2-alkyl, --SO.sub.2-substituted alkyl, --SO.sub.2-aryl and
--SO.sub.2-heteroaryl. Additionally, such substituted alkenylene
groups include those where 2 substituents on the alkenylene group
are fused to form one or more cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, aryl, heterocyclic or
heteroaryl groups fused to the alkenylene group.
[0128] The term "alkynyl" refers to a monoradical of an unsaturated
hydrocarbon preferably having from 2 to 40 carbon atoms, more
preferably 2 to 20 carbon atoms and even more preferably 2 to 6
carbon atoms and having at least 1 and preferably from 1-6 sites of
acetylene (triple bond) unsaturation. Preferred alkynyl groups
include ethynyl (--C.ident.CH), propargyl (--CH.sub.2C.ident.CH)
and the like.
[0129] The term "substituted alkynyl" refers to an alkynyl group as
defined above having from 1 to 5 substituents, and preferably 1 to
3 substituents, selected from the group consisting of alkoxy,
substituted alkoxy, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy,
amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl,
azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl,
carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,
thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy,
heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy,
hydroxyamino, alkoxyamino, nitro, --SO-alkyl, --SO-substituted
alkyl, --SO-aryl, --SO-heteroaryl, --SO.sub.2-alkyl,
--SO.sub.2-substituted alkyl, --SO.sub.2-aryl, and
--SO.sub.2-heteroaryl.
[0130] The term "alkynylene" refers to a diradical of an
unsaturated hydrocarbon preferably having from 2 to 40 carbon
atoms, more preferably 2 to 10 carbon atoms and even more
preferably 2 to 6 carbon atoms and having at least 1 and preferably
from 1-6 sites of acetylene (triple bond) unsaturation. Preferred
alkynylene groups include ethynylene (--C.ident.C--), propargylene
(--CH.sub.2C.ident.C--) and the like.
[0131] The term "substituted alkynylene" refers to an alkynylene
group as defined above having from 1 to 5 substituents, and
preferably 1 to 3 substituents, selected from the group consisting
of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy,
amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl,
azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl,
carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,
thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy,
heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy,
hydroxyamino, alkoxyamino, nitro, --SO-alkyl, --SO-substituted
alkyl, --SO-aryl, --SO-heteroaryl, --SO.sub.2-alkyl,
--SO.sub.2-substituted alkyl, --SO.sub.2-aryl and
--SO.sub.2-heteroaryl
[0132] The term "acyl" refers to the groups HC(O)--, alkyl-C(O)--,
substituted alkyl-C(O)--, alkenyl-C(O)--, substituted
alkenyl-C(O)--, cycloalkyl-C(O)--, substituted cycloalkyl-C(O)--,
cycloalkenyl-C(O)--, substituted cycloalkenyl-C(O)--, aryl-C(O)--,
heteroaryl-C(O)-- and heterocyclic-C(O)-- where alkyl, substituted
alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted
cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,
heteroaryl and heterocyclic are as defined herein.
[0133] The term "acylamino" or "aminocarbonyl" refers to the group
--C(O)NRR where each R is independently hydrogen, alkyl,
substituted alkyl, aryl, heteroaryl, heterocyclic or where both R
groups are joined to form a heterocyclic group (e.g., morpholino)
wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic
are as defined herein.
[0134] The term "sulfonylamino" refers to the group
--NRSO.sub.2R.sup.a where R is hydrogen, alkyl, substituted alkyl,
aralkyl, or heteroaralkyl, and R.sup.1 is alkyl, substituted alkyl,
amino, or substituted amino wherein alkyl, substituted alkyl,
aralkyl, heteroaralkyl and substituted amino are as defined
herein.
[0135] The term "aminoacyl" refers to the group --NRC(O)R where
each R is independently hydrogen, alkyl, substituted alkyl,
alkenyl, substituted alkenyl, amino, substituted amino, aryl,
heteroaryl, or heterocyclic wherein alkyl, substituted alkyl,
alkenyl, substituted alkenyl, aryl, heteroaryl and heterocyclic are
as defined herein.
[0136] The term "aminoacyloxy" or "alkoxycarbonylamino" 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.
[0137] The term "acyloxy" refers to the groups alkyl-C(O)O--,
substituted alkyl-C(O)O--, cycloalkyl-C(O)O--, substituted
cycloalkyl-C(O)O--, aryl-C(O)O--, heteroaryl-C(O)O--, and
heterocyclic-C(O)O-- wherein alkyl, substituted alkyl, cycloalkyl,
substituted cycloalkyl, aryl, heteroaryl, and heterocyclic are as
defined herein.
[0138] The term "aryl" refers to an unsaturated aromatic
carbocyclic group of from 6 to 20 carbon atoms having a single ring
(e.g., phenyl) or multiple condensed (fused) rings (e.g., naphthyl
or anthryl). The aryl group may optionally be fused to a
heterocyclic or cycloalkyl group. Preferred aryls include phenyl,
naphthyl and the like. Unless otherwise constrained by the
definition for the aryl substituent, such aryl groups can
optionally be substituted with from 1 to 5 substituents, preferably
1 to 3 substituents, selected from the group consisting of acyloxy,
hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, substituted alkyl, substituted alkoxy, substituted
alkenyl, substituted alkynyl, substituted cycloalkyl, substituted
cycloalkenyl, amino, substituted amino, aminoacyl, acylamino,
sulfonylamino, alkaryl, aryl, aryloxy, azido, carboxyl,
carboxylalkyl, cyano, halo, nitro, heteroaryl, heteroaryloxy,
heterocyclic, heterocyclooxy, aminoacyloxy, oxyacylamino,
thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy,
--SO-alkyl, --SO-substituted alkyl, --SO-aryl, --SO-heteroaryl,
--SO.sub.2-alkyl, --SO.sub.2-substituted alkyl, --SO.sub.2-aryl,
--SO.sub.2-heteroaryl and trihalomethyl. Preferred aryl
substituents include alkyl, alkoxy, halo, cyano, nitro,
trihalomethyl, and thioalkoxy.
[0139] 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.
[0140] The term "arylene" refers to the diradical derived from aryl
(including substituted aryl) as defined above and is exemplified by
1,2-phenylene, 1,3-phenylene, 1,4-phenylene, 1,2-naphthylene and
the like.
[0141] The term "amino" refers to the group --NH.sub.2.
[0142] The term "substituted amino" refers to the group --NRR where
each R is independently selected from the group consisting of
hydrogen, alkyl, substituted alkyl, acyl, cycloalkyl, substituted
cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted
cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl and
heterocyclic provided that both R's are not hydrogen.
[0143] The term "carboxyalkyl" or "alkoxycarbonyl" refers to the
groups "--C(O)O-alkyl", "--C(O)O-substituted alkyl",
"--C(O)O-cycloalkyl", "--C(O)O-substituted cycloalkyl",
"--C(O)O-alkenyl", "--C(O)O-substituted alkenyl", "--C(O)O-alkynyl"
and "--C(O)O-substituted alkynyl" where alkyl, substituted alkyl,
cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl,
alkynyl and substituted alkynyl are as defined herein.
[0144] The term "cycloalkyl" refers to cyclic alkyl groups of from
3 to 20 carbon atoms having a single cyclic ring or multiple
condensed rings, said cycloalkyl group may optionally be fused to
an aryl or heteroaryl group. 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.
[0145] The term "substituted cycloalkyl" refers to cycloalkyl
groups having from 1 to 5 substituents, and preferably 1 to 3
substituents, selected from the group consisting of alkyl,
substituted alkyl, alkenyl, substituted alkenyl, alkoxy,
substituted alkoxy, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy,
amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl,
azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl,
carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,
thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy,
heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy,
hydroxyamino, alkoxyamino, nitro, --SO-alkyl, --SO-substituted
alkyl, --SO-aryl, --SO-heteroaryl, --SO.sub.2-alkyl,
--SO.sub.2-substituted alkyl, --SO.sub.2-aryl and
--SO.sub.2-heteroaryl.
[0146] The term "cycloalkenyl" refers to cyclic alkenyl groups of
from 4 to 20 carbon atoms having a single cyclic ring and at least
one point of internal unsaturation. Examples of suitable
cycloalkenyl groups include, for instance, cyclobut-2-enyl,
cyclopent-3-enyl, cyclooct-3-enyl and the like.
[0147] The term "substituted cycloalkenyl" refers to cycloalkenyl
groups having from 1 to 5 substituents, and preferably 1 to 3
substituents, selected from the group consisting of alkyl,
substituted alkyl, alkenyl, substituted alkenyl, alkoxy,
substituted alkoxy, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy,
amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl,
azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl,
carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,
thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy,
heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy,
hydroxyamino, alkoxyamino, nitro, --SO-alkyl, --SO-substituted
alkyl, --SO-aryl, --SO-heteroaryl, --SO.sub.2-alkyl,
--SO.sub.2-substituted alkyl, --SO.sub.2-aryl and
--SO.sub.2-heteroaryl.
[0148] The term "halo" or "halogen" refers to fluoro, chloro, bromo
and iodo.
[0149] 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). The heteroaryl ring may optionally be fused to a
cycloalkyl or heterocyclyl ring. Unless otherwise constrained by
the definition for the heteroaryl substituent, such heteroaryl
groups can be optionally substituted with 1 to 5 substituents,
preferably 1 to 3 substituents, selected from the group consisting
of acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl,
cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy,
substituted alkenyl, substituted alkynyl, substituted cycloalkyl,
substituted cycloalkenyl, amino, substituted amino, aminoacyl,
acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl,
cyano, halo, nitro, heteroaryl, heteroaryloxy, heterocyclic,
heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted
thioalkoxy, thioaryloxy, thioheteroaryloxy, --SO-alkyl,
--SO-substituted alkyl, --SO-aryl, --SO-heteroaryl,
--SO.sub.2-alkyl, --SO.sub.2-substituted alkyl, --SO.sub.2-aryl,
--SO.sub.2-heteroaryl and trihalomethyl. Preferred heteroaryl
substituents include alkyl, alkoxy, halo, cyano, nitro,
trihalomethyl, and thioalkoxy. Such heteroaryl groups can have a
single ring (e.g., pyridyl or furyl) or multiple condensed rings
(e.g., indolizinyl or benzothienyl). Preferred heteroaryls include
pyridyl, pyrrolyl and furyl.
[0150] The term "heteroaryloxy" refers to the group
heteroaryl-O--.
[0151] The term "heteroarylene" refers to the diradical group
derived from heteroaryl (including substituted heteroaryl), as
defined above, and is exemplified by the groups 2,6-pyridylene,
2,4-pyridinylene, 1,2-quinolinylene, 1,8-quinolinylene,
1,4-benzofuranylene, 2,5-pyridinylene, 2,5-indolenyl, and the
like.
[0152] The term "cycloalkylene" refers to the diradical group
derived from cycloalkyl, as defined above, and is exemplified by
the groups 1,6-cyclohexylene, 1,3-cyclopentylene, and the like.
[0153] The term "substituted cycloalkylene" refers to the diradical
group derived from substituted cycloalkyl, as defined above.
[0154] The term "cycloalkenylene" refers to the diradical group
derived from cycloalkyl, as defined above.
[0155] The term "substituted cycloalkenylene" refers to the
diradical group derived from substituted cycloalkenyl, as defined
above.
[0156] The term "heterocycle" or "heterocyclyl" refers to a
monoradical saturated unsaturated group having a single ring or
multiple condensed rings, from 1 to 40 carbon atoms and from 1 to
10 hetero atoms, preferably 1 to 4 heteroatoms, selected from
nitrogen, sulfur, phosphorus, and/or oxygen within the ring and
further wherein one, two, or three of the ring carbon atoms may
optionally be replaced with a carbonyl group (i.e., a keto group).
Unless otherwise constrained by the definition for the heterocyclic
substituent, such heterocyclic groups can be optionally substituted
with 1 to 5, and preferably 1 to 3 substituents, selected from the
group consisting of alkoxy, substituted alkoxy, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl,
aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto,
thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,
thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy,
aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic,
heterocyclooxy, hydroxyamino, alkoxyamino, nitro, --SO-alkyl,
--SO-substituted alkyl, --SO-aryl, --SO-heteroaryl,
--SO.sub.2-alkyl, --SO.sub.2-substituted alkyl, --SO.sub.2-aryl and
--SO.sub.2-heteroaryl. Such heterocyclic groups can have a single
ring or multiple condensed rings. Preferred heterocyclics include
morpholino, piperidinyl, and the like.
[0157] Examples of heteroaryls and heterocycles include, but are
not limited to, pyrrole, thiophene, furan, 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, pyrrolidine, piperidine,
piperazine, indoline, morpholine, tetrahydrofuranyl,
tetrahydrothiophene, and the like as well as N-alkoxy-nitrogen
containing heterocycles.
[0158] The term "heterocyclooxy" refers to the group
heterocyclic-O--.
[0159] The term "thioheterocyclooxy" refers to the group
heterocyclic-S--.
[0160] The term "heterocyclene" refers to the diradical group
formed from a heterocycle, as defined herein, and is exemplified by
the groups 2,6-morpholino, 2,5-morpholino and the like.
[0161] The term "oxyacylamino" or "aminocarbonyloxy" refers to the
group --OC(O)NRR where each R is independently hydrogen, alkyl,
substituted alkyl, aryl, heteroaryl, or heterocyclic wherein alkyl,
substituted alkyl, aryl, heteroaryl and heterocyclic are as defined
herein.
[0162] The term "spiro-attached cycloalkyl group" refers to a
cycloalkyl group joined to another ring via one carbon atom common
to both rings.
[0163] The term "thiol" refers to the group --SH.
[0164] The term "thioalkoxy" or "alkylthio" refers to the group
--S-alkyl.
[0165] The term "substituted thioalkoxy" refers to the group
--S-substituted alkyl.
[0166] 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.
[0167] 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.
[0168] As to any of the above groups which contain one or more
substituents, it is understood, of course, that such groups do not
contain any substitution or substitution patterns which are
sterically impractical and/or synthetically non-feasible. In
addition, the compounds of this invention include all
stereochemical isomers arising from the substitution of these
compounds.
[0169] 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.
[0170] Pharmaceutically-acceptable base addition salts can be
prepared from inorganic and organic bases. Salts derived from
inorganic bases, include by way of example only, sodium, potassium,
lithium, ammonium, calcium and magnesium salts. Salts derived from
organic bases include, but are not limited to, salts of primary,
secondary and tertiary amines, such as alkyl amines, dialkyl
amines, trialkyl amines, substituted alkyl amines, di(substituted
alkyl)amines, tri(substituted alkyl)amines, alkenyl amines,
dialkenyl amines, trialkenyl amines, substituted alkenyl amines,
di(substituted alkenyl)amines, tri(substituted alkenyl)amines,
cycloalkyl amines, di(cycloalkyl)amines, tri(cycloalkyl)amines,
substituted cycloalkyl amines, disubstituted cycloalkyl amine,
trisubstituted cycloalkyl amines, cycloalkenyl amines,
di(cycloalkenyl)amines, tri(cycloalkenyl)amines, substituted
cycloalkenyl amines, disubstituted cycloalkenyl anine,
trisubstituted cycloalkenyl amines, aryl amines, diaryl amines,
triaryl amines, heteroaryl amines, diheteroaryl amines,
triheteroaryl amines, heterocyclic amines, diheterocyclic amines,
triheterocyclic amines, mixed di- and tri-amines where at least two
of the substituents on the amine are different and are selected
from the group consisting of alkyl substituted alkyl, alkenyl,
substituted alkenyl, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl,
heterocyclic, and the like. Also included are amines where the two
or three substituents, together with the amino nitrogen, form a
heterocyclic or heteroaryl group. Examples of suitable amines
include, by way of example only, isopropylamine, trimethyl amine,
diethyl amine, tri(iso-propyl)amine, tri(n-propyl)amine,
ethanolamine, 2-dimethylaminoethanol, tromethamine, lysine,
arginine, histidine, caffeine, procaine, hydrabamine, choline,
betaine, ethylenediamine, glucosamine, N-alkylglucamines,
theobromine, purines, piperazine, piperidine, morpholine,
N-ethylpiperidine, and the like. It should also be understood that
other carboxylic acid derivatives would be useful in the practice
of this invention, for example, carboxylic acid amides, including
carboxamides, lower alkyl carboxamides, dialkyl carboxamides, and
the like.
[0171] 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, maleic acid, fumaric acid, tartaric acid, citric
acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic
acid, ethanesulfonic acid, p-toluene-sulfonic acid, salicylic acid,
naphthoic acid, 2-hydroxynaphthoic acid, and the like.
[0172] The term "pharmaceutically-acceptable cation" refers to the
cation of a pharmaceutically-acceptable salt
[0173] The term "library" refers to at least 3, preferably from 102
to 109 and more preferably from 10.sup.2 to 10.sup.4 multimeric
compounds. Preferably, these compounds are prepared as a
multiplicity of compounds in a single solution or reaction mixture
which permits facile synthesis thereof. In one embodiment, the
library of multimeric compounds can be directly assayed for
multibinding properties. In another embodiment, each member of the
library of multimeric compounds is first isolated and, optionally,
characterized. This member is then assayed for multibinding
properties.
[0174] The term "collection" refers to a set of multimeric
compounds which are prepared either sequentially or concurrently
(e.g., combinatorially). The collection comprises at least 2
members; preferably from 2 to 10.sup.9 members and still more
preferably from 10 to 10.sup.4 members.
[0175] 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).
[0176] The term "pseudohalide" refers to functional groups which
react in displacement reactions in a manner similar to a halogen.
Such functional groups include, by way of example, mesyl, tosyl,
azido and cyano groups.
[0177] 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 (including intermediates thereof)
prevents reactions from occurring at these groups and which
protecting group can be removed by conventional chemical or
enzymatic steps to reestablish the hydroxyl, thiol, amino or
carboxyl group (See., T. W. Greene and P. G. H. Wuts, "Protective
Groups in Organic Synthesis", 2.sup.nd Ed.). The particular
removable blocking group employed is not critical and preferred
removable hydroxyl blocking groups include conventional
substituents such as allyl, benzyl, acetyl, chloroacetyl,
thiobenzyl, benzylidine, phenacyl, t-butyl-diphenylsilyl and any
other group that can be introduced chemically onto a hydroxyl
functionality and later selectively removed either by chemical or
enzymatic methods in mild conditions compatible with the nature of
the product. Preferred removable thiol blocking groups include
disulfide groups, acyl groups, benzyl groups, and the like.
[0178] Preferred removable amino blocking groups include
conventional substituents such as t-butyoxycarbonyl (t-BOC),
benzyloxycarbonyl (CBZ), fluorenylmethoxy-carbonyl (FMOC),
allyloxycarbonyl (ALOC), and the like which can be removed by
conventional conditions compatible With the nature of the
product.
[0179] Preferred carboxyl protecting groups include esters such as
methyl, ethyl, propyl, t-butyl etc. which can be removed by mild
conditions compatible with the nature of the product.
[0180] The term "optional" or "optionally" means that the
subsequently described event, circumstance or substituent may or
may not occur, and that the description includes instances where
said event or circumstance occurs and instances where it does
not.
[0181] The term "ligand" or "ligands" as used herein denotes a
compound that is a binding partner for a .beta.2 adrenergic
receptor and is bound thereto by complementarity. Preferred ligands
are those that are either .beta.2 adrenergic receptor agonists,
partial agonists, or antagonists. The specific region or regions of
the ligand that is (are) recognized by the receptor is designated
as the "ligand domain". A ligand may be either capable of binding
to the receptor by itself, or may require the presence of one or
more non-ligand components for binding (e.g., Ca.sup.+2, Mg.sup.+2
or a water molecule is required for the binding of a ligand to
various ligand binding sites). Examples of ligands useful in this
invention are described herein. Those skilled in the art will
appreciate that portions of the ligand structure that are not
essential for specific molecular recognition and binding activity
may be varied substantially, replaced or substituted with unrelated
structures (for example, with ancillary groups as defined below)
and, in some cases, omitted entirely without affecting the binding
interaction. The primary requirement for a ligand is that it has a
ligand domain as defined above. It is understood that the term
ligand is not intended to be limited to compounds known to be
useful in binding to .beta.2 adrenergic receptor (e.g., known
drugs). Those skilled in the art will understand that the term
ligand can equally apply to a molecule that is not normally
associated with .beta.2 adrenergic receptor binding properties. In
addition, it should be noted that ligands that exhibit marginal
activity or lack useful activity as monomers can be highly active
as multivalent compounds because of the benefits conferred by
multivalency.
[0182] The term "ligand" or "ligands" as used herein is intended to
include the racemic forms of the ligands as well as individual
enantiomers and diasteromers and non-racemic mixtures thereof.
[0183] The term "multibinding compound or agent" refers to a
compound that is capable of multivalency, as defined below, and
which has 2-10 ligands covalently bound to one or more linkers. In
all cases, each ligand and linker in the multibinding compound is
independently selected such that the multibinding compound includes
both symmetric compounds (i.e., where each ligand as well as each
linker is identical) and asymmetric compounds (i.e., where at least
one of the ligands is different from the other ligand(s) and/or at
least one linker is different from the other linker(s)).
Multibinding compounds provide a biological and/or therapeutic
effect greater than the aggregate of unlinked ligands equivalent
thereto which are made available for binding. That is to say that
the biological and/or therapeutic effect of the ligands attached to
the multibinding compound is greater than that achieved by the same
amount of unlinked ligands made available for binding to the ligand
binding sites (receptors). The phrase "increased biological or
therapeutic effect" includes, for example: increased affinity,
increased selectivity for target, increased specificity for target,
increased potency, increased efficacy, decreased toxicity, improved
duration of activity or action, increased ability to kill cells
such as fungal pathogens, cancer cells, etc., decreased side
effects, increased therapeutic index, improved bioavailibity,
improved pharmacokinetics, improved activity spectrum, and the
like. The multibinding compounds of this invention will exhibit at
least one and preferably more than one of the above-mentioned
affects.
[0184] The term "univalency" as used herein refers to a single
binding interaction between one ligand as defined herein with one
ligand binding site as defined herein. It should be noted that a
compound having multiple copies of a ligand (or ligands) exhibit
univalency when only one ligand is interacting with a ligand
binding site. Examples of univalent interactions are depicted
below.
##STR00016##
[0185] The term "multivalency" as used herein refers to the
concurrent binding of from 2 to 10 linked ligands (which may be the
same or different) and two or more corresponding receptors (ligand
binding sites) which may be the same or different.
