WO2007096186A1

WO2007096186A1 - Substituted phenylpiperidine derivatives as melanocortin-4 receptor modulators

Info

Publication number: WO2007096186A1
Application number: PCT/EP2007/001595
Authority: WO
Inventors: Michael Soeberdt; Philipp Weyermann; Holger Deppe; Andreas Von Sprecher; Achim Feurer; Cyrille Lescop; Marco Henneböhle
Original assignee: Santhera Pharmaceuticals (Schweiz) Ag
Priority date: 2006-02-23
Filing date: 2007-02-23
Publication date: 2007-08-30

Abstract

The present invention relates to substituted phenylpiperidine derivatives as melanocortin-4 receptor modulators. Depending on the structure and the stereochemistry the compounds of the invention are either selective agonists or selective antagonists of the human melanocortin-4 receptor (MC-4R). The agonists can be used for the treatment of disorders and diseases such as obesity, diabetes and sexual dysfunction, whereas the antagonists are useful for the treatment of disorders and diseases such as cancer cachexia, muscle wasting, anorexia, anxiety and depression. Generally all diseases and disorders where the regulation of the MC-4R is involved can be treated with the compounds of the invention formula (I).

Description

Substituted Phenylpiperidine derivatives as melanocortin-4 receptor modulators

Field of the Invention

The present invention relates to substituted phenylpiperidine derivatives as melano- cortin-4 receptor modulators. Depending on the structure and the stereochemistry the compounds of the invention are either selective agonists or selective antagonists of the human melanocortin-4 receptor (MC-4R). The agonists can be used for the treatment of disorders and diseases such as obesity, diabetes and sexual dysfunction, whereas the antagonists are useful for the treatment of disorders and diseases such as cancer cachexia, muscle wasting, anorexia, anxiety and depression. Generally all diseases and disorders where the regulation of the MC-4R is involved can be treated with the compounds of the invention.

Background of the Invention

Melanocortins (MCs) stem from proopiomelanocortin (POMC) via proteolytic cleavage. These peptides, adrenocorticotropic hormone (ACTH), α-melanocyte-stimulating hor- mone (α-MSH), β-MSH and γ-MSH, range in size from 12 to 39 amino acids. The most important endogenous agonist for central MC-4R activation appears to be the tride- capeptide α-MSH. Among MCs, it was reported that α-MSH acts as a neurotransmitter or neuromodulator in the brain. MC peptides, particularly α-MSH, have a wide range of effects on biological functions including feeding behavior, pigmentation and exocrine function. The biological effects of α-MSH are mediated by a sub-family of 7- transmembrane G-protein-coupled receptors, termed melanocortin receptors (MC-Rs). Activation of any of these MC-Rs results in stimulation of cAMP formation.

To date, five distinct types of receptor subtype for MC (MC-1R to MC-5R) have been identified and these are expressed in different tissues. MC-1R was first found in melanocytes. Naturally occurring inactive variants of MC-1R in animals were shown to lead to alterations in pigmentation and a subsequent lighter coat color by controlling the conversion of phaeomelanin to eumelanin through the con- trol of tyrosinase. From these and other studies, it is evident that MC-1R is an impor- tant regulator of melanin production and coat color in animals and skin color in hu- mans.

The MC-2R is expressed in the adrenal gland representing the ACTH receptor. The MC-2R is not a receptor for α -MSH but is the receptor for the adrenocorticotropic hormone I (ACTH I).

The MC-3R is expressed in the brain (predominately located in the hypothalamus) and peripheral tissues like gut and placenta, and knock-out studies have revealed that the MC-3R may be responsible for alterations in feeding behavior, body weight and ther- mogenesis.

The MC-4R is primarily expressed in the brain. Overwhelming data support the role of MC-4R in energy homeostasis. Genetic knock-outs and pharmacologic manipulation of MC-4R in animals have shown that agonizing the MC-4R causes weight loss and an- tagonizing the MC-4R produces weight gain (A. Kask, et al., "Selective antagonist for the melanocortin-4 receptor (HS014) increases food intake in free-feeding rats," Bio- chem. Biophys. Res. Commun., 245: 90-93 (1998)).

MC-5R is ubiquitously expressed in many peripheral tissues including white fat, pla- centa and a low level of expression is also observed in the brain. However its expres- sion is greatest in exocrine glands. Genetic knock-out of this receptor in mice results in altered regulation of exocrine gland function, leading to changes in water repulsion and thermoregulation. MC-5R knockout mice also reveal reduced sebaceous gland lipid production (Chen et al., Cell, 91: 789-798 (1997)).

Attention has been focused on the study of MC-3R and MC-4R modulators and their use in treating body weight disorders, such as obesity and anorexia. However, evi- dence has shown that the MC peptides have potent physiological effects besides their role in regulating pigmentation, feeding behavior and exocrine function. In particular, α- MSH recently has been shown to induce a potent anti-inflammatory effect in both acute and chronic models of inflammation including inflammatory bowel-disease, renal ischemia/reperfusion injury and endotoxin-induced hepatitis. Administration of α-MSH in these models results in substantial reduction of inflammation-mediated tissue dam- age, a significant decrease in leukocyte infiltration and a dramatic reduction in elevated levels of cytokines and other mediators to near baseline levels. Recent studies have demonstrated that the anti-inflammatory actions of α-MSH are mediated by MC-1R. The mechanism by which agonism of MC-1 R results in an anti-inflammatory response is likely through inhibition of the pro-inflammatory transcription activator, NF-κB. NF-κB is a pivotal component of the pro-inflammatory cascade, and its activation is a central event in initiating many inflammatory diseases. Additionally, anti-inflammatory actions of α-MSH may be, in part, mediated by agonism of MC-3R and/or MC-5R.

A specific single MC-R that may be targeted for the control of obesity has not yet been identified, although evidence has been presented that MC-4R signaling is important in mediating feeding behavior (S.Q. Giraudo et al., "Feeding effects of hypothalamic injec- tion of melanocortin-4 receptor ligands," Brain Research, 80: 302-306 (1998)). Further evidence for the involvement of MC-Rs in obesity includes: 1 ) the agouti (A^vy) mouse which ectopically expresses an antagonist of the MC-1 R, MC-3R and MC-4R is obese, indicating that blocking the action of these three MC-R's can lead to hyperphagia and metabolic disorders; 2) MC-4R knockout mice (D. Huszar et al., Cell, 88: 131-141 (1997)) recapitulate the phenotype of the agouti mouse and these mice are obese; 3) the cyclic heptapeptide melanotanin Il (MT-II) (a non-selective MC-1 R, -3R, -4R, and - 5R agonist) injected intracerebroventricularly (ICV) in rodents, reduces food intake in several animal feeding models (NPY, ob/ob, agouti, fasted) while ICV injected SHU- 9119 (MC-3R and 4R antagonist; MC-1 R and -5R agonist) reverses this effect and can induce hyperphagia; 4) chronic intraperitoneal treatment of Zucker fatty rats with an α- NDP-MSH derivative (HP-228) has been reported to activate MC-1 R, -3R, -4R, and - 5R and to attenuate food intake and body weight gain over a 12 week period (I. Corcos et al., "HP-228 is a potent agonist of melanocortin receptor-4 and significantly attenu- ates obesity and diabetes in Zucker fatty rats," Society for Neuroscience Abstracts, 23: 673 (1997)).

MC-4R appears to play a role in other physiological functions as well, namely control- ling grooming behavior, erection and blood pressure. Erectile dysfunction denotes the medical condition of inability to achieve penile erection sufficient for successful inter- course. The term "impotence" is often employed to describe this prevalent condition. Synthetic melanocortin receptor agonists have been found to initiate erections in men with psychogenic erectile dysfunction (H. Wessells et al., "Synthetic Melanotropic Pep- tide Initiates Erections in Men With Psychogenic Erectile Dysfunction: Double-Blind, Placebo Controlled Crossover Study," J. Urol., 160: 389-393, 1998). Activation of melanocortin receptors of the brain appears to cause normal stimulation of sexual arousal. Evidence for the involvement of MC-R in male and/or female sexual dysfunc- tion is detailed in WO 00/74679.

Diabetes is a disease in which a mammal's ability to regulate glucose levels in the blood is impaired because the mammal has a reduced ability to convert glucose to gly- cogen for storage in muscle and liver cells. In Type I diabetes, this reduced ability to store glucose is caused by reduced insulin production. "Type Il diabetes" or "Non- Insulin Dependent Diabetes Mellitus" (NIDDM) is the form of diabetes which is due to a profound resistance to insulin stimulating or regulatory effect on glucose and lipid me- tabolism in the main insulin-sensitive tissues, muscle, liver and adipose tissue. This resistance to insulin responsiveness results in insufficient insulin activation of glucose uptake, oxidation and storage in muscle, and inadequate insulin repression of lipolysis in adipose tissue and of glucose production and secretion in liver. When these cells become desensitized to insulin, the body tries to compensate by producing abnormally high levels of insulin and hyperinsulemia results. Hyperinsulemia is associated with hypertension and elevated body weight. Since insulin is involved in promoting the cellu- lar uptake of glucose, amino acids and triglycerides from the blood by insulin sensitive cells, insulin insensitivity can result in elevated levels of triglycerides and LDL which are risk factors in cardiovascular diseases. The constellation of symptoms which in- eludes hyperinsulemia combined with hypertension, elevated body weight, elevated triglycerides and elevated LDL, is known as Syndrome X. MC-4R agonists might be useful in the treatment of NIDDM and Syndrome X.

Among MC receptor subtypes, the MC4 receptor is also of interest in terms of the rela- tionship to stress and the regulation of emotional behavior, as based on the following findings. Stress initiates a complex cascade of responses that include endocrine, bio- chemical and behavioral events. Many of these responses are initiated by release of corticotropin-releasing factor (CRF) (Owen MJ and Nemeroff CB (1991) Physiology and pharmacology of corticotrophin releasing factor. Pharmacol Rev 43: 425-473). In addition to activation of the brain CRF system, there are several lines of evidence that melanocortins (MCs), which stem from proopiomelanocortin by enzymatic processing, mediate important behavioral and biochemical responses to stress and, consequently, stress-induced disorders like anxiety and depression (Anxiolytic-Like and Antidepres- sant-Like Activities of MCL0129 (1-[(S)-2-(4-Fluorophenyl)-2-(4-isopropyl-piperazin-1- yl)ethyl]-4- [4-(2-methoxynaphthalen-1-yl)butyl]piperazine), a Novel and Potent Non- peptide Antagonist of the Melanocortin-4 Receptor; Shigeyuki Chaki et al, J. Pharm. Exp. Ther. (2003) 304(2), 818-26).

Chronic diseases, such as malignant tumors or infections, are frequently associated with cachexia resulting from a combination of a decrease in appetite and a loss of lean body mass. Extensive loss of lean body mass is often triggered by an inflammatory process and is usually associated with increased plasma levels of cytokines (e.g. TNF- α), which increase the production of α-MSH in the brain. Activation of MC4 receptors in the hypothalamus by α-MSH reduces appetite and increases energy expenditure. Ex- perimental evidence in tumor bearing mice suggests that cachexia can be prevented or reversed by genetic MC4 receptor knockout or MC4 receptor blockade. The increased body weight in the treated mice is attributable to a larger amount of lean body mass, which mainly consists of skeletal muscle (Marks D.L. et al. Role of the central melano- cortin system in cachexia. Cancer Res. (2001) 61: 1432-1438).

Modulators of the melanocortin receptor have been previously described in the litera- ture.

WO 02/059117 relates to piperazine and piperidine derivatives as melanocortin recep- tor agonists. The compounds exhibit a mono- or bicyclic heterocyclic system connected via an substituted alkylamino chain with the piperazine or piperidine moiety. Pharmacological data of the compounds are not reported.

WO 03/009847 A1 describes substituted phenylpiperidines derivatives which are con- sidered to be useful in the treatment of obesity.

WO 04/083209 A1 describes substituted piperidine and piperazine derivatives which can be used in the treatment of disorders and diseases responsive to the modulation of the melanocortin-4 receptor. WO 04/024720 A1 describes piperazine urea derivatives as melanocortin- 4 receptor agonists. The compounds are prepared by the reaction of a 4-substituted piperidine with an N-protected amino acid derivative followed by removal of the amine protecting group to afford an intermediate amine. Said amine is then converted into the desired unsymmetrical piperazine urea compound. No pharmacological acitivity of the interme- diate amine is reported.

WO 05/047253 reports on compounds effective as agonists of the melanocortin recep- tor. The compounds have a substituted piperidine ring as the core moiety which is mandatorily disubstituted in the para position to the nitrogen atom. As agonists, these compounds are described to be useful in the prevention and treatment of obesity, dia- betes, inflammation and erectile dysfunction.

In view of the unresolved deficiencies in treatment of various diseases and disorders as discussed above, it is an object of the present invention to provide novel substituted phenylpiperidine derivatives with improved ability to cross the blood brain barrier, which are useful as melanocortin-4 receptor modulators to treat cancer cachexia, mus- cle wasting, anorexia, anxiety, depression, obesity, diabetes, sexual dysfunction and other diseases with MC-4R involvement.

Summary of the Invention

The present invention relates to substituted phenylpiperidine derivatives of structural formula (I) and structural formula (II)

(I)

wherein R¹ , R² _, R³ , R and n are defined as described below.

The phenylpiperidine derivatives of structural formulas (I) and (II) are effective as melanocortin receptor modulators and are particularly effective as selective melano- cortin-4 receptor (MC-4R) modulators. They are therefore useful for the treatment of disorders where the activation or inactivation of the MC-4R are involved. Agonists can be used for the treatment of disorders and diseases such as obesity, diabetes and sexual dysfunction, whereas the antagonists are useful for the treatment of disorders and diseases such as cancer cachexia, muscle wasting, anorexia, anxiety and depression.

The present invention also relates to pharmaceutical compositions comprising the compounds of the present invention and a pharmaceutically acceptable carrier.

Detailed Description of the Invention

The present invention relates to substituted phenylpiperidine derivatives useful as melanocortin receptor modulators, in particular, selective MC-4R agonists and MC-4R antagonists.

The compounds of the present invention are represented by structural formula (I)

and enantiomers, diastereomers, tautomers, solvates and pharmaceutically acceptable salts thereof, wherein R¹ is -(C(R⁸)2)l-T,

-O-(C(R⁸)₂)_m-T, T is NR⁵R⁶, morpholine,

.

R⁵ and R⁶ are independently

H C_1-6-alkyl,

C₂.₆-alkenyl,

C_2-6-alkinyl,

C_2-6-alkylene-O-C_1-6-alkyl wherein each alkyl, alkenyl and alkinyl is optionally substituted by one or more substituents selected from halogen atoms, CN and OH,

R⁷ is halogen,

CN,

OH,

C_1-6-alkyl, optionally substituted with 1 to 3 substituents selected from halo- gen, CN and OH,

O-C_1-6-alkyl optionally substituted with 1 to 3 substituents selected from halogen, CN and OH,

C_1-6-alkylene-O-C_1-6-alkyl optionally substituted with 1 to 3 substituents selected from halogen, CN and OH, R⁸ is independently

H,

F, OH,

OMe,

C_1-6-alkyl, optionally substituted with 1 to 3 substituents selected from halogen, CN, OH and OMe,

C_3-6-cycloalkyl, optionally substituted with 1 to 3 substituents selected from halogen, CN, OH and OMe, R⁹ is independently H,

C_1-6-alkyl, optionally substituted with 1 to 3 substituents selected from halo- gen, CN, OH and OMe,

C_3-6-cycloalkyl, optionally substituted with 1 to 3 substituents selected from halogen, CN₁ OH and OMe, X is CH, N, Yis CH, N, Zis CH₁N,

R² is F, Cl₁ methyl, R³ is Cl, methyl,

I is 3, 4, m is 2, 3, 4, nis 0,1,2,3,4, ois 0,1,2, pis 0, 1,2,3,4.

Preferably, the compounds according to formula (I) adopt the structural conformation of the following stereoisomer (formula (I'):

In a preferred embodiment, R² represents Cl. Preferably, the phenyl ring is monosubsti- tuted by a chlorine atom in the meta or para-position. In a further preferred embodiment, the phenyl ring is disubstituted by fluorine atoms.

In a further preferred embodiment, R³ represents Cl.

The variant I is preferably 3.

The variant m is preferably selected from 2 or 3.

In a preferred embodiment, R⁸ is independently selected from H, F, OH, OMe, C_1-6- alkyl, and C_3-6-cycloalkyl, wherein alkyl and cycloalkyl are optionally substituted with 1 to 3 substituents selected from halogen, CN, OH and OMe, provided that at least one R⁸ is not hydrogen, and R⁹ is hydrogen.

In a further preferred embodiment, R⁸ is independently selected from H₁ F, OH₁ OMe, C₁-₆-alkyl, and C_3-6-cycloalkyl, wherein alkyl and cycloalkyl are optionally substituted with 1 to 3 substituents selected from halogen, CN, OH and OMe, and R⁹ is H, C_1-6- alkyl, and C_3-6 -cycloalkyl, wherein alkyl and cycloalkyl are optionally substituted with 1 to 3 substituents selected from halogen, CN, OH and OMe with the proviso that at least one R⁹ is not hydrogen.

It is further preferred that R⁸ and R⁹ are both hydrogen, R⁵ and R⁶ are independently selected from C_1-6-alkyl, C_2-6-alkenyl, C₂-β-alkinyl, C_2-6-alkylene-O- C_{1 -6}alkyl, and R⁷ is halogen, CN, OH₁ C_{1 -6}-alkyl, and O-C_1-6-alkyl wherein each alkyl is optionally substituted with 1 to 3 substituents selected from halogen, CN and OH.

The present invention further relates to compounds according to formula (II)

95

11

and enantiomers, diastereomers, tautomers, solvates and pharmaceutically acceptable salts thereof, wherein R¹ is -(C(R⁸)₂)rT,

-O-(C(R⁸)₂)_m-T, T is NR⁵R⁶, morpholine,

R⁵ and R⁶ are independently H

C_{1 -6}alkyl,

C_2-6-alkenyl,

C_2-6-alkinyl,

C_2-6-alkylene-O- C_{1 -6}-alkyl wherein each alkyl, alkenyl and alkinyl is optionally substituted by one or more substituents selected from halogen atoms, CN and OH, R⁷ is halogen,

CN,

OH, C_1-6-alkyl optionally substituted with 1 to 3 substituents selected from halogen, CN and OH,

C

_1-6 alkylene-O- C_1-6-alkyl optionally substituted with 1 to 3 substituents selected from halogen, CN and OH,

R⁸ is independently

H,

F,

OH, OMe, C_1-6alkyl, optionally substituted with 1 to 3 substituents selected from halogen, CN, OH and OMe,

C₃-₆-cycloalkyl, optionally substituted with 1 to 3 substituents selected from halogen, CN, OH and OMe, R⁹ is independently

H,

C_3-6-cycloalkyl, optionally substituted with 1 to 3 substituents selected from halogen, CN, OH and OMe

Xis CH₁N, Y is CH, N, Zis CH₁N, R² is F, Cl₁ methyl, R³ is H,

F₁

Cl, with the proviso that R³ and R⁴ cannot simultaneously be Cl,

R⁴ is H,

Cl,

F,

I is 3, 4, m is 2, 3, 4, nis 0,1,2,3,4, ois 0,1,2, pis 0,1,2,3,4.

