U.S. patent application number 09/423420 was filed with the patent office on 2002-06-27 for beta lactams as antiproliferative agents.
Invention is credited to CHILTON III, FLOYD HAROLD, WINKLER, JAMES DAVID.
Application Number | 20020082250 09/423420 |
Document ID | / |
Family ID | 21932091 |
Filed Date | 2002-06-27 |
United States Patent
Application |
20020082250 |
Kind Code |
A1 |
WINKLER, JAMES DAVID ; et
al. |
June 27, 2002 |
BETA LACTAMS AS ANTIPROLIFERATIVE AGENTS
Abstract
This invention relates to a method of treating or reducing cell
proliferation in a mammal in need thereof which comprises
administering to said mammal an effective CoA-independent
transacylase (CoA-IT) inhibiting amount of a compound of formula
(I), wherein the variables of Y, X, m, R 3, R 4, R 10, R 20, R 5, R
6 and R 7 are defined in the specification. FIG. 3 demonstrates the
ability of SB216754,
((3S,4R)-4-(isobutenyloxy)-3-triphenylmethylamino)azetidin-2-one)
to decrease the viability of HL-60 cells.
Inventors: |
WINKLER, JAMES DAVID; (FORT
WASHINGTON, PA) ; CHILTON III, FLOYD HAROLD; (PILOT
MOUNTAIN, NC) |
Correspondence
Address: |
SMITHKLINE BEECHAM CORPORATION
CORPORATE INTELLECTUAL PROPERTY-US, UW2220
P. O. BOX 1539
KING OF PRUSSIA
PA
19406-0939
US
|
Family ID: |
21932091 |
Appl. No.: |
09/423420 |
Filed: |
January 28, 2000 |
PCT Filed: |
May 8, 1998 |
PCT NO: |
PCT/US98/09481 |
Current U.S.
Class: |
514/210.02 |
Current CPC
Class: |
A61K 31/00 20130101;
A61K 31/4436 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61P 35/00 20180101; A61P 43/00 20180101; A61P 9/10
20180101; A61P 29/00 20180101; A61P 17/06 20180101; A61K 31/4436
20130101; A61K 31/397 20130101; A61K 31/397 20130101; A61P 35/02
20180101 |
Class at
Publication: |
514/210.02 |
International
Class: |
A61K 031/397; A01N
043/00 |
Claims
What is claimed is:
1. A method of inhibiting or reducing cell proliferation in a
mammal, in need of such treatment, which method comprises
administering to said human or mammal an effective amount of a
CoA-independent transacylase (CoA-IT) inhibiting amount of a
compound of the Formula: 4wherein Y is NH; X is O or S(O)m; m is 0
or an integer having a value of 1, or 2; R.sub.3 is optionally
substituted triphenylmethyl; R.sub.4 is optionally substituted
C.sub.1-10 alkyl, (CR.sub.10R.sub.20)n--C.ident.C--R.sub.5, or
(CR.sub.1-10R.sub.20).sub.nC(R.sub.10).dbd.C(R.sub.7).sub.2; n is
an integer having a value of 1 to 4; R.sub.10 and R.sub.20 are
independently hydrogen or C.sub.1-4 alkyl; R.sub.5 is hydrogen,
alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl,
C(O).sub.2R.sub.6, or C(O)R.sub.6, wherein the alkyl, aryl,
arylalkyl, heteroaryl, or heteroarylalkyl moieties may be
optionally substituted; R.sub.6 is C.sub.1-10 alkyl, aryl,
arylalkyl, heteroaryl, heteroarylalkyl, heterocyclic, or
heterocyclicalkyl, wherein the alkyl, aryl, arylalkyl, heteroaryl,
heteroarylalkyl, heterocyclic, or heterocyclicalkyl moieties may be
optionally substituted; R.sub.7 is independently hydrogen,
C.sub.1-10 alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl,
heterocyclic, or heterocyclicalkyl, wherein the alkyl, aryl,
arylalkyl, heteroaryl, heteroarylalkyl, heterocyclic, or
heterocyclicalkyl moieties may be optionally substituted; or a
pharmaceutically acceptable salt thereof.
2. The method according to claim 1 wherein the CoA-IT inhibitor is
co-administered with an effective amount of ET-18-O-CH.sub.3 or an
or an alkyl lysophospholipid analog, or other anti-proliferative or
chemotherapeutic agent.
3. The method according to claim 1 wherein the mammal is afflicted
with psoriasis, rheumatoid arthritis, or atherosclerosis, or other
disease characterized by abnormal cell proliferation.
4. A method of treating a cancerous cell growth in a mammal, in
need of such treatment, which method comprises administering to
said mammal an effective amount of a compound of the formula:
5wherein Y is NH; X is O or S(O)m; m is 0 or an integer having a
value of 1, or 2; R.sub.3 is optionally substituted
triphenylmethyl; R.sub.4 is optionally substituted C.sub.1-10
alkyl, (CR.sub.10R.sub.20)n--C.ident.C--R.sub.5, or
(CR.sub.10R.sub.20).sub.nC(R.sub.10).dbd.C(R.sub.7).sub.2; n is an
integer having a value of 1 to 4; R.sub.10 and R.sub.20 are
independently hydrogen or C.sub.1-4 alkyl; R.sub.5 is hydrogen,
alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl,
C(O).sub.2R.sub.6, or C(O)R.sub.6, wherein the alkyl, aryl,
arylalkyl, heteroaryl, or heteroarylalkyl moieties may be
optionally substituted; R.sub.6 is C.sub.1-10 alkyl, aryl,
arylalkyl, heteroaryl, heteroarylalkyl, heterocyclic, or
heterocyclicalkyl, wherein the alkyl, aryl, arylalkyl, heteroaryl,
heteroarylalkyl, heterocyclic, or heterocyclicalkyl moieties may be
optionally substituted; .sub.R.sub.7 is independently hydrogen,
C.sub.1-10 alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl,
heterocyclic, or heterocyclicalkyl, wherein the alkyl, aryl,
arylalkyl, heteroaryl, heteroarylalkyl, heterocyclic, or
heterocyclicalkyl moieties may be optionally substituted; or a
pharmaceutically acceptable salt thereof.
5. The method according to claim 4 wherein the inhibitor is
co-administered with an effective amount of ET-18-O-CH.sub.3 or an
alkyl lysophospholipid analog or an alkyl lysophospholipid analog,
or other anti-proliferative or chemotherapeutic agent.
6. The method according to claim 4 wherein the mammal is afflicted
with leukemia, or other cancers.
7. A method of inducing apoptosis in a mammal, in need of such
treatment which method comprises administering to said human or
mammal an effective CoA-independent transacylase (CoA-IT)
inhibiting amount of a compound of the formula: 6wherein Y is NH; X
is O or S(O)m; m is 0 or an integer having a value of 1, or 2;
R.sub.3 is optionally substituted triphenylmethyl; R.sub.4 is
optionally substituted C.sub.1-10 alkyl,
(CR.sub.10R.sub.20)n--C.ident.C--R.sub.5, or
(CR.sub.10R.sub.20).sub.nC(R- .sub.10).dbd.C(R.sub.7).sub.2; n is
an integer having a value of 1 to 4; R.sub.10 and R.sub.20 are
independently hydrogen or C.sub.1-4 alkyl; .sub.R.sub.5 is
hydrogen, alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl,
C(O).sub.2R.sub.6, or C(O)R.sub.6, wherein the alkyl, aryl,
arylalkyl, heteroaryl, or heteroarylalkyl moieties may be
optionally substituted; R.sub.6 is C.sub.1-10 alkyl, aryl,
arylalkyl, heteroaryl, heteroarylalkyl, heterocyclic, or
heterocyclicalkyl, wherein the alkyl, aryl, arylalkyl, heteroaryl,
heteroarylalkyl, heterocyclic, or heterocyclicalkyl moieties may be
optionally substituted; R.sub.7 is independently hydrogen,
C.sub.1-10 alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl,
heterocyclic, or heterocyclicalkyl, wherein the alkyl, aryl,
arylalkyl, heteroaryl, heteroarylalkyl, heterocyclic, or
heterocyclicalkyl moieties may be optionally substituted; or a
pharmaceutically acceptable salt thereof.
Description
FIELD OF THE INVENTION
[0001] This invention relates to novel compounds, pharmaceutical
compositions thereof, and their use as anti-inflammatory agents and
for the treatment of cell proliferation and cancer in mammals.
BACKGROUND OF THE INVENTION
[0002] Over the past three decades, it has been firmly established
that the in vivo modulation of levels of the polyunsaturated fatty
acid, arachidonic acid (AA) and oxygenated metabolites of
arachidonic acid is intimately linked to human disease. Our
laboratories have focused on the pathways and enzyme activities
that acylate AA into membrane phospholipid of inflammatory and
neoplastic cells (for a review, see Chilton et al., Biochim.
Biophys. Acta 1299: 1-15 (1996)). These studies indicate that there
are as many as 20 different arachidonate-containing phospholipid
molecular species in any given inflammatory or neoplastic cell
type. We have demonstrated that AA moves through different
AA-containing phospholipid molecular species in a sequential
fashion requiring several different enzyme activities (FIG. 1).
Initially AA is converted to AA-CoA by fatty acyl CoA synthase(s).
There appears to be specific fatty acyl CoA synthase(s) which
utilize AA but not other fatty acids. Once formed, arachidonoyl-CoA
may be incorporated into 1-acyl-2-lyso-sn-glycero-3-phos-
phocholine by CoA-dependent acyl transferase(s). This AA in
1-acyl-2-AA-sn-glycero-3-phosphocholine is then transferred to
1-ether-linked phospholipids such as
1-alkyl-2-AA-sn-glycero-3-phosphocho- line and
1-alk-1-enyl-2-AA-sn-glycero-phosphoethanolamine. 1-Ether-linked
phospholipids contain the bulk of AA found in most inflammatory and
neoplastic cells. This latter reaction is orchestrated by the
action of the enzyme CoA-independent transacylase (CoA-IT).
