U.S. patent application number 10/217459 was filed with the patent office on 2003-04-10 for biological reagents and methods for determining the mechanism in the generation of beta-amyloid peptide.
Invention is credited to Audia, James E., Hyslop, Paul A., Nissen, Jeffrey S., Tanner, Laura I., Thompson, Richard C., Tung, Jay S..
Application Number | 20030069445 10/217459 |
Document ID | / |
Family ID | 22575443 |
Filed Date | 2003-04-10 |
United States Patent
Application |
20030069445 |
Kind Code |
A1 |
Audia, James E. ; et
al. |
April 10, 2003 |
Biological reagents and methods for determining the mechanism in
the generation of beta-amyloid peptide
Abstract
Disclosed are biological reagents which comprise compounds that
inhibit .beta.-amyloid peptide release and/or its synthesis, and,
accordingly, have utility in determining the cellular mechanism
involved in the generation of .beta.-amyloid peptide.
Inventors: |
Audia, James E.;
(Indianapolis, IN) ; Hyslop, Paul A.;
(Indianapolis, IN) ; Nissen, Jeffrey S.;
(Indianapolis, IN) ; Thompson, Richard C.;
(Frankfort, IN) ; Tung, Jay S.; (Belmont, CA)
; Tanner, Laura I.; (San Francisco, CA) |
Correspondence
Address: |
Gerald F. Swiss
BURNS, DOANE, SWECKER & MATHIS, L.L.P.
P.O. Box 1404
Alexandria
VA
22313-1404
US
|
Family ID: |
22575443 |
Appl. No.: |
10/217459 |
Filed: |
August 14, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10217459 |
Aug 14, 2002 |
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09408283 |
Sep 29, 1999 |
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6486350 |
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60160082 |
Sep 30, 1998 |
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Current U.S.
Class: |
564/59 ; 530/333;
560/157; 564/152 |
Current CPC
Class: |
C07C 235/34 20130101;
A61P 25/28 20180101; C07C 271/20 20130101; C07C 271/16 20130101;
G01N 33/6896 20130101; C07K 5/06191 20130101 |
Class at
Publication: |
564/59 ; 530/333;
560/157; 564/152 |
International
Class: |
C07K 007/00; C07C
275/14; C07C 271/20 |
Claims
What is claimed is:
1. A biological reagent defined by formula I below: A-B-C I
wherein: A is selected from the group consisting of formulas II,
II, IV and V below; B is selected from the group consisting of a)
(CH.sub.2CH(R)Q).sub.n b) alkylene-Q c) substituted alkylene-Q
wherein R is selected from hydrogen, alkyl, aryl and Q is selected
from the group consisting of --O--, --S--, --NH--, --OC(O)NH--,
--NHC(O)O--, --NHC(O)NH--, --NHC(O)-- and --C(O)NH--; and C is
selected from the group consisting of a solid support and a
detectable marker wherein C is optionally linked to Q through a
linking arm; wherein Formula II is defined as follows: of: 48
wherein R.sup.11 is selected from the group consisting (a) a
substituted phenyl group of the formula: 49wherein R.sup.c is
selected from the group consisting of acyl, alkyl, alkoxy,
alkoxycarbonyl, alkylalkoxy, azido, cyano, halo, hydrogen, nitro,
trihalomethyl, thioalkoxy, and wherein R.sup.b and R.sup.c are
fused to form a heteroaryl or heterocyclic ring with the phenyl
ring wherein the heteroaryl or heterocyclic ring contains from 3 to
8 atoms of which from 1 to 3 are heteroatoms independently selected
from the group consisting of oxygen, nitrogen and sulfur; R.sup.b
and R.sup.b' are independently selected from the group consisting
of hydrogen, halo, nitro, cyano, trihalomethyl, alkoxy, and
thioalkoxy with the proviso that R.sup.b, R.sup.b' and R.sup.c are
not all hydrogen and with the further proviso that when R.sup.c is
hydrogen, then neither R.sup.b nor R.sup.b' are hydrogen; (b)
2-naphthyl; and (c) 2-naphthyl substituted at the 4, 5, 6, 7 and/or
8 positions with 1 to 5 substituents selected from the group
consisting of alkyl, alkoxy, halo, cyano, nitro, trihalomethyl, and
thioalkoxy; R.sup.12 is selected from the group consisting of
hydrogen, alkyl of from 1 to 4 carbon atoms, alkylalkoxy of from 1
to 4 carbon atoms and alkylthioalkoxy of from 1 to 4 carbon atoms;
and R.sup.13 is selected from the group consisting of --O--, --S--,
--O--(CH.sub.2).sub.pC(O)--, --O--(CH.sub.2).sub.pC(O)O-- and
--O--(CH.sub.2).sub.pC(O)NH--, wherein p is an integer of from 1 to
2; Formula III is defined as follows: 50 wherein R.sup.21 is
selected from the group consisting of a) alkyl, alkenyl,
alkcycloalkyl, phenyl-(R.sup.d).sub.m--, naphthyl-(R.sup.d).sub.-
m-- wherein R.sup.d is an alkylene group of from 1 to 8 carbon
atoms and m is an integer equal to 0 or 1, cycloalkyl,
cycloalkenyl, 3-pyridyl, 4-pyridyl and heteroaryl, other than 3-
and 4-pyridyl, of 3 to 10 atoms and 1 to 4 heteroatoms selected
from oxygen, sulfur and nitrogen wherein the heteroaryl group is
optionally substituted with 1 to 3 substituents selected from the
group consisting of alkyl, alkoxy, aryl, aryloxy, halo, nitro,
thioalkoxy, and thioaryloxy with the proviso that for such
heteroaryls when there is at least one nitrogen heteroatom, there
is also at least one oxygen and/or sulfur heteroatom; (b) a
substituted phenyl group of the formula: 51wherein R is alkylene of
from 1 to 8 carbon atoms, m is an integer equal to 0 or 1, R.sup.e
and R.sup.f are independently selected from the group consisting of
hydrogen, hydroxy, fluoro and methyl; R.sup.g and R.sup.h are
independently selected from the group consisting of hydrogen,
alkyl, alkoxy, aryl, cyano, cycloalkyl, halo, heteroaryl,
heterocyclic, nitro, trihalomethyl, thioalkoxy, thioaryloxy,
thioheteroaryloxy, and --C(O)R.sup.j where R.sup.j is selected from
the group consisting of alkyl, aryl, alkoxy and aryloxy; and
R.sup.i is selected from the group consisting of hydrogen, alkyl,
aryl, cyano, halo, nitro, and where R.sup.g and R.sup.i are fused
to form a methylenedioxy ring with the phenyl ring; and when
R.sup.g and/or R.sup.h and/or R.sup.i is fluoro, chloro, bromo
and/or nitro, then R.sup.e and/or R.sup.f can also be chloro; and
(c) 1- or 2-naphthyl-(R.sup.k).sub.m-- substituted at the 5, 6, 7
and/or 8 positions with 1 to 4 substituents selected from the group
consisting alkyl, alkoxy, halo, cyano, nitro, trihalomethyl, and
thioalkoxy wherein R.sup.k is an alkylene group of from 1 to 8
carbon atoms and m is an integer equal to 0 or 1; R.sup.22 is
selected from the group consisting of hydrogen, alkyl, phenyl,
alkylalkoxy, alkylthioalkoxy; X is oxygen or sulfur; X' is
hydrogen, hydroxy or fluoro; and X" is hydrogen, hydroxy or fluoro,
or X' and X" together form an oxo group; Formula IV is defined as
follows: 52wherein: R.sup.31 is selected from the group consisting
of (a) phenyl, (b) a substituted phenyl group of the formula:
53wherein R.sup.o is selected from the group consisting of acyl,
alkyl, alkoxy, alkylalkoxy, azido, cyano, halo, hydrogen, nitro,
trihalomethyl, thioalkoxy, and wherein R.sup.o and R.sup.m are
fused to form a heteroaryl or heterocyclic ring with the phenyl
ring, R.sup.m and R.sup.n are independently selected from the group
consisting of hydrogen, halo, nitro, cyano, trihalomethyl, alkoxy,
and thioalkoxy with the proviso that when R.sup.o is hydrogen, then
R.sup.m and R.sup.n are either both hydrogen or both substituents
other than hydrogen, (c) 2-naphthyl, (d) 2-naphthyl substituted at
the 4, 5, 6, 7 and/or 8 positions with 1 to 5 substituents selected
from the group consisting of alkyl, alkoxy, halo, cyano, nitro,
trihalomethyl, thioalkoxy, aryl, and heteroaryl, (e) heteroaryl,
and (f) substituted heteroaryl containing 1 to 3 substituents
selected from the group consisting of alkyl, alkoxy, aryl, aryloxy,
cyano, halo, nitro, heteroaryl, thioalkoxy and thioaryloxy provided
that said substituents are not ortho (adjacent) to the heteroaryl
attachment to the --NH group; R.sup.32 is selected from the group
consisting of hydrogen, alkyl of from 1 to 4 carbon atoms,
alkylalkoxy of from 1 to 4 carbon atoms, alkylthioalkoxy of from 1
to 4 carbon atoms, aryl, heteroaryl, substituted aryl and
substituted heteroaryl provided that the substituents are not ortho
(adjacent) to the attachment of the aryl or heteroaryl atom to the
carbon atom; R.sup.33 is selected from the group consisting of
alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl,
heteroaryl, substituted alkyl, substituted alkenyl, substituted
alkynyl, and heterocyclic; and X is selected from the group
consisting of oxygen and sulfur; and Formula V is defined as
follows: 54wherein R.sup.41 is selected from the group consisting
of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted
alkyl, substituted alkenyl, substituted alkynyl, aryl, heteroaryl
and heterocyclic; R.sup.42 is selected from the group consisting of
hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, cycloalkyl, aryl, heteroaryl and
heterocyclic; each R.sup.43 is independently selected from the
group consisting of hydrogen and methyl and R.sup.43 together with
R.sup.44 can be fused to form a cyclic structure of from 3 to 8
atoms which is optionally fused with an aryl or heteroaryl group;
each R.sup.44 is independently selected from the group consisting
of hydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,
cycloalkenyl, heteroaryl, heterocyclic, substituted alkyl,
substituted alkenyl and substituted alkynyl; each R.sup.45 is
selected from hydrogen and methyl or together with R.sup.43 forms a
cycloalkyl group of from 3 to 6 carbon atoms; X is selected from
oxygen, sulfur and NH; X' is hydrogen, hydroxy or fluoro; and X" is
hydrogen, hydroxy or fluoro, or X' and X" together form an oxo
group; Z is selected from the group consisting of a bond covalently
linking R.sup.41 to --CX'X"--, oxygen and sulfur; and n is an
integer equal to 1 to 3; wherein the compounds of formulas II, III,
IV and V are effective in inhibiting the cellular release and/or
synthesis of .beta.-amyloid peptide.
2. The biological reagent of claim 1 wherein A comprises Formula
II.
3. The biological reagent of claim 1 wherein A comprises Formula
III.
4. The biological reagent of claim 1 wherein A comprises Formula
IV.
5. The biological reagent of claim 1 wherein A comprises Formula
V.
6. An intermediate useful in the synthesis of compounds of formula
I wherein said intermediate is represented by formula VI A-B' VI
wherein: A is selected from the group consisting of formulas II,
III, IV and V as defined in claim 1; and B' is selected from the
group consisting of a) (CH.sub.2CH(R)Q).sub.nH b) alkylene-Q-H c)
substituted alkylene-Q-H wherein R is selected from hydrogen,
alkyl, aryl and Q is selected from the group consisting of --O--,
--S--, --NH--.
7. A compound selected from the group consisting of: Tert-butyl
N-(8-amino-3,6-dioxaoctyl) carbamate Methyl
N-2-aminoethyl-N'-2-t-butylca- rbamoylethyl amine Tert-butyl
N-(8-N'-(N"-benzyloxycarbonyl-L-phenylglycin- e)-3,6-dioxaoctyl)
carbamate Methyl N-(2-N'-(N"-benzyloxycarbamoyl-L-pheny-
lglycine)ethyl)-N'-2-t-butylcarbamoylethyl amine Tert-butyl
N-(2-N'-(N"-benzyloxycarbonyl-L-phenylglycine)-ethyl) carbamate
Tert-butyl N-(8-N'-L-phenylglycine-3,6-dioxaoctyl) carbamate Methyl
N-(2-N-(L-phenylglycine)ethyl)-N'-2-t-butylcarbamoylethyl amine
Tert-butyl N-(2-N'-L-phenylglycine)-ethyl carbamate Tert-butyl
N-[8-N'-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl-L-alaninyl)-L-phe-
nylglycine)-3,6-dioxaoctyl]carbamate Methyl
N-[2-N-((2-(R/S)-hydroxy-2-(3,-
5-difluorophenyl)-acetyl-L-alaninyl)-L-phenylglycine)ethyl]-N'-2-t-butylca-
rbamoylethyl amine Tert-butyl
N-[2-N'-((2-(R/S)-hydroxy-2-(3,5-difluorophe-
nyl)-acetyl-L-alaninyl)-L-phenylglycine)-ethyl]carbamate
Tert-butyl-2-(2'-(N'-benzyloxycarbonyl-L-phenylglycine)
aminoethoxy)ethylcarbamate
Tert-butyl-2-(2'-(L-phenylglycine)aminoethoxy)- ethylcarbamate.
N-((R/S)-3,5-difluorophenyl-.alpha.-hydroxyacetyl)-L-alani- ne,
methyl ester N-((R/S
-3,5-difluorophenyl-.alpha.-hydroxyacetyl)-L-alan- ine
Tert-butyl-2-(2'-(N'-(((R/S)-3,5-difluorophenyl-.alpha.-hydroxyacetyl)-
-L-alanine)-L-phenylglycine)aminoethoxy)ethylcarbamate
8-N'-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl-L-alaninyl)-L-phenyl-
glycinyl)amino-3,6-dioxaoctylamine hydrochloride Methyl
N-[2-N-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl-L-alaninyl)-L-phen-
ylglycinyl)aminoethyl]-N'-2-aminoethyl amine hydrochloride
2-N'-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl-L-alaninyl)-L-phenyl-
glycine)-ethylamine hydrochloride
N-(8-N'-((2-(R/S)-hydroxy-2-(3,5-difluor-
ophenyl)-acetyl-L-alaninyl)-L-phenylglycine)-3,6-dioxaoctyl)-biotinamide
2-(2'-(N'-(((R/S)-3,5-difluorophenyl-a-hydroxyacetyl)-L-alanine)-L-phenyl-
glycine)aminoethoxy)ethylamine, hydrochloride salt
1-[N-(3,5-difluoropheny-
lacetyl)-L-alaninyl)-(L-phenylglycinyl)]amino-6-(N-biotinyl)aminohexane
1-[N-(3,5-difluorophenylacetyl)-L-alaninyl)-(L-phenylglycinyl)]amino-3-(4-
-iodophenoxy)propane
1-[N-(3,5-difluorophenylacetyl)-L-alaninyl)-(L-phenyl-
glycinyl)]amino-6-[N-(4-amino-7-nitrobenzofurazanyl)]hexane
1-[N-(3,5-difluorophenylacetyl)-L-alaninyl)-(L-phenylglycinyl)]amino-3-(4-
-trimethylstannylphenoxy) propane
N-{1-N-[N-(3,5-difluorophenylacetyl)-L-a-
laninyl-(L-phenylglycinyl)amino-6-hexyl]}-4,4-difluoro-5,7-dimethyl-4-bora-
-3.alpha.,4.alpha.-diaza-s-indacene-3-propionamide N-{methyl
N-[2-N-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl-L-alaninyl)-L-phen-
ylglycinyl)aminoethyl]-N'-2-aminoethyl}-4,4-difluoro-5,7-dimethyl-4-bora-3-
.alpha.,4.alpha.-diaza-s-indacene-3-propionamide
N-[8-N'-((2-(R/S)-hydroxy-
-2-(3,5-difluorophenyl)-acetyl-L-alaninyl)-L-phenylglycinyl)amino-3,6-diox-
aoctyl]-4,4-difluoro-5,7-dimethyl-4-bora-,
3.alpha.,4.alpha.-diaza-s-indac- ene-3-propionamide
5-(S)-[N'-((S)-3,5-Difluorophenyl-.alpha.-hydroxyacetyl-
)-L-alaninyl]amino-7-methyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one
5-(S)-[N'-((R)-3,5-Difluorophenyl-.alpha.-hydroxyacetyl)-L-alaninyl]amino-
-7-methyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one
5-(S)-(N'-((S)-(+)-2-Hydr-
oxy-3-methylbutyryl)-L-alaninyl)amino-7-methyl-5,7-dihydro-6H-dibenz[b,d]a-
zepin-6-one
1-[N-(3,5-difluorophenylacetyl)-L-alaninyl)-(L-phenylglycinyl)-
]amino-4-p-azidosalicylamidobutane
8. The biological reagent as defined in claim 1 wherein C is a
detectable marker.
9. The biological reagent as defined in claim 8 wherein the
detectable marker is selected from the group consisting of
radioactive markers and fluorescent markers.
10. The biological reagent as defined in claim 9 wherein the
radioactive marker is .sup.125I.
11. The biological reagent as defined in claim 1 wherein C is a
solid support.
12. A method for determining the mechanism in the generation of
.beta.-amyloid peptide, which method comprises contacting a
biological reagent of claim 1 with a biological solution from a
cell capable of generating the .beta.-amyloid peptide under
conditions whereby the cellular factor involved in the generation
of the .beta.-amyloid peptide attaches to the biological reagent;
removing the complex of the biological reagent and the cellular
factor from the biological solution; and identifying the cellular
factor attached to the biological reagent.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] This invention relates to biological reagents which comprise
compounds that inhibit .beta.-amyloid peptide release and/or its
synthesis, and, accordingly, have utility in determining the
cellular mechanism involved in the generation of .beta.-amyloid
peptide.
