U.S. patent application number 10/706877 was filed with the patent office on 2006-03-09 for lactacystin analogs.
This patent application is currently assigned to President and Fellows of Harvard College. Invention is credited to Gabriel Fenteany, Timothy F. Jamison, Stuart L. Schreiber, Robert F. Standaert.
Application Number | 20060052424 10/706877 |
Document ID | / |
Family ID | 27025297 |
Filed Date | 2006-03-09 |
United States Patent
Application |
20060052424 |
Kind Code |
A1 |
Schreiber; Stuart L. ; et
al. |
March 9, 2006 |
Lactacystin analogs
Abstract
Compounds related to lactacystin and lactacystin .beta.-lactone,
pharmaceutical compositions containing the compounds, and methods
of use.
Inventors: |
Schreiber; Stuart L.;
(Boston, MA) ; Standaert; Robert F.; (Bryan,
TX) ; Fenteany; Gabriel; (Cambridge, MA) ;
Jamison; Timothy F.; (Cambridge, MA) |
Correspondence
Address: |
WILMER CUTLER PICKERING HALE AND DORR LLP
60 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
President and Fellows of Harvard
College
Cambridge
MA
|
Family ID: |
27025297 |
Appl. No.: |
10/706877 |
Filed: |
November 12, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09924993 |
Aug 8, 2001 |
6838477 |
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10706877 |
Nov 12, 2003 |
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08945092 |
Jan 26, 1998 |
6645999 |
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PCT/US96/05072 |
Apr 12, 1996 |
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09924993 |
Aug 8, 2001 |
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08421583 |
Apr 12, 1995 |
6335358 |
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08945092 |
Jan 26, 1998 |
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Current U.S.
Class: |
514/369 ;
514/376; 514/389; 514/412 |
Current CPC
Class: |
A61K 31/4015 20130101;
A61K 31/4166 20130101; C07D 491/10 20130101; C12N 9/6421 20130101;
A61K 31/407 20130101; A61K 31/421 20130101; C07D 487/04 20130101;
A61K 31/426 20130101; C07D 495/10 20130101 |
Class at
Publication: |
514/369 ;
514/412; 514/389; 514/376 |
International
Class: |
A61K 31/426 20060101
A61K031/426; A61K 31/421 20060101 A61K031/421; A61K 31/4166
20060101 A61K031/4166 |
Claims
1. A method of treating cancer, comprising administering to a
subject an effective anti-cancer amount of a pharmaceutical
composition having the formula: ##STR69## wherein Z.sup.1 is O, S,
SO.sub.2, NH, or NR.sub.a, R.sub.a being C.sub.1-6 alkyl; X.sup.1
is O, S, CH.sub.2, two singly bonded H, CH(R.sub.b) in the E or Z
configuration, or C(R.sub.b)(R.sub.c) in the E or Z configuration,
each of R.sub.b and R.sub.d, independently, being C.sub.1-6 alkyl,
C.sub.6-12 aryl, C.sub.3-8 cycloalkyl, C.sub.3-8 heteroaryl,
C.sub.3-8 heterocyclic radical, or halogen, X.sup.1 being two
singly bonded H when Z.sup.1 is SO.sub.2; Z.sup.2is O, S, NH,
NR.sub.d, CHR.sup.1, or CHOR.sup.1 in the (R) or (S) configuration,
wherein R.sub.d is C.sub.1-6 alkyl and R.sup.1 is H, halogen,
C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, NR.sub.dR.sub.e (except where Z.sup.2 is CHOR.sup.1), or
the side chain of any naturally occurring .alpha.-amino acid, or
R.sup.1 and R.sup.2 taken together are a bivalent moiety, provided
that when R.sup.1 and R.sup.2 are taken together, Z.sup.1 is NH or
NR.sub.a and Z.sup.2 is CHR.sup.1; R.sup.e being H, C.sub.1-6
alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, or C.sub.2-6
alkynyl, and the bivalent moiety forming a C.sub.3-8 cycloalkyl,
C.sub.3-8 heteroaryl, C.sub.3-8 heterocyclic radical, or C.sub.6-12
aryl, where the H in CHR.sup.1 is deleted when R.sup.1 and R.sub.2
taken together form a C.sub.3-8 heteroaryl or C.sub.6-12 aryl;
R.sup.2 is C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl,
azido, C.sub.2-6 alkynyl, halogen, OR.sub.f, SR.sub.f,
NR.sub.fR.sub.g, --ONR.sub.fR.sub.g, --NR.sub.g(OR.sub.f), or
--NR.sub.g(SR.sub.f) (each of R.sub.f and R.sub.g, independently,
being H, C.sub.1-6, alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl,
or C.sub.2-6 alkynyl), or R.sup.1 and R.sup.2 taken together are a
bivalent moiety, the bivalent moiety forming a C.sub.3-8
cycloalkyl, C.sub.3-8 heteroaryl, C.sub.3-8 heterocyclic radical,
or C.sub.6-12 aryl, where the H in CHR.sup.1 is deleted when
R.sub.1 and R.sub.2 taken together form a C.sub.3-8 heteroaryl or
C.sub.6-12 aryl; A.sup.1 is H, the side chain of any naturally
occurring .alpha.-amino acid, or is of the following formula,
--(CH.sub.2).sub.m--Y--(CH.sub.2).sub.n--R.sup.3X.sup.3 wherein Y
is O, S, C.dbd.O, C.dbd.S, --(CH.dbd.CH)--, vinylidene,
--C.dbd.NOR.sub.h, --C.dbd.NNR.sub.iR.sub.i', sulfonyl, methylene,
CHX.sup.4 in the (R) or (S) configuration, or deleted, X.sup.4
being halogen, methyl, halomethyl, OR.sub.h, SR.sub.h,
NR.sub.iR.sub.i', --NR.sub.i(OR.sub.h), or
--NR.sub.i(NR.sub.iR.sub.i'), wherein R.sub.h is selected from H,
C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, C.sub.1-10 acyl, C.sub.1-6 alkylsulfonyl, and C.sub.6-10
arylsulfonyl, and each of R.sub.i and R.sub.i', independently is
selected from H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, and C.sub.1-10 acyl; m is 0, 1, 2, or
3, and n is 0, 1, 2, or 3; and R.sup.3 is straight chain or
branched C.sub.1-8 alkylidene, straight chain or branched C.sub.1-8
alkylene, C.sub.3-10 cycloalkylidene, C.sub.3-10 cycloalkylene,
phenylene, C.sub.6-14 arylalkylidene, C.sub.6-14 arylalkylene, or
deleted, and X.sup.3 is H hydroxyl, thiol, carboxyl, amino,
halogen, (C.sub.1-6 alkyl)oxycarbonyl, (C.sub.7-14
arylalkyl)oxycarbonyl, or C.sub.6-14 aryl; or R.sup.3 and X.sup.3
taken together are the side chain of any naturally occurring
.alpha.-amino acid; and L.sup.O is H or an organic moiety having 1
to 25 carbon atoms, 0 to 10 heteroatoms, and 0 to 6 halogen atoms;
and a pharmaceutically acceptable carrier.
2. A method of treating cancer, comprising administering to a
subject an effective anti-cancer amount of a pharmaceutical
composition having the formula: ##STR70## wherein Z.sup.1 is O, S,
SO.sub.2, NH, or NR.sub.a, R.sub.a being C.sub.1-6 alkyl; X.sup.1
is O, S, CH.sub.2, two singly bonded H, CH(R.sub.b) in the E or Z
configuration, or C(R.sub.b)(R.sub.c) in the E or Z configuration,
each of R.sub.b and R.sub.c, independently, being C.sub.1-6 alkyl,
C.sub.6-12 aryl, C.sub.3-8 cycloalkyl, C.sub.3-8 heteroaryl,
C.sub.3-8 heterocyclic radical, or halogen, provided that when
Z.sup.1 is SO.sub.2, X.sup.1 is two singly bonded H; Z.sup.2 is
CHR.sup.1 in the (R) or (S) configuration, R.sup.1 being H,
C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, hydroxyl, halogen, a side chain of a naturally occuring
.alpha.-amino acid, OR.sub.d, SR.sub.d, or NR.sub.dR.sub.e (each of
R.sub.d and R.sub.e, independently, being H, C.sub.1-6 alkyl,
C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, or C.sub.2-5 alkynyl);
Z.sup.3 is O, S, NH, or NR.sub.j, wherein R.sub.j is C.sub.1-6
alkyl; X.sup.2 is O or S; and A.sup.1 is H, the side chain of any
naturally occurring .alpha.-amino acid, or is of the following
formula, --(CH.sub.2).sub.m--Y--(CH.sub.2).sub.n--R.sup.3X.sup.3
wherein Y is O, S, C.dbd.O, C.dbd.S, --(CH.dbd.CH)--, vinylidene,
--C.dbd.NOR.sub.h, --C.dbd.NNR.sub.iR.sub.i', sulfonyl, methylene,
CHX.sup.4 in the (R) or (S) configuration, or deleted, X.sup.4
being halogen, methyl, halomethyl, OR.sub.h, SR.sub.h,
NR.sub.iR.sub.i', --NR.sub.i(OR.sub.h), or
--NR.sub.i(NR.sub.iR.sub.i'), wherein R.sub.h is selected from H,
C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, C.sub.1-10 acyl, C.sub.1-6 alkylsulfonyl, and C.sub.6-10
arylsulfonyl; and each of R.sub.i and R.sub.i', independently is
selected from H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, and C.sub.1-10 acyl; m is 0, 1, 2, or
3, and n is 0, 1, 2, or 3; and R.sup.3 is straight chain or
branched C.sub.1-8 alkylidene, straight chain or branched C.sub.1-8
alkylene, C.sub.3-10 cycloalkylidene, C.sub.3-10 cycloalkylene,
phenylene, C.sub.6-14 arylalkylidene, C.sub.6-14 arylalkylene, or
deleted, and X.sup.3 is H, hydroxyl, thiol, carboxyl, amino,
halogen, (C.sub.1-6 alkyl)oxycarbonyl, (C.sub.7-14
arylalkyl)oxycarbonyl, or C.sub.6-14 aryl; or R.sup.3 and X.sup.3
taken together are the side chain of any naturally occurring
.alpha.-amino acid; and a pharmaceutically acceptable carrier.
3. The method of claim 1 or 2, wherein the cancer is selected from
carcinoma, lymphoma, sarcoma, and myeloma.
4. The method of claim 1 or 2, wherein said cancer is selected from
adenocarcinoma, acinic cell adenocarcinoma, adrenal cortical
carcinomas, alveoli cell carcinoma, anaplastic carcinoma, basaloid
carcinoma, basal cell carcinoma, bronchiolar carcinoma,
bronchogenic carcinoma, renaladinol carcinoma, embryonal carcinoma,
anometroid carcinoma, fibrolamolar liver cell carcinoma, follicular
carcinomas, giant cell carcinomas, hepatocellular carcinoma,
intraepidermal carcinoma, intraepithelial carcinoma, leptomanigio
carcinoma, medullary carcinoma, melanotic carcinoma, menigual
carcinoma, mesometonephric carcinoma, oat cell carcinoma, squamal
cell carcinoma, sweat gland carcinoma, transitional cell carcinoma,
tubular cell carcinoma, amelioblastic sarcoma, angiolithic sarcoma,
botryoid sarcoma, endometrial stroma sarcoma, ewing sarcoma,
fascicular sarcoma, giant cell sarcoma, granulositic sarcoma,
immunoblastic sarcoma, juxaccordial osteogenic sarcoma, Kaposi's
sarcoma, leukocytic sarcoma, lymphatic sarcoma, medullary sarcoma,
myeloid sarcoma, austiogenci sarcoma, periosteal sarcoma, reticulum
cell sarcoma, round cell sarcoma, spindle cell sarcoma, synovial
sarcoma, and telangiectatic audiogenic sarcoma, neural blastoma,
glioblastoma, astrocytoma, melanoma, leiomyo sarcoma, multiple
myeloma, Hemangioma, Hodgkin's disease, Burkitt's lymphoma, and
nodular poorly-differentiated lymphocytic lymphoma, nodular mixed
lymphocytic lymphoma, nodular histiocytic lymphoma, and diffuse
lymphomas.
5. The method of claim 1 or 2, wherein Z.sup.1 is NH or
NR.sub.a.
6. The method of claim 1 or 2, wherein A.sup.1 is
--(CH.sub.2).sub.m--Y--(CH.sub.2).sub.n--R.sup.3X.sup.3 and Y is
CHX.sup.4 in the (R) or (S) configuration.
7. The method of claim 6, wherein Y is CHX.sup.4 in the (S)
configuration and X.sup.3 is H.
8. The method of claim 7, wherein m and n are each 0.
9. The method of claim 1 or 2, wherein Z.sup.2 is CHR.sup.1 in the
(R) configuration and R.sup.1 is C.sub.1-6 alkyl.
10. The method of claim 2, wherein X.sup.2 is O and Z.sup.3 is
O.
11. The method of claim 1, wherein R.sup.2 is OR.sub.f and R.sub.f
is H.
Description
BACKGROUND OF THE INVENTION
[0001] The invention relates generally to lactacystin and analogues
thereof.
[0002] Eukaryotic cells contain multiple proteolytic systems,
including lysosomal proteases, calpains, ATP-ubiquitin-proteasome
dependent pathway, and an ATP-independent nonlysosomal process. The
major neutral proteolytic activity in the cytosol and nucleus is
the proteasome, a 20S (700 kDa) particle with multiple peptidase
activities. The 20S complex is the proteolytic core of a 26S (1500
kDa) complex that degrades or processes ubiquitin-conjugated
proteins. Ubquitination marks a protein for hydrolysis by the 26S
proteasome complex. Many abnormal or short-lived normal
polypeptides are degraded by the ubiquitin-proteasome-dependent
pathway. Abnormal peptides include oxidant-damaged proteins (e.g.,
those having oxidized disulfide bonds), products of premature
translational termination (e.g., those having exposed hydrophobic
groups which are recognized by the proteasome), and stress-induced
denatured or damaged proteins (where stress is induced by, e.g.,
changes in pH or temperature, or exposure to metals). In addition,
some proteins, such as casein, do not require ubquitination to be
hydrolyzed by the proteasome.
[0003] The proteasome has chymotryptic, tryptic, and
peptidyl-glutamyl peptide hydrolizing activities, i.e., the
proteasome can cleave peptides on the carboxyl side of hydrophobic,
basic, and acidic residues, respectively.
SUMMARY OF THE INVENTION
[0004] The invention relates to novel compounds structurally
related to lactacystin and lactacystin .beta.-lactone. The
invention also relates to pharmaceutical compositions including
lactacystin and lactacystin analogs.
[0005] One aspect of the invention is a pharmaceutical composition
containing a compound having the formula (I) ##STR1## wherein
Z.sup.1 is O, S, SO.sub.2, NH, or NR.sub.a, R.sub.a being C.sub.1-6
alkyl; X.sup.1 is O, S, CH.sub.2, two singly bonded H, CH(R.sub.b)
in the E or Z configuration, or C(R.sub.b)(R.sub.c) in the E or Z
configuration, each of R.sub.b and R.sub.c, independently, being
C.sub.1-6 alkyl, C.sub.6-12 aryl, C.sub.3-8 cycloalkyl, C.sub.3-8
heteroaryl, C.sub.3-8 heterocyclic radical, or halogen, X.sup.1
being two singly bonded H when Z.sup.1 is SO.sub.2; Z.sup.2 is O,
S, NH, NR.sub.d, CHR.sup.1, or CHOR.sup.1 in the (R) or (S)
configuration, wherein R.sub.d is C.sub.1-6 alkyl and R.sup.1 is H,
halogen, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, NR.sub.dR.sub.e (except where Z.sup.2 is
CHOR.sup.1), or the side chain of any naturally occurring
.alpha.-amino acid, or R.sup.1 and R.sup.2 taken together are a
bivalent moiety, provided that when R.sup.1 and R.sup.2 are taken
together, Z.sup.1 is NH or NR.sub.a and Z.sup.2 is CHR.sup.1;
R.sub.e being H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6
alkenyl, or C.sub.2-6 alkynyl, and the bivalent moiety forming a
C.sub.3-8 cycloalkyl, C.sub.3-8 heteroaryl, C.sub.3-8 heterocyclic
radical, or C.sub.6-12 aryl, where the H in CHR.sup.1 is deleted
when R.sub.1 and R.sub.2 taken together form a C.sub.3-8 heteroaryl
or C.sub.6-12 aryl; R.sup.2 is C.sub.1-6 alkyl, C.sub.1-6
haloalkyl, C.sub.2-6 alkenyl, azido, C.sub.2-6 alkynyl, halogen,
OR.sub.f, SR.sub.f, NR.sub.fR.sub.g, --ONR.sub.fR.sub.g,
--NR.sub.g(OR.sub.f), or --NR.sub.g(SR.sub.f) (each of R.sub.f and
R.sub.g, independently, being H, C.sub.1-6, alkyl, C.sub.1-6
haloalkyl, C.sub.2-6 alkenyl, or C.sub.2-6 alkynyl), or R.sup.1 and
R.sup.2 taken together are a bivalent moiety, the bivalent moiety
forming a C.sub.3-8 cycloalkyl, C.sub.3-8 heteroaryl, C.sub.3-8
heterocyclic radical, or C.sub.6-12 aryl, where the H in CHR.sup.1
is deleted when R.sub.1 and R.sub.2 taken together form a C.sub.3-8
heteroaryl or C.sub.6-12 aryl; A.sup.1 is H, the side chain of any
naturally occurring .alpha.-amino acid, or is of the following
formula, --(CH.sub.2).sub.m--Y--(CH.sub.2).sub.n--R.sup.3X.sup.3
wherein Y is O, S, C.dbd.O, C.dbd.S, --(CH.dbd.CH)--, vinylidene,
--C.dbd.NOR.sub.h, --C.dbd.NNR.sub.iR.sub.i', sulfonyl, methylene,
CHX.sup.4 in the (R) or (S) configuration, or deleted, X.sup.4
being halogen, methyl, halomethyl, OR.sub.h, SR.sub.h,
NR.sub.iR.sub.i', --NR.sub.i(OR.sub.h), or
--NR.sub.i(NR.sub.iR.sub.i'), wherein R.sub.h is selected from H,
C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, C.sub.1-10 acyl, C.sub.1-6 alkylsulfonyl, and C.sub.6-10
arylsulfonyl; and each of R.sub.i and R.sub.i', independently is
selected from H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, and C.sub.1-10 acyl; m is 0, 1, 2, or
3, and n is 0, 1, 2, or 3; and R.sup.3 is straight chain or
branched C.sub.1-8 alkylidene, straight chain or branched C.sub.1-8
alkylene, C.sub.3-10 cycloalkylidene, C.sub.3-10 cycloalkylene,
phenylene, C.sub.6-14 arylalkylidene, C.sub.6-14 arylalkylene, or
deleted, and X.sup.3 is hydroxyl, thiol, carboxyl, amino, halogen,
(C.sub.1-6 alkyl)oxycarbonyl, (C.sub.7-14 arylalkyl)oxycarbonyl, or
C.sub.6-14 aryl; or R.sup.3 and X.sup.3 taken together are the side
chain of any naturally occurring .alpha.-amino acid; and L.sup.O is
an organic moiety having 1 to 25 carbon atoms, 0 to 10 heteroatoms,
and 0 to 6 halogen atoms; and a pharmaceutically acceptable
carrier.
[0006] A second aspect is a pharmaceutical composition comprising a
compound having the following formula (II) ##STR2##
[0007] wherein Z.sup.1 is O, S, SO.sub.2, NH, or NR.sub.a, R.sub.a
being C.sub.1-6 alkyl; X.sup.1 is O, S, CH.sub.2, two singly bonded
H, CH(R.sub.b) in the E or Z configuration, or C(R.sub.b)(R.sub.c)
in the E or Z configuration, each of R.sub.b and R.sub.c,
independently, being C.sub.1-6 alkyl, C.sub.6-12 aryl, C.sub.3-8
cycloalkyl, C.sub.3-8 heteroaryl, C.sub.3-8 heterocyclic radical,
or halogen, provided that when Z.sup.1 is SO.sub.2, X.sup.1 is two
singly bonded H; Z.sup.2 is CHR.sup.1 in the (R) or (S)
configuration, R.sup.1 being H, C.sub.1-6 alkyl, C.sub.1-6
haloalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, hydroxyl, halogen,
a side chain of a naturally occuring .alpha.-amino acid, OR.sub.d,
SR.sub.d, or NR.sub.dR.sub.e (each of R.sub.d and R.sub.e,
independently, being H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl,
C.sub.2-6 alkenyl, or C.sub.2-5 alkynyl); Z.sup.3 is O, S, NH, or
NR.sub.j, wherein R.sub.j is C.sub.1-6 alkyl; X.sup.2 is O or S;
and A.sup.1 is H, the side chain of any naturally occurring a-amino
acid, or is of the following formula,
--(CH.sub.2).sub.m--Y--(CH.sub.2).sub.n--R.sup.3X.sup.3 wherein Y
is O, S, C.dbd.O, C.dbd.S, --(CH.dbd.CH)--, vinylidene,
--C.dbd.NOR.sub.h, --C.dbd.NNR.sub.iR.sub.i', sulfonyl, methylene,
CHX.sup.4 in the (R) or (S) configuration, or deleted, X.sup.4
being halogen, methyl, halomethyl, OR.sub.h, SR.sub.h,
NR.sub.iR.sub.i', --NR.sub.i(OR.sub.h), or
--NR.sub.i(NR.sub.iR.sub.i'), wherein R.sub.h is selected from H,
C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, C.sub.1-10 acyl, C.sub.1-6 alkylsulfonyl, and C.sub.6-10
arylsulfonyl; and each of R.sub.i and R.sub.i', independently is
selected from H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, and C.sub.1-10 acyl; m is 0, 1, 2, or
3, and n is 0, 1, 2, or 3; R.sup.3 is straight chain or branched
C.sub.1-8 alkylidene, straight chain or branched C.sub.1-8
alkylene, C.sub.3-10 cycloalkylidene, C.sub.3-10 cycloalkylene,
phenylene, C.sub.6-14 arylalkylidene, C.sub.6-14 arylalkylene, or
deleted; and X.sup.3 is H, hydroxyl, thiol, carboxyl, amino,
halogen, (C.sub.1-6 alkyl)oxycarbonyl, (C.sub.7-14
arylalkyl)oxycarbonyl, or C.sub.6-14 aryl; or R.sup.3 and X.sup.3
taken together are the side chain of any naturally occurring
.alpha.-amino acid; and a pharmaceutically acceptable carrier.