[0186] For example, two ligands connected through a linker that
bind concurrently to two ligand binding sites would be considered
as bivalency; three ligands thus connected would be an example of
trivalency. An example of trivalent binding, illustrating a
multibinding compound bearing three ligands versus a monovalent
binding interaction, is shown below:
##STR00017##
[0187] It should be understood that not all compounds that contain
multiple copies of a ligand attached to a linker or to linkers
necessarily exhibit the phenomena of multivalency, i.e., that the
biological and/or therapeutic effect of the multibinding agent is
greater than the sum of the aggregate of unlinked ligands made
available for binding to the ligand binding site (receptor). For
multivalency to occur, the ligands that are connected by a linker
or linkers have to be presented to their ligand binding sites by
the linker(s) in a specific manner in order to bring about the
desired ligand-orienting result, and thus produce a multibinding
event.
[0188] Furthermore, the multibinding compound of the present
invention can be composed of ligands that are all .beta.2
adrenergic receptor agonists, partial agonists, or it can be
composed of ligands that are selected from .beta.2 adrenergic
receptor agonists and antagonists provided that the multi-binding
compounds exhibits an overall .beta.2 adrenergic receptor agonistic
or partial agonistic activity. A multibinding compound that
exhibits partial agonist activity at adrenoceptors may provide
advantages over a compound that exhibits full agonism. Partial
agonism may result in a reduction of the rate of receptor
desensitization, receptor recycling, or receptor expression in
mammalian tissue. This may result in increased therapeutic benefits
from using such an agonist versus a compound which behaves as a
full agonist for the chronic treatment of pathological conditions
or diseases. A multibinding compound may also, or separately, act
as a tissue-selective partial agonist. For example, a multibinding
compound with .beta.2 adrenoceptor agonist activity may exhibit a
full maximal response in relaxing smooth muscle cells in bronchial
tissue but exhibit a partial maximal response to adrenoceptor
agonism in cardiac tissue. Thus, a multibinding compound that is a
tissue-selective partial agonist may provide a lower incidence of
undesired side effects such as positive chronotropism and increases
in cardiac output.
[0189] The term "potency" refers to the minimum concentration at
which a ligand is able to achieve a desirable biological or
therapeutic effect. The potency of a ligand is typically
proportional to its affinity for its ligand binding site. In some
cases, the potency may be non-linearly correlated with its
affinity. In comparing 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 a human patient). The finding that
the multibinding agent produces an equivalent biological or
therapeutic effect at a lower concentration than the aggregate
unlinked ligand is indicative of enhanced potency.
[0190] The term "selectivity" or "specificity" is a measure of the
binding preferences of a ligand for different ligand binding sites
(receptors). The selectivity of a ligand with respect to its target
ligand binding site relative to another ligand binding site is
given by the ratio of the respective values of K.sub.d (i.e., the
dissociation constants for each ligand-receptor complex) or, in
cases where a biological effect is observed below the K.sub.d, the
ratio of the respective EC.sub.50's (i.e., the concentrations that
produce 50% of the maximum response for the ligand interacting with
the two distinct ligand binding sites (receptors)).
[0191] The term "ligand binding site" denotes the site on the
.beta.-adrenergic receptor that recognizes a ligand domain and
provides a binding partner for the ligand. The ligand binding site
may be defined by monomeric or multimeric structures. This
interaction may be capable of producing a unique biological effect,
for example, agonism, antagonism, and modulatory effects or it may
maintain an ongoing biological event, and the like.
[0192] It should be recognized that the ligand binding sites of the
receptor that participate in biological multivalent binding
interactions are constrained to varying degrees by their intra- and
inter-molecular associations. For example, ligand binding sites may
be covalently joined to a single structure, noncovalently
associated in a multimeric structure, embedded in a membrane or
polymeric matrix, and so on and therefore have less translational
and rotational freedom than if the same structures were present as
monomers in solution.
[0193] The terms "agonism", "partial agonism", and "antagonism" are
well known in the art. The term "modulatory effect" refers to the
ability of the ligand to change the activity of an agonist or
antagonist through binding to a ligand binding site.
[0194] The term "inert organic solvent" or "inert solvent" means a
solvent which is inert under the conditions of the reaction being
described in conjunction therewith including, by way of example
only, benzene, toluene, acetonitrile, tetrahydrofuran,
dimethylformamide, chloroform, methylene chloride, diethyl ether,
ethyl acetate, acetone, methylethyl ketone, methanol, ethanol,
propanol, isopropanol, t-butanol, dioxane, pyridine, and the like.
Unless specified to the contrary, the solvents used in the
reactions described herein are inert solvents.
[0195] The term "treatment" refers to any treatment of a pathologic
condition in a mammal, particularly a human, and includes:
[0196] (i) preventing the pathologic condition from occurring in a
subject which may be predisposed to the condition but has not yet
been diagnosed with the condition and, accordingly, the treatment
constitutes prophylactic treatment for the disease condition;
[0197] (ii) inhibiting the pathologic condition, i.e., arresting
its development;
[0198] (iii) relieving the pathologic condition, i.e., causing
regression of the pathologic condition; or
[0199] (iv) relieving the conditions mediated by the pathologic
condition.
[0200] The term "pathologic condition which is modulated by
treatment with a ligand" covers all disease states (i.e.,
pathologic conditions) which are generally acknowledged in the art
to be usefully treated with a ligand for the .beta.2-adrenergic
receptor in general, and those disease states which have been found
to be usefully treated by a specific multibinding compound of our
invention. Such disease states include, by way of example only, the
treatment of a mammal afflicted with asthma, chronic bronchitis,
chronic pulmonary obstructive disease, and the like.
[0201] The term "therapeutically effective amount" refers to that
amount of multibinding compound which is sufficient to effect
treatment, as defined above, when administered to a mammal in need
of such treatment. The therapeutically effective amount will vary
depending upon the subject and disease condition being treated, the
weight and age of the subject, the severity of the disease
condition, the manner of administration and the like, which can
readily be determined by one of ordinary skill in the art.
[0202] The term "linker", identified where appropriate by the
symbol `X`, refers to a group or groups that covalently attaches
from 2 to 10 ligands (as identified above) in a manner that
provides for a compound capable of multivalency. Among other
features, the linker is a ligand-orienting entity that permits
attachment of at least two copies of a ligand (which may be the
same or different) thereto. Additionally, the linker can be either
a chiral or achiral molecule. In some cases, the linker maybe a
covalent bond that attaches the ligands in a manner that provides
for a compound capable of multivalency. Additionally, in some
cases, the linker may itself be biologically active. The term
"linker" does not, however, extend to cover solid inert supports
such as beads, glass particles, fibers, and the like. But it is
understood that the multibinding compounds of this invention can be
attached to a solid support if desired. For example, such
attachment to solid supports can be made for use in separation and
purification processes and similar applications.
[0203] The extent to which multivalent binding is realized depends
upon the efficiency with which the linker or linkers that joins the
ligands presents these ligands to the array of available ligand
binding sites. Beyond presenting these ligands for multivalent
interactions with ligand binding sites, the linker or linkers
spatially constrains these interactions to occur within dimensions
defined by the linker or linkers. Thus, the structural features of
the linker (valency, geometry, orientation, size, flexibility,
chemical composition, etc.) are features of multibinding agents
that play an important role in determining their activities.
[0204] The linkers used in this invention are selected to allow
multivalent binding of ligands to the ligand binding sites of a
.beta.2 adrenergic receptor, whether such sites are located
interiorly, both interiorly and on the periphery of the receptor
structure, or at any intermediate position thereof.
Representative Compounds of Formula (I):
I. Representative Multibinding Compounds of Formula (I)
[0205] wherein p is 2, q is 1, Ar.sup.1 is
4-hydroxy-3-hydroxymethylphenyl, Ar.sup.2 is 1,4-phenylene, R.sup.1
and R.sup.2 are hydrogen, X, W, Q, and Ar.sup.3 are as defined in
Table A below are:
TABLE-US-00001 TABLE A ##STR00018## Stereo- Cpd. chem. at # *C W X
--Q--Ar.sup.3 (** = stereochem) 1A (RS) --(CH.sub.2).sub.2-- bond
--NH--CH.sub.2--** CH(OH)phenyl ** = (S) 2A (RS)
--(CH.sub.2).sub.2-- bond --NH--CH.sub.2--** CH(OH)phenyl ** = (R)
3A (RS) --(CH.sub.2).sub.2-- bond --NH--CH.sub.2--** CH(OH)phenyl
** = (RS) 4A (RS) --(CH.sub.2).sub.2-- bond --NH--CH.sub.2--**
CH(OH)-(4-hydroxy-3- hydroxy-methyl)phenyl ** = (RS) 5A (RS)
--(CH.sub.2).sub.6O-- bond
--(CH.sub.2).sub.3--O--(CH.sub.2).sub.6--NH--CH.sub.2--**
CH(OH)-(4-hydroxy-3- hydroxyethyl)phenyl ** = (RS) 6A (RS)
--CH.sub.2-- bond --NH--CH.sub.2--** CH(OH)-(4-hydroxy-3-
hydroxy-methyl)phenyl ** = (RS) 7A (R) --(CH.sub.2).sub.2-- bond
--NH--CH.sub.2--** CH(OH)phenyl ** = (S) 8A (R)
--(CH.sub.2).sub.2-- bond --NH--CH.sub.2--** CH(OH)phenyl ** = (R)
9A (RS) --(CH.sub.2).sub.6--O--(CH.sub.2).sub.3 bond
--O--(CH.sub.2).sub.6--O-[4-(3-hydroxypropyl)]- phenyl 10A (RS)
--CH.sub.2*CH(OH)--CH.sub.2--O-- bond --O--(CH.sub.2)--**
CH(OH)--(CH.sub.2)--NH--CH.sub.2-- * = (RS) **
CH(OH)-(4-hydroxy-3-hydroxy- methyl)phenyl ** = (RS) 11A (RS)
--(CH.sub.2).sub.2-- bond --NH--CH.sub.2--** CH(OH)--O-naphth-1-yl
** = (RS)
II. Representative multibinding compounds of Formula (I) wherein p
is 2, q is 1, Ar.sup.1 is 4-hydroxy-3-hydroxymethylphenyl, Ar.sup.2
is 1,4-phenylene, R.sup.1 and R.sup.2 are hydrogen, X, W, Q, and
Ar.sup.3, are as defined Table B below are:
TABLE-US-00002 TABLE B ##STR00019## Stereo- Cpd chem. at # *C W X Q
--Ar.sup.3 1B (RS) bond --O-(p-C.sub.6H.sub.4)--NH--CH.sub.2-- bond
4-hydroxy-3- ** CH(OH)-- ** = (RS) hydroxymethyl- phenyl 2B (RS)
bond --O-- bond 4-aminophenyl 3B (RS)
--(CH.sub.2).sub.6--O--(CH.sub.2).sub.3--
--O--(CH.sub.2).sub.10--O-(p-C.sub.6H.sub.4)--(CH.sub.2).sub.3)--O--(CH.s-
ub.2).sub.6--NH--CH.sub.2--** CH(OH)-- bond 4-hydroxy-3- ** = (RS)
stereochem. hydroxy- methylphenyl 4B (RS)
--(CH.sub.2).sub.6--O--(CH.sub.2).sub.3--
--O--(CH.sub.2).sub.6--O-(p-C.sub.6H.sub.4)--(CH.sub.2).sub.3--O--(CH.sub-
.2).sub.5--NH--CH.sub.2--** CH(OH)-- bond 4-hydroxy-3- ** = (RS)
stereochem. hydroxy- methylphenyl 5B (RS) --(CH.sub.2).sub.2--
--O--(CH.sub.2).sub.4-- bond phenyl
III. Representative multibinding compounds of Formula (I) wherein p
is 2, q is 1, Ar.sup.1 is 4-hydroxy-3-hydroxy-methylphenyl, R.sup.1
and R.sup.2 are hydrogen, Ar.sup.3 is
(4-hydroxy-3-hydroxymethyl)phenyl, X, W, Q, and Ar.sup.2 are as
defined in Table C below are:
TABLE-US-00003 TABLE C ##STR00020## Cpd. Stereochem. # at *C W X
Ar.sup.2 Q 1C (RS) bond bond trans-1,4- --NH--CH.sub.2--** CH(OH)--
cyclohexane ** = (RS) 2C (RS) --CH.sub.2-- bond 1,3-
--CH.sub.2--NH--CH.sub.2--** cyclohexane CH(OH)-- ** = (RS) 3C (RS)
--(CH.sub.2).sub.3-- bond 1,4-piperazine
--(CH.sub.2).sub.3--NH--CH.sub.2--** CH(OH)-- ** = (RS) 4C (RS)
bond bond p-menthane --NH--CH.sub.2--** CH(OH)-- ** = (RS) 5C (RS)
bond bond 1,2-phenylene --CH.sub.2--NH--CH.sub.2--** CH(OH)-- ** =
(RS)
IV. Representative multibinding compounds of Formula (I) wherein p
is 2, q is 1, Ar.sup.1 and Ar.sup.3 are
4-hydroxy-3-hydroxymethylphenyl, R.sup.1 and R.sup.2 are hydrogen,
Q is a bond, and W, Ar.sup.2, and X are as defined in Table D below
are:
TABLE-US-00004 TABLE D ##STR00021## Cpd. Stereochem. # at *C W
Ar.sup.2 X 1D (RS) bond 1,4-
--(CH.sub.2)-(p-C.sub.6H.sub.10)--NH--CH.sub.2-- cyclo- ** CH(OH)--
hexane ** = (RS) stereochem.
V. Representative multibinding compounds of Formula (I) wherein p
is 2, q is 1, R.sup.1 and R.sup.2 are hydrogen, W is
--(CH.sub.1).sub.2--, Ar.sup.2 is 1,4-phenylene, -Q-Ar.sup.3, is
[2-hydroxy-2-phenyl]ethylamino, X is a bond and Ar.sup.1 is as
defined below are as shown in Table E below:
TABLE-US-00005 TABLE E ##STR00022## Cpd. Stereochem. Stereochem. #
Ar.sup.1 at *C at **C 1E phenyl (RS) (RS) 2E phenyl (R) (S) 3E
phenyl (R) (R) 4E 4-amino-3,5-dichlorophenyl (RS) (RS) 5E
4-amino-3,5-dichlorophenyl (R) (R) 6E 4-amino-3,5-dichlorophenyl
(S) (S) 7E 4-amino-3,5-dichlorophenyl (R) (S) 8E
4-amino-3,5-dichlorophenyl (S) (R) 9E
3-formyl-amino-4-hydroxyphenyl (RS) (RS) 10E
3-formyl-amino-4-hydroxyphenyl (R) (R) 11E
3-formyl-amino-4-hydroxyphenyl (S) (S) 12E
3-formyl-amino-4-hydroxyphenyl (R) (S) 13E
3-formyl-amino-4-hydroxyphenyl (S) (R)
VI. Miscellaneous Compounds:
##STR00023##
[0206] Preferred Embodiments
[0207] While the broadest definition of this invention is set forth
in the Summary of the Invention, certain compounds of Formula (I)
are preferred.
(A) A Preferred Group is a Multibinding Compound of Formula
(II):
##STR00024##
[0208] where *C has RS, R, or S stereochemistry; Within this group
(A) a more preferred group of compounds is that wherein:
[0209] (i) Ar.sup.1 is aryl, more preferably Ar.sup.1 is a phenyl
ring of formula (c):
##STR00025##
wherein:
[0210] R.sup.4 is hydrogen, alkyl, halo, or alkoxy, preferably
hydrogen, methyl, fluoro, chloro, or methoxy;
[0211] R.sup.5 is hydrogen, hydroxy, halo, halo, amino, or
--NHSO.sub.2R.sup.a where R.sup.a is alkyl, preferably hydrogen,
hydroxy, fluoro, chloro, amino, or --NHSOCH.sub.3; and
[0212] R.sup.6 is hydrogen, halo, hydroxy, alkoxy, substituted
alkyl, sulfonylamino, aminoacyl, or acylamino; preferably hydrogen,
chloro, fluoro, hydroxy, methoxy, hydroxymethyl,
--CH.sub.2SO.sub.2CH.sub.3, --NHSO.sub.2CH.sub.3, --NHCHO,
--CONH.sub.2, or --NHCONH.sub.2;
(ii) Ar.sup.1 is heteroaryl, more preferably Ar.sup.1 is
2,8-dihydroxyquinolin-5-yl or 3-bromoisoxazol-5-yl; or (iii)
Ar.sup.1 is heterocyclyl, more preferably Ar.sup.1 is heterocyclyl
fused to an aryl ring, most preferably 6-fluorochroman-2-yl;
[0213] W is a bond linking the --NR.sup.2-- group to Ar.sup.2,
alkylene, or a substituted alkylene group wherein one or more of
the carbon atoms in the alkylene and the substituted alkylene group
is optionally replaced by --O--, preferably a covalent bond,
methylene, ethylene, propylene,
--(CH.sub.2).sub.6--O--(CH.sub.2).sub.3--, --(CH.sub.2).sub.6--O--,
or --CH.sub.2CH(OH)CH.sub.2--O--; and
[0214] Ar.sup.2 is phenyl wherein the W and the X groups are
attached at the 1,2-, 1,3-, and 1,4-positions of the phenyl ring;
cyclohexyl optionally substituted with methyl and wherein the W and
the X groups are attached at the 1,3- and 1,4-positions of the
cyclohexyl ring; or piperazine wherein the W and the X groups are
attached at the 1,4-positions of the piperazine ring, preferably
1,4-phenylene.
[0215] Within the above more preferred groups, even more preferred
groups of compounds are wherein:
(a) X is --O--, --O-alkylene, --O-(arylene)-NH-(substituted
alkylene)-,
--O-(alkylene)-O-(arylene)-(alkylene)-O-(alkylene)-NH-(substituted
alkylene)-, --O-(alkylene)-O-(arylene)-, or
-(alkylene)-(cycloalkylene)-NH-(substituted alkylene)-, preferably
--O--(CH.sub.2).sub.4--;
--CH.sub.2-(1,4-cyclohexyl)-NH--CH.sub.2--CH(OH)--;
--O-(1,4-phenylene)-NH--CH.sub.2--CH(OH)--; --O--(CH.sub.2),
--O-(1,4-phenylene)-(CH.sub.2).sub.3--O--(CH.sub.2).sub.6--NH--CH.sub.2---
CH(OH)--;
--O--(CH.sub.2).sub.6--O-(1,4-phenylene)-(CH.sub.2).sub.3--(CH.s-
ub.2).sub.5--NH--CH.sub.2--CH(OH)--; or
--O--(CH.sub.2).sub.6--O-(1,4-phenylene)-; and
[0216] Q is a covalent bond; or
(b) X is a bond; and
[0217] Q is a substituted alkylene group wherein one or more of the
carbon atoms in said substituted alkylene group is optionally
replaced by a heteroatom selected from the group consisting of
--NR.sup.a-- (where R.sup.a is hydrogen, alkyl, or acyl) and --O--,
preferably --NH--CH.sub.2--**CH(OH)--;
--NH--CH.sub.2--**CH(OH))--CH.sub.2--O--; --NH--**CH(CH.sub.2O)--;
--CH.sub.2--NH--CH.sub.2--**CH(OH)--;
--C(CH.sub.3).sub.2--NH--CH.sub.2--**CH(OH)--;
--(CH.sub.2).sub.3--NH--CH.sub.2--**CH(OH)--;
--(CH.sub.2).sub.3--(CH.sub.2).sub.6NH--CH.sub.2--CH(OH)--;
--(CH.sub.2)--NH--CH.sub.2--**CH(OH)--;
--O--(CH.sub.2)--**CH(OH)--CH.sub.2--NH--CH.sub.2--**CH(OH)--; or
--NH--CH.sub.2--**CH(OH)--CH.sub.2--O--; more preferably
--NH--CH.sub.2--**CH(OH)--; --NH--**CH(CH.sub.2OH)--;
--(CH.sub.2).sub.3--O--(CH.sub.2).sub.6--NH--CH.sub.2--**CH(OH)--;
or --NH--CH.sub.2--**CH(OH)--CH.sub.2--O-- (where ** is RS, R or S
stereochemistry), most preferably --NH--**CH(CH.sub.2OH)-- where **
is RS, R or S stereochemistry;
[0218] Within the above preferred, more preferred group of
compounds, a particularly preferred group of compounds is that
wherein: [0219] (i) Ar.sup.3 is same as Ar.sup.1 as defined in
preferred embodiments (A)(i)-(iii) above. Another particularly
preferred group of compounds is that wherein: [0220] (ii) Ar.sup.3
is a phenyl ring of formula (d):
##STR00026##
[0220] wherein:
[0221] R.sup.7 is hydrogen, alkyl, alkenyl, substituted alkyl,
halo, alkoxy, substituted alkoxy, hydroxy, aminoacyl, or
heteroaryl, preferably hydrogen, methyl, propen-2-yl, fluoro,
chloro, methoxy, --OCH.sub.2CQ.sub.2Me, --OCON(CH.sub.3).sub.2,
hydroxy, --CH.sub.2CONH.sub.2, --NHCOCH.sub.3, --NHCHO,
imidazol-1-yl, or 1-methyl-4-trifluoromethyldazol-2-yl; and
[0222] R.sup.8 is hydrogen, halo, alkoxy, substituted alkoxy, or
acylamino, preferably hydrogen, fluoro, chloro, methoxy,
--OCH.sub.2CO.sub.2Me, --OCON(CH.sub.3).sub.2, --NHCHO, or
--CONH.sub.2.
(iii) Yet another particularly preferred group of compounds is that
wherein:
[0223] Ar.sup.3 is naphthyl, pyridyl, benzimidazol-1-yl, indolyl,
2-cyanoindolyl, carbazolyl, 4-methylindanyl,
5-(CH.sub.3CO.sub.2CH.sub.2O--)-1,2,3,4-tetrahydronaphthyl,
1H-2-oxoindole, 2,3,4-trihydrothianaphthalene,
4-hydroxy-2-benzothiazolinone, or
4-oxo-2,3-dihydrothianapthalene.
[0224] Within the above preferred, more preferred, and particularly
preferred groups, even more particularly preferred group is that
wherein:
[0225] Ar.sup.1 is phenyl, 4-hydroxyphenyl, 3,4-dihydroxyphenyl,
3,4-dichlorophenyl, 3,5-dihydroxyphenyl,
2-chloro-3,4-dihydroxyphenyl, 2-fluoro-3,4-dihydroxyphenyl,
2-chloro-3,5-dihydroxyphenyl, 2-fluoro-3,5-dihydroxyphenyl,
4-hydroxy-3-methoxyphenyl, 4-hydroxy-3-hydroxymethylphenyl,
4-hydroxy-3-(HCONH-)phenyl, 4-hydroxy-3-(NH.sub.2CO--)phenyl,
3-chlorophenyl, 2,5-dimethoxyphenyl,
4-(CH.sub.3SO.sub.2NH--)-phenyl,
4-hydroxy-3-(CH.sub.3SO.sub.2CH.sub.2-)phenyl,
4-hydroxy-3-(CH.sub.3SO.sub.2NH-)phenyl,
4-hydroxy-3-(NH.sub.2CONH-)phenyl, 3,5-dichloro-4-aminophenyl,
##STR00027##
preferably 4-hydroxy-3-hydroxymethylphenyl,
4-hydroxy-3-(HCONH-)phenyl, 3,5-dichloro-4-aminophenyl, or
##STR00028##
most preferably 4-hydroxy-3-(HCONH-)phenyl or
3,5-dichloro-4-aminophenyl; and
[0226] Ar.sup.3 is:
##STR00029## ##STR00030## ##STR00031##
preferably, phenyl or 4-hydroxy-3-hydroxymethylphenyl, more
preferably phenyl.
