Cyclic peptide analogs of melanocortin and uses thereof

Abstract

This disclosure relates cyclic peptide analogs of melanocortins. This disclosure further relates to use of such peptide analogs for therapy and imaging. The disclosure also relates to methods of synthesizing such peptide analogs using a peptide stapling method.

Description

CYCLIC PEPTIDE ANALOGS OF MELANOCORTIN AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 546,610, filed on October 31, 2023, the contents of which is hereby incorporated by reference in its entirety for all purposes.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (A9TH_023_01WO_Seq_List_ST26.xml; Size: 86,245 bytes; and Date of Creation: October 30, 2024) are herein incorporated by reference in its entirety.TECHNICAL FIELD

[0003] This disclosure relates cyclic peptide analogs of melanocortins. This disclosure further relates to use of such peptide analogs for therapy and imaging. The disclosure also relates to methods of synthesizing such peptide analogs using a peptide stapling method.BACKGROUND

[0004] The pursuit of secondary structure stabilization is a ubiquitous goal in peptide chemistry and of central importance to the utilization of peptides as medicinal probes and high affinity ligandsJ. To this end, Nature typically employs oxidative disulfide formation from two cysteine thiols2Disulfide crosslinks are also exploited in the development of synthetic peptides as drugs and imaging agents3. Since disulfide linkages are labile under reducing conditions, Nature uses redox-inert crosslinks in natural products, most notably, thioethers, as seen in the lanthionine antibiotics4, tryptathionine linkages as seen amatoxins and phallotoxins5, and heteroaryl crosslinks between tryptophan and histidine as seen in celogentin6, in addition to several that have been recently reviewed7. These chemical crosslinks are critical to the biological activity of these molecules, whose syntheses are of enduring interest8. In considering the potency of these peptide natural products, the development of facile, synthetic crosslinking methodologies continues to be an active field of study. Such strategies offer the potential for truncating longer natural peptide sequences, thus facilitating scalable synthesis and improving pharmacokinetics9. More importantly, crosslinks rigidity the scaffold by reducing flexibility to favor a limited envelope of secondarystructures. When applied properly, conformational restriction may dramatically improve both affinity and specificity9e’10.

[0005] Towards these ends, many well-known reactions within the synthetic repertoire have been repurposed for novelty in peptide crosslinking as reviewed extensively.11With over 150 peptides entering various stages of clinical trials since 201814, new stapling methods are likely to contribute significantly to peptide-based drugs. Notwithstanding that these novel approaches generally lack biological precedence, natural products continue to be a prime source of drugs as a majority of FDA-approved drugs from 1980-2014 have been derived from or inspired by natural products15. To wit, peptide stapling methods may derive inspiration from natural products, as recently reviewed16.

[0006] Among a number of peptide natural products, the tryptathionine staple is found exclusively in two classic bicyclic toxic peptides, amatoxins and phallotoxins, which target protein folds in RNA polymerase II and F-actin respectively. Besides comprising the staple on these classic natural products, the indole ring itself enhances target affinity by engaging in hydrophobic, 7i-stacking, and H-bonding interactions with the target. Renewed interest in amanitin has in turn inspired recent methodological reports for installing tryptathionine staples at will17, with very recent investigations focused on helix stabilization and [3-tum setting18. Typically, the tryptathionine staple is installed either by use of an electrophilic cysteine sulfenyl halide that electrophilically substitutes (sic oxidizes) the indole17a’19, or use of an oxidized tryptophan e.g. hydroxypyrroloindoline that in acid is converted to an electrophilic indolium cation that traps the cysteine thiol20.

[0007] Melanotan-II is a melanocyte stimulating hormone (MSH)-analog, used illicitly for cosmetic tanning, that is now of considerable interest to imaging and treating melanoma through the use of targeted radiotracers21as reviewed22. Besides its clinical significance, a chemical perspective MSH features a clear challenge: the indole of a nearby tryptophan could react competitively with a proximal hydroxy indolium cation to generate a 2,2'-bis-indole peptide staple, of which there are limited examples in peptides23. Thus, there is a need for chemoselective stapling methods that can be utilized in preparation of biologically active compounds, such as high-affinity a-MSH derivatives.SUMMARY OF THE DISCLOSURE

[0008] The present disclosure relates to Compounds 1-21, or a salt thereof.

[0009] The present disclosure further relates to a compound having the structure of formula (I) or formula (II), or salt thereof,Nle-cyclo(bis-indolo)-[Xaa1-His-D-Phe-Arg-Xaa5-Xaa6]-NH2 (I) (SEQ ID NO: 1), or Nle-cyclo(tryptathio)-[Xaa1-His-D-Phe-Arg-Xaa5-Xaa6]-NH2 (II) (SEQ ID NO: 2),

[0010] wherein:

[0011] Xaa1is Trp, D-Trp, Cys, D-Cys, Pen, or HomoCys;

[0012] Xaa5is 2-Nal, Trp, or D-Trp;

[0013] Xaa6is Trp, D-Trp, Cys, or D-Cys;

[0014] one or more amino acid residues of the compound is optionally alpha N- methylated; and

[0015] the compound is optionally N-terminally acylated.

[0016] The present disclosure further relates to a compound comprising a melanocortin 1 receptor (MC1R) targeting peptide (MC1RTP), a radiolabeling group, and a linker joining the MC1R targeting compound to the radiolabeling group, wherein:

[0017] the MC1RTP is any one of the compounds disclose herein, or a salt thereof;

[0018] the MC1RTP is optionally C-terminally ami dated; and

[0019] one or more amino acid residues of the MC1RTP is optionally alpha N- methylated.

[0020] The present disclosure further relates to a method for treating cancer, comprising administering any one of the compounds disclosed herein.

[0021] The present disclosure further relates to a method for positron emission tomography (PET) or single photon emission computed tomography (SPECT) imaging of melanoma, comprising administering any one of the compounds disclosed herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0022] These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings.

[0023] Fig. 1 shows a PET image of68Ga-labeled Compound 23 in using male C57BL / 6J mice bearing B16F10 (melanoma) cells at 1 h p.i. (Scale bar unit is %ID / g).DETAILED DESCRIPTION

[0024] Definitions

[0025] As used herein, the terms “comprising,” “having”, “including” and “containing,” and grammatical variations thereof, are inclusive or open-ended and do not exclude additional, nonrecited elements and / or method steps. The term “consisting essentially of’ if used herein in connection with a compound, composition, use or method, denotes that additional elementsand / or method steps may be present, but that these additions do not materially affect the manner in which the recited compound, composition, method or use functions. The term “consisting of’ when used herein in connection with a composition, use or method, excludes the presence of additional elements and / or method steps. A compound, composition, use or method described herein as comprising certain elements and / or steps may also, in certain embodiments, consist essentially of those elements and / or steps, and in other embodiments consist of those elements and / or steps, whether or not these embodiments are specifically referred to. A use or method described herein as comprising certain elements and / or steps may also, in certain embodiments, consist essentially of those elements and / or steps, and in other embodiments, consist of those elements and / or steps, whether or not these embodiments are specifically referred to.

[0026] A reference to an element by the indefinite article “a” does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. The use of the word “a” or “an” when used herein in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one” and “one or more than one.” The term “plurality” if used herein means more than one, for example, two or more, three or more, four or more, and the like.

[0027] In this disclosure, the recitation of numerical ranges by endpoints includes all numbers subsumed within that range including all whole numbers, all integers and, where suitable, all fractional intermediates (e.g., 1 to 5 may include 1, 1.5, 2, 2.75, 3, 3.80, 4, and 5 etc.).

[0028] Unless otherwise specified, “certain embodiments”, “various embodiments”, “an embodiment” and similar terms includes the particular feature(s) described for that embodiment either alone or in combination with any other embodiment or embodiments described herein, whether or not the other embodiments are directly or indirectly referenced and regardless of whether the feature or embodiment is described in the context of a compound, method, product, use, composition, et cetera.

[0029] The term “subject” refers to an animal (e.g. a mammal or a non-mammal animal). The subject may be a human or a non-human primate. The subject may be a laboratory mammal (e.g., mouse, rat, rabbit, hamster and the like). The subject may be an agricultural animal (e.g., equine, ovine, bovine, porcine, camelid and the like) or a domestic animal (e.g., canine, feline and the like). In some embodiments, the subject is a human.

[0030] The compounds disclosed herein may also include base-free forms, prodrugs, salts or pharmaceutically acceptable salts thereof. Unless otherwise specified, the compounds claimed and described herein are meant to include all racemic mixtures and all individual enantiomers or combinations thereof, whether or not they are explicitly represented herein.

[0031] The compounds disclosed herein may be shown as having one or more charged groups, may be shown with ionizable groups in an uncharged (e.g. protonated) state or may be shown without specifying formal charges. As will be appreciated by the person of skill in the art, the ionization state of certain groups within a compound (e.g. without limitation, CO2H, PO3H2, SO2H, SO3H, SO4H, OPO3H2 and the like) is dependent, inter alia, on the pKa of that group and the pH at that location. For example, but without limitation, a carboxylic acid group (i.e. COOH) would be understood to usually be deprotonated (and negatively charged) at neutral pH and at most physiological pH values, unless the protonated state is stabilized (e.g. due to intramolecular H-bonding). Likewise, OSO3H (i.e. SO4H) groups, SO2H groups, SO3H groups, OPO3H2 (i.e. PO4H2) groups and PO3H groups would generally be deprotonated (and negatively charged) at neutral and physiological pH values.

[0032] As used herein, the terms “salt” and “solvate” have their usual meaning in chemistry. As such, when the compound is a salt or solvate, it is associated with a suitable counter-ion. It is well known in the art how to prepare salts or to exchange counter-ions. Generally, such salts can be prepared by reacting free acid forms of these compounds with a stoichiometric amount of a suitable base (e.g. without limitation, Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate, or the tike), or by reacting free base forms of these compounds with a stoichiometric amount of a suitable acid. Such reactions are generally carried out in water or in an organic solvent, or in a mixture of the two. Counter-ions may be changed, for example, by ion-exchange techniques such as ion-exchange chromatography. All zwitterions, salts, solvates and counterions are intended, unless a particular form is specifically indicated.

[0033] In certain embodiments, the salt or counter-ion may be pharmaceutically acceptable, e.g. for administration to a subject. More generally, with respect to any pharmaceutical composition disclosed herein, non-limiting examples of suitable excipients include any suitable buffers, stabilizing agents, salts, antioxidants, complexing agents, tonicity agents, cryoprotectants, lyoprotectants, suspending agents, emulsifying agents, antimicrobial agents, preservatives, chelating agents, binding agents, surfactants, wetting agents, non-aqueous vehicles such as fixed oils, or polymers for sustained or controlled release. See, for example, Berge et al. 1977. (J. Pharm Sci. 66:1-19), or Remington- The Science and Practice of Pharmacy, 21st edition (Gennaro et al editors. Lippincott Williams & Wilkins Philadelphia).

[0034] As used herein, the expression “Xy-Xz”, where y and z are integers (e.g. X1-X15, Xi- X30, X1-X100, and the like), refers to the number of carbons (for alkyls and aryls, whether saturated or unsaturated) in a compound, R-group or substituent, or refers to the number of carbons and heteroatoms (for heteroalkyls and heteroaryls, whether saturated or unsaturated) in a compound, R-group or substituent. Heteroatoms may include any, some or all possible heteroatoms. For example, in some embodiments, the heteroatoms are selected from N, O, S, P and Se. In some embodiments, the heteroatoms are selected from N, O, S and P. Such embodiments are non-limiting. Alkyls and aryls may alternatively be referred to using the expression “Cy-Cz”, where y and z are integers (e.g. C3-C15 and the like).