[0003] Coenzyme A-independent transacylase (CoA-IT) is an enzyme
responsible for the movement of arachidonate between phospholipid
molecular species of inflammatory cells. CoA-IT removes
arachidonate from the sn-2 position of 1-acyl-containing
phospholipids, such as
1-acyl-2-arachidonoyl-sn-glycero-3-phosphocholine
(1-acyl-2-arachidonoyl-- GPC). It then transfers that arachidonate
to a suitable lyso-phospholipid acceptor, such as
1-alkyl-2-lyso-GPC and 1-alkenyl-2-lyso-sn-glycero-3-ph-
ospho-ethanolamine (Sugiura et al., J. Biol. Chem. 262: 1199-1205
(1987); Kramer and Deykin, Biol. Chem. 258: 13806-13811 (1983);
Chilton et al., J. Biol. Chem. 258: 7268-7271 (1983)). This
activity is selective for 20 carbon fatty acyl groups and is the
mechanism by which inflammatory cells move arachidonate into
specific phospholipid pools prior to its release (Winkler and
Chilton, Drug News Perspec. 6: 133-138 (1993); Snyder et al., J.
Lipid Mediat. 10: 25-31 (1994)).
[0004] Although the pathway for the incorporation and remodeling of
AA among phospholipids have been worked out within inflammatory
cells and neoplastic cells, we have only recently begun to
understand the functional significance of this pathway. We have
recently discovered structurally distinct families of molecules
(characterized by SK&F 45905
(2-[2-[3-(4-Chloro-3-trifluoromethylphneyl)ureido]-4-tifluromethylphenoxy-
]-4,5-dichlorobenzene sulfonic acid) and SK&F 98625 (Diethyl
7-(3,4,5-triphenyl-2-oxo-2,3-dihydroimidazol-1yl)heptane
phosphonate)) which are capable of inhibiting CoA-IT (Chilton et
al., Biochemistry 34: 5403-5410 (1995); Winkler et al., J.
Pharmacol. Exp. Ther. 274: 1338-1347 (1995)). When provided acutely
or chronically to inflammatory or neoplastic cells, these
inhibitors attenuate the movement labeled AA from 1-acyl-linked
phospholipids to 1-alkyl- and 1-alkenyl-linked phospholipids
(Chilton et al., Biochemistry 34: 5403-5410 (1995)). In addition to
SK&F 45905 and SK&F 98625, it has been established that the
antineoplastic agent 1-0-octadecyl-2-0-methyl-phosphocholine
(ET-18-0-CH.sub.3) is also a potent inhibitor of the enzyme CoA-IT
(Winkler et al., J. Pharmacol. Exp. Ther. 279: 956-966 (1996)).
Moreover, other studies revealed that the CoA-IT inhibitors
SK&F 45905 and SK&F 98625 possess antiproliferative
properties (Surette et al., Biochemistry 35: 9187-9196 (1996);
Winkler et al., J. Pharmacol. Exp. Ther. 279: 956-966 (1996)). More
specifically, all CoA-IT inhibitors, including ET-18-0-CH3,
attenuate cell proliferation and induce apoptosis in several
neoplastic cell lines. Structurally related compounds which possess
no inhibitory activity toward CoA-IT do not induce apoptosis. In
addition, inhibitors of phospholipase A.sub.2, 5-lipoxygenase, and
cyclooxygenase do not induce apoptosis suggesting that free AA or
its metabolites are not responsible for this process. The
aforementioned discoveries indicate that blockage of the enzyme
CoA-IT is a novel chemotherapeutic approach for the treatment of
proliferative disorders such as cancer.
[0005] There exists a need to find other CoA-IT inhibitors with
better in vivo pharmacological and toxicological profiles. These
compounds should block cell proliferation of diseased cells and
hence provide potential treatment for cancers, such as leukemia and
other proliferative diseases and conditions such as psoriasis.
SUMMARY OF THE INVENTION
[0006] The present invention is a method to decreasing, inhibiting
or reducing disease or disorders of cell proliferation in a mammal,
and inducing apoptosis in a mammal, preferably in a human, by
inhibition of the enzyme CoA-IT. Therefore the present invention is
to the use of an effective amount of a CoA-IT inhibitor of Formula
(I) for the treatment of said cell proliferation, in mammals,
preferably humans, in need of such treatment, by administering to
said human an effective amount of a CoA-IT inhibitory compound, or
pharmaceutically acceptable salt thereof. A preferred disease
state, for treatment associated with cell proliferation, is
psoriasis, rheumatoid arthritis or atherosclerosis.
[0007] Another aspect of the present invention is to a method of
treating a cancerous cell growth in a mammal, preferably a human,
in need of such treatment, which method comprises administering to
said mammal an effective amount of a compound of Formula (I). A
preferred disease state for treatment associated with cancerous
cell growth is leukemia.
[0008] Another aspect of the present invention is to a method of
inducing apoptosis in a mammal, in need of such treatment which
method comprises administering to said human or mammal an effective
amount of a CoA-independent transacylase (CoA-IT) inhibitor, of
Formula (I).
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 is a scheme of the movement of arachidonic acid that
occurs and this relationship to apoptosis.
[0010] FIG. 2 demonstrates the time dependent inhibition of CoA-IT
by
(3S,4R)-4-(isobutenyloxy)-3-triphenylmethylamino)azetidin-2-one.
[0011] FIG. 3 demonstrates the ability of SB 216754,
((3S,4R)4-(isobutenyloxy)-3-triphenylmethylamino)azetidin-2-one) to
decrease viability of HL-60 cells.
[0012] FIG. 4 demonstrates the ability of
((3S,4R)-4-(isobutenyloxy)-3-tri- phenylmethylamino)azetidin-2-one)
to block the incorporation of thymidine into P388 cells.
[0013] FIG. 5 demonstrates the ability of
((3S,4R)-4-(isobutenyloxy)-3-tri- phenylmethylamino)azetidin-2-one)
to induce DNA fragmenatation in P388 cells.
[0014] FIG. 6 demonstrates the ability of
((3S,4R)-4-(isobutenyloxy)-3-tri- phenylmethylamino)azetidin-2-one)
to block the incorporation of thymidine into chronic myelogenous
leukemia cells.
[0015] FIG. 7 demonstrates the ability of
((3S,4R)-4-(isobutenyloxy)-3-tri- phenylmethylamino)azetidin-2-one)
to induce DNA fragmenatation in chronic myelogenous leukemia
cells.
DETAILED DESCRIPTION OF THE INVENTION
[0016] Inhibition of CoA-IT activity results in a decrease in cell
growth and proliferation, in induction of apoptosis, thereby
providing therapeutic utility in proliferative and cancerous
disorders. The present invention therefore provides a method for
treating proliferative and cancerous disorders by CoA-IT
inhibition. This inhibition is by administering to a mammal, in
need of such treatment an effective amount of a compound according
to Formula (I). This inhibition will result in the treatment, and
potential prophylaxis, of proliferative occurrences in mammals,
preferably humans. Such proliferative states in mammals may
include, but are not limited to, diseases (such as cancers),
dermatological diseases (such as psoriasis) and inflammatory
diseases (such as, rheumatoid arthritis). Treatment of both acute
and chronic diseases are thereby possible. For the purposes herein,
the compounds of Formula (I) are preferential and selective
inhibitors of CoA-IT.
[0017] Compounds of Formula (I) are represented by the structure:
1
[0018] wherein
[0019] Y is NH;
[0020] X is O or S(O)m;
[0021] m is 0 or an integer having a value of 1, or 2;
[0022] R.sub.3 is optionally substituted triphenylmethyl;
[0023] R.sub.4 is optionally substituted C.sub.1-10 alkyl,
(CR.sub.10R.sub.20)n--C.ident.C--R.sub.5, or
(CR.sub.10R.sub.20).sub.nC(R- .sub.10).dbd.C(R.sub.7).sub.2;
[0024] n is an integer having a value of 1 to 4;
[0025] R.sub.10 and R.sub.20 are independently hydrogen or
C.sub.1-4 alkyl;
[0026] R.sub.5 is hydrogen, alkyl, aryl, arylalkyl, heteroaryl,
heteroarylalkyl, C(O).sub.2R.sub.6, or C(O)R.sub.6 wherein the
alkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl moieties
may be optionally substituted;
[0027] R.sub.6 is C.sub.1-10 alkyl, aryl, arylalkyl, heteroaryl,
heteroarylalkyl, heterocyclic, or heterocyclicalkyl moiety, wherein
the alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl,
heterocyclic, or heterocyclicalkyl moieties may be optionally
substituted;
[0028] R.sub.7 is independently hydrogen, C.sub.1-10 alkyl, aryl,
arylalkyl, heteroaryl, heteroarylalkyl, heterocyclic, or
heterocyclicalkyl, wherein the alkyl, aryl, arylalkyl, heteroaryl,
heteroarylalkyl, heterocyclic, or heterocyclicalkyl moieties may be
optionally substituted; or a pharmaceutically acceptable salt
thereof.
[0029] Suitably, X is O or S(O)m; and m is 0 or an integer having a
value of 1 or 2. Preferably m is 0 or 2.
[0030] Suitably, R.sub.3 is optionally substituted triphenylmethyl
group. The phenyl rings may be independently substituted one to
three times by halogen, such as fluorine, chlorine, bromine or
iodine; hydroxy; hydroxy substituted C.sub.1-10alkyl; C.sub.1-10
alkoxy, such as methoxy or ethoxy; halosubstituted C.sub.1-10
alkoxy; S(O)m alkyl, such as methyl thio, methylsulfinyl or methyl
sulfonyl; S(O)m aryl; amino, mono & di-C.sub.1-10 alkyl
substituted amino; C.sub.1-10 alkyl; halosubstituted C.sub.1-10
alkyl, such as CF3; CHO, C(O)C.sub.1-10 alkyl, C(O) aryl,
C(0).sub.2R.sub.8, wherein R8 is C.sub.1-10 alkyl, aryl, or
arylalkyl; C(O)NR.sub.9R.sub.11; cyano; S(O).sub.2
NR.sub.9R.sub.11; N(R.sub.10)C(O)R.sub.6; N(R.sub.10)C(O)
NR.sub.9R.sub.11; N(R.sub.10)C(O)OR.sub.6; or
N(R.sub.10)S(O).sub.2R.sub.6.
[0031] Suitably, R.sub.4 is optionally substituted C.sub.1-10
alkyl, (CR.sub.10R.sub.20)n--C.ident.C--R.sub.5,
(CR.sub.10R.sub.20).sub.nC(R.su- b.10).dbd.C(R.sub.7).sub.2;
wherein n is an integer having a value of 1 to 4. Preferably n is
1.
