U.S. patent application number 11/312063 was filed with the patent office on 2006-07-27 for pyrrolidine inhibitors of iap.
This patent application is currently assigned to Genentech, Inc.. Invention is credited to Frederick Cohen, John A. Flygare, Cuong Ly, Vickie Hsiao-Wei Tsui.
Application Number | 20060167066 11/312063 |
Document ID | / |
Family ID | 36129810 |
Filed Date | 2006-07-27 |
United States Patent
Application |
20060167066 |
Kind Code |
A1 |
Cohen; Frederick ; et
al. |
July 27, 2006 |
Pyrrolidine inhibitors of IAP
Abstract
The invention provides novel inhibitors of IAP that are useful
as therapeutic agents for treating malignancies where the compounds
have the general formula I: ##STR1## wherein A, Q, X.sub.1,
X.sub.2, Y, R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.4', R.sub.5,
R.sub.6' and R.sub.6' are as described herein.
Inventors: |
Cohen; Frederick; (San
Francisco, CA) ; Tsui; Vickie Hsiao-Wei; (San
Francisco, CA) ; Ly; Cuong; (Daly City, CA) ;
Flygare; John A.; (Burlingame, CA) |
Correspondence
Address: |
GENENTECH, INC.
1 DNA WAY
SOUTH SAN FRANCISCO
CA
94080
US
|
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
36129810 |
Appl. No.: |
11/312063 |
Filed: |
December 19, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60638202 |
Dec 20, 2004 |
|
|
|
Current U.S.
Class: |
514/365 ;
514/372; 514/374; 548/200; 548/206; 548/215 |
Current CPC
Class: |
C07D 417/04 20130101;
A61P 35/00 20180101; A61P 43/00 20180101; C07D 471/04 20130101;
C07D 417/14 20130101 |
Class at
Publication: |
514/365 ;
514/372; 514/374; 548/200; 548/206; 548/215 |
International
Class: |
A61K 31/427 20060101
A61K031/427; A61K 31/422 20060101 A61K031/422; C07D 417/02 20060101
C07D417/02; C07D 413/02 20060101 C07D413/02 |
Claims
1. A compound of formula I: ##STR273## wherein A is a 5-member
aromatic heterocycle incorporating 1 to 4 heteroartoms N, O or S
and is optionally substituted with one or more R.sub.7 and R.sub.8
groups; Q is H, alkyl, a carbocycle, a heterocycle; wherein one or
more CH.sub.2 or CH groups of an alkyl is optionally replaced with
--O--, --S--, --S(O)--, S(O).sub.2, --N(R.sub.8)--, --C(O)--,
--C(O)--NR.sub.8--, --NR.sub.8--C(O)--, --SO.sub.2--NR.sub.8--,
--NR.sub.8--SO.sub.2--, --NR.sub.8--C(O)--NR.sub.8--,
--NR.sub.8--C(NH)--NR.sub.8--, --NR.sub.8--C(NH)--, --C(O)--O-- or
--O--C(O)--; and an alkyl, carbocycle and heterocycle is optionally
substituted with one or more hydroxyl, alkoxy, acyl, halogen,
mercapto, oxo, carboxyl, halo-substituted alkyl, amino, cyano,
nitro, amidino, guanidino an optionally substituted carbocycle or
an optionally substituted heterocycle; X.sub.1 and X.sub.2 are each
independently O or S; Y is a bond, (CR.sub.7R.sub.7).sub.n, O or S;
wherein n is 1 or 2 and R.sub.7 is H, halogen, alkyl, aryl,
aralkyl, amino, arylamino, alkylamino, aralkylamino, alkoxy,
aryloxy or aralkyloxy; R.sub.1 is H or R.sub.1 and R.sub.2 together
form a 5-8 member ring; R.sub.2 is alkyl, a carbocycle,
carbocyclylalkyl, a heterocycle or heterocyclylalkyl each
optionally substituted with halogen, hydroxyl, oxo, thione,
mercapto, carboxyl, alkyl, haloalkyl, alkoxy, alkylthio, sulfonyl,
amino and nitro; R.sub.3 is H or alkyl optionally substituted with
halogen or hydroxyl; or R.sub.3 and R.sub.4 together form a 3-6
heterocycle; R.sub.3' is H, or R.sub.3 and R.sub.3' together form a
3-6 carbocycle; R.sub.4 and R.sub.4' are independently H, hydroxyl,
amino, alkyl, carbocycle, carbocycloalkyl, carbocycloalkyloxy,
carbocycloalkyloxycarbonyl, heterocycle, heterocycloalkyl,
heterocycloalkyloxy or heterocycloalkyloxycarbonyl; wherein each
alkyl, carbocycloalkyl, carbocycloalkyloxy,
carbocycloalkyloxycarbonyl, heterocycle, heterocycloalkyl,
heterocycloalkyloxy and heterocycloalkyloxycarbonyl is optionally
substituted with halogen, hydroxyl, mercapto, carboxyl, alkyl,
alkoxy, amino, imino and nitro; or R.sub.4 and R.sub.4' together
form a heterocycle;; R.sub.5is H or alkyl; R.sub.6, and R.sub.6'
are each independently H, alkyl, aryl or aralkyl; R.sub.7 in each
instance is independently H, cyano, hydroxyl, mercapto, halogen,
nitro, carboxyl, amidino, guanidino, alkyl, a carbocycle, a
heterocycle or --U--V; wherein U is --O--, --S--, --S(O)--,
S(O).sub.2, --N(R.sub.8)--, --C(O)--, --C(O)--NR.sub.8--,
--NR.sub.8--C(O)--, --SO.sub.2--NR.sub.8--, --NR.sub.8--SO.sub.2--,
--NR.sub.8--C(O)--NR.sub.8--, --NR.sub.8--C(NH)--NR.sub.8--,
--NR.sub.8--C(NH)--, --C(O)--O-- or --O--C(O)--and V is alkyl, a
carbocycle or a heterocycle; and wherein one or more CH.sub.2 or CH
groups of an alkyl is optionally replaced with --O--, --S--,
--S(O)--, S(O).sub.2, --N(R.sub.8)--, --C(O)--, --C(O)--NR.sub.8--,
--NR.sub.8--C(O)--, --SO.sub.2--NR.sub.8--, --NR.sub.8--SO.sub.2--,
--NR.sub.8--C(O)--NR.sub.8--, --NR.sub.8--C(NH)--NR.sub.8--,
--NR.sub.8--C(NH)--, --C(O)--O-- or --O--C(O)--; and an alkyl,
carbocycle and heterocycle is optionally substituted with hydroxyl,
alkoxy, acyl, halogen, mercapto, oxo, carboxyl, acyl,
halo-substituted alkyl, amino, cyano nitro, amidino, guanidino an
optionally substituted carbocycle or an optionally substituted
heterocycle; R.sub.8 is H, alkyl, a carbocycle or a heterocycle
wherein one or more CH.sub.2 or CH groups of said alkyl is
optionally replaced with --O--, --S--, --S(O)--, S(O).sub.2,
--N(R.sub.8), or --C(O)--; and said alkyl, carbocycle and
heterocycle is optionally substituted with hydroxyl, alkoxy, acyl,
halogen, mercapto, oxo (.dbd.O), carboxyl, acyl, halo-substituted
alkyl, amino, cyano nitro, amidino, guanidino an optionally
substituted carbocycle or an optionally substituted heterocycle;
and salts and solvates thereof.
2. The compound of claim 1, wherein ring A has the general formula
II or II': ##STR274## wherein Z, is NR.sub.8, O or S; and Z.sub.2,
Z.sub.3 and Z.sub.4 are each independently N or CR.sub.7; wherein
R.sub.8 is H, alkyl or acyl; and R.sub.7 is H, halogen, amino,
hydroxyl, carboxyl, alkyl, haloalkyl or aralkyl.
3. The compound of claim 1, wherein ring A and Q together are
selected from the group consisting of IIa-IIz: ##STR275##
##STR276## ##STR277## wherein R.sub.8 is H, alkyl or acyl; and
R.sub.7 is H, halogen, amino, hydroxyl, carboxyl, alkyl, haloalkyl
or aralkyl.
4. The compound of claim 1, wherein Q is a carbocycle or
heterocycle optionally substituted with halogen, amino, oxo, alkyl,
a carbocycle or a heterocycle; wherein one or more CH.sub.2 or CH
groups of an alkyl is optionally replaced with --O--, --S--,
--S(O)--, S(O).sub.2, --N(R.sub.8)--, --C(O)--, --C(O)--NR.sub.8--,
--NR.sub.8--C(O)--, --SO.sub.2--NR.sub.8--, --NR.sub.8--SO.sub.2--,
--NR.sub.8--C(O)--NR.sub.8--, --NR.sub.8--C(NH)--NR.sub.8--,
--NR.sub.8--C(NH)--, --C(O)--O-- or --O--C(O)--; and wherein said
alkyl, carbocycle or heterocycle is optionally substituted with
halogen, amino, hydroxyl, mercapto, carboxyl, alkoxy, alkoxyalkoxy,
hydroxyalkoxy, alkylthio, acyloxy, acyloxyalkoxy, alkylsulfonyl,
alkylsulfonylalkyl, alkylsulfinyl, and alkylsulfinylalkyl.
5. The compound of claim 1, wherein Q is a carbocycle or
heterocycle selected from the group consisting of IIIa-IIIs:
##STR278## ##STR279## ##STR280## wherein n is 1-4; T is O, S,
NR.sub.8 or CR.sub.7R.sub.7; and W is O, NR.sub.8 or
CR.sub.7R.sub.7.
6. The compound of claim 1, wherein R.sub.1 is H.
7. The compound of claim 1, wherein R.sub.2 is alkyl, cycloalkyl or
a heterocycle.
8. The compound of claim 1, wherein R.sub.2 is is selected from the
group consisting of t-butyl, isopropyl, cyclohexyl,
tetrahydropyran-4-yl, N-methylsulfonylpiperidin-4-yl,
tetrahydrothiopyran-4-yl, tetrahydrothiopyran-4-yl (in which the S
is in oxidized form SO or SO.sub.2), cyclohexan-4-one,
4-hydroxycyclohexane, 4-hydroxy-4-methylcyclohexane,
1-methyl-tetrahydropyran-4-yl, 2-hydroxyprop-2-yl, but-2-yl, phenyl
and 1-hydoxyeth-1-yl.
9. The compound of claim 1, wherein R.sub.3 is methyl.
10. The compound of claim 1, wherein R.sub.4 is H or methyl, and
R.sub.4' is H.
11. The compound of claim 1, wherein R.sub.5 is H or methyl.
12. The compound of claim 1, wherein R.sub.6 and R.sub.6' are
independently H or methyl.
13. The compound of claim 1, wherein X.sub.1 and X.sub.2 are
independently O.
14. The compound of claim 2, wherein R.sub.1 is H; R.sub.2 is
isopropyl, t-butyl, cyclohexyl or pyran; R.sub.3 is methyl; R.sub.4
is H or methyl, R.sub.4' is H; R.sub.5 is H or methyl; and X.sub.1
and X.sub.2 are both O.
15. A method of inducing apoptosis in a cell comprising introducing
into said cell a compound of claim 1.
16. A method of sensitizing a cell to an apoptotic signal
comprising introducing into said cell a compound of claim 1.
17. The method of claim 16, wherein said apoptotic signal is
induced by contacting said cell with a compound selected from the
group consisting of cytarabine, fludarabine,
5-fluoro-2'-deoxyuiridine, gemcitabine, methotrexate, bleomycin,
cisplatin, cyclophosphamide, adriamycin (doxorubicin),
mitoxantrone, camptothecin, topotecan, colcemid, colchicine,
paclitaxel, vinblastine, vincristine, tamoxifen, finasteride,
taxotere and mitomycin C or radiation.
18. The method of claim 16, wherein said apoptotic signal is
induced by contacting said cell with Apo2L/TRAIL.
19. A method for inhibiting the binding of an IAP protein to a
caspase protein comprising contacting said IAP protein with a
compound of claim 1.
20. A method for treating a disease or condition associated with
the overexpression of an IAP in a mammal, comprising administering
to said mammal an effective amount of a compound of claim 1.
21. A method for treating cancer, comprising administering to said
mammal an effective amount of a compound of claim 1.
Description
[0001] This application claims priority under 35 U.S.C. .sctn.
119(e)(1) to United States provisional application 60/638,202 filed
on Dec. 20, 2004 which is incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to organic compounds useful
for therapy and/or prophylaxis in a mammal, and in particular to
inhibitors of IAP proteins useful for treating cancers.
BACKGROUND OF THE INVENTION
[0003] Apoptosis or programmed cell death is a genetically and
biochemically regulated mechanism that plays an important role in
development and homeostasis in invertebrates as well as
vertebrates. Aberrancies in apoptosis that lead to premature cell
death have been linked to a variety of developmental disorders.
Deficiencies in apoptosis that result in the lack of cell death
have been linked to cancer and chronic viral infections (Thompson
et al., (1995) Science 267, 1456-1462).
[0004] One of the key effector molecules in apoptosis are the
caspases (cysteine containing aspartate specific proteases).
Caspases are strong proteases, cleaving after aspartic acid
residues and once activated, digest vital cell proteins from within
the cell. Since caspases are such strong proteases, tight control
of this family of proteins is necessary to prevent premature cell
death. In general, caspases are synthesized as largely inactive
zymogens that require proteolytic processing in order to be active.
This proteolytic processing is only one of the ways in which
caspases are regulated. The second mechanism is through a family of
proteins that bind and inhibit caspases.
[0005] A family of molecules that inhibit caspases are the
Inhibitors of Apoptosis (IAP) (Deveraux et al., J Clin Immunol
(1999), 19:388-398). IAPs were originally discovered in baculovirus
by their functional ability to substitute for P35 protein, an
anti-apoptotic gene (Crook et al. (1993) J Virology 67, 2168-2174).
IAPs have been described in organisms ranging from Drosophila to
human. Regardless of their origin, structurally, IAPs comprise one
to three Baculovirus IAP repeat (BIR) domains, and most of them
also possess a carboxyl-terminal RING finger motif. The BIR domain
itself is a zinc binding domain of about 70 residues comprising 4
alpha-helices and 3 beta strands, with cysteine and histidine
residues that coordinate the zinc ion (Hinds et al., (1999) Nat.
Struct. Biol. 6, 648-651). It is the BIR domain that is believed to
cause the anti-apoptotic effect by inhibiting the caspases and thus
inhibiting apoptosis. As an example, human X-chromosome linked IAP
(XIAP) inhibits caspase 3, caspase 7 and the Apaf-1-cytochrome C
mediated activation of caspase 9 (Deveraux et al., (1998) EMBO J.
17, 2215-2223). Caspases 3 and 7 are inhibited by the BIR2 domain
of XIAP, while the BIR3 domain of XIAP is responsible for the
inhibition of caspase 9 activity. XIAP is expressed ubiquitously in
most adult and fetal tissues (Liston et al, Nature, 1996,
379(6563):349), and is overexpressed in a number of tumor cell
lines of the NCI 60 cell line panel (Fong et al, Genomics, 2000,
70:113; Tamm et al, Clin. Cancer Res. 2000, 6(5):1796).
Overexpression of XIAP in tumor cells has been demonstrated to
confer protection against a variety of pro-apoptotic stimuli and
promotes resistance to chemotherapy (LaCasse et al, Oncogene, 1998,
17(25):3247). Consistent with this, a strong correlation between
XIAP protein levels and survival has been demonstrated for patients
with acute myelogenous leukemia (Tamm et al, supra).
Down-regulation of XIAP expression by antisense oligonucleotides
has been shown to sensitize tumor cells to death induced by a wide
range of pro-apoptotic agents, both in vitro and in vivo (Sasaki et
al, Cancer Res., 2000, 60(20):5659; Lin et al, Biochem J., 2001,
353:299; Hu et al, Clin. Cancer Res., 2003, 9(7):2826).
Smac/DIABLO-derived peptides have also been demonstrated to
sensitize a number of different tumor cell lines to apoptosis
induced by a variety of pro-apoptotic drugs (Arnt et al, J. Biol.
Chem., 2002, 277(46):44236; Fulda et al, Nature Med., 2002,
8(8):808; Guo et al, Blood,2002, 99(9):3419; Vucic et al, J. Biol.
Chem.,2002, 277(14):12275; Yang et al, Cancer Res., 2003,
63(4):831).
[0006] Melanoma IAP (ML-IAP) is an IAP not detectable in most
normal adult tissues but is strongly upregulated in melanoma (Vucic
et al., (2000) Current Bio 10: 1359-1366). Determination of protein
structure demonstrated significant homology of the ML-IAP BIR and
RING finger domains to corresponding domains present in human XIAP,
C-IAP1 and C-IAP2. The BIR domain of ML-IAP appears to have the
most similarities to the BIR2 and BIR3 of XIAP, C-IAP1 and C-IAP2,
and appears to be responsible for the inhibition of apoptosis, as
determined by deletional analysis. Furthermore, Vucic et al.,
demonstrated that ML-IAP could inhibit chemotherapeutic agent
induced apoptosis. Agents such as adriamycin and 4-tertiary
butylphenol (4-TBP) were tested in a cell culture system of
melanomas overexpressing ML-IAP and the chemotherapeutic agents
were significantly less effective in killing the cells when
compared to a normal melanocyte control. The mechanism by which
ML-IAP produces an anti-apoptotic activity is in part through
inhibition of caspase 3 and 9. ML-IAP did not effectively inhibit
caspases 1, 2, 6, or 8.
[0007] Since apoptosis is a strictly controlled pathway with
multiple interacting factors, the discovery that IAPs themselves
are regulated was not unusual. In the fruit fly Drosophila, the
Reaper (rpr), Head Involution Defective (hid) and GRIM proteins
physically interact with and inhibit the anti-apoptotic activity of
the Drosophila family of IAPs. In the mammal, the proteins
SMAC/DIABLO act to block the IAPs and allow apoptosis to proceed.
It was shown that during normal apoptosis, SMAC is processed into
an active form and is released from the mitochondria into the
cytoplasm where it physically binds to IAPs and prevents the IAP
from binding to a caspase. This inhibition of the IAP allows the
caspase to remain active and thus proceed with apoptosis.
Interestingly, sequence homology between the IAP inhibitors shows
that there is a four amino acid motif in the N-terminus of the
processed, active proteins. This tetrapeptide appears to bind into
a hydrophobic pocket in the BIR domain and disrupts the BIR domain
binding to caspases (Chai et al., (2000) Nature 406:855-862, Liu et
al., (2000) Nature 408:1004-1008, Wu et al., (2000) Nature 408
1008-1012).
SUMMARY OF THE INVENTION
[0008] In one aspect of the present invention there is provided
novel inhibitors of IAP proteins having the general formula I:
##STR2## wherein [0009] A is a 5-member aromatic heterocycle
incorporating 1 to 4 heteroartoms N, O or S and is optionally
substituted with one or more R.sub.7 and R.sub.8 groups; [0010] Q
is H, alkyl, a carbocycle, a heterocycle; wherein one or more
CH.sub.2 or CH groups of an alkyl is optionally replaced with
--O--, --S--, --S(O)--, S(O).sub.2, --N(R.sub.8)--, --C(O)--,
--C(O)--NR.sub.8--, --NR.sub.8--C(O)--, --SO.sub.2--NR.sub.8--,
--NR.sub.8--SO.sub.2--, --NR.sub.8--C(O)--NR.sub.8`,
--NR.sub.8--C(NH)--NR.sub.8--, --NR.sub.8--C(NH)--, --C(O)--O-- or
--O--C(O)--; and an alkyl, carbocycle and heterocycle is optionally
substituted with one or more hydroxyl, alkoxy, acyl, halogen,
mercapto, oxo, carboxyl, halo-substituted alkyl, amino, cyano,
nitro, amidino, guanidino an optionally substituted carbocycle or
an optionally substituted heterocycle; [0011] X.sub.1 and X.sub.2
are each independently O or S; [0012] Y is a bond,
(CR.sub.7R.sub.7).sub.n, O or S; wherein n is 1 or 2 and R.sub.7 is
H, halogen, alkyl, aryl, aralkyl, amino, arylamino, alkylamino,
aralkylamino, alkoxy, aryloxy or aralkyloxy; [0013] R.sub.1 is H or
R.sub.1 and R.sub.2 together form a 5-8 member heterocycle; [0014]
R.sub.2 is alkyl, a carbocycle, carbocyclylalkyl, a heterocycle or
heterocyclylalkyl each optionally substituted with halogen,
hydroxyl, oxo, thione, mercapto, carboxyl, alkyl, haloalkyl,
alkoxy, alkylthio, sulfonyl, amino and nitro; [0015] R.sub.3 is H
or alkyl optionally substituted with halogen or hydroxyl; or
R.sub.3 and R.sub.4 together form a 3-6 heterocycle; [0016]
R.sub.3' is H, or R.sub.3 and R.sub.3' together form a 3-6
carbocycle; [0017] R.sub.4 and R.sub.4' are independently H,
hydroxyl, amino, alkyl, carbocycle, carbocycloalkyl,
carbocycloalkyloxy, carbocycloalkyloxycarbonyl, heterocycle,
heterocycloalkyl, heterocycloalkyloxy or
heterocycloalkyloxycarbonyl; wherein each alkyl, carbocycloalkyl,
carbocycloalkyloxy, carbocycloalkyloxycarbonyl, heterocycle,
heterocycloalkyl, heterocycloalkyloxy and
heterocycloalkyloxycarbonyl is optionally substituted with halogen,
hydroxyl, mercapto, carboxyl, alkyl, alkoxy, amino, imino and
nitro; or R.sub.4 and R.sub.4' together form a heterocycle; [0018]
R.sub.5 is H or alkyl; [0019] R.sub.6, and R.sub.6' are each
independently H, alkyl, aryl or aralkyl; [0020] R.sub.7 is H,
cyano, hydroxyl, mercapto, halogen, nitro, carboxyl, amidino,
guanidino, alkyl, a carbocycle, a heterocycle or --U--V; wherein U
is --O--, --S--, --S(O)--, S(O).sub.2, --N(R.sub.8)--, --C(O)--,
--C(O)--NR.sub.8--, --NR.sub.8--C(O)--, --SO.sub.2--NR.sub.8--,
--NR.sub.8--SO.sub.2--, --NR.sub.8--C(O)--NR.sub.8--,
--NR.sub.8--C(NH)--NR.sub.8--, --NR.sub.8--C(NH)--, --C(O)--O-- or
--O--C(O)-- and V is alkyl, a carbocycle or a heterocycle; and
wherein one or more CH.sub.2 or CH groups of an alkyl is optionally
replaced with --O--, --S--, --S(O)--, S(O).sub.2, --N(R.sub.8)--,
--C(O)--, --C(O)--NR.sub.8--, --NR.sub.8--C(O)--,
--SO.sub.2--NR.sub.8--, --NR.sub.8--SO.sub.2--,
--NR.sub.8--C(O)--NR.sub.8--, --C(O)--O-- or --O--C(O)--; and an
alkyl, carbocycle and heterocycle is optionally substituted with
hydroxyl, alkoxy, acyl, halogen, mercapto, oxo, carboxyl, acyl,
halo-substituted alkyl, amino, cyano nitro, amidino, guanidino an
optionally substituted carbocycle or an optionally substituted
heterocycle; [0021] R.sub.8 is H, alkyl, a carbocycle or a
heterocycle wherein one or more CH.sub.2 or CH groups of said alkyl
is optionally replaced with --O--, --S--, --S(O)--, S(O).sub.2,
--N(R.sub.8), or --C(O)--; and said alkyl, carbocycle and
heterocycle is optionally substituted with hydroxyl, alkoxy, acyl,
halogen, mercapto, oxo (.dbd.O), carboxyl, acyl, halo-substituted
alkyl, amino, cyano nitro, amidino, guanidino an optionally
substituted carbocycle or an optionally substituted heterocycle;
[0022] and salts and solvates thereof.
[0023] In another aspect of the invention, there are provided
compositions comprising compounds of formula I and a carrier,
diluent or excipient.
[0024] In another aspect of the invention, there is provided a
method of inducing apoptosis in a cell comprising introducing into
said cell a compound of formula I.
[0025] In another aspect of the invention, there is provided a
method of sensitizing a cell to an apoptotic signal comprising
introducing into said cell a compound of formula I.
[0026] In another aspect of the invention, there is provided a
method for inhibiting the binding of an IAP protein to a caspase
protein comprising contacting said IAP protein with a compound of
formula I.
[0027] In another aspect of the invention, there is provided a
method for treating a disease or condition associated with the
overexpression of an IAP protein in a mammal, comprising
administering to said mammal an effective amount of a compound of
formula I.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0028] "Acyl" means a carbonyl containing substituent represented
by the formula --C(O)--R in which R is H, alkyl, a carbocycle, a
heterocycle, carbocycle-substituted alkyl or
heterocycle-substituted alkyl wherein the alkyl, alkoxy, carbocycle
and heterocycle are as defined herein. Acyl groups include alkanoyl
(e.g. acetyl), aroyl (e.g. benzoyl), and heteroaroyl.
[0029] "Alkyl" means a branched or unbranched, saturated or
unsaturated (i.e. alkenyl, alkynyl) aliphatic hydrocarbon group,
having up to 12 carbon atoms unless otherwise specified. When used
as part of another term, for example "alkylamino", the alkyl
portion may be a saturated hydrocarbon chain, however also includes
unsaturated hydrocarbon carbon chains such as "alkenylamino" and
"alkynylamino. Examples of particular alkyl groups are methyl,
ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl,
tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl,
2-methylpentyl, 2,2-dimethylbutyl, n-heptyl, 3-heptyl,
2-methylhexyl, and the like. The terms "lower alkyl"
"C.sub.1-C.sub.4 alkyl" and "alkyl of 1 to 4 carbon atoms" are
synonymous and used interchangeably to mean methyl, ethyl,
1-propyl, isopropyl, cyclopropyl, 1-butyl, sec-butyl or t-butyl.
Unless specified, substituted, alkyl groups may contain one, for
example two, three or four substituents which may be the same or
different. Examples of substituents are, unless otherwise defined,
halogen, amino, hydroxyl, protected hydroxyl, mercapto, carboxy,
alkoxy, nitro, cyano, amidino, guanidino, urea, sulfonyl, sulfinyl,
aminosulfonyl, alkylsulfonylamino, arylsulfonylamino,
aminocarbonyl, acylamino, alkoxy, acyl, acyloxy, a carbocycle, a
heterocycle. Examples of the above substituted alkyl groups
include, but are not limited to; cyanomethyl, nitromethyl,
hydroxymethyl, trityloxymethyl, propionyloxymethyl, aminomethyl,
carboxymethyl, carboxyethyl, carboxypropyl, alkyloxycarbonylmethyl,
allyloxycarbonylaminomethyl, carbamoyloxymethyl, methoxymethyl,
ethoxymethyl, t-butoxymethyl, acetoxymethyl, chloromethyl,
bromomethyl, iodomethyl, trifluoromethyl, 6-hydroxyhexyl,
2,4-dichloro(n-butyl), 2-amino(iso-propyl), 2-carbamoyloxyethyl and
the like. The alkyl group may also be substituted with a carbocycle
group. Examples include cyclopropylmethyl, cyclobutylmethyl,
cyclopentylmethyl, and cyclohexylmethyl groups, as well as the
corresponding -ethyl, -propyl, -butyl, -pentyl, -hexyl groups, etc.
Substituted alkyls include substituted methyls e.g. a methyl group
substituted by the same substituents as the "substituted
C.sub.n-C.sub.m alkyl" group. Examples of the substituted methyl
group include groups such as hydroxymethyl, protected hydroxymethyl
(e.g. tetrahydropyranyloxymethyl), acetoxymethyl,
carbamoyloxymethyl, trifluoromethyl, chloromethyl, carboxymethyl,
bromomethyl and iodomethyl.
[0030] "Amidine" means the group --C(NH)--NHR wherein R is H or
alkyl or aralkyl. A particular amidine is the group
--NH--C(NH)--NH.sub.2.
[0031] "Amino" means primary (i.e. --NH.sub.2), secondary (i.e.
--NRH) and tertiary (i.e. --NRR) amines. Particular secondary and
tertiary amines are alkylamine, dialkylamine, arylamine,
diarylamine, aralkylamine and diaralkylamine wherein the alkyl is
as herein defined and optionally substituted. Particular secondary
and tertiary amines are methylamine, ethylamine, propylamine,
isopropylamine, phenylamine, benzylamine dimethylamine,
diethylamine, dipropylamine and disopropylamine.
[0032] "Amino-protecting group" as used herein refers to a
derivative of the groups commonly employed to block or protect an
amino group while reactions are carried out on other functional
groups on the compound. Examples of such protecting groups include
carbamates, amides, alkyl and aryl groups, imines, as well as many
N-heteroatom derivatives which can be removed to regenerate the
desired amine group. Particular amino protecting groups are Boc,
Fmoc and Cbz. Further examples of these groups are found in T. W.
Greene and P. G. M. Wuts, "Protective Groups in Organic Synthesis",
2.sup.nd ed., John Wiley & Sons, Inc., New York, N.Y., 1991,
chapter 7; E. Haslam, "Protective Groups in Organic Chemistry", J.
G. W. McOmie, Ed., Plenum Press, New York, N.Y., 1973, Chapter 5,
and T. W. Greene, "Protective Groups in Organic Synthesis", John
Wiley and Sons, New York, N.Y., 1981. The term "protected amino"
refers to an amino group substituted with one of the above
amino-protecting groups.
[0033] "Aryl" when used alone or as part of another term means a
carbocyclic aromatic group whether or not fused having the number
of carbon atoms designated or if no number is designated, up to 14
carbon atoms. Particular aryl groups are phenyl, naphthyl,
biphenyl, phenanthrenyl, naphthacenyl, and the like (see e.g.
