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Definitions

• the present invention relates to new melanocortin (MC) receptor ligands. These ligands preferably exhibit selectivity for the MC-4 and/or the MC-3 receptors relative to the other melanocortin receptors (in particular the MC-1 receptor).
• MC melanocortin
• Melanocortin peptides are natural peptide hormones in animals and man that bind to and stimulate MC receptors.
• melanocortins are ⁇ -MSH (melanocyte stimulating hormone), ⁇ -MSH, ⁇ -MSH, ACTH (adrenocorticotropic hormone) and their peptide fragments.
• MSH is mainly known for its ability to regulate peripheral pigmentation (Eberle 1988), whereas ACTH is known to induce steroidoneogenesis (Simpson and Waterman, 1988).
• the melanocortin peptides also mediate a number of other physiological effects.
• MC-1, MC-2, MC-3, MC-4 and MC-5 all couple in a stimulatory fashion to cAMP.
• the MC-2 receptor is the ACTH receptor, whereas the others constitute subtypes of MSH receptors.
• the MC-1 receptor is present on melanocytes and melanoma.
• the MC-2 receptor is present predominantly in the adrenal gland.
• the mRNA for the MC-3 receptor has been found in the brain, as well as in placental and gut tissues (Gantz et al. 1993a, Desarnaud et al. 1994, Roselli Rehfuss et al. 1993).
• the MC-4 receptor has been found primarily in the brain (Gantz et al. 1993b; Mountjoy et al 1994).
• the MC-5 receptor is expressed in the brain, as well as in several peripheral tissues (Chhajlani et al 1993; Gantz et al 1994; Griffon et al 1994; Labbu et al. 1994; Barrett et al. 1994; Fathi et al.1995). More recent data from humans indicate that all of the cloned MC-receptors have a wider tissue distribution (Chhajlani, 1996) than originally thought.
• the members of the melanocortin receptor family can be differentiated on the basis of their tissue distribution.
• Both the MC-4 and MC-3 receptors have been localized to the hypothalamus, a region of the brain believed to be involved in the modulation of feeding behavior.
• Compounds showing selectivity for the MC-4/MC-3 receptors have been shown to alter food intake following intracerebro ventricular and peripheral injection in rodents. Specifically, agonists have been shown to reduce feeding, while antagonists have been shown to increase feeding. See, Fan, W. et al., "Role of Melanocortinergic Neurons in Feeding and the Agouti Obesity Syndrome", Nature, 385(6612), pp. 165-8 (Jan. 9, 1997).
• the Applicants have discovered a class of compounds that surprisingly have high affinity for the MC-4 and/or the MC-3 receptor subtypes, and that are typically selective for these MC receptors relative to the other melanocortin receptor subtypes, particularly the MC-1 subtype. It is therefore an object of this invention to provide compounds that have affinity for the MC-4 and/or the MC-3 receptor subtypes. It is a further object of the invention to provide means for administration of said compounds to animals or man. Still other objects of the invention will be evident from the following disclosure of the invention.
• the invention relates to a class of compounds that are ligands for receptors of the MC-4 and/or the MC-3 subtype.
• the invention relates to a compound having a structure according to Formula (I):
• (A) X is selected from hydrogen, fluoro, aryloxy, acyloxy, OR , SR 1 , -NR ⁇ 1' and -CHOKER 1' , where R 1 and R 1 are independently selected from the group consisting of hydrogen, alkyl and acyl;
• each R 2 is independently selected from the group consisting of hydrogen, alkyl halo and heteroalkyl; or
• the R 2 bonded to the carbon atom that is bonded to X and Z 1 and an R 5 moiety can optionally join to form a carbocyclic or heterocyclic ring that is fused to phenyl ring J; or
• the R 2 bonded to the carbon atom that is bonded to Z 2 and Z 3 can optionally join with R 8 to form a carbocyclic or heterocyclic ring; or (e) . the R 2 bonded to the carbon atom that is bonded to Z 3 and D can optionally join with R 10 to form a carbocyclic or heterocyclic ring;
• each of Z 1 , Z 2 and Z 3 is independently selected from -OC(R 3 )(R 3a )-; -C(R 3 )(R 3a )0- S(0) ⁇ C(R 3 )(R 3a )-, where a is 0, 1 or 2; -C(R 3 )(R 3a )S(OV, where b is 0, 1 or 2 N(R 3e )C(R 3 )(R 3a )-; -C(R 3 )(R 3a )N(R 3e )-; -C(0)N(R 3d )-; -N(R 3d )C(0)-; -C(0)C(R 3 )(R 3a )- C(R 3 )(R 3a )C(0)-; -C(R 3 )(R 3a )C(0)-; -C(R 3 )(R 3a )C(0)-; -C(R 3 )(R 3a )C(
• each of R 3 , R 3a R 3b and R 3c when present, is independently selected from hydrogen, hydroxy, alkoxy, aryloxy, acyloxy, thiol, alkylthio, acylthio, arylthio, amino, alkylamino, acylamino, and alkyl;
• R 3d when present, is selected from hydrogen, alkyl and aryl;
• R 3e when present, is selected from hydrogen, alkyl, aryl and acyl;
• R 3f when present, is selected from hydrogen and alkyl; (D) p is 0, 1, 2, 3, 4 or 5; wherein
• each R 4 and R 4 is independently selected from hydrogen, alkyl, aryl, halo, hydroxy, alkoxy, amino and acylamino; (2) when p is greater than 1, two R 4 moieties, together with the carbon atoms to wliich they are bonded, can join to form a heterocycloalkyl, cycloalkyl or aryl ring; and
• the R 4 moieties on two adjacent carbon atoms can both be nil such that a double bond is formed between the two adjacent carbon atoms, or both the R 4 and R 4 moieties on two adjacent carbon atoms can all be nil such that a triple bond is formed between the two adjacent carbon atoms;
• R 5 represents the 5 substituents (i.e., positions 2-6) on phenyl ring J, wherein each R 5 is independently selected from hydrogen, hydroxy, halo, thiol, -OR 12 , -SR 12 , - S0 2 N(R 12 )(R 12' ), -N(R 12 )(R 12' ), alkyl, acyl, alkene, alkyne, cyano, nitro, aryl, heteroaryl, cycloalkyl, and heterocycloalkyl; where each R 12 and R 12 is independently selected from hydrogen, alkyl, acyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, and heterocycloalkyl; or two R 5 moieties can optionally join to form a carbocyclic or a heterocyclic ring that is fused to phenyl ring J;
• each R 6 and R 6 is independently selected from hydrogen, alkyl, aryl, halo, hydroxy, alkoxy, amino and acylamino;
• the R 6 moieties on two adjacent carbon atoms can be nil such that a double bond is formed between the two adjacent carbon atoms, or both the R 6 and R 6 moieties on two adjacent carbon atoms can all be nil such that a triple bond is formed between the two adjacent carbon atoms;
• Ar is an aryl or heteroaryl ring selected from the group consisting of phenyl, thiophene, furan, oxazole, thiazole, pyrrole and pyridine;
• R 7 represents all the substituents on ring Ar, wherein each R 7 is independently selected from hydrogen, halo, -NR 13 R 13 , alkyl, acyl, alkene, alkyne, cyano, nitro, aryl, heteroaryl, cycloalkyl, and heterocycloalkyl; where each R 13 and R 13 is independently selected from hydrogen, alkyl, acyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, and heterocycloalkyl; or two R 7 moieties can optionally join to form a carbocyclic or a heterocyclic ring fused to ring Ar;
• each R 8 and R 8 is independently selected from hydrogen, alkyl, halo, hydroxy, alkoxy and amino; (2) when r is greater than 1, two R 8 moieties, together with the carbon atoms to which they are bonded, can join to form a heterocycloalkyl, cycloalkyl or aryl ring; and
• R 8 moieties on two adjacent carbon atoms can be nil such that a double bond is formed between the two adjacent carbon atoms, or both the R 8 and R 8 moieties on two adjacent carbon atoms can all be nil such that a triple bond is formed between the two adjacent carbon atoms;
• each R 9 and R 9 is independently selected from hydrogen, alkyl, aryl, halo, hydroxy, alkoxy, amino and acylamino; (2) when s is greater than 1, two R 9 moieties, together with the carbon atoms to which they are bonded, can join to form a heterocycloalkyl, cycloalkyl or aryl ring; and (3) when s is greater than 1, the R 9 moieties on two adjacent carbon atoms can be nil such that a double bond is formed between the two adjacent carbon atoms, or both the R 9 and R 9 moieties on two adjacent carbon atoms can all be nil such that a triple bond is formed between the two adjacent carbon atoms;
• R 10 is selected from the group consisting of an optionally substituted bicyclic aryl ring and an optionally substituted bicyclic heteroaryl ring; and (M) D is independently selected from hydrogen, fluoro, hydroxy, thiol, acylthio, alkoxy, aryloxy, alkylthio, acyloxy, cyano, amino, acylamino, -C(0)R u and -C(S)R ; wherein R 11 is selected from the group consisting of hydroxy; alkoxy; amino; alkylamino; -
• L linking moiety
• the invention also relates to pharmaceutical compositions comprising the above compounds, and to methods of treating disorders mediated by the MC-3 or MC-4 receptor by administering these compounds.
• Amino acid refers to alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C), glutamic acid (Glu; Q), glutamine (Gin; E), glycine (Gly; G), histidine (His; H), isoleucine (lie; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).
• Alkoxy is an oxygen radical having a hydrocarbon chain substituent, where the hydrocarbon chain is an alkyl or alkene (i.e., -O-alkyl or -O-alkene). Preferred alkoxy groups include (for example) methoxy (MeO), ethoxy, propoxy and allyloxy.
• Alkyl is a saturated hydrocarbon chain having 1 to 15 carbon atoms, preferably 1 to 10, more preferably 1 to 4 carbon atoms.
• Alkene is a hydrocarbon chain having at least one (preferably only one) carbon-carbon double bond and having 2 to 15 carbon atoms, preferably 2 to 10, more preferably 2 to 4 carbon atoms.
• Alkyne is a hydrocarbon chain having at least one
• Alkyl, alkene and alkyne chains may be straight or branched and may be unsubstituted or substituted. Preferred branched alkyl, alkene and alkyne chains have one or two branches, preferably one branch. Preferred chains are alkyl. Alkyl, alkene and alkyne hydrocarbon chains each may be unsubstituted or substituted with from 1 to 4 substituents; when substituted, preferred chains are mono-, di-, or tri-substituted.