[0008] A third aspect is a pharmaceutical composition comprising a
compound having one of the following formulae (II) or (IV)
##STR3##
[0009] wherein Z.sup.1 is NH or NR.sub.a, R.sub.a being C.sub.1-6
alkyl; X.sup.1 is O, S, CH.sub.2, two singly bonded H, CH(R.sub.b)
in the E or Z configuration, or C(R.sub.b)(R.sub.e) in the E or Z
configuration, each of R.sub.b and R.sub.c, independently, being
C.sub.1-6 alkyl, C.sub.6-12 aryl, C.sub.3-8 cycloalkyl, C.sub.3-8
heteroaryl, C.sub.3-8 heterocyclic radical, or halogen; Z.sup.2 is
O, S, NH, or NR.sub.j, wherein R.sub.j is C.sub.1-6 alkyl; R.sup.1
is in the (R) or (S) configuration, and is H, C.sub.1-6 alkyl,
C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl,
hydroxyl, halogen, a side chain of a naturally occurring
.alpha.-amino acid, OR.sub.d, SR.sub.d, or NR.sub.dR.sub.e (each of
R.sub.d and R.sub.e, independently, being H, C.sub.1-6 alkyl,
C.sub.1-6 haloalkyl, C.sub.6-12 aryl, C.sub.3-8 cycloalkyl,
C.sub.3-8 heteroaryl, C.sub.3-8 hetero-cyclic radical, or halogen);
X.sup.2 is O or S; and
[0010] A.sup.1 is H, the side chain of any naturally occurring
a-amino acid, or is of the following formula,
--(CH.sub.2).sub.m--Y--(CH.sub.2).sub.n--R.sup.3X.sup.3 wherein Y
is O, S, C.dbd.O, C.dbd.S, --(CH.dbd.CH)--, vinylidene,
--C.dbd.NOR.sub.h, --C.dbd.NNR.sub.iR.sub.i', sulfonyl, methylene,
CHX.sup.4 in the (R) or (S) configuration, or deleted, X.sup.4
being halogen, methyl, halomethyl, OR.sup.h, SR.sub.h,
NR.sup.iR.sup.i', --NR.sup.i(OR.sup.h), or
--NR.sup.i(NR.sup.iR.sup.i'), wherein R.sub.h is selected from H,
C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, and
C.sub.2-6 alkynyl, and each of R.sub.i and R.sub.i', independently
is selected from H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, and C.sub.1-10 acyl; m is 0, 1, 2, or
3, and n is 0, 1, 2, or 3; and R.sup.3 is straight chain or
branched C.sub.1-8 alkylidene, straight chain or branched C.sub.1-8
alkylene, C.sub.3-10 cycloalkylidene, C.sub.3-10 cycloalkylene,
phenylene, C.sub.6-14 arylalkylidene, C.sub.6-14 arylalkylene, or
deleted, and X.sup.3 is H, hydroxyl, thiol, carboxyl, amino,
halogen, (C.sub.1-6 alkyl)oxycarbonyl, (C.sub.7-14
arylalkyl)oxycarbonyl, or C.sub.6-14 aryl; or R.sup.3 and X.sup.3
taken together are the side chain of any naturally occurring
.alpha.-amino acid; and a pharmaceutically acceptable carrier.
[0011] A fourth aspect is a pharmaceutical composition containing a
compound having the following formula (V) ##STR4##
[0012] wherein Z.sup.1 is O, S, NH or NR.sub.j, R.sub.j being
C.sub.1-6 alkyl; X.sup.1 is O or S; R.sup.1 is in the (R) or (S)
configuration, and is H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl,
C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, hydroxyl, halogen, a side
chain of a naturally occuring .alpha.-amino acid, OR.sub.d,
SR.sub.d, or NR.sub.dR.sub.e (each of R.sub.d and R.sub.e,
independently, being H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl,
C.sub.2-6 alkenyl, or C.sub.2-5 alkynyl); R.sup.2 is H, C.sub.1-6
alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl,
C.sub.6-12 aryl, C.sub.3-8 heteroaryl, or halogen; X.sup.2 is O or
S; and A.sup.1 is H, the side chain of any naturally occurring
.alpha.-amino acid, or is of the following formula,
--(CH.sub.2).sub.m--Y--(CH.sub.2).sub.n--R.sup.3X.sup.3 wherein Y
is O, S, C.dbd.O, C.dbd.S, --(CH.dbd.CH)--, vinylidene,
--C.dbd.NOR.sub.h, --C.dbd.NNR.sub.iR.sub.i', sulfonyl, methylene,
CHX.sup.4 in the (R) or (S) configuration, or deleted, X.sup.4
being halogen, methyl, halomethyl, OR.sub.h, SR.sub.h,
NR.sub.iR.sub.i', --NR.sub.i(OR.sub.h), or
--NR.sub.i(NR.sub.iR.sub.i'), wherein R.sub.h is selected from H,
C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, and
C.sub.2-6 alkynyl, and each of R.sub.i and R.sub.i', independently
is selected from H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, and C.sub.1-10 acyl; m is 0, 1, 2, or
3, and n is 0, 1, 2, or 3; and R.sup.3 is straight chain or
branched C.sub.1-8 alkylidene, straight chain or branched C.sub.1-8
alkylene, C.sub.3-10 cycloalkylidene, C.sub.3-10 cycloalkylene,
phenylene, C.sub.6-14 arylalkylidene, C.sub.6-14 arylalkylene, or
deleted, and X.sup.3 is H, hydroxyl, thiol, carboxyl, amino,
halogen, (C.sub.1-6 alkyl)oxycarbonyl, (C.sub.7-14
arylalkyl)-oxycarbonyl, or C.sub.6-14 aryl; or R.sup.3 and X.sup.3
taken together are the side chain of any naturally occurring
.alpha.-amino acid; and a pharmaceutically acceptable carrier.
[0013] A fifth aspect is a pharmaceutical composition comprising a
compound having the following formula (VI) ##STR5##
[0014] wherein Z.sup.1 is O, S, NH or NR.sub.j, R.sub.j being
C.sub.1-6 alkyl; X.sup.1 is O or S; R.sup.1 is H, C.sub.1-6 alkyl,
C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl,
hydroxyl, halogen, a side chain of a naturally occuring
.alpha.-amino acid, OR.sub.d, SR.sub.d, or NR.sub.dR.sub.e (each of
R.sub.d and R.sub.e, independently, being C.sub.1-6 alkyl,
C.sub.1-6 haloalkyl, C.sub.6-12 aryl, C.sub.3-8 cycloalkyl,
C.sub.3-8 heteroaryl, C.sub.3-8 hetero-cyclic radical, or halogen);
R.sup.2 is H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, C.sub.6-12 aryl, C.sub.3-8 hetroaryl,
or halogen; R.sub.a is C.sub.1-6 alkyl; and A.sup.1 is H, the side
chain of any naturally occurring a-amino acid, or is of the
following formula,
--(CH.sub.2).sub.m--Y--(CH.sub.2).sub.n--R.sup.3X.sup.3 wherein Y
is O, S, C.dbd.O, C.dbd.S, --(CH.dbd.CH)--, vinylidene,
--C.dbd.NOR.sub.h, --C.dbd.NNR.sub.iR.sub.i', sulfonyl, methylene,
CHX.sup.4 in the (R) or (S) configuration, or deleted, X.sup.4
being halogen, methyl, halomethyl, OR.sub.h, SR.sub.h,
NR.sub.iR.sub.i', --NR.sub.i(OR.sub.h), or
--NR.sub.i(NR.sub.iR.sub.i'), wherein R.sub.h is selected from H,
C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, and
C.sub.2-6 alkynyl, and each of R.sub.i and R.sub.i', independently
is selected from H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, and C.sub.1-10 acyl; m is 0, 1, 2, or
3, and n is 0, 1, 2, or 3; and R.sup.3 is straight chain or
branched C.sub.1-8 alkylidene, straight chain or branched C.sub.1-8
alkylene, C.sub.3-10 cycloalkylidene, C.sub.3-10 cycloalkylene,
phenylene, C.sub.6-14 arylalkylidene, C.sub.6-14 arylalkylene, or
deleted, and X.sup.3 is H, hydroxyl, thiol, carboxyl, amino,
halogen, (C.sub.1-6 alkyl)oxycarbonyl, (C.sub.7-14
arylalkyl)-oxycarbonyl, or C.sub.6-14 aryl; or R.sup.3 and X.sup.3
taken together are the side chain of any naturally occurring
.alpha.-amino acid; and a pharmaceutically acceptable carrier.
[0015] A sixth aspect is a pharmaceutical composition containing a
compound having the formula (VII) ##STR6##
[0016] wherein X.sup.1 is O, S, CH.sub.2, two singly bonded H,
CH(R.sub.b) in the E or Z configuration, or C(R.sub.b)(R.sub.c) in
the E or Z configuration, each of R.sub.b and R.sub.c,
independently, being C.sub.1-6 alkyl, C.sub.6-12 aryl, C.sub.3-8
cycloalkyl, C.sub.3-8 heteroaryl, C.sub.3-8 heterocyclic radical,
or halogen; Z.sup.1 is O, S, NH, o NR.sub.a, R.sub.a being
C.sub.1-6 alkyl; R.sup.1 is H, C.sub.1-6 alkyl, C.sub.1-6
haloalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, hydroxyl, halogen,
a side chain of any naturally occuring .alpha.-amino acid,
OR.sub.d, SR.sub.d, or NR.sub.dR.sub.e (each of R.sub.d and
R.sub.e, independently, being H, C.sub.1-6 alkyl, C.sub.1-6
halo-alkyl, C.sub.6-12 aryl, C.sub.3-8 cycloalkyl, C.sub.3-8
heteroaryl, C.sub.3-8 heterocyclic radical, or halogen); or R.sup.1
and R.sup.2 taken together are a bivalent moiety which forms a
C.sub.3-8 cyclo-alkyl, C.sub.3-8 heteroaryl, C.sub.3-8 heterocyclic
radical, or C.sub.6-12 aryl; R.sup.2 is H, C.sub.1-6 alkyl,
C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, azido, C.sub.2-6 alkynyl,
halogen, OR.sub.f, SR.sub.f, NR.sub.fR.sub.g, --ONR.sub.fR.sub.g,
--NR.sub.g(OR.sub.f), or --NR.sub.g(SR.sub.f) (each of R.sub.f and
R.sub.g, independently, being H, C.sub.1-6, alkyl, C.sub.1-6
haloalkyl, C.sub.2-6 alkenyl, or C.sub.2-6 alkynyl), or R.sup.1 and
R.sup.2 taken together are a bivalent moiety, the bivalent moiety
forming a C.sub.3-8 cycloalkyl, C.sub.3-8 heteroaryl, C.sub.3-8
heterocyclic radical, or C.sub.6-12 aryl; X.sup.2 is O or S; and
A.sup.1 is in the (R) or (S) configuration, and each of A.sup.1 and
A.sup.2 is independently H, the side chain of any naturally
occurring amino .alpha.-acid, or is of the following formula,
--(CH.sub.2).sub.m--Y--(CH.sub.2).sub.n--R.sup.3X.sup.3 wherein Y
is O, S, C.dbd.O, C.dbd.S, --(CH.dbd.CH)--, vinylidene,
--C.dbd.NOR.sub.h, --C.dbd.NNR.sub.iR.sub.i', sulfonyl, methylene,
CHX.sup.4 in the (R) or (S) configuration, or deleted, X.sup.4
being halogen, methyl, halomethyl, OR.sub.h, SR.sub.h,
NR.sub.iR.sub.i', --NR.sub.i(OR.sub.h), or
--NR.sub.i(NR.sub.iR.sub.i'), wherein R.sub.h is selected from H,
C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, and
C.sub.2-6 alkynyl, and each of R.sub.i and R.sub.i', independently
is selected from H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, and C.sub.1-10 acyl; m is 0, 1, 2, or
3, and n is 0, 1, 2, or 3; and R.sup.3 is straight chain or
branched C.sub.1-8 alkylidene, straight chain or branched C.sub.1-8
alkylene, C.sub.3-10 cycloalkylidene, C.sub.3-10 cycloalkylene,
phenylene, C.sub.6-14 arylalkylidene, C.sub.6-14 arylalkylene, or
deleted, and X.sup.3 is H, hydroxyl, thiol, carboxyl, amino,
halogen, (C.sub.1-6 alkyl)oxycarbonyl, (C.sub.7-14
arylalkyl)-oxycarbonyl, or C.sub.6-14 aryl; or R.sup.3 and X.sup.3
taken together are the side chain of any naturally occurring
.alpha.-amino acid; and a pharmaceutically acceptable carrier.
[0017] A seventh aspect is a pharmaceutical composition containing
a compound having the following formula (VIII) ##STR7##
[0018] wherein Z.sup.1 is NH or NR.sup.a, NR.sup.a being C.sub.1-6
alkyl;
[0019] X.sup.1 is O, S, CH.sub.2, two singly bonded H, CH(R.sub.b)
in the E or Z configuration, or C(R.sub.b)(R.sub.c) in the E or Z
configuration, each of R.sub.b and R.sub.c, independently, being
C.sub.1-6 alkyl, C.sub.6-12 aryl, C.sub.3-8 cycloalkyl, C.sub.3-8
heteroaryl, C.sub.3-8 heterocyclic radical, or halogen;
[0020] Z.sup.2 is O, S, NH, or NR.sub.j, R.sub.j being C.sub.1-6
alkyl;
[0021] R.sup.1 is in the (R) or (S) configuration, and is H,
C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, hydroxyl, halogen, a side chain of any naturally occuring
amino acid, OR.sub.d, SR.sub.d, or NR.sub.dR.sub.e (each of R.sub.d
and R.sub.e, independently, being H, C.sub.1-6 alkyl, C.sub.1-6
haloalkyl, C.sub.6-12 aryl, C.sub.3-8 cycloalkyl, C.sub.3-8
heteroaryl, C.sub.3-8 heterocyclic radical, or halogen); or R.sup.1
and R.sup.2 taken together are a bivalent moiety which forms a
C.sub.3-8 cycloalkyl, C.sub.3-8 heteroaryl, C.sub.3-8 heterocyclic
radical, or C.sub.6-12 aryl; R.sup.2 is H, C.sub.1-6 alkyl,
C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, azido, C.sub.2-6 alkynyl,
halogen, OR.sub.f, SR.sub.f, NR.sub.fR.sub.g, --ONR.sub.fR.sub.g,
--NR.sub.g(OR.sub.f), or --NR.sub.g(SR.sub.f) (each of R.sub.f and
R.sub.g, independently, being H, C.sub.1-6, alkyl, C.sub.1-6
haloalkyl, C.sub.2-6 alkenyl, or C.sub.2-6 alkynyl), or R.sup.1 and
R.sup.2 taken together are a bivalent moiety, the bivalent moiety
forming a C.sub.3-8 cycloalkyl, C.sub.3-8 heteroaryl, C.sub.3-8
heterocyclic radical, or C.sub.6-12 aryl; X.sup.2 is O or S; and
each of A.sup.1 and A.sup.2 is independently in the (R) or (S)
configuration, and is independently H, the side chain of any
naturally occurring a-amino acid, or is of the following formula,
--(CH.sub.2).sub.m--Y--(CH.sub.2).sub.n--R.sup.3X.sup.3 wherein Y
is O, S, C.dbd.O, C.dbd.S, --(CH.dbd.CH)--, vinylidene,
--C.dbd.NOR.sub.h, --C.dbd.NNR.sub.iR.sub.i', sulfonyl, methylene,
CHX.sup.4 in the (R) or (S) configuration, or deleted, X.sup.4
being halogen, methyl, halomethyl, OR.sub.h, SR.sub.h,
NR.sub.iR.sub.i', --NR.sub.i(OR.sub.h), or
--NR.sub.i(NR.sub.iR.sub.i'), wherein R.sub.h is selected from H,
C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, and
C.sub.2-6 alkynyl, and each of R.sub.i and R.sub.i', independently
is selected from H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, and C.sub.1-10 acyl; m is 0, 1, 2, or
3, and n is 0, 1, 2, or 3; and R.sup.3 is straight chain or
branched C.sub.1-8 alkylidene, straight chain or branched C.sub.1-8
alkylene, C.sub.3-10 cycloalkylidene, C.sub.3-10 cycloalkylene,
phenylene, C.sub.6-14 arylalkylidene, C.sub.6-14 arylalkylene, or
deleted, and X.sup.3 is H, hydroxyl, thiol, carboxyl, amino,
halogen, (C.sub.1-6 alkyl)oxycarbonyl, (C.sub.7-14
arylalkyl)-oxycarbonyl, or C.sub.6-14 aryl; or R.sup.3 and X.sup.3
together are the side chain of any naturally occurring a-amino
acid; and a pharmaceutically acceptable carrier.
[0022] An eighth aspect is a pharmaceutical composition containing
a compound of the formula (IX) ##STR8## wherein Z.sup.1 is O, NH,
or NR.sup.a, NR.sup.a being C.sub.1-6 alkyl; X.sup.1 is O, S,
CH.sub.2, or two singly bonded H; each of A.sup.1 and A.sup.2 is
independently H, the side chain of any naturally occurring
.alpha.-amino acid, or is of the following formula,
--(CH.sub.2).sub.m--Y--(CH.sub.2).sub.n--R.sup.3X.sup.3 wherein Y
is O, S, C.dbd.O, C.dbd.S, --(CH.dbd.CH)--, vinylidene,
--C.dbd.NOR.sub.h, --C.dbd.NNR.sup.iR.sup.i', sulfonyl, methylene,
CHX.sup.4 in the (R) or (S) configuration, or deleted, X.sup.4
being halogen, methyl, halomethyl, OR.sub.h, SR.sub.h,
NR.sub.iR.sub.i', --NR.sub.i(OR.sub.h), or
--NR.sub.i(NR.sub.iR.sub.i'), wherein R.sub.h is selected from H,
C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, and
C.sub.2-6 alkynyl, and each of R.sub.i and R.sub.i', independently
is selected from H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, and C.sub.1-10 acyl; m is 0, 1, 2, or
3, and n is 0, 1, 2, or 3; and R.sup.3 is straight chain or
branched C.sub.1-8 alkylidene, straight chain or branched C.sub.1-8
alkylene, C.sub.3-10 cycloalkylidene, C.sub.3-10 cycloalkylene,
phenylene, C.sub.6-14 arylalkylidene, C.sub.6-14 arylalkylene, or
deleted, and X.sup.3 is H, hydroxyl, thiol, carboxyl, amino,
halogen, (C.sub.1-6 alkyl)oxycarbonyl, (C.sub.7-14
arylalkyl)-oxycarbonyl, or C.sub.6-14 aryl; or R.sup.3 and X.sup.3
taken together are the side chain of any naturally occurring
.alpha.-amino acid; and R.sub.12 is H, C.sub.1-6 alkyl, C.sub.1-6
haloalkyl, C.sub.2-6 alkenyl, or C.sub.2-6 alkynyl; and a
pharmaceutically acceptable carrier.
[0023] A ninth aspect is a pharmaceutical composition comprising a
compound having the formula (X) ##STR9##
[0024] wherein Z.sup.1 is NH, or NR.sub.a, NR.sub.a being C.sub.1-6
alkyl; each of X.sup.1 and X.sup.2, independently, is O or S; each
of A.sup.1 and A.sup.2 is independently in the (R) or (S)
configuration, and is independently H, the side chain of any
naturally occurring amino acid, or is of the following formula,
--(CH.sub.2).sub.m--Y--(CH.sub.2).sub.n--R.sup.3X.sup.3 wherein Y
is O, S, C.dbd.O, C.dbd.S, --(CH.dbd.CH)--, vinylidene,
--C.dbd.NOR.sub.h, --C.dbd.NNR.sub.iR.sub.i', sulfonyl, methylene,
CHX.sup.4 in the (R) or (S) configuration, or deleted, X.sup.4
being halogen, methyl, halomethyl, OR.sub.h, SR.sub.h,
NR.sub.iR.sub.i', --NR.sub.i(OR.sub.h), or
--NR.sub.i(NR.sub.iR.sub.i'), wherein R.sub.h is selected from H,
C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, and
C.sub.2-6 alkynyl, and each of R.sub.i and R.sub.i', independently
is selected from H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, and C.sub.1-10 acyl; m is 0, 1, 2, or
3, and n is 0, 1, 2, or 3; and R.sup.3 is straight chain or
branched C.sub.1-8 alkylidene, straight chain or branched C.sub.1-8
alkylene, C.sub.3-10 cycloalkylidene, C.sub.3-10 cycloalkylene,
phenylene, C.sub.6-14 arylalkylidene, C.sub.6-14 arylalkylene, or
deleted, and X.sup.3 is H, hydroxyl, thiol, carboxyl, amino,
halogen, (C.sub.1-6 alkyl)oxycarbonyl, (C.sub.7-14
arylalkyl)-oxycarbonyl, or C.sub.6-14 aryl; or R.sup.3 and X.sup.3
taken together are the side chain of any naturally occurring
.alpha.-amino acid; and a pharmaceutically acceptable carrier.
[0025] Many of the compounds described above are novel compounds;
the novel compounds are also claimed. The invention also
encompasses lactacystin analogues that can be made by the synthetic
routes described herein, and methods of treating a subject having a
condition mediated by proteins processed by the proteasome by
administering (e.g., orally) to a subject an effective amount of a
pharmaceutical composition disclosed herein. These methods include
treatments for cancer, inflammation (e.g., inflammatory responses
associated with allergies, bone marrow or solid organ
transplantation, or disease states), psoriasis, and restenosis.
[0026] The compounds disclosed herein are highly selective for the
proteasome, and do not inhibit other proteases such as trypsin,
a-chymotrypsin, calpain I, calpain II, papain, and cathepsin B.
[0027] Other features or advantages of the present invention will
be apparent from the following detailed description, and also from
the appending claims.
DETAILED DESCRIPTION OF THE INVENTION
Terms
[0028] The term "naturally occurring amino acid" is meant to
include the 20 common .alpha.-amino acids (Gly, Ala, Val, Leu, Ile,
Ser, Thr, Asp, Asn, Lys, Glu, Gln, Arg, His, Phe, Cys, Trp, Tyr,
Met and Pro), and other amino acids that are natural products, such
as norleucine, ethylglycine, ornithine,
methylbutenylmethylthreonine, and phenylglycine. Examples of amino
acid side chains include H (glycine), methyl (alanine),
--(CH.sub.2--(C.dbd.O)--NH.sub.2 (asparagine), --CH.sub.2--SH
(cysteine), and --CH(OH)CH.sub.3 (threonine).
[0029] The term "inhibitor" is meant to describe a compound that
blocks or reduces the activity of an enzyme (e.g. the proteasome,
or the X/MB1 subunit of .alpha.-chain of the 20S proteasome). An
inhibitor may act with competitive, uncompetitive, or
noncompetitive inhibition. An inhibitor may bind reversibly or
irreversibly, and therefore the term includes compounds which are
suicide substrates of an enzyme. An inhibitor may modify one or
more sites on or near the active site of the enzyme, or it may
cause a conformational change elsewhere on the enzyme. Thus, some
compounds disclosed herein (e.g., where L.sup.O is an oxiranyl or
aldehyde group) react with the enzyme by bonding to the carbon atom
corresponding to C4 of lactacystin (e.g., resulting in a C4 having
a hydroxyl or thiol), while other compounds react with the enzyme
to release a leaving group (e.g., L.sup.1), corresponding to an
acylation.
[0030] An alkyl group is a branched or unbranched hydrocarbon that
may be substituted or unsubstituted. Examples of branched alkyl
groups include isopropyl, sec-butyl, isobutyl, tert-butyl,
sec-pentyl, isopentyl, tert-pentyl, isohexyl. Substituted alkyl
groups may have one, two, three or more substituents, which may be
the same or different, each replacing a hydrogen atom. Substituents
are halogen (e.g., F, Cl, Br, and I), hydroxyl, protected hydroxyl,
amino, protected amino, carboxy, protected carboxy, cyano,
methylsulfonylamino, alkoxy, acyloxy, nitro, and lower haloalkyl.
Similarly, cycloalkyl, aryl, arylalkyl, alkylaryl, heteroaryl, and
heterocyclic radical groups may be substituted with one or more of
the above substituting groups. Examples of cycloalkyl groups are
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and
cyclooctyl. An aryl group is a C.sub.6-20 aromatic ring, wherein
the ring is made of carbon atoms (e.g., C.sub.6-14, C.sub.6-10 aryl
groups, wherein C.sub.8 aryl may be an alkylaryl such as
2,3-dimethylphenyl or an arylalkyl such as phenylethyl, or an
alkylarylalkyl such as o-methyl-benzyl). Examples of haloalkyl
include fluoromethyl, dichioromethyl, trifluoromethyl,
1,1-difluoroethyl, and 2,2-dibromoethyl.
[0031] A heterocyclic radical contains at least one ring structure
which contains carbon atoms and at least one heteroatom (e.g., N,
O, S, or P). A heteroaryl is an aromatic heterocyclic radical.
Examples of heterocyclic radicals and heteroaryl groups include:
thiazolyl, thienyl, furyl, 1-isobenzofuranyl, 2H-chromen-3-yl,
2H-pyrrolyl, N-pyrrolyl, imidazolyl, pyrazolyl, isothiazolyl,
isooxazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyradazinyl,
indolizinyl, isoindolyl, indolyl, indazolyl, purinyl, phthalazinyl,
cinnolinyl, and pteridinyl. A heterocylic radical may be attached
to another moiety via a carbon atom or a heteroatom of the
heterocyclic radical.
