Patent Application
20190106460
The Sequence Listing written in file SEQ_098184-1092172.txt created on Sep. 11, 2018, 40,893 bytes, machine format IBM-PC, MS-Windows operating system, in accordance with 37 C.F.R. §§ 1.821 to 1.825, is hereby incorporated by reference in its entirety for all purposes.
The present invention relates to compounds which bind to Beta Transducin repeat-containing protein (βTrCP), and modulate the activity of βTrCP. In particular, the invention relates to compounds which demonstrate optimised binding to βTrCP. The invention also relates to pharmaceutical compositions comprising such compounds and the use of such compounds as medicaments, specifically for the treatment of disorders associated with aberrant protein degradation, such as cancer.
In order to maintain the delicate homeostatic balance of a cell, unneeded or damaged proteins must be degraded. Protein degradation is performed by the proteasome, which dismantles unwanted proteins into small peptides of about eight amino acids in length. These peptides are then further degraded by proteases in the cell, and the resulting amino acids are used to synthesise new proteins.
To ensure that only unwanted proteins are degraded, and that healthy functioning proteins remain intact, target proteins are tagged for degradation by the ubiquitin proteasome system (UPS). The aim of the UPS is to attach a chain of approximately four ubiquitin monomers to any unwanted proteins in order to direct entry of the target protein into the proteasome.
The major components of the UPS are ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2) and ubiquitin ligases (E3). There are several members of each of these groups of enzymes, which generally recognise different groups of target proteins.
The first step of the UPS is the hydrolysation of ATP by an ubiquitin-activating enzyme in order to facilitate the adenylation of an ubiquitin molecule. Following this, the ubiquitin molecule is transferred to a cysteine residue in the active site of the ubiquitin-activating enzyme at the same time as a second ubiquitin molecule is adenylated. The second adenylated ubiquitin molecule is subsequently transferred to a cysteine residue in the active site of an ubiquitin-conjugating enzyme. The final step requires the recognition of the target protein by an ubiquitin ligase, which catalyses the transfer of the ubiquitin molecule from the ubiquitin-conjugating enzyme to the target protein. Following the addition of four ubiquitin molecules, the target protein is recognised by the proteasome, and sent for degradation.
βTrCP is an E3 ubiquitin ligase forming part of the UPS. It recognises a variety of target proteins, including inhibitor of nuclear factor κB (IκB), β-catenin, REST (repressor-element-1-silencing transcription factor), CDC25A/B, ATF4 (Activating Transcription Factor 4), and pro-caspase 3 and is known to function by binding to a phosphodegeneron motif DSGXXS in which the two serines are phosphorylated.
βTrCP is involved in apoptotic regulation through the targeted degradation of pro-apoptotic factors. βTrCP has been shown to be over-expressed in a variety of cancers including colorectal cancer, chemoresistant pancreatic cancer, hepatoblastomas and breast cancer. Human hepatocellular carcinomas (HCCs), pancreatic tumours and melanomas have also been shown to display an aberrant loss of IκB, which is thought to be caused by βTrCP over-expression. This over-expression increases the degradation of pro-apoptotic factors, leading to a reduction in apoptotic cell death and subsequent aberrant cell growth.
Inhibition of βTrCP prevents the degradation of pro-apoptotic factors such as IκB and programmed cell death 4 (PDCD4). This has been shown to induce apoptosis in human malignant melanoma, breast cancer and prostate cancer cells, augmenting the cytotoxic effects of anticancer drugs and ionizing radiation.
The inventors hypothesised that compounds that bond βTrCP may be able to prevent βTrCP binding to its substrates, thus preventing the ubiquitination of target proteins. The prolonged presence in a cell of pro-apoptotic factors will increase cellular apoptosis, providing a useful tool for the treatment of disorders associated with aberrant protein degradation such as hyperproliferative disorders including cancer.
The inventors have therefore designed a series of compounds which bind βTrCP and which will be therapeutically useful.
Compounds and Modified Peptides
The present invention relates to compounds which bind βTrCP.
Accordingly, in the first aspect, the invention provides a compound of Formula Ia:
X1-X2-X3-X4-X5-X6-X7 Formula Ia
wherein,
X1 is a group A1-B—Z1—;
X2 is a group —N(Ra)—Y1(-L1-A2)-Z2—;
X3 is a group —N(Rb)—Y2—Z3—;
X4 is a group —N(Rc)—Y3(-L2-A3)-Z4—; or X4 is a group —N(Rc)—Y3(-L2)-Z4—
X5 is a group —N(Rd)—Y4(-L3-A4)-Z5—;
X6 is a group —N(Re)—Y5(-L4-A5)-Z6—;
X7 is a group —N(RN1)(RN2);
wherein,
B is C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl or aryl;
wherein, B may be substituted with one or more RE, wherein RE is selected from the group consisting of C1-C4 alkyl, —NH2, —NH(RN2) and —N(RN2)2;
Ra, Rb, Rc, Rd, and Re are each independently selected from the group consisting of —H, C1-C10 alkyl, aryl and heteroaryl;
L1, L2, L3 and L4 are each independently C0-C5 alkyl, C2-C5 alkenyl or C2-C5 alkynyl;
wherein,
L1 may be substituted with one or more RL1, wherein RL1 is C1-C4 alkyl;
L2 may be substituted with one or more RL2, wherein RL2 is C1-C4 alkyl or C2-C4 alkenyl;
L3 may be substituted with one or more RL3, wherein RL3 is C1-C4 alkyl;
L4 may be substituted with one or more RL4, wherein RL4 is C1-C4 alkyl;
Y1, Y3, Y4 and Y5 are each independently CH or N;
Y2 is CF2, CH2, N(RY2) or O; wherein, RY2 is —H or C1-C4 alkyl;
Z1 is a bond, C═O, C═S, CH2, S═O, S(O)2, C═N(C1-C4 alkyl) or C═NH;
Z2, Z3, Z4, Z5, and Z6 are independently selected from the group consisting of C═O, C═S, CH2, S═O, S(O)2, C═N(C1-C4 alkyl) and C═NH;
A1 and A5 are each independently carboxylic acid (—CO2H) or a bioisostere thereof and A2 is a carboxylic acid (—CO2H) or a bioisostere thereof or —C(O)N(RN1)2;
wherein,
A1 may be substituted with one or more RA1, wherein RA1 is selected from the group consisting of —H, C1-C4 alkyl, C2-C4 alkenyl and aryl;
A2 may be substituted with one or more RA2, wherein RA2 is selected from the group consisting of —H, C1-C4 alkyl, C2-C4 alkenyl and aryl;
A5 may be substituted with one or more RA5, wherein RA5 is selected from the group consisting of —H, C1-C4 alkyl, C2-C4 alkenyl and aryl;
A3 and A4 are each independently aryl or heteroaryl; wherein, A3 may be substituted with one or more RA3, wherein, RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —CN, —NO2, —CF3, —OCF3, —CO2H, —C1-C10 alkyl, —NH2, —NH(C1-C2 alkyl) and —N(C1-C2 alkyl)2;
A4 may be substituted with one or more RA4, wherein RA4 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —CN, —NO2, —CF3, —OCF3, —CO2H, —C1-C10 alkyl, —NH2, —NH(C1-C2 alkyl), —N(C1-C2 alkyl)2;
RN1 is selected from the group consisting of —H, C1-C10 alkyl and aryl;
RN2 is selected from the group consisting of RN1, —(CH2)0-10-(Z7)0-1-Aa, —(CH2O)0-10-CH2-(Z7)0-1-Aa, —(CH2CH2O)1-10-CH2CH3, —(CH2CH2O)1-10-(CH2)1-3-(Z7)0-1-Aa;
wherein,
Z7 is (C═O);
Aa is —OH, —NH2, —C(O)NH2, a cholesteryl derivative, a chain of one or more non-naturally occurring amino acids, or a chain of one or more naturally occurring amino acids or a chain of a mixture of one or more naturally occurring amino acids and one or more non-naturally occurring amino acids; wherein the one or more non-naturally occurring or naturally occurring amino acids are independently selected from the group consisting of L-amino acids, D-amino acids and aza-amino acids;
wherein,
when the compound of Formula Ia is substituted with an amino/amine group, said amino/amine group may be optionally capped, by replacement of a H atom, with a capping group.
Formula Ia may also be represented by Formula Ib:
In a second aspect, the invention provides a modified peptide comprising a sequence of amino acids:
X1-E/D/pS-G-X4-X5-E/D/pS-NHRN2 Formula Ic
wherein,
each of the amino acids are selected from L-amino acids, D-amino acids, aza-amino acids and substituted amino acids; and
wherein,
X1 is a group A1-B—Z1—;
X4 is a group —N(Rc)—Y3(-L2-A3)-Z4—;
X5 is a group —N(Rd)—Y4(-L3-A4)-Z5—;
wherein,
B, Rc, Rd, L2, L3, Y3, Y4, Z1, Z4, Z5, A1, A3, A4, and RN2 are as previously defined.
In a third aspect, the invention provides a prodrug comprising a methyl, ethyl, propyl, butyl, pentyl, cyclopentyl, hexyl, benzyl, aryl or heteroaryl ester of a compound of Formula Ia or a modified peptide of Formula Ic.
In a fourth aspect, the invention provides a prodrug comprising a —CO2(CH2CH2O)1-10CH2CH3 ester of a compound of Formula Ia or a modified peptide of Formula Ic.
In a fifth aspect, the invention provides a pharmaceutical composition comprising a compound of Formula Ia or a modified peptide of Formula Ic; or a prodrug of a compound of Formula Ia or a modified peptide of Formula Ic.
In a sixth aspect, the invention provides a compound of Formula Ia, a modified peptide of Formula Ic, a prodrug of a compound of Formula Ia, a prodrug of a modified peptide of Formula Ic, or a pharmaceutical composition comprising a compound of Formula Ia or a modified peptide of Formula Ic, for use in medicine.
In a seventh aspect, the invention provides a compound of Formula Ia, a modified peptide of Formula Ic, a prodrug of a compound of Formula Ia, a prodrug of a modified peptide of Formula Ic, or a pharmaceutical composition comprising a compound of Formula Ia or a modified peptide of Formula Ic, for use in the treatment of a disease associated with aberrant protein degradation.
In an eighth aspect, the invention provides a method of treating a disease associated with aberrant protein degradation comprising administering a compound of Formula Ia, a modified peptide of Formula Ic, a prodrug of a compound of Formula Ia, a prodrug of a modified peptide of Formula Ic, or a pharmaceutical composition comprising a compound of Formula Ia or a modified peptide of Formula Ic, in a pharmaceutically effective amount.
In a ninth aspect, the invention provides a diagnostic kit comprising a compound of Formula Ia, a modified peptide of Formula Ic, a prodrug of a compound of Formula Ia, or a prodrug of a modified peptide of Formula Ic.
Embodiments of Compounds of Formula Ia
Various embodiments of the invention are described herein. It will be recognised that features specified in each embodiment may be combined with other specified features to provide further embodiments.
In the first aspect, the invention provides a compound of Formula Ia:
X1-X2-X3-X4-X5-X6-X7 Formula Ia
wherein, X1, X2, X3, X4, X5, X6 and X7 are as hereinbefore defined.
Carboxylic acid isosteres
Groups A1, A2 and A5 are each independently carboxylic acid (—CO2H) groups or bioisosteres thereof (and A2 can also be (˜C(O)N(RN1)2) “Bioisostere” is a term with which the skilled person will be familiar. In particular, bioisosteres (also known as non-classical isosteres) are functional groups or molecules which have chemical and physical similarities producing broadly similar biological properties to those of the replaced moiety (Stocks et al. On Medicinal Chemistry, 2007).
Carboxylic acids are weak organic acids with pKas in the range of 0-5, although this can be affected by the electronegative or electropositive nature of any substituents. For example, acetic acid (CH3CO2H) has a pKa of 4.8. Bioisosteres of carboxylic acids may have comparable pKa values to those of carboxylic acids, i.e. they may be deprotonated at physiological pH (pH 7.3-7.5, Werle et al. British Journal of Cancer, 1997).
Common bioisosteric replacements for carboxylic acids include functional groups such as sulfonamides (pKa ˜4-9), sulfamides (pKa ˜6-10), acylsulfonamides (pKa ˜5), sulfonyl ureas (pKa ˜3-5), hydroxaminc acids (pKa ˜9), acylcyanamides (pKa ˜8), sulfonic acids (pKa ˜2), sulfonates (pka ˜1-2), phosphates (pKa ˜2), phosphonic acids/phosphonates (pKa ˜6.5) and phosphinic acids (pKa ˜4). Heterocycles with intrinsic acidity may also be used as bioisosteres for carboxylic acids. Common heterocyclic bioisosteric replacements for carboxylic acids include tetrazoles (pKa ˜4-8), triazoles (pKa ˜9), isoxazolones (pKa ˜5), 1,2,4-oxadiazolones (pKa ˜6), and 1,2-dihydro-pyrazolones (pKa ˜8). Examples of carboxylic acid bioisosteric functional groups include:
wherein, R1 is RA1, RA2 or RA5 respectively, wherein RA1, RA2 and RA5 are as previously defined.
Examples of heterocyclic carboxylic acid bioisosteres include:
wherein, R1 is RA1, RA2 or RA5 respectively, wherein RA1, RA2 and RA5 are as previously defined.
Group X1
It is believed that the side chain of group X1 interacts with the βTrCP binding domain to form an ionic bridge. Accordingly, X1 is a functional group which is ionisable at physiological pH, in particular a carboxylic acid group or bioisostere thereof, in order to sustain such a binding interaction with the βTrCP binding domain.
X1 is a group A1-B—Z1—;
wherein,
A1 is carboxylic acid (˜CO2H) or a bioisostere thereof;
wherein, A1 may be substituted with one or more RA1, wherein RA1 is selected from the group consisting of —H, C1-C4 alkyl, C2-C4 alkenyl and aryl;
B is C1-C10 alkyl, C2-C10 alkenyl or C2-C10 alkynyl or aryl;
wherein, B may be substituted with one or more RE, wherein RE is selected from the group consisting of C1-C4 alkyl, —NH2, —NH(RN2) and —N(RN2)2; and
Z1 is a bond, C═O, C═S, CH2, S═O, S(O)2, C═N(C1-C4 alkyl) or C═NH.
The term bioisostere is as hereinbefore described. In one embodiment, A1 is carboxylic acid. In one embodiment, A1 is selected from the group consisting of
wherein, R1 is RA1.
In one embodiment, A1 is selected from the group consisting of
wherein, R1 is RA1.
In one embodiment, A1 is selected from the group consisting of
wherein, R1 is RA1.
In one embodiment, A1 is selected from the group consisting of carboxylic acid (—CO2H), phosphate, phosphonate, phosphonic acid, tetrazole and sulphate.
In one embodiment, A1 is selected from the group consisting of carboxylic acid and phosphate. In one embodiment, preferably A1 is carboxylic acid.
In one embodiment, A1 is substituted by RA1, wherein RA1 is selected from the group consisting of —H, C1-C4 alkyl, C2-C4 alkenyl and aryl.
In one embodiment, B is C1-C4 alkyl, C2-C4 alkenyl or C2-C4 alkynyl. In one embodiment, B is C1-C2 alkyl or C2 alkenyl. In one embodiment B is aryl, particularly B is phenyl.
In one embodiment, B is substituted with one or more RE, wherein RE is selected from the group consisting of C1-C4 alkyl, —NH2, —NH(RN2) and —N(RN2)2. In one embodiment, B is substituted with —NH2. In one embodiment, B is substituted with —NH(RN2), wherein RN2 is a chain of one or more naturally or non-naturally occurring amino acids.
In one embodiment, B is substituted with —N(RN2)2. In one embodiment, B is substituted with —N(RN2)2, wherein both RN2 are RN1, wherein one RN1 is —H and the other RN1 is C1-C10 alkyl, aryl or heteroaryl.
In one embodiment, Z1 is C═O, C═S, or CH2. In one embodiment, Z1 is C═O.