• Alkyl, alkene and alkyne hydrocarbon chains each may be substituted with halo, hydroxy, aryloxy (e.g., phenoxy), heteroaryloxy, acyloxy (e.g., acetoxy), carboxy, aryl (e.g., phenyl), heteroaryl, cycloalkyl, heterocycloalkyl, spirocycle, amino, amido, acylamino, keto, thioketo, cyano, or any combination thereof.
• Preferred hydrocarbon groups include methyl (Me), ethyl, propyl, isopropyl, butyl, vinyl, allyl and butenyl.
• a “lower” alkyl, alkene or alkyne moiety is a chain comprised of 1 to 6, preferably from 1 to 4, carbon atoms in the case of alkyl and 2 to 6, preferably 2 to 4, carbon atoms in the case of alkene and alkyne.
• Alkylthio is a sulfur radical having a hydrocarbon chain substituent, where the hydrocarbon chain is an alkyl or alkene (i.e., -S-alkyl or -S-alkene).
• Preferred alkylthio groups include (for example) methylthio (MeS) and ethylthio.
• Aryl is an aromatic hydrocarbon ring.
• Aryl rings are monocyclic or fused bicyclic ring systems.
• Monocyclic aryl rings contain 6 carbon atoms in the ring.
• Monocyclic aryl rings are also referred to as phenyl rings.
• Bicyclic aryl rings contain from about 8 to about 17 carbon atoms, preferably about 9 to about 12 carbon atoms in the ring.
• Bicyclic aryl rings include ring systems wherein one ring is aryl and the other ring is aryl, cycloalkyl, or heterocycloalkyl.
• Preferred bicyclic aryl rings comprise 5-, 6- or 7-membered rings fused to 5-, 6-, or 7-membered rings.
• Aryl rings may be unsubstituted or substituted with from 1 to 4 substituents on the ring.
• Aryl may be substituted with halo, cyano, nitro, hydroxy, carboxy, amino, acylamino, alkyl, heteroalkyl, haloalkyl, phenyl, aryloxy, heteroaryloxy, or any combination thereof.
• Preferred aryl rings include naphthyl, tolyl, xylyl, and phenyl. The most preferred aryl ring radical is phenyl.
• Aryloxy is an oxygen radical having an aryl substituent (i.e., -O-aryl).
• Preferred aryloxy groups include (for example) phenoxy, naphthyloxy, methoxyphenoxy, and methylenedioxyphenoxy.
• basic amino acids refers to His, Lys, and Arg.
• Cycloalkyl is a saturated or unsaturated hydrocarbon ring. Cycloalkyl rings are not aromatic. Cycloalkyl rings are monocyclic, or are fused, spiro, or bridged bicyclic ring systems. Monocyclic cycloalkyl rings contain from about 3 to about 9 carbon atoms, preferably from 3 to 7 carbon atoms in the ring. Bicyclic cycloalkyl rings contain from 7 to 17 carbon atoms, preferably from about 7 to about 12 carbon atoms in the ring. Preferred bicyclic cycloalkyl rings comprise 4-, 5-, 6- or 7-membered rings fused to 5-, 6-, or 7-membered rings.
• Cycloalkyl rings may be unsubstituted or substituted with from 1 to 4 substituents on the ring. Cycloalkyl may be substituted with halo, cyano, alkyl, heteroalkyl, haloalkyl, phenyl, keto, hydroxy, carboxy, amino, acylamino, aryloxy, heteroaryloxy, or any combination thereof. Preferred cycloalkyl rings include cyclopropyl, cyclopentyl, and cyclohexyl.
• fused refers to cyclic moieties having at least two common ring atoms, the preferred maximum number of fused cycles being three.
• Halo is fluoro (F), chloro (Cl), bromo (Br) or iodo (I).
• Heteroatom is a nitrogen, sulfur, or oxygen atom, to which one or more moieties may be connected according to heteroatom valence; in the case of nitrogen, one oxygen atom may be optionally connected to it by a coordinate covalent bond, such as forming an N-oxide. Groups containing more than one heteroatom may contain different heteroatoms.
• Heteroalkyl is a saturated or unsaturated chain containing carbon and at least one heteroatom, wherein no two heteroatoms are adjacent. Heteroalkyl chains contain from 2 to about 15 member atoms (carbon and heteroatoms) in the chain, preferably 2 to about 10, more preferably 2 to about 5. For example, alkoxy (i.e., -O-alkyl or -O-heteroalkyl) radicals are included in heteroalkyl. Heteroalkyl chains may be straight or branched. Preferred branched heteroalkyl have one or two branches, preferably one branch. Preferred heteroalkyl are saturated.
• Unsaturated heteroalkyl have one or more double bonds (also referred to herein as "heteroalkenyl") and/or one or more triple bonds (also referred to herein as “heteroalkynyl”).
• Preferred unsaturated heteroalkyl have one or two double bonds or one triple bond, more preferably one double bond.
• Heteroalkyl chains may be unsubstituted or substituted with from 1 to 4 substituents.
• Preferred substituted heteroalkyl are mono-, di-, or tri-substituted.
• Heteroalkyl may be substituted with lower alkyl, halo, hydroxy, aryloxy, heteroaryloxy, acyloxy, carboxy, monocyclic aryl, heteroaryl, cycloalkyl, heterocycloalkyl, spirocycle, amino, acylamino, amido, keto, thioketo, cyano, or any combination thereof.
• Heterocycloalkyl is a saturated or unsaturated, non-aromatic ring containing carbon and from 1 to about 4 (preferably 1 to 3) heteroatoms in the ring, wherein no two heteroatoms are adjacent in the ring and no carbon in the ring that has a heteroatom attached to it also has a hydroxyl, amino, or thiol radical attached to it.
• Heterocycloalkyl rings are monocyclic, or are fused, bridged, or spiro bicyclic ring systems.
• Monocyclic heterocycloalkyl rings contain from about 4 to about 9 member atoms (carbon and heteroatoms), preferably from 5 to 7 member atoms in the ring.
• Bicyclic heterocycloalkyl rings contain from about 7 to about 17 atoms, preferably from 7 to 12 atoms. Bicyclic heterocycloalkyl rings may be fused, spiro, or bridged ring systems. Preferred bicyclic heterocycloalkyl rings comprise 5-, 6- or 7-membered rings fused to 5-, 6-, or 7-membered rings. Heterocycloalkyl rings may be unsubstituted or substituted with from 1 to 4 substituents on the ring.
• Heterocycloalkyl may be substituted with halo, cyano, hydroxy, carboxy, keto, thioketo, amino, acylamino, acyl, amido, alkyl, heteroalkyl, haloalkyl, phenyl, phenoxy or any combination thereof.
• Preferred substituents on heterocycloalkyl include fluoro and alkyl.
• Heteroaryl is an aromatic ring containing carbon and from 1 to about 4 heteroatoms in the ring. Heteroaryl rings are monocyclic or fused bicyclic ring systems. Monocyclic heteroaryl rings contain from about 5 to about 9 member atoms (carbon and heteroatoms), preferably 5 or 6 member atoms in the ring. Bicyclic heteroaryl rings contain from about 8 to about 17 member atoms, preferably about 8 to about 12 member atoms in the ring. Bicyclic heteroaryl rings include ring systems wherein one ring is heteroaryl and the other ring is aryl, heteroaryl, cycloalkyl, or heterocycloalkyl.
• Preferred bicyclic heteroaryl ring systems comprise 5-, 6- or 7- membered rings fused to 5-, 6-, or 7-membered rings.
• Heteroaryl rings may be unsubstituted or substituted with from 1 to 4 substituents on the ring.
• Heteroaryl may be substituted with halo, cyano, nitro, hydroxy, carboxy, amino, acylamino, alkyl, heteroalkyl, haloalkyl, phenyl, aryloxy, heteroaryloxy, or any combination thereof.
• Preferred heteroaryl rings include thienyl, thiazolo, imidazyl, purinyl, pyrimidyl, pyridyl, and furanyl.
• MC-4 agonist and “MC-3 agonist” refers to a compound with affinity for the MC-4 receptor or MC-3 receptor, respectively, that results in measurable biological activity in cells, tissues, or organisms which contain the MC-4 or MC-3 receptor.
• an "MC-4/MC-3 agonist” refers to a compound that is both an MC-4 agonist and an MC-3 agonist, as those terms are defined herein. Assays which demonstrate MC-4 and/or MC-3 agonistic activity of compounds are well known in the art.
• EIA BioTrak TM cAMP direct enzymeimmunoassay
• Tropix cAMP ScreenTM Another useful assay. These systems allow the simple quantitation of total cellular cAMP measurement in cells exposed to selective ligands. Briefly summarized: HEK cells stably transfected with the MC-1, MC-3 or MC-4 receptors are plated into 96 well microtiter plates and grown overnight. Cells are dosed with the appropriate MC ligand for 1 hour and then lysed.
• a fraction of the lysed cell extract is transferred to the assay plate.
• the ELISA assay is performed according to kit instructions.
• Each plate contains a series of cAMP standards for calculating a standard curve, as well as a full MC agonist as a positive control for each MC receptor.
• cAMP activity is calculated as a % of the maximum cAMP activity of the full MC agonist control.
• MC-4 antagonist and “MC-3 antagonist” refer to compounds with affinity for the MC-4 receptor or MC-3 receptor, respectively, and blocks stimulation by a known MC agonist.
• an "MC-4/MC-3 antagonist” refers to a compound that is both an MC-4 antagonist and an MC-3 antagonist, as those terms are defined herein.
• HEK cells stably transfected with the MC-1, MC-3, MC-4 or MC-5 receptors are plated into 96-well microtiter plates and grown overnight. Cells are dosed with the appropriate MC ligand in the presence of europilated-NDP-MSH for 60 min., cells are washed several times, enhancement solution is added and fluorescence is measured. IC 50 and Ki values can be calculated at each receptor for each MC ligand using standard graphing programs such as GraphPad PrismTM, (GraphPad Software Inc., San Diego, CA).
• MC-3 receptor and MC-4 receptor mean the known MC-3 and MC-4 receptors, their splice variants, and undescribed receptors.
• MC-3 receptors are described by Gantz et al., supra (human MC-3); Desarnaud et al., supra (mouse MC-3) and L. Reyfuss et al., "Identification of a Receptor for Gamma Melanotropin and Other Proopiomelanocortin Peptides in the Hypothalamus and Limbic System., Proc. Natl. Acad. Sci. USA, vol. 90, pp. 8856-8860 (1993) (rat MC-3).
• MC-4 receptors are described by Gantz et al., supra (human MC-4), J.D. Alvaro et al., "Morphine Down-Regulates Melanocortin-4 Receptor Expression in Brain Regions that Mediate Opiate Addiction", Mol-Pharmacol. Sep, vol. 50(3), pp. 583-91 (1996) (rat MC-4) and Takeuchi, S. and Takahashi, S., "Melanocortin Receptor Genes in the Chicken-Tissue Distributions", Gen-Comp-Endocririol., vol. 112(2), pp 220-31 (Nov. 1998) (chicken MC-4).