Lang's Handbook of Chemistry (Dean, J. A., ed) 13.sup.th ed. Table
7-2 [1985]). A particular aryl is phenyl. Substituted phenyl or
substituted aryl means a phenyl group or aryl group substituted
with one, two, three, four or five, for example 1-2, 1-3 or 1-4
substituents chosen, unless otherwise specified, from halogen (F,
Cl, Br, I), hydroxy, protected hydroxy, cyano, nitro, alkyl (for
example C.sub.1-C.sub.6 alkyl), alkoxy (for example C.sub.1-C.sub.6
alkoxy), benzyloxy, carboxy, protected carboxy, carboxymethyl,
protected carboxymethyl, hydroxymethyl, protected hydroxymethyl,
aminomethyl, protected aminomethyl, trifluoromethyl,
alkylsulfonylamino, alkylsulfonylaminoalkyl, arylsulfonylamino,
arylsulonylaminoalkyl, heterocyclylsulfonylamino,
heterocyclylsulfonylaminoalkyl, heterocyclyl, aryl, or other groups
specified. One or more methyne (CH) and/or methylene (CH.sub.2)
groups in these substituents may in turn be substituted with a
similar group as those denoted above. Examples of the term
"substituted phenyl" includes but is not limited to a mono- or
di(halo)phenyl group such as 2-chlorophenyl, 2-bromophenyl,
4-chlorophenyl, 2,6-dichlorophenyl, 2,5-dichlorophenyl,
3,4-dichlorophenyl, 3-chlorophenyl, 3-bromophenyl, 4-bromophenyl,
3,4-dibromophenyl, 3-chloro4-fluorophenyl, 2-fluorophenyl and the
like; a mono- or di(hydroxy)phenyl group such as 4-hydroxyphenyl,
3-hydroxyphenyl, 2,4-dihydroxyphenyl, the protected-hydroxy
derivatives thereof and the like; a nitrophenyl group such as 3- or
4-nitrophenyl; a cyanophenyl group, for example, 4-cyanophenyl; a
mono- or di(lower alkyl)phenyl group such as 4-methylphenyl,
2,4-dimethylphenyl, 2-methylphenyl, 4-(iso-propyl)phenyl,
4-ethylphenyl, 3-(n-propyl)phenyl and the like; a mono or
di(alkoxy)phenyl group, for example, 3,4-dimethoxyphenyl,
3-methoxy4-benzyloxyphenyl,
3-methoxy4-(1-chloromethyl)benzyloxy-phenyl, 3-ethoxyphenyl,
4-(isopropoxy)phenyl, 4-(t-butoxy)phenyl, 3-ethoxy4-methoxyphenyl
and the like; 3- or 4-trifluoromethylphenyl; a mono- or
dicarboxyphenyl or (protected carboxy)phenyl group such
4-carboxyphenyl, ; a mono- or di(hydroxymethyl)phenyl or (protected
hydroxymethyl)phenyl such as 3-(protected hydroxymethyl)phenyl or
3,4-di(hydroxymethyl)phenyl; a mono- or di(aminomethyl)phenyl or
(protected aminomethyl)phenyl such as 2-(aminomethyl)phenyl or
2,4-(protected aminomethyl)phenyl; or a mono- or
di(N-(methylsulfonylamino))phenyl such as
3-(N-methylsulfonylamino))phenyl. Also, the term "substituted
phenyl" represents disubstituted phenyl groups where the
substituents are different, for example, 3-methyl4-hydroxyphenyl,
3-chloro-4-hydroxyphenyl, 2-methoxy-4-bromophenyl,
4-ethyl-2-hydroxyphenyl, 3-hydroxy-4-nitrophenyl,
2-hydroxy-4-chlorophenyl, and the like, as well as trisubstituted
phenyl groups where the substituents are different, for example
3-methoxy4-benzyloxy-6-methyl sulfonylamino,
3-methoxy4-benzyloxy-6-phenyl sulfonylamino, and tetrasubstituted
phenyl groups where the substituents are different such as
3-methoxy-4-benzyloxy-5-methyl-6-phenyl sulfonylamino. Particular
substituted phenyl groups include the 2-chlorophenyl,
2-aminophenyl, 2-bromophenyl, 3-methoxyphenyl, 3-ethoxy-phenyl,
4-benzyloxyphenyl, 4-methoxyphenyl, 3-ethoxy4-benzyloxyphenyl,
3,4-diethoxyphenyl, 3-methoxy-4-benzyloxyphenyl, 3-methoxy4-(
1-chloromethyl)benzyloxy-phenyl,
3-methoxy-4-(1-chloromethyl)benzyloxy -6- methyl sulfonyl
aminophenyl groups. Fused aryl rings may also be substituted with
any, for example 1, 2 or 3, of the substituents specified herein in
the same manner as substituted alkyl groups.
[0034] "Carbocyclyl", "carbocyclylic", "carbocycle" and
"carbocyclo" alone and when used as a moiety in a complex group
such as a carbocycloalkyl group, refers to a mono-, bi-, or
tricyclic aliphatic ring having 3 to 14 carbon atoms, for example 3
to 7 carbon atoms, which may be saturated or unsaturated, aromatic
or non-aromatic. Particular saturated carbocyclic groups are
cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl groups. A
particular saturated carbocycle is cyclopropyl. Another particular
saturated carbocycle is cyclohexyl. Particular unsaturated
carbocycles are aromatic e.g. aryl groups as previously defined,
for example phenyl. The terms "substituted carbocyclyl",
"carbocycle" and "carbocyclo" mean these groups substituted by the
same substituents as the "substituted alkyl" group.
[0035] "Carboxy-protecting group" as used herein refers to one of
the ester derivatives of the carboxylic acid group commonly
employed to block or protect the carboxylic acid group while
reactions are carried out on other functional groups on the
compound. Examples of such carboxylic acid protecting groups
include 4-nitrobenzyl, 4-methoxybenzyl, 3,4-dimethoxybenzyl,
2,4-dimethoxybenzyl, 2,4,6-trimethoxybenzyl, 2,4,6-trimethylbenzyl,
pentamethylbenzyl, 3,4-methylenedioxybenzyl, benzhydryl,
4,4'-dimethoxybenzhydryl, 2,2',4,4'-tetramethoxybenzhydryl, alkyl
such as t-butyl or t-amyl, trityl, 4-methoxytrityl,
4,4'-dimethoxytrityl, 4,4',4''-trimethoxytrityl, 2-phenylprop-2-yl,
trimethylsilyl, t-butyldimethylsilyl, phenacyl,
2,2,2-trichloroethyl, beta-(trimethylsilyl)ethyl,
beta-(di(n-butyl)methylsilyl)ethyl, p-toluenesulfonylethyl,
4-nitrobenzylsulfonylethyl, allyl, cinnamyl,
1-(trimethylsilylmethyl)prop-1-en-3-yl, and like moieties. The
species of carboxy-protecting group employed is not critical so
long as the derivatized carboxylic acid is stable to the condition
of subsequent reaction(s) on other positions of the molecule and
can be removed at the appropriate point without disrupting the
remainder of the molecule. In particular, it is important not to
subject a carboxy-protected molecule to strong nucleophilic bases,
such as lithium hydroxide or NaOH, or reductive conditions
employing highly activated metal hydrides such as LiAlH.sub.4.
(Such harsh removal conditions are also to be avoided when removing
amino-protecting groups and hydroxy-protecting groups, discussed
below.) Particular carboxylic acid protecting groups are the alkyl
(e.g. methyl, ethyl, t-butyl), allyl, benzyl and p-nitrobenzyl
groups. Similar carboxy-protecting groups used in the
cephalosporin, penicillin and peptide arts can also be used to
protect a carboxy group substituents. Further examples of these
groups are found in T. W. Greene and P. G. M. Wuts, "Protective
Groups in Organic Synthesis", 2.sup.nd ed., John Wiley & Sons,
Inc., New York, N.Y., 1991, chapter 5; E. Haslam, "Protective
Groups in Organic Chemistry", J. G. W. McOmie, Ed., Plenum Press,
New York, N.Y., 1973, Chapter 5, and T. W. Greene, "Protective
Groups in Organic Synthesis", John Wiley and Sons, New York, N.Y.,
1981, Chapter 5. The term "protected carboxy" refers to a carboxy
group substituted with one of the above carboxy-protecting
groups.
[0036] "Guanidine" means the group --NH--C(NH)--NHR wherein R is H
or alkyl or aralkyl. A particular guanidine is the group
--NH--C(NH)--NH.sub.2.
[0037] "Hydroxy-protecting group" as used herein refers to a
derivative of the hydroxy group commonly employed to block or
protect the hydroxy group while reactions are carried out on other
functional groups on the compound. Examples of such protecting
groups include tetrahydropyranyloxy, benzoyl, acetoxy,
carbamoyloxy, benzyl, and silylethers (e.g. TBS, TBDPS) groups.
Further examples of these groups are found in T. W. Greene and P.
G. M. Wuts, "Protective Groups in Organic Synthesis", 2.sup.nd ed.,
John Wiley & Sons, Inc., New York, N.Y., 1991, chapters 2-3; E.
Haslam, "Protective Groups in Organic Chemistry", J. G. W. McOmie,
Ed., Plenum Press, New York, N.Y., 1973, Chapter 5, and T. W.
Greene, "Protective Groups in Organic Synthesis", John Wiley and
Sons, New York, N.Y., 1981. The term "protected hydroxy" refers to
a hydroxy group substituted with one of the above
hydroxy-protecting groups.
[0038] "Heterocyclic group", "heterocyclic", "heterocycle",
"heterocyclyl", or "heterocyclo" alone and when used as a moiety in
a complex group such as a heterocycloalkyl group, are used
interchangeably and refer to any mono-, bi-, or tricyclic,
saturated or unsaturated, aromatic (heteroaryl) or non-aromatic
ring having the number of atoms designated, generally from 5 to
about 14 ring atoms, where the ring atoms are carbon and at least
one heteroatom (nitrogen, sulfur or oxygen), for example 1 to 4
heteroatoms. Typically, a 5-membered ring has 0 to 2 double bonds
and 6- or 7-membered ring has O to 3 double bonds and the nitrogen
or sulfur heteroatoms may optionally be oxidized (e.g. SO,
SO.sub.2), and any nitrogen heteroatom may optionally be
quaternized. Particular non-aromatic heterocycles are morpholinyl
(morpholino), pyrrolidinyl, oxiranyl, oxetanyl, tetrahydrofuranyl,
2,3-dihydrofuranyl, 2H-pyranyl, tetrahydropyranyl, thiiranyl,
thietanyl, tetrahydrothietanyl, aziridinyl, azetidinyl,
1-methyl-2-pyrrolyl, piperazinyl and piperidinyl. A
"heterocycloalkyl" group is a heterocycle group as defined above
covalently bonded to an alkyl group as defined above. Particular
5-membered heterocycles containing a sulfur or oxygen atom and one
to three nitrogen atoms are thiazolyl, in particular thiazol-2-yl
and thiazol-2-yl N-oxide, thiadiazolyl, in particular
1,3,4-thiadiazol-5-yl and 1,2,4-thiadiazol-5-yl, oxazolyl, for
example oxazol-2-yl, and oxadiazolyl, such as 1,3,4-oxadiazol-5-yl,
and 1,2,4-oxadiazol-5-yl. Particular 5-membered ring heterocycles
containing 2 to 4 nitrogen atoms include imidazolyl, such as
imidazol-2-yl; triazolyl, such as 1,3,4-triazol-5-yl;
1,2,3-triazol-5-yl, 1,2,4-triazol-5-yl, and tetrazolyl, such as
1H-tetrazol-5-yl. Particular benzo-fused 5-membered heterocycles
are benzoxazol-2-yl, benzthiazol-2-yl and benzimidazol-2-yl.
Particular 6-membered heterocycles contain one to three nitrogen
atoms and optionally a sulfur or oxygen atom, for example pyridyl,
such as pyrid-2-yl, pyrid-3-yl, and pyrid4-yl; pyrimidyl, such as
pyrimid-2-yl and pyrimid4-yl; triazinyl, such as 1,3,4-triazin-2-yl
and 1,3,5-triazin-4-yl; pyridazinyl, in particular pyridazin-3-yl,
and pyrazinyl. The pyridine N-oxides and pyridazine N-oxides and
the pyridyl, pyrimid-2-yl, pyrimid4-yl, pyridazinyl and the
1,3,4-triazin-2-yl groups, are a particular group. Substituents for
"optionally substituted heterocycles", and further examples of the
5- and 6-membered ring systems discussed above can be found in W.
Druckheimer et al., U.S. Pat. No. 4,278,793. In a particular
embodiment, such optionally substittuted heterocycle groups are
substituted with hydroxyl, alkyl, alkoxy, acyl, halogen, mercapto,
oxo, carboxyl, acyl, halo-substituted alkyl, amino, cyano, nitro,
amidino and guanidino.
[0039] "Heteroaryl" alone and when used as a moiety in a complex
group such as a heteroaralkyl group, refers to any mono-, bi-, or
tricyclic aromatic ring system having the number of atoms
designated where at least one ring is a 5-, 6- or 7-membered ring
containing from one to four heteroatoms selected from the group
nitrogen, oxygen, and sulfur, and in a particular embodiment at
least one heteroatom is nitrogen (Lang's Handbook of Chemistry,
supra). Included in the definition are any bicyclic groups where
any of the above heteroaryl rings are fused to a benzene ring.
Particular heteroaryls incorporate a nitrogen or oxygen heteroatom.
The following ring systems are examples of the heteroaryl (whether
substituted or unsubstituted) groups denoted by the term
"heteroaryl": thienyl, furyl, imidazolyl, pyrazolyl, thiazolyl,
isothiazolyl, oxazolyl, isoxazolyl, triazolyl, thiadiazolyl,
oxadiazolyl, tetrazolyl, thiatriazolyl, oxatriazolyl, pyridyl,
pyrimidyl, pyrazinyl, pyridazinyl, thiazinyl, oxazinyl, triazinyl,
thiadiazinyl, oxadiazinyl, dithiazinyl, dioxazinyl, oxathiazinyl,
tetrazinyl, thiatriazinyl, oxatriazinyl, dithiadiazinyl,
imidazolinyl, dihydropyrimidyl, tetrahydropyrimidyl,
tetrazolo[1,5-b]pyridazinyl and purinyl, as well as benzo-fused
derivatives, for example benzoxazolyl, benzofuryl, benzothiazolyl,
benzothiadiazolyl, benzotriazolyl, benzoimidazolyl and indolyl. A
particular "heteroaryl" is: 1,3-thiazol-2-yl,
4-(carboxymethyl)-5-methyl- 1,3-thiazol-2-yl,
4-(carboxymethyl)-5-methyl-1,3-thiazol-2-yl sodium salt,
1,2,4-thiadiazol-5-yl, 3-methyl- 1,2,4-thiadiazol-5-yl,
1,3,4-triazol-5-yl, 2-methyl-1,3,4-triazol-5-yl,
2-hydroxy-1,3,4-triazol-5-yl, 2-carboxy-4-methyl-1,3,4-triazol-5-yl
sodium salt, 2-carboxy4-methyl-1,3,4-triazol-5-yl, 1,3-oxazol-2-yl,
1,3,4-oxadiazol-5-yl, 2-methyl-1,3,4-oxadiazol-5-yl,
2-(hydroxymethyl)-1,3,4-oxadiazol-5-yl, 1,2,4-oxadiazol-5-yl,
1,3,4-thiadiazol-5-yl, 2-thiol-1,3,4-thiadiazol-5-yl,
2-(methylthio)-1,3,4-thiadiazol-5-yl,
2-amino-1,3,4-thiadiazol-5-yl, 1H-tetrazol-5-yl,
1-methyl-1H-tetrazol-5-yl,
1-(1-(dimethylamino)eth-2-yl)-1H-tetrazol-5-yl,
1-(carboxymethyl)-1H-tetrazol-5-yl,
1-(carboxymethyl)-1H-tetrazol-5-yl sodium salt, 1-(methylsulfonic
acid)-1H-tetrazol-5-yl, 1-(methylsulfonic acid)-1H-tetrazol-5-yl
sodium salt, 2-methyl-1H-tetrazol-5-yl, 1,2,3-triazol-5-yl,
1-methyl-1,2,3-triazol-5-yl, 2-methyl-1,2,3-triazol-5-yl,
4-methyl-1,2,3-triazol-5-yl, pyrid-2-yl N-oxide,
6-methoxy-2-(n-oxide)-pyridaz-3-yl, 6-hydroxypyridaz-3-yl,
1-methylpyrid-2-yl, 1-methylpyrid-4-yl, 2-hydroxypyrimid-4-yl,
1,4,5,6-tetrahydro-5,6-dioxo-4-methyl-as-triazin-3-yl,
1,4,5,6-tetrahydro-4-(formylmethyl)-5,6-dioxo-as-triazin-3-yl,
2,5-dihydro-5-oxo-6-hydroxy-astriazin-3-yl,
2,5-dihydro-5-oxo-6-hydroxy-as-triazin-3-yl sodium salt,
2,5-dihydro-5-oxo-6-hydroxy-2-methyl-astriazin-3-yl sodium salt,
2,5-dihydro-5-oxo-6-hydroxy-2-methyl-as-triazin-3-yl,
2,5-dihydro-5-oxo-6-methoxy-2-methyl-as-triazin-3-yl,
2,5-dihydro-5-oxo-as-triazin-3-yl,
2,5-dihydro-5-oxo-2-methyl-as-triazin-3-yl,
2,5-dihydro-5-oxo-2,6-dimethyl-as-triazin-3-yl,
tetrazolo[1,5-b]pyridazin-6-yl and
8-aminotetrazolo[1,5-b]-pyridazin-6-yl. An alternative group of
"heteroaryl" includes; 4-(carboxymethyl)-5-methyl-1,3-thiazol-2-yl,
4-(carboxymethyl)-5-methyl-1,3-thiazol-2-yl sodium salt,
1,3,4-triazol-5-yl, 2-methyl-1,3,4-triazol-5-yl, 1H-tetrazol-5-yl,
1-methyl-1H-tetrazol-5-yl,
1-(1-(dimethylamino)eth-2-yl)-1H-tetrazol-5-yl,
1-(carboxymethyl)-1H-tetrazol-5-yl,
1-(carboxymethyl)-1H-tetrazol-5-yl sodium salt, 1-(methylsulfonic
acid)-1H-tetrazol-5-yl, 1-(methylsulfonic acid)-1H-tetrazol-5-yl
sodium salt, 1,2,3-triazol-5-yl,
1,4,5,6-tetrahydro-5,6-dioxo4-methyl-as-triazin-3-yl,
1,4,5,6-tetrahydro-4-(2-formylmethyl)-5,6-dioxo-as-triazin-3-yl,
2,5-dihydro-5-oxo-6-hydroxy-2-methyl-as-triazin-3-yl sodium salt,
2,5-dihydro-5-oxo-6-hydroxy-2-methyl-as-triazin-3-yl,
tetrazolo[1,5-b]pyridazin-6-yl, and
8-aminotetrazolo[1,5-b]pyridazin-6-yl. Heteroaryl groups are
optionally substituted as described for heterocycles.
[0040] "Inhibitor" means a compound which reduces or prevents the
binding of IAP proteins to caspase proteins or which reduces or
prevents the inhibition of apoptosis by an IAP protein.
Alternatively, "inhibitor" means a compound which prevents the
binding interaction of X-IAP with caspases or the binding
interaction of ML-IAP with SMAC.
[0041] "Optionally substituted" unless otherwise specified means
that a group may be substituted by one or more (e.g. 0, 1, 2, 3 or
4) of the substituents listed for that group in which said
substituents may be the same or different. In an embodiment an
optionally substituted group has 1 substituent. In another
embodiment an optionally substituted group has 2 substituents. In
another embodiment an optionally substituted group has 3
substituents.
[0042] "Pharmaceutically acceptable salts" include both acid and
base addition salts. "Pharmaceutically acceptable acid addition
salt" refers to those salts which retain the biological
effectiveness and properties of the free bases and which are not
biologically or otherwise undesirable, formed with inorganic acids
such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric
acid, carbonic acid, phosphoric acid and the like, and organic
acids may be selected from aliphatic, cycloaliphatic, aromatic,
araliphatic, heterocyclic, carboxylic, and sulfonic classes of
organic acids such as formic acid, acetic acid, propionic acid,
glycolic acid, gluconic acid, lactic acid, pyruvic acid, oxalic
acid, malic acid, maleic acid, maloneic acid, succinic acid,
fumaric acid, tartaric acid, citric acid, aspartic acid, ascorbic
acid, glutamic acid, anthranilic acid, benzoic acid, cinnamic acid,
mandelic acid, embonic acid, phenylacetic acid, methanesulfonic
acid, ethanesulfonic acid, p-toluenesulfonic acid, salicyclic acid
and the like.
[0043] "Pharmaceutically acceptable base addition salts" include
those derived from inorganic bases such as sodium, potassium,
lithium, ammonium, calcium, magnesium, iron, zinc, copper,
manganese, aluminum salts and the like. Particularly base addition
salts are the ammonium, potassium, sodium, calcium and magnesium
salts. Salts derived from pharmaceutically acceptable organic
nontoxic bases includes salts of primary, secondary, and tertiary
amines, substituted amines including naturally occurring
substituted amines, cyclic amines and basic ion exchange resins,
such as isopropylamine, trimethylamine, diethylamine,
triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol,
trimethamine, dicyclohexylamine, lysine, arginine, histidine,
caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine,
glucosamine, methylglucamine, theobromine, purines, piperizine,
piperidine, N-ethylpiperidine, polyamine resins and the like.
Particularly organic non-toxic bases are isopropylamine,
diethylamine, ethanolamine, trimethamine, dicyclohexylamine,
choline, and caffeine.
[0044] "Sulfonyl" means a --SO.sub.2--R group wherein R is alkyl,
carbocycle, heterocycle, carbocycloalkyl or heterocycloalkyl.
Particular sulfonyl groups are alkylsulfonyl (i.e.
--SO.sub.2-alkyl), for example methylsulfonyl; arylsulfonyl, for
example phenylsulfonyl; aralkylsulfonyl, for example
benzylsulfonyl.
[0045] The phrase "and salts and solvates thereof" as used herein
means that compounds of the inventions may exist in one or a
mixture of salts and solvate forms. For example a compound of the
invention may be substantially pure in one particular salt or
solvate form or else may be mixtures of two or more salt or solvate
forms.
[0046] The present invention provides novel compounds having the
general formula I: ##STR3## wherein A, Q, X.sub.1, X.sub.2, Y,
R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.4', R.sub.5, R.sub.6 and
R.sub.6' are as described herein.
[0047] Ring A is a 5-member aromatic heterocycle incorporating 1 to
4 heteroartoms N, O or S which is substituted with group Q and is
optionally further substituted with one or more R.sub.7 (for
substitutions at a ring carbon atom) and one or more R.sub.8 (for
substitutions at a ring nitrogen).
[0048] R.sub.7 in each occurrence is independently H, cyano,
hydroxyl, mercapto, halogen, nitro, carboxyl, amidino, guanidino,
alkyl, a carbocycle, a heterocycle or --U--V; wherein U is --O--,
--S--, --S(O)--, S(O).sub.2, --N(R.sub.8)--, --C(O)--,
--C(O)--NR.sub.8--, --NR.sub.8--C(O)--, --SO.sub.2--NR.sub.8--,
--NR.sub.8--SO.sub.2--, --NR.sub.8--C(O)--NR.sub.8--,
--NR.sub.8--C(NH)--NR.sub.8--, --NR.sub.8--C(NH)--, --C(O)--O-- or
--O--C(O)-- and V is alkyl, a carbocycle or a heterocycle; and
wherein one or more CH.sub.2 or CH groups of an alkyl is optionally
replaced with --O--, --S--, --S(O)--, S(O).sub.2, --N(R.sub.8)--,
--C(O)--, --C(O)--NR.sub.8--, --NR.sub.8--C(O)--,
--SO.sub.2--NR.sub.8--, --NR.sub.8--SO.sub.2--,
--NR.sub.8--C(O)--NR.sub.8--, --NR.sub.8--C(NH)--NR.sub.8--,
--NR.sub.8--C(NH)--, --C(O)--O-- or --O--C(O)--; and an alkyl,
carbocycle and heterocycle is optionally substituted with hydroxyl,
alkoxy, acyl, halogen, mercapto, oxo, carboxyl, acyl,
halo-substituted alkyl, amino, cyano, nitro, amidino, guanidino an
optionally substituted carbocycle or an optionally substituted
heterocycle. Substituents of the "optionally substituted
carbocycle" and "optionally substituted heterocycle" are as defined
herein. In a particular embodiment such carbocycle and heterocycle
groups are substituted with hydroxyl, alkyl, alkoxy, acyl, halogen,
mercapto, oxo, carboxyl, acyl, halo-substituted alkyl, amino,
cyano, nitro, amidino and guanidino. In an embodiment R.sub.7 is H,
halogen, cyano, alkyl, hydroxyalkyl or alkoxyalkyl.
[0049] R.sub.8 is H, alkyl, a carbocycle or a heterocycle wherein
one or more CH.sub.2 or CH groups of said alkyl is optionally
replaced with --O--, --S--, --S(O)--, S(O).sub.2, --N(R.sub.8), or
--C(O)--; and said alkyl, carbocycle and heterocycle is optionally
substituted with hydroxyl, alkoxy, acyl, halogen, mercapto, oxo
(.dbd.O), carboxyl, acyl, halo-substituted alkyl, amino, cyano
nitro, amidino, guanidino an optionally substituted carbocycle or
an optionally substituted heterocycle. Substituents of the
"optionally substituted carbocycle" and "optionally substituted
heterocycle" are as defined herein. In a particular embodiment such
carbocycle and heterocycle groups are substituted with hydroxyl,
alkyl, alkoxy, acyl, halogen, mercapto, oxo, carboxyl, acyl,
halo-substituted alkyl, amino, cyano, nitro, amidino and guanidino.
In a particular embodiment R.sub.8 is H, alkyl, or acyl. In an
embodiment R.sub.8 is methyl. In another embodiment R.sub.8 is
acetyl. In a particular embodiment R.sub.8 is H. In an embodiment
R.sub.7 is H, halogen, amino, hydroxyl, carboxyl, alkyl, haloalkyl
or aralkyl. In a particular embodiment R.sub.7 is halogen, for
example Cl or F. In a particular embodiment R.sub.7 is H. It is
understood that substitutions defined for R.sub.7 and R.sub.8 as
well as all other variable groups herein are subject to permissible
valency.
[0050] In a particular embodiment ring A has the general formula
II: ##STR4## wherein Z.sub.1 is NR.sub.8, O or S; and Z.sub.2,
Z.sub.3 and Z.sub.4 are each independently N or CR.sub.7. Group Q
is attached to ring A of formula II and II' at the ring member
between Z.sub.2 and Z.sub.3. In a particular embodiment Z.sub.1 is
S. In a particular embodiment Z.sub.1 is O. In another particular
embodiment Z.sub.1 is NR.sub.8 wherein R.sub.8 is as defined
herein. In a particular embodiment Z.sub.1 is NR.sub.8 wherein
R.sub.8 is H. In another particular embodiment Z.sub.1 is NR.sub.8
wherein R.sub.8 is Me. In another embodiment Z.sub.1 is O or S
while Z.sub.2 is N and Z.sub.3 is N or CR.sub.7. In a particular
embodiment Z.sub.1 is S while Z.sub.2 is N and Z.sub.3 is CR.sub.7.
In a particular embodiment Z.sub.1 is S while Z2 is N and Z.sub.3
is CH.
[0051] In a particular embodiment, ring A is an aromatic heterocyle
selected from the group consisting of IIa-IIcc: ##STR5## ##STR6##
##STR7## wherein R.sub.7 and R.sub.8 are as defined herein. Q is
not part of ring A and is shown for positional purposes. In a
particular embodiment, ring A is any one of the groups IIa-IIz
wherein R.sub.8 is H and R.sub.7 is H, Cl, or hydroxypropynyl. In
another particular embodiment, ring A is any one of the groups
IIa-IIz wherein R.sub.7 and R.sub.8 are both H. In another
embodiment, ring A is the IIg. In another embodiment, ring A is IIg
and R.sub.7 is H.
[0052] Q is H, alkyl, a carbocycle, a heterocycle; wherein one or
more CH.sub.2 or CH groups of an alkyl is optionally replaced with
--O--, --S--, --S(O)--, S(O).sub.2, --N(R.sub.8)--, --C(O)--,
--C(O)--NR.sub.8--, --NR.sub.8--C(O)--, --SO.sub.2--NR.sub.8--,
--NR.sub.8--SO.sub.2--, --NR.sub.8--C(O)--NR.sub.8--,
--NR.sub.8--C(NH)--NR.sub.8--, --NR.sub.8--C(NH)--, --C(O)--O-- or
--O--C(O)--; and wherein any of the foregoing alkyl, carbocycle and
heterocycle is optionally substituted with one or more hydroxyl,
alkoxy, acyl, halogen, mercapto, oxo, carboxyl, acyl,
halo-substituted alkyl, amino, cyano nitro, amidino, guanidino an
optionally substituted carbocycle or an optionally substituted
heterocycle. Substituents of the "optionally substituted
carbocycle" and "optionally substituted heterocycle" are as defined
herein. In a particular embodiment such carbocycle and heterocycle
groups are substituted with hydroxyl, alkyl, alkoxy, acyl, halogen,
mercapto, oxo, carboxyl, acyl, halo-substituted alkyl, amino,
cyano, nitro, amidino and guanidino. In a particular embodiment Q
is a carbocycle or heterocycle optionally substituted with halogen,
amino, oxo, alkyl, a carbocycle or a heterocycle; wherein one or
more CH.sub.2 or CH groups of an alkyl is optionally replaced with
--O--, --S--, --S(O)--, S(O).sub.2, --N(R.sub.8)--, --C(O)--,
--C(O)--NR.sub.8--, --NR.sub.8--C(O)--, --SO.sub.2--NR.sub.8--,
--NR.sub.8--SO.sub.2--, --NR.sub.8--C(O)--NR.sub.8--,
--NR.sub.8--C(NH)--NR.sub.8--, --NR.sub.8--C(NH)--, --C(O)--O-- or
--O--C(O)--; and wherein said alkyl, carbocycle or heterocycle is
optionally substituted with halogen, amino, hydroxyl, mercapto,
carboxyl, alkoxy, alkoxyalkoxy, hydroxyalkoxy, alkylthio, acyloxy,
acyloxyalkoxy, alkylsulfonyl, alkylsulfonylalkyl, alkylsulfinyl,
and alkylsulfinylalkyl.
[0053] In a particular embodiment, Q is a carbocycle or heterocycle
selected from the group consisting of IIIa-IIIs: ##STR8## ##STR9##
wherein n is 1-4, for example 1-3, for example 1-2, for example 1;
T is O, S, NR.sub.8 or CR.sub.7R.sub.7; W is O, NR.sub.8 or
CR.sub.7R.sub.7; and R.sub.7 and R.sub.8 are as defined herein. In
a particular embodiment Q is any one of IIIa-IIIi wherein R.sub.8
is H and R.sub.7 is selected from the group consisting of H, F, Cl,
Me, methoxy, hydroxyethoxy, methoxyethoxy, acetoxyethoxy,
methylsulfonyl methylsulfonylmethyl, phenyl and morpholin4-yl. In
another particular embodiment Q is IIId. In a particular embodiment
Q is IIId which is substituted at the 4-position with R.sub.7. In
another particular embodiment Q is IIId which is substituted at the
5-position with R.sub.7.
[0054] X.sub.1 and X.sub.2 are each independently O or S. In a
particular embodiment, X.sub.1 and X.sub.2 are both O. In another
particular embodiment X.sub.1 and X.sub.2 are both S. In another
particular embodiment, X.sub.1 is S while X.sub.2 is O. In another
particular embodiment, X.sub.1 is O while X.sub.2 is S.
[0055] Y is a bond, (CR.sub.7R.sub.7).sub.n, O or S; wherein n is 1
or 2 and R.sub.7 is H, halogen, alkyl, aryl, aralkyl, amino,
arylamino, alkylamino, aralkylamino, alkoxy, aryloxy or aralkyloxy.
In a particular embodiment, Y is (CHR.sub.7).sub.n, O or S; wherein
n is 1 or 2 and R.sub.7 is H, halogen, alkyl, aryl, aralkyl, amino,
arylamino, alkylamino, aralkylamino, alkoxy, aryloxy or aralkyloxy.
In a particular embodiment, Y is CH.sub.2. In a particular
embodiment n is 1. In a particular embodiment Y is a bond. In a
particular embodiment n is 1 and Y is CHR.sub.7 wherein R.sub.7 is
aralkyloxy, for example benzyloxy. In a particular embodiment n is
1 and Y is CHR.sub.7 wherein R.sub.7 is F. In a particular
embodiment n is 1 and Y is CHR.sub.7 wherein R.sub.7 is
aralkylamino, for example benzylamino. In another particular
embodiment Y is O. In another particular embodiment Y is S. [0056]
R.sub.1 is H or R.sub.1 and R.sub.2 together form a 5-8 member
ring. In a particular embodiment, R.sub.1is H. In a particular
embodiment, R.sub.1 and R.sub.2 together form a 6-member ring. In a
particular embodiment, R.sub.1 and R.sub.2 together form a 7-member
ring. In another particular embodiment, R.sub.1 and R.sub.2
together form an 8-member ring. In another particular embodiment,
R.sub.1 and R.sub.2 together form a 7-member ring while Y is S. In
another particular embodiment, R.sub.1 is H, while Y is CH.sub.2.
