COMPOUNDS

Abstract

The invention provides cyclic peptides that are able to disrupt the typical response to hypoxia and which have particular utility in the treatment of cancers and von Hippel-Lindau disease.

Claims

1. A cyclic peptide, wherein the cyclic peptide has the following sequence: TABLE-US-00074 [Formula1] [SEQIDNO:1] CX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5 wherein: i) C is selected from the group comprising or consisting of cys, h-cys and d-cys, optionally is cys; ii) X.sub.1 is selected from the group comprising or consisting of leu, lys and glu, optionally is lys or leu, optionally is lys; iii) X.sub.2 is: ##STR00097## wherein: R.sub.1 is H or methyl; R.sub.2 is C.sub.1-C.sub.6 linear or branched alkyl, optionally substituted with OC.sub.1-3 alkyl iv) X.sub.3 is: ##STR00098## wherein: L.sub.1 is C.sub.1-C.sub.3 alkylene; R.sub.3 is selected from the group consisting of a C.sub.3-C.sub.6 cycloalkyl, a 5-6 membered ring and 8-10 membered bicyclic ring, wherein the rings are optionally substituted with phenyl, halogen, OH, O(C.sub.1-C.sub.3 alkyl), C.sub.1-C.sub.4 alkyl or C.sub.1-C.sub.4 haloalkyl; v) X.sub.4 is: ##STR00099## wherein: R.sub.4 is H or methyl; R.sub.5 is C.sub.1-C.sub.6 linear or branched alkyl, optionally substituted with OC.sub.1-3 alkyl; vi) X.sub.5 is: ##STR00100## wherein: L.sub.2 is C.sub.1-C.sub.3 alkylene or a direct bond; R.sub.6 is selected from the group consisting of a C.sub.3-C.sub.6 cycloalkyl, 5-6 membered ring and 8-10 membered bicyclic ring, wherein the rings are optionally substituted with phenyl, halogen, NO.sub.2, OH, O(C.sub.1-C.sub.3 alkyl), C.sub.1-C.sub.4 alkyl or C.sub.1-C.sub.4 haloalkyl; wherein: ##STR00101##

2. The cyclic peptide according to claim 1 wherein the peptide does not comprise the sequence: Cys leu leu phe val tyr [SEQ ID NO: 44].

3. The cyclic peptide according to claim 1 wherein the cyclic peptide comprises at least one of: X.sub.2 wherein: (i) R.sub.1 and R.sub.2 are methyl (aib); or (ii) R.sub.1 is H and R.sub.2 is selected from the group consisting of (S)-isopentyl (h-leu), (S)-n-propyl (n-val), (S)-methoxymethyl (ser(OMe)), and (S)-n-butyl (n-leu), preferably (iii) R.sub.1 is H and R.sub.2 is selected from the group consisting of (S)-isopentyl (h-leu), (S)-n-propyl (n-val), (S)-methoxymethyl (ser(OMe)), and (S)-n-butyl (n-leu), more preferably; (iv) R.sub.1 is H and R.sub.2 is (S)-n-butyl (n-leu); or X.sub.3 wherein: (i) L.sub.1 is (S)-methylene and R.sub.3 is selected from the group consisting of cyclohexyl (Cha), 4-iodophenyl (phe(4-I)), 4-chlorophenyl (phe(4-Cl)), 4-bromophenyl (phe(4-Br)), 4-phenylphenyl (phe(4-Ph)), 4-trifluoromethylphenyl (phe(4-CF.sub.3)) and 1-naphtyl (NaI), or (ii) L.sub.1 is (R)-methylene and R.sub.3 is phenyl (d-Phe), or (iii) L.sub.1 is ethylene, R.sub.3 is selected from the group consisting of phenyl (h-Phe) and 7-hydroxy-4-coumarinyl (Cou), preferably (iv) L.sub.1 is (S)-methylene and 0.3 is selected from the group consisting of 4-iodophenyl (phe(4-I)), 4-chlorophenyl (phe(4-Cl)), 4-bromophenyl (phe(4-Br)), 4-phenylphenyl (phe(4-Ph)), and 4-trifluoromethylphenyl (phe(4-CF3)), 1-naphtyl (NaI), or (v) L.sub.1 is (R)-methylene and R.sub.3 is phenyl (d-Phe), or (vi) L.sub.1 is ethylene and R.sub.3 is 7-hydroxy-4-coumarinyl (Cou), more preferably (vii) L.sub.1 is (S)-methylene and R.sub.3 is 4-trifluoromethylphenyl (phe(4-CF.sub.3)); or X.sub.4 wherein: (i) R.sub.4 and R.sub.5 are methyl (aib), (ii) R.sub.4 is H and R.sub.5 is selected from the group consisting of ethyl (abu), (S)-n-butyl (n-leu) and (S)-methoxymethyl (ser(OMe)), preferably (iii) R.sub.4 is H and R.sub.5 is selected from (S)-n-butyl (n-leu) and (S)-methoxymethyl (ser(OMe)); or X.sub.5 wherein: (i) L.sub.2 is (S)-methylene and R.sub.6 is selected from the group consisting of 4-pyrid-tyrl (4-Pal), cyclohexyl (Cha), 4-phenylphenyl (phe(4-Ph)), 4-trifluoromethylphenyl (phe(4-CF.sub.3)), 4-iodophenyl (phe(4-I)), 4-fluorophenyl (phe(4-F)), 4-chlorophenyl (phe(4-Cl)), 4-bromophenyl (phe(4-Br)), 4-nitrophenyl (phe(4-NO.sub.2)), 1-naphtyl (NaI) and 4-methoxyphenyl (tyr(Me)), or (ii) L.sub.2 is (R)-methylene and R.sub.6 is 4-hydroxyphenyl (d-tyr), or (iii) L.sub.2 is a direct bond and R.sub.6 is phenyl (phg), or (iv) L.sub.2 is ethylene and R.sub.6 is phenyl (h-phe), preferably (v) L.sub.2 is (S)-methylene and R.sub.6 is selected from the group consisting of cyclohexyl (Cha), 4-phenylphenyl (phe(4-Ph)), 4-trifluoromethylphenyl (phe(4-CF.sub.3)), 4-iodophenyl (phe(4-I)), 4-fluorophenyl (phe(4-F)), 4-chlorophenyl (phe(4-Cl)), 4-bromophenyl (phe(4-Br)), 4-nitrophenyl (phe(4-NO.sub.2)), 1-naphtyl (NaI) and 4-methoxyphenyl (tyr(Me)), or (vi) L.sub.2 is (R)-methylene and R.sub.6 is 4-hydroxyphenyl (d-tyr), or (vii) L.sub.2 is a direct bond and R.sub.6 is phenyl (phg), or (viii) L.sub.2 is ethylene and R.sub.6 is phenyl (h-phe), more preferably (ix) L.sub.2 is (S)-methylene and R.sub.6 is selected from the group consisting of 4-iodophenyl (phe(4-I)), 4-fluorophenyl (phe(4-F)), 4-chlorophenyl (phe(4-Cl)), 4-bromophenyl (phe(4-Br)) and 4-nitrophenyl (phe(4-NO.sub.2)), or (x) L.sub.2 is ethylene and R.sub.6 is phenyl (h-phe), most preferably (xi) L.sub.2 is (S)-methylene and R.sub.6 is 4-bromophenyl (phe(4-Br)).

4. The cyclic peptide according to claim 1 wherein R.sub.4 is H and R.sub.5 is (S,S)-2-butyl (ile).

5. The cyclic peptide according to claim 1 wherein where R.sub.4 is H and R.sub.5 is (S,S)-2-butyl (ile), then X.sub.2 selected from Aib, h-leu, n-val, Prop, or n-leu, optionally selected from hL, n-val, Ser(OMe) or n-leu, optionally n-leu; and X.sub.3 selected from cha, h-phe, phe(4-I), phe(4-Cl), d-phe, NaI, phe(4-Br), phe(4-Ph), phe(CF3) and Cou; optionally phe(4-I), phe(4-Cl), d-phe, NaI, phe(4-Br), phe(4-Ph), phe(CF3) and Cou; optionally phe(CF3); and X.sub.5 is selected from Pal, Cha, phe(4-Ph), d-tyr, Phg, phe(CF3), tyr(Me), phe(4-I), h-phe, phe(NO2), NaI, phe(4-F), phe(4-Cl), or phe(4-Br); optionally Cha, phe(4-Ph), d-tyr, Phg, phe(CF3), tyr(Me), phe(4-I), h-phe, phe(NO2), NaI, phe(4-F), phe(4-Cl), or phe(4-Br); optionally phe(4-I), h-phe, phe(NO2), NaI, phe(4-F), phe(4-Cl), or phe(4-Br); optionally phe(4-Br).

6. The cyclic peptide according to claim 1 wherein: i) C is selected from the group comprising or consisting of cys, h-cys and d-cys, optionally is cys; ii) X.sub.1 is selected from the group comprising or consisting of leu, lys and glu, optionally is lys or leu, optionally is lys; iii) X.sub.2 is selected from the group comprising or consisting leu, val, aib, h-leu, n-val, ser(OMe), ile, n-leu, optionally selected from the group comprising or consisting of h-leu, n-val, ser(OMe), ile, n-leu, optionally selected from the group comprising or consisting of ser(OMe) or ile; iv) X.sub.3 is selected from the group comprising or consisting of phe, Cha and h-phe, phe(4-I), phe(4-Cl), d-phe, NaI, phe(4-Br), phe(4-Ph) and phe(4-CF.sub.3), Cou; optionally selected from the group comprising or consisting of phe(4-I), phe(4-Cl), d-phe, NaI, phe(4-Br), phe(4-Ph) and phe(4-CF.sub.3), Cou; optionally is phe(4-CF.sub.3); v) X.sub.4 is selected from the group comprising or consisting val, aib, abu, n-leu, ser(OMe), leu, ile; optionally selected from the group comprising or consisting of n-leu, ser(OMe), leu, ile; optionally selected from the group comprising or consisting of leu, ile; optionally is ile; and vi) X.sub.5 is selected from the group comprising or consisting tyr, 4-Pal, Cha, phe(4-Ph), d-tyr, phg, phe(4-CF.sub.3), phe, tyr(Me), phe(4-I), h-phe, phe(4-NO.sub.2), Nal, phe(4-F), phe(4-Cl), phe(4-Br); optionally selected from the group comprising or consisting of Cha, phe(4-Ph), d-tyr, phg, phe(4-CF.sub.3), phe, tyr(Me), phe(4-I), h-phe, phe(4-NO.sub.2), Nal, phe(4-F), phe(4-Cl), phe(4-Br); optionally selected from the group comprising or consisting of phe(4-I), h-phe, phe(4-NO.sub.2), Nal, phe(4-F), phe(4-Cl), phe(4-Br); optionally is phe(4-Br); wherein: ##STR00102## ##STR00103## ##STR00104## ##STR00105##

7. The cyclic peptide according to claim 1 wherein the cyclic peptide comprises at least one of: X.sub.2 selected from aib, h-leu, n-val, ser(OMe), n-leu, optionally selected from h-leu, n-val, ser(OMe), and n-leu, optionally n-leu; or X.sub.3 selected from Cha, h-phe, phe(4-I), phe(4-Cl), d-phe, NaI, phe(4-Br), phe(4-Ph), phe(4-CF.sub.3), phe(4-tBu) and Cou, optionally selected from phe(4-I), phe(4-Cl), d-phe, NaI, phe(4-Br), phe(4-Ph), phe(4-CF.sub.3), phe(4-tBu) and Cou, optionally is phe(4-CF.sub.3); or X.sub.4 is selected from aib, abu, n-leu, n-val, or ser(OMe), optionally selected from n-leu or ser(OMe); or X.sub.5 is selected from 4-Pal, Cha, phe(4-Ph), d-tyr, phg, phe(4-CF.sub.3), tyr(Me), phe(4-I), h-phe, phe(4-NO.sub.2), Nal, phe(4-F), phe(4-Cl), phe(4-tBu) or phe(4-Br), optionally selected from Cha, phe(4-Ph), d-tyr, phg, phe(4-CF.sub.3), tyr(Me), phe(4-I), h-phe, phe(4-NO.sub.2), Nal, phe(4-F), phe(4-Cl), phe(4-tBu) and phe(4-Br), optionally selected from phe(4-I), h-phe, phe(4-NO.sub.2), Nal, phe(4-F), phe(4-Cl), phe(4-Br), optionally is phe(4-Br).