• measurable means the biologic effect is both reproducible and significantly different from the baseline variability of the assay.
• a "pharmaceutically-acceptable salt” is a cationic counterion formed at any acidic (carboxylic acid) group, or an anionic counterion formed at any basic (e.g., amino) group.
• Preferred cationic salts include the alkali metal salts (such as sodium and potassium), and alkaline earth metal salts (such as magnesium and calcium) and organic salts.
• Preferred anionic salts include the halides (such as chloride salts), sulfonates, carboxylates, phosphates, trifluoroacetate (TFA) and the like.
• addition salts that may provide an optical center where once there is none.
• a chiral tartrate salt may be prepared from the compounds of the invention, and this definition includes such chiral salts.
• Such salts are well understood by the skilled artisan, and the skilled artisan is able to prepare any number of salts given the knowledge in the art. Furthermore, it is recognized that the skilled artisan may prefer one salt over another for reasons of solubility, stability, formulation ease and the like. Determination and optimization of such salts is within the purview of the skilled artisan's practice.
• selective means having an activation preference for a specific receptor over other receptors which can be quantified based upon whole cell, tissue, or organism assays which demonstrate receptor activity, such as the cAMP enzyme immunoassay (EIA) system discussed above.
• EIA enzyme immunoassay
• a compound's selectivity is detemiined from a comparison of its EC 5 0 values at the relevant receptors being referenced.
• use of the term "selective over the other MC receptors” means selective with respect to the other melanocortin receptors, including the MC-1, MC-2 and MC-5 receptors.
• a compound having an EC 50 of 8 nM at the MC-4 receptor and an EC 50 of > 80 nM at the MC-1, MC-2 and MC-5 receptors has a selectivity ratio for the MC-4 receptor over the other MC receptors of at least 1: 10.
• selectivity may also refer to one of the MC-1, MC-2 or MC-5 receptors individually.
• a compound having an EC 50 of 8 nM at the MC-4 receptor and an EC 50 of 80 nM at the MC-1 receptor has a selectivity ratio for the MC-4 receptor over the MC-1 receptor of 1: 10.
• Such a compound is selective over the MC-1 receptor, regardless of its EC 50 value for MC-2 or MC-5. Selectivity is described in more detail below and may be determined by using, for example, the software Prism v 2.0 which is availablp from GraphPad, Inc.
• a “solvate” is a complex formed by the combination of a solute (e.g., a MC-4/MC-3 receptor ligand of the present invention) and a solvent (e.g., water).
• a solute e.g., a MC-4/MC-3 receptor ligand of the present invention
• a solvent e.g., water
• Pharmaceutically-acceptable solvents used according to this invention include those that do not interfere with the biological activity of the compound (e.g., water, ethanol, ethers, acetic acid, N,N-dimethylformamide and others known or readily determined by the skilled artisan).
• “Spirocycle” is an alkyl or heteroalkyl diradical substituent of an alkyl or heteroalkyl, wherein said diradical substituent is attached geminally and wherein said diradical substituent forms a ring, said ring containing about 4 to about 8 member atoms (carbon or heteroatoms), preferably 5 or 6 member atoms.
• the compounds of the present invention are MC-4 and/or MC-3 receptor ligands having a structure according to Formula (I):
• R 2 , R 4 , R 4' , R 5 , R 6 , R 6' , R 7 , R 8 , R 8' , R 9 , R 9' , R 10 , Ar, Z 1 , Z 2 , Z 3 , X, B, D, p, q, r and s are as described in the Disclosure of the Invention section above.
• the core peptidic residues can be pegylated to provide enhanced therapeutic benefits such as, for example, increased efficacy by extending half-life in vivo. Peptide pegylation methods are well known in the literature.
• pegylation of peptides is described in the following references, the disclosure of each of which is incorporated herein by reference: Lu, Y.A. et al., "Pegylated peptides. H Solid-phase synthesis of amino-, carboxy- and side-chain pegylated peptides", Int. J. Pept. Protein Res., Vol. 43(2), pp. 127-38 (1994); Lu, Y.A. et al., "Pegylated peptides. I. Solid- phase synthesis of N alpha-pegylated peptides using Fmoc strategy", Pept. Res., Vol. 6(3), pp. 140-6 (1993); Felix, A.M.
• X is selected from hydrogen, fluoro, aryloxy, acyloxy, OR 1 , SR 1 , -N ⁇ 1 and -CBOR R 1 .
• Preferred are hydrogen (when D is not also hydrogen), -NR ⁇ 1 and -CBDR R 1 . More preferred are -NR ⁇ 1' and -CHR 1 ⁇ ' . Still more preferred is -NR .
• R 1 and R 1 are independently selected from the group consisting of hydrogen, alkyl and acyl. Preferred is where R 1 is hydrogen or alkyl and R 1 is acyl. .
• R 2 is independently selected from the group consisting of hydrogen, alkyl halo and heteroalkyl. Preferred is hydrogen.
• two consecutive R 2 moieties, or consecutive R 2 and R 3 moieties may join to form a 3 to 8 membered carbocyclic or heterocyclic ring.
• the R 2 bonded to the carbon atom that is bonded to X and Z 1 and an R 5 moiety can optionally join to form a carbocyclic or heterocyclic ring that is fused to phenyl ring J.
• the R 2 bonded to the carbon atom that is bonded to ring Ar can join with an R 7 to form a ring fused to ring Ar.
• R 2 bonded to the carbon atom that is bonded to Z 2 and Z 3 can optionally join with R 8 to form a carbocyclic or heterocyclic ring.
• R 2 bonded to the carbon atom that is bonded to Z 3 and D can optionally join with R 10 to form a carbocyclic or heterocyclic ring.
• R 2 does not form a ring with another R 2
• R 2 does not form a ring with R 3 , R 7 or R 8
• More preferred is where R 2 also does not form a ring with R 10 .
• rings formed between R 2 and another moiety will have 5 to 8 ring atoms.
• Each of Z 1 , Z 2 and Z 3 is independently selected from -OC(R 3 )(R 3a )-; -C(R 3 )(R 3a )0-; - S(0) a C(R 3 )(R 3a )-, where a is 0, 1 or 2; -C(R 3 )(R 3a )S(0) a -, where b is 0, 1 or 2; -N(R 3e )C(R 3 )(R 3a )-; - C(R 3 )(R 3a )N(R 3e ); -C(0)N(R 3d )-; -N(R 3d )C(0)-; -C(0)C(R 3 )(R 3a )-; -C(R 3 )(R 3a )C(0)-; - C(R 3 )(R 3a )C(0)-; - C(R 3 )(R 3a )C(0)-; - C(R 3
• Preferred are -OC(R 3 )(R 3a )-; -C(R 3 )(R 3a )0-; - S(0) « C(R 3 )(R 3a )-, where a is 2; -C(R 3 )(R 3a )S(0) ⁇ -, where b is 2; -N(R 3e )C(R 3 )(R 3a )-; - C(R 3 )(R 3a )N(R 3e )-; -C(O)N(R 3d )-; -N(R 3d )C(0)-; -C(R 3 )(R 3a )C(R 3b )(R 3c )-; -C(R 3 ) C(R 3a )-; -C ⁇ C-; - S0 2 N(R 3d )-; -N(R 3d )S0 2 -; -C(R 3 )(R 3
• preferred compounds are those where at least one of Z 1 , Z 2 or Z 3 is other than -C(0)N(R 3d )-. More preferred are compounds where at least two of Z 1 , Z 2 or Z 3 are other than -C(0)N(R 3d )-.
• R 3 , R 3a R 3b and R 3c when present, is independently selected from hydrogen, hydroxy, alkoxy, aryloxy, acyloxy, thiol, alkylthio, acylthio, arylthio, amino, alkylamino, acylamino, and alkyl.
• Preferred are hydrogen, hydroxy, alkoxy, aryloxy and alkyl. Most preferred is where each of R 3 , R 3a R 3b and R 3c is hydrogen.
• R 3d when present, is selected from hydrogen, alkyl and aryl. Preferred is where R 3d is selected from hydrogen and alkyl.
• R 3e when present, is selected from hydrogen, alkyl, aryl and acyl. Preferred is where R 3e is selected from hydrogen and alkyl
• R R 33ff i iss sseelleecctteed from hydrogen and alkyl.
• R 3f is alkyl
• p is 0, 1, 2, 3, 4 or 5.
• Preferably p is 1 or 2, more preferably 1.
• each R 4 and R 4 is independently selected from hydrogen, alkyl, aryl, halo (preferably fluoro), hydroxy, alkoxy, amino and acylamino.
• two R 4 moieties, together with the carbon atoms to which they are bonded, may join to form a heterocycloalkyl, cycloalkyl or aryl ring.
• the R 4 moieties on two adjacent carbon atoms can both be nil such that a double bond is formed between the two adjacent carbon atoms, or both the R 4 and R 4 moieties on two adjacent carbon atoms can all be nil such that a triple bond is formed between 'the two adjacent carbon atoms.
• each R 4 when present, is hydrogen and each R , when present, is hydrogen or alkyl. Most preferably there is no unsaturation in the chain linking ring J to the X-containing carbon atom of Formula (I).
• R represents the 5 substituents (i.e., positions 2-6) on phenyl ring J, wherein each R is independently selected from hydrogen, hydroxy, halo, thiol, -OR 12 , -SR 12 , -S0 2 N(R 12 )(R 12' ), - N(R 12 )(R 12 ), alkyl, acyl, alkene, alkyne, cyano, nitro, aryl, heteroaryl, cycloalkyl, and heterocycloalkyl.
• Each R 12 and R 12 is independently selected from hydrogen, alkyl, acyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, and heterocycloalkyl; or two R 5 moieties can optionally join to form a carbocyclic or a heterocyclic ring that is fused to phenyl ring J.
• R 5 moieties are hydrogen, hydroxy, halo, thiol, -OR 12 where R 12 is lower alkyl or acyl, -SR 12 where R 12 is lower alkyl or acyl, -S0 2 N(R 12 )(R 12' ), -N(R 12 )(R 12' ), alkyl, cyano, nitro, aryl, heteroaryl, cycloalkyl, and heterocycloalkyl.