In one embodiment, X1 is of Formula HO2C—B—Z1—; wherein Z1 is a bond, C═O, C═S, CH2, S═O or S(O)2; and B is C1-C10 alkyl, C2-C10 alkenyl or C2-C10 alkynyl. In one embodiment, X1 is of Formula HO2C—B—Z—, wherein Z1 is C═O and B is C1-C4 alkyl, C2-C4 alkenyl or C2-C4 alkynyl. In one embodiment, X1 is of Formula HO2C—B—Z1—, wherein Z1 is C═O and B is C1 alkyl. In one embodiment, X1 is of Formula HO2C—B—Z1—, wherein Z1 is C═O and B is C2 alkyl, C2 alkenyl or C2 alkynyl. In one embodiment, X1 is of Formula HO2C—B—Z1—, wherein Z1 is C═O and B is C3 alkyl, C3 alkenyl or C3 alkynyl. In one embodiment, X1 is of Formula HO2C—B—Z1—, wherein Z1 is C═O and B is C4 alkyl, C4 alkenyl or C4 alkynyl. In one embodiment, X1 is of Formula HO2C—B—Z1—, wherein Z1 is C═O and B is C5 alkyl, C5 alkenyl or C5 alkynyl. In one embodiment, X1 is of Formula HO2C—B—Z—, wherein Z1 is C═O and B is C6 alkyl, C6 alkenyl or C6 alkynyl. In one embodiment, X1 is of Formula HO2C-E-Z1—, wherein Z1 is C═O and B is C2-alkyl. In one embodiment, X1 is of Formula HO2C—B—Z1—, wherein Z1 is C═O and B is C2 alkenyl. In one embodiment, X1 is of Formula HO2C—B—Z1—, wherein Z1 is C═O and B is C2 alkyl; wherein C2 alkyl is substituted with one or more RE. In one embodiment, X1 is of Formula HO2C—B—Z1—, wherein Z1 is C═O and B is C2 alkyl; wherein C2 alkyl is substituted with one or more —N(RN2)2. In one embodiment, X1 is of Formula HO2C—B—Z1—, wherein Z1 is C═O and B is C2 alkenyl; wherein C2 alkenyl is substituted with one or more —N(RN2)2. In one embodiment, X1 is of Formula HO2C—B—Z1—, wherein Z1 is C═O and B is C3 alkyl; wherein C3 alkyl is substituted with one or more —N(RN2)2. In one embodiment, X1 is of Formula HO2C—B—Z1—, wherein Z1 is C═O and B is C3 alkyl; wherein C3 alkyl is substituted with one or more —N(RN1)(RN2). In one embodiment, X1 is of Formula HO2C—B—Z1—, wherein Z1 is C═O and B is C3 alkenyl; wherein C3 alkenyl is substituted with one or more —N(RN1)(RN2). In one embodiment, X1 is of Formula HO2C—B—Z1—, wherein Z1 is C═O and B is C3 alkyl; wherein C3 alkyl is substituted with —NH2. In one embodiment, X1 is of Formula HO2C—B—Z1—, wherein Z1 is C═O and B is C2 alkyl; wherein C2 alkyl is substituted with —NH2 at the carbon atom adjacent to Z1. In one embodiment, X1 is of Formula HO2C—B—Z1—, wherein Z1 is C═O and B is C2 or C3 alkyl, wherein said C2 or C3 alkyl is substituted with one or more —N(RN1)(RN2) wherein RN2 is a chain of one or more amino acids. In one embodiment, X1 is of Formula HO2C—B—Z1—, wherein Z1 is C═O and B is C2 or C3 alkyl, wherein said C2 or C3 alkyl is substituted with one or more —N(RN1)(RN2) wherein RN1 is —H and RN2 is a chain of one or more naturally or non-naturally occurring amino acids.
In another embodiment, X1 may be selected from the group consisting of:
In one embodiment, X1 is aspartyl, succinyl or maleyl. In one embodiment, preferably X1 is aspartyl. In one embodiment, X1 is aspartyl or glutamyl, and is substituted at the N-terminus with a chain of one or more naturally or non-naturally occurring amino acids.
When X1 is aspartyl, it is preferably L-aspartyl (D) or D-aspartyl (d). When X1 is glutamyl it is preferably L-glutamyl (E) or D-glutamyl (e).
Group X2
It is believed that the side chain of group X2 interacts with the βTrCP binding domain to form an ionic bridge. Accordingly, X2 is a functional group which is ionisable at physiological pH, in particular carboxylic acid groups or bioisosteres thereof, in order to sustain such a binding interaction with the βTrCP binding domain.
X2 is a group —N(Ra) —Y1(-L1-A2)-Z2—;
wherein;
Ra is selected from the group consisting of —H, C1-C10 alkyl, aryl and heteroaryl;
L1 is C0-C5 alkyl, C2-C5 alkenyl or C2-C5 alkynyl; wherein, L1 may be substituted with one or more RL1, wherein RL1 is C1-C4 alkyl;
Y1 is CH or N;
Z2 is selected from the group consisting of C═O, C═S, CH2, S═O, S(O)2, C═N(C1-C4 alkyl) and C═NH; and
A2 is carboxylic acid (—CO2H) or a bioisostere thereof or —C(O)N(RN1)2; wherein, A2 may be substituted with one or more RA2, wherein RA2 is selected from the group consisting of —H, C1-C4 alkyl, C2-C4 alkenyl and aryl.
In one embodiment, Ra is —H. In one embodiment, Ra is C1-C10 alkyl.
In one embodiment, Y1 is CH. In one embodiment, Y1 is N.
In one embodiment, A2 is carboxylic acid. In one embodiment, A2 is selected from the group consisting of
wherein, R1 is RA2.
In one embodiment, A2 is selected from the group consisting of
wherein, R1 is RA2.
In one embodiment, A2 is selected from the group consisting of
wherein, R1 is RA2.
In one embodiment, A2 is selected from the group consisting of carboxylic acid (—CO2H), phosphate, phosphonate, phosphonic acid, tetrazole and sulphate.
In one embodiment, A2 is selected from the group consisting of carboxylic acid and phosphate. In one embodiment, preferably A2 is carboxylic acid.
In one embodiment, A2 is substituted with one or more RA2, wherein RA2 is selected from the group consisting of —H, C1-C4 alkyl, C2-C4 alkenyl and aryl. In one embodiment, A2 is substituted with one or more RA2, wherein RA2 is methyl or ethyl. In one embodiment, A2 is substituted with one or more RA2, wherein RA2 is methyl.
In one embodiment A2 is —C(O)N(RN1)2 wherein each RN1 may be the same or different. Particularly A2 is C(O)NH(RN1), more particularly A2 is C(O)NH2
In one embodiment, L1 is C0-C5 alkyl or C2-C5 alkenyl. In one embodiment, L1 is C0-C5 alkyl. In one embodiment, L1 is preferably C1-C2 alkyl.
In one embodiment, L1 is substituted with one or more RL1, wherein RL1 is C1-C4 alkyl. In one embodiment, L1 is substituted with one or more RL1, wherein RL1 is methyl.
In one embodiment, X2 is of Formula —NH—Y1(-L1-A2)-Z2—; wherein L1 is C1-C5 alkyl, Y1 is CH, Z2 is C═O and A2 is carboxylic acid (—CO2H) or phosphate. In one embodiment, X2 is of Formula —NH—Y1(-L1-A2)-Z2—; wherein L1 is C1-C2 alkyl, Y1 is CH, Z2 is C═O and A2 is carboxylic acid (—CO2H) or phosphate. In one embodiment, X2 is of Formula —NH—Y1(-L1-A2)-Z2—; wherein L1 is C1 alkyl, Y1 is CH, Z2 is C═O and A2 is carboxylic acid (—CO2H) or phosphate. In one embodiment, X2 is of Formula —NH—Y1(-L1-A2)-Z2—; wherein L1 is C2 alkyl, Y1 is CH, Z2 is C═O and A2 is carboxylic acid (—CO2H) or phosphate. In one embodiment, X2 is of Formula —NH—Y1(-L1-A2)-Z2—; wherein L1 is C1 alkyl, Y1 is CH, Z2 is C═O and A2 is carboxylic acid (—CO2H). In one embodiment, X2 is of Formula —NH—Y1(-L1-A2)-Z2—; wherein L1 is C1 alkyl, Y1 is CH, Z2 is C═O and A2 is phosphate. In one embodiment, X2 is of Formula —NH—Y1(-L1-A2)-Z2—; wherein L1 is C2 alkyl, Y1 is CH, Z2 is C═O and A2 is carboxylic acid (—CO2H). In one embodiment, X2 is of Formula —NH—Y1(-L1-A2)-Z2—; wherein L1 is C2 alkyl, Y1 is CH, Z2 is C═O and A2 is phosphate. In one embodiment, X2 is of Formula —NH—Y1(-L1-A2)-Z2—; wherein L1 is C1 alkyl substituted with RL1, wherein RL1 is methyl, Y1 is CH, Z2 is C═O and A2 is carboxylic acid (—CO2H). In one embodiment, X2 is of Formula —NH—Y1(-L1-A2)-Z2—; wherein L1 is C1 alkyl substituted with RL1, wherein RL1 is methyl, Y1 is CH, Z2 is C═O and A2 is phosphate.
In one embodiment, group X2 may be a glutamate, an aspartate, or a phosphorylated serine residue. In one embodiment, preferably, X2 is glutamate or aspartate. In one embodiment, the glutamate, aspartate, or phosphorylated serine residue of group X2 is an
In one embodiment, preferably, group X2 is a glutamate or aspartate residue. This eliminates the requirement for phosphorylated serine residues, which are naturally present within the phosphodegeneron sequence, whilst retaining binding. The negatively charged phosphorylated serine residues are not synthetically desirable.
In one embodiment, the glutamate, aspartate or phosphorylated serine residue of X2 may be substituted with methyl. In another embodiment, the glutamate, aspartate or phosphorylated serine residue of group X2 may be substituted with ethyl.
Group X3
It is believed that group X3 associates with an area in the βTrCP binding domain which may accommodate a compound/modified peptide with a beta-turn.
Accordingly, X3 is a functional group which is suitably configured to reside in this area of the βTrCP binding domain.
X3 is a group —N(Rb)—Y2—Z3—;
wherein,
Rb is selected from the group consisting of —H, C1-C10 alkyl, aryl and heteroaryl;
Y2 is CF2, CH2, N(RY2) or O; wherein, RY2 is —H or C1-C4 alkyl; and
Z3 is selected from the group consisting of C═O, C═S, CH2, S═O, S(O)2, C═N(C1-C4 alkyl) and C═NH.
In one embodiment, Rb is —H or C1-C10 alkyl. In one embodiment, preferably Rb is —H.
In one embodiment, Y2 is CH2 or N(RY2). In one embodiment, Y2 is preferably CH2.
In one embodiment, Y2 is N(RY2). In one embodiment, Y2 is N(RY2), wherein RY2 is methyl.
In one embodiment, X3 is of Formula —NH—Y2—Z3—, wherein Y2 is CH2, N(RY2) or O and Z3 is selected from the group consisting of C═O, C═S, CH2, S═O and S(O)2. In one embodiment, preferably, X3 is of Formula —NH—Y2—Z3—, wherein Y2 is CH2 and Z3 is C═O. In one embodiment, X3 is of Formula —NH—Y2—Z3—, wherein Y2 is NH and Z3 is C═O.
In one embodiment, preferably group X3 is a glycine residue.
In one embodiment the glycine residue of group X3 is an aza glycine residue, wherein an “aza amino acid” is an
In one embodiment the glycine residue of group X3 is an oxo glycine residue, wherein an “oxo amino acid” is an
Group X4
Without wishing to be bound by theory, it is believed that the side chain of group X4 sustains a Van der Waals interaction with the βTrCP binding domain.
X4 is a group —N(Rc)—Y3(-L2-A3)-Z4—; or —N(Rc)—Y3(-L2)-Z4—
wherein,
Rc is selected from the group consisting of —H, C1-C10 alkyl, aryl and heteroaryl;
L2 is C0-C5 alkyl, C2-C5 alkenyl or C2-C5 alkynyl; wherein, L2 may be substituted with one or more RL2, wherein RL2 is C1-C4 alkyl or C2-C4 alkenyl;
Y3 is CH or N;
Z4 is selected from the group consisting of C═O, C═S, CH2, S═O, S(O)2, C═N(C1-C4 alkyl) and C═NH; and
A3 is aryl or heteroaryl; wherein, A3 may be substituted with one or more RA3, wherein, RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —CN, —NO2, —CF3, —OCF3, —CO2H, —C1-C10 alkyl, —NH2, —NH(C1-C2 alkyl) and —N(C1-C2 alkyl)2;
In one embodiment, X4 is a group —N(Rc)—Y3(-L2-A3)-Z4—.
In one embodiment, X4 is a group —N(Rc)—Y3(-L2)-Z4—.
In one embodiment, Rc is —H or C1-C10 alkyl. In one embodiment, preferably Rc is —H.
In one embodiment, Y3 is CH. In one embodiment, Y3 is N.
In one embodiment, L2 is C0-C5 alkyl or C2-C5 alkenyl. In one embodiment, L2 is C0-C5 alkyl. In one embodiment, L2 is C1-C2 alkyl. In one embodiment, preferably L2 is C1 alkyl.
In one embodiment, L2 is substituted with one or more RL2, wherein RL2 is C1-C4 alkyl or C2-C4 alkenyl. In one embodiment, L2 is substituted with RL2, wherein RL2 is methyl.
In one embodiment, A3 is aryl. In one embodiment, A3 is phenyl. In one embodiment, A3 is heteroaryl. In one embodiment, A3 is aryl or heteroaryl substituted with one or more RA3, wherein, RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —CN, —NO2, —CF3, —OCF3, —CO2H, —C1-C10 alkyl, —NH2, —NH(C1-C2 alkyl) and —N(C1-C2 alkyl)2.
In one embodiment, A3 is aryl or heteroaryl, wherein said aryl or heteroaryl is substituted with one or more RA3, wherein RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl.
In one embodiment, A3 is aryl or heteroaryl, wherein said aryl or heteroaryl is substituted with one or more RA3, wherein RA3 is selected from the group consisting of —F, —Cl, —OH and —NO2.
In one embodiment, A3 is phenyl substituted at one or more of the 2-, 3- or 4-positions, with RA3, wherein RA3 is a substituent selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —CN, —NO2, —CF3, —OCF3, —CO2H and —C1-C10 alkyl.
In one embodiment, preferably A3 is phenyl substituted with one or more RA3, wherein RA3 is selected from the group consisting of —F, —Cl, —NO2 and —OH.
In another embodiment, preferably A3 is phenyl substituted with RA3, wherein RA3 is chlorine and/or fluorine.
In one embodiment, Z4 is selected from the group consisting of C═O, C═S and CH2.
In one embodiment, Z4 is C═O. In one embodiment, X4 is of Formula —NH—Y3(-L2-A3)-Z4—; wherein Y3 is CH, Z4 is C═O, L2 is C1-C5 alkyl and A3 is aryl or heteroaryl, wherein said aryl or heteroaryl is substituted with one or more RA3.
In one embodiment, X4 is of Formula —NH—Y3(-L2-A3)-Z4—; wherein Y3 is CH, Z4 is C═O, L2 is C1-C5 alkyl and A3 is aryl or heteroaryl, wherein said aryl or heteroaryl is substituted with one or more RA3, wherein RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —CN, —NO2, —CF3, —OCF3, —CO2H, —C1-C10 alkyl, —NH2, —NH(C1-C2 alkyl) and —N(C1-C2 alkyl)2.
In one embodiment, X4 is of Formula —NH—Y3(-L2-A3)-Z4—; wherein Y3 is CH, Z4 is C═O, L2 is C1-C5 alkyl and A3 is aryl or heteroaryl, wherein said aryl or heteroaryl is substituted with one or more RA3, wherein RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), C1-C10 alkyl and —NO2.
In one embodiment, X4 is of Formula —NH—Y3(-L2-A3)-Z4—; wherein Y3 is CH, Z4 is C═O, L2 is C1 alkyl and A3 is phenyl, wherein said phenyl is substituted with one or more RA3, wherein RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl) and —NO2.
In one embodiment, X4 is of Formula —NH—Y3(-L2-A3)-Z4—; wherein Y3 is CH, Z4 is C═O, L2 is C1 alkyl and A3 is selected from the group consisting of indole and imidazole, wherein said indole or imidazole is substituted with one or more RA3, wherein RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), C1-C10 alkyl and —NO2.
In one embodiment, X4 is of Formula —NH—Y3(-L2-A3)-Z4—; wherein Y3 is CH, Z4 is C═O, L2 is C1 alkyl and A3 is phenyl, wherein said phenyl is substituted at one or more of the 2-, 3- or 4-positions with RA3, wherein RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), C1-C10 alkyl and —NO2.
In one embodiment, X4 is of Formula —NH—Y3(-L2-A3)-Z4—; wherein Y3 is CH, Z4 is C═O, L2 is C1 alkyl and A3 is phenyl, wherein said phenyl is substituted at one or more of the 2-, 3- or 4-positions with RA3, wherein RA3 is selected from the group consisting of —F, —Cl, —NO2 and —OH.
In one embodiment, group X4 is an aromatic alanine derivative.
In one embodiment, preferably group X4 is selected from the group consisting of phenylalanine, tyrosine, tryptophan and histidine. In one embodiment, X4 is phenylalanine. In one embodiment, group X4 may be an L amino acid. In one embodiment, group X4 is selected from the group consisting of phenylalanine, tyrosine, tryptophan and histidine, wherein said phenylalanine, tyrosine, tryptophan and histidine is substituted with one or more RA3.
In one embodiment, group X4 is selected from the group consisting of:
Group X5
It is believed that the side chain of group X5 interacts with an alkyl portion within the βTrCP binding domain. Accordingly, X5 is a functional group which is capable of sustaining such an interaction within the βTrCP binding domain.
X5 is a group —N(Rd)—Y4(-L3-A4)-Z5—;
wherein;
Rd is selected from the group consisting of —H, C1-C10 alkyl, aryl and heteroaryl;
Y4 is CH or N;
L3 is C0-C5 alkyl, C2-C5 alkenyl or C2-C5 alkynyl; wherein, L3 may be substituted with one or more RL3, wherein RL3 is C1-C4 alkyl;
A4 is aryl or heteroaryl; wherein, A4 may be substituted with one or more RA4, wherein RA4 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —CN, —NO2, —CF3, —OCF3, —CO2H, —C1-C10 alkyl, —NH2, —NH(C1-C2 alkyl) and —N(C1-C2 alkyl)2; and
Z5 is selected from the group consisting of C═O, C═S, CH2, S═O, S(O)2, C═N(C1-C4 alkyl) and C═NH.