8. The cyclic peptide according to claim 1 wherein where X.sub.4 is ile, then: X.sub.2 is selected from aib, h-leu, n-val, ser(OMe), n-leu, optionally selected from h-leu, n-val, ser(OMe), and n-leu, optionally n-leu; or X.sub.3 is selected from Cha, h-phe, phe(4-I), phe(4-Cl), d-phe, NaI, phe(4-Br), phe(4-Ph), phe(4-CF.sub.3) and Cou, optionally selected from phe(4-I), phe(4-Cl), d-phe, Nal, phe(4-Br), phe(4-Ph), phe(4-CF.sub.3) and Cou, optionally is phe(4-CF.sub.3); or X.sub.5 is selected from 4-Pal, Cha, phe(4-Ph), d-tyr, phg, phe(4-CF.sub.3), tyr(Me), phe(4-I), h-phe, phe(4-NO.sub.2), Nal, phe(4-F), phe(4-Cl) or phe(4-Br), optionally selected from Cha, phe(4-Ph), d-tyr, phg, phe(4-CF.sub.3), tyr(Me), phe(4-I), h-phe, phe(4-NO.sub.2), Nal, phe(4-F), phe(4-Cl) and phe(4-Br), optionally selected from phe(4-I), h-phe, phe(4-NO.sub.2), Nal, phe(4-F), phe(4-Cl), phe(4-Br), optionally is phe(4-Br).

9. The cyclic peptide according to claim 1 wherein where X.sub.4 is Ile, then X.sub.2 is selected from Aib, h-leu, n-val, Prop, or n-leu, optionally selected from h-leu, N-VAL, Prop or n-leu, optionally n-leu; and X.sub.3 is selected from ha, h-phe, phe(I), phe(4-Cl), d-phe, NaI, phe(4-Br), phe(4-Ph), phe(CF3) and Cou; optionally phe(I), phe(Cl), dphe, NaI, phe(Br), phe(4-Ph), phe(CF3) and Cou; optionally phe(CF3); and X.sub.4 is selected from Abu, Aib, n-leu, or Prop; optionally n-leu or Prop; and X.sub.5 is selected from Pal, Cha, phe(4-Ph), d-tyr, Phg, phe(CF3), tyr(Me), F(I), hF, phe(NO2), NaI, phe(F), phe(Cl), or phe(Br); optionally Cha, phe(4-Ph), d-tyr, Phg, phe(CF3), tyr(Me), F(I), hF, phe(NO2), NaI, phe(F), phe(Cl), or phe(Br); optionally F(I), hF, phe(NO2), NaI, phe(F), phe(Cl), or phe(Br); optionally phe(Br).

10. The cyclic peptide according to claim 1 wherein the peptide comprises, or consists of, one of the following sequences: TABLE-US-00075 [SEQIDNO:2] CysleuleuChavaltyr [SEQIDNO:3] Cysleuleuhphevaltyr [SEQIDNO:4] Cysleuleuphe(I)valtyr [SEQIDNO:5] Cysleuleuphe(Cl)valtyr [SEQIDNO:6] Cysleuleudphevaltyr [SEQIDNO:7] CysleuleuNalvaltyr [SEQIDNO:8] Cysleuleuphe(Br)valtyr [SEQIDNO:9] Cysleuleuphe(4-Ph)valtyr [SEQIDNO:10] Cysleuleuphe(CF3)valtyr [SEQIDNO:11] Cysleuleucouvaltyr [SEQIDNO:56] Cysleuleuphe(4-tBu)valtyr optionally comprises or consists of one of the following sequences: TABLE-US-00076 [SEQIDNO:4] Cysleuleuphe(I)valtyr [SEQIDNO:5] Cysleuleuphe(Cl)valtyr [SEQIDNO:6] Cysleuleudphevaltyr [SEQIDNO:7] CysleuleuNalvaltyr [SEQIDNO:8] Cysleuleuphe(Br)valtyr [SEQIDNO:9] Cysleuleuphe(4-Ph)valtyr [SEQIDNO:10] Cysleuleuphe(CF3)valtyr [SEQIDNO:39] Cysleuleucouvaltyr [SEQIDNO:56] Cysleuleuphe(4-tBu)valtyr optionally comprises or consists of the following sequence: TABLE-US-00077 [SEQIDNO:10] Cysleuleuphe(CF.sub.3)valtyr [SEQIDNO:56] Cysleuleuphe(4-tBu)valtyr optionally comprises or consists of the following sequence: TABLE-US-00078 [SEQIDNO:10] Cysleuleuphe(CF.sub.3)valtyr.

11. The cyclic peptide according to claim 1 wherein the peptide comprises, or consists of, one of the following sequences: TABLE-US-00079 [SEQIDNO:11] Cysleuleuphe(CF.sub.3)valPal [SEQIDNO:12] Cysleuleuphe(CF.sub.3)valCha [SEQIDNO:13] Cysleuleuphe(CF.sub.3)valphe(4-Ph) [SEQIDNO:14] Cysleuleuphe(CF.sub.3)vald-tyr [SEQIDNO:15] Cysleuleuphe(CF.sub.3)valPhg [SEQIDNO:16] Cysleuleuphe(CF3)valphe(4-CF3) [SEQIDNO:17] Cysleuleuphe(CF.sub.3)valphe [SEQIDNO:18] Cysleuleuphe(CF.sub.3)valtyr(Me) [SEQIDNO:19] Cysleuleuphe(CF.sub.3)valphe(I) [SEQIDNO:20] Cysleuleuphe(CF.sub.3)valh-phe [SEQIDNO:21] Cysleuleuphe(CF.sub.3)valphe(NO.sub.2) [SEQIDNO:22] Cysleuleuphe(CF.sub.3)valNal [SEQIDNO:23] Cysleuleuphe(CF.sub.3)valphe(F) [SEQIDNO:24] Cysleuleuphe(CF.sub.3)valphe(Cl) [SEQIDNO:25] Cysleuleuphe(CF.sub.3)valphe(Br) [SEQIDNO:57] Cysleuleuphe(CF.sub.3)valphe(4-tBu) optionally comprises or consists of one of the following sequences: TABLE-US-00080 [SEQIDNO:12] Cysleuleuphe(CF.sub.3)valCha [SEQIDNO:13] Cysleuleuphe(CF.sub.3)valphe(4-Ph) [SEQIDNO:14] Cysleuleuphe(CF.sub.3)vald-tyr [SEQIDNO:15] Cysleuleuphe(CF.sub.3)valPhg [SEQIDNO:16] Cysleuleuphe(CF3)valphe(4-CF3) [SEQIDNO:17] Cysleuleuphe(CF.sub.3)valphe [SEQIDNO:18] Cysleuleuphe(CF.sub.3)valtyr(Me) [SEQIDNO:19] Cysleuleuphe(CF.sub.3)valphe(I) [SEQIDNO:20] Cysleuleuphe(CF.sub.3)valh-phe [SEQIDNO:21] Cysleuleuphe(CF.sub.3)valphe(NO.sub.2) [SEQIDNO:22] Cysleuleuphe(CF.sub.3)valNal [SEQIDNO:23] Cysleuleuphe(CF.sub.3)valphe(F) [SEQIDNO:24] Cysleuleuphe(CF.sub.3)valphe(Cl) [SEQIDNO:25] Cysleuleuphe(CF.sub.3)valphe(Br) [SEQIDNO:57] Cysleuleuphe(CF.sub.3)valphe(4-tBu) optionally comprises or consists of one of the following sequences: TABLE-US-00081 [SEQIDNO:19] Cysleuleuphe(CF.sub.3)valphe(I) [SEQIDNO:20] Cysleuleuphe(CF.sub.3)valh-phe [SEQIDNO:21] Cysleuleuphe(CF.sub.3)valphe(NO.sub.2) [SEQIDNO:22] Cysleuleuphe(CF.sub.3)valNal [SEQIDNO:23] Cysleuleuphe(CF.sub.3)valphe(F) [SEQIDNO:24] Cysleuleuphe(CF.sub.3)valphe(Cl) [SEQIDNO:25] Cysleuleuphe(CF.sub.3)valphe(Br) [SEQIDNO:57] Cysleuleuphe(CF.sub.3)valphe(4-tBu) optionally comprises or consists of the following sequence: TABLE-US-00082 [SEQIDNO:25] Cysleuleuphe(CF.sub.3)valphe(Br).

12. The cyclic peptide according to claim 1 wherein the peptide comprises, or consists of, one of the following sequences: TABLE-US-00083 [SEQIDNO:26] Cysleuleuphe(CF.sub.3)Abuphe(4-Br) [SEQIDNO:27] Cysleuleuphe(CF.sub.3)Aibphe(4-Br) [SEQIDNO:28] Cysleuleuphe(CF.sub.3)n-leuphe(Br) [SEQIDNO:29] Cysleuleuphe(CF.sub.3)Propphe(Br) [SEQIDNO:30] Cysleuleuphe(CF.sub.3)leuphe(4-Br) [SEQIDNO:31] Cysleuleuphe(CF.sub.3)ilephe(Br) [SEQIDNO:58] Cysleuleuphe(CF.sub.3)n-valphe(4-Br) [SEQIDNO:59] Cysleuleuphe(CF.sub.3)h-leuphe(4-Br) optionally comprises or consists of one of the following sequences: TABLE-US-00084 [SEQIDNO:28] Cysleuleuphe(CF.sub.3)n-leuphe(Br) [SEQIDNO:29] Cysleuleuphe(CF.sub.3)Propphe(Br) [SEQIDNO:30] Cysleuleuphe(CF.sub.3)leuphe(4-Br) [SEQIDNO:31] Cysleuleuphe(CF.sub.3)ilephe(Br) [SEQIDNO:59] Cysleuleuphe(CF.sub.3)h-leuphe(4-Br) optionally comprises or consists of one of the following sequences: TABLE-US-00085 [SEQIDNO:30] Cysleuleuphe(CF.sub.3)leuphe(4-Br) [SEQIDNO:31] Cysleuleuphe(CF.sub.3)ilephe(Br) [SEQIDNO:59] Cysleuleuphe(CF.sub.3)h-leuphe(4-Br) optionally comprises or consists of the following sequence: TABLE-US-00086 [SEQIDNO:31] Cysleuleuphe(CF.sub.3)ilephe(Br).

13. The cyclic peptide according to claim 1 wherein the peptide comprises, or consists of, one of the following sequences: TABLE-US-00087 [SEQIDNO:32] Cysleuvalphe(CF.sub.3)ilephe(Br) [SEQIDNO:33] CysleuAibphe(CF.sub.3)ilephe(Br) [SEQIDNO:34] Cysleuh-leuphe(CF.sub.3)ilephe(Br) [SEQIDNO:35] Cysleun-valphe(CF.sub.3)ilephe(Br) [SEQIDNO:36] CysleuPropphe(CF.sub.3)ilephe(Br) [SEQIDNO:37] Cysleuilephe(CF.sub.3)ilephe(Br) [SEQIDNO:38] Cysleun-leuphe(CFCF.sub.33)ilephe(Br); optionally comprises or consists of one of the following sequences: TABLE-US-00088 [SEQIDNO:34] Cysleuh-leuphe(CF.sub.3)ilephe(Br) [SEQIDNO:35] Cysleun-valphe(CF.sub.3)ilephe(Br) [SEQIDNO:36] CysleuPropphe(CF.sub.3)ilephe(Br) [SEQIDNO:37] Cysleuilephe(CF.sub.3)ilephe(Br) [SEQIDNO:38] Cysleun-leuphe(CFCF.sub.33)ilephe(Br) optionally comprises or consists of the following sequence: TABLE-US-00089 [SEQIDNO:38] Cysleun-leuphe(CFCF.sub.33)ilephe(Br)

14. The cyclic peptide according to claim 1 wherein the peptide comprises, or consists of, one of the following sequences: TABLE-US-00090 Cyslysn-leuphe(CF.sub.3)ilephe(Br) Cysglun-leuphe(CF.sub.3)ilephe(Br).

