Organic compounds -- part of the class 532-570 series – Organic compounds – Four or more ring nitrogens in the bicyclo ring system
Reexamination Certificate
1999-11-10
2003-02-11
Raymond, Richard L. (Department: 1624)
Organic compounds -- part of the class 532-570 series
Organic compounds
Four or more ring nitrogens in the bicyclo ring system
C544S319000, C544S326000, C514S256000, C514S269000
Reexamination Certificate
active
06518424
ABSTRACT:
FIELD OF THE INVENTION
The present invention provides novel substituted pyrimidines. These compounds have been found to inhibit phospholipase A
2
activity, in particular cPLA
2
(cytosolic phospholipase A
2
).
BACKGROUND OF THE INVENTION
Phospholipases A
2
(PLA
2
s; EC 3.1.1.4) are enzymes that hydrolyze the 2-acyl ester bond of phosphoglycerides generating free fatty acids and lysophospholipids (for review, see, Kramer, R M (1993)
Advances in Second Messenger and Phosphoprotein Research
28: 81; Glaser et al. (1993) TiPS 14: 92; Dennis E A (1994)
J. Biol. Chem
. 269: 13057). PLA
2
s are a diverse class of enzymes with regard to function, localization, regulation, mechanism, sequence, structure, and role of divalent metal ions. A variety of polypeptide species can exhibit PLA
2
activity; for purposes of this specification, these polypeptides are considered PLA
2
isozymes.
In general, PLA
2
enzymes catalyze the hydrolysis of the fatty acid ester bond at the sn-2 position of membrane phospholipids to produce arachidonic acid and its metabolites. Group I, II, and III PLA
2
s are extracellular enzymes of approximately 14-18 kD in humans, and are designated sPLA
2
s, in recognition of their secretion. sPLA
2
s are found in many extracellular fluids and have a broad substrate specificity for many types of phospholipids.
Group IV PLA
2
is a cytosolic enzyme of approximately 85 kD (based on deduced cDNA coding sequence) to 110 kD (based on SDS-PAGE of purified protein), and is designated cPLA
2
to indicate its cytosolic location. Unlike sPLA
2
s, the cPLA
2
enzyme exhibits preferential catalysis of phospholipids which contain arachidonic acid, and is most likely the enzyme responsible for arachidonic acid release which is the rate-limiting step for subsequent eicosanoid biosynthesis of pro-inflammatory lipid mediators (prostaglandins, leukotrienes, lipoxins, and platelet-activating factor: “PAF”). cPLA
2
is present in the cytosol of a variety of species and cell types, including human U937 cells (monocytes), platelets, kidney, and macrophages, among others, and is implicated in controlling arachidonic acid metabolism and eicosanoid production.
Some cells contain calcium independent phospholipase A
2
/B enzyme. The phospholipase A
2
/B purified enzyme is characterized by activity in the absence of calcium and having a molecular weight of 86 kD on SDS-PAGE (see, U.S. Pat. Nos. 5,554,511 and 5,466,595).
Of particular interest in the present invention is the cPLA
2
enzyme. Human cPLA
2
has been cloned as a cDNA isolated from mRNA of a human monocytic cell line, (U.S. Pat. Nos. 5,354,677 and 5,328,842; Sharp et al. (1991)
J. Biol. Chem
. 266: 14850; Clark et al. (1991)
Cell
65: 1043) and the mRNA encodes a protein of 749 amino acids which has little detectable homology with the secreted sPLA
2
s or any other protein in known sequence databases. The cPLA
2
cDNA identifies a single copy gene in the human genome, with no detectable closely related genes based on Southern blotting experiments. A suitable source of cPLA
2
can be obtained, if desired, by expression of a recombinant expression vector in a suitable host cell, as described in U.S. Pat. No. 5,354,677, or by conventional biochemical purification from mammalian cells, as is known in the art.
Moreover, cPLA
2
contains an amino-terminal domain which binds calcium and similar divalent cations, and cPLA
2
binds to membrane vesicles at submicromolar concentrations of Ca
+2
in a calcium-dependent fashion. cPLA
2
can translocate to membranes when activated in the presence of calcium. Presumably, cPLA
2
associates with membrane components in vivo under suitable calcium concentrations. Agents that stimulate the release of arachidonic acid (ATP, thrombin, phorbol ester, calcium ionophore) can cause increased serine phosphorylation of cPLA
2
which increases the enzymatic activity of cPLA
2
(Lin et al. (1993)
Cell
72: 269). Phosphorylation is believed to contribute to the control of cPLA
2
activity in vivo (Lin et al. (1992)
Proc. Natl. Acad. Sci
. (
USA
) 89: 6147; Lin et al. (1993)
Cell
72: 269; Qiu et al. (1993)
J. Biol. Chem
. 268: 24506; Kramer et al. (1993)
J Biol. Chem
. 268: 26796).