[0032] A (C.sub.n alkyl)oxycarbonyl group has the formula
R--O--(C.dbd.O)--. (C.sub.1-6 alkyl)oxycarbonyl, therefore,
includes methoxycarbonyl and hexyloxycarbonyl. A C.sub.1-10 acyl
group as used herein is of the formula --(C.dbd.O)--L.sub.3 and
contains 1 to 10 carbon atoms (e.g., C.sub.1-6) and 1-5 heteroatoms
(at least one oxygen). Examples of such acyl groups include formyl,
acetyl, benzyloxycarbonyl, tert-butoxycarbonyl,
trifluoroethyloxycarbonyl, thiobenzoyl, phenylamidocarbonyl, and
4-nitrophenoxycarbonyl.
[0033] An alkylene is a bivalent radical derived from alkanes by
removing two hydrogens from two different carbon atoms. Examples of
alkylenes include --CH.sub.2--CH(R)--CH.sub.2 and
1,4-cyclohexylene. An alkylidene is a bivalent radical derived from
alkenes by removing two hydrogens from the same carbon atom, such
as 1-propanyl-3-ylidene (.dbd.CH--CH.sub.2--CH.sub.2--).
[0034] An aromatic carbon atom, as used herein, is a carbon atom
within an aromatic system such as benzene or naphthalene or a
heteroaromatic system such as quinoline. Examples of nonaromatic
carbon atoms include the carbons atoms in R--(C.dbd.O)--R,
--CH.sub.2Cl, --CH.sub.2-- and R--(C.dbd.O)--O--R. A fragment
formula weight is the combined atomic weight of the fragment or
moiety indicated. For example, the fragment formula weight of
methyl is 15 and the fragment formula weight of hydroxyl is 17.
[0035] A leaving group departs from a substrate with the pair of
electrons of the covalent bond between the leaving group and the
substrate; preferred leaving groups stabilize those electrons via
the presence of electron withdrawing groups, aromaticity, resonance
structures, or a combination thereof. Examples include halide (I
and Br are preferred), mesylate, trifluoromethanesulfonate,
p-toluenesulfonate, p24-nitrobenzensulfonate, benzoate,
p-nitrobenzoate, p-nitrobenzyl, and C.sub.2-5 haloalkylcarbonyl-oxy
such as trifluoroacetate.
[0036] Numerous thiol-, amino-, hydroxy- and carboxy-protecting
groups are well-known to those in the art. In general, the species
of protecting group is not critical, provided that it is stable to
the conditions of any subsequent reaction(s) on other positions of
the compound and can be removed at the appropriate point without
adversely affecting the remainder of the molecule. In addition, a
protecting group may be substituted for another after substantive
synthetic transformations are complete. Clearly, where a compound
differs from a compound disclosed herein only in that one or more
protecting groups of the disclosed compound has been substituted
with a different protecting group, that compound is within the
invention. For example, where a compound differs from a compound
disclosed herein only in that one or more hydrogens from a thiol-,
amino-, or hydroxyl-moiety (or a hydrogen or hydroxyl from a
carboxyl moiety) has been substituted with a protecting group to
form a carboxyl or sulfonyl ester, that protected compound is
within the invention. Sulfonyl esters include alkylsulfonyls (e.g.,
methylsulfonyl or mesyl) and arylsulfonyls (e.g., tosyl). Further
examples and conditions are found in T. W. Greene, Protective
Groups in Organic Chemistry, (1st ed., 1981; 2nd ed., 1991, T. W.
Greene and P. G. M. Wuts).
[0037] The invention also encompasses isotopically-labelled
counterparts of compounds disclosed herein. An
isotopically-labelled compound of the invention has one or more
atoms replaced with an isotope having a detectable particle- or
x-ray-emitting (radioactive) nucleus or a magnetogyric nucleus.
Examples of such nuclei include .sup.2H, .sup.3H, .sup.13C,
.sup.15N, .sup.19F, .sup.29Si, .sup.31P, .sup.32P and .sup.125I.
Isotopically-labelled compounds of the invention are particularly
useful as probes or research tools for spectrometric analyses,
radioimmunoassays, binding assays based on .gamma.- or
.beta.-scintillation, fluorography, autoradiography, and kinetic
studies such as inhibition studies or determination of primary and
secondary isotope effects.
[0038] The following abbreviations are used in the synthetic
description:
[0039] AIBN, 2,2'-azobis(isobutyronitrile); Bn, benzyl; BOP-Cl,
bis(2-oxo-3-oxazolidinyl)phosphinic chloride; Bu.sub.2BOTf,
dibutylboron triflate; CDI, N,N'-carbonyl-diimidazole; Cp,
cyclopentadienyl; DBU, 1,8-diazabicyclo-[5.4.0]undec-7-ene; DCC
dicyclohexylcarbodiimide; DDQ,
2,3-dichloro-5,6-dicyano-1,4-benzoquinone; DEAD,
diethylazodicarboxylate; DIBAL-H diisobutylaluminum hydride; DMF,
dimethylformamide; Gilbert reagent, dimethyl
diazomethylphosphonate; LDA, lithium diisopropylamide; LiHMDS,
lithium hexamethyldisilazamide; mesylate, methanesulfonic acid
ester; Mitsunobu reagents, (DEAD, PPh.sub.3, and nucleophile); NMO,
N-methylmorpholine-N-oxide; Ph, phenyl; PHFl, 9-phenyl-9-fluorenyl;
Ph.sub.2NTf, N-phenyltrifluoromethanesulfonimide; Swem oxidation
reagents ((COCl).sub.2, DMSO, Et.sub.3N); TBAF, tetrabutylammonium
fluoride; TBS, tert-butyldimethylsilyl; TCDI,
thiono-N,N'-carbonyldiimidazole; Tf.sub.2O,
trifluoromethane-sulfonic acid anhydride; TMS, trimethylsilyl;
triflate, trifluromethane sulfonate ester; and TsCl,
p-toluene-sulfonyl chloride.
[0040] The following reagents are also used: ##STR10##
[0041] The invention is based, in part, on the structure-function
information disclosed herein which suggests the following preferred
stereochemical relationships. Note that a preferred compound may
have one, two, three, or more stereocenters having the indicated
up-down (or .beta.-.alpha. where .beta. as drawn herein is above
the plane of the page) or (R)-(S) relationship. In some
embodiments, preferred configurations are 7(R), 9(S), 6(S), or
combinations thereof. In lactacystin analogs, R.sup.2 and L.sup.0
preferably have a syn relationship to allow formation of a lactone
or lactam-type intermediate, which is believed to be related to the
activity of the compounds disclosed herein as proteasome
inhibitors. However, it is not required that every stereocenter
conform to the structures below. ##STR11##
[0042] A person of skill will recognize that the compounds
described herein preserve certain stereochemical and electronic
characteristics found in either lactacystin or lactacystin
.beta.-lactone. For example, the hydroxyisobutyl group and the
configuration of the hydroxyl group on C9 are believed to be
important for recognition of the target, as are the configurations
of the C6 hydroxyl and the C7 methyl of the .gamma.-lactam ring.
However, the N-acetylcysteine moiety is not required for activity.
Moieties such as R.sup.2, R.sup.1, and particularly A.sup.1 (e.g.,
where A.sup.1 is a side chain of a naturally occurring
.alpha.-amino acid such as Val, Leu, Lys, and Phe) can be modified
to control selectivity for the proteasome, and selectivity for a
particular peptidase activity of the proteasome. In combination,
these moieties simulate certain peptides or proteins processed or
degraded by the proteasome (i.e., are peptidomimetics).
[0043] The invention is also based, in part, on the finding that
lactacystin and lactacystin .beta.-lactone are highly selective for
the X/MB1 subunit and .alpha.-chain of the proteasome and do not
inhibit the activity of proteases such as trypsin,
.alpha.-chymotrypsin, calpain I, calpain II, cathepsin, and papain.
Such selectivity is useful to formulate a pharmaceutical
composition with fewer side effects and to evaluate basic research
results involving any subunit of the proteasome in conjunction with
the selective inhibition of the X/MB1 subunit and
.alpha.-chain.
[0044] Turning to the novel compounds described by the formulas of
compounds contained within the pharmaceutical compositions, several
embodiments are next considered.
[0045] Embodiments of the first aspect include compounds having one
or more of the following limitations. In one limitation L.sup.1 is
linked by an oxygen or preferably sulfur atom to the carbon atom
bonded to X.sup.2. Such an L.sup.1 forms a thioester, thus
providing a good leaving group. The precise nature of the thioester
group is generally not critical. In initial experiments, the
magnitude of activity measured by K.sub.obs
[0046] /[I] for both large and small thioesters was about the same,
whether the thioester terminus was a simple arylalkyl group or a
more complex, branched moiety with intervening amide linkages and
terminal carboxylic acid, esters, or aminocarbonyl groups. In one
embodiment, Z.sup.1 is NH or NR.sub.a; X.sup.1 is O or S; Z.sup.2
is CHR.sup.1 or CHOR.sup.1, R.sup.1 being H, halogen, C.sub.1-6
alkyl, or C.sub.1-6 haloalkyl; R.sup.2 is OR.sub.f; and L.sup.0 is
--(C.dbd.X.sup.2)-L.sup.1, X.sup.2 being O and L.sup.1 being linked
by a sulfur atom to the carbon atom bonded to X.sup.2. In another
embodiment, L.sup.1 is selected from substituted or unsubstituted
C.sub.6-20 arylalkylcarbonylthio, C.sub.6-20 arylthio, C.sub.2-8
alkylcarbonylthio, and C.sub.1-12 alkylthio. As defined above in
the "Terms" section, arylthio includes arylthio, alkylarylthio,
arylalkylthio, and alkylarylalkylthio groups.
[0047] In other limitations, A.sup.1 is C.sub.5-20 alkyl when
L.sup.O is carboxyl or (C.sub.1-4 alkyl)oxycarbonyl and A.sup.1 is
alkyl; only one of A.sup.1 and L.sup.O is selected from carboxyl
and (C.sub.1-4 alkyl)oxycarbonyl; A.sup.1 cannot be H; L.sup.1 has
at least 3 carbon atoms when Z.sup.1 and Z.sup.2 are both NH, and
A.sup.1 is methoxy; when R.sup.1 and R.sup.2 are taken together,
Z.sup.2 is CR.sup.1; and when one of A.sup.1 and L.sup.O is
(C.sub.1-4 alkyl)oxycarbonyl or carboxyl, the other of A.sup.1 and
L.sup.O has a fragment formula weight of at least 20. Novel
compounds of the first, fifth, and seventh aspects generally do not
have A.sup.1 being H.
[0048] Further embodiments of the first aspect, when Z.sup.1 is NH
and Z.sup.2 is (R) CHR.sup.1, are compounds wherein: L.sup.1 has
between 0 and 3 nonaromatic acyclic carbon atoms when there are 5
heteroatoms; L.sup.1 has between 6 and 15 nonaromatic acyclic
carbon atoms when there is only one oxygen atom; and L.sup.1 has 0,
1, 3, or 5 nonaromatic carbon atoms when there are three halogen
atoms.
[0049] Further embodiments of the first aspect include a compound
wherein Z.sup.2 is CHR.sup.1 and Z.sup.1 is NH or NR.sup.a; wherein
L.sup.O is C.sub.2-16 oxiranyl; L.sup.1 is hydroxy, C.sub.1-12
alkoxy, C.sub.1-12 alkyl-sulfonyloxy, C.sub.6-20 arylsulfonyloxy,
C.sub.7-20 arylalkyl, C.sub.6-20 aryloxy, C.sub.6-20
arylalkylcarbonyloxy, C.sub.2-8 alkyl-carbonyloxy, C.sub.2-8
alkylcarbonylthio, C.sub.1-12 alkylthio, C.sub.6-20
arylalkylcarbonylthio, or C.sub.6-20 arylthio; or L.sup.2 is H,
C.sub.1-2 haloalkyl, or C.sub.1-6 alkyl. In addition, Z.sup.2 is O,
S, NH, or NR.sub.d. In another embodiment, Z.sup.2 is O, S, NHJ,
NR.sub.d, or CHR.sup.1; and R.sub.h is H, C.sub.1-6 alkyl,
C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, or C.sub.2-6 alkynyl.
[0050] Further embodiments of the first aspect include compounds
wherein Z.sup.1 is O, S, or SO.sub.2; wherein Z.sup.2 is O, S, NH,
or NR.sub.d; wherein X.sup.1 is CH.sub.2, CHR.sup.b, or
C(R.sup.b)(R.sup.c); wherein R.sup.2 is C.sub.1-6 alkyl, C.sub.1-6
haloalkyl, C.sub.2-6 alkenyl, or halogen; wherein R.sub.2 is
OR.sub.f, SR.sub.f, or NR.sub.fR.sub.g; wherein Z.sub.2 is in the
beta (above plane of page) configuration; wherein CHX.sup.4 is in
the alpha (below plane of paper) configuration; wherein R.sup.2 is
in the beta configuration; or combinations thereof.
[0051] The invention also provides a pharmaceutical composition
wherein L.sup.O is H or --(C.dbd.X.sup.2)-L.sup.1, X.sup.2 being O
and L.sup.1 being linked by an oxygen or sulfur atom to the carbon
atom bonded to X.sup.2; where only one of A.sup.1 and L.sup.O is
selected from carboxyl and (C.sub.1-4 alkyl)oxycarbonyl; wherein
L.sup.1 is hydroxy, C.sub.1-12 alkoxy, C.sub.1-12 alkylsulfonyloxy,
C.sub.6-20 arylsulfonyloxy, C.sub.7-20 arylalkyl, C.sub.6-20
aryloxy, C.sub.6-20 arylalkylcarbonyloxy, C.sub.2-8
alkylcarbonyloxy, C.sub.6-20 arylalkylcarbonylthio, C.sub.6-20
arylthio, C.sub.2-8 alkyl-carbonylthio, or C.sub.1-12
alkylthio.
[0052] The invention also provides embodiments wherein Z.sup.1 is
NH or NR.sub.a; X.sup.1 is O or S; Z.sup.2 is CHR.sup.1 or
CHOR.sup.1, R.sup.1 being H, halogen, C.sub.1-6 alkyl, or C.sub.1-6
haloalkyl; R.sup.2 is OR.sub.f; and L.sup.0 is
--(C.dbd.X.sup.2)-L.sup.1, X.sup.2 being O and L.sup.1 being linked
by a sulfur atom to the carbon atom bonded to X.sup.2. Individual
compounds include lactacystin, clasto-lactacystin trifluoroethyl
ester, descarboxylactacystin, lactacystin amide, and phenylacetyl
lactacystin.
[0053] Embodiments of the second aspect include compounds wherein
CHX.sup.4 is in the (S) configuration when X.sup.4 is hydroxyl and
--(CH.sub.2).sub.n--R.sup.3X.sup.3 is isopropyl; wherein the moiety
--(CH.sub.2).sub.n--R.sup.3X.sup.3 has between 5 and 20 carbon
atoms when X.sup.4 is hydroxyl, m is 0, and Z.sup.1 is NH; wherein
Z.sup.1 is NH or NR.sub.a; wherein Z.sup.1 is O, S, or SO.sub.2;
wherein Z.sup.1 is NR.sup.a; wherein X.sup.1 is CH.sub.2,
CH(R.sub.b), or C(R.sub.b)(R.sub.c); wherein Z.sup.2 is in the beta
(above plane of page) configuration; wherein CHX.sup.4 is in the
alpha (below plane of paper) configuration; wherein R.sup.1 is H,
C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, or halogen; wherein R.sup.1 is a side chain of a naturally
occurring .alpha.-amino acid, OR.sub.d, SR.sub.d, or
NR.sub.dR.sub.e; or combinations of the above.
[0054] Another embodiment of the second aspect includes compounds
wherein Z.sup.1 is NH or NR.sub.a; X.sup.1 is O or S; Z.sup.2 is
CHR.sup.1 in the (R) or (S) configuration, wherein R.sup.1 is H,
C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, hydroxyl, halogen, a side chain of a naturally occurring
amino acid, OR.sub.d, SR.sub.d, or NR.sub.dR.sub.e (each of R.sub.d
and R.sub.e, independently, being H, C.sub.1-6 alkyl, C.sub.1-6
haloalkyl, C.sub.2-6 alkenyl, or C.sub.2-5 alkynyl); Z.sup.3 is O
or NH; X.sup.2 is O or S; and A.sup.1 is the side chain is any
naturally occurring .alpha.-amino acid, or is of the following
formula, --Y--(CH.sub.2).sub.n--R.sup.3X.sup.3 wherein Y is C.dbd.O
or CHX.sup.4 in the (R) or (S) configuration, or deleted, X.sup.4
being halogen, methyl, halomethyl, or OR.sub.h, wherein R.sub.h is
selected from H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, C.sub.1-10 acyl, C.sub.1-6
alkylsulfonyl, and C.sub.6-10 arylsulfonyl; n is 0, 1, 2, or 3; and
R.sup.3 is straight chain or branched C.sub.1-8 alkylene,
C.sub.3-10 cycloalkylene, phenylene, C.sub.6-14 arylalkylene, or
deleted; and X.sup.3 is H, hydroxyl, thiol, carboxyl, amino,
halogen, or C.sub.6-10 aryl; or R.sup.3 and X.sup.3 taken together
are the side chain of any naturally occurring .alpha.-amino
acid.
[0055] The invention also provides a compound wherein Z.sup.1 is NH
and Z.sup.3 is O; or a compound wherein each of Z.sup.1 and Z.sup.3
is NH; or a compound wherein at least four (e.g., at least 5, or
all) of the following limitations apply: Z.sup.1 is NH or NR.sub.a;
X.sup.1 is O or S; Z.sup.2 is CHR.sup.1 in the (R) or (S)
configuration, wherein R.sup.1 is H, C.sub.1-6 alkyl, C.sub.1-6
haloalkyl, hydroxyl, or OR.sub.d, R.sub.d being C.sub.1-6 alkyl;
Z.sup.3 is O or NH; X.sup.2 is O; Z.sup.3 is O or NH; and A.sup.1
is the side chain of any naturally occurring .alpha.-amino acid, or
CHX.sup.4, X.sup.4 being halogen, methyl, halomethyl, or OR.sub.h,
R.sub.h being selected from H, C.sub.1-6 alkyl, C.sub.1-6
haloalkyl, C.sub.1-10 acyl, C.sub.1-6 alkylsulfonyl, and C.sub.6-10
arylsulfonyl; R.sup.3 is straight chain or branched C.sub.1-8
alkylene, C.sub.3-10 cycloalkylene, phenylene, C.sub.6-14
arylalkylene, or deleted; m is 0, 1, or 2 and n is 0 or 1; and
X.sup.3 is H.
[0056] The invention also provides a pharmaceutical composition
wherein Z.sup.1 is NH or NR.sub.a; X.sup.1 is O or S; Z.sup.2 is
CHR.sup.1 in the (R) or (S) configuration, wherein R.sup.1 is H,
C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, hydroxyl, halogen, a side chain of a naturally occuring
.alpha.-amino acid, OR.sub.d, SR.sub.d, or NR.sub.dR.sub.e (each of
R.sub.d and R.sub.e, independently, being H, C.sub.1-6 alkyl,
C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, or C.sub.2-5 alkynyl);
Z.sup.3 is O or NH; X.sup.2 is O or S; and A.sup.1 is the side any
naturally occurring a-amino acid, or is of the following formula,
--Y--(CH.sub.2).sub.n--R.sup.3X.sup.3 wherein Y is C.dbd.O or
CHX.sup.4 in the (R) or (S) configuration, or deleted, X.sup.4
being halogen, methyl, halomethyl, or OR.sub.h, wherein R.sub.h is
selected from H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, C.sub.1-10 acyl, C.sub.1-6
alkylsulfonyl, and C.sub.6-10 arylsulfonyl; n is 0, 1, 2, or 3; and
R.sup.3 is straight chain or branched C.sub.1-8 alkylene,
C.sub.3-10 cycloalkylene, phenylene, C.sub.6-14 arylalkylene, or
deleted; and X.sup.3 is H, hydroxyl, thiol, carboxyl, amino,
halogen, or C.sub.6-10 aryl; or R.sup.3 and X.sup.3 taken together
are the side chain of any naturally occurring .alpha.-amino
acid.
[0057] Another embodiment includes a compound wherein X.sup.2 is O,
and A.sup.1 is the side chain of any naturally occurring
.alpha.-amino acid, or X.sup.4 is halogen, methyl, halomethyl, or
OR.sup.h, R.sup.h being selected from H, C.sub.1-6 alkyl, C.sub.1-6
haloalkyl, C.sub.1-10 acyl, C.sub.1-6 alkylsulfonyl, and C.sub.6-10
arylsulfonyl; R.sup.3 is straight chain or branched C.sub.1-8
alkylene, C.sub.3-10 cycloalkylene, phenylene, C.sub.6-14
arylalkylene, or deleted; m is 0, 1, or 2 and n is 0 or 1; and
X.sup.3 is H. Individual lactone analogs include clasto-lactacystin
.beta.-lactone.
[0058] Embodiments of the third aspect include compounds wherein:
when X.sup.1 is 2 singly bonded H and R.sup.1 has only one oxygen,
then the fragment formula weight of R.sup.1 is at least 30 atomic
mass units; when X.sup.1 is 2 singly bonded H and R.sup.1 is 2 H,
and A.sup.1 is alkyl, then A.sup.1 has at least 3 carbon atoms;
A.sup.1, R.sup.1 and X.sup.1 taken together have at least one
carbon atom, halogen, or heteratom; when X.sup.1 is two singly
bonded H, and R.sup.1 is H, X.sup.2 is O, and R.sub.h is alkyl,
then R.sub.h is C.sub.4-6 alkyl; and when n is 2, R.sub.h is
C.sub.1-6 haloalkyl.
[0059] Further embodiments of the third aspect include compounds
wherein: X.sup.1 is CH.sub.2, two singly bonded H, CH(R.sub.b) or
C(R.sub.b)(R.sub.c); Z.sup.2 is O or S; Z.sup.2 is NH or NR.sub.j;
R.sup.1 is C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, or halogen; R.sup.1 is a side chain of a
naturally occuring .alpha.-amino acid, OR.sub.d, SR.sub.d, or
NR.sub.dR.sub.e; A.sup.1 cannot be H; R.sup.1 is in the beta
configuration; X.sup.4 is in the alpha configuration; or
combinations thereof.
[0060] Embodiments of the fourth aspect include compounds wherein:
Z.sup.2 is O or S; Z.sup.2 is NH or NR.sub.j; X.sup.1 is O or S;
R.sup.1 is C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, or halogen; R.sup.1 is a side chain of a
naturally occurring .alpha.-amino acid, OR.sub.d, SR.sub.d, or
NR.sub.dR.sub.e; R.sup.2 is H, C.sub.1-3 alkyl, C.sub.1-3
haloalkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl, C.sub.6-12 aryl,
C.sub.3-8 heteroaryl, or halogen; R.sup.2 is H, C.sub.3-6 alkyl,
C.sub.3-6 haloalkyl, C.sub.4-6 alkenyl, C.sub.4-6 alkynyl,
C.sub.6-12 aryl, or C.sub.3-8 heteroaryl; A.sup.1 cannot be H;
R.sup.1 is in the beta configuration; X.sup.4 is in the alpha
configuration; or combinations of the above.