[0035] Unless explicitly stated otherwise, the terms “alkyl” and “heteroalkyl” each includes any reasonable combination of the following: (1) saturated alkyls as well as unsaturated alkyls (e.g. alkenyls and alkynyls); (2) linear or branched; (3) acyclic or cyclic (aromatic or nonaromatic), the latter of which may include multi-cyclic (fused rings, multiple non-fused rings or a combination thereof); and (4) unsubstituted or substituted. For example, an alkyl or heteroalkyl (i.e. “alkyl / heteroalkyl”) may be saturated, branched and cyclic, or unsaturated, branched and cyclic, or linear and unsaturated, or any other reasonable combination according to the skill of the person of skill in the art. If unspecified, the size of the alkyl / heteroalkyl is what would be considered reasonable to the person of skill in the art. For example, but without limitation, if unspecified, the size of an alkyl may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more than 100 carbons in length, subject to the common general knowledge of the person of skill in the art. Further, but without limitation, if unspecified, the size of a heteroalkyl may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100 or more than 100 carbons and heteroatoms in length, subject to the common general knowledge of the person of skill in the art.

[0036] As used herein, in the context of an alkyl / heteroalkyl group of a compound, the term “linear” may be used as it is normally understood to a person of skill in the art and generally refers to a chemical entity that comprises a skeleton or main chain that does not split off intomore than one contiguous chain. Non-limiting examples of linear alkyls include methyl, ethyl, n-propyl, and n-butyl.

[0037] As used herein, a “cyclic” peptide / polypeptide may be used as it is normally understood to a person of skill in the art and generally refers to the peptide or polypeptide having a covalent bond between two amino acids within the peptide or polypeptide to form a ring structure, e.g. between carboxyl and amino termini, between carboxyl terminus and side chain amino, between amino terminus and side chain carboxyl, or between side chains. For example, but without limitation, a peptide may be cyclized by forming a lactam bridge between the sidechain carboxylate of one amino acid residue in the peptide (e.g. Asp, Glu) and the amine of another amino acid residue in the peptide (e.g. Dap, Dab, Om, Lys). Further details are provided in Section II.

[0038] As used herein, the term “branched” may be used as it is normally understood to a person of skill in the art and generally refers to a chemical entity that comprises a skeleton or main chain that splits off into more than one contiguous chain. The portions of the skeleton or main chain that split off in more than one direction may be linear, cyclic or any combination thereof. Non-limiting examples of a branched alkyl group include tert-butyl and isopropyl.

[0039] As used herein, the term “saturated” when referring to a chemical entity may be used as it is normally understood to a person of skill in the art and generally refers to a chemical entity that comprises only single bonds. Non-limiting examples of a saturated C1-C15 alkyl group may include methyl, ethyl, n-propyl, i-propyl, sec-propyl, n-butyl, i-butyl, sec-butyl, t- butyl, n-pentyl, i-pentyl, sec-pentyl, t-pentyl, n-hexyl, i-hexyl, 1 ,2-dimethylpropyl, 2- ethylpropyl, 1 -methyl-2-ethylpropyl, l-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1,2- triethylpropyl, 1,1 -dimethylbutyl, 2,2-dimethylbutyl, 2-ethylbutyl, 1,3-dimethylbutyl, 2- methylpentyl, 3-methylpentyl, sec-hexyl, t-hexyl, n-heptyl, i-heptyl, sec-heptyl, t-heptyl, n- octyl, i-octyl, sec-octyl, t-octyl, n-nonyl, i-nonyl, sec-nonyl, t-nonyl, n-decyl, i-decyl, sec-decyl and t-decyl. Non-limiting examples of C2-C15 alkenyl group may include vinyl, allyl, isopropenyl, l-propene-2-yl, 1-butene-l-yl, l-butene-2-yl, l-butene-3-yl, 2-butene-l-yl, 2- butene-2-yl, octenyl and decenyl. Non-limiting examples of C2-C15 alkynyl group may include ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl and decynyl. Without limitation, the above-defined saturated C1-C15 alkyls, C2-C15 alkenyls and C2-C15 alkynyls are all encompassed within the term “X1-X15 alkyl”, as used herein. Without limitation, the term “X1-X15 heteroalkyl” would encompass each of the above-defined saturated C1-C15 alkyls, C2-C15 alkenyls and C2-C15 alkynyls, where one or more of the carbon atoms isindependently replaced with a heteroatom. The person of skill in the art would understand that various combinations of different heteroatoms may be used.

[0040] Unless explicitly stated otherwise, the terms “aryl” and “heteroaryl” each includes any reasonable combination of the following: (1) cyclic or multi-cyclic (fused rings, multiple nonfused rings or a combination thereof); (2) aromatic (i.e. unsaturated rings) or nonaromatic (i.e. saturated rings); and (3) unsubstituted or substituted. Non-limiting examples of aryls or heteroaryls (i.e. “aryl / heteroaryl”) include: phenyl, naphthyl, thienyl, indolyl, pyridyl and the like. If unspecified, the size of the aryl / heteroaryl is what would be considered reasonable to the person of skill in the art. For example, but without limitation, if unspecified, the size of an aryl may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more than 100 carbons in length, subject to the common general knowledge of the person of skill in the art. Further, but without limitation, if unspecified, the size of a heteroaryl may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more than 100 carbons and heteroatoms in length, subject to the common general knowledge of the person of skill in the art. It is noted that an aryl or heteroaryl may have all or only a portion of its skeleton or main chain bonded in such a way so as to form a 'loop', circle or ring of atoms bonded together. That is, the aryl / heteroaryl may comprise linear or branched chains of carbons / heteroatoms that are not part of a ring or loop.

[0041] For example, a X3-X18 aryl / heteroaryl may include, without limitation, a saturated C3- Cis cycloalkyl group, a C3-C18 cycloalkenyl group, a C3-C18 cycloalkynyl group, a C3-C18 aromatic aryl group, a X3-X18 non-aromatic heterocyclic group where each X may independently be C, N, S, P, O or Se, and a X3-X18 aromatic heterocyclic group where each X may independently be C, N, S, P, O or Se. Non-limiting examples of the saturated C3-C18 cycloalkyl group may include cyclopropanyl, cyclobutanyl, cyclopentanyl, cyclohexanyl, cycloheptanyl, cyclooctanyl, cyclononanyl and cyclodecanyl. Non-limiting examples of the C3-C18 cycloalkenyl group may include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, cyclononanenyl and cyclodecanenyl. Non-limiting examples of the C3-C18 aromatic aryl group may include phenyl (Ph), pentalenyl, indenyl,naphthyl and azulenyl. Non-limiting examples of the X3-X18 non-aromatic heterocyclic group may include aziridinyl, azetidinyl, diazetidinyl, pyrrolidinyl, pyrrolinyl, piperidinyl, piperazinyl, imidazolinyl, pyrazolidinyl, imidazolydinyl, phthalimidyl, succinimidyl, oxiranyl, tetrahydropyranyl, oxetanyl, dioxanyl, thietanyl, thiepinyl, morpholinyl, and oxathiolanyl. Non-limiting examples of the X3-X18 aromatic heterocyclic group may include pyrrolyl, imidazolyl, pyrazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pirazinyl, quinolinyl, isoquinolinyl, acridinyl, indolyl, isoindolyl, indolizinyl, purinyl, carbazolyl, indazolyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, phenanthridinyl, phenazinyl, phenanthrolinyl, perimidinyl, furyl, dibenzofuryl, xanthenyl, benzofuryl, thiophenyl, thianthrenyl, benzothiophenyl, phosphorinyl, phosphinolinyl, phosphindolyl, thiazolyl, oxazolyl, and isoxazolyl. Unless otherwise specified, Xi-Xis alkyl / heteroalkyl would encompass, among others, X3-X18 aryl / heteroaryl, including the groups defined above.

[0042] As used herein, the term “substituted” is used as it would normally be understood to a person of skill in the art and generally refers to a compound or chemical entity that has one chemical group replaced with a different chemical group. Unless otherwise specified, a substituted alkyl is an alkyl in which one or more hydrogen atom(s) are independently each replaced with an atom that is not hydrogen. For example, chloromethyl is a non-limiting example of a substituted alkyl, more particularly an example of a substituted methyl. Aminoethyl is another non-limiting example of a substituted alkyl, more particularly an example of a substituted ethyl. Unless otherwise specified, a substituted compound or group (e.g. alkyl, heteroalkyl, aryl, heteroaryl and the like) may be substituted with any chemical group reasonable to the person of skill in the art. For example, but without limitation, a hydrogen bonded to a carbon or heteroatom (e.g. N) may be substituted with halide (e.g. F, I, Br, Cl), amine, amide, oxo, hydroxyl, thiol, phosphate, phosphonate, sulfate, SO2H, SO3H, alkyls, heteroalkyls, aryl, heteroaryl, ketones, carboxaldehyde, carboxylates, carboxamides, nitriles, monohalomethyl, dihalomethyl or trihalomethyl.

[0043] As used herein, the term “unsubstituted” is used as it would normally be understood to a person of skill in the art. Non-limiting examples of unsubstituted alkyls include methyl, ethyl, tert-butyl, pentyl and the like. The expression “optionally substituted” is used interchangeably with the expression “unsubstituted or substituted”.

[0044] In the structures provided herein, hydrogen may or may not be shown. In some embodiments, hydrogens (whether shown or implicit) may be protium (i.e.1H), deuterium (i.e.2H) or combinations ofJH and2H. Methods for exchangingJH with2H are well known in the art. For solvent-exchangeable hydrogens, the exchange ofTH with2H occurs readily in thepresence of a suitable deuterium source, without any catalyst. The use of acid, base or metal catalysts, coupled with conditions of increased temperature and pressure, can facilitate the exchange of non-exchangeable hydrogen atoms, generally resulting in the exchange of allJH to2H in a molecule.

[0045] Unless otherwise specified a “peptide” as referred to herein may comprise proteinogenic and / or non-proteinogenic amino acid residues. Non-limiting examples of nonproteinogenic amino acids include: D-amino acids (including without limitation any D- form of the following amino acids), ornithine (Om), 3-(l-naphtyl)alanine (Nal), 3-(2- naphtyl)alanine (2 -Nal), a-aminobutryic acid, norvaline, norleucine (Nle), homonorleucine, beta-(l,2,3-triazol-4-yl)-L-alanine, l,2,4-triazole-3-alanine, Phe(4-F), Phe(4-Cl), Phe(4-Br), Phe(4-I), Phe(4-NH2), Phe(4-NO2), homoarginine (hArg), 2-amino-4-guanidinobutyric acid (Agb), 2-amino-3-guanidinopropionic acid (Agp), Trp(5-Br), Trp(5-OCH3), Trp(6-F), Trp(5- OH) or Trp(CHO), 2-aminoadipic acid (2-Aad), 3-aminoadipic acid (3-Aad), propargylglycine (Pra), homopropargylglycine (Hpg), beta-homopropargylglycine (Bpg), 2,3-diaminopropionic acid (Dap), 2,4-diaminobutyric acid (Dab), azidolysine (Lys(Ns)), azido-omithine (OmfNs)), 2-amino-4-azidobutanoic acid Dab(Ns), Dap(Ns), 2-(5'-azidopentyl)alanine, 2-(6'- azidohexyl)alanine, 4-amino-l-carboxymethyl-piperidine (Pip or APip), 4-(2-aminoethyl)-l- carboxymethyl-piperazine (Acp).

[0046] If not specified as an L- or D-amino acid, an amino acid shall be understood to be an L-amino acid.