[0032] When R.sub.4 is an optionally substituted C.sub.1-10 alkyl,
the alkyl moiety may be straight or branched, and may be
substituted one or more times, independently by halogen, such as
fluorine; hydroxy; C.sub.1-10 alkoxy; S(O)m alkyl, wherein m is 0,
1 or 2; amino, mono & di-substituted amino, such as
NR.sub.9R.sub.11 group; wherein R.sub.9 and R.sub.11 are as defined
below or R.sub.9 and R.sub.11 together with the nitrogen to which
they are attached cyclize to form a 5 to 7 membered ring which
optionally includes an additional heteroatom selected from O/N/S;
--O(CR.sub.10R.sub.20).sub.sO-- wherein s is an integer having a
value of 2 to 4 and both oxygens are attached to the same carbon in
R.sub.4; --S(CR.sub.10R.sub.20).sub.sS-- wherein s is as previously
defined and both sulfurs are attached to the same carbon in
R.sub.4; cycloalkyl, or cycloalkyl alkyl group; a halosubstituted
C.sub.1-4 alkyl, such as CF.sub.3; an optionally substituted aryl,
such as phenyl, or an optionally substituted arylalkyl, such as
benzyl or phenethyl, heteroaryl, or heteroarylalkyl, wherein these
aryl or heteoraryl moieties may also be substituted one to two
times by halogen; hydroxy; hydroxy substituted alkyl; C.sub.1-10
alkoxy; S(O)m alkyl; amino, mono & di-C.sub.1-4 alkyl
substituted amino, such as in the NR.sub.9R.sub.11 group (wherein
R.sub.9 and R.sub.11 are as defined below); C.sub.1-10 alkyl, or
CF.sub.3.
[0033] Preferably, R.sub.4 is a C.sub.14 alkyl, such as isobutyl,
or an alkenyl, such as isobutenyl.
[0034] Suitably, R.sub.9 and R.sub.11 are independently hydrogen,
C.sub.1-10 alkyl, aryl, or arylalkyl.
[0035] Suitably, R.sub.10 and R.sub.20 are independently hydrogen
or C.sub.1-4 alkyl.
[0036] Suitably, R.sub.5 is hydrogen, C.sub.1-10 alkyl, aryl,
arylalkyl, heteroaryl, heteroarylalkyl, C(O).sub.2R.sub.6,
C(O)R.sub.6. Preferably R.sub.5 is hydrogen, C(O).sub.2R.sub.6, or
a heteroaryl ring, and preferably R.sub.6 therein is a C.sub.1-4
alkyl, such as methyl. If R.sub.5 is a heteroaryl ring, it is
preferably a 2-, 3,- or 4-pyridyl. The alkyl, aryl, arylalkyl,
heteroaryl, and heteroarylalkyl may be optionally substituted as
herein defined.
[0037] Suitably, R.sub.6 is C.sub.1-10 alkyl, aryl, arylalkyl,
heteroaryl, heteroarylalkyl, heterocyclic, or heterocyclicalkyl.
The aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclic, and
heterocyclicalkyl moieties may be optionally substituted as herein
defined.
[0038] Suitably, R.sub.7 is independently hydrogen, C.sub.1-10
alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclic,
or heterocyclicalkyl. The alkyl, aryl, arylalkyl, heteroaryl,
heteroarylalkyl, heterocyclic, and heterocyclicalkyl moieties may
be optionally substituted as herein defined.
[0039] Suitable pharmaceutically acceptable salts are well known to
those skilled in the art and include basic salts of inorganic and
organic acids, such as hydrochloric acid, hydrobromic acid,
sulphuric acid, phosphoric acid, methane sulphonic acid, ethane
sulphonic acid, acetic acid, malic acid, tartaric acid, citric
acid, lactic acid, oxalic acid, succinic acid, fumaric acid, maleic
acid, benzoic acid, salicylic acid, phenylacetic acid and mandelic
acid.
[0040] The following terms, as used herein, refer to:
[0041] "halo"--all halogens, that is chloro, fluoro, bromo and
iodo;
[0042] "C.sub.1-10 alkyl" or "alkyl"--both straight and branched
chain radicals of 1 to 10 carbon atoms, unless the chain length is
otherwise limited, including, but not limited to, methyl, ethyl,
n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl,
and the like;
[0043] "cycloalkyl" is used herein to mean cyclic radicals,
preferably of 3 to 8 carbons, including but not limited to
cyclopropyl, cyclopentyl, cyclohexyl, and the like;
[0044] "alkenyl" is used herein at all occurrences to mean straight
or branched chain radical of 2-10 carbon atoms, unless the chain
length is limited thereto, including, but not limited to ethenyl,
1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl
and the like;
[0045] "aryl"--phenyl and naphthyl;
[0046] "heteroaryl" (on its own or in any combination, such as
"heteroaryloxy", or "heteroaryl alkyl")--a 5-10 membered aromatic
ring system in which one or more rings contain one or more
heteroatoms selected from the group consisting of N, O or S, such
as, but not limited, to pyrrole, pyrazole, furan, thiophene,
quinoline, isoquinoline, quinazolinyl, pyridine, pyrimidine,
oxazole, thiazole, thiadiazole, triazole, imidazole, or
benzimidazole;
[0047] "heterocyclic" (on its own or in any combination, such as
"heterocyclylalkyl")--a saturated or partially unsaturated 4-10
membered ring system in which one or more rings contain one or more
heteroatoms selected from the group consisting of N, O, or S; such
as, but not limited to, pyrrolidine, piperidine, piperazine,
morpholine, tetrahydropyran, or imidazolidine;
[0048] The term "aralkyl" or "heteroarylalkyl" or
"heterocyclicalkyl" is used herein to mean C.sub.1-4 alkyl as
defined above attached to an aryl, heteroaryl or heterocyclic
moiety as also defined herein unless otherwise indicate;
[0049] "sulfinyl"--the oxide S(O) of the corresponding sulfide, the
term "thio" refers to the sulfide, and the term "sulfonyl" refers
to the fully oxidized S(o).sub.2 moiety;
[0050] "aroyl"--a C(O)Ar, wherein Ar is as phenyl, naphthyl, or
aryl alkyl derivative such as defined above, such group include but
are note limited to benzyl and phenethyl;.
[0051] "alkanoyl"--a C(O)C.sub.1-10 alkyl wherein the alkyl is as
defined above.
[0052] "optionally substituted" unless specifically defined herein,
shall mean such groups as halogen, such as fluorine, chlorine,
bromine or iodine; hydroxy; hydroxy substituted C.sub.1-10alkyl;
C.sub.1-10 alkoxy, such as methoxy or ethoxy; S(O)m alkyl, wherein
m is 0, 1 or 2, such as methyl thio, methylsulfinyl or methyl
sulfonyl; amino, mono & di-substituted amino, such as in
NR.sub.9R.sub.11 group; C.sub.1-10 alkyl, cycloalkyl, or cycloalkyl
alkyl group, such as methyl, ethyl, propyl, isopropyl, t-butyl,
etc. or cyclopropyl methyl; halosubstituted C.sub.1-10 alkyl, such
as CF.sub.3; an optionally substituted aryl, such as phenyl, or an
optionally substituted arylalkyl, such as benzyl or phenethyl,
wherein these aryl moieties may also be substituted one to two
times by halogen; hydroxy; hydroxy substituted alkyl; C.sub.1-10
alkoxy; S(O)m alkyl; amino, mono & di-C.sub.1-4 alkyl
substituted amino, such as in the NR.sub.9R.sub.11 group;
C.sub.1-10 alkyl, or CF.sub.3.
[0053] As used herein, the terms "alkyl lysophospholipid" (ALP),
"alkyl lysophospholipid analog" and "ether-linked phospholipid and
analogs thereof" are well known terms to those of skill in the art
and need no additional definitions. Generally these terms will
encompass compounds that are ether linked phospholipids with alkyl
chains in the sn-1 position of the glycerol backbone. These
compounds may be derivatives of
1-O-alkyl-2-O-methyl-rac-glycero-3-phosphocholine, derivatives of
1-O-hexadecyl-2-acetal-sn-glycero-3-phosphocholine (PAF),
derivatives of ester-lysophospholipid (2-PLC), or derivatives of
lysophosphatidylcholine (2-LPC). Ether-lysophospholipids are also
referred to as alkyllysoPC's, or ALPs; alkylphosphocholines are
also referred to as APCs; a hexadecyl phosphocholine is referred to
as HPC. Suitable compounds encompassed by this term, include but
are not limited to, those described in Schick et al., Lipids, 22
(11) 904-910 (1987); such as those described in Morris-Natschke et
al., J. Med. Chem., 29 (10) 2114-2117 (1986); Goto et al.,
Anticancer Research, 14:357362 (1994); Langen et al., Anticancer
Research, 12:2109-2112 (1992); Volger et al., Lipids, 28(6) 511-516
(1993); Workman et al., Biochem. Pharm., 41(2) 319-22 (1991);
Kohler et al., Inflammation, 17(3), 245-261 (1993); Brachwitz et
al., Lipids, 22 (11) 897-903 (1987); Berdel, W., Lipids, 22(11)
970-973 (1987); Danhauser et al., Lipids, 22(11) 911-915 (1987);
Danhauser-Riedl et al., J. Lipid Metabolism, 2, 271-280 (1990);
Lohmeyer et al., Biochem. Pharm., 45 (1) 77086 (1993); Ries, et
al., Chemistry and Physics of Lipids, 61, 225-234 (1992); as well
as derivatives having sulfur and nitrogen linkages, such as
Guivisdalsky et al., J. Med. Chem., 33 (9), 2614-2621 (1990); and
Pignol et al., Anti-Cancer Drugs, 3, 599-608 (1992) whose
disclosures are incorporated by reference in their entirety
herein.
[0054] It is recognized that the compounds of the present invention
may exist as stereoisomers, regioisomers, or diastereoisomers.
These compounds may contain one or more asymmetric carbon atoms and
may exist in racemic and optically active forms. All of these
compounds are included within the scope of the present
invention.