[0061] Further embodiments of the fifth aspect are compounds
wherein: Z.sup.1 is O or S; Z.sup.2 is NH or NR.sub.j; R.sup.1 is
H, C.sub.1-3 alkyl, C.sub.1-3 haloalkyl, C.sub.2-3 alkenyl,
C.sub.2-3 alkynyl, or a side chain of a naturally occuring
.alpha.-amino acid, OR.sub.d, SR.sub.d, or NR.sub.dR.sub.e; R.sup.1
is H, C.sub.4-6 alkyl, C.sub.4-6 haloalkyl, C.sub.4-6 alkenyl,
C.sub.4-6 alkynyl, halogen, or a side chain of a naturally occuring
.alpha.-amino acid, OR.sub.d, SR.sub.d, or NR.sub.dR.sub.e; R.sup.2
is H, C.sub.1-3 alkyl, C.sub.1-3 halo-alkyl, C.sub.2-3 alkenyl,
C.sub.2-3 alkynyl, C.sub.6-12 aryl, C.sub.3-8 heteroaryl, or
halogen; R.sup.2 is H, C.sub.4-6 alkyl, C.sub.4-6 haloalkyl,
C.sub.4-6 alkenyl, C.sub.4-6 alkynyl, C.sub.6-12 aryl, or C.sub.3-8
heteroaryl;n R.sub.a is C.sub.1-3 alkyl; A.sup.1 cannot be H;
X.sup.4 is in the alpha configuration; or combinations thereof.
[0062] Embodiments of the sixth aspect are compounds wherein:
X.sup.1 is O or S; X.sup.1 is CH.sub.2, two singly bonded H,
CH(R.sub.b), or C(R.sub.b)(R.sub.c); Z.sup.1 is O or S; Z.sup.1 is
NH or NR.sub.a; R.sup.1 is C.sub.1-3 alkyl, C.sub.1-3 haloalkyl,
C.sub.2-3 alkenyl, C.sub.2-3 alkynyl, halogen, or a side chain of
any naturally occuring .alpha.-amino acid; R.sup.1 is OR.sub.d,
SR.sub.d, or NR.sub.dR.sub.e; R.sup.1 is C.sub.4-6 alkyl, C.sub.4-6
haloalkyl, C4-6 alkenyl, C.sub.4-6 alkynyl, a side chain of any
naturally occuring .alpha.-amino acid, OR.sub.d, SR.sub.d, or
NR.sub.dR.sub.e; R.sup.1 and R.sup.2 taken together are a bivalent
moiety; R.sup.1 is in the beta configuration; R.sup.2 is in the
beta configuration; R.sup.2 is C.sub.1-3 alkyl, C.sub.1-3
haloalkyl, C.sub.6-12 aryl, C.sub.3-8 cycloalkyl, or halogen;
R.sup.2 is C.sub.4-6 alkyl, C.sub.4-6 haloalkyl, C.sub.6-12 aryl,
C.sub.3-8 cycloalkyl, C.sub.3-8 heteroaryl, or C.sub.3-8
heterocyclic radical; A.sup.1 has a higher fragment formula weight
than A.sup.2; A.sup.1 has a lower fragment formula weight than
A.sup.2; A.sup.1 is in the (R) configuration; A.sup.1 is in the (S)
configuration; only one of A.sup.1 and A.sup.2 is H; A.sup.1 is a
side chain of any naturally occuring .alpha.-amino acid; or
combinations thereof.
[0063] Embodiments of the seventh aspect are compounds wherein:
X.sup.1 is O or S; X.sup.1 is CH.sub.2, two singly bonded H,
CH(R.sub.b) or C(R.sub.b)(R.sub.c); Z.sup.2 is O or S; Z.sup.2 is
NH or NR.sub.j; R.sup.1 is C.sub.1-3 alkyl, C.sub.1-3 haloalkyl,
C.sub.2-3 alkenyl, C.sub.2-3 alkynyl, hydroxyl, halogen, or a side
chain of any naturally occuring .alpha.-amino acid; R.sup.1 is
OR.sub.d, SR.sub.d, or NR.sub.dR.sub.e; R.sup.1 is C.sub.4-6 alkyl,
C.sub.4-6 haloalkyl, C.sub.4-6 alkenyl, C.sub.4-6 alkynyl, a side
chain of any naturally occuring .alpha.-amino acid; R.sup.1 and
R.sup.2 taken together are a bivalent moiety; R.sup.2 is C.sub.1-3
alkyl, C.sub.1-3 haloalkyl, C.sub.2-3 alkenyl, azido, C.sub.2-3
alkynyl, hydroxyl, or halogen; R.sup.2 is OR.sub.f, SR.sub.f,
NR.sub.fR.sub.fR.sub.g, --ONR.sub.fR.sub.g, --NR.sub.g(OR.sub.f),
or --NR.sub.g(RS.sub.f); R.sup.2is C.sub.4-6 alkyl, C.sub.4-6
haloalkyl, C.sub.4-6 alkenyl, azido, C.sub.4-6 alkynyl, OR.sub.f,
SR.sub.f, NR.sub.fR.sub.fR.sub.g, --ONR.sub.fR.sub.g,
--NR.sub.g(OR.sub.f), or --NR.sub.g(RS.sub.f); R.sup.1 and R.sup.2
taken together are a bivalent moiety; R.sup.1 is in the alpha
configuration; R.sup.1 is in the beta configuration; R.sup.2 is in
the alpha configuration; R.sup.2 is in the beta configuration;
A.sup.1 is in the alpha configuration; A.sup.2 is in the beta
configuration; or combinations thereof.
[0064] One embodiment of the eighth aspect is a compound where
A.sup.1, A.sup.2, R.sup.12, and X.sup.1 taken together have at
least one carbon atom and one heteratom. Further embodiments of the
eighth aspect are compounds wherein: Z.sup.1 is NH, or NR.sub.a;
X.sup.1 is O or S; X.sup.1 is CH.sub.2, or two singly bonded H;
R.sup.12is H, C.sub.1-3 alkyl, C.sub.1-3 haloalkyl, C.sub.2-3
alkenyl, or C.sub.2-3 alkynyl; R.sup.12 is C.sub.4-6 alkyl,
C.sub.4-6 haloalkyl, C.sub.4-6 alkenyl, or C.sub.4-6 alkynyl; where
A.sup.1, A.sup.2, R.sup.12, and X.sup.1 taken together have at
least one carbon atom and one heteratom; where the fragment formula
weight of A.sup.1 is greater than the fragment formula weight of
A.sup.2 (e.g., by at least 30 or 60); where the fragment formula
weight of A.sup.2 is greater than the fragment formula weight of
A.sup.1 (e.g., by at least 15 or 50); or combinations thereof.
[0065] Embodiments of the ninth aspect are compounds wherein
A.sup.1 is a side chain of any naturally occurring .alpha.-amino
acid; wherein A.sup.1 has a fragment formula weight of at least 50
(e.g., 70, 100, or 120).
[0066] Examples of individual compounds described herein are
provided in Tables A, B, and C. TABLE-US-00001 TABLE A ##STR12##
##STR13## ##STR14## ##STR15## ##STR16## ##STR17## ##STR18##
##STR19## ##STR20## ##STR21## ##STR22##
[0067] TABLE-US-00002 TABLE B ##STR23## ##STR24## ##STR25##
##STR26## ##STR27## ##STR28## ##STR29## ##STR30##
[0068] TABLE-US-00003 TABLE C ##STR31## ##STR32## ##STR33##
##STR34## ##STR35## ##STR36## ##STR37##
Synthesis
[0069] The syntheses disclosed herein are organized by structure
groups. A person of skill will recognize that the claimed compounds
are related to the compounds in the compositions claimed. The
synthesis of the compounds of formula (I) rely primarily on the
results reported by Uno, et al. in their enantiospecific synthesis
of (+)-lactacystin from (R)-glutamate [Uno, et al., J. Am. Chem.
Soc. (1994) 116:2139]. Note that in all schemes relating to
compounds of formula (I), when a straight line is used to connect
A.sup.1 (or anything corresponding to A.sup.1) to the rest of the
molecule, a dashed line should be assumed. The straight line is
used simply for clarity. All A.sup.1 are attached to the rest of
the molecule on the "alpha," i.e., the bottom, face.) Compound A-1
(Scheme 1) serves as the starting material for all synthetic routes
proposed for compounds of formula (I) wherein Z.sup.2 is CHR.sup.1
and Z.sup.1 is NH or NR.sub.a for example. Thus, conversion of A-1
to A-2, in analogy to the above-mentioned work allows for
introduction of R.sup.1 via standard alkylation chemistry. In the
cases where R.sup.1 is hydroxyl, halide, --SR.sub.d, or
--NR.sub.dR.sub.e, wherein R.sub.d and R.sub.e each, independently,
is selected from H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, the electrophile in step b is,
respectively, a N-arylsulfonyl-3-phenyloxaziridine [Davis, F. A. et
al., J. Org. Chem. (1984), 49:3241], a N-halosuccinimide [Stotter,
P. A. et al., J. Org. Chem. (1973) 38:2576], R.sub.dS--SR.sub.d or
elemental sulfur [Zoretic, P. A. et al., J. Org. Chem. (1976)
41:3587] [Gassman, P. G. et al., J. Org. Chem. (1977) 42:3236], a
N-arylsulfonylazide (followed by reduction of the azide to the
primary amine and elaboration to NR.sub.dR.sub.e) [Evans, D. A. et
al., J. Am. Chem. (1987) 109:6881]. If R.sup.1 is labile or
reactive, or both, a protecting group would be introduced at this
point and removed at the end of the synthesis. The preparation of
compounds of type A-2 is then completed with the two-step (steps b
and c) process reported by Uno [Uno, et al., (1994)].
[0070] Introduction of A.sup.1 is the next task. Uno [Uno, et al.,
(1994)] showed that under Lewis-acid mediated aldol addition
conditions, A.sup.1 is introduced with very high facial selectivity
with respect to the bicyclic aminoacid derivative A-2. Accordingly,
conversion of A-2 to A-3, as described by Uno, followed by
treatment of A-3 with SnCl.sub.4 and an aldehyde (R.sub.fCHO, where
R.sub.f corresponds to H or to the rest of A.sup.1), in
diethylether yields aldol products A-4 (or A-5 if R.sub.f is H),
following suitable protection of the hydroxyl group with a group
designated here as P.sub.o. [Greene and Wuts, et al., Protective
Groups in Organic Synthesis, 2nd ed., (1991)]. ##STR38##
[0071] Introduction of R.sup.2 by a stereoselective, conjugate
nucleophilic addition to the unsaturated carbonyl system of A-4 or
A-5, followed by quenching of the resulting enolate with acidic
water gives access to compounds A-6 and A-7, respectively. This
process is analogous to the "three-component coupling" strategy for
prostaglandin synthesis of [Suzuki, et al. (1988)]. In this case,
the three components are A-4 (or A-5), R.sup.2, and a proton.
[0072] The diastereomers shown for A-6 and A-7 are predicted to be
the major ones based on several facts. The Uno synthesis [Uno, H.,
et al., (1994)] used an OsO.sub.4-catalyzed dihydroxylation of a
compound similar to A-4 (R.sup.1=methyl, R.sub.f=iso-propyl) to
introduce a hydroxyl group corresponding to R.sup.2. This reaction
proceeded with complete facial selectivity. Thus, in general,
groups with appropriate reactivity (nucleophiles or electrophiles)
similarly approach preferentially the "beta" face (from the top) of
A-4 or A-5.
[0073] Quenching of the enolate with acidic water proceeds
stereoselectively. In Suzuki's three-component coupling strategy,
[Suzuki, et al., (1988)] the nucleophile and electrophile are
introduced trans to each other. In our case, R.sup.2 and the proton
are introduced trans to each other. In the event that compounds in
which R.sup.1 and R.sup.2 are trans to each other are desired, as
depicted in compounds A-12 and A-13, a similar strategy has been
devised.
[0074] The basic strategy has one exception to that detailed above:
The order of introduction of R.sup.1 and proton at the carbon alpha
to the carbonyl is reversed. Thus, conversion of A-1 to A-9 is
carried out using the same procedures as those for the preparation
of A-3, omitting the alkylation step (step a). Elaboration of A-9
to A-10 and A-11 utilizes the same conditions as those which afford
A-4 and A-5. Addition of R.sup.2 still occurs from the top face,
but the quenching step with an electrophile corresponding to
R.sup.1 now gives a trans relationship of R.sup.1 and R.sup.2, as
shown in compounds A-12 and A-13.
[0075] Another advantage of these approaches to A-6, A-7, A-12, and
A-13 is as follows. Should the quenching step introduce the
electrophile (proton or R.sup.1) cis to R.sup.2 (opposite that
predicted above), then one still has stereospecific routes to the
desired compounds, except that A-4 and A-5 give A-12 and A-13,
respectively, and A-10 and A-11 give A-6 and A-7.
[0076] Compounds in which A.sup.1 is H are prepared by a different
strategy (Scheme 2). For example, it has been shown that when
A.sup.1 is H, nucleophiles [Hanessian, et al., Synlett (1991)
10:222] and electrophiles [Griffart-Brunet, et al., Tet. Lett.
(1991) 35:119] approach from the bottom face, cis to the hydrogen
at A.sup.1. Thus, the key to our strategy above is that the
stereoselectivity of addition is reversed when A.sup.1 is not H.
The rapid preparation described below of compounds in which A.sup.1
is H takes advantage of this reversal of stereoselectivity.
[0077] Thus, bromination of A-2 by standard methods [Caine, et al.,
J. Org. Chem. (1985) 50:2195] gives A-14, which is utilized in
conjugate addition-elimination procedures to give compounds of type
A-15. Since A.sup.1 is H, catalytic hydrogenation of the olefin
should install R.sup.1 and R.sup.2 in the desired cis configuration
shown in compound A-16. Cleavage of the N,O-aminal may occur under
such conditions, giving compound A-17. Such an occurrence is not
disadvantageous since such a deprotection is the next step in the
synthesis anyway. Compounds A-18 and A-19 are accessed by
base-catalyzed epimerization of A-16 and A-17, respectively.
##STR39##
[0078] The above strategies allow for the preparation of all
compounds of formula I wherein Z.sup.2 is CHR.sup.1 and Z.sup.1 is
NH or NR.sub.a except compounds where an additional ring is fused
to the lactam ring (specifically, compounds in which R.sup.1 is
taken together with R.sup.2, giving a bivalent moiety which forms a
C.sub.3-8 cycloalkyl, C.sub.3-8 heteroaryl, C.sub.3-8 heterocyclic
radical, or C.sub.6-12 aryl). Their preparation will be discussed
below (Schemes 3 and 4). Note that while only carbocyclic systems
are depicted in Schemes 3 and 4, both the alicyclic and
heterocyclic (aromatic and non-aromatic in both cases) classes of
compounds can be prepared in this manner by choice of the
appropriate reagents. Further, all substitution patterns on these
rings that are normally accessible by such processes are accessible
by the methods proposed below. The simplest carbocyclic variants
are shown for clarity and to illustrate the general strategy to
these compounds.
[0079] The synthesis of these compounds relies on the diverse
chemistry of cycloaddition [Carruthers, et al., Cycloaddition
Reactions in Organic Synthesis (1990) and J. March, Advanced
Organic Chemistry, (1992) pp. 826-877] to alpha-beta unsaturated
olefins. Thus, as shown in Scheme 3, [1+2] processes (e.g.
diazoinsertion, cyclopropanation, epoxidation, and aziridination)
with compound A-20 (see Scheme 1 for preparation of compounds of
this type, e.g. A-10 and A-11) gives access to compounds of type
A-21. Compounds of type A-22 are prepared with a variety of [2+2]
cycloaddition processes (e.g. photocycloadditions and ketene
additions). 1,3-Dipolar cycloaddition chemistry ([3+2] cycloaddtion
reagents, such as nitrile oxides and azomethine ylides) prepares a
wide variety compounds of the type A-23. The preparation of
compounds of type A-24 is perhaps the most flexible and
far-reaching of the cycloaddition chemistry ##STR40## ##STR41##
because it relies on the Diels-Alder reaction, one of the most
utilized and studied reactions in all of organic chemistry. The
variety of compounds of type A-24 that are prepared by this method
is far too great to detail; [Carruthers, (1990)] however, notably,
bridged compounds (type A-25) are prepared by this method. [5+2]
cycloadditions to give A-26 are less common, but some intriguing
examples have been reported. [Wender, et al., J. Org. Chem. (1991)
56:6267 and Wender, et al., Tet. Lett. (1992) 32:6115].
[0080] While all these procedures introduce R.sup.1 and R.sup.2 cis
to each other, base-catalyzed epimerization in the cases of A-24
and A-26 gives access to the trans-fused versions of these
compounds, A-28 and A-29, respectively. (A-21, A-22, A-23, and A-25
can not be epimerized in this fashion, as these trans-fused rings
suffer from much more ring strain than do the cis-fused
systems.)
[0081] Cycloheptyl (A-26) and cyclooctyl (A-27) compounds are
prepared with a slightly different process, one that is akin to
that shown in Scheme 1. Fundamentally, one adds a nucleophile
(abbreviated as "Nu") to A-20 which also has a leaving group
(abbreviated as "LG") attached to the other end of the incoming
nucleophile, separated by the necessary number of atoms (5 in the
case of A-26, 6 in the case of A-27). Thus, following the addition
of the nucleophile, the resulting enolate displaces the leaving
group, forming the ring. Alternatively, such a process is done
stepwise in order to minimize undesired intramolecular side
reactions of the nucleophile and electrophile. These and other
methods for making cycloheptyl and cyclooctyl compounds from
alpha-beta unsaturated carbonyl compounds have been reviewed in the
chemical literature. [Petasis, N. A., et al., Tetrahedron (1992)
48:5757].
[0082] Aryl and heteroaryl compounds (e.g. compounds A-30 and A-31,
Scheme 4) are prepared by [4+2] cycloaddition chemistry, followed
by oxidation of the resultant cycloalkene or heterocycloalkene with
dichlorodicyanoquinone (DDQ). [Pizey, N., Synth. Reagents (1977)
3:193-225]. In the case of compounds of type A-31, other types of
dienes that are utilized are the hydroxyorthoxylylenes, formed by
the ring opening reaction of hydroxybenzocyclobutenes. [Arnold, B.
J., et al., J. Chem. Soc., (1974) 409-415].
[0083] Thus, having prepared all the variations of R.sup.1 and
R.sup.2, we turn now to the elaboration of these compounds to
include all of the variations of A.sup.1 (Scheme 5).
[0084] Based on the results of [Uno, et al., (1994)] and as shown
in Scheme 1, Lewis-acid catalyzed aldol additions of A-3 to
R.sub.fCHO, followed by further elaboration, provide compounds of
the type A-33 (Scheme 5) with the stereocenters in the
configurations shown, including both configurations of R.sup.1. (A
straight bond (i.e. neither bold or dashed) is shown here to
represent both diastereomers at this position.) Here we will focus
on the stereochemistry of the secondary hydroxyl that is included
in A.sup.1, and related derivatives. While modification of
conditions in the aldol addition (Scheme 1) reaction can provide
the other diastereomer at this position, another method of
inverting this center is to first oxidize to the ketone under
standard conditions (e.g. Swern, Dess-Martin periodinane, etc.) and
then reduce with the appropriate reducing agent (e.g. NaBH.sub.4,
etc.) whose identity is determined by screening a number of
reductants. This two-step procedure provides compounds of type
A-35. Analogously, reductive amination of ketone A-34 with an amine
or hydroxylamine (represented in Scheme 5 as R.sub.nNH.sub.2) and
sodium cyanoborohydride (NaCNBH.sub.3) provides compounds of type
##STR42## ##STR43## ##STR44## A-36. The sense of stereochemical
induction in this step can be altered by using a different reducing
agent. Alternatively, by first preparing the methanesulfonate ester
("mesylate") of A-33 or A-35, and then displacing the mesylate with
a nucleophile (e.g. RNH.sub.2, RS--, RO--, halide, hydroxylamine,
Grignard reagents, etc.) one obtains a wide variety of Al
derivatives with complete control of stereochemistry at the carbon
corresponding to the secondary hydroxyl in A-33. Reduction of the
mesylate (with lithium triethylborohydride (LiEt.sub.3BH)) or of a
halide (with, e.g., zinc in hydrochloric acid) provides compounds
of the type A-38.
[0085] Another method to produce the various A.sup.1 derivatives is
depicted in Scheme 6. Compounds of the type A-39, which are
prepared according to Scheme 1, are converted to those of type A-40
where X.sub.a is a halide or trifluoromethanesulfonate ester
("triflate"), for example, using standard functional group
manipulation. Displacement of these leaving groups with
nucleophiles gives another method for the preparation of compounds
of type A-38. Subjecting iodide A-40 (X.sub.a is I) to
metal-halogen exchange conditions (e.g. activated Mg metal or
tert-butyllithium) provides nucleophiles to which a variety of
nucleophiles can be added, providing yet another route to compounds
of type A-38.
[0086] Another strategy for the preparation of a subset of
compounds of type 33 is worth noting (Scheme 7). This strategy
allows for the preparation of compounds of type A-33 for all
R.sup.2 discussed above and for all R.sup.1 other than hydroxyl,
halide, --SR.sub.d,--NR.sub.dR.sub.e. Compounds of the type A-4,
A-5, A-10, and A-11 as shown in Scheme 1 (represented as compound
A-41 in Scheme 7), in which R.sup.1 is attached to the lactam ring
with a carbon-carbon bond, can be dihydroxylated to give compounds
of type A-42, in which the hydroxyl groups have been added to the
top face exclusively. [Uno, et al., (1994)] (The reasons for this
stereoselectivity have been discussed above.) Following the
procedure of [Uno, et al., 1994)] the tertiary hydroxyl can be
removed selectively, yielding a mixture of R.sup.1 diastereomers
A-43, which can be separated by a variety of chromatographic
methods. Mitsunobu inversion of the secondary hydroxyl, followed by
displacement of a suitably derived leaving group by any nucleophile
corresponding to R.sup.2 (e.g. hydroxide, alkoxide, sulfide,
Grignard reagents, amine, halide, etc.) gives compounds of the type
A-44, which are a subset of compounds of the type A-33, and can
therefore be elaborated into their corresponding A.sup.1
derivatives as described above.
[0087] Thus, having provided viable routes to all variations of
R.sup.1, R.sup.2, and now A.sup.1 in formula (I) we now turn our
attention to the installation of X.sup.1, L.sup.1, and X.sup.2
(Scheme 8).
[0088] The compound of general structure A-45 can be converted to
A-46 in a two-step process involving first catalytic hydrogenolysis
or mild acidic hydrolysis [Corey, E. J., et al., J. Am. Chem. Soc.
(1992a), 114:10677] and then protection of the primary hydroxyl
group as its tert-butyldimethylsilyl (TBS) ether under standard
conditions. [Corey, E. J. and Venkateswarlu, A., J. Am. Chem. Soc.
(1972), 94:6190] N-Alkylation of the amide provides access to
compounds A-47. [Challis, N., The Chemistry of Amides, (1970)
734-754]. (In the cases where R.sub.b is desired to be H, the amide
can be blocked with a suitable protecting group that will be
removed at the end of the synthesis. [Greene, T. W., et al.,
Protective Groups in Organic Synthesis (1991)].
[0089] Enamine A-48 can be prepared from A-47 under Tebbe
olefination conditions. [Pine, S. H., et al., J. Org. Chem. (1985)
50:1212]. Thioamide A-49 can be prepared by treatment of A-47 with
bis(tricyclohexyltin)sulfide and ##STR45## ##STR46## boron
trichloride. [Steliou, K., et al., J. Am. Chem. Soc., (1982)
104:3104]. Substituted enamines A-50 can be prepared by addition of
the appropriate Grignard reagent or alkyllithium to A-47 or A-49,
followed by acidic hydrolysis, and separation of the E and Z olefin
isomers. [Aguerro, A., et al., J. Chem. Soc., Chem. Commun. (1986)
531 and Schrock, R. R., J. Am. Chem. Soc., (1976) 98:5399, and
Hansen, C., J. Org. Chem. (1973) 38:3074]. Pyrrolidines of type 51
can be prepared by reduction of A-47 with lithium aluminum hydride
[March, J., Advanced Organic Chemistry (1992) 826-877 and Gaylord,
J., Reduction With Complex Metal Hydrides (1956)] 544-636] or
alternatively by reduction of A-49 with Raney nickel. [Belen'kii,
W., Chemistry of Organosulfur Compounds 91990) 193-228].