REFERENCES
[0003] The following publications, patents and patent applications
are cited in this application as superscript numbers:
[0004] .sup.1 Glenner, et al., "Alzheimer's Disease: Initial Report
of the Purification and Characterization of a Novel Cerebrovascular
Amyloid Protein", Biochem. Biophys. Res. Commun., 120:885-890
(1984).
[0005] .sup.2 Glenner, et al., "Polypeptide Marker for Alzheimer's
Disease and its Use for Diagnosis", U.S. Pat. No. 4,666,829 issued
May 19, 1987.
[0006] .sup.3 Selkoe, "The Molecular Pathology of Alzheimer's
Disease", Neuron, 6:487-498 (1991).
[0007] .sup.4 Goate, et al., "Segregation of a Missense Mutation in
the Amyloid Precursor Protein Gene with Familial Alzheimer's
Disease", Nature, 349:704-706 (1990).
[0008] .sup.5 Chartier-Harlan, et al., "Early-Onset Alzheimer's
Disease Caused by Mutations at Codon 717 of the .beta.-Amyloid
Precursor Proteing Gene", Nature, 353:844-846 (1989).
[0009] .sup.6 Murrell, et al., "A Mutation in the Amyloid Precursor
Protein Associated with Hereditary Alzheimer's Disease", Science,
254:97-99 (1991).
[0010] .sup.7 Mullan, et al., "A Pathogenic Mutation for Probable
Alzheimer's Disease in the APP Gene at the N-Terminus of
.beta.-Amyloid, Nature Genet., 1:345-347 (1992).
[0011] .sup.8 International Patent Application No. PCT/US97/20356;
Publication No. WO98/22441
[0012] .sup.9 International Patent Application No. PCT/US97/20355;
Publication No. WO98/22430
[0013] .sup.0 International Patent Application No. PCT/US97/18704;
Publication No. WO98/22493
[0014] .sup.111 International Patent Application No.
PCT/US97/20804; Publication No. WO98/22494
[0015] .sup.12 Bioconjugate Chemistry (1990) 1(6) 431-437
[0016] .sup.13 International Patent Application No. PCT/US97/22986;
Publication No. WO98/28268
[0017] All of the above publications, patents and patent
applications are herein incorporated by reference in their entirety
to the same extent as if each individual publication, patent or
patent application was specifically and individually indicated to
be incorporated by reference in its entirety.
[0018] 2. State of the Art
[0019] Alzheimer's Disease (AD) is a degenerative brain disorder
characterized clinically by progressive loss of memory, cognition,
reasoning, judgment and emotional stability that gradually leads to
profound mental deterioration and ultimately death. AD is a very
common cause of progressive mental failure (dementia) in aged
humans and is believed to represent the fourth most common medical
cause of death in the United States. AD has been observed in races
and ethnic groups worldwide and presents a major present and future
public health problem. The disease is currently estimated to affect
about two to three million individuals in the United States alone.
AD is at present incurable. No treatment that effectively prevents
AD or reverses its symptoms and course is currently known.
[0020] The brains of individuals with AD exhibit characteristic
lesions termed senile (or amyloid) plaques, amyloid angiopathy
(amyloid deposits in blood vessels) and neurofibrillary tangles.
Large numbers of these lesions, particularly amyloid plaques and
neurofibrillary tangles, are generally found in several areas of
the human brain important for memory and cognitive function in
patients with AD. Smaller numbers of these lesions in a more
restrictive anatomical distribution are also found in the brains of
most aged humans who do not have clinical AD. Amyloid plaques and
amyloid angiopathy also characterize the brains of individuals with
Trisomy 21 (Down's Syndrome) and Hereditary Cerebral Hemorrhage
with Amyloidosis of the Dutch Type (HCHWA-D). At present, a
definitive diagnosis of AD usually requires observing the
aforementioned lesions in the brain tissue of patients who have
died with the disease or, rarely, in small biopsied samples of
brain tissue taken during an invasive neurosurgical procedure.
[0021] The principal chemical constituent of the amyloid plaques
and vascular amyloid deposits (amyloid angiopathy) characteristic
of AD and the other disorders mentioned above is an approximately
4.2 kilodalton (kD) protein of about 39-43 amino acids designated
the .beta.-amyloid peptide (.beta.AP) or sometimes A.beta.,
A.beta.P or .beta./A4. .beta.-Amyloid peptide was first purified
and a partial amino acid sequence was provided by Glenner, et
al..sup.1 The isolation procedure and the sequence data for the
first 28 amino acids are described in U.S. Pat. No.
4,666,829.sup.2.
[0022] Molecular biological and protein chemical analyses have
shown that the .beta.-amyloid peptide is a small fragment of a much
larger precursor protein (APP), that is normally produced by cells
in many tissues of various animals, including humans. Knowledge of
the structure of the gene encoding the APP has demonstrated that
.beta.-amyloid peptide arises as a peptide fragment that is cleaved
from APP by protease enzymes. The precise biochemical mechanism by
which the .beta.-amyloid peptide fragment is cleaved from APP and
subsequently deposited as amyloid plaques in the cerebral tissue
and in the walls of the cerebral and mningeal blood vessels is
currently unknown.
[0023] Several lines of evidence indicate that progressive cerebral
deposition of .beta.-amyloid peptide plays a seminal role in the
pathogenesis of AD and can precede cognitive symptoms by years or
decades. See, for example, Selkoe.sup.3. The most important line of
evidence is the discovery that missense DNA mutations at amino acid
717 of the 770-amino acid isoform of APP can be found in affected
members but not unaffected members of several families with a
genetically determined (familial) form of AD (Goate, et al..sup.4;
Chartier Harlan, et al..sup.5; and Murrell, et al..sup.6) and is
referred to as the Swedish variant. A double mutation changing
lysine.sup.595-methionine.sup.596 to
asparagine.sup.595-leucine.sup.596 (with reference to the 695
isoform) found in a Swedish family was reported in 1992 (Mullan, et
al..sup.7). Genetic linkage analyses have demonstrated that these
mutations, as well as certain other mutations in the APP gene, are
the specific molecular cause of AD in the affected members of such
families. In addition, a mutation at amino acid 693 of the
770-amino acid isoform of APP has been identified as the cause of
the .beta.-amyloid peptide deposition disease, HCHWA-D, and a
change from alanine to glycine at amino acid 692 appears to cause a
phenotype that resembles AD is some patients but HCHWA-D in others.
The discovery of these and other mutations in APP in genetically
based cases of AD prove that alteration of APP and subsequent
deposition of its .beta.-amyloid peptide fragment can cause AD.
[0024] Despite the progress which has been made in understanding
the underlying mechanisms of AD and other .beta.-amyloid peptide
related diseases, there still remains a need to determine the
precise mechanism for the generation of .beta.-amyloid peptide.
Biological reagents comprising compounds known to inhibit the
generation of .beta.-amyloid peptide would be useful in determining
the mechanism for the generation of .beta.-amyloid peptide and thus
AD. Knowledge of the disease mechanism would, in turn, allow
rationale drug design of novel entities which specifically target
AD.
SUMMARY OF THE INVENTION
[0025] This invention is directed to the discovery of a class of
biological reagents which comprise compounds that inhibit
.beta.-amyloid peptide release and/or its synthesis and, therefore,
are useful in determining the underlying cellular mechanism leading
to AD in patients susceptible to AD. The class of biological
reagents having the described properties are defined by formula I
below:
A-B-C I
[0026] wherein:
[0027] A is selected from the group consisting of formulas II, III,
IV and V below;
[0028] B is selected from the group consisting of
[0029] a) (CH.sub.2CH(R)Q).sub.n
[0030] b) alkylene-Q
[0031] c) substituted alkylene-Q
[0032] wherein R is selected from hydrogen, alkyl, aryl and Q is
selected from the group consisting of --O--, --S--, --NH--,
--OC(O)NH--, --NHC(O)O--, --NHC(O)NH--, --NHC(O)-- and --C(O)NH--;
and
[0033] C is selected from the group consisting of a solid support
and a detectable marker wherein C is optionally linked to Q through
a linking arm;
[0034] wherein Formula II is defined as follows: 1
[0035] wherein R.sup.11 is selected from the group consisting
of:
[0036] (a) a substituted phenyl group of the formula: 2
[0037] wherein R.sup.c is selected from the group consisting of
acyl, alkyl, alkoxy, alkoxycarbonyl, alkylalkoxy, azido, cyano,
halo, hydrogen, nitro, trihalomethyl, thioalkoxy, and wherein
R.sup.b and R.sup.c are fused to form a heteroaryl or heterocyclic
ring with the phenyl ring wherein the heteroaryl or heterocyclic
ring contains from 3 to 8 atoms of which from 1 to 3 are
heteroatoms independently selected from the group consisting of
oxygen, nitrogen and sulfur;
[0038] R.sup.b and R.sup.b are independently selected from the
group consisting of hydrogen, halo, nitro, cyano, trihalomethyl,
alkoxy, and thioalkoxy with the proviso that R.sup.b, R.sup.b' and
R.sup.c are not all hydrogen and with the further proviso that when
R.sup.c is hydrogen, then neither R.sup.b nor R.sup.b' are
hydrogen;
[0039] (b) 2-naphthyl; and
[0040] (c) 2-naphthyl substituted at the 4, 5, 6, 7 and/or 8
positions with 1 to 5 substituents selected from the group
consisting of alkyl, alkoxy, halo, cyano, nitro, trihalomethyl, and
thioalkoxy;
[0041] R.sup.12 is selected from the group consisting of hydrogen,
alkyl of from 1 to 4 carbon atoms, alkylalkoxy of from 1 to 4
carbon atoms and alkylthioalkoxy of from 1 to 4 carbon atoms;
and
[0042] R.sup.13 is selected from the group consisting of --O--,
--S--, --O--(CH.sub.2).sub.pC(O)--, --O--(CH.sub.2).sub.pC(O)O--
and --O--(CH.sub.2).sub.pC(O)NH--, wherein p is an integer of from
1 to 2;
[0043] wherein Formula III is defined as follows: 3
[0044] wherein R.sup.21 is selected from the group consisting
of
[0045] a) alkyl, alkenyl, alkcycloalkyl, phenyl-(R.sup.d).sub.m--,
naphthyl-(R.sup.d).sub.m-- wherein R.sup.d is an alkylene group of
from 1 to 8 carbon atoms and m is an integer equal to 0 or 1,
cycloalkyl, cycloalkenyl, 3-pyridyl, 4-pyridyl and heteroaryl,
other than 3- and 4-pyridyl, of 3 to 10 atoms and 1 to 4
heteroatoms selected from oxygen, sulfur and nitrogen wherein the
heteroaryl group is optionally substituted with 1 to 3 substituents
selected from the group consisting of alkyl, alkoxy, aryl, aryloxy,
halo, nitro, thioalkoxy, and thioaryloxy with the proviso that for
such heteroaryls when there is at least one nitrogen heteroatom,
there is also at least one oxygen and/or sulfur heteroatom;
[0046] (b) a substituted phenyl group of the formula: 4
[0047] wherein
[0048] R is alkylene of from 1 to 8 carbon atoms,
[0049] m is an integer equal to 0 or 1,
[0050] R.sup.e and R.sup.f are independently selected from the
group consisting of hydrogen, hydroxy, fluoro and methyl;
[0051] R.sup.g and R.sup.h are independently selected from the
group consisting of hydrogen, alkyl, alkoxy, aryl, cyano,
cycloalkyl, halo, heteroaryl, heterocyclic, nitro, trihalomethyl,
thioalkoxy, thioaryloxy, thioheteroaryloxy, and --C(O)R.sup.j where
R.sup.j is selected from the group consisting of alkyl, aryl,
alkoxy and aryloxy; and
[0052] R.sup.i is selected from the group consisting of hydrogen,
alkyl, aryl, cyano, halo, nitro, and where R.sup.g and R.sup.i are
fused to form a methylenedioxy ring with the phenyl ring; and
[0053] when R.sup.g and/or R.sup.h and/or R.sup.i is fluoro,
chloro, bromo and/or nitro, then R.sup.e and/or R.sup.f can also be
chloro; and
[0054] (c) 1- or 2-naphthyl-(R.sup.k).sub.m-- substituted at the 5,
6, 7 and/or 8 positions with 1 to 4 substituents selected from the
group consisting alkyl, alkoxy, halo, cyano, nitro, trihalomethyl,
and thioalkoxy wherein R.sup.k is an alkylene group of from 1 to 8
carbon atoms and m is an integer equal to 0 or 1;
[0055] R.sup.22 is selected from the group consisting of hydrogen,
alkyl, phenyl, alkylalkoxy, alkylthioalkoxy;
[0056] X is oxygen or sulfur;
[0057] X' is hydrogen, hydroxy or fluoro; and
[0058] X" is hydrogen, hydroxy or fluoro, or X' and X" together
form an oxo group;
[0059] wherein Formula IV is defined as follows: 5
[0060] R.sup.31 is selected from the group consisting of
[0061] (a) phenyl,
[0062] (b) a substituted phenyl group of the formula: 6
[0063] wherein
[0064] R.sup.p is selected from the group consisting of acyl,
alkyl, alkoxy, alkylalkoxy, azido, cyano, halo, hydrogen, nitro,
trihalomethyl, thioalkoxy, and wherein R.sup.p and R.sup.m are
fused to form a heteroaryl or heterocyclic ring with the phenyl
ring,
[0065] R.sup.m and R.sup.n are independently selected from the
group consisting of hydrogen, halo, nitro, cyano, trihalomethyl,
alkoxy, and thioalkoxy with the proviso that when R.sup.p is
hydrogen, then R.sup.m and R.sup.n are either both hydrogen or both
substituents other than hydrogen,
[0066] (c) 2-naphthyl,
[0067] (d) 2-naphthyl substituted at the 4, 5, 6, 7 and/or 8
positions with 1 to 5 substituents selected from the group
consisting of alkyl, alkoxy, halo, cyano, nitro, trihalomethyl,
thioalkoxy, aryl, and heteroaryl,
[0068] (e) heteroaryl, and
[0069] (f) substituted heteroaryl containing 1 to 3 substituents
selected from the group consisting of alkyl, alkoxy, aryl, aryloxy,
cyano, halo, nitro, heteroaryl, thioalkoxy and thioaryloxy provided
that said substituents are not ortho (adjacent) to the heteroaryl
attachment to the --NH group;
[0070] R.sup.32 is selected from the group consisting of hydrogen,
alkyl of from 1 to 4 carbon atoms, alkylalkoxy of from 1 to 4
carbon atoms, alkylthioalkoxy of from 1 to 4 carbon atoms, aryl,
heteroaryl, substituted aryl and substituted heteroaryl provided
that the substituents are not ortho (adjacent) to the attachment of
the aryl or heteroaryl atom to the carbon atom;
[0071] R.sup.33 is selected from the group consisting of alkyl,
alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl,
substituted alkyl, substituted alkenyl, substituted alkynyl, and
heterocyclic; and
[0072] X is selected from the group consisting of oxygen and
sulfur; and
[0073] wherein Formula V is defined as follows: 7
[0074] wherein
[0075] R.sup.41 is selected from the group consisting of alkyl,
alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl,
substituted alkenyl, substituted alkynyl, aryl, heteroaryl and
heterocyclic;
[0076] R.sup.42 is selected from the group consisting of hydrogen,
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, cycloalkyl, aryl, heteroaryl and
heterocyclic;
[0077] each R.sup.43 is independently selected from the group
consisting of hydrogen and methyl and R.sup.43 together with
R.sup.44 can be fused to form a cyclic structure of from 3 to 8
atoms which is optionally fused with an aryl or heteroaryl
group;
[0078] each R.sup.44 is independently selected from the group
consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,
cycloalkenyl, heteroaryl, heterocyclic, substituted alkyl,
substituted alkenyl and substituted alkynyl;
[0079] each R.sup.45 is selected from hydrogen and methyl or
together with R.sup.43 forms a cycloalkyl group of from 3 to 6
carbon atoms;
[0080] X is selected from oxygen, sulfur and NH;
[0081] X' is hydrogen, hydroxy or fluoro; and
[0082] X" is hydrogen, hydroxy or fluoro, or X' and X" together
form an oxo group;
[0083] Z is selected from the group consisting of a bond covalently
linking R.sup.41 to --CX'X"--, oxygen and sulfur; and
[0084] n is an integer equal to 1 to 3;
[0085] wherein the compounds of formulas II, III, IV and V are
effective in inhibiting the cellular release and/or synthesis of
.beta.-amyloid peptide.
[0086] Accordingly, in one of its method aspects, this invention is
directed to a method for determining the proteins involved in
.beta.-amyloid peptide release and/or its synthesis in a cell which
method comprises contacting the components of a cell with an
effective amount of a biological reagent or a mixture of biological
reagents of formula I above.
[0087] In another of its composition aspects, this invention is
directed to intermediates useful in the synthesis of compounds of
formula I wherein said intermediates are represented by formula
VI
A-B' VI
[0088] wherein:
[0089] A is selected from the group consisting of formulas II, III,
IV and V as described above; and
[0090] B' is selected from the group consisting of
[0091] a) (CH.sub.2CH(R)Q).sub.nH
[0092] b) alkylene-Q-H
[0093] c) substituted alkylene-Q-H
[0094] wherein R is selected from hydrogen, alkyl, aryl and Q is
selected from the group consisting of --O--, --S--, --NH--.