Preferably, the compounds according to formula (II) adopt the structural conformation of the following stereoisomer (formula (II'):

In a preferred embodiment, R² represents Cl and/or F. Preferably, the phenyl ring monosubstituted by a fluorine atom in the ortho, meta or para-position, most preferably in the para-position. It is further preferred that the phenyl ring is monosubstituted by a chlorine atom in the meta or para-position. In a further preferred embodiment, the phenyl ring is disubstituted by fluorine atoms.

In a further preferred embodiment, R³ represents H or F.

The variant I is preferably 3.

The variant m is preferably selected from 2 or 3.

In a preferred embodiment, R⁸ is independently selected from H, F, OH, OMe, C_{1 -6}- alky!, and C_3-6-cycloaIkyl, wherein alkyl and cycloalkyl are optionally substituted with 1 to 3 substituents selected from halogen, CN, OH and OMe, provided that at least one R⁸ is not hydrogen, and R⁹ is hydrogen.

In a further preferred embodiment, R⁸ is independently selected from H, F, OH, OMe, C₁-₆-alkyl, and C_3-6-cycloalkyl, wherein alkyl and cycloalkyl are optionally substituted with 1 to 3 substituents selected from halogen, CN, OH and OMe, and R⁹ is independently selected from H, C_{1 -6}-alkyl, and C_3-6-cycloalkyl, wherein alkyl and cycloalkyl are optionally substituted with 1 to 3 substituents selected from halogen, CN₁ OH and OMe, with the proviso that at least one R⁹ is not hydrogen.

It is further preferred that R⁸ and R⁹ are both hydrogen, R⁵ and R⁶ are independently selected from C_1-6-alkyl, C_2-6-alkenyl, C_2-6-alkinyl, C₂-₆-alkylene-O- C_{1 -6}-alkyl, R⁷ is selected from halogen, CN, OH, C_{1 -6}-alkyI, O- C_{1 -6}-alkyl wherein alkyl is optionally substi- tuted with 1 to 3 substituents selected from halogen, CN and OH, R³ is selected from H, and F, and R⁴ is selected from Cl₁ and F.

As regards compounds of formulas (I) or (II), R¹ is preferably selected from the follow- ing radicals:

In a further preferred embodiment of compounds of formula (I) as well as of com- pounds of formula (II), T is preferably selected from the following radicals:

Compounds of the formula (I) or (II) in which some or all of the above-mentioned groups have the preferred or more preferred meanings are also an object of the present invention.

In the above and the following, the employed terms have the meaning as described below:

Alkyl is a straight chain or branched alkyl having 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, or hexyl.

Alkenyl is a straight chain or branched alkyl having 2 to 6 carbon atoms and which contains at least one carbon-carbon double bond, such as vinyl, allyl, 1-propenyl, 2-butenyl, 2-methyl-2-butenyl, isopropenyl, pentenyl, or hexenyl.

Alkinyl is a straight chain or branched alkyl having 2 to 6 carbon atoms and which contains at least one carbon-carbon triple bond, such as ethinyl, 1-propinyl, 1-butinyl, 2- butinyl, pentinyl or hexinyl.

Cycloalkyl is an saturated alkyl ring having 3 to 6 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.

The compounds of structural formulas (I) and (II) are effective as melanocortin receptor modulators and are particularly effective as selective modulators of MC-4R. They are therefore useful for the treatment and/or prevention of disorders responsive to the acti- vation and inactivation of MC-4R, such as cancer cachexia, muscle wasting, anorexia, anxiety, depression, obesity, diabetes, sexual dysfunction and other diseases with MC- 4R involvement.

Optical Isomers - Diastereomers - Geometric Isomers - Tautomers

Compounds of structural formulas (I) and (II) contain one or more asymmetric centers and can occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. The present invention is meant to comprehend all such isomeric forms of the compounds of structural formulas (I) and (II). Compounds of structural formulas (I) and (II) may be separated into their individual di- astereoisomers by, for example, fractional crystallization from a suitable solvent, for example methanol or ethyl acetate or a mixture thereof, or via chiral chromatography using an optically active stationary phase. Absolute stereochemistry may be deter- mined by X-ray crystallography of crystalline products or crystalline intermediates which are derivatized, if necessary, with a reagent containing an asymmetric center of known absolute configuration.

Alternatively, any stereoisomer of a compound of the general formulas (I) and (II) may be obtained by stereospecific synthesis using optically pure starting materials or re- agents of known absolute configuration.

Salts

The term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceu- tically acceptable non-toxic bases or acids including inorganic or organic bases and inorganic or organic acids. Salts derived from inorganic bases include aluminum, am- monium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, man- ganous, potassium, sodium, zinc and the like. Particularly preferred are the ammonium, calcium, lithium, magnesium, potassium and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as arginine, betaine, caffeine, cho- line, N.N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2- dimethylamino-ethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N- ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, ly- sine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like.

When the compound of the present invention is basic, salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethane- sulfonic, formic, furnaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lac- tic, maleic, malic, mandelic, methanesulfonic, malonic, mucic, nitric, parnoic, pan- tothenic, phosphoric, propionic, succinic, sulfuric, tartaric, p-toluenesulfonic, trifluoroacetic acid and the like. Particularly preferred are citric, fumaric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric and tartaric acids.

It will be understood that, as used herein, references to the compounds of formulas (I) and (II) are meant to also include the pharmaceutically acceptable salts.

Utility

Compounds of formula (I) are melanocortin receptor antagonists and as such are use- ful in the treatment, control or prevention of diseases, disorders or conditions respon- sive to the inactivation of one or more of the melanocortin receptors including, but not limited to, MC-1R, MC-2R, MC-3R, MC-4R or MC-5R. Such diseases, disorders or conditions include, but are not limited to, cancer cachexia, muscle wasting, anorexia, anxiety and depression.

Compounds of formula (II) are melanocortin receptor agonists and as such are useful in the treatment, control or prevention of diseases, disorders or conditions responsive to the activation of one or more of the melanocortin receptors including, but not limited to, MC-1 R, MC-2R, MC-3R, MC-4R or MC-5R. Such diseases, disorders or conditions include, but are not limited to, obesity (by reducing appetite, increasing metabolic rate, reducing fat intake or reducing carbohydrate craving), diabetes mellitus (by enhancing glucose tolerance, decreasing insulin resistance) and male and female sexual dysfunc- tion (including impotence, loss of libido and erectile dysfunction).

The compounds of formulas (I) and (II) can be further used in the treatment, control or prevention of diseases, disorders or conditions which are responsive to the activation or inactivation of one or more melanocortin receptors including, but not limited to, MC-

1 R, MC-2R, MC-3R, MC-4R or MC-5R. Such diseases, disorders or conditions include, but are not limited to, hypertension, hyperlipidemia, osteoarthritis, cancer, gall bladder disease, sleep apnea, compulsion, neuroses, insomnia/sleep disorder, substance abuse, pain, fever, inflammation, immune-modulation, rheumatoid arthritis, skin tan- ning, acne and other skin disorders, neuroprotective and cognitive and memory en- hancement including the treatment of Alzheimer's disease.

Administration and Dose Ranges

Any suitable route of administration may be employed for providing a mammal, espe- cially a human with an effective dosage of a compound of the present invention. For example, oral, rectal, topical, parenteral, ocular, pulmonary, nasal and the like may be employed. Dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols and the like. Preferably compounds of formulas (I) and (II) are administered orally or topically.

The effective dosage of active ingredient employed may vary depending on the particu- lar compound employed, the mode of administration, the condition being treated and the severity of the condition being treated. Such dosage may be ascertained readily by a person skilled in the art.

When treating cancer cachexia, muscle wasting or anorexia generally satisfactory re- suits are obtained when the compounds of the present invention are administered at a daily dosage of from about 0.001 milligram to about 100 milligrams per kilogram of body weight, preferably given in a single dose or in divided doses two to six times a day, or in sustained release form. In the case of a 70 kg adult human, the total daily dose will generally be from about 0.07 milligrams to about 3500 milligrams. This dos- age regimen may be adjusted to provide the optimal therapeutic response.

When treating obesity, in conjunction with diabetes and/or hyperglycemia, or alone, generally satisfactory results are obtained when the compounds of the present inven- tion are administered at a daily dosage of from about 0.001 milligram to about 100 mil- ligrams per kilogram of body weight, preferably given in a single dose or in divided doses two to six times a day, or in sustained release form. In the case of a 70 kg adult human, the total daily dose will generally be from about 0.07 milligrams to about 3500 milligrams. This dosage regimen may be adjusted to provide the optimal therapeutic response. When treating diabetes mellitus and/or hyperglycemia, as well as other diseases or disorders for which compounds of formulas (I) and (II) are useful, generally satisfactory results are obtained when the compounds of the present invention are administered at a daily dosage of from about 0.001 milligram to about 100 milligram per kilogram of animal body weight, preferably given in a single dose or in divided doses two to six times a day, or in sustained release form. In the case of a 70 kg adult human, the total daily dose will generally be from about 0.07 milligrams to about 3500 milligrams. This dosage regimen may be adjusted to provide the optimal therapeutic response.

For the treatment of sexual dysfunction, compounds of the present invention are given in a dose range of 0.001 milligram to about 100 milligram per kilogram of body weight, preferably as a single dose orally or as a nasal spray.

Formulation

The compounds of formulas (I) and (II) are preferably formulated into a dosage form prior to administration. Accordingly the present invention also includes a pharmaceuti- cal composition comprising a compound of formulas (I) or (II) and a suitable pharma- ceutical carrier.

The present pharmaceutical compositions are prepared by known procedures using well-known and readily available ingredients. In making the formulations of the present invention, the active ingredient (a compound of formulas (I) or (II)) is usually mixed with a carrier, or diluted by a carrier, or enclosed within a carrier, which may be in the form of a capsule, sachet, paper or other container. When the carrier serves as a diluent, it may be a solid, semisolid or liquid material which acts as a vehicle, excipient or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosol (as a solid or in a liquid medium), soft and hard gelatin capsules, sup- positories, sterile injectable solutions and sterile packaged powders. Some examples of suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tra- gacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellu- lose, water syrup, methyl cellulose, methyl and propylhydroxybenzoates, talc, magne- sium stearate and mineral oil. The formulations can additionally include lubricating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweet- ening agents or flavoring agents. The compositions of the invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after ad- ministration to the patient.

Preparation of Compounds of the Invention

When describing the preparation of the present compounds of formulas (I) and (II), the terms "A moiety" and "B moiety" are used below. This moiety concept is illustrated be- low with formula (I) but also applies to formula (II):

The skilled artisan will recognize that, in general, the two moieties of a compound of formulas (I) and (II) are connected via an amide bond. The skilled artist can, therefore, readily envision numerous routes and methods of connecting the two moieties via standard peptide coupling reaction conditions.

The phrase "standard peptide coupling reaction conditions" means coupling a carbox- ylic acid with an amine using an acid activating agent such as EDCI, dicyclo- hexylcarbodiimide and benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluoro-phosphate, in a inert solvent such as DCM, in the presence of a catalyst such as HOBt. The uses of protective groups for amine and carboxylic acids to facili- tate the desired reaction and minimize undesired reactions are well documented. Con- ditions required to remove protecting groups which may be present can be found in Greene, et al., Protective Groups in Organic Synthesis, John Wiley & Sons, Inc., New York, NY 1991. Protecting groups like Z, Boc and Fmoc are used extensively in the synthesis, and their removal conditions are well known to those skilled in the art. For example, removal of Z groups can he achieved by catalytic hydrogenation with hydrogen in the presence of a noble metal or its oxide, such as palladium on activated carbon in a protic solvent, such as ethanol. In cases where catalytic hydrogenation is contraindicated by the pres- ence of other potentially reactive functionality, removal of Z can also be achieved by treatment with a solution of hydrogen bromide in acetic acid, or by treatment with a mixture of TFA and dimethylsulfide. Removal of Boc protecting groups is carried out in a solvent such as methylene chloride, methanol or ethyl acetate with a strong acid, such as TFA or HCI or hydrogen chloride gas.

The compounds of formulas (I) and (II), when existing as a diastereomeric mixture, may be separated into diastereomeric pairs of enantiomers by fractional crystallization from a suitable solvent such as methanol, ethyl acetate or a mixture thereof. The pair of enantiomers thus obtained may be separated into individual stereoisomers by conven- tional means by using an optically active acid as a resolving agent. Alternatively, any enantiomer of a compound of the formulas (I) and (II) may be obtained by stereospeci- fic synthesis using optically pure starting materials or reagents of known configuration.

The compounds of formulas (I) and (II) of the present invention can be prepared ac- cording to the procedures of the following Schemes and Examples, using appropriate materials and are further exemplified by the following specific examples. Moreover, by utilizing the procedures described herein, in conjunction with ordinary skills in the art, additional compounds of the present invention claimed herein can be readily prepared. The compounds illustrated in the examples are not, however, to be construed as form- ing the only genus that is considered as the invention. The Examples further illustrate details for the preparation of the compounds of the present invention. Those skilled in the art will readily understand that known variations of the conditions and processes of the following preparative procedures can be used to prepare these compounds. The instant compounds are generally isolated in the form of their pharmaceutically acceptable salts, such as those described previously. The free amine bases corresponding to the isolated salts can be generated by neutralization with a suitable base, such as aqueous sodium hydrogencarbonate, sodium carbonate, sodium hydroxide and potas- sium hydroxide, and extraction of the liberated amine free base into an organic solvent followed by evaporation. The amine free base isolated in this manner can be further converted into another pharmaceutically acceptable salt by dissolution in an organic solvent followed by addition of the appropriate acid and subsequent evaporation, pre- cipitation or crystallization. All temperatures are degrees Celsius.

In the schemes, preparations and examples below, various reagent symbols and ab- -reviations have the following meanings:

AcOH acetic acid

Boc tert-butoxycarbonyl Bz₂O₂ dibenzoylperoxide

DCM dichloromethane

DEAD diethyl azodicarboxylate

DIAD diisopropyl azodicarboxylate

DIEA ethyl-diisopropylamine DMA N,N-dimethylacetamide

DMAP 4-dimethylaminopyridine

DMF N,N-dimethylformamide

DMSO dimethylsulfoxide dppf 1 ,1 '-bis(diphenylphosphino)-ferrocene EDCI 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

Et₂O diethyl ether

EtOAc ethyl acetate

EtOH ethanol

HOBt 1-hydroxybenzotriazole h hour(s)

MeCN acetonitrile

MeOH methanol

NBS N-bromosuccinimide

NMM N-methylmorpholine PPh₃ triphenylphosphine

TEBAC benzyltriethylammonium chloride

TFA trifluoroacetic acid

THF tetrahydrofurane

TMSCI trimethylsilylchloride The following amino acid derivatives were custom synthesized by PepTech Corporation, 20 Mall Road, Suite 460, Burlington, MA 01803 USA: Boc-D-2-chloro-4- fluorophenylalanine, Boc-D-4-chloro-2-fluorophenylalanine, and Boc-D-2,4-difluoro- phenylalanine.

Reaction scheme 1 :

Synthesis of A Moieties with Alkylether Spacer (R¹ = -O(C(R⁸)₂)_m-T)

As shown in Reaction Scheme 1 , optionally substituted 2-bromo-phenol and 4-(4,4,5,5- tetramethyl-[1 ,3,2]dioxaborolan-2-yl)-3,6-dihydro-2H-pyridine-i-carboxylic acid tert- butyl ester (Tetrahedron Lett. 2000, 41, 3705-3708) are reacted in a Suzuki coupling in the presence of a base such as K₂CO₃ and a catalyst such as dichloro(1,1'- bis(diphenyl-phosphino)-ferrocene)palladiurn(ll) DCM adduct, in an organic solvent such as DMF or toluene, at a suitable temperature. The resulting tetrahydropyridine can be hydrogenated in the presence of a catalyst, such as PtO₂ or Pd/C, to yield the protected piperidine. The piperidine is further reacted with an alkylchloride or alkylbro- mide bearing the capping group T in the presence of a base such as Cs₂CO₃ or NaH in an appropriate solvent such as DMF to give the Boc-protected A moiety. Reaction scheme 2:

Alternative Synthesis of A Moieties with Alkylether Spacer (R¹ = -O(C(R⁸)₂)_m-T)

The synthesis of A Moieties bearing an alkylether spacer (R¹ = -O(C(R⁸)₂)_m-T) can alternatively be performed starting from optionally substituted 2-bromoanisole (see Reaction scheme 2). A Suzuki coupling with 4-(4,4,5₎5-tetramethyl-[1,3,2]dioxaborolan-2-yl)- 3,6-dihydro-2H-pyridine-1-carboxylic acid tert-butyl ester in the presence of a base such as K₂CO₃ and a catalyst such as dichloro(1,1'-bis(diphenylphosphino)- ferrocene)palladium(ll) DCM adduct, in an organic solvent such as DMF or toluene, at a suitable temperature leads to the corresponding tetrahydropyridine. The resulting tetrahydropyridine can be hydrogenated in the presence of a catalyst, such as PtO₂ or Pd/C, to yield the protected piperidine. The methylether can be cleaved with a reagent such as aqueous hydroiodic acid in acetic acid or trimethylsilyl iodide in chloroform, at a suitable temperature to get access to the corresponding phenol as hydroiodide. The Boc-protecting group, which is lost during this process, can subsequently be reintroduced by using a reagent such as BoC₂O in the presence of a base such as DIEA in an appropriate solvent such as DCM or DMF. The Boc-protected piperidine is further re- acted with an alkylchloride or alkylbromide bearing the capping group T in the presence of a base such as Cs₂CO₃ or NaH in an appropriate solvent such as DMF to give the Boc-protected A moiety.

Reaction scheme 3:

Synthesis of A Moieties with Aikylether Spacer (R¹ = -O(C(R⁸)₂)_m-T) Using Mit- sunobu Conditions

As shown in Reaction scheme 3, the intermediate product from Reaction schemes 1 and 2, optionally substituted 1-Boc-4-(2-hydroxy-phenyl)-piperidine, can also be alkylated with an ω-T-capped alkylalcohol in the presence of a reagent such as DEAD or DIAD and a phosphine such as PPh₃ in a suitable solvent such as THF to give the Boc- protected A moieties.