[0090] Taken together, these procedures constitute a synthesis of
compounds of the general structure A-52, which are converted to
A-53 following, for example, the procedure of Uno, et al. [Uno, H.,
et al., (1994)] (Scheme 9). Compounds A-53 are converted to
analogues A-54a via oxidation to the acid and coupling with L.sup.1
(all of which can be prepared by standard methods) under the
conditions utilized by Corey and Reichard [Corey, E. J., et al.,
(1992a]. Sulfurization of these compounds with Lawesson's reagent
[Cava, M. P., et al., Tetrahedron (1985) 41:5061] gives thiono
analogues A-55a. Compounds A-54b are prepared via addition of a
nucleophile (e.g. CF.sub.3) [Francese, C., et al., J. Chem. Soc.,
Chem. Commun (1987) 642], corresponding to L.sup.2 to the aldehydes
A-54b followed by oxidation of the alcohol with Dess-Martin
periodinane. [Linderman, R. J., et al., Tet. Lett. (1987) 28:4259].
Sulfurization of these compounds with Lawesson's reagent [Cava, M.
P., et al., (1985)] gives thiono analogues A-55b. Epoxides of the
type A-55c are prepared from A-53 or A-54b by the method of Johnson
##STR47## [Johnson, C. R., Acc. Chem. Res. (1973) 6:341], thus
completing the synthesis of all the analogues detailed in this
section.
[0091] The preparation of the compounds of formula (I) wherein
Z.sup.2 is O, S, NH or NR.sub.d relies primarily on the methods of
Seebach and of Corey. Thus, as shown in Scheme 10, epoxides of the
type A-58 are prepared according to the procedure of Corey, et al.,
[Corey, E. J., et al., (1992b)] from 61 via compound 62. R.sup.2 is
thus installed by choosing the appropriate aldehyde for the aldol
reaction in step a. Corey [Corey, E. J., et al., (1992b)] showed
that epoxides A-58 are opened stereospecifically to give A-59.
Thus, Z.sup.1 is installed by treating A-58 with a nucleophile
corresponding to Z.sup.1. (P.sub.z refers to a protecting group for
Z.sup.1.) Installation of Z.sup.2 is accomplished by conversion of
the hydroxyl A-59 to a leaving group, for example, a tosylate, and
displacement of the leaving group with a nucleophile corresponding
to Z.sup.2. (P.sub.z, refers to a protecting group for
Z.sup.2).
[0092] The results of Seebach [Seebach, D., et al., Helv. Chim.
Acta. (1987) 70:1194] are used in the next part of the synthesis.
Conversion of A-60 to A-61 allows for enolization and alkylation
with an electrophile corresponding to A.sup.1, yielding A-62.
Seebach showed that such alkylations give predominantly the
diastereomer shown.
[0093] After reduction of the ester and protection of the resulting
primary alcohol as the TBS ether, removal of the
tert-butylmethylene protecting group, enables conversion to
compounds of either the type A-63 (by treatment with, for example,
formaldehyde and an acid catalyst) or the type A-64 (by treatment
with, for example, CDI) is effected.
[0094] Compound 64 is a key intermediate in the completion of the
synthesis of the compounds of formula (I) wherein Z.sup.2 is O, S,
NH or NR.sub.d, (Scheme 11). Compounds of the type A-65 are
prepared from A-64 under Tebbe olefination conditions. [Pine, S.
H., et al., (1985)]. Compounds of the type A-66 are prepared by
treatment of A-64 with bis(tricyclohexyltin)sulfide and boron
trichloride. [Steliou, K., et al., (1982)]. Compounds of the type
A-67 are prepared by addition of the appropriate Grigard reagent or
alkyllithium to A-64 or A-66, followed by acidic hydrolysis, and
separation of the E and Z olefin isomers. [Aguerro, A., et al.,
(1986) and Schrock, R. R., (1976), and Hansen, C., et al., (1973)].
Compounds of the type A-63, prepared in Scheme 10, are also
prepared by reduction of A-64 with lithium aluminum hydride [March,
J., (1992), and Gaylord, J. (1956)] or alternatively by reduction
of A-66 with Raney nickel. [Belen'kii, W., (1990)].
[0095] Taken together, compounds of the type A-63, A-64, A-65,
A-66, and A-67 are of the general class A-68, which are converted
the general class of lactacystin analogues A-69 by fluoride
deprotection of the TBS ether, oxidation of the resulting primary
alcohol to the carboxylic acid, via the aldehyde analogues, in, for
example, the two-step process shown, and coupling with L.sup.1
using the method of Corey and Reichard, [Corey, E. J., et al.,
(1992a)] and removal of the protecting groups on Z.sup.1 and
Z.sup.2 (if necessary). Lactacystin analogues A-71 are prepared by
treating A-69 with Lawesson's reagent [Cava, M. P., et al.,
(1985)]. Analogues A-70 are also prepared from A-68 by fluoride
deprotection of the TBS ether, oxidation of the resulting primary
alcohol to the aldehyde, addition of nucleophile (e.g., CF.sub.3)
[Francese, C. et al. 1987, supra] Dess-Martin periodinane
oxidation, sulfurization with Lawesson's reagent (if desired)
[Cava, M. P., 91985)], and deprotection of Z.sup.2 and Z.sup.2, if
necessary. Epoxides of the type A-72 are also prepared from A-68 by
fluoride deprotection of the TBS ether, oxidation of the ##STR48##
resulting primary alcohol to the aldehyde, addition of a
nucleophile corresponding to L.sup.2, Dess-Martin periodinane
oxidation, addition of dimethyloxosulfonium methylide, and
deprotection of Z.sup.1 and Z.sup.2 (if necessary). Thus, the above
strategy prepares all the compounds of formula (1) wherein Z.sup.2
is O, S, NH or NR.sub.d.
[0096] The preparation of the compounds of formula (I) relies
primarily on the methods of Evans and of Corey. Thus, as shown in
Scheme 12, chiral oxazolidinones of the type A-73 are enolized and
alkylated with benzyloxy-bromomethane, thereby installing R.sup.1.
[Evans, D. A., et al. J. Am. Chem. Soc., (1982) 104:1737]. (Only
one configuration of R.sup.1 is obtained in the reaction. The other
configuration of R.sup.1 is obtained by using the opposite
enantiomer of the chiral oxazolidinone.) Conversion of A-74 to
aldehydes of the type A-75, and chiral, boron-mediated (using A-76)
aldol condensation with tert-butylbromoacetate gives [Corey, E. J.,
et al., (1992b)] a secondary alcohol that is protected as the
tert-butyldimethylsilyl (TBS) ether to give compounds of the type
A-77. Displacement of the bromide with a nucleophile [Corey, E. J.,
et al. (1992b)] corresponding to Z.sup.1 yields A-78, which can be
converted to compounds of the type A-79. As discussed above
regarding claim 4, such compounds can be enolized and alkylated
stereospecifically with electrophiles corresponding to A.sup.1,
giving A-80. Standard functional group manipulation yields
tosylates of the type A-81. Displacement of the tosylate with a
nucleophile corresponding to R.sup.2, [Hanessian, S., et al., J.
Org. Chem. (1989) 54:5831] gives compounds of the type A-82, the
primary alcohol of which is deprotected via, for example, catalytic
hydrogenolysis, and cyclized to compounds A-83 after oxidation of
the primary alcohol to the carboxylic acid. ##STR49##
[0097] As shown in Scheme 13, compounds of the type A-84, in which
R.sup.1 is in either the (R) or (S) configuration, are converted
into a variety of X.sup.1 variants. Compounds of the type A-85 are
prepared from A-84 under Tebbe olefination conditions. [Pine, et
al., (1985)] Compounds of the type A-86 are prepared by treatment
of A-84 with bis(tricyclohexyltin)sulfide and boron trichloride.
[Stelious, K., et al., (1982)]. Compounds of the type A-87 are
prepared by addition of the appropriate Grignard reagent or
alkyllithium to A-84 or A-86 followed by acidic hydrolysis, and
separation of the E and Z olefin isomers. [Aguerro, A., et al.,
(1986), Schrock, R. R., (1976) and Hansen C., et al., (1973)].
Compounds of the type A-88 are prepared by reduction of A-84 with
lithium aluminum hydride [March, J. (1992), and Gaylord, J.,
(1956)] or alternatively by reduction of A-86 with Raney nickel.
[Belen'kii, W., (1990)]. When Z.sup.1 is S in A-88, oxidation to
the cyclic sulfone variants is effected with, for example, KHSO.
[Trost, B. M., et al., Tet. Lett. (1981) 22:1287].
[0098] Taken together, compounds of the type A-84, A-85, A-86,
A-87, A-88, and A-89 are of the general class A-90, which are
converted analogues A-91, following for example, the procedure of
Uno, et al. [Uno, H., et al., (1994)] (Scheme 13b). Compounds A-91
are converted to analogues A-92 via oxidation to the acid and
coupling with L.sup.1 (all of which can be prepared by standard
methods) under the conditions utilized by Corey and Reichard.
[Corey, E. J., et al., (1992a)]. Sulfurization of these compounds
with Lawesson's reagent gives thiono analogues A-93. Compounds A-94
are prepared via addition of a nucleophile (e.g. CF.sub.3)
[Francese, C., et al., (1987)] corresponding to L.sup.2 to the
aldehydes A-91 followed by oxidation of the alcohol with
Dess-Martin periodinane. Sulfurization of these compounds ##STR50##
##STR51## with Lawesson's reagent gives thiono analogues A-95.
Epoxides of the type A-96 are prepared from A-91 or A-94 by the
method of Johnson, thus completing the synthesis of all the
analogues detailed in this section. Note: As above for compounds of
formula (I), in all Schemes relating to compounds of forumal (II),
when a straight line is used to connect A.sup.2 (or anything
corresponding to A.sup.2) to the rest of the molecule, a dashed
line should be assumed. The straight line is used simply for
clarity. All A.sup.2 are attached to the rest of the molecule on
the "alpha" face (i.e. the bottom face).
[0099] The proposed syntheses of all of the compounds P1-P7 (Table
A) and J1-J15 (Tables B and C) rely on a key intermediate from
Scheme 9. As shown in Scheme 14, compounds of type B-1 are
analogous to a subset of compounds A-53, whose various preparations
were described in Schemes 1, 2, and 5-8, and described in the
accompanying text.
[0100] In order to prepare analog B-2, the method of Corey, et al.
is utilized. [Corey, E. J., et al., Tet. Lett. (1993) 34:6977].
Sulfurization of B-2 with, for example, Lawesson's reagent [Cava,
M. P., et al., (1985)] provides analogs B-3.
[0101] The preparation of compounds of type B-1 is summarized in
Scheme 15. Thus the same starting material (A-1) as that used in
Scheme 1 is elaborated to all of the compounds B-2 by the same
methods as those used in Schemes 1, 2, 5, 6, and 8. There are then
two possible general strategies for the conversion of these
intermediates to B-2. The first is analogous to that detailed in
Schemes 1, 2, 5, 6, 8, and 9, in which Z.sup.5 (suitably activated
or protected) serves as the nucleophile in the relevant
reactions.
[0102] The other strategy, based on that shown in Scheme 7 is also
depicted in Scheme 15. Thus, dihydroxylation of B-4 gives B-5,
which is then be deoxygenated as before, giving B-6. Separation of
the diastereomers, Mitsunobu ##STR52## ##STR53## inversion of the
secondary hydroxyl, and displacement of a leaving group derived
from the Mitsunobu product provides compounds of the type B-7,
which are then elaborated to B-1 as described in Schemes 8 and
9.
[0103] The proposed syntheses of all of the compounds of formula
(II) rely on intermediate A-81 from Scheme 12. As shown in Scheme
16, compounds of type B-11 are analogous to a subset of compounds
A-81, whose various preparations are illustrated in Scheme 12, and
described in the accompanying text. Conversion of compounds of the
type B-11 to B-12 is performed in analogy to Scheme 12. Compounds
of the type B-13 are prepared from B-12 following the relevant
steps in Schemes 13a and 13b.
[0104] In order to prepare analogs B-14, the method of Corey, et
al. is utilized. [Corey, E. J., et al., (1993)] Sulfurization of
B-14 with, for example, Lawesson's reagent [Cava, M. P., et al.,
(1985)] provides analogs B-15.
[0105] The preparation of the compounds of formula (II) relies on a
key intermediate from Scheme 7. As shown in Scheme 17, compound C-1
is analogous to A-41, whose preparation is illustrated in Scheme 7
and described in the accompanying text. Following the work of
Fuchs, [Hutchinson, D. K., et al., J. Am. Chem. Soc. (1987)] C-1 is
treated sequentially with bis(benzyloxymethyl)lithium cuprate and
acidic water, giving compounds of the type C-2. Both configurations
of R.sup.7 can be accessed. See Schemes 1 and 2 for details. As in
Scheme 8, mild acidic hydrolysis, protection of the primary
hydroxyl, and alkylation or protection of the nitrogen gives
compounds C-2.
[0106] Deprotection of the benzyl group by, for example, catalytic
hydrogenolysis followed by displacement of the derived tosylate
with a nucleophile corresponding to Z.sup.2, accesses compounds
C-3, in which Z.sup.2 is protected, if ##STR54## necessary.
Elaboration to carboxylic acids C-4 follows the same set of
reactions in Schemes 8 and 9, allowing the preparation of all
X.sup.9 variants. Deprotection of Z.sup.2, if necessary, followed
by cyclization with, for example, DCC, yields bicyclic structures
C-5 which if desired are sulfurized with Lawesson's reagent [Cava,
M. P., et al., (1985)] to give C-6.
[0107] The preparation of the compounds of formula IV relies on a
key intermediate from Scheme 9. As shown in Scheme 18, compound
C-11 is analogous to a subset, defined by Z.sup.2, of the class of
compounds A-52, whose preparation is illustrated in Schemes 1-8,
and described in the accompanying text. Deprotection of the primary
hydroxyl with, for example, TBAF in THF and oxidation via, for
example, under Swern oxidation conditions to the aldehydes C-12.
Homologation of the aldehyde with, for example, the phosphorous
ylide derived from triphenylmethoxymethylphosphonium chloride
[Jamison, T. F., et al., J. Am. Chem. Soc., (1994) 116:5505] yields
enolethers of the type C-13, which are converted to carboxylic
acids C-14 with acidic hydrolysis and oxidation of the resulting
aldehyde with, for example, buffered NaClO.sub.2 oxidation. [Corey,
E. J., et al., (1992a)].
[0108] Deprotection of Z.sup.2, if necessary, followed by
cyclization with, for example, DCC, [Klausner, Y. S. et al.,
Synthesis (1972) 453] yields bicyclic structures C-15 which if
desired are sulfurized with Lawesson's reagent [Cava, M. P.,
(1985)] to give C-16.
[0109] The preparation of the compounds of formula V relies
primarily on the boron-enolate mediated asymmetric aldol procedures
of Evans [Evans, D. A., et al., J. Am. Chem. Soc., (1981) 103:2127]
and on the results of studies on intramolecular radical ##STR55##
##STR56## ##STR57## ##STR58## cyclizations. [Curran, D. P.,
Comprehensive Organic Synthesis, (1991) 4:779-831].
[0110] Esters C-21 (Scheme 19), prepared by standard methods, are
deprotonated and then alkylated with benzyloxybromo-methane and
reduced with DIBAL-H to give aldehydes C-22 as a racemic mixture.
Addition of C-22 to an boron enolate derived from chiral
oxazolidinone imides C-23 [Evans, D. A., et al., (1981)] yields
C-24 as a mixture of A.sup.3 diastereomers. The configurational
identity of the other two new stereocenters is established in this
reaction and is not affected by the configuration of A.sup.3.
[0111] Conversion to aldehydes C-25 in a six-step procedure (for
example, tosylation of the secondary hydroxyl, displacement with a
nucleophile corresponding to Z.sup.7, catalytic hydrogenolysis of
the benzyl group, oxidation of the alcohol to the carboxylic acid
and esterification) allows for preparation of the substrate for the
intramolecular radical cycloaddition. Thus, treatment of the
aldehyde with the Gilbert reagent [Gilbert, J. C., et al., J. Org.
Chem. (1982) 47:1837] and replacement of the enolizable hydrogen
with iodine gives an acetylenes C-26, which, after deprotection of
Z.sup.7, if necessary, and cyclization with, for example, BOP-Cl,
affords C-27.
[0112] Exposure of C-27 to atom-transfer, intramolecular radical
cyclization conditions [Curran, D. P., et al., Tet. Lett. (1987)
28:2477 and Curran, D. P., et al., J. Org. Chem. (1989) 54:3140]
gives compounds C-28. This reaction deserves further comment. The
stereochemistry of A.sup.1 and the iodine atom are inconsequential
because the radical generated from the iodine can interconvert
under the reaction conditions. Further, only one of the radical
diastereomers will be able to cyclize--that giving the cis-4,5 ring
system depicted, because the trans-4,5 suffers from much greater
ring strain. ##STR59## ##STR60##
[0113] This cyclization is favored for two other reasons. First, it
is a 5-endo-dig type cyclization and therefore favored according to
Baldwin's rules for cyclization. [Baldwin, J. E., J. Chem. Soc.,
Chem. Commun. (1976) 734]. Second, the atom transfer conditions
used are ideal because the resulting vinylic radical is very
reactive and is rapidly quenched by the iodine radical. [Curran, D.
P., et al., J. Am. Chem. Soc., (1986) 108:2489].
[0114] Elaboration of C-28 to C-29 is accomplished using, for
example, standard Stille [Stille, J. K., Angew. Chem., Int. Ed.
Engl. (1986) 25:504] or Heck [Heck, R. F., Comprehensive Organic
Synthesis, (1991) 4:833-863] type coupling conditions using a
suitable metallic derivative (M is, e.g., tributylstannyl) of
R.sup.9. Sulfurization with Lawesson's reagent [Cava, M. P., et
al., (1985)] gives compounds C-30, thus completing the preparation
of all the compounds of formula V.
[0115] The preparation of the compounds of formula VI relies
primarily on intermediate B-2, whose preparation is described in
Schemes 13a, 13b and 14 and discussed in the accompanying text. As
shown in Scheme 20, C-31 is deprotonated with a suitable base, and
the resulting enolate is treated with, for example, the triflate
source PhN(Tf).sub.2 [McMurry, J. E., et al., Tet. Lett. (1983)
24:979]. The vinyl triflates C-32 are treated with, for example, a
catalytic amount of (Ph.sub.3P).sub.4Pd and a suitable metallic
derivative [Stille, J. K. (1986) and Heck, R. F. (1991)] (M is,
e.g., tributylstannyl) of R.sup.9, giving analogues C-33, which can
be treated with Lawesson's reagent [Cava, M. P., et al., (1985)]
gives compounds C-34, thus completing the preparation of all the
compounds in this section.
[0116] The preparation of the compounds of formula VII relies
primarily on intermediate A-38, whose ##STR61## preparation is
illustrated in Schemes 1, 5, and 6, and discussed in the
accompanying text. Compounds of the type C-41 (Scheme 21) can
therefore be prepared with either the (R) or (S) configuration of
Z.sup.7 by following the procedures illustrated in Schemes 1-6, and
discussed in the accompanying text.
[0117] Mild acidic hydrolysis of C-41, [Corey, E. J., et al.,
(1992a)] followed by protection of the primary hydroxyl [Corey, E.
J., et al., (1972)] allows for installation of A.sup.4 via standard
alkylation procedures, [Challis, N. (1970)] giving C-42.
Elaboration of these intermediates to C-43 is performed as
described for the analogous compounds in Scheme 8. Deprotection of
the primary hydroxyl allows for oxidation to the carboxylic acids
C-44, which are cyclized with, for example, BOP-Cl, giving
analogues C-45, which can be treated with Lawesson's reagent [Cava,
M. P., et al., (1985)] gives compounds C-46, thus completing the
preparation of all the compounds of formula VII.
[0118] The preparation of the compounds of formula VIf follows the
strategy illustrated in Schemes 1, 3, 4, 5, 8, and 9, and described
in the accompanying text. Thus, as shown in Scheme 22, C-51, being
analogous to A-2 is elaborated to C-52 following the same methods
used in Scheme 1 using an aldehyde containing As and a protected
form of Z.sup.7 in either the (R) or (S) configuration. Protection
of the secondary hydroxyl gives compounds C-52, [Corey, E. J., et
al., (1972)] which can be treated with a nucleophile corresponding
to R.sup.9 and subsequent quenching with acidic water, giving C-53.
The reasons governing the indicated stereoselectivity of the
nucleophilic addition and aqueous quenching have been discussed in
the text accompanying Scheme 1.
[0119] Deprotection and subsequent conversion of the secondary
hydroxyl to the tosylate allows for ##STR62## ##STR63## ##STR64##
##STR65## introduction of A.sup.3 via displacement of the tosylate
with a nucleophile corresponding to A.sup.3 to give compounds of
the type C-54. [Hanessian, S., (1980)]. Elaboration of these
compounds to C-55 is performed with the methods illustrated in
Scheme 8 and described in the accompanying text. Deprotection of
the primary hydroxyl allows for oxidation to the carboxylic acids
C-56, which are cyclized with, for example, DCC, [Klausner, Y. S.,
et al., (1972)] following deprotection of Z.sup.7, if necessary, to
give analogues C-57. Sulfurization with Lawesson's reagent [Cava,
M. P., (1985)] gives compounds C-58, thus completing the
preparation of all the compounds of formula VII.
[0120] The preparation of the compounds of formula IX relies
primarily on the results of Lubell, et al., J. Org. Chem., 1990
55:3511. Thus, as shown in Scheme 23, L-serine (C-61) is converted
to ketones C-62 by known procedures. (PhFl is an abbreviation of
9-phenyl-9-fluorenyl.) Standard Wadsworth-Emmons olefination of
C-62 yields either C-63 or C-64, or a mixture thereof. The
composition of the product mixture is of no consequence, since both
olefin isomers yield the same diastereomer in the next
reaction.
[0121] Thus, a cuprate reagent derived from A.sup.1 approaches from
the back face of both C-63 or C-64 since the very bulky PhFl group
blocks the other face completely, giving C-65 as the major
diastereomer. C-65 is converted to C-66 in a five-step procedure
(reduction of the methyl ester to the aldehyde and protection as
the dimethyl acetal, deprotection of the oxazolidinone, and
oxidation of the primary alcohol to the carboxylic acid.)
[0122] As depicted in Scheme 24, DCC-mediated coupling, for
example, of C-66 with an amine, yields amides C-67. Taken together,
C-66 and C-67 comprise the general class of compounds C-88. The
PhFl group is removed by ##STR66## catalytic hydrogenolysis, and
the free amine alkylated with an electrophile corresponding to
R.sup.12 (reductive amination with an aldehyde or ketone and
NaCNBH.sub.3 is another option) to give compounds C-69. Acidic
hydrolysis of the dimethyl acetal yields intermediate aldehyde
C-70, which cyclizes to analogues C-73.
[0123] Tebbe olefination of C-71 provides compounds C-73. Treatment
of C-71 with Lawesson's reagent provides compounds C-72, which can
be desulfurized with Raney Nickel to give compounds C-74, thus
completing the synthesis of all the compounds of formula IX.
[0124] The preparation of the compounds of formula X relies
primarily on the results of Dener, et al., [J. Org. Chem., 1993,
58:1159], and on those of Evans et al.,[J. Am. Chem. Soc., 1981,
103:2127]. Thus, as shown in Scheme 25, D-aspartic acid (C-81) is
protected and alkylated with an electrophile corresponding to
A.sup.6 to give compounds C-82, with the major diastereomer to be
that shown. Dener et al.,[J. Org. Chem., 1993, 58:1159]. (PhFl is
an abbreviation of 9-phenyl-9-fluorenyl.) Site-specific DIBAL-H
reduction (owing to the extreme size of the PhFl group) affords
aldehyde C-83.