[0095] Suitable compounds described by formula I and VI above
include, by way of example, the following:
[0096] Tert-butyl N-(8-amino-3,6-dioxaoctyl) carbamate
[0097] Methyl N-2-aminoethyl-N'-2-t-butylcarbamoylethyl amine
[0098] Tert-butyl
N-(8-N'-(N"-benzyloxycarbonyl-L-phenylglycine)-3,6-dioxa- octyl)
carbamate
[0099] Methyl
N-(2-N'-(N"-benzyloxycarbamoyl-L-phenylglycine)ethyl)-N'-2-t-
-butylcarbamoylethyl amine
[0100] Tert-butyl
N-(2-N'-(N"-benzyloxycarbonyl-L-phenylglycine)-ethyl) carbamate
[0101] Tert-butyl N-(8-N'-L-phenylglycine-3,6-dioxaoctyl)
carbamate
[0102] Methyl
N-(2-N-(L-phenylglycine)ethyl)-N'-2-t-butylcarbamoylethyl amine
[0103] Tert-butyl N-(2-N'-L-phenylglycine)-ethyl carbamate
[0104] Tert-butyl
N-[8-N'-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl--
L-alaninyl)-L-phenylglycine)-3,6-dioxaoctyl]carbamate
[0105] Methyl
N-[2-N-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl-L-ala-
ninyl)-L-phenylglycine)ethyl]-N'-2-t-butylcarbamoylethyl amine
[0106] Tert-butyl
N-[2-N'-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl--
L-alaninyl)-L-phenylglycine)-ethyl]carbamate
[0107] Tert-butyl-2-(2'-(N'-benzyloxycarbonyl-L-phenylglycine)
aminoethoxy)ethylcarbamate
[0108]
Tert-butyl-2-(2'-(L-phenylglycine)aminoethoxy)ethylcarbamate.
[0109]
N-((R/S)-3,5-difluorophenyl-.alpha.-hydroxyacetyl)-L-alanine,
methyl ester
[0110] N-((R/S
-3,5-difluorophenyl-.alpha.-hydroxyacetyl)-L-alanine
[0111]
Tert-butyl-2-(2'-(N'-(((R/S)-3,5-difluorophenyl-.alpha.-hydroxyacet-
yl)-L-alanine)-L-phenylglycine)aminoethoxy)ethylcarbamate
[0112]
8-N'-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl-L-alaninyl)-L--
phenylglycinyl)amino-3,6-dioxaoctylamine hydrochloride
[0113] Methyl
N-[2-N-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl-L-ala-
ninyl)-L-phenylglycinyl)aminoethyl]-N'-2-aminoethyl amine
hydrochloride
[0114]
2-N'-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl-L-alaninyl)-L--
phenylglycine)-ethylamine hydrochloride
[0115]
N-(8-N'-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl-L-alaninyl)-
-L-phenylglycine)-3,6-dioxaoctyl)-biotinamide
[0116]
2-(2'-(N'-(((R/S)-3,5-difluorophenyl-.alpha.-hydroxyacetyl)-L-alani-
ne)-L-phenylglycine)aminoethoxy)ethylamine, hydrochloride salt
[0117]
1-[N-(3,5-difluorophenylacetyl)-L-alaninyl)-(L-phenylglycinyl)]amin-
o-6-(N-biotinyl)aminohexane
[0118]
1-[N-(3,5-difluorophenylacetyl)-L-alaninyl)-(L-phenylglycinyl)]amin-
o-3-(4-iodophenoxy)propane
[0119]
1-[N-(3,5-difluorophenylacetyl)-L-alaninyl)-(L-phenylglycinyl)]amin-
o-6-[N-(4-amino-7-nitrobenzofurazanyl)]hexane
[0120]
1-[N-(3,5-difluorophenylacetyl)-L-alaninyl)-(L-phenylglycinyl)]amin-
o-3-(4-trimethylstannylphenoxy) propane
[0121]
N-{1-N-[N-(3,5-difluorophenylacetyl)-L-alaninyl-(L-phenylglycinyl)a-
mino-6-hexyl]}-4,4-difluoro-5,7-dimethyl-4-bora-3.alpha.,4.alpha.-diaza-s--
indacene-3-propionamide
[0122] N-{methyl
N-[2-N-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl-L--
alaninyl)-L-phenylglycinyl)aminoethyl]-N'-2-aminoethyl}-4,4-difluoro-5,7-d-
imethyl-4-bora-3.alpha.,4.alpha.-diaza-s-indacene-3-propionamide
[0123]
N-[8-N'-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl-L-alaninyl)-
-L-phenylglycinyl)amino-3,6-dioxaoctyl]-4,4-difluoro-5,7-dimethyl-4-bora-,
3.alpha.,4.alpha.-diaza-s-indacene-3-propionamide
[0124]
5-(S)-[N'-((S)-3,5-Difluorophenyl-.alpha.-hydroxyacetyl)-L-alaninyl-
]amino-7-methyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one
[0125]
5-(S)-[N'-((R)-3,5-Difluorophenyl-.alpha.-hydroxyacetyl)-L-alaninyl-
]amino-7-methyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one
[0126]
5-(S)-(N'-((S)-(+)-2-Hydroxy-3-methylbutyryl)-L-alaninyl)amino-7-me-
thyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one
[0127]
1-[N-(3,5-difluorophenylacetyl)-L-alaninyl)-(L-phenylglycinyl)]amin-
o-4-p-azidosalicylamidobutane
DETAILED DESCRIPTION OF THE INVENTION
[0128] As above, this invention relates to biological reagents
comprising compounds which inhibit .beta.-amyloid peptide release
and/or its synthesis, and, accordingly, have utility in determining
the mechanism for the generation of .beta.-amyloid peptide.
However, prior to describing this invention in further detail, the
following terms will first be defined.
[0129] Definitions
[0130] The term ".beta.-amyloid peptide" refers to a 39-43 amino
acid peptide having a molecular weight of about 4.2 kD, which
peptide is substantially homologous to the form of the protein
described by Glenner, et al..sup.1 including mutations and
post-translational modifications of the normal .beta.-amyloid
peptide. In whatever form, the .beta.-amyloid peptide is
approximately a 39-43 amino acid fragment of a large
membrane-spanning glycoprotein, referred to as the .beta.-amyloid
precursor protein (APP). Its 43-amino acid sequence is:
[0131] 1
[0132] Asp Ala Glu Phe Arg His Asp Ser Gly Tyr
[0133] 11
[0134] Glu Val His His Gln Lys Leu Val Phe Phe
[0135] 21
[0136] Ala Glu Asp Val Gly Ser Asn Lys Gly Ala
[0137] 31
[0138] Ile Ile Gly Leu Met Val Gly Gly Val Val
[0139] 41
[0140] Ile Ala Thr (SEQ ID NO: 1)
[0141] or a sequence which is substantially homologous thereto.
[0142] "Alkyl" refers to monovalent alkyl groups preferably having
from 1 to 10 carbon atoms and 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, and the like.
[0143] "Alkylene" refers to divalent alkylene groups preferably
having from 1 to 10 carbon atoms and 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.
[0144] "Alkaryl" refers to -alkylene-aryl groups preferably having
from 1 to 10 carbon atoms in the alkylene moiety and from 6 to 10
carbon atoms in the aryl moiety. Such alkaryl groups are
exemplified by benzyl, phenethyl and the like.
[0145] "Alkoxy" refers to the group "alkyl-O--" where alkyl is as
defined herein. Preferred alkoxy groups 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.
[0146] "Alkoxycarbonyl" refers to the group "alkyl-O--C(O)--"
wherein alkyl is as defined herein. Such groups include, by way of
example, methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl,
iso-propoxycarbonyl, n-butoxycarbonyl, tert-butoxycarbonyl,
sec-butoxycarbonyl, n-pentoxycarbonyl, n-hexoxycarbonyl, and the
like.
[0147] "Alkylalkoxy" refers to the group "-alkylene-O-alkyl"
wherein alkylene and alkoxy are as defined herein. Such groups
include, by way of example, methylmethoxy (--CH.sub.2OCH.sub.3),
ethylmethoxy (--CH.sub.2CH.sub.2OCH.sub.3), n-propyl-iso-propoxy
(--CH.sub.2CH.sub.2CH.sub.2OCH(CH.sub.3).sub.2), methyl-tert-butoxy
(--CH.sub.2--O--C(CH.sub.3).sub.3) and the like.
[0148] "Alkylthioalkoxy" refers to the group "-alkylene-S-alkyl"
wherein alkylene and alkoxy are as defined herein. Such groups
include, by way of example, methylthiomethoxy
(--CH.sub.2SCH.sub.3), ethylthiomethoxy
(--CH.sub.2CH.sub.2SCH.sub.3), n-propyl-iso-thiopropoxy
(--CH.sub.2CH.sub.2CH.sub.2SCH(CH.sub.3).sub.2),
methyl-tert-thiobutoxy (--CH.sub.2SC(CH.sub.3).sub.3) and the
like.
[0149] "Alkenyl" refers to alkenyl groups preferably having from 2
to 10 carbon atoms and more preferably 2 to 6 carbon atoms and
having at least 1 and preferably from 1-2 sites of alkenyl
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.2), but-2-enyl
(--CH.sub.2CH.dbd.CHCH.sub.3) and the like.
[0150] "Alkynyl" refers to alkynyl groups preferably having from 2
to 10 carbon atoms and more preferably 2 to 6 carbon atoms and
having at least 1 and preferably, from 1-2 sites of alkynyl
unsaturation. Preferred alkynyl groups include ethynyl
(--C.ident.CH), propargyl (--CH.sub.2C.ident.CH) and the like.
[0151] "Acyl" refers to the groups alkyl-C(O)--, aryl-C(O)--, and
heteroaryl-C(O)-- where alkyl, aryl and heteroaryl are as defined
herein.
[0152] "Acylamino" refers to the group --C(O)NRR where each R is
independently hydrogen or alkyl where alkyl is as defined
herein.
[0153] Alkcycloalkyl" refers to the group-alkylene-cycloalkyl
wherein alkylene and cycloalkyl are as defined herein.
[0154] "Aminoacyl" refers to the group --NRC(O)R where each R is
independently hydrogen or alkyl where alkyl is as defined
herein.
[0155] "Acyloxy" refers to the groups alkyl-C(O)O--, aryl-C(O)O--,
heteroaryl-C(O)O--, and heterocyclic-C(O)O-- where alkyl, aryl,
heteroaryl and heterocyclic are as defined herein.
[0156] "Aminoacyloxy" refers to the groups --NRC(O)O-alkyl,
--NRC(O)O-substituted alkyl, --NRC(O)O-cycloalkyl, --NRC(O)O-aryl,
--NRC(O)O-heteroaryl-, and --NRC(O)O-heterocyclic where R is
hydrogen, alkyl, substituted alkyl, cycloalkyl, aryl, heteroaryl,
and heterocyclic and where each of alkyl, substituted alkyl,
cycloalkyl, aryl, heteroaryl and heterocyclic are as defined
herein.
[0157] "Oxyacylamino" refers to the groups --OC(O)NR-alkyl,
--OC(O)NR-substituted alkyl, --OC(O)NR-aryl, --OC(O)NR-heteroaryl-,
and --OC(O)NR-heterocyclic where R is hydrogen, alkyl, substituted
alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclic and where
each of alkyl, substituted alkyl, cycloalkyl, aryl, heteroaryl and
heterocyclic are as defined herein.
[0158] "Aryl" refers to an unsaturated aromatic carbocyclic group
of from 6 to 14 carbon atoms having a single ring (e.g., phenyl) or
multiple condensed rings (e.g., naphthyl or anthryl). Preferred
aryls include phenyl, naphthyl and the like.
[0159] Unless otherwise constrained by the definition for the aryl
substituent, such aryl groups can optionally be substituted with
from 1 to 3 substituents selected from the group consisting of
hydroxy, acyl, acyloxy, alkyl, alkoxy, alkenyl, alkynyl, amino,
aminoacyl, aryl, aryloxy, carboxyl, alkoxycarbonyl, acylamino,
cyano, halo, nitro, heteroaryl, trihalomethyl and the like.
Preferred substituents include alkyl, alkoxy, halo, cyano, nitro,
trihalomethyl, and thioalkoxy.
[0160] "Aryloxy" refers to the group aryl-O-- wherein the aryl
group is as defined above including optionally substituted aryl
groups as also defined above.
[0161] "Caboxylalkyl" refers to the group --C(O)(O)-alkyl where
alkyl is as defined herein.
[0162] "Cycloalkyl" refers to cyclic alkyl groups of from 3 to 10
carbon atoms having a single cyclic ring or multiple condensed
rings which can be optionally substituted with from 1 to 3 alkyl
groups. Such cycloalkyl groups include, by way of example, single
ring structures such as cyclopropyl, cyclobutyl, cyclopentyl,
cyclooctyl, 1-methylcyclopropyl, 2-methylcyclopentyl,
2-methylcyclooctyl, and the like, or multiple ring structures such
as adamantanyl, and the like.
[0163] "Cycloalkenyl" refers to cyclic alkenyl groups of from 4 to
8 carbon atoms having a single cyclic ring and at least one point
of internal unsaturation which can be optionally substituted with
from 1 to 3 alkyl groups. Examples of suitable cycloalkenyl groups
include, for instance, cyclobut-2-enyl, cyclopent-3-enyl,
cyclooct-3-enyl and the like.
[0164] "Halo" or "halogen" refers to fluoro, chloro, bromo and iodo
and preferably is either chloro or fluoro.
[0165] "Heteroaryl" refers to a monovalent aromatic group of from 2
to 8 carbon atoms and 1 to 4 heteroatoms selected from oxygen,
nitrogen and sulfur within the ring.
[0166] Unless otherwise constrained by the definition for the
heteroaryl substituent, such heteroaryl groups can be optionally
substituted with 1 to 3 substituents selected from the group
consisting of alkyl, alkoxy, aryl, aryloxy, halo, nitro,
heteroaryl, thioalkoxy, thioaryloxy and the like. 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.
[0167] "Heterocycle" or "heterocyclic" refers to a monovalent
saturated or unsaturated group having a single ring or multiple
condensed rings, from 1 to 8 carbon atoms and from 1 to 4 hetero
atoms selected from nitrogen, sulfur or oxygen within the ring.
[0168] Unless otherwise constrained by the definition for the
heterocyclic substituent, such heterocyclic groups can be
optionally substituted with 1 to 3 substituents selected from the
group consisting of alkyl, alkoxy, aryl, aryloxy, halo, nitro,
heteroaryl, thioalkoxy, thioaryloxy and the like. Such heterocyclic
groups can have a single ring (e.g., piperidinyl or
tetrahydrofuryl) or multiple condensed rings (e.g., indolinyl,
dihydrobenzofuran or quinuclidinyl). Preferred heterocycles include
piperidinyl, pyrrolidinyl and tetrahydrofuryl.
[0169] Examples of heterocycles and heteroaryls include, but are
not limited to, furan, thiophene, thiazole, oxazole, pyrrole,
imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine,
indolizine, isoindole, indole, indazole, purine, quinolizine,
isoquinoline, quinoline, phthalazine, naphthylpyridine,
quinoxaline, quinazoline, cinnoline, pteridine, carbazole,
carboline, phenanthridine, acridine, phenanthroline, isothiazole,
phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine,
imidazoline, piperidine, piperazine, pyrrolidine, indoline and the
like.
[0170] "Thiol" refers to the group --SH.
[0171] "Thioalkoxy" refers to the group --S-alkyl.
[0172] "Thioaryloxy" refers to the group aryl-S-- wherein the aryl
group is as defined above including optionally substituted aryl
groups as also defined above.
[0173] "Thioheteroaryloxy" refers to the group heteroaryl-S--
wherein the heteroaryl group is as defined above including
optionally substituted aryl groups as also defined above.
[0174] The class of biological reagent having the described
properties are defined by formula I below:
A-B-C I
[0175] wherein A is selected from the group consisting of compounds
defined by formulas II, III, IV and V as described below;
[0176] B is selected from the group consisting of
[0177] a) (CH.sub.2CH(R)Q).sub.n
[0178] b) alkylene-Q
[0179] c) substituted alkylene-Q
[0180] wherein R is selected from hydrogen, alkyl, aryl and Q is
selected from --O--, --S--, --NH--, --OC(O)NH--, --NHC(O)O--,
--NHC(O)NH--, --NHC(O)-- and --C(O)NH-- and
[0181] C is selected from the group consisting of a solid support
and a detectable marker optionally linked to Q through a linking
arm.
[0182] In one embodiment, A comprises a group defined by Formula II
below:: 8
[0183] wherein R.sup.11 is selected from the group consisting
of:
[0184] (a) a substituted phenyl group of the formula: 9
[0185] wherein R.sup.c is selected from the group consisting of
acyl, alkyl, alkoxy, alkoxycarbonyl, alkylalkoxy, azido, cyano,
halo, hydrogen, nitro, trihalomethyl, thioalkoxy, and wherein
R.sup.b and R.sup.c are fused to form a heteroaryl or heterocyclic
ring with the phenyl ring wherein the heteroaryl or heterocyclic
ring contains from 3 to 8 atoms of which from 1 to 3 are
heteroatoms independently selected from the group consisting of
oxygen, nitrogen and sulfur;
[0186] R.sup.b and R.sup.b' are independently selected from the
group consisting of hydrogen, halo, nitro, cyano, trihalomethyl,
alkoxy, and thioalkoxy with the proviso that R.sup.b, R.sup.b' and
R.sup.c are not all hydrogen and with the further proviso that when
R.sup.c is hydrogen, then neither R.sup.b nor R.sup.b' are
hydrogen;
[0187] (b) 2-naphthyl; and
[0188] (c) 2-naphthyl substituted at the 4, 5, 6, 7 and/or 8
positions with 1 to 5 substituents selected from the group
consisting of alkyl, alkoxy, halo, cyano, nitro, trihalomethyl, and
thioalkoxy;
[0189] R.sup.12 is selected from the group consisting of hydrogen,
alkyl of from 1 to 4 carbon atoms, alkylalkoxy of from 1 to 4
carbon atoms and alkylthioalkoxy of from 1 to 4 carbon atoms;
and
[0190] R.sup.13 is selected from the group consisting of --O--,
--S--, --O--(CH.sub.2).sub.pC(O)--, --O--(CH.sub.2).sub.pC(O)O--
and --O--(CH.sub.2).sub.pC(O)NH--, wherein p is an integer of from
1 to 2.
[0191] In formula II above, R.sup.11 substituted phenyls are
preferably 4-substituted, 3,5-disubstituted or 3,4-disubstituted
phenyl substituents wherein the substituents at the 3 and/or 5
positions are defined by R.sup.b, R.sup.b' as above and the
substituent at the 4 position is defined by R.sup.c as above.
