Modulators of SREBP processing

Details Modulators of SREBP processing Modulators of SREBP processing

2000-10-06

2003-11-18

Weber, Jon P. (Department: 1651)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C530S331000

Reexamination Certificate

active

06649593

ABSTRACT:

BACKGROUND OF THE INVENTION
The lipid composition of animal cells is controlled by transcriptions factors known as sterol regulatory element binding proteins (SREBPs). These transcription factors are bound to membranes of the endoplasmic reticulum (ER) and nuclear envelope, and are released by sterol-regulated proteolysis. SREBP release is initiated by Site-1protease (S1P), which cleaves SREBPs in the ER luminal loop between two membrane spanning regions (see, for example, Sakai, et al., Cell 85:1037-1046 (1996); Duncan, et al.,
J. Biol. Chem
. 272:12778-12785 (1997); and copending application Ser. No. 09/360,237, entitled “cDNA Cloning of Site-1 Protease for SREBPs”).
S1P has features characteristic of a superfamily of serine proteases, broadly characterized as subtilisins, which are found in all living organisms from bacteria to humans (see Siezen and Leunissen,
Prot. Sci
. 6:501-523 (1997)). Human S1P consists of about 1052 amino acids. Based on its resemblance to other serine proteases, it has been postulated that the catalytic triad should consist of Asp218, His249 and Ser414 (ibid.). Additionally, the S1P sequence consists of six potential sites of N-linked glycosylation, an unbroken stretch of 25 nonpolar residues near the carboxy terminus (consistent with a membrane-spanning sequence), and a COOH terminal sequence of 30 amino acids that is strikingly rich in prolines and basic residues (including a complete absence of acidic residues).
From a functional viewpoint, the action of S1P most resembles that of the furins, which are subtilases of the Kex2p-like subfamily that process proteins such as the insulin pro-receptor and pro-endothelin-1. S1P differs from the furins in two respects: (1) substrate recognition (furins always cleave after dibasic residues and S1P may cleave after nonbasic residues, such as cleavage after the RSVL (SEQ ID NO:1) sequence; and (2) cellular location (furins act in post-Golgi secretory vesicles, and S1P acts in a pre-Golgi compartment, thought to be the ER). If S1P does function in the ER, its activity must be regulated so as to prevent, it from degrading nascent polypeptides nonspecifically.
In view of the role of S1P in regulating SREBPs, and thereby regulating the synthesis of fatty acids and cholesterol, there exists a need for compounds that can inhibit the activity of S1P. The present invention provides such compounds, as well as methods for the modulation of cholesterol homeostasis in animal cells.
SUMMARY OF THE INVENTION
The present invention provides compounds, compositions and methods useful for inhibiting of S
1
protease, and for modulating cholesterol homeostasis in cells. The compounds provided herein, and which are useful in the present compositions and methods are those having the formula:
R
1
—C(═O)—(Aa
1
)
n
—R
2
.
In the general formula above, the symbol R
1
represents (C
1
-C
6
)alkyl, aryl(C
1
-C
6
)alkyl, aryl, (C
1
-C
6
)alkylamino, aryl(C
1
-C
6
)alkylamino, (C
1
-C
6
)alkoxy, or aryl(C
1
-C
6
)alkoxy.
The symbol Aa represents a divalent amino acid residue, connected via its amino and acyl groups, or a linking group. The superscript i is an integer denoting the position downstream from —C(═O)— and the subscript n is an integer of from 2 to 10, such that Aa at any position can be the same as or different from Aa at any other position, with the proviso that at least two of Aa are amino acid residues.
The symbol R
2
represents any one of:
in which X is O, NH or N—(C
1
-C
4
)alkyl and the subscript n′ is an integer of from 0 to 4. Additionally, the symbols R
11
and R
12
independently represent H, (C
1
-C
8
)alkyl, aryl, or aryl(C
1
-C
6
)alkyl, or taken together, R
11
and R
12
form a five- to seven-membered ring; R
13
represents H, CF
3
, CH
2
Y, heteroaryl, CONHR
16
and CO
2
R
16
; wherein Y is a leaving group and R
16
is H or (C
1
-C
4
)alkyl. The symbols R
14
and R
15
independently represent H, (C
1
-C
6
)alkyl, aryl, and aryl(C
1
-C
6
)alkyl; or taken together, R
14
and R
15
form a five- to seven-membered ring.
BRIEF DESCRIPTION OF THE DRAWINGS
Not applicable
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Abbreviations and Definitions
The following abbreviations are used herein:
Ac, acetyl; AMC, 7-amino-4-methylcoumarin; ALLN, N-acetyl-leucinal-leucinal-norleucinal; Bn, benzyl; BOC, t-butyloxycarbonyl; Cbz, benzyloxycarbonyl; CHO, Chinese hamster ovary; CMV, cytomegalovirus; DMSO: dimethylsulfoxide; Et
3
N, triethylamine; Fmoc, fluorenylmethoxy; MCA, 4-methyl-coumaryl-7-amide; MeOH, methanol; S1P, Site 1 protease; SREBP, sterol regulatory element-binding protein; and TFA, trifluoroacetic acid.
The term “alkyl,” by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain, or cyclic hydrocarbon radical, or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent radicals, having the number of carbon atoms designated (i.e. C
1
-C
10
means one to ten carbon atoms). Examples of saturated hydrocarbon alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers. The term “alkyl,” unless otherwise noted, is also meant to include those derivatives of alkyl defined in more detail below as “heteroalkyl.” Alkyl groups which are limited to hydrocarbon groups are termed “homoalkyl”.
The term “alkylene” by itself or as part of another substituent means a divalent radical derived from an alkane, as exemplified by —CH
2
CH
2
CH
2
CH
2
—, and further includes those groups described below as “heteroalkylene.” Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the present invention. A “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.
The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) are used in their conventional sense, and refer to those alkyl groups attached to the remainder of the molecule via an oxygen atom, an amino group, or a sulfur atom, respectively.
The term “heteroalkyl,” by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or cyclic hydrocarbon radical, or combinations thereof, consisting of the stated number of carbon atoms and from one to three heteroatoms selected from the group consisting of O, N, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N and S may be placed at any interior position of the heteroalkyl group. The heteroatom Si may be placed at any position of the heteroalkyl group, including the position at which the alkyl group is attached to the remainder of the molecule. Examples include —CH
2
—CH
2
—O—CH
3
, —CH
2
—CH
2
—NH—CH
3
, —CH
2
—CH
2
—N(CH
3
)—CH
3
, —CH
2
—S—CH
2
—CH
3
, —CH
2
—CH
2
, —S(O)—CH
3
, —CH
2
—CH
2
—S(O)
2
—CH
3
, —CH═CH—O—CH
3
, —Si(CH
3
)
3
, —CH
2
—CH═N—OCH
3
, and —CH═CH—N(CH
3
)—CH
3
. Up to two heteroatoms may be consecutive, such as, for example, —CH
2
—NH—OCH
3
and —CH
2
—O—Si(CH
3
)
3
. Similarly, the term “heteroalkylene” by itself or as part of another substituent means a divalent radical derived from heteroalkyl, as exemplified by —CH
2
—CH
2
—S—CH
2
CH
2
—and —CH
2
—S—CH
2
—CH
2
—NH—CH
2
—. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking group

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