[0125] A diastereoselective boron-mediated aldol addition is the
next step. Following the procedure of Evans, et al., J. Am. Chem.
Soc., (1981) 103:2127, one obtains C-85, after TBS protection of
the secondary hydroxyl. The oxazolidinone is removed with lithium
hydroperoxide, the PhFl group removed with catalytic
hydrogenolysis, and lactam formation accomplished under, for
example, DCC conditions, to give C-86. Removal of the TBS group
with TBAF and lactone formation (saponification of the methyl ester
with LiOH, followed by DCC coupling may be necessary) yields C-87,
which is elaborated to C-88 by alkylating the amide nitrogen with a
nucleophile corresponding to R.sub.d. Epimerization with DBU
provides access to compounds C-89. Treatment of C-89 with
Lawesson's reagent, Cava et al., Tetrahedron, 1985, 41: 5061,
yields compounds C-90, thus completing the preparation of all
compounds in this section. Examples 13-21 below are individual
syntheses. ##STR67## ##STR68## Use
[0126] The disclosed compounds are used to treat conditions
mediated directly by the proteolytic function of the proteasome
such as muscle wasting, or mediated indirectly via proteins which
are processed by the proteasome such as NF-.kappa.B. The proteasome
participates in the rapid elimination and post-translational
processing of proteins (e.g., enzymes) involved in cellular
regulation (e.g., cell cycle, gene transcription, and metabolic
pathways), intercellular communication, and the immune response
(e.g., antigen presentation). Specific examples discussed below
include .beta.-amyloid protein and regulatory proteins such as
cyclins and transcription factor NF-.kappa.B. Treating as used
herein includes reversing, reducing, or arresting the symptoms,
clinical signs, and underlying pathology of a condition in manner
to improve or stabilize the subject's condition.
[0127] Alzheimer's disease is characterized by extracellular
deposits of .beta.-amyloid protein (.beta.-AP) in senile plaques
and cerebral vessels. .beta.-AP is a peptide fragment of 39 to 42
amino acids derived from an amyloid protein precursor (APP). At
least three isoforms of APP are known (695, 751, and 770 amino
acids). Alternative splicing of mRNA generates the isoforms; normal
processing affects a portion of the .beta.-AP sequence, thereby
preventing the generation of .beta.-AP. It is believed that
abnormal protein processing by the proteasome contributes to the
abundance of .beta.-AP in the Alzheimer brain. The APP-processing
enzyme in rats contains about ten different subunits (22 kDa-32
kDa). The 25 kDa subunit has an N-terminal sequence of
X-Gln-Asn-Pro-Met-X-Thr-Gly-Thr-Ser, which is identical to the
.beta.-subunit of human macropain. Kojima, S. et al., Fed. Eur.
Biochem. Soc., (1992) 304:57-60. The APP-processing enzyme cleaves
at the Gln.sup.15-Lys.sup.16 bond; in the presence of calcium ion,
the enzyme also cleaves at the Met.sup.-1-Asp.sup.1 bond, and the
Asp.sup.1-Ala.sup.2 bonds to release the extracellular domain of
.beta.-AP.
[0128] One embodiment, therefore, is a method of treating
Alzheimer's disease, including administering to a subject an
effective amount of a compound (e.g., pharmaceutical composition)
having a formula disclosed herein. Such treatment includes reducing
the rate of .beta.-AP processing, reducing the rate of .beta.-AP
plaque formation, and reducing the rate of .beta.-AP generation,
and reducing the clinical signs of Alzheimer's disease.
[0129] Other embodiments of the invention relate to cachexia and
muscle-wasting diseases. The proteasome degrades many proteins in
maturing reticulocytes and growing fibroblasts. In cells deprived
of insulin or serum, the rate of proteolysis nearly doubles.
Inhibiting the proteasome reduces proteolysis, thereby reducing
both muscle protein loss and the nitrogenous load on kidneys or
liver. Proteasome inhibitors are useful for treating conditions
such as cancer, chronic infectious diseases, fever, muscle disuse
(atrophy) and denervation, nerve injury, fasting, renal failure
associated with acidosis, and hepatic failure. See, e.g., Goldberg,
U.S. Pat. No. 5,340,736 (1994). Embodiments of the invention
therefore encompass methods for: reducing the rate of muscle
protein degradation in a cell, reducing the rate of intracellular
protein degradation, reducing the rate of degradation of p53
protein in a cell, and inhibiting the growth of p53-related
cancers). Each of these methods includes the step of contacting a
cell (in vivo or in vitro, e.g., a muscle in a subject) with an
effective amount of a compound (e.g., pharmaceutical composition)
of a formula disclosed herein.
[0130] Another protein processed by the proteasome is NF-.kappa.B,
a member of the Rel protein family. The Rel family of
transcriptional activator proteins can be divided into two groups.
The first group requires proteolytic processing, and includes p50
(NF-.kappa.B1, 105 kDa) and p52 (NF-.kappa.2, 100 kDa). The second
group does not require proteolytic processing, and includes p65
(RelA, Rel (c-Rel), and RelB). Both homo- and heterodimers can be
formed by Rel family members; NF-.kappa.B, for example, is a
p50-p65 heterodimer. After phosphorylation and ubiquitination of
I.kappa.B and p105, the two proteins are degraded and processed,
respectively, to produce active NF-.kappa.B which translocates from
the cytoplasm to the nucleus. Ubiquitinated p105 is also processed
by purified proteasomes. Palombella et al., Cell (1994) 78:773-785.
Active NF-.kappa.B forms a stereospecific enhancer complex with
other transcriptional activators and, e.g., HMG I(Y), inducing
selective expression of a particular gene.
[0131] NF-.kappa.B regulates genes involved in the immune and
inflammatory response, and mitotic events. For example, NF-.kappa.B
is required for the expression of the immunoglobulin light chain K
gene, the IL-2 receptor .alpha.-chain gene, the class I major
histocompatibility complex gene, and a number of cytokine genes
encoding, for example, IL-2, IL-6, granulocyte colony-stimulating
factor, and IFN-.beta.. Palombella et al., (1994). Some embodiments
of the invention include methods of affecting the level of
expression of IL-2, MHC-I, IL-6, IFN-.beta. or any of the other
previously-mentioned proteins, each method including administering
to a subject an effective amount of a compound of a formula
disclosed herein.
[0132] NF-.kappa.B also participates in the expression of the cell
adhesion genes that encode E-selectin, P-selectin, ICAm, and
VCAM-1, Collins, T., Lab. Invest. (1993) 68:499-508. One embodiment
of the invention is a method for inhibiting cell adhesion (e.g.,
cell adhesion mediated by E-selectin, P-selectin, ICAm, or VCAM-1),
including contacting a cell with (or administering to a subject) an
effective amount of a compound (e.g., pharmaceutical composition)
having a formula disclosed herein.
[0133] NF-.kappa.B also binds specifically to the
HIV-enhancer/promoter. When compared to the Nef of mac239, the HIV
regulatory protein Nef of pbj 14 differs by two amino acids in the
region which controls protein kinase binding. It is believed that
the protein kinase signals the phosphorylation of I-.kappa.B,
triggering I.kappa.B degradation through the ubiquitin-proteasome
pathway. After degradation, NF-.kappa.B is released into the
nucleus, thus enhancing the transcription of HIV. Cohen, J.,
Science, (1995) 267:960. Two embodiments of the invention are a
method for inhibiting or reducing HIV infection in a subject, and a
method for decreasing the level of viral gene expression, each
method including administering to the subject an effective amount
of a compound of a formula disclosed herein.
[0134] Complexes including p50 are rapid mediators of acute
inflammatory and immune responses. Thanos, D. and Maniatis, T.,
Cell (1995) 80:529-532. Intracellular proteolysis generates small
peptides for presentation to T-lymphocytes to induce MHC class
I-mediated immune responses. The immune system screens for
autologous cells that are virally infected or have undergone
oncogenic transformation. Two embodiments of the invention are a
method for inhibiting antigen presentation in a cell, including
exposing the cell to a compound of a formula described herein, and
a method for suppressing the immune system of a subject (e.g.,
inhibiting transplant rejection), including administering to the
subject an effective amount of a compound of a formula described
herein.
[0135] In addition, the invention provides a method for treating
inflammation, wherein the method includes administering to a
subject an effective anti-inflammatory amount of a pharmaceutical
composition containing a compound of a formula described herein.
Inflammation can be a primary or secondary responses associated
with
[0136] (a) injury such as a cut, laceration, puncture wound,
[0137] (b) infection (including infected surgical incisions) by one
or more viruses, bacteria, mycobacteria, microorganisms, parasites,
and fungi, (c) allergies, (d) a disease state, (e) surgery (e.g.,
transplantation), or (f) a combination thereof.
[0138] Allergies are primary inflammatory responses to antigens or
allergens. Sources of allergens include plants (e.g., grass or tree
pollen), animals (e.g., dander, venom, urine, execreta from dogs,
cats, insects, and snakes), and fungi. In addition to allergens
such as rye grass, ragweed, and Japanese cedar pollen, certain
foods or food components (e.g., eggs, milk, shellfish,
strawberries, chocolate), vaccines, and drugs (e.g., penicillin)
can induce allergic reactions in certain individuals.
[0139] Disease states include rheumatoid arthritis, scleroderma,
rheumatic fever, inflammatory bowel disease (e.g., Crohn's disease
and ulcerative colitis), diabetes mellitus, myasthenia gravis,
multiple sclerosis, Guillain-Barre syndrome, conjuctiva of the eye,
systemic lupus erythematosus, encephalitis, Adult Respiratory
Distress Syndrome, psoriasis, emphysema, Alzheimer's disease, and
muscular dystrophy.
[0140] The invention provides a method of treating inflammation
induced by organ or tissue transplantation. This method includes
administering to a patient who has undergone or is about to undergo
transplantation a composition containing a compound having a
formula disclosed herein. Transplantations include bone marrow,
solid organ (e.g., kidney, lungs, heart, pancreas, liver, and
skin), or tissues.
[0141] Certain proteasome inhibitors block both degradation and
processing of ubquitinated NF-.kappa.B in vitro and in vivo.
Proteasome inhibitors also block I.kappa.B-.alpha. degradation and
NF-.kappa.B activation, Palombella, et al.; and Traenckner, et al.,
EMBO J. (1994) 13:5433-5441. One embodiment of the invention is a
method for inhibiting I.kappa.B-.alpha. degradation, including
contacting the cell with a compound of a formula described herein.
A further embodiment is a method for reducing the cellular content
of NF-.kappa.B in a cell, muscle, organ, or subject, including
contacting the cell, muscle, organ, or subject with a compound of a
formula described herein.
[0142] Other eukaryotic transcription factors that require
proteolytic processing include the general transcription factor
TFIIA, herpes simplex virus VP16 accessory protein (host cell
factor), virus-inducible IFN regulatory factor 2 protein, and the
membrane-bound sterol regulatory element-binding protein 1.
[0143] Other embodiments of the invention are methods for affecting
cyclin-dependent eukaryotic cell cycles, including exposing a cell
(in vitro or in vivo) to a compound of a formula disclosed herein.
Cyclins are proteins involved in cell cycle control. The proteasome
participates in the degradation of cyclins. Examples of cyclins
include mitotic cyclins, G1 cyclins, (cyclin B). Degradation of
cyclins enables a cell to exit one cell cycle stage (e.g., mitosis)
and enter another (e.g., division). It is believed all cyclins are
associated with p34.sup.cdc2 protein kinase or related kinases. The
proteolysis targeting signal is localized to amino acids
42-RAALGNISEN-50 (destruction box). There is evidence that cyclin
is converted to a form vulnerable to a ubiquitin ligase or that a
cyclin-specific ligase is activated during mitosis. Ciechanover,
A., Cell, (1994) 79:13-21. Inhibition of the proteasome inhibits
cyclin degradation, and therefore inhibits cell proliferation
(e.g., cyclin-related cancers). Kumatori et al., Proc. Natl. Acad.
Sci. USA (1990) 87:7071-7075. One embodiment of the invention is a
method for treating a proliferative disease in a subject (e.g.,
cancer, psoriasis, or restenosis), including administering to the
subject an effective amount of a compound of a formula disclosed
herein. Chronic or acute inflammation can result from
transplantation rejection, arthritis, rheumatoid arthritis,
infection, dermatosis, inflammatory bowel disease, asthma,
osteoporosis, and autoimmune diseases. Rejection or inflammation
can occur in transplanted tissues or organs of any type, including
heart, lung, kidney, liver, skin grafts, and tissue grafts. The
invention also encompasses a method for treating cyclin-related
inflammation in a subject, including adminstering to a subject an
effective amount of a compound of a formula described herein.
[0144] Additional embodiments are methods for affecting the
proteasome-dependent regulation of oncoproteins and methods of
treating or inhibiting cancer growth, each method including
exposing a cell (in vivo, e.g., in a subject or in vitro) to a
compound of a formula disclosed herein. HPV-16 and HPV-18-derived
E6 proteins stimulate ATP- and ubiquitin-dependent conjugation and
degradation of p53 in crude reticulocyte lysates. The recessive
oncogene p53 has been shown to accumulate at the nonpermissive
temperature in a cell line with a mutated thermolabile E1. Elevated
levels of p53 may lead to apoptosis. Examples of proto-oncoproteins
degraded by the ubiquitin system include c-Mos, c-Fos, and c-Jun.
One embodiment is a method for treating p53-related apoptosis,
including administering to a subject an effective amount of a
compound of a formula disclosed herein.
[0145] Treatment of cancer prevents, alleviates, or ameliorates one
or more primary or secondary phenomena associated with the
initiation, progression, and metastasis of tumors, especially
malignant tumors, e.g., a growth of tissue wherein cell
multiplication is uncontrolled. Malignant tumors show a greater
degree of anaplasia than do benign tumors. The invention provides a
method of treating cancer including administering to a subject an
effective anti-cancer amount of a pharmaceutical composition
described herein, wherein the cancer is selected from carcinoma,
lymphoma, sarcoma, and myeloma.
[0146] Examples of carcinomas include adenocarcinoma, acinic cell
adenocarcinoma, adrenal cortical carcinomas, alveoli cell
carcinoma, anaplastic carcinoma, basaloid carcinoma, basal cell
carcinoma, bronchiolar carcinoma, bronchogenic carcinoma,
renaladinol carcinoma, embryonal carcinoma, anometroid carcinoma,
fibrolamolar liver cell carcinoma, follicular carcinomas, giant
cell carcinomas, hepatocellular carcinoma, intraepidermal
carcinoma, intraepithelial carcinoma, leptomanigio carcinoma,
medullary carcinoma, melanotic carcinoma, menigual carcinoma,
mesometonephric carcinoma, oat cell carcinoma, squamal cell
carcinoma, sweat gland carcinoma, transitional cell carcinoma, and
tubular cell carcinoma. Examples of sarcoma include amelioblastic
sarcoma, angiolithic sarcoma, botryoid sarcoma, endometrial stroma
sarcoma, ewing sarcoma, fascicular sarcoma, giant cell sarcoma,
granulocytic sarcoma, immunoblastic sarcoma, juxaccordial
osteogenic sarcoma, Kaposi's sarcoma, leukocytic sarcoma (also
known as leukemia), lymphatic sarcoma (also known as lympho
sarcoma), medullary sarcoma, myeloid sarcoma (also known as
granulocytic sarcoma), austiogenci sarcoma, periosteal sarcoma,
reticulum cell sarcoma (also known as histiocytic lymphoma), round
cell sarcoma, spindle cell sarcoma, synovial sarcoma, and
telangiectatic audiogenic sarcoma. Examples of lymphomas include
Hodgkin's disease and lymphocytic lymphomas, such as Burkitt's,
nodular poorly-differentiated lymphocytic (NPDL), nodular mixed
lymphocytic (NML), NH (nodular histiocytic), and diffuse lymphomas.
Additional carcinomas include neural blastoma, glioblastoma,
astrocytoma, melanoma, leiomyo sarcoma, multiple myeloma, and
Hemangioma.
[0147] A tripeptide aldehyde protease inhibitor (benzyloxycarbonyl
(Z)-Leu-Leu-leucinal induces neurite outgrowth in PC12 cells at an
optimal concentration of 30 nM, Tsubuki et al., Biochem. and
Biophys. Res. Comm. (1993) 196:1195-1201. Peptide aldehydes have
been shown to inhibit the chymotryptic activity of the proteasome.
Vinitsky, et al., 1992, Tsubuki et al., 1993. One embodiment of the
invention is a method of promoting neurite outgrowth, including
administering to the subject a compound of a formula disclosed
herein.
[0148] Finally, the disclosed compounds are also useful as
diagnostic agents (e.g., in diagnostic kits or for use in clinical
laboratories) for screening for proteins (e.g., enzymes,
transcription factors) processed by the proteasome. The disclosed
compounds are also useful as research reagents for specifically
binding the X/MB1 subunit or .alpha.-chain and inhibiting the
proteolytic activities associated with it. For example, the
activity of (and specific inhibitors of) other subunits of the
proteasome can be determined.
[0149] Most cellular proteins are subject to proteolytic processing
during maturation or activation. Lactacystin can be used to
determine whether a cellular, developmental, or physiological
process or output is regulated by the proteolytic activity of the
proteasome. One such method includes obtaining an organism, an
intact cell preparation, or a cell extract; exposing the organism,
cell preparation, or cell extract to a compound of a formula
disclosed herein; exposing the compound-exposed organism, cell
preparation, or cell extract to a signal, and monitoring the
process or output. The high selectivity of the compounds disclosed
herein permits rapid and accurate elimination or implication of the
proteasome in a given cellular, developmental, or physiological
process.
[0150] The compounds and compositions of the invention are useful
in several methods including a method of treating inflammation,
comprising administering to a subject an effective
anti-inflammatory amount of a pharmaceutical composition described
herein (e.g., a lactacystin thioester, a lactone, or a lactam);
wherein the inflammation is associated with injury or infection;
wherein the inflammation is associated with an allergy or asthma;
wherein the inflammation is associated with a disease state
selected from rheumatoid arthritis, scleroderma, rheumatic fever,
inflammatory bowel disease, diabetes mellitus, myasthenia gravis,
multiple sclerosis, Guillan-Barre syndrome, conjunctiva of the eye,
systemic lupus erythematosus, encephalitis, Adult Respiratory
Distress Syndrome, emphysema, Alzheimer's disease, and muscular
dystrophy; wherein the inflammation is associated with
transplantation of bone marrow or a solid organ selected from
kidney, lung, heart, pancreas, liver, and skin, and the composition
is administered before, during, or after transplantation; wherein
the pharmaceutical composition is administered orally; or
combinations thereof.
[0151] The invention also provides a method of treating cancer,
comprising administering to a subject an effective anti-cancer
amount of a pharmaceutical composition described herein; a method
for treating psoriasis, comprising administering to a subject an
effective amount of a pharmaceutical composition described herein;
and a method for treating restenosis, comprising administering to a
subject an effective amount of a pharmaceutical composition
described herein. Preferred compounds and compositions include
various lactacystin thioesters, and lactacystin .beta.-lactone
analogs, including the .beta.-lactone itself.
Formulation and Administration
[0152] The methods of the invention contemplate treatment of animal
subjects, such as mammals (e.g., higher primates, and especially
humans). The invention encompasses pharmaceutical compositions
which include novel compounds described herein, and pharmaceutical
compositions which include compounds described and first recognized
herein as proteasome inhibitors, such as lactacystin.
[0153] Pharmaceutically acceptable salts may be formed, for
example, with 1, 2, 3, or more equivalents of hydrogen chloride,
hydrogen bromide, trifluoroacetic acid, and others known to those
in the art of drug formulation. Compounds of the invention can be
formulated into pharmaceutical compositions by admixture with
pharmaceutically acceptable non-toxic excipients and carriers. A
pharmaceutical composition of the invention may contain more than
one compound of the invention, and/or may also contain other
therapeutic compounds not encompassed by the invention, such as
anti-inflammatory, anti-cancer, or other agents. A subject may have
more than one type of inflammation, or more than one type of
cancer, a combination of allergies, or a mixture of the above
conditions for which the disclosed compounds are useful. A compound
of the invention may be administered in unit dosage form, and may
be prepared by any of the methods well known in the pharmaceutical
art, for example, as described in Remington's Pharmaceutical
Sciences (Mack Pub. Co., Easton, Pa., 1980). The invention also
encompasses a packaged drug, containing a pharmaceutical
composition formulated into individual dosages and printed
instructions for self-administration.
[0154] Compounds disclosed herein as proteasome inhibitors may be
prepared for use in parenteral administration in the form of
solutions or liquid suspensions; for oral administration
(preferable), particularly in the form of tablets or capsules; or
intranasally, particularly in the form of powders, gels, oily
solutions, nasal drops, aerosols, or mists. Formulations for
parenteral administration may contain as common excipients sterile
water or sterile saline, polyalkylene glycols such as polyethylene
glycol, oils of vegetable origin, hydrogenated naphthalenes, and
the like. Controlled release of a compound of the invention may be
obtained, in part, by use of biocompatible, biodegradable polymers
of lactide, and copolymers of lactide/glycolide or
polyoxyethylene/polyoxypropylene. Additional parental delivery
systems include ethylene-vinyl acetate copolymer particles, osmotic
pumps, implantable infusion systems, and liposomes. Formulations
for inhalation administration contain lactose,
polyoxyethylene-9-lauryl ether, glycocholate, or deoxycholate.
Formulations for buccal administration may include glycocholate;
formulations for vaginal administration may include citric
acid.
[0155] The concentration of a disclosed compound in a
pharmaceutically acceptable mixture will vary depending on several
factors, including the dosage of the compound to be administered,
the pharmacokinetic characteristics of the compound(s) employed,
and the route of administration. In general, the compounds of this
invention may be provided in an aqueous physiological buffer
solution containing about 0.1-10% w/v of compound for parenteral
administration. Typical dose ranges are from about 0.1 to about 50
mg/kg of body weight per day, given in 1-4 divided doses. Each
divided dose may contain the same or different compounds of the
invention. The dosage will be an effective amount depending on
several factors including the overall health of a patient, and the
formulation and route of administration of the selected
compound(s).
[0156] The effective amount of the active compound used to practice
the present invention for treatment of conditions directly or
indirectly mediated by the proteasome varies depending upon the
manner of administration, the age and the body weight of the
subject and the condition of the subject to be treated, and
ultimately will be decided by the attending physician or
veterinarian. Such amount of the active compound as determined by
the attending physician or veterinarian is referred to herein as
"effective amount".
[0157] Without further elaboration, it is believed that one skilled
in the art can, based on the description herein, utilize the
present invention to its fullest extent. The following specific
examples are, therefore, to be construed as merely illustrative,
and not limitative of the remainder of the disclosure in any way
whatsoever. All publications cited herein are hereby incorported by
reference.
EXAMPLES
Example 1
Synthesis of [.sup.3H] lactacystin
[0158] Lactacystin was prepared from the .beta.-lactone, which was
tritiated by an oxidation-reduction sequence. Unlabeled P-lactone
was oxidized at C9 to the ketone with the Dess-Martin periodinane
in dichloromethane and then reduced with [.sup.3H]NaBH4 (NEN, 13.5
Ci/mmol) in 1,2-dimethoxyethane/1% H.sub.2O to afford tritiated
.beta.-lactone along with its C9-epimer (2:3 ratio). The isomeric
2-lactones were separated by HPLC (Rainin Microsorb SiO.sub.2
column, 4.6.times.100 mm, 10% i-Pr-OH in hexane) and reacted
separately with N-acetylcysteine (0.5 M) and Et.sub.3N (1.5 M) in
CH.sub.3CN to afford the lactacystin (9S) and its C9-epimer (9R),
which were purified from their respective reaction mixtures by
reverse-phase HPLC (TSK ODS-80T.sub.m column, 4.6.times.250 mm, 10%
CH.sub.3CN/0.1% CF.sub.3CO.sub.2H in H.sub.2O).