[0055] Specifically exemplified compounds of Formula (I) are:
[0056]
(3RS,4RS)-4-(Isobutoxy)-3-(triphenylmethylamino)azetidin-2-one
[0057]
(3R,4R)-4-(Isobutoxy)-3-(triphenylmethylamino)azetidin-2-one
[0058]
(3S,4S)-4-(Isobutoxy)-3-(triphenylmethylamino)azetidin-2-one
[0059]
(3R,4R)-4-(Isobutylthio)-3-(triphenylmethylamino)azetidin-2-one
[0060]
(3R,4R)-4-(Isobutylsulfonyl)-3-(triphenylmethylamino)azetidin-2-one
[0061]
(3S,4R)-4-(Isobutoxy)-3-(triphenylmethylamino)azetidin-2-one
[0062]
(3S,4R)-4-(Propoxy)-3-(triphenylmethylamino)azetidin-2-one
[0063]
(3S,4S)-4-(Propoxy)-3-(triphenylmethylamino)azetidin-2-one
[0064]
(3S,4R)-4-(Benzyloxy)-3-(triphenylmethylamino)azetidin-2-one
[0065]
(3S,4S)-4-(Benzyloxy)-3-(triphenylmethylamino)azetidin-2-one
[0066] (3S,4R)-4-Methoxy-3-(triphenylmethylamino)azetidin-2-one
[0067]
(3S,4R)-4-(Isobutenyloxy)-3-(triphenylmethylamino)azetidin-2-one
[0068]
(3S,4R)-4-Octyloxy-3-(triphenylmethylamino)azetidin-2-one
[0069] (3S,4R)-4-Phenoxy-3-(triphenylmethylamino)azetidin-2-one
[0070] (3S,4S)-4-Phenoxy-3-(triphenylmethylamino)azetidin-2-one
[0071]
(3S,4R)-3-[[(4-Iodophenyl)diphenylmethyl]amino]-4-(isobutoxy)azetid-
in-2-one
[0072]
(3S,4S)-4-[3-(Methoxycarbonyl)propoxy]-3-(triphenylmethylamino)azet-
idin-2-one
[0073]
(3S,4R)-4-[[2-(3-Pyridylmethyl)-1,3-dithian-2-yl]methoxy]-3-(triphe-
nylmethylamino)-azetidin-2-one
[0074]
(3S,4S)-4-(Prop-2-ynyloxy)-3-(triphenylmethylamino)azetidin-2-one
[0075] Methyl
4-[(3S,4S)-2-oxo-3-(triphenylmethylamino)azetidin-4-yloxy]bu-
t-2-ynoate
[0076] Methyl
4-[(3S,4R)-2-oxo-3-(triphenylmethylamino)azetidin-4-yloxy]bu-
t-2-ynoate
[0077]
(3S,4R)-4-[(2(5H)Furanon-4-yl)methoxy]-3-(triphenylmethylamino)azet-
idin-2-one
[0078] (S)-3-(Triphenylmethylamino)azetidin-2-one
[0079] (RS)-3-(Triphenylmethylamino)azetidin-2-one
[0080]
(3R,4R)-4-(Methylsulfonyl)-3-(triphenylmethylamino)azetidin-2-one
[0081] Compounds of formula (I) where 4-(X--R.sup.4) is
4-(O--R.sup.4) or 4-(S--R.sup.4) can be prepared according to
Scheme I from 1, where R.sup.3 is defined as in formula (I) and Y
is a suitable leaving group such as methylsulfonyl, acyloxy, or
chloro. Compounds 1, where Y is methylsulfonyl, are obtained as
described in J. Chem. Soc. Perkin I, 447, 1976 whose disclosure is
incorporated herein by reference. The displacement of group Y in 1
with HO-R.sup.4 or HS-R.sup.4 may be carried out with a suitable
catalyst such as zinc acetate in a suitable solvent such as toluene
at a suitable temperature such at 90.degree. C. Alternatively, with
phenols, the displacement may be carried out in the presence of a
suitable base, such as aqueous sodium hydroxide, in a suitable
solvent such as acetone.
[0082] Compound 3 where 4-(X--R.sup.4) is 4-(SO--R.sup.4) and
4-(SO.sub.2--R.sup.4) and R.sup.3, R.sup.4 are defined as in
formula (I), can be obtained by further oxidation of 2 where
4-(X--R.sup.4) is 4-(S--R.sup.4) with a suitable organic oxidizing
agent such as m-chloroperbenzoic, peracetic acid, etc. in a
suitable solvent such as dichloromethane, or by further oxidation
with a suitable inorganic oxidizing agent such as sodium periodate
or potassium permangante in a solvent such as water, acetone or
acetic acid. 2
[0083] Alternatively, compounds of formula (I) may be prepared
according to Scheme 11 from 4. Compound 4, wherein X is O--R.sup.4
or S--R.sup.4 and R.sup.4 is as defined in formula (I) and R.sup.1
is hydrogen, can be prepared from 4-acetoxy or
4-benzoyloxy-azetidin-2-one as described in Synthetic
Communications, 24, 131-135 (1994) whose disclosure is incorporated
herein by reference. Treatment of 4, where R.sup.1 is hydrogen,
with a suitable silylating group such as tert-butyldimethylsilyl
chloride and a suitable base such as triethylamine in a suitable
solvent such as tetrahydrofuran gives 4 where R.sup.1 is
tert-butyldimethylsilyl. Treatment of 4, where R.sup.1 is
tert-butyldimethylsilyl, with a suitable base such as lithium
diisopropylamide in a suitable solvent such as tetrahydrofuran at a
suitable temperature such as -50.degree. C., followed by addition
to a solution of a suitable azidating reagent such as tosyl azide
in a suitable solvent such as tetrahydrofuran, followed by
treatment with trimethylsilyl chloride gives 5 where R.sup.1 is
tert-butyldimethylsilyl. Reduction of the azido group in 5, where
R.sub.1 is tert-butyldimethylsilyl, with a suitable reducing agent
such as hydrogen sulfide in a suitable solvent such as
dichloromethane containing a suitable base such as triethylamine
gives 6 where R.sup.1 is tert-butyldimethylsilyl. Treatment of 6,
where R.sub.1 is tert-butyldimethylsilyl, with a suitable
alkylating agent R.sup.3Z, where R.sup.3 is as defined in formula
(I) and Z is a suitable leaving group such as chloro, in a suitable
solvent such as dimethylformamide containing a suitable base such
as diisopropylethylamine, gives 2 where R.sup.1 is
tert-butyldimethylsilyl. Treatment of 2, where R.sub.1 is
tert-butyldimethylsilyl, with a suitable inorganic fluoride, such
as tetrabutylammonium fluroride, in a suitable solvent such as
tetrahydrofuran and acetic acid gives 2 where R.sub.1 is hydrogen.
3
[0084] Alternatively, compounds II-5 may be prepared using [2+2]
cycloaddition reactions, for example, by following the general
procedures described in Cama et. al., Tetrahedron Letters, 4233,
1978, whose disclosure is incorporated herein by reference.
[0085] Synthetic Chemistry
[0086] The invention will now be described by reference to the
following examples which are merely illustrative and are not to be
construed as a limitation of the scope of the present invention.
All temperatures are given in degrees centigrade, all solvents are
highest available purity and all reactions run under anhydrous
conditions in an argon atmosphere unless otherwise indicated.
[0087] In the Examples, all temperatures are in degrees Centigrade
(.degree. C.). Mass spectra were performed upon a VG Zab mass
spectrometer using fast atom bombardment, unless otherwise
indicated. .sup.1H-NMR (hereinafter "NMR") spectra were recorded at
250 MHz using a Bruker AM 250 or Am 400 spectrometer.
Multiplicities indicated are: s=singlet, d=doublet, t=triplet,
q=quartet, m=multiplet and br indicates a broad signal. Sat.
indicates a saturated solution, eq indicates the proportion of a
molar equivalent of reagent relative to the principal reactant.
[0088] Flash chromatography is run over Merck Silica gel 60
(230-400 mesh).
EXAMPLE 1
Preparation of
(3RS,4RS)-4-(Isobutoxy)-3-(triphenylmethylamino)azetidin-2--
one
[0089] a). 4-(Isobutoxy)azetidin-2-one
[0090] A mixture of 4-(benzoyloxy)azetidin-2-one (15.2 g, 80 mmol)
in toluene (150 mL) was treated with isobutanol (12 g, 0.16 mol),
triethylamine (16 g, 0.16 mol) and palladium acetate (3.6 g, 16
mmol), stirred in an ice bath for several hours, allowed to warm to
RT and stirred for 16 h. The mixture was filtered through Supercel,
concentrated and the residue was chromatographed on silica gel
eluted with 10-35% ethyl acetate:hexane. Fractions containing the
product were combined, concentrated and rechromatographed on silica
gel eluted with 10-25% ethyl acetate:hexane to give the title
compound (4.9 g).
[0091] b).
1-tert-butyldimethylsilyl-4-(isobutoxy)azetidin-2-one
[0092] A solution of 4-(isobutoxy)azetidin-2-one (4.8 g, 33 mmol)
in tetrahydrofuran (50 mL) was stirred in an ice bath and treated
with triethylamine (6.6 mL, 66 mmol) followed by dropwise addition
of a solution of tert-butyldimethylsilyl chloride (6.5 g, 43 mmol)
in tetrahydrofuran (15 mL). The mixture was stirred for 5 h in the
cold and stored in the refrigerator for 64 h. The mixture was
poured into cold water, extracted with ethyl acetate, and the
combined organic phase was washed with brine, dried (magnesium
sulfate), and concentrated. The residue was chromatographed on
silica gel eluted with 10% ethyl acetate:hexane. Fractions
containing the product were pooled and concentrated to give the
title compound (6.6 g, 79%).
[0093] c).
(3RS,4RS)-3-azido-1-(tert-butyldimethylsilyl)-4-(isobutoxy)azet-
idin-2-one
[0094] A solution of N-(isopropyl)cyclohexylamine (1.5 g, 10 mmol)
in tetrahydrofuran (18 mL) was cooled to -15.degree. C. and 2M
butyllithium (48 mL, 10 mmol) was added dropwise. The reaction
mixture was stirred for 40 min, the temperature was lowered to
-70.degree. C., and the mixture was treated dropwise over 10 min
with a solution of
1-(tert-butyldimethylsilyl)-4-(isobutoxy)-azetidin-2-one (1.8 g, 7
mmol) in tetrahydrofuran (7 mL). The mixture was stirred for 1 h,
transferred to a jacketed addition funnel maintained at -70.degree.
C., and added over 40 min to a solution of p-toluenesulfonyl azide
(1.8 g, 9 mmol) in tetrahydrofuran (8 mL) containing
hexamethylphosphoramide (2 mL) maintained at -70.degree. C. The
reaction was stirred for 1 h and at -50.degree. C. for 4.5 h. The
mixture was stirred at -28.degree. C. for 16 h, and trimethylsilyl
chloride (5 mL) was added and the mixture was stirred at RT for 45
min. The mixture was diluted with water and extracted with ethyl
acetate. The combined organic phases were washed with brine, dried
(magnesium sulfate) and concentrated. The residue was
chromatographed on silica gel eluted with 10% ethyl acetate:hexane
to give the title compound (0.6 g, 30%). MS(ES) m/e 299
[M+H].sup.+.