15. The cyclic peptide according to claim 1 wherein the peptide comprises, or consists of, one of the following sequences: TABLE-US-00091 [SEQIDNO:40] h-cysleuleuphevaltyr [SEQIDNO:41] d-cysleuleuphevaltyr.

16. The cyclic peptide according to claim 1 wherein any one or more of C X.sub.1 X.sub.2 X.sub.3 X.sub.4 X.sub.5 is a D amino acid.

17. The cyclic peptide according to claim 1 wherein any one or more of C X.sub.1 X.sub.2 X.sub.3 X.sub.4 X.sub.5 is an L amino acid.

18. The cyclic peptide according to claim 1 wherein the cyclic peptide is capable of binding to HIF-1, optionally capable of binding to recombinantly expressed PAS-B domain of HIF-1.

19. The cyclic peptide according to claim 1 wherein the cyclic peptide is capable of binding to recombinantly expressed PAS-B domain of HIF-1 with a Kd of: less than 40 M, 37 M, 36 M, 35 M, 30 M, 25 M, 20 M, 18 M, 16 M, 15 M, 14 M, 13 M, 12 M, 11 M, 10 M, 9 M, 8 M, 7 M, 6 M, 5 M, 4 M, 3 M, 2.5 M, 2 M, 1.5 M, 1 M, 0.8 M, 0.6 M, 0.5 M, 0.4 M, 0.3 M, 0.2 M, 0.1 M; and/or between 0.1 M and 10 M, 0.2 M and 9 M, 0.3 M and 8 M, 0.4 M and 7 M, 0.5 M and 6 M, 0.6 and 5.5 M, 0.7 and 5 M, 0.8 and 4.5 M, 0.9 M and 4 M, 1 and 3.5 M, 1.25 M and 3 M, 1.5 M and 2.75 M, 1.75 M and 2.5 M, 2 M and 2.25 M; and/or between 0.1 M and 40 M, 0.2 M and 37 M, 0.3 M and 36 M, 0.4 M and 35 M, 0.5 M and 30 M, 0.6 M and 25 M, 0.7 M and 20 M, 0.8 M and 18 M, 0.9 M and 16 M, 1 M and 15 M, 2 M and 14 M, 3 M and 13 M, 4 M and 12 M, 5 M and 11 M, 6 M and 10 M, 7 M and 9 M, optionally wherein the affinity is determined against the recombinantly expressed PAS-B domain of HIF-1 using microscale thermophoresis.

20. The cyclic peptide according to claim 1 wherein the cyclic peptide has a solubility profile of: less than 10 C Log P, optionally less than 9.5, 9.0, 8.5, 8.0, 7.5, 7.0, 6.5, 6.0, 5.5, 5.0 C Log P.

21. The cyclic peptide according to claim 1, wherein the cyclic peptide is able to disrupt the interaction between recombinantly expressed PAS-B domains from HIF-1-HIF-1 with an IC50 of: less than 50 M, optionally less than 45 M, 40 M, 35 M, 30 M, 25 M, 20 M, 15 M, 10 M or less than 5 M.

22. The cyclic peptide according to claim 1, wherein the cyclic peptide is able to disrupt the interaction between recombinantly expressed HIF-1 and a HRE element in a section of DNA with an IC50 of: less than 75 M, optionally less than 70 M, 65 M, 60 M, 55 M, 50 PM, 45 M, 40 M, 35 M, 30 M, 25 M, 20 M, 15 M, 10 M or less than 5 M.

23. The cyclic peptide according to claim 1 wherein the cyclic peptide prevents or reduces the hypoxia induced expression from a promoter that comprises one or more hypoxia-responsive elements under hypoxic conditions.

24. The cyclic peptide according to claim 1 wherein the cyclic peptide reduces the hypoxia induced expression from a promoter that comprises one or more hypoxia-responsive elements under hypoxic conditions to less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%0, 10%, 5%, 4%, 3%, 2%, 1% of the expression obtained in the absence of the cyclic peptide.

25. A polynucleotide comprising or consisting of a sequence that encodes a cyclic peptide according to claim 1.

26. A polynucleotide comprising or consisting of a sequence that encodes an N-terminal intein fragment, followed by a sequence encoding a cyclic polypeptide according to claim 1, followed by a sequence encoding a C-terminal intein fragment.

27. A cell comprising the polynucleotide according to claim 25.

28. A pharmaceutical composition comprising one or more of the cyclic peptides according to claim 1, and/or a polynucleotide comprising or consisting of a sequence that encodes a cyclic peptide according to claim 1 and/or a cell comprising a polynucleotide comprising or consisting of a sequence that encodes a cyclic peptide according to claim 1, optionally wherein the pharmaceutical composition is formulated with one or more further therapeutic agents, optionally one or more further anti-cancer therapeutic agents.

29. (canceled)

30. (canceled)

31. (canceled)

32. (canceled)

33. (canceled)

34. (canceled)

35. A method for the treatment or prevention of a disease, wherein the method comprises: (a) administration of one or more cyclic peptides according to claim 1; (b) a polynucleotide comprising or consisting of a sequence that encodes a cyclic peptide according to claim 1 and/or (c) a cell comprising a polynucleotide comprising or consisting of a sequence that encodes a cyclic peptide according to claim 1; or (d) a pharmaceutical comprising one or more of the cyclic peptides according to claim 1, a polynucleotide comprising or consisting of a sequence that encodes a cyclic peptide according to claim 1, and/or a cell comprising a polynucleotide comprising or consisting of a sequence that encodes a cyclic peptide according to claim 1, optionally wherein the pharmaceutical composition is formulated with one or more further therapeutic agents, optionally one or more further anti-cancer therapeutic agents.

36. The method of claim 35, wherein the disease, disorder or condition is a disease, disorder or condition that experiences a hypoxic environment and requires the typical hypoxia response for maintenance; that is treatable or preventable by inhibition of dimerization of HIF-1a with HIF1-b and HIF2a with HIF1b and/or inhibits the activity of HIF-1 and HIF-2 and/or HIF-1 or HIF-2 signalling; and/or in which it is desirable to repress hypoxia induced gene expression.

37. (canceled)

38. (canceled)

39. (canceled)

40. A fluorescent probe wherein the fluorescent probe is a cyclic peptide according to claim 1 wherein X.sub.3 is Cou.

41. The fluorescent probe according to claim 40 wherein the cyclic peptide has a sequence of: Cys leu leu cou val tyr [SEQ ID NO: 11].

42. A kit comprising one or more of: a cyclic peptide according to claim 1; a polynucleotide comprising or consisting of a sequence that encodes a cyclic peptide according to claim 1 according to claim 21; a cell comprising a polypeptide comprising or consisting of a sequence that encodes a cyclic peptide according to claim 1; a pharmaceutical composition comprising one or more of the cyclic peptides according to claim 1, a polynucleotide comprising or consisting of a sequence that encodes a cyclic peptide according to claim 1, and/or a cell comprising a polynucleotide comprising or consisting of a sequence that encodes a cyclic peptide according to claim 1, optionally wherein the pharmaceutical composition is formulated with one or more further therapeutic agents, optionally one or more further anti-cancer therapeutic agents; or a fluorescent probe, wherein the fluorescent probe is a cyclic peptide according to claim 1, wherein X.sub.3 is Cou.

43. the method of claim 35, wherein the pharmaceutical composition is administered as part of a combination therapy, optionally wherein the cyclic peptide, polynucleotide or pharmaceutical composition is administered prior to, subsequent to, or simultaneously with one or more further therapeutic agents.

44. The method of claim 35, wherein the diseases is cancer, wherein the cancer is selected from the group comprising or consisting of: acute lymphoblastic leukemia (ALL), Acute myeloid leukemia, Adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphoma, Anal cancer, Appendix cancer, Astrocytoma, childhood cerebellar or cerebral, Basal-cell carcinoma, Bile duct cancer, extrahepatic (see cholangiocarcinoma), Bladder cancer, Bone tumor, osteosarcoma/malignant fibrous histiocytoma, Brainstem glioma, Brain cancer, cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, epend-tyrmoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, Breast cancer, Bronchial adenomas/carcinoids, Burkitt's lymphoma, Carcinoid tumor, childhood, gastrointestinal, Carcinoma of unknown primary, Cerebellar astrocytoma, Cerebral astrocytoma/malignant glioma, Cervical cancer, Chondrosarcoma, Chronic lymphocytic leukemia, Chronic myelogenous leukemia, Chronic myeloproliferative disorders, Colon cancer, Cutaneous T-cell lymphoma, Desmoplastic small round cell tumor, Endometrial cancer, Epend-tyrmoma, Esophageal cancer, Ewing's sarcoma, Extracranial germ cell tumor, Extragonadal germ cell tumor, Extrahepatic bile duct cancer, intraocular melanoma, retinoblastoma, Gallbladder cancer, Gastric (stomach) cancer, Gastrointestinal carcinoid tumor, Gastrointestinal stromal tumor (GIST), Germ cell tumor: extracranial, extragonadal, or ovarian, Gestational trophoblastic tumor, Glioma of the brain stem, Glioma, childhood cerebral astrocytoma, Glioma, childhood visual pathway and hypothalamic, Gastric carcinoid, Hairy cell leukemia, Hepatocellular (liver) cancer, Hodgkin lymphoma, Hypopharyngeal cancer, Hypothalamic and visual pathway glioma, childhood, Intraocular melanoma, Islet cell carcinoma (endocrine pancreas), Kaposi sarcoma, Kidney cancer (renal cell cancer), Laryngeal cancer, Leukaemia, acute lymphoblastic (also called acute lymphocytic leukaemia), acute myeloid (also called acute myelogenous leukemia), chronic lymphocytic (also called chronic lymphocytic leukemia), Leukemia, chronic myelogenous (also called chronic myeloid leukemia), Leukemia, hairy cell, Lip and oral cavity cancer, Liposarcoma, Liver cancer (primary), Lung cancer, non-small cell, Lung cancer, small cell, Lymphomas, Lymphoma, AIDS-related, Lymphoma, Burkitt, Lymphoma, cutaneous T-Cell, Lymphoma, Hodgkin, Lymphomas, Non-Hodgkin, Lymphoma, primary central nervous system, Macroglobulinemia, Waldenstrom, Malignant fibrous histiocytoma of bone/osteosarcoma, Medulloblastoma, Melanoma, Merkel cell cancer, Mesothelioma, Mouth cancer, Multiple endocrine neoplasia syndrome, Multiple myeloma/plasma cell neoplasm, Mycosis fungoides, Myelod-tyrsplastic syndromes Myelod-tyrsplastic/myeloproliferative diseases, Myelogenous leukemia, chronic Myeloid leukemia, adult acute, Myeloid leukemia, childhood acute, Myeloma, multiple (cancer of the bone-marrow), Myeloproliferative disorders, chronic, Myxoma, Nasal cavity and paranasal sinus cancer, Nasopharyngeal carcinoma, Neuroblastoma, Non-Hodgkin lymphoma, Non-small cell lung cancer, Oligodendroglioma, Oral cancer, Oropharyngeal cancer, Osteosarcoma/malignant fibrous histiocytoma of bone, Ovarian cancer, Ovarian epithelial cancer (surface epithelial-stromal tumor), Ovarian germ cell tumor, Ovarian low malignant potential tumor, Pancreatic cancer, Pancreatic cancer, islet cell, Paranasal sinus and nasal cavity cancer, Parathyroid cancer, Penile cancer, Pharyngeal cancer, Pheochromocytoma, Pineal astrocytoma, Pineal germinoma, Pineoblastoma and supratentorial primitive neuroectodermal tumors, childhood, Pituitary adenoma, Plasma cell neoplasia/Multiple myeloma, Pleuropulmonary blastoma, Primary central nervous system lymphoma, Prostate cancer, Rectal cancer, Renal cell carcinoma, Renal pelvis and ureter, transitional cell cancer, Rhabdomyosarcoma, childhood, Salivary gland cancer, Sarcoma, Ewing family of tumors, Sarcoma, Kaposi, Sarcoma, soft tissue, Sarcoma, uterine, Sezary syndrome, Skin cancer (non-melanoma), Skin cancer (melanoma), Skin carcinoma, Merkel cell, Small cell lung cancer, Small intestine cancer, Soft tissue sarcoma, Squamous cell carcinomasee skin cancer (non-melanoma), Squamous neck cancer with occult primary, metastatic, Stomach cancer, Supratentorial primitive neuroectodermal tumor, childhood, T-Cell lymphoma, cutaneous, Testicular cancer, Throat cancer, Thymoma, Thymoma and thymic carcinoma, Thyroid cancer, Thyroid cancer, Transitional cell cancer of the renal pelvis and ureter, Trophoblastic tumor, gestational, Unknown primary site, Ureter and renal pelvis, transitional cell cancer, Urethral cancer, Uterine cancer, endometrial, Uterine sarcoma, Vaginal cancer, Visual pathway and hypothalamic glioma, Vulvar cancer, Waldenstrom macroglobulinemia and/or Wilms tumor (kidney cancer).