[0047] Unless otherwise specified, amino acids may be modified by any modifications known in the art subject to the common general knowledge of the person of skill in the art. For example, but without limitation, a C-terminal amino acid residue may be amidated, which refers to replacement of the C-terminal carboxylate with an amide, i.e. -C(O)NH2 instead of -C(O)OH. Amidated residues are identified with -NEU (e.g. Lys-NFB, Trp-NFh and the like). As a further non-limiting example, amino acids may be methylated, e.g. N-methylated or alpha N-methylated.

[0048] The term “Xaa” refers to any amino acid. The term “-Xaa8-Xaa9-Xaa10-” refers to three amino acids, Xaa8, Xaa9, and Xaa10chained together with peptide bonds. The term Xaa9(Xaa8)-Xaa10-” refers to the same three amino acids, but chained together with an amide bond between the C-terminus of Xaa9and the N-terminus of Xaa10with a further amide bond formed between the C-terminus of Xaa8and the side chain amine of Xaa9. Unless otherwise indicated, “Xaa” may be any amino acid commonly known to the person of skill in the art,including proteinogenic and nonproteinogenic amino acids (e.g. but not limited to the nonproteinogenic amino acids listed above).

[0049] For cyclized peptides, the formula Xaa1-cyclo(Xaa2-Xaa3-Xaa4)-Xaa5and the like, refers to a covalent linkage between the side chains of Xaa2and Xaa4(i.e. between the first and last amino acids in the parentheses). Similarly, the formula Xaa1-cyclo(Xaa2-Xaa3-Xaa4-Xaa5) refers to a cyclized peptide having a covalent linkage between the side chains of Xaa2and Xaa5.

[0050] Compounds

[0051] The compounds presented herein incorporate peptides, which may be synthesized by any of a variety of methods established in the art. This includes but is not limited to liquidphase as well as solid-phase peptide synthesis using methods employing 9- fluorenylmethoxycarbonyl (Fmoc) and / or t-butyloxycarbonyl (Boc) chemistries, and other synthetic approaches.

[0052] Solid-phase peptide synthesis methods and technology are well-established in the art. For example, peptides may be synthesized by sequential incorporation of the amino acid residues of interest one at a time. In such methods, peptide synthesis is typically initiated by attaching the C-terminal amino acid of the peptide of interest to a suitable resin. Prior to this, reactive side chain and alpha amino groups of the amino acids are protected from reaction by suitable protecting groups, allowing only the alpha carboxyl group to react with a functional group such as an amine group, a hydroxyl group, or an alkyl halide group on the solid support. Following coupling of the C-terminal amino acid to the support, the protecting group on the side chain and / or the alpha amino group of the amino acid is selectively removed, allowing the coupling of the next amino acid of interest. This process is repeated until the desired peptide is fully synthesized, at which point the peptide can be cleaved from the support and purified. A non-limiting example of an instrument for solid-phase peptide synthesis is the Aapptec Endeavor 90 peptide synthesizer.

[0053] The choice of resin determines whether the peptide will have either a C-terminal carboxylate or a C-terminal amide (i.e. whether or not the C-terminus of the peptide is amidated). For example, but without limitation, 4-(2',4'- Dimethoxyphenyl-Fmoc-aminomethyl)phenoxy resin (Rink Amide Resin) or 9-Fmoc- aminoxanthen-3-yloxy-Merrifield resin (Sieber Amide resin) may be used for a C-terminal amide once the peptide is cleaved. Without limitation, p-benzyloxybenzyl alcohol resin (Wang resin) or 2-chlorotrityl chloride resin may be used for a C-terminal carboxylate once thepeptide is cleaved. During use, the resin is swelled in solvents, such as N,N-dimethylformamide (DMF), di chloromethane (DCM) or l-methyl-2 -pyrrolidone (NMP), and the like.

[0054] To allow coupling of additional amino acids, Fmoc protecting groups may be removed from the amino acid on the solid support, e.g. under mild basic conditions, such as piperidine (20-50% v / v) in DMF. The amino acid to be added must also have been activated for coupling (e.g. at the alpha carboxylate). Non-limiting examples of activating reagents include without limitation 2-(lH-benzotriazol-l-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 2-(lH-benzotriazol-l-yl)- 1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), 2-(7-Aza-lH- benzotriazole-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (HATU), benzotriazole- 1 -yl-oxy-tris(dimethylamino)phosphoniumhexafluorophosphate (BOP), benzotriazole- 1 -yl- oxy-tris(pyrrolidino)phosphoniumhexafluorophosphate (PyBOP). Racemization is minimized by using triazoles, such as 1-hydroxy-benzotriazole (HOBt) and 1 -hydroxy-7 -azabenzotriazole (HO At). Coupling may be performed in the presence of a suitable base, such as N,N-diisopropylethylamine (DIPEA / DIEA) and the like. For long peptides, peptide synthesis and ligation may be used.

[0055] Cyclization of the peptide may be performed by any known method. Cyclization may be performed on-resin or off-resin. Non-limiting examples of peptide cyclization include: forming a lactam bridge between an amino acid side chain containing a carboxyl group (e.g. Asp, D-Asp, Glu, D-Glu, and the like) and an amino acid side chain containing an amino group (e.g. Lys, D-Lys, Om, D-Om, Dab, D-Dab, Dap, D-Dap, and the like); forming a 1, 2, 3-triazole via click chemistry between an amino acid side chain containing an azide group (e.g. Lys(N3), D-Lys(N3), and the like) and an alkyne group (e.g. Pra, D-Pra, and the like); and forming a disulfide bridge between side chains of Cys residues. Since cyclization occurs between amino acid side chains, the protecting groups on these amino acids must be selectively removed before cyclization except the reaction between an alkyne and an azido groups via the click reaction to form an 1,2, 3-triazole. Non-limiting examples of selectively removable protecting groups include acetaminomethyl (Acm) (e.g. on Cys), 2-phenylisopropyl esters (O-2-PhiPr) (e.g. on Asp / Glu) as well as 4-methyltrityl (Mtt), allyloxy carbonyl (alloc), l-(4,4-dimethyl-2,6- dioxocyclohex-l-ylidene))ethyl (Dde), and l-(4,4-dimethyl-2,6-dioxocyclohex-l-ylidene)-3- methylbutyl (ivDde) (e.g. on Lys / Om / Dab / Dap). The Acm group on Cys can be selectively removed by 2 eq. of thallium (III) trifluoroacetate in DMF, which simultaneously induces cyclization via the formation of the disulfide bridge. O-2-PhiPr and Mtt protecting groups can be selectively deprotected under mild acidic conditions, such as 2.5% trifluoroacetic acid(TFA) in DCM. Alloc protecting groups can be selectively deprotected using tetrakis(triphenylphosphine)palladium(0) and phenyl silane in DCM. Dde and ivDde protecting groups can be selectively deprotected using 2-5% of hydrazine in DMF. Deprotected side chains of Asp / Glu (L- or D-forms) and Lys / Om / Dab / Dap (L- or D-forms) can then be cyclized, e.g. by using the coupling reaction conditions described above.

[0056] Peptide backbone amides may be N-methylated (i.e. alpha amino methylated). This may be achieved by directly using Fmoc-N-methylated amino acids during peptide synthesis. Alternatively, N-methylation under Mitsunobu conditions may be performed. First, a free primary amine group is protected using a solution of 4-nitrobenzenesulfonyl chloride (Ns-Cl) and 2,4,6-trimethylpyridine (collidine) in NMP. N-methylation may then be achieved in the presence of triphenylphosphine, diisopropyl azodicarboxylate (DIAD) and methanol. Subsequently, N-deprotection may be performed using mercaptoethanol and 1,8- diazabicyclo[5.4.0]undec-7-ene (DBU) in NMP. For coupling protected amino acids to N- methylated alpha amino groups, HATU, HO At and DIEA may be used.XH K?

[0057] Peptide backbone amides may be replaced with amidine, or thioamide. In some embodiments, 0-3 peptide backbone amides are independently replaced . ,. , . . ,amidine, or thioamide.

[0058] As described further below, chelators, linkers (peptide or non-peptide linkers) and / or albumin-binding groups may be coupled to the peptide N-terminus while the peptide is attached to the solid support. This is facile when the chelator, linker and / or albumin-binding groups comprise an activated carboxylate (and protected groups if necessary) so that coupling can be performed on resin.

[0059] When the peptide has been fully synthesized on the solid support, the desired peptide may be cleaved from the solid support using suitable reagents, such as TFA, tri -isopropylsilane (TIS) and water. Side chain protecting groups, such as Boc, pentamethyldihydrobenzofuran-5- sulfonyl (Pbf), trityl (Trt) and tert-butyl (tBu) are simultaneously removed (i.e. deprotection). The crude peptide may be precipitated and collected from the solution by adding cold ether followed by centrifugation. Purification and characterization of the peptides may be performed by standard separation techniques, such as high performance liquid chromatography (HPLC) based on the size, charge and polarity of the peptides. The identity of the purified peptides may be confirmed by mass spectrometry or other similar approaches.

[0060] In some embodiments, the compound of the disclosure is a compound selected from Table A, or a salt thereof.

[0061] Table A. Compounds

[0062] In some embodiments, the compound of the disclosure has a formula (I) or formula (II): Nle-cyclo(bis-indolo)-[Xaa1-His-D-Phe-Arg-Xaa5-Xaa6]-NH2 (I) (SEQ ID NO: 1), or Nle-cyclo(tryptathio)-[Xaa1-His-D-Phe-Arg-Xaa5-Xaa6]-NH2 (II) (SEQ ID NO: 2), or salt thereof, wherein:

[0063] Xaa1is Trp, D-Trp, Cys, D-Cys, Pen, or HomoCys;

[0064] Xaa5is 2-Nal, Trp, or D-Trp;

[0065] Xaa6is Trp, D-Trp, Cys, or D-Cys;

[0066] one or more amino acid residues of formula (I) or formula (II) is optionally alpha N-methylated; and

[0067] the compound is optionally N-terminally acylated.

[0068] As used herein, Nle is norleucine, 2-Nal is 3-(2-naphthyl)alanine, and HomoCys is homocysteine.

[0069] In some embodiments of the compounds of formula (I) or formula (II), or salt thereof, Xaa5is 2-Nal or Trp.

[0070] In some embodiments of the compounds of formula (I) or formula (II), or salt thereof, Xaa6is Trp, D-Trp, or Cys.