Particularly preferred 3,5-disubstituted phenyls include, by way of
example, 3,5-dichlorophenyl, 3,5-difluorophenyl,
3,5-di(trifluoromethyl)phenyl, 3,5-dimethoxyphenyl, and the like.
Particularly, preferred 3,4-disubstituted phenyls include, by way
of example, 3,4-dichlorophenyl, 3,4-difluorophenyl,
3-(trifluoromethyl)-4-chlorophenyl, 3-chloro-4-cyanophenyl,
3-chloro-4-iodophenyl, 3,4-methylenedioxyphenyl,
3,4-ethylenedioxyphenyl, and the like. Particularly preferred
4-substituted phenyls include, by way of example, 4-azidophenyl,
4-bromophenyl, 4-chlorophenyl, 4-cyanophenyl, 4-ethylphenyl,
4-fluorophenyl, 4-iodophenyl, 4-(phenylcarbonyl)phenyl,
4-(1-ethoxy)ethylphenyl, 4-(ethoxycarbonyl)phenyl, and the
like.
[0192] In the compounds of formula II, R.sup.b and R.sup.c can be
fused to form a heteroaryl or heterocyclic ring with the phenyl
ring. Fusion in this manner results in a fused bicyclic ring
structure of the formula: 10
[0193] where R.sup.b' is as defined above and A is the fused
heteroaryl or heterocyclic group containing from 3 to 8 atoms of
which from 1 to 3 are heteroatoms independently selected from the
group consisting of oxygen, nitrogen and sulfur wherein the two
atoms of the phenyl ring are included in the total atoms present in
the heteroaryl or heterocyclic group. Examples of such fused ring
systems include, for instance, indol-5-yl, indol-6-yl,
thionaphthen-5-yl, thionaphthen-6-yl, isothionaphthen-5-yl,
isothionaphthen-6-yl, indoxazin-5-yl, indoxazin-6-yl,
benzoxazol-5-yl, benzoxazol-6-yl, anthranil-5-yl, anthranil-6-yl,
quinolin-6-yl, quinolin-7-yl, isoquinolin-6-yl, isoquinolin-7-yl,
cinnolin-6-yl, cinnolin-7-yl, quinazolin-6-yl, quinazolin-7-yl,
benzofuran-5-yl, benzofuran-6-yl, isobenzofuran-5-yl,
isobenzofuran-6-yl, and the like.
[0194] Other preferred R.sup.11 substituents include, by way of
example, 2-naphthyl, 2-methylquinolin-6-yl, benzothiazol-6-yl,
5-indolyl, and the like.
[0195] Preferably R.sup.12 is selected from the group consisting of
alkyl of from 1 to 4 carbon atoms, alkylalkoxy of from 1 to 4
carbon atoms and alkylthioalkoxy of from 1 to 4 carbon atoms.
Particularly preferred R.sup.12 substituents include, by way of
example, methyl, ethyl, n-propyl, iso-butyl, and the like.
[0196] Preferred R.sup.13 substituents include methoxy, ethoxy,
iso-propoxy, n-propoxy, n-butoxy, iso-butoxy, cyclopentoxy,
allyloxy, 4-methylpentoxy,
--O--CH.sub.2-(2,2-dimethyl-1,3-dioxolan-4-yl),
--O--CH.sub.2-cyclohexyl, --O--CH.sub.2-(3-tetrahydrofuranyl),
--O--CH.sub.2--C(O)O-tert-butyl, --O--CH.sub.2--C(CH.sub.3).sub.3,
--O--CH.sub.2-.phi., --OCH.sub.2CH(CH.sub.2CH.sub.3).sub.2,
--O(CH.sub.2).sub.3CH(CH.sub.3).sub.2, --ON.dbd.C(NH.sub.2).phi.,
--ON.dbd.C(NH.sub.2)CH.sub.3, --ON.dbd.C(NH.sub.2)CH.sub.2CH.sub.3,
--ON.dbd.C(NH.sub.2)CH.sub.2CH.sub.2CH.sub.3,
--ON.dbd.C(NH.sub.2)-cyclop- ropyl,
--ON.dbd.C(NH.sub.2)--CH.sub.2-cyclopropyl,
--ON.dbd.C(NH.sub.2)-cy- clopentyl,
--ON.dbd.C(NH.sub.2)CH.sub.2CH(CH.sub.3).sub.2, and the like.
[0197] In another embodiment, A comprises the compounds of Formula
III set forth below: 11
[0198] wherein R.sup.21 is selected from the group consisting
of
[0199] a) alkyl, alkenyl, alkcycloalkyl, phenyl-(R.sup.d).sub.m--,
naphthyl-(R.sup.d).sub.m-- wherein R.sup.d is an alkylene group of
from 1 to 8 carbon atoms and m is an integer equal to 0 or 1,
cycloalkyl, cycloalkenyl, 3-pyridyl, 4-pyridyl and heteroaryl,
other than 3- and 4-pyridyl, of 3 to 10 atoms and 1 to 4
heteroatoms selected from oxygen, sulfur and nitrogen wherein the
heteroaryl group is optionally substituted with 1 to 3 substituents
selected from the group consisting of alkyl, alkoxy, aryl, aryloxy,
halo, nitro, thioalkoxy, and thioaryloxy with the proviso that for
such heteroaryls when there is at least one nitrogen heteroatom,
there is also at least one oxygen and/or sulfur heteroatom;
[0200] (b) a substituted phenyl group of the formula: 12
[0201] wherein R is alkylene of from 1 to 8 carbon atoms,
[0202] m is an integer equal to 0 or 1,
[0203] R.sup.e and R.sup.f are independently selected from the
group consisting of hydrogen, hydroxy, fluoro and methyl;
[0204] R.sup.g and R.sup.h are independently selected from the
group consisting of hydrogen, alkyl, alkoxy, aryl, cyano,
cycloalkyl, halo, heteroaryl, heterocyclic, nitro, trihalomethyl,
thioalkoxy, thioaryloxy, thioheteroaryloxy, and --C(O)R.sup.j where
R.sup.j is selected from the group consisting of alkyl, aryl,
alkoxy and aryloxy; and
[0205] R.sup.i is selected from the group consisting of hydrogen,
alkyl, aryl, cyano, halo, nitro, and where R.sup.g and R.sup.i are
fused to form a methylenedioxy ring with the phenyl ring; and
[0206] when R.sup.g and/or R.sup.h and/or R.sup.i is fluoro,
chloro, bromo and/or nitro, then R.sup.e and/or R.sup.f can also be
chloro; and
[0207] (c) 1- or 2-naphthyl-(R.sup.k).sub.m-- substituted at the 5,
6, 7 and/or 8 positions with 1 to 4 substituents selected from the
group consisting alkyl, alkoxy, halo, cyano, nitro, trihalomethyl,
and thioalkoxy wherein R.sup.k is an alkylene group of from 1 to 8
carbon atoms and m is an integer equal to 0 or 1;
[0208] R.sup.22 is selected from the group consisting of hydrogen,
alkyl, phenyl, alkylalkoxy, alkylthioalkoxy;
[0209] X is oxygen or sulfur;
[0210] X' is hydrogen, hydroxy or fluoro; and
[0211] X" is hydrogen, hydroxy or fluoro, or X' and X" together
form an oxo group.
[0212] In formula III above, preferred R.sup.21 unsubstituted aryl
groups include, for example, phenyl, 1-naphthyl, 2-naphthyl, and
the like.
[0213] Preferred R.sup.21 substituted aryl groups include, for
example, monosubstituted phenyls having a single substitution at
the 2, 3 or 4 positions where each of the particular subsituents is
governed by the respective R.sup.e/R.sup.f, R.sup.g/R.sup.h and
R.sup.i groups; disubstituted phenyls which include those having
two substituents at the 2,3-positions, 2,4-positions,
2,5-positions, 2,6-positions, 3,4-positions, 3,5-positions or
3,6-positions where each of these substituents is governed by the
respective R.sup.e, R.sup.f, R.sup.g, R.sup.h and R.sup.i groups;
and trisubstituted phenyls which include those having three
substituents at the 2,3,4-positions, 2,3,5-positions,
2,3,6-positions, 3,4,5-positions and 3,4,6-positions again where
each of these substituents is governed by the respective R.sup.e,
R.sup.f, R.sup.g, R.sup.h and R.sup.i groups. Preferably, the
substituted phenyl groups do not include more than 3
substituents.
[0214] Examples of substituted phenyls include, for instance,
4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, 4-nitrophenyl,
4-methylphenyl, 3-methoxyphenyl, 3-nitrophenyl, 3-fluorophenyl,
3-chlorophenyl, 3-bromophenyl, 3-thiomethoxyphenyl, 3-methylphenyl,
3-trifluoromethylphenyl, 2-hydroxyphenyl, 2-methylphenyl,
2-fluorophenyl, 3,4-dichlorophenyl, 3,4-methylene-dioxyphenyl,
3,5-difluorophenyl, 3,5-dichlorophenyl, 2,4-dichlorophenyl, and
2,5-difluorophenyl.
[0215] Preferred R.sup.21 groups represented by phenyl-R-- include,
by way of example, benzyl, 3-phenylethyl, 4-phenyl-n-propyl, and
the like.
[0216] Preferred R.sup.21 alkyl, alkcycloalkyl, cycloalkyl and
cycloalkenyl groups include, by way of example, sec-butyl,
cyclopropyl, cyclobutyl, cyclohexyl, cyclopentyl, cyclohex-1-enyl,
--CH.sub.2-cyclopropyl, --CH.sub.2-cyclobutyl,
--CH.sub.2-cyclohexyl, --CH.sub.2-cyclopentyl,
--CH.sub.2CH.sub.2-cyclopropyl, --CH.sub.2CH.sub.2-cyclobutyl,
--CH.sub.2CH.sub.2-cyclohexyl, --CH.sub.2CH.sub.2-cyclopentyl, and
the like.
[0217] Preferred R.sup.21 heteroaryls and substituted heteroaryls
include, by way of example, pyrid-3-yl, pyrid-4-yl, thien-2-yl,
thien-3-yl, benzothiazol-4-yl, 2-phenylbenzoxazol-5-yl, furan-2-yl,
benzofuran-2-yl, benzothiophen-3-yl, 2-chlorothien-5-yl,
3-methylisoxazol-5-yl, 2-(phenylthio)thien-5-yl,
6-methoxythiophen-2-yl, 3-phenyl-1,2,4-thiooxad- iazol-5-yl,
2-phenyloxazol-4-yl, and the like.
[0218] Preferably R.sup.22 is selected from the group consisting of
alkyl of from 1 to 4 carbon atoms, phenyl, alkylalkoxy of from 1 to
4 carbon atoms and alkylthioalkoxy of from 1 to 4 carbon atoms.
Particularly preferred R.sup.22 substituents include, by way of
example, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl,
--CH.sub.2CH.sub.2SCH.su- b.3, cyclohexyl and phenyl.
[0219] When X is oxygen, preferred R.sup.23 substituents include,
for example, methyl, ethyl, iso-propyl, n-propyl, n-butyl,
iso-butyl, cyclopentyl, allyl, iso-but-2-enyl, 3-methylpentyl,
--CH.sub.2-cyclopropyl, --CH.sub.2-cyclohexyl,
--CH.sub.2-(3-tetrahydrofu- ranyl), --CH.sub.2-thien-2-yl,
--CH.sub.2(1-methyl)cyclopropyl, --CH.sub.2-thien-3-yl,
--CH.sub.2--C(O)O-t-butyl, --CH.sub.2--C(CH.sub.3)- .sub.3,
--CH.sub.2CH(CH.sub.2CH.sub.3).sub.2, -2-methylcyclopentyl,
-cyclohex-2-enyl, --CH[CH(CH.sub.3).sub.2]COOCH.sub.3,
--CH.sub.2CH.sub.2N(CH.sub.3).sub.2,
--CH.sub.2C(CH.sub.3).dbd.CH.sub.2,
--CH.sub.2CH.dbd.C(CH.sub.3).sub.2 and the like.
[0220] When X is sulfur, preferred R.sup.23 substituents include,
for example, iso-but-2-enyl and iso-butyl.
[0221] In another embodiment, A comprises the compounds set forth
in Formula IV below: 13
[0222] wherein R.sup.31 is selected from the group consisting
of
[0223] (a) phenyl,
[0224] (b) a substituted phenyl group of the formula: 14
[0225] wherein R.sup.p is selected from the group consisting of
acyl, alkyl, alkoxy, alkylalkoxy, azido, cyano, halo, hydrogen,
nitro, trihalomethyl, thioalkoxy, and wherein R.sup.p and R.sup.m
are fused to form a heteroaryl or heterocyclic ring with the phenyl
ring,
[0226] R.sup.m and R.sup.n are independently selected from the
group consisting of hydrogen, halo, nitro, cyano, trihalomethyl,
alkoxy, and thioalkoxy with the proviso that when R.sup.p is
hydrogen, then R.sup.m and R.sup.n are either both hydrogen or both
substituents other than hydrogen,
[0227] (c) 2-naphthyl,
[0228] (d) 2-naphthyl substituted at the 4, 5, 6, 7 and/or 8
positions with 1 to 5 substituents selected from the group
consisting of alkyl, alkoxy, halo, cyano, nitro, trihalomethyl,
thioalkoxy, aryl, and heteroaryl,
[0229] (e) heteroaryl, and
[0230] (f) substituted heteroaryl containing 1 to 3 substituents
selected from the group consisting of alkyl, alkoxy, aryl, aryloxy,
cyano, halo, nitro, heteroaryl, thioalkoxy and thioaryloxy provided
that said substituents are not ortho (adjacent) to the heteroaryl
attachment to the --NH group;
[0231] R.sup.32 is selected from the group consisting of hydrogen,
alkyl of from 1 to 4 carbon atoms, alkylalkoxy of from 1 to 4
carbon atoms, alkylthioalkoxy of from 1 to 4 carbon atoms, aryl,
heteroaryl, substituted aryl and substituted heteroaryl provided
that the substituents are not ortho (adjacent) to the attachment of
the aryl or heteroaryl atom to the carbon atom;
[0232] R.sup.33 is selected from the group consisting of alkyl,
alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl,
substituted alkyl, substituted alkenyl, substituted alkynyl, and
heterocyclic; and
[0233] X is selected from the group consisting of oxygen and
sulfur.
[0234] In formula IV above, R.sup.31 substituted phenyls are
preferably 4-substituted, 3,5-disubstituted or 3,4-disubstituted
phenyl substituents wherein the substituents at the 3 and/or 5
positions are defined by R.sup.m, R.sup.m' as above and the
substituents at the 4 position is defined by R.sup.1 as above.
Particularly preferred 3,5-disubstituted phenyls include, by way of
example, 3,5-dichlorophenyl, 3,5-difluorophenyl,
3,5-di(trifluoromethyl)phenyl, 3,5-dimethoxyphenyl, and the like.
Particularly, preferred 3,4-disubstituted phenyls include, by way
of example, 3,4-dichlorophenyl, 3,4-difluorophenyl,
3-(trifluoromethyl)-4-chlorophenyl, 3-chloro-4-cyanophenyl,
3-chloro-4-iodophenyl, 3,4-methylenedioxyphenyl, and the like.
Particularly preferred 4-substituted phenyls include, by way of
example, 4-azidophenyl, 4-bromophenyl, 4-chlorophenyl,
4-cyanophenyl, 4-ethylphenyl, 4-fluorophenyl, 4-iodophenyl,
4-(phenylcarbonyl)phenyl, 4-(1-ethoxy)ethylphenyl, and the
like.
[0235] Other preferred R.sup.31 substituents include, by way of
example, 2-naphthyl, quinolin-3-yl, 2-methylquinolin-6-yl,
benzothiazol-6-yl, benzothiazol-2-yl, 5-indolyl, phenyl,
2-naphthyl, and the like.
[0236] In the compounds of formula IV, R.sup.p and R.sup.m can be
fused to form a heteroaryl or heterocyclic ring with the phenyl
ring. Fusion in this manner results in a fused bicyclic ring
structure of the formula: 15
[0237] where R.sup.n is as defined above and A is the fused
heteroaryl or heterocyclic group as these terms are as defined
above wherein the two atoms of the phenyl ring are included in the
total atoms present in the heteroaryl or heterocyclic group.
Examples of such fused ring systems include, for instance,
indol-5-yl, indol-6-yl, thionaphthen-5-yl, thionaphthen-6-yl,
isothionaphthen-5-yl, isothionaphthen-6-yl, indoxazin-5-yl,
indoxazin-6-yl, benzoxazol-5-yl, benzoxazol-6-yl, anthranil-5-yl,
anthranil-6-yl, quinolin-6-yl, quinolin-7-yl, isoquinolin-6-yl,
isoquinolin-7-yl, cinnolin-6-yl, cinnolin-7-yl, quinazolin-6-yl,
quinazolin-7-yl, benzofuran-5-yl, benzofuran-6-yl,
isobenzofuran-5-yl, isobenzofuran-6-yl, and the like.
[0238] Preferably R.sup.32 is selected from the group consisting of
alkyl of from 1 to 4 carbon atoms, alkylalkoxy of from 1 to 4
carbon atoms, alkylthioalkoxy of from 1 to 4 carbon atoms, aryl,
heteroaryl, substituted aryl and substituted heteroaryl provided
that the substituents are not ortho to the attachment of the aryl
or heteroaryl atom to the carbon atom. Particularly preferred
R.sup.32 substituents include, by way of example, methyl, ethyl,
n-propyl, iso-propyl, n-butyl, iso-butyl,
--CH.sub.2CH.sub.2SCH.sub.3, phenyl and the like.
[0239] Preferred R.sup.33 substituents include alkyl groups such as
methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl,
and the like; substituted alkyl groups such as
.alpha.-hydroxyethyl, --CH.sub.2-cyclohexyl, benzyl,
p-hydroxybenzyl, 3-iodo-4-hydroxybenzyl,
3,5-diiodo-4-hydroxybenzyl, --CH.sub.2-indol-3-yl, phenyl,
--(CH.sub.2).sub.4--NH--BOC, --(CH.sub.2).sub.4--NH.sub.2,
--CH.sub.2-(1-N-benzyl-imidazol-4-yl), --CH.sub.2-imidazol-4-yl,
--CH.sub.2CH.sub.2SCH.sub.3,
--(CH.sub.2).sub.4NHC(O)(CH.sub.2).sub.4CH.s- ub.3,
--(CH.sub.2).sub.yC(O)OR.sup.35 where y is 1 or 2 and R.sup.35 is
hydrogen, methyl, tert-butyl, phenyl, and the like.