Description

FIGURE LEGENDS

[0279] FIG. 1Assessing the activity of cyclo-CLLFVY (SEQ ID NO: 44 Cys leu leu phe val tyr) via MST shows that it binds with a Kd 36.043.6 M.

[0280] FIG. 2A) Bar Chart showing the Kds obtained when replacing cysteine with various amino acid analogues. The data shows that all analogues resulted in poorer binding than the parent compound and only thiol containing amino acids retain any binding activity. B) Bar Chart showing the affinity of Cyclo-CLLFVY for the three cysteine mutant proteins (C255A, C334A and C337A) C) Cysteine derivative MST curves. Tp=Thioproline, M=methionine, A=alanine, S=serine, T=threonine.

[0281] FIG. 3Cysteine mutant HIF-1a Pas-B domains C255A, C334A and C337A.

[0282] FIG. 4A) Bar Chart showing the Kds obtained when replacing phenylalanine with various amino acid analogues. B) MST traces of phenylalanine derivatives with a KD<10 M. Error bars represent the standard deviation of n=3.

[0283] FIG. 5A) Bar Chart showing the Kds obtained when replacing tyrosine with various amino acid analogues. B) MST traces of tyrosine derivatives with a KD<2.36 M. Error bars represent the standard deviation of n=3.

[0284] FIG. 6Bar Chart showing the Kds obtained upon substitution of the valine residue.

[0285] FIG. 7A) Bar Chart showing the Kds obtained when replacing the leucine at position 2 various amino acid analogues. B) MST traces of leucine derivatives.

[0286] FIG. 8MST analysis of CK-n-leu-F(CF3)IF(Br) showed that the peptide bound with a Kd of 1.660.3 M and therefore that the incorporation of the lysine residue is well tolerated.

[0287] FIG. 9Effects of (CONC) CK-n-leu-F(CF3)IF(Br) [SEQ ID NO: 40 Cys lys NL phe(CF3) ile phe(Br)] on the heterodimerization of His-HIF-1 PAS-B domain with FLAG-HIF-1 PAS-B domain analysed by ELISA. CK-n-leu-F(CF3)IF(Br) disrupts this interaction with an IC50 of 27.714 M.

[0288] FIG. 10The parent peptide CLLFVY was found to inhibit DNA binding with an IC50 of 76.5=8.0 M and the peptide CLLF(CF3)IF(Br) [SEQ ID NO: 31 Cys leu leu phe(CF3) ile phe(Br)] was found to inhibit FAM-HRE binding with an IC50 of 11.761.1 M demonstrating that the improvements seen during the SAR study translates to increased potency when inhibiting DNA binding.

[0289] FIG. 11A) Structure of cyclic peptide that incorporates a coumaryl motif at the phenylalanine position CLL-Cou-VY [SEQ ID NO: 39 Cys leu leu cou val tyr]. B) MST analysis of CLL-Cou-VY. C) The peptide was seen to be internalized in HeLa cells upon visualization with PMT detection at 405 nm. D) HIF-1 immunofluorescence was then used to show that the compound is preventing HIF-1 nuclear localization. E) HIF-1 immunofluorescence under control conditions showing that without treatment HIF-1a localizes to the nucleus.

[0290] FIG. 12Fluorescent Polarization Assays

[0291] HIF-1 three-domain protein was thawed on ice and diluted to 150 nM in HIF FP buffer. Compounds for analysis were serially diluted in DMSO to give 10 stocks. The compounds (10 L, 100% DMSO) were then added to the protein (80 L). The samples were then incubated at room temperature for 30 minutes. FAM-HRE (10 L, 20 nM) was then added and the samples loaded into a 384 well solid black non-binding microplate (Corning, USA). The fluorescence polarization of each well was then measured using gain and focus settings optimized by the used software set to a signal of 400 mP on a control well.

[0292] FIG. 13A Panc HRE readout cell line was then used to show that CK-n-leu-F(CF3)IF(Br) [SEQ ID NO: 40 Cys lys n-leu phe(CF3) ile phe(Br)] inhibited the expression of EYFP and therefore inhabits HIF-1a driven transcription in this cell line. B) A Cytotox-Glo assay was used to assess the toxicity of CK-n-leu-F(CF3)IF(Br) and showed that the peptide was not toxic at doeses 50 uM. C) A U2OS HRE readout assay was used to show that the peptide CL-n-leu-F(CF3)IF(Br) [SEQ ID NO: 35 Cys leu n-leu phe(CF3) He phe(Br)] inhibited the expression of EYFP and therefore inhabits HIF-1a driven transcription in this cell line.

EXAMPLES

Example 1

[0293] Cyclo-CLLFVY was obtained by Fmoc solid phase peptide synthesis, and its binding affinity for the HIF-1 PAS B domain determined via microscale thermophoresis (MST). The peptide was found to bind with a Kd of 36.043.6 M (FIG. 1).

[0294] A series of modifications were then carried out whereby the amino acids of the peptide were sequentially replaced with a variety of natural and unnatural amino acid analogues. The impact of these substitutions on the binding affinity was then assessed by MST.

Modifications at the Cysteine Position

[0295] The first position investigated was the cysteine, with seven analogues substituted into this position. The affinity of the new peptides was determined by MST and the resulting KD values are given in Error!Reference source not found.

[0296] The majority of substitutions had no detectable binding activity, and none of the substitutions led to a peptide with a higher affinity than cyclo-CLLFVY. D-cysteine and homocysteine gave comparable affinities to the unsubstituted peptide.

[0297] Notably both the analogues that did show binding contained a free thiol group. This is suggestive of a key interaction between this functionality and the protein. One rationalization would be that the peptide binding is disulphide-mediated. Whilst this was thought to be unlikely as all assays were performed in the presence of the reducing agent TCEP, it was decided that the possibility would be further investigated via site-directed mutagenesis (SDM). The three cysteine residues within the HIF-1 PAS-B domain were sequentially mutated to alanine. The affinity of cyclo-CLLFVY for the resultant mutant proteins C255A, C344A and C337A was then assessed by MST.

[0298] The SDM experiments show that binding activity is retained for all three mutant HIF-1 proteins. Whilst some variation is seen this is likely attributable to changes in protein stability brought about by the mutations which is corroborated by the thermal shift profiles of the three mutants (FIG. 3). If the peptide binding was disulfide mediated, then it would be expected that one of the mutant proteins would have no detectable binding activity as was seen for the derivatives lacking a thiol moiety. It was decided that as the cysteine had the best affinity and was not forming a covalent link that it would be retained, and the next position of the peptide investigated.

Modifications at the Phenylalanine Position

[0299] A series of 16 phenylalanine analogues were synthesized and their affinity for the HIF-1 PAS-B domain determined by MST. Initially a single 16-point run was carried out for each compound after which any peptide with a Kd<10 M was run in triplicate. The resulting Kd values are given in Error!Reference source not found.4. Within this data set we see a preference for large hydrophobic substituents (Nal, phe(4-Ph), phe(CF.sub.3)), with phe(CF.sub.3) yielding the best affinity with a kd of 2.360.2 M, representing a 15-fold improvement in activity compared to the parent peptide.

Modifications at the Tyrosine Position

[0300] CLLF(CF3)VY [SEQ ID NO: 10 Cys leu leu phe(CF3) val tyr] was taken into the next round of analysis.

[0301] We next used the same amino acid series at the remaining aromatic position, with the exception of d-phe being replaced with d-tyr. This yielded the compound series shown in FIG. 5.

[0302] Again the peptides were initially assessed by a single MST run after which any peptide binding with an affinity greater than CLLF(CF.sub.3)VY [SEQ ID NO: 10] was run in triplicate. This series yielded more modest changes in affinity and suggests that the tyrosine position of cyclo-CLLF(CF.sub.3)VY is not forming a critical interaction with the HIF-1 PAS-B protein. As such there is not as a clear a trend observed in the SAR of these derivatives though it is noteworthy how more polar derivatives (F(NO.sub.2)) are tolerated at this position as predicted through the SICLOPPS screening selecting tyrosine over phenylalanine at this position. As was seen for the phenylalanine position extended aromatic systems were well tolerated (Nal) as well as large substituents (phe(4-Br), phe(4-tBu). The amino acid resulting in the greatest affinity for binding was phe(4-Br) with a Kd of 1.350.3 M and so the CLLF(CF.sub.3)VF(Br) [SEQ ID NO: 25 Cys leu leu phe(CF.sub.3) val phe(4-Br)] peptide was carried forward for further optimization.

Modifications at the Valine Position

[0303] The next position investigated was the valine, a series of aliphatic amino acids were selected and substituted into this position. The resulting peptides were assessed by MST and the resulting Kds of this compound series are shown in FIG. 6. The majority of substitutions yielded minor variations in binding affinity with most of the peptides having a Kd of approximately 1 M. A modest increase in affinity was seen upon substitution with amino acids with branched side chains with hL, L and I yielded the best affinities of the series. Isoleucine gave the best affinity and so the CLLF(CF.sub.3)IF(Br) [SEQ ID NO: 31 Cys leu leu phe(CF.sub.3) ile phe(4-Br)] peptide was carried forward.

Modifications at the Leucine 2 Position

[0304] The same aliphatic derivatives (replacing leu for val) were then used to investigate the second leucine of the peptide (CLLF(CF.sub.3)IF(Br)). Upon running the MSTs, it was noted that the error of the binding curves in this derivative series had increased. This was hypothesized to be the result of a deteriorating solubility profile, though no clear precipitation was observed upon sample preparation, there is a clear correlation between increasing C log P of the peptides and their affinity. This highlights a challenge in the development of inhibitors targeting PPIs as the relatively flat and featureless binding surfaces lead to an increasing reliance on weaker Van der Waals forces, that pushes hit compounds to become increasingly hydrophobic.

TABLE-US-00022 Peptide Kd (M) CLogP CLLFVY 6.47 CLLF(CF.sub.3)VY 7.36 CLLF(CF.sub.3)VF(Br) 8.89 CLLF(CF.sub.3)IF(Br) 9.42

[0305] CL-NL-F(CF.sub.3)IF(Br) [SEQ ID NO: 38 Cys leu n-leu phe(CF.sub.3) ile phe(4-Br)] was taken forward for further analysis.

Incorporation of Lysine

[0306] It was noted that during SICLOPPS screening against HIF a number of the resultant hit compounds contained an amino acid bearing a positive charge in the second position immediately after the cysteine. In an attempt to improve the solubility of the CL-n-leu-F(CF.sub.3)IF(Br) peptide its tolerance for incorporation of a positive charge was investigated. This was achieved by synthesizing the peptide CK-n-leu-F(CF.sub.3)IF(Br) [SEQ ID NO: 40 Cys lys n-leu phe(CF.sub.3) ile phe(4-Br)]. MST analysis of CK-n-leu-F(CF.sub.3)IF(Br) showed that the peptide bound with a Kd of 1.660.3 M and therefore that the incorporation of the lysine residue is well tolerated.

[0307] Solubility of CK-n-leu-F(CF.sub.3)IF(Br) compound C log P=7.79

ELISAs

[0308] The ability of CK-n-leu-F(CF.sub.3)IF(Br) to disrupt the HIF-1-HIF-1 PAS-B-PAS-B PPI was then assessed via an ELISA assay. CK-n-leu-F(CF.sub.3)IF(Br) was seen to inhibit dimer formation with an IC50 of 27.714 MFIG. 9.