General Synthetic Scheme
[0227] Compounds of this invention can be made by the methods
depicted in the reaction schemes shown below.
[0228] The starting materials and reagents used in preparing these
compounds are either available from commercial suppliers such as
Aldrich Chemical Co., (Milwaukee, Wis., USA), Bachem (Torrance,
Calif., USA), Emka-Chemie, or Sigma (St. Louis, Mo., USA) or are
prepared by methods known to those skilled in the art following
procedures set forth in references such as Fieser and Fieser's
Reagents for Organic Synthesis, Volumes 1-15 (John Wiley and Sons,
1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and
Supplementals (Elsevier Science Publishers, 1989), Organic
Reactions, Volumes 1-40 (John Wiley and Sons, 1991), March's
Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition), and
Larock's Comprehensive Organic Transformations (VCH Publishers
Inc., 1989).
[0229] The starting materials and the intermediates of the reaction
may be isolated and purified if desired using conventional
techniques, including but not limited to filtration, distillation,
crystallization, chromatography, and the like. Such materials may
be characterized using conventional means, including physical
constants and spectral data.
[0230] Furthermore, it will be appreciated that where typical or
preferred process conditions (i.e., reaction temperatures, times,
mole ratios of reactants, solvents, pressures, etc.) are given,
other process conditions can also be used unless otherwise stated.
Optimum reaction conditions may vary with the particular reactants
or solvent used, but such conditions can be determined by one
skilled in the art by routine optimization procedures.
[0231] Additionally, as will be apparent to those skilled in the
art, conventional protecting groups may be necessary to prevent
certain functional groups from undergoing undesired reactions. The
choice of a suitable protecting group for a particular functional
group as well as suitable conditions for protection and
deprotection are well known in the art. For example, numerous
protecting groups, and their introduction and removal, are
described in T. W. Greene and G. M. Wuts, Protecting Groups in
Organic Synthesis, Second Edition, Wiley, New York, 1991, and
references cited therein.
[0232] These schemes are merely illustrative of some methods by
which the compounds of this invention can be synthesized, and
various modifications to these schemes can be made and will be
suggested to one skilled in the art having referred to this
disclosure.
Preparation of a Multibinding Compound of Formula (I)
[0233] In general, a multibinding compound of Formula (I) where p
is 2 and q is I can be prepared as illustrated and described in
Schemes A-D below.
[0234] A multibinding compound of Formula (I) where p is 2 and q is
1 can be prepared by covalently attaching the ligands, L, wherein
at least one of the ligand is selected from a compound of formula
(a) as defined in the Summary of the Invention, to a linker, X, as
shown in Scheme A below.
##STR00032##
[0235] In method (a), a multibinding compound of Formula (I) where
p is 2 and q is 1 is prepared in one step, by covalently attaching
the ligands, L, to a linker, X, where FG.sup.1 and FG.sup.2
represent a functional group such as halo, amino, hydroxy, thio,
aldehyde, ketone, carboxy, carboxy derivatives such as acid halide,
ester, amido, and the like. This method is preferred for preparing
compounds of Formula (I) where the ligands are the same
[0236] In method (b), the compounds of Formula (I) are prepared in
a stepwise manner by covalently attaching one equivalent of a
ligand, L.sub.1, with a ligand X where FG.sup.1 and FG.sup.2
represent a functional group as defined above, and FG.sup.2PG is a
protected functional group to give an intermediate of formula (II).
Deprotection of the second functional group on the ligand, followed
by reaction with a ligand L.sub.2, which may be same or different
than ligand L.sub.1, then provides a compound of Formula (I). This
method is suitable for preparing compounds of Formula (I) where the
ligands are the non-identical.
[0237] The ligands are covalently attached to the linker using
conventional chemical techniques providing for covalent linkage of
the ligand to the linker. Reaction chemistries resulting in such
linkages are well known in the art and involve the use of
complementary functional groups on the linker and ligand as shown
in Table I below.
TABLE-US-00006 TABLE I Representative Complementary Binding
Chemistries First Reactive Group Second Reactive Group Linkage
carboxyl amine amide sulfonyl halide amine sulfonamide hydroxyl
alkyl/aryl halide ether hydroxyl isocyanate urethane amine epoxide
.beta.-hydroxyamine amine alkyl/aryl halide alkylamine amine
isocyanate urea hydroxyl carboxyl ester amine aldehyde amine
[0238] 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
such as dicyclohexylcarbodiimide, results m-formation of an amide
bond covalently linking the ligand to the linker; reaction between
an amine group of either the linker or the ligand and a sulfonyl
halide of the ligand or the linker, in the presence of a base such
as triethylamine, pyridine, an the like results in formation of a
sulfonamide bond covalently linking the ligand to the linker, and
reaction between an alcohol or phenol group of either the linker or
the ligand and an alkyl or aryl halide of the ligand or the linker
in the presence of a base such as triethylamine, pyridine, and the
like, results in formation of an ether bond covalently linking the
ligand to the linker.
[0239] A multibinding compound of Formula (I) where p is 2 q is 1,
the second ligand Ar.sup.3 is the same as Ar.sup.1, X is a bond,
and Q is 2-hydroxyethylamino group, and the ligands are linked
through the Ar.sup.2 group can be prepared from an aryl glyoxal
derivative of formula 1 as shown in Scheme B below.
##STR00033##
[0240] Condensation of an acetophenone derivative of formula 1 with
a diamine of formula 2 in an ethereal solution such as
tetrahydrofuran provides an imine of formula 3. Reduction of the
imine with a suitable reducing agent such as diborane provides a
compound of Formula (I). Suitable reaction solvents are
tetrahydrofuran, and the like. Compound 1 where Ar.sup.1 is phenyl
is prepared by heating acetophenone in 48% hydrobromic acid in
dimethylsulfoxide.
[0241] Compounds of formula 1 can be prepared by methods well known
in the art. For example,
.alpha.,.alpha.-dihydroxy-4-hydroxy-3-methoxycarbonylacetophenone
can be prepared by heating 5-acetylsalicylic acid methyl ester in
48% hydrobromic acid.
[0242] Alternatively, a multibinding compound of Formula (I) where
p is 2, q is 1, the second ligand Ar.sup.3 is the same as Ar.sup.1,
X is a bond, and Q is 2-hydroxyethylamino group, and the ligands
are linked through the Ar.sup.2 group can be prepared from an aryl
epoxide of formula 4 as shown in Scheme C below.
##STR00034##
[0243] A compound of Formula (I) can be prepared by reacting an
epoxide of formula 4 with a diamine of formula 2. Epoxides 4 are
either commercially available or they can be prepared by the
methods described in Kierstead, R. W. et. al. J. Med. Chem. 26,
1561-1569, (1983) or Hett, R. et. al. Tet. Lett. 35, 9345-9348
(1994).
[0244] Another method of preparing a multibinding compound of
Formula (I) where p is 2, q is 1, the second ligand Ar.sup.3 is the
same as Ar.sup.1, X is a bond, and Q is 2-hydroxyethylamino group,
and the ligands are linked through the Ar.sup.2 group can be
prepared from an acetophenone derivative of formula 5 as shown in
Scheme D below.
##STR00035##
[0245] Bromination of an acetophenone derivative of formula 5 with
bromine in a halogenated organic solvent such as chloroform
provides an .alpha.-bromoacetophenone derivative of formula 6.
Treatment of 6 with sodium azide followed by reduction of the
resulting azide 7 with a suitable reducing agent such as lithium
aluminum hydride provides ethanolamine derivative of formula 8.
Condensation of two equivalents of 8 with a dialdehyde compound of
formula 2 provides an imine of formula 10 which is converted to a
compound of Formula (I) as described in Scheme A above.
[0246] Another method of preparing a multibinding compound of
Formula (I) where p is 2, q is 1, Ar.sup.1 and Ar.sup.3 are
different, X is a bond, and Q is 2-hydroxyethylamino group, and the
ligands are linked through the Ar.sup.1 group can be prepared as
shown in Scheme E below.
##STR00036##
[0247] Condensation of a diamine of formula 11 (where PG.sub.1 and
PG.sub.2 are suitable amino protecting groups which can be
selectively removed) with a glyoxal of formula 2 followed by
reduction of the resulting imine of formula 12 with a suitable
reducing agent such as diborane in a suitable reaction solvents
such as tetrahydrofuran provides a compound of formula 11.
Compounds of formula 11 can be prepared by methods described in
FIG. 14.
[0248] Reaction of compound 15 with an alpha bromoacetophenone
compound of formula 6 followed by reduction of the keto group
provides a compound of formula 16. The reaction is carried out
under conditions well known in the art. Deprotection of the amino
protecting group then provides a compound of Formula (I). The
deprotection reaction conditions depend on the nature of the
protecting group. For example, if the protecting group is benzyl,
it is removed under catalytic hydrogenation reaction
conditions.
[0249] Any compound which is a .beta.2 adrenergic receptor agonist
can be used as a ligand in this invention. Typically, a compound
selected for use as a ligand will have at least one functional
group, such as an amino, hydroxyl, thiol or carboxyl group and the
like, which allows the compound to be readily coupled to the
linker. Compounds having such functionality are either known in the
art or can be prepared by routine modification of known compounds
using conventional reagents and procedures.
[0250] Linkers can be attached to different positions on the ligand
molecule to achieve different orientations of the ligand domains,
and thereby facilitate multivalency. While a number of positions on
.beta.-adrenergic-modulating ligands are synthetically practical
for linking, it is preferred to preserve those ligand substructures
which are most important for ligand-receptor binding. At present,
the aryl group and the sidechain nitrogen are preferred points of
attachment.
[0251] It will be apparent to one skilled in the art that the above
chemistries are not limited to preparing bivalent multibinding
compounds of Formula (I) and can be used to prepare tri-, tetra-,
etc., multibinding compounds of Formula (I).
[0252] 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 that is not considered to be part
of the ligand.
[0253] The relative orientation in which the ligand domains are
displayed derives from the particular point or points of attachment
of the ligands to the linker, and on the framework geometry. The
determination of where acceptable substitutions can be made on a
ligand is typically based on prior knowledge of structure-activity
relationships (SAR) of the ligand and/or congeners and/or
structural information about ligand-receptor complexes (e.g., X-ray
crystallography, NMR, and the like). Such positions and the
synthetic methods for covalent attachment are well known in the
art. Following attachment to the selected linker (or attachment to
a significant portion of the linker, for example 2-10 atoms of the
linker), the univalent linker-ligand conjugate may be tested for
retention of activity in the relevant assay.
[0254] The linker, when covalently attached to multiple copies of
the ligands, provides a biocompatible, substantially
non-immunogenic multibinding compound. The biological activity of
the multibinding compound is highly sensitive to the valency,
geometry, composition, size, flexibility or rigidity, etc. of the
linker and, in turn, on the overall structure of the multibinding
compound, as well as the presence or absence of anionic or cationic
charge, the relative hydrophobicity/hydrophilicity of the linker,
and the like on the linker. Accordingly, the linker is preferably
chosen to maximize the biological activity of the multibinding
compound. The linker may be chosen to enhance the biological
activity of the molecule. In general, the linker may be chosen from
any organic molecule construct that orients two or more ligands to
their ligand binding sites to permit multivalency. In this regard,
the 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.
[0255] For example, different orientations can be achieved by
including in the framework groups containing mono- or polycyclic
groups, including aryl and/or heteroaryl groups, or structures
incorporating one or more carbon-carbon multiple bonds (alkenyl,
alkenylene, alkynyl or alkynylene groups). Other groups can also
include oligomers and polymers which are branched- or
straight-chain species. In preferred embodiments, rigidity is
imparted by the presence of cyclic groups (e.g., aryl, heteroaryl,
cycloalkyl, heterocyclic, etc.). In other preferred embodiments,
the ring is a six or ten member ring. In still further preferred
embodiments, the ring is an aromatic ring such as, for example,
phenyl or naphthyl.
[0256] 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
(H.sub.2N(CH.sub.2).sub.6NH.sub.2) or related polyamines can be
modified to be substantially more hydrophilic by replacing the
alkylene group with a poly(oxyalkylene) group such as found in the
commercially available "Jeffamines".
[0257] Different frameworks can be designed to provide preferred
orientations of the ligands. Such frameworks may be represented by
using an array of dots (as shown below) wherein each dot may
potentially be an atom, such as C, O, N, S, P, H, F, Cl, Br, and F
or the dot may alternatively indicate the absence of an atom at
that position. To facilitate the understanding of the framework
structure, the framework is illustrated as a two dimensional array
in the following diagram, although clearly the framework is a three
dimensional array in practice:
TABLE-US-00007 . . . . . . . . . . . . . . . . . . . . . . . . 8
.cndot. .cndot. .cndot. .cndot. .cndot. .cndot. .cndot. .cndot.
.cndot. . . . 7 .cndot. .cndot. .cndot. .cndot. .cndot. .cndot.
.cndot. .cndot. .cndot. . . . 6 .cndot. .cndot. .cndot. .cndot.
.cndot. .cndot. .cndot. .cndot. .cndot. . . . 5 .cndot. .cndot.
.cndot. .cndot. .cndot. .cndot. .cndot. .cndot. .cndot. . . . 4
.cndot. .cndot. .cndot. .cndot. .cndot. .cndot. .cndot. .cndot.
.cndot. . . . 3 .cndot. .cndot. .cndot. .cndot. .cndot. .cndot.
.cndot. .cndot. .cndot. . . . 2 .cndot. .cndot. .cndot. .cndot.
.cndot. .cndot. .cndot. .cndot. .cndot. . . . 1 .cndot. .cndot.
.cndot. .cndot. .cndot. .cndot. .cndot. .cndot. .cndot. . . . 0
.cndot. .cndot. .cndot. .cndot. .cndot. .cndot. .cndot. .cndot.
.cndot. . . . 0 1 2 3 4 5 6 7 8
[0258] Each dot is either an atom, chosen from carbon, hydrogen,
oxygen, nitrogen, sulfur, phosphorus, or halogen, or the dot
represents a point in space (i.e., an absence of an atom). As is
apparent to the skilled artisan, only certain atoms on the grid
have the ability to act as an attachment point for the ligands,
namely, C, O, N, S and P.
[0259] Atoms can be connected to each other via bonds (single,
double or triple bonds with acceptable resonance and tautomeric
forms), with regard to the usual constraints of chemical bonding.
Ligands may be attached to the framework via single, double or
triple bonds (with chemically acceptable tautomeric and resonance
forms). Multiple ligand groups (2 to 10) can be attached to the
framework such that the minimal, shortest path distance between
adjacent ligand groups does not exceed 100 atoms. Preferably, the
linker connections to the ligand is selected such that the maximum
spatial distance between two adjacent ligands is no more than 100
.ANG..
[0260] An example of a linker as presented by the grid is shown
below for a biphenyl construct
##STR00037##
Nodes (1,2), (2,0), (4,4), (5,2), (4,0), (6,2), (7,4), (9,4),
(10,2), (9,0), (7,0) all represent carbon atoms Node (10,0)
represents a chlorine atom. All other nodes (or dots) are points in
space (i.e., represent an absence of atoms).
[0261] Nodes (1,2) and (9,4) are attachment points. Hydrogen atoms
are affixed to nodes (2,4), (4,4), (4,0), (2,0), (7,4), (10,2) and
(7,0). Nodes (5,2) and (6,2) are connected by a single bond.
[0262] The carbon atoms present are connected by either a single or
double bonds, taking into consideration the principle of resonance
and/or tautomerism.
[0263] The intersection of the framework (linker) and the ligand
group, and indeed, the framework (linker) itself can have many
different bonding patterns. Examples of acceptable patterns of
three contiguous atom arrangements are shown in the following
diagram:
TABLE-US-00008 C C C N C C O C C S C C P C C C C N N C N O C N S C
N P C N C C O N C O O C O S C O P C O C C S N C S O C S S C S P C S
C C P N C P O C P S C P P C P C N C N N C O N C S N C P N C C N N N
N N O N N S N N P N N C N O N N O O N O S N O P N O C N S N N S O N
S S N S P N S C N P N N P O N P S N P P N P C O C N O C O O C S O C
P O C C O O N O N O O N S O N P O N C O C N O O O O O S O O P O O C
O P N O P O O S S O S P O S O O P S O P P O P C S C N S C O S C S S
C P S C C S N N S N O S N S S N P S N C S O N S O O S O S S O P S O
C S S N S S O S S S S S P S S C S P N S P O S P S S P P S P C P C N
P C C P N N P N O P C S P C P P C C P O N P O O P N S P N P P N C P
S N P S O P O S P O P P O C P P N P P O P S S P S P P S O P P S P P
P P P
[0264] One skilled in the art would be able to identify bonding
patterns that would produce multivalent compounds. Methods for
producing these bonding arrangements are described in March,
"Advanced Organic Chemistry", 4th Edition, Wiley-Interscience, New
York, N.Y. (1992). These arrangements are described in the grid of
dots shown in the scheme above. All of the possible arrangements
for the five most preferred atoms are shown. Each atom has a
variety of acceptable oxidation states. The bonding arrangements
underlined are less acceptable and are not preferred.
[0265] Examples of molecular structures in which the above bonding
patterns could be employed as components of the linker are shown
below.
##STR00038##
The identification of an appropriate framework geometry and size
for ligand domain presentation are important steps in the
construction of a multibinding compound with enhanced activity.
Systematic spatial searching strategies can be used to aid in the
identification of preferred frameworks through an iterative
process. FIG. 3 illustrates a useful strategy for determining an
optimal framework display orientation for ligand domains. Various
other strategies are known to those skilled in the art of molecular
design and can be used for preparing compounds of this
invention.
[0266] As shown in FIG. 1, display vectors around similar central
core structures such as a phenyl structure (Panel A) and a
cyclohexane structure (Panel B) can be varied, as can the spacing
of the ligand domain from the core structure (i.e., the length of
the attaching moiety). It is to be noted that core structures other
than those shown here can be used for determining the optimal
framework display orientation of the ligands. The process may
require the use of multiple copies of the same central core
structure or combinations of different types of display cores.
[0267] The above-described process can be extended to trimers (FIG.
2) and compound of higher valency (FIGS. 3 and 4).
[0268] Assays of each of the individual compounds of a collection
generated as described above will lead to a subset of compounds
with the desired enhanced activities (e.g., potency, selectivity,
etc.). The analysis of this subset using a technique such as
Ensemble Molecular Dynamics will provide a framework orientation
that favors the properties desired. A wide diversity of linkers is
commercially available (see, e.g., Available Chemical Directory
(ACD)). Many of the linkers that are suitable for use in this
invention fall into this category. Other can be readily synthesized
by methods well known in the art and/or are described below.
[0269] Having selected a preferred framework geometry, the physical
properties of the linker can be optimized by varying the chemical
composition thereof. The composition of the linker can be varied in
numerous ways to achieve the desired physical properties for the
multibinding compound.
[0270] It can therefore be seen that there is a plethora of
possibilities for the composition of a linker. Examples of linkers
include aliphatic moieties, aromatic moieties, steroidal moieties,
peptides, and the like. Specific examples are peptides or
polyamides, hydrocarbons, aromatic groups, ethers, lipids, cationic
or anionic groups, or a combination thereof.
[0271] Examples are given below, but it should be understood that
various changes may be made and equivalents may be substituted
without departing from the true spirit and scope of the invention.
For example, properties of the linker can be modified by the
addition or insertion of ancillary groups into or onto the linker,
for example, to change the solubility of the multibinding compound
(in water, fats, lipids, biological fluids, etc.), hydrophobicity,
hydrophilicity, linker flexibility, antigenicity, stability, and
the like. For example, the introduction of one or more
poly(ethylene glycol) (PEG) groups onto or into the linker enhances
the hydrophilicity and water solubility of the multibinding
compound, increases both molecular weight and molecular size and,
depending on the nature of the unPEGylated linker, may increase the
in vivo retention time. Further PEG may decrease antigenicity and
potentially enhances the overall rigidity of the linker.
[0272] Ancillary groups which enhance the water
solubility/hydrophilicity of the linker and, accordingly, the
resulting multibinding compounds are useful in practicing this
invention. Thus, it is within the scope of the present invention to
use ancillary groups such as, for example, small repeating units of
ethylene glycols, alcohols, polyols (e.g., glycerin, glycerol
propoxylate, saccharides, including mono-oligosaccharides, etc.),
carboxylates (e.g., small repeating units of glutamic acid, acrylic
acid, etc.), amines (e.g., tetraethylenepentamine), and the like)
to enhance the water solubility and/or hydrophilicity of the
multibinding compounds of this invention. In preferred embodiments,
the ancillary group used to improve water solubility/hydrophilicity
will be a polyether.
[0273] The incorporation of lipophilic ancillary groups within the
structure of the linker to enhance the lipophilicity and/or
hydrophobicity of the multibinding compounds described herein is
also within the scope of this invention. Lipophilic groups useful
with the linkers of this invention include, by way of example only,
aryl and heteroaryl groups which, as above, may be either
unsubstituted or substituted with other groups, but are at least
substituted with a group which allows their covalent attachment to
the linker. Other lipophilic groups useful with the linkers of this
invention include fatty acid derivatives which do not form bilayers
in aqueous medium until higher concentrations are reached.
[0274] Also within the scope of this invention is the use of
ancillary groups which result in the multibinding compound being
incorporated or anchored into a vesicle or other membranous
structure such as a liposome or a micelle. The term "lipid" refers
to any fatty acid derivative that is capable of forming a bilayer
or a micelle such that a hydrophobic portion of the lipid material
orients toward the bilayer while a hydrophilic portion orients
toward the aqueous phase. Hydrophilic characteristics derive from
the presence of phosphato, carboxylic, sulfato, amino, sulfhydryl,
nitro and other like groups well known in the art. Hydrophobicity
could be conferred by the inclusion of groups that include, but are
not limited to, long chain saturated and unsaturated aliphatic
hydrocarbon groups of up to 20 carbon atoms and such groups
substituted by one or more aryl, heteroaryl, cycloalkyl, and/or
heterocyclic group(s). Preferred lipids are phosphglycerides and
sphingolipids, representative examples of which include
phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,
phosphatidylinositol, phosphatidic acid, palmitoyleoyl
phosphatidylcholine, lysophosphatidylcholine,
lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine,
dioleoyl-phosphatidylcholine, distearoyl-phosphatidylcholine or
dilinoleoylphosphatidyl-choline could be used. Other compounds
lacking phosphorus, such as sphingolipid and glycosphingolipid
families are also within the group designated as lipid.