Similarly, the same intermediate can be reacted with an ω-bromo alkylalcohol, using the reaction conditions described above, to give access to the corresponding phe- nolether which subsequently can be used to alkylate the capping group T in the presence of a suitable base such as K₂CO₃ or NaH, in an appropriate solvent such as MeCN₁ THF, or DMF, at a suitable temperature, to yield the Boc-protected A moieties. Reaction scheme 4:

Synthesis of A Moieties with Alkylene Spacer (R¹ = -(C{^R8)₂)ι-T)

The first route for the synthesis of A moieties bearing an alkylene spacer (R¹ = - (C(R⁸)₂)ι-T) is depicted in Reaction scheme 4. Optionally substituted 2-bromotoluene is brominated with NBS in the presence of a radical starter such as Bz₂O₂ in an appropriate solvent such as CCI₄ at a suitable temperature to yield the corresponding benzyl- bromide. The benzylbromide is reacted with optionally substituted diethyl malonate in the presence of a base such as sodium ethoxide in a suitable solvent such as ethanol. Subsequent saponification with a base such as KOH in an appropriate solvent such as water-ethanol mixture followed by a second saponification step with a suitable base such as KOH in a solvent such as water leads to the alkylated malonic acid which is decarboxylated at an appropriate temperature. The product of this reaction, optionally substituted 3-(2-bromophenyl)propionic acid, is converted to the acid chloride using a reagent such as oxalyl chloride or thionyl chloride in an inert solvent such as DCM with a catalytic amount of DMF, and reacted with the capping group T to form the corresponding amide. Optionally substituted 3-(2-bromophenyl)propionic acid amide can be reacted with 4-(4,4,5,5-tetramethyl-[1 ,3,2]dioxaborolan-2-yl)-3,6-dihydro-2H-pyridine-1- carboxylic acid tert-butyl ester in the presence of a base such as K₂CO₃ and a catalyst such as dichloro(1,1'-bis(diphenylphosphino)-ferrocene)palladium(ll) DCM adduct, in an organic solvent such as DMF or toluene, at a suitable temperature to lead to the corresponding tetrahydropyridine. The resulting tetrahydropyridine can be hydrogen- ated in the presence of a catalyst, such as PtO₂ or Pd/C, to yield the protected piperidine. The side chain amide function can be reduced using a reagent such as Li- AIH₄ or borane-THF complex in an appropriate inert solvent such as diethyl ether or THF at a suitable temperature to yield the Boc-protected A moiety.

Reaction scheme 5:

Alternative Route for the Synthesis of A Moieties with Alkylene Spacer (R¹ (CH₂),-T)

An alternative approach for the synthesis of of A moieties bearing an alkylene spacer (R¹ = -(CH₂)_I-T) starts with optionally substituted 2-bromobenzaldehyde (see Reaction scheme 5). Reaction with malonic acid in an appropriate solvent such as ethanol, in the presence of a base such as pyridine, at a suitable temperature, leads to the corresponding 2'-bromo-cinnamic acid. Said acid is activated with a reagent such as EDCI in the presence of a catalyst such as DMAP and a base such as NMM in DCM, and reacted with the capping group T to form the corresponding amide. Optionally substituted 2'-bromo-cinnamic acid amide can be reacted with 4-(4,4,5,5-tetramethyl- [1 ,3,2]dioxaborolan-2-yl)-3_I6-dihydro-2H-pyridine-1-carboxylic acid tert-butyl ester in the presence of a base such as K₂CO₃ and a catalyst such as dichloro(1,1'- bis(diphenylphosphino)-ferrocene)palladium(ll) DCM adduct, in an organic solvent such as DMF or toluene, at a suitable temperature to lead to the corresponding tetrahydro- pyridine. The resulting tetrahydropyridine and the cinnamic acid amide double bond can be hydrogenated in the presence of a catalyst, such as PtO₂ or Pd/C, to yield the protected piperidine. The side chain amide function can be reduced using a reagent such as LiAIH₄ or borane-THF complex in an appropriate inert solvent such as diethyl ether or THF at a suitable temperature to yield the Boc-protected A moiety.

Reaction scheme 6: Alternative Route for the Synthesis of A Moieties with Alkylene Spacer (R¹ -(C(R⁸)₂)rT)

As shown in Reaction scheme 6, optionally substituted 3-(2-bromophenyl)propionic acid, is reacted with methanol in the presence of a catalyst such as sulfuric acid to form the corresponding methyl ester. Optionally substituted 3-(2-bromophenyl)propionic acid ester can be reacted with 4-(4,4,5_I5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-3,6-dihydro- 2H-pyridine-1-carboxylic acid tert-butyl ester in the presence of a base such as K₂CO₃ and a catalyst such as dichloro(1,1'-bis(diphenylphosphino)-ferrocene)palladium(ll) DCM adduct, in an organic solvent such as DMF or toluene, at a suitable temperature to lead to the corresponding tetrahydropyridine. The resulting tetrahydropyridine can be hydrogenated in the presence of a catalyst, such as PtO₂ or Pd/C, to yield the pro- tected piperidine. The side chain ester function can be reduced using a reagent such as LiAIH₄ or borane-THF complex in an appropriate inert solvent such as diethyl ether or THF at a suitable temperature to yield the corresponding alcohol which can subsequently be oxidized using a reagent such as Dess-Martin periodinane in an appropriate solvent such as DCM or using sulfurtrioxide-pyridine complex with a base such as triethylamine in a suitable solvent such as DCM. Optionally substituted 3-(2- bromophenyl)propionyl aldehyde is reacted with the capping group T in the presence of a reducing agent such as sodium triacetoxyborohydride in an appropriate solvent such as 1 ,2-dichloroethane to form the corresponding Boc-protected A moiety.

Reaction scheme 7:

Synthesis of A Moieties with Alkylene Spacer (R¹ = -(C(R⁸)₂)ι-T) Using a Negishi Coupling Reaction

As shown in Reaction scheme 7, the intermediate product from Reaction scheme 6, optionally substituted 3-(2-bromophenyl)propionic acid ester can also be subjected to a Negishi coupling with (1-tert-butoxycarbonylpiperidin-4-yl)(iodo)zinc {J. Org. Chem. 2004, 69, 5120-5123) in the presence of copper(l) iodide and dichloro(1,1'- bis(diphenyl-phosphino)-ferrocene)palladium(ll) DCM adduct in an inert solvent such as DMA to yield the resulting phenylpiperidine which can be further processed as shown in Reaction scheme 6.

Reaction scheme 8: Synthesis of A Moieties with Alkylene Spacer (R¹ = -(C(R⁸)₂),-T)

NaH

THF

A route for the synthesis of A moieties bearing an C₄-alkylene spacer (R¹ = -(C(R⁸)₂)ι-T) is depicted in Reaction scheme 8. Optionally substituted 2-bromophenylacetic acid is reduced with sodium borohydride in the presence of a reagent such like boron trifluoride diethyl etherate in an appropriate solvent such as THF at a suitable temperature to yield the corresponding phenylethylalcohol. Reaction of the alcohol with a bro- mination reagent such as phosphorous tribromide in the presence of a base such as pyridine in an appropriate solvent like toluene at a suitable temperature leads to the phenylethylbromide. The phenethylbromide is reacted with optionally substituted diethyl malonate in the presence of a base such as sodium hydride in a suitable solvent such as THF. Subsequent saponification with a base such as KOH in an appropriate solvent such as water-ethanol mixture followed by a second saponification step with a suitable base such as KOH in a solvent such as water leads to the alkylated malonic acid which is decarboxylated at an appropriate temperature. The product of this reaction, option- ally substituted 3-(2-bromophenyl)butanoic acid, is converted to the acid chloride using a reagent such as oxalyl chloride or thionyl chloride in an inert solvent such as DCM with a catalytic amount of DMF, and reacted with the capping group T to form the cor- responding amide. Optionally substituted 3-(2-bromophenyl)butanoic acid amide can be reacted with 4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-3,6-dihydro-2H- pyridine-1-carboxylic acid tert-butyl ester in the presence of a base such as K₂CO₃ and a catalyst such as dichloro(1,1'-bis(diphenylphosphino)-ferrocene)palladium(ll) DCM adduct, in an organic solvent such as DMF or toluene, at a suitable temperature to lead to the corresponding tetrahydropyridine. The resulting tetrahydropyridine can be hydro- genated in the presence of a catalyst, such as PtO₂ or Pd/C, to yield the protected piperidine. The side chain amide function can be reduced using a reagent such as Li- AIH₄ or borane-THF complex in an appropriate inert solvent such as diethyl ether or THF at a suitable temperature to yield the Boc-protected A moiety.

Reaction scheme 9: Deprotection of A Moiety

Generally, the starting material, a Boc-protected phenylpiperidine (A moiety) can be deprotected in the presence of TFA/CH₂CI₂₎ HCI/EtOAc, HCI/dioxane or HCI in MeOH/dioxane with or without a cation scavenger, such as dimethyl sulfide (DMS) be- fore being subjected to the coupling procedure. It can be converted to the free base before being subjected to the coupling procedure or be used as the salt. Reaction scheme 10:

Coupling of A Moiety with B Moiety and Subsequent Deprotection of the B Moiety

In coupling the A moiety with the protected B moiety shown in reaction scheme 10, an appropriate "A moiety" is coupled to a Boc-protected "B moiety" in the presence of EDCI/HOBt, a base such as N-methylmorpholine (NMM) and a solvent such as di- chloromethane (DCM) followed by Boc deprotection with the aid of hydrochloric acid in an appropriate solvent such as dioxane or a mixture of dioxane and methanol.

A suitable solvent, such as DCM, DMF, THF or a mixture of the above solvents, can be used for the coupling procedure. Suitable bases include triethylamine (TEA), diisopro- pyethylamine (DIEA), N-methylmorpholine (NMM), collidine, and 2,6-lutidine.

A base may not be needed when EDCI/HOBt is used.

Generally after the reaction is completed, the reaction mixture can be diluted with an appropriate organic solvent, such as EtOAc, DCM or Et₂O, which is then washed with aqueous solutions, such as water, HCI, NaHSO₄, bicarbonate, NaH₂PO₄, phosphate buffer (pH 7), brine or any combination thereof. The reaction mixture can be concentrated and then be partitioned between an appropriate organic solvent and an aqueous solution. Alternatively, the reaction mixture can be concentrated and subjected to chromatography without aqueous workup. Reaction scheme 11 : B Moiety N-Alkylation

As shown in Scheme 11, optionally substituted phenylalanine phenylpiperidylamide can be N-alkylated with aqueous formaldehyde solution in the presence of a reducing agent such as sodium triacetoxyborohydride in an appropriate solvent such as 1 ,2- dichloroethane to yield the N,N-dialkylated amine.

Analytical LC-MS

The compounds of the present invention according to formulas (I) and (II) were analyzed by analytical LC-MS. The conditions are summarized below.

Analytical conditions summary:

LC10Advp-Pump (Shimadzu) with SPD-M10Avp UV/Vis diode array detector and

QP2010 MS-detector in ESI+ modus with UV-detection at 214, 254 and 275 nm, Column: Waters XTerra MS C18, 3.5 μm, 2.1 * 100 mm, linear gradient with acetonitrile in water (0.1% HCOOH)

Flow rate of 0,4 ml/min;

Mobile Phase A: water (0.1 % HCOOH)

Mobile Phase B: acetonitrile (0.1 % HCOOH)

Gradient A: linear gradient from 1% to 95% acetonitrile in water (0.1% HCOOH)

0.00 m in 1% B

10.00 min 95% B 10.10 min 99% B

11.40 min 99% B 11.50 min 1% B 13.00 min Pump STOP

Gradient B: linear gradient from 1% to 95% acetonitrile in water (0.1% HCOOH)

0.00 min 1% B

5.00 min 95 % B

5.10 min 99 % B

6.40 min 99 % B

6.50 min 1 % B

8.00 min Pump STOP

Gradient C: linear gradient from 5% to 95% acetonitrile in water (0.1% HCOOH)

0.00 min 5% B

10.00 min 95% B

10.10 min 99% B

11.40 min 99% B

11.50 min 1% B

13.00 min Pump STOP

Gradient D: linear gradient from 5% to 95% acetonitrile in water (0.1% HCOOH)

0.00 min 5% B 5.00 min 95 % B

5.10 min 99 % B

6.40 min 99 % B

6.50 min 1 % B

8.00 min Pump STOP

Gradient E: linear gradient from 10% to 60% acetonitrile in water (0.1% HCOOH)

0.00 min 10% B

10.00 min 60% B 10.10 min 99% B 11.40 min 99% B 11.50 min 1% B 13.00 min Pump STOP

Gradient F: linear gradient from 1% to 30% acetonitrile in water (0.1% HCOOH)

0.00 min 1% B

10.00 min 30% B

10.10 min 99% B

11.40 min 99% B

11.50 min 1% B

13.00 min Pump STOP

Gradient G: linear gradient from 1 % to 70% acetonitrile in water (0.1 % HCOOH)

0.00 min 1% B

10.00 min 70% B

10.10 min 99% B

11.40 min 99% B

11.50 min 1% B

13.00 min Pump STOP

Gradient H: linear gradient from 1% to 60% acetonitrile in water (0.1% HCOOH)

0.00 min 1% B

10.00 min 60% B

10.10 min 99% B

11.40 min 99% B

11.50 min 1% B

13.00 min Pump STOP

The following describes the detailed examples of the invention which can be prepared via the reaction schemes 1 to 11. Table 1 : Antagonists

Table 2: Antagonists

Table 3: Antagonists

Table 4: Antagonists

Table 5: Agonists

Table 6: Agonists

Table 7: Agonists

Table 8: Agonists

Table 9: Agonists

EP2007/001595

50

Table 10: Agonists

Table 11 : Agonists

Table 12: Agonists

7001595

Table 13: Agonists

The following examples are provided to illustrate the invention and are not limiting the scope of the invention in any manner.

Synthesis of Example 1 : Intermediate 1a):

To a solution of 1-Boc-4-(2-hydroxy-phenyl)-piperidine (789 mg) in DMF (15 ml) was added 1-(2-chloroethyl)pyrrolidine hydrochloride (605 mg) and Cs₂CO₃ (3243 mg). The reaction was stirred at room temperature for 18 h. An additional amount of 1-(2- chloroethyl)pyrrolidine hydrochloride (483 mg) and Cs₂CO₃ (926 mg) were added and stirring at room temperature was continued for another 6 h. The reaction mixture was evaporated at 50⁰C in vacuo to dryness and the residue was partitioned between Et₂O (75 ml) and water (25 ml). The aqueous layer was extracted with Et₂O (25 ml). The combined organic layer was washed with water (10 ml) and brine (15 ml). The organic layer was dried over Na₂Sθ₄ and evaporated in vacuo to dryness. The residue was finally dried under high vacuum at room temperature overnight.

Intermediate 1b):

To Boc-protected intermediate 1a) (1002 mg) in methanol (5 ml) was added hydrogen chloride, 4.0 M sol. in 1,4-dioxane (25 ml) and the solution was stirred at room temperature for 2 h. The solvent was removed under reduced pressure. The residue was triturated in acetone (30 ml), filtered off, and washed with acetone (2 x 5 ml). Finally it was dried in vacuo at room temperature over P₂O₅ overnight to yield a white solid.

Intermediate 1c):

To Boc-D-2,4-dichlorophenylalanine (685 mg) in DCM (20 ml) was added the amine hydrochloride from 1b) (347 mg), N-methylmorpholine (311 μl), HOBt (230 mg) and the mixture was stirred for 30 min. EDCI (351 mg) was added and stirring was continued for 1 h. An additional amount of N-methylmorpholine (91 μl) was added and stirred overnight. The reaction mixture was evaporated in vacuo, diluted with EtOAc, washed with sat. Na₂CO₃, H₂O and brine. The aqueous layers were extracted with EtOAc. The combined organic layers were dried over Na₂SO₄, filtered and evaporated in vacuo to dryness. Purification by flash chromatography yielded the title compound as white foam.

Example 1:

To Boc-protected intermediate 1c) (560 mg) in methanol (5 ml) was added hydrogen chloride, 4.0 M sol. in 1 ,4-dioxane (9 ml) and the solution was stirred for 90 min at room temperature. The reaction mixture was evaporated in vacuo to dryness. The residue was triturated in Et₂θ, filtered off and washed with Et₂O to yield a beige solid.

Synthesis of Example 8: lntermediate 8a):

2-Bromo-4,5-difluorophenol (2857 μl), N-Boc-1,2,3,6-tetrahydropyridine-4-boronic acid pinacol ester (7.73 g), potassium carbonate (10.36 g) and dichloro(1,1'-bis(diphenyl- phosphino)-ferrocene)palladium(ll) DCM adduct (1.22 g) were dissolved in DMF (150 ml) in a dry apparatus under argon and the mixture was degassed by bubbling with argon for 30 min. The orange suspension was then heated under argon in an oil bath at

85°C for 1 d to give a dark purple suspension. The reaction mixture was filtered through Celite and evaporated to dryness in vacuo. The crude product was purified by flash chromatography to yield pale green crystals.

Intermediate 8b):

Intermediate 8a) (1404 mg) was dissolved in EtOH (50 ml) and AcOH (50 ml) and platinum(IV) oxide (102 mg) was added. The reaction mixture was evacuated three times and purged with hydrogen. The reaction mixture was then stirred at room temperature for 2 h. The reaction mixture was filtered and evaporated to dryness in vacuo. The residue was coevaporated with toluene (3 x 75 ml) and was finally dried under high vacuum at room temperature overnight to yield a beige solid.

Intermediate 8c):

To a solution of intermediate 8b) (674 mg) in DMF (15 ml) was added 1-(2- chloroethyl)piperidine hydrochloride (605 mg) and Cs₂CO₃ (2453 mg). The reaction was stirred at room temperature for 2 d. An additional amount of 1-(2- chloroethyl)piperidine hydrochloride (199 mg) and CS2CO₃ (352 mg) was added and stirring at room temperature was continued for another 3 d. The reaction mixture was evaporated at 50⁰C in vacuo to dryness and the residue was partitioned between Et₂O (75 ml) and water (25 ml). The aqueous layer was extracted with Et₂O (25 ml). The combined organic layer was washed with water (10 ml) and brine (15 ml). The organic layer was dried over Na₂SO₄ and evaporated in vacuo to dryness. The residue was finally dried under high vacuum at room temperature overnight. The crude product was purified by flash chromatography.