FP

[0309] We next sought to show that inhibition of the interaction between the HIF-1 and HIF-1 PAS-B domains by CK-n-leu-F(CF.sub.3)IF(Br) prevents HIF from binding to a hypoxia response element. An FP assay utilizing a FAM labelled HRE was used and CK-NL-F(CF.sub.3)IF(Br) assessed. However, the peptide was found to be incompatible with this assay as it was found to bind to the FAM-HRE directly, most likely as a result of the positively charged peptide interacting favorably with the negatively charged DNA backbone.

[0310] To avoid this the peptide CLLF(CF.sub.3)IF(Br) was also assessed and found to inhibit HIF-DNA binding with an IC50

[0311] The parent peptide CLLFVY was found to inhibit DNA binding with an IC50 of 76.58.0 M and the peptide CLLF(CF.sub.3)IF(Br) was found to inhibit FAM-HRE binding with an IC50 of 11.761.1 M demonstrating that the improvements seen during the SAR study translates to increased potency when inhibiting DNA binding.

Coumarin

[0312] During the investigation of the SAR at the phenylalanine position it was noted that the extended aromatic derivative, Nal, was well tolerated, leading to a 6-fold increase in affinity compared to CLLFVY. The fluorescent molecule coumarin is structurally similar to Nal and has been demonstrated to be well suited for biological applications. We therefore made a peptide with a coumaryl motif incorporated at the phenylalanine position the compound, CLL-Cou-VY, shown in FIG. 11 was seen to be internalized in HeLa cells upon visualization with PMT detection at 405 nm.

[0313] HIF-1 immunofluorescence was then used to show that the compound is preventing HIF-1 nuclear localization.

Methods

HIF PAS-B MSTs

[0314] An aliquot of labelled HIF-1 PAS-B was thawed on ice and diluted to 50 nM with HIF MST assay buffer, before being centrifuged at 12,000 rpm for 15 minutes. The compound was then serially diluted in DMSO to give 10 stocks. These were then diluted 1:5 in HIF MST assay buffer to give 2 stocks, before finally being mixed 1:1 (v/v) with the labelled protein to give a final concertation of 25 nM protein and a final DMSO content of 10%. Samples were loaded into NanoTemper Monolith NT.115 premium treated capillaries and measurements were carried out using a Monolith NT.115 system at 25 C. using 50% MST power and 50% LED power. Data was analysed by NanoTemper analysis software. Binding curves were fitted using GraphPad Prism 8 software.

Enzyme-Linked Immunosorbent Assay

[0315] To each well of a clear Pierce nickel coated 96-well plate (Thermo Scientific), purified Hiss-HIF-1 PAS-B protein in HIP buffer was added (25 L, 0.1 M), the plate was then incubated for 1 hr at RT with rocking. The wells were then washed 2 with HIF buffer (150 L) and 1 with PBS Tween (0.05% Tween 20, 150 L). 2% milk in PBS Tween (150 L) was then added to each well and incubated for 1 hr at RT with rocking as a blocking step. The wells were then washed 2PBS Tween (150 L) and 1 with HIF buffer (150 L). For HIF-B dimerisation a serial dilution of HIF-1 PAS-B ranging from 250 M to 11 nM in HIF buffer (100 L) was then added. For CLLFVY inhibition a serial dilution of CLLFVY ranging from 500 M to 15 nM in 100% DMSO (10 L) was added at the same time as HIF-1 PAS-B (10 M, 90 L) for a 10% final concentration of DMSO. The plate was then incubated at RT for 1 hr with rocking. The wells were then washed 2 with HIF buffer (150 L) and 1 with PBS Tween (150 L), A 1:1000 dilution of mouse anti-flag antibody in 2% milk PBS Tween (50 L) was then added to each well and the plate incubated for a further hour at RT with rocking. The wells were then washed with 3PBS Tween (150 L) with 5 minutes incubation per wash. A 1:6000 dilution of sheep anti mouse linked to horse radish peroxidase in 2% milk PBS Tween (50 L) was then added and a further incubation of 1 hr at RT with rocking carried out. The plate was then washed as before and 1-Step TMB Ultra Solution (Thermo Scientific) (100 L) was added to each well and incubated for 15 minutes. H.sub.2SO.sub.4(aq) (2 M, 100 L) was then added to each well and the absorbance at 450 nm determined on a BMG Clariostar plate reader.

Fluorescent Polarization Assays

[0316] HIF-1 three-domain protein was thawed on ice and diluted to 150 nM in HIF FP buffer. Compounds for analysis were serially diluted in DMSO to give 10 stocks. The compounds (10 l, 100% DMSO) were then added to the protein (80 L). The samples were then incubated at room temperature for 30 minutes. FAM-HRE (10 L, 20 nM) was then added and the samples loaded into a 384 well solid black non-binding microplate (Corning, USA). The fluorescence polarization of each well was then measured using gain and focus settings optimized by the used software set to a signal of 400 mP on a control well.

Example 2Compound Synthesis and Characterization

CLLFVY Phenylalanine Derivatives

[0317] The linear resin bound sequence resin-LLCYV-NH.sub.2 was synthesised using Wang resin SPPS (0). The resin was then split equally into 10 syringes and swelled in a small amount of DMF. A solution of unnatural amino acid (1 eq.), Oxyma pure (3 eq.) and DIC (3 eq.) was added and the syringes placed on a rocker for 1 hour. The reaction mixtures were then removed via vacuum filtration and the resins washed with DMF2, DCM2 and Et.sub.2O1. Fmoc protected amino acids were then deprotected via the addition of piperidine 20% in DMF and rocking for 30 minutes. The resins were then washed as before. The linear sequences were then cleaved from the resin as described in section 0, and cyclised using HATU, HOAt and DiPEA as described in section 0. The StBu group was then removed (section 0) and the peptide purified via reverse-phase HPLC (section 0), like fractions were combined and concentrated under reduced pressure, then lyophilised to yield the desired cyclic peptides.

TABLE-US-00023 Cyclo-CLLY(Me)VY- SEQIDNO:50 CysleuleuTyr(me)valtyr

##STR00045##

Cyclo-CLLY(Me)VY was obtained as a white solid yield (3.7 mg, 3.3% overall yield). Analytical HPLC R.sub.t=13.419 min (90% purity). m/z (ESI.sup.+): 769.8 [M+H].sup.+ (100%), 791.8 [M+Na].sup.+ (25%). HRMS (ESI.sup.+) found: 769.3944, [M+H].sup.+ calculated: 769.3953.

TABLE-US-00024 Cyclo-CLLYVY SEQIDNO:51 Cysleuleutyrvaltyr

##STR00046##

Cyclo-CLLYVY was obtained as white solid (25.1 mg, 22.2 Va overall yield). Analytical HPLC R.sub.t=11.830 min (99% purity). m/z (ESI.sup.+): 755.7 [M+H].sup.+ (100%). HRMS (ESI.sup.+) found: 755.3791, [M+H].sup.+ calculated: 755.3797.

TABLE-US-00025 Cyclo-CLL-PAL-VY SEQIDNO:53 Cysleuleu4-Palvaltyr

##STR00047##

Cyclo-CLL-PAL-VY was obtained as white solid (16.2 mg, 15.1% overall yield). Analytical HPLC R.sub.t=9.721 min (89% purity). m/z (ESI.sup.+): 740.8 [M+H].sup.+ (100%). HRMS (ESI.sup.+) found: 740.3817, [M+H].sup.+ calculated: 740.3800.

TABLE-US-00026 Cyclo-CLLF(4-NO.sub.2)VY SEQIDNO:54 Cysleuleuphe(NO2)valtyr

##STR00048##

Cyclo-CLLF(4-NO.sub.2)VY was obtained as a white solid (11.5 mg, 10.1% overall yield). Analytical HPLC R.sub.t=13.580 min (93% purity). m/z (ESI.sup.+): 784.8 [M+H].sup.+ (100%). HRMS (ESI.sup.+) found: 784.3695, [M+H].sup.+ calculated: 784.3698.

TABLE-US-00027 Cyclo-CLL-Cha-VY SEQIDNO:2 CysleuleuChavaltyr

##STR00049##

Cyclo-CLL-Cha-VY was obtained as a white solid (3.0 mg, 2.9% overall yield). Analytical HPLC R.sub.t=15.188 min (99% purity). m/z (ESI.sup.+): 745.8 [M+H].sup.+ (100%), 1491.6 [2 M+H].sup.+ (90%). HRMS (ESI.sup.+) found: 745.4309, [M+H].sup.+ calculated: 745.4317.

TABLE-US-00028 Cyclo-CLL-Nal-VY SEQIDNO:7 CysleuleuNalvaltyr

##STR00050##

Cyclo-CLL-Nal-VY yield was obtained as a white solid (14.0 mg, 8.9% overall yield). Analytical HPLC R.sub.t=15.317 min (92% purity). m/z (ESI.sup.+): 789.7 [M+H].sup.+ (100%), 811.6 [M+Na].sup.+ (35%). HRMS (ESI.sup.+) found: 789.3997, [M+H].sup.+ calculated: 789.4004.

TABLE-US-00029 Cyclo-CLL-Phg-VY SEQIDNO:52 CysleuleuPhGvaltyr

##STR00051##

Cyclo-CLL-Phg-VY was obtained as a white solid (22.3 mg, 15.4% overall yield). Analytical HPLC R.sub.t=12.903 min (87% purity). m/z (ESI.sup.+): 725.7 [M+H].sup.+ (100%), 1450.1 [2 M+H].sup.+ (45%). HRMS (ESI.sup.+) found: 725.3687, [M+H].sup.+ calculated: 725.3691.

TABLE-US-00030 Cyclo-CLL-hF-VY SEQIDNO:3 Cysleuleuhphevaltyr

##STR00052##

Cyclo-CLL-hF-VY was obtained as a white solid (29.5 mg, 19.3% overall yield). Analytical HPLC R.sub.t=14.175 min (87% purity). m/z (ESI.sup.+): 753.7 [M+H].sup.+ (100%), 1149.6 [2 M+H].sup.+ (45%). HRMS (ESI.sup.+) found: 753.3991, [M+H].sup.+ calculated: 753.4004.

TABLE-US-00031 Cyclo-CLL-dF-VY SEQIDNO:6 Cysleuleudphevaltyr

##STR00053##

Cyclo-CLL-dF-VY was obtained as a white solid (17.9 mg, 14.8% overall yield). Analytical HPLC R.sub.t=13.746 min (94% purity). m/z (ESI.sup.+): 739.8 [M+H].sup.+ (100%). HRMS (ESI.sup.+) found: 739.3846, [M+H].sup.+ calculated: 739.3847.

TABLE-US-00032 Cyclo-CLLF(F)VY SEQIDNO:55 Cysleuleuphe(4-F)valtyr

##STR00054##

Cyclo-CLLF(F)VY was obtained as a white solid (11.9 mg, 10.9% overall yield). Analytical HPLC R.sub.t=13.905 min (92 % purity). m/z (ESI.sup.+): 757.8 [M+H].sup.+ (100%). HRMS (ESI.sup.+) found: 757.3751, [M+H].sup.+ calculated: 757.3753.

TABLE-US-00033 Cyclo-CLLF(Cl)VY SEQIDNO:5 Cysleuleuphe(Cl)valtyr

##STR00055##

Cyclo-CLLF(Cl)VY was obtained as a white solid (8.5 mg, 7.1 V % overall yield). Analytical HPLC R.sub.t=14.519 min (94% purity). m/z (ESI.sup.+): 773.7 [M+H].sup.+ (100%), 795.7 [M+Na]'.sup.0 (30% a). HRMS (ESI.sup.+) found: 773.3452, [M+H].sup.+ calculated: 773.3458.

TABLE-US-00034 Cyclo-CLLF(Br)VY SEQIDNO:8 Cysleuleuphe(Br)valtyr

##STR00056##

Cyclo-CLLF(Br) VY was obtained as a white solid (12.1 mg, 7.4% overall yield). Analytical HPLC R.sub.t=14.657 min (90% purity). m/z (ESI.sup.+): 819.5 [M+H].sup.+ (100%). HRMS (ESI.sup.+) found: 817.2926, [M+H].sup.+ calculated: 817.2953.

TABLE-US-00035 Cyclo-CLLF(I)VY SEQIDNO:4 Cysleuleuphe(I)valtyr

##STR00057##

Cyclo-CLLF(I)VY was obtained as a white solid (15.8 mg, 9.1% overall yield). Analytical HPLC R.sub.t=15.144 min (90% purity). m/z (ESI.sup.+): 865.7 [M+H].sup.+ (100%). HRMS (ESI.sup.+) found: 865.2803, [M+H].sup.+ calculated: 865.2814.