In another particular embodiment, R.sub.1 is H, while Y is S. In
another particular embodiment, R.sub.1 is H, while Y is O.
[0057] R.sub.2 is alkyl, a carbocycle, carbocyclylalkyl, a
heterocycle or heterocyclylalkyl each optionally substituted with
halogen, hydroxyl, oxo, thione, mercapto, carboxyl, alkyl,
haloalkyl, alkoxy, alkylthio, sulfonyl, amino and nitro. In a
particular embodiment R.sub.2 is alkyl, a carbocycle,
carbocyclylalkyl, a heterocycle or heterocyclylalkyl each
optionally substituted with halogen, hydroxyl, oxo, mercapto,
thione, carboxyl, alkyl, haloalkyl, alkoxy, alkylthio, sulfonyl,
amino and nitro. In an embodiment R.sub.2 is alkyl, a carbocycle,
carbocyclylalkyl, a heterocycle or heterocyclylalkyl each
optionally substituted with halogen, hydroxyl, mercapto, carboxyl,
alkyl, alkoxy, amino and nitro. In a particular embodiment R.sub.2
is alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, a heterocycle
or heterocyclylalkyl. In a particular embodiment R.sub.2 is alkyl,
cycloalkyl or a heterocycle. In a particular embodiment R.sub.2 is
selected from the group consisting of t-butyl, isopropyl,
cyclohexyl, tetrahydropyran-4-yl, N-methylsulfonylpiperidin-4-yl,
tetrahydrothiopyran-4-yl, tetrahydrothiopyran-4-yl (in which the S
is in oxidized form SO or SO.sub.2), cyclohexan-4-one,
4-hydroxycyclohexane, 4-hydroxy-4-methylcyclohexane,
1-methyl-tetrahydropyran-4-yl, 2-hydroxyprop-2-yl, but-2-yl, phenyl
and 1-hydoxyeth-1-yl. In an embodiment of the invention R.sub.2 is
t-butyl, isopropyl, cyclohexyl, cyclopentyl, phenyl or
tetrahydropyran-4-yl. In a particular embodiment, R.sub.2 is
phenyl. In a particular embodiment, R.sub.2 is cyclohexyl. In
another embodiment R.sub.2 is tetrahydropyran-4-yl. In another
particular embodiment, R.sub.2 is isopropyl (i.e. the valine amino
acid side chain). In another particular embodiment, R.sub.2 is
t-butyl. In a particular embodiment R.sub.2 is oriented such that
the amino acid, or amino acid analogue, which it comprises is in
the L-configuration.
[0058] R.sub.3 is H or alkyl optionally substituted with halogen or
hydroxyl; or R.sub.3 and R4 together form a 3-6 heterocycle. In an
embodiment R.sub.3 is H or alkyl; or R.sub.3 and R.sub.4 together
form a 3-6 heterocycle. In an embodiment R.sub.3 is H or methyl,
ethyl, propyl or isopropyl. In a particularly particular embodiment
R.sub.3 is H or methyl. In a another particular embodiment R.sub.3
is methyl. In another particular embodiment, R.sub.3 is ethyl. In a
particular embodiment R.sub.3 is fluoromethyl. In a particular
embodiment R.sub.3 is hydroxyethyl. In another embodiment R.sub.3
is oriented such that the amino acid, or amino acid analogue, which
it comprises is in the L-configuration. In a particular embodiment
R.sub.3 and R.sub.4 together with the atoms from which they depend
form a 3-6 heterocycle. In a particular embodiment R.sub.3 and
R.sub.4 together form an azetidine ring. In a particular embodiment
R.sub.3 and R.sub.4 together form a pyrrolidine. R.sub.3' is H, or
R.sub.3 and R.sub.3' together form a 3-6 carbocycle. In an
embodiment, R.sub.3' is H. In another embodiment R.sub.3 and
R.sub.3' together form a 3-6 carbocycle, for example a cyclopropyl
ring. In a particular embodiment R.sub.3 and R.sub.3' are both
methyl.
[0059] R.sub.4 and R.sub.4' are independently H, hydroxyl, amino,
alkyl, carbocycle, carbocycloalkyl, carbocycloalkyloxy,
carbocycloalkyloxycarbonyl, heterocycle, heterocycloalkyl,
heterocycloalkyloxy or heterocycloalkyloxycarbonyl; wherein each
alkyl, carbocycloalkyl, carbocycloalkyloxy,
carbocycloalkyloxycarbonyl, heterocycle, heterocycloalkyl,
heterocycloalkyloxy and heterocycloalkyloxycarbonyl is optionally
substituted with halogen, hydroxyl, mercapto, carboxyl, alkyl,
alkoxy, amino, imino and nitro; or R.sub.4 and R.sub.4' together
form a heterocycle. In an embodiment R.sub.4 and R.sub.4' are
independently H, hydroxyl, amino, alkyl, aryl, aralkyl, cycloalkyl,
cycloalkylalkyl, heteroaryl, or heteroarylalkyl wherein each alkyl,
aryl, aralkyl, cycloalkyl, cycloalkylalkyl, heteroaryl and
heteroarylalkyl is optionally substituted with halogen, hydroxyl,
mercapto, carboxyl, alkyl, alkoxy, amino and nitro; or R.sub.4 and
R.sub.4' together form a heterocycle. In a particular embodiment
R.sub.4 and R.sub.4' together form a heterocycle, for example an
azetidine ring, or a pyrrolidine ring. In a particular embodiment
R.sub.4 and R.sub.4' are both H. In another particular embodiment
R.sub.4 is methyl and R.sub.4' is H. In a particular embodiment one
of R.sub.4 and R.sub.4' is hydroxyl (OH) while the other is H. In
another embodiment, one of R.sub.4 and R.sub.4' is amino, such as
NH.sub.2, NHMe and NHEt, while the other is H. In a particular
embodiment, R.sub.4' is H and R.sub.4 is H, alkyl, aryl, aralkyl,
cycloalkyl, cycloalkylalkyl, heteroaryl or heteroarylalkyl. In a
particular embodiment R.sub.4 is a group selected from the group
consisting of: ##STR10## R.sub.5 is H or alkyl. In a particular
embodiment, R.sub.5 is H or methyl. In a particular embodiment,
R.sub.5 is H. In another particular embodiment, R.sub.5 is
methyl.
[0060] R.sub.6, and R.sub.6' are each independently H, alkyl, aryl
or aralkyl. In a particular embodiment, R.sub.6 is alkyl, for
example methyl. In another particular embodiment R.sub.6 is aryl,
for example phenyl. In another particular embodiment R.sub.6 is
aralkyl, for example benzyl. In a particular embodiment R.sub.6 and
R.sub.6' are the same, for example both alkyl, e.g. both methyl. In
another particular embodiment R.sub.6 is methyl and R.sub.6' is H.
In another embodiment R6 and R.sub.6 are both H.
[0061] Compounds of the invention contain one or more asymmetric
carbon atoms. Accordingly, the compounds may exist as
diastereomers, enantiomers or mixtures thereof. The syntheses of
the compounds may employ racemates, diastereomers or enantiomers as
starting materials or as intermediates. Diastereomeric compounds
may be separated by chromatographic or crystallization methods.
Similarly, enantiomeric mixtures may be separated using the same
techniques or others known in the art. Each of the asymmetric
carbon atoms may be in the R or S configuration and both of these
configurations are within the scope of the invention. In a
particular embodiment, compounds of the invention have the
following stereochemical configuration of formula I' ##STR11##
wherein ring A, Q, X.sub.1, X.sub.2, Y, R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.4', R.sub.5, R.sub.6 and R.sub.6' are as described
herein.
[0062] In an embodiment, compounds of the invention have the
general formula IV ##STR12## wherein Q, X.sub.1, X.sub.2, Y,
R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.4', R.sub.5, R.sub.6,
R.sub.6 and R.sub.7 are as described herein. In a particular
embodiment Q is a carbocycle or a heterocycle optionally
substituted with one or more hydroxyl, alkyl, alkoxy, acyl,
halogen, mercapto, oxo, carboxyl, amino, cyano, nitro, amidino,
guanidino an optionally substituted carbocycle or an optionally
substituted heterocycle wherein one or more CH.sub.2 or CH groups
of said alkyl is optionally replaced with --O--, --S--, --S(O)--,
S(O).sub.2, --N(R.sub.8)--, --C(O)--, --C(O)--NR.sub.8--,
--NR.sub.8--C(O)--, --SO.sub.2--NR.sub.8--, --NR.sub.8--SO.sub.2--,
--NR.sub.8--C(O)--NR.sub.8--, --NR.sub.8--C(NH)--NR.sub.8--,
--NR.sub.8--C(NH)--, --C(O)--O-- or --O--C(O)--. In a particular
embodiment, Q is aryl or heteroaryl optionally substituted with one
or more hydroxyl, alkyl, alkoxy, acyl, halogen, mercapto, oxo,
carboxyl, amino, cyano, nitro, amidino, guanidino an optionally
substituted carbocycle or an optionally substituted heterocycle
wherein one or more CH.sub.2 or CH groups of said alkyl is
optionally replaced with --O--, --S--, --S(O)--, S(O).sub.2,
--N(R.sub.8)--, --C(O)--, --C(O)--NR.sub.8--, --NR.sub.8--C(O)--,
--SO.sub.2--NR.sub.8--, --NR.sub.8--SO.sub.2--,
--NR.sub.8--C(O)--NR.sub.8--, --NR.sub.8--C(NH)--NR.sub.8--,
--NR.sub.8--C(NH)--, --C(O)--O-- or --O--C(O)--. In a particular
embodiment Q is aryl or heteroaryl optionally substituted with with
one or more hydroxyl, alkyl, alkoxy, alkoxyalkoxy, acyl, halogen,
mercapto, carboxyl, acyl, halo-substituted alkyl, amino, cyano,
nitro, amidino, guanidino. In a particular embodiment Q is aryl or
heteroaryl optionally substituted with halogen, alkyl, alkoxy,
alkoxyalkoxy, cyano. In an embodiment Q is IIIa to IIIs wherein
R.sub.7, R.sub.8 and n are as defined herein. In a particular
embodiment Q is IIIq. In a particular embodiment Q is IIId. In a
particular embodiment Q is IIIb, IIIc, IIIe, IIIf, IIIj, IIIk,
IIIl, IIIn, IIIo, IIIq, IIIr or IIIs.
[0063] In an embodiment when compounds of the invention have the
general formula IV, R.sub.1 is H. In an embodiment when compounds
of the invention have the general formula IV, R.sub.2 is alkyl, a
carbocycle, carbocyclylalkyl, a heterocycle or heterocyclylalkyl
each optionally substituted with halogen, hydroxyl, mercapto,
carboxyl, alkyl, alkoxy, amino and nitro. In an embodiment when
compounds of the invention have the general formula IV, R.sub.3 is
H or methyl, ethyl, propyl or isopropyl. In an embodiment when
compounds of the invention have the general formula IV, R.sub.4 is
methyl and R.sub.4' is H. In an embodiment when compounds of the
invention have the general formula IV, R.sub.5 is H. In an
embodiment when compounds of the invention have the general formula
IV, R.sub.6 and R.sub.6', are both H. In an embodiment, when
compounds of the invention have the general formula IV R.sub.7 is
is H, halogen, cyano, alkyl, hydroxyalkyl or alkoxyalkyl. In an
embodiment, when compounds of the invention have the general
formula IV, X.sub.1 and X.sub.2 are both O. In an embodiment, when
compounds of the invention have the general formula IV, Y is
CH.sub.2.
[0064] The invention also encompasses prodrugs of the compounds
described above. Suitable prodrugs where applicable include known
amino-protecting and carboxy-protecting groups which are released,
for example hydrolyzed, to yield the parent compound under
physiologic conditions. A particular class of prodrugs are
compounds in which a nitrogen atom in an amino, amidino,
aminoalkyleneamino, iminoalkyleneamino or guanidino group is
substituted with a hydroxy (OH) group, an alkylcarbonyl (--CO--R)
group, an alkoxycarbonyl (--CO--OR), an acyloxyalkyl-alkoxycarbonyl
(--CO--O--R--O--CO--R) group where R is a monovalent or divalent
group and as defined above or a group having the formula
--C(O)--O--CP1P2-haloalkyl, where P1 and P2 are the same or
different and are H, lower alkyl, lower alkoxy, cyano, halo lower
alkyl or aryl. In a particular embodiment, the nitrogen atom is one
of the nitrogen atoms of the amidino group of the compounds of the
invention. These prodrug compounds are prepared reacting the
compounds of the invention described above with an activated acyl
compound to bond a nitrogen atom in the compound of the invention
to the carbonyl of the activated acyl compound. Suitable activated
carbonyl compounds contain a good leaving group bonded to the
carbonyl carbon and include acyl halides, acyl amines, acyl
pyridinium salts, acyl alkoxides, in particular acyl phenoxides
such as p-nitrophenoxy acyl, dinitrophenoxy acyl, fluorophenoxy
acyl, and difluorophenoxy acyl. The reactions are generally
exothermic and are carried out in inert solvents at reduced
temperatures such as -78 to about 50C. The reactions are usually
also carried out in the presence of an inorganic base such as
potassium carbonate or sodium bicarbonate, or an organic base such
as an amine, including pyridine, triethylamine, etc. One manner of
preparing prodrugs is described in U.S. Ser. No. 08/843,369 filed
Apr. 15, 1997 (corresponding to PCT publication WO9846576) the
contents of which are incorporated herein by reference in their
entirety.
[0065] Particular compounds of formula I include the following:
##STR13## ##STR14## ##STR15## ##STR16## ##STR17## ##STR18##
##STR19## ##STR20## ##STR21## ##STR22## ##STR23## ##STR24##
##STR25## ##STR26## ##STR27## ##STR28## ##STR29## ##STR30##
##STR31## ##STR32## ##STR33## ##STR34## ##STR35## ##STR36##
##STR37## ##STR38## ##STR39## ##STR40## ##STR41## ##STR42##
##STR43## ##STR44## ##STR45## ##STR46## ##STR47## ##STR48##
##STR49## Synthesis
[0066] Compounds of the invention are prepared using standard
organic synthetic techniques from commercially available starting
materials and reagents. It will be appreciated that synthetic
procedures employed in the preparation of compounds of the
invention will depend on the particular substituents present in a
compound and that various protection and deprotection steps that
are standard in organic synthesis may be required but may not be
illustrated in the following general schemes. In a particular
general synthetic scheme compounds of the invention may be prepared
using typical peptide chemistry techniques by coupling amino acid
residue analogues employing typical amide coupling procedures. In
scheme 1, amine-protected amino acid residue analogues are coupled
and deprotected sequentially to give the final compounds.
##STR50##
[0067] It will be appreciated that the amino acid analogs may be
coupled in any order and may be prepared using solid phase support
which is routine in the art. For example, Scheme2 illustrates an
alternative amino acid residue analogue coupling route.
##STR51##
[0068] The intermediate incorporating ring A is prepared from
commercially available reagents employing standard organic
chemistry techniques. For example, when ring A is thiazole, the
intermediate may be prepared according to scheme 3. ##STR52##
wherein Q, Y, R.sub.1, R.sub.6, and R.sub.6' are as defined herein
and Pr is an amine protecting group. A proline analogue wherein the
alpha nitrogen is protected (Pr), for example with Boc or Cbz, and
amidated is converted to the corresponding thioamide, for example
using Lawesson's reagent according to the procedures described in
Williams et al ( J. Org. Chem, 2001, 66:8463). The thiamide is then
cyclized with an appropriate bromide to give the desired thiazole
substituted with group Q, for example using the procedures
described in Ciufolini et al, (J. Org. Chem. 1997, 62: 3804).
Alternatively, the bromide in the present scheme may incorporate a
functional group which may be used to couple a desired group Q to
the thiazole formed from the cyclization step.
[0069] For compounds of the invention in which ring A is an
oxazole, the intermediate may be prepared according to scheme 4.
##STR53## wherein Q, Y, R.sub.1, R.sub.6, and R.sub.6' are as
defined herein and Pr is an amine protecting group. The starting
proline analogue is reacted with an appropriate amine using
standard amide forming procedures. The resulting amide is cyclized,
for example using Burgess Reagent according to the procedures
described in Pihko et al (J. Org. Chem., 1999, 64:652), to give the
dihydro-oxazole. The dihydro-oxazole is then reduced to give the
desired oxazole substituted with group Q. Alternatively, the amine
of the first step in the present scheme may incorporate a
functional group in place of Q which may be used directly or
indirectly to couple a desired group Q to the thiazole formed from
the cyclization step.
[0070] Compounds of the invention in which R.sub.4 or R.sub.4' are
other than H may be prepared according to standard organic
chemistry techniques, for example by reductive amination in which a
starting amino acid residue analog e.g.
NH.sub.2--CH(R.sub.3)--C(O)--OH is reacted with a suitable aldehyde
or ketone to give the desired R.sub.4 and R.sub.4' substituents.
See scheme 5. The resulting R.sub.4/R.sub.4' substituted amino acid
intermediate can then be conjugated to the next amino acid
intermediate or the remainder of the compound using standard
peptide coupling procedures. ##STR54##
[0071] In a particular embodiment, alanine is reacted with
1-methylindole-2-carboxaldehyde and reduced with sodium
cyanoborohydride dissolved in 1% HOAc/DMF to give the N-substituted
alanine residue which may be used in preparing compounds of the
invention. See scheme 6. ##STR55##
[0072] Alternatively, the reductive amination procedure to
introduce R.sub.4/R.sub.4' substituents is the final step in the
preparation of the compound.
[0073] When compounds of the invention incorporate R.sub.4 or
R.sub.4' substituents other than H, they may also be prepared by
substitution of a suitable acid intermediate which incorporates a
leaving group with a desired amine. For example
Br--CH(R.sub.3)--C(O)--OH is substituted with an amine
R.sub.4--NH.sub.2 or R.sub.4--NH--R.sub.4' according to scheme 7.
##STR56##
[0074] Alternatively, the substitution reaction introducing R.sub.4
or R.sub.4' substituents may be performed as a final step in the
preparation of the compound as illustrated in scheme 8.
##STR57##
[0075] In a particular embodiment, 2-bromopropionic acid is reacted
with the following amines dissolved in DMF and bubbled for until
substitution is complete to form N-substituted alanine residues:
##STR58##
[0076] Compounds of the invention in which either X.sub.1 or
X.sub.2 is sulfur, i.e. the compound incorporates a thioamide, may
be prepared according to established organic chemistry techniques.
For example, compounds in which X.sub.2 is sulfur can be prepared
according to scheme 9 starting from an Fmoc protected amino acid
residue analog NH.sub.2--CH(R.sub.2)--COOH which is dissolved in
THF and cooled to -25.degree. C., with addition of DIPEA followed
by addition of isobutylchloroformate. After 10 minutes, the
diamine, 4-nitrobenzene-1,2-diamine, is added and the reaction
mixture is continuously stirred at -25.degree. C. for 2 hours, then
at room temperature overnight. THF is vacuumed off and the mixture
is then subjected to flash chromatography using 50% EtOAc/Hexane to
yield the product. The Fmoc-alanine derivative, phosphorus
pentasulfide and sodium carbonate are mixed in THF and stirred
overnight. The solution is concentrated and direct chromatography
using 80% EtOAc/Hexane yields the activated thioalanine. The
activated thioalanine and sodium nitrite are then mixed in acetic
acid and diluted with H.sub.2O. The resulting precipitant is
filtered and dried to yield the product. The thioalanine is coupled
to an A ring substitued proline amino acid residue analog by
dissolving both in DMF. The thioamide product may then be
deprotected with 20% PIP/DMA for 15 minutes and used to conjugate
to the R.sub.4/R.sub.4'--N--C(R.sub.3)(R.sub.3')--COOH.
Alternatively the Fmoc-protected thioamide is first coupled to the
A ring substituted proline amino acid residue analog followed by
Fmoc deprotection and subsequent coupling to the
R.sub.4/R.sub.4'--R.sub.4/R.sub.4'--N--C(R.sub.3)(R.sub.3')--COOH
amino acid residue analog. ##STR59## Utility
[0077] The compounds of the invention inhibit the binding of IAP
proteins to caspases, in particular X-IAP binding interaction with
caspases 3 and 7. The compounds also inhibit the binding of ML-IAP
to Smac protein. Accordingly, the compounds of the invention are
useful for inducing apoptosis in cells or sensitizing cells to
apoptotic signals, in particular cancer cells. Compounds of the
invention are useful for inducing apoptosis in cells that
overexpress IAP proteins. Alternatively, compounds of the invention
are useful for inducing apoptosis in cells in which the
mitochondrial apoptotic pathway is disrupted such that release of
Smac from ML-IAP proteins is inhibited, for example by up
regulation of Bcl-2 or down regulation of Bax/Bak. More broadly,
the compounds can be used for the treatment of all cancer types
which fail to undergo apoptosis. Examples of such cancer types
include neuroblastoma, intestine carcinoma such as rectum
carcinoma, colon carcinoma, familiary adenomatous polyposis
carcinoma and hereditary non-polyposis colorectal cancer,
esophageal carcinoma, labial carcinoma, larynx carcinoma,
hypopharynx carcinoma, tong carcinoma, salivary gland carcinoma,
gastric carcinoma, adenocarcinoma, medullary thyroidea carcinoma,
papillary thyroidea carcinoma, renal carcinoma, kidney parenchym
carcinoma, ovarian carcinoma, cervix carcinoma, uterine corpus
carcinoma, endometrium carcinoma, chorion carcinoma, pancreatic
carcinoma, prostate carcinoma, testis carcinoma, breast carcinoma,
urinary carcinoma, melanoma, brain tumors such as glioblastoma,
astrocytoma, meningioma, medulloblastoma and peripheral
neuroectodermal tumors, Hodgkin lymphoma, non-Hodgkin lymphoma,
Burkitt lymphoma, acute lymphatic leukemia (ALL), chronic lymphatic
leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid
leukemia (CML), adult T-cell leukemia lymphoma, hepatocellular
carcinoma, gall bladder carcinoma, bronchial carcinoma, small cell
lung carcinoma, non-small cell lung carcinoma, multiple myeloma,
basalioma, teratoma, retinoblastoma, choroidea melanoma, seminoma,
rhabdomyo sarcoma, craniopharyngeoma, osteosarcoma, chondrosarcoma,
myosarcoma, liposarcoma, fibrosarcoma, Ewing sarcoma and
plasmocytoma.
[0078] Compounds of the invention are useful for sensitizing cells
to apoptotic signals. Accordingly, the compounds may be
administered prior to, concomitantly with, or following
administration of radiation therapy or cytostatic or antineoplastic
chemotherapy. Suitable cytostatic chemotherapy compounds include,
but are not limited to (i) antimetabolites, such as cytarabine,
fludarabine, 5-fluoro-2'-deoxyuiridine, gemcitabine, hydroxyurea or
methotrexate; (ii) DNA-fragmenting agents, such as bleomycin, (iii)
DNA-crosslinking agents, such as chlorambucil, cisplatin,
cyclophosphamide or nitrogen mustard; (iv) intercalating agents
such as adriamycin (doxorubicin) or mitoxantrone; (v) protein
synthesis inhibitors, such as L-asparaginase, cycloheximide,
puromycin or diphtheria toxin; (Vi) topoisomerase I poisons, such
as camptothecin or topotecan; (vii) topoisomerase II poisons, such
as etoposide (VP-16) or teniposide; (viii) microtubule-directed
agents, such as colcemid, colchicine, paclitaxel, vinblastine or
vincristine; (ix) kinase inhibitors such as flavopiridol,
staurosporin, STI571 (CPG 57148B) or UCN-01
(7-hydroxystaurosporine); (x) miscellaneous investigational agents
such as thioplatin, PS-341, phenylbutyrate, ET-18-OCH.sub.3, or
farnesyl transferase inhibitors (L-739749, L-744832); polyphenols
such as quercetin, resveratrol, piceatannol, epigallocatechine
gallate, theaflavins, flavanols, procyanidins, betulinic acid and
derivatives thereof; (xi) hormones such as glucocorticoids or
fenretinide; (xii) hormone antagonists, such as tamoxifen,
finasteride or LHRH antagonists. In a particular embodiment,
compounds of the present invention are coadministered with a
cytostatic compound selected from the group consisting of
cisplatin, doxorubicin, taxol, taxotere and mitomycin C. In a
particular embodiment, the cytostatic compound is doxorubicin.
[0079] Another class of active compounds which can be used in the
present invention are those which are able to sensitize for or
induce apoptosis by binding to death receptors ("death receptor
agonists"). Such agonists of death receptors include death receptor
ligands such as tumor necrosis factor a (TNF-.alpha.), tumor
necrosis factor .beta. (TNF-.beta., lymphotoxin-.alpha.), LT-.beta.
(lymphotoxin-.beta.), TRAIL (Apo2L, DR4 ligand), CD95 (Fas, APO-1)
ligand, TRAMP (DR3, Apo-3) ligand, DR6 ligand as well as fragments
and derivatives of any of said ligands. In an embodiment, the death
receptor ligand is TNF-.alpha.. In a particular embodiment, the
death receptor ligand is Apo2L/TRAIL. Furthermore, death receptors
agonists comprise agonistic antibodies to death receptors such as
anti-CD95 antibody, anti-TRAIL-R1 (DR4) antibody, anti-TRAIL-R2
(DR5) antibody, anti-TRAIL-R3 antibody, anti-TRAIL-R4 antibody,
anti-DR6 antibody, anti-TNF-R1 antibody and anti-TRAMP (DR3)
antibody as well as fragments and derivatives of any of said
antibodies.
[0080] For the purpose of sensitizing cells for apoptosis, the
compounds of the present invention can be also used in combination
with radiation therapy. The phrase "radiation therapy" refers to
the use of electromagnetic or particulate radiation in the
treatment of neoplasia. Radiation therapy is based on the principle
that high-dose radiation delivered to a target area will result in
the death of reproducing cells in both tumor and normal tissues.
The radiation dosage regimen is generally defined in terms of
radiation absorbed dose (rad), time and fractionation, and must be
carefully defined by the oncologist. The amount of radiation a
patient receives will depend on various consideration but the two
most important considerations are the location of the tumor in
relation to other critical structures or organs of the body, and
the extent to which the tumor has spread. Examples of
radiotherapeutic agents are provided in, but not limited to,
radiation therapy and is known in the art (Hellman, Principles of
Radiation Therapy, Cancer, in Principles I and Practice of
Oncology, 24875 (Devita et al., 4th ed., vol 1, 1993). Recent
advances in radiation therapy include three-dimensional conformal
external beam radiation, intensity modulated radiation therapy
(IMRT), stereotactic radiosurgery and brachytherapy (interstitial
radiation therapy), the latter placing the source of radiation
directly into the tumor as implanted "seeds". These newer treatment
modalities deliver greater doses of radiation to the tumor, which
accounts for their increased effectiveness when compared to
standard external beam radiation therapy.
[0081] Ionizing radiation with beta-emitting radionuclides is
considered the most useful for radiotherapeutic applications
because of the moderate linear energy transfer (LET) of the
ionizing particle (electron) and its intermediate range (typically
several millimeters in tissue). Gamma rays deliver dosage at lower
levels over much greater distances. Alpha particles represent the
other extreme, they deliver very high LET dosage, but have an
extremely limited range and must, therefore, be in intimate contact
with the cells of the tissue to be treated. In addition, alpha
emitters are generally heavy metals, which limits the possible
chemistry and presents undue hazards from leakage of radionuclide
from the area to be treated. Depending on the tumor to be treated
all kinds of emitters are conceivable within the scope of the
present invention.
[0082] Furthermore, the present invention encompasses types of
non-ionizing radiation like e.g. ultraviolet (UV) radiation, high
energy visible light, microwave radiation (hyperthermia therapy),
infrared (IR) radiation and lasers. In a particular embodiment of
the present invention UV radiation is applied.
[0083] The invention also includes pharmaceutical compositions or
medicaments containing the compounds of the invention and a
therapeutically inert carrier, diluent or excipient, as well as
methods of using the compounds of the invention to prepare such
compositions and medicaments. Typically, the compounds of formula I
used in the methods of the invention are formulated by mixing at
ambient temperature at the appropriate pH, and at the desired
degree of purity, with physiologically acceptable carriers, i.e.,
carriers that are non-toxic to recipients at the dosages and
concentrations employed into a galenical administration form. The
pH of the formulation depends mainly on the particular use and the
concentration of compound, but may range anywhere from about 3 to
about 8. Formulation in an acetate buffer at pH 5 is a suitable
embodiment. In an embodiment, the inhibitory compound for use
herein is sterile. The compound ordinarily will be stored as a
solid composition, although lyophilized formulations or aqueous
solutions are acceptable.
[0084] The composition of the invention will be formulated, dosed,
and administered in a fashion consistent with good medical
practice. Factors for consideration in this context include the
particular disorder being treated, the particular mammal being
treated, the clinical condition of the individual patient, the
cause of the disorder, the site of delivery of the agent, the
method of administration, the scheduling of administration, and
other factors known to medical practitioners. The "effective
amount" of the compound to be administered will be governed by such
considerations, and is the minimum amount necessary to inhibit IAP
interaction with caspases, induce apoptosis or sensitize a
malignant cell to an apoptotic signal. Such amount is may be below
the amount that is toxic to normal cells, or the mammal as a
whole.
[0085] Generally, the initial pharmaceutically effective amount of
the compound of the invention administered parenterally per dose
will be in the range of about 0.01-100 mg/kg, for example about 0.1
to 20 mg/kg of patient body weight per day, with the typical
initial range of compound used being 0.3 to 15 mg/kg/day. Oral unit
dosage forms, such as tablets and capsules, may contain from about
25 to about 1000 mg of the compound of the invention.
[0086] The compound of the invention may be administered by any
suitable means, including oral, topical, transdermal, parenteral,
subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and,
if desired for local treatment, intralesional administration.
Parenteral infusions include intramuscular, intravenous,
intraarterial, intraperitoneal, or subcutaneous administration. An
example of a suitable oral dosage form is a tablet containing about
25 mg, 50 mg, 100 mg, 250 mg, or 500 mg of the compound of the
invention compounded with about 90-30 mg anhydrous lactose, about
5-40 mg sodium croscarmellose, about 5-30 mg polyvinylpyrrolidone
(PVP) K30, and about 1-10 mg magnesium stearate. The powdered
ingredients are first mixed together and then mixed with a solution
of the PVP. The resulting composition can be dried, granulated,
mixed with the magnesium stearate and compressed to tablet form
using conventional equipment. An aerosol formulation can be
prepared by dissolving the compound, for example 5-400 mg, of the
invention in a suitable buffer solution, e.g. a phosphate buffer,
adding a tonicifier, e.g. a salt such sodium chloride, if desired.
The solution is typically filtered, e.g. using a 0.2 micron filter,
to remove impurities and contaminants.
EXAMPLES
[0087] The invention will be more fully understood by reference to
the following examples. They should not, however, be construed as
limiting the scope of the invention. Reagents and solvents were
obtained from commercial sources and used as received. ISCO
chromatography refers to use of a pre-packed silica gel columns on
a Companion system by Teledyne-Isco, Inc. Lincoln, Nebr. The
identity and purity of all compounds were checked by LCMS and
.sup.1H NMR analysis.