Intermediate 8d):

To Boc-protected intermediate 8c) (490 mg) in methanol (2 ml) and dioxane (10 ml) was added hydrogen chloride, 4.0 M sol. in 1,4-dioxane (10 ml) and the solution was stirred at room temperature for 30 min. The solvent was removed under reduced pressure. The residue was triturated in acetone and Et₂O, filtered off, and washed with Et₂O. Finally it was dried in vacuo at room temperature over P₂O₅ overnight to yield an off-white solid.

Intermediate Be):

To Boc-D-2,4-dichlorophenylalanine (90 mg) in DCM (2 ml) was added amine hydrochloride from 8d) (99 mg), N-methylmorpholine (41 μl), HOBt (42 mg) and the mixture was stirred for 30 min. EDCI (72 mg) was added and stirring was continued for 1 h. An additional amount of N-methylmorpholine (20 μl) was added and stirred overnight. The reaction mixture was diluted with EtOAc, washed with sat. Na₂CO₃ and brine. The organic layer was dried over Na₂SO₄, filtered and evaporated in vacuo to dryness. Purification by flash chromatography yielded the title compound as colorless glassy solid.

Example 8:

To Boc-protected intermediate 8e) (128 mg) in methanol (1 ml) and dioxane (5 ml) was added hydrogen chloride, 4.0 M sol. in 1,4-dioxane (5 ml) and the solution was stirred for 30 min at room temperature. The reaction mixture was evaporated in vacuo to dryness. The residue was triturated in Et₂O, filtered off and washed with Et₂O to yield an off-white solid. Synthesis of Example 11: Intermediate 11a):

2-Bromo-5-chloroanisole (5.54 g), 1-(2(H)-pyridine-carboxylic acid-3,6-dihydro-4- (4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)-tert.-butyl ester (7.73 g), dichloro(1,1'- bis(diphenyl-phosphino)-ferrocene)palladium(ll) DCM adduct (1.22 g) and K₂CO₃ (10.36 g) were dissolved in degassed DMF in a dry apparatus under argon and the mixture was degassed again by evacuation followed by refilling with argon. The resulting suspension was heated in an oil bath at 85⁰C overnight. The mixture was cooled, filtered through Celite and evaporated to dryness. The crude product was purified by flash chromatography to yield a clear yellowish oil.

Intermediate 11b):

Intermediate 11a) (2.18 g) was dissolved in EtOH (80 ml) and AcOH (80 ml) under argon. Platinum(IV) oxide (0.23 g) was added and the reaction mixture was placed under an H₂ atmophere using a balloon. The reaction mixture was then stirred at room temperature for 120 min. The reaction mixture was filtered through Celite and evaporated to dryness in vacuo. The residue was coevaporated with toluene (3 x 40 ml). The crude product was purified by flash chromatography to yield a clear colorless oil. T/EP2007/001595

60

Intermediate 11c):

To a solution of intermediate 11b) (1.56 g) in AcOH (6.5 ml) was added hydroiodic acid (5.2 ml of a 57 wt.% aq. solution) and the mixture was heated under reflux (oil bath at 140⁰C) in an argon atmosphere for 2 h. The reaction mixture was cooled to room tem- perature and then evaporated to dryness in vacuo. The residue was coevaporated with toluene (3 x 30 ml). The crude product was triturated in Et₂O (40 ml), the insoluble compound was filtered off and washed with Et₂O (10 ml). Finally the product was dried in vacuo over P₂O₅ at room temperature overnight to yield a white solid.

Intermediate 11d):

To a solution of intermediate 11c) (1.55 g) in DMF (10 ml) was added DIEA (0.88 ml) followed by di-tert.-butyl-dicarbonate (1.01 g). The reaction mixture was stirred at room temperature for 4 h. The mixture was evaporated in vacuo to dryness and partitioned between 0.5 M HCI (50 ml) and EtOAc (100 ml). The organic layer was washed with water (25 ml) and brine (30 ml). The organic layer was dried with MgSO₄ and evapo- rated in vacuo to dryness to yield a yellowish solid. The solid residue was triturated in EtOAc (1 ml) and Et₂O (10 ml), and left in the fridge overnight to complete crystallization of the product. The precipitate was then filtered off, washed with cold Et₂O (1 ml), and finally dried in vacuo at room temperature over P₂O₅ overnight. The product was obtained in form of a white solid. Intermediate 11e):

To a solution of intermediate 11d) (468 mg) in DMF (8 ml) was added 1-(3- chloropropyl)piperidine hydrochloride (373 mg) and Cs₂CO₃ (1710 mg). The reaction was stirred at room temperature for 18 h. An additional amount of 1-(3- chloropropyl)piperidine hydrochloride (297 mg) and CS₂CO₃ (489 mg) were added and stirring at room temperature was continued for 3 d. The reaction mixture was evaporated at 40 ⁰C in vacuo to dryness and the residue was partitioned between EtOAc and water. The aqueous layer was extracted with EtOAc. The combined organic layer was washed with water and brine. The organic layer was dried over Na₂SO₄ and evaporated in vacuo to dryness. The crude product was purified using flash chromatography.

Intermediate 11f):

To Boc-protected intermediate 11e) (651 mg) in methanol (3 ml) was added hydrogen chloride, 4.0 M sol. in 1,4-dioxane (11 ml) and the solution was stirred at room temperature for 90 min. The solvent was removed under reduced pressure. The residue was triturated in acetone and Et₂O, filtered off, and washed with Et₂O. Finally it was dried in vacuo at room temperature over P₂O₅ overnight to yield a white solid. Intermediate 11g):

To Boc-D-2,4-dichlorophenylalanine (33 mg) in DCM (2 ml) was added amine hydrochloride from 11f) (30 mg), N-methylmorpholine (23 μl), HOBt (17 mg) and the mixture was stirred for 30 min. EDCI (26 mg) was added and stirring was continued for 1 h. An additional amount of N-methylmorpholine (7 μl) was added and stirred overnight. The reaction mixture was evaporated in vacuo, diluted with EtOAc, washed with sat. Na₂CO₃, H₂O and brine. The aqueous layers were extracted with EtOAc. The combined organic layers were dried over Na₂SO₄, filtered and evaporated in vacuo to dryness. Purification by flash chromatography yielded the title compound as white foam.

Example 11:

To Boc-protected intermediate 11g) (40 mg) in methanol (0.3 ml) was added hydrogen chloride, 4.0 M sol. in 1,4-dioxane (0.6 ml) and the solution was stirred at room temperature for 2 h. The reaction mixture was evaporated in vacuo to dryness. The residue was triturated in acetone and Et₂O, filtered off and washed with Et₂O to yield a white solid. Synthesis of Example 12: Intermediate 12a):

A mixture of 2-bromo-5-fluorobenzaldehyde (10.15 g), malonic acid (5.72 g) and pyridine (1.5 ml) in ethanol (25 ml) was kept under reflux for 7.5 h. After cooling in an ice bath the crystal mass was filtered off. The crystals were washed with cold ethanol (10 ml) and then twice with diethyl ether (10 ml each). The residue was suspended in ethanol (60 ml) and kept under reflux for 2-3 h. The mixture was cooled and filtered and the solid was dried under reduced pressure. The product was obtained in form of colorless needles.

Intermediate 12b):

Intermediate 12a) (2451 mg), pyrrolidine (918 μl), EDCI (2109 mg) and DMAP (100 mg) were dissolved in DCM (100 ml) and stirred overnight. The reaction mixture was poured into water (100 ml) and the organic layer was separated. The aqueous layer was extracted twice with DCM. The combined organics were washed three times with with 0.5 N HCI (30 ml each), three times with 1 M sodium hydroxide solution and brine, dried over Na₂SCv and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography to yield a white solid. Intermediate 12c):

Intermediate 12b) (1130 mg), N-Boc-1,2,3,6-tetrahydropyridine-4-boronic acid pinacol ester (1231 mg), potassium carbonate (1571 mg) and dichloro(1,1'-bis(diphenyl- phosphino)-ferrocene)palladium(ll) DCM adduct (188 mg) were dissolved in DMF (50 ml) in a dry apparatus under argon and the mixture was degassed by bubbling with argon for 30 min. The orange suspension was then heated under argon in an oil bath at

85°C for 1 d. The reaction mixture was filtered through Celite and evaporated to dry- ness in vacuo. The crude product was purified by flash chromatography to yield a beige solid.

Intermediate 12d):

Intermediate 12c) (1459 mg) was dissolved in ethanol (50 ml) and 10% palladium on activated carbon (150 mg) was added. The reaction mixture was purged three times with hydrogen (5 bar) and stirred under hydrogen atmosphere (10 bar) for 3 d. The crude mixture was filtered through Celite and the solvent was removed under reduced pressure to yield a yellow-grey oil. Intermediate 12e):

Intermediate 12d) (1472 mg) in diethyl ether (20 ml) was slowly added to a mixture of lithium aluminum hydride (207 mg) and diethyl ether (30 ml) at O⁰C. After addition the reaction mixture was stirred at 0⁰C for 1 h. The reaction mixture was hydrolyzed with a minimum amount of water. The inorganic precipitate was filtered off and washed twice with diethyl ether. The combined filtrates were dried over sodium sulfate, filtered, and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography to yield a pale yellow oil.

Intermediate 12f):

To intermediate 12e) (654 mg) in dioxane (10 ml) and methanol (2 ml) was added hydrogen chloride, 4.0 M sol. in 1 ,4-dioxane (10 ml) and the solution was stirred for 30 min at room temperature. The solvent was removed under reduced pressure, the residue was triturated with acetone (5 ml) and diethyl ether (50 ml) and the product was filtered off to yield an off-white solid. Intermediate 12g):

To Boc-D-2,4-dichlorophenylalanine (90 mg) in DCM (2 ml) was added amine hydrochloride from 12f) (91 mg), N-methylmorpholine (41 μl), HOBt (42 mg) and the mixture was stirred for 30 min. EDCI (72 mg) was added and stirring was continued for 1 h. An additional amount of N-methylmorpholine (20 μl) was added and stirred overnight. The reaction mixture was diluted with EtOAc, washed with sat. Na₂CO₃ and brine. The organic layers was dried over Na₂SO₄, filtered and evaporated in vacuo to dryness. Purification by flash chromatography yielded the title compound as colorless glassy solid.

Example 12:

To Boc-protected intermediate 12g) (83 mg) in methanol (1 ml) and dioxane (5 ml) was added hydrogen chloride, 4.0 M sol. in 1,4-dioxane (5 ml) and the solution was stirred for 30 min at room temperature. The reaction mixture was evaporated in vacuo to dryness. The residue was triturated in Et₂O, filtered off and washed with Et₂O to yield an off-white solid. Synthesis of Example 15: Intermediate 15a):

2-Bromo-5-chlorotoluene (8.26 ml) and N-bromosuccinimide (11.03 g) in carbontetra- chloride (50 ml) were treated with a catalytic amount of benzoylperoxide (100 mg) and heated under reflux until the reaction had reached completion as monitored by TLC. The reaction mixture was then allowed to cool and filtered. The filtrate was washed twice with water and brine, dried over sodium sulfate and concentrated in vacuo.

Intermediate 15b):

To a solution of sodium ethoxide (2.79 g) in ethanol (30 ml) was added diethyl malo- nate (6.54 ml) and the mixture was stirred for 1 h at room temperature. The mixture was cooled in an ice-bath and intermediate 15a) (11.67 g) was slowly added and the reaction mixture was kept under reflux overnight. The reaction mixture was evaporated in vacuo and the residue was partitioned between diethyl ether and water and the aqueous layer extracted two times with diethyl ether. The combined organic layer was washed twice with water and brine. The combined organic layer was dried over Na₂SC>₄ and evaporated in vacuo to dryness. The product was purified by Kugelrohr distillation. The fractions which distilled off between 160 and 230⁰C at 0.2-0.3 mbar were collected.

Intermediate 15c):

Intermediate 15b) (8.98 g) was heated under reflux in 1.8 M KOH in H₂O/EtOH (60 ml) for 5 h. After evaporation of the ethanol an additional amount of KOH (18 g) was added to the residue, and the reaction mixture was stirred for 2 h at 100⁰C. The reaction mixture was diluted with 100 ml of H₂O, extracted with Et₂O and the organic layer was washed with H₂O. The combined aqueous layer was cooled with ice/H₂0 and acidified with 50% H₂SO₄ to pH 1. The precipitate was extracted twice with Et₂O (100 ml each) and the organic layers were washed with water and brine. The combined organic layers were dried over Na₂SO₄ and evaporated in vacuo to dryness. The residue was triturated in hexane and less Et₂O₁ then filtered and washed with hexane and less Et₂O. The solid residue was decarboxylated by heating at 200⁰C. The development of CO₂ ceased after 20 min and the melt was cooled to room temperature. The residue was crushed with a glass rod to get a homogeneous beige solid.

Intermediate 15d):

Intermediate 15c) (2320 mg), pyrrolidine (808 μl), EDCI (1856 mg) and DMAP (100 mg) were dissolved in DCM (100 ml) and stirred overnight. The reaction mixture was poured into water (100 ml) and the organic layer was separated. The aqueous layer was extracted twice with DCM. The combined organics were washed three times with with 0.5 N HCI (30 ml each), three times with 1 M sodium hydroxide solution and brine, dried over Na₂SO₄ and the solvent was removed under reduced pressure. The product was obtained as off-white solid after purification by flash chromatography.

Intermediate 15e):

Intermediate 15d) (1901 mg), N-Boc-1,2,3,6-tetrahydropyridine-4-boronic acid pinacol ester (1948 mg), potassium carbonate (3317 mg) and dichloro(1,1'-bis(diphenyl- phosphino)-ferrocene)palladium(ll) DCM adduct (294 mg) were dissolved in DMF (70 ml) in a dry apparatus under argon and the mixture was degassed by bubbling with argon for 30 min. The orange suspension was then heated under argon in an oil bath at 85⁰C for 3 d to give a dark purple suspension. The reaction mixture was filtered through Celite and evaporated to dryness in vacuo. The crude product was purified by column chromatography.

Intermediate 15f):

Intermediate 15e) (2372 mg) was dissolved in EtOH (50 ml) and AcOH (50 ml) and platinum(IV) oxide (129 mg) was added. The reaction mixture was evacuated three times and purged with hydrogen. The reaction mixture was then stirred at room temperature for 2 h. The reaction mixture was filtered and evaporated to dryness in vacuo. The residue was coevaporated with toluene (3 x 75 ml) and was finally dried under high vacuum at RT overnight.

Intermediate 15g):

Intermediate 15f) (1687 mg) in diethyl ether (20 ml) was slowly added to a mixture of lithium aluminum hydride (228 mg) and diethyl ether (30 ml) at 0⁰C. After addition, the reaction mixture was stirred at 0⁰C for 1 h. The reaction mixture was hydrolyzed with a minimum amount of water. The inorganic precipitate was filtered off and washed twice with diethyl ether. The combined filtrates were dried over sodium sulfate, filtered, and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography to yield a colorless oil.

Intermediate 15h):

To intermediate 15g) (864 mg) in dioxane (10 ml) and methanol (2 ml) was added hy- drogen chloride, 4.0 M sol. in 1,4-dioxane (10 ml) and the solution was stirred for 30 min at room temperature. The solvent was removed under reduced pressure, the resi- due was triturated with acetone (5 ml) and diethyl ether (50 ml) and the product was filtered off. The product was obtained as off-white solid.

Intermediate 15i):

To Boc-D-2,4-dichlorophenylalanine (90 mg) in DCM (2 ml) was added amine hydro- chloride from 15h) (95 mg), N-methylmorpholine (41 μl), HOBt (42 mg) and the mixture was stirred for 30 min. EDCI (72 mg) was added and stirring was continued for 1 h. An additional amount of N-methylmorpholine (20 μl) was added and stirred overnight. The reaction mixture was diluted with EtOAc, washed with sat. Na₂CO₃ and brine. The or- ganic layers was dried over Na₂SO₄, filtered and evaporated in vacuo to dryness. Purification by flash chromatography yielded the title compound as colorless glassy solid. Example 15:

To Boc-protected intermediate 15i) (84 mg) in methanol (1 ml) and dioxane (5 ml) was added hydrogen chloride, 4.0 M sol. in 1,4-dioxane (5 ml) and the solution was stirred for 30 min at room temperature. The reaction mixture was evaporated in vacuo to dryness. The residue was triturated in Et₂O, filtered off and washed with Et₂O to yield an off-white solid.

Synthesis of Example 16: Intermediate 16a):

A solution of 2-bromo-5-chlorophenylacetic acid (20.0 g) in THF (200 ml) was added to a suspension of sodium borohydride (3.18 g) in THF (50 ml) over 30 min at room temperature. After being stirred for 15 min, boron trifluoride diethyl etherate (13.2 ml) was added over 30 min while the temperature was maintained at 25-35°C. The resulting slurry was stirred for 1 h at room temperature, then cooled to 0⁰C and carefully hydro- lyzed by addition of sat. NH₄CI (50 ml). The main part of the THF was removed in vacuo, the residue was diluted with sat. NH₄CI and water (each with 50 ml), followed by extraction with diethyl ether (3 x 100 ml). The combined ether layer was washed with 1 M NaOH (2 x 100 ml) and water (100 ml). All aqueous phases were combined and extracted with diethyl ether (2 x 100 ml). All organic layers were combined, washed with brine (100 ml), and dried (Na₂SO₄). Evaporation of the solvent afforded the desired product as a yellowish oil. Intermediate 16b):

Phosphorous tribromide (3.71 ml) was dissolved in toluene (30 ml) and cooled to 0⁰C. Then pyridine (1.68 ml) was added. To the suspension thus obtained, a solution of in- termediate 16a) (18.6 g) and pyridine (0.56 ml) in toluene (30 ml) was added over 15 min. The cooling bath was removed and stirring was continued at room temperature for 1 h. Then the reaction mixture was heated to 100⁰C for another hour. The reaction mixture was cooled to ambient temperature, diluted with EtOAc (300 ml) and washed with water (2 x 100 ml). The combined aqueous layer was extracted with EtOAc (100 ml), all organic extracts were merged and washed with brine (100 ml), dried (Na₂SO₄), and concentrated in vacuo. The crude product was purified by column chromatography to furnish the desired compound as a colorless oil.