[0071] In some embodiments of the compounds of formula (I) or formula (II), or salt thereof,0-3 peptide backbone amides are independently replaced withamidine, or thioamide. In some embodiments, the compound is:

[0072] H-Nle-cyclo(tryptathio)-[Trp-His-D-Phe-Arg-Trp-Cys]-NH2 (SEQ ID NO: 3);

[0073] H-Nle-cyclo(bis-indolo)-[Trp-His-D-Phe-Arg-2-Nal-Trp]-NH2(SEQ ID NO: 4);

[0074] H-Nle-cyclo(bis-indolo)-[Trp-His-D-Phe-Arg-2-Nal-D-Trp]-NH2(SEQ IDNO: 5);

[0075] H-Nle-cyclo(bis-indolo)-[D-Trp-His-D-Phe-Arg-2-Nal-Trp]-NH2(SEQ IDNO: 6);

[0076] H-Nle-cyclo(bis-indolo)-[D-Trp-His-D-Phe-Arg-2-Nal-D-Trp]-NH2(SEQ IDNO: 7);

[0077] H-Nle-cyclo(tryptathio)-[Trp-His-D-Phe-Arg-2-Nal-Cys]-NH2(SEQ ID NO:8);

[0078] H-Nle-cyclo(tryptathio)-[Trp-His-D-Phe-Arg-2-Nal-D-Cys]-NH2(SEQ IDNO: 9);

[0079] H-Nle-cyclo(tryptathio)-[D-Trp-His-D-Phe-Arg-2-Nal-Cys]-NH2(SEQ IDNO: 10);

[0080] H-Nle-cyclo(tryptathio)-[D-Trp-His-D-Phe-Arg-2-Nal-D-Cys]-NH2(SEQ IDNO: 11);

[0081] H-Nle-cyclo(tryptathio)-[Cys-His-D-Phe-Arg-2-Nal-Trp]-NH2(SEQ ID NO:12);

[0082] H-Nle-cyclo(tryptathio)-[D-Cys-His-D-Phe-Arg-2-Nal-Trp]-NH2(SEQ IDNO: 13);

[0083] H-Nle-cyclo(tryptathio)-[Cys-His-D-Phe-Arg-2-Nal-D-Trp]-NH2(SEQ IDNO: 14);

[0084] H-Nle-cyclo(tryptathio)-[D-Cys-His-D-Phe-Arg-2-Nal-D-Trp]-NH2(SEQ IDNO: 15);

[0085] Ac-Nle-cy clo(tryptathio)-[Cys-His-D-Phe- Arg-2 -Nal-Trp]-NH2(SEQ ID NO:16);

[0086] H-Nle-cyclo(tryptathio)-[Pen-His-D-Phe-Arg-2-Nal-Trp]-NH2(SEQ ID NO:17);

[0087] H-Nle-cyclo(tryptathio)-[Pen-His-D-Phe-Arg-2-Nal-D-Trp]-NH2(SEQ ID NO:18);

[0088] H-Nle-cyclo(tryptathio)-[HomoCys-His-D-Phe-Arg-2-Nal-Trp]-NH2(SEQ IDNO: 19);

[0089] H-Nle-cyclo(tryptathio)-[HomoCys-His-D-Phe-Arg-2-Nal-D-Trp]-NH2(SEQID NO: 20); or

[0090] H-Nle-cyclo(tryptathio)-[HomoCys-His-D-Phe-Arg-Trp-Trp]-NH2 (SEQ ID NO: 21); or a salt thereof.

[0091] In some embodiments, the disclosure relates to a compound comprising a melanocortin 1 receptor (MC1R) targeting peptide (MC1RTP), a radiolabeling group, and a linker joining the MC1R targeting compound to the radiolabeling group, wherein the MC1RTP is optionally C-terminally ami dated, and one or more amino acid residues of the MC1RTP is optionally alpha N-methylated.

[0092] In some embodiments, the MC1RTP is selected from Table A, or salt thereof. In some embodiments, the M1RTP is selected from:

[0093] H-Nle-cyclo(tryptathio)-[Trp-His-D-Phe-Arg-Trp-Cys]-NH2 (SEQ ID NO: 3);

[0094] H-Nle-cyclo(bis-indolo)-[Trp-His-D-Phe-Arg-2-Nal-Trp]-NH2(SEQ ID NO:4);

[0095] H-Nle-cyclo(bis-indolo)-[Trp-His-D-Phe-Arg-2-Nal-D-Trp]-NH2(SEQ IDNO: 5);

[0096] H-Nle-cyclo(bis-indolo)-[D-Trp-His-D-Phe-Arg-2-Nal-Trp]-NH2 (SEQ IDNO: 6);

[0097] H-Nle-cyclo(bis-indolo)-[D-Trp-His-D-Phe-Arg-2-Nal-D-Trp]-NH2 (SEQ IDNO: 7);

[0098] H-Nle-cyclo(tryptathio)-[Trp-His-D-Phe-Arg-2-Nal-Cys]-NH2 (SEQ ID NO:8);

[0099] H-Nle-cyclo(tryptathio)-[Trp-His-D-Phe-Arg-2-Nal-D-Cys]-NH2 (SEQ IDNO: 9);

[0100] H-Nle-cyclo(tryptathio)-[D-Trp-His-D-Phe-Arg-2-Nal-Cys]-NH2 (SEQ IDNO: 10);

[0101] H-Nle-cyclo(tryptathio)-[D-Trp-His-D-Phe-Arg-2-Nal-D-Cys]-NH2 (SEQ IDNO: 11);

[0102] H-Nle-cyclo(tryptathio)-[Cys-His-D-Phe-Arg-2-Nal-Trp]-NH2 (SEQ ID NO:12);

[0103] H-Nle-cyclo(tryptathio)-[D-Cys-His-D-Phe-Arg-2-Nal-Trp]-NH2 (SEQ IDNO: 13);

[0104] H-Nle-cyclo(tryptathio)-[Cys-His-D-Phe-Arg-2-Nal-D-Trp]-NH2 (SEQ IDNO: 14);

[0105] H-Nle-cyclo(tryptathio)-[D-Cys-His-D-Phe-Arg-2-Nal-D-Trp]-NH2 (SEQ IDNO: 15);

[0106] Ac-Nle-cy clo(tryptathio)-[Cys-His-D-Phe- Arg-2 -Nal-Trp]-NH2 (SEQ ID NO:16);

[0107] H-Nle-cyclo(tryptathio)-[Pen-His-D-Phe-Arg-2-Nal-Trp]-NH2 (SEQ ID NO:17);

[0108] H-Nle-cyclo(tryptathio)-[Pen-His-D-Phe-Arg-2-Nal-D-Trp]-NH2 (SEQ ID NO:18);

[0109] H-Nle-cyclo(tryptathio)-[HomoCys-His-D-Phe-Arg-2-Nal-Trp]-NH2 (SEQ IDNO: 19);

[0110] H-Nle-cyclo(tryptathio)-[HomoCys-His-D-Phe-Arg-2-Nal-D-Trp]-NH2 (SEQID NO: 20); or

[0111] H-Nle-cyclo(tryptathio)-[HomoCys-His-D-Phe-Arg-Trp-Trp]-NH2 (SEQ IDNO: 21); or a salt thereof.

[0112] In some embodiments, the MC1RTP is connected to the linker at the N-terminus. In some embodiments the MC1RTP is connected to the linker at the N-terminus of Nle.

[0113] In some embodiments of the compounds of the disclosure, 0-3 peptide backbone amides are independently replaced with, amidine, or thioamide.

[0114] In some embodiments of the compound of the disclosure, the linker comprises an albumin binding group.

[0115] In some embodiment, the linker is H,and the albumin-binding group is N-[4-(p- tolyl)butanoyl].

[0116] In some embodiments, the linker isH. In some embodiments, thelinker isH, wherein * indicates the connectivity to MCI RTP.

[0117] In some embodiments, the linker is X1!?, X^X1!?, or X^X^X1!?;

[0119] L1is each independently -NH-, -C(O)-, -NHC(O)-, -C(O)NH-, - N(CH3)C(O)-, or -C(O)N(CH3)-;

[0120] R11is each independently a carboxylic acid, a sulfonic acid, a sulfinic acid, or a phosphoric acid; and

[0121] Rzis each independently an albumin binder.

[0122] In some embodiments, the linker is XlaLlaXlbLlb;

[0124] Llais -NH- or -NHC(O)-;

[0127] In some embodiments, the linker is XlaLlaXlbLlb;

[0131] Llbis -NH- or -NHC(O)-.

[0132] In some embodiments, RB10ais -NHC(O)-[linker]-Rxni, the linker is XlaLlaXlbLlb,

[0133] Xlais C1-C2 alkenyl;R11is independently carboxylic acid, sulfonic acid, sulfinic acid, or phosphoric acid. In some embodiments, R11is sulfonic acid (-SO3H).

[0134] In some embodiments, the linker comprises 2-7 amino acid residues. In some embodiment, the linker comprises 2-7 amino acid residues selected from one or a combination of: Glu, Asp, and / or 2-aminoadipic acid (2-Aad). In some embodiments, the linker comprises 2 amino acid residues selected from one or a combination of: Glu, Asp, and / or 2-Aad. In some embodiments, the linker comprises 3 amino acid residues selected from one or a combination of: Glu, Asp, and / or 2-Aad. In some embodiments, the linker comprises 4 amino acid residues selected from one or a combination of: Glu, Asp, and / or 2-Aad. In some embodiments, the linker comprises 5 amino acid residues selected from one or a combination of: Glu, Asp, and / or 2-Aad. In some embodiments, the linker comprises 2 or 3 consecutive Glu, Asp, and / or 2-Aad residues. In some embodiments, the linker comprises 3 consecutive Glu residues. In some embodiments, the linker (when including the -C(O)- of L1) consists of a linear peptide of 3 Glu / Asp / 2-Aad residues.

[0135] The albumin-binding group may have the following structure, wherein each R4is independently H, halogen, C1-5 alkyl, C1-5 alkoxyl or nitro group:

[0136] In some embodiments, three R4groups are H and two R4group are independently I, F, Br, Cl or methyl in meta / meta, ortho / ortho, para / meta, meta / para, para / ortho, ortho / para, meta / ortho or ortho / meta. In some embodiments, four R4groups are H and one R4group is I, F, Br, Cl or methyl in para, meta or ortho position. In some embodiments, the albuminbinding group is JV-[4-(iodophenyl)butanoyl], JV-[4-(fluorophenyl)butanoyl ], 7V-[4- (bromophenyl)butanoyl], JV-[4-(chlorophenyl) butanoyl], or JV-[4-(tolyl)butanoyl], In some embodiments, the albumin-binding group is N- [4-(tolyl)butanoyl], In some embodiments, the albumin-binding group is 7V-[4-(p- iodophenyl)butanoyl], JV-[4-(p-fluorophenyl)butanoyl], JV-[4-(p-bromophenyl) butanoyl], JV-[4-( / 7-chlorophenyl)butanoyl], or JV-[4-(p-tolyl)butanoyl]. In some embodiments, the albumin-binding group is JV-[4-(p-tolyl)butanoyl].

[0137] In some embodiments, the linker is: (albumin-binding group)albumin-binding group has the following structure: , wherein each R4is independently H, halogen, C1-5 alkyl, C1-5 alkoxy, or nitro

[0138] In some embodiments of the compound of the disclosure, the radiolabeling group is conjugated to a radioisotope.

[0139] In some embodiments of the compound of the disclosure, the radiolabeling group comprises a radioisotope chelator. Chelators may be incorporated into the compound by coupling to a peptide portion of the compound. Non-limiting examples of radioisotope chelators include chelators selected from DOTA; DOTAGA; NOTA; NOD AGA; NODASA; CB-DO2A; 3p-C-DEPA; TCMC; DO3A; DTPA and DTPA analogues optionally selected from CHX-A”-DTPA and 1B4M-DTPA; TETA; NOPO; Me-3,2-HOPO; CB-TE1A1P; CB- TE2P; MM-TE2A; DM-TE2A; sarcophagine and sarcophagine derivatives optionally selected from SarAr, SarAr-NCS, diamSar, AmBaSar, and BaBaSar; TRAP; AAZTA; DATA and DATA derivatives; macropa; H2dedpa, H4octapa, H4py4pa, H4Pypa, H2azapa, H5decapa, H4py4pa-phenyl-NCS, and other picolinic acid derivatives; CP256; PCTA; C-NETA; C- NE3TA; HBED; HBED-CC; SHBED; BCPA; CP256; YM103; desferrioxamine (DFO) and DFO derivatives; H6phospa; CROWN; HYNIC; and N4. In some embodiments, the radioisotope chelator is DOTA, macropa, or CROWN.