In one embodiment, Rd is —H or C1-C10 alkyl. In one embodiment, preferably Rd is —H.
In one embodiment, Y4 is CH. In one embodiment, Y4 is N.
In one embodiment, L3 is C0-C5 alkyl or C2-C5 alkenyl. In one embodiment, L3 is C0-C5 alkyl. In one embodiment, L3 is C1-C2 alkyl. In one embodiment, preferably L3 is C1 alkyl.
In one embodiment, L3 is substituted with RL3, wherein RL3 is C1-C4 alkyl. In one embodiment, L3 is substituted with RL3, wherein RL3 is methyl.
A4 is aryl or heteroaryl. In one embodiment, A4 is aryl. In one embodiment, A4 is bi-aryl, monocyclic aryl or polycyclic fused ring aryl. In one embodiment, A4 is heteroaryl. In one embodiment, A4 is monocyclic heteroaryl. In one embodiment, A4 is polycyclic fused ring heteroaryl.
In one embodiment, A4 is selected from the group consisting of:
In one embodiment, A4 is selected from the group consisting of phenyl, biphenyl, naphthyl, indenyl, fluorenyl, anthracyl and phenanthryl. In one embodiment, A4 is selected from the group consisting of pyridyl, thienyl, furanyl, pyrrolyl, pyrazolyl, imidazoyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazoyl, oxadiazolyl, thiadiazolyl and tetrazolyl. In one embodiment, A4 is selected from the group consisting of indolyl, benzofuranyl, quinolyl, isoquinolyl, indazolyl, indolinyl, isoindolyl, indolizinyl, benzimidazolyl or quinolinyl. In one embodiment, A4 is selected from the group consisting of phenyl, naphthyl, indolyl and imidazoyl.
In one embodiment, A4 is aryl or heteroaryl, wherein said aryl or heteroaryl are substituted with one or more RA4, wherein RA4 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —CN, —NO2, —CF3, —OCF3, —CO2H, —C1-C10 alkyl, —NH2, —NH(C1-C2 alkyl) and —N(C1-C2 alkyl)2.
In one embodiment, X5 is of Formula —NH—Y4(-L3-A4)-Z5—; wherein Y4 is CH, Z5 is C═O, L3 is C1-C5 alkyl and A4 is aryl. In one embodiment, X5 is of Formula —NH—Y4(-L3-A4)-Z5—; wherein Y4 is CH, Z5 is C═O, L3 is C1-C5 alkyl and A4 is heteroaryl.
In one embodiment, preferably, X5 is of Formula —NH—Y4(-L3-A4)-Z5—; wherein Y4 is CH, Z5 is C═O, L3 is C1 alkyl and A4 is selected from the group consisting of phenyl, naphthyl, indolyl and imidazoyl.
In one embodiment, preferably group X5 is selected from the group consisting of tryptophan, naphthyl-alanine, histidine and phenylalanine, wherein X5 may be substituted at one or more positions with RA4. In one embodiment, group X5 is selected from the group consisting of tryptophan, 1 naphthyl-alanine, 2 napthyl-alanine, histidine and F(4NO2). In one embodiment, group X5 is tryptophan. In one embodiment, group X5 is tryptophan, wherein the nitrogen of the indole group is substituted with methyl. In one embodiment, group X5 is naphthyl-alanine. In one embodiment, group X5 is 2 naphthyl-alanine. In one embodiment, group X5 is 1 naphthyl-alanine. In one embodiment, group X5 is histidine. In one embodiment, group X5 is phenylalanine. In one embodiment, group X5 is phenylalanine, wherein in phenyl is substituted with one or more RA4. In one embodiment, group X5 is F(4NO2) or F(3NO2). In one embodiment, group X5 is an L amino acid. In one embodiment, the tryptophan, naphthyl-alanine, histidine or phenylalanine residue of group X5 may be substituted at one or more positions with RA4.
Group X6
It is believed that the side chain of group X6 interacts with the βTrCP binding domain to form an ionic bridge. Accordingly, X6 is a functional group which is ionisable at physiological pH, in particular a carboxylic acid group or bioisostere thereof, in order to sustain such a binding interaction with the βTrCP binding domain.
X6 is a group —N(Re)—Y5(-L4-A5)-Z6—;
wherein;
Re is selected from the group consisting of —H, C1-C10 alkyl, aryl and heteroaryl;
L4 is C0-C5 alkyl, C2-C5 alkenyl or C2-C5 alkynyl; wherein L4 may be substituted with one or more RL4, wherein RL4 is C1-C4 alkyl;
Y5 is CH or N;
Z6 is selected from the group consisting of C═O, C═S, CH2, S═O, S(O)2, C═N(C1-C4 alkyl) and C═NH; and
A5 is carboxylic acid (—CO2H) or a bioisostere thereof; wherein, A5 may be substituted with one or more RA5, wherein RA5 is selected from the group consisting of —H, C1-C4 alkyl, C2-C4 alkenyl and aryl.
In one embodiment, Re is —H. In one embodiment, Re is C1-C10 alkyl. In one embodiment, Re is C1-C4 alkyl. In one embodiment, Re is methyl or ethyl. In one embodiment, Re is methyl. In one embodiment, Re is aryl or heteroaryl. In one embodiment, Re is phenyl.
In one embodiment, Y5 is CH. In one embodiment, Y5 is N.
In one embodiment, L4 is C0-C5 alkyl or C2-C5 alkenyl. In one embodiment, L4 is C0-C5 alkyl. In one embodiment, L4 is preferably C1-C2 alkyl.
In one embodiment, L4 is substituted with one or more RL4, wherein RL4 is C1-C4 alkyl.
In one embodiment, A5 is carboxylic acid. In one embodiment, A5 is selected from the group consisting of
wherein, R1 is RA5.
In one embodiment, A5 is selected from the group consisting of
wherein, R1 is RA5.
In one embodiment, A5 is selected from the group consisting of
wherein, R1 is RA5.
In one embodiment, A5 is selected from the group consisting of carboxylic acid (—CO2H), phosphate, phosphonate, phosphonic acid, tetrazole and sulphate.
In one embodiment, A5 is selected from the group consisting of carboxylic acid and phosphate. In one embodiment, preferably, A5 is carboxylic acid.
In one embodiment, A5 is substituted with one or more RA5, wherein RA5 is selected from the group consisting of —H, C1-C4 alkyl, C2-C4 alkenyl and aryl.
In one embodiment, X6 is of Formula —N(Re)—Y5(-L4-A5)-Z6—; wherein Re is —H, C1-C10 alkyl, aryl or heteroaryl, L4 is C1-C5 alkyl, Y5 is CH, Z6 is C═O and A5 is carboxylic acid (—CO2H) or phosphate. In one embodiment, X6 is of Formula —N(Re)—Y5(-L4-A5)-Z6—; wherein Re is —H, C1-C10 alkyl, aryl or heteroaryl, L4 is C1-C2 alkyl, Y5 is CH, Z6 is C═O and A5 is carboxylic acid (—CO2H) or phosphate. In one embodiment, X6 is of Formula —N(Re)—Y5(-L4-A5)-Z6—; wherein Re is —H, C1-C4 alkyl or aryl, L4 is C1-C2 alkyl, Y5 is CH, Z6 is C═O and A5 is carboxylic acid (—CO2H) or phosphate. In one embodiment, X6 is of Formula —N(Re)—Y5(-L4-A5)-Z6—; wherein Re is methyl, L4 is C1-C2 alkyl, Y5 is CH, Z6 is C═O and A5 is carboxylic acid (—CO2H) or phosphate. In one embodiment, X6 is of Formula —NH—Y5(-L4-A5)-Z6—; wherein L4 is C1 alkyl, Y5 is CH, Z6 is C═O and A5 is carboxylic acid (—CO2H) or phosphate. In one embodiment, X6 is of Formula —NH—Y5(-L4-A5)-Z6—; wherein L4 is C2 alkyl, Y5 is CH, Z6 is C═O and A5 is carboxylic acid (—CO2H) or phosphate. In one embodiment, X6 is of Formula —NH—Y5(-L4-A5)-Z6—; wherein L4 is C1 alkyl, Y5 is CH, Z6 is C═O and A5 is carboxylic acid (—CO2H). In one embodiment, X6 is of Formula —NH—Y5(-L4-A5)-Z6—; wherein L4 is C1 alkyl, Y5 is CH, Z6 is C═O and A5 is phosphate. In one embodiment, X6 is of Formula —N(CH3)—Y5(-L4-A5)-Z6—; wherein L4 is C1 alkyl, Y5 is CH, Z6 is C═O and A5 is carboxylic acid (—CO2H) or phosphate. In one embodiment, X6 is of Formula —N(CH3)—Y5(-L4-A5)-Z6—; wherein L4 is C2 alkyl, Y5 is CH, Z6 is C═O and A5 is carboxylic acid (—CO2H) or phosphate. In one embodiment, X6 is of Formula —N(CH3)—Y5(-L4-A5)-Z6—; wherein L4 is C1 alkyl, Y5 is CH, Z6 is C═O and A5 is carboxylic acid (—CO2H). In one embodiment, X6 is of Formula —N(CH3)—Y5(-L4-A5)-Z6—; wherein L4 is C1 alkyl, Y5 is CH, Z6 is C═O and A5 is phosphate. In one embodiment, X6 is of Formula —NH—Y5(-L4-A5)-Z6—; wherein L4 is C2 alkyl, Y5 is CH, Z6 is C═O and A5 is carboxylic acid (—CO2H). In one embodiment, X6 is of Formula —NH—Y5(-L4-A5)-Z6—; wherein L4 is C2 alkyl, Y5 is CH, Z6 is C═O and A5 is phosphate. In one embodiment, X6 is of Formula —NH—Y5(-L4-A5)-Z6—; wherein L4 is C1 alkyl substituted by methyl, Y5 is CH, Z6 is C═O and A5 is carboxylic acid (—CO2H). In one embodiment, X6 is of Formula —NH—Y5(-L4-A5)-Z6—; wherein L4 is C1 alkyl substituted by methyl, Y5 is CH, Z6 is C═O and A5 is phosphate.
In one embodiment, group X6 may be a glutamate, an aspartate or a phosphorylated serine residue. In one embodiment, the glutamate, aspartate or phorphorylated serine residue of group X6 is an L amino acid. In a further embodiment, X6 may be phosphorylated threonine.
In one embodiment, preferably group X6 is a glutamate or aspartate residue. This eliminates the requirement for phosphorylated serine residues, which are naturally present within the phosphodegeneron sequence, whilst retaining binding. The negatively charged phosphorylated serine residues are not synthetically desirable.
In one embodiment, the glutamate, aspartate or phosphorylated serine residue of X6 may be substituted with methyl. In another embodiment, the glutamate, aspartate or phosphorylated serine residue of group X6 may be substituted with ethyl.
Group X7
It is believed that group X7 forms a hydrogen bond with the βTrCP binding domain. Accordingly, X7 is a functional group which is capable of forming such a hydrogen bond with the βTrCP binding domain.
X7 is a group —N(RN1)(RN2); wherein RN1 and RN2 are as previously defined. In one embodiment, preferably X7 is —NH2. In one embodiment, X7 is —N(RN1)2, wherein one RN1 is —H and the other RN1 is C1-C10 alkyl or aryl. In one embodiment, X7 is —N(RN1)2, wherein both RN1 are independently C1-C10 alkyl or aryl.
In one embodiment, X7 is —N(RN1)(RN2), wherein RN2 is —(CH2)0-10-(Z)0-1-Aa, and wherein Aa is —OH. In one embodiment, X7 is —N(RN1)(RN2), wherein RN2 is —(CH2)0-10-(Z7)0-1-Aa, and wherein Aa is —NH2. In one embodiment, X7 is —N(RN1)(RN2), wherein RN2 is —(CH2)4-8-(Z7)0-1-Aa, and wherein Aa is —NH2. In one embodiment, X7 is —N(RN1)(RN2), wherein RN2 is —(CH2)0-10-(Z7)0-1-Aa, and wherein Aa is —C(O)NH2. In one embodiment, X7 is —N(RN1)(RN2), wherein RN2 is —(CH2)0-10-(Z7)0-1-Aa, and wherein Aa is a chain of one or more naturally occurring amino acids. In one embodiment, X7 is —N(RN1)(RN2), wherein RN2 is —(CH2)0-10-(Z7)0-1-Aa, and wherein Aa is a chain of one or more non-naturally occurring amino acids. In one embodiment, X7 is —N(RN1)(RN2), wherein RN2 is —(CH2)0-10-(Z)0-1-Aa, and wherein Aa is a chain of a mixture of one or more naturally occurring amino acids and one or more non-naturally occurring amino acids. In one embodiment, X7 is —N(RN1)(RN2), wherein RN2 is —(CH2)0-10—(Z7)0-1-Aa, and wherein Aa is a cholesteryl derivative.
In one embodiment, X7 is —N(RN1)(RN2), wherein RN2 is —(CH2CH2O)1-10-CH2CH3. In one embodiment, X7 is —N(RN1)(RN2), wherein RN2 is —(CH2CH2O)1-10-(CH2)1-3-(Z7)0-1-Aa, and wherein Aa is —NH2 or —C(O)NH2. In one embodiment, X7 is —N(RN1)(RN2), wherein RN2 is —(CH2CH2O)1-10-(CH2)1-3-(Z7)0-1-Aa, and wherein Aa is a chain of one or more naturally occurring amino acids. In one embodiment, X7 is —N(RN1)(RN2), wherein RN2-(CH2CH2O)4-8-(CH2)1-3-(Z7)0-1-Aa, and wherein Aa is a chain of one or more naturally occurring amino acids. In one embodiment, X7 is —N(RN1)(RN2), wherein RN2-(CH2CH2O)1-10-(CH2)1-3-(Z7)0-1-Aa, and wherein Aa is a chain of one or more non-naturally occurring amino acids. In one embodiment, X7 is —N(RN1)(RN2), wherein RN2-(CH2CH2O)4-8-(CH2)1-3-(Z7)0-1-Aa, and wherein Aa is a chain of one or more non-naturally occurring amino acids. In one embodiment, X7 is —N(RN1)(RN2), wherein RN2-(CH2CH2O)1-10-(CH2)1-3-(Z7)0-10-Aa, and wherein Aa is a cholesteryl derivative. In one embodiment RN2 is —(CH2)0-10-(Z)0-1-Aa and Aa is a cholesteryl derivative, in particular the cholesteryl derivative is:
wherein RN1 is as previously defined, Y6 is CH or N and Z8 is selected from the group consisting of C═O, C═S, CH2, S═O, S(O)2, C═N(C1-C4) alkyl) and C═NH. In particular, the cholesteryl derivative is:
It is believed that the cholesteryl group enhances the cell penetration of the compounds and modified peptides of the invention, without affecting the activity of the compounds and modified peptides of the invention against the targets of the invention.
Additional Amino Acids/Chains of Substituents
Compounds of the invention may additionally contain one or two chains of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids. For example, in one embodiment, group B may be substituted with —NH(RN2) or —N(RN2)2, wherein one RN2 is a chain of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more naturally occurring or non-naturally occurring amino acids. Alternatively, group X7 may be of Formula —N(RN1)(RN2), wherein RN1 is preferably —H and RN2 is a chain of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more naturally or non-naturally occurring amino acids.
Alternatively, group E may be substituted with —NH(RN2) or —N(RN2)2, wherein one RN2 is a chain of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more naturally occurring or non-naturally occurring amino acids and X7 may be of Formula —N(RN1)(RN2), wherein RN2 is a chain of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids, thus providing a compound containing two additional chains of amino acids. The one or more naturally occurring or non-naturally occurring amino acids are independently selected from the group consisting of L-amino acids, D-amino acids and aza-amino acids.
Chains of non-naturally occurring amino acids may include peptoids, which are peptidomimetics whose side chains are appended to the nitrogen atom of the peptide, rather than to the alpha-carbons.
Modified Peptide Embodiment of Formula Ia
In one embodiment, the compound of Formula Ia may comprise a sequence of amino acids according to the following Formula Ic:
X1-E/D/pS-G-X4-X5-E/D/pS-NHRN2 Formula Ic
X1, X4, X5 and RN2 are as previously defined.
In one embodiment, the compound of Formula Ia may comprise a sequence of amino acids according to the following Formula Iv:
d-E-G-F(3F)—W-E-NHRN2 Formula Iv
Herein “comprise” is used in the open sense to indicate that additional amino acids may also be present in the sequence.
The amino acids of the Formula depicted above preferably form a contiguous sequence.
In order to arrive at Formula Ic, the inventors used the phosphodegeneron sequence as a starting point, and systematically substituted each of the amino acids to alternative natural and non-natural amino acids. At each stage the binding of the substituted peptides to βTrCP was assessed and further substituted peptides were designed in order to maximise binding.