Additionally, the amphipathic lipids described above may be mixed
with other lipids including triglycerides and sterols.
[0275] The flexibility of the linker can be manipulated by the
inclusion of ancillary groups which are bulky and/or rigid. The
presence of bulky or rigid groups can hinder free rotation about
bonds in the linker or bonds between the linker and the ancillary
group(s) or bonds between the linker and the functional groups.
Rigid groups can include, for example, those groups whose
conformational lability is restrained by the presence of rings
and/or multiple bonds within the group, for example, aryl,
heteroaryl, cycloalkyl, cycloalkenyl, and heterocyclic groups.
Other groups which can impart rigidity include polypeptide groups
such as oligo- or polyproline chains.
[0276] Rigidity can also be imparted electrostatically. Thus, if
the ancillary groups are either positively or negatively charged,
the similarly charged ancillary groups will force the presenter
linker into a configuration affording the maximum distance between
each of the like charges. The energetic cost of bringing the
like-charged groups closer to each other will tend to hold the
linker in a configuration that maintains the separation between the
like-charged ancillary groups. Further ancillary groups bearing
opposite charges will tend to be attracted to their oppositely
charged counterparts and potentially may enter into both inter- and
intramolecular ionic bonds. This non-covalent mechanism will tend
to hold the linker into a conformation which allows bonding between
the oppositely charged groups. The addition of ancillary groups
which are charged, or alternatively, bear a latent charge when
deprotected, following addition to the linker, include
deprotonation of a carboxyl, hydroxyl, thiol or amino group by a
change in pH, oxidation, reduction or other mechanisms known to
those skilled in the art which result in removal of the protecting
group, is within the scope of this invention.
[0277] Rigidity may also be imparted by internal hydrogen bonding
or by hydrophobic collapse.
[0278] Bulky groups can include, for example, large atoms, ions
(e.g., iodine, sulfur, metal ions, etc.) or groups containing large
atoms, polycyclic groups, including aromatic groups, non-aromatic
groups and structures incorporating one or more carbon-carbon
multiple bonds (i.e., alkenes and alkynes). Bulky groups can also
include oligomers and polymers which are branched- or
straight-chain species. Species that are branched are expected to
increase the rigidity of the structure more per unit molecular
weight gain than are straight-chain species.
[0279] In preferred embodiments, rigidity is imparted by the
presence of cyclic groups (e.g., aryl, heteroaryl, cycloalkyl,
heterocyclic, etc.). In other preferred embodiments, the linker
comprises one or more six-membered rings. In still further
preferred embodiments, the ring is an aryl group such as, for
example, phenyl or naphthyl.
[0280] In view of the above, it is apparent that the appropriate
selection of a linker group providing suitable orientation,
restricted/unrestricted rotation, the desired degree of
hydrophobicity/hydrophilicity, etc. is well within the skill of the
art. Eliminating or reducing antigenicity of the multibinding
compounds described herein is also within the scope of this
invention. In certain cases, the antigenicity of a multibinding
compound may be eliminated or reduced by use of groups such as, for
example, poly(ethylene glycol).
[0281] As explained above, the multibinding compounds described
herein comprise 2-10 ligands attached to a linker that attaches the
ligands in such a manner that they are presented to the enzyme for
multivalent interactions with ligand binding sites thereon/therein.
The linker spatially constrains these interactions to occur within
dimensions defined by the linker. This and other factors increases
the biological activity of the multibinding compound as compared to
the same number of ligands made available in monobinding form.
[0282] The compounds of this invention are preferably represented
by the empirical Formula (L).sub.p(X).sub.q where L, X, p and q are
as defined above. This is intended to include the several ways in
which the ligands can be linked together in order to achieve the
objective of multivalency, and a more detailed explanation is
described below.
[0283] 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.
[0284] The simplest and most preferred multibinding compound is a
bivalent compound which can be represented as L-X-L, where each L
is independently a ligand which may be the same or different and
each X is independently the linker. Examples of such bivalent
compounds are provided in FIG. 1 where each shaded circle
represents a ligand. A trivalent compound could also be represented
in a linear fashion, i.e., as a sequence of repeated units
L-X-L-X-L, in which L is a ligand and is the same or different at
each occurrence, as can X. However, a trimer can also be a radial
multibinding compound comprising three ligands attached to a
central core, and thus represented as (L).sub.3X, where the linker
X could include, for example, an aryl or cycloalkyl group.
Illustrations of trivalent and tetravalent compounds of this
invention are found in FIGS. 2 and 3 respectively where, again, the
shaded circles represent ligands. Tetravalent compounds can be
represented in a linear array, e.g.,
L-X-L-X-L-X-L
in a branched array, e.g.,
##STR00039##
(a branched construct analogous to the isomers of butane--n-butyl,
iso-butyl, sec-butyl, and t-butyl) or in a tetrahedral array,
e.g.,
##STR00040##
where X and L are as defined herein. Alternatively, it could be
represented as an alkyl, aryl or cycloalkyl derivative as above
with four (4) ligands attached to the core linker.
[0285] The same considerations apply to higher multibinding
compounds of this invention containing 5-10 ligands as illustrated
in FIG. 4 where, as before, the shaded circles represent ligands.
However, for multibinding agents attached to a central linker such
as aryl or cycloalkyl, there is a self-evident constraint that
there must be sufficient attachment sites on the linker to
accommodate the number of ligands present; for example, a benzene
ring could not directly accommodate more than 6 ligands, whereas a
multi-ring linker (e.g., biphenyl) could accommodate a larger
number of ligands.
[0286] The above described compounds may alternatively be
represented as cyclic chains of the form:
##STR00041##
and variants thereof.
[0287] All of the above variations are intended to be within the
scope of the invention defined by the Formula
(L).sub.p(X).sub.q.
[0288] With the foregoing in mind, a preferred linker may be
represented by the following formula:
--X.sup.a-Z-(Y.sup.a-Z).sub.m-X.sup.a--
wherein
[0289] m is an integer of from 0 to 20;
[0290] X.sup.a at each separate occurrence is selected from the
group consisting of --O--, --S--, --NR--, --C(O)--, --C(O)O--,
--OC(O)--, --C(O)NR--, --NRC(O)--, C(S), --C(S)O--, --C(S)NR--,
--NRC(S)--, or a covalent bond where R is as defined below;
[0291] Z at each separate occurrence is selected from the group
consisting of alkylene, substituted alkylene, cycloalkylene,
substituted cylcoalkylene, alkenylene, substituted alkenylene,
alkynylene, substituted alkynylene, cycloalkenylene, substituted
cycloalkenylene, arylene, heteroarylene, heterocyclene, or a
covalent bond;
[0292] each Y.sup.a at each separate occurrence is selected from
the group consisting of --O--, --C(O)--, --OC(O)--, --C(O)O--,
--NR--, --S(O)n--, --C(O)NR'--, --NR'C(O)--, --NR'C(O)NR'--,
--NR'C(S)NR'--, --C(.dbd.NR')--NR'--, --NR'--C(.dbd.NR')--,
--OC(O)--NR'--, --NR'--C(O)--O--, --N.dbd.C(X.sup.a)--NR'--,
--NR'--C(O).dbd.N--, --P(O)(OR')--O--, --O--P(O)(OR')--,
--S(O).sub.nCR'R''--, --S(O).sub.n--NR'--, --NR'--S(O).sub.n--,
--S--S--, and a covalent bond; where n is 0, 1 or 2; and R, R' and
R'' at each separate occurrence are selected from the group
consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl,
substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl,
substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl,
heteroaryl and heterocyclic.
[0293] Additionally, the linker moiety can be optionally
substituted at any atom therein by one or more alkyl, substituted
alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted
alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl,
substituted alkynyl, aryl, heteroaryl and heterocyclic group.
[0294] In view of the above description of the linker, it is
understood that the term "linker" when used in combination with the
term "multibinding compound" includes both a covalently contiguous
single linker (e.g., L-X-L) and multiple covalently non-contiguous
linkers (L-X-L-X-L) within the multibinding compound.
Combinational Libraries
[0295] The methods described above lend themselves to combinatorial
approaches for identifying multimeric compounds which possess
multibinding properties.
[0296] Specifically, factors such as the proper juxtaposition of
the individual ligands of a multibinding compound with respect to
the relevant array of binding sites on a target or targets is
important in optimizing the interaction of the multibinding
compound with its target(s) 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, (5) linker geometry, (6) linker
physical properties, and (7) linker chemical functional groups.
[0297] 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:
Selection of Ligand(s):
[0298] A single ligand or set of ligands is (are) selected for
incorporation into the libraries of candidate multibinding
compounds which library is directed against a particular biological
target or targets e.g., .beta.2 adrenergic receptor. The only
requirement for the ligands chosen is that they are capable of
interacting with the selected target(s). Thus, ligands may be known
drugs, modified forms of known drugs, substructures of known drugs
or substrates of modified forms of known drugs (which are competent
to interact with the target), or other compounds. Ligands are
preferably chosen based on known favorable properties that may be
projected to be carried over to or amplified in multibinding forms.
Favorable properties include demonstrated safety and efficacy in
human patients, appropriate PK/ADME profiles, synthetic
accessibility, and desirable physical properties such as
solubility, log P, etc. However, it is crucial to note that ligands
which display an unfavorable property from among the previous list
may obtain a more favorable property through the process of
multibinding compound formation; i.e., ligands should not
necessarily be excluded on such a basis. For example, a ligand that
is not sufficiently potent at a particular target so as to be
efficacious in a human patient may become highly potent and
efficacious when presented in multibinding form. A ligand that is
potent and efficacious but not of utility because of a
non-mechanism-related toxic side effect may have increased
therapeutic index (increased potency relative to toxicity) as a
multibinding compound. Compounds that exhibit short in vivo
half-lives may have extended half-lives as multibinding compounds.
Physical properties of ligands that limit their usefulness (e.g.
poor bioavailability due to low solubility, hydrophobicity,
hydrophilicity) may be rationally modulated in multibinding forms,
providing compounds with physical properties consistent with the
desired utility.
Orientation: Selection of Ligand Attachment Points and Linking
Chemistry:
[0299] Several points are chosen on each ligand at which to attach
the ligand to the linker. The selected points on the ligand/linker
for attachment are functionalized to contain complementary reactive
functional groups. This permits probing the effects of presenting
the ligands to their receptor(s) in multiple relative orientations,
an important multibinding design parameter. The only requirement
for choosing attachment points is that attaching to at least one of
these points does not abrogate activity of the ligand. Such points
for attachment can be identified by structural information when
available. For example, inspection of a co-crystal structure of a
protease inhibitor bound to its target allows one to identify one
or more sites where linker attachment will not preclude the
enzyme:inhibitor interaction. Alternatively, evaluation of
ligand/target binding by nuclear magnetic resonance will permit the
identification of sites non-essential for ligand/target binding.
See, for example, Fesik, et al., U.S. Pat. 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.
[0300] It is important to emphasize that positions of attachment
that do abrogate the activity of the monomeric ligand may also be
advantageously included in candidate multibinding compounds in the
library provided that such compounds bear at least one ligand
attached in a manner which does not abrogate intrinsic activity.
This selection derives from, for example, heterobivalent
interactions within the context of a single target molecule. For
example, consider a 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 and/or 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
site. Another way to consider this is that the SAR of individual
ligands within the context of a multibinding structure is often
different from the SAR of those same ligands in monomeric form.
[0301] The foregoing discussion focused on bivalent interactions of
dimeric compounds bearing two copies of the same ligand joined to a
single linker through different attachment points, one of which may
abrogate the binding/activity of the monomeric ligand. It should
also be understood that bivalent advantage may also be attained
with heterodimeric constructs bearing two different ligands that
bind to common or different targets. For example, a 5HT.sub.4
receptor antagonist and a bladder-selective muscarinic M.sub.3
antagonist may be joined to a linker through attachment points
which do not abrogate the binding affinity of the monomeric ligands
for their respective receptor sites. The dimeric compound may
achieve enhanced affinity for both receptors due to favorable
interactions between the 5HT.sub.4 ligand and elements of the
M.sub.3 receptor proximal to the formal M.sub.3 antagonist binding
site and between the M.sub.3 ligand and elements of the 5HT.sub.4
receptor proximal to the formal 5HT.sub.4 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.
[0302] Once the ligand attachment points have been chosen, one
identifies the types of chemical linkages that are possible at
those points. The most preferred types of chemical linkages are
those that are compatible with the overall structure of the ligand
(or protected forms of the ligand) readily and generally formed,
stable and intrinsically inocuous under typical chemical and
physiological conditions, and compatible with a large number of
available linkers. Amide bonds, ethers, amines, carbamates, ureas,
and sulfonamides are but a few examples of preferred linkages.
Linkers: spanning relevant multibinding parameters through
selection of valency, linker length, linker geometry, rigidity,
physical properties and chemical functional groups.
[0303] In the library of linkers employed to generate the library
of candidate multibinding compounds, the selection of linkers
employed in this library of linkers takes into consideration the
following factors:
Valency:
[0304] 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.
Linker Length:
[0305] 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 .ANG., with more preferred linker
distances of 3-12 .ANG.. In situations where two binding sites
reside on separate (e.g., protein) target sites, preferred linker
distances are 20-100 .ANG., with more preferred distances of 30-70
.ANG..
Linker Geometry and Rigidity
[0306] The combination of ligand attachment site, linker length,
linker geometry, and linker rigidity determine the possible ways in
which the ligands of candidate multibinding compounds may be
displayed in three dimensions and thereby presented to their
binding sites. Linker geometry and rigidity are nominally
determined by chemical composition and bonding pattern, which may
be controlled and are systematically varied as another spanning
function in a multibinding array. For example, linker geometry is
varied by attaching two ligands to the ortho, meta, and para
positions of a benzene ring, or in cis- or trans-arrangements at
the 1,1-vs. 1,2-vs. 1,3-vs. 1,4-positions around a cyclohexane core
or in cis- or trans-arrangements at a point of ethylene
unsaturation. Linker rigidity is varied by controlling the number
and relative energies of different conformational states possible
for the linker. For example, a divalent compound bearing two
ligands joined by 1,8-octyl linker has many more degrees of
freedom, and is therefore less rigid than a compound in which the
two ligands are attached to the 4,4' positions of a biphenyl
linker.
Linker Physical Properties:
[0307] The physical properties of linkers are nominally determined
by the chemical constitution and bonding patterns of the linker,
and linker physical properties impact the overall physical
properties of the candidate multibinding compounds in which they
are included. A range of linker compositions is typically selected
to provide a range of physical properties (hydrophobicity,
hydrophilicity, amphiphilicity, polarization, acidity, and
basicity) in the candidate multibinding compounds. The particular
choice of linker physical properties is made within the context of
the physical properties of the ligands they join and preferably the
goal is to generate molecules with favorable PK/ADME properties.
For example, linkers can be selected to avoid those that are too
hydrophilic or too hydrophobic to be readily absorbed and/or
distributed in vivo.
Linker Chemical Functional Groups:
[0308] 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.
Combinatorial Synthesis
[0309] Having chosen a set of n ligands (n being determined by the
sum of the number of different attachment points for each ligand
chosen) and m linkers by the process outlined above, a library of
(n!)m candidate divalent multibinding compounds is prepared which
spans the relevant multibinding design parameters for a particular
target. For example, an array generated from two ligands, one which
has two attachment points (A1, A2) and one which has three
attachment points (B1, B2, B 3) joined in all possible combinations
provide for at least 15 possible combinations of multibinding
compounds:
TABLE-US-00009 A1-A1 A1-A2 A1-B1 A1-B2 A1-B3 A2-A2 A2-B1 A2- B2
A2-B3 B1-B1 B1-B2 B1-B3 B2-B2 B2-B3 B3-B3
[0310] When each of these combinations is joined by 10 different
linkers, a library of 150 candidate multibinding compounds
results.
[0311] 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 and/or linker
is attached to a solid support. Alternatively and preferably, the
combinatorial library is prepared in the solution phase. After
synthesis, candidate multibinding compounds are optionally purified
before assaying for activity by, for example,
chromatographic-methods (e.g., HPLC).
Analysis of Array by Biochemical Analytical, Pharmacological, and
Computational Methods:
[0312] Various methods are used to characterize the properties and
activities of the candidate multibinding compounds in the library
to determine which compounds possess multibinding properties.
Physical constants such as solubility under various solvent
conditions and logD/clogD values can be determined. A combination
of NMR spectroscopy and computational methods is used to determine
low-energy conformations of the candidate multibinding compounds in
fluid media. The ability of the members of the library to bind to
the desired target and other targets is determined by various
standard methods, which include radioligand displacement assays for
receptor and ion channel targets, and kinetic inhibition analysis
for many enzyme targets. In vitro efficacy, such as for receptor
agonists and antagonists, ion channel blockers, and antimicrobial
activity, can also be determined. Pharmacological data, including
oral absorption, everted gut penetration, other pharmacokinetic
parameters and efficacy data can be determined in appropriate
models. In this way, key structure-activity relationships are
obtained for multibinding design parameters which are then used to
direct future work.
[0313] 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).
[0314] Second, ascertaining the structure of those compounds which
exhibit multibinding properties can be accomplished via art
recognized procedures. For example, each member of the library can
be encrypted or tagged with appropriate information allowing
determination of the structure of relevant members at a later time.
See, for example, Dower, et al., International Patent Application
Publication No. WO 93/06121; Brenner, et al., Proc. Natl. Acad.
Sci., USA, 89:5181 (1992); Gallop, et al., U.S. Pat. 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 libraries of candidate
multivalent compounds by methods known in the art such as those
described by Hindsgaul, et al., Canadian Patent Application No.
2,240,325 which was published on Jul. 11, 1998. Such methods couple
frontal affinity chromatography with mass spectroscopy to determine
both the structure and relative binding affinities of candidate
multibinding compounds to receptors.
[0315] The process set forth above for dimeric candidate
multibinding compounds can, of course, be extended to trimeric
candidate compounds and higher analogs thereof.
Follow-Up Synthesis and Analysis of Additional Array(s):
[0316] 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
(antagonisin, partial agonism, etc.), and/or alter physical
properties. By iterative redesign/analysis using the novel
principles of multibinding design along with classical medicinal
chemistry, biochemistry, and pharmacology approaches, one is able
to prepare and identify optimal multibinding compounds that exhibit
biological advantage towards their targets and as therapeutic
agents.
[0317] To further elaborate upon this procedure, suitable divalent
linkers include, by way of example only, those derived from
dicarboxylic acids, disulfonylhalides, dialdehydes, diketones,
dihalides, diisocyanates, diamines, diols, mixtures of carboxylic
acids, sulfonylhalides, aldehydes, ketones, halides, isocyanates,
amines and diols. In each case, the carboxylic acid,
sulfonylhalide, aldehyde, ketone, halide, isocyanate, amine and
diol functional group is reacted with a complementary functionality
on the ligand to form a covalent linkage. Such complementary
functionality is well known in the art as illustrated in the
following table:
TABLE-US-00010 COMPLEMENTARY BINDING CHEMISTRIES First Reactive
Group Second Reactive Group Linkage hydroxyl isocyanate urethane
amine epoxide .beta.-hydroxyamine hydroxyamine sulfonyl halide
sulfonamide carboxyl acid amine amide hydroxyl alkyl/aryl halide
ether aldehyde amine/NaCNBH.sub.3 amine ketone amine/NaCNBH.sub.3
amine amine isocyanate urea
[0318] The following table illustrates, by way of examples,
starting materials (identified as X-1 through X-418) that can be
used to prepare linkers incorporated in the multibinding compounds
of this invention utilizing the chemistry described above. For
example, 1,10-decanedicarboxylic acid, X1, can be reacted with 2
equivalents of a ligand carrying an amino group in the presence of
a coupling reagent such as DCC to provide a bivalent multibinding
compound of formula a) wherein the ligands are linked via
1,10-decanediamido linking group.
##STR00042## ##STR00043## ##STR00044## ##STR00045## ##STR00046##
##STR00047## ##STR00048## ##STR00049## ##STR00050## ##STR00051##
##STR00052## ##STR00053## ##STR00054## ##STR00055## ##STR00056##
##STR00057## ##STR00058## ##STR00059## ##STR00060## ##STR00061##
##STR00062## ##STR00063## ##STR00064## ##STR00065## ##STR00066##
##STR00067## ##STR00068## ##STR00069## ##STR00070## ##STR00071##
##STR00072## ##STR00073## ##STR00074## ##STR00075## ##STR00076##
##STR00077## ##STR00078## ##STR00079## ##STR00080##
##STR00081##
[0319] Representative ligands for use in this invention include, by
way of example, L-1 and L-2 as identified above wherein L-1 is
selected from a compound of formula (a) and L-2 is selected from a
compound of formula (b).