[0140] Exemplary non-limiting examples of radioisotope chelators and radioisotopes chelated by the chelators are shown in Table B. In alternative embodiments, the radioisotope chelator in the compound is one of those listed above or in Table B, or is any other radioisotope chelator. One skilled in the art could replace any of the chelators listed herein with another chelator

[0141] Table B: Exemplary chelators and exemplary isotopes which bind said chelators

[0142] In some embodiments, the DOTA chelator has the following connectivity to the linker( J shows the connectivity to the linker):

[0144] In some embodiments, the radioisotope chelator is conjugated with a radioisotope. The conjugated radioisotope may be, without limitation,68Ga,61Cu,64Cu,67Ga, "mTc,mIn,114mIn,44Sc,47Sc,86Y,90Y,89Zr,90Nb,177Lu,117mSn,165Er,227Th,225Ac,213Bi,212Bi,211As,203Pb,212Pb,166HO,188Re,186Re,149Pm,159Gd,105Rh,109Pd,198Au,199Au,175Yb,142Pr,149Tb,152Tb,155Tb,161Tb, and the like. In some embodiments, the radioisotope is68Ga,61Cu,64Cu,67Ga,99mTc, '" In.44Sc,86Y,89Zr,90Nb,177Lu,117mSn,203Pb,188Re,186Re,149Pm,105Rh,198Auor199Au. In some embodiments, the chelator is a chelator from Table B and the conjugated radioisotope is a radioisotope indicated in Table B as a binder of the chelator.

[0145] In some embodiments, the conjugated radioisotope is a therapeutic radioisotope (e.g. a beta emitter or an alpha emitter). Non-limiting examples of therapeutic radioisotopes include,165Er,212Bi,213Bi,211At,166Ho,149Pm,159Gd,105Rh,109Pd,198Au,199Au,175Yb,142Pr,177LU,mIn,203Pb,212Pb,47Sc,90Y,117mSn,153Sm,149Tb,152Tb,155Tb,161Tb,224Ra,225Ac,227Th,223Ra,77As,1311,64Cu,67Cu, and the like. In some embodiments, the radioisotope is225Ac.

[0146] In some embodiments, the conjugated radioisotope is suitable for PET or SPECT imaging (e.g. a positron emitter or a gamma emitter). Non-limiting examples of positron or gamma emitting radioisotopes include68Ga,67Ga,61Cu,64Cu, "mTc,110mIn,mIn,44Sc,86Y,89Zr,90Nb,18F,1311,1231,124I or72As.

[0147] In some embodiments, the conjugated radioisotope is177Lu,mIn,213Bi,68Ga,67Ga,159Gd,203Pb,212Pb,44Sc,90Y,86Y,175Yb,166Ho,211As,44Sc,47Sc,149Pm,142Pr,225Ac,64Cu or67Cu. In some embodiments, the conjugated radioisotope is225Ac or227Th. In some embodiments, the conjugated radioisotope is213Bi,177Lu,225Ac,149Tb,152Tb,155Tb,161Tb, or227Th.

[0148] In some embodiments, the radioisotope chelator is DOTA and the compound is conjugated with177Lu,mIn,213Bi,68Ga,67Ga,159Gd,203Pb,212Pb,44Sc,90Y,86Y,175Yb,166Ho,211AS,44SC,47SC,149Pm,142Pr,225Ac,64Cu or67Cu. In some embodiments, the radioisotope chelator is macropa (H2-MACROPA) and the compound is conjugated with225Ac or227Th. In some embodiments, the radioisotope chelator is CRWON and the compound is conjugated with213BI,177LU,225AC,149Tb,152Tb,155Tb,161Tb, or227Th.

[0149] In some embodiments, the compound issalt thereof, optionally conjugated with a radioisotope.

[0150] In some embodiments, the compound isor salt thereof. In some embodiments, Ga isnatGa or68Ga. In some embodiments, Ga is68Ga.

[0151] In some embodiments, the compound is, or salt thereof, optionally conjugated with a radioisotope.

[0152] In some embodiments, the compoundor salt thereof. In some embodiments, Ga isnatGa or68Ga. In some embodiments, Ga is68Ga.

[0153] Uses and Methods

[0154] This section incorporates all embodiments and combinations of features of the compounds described in the previous section.

[0155] There is also disclosed a pharmaceutical composition comprising a compound as described herein and a pharmaceutically acceptable excipient, carrier or diluent. The compound may be any of the embodiments (or combination of features thereof) defined in the previous section. The compound may be conjugated with a therapeutic radioisotope (as defined in the previous section) or may be conjugated with a radioisotope that is suitable for PET or SPECT imaging (as defined in the previous section).

[0156] The compounds may be useful in preparing a radioisotope-conjugated compound. There is therefore provided a method of preparing a radioisotope-conjugated compound comprising conjugating a radioisotope as defined in the previous section to the radioisotope chelator of a compound as defined in the previous section. The compound may be any of the embodiments (or combination of features thereof) defined in the previous section. The radioisotope may be a therapeutic radioisotope (as defined in the previous section) or may be a radioisotope that is suitable for PET or SPECT imaging (as defined in the previous section).

[0157] In some embodiments, the present disclosure provides a method of treating cancer.

[0158] In some embodiments, the present disclosure provides a method of treating melanoma.

[0159] In some embodiments, the present disclosure provides a method of treating an MC1R- related disease, comprising administering any one of the compounds as disclosed herein to a subject. In some embodiments, the present disclosure provides a use of any one of the compounds disclosed herein in manufacture of a medicament for treatment of an MClR-related disease or condition.

[0160] In some embodiments, the MClR-related disease or condition is an MClR-expressing cancer. In some embodiment, cancer is melanoma or non-melanoma skin cancer. For example, but without limitation, MC1R is specifically expressed in cutaneous, amelanotic and uveal melanomas. In some embodiments, the MClR-expressing cancer is melanoma or nonmelanoma skin cancer. In some embodiments, the MClR-expressing cancer is primary melanoma or metastatic melanoma. In some embodiments, the MClR-expressing cancer is uveal melanoma. In some embodiments, the MClR-expressing cancer is melanoma.

[0161] In some embodiments, the present disclosure provides a method of imaging MClR- expressing tissues in a subject, in which the method comprises: administering to the subject any one of the compounds as described herein; and imaging tissue of the subject using PET or SPECT. When the tissue is a diseased tissue (e.g. an MClR-expressing cancer), MC1R- targeted treatment may then be selected for treating the subject. In some embodiment of anyone of the methods as disclosed herein, the compound is administered as a pharmaceutical composition. In some embodiments, the MClR-expressing tissues are cancer tissues. In some embodiments, cancer is melanoma or non-melanoma skin cancer. In some embodiments, cancer is primary melanoma or metastatic melanoma. In some embodiments, cancer is uveal melanoma.

[0162] Without limitation, the compound of the disclosure or a pharmaceutical composition comprising the compound may be administered to the subject intravenously, or as a subcutaneous or intradermal injection surrounding the site of the initial tumour (e.g. to detect lymphatic spread).

[0163] In some embodiments, the present disclosure provides a method of preparing the compounds as disclosed herein. In some embodiments, the method utilizes tryptathionylation. In some embodiments, the method utilizes tryptathionylation as described in Example 1. In some embodiments, tryptathionylation is carried out on a substrate with a Mmt-protected cysteine and a Boc-protected indole. In some embodiments, the method utilizes bis-indole formation. In some embodiments, the method utilizesbis-indole formation as described in Example 1. In some embodiments, bis-indole formation is carried out on a substrate with a Trt- protected cysteine.EXAMPLES

[0164] Various alternative embodiments and examples are described herein. These embodiments and examples are illustrative and should not be construed as limiting the scope of the invention.

[0165] General Methods

[0166] HPLC Purification: All HPLC purifications were performed on an Agilent 1260 Infinity II (HPLC 1) Prep HPLC outfitted with the following modules: G7161A Prep Pump, G7157A Prep Autosampler, G7115A DAD, G1364E Fraction Collector One of the following columns was used; Column 1: Agilent 5 Prep C18 50 x 21.2 mm (L x ID), Column 2: Agilent 5 Prep C18 50 x 10 mm (L x ID). Or an Agilent 1100 Series (HPLC 2) outfitted with the following modules: G1379A Degasser, G1311A Quat Pump, G1313 A Autosampler, G1316A COLCOM, G1315B DAD, G1364C Analytical Fraction Collector. Column 3, an Agilent Eclipse XDB- C18 9.4 x 250 mm, was used. Solvent A was 0.1% formic acid H2O (filtered through a 0.2 pm PALL filter). Solvent B was 0.1% formic acid MeCN (purchased at HPLC grade, formic acid then added. Absorbance was monitored simultaneously at 230 nm, 260 nm and 290 nm.

[0167] HPLC Reinjection: All analytical HPLC chromatographs were acquired on an Agilent 1100 Series (HPLC 2) outfitted with the following modules: G1379A Degasser, G1311A Quat Pump, G1313A Autosampler, G1316A COLCOM, G1315B DAD, G1364C Analytical Fraction Collector. Column 3, an Agilent Eclipse XDB-C18 9.4 x 250 mm, was used. Solvent A was 0.1% formic acid H2O (filtered through a 0.2 pm PALL filter). Solvent B was 0.1% formic acid MeCN (purchased at HPLC grade, formic acid then added. Absorbance was monitored simultaneously at 230 nm, 260 nm and 290 nm.

[0168] HPLC Method A: HPLC 2, 2 mL / min, Column 3. Solvent A: 0.1% formic acid H2O, Solvent B: 0.1% formic acid MeCN

[0169] 0 min -19 min 90: 10 A / B - 60:40 A / B

[0170] 19 min-22 min 60:40 A / B - 0: 1 A / B

[0171] 22 min-28 min 0: 1 A / B

[0172] 28 min-31 min 0:1 A / B - 90:10 A / B

[0173] 31 min-38 min 90:10 A / B

[0174] HPLC Method B: HPLC 1, 15mL / min, Column 1. Solvent A: 0.1% formic acid H2O, Solvent B: 0.1% formic acid MeCN

[0175] 0 min-6.5 min 95:5 A / B - 65:35 A / B

[0176] 6.5 min-7.5 min 65:35 A / B - 0:1 A / B

[0177] HPLC Method C: Solvent A: 0.1% TFA H2O, Solvent B: 0.1% TFA MeCN

[0178] 0 min -10 min 90: 10 A / B - 50:50 A / B

[0179] 10 min-10.5 min 50:50 A / B - 0:1 A / B

[0180] 10.5 min-14 min 0:1 A / B

[0181] HPLC Method D: Solvent A: 0.1% TFA H2O, Solvent B: 0.1% TFA MeCN

[0182] 0 min -8 min 85:15 A / B - 45:55 A / B

[0183] 8 min-8.5 min 45:55 A / B - 0:1 A / B

[0184] 8.5 min-14 min 0: 1 A / B

[0185] Peptide quantification: All peptides were quantified by UV spectrophotometry on a Carry 5000 UV-Vis-NIR Spectrophotometer. Lyophilized peptides were dissolved in a known volume of H2O or 0.1% formic acid H2O, transferred to a quartz cuvette and absorbance was measured at a Zmax of 290 nm for peptides with unsubstituted tryptathionine staples12,13or at 320 nm for peptides with 2-2’ bisindole staples.24

[0186] CD Spectral Acquisition: All CD spectra were acquired on a JASCO J-815 CD Spectrometer. All peptides were dissolved in MeOH to a concentration of 50 mM and read atroom temperature. 3 scans were accumulated for each peptide and a smoothing algorithm was applied.