Within the meaning of the present invention, the term “modified” indicates that the peptide is not naturally occurring. A modified peptide may contain one or more non-naturally occurring amino acids, and/or may include one or more moieties which are not classified as amino acids.
Within the present invention, the term “residue” will be used to refer to each of the component moieties of the modified peptide, whether these are amino acids or other chemical moieties. Within Formula Ic, the individual residues are shown separated by hyphens (“-”).
Where the residues of the modified peptide are amino acids, each of the amino acids may be independently selected from an L-amino acid, a D-amino acid and an aza-amino acid. One or more of the residues may additionally be independently substituted at one or more positions irrespective of which subtype of amino acid forms the basis for the residue.
An “L amino acid” is defined as an amino acid which can theoretically be synthesised from levorotatory glyceraldehyde. Amino acids found in naturally occurring proteins are usually L amino acids. According to generally accepted notation, L amino acids are depicted herein using the capital letter single letter amino acid code.
A “D amino acid” is the stereoisomer of an L amino acid and is defined as an amino acid which can theoretically be synthesised from dextrorotary glyceraldehyde.
According to generally accepted notation, D amino acids are depicted herein using the lower case single letter amino acid code.
An “aza amino acid” is an L amino acid in which the α-carbon atom has been replaced by a nitrogen atom. The replacement of the αC—COOH bond found in naturally occurring amino acids with an αN—COOH bond can increase the stability of a peptide.
Herein, the suffix “p” is used to denote a phosphorylated residue, e.g. “pS” denotes phosphorylated serine.
The compounds of the present invention bind to βTrCP. In one embodiment the compounds are considered to “bind to βTrCP” if they bind with an affinity of less than about 10M. In some embodiments the compounds/peptides may bind to βTrCP with an affinity of less than about 900 nM, less than about 800 nM, less than about 700 nM, less than about 600 nM, less than about 500 nM, less than about 400 nM, less than about 300 nM, less than about 200 nM, less than about 150 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 9 nM, less than about 8 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM or less.
Capping Groups
The compounds or modified peptides of the present invention may comprise a capping group. The function of the capping group is to increase the stability of the compound towards enzymic degradation, thus improving cell penetration, and any groups which are known to perform this function may be used as capping groups. In embodiments where a capping group is present, the definitions given for compounds of Formula Ia, Ib and Ic above equally apply.
In one embodiment, any amino/amine group, in particular an —NH2, —NH(RN1), or —NH(RN2) group which is present in a compound of the present invention may be capped, by replacement of a H atom with a capping group. Suitable capping groups include any groups which are known to prevent the compound from being degraded on entry into a cell.
In one embodiment, the capping group may be selected from the group consisting of
and the thiocarbonyl derivatives of any of these capping groups.
The person skilled in the art will recognise that R* is used to indicate a generic structure for the purposes of illustrating the various functional groups which may be suitable as amine/amino capping groups. Specific examples of capping groups are illustrated below.
In one embodiment, a compound of the present invention is substituted by an amino/amine group, in particular an —NH2, —NH(RN1), or —NH(RN2) group as defined previously, wherein said amino/amine group, in particular the —NH2, —NH(RN1), or —NH(RN2) group, is capped, by the replacement of a H atom with a capping group selected from the group consisting of:
wherein,
Rcg is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —CN, —NO2, —CF3, —OCF3, —CO2H, —NH2, —NH(C1-C2 alkyl), —N(C1-C2 alkyl)2, —C1-C10 alkyl, aryl and heteroaryl.
In a further embodiment, the capping group may be selected from the group consisting of:
Further capping groups which may be used in the synthesis of compounds of the present invention are:
Acidic capping groups which may be used in the synthesis of compounds of the present invention are:
In one embodiment, group B is substituted by a substituent of Formula —NH2, —NH(RN1), or —NH(RN2), wherein the —NH2, —NH(RN1) or —NH(RN2) substituent is capped, by the replacement of a H atom, with a capping group selected from the group consisting of:
wherein, Rcg is as previously defined.
In a further embodiment, group B is substituted by a substituent of Formula —NH2, —NH(RN1), —NH(RN2), wherein the —NH2, —NH(RN1), —NH(RN2) substituent is capped, by the replacement of a H atom, with a capping group selected from the group consisting of:
In one embodiment, X1 is aspartyl or glutamyl and comprises a capping group. In one embodiment, X1 is aspartyl and comprises a capping group on the N-terminus. In one embodiment, X1 is aspartyl or glutamyl and comprises a capping group on the N-terminus, wherein the capping group is selected from the group consisting of:
wherein, Rcg is as previously defined.
In a further embodiment, X1 is aspartyl or glutamyl and comprises a capping group on the N-terminus, wherein the capping group is selected from the group consisting of:
In one embodiment, X1 is aspartyl and comprises a capping group on the N-terminus, wherein the capping group is selected from the group consisting of:
wherein, Rcg is as previously defined.
In one embodiment, X1 is aspartyl and comprises a capping group on the N-terminus, wherein the capping group is selected from the group consisting of:
Preferred capping groups are those selected from List 1:
In a further embodiment, group B is substituted by a substituent of Formula —NH2, —NH(RN1), —NH(RN2), wherein the —NH2, —NH(RN1), —NH(RN2) substituent is capped, by the replacement of a H atom, with a capping group selected from the group consisting of those selected from List 2:
In such an embodiment, X1 may be aspartyl or glutamyl, in particularly aspartyl, which comprises a capping group on the N-terminus, wherein the capping group is selected from the group consisting of List 2.
As described above, in some embodiments RN2 is —(CH2)0-10-(Z7)0-1-Aa, wherein Z7 is C═O, and Aa is a chain of one or more non-naturally occurring amino acids, or a chain of one or more naturally occurring amino acids, or a chain of a mixture of one or more naturally occurring amino acids and one or more non-naturally occurring amino acids; wherein the one or more non-naturally occurring or naturally occurring amino acids are independently selected from the group consisting of L-amino acids, D-amino acids and aza-amino acids and comprises a capping group, wherein the capping group is selected from the group consisting of List 3:
In particularly Aa may be lysyl, with a capping group on an N as illustrated below (Formula M), in particular where the capping group is a capping group selected from List 3.
Advantageously, the capping group on Aa does not have a detrimental effect on the activity of the compounds or modified peptides of the invention.
Where the compounds or modified peptides of the present invention include more than one capping group, all combinations of the capping groups described herein are envisaged. In particular, when group B has a capping group selected from List 2, and RN2 is as defined above in association with List 3, and has a capping group selected from List 3, all combinations of capping groups from List 2 and List 3 are envisaged.
Particular combinations of capping groups may increase the ability of the compounds and modified peptides of the invention to penetrate cells.
Exemplary combinations of capping groups are as follows;
X1 is aspartyl or glutamyl and comprises a capping group on the N-terminus, wherein the capping group is:
and;
RN2 is, —(CH2)0-10-(Z7)0-1-Aa, wherein Z7 is C═O, and Aa is lysyl and comprises a capping group as illustrated in Formula M, wherein the capping group is;
X1 is aspartyl or glutamyl and comprises a capping group on the N-terminus, wherein the capping group is:
and;
RN2 is, —(CH2)0-10-(Z7)0-1-Aa, wherein Z7 is C═O, and Aa is lysyl and comprises a capping group as illustrated in Formula M, wherein the capping group is;
X1 is aspartyl or glutamyl and comprises a capping group on the N-terminus, wherein the capping group is:
and;
RN2 is, —(CH2)0-10-(Z7)0-1-Aa, wherein Z7 is C═O, and Aa is lysyl and comprises a capping group as illustrated in Formula M, wherein the capping group is;
X1 is aspartyl or glutamyl and comprises a capping group on the N-terminus, wherein the capping group is:
and;
RN2 is, —(CH2)0-10-(Z7)0-1-Aa, wherein Z7 is C═O, and Aa is lysyl and comprises a capping group as illustrated in Formula M, wherein the capping group is;
X1 is aspartyl or glutamyl and comprises a capping group on the N-terminus, wherein the capping group is:
and;
RN2 is, —(CH2)0-10-(Z7)0-1-Aa, wherein Z7 is C═O, and Aa is lysyl and comprises a capping group as illustrated in Formula M, wherein the capping group is;
X1 is aspartyl or glutamyl and comprises a capping group on the N-terminus, wherein the capping group is:
and;
RN2 is, —(CH2)0-10-(Z7)0-1-Aa, wherein Z7 is C═O, and Aa is lysyl and comprises a capping group as illustrated in Formula M, wherein the capping group is;
X1 is aspartyl or glutamyl and comprises a capping group on the N-terminus, wherein the capping group is:
RN2 is, —(CH2)0-10-(Z7)0-1-Aa, wherein Z7 is C═O, and Aa is lysyl and comprises a capping group as illustrated in Formula M, wherein the capping group is;
A capping group can be added to a compound according to any one of the above-described aspects of the invention. For example, the compound may be of Formula Id:
Capping group-X1-X2-X3-X4-X5-X6-X7 Formula Id
In one embodiment, the compound may be of Formula Ie
X1-X2-X3-X4-X5-X6-X7-Capping group Formula Ie
In one embodiment, the compound may be of Formula If
Capping group-X1-X2-X3-X4-X5-X6-X7-Capping group Formula If
In one embodiment, the compound may be a modified peptide of Formula Ig:
Capping group-X1-E/D/pS-G-X4-X5-E/D/pS-NHRN2 Formula Ig
In particular, the compound may be a modified peptide of Formula Iw:
Capping group-d-E-G-F(3F)—W-E-NHRN2
In particular, the compound may be a modified peptide of Formula Ix:
Capping group-d-E-G-F(3F)—W-E-NHRN2-Capping group
Cyclised Peptides
In one embodiment of the present invention, the compound may be a modified peptide, wherein said modified peptide may be cyclised.
Cyclisation of the modified peptide may require the addition of one or more additional residues to the peptide sequences described above. In particular, enough additional residues are required to enable a carboxy-terminal group at one end of the linear sequence to bind to the amino-terminal group at the other end of the sequence and form a cyclised peptide. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional residues may be required for this purpose.
Chemical Groups
Halo
The term “halogen” (or “halo”) is used herein to refer to fluorine, chlorine, bromine and iodine. In one embodiment, “halogen” is fluorine. In another embodiment, “halogen” is chlorine.
Carbonyl and Carboxy
Structure C═O represents a carbonyl group, which is a carbon atom connected with a double bond to an oxygen atom, and tautomeric forms thereof. A carbonyl group may also be denoted as —C(O)—. Examples of moieties that contain a carbonyl include but are not limited to aldehydes —C(O)H, ketones —C(O)—(C1-C10 alkyl)-, carboxylic acids —CO2H, amides —C(O)NH2, —C(O)—NH(C1-C10 alkyl), —C(O)—N(C1-C10 alkyl)2, —NH—C(O)—(C1-C10 alkyl) and esters —C(O)—O(C1-C10 alkyl).
Amine, Amino Etc.
An amine group is denoted by —NH2, in which a nitrogen atom is covalently bonded to two hydrogen atoms. An alkylamino group is denoted by —NH(C1-C10 alkyl), in which a nitrogen atom is covalently bonded to one hydrogen atom and one (C1-C10 alkyl) group. A dialkylamino group is denoted by —N(C1-C10 alkyl)2, in which a nitrogen atom is bonded to at least two additional (C1-C10 alkyl) groups. Amines may be named in several ways. Typically, a compound is given the prefix “amino” or the suffix “amine”.
Alkyl, Cycloalkyl, Heterocyclyl, Alkenyl, Alkynyl
The term “alkyl” is used herein to refer to monovalent, divalent or trivalent straight or branched, saturated, acyclic hydrocarbyl groups. In one embodiment, alkyl is C1-C10 alkyl, in another embodiment C1-C6 alkyl, in another embodiment C1-C4 alkyl, such as methyl, ethyl, n-propyl, i-propyl, n-butyl or t-butyl groups.
The term “cycloalkyl” is used herein to refer to monovalent, divalent or trivalent saturated, cyclic hydrocarbyl groups. In one embodiment cycloalkyl is C3-10cycloalkyl, in another embodiment, C3-6cycloalkyl, such as cyclopentyl and cyclohexyl.
The term “heterocyclyl” is used herein to refer to monovalent, divalent or trivalent cycloalkyl groups in which up to three carbon atoms, in one embodiment up to two carbon atoms, in another embodiment one carbon atom, are each replaced independently by O, S(O)1-2 or N, provided at least one of the cycloalkyl carbon atoms remains.
Examples of heterocyclyl groups include oxiranyl, thiaranyl, aziridinyl, oxetanyl, thiatanyl, azetidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperidinyl, 1,4-dioxanyl, 1,4-oxathianyl, morpholinyl, 1,4-dithianyl, piperazinyl, 1,4-azathianyl, oxepanyl, thiepanyl, azepanyl, 1,4-dioxepanyl, 1,4-oxathiepanyl, 1,4-oxaazepanyl, 1,4-dithiepanyl, 1,4-thieazepanyl and 1,4-diazepanyl. Other examples include cyclic imides, cyclic anhydrides and thiazolidindiones. The heterocyclyl group may be C-linked or N-linked, i.e. it may be linked to the remainder of the molecule through a carbon atom or through a nitrogen atom.
The term “alkenyl” is used herein to refer to monovalent, divalent or trivalent straight or branched, unsaturated, acyclic hydrocarbyl groups having at least one carbon-carbon double bond and, in one embodiment, no carbon-carbon triple bonds. In one embodiment, alkenyl is C2-C10 alkenyl, in another embodiment, C2-C6 alkenyl, in another embodiment C2-C4 alkenyl.
The term “alkynyl” is used herein to refer to monovalent or divalent unsaturated, acyclic hydrocarbyl groups having at least one carbon-carbon triple bond. In one embodiment alkynyl is C2-C10 alkynyl, in another embodiment, C2-C6 alkynyl, in another embodiment C2-C4 alkynyl.
Aryl
The term “aryl” is used herein to refer to monovalent, divalent or trivalent, aromatic, cyclic hydrocarbyl groups, such as phenyl or naphthyl (e.g. 1-naphthyl or 2-naphthyl). In general, the aryl group may be a monocyclic or polycyclic fused ring aromatic group. Preferred aryl groups are C6-C14aryl. Aryl groups include phenyl, biphenyl, naphthyl, indenyl, fluorenyl, anthracyl and phenanthryl.
Heteroaryl
The term “heteroaryl” is used herein to refer to monovalent, divalent or trivalent, heteroaromatic, cyclic hydrocarbyl groups additionally containing one or more heteroatoms independently selected from O, S, N and NRT, wherein RT is preferably H or C1-C10 alkyl. In general, the heteroaryl group may be a monocyclic or polycyclic fused ring heteroaromatic group. Examples of monocyclic heteroaromatic groups are pyridyl, thienyl, furanyl, pyrrolyl, pyrazolyl, imidazoyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazoyl, oxadiazolyl, thiadiazolyl and tetrazolyl. Examples of polycyclic heteroaromatic groups are indolyl, benzofuranyl, benzothienyl, quinolyl, isoquinolyl, indazolyl, indolinyl, isoindolyl, indolizinyl, benzimidazolyl, quinolinyl and isoquinolinyl. Further examples of heteroaromatic groups include:
Isomeric Forms
Compounds of the invention may exist in one or more geometrical, optical, enantiomeric, diastereomeric and tautomeric forms, including but not limited to cis- and trans-forms, E- and Z-forms, R-, S- and meso-forms, keto-, and enol-forms. All such isomeric forms are included within the invention. The isomeric forms may be in isomerically pure or enriched form, as well as in mixtures of isomers (e.g. racemic or diastereomeric mixtures).