[0095] d).
(3RS,4RS)-3-amino-1-(tert-butyldimethylsilyl)-4-(isobutoxy)azet-
idin-2-one
[0096] A solution of
(3RS,4RS)-3-azido-1-(tert-butyldimethylsilyl)-4-(isob-
utoxy)azetidin-2-one (0.2 g, 0.67 mmol) in dichloromethane (15 mL)
containing triethylamine (0.07 g, 0.7 mmol) was cooled in an ice
bath and hydrogen sulfide was bubbled through the solution gently
for 10 min. The mixture was stirred in the cold for 4 h,
concentrated, and then treated with dichloromethane and
concentrated four times to give the title compound.
[0097] e).
(3RS,4RS)-1-(tert-butyldimethylsily)-4-(isobutoxy)-3-(triphenyl-
methylamino)azetidin-2-one
[0098] A solution of
(3RS,4RS)-3-amino-1-(tert-butyldimethylsilyl)-4-(isob-
utoxy)azetidin-2-one (0.2 g) was dissolved in dimethylformamide (8
mL), cooled in an ice bath, and treated with diisopropylethylamine
(0.1 mL) followed by trityl chloride (167 mg, 0.6 mmol). The
mixture was stirred for 18 h, diluted with water (40 mL) and
extracted with ethyl acetate. The combined organic phase was dried
(magnesium sulfate), concentrated, and the residue was
chromatographed on silica gel eluted with 20% ethyl acetate:hexane
to give the title compound. MS(ES) m/e 515 [M+H].sup.+.
[0099] f).
(3RS,4RS)-4-(Isobutoxy)-3-(triphenylmethylamino)azetidin-2-one
[0100] A solution of
(3RS,4RS)-1-(tert-butyldimethylsilyl)-4-(isobutoxy)-3-
-(triphenylmethylamino)azetidin-2-one (160 mg, 0.3 mmol) in
tetrahydrofuran (4 mL) was cooled in an ice bath and treated with
acetic acid (25 mg, 0.4 mmol) followed by dropwise addition of 1 M
tetrabutylammonium fluoride (0.6 mL, 0.6 mmol). The mixture was
stirred for 20 min and passed through silica gel (10 g) eluted with
ethyl acetate. The eluate was concentrated and the residue was
chromatographed on silica gel eluted with 20% ethyl acetate:hexane
to give the title compound. MS(ES) m/e 423 [M+Na].sup.+.
EXAMPLE 2
[0101] Preparation of (3R,4R)- and
(3S,4S)-4-(Isobutoxy)-3-(triphenylmethy- lamino)azetidin-2-one
[0102]
(3RS,4RS)-4-(Isobutoxy)-3-(triphenylmethylamino)azetidin-2-one was
resolved by HPLC (Chiralcel OD, 21.times.250 mm, 10 mL/min,
gradient, A:ethanol B:hexane, 0.5-2.5% A during 20 min, UV
detection at 254 nm) to afford the title compounds:
[0103]
(3R,4R)-4-(isobutoxy)-3-(triphenylmethylamino)azetidin-2-one,
t.sub.R 35 min. MS(ES) m/e 801 [2M+H].sup.+, and
[0104]
(3S,4S)-4-(isobutoxy)-3-(triphenylmethylamino)azetidin-2-one,
t.sub.R 39.9 min. MS(ES) m/e 801 [2M+H].sup.+.
EXAMPLE 3
[0105] Preparation of
(3R,4R)-4-(Isobutylthio)-3-(triphenylmethylamino)aze-
tidin-2-one
[0106] A solution of zinc acetate (0.7 g, 3 mmol) in toluene (15
mL) and 2-methyl-propanethiol (0.72 g, 8 mmol) was refluxed for 45
min in an apparatus equipped with a Dean-Stark trap to azeotrope
water.
(3R,4R)-4-(Methylsulfonyl)-3-(triphenylmethylamino)azetidin-2-one
(1.5 g, 36 mmol) was added and the mixture was heated to 90.degree.
C. for 2 h. The mixture was concentrated and the residue was
triturated with ethyl acetate and the insoluble material was
removed by filtration. The filtrate was concentrated and the
residue was chromatographed on silica gel eluted with 20% ethyl
acetate:hexane. The fractions containing the product were combined,
concentrated to give the title compound. MS(ES) mle 417
[M+H].sup.+.
EXAMPLE 4
[0107] Preparation of
(3R,4R)-4-(Isobutylsulfonyl)-3-(triphenylmethylamino-
)azetidin-2-one
[0108] A solution of
(3R,4R)-4-(isobutylthio)-3-(triphenylmethylamino)azet- idin-2-one
(50 mg, 0.12 mmol) in dichloromethane (2 mL) was cooled in an ice
bath and treated with m-chloroperbenzoic acid (44 mg, 0.25 mmol).
The mixture was stirred for 2.5 h in the cold and partitioned
between 5% sodium carbonate (5 mL) and dichloromethane. The organic
phase was washed with 5% sodium carbonate and with brine, dried
(magnesium sulfate), filtered and concentrated. The residue was
chromatographed on silica gel eluted with 20% ethyl acetate:hexane
and fractions containing the product were pooled and concentrated
to give the title compound (25 mg, 47%). MS(ES) m/e 447
[M-H].sup.+.
EXAMPLES 5-13
[0109] The following compound have been prepared using the
procedure of Example 3, except substituting isobutanol, propanol,
benzyl alcohol, methanol, isobutenol, or octanol for
2-methyl-propanethiol gave:
Example 5
[0110]
(3S,4S)-4-(Isobutoxy)-3-(triphenylmethylamino)azetidin-2-one:
[0111] MS(ES) m/e 801 [2M+H].sup.+;
Example 6
[0112]
(3S,4R)-4-(Isobutoxy)-3-(triphenylmethylamino)azetidin-2-one:
[0113] MS(ES) m/e 401 [M+H].sup.+;
Example 7
[0114]
(3S,4R)-4-(Propoxy)-3-(triphenylmethylamino)azetidin-2-one:
[0115] MS(ES) m/e 387 [M+H].sup.+;
Example 8
[0116]
(3S,4S)-4-(Propoxy)-3-(triphenylmethylamino)azetidin-2-one:
[0117] MS(ES) m/e 773 [2M+H].sup.+;
Example 9
[0118]
(3S,4R)-4-(Benzyloxy)-3-(triphenylmethylamino)azetidin-2-one:
[0119] MS(ES) m/e 435 [M+H].sup.+;
Example 10
[0120]
(3S,4S)-4-(Benzyloxy)-3-(triphenylmethylamino)azetidin-2-one:
[0121] MS(ES) m/e 435 [M+H].sup.+;
Example 11
[0122]
(3S,4R)-4-Methoxy-3-(triphenylmethylamino)azetidin-2-one:
[0123] MS(ES) m/e 359 [M+H].sup.+;
Example 12
[0124]
(3S,4R)-4-(Isobutenyloxy)-3-(triphenylmethylamino)azetidin-2-one:
[0125] MS(ES) m/e 399 [M+H].sup.+;
Example 13
[0126]
(3S,4R)-4-Octyloxy-3-(triphenylmethylamino)azetidin-2-one:
[0127] MS(ES) m/e 457 [M+H].sup.+;
EXAMPLES 14-15
Preparation of
(3S,4R)-4-phenoxy-3-(triphenylmethylamino)azetidin-2-and
(3S,4S)-4-phenoxy-3-(triphenylmethylamino)azetidin-2-one
[0128] A solution of phenol (0.3 g, 3.2 mmol) in acetone (3 mL) was
treated with 1N sodium hydroxide (3.2 mL, 3.2 mmol), stirred 10 min
and treated dropwise with a solution of
(3R,4R)-4-methylsulfonyl-3-(triphenyl- methylamino)azetidin-2-one
(1.2 g, 3 mmol) in acetone (2 mL). The mixture was stirred for 1.5
h, partitioned between water and diethyl ether, and the combined
organic phase was washed with brine, dried (magnesium sulfate), and
concentrated. The residue was chromatographed on silica gel eluted
with 20% ethyl acetate:hexane to give the title compounds:
[0129] (3S,4R)-4-phenoxy-3-(triphenylmethylamino)azetidin-2-one:
MS(ES) m/e 443 [M+Na].sup.+; 421 [M+H].sup.+;
[0130] (3S,4S)-4-phenoxy-3-(triphenylmethylamino)azetidin-2-one:
MS(ES) m/e 841 [2M+H].sup.+; 419 [M-H].sup.-.
EXAMPLE 16
[0131] Preparation of
(3S,4R)-3-[[(4-iodophenyl)diphenylmethyl]amino]-4-(i-
sobutoxy)azetidin-2-one
[0132] a). (3S,4R)-3-Amino-4-(isobutoxy)azetidin-2-one
para-toluenesulfonate
[0133] A solution of para-toluenesulfonic acid hydrate (190 mg) in
acetone (20 mL) was added to a solution of
(3S,4R)-4-isobutoxy-3-(triphenylmethyl- amino)azetidin-2-one (400
mg, 1 mmol) in acetone (20 mL), stirred for 1 h, and concentrated.
The residue was triturated with diethyl ether and the resulting
solid isolated by filtration to give the title compound (180 mg,
55%).
[0134] b).
(3S,4R)-3-[[(4-iodophenyl)diphenylmethyl]amino]-4-(isobutoxy)az-
etidin-2-one
[0135] A solution of (3S,4R)-3-amino-4-(isobutoxy)azetidin-2-one
para-toluenesulfonate (180 mg, 0.54 mmol) in acetone (8 mL)
containing diisopropylethylamine (1.1 mmol) was treated with a
solution of (4-iodophenyl)diphenylmethyl chloride (218 mg, 0.54
mmol), prepared as described by Tschitschibabin, Chem. Ber. 44, 450
(1911), in acetone (98 mL). The solution was stirred for 6 h and
partitioned between water and dichloromethane. The combined organic
phases were dried (magnesium sulfate), concentrated, and the
residue was chromatographed on silica gel eluted with 15% ethyl
acetate:hexane to give the title compound (190 mg, 67%). MS(ES) m/e
527 [M+H].sup.+.
EXAMPLE 17
[0136] Preparation of
(3S,4S)-4-[3-(Methoxycarbonyl)propoxy]-3-(triphenylm-
ethylamino)azetidin-2-one
[0137] a).