[0320] Combinations of ligands (L) and linkers (X) per this
invention include, by way example only, homo- and hetero-dimers
wherein a first ligand is selected from L-1 and the second ligand
and linker is selected from the following:
TABLE-US-00011 L-2/X-1- L-2/X-2- L-2/X-3- L-2/X-4- L-2/X-5-
L-2/X-6- L-2/X-7- L-2/X-8- L-2/X-9- L-2/X-10- L-2/X-11- L-2/X-12-
L-2/X-13- L-2/X-14- L-2/X-15- L-2/X-16- L-2/X-17- L-2/X-18-
L-2/X-19- L-2/X-20- L-2/X-21- L-2/X-22- L-2/X-23- L-2/X-24-
L-2/X-25- L-2/X-26- L-2/X-27- L-2/X-28- L-2/X-29- L-2/X-30-
L-2/X-31- L-2/X-32- L-2/X-33- L-2/X-34- L-2/X-35- L-2/X-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-2/X-51- L-2/X-52- L-2/X-53- L-2/X-54-
L-2/X-55- L-2/X-56- L-2/X-57- L-2/X-58- L-2/X-59- L-2/X-60-
L-2/X-61- L-2/X-62- L-2/X-63- L-2/X-64- L-2/X-65- L-2/X-66-
L-2/X-67- L-2/X-68- L-2/X-69- L-2/X-70- L-2/X-71- L-2/X-72-
L-2/X-73- L-2/X-74- L-2/X-75- L-2/X-76- L-2/X-77- L-2/X-78-
L-2/X-79- L-2/X-80- L-2/X-81- L-2/X-82- L-2/X-83- L-2/X-84-
L-2/X-85- L-2/X-86- L-2/X-87- L-2/X-88- L-2/X-89- L-2/X-90-
L-2/X-91- L-2/X-92- L-2/X-93- L-2/X-94- L-2/X-95- L-2/X-96-
L-2/X-97- L-2/X-98- L-2/X-99- L-2/X-100- L-2/X-101- L-2/X-102-
L-2/X-103- L-2/X-104- L-2/X-105- L-2/X-106- L-2/X-107- L-2/X-108-
L-2/X-109- L-2/X-110- L-2/X-111- L-2/X-112- L-2/X-113- L-2/X-114-
L-2/X-115- L-2/X-116- L-2/X-117- L-2/X-118- L-2/X-119- L-2/X-120-
L-2/X-121- L-2/X-122- L-2/X-123- L-2/X-124- L-2/X-125- L-2/X-126-
L-2/X-127- L-2/X-128- L-2/X-129- L-2/X-130- L-2/X-131- L-2/X-132-
L-2/X-133- L-2/X-134- L-2/X-135- L-2/X-136- L-2/X-137- L-2/X-138-
L-2/X-139- L-2/X-140- L-2/X-141- L-2/X-142- L-2/X-143- L-2/X-144-
L-2/X-145- L-2/X-146- L-2/X-147- L-2/X-148- L-2/X-149- L-2/X-150-
L-2/X-151- L-2/X-152- L-2/X-153- L-2/X-154- L-2/X-155- L-2/X-156-
L-2/X-157- L-2/X-158- L-2/X-159- L-2/X-160- L-2/X-161- L-2/X-162-
L-2/X-163- L-2/X-164- L-2/X-165- L-2/X-166- L-2/X-167- L-2/X-168-
L-2/X-169- L-2/X-170- L-2/X-171- L-2/X-172- L-2/X-173- L-2/X-174-
L-2/X-175- L-2/X-176- L-2/X-177- L-2/X-178- L-2/X-179- L-2/X-180-
L-2/X-181- L-2/X-182- L-2/X-183- L-2/X-184- L-2/X-185- L-2/X-186-
L-2/X-187- L-2/X-188- L-2/X-189- L-2/X-190- L-2/X-191- L-2/X-192-
L-2/X-193- L-2/X-194- L-2/X-195- L-2/X-196- L-2/X-197- L-2/X-198-
L-2/X-199- L-2/X-200- L-2/X-201- L-2/X-202- L-2/X-203- L-2/X-204-
L-2/X-205- L-2/X-206- L-2/X-207- L-2/X-208- L-2/X-209- L-2/X-210-
L-2/X-211- L-2/X-212- L-2/X-213- L-2/X-214- L-2/X-215- L-2/X-216-
L-2/X-217- L-2/X-218- L-2/X-219- L-2/X-220- L-2/X-221- L-2/X-222-
L-2/X-223- L-2/X-224- L-2/X-225- L-2/X-226- L-2/X-227- L-2/X-228-
L-2/X-229- L-2/X-230- L-2/X-231- L-2/X-232- L-2/X-233- L-2/X-234-
L-2/X-235- L-2/X-236- L-2/X-237- L-2/X-238- L-2/X-239- L-2/X-240-
L-2/X-241- L-2/X-242- L-2/X-243- L-2/X-244- L-2/X-245- L-2/X-246-
L-2/X-247- L-2/X-248- L-2/X-249- L-2/X-250- L-2/X-251- L-2/X-252-
L-2/X-253- L-2/X-254- L-2/X-255- L-2/X-256- L-2/X-257- L-2/X-258-
L-2/X-259- L-2/X-260- L-2/X-261- L-2/X-262- L-2/X-263- L-2/X-264-
L-2/X-265- L-2/X-266- L-2/X-267- L-2/X-268- L-2/X-269- L-2/X-270-
L-2/X-271- L-2/X-272- L-2/X-273- L-2/X-274- L-2/X-275- L-2/X-276-
L-2/X-277- L-2/X-278- L-2/X-279- L-2/X-280- L-2/X-281- L-2/X-282-
L-2/X-283- L-2/X-284- L-2/X-285- L-2/X-286- L-2/X-287- L-2/X-288-
L-2/X-289- L-2/X-290- L-2/X-291- L-2/X-292- L-2/X-293- L-2/X-294-
L-2/X-295- L-2/X-296- L-2/X-297- L-2/X-298- L-2/X-299- L-2/X-300-
L-2/X-301- L-2/X-302- L-2/X-303- L-2/X-304- L-2/X-305- L-2/X-306-
L-2/X-307- L-2/X-308- L-2/X-309- L-2/X-310- L-2/X-311- L-2/X-312-
L-2/X-313- L-2/X-314- L-2/X-315- L-2/X-316- L-2/X-317- L-2/X-318-
L-2/X-319- L-2/X-320- L-2/X-321- L-2/X-322- L-2/X-323- L-2/X-324-
L-2/X-325- L-2/X-326- L-2/X-327- L-2/X-328- L-2/X-329- L-2/X-330-
L-2/X-331- L-2/X-332- L-2/X-333- L-2/X-334- L-2/X-335- L-2/X-336-
L-2/X-337- L-2/X-338- L-2/X-339- L-2/X-340- L-2/X-341- L-2/X-342-
L-2/X-343- L-2/X-344- L-2/X-345- L-2/X-346- L-2/X-347- L-2/X-348-
L-2/X-349- L-2/X-350- L-2/X-351- L-2/X-352- L-2/X-353- L-2/X-354-
L-2/X-355- L-2/X-356- L-2/X-357- L-2/X-358- L-2/X-359- L-2/X-360-
L-2/X-361- L-2/X-362- L-2/X-363- L-2/X-364- L-2/X-365- L-2/X-366-
L-2/X-367- L-2/X-368- L-2/X-369- L-2/X-370- L-2/X-371- L-2/X-372-
L-2/X-373- L-2/X-374- L-2/X-375- L-2/X-376- L-2/X-377- L-2/X-378-
L-2/X-379- L-2/X-380- L-2/X-381- L-2/X-382- L-2/X-383- L-2/X-384-
L-2/X-385- L-2/X-386- L-2/X-387- L-2/X-388- L-2/X-389- L-2/X-390-
L-2/X-391- L-2/X-392- L-2/X-393- L-2/X-394- L-2/X-395- L-2/X-396-
L-2/X-397- L-2/X-398- L-2/X-399- L-2/X-400- L-2/X-401- L-2/X-402-
L-2/X-403- L-2/X-404- L-2/X-405- L-2/X-406- L-2/X-407- L-2/X-408-
L-2/X-409- L-2/X-410- L-2/X-411- L-2/X-412- L-2/X-413- L-2/X-414-
L-2/X-415- L-2/X-416- L-2/X-417- L-2/X-418-
and so on.
Utility, Testing, and Administration
Utility
[0321] The multibinding compounds of this invention are .beta.2
adrenergic receptor agonists or partial agonists. Accordingly, the
multibinding compounds and pharmaceutical compositions of this
invention are useful in the treatment and prevention of diseases
mediated by .beta..sub.2 adrenergic receptor such as asthma,
chronic obstructive pulmonary disease, bronchitis, and the like.
They are also useful in the treatment of nervous system injury and
premature labor. It is also contemplated that the compounds of this
invention are useful for treating metabolic disorders such as
obesity, diabetes, and the like.
Testing
[0322] The .beta.2 adrenergic receptor agonistic activity of the
compounds of Formula (I) to may be demonstrated by a variety of in
vitro assays known to those of ordinary skill in the art, such as
the assay described in the biological Examples 1 and 2. It may also
be assayed by the ex vivo assays described in Ball, D. I. et al.,
"Salmeterol a Novel, Long-acting beta 2-Adrenergic Agonist:
Characterization of Pharmacological Activity in Vitro and in Vivo"
Br. J. Pharmacol., 104, 665-671 (1991); Linden, A. et al.,
"Salmeterol, Formoterol, and Salbutamol in the Isolated Guinea-Pig
Trachea: Differences in Maximum Relaxant Effect and Potency but not
in Functional Atagonism. Thorax, 48, 547-553, (1993); and Bials, A.
T. et al., Investigations into Factors Determining the Duration of
Action of the Beta 2-Adrenoceptor Agonist, Salmeterol. Br. J.
Pharmacol., 108, 505-515 (1993); or in vivo assays such as those
described in Ball, D. I. et al., "Salmeterol a Novel, Long-acting
beta 2-Adrenergic Agonist: Characterization of Pharmacological
Activity in Vitro and in Vivo" Br. J. Pharmacol., 104, 665-671
(1991); Kikkawa, H. et al., "TA-2005, a Novel, Long-acting, and
Selective Beta 2-Adrenoceptor Agonist: Characterization of its in
vivo Bronchodilating Action in Guinea Pigs and Cats in Comparison
with other Beta 2-Agonists". Biol. Pharm. Bull., 17, 1047-1052,
(1994); and Anderson, G. P., "Formoterol: Pharmacology, Molecular
basis of Agonism and Mechanism of Long Duration of a Highly Potent
and Selective Beta 2-Adrenoceptor Agonist Bronchodilator, Life
Sciences, 52, 2145-2160, (1993).
Pharmaceutical Formulations
[0323] When employed as pharmaceuticals, the compounds of this
invention are usually administered in the form of pharmaceutical
compositions. These compounds can be administered by a variety of
routes including oral, rectal, transdermal, subcutaneous,
intravenous, intramuscular, and inhalation (e.g., intranasal or
oral inhalation). These compounds are effective as injectable,
inhaled and oral compositions. Such compositions are prepared in a
manner well known in the pharmaceutical art and comprise at least
one active compound. A preferred manner for administering compounds
of this invention is inhalation. This is an effective means for
delivering a therapeutic agent directly to the respiratory tract
for the treatment of diseases such as asthma and other similar or
related respiratory tract disorders (see U.S. Pat. No.
5,607,915).
[0324] This invention also includes pharmaceutical compositions
which contain, as the active ingredient, one or more of the
compounds described herein associated with pharmaceutically
acceptable carriers. In making the compositions of this invention,
the active ingredient is usually mixed with an excipient, diluted
by an excipient or enclosed within such a carrier which can be in
the form of a capsule, sachet, paper or other container. When the
excipient serves as a diluent, it can be a solid, semi-solid, or
liquid material, which acts as a vehicle, carrier or medium for the
active ingredient. Thus, the compositions can be in the form of
tablets, pills, powders, lozenges, sachets, cachets, elixirs,
suspensions, emulsions, solutions, syrups, aerosols (as a solid or
in a liquid medium), ointments containing, for example, up to 100%
by weight of the active compound, soft and hard gelatin capsules,
suppositories, sterile injectable solutions, and sterile packaged
powders.
[0325] In preparing a formulation, it may be necessary to mill the
active compound to provide the appropriate particle size prior to
combining with the other ingredients. If the active compound is
substantially insoluble, it ordinarily is milled to a particle size
of less than 200 mesh. If the active compound is substantially
water soluble, the particle size is normally adjusted by milling to
provide a substantially uniform distribution in the formulation,
e.g. about 40 mesh.
[0326] 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.
[0327] 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 produce the desired therapeutic effect, in
association with a suitable pharmaceutical excipient. Preferably,
the compound of Formula (I) above is employed at no more than about
20 weight percent of the pharmaceutical composition, more
preferably no more than about 15 weight percent, with the balance
being pharmaceutically inert carrier(s).
[0328] The active compound is effective over a wide dosage range
and is generally administered in a pharmaceutically effective
amount. For example, when the drug is administered via-inhalation,
each dosage contains from about 1 .mu.g to about 1000 .mu.g,
preferably about 2 .mu.g to about 500 .mu.g, more preferably about
5 .mu.g to about 100 .mu.g, even more preferably about 5 .mu.g to
about 60 .mu.g, of the active ingredient. 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
route of administration, the actual compound administered and its
relative activity, the age, weight, and response of the individual
patient, the severity of the patient's symptoms, and the like.
Furthermore, the compound of this invention may be administered
prophylactically, for example, a pharmaceutical composition
containing a compound of this invention may be administered before
the bronchospasm begins in an asthma attack, to prevent its
occurrence or to reduce the extent to which it occurs.
[0329] 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 pre-formulation 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. This solid pre-formulation is then subdivided into unit
dosage forms of the type described above containing the active
ingredient of the present invention.
[0330] The tablets or pills of the present invention may be coated
or otherwise compounded to provide a dosage form affording the
advantage of prolonged action. For example, the tablet or pill can
comprise an inner dosage and an outer dosage component, the latter
being in the form of an envelope over the former. The two
components can be separated by an enteric layer which serves to
resist disintegration in the stomach and permit the inner component
to pass intact into the duodenum or to 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.
[0331] 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.
[0332] Compositions for inhalation or insufflation include
solutions and suspensions in pharmaceutically acceptable, aqueous
or organic solvents, or mixtures thereof, and powders (see U.S.
Pat. No. 5,983,956). The liquid or solid compositions may contain
suitable pharmaceutically acceptable excipients as described supra.
Preferably the compositions are administered by the oral or nasal
respiratory route for local or systemic effect. Compositions in
preferably pharmaceutically acceptable solvents may be nebulized by
use of inert gases. Nebulized solutions may be inhaled directly
from the nebulizing device or the nebulizing device may be attached
to a face mask tent, or intermittent positive pressure breathing
machine. Solution, suspension, or powder compositions may be
administered, preferably orally or nasally, from devices which
deliver the formulation in an appropriate manner (see U.S. Pat.
Nos. 5,919,827 and 5,972,919).
[0333] Furthermore, the pharmaceutical compositions containing one
or more compound(s) of this invention can be administered in
combination any other suitable drug, for example, with a suitable
steroidal anti-inflammatory drug, e.g., budesonide, flucatisone,
beclamethasone, for the treatment of respiratory disorders. When
the combination therapy is employed, the pharmaceutical composition
containing the compound(s) of this invention and the steroidal
anti-inflammatory drug may be administered simultaneously,
sequentially or separately. Each component used in the combination
therapy is employed in an amount sufficient for its intended
purpose. For example, the steriodal anti-inflammatory drugs are
employed in sufficient amounts to effect reduction in inflammation
in vivo. The .beta.-2 adrenergic receptor agonist/partial agonist
compounds of this invention are employed in an amount sufficient to
cause relaxation of smooth muscle tissue, for example, in the
bronchial system.
[0334] Preferably, the dose range for compounds of this invention
is from about 1 .mu.g to about 1000 .mu.g per dose, more preferably
about 2 .mu.g to about 500 .mu.g, even more preferably about 5
.mu.g to about 100 .mu.g, and still more preferably about 5 .mu.g
to about 60 .mu.g. The preferred dosage range for a steroidal
anti-inflammatory drug is from about 50 to 4800 .mu.g and more
preferably about 100 .mu.g to about 1600 .mu.g. Again, the
particular dose used will depend on the patient (age, weight,
etc.), and the severity of the disease (mild, moderate, severe).
Lastly, a pharmaceutical composition containing the two active
ingredients can also be prepared for administering the drugs
simultaneously.
EXAMPLES
[0335] The following preparations and examples are given to enable
those skilled in the art to more clearly understand and to practice
the present invention. They should not be considered as limiting
the scope of the invention, but merely as being illustrative and
representative thereof.
[0336] In the examples below, the following abbreviations have the
following meanings. Unless otherwise stated, all temperatures are
in degrees Celsius. If an abbreviation is not defined, it has its
generally accepted meaning
[0337] .ANG.=Angstroms
[0338] cm=centimeter
[0339] DCC=dicyclohexyl carbodiimide
[0340] DMF=N,N-dimethylformamide
[0341] DMSO=dimethylsulfoxide
[0342] g=gram
[0343] HPLC=high performance liquid chromatography
[0344] MEM=minimal essential medium
[0345] mg=milligram
[0346] MIC=minimum inhibitory concentration
[0347] min=minute
[0348] mL=milliliter
[0349] mm=millimeter
[0350] mmol=millimol
[0351] N=normal
[0352] THF=tetrahydrofuran
[0353] .mu.L=microliters
[0354] .mu.m=microns
[0355] rt=room temperature
[0356] R.sub.f=retention faction
[0357] NMR=nuclear magnetic resonance
[0358] ESMS=electrospray mass spectrum
[0359] ppm=parts per million
Synthetic Examples
Example 1
Synthesis of
trans-1,4-bis{N-[2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino-
}cyclohexane (following FIG. 5)
##STR00082##
[0360] Step 1
[0361] To a solution of 5-acetylsalicylic acid methyl ester 11 (5.0
g, 25.7 mmole) in dimethylsulfoxide (44 mL) was added 48%
hydrobromic acid. The resulting mixture was stirred at 55.degree.
C. for 24 h, and poured into a slurry of ice-water (200 mL),
precipitating a pale yellow solid. The solid was filtered, washed
with water (200 mL), and dried to give
.alpha.,.alpha.-dihydroxy-4-hydroxy-3-methoxycarbonyl-acetophenone
12. The product was re-suspended in ethyl ether (200 mL), filtered
and dried to give (3.41 g, 59%) of pure product. R.sub.f=0.8 (10%
MeOH/CH.sub.C12).
[0362] H.sup.1-NMR (4/1 CDCl.sub.3/CD.sub.3OD, 299.96 MHz): .delta.
(ppm) 8.73-8.72 (d, 1H), 8.28-8.24 (dd, 1H), 7.08-7.05 (d, 1H),
5.82 (s, 1H), 4.01 (s, 3H).
Step 2
[0363] To a suspension of
.alpha.,.alpha.-dihydroxy-4-hydroxy-3-methoxycarbonyl-acetophenone
12 (0.3 g, 1.33 mmole) in THF (10 mL) was added a solution of
trans-1,4-diaminocyclohexane (76 mg, 0.66 mmole) in THF (5 mL). The
resulting suspension was stirred for 3 h at ambient temperature
under nitrogen atmosphere, at which formation of an imine was
completed judged by TLC analysis. After cooling of the resulting
solution at ice bath, an excess amount of 2M BH.sub.3-Me.sub.2S in
hexane (4 mL, 8 mmole) was added to the previous solution. The
resulting mixture was slowly warmed to rt and refluxed for 4 h
under N.sub.2 stream. After cooling the reaction mixture, MeOH (5
mL) was added to quench excess amount of 2M BH.sub.3-Me.sub.2S.
After stirring for 30 min., the final solution (or cloudy solution)
was evaporated in vacuo, yielding a pale brown solid. The solid was
washed with EtOAc/hexane (1/2; 20 mL), and dried. The crude product
was dissolved in 50% MeCN/H.sub.2O containing 0.5% TFA, and
purified by prep-scale high performance liquid chromatography
(HPLC) using a linear gradient (5% to 50% MeCN/H.sub.2O over 50
min, 20 mL/min; detection at 254 nM). Fractions with UV absorption
were analyzed by LC-MS to isolate
trans-1,4-bis{N-[2-(4-hydroxy-3-hydroxymethyl-phenyl)-2-hydroxyethyl]amin-
o}cyclohexane 13.
[0364] H.sup.1-NMR (CD.sub.3OD, 299.96 MHz): .delta. (ppm) 7.35 (d,
2H), 7.18 (dd, 2H), 6.80-6.78 (d, 2H), 4.88-4.86 (m, 2H), 4.65 (s,
4H), 3.15 (br s, 4H), 2.89 (m, 2H), 1.68-1.55 (br m, 4H); ESMS
(C.sub.24H.sub.34N.sub.2O.sub.6): calcd. 446.5, obsd. 447.5
[M+H].sup.+.
Compound 14:
[0365] Proceeding as described above but substituting
trans-1,4-diamino-cyclohexane with
4,4'-methylenebis(cyclohexylamine) gave
bis(4,4'-[N-[2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amin-
o]cyclohexane) methane.
[0366] ESMS (C.sub.31H.sub.46N.sub.2O.sub.6): calcd. 542.7, obsd.
543.6 [M+H].sup.+.
Compound 15:
[0367] Proceeding as described above but substituting
trans-1,4-diamino-cyclohexane with 1,3-cyclohexanebis(methylamine)
gave
1,3-bis{N-[2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]aminomethyl-
}cyclohexane.
[0368] ESMS (C.sub.27H.sub.38N.sub.2O.sub.6: calcd. 474.6, obsd.
475.3 [M+H].sup.+.
Compound 16:
[0369] Proceeding as described above but substituting
trans-1,4-diamino-cyclohexane with 1,8-diamino-p-menthane gave
1,8-bis{N-[2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino}-p-me-
nthane.
[0370] ESMS (C.sub.28H.sub.42N.sub.2O): calcd. 502.6, obsd. 503.3
[M+H].sup.+.
Compound 17:
[0371] Proceeding as described above but substituting
trans-1,4-diamino-cyclohexane with 1,4-bis(3-aminopropyl)piperazine
gave
1,4-bis{3-[[N-2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino]pr-
opyl}piperazine.
[0372] ESMS (C.sub.28H.sub.44N.sub.4O.sub.6): calcd. 532.6, obsd
533.3 [M+H].+-.555.0 [M+Na].sup.+.
Compound 18:
[0373] Proceeding as described above but substituting
trans-1,4-diamino-cyclohexane with p-xylylenediamine gave
1,4-bis{N-[2-(4-hydroxy-3-hydroxy-methylphenyl)-2-hydroxyethyl]aminomethy-
l}benzene. ESMS (C.sub.26H.sub.32N.sub.2O.sub.6): calcd. 468.5,
obsd, 469.3 [M+H].sup.+, 492.0 [M+Na].sup.+.
Compound 19:
[0374] Proceeding as described above but substituting
trans-1,4-diamino-cyclohexane with m-xylylenediamine gave
1,3-bis{N-[2-(4-hydroxy-3-hydroxy-methylphenyl)-2-hydroxyethyl]aminomethy-
l}benzene. ESMS (C.sub.26H.sub.32N.sub.2O.sub.6): calcd. 468.5,
obsd. 469.3 [M+H].sup.+, 492.0 [M+Na].sup.+.
Compound 20:
[0375] Proceeding as described above but substituting
trans-1,4-diamino-cyclohexane with 2-aminobenzylamine gave
1-{N-[2-(4-hydroxy-3-hydroxy-methylphenyl)-2-hydroxyethyl]aminomethyl}-2--
(N-[2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino
benzene. ESMS (C.sub.25H.sub.30N.sub.2O.sub.6): calcd. 454.5, obsd.
455.3 [M+H].sup.+.