Exemplary Compounds
In one embodiment, the compounds of the invention comprise a sequence of amino acids according to the following Formula Ic:
X1-E/D/pS-G-X4-X5-E/D/pS-NHRN2 Formula Ic
In a further embodiment, a compound of the invention may be a modified peptide of Formula Ig:
Capping group-X1-E/D/pS-G-X4-X5-E/D/pS-NHRN2 Formula Ig
In one embodiment, the compound of the invention may be of Formula (IA)
wherein each R4 is independently —CO2H, —CH2CO2H—OP(O)(OH)2, triazole, tetrazole, sulfonamide or sulphate;
R3 is
and
RA3, RA4 and X1 are as previously defined. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of Formula (IAA)
wherein each R4 is independently —CO2H, —CH2CO2H—OP(O)(OH)2, triazole, tetrazole, sulfonamide or sulphate;
R3 is
RA3, RA4 and X1 are as previously defined, and CG is a capping group. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of formula (IAAA)
wherein each R4 is independently —CO2H, —CH2CO2H—OP(O)(OH)2, triazole, tetrazole, sulfonamide or sulphate;
R3 is
RA3, RA4 and X1 are as previously defined, RN1 and RN2 are as previously defined, and CG is a capping group. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of formula (IAAAA)
wherein each R4 is independently —CO2H, —CH2CO2H—OP(O)(OH)2, triazole, tetrazole, sulfonamide or sulphate;
R3 is
RA3, RA4 and X1 are as previously defined, RN1 is as previously defined, Aa is a chain of one or more non-naturally occurring amino acids, or a chain of one or more naturally occurring amino acids, or a chain of a mixture of one or more naturally occurring amino acids and one or more non-naturally occurring amino acids; wherein the one or more non-naturally occurring or naturally occurring amino acids are independently selected from the group consisting of L-amino acids, D-amino acids and aza-amino acids, in particular Aa is lysyl, and CG is a capping group. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of formula (IAAAAA)
wherein each R4 is independently —CO2H, —CH2CO2H—OP(O)(OH)2, triazole, tetrazole, sulfonamide or sulphate;
R3 is
RA3, RA4 and X1 are as previously defined, RN1 is as previously defined, Aa is a chain of one or more non-naturally occurring amino acids, or a chain of one or more naturally occurring amino acids, or a chain of a mixture of one or more naturally occurring amino acids and one or more non-naturally occurring amino acids; wherein the one or more non-naturally occurring or naturally occurring amino acids are independently selected from the group consisting of L-amino acids, D-amino acids and aza-amino acids, in particular Aa is lysyl, and CG is a capping group. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of formula (IAAAAA), wherein each R4 is independently —CO2H, —CH2CO2H—OP(O)(OH)2, triazole, tetrazole, sulfonamide or sulphate;
R3 is
RA3, RA4 and X1 are as previously defined, RN1 is as previously defined, Aa is a chain of one or more non-naturally occurring amino acids, or a chain of one or more naturally occurring amino acids, or a chain of a mixture of one or more naturally occurring amino acids and one or more non-naturally occurring amino acids; wherein the one or more non-naturally occurring or naturally occurring amino acids are independently selected from the group consisting of L-amino acids, D-amino acids and aza-amino acids, in particular Aa is lysyl, and CG is a capping group, wherein the capping group is selected from List 1. In one embodiment, the capping group on X1 is selected from List 2 and/or the capping group on Aa is selected from List 3. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of Formula (IB)
wherein each R4 is independently —CO2H, —CH2CO2H, —OP(O)(OH)2, triazole, tetrazole, sulfonamide or sulphate;
R3 is
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; and RA4 and X1 are as previously defined. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of Formula (IBB)
wherein each R4 is independently —CO2H, —CH2CO2H, —OP(O)(OH)2, triazole, tetrazole, sulfonamide or sulphate;
R3 is
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; RA4 and X1 are as previously defined, and CG is a capping group. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of Formula (IBBB)
wherein each R4 is independently —CO2H, —CH2CO2H, —OP(O)(OH)2, triazole, tetrazole, sulfonamide or sulphate;
R3 is
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; RA4 and X1 are as previously defined, RN1 and RN2 are as previously defined, and CG is a capping group. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of formula (IBBBB)
wherein each R4 is independently —CO2H, —CH2CO2H, —OP(O)(OH)2, triazole, tetrazole, sulfonamide or sulphate;
R3 is
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; RA4 and X1 are as previously defined, RN1 is as previously defined, Aa is a chain of one or more non-naturally occurring amino acids, or a chain of one or more naturally occurring amino acids, or a chain of a mixture of one or more naturally occurring amino acids and one or more non-naturally occurring amino acids; wherein the one or more non-naturally occurring or naturally occurring amino acids are independently selected from the group consisting of L-amino acids, D-amino acids and aza-amino acids, in particular Aa is lysyl, and CG is a capping group. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of formula (IBBBBB)
wherein each R4 is independently —CO2H, —CH2CO2H, —OP(O)(OH)2, triazole, tetrazole, sulfonamide or sulphate;
R3 is
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; RA4 and X1 are as previously defined, RN1 is as previously defined, Aa is a chain of one or more non-naturally occurring amino acids, or a chain of one or more naturally occurring amino acids, or a chain of a mixture of one or more naturally occurring amino acids and one or more non-naturally occurring amino acids; wherein the one or more non-naturally occurring or naturally occurring amino acids are independently selected from the group consisting of L-amino acids, D-amino acids and aza-amino acids, in particular Aa is lysyl, and CG is a capping group. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of formula (IBBBBB), wherein each R4 is independently —CO2H, —CH2CO2H, —OP(O)(OH)2, triazole, tetrazole, sulfonamide or sulphate;
R3 is
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; RA4 and X1 are as previously defined, RN1 is as previously defined, Aa is a chain of one or more non-naturally occurring amino acids, or a chain of one or more naturally occurring amino acids, or a chain of a mixture of one or more naturally occurring amino acids and one or more non-naturally occurring amino acids; wherein the one or more non-naturally occurring or naturally occurring amino acids are independently selected from the group consisting of L-amino acids, D-amino acids and aza-amino acids, in particular Aa is lysyl, and CG is a capping group, wherein the capping group is selected from List 1. In one embodiment, the capping group on X1 is selected from List 2 and/or the capping group on Aa is selected from List 3. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of Formula (IC)
wherein each R4 is independently —CO2H, —CH2CO2H or —OP(O)(OH)2,
R3 is
RA3 selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; and RA4 and X1 are as previously defined. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of Formula (ICC)
wherein each R4 is independently —CO2H, —CH2CO2H or —OP(O)(OH)2;
R3 is
RA3 selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; RA4 and X1 are as previously defined, RN1 and RN2 are as previously defined, and CG is a capping group. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of Formula (ICCCC)
wherein each R4 is independently —CO2H, —CH2CO2H or —OP(O)(OH)2;
R3 is
RA3 selected from the group consiting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; RA4 and X1 are as previously defined, RN1 and RN2 are as previously defined, and CG is a capping group. In one embodiment, R3 may be substituted at one or more positions with RA4. In one embodiment, the compound of the invention may be of Formula (ICCCC)
wherein each R4 is independently —CO2H, —CH2CO2H or —OP(O)(OH)2;
R3 is
RA3 selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; RA4 and X1 are as previously defined, RN1 is as previously defined, Aa is a chain of one or more non-naturally occurring amino acids, or a chain of one or more naturally occurring amino acids, or a chain of a mixture of one or more naturally occurring amino acids and one or more non-naturally occurring amino acids; wherein the one or more non-naturally occurring or naturally occurring amino acids are independently selected from the group consisting of L-amino acids, D-amino acids and aza-amino acids, in particular Aa is lysyl, and CG is a capping group. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of Formula (ICCCCC)
wherein each R4 is independently —CO2H, —CH2CO2H or —OP(O)(OH)2;
R3 is
RA3 selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; RA4 and X1 are as previously defined, NR1 is as previously defined, Aa is a chain of one or more non-naturally occurring amino acids, or a chain of one or more naturally occurring amino acids, or a chain of a mixture of one or more naturally occurring amino acids and one or more non-naturally occurring amino acids; wherein the one or more non-naturally occurring or naturally occurring amino acids are independently selected from the group consisting of L-amino acids, D-amino acids and aza-amino acids, in particular Aa is lysyl, and CG is a capping group. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of Formula (ICCCCC) wherein each R4 is independently —CO2H, —CH2CO2H or —OP(O)(OH)2;
R3 is
RA3 selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; RA4 and X1 are as previously defined, NR1 is as previously defined, Aa is a chain of one or more non-naturally occurring amino acids, or a chain of one or more naturally occurring amino acids, or a chain of a mixture of one or more naturally occurring amino acids and one or more non-naturally occurring amino acids; wherein the one or more non-naturally occurring or naturally occurring amino acids are independently selected from the group consisting of L-amino acids, D-amino acids and aza-amino acids, in particular Aa is lysyl, and CG is a capping group, wherein the capping group is selected from List 1. In one embodiment, the capping group on X1 is selected from List 2, and the caping group on Aa is selected from List 3. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of Formula (ID)
wherein each R4 is independently —CO2H, —CH2CO2H or —OP(O)(OH)2;
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; and X1 is as previously defined.
In one embodiment, the compound of the invention may be of Formula (IDD)
wherein each R4 is independently —CO2H, —CH2CO2H or —OP(O)(OH)2;
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 or C1-C10 alkyl; X1 is as previously defined and CG is a capping group.
In one embodiment, the compound of the invention may be of Formula (IDDD)
wherein each R4 is independently —CO2H, —CH2CO2H or —OP(O)(OH)2;
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 or C1-C10 alkyl; X1 is as previously defined, RN1 and RN2 are as previously defined, and CG is a capping group;
In one embodiment, the compound of the invention may be of Formula (IDDDD)
wherein each R4 is independently —CO2H, —CH2CO2H or —OP(O)(OH)2;
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 or C1-C10 alkyl; X1 is as previously defined, RN1 is as previously defined, Aa is a chain of one or more non-naturally occurring amino acids, or a chain of one or more naturally occurring amino acids, or a chain of a mixture of one or more naturally occurring amino acids and one or more non-naturally occurring amino acids; wherein the one or more non-naturally occurring or naturally occurring amino acids are independently selected from the group consisting of L-amino acids, D-amino acids and aza-amino acids, in particular Aa is lysyl, and CG is a capping group.
In one embodiment, the compound of the invention may be of Formula (IDDDDD)
wherein each R4 is independently —CO2H, —CH2CO2H or —OP(O)(OH)2;
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 or C1-C10 alkyl; X1 is as previously defined, RN1 is as previously defined, Aa is a chain of one or more non-naturally occurring amino acids, or a chain of one or more naturally occurring amino acids, or a chain of a mixture of one or more naturally occurring amino acids and one or more non-naturally occurring amino acids; wherein the one or more non-naturally occurring or naturally occurring amino acids are independently selected from the group consisting of L-amino acids, D-amino acids and aza-amino acids, in particular Aa is lysyl, and CG is a capping group.
In one embodiment, the compound of the invention may be of Formula (IDDDDD), wherein each R4 is independently —CO2H, —CH2CO2H or —OP(O)(OH)2;
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 or C1-C10 alkyl, X1 is as previously defined, RN1 is as previously defined, Aa is a chain of one or more non-naturally occurring amino acids, or a chain of one or more naturally occurring amino acids, or a chain of a mixture of one or more naturally occurring amino acids and one or more non-naturally occurring amino acids; wherein the one or more non-naturally occurring or naturally occurring amino acids are independently selected from the group consisting of L-amino acids, D-amino acids and aza-amino acids, in particular Aa is lysyl, and CG is a capping group, wherein the capping group is selected from List 1. In one embodiment the capping group on X1 is selected from List 2 and/or the capping group on Aa is selected from List 3.
In one embodiment, the compound of the invention may be of Formula (IE)
wherein each R4 is independently —CO2H, —CH2CO2H or —OP(O)(OH)2;
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; and X1 is as previously defined.
In one embodiment, the compound of the invention may be of Formula (IEE)
wherein each R4 is independently —CO2H, —CH2CO2H or —OP(O)(OH)2;
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; X1 is as previously defined and CG is a capping group.
In one embodiment, the compound of the invention may be of Formula (IF)
wherein each R4 is independently —CO2H, —CH2CO2H or —OP(O)(OH)2;
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; and X1 is as previously defined.
In one embodiment, the compound of the invention may be of Formula (IFF)
wherein each R4 is independently —CO2H, —CH2CO2H or —OP(O)(OH)2;
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; X1 is as previously defined and CG is a capping group.
In one embodiment, the compound of the invention may be of Formula (IG)
wherein each R4 is independently —CO2H, —CH2CO2H or —OP(O)(OH)2;
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; and RA4 and X1 are as previously defined.
In one embodiment, the compound of the invention may be of Formula (IGG)
wherein each R4 is independently —CO2H, —CH2CO2H or —OP(O)(OH)2;
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; RA4 and X1 are as previously defined, and CG is a capping group.
In one embodiment, the compound of the invention may be of Formula (IH)
wherein each R4 is independently —CO2H, —CH2CO2H or —OP(O)(OH)2,
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; and X1 is as previously defined.
In one embodiment, the compound of the invention may be of Formula (IHH)
wherein each R4 is independently —CO2H, —CH2CO2H or —OP(O)(OH)2;
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; X1 is as previously defined and CG is a capping group.
In one embodiment, the compound of the invention may be of Formula (II)
wherein R4 is —CO2H, —CH2CO2H or —OP(O)(OH)2;
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; and RA4 and X1 are as previously defined. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of Formula (III)
wherein R4 is —CO2H, —CH2CO2H or —OP(O)(OH)2;
R3 is
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; and RA4 and X1 are as previously defined. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of Formula (IJ)
wherein R4 is —CO2H, —CH2CO2H or —OP(O)(OH)2;
R3 is
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; and RA4 and X1 are as previously defined. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of Formula (IJJ)
wherein R4 is —CO2H, —CH2CO2H or —OP(O)(OH)2;
R3 is
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; RA4 and X1 are as previously defined, and CG is a capping group. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of Formula (IJJJ)
wherein R4 is —CO2H, —CH2CO2H or —OP(O)(OH)2;
R3 is
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; RA4 and X1 are as previously defined, RN1 and RN2 are as previously defined, and CG is a capping group. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of Formula (IJJJJ)
wherein R4 is —CO2H, —CH2CO2H or —OP(O)(OH)2;
R3 is
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; RA4 and X1 are as previously defined, RN1 is as previously defined, Aa is a chain of one or more non-naturally occurring amino acids, or a chain of one or more naturally occurring amino acids, or a chain of a mixture of one or more naturally occurring amino acids and one or more non-naturally occurring amino acids; wherein the one or more non-naturally occurring or naturally occurring amino acids are independently selected from the group consisting of L-amino acids, D-amino acids and aza-amino acids, in particular Aa is lysyl, and CG is a capping group. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of Formula (IJJJJJ)
wherein R4 is —CO2H, —CH2CO2H or —OP(O)(OH)2;
R3 is
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; RA4 and X1 are as previously defined, RN1 is as previously defined, Aa is a chain of one or more non-naturally occurring amino acids, or a chain of one or more naturally occurring amino acids, or a chain of a mixture of one or more naturally occurring amino acids and one or more non-naturally occurring amino acids; wherein the one or more non-naturally occurring or naturally occurring amino acids are independently selected from the group consisting of L-amino acids, D-amino acids and aza-amino acids, in particular Aa is lysyl, and CG is a capping group. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of Formula (IJJJJJ), wherein R4 is —CO2H, —CH2CO2H or —OP(O)(OH)2;
R3 is
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; RA4 and X1 are as previously defined, RN1 is as previously defined, Aa is a chain of one or more non-naturally occurring amino acids, or a chain of one or more naturally occurring amino acids, or a chain of a mixture of one or more naturally occurring amino acids and one or more non-naturally occurring amino acids; wherein the one or more non-naturally occurring or naturally occurring amino acids are independently selected from the group consisting of L-amino acids, D-amino acids and aza-amino acids, in particular Aa is lysyl, and CG is a capping group, wherein the capping group is selected from List 1. In one embodiment, the capping group on X1 is selected from List 2 and/or the capping group on Aa is selected from List 3. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of Formula (IK)
wherein R4 is —CO2H, —CH2CO2H or —OP(O)(OH)2;
R3 is
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; and RA4 and X1 are as previously defined. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of Formula (IKK)
wherein R4 is —CO2H, —CH2CO2H or —OP(O)(OH)2;
R3 is
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; RA4 and X1 are as previously defined and CG is a capping group. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of Formula (IL)
wherein R4 is —CO2H, —CH2CO2H or —OP(O)(OH)2,
R3 is
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; and RA4 and X1 are as previously defined. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of Formula (ILL)
wherein R4 is —CO2H, —CH2CO2H or —OP(O)(OH)2;
R3 is
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; RA4 and X1 are as previously defined, and CG is a capping group. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of Formula (IM)
wherein R4 is —CO2H, —CH2CO2H or —OP(O)(OH)2;
R3 is
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; and RA4 and X1 are as previously defined. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of Formula (IMM)
wherein R4 is —CO2H, —CH2CO2H or —OP(O)(OH)2;
R3 is
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; RA4 and X1 are as previously defined and CG is a capping group. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of Formula (IMMM)
wherein R4 is —CO2H, —CH2CO2H or —OP(O)(OH)2;
R3 is
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; RA4 and X1 are as previously defined, RN1 and RN2 are as previously defined, and CG is a capping group. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of Formula (IMMMM)
wherein R4 is —CO2H, —CH2CO2H or —OP(O)(OH)2;
R3 is
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl, RA4 and X1 are as previously defined, RN1 is as previously defined, Aa is a chain of one or more non-naturally occurring amino acids, or a chain of one or more naturally occurring amino acids, or a chain of a mixture of one or more naturally occurring amino acids and one or more non-naturally occurring amino acids; wherein the one or more non-naturally occurring or naturally occurring amino acids are independently selected from the group consisting of L-amino acids, D-amino acids and aza-amino acids, in particular Aa is lysyl, and CG is a capping group. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of Formula (IMMMMM)
wherein R4 is —CO2H, —CH2CO2H or —OP(O)(OH)2;
R3 is
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; RA4 and X1 are as previously defined, RN1 is as previously defined, Aa is a chain of one or more non-naturally occurring amino acids, or a chain of one or more naturally occurring amino acids, or a chain of a mixture of one or more naturally occurring amino acids and one or more non-naturally occurring amino acids; wherein the one or more non-naturally occurring or naturally occurring amino acids are independently selected from the group consisting of L-amino acids, D-amino acids and aza-amino acids, in particular Aa is lysyl, and CG is a capping group. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of Formula (IMMMMM), wherein R4 is —CO2H, —CH2CO2H or —OP(O)(OH)2;
R3 is
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; RA4 and X1 are as previously defined, RN1 is as previously defined, Aa is a chain of one or more non-naturally occurring amino acids, or a chain of one or more naturally occurring amino acids, or a chain of a mixture of one or more naturally occurring amino acids and one or more non-naturally occurring amino acids; wherein the one or more non-naturally occurring or naturally occurring amino acids are independently selected from the group consisting of L-amino acids, D-amino acids and aza-amino acids, in particular Aa is lysyl, and CG is a capping group, wherein the capping group is selected from List 1. In one embodiment, the capping group on X1 is selected from List 2 and/or the capping group on Aa is selected from List 3. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of Formula (IN)
wherein R4 is —CO2H, —CH2CO2H or —OP(O)(OH)2;
R3 is
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; and RA4 and X1 are as previously defined. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of Formula (INN)
wherein R4 is —CO2H, —CH2CO2H or —OP(O)(OH)2;
R3 is
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; RA4 and X1 are as previously defined; and CG is a capping group. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of Formula (IO)
wherein each R4 is independently —CO2H, —CH2CO2H or —OP(O)(OH)2,
R3 is
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; RA4 is as previously defined and CG is a capping group. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of Formula (IOO)
wherein each R4 is independently —CO2H, —CH2CO2H or —OP(O)(OH)2,
R3 is
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; RA4 is as previously defined, RN1 and RN2 are as previously defined, and CG is a capping group. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of Formula (IOOO)
wherein each R4 is independently —CO2H, —CH2CO2H or —OP(O)(OH)2,
R3 is
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; RA4 is as previously defined, RN1 is as previously defined; Aa is a chain of one or more non-naturally occurring amino acids, or a chain of one or more naturally occurring amino acids, or a chain of a mixture of one or more naturally occurring amino acids and one or more non-naturally occurring amino acids; wherein the one or more non-naturally occurring or naturally occurring amino acids are independently selected from the group consisting of L-amino acids, D-amino acids and aza-amino acids, in particular Aa is lysyl, and CG is a capping group. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of Formula (IOOOO)
wherein each R4 is independently —CO2H, —CH2CO2H or —OP(O)(OH)2,
R3 is
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; RA4 is as previously defined, RN1 is as previously defined; Aa is a chain of one or more non-naturally occurring amino acids, or a chain of one or more naturally occurring amino acids, or a chain of a mixture of one or more naturally occurring amino acids and one or more non-naturally occurring amino acids; wherein the one or more non-naturally occurring or naturally occurring amino acids are independently selected from the group consisting of L-amino acids, D-amino acids and aza-amino acids, in particular Aa is lysyl, and CG is a capping group. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of Formula (IOOOO), wherein each R4 is independently —CO2H, —CH2CO2H or —OP(O)(OH)2,
R3 is
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; RA4 is as previously defined, RN1 is as previously defined; Aa is a chain of one or more non-naturally occurring amino acids, or a chain of one or more naturally occurring amino acids, or a chain of a mixture of one or more naturally occurring amino acids and one or more non-naturally occurring amino acids; wherein the one or more non-naturally occurring or naturally occurring amino acids are independently selected from the group consisting of L-amino acids, D-amino acids and aza-amino acids, in particular Aa is lysyl, and CG is a capping group, wherein the capping group is selected from List 1. In one embodiment, the capping group on X1 is selected from List 2 and/or the capping group on Aa is selected from List 3. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of Formula (IP)
wherein each R4 is independently —CO2H, —CH2CO2H or —OP(O)(OH)2;
R3 is
RA3 is selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 and C1-C10 alkyl; and RA4 is as previously defined. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of Formula (IQ)
wherein each R4 is independently —CO2H, —CH2CO2H or —OP(O)(OH)2;
R3 is
RA3 is —H, —F, —Cl, —Br, —I, —OH, —O(C1-C10 alkyl), —NO2 or C1-C10 alkyl; and RA4 is as previously defined. In one embodiment, R3 may be substituted at one or more positions with RA4.