[0088] Abbreviations used herein are as follows: [0089] ACN:
acetonitrile; [0090] Chg: cyclohexylglycine; [0091] DCM:
dichloromethane [0092] DIPEA: diisopropylethylamine; [0093] DMAP:
4- dimethylaminopyridine; [0094] DME: 1,2-dimethoxyethane; [0095]
DMF: dimethylformamide; [0096] DMSO: dimethylsulfoxide [0097] EDC:
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; [0098] EEDQ:
2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline [0099] LCMS: liquid
chromatography mass spectrometry; [0100] HATU:
O-(7-Azobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate; [0101] HOBt: N-Hydroxybenzotriazole [0102]
HBTU: 2-(1H-Benzotriazol-1-yl)-1,1,3,3-Tetramethyl-uronium
Hexafluorophosphate [0103] HPLC: high performance liquid
chromatography; [0104] NBS: N-bromosuccinamide; [0105] TASF:
tris(dimethylamino)sulfonium difluorotrimethylsilicate; [0106] TEA:
triethylamine; [0107] TFA: trifluoroacetate; [0108] THF:
tetrahydrofuran;
Example 1
2-[tert-Butoxycarbonyl-(1H-pyrrol-2-ylmethyl)-amino]-propionic
acid
[0109] ##STR60##
[0110] Alanine ethyl ester b (5 g, 32.5 mmol),
pyrrole-2-carboxaldehyde a (3.1 g, 32.5 mmol), sodium
cyanoborohydride (2.04 g, 32.5 mmol) and AcOH (1%) were mixed in
DMF and stirred overnight. The reaction was quenched with H.sub.2O,
and DMF was evaporated. The mixture was diluted with EtOAc, washed
by 0.1N NaOH, dried and concentrated to yield product c 2.5 g. The
resulting ester c (2.5 g, 12.8 mmol), di-tert-butyldicarbonate
(3.06 g, 14 mmol) were mixed in THF, H.sub.2O with NaHCO.sub.3 and
stirred overnight. THF was evaporated, and the mixture was diluted
with EtOAc, washed by 1N NaOH, sat. NH.sub.4Cl and brine. After
dried, the mixture was concentrated to yield the Boc-protected
ester d 3.3 g. The Boc-protected ester d (1.67 g, 5.6 mol), lithium
hydroxide mono hydrate (284 mg, 6.77 mmol) were mixed in THF and
H.sub.2O at 0.degree. C. THF was vacuumed off, and the solution was
acidified by dilute H.sub.2SO.sub.4, extracted by EtOAc twice.
Organic layers were combined, dried and evaporated giving product
2-[tert-butoxycarbonyl-(1H-pyrrol-2-ylmethyl)-amino]-propionic acid
e.
Example 2
Thiazole Substituted Pyrrolidine
[0111] ##STR61##
[0112] Following the general procedure of Williams (Williams, D. R.
et al, M. J. Org. Chem. 2001, 66, 8463), a mixture of N-Cbz-proline
amide a (500 mg, 2.0 mmol) Lawesson's reagent (420 mg, 1.05 mmol)
and toluene (5 mL) was heated at reflux for 2 h. The solution was
concentrated, adsorbed onto Celite, and purified by flash
chromatography (SiO.sub.2, 40% ethyl acetate-hexanes) to afford 393
mg (74%) of compound b as a colorless solid. ##STR62##
[0113] Following the general procedure of Ciufolini (Ciufolini, M.
A. et al, J. Org. Chem. 1997, 62, 3804), ethyl bromopyruvate (200
.mu.l, 1.43 mmol) was added to a suspension of thioamide b (378 mg,
1.43 mmol) in ethanol (5 mL), and the mixture heated at 80.degree.
C. for 5 min. The solvent was evaporated under reduced pressure,
and the residue purified by flash chromatography (SiO.sub.2,
gradient elution, 30-40-50% ethyl acetate-hexanes) to afford 393 mg
(74%) of thiazole c as a colorless solid. ##STR63##
[0114] Phenyl magnesium bromide (2.1 mL of 1.0 M solution in THF,
2.1 mmol) was added dropwise to a cold (-78.degree. C.) solution of
ester c (360 mg, 1.0 mmol) in THF (5 mL) over 5 min. The cooling
bath was removed and the solution allowed to reach room
temperature, at which time it was poured into saturated aqueous
NH.sub.4Cl (50 mL). The aqueous layer was extracted with 50% ethyl
acetate-hexanes (3.times.10 mL). The combined organic layers were
dried (Na.sub.2SO.sub.4), filtered, and concentrated. The residue
was purified by flash chromatography (SiO.sub.2, gradient elution,
30-40% ethyl acetate-hexanes) to afford 404 mg (84%) of thiazole d
as a colorless solid. ##STR64##
[0115] Triethylsilane (850 .mu.l, 5.3 mmol) and TFA (5 mL) were
added sequentially to alcohol d, and the resulting solution was
allowed stand at rt for 1 h. The solvent was evaporated, and the
residue purified by flash chromatography (SiO.sub.2, 30% ethyl
acetate-hexanes) to afford a quantitative yield of compound e as a
colorless oil. ##STR65##
[0116] Following the general procedure of Thurston (Bose, S. D.;
Thurston, D. E. Tetrahedron Lett. 1990, 31, 6903),
BF.sub.3.Et.sub.2O (0.78 mL, 6.2 mmol) was added to a solution of
carbamate e (280 mg, 0.62 mmol), propanethiol (560 .mu.l, 6.2 mmol)
and CH.sub.2Cl.sub.2 (3 mL) at rt. After 1 day at rt, the reaction
was poured into 1 N NaOH (50 mL) and stirred vigorously for 1 h.
The layers were separated and the organic phase was washed with 1 N
NaOH (2.times.5 mL). The combined aqueous layers were extracted
with CH.sub.2Cl.sub.2 (2.times.5 mL), and the combined organic
layers were dried (K.sub.2CO.sub.3), filtered and concentrated. The
residue was purified by flash chromatography (SiO.sub.2, gradient
elution, 40-50-60% ethyl acetate-hexanes, 1% TEA) to afford 122 mg
(61%) of amine f as a colorless solid.
Example 3
Oxazole Substituted Pyrrolidine
[0117] ##STR66##
[0118] A mixture of N-Boc-proline a (5.35 g, 24.9 mmol) serine
methyl ester hydrochloride b (3.50 g, 22.5 mmol), EDC (4.76 g,
24.85 mmol), DIPEA (4.0 mL, 22.5 mmol) and CH.sub.2Cl.sub.2 (90 mL)
was maintained overnight. The mixture was diluted with
CH.sub.2Cl.sub.2 (200 mL) and washed with 1 N HCl (3.times.100 mL),
0.1 N NaOH (3.times.100 mL) and brine (1.times.100 mL). The organic
layer was dried (Na.sub.2SO.sub.4), filtered, and concentrated to
afford 5.2 g (73%) of dipeptide c as a colorless foam.
##STR67##
[0119] To a cool (0.degree. C.) solution of dipeptide c (4.57 g,
14.4 mmol) and THF (100 mL) was added Burgess Reagent (Pihko, P.
M.; Koskinen, A. M. P.; Nissinen, M. J.; Rissanen, K. J. Org. Chem.
1999, 64, 652, and references therein) (3.77 g, 15.8 mmol) in 3
portions over 30 min. The cooling bath was removed and the reaction
allowed to reach rt, then heated at reflux for 1 h. After cooling
to rt, the THF was removed under reduced pressure and the residue
was partitioned between EtOAc (200 mL) and saturated aqueous
NH.sub.4Cl (200 mL). The organic layer was washed with saturated
aqueous NH.sub.4Cl (2.times.50 mL). The combined aqueous phases
were extracted with EtOAc (1.times.50 mL) and the combined organic
phases were washed with brine, dried (Na.sub.2SO.sub.4), filtered,
and concentrated. The residue was purified by flash chromatography
(SiO.sub.2, 50-75-100% ethyl acetate-hexanes) to afford 2.94 g
(68%) of compound d as a colorless solid. ##STR68##
[0120] Following the general procedure of Koskinen (Pihko, P. M.;
Koskinen, A. M. P.; Nissinen, M. J.; Rissanen, K. J. Org. Chem.
1999, 64, 652, and references therein), to degassed
CH.sub.2Cl.sub.2 (25 mL), was added CuBr (8.79 g, 39.3 mmol),
hexamethylene tetraamine (5.51 g, 39.3 mmol) and DBU (5.9 mL, 39.3
mmol) and the resulting dark mixture stirred vigorously while it
was cooled to 0.degree. C. To this mixture was added a degassed
solution of d (2.94 g, 9.83 mmol) and CH.sub.2Cl.sub.2 (25 mL) over
5 min. The cooling bath was removed and the mixture stirred
vigorously for 2 h. The reaction was then poured into 1:1 saturated
aqueous NH.sub.4Cl: conc. NH.sub.4OH (200 mL), stirred for 30 min,
then extracted with EtOAc (3.times.50 mL). The combined organic
phases were washed with saturated aqueous NH.sub.4Cl (2.times.50
mL), brine, dried (Na.sub.2SO.sub.4), filtered, and concentrated.
The residue was purified by flash chromatography (SiO--.sub.2,
40-50% ethyl acetate-hexanes) to afford 1.1 g (38%) of oxazole e as
a colorless solid. ##STR69##
[0121] Phenylmagnesium bromide (4.4 mL of 1.0 M solution in THF,
4.4 mmol) was added dropwise to a cold (-78.degree. C.) solution of
ester e (600 mg, 2.0 mmol) in THF (10 mL) over 5 min. The cooling
bath was removed and the solution allowed to reach rt, at which
time it was poured into saturated aqueous NH.sub.4Cl (50 mL). The
aqueous layer was extracted with 50% ethyl acetate-hexanes
(3.times.10 mL). The combined organic layers were dried
(Na.sub.2SO.sub.4), filtered, and concentrated. The residue was
purified by flash chromatography (SiO.sub.2, gradient elution,
20-30-40% ethyl acetate-hexanes) to afford 443 mg (52%) of oxazole
f as a colorless solid. ##STR70##
[0122] Triethylsilane (20 .mu.l) and TFA (1 mL) were added
sequentially to a solution of alcohol f (50 mg, 0.1 mmol) and
CH.sub.2Cl.sub.2 (1 mL). The resulting solution was allowed stand
at rt for 1 h. The solvent was evaporated, and the residue
partitioned between EtOAc (20 mL) and 1N NaOH (20 mL). The organic
phase was washed with 1N NaOH (2.times.20 mL). The combined aqueous
phases were extracted with EtOAc (1.times.20 mL), and the combined
organic phases were washed with brine (1.times.20 mL) dried
(Na.sub.2SO.sub.4), filtered, and concentrated to afford amine g as
a colorless oil, contaminated with residual triethylsilane. This
material was used directly in the next coupling.
Example 4
Synthesis of Methyl Ketones
[0123] ##STR71##
[0124] A mixture of dihydrobezofuran a (Davies, H. M. L.; Grazini,
M. V. A.; Aouad, E. Org. Lett. 2001, 3, 1475) (160 mg, 0.9 mmol)
DDQ (300 mg) and CH.sub.2Cl.sub.2 (11 mL) was maintained at room
temp. for 2 days. The solution was diluted with 50% ethyl
acetate-hexanes and washed with 0.5 N NaOH (3.times.10 mL), brine
(1.times.10 mL), dried (Na.sub.2SO.sub.4), filtered, and
concentrated to afford 150 mg (93%) of bezofuran b. ##STR72##
[0125] Isopropylmagnesium chloride (7.1 mL of a 2.0 M solution in
THF, 14.2 mmol) was added dropwise to a mixture of benzofuran
methyl ester b (500 mg, 2.84 mmol) and N,O-dimethyl hydroxyl amine
hydrochloride (690 mg, 7.1 mmol) and THF (8 mL) maintained
.rarw.20.degree. C. The mixture was allowed to warm to 0.degree. C.
over 20 min, then poured into 50 mL of saturated aqueous
NH.sub.4Cl. The aqueous phase was extracted with EtOAc (3.times.20
mL), the combined organic phases were washed with brine (1.times.50
mL), dried (Na.sub.2SO.sub.4), filtered, and concentrated to afford
577 mg (85%) of amide c, as a clear oil. ##STR73##
[0126] To a solution of amide c (660 mg, 3.22 mmol) and THF (6 mL)
was added MeMgBr (3 mL of a 3.0 M solution in THF, 9 mmol) at
0.degree. C. The solution was maintained at 0.degree. C. for 30
min, then allowed to warm to 20.degree. C. for 30 min, at which
time a precipitate forms. The mixture was poured into 100 mL of
saturated aqueous NH.sub.4Cl. The aqueous phase was extracted with
EtOAc (3.times.50 mL), the combined organic phases were washed with
brine (1.times.50 mL), dried (Na.sub.2SO.sub.4), filtered, and
concentrated to afford 460 mg (89%) of ketone d, as a clear oil.
##STR74##
[0127] A mixture of potassium tert-butoxide (2.2 g, 17.5 mmol),
fluoro ketone e (3.0 g, 15.9 mmol) and ethylene glycol (30 mL) was
heated at 50.degree. C. for 1 h, then 60.degree. C. for 2 h. The
mixture was then poured into 500 mL of saturated aqueous
NH.sub.4Cl. The aqueous phase was extracted with Et.sub.2O
(3.times.150 mL), the combined organic phases were washed with
water (3.times.150 mL), brine (1.times.50 mL), dried
(Na.sub.2SO.sub.4). The mixture was adsorbed onto Celite, and
chromatographed (ISCO, 120 g silica column, 10-60% EtOAc-hexanes)
to afford 2.23 g (61 %) of the hydroxy ether f as a colorless
solid. ##STR75##
[0128] A mixture of potassium cyanide (6.9 g, 106 mmol), fluoro
ketone e (2.0 g, 10.6 mmol) and DMSO (20 mL) was maintained at rt
for 4 days, then heated at 50.degree. C. for 1 day. The mixture was
then poured into 500 mL of 1 N NaOH. The aqueous phase was
extracted with Et.sub.2O (3.times.150 mL), the combined organic
phases were washed with water (3.times.150 mL), brine (1.times.50
mL), dried (Na.sub.2SO.sub.4). The mixture was adsorbed onto
Celite, and chromatographed (ISCO, 120 g silica column, 0-20%
EtOAc-hexanes) to afford 1.15 g (55%) of the nitrile g as a yellow
solid. ##STR76##
[0129] A mixture of dibromide h (2.33 g, 7.78 mmol), NaSMe (600 mg,
8.56 mmol), and EtOH (5 mL) was maintained at rt 18 h. The mixture
was poured into 75 mL of 1 N NaOH and extracted with EtOAc
(3.times.50 mL). The combined organic phases were washed with 1 N
NaOH (1.times.50 mL), brine (3.times.50 mL), dried
(Na.sub.2SO.sub.4), filtered and concentrated to afford 2.06 g
(98%) of thioether i as a colorless oil. ##STR77##
[0130] To a -78.degree. C. solution of bromide i (500 mg, 1.87
mmol) and THF (15 mL) was added sec-BuLi (1.6 mL of 1.4 M solution
in cyclohexane, 2.25 mmol) over 5 min. After 5 min at -78.degree.
C., the dark purple solution was quenched rapidly with DMF (0.5 mL)
and the solution warmed to 0.degree. C. and maintained at that
temp. for 5 min. The solution was then poured into saturated
aqueous NH.sub.4Cl (50 mL). The aqueous phase was extracted with
EtOAc (3.times.25 mL), the combined organic phases were washed
brine (1.times.50 mL), dried (Na.sub.2SO.sub.4) and filtered. The
mixture was adsorbed onto Celite, and chromatographed (ISCO, 12 g
silica column, 0-10% EtOAc-hexanes) to afford 260 mg (64%) of
aldehydei as a clear oil. ##STR78##
[0131] To a solution of aldehyde j (400 mg, 1.86 mmol) and THF (5
m-L) was added MeMgCl (0.9 mL of a 3.0M solution in THF, 2.8 mmol)
at 0.degree. C. The solution was maintained at 0.degree. C. for 30
min, then allowed to warm to 20.degree. C. for 30 min. The mixture
was poured into 50 mL of saturated aqueous NH.sub.4Cl. The aqueous
phase was extracted with EtOAc (3.times.25 mL), the combined
organic phases were washed with brine (1.times.50 mL), dried
(Na.sub.2SO.sub.4), filtered, and concentrated to afford crude
alcohol k as a clear oil, which was used without further
purification. ##STR79##
[0132] To a solution of crude sulfide k and MeOH (5 mL) at
0.degree. C. was added a suspension of Oxone (1.3 g, 2.1 mmol) in
water (5 mL) over 20 min. The mixture was allowed to reach room
temp and then poured into 50 mL of saturated aqueous NH.sub.4Cl.
The aqueous phase was extracted with EtOAc (3.times.25 mL), the
combined organic phases were washed with brine (1.times.50 mL),
dried (Na.sub.2SO.sub.4), filtered, and concentrated. This residue
was dissolved in MeOH (10 mL), cooled to 0.degree. C., and to it
was added a suspension of Oxone (2.6 g, 4.2 mmol) in water (10 mL)
over 20 min. The mixture was stirred at rt overnight then poured
into 50 mL of saturated aqueous NH.sub.4Cl. The aqueous phase was
extracted with EtOAc (3.times.25 mL), the combined organic phases
were washed with brine (1.times.50 mL), dried (Na.sub.2SO.sub.4),
filtered, and concentrated to afford 550 mg (100 % for two steps)
of sulfone l as a clear oil. ##STR80##
[0133] A mixture of alcohol l (550 mg, 2.1 mmol), Celite (680 mg),
and PCC (500 mg, 2.31 mmol) was stirred vigorously at rt for 6 h.
More PCC (200 mg) was added and the mixture was stirred overnight.
The mixture was adsorbed onto more Celite (5 g) and chromatographed
(ISCO, 12 g silica column 0-50% EtOAc-hexanes) to afford 380 mg
(69%) of ketone m as a colorless solid. ##STR81##
[0134] Thionyl chloride (26 mL, 365 mmol) was added to a mixture of
2-methoxy-1-naphthoic acid n (4.5 g, 22.3 mmol) and toluene (45
mL). The resulting mixture was heated at 75.degree. C. for 3 h. The
solvent was removed under reduced pressure, and the intermediate
acid chloride was dried under high vacuum for 1 h. It was dissolved
in THF (50 mL) and cooled to 0.degree. C. under N.sub.2.
Dimethylzinc (45 mL of 1.0 M solution in heptane, 44.6 mmol) was
added over 15 min. The reaction mixture was kept at 0.degree. C.
for 5 min, allowed to warm to room temperature. The reaction was
quenched with slow addition of saturated NH.sub.4Cl (200 mL). The
aqueous phase was extracted with EtOAc (3.times.100 mL), and the
combined organic phases were washed with brine (1.times.100 mL),
dried (MgSO.sub.4), filtered, and concentrated in vacuo. The crude
product was adsorbed on to Celite and purified by ISCO CombiFlash
40 g column (5-15% ethyl acetate-hexane) to afford 1.96 g (44%) of
ketone o as a white solid. ##STR82##
[0135] Following the general procedure of Caldwell (Ichinose, N.;
Mizuno, K.; Otsuji, Y.; Caldwell, R. A.; Helms, A. M. J. Org. Chem.
1998, 63, 3176-84), to a solution of CH.sub.3MgCl (3.4 mL of 3.0 M
solution in THF, 10.0 mmol) in THF (20 mL) was added dropwise a
solution of 4-methoxy-1-naphthalenecarbonitrile p (0.5 g, 2.7 mmol)
in toluene (10 mL). After the addition, toluene (10 mL) was added
to the mixture. The resulting solution was heated to reflux for 8
h. Aqueous AcOH (50%, 10 mL) was added, and the mixture was heated
to reflux for 4 h. After cooling, the mixture was diluted with
water, and the organic phase separated, dried (MgSO.sub.4),
filtered, and concentrated in vacuo to afford 0.5 g (93%) of ketone
q as a yellow oil, which was used without further purification.
##STR83##
[0136] Following the general procedure of Boswell (Boswell, E.G.;
Licause, J. F. J. Org. Chem. 1995, 60, 6592-94), to a solution of
sodium thiomethoxide (0.41 g, 5.8 mmol) in anhydrous DMSO (8 mL) at
0.degree. C. under N.sub.2 was added dropwise a solution of
4-fluoro-1-acetylnaphthalene e (1.0 g, 5.3 mmol) in DMSO (8 mL).
After stirring at room temperature for 1.5 h, the mixture was
diluted with water, extracted with CH.sub.2Cl.sub.2 (3.times.20
mL), and the combined organic phases were dried (MgSO.sub.4),
filtered, and concentrated in vacuo to afford 1.0 g (88%) of
sulfide r as a light yellow solid, which was carried on without
further purification. ##STR84##
[0137] Following the general procedure of Trost (Trost, B. M.;
Curran, D. P. Tetrahedron Lett. 1981, 22, 1287-90), to a cold
(0.degree. C.) solution of sulfide r (2.3 g, 10.6 mmol) in methanol
(50 mL) was added dropwise a solution of potassium hydrogen
persulfate (Oxone, 22.8 g, 37.1 mmol) in water (75 mL) keeping the
reaction temperature below 5.degree. C. The resulting slurry was
stirred at room temperature for 72 h, diluted with water and
extracted with CH.sub.2Cl.sub.2 (2.times.100 mL). The combined
organics were washed with brine, dried (MgSO.sub.4), filtered, and
concentrated to afford crude product. The residue was adsorbed on
to Celite and purified by ISCO CombiFlash 40 g column (10-40% ethyl
acetate-hexane) to afford 2.32 g (88%) of sulfone s as an off white
solid. ##STR85##
[0138] A mixture of 4-fluoro-1-acetylnaphthalene e (4.75 g, 25.2
mmol), morpholine (6.60 mL, 75.8 mmol), K.sub.2CO.sub.3 (5.21 g,
37.8 mmol), DMSO (30 mL), and water (12 mL) was heated at
90.degree. C. for 8 h. The reaction mixture was diluted with water,
extracted with CH.sub.2Cl.sub.2 (2.times.100 mL). The combined
organic layers were washed with brine, dried (MgSO.sub.4),
filtered, and concentrated in vacuo to afford crude product. It was
triturated with water, filtered, washed with water, dried to afford
6.40 g (99%) of morpholinyl ketone t as a yellow solid.
##STR86##
[0139] 2'-Hydroxy-l'-acetonaphthone u (5.0 g, 26.9 mmol) and
K.sub.2CO.sub.3 (11.1 g, 81.0 mmol) in acetone (150 mL) were
stirred for 20 min. To this mixture was added bromoethyl methyl
ether (3.8 mL, 39.5 mmol) and catalytic KI. The resulting mixture
was heated to reflux for 72 h. After cooling, the solvent was
removed in vacuo. The residue was dissolved in EtOAc, washed with 1
N aqueous NaOH, brine, dried (MgSO.sub.4), filtered, and
concentrated in vacuo. The crude product was adsorbed on to Celite
and purified by ISCO CombiFlash 120 g column (5-25% ethyl
acetate-hexane) to afford 3.21 g (49%) of ether v as an oil.
##STR87##
[0140] Following the general procedure of Short (Short, W. F.;
Wang, H. J. Chem. Soc. 1950, 9914), to a three-necked
round-bottomed flask equipped with a reflux condenser, a dropping
funnel, and an aqueous NaOH trap was added 1-naphthoic acid w (10.0
g, 58.0 mmol) and AcOH (35 mL). This solution was heated at
110.degree. C. and stirred during the addition of bromine (3.12 mL,
61.0 mmol). After the addition, the mixture was heated for another
1.5 h (A yellow solid precipitated during the heating), and then
stirred at room temperature for 24 h. The mixture was poured into
ice water. The solid was filtered, washed with water, and
crystallized from acetic acid (250 mL) to afford 8.9 g (61 %) of
bromo acid x as a white solid. ##STR88##
[0141] A solution of bromo acid x (6.0 g, 23.9 mmol),
N,O-dimethylhydroxyl amine hydrochloride (2.33 g, 23.9 mmol), EDC
(4.6 g, 23.9 mmol), and DIPEA (6.3 mL, 35.8 mmol) in DMF (35 mL)
was stirred at room temperature for 4 h. The mixture was poured
into water, extracted with CH.sub.2Cl.sub.2 (2.times.200 mL). The
combined organic layers were washed with 0.5 N aqueous HCl, 0.5 N
aqueous NaOH, dried (MgSO.sub.4), filtered, and concentrated in
vacuo. The crude product was adsorbed on to Celite and purified by
ISCO CombiFlash 120 g column (2-10% ethyl acetate-CH.sub.2Cl.sub.2)
to afford 4.6 g (65%) of amide y as an oil. ##STR89##
[0142] Methylmagnesium chloride (8.5 mL of 3 M solution in THF,
25.5 mmol) was added dropwise to a cold (0.degree. C.) solution of
amide z (2.5 g, 8.5 mmol) and THF (80 mL). The resulting solution
was stirred at 0.degree. C. for 1 h, then allowed to warm to room
temperature. After 2.5 h, it was quenched by slow addition of
aqueous AcOH (50%, 10 mL), diluted with water (100 mL), and
separated. The aqueous layer was extracted with EtOAc (1.times.100
mL). The combined organic layers were washed with brine, dried
(MgSO.sub.4), filtered, and concentrated in vacuo to afford 1.9 g
(90%) of ketone a' as a yellow solid. ##STR90##
[0143] A mixture of 2'-hydroxy-1'-acetonaphthone u (5.0 g, 26.9
mmol), K.sub.2CO.sub.3 (7.41 g, 53.7 mmol), and
1-bromo-2-chloroethane (4.4 mL, 53.7 mmol) in DMF (70 mL) was
heated at 80.degree. C. for 24 h. The cooled mixture was diluted
with water, and extracted with CH.sub.2Cl.sub.2 (2.times.100 mL).
The combined organic phases were washed with 0.5 N aqueous NaOH,
brine, dried (MgSO.sub.4), filtered, and concentrated to afford
crude product. The residue was adsorbed on to Celite and purified
by ISCO CombiFlash 40 g column (5-25% ethyl acetate-hexane) to
afford 1.6 g (24%) of chloroethoxy ketone b' as a light yellow
solid. ##STR91##
[0144] A mixture of chloroethoxy ketone b' (3.0 g, 12.1 mmol),
benzoic acid (1.47 g, 12.1 mmol), and Cs.sub.2CO.sub.3 (4.73 g,
14.5 mmol) in DMF (25 mL) was heated at 50.degree. C. for 16 h.
Benzoic acid (0.735 g, 6.0 mmol) and Cs.sub.2CO.sub.3 (2.36 g, 7.2
mmol) were added, and the mixture heated at 80.degree. C. for 24 h.
The mixture was filtered, diluted with EtOAc (100 mL), washed with
water, dried (MgSO.sub.4), filtered, and concentrated in vacuo to
afford 3.95 g (98%) of ketone c' as a yellow oil. ##STR92##
[0145] Following the general procedure of Oda (Oda, M.; Yamamuro,
A.; Watabe, T. Chem. Lett. 1979, 1427-30), Trimethylsilyl cyanide
(4.5 mL, 34.1 mmol) was added slowly into a mixture of
5-methoxy-l-tetralone d' (5.0 g, 28.4 mmol), catalytic ZnI.sub.2 in
toluene (12 mL). The resulting mixture was stirred at room
temperature for 24 h. Pyridine (40 mL) and POCl.sub.3 (8.0 mL, 85.2
mmol) were added, and the mixture was heated to reflux for 8 h. The
cooled dark solution was poured into ice water (300 mL) and conc.
HCl (10 mL) with stirring, extracted with EtOAc (3.times.400 mL).
The combined organic layers were washed with brine, dried
(MgSO.sub.4), filtered, and concentrated to afford 4.78 g of crude
unsaturated nitrile e' as a brown solid.
[0146] A mixture of the above unsaturated nitrile e' (4.78 g, 25.8
mmol) and DDQ (5.86 g, 25.8 mmol) in toluene (100 mL) was heated at
100.degree. C. for 3.5 h. After cooling, the precipitate was
removed by filtration, and washed with toluene. The combined
toluene layers were washed with 0.5 N NaOH (2.times.100 mL), dried
(MgSO.sub.4), and concentrated in vacuo to afford 4.22 g (81%) of
nitrile f' as a yellow solid, which was carried on without further
purification. ##STR93##
[0147] Following the general procedure for conversion of p to q,
nitrile f' (2.20 g, 12.0 mmol) afforded 1.64 g (68%) of ketone g as
a brown oil. ##STR94##
[0148] (2-Chloroethoxy)trimethylsilane (8.70 mL, 53.8 mmol) was
added to the mixture of 2'-hydroxy-1'-acetonaphthone u (5.0 g, 26.9
mmol), KOH (3.0 g, 53.8 mmol) in DMSO (60 mL) and water (20 mL).
The resulting mixture was heated at 80.degree. C. for 24 h. The
mixture was diluted with water (400 mL). The crystalline
precipitate was collected by filtration, washed with water, dried
to afford 5.21 g (84%) of hydroxy ketone h' as a brown solid.
##STR95##
[0149] Bromine (610 .mu.l, 11.9 mmol) was added over 10 min to a
solution of hydroxy ketone h' (2.50 g, 10.9 mmol) in
CH.sub.2Cl.sub.2 (30 mL) and AcOH (8.0 mL) at room temperature.
After 2 h, it was quenched with 10% aqueous Na.sub.2S.sub.2O.sub.3
(5 mL), diluted with CH.sub.2Cl.sub.2 (50 mL). The layers were
separated and the aqueous layer was extracted with 50 mL of
CH.sub.2Cl.sub.2. The combined organics were washed with 0.5 N
aqueous NaOH until the aqueous washes are basic, dried
(MgSO.sub.4), filtered, and concentrated to afford 3.70 g (96%) of
bromo ketone i' as a dark brown oil. ##STR96##
[0150] A mixture of 2-methoxyethanol (3.35 mL, 42.5 mmol) and
potassium t-butoxide (4.76 g, 42.5 mmol) in THF (80 mL) was stirred
at room temperature for 10 min. To this mixture was added dropwise
a solution of 4-fluoro-1-acetylnaphthalene (4.0 g, 21.3 mmol) in
THF (20 mL), and the mixture was stirred at room temperature for 24
h. The mixture was diluted with water (50 mL), and the phases
separated. The organic layer was washed with 0.5 N NaOH, brine,
dried (MgSO.sub.4), filtered, and concentrated to afford 5.6 g
(106%, excess wt. is solvent) of ketone j' as a brown liquid which
solidified under high vacuum. ##STR97##
[0151] Following the general procedure of Tagat (Tagat, J. R.;
McCombie, S. W.; Nazareno, D. V.; Boyle, C. D.; Kozlowski, J. A.;
Chackalamannil, S.; Josien, H.; Wang, Y.; Zhou, G. J. Org. Chem.
2002, 67, 1171-77), a suspension of the bromo acid x (3.0 g, 12.0
mmol) in toluene (18 mL) was heated at 80.degree. C. To this
reaction mixture was added dropwise N,N-dimethylformamide
di-tert-butyl acetal (10.0 mL, 42 mmol), and the resulting mixture
was heated for an additional 30 min. It was cooled to rt, washed
with water, saturated aqueous NaHCO.sub.3, brine, dried
(Na.sub.2SO.sub.4), filtered, and concentrated in vacuo to afford
2.87 g (78%) of t-butyl ester k' as a yellow oil, which was carried
on without further purification. ##STR98##
[0152] Following the general procedure of Tagat, a stirred solution
of t-butyl ester k' (1.4 g, 4.5 mmol) in anhydrous THF (30 mL) was
cooled to -78.degree. C. under N.sub.2. n-BuLi (3.65 mL of 1.6 M
solution in hexane, 5.85 mmol) was added, and the resulting
solution was stirred for 2 min, followed by addition of a solution
of N-fluorobenzenesulfonimide (2.83 g, 9.0 mmol) in THF (10 mL).