Intermediate 16c):

Diethyl malonate (9.59 ml) was added dropwise to a suspension of sodium hydride, 60% dispersion in mineral oil (2.42 g) in THF (50 ml) at 0⁰C. The cooling bath was re- moved and stirring was continued for 30 min at room temperature. Then a solution of intermediate 16b) (15.7 g) in THF (50 ml) was added and the reaction mixture was kept under reflux overnight. The suspension was concentrated in vacuo. The residue was diluted with water (300 ml) and extracted with diethyl ether (3 x 200 ml). The combined organic layer was washed with brine (200 ml), dried (Na₂SO₄), and concentrated in vacuo. The crude product was purified by column chromatography to furnish the de- sired compound as a colorless oil. Intermediate 16d):

An emulsion of intermediate 16c) and potassium hydroxide (11.6 g) in a 1:1 mixture of EtOH and water (100 ml) was heated under reflux for 5 h. The main part of EtOH was evaporated, more potassium hydroxide (19.3 g) was added and the reaction mixture was heated to 100⁰C for 45 min. After dilution with water (150 ml), the solution was extracted with diethyl ether (2 x 50 ml). The combined ether layer was re-extracted with water (50 ml). The two water layers were combined and acidified with 50% H₂SO₄ to pH 1 , extracted with diethyl ether (3 x 100 ml), re-extracted with water (100 ml). Following drying over Na₂SO₄ the solvent was evaporated to afford the corresponding malo- nic acid derivative in form of a white solid. Decarboxylation was achieved by heating the product at 200⁰C until evolution of carbon dioxide ceased to provide the desired compound as a slightly brownish solid.

Intermediate 16e):

A solution of intermediate 16d) (5.03 g) in DCM (130 ml) was cooled to 0°C. To this solution a solution of oxalyl chloride (1.69 ml) in DCM (20 ml) was added dropwise. DMF (5 drops) was added and the reaction mixture was stirred at 0°C for 1 h and then at room temperature until the gas formation ceased. Concentration in vacuo furnished the desired product in form of a yellowish oil.

Intermediate 16f):

007/001595

A solution of intermediate 16e) (5.31 g) in DCM (70 ml) was cooled to 0⁰C. Pyrrolidine (4.49 ml) was added dropwise and stirring was continued at O⁰C for 45 min. The ice bath was removed and stirring was continued at room temperature. After being stirred for 2 h in total the reaction mixture was diluted with DCM (100 ml) and washed with 1 M HCI, 1 M NaOH (each with 3 x 50 ml), water and brine (each with 50 ml). The organic phase was dried (Na₂SO₄) and evaporated to afford the desired product as a yellow oil.

Intermediate 16g):

A mixture of intermediate 16f) (5.50 g), Λ/-Boc-1,2,3,6-tetrahydropyridine-4-boronic acid pinacol ester (5.40 g), potassium carbonate (6.90 g), and dichloro(1,1'-bis(diphenyl- phosphino)-ferrocene)palladium(ll) DCM adduct (0.82 g) in degassed DMF (100 ml) was heated under argon at 85°C overnight. The reaction mixture was evaporated. The residue was partitioned between EtOAc and water (each with 100 ml) and filtered through Celite. The filtrate was extracted with EtOAc (2 x 100 ml) and the combined organic layer re-extracted with water and brine (100 ml each), dried (Na₂SO₄) and concentrated in vacuo. The crude product was purified by column chromatography to fur- nish the desired compound as a brownish resin.

Intermediate 16h):

To a solution of intermediate 16g) (6.06 g) in EtOH and AcOH (100 ml each) plati- num(IV) oxide (0.32 g) was added. The flask was purged with H₂ at atmospheric pres- sure. The reaction mixture was then vigorously stirred for 3 h at room temperature. Fil- tration through Celite, evaporation of the solvent and purification of the residue by col- umn chromatography afforded the desired product in form of a colorless oil.

Intermediate 16i):

Under vigorous stirring a solution of intermediate 16h) (2.16 g) in diethyl ether (20 ml) was added to a suspension of lithium aluminum hydride (0.28 g) in diethyl ether (30 ml) at O⁰C under argon. The reaction mixture was stirred for 1 h at this temperature and was then hydrolyzed by addition of water (0.5 ml). The inorganic precipitate thus ob- tained was filtered off and washed with diethyl ether (3 x 40 ml). The filtrate was dried (Na₂SO₄) and concentrated in vacuo. Purification of the crude product by column chromatography furnished the desired compound as a colorless resin.

Intermediate 16j):

To Boc-protected intermediate 16i) (670 mg) in MeOH (5 ml) was added hydrogen chloride, 4.0 M sol. in 1 ,4-dioxane (5 ml) and the solution was stirred at room tempera- ture for 30 min. The solvent was completely removed in vacuo to afford the desired compound as a white solid. Intermediate 16k):

Intermediate 16j) (74 mg), Boc-D-2,4-dichlorophenylalanine (82 mg), 1-hydroxy- benzotriazole hydrate (43 mg) and N-methylmorpholine (58 μl) were dissolved in DMF (5 ml). After being stirred for 30 min at room temperature, Λ/-(3-dimethylaminopropyl)- /V-ethylcarbodiimide hydrochloride (65 mg) was added and stirring was continued for another hour. An additional amount of Nnethylmorpholine (17 μl) was added and stirring was continued overnight. The reaction mixture was diluted with EtOAc (70 ml), washed with sat. Na₂CO₃ (3 x 25 ml), H₂O and brine (each with 25 ml). The organic layer was dried (Na₂SO₄) and the solvent removed in vacuo. Purification of the crude product by column chromatography furnished the desired compound in form of a yellowish oil.

Example 16:

To Boc-protected intermediate 16k) (86 mg) in DCM (3 ml) was added hydrogen chloride, 4.0 M sol. in 1,4-dioxane (3 ml) and the solution was stirred at room temperature for 1.5 h. All volatiles were evaporated and the residue was suspended in a 1 :1 mixture of Et₂O and hexane (5 ml). After filtration, washing with hexane and drying in vacuo overnight (desiccator, sicapent) the desired product was isolated in form of a white solid. Synthesis of Examples 17, 18 and 19: Intermediate 17-19a):

To a solution of DL-2-amino-1-propanol (5.34 ml) and 1 ,4-dibromobutane (7.91 ml) in acetonitrile (67 ml) was added potassium carbonate (18.52 g) and the resulting solution was stirred at reflux temperature for 20 h. The reaction mixture was evaporated in vacuo and the residue was partioned between EtOAc and water. The organic layer was washed with water and brine. The aqueous layers were re-extracted with EtOAc. The combined organic layer was dried over Na₂SO₄ and evaporated in vacuo to dryness. The crude product was purified by Kugelrohr distillation (20 mbar, 103-112°C) to yield a clear colorless oil.

Intermediate 17-19b):

A solution of intermediate 11d) (1.21 g), intermediate 17-19a) (1.00 g), and triphenyl- phosphine (2.03 g) in THF (30 ml) under argon, was cooled in ice/H₂O. DEAD (ca. 40% in toluene, 1.42 ml) was added dropwise, at a rate to keep the temperature below 5⁰C (ca. 15 min). After stirring for another 10 min in ice/H₂O, the cooling bath was removed and the mixture was stirred at room temperature overnight. The reaction mixture was evaporated to dryness in vacuo at 40⁰C. The crude product was purified by flash chromatography. Intermediate 17-19c):

To Boc-protected intermediate 17-19b) (1.25 g) in dioxane (5 ml) was added hydrogen chloride, 4.0 M sol. in 1 ,4-dioxane (20 ml) and the solution was stirred at room temperature for 60 min. The solvent was removed under reduced pressure. The residue was triturated in acetone and Et₂O, filtered off, and washed with Et₂O. Finally, it was dried in vacuo at room temperature over P₂O₅ overnight to yield a white solid.

Intermediate 17-19d)

To Boc-D-2,4-dichlorophenylalanine (106 mg) in DCM (7 ml) was added amine hydrochloride from 17-19c) (100 mg), N-methylmorpholine (77 μl), HOBt (58 mg) and the mixture was stirred for 30 min. EDCI (85 mg) was added and stirring was continued for 1 h. An additional amount of N-methylmorpholine (21 μl) was added and the mixture was stirred overnight. The reaction mixture was evaporated in vacuo, diluted with EtOAc, and washed with sat. Na₂CO₃, H₂O and brine. The aqueous layers were re- extracted with EtOAc. The combined organic layer was dried over Na₂SO₄, filtered, and evaporated in vacuo to dryness.

Purification by flash chromatography yielded the title compounds as a clear colorless oil.

To Boc-protected intermediate 17-19d) (159 mg) in dioxane (1 ml) was added hydro- gen chloride, 4.0 M sol. in 1 ,4-dioxane (3 ml) and the solution was stirred at room tem- perature for 2 h. The reaction mixture was evaporated in vacuo to dryness. The three products were separated with preparative HPLC-MS.

Synthesis of Example 23: Intermediate 23a):

Phosphorous tribromide (735 μl) was added to a stirred solution of 4-chloro-2- methylbenzyl alcohol (3.5 g) in toluene (30 ml) at 4O⁰C. The solution was heated to

100⁰C for 30 min, and the reaction was cooled to ambient temperature. The liquid was decanted and washed with water (2 x 50 ml) and brine (50 ml). The combined aqueous layer was extracted with diethyl ether (70 ml) and the combined organic layer was evaporated to yield a semisolid residue. The residue was dissolved in diethyl ether (350 ml) and washed with water (2 x 100 ml) and brine (100 ml). The organic phase was dried over Na₂SO₄, filtered and evaporated to yield a light yellow oil. Intermediate 23b):

N-(Diphenylmethylene) glycine ethyl ester (5.27 g), intermediate 23a) (4.81 g) and benzyltriethylammonium chloride (TEBAC) (4.49 g) were dissolved in DCM (52 ml) and 10% aqueous KOH (52 ml) was added. The resulting two-phase mixture was stirred at room temperature for 24 h. The organic layer was separated and concentrated. The residue was taken up with diethyl ether (125 ml) and washed with water (100 ml) followed by brine (100 ml) and dried over Na₂SO₄. The solvent was removed to give the crude product as a yellow oil. The crude product was purified by flash column chromatography.

Intermediate 23c):

5% Aqueous HCI (50 ml) was added portionwise to a solution of intermediate 23b) (5.6 g) in THF (20 ml) at 0⁰C. The mixture was then stirred at room temperature for 2 h. The solvent was evaporated under reduced pressure and water (200 ml) was added to the residue. The aqueous phase was washed with diethyl ether (3 x 250 ml) and DCM (250 ml). The aqueous phase was then basified with 5N aqueous NaOH (11 ml) to get pH 7/8 and extracted with DCM (8 x 400 ml). The aqueous phase was concentrated to a volume of 150 ml and extracted with DCM (11 x 150 ml). The combined organic layer was dried over Na₂SO₄, filtered and the solvent was removed under reduced pressure to give a yellow oil. The crude product was dissolved in THF (20 ml) and 1N aqueous sodium hydroxide (12.1 ml) was added portionwise at 0⁰C. The reaction mixture was stirred at room temperature overnight. The solvent was removed under reduced pressure and water (50 ml) was added to the residue. The aqueous phase was washed with diethyl ether (2 x 100 ml) and DCM (2 x 100 ml) and then neutralized to pH 7 with 5N aqueous HCI (2 ml). The aqueous phase was evaporated under reduced pressure to yield the product as a white solid.

Intermediate 23d):

Intermediate 23 c) (2.96 g) was dissolved in Tris-maleate buffer (125 mL) containing 0.1 M KCI. To the turbid solution was added L-amino acid oxidase (sigma Type I, 85 mg) and catalase (8.5 mg). After 84 h of vigorous stirring at 35⁰C, the reaction was brought to pH 7 with 0.5N HCI (4.5 ml). The aqueous solution was reduced to a volume of 10 ml and then purified by ion exchange using Dowex 50 (60 ml). The product was eluted with water (500 ml), then 1N ammonia (800 ml). The combined eluants were evaporated under reduced pressure to yield the title compound.

Intermediate 23e):

Intermediate 23d) (88 mg) was dissolved in 2N aqueous sodium hydroxide (0.58 ml) and cooled to 0°C. Di-tert-butyl dicarbonate (101 mg) was slowly added followed by dioxane (0.5 ml). After half an hour, the reaction mixture was warmed to room temperature and allowed to stir for 12 h. Di-tert-butyl-dicarbonate (101 mg) and 1N aqueous NaOH (0.29 ml) were added. The reaction was stirred at room temperature for another 20 h. The reaction mixture was evaporated to dryness and water (2 ml) was added. The reaction mixture was acidified to pH 2 using 1N aqueous hydrochloric acid (1.3 ml) and extracted with DCM (3 x 20 ml). The combined organic layer was dried over Na₂SO₄, filtered and concentrated in vacuo to give a colorless wax. The crude product was purified by column chromatography. Intermediate 23f):

To a solution of intermediate 23e) (35 mg) in dry DMF (2 ml) was added HOBt (19 mg), EDCI (23 mg) and NMM (49 μl). The reaction mixture was stirred for 5 min and then intermediate 24b) (47 mg) was added as a solid at 0⁰C. The reaction stirred at room temperature overnight. The solvent was evaporated and the residue was diluted with ethyl acetate (25 ml) and successively washed with water (15 ml), followed by saturated NaHCO₃ (15 ml) and brine (25 ml). The aqueous phases were extracted again with ethyl acetate (15 ml). The combined organic layer was dried over Na₂SO₄, filtered and evaporated. The crude compound was purified by column chromatography.

Example 23:

Intermediate 23f) (23 mg) was dissolved in dioxane (0.4 ml) and 4M HCI in dioxane (0.28 ml) was added at 0⁰C. The reaction mixture was stirred at room temperature for 90 min. The reaction mixture was evaporated to dryness in vacuo. The residue was dissolved in MeOH (0.1 ml) and triturated with Et₂O. A beige solid compound was obtained. It was filtered off, rinsed with Et₂O and dried under high vacuum. Synthesis of Example 24: Intermediate 24a):

To a solution of intermediate 11d) (887 mg) in DMF (15 ml) was added 1-(3- chloroethyl)pyrrolidine hydrochloride (605 mg) and Cs₂CO₃ (3243 mg). The reaction was stirred at room temperature for 18 h. An additional amount of 1-(3- chloroethyOpyrrolidine hydrochloride (483 mg) and Cs₂CO₃ (926 mg) were added and stirring at room temperature was continued for 6 h. The reaction mixture was evapo- rated at 40⁰C in vacuo to dryness and the residue was partitioned between EtOAc and water. The aqueous layer was extracted with EtOAc. The combined organic layer was washed with water and brine. The organic layer was dried over Na₂SO₄ and evaporated in vacuo to dryness. The crude product was purified using flash chromatography.

Intermediate 24b):

To Boc-protected intermediate 24a) (1170 mg) in dioxane (5 ml) was added hydrogen chloride, 4.0 M sol. in 1,4-dioxane (20 ml) and the solution was stirred at room tem- perature for 2 h. The solvent was removed under reduced pressure. The residue was triturated in acetone and Et₂O, filtered off, and washed with Et₂O. Finally, it was dried in vacuo at room temperature over P₂O₅ overnight to yield a white solid. Intermediate 24c):

Boc-D-2,4-dichlorophenylalanine (1003 mg) and methyl iodide (1495 μl) were dissolved in dry THF under argon atmosphere. The solution was cooled to 0⁰C and sodium hy- dride (360 mg) was added in three portions. The methylation reaction was stirred at room temperature overnight. It was quenched by dropwise addition of ethyl acetate (1 ml) and 3% citric acid (2 ml). The reaction mixture was evaporated in vacuo and the residue was taken up with ethyl acetate (100 ml). The solution was cooled to 0⁰C, wa- ter (30 ml) was added and the pH was brought to 2 by dropwise addition of 1N HCI. The two phases were separated and the organic phase was extracted twice with brine, dried over sodium sulfate and the solvent was removed under reduced pressure. Puri- fication by flash chromatography yielded the title compound as a white foam.

Intermediate 24d):

To intermediate 24c) (132 mg) in DMF (2 ml) was added the amine hydrochloride from 24b) (95 mg), N-methylmorpholine (41 μl), HOBt (42 mg) and the mixture was stirred for 30 min. EDCI (53 mg) was added and stirring was continued for 1 h. An additional amount of N-methylmorpholine (20 μl) was added and stirred overnight. The reaction mixture was poured into brine (20 ml), diluted with ethyl acetate and the organic phase was separated. The aqueous phase was extracted two times with ethyl acetate. The combined organic layer was washed twice with sat. Na₂CO₃, twice with H₂O and brine. The organic layer was dried over Na₂SO₄, filtered and evaporated in vacuo to dryness. The product was purified by flash chromatography. Example 24:

To Boc-protected intermediate 24d) (101 mg) in dioxane (5 ml) and methanol (1 ml) was added hydrogen chloride, 4.0 M sol. in 1 ,4-dioxane (5 ml) and the solution was stirred for 30 min at room temperature. The reaction mixture was evaporated in vacuo to dryness. The product was purified by preparative HPLC-MS.

Synthesis of Example 25: Intermediate 25a):

To intermediate 24c) (132 mg) in DMF (2 ml) was added the amine hydrochloride from 15h) (95 mg), N-methylmorpholine (41 μl), HOBt (42 mg) and the mixture was stirred for 30 min. EDCI (53 mg) was added and stirring was continued for 1 h. An additional amount of N-methylmorpholine (20 μl) was added and stirred overnight. The reaction mixture was poured into brine (20 ml), diluted with ethyl acetate and the organic phase was separated. The aqueous phase was extracted two times with ethyl acetate. The combined organic layer was washed twice with sat. Na₂CC>₃, twice with H₂O and brine.

The organic layer was dried over Na₂SO₄, filtered and evaporated in vacuo to dryness.

The product was purified by flash chromatography. Example 25:

To Boc-protected intermediate 25a) (120 mg) in dioxane (5 ml) and methanol (1 ml) was added hydrogen chloride, 4.0 M sol. in 1 ,4-dioxane (5 ml) and the solution was stirred for 30 min at room temperature. The reaction mixture was evaporated in vacuo to dryness. The product was purified by preparative HPLC-MS.

Example 26:

Formaldehyde solution (7 μl) was added to a solution of Example 24 (42 mg) and DIEA (23 μl) in 1 ,2-dichloroethane (2 ml). The reaction mixture was cooled in an ice/H₂O- bath and sodium triacetoxyborohydride (114 mg, 0.536 mmol) was added in one por- tion. After 5 minutes the ice/H₂O-bath was removed and the reaction mixture was stirred at room temperature for 3.5 h. The reaction mixture was quenched by adding aqueous sat. NaHCO₃ and the product was extracted with EtOAc. The aqueous layer was extracted with EtOAc. The combined organic layer was washed with brine, dried over Na₂Sθ₄ and the solvent was evaporated in vacuo. The product was purified by preparative HPLC-MS. Synthesis of Example 28: Intermediate 28a):

To Boc-D-4-chlorophenylalanine (574 mg) in DCM (30 ml) was added amine hydrochloride from 1b) (500 mg), N-methylmorpholine (448 μl), HOBt (330 mg) and the mixture was stirred for 30 min. EDCI (505 mg) was added and stirring was continued for 1 h. An additional amount of N-methylmorpholine (131 μl) was added and stirred overnight. The reaction mixture was evaporated in vacuo, diluted with EtOAc, washed with sat. Na₂CO₃, H₂O and brine. The aqueous layers were extracted with EtOAc. The combined organic layers were dried over Na₂SO₄, filtered and evaporated in vacuo to dryness. Purification by flash chromatography yielded the title compound as white foam.