In one embodiment, the compound of the invention may be of Formula (IS)
wherein CG is a capping group.
In one embodiment, the compound of the invention may be of Formula (ISS)
wherein RN1 and RN2 are as previously defined, and CG is a capping group.
In one embodiment, the compound of the invention may be of Formula (ISSS)
wherein RN1 is as previously defined and CG is a capping group;
Aa is a chain of one or more non-naturally occurring amino acids, or a chain of one or more naturally occurring amino acids, or a chain of a mixture of one or more naturally occurring amino acids and one or more non-naturally occurring amino acids; wherein the one or more non-naturally occurring or naturally occurring amino acids are independently selected from the group consisting of L-amino acids, D-amino acids and aza-amino acids, in particular Aa is lysyl.
In one embodiment, the compound of the invention may be of Formula (ISSSS)
Wherein RN1 is as previously defined and CG is a capping group;
Aa is a chain of one or more non-naturally occurring amino acids, or a chain of one or more naturally occurring amino acids, or a chain of a mixture of one or more naturally occurring amino acids and one or more non-naturally occurring amino acids; wherein the one or more non-naturally occurring or naturally occurring amino acids are independently selected from the group consisting of L-amino acids, D-amino acids and aza-amino acids, in particular Aa is lysyl.
In one embodiment, the compound of the invention may be of Formula (ISSSSS)
Wherein CG is a capping group.
In one embodiment, the compound of the invention may be of Formula (ISSSSS), wherein CG is a capping group selected from List 1. In one embodiment, the capping group on the “d” terminus is selected from List 2 and/or the capping group on the “K” terminus is selected from List 3.
In one embodiment, the compound of the invention may comprise or consist of a sequence selected from the group consisting of:
In one embodiment, any one or more of the residues included in the exemplary compounds described above may be an aza amino acid, wherein an “aza amino acid” is an L amino acid in which the α-carbon atom has been replaced by a nitrogen atom.
Prodrugs
In one embodiment, the compound may be formulated for administration to a patient as a prodrug. The term “prodrug” means a precursor of a designated compound that, following administration to a subject yields the compound in vivo via a chemical or physiological process such as solvolysis or enzymatic cleavage, or under physiological conditions (e.g., a prodrug, on being brought to physiological pH is converted to a compound of Formula Ia, Ib, Ic, Id, Ie, If or Ig). Illustrative procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in “Design of prodrugs”, H. Bundgaard et al.
Prodrugs may be produced, for example, by derivatising free carboxylic acid groups of structures of Formula Ia, Ib, Ic, Id, Ie, If or Ig as amides or esters. In one embodiment, the prodrug is an alkyl, aryl, or heteroaryl ester of a compound of the invention. In another embodiment the prodrug may comprise or consist of a methyl, ethyl, propyl, butyl, pentyl, hexyl, or benzyl ester of a compound of the invention.
In a further embodiment, the prodrug may comprise a cycloalkyl ester, preferably a cyclopentyl ester of a compound of the present invention. In a further embodiment, the prodrug may comprise a —CO2CH2CH2(heterocyclyl) ester, wherein heterocyclyl is preferably morpholino, of a compound of the present invention. In a further embodiment, the prodrug may comprise a —C02(CH2CH2O)1-10—CH2CH3 (polyethylene glycol or PEG) ester of a compound of the present invention.
A prodrug may be a compound comprising an alcohol functionality, which when phosphorylated in vivo produces the active compound. For example, a compound comprising a serine residue may be a prodrug which, when subjected to physiological conditions is phosphorylated to form the corresponding phosphorylated serine residue, thereby producing the active compound.
Methods of Manufacture
Compounds of the invention were synthesised by the coupling of smaller fragments/subunits, usually amino acids.
Amino acids that were commercially available were purchased and used directly (following any appropriate protecting group modification). Unnatural amino acids were synthesised starting from the appropriate amino acid precursor.
Carboxylic Acid Bioisostere Synthesis
Compounds of the present invention may comprise carboxylic acid bioisosteres. Such carboxylic acid bioisosteres may be synthesised by modification of the functionality of the side chain of an amino acid. Such functionality may be, for example, a carboxylic acid or amide. In this case, appropriate starting amino acids would include aspartic acid, glutamic acid, asparagine and glutamine. A representative scheme for the conversion of an amide functionality of the side chain of an amino acid into a tetrazole group is illustrated below (Tetrazole amino acids as competitive NMDA antagonists, Bioorganic & Medicinal Chemistry Letters, 1993):
As will be appreciated by the skilled person, the scheme above is a representative procedure for the conversion of a natural amino acid into an unnatural amino acid.
Using standard synthetic procedures, the person skilled in the art would be able to synthesise other unnatural amino acids in an analogous manner to that shown above.
Once synthesised, the smaller fragments/subunits (usually amino acids —natural/unnatural) are coupled together to form compounds of the present invention.
The smaller fragments/subunits are coupled together using a solid-phase peptide synthesis. Reagents and conditions for this technique are illustrated in
Further experimental procedures are provided in the Examples section.
Pharmaceutical Compositions
The compound, modified peptide or prodrug of the invention may be formulated into a pharmaceutical composition. The invention therefore includes a pharmaceutical composition comprising one or more of the compounds, modified peptides or produgs of the invention.
In one embodiment the pharmaceutical composition may additionally comprise a pharmaceutically-acceptable carrier, excipient, diluent or buffer. Suitable pharmaceutically acceptable carriers, excipients, diluents or buffers may include liquids such as water, saline, glycerol, ethanol or auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like. Excipients may enable the pharmaceutical compositions to be formulated into tablets, pills, capsules, liquids, gels, or syrups to aid intake by the subject. A thorough discussion of pharmaceutically acceptable carriers is available in Remington's Pharmaceutical Sciences.
The pharmaceutical composition may include a therapeutically effective amount of one or more of the compounds, modified peptides or produgs of the invention. A pharmaceutically effective amount is an amount able to treat the disease for which the composition is intended. The actual amount will depend on a number of factors including the size, weight, age, gender, and health of an individual, and the rate of blood clearance, and will be decided by a clinical practitioner. Generally a pharmaceutically effective amount will be between 1 g/kg body weight and 1 mg/kg body weight or less.
In one embodiment the pharmaceutical composition may include an additional pharmaceutically active agent such as a therapeutic component, in particular, a component useful for the treatment of hyperproliferative disorders such as cancer, inflammatory disorders involving the NFkB signalling pathway such as arthritis, osteoarthritis, rheumatoid arthritis, Crohn's Disease and Irritable Bowel Syndrome (IBS), infectious disorders or neurodegenerative disorders and may include chemotherapeutics, ERMs, SERMs, other E1, E3, E3 and deubiquitinating enzyme inhibitors, proteasome inhibitors, kinase inhibitors, HDAC inhibitors, PPAR inhibitors or specific biological targeted therapies e.g. Herceptin.
The invention also includes any medical device which may have the pharmaceutical composition of the invention inserted into it or coated onto it. Such devices include but are not limited to stents, pins, rods, meshes, beads, syringes, plasters, microchips, micro fluidic devices, and stitches.
Methods of Treatment
In another aspect, the invention includes a compound, modified peptide, prodrug or pharmaceutical composition of the invention for use in medicine.
In one embodiment the invention provides a compound, modified peptide, prodrug or pharmaceutical composition of the invention for use in the treatment of a disease associated with aberrant protein degradation.
In one embodiment the invention provides a compound, modified peptide, prodrug or pharmaceutical composition of the invention for use in the treatment of a hyperproliferative disorder such as cancer, inflammatory disorders involving the NFkB signalling pathway such as arthritis, osteoarthritis, rheumatoid arthritis, Crohn's Disease and Irritable Bowel Syndrome (IBS), infectious disorders or neurodegenerative disorders.
In another embodiment the invention includes a method of treating a hyperproliferative disorder such as cancer, inflammatory disorders involving the NFkB signalling pathway such as arthritis, osteoarthritis, rheumatoid arthritis, Crohn's Disease and Irritable Bowel Syndrome (IBS), infectious disorders or neurodegenerative disorders comprising administering a pharmaceutically effective amount of a compound, modified peptide, prodrug or pharmaceutical composition of the invention to a patient in need of treatment.
In a particular embodiment, the invention includes a method of treating breast cancer or prostate cancer comprising administering a pharmaceutically effective amount of a compound, modified peptide, prodrug or pharmaceutical composition of the invention to a patient in need of treatment.
As used herein, the term “treatment” encompasses therapy, and can be prophylactic or therapeutic.
A pharmaceutically effective amount is an amount able to treat the disease for which the compound, modified peptide, prodrug or pharmaceutical composition has been administered. The actual amount will depend on a number of factors including the size, weight, age, gender, health of an individual, and the rate of blood clearance, and will be decided by a clinical practitioner. Generally a pharmaceutically effective amount will be between 1 g/kg body weight and 1 mg/kg body weight or less.
In another embodiment the invention includes the use of a compound, modified peptide, prodrug or pharmaceutical composition of the invention in the manufacture of a medicament for the treatment of a hyperproliferative disorder such as cancer, inflammatory disorders involving the NFkB signalling pathway such as arthritis, osteoarthritis, rheumatoid arthritis, Crohn's Disease and Irritable Bowel Syndrome (IBS), infectious disorders or neurodegenerative disorders.
In a particular embodiment, the invention includes the use of a compound, modified peptide, prodrug or pharmaceutical composition of the invention in the manufacture of a medicament for the treatment of breast cancer or prostate cancer.
The compound, modified peptide, prodrug or pharmaceutical composition of the invention may be used for the treatment of disease in any animal. The animal may be a mammal such as a camel, dog, cat, horse, cow, pig, sheep, camelid, mouse, rat, rabbit, hamster, guinea pig, pig, or sheep. In one embodiment, the mammal may be a human.
The compound, modified peptide, prodrug or pharmaceutical composition of the invention may be administered to a patient using any one or more of a number of modes of administration which will be known to a person skilled in the art. Such modes of administration may include parenteral injection (e.g. intravenously, subcutaneously, intraperitoneally, intramuscularly, or to the interstitial space of a tissue), or by rectal, oral, vaginal, topical, transdermal, intradermal, intrathecal, intranasal, ocular, aural, pulmonary or other mucosal administration. The precise mode of administration will depend on the disease or condition to be treated.
Diagnostic Kits
In another aspect, the invention includes a diagnostic kit comprising a compound, modified peptide or prodrug of the invention. Within this aspect, the compound, modified peptide or prodrug may be labelled to allow its identification. Suitable labels may include, coloured labels, fluorescent labels, and radioactive labels. Detection may be performed by FACS, Western blot, immunoblot or any other technique known to be useful for the identification of labelled molecules.
Diagnostics kits may be used to identify patients having increased βTrCP expression.
As discussed above, increased βTrCP expression can be associated with aberrant protein degradation mechanisms, which can lead to hyperproliferative disorders such as cancer through the increased degradation of pro-apoptotic factors. Increased βTrCP expression can also lead to inflammatory disorders involving the NFkB signalling pathway such as arthritis, osteoarthritis, rheumatoid arthritis, Crohn's Disease and Irritable Bowel Syndrome (IBS), infectious disorders and neurodegenerative disorders.
Diagnostics kits may also comprise instructions.
Various aspects and embodiments of the present invention will now be described in more detail by way of example. It will be appreciated that modification of detail may be made without departing from the scope of the invention.
Reagents and Conditions: a) Rink amide linker (3 equiv), oxyma (3 equiv), DIC (3 equiv), 0.1 M in DMF, 30 min; b) 20% piperidine in DMF (2×5 min); c) Amino acid (3 equiv), HBTU (3 equiv), DIPEA (6 equiv) 0.1 M in DMF, 40 min; d) TsCl (5 equiv), DMAP (0.1 equiv), DIPEA (10 equiv), 0.1 M in DMF, 40 min; e) TFA, 5% TIS, 5% DCM, 3h.
Reagents and Conditions: a) Rink amide linker (3 equiv), oxyma (3 equiv), DIC (3 equiv), 0.1 M in DMF, 30 min; b) 20% piperidine in DMF (2×5 min); c) Amino acid (3 equiv), HBTU (3 equiv), DIPEA (6 equiv) 0.1 M in DMF, 40 min; d) TsCl (5 equiv), DMAP (0.1 equiv), DIPEA (10 equiv), 0.1 M in DMF, 40 min; e) 2% Hydrazine in DMF (6×15 mins); f) BzCl (5 equiv), DMAP (0.1 equiv), DIPEA (10 equiv), 0.1 M in DMF, 40 min; g) TFA, 5% TIS, 5% DCM, 3h.
Peptides are numbered according to Table 11: DSGIFE (SEQ ID NO: 119); DpSGIFE (SEQ ID NO: 12); and succ-EGFFE (SEQ ID NO: 57).
The following experimental conditions were used throughout the examples unless other details are provided.
General Conditions for the Solid-Phase Synthesis
All the coupling reactions were carried out at room temperature if no specifications are given. Solid-phase synthesis was performed manually using Isolute filtration reservoirs as the reaction vessel, fitted with polyethylene frits (Argonaut Technologies Inc). Amino acids are Fmoc protected at the N terminus, with suitable acid labile protecting groups on the side chains. For C-terminal modified peptides Fmoc-Lys(Dde)-OH was used to allow selective modification of the Lys side chain. Each coupling step of the synthesis was assessed for completion using either the Kaiser test for primary amines, or the chloranil test for secondary amines.