Compound 21:
[0376] Proceeding as described above but substituting
trans-1,4-diamino-cyclohexane with 2-(4-aminophenyl)ethylamine gave
1-{2-[N2-[(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino]ethyl}-2-
-{N-[2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino]benzene.
ESMS (C.sub.26H.sub.32N.sub.2O.sub.6): calcd. 468.5, obsd. 469.3
[M+H].sup.+.
Compound 22:
[0377] Proceeding as described above but substituting
trans-1,4-diamino-cyclohexane with 4,4'-oxydianiline gave
4,4'-bis{N-[2-(4-hydroxy-3-hydroxy-methylphenyl)-2-hydroxyethyl]amino}phe-
nylether. ESMS (C.sub.30H.sub.32N.sub.2O.sub.7): calcd. 532.6,
obsd. 533.3 [M+H].sup.+, 556.1 [M+Na].sup.+.
Compound 23:
[0378] Proceeding as described above but substituting
trans-1,4-diamino-cyclohexane with 2-aminobenzylamine gave
1-{N-[2-(4-hydroxy-3-hydroxy-methylphenyl)-2-hydroxyethyl]aminomethyl}-4--
{N-[2-(4-hydroxy-3-hydroxy-methylphenyl)-2-hydroxyethyl]amino}benzene.
ESMS (C.sub.25H.sub.3N.sub.2O.sub.6): calcd. 454.5, obsd. 455.5
[M+H].sup.+, 477.3 [M+Na].sup.+.
Example 2
Synthesis of
1-{2-[N-2-[(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino]ethyl}--
4-{N-[2-phenyl-2-hydroxyethyl]amino]benzene (following FIG. 6)
##STR00083##
[0380] To a suspension of
.alpha.,.alpha.-dihydroxy-4-hydroxy-3-methoxycarbonyl-acetophenone
12, prepared in Example 1, Step 1 above, (0.3 g, 1.33 mmole) in THF
(10 mL) was added a solution of 2-(4-aminophenyl)ethylamine 25
(0.181 g, 1.33 mmol) in THF (5 mL). The resulting suspension was
stirred for 3 h at ambient temperature under nitrogen atmosphere,
followed by addition .alpha.,.alpha.-dihydroxy-acetophenone 24 (0.2
g, 1.32 mmole). The reaction mixture was stirred for 3 h at RT, at
which formation of the imine was completed as judged by TLC
analysis. The reaction mixture was cooled in an ice bath and an
excess amount of 2M BH.sub.3-Me.sub.2S in hexane (9 mL; 18 mmole)
was added. The resulting mixture was slowly warmed to rt, and
refluxed for 4 h under N2 stream. After cooling, MeOH (10 mL) was
added to quench excess amount of BH.sub.3-Me.sub.2S. After stirring
30 min., at rt, the final solution (or cloudy suspension) was
evaporated in vacuo, to give a pale brown solid. The solid was
washed with EtOAc/hexane (1/2; 20 mL), and dried. The crude product
was dissolved in 50% MeCN/H.sub.2O containing 0.5% TFA, and
purified by prep-scale high performance liquid chromatography
(HPLC) using a linear gradient (5% to 50% MeCN/H.sub.2O over 50
min, 20 ml/min; detection at 254 nM). Fractions with UV absorption
were analyzed by LC-MS to locate
1-{2-[N-2-[(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino]-ethyl}-
-4-(N-[2-phenyl-2-hydroxyethyl]amino]benzene 26. ESMS
(C.sub.25H.sub.30N.sub.2O.sub.4): calcd. 422.5, obsd. 423.3
[M+H].sup.+.
Compound 27:
[0381] Proceeding as described above, but substituting
.alpha.,.alpha.-dihydroxy-4-hydroxy-3-methoxycarbonylacetophenone
with .alpha.,.alpha.-dihydroxyacetophenone gave
1-{2-[N-[2-phenyl-2-hydroxyethyl]aminoethyl}-4-[N-(2-phenyl-2-hydroxyethy-
l)amino]-benzene. ESMS (C.sub.24H.sub.28N.sub.2O.sub.8): calcd.
376.5, obsd. 377.0 [M+H].sup.+.
Example 3
Synthesis of
1-{2-[N-2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]-amino]ethyl}--
4-[N-(2-phenyl-2-hydroxyethyl)amino]benzene (following FIG. 7)
##STR00084##
[0382] Step 1
[0383] To a solution of 4-(2-aminoethyl)aniline 25 (20 g, 147
mmole) in methanol (250 mL) was added (Boc).sub.2O (32.4 g, 148
mmole) in methanol (50 mL) at rt. After stirring for 24 h, the
reaction mixture was concentrated to dryness to afford a pale
yellow oily residue. The oily material solidified slowly; thus it
was dissolved in 5% MeOH/CH.sub.2Cl.sub.2, and subsequently applied
to flash silica column chromatography (3 to 10%
MeOH/CH.sub.2Cl.sub.2). After purification,
4-(N-Boc-2-aminoethyl)aniline 28 was obtained as a pale yellow
solid (32.95 g, 95%): R.sub.f 0.6 in 10% MeOH/CH.sub.2Cl.sub.2.
.sup.1H-NMR (CD.sub.3OD, 299.96 MHz): .delta. (ppm) 6.96-6.93 (d,
2H), 6.69-6.65 (d, 2H), 3.20-3.13 (q, 2H), 2.63-2.58 (t, 2H), 1.41
(s, 9H).
Step 2
4-(N-Boc-2-aminoethyl)aniline 28 (1.25 g, 5.29 mmole) was dissolved
in methanol (30 mL), followed by addition of phenyl glyoxal 24
(0.708 g, 5.28 mmole). The reaction mixture was stirred for 1 h at
rt, prior to addition of NaCNBH.sub.3 (0.665 g, 10.6 mmole). The
final mixture was stirred for 12 h at rt, concentrated, and
purified by flash silica column chromatography (2 to 5%
MeOH/CH.sub.2Cl.sub.2) to give
N-(2-phenyl-2-hydroxyethyl)-4-(N-Boc-2-aminoethyl)-aniline as a
pale yellow oil (1.71 g, 91%): R.sub.f=0.18 in 5%
MeOH/CH.sub.2Cl.sub.2. .sup.1H-NMR (CD.sub.3OD, 299.96 MHz):
.delta. (ppm) 7.4-7.25 (m, 5H), 7.0-6.95 (d, 2H), 6.63-6.60 (d,
2H), 4.85-4.79 (dd, 1H), 3.3-3.21 (t, 2H), 3.2-3.15 (m, 2H),
2.64-2.5 (t, 2H), 1.42 (s, 9H).
Step 3
[0384] A solution of
N-(2-phenyl-2-hydroxyethyl)-4-(N-Boc-2-aminoethyl)aniline (1.7 g,
4.77 .mu.mmole) in methylene chloride (10 mL) was cooled in ice
bath, and TFA (10 mL) was slowly added under a stream of nitrogen
gas. The reaction mixture was stirred for 1 h, and concentrated to
yield a pale yellow oil. The crude material was purified by
reversed phase HPLC (10% to 40% MeCN/H.sub.2O over 50 min; 20
mL/min) to give
--N-(2-phenyl-2-hydroxyethyl)-4-(2-aminoethyl)aniline 29 as the TFA
salt (1.1 g). .sup.1H-NMR (CD.sub.3OD, 299.96 MHz): .delta. (ppm)
7.42-7.3 (m, 5H), 7.29-7.25 (d, 2H), 7.12-7.0 (d, 2H), 4.85-4.82
(m, 1H), 3.45-3.35 (m, 2M), 3.18-3.1 (t, 2H), 2.98-2.94 (t, 2H);
ESMS (C.sub.16H.sub.2N.sub.2O.sub.1): calcd. 256.4, obsd. 257.1
[M+H].sup.+, 278.8 [M+Na].sup.+, 513.4 [2M+H].sup.+.
Step 4
[0385] To a solution of
N-(2-phenyl-2-hydroxyethyl)-4-(2-aminoethyl)aniline
trifluoroacetate salt 29 (1.1 g, 2.3 mmole) in methanol (10 mL) was
added 5 M NaOH solution (0.93 mL). After stirring for 10 min., the
solution was concentrated to dryness. The residue was dissolved in
THF (25 mL), and
.alpha.,.alpha.-dihydroxy-4-hydroxy-3-methoxy-carbonylacetophenone
12 (0.514 g, 2.27 mmole) was added. The reaction mixture was
stirred for 12 h at rt, cooled to 0.degree. C., and
BH.sub.3/Me.sub.2S (1.14 mL, 10 M) was added under nitrogen
atmosphere. The reaction mixture was gradually warmed to rt,
stirred for 2 h at rt, and refluxed for 4 h. The reaction mixture
was cooled and methanol (10 mL) was added slowly. After stirring
for 30 min., at rt, the reaction mixture was concentrated to afford
a solid residue, which was dissolved in MeOH (20 mL) containing 10%
TFA. Evaporation of the organics yielded a pale yellow oil which
was purified by reversed phase HPLC: 10% to 30% MeCN/H.sub.2O over
50 min; 20 mL/min to give
1-{2-[N-2-(4-hydroxy-3-hydroxy-methylphenyl)-2-hydroxyethyl]-amin-
o]ethyl}+[N-2-phenyl-2-hydroxyethyl)-amino]benzene 30 as the TFA
salt (0.65 g). .sup.1H-NMR (CD.sub.3OD, 299.96 MHz): .delta. (ppm)
7.42-7.3 (m, 6H), 7.28-7.24 (d, 2H), 7.18-7.14 (dd, 1H), 7.1-7.07
(d, 2H), 6.80-6.77 (d, 1H), 4.86-4.82 (m, 2H), 4.65 (s, 2H),
3.44-3.34 (m, 2H), 3.28-3.22 (m, 2H), 3.20-3.14 (m, 2H), 3.04-2.96
(m, 2H); ESMS (C.sub.25H.sub.30N.sub.2O.sub.4): calcd 422.5, obsd
423.1 [M+H].sup.+, 404.7 [M-1H.sub.2O].sup.+, 387.1
[M-2H.sub.2O].sup.+.
Example 4
Synthesis of
1-{2-[N-2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]-aminoethyl}-4-
-[N-(2-phenyl-2-(S)-hydroxyethyl)amino]benzene (following FIG.
8)
##STR00085##
[0386] Step 1
[0387] A solution of 4-(N-Boc-2-aminoethyl)aniline 28 (7.0 g, 29.6
mmole) in ethanol (100 mL) and (R)-styreneoxide (3.56 g, 29.6
mmole) was refluxed for 24 h. The organics were removed to give a
pale yellow solid.
N-(2-phenyl-2-(S)-hydroxyethyl)4-(N-Boc-2-aminoethyl)aniline was
separated by flash silica column chromatography: 1/2 EtOAc/hexane
to 3/1 EtOAc/hexane to 3% MeOH in 3/1 EtOAc/hexane: Rf=0.39 in 3%
MeOH/CH.sub.2Cl.sub.2.
Step 2
[0388] A solution of
N-(2-phenyl-2-(S)-hydroxyethyl)-4-(N-Boc-2-aminoethyl)-aniline (2.5
g, 7.0 mmole) in CH.sub.2Cl.sub.2 (15 mL) was cooled in an ice bath
under stream of nitrogen and TFA (15 mL) was slowly added. The
reaction mixture was stirred for 2 h at 0.degree. C. and then
concentrated in vacuo. The crude product was dissolved in 20%
MeCN/H.sub.2O and purified by preparative reversed phase HPLC (5 to
2% MeCN/H.sub.2O over 50 min; 254 nm; 20 mL/min.), to give
N-(2-phenyl-2-(S)-hydroxyethyl)-4-(2-aminoethyl)aniline
trifluoroacetate salt 31 as a colorless oil. .sup.1H-NMR
(CD.sub.3OD, 299.96 MHz): .delta. (ppm); 7.45-7.25 (m, 9H), 4.9
(dd, 1H), 3.55-3.45 (m, 2H), 3.21-3.15 (t, 2M), 3.05-2.95 (t, 2H)
ESMS (C.sub.16H.sub.20N.sub.2O.sub.1): calcd. 256.4, obsd. 257.1
[M+H].sup.+, 280.2 [M+Na].sup.+.
Step 3
[0389] To a solution of
N-(2-phenyl-2-(S)-hydroxyethyl)-4-(2-aminoethyl)aniline
trifluoroacetate 31 (0.144 g, 0.3 mmole) in methanol (10 mL) was
added aq. NaOH solution (1.0 M, 0.625 mL). The solution was
concentrated to dryness and the residue was dissolved in anhydrous
THF (5 mL).
.alpha.,.alpha.-Dihydroxy-4-hydroxy-3-methoxycarbonylacetophenone
12 (0.067 g, 0.3 mmole) was added and the reaction mixture was
stirred for 12 h at rt. BH.sub.3-Me.sub.2S (0.2 mL, 2M) was added
at 0.degree. C. and the reaction mixture was heated at 75.degree.
C. for 6 h. After cooling the reaction mixture in ice bath, MeOH (5
mL) was slowly added to it to quench the reaction, and the reaction
mixture was stirred for 30 min., at rt. The organics were removed
and the residue was dissolved in TFA/MeOH (1/9; 20 mL), and
concentrated. The crude product was dissolved in 20% MeCN/H.sub.2O,
and purified by preparative HPLC: 5 to 20% MeCN/H.sub.2O; 20
mL/min; 254 nm.) to give
1-{2-[N-2-(4-hydroxy-3-hydroxy-methylphenyl)-2-hydroxyethyl]amino]ethyl}--
4-[N-(2-phenyl-2-(S)-hydroxyethyl)-amino]benzene 33.
[0390] .sup.1H-NMR (CD.sub.3OD, 299.96 MHz): .delta. (ppm)
7.42-7.29 (m, 8H), 7.22-7.18 (d, 2H); 7.17-7.14 (dd, 1H), 6.80-6.77
(d, 1H), 4.9-4.85 (m, 2H), 4.65 (s, 2H), 3.5-3.34 (m, 2H),
3.28-3.25 (m, 2H), 319-3.14 (m, 2H), 3.04-2.98 (m, 2H); ESMS
(C.sub.25H.sub.30N.sub.2O.sub.4): calcd. 422.5, obsd. 423.1
[M+H].sup.+, 446.1 [M+Na].sup.+.
[0391] Proceeding as described in Example 4 above but substituting
(R)-styreneoxide with (S)-styreneoxide gave
1-{2-[N-2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino]ethyl}-4-
-[N-(2-phenyl-2-(R)-hydroxyethyl)amino]benzene 34.
[0392] .sup.1H-NMR (CD.sub.3OD, 299.96 MHz): .delta. (ppm)
7.42-7.28 (m, 8H), 7.20-7.1 (m, 3H), 6.80-6.77 (d, 1H), 4.9-4.85
(m, 2H), 4.65 (s, 2), 3.45-3.34 (m, 2H), 3.28-3.25 (m, 2H),
3.19-3.15 (m, 2), 3.04-2.98 (m, 2H); ESMS
(C.sub.25H.sub.30N.sub.2O.sub.4): calcd. 422.5, obsd. 423.1
[M+H].sup.+, 446.1 [M+Na].sup.+.
Example 5
Synthesis of
1,6-bis{4-(N-[2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]aminohex-
yloxypropyl]phenoxy}hexane (following FIG. 9)
##STR00086##
[0394] A solution of 3-(4-hydroxyphenyl)-1-propanol 35 (3.3 g, 21.7
mmole) and 1,6-di-iodohexane (3.5 g, 8.88 mmole) in
dimethylsulfoxide (40 mL) was degassed and saturated with N.sub.2
gas and potassium carbonate (4.5 g, 32.56 mmole) was added. The
reaction mixture was stirred at 80.degree. C. for 18 h under
nitrogen atmosphere and then quenched with brine (150 mL). The
product was extracted with EtOAc (200 mL) and the organic extracts
were washed with 0.1 M NaOH and brine, and dried with MgSO.sub.4.
The organics were removed in vacuo to give a pale brown solid. The
solid was purified by flash silica column chromatography: 411
hexane/EtOAc to 5% MeOH in 1/1 hexane/EtOAc to give
1,6-bis[4-(3-hydroxypropyl)phenoxy]hexane 36 (R.sub.f=0.17 in 1/1
hexane/EtOAc) in 65% yield (2.23 g). .sup.1H-NMR (CD.sub.3OD,
299.96 MHz): .delta. (ppm) 7.08-7.05 (d, 4H), 6.80-6.77 (d, 4H),
3.93-3.89 (t, 4H), 3.56-3.52 (t, 4H), 2.64-2.56 (t, 4H), 1.81-1.69
(m, 8H), 1.44-1.21 (m, 4H).
Step 2
[0395] A solution of 1,6-bis[4-(3-hydroxypropyl)phenoxy]hexane 36
(2.2 g, 5.69 mmole) in DMF (10 mL) was added to a solution of DMF
(40 mL) containing NaH (0.57 g; 60% dispersion in mineral oil) at
0.degree. C. under nitrogen atmosphere and the reaction mixture was
heated at 50.degree. C. After 1 h, 6-bromohexanenitrile (2.26 mL,
17 mmole) was added and the reaction mixture was heated at
80.degree. C. for 24 h. The reaction mixture was quenched with
brine solution (100 mL) and was extracted with EtOAc (250 mL). The
organic phase was washed with brine, dried with MgSO.sub.4, and
evaporated in vacuo, to give a pale yellow oil. Purification by
flash silica column chromatography: 4/1 to 1/1 hexane/EtOAc
afforded 1,6-bis[4-(5-cyanopentyloxypropyl)]phenoxy]hexane 37
product (R.sub.f=0.6 in 1/1 EtOAc/hexane). .sup.1H-NMR (CDCl.sub.3,
299.96 MHz): .delta. (ppm) 7.09-7.06 (d, 4H), 6.82-6.79 (d, 4H),
3.94-3.90 (t, 4H), 3.42-3.37 (m, 8H), 2.64-2.58 (t, 4H), 2.40-2.32
(m, 8H), 1.90-1.26 (m, 24H).
Step 3
[0396] The 1,6-bis[4-(5-cyanopentyloxypropyl)]phenoxy]hexane 37
(0.278 g, 0.48 mmole) obtained in Step 2 above was added to a
mixture of conc. HCl (10 mL) and AcOH (2 mL) and the reaction
mixture was heated at 90.degree. C. After 15 h, the reaction
mixture was diluted with brine (50 mL), extracted with EtOAc (100
mL), and dried with MgSO.sub.4. Evaporation of the organic phase
afforded the 1,6-bis[4-(5-carboxypentyl-oxypropyl)]phenoxy]hexane
38 as a pale yellow oily residue, which was used in next step
without further purification. .sup.1H-NMR (CDCl.sub.3, 299.96 MHz):
.delta. (ppm) 7.09-7.07 (d, 4H), 6.82-6.79 (d, 4H), 3.96-3.92 (t,
4H), 3.42-3.56 (m, 8H), 2.64-2.59 (t, 4H), 2.39-2.32 (m, 4H),
1.91-1.40 (m, 24H).
Step 4
[0397] To a solution of
2-hydroxy-2-(4-benzyloxy-3-hydroxymethylphenyl)-ethylamine 39
(0.263 g, 0.96 mmole) in DMF (8 mL) was added
1,6-bis[4-(5-carboxypentyloxypropyl)phenoxy]hexane (.about.0.48
mmole), obtained in Step 3 above, HOBt (0.13 g, 0.96 mmole), DIPEA
(0.21 mL, 1-20 mmole), and PyBOP (0.502 g, 0.96 mmole). After
stirring for 24 h at rt, the reaction mixture was diluted with
brine (20 mL) and extracted with EtOAc (50 mL). The organic layer
was washed with 0.1 M NaOH, 0.1 M HCl, and brine, and dried over
MgSO.sub.4. The organic solvents were removed in vacuo to give
1,6-bis[4-(5-amidopentyloxypropyl)-phenoxy]hexane as a pale yellow
oily residue (0.45 g).
Step 5
[0398] A solution of
1,6-bis[4-(5-amidopentyloxypropyl)-phenoxy]hexane (0.45 g, 0.4
mmole) obtained in Step 4 above, in anhydrous THF (10 mL) was added
to a solution of LiAlH.sub.4 (0.16 g, 4.22 mmole) in anhydrous THF
(40 mL) at 0.degree. C. The reaction mixture was stirred for 4 h at
80.degree. C. under nitrogen atmosphere and then quenched by with
10% NaOH (1 mL) at 0.degree. C. After 30 min., the reaction mixture
was filtered and the precipitate was washed with 10% MeOH in THF
(50 mL). The filtrates were combined and evaporated in vacuo to
give a pale yellow oily residue. Purification by flash silica
column chromatography: 5% MeOH/CH.sub.2Cl.sub.2 to 3% i-PrNH.sub.2
in 10% MeOH/CH.sub.2Cl.sub.2 gave the
1,6-bis[4-(6-aminohexyloxypropyl)-phenoxy]hexane. .sup.1H-NMR
(CDCl.sub.3, 299.96 MHz): .delta. (ppm) 7.40-7.25 (m, 12H),
7.22-7.18 (d, 2H), 7.09-7.02 (d, 4H), 6.91-6.88 (d, 2H), 6.81-6.75
(d, 4H), 5.01 (s, 4H), 4.8-4.75 (m, 2H), 4.70 (s, 4H), 3.96-3.83
(q, 4H), 3.42-3.34 (m, 8H), 2.84-2.64 (m, 8H), 2.62-2.56 (t, 4H),
1.84-1.75 (m, 8H), 1.57-1.50 (m, 10H), 1.34-1.23 (m, 10H).
Step 6
[0399] A solution of
1,6-bis[4-(6-aminohexyloxypropyl)-phenoxy]hexane (0.16 g, 0.15
mmole) obtained in Step 5 above, in EtOH (40 mL) was hydrogenated
under H.sub.2 (1 atm) atmosphere with 10% Pd/C catalyst (100 mg) at
rt for 24 h. The catalyst was filtered and the filtrate was
concentrated to afford crude product as a pale yellow oil.
Purification by reversed phase HPLC: 10 to 50% MeCN/H.sub.2O over
40 min; 20 nm/min; 254 nm provides
1,6-bis{4-(N-[2-(4-hydroxy-3-hydroxymethyl-phenyl)-2-hydroxyethyl]aminohe-
xyloxypropyl]-phenoxy}hexane 40. H.sup.1-NMR (CD.sub.3OD, 299.96
MHz): .delta. (ppm) 7.35 (d, 2H), 7.18-7.15 (dd, 2H), 7.08-7.05 (d,
4H), 6.82-6.77 (m, 6H), 4.65 (s, 4H), 3.96-3.92 (t, 4H), 3.45-3.34
(m, 8H), 3.12-3.01 (m, 6H), 2.94-2.89 (t, 2H), 2.62-2.57 (t, 4H),
1.86-1.43 (m, 28H); ESMS (C.sub.54H.sub.80N.sub.2O.sub.10): calcd.