[0240] In another embodiment, A comprises the compounds represented
by Formula V set forth below: 16
[0241] wherein R.sup.41 is selected from the group consisting of
alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted
alkyl, substituted alkenyl, substituted alkynyl, aryl, heteroaryl
and heterocyclic;
[0242] R.sup.42 is selected from the group consisting of hydrogen,
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, cycloalkyl, aryl, heteroaryl and
heterocyclic;
[0243] each R.sup.43 is independently selected from the group
consisting of hydrogen and methyl and R.sup.43 together with
R.sup.44 can be fused to form a cyclic structure of from 3 to 8
atoms which is optionally fused with an aryl or heteroaryl
group;
[0244] each R.sup.44 is independently selected from the group
consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,
cycloalkenyl, heteroaryl, heterocyclic, substituted alkyl,
substituted alkenyl and substituted alkynyl;
[0245] each R.sup.45 is selected from hydrogen arid methyl or
together with R.sup.43 forms a cycloalkyl group of from 3 to 6
carbon atoms;
[0246] X is selected from oxygen, sulfur and NH;
[0247] X' is hydrogen, hydroxy or fluoro; and
[0248] X" is hydrogen, hydroxy or fluoro, or X' and X" together
form an oxo group;
[0249] Z is selected from the group consisting of a bond covalently
linking R.sup.41 to --CX'X"--, oxygen and sulfur; and
[0250] n is an integer equal to 1 to 3.
[0251] In formula V above, X" is preferably hydrogen and X' is
preferably hydrogen or fluoro.
[0252] In formula V above, Z is preferably a covalent bond linking
R.sup.1 to --CX'X"--.
[0253] In formula V above, preferred R.sup.41 unsubstituted aryl
groups include, for example, phenyl, 1-naphthyl, 2-naphthyl, and
the like.
[0254] Preferred R.sup.41 substituted aryl groups include, for
example, monosubstituted phenyls (preferably 3 or 5 substituents);
disubstituted phenyls (preferably 3,5 substituents); and
trisubstituted phenyls (preferably 3,4,5 substituents). Preferably,
the substituted phenyl groups do not include more than 3
substituents.
[0255] Examples of substituted phenyls include, for instance,
4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, 4-nitrophenyl,
4-methylphenyl, 3-methoxyphenyl, 3-nitrophenyl, 3-fluorophenyl,
3-chlorophenyl, 3-bromophenyl, 3-thiomethoxyphenyl, 3-methylphenyl,
3-trifluoromethylphenyl, 2-hydroxyphenyl, 2-methylphenyl,
2-fluorophenyl, 2-chlorophenyl, 3,4-difluorophenyl,
2,3,4,5,6-pentafluorophenyl, 3,4-dibromophenyl, 3,4-dichlorophenyl,
3,4-methylene-dioxyphenyl, 3,5-difluorophenyl, 3,5-dichlorophenyl,
2,4-dichlorophenyl, and 2,5-difluorophenyl.
[0256] Preferred R.sup.41 alkaryl groups include, by way of
example, benzyl, 2-phenylethyl, 3-phenyl-n-propyl, and the
like.
[0257] Preferred R.sup.41 alkyl, substituted alkyl, alkenyl,
cycloalkyl and cycloalkenyl groups include, by way of example,
iso-propyl, n-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl,
--CH.sub.2CH.dbd.CH.sub.2,
--CH.sub.2CH.dbd.CH(CH.sub.2).sub.4CH.sub.3, cyclopropyl,
cyclobutyl, cyclohexyl, cyclopentyl, cyclohex-1-enyl,
--CH.sub.2-cyclopropyl, --CH.sub.2-cyclobutyl,
--CH.sub.2-cyclohexyl, --CH.sub.2-cyclopentyl,
--CH.sub.2CH.sub.2-cyclopropyl, --CH.sub.2CH.sub.2-cyclobutyl,
--CH.sub.2CH.sub.2-cyclohexyl, --CH.sub.2CH.sub.2-cyclopentyl,
aminomethyl, N-tert-butoxycarbonylaminomethyl, and the like.
[0258] Preferred R.sup.41 heteroaryls and substituted heteroaryls
include, by way of example, pyrid-2-yl, pyrid-3-yl, pyrid-4-yl,
fluoropyridyls (including 5-fluoropyrid-3-yl), chloropyridyls
(including 5-chloropyrid-3-yl), thien-2-yl, thien-3-yl,
benzothiazol-4-yl, 2-phenylbenzoxazol-5-yl, furan-2-yl,
benzofuran-2-yl, thionaphthen-2-yl, 2-chlorothiophen-5-yl,
3-methylisoxazol-5-yl, 2-(thiophenyl)thiophen-5-yl- ,
6-methoxythionaphthen-2-yl, 3-phenyl-1,2,4-thiooxadiazol-5-yl,
2-phenyloxazol-4-yl, and the like.
[0259] Preferably R.sup.42 is selected from the group consisting of
hydrogen, alkyl, substituted alkyl, cycloalkyl, aryl, heteroaryl
and heterocyclic. Particularly preferred R.sup.42 substituents
include, by way of example, methyl, ethyl, n-propyl, iso-propyl,
n-butyl, iso-butyl, sec-butyl, phenyl, 4-fluorophenyl,
3,5-difluorophenyl, 4-methoxyphenyl, benzyl, cyclopropyl,
cyclohexyl, cyclopentyl, cycloheptyl, thien-2-yl, thien-3-yl,
--CH.sub.2CH.sub.2SCH.sub.3, --CH.sub.2OCH.sub.2.phi.,
--CH(CH.sub.3)OCH.sub.2.phi., --CH(OH)CH.sub.3, --CH.sub.2OH and
the like. As noted below, R.sup.42 (as well as R.sup.44) is
preferably the side chain of an L-amino acid.
[0260] Preferably, R.sup.43 is hydrogen, methyl or together with
R.sup.44 and the nitrogen to which R.sup.43 is attached forms
pyrrolidin-2-yl, 2,3-dihydroindol-2-yl, piperidin-2-yl,
4-hydroxy-pyrrolidin-2-yl, 1,2,3,4-tetrahydroisoquinolin-3-yl, and
the like.
[0261] Preferred R.sup.44 substituents include, for example,
hydrogen, methyl, ethyl, iso-propyl, n-propyl, n-butyl, sec-butyl,
iso-butyl, cyclopentyl, cyclohexyl, allyl, iso-but-2-enyl,
3-methylpentyl, --CH.sub.2-cyclopropyl, --CH.sub.2-cyclohexyl,
--CH.sub.2-indol-3-yl, phenyl, p-(phenyl)phenyl, m-(phenyl)phenyl
o-fluorophenyl, m-fluorophenyl, p-fluorophenyl, p-bromophenyl,
m-methoxyphenyl, p-methoxyphenyl, phenethyl, benzyl,
m-hydroxybenzyl, p-hydroxybenzyl, p-nitrobenzyl,
m-trifluoromethylphenyl, p-(CH.sub.3).sub.2NCH.sub.2CH.sub-
.2CH.sub.2O-benzyl, p-(CH.sub.3).sub.3COC(O)CH.sub.2O-benzyl,
p-phenylphenyl, 3,5-difluorophenyl, p-(HOOCCH.sub.2O)-benzyl,
2-aminopyrid-6-yl, 4-(N-morpholino-CH.sub.2CH.sub.2O)-benzyl,
--CH.sub.2CH.sub.2C(O)NH.sub.2, --CH.sub.2-imidazol-4-yl,
--CH.sub.2-(3-tetrahydrofuranyl), --CH.sub.2-thien-2-yl,
--CH.sub.2-thiazol-4-yl, --CH.sub.2(1-methyl)cyclopropyl,
--CH.sub.2-thien-3-yl, thien-3-yl, thien-2-yl,
--CH.sub.2--C(O)O-t-butyl, --CH.sub.2--C(CH.sub.3).sub.3,
--CH.sub.2CH(CH.sub.2CH.sub.3).sub.2, 2-methylcyclopentyl,
-cyclohex-2-enyl, --CH[CH(CH.sub.3).sub.2]COOCH.sub.- 3,
--(CH.sub.2).sub.2SCH.sub.3, --CH.sub.2CH.sub.2N(CH.sub.3).sub.2,
--CH.sub.2C(CH.sub.3).dbd.CH.sub.2, --CH.sub.2CH.dbd.CHCH.sub.3
(cis and trans), --CH.sub.2OH, --CH(OH)CH.sub.3,
--CH(O-t-butyl)CH.sub.3, --CH.sub.2OCH.sub.3,
--(CH.sub.2).sub.4NH-Boc, --(CH.sub.2).sub.4NH.sub.2- ,
--(CH.sub.2).sub.4N(CH.sub.3).sub.2, --CH.sub.2-pyridyl (e.g.,
2-pyridyl, 3-pyridyl and 4-pyridyl), pyridyl (2-pyridyl, 3-pyridyl
and 4-pyridyl), --CH.sub.2-naphthyl (e.g., 1-naphthyl and
2-naphthyl), --CH.sub.2-(N-morpholino),
p-(N-morpholino-CH.sub.2CH.sub.2O)-benzyl, benzo[b]thiophen-2-yl,
benzo[b]thiophen-3-yl, 5-chlorobenzo[b]thiophen-2-- yl,
4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl, benzo[b]thiophen-3-yl,
tetrazol-5-yl, 5-chlorobenzo[b]thiophen-3-yl,
benzo[b]thiophen-5-yl, 6-methoxynaphth-2-yl,
--CH.sub.2--N-phthalimidyl, 2-methylthiazol-4-yl, and
thieno[2,3-b]thiophen-2-yl, 5-bromothien-2-yl, 4-bromothien-2-yl,
5-chlorothien-2-yl, 3-phenoxyphenyl, 2-phenoxyphenyl,
4-ethylphenyl, 2-benzylphenyl, (4-ethylphenyl)phenyl,
4-tert-butylphenyl, 4-n-butylphenyl, o-(4-chlorophenoxy)phenyl,
furan-2-yl, 4-phenylacetylenylphenyl and the like.
[0262] Preferably, R.sup.45 is hydrogen. However, in another
embodiment, R.sup.44 and R.sup.45 are fused to form a cycloalkyl
group including, for example, cyclopropyl, cyclobutyl, and the
like.
[0263] "Detectable marker" means a radioactive label, a fluorescent
label, a chemiluminescer, a heavy metal ion, an antibody, an
enzyme, biotin, an azido group, an immunobiotin and the like.
[0264] Conveniently, a radioactive label may be employed.
Radioactive labels include .sup.125I, .sup.32P, .sup.3H, .sup.14C
and the like. Any radioactive label may be employed which provides
for an adequate signal and has sufficient half-life.
[0265] Further, a fluorescent label may be employed. Fluorescent
labels include fluorescein, rhodamine and its derivatives, dansyl,
umbelliferone, BODIPY.RTM., and the like. Any fluorescent label may
be employed with provides for an adequate signal. A chemiluminescer
may be employed. Suitable chemiluminescers include luciferin, and
2,3-dihydrophthalazinediones, i.e. luminol.
[0266] Other labels include ligands, which can serve as a specific
binding pair member to a labeled antibody, fluorescers,
chemiluminescers, enzymes, photoaffinity probes, antibodies which
can serve as a specific binding pair member for a labeled ligand
and the like. A wide variety of labels have been employed in
immunoassays which can be readily employed in the present methods.
The choice of label will be governed by the effect of the label on
the rate of binding of the biological reagent to the target. It
will be necessary that the label provide sufficient sensitivity to
detect the target.
[0267] "Solid support" means any solid support in which a compound
can be affixed. Examples of these supports include glass, test
tubes, microtiter plates, nylon beads, agarose beads, magnetic
beads, glass beads, teflon, polystyrene beads, photodetectable
chips and the like.
[0268] Preferably, the solid support or the detectable marker
contain a reactive functional group which is complementary to and
reacts with the compounds of Formula VI to form the compounds of
Formula I.
[0269] "Linking arms" are well known in the art and include, by way
of example only, conventional linking arms such as those comprising
ester, amide, carbamate, ether, thio ether, urea, amine groups and
the like. The linking arm can be cleavable or non-cleavable.
[0270] Cleavable linking arms refer to linking arms wherein at
least one of the covalent bonds of the linking arm which attaches
the compound to the solid support can be readily broken by specific
chemical reactions thereby providing for compounds free of the
solid support or detectable marker. The chemical reactions employed
to break the covalent bond of the linking arm are selected so as to
be specific for bond breakage thereby preventing unintended
reactions occurring elsewhere on the compound. The cleavable
linking arm is selected relative to the synthesis of the compounds
to be formed on the solid support so as to prevent premature
cleavage of this compound from the solid support as well as not to
interfere with any of the procedures employed during compound
synthesis on the support. Suitable cleavable linking arms are well
known in the art.
[0271] "Non-cleavable linking arms" refer to linking arms wherein
the covalent bond(s) linking the activated ketone compound to the
solid support can only be cleaved under conditions which chemically
alters unintended parts of the structure of the compound attached
thereto.
[0272] Compound Preparation
[0273] The compounds of formula I above are readily prepared via
several divergent synthetic routes with the particular route
selected relative to the ease of compound preparation, the
commercial availability of starting materials, and the like.
[0274] The starting materials for compounds comprising Formula II
are readily prepared by the methods set forth in International
Patent Application No. PCT/US97/20356.sup.8, which is incorporated
by reference herein in its entirety.
[0275] The starting materials for compounds comprising Formula III
are readily prepared by the methods set forth in International
Patent Application No. PCT/US97/20355.sup.9, which is incorporated
by reference herein in its entirety.
[0276] The starting materials for compounds comprising Formula IV
are readily prepared by the methods set forth in International
Patent Application No. PCT/US97/18704.sup.10, which is incorporated
by reference herein in its entirety.
[0277] The starting materials for compounds comprising Formula V
are readily prepared by the methods set forth in International
Patent Application No. PCT/US97/20804.sup.11, which is incorporated
by reference herein in its entirety.
[0278] The manner in which the label is bound to the compound will
vary depending upon the nature of the label. For a radioactive
label, a wide variety of techniques can be employed which are known
in the art.
[0279] The manner for attaching the biological reagent to a solid
support is well known in the art. One method is by derivatizing the
end of the linker having a carboxyl or amino group. Another example
of attachment is by attaching a biotin molecule to the biological
reagent and then attaching the biological reagent to a solid
support bearing avidin molecules.
[0280] It is recognized that the biological reagent should be
attached to the solid support in an orientation which will allow
binding of the biological reagent to the protein or peptide of
interest.
[0281] Methods
[0282] The biological reagents of the present invention are useful
in determining the mechanism of .beta.-amyloid peptide release
and/or its synthesis, and, accordingly, have utility in future drug
discovery.
[0283] Without being limited to a theory, it is thought that the
.beta.-amyloid peptide is generated by the actions of two enzymes,
a .beta.-secretase which cleaves the parent protein at the amino
terminus of the .beta.-amyloid peptide and a .gamma.-secretase
which cleaves the parent protein at the carboxyl terminus of the
.beta.-amyloid peptide. It is thought that the compounds of formula
I, II, III and IV act to inhibit the cleavage by the
.gamma.-secretase, either directly or indirectly via a protein
modulating .gamma.-secretase activity.
[0284] The biological reagents are useful in identifying the
cellular factor (either peptide, protein etc.) whose activity is
inhibited by the compounds of formula I, II, III and IV. Such
biological reagents can be used is a variety of ways depending on
the reactive group which comprises C in Formula I.
[0285] In one embodiment, cells known to produce .beta.-amyloid
peptide are disrupted to produce a cell membrane suspension. Such
cellular membrane suspensions are passed over the biological
reagents of the present invention, wherein C of formula I comprises
a solid support. The cellular factor or factors which interacts
with the compounds of formula II, III, IV and V, will bind to the
biological reagents of the present invention. The remainder of the
cellular biological solution will be washed from the biological
reagent comprising the solid support. The cellular factor can then
be identified.
[0286] In another embodiment, cells known to produce .beta.-amyloid
peptide are disrupted to produce a cellular membrane suspension.
Such cellular membrane suspensions are mixed with the biological
reagents of the present invention, wherein C of formula I comprises
a photoaffinity reagent, for example an azido group under
conditions whereby the cellular factor or factor which interact
with the compounds of formula II, III, IV and V will bind. The
mixture is then subjected to light, for example, ultraviolet light
which covalently links the biiological reagents of the present
invention to the cellular factor. The cellular factor bound to the
photoaffinity reagent is then removed/purified from the remainder
of the biological solution and the cellular factor identified.
[0287] In another embodiment, cells known to produce .beta.-amyloid
peptide are mixed with the biological reagent of the present
invention wherein C of Formula I comprises a fluorescent dye under
conditions wherein the biological reagent of the present invention
binds to cells comprising the cellular factor or factors involved
in the production of .beta.-amyloid peptide. Such cells are then
identified by their fluorescence. Methods of identification
include, but are not limited to, cell sorting.
[0288] It is contemplated that the cells known to produce
.beta.-amyloid peptide may include a library of eukaryotic or
prokaryotic cells transformed with genes under the control of an
expression vector, such that the genes are expressed in the cells.
Cells expressing the cellular factor or factors involved in the
production of the .beta.-amyloid peptide may be identified by
fluorescence. The isolated cells may then be disrupted and the
expressed gene and or cellular factor identified.
[0289] The cellular factor may be identified by an number of
methods, including, without being limited to, peptide sequencing,
binding to known antibodies and the like. The gene coding for the
cellular factor may be sequenced. By these methods the cellular
factor involved in .beta.-amyloid peptide release and/or its
synthesis may be identified.