[0187] General Procedure for Solid Phase Peptide Synthesis

[0188] i) Manual Solid Phase Peptide Synthesis: The dried resin contained in a ZEBA Desalt spin column was solvated in 5 mL DMF and shaken for at least 30 minutes. The solvent was drained and the resin was resuspended in 5 mL 2:8 Piperidine / DMF (0.5M Oxyma) and shaken for 5 minutes. The solvent was drained and the resin was resuspended in 5 mL 2:8 Piperidine / DMF (0.5M Oxyma) and shaken for an additional 10 minutes. The solvent was drained and the resin was washed with seven 5 mL portions of DMF, with shaking and draining. The resin was then Kaiser tested and if a positive result (purple or brown) was observed, the beads were resuspended in a 5mL solution of DMF which already contained 4 equivalents each of Fmoc-Xaa, Oxyma and COMU and ~11 equivalents DIEA. The resin was then shaken for 1-3 hours at room temperature. The solvent was drained and the resin was washed with five 5 mL portions of DMF, with shaking and draining. The resin was then Kaiser tested and if a negative result (no change in colouration) was observed, the beads were then resuspended in a 5 mL solution of 1 :2:2 Ac2O / Collidine / EtOAc and shaken for 20 minutes at room temperature. If the result of the Kaiser test remained positive, another coupling with same Fmoc amino acid was performed. Once the capping was complete, the solvent was drained and the resin was washed with five 5 mL portions of DMF, with shaking and draining. The resin was then washed with DCM and left to dry under reduced pressure if couplings were complete for the day or resuspended in 2:8 Piperidine / DMF (0.5 M Oxyma) for the next Fmoc deprotection.

[0189] ii) Automatic Solid Phase Peptide Synthesis: Using a Gyros PurePep Chorus GT outfitted with induction heating, preloaded CTC resin was solvated in 3mL of DMF and mixed by bubbling nitrogen for 20 minutes and then drained. Resin was resuspended in 3 mL 2:8 Piperidine / DMF and mixed by bubbling nitrogen for 1 minute at 50°C, solution was drained and process was repeated with a fresh 3 mL portion of DMF. Resin was then washed three times with 3 mL portions of DMF (mixed by bubbling with nitrogen and then drained.) Resin was then resuspended in 3 mL DMF containing Fmoc-Xaa (100 mM), HCTU (100 mM), N-methyl-morpholine (200 mM) with at least 7.5 equivalents of amino acid and coupling agent to initial resin loading. Resin suspended in coupling solution was mixed by bubbling with nitrogen for 5 minutes at 50°C. Exceptions were as follows: Fmoc-Hpi-OH or Boc- Fpi(5OTBS)-OH were mixed by bubbling with nitrogen at room temperature for 1 hour. The resin was then washed three times with 3 mL portions of DMF (mixed by bubbling with nitrogen and then drained.) After the last coupling, resin was then washed four times with 3 mL portions of DCM (mixed by bubbling with nitrogen and then drained.) The resin was then dried by flushing with nitrogen for 20 minutes. Fmoc-Hpi-OH was prepared according to Blanc, A. et al. Amino Acids 2017, 49 (2), 407-414.

[0190] General Procedure for Savige-Fontana Tryptathionylation / BiTe Formation

[0191] i) Cleavage Procedure from Manual Synthesis: To the spin column containing the free- base resin bound peptide was added 5mL TFA and the beads were shaken at room temperature for 2 hours. The solution was then fdtered into a round bottom flask containing 0.25 mL 1:1 v / v TIS / H2O and the fdtrate was evaporated under reduced pressure to yield a brown or pinkish solid. The last traces of TFA were removed by two successive rounds of co-evaporation with DCM. The crude peptide was then triturated with three 10 mL portions of Et20 and allowed to air dry overnight to yield an off-white powder.

[0192] ii) Cleavage Procedure from Automatic Peptide Synthesis: Using a Gyros PurePep Chorus GT outfitted with induction heating, free amine or Boc protected Hpi bearing resin was suspended in TFA (3 mL) and mixed by gentle bubbling with nitrogen for 2 hours at room temperature. Following reaction, the cleaved peptide in TFA was collected in a 15 mL Falcon tube, and 75 pL of TIS and 75 pL of H2O were added and the solution was vortexed and allowed to react until the yellow colour from trityl cation was no longer visible (around 30 minutes). The light orange / brown solution was diluted with Et20, and cooled to -20°C resulting in a white precipitate. The suspension was spun down on a clinical centrifuge, the supernatant was discarded, and the pellet was resuspended in Et20, vortexed and then spun down on clinical centrifuge and process of resuspension in Et20 was repeated. The resulting white pellet was allowed to dry under ambient atmosphere.

[0193] Gallium Labeling Protocol: Purification of 68Ga eluted from 68Ge / 68Ga generator and labeling experiments was performed according to standard protocols. Briefly, the 68Ga generator was activated by injecting with HC1 (0.05 M, 5 mL), the solution was subsequently passed through an anion exchange column and rinsed with more HC1 (5 N, 3 mL). Following this elution of the 68Ga was achieved with H2O (0.5 mL) and collected in a vial containing 22(10 nmol) dissolved in HEPES buffer (2 M, 0.7 mL, pH 5). The reaction vessel was then placed in the microwave and heated for 1 minute at 20% power. The resulting mixture was then purified via semi-prep HPLC, via method C; the desired radiolabeled peptide eluted at Rt = 9.99 minutes. The eluted fractions containing the radiolabeled product was further diluted with water (50 mL) and passed through a C18 Sep-pak cartridge that was previously activated with EtOH (3 mL) and water (3 mL). The cartridge was rinsed with H2O (2 mL) and the product was eluted with 0.4 mL of EtOH and collected in PBS (phosphate buffered saline) (4 mL) and analyzed for purity via analytical HPLC method D; the peak eluting at Rt = 6.99 mins at > 99% radiochemical purity, was isolated in radiochemical yield of 74.8%, and molar activity was measured to be 28.9 MBq / pmol (0.78 Ci / pmol). The radiochemically pure product was further diluted as necessary with PBS and transferred to the animal facility for PET imaging and biodistribution studies.

[0194] Animal Care: All animal experiments were conducted according to the guidelines established by Canadian Council on Animal Care and approved by Animal Ethics Committee of the University of British Columbia. Mice were housed under pathogen-free conditions and kept on twelve-hour light and dark cycles in the Animal Resource Centre, BC Cancer Research Centre, Vancouver, Canada. PET imaging and biodistribution studies were performed using male C57BL / 6J mice. For tumor implantation, mice were anesthetized by inhalation with 2% isoflurane in 2.0 L / min of oxygen, and 1 X 106B16F10 cells were implanted subcutaneously on the right back at the level of the forelimbs. Mice were imaged or used in biodistribution studies once the tumor grew to reach 8-10 mm in diameter in 8-10 days.

[0195] Example 1: Competition Experiments

[0196] While the nucleophilic addition of thiol to a hydroxyindolinium cation is a well- established reaction in the case of amanitin and phalloidin syntheses, the potential for competitive indole attack has been unknown. Previously, acid-promoted bis-indolyl-indole formation has been followed by DDQ oxidation25. Surprisingly, our initial attempts at tryptathionylation in the presence of a nearby tryptophan competitive reaction that gave both the bis-indole and tryptathionine crosslinks in roughly equal proportions (data not shown). To control the outcome of this reaction, two linear peptides were synthesized, one with a Mmt- protected cysteine and a Boc-protected indole la and the other with a Trt-protected cysteine lb (Scheme 1). The choice of protecting groups was rationalized accordingly: i) to install the 2,2'-bis-indole staple, the S-Trt would be sufficiently long-lived under the acidic conditions toenable the unprotected indole to react as a nucleophile; ii) to install the tryptathionine, the Mmt would be removed more rapidly in acid while the Boc-indole would be sufficiently stable such that its removal would occur following tryptathionylation.

[0197] Scheme 1.

[0198] In each case, cyclization proceeded concomitantly with resin cleavage and thiol detritylation. Addition of tri-isopropylsilane (TIS) as a cation scavenger after cyclization was completed but prior to concentration in vacuo prevented the formation of alkylated byproducts. In accord with this anticipated reaction sequence, peptide lb afforded significant amounts of 2,2'-bis-indole stapled peptide 2a whilst peptide la yielded only the tryptathionine stapled peptide 3a.

[0199] Compared to the use of acid-catalyzed bis-indolyl-indole formation followed by the use of DDQ or other oxidants, this result, without bound to any theory, represents a milder and more selective route to 2-2 ’-bis-indole staples whereby indole-oxidation is effected prior to reaction, thereby enabling one to designate a specific indole to serve as the latent electrophilerevealed in acid. Notably, a bis-indole staple is known to constrain a-helices and affords the added value of inherent fluorescence.

[0200] Because significant chemoselectivity in this test sequence was observed, additional stapled MSH analogs were synthesized and evaluated for its biological activities.

[0201] Example 2: Synthesis of Compound 1 and Compound 2

[0202] H-Hpi-His(Trt)-D-Phe-Arg(Pbf)-2-Nal-Cys(Trt)-NHRAM (SEQ ID NO: 22) (~40mg) was resuspended in 5mL DMF swelled by bubbling with N2 on peptide synthesizer for 20 minutes. The DMF was then drained and the resin was resuspended in a wine-red solution (called “solution A”) of COMU (102 mg, 0.238 mmol), Fmoc-Nle-OH (84 mg, 0.238 mmol) and DIEA (0.12 mL, 0.689 mmol) in DMF (ImL) (Final Concentration 0.24M COMU and Fmoc-Nle-OH, 0.69M DIEA). The resulting solution was mixed by bubbling with N2 on peptide synthesizer for 1 hour, then drained. The resin was then resuspended in 5mL DMF, bubbled with N2 for 30 seconds and then drained. This was repeated two more times (three washes total). The resin was then resuspended in 5mL DCM, bubbled with N2 for 30 seconds and then drained. A sample was then removed (approximately the amount of beads on the tip of a spatula) and set aside (labelled “lh”). The remaining bulk resin was then resuspended in ImL of solution A and mixed by bubbling with N2 for lh. The washing procedure described above was then performed and sample was taken and set aside (labelled “2h”). This procedure was repeated a further three times to result in 5 resin samples labelled lh, 2h, 3h, 4h, and 5.5h.These five resin samples were each separately resuspended in 5mL DMF swelled by bubbling with N2 on peptide synthesizer for 20 minutes. The DMF was then drained and the resins samples were resuspended in a 3mL solution of 2:8 v / v solution of Piperidine:DMF and mixed by bubbling with N2 on peptide synthesizer for 3 minutes. The solution was drained and the resin samples were then resuspended in 5mL DMF, bubbled with N2 for 30 seconds and then drained. This was repeated two more times (three washes total). The resin was then resuspended in 5mL DCM, bubbled with N2 for 30 seconds and then drained. The resin samples were then dried on the peptide synthesizer for 20 minutes. The resin samples were then resuspended in 3mL TFA and mixed by bubbling with N2 on peptide synthesizer for 2 hours. The resulting solutions were then drained by filtration on the peptide synthesizer and collected in 50mL Falcon tubes for collection. To each solution 75pL of TIS and 75pL of H2O were added and the solutions were mixed by shaking on vortex and allowed to react at room temperature for ~15 minutes. The reaction mixtures were then transferred to 15mL falcon tubes and then ~1 ImL Et2O added to each falcon tube, shaken and placed in -20°C freezer for ~lh to precipitate peptides. The suspension was spun down in clinical centrifuge and then triturated with Et2O two more times. The resulting pellet was allowed to dry in air overnight to afford crude peptide samples, which were then resuspended in 0.1% FEO / MeCN (—2: 1) and injected onto HPLC using method B.