After stirring at -78.degree. C. for 30 min, the reaction was
quenched at -78.degree. C. with saturated aqueous NH.sub.4Cl. The
aqueous layer was extracted with Et.sub.2O (2.times.50 mL), dried
(MgSO.sub.4), filtered, and concentrated in vacuo. The crude
material was adsorbed on to Celite and purified by ISCO CombiFlash
40 g column (2-20%, EtOAc-hexane) to afford 0.57 g (52%) of fluoro
compound l' as colorless liquid. ##STR99##
[0153] Trifluoroacetic acid (3.85 mL, 50 mmol) was added to a
stirred solution of fluoro compound l' (1.23 g, 5.0 mmol) in
CH.sub.2Cl.sub.2 (50 mL) at rt. After stirring for 3 h, the
solution was concentrated in vacuo to afford 0.95 g (100%) of
fluoro acid m' as an oil, which was carried on. ##STR100##
[0154] A mixture of fluoro acid m' (820 mg, 4.3 mmol),
N,O-dimethylhydroxyl amine hydrochloride (420 mg, 4.3 mmol), EDC
(825 mg, 4.3 mmol), and DIPEA (750 .mu.l, 4.3 mmol) in DMF (12 mL)
was stirred at rt for 3 h. The mixture was diluted with EtOAc (50
mL), washed with 10% citric acid, 0.5 N NaOH, dried (MgSO.sub.4),
filtered, adsorbed on to Celite, and purified by ISCO CombiFlash 12
g column (2-10%, EtOAc-hexane) to afford 0.48 g (48%) of fluoro
amide n' as an oil. ##STR101##
[0155] To a solution of fluoro amide n' (1.07 g, 4.6 mmol) in THF
at 0.degree. C. was added dropwise a solution of CH.sub.3MgCl (4.6
mL of 3 M solution in THF, 13.8 mmol). The resulting mixture was
stirred at 0.degree. C. for 1 h, then 2 h at rt. The mixture was
quenched with 50% aqueous AcOH (10 mL), diluted with water (50 mL),
EtOAc (50 mL), and separated. The aqueous layer was extracted with
EtOAc (50 mL). The combined EtOAc layers were dried (MgSO.sub.4),
filtered, and concentrated to afford 0.77 g (89%) of fluoro ketone
o' as an oil. ##STR102##
[0156] Following the general procedure of Coudret (Hortholary, C.;
Coudret, C. J. Org. Chem. 2003, 68, 2167-74), to a solution of
4-amino-1-naphthalenecarbonitrile p' (5.0 g, 29.7 mmol) in conc.
HCl (50 mL) at 0.degree. C. was carefully added sodium nitrite
(3.07 g, 44.5 mmol). The mixture was stirred at 0.degree. C. for 1
h, then transferred into an additional funnel, and added dropwise
to an ice-cold solution of CuCl (5.3 g, 53.5 mmol) in water (150
mL). After addition, CH.sub.2Cl.sub.2 (80 mL) was added to the
reaction mixture. The resulting mixture was allowed to warm to rt
and was stirred for 4 h. The mixture was diluted with
CH.sub.2Cl.sub.2, and the phases separated. The aqueous phase was
carefully extracted with CH.sub.2Cl.sub.2 (2.times.150 mL). The
combined CH.sub.2Cl.sub.2 phases were washed once with saturated
sodium thiosulfate, dried (MgSO.sub.4), filtered, adsorbed on to
Celite, and purified by ISCO CombiFlash 120 g column (2-12%,
EtOAc-hexane) to afford 2.63 g (46%) of chloro compound q as white
solid. ##STR103##
[0157] Following the general procedure for conversion of p to q,
chloro compound q' (2.63 g, 14.1 mmol) afforded 2.1 g (74%) of
chloro ketone r' as a yellow liquid. ##STR104##
[0158] Following the procedure of Hallberg (Alterman, M., Hallberg,
A. J. Org. Chem. 2000, 68, 7984-89) a mixture of bromo ketone x
(1.40 g, 5.62 mmol), Zn(CN).sub.2 (790 mg, 6.74 mmol),
Pd(PPh.sub.3).sub.4 (216 mg, 0.19 mmol) and DMF (8 mL) was heated
in a microwave reactor (Emry's Optimizer) in a sealed heavy-walled
tube at 180.degree. C. for 5 min. After cooling, it was diluted
with water (30 mL), extracted with EtOAc (50 mL), dried
(MgSO.sub.4), filtered, adsorbed on to Celite, and purified by ISCO
CombiFlash 40 g column (5-20%, EtOAc-hexane) to afford 900 mg (83%)
of nitrile ketone s' as white solid. ##STR105##
[0159] Following the procedure of Leadbeater (Arvela, R.;
Leadbeater, N. E. SynLett. 2003, 8, 1145-48), a mixture of bromo
ketone x (100 mg, 0.40 mmol), NiCl.sub.2 (103 mg, 0.80 mmol) and
DMF (2 mL) was heated in a microwave reactor (Emry's Optimizer) in
a sealed heavy-walled tube at 200.degree. C. for 8 min. After
cooling, it was diluted with water (15 mL), extracted with EtOAc
(20 mL), dried (MgSO.sub.4), filtered, adsorbed on to Celite, and
purified by ISCO CombiFlash 4 g column (5-15%, EtOAc-hexane) to
afford 55 mg (68%) of chloro ketone t' as off white solid.
Example 5
Bromination of Methyl Ketones and Preparation of Thiazoles
[0160] ##STR106##
[0161] Bromine (260 .mu.l, 5.07 mmol), was added over 20 min to a
solution of ketone a (784 mg, 4.6 mmol) in CH.sub.2Cl.sub.2 (10
mL). The solution was maintained at rt for I h, then quenched with
10% aqueous Na.sub.2S.sub.2O.sub.3 (10 mL) and stirred vigorously
for 20 min. The layers were separated and the organic phase washed
with saturated aqueous NaHCO.sub.3 (1.times.10 mL), brine
(1.times.10 mL), dried (Na.sub.2SO.sub.4), filtered, and
concentrated to afford 1.15 g of bromo ketone b as a yellow oil.
Analysis by .sup.1H NMR indicates a 70:15:15 mixture of product to
starting ketone and dibrominated material. ##STR107##
[0162] A particular procedure: A mixture of Boc-proline-amide c
(8.4 g, 39.2 mmol), Lawesson's reagent (8.25 g, 20.4 mmol) and
toluene was heated at 50.degree. C. for 1 h (use of higher
temperatures results in loss of enantiopurity). The mixture was
then adsorbed onto Celite, and purified by chromatography (ISCO,
120 g silica column, gradient elution 10-70% EtOAc-hexanes) to
afford 7.6 g (84%) of the thioamide d as a colorless solid.
##STR108##
[0163] A particular procedure for thiazole formation: A mixture of
thioamide d (7.81 g, 34 mmol), bromoketone b (7.05 g, 80% pure by
.sup.1H NMR, 22.6 mmol), pyridine (1.76 mL, 20.3 mmol) and ethanol
(75 mL) was heated at 80.degree. C. for 1 h. The ethanol was
removed under reduced pressure, and the residue was adsorbed onto
Celite. The residue was chromatographed (SiO.sub.2, gradient
elution 0-2.5-5% EtOAc/CH.sub.2Cl.sub.2) to afford 6.3 g (73%) of
thiazole e as a colorless solid. ##STR109##
[0164] A mixture of bromide f (145 mg, 0.33 mmol), PhB(OH).sub.2
(107 mg, 0.88 mmol), K.sub.2CO.sub.3 (825 .mu.l of 2.0 M aqueous
solution, 1.65 mmol), Pd(PPh.sub.3).sub.4 (15 mg, 0.13 mmol), and
20% EtOH-toluene (2.5 mL) was maintained at 80.degree. C. for 3 h.
The mixture was diluted with CH.sub.2Cl.sub.2 (10 mL), and washed
with 1 N NaOH (2.times.5 mL). The combined aqueous layers were
extracted with CH.sub.2Cl.sub.2 (1.times.10 mL). The combined
organic phases were washed with brine (1.times.10 mL), dried
(Na.sub.2SO.sub.4), filtered, adsorbed on to Celite, and purified
by flash chromatography (SiO.sub.2, 10-15-20% acetone-hexanes) to
afford 74 mg (52%) of thiazole g as a colorless solid.
##STR110##
[0165] Thiazole e (70 mg, 0.18 mmol) in 1:1 dichloromethane:hexanes
(1.5 mL), was treated with N-chlorosuccinimide (30 mg 0.22 mmol).
The reaction mixture was stirred at rt for 2 h, at which point
additional NCS (10 mg) was added and the mixture stirred overnight.
Celite was added, and the dichloromethane was removed under reduced
pressure. The product was purified by chromatorgraphy (ISCO, 12 g
silica column, gradient elution 0-30% EtOAc/hexanes) to afford 70
mg (99%) of chlorothiazole h. ##STR111##
[0166] Thiazole e (120 mg, 0.31 mmol) in dichloromethane (1.5 mL),
was treated with N-bromosuccinimide (65 mg 0.37 mmol). The reaction
mixture was stirred at room temperature for 3 h. After this period,
Celite was added, and the dichloromethane was removed under reduced
pressure. The product was purified by chromatorgraphy (ISCO, 12 g
silica column, column was first flushed with CH.sub.2Cl.sub.2 for 7
minutes and then a gradient of 0-9% EtOAc/ CH.sub.2Cl.sub.2
gradient over 9 minutes.) to afford 128 mg (90%) of bromide i.
##STR112##
[0167] Following literature precedent ((1) Maguire, M. P.; Sheets,
K. R.; McVety, K.; Spada, A. P.; Zilberstein, A. J. Med. Chem.
1994, 37, 2129-2137; (2) Moreno, I.; Tellitu, I.; Dominguez, E.;
SanMartin, R.; Eur. J. Org. Chem. 2002, 2126-2135) a mixture of
bromothiazole i, (280 mg, 0.61 mmol) and alkynylstannane i
(Dabdoub, M. J.; Dabdoub, V. B.; Baroni, A. C. M. J. Am. Chem. Soc.
2001, 123, 9694-9695) (250 mg, 0.73 mmol), LiCl (approximately 50
mg, 120 mmol) and toluene (6 mL) was degassed with nitrogen for 30
min. Tetrakis(triphenylphosphine)palladium(0) (28 mg, 0.02 mmol),
was added and the mixture was heated at 100.degree. C. 3 h. After
cooling, Celite was added to the mixture, and the solvents were
removed under reduced pressure. The residue was purified by
chromatography (ISCO, 12 g silica column, column was first flushed
with CH.sub.2Cl.sub.2 for 5 minutes and then a gradient of 0-20%
EtOAc/ CH.sub.2Cl.sub.2 gradient over 10 minutes.) to afford 160 mg
(60%) of alcohol k. ##STR113##
[0168] Following literature precedent (Neidlein, R.; Nussbaumer, T
Heterocycles, 2000, 52, 349), bromide i (600 mg, 1.3 mmol),
TMS-acetylene 1(1.8 mL, 13 mmol) and TMG (0.6 mL, 5 mmol), were
dissolved in dimethylacetamide (6 mL). This mixture was degassed
with nitrogen for 30 min. Bis(triphenylphosphine)palladium
dichloride (46 mg, 0.07 mmol) and copper(I) iodide (62 mg, 0.3
mmol) were added and the mixture was sealed and heated at
70.degree. C. for 30 minutes. The mixture was diluted with
1/2-saturated ammonium chloride and filtered through a pad of
celite. The aqueous mixture was extracted with 70% diethyl ether in
hexane (3.times.20 mL), dried (Na.sub.2SO.sub.4), filtered,
adsorbed on to Celite, and chromatographed (ISCO, 40 g column and a
solvent gradient of 0-11% ethyl acetate in hexane after flushing
with hexane for 3 minutes). Terminal alkyne product 27 mg (5%) was
isolated along with 200 mg of silyl derivative. The TMS group was
removed from this material by treatment with potassium carbonate
(200 mg) in methanol (5 mL) for 3 hours at rt. Celite and toluene
(1 mL) were added to the mixture, and the solvents were removed
under reduced pressure. The product was purified by chromatography
(ISCO 40 g column, solvent gradient of 0-11% ethyl acetate/hexane
after flushing with pure hexane for 3 minutes), to afford a further
110 mg of terminal alkyne m (26% combined). ##STR114##
[0169] Terminal alkyne m (50 mg, 0.12 mmol) was dissolved in THF
(0.3 mL) and cooled to -78.degree. C. LHMDS (0.15 mL of a 1.0 M
solution of in THF, 0.15 mmol) was added dropwise and allowed to
stir for 10 minutes. Methyl iodide (0.1 mL, excess) was added, the
reaction was stirred for 10 minutes at -78.degree. C. and then
allowed to gradually warm to rt, over 45 minutes. Celite was then
added to the reaction mixture, the solvents were evaporated under
reduced pressure, and the residue purified by chromatography (ISCO,
12 g, column gradient elution 0-18% ethyl acetate in hexane) to
afford 25 mg (63%) of the methyl alkyne n. ##STR115##
[0170] Typical Boc deprotection: Carbamate o (75 mg, 0.18 mmol) was
treated with TFA (2 mL) and water (2 drops), in CH.sub.2Cl.sub.2 (2
mL) for 2 h. The volatiles were removed under reduced pressure, the
residue dissolved in ethyl acetate (10 mL) and washed with 1 N NaOH
(3.times.3 mL). The combined aqueous layers were extracted with
ethyl acetate (1.times.2 mL). The combined organic phases were
washed with brine (1.times.3 mL), dried (Na.sub.2SO.sub.4),
filtered, and concentrated to provide quantitative yield of amine
p.
Example 6
Linear Coupling Procedure
[0171] ##STR116##
[0172] Typical HATU coupling: A mixture of amine a (169 mg, 0.59
mmol), N-Boc-t-butly glycine (150 mg, 0.65 mmol), HATU (450 mg,
1.18 mmol), DIPEA (200 .mu.l, 1.18 mmol) and DMF (2 mL) was
maintained at rt for 2 h. The solution was diluted with ethyl
acetate (50 mL) and washed with 1 N HCl (3.times.10 mL), 1 N NaOH
(3.times.5 mL), brine (1.times.10 mL), dried (Na.sub.2SO.sub.4),
filtered, and concentrated. The residue was purified by flash
chromatography (SiO--.sub.2, 10-15-20% ethyl acetate-hexanes) to
afford 286 mg (97%) of amide b as a colorless solid. ##STR117##
[0173] Following the general Boc deprotection procedure described
above, Boc amine b (317 mg, 0.64 mmol) afforded a quantitative
yield of amine c as a colorless solid. ##STR118##
[0174] Typical EDC coupling: A solution of amine c (300 mg, 0.76
mmol), N-Boc-alanine (158 mg, 0.84 mmol), EDC (161 mg, 0.84 mmol),
catalytic DMAP and MeCN (3 mL) was maintained at rt for 3 h. The
solution was diluted with ethyl acetate (50 mL) and washed with 1 N
HCl (3 .times.10 mL), 1 N NaOH (3.times.5 mL), brine (1.times.10
mL), dried (Na.sub.2SO.sub.4), filtered, and concentrated to
provide 453 mg of crude residue d, which was carried on directly:
##STR119##
[0175] Typical final Boc removal and purification: The crude
residue d from above was treated with TFA (2 mL) and water (2
drops), in CH.sub.2Cl.sub.2 (2 mL) for 2 h. The volatiles were
removed under reduced pressure. The residue was purified by
reverse-phase HPLC (C.sub.18, MeCN--H.sub.2O, 0.1% TFA) and the
solvents removed by lyophylization to provide 166 mg (38% for 2
steps) of amine e as a colorless powder.
Example 7
N-Boc-N-methyl-L-alanine-L-cyclohexylglycine
[0176] ##STR120##
[0177] A solution of Fmoc-L-cyclohexylglycine (3.6 g, 9.6 mmol)
dissolved in DCM (50 mL) and DIPEA (5.6 mL, 32 mmol) was added to
2-chlorotrityl chloride resin (5 g, 8 mmol) and gently agitated for
3 hours at room temperature. The resin was washed with DCM 4 times,
DCM/MeOH/DIPEA (17:2:1) 3 times, DCM 3 times, and 2 times
dimethylacetamide (DMA). The Fmoc group was removed by treating the
resin with 20% piperidine/DMA (50 mL) for 15 minutes. The resin was
washed with DMA 6 times. A solution of Boc-N-methylalanine (3.3 g,
16 mmol), HBTU (6.1 g, 16 mmol), and DIPEA (5.6 mL, 32 mmol) and
DMA/DCM (1:1, 50 mL) was added to the resin and gently agitated for
2 hours at room temperature. The resin was washed with DMA 5 times,
DCM 2 times, and dried under reduced pressure. The dipeptide was
cleaved from the resin by gentle agitation with HOAc/TFE/DCM
(1:1:3, 100 mL) for 2 hours at room temperature. The resin was
removed by filtration and the solution concentrated. Residual AcOH
was removed by azeotroping with hexanes (15 times volume). The
solid residue was purified by reverse-phase HPLC (C.sub.18,
MeCN--H.sub.2O, 0.1% TFA) and the solvents removed by
lyophylization to provide 1.2 g (43%) of dipeptide
N-Boc-N-methyl-L-alanine-L-cyclohexylglycine as a white powder.
Example 8
N-Boc-N-methyl-L-alanine-L-dehydropyranylglycine
[0178] ##STR121##
[0179] A mixture of N-Cbz-dehydropyranylglycine methyl ester a
(Burk, M. J.; Gross, M. F.; Martinez, J. P. J. Am Chem. Soc. 1995,
117, 9375, and references therein) (5.2 g, 17 mmol), 5% Pd.C (500
mg), MeOH (75 mL) and THF (25 mL) was maintained under an
atmosphere of H.sub.2 for 24 h. The mixture was filtered through
Celite and the Celite washed with MeOH, and concentrated under
reduced pressure to afford a quantitative yield of amine b as a
colorless oil, which was carried on directly. ##STR122##
[0180] The amine b prepared above was combined with
CH.sub.2Cl.sub.2 (40 mL), saturated aqueous NaHCO.sub.3 (40 mL) and
cooled to 0.degree. C. Benzyloxy carbonyl chloride (3.0 mL) was
then added dropwise and the mixture stirred vigorously overnight.
The phases were separated and the aqueous phase extracted with
CH.sub.2Cl.sub.2 (3.times.20 mL). The combined organic phases were
washed with brine (1.times.50 mL), dried (Na.sub.2SO.sub.4),
filtered, adsorbed onto Celite and chromatographed (ISCO, 120 g
silica column, gradient elution 5-55% EtOAc-hexanes) to afford 4.15
g (80%) of racemic Cbz-pyranylglycine methyl ester. The enantiomers
were separated on a Chiracel OD column eluting with 10%
EtOH-hexanes. The desired S-enantiomer c elutes first under these
conditions. ##STR123##
[0181] A mixture of (S)-N-Cbz-pyranyl glycine c methyl ester (2.4
g, 7.82 mmol) 10% Pd.C (700 mg), MeOH (80 mL) was maintained under
1 atmosphere of H.sub.2 for 24 h. The mixture was filtered through
Celite with MeOH, and concentrated under reduced pressure to afford
1.35 g (100%) of amine d as a colorless oil. Alternatively, pyranyl
glycine can be synthesized in enantiopure form following the
procedure of Ghosh (Ghosh, A. K.; Thompson, W. J.; Holloway, M. K.;
McKee, S. P.; Duong, T. T.; Lee, H. Y.; Munson, P. M.; Smith, A.
M.; Wai, J. M.; Darke, P. L.; Zugay, J. A.; Imini, E. A.; Schleif,
W. A.; Huff, J. R.; Anderson, P. S. J. Med. Chem., 1993, 36, 2300).
##STR124##
[0182] A mixture of amine d (1.35 g, 7.8 mmol), N-Boc-N-methyl
alanine e (1.74 g, 8.6 mmol), EDC (1.65 g 8.8 mmol) and MeCN (50
mL) was maintained at rt overnight. The MeCN was removed under
reduced pressure, and the residue diluted with EtOAc, washed with
0.5 N HCl (3.times.10 mL), 0.5 N NaOH (3.times.10 mL), dried
(MgSO.sub.4), filtered, and concentrated to provide 2.1 g (75%) of
protected dipeptide f, as a clear oil. ##STR125##
[0183] To a 0.degree. C. solution of ester f (2.10 g, 5.86 mmol)
and THF (50 mL) were added LiOH.H.sub.2O (1.23 g, 29.3 mmol) and
water (2 mL). The mixture was maintained at 0.degree. C. for 2 h,
then the cooling bath was removed and the mixture was stirred
overnight. Most of the THF was then removed under reduced pressure
and the residue was diluted with CH.sub.2Cl.sub.2, washed with 0.5
N HCl, dried (MgSO.sub.4), filtered, and concentrated to provide
1.53 g (78%) of dipeptide
N-Boc-N-methyj-L-alanine-L-dehydropyranylglycine g, as a colorless
solid. ##STR126##
[0184] A particular procedure for convergent coupling: A mixture of
amine h (69 mg, 0.26 mmol), dipeptide
N-Boc-N-methyl-L-alanine-L-cyclohexylglycine from example 7 (60 mg,
0.23 mmol), HOAt (Carpino, L. A.; El-Faham, A. Tetrahedron, 1999,
55, 6813) (47 mg, 0.24 mmol), DIC (53 .mu.l, 0.34 mmol) and
CH.sub.2Cl.sub.2 (2 mL) was maintained at rt overnight. The mixture
was adsorbed onto Celite and purified by chromatography (ISCO, 4 g
silica column, gradient elution 5-50% EtOAc-hexanes) to afford 94
mg of the product i as a colorless solid contaminated with
diisopropyl urea. The mixture was carried on directly to the next
step. ##STR127##
[0185] The crude residue i from above was treated with TFA (2 mL)
and water (2 drops), in CH.sub.2Cl.sub.2 (2 mL) for 2 h. The
volatiles were removed under reduced pressure. The residue was
purified by reverse-phase HPLC (C.sub.18, MeCN--H.sub.2O, 0.1 %TFA)
and the solvents removed by lyophylization to provide 77 mg (54%
for 2 steps) of amine salt i as a colorless powder. ##STR128##
[0186] A mixture of the Acetate product k (228 mg, 0.32 mmol),
K.sub.2CO.sub.3 (53 mg, 0.38 mmol) in aqueous methanol (1:2, v:v,
15 mL) was stirred at rt for 1 h. Methanol was removed in vacuo.
The residue was diluted with water, extracted with CH.sub.2Cl.sub.2
(1.times.50 mL), and the organic phase dried (MgSO.sub.4), and
concentrated in vacuo to afford a crude product. Conversion to the
amine salt l was accomplished in 18% yield (3 steps) following the
general procedure.
Example 8
[0187] ##STR129##
[0188] A mixture of acid a (1.5 g, 6.9 mmol) prepared according to
the procedures of described in Kice et al. (J. Org. Chem. 1989, 54,
3596-3602), amine HCl salt (868 mg, 8.9 mmol), EDC (1.3 g, 6.9
mmol), and DIPEA (1.2 mL, 6.9 mmol) in DMF (17 mL) was stirred at
RT for overnight. The mixture was diluted with EtOAc (50 mL),
washed with 0.5 N HCl, 0.5 N NaOH, dried (MgSO.sub.4), filtered,
concentrated in vacuo to afford 1.3 g (74%) of amtide b as a yellow
solid, which was carried on without further purification.
##STR130##
[0189] 10% Pd/C (200 mg) was added into a solution of acid a (500
mg, 2.3 mmol), NaOH (92 mg, 2.3 mmol) in EtOH (25 mL) and water (5
mL) in a Parr reactor. This mixture was purged with N.sub.2 for 10
min, then hydrogenated with a Parr hydrogenator at 50 psi at RT for
2.5 h. The resulting mixture was filtered through Celite,
concentrated in vacuo to afford 50 mg (104%) of amine salt b as a
greenish-brown solid.
Example 10
[0190] ##STR131##
[0191] To a stirred mixture of the amine salt a (500 mg, 2.4 mmol)
in 6N HCl (30 mL), cooled at 0.degree. C., was added NaNO.sub.2
(248 mg, 3.6 mmol) in one portion (caution was used due to elevated
reaction temperature). After stirred at 0.degree. C. for 1 h, this
solution was added drop wise, via a dropping funnel, over 20 min to
an ice-water cold solution of CuBr (618 mg, 4.3 mmol) in water (30
mL). Then dichloromethane (40 mL) was added to slowly to the
reaction mixture (caution was used due to foaming). The resulting
mixture was allowed to reach RT and was stirred for 4 h. It was
diluted with CH.sub.2Cl.sub.2 (100 mL), separated, washed the
aqueous layer with another portion of CH.sub.2Cl.sub.2 (100 mL).
The combined CH.sub.2Cl.sub.2 were washed once with sat. aq.
Na.sub.2S.sub.2O.sub.3, dried (MgSO.sub.4), and concentrated in
vacuo. The crude product was adsorbed on to Celite and purified by
ISCO CombiFlash 12 g column (20-100% ethyl acetate-hexane) to
afford 175 mg (29%) of bromo acid b as a light yellow solid.
Example 11
[0192] ##STR132##
[0193] A mixture of bromo acid a (680 mg, 2.7 mmol), amine HCl salt
(264 mg, 2.7 mmol), EDC (520 mg, 2.7 mmol), and DIPEA (472 .mu.L,
2.7 mmol) in DMF (10 mL) was stirred at RT for overnight. The
mixture was partition between water (50 mL) and EtOAc (100 mL),
separated, washed the aqueous layer with another portion of EtOAc
(100 mL). The combined organic were washed with 1N HCl (50 mL), 1N
NaOH (50 mL), dried (MgSO.sub.4), filtered, concentrated in vacuo.
The crude product was adsorbed on to Celite and purified by ISCO
CombiFlash 12 g column (10-50% ethyl acetate-hexane) to afford 300
mg (38%) of bromo amide b as a yellow oil.
Example 12
[0194] ##STR133##
[0195] To a solution of bromo amide a (300 mg, 1.0 mmol) in THF (8
mL) at 0.degree. C. was added drop wise a solution of CH.sub.3MgCl
(2.0 mL of 3 M solution in THF, 6.0 mmol). The resulting mixture
was stirred for 1 h, and then allowed to warm to RT for 2 h. The
mixture was quenched with 50% aq. AcOH (4 niL), diluted with water
(50 mL) and EtOAc (50 mL), separated. The aqueous layer was
extracted with EtOAc (50 mL). The combined EtOAc were dried
(MgSO.sub.4), filtered, and concentrated. The crude product was
adsorbed on to Celite and purified by ISCO CombiFlash 12 g column
(2-10% ethyl acetate-hexane) to afford 150 mg (60%) of bromo ketone
b as light yellow oil. Compound b may also be prepared employing
the procedures described by Alvaro et al. (WO 2004099143) and and
Tsuno et al. (Bull. Chem. Soc. of Japan 1975, 48(11), 3347-55).
Example 13
[0196] ##STR134##
[0197] The starting acid a (6.6 g, 30.4 mmol) was treated with
thionyl chloride (50 mL), CHCl.sub.3 (50 mL), and 1 drop of DMF at
78.degree. C. for 5 h. The resulting mixture was concentrated in
vacuo, dried under high vacuum for overnight. The resulting yellow
solid was cooled in an ice-water bath and MeOH (200 mL) was added
slowly. This was then refluxed for 1 h. After cooled to RT, the
resulting precipitate was collected by filtration, washed with cold
MeOH, and dried to afford 5.0 g (71%) of ester b as a yellow
solid.
Example 14
[0198] ##STR135##
[0199] A suspension of ester a (5.0 g, 21.6 mmol) and 10% Pd/C (1.2
g) in MeOH (200 mL) was purged with N.sub.2 for 5 min, then treated
with a balloon of H.sub.2 at RT until the reaction is completed
(checked by LCMS). After purging the reaction mixture with N.sub.2
for 10 mim, the mixture was filtered through Celite, washed with
MeOH, concentrated in vacuo, and high vacuum dried to afford 4.2 g
(97%) of amine b as a brown oil.
Example 15
[0200] ##STR136##
[0201] A mixture of bromo ketone a (285 mg, 1.1 mmol), Zn(CN).sub.2
(400 mg, 3.4 mmol), and Pd(PPh.sub.3).sub.4 (88 mg, 0.076 mmol) in
DMF (2 mL) was heated in a microwave at 200.degree. C. for 600 sec.
After cooled, diluted with water (10 mL), extracted with EtOAc
(2.times.10 mL). The insoluble material was removed by filtration;
the solvent was dried (MgSO.sub.4), and concentrated. The crude
product was adsorbed on to Celite and purified by ISCO CombiFlash
12 g column (5-20% ethyl acetate-hexane) to afford 147 mg (66%) of
nitrile ketone b as a white solid. Compound a may also be prepared
employing the procedures described by Alvaro et al. (WO 2004099143)
and and Tsuno et al. (Bull. Chem. Soc. of Japan 1975, 48(11),
3347-55).
Example 16
[0202] ##STR137##
[0203] To a suspension of amine a (4.2 g, 20.9 mmol) in 6 N HCl
(100 mL), cooled in ice-water bath, was added NaNO.sub.2 (2.2 g,
31.4 mmol) in portions (caution was used due to elevated reaction
temperature). After stirred at ice-water temperature for 1 h, this
cold solution was added drop wise onto an ice-cold solution of CuBr
(5.4 g, 37.6 mmol) in water (150 mL). After addition,
CH.sub.2Cl.sub.2 (80 mL) was added slowly to the mixture. The
reaction mixture was allowed to reach RT and was stirred for 4 h.
It was diluted with more CH.sub.2Cl.sub.2 (50 mL). The phases were
separated. The aqueous phase was extracted with CH.sub.2Cl.sub.2
(2.times.50 mL). The combined CH.sub.2Cl.sub.2 were washed with
sat. sodium thiosulfate (100 mL), dried (MgSO.sub.4), and
concentrated. The crude product was adsorbed on to Celite and
purified by ISCO CombiFlash 120 g column (1-8% ethyl
acetate-hexane) to afford 3.1 (66%) of chloro ester b as a white
solid.
Example 17
[0204] ##STR138##
[0205] Following the general procedure of Williams et al.
(Tetrahedron Lett. 1995, 36(31), 5461-5464), to a stirred
suspension of ester a (1.5 g, 5.7 mmol) and amine (700 mg, 7.1
mmol) in THF (30 mL) at -5.degree. C. under N.sub.2 was added
CH.sub.3MgCl (16.1 mL of 3 M solution in THF, 48.4 mmol) over 20
min while keeping the temperature below 0.degree. C. After 0.5 h
at-5.degree. C, the reaction mixture was allowed to warm to RT and
stirred for overnight. The reaction was quenched with 1 N HCl,
diluted with 1 N HCl (100 mL), heated the mixture at 35.degree. C.
for 3 h, then cooled, diluted with EtOAc (150 mL), dried
(MgSO.sub.4), and concentrated in vacuo. The crude product was
adsorbed on to Celite and purified by ISCO CombiFlash 40 g column
(1-10% ethyl acetate-hexane) to afford 900 mg (64%) of chloro
ketone b as a clear liquid. Compound b may also be prepared
employing the procedures described by Alvaro et al. (WO 2004099143)
and and Tsuno et al. (Bull. Chem. Soc. of Japan 1975, 48(11),
3347-55).
Example 18
[0206] ##STR139##
[0207] Concentrated sulfuric acid (2 mL) was added slowly to a
stirred solution of 2-fluorophenylacetic acid a (10.0 g, 65.0 mmol)
in toluene (100 mL) and EtOH (7.6 mL, 130 mmol). The resulting
mixture was heated at 100.degree. C. for 1.5 h. It was concentrated
in vacuo, diluted with EtOAc (200 mL), washed with 10%
K.sub.2CO.sub.3 until the washes were basic, dried (MgSO.sub.4),
and concentrated in vacuo to afford 9.9 g (84%) of ester b as a
light yellow oil.