[0290] Utility
[0291] The compounds of the invention are useful in determining the
mechanism for .beta.-amyloid peptide release and/or its synthesis,
and, accordingly, have utility in future drug discovery.
[0292] In particular the biological reagents may be used to
determine cellular agents, such as peptides or proteins involved
with .beta.-amyloid peptide release and/or its synthesis. Such
identification involves the binding of the biological reagent to
the cellular agent in a solution and the extraction of the
biological reagent/cellular agent from the solution and the
identification of the cellular agent.
[0293] The biological reagents may also be used to identify cells
which express the cellular agent, such as peptides or protein
involved with .beta.-amyloid peptide release and/or its synthesis.
Such identification would involve labeling of cells expressing the
cellular agent by attachment of detectably labelled biological
reagents of the present invention to such cells.
[0294] The following synthetic and biological examples are offered
to illustrate this invention and are not to be construed in any way
as limiting the scope of this invention. Unless otherwise stated,
all temperatures are in degrees Celsius.
EXAMPLES
[0295] In the examples below, the following abbreviations have the
following meanings. If an abbreviation is not defined, it has its
generally accepted meaning.
[0296] BOC=tert-butoxycarbonyl
[0297] bd=broad doublet
[0298] bs=broad singlet
[0299] cc=cubic centimeter
[0300] d=doublet.sup.+
[0301] dd=doublet of doublets
[0302] DMF=dimethylformamide
[0303] DMSO=dimethyl sulfoxide
[0304] EDC=1-(3-dimethyaminopropyl)-ethylcarbodiimide
hydrochloride
[0305] EDTA=ethylene diamine tetraacetic acid
[0306] eq.=equivalents
[0307] ether=diethyl ether
[0308] g=grams
[0309] hept.=heptuplet
[0310] m=multiplet
[0311] M=molar
[0312] max=maximum
[0313] mg=milligram
[0314] min.=minutes
[0315] mL=milliliter
[0316] mM=millimolar
[0317] mmol=millimole
[0318] N=normal
[0319] ng=nanogram
[0320] nm=nanometers
[0321] OD=optical density
[0322] pg=picogram
[0323] pM=picomolar
[0324] .phi.=phenyl
[0325] psi=pounds per square inch
[0326] q=quartet
[0327] quint.=quintuplet
[0328] rpm=rotations per minute
[0329] s=singlet
[0330] sept=septuplet
[0331] t=triplet
[0332] THF=tetrahydrofuran
[0333] TLC=thin layer chromatography
[0334] .mu.L=microliter
[0335] UV=ultraviolet
[0336] w/v=weight to volume
[0337] Additionally, the term "Aldrich" indicates that the compound
or reagent used in the following procedures is commercially
available from Aldrich Chemical Company, Inc., 1001 West Saint Paul
Avenue, Milwaukee, Wis. 53233 USA; the term "Fluka" indicates the
compound or reagent is commercially available from Fluka Chemical
Corp., 980 South 2nd Street, Ronkonkoma, N.Y. 11779 USA; the term
"Lancaster" indicates the compound or reagent is commercially
available from Lancaster Synthesis, Inc., P.O. Box 100, Windham,
N.H. 03087 USA; and the term "Sigma" indicates the compound or
reagent is commercially available from Sigma, P.O. Box 14508, St.
Louis, Mo. 63178 USA; the term "Bachem" indicates that the compound
or reagent is commercially available from Bachem Biosciences Inc.,
3700 Horizon Drive, Renaissance at Gulph Mills, King of Prussia,
Pa. 19406 USA; the term "Engelhard" indicates the compound or
reagent is commercially available from Engelhard Catalysts &
Chemicals Division, 554 Mormon Church Road, Seneca, S.C. 29678; the
term "TCI-US" indicates that the compound or reagent is
commercially available from TCI America, 9211 North Harborgate
Street, Portland Oreg. 97203; the term "Advanced Chemtech"
indicates that the compound or reagent is commercially available
from Advanced Chemtech, Louisville, Ky.; the term "Molecular
Probes" indicates the compound or reagent is commercially available
from Molecular Probes, Inc., Eugene, Oreg.; and the term "Pierce"
indicates the compound or reagent is commercially available from
Pierce Chemical Company P.O. 117 Rockford Ill. 61105.
[0338] In the examples below, all temperatures are in degrees
Celsius (unless otherwise indicated) and the following general
procedures were used to prepare the compounds as indicated.
General Procedure A
Boc Protection of Diamine
[0339] A solution of the appropriate diamine (8 eq.) dissolved in
dioxane (1 M) was treated with a dioxane solution (0.3 M) of
di-tert-butyl dicarbonate (1 eq., Aldrich), added over 2.5 hours.
The resulting mixture was stirred at room temperature 24 hours then
concentrated in vacuo. The crude oil was dissolved in water and
extracted with three portions of methylene chloride. The combined
organic extracts were washed with water then dried over
Na.sub.2SO.sub.4, filtered and concentrated to provide the desired
mono-Boc protected diamine.
General Procedure B
HOAt Coupling Reaction
[0340] A solution of the carboxylic acid component (1 eq.) was
dissolved in tetrahydrofuran (THF, 0.1M) and treated sequentially
with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride
(EDC-HCl, 1.1 eq., Bachem) and 1-hydroxy-7-azabenzotriazole (HOAt,
1.1 eq., Aldrich). To the resulting solution was added the amine
component (1 eq) after which the mixture was stirred overnight, or
until judged complete by TLC analysis. The solvent was then removed
in vacuo. The crude oil was diluted with water and extracted twice
with methylene chloride. The combined organic extracts were dried
over Na.sub.2SO.sub.4, filtered and concentrated. Purification of
the desired product was accomplished by flash chromatography.
General Procedure C
Cbz Removal by Hydrogenolysis
[0341] The Cbz-protected amine was dissolved in methanol (0.4 M)
and the resulting solution was purged of oxygen after which an
atmosphere of nitrogen was established. The reaction mixture was
charged with 5% Pd/C (62 mg/mmol of substrate, Engelhard) then
purged with hydrogen. An atmosphere of hydrogen (1 atm., balloon)
was maintained overnight. The mixture was then filtered through a
pad of celite and the solvent removed in vacuo to afford the crude
product. Purification by flash chromatography afforded the desired
product.
General Procedure D
Boc Removal
[0342] A stream of anhydrous HCl gas was passed through a solution
of the Boc-protected amine in 1,4-dioxane at room temperature for
10-15 minutes. A CaSO4 drying tube was placed on the flask and the
reaction mixture was stirred overnight. The solvent was removed in
vacuo to afford the final product as the HCl-salt.
General Procedure E
EDC Coupling Procedure
[0343] A round bottom flask was charged with the corresponding
carboxylic acid (1.0 eq.), hydroxybenzotriazole hydrate (1.1 eq.)
and the corresponding amine (1.0 eq.) in THF under nitrogen
atmosphere. An appropriate amount (1.1 eq for free amines and 2.2
eq. for hydrochloride amine salts) of base, such as Hunig's base
was added to the well stirred mixture followed by EDC (1.1 eq.).
After stirring from 4 to 17 hours at room temperature the solvent
was removed at reduced pressure, the residue taken up in ethyl
acetate (or similar solvent) and water, washed with saturated
aqueous sodium bicarbonate solution, 1 N HCl, brine, dried over
anhydrous sodium sulfate and the solvent removed at reduced
pressure to provide the product.
General Procedure F
Ester Hydrolysis to Free Acid
[0344] Ester hydrolysis to the free acid was conducted by
conventional methods. Below are two examples of such conventional
de-esterification methods.
[0345] Method A: To a carboxylic ester compound in a 1:1 mixture of
CH.sub.3OH/H.sub.2O was added 2-5 equivalents of K.sub.2CO.sub.3.
The mixture was heated to 50.degree. C. for 0.5 to 1.5 hours until
tic showed complete reaction. The reaction was cooled to room
temperature and the methanol was removed on a rotary evaporator.
The pH of the remaining aqueous solution was adjusted to .about.2,
and ethyl acetate was added to extract the product. The organic
phase was then washed with saturated aqueous NaCl and dried over
MgSO.sub.4. The solution was stripped free of solvent on a rotary
evaporator to yield the product.
[0346] Method B: The amino acid ester was dissolved in
dioxane/water (4:1) to which was added LiOH (.about.2 eq.) that was
dissolved in water such that the total solvent after addition was
about 2:1 dioxane:water. The reaction mixture was stirred until
reaction completion and the dioxane was removed under reduced
pressure. The residue was dissolved in water and washed with ether.
The layers were separated and the aqueous layer was acidified to pH
2. The aqueous layer was extracted with ethyl acetate. The ethyl
acetate extracts were dried over Na.sub.2SO.sub.4 and the solvent
was removed under reduced pressure after filtration. The residue
was purified by conventional methods (e.g., recrystallization).
General Procedure G
BOC Removal Procedure
[0347] A stream of anhydrous HCl gas was passed through a stirred
solution of the N-t-Boc protected amino acid in 1,4-dioxane
(0.03-0.09 M), chilled in a ice bath to .about.10.degree. C. under
N.sub.2, for 10-15 minutes. The solution was capped, the cooling
bath removed, and the solution was allowed to warm to room
temperature with stirring for 2-8 hours, monitoring by TLC for the
consumption of starting material. The solution was concentrated
(and in some instances dissolved in CH.sub.2Cl.sub.2 then
re-concentrated and placed in vacuum oven at 60-70.degree. C. to
remove most of the residual dioxane) and used without further
purification.
General Procedure H
Synthesis of S-(+)-3,5-Difluoromandelic Acid
[0348] Step A.--Preparation of Methyl
S-(.+-.)-3,5-difluoromandelate
[0349] To a solution of 3,5-difluorobenzaldehyde (Aldrich) in
CH.sub.2Cl.sub.2 (100 mL) was added ZnCl.sub.2 (6.7 g, 21.1 mmol)
to form a slurry. Trimethysilyl cyanide (21.0 g, 211.2 mmol)
dissolved in CH.sub.2Cl.sub.2 (100 mL) was slowly added to the
slurry at 0.degree. C. The resulting solution was stirred at room
temperature for 4 h. The reaction mixture was then diluted with
water and the organic layer separated. The combined organic layers
were concentrated to a residue. The residue was dissolved with MeOH
(200 mL) at 0.degree. C. and anhydrous HCl gas bubbled into the
solution for 10 min. After stirring at room temperature for 18 h,
the solution was concentrated to a solid. The solid was dissolved
in CH.sub.2Cl.sub.2/H.sub.2O and the aqueous portion extracted with
CH.sub.2Cl.sub.2. The combined organics were washed with brine,
dried over anhydrous MgSO.sub.4 and concentrated to a solid (37.4
g, 87.6%), mp=77-78.degree. C.
[0350] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=6.97 (dd, J=9.6
Hz, J=1.79 Hz, 2H), 6.74 (dt, J=8.82, J=2.28 Hz, 1H), 5.14 (d,
J=4.64 Hz, 1H), 3.78 (s, 3H), 3.54 (d, J=5.1 Hz, 1H).
[0351] Step B--Preparation of Methyl
S-(+)-3,5-difluoromandelate
[0352] Methyl (.+-.)-3,5-difluoromandelate was separated via
preparative chiral HPLC to give a white solid having a melting
point of 70-71.degree. C.
[0353] C.sub.9H.sub.8F.sub.2O.sub.3 (MW=202.17); mass spectroscopy
found (M+NH.sub.4.sup.+) 220.0.
[0354] Anal. calcd for C.sub.9H.sub.8F.sub.2O.sub.3: C, 53.47; H,
3.99. Found: C, 53.40; H, 3.89.
[0355] Step C--Preparation of S-(+)-3,5-Difluoromandelic Acid
[0356] A solution of methyl S-(+)-3,5-difluoromandelate (1 eq.) in
74% aqueous THF was cooled to 0.degree. C. and treated with lithium
hydroxide. After 40 minutes at 0.degree. C. the reaction was
complete by TLC. The contents were transferred to a separatory
funnel and partitioned between CH.sub.2Cl.sub.2 and saturated
aqueous NaHCO.sub.3. The aqueous layer was acidified with 0.5 N
NaHSO.sub.4 and extracted thrice with ethyl acetate. The combined
extracts were washed with brine, dried over Na.sub.2SO.sub.4,
filtered, and concentrated to a white solid having a melting point
of 119-122.degree. C. The .sup.1H NMR was consistent with known
3,5-difluoromandelic acid.
General Procedure I
Synthesis of BODIPY-Containing Compounds
[0357] An appropriate amine starting material (2-5 eq.) and Bodipy
N-hydroxysuccinimide ester (1 eq., Molecular Probes) was stirred in
DMF for 48 hours. The mixture was concentrated to provide a crude
solid which was purified by silica gel chromatography.
Example 1
Synthesis of Tert-Butyl N-(8-amino-3,6-dioxaoctyl) Carbamate
[0358] 17
[0359] Following general procedure A and using
1,8-diamino-3,6-dioxaoctane (Aldrich) the title compound was
prepared.
[0360] C.sub.11H.sub.24N.sub.2O.sub.4 (MW=248.3); mass spectroscopy
(MH.sup.+) 249.3.
[0361] Anal. Calcd for C.sub.11H.sub.24N.sub.2O.sub.4: C, 53.21; H,
9.74; N, 11.28. Found: C, 52.29; H, 9.07; N, 10.32.
Example 2
Synthesis of Methyl N-2-aminoethyl-N'-2-t-butylcarbamoylethyl
Amine
[0362] 18
[0363] Following general procedure A and using
N'-methyl-2,2'-diaminodieth- ylamine (TCI-US) the title compound
was prepared.
[0364] C.sub.10H.sub.23N.sub.3O.sub.2 (MW=217.3); mass spectroscopy
(MH.sup.+) 218.2.
[0365] Anal. Calcd for C.sub.10H.sub.23N.sub.3O.sub.2: C, 55.27; H,
10.67; N, 19.34. Found: C, 54.54; H, 10.13; N, 18.07.
Example 3
Synthesis of Tert-Butyl
N-(8-N'-(N"-benzyloxycarbonyl-L-phenylglycine)-3,6- -dioxaoctyl)
Carbamate
[0366] 19
[0367] Following general procedure B and using
N-Cbz-L-phenylglycine (Bachem) as the carboxylic component and
tert-butyl N-(8-amino-3,6-dioxaoctyl) carbamate (from Example 1
above) as the amine component, the title compound was prepared.
Purification by flash chromatography (2:1 ethyl acetate/hexanes)
afforded the pure product.
[0368] C.sub.27H.sub.37N.sub.3O.sub.7 (MW=515.6); mass spectroscopy
(MH.sup.+) 516.0.
[0369] Anal. Calcd for C.sub.27H.sub.37N.sub.3O.sub.7: C, 62.90; H,
7.23; N, 8.15. Found: C, 62.93; H, 7.39; N, 8.06.
Example 4
Synthesis of Methyl
N-(2-N'-(N"-benzyloxycarbamoyl-L-phenylglycine)ethyl)--
N'-2-t-butylcarbamoylethyl Amine
[0370] 20
[0371] Following general procedure B and using
N-Cbz-L-phenylglycine (Bachem) as the carboxylic component and
methyl N-2-aminoethyl-N'-2-t-but- ylcarbamoylethyl amine (from
Example 2 above) as the amine component, the title compound was
prepared. Purification by flash chromatography (5:1 ethyl
acetate/methanol) afforded the pure product.
[0372] C.sub.26H.sub.36N.sub.4O.sub.5 (MW=484.6); mass spectroscopy
(MH.sup.+) 485.6.
[0373] Anal. Calcd for C.sub.26H.sub.36N.sub.4O.sub.5: C, 64.44; H,
7.49; N, 11.56. Found: C, 64.33; H, 7.24; N, 10.75.
Example 5
Synthesis of Tert-Butyl
N-(2-N'-(N"-benzyloxycarbonyl-L-phenylglycine)-eth- yl)
Carbamate
[0374] 21
[0375] Following general procedure E and using
N-Cbz-L-phenylglycine (Bachem) as the carboxylic component and
tert-butyl-N-(2-aminoethyl) carbamate (Fluka) as the amine
component, the title compound was prepared. Purification by flash
chromatography (9:1 methylene chloride/methanol) afforded the pure
product.
[0376] C.sub.23H.sub.29N.sub.3O.sub.5 (MW=427.5); mass spectroscopy
(MH.sup.+) 428.2
[0377] Anal. Calcd for C.sub.23H.sub.29N.sub.3O.sub.5: C, 64.62; H,
6.84; N, 9.83. Found: C, 64.34; H, 6.61; N, 9.81.
Example 6
Synthesis of Tert-Butyl N-(8-N'-L-phenylglycine-3,6-dioxaoctyl)
Carbamate
[0378] 22
[0379] Following general procedure C and using tert-butyl
N-(8-N'-(N"-benzyloxycarbonyl-L-phenylglycine)-3,6-dioxaoctyl)
carbamate (set forth in Example 3) as the substrate, the title
compound was prepared. Purification by flash chromatography (3:1
ethyl acetate/methanol) afforded the pure product.
[0380] C.sub.19H.sub.31N.sub.3O.sub.5 (MW=381.5); mass spectroscopy
(MH.sup.+) 382.4
[0381] NMR data was as follows:
[0382] .sup.1H-nmr (CD.sub.3OD, 250 MHz) .delta.=7.47-7.24 (m, 5H),
4.47 (s, 1H), 3.60-3.16 (m, 12H), 1.44 (s, 9H)
Example 7
Synthesis of Methyl
N-(2-N-(L-phenylglycine)ethyl)-N'-2-t-butylcarbamoylet- hyl
Amine
[0383] 23
[0384] Following general procedure C and using methyl
N-(2-N'-(N"-benzyloxycarbamoyl-L-phenylglycine)ethyl)-N'-2-t-butylcarbamo-
ylethyl amine (set forth in Example 4 above) as the substrate, the
title compound was prepared. Purification by flash chromatography
(5:1 ethyl acetate/methanol) afforded the pure product.