• R 5 moieties are hydrogen, hydroxy, halo, thiol, -S0 2 N(R 12 )(R 12' ) where R 12 and R 12' both are hydrogen, -N(R 12 )(R 12' ) where R 12 and R 12 each are hydrogen or alkyl. Still more preferred R 5 moieties are hydrogen, hydroxy, cchhlloorroo,, flfluuoorroo,, --NN((RR 1122 ))((RR 1122' )) wwhheerree RR 1122 aanndd RR 1122' eeaacchh aaire hydrogen or alkyl. Most preferred R : 5 moieties are hydrogen, hydroxy, chloro, fluoro and nitro.
• R 5 moieties are hydrogen.
• the 4-position is other than hydrogen. Most preferred is where the 4-position is other than hydrogen and the remaining 4 substituents are hydrogen.
• q is 0, 1, 2, 3, 4 or 5.
• q is 0, 1 or 2, more preferably q is 1.
• each R 6 and R 6 is independently selected from hydrogen, alkyl, aryl, halo (preferably fluoro), hydroxy, alkoxy, amino and acylamino.
• q is greater than 1
• two R 6 moieties, together with the carbon atoms to which they are bonded, can join to form a heterocycloalkyl, cycloalkyl or aryl ring.
• the R 6 moieties on two adjacent carbon atoms can be nil such that a double bond is formed between the two adjacent carbon atoms, or both the R 6 and R 6 moieties on two adjacent carbon atoms can all be nil such that a triple bond is formed between the two adjacent carbon atoms.
• each R 6 when present, is hydrogen and each R ⁇ , when present, is hydrogen or alkyl. Most preferably there is no unsaturation in the chain linking the ring Ar to the carbon atom of Formula (I) that is bonded to Z 1 and Z 2 .
• Ar is an aryl or heteroaryl ring selected from the group consisting of phenyl, thiophene, furan, oxazole, thiazole, pyrrole and pyridine.
• Ar is preferably phenyl, thiophene or furan.
• Ar is most preferably phenyl.
• R 7 represents the substituents on the Ar ring, wherein each R 7 is independently selected from hydrogen; halo; -NR R where R and R each are hydrogen or alkyl; alkyl; acyl; alkene; alkyne; cyano; nitro; aryl; heteroaryl; cycloalkyl and heterocycloalkyl.
• R and R each are hydrogen or alkyl; alkyl; acyl; alkene; alkyne; cyano; nitro; aryl; heteroaryl; cycloalkyl and heterocycloalkyl.
• two R 7 moieties can join to form a carbocyclic or a heterocyclic ring fused to ring Ar.
• Ar is phenyl
• R 7 moieties are selected from fluoro, chloro, cyano, bromo, iodo, nitro, alkoxy and alkyl; or two R 7 moieties join to form a carbocyclic or a heterocyclic ring fused to the phenyl ring. More preferred is where the 4-position of the phenyl ring is hydrogen, fluoro, chloro, cyano,- bromo, iodo, nitro and alkyl and the remaining four positions are hydrogen. Most preferred is where the 4-position of the phenyl ring is hydrogen or fluoro and the remaining four substituents are hydrogen.
• Z 1 and Z 2 are both -C(0)N(R 3d )-, peferred are compounds where the carbon atom that is bonded to Z 1 and Z 2 is assigned an R configuration according to Cahn-Ingold-Prelog rules of nomenclature.
• r is 0, 1, 2, 3, 4, 5, 6 or 7.
• Preferred r is 2, 3, 4 or 5. More preferred r is 2, 3 or 5.
• Most preferred r is 3.
• Each R 8 and R 8 is independently selected from hydrogen, alkyl, halo (preferably fluoro), hydroxy, alkoxy and amino.
• each R 8 and R 8 is independently selected from hydrogen and alkyl. Most preferably, each R 8 and R 8 is hydrogen.
• the R 8 moieties on two adjacent carbon atoms can be nil such that a double bond is formed between the two adjacent carbon atoms, or both the R 8 and R 8 moieties on two adjacent carbon atoms can all be nil such that a triple bond is formed between the two adjacent carbon atoms.
• thiophene an alkyl or dialkyl amine, a heteroaryl ring containing at least one ring nitrogen atom and a heterocycloalkyl ring containing at least one ring nitrogen atom.
• R 14 and R 15 are independently selected from hydrogen, alkyl, alkene, and alkyne.
• R 16 and R 17 are independently selected from hydrogen, alkyl, alkene, and alkyne. Preferred are hydrogen and alkyl.
• R 20 and R 21 are independently selected from hydrogen, alkyl, alkene, and alkyne. Preferred are hydrogen and alkyl.
• R 14 , R 15 , R 16 and R 17 combine to form a monocyclic or bicyclic ring.
• R 14 and R 15 together with the atoms to which they are bonded, can join to form a heterocycloalkyl or a heteroaryl.
• R 14 and R 16 together with the atoms to which they are bonded, can join to form a heterocycloalkyl or a heteroaryl.
• R 15 and R 16 together with the atoms to which they are bonded, can join to form a heterocycloalkyl or a heteroaryl.
• R 16 and R 17 can optionally join to form a heteroaryl or heterocycloalkyl ring.
• Preferred is where R 15 and R 16 join to form a ring.
• s is 0, 1, 2, 3, 4 or 5.
• s is 1 or 2, more preferably 1.
• each R 9 and R 9 ' is independently selected from hydrogen, alkyl, aryl, halo (preferably fluoro), hydroxy, alkoxy, amino and acylamino.
• each R 9 when present, is hydrogen and each R 9 , when present, is hydrogen or alkyl.
• the R 9 moieties on two adjacent carbon atoms can be nil such that a double bond is formed between the two adjacent carbon atoms.
• both the R 9 and R 9 moieties on two adjacent carbon atoms can all be nil such that a triple bond is formed between the two adjacent carbon atoms. Most preferably there is no unsaturation in the chain linking R 10 to the D- containing carbon atom of Formula (I).
• R 10 is selected from the group consisting of an optionally substituted bicyclic aryl ring and an optionally substituted bicyclic heteroaryl ring.
• Preferred bicyclic aryl rings include 1- naphthyl, 2-naphthyl, indan, lH-indene, benzocylcobutane and benzocylcobutene.
• Preferred bicyclic heteroaryl rings include indole, indoline, pyrindine, dihydropyrindine, octahydropyrindine, benzothiophene, benzofuran, benzimidozole, benzopyran, quinoline, quinolone and isoquinoline.
• R 10 is 1-naphthyl, 2-naphthyl, indole, indan, lH-indene, benzothiophene, benzofuran and benzopyran. Most preferred is where R 10 is 1- naphthyl, 2-naphthyl or indole (particularly 3-indole).
• D is selected from hydrogen, fluoro, hydroxy, thiol, alkoxy, aryloxy, alkylthio, acyloxy, cyano, amino, acylamino, -C(0)R u and -C(S)R U .
• Preferred are fluoro, hydroxy, thiol, alkoxy, aryloxy, alkylthio, acyloxy, cyano, amino, acylamino, -C(0)R n and -C(S)R ⁇ .
• More preferred are alkoxy, cyano, amino, acylamino, -C(0)R u and -C(S)R n .
• Still more preferred are -C(0)R u and - C(S)R ⁇ .
• Most preferred is -C(0)R".
• R 11 are amino; alkylamino; -NHOR 18 , where R 18 is selected from hydrogen and alkyl (preferably hydrogen); -N(R 19 )CH 2 C(0)NH 2 , where R 19 is alkyl (preferably lower alkyl); -N ⁇ CH 2 CH 2 OH; and -N(CH 3 )CH 2 CH 2 OH. More preferred R 11 are amino; alkylamino; -NHOR 18 , where R 18 is selected from hydrogen and alkyl (preferably hydrogen); and -N(R 19 )CH 2 C(O)NH 2 , where R 19 is alkyl. Most preferred are amino and alkylamino.
• X and D may optionally be linked together via a linking moiety, L, that contains all covalent bonds or covalent bonds and an ionic bond so as to form a cyclic peptide analog.
• L linking moiety
• the bridge connecting X and D can be in the form of covalent bond linkages or alternatively can include a salt bridge resulting from the formation of ionic bonds.
• the linking moiety can be wholly peptidic in nature (i.e., containing amino acids only), non-peptidic (i.e., containing no amino acids) in nature, or it can include both peptidic and non-peptidic moieties introduced using well-known chemistry.
• the linking moiety can comprise aliphatic residues, aromatic residues or heteroaromatic residues, or any combination thereof.
• the linking moiety will preferably comprise long chain omega-amino acids in wliich amino and carboxyl groups are separated by from about 4 to about 24 methylene groups or a combination of said omega-amino acids and aminobenzoic acids.
• the linking moiety will contain all covalent bonds, such as amide bonds.
• the linking moiety can comprise an amide formed through the chemical coupling of a side-chain amino group of amino acids such as Lys or Orn, and a side-chain carboxyl group of the amino acid residue such as Asp or Glu.
• the linking moiety can comprise an amide formed between the amino and carboxylate groups attached to the ⁇ -carbon of the bridging moiety amino acids.
• the linking moiety can comprise an amide formed between any combination of the side-chain amino group or side-chain carboxyl group and the ⁇ -amino and the ⁇ -carboxyl moieties.
• the linking residues may be amine- or carboxyl-containing structures other than natural amino acids, including, e.g., 6-aminohexanoic acid as an amine-containing residue and succinic acid as a carboxyl-containing residue.
• the invention allows for linking using other types of chemical functionalities.
• linking residues may contain a variety of groups and substituents, including aliphatic, heteroalkyl, aromatic and heterocyclic moieties.
• the linking moiety can include but is not limited to amide, ester, ether, thioether, aminoalkyl, aminoaryl, alkyl, other heteroalkyl, alkene, alkyne, heterocycloalkyl, aryl, and heteroaryl.
• the linking moiety can include ether, aminoalkyl, aminoaryl, alkyl, other heteroalkyl, alkene, alkyne, heterocycloalkyl, aryl, and heteroaryl.
• the linking moiety can include ether, aminoalkyl, alkyl, alkene, and alkyne.
• L contains only covalent bonds, preferred are compounds having from about 12 to about 32 ring atoms, more preferred are compounds having from about 22 to about 28 ring atoms.
• the linking moiety can alternatively include an ionic bond/association that favors a cyclic structure.
• This "ionic" bridge is comprised of salt-forming basic and acid functionalities.
• the link can comprise an ionic bond formed between the side-chain amino group of amino acids such as Lys or Om, and the side-chain carboxyl group of the amino acid residue such as Asp or Glu.
• the linking moiety can comprise an ionic bond formed between the amino and carboxylate groups attached to the ⁇ -carbon of the linking moiety amino acids.
• the linking moiety may comprise an amide formed between any combination of the side-chain amino group or side-chain carboxyl and the ⁇ -amino and the ⁇ -carboxyl moieties.