[0203] Example 3: Synthesis of Compounds 3-11

[0204] General Procedure for Savige-Fontana Tryptathionylation / BiTe Formation, Cleavage Procedure from Manual Peptide Synthesis was used to prepare Compounds 3-11.

[0205] Compound 3 (H-Nle-cyclo(tryptathio)-[Trp-His-D-Phe-Arg-Trp-Cys]-NH2 (SEQ ID NO: 3)): HPLC 2 (HPLC Method A): tR= 11.2min; Xmax290 nm. HRMS-ESI (m / z) [M+H]+calcd. for C52H66N15O7S, 1044.4990; found 1044.4987.

[0206] Compound 4 (H-Nle-cyclo(bis-indolo)-[Trp-His-D-Phe-Arg-2-Nal-Trp]-NH2 (SEQ ID NO: 4)): HPLC 2 (HPLC Method A): tR= 14.3min; / .max320 nm. HRMS-ESI (m / z) [M+H]+calcd. for C62H72N15O7, 1138.5739; found 1138.5736.

[0207] Compound 5 (H-Nle-cyclo(bis-indolo)-[Trp-His-D-Phe-Arg-2-Nal-D-Trp]-NH2(SEQ ID NO: 5)): HPLC 2 (HPLC Method A): tR= 15.9min; / .max320 nm. HRMS-ESI (m / z) [M+H]+calcd. for C62H72N15O7, 1138.5739; found 1138.5748.

[0208] Compound 6 (H-Nle-cyclo(bis-indolo)-[D-Trp-His-D-Phe-Arg-2-Nal-Trp]-NH2(SEQ ID NO: 6)): HPLC 2 (HPLC Method A): tR= 16. Imin; / .max320 nm. HRMS-ESI (m / z) [M+H]+calcd. for C62H72N15O7, 1138.5739; found 1138.5732.

[0209] Compound 7: (H-Nle-cyclo(bis-indolo)-[D-Trp-His-D-Phe-Arg-2-Nal-D-Trp]-NH2 (SEQ ID NO: 7)): HPLC 2 (HPLC Method A): tR= 17.8min; / .max320 nm. HRMS-ESI (m / z) [M+H]+calcd. for C62H72N15O7, 1138.5739; found 1138.5731.

[0210] Compound 8 (H-Nle-cyclo(tryptathio)-[Trp-His-D-Phe-Arg-2-Nal-Cys]-NH2 (SEQ ID NO: 8)): HPLC 2 (HPLC Method A): tR= 13. Imin; Xmax290 nm HRMS-ESI (m / z) [M+H]+calcd. for C54H67N14O7S, 1055.5038; found 1055.5036.

[0211] Compound 9 (H-Nle-cyclo(tryptathio)-[Trp-His-D-Phe-Arg-2-Nal-D-Cys]-NH2(SEQ ID NO: 9)): HPLC 2 (HPLC Method A): tR= 14.4min; / .max290 nm. HRMS-ESI (m / z) [M+H]+calcd. for C54H67N14O7S, 1055.5038; found 1055.5037.

[0212] Compound 10 (H-Nle-cyclo(tryptathio)-[D-Trp-His-D-Phe-Arg-2-Nal-Cys]-NH2 (SEQ ID NO: 10)): HPLC 2 (HPLC Method A): tR= 15.2min; Xmax290 nm. HRMS-ESI (m / z) [M+H]+calcd. for C54H67N14O7S, 1055.5038; found 1055.5035.

[0213] Compound 11 (H-Nle-cyclo(tryptathio)-[D-Trp-His-D-Phe-Arg-2-Nal-D-Cys]-NH2 (SEQ ID NO: 11)): HPLC 2 (HPLC Method A): tR= 15.2min; Xmax290 nm. HRMS-ESI (m / z) [M+H]+calcd. for C54H67N14O7S, 1055.5038; found 1055.5034.

[0214] Example 4: Synthesis of Compounds 12-20

[0215] General Procedure for Savige-Fontana Tryptathionylation / BiTe Formation, Cleavage Procedure from Automatic Peptide Synthesis was used to prepare Compounds 12-20.

[0216] Compound 12 (H-Nle-cyclo(tryptathio)-[Cys-His-D-Phe-Arg-2-Nal-Trp]-NH2 (SEQ ID NO: 12)): HPLC 2 (HPLC Method A): tR= 11.3min; / .max290 nm. HRMS-ESI (m / z) [M+H]+calcd. for C54H67N14O7S, 1055.5038; found 1055.5039. 1H NMR (600 MHz, CD3CN) δ 7.82 - 7.74 (m, 1H), 7.70 - 7.62 (m, 2H), 7.56 - 7.46 (m, 3H), 7.45 - 7.37 (m, 2H), 7.30 (d, J = 8.1 Hz, 1H), 7.25 (t, J = 7.6 Hz, 2H), 7.22 - 7.16 (m, 2H), 7.16 - 7.12 (m, 2H), 7.10 (t, J = 7.4 Hz, 1H), 7.00 (t, J = 7.4 Hz, 1H), 4.85 (dd, J = 7.6, 5.8 Hz, 1H), 4.77 (dd, J = 9.7, 5.7 Hz, 1H), 4.53 (t, J = 7.2 Hz, 1H), 4.34 (dd, J = 8.7, 6.8 Hz, 1H), 4.24 (t, J = 8.0 Hz, 1H), 3.76 (dd, J = 9.7, 4.4 Hz, 1H), 3.27 - 3.21 (m, 1H), 3.16 (dd, J = 14.1, 5.7 Hz, 1H), 3.11 - 2.95 (m, 4H), 2.92 - 2.81 (m, 3H), 2.76 (dd, J = 14.6, 6.9 Hz, 1H), 2.63 (ddd, J = 29.1, 13.5, 6.8 Hz, 2H), 2.11 (q, J = 5.6 Hz, 1H), 1.61 - 1.52 (m, 1H), 1.21 - 1.11 (m, 9H), 0.77 (t, J = 7.2 Hz, 3H).

[0217] Compound 13 (H-Nle-cyclo(tryptathio)-[D-Cys-His-D-Phe-Arg-2-Nal-Trp]-NH2 (SEQ ID NO: 13)): HPLC 2 (HPLC Method A): tR= 14.0min; Xmax290 nm. HRMS-ESI (m / z) [M+H]+calcd. for C54H67N14O7S, 1055.5038; found 1055.5037.

[0218] Compound 14 (H-Nle-cyclo(tryptathio)-[Cys-His-D-Phe-Arg-2-Nal-D-Trp]-NH2 (SEQ ID NO: 14)): HPLC 2 (HPLC Method A): tR= 12.7min; / .max290 nm. HRMS-ESI (m / z) [M+H]+calcd. for C54H67N14O7S, 1055.5038; found 1055.5040.

[0219] Compound 15 (H-Nle-cyclo(tryptathio)-[D-Cys-His-D-Phe-Arg-2-Nal-D-Trp]-NH2 (SEQ ID NO: 15)): HPLC 2 (HPLC Method A): tR= 16.1min; Xmax290 nm HRMS-ESI (m / z) [M+H]+ calcd. for C54H67N14O7S, 1055.5038; found 1055.5039.

[0220] Compound 16 (Ac-Nle-cyclo(tryptathio)-[Cys-His-D-Phe-Arg-2-Nal-Trp]-NH2 (SEQ ID NO: 16)): HPLC 2 (HPLC Method A): tR= 18.3min; / .max290 nm. HRMS-ESI (m / z) [M+H]+calcd. for C56H69N14O8S, 1097.5144; found 1097.5145.

[0221] Compound 17 (H-Nle-cyclo(tryptathio)-[Pen-His-D-Phe-Arg-2-Nal-Trp]-NH2 (SEQ ID NO: 17)): HPLC 2 (HPLC Method A): tR= 12.3min; Xmax290 nm. HRMS-ESI (m / z) [M+H]+calcd. for C56H71N14O7S, 1083.5351; found 1083.5355.

[0222] Compound 18 (H-Nle-cyclo(tryptathio)-[Pen-His-D-Phe-Arg-2-Nal-D-Trp]-NH2 (SEQ ID NO: 18)): HPLC 2 (HPLC Method A): tR= 13.5min; Xmax290 nm HRMS-ESI (m / z) [M+H]+calcd. for C56H71N14O7S, 1083.5351; found 1083.5349.

[0223] Compound 19 (H-Nle-cyclo(tryptathio)-[HomoCys-His-D-Phe-Arg-2-Nal-Trp]-NH2 (SEQ ID NO: 19)): HPLC 2 (HPLC Method A): tR= 12. Imin; U, 290 nm LRMS-ESI (m / z) [M+H]+calcd. for C55H69N14O7S, 1069.5194; found 1069.8.

[0224] Compound 20 (H-Nle-cyclo(tryptathio)-[HomoCys-His-D-Phe-Arg-2-Nal-D-Trp]- NH2(SEQ ID NO: 20)): HPLC 2 (HPLC Method A): tR= 13.5min; Xmax290 nm. LRMS-ESI (m / z) [M+H]+calcd. for C55H69N14O7S, 1069.5194; found 1069.8.

[0225] Compound 21 (H-Nle-cyclo(tryptathio)-[HomoCys-His-D-Phe-Arg-Trp-Trp]-NH2 (SEQ ID NO: 21))

[0226] Example 5: Synthesis of Compounds 22 and 23

[0227] Compound 21 was coupled with Fmoc-Pip-OH and Fmoc was removed to provide Compound 21-4APip (4-Apip-Nle-cyclo(tryptathio)-[Homocys-His-D-Phe-Arg-Trp-Trp]- NH2 (SEQ ID NO: 23)). Using a Gyros PurePep Chorus GT outfitted with induction heating, Fmoc-Rink Amide resin was solvated in 3 mL of DMF and mixed by bubbling nitrogen for 20 minutes and then drained. Resin was resuspended in 3 mL 2:8 piperidine / DMF and mixed bybubbling nitrogen for 3 minutes at room temperature, solution was drained and process was repeated with a fresh 3 mL portion of DMF. Resin was then washed three times with 3 mL portions of DMF (mixed by bubbling with nitrogen and then drained.) Resin was then resuspended in 3 mL DMF containing Fmoc-Xaa (100 mM), HCTU (100 mM), N-methyl- morpholine (200 mM) with at least 7.5 equivalents of amino acid and coupling agent to initial resin loading. Resin suspended in coupling solution was mixed by bubbling with nitrogen for 20 minutes at room temperature. Exceptions were as follows: Fmoc-Hpi-OH was mixed by bubbling with nitrogen at room temperature for 1 hour. The amino acid immediately following Hpi was coupled at room temperature, (240 mM COMU and Fmoc-Xaa-OH, 690 mM DIPEA) for 1 hour. Coupling was repeated 3 times (4 hours total) The resin was then washed three times with 3 mL portions of DMF (mixed by bubbling with nitrogen and then drained.) After the last coupling and deprotection, resin was then washed four times with 3 mL portions of DCM (mixed by bubbling with nitrogen and then drained.) The resin was then dried by flushing with nitrogen for 20 minutes. The resin was then suspended in TFA (3 mL) and mixed by gentle bubbling with nitrogen for 2 hours at room temperature. Following reaction, the cleaved peptide in TFA was collected in a 15 mL Falcon tube, and 75 pL of TIS and 75 pL of H2O were added and the solution was vortexed and allowed to react until the yellow colour from trityl cation was no longer visible (around 30 minutes). The light orange / brown solution was diluted with Et20, and cooled to -20°C resulting in a white precipitate. The suspension was spun down on a clinical centrifuge, the supernatant was discarded, and the pellet was resuspended in Et20, vortexed and then spun down on clinical centrifuge and process of resuspension in Et20 was repeated. The resulting white pellet was allowed to dry under ambient atmosphere. The compound was then used in the next step with no further purification.