TABLE-US-00036 Cyclo-CLLF(CF.sub.3)VY SEQIDNO:10 Cysleuleuphe(CF3)valtyr

##STR00058##

Cyclo-CLLF(CF.sub.3)VY was obtained as a white solid (29.4 mg, 18.2% overall yield). Analytical HPLC R.sub.t=14.840 min (90% purity). m/z (ESI.sup.+): 807.7 [M+H].sup.+ (100%), 829.7 [M+Na].sup.+ (60%). HRMS (ESI.sup.+) found: 807.3720, [M+H].sup.+ calculated: 807.3721.

TABLE-US-00037 Cyclo-CLLF(tBu)VY [SEQIDNO:56]

##STR00059##

Cyclo-CLLF(4-tBu)VY was obtained as a white solid (16.6 mg, 10.4% overall yield). Analytical HPLC R.sub.t=16.384 min (93% purity). m/z (ESI.sup.+): 795.87 [M+H].sup.+ (100%). HRMS (ESI.sup.+) found: 795.4449, [M+H].sup.+ calculated: 795.4473.

CLLF(CF.SUB.3.)VY Tyrosine Derivatives

[0318] The linear resin bound sequence resin-LC-NH.sub.2 was synthesised using Wang resin SPPS (section 0). The resin was then split equally into 10 syringes and swelled in a small amount of DMF. Standard SPPS protocols (section 0) were then followed to yield the resin bound linear sequence resin-LCXVF(CF.sub.3)L where X is the unnatural amino acid. The linear sequences were then cleaved from the resin as described in section 0, and cyclised using HATU, HOAt and DIPEA as described in section 0. The StBu group was then removed (section 0) and the peptide purified via reverse-phase HPLC (section 0), like fractions were combined and concentrated under reduced pressure, then lyophilised to yield the desired cyclic peptides.

TABLE-US-00038 CLLF(CF.sub.3)VY(Me) SEQIDNO:18 Cysleuleuphe(CF3)valTyr(me)

##STR00060##

Cyclo-CLLF(CF.sub.3)VY(Me) was obtained as a white solid (9.2 mg, 5.6% overall yield). Analytical HPLC R.sub.t=16.557 min (99% purity). m/z (ESI.sup.+): 821.7 [M+H].sup.+ (100%). HRMS (ESI.sup.+) found: 821.3866, [M+H].sup.+ calculated: 821.3878.

TABLE-US-00039 CLLF(CF.sub.3)VF SEQIDNO:17 Cysleuleuphe(CF3)valphe

##STR00061##

Cyclo-CLLF(CF.sub.3)VF was obtained as a white solid (62.7 mg, 39.7% overall yield). Analytical HPLC R.sub.t=16.769 min (95% purity). m/z (ESI.sup.+): 791.6 [M+H].sup.+ (100%), 813.6 [M+Na].sup.+ (20%). HRMS (ESI.sup.+) found: 791.3779, [M+H].sup.+ calculated: 791.3772.

TABLE-US-00040 CLLF(CF.sub.3)V-Pal SEQIDNO:11 Cysleuleuphe(CF3)valPal

##STR00062##

Cyclo-CLLF(CF.sub.3)V-4-Pal was obtained as a white solid (24.7 mg, 15.6% overall yield). Analytical HPLC R.sub.t=12.770 min (97% purity), m/z (ESI.sup.+): 792.6 [M+H].sup.+ (100%). HRMS (ESI.sup.+) found: 792.3716, [M+H].sup.+ calculated: 792.3725.

TABLE-US-00041 CLLF(CF.sub.3)VF(NO.sub.2) SEQIDNO:21 Cysleuleuphe(CF3)valphe(NO2)

##STR00063##

Cyclo-CLLF(CF.sub.3)VF(NO.sub.2) was obtained as a white solid (20.0 mg, 12.0%). Analytical HPLC R.sub.t=12.877 min (99% purity). m/z (ESI.sup.+): 836.6 [M+H].sup.+ (100%). HRMS (ESI.sup.+) found: 836.3616, [M+H].sup.+ calculated: 836.3623.

TABLE-US-00042 CLLF(CF.sub.3)V-Cha SEQIDNO:12 Cysleuleuphe(CF3)valCha

##STR00064##

Cyclo-CLLF(CF.sub.3)V-Cha was obtained as a white solid (16.2 mg, 10.2% overall yield). Analytical HPLC R.sub.t=18.111 min (97% purity). m/z (ESI.sup.+): 797.7 [M+H].sup.+ (100%). HRMS (ESI.sup.+) found: 797.4242, [M+H].sup.+ calculated: 797.4242.

TABLE-US-00043 CLLF(CF.sub.3)V-Nal SEQIDNO:22 Cysleuleuphe(CF3)valNal

##STR00065##

Cyclo-CLLF(CF.sub.3)V-Nal was obtained as white solid (31.9 mg, 19.0% overall yield). Analytical HPLC R.sub.t=17.959 min (99% purity). m/z (ESI.sup.+): 841.6 [M+H].sup.+ (100%). HRMS (ESI.sup.+) found: 841.3919, [M+H].sup.+ calculated: 841.3929.

TABLE-US-00044 CLLF(CF.sub.3)V-Phg SEQIDNO:15 Cysleuleuphe(CF3)valPhg

##STR00066##

Cyclo-CLLF(CF.sub.3)V-Phg was obtained as a white solid (16.1 mg, 10.4% overall yield). Analytical HPLC R.sub.t=16.544 min (92% purity). m/z (ESI.sup.+): 777.7 [M+H].sup.+ (100%). HRMS (ESI.sup.+) found: 777.3627, [M+H].sup.+ calculated: 777.3616.

TABLE-US-00045 CLLF(CF.sub.3)V-hF SEQIDNO:20 Cysleuleuphe(CF3)valh-phe

##STR00067##

Cyclo-CLLF(CF.sub.3)V-hF was obtained as a white solid (2.7 mg, 1.7% overall yield). Analytical HPLC R.sub.t=17.200 min (90% purity). m/z (ESI.sup.+): 806.7 [M+H].sup.+ (100%). HRMS (ESI.sup.+) found: 805.3933, [M+H].sup.+ calculated: 805.3929.

TABLE-US-00046 CLLF(CF.sub.3)V-dY SEQIDNO:14 Cysleuleuphe(CF3)vald-tyr

##STR00068##

Cyclo-CLLF(CF.sub.3)V-dY was obtained as a white solid (12.1 mg, 7.5% overall yield). Analytical HPLC R.sub.t=14.683 min (97% purity). m/z (ESI.sup.+): 807.7 [M+H].sup.+ (100%). HRMS (ESI.sup.+) found: 807.3720, [M+H].sup.+ calculated: 807.3721.

TABLE-US-00047 CLLF(CF.sub.3)VF(F) SEQIDNO:23 Cysleuleuphe(CF3)valphe(4-F)

##STR00069##

Cyclo-CLLF(CF.sub.3)VF(F) was obtained as a white solid (28.4 mg, 17.6% overall yield). Analytical HPLC R.sub.t=16.665 min (95% purity). m/z (ESI.sup.+): 809.7 [M+H].sup.+ (100%). HRMS (ESI.sup.+) found: 809.3672, [M+H].sup.+ calculated: 809.3678.

TABLE-US-00048 CLLF(CF.sub.3)VF(Cl) SEQIDNO:24 Cysleuleuphe(CF3)valphe(4-Cl)

##STR00070##

Cyclo-CLLF(CF.sub.3)VF(C) was obtained as a white solid (28.4 mg, 17.2% overall yield). Analytical HPLC R.sub.t=17.376 min (99% purity), m/z (ESI.sup.+): 825.6 [M+H].sup.+ (100%). HRMS (ESI.sup.+) found: 825.3382, [M+H].sup.+ calculated: 825.3382.

TABLE-US-00049 CLLF(CF.sub.3)VF(Br) SEQIDNO:25 Cysleuleuphe(CF3)valphe(4-Br)

##STR00071##

Cyclo-CLLF(CF.sub.3)VF(Br) was obtained as a white solid (8.3 mg, 4.8% overall yield). Analytical HPLC R.sub.t=17.580 min (99% purity). m/z (ESI.sup.+): 871.6 [M+H].sup.+ (100%). HRMS (ESI.sup.+) found: 869.2877, [M+H].sup.+ calculated: 869.2881.

TABLE-US-00050 CLLF(CF.sub.3)VF(I) SEQIDNO:19 Cysleuleuphe(CF3)valphe(4-I)

##STR00072##

Cyclo-CLLF(CF.sub.3)VF(I) was obtained as a white solid (19.8 mg, 10.8% overall yield). Analytical HPLC R.sub.t=17.893 min (91% purity). m/z (ESI.sup.+): 917.7 [M+H].sup.+ (100%). HRMS (ESI.sup.+) found: 917.2728, [M+H].sup.+ calculated: 917.2739.

TABLE-US-00051 CLLF(CF.sub.3)VF(CF.sub.3) SEQIDNO:16 Cysleuleuphe(CF3)valphe(4-CF3)

##STR00073##

Cyclo-CLLF(CF.sub.3)VF(CF.sub.3) was obtained as a white solid (31.6 mg, 18.4%). Analytical HPLC R.sub.t=16.284 min (99% purity). m/z (ESI.sup.+): 859.7 [M+H].sup.+. HRMS (ESI.sup.+) found: 859.3640, [M+H].sup.+ calculated: 859.3646.

TABLE-US-00052 CLLF(CF.sub.3)VF(tBu) [SEQIDNO:57]

##STR00074##

Cyclo-CLLF(CF.sub.3)V-dY was obtained as a white solid (24.1 mg, 14.2%). Analytical HPLC R.sub.t=17.333 min 99% purity). m/z (ESI.sup.+): 847.7 [M+H].sup.+ (100%), 869.7 [M+Na].sup.+ (60%). HRMS (ESI.sup.+) found: 847.4391, [M+H].sup.+ calculated: 847.4398.

TABLE-US-00053 CLLF(CF.sub.3)VF(Ph) SEQIDNO:13 Cysleuleuphe(CF3)valphe(4-Ph)

##STR00075##

Cyclo-CLLF(CF.sub.3)V-dY was obtained as a white solid (29.1 mg, 16.8%). Analytical HPLC R.sub.t=18.351 min (98% purity). m/z (ESI.sup.+): 867.7 [M+H].sup.+ (100%), 889.7 [M+Na].sup.+ (30%). HRMS (ESI.sup.+) found: 867.4078, [M+H].sup.+ calculated: 867.4085.

CLLF(CF.SUB.3.)VF(Br) Valine Derivatives

[0319] Fmoc-Leu-Wang resin (0.1 mmol) was added to 8 syringes and the Fmoc group removed via exposure to 20% piperidine in DMF. A solution of amino acid (1 eq.), Oxyma pure (3 eq.) and DIC (3 eq.) were added and the syringes placed on a rocker for 1 hour. The reaction mixtures were then removed via vacuum filtration and the resins washed with DMF2, DCM2 and Et.sub.2O1. Fmoc protected amino acids were then deprotected via the addition of piperidine 20% in DMF and being placed on a rocker for 30 minutes. The resins were then washed as before. These steps were repeated until the desired linear sequence was synthesised. The linear sequences were then cleaved from the resin using a TFA cleavage cocktail (section 0) and cyclised using HATU and DIPEA (section 0) excess DMF was removed under reduced pressure and the crude cyclic peptide used without further purification. The StBu group was then removed (section 0) and the peptide purified via reverse-phase HPLC (section 0), like fractions were combined and concentrated under reduced pressure, then lyophilised to yield the desired cyclic peptides.

TABLE-US-00054 CLLF(CF.sub.3)-Aib-F(Br) SEQIDNO:27 Cysleuleuphe(CF3)Aibphe(4-Br)

##STR00076##

Cyclo-CLLF(CF.sub.3)-Aib-F(Br) was obtained as a white solid (3.3 mg, 3.86%). Analytical HPLC R.sub.t=16.835 min (89% purity), m/z (ESI.sup.+): 857.4 [M+H].sup.+ (100%). HRMS (ESI.sup.+) found: 877.2460, [M+Na].sup.+ calculated: 877.2546.