Example 28:

To Boc-protected intermediate 28a) (721 mg) in methanol (4 ml) was added hydrogen chloride, 4.0 M sol. in 1 ,4-dioxane (10 ml) and the solution was stirred for 90 min at room temperature. The reaction mixture was evaporated in vacuo to dryness. The residue was triturated in Et₂O, filtered off and washed with Et₂O to yield a beige solid. Synthesis of Example 36: Intermediate 36a):

To Boc-D-4-chlorophenylalanine (574 mg) in DCM (30 ml) was added amine hydrochloride from 24b) (500 mg), N-methylmorpholine (448 μl), HOBt (330 mg) and the mixture was stirred for 30 min. EDCI (505 mg) was added and stirring was continued for 1 h. An additional amount of N-methylmorpholine (131 μl) was added and stirred overnight. The reaction mixture was evaporated in vacuo, diluted with EtOAc, washed with sat. Na₂CO₃, H₂O and brine. The aqueous layers were extracted with EtOAc. The combined organic layer was dried over Na₂SO₄, filtered, and evaporated in vacuo to dryness. Purification by flash chromatography yielded the title compound as a white foam.

Example36:

To Boc-protected intermediate 36a) (721 mg) in methanol (4 ml) was added hydrogen chloride, 4.0 M sol. in 1,4-dioxane (10 ml) and the solution was stirred for 90 min at room temperature. The reaction mixture was evaporated in vacuo to dryness. The residue was triturated in Et₂O, filtered off, and washed with Et₂O to yield a beige solid. Synthesis of Example 41 : Intermediate 41a):

To Boc-D-4-chlorophenylalanine (29 mg) in DCM (2 ml) was added amine hydrochloride from 11f) (30 mg), N-methylmorpholine (23 μl), HOBt (17 mg) and the mixture was stirred for 30 min. EDCI (26 mg) was added and stirring was continued for 1 h. An additional amount of N-methylmorpholine (7 μl) was added and stirred overnight. The reaction mixture was evaporated in vacuo, diluted with EtOAc, washed with sat. Na₂CO_3l H₂O and brine. The aqueous layers were extracted with EtOAc. The combined organic layers were dried over Na₂SO₄, filtered and evaporated in vacuo to dryness. Purification by flash chromatography yielded the title compound as white foam.

Example 41:

To Boc-protected intermediate 41a) (39 mg) in methanol (0.3 ml) was added hydrogen chloride, 4.0 M sol. in 1,4-dioxane (0.6 ml) and the solution was stirred at room temperature for 2 h. The reaction mixture was evaporated in vacuo to dryness. The residue was triturated in acetone and Et₂O, filtered off and washed with Et₂O to yield a white solid. Synthesis of Example 42: Intermediate 42a):

To a mixture of 48% aqueous HBr (89.5 ml) and water (90 ml) was added 6-amino-m- cresol (10.0 g). The mixture was kept under reflux for 10 min and then stirred at room temperature for 2 h. The suspension was cooled down to -15⁰C (ice/NaCI) and NaNO₂ (5.49 g) dissolved in water (90 ml) was added dropwise in a way to keep the temperature below -5°C. The mixture was stirred for 10 min and was added dropwise to an ice- cooled mixture of 48% aqueous HBr (53.7 ml), EtOAc (300 ml) and CuBr (22.8 g) during a period of 15 min. The resulting brown suspension was stirred at room temperature for 1 h and at 40⁰C for 3 h. The reaction mixture was diluted with EtOAc (300 ml), the organic phase was removed and the aqueous phase was extracted with diethyl ether (3 x 200ml). The combined organic layer was washed with 48% aqueous HBr (2 x 50 ml) followed by water (5 x 100 ml) and brine (70 ml), dried over Na₂SO₄ and evaporated to give a brown oil. The crude product was purified by distillation. All fractions which distilled of at normal pressure up to 60⁰C were discarded. Vacuum was applied and the fraction distilling off at 45°C was collected. This fraction was further purified by column chromatography.

Intermediate 42b):

Intermediate 42a) (2.07 g), Λ/-Boc-1 ,2,3,6-tetrahydropyridine-4-boronic acid pinacol ester (3.42 g) and potassium carbonate (4.59 g) were dissolved in DMF (58 ml). The solution was degassed by bubbling with argon for 1 h. Then [Pd(dppf)CI₂] (542 mg) was added. The brown suspension was then heated under argon in an oil bath at 85°C for 3 d. Another load of catalyst was added (220 mg) and the reaction mixture was stirred at 85⁰C for 7 h. The reaction mixture was filtered through Celite and rinsed with ethyl ace- tate. The combined filtrates were evaporated and the residue was partitioned between ethyl acetate (150 ml) and water (150 ml). The mixture was filtered through Celite again and rinsed with ethyl acetate. The phases were separated and the aqueous layer was extracted with ethyl acetate (2 x 60 ml). The combined organic layer was washed with water (60 ml) and brine (60 ml), dried over Na2SO₄, filtered and evaporated in vacuo to dryness. The residue was purified by flash chromatography.

Intermediate 42c):

Intermediate 42b) (438 mg) was dissolved in dry ethanol (18 ml) and acetic acid (18 ml) and the solution was degassed by bubbling with argon. Platinum(IV) oxide (120 mg) was added and the reaction mixture was placed under a H₂ atmosphere using a bal- loon. The reaction mixture was then stirred at room temperatur for 2 h. The reaction mixture was filtered through Celite, rinsed with EtOAc and evaporated to dryness in vacuo. The residue was coevaporated with toluene (3x 40 ml) and finally dried under high vacuum overnight to give a yellow oil that started to crystallize on standing.

Intermediate 42d):

To a solution of intermediate 42c) (256 mg) in DMF (5.0 ml) was added 1-(2- chloroethyl)-pyrrolidine hydrochloride (179 mg) and cesium carbonate (958 mg). The reaction mixture was stirred at room temperature for 36 h. The reaction mixture was evaporated and the residue was partitioned between ethyl acetate (50 ml) and water (40 ml). The organic layer was washed with water (20 ml) and brine (20 ml), dried over Na₂SO₄ and evaporated in vacuo to dryness. The crude product was purified by flash chromatography.

Intermediate 42e):

Intermediate 42d) (408 mg) was dissolved in dioxane (2.0 ml) and 4M HCI in dioxane (20 ml) was added at O⁰C. The reaction mixture was stirred at room temperature for 90 min. The reaction mixture was evaporated to dryness in vacuo and the residue triturated in acetone (1 ml), ethyl acetate (1 ml) and Et₂O (1 ml). A beige sticky compound was obtained. A few drops of MeOH were added to get a beige powder that was fil- tered off, rinsed with Et₂O and dried.

Intermediate 42f):

To a solution of Boc-D-4-chlorophenylalanine (37mg) in dry DMF (3 ml) was added HOBt (22 mg), EDCI (28 mg) and NMM (49 μl). The reaction mixture was stirred for 5 min and then amine intermediate 42e) (40 mg) was added as a solid at 0⁰C. The reaction was stirred at room temperature for 6 h. The solvent was evaporated and the orange residue was diluted with ethyl acetate (50 ml) and successively washed with wa- ter (25 ml), followed by sat. NaHCO₃ (25 ml) and brine (25 ml). The aqueous phases were extracted again with ethyl acetate (20 ml). The combined organic layer was dried over Na₂SO₄, filtered and evaporated. The crude compound was purified by column chromatography.

Example 42:

Intermediate 42f) (27 mg) was dissolved in dioxane (500 μl) and 4M HCI in dioxane (350 μl) was added at 0⁰C. The reaction mixture was stirred at room temperature for 90 min. The reaction mixture was evaporated to dryness in vacuo and the residue was dissolved in MeOH (100 μl) and triturated with Et₂O and acetone. A beige solid compound was obtained. It was filtered off, rinsed with Et₂O and dried under high vacuum.

Synthesis of Example 44: Intermediate 44a):

To Boc-D-4-chlorophenylalanine (80 mg) in DCM (2 ml) was added amine hydrochloride from 8d) (99 mg), N-methylmorpholine (41 μl), HOBt (42 mg) and the mixture was stirred for 30 min. EDCI (72 mg) was added and stirring was continued for 1 h. An additional amount of N-methylmorpholine (20 μl) was added and stirred overnight. The reaction mixture was diluted with EtOAc, washed with sat. Na₂CO₃ and brine. The organic layer was dried over Na₂SO₄, filtered and evaporated in vacuo to dryness. Purification by flash chromatography yielded the title compound as colorless glassy solid. Example 44:

To Boc-protected intermediate 44a) (119 mg) in methanol (1 ml) and dioxane (5 ml) was added hydrogen chloride, 4.0 M sol. in 1,4-dioxane (5 ml) and the solution was stirred for 30 min at room temperature. The reaction mixture was evaporated in vacuo to dryness. The residue was triturated in Et₂O, filtered off and washed with Et₂O to yield an off-white solid.

Synthesis of Example 46: Intermediate 46a):

To Boc-D-4-chlorophenylalanine (80 mg) in DCM (2 ml) was added amine hydrochloride from 12f) (91 mg), N-methylmorpholine (41 μl), HOBt (42 mg) and the mixture was stirred for 30 min. EDCI (72 mg) was added and stirring was continued for 1 h. An additional amount of N-methylmorpholine (20 μl) was added and stirred overnight. The reaction mixture was diluted with EtOAc, washed with sat. Na₂CO₃ and brine. The or- ganic layer was dried over Na₂SO₄, filtered and evaporated in vacuo to dryness.

Purification by flash chromatography yielded the title compound as colorless glassy solid. Example 46:

To Boc-protected intermediate 46a) (81 mg) in methanol (1 ml) and dioxane (5 ml) was added hydrogen chloride, 4.0 M sol. in 1 ,4-dioxane (5 ml) and the solution was stirred for 30 min at room temperature. The reaction mixture was evaporated in vacuo to dryness. The residue was triturated in Et₂O, filtered off and washed with Et₂O to yield an off-white solid.

Synthesis of Example 47: Intermediate 47a):

To Boc-D-4-chlorophenylalanine (80 mg) in DCM (2 ml) was added amine hydrochloride from 15h) (95 mg), N-methylmorpholine (41 μl), HOBt (42 mg) and the mixture was stirred for 30 min. EDCI (72 mg) was added and stirring was continued for 1 h. An additional amount of N-methylmorpholine (20 μl) was added and stirred overnight. The reaction mixture was diluted with EtOAc, washed with sat. Na₂CO₃ and brine. The or- ganic layer was dried over Na₂SO₄, filtered and evaporated in vacuo to dryness.

Purification by flash chromatography yielded the title compound as colorless glassy solid. Example 47:

To Boc-protected intermediate 47a) (83 mg) in methanol (1 ml) and dioxane (5 ml) was added hydrogen chloride, 4.0 M sol. in 1 ,4-dioxane (5 ml) and the solution was stirred for 30 min at room temperature. The reaction mixture was evaporated in vacuo to dryness. The residue was triturated in Et₂O, filtered off and washed with Et₂O to yield an off-white solid.

Synthesis of Examples 51 , 52 and 53: Intermediate 51-53a):

To Boc-D-4-chlorophenylalanine (95 mg) in DCM (7 ml) was added amine hydrochloride from 17-19c) (100 mg), N-methylmorpholine (77 μl), HOBt (58 mg) and the mixture was stirred for 30 min. EDCI (85 mg) was added and stirring was continued for 1 h. An additional amount of N-methylmorpholine (21 μl) was added and the mixture was stirred overnight. The reaction mixture was evaporated in vacuo, diluted with EtOAc, and washed with sat. Na₂Cθ3, H₂O and brine. The aqueous layers were re-extracted with EtOAc. The combined organic layer was dried over Na₂SO₄, filtered, and evaporated in vacuo to dryness. Purification by flash chromatography yielded the title compounds as clear colorless oil.

53

To Boc-protected intermediate 51 -53a) (132 mg) in dioxane (0.5 ml) was added hydrogen chloride, 4.0 M sol. in 1 ,4-dioxane (2 ml) and the solution was stirred at room temperature for 2 h. The reaction mixture was evaporated in vacuo to dryness. The three products were separated with preparative HPLC-MS.

Synthesis of Example 60: Intermediate 60a):

A mixture of (R)-(-)-2-amino-1-butanol (4.24 ml), formaldehyde solution (36.5% in H₂O, 10.87 ml) and formic acid (6.79 ml) in water (34.06 ml) was heated under reflux for 24 h. The reaction mixture was concentrated in vacuo, the residue was diluted with water (80 ml) and made alkaline by addition of NaOH 1N (pH 14). The aqueous solution was extracted with CH₂CI₂ (3 x 60 ml) and the organic layers were washed with water (25 ml) and brine (25 ml). The combined organic layer was dried over Na₂SO₄ and evapo- rated in vacuo at 4O⁰C. The crude product was purified by Kugelrohr distillation (atmospheric pressure / 155 - 250⁰C) to yield a clear colorless oil. Intermediate 60b):

B

A solution of intermediate 11d) (1.00 g), intermediate 60a) (0.75 g), and triphenyl- phosphine (1.68 g) in THF (30 ml) under argon, was cooled in ice/H₂O. DEAD (ca. 40% in toluene, 2.94 ml) was added dropwise, at a rate to keep the temperature below 5°C (ca. 15 min). After stirring for another 10 min in ice/H₂O, the cooling bath was removed and the mixture was stirred at room temperature overnight. The reaction mixture was evaporated to dryness in vacuo at 40⁰C. The two regioisomeric products were separated by flash chromatography.

Intermediate 60c):

To Boc-protected intermediate 60b) (product A) (825 mg) in dioxane (2.5 ml) was added hydrogen chloride, 4.0 M sol. in 1,4-dioxane (11 ml) and the solution was stirred at room temperature for 120 min. The solvent was removed under reduced pressure. The residue was triturated in a mixture of acetone, methanol and Et₂O, filtered off, and washed with Et₂O. Finally it was dried in vacuo at room temperature over P₂O₅ overnight to yield a white solid. Intermediate 6Od):

To Boc-D-4-chlorophenylalanine (29 mg) in DCM (2 ml) was added amine hydrochlo- ride 60c) (30 mg), N-methylmorpholine (24 μl), HOBt (18 mg) and the mixture was stirred for 30 min. EDCI (26 mg) was added and stirring was continued for 1 h. An additional amount of N-methylmorpholine (6 μl) was added and the mixture was stirred overnight. The reaction mixture was evaporated in vacuo, diluted with EtOAc, and washed with sat. Na₂Cθ₃, H₂O and brine. The aqueous layer was re-extracted with EtOAc. The combined organic layer was dried over Na₂SO₄, filtered and evaporated in vacuo to dryness. The product was purified by flash chromatography.

Example 60:

To Boc-protected intermediate 6Od) (32 mg) in dioxane (0.25 ml) was added hydrogen chloride, 4.0 M sol. in 1,4-dioxane (1.5 ml) and the solution was stirred at room temperature for 2 h. The reaction mixture was evaporated in vacuo to dryness and the residue was triturated in acetone and Et₂O. The solid was filterred off and washed with Et₂O. The product was dried in the exsiccator in vacuo over Sicapent for 30 min and in vacuo at 40⁰C for 1 hour. Synthesis of Example 61 : Intermediate 61a):

To Boc-protected intermediate 60b) (product B) (251 mg) in dioxane (0.75 ml) was added hydrogen chloride, 4.0 M sol. in 1 ,4-dioxane (3.5 ml) and the solution was stirred at room temperature for 120 min. The reaction mixture was diluted with Et₂O, the precipitated salt was filtered off, and washed with Et₂O. Finally it was dried in vacuo at room temperature over P₂O₅ overnight to yield a white solid.

Intermediate 61b):

To Boc-D-4-chlorophenylalanine (29 mg) in DCM (2 ml) was added amine hydrochlo- ride from 61a) (30 mg), N-methylmorpholine (24 μl), HOBt (18 mg) and the mixture was stirred for 30 min. EDCI (26 mg) was added and stirring was continued for 1 h. An additional amount of N-methylmorpholine (6 μl) was added and the mixture was stirred overnight. The reaction mixture was evaporated in vacuo, diluted with EtOAc, and washed with sat. Na₂CO₃, H₂O and brine. The aqueous layers were re-extracted with EtOAc. The combined organic layer was dried over Na₂Sθ4, filtered and evaporated in vacuo to dryness. The product was purified by flash chromatography. Example 61:

To Boc-protected intermediate 61b) (29 mg) in dioxane (0.25 ml) was added hydrogen chloride, 4.0 M sol. in 1,4-dioxane (1.5 ml) and the solution was stirred at room tem- perature for 2 h. The reaction mixture was evaporated in vacuo to and the residue was triturated in acetone and Et₂O. The solid was filterred off and washed with Et₂O. The residue was dried in the exsiccator in vacuo over Sicapent for 30 min and in vacuo at 40⁰C for 1 hour.

Synthesis of Example 64: Intermediate 64a):

To a solution of sodium ethoxide (1.26 g) in ethanol (30 ml) was added diethyl ethyl- malonate (3.64 ml) followed by 2-bromo-5-fluorobenzylbromide (4.96 g) and the reaction mixture was kept under reflux overnight. The reaction mixture was evaporated in vacuo and the residue was partitioned between diethyl ether and water and the aqueous layer was extracted two times with diethyl ether. The combined organic layer was washed twice with water and brine. The organic layer was dried over Na₂Sθ₄ and evaporated in vacuo to dryness. The product was purified using flash chromatography.

Intermediate 64b):

Intermediate 64a) (5.27 g) was heated under reflux in 1.8 M KOH in H₂O/EtOH (60 ml) for 5 h. After evaporation of the ethanol an additional amount of KOH (18 g) was added to the residue, and the reaction mixture was stirred at 100⁰C for 2 h. The reaction mixture was diluted with H₂O (100 ml), extracted with Et₂O and the organic layer was washed with H₂O. The combined aqueous layer was cooled in ice/H₂O and acidified with 50% H₂SO₄ to pH 1. The resulting suspension was extracted twice with Et₂O (100 ml each) and the organic layers were washed with water and brine. The combined organic layer was dried over Na₂SO₄ and evaporated in vacuo to dryness. The residue was triturated in hexane and less Et₂O, then filtered and washed with hexane and less Et₂O. The solid residue was decarboxylated by heating at 200⁰C. The evolution of CO₂ ceased after 20 min and the melt was left to cool to room temperature to yield beige needles.