• L contains an ionic bond
• the ring formed will preferably contain from about 22 to about 28 ring atoms.
• any free peptidic ⁇ -carboxy and ⁇ -amino groups i.e., amino acid ⁇ -carboxy and ⁇ -amino groups
• the most preferred L- containing compounds are analogs wherein X and D form covalently bonded cyclic structures.
• alkyl, heteroalkyl, cycloalkyl, and heterocycloalkyl groups may be substituted with hydroxy, amino, and amido groups as stated above, the following are not envisioned in the invention: 1. Enols (OH attached to a carbon bearing a double bond).
• a preferred subclass of compounds of Formula (I) wherein there is no linking moiety L to form a macrocyclic ring are compounds having a structure of Formula (A) as follows:
• the moieties R 1 , R 1 , Z 1 , R 4 , R 4 , R 5 , R 6 , R 6 , R 7 , B, R 10 and R 11 are as defined with respect to Formula (I).
• the compounds of Formula (A) are those where ring J of Formula is a phenyl ring wherein all of positions 2, 3, 5 and 6 are hydrogen, such that the ring is only substituted at the 4-position with the R 5 moiety, which is as defined with respect to Formula (I).
• the R 5 ring moiety and the R 2 substituent can optionally join to form a ring fused to the depicted phenyl ring.
• the fused ring may join the phenyl ring at a position other than the 4-position.
• ring Ar of Formula (I) is a phenyl ring where all of positions 2', 3', 5' and 6' are hydrogen and position 4' is R 7 which is as defined above. In this regard, preferred are where R 7 is selected from hydrogen and fluoro.
• a preferred sub-class of compounds of Formula (II) are compounds having a structure according to Formula (B), as follows:
• R 9 , R 9' and R 10 are as defined above and p is 1 or 2.
• R 6 and R 6 are both hydrogen.
• R 8 , R 8' , R 9 and R 9 are all hydrogen.
• R 7 is selected from hydrogen and fluoro.
• the compounds of the present invention can be prepared using a variety of procedures, including solid phase and solution phase techniques.
• solid phase and solution phase techniques A general description of both the solid and solution phase techniques is set forth below.
• Section VII several representative examples are set forth for each of these synthetic techniques.
• the general Fmoc chemistry protocol for SPPS solid phase peptide synthesis
• HBTU 2-(lH-benzotriazol- l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate
• the acetyl or butyl group is added on the N-terminal side of the peptide after the full length of the peptide chain is made. It is accomplished by reaction of acetic anhydride or butyric anhydride (4.75% V:V acetic anhydride or butyric anhydride, 0.2% HOBt W:V, 2.25% DIEA in NMP) with the ⁇ -amino group on N- terminal side of residue. The final synthesis product is washed extensively with NMP and dichloromethane (DCM).
• DCM dichloromethane
• HATU 0-(7-azabenzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluoraphosphate
• the cleavage solution is separated from the resin by filtration.
• the peptide in the filtrate is then precipitated by adding 40 ml cold ether.
• Peptide precipitate is filtered and washed with 4 x 40 ml of cold ether.
• nitrogen stream is delivered to evaporate the ether.
• the peptides are then frozen and lyophilized for more than 24hrs.
• the peptides are recovered into the solution by adding acetic acid. Purification and Characterization: The peptide powder along with other by-products are re-dissolved in 50% acetic acid solution and injected onto a Vydac 1.0 cm ID.
• Solvents and reagents commercially obtained are used without purification. Reaction mixtures are stirred magnetically and are monitored by either analytical high performance liquid chromatography (HPLC) or thin-layer chromatography (TLC). Solutions are routinely concentrated using a B ⁇ chi rotary evaporator at 15-25 mm Hg. TLC is performed using silica gel 60 F 2 5 precoated plates with a fluorescent indicator. Visualization is accomplished routinely with UV light (254 nm.) Flash column chromatography is carried out with E. Merck silica gel 60 (230-400 mesh) using the eluants indicated; chromatographic separations are monitored by TLC analyses.
• HPLC high performance liquid chromatography
• TLC thin-layer chromatography
• Analytical HPLC is carried out on either 4.6 x 250 mm MetaChem Kromasil C - or Polaris C I8 -reverse-phase columns (3.5 ⁇ or 3.0 ⁇ particle sizes for C 4 or s, respectively) using a 0.1% phosphoric acid in water (A)/acetonitrile (B) gradient (5% B for C or 20% B for C ls to 100 % B over 20 min, hold 5 min) with a flow rate of 1.0 ml/min; detection is by UV light at both 214 and 254 nm.
• Preparative HPLC is conducted on either a 50 x 250 mm Polaris Cis-reverse- phase column (10 ⁇ particle size, 100 A pore size) or a 41.4 x 250 mm Rainin Dynamax C 4 - reverse-phase column (8 ⁇ particle size, 300 A pore size) using a 0.1% trifluoroacetic acid in water(A)/acetonitrile(B) gradient (5% to 100% B over 55 min, hold 10 min); detection is by UV light at 214 nm.
• Functional activity can be evaluated using various methods known in the art. Examples of such methods are measurement of second messenger responses, in particular cAMP, the use of modified cell systems yielding color reaction upon accumulation of second messenger elements such as cAMP, e.g. as described by Chen et al. 1995 (Anal Biochem. 1995, 226, 349-54), Cytosensor Microphysiometer techniques (see Boyfield et al. 1996), or the study of physiological effects caused by the compounds of the invention may be applied by using the compounds of the invention alone, or in combination with natural or synthetic MSH-peptides.
• MC-3/MC-4 selectivity of a compound is defined herein as the ratio of the EC 50 of the compound for an MC-1 receptor ("EC 50 -MC-I") over the EC 5 0 of the compound for the MC-3 (EC 50 -MC-3) / MC-4 (EC 50 -MC-4) receptor, the EC 50 values being measured as described above.
• the formulas are as follows:
• MC-3 selectivity [EC 50 -MC-1] / [EC 50 -MC-3]
• MC-4 selectivity [EC 50 -MC-1] / [EC 50 -MC-4] *
• a compound is defined herein as being “selective for the MC-3 receptor" when the above mentioned ratio "MC-3-selectivity" is at least about 10, preferably at least about 100, and more preferably at least about 500.
• a compound is defined herein as being “selective for the MC-4 receptor” when the above mentioned ratio "MC-4-selectivity" is at least about 10, preferably at least about 100, and more preferably at least about 500.
• the present invention Based on their ability to agonize or antagonize the MC-4 and/or MC-3 receptor, the present invention also relates to the use of the ligands described herein in methods for treating obesity and other body weight disorders, including, for example, anorexia and cachexia.
• the compounds may also be used in methods for treating disorders that result from body weight disorders, including but not limited to insulin resistance, glucose intolerance, Type-2 diabetes mellitus, coronary artery disease, elevated blood pressure, hypertension, dyslipidaemia, cancer
• the invention further relates to the treatment of disorders relating to behavior, memory (including learning), cardiovascular function, inflammation, sepsis, cardiogenic and hypovolemic shock, sexual dysfunction, penile erection, muscle atrophy, nerve growth and repair, intrauterine fetal growth, and the like.
• treating and treatment are used herein to mean that, at a minimum, administration of a compound of the present invention mitigates a disorder by acting via the MC- 3 or MC-4 receptor.
• the terms include: preventing a disease state from occurring in a mammal, particularly when the mammal is predisposed to acquiring the disease, but has not yet been diagnosed with the disease; inhibiting progression of the disease state; and/or alleviating or reversing the disease state.
• the invention compounds can therefore be formulated into pharmaceutical compositions for use in treatment or prophylaxis of these conditions.
• Standard pharmaceutical formulation techniques are used, such as those disclosed in Remington's Pharmaceutical Sciences, Mack
• compositions of the invention comprise: a. a safe and effective amount of a compound of Formula (I); and b. a pharmaceutically-acceptable excipient.
• a "safe and effective amount" of a Formula (I) compound is an amount that is effective to interact with the MC-4 and/or MC-3 receptor, in an animal, preferably a mammal, more preferably a human subject, without undue adverse side effects (such as toxicity, irritation, or allergic response), commensurate with a reasonable benefit/risk ratio when used in the manner of this invention.
• the specific "safe and effective amount” will, obviously, vary with such factors as the particular condition being treated, the physical condition of the patient, the duration of treatment, the nature of concurrent therapy (if any), the specific dosage form to be used, the excipient employed, the solubility of the Formula (I) compound therein, and the dosage regimen desired for the composition.
• compositions of the subject invention contain one or more pharmaceutically-acceptable excipients.
• pharmaceutically-acceptable excipient means one or more compatible solid or liquid ingredients which are suitable for administration to an animal, preferably a mammal, more preferably a human.
• compatible means that the components of the composition are capable of being commingled with the subject compound, and with each other, in a manner such that there is no interaction which would substantially reduce the pharmaceutical efficacy of the composition under ordinary use situations.
• Pharmaceutically-acceptable excipients must, of course, be of sufficiently high purity and sufficiently low toxicity to render them suitable for administration to the animal, preferably a mammal, more preferably a human being treated.
• substances which can serve as pharmaceutically-acceptable excipients or components thereof are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and methyl cellulose; powdered tragacanth; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil of theobroma; polyols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; agar; alginic acid; wetting agents and lubricants, such as sodium lauryl sulfate; coloring agents; flavoring agents; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline
• the choice of pharmaceutically-acceptable excipients to be used in conjunction with the subject compound is basically determined by the way the compound is to be administered. If the subject compound is to be injected, the preferred pharmaceutically-acceptable excipient is sterile water, physiological saline, or mixtures thereof, the pH of which has preferably been adjusted to about 4-10 with a pharmaceutical buffer; a compatible suspending agent may also be desirable.
• pharmaceutically-acceptable excipients for systemic administration include sugars, starches, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, lactose, vegetable oils, synthetic oils, polyols, alginic acid, phosphate, acetate and citrate buffer solutions, emulsifiers, isotonic saline, and pyrogen-free water.
• Preferred excipients for parenteral administration include propylene glycol, ethyl oleate, pyrrolidone, ethanol, and sesame oil.
• the pharmaceutically-acceptable excipient, in compositions for parenteral administration comprises at least about 90% by weight of the total composition.
• compositions of this invention are preferably provided in unit dosage form.
• a "unit dosage form” is a composition of this invention containing an amount of a Formula (I) compound that is suitable for administration to an animal, preferably a mammal, more preferably a human subject, in a single dose, according to good medical prac- tice.
• These compositions preferably contain from about 1 mg to about 750 mg, more preferably from about 3 mg to about 500 mg, still more preferably from about 5 mg to about 300 mg, of a Formula (I) compound.