(3S,4S)-3-Amino-4-[3-(methoxycarbonyl)propoxy]azetidin-2-one
[0138] A solution of methyl
4-[(3S,4S)-2-oxo-3-(triphenylmethylamino)azeti-
din-4-yloxy]but-2-ynoate (75mg, 0.17 mmol) in absolute ethanol (6
mL) was treated with 10% palladium-on-carbon (35mg) and stirred
under hydrogen overnight. The catalyst was removed by filtration
and the filtrate concentrated. The residual oil was purified on by
preparative thin layer chromatography (Whatman, silica gel 60A,
20.times.20 cm, 1000 um, 20% ethyl acetate:hexane). The origin band
contained the title compound (30mg). MS(ES) m/e 203.0 [M+H].
[0139] b).
(3S,4S)-4-[3-(Methoxycarbonyl)propoxy]-3-(triphenylmethylamino)-
azetidin-2-one
[0140] (3S,4S)-3-Amino-4-[3-(methoxycarbonyl)propoxy]azetidin-2-one
(30mg, 0.148 mmol) was dissolved in dry dichloromethane (2 mL) and
treated with triphenylmethyl chloride (41 mg, 0.148 mmol) followed
by diisopropylethylamine (19 mg, 0.148 mmol). The solution was
stirred at RT under argon for 5 h, diluted with water and extracted
with dichloromethane. The organic phases were combined, washed with
water and brine, dried (magnesium sulfate), filtered, and
concentrated. The residue was purified by preparative thin layer
chromatography (Whatman, silica gel 60A, 20.times.20 cm, 1000 um,
40% ethyl acetate:hexane) to give the title compound (7mg). MS(ES)
m/e 445.2 [M+H].
EXAMPLES 18-21
[0141] The following compounds were prepared using the general
procedure of Example 3, except substituting:
[0142] Examples 18 and 19: methyl 4-hydroxy-2-butynoate (Zh.
Obshch. Khim. 66, 106, 1996),
[0143] Example 20: 4-hydroxymethyl-2(5H)-furanone, (J. Chem. Res.,
Synop. 222, 1986), or
[0144] Example 21: 2-(3-pyridylmethyl)-1,3-dithianyl-2-methanol for
2-methyl-propanethiol used therein.
[0145] Preparation of
Example 18
[0146] Methyl
4-[(3S,4S)-2-oxo-3-(triphenylmethylamino)azetidin-4-yloxy]bu-
t-2-ynoate
[0147] Methyl
4-[(3S,4S)-2-oxo-3-(triphenylmethylamino)azetidin-4-yloxy]bu-
t-2-ynoate: .sup.1H NMR(270 MHz, CDCl.sub.3) .delta. 3.54 (d, J=17
Hz, 1H), 3.77 (s, 3H), 3.83 (d, J=17 Hz, 1H), 4.08 (s, 1H), 4.11
(d, J=1.1 Hz, 1H), 6.39 (s, 1H), 7.19-7.53 (m, 15H); IR(CHCl.sub.3)
3330, 2240, 1770, 1718, 1477, 1445, 1434, 1260, 1194,
1166,940,759,706 cm.sup.-1.
Example 19
[0148] Methyl
4-[(3S,4R)-2-oxo-3-(triphenylmethylamino)azetidin-4-yloxy]bu-
t-2-ynoate
[0149] Methyl
4-[(3S,4R)-2-oxo-3-(triphenylmethylamino)azetidin-4-yloxy]bu-
t-2-ynoate: 1H NMR(60 MHz, CDCl.sub.3) .delta. 2.9 (d, J=9 Hz, 1H),
3.70 (s, 3H), 3.60 (d, J=17 Hz, 1H), 3.93 (d, J=17 Hz, 1H), 4.0-4.3
(m, 2H), 6.8 (s, 1H), 7.2-7.8 (m, 15H); IR(CHCl.sub.3) 3350, 1775,
1715 cm.sup.-1.
Example 20
[0150]
(3S,4R)-4-[(2(5H)Furanon-4-yl)methoxy]-3-(triphenylmethylamino)azet-
idin-2-one
[0151]
(3S,4R)-4-[(2(5H)furanon-4-yl)methoxy]-3-(triphenylmethylamino)azet-
idin-2-one: .sup.1H NMR(60 MHz, CDCl.sub.3) .delta. 2.8 (d, J=9 Hz,
1H), 3.9 (m, 2H), 4.2 (m, 2H), 4.53 (m, 2H), 5.87 (m, 1H), 7.0-8.7
(m, 17H); IR(CHCl.sub.3) 1785, 1755, 1650 cm.sup.-1.
Example 21
[0152]
(3S,4R)-4-[[2-(3-Pyridylmethyl)-1,3-dithian-2-yl]methox]-3-(triphen-
ylmethylamino)azetidin-2-one
[0153]
(3S,4R)-4-[[2-(3-pyridylmethyl)-1,3-dithian-2-yl]methoxy]-3-(triphe-
nylmethylamino)azetidin-2-one: IR(CHCl.sub.3) 3370, 1775
cm.sup.-1.
[0154] Using analagous procedures to those indicated above, or as
indicated, the following compounds have been syntheized:
Example 22
[0155]
(3S,4S)-4-(Prop-2-ynyloxy)-3-(triphenylmethylamio)azetidin-2-one:
J. Chem. Soc. Perkin Trans. I 2268, 1979;
Example 23
[0156]
(3R,4R)-4-(Methylsulfonyl)-3-(triphenylmethylamino)azetidin-2-one,
J. Chem. Soc. Perkin Trans. I 2268, 1979;
Example 24
[0157]
(3S,4R)-4-(Isobutenyloxy)-3-(triphenylmethylamino)azetidin-2-one,
J. Chem. Soc. Perkin Trans. I 2268, 1979;
Example 25
[0158] (S)-3-(Triphenylmethylamino)azetidin-2-one: Tetr. Letters
30, 3219, 1989.
Example 26
[0159] Using analagous procedures to those noted therein, the
racemic mixture of (RS)-3-(Triphenylmethylamino)azetidin-2-one may
be produced.
[0160] Methods of Treatment
[0161] The compounds of Formula (I) or pharmaceutically acceptable
salts thereof can be used in the manufacture of a medicament for
the prophylactic or therapeutic treatment of an proliferative
disease state in a mammal, preferably a human. Disease states which
could benefit from the inhibition of cell proliferation include,
but are not limited to, rheumatoid arthritis, psoriasis,
atherosclerosis, and cancers such as leukemia or solid tumors.
[0162] Inhibition of CoA-IT and the blockade of proliferation in
diseased cells according to this invention is of therapeutic
benefit in a broad range of diseases or disorders. The invention
herein is therefore useful to treat such disease states both in
humans and in other mammals.
[0163] The compounds of Formula (I) or pharmaceutically acceptable
salts thereof can be used in the treatment of proliferative
diseases either as sole treatment or in combination with other
treatments that also inhibit proliferation. Examples of such
treatments include, but are not limited to, cancer chemotherapeutic
drugs and radiation therapy. Many antiproliferative drugs show
additive or potentiating effectiveness when used in combination
with other antiproliferative drugs, as in the standard therapies of
numerous cancers.
[0164] In order to use a compound of formula (I) or a
pharmaceutically acceptable salt thereof in therapy, it will
normally be formulated into a pharmaceutical composition in
accordance with standard pharmaceutical practice. This invention,
therefore, also relates to a pharmaceutical composition comprising
an effective, non-toxic amount of a compound of formula (I) and a
pharmaceutically acceptable carrier or diluent.
[0165] Compounds of formula (I), pharmaceutically acceptable salts
thereof and pharmaceutical compositions incorporating such may
conveniently be administered by any of the routes conventionally
used for drug administration, for instance, orally, topically,
parenterally or by inhalation. The compounds of formula (I) may be
administered in conventional dosage forms prepared by combining a
compound of formula (I) with standard pharmaceutical carriers
according to conventional procedures. Such pharmaceutically
acceptable carriers or diluents and methods of making are well
known to those of skill in the art, and reference can be found in
such texts as Remington's Pharmaceutical Sciences, 18th Ed.,
Alfonso R. Genarao, Ed., 1990, Mack Publishing Co. and the Handbook
of Pharmaceutical Excipents, APhA Publications, 1986.
[0166] The compounds of formula (I) may also be administered in
conventional dosages in combination with known second
therapeutically active compounds, such as anticancer, or
antiproliferative drugs for instance. These procedures may involve
mixing, granulating and compressing or dissolving the ingredients
as appropriate to the desired preparation. It will be appreciated
that the form and character of the pharmaceutically acceptable
carrier or diluent is dictated by the amount of active ingredient
with which it is to be combined, the route of administration and
other well-known variables. The carrier(s) must be "acceptable" in
the sense of being compatible with the other ingredients of the
formulation and not deleterious to the recipient thereof.
[0167] The pharmaceutical carrier employed may be, for example,
either a solid or liquid. Exemplary of solid carriers are lactose,
terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium
stearate, stearic acid and the like. Exemplary of liquid carriers
are syrup, peanut oil, olive oil, water and the like. Similarly,
the carrier or diluent may include time delay material well known
to the art, such as glyceryl mono-stearate or glyceryl distearate
alone or with a wax.
[0168] A wide variety of pharmaceutical forms can be employed.
Thus, if a solid carrier is used, the preparation can be tableted,
placed in a hard gelatin capsule in powder or pellet form or in the
form of a troche or lozenge. The amount of solid carrier will vary
widely but preferably will be from about 25mg. to about 1 g. When a
liquid carrier is used, the preparation will be in the form of a
syrup, emulsion, soft gelatin capsule, sterile injectable liquid
such as an ampule or nonaqueous liquid suspension.
[0169] Compounds of formula (I) may be administered topically, that
is by non-systemic administration. This includes the application of
a compound of formula (I) externally to the epidermis or the buccal
cavity and the instillation of such a compound into the ear, eye
and nose, such that the compound does not significantly enter the
blood stream. In contrast, systemic administration refers to oral,
intravenous, intraperitoneal and intramuscular administration.
[0170] Formulations suitable for topical administration include
liquid or semi-liquid preparations suitable for penetration through
the skin to the site of inflammation such as liniments, lotions,
creams, ointments or pastes, and drops suitable for administration
to the eye, ear or nose. The active ingredient may comprise, for
topical administration, from 0.001% to 10% w/w, for instance from
1% to 2% by weight of the formulation. It may however comprise as
much as 10% w/w but preferably will comprise less than 5% w/w, more
preferably from 0.1% to 1% w/w of the formulation.