The art generally recognizes the physiologic role of cPLA
2
to be in the mediation of inflammation via its role in arachidonic acid metabolism and lipid/lipoprotein metabolism, such as cell membrane homeostasis. Roshak et al. (1994)
J. Biol. Chem
. 269: 25999 used antisense oligonucleotides complementary to the cPLA
2
mRNA to inhibit prostaglandin production in LPS-induced monocytes, indicating a potential role for cPLA
2
in generating inflammatory regulators in monocytes. Verity MA (1993)
Ann. N.Y. Acad. Sci
. 679:110 speculates that “abusive activation” of PLA
2
via uncontrolled Ca
+2
influx might produce irreversible cell injury of neurons via extensive localized lipid peroxidation and subsequent membrane disintegration. U.S. Pat. Nos. 5,354,677 and 5,328,842 indicates that cPLA
2
inhibitors are expected to be used to treat inflammatory conditions, such as psoriasis, asthma, and arthritis (see, col. 15), and prophesizes that such anti-inflammatory compounds can be identified as cPLA
2
inhibitors.
In addition to the roles mentioned above, PLA
2
activity has been implicated as a contributor to destructive cellular processes in various tissues including, but not limited to, the central nervous system. PLA
2
activity has also been reportedly involved in ischemic injury and pathological nervous system conditions.
A number of inhibitors of PLA
2
activity have been reported. Bromoenol lactone and trifluoromethyl ketones (e.g., palmitoyl trifluoromethyl ketone, arachidonyl trifluoromethyl ketone) have been reported to be capable of inhibiting a Ca
+2
-independent PLA
2
activity (Ackermann et al. (1995)
J. Biol. Chem
. 270: 445) as well as cPLA
2
(Street et al. (1993)
Biochemistry
32: 5935). Several benzenesulfonamide derivatives have also been reported to be capable of inhibiting PLA
2
activity (European Patent Application 468 054; Oinuma et al. (1991)
J. Med. Chem
. 34: 2260).
In view of the role PLA
2
can play in destructive cellular processes, there is a need in the art for new compounds that are inhibitors of PLA
2
. These compounds can then be used to treat or prevent PLA
2
-mediated diseases. The present invention provides such new compounds, compositions and methods of treatment.
SUMMARY OF THE INVENTION
The present invention provides substituted pyrimidines which are effective inhibitors of PLA
2
, more particularly cPLA
2
. As such, the present invention provides novel substituted pyrimidines which have the general structure:
with the symbols R
1
, R
2
, R
3
, R
4
and R
5
representing the groups provided in the detailed description below. These compounds have activity as inhibitors of phospholipase A
2
, and in particular cytosolic phospholipase A
2
.
In other aspects, this invention provides pharmaceutical compositions of the substituted pyrimidine compounds of formula I or a pharmaceutically acceptable salt thereof with a pharmaceutically acceptable carrier.
As described in detail below, the compounds of formula I are useful in the treatment of conditions associated with PLA
2
-mediated conditions, such as inflammation and Alzheimer's disease. As such, the present invention provides a method of treating cPLA
2
-dependent diseases. In this aspect, the method comprising administering to a subject having at least one of cPLA
2
-mediated disease with an effective amount of a compound of formula I.
The compounds and pharmaceutical compositions of compounds of formula I are also useful for in vitro assays for PLA
2
inhibitors. As such, in another aspect, the present invention provides a method of inhibiting PLA
2
activity in vitro, comprising contacting a cell having PLA
2
activity with a compound of formula I and assaying the PLA
2
activity.
In another embodiment, the present invention provides the use for the manufacture of a medicament for the treatment of inhibiting PLA
2
act
Dappen Michael S.
John Varghese
Rydel Russell E.
Thorsett Eugene D.
Balasubramanian Venkataraman
Elan Pharmaceuticals Inc.
Raymond Richard L.
Townsend and Townsend / and Crew LLP
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