Coupling the Linker to the Resin
Aminomethyl PS resin (loading 1.23 mmol/g, 0.30 g, 0.369 mmol) in a 6 mL reaction vessel was swollen for 5 minutes in DCM (3 mL), then washed with DCM (3×3 mL). To a solution of Rink amide linker (598 mg, 1.11 mmol) in DMF (3.69 mL) was added oxyma (157 mg, 1.11 mmol) and the solution shaken for 10 minutes. DIC (173 uL, 1.11 mmol) was added and the solution shaken for 2 minutes. The mixture was added to the resin and shaken for 30 minutes. The resin was filtered and washed with DMF (3×4 mL), DCM (3×4 mL) and MeOH (3×4 mL). Kaiser test negative. The resin was washed with Et2O (3×4 mL) and dried under vacuum for storage.
Coupling of Amino Acids/Spacer
Resin (˜0.049 mmol) in a 3 mL reaction vessel was swollen for 5 minutes in DCM (1.5 mL) and filtered. A solution of 20% piperidine in DMF (1.5 mL) was added, the vessel was shaken for 5 mins and the resin was filtered and washed with DMF (3×1.5 mL) and DCM (3×1.5 mL). Piperidine deprotection and washing cycle was repeated and the resin was dried under vacuum, Kaiser test positive. To a solution of the appropriate amino acid/spacer (0.15 mmol, 3 equiv) in DMF (0.49 mL) was added HBTU (0.15 mmol, 3 equiv) and the solution shaken for 2 minutes. DIPEA (0.30 mmol, 6 equiv) was added and the solution shaken for 1 minute. The mixture was added to the resin and shaken for 40 minutes. The resin was filtered and washed with DMF (3×1.5 mL), DCM (3×1.5 mL) and MeOH (3×1.5 mL). Kaiser test negative, otherwise treatment of activated amino acid repeated.
Coupling to N-Alkylated Amino Acids
Resin (˜0.049 mmol) in a 3 mL reaction vessel was swollen for 5 minutes in DCM (1.5 mL) and filtered. A solution of 20% piperidine in DMF (1.5 mL) was added, the vessel was shaken for 5 mins and the resin was filtered and washed with DMF (3×1.5 mL) and DCM (3×1.5 mL). Piperidine deprotection and washing cycle was repeated and the resin was dried under vacuum, Choranil test positive. To a solution of the appropriate amino acid (0.15 mmol, 3 equiv) in DMF (0.49 mL) was added oxyma (0.15 mmol, 3 equiv) and the solution shaken for 10 minutes. DIC (0.15 mmol, 3 equiv) was added and the solution shaken for 2 minutes. The mixture was added to the resin and heated in a microwave at 60° C. for 20 minutes. The mixture was then shaken for an additional 20 minutes. The resin was filtered and washed with DMF (3×4 mL), DCM (3×4 mL) and MeOH (3×4 mL). Chloranil test negative, otherwise treatment of activated amino acid repeated.
Example of the N-Terminus Capping
Resin (˜0.049 mmol) in a 3 mL reaction vessel was swollen for 5 minutes in DCM (1.5 mL) and filtered. A solution of 20% piperidine in DMF (1.5 mL) was added, the vessel was shaken for 5 mins and the resin was filtered and washed with DMF (3×1.5 mL) and DCM (3×1.5 mL). Piperidine addition and washing cycle was repeated and the resin was dried under vacuum, Kaiser test positive. To a solution of 4-toluenesulfonyl chloride (0.25 mmol, 5 equiv) in DCM:DMF (1:1, 0.49 mL) was added DMAP (0.005 mmol, 0.1 equiv) and the solution shaken for 2 minutes. DIPEA (0.50 mmol, 10 equiv) was added and the solution shaken for 1 minute. The mixture was added to the resin and shaken for 40 minutes. The resin was filtered and washed with DMF (3×1.5 mL), DCM (3×1.5 mL) and MeOH (3×1.5 mL). Kaiser test negative, otherwise treatment of with the capping group was repeated.
Example of the C-Terminus Capping
After N-terminus capping, resin (˜0.049 mmol) in a 3 mL reaction vessel was swollen for 5 minutes in DCM (1.5 mL) and filtered. A solution of 2% hydrazine monohydrate in DMF (1.5 mL) was added, the vessel was shaken for 15 mins and the resin was filtered and washed with DMF (3×1.5 mL) and DCM (3×1.5 mL). Hydrazine addition and washing cycle was repeated (×5) and the resin was dried under vacuum, Kaiser test positive. To a solution of benzoyl chloride (0.25 mmol, 5 equiv) in DCM:DMF (1:1, 0.49 mL) was added DMAP (0.005 mmol, 0.1 equiv) and the solution shaken for 2 minutes. DIPEA (0.50 mmol, 10 equiv) was added and the solution shaken for 1 minute. The mixture was added to the resin and shaken for 40 minutes. The resin was filtered and washed with DMF (3×1.5 mL), DCM (3×1.5 mL) and MeOH (3×1.5 mL). Kaiser test negative, otherwise treatment of with the capping group was repeated.
Characterization of selected examples:
aMass identified as [M − H]−;
bMass identified as [M + H]+;
cMass identified as [M + Na]+;
dMass identified as [M + K]+;
f5% to 95% MeCN (+0.1% formic acid) in H2O (+0.1% formic acid) over 10 minutes, 4 minute hold, then 1 minute at 5% MeCN (+0.1% formic acid);
g5% to 95% MeOH (+0.1% formic acid) in H2O (+0.1% formic acid) over 10 minutes, 4 minute hold, then 1 minute at 5% MeOH (+0.1% formic acid);
Cleavage from Resin
Resin (˜0.0.49 mmol) in a 3 mL reaction vessel was swollen for 5 minutes in DCM (2 mL) and filtered. A solution of TFA:TIS:DCM (90:5:5 0.49 mL) was added, and the vessel was shaken for 3 h. The resin was removed by filtration, and ice-cold Et2O (10 mL) was added to the filtrate. The resultant solid was pelleted by centrifuge, and the solvent removed by decantation. Solid was dried under vacuum.
The experimental scheme for solid phase synthesis is shown in
Fluorescence Polarization Screening of βTrCP
Assay Components
0.035 μM βTrCP (tag cleaved and complexed with Skp1)
10 nM fluorescein-RHDpSGLDpSMKD (SEQ ID NO:68)
50 mM Hepes pH 7.5
50 mM NaCl
1 mM DTT
0.1 mg/ml BSA (Bovine Serum Albumin)
50 μM compound (in DMSO)
Assay Protocol
Assay components (without compound) were premixed in a microcentrifuge tube and incubated for 1 hour to ensure equilibrium was achieved. Each compound was then added to one tube, mixed by vortexing, and then dispensed into 3 wells of a black 384-well plate and incubated for 30 minutes. Fluorescence polarization was then read (excitation 485 nM, emission 530 nM) using an Analyst-AD from Molecular Devices.
For dose-response curves to determine Ki, 10 different concentrations of compound were tested at equally spaced intervals. DMSO was added such that final concentration was 2%. Conditions are very tolerant to DMSO. Up to 10% DMSO has been tested previously, with no significant change to Kd values.
Surface Plasmon Resonance (SPR)
Experiments were carried out using the Biacore T200 SPR detection system. This system exploits the phenomenon of surface plasmon resonance (SPR) to monitor interactions between molecules. The system involves the attachment of one interacting partner to a surface (an appropriate sensor chip) while the other interacting partner is passed over it in solution. The binding of molecules to the surface generates an SPR response (measured in response units (RU)) that is proportional to the mass and amount of the biomolecule (in this case βTrCP/Skp1) bound to the chip. The relative responses obtained are dependent on the concentration of the molecule binding. At RUmaximum, the attached protein's binding sites are saturated. Binding events can be followed in real time and a range of interaction characteristics can be determined including kinetics, specificity of interactions and the concentration of specific molecules in a sample.
As βTrCP was His tagged, an NTA sensor chip was used. This sensor chip has a dextran surface matrix with immobilized nitrilotriacetic acid (NTA) which provides a means of capturing polyHis-tagged proteins through Nickel chelation. It was hoped that addition of Ni+ would orient the protein in a specific (and hopefully active) manner as it is covalently immobilised via amine coupling. Addition of EDC:NHS (N-ethyl-N′-(3-diethylaminopropyl)-carbodiimide:N-hydroxysuccinimide) converts carboxyl groups on the dextran sensor chip surface to succinamide esters which readily form covalent bonds with primary amines. Each chip contains four flow cells (each a separate surface) which means that compounds/peptides can be passed over different forms of βTrCP and a reference surface simultaneously. If binding to βTrCP is occurring, responses should be the same (accounting for differences in density of surface etc) on each surface.
Two different βTrCP protein complexes were immobilised on to an NTA biacore sensor chip. One included the GSTSkp1 fusion (HisβTrCP/GSTSkp1) while the other was immobilised after GST Removal by Thrombin (HisβTrCP/Skp1). To ensure that the GST moiety is completely removed from Skp1, βTrCP/GSTSkp1 was incubated with thrombin ((10units/mg protein) for at least 16 hours at room temperature in 10mMHEPES 150mMNaCl pH7.4+2 mM CaCl2. Thrombin and GST were removed from βTrCP/Skp1 by buffer exchange through a 50 KDa MWCO vivaspin concentrator.
Protein Immobilisation Procedure for NTA Chip
Ni+ (500 μM NiCl) loaded on to surface at a flow rate of 5 μl/min for 60s. EDC/NHS (activates dextran carboxylates) loaded at a flow rate of 5 μl/min for 240s. Protein ([protein]=100 nM to 1 μM) loaded at a flow rate of 10 μl/min for 180s. Strip solution (350 μM EDTA/1M NaCl) added at flow rate of 10 μl/min for 30s (to chelate excess Ni+ and remove non-covalently bound protein e.g. protein oligomers). Quench solution (ethanolamine) at flow rate of 5 μl/min for 240s (to deactivate surface molecules on the chip that have not crosslinked protein). The immobilisation buffer used was 10 mM HEPES pH 7.4, 150 mM NaCl,
GST Capture Procedure (for Immobilisation Via GSTSkp1)
EDC:NHS was injected at 5 μl/min for 4 min to activate surface for amine coupling (this converts carboxyl groups on the surface of the chip to succinamide esters that react with primary amines)
Anti-GST (60 μg/ml). was loaded at 10 μl/min for 4 min resulting in an increase in response units of 6730 (Biacore manual states it should result in ˜7000)
Ethanolamine was injected at 5 μl/min for 5 min to deactivate remaining unreacted esters at surface (quenching).
Injection of a low concentration of purified GST (from kit) was injected for 3 mins at 5 μl/min before running a regeneration cycle with glycine pH2.0 that disrupts the antibody-GST interaction. This step is recommended in the Biacore manual in order to “block” a minority of high affinity GST binding sites that may prevent regeneration and therefore reloading of fresh GST-protein of interest.
GSTSkp1/βTrCP (0.16 mg/ml) was then loaded at 10 μl/min for 4 min resulting in an increase of 1550 RU (2000 RU is about the maximum to expect according to Biacore manual).
SPR Assay Conditions
Small molecule/peptide samples to be assayed for binding to βTrCP surfaces, were provided as 10 mM stocks in 100% DMSO. All samples were tested in running buffer composed of 10 mM HEPES pH 7.4, 150 mM NaCl, 50 μM EDTA, 0.005% p20, and 1% DMSO. Serial dilutions were made using running buffer. Samples were tested over varying concentrations up to a maximum of 100 μM. Two methods of measuring the SPR response were employed: single cycle kinetics which measures the response across different concentrations of sample within a single cycle (no regeneration of surface) and a method that measures the response at a given concentration in each cycle and includes a regeneration wash after each sample injection. The regeneration solution used was the same as running buffer, but included 500 mM NaCl. Data was fitted using Biacore T200 evaluation software. KD values calculated from binding curves from both surfaces (with or without the GST moiety) were averaged to produce apparent KD's for each sample tested.
Biotin Pull Down Assay
Assay components
150 mM NaCl
10.01% NP40
1 mM DTT
0.3 μM βTrCP (1 μg)
0.3 μM biotinylated IκB peptide (KKERLLDDRHDpSGLDpSMKDEE)
100 μM compound (mabridge library: 50 μM)
Protocol
βTrCP1 and biotinylated peptide were incubated in a volume of 25 μl at a final concentration of DMSO of 1% for 30 minutes to achieve equilibrium. Compounds were then added to a final concentration of 100 μM and allowed to incubate for an additional 30 minutes. 7.5 μl of streptavidin-agarose beads were then added to the reaction mix and allowed to incubate at room temperature for 30 minutes with gentle rocking. Beads were spun down and washed in buffer 3 times and then loaded onto a 10% SDS PAGE gel and visualized by GelCode blue staining.
βTrCP Ubiquitination Assay and Selectivity Assays
Assay Components
0.2 μM E1 (Ube1)
2 μM E2 (UbcH5C)
0.25 μM E3 (Cul1/Rbx1)
0.25 μM E3 (β-TrCP1/Skp1)
12 μM Ubiquitin
0.5 μM Peptide substrate (biotin)
10 mM MgCl2
2 mM ATP
Protocol
Master mixes were prepared in a 50 mM Herpes buffer at pH 7.5, in 75 mM NaCl and 1 mM DTT without Mg or ATP and peptides were added to a final concentration of 100 μM. Reactions were incubated at room temperature for 30 minutes and then Mg/ATP was added to the mix. Reactions were further incubated for an additional 60 minutes and then stopped by adding SDS gel loading buffer and boiled for 5 minutes. Reactions were run on SDS-PAGE (10%) and transferred to nitrocellulose membranes and probed with HRP-Streptavidin.
FBW7 selectivity assays were performed in the same manner except using FBW7/Skp1 as E3 component and cyclin E as the substrate. Blots were probed using anti-cyclin E antibody.
The following Examples illustrate the experiments performed by the inventors to arrive at the preset invention. It will be appreciated that modification of detail may be made without departing from the scope of the invention.
A consensus binding motif is known to be present in IkBa, Vpu and β-catenin, all of which bind βTrCP (J. Pons et al., Biochemistry, 2008, 47 (1), 14-29). The consensus motif has the sequence DpSGXXpS (SEQ ID NO:69), wherein the two serine residues are phosphorylated.
A selection of compounds were prepared using the synthesis procedure described above, and shown in
Binding of the compounds to βTrCP was assessed using a fluorescence polarisation (FP) binding assay. The FP assay is an in vitro binding assay using a fluorescein-tagged IkB peptide at 10 nM to mimic substrate binding to βTrCP, and was performed as described above.
Dose response curves for a number of representative peptides are shown in
The peptide DEGFFE-NH2, having an IC50 of 43.6 μM, was selected as a suitable non-phosphorylated candidate for further progression.
Starting from DEGFFE-NH2, an array of compounds was prepared, as shown in Table 2, in which the N-terminal amino acid (D) was replaced with various capping groups. All of these compounds have an aminde at the C-terminus, as do those shown in Tables 5 and 6.
Four sequences were selected for re-synthesis and testing in the FP assay, which was performed as described above along with 4 negative controls. The results of the FP assay are shown in Table 3.
The FP assay identified Suc-EGFFE-NH2 as a low μM inhibitor for further optimisation.
Further peptides were synthesised as described above by replacing residues in the Suc-EGFFE-NH2 sequence with alternative acidic capping groups and non-natural amino acids. The sequences and abbreviations of the acidic capping groups and non-natural amino acids are shown in
Selected peptides from the arrays shown in Tables 5 and 6 were re-synthesised and analysed using the FP assay described above. The results of this assay are shown in Table 7.
X-EGXXE-NH2 was identified as a useful consensus binding motif and further peptides were designed and synthesised to increase potency. These peptides were tested in the FP assay described above and the results are shown in Table 8.
A non-fluorescent based assay was used to validate potential binding peptides. The phosphopeptide substrate KKERLLDDRHDpSGLDpSMKDEE was biotinylated by coupling a biotinamido-hexanoic acid succinimide ester to a lysine in the peptide. 0.5 μg of each protein was mixed with 50 picomoles of biotinylated peptide in a total volume of 50 μl. The buffer conditions were 50 mM Hepes 7.5 and 100 mM NaCl. Compounds were then added to a final concentration of 60 μM. The reaction was allowed to incubate for 1 hour at room temperature and then 5 μl of streptavidin-agarose beads was added and incubated for 1 hour. Beads were washed twice with buffer and run on an SDS-PAGE gel. Proteins were visualized by anti-His antibody. The results of this assay are shown in
Binding of SucEGF(4NO2)1NalENH2 to βTrCP1 was tested using the Surface Plasmon Resonance (SPR) method described above.