TABLE-US-00055 CLLF(CF.sub.3)-Abu-F(Br) SEQIDNO:26 Cysleuleuphe(CF3)Abuphe(4-Br)

##STR00077##

Cyclo-CLLF(CF.sub.3)-Abu-F(Br) was obtained as a white solid (32.9 mg, 38.5%). Analytical HPLC R.sub.t=16.609 min (99% purity). m/z (ESI.sup.+): 857.4 [M+H].sup.+ (100%), 879.5 [M+Na].sup.+ (40%). HRMS (ESI.sup.+) found: 877.2537, [M+H].sup.+ calculated: 877.2546.

TABLE-US-00056 CLLF(CF.sub.3)LF(Br) SEQIDNO:30 Cysleuleuphe(CF3)leuphe(4-Br)

##STR00078##

Cyclo-CLLF(CF.sub.3)LF(Br) was obtained as a white solid (25.9 mg, 29.4%). Analytical HPLC R.sub.t=17.873 min (99% purity). m/z (ESI.sup.+): 885.5 [M+H].sup.+ (100%). HRMS (ESI.sup.+) found: 905.2863, [M+Na].sup.+ calculated: 905.2859.

TABLE-US-00057 CLLF(CF.sub.3)-NL-F(Br) SEQIDNO:28 Cysleuleuphe(CF3)n-leuphe(Br)

##STR00079##

Cyclo-CLLF(CF.sub.3)N-LEU-F(Br) was obtained as a white solid (15.2 mg, 17.2%). Analytical HPLC R.sub.t=18.107 min (95% purity). m/z (ESI.sup.+): 885.5 [M+H].sup.+ (100%), 905.4 [M+Na].sup.+ (60%). HRMS (ESI.sup.+) found: 883.3034, [M+H].sup.+ calculated: 883.3034.

TABLE-US-00058 CLLF(CF.sub.3)-NV-F(Br) [SEQIDNO:58]

##STR00080##

Cyclo-CLLF(CF.sub.3)NVF(Br) was obtained as a white solid (23.8 mg, 27.4%). Analytical HPLC R.sub.t=17.340 min (98% purity). m/z (ESI.sup.+): 893.5 [M+Na].sup.+ (100%), 869.5 [M+H].sup.+ (90%). HRMS (ESI.sup.+) found: 869.2873, [M+H].sup.+ calculated: 869.2877.

TABLE-US-00059 CLLF(CF.sub.3)-hL-F(Br) [SEQIDNO:59]

##STR00081##

Cyclo-CLLF(CF.sub.3)-hL-F(Br) was obtained as a white solid (33.3 mg, 18.6%). Analytical HPLC R.sub.t=18.577 min (99% purity). m/z (ESI.sup.+): 899.7 [M+H].sup.+ (100%). HRMS (ESI.sup.+) found: 897.3166, [M+H].sup.+ calculated: 897.3190

TABLE-US-00060 CLLF(CF.sub.3)IF(Br) SEQIDNO:31 Cysleuleuphe(CF3)ilephe(4-Br)

##STR00082##

Cyclo-CLLF(CF.sub.3)IF(Br) was obtained as a white solid (14.2 mg, 8.0%). Analytical HPLC R.sub.t=18.149 min (99% purity). m/z (ESI.sup.+): 885.6 [M+H].sup.+ (100%), 907.6 [M+Na].sup.+ (40%). HRMS (ESI.sup.4) found: 883.3020, [M+H].sup.+ calculated: 883.3034.

TABLE-US-00061 CLLF(CF.sub.3)-Prop-F(Br) SEQIDNO:29 Cysleuleuphe(CF3)Propphe(4-Br)

##STR00083##

Cyclo-CLLF(CF.sub.3)IF(Br) was obtained as a white solid (14.2 mg, 8.0%). Analytical HPLC R.sub.t=16.822 min (98% purity). m/z (ESI.sup.+873.5 [M+H].sup.+ (100%), 893.4 [M+Na].sup.+ (99%). HRMS (ESI.sup.+) found: 871.2660, [M+H].sup.+ calculated: 871.2670.

CLLF(CF.SUB.3.)IF(Br) Leucine Derivatives

TABLE-US-00062 CLVF(CF.sub.3)IF(Br) SEQIDNO:32 Cysleuvalphe(CF3)ilephe(4-Br)

##STR00084##

Cyclo-CLVF(CF.sub.3)IF(Br) was obtained as a white solid (0.4 mg, 0.5%). Analytical HPLC R.sub.t=18.116 min (97% purity). m/z (ESI.sup.+): 871.6 [M+H].sup.+ (100%), 890.6 [M+Na].sup.+ (45%). HRMS (ESI.sup.+) found: 869.2850, [M+H].sup.+ calculated: 869.2877.

TABLE-US-00063 CL-Prop-F(CF.sub.3)IF(Br) SEQIDNO:36 CysleuPropphe(CF3)ilephe(4-Br)

##STR00085##

Cyclo-CL-Prop-F(CF.sub.3)IF(Br) was obtained as white solid (6.1 mg, 7.0%). Analytical HPLC R.sub.t=17.001 min (95% purity). m/z (ESI.sup.+): 873.5 [M+H].sup.+ (100%). HRMS (ESI.sup.+) found: 871.2670, [M+H].sup.+ calculated: 871.2670.

TABLE-US-00064 CL-NV-F(CF.sub.3)IF(Br) SEQIDNO:35 Cysleun-valphe(CF3)ilephe(4-Br)

##STR00086##

Cyclo-CL-NVF(CF.sub.3)IF(Br) was obtained as a white solid (6.7 mg, 7.7%). Analytical HPLC R.sub.t=17.729 min (99% purity). m/z (ESI.sup.+): 871.6 [M+H].sup.+ (100%). HRMS (ESI.sup.+) found: 869.2853, [M+H].sup.+ calculated: 8692877.

TABLE-US-00065 CL-hL-F(CF.sub.3)IF(Br) SEQIDNO:34 Cysleuh-leuphe(CF3)ilephe(4-Br)

##STR00087##

Cyclo-CL-hL-F(CF.sub.3)IF(Br) was obtained as a white solid (16.2 mg, 18.1%). Analytical HPLC R.sub.t=18.838 min (99% purity). m/z (ESI.sup.+): 899.7 [M+H].sup.+ (100%). HRMS (ESI.sup.+) found: 897.3166, [M+H].sup.+ calculated: 897.3190.

TABLE-US-00066 CL-Aib-F(CF.sub.3)IF(Br) SEQIDNO:33 CysleuAibphe(CF3)ilephe(4-Br)

##STR00088##

Cyclo-CL-Aib-F(CF.sub.3)IF(Br) was obtained as a white solid (24.7 mg, 28.9%). Analytical HPLC R.sub.t=17.737 min (96% purity). m/z (ESI.sup.+) 857.6 [M+H].sup.+ (100%). HRMS (ESI.sup.+) found: 855.2721, [M+H].sup.+ calculated: 855.2721.

TABLE-US-00067 CLIF(CF.sub.3)IF(Br) SEQIDNO:37 Cysleuilephe(CF3)ilephe(4-Br)

##STR00089##

Cyclo-CLIF(CF)IF(Br) was obtained as a white solid (4.5 mg, 5.1%). Analytical HPLC R.sub.t=18.566 min (96% purity). m/z (ESI.sup.+): 885.6 [M+H].sup.+ (100%). HRMS (ESI.sup.+) found: 883.3020, [M+H].sup.+ calculated: 883.3034.

TABLE-US-00068 CL-NL-F(CF.sub.3)IF(Br) SEQIDNO:38 Cysleun-leuphe(CF3)ilephe(4-Br)

##STR00090##

Cyclo-CL-NL-F(CF.sub.3)IF(Br) was obtained as a white solid (12.5 mg, 14.2%). Analytical HPLC R.sub.t=18.310 min (99% purity). m/z (ESI.sup.+): 885.6 [M+H].sup.+ (100%). HRMS (ESI.sup.+) found: 883.3021, [M+H].sup.+ calculated: 883.3034.

Polar Derivatives

TABLE-US-00069 CK-NL-F(CF.sub.3)IF(Br) SEQIDNO:40 Cyslysn-leuphe(CF3)ilephe(4-Br)

##STR00091##

2-chlorotrityl chloride resin was loaded with Fmoc-Lys(Boc) as detailed in section 0 and the resin loading determined (section 0). The linear sequence K(Boc)C(trt)F(Br)IF(CF.sub.3)-NL was then synthesised via Fmoc SPPS (section 0). The linear sequence was then cleaved from the resin using HFIP, leaving the protecting groups intact (section 0). The protected linear was then cyclised with HATU (section 0) to yield the protected cyclic peptide that was immediately globally deprotected with a TFA cleavage cocktail (section 0). The crude product was then dissolved in DMF before being purified via reverse-phase HPLC (section 0) to yield the desired cyclic peptide sequence cyclo-CK-NL-F(CF.sub.3)IF(Br) as a white powder (51.2 mg, 28.5%). Analytical HPLC R.sub.t=14.309 min (90.0% purity). m/z (ESI.sup.+): 900.5 [M+H].sup.+ (100%). HRMS (ESI.sup.+) found: 898.3061, [M+H].sup.+ calculated: 898.3143.

TABLE-US-00070 CE-NL-F(CF.sub.3)IF(Br) SEQIDNO:41 Cysglun-leuphe(CF3)ilephe(4-Br)

##STR00092##

2-chlorotrityl chloride resin was loaded with Fmoc-Glu(OtBu) as detailed in section 0 and the resin loading determined (section 0). The linear sequence E(OtBu)C(trt)F(Br)IF(CF.sub.3)-NL was then synthesised via Fmoc SPPS (section 0). The linear sequence was then cleaved from the resin using HFIP, leaving the protecting groups intact (section 0). The protected linear was then cyclised with HATU (section 0) to yield the protected cyclic peptide that was immediately globally deprotected with a TFA cleavage cocktail (section 0). The crude product was then dissolved in DMF before being purified via reverse-phase HPLC (section 0) to yield the desired cyclic peptide sequence cyclo-CE-NL-F(CF.sub.3)IF(Br) as a white powder (5.7 mg, 3.2%). Analytical HPLC R.sub.t=15.749 min (97% purity). m/z (ESI.sup.+): 901.4 [M+H].sup.+ (100%). HRMS (ESI.sup.+) found: 899.2605, [M+H].sup.+ calculated: 899.2619.