Intermediate 64c):

Intermediate 64b) (2.71 g) was dissolved in methanol (7.53 ml). Then sulfuric acid (100 μl) was added, and the reaction mixture was heated under reflux overnight (oil bath temperature 85⁰C) with exclusion of humidity by means of a drying tube (blue silica gel). The reaction mixture was evaporated in vacuo at 40⁰C and the colorless oily residue was poured into ice-water (50 ml). The resulting white emulsion was extracted with Et₂O (75 ml), and the organic phase was washed with sat. Na₂CO₃ (3 x 20 ml), H₂O (15 ml), and brine (15 ml). The organic phase was then dried over MgSO₄ and evaporated in vacuo.

Intermediate 64d):

Intermediate 64c) (2520 mg), N-Boc-1,2,3,6-tetrahydropyridine-4-boronic acid pinacol ester (2829 mg), potassium carbonate (3611 mg) and dichloro(1,1'-bis(diphenyl- phosphino)-ferrocene)palladium(ll) DCM adduct (425 mg) were dissolved in DMF (100 ml) in a dry apparatus under argon and the mixture was degassed by bubbling with argon for 30 min. The orange suspension was then heated under argon in an oil bath at 85⁰C overnight to give a dark purple suspension. The reaction mixture was filtered through Celite and evaporated to dryness in vacuo. The crude product was purified by column chromatography.

Intermediate 64e):

Intermediate 64d) (1453 mg) was dissolved in ethanol (50 ml) and 10% palladium on activated carbon (150 mg) was added. The reaction mixture was purged three times with hydrogen (5 bar) and stirred under a hydrogen atmosphere (25 bar) overnight. The crude mixture was filtered through Celite and the solvent was removed under reduced pressure to yield a beige oil.

Intermediate 64f):

Intermediate 64e) (1288 mg) in diethyl ether (20 ml) was slowly added to a mixture of lithium aluminum hydride (186 mg) and diethyl ether (30 ml) at 0⁰C. After addition, the reaction mixture was stirred at 0⁰C for 2 h. The reaction mixture was hydrolyzed with a minimum amount of water. The inorganic precipitate was filtered off and washed twice with diethyl ether. The combined filtrate was dried over sodium sulfate, filtered again, and the solvent was removed under reduced pressure.

Intermediate 64g):

To a solution of intermediate 64f) (1001 mg) in DCM (20 ml) was slowly added Dess- Martin periodinane (1510 mg). After addition, the reaction mixture was stirred at room temperature for 3 h. TLC indicated that the reaction was not complete. A second batch of Dess-Martin periodinane (755 mg) was added and the reaction was stirred overnight. The reaction mixture was diluted with DCM and washed three times with saturated so- dium bicarbonate solution and brine. The organic phase was dried over sodium sulfate, filtered, and the solvent was removed under reduced pressure. The product was puri- fied by flash chromatography.

Intermediate 64h):

To a solution of intermediate 64g) (91 mg) and dimethylamine, 2.0 M solution in THF (250 μl) in 1 ,2-dichloroethane (3 ml), sodium triacetoxyborohydride (74 mg) was added. The reaction mixture was then stirred at room temperature overnight. The mixture was diluted with EtOAc (70 ml) and washed two times with sat. NaHCO₃ (25 ml), water and brine (25 ml each). The organic phase was dried over Na2SO₄ and concentrated. The product was purified by flash chromatography. Intermediate 64i):

To intermediate 64h) (74 mg) in dioxane (5 ml) and methanol (1 ml) was added hydrogen chloride, 4.0 M sol. in 1 ,4-dioxane (5 ml) and the solution was stirred for 60 min at room temperature. The solvent was removed under reduced pressure to yield the product as a colorless glassy solid.

Intermediate 64j):

To Boc-D-4-chlorophenylalanine (31 mg) in DMF (1 ml) was added amine hydrochloride from 64i) (35 mg), N-methylmorpholine (16 μl), HOBt (16 mg) and the mixture was stirred for 30 min. EDCI (20 mg) was added and stirring was continued for 1 h. An additional amount of N-methylmoφholine (10 μl) was added and the mixture was stirred overnight. The reaction mixture was diluted with EtOAc₁ and washed twice with sat. Na₂CO₃, twice with water and once with brine. The organic layer was dried over Na₂SO₄, filtered and evaporated in vacuo to dryness. Purification by flash chromatography yielded the title compound as a colorless glassy solid. Example 64:

To Boc-protected intermediate 64j) (17 mg) in methanol (1 ml) and dioxane (5 ml) was added hydrogen chloride, 4.0 M sol. in 1 ,4-dioxane (5 ml) and the solution was stirred for 30 min at room temperature. The reaction mixture was evaporated in vacuo to dryness. The crude product was purified using preparative HPLC-MS.,

Synthesis of Example 66: Intermediate 66a):

To a solution of sodium ethoxide (1.26 g) in ethanol (30 ml) was added diethyl methyl- malonate (3.31 ml) followed by intermediate 15a) (5.26 g) and the reaction mixture was kept under reflux overnight. The reaction mixture was evaporated in vacuo and the residue was partitioned between diethyl ether and water. The aqueous layer was extracted two times with diethyl ether. The combined organic layer was washed twice with water and brine. The organic layer was dried over Na₂SO₄ and evaporated in vacuo to dryness. The product was purified by distillation. The fractions which distilled off between 140 and 220⁰C at 0.2 mbar were collected.

Intermediate 66b):

Intermediate 66a) (5.89 g) was heated under reflux in 1.8 M KOH in H₂O/EtOH (60 ml) for 5 h. After evaporation of the ethanol, an additional amount of KOH (18 g) was added to the residue, and the reaction mixture was stirred at 100⁰C for 2 h. The reaction mixture was diluted with 100 ml of H₂O, extracted with Et₂O, and the organic layer was washed with H₂O. The combined aqueous layer was cooled in ice/H₂O and acidified with 50% H₂SO₄ to pH 1. The resulting suspension was extracted twice with Et₂O (100 ml each) and the organic layers were washed with water and brine. The combined organic layer was dried over Na₂SO₄ and evaporated in vacuo to dryness. The residue was triturated in hexane and less Et₂O₁ then filtered and washed with hexane and less Et₂O. The solid residue was decarboxylated by heating at 200⁰C. The evolution of CO₂ ceased after 20 min and the product was left to cool to room temperature to yield a brown oil.

Intermediate 66c):

Intermediate 66b) (3.08 g mmol) was dissolved in dry DCM (85 ml) and cooled to 0⁰C in an ice-water bath. Oxalyl chloride (1.03 ml) in DCM (15 ml) was added dropwise followed by the addition of 1-2 drops of DMF. This mixture was stirred at 0⁰C for 1.5 h and at room temperature for 2 h (no more gas evolution, clear solution obtained) and then concentrated. The product was dried under reduced pressure.

Intermediate 66d):

To a solution of intermediate 66c) (3.24 g) in DCM (70 ml) at 0⁰C was added pyrrolidine (2.73 ml) dropwise and the reaction mixture was stirred at O⁰C for 2 h. The reaction mixture was diluted with DCM (100 ml) and washed twice each with 1 M HCI, 1 M NaOH and once with brine. The organic phase was dried over sodium sulfate and the solvent was distilled off.

Intermediate 66e):

Intermediate 66d) (3226 mg), N-Boc-1 ,2,3,6-tetrahydropyridine-4-boronic acid pinacol ester (3170 rηg), potassium carbonate (4047 mg) and dichloro(1,1'-bis(diphenyl- phosphino)-ferrocene)palladium(ll) DCM adduct (482 mg) were dissolved in DMF (100 ml) in a dry apparatus under argon and the mixture was degassed by bubbling with argon for 30 min. The orange suspension was then heated under argon in an oil bath at 85°C overnight to give a dark purple suspension. The reaction mixture was filtered through Celite and evaporated to dryness in vacuo. The crude product was purified by column chromatography.

Intermediate 66 f):

Intermediate 66e) (2793 mg) was dissolved in EtOH (50 ml) and AcOH (50 ml) and platinum(IV) oxide (148 mg) was added. The reaction mixture was evacuated three times and purged with hydrogen. The reaction mixture was then stirred at room temperature under hydrogen for 2 h. The reaction mixture was filtered and evaporated to dryness in vacuo. The residue was coevaporated with toluene (3 x 75 ml) and was finally dried under high vacuum at RT overnight. The crude product was purified by col- umn chromatography. Intermediate 66g):

Intermediate 66f) (2092 mg) in diethyl ether (20 ml) was slowly added to a mixture of lithium aluminum hydride (274 mg) and diethyl ether (30 ml) at 0⁰C. After addition, the reaction mixture was stirred at 0⁰C for 2 h. The reaction mixture was hydrolyzed with a minimum amount of water. The inorganic precipitate was filtered off and washed twice with diethyl ether. The combined filtrate was dried over sodium sulfate, filtered again, and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography to yield a colorless oil.

Intermediate 66h):

To intermediate 66g) (901 mg) in dioxane (10 ml) and methanol (2 ml) was added hydrogen chloride, 4.0 M sol. in 1,4-dioxane (10 ml) and the solution was stirred for 30 min at room temperature. The solvent was removed under reduced pressure, the residue was triturated with acetone (5 ml) and diethyl ether (50 ml), and the product was filtered off. The product was obtained as a white solid. Intermediate 66i):

To Boc-D-4-chlorophenylalanine (80 mg) in DMF (2 ml) was added amine hydrochlo- ride from 66h) (98 mg), N-methylmorpholine (41 μl), HOBt (42 mg) and the mixture was stirred for 30 min. EDCI (53 mg) was added and stirring was continued for 1 h. An additional amount of N-methylmorpholine (20 μl) was added and the mixture was stirred overnight. The reaction mixture was diluted with EtOAc, and washed twice with sat. Na₂CO₃, twice with water and brine. The organic layer was dried over Na₂SO₄, filtered and evaporated in vacuo to dryness.

Purification by flash chromatography yielded the title compound as a pale yellow glassy solid.

Example 66:

To Boc-protected intermediate 66i) (105 mg) in methanol (1 ml) and dioxane (5 ml) was added hydrogen chloride, 4.0 M sol. in 1 ,4-dioxane (5 ml) and the solution was stirred at room temperature for 30 min. The reaction mixture was evaporated in vacuo to dry- ness. The residue was triturated in Et₂O, filtered off, and washed with Et₂O to yield a beige solid. Synthesis of Example 68: Intermediate 68a):

To a solution of sodium ethoxide (2.72 g) in ethanol (60 ml) was added diethyl methyl- malonate (7.87 ml) followed by intermediate 15a) (11.38 g) and the reaction mixture was kept under reflux overnight. The reaction mixture was evaporated in vacuo and the residue was partitioned between diethyl ether and water. The aqueous layer was ex- tracted two times with diethyl ether. The combined organic layer was washed twice with water and brine. The organic layer was dried over Na₂SO₄ and evaporated in vacuo to dryness. The product was purified by distillation. The fractions which distilled off be- tween 140 and 220⁰C at 0.2 mbar were collected.

Intermediate 68b):

Intermediate 68a) (12.11 g) was heated under reflux in 1.8 M KOH in H₂O/EtOH (150 ml) for 5 h. After evaporation of the ethanol, an additional amount of KOH (54 g) was added to the residue, and the reaction mixture was stirred at 100⁰C for 2 h. The reac- tion mixture was diluted with H₂O (200 ml), extracted with Et₂O, and the organic layer was washed with H₂O. The combined aqueous layer was cooled in ice/H₂0 and acidi- fied with 50% H₂SO₄ to pH 1. The resulting suspension was extracted twice with Et₂O (200 ml each) and the organic layers were washed with water and brine. The combined organic layer was dried over Na₂SO₄ and evaporated in vacuo to dryness. The residue was triturated in hexane and less Et₂O, then filtered and washed with hexane and less Et₂O. The solid residue was decarboxylated by heating at 200°C. The evolution of CO₂ ceased after 20 min and the product was left to cool to room temperature to yield a brown oil. Intermediate 68c):

Intermediate 68b) (5.43 g) was dissolved in methanol (11.93 ml). Then sulfuric acid (365 μl) was added, and the reaction mixture was heated under reflux overnight (oil bath temperature 85⁰C) with exclusion of humidity by means of a drying tube (blue silica gel). The reaction mixture was evaporated in vacuo at 4O⁰C and the colorless oily residue was poured into ice-water (100 ml). The resulting white emulsion was extracted with Et₂O (100 ml), and the organic phase was washed with sat. Na₂CO₃ (3 x 30 ml), H₂O (20 ml), and brine (20 ml). The organic phase was then dried over MgSO₄ and evaporated in vacuo.

Intermediate 68d):

Zinc activation. Celite (174 mg) was added into a flame dried 50 ml Schlenk flask and dried by heating in vacuo. Then zinc dust (883 mg) and dry DMA (1.5 ml) were added under argon. The mixture was stirred at room temperature while a 7:5 v/v mixture of TMSCI/1 ,2-dibromoethane (153 μl TMSCI, 109 μl 1 ,2-dibromoethane, solution in 0.7 ml of DMA) was added at a rate to maintain the temperature below 65⁰C. The resulting slurry was aged for 15 min.

Zink insertion. A solution of Boc-4-iodopiperidine (3364 mg) in dry DMA (6.8 ml) was slowly added under argon atmosphere to the mixture described above at a rate to maintain the temperature below 65°C. The reaction mixture was then aged for 30 min at room temperature.

Coupling. A 50 ml three-necked flask was charged with dichloro-1,1'- bis(diphenylphosphino)-ferrocene-palladium(ll) DCM adduct (188 mg), copper iodide (88 mg) and intermediate 68c) (2.36 g) in DMA (11 ml) The resulting mixture was degassed three times and the filtrate of the zinc insertion reaction was then added. The reaction mixture was degassed two times, then heated to 80⁰C and stirred overnight. The reaction mixture was concentrated under high vacuum at 60⁰C and the remainig black oil was taken up in a mixture of ethyl acetate and water. The mixture was filtered through Celite and the phases were separated. The aqueous phase was extracted twice with ethyl acetate. The combined organic layer was washed with water and brine, dried over sodium sulfate and the solvent was removed under reduced pressure. The product was purified using flash chromatography.

Intermediate 68e):

Intermediate 68d) (571 mg) in diethyl ether (20 ml) was slowly added to a mixture of lithium aluminum hydride (79 mg) and diethyl ether (30 ml) at O⁰C. After addition the reaction mixture was stirred at 0⁰C for 2 h. The reaction mixture was hydrolyzed with a minimum amount of water. The inorganic precipitate was filtered off and washed twice with diethyl ether. The combined filtrate was dried over sodium sulfate, filtered, and the solvent was removed under reduced pressure.

Intermediate 6Bf):

Intermediate 68e) (450 mg) was dissolved in dry DMSO (6 ml) and triethylamine (1151 μl). A solution of sulfurtrioxide-pyridine complex (563 mg) in dry DMSO (6 ml) was slowly added while the reaction mixture was maintained at 25°C. The reaction mixture was stirred for 4 h. After acidification of the reaction mixture to pH 4.5-5 with 1N HCI, it was poured into water. The resulting emulsion was extracted three times with diethyl ether. The combined organic layer was washed with water and brine, dried over sodium sulfate, and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography.

Intermediate 68g):

To a solution of intermediate 68f) (97 mg) and (R)-3-fluoropyrrolidine hydrochloride (33 mg) in 1 ,2-dichloroethane (3 ml), DIEΞA (67 μl) was added followed by sodium tri- acetoxyborohydride (74 mg). The reaction mixture was then stirred at room temperature overnight. The mixture was diluted with EtOAc (70 ml) and washed two times with sat. NaHCθ₃ (25 ml), water and brine (25 ml each). The organic phase was dried over Na₂Sθ₄ and concentrated. The product was purified with flash chromatography.

Intermediate 68h):

To intermediate 68g) (101 mg) in dioxane (5 ml) and methanol (1 ml) was added hydrogen chloride, 4.0 M sol. in 1 ,4-dioxane (5 ml) and the solution was stirred for 60 min at room temperature. Then the solvent was removed under reduced pressure to yield the product as a colorless glassy solid. Intermediate 68i):

To Boc-D-4-chlorophenylalanine (35 mg) in DMF (1 ml) was added amine hydrochloride from 68h) (47 mg), N-methylmorpholine (18 μl), HOBt (19 mg) and the mixture was stirred for 30 min. EDCI (23 mg) was added and stirring was continued for 1 h. An additional amount of N-methylmorpholine (10 μl) was added and the reaction mixture was stirred overnight. The reaction mixture was diluted with EtOAc, and washed twice with sat. Na₂Cθ₃, twice with water and once with brine. The organic layer was dried over Na₂SO₄, filtered and evaporated in vacuo to dryness. Purification by flash chromatography yielded the title compound as a beige glassy solid.

Example 68:

To Boc-protected intermediate 68i) (110 mg) in methanol (1 ml) and dioxane (5 ml) was added hydrogen chloride, 4.0 M sol. in 1,4-dioxane (5 ml) and the solution was stirred for 30 min at room temperature. The solvent was removed under reduced pressure, the residue was triturated with acetone (5 ml) and hexane (50 ml) and the solid product was filtered off. Synthesis of Example 74, 75, 76 and 77: Intermediate 74-77a):

N-(Dipheπylmethylene) glycine ethyl ester (9.29 g), 1-(bromomethyl)-4-chloro-2- fluorobenzene (8.63 g) and benzyltriethylammonium chloride (TEBAC) (7.91 g) were dissolved in DCM (100 ml) and 10% aqueous KOH (91 ml) was added. The resulting two-phase mixture was stirred at room temperature for 24 hours. Then the organic layer was separated and concentrated. The residue was taken up in diethyl ether (200 ml) and washed with water (150 ml) followed by brine (100 ml) and the organic layer was dried over Na₂SO₄. The solvent was removed under reduced pressure. The product was purified by flash chromatography.

Intermediate 74-77b):

5% aq. HCI in H₂O (9 ml) was added portionwise to a solution of intermediate 74-77a) (1 g) in THF (3.4 ml) at 0⁰C. A precipitate appeared after the addition. The mixture was stirred at room temperature for 1 h. THF was evaporated under reduced pressure. Wa- ter (50 ml) was added to the residue. The aqueous solution was washed with diethyl ether (3 x 75 ml) and with DCM (75 ml). The aqueous layer was then basified with 5 N aqueous NaOH (2 ml) and 1 N aqueous NaOH (12 ml) to get pH7/8. The compound was extracted with DCM (5 x 100 ml). The combined organic layer was dried over Na₂SO₄, filtered and evaporated under reduced pressure. The crude compound was purified by column chromatography. Intermediate 74-77c):

1 N Aqueous sodium hydroxide (5.5 ml) was added portionwise to a solution of inter- mediate 74-77b) (446 mg) in THF (5 ml) at 0⁰C. The reaction mixture was stirred at room temperature overnight. The solvent was evaporated under reduced pressure and water (60 ml) was added to the residue. The aqueous phase was washed with diethyl ether (2 x 90 ml; 3 x 30 ml) and DCM (2 x 100 ml). The aqueous phase was neutralized with 5 N aqueous HCI (1 ml). The impurities were extracted with diethyl ether (2 x 100 ml). The aqueous phase was evaporated to dryness and water (40 ml) was added. The suspension was put in the fridge overnight, filtered and the product, a white solid, was rinsed with water and diethyl ether. The filtrate was evaporated again to dryness and water (10 ml) was added. The suspension was put in the fridge overnight, filtered and the second batch of product was rinsed with water and diethyl ether. The solids from the two batches were combined and dried in vacuo.