[0385] C.sub.18H.sub.30N.sub.4O.sub.3 (MW=350.5); mass spectroscopy
(MH.sup.+) 351.5
[0386] Anal. Calcd for C.sub.18H.sub.30N.sub.4O.sub.3: C, 61.69; H,
8.63; N, 15.99. Found: C, 61.63; H, 8.52; N, 15.85.
Example 8
Synthesis of Tert-Butyl N-(2-N'-L-phenylglycine)-ethyl
Carbamate
[0387] 24
[0388] Following general procedure C and using tert-butyl
N-(2-N'-(N"-benzyloxycarbonyl-L-phenylglycine)-ethyl) carbamate (as
set forth in Example 5 above) as the substrate, the title compound
was prepared. Purification by flash chromatography (10:1 to 5:1
ethyl acetate/methanol gradient) afforded the pure product.
[0389] C.sub.15H.sub.23N.sub.3O.sub.3 (MW=293.37); mass
spectroscopy (MH.sup.+) 294.3
[0390] Anal. Calcd for C.sub.15H.sub.23N.sub.3O.sub.3: C, 61.41; H,
7.90; N, 14.32. Found: C, 61.18; H, 7.98; N, 14.09.
Example 9
Synthesis of Tert-Butyl
N-[8-N'-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-a-
cetyl-L-alaninyl)-L-phenylglycine)-3,6-dioxaoctyl]Carbamate
[0391] 25
[0392] Following general procedure B and using
N-((R/S)-3,5-difluorophenyl- -.alpha.-hydroxyacetyl)-L-alanine (as
set forth in Example 15 below) as the carboxylic component and
tert-butyl N-(8-N'-L-phenylglycine-3,6-dioxa- octyl) carbamate (set
forth in Example 6 above) as the amine component, the title
compound was prepared. Purification by flash chromatography (10:1
ethyl acetate/methanol) afforded the pure product.
[0393] C.sub.30H.sub.40F.sub.2N.sub.4O.sub.8 (MW=622.67); mass
spectroscopy (MH.sup.+)=623.5
[0394] Anal. Calcd for C.sub.30H.sub.40F.sub.2N.sub.4O.sub.8: C,
57.87; H, 6.48; N, 9.00. Found: C, 57.86; H, 6.30; N, 8.86.
Example 10
Synthesis of Methyl
N-[2-N-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl-
-L-alaninyl)-L-phenylglycine)ethyl]-N'-2-t-butylcarbamoylethyl
Amine
[0395] 26
[0396] Following general procedure B and using
N-((R/S)-3,5-difluorophenyl- -a-hydroxyacetyl)-L-alanine (set forth
in Example 15 below) as the carboxylic component and methyl
N-(2-N-(L-phenylglycine)ethyl)-N'-2-t-but- ylcarbamoylethyl amine
(set forth in Example 7 above) as the amine component, the title
compound was prepared. Purification by flash chromatography (5:1
ethyl acetate/methanol) afforded the pure product.
[0397] C.sub.29H.sub.39F.sub.2N.sub.5O.sub.6 (MW=591.7); mass
spectroscopy (MH.sup.+)=592.7
[0398] Anal. Calcd for C.sub.29H.sub.39F.sub.2N.sub.5O.sub.6: C,
58.87; H, 6.64; N, 11.84. Found: C, 57.47; H, 6.59; N, 11.68.
Example 11
Synthesis of Tert-Butyl
N-[2-N'-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-a-
cetyl-L-alaninyl)-L-phenylglycine)-ethyl]Carbamate
[0399] 27
[0400] Following general procedure B and using
N-((R/S)-3,5-difluorophenyl- -.alpha.-hydroxyacetyl)-L-alanine (set
forth in Example 15 below) as the carboxylic component and
tert-butyl N-(2-N'-L-phenylglycine)-ethyl carbamate (set forth in
Example 8 above) as the amine component, the title compound was
prepared. Purification by flash chromatography (10:1 ethyl
acetate/methanol) afforded the pure product.
[0401] C.sub.26H.sub.32F.sub.2N.sub.4O.sub.6 (MW=534.6); mass
spectroscopy (MH.sup.+) 535
[0402] Anal. Calcd for C.sub.26H.sub.32F.sub.2N.sub.4O.sub.6: C,
58.42; H, 6.03; N, 10.48. Found: C, 58.19; H, 6.13; N, 10.77.
Example 12
Synthesis of
Tert-butyl-2-(2'-(N'-benzyloxycarbonyl-L-phenylglycine)aminoe-
thoxy)ethylcarbamate
[0403] 28
[0404] Following General Procedure E and using
tert-butyl-2-(2'-aminoethox- y)ethylcarbamate (as described in
Bioconjugate Chem. (1990), 1 (6), 431-437.sup.12) and
benzyloxycarbonyl-L-phenylglycine (Aldrich) the title compound was
made.
[0405] C.sub.25H.sub.35N.sub.3O.sub.6 (MW=473.57); mass
spectroscopy (MH.sup.+)=474.
[0406] Anal. Calcd. For C.sub.25H.sub.35N.sub.3O.sub.6, C 63.41; H
7.45, N 8.87. Found: C 63.09, H 7.13, N 8.56.
Example 13
Synthesis of
Tert-butyl-2-(2'-(L-phenylglycine)aminoethoxy)ethylcarbamate
[0407] 29
[0408]
Tert-butyl-2-(2'-(N'-benzyloxycarbonyl-L-phenylglycine)aminoethoxy)
ethylcarbamate, (as set forth in Example 12 above), was suspended
in MeOH at room temperature under a nitrogen atmosphere. 5% Pd/C,
25 mg catalyst/1 mmol substrate, was added under nitrogen, and the
reaction mixture was stirred under a hydrogen atmosphere (balloon)
for 17 hours. The mixture was filtered through celite, and the
solvents removed in vacuo. The residue was purified via radial
chromatography using 50% ethyl acetate in acetonitrile to afford
the title compound.
[0409] C.sub.17H.sub.27N.sub.3O.sub.4 (MW=337.42); mass
spectroscopy (MH.sup.+)=338.
[0410] Anal. calcd. For C.sub.17H.sub.27N.sub.3O.sub.4: C 60.51, H
8.07, N 12.45. Found: C 59.12, H 7.86, N 12.26.
Example 14
Synthesis of
N-((R/S)-3,5-difluorophenyl-.alpha.-hydroxyacetyl)-L-alanine,
Methyl Ester
[0411] 30
[0412] Following General Procedure E and using
(R/S)-3,5-difluoromandelic acid (as set forth in General Procedure
H, step A) and L-alanine, methyl ester (Bachem) the title compound
was synthesized.
[0413] C.sub.12H.sub.13F.sub.2NO.sub.4 (MW=273.23) mass
spectroscopy (M+)=273.
[0414] NMR data was as follows:
[0415] .sup.1H-nmr (400 MHz, CDCl.sub.3) .delta.=7.03-6.76 (3H, m),
5.08 (1H, s), 4.61-4.54 (1H, m), 3.75 (3H, s), 1.43 (3H, d, J=5.6
Hz).
Example 15
Synthesis of N-((R/S
-3,5-difluorophenyl-.alpha.-hydroxyacetyl)-L-alanine
[0416] 31
[0417] Following General Procedure F, Method B and using
N-((R/S)-3,5-difluorophenyl-.alpha.-hydroxyacetyl)-L-alanine,
methyl ester (set forth in Example 14 above) afforded the title
compound.
[0418] C.sub.11H.sub.11F.sub.2NO.sub.4 (MW=259.21); mass
spectroscopy (MH.sup.+)=260.
[0419] NMR data was as follows:
[0420] .sup.1H-nmr (400 MHz, CDCl.sub.3) .delta.=7.02-7.00 (2H, m),
6.80-6.76 (1H, m), 5.11 (1H, s), 4.61-4.52 (1H, m), 1.48 (3H, d,
J=5.2 Hz).
Example 16
Synthesis of
tert-butyl-2-(2'-(N'-(((R/S)-3,5-difluorophenyl-.alpha.-hydro-
xyacetyl)-L-alanine)-L-phenylglycine)aminoethoxy)ethylcarbamate
[0421] 32
[0422] Following General Procedure E and using
N-((R/S)-3,5-difluorophenyl- -.alpha.-hydroxyacetyl)-L-alanine (set
forth in Example 15 above) and
tert-butyl-2-(2'-(L-phenylglycine)aminoethoxy)ethylcarbamate (set
forth in Example 13 above), the title intermediate was
synthesized.
[0423] C.sub.28H.sub.36F.sub.2N.sub.4O.sub.7 (MW=578.61); mass
spectroscopy (MH.sup.+)=579.
[0424] NMR data was as follows:
[0425] .sup.1H-nmr (400 MHz, CDCl.sub.3) .delta.=8.01-8.58 (1H, m),
7.41-7.23 (5H, m), 7.10-6.96 (2H, m), 6.75-6.65 (1H, m), 5.63-5.50
(1H, m), 5.17-5.05 (1H, m), 4.78-4.62 (1H, m), 3.49-2.25 (6H, m),
3.22-3.16 (2H, m), 1.50 (9H, s), 1.37 (3H, d, J=5.7 Hz).
Example 17
Synthesis of
8-N'-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl-L-alanin-
yl)-L-phenylglycinyl)amino-3,6-dioxaoctylamine Hydrochloride
[0426] 33
[0427] Following general procedure D and using tert-butyl
N-[8-N'-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl-L-alaninyl)-L-phe-
nylglycine)-3,6-dioxaoctyl]carbamate (set forth in Example 9 above)
as the substrate, the title compound was prepared.
[0428] C.sub.25H.sub.32F.sub.2N.sub.4O.sub.6--HCl (MW=558.2); mass
spectroscopy (MH.sup.+)=557.1
[0429] Anal. Calcd for C.sub.25H.sub.32F.sub.2N.sub.4O.sub.6--HCl:
C, 53.72; H, 5.95; N, 10.02. Found: C, 49.63; H, 5.68; N, 8.37.
Example 18
Synthesis of Methyl
N-[2-N-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl-
-L-alaninyl)-L-phenylglycinyl)aminoethyl]-N'-2-aminoethyl Amine
Hydrochloride
[0430] 34
[0431] Following general procedure D and using methyl
N-[2-N-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl-L-alaninyl)-L-phen-
ylglycine)ethyl]-N'-2-t-butylcarbamoylethyl amine (set forth in
Example 10 above) as the substrate, the title compound was
prepared.
[0432] C.sub.24H.sub.31F.sub.2N.sub.5O.sub.4--HCl (MW=528.0); mass
spectroscopy (MH.sup.+)=526.3
[0433] NMR data was as follows:
[0434] .sup.1H-nmr (CD.sub.3OD, 250 MHz) .delta.=7.51-7.29 (m, 5H),
7.22-7.04 (m, 2H), 7.00-6.87 (m, 1H), 5.33-5.09 (m, 2H), 4.49-4.33
(m, 1H), 3.79-3.36 (m, 8H), 3.07-2.96 (m, 3H), 1.27-1.36 (m,
3H).
Example 19
Synthesis of
2-N'-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl-L-alanin-
yl)-L-phenylglycine)-ethylamine Hydrochloride
[0435] 35
[0436] Following general procedure D and using tert-butyl
N-[2-N'-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl-L-alaninyl)-L-phe-
nylglycine)-ethyl]carbamate (set forth in Example 11 above) as the
substrate, the title compound was prepared.
[0437] C.sub.21H.sub.24F.sub.2N.sub.4O.sub.4--HCl (MW=470.9); mass
spectroscopy (MH.sup.+)=469.2
[0438] Anal. Calcd for C.sub.21H.sub.24F.sub.2N.sub.4O.sub.4--HCl:
C, 53.56; H, 5.35; N, 11.90. Found: C, 54.58; H, 5.65; N,
10.99.
Example 20
Synthesis of
N-(8-N'-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl-L-ala-
ninyl)-L-phenylglycine)-3,6-dioxaoctyl)-biotinamide
[0439] 36
[0440] General procedure B was used with the exception that
dimethylformamide was used in place of tetrahydrofuran and 2 eq.
diisopropylethylamine was added to the reaction mixture. Using
d-biotin (Sigma) as the carboxylic acid component and
8-N'-((2-(R/S)-hydroxy-2-(3,-
5-difluorophenyl)-acetyl-L-alaninyl)-L-phenylglycine)-3,6-dioxaoctylamine
hydrochloride (set forth in Example 17 above) as the amine
component, the title compound was prepared. Purification by flash
chromatography (3:1 ethyl acetate/methanol) afforded the pure
product.
[0441] C.sub.35H.sub.46F.sub.2N.sub.6O.sub.8S (MW=748.9); mass
spectroscopy (MH.sup.+)=749.3
[0442] Anal. Calcd for C.sub.35H.sub.46F.sub.2N.sub.6O.sub.8S: C,
56.14; H, 6.19; N, 11.22. Found: C, 55.45; H, 6.33; N, 10.85.
Example 21
Synthesis of
2-(2'-(N'-(((R/S)-3,5-difluorophenyl-.alpha.-hydroxyacetyl)-L-
-alanine)-L-phenylglycine)aminoethoxy)ethylamine, Hydrochloride
Salt
[0443] 37
[0444] Following General Procedure G using no cooling bath, and
using
tert-butyl-2-(2'-(N'-(((R/S)-3,5-difluorophenyl-.alpha.-hydroxyacetyl)-L--
alanine)-L-phenylglycine)aminoethoxy)ethylcarbamate (set forth in
Example 16), the title compound was prepared.
[0445] C.sub.23H.sub.28F.sub.2N.sub.4O.sub.5. Cl H (MW=478.50);
mass spectroscopy (MH.sup.+)=479.
[0446] NMR data was as follows:
[0447] .sup.1H-nmr (400 MHz, CD.sub.3OD) .delta.=7.42-7.30 (5H, m),
7.18-7.05 (2H, m), 6.99-6.82 (1H, m), 5.40-5.22 (1H, m), 5.06 (1H,
d, J=9.8 Hz), 4.50-4.36 (1H, m), 3.60-3.23 (6H, m), 3.01 (2H, br
s), 1.40-1.32 (3H, d, J=6.1 Hz).
Example 22
Synthesis of
1-[N-(3,5-difluorophenylacetyl)-L-alaninyl)-(L-phenylglycinyl-
)]amino-6-(N-biotinyl)aminohexane
[0448] 38
[0449] Step 1
[0450] Following General Procedure E and General Procedure G and
using biotin and tert-butyl N-(6-aminohexyl)carbamate hydrochloride
(Fluka), N-biotinyl-1,6-hexanediamine hydrochloride was prepared.
The free base form was obtained by purification using SCX-silica
gel chromatography.
[0451] Step 2
[0452] Following General Procedure E and using
N(3,5-difluorophenylacetyl)- -L-alaninyl-phenylglycine (Example D25
of International Patent Application No. PCT/US98/20804.sup.11) and
N-biotinyl-1,6-hexanediamine hydrochloride (prepared in Step 1
above), the title compound was prepared.
[0453] C.sub.35H.sub.46F.sub.2N.sub.6O.sub.5S (MW 700.855); mass
spectroscopy (MH.sup.+)=701.
[0454] NMR data was as follows:
[0455] .sup.1H-nmr (.delta..sup.6-DMSO) .delta.=5.40 (t, 1H); 6.36
(s, 1H); 6.43 (s, 1H).
Example 23
Synthesis of
1-[N-(3,5-difluorophenylacetyl)-L-alaninyl)-(L-phenylglycinyl-
)]amino-3-(4-iodophenoxy)propane
[0456] 39
[0457] Step 1
[0458] N-(3-bromopropyl)phthalimide (1 eq., Aldrich), 4-iodophenol
(1 eq., Aldrich) and potassium carbonate (2 eq.) were stirred in
acetonitrile. The mixture was heated at reflux. After 64 hours, the
reaction mixture was concentrated to a thick mixture which was
slurried in water. A white solid was collected, washed with water
and vacuum dried to provide
1-(4-iodophenoxy)-3-(phthalimido)propane.
[0459] mass spectroscopy (MH.sup.+)=407.
[0460] Step 2
[0461] 1-(4-Iodophenoxy)-3-(phthalimido)propane was stirred in
ethanol. Anhydrous hydrazine (2 eq.) was added and the mixture was
heated at reflux for 18 hours. The reaction mixture was
concentrated to yield a solid which was treated with 1N NaOH and
extracted with CHCl.sub.3. The organic portion was dried,
concentrated then diluted with ether. The mixture was treated with
dry HCl. A white solid was collected and vacuum dried to provide
1-amino-3-(4-iodophenoxy)propane hydrochloride.
[0462] mass spectroscopy (MH.sup.+)=277.
[0463] Step 3
[0464] Following General Procedure E and using
N-t-Boc-phenylglycine (Advanced Chemtech) and
1-amino-3-(4-iodophenoxy)propane hydrochloride,
1-(N-t-Boc-phenylglycinyl)amino-3-(4-iodophenoxy)propane was
prepared.
[0465] Step 4
[0466] Following General Procedure G and using
1-(N-t-Boc-phenylglycinyl)a- mino-3-(4-iodophenoxy)propane,
1-(phenylglycinyl)amino-3-(4-iodophenoxy)pr- opane hydrochloride
was prepared.
[0467] Step 5 Synthesis of
N-(3,5-Difluorophenylacetyl)-L-alanine
[0468] Following the procedure set forth in Example B2 of
International Patent Application No. PCT/US97/20804,
3,5-difluorophenylacetic acid (30 g, 0.174 mol) (Aldrich) was
dissolved in dichloromethane and this solution was cooled to
0.degree. C. DMF (0.5 mL, catalytic) was added followed by the
dropwise addition of oxalyl chloride (18 mL, 0.20 mol) over a 5
minute period. The reaction was stirred for 3 h and then
rotoevaporated at reduced pressure to a residue which was placed on
a high vacuum pump for 1 h to afford 3,5-difluorophenylacetyl
chloride as a thin yellow oil.