Chapter 1 Coumarin Synthesis

1-Benzyl 7-ethyl (S)-2-(((benzyloxy)carbonyl)amino)-5-oxoheptanedioate

##STR00093##

Cbz-Glu-OBn (2.00 g, 5.39 mmol, 1 eq.) was dissolved in THF (25 mL) and CDI (0.96 g, 5.92 mmol, 1.1 eq.) was added. The mixture was then stirred at RT for 2 hours under argon. Ethyl magnesium malonate (0.95 g, 2.96 mmol, 0.55 eq.) was then added and the mixture stirred under argon for a further 16 hours. The product was extracted with diethyl ether (350 mL) and washed with NaHCO.sub.3 (350 mL), water (350 mL) and brine (350 mL). The organic layer was then dried with anhydrous MgSO.sub.4 and concentrated under reduced pressure to yield a clear oil. The crude product was purified by silica column chromatography with an eluent of 1:1 PE:EtOAc to yield the -ketoester (2.24 g, 94.1%) as a white solid. m/z (ESI.sup.+): 464.4 [M+Na].sup.+ (100%). .sup.1H NMR (400 MHz, CDCl.sub.3) ppm 1.28 (t, J=7.09 Hz, OCH.sub.2CH.sub.3, 3H) 1.90-2.28 (m, NHCHCH.sub.2CH.sub.2, 2H) 2.46-2.70 (m, NHCHCH.sub.2CH.sub.2, 2H) 3.39 (s, COCH.sub.2COO, 2H) 4.17 (q, 3=7.09 Hz, OCH.sub.2CH.sub.3, 2H) 4.43 (td, 3=8.01, 5.26 Hz, NHCHCH.sub.2CH.sub.2, 1H) 5.12 (s, PhCH2OCONH, 2H) 5.19 (s, COOCH2Ph, 2H) 5.44 (br d, J=8.07 Hz, NHCHCH2CH2, 1H) 7.28-7.39 (m, ArH, 10H). Spectral data matches literature.sup.227

(S)-2-Amino-4-(7-hydroxy-2-oxo-2H-chromen-4-yl)butanoic acid (H2N-hCou-OH)

##STR00094##

The -ketoester (1.0 g, 2.27 mmol, 1 eq.) was added slowly to a solution of resorcinol (1.25 g, 11.35 mmol, 5 eq.) in methanesulfonic acid (5 mL). The solution was stirred at RT for 2 hours before being diluted with ice-cold ether (300 mL). The precipitate was isolated via filtration and then dissolved in water before being filtered again and lyophilised. The resulting residue was purified via RP-chromatography to give the coumaryl amino acid (0.170 g, 28.5%) as an off-white solid. m/z (ESI.sup.+): 264.2 [M+H].sup.+ (100%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) ppm 7.65 (d, J=8.80 Hz, 1H) 6.83 (dd, J=8.68, 2.32 Hz, 1H) 6.75 (d, J=2.45 Hz, 1H) 6.12 (s, 1H) 3.85 (br t, J=5.99 Hz, 1H) 2.80-2.97 (m, 2H) 2.00-2.19 (m, 2H). Spectral data matches literature.sup.227

(S)-2-((Tert-butoxycarbonyl)amino)-4-(7-((tert-butoxycarbonyl)oxy)-2-oxo-2H-chromen-4-yl)butanoic acid (Boc-hCou(Boc)-OH)

##STR00095##

The coumaryl amino acid (0.4 g, 1.5 mmol, 1 eq.) was dissolved in 5% aqueous NaHCO.sub.3/dioxane (1:1 v/v) (40 mL) and placed in an ice bath. Boc anhydride (3.27 g, 15 mmol, 10 eq.) was then added and the reaction stirred on ice for 1 hour before being warmed up to RT over 16 hours. The mixture was then acidified to pH 3 with 10% (w/v) citric acid.sub.(aq) (60 mL) and the resulting solution extracted with EtOAc (375 mL). The extract was then washed with water (3100 mL) and brine (3100 mL) before being dried over anhydrous MgSO.sub.4 and concentrated under reduced pressure. The resulting oil was purified via RP-chromatography to yield a mixture of the mono-boc coumaryl amino acid (17.4 mg, 3.2%) and di-boc coumaryl amino acid and (379.4, 54.6%). m/z (ESI.sup.+): 408.3 [MtBu+H].sup.+ (100%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) ppm 7.92 (d, J=8.80 Hz, 1H), 7.38 (d, J=2.45 Hz, 1H), 7.32 (d, J=8.07 Hz, 1H), 7.26 (dd, J=8.68, 2.32 Hz, 1H), 6.34 (s, 1H), 3.86-4.10 (m, 1H), 2.87 (br t, J=7.58 Hz, 2H), 1.85-2.10 (m, 2H), 1.52 (s, 9H), 1.41 (s, 9H).

CLL-hCou-VY SEQ ID NO: 39 Cys Leu Leu Cou Val Tyr

##STR00096##

Cyclo-CLL-hCou-VY was obtained as a white solid (9.4 mg, 11.2%). Analytical HPLC R.sub.t=10.990 min (91% purity). m/z (ESI.sup.+): 837.5 [M+H].sup.+ (100%), 859.5 [M+Na].sup.+ (100%). HRMS (ESI.sup.+) found: 836.3769, [M+H].sup.+ calculated: 836.3779.

[0320] (ESI.sup.+) found: 889.3768, [M+H].sup.+ calculated: 889.3776.

Chemical Procedures

Wang Resin SPPS

[0321] Wang resins pre-loaded with Fmoc amino acids were swollen in DMF for 10 minutes with agitation via argon flow. Fmoc deprotection was achieved via the addition of piperidine 20% (v/v) in DMF. Fmoc protected amino acids were then coupled to the resin via the addition of a solution of the amino acid (3 eq.), Oxyma Pure (3 eq.) and DIC (3 eq.) in DMF and leaving the mixture with agitation for 1 hour. The resin was then washed with 3DMF, 3DCM and 3Et.sub.2O and coupling confirmed via Kaiser test. Deprotection of the Fmoc group was again achieved via the addition of piperidine 20% (v/v) in DMF with agitation for 30 minutes. The resin was washed as previously described and deprotection confirmed via Kaiser Test. These steps were repeated until the desired resin bound sequence was achieved.

Cleavage of Wang Resin

[0322] Cleavage of the linear sequence and simultaneous deprotection of side chain protecting groups was achieved using a cleavage cocktail of TFA (4.75 mL), H.sub.2O (0.125 mL) and TIS (0.125 mL) and stirring for 2 hours at RT. The resin was then removed via filtration and the TFA removed under reduced pressure. The peptide was then precipitated with ice cold (80 C.) diethyl ether. The ether was decanted and the peptide dried under reduced pressure.

2-Chlorotrityl Chloride Resin Loading

[0323] 2-Chlorotrityl chloride resin (1 eq.) was placed in a sinter funnel and DCM (10 mL) added. The resin was then agitated via argon flow for 15 minutes to swell the resin. The DCM was then removed from the funnel and the Fmoc amino acid to be loaded (1 eq.) was dissolved in DCM (10 mL) and added to the resin. DIPEA (3 eq.) was then added and the resin agitated via argon flow for 2 hours. MeOH (1 mL) was then added to the funnel to end cap any unreacted sites and the resin agitated with argon flow for a further 15 minutes. The reaction mixture was then removed under reduced pressure and the resin washed with 3DCM, 3DMF and 3Et.sub.2O. The resin was then dried under reduced pressure.

Determining Fmoc-AA Resin Loading

[0324] Approximately 10 mg of dry resin was accurately weighed into a glass vial and DMF (800 L) added. The resin was then left to swell for 15 minutes. Piperidine (200 L) was then added and the resin left for a further 15 minutes. An aliquot of the reaction mixture (100 L) was then diluted with DMF (900 L). The resin loading was then determined from the UV absorbance of the dibenzofulvene-piperidine adduct at a wavelength of 301 nm as determined using a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, USA).

2-Chlorotrityl Chloride Resin SPPS

[0325] Pre-loaded 2-chlorotirityl chloride resins were deprotected with 20% piperidine in DMF. The linear sequence was then synthesised as described for Fmoc-Wang resin synthesis.

2-Chlorotrityl Chloride Resin Cleavage

[0326] To yield protected linear peptides, linear bound 2-chlorotrityl chloride resin was transferred to a vial and 1,1,1,3,3,3-hexafluroisopropanol (HFIP) (5 mL) and DCM (5 mL) added. The mixture was then stirred at RT for 2 hrs. The resin was then removed via filtration and the solvent removed under reduced pressure. The peptide was then precipitated using ice cold (80 C.) diethyl ether. The ether was decanted and the peptide dried under reduced pressure.

Cyclisation via EDC/Oxyma Pure

[0327] Linear peptides were dissolved in DMF (1 mL/mg) and EDC (3 eq.) and Oxyma pure (3 eq.) were added. The mixture was then stirred at RT overnight. Excess DMF was removed under reduced pressure and the crude solution purified via reverse phase flash chromatography.

Cyclisation Via HATU

[0328] Linear peptides were dissolved in DMF (1 mL/mg) and HATU (3 eq.) DIPEA (3 eq.) and HOAt (1 eq.) were added. The mixture was then stirred at RT overnight. Excess DMF was removed under reduced pressure and the crude solution purified via reverse phase flash chromatography.

Cys(StBu) Deprotection

[0329] Cys(StBu) containing sequences were deprotected by dissolving the cyclic peptide in DMF (1 mL) and adding DTT (10 eq.) and DIPEA (10 eq.) the mixture was then stirred for 30 minutes at RT. The mixture was then filtered through a 0.22 m PTFE syringe filter and purified via reverse-phase HPLC.

Deprotection of Protected Cyclic Peptides

[0330] Protected cyclic peptides were dissolved in TFA cleavage cocktail (TFA (4.75 mL), H.sub.2O (0.125 mL) and TIS (0.125 mL)) and stirred at room temperature for 1 hour. Excess TFA was then removed under reduced pressure.

Kaiser Free Amine Test

[0331] After each amino acid coupling step and Fmoc deprotection step, a few beads were transferred into a vial and 10 drops of KCN (0.02 mM) in pyridine and 3 drops of ninhydrin (0.30 M) in ethanol were added. The mixture was then heated to 130 C. for 2 minutes and the presence or absence of blue colour determined if the step was complete.

Purification via HPLC

[0332] Peptides were manually injected as solutions in either DMF or ACN via a Waters Flex inject system into a RP-HPLC system consisting of a Waters 1525 binary pump coupled to a Waters 2998 photodiode array detector. Peptides were then purified using a Waters X Select CSH prep C18 5 m OBD 19250 mm column. A binary solvent system consisting of solution A (0.1% TFA/water) and B (0.1% TFA/MeCN) was run at the gradient shown below and elution monitored at 220 nm (peptide backbone). Fractions were collected automatically when the UV trace was in excess of a set threshold. Like fractions were combined and the ACN removed under reduced pressure. The sample was the lyophilised to yield the desired product.

TABLE-US-00071 % A % B Time (min) 95 5 0 10 90 0-15 10 90 15-20

Analytical HPLC

[0333] Analytical HPLCs were carried out on an Agilent 1260 Infinity II HPLC system running a binary solvent system consisting of solution A (0.1% TFA/water) and B (0.1% TFA/MeCN). Peptides were injected onto a Poroshell 120 EC-C18 column (2.7 m particle size, 3.0100 mm) and the gradient shown below run at a flow rate of 0.625 mL/min. UV absorbance was recorded at both 220 and 280 nm.

TABLE-US-00072 % A % B Time (min) 95 5 0 0 100 0-20 0 100 20-22

Purification via Flash Chromatography

[0334] A Biotage Isolera One system equipped with a SNAP Ultra 30 g HP-Sphere C18 25 m column was used. A binary solvent system consisting of solution A (0.1% TFA/water) and B (0.1% TFA/MeCN) was run at 80% A 20% B for 3CV at 25 mL/min the peptide was then injected directly onto the column as a solution in either DMF or ACN.

[0335] A gradient was then run with the solvent steps shown below. Fractions were collected automatically when UV.sub.220 nm exceeded 40 mAu. Collected fractions were analysed via mass spectrometry to confirm their identity and the desired fractions were concentrated under reduced pressure and then lyophilised to yield the desired product.

TABLE-US-00073 % A % B Number of column volumes 80 20 2 10 90 7 10 90 2

ESI.SUP.+ MS

[0336] Samples were analysed using a Waters (Manchester, UK) TQD mass spectrometer equipped with a triple quadrupole analyser. Samples were introduced to the mass spectrometer via an Acquity H-Class quaternary solvent manager (with TUV detector at 254 nm, sample and column manager). Ultra-performance liquid chromatography was undertaken via a Waters BEH C18 column (50 mm2.1 mm 1.7 m).

[0337] Gradient 20% acetonitrile (0.2% formic acid) to 100% acetonitrile (0.2% formic acid) in five minutes at a flow rate of 0.6 mL/min. Low-resolution mass spectra were recorded using positive ion electrospray ionisation.

High Resolution Mass Spectrometry (HRMS)

[0338] Samples were analysed using a MaXis (Bruker Daltonics, Bremen, Germany) mass spectrometer equipped with a Time of Flight (TOF) analyser. Samples were introduced to the mass spectrometer via a Dionex Ultimate 3000 autosampler and uHPLC pump. Gradient 20% acetonitrile (0.2% formic acid) to 100% acetonitrile (0.2% formic acid) in five minutes at 0.6 mL/min. Column, Acquity UPLC BEH C18 (Waters) 1.7 micron 502.1 mm. High resolution mass spectra were recorded using positive ion electrospray ionisation.

.sup.1H and .sup.13C NMR

[0339] NMR spectra were recorded on Bruker AVII400 or Bruker AVIIIHD400 FT-NMR spectrometers in the indicated solvent at 298 K. Chemical shifts for proton and carbon spectra are reported on the delta scale in ppm and were referenced to residual solvent references or internal TMS reference.