917.1, obsd. 917.5 mM.sup.+, 940.8 [M+Na].sup.+.
Example 6
Synthesis of
1-{2-[N-2-(4-hydroxy-3-hydroxymethylphenyl)-2-(R)-hydroxy-ethyl]aminoethy-
l}-4-[N-(2-phenyl-2-(S)-hydroxyethyl)amino]phenyl (following FIG.
10)
##STR00087##
[0400] Step 1
[0401] A mixture of 4-(N-Boc-2-aminoethyl)aniline 28 (10 g, 42.34
mmole), benzaldehyde (4.52 mL, 44.47 mmole), and molecular sieves
4A (10 g) in toluene (100 mL) was refluxed at 95.degree. C. for 15
h. The reaction mixture was filtered and the filtrate was
concentrated in vacuo to give a colorless oil. The oil was
dissolved in MeOH (150 mL) and AcOH (0.5 mL) and NaCNBH.sub.3 (2.79
g, 44.4 mmole) were added. The reaction mixture was stirred at
0.degree. C. for 1 h and at rt for 2 h and then concentrated in
vacuo to give a pale yellow oily residue. Purification by flash
silica column chromatography: 1/1 hexane/EtOAc gave
N-benzyl-4-(N-Boc-2-aminoethyl)aniline 41 as colorless oil (11.5 g,
83%) R.sub.f=0.75 in 1/1 hexane/EtOAc. H.sup.1-NMR (CD.sub.3OD,
299.96 MHz): 6 (ppm) 7.38-7.2 (m, 5H), 6.87-6.84 (d, 2H), 6.58-6.55
(d, 2H), 4.27 (s, 2H), 3.2-3.15 (m, 2H), 2.6-2.56 (t, 2H), 1.41 (s,
9H); ESMS (C.sub.20H.sub.26N.sub.2O): calcd. 326.4, obsd. 328
[M+H].sup.+.
Step 2
[0402] A mixture of N-benzyl-4-(N-Boc-2-aminoethyl)aniline 41 (10
g, 30.7 mmole) and (R)-styreneoxide (3.51 mL, 30.7 mmole) in EtOH
(100 mL) was refluxed for 48 h. A small aliquot of the reaction
mixture was taken out for liquid chromatographic analysis, which
indicated that the desired adduct
2-[(N-benzyl-4-[2-N-Boc-aminoethyl)anilino]-1-phenylethanol was
formed as a minor product along with another regio-isomer
2-[(N-benzyl-4-[2-N-Boc-aminoethyl)anilino]-2-phenyl-ethanol in a
ratio of .about.1/2. Evaporation of the solution afforded thick,
pale yellow oil, which was purified by flash silica column
chromatography: 4/1 to 2/1 hexane/EtOAc. After repeated
chromatography,
2-[(N-benzyl-4-[2-N-Boc-aminoethyl)anilino]-1-phenyl-ethanol was
obtained as a colorless oil (4.01 g, 29%) (R.sub.f=0.76 in 2/1
hexane/EtOAc). H.sup.1-NMR (CD.sub.3OD, 299.96 MHz): .delta. (ppm)
7.4-7.1 (m, 10H), 7.1-7.06 (d, 2H), 6.68-6.65 (d, 2H), 5.0 (t, 1H),
4.52-4.46 (d, 10H), 4.26-4.22 (d, 1H), 3.76-3.68 (dd, 1H),
3.56-3.48 (dd, 1H), 3.22-3.12 (m, 2H), 2.68-2.56 (m, 2H), 1.41 (s,
9H); ESMS (C.sub.28H.sub.34N.sub.2O.sub.3): calcd. 446.6, obsd.
447.1 [M+H].sup.+, 893.4 [2M+H].sup.+.
Step 3
[0403] To a solution of
2-[(N-benzyl-4-[2-N-Boc-aminoethyl)anilino]-1-phenyl-ethanol (4.01
g, 8.99 mmole) in CH.sub.2Cl.sub.2 (15 mL) maintained in an ice
bath was added TFA (15 m/L) under stream of nitrogen atmosphere.
After stirring at 0.degree. C. for 30 min., the reaction mixture
was concentrated in vacuo, yielding a pale yellow oil. Purification
by flash silica column chromatography: (1/2 hexane/EtOAc to 5%
i-PrNH.sub.2 in 1/2 hexane/EtOAc) gave
2-[(N-benzyl-4-[2-aminoethyl)anilino]-1-phenyl-ethanol 42 as a pale
yellow oil from such fractions with R of 0.2 (5% i-PrNH.sub.2 in
1/2 hexane/EtOAc) in 74% yield (2.29 g). H.sup.1-NMR (CD.sub.3OD,
299.96 MHz): .delta. (.mu.m) 7.38-7.06 (m, 10H), 7.01-6.98 (d, 2H),
6.71-6.68 (d, 2H), 5.02-4.96 (dd, 1H), 4.54-4.48 (d, 10H),
4.29-4.23 (d, 1H), 3.76-3.67 (dd, 1H), 3.58-3.50 (dd, 1H),
2.82-2.74 (t, 2H), 2.64-2.59 (t, 2H); ESMS
(C.sub.23H.sub.26N.sub.2O.sub.1): calcd. 346.5, obsd.
346.3[M].sup.+,
Step 4
[0404] A mixture of
2-[(N-benzyl-4-[2-aminoethyl)anilino]-1-phenylethanol 42 (2.28 g,
6.59 mmole), benzaldehyde (0.74 mL, 7.28 mmole), and molecular
sieves 4A (4 g) in toluene (40 mL) was heated at 90.degree. C. for
14 h. The reaction mixture was cooled and filtered, and the sieves
were rinsed with toluene. The combined filtrates were concentrated
to give an oily residue which was washed with hexane, and dried.
The residue was dissolved in MeOH (40 mL) containing AcOH (0.4 mL)
and the reaction mixture was cooled in an ice bath. NaCNBH.sub.3
(0.62 g, 9.87 mmole) was added and the reaction mixture was stirred
for 2 h at rt, and then concentrated. The oily residue was
dissolved in 60% MeCN/H.sub.2O, and purified by reversed phase
preparative liquid chromatography (40 to 80% MeCN/H.sub.2O over 30
min; 30 mL/min) to give
2-[(N-benzyl-4-[2-N-benzylaminoethyl)anilino]-1-phenylethanol as
the TFA salt. The product was treated with alkaline brine solution,
and extracted with ether (200 mL). The organic layer was dried with
NaSO.sub.4, and concentrated, to give
2-[(N-benzyl-4-[2-N-benzylaminoethyl)anilino]1-phenylethanol 43 as
a colorless oil (1.36 g). H.sup.1-NMR (CD.sub.3OD, 299.96 MHz):
.delta. (ppm) 7.36-7.06 (m, 15H), 6.98-6.95 (d, 2H), 6.69-6.60 (d,
2H), 5.01-4.96 (t, 1H), 4.54-4.47 (d, 1H), 4.29-4.24 (d, 1H), 3.73
(s, 2H), 3.72-3.68 (dd, 1H), 3.59-3.54 (dd, 1H), 2.80-2.74 (m, 2H),
2.70-2.64 (m, 2H); ESMS (C.sub.30H.sub.32N.sub.2O.sub.1): calcd.
436.6, obsd. 437.2 [M+H].sup.+.
Step 5
[0405] A concentrated solution of
2-[(N-benzyl-4-[2-N-benzylaminoethyl)anilino]-1-phenylethanol (1.36
g, 3.12 mmole) and compound
(S)-4-benzyloxy-3-methoxycarbonylstyreneoxide 44 (0.887 g, 3.12
mmole; .about.95% cc) (prepared as described in R. Hett, R. Stare,
P. Helquist, Tet. Lett., 35, 9375-9378, (1994)) in toluene (1 mL)
was heated at 105.degree. C. for 72 h under nitrogen atmosphere.
The reaction mixture was purified by flash silica column
chromatography (2/1 hexane/EtOAc to 3% MeOH in 1/1 hexane/EtOAc) to
give
1-{2-[N-benzyl-N-2-(4-benzyloxy-3-methoxycarbonylphenyl)-2-(R)-hydroxy]et-
hylaminoethyl}4-[N-(2-phenyl-2-(S)-hydroxy)ethylamino]benzene 45.
R.sub.f=0.62 in 3% MeOH in 1/1 hexane/EtOAc) was obtained as a pale
yellow foam (2.0 g, 89%).
[0406] H.sup.1-NMR (CD.sub.3OD, 299.96 MHz): .delta. (ppm)
7.67-7.66 (d, 1H), 7.49-7.42 (m, 2H), 7.38-7.0 (m, 20H), 6.88-6.85
(d, 2H), 6.65-6.62 (d, 2H), 5.15 (s, 214), 5.05-4.98 (t, 1H),
4.6-4.54 (t, 1H), 4.53-4.46 (d, 1H), 4.28-4.22 (d, 1H), 3.84 (s,
3H), 3.72-3.64 (m, 3), 3.56-3.46 (dd, 10H), 2.74-2.56 (m, 6H); ESMS
(C.sub.47H.sub.48N.sub.2O.sub.5): calcd. 720.9, obsd. 721.4
[M+H].sup.+, 743.3 [M+Na].sup.+.
Step 6
[0407] To a suspension of LiAlH.sub.4 (0.211 g, 5.56 mmole) in THF
(40 mL) cooled with ice bath was added
1-{2-[N-benzyl-N-2-(4-benzyloxy-3-methoxycarbonylphenyl)-2-(R)-hydroxyeth-
yl]aminoethyl}-4-[N-(2-phenyl-2-(S)-hydroxyethyl)amino]benzene 45
(2.0 g, 2.78 mmole) in THF (10 mL) under nitrogen atmosphere. The
reaction mixture was warmed slowly to rt and the stirring was
continued for 5 h. The reaction was cooled to 0.degree. C., and 10%
NaOH (0.5 mL) was slowly added. After 30 min., a thick gel formed.
The gel was diluted with THF (300 mL), filtered, and the solid mass
was rinsed with THF (50 mL). The filtrates were combined, and
concentrated in vacuo, yielding an oily residue. The residue was
purified by flash silica column chromatography (2/1 hexane/EtOAc to
3% MeOH in 1/1 hexane/EtOAc) to give
1-{2-[N-benzyl-N-2-(4-benzyloxy-3-hydroxymethylphenyl)-2-(R)-hydroxyethyl-
]aminoethyl}-4-[N-(2-phenyl-2-(S)-hydroxyethyl)amino]benzene as a
colorless oil (1.28 g, 67%). H.sup.1-NMR (CD.sub.3OD, 299.96 MHz):
.delta. (ppm) 7.4-7.0 (m, 22H), 6.85-6.82 (m, 3H), 6.63-6.60 (d,
2H), 5.02-4.94 (m, 3H), 4.66 (s, 2H), 4.59-4.54 (dd, 1H), 4.48-4.4
(d, 1H), 4.24-4.16 (d, 1H), 3.76-3.7 (d, 1H), 3.69-3.62 (dd, 1H),
3.58-3.52 (d, 1H), 3.50-3.44 (dd, 1H), 2.76-2.54 (m, 6H); ESMS
(C.sub.46H.sub.48N.sub.2O.sub.4): calcd. 692.90, obsd. 693.5
[M+H].sup.+.
Step 7
[0408] A solution of
1-{2-[N-benzyl-N-2-(4-benzyloxy-3-hydroxymethylphenyl)-2-(R)-hydroxyethyl-
]amino]ethyl}-4-[N-(2-phenyl-2-(S)-hydroxyethyl)amino]-benzene
(1.28 g, 1.85 mmole) in EtOH (80 mL) was hydrogenated under H.sub.2
(1 atm) with 10% Pd/C (0.6 g) for 36 h. After filtration and
rinsing of the catalyst with EtOH (50 mL), the filtrates were
combined, and evaporated in vacuo, yielding pale yellow foam which
was dissolved in 10% MeCN/H.sub.2O, and purified by reversed phase
preparative liquid chromatography (10 to 30% MeCN/H.sub.2O
(containing 0.3% TFA) over 50 min; 30 mL/min; 254 nm) to give
1-{2-[N-2-(4-hydroxy-3-hydroxymethyl-phenyl)-2-(R)-hydroxyethyl]amin-
oethyl}-4-1N{2-phenyl-2-(S)-hydroxyethyl)-amino]benzene as the TFA
salt (0.6 g, 50%). Optical purity of
1-{2-[N-2-(4-hydroxy-3-hydroxymethylphenyl)-2-(R)-hydroxyethyl]aminoethyl-
}-4-[N-(2-phenyl-2-(S)-hydroxyethyl)amino]benzene 46 which was
analyzed with capillary electrophoresis by using a chiral medium,
and estimated to be .about.93%.
[0409] H.sup.1-NMR (CD.sub.3OD, 299.96 MHz): .delta. (ppm)
7.42-7.28 (m, 8H), 7.26-7.22 (d, 2H), 7.18-7.14 (dd, 1H), 6.80-6.77
(d, 1H), 4.88-4.82 (m, 2H), 4.65 (s, 2H), 3.5-3.43 (m, 2H),
3.29-3.26 (m, 2H), 3.19-4.14 (m, 2H), 3.06-3.0 (m, 2H); ESMS
(C.sub.25H.sub.30N.sub.2O.sub.4): calcd. 422.5, obsd. 423.1
[M+H].sup.+, 445.4 [M+Na].sup.+.
Example 7
Synthesis of
1-{6-[N-[2-(4-hydroxy-3-hydroxymethylphenyl)-2-[hydroxyethyl]-amino]hexyl-
oxy}-4-{6-[N-[2-(4-hydroxy-3-hydroxy-methylphenyl)-2-hydroxyethyl]amino]he-
xyloxypropyl}benzene (following FIG. 11)
##STR00088##
[0410] Step 1
[0411] A solution of 3-(4-hydroxyphenyl)-1-propanol (2.0 g, 13.1
mmole) in DMF (5 mL) was added to a solution of DMF (35 mL)
containing NaH (1.31 g, 60% in mineral oil) at 0.degree. C. under
nitrogen atmosphere. The reaction mixture was slowly warmed to
80.degree. C. After stirring for 1 h at 80.degree. C., the reaction
mixture was cooled to 0.degree. C., and 6-bromohexanenitrile (5.78
g, 32.83 mmole) was added. The final mixture was re-heated to
80.degree. C., and stirred for 24 h. The reaction mixture was
quenched with saturated NaCl solution (200 mL), and the product was
extracted with EtOAc (300 mL). The organic layer was washed with
brine solution, dried with Na.sub.2SO.sub.4, and evaporated to
dryness, yielding a pale yellow solid. Purification of the crude
product by flash silica column chromatography: 4/1 to 1/1
hexane/EtOAc provided
6-(3-[4-(5-cyanopentyloxy)phenyl]propoxy}hexanenitrile in 30% yield
(1.33 g). R.sub.f=0.63 in 1/1 EtOAc/hexane. .sup.1H-NMR
(CDCl.sub.3, 299.96 MHz): .delta. (ppm) 7.09-7.01 (d, 2H),
6.81-6.78 (d, 2H), 3.96-3.92 (t, 2H), 3.42-3.37 (m, 4H), 2.64-2.58
(t, 2H), 2.39-2.32 (m, 4H), 1.87-1.52 (m, 14H).
Step 2
[0412] A solution of
6-{3-[4-(5-pentyloxy)phenyl]propoxy}hexanenitrile (1.33 g, 3.88
mmole) in THF (10 mL) was added to a solution of LiAlH.sub.4 (0.442
g, 11.65 mmole) in THF (50 mL) at 0.degree. C. under nitrogen
atmosphere. The reaction mixture was heated slowly to reflux, and
stirred for 2 h. The reaction mixture was cooled to 0.degree. C.,
and 10% NaOH solution (5 mL) was slowly added. After 30 min., the
reaction mixture was filtered, and the collected solids were washed
with THF (100 mL). The filtrate was concentrated to yield a pale
yellow oil which was purified by flash silica column
chromatography: 5% MeOH/CH.sub.2Cl.sub.2 to 3% i-PrNH.sub.2/20%
MeOH/CH.sub.2Cl.sub.2 to give
6-(3-[4-(6-aminohexyloxy)-phenyl]propoxy)-hexylamine as a colorless
oil (0.5 g, 37%) which was converted to the desired compound by
proceeding as described in Example 1, step 2 above. The crude
product was purified by preparatory reversed phase HPLC: 10 to 40%
MeCN/H.sub.2O over 40 min; 20 mL/min; 254 nm. ESMS
(C.sub.39H.sub.58N.sub.2O.sub.8): calcd. 682.8, obsd. 683.6
[M+H].sup.+, 797.5 [M+CF.sub.3CO.sub.2H].sup.+.
Example 8
Synthesis of
bis{2-{2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxy]ethyamino}-2-hydroxy-
ethoxy}benzene (following FIG. 12)
##STR00089##
[0413] Step 1
[0414] To a N.sub.2-saturated solution of acetonitrile (300 mL)
containing methyl 5-acetylsalicylate 50 (20 g, 0.1 mole) and
benzylbromide (13.5 mL, 0.11 mole) was added K.sub.2CO.sub.3 (28.5
g, 0.21 mole). The reaction mixture was stirred at 90.degree. C.
for 5 h. After cooling, the reaction mixture was filtered, and the
filtrate was concentrated, in vacuo, yielding a white solid which
was suspended in hexane (300 mL), and collected on Buchner funnel
to give methyl O-benzyl-5-acetylsalicylate 51 as colorless to white
crystals (28.1 g, 96%). R.sub.f=0.69 in 1/1 EtOAc/hexane.
H.sup.1-NMR (CDCl.sub.3, 299.96 MHz): .delta. (ppm) 7.8.43-8.42 (d,
1H), 8.1-8.04 (dd, 1H), 7.5-7.28 (m, 5H), 7.08-7.04 (d, 1H), 5.27
(s, 2H), 3.93 (s, 3H), 2.58 (s, 3H).
Step 2
[0415] To a solution of methyl O-benzyl-5-acetylsalicylate 51
(14.15 g, 0.05 mole) in CHCl (750 mL) was added bromine (2.70 mL,
0.052 mole). The reaction mixture was stirred at rt. While being
stirred, the reaction mixture gradually turned from red-brown to
colorless. The mixture was stirred for 2 h at rt, and quenched by
adding brine solution (300 mL). After shaking the mixture in a
separatory funnel, organic layer was collected, washed with brine,
and dried under Na.sub.2SO.sub.4. The organic solution was
concentrated in vacuo, yielding white solid. It was washed with
ether (200 mL). After drying in air, 15 g (83%) of methyl
O-benzyl-5-(bromoacetyl)-salicylate 52 was obtained. R.sub.f=0.76
in 1/1 EtOAc/hexane. H.sup.1-NMR (CDCl.sub.3, 299.96 MHz): .delta.
(ppm) 8.48-8.46 (d, 1H), 8.14-8.08 (dd, 1H), 7.51-7.3 (m, 5H),
7.12-7.09 (d, 1H), .delta. 29 (s, 2H), 4.42 (s, 2H), 3.94 (s,
3H).
Step 3
[0416] To a solution of DMF (60 mL) containing methyl
O-benzyl-5-(bromoacetyl)-salicylate 52 (7.08 g, 0.019 mole) was
added NaN.sub.3 (1.9 g, 0.029 mole). After stirring at rt for 24 h
in the dark, the mixture was diluted with EtOAc (200 mL), and
washed with brine solution (3.times.200 mL) in a separatory funnel.
The organic phase was dried under MgSO.sub.4, and concentrated to
afford pale red solid. It was purified by flash silica column
chromatography: 10 to 50% EtOAc in hexane. The desired product
methyl O-benzyl-5-(azidoacetyl)salicylate 53 was obtained as white
crystals (4.7 g, 74%). R.sub.f=0.68 in 1/1 EtOAc/hexane.
H.sup.1-NMR (CDCl.sub.3, 299.96 MHz): .delta. (ppm) 8.38-8.36 (d,
1H), 8.08-8.04 (dd, 1H), 7.5-7.3 (m, 5H), 7.12-7.09 (d, 1H), 5.29
(s, 2H), 4.53 (s, 2H), 3.94 (s, 3H).
Step 4
[0417] To a gray suspension of LiAlH.sub.4 (2.74 g, 0.072 mole) in
THF (400 mL) cooled in ice bath was added methyl
O-benzyl-5-(azidoacetyl)salicylate 53 (4.7 g, 0.014 mole) under
nitrogen atmosphere. The reaction mixture was stirred at 0.degree.
C. for 1 h, and gradually warmed to rt. After stirring for 16 h at
rt, the mixture was heated at 75.degree. C. for 3 h. The reaction
mixture was cooled in ice bath, and quenched by slowly adding 10%
NaOH (10 mL). After stirring for 1 h, precipitates were filtered,
and rinsed with 5% MeOH in THF (200 mL). Filtrates were combined,
and concentrated in vacuo, yielding pale yellow oily residue. The
crude product was purified by flash silica column chromatography:
10% MeOH/CH.sub.2Cl.sub.2 to 5% i-PrNH.sub.2 in 30%
MeOH/CH.sub.2Cl.sub.2 to give
2-(4-benzyloxy-3-hydroxymethylphenyl)-2-hydroxyethylamine 39 as a
pale yellow solid (2.6 g, 66%). R.sub.f=0.63 in 5% i-PrNH.sub.2 in
30% MeOH/CH.sub.2Cl.sub.2. H.sup.1-NMR (CD.sub.3OD, 299.96 MHz): 6
(ppm) 7.46-7.28 (m, 6H), 7.24-7.20 (dd, 1H), 7.0-6.96 (d, 1H), 5.11
(s, 2H), 4.70 (s, 2H), 4.65-4.60 (t, 1H), 2.83-2.81 (d, 2H); ESMS
(C.sub.16H.sub.19N.sub.1O.sub.3): calcd. 273.3, obsd. 274.7
[M+H].sup.+, 547.3 [2M+H].sup.+.
Step 5
[0418] To a solution of EtOH (15 mL) containing compound
2-(4-benzyloxy-3-hydroxymethylphenyl)-2-hydroxyethylamine 39 (0.3
g, 1.1 mmole) was added resorcinol diglycidyl ether (0.122 g, 0.55
mmole) dissolved in EtOH (5 mL). The reaction mixture was refluxed
for 20 h. After cooling down to rt, the reaction mixture was
degassed with nitrogen and hydrogenated with 10% Pd/C (0.3 g, 10%)
under H.sub.2 (1 atm) atmosphere for 24 h. After filtration of the
catalyst, the filtrate was concentrated to dryness, yielding a
colorless oily residue which was purified by preparatory reversed
phase HPLC (10 to 50% MeCN/H.sub.2O over 40 min; 20 mL/min; 254 mm)
to give
bis(2-(2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxy]-ethyamino}-2-hydrox-
yethoxy}benzene 54. ESMS (C.sub.30H.sub.40N.sub.2O.sub.10): calcd.