• compositions of this invention may be in any of a variety of forms, suitable (for example) for oral, rectal, topical, nasal, ocular, transdermal, pulmonary or parenteral administration.
• a variety of pharmaceutically-acceptable excipients well-known in the art may be used. These include solid or liquid fillers, diluents, hydrotropes, surface-active agents, and encapsulating substances.
• Optional pharmaceutically-active materials may be included, which do not substantially interfere with the inhibitory activity of the Formula (I) compound.
• the amount of excipient employed in conjunction with the Formula (I) compound is sufficient to provide a practical quantity of material for administration per unit dose of the compound.
• oral dosage forms can be used, including such solid forms as tablets, capsules, granules and bulk powders. These oral forms comprise a safe and effective amount, usually at least about 5%, and preferably from about 25% to about 50%, of the Formula (I) compound. Tablets can be compressed, tablet triturates, enteric -coated, sugar- coated, film-coated, or multiple-compressed, containing suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents.
• Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules, and effervescent preparations reconstituted from effervescent granules, containing suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, melting agents, coloring agents and flavoring agents.
• suitable solvents preservatives, emulsifying agents, suspending agents, diluents, sweeteners, melting agents, coloring agents and flavoring agents.
• the pharmaceutically-acceptable excipient suitable for the preparation of unit dosage forms for peroral administration are well-known in the art.
• Tablets typically comprise conventional pharmaceutically-compatible adjuvants as inert diluents, such as calcium carbonate, sodium carbonate, mannitol, lactose and cellulose; binders such as starch, gelatin, polyvinylpyrrolidone and sucrose; disintegrants such as starch, alginic acid and croscarmelose; lubricants such as magnesium stearate, stearic acid and talc. Glidants such as silicon dioxide can be used to improve flow characteristics of the powder mixture. Coloring agents, such as the FD&C dyes, can be added for appearance.
• inert diluents such as calcium carbonate, sodium carbonate, mannitol, lactose and cellulose
• binders such as starch, gelatin, polyvinylpyrrolidone and sucrose
• disintegrants such as starch, alginic acid and croscarmelose
• lubricants such as magnesium stearate, stearic acid and
• Sweeteners and flavoring agents such as aspartame, saccharin, menthol, peppermint, and fruit flavors, are useful adjuvants for chewable tablets.
• Capsules typically comprise one or more solid diluents disclosed above. The selection of excipient components depends on secondary considerations like taste, cost, and shelf stability, which are not critical for the purposes of the subject invention, and can be readily made by a person skilled in the art.
• Peroral compositions also include liquid solutions, emulsions, suspensions, and the like.
• the pharmaceutically-acceptable excipients suitable for preparation of such compositions are well known in the art.
• Typical components of excipients for syrups, elixirs, emulsions and suspensions include ethanol, glycerol, propylene glycol, polyethylene glycol, liquid sucrose, sorbitol and water.
• typical suspending agents include methyl cellulose, sodium carboxymethyl cellulose, Avicel® RC-591, tragacanth and sodium alginate;
• typical wetting agents include lecithin and polysorbate 80; and typical preservatives include methyl paraben, propyl paraben and sodium benzoate.
• Peroral liquid compositions may also contain one or more components such as sweeteners, flavoring agents and colorants disclosed above.
• compositions may also be coated by conventional methods, typically with pH or time-dependent coatings, such that the subject compound is released in the gastrointestinal tract in the vicinity of the desired topical application, or at various times to extend the desired action.
• dosage forms typically include, but are not limited to, one or more of cellulose acetate phthalate, polyvinylacetate phthalate, hydroxypropyl methyl cellulose phthalate, ethyl cellulose,
• a preferred mode of administration is parenteral (more preferably intravenous injection) or nasal administration, in the form of a unit dose form.
• Preferred unit dose forms include suspensions and solutions, comprising a safe and effective amount of a Formula I compound.
• the unit dose form When administered parenterally, the unit dose form will typically comprise from about 1 mg to about 3 g, more typically from about 10 mg to about 1 g, of the Formula (I) compound, although the amount of compound administered will depend, for example, on its relative affinity for the MC-4/MC-3 receptor subtypes, its selectivity over other receptors, including the other melanocortin receptors, etc.
• Compositions of the subject invention may optionally include other drug actives.
• compositions useful for attaining systemic delivery of the subject compounds include subling ⁇ al and buccal dosage forms.
• Such compositions typically comprise one or more of soluble filler substances such as sucrose, sorbitol and mannitol; and binders such as acacia, microcrystalline cellulose, carboxymethyl cellulose and hydroxypropyl methyl cellulose. Glidants, lubricants, sweeteners, colorants, antioxidants and flavoring agents disclosed above may also be included.
• compositions of this invention can be administered topically or systemically.
• Systemic application includes any method of introducing a Formula (I) compound into the tissues of the body, e.g., intra-articular, intrathecal, epidural, intramuscular, transdermal, intravenous, intraperitoneal, subcutaneous, sublingual, rectal, nasal, pulmonary, and oral administration.
• the Formula (I) compounds of the present invention are preferably administered systemically, more preferably parenterally and most preferably via intravenous injection.
• the specific dosage of compound to be administered, as well as the duration of treatment, and whether the treatment is topical or systemic are interdependent.
• the dosage and treatment regimen will also depend upon such factors as the specific Formula (I) compound used, the treatment indication, the personal attributes of the subject (such as weight), compliance with the treatment regimen, and the presence and severity of any side effects of the treatment.
• a preferred method of systemic administration is intravenous delivery. Individual doses of from about 0.01 mg to about 100 mg, preferably from about 0.1 mg to about 100 mg are preferred when using this mode of delivery.
• the compounds of the invention can be administered alone or as mixtures, and the compositions may further include additional drugs or excipients as appropriate for the indication.
• the compound of the invention can be delivered to the preferred site in the body by using a suitable drug delivery system.
• Drug delivery systems are well known in the art.
• a drug delivery technique useful for the compounds of the present invention is the conjugation of the compound to an active molecule capable of being transported through a biological barrier (see e.g. Zlokovic, BN., Pharmaceutical Research, Vol. 12, pp. 1395-1406 (1995)).
• a specific example constitutes the coupling of the compound of the invention to fragments of insulin to achieve transport across the blood brain barrier (Fukuta, M., et al. Pharmaceutical Res., Vol. 11, pp. 1681-1688 (1994)).
• DME 1,2-dimethoxyethane
• DIEA diisopropylethylamine
• Pbf 2,2,4,6,7- ⁇ entamethyl-dihydrobenzofurane-5-sulfonyl-
• Pmc 2,2,5,7,8-pentamethyl-6-chromansulfonyl- p-TSA: 7-toluenesulfonate
• PyBOP benzotriazole-lyl-oxy-tris-pyrrolidinophosphonium hexafluorophosphate
• TFA trifluoroacetic acid
• THF tetrahydrofuran
• synthesis reagents and solvents are purchased commercially and loaded on the instrument according to the instrument's instruction.
• a chemistry program called NAc- 0.25mmole MonPrePk is used for synthesizing this peptide.
• the Fmoc deprotection is monitored and controlled by conductivity measurement with set criteria of 5% or less conductivity comparing to previous deprotection cycle.
• the resin is air-dried and transferred into a glass vial and a freshly prepared cleavage reagent (10 ml) is added.
• the deprotection reaction is carried out for 2 hours at room temperature with constant stirring.
• the supernatant is then separated from the resin by filtration.
• the synthesized peptide is then precipitated in ether layer by adding 40 ml cold ether.
• the peptide precipitates are centrifuged (Heraeus Labofuge 400, Rotor #8179) at 3,500 rpm for four minutes.
• the ether is discarded and 40 ml of fresh cold ether is added to wash the peptide precipitates.
• the washing steps are repeated for three times to remove the deprotection byproducts.
• the final peptide precipitates are freeze-dried overnight.
• the identity and purity of the linear peptide is determined by both MS and HPLC. Expected peptide molecular weight is detected. Peptide is re-dissolved in 50% acetic acid and purified by a C8 reverse phase HPLC using a linear gradient of 0-70% solvent B with solvent A in 70 min at a flow rate 3 ml/min.
• the composition of solvents A and B are as follows: A: 0.1% TFA, 2% acetonitrile in water; B: 0.1% TFA in 95% acetonitrile.
• the fractions are collected at every 0.25 min. Aliquots of each fraction are analyzed by both MS and analytical RP-HPLC. The fractions that contain a single u.v. 220 nm absorbance peak with expected mass unit for the peptide are combined and lyophilized.
• the final purity of the peptide is determined by an analytical RP-HPLC of the combined fractions.
• Example 13 Synthesis of Ac-YF(L-N-Me-Arg)W-NH Prepared according to Example 1, except Fmoc-L-N-Me-Arg(Mtr) is used instead Fmoc-L- Arg(Pmc), and Fmoc-L-Phe is used instead of Fmoc-D-Phe.
• Example 18 Synthesis of Ac-Y(D-4-Fluoro-Phe)RW-NH 2 Prepared according to Example 1, except Fmoc-D-4-Fluoro-Phe is used instead Fmoc-D-Phe.
• Example 26 Synthesis of Ac-Y(D-3-Fluoro-Phe)RW-NH 2 Prepared according to Example 1, except Fmoc-D-3-Fluoro-Phe is used instead Fmoc-D-Phe.
• Example 29 Synthesis of Ac-(L-4-Chloro-Phe)(D-4-Fluoro-Phe)RW-NH 2 Prepared according to Example 1, except Fmoc-L-4-Chloro-Phe is used instead Fmoc-L- Tyr(OtBu), and Fmoc-D-4-Fluoro-Phe is used instead of Fmoc-D-Phe.
• Example 32 Synthesis of Ac-(L-4-Chloro-Phe)(D-4-Fluoro-Phe)R-NH 2 Prepared according to Example 1, except Fmoc-L-4-Chloro-Phe is used instead Fmoc-L- Tyr(OtBu), Fmoc-D-4-Fluoro-Phe is used instead of Fmoc-D-Phe, and Fmoc-L-Trp(Boc) is not used.
• Example 38 Synthesis of Ac-(L-Pentafluoro-Phe)fRW-NH 2 Prepared according to Example 1, except Fmoc-L-Pentafluoro-Phe is used instead Fmoc-L- Tyr(OtBu).
• Example 40 Synthesis of Ac-(L-Aminomethyl-Phe)fRW-NH 2 Prepared according to Example 1, except Fmoc-L-Aminomethyl-Phe(Boc) is used instead Fmoc- L-Tyr(OtBu).