[0171] Lotions according to the present invention include those
suitable for application to the skin or eye. An eye lotion may
comprise a sterile aqueous solution optionally containing a
bactericide and may be prepared by methods similar to those for the
preparation of drops. Lotions or liniments for application to the
skin may also include an agent to hasten drying and to cool the
skin, such as an alcohol or acetone, and/or a moisturizer such as
glycerol or an oil such as castor oil or arachis oil.
[0172] Creams, ointments or pastes according to the present
invention are semi-solid formulations of the active ingredient for
external application. They may be made by mixing the active
ingredient in finely-divided or powdered form, alone or in solution
or suspension in an aqueous or non-aqueous fluid, with the aid of
suitable machinery, with a greasy or non-greasy base. The base may
comprise hydrocarbons such as hard, soft or liquid paraffin,
glycerol, beeswax, a metallic soap; a mucilage; an oil of natural
origin such as almond, corn, arachis, castor or olive oil; wool fat
or its derivatives or a fatty acid such as steric or oleic acid
together with an alcohol such as propylene glycol or a macrogel.
The formulation may incorporate any suitable surface active agent
such as an anionic, cationic or non-ionic surfactant such as a
sorbitan esteror a polyoxyethylene derivative thereof. Suspending
agents such as natural gums, cellulose derivatives or inorganic
materials such as silicaceous silicas, and other ingredients such
as lanolin, may also be included.
[0173] Drops according to the present invention may comprise
sterile aqueous or oily solutions or suspensions and may be
prepared by dissolving the active ingredient in a suitable aqueous
solution of a bactericidal and/or fungicidal agent and/or any other
suitable preservative, and preferably including a surface active
agent. The resulting solution may then be clarified by filtration,
transferred to a suitable container which is then sealed and
sterilized by autoclaving or maintaining at 98-100.degree. C. for
half an hour. Alternatively, the solution may be sterilized by
filtration and transferred to the container by an aseptic
technique. Examples of bactericidal and fungicidal agents suitable
for inclusion in the drops are phenylmercuric nitrate or acetate
(0.002%), benzalkonium chloride (0.01%) and chlorhexidine acetate
(0.01%). Suitable solvents for the preparation of an oily solution
include glycerol, diluted alcohol and propylene glycol.
[0174] Each dosage unit for oral administration contains preferably
from 1 to 250 mg (and for parenteral administration contains
preferably from 0.1 to 25 mg) of a compound of formula (I) or a
pharmaceutically acceptable salt thereof calculated as the free
base.
[0175] The pharmaceutically acceptable compounds of the invention
will normally be administered to a subject in a daily dosage
regimen. For an adult patient this may be, for example, an oral
dose of between 1 mg and 500 mg, preferably between 1 mg and 250
mg, or an intravenous, subcutaneous, or intramuscular dose of
between 0.1 mg and 100 mg, preferably between 0.1 mg and 25 mg, of
the compound of the Formula (I) or a pharmaceutically acceptable
salt thereof calculated as the free base, the compound being
administered from 1 to 4 times per day.
[0176] The choice of form for administration, as well as effective
dosages, will vary depending, inter alia, on the condition being
treated. The choice of mode of administration and dosage is within
the skill of the art.
[0177] Biological Methods:
[0178] To determine activity of the compounds of Formula (I)
various cellular assays can be used to determine in vitro activity.
Described herein is an in vitro assay for CoA-IT enzyme activity.
Several assays for measuring cell proliferation and/or apoptosis
are also described, both for in vitro and ex vivo.
In Vitro Assays
[0179] Assay: CoA-IT Activity
[0180] The following is a method to measure CoA-IT activity and the
effects of compounds on CoA-IT activity. The assay is based upon
mixing cellular material containing CoA-IT activity with a stable
lyso phospholipid such as l-alkyl-2-acyl-GPC and measuring the
production of phospholipid product such as 1-alkyl-2-acyl-GPC
occurring in the absence of added CoA or CoA-fatty acids.
[0181] Cell Preparation
[0182] Any inflammatory cell that contains high levels of CoA-IT
activity can be used, such as neutrophils, macrophages or cell
lines such as U937 cells. U937 cells were obtained from American
Type Culture Collection and grown in RPMI-1640 media (Gibco, Grand
Island, N.Y.) supplemented with 10% fetal bovine serum (Hyclone,
Logan, Utah) at 37.degree. C., 5%C02. Cells were grown without
differentiation (basal state) by any agent, such as dimethyl
sulfoxide. As used herein, "inflammatory cells" include, but are
not limited to neutrophils, macrophages, monocytes, lymphocytes,
eosinophils, basophils, and mast cells.
[0183] Microsomal preparation
[0184] Microsomes were prepared using standard techniques. In this
case, cells were washed with a buffer of 250 mM sucrose, 10 mM
Tris, 1 mM EGTA, 1 mM MgCl.sub.2, pH 7.4 and ruptured by N.sub.2
cavitation (750 psi, 10 minutes). The ruptured cells were
centrifuged 1000.times.g, 5 minutes. The resulting supernatant was
centrifuged at 20,000.times.g,.about.20 minutes. Microsomes were
prepared from this supernatant by centrifugation at
100,000.times.g, 60 minutes. The resulting pellet was washed once
with assay buffer (150 mM NaCl, 10 mM Na.sub.2KPO.sub.4, 1 mM EGTA,
pH 7.4), recentrifuged and the pellet resuspended in assay buffer
(4-20 mg protein/ml) and was stored at -80.degree. C. until
assayed.
[0185] CoA-IT activity
[0186] CoA-IT activity was measured in 1.5 ml centrifuge tubes in a
total volume of 100 ul. Microsomes were diluted in assay buffer to
the desired protein concentration (6-20 ug/tube). The reaction was
initiated by addition of [3H ]
1-alkyl-2-lyso-sn-glycero-3-phosphocholine (GPC) (.about.0.1
uCi/tube) and 1 .mu.M final cold 1-alkyl-2-lyso-GPC in assay buffer
with 0.25 mg/ml fatty acid-poor bovine serum albumin (BSA)
(Calbiochem, La Jolla, Calif.). [3H] 1-alkyl-2-lyso-GPC,
approximately 50 Ci/mmol, was from NEN-Dupont (Boston, Mass.) and
cold l-alkyl-2-lyso-GPC was from Biomol (Plymouth Meeting, Pa.).
Microsomes were pretreated with desired agents for the desired time
(10 minutes) before the addition of [3H]1-alkyl-2-lyso-GPC. The
reaction was run for the desired time (10 minutes) at 37.degree. C.
The reaction was stopped and the lipids extracted by addition of
100 ul of chloroform:methanol (1:2, v/v) followed by 100 ul of
chloroform and 100 ul of 1 M KCl. The samples were vortexed and
centrifuged at high speed in a microfuge for 2-3 minutes. An
aliquot of the chloroform-extracted materials were separated,
usually by TLC in chloroform/methanol/acetic acid/water (50:25:8:4,
v/v), visualized by radioscanning (Bioscan) and the product, [3H]
1-alkyl-2-acyl-GPC, was scraped and quantified by liquid
scintillation spectroscopy. With this TLC system, the synthetic
standards of 1-alkyl-2-lyso-GPC and 1-alkyl-2-acyl-GPC were well
separated, with Rf values of approximately 0.25 and 0.65,
respectively. Other methods can be used to separate substrate from
product, including but not limited to column chromatography,
affinity chromatography and post reaction derivitization.
[0187] Protein concentration were assessed using the protein assay
reagents from Bio-Rad (Richmond, Calif.).
[0188] Results
[0189] A variety of compounds have been tested in this assay to
determine its selectivity and inability to detect trivial,
non-selective inhibitors. Inhibitors of 5-lipoxygenase (5-LO) and
cyclooxygenase (CO), such as indomethicin, naproxen,
6-(4'-Fluorophenyl)-5-(4-pyridyl)-2,3-dih-
ydroimidzo-[2,1-b]thiazole and
6-(4'-Fluorophenyl)-5-(4-pyridyl)2,3-dihydr-
oimidzo-[2,1-b]thiazole-dioxide had no effect on CoA-IT activity at
concentrations up to 100 .mu.M. The anti-oxidant BHT also has no
effect at concentrations up to 100 .mu.M. Compounds which complex
with phospholipids and inhibit PLA.sub.2 activity, such as
quinacrine and aristolochic acid have no effect on CoA-IT activity
at concentrations up to 500 .mu.M. Doxepine, a compound reported to
inhibit PAF release did not inhibit CoA-IT at concentrations up to
100 .mu.M. Sodium diclofenac, reported to decrease leukotriene
production by altering arachidonic acid metabolism, had no effect
on CoA-IT activity at concentrations up to 500 .mu.M. These results
show that the assay for CoA-IT activity is sensitive and
selective.
[0190] FIG. 2 demonstrates the inhibition of CoA-IT activity in a
time dependent manner using a representative compound of Formula
(I), Example 24,
(3S,4R)-4-(isobutenyloxy)-3-(triphenylmethylamino)azetidin-2-one.