Example 19
[0208] ##STR140##
[0209] Concentrated sulfuric acid (3 mL) was added slowly to a
stirred solution of 2,4 di-fluorophenylacetic acid a (10.0 g, 58.1
mmol) in EtOH (100 mL), stirred at RT for 2 d. It was concentrated,
diluted with EtOAc (200 mL), washed with 10% K.sub.2CO.sub.3 until
the washes were basic, dried (MgSO.sub.4), and concentrated in
vacuo to afford 1.1 g (95.5%) of difluoro ester b as a white
solid.
Example 20
[0210] ##STR141##
[0211] Following the procedure for preparing ester from example 19,
2-nitrophenylacetic acid a (10.0 g, 55.2 mmol) afforded 10.9 g
(95%) of nitro ester b as a light yellow solid.
Example 21
[0212] ##STR142##
[0213] Following the general procedure described by Kemp et al. (J.
Am. Chem. Soc. 1975, 97, 7305-7312), reaction of difluoro-ester a
(4.0 g, 20.0 mmol), isoamyl-nitrite (3.2 mL, 24.0 mmol), and NaOEt
(1.4 g, 20.0 mmol) in EtOH (40 mL), after purified by ISCO
CombiFlash 80 g column (2-30% ethyl acetate-hexane) to afford 2.1 g
(47%) of difluoro oxime b as a light yellow solid.
Example 22
[0214] ##STR143##
[0215] A solution of the nitro oxime a (5.0 g, 21.0 mmol) prepared
according to the procedures described by Kemp et al. (J. Am. Chem.
Soc. 1975, 97, 7305-7312) in DMF (30 mL) was added drop wise over
25 min to a vigorously stirred suspension of hexane-washed NaH (60%
in mineral oil, 840 mg, 21.0 mmol) in DMF (40 mL) under N.sub.2.
The resulting dark colored solution was heated slowly to
130.degree. C. for 8 h. It was diluted with water (200 mL),
extracted with EtOAc (2.times.200 mL), washed the EtOAc with brine,
dried (MgSO.sub.4), and concentrated in vacuo. The crude product
was adsorbed on to Celite and purified by ISCO CombiFlash 120 g
column (1-10% ethyl acetate-hexane) to afford 1.6 g (41%) of
benzisoxazole b as an off white solid.
Example 23
[0216] ##STR144##
[0217] A mixture of benzisoxazole acid a (1.23 g, 7.5 mmol)
prepared according to the procedures described by Kemp et al. (J.
Am. Chem. Soc. 1975, 97, 7305-7312), amine HCl salt (736 mg, 7.5
mmol), EDC (1.44 g, 7.5 mmol), and DIPEA (1.2 mL, 6.7 mmol) in MeCN
(50 mL) was stirred at RT for overnight. It was concentrated in
vacuo, dissolved in EtOAc (200 mL), washed with 0.5 N HCl and
water, dried (MgSO.sub.4), and concentrated to afford 1.4 g (88%)
of benzisoxazole amide b as an off white solid.
Example 24
[0218] ##STR145##
[0219] Following the procedure for preparing bromo ketone from
example 12, to benzisoxazole amide a (1.2 g, 5.9 mmol) was added
CH.sub.3MgCl (6.0 mL of 3 M solution in THF, 17.8 mmol). The crude
product was adsorbed on to Celite and purified by ISCO CombiFlash
40 g column (1-5% ethyl acetate-hexane) to afford 670 mg (71%) of
benzisoxazole ketone b as a white crystalline. Compound b may also
be prepared according to the procedures described by Smalley et al.
(Science of Synthesis 2002, 11, 289-335) and Farooq et al. (WO
9614305).
Example 25
[0220] ##STR146##
[0221] Bromine (184 .mu.L, 3.6 mmol) was added drop wise to a
solution of benzisoxazole ketone a (525 mg, 3.3 mmol) in AcOH (1.5
mL) and CH.sub.2Cl.sub.2 (6.0 mL). After 1 h at RT, LCMS indicated
no reaction. Five drops of conc. HCl were added to the reaction
mixture and stirred at RT for overnight. It was quenched with 10%
Na.sub.2S.sub.2O.sub.3, diluted with CH.sub.2Cl.sub.2 (100 mL),
washed with 5% NaHCO.sub.3, separated, dried (MgSO.sub.4), and
concentrated in vacuo to afford 820 mg (105%) of bromo ketone b as
a brown oil.
Example 26
[0222] ##STR147##
[0223] Following the general procedure of Strupczewski et al. (J.
Med. Chem. 1985, 28, 761-769), to a suspension of NaH (60% in
mineral oil, 37 mg, 0.92 mmol) in THF (3.0 mL) was added drop wise
a solution of difluoro oxime a (140 mg, 0.61 mmol) in DMF (1.5 mL).
The resulting mixture was heated at 70.degree. C. for 4 h. It was
cooled, poured onto water (30 mL), extracted with EtOAc (2.times.50
mL). The EtOAc was washed with water, dried (MgSO.sub.4), and
concentrated. The crude product was adsorbed on to Celite and
purified by ISCO CombiFlash 12 g column (1-5% ethyl acetate-hexane)
to afford 60 mg (47%) of benzisoxazole ester b as an off white
solid.
Example 27
[0224] ##STR148##
[0225] A suspension of benzisoxazole a (1.6 g, 7.8 mmol) in 70%
H.sub.2SO.sub.4 (30 mL) was heated at 80.degree. C. for 4 h. It was
cooled, poured onto crushed ice. The solid was collected by
filtration, washed with water, and dried to afford 1.3 g (89%) of
benzisoxazole acid b as a white solid.
Example 28
[0226] ##STR149##
[0227] Following the procedure for preparing amide from example 23,
benzisoxazole acid a (1.3 g, 7.0 mmol) afforded, after purified by
ISCO CombiFlash 12 g column (2-15% ethyl acetate-hexane), 740 mg
(47%) of benzisoxazole amide b as a white solid.
Example 29
[0228] ##STR150##
[0229] Following the procedure for preparing of ketone from example
24, benzisoxazole amide a (740 mg, 3.3 mmol) afforded 390 mg (66%)
of benzisoxazole ketone b as an off white solid.
Example 30
[0230] ##STR151##
[0231] Following the procedure for preparing ester from example 19,
2,5-difluorophenylacetic acid a (9.56 g, 55.6 mmol) afforded 9.24 g
(83%) of difluoro ester b as a clear liquid.
Example 31
[0232] ##STR152##
[0233] Following the procedure for preparing ester from example 19,
2,3-difluorophenylacetic acid a (10.0 g, 58.1 mmol) afforded 10.8 g
(93%) of difluoro ester b as a clear liquid.
Example 32
[0234] ##STR153##
[0235] Following the procedure for preparing oxime from example 21,
difluoro ester a (9.2 g, 46.0 mmol) afforded 5.57 g (53%) of
difluoro oxime b as a white solid.
Example 33
[0236] ##STR154##
[0237] Following the procedure for preparing oxime from example 21,
difluoro ester a (10.8 g, 54 mmol) afforded 4.9 g (40%) of difluoro
oxime b as a white solid.
Example 34
[0238] ##STR155##
[0239] Following the procedure for preparing ester from example 26,
difluoro oxime a (5.5 g, 24.0 mmol) afforded, after purified by
ISCO CombiFlash 40 g column (1-5% ethyl acetate-hexane), 2.66 g
(53%) of benzisoxazole ester b as an off white solid.
Example 35
[0240] ##STR156##
[0241] Following the procedure for preparing ester from example 26,
difluoro oxime a (4.9 g, 21.4 mmol) afforded 2.9 g (65%) of
benzisoxazole ester b as a light yellow crystalline.
Example 36
[0242] ##STR157##
[0243] Following the procedure for preparing acid from example 27,
benzisoxazole ester a (2.1 g, 10.0 mmol) afforded 1.92 g (86%) of
benzisoxazole acid b as an off white solid.
Example 37
[0244] ##STR158##
[0245] Following the procedure for preparing acid from example 27,
benzisoxazole ester a (2.4 g, 11.5 mmol) afforded 1.92 g (76%) of
benzisoxazole acid b as an off white solid.
Example 38
[0246] ##STR159##
[0247] Following the procedure for preparing amide from example 23,
benzisoxazole acid a (1.4 g, 7.73 mmol) afforded 1.95 g (83%) of
benzisoxazole amide b as a yellow solid.
Example 39
[0248] ##STR160##
[0249] (Following the procedure for preparing amide from example
23, benzisoxazole acid a (1.9 g, 10.5 mmol) afforded 1.7 g (72%) of
benzisoxazole amide b as a brown solid.
Example 40
[0250] ##STR161##
[0251] Following the procedure for preparing ketone from example
12, benzisoxazole amide a (1.95 g, 8.7 mmol) afforded 448 mg (30%)
of benzisoxazole ketone b as a brown oil. Compound b may also be
prepared according to the procedures described by Farooq et al. (WO
9614305).
Example 41
[0252] ##STR162##
[0253] Following the procedure for preparing ketone from example
12, benzisoxazole amide a (1.70 g, 7.6 mmol) afforded 192 mg (14%)
of benzisoxazole ketone b as a white crystalline solid.
Example 42
[0254] ##STR163##
[0255] A suspension of 1,4-naphthalenedicarboxylic acid a (10.0 g,
46.3 mmol) in MeOH (70 mL) and H.sub.2SO.sub.4 (5 mL) was stirred
at RT for 2 days, and then heated at 50.degree. C. for 10 h. It was
concentrated in vacuo, re-dissolved in CH.sub.2Cl.sub.2 (300 mL),
washed with 10% K.sub.2CO.sub.3 (200 mL), dried (MgSO.sub.4), and
concentrated to give 6.6 g (60%) of di-ester b as yellow solid.
Example 43
[0256] ##STR164##
[0257] A mixture of the di-ester a (2.0 g, 8.2 mmol), LiOH (344 mg,
8.2 mmol) in THF (40 mL), water (5 mL), and MeOH (1 mL) was stirred
at RT overnight. LCMS indicated some starting di-ester still
remains un-reacted. Extra LiOH (84 mg, 2.0 mmol) was added to the
reaction mixture. After 4 h, it was diluted with 0.5 N HCl (100
mL), extracted with EtOAc (100 mL), dried (MgSO.sub.4),
concentrated to afford 1.4 g (74%) of monoacid b as a light yellow
solid.
Example 44
[0258] ##STR165##
[0259] A mixture of monoacid a (1.4 g, 6.1 mmol) and SOCl.sub.2 (9
mL) in toluene (15 mL) was heated at 75.degree. C. for 4 h. The
solvent was removed in vacuo, diluted with toluene (50 mL) and
concentrated, and dried under high vacuum overnight. The residue
was suspended in toluene (30 mL) and cooled in ice-water bath.
Me.sub.2Zn (12 mL of 1 M solution in heptane, 12.0 mmol) was added
slowly, stirred at RT for 3.5 h. The reaction was quenched with
sat. NH.sub.4Cl, diluted with water (100 mL), extracted with EtOAc
(2.times.100 mL), dried (MgSO.sub.4), and concentrated in vacuo.
The crude product was adsorbed on to Celite and purified by ISCO
CombiFlash 40 g column (1-10% ethyl acetate-hexane) to afford 890
mg (86%) of ketone ester b as an off white solid. Compound b may
also be prepared accrording to the procedures described by Uehata
et al. (JP 2003073357).
Example 45
[0260] ##STR166##
[0261] Bromine (1.49 g, 9.3 mmol) was added slowly to a stirred
solution of ketone a (1.47 g, 8.5 mmol) prepared according to the
procedures described by Berg et al. (WO 02066480) in 33% HBr/AcOH
(20 mL) at RT. It was stirred for 1 h, diluted with ether (65 mL),
and stirred vigorously for 1 h. The solid was collected by
filtration, washed with ether, high vacuum dried to afford 2.88 g
(100%) of bromo methyl ketone b as a yellow solid.
Example 46
[0262] ##STR167##
[0263] A mixture of 2-hydroxyquinoline4-carboxylic acid a (6.25 g,
33.1 mmol), MeI (10.33 g, 72.7 mmol), and K.sub.2CO.sub.3 (10.0 g,
72.7 mmol) in DMF (110 mL) was heated at 80.degree. C. for 16 h
overnight. LCMS indicated incomplete reaction. Extra MeI (4.69 g,
33.1 mmol) was added to the reaction mixture and heated at
100.degree. C. for 3 h. It was cooled, poured into ice water, and
10% K.sub.2CO.sub.3 (50 mL), extracted with EtOAc (2.times.150 mL).
The combined EtOAc were washed with water, dried (MgSO.sub.4),
concentrated in vacuo. The crude product was adsorbed on to Celite
and purified by ISCO CombiFlash 80 g column (2-50% ethyl
acetate-hexane) to afford 4.68 g (65%) of dihydroquinoline ester b
as an off white solid.
Example 47
[0264] ##STR168##
[0265] Lithium hydroxide (1.45 g, 34.5 mmol) was added to a
solution of dihydroquinoline ester a (1.50 g, 6.91 mmol) in THF (40
mL), followed by water (10 mL). The mixture was stirred at RT for
16 h. It was concentrated in vacuo, diluted with EtOAc (100 mL) and
0.5 N HCl (100 mL). A white solid precipitated and was collected by
filtration, washed with water. The EtOAc was separated, dried
(MgSO.sub.4), concentrated in vacuo to afford a white solid
product. A combined yields of 1.41 g (100%) of dihydroquinoline
acid b as a white solid, which was used with out further
purification.
Example 48
[0266] ##STR169##
[0267] N,N-Diisopropylethylamine (1.37 g, 10.7 mmol) was added to a
suspension of dihydroquinoline acid a (2.42 g, 11.9 mmol), amine
(1.40 g, 14.3 mmol), and EDC (2.28 g, 11.9 mmol) in MeCN (80 mL),
stirred at RT for 2 h. It was concentrated in vacuo, diluted with
CH.sub.2Cl.sub.2 (100 mL), washed with 0.5 N HCl (50 mL) and 0.5 N
NaOH (50 mL), dried (MgSO.sub.4), concentrated to afford 1.98 g
(67%) of dihydroquinoline amide b as a white solid, which was used
with out further purification.
Example 49
[0268] ##STR170##
[0269] Following the procedure for preparing ketone from example
12, dihydroquinoline amide a (2.17 g, 8.82 mmol), CH.sub.3MgCl
(8.82 mL of 3 M solution in THF, 26.5 mmol) afforded 766 mg (43%)
of dihydroquinoline ketone b as a yellow solid. Compound b may also
be prepared according to the procedures described by Fujita et al.
(Chem. & Pharm. Bull. 2001, 49(7), 900-904 and Chem. &
Pharm. Bull. 2001, 49(4), 407-412).
Example 50
[0270] ##STR171##
[0271] Following the general procedure of Legros et al.
(Tetrahedron 2001, 57, 2507-2514), 4-bromoisoquinoline a (1.0 g,
4.8 mmol) afforded 707 mg (86%) of ketone b as a light yellow
solid.
Example 51
[0272] ##STR172##
[0273] Following the procedure for preparing amide from example 48,
dihydroisoquinoline acid a (1.25 g, 6.2 mmol) prepared according to
the procedures described by Deady et al. (J. Heterocyclic Chem.
2001, 38, 1185) afforded 754 mg (50%) of dihydroisoquinoline amide
b as a yellow gum.
Example 52
[0274] ##STR173##
[0275] Following the procedure for preparing ketone from example
49, dihydroisoquinoline aimde a (0.754 g, 3.06 mmol) afforded 510
mg (85%) of dihydroisoquinoline ketone b as a yellow solid.
Compound b may also be prepared according to the procedures
desribed by Alvarez et al. (Science of Synthesis 2005, 15,
839-906), Kimura et al. (Chem. & Pharm. Bull. 1983, 31(4),
1277-82), Tomisawa et al. (Chem. & Pharm. Bull. 1975, 23(3),
592-6) and Dyke et al. (Tetrahedron 1973, 29(23), 3881-8).
Example 53
[0276] ##STR174##
[0277] A mixture of 2-hydroxyquinoline-4-carboxylic acid a (4.0 g,
21.2 mmol), POBr.sub.3 (25.0 g, 87.2 mmol) in toluene (40 mL) was
heated at 100.degree. C. for 3 h. It was cooled to RT, carefully
poured onto crushed ice, extracted with EtOAc (2.times.250 mL),
dried (MgSO.sub.4), concentrated in vacuo. The residue was
dissolved in 1 N NaOH (150 mL), extracted with EtOAc (2.times.100
mL). The aqueous layer was then acidified with 1 N HCl to pH 3. The
white solid was collected by filtration, washed with water, dried
to afford 3.0 g (56%) of bromo acid b as a white solid.
Example 54
[0278] ##STR175##
[0279] Following the procedure for preparing amide from example 48,
bromo acid a (3.0 g, 11.9 mmol) afforded 2.67 g (77%) of bromo
amide b as a white solid.
Example 55
[0280] ##STR176##
[0281] (46801-78) Following the procedure for preparing ketone from
example 49, bromo amide a (1.0 g, 3.4 mmol) afforded 800 mg (94%)
of bromo ketone b as an off white solid.
Example 56
[0282] ##STR177##
[0283] Trifluoromethane sulfonic anhydride (6.82 g, 24.2 mmol) was
added drop wise onto to a mixture of ethyl-4-hydroxyquinoline
carboxylate a (5.0 g, 23.0 mmol) and pyridine (1.95 mL, 24.2 mmol)
in CH.sub.2Cl.sub.2 (100 mL) at ice water bath temperature under
N.sub.2. The mixture was stirred at RT overnight. It was diluted
with CH.sub.2Cl.sub.2 (100 mL), washed with 0.5 N NaOH (100 mL),
dried (MgSO.sub.4), concentrated in vacuo. The crude product was
adsorbed on to Celite and purified by ISCO CombiFlash 80 g column
(2-15% ethyl acetate-hexane) to afford 7.4 g (93%) of triflate b as
a white solid.
Example 57
[0284] ##STR178##
[0285] Following the general procedure of Legros et al.
(Tetrahedron 2001, 57, 2507-2514), a mixture of triflate a (1.0 g,
2.86 mmol), bis(dibenzylideneacetone)palladium(0) (82 mg, 0.14
mmol), 1,3-bis(diphenylphosphino)propane (65 mg, 0.16 mmol), and
Et.sub.3N (1.19 mL, 8.58 mmol) in DMF (10 mL) were stirred at RT
for 15 min under N.sub.2. n-Butyl vinyl ether (1.43 g, 14.3 mmol)
in DMF (5 mL) was added and the resulting mixture was stirred at
80.degree. C. for 24 h. It was cooled to RT, 1 N HCl (30 mL) were
added slowly, stirred at RT for 24 h. The mixture was neutralized
with 1 N NaOH and extracted with ether (2.times.100 mL), dried
(MgSO.sub.4), and concentrated. The crude product was adsorbed on
to Celite and purified by ISCO CombiFlash 12 g column (2-20% ethyl
acetate-hexane) to afford 460 mg (66%) of ketone b as a white
solid.
Example 58
[0286] ##STR179##
[0287] A mixture of ketone ester a (1.50 g, 6.17 mmol), KOH (640
mg, 11.35 mmol) in EtOH (30 mL) was stirred at RT overnight. The
reaction mixture was diluted with water (150 mL), extracted with
EtOAc (100 mL). The aqueous layer was then acidified with 1 N HCl,
extracted with EtOAc (2.times.100 mL), dried (MgSO.sub.4),
concentrated in vacuo afforded 1.53 g (100%) of ketone acid b as a
yellow solid. Compound b may also be prepared according to the
procedures described by Priestly et al. (Bioorg. & Med. Chem.
1996, 4(7), 1135-1147).
Example 59
[0288] ##STR180##
[0289] A mixture of the ketone acid a (1.30 g, 5.16 mmol),
dimethylamine hydrochloride (480 mg, 5.93 mmol), EDC (1.14 g, 5.93
mmol), and DIPEA (765 mg, 5.93 mmol) in MeCN (30 mL) was stirred at
RT overnight. Solvent was removed. The residue was dissolved in
CH.sub.2Cl.sub.2 (100 mL), washed with 1 N HCl (50 mL) and 1 N NaOH
(50 mL), dried (MgSO.sub.4), and concentrated. The crude product
was adsorbed on to Celite and purified by ISCO CombiFlash 40 g
column (2-20% ethyl acetate-dichloromethane) to afford 656 mg (53%)
of ketone amide b as a light yellow gum.
Example 60
[0290] ##STR181##
[0291] Following the procedure for preparing triflate from example
56, 5-hydroxyquinoline a (3.42 g, 23.6 mmol) afforded 6.0 g (92%)
of triflate b as light yellow liquid.
Example 61
[0292] ##STR182##
[0293] Following the procedure for preparing ketone from example
57, triflate a (6.0 g, 21.7 mmol) afforded 3.58 g (97%) of ketone b
as a brown oil.
Example 62
[0294] ##STR183##
[0295] Following the procedure for preparing triflate from example
56, 2-(trifluoromethyl)-4-hydroxyquinoline a (6.87 g, 32.3 mmol)
afforded 9.31 g (84%) of triflate b as a yellow solid.
Example 63
[0296] ##STR184##
[0297] Following the procedure for preparing ketone from example
57, triflate a (7.35 g, 21.3 mmol) afforded 1.79 g (35%) of ketone
b as a yellow solid.
Example 64
[0298] ##STR185##
[0299] Following the procedure for preparing amide from example 59,
2-phenyl-4-quinolinecarboxylic acid a (5.0 g, 20.1 mmol) afforded
3.29 g (56%) of amide b as an off white solid.
Example 65
[0300] ##STR186##
[0301] Following the procedure for preparing ketone from example
49, amide a (3.29 g, 11.26 mmol) afforded 3.29 g (118%) of ketone b
as a yellow solid. Compound b may also be prepared accoridng to the
procedures described by Sato et al. (JP 2002371078), Wong et al (WO
9846572), Leardini et al. (J. Chem. Soc., Chem. Communications
1984, 20, 1320-1), Kaneko et al. (Chem. & Pharm. Bull. 1982,
30(1), 74-85), Schwenk et al. (J. Org. Chem. 1946, 11, 798-802) and
Shivers et al (J. Am. Chem. Soc. 1947, 69, 119-23).
Example 66
[0302] ##STR187##
[0303] Following the procedure for preparing amide from example 59,
2-hydroxy-4-quinolinecarboxylic acid a (5.0 g, 26.4 mmol) afforded
1.88 g (30%) of amide b as a cream color solid.
Example 67
[0304] ##STR188##
[0305] Following the procedure for preparing ketone from example
49, amide a (1.88 g, 8.1 mmol) afforded 993 mg (66%) of ketone b as
a light yellow solid. Compound b may also be prepared accoridn to
the procedures described by Wetzel et al. (J. Med. Chem. 1973,
16(5), 528-32), Jones et al. (J. Chem. Soc. [Section C]: Organic
1967, 19, 1808-13) and Ochia et al. (Chem. & Pharm. Bull. 1963,
11, 137-8).
Example 68
[0306] ##STR189##
[0307] Boc-ester a (900 mg, 2.0 mmol) was dissolved in THF (20 mL)
and MeOH (1 mL) at ice water bath temperature. NaBH.sub.4 (300 mg,
8.0 mmol) was added and the mixture was stirred for 1 h then
another 1 h at RT. Reaction was quenched with the addition of few
drops of water, then diluted with more water (100 mL), extracted
with EtOAc (2.times.100 mL), dried (MgSO.sub.4), and concentrated
in vacuo to afford 739 mg (90%) of alcohol b as a yellow foamy
solid.
Example 69
[0308] ##STR190##
[0309] To the mixture of amine a (159 mg, 0.46 mmol),
Boc-cyclohexyl-Gly-OH b (129 mg, 0.50 mmol), and HATU (350 mg, 0.92
mmol) in MeCN (4 mL) was added DIPEA (162 .mu.L, The resulting
mixture was stirred at RT for 2 h. LCMS indicated incomplete
reaction. An extra equivalence of HATU (175 mg, 0.46 mmol) and
DIPEA (81 .mu.L, 0.46 mmol) were added and stirred for another 1 h.
Solvent was removed in vacuo, diluted with CH.sub.2Cl.sub.2 (10
mL), washed with 0.5 N HCl (10 mL) and with water, dried
(MgSO.sub.4), and concentrated. The crude product was adsorbed on
to Celite and purified by ISCO CombiFlash 12 g column (0-5%
MeOH/CH.sub.2Cl.sub.2) to afford 187 mg (74%) of product c as a
brown solid.
Example 70
[0310] ##STR191##
[0311] To the mixture of amine a (62 mg, 0.13 mmol),
Boc-N-Me-Ala-OH b (27 mg, 0.13 mmol), and HATU (99 mg, 0.26 mmol)
in MeCN (2 mL) was added DIPEA (46 .mu.L, 0.26 mmol). The resulting
mixture was stirred at RT for 2 h. Solvent was removed in vacuo,
diluted with CH.sub.2Cl.sub.2 (10 mL), washed with 0.5 N HCl (10
mL) and with water, dried (MgSO.sub.4), and concentrated to afford
69 mg (85%) of product c as a clear oil.
Example 71
[0312] ##STR192##
[0313] A mixture of di-peptide a (100 mg, 0.29 mmol), thiazole
amine b (113 mg, 0.32 mmol), HOAt (59 mg, 0.435 mmol), and DIC (67
.mu.L, 0.435 mmol) in CH.sub.2Cl.sub.2 (3 mL) was stirred at RT for
overnight. The mixture was diluted with CH.sub.2Cl.sub.2 (10 mL),
washed with 0.5 N HCl (10 mL) and 0.5 N NaOH (10 mL), dried
(MgSO.sub.4), and concentrated. The crude product was adsorbed on
to Celite and purified by ISCO CombiFlash 12 g column (10-90% ethyl
acetate-hexane) to afford 175 mg (89%) of product c as a clear
oil.
Example 72
[0314] ##STR193##
[0315] Boc-amine a (175 mg, 0.26 mmol) was treated with (1:1)
TFA/CH.sub.2Cl.sub.2 (8 mL), catalytic toluene at RT for 1 h.
Solvent was removed in vacuo. The residue was purified by
reverse-phase HPLC (C.sub.18, MeCN--H.sub.2O, 0.1% TFA) and
lyophilized to afford 98 mg (49%, in 2 steps) of desired product b
as a hygroscopic white solid.
Example 73
[0316] ##STR194##
[0317] A mixture of the Boc-ester a (200 mg, 0.30 mmol), LiOH (200
mg, 0.30 mmol) in THF (2 mL), and water (25 .mu.L) was stirred at
RT for 1 h. MeOH (500 .mu.L) was added and stirred at RT overnight.
It was diluted with EtOAc (10 mL), washed with 0.5 N HCl (10 mL),
dried (MgSO.sub.4), concentrated in vacuo to afford 160 mg (82%) of
Boc acid 187 as a white solid.
Example 74
[0318] ##STR195##
[0319] A mixture of the Boc nitrile a (200 mg, 0.31 mmol),
NaN.sub.3 (309 mg, 4.75 mmol), and NH.sub.4Cl (252 mg, 4.75 mmol)
in DMF (3 mL) was heated at 100.degree. C. for 3.5 d. It was cooled
to RT, diluted with water, extracted with EtOAc (2.times.50 mL),
washed with brine, dried (MgSO.sub.4), concentrated in vacuo. The
crude product was adsorbed on to Celite and purified by ISCO
CombiFlash 4 g column (10-90% ethyl acetate-hexane) to afford 37 mg
of tetrazole # as a brown oil. More material was recovered by
extracting the aqueous layer with CH.sub.2Cl.sub.2 to recover 65 mg
of product. A combined 102 mg (49%) of tetrazole b was
isolated.
Example 75
[0320] ##STR196##
[0321] A mixture of the Boc ester a (135 mg, 0.20 mmol), KOH (14.5
mg, 0.26 mmol) in EtOH (4 mL) was stirred at RT for 2 h, then
diluted with EtOAc (8 mL), acidified with 1N HCl, separated, dried
(MgSO.sub.4), concentrated in vacuo, and high vacuum dried. The
crude product b was carried on without further purification.
Example 76
[0322] ##STR197##
[0323] Following the general procedure of Yamamoto [Asao, N; Lee,
S.; Yamamoto,Y. Tetrahedron Letters, 2003, 4265-4266.], MeLi (20 mL
of 1.6M solution in ether, 30.2 mmol) was added to a 0.degree. C.
suspension of powdered CuI (2.90 g, 15.1 mmol) and ether (5 mL).
The resulting grey solution was stirred vigorously for 10 min then
concentrated under reduced pressure at 0.degree. C. Dichloromethane
(20 mL, precooled to 0.degree. C.) was added, then the suspension
was cooled to -78.degree. C. and TMSCl (1.9 mL, 15.1 mmol) was
added followed quickly by a solution of ester 192 [WO 01168603]
(1.0 g, 5.0 mmol) and CH.sub.2Cl.sub.2 (50 mL), the mixture was
stirred vigorously at -78.degree. C. for 30 min, then at 0.degree.
C. for 2h. The mixture was then poured into 200 mL of 1:1 saturated
NH.sub.4Cl:NH.sub.4OH. The layers were separated and the aqueous
phase was extracted with CH.sub.2Cl.sub.2 (2.times.20 mL). The
combined organic phases were washed with brine (1.times.20 mL),
dried (Na.sub.2SO.sub.4), adsorbed onto Celite and purified by
chromatography ISCO CombiFlash 12 g column, 0-8% ethyl
acetate-hexanes to afford 783 mg (72%) of ester 193 as a clear
oil.
Example 77
[0324] ##STR198##
[0325] A solution of ester a (780 mg), CH.sub.2Cl.sub.2 (5 mL), and
TFA (2 mL) was maintained at rt overnight. The solvents were
removed under reduced pressure to afford quantitative yield acid b
as a colorless oil which was used without further purification.
Example 78
[0326] ##STR199##
[0327] Following the general procedure of Evans [Evans, D. A.;
Britton, T. C.; Ellman, J. A.; Dorow, R. L. J. Am. Chem. Soc. 1990,
112, 4011-4030], pivaloyl chloride (3.2 mL, 26 mmol) was added to a
-10.degree. C. solution of acid a (3.72 g, 23.5 mmol), TEA (4.3 mL,
31 mmol) and THF (50 mL). The resulting white slurry was allowed to
warm to -5.degree. C. over 20 min with vigorous stirring. The
mixture was then cooled to -78.degree. C. and a solution of the
lithium salt of (S)-4-benzyl-2-oxazolidinone (prepared from
(S)-4-benzyl-2-oxazolidinone (7.5 g, 42.3 mmol), n-BuLi (26 mL of
1.6M solution in hexanes, 42.3 mmol) and THF (150 mL) at
-78.degree. C.) was added via cannula over 10 min. The mixture was
maintained at -78.degree. C. for 1 h, then quenched with saturated
NH.sub.4Cl (200 mL) and the THF removed under reduced pressure. The
aqueous phase was extraced with EtOAc (3.times.50 mL). The combined
organic phases were washed with brine (1.times.50 mL), dried
(Na.sub.2SO.sub.4), adsorbed onto Celite and purified by
chromatography ISCO CombiFlash 120 g column, 540% ethyl
acetate-hexanes to afford 5.7 g (76%) of imide b as a clear
oil.