• Example 44 Synthesis of Ac-(L-4-SO 3 -Phe)fRW-NH 2 Prepared according to Example 1, except Fmoc-L-4-S0 3 -Phe is used instead Fmoc-L-Tyr(OtBu).
• the resulting solution is stirred at room temperature for 20 hours, and then the solvents are removed under reduced pressure.
• the resulting residue is partitioned between 10% sodium carbonate (100ml) and methylene chloride (100ml). The organics are dried over anhydrous magnesium sulfate, filtered and the solvent removed under reduced pressure.
• the resulting crude material is purified by flash chromatography on silica gel (90:9:1 chloroform:methanol:ammonium hydroxide) to afford the title compound.
• the aqueous layer is extracted with ethyl acetate (3 x 250ml), the organics are pooled and dried over anhydrous magnesium sulfate, filtered and the solvent removed under reduced pressure. The crude material is used without further purification.
• the resulting suspemsion is stirred at room temperature for 24 hours, and then the pH is adjusted to 5 with acetic acid.
• a 1.0M solution of sodium cynanoborohydride in tetrahydrofuran (1.44ml, 1.2eq) is then added at a rate of 0.2ml/min with a syringe pump.
• the resulting suspension is stirred at room temperature for 24 hours, filtered through celite, and the solvents removed under reduced pressure.
• the crude material is purified by reverse-phase HPLC to afford the title compound.
• the resulting suspension is stirred at room temperature for twenty-four hours, and the solvents are then removed under reduced pressure.
• the crude material is partitioned between 10% sodium carbonate (75ml) and methylene chloride (75ml). The organics are dried over anhydrous magnesium sulfate, filtered and solvents removed under reduced pressure.
• the crude material is purified by flash chromatography on silica gel (90:9:1 chloroform:methanol: ammonium hydroxide) to afford the title compound.
• the resulting suspension is stirred at room temperature for twenty-four hours, and then the solvents are removed under reduced pressure.
• the residue is partitioned between methylene chloride and 10% sodium carbonate, the organics dried over anhydrous magnesium sulfate, filtered and the solvent removed under reduced pressure to give the crude product.
• the product is purified by flash chromatography on silica gel (90:9:1 chloroform: methanol: ammonium hydroxide) to afford the title compound.
• the resulting solution is stirred at room temperature for twenty-four hours, and then the solvents are removed under reduced pressure.
• the residue is partitioned between ethyl acetate (75ml) and water (75ml), the organics dried over magnesium sulfate, filteed and the solvents removed under reduced pressure.
• the crude product is purified be reverse-phase HPLC to afford the title compound.
• the resulting suspension is stirred at room temperature for twenty-four hours, and then the pH is adjusted to 5 with acetic acid.
• Sodium cyanoborohydride (0.70ml, l.OM solution in tetrahydorofuran, leq) is then slowly added (0.2ml hour) with a syringe pump. After twenty-four hours the suspension is filtered through celite and the solvents removed under reduced pressure. The crude product is purified by reverse-phase HPLC to afford the title compound.
• the resulting solution is stirred at room temperature for twenty-four hours and then the solvents are removed under reduced pressure.
• the residue is partitioned between ethyl acetate (75ml) and 10% sodium carbonate (75ml).
• the organics are dried over anhydrous magnesiumn sulfate, filtered and the solvents removed under reduced pressure.
• the crude product is purified by flash chormatography on silica gel (90:9:1 chloroform:methanol: ammonium hydroxide) to afford the title compound.
• Triethylsilane is then added dropwise until the bright yellow color just disappeared.
• the resulting solution is stirred at room temperature for twenty-four hours and then the solvents are removed under reduced pressure.
• the crude product is purified by flash chromatography on silica gel (90:9:1 chloroform:methanol: ammonium hydroxide) to afford the title compound as a mixture of diastereomers.
• the diastereomers are separated by reverse-phase HPLC to afford an earlier eluting diastereomer and a later eluting diastereomer.
• the resulting solution is stirred at room temperature for twenty-four hours, and then the solvents are removed under reduced pressure.
• the residue is partitioned between ethyl acetate and 10% sodium carbonate, the organics are dried over anhydrous magnesium sulfate, filtered and the solvents removed under reduced pressure.
• the crude product is purified by flash chromatography on silica gel (90:9:1 chloroform:methanol: ammonium hydroxide) to afford the title compound.
• HOBt (1.0 eq.) is added to a well-stirred solution of Boc-R(N0 2 )-OH (0.639 g, 2.0 mmol) and H- W-NH 2 » HCl (0.479 g, 2.0 mmol) in DMF (10 ml) at 0°C followed by addition of EDCI (1.1 eq.) and NMM (0.484 ml, 2.2 eq.). The resulting mixture is stirred at 0°C for 1 h and then at room temperature for 4-18 h.
• HOBt (1.0 eq.) and EDCI (0.949 g, 4.95 mmol, 1.1 eq.) are added sequentially to a well-stirred mixture of Boc-R(N0 2 )-OH (1.437 g, 4.5 mmol) and tryptamine (0.721 g, 4.5 mmol) in DMF (20 ml) at 0°C. The resulting mixture is stirred for 30 minutes at 0°C and then at room temperature for -20 h.
• the mixture is diluted with EtOAc (100 ml) and is then washed successively with water (2 x 20 ml), 5 % aqueous citric acid (3 x 10 ml), 5% aqueous sodium bicarbonate (2 x 10 ml), and brine (2 x 20 ml).
• the organic layer is dried over anhydrous sodium sulfate and concentrated by rotary evaporation to yield 12, which is used directly in the next step.
• HOAt (0.409 g, 3.005 mmol), EDCI (0.632 g, 3.306 mmol, 1.1 eq.), and TEA (l.leq.) are added sequentially to a well-stirred solution of 13 (1.086 g, 3.005 mmol) and Fmoc-Atc-OH (1.242 g, 3.005 mmol) in DMF (15 ml) at 0°C. The resulting mixture is stirred for 30 minutes at 0°C and then at room temperature for 21 h. Work-up as described above for 12 gives crude 14. This material is used directly in the next step without further purification.
• DIEA (0.310 ml, 1.772 mmol, 2.0 eq.) is added to a well-stirred mixture of 21 (0.4293 g, 0.886 mmol), Boc-f-OH (0.2351 g, 0.886 mmol), and PyBrop (0.3795 g, 0.886 mmol) in DCM (10 ml) at 0°C.
• the resulting reaction mixture is stirred at room temperature for 16 h and then is diluted with EtOAc (50 ml).
• the organic layer is washed successively with water (1 x 10 ml), 5% aqueous sodium bicarbonate (2 x 10 ml), and brine ( 2 x 10 ml) and is dried over anhydrous sodium sulfate. Removal of the dessicant and evaporation of the volatiles under reduced pressure yields crude 22.
• TEA 0.209 mmol, 1.1 eq.
• DMF 3 ml
• the reaction mixture is diluted with EtOAc (30 ml) and is washed successively with water (2 x 5 ml), 5% aqueous citric acid (3 x 3 ml), 5% aqueous sodium bicarbonate (2x 3 ml), and brine (2 x 5 ml).
• the organic layer is dried over anhydrous sodium sulfate, concentrated, and then triturated with ether to yield crude 24.
• Boc-f-OH (7.64 g, 28.8 mmol), H-W(N0 2 )-OMe ⁇ Cl (6.72 g, 28.8 mmol), HOBt (3.94 g, 29.2 mmol), TEA (8 ml, 57.6 mmol) and DMF (110 ml, anhydrous) are combined and cooled at 0°C and EDCI (5.89 g, 30.8 mmol) is added with stirring. After stirring at room temperature for 18 h, the mixture is concentrated by rotary evaporation, diluted with water (350 ml), and extracted with EtOAc (4x80 ml).
• the combined organic extract is washed with aqueous 1 N HCl (3x20 ml), saturated aqueous NaHC0 3 (2x25 ml), brine (25 ml), and is then dried with anhydrous Na 2 S0 . After filtration, the filtrate is concentrated by rotary evaporation to give 5.
• Boc-YfR(N0 2 )-OH (29, 0.99 g, 1.56 mmol), H-W-NH 2 ⁇ C1 (0.43 g, 1.8 mmol), HOBt (0.25 g,
• H-YfRW-NH 2 (32) Peptide 31 (0.25 g, 0.35 mmol), 5% Pd/BaS0 4 (0.25 g, umeduced) and MeOH (15 ml) are combined and hydrogenated under 40 psi of hydrogen at room temperature for 48 h. After filtration, the filtrate is concentrated by rotary evaporation and the residue purified by preparative HPLC to yield H-YfRW-NH 2 (32).
• the procedure of preparing 28 is used followed by purification by preparative HPLC and 35 is obtained from H-YfR(N0 2 )W-NH 2 (31, 0.26 g, 0.36 mmol), decanoic acid (0.071 g, 0.41 mmol), HOBt (0.057 g, 0.42 mmol), TEA (0.1 ml, 0.73 mmol), EDCI (0.083 g, 0.43 mmol), and DMF (12 ml).
• Boc-YfR(NQ 2 )-trvptamide (37) The procedure of preparing 30 is used and 37 is obtained from Boc-YfR(N0 2 )-OH (29, 0.95 g, 1.5 mmol), tryptamine (0.276 g, 1.73 mmol), HOBt (0.239 g, 1.77 mmol), TEA (0.5 ml, 3.6 mmol), EDCI (0.34 g, 1.8 mmol), and DMF (18 ml).
• Boc-fR(N0 2 )-OH (43) The procedure of preparing 29 is followed and 43 is obtained from Boc-fR(N0 2 )-OMe (26, 2.8 g, 5.8 mmol), LiOH (0.27 g, 11 mmol) and MeOH (15 ml).
• Boc-YfRW-OCH (56) The procedure of preparing compound 28 is followed and 2. Ig (95%) of 56 is obtained from Boc- YfR(N0 2 )-OH (29, 1.7 g, 2.6 mmol), H-W-OCH 3 ⁇ Cl (0.68 g, 2.7 mmol), HOBt (0.42 g, 3.1 mmol), TEA (0.8 ml, 5.8 mmol), EDCI (0.63 g, 3.3 mmol), and DMF(25 ml).
• the procedure of preparing compound 28 is followed to give a coupling product 60 from 59 (0.1 g, 0.125 mmol), methyl amine (0.006 ml, 0.15 mmol), HOBt (0.02 g, 0.15 mmol), TEA (0.04 ml, 0.3 mmol), EDCI (0.03g, 0.16 mmol), and DMF (3 ml).
• This product is hydrogenated according to the procedure of preparing compound 32 with 5% Pd/BaS0 (0.1 g, unreduced) in MeOH (6 ml) to give 63.