[0191] Other representative compounds of Formula (I) which
inhibited CoA-IT activity, as a time-dependent inhibitor, in the
microsomal CoA-IT assay described above [generally at an IC.sub.50
of <50 .mu.M or less, at a 10 min preincubation time] are:
[0192]
(3RS,4RS)-4-(Isobutoxy)-3-(triphenylmethylamino)azetidin-2-one
[0193]
(3R,4R)-4-(Isobutoxy)-3-(triphenylmethylamino)azetidin-2-one
[0194]
(3S,4S)-4-(Isobutoxy)-3-(triphenylmethylamino)azetidin-2-one
[0195]
(3R,4R)-4-(Isobutylthio)-3-(triphenylmethylamino)azetidin-2-one
[0196]
(3R,4R)-4-(Isobutylsulfonyl)-3-(triphenylmethylamino)azetidin-2-one
[0197]
(3S,4R)-4-(Isobutoxy)-3-(triphenylmethylamino)azetidin-2-one
[0198]
(3S,4R)-4-(Propoxy)-3-(triphenylmethylamino)azetidin-2-one
[0199]
(3S,4S)-4-(Propoxy)-3-(triphenylmethylamino)azetidin-2-one
[0200]
(3S,4R)-4-(Benzyloxy)-3-(triphenylmethylamino)azetidin-2-one
[0201]
(3S,4S)-4-(Benzyloxy)-3-(triphenylmethylamino)azetidin-2-one
[0202] (3S,4R)-4-Methoxy-3-(triphenylmethylamino)azetidin-2-one
[0203]
(3S,4R)-4-(Isobutenyloxy)-3-(triphenylmethylamino)azetidin-2-one
[0204]
(3S,4R)-4-Octyloxy-3-(triphenylmethylamino)azetidin-2-one
[0205] (3S,4R)-4-Phenoxy-3-(triphenylmethylamino)azetidin-2-one
[0206] (3S,4S)4-Phenoxy-3-(triphenylmethylamino)azetidin-2-one
[0207]
(3S,4R)-3-[[(4-iodophenyl)diphenylmethyl]amino]-4-(isobutoxy)azetid-
in-2-one
[0208]
(3S,4R)-4-[[2-(Pyrid-3-yl)-1,3-dithian-2-yl]methoxy]-3-(triphenylme-
thylamino)azetidin-2-one
[0209]
(3S,4S)-4-(Prop-2-ynyloxy)-3-(triphenylmethylamino)azetidin-2-one
[0210] Methyl
4-[(3S,4S)-2-oxo-3-(triphenylmethylamino)azetidin-4-yloxy]bu-
t-2-ynoate
[0211] Methyl 4-[(3S,4R)-2-
oxo-3-(triphenylmethylamino)azetidin-4-yloxy]b- ut-2-ynoate
[0212]
(3S,4R)-4-[(2(5H)Furanon-4-yl)methoxy]-3-(triphenylmethylamino)azet-
idin-2-one
[0213] (S)-3-(Triphenylmethylamino)azetidin-2-one
[0214] (RS)-3-(Triphenylmethylamino)azetidin-2-one
[0215]
(3S,4S)-4-[3-(Methoxycarbonyl)propoxy]-3-(triphenylmethylamino)azet-
idin-2-one
[0216]
(3R,4R)-4-(Methylsulfonyl)-3-(triphenylmethylamino)azetidin-2-one.
[0217] The following compound was found active at either increased
uM or longer pretreatment time:
[0218]
(3S,4S)-4-[3-(Methoxycarbonyl)propoxy]-3-(triphenylmethylamino)azet-
idin-2-one
[0219] Apoptosis Assay:
[0220] Materials: Compounds
[0221] Compounds were made as stocks (5-100 mM) in
dimethylsulfoxide (DMSO) and diluted in DMSO to provide final
concentrations, with DMSO concentrations ranging from 0.1-1%.
[0222] Preparation of cells
[0223] HL-60 cells were obtained from American Type Culture
Collection and grown in RPMI-1640 media supplemented with 10% fetal
bovine serum at 370, 5% CO.sub.2. Cells were seeded in T-flasks at
0.03 to 0.08.times.10.sub.6 cells/ml and used for experiments at
0.5 to 0.6.times.10.sub.6 cells/ml. Other proliferative cells can
be used. In addition, leukemic cells can be isoloted from leukemic
patients for ex vivo use.
[0224] Proliferation and apoptosis
[0225] The functional status of cells was measured in one or more
ways. First cell viability was assessed by the ability of the cells
to exclude trypan blue stain.
[0226] A second, more sensitive and quantitative method was to
measure DNA synthesis, using the incorporation of
[.sup.3H]thymidine into newly-formed DNA as the index of synthesis.
In these assays, cells were treated with various concentrations of
compounds or vehicle and kept in cell culture for different lengths
of time (1 to 3 days). In a typical experiment, cells were then
given a pulse of [.sup.3H]thymidine (0.1-1 .mu.Ci) then removed
from culture 6-48 hours later, washed 2.times. with PBS and then
treated with 0.2 N NaOH for 30 min followed by addition of 15%
trichloroacetic acid for 2 hrs. The total sample from each well was
filtered through GF/C Watman glass microfilters under vacuum and
washed 3.times. with 5% trichloroacetic acid. The incorporation of
[.sup.3H]thymidine into DNA bound to the filter was determined
using liquid scintillation spectroscopy.
[0227] Measurement of DNA fragmentation, determines not only the
amount of cell death, but also provides insight into mechanism.
When cells undergo programmed cell death or apoptosis, but not
necrosis, cellular DNA is broken down into oligonucleosomal
fragments which can be discerned on gels (Kerr et al., Cancer 73:
2013-2026, (1994)). Cells were treated for various times (3-24
hours) with different concentrations of compounds or vehicle. The
washed cells were lysed by resuspending them in 200 .mu.L of cold,
sterile detergent buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.2%
Triton X-l00) and incubated on ice 30 min. Cellular protein and RNA
were enzymatically degraded by incubation with 75 .mu.g/mL RNAse A
for 1 hour at 370, then 200 .mu.g/mL Proteinase K and 0.5% SDS
(final) for 1 hour at 370. An internal standard of DNA was added
during the cell lysis and its recovery used as a confirmation of
efficient extraction of cellular DNA fragments. The samples were
extracted twice with an equal volume of cold, buffer saturated
phenol, once with phenol/chloroform/isoamyl alcohol (25:24:1, v/v),
and once with chloroform. NaCl (300 mM final) and cold ethanol were
added, the solution was vortexed and let stand overnight. The
ethanol was removed and the samples and DNA bp standards (Sigma)
were run on a 1% agarose gel with TBE buffer with ethidium bromide
(45 mM Tris, pH 8.0, 45 mM boric acid, 1 mM EDTA). The DNA was
visualized under UV light (UVP Gel Documentation system) and the
size of DNA fragments noted by comparison to molecular weight
standards.
[0228] Protein analysis
[0229] Protein concentrations were determined using the method of
Bradford. M., Anal. Biochem. 72: 248-254, (1976), with reagents
purchased from Bio-Rad (Hercules, Calif.).
[0230] Results
[0231] As mentioned in the "Background of the Invention", we have
recently demonstrated that CoA-IT inhibitors block the
proliferation and induce apoptosis of several neoplastic cell
lines. Data below shows that compounds of formula I have the same
effects. For example, FIG. 3 demonstrates that compounds of formula
1 cause a time and dose-dependent decrease in cell viability of
cultured HL-60 cells. A concentration of 50 uM of Compound 1,
Example 24, caused a 70% and 97% decrease in cell viability at 24
and 48 hr, respectively, after additions. The next studies were
designed to determine if compounds of formula I would have similar
effects on other neoplastic cells. Two other neoplastic cell types
were examined. The first were P388 murine leukemia cells. These
cells are utilized as a key test for antitumor activity by the
National Cancer Institute in cancer chemotherapy screening studies.
In P388 cells, we monitored the synthesis of new DNA by measuring
thymidine incorporation into DNA as a measure of cell
proliferation. Compound 1 caused a marked reduction (at 72 hr at 50
uM) in the capacity of these cells to incorporate thymidine into
newly-formed DNA (FIG. 4). A major mechanism that blocks cell
proliferation and reduces cell numbers is the induction of
apoptosis. Apoptosis was determined in these studies by examining
internucleosomal DNA fragmentation. Compound 1 induced apoptosis of
P388 cells as determined by the marked appearance of
internucleosomal DNA fragments (DNA ladders) after addition of
compound (FIG. 5).
[0232] Finally, Compound 1 was examined for its capacity to
influence new DNA synthesis and DNA fragmentation in cells from
patients with acute myelogenous leukemia (AML). FIG. 6 shows that
Compound 1 prevented thymidine incorporation into AML cells. At
similar concentrations and time points, this compound caused DNA
"ladder" formation in AML cells from patients (FIG. 7). Taken
together these data reveal that compounds of formula I inhibit cell
proliferation and induce apoptosis of a variety of neoplastic
cells. Moreover, these studies point out the utility of these
compounds as chemotherapeutic agents in proliferative disorders
[0233] As used herein, various abbreviations and explanations are
as follows: [.sup.3H], a molecule that contains tritium atoms, a
radioactive isotope; A23 187, a compound that allows free entry of
calcium into a cell; AA, arachidonic acid, 5-8-11-14
eicosatetraenoic acid; arachidonate, arachidonic acid contained
within a phospholipid; free arachidonic acid, arachidonic acid that
is not contained within a phospholipid; [.sup.2H.sub.8] arachidonic
acid, the form of arachidonic acid labeled with 8 deuterium atoms,
a stable isotope; 1-alkyl, 1-O-alkyl; 1-alkenyl, 1-O-alk-1'-enyl;
BSA, bovine serum albumin; CoA, coenzyme A; CoA-IT, CoA-independent
transacylase; COX, cyclooxygenase; DTT, dithiothreitol; EGTA,
[ethylenebis(oxyethylenenitrilo)]tetra acetic acid, a calcium
chelator; GPC, sn-glycero-3-phosphocholine; EDTA, a metal ion
chelator; GPE, sn-glycero-3-phosphoethanolamine; GC/MS, gas
chromatography and mass spectrometry; 5HETE,
5(S)-hydroxyeicosa-6,8,11,14- -tetraenoic acid; 15HETE,
15(S)-hydroxyeicosa-5,8,11,13-tetraenoic acid; HL-60, American Type
Tissue Culture designated cell line similar to a monocyte; 5LO,
5-lipoxygenase; LTB.sub.4, leukotriene B.sub.4; LTC.sub.4,
leukotriene C.sub.4; LTD.sub.4, leukotriene D.sub.4; lyso PAF,
1-alkyl-2-lyso-GPC, lyso platelet-activating factor; PLA.sub.2,
phospholipase A.sub.2; PBS, phosphate buffered saline; PAF,
platelet activating factor, 1-alkyl-2-acetyl-GPC; PL, phospholipid;
PC, phosphatidylcholine; PE, phosphatidylethanolamine, PI,
phosphatidylinositol; PMN, polymorphonuclear neutrophilic cell,
neutrophil; PS phosphatidylserine; Rf, the distance a compound
travels as a fraction of the solvent front; TLC, thin layer
chromatography; U937, American Type Tissue Culture designated cell
line similar to a monocyte.
[0234] All publications, including but not limited to patents and
patent applications, cited in this specification are herein
incorporated by reference as if each individual publication were
specifically and individually indicated to be incorporated by
reference herein as though fully set forth.
[0235] The above description fully discloses the invention
including preferred embodiments thereof. Modifications and
improvements of the embodiments specifically disclosed herein are
within the scope of the following claims. Without further
elaboration, it is believed that one skilled in the art can, using
the preceding description, utilize the present invention to its
fullest extent. Therefore, the Examples herein are to be construed
as merely illustrative and not a limitation of the scope of the
present invention in any way. The embodiments of the invention in
which an exclusive property or privilege is claimed are defined as
follows.
* * * * *