Candidate compounds were tested in duplicate at both 10 and 100 μM against the E3 ligase cascades βTrCP(IκBα) and βTrCP(β-catenin). The E3 assays were carried out according to the component concentrations detailed in Table 9. In each case the E2 was HA,6His-UbcH3-(hu,FL), the E1 was 6His-UBE1-(hu,FL) and the ubiquitin (Sigma U6253) was biotinylated at a 5:1 ratio. The E3 tetramer constructs and substrate pairings are shown in Table 10. Substrate phosphorylation was performed in the absence of compound; consequently any observed signal modulation should not reflect inhibition of the up-stream kinase reaction. All other steps (E1, E2 and substrate ubiquitination) were carried out in the presence of compound. The stopped reaction mix (10 μl) was added to an ECL plate loaded with anti c-Myc ( 1/500 Dilution, Millipore 05-724) and blocked with 5% BSA. Binding was allowed to proceed for 1 hour at RT before a wash step (3 40 μl PBST washes). Detection was achieved by binding SA-Ru TAG at 1 μg/ml (1 hour @ RT, 3 40 μl PBST washes) before reading on an MSD Sector Imager 6000.
Assay results are shown in
Candidate compounds were tested for inhibition of Fbw7 and Skp2 as described above. As shown in
Table 11 (below) shows the collation of all data points collected for the most promising compounds.
The activity of 4-(MeO)-PhSO2-dEGF(3F)WE-NH2 in a cell was investigated as an example of activity seen with this family of compounds.
In-Cell Western Assay for PDCD4 Accumulation.
PDCD4 is a substrate of βTrCP and so an inhibitor of βTrCP should result in the stabilisation and accumulation of PDCD4 in cells. To measure this an in-cell western assay was used and the peptidomimetics were delivered to the cells by nucleofection.
Nucleofection
MCF7 cells were grown in 10 cm dishes in 10 ml DMEM+10% FBS and 1% Pen/Strep at 37° C./5% CO2. On the day of nucleofection, the dish was washed with 90% confluent MCF7 cells with 5 ml PBS, then 3 ml of Trypsin/EDTA was added and incubated at 37° C./5% CO2 for approximately 5 mins until the cells detached from the plastic. 7 ml of normal growth media was added and the number of cells present was counted using a haemocytometer.
The cells were centrifuged cells at 90 g for 10 mins at RT and the supernatant was removed. 100 μl of Nucleofection Buffer V+ Supplement was added (Lonza Biologics Cat no VCA-1003) per 8×105 cells, and the cells were added to peptidomimetics dissolved in <3% DMSO.
The cell/peptidomimetic mix was added to cuvettes supplied for the nucleofector and the sample was nucleofected using the recommended MCF7 programme for high cell viability (i.e. E-014). 500 μl of TPA (12-O-tetradecanoylphorbol-13-acetate)-supplemented growth media (i.e. DMEM/10% FBS/1% Pen-Strep/10 nM TPA) was added to the cuvette and 100 μl of this solution was transferred to a well of a 96 well plate and incubated at 37° C./5% CO2 for 8 hours.
In Cell Western
The celled were fixed by removing the growth media, adding 3.7% Formaldehyde in PBS and incubating at RT for 20 mins. The plate was washed three times in PBS. And the cells were permeabilised by washing 5× for 5 mins each in PBS+0.1% Triton X-100.
The cells were blocked by adding 3% BSA in PBS-Tween to the cells and incubating at RT for 1.5 hours. An anti-PDCD4 antibody (Abcam cat no: ab80590) was added at a concentration of 1:1000 in 3% BSA in PBS-T (50 ul/well) and incubated at RT for 2.5 hours. The cells were washed 5× for 5 mins each with PBS-T before the secondary antibody (LiCor Biosciences Donkey Anti-Rabbit IRDye 800CW cat no 926-32213) was added at a concentration of 1:1000, along with the DNA stain DRAQ5 at a concentration of 1:10000, in 3% BSA in PBS-T (50 ul/well) and incubated at RT for 1 hour, protected from light.
The cells were washed cells 5× for 5 mins each with PBS-T and all the liquid was removed from the wells before the plate was read on the LiCor Biosciences Odyssey at 700 nm and 800 nm. The reading was normalised in the 800 nm channel to that in the 700 nm channel. The data are shown in
Fluorescence Reporter Assay for PDCD4 Accumulation
This assay uses two stable cell lines expressing PDCD4 fused to a GFP tag. One cell line (MCF7:GFP-PDCD4WT) shows an increase in nuclear fluorescence when βTrCP is inhibited due to the accumulation of GFP-PDCD4 in the nucleus. The other cell line (MCF7:GFP-PDCD4S71A/S76A or or MCF7:GFP-PDCD4Mut) does not show an increase in nuclear fluorescence when βTrCP is inhibited because of a mutation of two serine residues in the phosphodegron of PDCD4 that are required for βTrCP recognition. This allows false positives to be identified, where accumulation of PDCD4 is not due to stabilisation by inhibiting βTrCP.
Nucleofection
The two stable cell lines MCF7:GFP-PDCD4WT and MCF7:GFP-PDCD4S71A/S76Awere grown in 10 ml DMEM+10% FBS and 1% Pen/Strep supplemented with 2 mg/ml Geneticin at 37° C./5% CO2. These cell lines were nucleofected as described above except for the additional supplementation of all media with 2 mg/ml Geneticin.
Fluorescent Reporter Assay
The cells were fixed by removing the growth media and adding 3.7% Formaldehyde in PBS to the cells. The cells were then incubated at RT for 20 mins and the plates washed 3× in PBS.
DRAQ5 DNA stain was added to the cells in PBS at a concentration of 1:10000 and incubated at RT for 1 hour protecting from light. The plate was washed 3× in PBS and read on the Perkin Elmer OPERA platform using the nuclei counting algorithm F in both the 488 nm channel (GFP) and 640 nm channel (DRAQ5). The number of GFP-positive nuclei was expressed as a percentage of total number of nuclei, and the data are shown in
The compounds shown in
Testing of UBP037 and UBP038 by in cell western provided inconclusive results. The fluorescence readings were consistently under those of the DMSO negative control. This suggested that the compounds were interfering in some way with the fluorescence readout assay. When these compounds were tested by traditional western blot however, they exhibited cellular activity. The results for PDCD4 accumulation in MCF7 cells are shown in
β-Catenin in Cell Western
Plating of the MCF7 cells onto 96 well plates—this step is carried out 1 day before the treatment of the cells to allow the cells to adhere well to the tissue culture plastic. MCF7 cells to be used for seeding should be less than 100% confluent in a 10 cm dish. Add 3 ml of RT trypsin/EDTA to the cells. Incubate at 37° C./5% CO2 for a few minutes until the cells easily come away from the plastic by gentle swirling. Add 7 ml of media to the cells. Wash the bottom of the plate gently with the 10 ml to ensure all cells are captured and dispense into a 15 ml falcon. Add 1001l of the cells to 1001l of Trypan blue and add to haemocytometer to count the number of cells present. Make up approx 10 ml of cells in media at 2×104 cells/1001l (well) with fresh media. Add this correct seeding density to a 10 cm dish. Dispense 1001l of cells/well into a 96 well plate without using the outside wells. Incubate at 37° C./5% CO2 overnight
Treatment of MCF7 cells with compound of interest (COI) and controls—Put OptiMEM into 37° C. water bath to warm. Add 4 ul of 50 mM compound X for testing to eppendorf—this is tube 1. Add 2 al DMSO to 6 tubes marked tube 2-7. Take 21 al of 50 mM (COI) and add to 2 al of DMSO in tube 2 and pipette up and down to mix. Take 21 al from tube 2 and add to 2 μl DMSO in next tube and mix. Repeat until 2 μl in all 7 tubes and have 1:2 serial dilutions from tube 1 to 7 (final conc.: 250 PM to 2 μM). Add 3.5 μl DMSO in tube marked “DMSO” (final percentage 0.5% as for all compounds). Add 0.7 μl of 10 mM MG132 and 2.8pal DMSO in tube marked “MG132” (final conc.: 10 μM). Add 0.7pal of 10 mM MLN4924 and 2.8 μM DMSO in tube marked “MLN4924” (final conc. 10 uM). Add 4001l of pre-warmed OptiMEM to each of tubes 1-7 with serial dilution of compound X. Add 700 ul of pre-warmed OptiMEM to tubes marked “DMSO”, “MG132” and “MLN4924”. Take plate with seeded MCF7 cells and remove the media from the first column of wells. Add 100 μl of “DMSO” to each of six wells in first column of wells. Remove media from the second column of wells and add 100 μl of “MG132” to each of six wells in second column of wells. Remove media from the third column of wells and add 100 μl of “MLN4924” to each of six wells in third column of wells. Remove media from the remaining columns of wells in small batches and add 100 μl of each dilution of compound X to three wells in fourth to tenth column of wells. Incubate at 37° C./5% CO2 for 8 hrs. Add 1 ml of 37% formaldehyde to 9 ml PBS. Remove the media from the plate and add 100 μl of 3.7% formaldehyde to each well. Incubate at RT for 20 mins. Remove formaldehyde and add 100 μl of PBS to each well. Remove PBS and add another 100 μl PBS to each well. Store at 4° C. overnight.
Immunostaining of β-catenin using In Cell Western (ICW) protocol—Add 100 μl of PBS+0.1% triton to each well and incubate at RT with gentle mixing for 5 mins. Replace with fresh PBS+0.1% triton and repeat 4 times. Note—all washing steps must be carried out gently to avoid dislodging cells. Add 100 μl of 3% BSA in PBS-Tween to each well and incubate at RT with gentle mixing for 1 hour. Add 6.51 al of β-catenin antibody to 6.5 ml of 3% BSA in PBS-Tween. Add 100 μl of primary antibody solution to all bar one of the DMSO-treated wells. This will act as a negative control. It is also possible to not add primary antibody to one well of each treated column of wells in order to have a no primary control for all positive controls and each concentration of COI. Incubate at RT with gentle mixing for 2.5 hours or overnight at 4° C. Add 100 μl of PBS-Tween to each well and incubate at RT with gentle mixing for 5 mins. Replace with fresh PBS-Tween and repeat 4 times. Spin down the vial of anti-rabbit IR800 (LI-COR Biosciences: cat no 926-32213 Donkey Anti-Rabbit IRDye 800CW) at top speed for a few seconds and add 6.5 μl of this to 6.5 ml of 3% BSA in PBS-Tween. Add 50 μl of this secondary antibody solution to one well as a control for DRAQ5. Add 0.65pal of DRAQ5 to the secondary antibody solution and add 100 μl of this to all other wells. Incubate at RT with gentle mixing for 1 hr with the plates protected from light with tin foil. Add 100 μl of PBS-Tween to each well and incubate at RT with gentle mixing for 5 mins. Replace with fresh PBS-Tween and repeat 4 times
Traditional Western Blot
The effects of the compounds shown in
Different doses of the compounds were tested on MCF7 cells to see if there would be an effect on their cell viability.
In order to prove that this activity was specific to the active compounds of the invention, a control compound was developed that is identical to the active compound except for the amino acid sequence that confers the specificity of the active compound. The control compound was compared to its partner active compound (UBP037).
xCELLigence Method
The susceptibility of cancer cell lines versus non-cancer cell lines following treatment with the compounds was also investigated.
Analysis
1. Demonstration of Cell Based Activity by Active Compounds in Comparison to an Inactive Compound of a Similar Physicochemical Nature
This has been demonstrated by the xCELLigence assay that compared UBP037 with the control compound (Ts-EdFEGW-Ahx-K(Stearic)-NH2, a scrambled version of a compound of the present invention (see
2. Demonstration of Target-Specific Activity in a Cell Based Assay Format
This is illustrated for compound UBP036 when examined in the GFP reporter (
In addition, the use of two βTrCP substrates; PDCD4 and 1-catenin, in the in cell western assay would also suggest that any effect seen is due to βTrCP inhibition.
Again the active species of all the compounds in
3. Demonstration of Activity in Several Cancer Cell Lines
In the development of the assays for a βTrCP inhibitor a number of cell-line —biomarker combinations were surveyed to identify the most sensitive assay for βTrCP inhibitors. The breast cancer cell-line MCF7 proved extremely responsive to βTrCP inhibition and this could be measured by the rapid and robust accumulation of the βTrCP substrates PDCD4 and 1-catenin.
As can been seen from the data presented here all compounds exhibit this activity (to various degrees) in MCF7 cells.
The next phase of development involved addressing the potential therapeutic benefit of βTrCP inhibition in a cell line that could be replicated in an animal model. Here LNCaPs were chosen given previous evidence that βTrCP inhibition inhibited cell proliferation both in vitro and in vivo (PLoS One. 2010 Feb. 5; 5(2):e9060.) Again it can be seen from the data that all compounds tested exhibit a reduction in proliferation (to various degrees) in LNCaP cells.
In addition to the cellular activity seen in each of these cell lines, it is also becoming apparent that the biomarker activity assays in MCF7 cells appear to predict the therapeutic activity seen in LNCaP cells.
There is also cell viability inhibition in MCF7 cells upon treatment with these compounds (see
Medical Applications of βTrCP Inhibitors
There are a number of potential indications for βTrCP inhibitors including many forms of cancer.
Two key indications exemplified are prostate cancer and breast cancer.
Breast Cancer.
There is clinical evidence that βTrCP2 is over expressed in a number of cancers including breast cancer [J Biol Chem. 2002, 277, 36624-30]. This study also demonstrated that the cell lines used to model breast cancer such as MCF7 cells also display this same overexpression when compared to non-cancer cell lines such as MCF10A. This implies that work done on βTrCP inhibition in these cancer cell lines could indeed reflect potential outcomes in a clinical setting.
There have been numerous in vivo studies to demonstrate the importance of βTrCP in mammary development. In βTrCP1−/− mice there is a hypoplastic phenotype observed where cell proliferation is reduced by 50% in the mammary gland with other organs unaffected. Furthermore, when there is exogenous high expression of βTrCP1 introduced in the mammary epithelia, approx 40% of mice develop carcinomas. [Mol Cell Biol. 2004, 24, 8184-94.]. The value of this study is two-fold. It demonstrates that despite the widespread expression of βTrCP, a systemic reduction in βTrCP levels (via the genetic ablation of βTrCP1) has a preferential effect on the mammary gland. Also it reveals that overexpression of βTrCP1 can in itself result in an increased cancer risk in this tissue. This suggests inhibition of βTrCP may be of value in both breast cancers that do not display βTrCP overexpression (as inhibition of βTrCP in healthy animals appears to preferentially target the mammary gland for reduced cell proliferation) and those that do (due to the potential causative effect of βTrCP mis-regulation).
In addition to the value of inhibiting βTrCP alone to affect favourable outcomes in breast cancer, there is also work to suggest that combining βTrCP inhibition with some of the current therapies for breast cancer could improve the outcome of these therapies. Inhibition of βTrCP by an RNAi approach suppressed growth and survival of human breast cancer cells [Cancer Res. 2005 Mar. 1; 65(5):1904-8]. It is worth noting that these experiments were carried out on both ER-positive and ER-negative breast cancer cell lines with βTrCP inhibition having a similar impact on both. In addition, inhibition of βTrCP augmented the anti-proliferative effects of anticancer drugs such as doxorubicin, tamoxifen, and paclitaxel on human breast cancer cells. These data suggest that βTrCP inhibition could be effective as a front line adjuvant therapy or in combination with an existing breast cancer treatment regime.
We have shown that the βTrCP inhibitors described here inhibit binding of βTrCP to IκBα (a well-known βTrCP substrate) in in vitro binding assays and stabilise several βTrCP substrates in cell based assays. In addition this inhibitor can reduce the cell viability of a breast cancer cell line in a similar fashion to βTrCP RNAi.
These data and the studies described above show that βTrCP is a validated, novel target in breast cancer, and that its inhibition is tractable and of clinical significance.
Prostate Cancer.
Here the main evidence for the role of βTrCP is from the work of Yinon Ben-Neriah and Eli Pikarsky [PLoS One. 2010 Feb. 5; 5(2):e9060.] Their key finding here was not only that inhibition of βTrCP resulted in the loss of cell viability of LNCaP cells in vitro—but also that when this inhibition is transferred from cells to animals through the use of LNCaP xenografts—it results in a loss of growth of prostate tumours and in combination with androgen ablation—the lack of tumour growth entirely.
A number of different C-terminal and N-terminal capping groups were added to peptide d-E-G-F(3F)—W-E-NH2 in order to demonstrate how the inhibitory activity of d-E-G-F(3F)—W-E-NH2 is affected by particular capping groups, which act to increase cell penetration (as illustrated by Δc Log P, relative to UBP022). Ki and Δc Log P values for the the capped compounds are shown in Table 12.
As suggested by the data in table 12, modification of the C-terminal capping group has little effect upon activity. Modification of the N-terminal capping group has a more profound effect. The function of the capping groups is to aid cell penetration as demonstrated by the Δc Log P values. c Log P values are calculated by means well known to the person skilled in the art.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1110938.6 | Jun 2011 | GB | national |
This application is a continuation of U.S. application Ser. No. 14/127,430, filed Jun. 30, 2014, which is a national stage of PCT Application No. PCT/GB2012/051507, filed on Jun. 27, 2012. This application claims priority under 35 USC § 119 to British Application No. 1110938.6, filed on Jun. 27, 2011.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14127430 | Jun 2014 | US |
| Child | 16129700 | US |