• Hvdrocinnamoyl-fRW-NB (77) The procedure of preparing compound 28 is followed to give a crude coupling product 76 from 75 (0.2 g, 0.4 mmol), H-W-NH 2 ⁇ C1 (0.1 g, 0.42 mmol), HOBt (0.068 g, 0.5 mmol), TEA (0.14 ml, 1 mmol), EDCI (0.1 g, 0.52 mmol), and DMF (12 ml). This product is hydrogenated using the procedure of preparing compound 32 using 5% Pd7BaS0 4 (0.2 g, unreduced) and MeOH (10 ml) to give 77.
• Boc-fR(N0 2 )-OH 43, 507.9 mg, 1.09 mmol
• tryptamine (172.9 mg, 1.08 mmol
• HOBt 163.3 g, 1.08 mmol
• TEA TEA
• EDCI 226.9 mg, 1.18 mmol
• Boc-Me-f-OH (1.0008 g, 3.6 mmol), H-R(N0 2 )-OMe • HCl (0.9659 g, 3.6 mmol), HOBt (0.5515 g, 3.6 mmol), TEA (0.525 ml, 3.8 mmol) and DMF (20 ml, anhydrous) are combined and cooled at 0°C and EDCI (0.7211 g, 3.8 mmol) is added with stirring. After stirring at 0°C for 1 h the ice bath is removed and the mixture is warmed and stirred at room temperature for 23 h.
• the mixture is worked up as in the procedure for preparing compound 85 to give a crude coupling product which is purified by preparative HPLC(C 4 ).
• the purified product is then dissolved in 10% acetic acid-MeOH (20 ml) and is hydrogenated under 40 psi of H 2 at room temperature for 15 h using 5% Pd-BaS0 4 (31.3 mg, unreduced) as catalyst.
• the catalyst is removed by filtration through Celite, the volatiles are removed in vacuo, the residue is re- dissolved in 10% acetonitrile-water (15 ml), and the mixture is frozen and lyophilized to yield 95.
• Boc-f-OH (5.30 g, 20 mmol) in dry THF (100 ml) are added TEA (2.02 g, 20 mmol) and ethyl chloroformate (2.16 g, 20 mmol) at -15°C under argon atmosphere. The mixture is stirred at -15° C for 30 min, and is then warmed to 0°C.
• a solution of diazomethane in ether [100 ml, prepared from Me(NO)NCONH 2 (4.0 g, 40 mmol) and 50% aqueous KOH (20 ml)] is added. The mixture is warmed and stirred at room temperature for 3 h.
• the mixture is extracted with DCM (3 x 10 ml) and the combined organic phases are washed with brine (10 ml) and dried over Na 2 S0 4 .
• the solvents are removed by rotary evaporation to give the di-acid.
• the crude di-acid is suspended in toluene (10 ml) and the mixture is heated at reflux under an argon atmosphere for 3 h.
• the solvent is evaporated and the residue is purified by chromatography (DCM-MeOH, 98:2 to 95:5) to give a mixture of two diastereoisomers that are separable by preparative HPLC.
• ⁇ MM (0.48 ml, 4.4 mmol) is added to a mixture of Boc-R(Pbf)-OH (1.053 g, 2.0 mmol), H-W- OMe • HCl (0.509 g, 2.0 mmol), HOBt (0.270 g, 2.0 mmol), EDCI (0.422 g, 2.2 mmol) in DMF (10 ml) at 0°C and the resulting mixture is stirred at 0°C for 1 h and then at room temperature for 4 h.
• the reaction mixture is diluted with EtOAc (100 ml) and is washed successively with water (2 x 10 ml), 1 ⁇ HCl (2 x 10 ml), saturated ⁇ aHC0 3 , 2 x 10 ml), and brine (2 x 10 ml).
• the organic layer is dried over MgS0 , the dessicant removed by filtration, and the filtrate concentrated by rotary evaporation to yield 1.454 g (100%) of crude 104.
• Fmoc-1 l-Aun-(c rb ⁇ -4-Cl-Ff)-R(Pbf)W-OMe The procedure of making compound 104 is followed and crude 108 is obtained from Fmoc-11- Aun-OH (207 mg, 488 ⁇ mol), 107 (543 mg, 488 ⁇ mol), HOBt (66.0 mg, 488 ⁇ mol), EDCI (103.0 mg, 537 ⁇ mol), and NMM (0.118 ml, 1.07 mmol).
• ri l-Aun-(c rba-4-Cl-Ff)-R(Pbf)Wl (109)
• Solid PCI 5 is added to a solution of 3-(4-benzyloxy-phenyl)-propionic acid in toluene over a 1 hour period.
• the reaction mixture is stirred at room temperature for 3 hours and solvent is evaporated.
• the residue is stirred with hexanes overnight producing crystalline material, which is filtered and dried under vacuum.
• Li - (S)-(- )-4-benzyloxazolidinone salt is prepared at -65° to -72°C.
• a solution of decanoyl chloride in THF is cooled to -72°C and treated with Li - (S)-(-)-4-benzyloxazolidinone solution at this temperature.
• the reaction mixture is stirred at -70° to -75°C for 1 hour and overnight at room temperature, treated with NH C1 solution and extracted with ethyl acetate.
• the organic layer is washed with water/brine, dried with MgS0 , and evaporated.
• the residue is separated on a silica column using hexane / ethyl acetate 7 / 3 solution to afford the title compound.
• TEA is added to a solution of the remaining reactants in DCM at -2° to +3°C.
• the reaction mixture is stirred at room temperature for 4 hrs and diluted with 0.1N HCl.
• the product is extracted with DCM, washed with water, dried with MgS0 4 , and solvent is evaporated under reduced pressure. The residue is crystallized from hexanes to afford the title compound.
• Reactants are stirred at room temperature for 5 hrs. Solvents are evaporated under reduced pressure; the residue is diluted with water and acidified to pH about 2. Resulting precipitate of the product is filtered, washed with water until pH of the filtrate reaches about 6, and dried under vacuum overnight.
• a 2 M THF solution of BH 3 Me 2 S complex (40 ml) is added to a solution of the amide substrate (4.5 g, 8.4 mmol) in anhydrous THF (50 ml).
• the reaction mixture is heated at 75°C while slowly distilling liquid is collected through the condenser. After 2 hours, a new portion of 2 M THF solution of BH 3 Me 2 S complex (10 ml) is added and heating with simultaneous distillation is continued for additional 3 hours.
• the reaction mixture is cooled to room temperature and carefully treated with MeOH, until the release of gas ceases, and with 3 N NaOH.
• the crude product is extracted with ethyl acetate and purified on a silica column using a solution of 1.5 % MeOH in AcOEt following by a solution of EtOAc/DCM/MeO_H/Et 3 N 4/5/0.5/0.3 to afford the title compopund.
• the coupling procedure (CP) for the peptide bond formation in solution is used.
• the crude product is purified on a silica column using hexane / ethyl acetate 6 / 1 to afford the title compound.
• reaction mixture is stirred at room temperature for 4 hours and diluted with 1,2- dichloethane. Solvents are evaporated under reduced pressure; the residue is dried under vacuum overnight.
• the reaction mixture is hydrogenated at room temperature, 45 psi overnight.
• the catalyst is separated by filtration through Celite. Solvent is evaporated under reduced pressure.
• the crude product is purified by reverse phase preparative HPLC to afford the title compound.
• Example 95 A procedure analogous to that used to make Example 95 was used to prepare the compound of Examples 97-99.
• the crude product is cleaved from the resin and the protecting groups removed using 93% TFA and 2.3 % ethanedithiol in water for 3 h at room temperature. After removal of the resin by filtration and washing with 3% TFA (3 x 18 ml), the filtrate is extracted with ether (6 x 20 ml) and is frozen and lyophilized. The crude product is then dissolved in 30% aqueous acetic acid and the solution purified by preparative HPLC (lO ⁇ Vydac dg, 10 x 250 mm, 6 to 60% acetonitrile-water (0.1% TFA) gradient over 1.5 h.) The product-containing fractions are combined and lyophilized to give the purified peptides.
• Fmoc-W(Boc)-OH and Fmoc-R(pbf)-OH are attached sequentially to the Rink amide resin (4.28g, 3 mmol) using PyBOP (2-fold excess) and NMM (4- fold excess). After washing with DMF (3 x 35 ml), ether (4 x 35 ml), and drying in vacuo an increase in weight is achieved.
• the resin (1.17 g, 0.35 mmol) is suspended in DMF (10 ml), the Fmoc group is removed, PyBOP (0.6 g, 1.15 mmol), NMM (0.26 ml, 2.8 mmol), and Fmoc-(4- Py)ala-OH (0.447 g, 1.15 mmol) are added sequentially, and the mixture is shaken for 1 h. Following Fmoc-deprotection, the coupling procedure is again repeated with Fmoc Y(t-Bu)-OH (0.528 g, 1.1 mmol). The peptide is then deprotected, acetylated, and cleaved from the resin to give of product.
• Example 101 Example 101
• an obese human female subject weighing 130 kg is treated by this method to incur weight loss. Specifically, once each day for a period of 6 months, the subject is administered, via intravenous injection, 15 ml of an aqueous solution comprising the following:
• the patient At the end of the treatment period, the patient exhibits measurable weight loss.
• An obese human male subject weighing 150 kg is subjected to a weight-reduction program that achieves weight loss with reduced adiposity through a combination of a restricted diet and increased exercise. Specifically, once each day for a period of 6 months after weight loss, the subject is administered, via intravenous injection, 15 ml of an aqueous solution comprising the following:
• the patient At the end of the treatment period, the patient exhibits maintenance of weight loss and reduced adiposity.
• Example C An obese human male subject weighing 165 kg is subjected to a weight reduction program that achieves weight loss through a combination of restricted diet, increased exercise and subcutaneous injection daily of 15 is of an aqueous solution comprising:
• the patient's weight loss is maintained through continuation of the intravenous injection once each day for an additional period of 6 months. At the end of the treatment period the patient exhibits maintained weight loss and reduced adiposity.
• An obese human female subject weighing 140 kg is treated by the present method to incur weight loss. Specifically, she is treated with an implantable subcutaneous pump that delivers 0.1 mg/kg of the compound of Example 31 over a 24 hour period.
• the pump contains a solution of the compound dissolved in a solution of 50% propylene glycol and 50% sterile water. The pump is replaced monthly and treatment continues for a six-month period at which time the patient exhibits weight loss and reduced adiposity.
• Example E An obese male weighing 150 kg is treated by this method to incur weight loss. Specifically, he is treated with an oral tablet taken twice daily containing 300 mg of the compound in Example 29. The treatment continues for 12 months at which time the patient exhibits weight loss and reduced adiposity.

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