[0469] 3,5-Difluorophenylacetyl chloride was added dropwise to a
0.degree. C. solution of L-alanine (Aldrich) (16.7 g, 0.187 mol) in
2 N sodium hydroxide (215 mL, 0.43 mol). The reaction was stirred
for 1 h at 0.degree. C. and then overnight at room temperature. The
reaction was diluted with water (100 mL), then extracted with ethyl
acetate (3.times.150 mL). The organic layer was then washed with
brine (200 mL), dried over MgSO.sub.4, and rotoevaporated at
reduced pressure to a residue. Recrystallization of the residue
from ethyl acetate/hexanes afforded the desired product (34.5 g,
82% yield).
[0470] NMR data was as follows:
[0471] .sup.1H-nmr (CD.sub.3OD): .delta.=8.32 (br s, 0.3H), 6.71
(m, 2H), 6.60 (m, 1H), 4.74 (br s, 1.7H), 4.16 (m, 1H), 3.36 (s,
2H), 1.19 (d, J=7.3 Hz, 3H).
[0472] .sup.13C-nmr (CD.sub.3OD): .delta.=175.9, 172.4, 164.4 (dd,
J=13.0, 245.3 Hz), 141.1, 113.1 (dd, J=7.8, 17.1 Hz), 102.9 (t,
J=25.7 Hz), 49.5, 42.7, 17.5.
[0473] Step 6
[0474] Following General Procedure E and using
N-(3,5-difluorophenylacetyl- )-L-alanine (from step 5 above) and
1-(phenylglycinyl)amino-3-(4-iodopheno- xy)propane hydrochloride
(from step 4 above), the title compound was prepared.
[0475] mass spectroscopy (MH.sup.+)=635.
[0476] Calc: C, 68.62; H, 6.16; N, 8.28. Found: C, 68.50; H, 6.04;
N, 8.01.
Example 24
Synthesis of
1-[N-(3,5-difluorophenylacetyl)-L-alaninyl)-(L-phenylglycinyl-
)]amino-6-[N-(4-amino-7-nitrobenzofurazanyl)]hexane
[0477] 40
[0478] N-biotinyl-1,6-hexanediamine (as set forth in Example 22,
Step 1)(1 eq.), 4-chloro-7-nitrobenzofurazan (2 eq., Aldrich), and
triethylamine (2 eq.) were stirred in DMF for 16 hours. The
resulting mixture was concentrated to yield a crude solid which was
purified by silica gel chromatography to yield the title
compound.
[0479] C.sub.31H.sub.35F.sub.2N.sub.7O.sub.6 (MW 639.66); mass
spectroscopy (MH.sup.+)=638.3.
[0480] NMR data was as follows:
[0481] .sup.1H-nmr (.delta..sup.6-DMSO) .delta.=5.37 (d, 1H); 6.37
(d, 1H)
[0482] Rf (5% methanol/methylene chloride)=0.2.
Example 25
Synthesis of
1-[N-(3,5-difluorophenylacetyl)-L-alaninyl)-(L-phenylglycinyl-
)]amino-3-(4-trimethylstannylphenoxy) Propane
[0483] 41
[0484]
1-[N-(3,5-difluorophenylacetyl)-L-alaninyl)-(L-phenylglycinyl)]amin-
o-3-(4-iodophenoxy)propane (as set forth in Example 23) (1 eq.),
hexamethylditin (2 eq.), and
tetrakis(triphenylphosphine)palladium(Aldric- h) (0.1 eq) were
stirred in 1,4-dioxane. The mixture was heated at reflux for 16
hours. The mixture was allowed to cool then was diluted with
methylene chloride and filtered through Celite. The filtrate was
concentrated to provide a crude product which was purified by
silica gel chromatography.
[0485] NMR data was as follows:
[0486] .sup.1H-NMR (.delta..sup.6-DMSO) .delta.=4.28 (m, 1H); 5.25
(d, 1H)
[0487] Rf (10% methanol/methylene chloride)=0.5.
Example 26
Synthesis of
N-{1-N-[N-(3,5-difluorophenylacetyl)-L-alaninyl-(L-phenylglyc-
inyl)amino-6-hexyl]}-4,4-difluoro-5,7-dimethyl-4-bora-3.alpha.,4.alpha.-di-
aza-s-indacene-3-propionamide
[0488] 42
[0489] Step 1
[0490] Following General Procedure E and using Cbz-L-phenylglycine
(Advanced Chemtech) and N-t-Boc-1,6-hexanediamine (Fluka),
1-N-(Cbz-L-phenylglycinyl)-6-(N-t-Boc)-hexanediamine was
prepared.
[0491] Step 2
[0492] 1-N-(Cbz-L-phenylglycinyl)-6-(N-t-Boc)-hexanediamine was
stirred in ethanol with 5% palladium on carbon under an atmosphere
of hydrogen (60 psi) at ambient temperature for 16 hours. The
mixture was filtered and the filtrate was concentrated to provide
1-N-(L-phenylglycinyl)-6-(N-t-Bo- c)-hexanediamine.
[0493] Step 3
[0494] Following General Procedure E and using
N-(3,5-difluorophenylacetyl- )-L-alanine and
1-N-(L-phenylglycinyl)-6-(N-t-Boc)-hexanediamine,
1-N-[N-(3,5-difluorophenylacetyl)-L-alaninyl-(L-phenylglycinyl)-6-(N-t-Bo-
c)-hexanediamine was prepared.
[0495] Step 4
[0496] Following General Procedure G and using
1-N-[N-(3,5-difluorophenyla-
cetyl)-L-alaninyl-(L-phenylglycinyl)-6-(N-t-Boc)-hexanediamine,
1-N-[N-(3,5-difluorophenylacetyl)-L-alaninyl-(L-phenylglycinyl)-6-hexaned-
iamine was prepared.
[0497] Step 5
[0498] Following General Procedure I and using
1-N-[N-(3,5-difluorophenyla-
cetyl)-L-alaninyl-(L-phenylglycinyl)-6-hexanediamine, the title
compound was prepared.
[0499] Rf (5% methanol/methylene chloride)=0.25.
Example 27
Synthesis of N-{methyl
N-[2-N-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-ace-
tyl-L-alaninyl)-L-phenylglycinyl)aminoethyl]-N'-2-aminoethyl}-4,4-difluoro-
-5,7-dimethyl-4-bora-3.alpha.,4.alpha.-diaza-s-indacene-3-propionamide
[0500] 43
[0501] Following General Procedure I and using methyl
N-[2-N-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl-L-alaninyl)-L-phen-
ylglycinyl)aminoethyl]-N'-2-aminoethyl amine hydrochloride (as set
forth in Example 18), the title compound was prepared.
[0502] Mass Spectroscopy (MH.sup.+)=766.1.
Example 28
Synthesis of
N-[8-N'-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl-L-ala-
ninyl)-L-phenylglycinyl)amino-3,6-dioxaoctyl]-4,4-difluoro-5,7-dimethyl-4--
bora-3.alpha.,4.alpha.-diaza-s-indacene-3-propionamide
[0503] 44
[0504] Following General Procedure I and using
8-N'-((2-(R/S)-hydroxy-2-(3-
,5-difluorophenyl)-acetyl-L-alaninyl)-L-phenylglycinyl)amino-3,6-dioxaocty-
lamine hydrochloride (as set forth in Example 17), the title
compound was prepared. The title compound contains the C-terminal
fluorophore, coupled via its succinimidyl ester,
4,4-difluoro-5,7-dimethyl-4-bora-3.alpha.,4.a-
lpha.-diaza-s-indacene-3-propionic acid. The ester is coupled via
the C-terminal amine of a bis-ethylene amine ethylene glycol linker
attached to the C-terminal nitrogen of the dipeptide
difluoromandelate-ala-phenyl gly.
[0505] Mass Spectroscopy (MH.sup.+)=797.2.
Example 29
Synthesis of
5-(S)-[N'-((S)-3,5-Difluorophenyl-.alpha.-hydroxyacetyl)-L-al-
aninyl]amino-7-methyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one and
5-(S)-[N'-((R)-3,5-Difluorophenyl-.alpha.-hydroxyacetyl)-L-alaninyl]amino-
-7-methyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one
[0506] 45
[0507] Following General Procedure D (from International Patent
Application No. PCT/US97/22986.sup.13) above using
3,5-difluoromandelic acid and
5-(S)-[L-alaninyl]-amino-7-methyl-5,7-dihydro-6H-dibenz[b,d]azep-
in-6-one hydrochloride (Example 7-B of International Patent
Application No. PCT/US97/22986), the title compound was prepared as
a colorless solid. The diastereomers were purified by flash
chromatography using 98:2 CHCl.sub.3/MeOH.
[0508] Isomer 1:
[0509] NMR data was as follows:
[0510] .sup.1H-nmr (CDCl.sub.3): .delta.=7.67 (d, 1H), 7.60-7.28
(m, 8H), 7.15 (d, 1H), 6.98 (m, 2H), 6.74 (m, 1H), 5.21 (d, 1H),
4.94 (d, 1H), 4.61 (m, 1H), 4.56 (m, 1H), 3.34 (s, 3H), 1.42 (d,
3H).
[0511] Optical Rotation: [.alpha.].sub.20=-121 @589 nm (c=1,
MeOH).
[0512] C.sub.26H.sub.23F.sub.2N.sub.3O.sub.4 (MW=479.488); mass
spectroscopy (MH+) 479.
[0513] Anal. Calcd for C.sub.26H.sub.23F.sub.2N.sub.3O.sub.4; C,
65.13 H, 4.83 N, 8.76. Found: C, 65.42H, 4.73 N, 8.65.
[0514] Isomer 2:
[0515] NMR data was as follows:
[0516] .sup.1H-nmr (CDCl.sub.3): .delta.=7.78 (d, 1H), 7.66 (d,
1H), 7.54-7.28 (m, 8H), 6.89 (m, 2H), 6.71 (m, 2H), 5.22 (d 1H),
4.92 (m, 1H), 4.65 (m, 1H), 4.01 (m, 1H), 3.37 (s, 3H), 1.39 (d,
3H).
[0517] Optical Rotation: [.alpha.].sub.20=-146 589 nm (c=1,
MeOH).
[0518] C.sub.26H.sub.23F.sub.2N.sub.3O.sub.4 (MW=479.488); mass
spectroscopy (MH+) 479.
[0519] Anal. Calcd for C.sub.26H.sub.23F.sub.2N.sub.3O.sub.4; C,
65.13 H, 4.83 N, 8.76. Found: C, 65.18, 4.82, 8.65.
Example 30
Synthesis of
5-(S)-(N'-((S)-(+)-2-Hydroxy-3-methylbutyryl)-L-alaninyl)amin-
o-7-methyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one
[0520] 46
[0521] Following General Procedure H (from International Patent
Application No. PCT/US98/22986.sup.13) using
(S)-(+)-2-hydroxy-3-methylbu- tyric acid (Aldrich) and
5-S-(L-alaninyl)-amino-7-methyl-5,7-dihydro-6H-di-
benz[b,d]azepin-6-one (Example 7-B from International Patent
Application No. PCT/US98/22986.sup.13), the title compound was
prepared as a white solid. The product was purified by silica gel
chromatography using gradient elution of MeOH/CH.sub.2Cl.sub.2
(1:99-3:97).
[0522] NMR data was as follows:
[0523] .sup.1H-nmr (CDCl.sub.3): .delta.=7.94 (d, J=7.0 Hz, 1H),
7.55-7.22 (m, 9H), 5.25 (d, J=7.5 Hz, 1H), 4.79-4.75 (m, 1H), 3.83
(d, J=3.1 Hz, 1H), 3.78 (br s, 1H), 3.32 (s, 3H), 2.08-2.01 (m,
1H), 1.36 (d, J=7.0 Hz, 3H), 0.83 (d, J=7.0 Hz, 3H), 0.76 (d, J=6.5
Hz, 3H).
[0524] C.sub.23H.sub.27N.sub.3O.sub.4 (MW=409.48); mass
spectroscopy (MH.sup.+) 410.4.
[0525] Anal Calcd for C.sub.23H.sub.27N.sub.3O.sub.4, C, 67.46; H,
6.65; N, 10.26; Found: C, 67.59; H, 6.66; N, 10.34.
Example 31
Synthesis of Azido Compound
1-[N-(3,5-difluorophenylacetyl)-L-alaninyl)-(L-
-phenylglycinyl)]amino-4-p-azidosalicylamidobutane
[0526] 47
[0527] The title compound was made with
4-(p-azidosalicylamido)butylamine (Pierce) and
N-3,5-difluorophenylacetyl-alaninyl-phenylglycine according to the
following method except DMF was used in place of
CH.sub.2CL.sub.2.
[0528] To a 1:1 mixture of the corresponding carboxylic acid and
the corresponding amino acid ester or amide in CH.sub.2CL.sub.2 at
0.degree. C. was added 1.5 equivalents triethylamine, followed by
2.0 equivalents hydroxybenzotriazole monohydrate and then 1.25
equivalents of ethyl-3(3-dimethylamino propyl carbodiimide-HCl. The
reaction mixture was stirred overnight at room temperature and then
transferred to a separatory funnel. The mixture was washed with
water, saturated aqueous NaHCO.sub.3, 1N HCl and saturated aqueous
NaCl, and then dried over MgSO.sub.4. The resulting solution was
stripped free of solvent on a rotary evaporator to yield the crude
product.
[0529] NMR data was as follows:
[0530] .sup.1H-nmr (CDCl.sub.3 with 2 drops of CD.sub.3OD, ppm):
.delta.=1.35 (d, 2H), 1.57 (bs, 4H), 3.21 (bs, 2H), 3.31 (bs, 2H),
3.56 (s, 2H), 4.39 (q, 1H), 5.34 (s, 1H), 6.54 (m, 2H), 6.73 (m,
1H), 6.84 (m, 2H), 7.30 (m, 5H), 7.72 (d, 2H)
[0531] .sup.13C-nmr (CDCl.sub.3 with 2 drops of CD.sub.3OD, ppm):
.delta.=11.8, 21.6, 34.1, 34.2, 36.8, 44.7, 52.7, 97.3, 102.3,
105.1, 107.2, 107.5, 107.9, 122.5, 122.9, 123.5, 124.0, 124.6,
132.7, 140.6, 156.9, 164.5, 166.3, 166.8, 168.4, 168.8
[0532] C.sub.30H.sub.33F.sub.2N.sub.7O.sub.5 (MW=609.64)
Example 32
Biological Assay
[0533] The pharmacological profile of
.sup.3H-5-(S)-(N'-((S)-(+)-2-Hydroxy-
-3-methylbutyryl)-L-alaninyl)amino-7-methyl-5,7-dihydro-6H-dibenz[b,d]azep-
in-6-one (Compound 1 from Example 30) has been studied in 293
membranes. There are two distinct binding components in the
membrane preparations. Saturation binding analysis utilizing 0-50
pM and 0-150 nM .sup.3H-Compound 1 reveal two sites (K.sub.D1=52.7
pM [B.sub.max1=710 fmol/mg protein]; K.sub.D2=32.5 nM
(B.sub.max2=34.4 pmol/mg protein]).
[0534] The compound of Example 28 (Compound 3) was also tested. The
high affinity
.sup.3H--N-[8-N'-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl-
-L-alaninyl)-L-phenylglycine)-3,6-dioxaoctyl]-4,4-difluoro-5,7-dimethyl-4--
bora-3.alpha.,4.alpha.-diaza-s-indacene-3-propionamide (Compound 3)
binding site mediates the A.beta. lowering activity of Compound 1,
whereas the low affinity site is irrelevant to A.beta. lowering.
Compound 3 competes with .sup.3H-Compound 1 in binding to membranes
obtained from 293 cells with a K.sub.i of 74.5 pM (n=2). At the low
affinity Compound 1 site, Compound 3 competes with .sup.3H-Compound
1 with a K.sub.i of 573.5 nM (n=2). Compound 3 inhibits A.beta.
production in 293 cells with an IC.sub.50 of 585 pM. This value for
whole cell biological activity of Compound 3 we attribute to its
potency at the A.beta. lowering, high affinity Compound 1 binding
site, since the ratio of its A.beta. lowering to the K.sub.i at the
high affinity site is 7.9. For comparison, the ratio of the K.sub.i
at the low affinity site to the IC.sub.50 for A.beta. lowering at
the low affinity site is 980.3. This wide separation in high and
low affinity K.sub.i's (7741.5; n=2) affords a wide window of
concentrations of Compound 3 where binding of the compound is
almost exclusively to the high affinity Compound 1 site.
[0535] SWE293 cells were successfully labelled with 5 nM of
Compound 3 for flow cytometry analysis (excitation 488 nm, emission
510 (+/-10)nm) to give a four fold increase in fluorescence
intensity over background unlabeled cells, consistent with the
binding site being in relatively low abundance. At 5 nM
concentration this compound only labels the high affinity Compound
1 (A.beta. lowering) site. In the presence of 1 .mu.M
5-(S)-[N'-((S)-3,5-Difluorophenyl-.alpha.-hydroxyacetyl)-L-alaninyl]amino-
-7-methyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one (Example 29),
(which is sufficient to completely displace all specifically bound
Compound of Example 28 at >>5 nM)), the signal was reduced by
20% (p<0.001), after background subtraction. Compound 3 is
obviously a very hydrophobic molecule (log P>3), and the non
displaceable signal is almost certainly due to non-specific
partitioning into cell membranes.
[0536] Cells over-expressing the high affinity Compound 1 binding
site are amenable to cell sorting after transfection of 293T cells
with cDNA from a HEK 293 library. The strong induction of
transcription with this vector system should result in a 50-100
fold increase in expression of the transfected gene. Thus, cells
over-expressing the high affinity Compound 1 binding site are
amenable to sorting by FACS, since at 5 nM, the non-displaceable
binding will stay constant. The incorporated cDNA from positive
cells is amplified, cloned and sequenced as a means to identify the
protein mediating the functional .gamma.-secretase inhibition.
[0537] From the foregoing description, various modifications and
changes in the composition and method will occur to those skilled
in the art. All such modifications coming within the scope of the
appended claims are intended to be included therein.
* * * * *