Example 79
[0328] ##STR200##
[0329] Following the general procedure of Evans [Evans, D. A.;
Britton, T. C.; Ellman, J. A.; Dorow, R. L. J. Am. Chem. Soc. 1990,
112, 401 1-4030], a cold (-78.degree. C.) solution of imide a (5.7
g, 18 mmol) and THF (64 mL) was added to a cold (-78.degree. C.)
solution of KHMDS (20 mmol) and THF (120 mL) over 10 min. The
colorless solution was maintained at -78.degree. C. for 30 min.,
then a cold (-78.degree. C.) solution of TrsylN.sub.3 (6.9 g, 22.4
mmol) and THF (40 mL) was added via cannula over 5 min. Acetic acid
(5.3 mL, 90 mmol) was added and the mixture was immediately brought
to 30.degree. C. and held there for 1 h. The reaction was quenched
with brine (200 mL) and CH.sub.2Cl.sub.2 (200 mL). The phases were
separated and the aqeous phase was extraced with CH.sub.2Cl.sub.2
(2.times.50 mL). The combined organic phases were washed with
saturated NaHCO.sub.3 (1.times.50 mL), dried (Na.sub.2SO.sub.4),
adsorbed onto Celite and purified by chromatography ISCO CombiFlash
330 g column, 5-45% ethyl acetate-hexanes to afford 4.2 g (65%) of
azo imide b as a colorless solid.
Example 80
[0330] ##STR201##
[0331] A mixture of azido-imide a (4.7 g, 13 mmol) LiOH.H.sub.2O
(660 mg, 15.6 mmol) THF (93 mL) and water (31 mL) was maintained at
rt for 1 d. Further LiOH.H.sub.2O (200 mg) was added, and the
mixture stirred for 2 h. Solid NaHCO.sub.3 (2.18 g) was added and
the THF removed under reduced pressure. Following dilution with
water (150 mL), the aqueous phase was washed with CH.sub.2Cl.sub.2
(3.times.50 mL) and the combined organic phases were extracted with
saturated NaHCO.sub.3 (1.times.50 mL). The combined aqueous phases
were acidified with con. HCl to pH<2 and extracted with EtOAc
(4.times.50 mL). The combined organic phases were dried
(Na.sub.2SO.sub.4), and concentrated to yield 1.38 g (53%) of acid
b as a colorless solid.
Example 81
[0332] ##STR202##
[0333] Isoquinoline carboxylic acid a (5.0 g, 28.9 mmol),
N,O-dimethy-hydroxylamine hydrochloride (3.1 g, 31.8 mmol) EDC (6.1
g, 32 mmol), DIPEA (5.7 mL, 32 mmol) and MeCN (50 mL) were mixed
together and stirred at rt overnight. The MeCN was removed under
reduced pressure and the residue partitioned between water (200 mL)
and EtOAc (200 mL). The phases were separated and the aqueous phase
was extracted with EtOAc (2.times.50 mL). The combined organic
phases were b as a colorless solid.
Example 82
[0334] ##STR203##
[0335] Methyl magnesium chloride (12.3 mL of 3.0M THF) was added to
a 0.degree. C. solution of amide a (4.0 g, 18.5 mmol) and THF (40
mL). After 30 min at 0.degree. C., the cooling bath was removed for
40 min. The reaction was poured into cold saturated NH.sub.4Cl (200
mL), and extracted with EtOAc (3.times.50 mL). The combined organic
phases were washed with water, brine, dried (Na.sub.2SO.sub.4), and
concentrated to yield 3.15 g (100%) of ketone b as a colorless
oil.
Example 83
[0336] ##STR204##
[0337] Following the general procedure of Barlin [Barlin, G. A.;
Davies, L. P.; Ireland, S. J.; Ngu, M. M. L. Aust. J. Chem. 1989,
42, 1735-1748], Br.sub.2 (150 .mu.L, 2.92 mmol) was added in one
portion to a solution of ketone a (500 mg, 2.92 mmol) and 33%
HBr/AcOH (10 mL). After 1 h, ether (20 mL) was added, and the ppt
was collected on filter paper, washed with ether, and dried under
vacuum to afford 910 mg (94%) of bromide b as a yellow solid.
Example 84
[0338] ##STR205##
[0339] A mixture of 4-carboxy-2-hydroxyquinoline a (500 mg, 2.64
mmol) and POCl.sub.3 (5 mL) was heated at 100.degree. C. for 1 h.
The solvent was removed under reduced pressure, and the residue was
dissolved in CH.sub.2Cl.sub.2 (10 mL), and cooled to 0.degree. C.
Morpholine (1.0 mL, 13.2 mmol) was added dropwise, and the mixture
was allowed to come to rt. The mixture was then re-cooled to
0.degree. C. and more morpholine (1.0 mL, 13.2 mmol) was added
dropwise, and the mixture was allowed to come to rt overnight. The
mixture was then diluted with CH.sub.2Cl.sub.2 (50 mL), and washed
with saturated NH.sub.4Cl (3.times.20 mL). The combined aqueous
phases were extracted with CH.sub.2Cl.sub.2 (1.times.20 mL), and
the combined organic phases were dried (Na.sub.2SO.sub.4), adsorbed
onto Celite and purified by chromatography ISCO CombiFlash 12 g
column, 5-75% ethyl acetate-hexanes to afford 570 mg (78%) of amide
b as a colorless solid.
Example 85
[0340] ##STR206##
[0341] Diethyl zinc (2.3 mL of 1.1M solution in toluene, 2.5 mmol)
was added to a mixture of amide a (500 mg, 1.8 mmol),
NiCl.sub.2DPPP (100 mg, 0.18 mmol), and THF (5 mL) (caution was
used due to exothermic reaction). The dark solution was then heated
at 100.degree. C. in a .mu.W reactor for 15 min. The reaction was
then quenched into saturated NH.sub.4Cl (50 mL), and extracted with
EtOAc (3.times.20 mL). The combined organic phases were dried
(Na.sub.2SO.sub.4), adsorbed onto Celite and purified by
chromatography ISCO CombiFlash 12 g column, 0-75% ethyl
acetate-hexanes to afford 350 mg (71%) of amide b as a colorless
solid.
Example 86
[0342] ##STR207##
[0343] Diisopropyl zinc (3 ml of 1.0M solution in toluene, 2.5
mmol) was added to a mixture of chloride a (500 mg, 1.8 mmol),
NiCl.sub.2DPPP (115 mg, 0.18 mmol), and THF (3 mL) The dark
solution was then heated at 100.degree. C. in a .mu.W reactor for
15 min. The reaction was then quenched into saturated NH.sub.4Cl
(50 mL), and extracted with EtOAc (3.times.20 mL). The combined
organic phases were dried (Na.sub.2SO.sub.4), adsorbed onto Celite
and purified by chromatography ISCO CombiFlash 12 g column, 0-15%
ethyl acetate-hexanes to afford 383 mg (74%) of amide b as a
colorless solid.
Example 87
[0344] ##STR208##
[0345] Methyl magnesium chloride (12.3 mL of 3.0M in THF, 37 mmol)
was added to 0.degree. C. solution of amide a (2.84 g, 10.51 mmol)
and THF (20 mL). The solution was allowed to come to rt, then
maintained at that temp. for 4 h. The reaction was quenched into
cold saturated NH.sub.4Cl (100 mL), extracted with EtOAc
(3.times.50 mL). The combined organic phases were washed with brine
(1.times.50 mL), dried (Na.sub.2SO.sub.4), adsorbed onto Celite and
purified by chromatography ISCO CombiFlash 40 g column, 0-30% ethyl
acetate-hexanes to afford 1.88 g (86%) of ketone b as a colorless
oil.
Example 88
[0346] ##STR209##
[0347] Following the general procedure of Barlin [Barlin, G. A.;
Davies, L. P.; Ireland, S. J.; Ngu, M. M. L. Aust. J. Chem. 1989,
42, 1735-1748], Br.sub.2 (640 .mu.L, 2.92 mmol) was added in one
portion to a solution of ketone a (2.27 g, 11.4 mmol) and 33%
HBr/AcOH (40 mL). After 1 h, ether (50 mL) was added, and the ppt
was collected on filter paper, washed with ether, and dried under
vacuum to afford 3.88 g (94%) of bromide b as a yellow solid.
Example 89
[0348] ##STR210##
[0349] Following the general procedure of Rieke [Zhu, L.; Wehmeyer,
R. M.; Rieke, R. D. J. Org. Chem. 1991, 56, 1445-1453], propyl zinc
bromide (4.0 mL of 0.5M solution in THF, 2.0 mmol) was added to a
mixture of chloride a (500 mg, 1.81 mmol), Pd(PPh.sub.3).sub.4 (100
mg, 0.09 mmol) and THF (3 mL). The resulting solution was heated at
70.degree. C. in a .mu.W reactor for 15 min. The reaction was then
quenched into saturated NH.sub.4Cl (50 mL), and extracted with
EtOAc (3.times.20 mL). The combined organic phases were dried
(Na.sub.2SO.sub.4), adsorbed onto Celite and purified by
chromatography ISCO CombiFlash 12 g column, 0-75% ethyl
acetate-hexanes to afford 400 mg (77%) of amnide b as a colorless
solid.
Example 90
[0350] ##STR211##
[0351] Following the general procedure of Rieke [Zhu, L.; Wehmeyer,
R. M.; Rieke, R. D. J. Org. Chem. 1991, 56, 1445-1453], cyclopently
zinc bromide (4.0 mL of 0.5M solution in THF, 2.0 mmol) was added
to a mixture of chloride a (500 mg, 1.81 mmol), Pd(PPh.sub.3).sub.4
(100 mg, 0.09 mmol) and THF (3 mL). The resulting solution was
heated at 70.degree. C. in a .mu.W reactor for 15 min. The reaction
was then quenched into saturated NH.sub.4Cl (50 mL), and extracted
with EtOAc (3.times.20 mL). The combined organic phases were dried
(Na.sub.2SO.sub.4), adsorbed onto Celite and purified by
chromatography ISCO CombiFlash 12 g column, 0-75% ethyl
acetate-hexanes to afford 333 mg (59%) of amide b as a colorless
solid.
Example 91
[0352] ##STR212##
[0353] Methyl magnesium chloride (7.3 mL of 3.0M in THF, 22 mmol)
was added to 0.degree. C. solution of amide a (1.77 g, 6.2 mmol)
and THF (15 mL). The solution was allowed to come to rt, then
maintained at that temp. for 4h. The reaction was quenched into
cold saturated NH.sub.4Cl (100 mL), extracted with EtOAc
(3.times.50 mL). The combined organic phases were washed with brine
(1.times.50 mL), dried (Na.sub.2SO.sub.4), adsorbed onto Celite and
purified by chromatography ISCO CombiFlash 40 g column, 0-30% ethyl
acetate-hexanes to afford 1.14 g (85%) of ketone b as a colorless
oil.
Example 92
[0354] ##STR213##
[0355] Following the general procedure of Angibaud [Angibaud, P;
et. al, Bioorg. Med. Chem. Lett. 2003, 13, 4365-4369], a mixture of
chloride a (1.0 g, 5.0 mmol) NaN.sub.3 (1.6 g, 25 mmol) DMF (10 mL)
and water (1.0 mL) was heated at heated at 120.degree. C. in a
.mu.W reactor for 2 h. The reaction was then quenched into water
(50 mL), and extracted with EtOAc (3.times.20 mL). The combined
organic phases were dried (Na.sub.2SO.sub.4), adsorbed onto Celite
and purified by chromatography ISCO CombiFlash 40 g column, 0-50%
ethyl acetate-hexanes to afford 300 mg (28%) of tetrazole b as a
yellow solid.
Example 93
[0356] ##STR214##
[0357] Trifluoromethane sulfonic anhydride (5.0 g, 17.7 mmol) was
added drop wise to a mixture of 2-methyl-4-hydroxyquinoline a (2.56
g, 16.1 mmol) and pyridine (1.54 mL, 17.7 mmol) in CH.sub.2Cl.sub.2
(25 mL) at ice water bath temperature under N.sub.2. The mixture
was allowed to warm to 10.degree. C. It was diluted with
CH.sub.2Cl.sub.2 (100 mL), washed with saturated NaHCO.sub.3
(3.times.50 mL), dried (Na.sub.2SO.sub.4), adsorbed on to Celite
and purified by ISCO CombiFlash 40 g column (0-30% ethyl
acetate-hexane) to afford 2.57 g (54%) of triflate b as a dark
oil.
Example 94
[0358] ##STR215##
[0359] Following the general triflation procedure,
7-chloro-4-hydroxyquinoline a (10.0 g, 35.4 mmol) afforded 7.5 g
(68%) of triflate b as a colorless solid.
Example 95
[0360] ##STR216##
[0361] Follwing the general triflation procedure
6-flouro-4-hydroxyquinoline a (5.0 g, 28.2 mmol) afforded 6.6 g
(75%) of triflate b as a colorless solid.
Example 96
[0362] ##STR217##
[0363] Following the general procedure of Legros [Tetrahedron 2001,
57, 2507-2514], a mixture of triflate a (7.5 g, 24.1 mmol),
bis(dibenzylideneacetone)palladium(0) (690 mg, 1.2 mmol),
1,3-bis(diphenylphosphino)propane (546 mg, 1.33 mmol), and
Et.sub.3N (10 mL, 72.3 mmol) in DMF (50 mL) were stirred at RT for
15 min under N.sub.2. n-Butyl vinyl ether (15 mL, 120 mmol) in DMF
(15 mL) was added and the resulting mixture was stirred at
80.degree. C. for 24 h. It was cooled to RT, 1 N HCl (150 mL) were
added slowly, stirred at RT for 24 h. The mixture was neutralized
with 1 N NaOH and extracted with ether (3.times.100 mL), dried
(MgSO.sub.4), and concentrated. The crude product was adsorbed on
to Celite and purified by ISCO CombiFlash 120 g column (5-30% ethyl
acetate-hexane) to afford 1.62 g (32%) of ketone b as a white
solid.
Example 97
[0364] ##STR218##
[0365] Following the general procedure for preparing ketone from
example 96, 6.56 g of triflate a afforded 3.14 g (73%) of ketone b
as a colorless solid.
Example 98
[0366] ##STR219##
[0367] Following the general procedure for preparing ketone from
example 96, 2.57 g of triflate a afforded 820 mg (50%) of ketone b
as a colorless solid.
Example 99
[0368] ##STR220##
[0369] Methyl magnesium chloride (0.5 mL of 3.OM in THF, 1.5 mmol)
was added to 0.degree. C. solution of ester a (230 mg, 0.5 mmol)
and THF (5 mL). The solution was maintained at 0.degree. C. for 2h.
The reaction was quenched into cold saturated NH.sub.4Cl (50 mL),
extracted with EtOAc (3.times.20 mL). The combined organic phases
were washed with brine 1.times.50 mL), dried (Na.sub.2SO.sub.4),
adsorbed onto Celite and purified by chromatography ISCO CombiFlash
12 g column, 0-50% ethyl acetate-hexanes to afford 135 mg (61%) of
alcohol b as a colorless oil
Example 100
[0370] ##STR221##
[0371] Bromine (122 .mu.L, 2.4 mmol) was added to the solution of
benzisoxazole ketone a (390 mg, 2.2 mmol) in AcOH (1.5 mL) and
CH.sub.2Cl.sub.2 (6 mL). After 1 h at RT, LCMS indicated no
reaction. Four drops of conc. HCl were added to the reaction
mixture and stirred at RT overnight. It was quenched with 10%
Na.sub.2S.sub.2O.sub.3, diluted with CH.sub.2Cl.sub.2 (100 mL) and
water, separated, washed the organic layer with 5% NaHCO.sub.3,
dried (MgSO.sub.4), and concentrated to afford 537 mg (95%) of
bromo ketone b as an off white solid.
Example 101
[0372] ##STR222##
[0373] A mixture of bromo ketone a (537 mg, 2.1 mmol), thioamide b
(718 mg, 3.1 mmol), and pyridine (153 .mu.L, 1.9 mol) in EtOH (15
mL) was heated at 70.degree. C. for 1 h. It was concentrated in
vacuo. The crude product was adsorbed on to Celite and purified by
ISCO CombiFlash 40 g column (3-30% ethyl acetate-hexane) to afford
190 mg (23%) of thiazole c as a light yellow gum.
Example 102
Tetrahydropyranylglycine
[0374] ##STR223##
[0375] Tetrahydropyranylglycine was purchased from NovaBiochem, or
synthesized according to the literature: Ghosh, A. K.; Thompson, W.
J.; holloway, M. K.; McKee, S. P.; Duong, T. T.; Lee, H. Y.;
Munson, P. M.; Smith, A. M.; Wai, J. M; Darke, P. L.; Zugay, J. A.;
Emini, E. A.; Schleife, W. A.; Huff, J. R.; Anderson, P. S. J. Med.
Chem, 1993, 36, 2300-2310.
Example 103
Piperidinylglycine
[0376] ##STR224##
[0377] Piperidinylglycine was synthesized according to the
procedures described by Shieh et al. (Tetrahedron: Asymmetry, 2001,
12, 2421-2425.
Example 104
4,4-difluorocyclohexylglycine
[0378] ##STR225##
[0379] 4,4-difluorocyclohexylglycine was made according to the
procedures described in US 2003/0216325.
Example 105
Boc (S)-2-amino-2-(4-hydroxycyclohexyl)acetic acid
[0380] ##STR226##
[0381] Following the procedure described by Sheih et al.
(Tetrahedron: Asymmetry, 2001, 12, 2421-2425), a solution of ketone
a (8.4 g) and EtOAc (30 mL) was added to a solution of
N-Cbz-phosphonoglycine methyl ester b, TMG (4.5 mL) and EtOAc (30
mL). The solution was maintained at rt for 48 h, then washed with
1N HCl (3.times.50 mL), brine (1.times.50 mL) dried
(Na.sub.2SO.sub.4), filtered, and concentrated. The residue was
adsorbed onto Celite, and purified by chromatography, then further
purified by re-crystalization from EtOAc/hexanes to afford 5.2 g of
product c. ##STR227##
[0382] Following the procedure described by Sheih, (Tetrahedron:
Asymmetry, 2001, 12, 2421-2425), a solution of eneamide c (5.0 g),
(S,S)-Me-BPE-Rh(I) (1.5 g, Strem Chemicals, Newburyport, Mass.),
and MeOH (100 mL) was shaken virgorously under 70psi of H.sub.2 for
48 h. The solvent was removed under reduced pressure. The residue
was taken up in EtOAc, and filtered through SiO.sub.2 with more
EtOAc. The solvent was removed under reduced pressure to afford 4.0
g of product d as a colorless solid. ##STR228##
[0383] A mixture of Cbz-carbamate d, (4.0 g) Boc.sub.2O, (2.9 g),
20% Pd(OH).sub.2.C (1.0 g) and MeOH (30 mL) was maintained under an
atmosphear of H.sub.2 for 6 h. The mixture was filtered through
Celite with MeOH. The solvent was removed under reduced pressure to
afford 4.5 g of residue e, which was taken on directly.
##STR229##
[0384] The residue e from above was dissolved in H.sub.2O (10 mL),
AcOH (30 mL), THF (5 mL), and dichloroacetic acid (3 mL) and
maintained at rt overnight. Water (5 mL) was added and the solution
and maintaned until hyrolysis was complete, as monitored by
HPLC-MS. Solid Na.sub.2CO.sub.3 was added cautiously until gas
evolution ceased, the mixture was diluted with aq NaHCO.sub.3, and
extracted with 10% EtOAc/DCM. The combined organic phases were
washed once with brine, dried (Na.sub.2SO.sub.4), filtered, and
concentrated. The residue was purified by chromatography to afford
2.9 g of product f. ##STR230##
[0385] A mixture of ketone f (1.5 g) MeOH (50 ml) was treated with
NaBH4 (290 mg) at 0.degree. C. for 20 min. The mixture was acidifed
to .about.pH1 with 10% aq citric acid and the MeOH was removed
under reduced pressure. The residue was diluted with water and
extraced with 20% EtOAc/DCM. The combined organic phases were
washed once with brine, dried (Na.sub.2SO.sub.4), filtered, and
concentrated. The residue was purified by chromatography to afford
1.17 g of product g and 0.23 g of product h. ##STR231##
[0386] A mixture of ester g (1.17 g) LiOH.H2O (160 mg), THF (3 mL)
and water (4.5 mL) was stirred vigorously at rt overnight. The
mixture was diluted with brine and exaustivly extraced with EtOAc.
The combined organic phases were washed once with brine, dried
(Na.sub.2SO.sub.4), filtered, and concentrated to afford acid i
(525 mg).
Example 106
N-Boc-N-cyclopropylmethyl-L-alanine
[0387] ##STR232##
[0388] L-alanine methyl ester hydrochloride a (5 g, 35.8 mmol) and
cyclopropanecarboxaldehyde b (2.67 ml, 35.8 mmol) were suspended in
50 ml THF w/1% AcOH. Addition of 5 ml of CH.sub.3OH made the cloudy
solution turned to clear. NaCNBH.sub.4 (2.25 g, 35.8 mmol) was
added and the reaction mixture stirred overnight. The reaction was
quenched by addition of 1N aq. NaOH, extracted by EtOAc twice,
organic layers were dried over Na.sub.2SO.sub.4 and concentrated to
dryness. The crude material was purified by chromatography using
30% EtOAc/hexane (stained by ninhydrin) to obtain the compound c (1
g, 18%). The compound c (1 g, 6.37 mmol) and di-t-bocdicarbonate
(2.1 g, 9.55 mmol) were diluted in THF (20 ml) and H.sub.2O (20
ml), NaHCO.sub.3 (1.3 g, 15.9 mmol) was added. The reaction mixture
stirred overnight for completion. THF was removed under reduced
pressure, and the aqueous layer was extracted by EtOAc 3 times.
Combined organic layers were washed by 1N NaOH, sat, NH.sub.4Cl
followed by brine, the concentrated to dryness. The Boc-protected
compound d (1.39 g, 5.40 mmol) was stirred with LiOH.H.sub.2O (1.14
g, 27 mmol) in THF (20 ml) and H.sub.2O (20 ml) overnight at room
temperature. THF was stripped off, and the aqueous layer was
adjusted to pH=4 by adding 10% citric acid, then extracted by EtOAc
3 times. Combined organic layers were washed by brine and
concentrated. The crude was purified by reverse phase C-18 column
eluted by 0%-50% acetonitrile/H.sub.2O to give pure compound e as a
white solid (794 mg).
Example 107
[0389] ##STR233##
[0390] Phosphonate b (7.2 g, 21 mmol) was dissolved in THF (25 mL)
at room temperature, and TMG (3.6 mL, 29 mmol, 1.3 equiv) was added
dropwise. The mixture was stirred for 15 min at room temp.
Commercially available ketone a (6.7 g, 43 mmol) was dissolved in
THF (25 mL) and added dropwise to the mixture of phosphonate and
base. The reaction was stirred for 24 h at room temperature and
quenched by adding approx 200 mL of 1 N HCl. Organic products were
quickly extracted into 80% ethyl acetate-hexanes (400 mL total).
The combined organic phases were dried (Na.sub.2SO.sub.4), adsorbed
onto Celite and purified twice by chromatography ISCO CombiFlash
120 g column, 0-55% ethyl acetate-hexanes over 20 min. followed by
55% ethyl acetate-hexanes for 5 min. to afford 3.83 g (10.6 mmol,
50%) of the product amino ester c as a white solid.
Example 108
[0391] ##STR234##
[0392] Ketal a (1.56 g, 4.73 mmol) was dissolved in 6 mL of THF. To
this solution was added deionized water (15 mL), glacial acetic
acid (6 mL), and dichloroacetic acid (1 mL). The mixture was
stirred overnight at room temperature. Aqueous 1 N sodium hydroxide
(approx. 100 mL) was added, and crude product was extracted into
dichloromethane (approx. 200 mL). The organic product was adsorbed
onto Celite by evaporation of the solvent, and purified by
chromatography ISCO CombiFlash 80 g column with a solvent gradient
of 0-40% ethyl acetate-hexanes over 20 min to afford 452 mg (1.58
mmol, 33%) of ketone b.
Example 109
[0393] ##STR235##
[0394] Ester a (184 mg, 0.55 mmol) was dissolved in 2 mL of THF.
Deionized water was added (1 mL), followed by lithium hydroxide
monohydrate (42 mg, 1.0 mmol). The mixture was stirred at room
temperature overnight, then acidified using aqueous 1 N HCl and
extracted into dichloromethane. Drying (Na.sub.2SO.sub.4),
filtration and evaporation of the solvent yielded 175 mg
(quantitative yield) of the carboxylic acid b.
Example 110
[0395] ##STR236##
[0396] A small vial was charged with amine b (130 mg, 0.46 mmol),
acid a (175 mg, 0.55 mmol) and EDC.HCl (135 mg, 0.70 mmol). The
mixture was dissolved in dichloromethane (3 mL) and stirred
overnight at room temperature. Celite was added to the reaction,
and solvent was removed under reduced pressure. Crude product was
purified by chromatography ISCO CombiFlash 40 g column with a
solvent gradient of 0-45% ethyl acetate-hexanes over 10 min
followed by 45% ethyl acetate-hexanes for 5 min. The BOC-protected
amine obtained from this coupling reaction was dissolved in
dichloromethane (2 mL), deionized water (0.5 mL) and
trifluoroacetic acid (1 mL) and allowed to stir for 3 h at room
temperature. Organic solvents were removed under reduced pressure,
the aqueous layer was made basic using a small amount of 1 N NaOH,
and product was extracted into dichloromethane. Removal of organic
solvent yielded 110 mg (0.25 mmol, 45% amine #) of the free amine
#.
Example 111
[0397] ##STR237##
[0398] Standard EDC coupling procedure was performed using amine b
(110 mg, 0.25 mmol) L-BOC-N-methylalanine a (72 mg, 0.35 mmol) and
EDC (67 mg, 0.35 mmol). BOC-protected final product was purified by
chromatography ISCO CombiFlash 12 g column with a solvent gradient
of 5-55% ethyl acetate-dichloromethane over 15 min followed by 55%
ethyl acetate-dichloromethane for 4 min. BOC-deprotection was
performed using 2:1 DCM:TFA with few drops of water. Final product
c (54 mg, 66%) was purified by reverse-phase HPLC C.sub.18 column
with a solvent gradient of 5-50% acetonitrile-water over 20
min.
Example 112
[0399] ##STR238##
[0400] Following the general procedure of Burk [Burk, M. J.; Gross,
M. F.; Martinez, J. P. J. Am. Chem. Soc. 1995, 117, 9375-9376.],
5.0 g (13.8 mmol) of alkene a, 100 mL of dry methanol, and
[(S,S)-Me-BPE-Rh(COD)].sup.+OTf.sup.- (1.5 g, 2.4 mmol) were mixed
in a Parr shaker flask purged with nitrogen. Parr shaker was
evacuated and subsequently charged to 70 psi of hydrogen gas for 32
hours. Methanol was removed under reduced pressure, and crude
product was filtered through a small plug of silica gel using ethyl
acetate. Evaporation of the solvent gave 4.0 g (11 mmol, 80%) of
product b with >98% yield.
Example 113
[0401] ##STR239##
[0402] Z-protected amino ester a (4.0 g, 11 mmol) was dissolved in
methanol (30 mL). To this solution was added BOC-anhydride (2.9 g,
13.5 mmol), followed by 20% Pd(OH).sub.2.C (1.0 g). All air was
removed from the reaction flask by house vacuum, and the mixture
was stirred vigorously for 5 min. The flask was then filled with
hydrogen gas and allowed to stir vigorously at room temperature for
6 h. After evacuating the hydrogen atmosphere, the mixture was
filtered through Celite using methanol, and crude product was
obtained by evaporation of the solvent.
[0403] The product BOC-protected amine b was dissolved in 5 mL of
THF. The following solvents were then added sequentially: deionized
water (15 mL), glacial acetic acid (30 mL), and dichloroacetic acid
(3 mL). The mixture was stirred overnight at room temperature, and
the reaction was quenched by slowly adding solid sodium carbonate
with vigorous stirring until the release of gas was no longer
visible. Crude product was extracted into 10% ethyl
acetate-dichloromethane. The product was adsorbed onto Celite by
evaporation of the solvents, and purified by chromatography ISCO
CombiFlash 120 g column with a solvent gradient of 0-36% ethyl
acetate-hexanes over 20 min followed by flushing with 36% ethyl
acetate-hexanes for 5 min to afford 2.86 g (10.0 mmol, 91%) of
ketone b.
Example 114
[0404] ##STR240##
[0405] Standard EDC coupling was performed using amine b (46 mg,
0.15 mmol), carboxylic acid a, (42 mg, 0.15 mmol syn-diastereomer)
and EDC (33 mg, 0.17 mmol). BOC-protected final product was
purified by chromatography ISCO CombiFlash stacker 2.times.4 g
column with a solvent gradient of 0-28% ethyl
acetate-dichloromethane over 15 min, followed by 28% ethyl
acetate-dichloromethane for 3 min. Standard BOC-deprotection was
performed using 2:1 DCM:TFA with few drops of water. The TFA salt
was treated with base (aqueous 1 N NaOH) and extracted into
dichloromethane.
Example 115
[0406] ##STR241##
[0407] Coupling of the product primary amine a (20 mg, 0.044 mmol)
to L-BOC-N-methylalanine b (12 mg, 0.059 mmol) was performed by
adding EDC (10 mg 0.052 mmol) and dissolving in dichloromethane (1
mL). BOC-protected final product was purified by chromatography
ISCO CombiFlash stacker 2.times.12 g column with a solvent gradient
of 0-70% ethyl acetate-dichloromethane over 20 min followed by 70%
ethyl acetate-dichloromethane for 5 min. BOC-deprotection was
performed using 2:1 DCM:TFA with few drops of water. Final product
c was purified by reverse-phase HPLC C.sub.18 column with a
gradient of 5-50% acetonitrile-water over 20 min. Yield of product
anti-diastereomer c was 22 mg.
Example 116
[0408] ##STR242##
[0409] Standard EDC coupling was performed using amine b (110 mg,
0.38 mmol), carboxylic acid a, (105 mg, 0.38 mmol) and EDC (86 mg,
0.45 mmol). BOC-protected final product was purified by
chromatography ISCO CombiFlash stacker 2.times.4 g column with a
solvent gradient of 0-28% ethyl acetate-dichloromethane over 15
min, followed by 28% ethyl acetate-dichloromethane for 3 min.
Standard BOC-deprotection was performed using 2:1 DCM:TFA with few
drops of water. The TFA salt was treated with base (aqueous 1 N
NaOH) and extracted into dichloromethane.
Example 117
[0410] ##STR243##
[0411] Coupling of the product primary amine b (170 mg, 0.35 mmol)
to L-BOC-N-methylalanine a (81 mg, 0.40 mmol) was performed by
adding EDC (77 mg 0.40 mmol) and dissolving in dichloromethane (2
mL). BOC-protected final product was purified by chromatography
ISCO CombiFlash stacker 2.times.12 g column with a solvent gradient
of 0-70% ethyl acetate-dichloromethane over 20 min followed by 70%
ethyl acetate-dichloromethane for 5 min. Standard BOC-deprotection
was performed using 2:1 DCM:TFA with few drops of water. Final
product c was purified by reverse-phase HPLC C.sub.18 column with a
solvent gradient of 5-50% acetonitrile-water over 20 min. Yield of
anti-diastereomer product c was 106 mg.
Example 118
[0412] ##STR244##
[0413] Ketone a (1.45 g, 5.3 mmol), was dissolved in dry diethyl
ether (20 mL) and cooled to -78.degree. C. Methyllithium (1.6 M in
Et.sub.2O, 9.5 mL, 15 mmol) was added dropwise to the reaction
mixture and stirred vigorously at the reduced temperature for 1 h.