[0228] Crude, unpurified Compound 21-4APip (2 pmole), and DOTA-NHS (6 pmole, 4.6 mg) were dissolved in 50 pL of DMF. DIEA (3.5 pL, 20 pmole) was added to the reaction mixture, the pH was tested and enough DIEA was then added to register pH -9-10 on wetted pH paper. The reaction mixture was vortexed and allowed to proceed at room temperature overnight. After overnight reaction, the reaction mixture was diluted with 0.1 % formic acid H2O and purified directly by HPLC 1. The pure fractions were pooled, frozen and lyophilized, yielding Compound 22 (DOTA-4Apip-Nle-cyclo(tryptathio)-[HomoCys-His-D-Phe-Arg-Trp-Trp]- NH2 (SEQ ID NO: 24)) a white powder (0.4 pmole, 20 % yield from crude peptide). HPLC 1 (HPLC Method B): tR = 3.9min; Zmax 290 nm. LRMS-ESI (m / z) [M+2H]2+ / 2calcd. for C76H105N21O15S, 792.9; found 793.5.

[0229] Compound 22 (200 nmoles) was dissolved in 56pL 2M HEPES (pH 5) and 40pL Ga(NO2)s in IM HC1 (7.2mg / mL) (5eq.) The solution was transferred to a sealed glass vial and heated in micro wave for Imin at 20% power. Diluted with H2O and then purified directly by HPLC 1. The pure fractions were pooled, frozen and lyophilized, yielding Compound 23 (Ga- DOTA-4Apip-Nle-cyclo(tryptathio)-[HomoCys-His-D-Phe-Arg-Trp-Trp]-NH2 (SEQ ID NO: 25)) as a white powder (129 nmoles, 65 % yield). HPLC reinjection matched hot68Ga labelled version as described in gallium labelling procedure. LRMS-ESI (m / z) [M+2H]2+ / 2calcd. for C76Hio2N2iOi5SGa, 825.9; found 826.2.

[0230] Example 6: Receptor Binding Assay

[0231] Competitive receptor binding assays were performed using B16F10 cells; approximately 500,000 cells / well were seeded onto a 24-well poly-D-lysine coated plate (Coming) overnight. Growth media was removed, and reaction buffer containing 4.8 mg / mL HEPES, 100 U / mL penicillin, 1,000 pg / mL streptomycin and 2 mg / mL BSA was added, and allowed to incubate with cells at 37°C for at least an hour. Non-radioactive peptides and125I- [Nle4, D-Phe7]-alpha-MSH (PerkinElmer) were added to each well along with increasing concentrations of test peptide ( 0.5 pM to 5 pM). The reaction mixture was incubated at 25°C with gentle agitation for 1 h. After the incubation, the reaction mixture was removed, and cells were washed with ice-cold PBS twice. 0.25% Trypsin solution was used to harvest the cells and radioactivity was measured on a WIZARD 2480 gamma counter (PerkinElmer). Table 2 shows the binding affinities of the compounds of the disclosure.

[0232] Table 2. Binding Affinities

[0233] Example 7: PET / CT imaging and Biodistribution Studies

[0234] PET / CT imaging studies were carried out on a microPET / CT scanner (Inveon, Siemens) according to standard protocol for static PET scans; a tumor bearing mouse was injected with 4-6 MBq of68Ga-labeled Compound 23 for the one-hour time point, via the caudal lateral tail vein under isoflurane sedation. After injection, the mouse was allowed to recover and roam freely. After 30 min, the mouse was re-sedated and positioned in the scanner. A baseline CT scan was obtained for localization and attenuation correction. This was followed by a 10 min static PET scan. Following scanning, the mouse was euthanized using CO2 inhalation followed by biodistribution. The PET images were reconstructed using the ordered subset expectation maximization and maximum a posteriori algorithm (OSEM3D / MAP), using 2 OSEM3D iterations followed by 18 MAP iterations, with a requested resolution of 1.5 mm.

[0235] Four additional mice were anesthetized by 2% isoflurane inhalation, and injected with 1-2 MBq of68Ga-labeled Compound 23. After injection, the mice were allowed to recover and roam freely in their cages, and euthanized by CO2 inhalation 1 h. Blood was promptly withdrawn, and the organs of interest were harvested, rinsed with 1 * PBS (pH 7.4), and blotted dry. Each organ was weighed and the radioactivity of the collected tissue was measured using a WIZARD 2480 (PerkinElmer) or a Cobra II gamma counter (Packard), normalized to the injected dose using a standard curve and expressed as the percentage of the injected dose per gram of tissue (%ID / g). Table 3 summarizes the biodistribution data of68Ga-labeled Compound 23 at 1 h p.i.

[0236] Table 3 Biodistribution data (at 1 h post-injection, %ID / g) of68Ga-labeled Compound 23 (n = 4)

[0237] PET imaging of68Ga-labeled Compound 23 (68Ga-23) showed tumor uptake of ~2%ID / g (Fig. 1) at one-hour post injection. Interestingly, despite the general hydrophobicity of the tryptathionine staple, it is rather surprising that liver uptake was middling (~4%ID / g), and most of the peptide cleared substantially to the bladder.

[0238] Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range. The word “comprising” is used herein as an open-ended term, substantially equivalentto the phrase “including, but not limited to”, and the word “comprises” has a corresponding meaning. As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a thing” includes more than one such thing. Citation of references herein is not an admission that such references are prior art to an embodiment of the present invention. The invention includes all embodiments and variations substantially as hereinbefore described and with reference to the examples and drawings. Titles, headings, or the like are provided to enhance the reader’s comprehension of this document, and should not be read as limiting the scope of the present invention.REFERENCES

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Claims

WHAT IS CLAIMED IS:

1. A compound having the structure:

2. A compound having the structure of formula (I) or formula (II), or salt thereof, Nle-cyclo(bis-indolo)-[Xaa1-His-D-Phe-Arg-Xaa5-Xaa6]-NH2 (I) (SEQ ID NO: 1), orNle-cyclo(tryptathio)-[Xaa1-His-D-Phe-Arg-Xaa5-Xaa6]-NH2 (II) (SEQ ID NO: 2), wherein:Xaa1is Trp, D-Trp, Cys, D-Cys, Pen, or HomoCys;Xaa5is 2-Nal, Trp, or D-Trp;Xaa6is Trp, D-Trp, Cys, or D-Cys; one or more amino acid residues of the compound is optionally alpha N-methylated; and the compound is optionally N-terminally acylated.

3. A pharmaceutical composition comprising a compound of claim 1 or 2 and a pharmaceutically acceptable carrier or vehicle.

4. A compound comprising a melanocortin 1 receptor (MC1R) targeting peptide (MC1RTP), a radiolabeling group, and a linker joining the MC1R targeting compound to the radiolabeling group, wherein: the MC1RTP is a compound of claim 1 or 2, or a salt thereof; the MC1RTP is optionally C-terminally amidated; and one or more amino acid residues of the MC1RTP is optionally alpha N- methylated.

5. The compound of claims 4, wherein the linker comprises an albumin binding group.

6. The compound of claim 4 or 5, wherein the radiolabeling group is conjugated to a radioisotope.

7. The compound of any one of claims 4-6, wherein the linker is:

8. The compound of any one of claims 5-7, wherein the albumin-binding group has the following structure:wherein each R4is independently H, halogen, C1-5 alkyl, C1-5 alkoxy, or nitro.

9. The compound of any one of claims 4-7, wherein the linker is:H10. The compound of any one of claims 4-7, wherein the linker is:the albumin-binding group has the following structure:wherein each R4is independently H, halogen, C1-5 alkyl, C1-5 alkoxy, or nitro.

11. The compound of any one of claims 5-8 and 10, wherein the albumin-binding group is #-| 4-( / ?-toly I )butanoy 11.

12. The compound of any one of claims 1 to 11, wherein the radiolabeling group comprises a radioisotope chelator.

13. The compound of claim 12, wherein the radioisotope chelator is selected from the group consisting of: DOTA; DOTAGA; NOTA; NOD AGA; NODASA; CB-DO2A; 3p-C-DEPA; TCMC; DO3A; DTPA and DTPA analogues optionally selected from CHX-A”-DTPA and 1B4M-DTPA; TETA; NOPO; Me-3,2-HOPO; CB-TE1A1P; CB-TE2P; MM-TE2A; DM-TE2A; sarcophagine and sarcophagine derivatives optionally selected from SarAr, SarAr-NCS, diamSar, AmBaSar, and BaBaSar; TRAP; AAZTA; DATA and DATA derivatives; macropa; H2dedpa, H4octapa, H4py4pa, H4Pypa, H2azapa, H5decapa, H4py4pa-phenyl-NCS, and other picolinic acid derivatives; CP256; PCTA; C-NETA; C-NE3TA; HBED; HBED-CC; SHBED; BCPA; CP256; YM103; desferrioxamine (DFO) and DFO derivatives; H6phospa; CROWN; HYNIC; and N4.

14. The compound of claim 12 or 13, wherein the radioisotope chelator is DOTA, macropa, or CROWN.

15. The compound of any one of claims 12-14, wherein the radioisotope chelator is conjugated to a radioisotope.16 The compound of claim 15, wherein the radioisotope is668Ga,61Cu,64Cu,67Ga, "mTc, '" in.114mIn,44Sc,47Sc,86Y,90Y,89Zr,90Nb,177Lu,117mSn,165Er,227Th,225Ac,213Bi, 212Bi,211AS,203Pb,212Pb,166HO,188Re,186Re,149Pm,159Gd,105Rh,109Pd,198Au,199Au, 175Yb,142Pr,149Tb,152Tb,155Tb, or161Tb.

17. The compound of claim 12 or 13, wherein the radioisotope chelator is DOTA and the compound is conjugated with177Lu,niIn,213Bi,68Ga,67Ga,159Gd,203Pb,212Pb,44Sc, 90Y,86Y,175Yb,166HO,211AS,44SC,47SC,149Pm,142Pr,225Ac,64Cu or67Cu.

18. The compound of claim 12 or 13, wherein the radioisotope chelator is macropa and the compound is conjugated with225Ac or227Th.

19. The compound of claim 12 or 13, wherein the radioisotope chelator is CROWN and the compound is conjugated with213Bi,177Lu,225Ac,149Tb,152Tb,155Tb,161Tb, or 227Th.

20. The compound of claim 12 or 13, wherein the compound is conjugated with68Ga, 61Cu,64Cu,67Ga, "mTc,mIn,44Sc,86Y,89Zr,90Nb,177Lu,117mSn,203Pb,188Re,186Re, 149Pm,105Rh,198Au or199Au.

21. The compound of claim 4, wherein the compound isr salt thereof, wherein the compound is optionally conjugated with a radioisotope.

22. The compound of claim 4, wherein the compound is23. The compound of claim 22, wherein Ga is68Ga.

24. A pharmaceutical composition comprising a compound of any one of claims 4-23 and a pharmaceutically acceptable carrier or vehicle.

25. A method for treating cancer, comprising administering the compound of any one of claims 4-24 or the composition of claim 3 or 24 to a subject in need thereof.

26. The method of claim 25, wherein the cancer is melanoma.

27. The method of claim 26, wherein the melanoma is metastatic cutaneous melanoma or metastatic uveal melanoma.

28. A method for positron emission tomography (PET) or single photon emission computed tomography (SPECT) imaging of melanoma, comprising administering a compound of claim any one of claims 4-20 and 23 to a subject and imaging the melanoma.

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