Example 2
Fluorography of Crude Cell and Tissue Extracts
[0159] Crude Neuro 2A extracts (.about.11 .mu.g total protein/lane)
and crude bovine brain extracts (.about.54 .mu.g total
protein/lane) were treated as follows: (1) 10 .mu.M
[.sup.3H]lactacystin; (2) 10 .mu.M [.sup.3H]lactacystin and 1 mM
cold lactacystin; (3) 10 .mu.M [.sup.3H]lactacystin; (4) 10 .mu.M
[.sup.3H]lactacystin and 1 mM cold lactacystin; (5) 10 .mu.M
[.sup.3H]lactacystin and 1 mM cold P-lactone; (6) 10 .mu.M
[.sup.3H]lactacystin and 1 mM cold phenylacetyl lactacystin; (7) 10
.mu.M [.sup.3H]lactacystin and 1 mM cold lactacystin amide; (8) 10
.mu.M [.sup.3H]lactacystin and 1 mM cold dihydroxy acid; (9) 10
.mu.M [.sup.3H]lactacystin and 1 mM cold 6-deoxylactacystin; (10)
10 .mu.M [.sup.3H]lactacystin and 1 mM cold (6R,7S)-lactacystin
(6-epi, 7-epi); (11) 10 .mu.M [.sup.3H]lactacystin and 1 mM cold
des(hydroxyisobutyl)-lactacystin. Crude extracts from Neuro2A
neuroblastoma cells or bovine brain were incubated in the presence
of 10 .mu.M [.sup.3H]lactacystin in the presence or absence of 1 mM
cold competitor (added simultaneously) for 24 h at room
temperature, followed by SDS-polyacrylamide gel electrophoresis
(0.75-mm-thick 12% polyacrylamide gel). The gel was stained in 0.1%
Coomassie brilliant blue R-250, 12% acetic acid, 50% MeOH, and then
destained in 12% acetic acid, 50% MeOH, followed by impregnation
with EN.sup.3HANCE (NEN) and precipitation of the fluor with water.
The gel was dried on Whatman filter paper in a gel dryer at
65.degree. C. for 30 min and then exposed to Kodak SB film at
-78.degree. C.
Example 3
Separation of Purified Protein Complex (20S Proteasome)
[0160] Bovine brain was frozen in liquid N.sub.2. A total of two kg
of brain was dry homogenized in a Waring blender for one minute.
All operations were performed at 4.degree. C., except as noted. The
following buffer (6 liters total) was then added (pH 7.7): 18.25 mM
K.sub.2HPO.sub.4, 6.75 mM KH.sub.2PO.sub.4, 0.27 M sucrose, 2 mM
EDTA, 2 mM EGTA, 25 mM NaF, 5 mM tetrasodium pyrophosphate, 5
.mu.g/ml each of leupeptin and pepstatin A, and 5 mM
.beta.-mercaptoethanol. The tissue was wet homogenized in the
Waring blender for two minutes. The homogenate was centrifuged at
5,000 g for 15 min and then at 12,000 g for 30 min. Ammonium
sulfate was added to the supernatant to 50% saturation, and the
sample spun at 10,000 g for 20 min. The 50-60% saturation ammonium
sulfate fraction, containing the lactacystin-binding activity as
determined by SDS-PAGE and fluorography of samples incubated with
radioactive compound, was then dialyzed overnight against 20 mM
MES-NaOH, pH 5.6, 5 mM .beta.-mercaptoethanol, followed by
SP-sepharose chromatography (Pharmacia SP-sepharose, fast flow;
120-ml bed volume column) with 20 mM MES-NaOH, pH 5.6, 5 mM
.beta.-mercaptoethanol and an NaCl gradient from 0-0.3 M (500-ml
gradient; flow rate=2 ml/min).
[0161] After pooling and diluting the relevant fractions, the pH
was adjusted to 8 with 1 M Tris-HCl, pH 8. Q-sepharose
chromatography (Pharmacia Q-sepharose, fast flow; 16-ml bed volume
column) was performed with 20 mM Tris-HCl, pH 8, 5 mM
.beta.-mercaptoethanol and a NaCl gradient from 0-0.5 M (120-ml
gradient; flow rate=1 ml/min). The relevant fractions were pooled,
concentrated, and then applied to a Pharmacia Superose 6 gel
filtration column (10 mM Tris-HCl, 1 mM EDTA, pH 8, 5 mM
P-mercaptoethanol; flow rate=0.5 mi/min), with the
lactacystin-binding activity corresponding to a single high
molecular weight peak. This peak was isolated and treated with 10
.mu.M [.sup.3H]lactacystin or 10 .mu.M [.sup.3H].beta.-lactone for
24 h at 25.degree. C. Trifluoroacetic acid (TFA) was added to 0.1%,
and the sample was allowed to stand at room temperature for 20 min.
Reverse-phase HPLC was then carried out at room temperature using a
Vydac C4 column (300 .ANG./4.6.times.150 mm) with 20-40%
acetonitrile/0.1% TFA over 10 min and then 40-55% acetonitrile/0.1%
TFA over 30 min (flow rate=0.8 ml/min). An IN/US .beta.-RAM in-line
scintillation detector was used to monitor radioactivity.
[0162] The lactacystin-binding proteins were purified from bovine
brain, and both were found to reside in the same high-molecular
weight protein complex by gel-filtration chromatography. Treatment
of the complex with [.sup.3H]lactacystin did not cause its
dissociation, and the radioactivity uniquely comigrated with the
complex. The molecular weight of the complex was estimated to be
700 kDa, and SDS-PAGE revealed that it consisted of numerous
proteins with molecular weights of .about.24-32 kDa. Edman
degradation of blotted protein revealed the sequences of several
proteasome subunits in the 24 kDa band, leading to a tentative
identification of the complex as the 20S proteasome. After the
complex was treated with [.sup.3H]lactacystin and subjected to
reverse-phase HPLC to separate the proteasome subunits, eleven to
twelve distinct peaks were resolved. However, the radioactivity was
associated exclusively with the first two peaks, and predominantly
with the second. These first two peaks were judged to be
homogeneous by Coomassie blue staining of SDS-polyacrylamide gels
and by sequencing of tryptic fragments, while some of the
subsequently eluting, larger peaks were clearly not homogeneous.
The ratio of counts incorporated into the first peak versus the
second peak varied with the batch of protein, the length of
incubation, and the ligand. The first peak is labeled more slowly,
and the degree of labeling of the first peak relative to the second
is greater with [.sup.3H]lactacystin than with the
[.sup.3H].beta.-lactone at any given time. A one or two hour
reaction with [.sup.3H].beta.-lactone results in only trace
labeling of the first peak, while a 24 hour reaction with
[.sup.3H].beta.-lactone or [.sup.3H]lactacystin results in
significant labeling of this peak. The selectivity runs opposite to
the relative chemical reactivity of the two compounds, and this
finding suggests that the N-acetylcysteine moiety of lactacystin
may facilitate recognition by this secondary protein. The first
peak to elute from the reverse-phase HPLC column contained only a
.about.32 kDa protein, which corresponded to the 32 kDa secondary
lactacystin-binding protein identified earlier.
Example 4
Amino Acid Sequence of Purified Bovine Lactacystin-Binding
Proteins
[0163] Purified 20S proteasome (purified as described in Example 3)
was incubated for 24 h at room temperature with 10 .mu.M
lactacystin in 10 mM Tris-HCl, 1 mM EDTA (pH 8). The solution was
then diluted tenfold with 20% aqueous acetonitrile containing 0.1%
trifluoroacetic acid (TFA) and allowed to stand at room temperature
for 5 min. Reverse-phase HPLC was then performed at room
temperature using a Vydac C4 column (100 .ANG./4.6.times.150 mm)
with 20-40% acetonitrile/0.1% TFA over ten minutes and then 40-55%
acetonitrile/0.1% TFA over 30 min to elute the proteasome subunits
(flow rate=0.8 mi/min). Peaks were collected based on ultraviolet
light absorbance at 210 and 280 nm, with the primary
lactacystin-modified protein being the second to elute off the
column and the secondary lactacystin-binding protein being the
first. Protein from repeated injections was pooled, lyophilized and
subjected to Edman degradation following tryptic digestion and
reverse-phase HPLC separation of tryptic fragments. The putative
lactacystin-modified residue was identified by adding subunit X/MB1
isolated from [.sup.3H]lactacystin-treated proteasome to a sample
that had been treated with unlabeled lactacystin, and then
isolating and sequencing radioactive tryptic fragments. At one
position, the phenylthiohydantoin-amino acid derivative was not
identifiable, indicating possible modification of the residue.
[0164] Sequences from direct N-terminal sequencing and from tryptic
fragments derived from the primary bovine lactacystin-binding
protein aligned with sequences of human proteasome subunit X
(predicted from the cDNA clone), and human LMP7-E2 (predicted from
the exon 2-containing cDNA clone). Sequence 1 (from direct
N-terminal sequencing) is also aligned with sequences of
Saccharomyces cerevisiae Pre-2 (predicted from the genomic clone)
and Thermoplasma acidophilum .beta.-subunit (predicted from the
cloned gene). The N-terminal heptapeptide corresponds to the
tryptic fragment that appears to contain a lactacystin-modified
N-terminal threonine residue. The threonine at this position also
corresponds to the putative N-termini of the mature, processed
forms of all the homologs listed. Upper-case letters denote high
confidence sequence, while lower case letters indicate lower
confidence assignments. TABLE-US-00004 TABLE 1 DIRECT N-TERMINAL
SEQUENCES 1. Direct N-terminal sequence of primary bovine
lactacystin-binding protein TTTLAFKFRHggIIA SEQ ID NO: 1 Human
subunit X/MB1 5 TTTLAFKFRHGVIVA 19 SEQ ID NO: 2 Human LMP7-E2 74
TTTLAFKFQHGVIAA 88 SEQ ID NO: 3 S. cerevisiae Pre-2 76
TTTLAFRFQGGIIVA 90 SEQ ID NO: 4 T. acidopholum .beta.-subunit 9
TTTVGITLKDAVIHA 23 SEQ ID NO: 5 2. Primary bovine
lactacystin-binding protein DAYSGGSVSLY SEQ ID NO: 6 Human subunit
X 171 DAYSGGAVNLYHVR 184 SEQ ID NO: 7 Human LMP7-E2 239
DSYSGGVVNHYHMK 252 SEQ ID NO: 8 3. Primary bovine
lactacystin-binding protein VIEINPYLLGTMAGGAADCSF SEQ ID NO: 9
Human subunit X 38 VIEINPYLLGTLAGGAADCQFWER 61 SEQ ID NO: 10 Human
LMP7-E2 106 VIEINPYLLGTMSGCAADCQYWER 129 SEQ ID NO: 11 4. Primary
bovine lactacystin-binding protein GYSYDLEVEEAYDLAR SEQ ID NO: 12
Human subunit X 146 GYSDLEVEQAYDLAR 161 SEQ ID NO: 13 Human LMP7-E2
214 GYRPMLSPEEAYDLGR 229 SEQ ID NO: 14
[0165] Sequences of tryptic fragments derived from the secondary
lactacystin-binding protein aligned with N-terminal fragment
sequence of human erythrocyte proteasome .alpha. chain and of rat
liver proteasome chain. TABLE-US-00005 TABLE 2 TRYPTIC FRAGMENT
SEQUENCES Secondary bovine lactacystin-binding activity TTIAGVYK
DGIVLGADTR SEQ ID NO: 15 Human erythrocyte proteasome .alpha. chain
1 XXIAGVVYK DGIVLGADTR 19 SEQ ID NO: 16 Rat liver proteasome chain
1 1 TTIAGVVYK DGI 12 SEQ ID NO: 17.sup.1
Sequence analysis of tryptic fragments derived from this protein
showed it to be homologous to the proteasome a chain, a .about.30
kDa protein identified in purified human erythrocyte proteasome and
rat liver proteasome for which only an N-terminal fragment sequence
exists. The second peak to elute contained only a .about.24 kDa
protein, the primary lactacystin-binding protein. Sequence from the
N-terminus of the protein and from derived tryptic fragments showed
high identity to the recently discovered 20S proteasome subunit X,
also known as MB1, a homolog of the major histocompatibility
complex (MHC)-encoded LMP7 proteasome subunit.
Example 5
Kinetics of Inhibition of 20S Proteasome Peptidase Activities
[0166] Experiments involving the proteasome were performed as
follows: 20S proteasome (.about.5 ng/.mu.l) in 10 mM Tris-HCl, pH
7.5, 1 mM EDTA was incubated at 25.degree. C. in the presence of
lactacystin or lactacystin analogs in DMSO or MeOH [not exceeding
5% (v/v)]. Aliquots for fluorescence assay were removed at various
times following addition of compound and diluted in 10 mM Tris-HCl,
pH 7.5, 1 mM EDTA containing fluorogenic peptide substrates (100
.mu.M final). Suc-LLVY-AMC, Cbz-GGR-.beta.NA and Cbz-LLE-.beta.A
(AMC=7-amido-4-methylcoumarin; .beta.NA=.beta.-naphthylamide) were
used to assay for the chymotrypsin-like, trypsin-like and
peptidylglutamyl-peptide hydrolyzing activities, respectively.
[0167] Biological activity of compound refers to the ability of the
compound to induce neurite outgrowth in Neuro 2A neuroblastoma
cells and to inhibit cell cycle progression in MG-63 osteosarcoma
cells. TABLE-US-00006 TABLE 3 Kinetics of Inhibition
.kappa..sub.assoc = .kappa..sub.obs/[I] (s.sup.-1M.sup.-1)
Biological Chymotrypsin-like Trypsin-like Peptidylglutamyl-peptide
activity of activity (Suc- activity (Cbz- hydrolyzing activity
(Cbz- Compound (concentration) compound LLVY-AMC) GGR-.beta.NA)
LLE-.beta.NA) Lactacystin (10 .mu.M) + 194 .+-. 15 10.1 .+-. 1.8
Lactacystin (100 .mu.M) + 4.2 .+-. 0.6 .beta.-Lactone (1 .mu.M) +
3059 .+-. 478 .beta.-Lactone (5 .mu.M) + 208 .+-. 21 .beta.-Lactone
(50 .mu.M) + 59 .+-. 17 Dihydroxy acid (100 .mu.M) - No inhibition
No inhibition No inhibition Lactacystin amide (12.5 .mu.M) + 306
.+-. 99 Phenylacetyl lactacystin + 179 .+-. 19 (12.5 .mu.M)
6-Deoxylactacystin - No inhibition (12.5 .mu.M) (6R,7S)Lactacystin
(6-epi,7- - No inhibition epi) (12.5 .mu.M) Des(hydroxyisobutyl)- -
No inhibition lactacystin (12.5 .mu.M)
Example 6
Selectivity of Protease Inhibition
[0168] Experiments involving the other proteases were similar to
Example 5, except that buffers used for incubation with lactacystin
were as follows: .alpha.-Chymotrypsin: 10 mM Tris-HCL, pH 8, 1 mM
EDTA (plus 100 .mu.M Suc-LLVY-AMC for fluorescence assay); Trypsin:
10 mM Tris-HCL, pH 8, 20 mM CaCl.sub.2 (plus 100 .mu.M
Cbz-GGR-.beta.NA for assay); Calpain I: 20 mM Tris-HCL, pH 8, 1 mM
CaCl.sub.2, 1 mM DTT (plus 100 .mu.M Suc-LLVY-AMC for assay);
Calpain II: 20 mM Tris-HCL, pH 8, 10 mM CaCl.sub.2, 1 mM DTT (plus
100 .mu.M Suc-LLVY-AMC for assay); Papain: 50 mM MES-NaOH, pH 6.4,
1 mM DTT (plus 100 .mu.M Cbz-RR-AMC for assay); Cathepsin B: 100 mM
KH.sub.2PO.sub.4, pH 5.5, 2 mM EDTA, 1 mM DTT (plus 100 .mu.M
Cbz-RR-AMC for assay). Fluorescent emission at 460 nm with
excitation at 380 nm was measured for AMC-containing substrates,
and emission at 410 nm with excitation at 335 nm was measured for
.beta.NA-containing substrates. The fluorescence assays were
conducted at 25.degree. C., each experiment was performed at least
three times, and values represent mean.+-.standard deviation.
TABLE-US-00007 TABLE 4 Inhibition of Other Proteases Protease
tested Effect of lactacystin (100 .mu.M) .alpha.-Chymotrypsin No
inhibition Trypsin No inhibition Calpain I No inhibition Calpain II
No inhibition Papain No inhibition Cathepsin B No inhibition
[0169] The effects of lactacystin and the P-lactone on proteasame
peptitase on activities using fluorogenic peptide substrates were
assessed. All three peptidase activities were inhibited,
irreversibly in the case of the trypsin-like and chymotrypsin-like
activities and reversibly in the case of the PGPH activity. The
apparent second-order rate constant for association of each
inhibitor with the enzyme, k.sub.assoc, was determined for each of
the activities (Table 1A). Lactacystin inhibits the
chymotrypsin-like activity the fastest (k.sub.assoc=194.+-.15
M-1.sub.S-1), the trypsin-like activity 20-fold more slowly, and
the PGPH activity 50-fold more slowly. The fact that the inhibition
kinetics are different for the three activities lends further
support to the notion that the activities are due to separate
active sites. The P-lactone displays the same rank order but
inhibits each activity 15-20 times faster than does lactacystin
itself, in accord with the greater expected chemical reactivity of
the .beta.-lactone. It is also possible that, upon initial binding
to the protein target, the lactacystin thioester is cyclized in a
rate-limiting step to the .beta.-lactone, which serves as an
activated intermediate for attack by the nucleophile.
[0170] The reversibility of the inhibitory effects was assessed by
measuring residual peptidase activity after removal of excess
inhibitor by extensive serial dilution/ultrafiltration. The
trypsin-like and chymotrypsin-like activities were still completely
inhibited in the lactacystin-treated samples following
dilution/ultrafiltration, implying very low k.sub.off of inhibitor
from enzyme, whereas controls untreated with inhibitor maintained
activity (data not shown). In the case of the PGPH activity,
removal of the inhibitor was accompanied by a return of the
catalytic activity; inhibition of the PGPH activity could be due to
non-covalent association of lactacystin with the PGPH site or
covalent association with turnover.
[0171] Previously, we had shown that the ability of analogs to
cause neurite outgrowth in Neuro 2A cells was mirrored by their
ability to inhibit cell-cycle progression in MG-63 osteosarcoma
cells, and that modifications to the .gamma.-lactam part of the
molecule impared both activities whereas modifications of the
N-acetylcysteine moiety had little effect [Fenteany, 1994 3135]. We
therefore tested the ability of the analogs to inhibit the 20S
proteasome, but as our supplies of these materials were
insufficient to examine all three activities, we focused on the
chymotrypsin-like activity, which is inhibited most rapidly by
lactacystin. The same trends found in the biological studies were
apparent: the biologically active compounds inhibited the
chymotryptsin-like activity about as well as lactacystin itself,
and the biologically inactive compounds did not inhibit this
activity as all (Table 3). Modifications of the .gamma.-lactam core
of lactacystin mitigate its effect, but modifications of the
N-acetylcysteine moiety do not.
Example 7
Assay of the Ability of Lactacystin (.beta.-Lactone Form) Can Block
TNF-.alpha. Dependent Degradation of I.kappa.B-.alpha. In Vivo.
[0172] Hela cells were plated onto 6-well plates in DME plus 10%
fetal calf serum (3 mls/well). Cells were then pretreated with
0.125% DMSO 40 .mu.M G132
(carbobenzoxyl-leucinyl-leucinyl-leucinal-H)(40 .mu.M, lanes 103),
or 5 .mu.M .beta.-lactone for one hour, followed by treatment with
1,000 U/ml TNF-.alpha. or phosphate buffered saline (PBS). Cells
were harvested after 0 min, 20 min, or 40 min of further incubation
at 37.degree. C. Cells were then lysed in buffer containing NP-40
and protease inhibitors, and the proteins separated on a 10%
SDS-polyacrylamide gel. The proteins were then transferred to
nitrocellulose and probed with antibodies against the C-terminal 20
amino acids of human I.kappa.B-.alpha..
[0173] Previous reports have shown that the inducible degradation
of I.kappa.B is preceded by phosphorylation of the protein (Beg et
al., 1993, Mol. Cell. Biol. 13:3301; Traenckner et al., 1994, EMBO
J. 13:5433; Miyamoto et al., 1994, PNAS 91:12740; Lin et al., 1995,
PNAS 92:552; DiDonato et al., 1995, Mol. Cell. Biol. 15:1302; and
Alkalay et al., 1995, Mol. Cell. Biol. 15:1294). The phosphorylated
IKB is then preferentially degraded, apparently by the proteasome
(Palombella et al., 1994, Cell 78:773). Phosphorylation is
evidenced by a slightly heavier shift in the electrophoretic
mobility of I.kappa.B. I.kappa.B from cells induced with TNF show
the requisite pattern of phosphorylation and degradation over time.
Cells treated with PBS only show no change in the level or
modification of I.kappa.B. As was reported previously, (Palombella
et al, 1994), the peptide aldehyde proteasome inhibitor MG132
blocks the degradation of IKB and stabilizes the phosphorylated
form. .beta.-lactone also blocks degradation and stabilizes the
phosphorylated form of I.kappa.B. These results indicate that
lactacystin does not effect the TNF dependent signalling pathway
that leads to the phosphorylation of I.kappa.B, but does block the
degradation of the protein. Lactacystin inhibits the degradation of
I.kappa.B after TNF induction of Hela cells, most likely by
specifically blocking the action of the proteasome.
Example 8
Assay of the Effect of Lactacystin (.beta.-Lactone Form) on the
Proteasome Dependent p105 to p50 Processing In Vivo.
[0174] COS cells were plated onto 100 mm dishes, then, with
DEAE-Destran, either mock transfected, or transfected with 3 .mu.g
of pcDNA plasmid containing the human p105 cDNA. Forty-eight hrs
after transfection, cells were pretreated for 1 hour with 0.5%
DMSO, 50 .mu.M calpain inhibitor II, 50 .mu.M MG132, or 10 .mu.M
.beta.-lactone. Cells were then pulse labelled with 250 uCi/plate
of .sup.35S-methionine/cysteine for 15 minutes, and either
harvested immediately or followed by a 2 hour chase with excess
unlabelled methionine and cysteine. Cells were then lysed with
SDS/Tris, and proteins were immunoprecipitated with anti-p50
antibodies (recognize the N-terminal half of the p105 protein).
These proteins were then separated on a 10% SDS-polyacrylamide gel,
which was then fixed, dried, and exposed to autoradiographic
film.
[0175] An analysis of the proteins isolated immediately after the
pulse-labelling period reveals significant amounts of labelled p105
protein and very little p50. After the 2 hour chase period, the
levels of p105 were reduced, and a new band that corresponds to p50
protein is apparent, as was expected (Fan and Maniatis, 1991,
Nature 354:395; Palombella et al., 1994, Cell 78:773). Pretreatment
of cells with calpain inhibitor II has no effect on the processing
of p1O5 to p5O (lane 4). However, treatment of cells with the
peptide aldehyde proteasome inhibitor MG132 completely blocks the
appearance of p50 (lane 5), as has been reported previously
(Palombella et al., 1994). Low levels of .beta.-lactone (10 .mu.M)
have only a slight effect on the level of p50, but higher
concentrations (50 .mu.M) result in the complete inhibition of the
processing of p105 to p50. Lactacystin efficiently blocks the
proteasome dependent procession of p105 to p50 in vivo.
Example 9
Neurite Outgrowth Assay
[0176] Compounds are dissolved in the minimal amount of methanol
(MeOH) or dimethyl sulfoxide (DMSO) required for solubilization. No
more than 0.1% solvent is present in any assay. When necessary,
solutions are evaporated to dryness and resuspended in cell culture
medium to their final concentrations before use.
[0177] Neuro 2A, IMR-32, PC12, and MG-63 cells are obtained from
the American Type Culture Collection. Neuro 2A and IMR-32 cells are
cultured in Eagle's minimal essential medium (MEM) containing 10%
(vol/vol) fetal bovine serum (FBS). PC12 cells are grown in RPMI
1640 medium containing 10% (vol/vol) horse serum and 5% FBS, and
MG-63 cells are cultured in RPMI 1640 containing 10% FBS.