The reaction was quenched by pouring the cold mixture into
saturated aqueous ammonium chloride and extracting the organics
into dichloromethane. The organic layer was dried
(Na.sub.2SO.sub.4), filtered, adsorbed onto Celite and purified by
chromatography ISCO CombiFlash 120 g column, 0-50% ethyl
acetate-hexanes over 25 min, followed by flushing 50% ethyl
acetate-hexanes for 3 min, and 90% ethyl acetate-hexanes for 3 min.
This purification afforded 344 mg (1.1 mmol, 42%) of the
syn-diastereomer c and 299 mg (0.99 mmol, 37%) of the
anti-diastereomer b.
Example 119
[0414] ##STR245##
[0415] Hydrolysis of the methyl ester a (300 mg, 0.99 mmol) was
carried out by dissolving in THF (0.8 mL), adding deionized water
(1.2 mL) and LiOH.H.sub.2l O (47 mg, 1.1 mmol). The mixture was
stirred at room temperature for 2 h, then reacidified using aqueous
1 N HCl and extracted into 90% ethyl acetate-dichloromethane. Brine
was added to the aqueous acid layer to aid in the extraction.
Drying (Na.sub.2SO.sub.4), filtration, and evaporation of the
solvent yielded the carboxylic acid b (79 mg, 0.28 mmol).
Example 120
[0416] ##STR246##
[0417] Hydrolysis of the methyl ester a (340 mg, 1.1 mmol) was
carried out by dissolving in THF (0.9 mL), adding deionized water
(1.4 mL) and LIOH.H.sub.2O (50 mg, 1.2 mmol). The mixture was
stirred at room temperature for 2 h, then reacidified using aqueous
1 N HCl and extracted into 90% ethyl acetate-dichloromethane. Brine
was added to the aqueous acid layer to aid in the extraction.
Drying (Na.sub.2SO.sub.4), filtration, and evaporation of the
solvent yielded the carboxylic acid b (254 mg, 0.88 mmol), clean
enough to use in the next step without purification.
Example 121
[0418] ##STR247##
[0419] Standard EDC coupling was performed using amine b (62 mg,
0.21 mmol), the carboxylic acid a, (32 mg, 0.11 mmol) and EDC (21
mg, 0.11 mmol). BOC-protected final product was purified by
chromatography ISCO CombiFlash 12 g column with a solvent gradient
of 0-40% ethyl acetate-dichloromethane over 22 min, followed by 67%
ethyl acetate-dichloromethane for 3 min. Standard BOC-deprotection
was performed using 2:1 DCM:TFA with few drops of water. The TFA
salt was treated with base (aqueous 1 N NaOH) and extracted into
ethyl acetate with 10% dichloromethane.
Example 122
[0420] ##STR248##
[0421] Coupling of the primary amine b (47 mg, 0.1 mmol) to
L-BOC-N-methylalanine a (65 mg, 0.30 mmol) was performed by adding
EDC (61 mg, 0.32 mmol) and dissolving in dichloromethane (2 mL).
BOC-protected final product was purified by chromatography ISCO
CombiFlash 12 g column with a solvent gradient of 5-65% ethyl
acetate-dichloromethane over 25 min. Standard BOC-deprotection was
performed using 2:1 DCM:TFA+few drops of water. Final product c was
purified by reverse-phase HPLC C.sub.18 column with a solvent
gradient of 5-50% acetonitrile-water over 20 min. Yield of
anti-diastereomer product c was 22 mg (31 % from proline amine
starting material).
Example 123
[0422] ##STR249##
[0423] Standard EDC coupling was performed using amine b (82 mg,
0.27 mmol), the carboxylic acid a, (95 mg, 0.33 mmol) and EDC (65
mg, 0.34 mmol). BOC-protected final product was purified by
chromatography ISCO CombiFlash 12 g column with a solvent gradient
of 0-40% ethyl acetate-dichloromethane over 22 min, followed by 67%
ethyl acetate-dichloromethane for 3 min. Standard BOC-deprotection
was performed using 2:1 DCM:TFA with few drops of water. The TFA
salt was treated with base (aqueous 1 N NaOH) and extracted into
ethyl acetate with 10% dichloromethane.
Example 124
[0424] ##STR250##
[0425] Coupling of the product primary amine b (70 mg, 0.15 mmol)
to L-BOC-N-methylalanine a (37 mg, 0.18 mmol) was accomplished by
adding EDC (36 mg 0.19 mmol) and dissolving in dichloromethane (2
mL). BOC-protected final product was purified by chromatography
ISCO CombiFlash 12 g column with a solvent gradient of 1-51% ethyl
acetate-dichloromethane over 20 min followed by 51% ethyl
acetate-dichloromethane for 3 min. Standard BOC-deprotection was
performed using 2:1 DCM:TFA+few drops of water. Final product b was
purified by reverse-phase HPLC C.sub.18 column with a solvent
gradient of 5-50% acetonitrile-water over 20 min. Yield of product
b was 49 mg.
Example 125
[0426] ##STR251##
[0427] Sulfide a (810 mg, 2.5 mmol), synthesized according to the
general procedure of Shieh [Shieh, W-C.; Xue, S.; Reel, N.; Wu, R.;
Fitt, J.; Repic, O. Tetrahedron: Asymmetry, 2001, 12, 2421-2425],
was dissolved in methanol (25 mL). Oxone (4.5 g) was dissolved in
deionized water (25 mL). The methanol solution of substrate was
cooled to -10.degree. C., and the aqueous solution of Oxone was
added to the reaction slowly. The reaction was kept on ice and
gradually allowed to warm to room temperature while stirring
overnight. Deionized water was used to dilute the reaction to
approx 150 mL, which was poured into 90% ethyl acetate-hexanes for
extraction. The organic phase was dried (Na.sub.2SO.sub.4),
adsorbed onto Celite and purified by chromatography ISCO CombiFlash
40 g column, 5-90% ethyl acetate-hexanes over 30 min to afford 804
mg (2.27 mmol, 91%) of the product sulfone b.
Example 126
[0428] ##STR252##
[0429] Following the general procedure of Burk [Burk, M. J.; Gross,
M. F.; Martinez, J. P. J. Am. Chem. Soc. 1995, 117, 9375-9376.],
alkene a (774 mg 2.19 mmol), dry methanol (40 mL), and
[(S,S)-Me-BPE-Rh(COD)].sup.+OTf.sup.- (500 mg, 0.8 mmol) were mixed
in a Parr shaker flask purged with nitrogen. Parr shaker was
evacuated and subsequently charged to 60 psi of hydrogen gas and
shaken vigorously overnight. Methanol was removed under reduced
pressure, and crude product was filtered through a small plug of
silica gel using ethyl acetate. Evaporation of the solvent yielded
730 mg (2.0 mmol, 94%) of product b with >98% yield.
Example 127
[0430] ##STR253##
[0431] Z-protected amino ester a (804 mg, 2.27 mmol) was dissolved
in methanol (16 mL). To this solution was added BOC-anhydride (1.5
g, 6.8 mmol), followed by 20% Pd(OH).sub.2.C (250 mg). All air was
removed from the reaction flask by house vacuum, and the mixture
was stirred vigorously for 5 min. The flask was then filled with
hydrogen gas and allowed to stir vigorously at room temperature for
6 h. After evacuating the hydrogen atmosphere, the mixture was
filtered through Celite using methanol, and crude product b was
obtained by evaporation of the solvent (508 mg, 1.56 mmol, 70%
yield).
Example 128
[0432] ##STR254##
[0433] Ester a (508 mg, 1.56 mmol) was dissolved in 8 mL of THF.
Deionized water (4 mL) was added, followed by LiOH.H.sub.2O (120
mg, 2.8 mmol). The mixture was stirred at room temperature
overnight, acidified using aqueous 1 N HCl and extracted into ethyl
acetate. Drying (Na.sub.2SO.sub.4), filtration and evaporation of
the solvent yielded 372 mg (1.21 mmol, 78% yield) of the carboxylic
acid b, clean enough to use in the next step without
purification.
Example 129
[0434] ##STR255##
[0435] Standard EDC coupling was performed using amine b (100 mg,
0.2 mmol), the carboxylic acid a, (58 mg, 0.29 mmol) and EDC (56
mg, 0.29 mmol). BOC-protected final product was purified by
chromatography ISCO CombiFlash 12 g column with a solvent gradient
of 0-65% ethyl acetate-dichloromethane over 15 min. Standard
BOC-deprotection was performed using 2:1 DCM:TFA with few drops of
water. Final product c was purified by reverse-phase HPLC C.sub.18
column with a solvent gradient of 5-50% acetonitrile-water over 18
min. Yield of product c was 132 mg.
Example 130
[0436] ##STR256##
[0437] Standard EDC coupling was performed using amine b (130 mg,
0.3 mmol), the carboxylic acid a, (60 mg, 0.28 mmol) and EDC (60
mg, 0.3 mmol). BOC-protected final product was purified by
chromatography ISCO CombiFlash 12 g column with a solvent gradient
of 0-65% ethyl acetate-dichloromethane over 15 min. Standard
BOC-deprotection was performed using 2:1 DCM:TFA with few drops of
water. Final product c was purified by reverse-phase HPLC C.sub.18
column with a solvent gradient of 5-50% acetonitrile-water over 18
min. Yield of product c was 78 mg.
Example 131
[0438] ##STR257##
[0439] Following the general procedure of Grigg [Blaney, P.; Grigg,
R.; Rankovic, Z.; Thornton-Pett, M.; Xu, J. Tetrahedron, 2002, 58,
1719-1737] a roundbottom flask was charged with sodium hydride (480
mg 60% dispersion in oil, 12.0 mmol, 4.0 equiv) and purged with
nitrogen for 15 min. THF (6.0 mL) was added to the flask, and the
suspension was cooled to 0.degree. C. using an ice water bath. A
separate flask was charged with BOC-glycine a (525 mg, 3.0 mmol),
dry THF (6.0 mL) and ethyl iodide (1.0 mL, 12 mmol, 4 equiv). This
mixture was added dropwise to the NaH suspension in THF, with
vigorous stirring at 0.degree. C. After 1 h of stirring, the
reaction was warmed to room temperature and allowed to stir
overnight. The reaction was again cooled to 0.degree. C., and
methanol (4 mL) was added very slowly to quench the excess hydride.
Deionized water was added to dilute the mixture, and methanol was
removed under reduced pressure. Impurities were extracted into 90%
ethyl acetate-hexanes, the aqueous layer was then acidified by
adding solid citric acid until the pH reached 2-3. The product was
extracted into 90% ethyl acetate-hexanes. This organic layer was
dried (Na.sub.2SO.sub.4) and filtered. Removal of the solvents
under reduced pressure afforded a quantitative yield of the product
b.
Example 132
[0440] ##STR258##
[0441] Standard EDC coupling was performed using amine b (70 mg,
0.16 mmol), the carboxylic acid a, (49 mg, 0.24 mmol) and EDC (46
mg, 0.24 mmol). BOC-protected final product was purified by
chromatography ISCO CombiFlash 12 g column with a solvent gradient
of 0-55% ethyl acetate-dichloromethane over 15 min. Standard
BOC-deprotection was performed using 2:1 DCM:TFA with few drops of
water. Final product c was purified by reverse-phase HPLC C.sub.18
column with a solvent gradient of 5-50% acetonitrile-water over 18
min. Yield of of product c was 82 mg.
Example 133
[0442] ##STR259##
[0443] The free primary amine b (35 mg, 0.056 mmol), anhydrous
potassium carbonate (70 mg, 0.5 mmol) and formamidine hydrochloride
a (30 mg, 0.37 mmol) were mixed together in a vial and dissolved in
methanol (1.2 mL). The mixture was stirred at room temperature for
1.5 hr. Glacial acetic acid was added until gas release was no
longer visible, and the mixture was filtered. Reverse-phase HPLC,
using a C.sub.18 column and a solvent gradient of 5-50%
acetonitrile-water over 25 min with 0.1% TFA, separated the desired
product c, affording 8.2 mg (0.015 mmol, 27% yield) of the TFA salt
after lyophilization.
Example 134
[0444] ##STR260##
[0445] A mixture of unprotected amino acid a (775 mg, 7.24 mmol)
and sodium carbonate (1.69 g, 16.0 mmol) was dissolved in a 1:1
solution of deionized water and THF (15 mL each). To this mixture
was added BOC-anhydride b (1.73 g, 7.96 mmol). The mixture was
stirred at room temperature overnight, and THF was removed under
reduced pressure. The mixture was then acidified to pH 2-3 with
saturated aqueous citric acid, and product was extracted into 10%
ethyl acetate-dichloromethane. The organic layer was dried
(Na.sub.2SO.sub.4), filtered and concentrated under reduced
pressure to afford clean BOC-protected amino acid c (1.40 g, 6.7
mmol, 93%) to be used without further purification.
Example 135
[0446] ##STR261##
[0447] Standard EDC coupling was performed using amine b (64 mg,
0.14 mmol), the carboxylic acid a, (41 mg, 0.2 mmol) and EDC (38
mg, 0.2 mmol). BOC-protected final product was purified by
chromatography ISCO CombiFlash 12 g column with a solvent gradient
of 0-55% ethyl acetate-dichloromethane over 10 min, followed by a
steady flow of 55% ethyl acetate-dichloromethane for 3 min.
Standard BOC-deprotection was performed using 2:1 DCM:TFA+few drops
of water. Final product c was purified by reverse-phase HPLC
C.sub.18 column with a solvent gradient of 5-50% acetonitrile-water
over 18 min. Yield of product c was 70.2 mg.
Example 136
[0448] ##STR262##
[0449] Standard EDC coupling was performed using amine
hydrochloride b (250 mg, 0.67 mmol), the carboxylic acid a, (187
mg, 0.81 mmol), DIPEA (0.35 mL, 2.0 mmol) and EDC (157 mg, 0.81
mmol). Reaction was stirred at room temperature for 48 h.
BOC-protected final product was purified by chromatography ISCO
CombiFlash 12 g column with a solvent gradient of 0-25% ethyl
acetate-hexanes over 10 min, followed by a steady flow of 26% ethyl
acetate-hexanes for 3 min. Standard BOC-deprotection was performed
using HCl in dioxane (4.0 M, 3.0 mL).
[0450] To the primary amine hydrochloride c (170 mg, 0.38 mmol) and
L-BOC-N-methylalanine (91 mg, 0.45 mmol), was added dichloromethane
(2 mL), DIPEA (0.20 mL, 1.1 mmol) and EDC (86 mg, 0.45 mmol),
stirring at room temperature for 24 h. BOC-protected final product
was purified by chromatography ISCO CombiFlash 12 g column with a
solvent gradient of 0.5-52% ethyl acetate-hexanes over 13 min
followed by 52% ethyl acetate-hexanes for 3 min. Standard
BOC-deprotection was performed using 2:1 DCM:TFA with few drops of
water. Final product was purified by reverse-phase HPLC C.sub.18
column with a solvent gradient of 5-60% acetonitrile-water over 20
min. Yield of final product was 90 mg.
Example 137
[0451] ##STR263##
[0452] Standard EDC coupling was performed using amine
hydrochloride # (250 mg, 0.67 mmol), the carboxylic acid a, (187
mg, 0.81 mmol), DIPEA (0.350 mL, 2.0 mmol) and EDC (157 mg, 0.81
mmol). Reaction was stirred at room temperature for 3 h.
BOC-protected final product was purified by chromatography ISCO
CombiFlash 12 g column with a solvent gradient of 0-25% ethyl
acetate-hexanes over 10 min, followed by a steady flow of 26% ethyl
acetate-hexanes for 3 min. Standard BOC-deprotection was performed
using HCl in dioxane (4.0 M, 3.0 mL).
[0453] To the primary amine hydrochloride (160 mg, 0.35 mmol) and
L-BOC-N-methylalanine (91 mg, 0.45 mmol), was added dichloromethane
(2 mL), DIPEA (0.200 mL, 1.1 mmol) and EDC (86 mg, 0.45 mmol),
stirring at room temperature for 24 h. BOC-protected final product
was purified by chromatography ISCO CombiFlash 12 g column with a
solvent gradient of 0.5-52% ethyl acetate-hexanes over 13 min
followed by 52% ethyl acetate-hexanes for 3 min. Standard
BOC-deprotection was performed using 2:1 DCM:TFA with few drops of
water. Final product was purified by reverse-phase HPLC C.sub.18
column with a solvent gradient of 5-60% acetonitrile-water over 20
min. Yield of product c was 79 mg.
Example 138
[0454] ##STR264##
[0455] EDC coupling was performed using amine hydrochloride b (230
mg, 0.61 mmol), the carboxylic acid a, (165 mg, 0.75 mmol), DIPEA
(0.350 mL, 2.0 mmol) and EDC (157 mg, 0.81 mmol). Reaction was
stirred at room temperature for 3 h, LC/MS indicated only half
complete. More carboxylic acid (160 mg) and EDC (150 mg) was added
to the reaction, and the mixture was stirred overnight at room
temperature. BOC-protected final product was purified by
chromatography ISCO CombiFlash 40 g column with a solvent gradient
of 0-55% ethyl acetate-hexanes over 17 min, followed by a steady
flow of 56% ethyl acetate-hexanes for 5 min. Standard
BOC-deprotection was performed using 2:1 DCM:TFA+few drops of
water. Coupling of the product primary amine c (199 mg, 0.5 mmol)
to L-BOC-N-methylalanine (140 mg, 0.7 mmol) was performed with EDC
(135 mg, 0.7 mmol) and dichloromethane (3 mL). BOC-protected final
product was purified by chromatography ISCO CombiFlash 12 g column
with a solvent gradient of 0-40% ethyl acetate-dichloromethane over
15 min followed by 40% ethyl acetate-dichloromethane for 3 min.
Standard BOC-deprotection was performed using 2:1 DCM:TFA with few
drops of water. Final product was purified by reverse-phase HPLC
C.sub.18 column with a solvent gradient of 5-50% acetonitrile-water
over 20 min. Yield of final product was 178 mg.
Example 139
[0456] ##STR265##
[0457] Methyl ketone a (480 mg, 3.0 mmol), synthesized according to
the general procedure of Miki [Miki, Y.; Nakamura, N.; Hachiken,
H.; Takemura, S. J. Heterocyclic Chem., 1989, 26, 1739-1745], was
suspended in 33% HBr in acetic acid (6 mL). Elemental bromine was
added in six portions (6.times.0.025 mL, 0.15 mL total, 3.0 mmol)
with vigorous stirring at room temperature. The reaction appeared
to have a light color after 10 min of stirring, when diethyl ether
was added (10 mL). Stirring at room temperature was continued for
30 min. The mixture was filtered through a frit, and the solids
left behind were rinsed with 20 mL of ether, transferred to a vial,
and dried under high vacuum. The solid afforded (840 mg) was a
mixture of desired product b and the HBr salt of the starting
material, used without further purification in the thiazole-forming
step.
Example 140
[0458] ##STR266##
[0459] Thioamide a (2.26 mg, 9.8 mmol) was added to the mixture of
bromomethyl ketone b and methyl ketone (1.44 g) in a roundbottom
flask. Ethanol (30 mL) was added, dissolving the thioamide and
suspending the salts. Pyridine was then added dropwise (0.4 mL, 5.0
mmol) and the mixture was stirred at room temperature for 5 min.
The reaction flask was then heated to 70.degree. C. in an oil bath,
with vigorous stirring. After 10 min, the suspension of salts was
no longer visible and the reaction was homogeneous. The reaction
was allowed to cool to room temperatue for 45 min, and Celite was
added along with toluene (20 mL). Solvents were removed under
reduced pressure. The crude product adsorbed onto Celite was
purified by chromatography ISCO CombiFlash 120 g column, 0-30%
ethyl acetate-dichloromethane over 20 min, followed by a gradient
of 30-70% ethyl acetate-dichloromethane over 5 min, to afford 518
mg (1.4 mmol, 47%) of the product thiazole. Removal of BOC from the
proline amine was accomplished by dissolving the substrate in 2:1
DCM:TFA with few drops of water, following the standard procedure.
Free base was obtained by treating the TFA salt with 1 N aqueous
sodium hydroxide and extracting the amine into dichloromethane.
Drying of the organic layer (Na.sub.2SO.sub.4), filtering and
removing the solvent under reduced pressure afforded 356 mg (1.3
mmol, 93%) of free amine c.
Example 141
[0460] ##STR267##
[0461] HOAt, DIC procedure was used to couple the above dipeptide
to amine. Secondary amine b (65 mg, 0.25 mmol), carboxylic acid a,
(97 mg, 0.28 mmol) HOAt (53 mg, 0.4 mmol) and DIC (50 mg, 0.4
mmol). BOC-protected final product was purified by chromatography
ISCO CombiFlash 12 g column with a solvent gradient of 0-65% ethyl
acetate-dichloromethane over 15 min. Standard BOC-deprotection was
performed using 2:1 DCM:TFA+few drops of water. Final product c was
purified by reverse-phase HPLC C.sub.18 column with a solvent
gradient of 5-50% acetonitrile-water over 18 min. Yield of product
c was 98 mg.
Example 142
[0462] ##STR268##
[0463] HOAt, DIC procedure was used to couple the above dipeptide
to amine. Secondary amine b (50 mg, 0.2 mmol), carboxylic acid a,
(72 mg, 0.21 mmol) HOAt (40 mg, 0.3 mmol) and DIC (38 mg, 0.3
mmol). BOC-protected final product was purified by chromatography
ISCO CombiFlash 12 g column with a solvent gradient of 10-85% ethyl
acetate-dichloromethane over 20 min. Standard BOC-deprotection was
performed using 2:1 DCM:TFA+few drops of water. Final product c was
purified by reverse-phase HPLC C.sub.18 column with a solvent
gradient of 3-40% acetonitrile-water over 20 min. Yield of final
product c was 25 mg.
Example 143
[0464] ##STR269##
[0465] A mixture of unprotected amino acid a (1.1 g, 10 mmol) and
sodium carbonate (850 mg, 10 mmol) was dissolved in a 1:1 solution
of deionized water and THF (13 mL each). To this mixture was added
FMOC--OSu b (6.times.550 mg, total 3.3 g, 9.8 mmol) over a period
of 1 h. After each addition of FMOC--OSu was added 2-3 mL of 1 M
aqueous sodium bicarbonate to keep the reaction mixture at basic
pH. The mixture was stirred at room temperature overnight, and THF
was removed under reduced pressure. The mixture was then diluted
with deionized water, poured into ethyl acetate in a separatory
funnel, and made acidic by the addition of 6 N HCl. After
extracting into ethyl acetate, the organic layer was washed with
deionized water, followed by brine. The organic layer was dried
(Na.sub.2SO.sub.4), filtered and concentrated under reduced
pressure to afford clean FMOC-protected amino acid c (1.05 g, 3.19
mmol, 32%) to be used without further purification.
Example 144
[0466] ##STR270##
[0467] Following the general procedure of Freidinger [Freidinger,
R. M.; Hinkle, J. S.; Perlow, D. S.; Arison, B. H. J. Org. Chem.,
1983, 48, 77-81], the FMOC-protected primary amine a (1.04 g, 3.17
mmol) was dissolved in toluene (60 mL). Paraformaldehyde (630 mg)
was added, followed by a catalytic amount of p-toluenesulfonic acid
(70 mg, 0.37 mmol). The mixture was vigorously stirred at reflux
temperature for 45 min, collecting any generated water in a Dean
Stark trap. The reaction mixture was then allowed to cool to room
temperature, and washed with saturated aqueous sodium bicarbonate
(2.times.30 mL). The organic layer was dried (Na.sub.2SO.sub.4),
filtered, and concentrated under reduced pressure to afford 910 mg
(2.7 mmol) of oxazolidinone b. The oxazolidinone (337 mg, 0.99
mmol) was dissolved in dichloromethane (20 mL). To this solution
was added anhydrous aluminum trichloride (260 mg, 2.0 mmol),
followed by triethylsilane (0.32 mL, 2.0 mmol). The reaction
mixture was stirred for 5 h at room temperature and then quenched
with 20 mL of 1 N aqueous HCl. The product carboxylic acid was
extracted into 25% ethyl acetate-dichloromethane and washed with 1
N aqueous HCl (20 mL) followed by brine. The organic layer was
dried (Na.sub.2SO.sub.4) and filtered. Celite was added, and the
solvent was removed under reduced pressure. The crude product
adsorbed onto Celite was purified by chromatography ISCO CombiFlash
40 g column, 1-55% ethyl acetate-dichloromethane over 25 min, to
afford 272 mg (0.79 mmol, 25% yield from FMOC-primary amine) of the
FMOC protected N-methyl amino acid c.
Example 145
[0468] ##STR271##
[0469] Standard EDC coupling was performed using amine # (140 mg,
0.4 mmol), the crude carboxylic acid a, (176 mg, 0.4 mmol) and EDC
(80 mg, 0.4 mmol). BOC-protected final product was purified by
chromatography ISCO CombiFlash 40 g column with a solvent gradient
of 1-40% ethyl acetate-dichloromethane over 20 min. The desired
BOC-protected product was split into two portions for removing the
FMOC group. The first portion (50 mg, 0.065 mmol) was dissolved in
dichloromethane (1.0 mL), treated with piperidine (0.10 mL, 1.0
mmol), and allowed to stir at room temperature for 2 h. The second
portion (100 mg, 0.13 mmol) was dissolved in 20% piperidine in DMF
(1.0 mL) and allowed to stir at room temperature overnight. Both
reactions were quenched by adding few drops of TFA. Final product
was purified by reverse-phase HPLC C.sub.18 column with a solvent
gradient of 3-40% acetonitrile-water over 20 min. Combined yield of
final product c was 55 mg.
Example 146
IAP Inhibition Assays
[0470] In the following experiments was used a chimeric BIR domain
referred to as MLXBIR3SG in which 11 of 110 residues correspond to
those found in XIAP-BIR3, while the remainder correspond to
ML-IAP-BIR. The chimeric protein MLXBIR3SG was shown to bind and
inhibit caspase-9 significantly better than either of the native
BIR domains, but bound Smac-based peptides and mature Smac with
affinities similar to those of native ML-IAP-BIR. The improved
caspase-9 inhibition of the chimeric BIR domain MLXBIR3SG has been
correlated with increased inhibition of doxorubicin-induced
apoptosis when transfected into MCF7 cells. TABLE-US-00001
MLXBIR3SG sequence: MGSSHHHHHHSSGLVPRGSHMLETEEEEEEGAGATL (SEQ ID
NO.:1) SRGPAFPGMGSEELRLASFYDWPLTAEVPPELLAAA
GFFHTGHQDKVRCFFCYGGLQSWKRGDDPWTEHAKW FPGCQFLLRSKGQEYINNIHLTHSL
TR-FRET Peptide Binding Assay
[0471] Time-Resolved Fluorescence Resonance Energy Transfer
competition experiments were performed on the Wallac Victor2
Multilabeled Counter Reader (Perkin Elmer Life and Analytical
Sciences, Inc.) according to the procedures of Kolb et al (Journal
of Biomolecular Screening, 1996, 1(4):203). A reagent cocktail
containing 300 nM his-tagged MLXBIR3SG; 200 nM biotinylated SMAC
peptide (AVPI); 5 .mu.g/mL anti-his allophycocyanin (XL665) (CISBio
International); and 200 ng/mL streptavidin-europium (Perkin Elmer)
was prepared in reagent buffer (50 mM Tris [pH 7.2], 120 mM NaCl,
0.1% bovine globulins, 5 mM DTT and 0.05% octylglucoside).
(Alternatively, this cocktail can be made using europium-labeled
anti-His (Perkin Elmer) and streptavidin-allophycocyanin (Perkin
Elmer) at concentrations of 6.5 nM and 25nM, respectively). The
reagent cocktail was incubated at room temperature for 30 minutes.
After incubation, the cocktail was added to 1:3 serial dilutions of
an antagonist compound (starting concentration of 50 .mu.M) in
384-well black FIA plates (Greiner Bio-One, Inc.). After a 90
minute incubation at room temperature, the fluorescence was read
with filters for the excitation of europium (340 nm) and for the
emission wavelengths of europium (615 nm) and a allophycocyanin
(665 nm). Antagonist data were calculated as a ratio of the
emission signal of allophycocyanin at 665 nm to that of the
emission of europium at 615 nm (these ratios were multiplied by a
factor of 10,000 for ease of data manipulation). The resulting
values were plotted as a function of antagonist concentration and
fit to a 4-equation using Kaleidograph software (Synergy Software,
Reading, Pa.). Indications of antagonist potency were determined
from the IC50 values. Compounds of the invention that were tested
in this assay exhibited IC50 values of less than 200 .mu.M
indicating IAP inhibitory activity.
Fluorescence Polarization Peptide Binding Assay
[0472] Polarization experiments were performed on an Analyst HT
96-384 (Molecular Devices Corp.) according to the procedure of
Keating, S. M., Marsters, J, Beresini, M., Ladner, C., Zioncheck,
K., Clark, K., Arellano, F., and Bodary., S.(2000) in Proceedings
of SPIE: In Vitro Diagnostic Instrumentation (Cohn, G. E., Ed.) pp
128-137, Bellingham, Wash. Samples for fluorescence polarization
affinity measurements were prepared by addition of 1:2 serial
dilutions starting at a final concentration of 5 .mu.M of MLXBIR3SG
in polarization buffer (50 mM Tris [pH 7.2], 120 mM NaCl, 1% bovine
globulins 5 mM DTF and 0.05% octylglucoside) to
5-carboxyflourescein-conjugated AVPdi-Phe-NH.sub.2 (AVP-diPhe-FAM)
at 5 nM final concentration. ##STR272##
[0473] The reactions were read after an incubation time of 10
minutes at room temperature with standard cut-off filters for the
fluorescein fluorophore (.lamda..sub.ex=485 nm; .lamda..sub.em=530
nm) in 96-well black HE96 plates (Molecular Devices Corp.).
Fluorescence values were plotted as a function of the protein
concentration, and the IC50s were obtained by fitting the data to a
4-parameter equation using Kaleidograph software (Synergy software,
Reading, Pa.). Competition experiments were performed by addition
of the MLXBIR3SG at 30 nM to wells containing 5 nM of the
AVP-diPhe-FAM probe as well as 1:3 serial dilutions of antagonist
compounds starting at a concentration of 300 .mu.M in the
polarization buffer. Samples were read after a 10-minute
incubation. Fluorescence polarization values were plotted as a
function of the antagonist concentration, and the IC.sub.50 values
were obtained by fitting the data to a 4-parameter equation using
Kaleidograph software (Synergy software, Reading, Pa.). Inhibition
constants (K.sub.i) for the antagonists were determined from the
IC.sub.50 values. Compounds of the invention that were tested in
this assay exhibited a Ki or less than 100 .mu.M.
Sequence CWU 1
1
1 1 133 PRT Homo sapiens 1 Met Gly Ser Ser His His His His His His
Ser Ser Gly Leu Val 1 5 10 15 Pro Arg Gly Ser His Met Leu Glu Thr
Glu Glu Glu Glu Glu Glu 20 25 30 Gly Ala Gly Ala Thr Leu Ser Arg
Gly Pro Ala Phe Pro Gly Met 35 40 45 Gly Ser Glu Glu Leu Arg Leu
Ala Ser Phe Tyr Asp Trp Pro Leu 50 55 60 Thr Ala Glu Val Pro Pro
Glu Leu Leu Ala Ala Ala Gly Phe Phe 65 70 75 His Thr Gly His Gln
Asp Lys Val Arg Cys Phe Phe Cys Tyr Gly 80 85 90 Gly Leu Gln Ser
Trp Lys Arg Gly Asp Asp Pro Trp Thr Glu His 95 100 105 Ala Lys Trp
Phe Pro Gly Cys Gln Phe Leu Leu Arg Ser Lys Gly 110 115 120 Gln Glu
Tyr Ile Asn Asn Ile His Leu Thr His Ser Leu 125 130
* * * * *