588.6, obsd. 589.4 [M+H].sup.+, 610.7 [M+Na].sup.+.
Example 9
Synthesis of
1-[{2-[N-2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxy-ethyl]-amino]ethyl-
}-4-[N-(2-napth-1-yloxymethyl-2-hydroxyethyl)amino]benzene
(following FIG. 13)
##STR00090##
[0419] Step 1
[0420] A solution of EtOH (50 mL) containing
4-(N-Boc-2-aminoethyl)aniline 28 (0.4 g, 1.69 mmole) and
3-(1-naphthoxy)-1,2-epoxypropane 55 (0.33 g, 1.65 mmole) was
refluxed for 18 h, and concentrated in vacuo to dryness, yielding a
pale yellow oil. It was dissolved in 10 mL of CH.sub.2Cl.sub.2,
cooled in ice bath, and treated with TFA (5 mL). After stirring for
2 h at 0.degree. C., the mixture was evaporated, yielding a pale
red oil. It was dissolved in 30% aqueous acetonitrile, and purified
by preparatory HPLC; 10 to 30% MeCN/H.sub.2O over 30 min; 20
mL/min; 254 nm. The product 56 was obtained as colorless oil (260
mg; TFA salt). H.sup.1-NMR (CD.sub.3OD, 299.96 MHz): d (ppm)
8.88-8.25 (dd, 1H), 7.82-7.79 (dd, 1H), 7.51-7.42 (m, 3H),
7.39-7.38 (d, 1H), 7.33-7.30 (d, 2H), 7.25-7.23 (d, 2H), 6.91-6.89
(d, 1H), 4.37-4.31 (m, 1H), 4.22-4.19 (m, 2H), 3.69-3.63 (dd, 1H),
3.67-3.54 (dd, 1H), 3.17-3.11 (t, 2H), 2.96-2.91 (t, 2H); ESMS
(C.sub.21H.sub.24N.sub.2O.sub.2): calcd. 336.4, obsd. 337.5
[M+H].sup.+, 359.6 [M+Na].sup.+, 673.4 [2M+H].sup.+.
Step 2
[0421] To a solution of compound 56 (0.13 g, 0.023 mmole; TFA salt)
in 5 mL of MeOH was added 1.0 M NaOH (1.0 M, 0.46 mL). After
homogeneous mixing, the solution was evaporated to dryness. The
residue was dissolved in THF (10 mL), followed by addition of
glyoxal 12 (52 mg; 0.023 mmole). The resulting suspension was
stirred for 4 h at ambient temperature under nitrogen atmosphere.
After cooling of the resulting solution in ice bath, an excess
amount of 2M BH.sub.3-Me.sub.2S in THF (3 mL; 6 mmole) was added to
the previous reaction solution. The resulting mixture was slowly
warmed to rt, and refluxed for 4 h under N.sub.2 stream. After
cooling of the hot solution, 5 mL of MeOH was added to the cooled
mixture to quench the reaction mixture under nitrogen atmosphere.
After stirring 30 min at rt, the final solution was evaporated in
vacuo, yielding a pale brown solid. It was washed with EtOAc/hexane
(1/2; 20 mL), and dried. The crude product was dissolved in 50%
MeCN/H.sub.2O containing 0.5% TFA, and purified by prep-scale high
performance liquid chromatography (HPLC) using a linear gradient
(5% to 50% MeCN/H.sub.2O over 50 min, 20 mL/min; detection at 254
nM). Fractions with UV absorption were analyzed by LC-MS to locate
the desired product
1-{2-[N-2-(4-hydroxy-3-hydroxy-methylphenyl)-2-hydroxyethyl]amino]-ethyl}-
-4-[N-(2-napth-1-yloxymethyl-2-hydroxyethyl)amino]benzene 57. ESMS
(C.sub.30H.sub.34N.sub.2O.sub.5): calcd. 502.6, obsd. 503.2
[M+H].sup.+, 525.6 [M+Na].sup.+.
Example 10
Synthesis of
1,4,7-tris{N-[2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino}oc-
tane
##STR00091##
[0423] To a suspension of
.alpha.,.alpha.-dihydroxy-4-hydroxy-3-methoxycarbonyl-acetophenone
12 (0.45 g, 1.99 mmol) in tetrahydrofuran (15 mL) was added a
solution of 4-(aminomethyl)-1,8-octadiamine (115 mg, 0.66 mmol) in
tetrahydrofuran (5 mL). The resulting suspension was stirred for 12
h at ambient temperature under nitrogen atmosphere. After cooling
of the resulting solution in ice bath an excess amount of 2 M
BH.sub.3-Me.sub.2S in hexane (6 mL, 12 mmol) was added. The
resulting mixture was slowly warmed to rt, and refluxed for 6 h
under nitrogen atmosphere. After cooling, the reaction mixture was
quenched with methanol (5 mL). The resulting solution was stirred
at rt for 30 min., and then concentrated in vacuo to give a pale
brown solid. The solid was washed with ethyl acetate:hexane mixture
(1:2) and then dried. The crude product was dissolved in 50%
acetonitrile/water containing 0.5% TFA and purified by HPLC using a
linear gradient (5% to 50% MeCN/H.sub.2O over 50 min., 20 mL/min.;
detection at 254 nM). Fractions with UV absorption was analyzed by
LC-MS to locate the desired product. ESMS
(C.sub.36H.sub.13N.sub.3O.sub.9): Calcd. 671.8; Obsd. 671.7.
Example 11
Synthesis of
1-{2-[N-2-(4-amino-3,5-dichlorophenyl)-2-(RS)-hydroxyethyl]aminoethyl}-4--
N-(2-phenyl-2-(RS)-hydroxyethyl)amino]phenyl (following FIG.
14)
##STR00092##
[0424] Step 1
[0425] A solution of 2-(4-aminophenyl)ethylamine 25 (4.70 mL, 36.7
mmol), benzaldehyde (7.46 mL, 73.4 mmol) and 4A molecular sieves
(18 g) in toluene (180 mL) was refluxed for 3 h. The reaction
mixture was cooled and filtered. The filtrate was concentrated
under reduced pressure to afford compound 58 (95% yield).
Step 2
[0426] To a cooled a solution of 58 (2.00 g, 6.40 mmol) in ethanol
(150 mL) in ice bath was slowly added sodium borohydride (361 mg,
9.50 mmol) under a nitrogen atmosphere. The reaction mixture was
allowed to stir at 0.degree. C. for 1.5 h, and then warmed slowly
to room temperature. The reaction mixture was quenched by slowly
adding 50% methanol/TFA (5 mL) and then the mixture was
concentrated under reduced pressure. The resultant residue was
dissolved in EtOAc, and washed with 0.1 M NaOH. After drying over
Na.sub.2SO.sub.4, the organic phase was concentrated in vacuo, and
the residue was purified by flash silica gel chromatography using
ethyl acetate/hexanes as eluant to give compound 59 (80%
yield).
Step 3
[0427] To a solution of 59 (1.00 g, 3.20 mmol) in methanol cooled
with ice bath was slowly added di-tert-butyl dicarbonate (0.69 g,
3.2 mmol) in cold methanol (5 mL). The reaction mixture was allowed
to stir at 0.degree. C. for 0.5 h, and then warmed gradually to
room temperature. After stirring for 1 h, the reaction mixture was
concentrated under reduced pressure, and dried under high vacuum
overnight. The oily residue was purified by silica gel
chromatography using ethyl acetate/hexane (2:1) to afford compound
60 (80% yield).
Step 4
[0428] To a solution of 60 (2.09 g, 5.00 mmol) in methanol (45 mL)
was added phenylglyoxal (2.01 g, 15.0 mmol). The reaction mixture
was stirred at room temperature for 1 h, and followed by slow
addition of sodium cyanoborohydride (1.25 g, 20 mmol). The reaction
mixture was stirred at room temperature overnight. The reaction
mixture was concentrated under reduced pressure, and the residue
was dissolved in methanol. After filtration, the filtrate was
concentrated under reduced pressure. The residue was purified by
silica gel chromatography using hexane/ethyl acetate (9:1) to
afford compound 61 (26% yield).
Step 5
[0429] To a cooled a solution of 61 (3.19 g, 5.90 mmol) in
methylene chloride (12 mL) in an ice bath was added slowly
trifluoroacetic acid (12 .mu.L) under stream of nitrogen. After
stirring the reaction mixture for 1 h at the same temperature, the
mixture was concentrated under reduced pressure to yield compound
62 as an oily residue. The product was dried in vacuo overnight,
and was used in next step without further purification (90%
yield).
Step 6
[0430] To a mixture of 62 (1.87 g, 3-4 mmol) and
2,6-dichloro-4-(bromoacetyl)-aniline 63 (1.07 g, 3.8 mmol) in DMF
(50 mL) was added potassium carbonate (0.96 g, 6.90 mmol). The
reaction mixture was stirred at room temperature for 0.5 h and then
at 35.degree. C. for 1 h. The reaction mixture was cooled to room
temperature, and sodium borohydride (0.16 g, 4.10 mmol) in ethanol
(50 mL) was added slowly. The reaction continued overnight at room
temperature and then quenched with aqueous NH.sub.4Cl (sat'd).
After concentration of the reaction mixture under reduced pressure,
the residue was dissolved in ether, and washed with brine. After
drying over MgSO.sub.4, the organic layer was concentrated, and the
residue was purified by silica gel chromatography by eluting with
hexane/ethyl acetate (4:1). The product 64 was obtained in 50%
yield.
Step 7
[0431] A suspension of 64 (107 mg, 0.17 mmol) and palladium
hydroxide (25 mg) in ethanol (2.5 mL) was stirred overnight under
hydrogen atmosphere (1 atm) at room temperature. The mixture was
filtered, and the filtrate was concentrated to yield crude product
which was purified using silica gel chromatography with 6%
methanol/dichloromethane (10% yield) to give compound 65. ESMS
(CO.sub.24H.sub.27Cl.sub.2N.sub.3O.sub.2): calcd: 460.4; obsd: 460
[M+H].sup.-, 442 [M-H.sub.2O].sup.+, 921 [2M+H].sup.+.
Example 12
Synthesis of 1-{2-[N-2-(4-hydroxy-3-formylaminophenyl)-2-(RS)--
hydroxyethyl]aminoethyl}-4-[N-(2-phenyl-2-(RS)-hydroxyethyl)amino]phenyl
(following FIG. 15)
##STR00093##
[0432] Step 1
[0433] 1-Benzyloxy-4-bromoacetyl-2-nitrobenzene 66 (3-76 g, 10.8
mmol) [prepared as described in Chem. Bull., 25, 1368-1377,
(1977)], was added to a solution of compound 59 (3.4 g, 10.8 mmol)
in dimethylformamide (150 mL) at room temperature. After 28 h, the
reaction mixture was diluted with ether and washed with a dilute
solution of aqueous sodium chloride. The organic layer was
separated and dried over sodium sulfate, filtered and concentrated
to give a crude oil. Purification with column chromatography using
hexane:ethyl acetate as the eluent provided compound 67 (95%
yield).
Step 2
[0434] To a solution of 67 (4.0 g, 6.80 mmol) in ethanol (500 mL)
was slowly added sodium borohydride (1 g, 26.50 mmol) in portions
over 30 min., under a nitrogen atmosphere. After 6 h, the reaction
mixture was quenched by slowly adding saturated solution of aqueous
ammonium chloride. The solution was diluted with 1 N sodium
hydroxide and ethyl acetate and hexanes. The organic layer was
separated, dried over sodium sulfate, filtered and concentrated to
give compound 68 as an oil which was used in the next step without
further purification.
Step 3
[0435] To a mixture of 68 (3.6 g, 6.1 mmol) and potassium carbonate
(3.0 g, 9.2 mmol) in dimethylformamide (100 mL) was added
alpha-bromoacetophenone (1.28 g, 6.4 mmol) portionwise. The
reaction mixture was heated overnight at 65.degree. C. An
additional portion of alpha-bromoacetophenone (0.25 g, 1.25 mmol)
was added and heating was continued. After 18 h, the reaction
mixture was cooled to room temperature and ethanol (50 mL) was
added. Sodium borohydride (1.0 g, 26.5 mmol) was added and stirring
was continued for 2.5 h. The reaction mixture was concentrated and
then methanol (25 mL) was added and the excess hydride was quenched
with the addition of a saturated solution of ammonium chloride. The
reaction mixture was diluted with ethyl acetate and ether. The
organic layer was separated, dried over sodium sulfate, filtered,
and concentrated. The crude oil was purified with column
chromatography eluting with ethyl acetate/hexanes mixture to give
compound 69.
Step 4
[0436] To a solution of 69 (0.73 g, 1.0 mmol) in a mixture of
methanol (20 mL), 6 N hydrochloride acid (1 mL) and water (2 mL)
was added iron powder (0.56 g, 10.0 mmol). The reaction mixture was
heated at 90.degree. C. for 1.5 h. The reaction mixture was cooled
to room temperature and allowed to stand overnight. Methanol was
added and the brown precipitates and unreacted iron was filtered
off. The filtrate was concentrated under reduced pressure, to give
70 as a brown solid which was used in the next step without further
purification.
Step 5
[0437] Compound 70 was dissolved in a premixed solution of acetic
anhydride (5 mL) and formic acid (3 mL) at room temperature. After
3 h, the reaction mixture was diluted with ethyl acetate and
evaporated to dryness. A methanolic solution of 0.5 M sodium
hydroxide was added and the reaction mixture was stirred for 6 h at
room temperature. The reaction mixture was treated with methanolic
solution of 1 N hydrochloric acid and then concentrated to dryness.
The residue was redissolved in methanol and filtered. The filtrate
was concentrated to give compound 71 as a brown residue which was
used in the next step without further purification.
Step 6
[0438] Palladium on carbon (10%, 0.5 g) was added to a suspension
of 71 in methanol (120 mL) and dimethylformamide (80 mL). The
reaction mixture was purged with nitrogen gas and stirred overnight
under hydrogen atmosphere (1 atm) at room temperature. The mixture
was filtered, and the filtrate was concentrated to yield crude
product which was purified by HPLC (acetonitril/water/1% TFA
gradient) to give crude product which was purified with column
chromatography using methanol/methylene chloride/1% isopropylamine
as the eluent to give desired compound
1-{2-[N-2-(4-hydroxy-3-formylaminophenyl)-2-(RS)-hydroxyethyl]-aminoethyl-
}-4[N-(2-phenyl-2-(RS)-hydroxyethyl)amino]phenyl 72.
Formulation Examples
Example 1
[0439] Hard gelatin capsules containing the following ingredients
are prepared:
TABLE-US-00012 Quantity Ingredient (mg/capsule) Active Ingredient
30.0 Starch 305.0 Magnesium stearate 5.0
[0440] The above ingredients are mixed and filled into hard gelatin
capsules in 340 mg quantities
Example 2
[0441] A tablet formula is prepared using the ingredients
below:
TABLE-US-00013 Quantity Ingredient (mg/tablet) Active Ingredient
25.0 Cellulose, microcrystalline 200.0 Colloidal silicon dioxide
10.0 Stearic acid 5.0
[0442] The components are blended and compressed to form tablets,
each weighing 240 mg.
Example 3
[0443] A dry powder inhaler formulation is prepared containing the
following components:
TABLE-US-00014 Ingredient Weight % Active Ingredient 5 Lactose
95
[0444] The active ingredient is mixed with the lactose and the
mixture is added to a dry powder inhaling appliance.
Example 4
[0445] Tablets, each containing 30 mg of active ingredient, are
prepared as follows:
TABLE-US-00015 Quantity Ingredient (mg/tablet) Active Ingredient
30.0 mg Starch 45.0 mg Microcrystalline cellulose 35.0 mg
Polyvinylpyrrolidone 4.0 mg (as 10% solution in sterile water)
Sodium carboxymethyl starch 4.5 mg Magnesium stearate 0.5 mg Talc
1.0 mg Total 120.0 mg
[0446] 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.degree. to 60.degree. 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.
Example 5
[0447] Capsules, each containing 40 mg of medicament are made as
follows:
TABLE-US-00016 Quantity Ingredient (mg/capsule) Active Ingredient
40.0 mg Starch 109.0 mg Magnesium stearate 1.0 mg Total 150.0
mg
[0448] The active ingredient, starch, and magnesium stearate are
blended, passed through a No. 20 mesh U.S. sieve, and filled into
hard gelatin capsules in 150 mg quantities.
Example 6
[0449] Suppositories, each containing 25 mg of active ingredient
are made as follows:
TABLE-US-00017 Ingredient Amount Active Ingredient 25 mg Saturated
fatty acid glycerides to 2,000 mg
[0450] The active ingredient is passed through a No 60 mesh U.S.
sieve and suspended in the saturated fatty acid glycerides
previously melted using the minimum heat necessary. The mixture is
then poured into a suppository mold of nominal 2.0 g capacity and
allowed to cool.
Example 7
[0451] Suspensions, each containing 50 mg of medicament per 5.0 mL
dose are made as follows:
TABLE-US-00018 Ingredient Amount Active Ingredient 50.0 mg Xanthan
gum 4.0 mg Sodium carboxymethyl cellulose (11%) 50.0 mg
Microcrystalline cellulose (89%) Sucrose 1.75 g Sodium benzoate
10.0 mg Flavor and Color q.v. Purified water to 5.0 mL
[0452] 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.
Example 8
[0453] A formulation may be prepared as follows:
TABLE-US-00019 Quantity Ingredient (mg/capsule) Active Ingredient
15.0 mg Starch 407.0 mg Magnesium stearate 3.0 mg Total 425.0
mg
[0454] 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.
Example 9
[0455] A formulation may be prepared as follows:
TABLE-US-00020 Ingredient Quantity Active Ingredient 5.0 mg Corn
Oil 1.0 mL
Example 10
[0456] A topical formulation may be prepared as follows:
TABLE-US-00021 Ingredient Quantity Active Ingredient 1-10 g
Emulsifying Wax 30 g Liquid Paraffin 20 g White Soft Paraffin to
100 g
[0457] The white soft paraffin is heated until molten. The liquid
paraffin and emulsifying wax are incorporated and stirred until
dissolved. The active ingredient is added and stirring is continued
until dispersed. The mixture is then cooled until solid.
[0458] Another preferred formulation employed in the methods of the
present invention employs transdermal delivery devices ("patches").
Such transdermal patches may be used to provide continuous or
discontinuous infusion of the compounds of the present invention in
controlled amounts. The construction and use of transdermal patches
for the delivery of pharmaceutical agents is well known in the art.
See, e.g., U.S. Pat. No. 5,023,252, issued Jun. 11, 1991, herein
incorporated by reference in its entirety. Such patches may be
constructed for continuous, pulsatile, or on demand delivery of
pharmaceutical agents.
[0459] Other suitable formulations for use in the present invention
can be found in Remington's Pharmaceutical Sciences, edited by E.
W. Martin (Mack Publishing Company, 18th ed., 1990).
Biological Examples
Example 1
.beta.2-Adrenergic Receptor In Vitro Functional Assay
[0460] The .beta.2-adrenergic receptor functional activity of
compounds of the invention was tested follows.
Cell Seeding and Growth
[0461] Primary bronchial smooth muscle cells from a 21 yr. old male
(Clonetics, San Diego, Calif.) were seeded at 50,000 cells/well in
24-well tissue culture plates. The media used was Clonetic's SmBM-2
supplemented with hEGF, Insulin, hFGF, and Fetal Bovine Serum.
Cells were grown two days at 37.degree. C., 5% CO.sub.2 until
confluent monolayers were seen.
Agonist Stimulation of Cells
[0462] The media was aspirated from each well and replaced with 250
ml fresh media containing 1 mM IBMX, a phosphodiesterase inhibitor
(Sigma, St Louis, Mo.). Cells were incubated for 15 minutes at
37.degree. C., and then 250 ml of agonist at appropriate
concentration was added. Cells were then incubated for an
additional 10 minutes. Media was aspirated and 500 ml cold 70% EtOH
was added to cells, and then removed to an empty 96-well deep-well
plate after about 5 minutes. This step was then repeated. The
deep-well plate was then spun in a speed-vac until all EtOH dried
off, leaving dry pellets. cAMP (pmol/well) was quantitated using a
cAMP ELISA lit from Stratagene (La Jolla, Calif.). EC.sub.50 curves
were generated using the 4-parameter fit equation:
y=(a-d)/(1+(x/c).sup.b)+d,
[0463] where,
[0464] y=cpm a=total binding c=IC.sub.50
[0465] x=[compound] d=NS binding b=slope
[0466] Fix NS binding and allow all other parameters to float.
Example 2
.beta.2-Adrenergic Receptor In Vitro Radioligand Binding Assay
[0467] The .beta.1/2-adrenergic receptor binding activity of
compounds of the invention can be tested follows. SF9 cell
membranes containing either 1 or .beta.2-adrenergic receptor (NEN,
Boston, Mass.) were incubated with 0.07 nM
.sup.125I-iodocyanopindolol (NEN, Boston, Mass.) in binding buffer
containing 75 mM Tris-HCl (pH 7.4), 12.5 mM MgCl.sub.2 and 2 mM
EDTA and varying concentrations of test compounds or buffer only
(control) in 96-well plates. The plates were incubated at room
temperature with shaking for 1 hour. The receptor bound radioligand
was harvested by filtration over 96-well GF/B filter plates
(Packard, Meriden, Conn.) pre-blocked with 0.3% polyethylenimine
and washed twice with 200 .mu.l PBS using cell harvester. The
filters were washed thrice with 200 .mu.l PBS using cell harvester
and then resuspended in 40 .mu.l scintillation cocktail. The
filter-bound radioactivity was measured with a scintillation
counter and IC.sub.50 curves are generated using the standard
4-parameter fit equation described above.
[0468] The foregoing invention has been described in some detail by
way of illustration and example, for purposes of clarity and
understanding. It will be obvious to one of skill in the art that
changes and modifications may be practiced within the scope of the
appended claims. Therefore, it is to be understood that the above
description is intended to be illustrative and not restrictive. The
scope of the invention should, therefore, be determined not with
reference to the above description, but should instead be
determined with reference to the following appended claims, along
with the full scope of equivalents to which such claims are
entitled.
[0469] All patents, patent applications and publications cited in
this application are hereby incorporated by reference in their
entirety for all purposes to the same extent as if each individual
patent, patent application or publication were so individually
denoted.
* * * * *