[0178] Neuro 2A cells are plated at a density of 1.times.10.sup.4
cells per 1 ml per well in 12-well polystyrene dishes
(22-mm-diameter flat-bottom wells) and are grown for 24 h in MEM
with 10% FBS prior to any treatment. In the relevant experiments,
nocodazole, cytochalasin B, or cycloheximide is added 3 h before
addition of lactacystin. In the serum deprivation experiments,
cells are switched to serum-free MEM 24 h after plating and, when
relevant, incubated another 24 h before addition of lactacystin and
subsequently maintained in serum-free conditions.
Example 10
Cell Cycle Analysis
[0179] MG-63 cells are plated at 7.5.times.10.sup.4 cells per 3 ml
per 25-cm.sup.2 flask and grown for 24 h in RPMI containing 10%
FBS. These subconfluent MG-63 cultures are synchronized in
G.sub.0/G.sub.1 by changing the medium to RPMI containing 0.2% FBS
and incubating for 64 h. This is followed by stimulation with 2 ml
of RPMI containing 10% FBS and addition of compounds. Neuro 2A
cells are grown to .about.2.times.10.sup.7 in 175-cm.sup.2 flasks
in MEM with 10% FBS. Mitotic cells are harvested by shaking for 5
min at 100 rpm on a rotary shaker. The detached cells are replated
at 1.5.times.10.sup.5 cells per 2 ml per 25-cm.sup.2 flask and
incubated for 30 min to allow for reattachment prior to addition of
lactacystin. Cells are harvested for cell cycle analysis 21 h after
stimulation in the case of the MG-63 cells and 20 h after replating
in the case of the Neuro 2A cells and then were processed for flow
cytometry. DNA histograms are obtained using a Becton Dickinson
FACScan flow cytometer.
Example 11
Purification of 20S Proteasome and Proteasome Activator PA28
[0180] 20S proteasome was purified by a modification of procedures
by Hough et al. [Hough, R., et al., (1987) J. Biol. Chem., 262,
8303-8313] and Ganoth et al. [Ganoth, D., et al., (1988) J. Biol.
Chem., 263, 12412-12419. PA28 Activator was purified by a
modification of procedures of Chu-Ping et al. [Chu-Ping, M., et
al., (1992) J. Biol. Chem., 267, 10515-10523]. Rabbit reticulocytes
were obtained from Green Hectares (Oregon, Wis.). The reticulocytes
were washed three times by suspending them in ice cold PBS and
centrifuging at 2000.times. g for 15 min. After the final wash, the
cells were lysed in 1.5 volumes of cold distilled water containing
1 mM DTT. The mixture was then passed through a Glas-CO
Bio-Nebulizer at 250 psi to ensure complete cell lysis. The lysate
was then centrifuged at 100,000.times. g for one hour to remove
cell debris. The supernatant (extract) was brought to 20 mM HEPES
pH 7.6, 1 mM DTT (Buffer A), filtered through a 0.2 .mu.m filter,
and applied to a DE52 anion exchange column equilibrated in Buffer
A. The column was washed with 5 volumes of Buffer A and absorbed
proteins were eluted with 2.5 volumes of Buffer A containing 500 mM
NaCl. The eluate (Fraction II) was brought to 38% saturation with
ammonium sulfate and centrifuged at 10,000.times. g for 20 minutes.
The supernatant was brought to 85% saturation with ammonium sulfate
and centrifuged 10,000.times. g for 20 minutes. The resulting
pellets were resuspended in a minimal volume of Buffer A and
dialyzed vs. 4L of Buffer A overnight. The dialysate (Fraction
IIIB), which contains both 20S proteasome and PA28 activator, was
applied to a Mono Q anion exchange column. Elution was performed by
a 40 column volume gradient from 0-500 mM NaCl.
20S Proteasome
[0181] Column fractions were assayed for SDS-stimulated
Suc-Leu-Leu-Val-Tyr-AMC hydrolyzing activity. The active fractions
were pooled, diluted to 50 mM [NaCl], and applied to a 1 mL Heparin
Sephrose Hi-Trap column. Elution was performed with a 20 column
volume gradient from 50 mM-500 mM NaCl. Active fractions were
pooled and concentrated using a 30,000 MWCO Centricon centrifugal
concentrator. The concentrate was applied to a Superose 6 size
exclusion chromatography column and eluted with Buffer A containing
100 mM NaCl. The active fractions that were judged to be pure by
SDS-PAGE analysis were pooled and stored at -80.degree. C. for
subsequent use.
PA28
[0182] Column fractions (vide supra) were assayed for their ability
to stimulate the Suc-Leu-Leu-Val-Tyr-AMC hydrolysing activity of
exogenous 20S proteasome. The active fractions were pooled and
diluted to 10 mM NaCl. This material was then further purified and
concentrated on a 1 ml Resource Q anion exchange column with a 40
column volume gradient from 10-500 mM NaCl. Active fractions were
pooled and concentrated using a 10,000 MWCO Centricon centrifugal
concentrator. The concentrate was applied to a Superdex 200 size
exclusion chromatography column and eluted with Buffer A containing
100 mM NaCl. The active fractions that were judged to be pure by
SDS-PAGE analysis were pooled and stored at -80.degree. C. for
subsequent use.
Example 12
Lactacystin Inactivation of Proteasome Activity
[0183] 2 ml of assay buffer (20 mM HEPES, 0.5 mM EDTA, pH 8.0) and
Suc-Leu-Leu-Val-Tyr-AMC in DMSO were added to a 3 ml fluorescent
cuvette and the cuvette was placed in the jacketed cell holder of a
Hitachi F-2000 fluorescence spectrophotometer. The temperature was
maintained at 37.degree. C. by a circulating water bath. 0.34 .mu.g
of proteasome and 3.5 .mu.g of PA28 were added and the reaction
progress was monitored by the increase in fluorescence at 440 nm
(.lamda.ex=380 nm) that accompanies production of free AMC. The
progress curves exhibited a lag-phase lasting 1-2 minutes resulting
from the slow formation of the 20S-PA28 complex. After reaching a
steady-state of substrate hydrolysis, lactacystin was added to a
final concentration of 1 .mu.M and the reaction was monitored for 1
hr. The fluorescence (F) versus time (t) data were collected on a
microcomputer using LAB CALC (Galactic) software. k.sub.inact
values were estimated by a nonlinear least-squares fit of the data
to the first-order equation: F=A(1-e.sup.-kt)+C where C=F.sub.t=0
and A=F.sub.t=4-F.sub.t=0. Owing to the complex kinetics of
lactacystin hydrolysis and its mechanism of inactivation of the
proteasome the kinetics of proteasome inactivation are also complex
and this fact is reflected in the curve fitting. There was a small
systematic deviation of the measured progress curves from the
"best-fit" curves generated by fitting the data to the equation
above. Estimates of k.sub.inact were obtained using this simple
kinetic model.
Example 13
Inhibition of Protein Degradation in C2C12 Cells
[0184] C2C12 cells (a mouse myoblast line) were labeled for 48 hrs
with .sup.35S-methionine. The cells were then washed and
preincubated for 2 hrs in the same media supplemented with 2 mM
unlabeled methionine. The media was removed and replaced with a
fresh aliquot of the preincubation media containing 50% serum, and
a concentration of the compound to be tested. The media was then
removed and made up to 10% TCA and centrifuged. The TCA soluble
radioactivity was counted. Inhibition of proteolysis was calculated
as the percent decrease in TCA soluble radioactivity. From this
data, an IC.sub.50 for each compound was calculated. The compounds
can be qualitatively grouped into ++, +, and 0 categories, wherein
wherein ++ indicates better activity than +, and 0 indicates little
or no activity, due to insolubility, instability, or other reasons.
For example, in the lactacystin group compounds I-1, I-2, and I-3
were in the ++ category; compound I-4 was in the + category.
Turning to the P series of lactone-like compounds, P-1 through
[0185] P-4 were in the ++ category; compounds P-5 and P-6 were in
the + category; and compound P-7 was in the 0 category.
Example 14
Synthesis of Compound I-1
[0186]
(3R,4S,5R,1'S)-4-Hydroxy-5(1'-hydroxy-2'-methylpropyl)-3-methylpyr-
rolidin-2-one-5-carboxylic acid (10.0 mg, 43.2 mmol; made by the
route described by Corey, E. J.; Reichard, G. A., J. Am. Chem. Soc.
1992, 114, 10677) was dissolved in 0.4 mL anhydrous dichloromethane
and then treated, in order, with tri-ethylamine (18.0 mL, 0.13
mmol, 3.0 equiv.), 13.0 mg bis(2-oxo-3-oxazolidinyl)-phosphinic
chloride (51.1 mmol, 1.2 equiv.) and benzyl mercaptan (8.0 mL, 68.1
mmol, 1.58 equiv.). The solution was stirred for 5 hours at room
temperature and then diluted with dichloromethane and washed with
water. The aqueous layer was extracted with ethyl acetate and the
organic layers were combined and dried over magnesium sulfate.
Filtration followed by removal of the solvent afforded a crude
solid which was triturated with 19:1 hexane/ethyl acetate and
collected by filtration. The white solid obtained was then
dissolved in a minimum amount of acetonitrile and the clear
solution was diluted with water then frozen and lyophilized
overnight. This afforded 8.0 mg of the desired thioester I-1 (55%)
as a fluffly white solid. .sup.1H NMR (DMSO-d.sub.6, 300 MHz) d
8.34 (s, 1H, NH), 7.19-7.28 (m, 5H), 5.40 (d, 1H, .sup.3J=6.4 Hz,
OH), 5.11 (d, 1H, .sup.3J=7.4 Hz, OH), 4.39 (t, .sup.3J=6.4 Hz),
4.04 (s, 2H), 3.76 (t, 1H, .sup.3J=7.4 Hz), 2.62 (m, 1H), 1.55 (m,
1H), 0.90 (d, 3H, .sup.3J=7.5 Hz), 0.85 (d, 3H, .sup.3J=6.6 Hz),
0.66 (d, 3H, .sup.3J=6.8 Hz).
Example 15
Synthesis of Compound I-2
[0187] To a solution of glutathione (360 mg, 1.17 mmol) in 11 miL
of water was added 1.35 mL of 1.004 N sodium hydroxide in water.
This solution was then added to a freshly prepared solution of
clasto-lactacystin-b-lactone (49.0 mg, 0.23 mmol; made by the route
described by Corey, E. J.; Reichard, G. A. J. Am. Chem. Soc. 1992,
114, 10677) in 10 mL of acetonitrile. After stirring the solution
at room temperature for 4 hours, the pH was adjusted to about pH=4
by the slow addition of 1 N aqueous hydrochloric acid solution. The
acetonitrile was removed in vacuo and the aqueous phase was frozen
and lyophilized. The solid obtained was redissolved in water,
filtered and lyophilized again. The white solid obtained was
purified by reverse phase HPLC [Waters Prep Delta-Pak.RTM.
19.times.300 mm C18 column eluting with 98% 0.05% trifluoroacetic
acid/H.sub.2O and 2% 0.05% trifluoroacetic acid/methanol to 80%
0.05% trifluoroacetic acid/H.sub.2O and 20% 0.05% trifluoroacetic
acid/methanol in 65 min at 5 mL/min]. After lyophilizing the pure
fractions, glutathione thioester adduct I-2 (48 mg, 40%) was
obtained as a fluffly white solid. 1H NMR (DMSO-d6, 300 MHz) d
7.73-8.26 (m, 4H), 5.28 (bs, 1H, OH), 5.09 (bs, 1H, OH), 4.44 (m,
1H), 4.37 (bd, 1H, 3J=5.9 Hz), 3.74 (m, 2H), 3.23 (dd, 1H, J=13.3,
4.6 Hz), 2.95 (dd, 1H, J=13.3, 8.8 Hz), 2.62 (m, 1H), 2.29-2.32 (m,
2H), 1.98-2.08 (m, 2H), 1.51-1.63 (m, 1H), 0.92 (d, 3H, .sup.3J=7.5
Hz), 0.86 (d, 3H, .sup.3J=6.6 Hz), 0.71 (d, 3H, .sup.3J=6.7
Hz).
Example 16
Synthesis of Compound I-4
[0188] (3R,4S
,5R,1'S)-4-Hydroxy-5(1'-hydroxy-2'-methylpropyl)-3-methylpyrrolidin-2-one-
-5-carboxylic acid (20.0 mg, 86.5 mmol; made by the route described
by Corey, E. J.; Reichard, G. A. J. Am. Chem. Soc. 1992, 114,
10677) was dissolved in 1.0 m1L acetonitrile and then treated, at
0.degree. C., with 1,8-diazabicyclo[5.4.0]undec-7-ene (14.0 mL,
93.6 mmol, 1.08 equiv.) followed by benzyl bromide (12.0 mL, 101
mmol, 1.17 equiv.). The mixture was stirred at room temperature
overnight and was then partitioned between ethyl acetate and water.
The aqueous layer was extracted three times with ethyl acetate and
the organic layers were then combined and dried over magnesium
sulfate. Filtration followed by removal of the solvent afforded a
crude solid which was triturated with hexane and collected by
filtration. The white solid obtained was then dissolved in a
minimum amount of acetonitrile and the clear solution was diluted
with water then frozen and lyophilized overnight. This afforded 6.7
mg of the desired ester 1-4 (24%) as a fluffly white solid. 1H NMR
(DMSO-d6, 300 MHz) d 8.02 (s, 1H, NH), 7.31-7.44 (m, 5H), 5.45 (d,
1H, 3J=6.4 Hz, OH), 5.11 (d, 1H, 2J=12.5 Hz), 5.03 (d, 1H, 3J=7.2
Hz, OH), 5.00 (d, 1H, 2J=12.5 Hz), 4.31 (t, 3J=6.4 Hz), 3.76 (t,
1H, .sup.3J=7.2 Hz), 2.68 (m, 1H), 1.52 (m, 1H), 0.89 (d, 3H,
3J=7.4 Hz), 0.85 (d, 3H, 3J=6.6 Hz), 0.67 (d, 3H, 3J=6.7 Hz).
Example 17
Synthesis of Compound P-2
[0189]
(3S,7aR)-3-Phenyl-5,6,7,7a-tetrahydro-1H-pyrrolo[1,2-c]oxazol-5-on-
e was prepared in three steps from D-Pyroglutamic acid by a known
procedure (Thottathil, J. K. et al. J. Org. Chem. 1986, 51, 3140)
and was converted to
(3S,7aR)-3-Phenyl-1H-pyrrolo[1,2-c]oxazol-5-one in two steps
(selenylation and oxidative elimination) by the method reported by
Hamada et al. (J. Am. Chem. Soc. 1989, 111, 1524).
(3S,7aR)-3-Phenyl-1H-pyrrolo[1,2-c]oxazol-5-one was then converted
to
(3S,7S,7aS,1'S)-7a-(1'-acetoxy-2'-methylpropyl)-7-hydroxy-3-phenyl-5,6,7,-
7a-tetrahydro-1H-pyrrolo[1,2-c]oxazol-5-one in 6 steps by analogy
to the sequence reported by Uno et al. (J. Am. Chem. Soc. 1994,
116, 2139).
(3S,7S,7aS,1'S)-7a-(1'-acetoxy-2'-methylpropyl)-7-hydroxy-3-phenyl-5,6,7,-
7a-tetrahydro-1H-pyrrolo[1,2-c]oxazol-5-one (70 mg, 0.21 mmol) was
dissolved in 1.0 mL pyridine and treated at room temperature with
32.0 mL acetic anhydride (0.34 mmol, 1.6 equiv.). The mixture was
stirred at room temperature overnight and the solvent was removed
in vacuo affording 77 mg of desired
(3S,7S,7aS,1'S)-7a-(1'-acetoxy-2'-methylpropyl)-7-acetoxy-3-phenyl-5,6,7,-
7a-tetrahydro-1H-pyrrolo[1,2-c]oxazol-5-one (97%), a yellow oil
which was used as such in the following step.
[0190] The crude (3S,7S
,7aS,1'S)-7a-(1'-acetoxy-2'-methylpropyl)-7-acetoxy-3-phenyl-5,6,7,7a-tet-
rahydro-1H-pyrrolo[1,2-c]oxazol-5-one (75 mg, 0.20 mmol) was
dissolved in 5 mL methanol and placed under 1 atmosphere of
hydrogen in the presence of 50 mg 20% palladium hydroxide on
carbon. When a TLC analysis (1:1 hexane/ethyl acetate) showed the
reaction to be complete, the mixture was filtered and concentrated
in vacuo affording 55 mg (97%) of the desired primary alcohol which
without any purification was converted to
(4S,5R,1'S)-4-Hydroxy-5-(1'-hydroxy-2'-methylpropyl)-pyrrolidin-2-one-5-c-
arboxylic by a two step sequence (Jones' oxidation, saponification)
analogous to the sequence reported by Uno et al. (J. Am. Chem. Soc.
1994, 116, 2139).
[0191]
(4S,5R,1'S)-4-Hydroxy-5-(1'-hydroxy-2'-methylpropyl)-pyrrolidin-2--
one-5-carboxylic acid (7.0 mg, 32 mmol) was dissolved in 2.0 mL 1:1
acetonitrile/tetrahydrofuran and then treated, at room temperature,
with 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyl uronium
tetrafluoro-borate (11.0 mg, 34 mmol, 1.1 equiv.) followed by
triethylamine (0.36 mmol, 11 equiv.). The mixture was stirred for
30 min to room temperature and concentrated in vacuo. Flash
chromatography (230-400 mesh SiO, elution with ethyl acetate)
finally afforded 3 mg of the pure lac-tone P-2 (47%) obtained as
white solid. H NMR (DMSO-d6, 300 MHz) d 8.95 (bs, 1H, NH), 5.64 (d,
1H, 3J=6.7 Hz, OH), 5.26 (d, 1H, 3J=6.0 Hz, OH), 3.78 (dd, 1H,
3J=6.7, 3.5 Hz), 2.81 (dd, 1H, 2J=19.2 Hz, 3J=6.0 Hz), 2.60 (d, 1H,
2J=19.2 Hz), 1.74 (m, 1H), 0.90 (d, 3H, 3J=6.9 Hz), 0.68 (d, 3H,
.sup.3J=6.8 Hz).
Example 18
Synthesis of Compound P-3
[0192] Clasto-Lactacystin-.beta.-lactone (5.0 mg, 23.4 mmol; made
by the route described by Corey, E. J.; Reichard, G. A. J. Am.
Chem. Soc. 1992, 114, 10677) was dissolved in 1.5 mL pyridine and
treated, at room temperature with acetic anhydride (40 mL, 0.42
mmol, 18 equiv.). After stirring the solution at room temperature
overnight, the pyridine was removed in vacuo with the aid of 2 mL
of toluene affording a light brown solid. The solid was washed with
three portions of hexane and then filtered giving 5.5 mg of the
pure acetate P-3 (93%) obtained as a white solid. .sup.1H NMR
(DMSO-d.sub.6, 300 MHz) d 9.11 (bs, 1H, NH), 5.33 (d, 1H,
.sup.3J=6.1 Hz), 5.21 (d, 1H, .sup.3J=5.0 Hz), 2.84 (m, 1H), 2.06
(s, 3H), 1.90 (m, 1H), 1.08 (d, 3H, .sup.3J=7.5 Hz), 0.87 (d, 3H,
.sup.3J=6.9 Hz), 0.78 (d, 3H, .sup.3J=6.8 Hz).
Example 19
Synthesis of Compound P-4
[0193]
(3S,7aR)-3-Phenyl-5,6,7,7a-tetrahydro-1H-pyrrolo[1,2-c]oxazol-5-on-
e was prepared in three steps from D-Pyroglutamic acid by a known
procedure (Thottathil, J. K. et al. J. Org. Chem. 1986, 51, 3140)
and was converted to
(3S,7aR)-3-Phenyl-1H-pyrrolo[1,2-c]oxazol-5-one in two steps
(selenylation, and oxidative elimination) by the method reported by
Hamada et al. (J. Am. Chem. Soc. 1989, 111, 1524).
(3S,7aR)-3-Phenyl-1H-pyrrolo[1,2-c]oxazol-5-one was then converted
to
(3S,6R,7S,7aS,1'S)-7a-(1'-acetoxy-2'-methylpropyl)-6-hydroxy-7-hydroxy-3--
phenyl-5,6,7,7a-tetrahydro-1H-pyrrolo[1,2-c]oxazol-5-one in 4 steps
by analogy to the sequence reported by Uno et al. (J. Am. Chem.
Soc. 1994, 116, 2139).
[0194] (3S ,6R,7S
,7aS,1'S)-7a-(1'-acetoxy-2'-methylpropyl)-6-hydroxy-7-hydroxy-3-phenyl-5,-
6,7,7a-tetrahydro-1H-pyrrolo[1,2-c]oxazol-5-one was then converted
in 4 steps (acetylation, hydrogenolysis, oxidation and
saponification) to
(3R,4S,5R,1'S)-4-Hydroxy-5-(1'-hydroxy-2'-methylpropyl)-3-hydroxypyrrolid-
in-2-one-5-carboxylic acid in the same manner described for making
P-2.
[0195] (3R,4S
,5R,1'S)-4-Hydroxy-5-(1'-hydroxy-2'-methylpropyl)-3-hydroxypyrrolidin-2-o-
ne-5-carboxylic acid (20 mg, 86 mmol) was converted to the
corresponding .beta.-lactone by the method described for making
P-2. This afforded 3 mg of the pure lactone P-4 (16%) obtained as
white solid. H NMR (DMSO-d.sub.6, 300 MHz) d 8.96 (s, 1H, NH), 6.08
(bs, 1H, OH), 5.63 (d, 1H, .sup.3J=6.6 Hz, OH), 5.12 (d, 1H,
.sup.3J=5.7 Hz), 4.31 (d, 1H, .sup.3J=5.7 Hz), 3.72 (bt, 1H), 1.70
(m, 1H), 0.88 (d, 3H, .sup.3J=6.9 Hz), 0.72 (d, 3H, .sup.3J=6.8
Hz).
Example 20
Synthesis of Compound P-6
[0196] Clasto-Lactacystin-.beta.-lactone (11.8 mg, 55.3 mmol; made
by the route described by Corey, E. J.; Reichard, G. A. J. Am.
Chem. Soc. 1992, 114, 10677) was dissolved in 0.4 mL freshly
distilled pyridine and treated, at room temperature with
methanesulfonyl chloride (6.0 mL, 77.5 mmol, 1.4 equiv.). The
solution was stirred for 2 hours at room temperature, treated with
more methanesulfonyl chloride (10.0 mL, 129 mmol, 2.3 equiv.) and
stirred at room temperature overnight. The dark solution was then
concentrated in vacuo with 3.times.2 mL of toluene affording a
yellow solid. Flash chromatography (230-400 mesh SiO.sub.2, elution
with 1:1 hexane/ethyl acetate) finally afforded 12.6 mg of the pure
mesylate P-6 (78%) obtained as fluffy white solid. .sup.1H NMR
(CDCl.sub.3, 300 MHz) d 6.28 (bs, 1H, NH), 5.27 (d, 1H, .sup.3J=6.1
Hz), 5.06 (d, 1H, .sup.3J=4.0 Hz), 3.11 (s, 3H), 2.80-2.89 (m, 1H),
2.24-2.34 (m, 1H), 1.33 (d, 3H, .sup.3J=7.5 Hz), 1.14 (d, 3H,
.sup.3J=7.0 Hz), 1.05 (d, 3H, .sup.3J=6.8 Hz).
Other Embodiments
[0197] From the above description, one skilled in the art can
easily ascertain the essential characteristics of the present
invention, and without departing from the spirit and scope thereof,
can make various changes and modifications of the invention to
adapt it to various usages and conditions. Thus, other embodiments
are also within the claims.
Sequence CWU 1
1
* * * * *