Intermediate 74-77d):

HO

Racemic intermediate 74-77c) (313 mg) was dissolved in Tris-maleate buffer (26 ml, pH 7.8) containing 0.1 M KCI. To this solution was added L-amino acid oxidase (Sigma Type 1, activity 0.33 units/mg; 10 mg) and catalase (1 mg). After 84 h, the reaction mix- ture was brought to pH 7 with 0.5 N HCI and purified by ion-exchange chromatography over Dowex 50, eluting the amino acid with 1 N ammonia. The solvent was removed under reduced pressure and the product was dried in vacuo at room temperature over P₂O₅ overnight. Intermediate 74-77e):

Intermediate 74-77d) (360 mg) was dissolved in 2 M sodium hydroxide (0.7 ml) and cooled to 0⁰C. Di-tert-butyl dicarbonate (188 mg) in dioxaπe (1 ml) was slowly added. After half an hour, the reaction mixture was warmed to room temperature and allowed to stir overnight. A second batch of di-tert-butyl-dicarbonate (79 mg) was added and stirring was continued for another 4 h. The reaction mixture was evaporated to dryness and water (20 ml) was added. The aqueous phase was washed with diethyl ether (5 x 40 ml) and DCM (3 x 30 ml). The aqueous phase was acidified to pH 2 using 1 N aqueous hydrochloric acid and extracted with ethyl acetate (3 x 40 ml). The combined organic layer was then dried over Na₂SO₄, filtered and concentrated in vacuo.

Intermediate 74-77f):

To intermediate 74-77e) (100 mg) in DCM (7 ml) was added amine hydrochloride from 17-19c) (100 mg), N-methylmorpholine (77 μl), HOBt (58 mg) and the mixture was stirred for 30 min. EDCI (61 mg) was added and stirring was continued for 1 h. An additional amount of N-methylmorpholine (21 μl) was added and the mixture was stirred overnight. The reaction mixture was evaporated in vacuo, diluted with EtOAc, washed with sat. Na₂Cθ₃, H₂O and brine. The aqueous layers were extracted with EtOAc. The combined organic layer was dried over Na₂SO₄, filtered, and evaporated in vacuo to dryness.

Purification by flash chromatography yielded the title compounds as a clear colorless oil. Examples 74, 75, 76 and 77:

76 77

To Boc-protected intermediate 74-77f) (145 mg) in dioxane (0.5 ml) was added hydrogen chloride, 4.0 M sol. in 1 ,4-dioxane (2 ml) and the solution was stirred at room temperature for 2 h. The reaction mixture was evaporated in vacuo to dryness. The four products were separated using preparative HPLC-MS.

Synthesis of Example 82: Intermediate 82a):

Intermediate 24b) (33 mg), Boc-D-N-methyl-4-chlorophenylalanine (34 mg) and HOBt (20 mg) were dissolved in DCM (2.5 ml). N-Methylmorpholine (26 μl) was added and the mixture stirred at room temperature for 30 min. Then EDCI (29 mg) was added, and the reaction stirred at room temperature for another 60 min. An additional amount of N- methylmorpholine (7 μl) was added and stirring continued at room temperature overnight. The reaction mixture was diluted with EtOAc (40 ml), washed with sat. Na₂CO₃ (3 x 15 ml), H₂O (2 x 15 ml) and brine (10 ml). The organic layer was dried over Na₂SO₄, filtered and evaporated in vacuo to dryness. The product was purified by flash chromatography.

Example 82:

Intermediate 82a) (48 mg) was dissolved in dioxane (1 ml) and hydrogen chloride, 4.0 M sol. in 1,4-dioxane (5 ml) was added at room temperature. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was evaporated in vacuo to dryness. The residue was triturated in acetone (1 ml) and Et₂O (3 ml), filtered off and washed with Et₂O (2 x 1 ml). Finally, the product was dried in vacuo over P₂O₅ at room temperature overnight. The product was obtained as a white solid.

Synthesis of Example 83: Intermediate 83a):

To a solution of 2-amino-1-butanol (6.33 ml) and 1 ,4-dibromobutane (7.91 ml) in CH₃CN (70 ml) was added K₂CO₃ (18.52 g) and the resulting suspension was stirred at reflux temperature for 22 h. The reaction mixture was evaporated in vacuo and the residue was partitioned between EtOAc and water. The organic layer was washed with water and brine. The aqueous layers were extracted with EtOAc. The combined or- ganic layer was dried over Na₂SO₄ and evaporated in vacuo to dryness. The crude product was purified by Kugelrohr-distillation (15 mbar, 110-140⁰C) to yield a clear colorless oil. Intermediate 83b):

B

A solution of intermediate 11d) (1.09 g), intermediate 83a) (1.00 g) and triphenyl- phosphine (1.83 g) in dry THF (30 ml) under argon atmosphere, was cooled in an ice/H₂O bath. Then DEAD (ca. 40% in toluene, 3.20 ml) was added dropwise, at a rate to keep the temperature below 5°C. After stirring for another 10 min, the cooling bath was removed and the mixture was stirred at room temperature overnight. The reaction mixture was evaporated to dryness in vacuo at 40⁰C. The two regioisomeric products were separated by flash chromatography.

Intermediate 83c):

To intermediate 83b) product A (1.07 g) in dioxane (4 ml) was added hydrogen chloride, 4.0 M sol. in 1,4-dioxane (15 ml) and the solution was stirred for 2 h at room temperature. The solvent was removed under reduced pressure and the residue was triturated with acetone and diethyl ether. The product was filtered off and washed with ace- tone/diethyl ether. The product was obtained as an off-white solid. Intermediate 83d):

Intermediate 83c) (30 mg), Boc-D-N-methyl-4-chlorophenylalanine (29 mg) and HOBt (28 mg) were dissolved in DCM (2 ml). N-Methylmorpholine (22 μl) was added and the mixture stirred at room temperature for 30 min. Then EDCI (25 mg) was added, and the reaction stirred at room temperature for another 60 min. An additional amount of N- methylmorpholine (6 μl) was added and stirring continued at room temperature over- -ight. The reaction mixture was diluted with EtOAc (40 ml), washed with sat. Na₂Cθ₃ (3 x 15 ml), H₂O (2 x 15 ml) and brine (10 ml). The organic layer was dried over Na₂SO₄, filtered, and evaporated in vacuo to dryness. The product was purified by flash chroma- tography.

Example 83:

Intermediate 83d) (19 mg) was dissolved in dioxane (250 μl) and hydrogen chloride, 4.0 M sol. in 1,4-dioxane (500 μl) was added at room temperature. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was evaporated in vacuo to dryness. The residue was triturated in acetone, Et₂O and hexane, filtered off, and washed with Et₂O/hexane. Finally, the product was dried in vacuo at 40⁰C for 2 h. The product was obtained as a white solid. Synthesis of Example 84: Intermediate 84a):

To intermediate 83b) product B (370 mg) in dioxane (1 ml) was added hydrogen chloride, 4.0 M sol. in 1 ,4-dioxane (5 ml) and the solution was stirred at room temperature for 2 h. The solvent was removed under reduced pressure and the residue was triturated with acetone and diethyl ether. The product was filtered off and washed with ace- tone/diethyl ether. The product was obtained as an off-white solid.

Intermediate 84b):

Intermediate 84a) (50 mg), Boc-D-N-methyl-4-chlorophenylalanine (46 mg) and HOBt (28 mg) were dissolved in DCM (3 ml). N-Methylmorpholine (37 μl) was added and the mixture stirred at room temperature for 30 min. Then EDCI (41 mg) was added, and the reaction stirred at room temperature for another 60 min. An additional amount of N- methylmorpholine (10 μl) was added and stirring continued at room temperature overnight. The reaction mixture was evaporated in vacuo the residue taken up with EtOAc, washed with sat. Na₂CO₃, water and brine. The aqueous layers were extracted with EtOAc. The combined organic layer was dried over Na₂SO₄, filtered, and evaporated in vacuo to dryness. The product was purified by flash chromatography. Example 84:

Intermediate 84b) (58 mg) was dissolved in dioxane (500 μl) and hydrogen chloride, 4.0 M sol. in 1,4-dioxane (1000 μl) was added at room temperature. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was evaporated in vacuo to dryness. The residue was dissolved in isopropanol and precipitated by adding acetone, Et₂O and hexane. The solid was filtered off, washed with Et₂O, and dried in vacuo. The product was obtained as a white solid.

Synthesis of Example 86: Intermediate 86a):

To Boc-D-N-methyl-4-chlorophenylalanine (84 mg) in DMF (2 ml) was added amine hydrochloride from 8d) (99 mg), N-methylmorpholine (41 μl), HOBt (42 mg) and the mixture was stirred for 30 min. EDCI (53 mg) was added and stirring was continued for 1 h. An additional amount of N-methylmorpholine (20 μl) was added and the reaction was stirred overnight. The reaction mixture was diluted with EtOAc, washed three times with sat. Na₂CO₃, two times with water and with brine. The organic layer was dried over Na₂SO₄, filtered and evaporated in vacuo to dryness. Purification by flash chromatography yielded the title compound as colorless crystals. Example 86:

To Boc-protected intermediate 86a) (120 mg) in methanol (1 ml) and dioxane (5 ml) was added hydrogen chloride, 4.0 M sol. in 1,4-dioxane (5 ml) and the solution was stirred for 30 min at room temperature. The reaction mixture was evaporated in vacuo to dryness. The residue was triturated in acetone and hexane, filtered off, and washed with Et₂O to yield an off-white solid.

Synthesis of Example 89: Intermediate 89a):

To Boc-D-N-methyl-4-chlorophenylalanine (84 mg) in DMF (2 ml) was added amine hydrochloride from 66h) (98 mg), N-methylmorpholine (41 μl), HOBt (42 mg) and the mixture was stirred for 30 min. EDCI (53 mg) was added and stirring was continued for 1 h. An additional amount of N-methylmorpholine (20 μl) was added and the mixture was stirred overnight. The reaction mixture was diluted with EtOAc, and washed twice with sat. Na₂CO₃, and twice with water and brine. The organic layer was dried over Na₂SO₄, filtered, and evaporated in vacuo to dryness.

Purification by flash chromatography yielded the title compound as a colorless glassy solid. Example 89:

To Boc-protected intermediate 89a) (48 mg) in methanol (1 ml) and dioxane (5 ml) was added hydrogen chloride, 4.0 M sol. in 1 ,4-dioxane (5 ml) and the solution was stirred for 30 min at room temperature. The reaction mixture was evaporated in vacuo to dry- ness. The residue was triturated with acetone and hexane, filtered off, and washed with Et₂O to yield an off-white solid.

Synthesis of Example 91 : Example 91:

Formaldehyde solution (>36.5% in H₂O) (27 μl) was added to a solution of example 36 (50 mg) and N.N-diisopropylethylamine (31 μl) in 1 ,2-dichloroethane (2 ml). The reac- tion mixture was cooled in an ice/H₂O-bath and sodium triacetoxyborohydride (168 mg) was added in one portion. After 5 minutes the ice/H₂O-bath was removed and the reac- tion mixture was stirred at room temperature for 3.5 h. The reaction mixture was quenched by adding aqueous sat. NaHCO₃ and the product was extracted with EtOAc. The aqueous layer was extracted with EtOAc. The combined organic layer was dried over Na₂SO₄ and evaporated in vacuo to dryness. The crude product was purified by flash chromatography. The purified product was dissolved in CH₂CI₂, acidified with 1M HCI in Et₂O (148 μl), and evaporated in vacuo. The residue was triturated in CH₂CI₂/Et₂0 and the solid was filtered off, washed with Et₂O, and dried in vacuo at 40⁰C for 2 hours. The dihydrochlo- ride was obtained as a white solid.

Further examples are exemplified below.

BIOLOGICAL ASSAYS

A. Binding Assay

A membrane binding assay is used to identify competitive inhibitors of fluorescence labeled NDP-alpha-MSH binding to HEK293 cell membrane preparations expressing human melanocortin receptors.

The test compound or unlabeled NDP-alpha-MSH is dispensed at varying concentra- tions to a 384 well microtiter plate. Fluorescence labeled NDP-alpha-MSH is dispensed at a single concentration, followed by addition of membrane preparations. The plate is incubated for 5 h at room temperature.

The degree of fluorescence polarization is determined with a fluorescence polarization microplate reader.

B. Functional Assay

Agonistic activity of human melanocortin receptors is determined in a homogeneous membrane based assay. Competition between unlabeled cAMP and a fixed quantity of fluorescence labeled cAMP for a limited number of binding sites on a cAMP specific antibody is revealed by fluorescence polarization.

The test compound or unlabeled NDP-alpha-MSH is dispensed at varying concentra- tions to a 384 well microtiter plate. Membrane preparations from HEK293 cells ex- pressing the human melanocortin receptors are added. After a short preincubation pe- riod, an appropriate amount of ATP, GTP and the cAMP antibody is added and the plate is further incubated before the fluorescence labeled cAMP conjugate is dis- pensed. The plate is incubated for 2 h at 4°C before it is read on a fluorescence polari- zation microplate reader. The amount of cAMP produced as a response to a test com- pound is compared to the production of cAMP resulting from stimulation with NDP- alpha-MSH. Representative compounds of the present invention were tested and found to bind to the melanocortin-4 receptor. These compounds were generally found to have IC₅₀ values less than 2 μM. Representative compounds of the present invention were also tested in the functional assay and found generally to activate the melanocortin-4 receptor with EC₅₀ values less than 2 μM.

Table 3: Biological data for selected examples of the invention

In the table are listed the IC₅₀ values of the hMC-4R binding assay and the EC₅₀ values of the functional assay. The IC_5O and EC₅O values were grouped in 3 classes: a ≤ 0.1 μM; b > 0.1 μM and < 1.0 μM; c > 1.0 μM

C. In Vivo Food Intake Models

1. Spontaneous Feeding Paradigm

Food intake in rats is measured after i.p. or p.o. administration of the test compound (see e.g. Chen, A.S. et ai. Transgenic Res 2000 Apr;9(2): 145-54).

2. Models of LPS-lnduced Anorexia and Tumor-Induced Cachexia

Prevention or amelioration of anorexia induced by lipopolysaccharide (LPS) administration or cachexia induced by tumor growth is determined upon i.p. or p.o. administration of test compounds to rats (see e.g. Marks, D.L.; Ling, N and Cone, R.D. Cancer Res 2001 Feb 15;61 (4): 1432-8).

D. Rat Ex Copula Assay

Sexually mature male Caesarian Derived Sprague Dawley (CD) rats (over 60 days old) are used with the suspensory ligament surgically removed to prevent retraction of the penis back into the penile sheath during the ex copula evaluations. Animals receive food and water ad lib and are kept on a normal light/dark cycle. Studies are conducted during the light cycle.

1. Conditioning to Supine Restraint for Ex Copula Reflex Tests

This conditioning takes about 4 days. Day 1 , the animals are placed in a darkened re- strainer and left for 15 - 30 minutes. Day 2, the animals are restrained in a supine posi- tion in the restrainer for 15 - 30 minutes. Day 3, the animals are restrained in the su- pine position with the penile sheath retracted for 15 - 30 minutes. Day 4, the animals are restrained in the supine position with the penile sheath retracted until penile re- sponses are observed. Some animals require additional days of conditioning before they are completely acclimated to the procedures; non-responders are removed from further evaluation. After any handling or evaluation, animals are given a treat to ensure positive reinforcement.

2. Ex Copula Reflex Tests

Rats are gently restrained in a supine position with their anterior torso placed inside a cylinder of adequate size to allow for normal head and paw grooming. For a 400 - 500 gram rat, the diameter of the cylinder is approximately 8 cm. The lower torso and hind limbs are restrained with a nonadhesive material (vetrap). An additional piece of vetrap with a hole in it, through which the glans penis will be passed, is fastened over the animal to maintain the preputial sheath in a retracted position. Penile responses will be observed, typically termed ex copula genital reflex tests. Typically, a series of penile erections will occur spontaneously within a few minutes after sheath retraction. The types of normal reflexogenic erectile responses include elongation, engorgement, cup and flip. An elongation is classified as an extension of the penile body. Engorgement is a dilation of the glans penis. A cup is defined as an intense erection where the distal margin of the glans penis momentarily flares open to form a cup. A flip is a dorsiflexion of the penile body.

Baseline and or vehicle evaluations are conducted to determine how, and if, an animal will respond. Some animals have a long duration until the first response while others are non-responders altogether. During this baseline evaluation latency to first re- sponse, number and type of responses are recorded. The testing time frame is 15 min- utes after the first response.

After a minimum of 1 day between evaluations, these same animals are administered the test compound at 20 mg/kg and evaluated for penile reflexes. All evaluations are videotaped and scored later. Data are collected and analyzed using paired 2 tailed t- tests to compared baseline and/or vehicle evaluations to drug treated evaluations for individual animals. Groups of a minimum of 4 animals are utilized to reduce variability.

Positive reference controls are included in each study to assure the validity of the study. Animals can be dosed by a number of routes of administration depending on the nature of the study to be performed. The routes of administration includes intravenous (IV), intraperitoneal (IP), subcutaneous (SC) and intracerebral ventricular (ICV).

E. Models of Female Sexual Dysfunction

Rodent assays relevant to female sexual receptivity include the behavioral model of lordosis and direct observations of copulatory activity. There is also a urethrogenital reflex model in anesthetized spinally transected rats for measuring orgasm in both male and female rats. These and other established animal models of female sexual dysfunction are described in McKenna KE et al, A Model For The Study of Sexual Function In Anesthetized Male And Female Rats, Am. J. Physiol. (Regulatory Integra- tive Comp. Physiol 30): R1276-R1285, 1991; McKenna KE et al, Modulation By Peripheral Serotonin of The Threshold For Sexual Reflexes In Female Rats, Pharm. Bioch. Behav., 40:151-156, 1991 ; and Takahashi LK et al, Dual Estradiol Action In The Diencephalon And The Regulation of Sociosexual Behavior In Female Golden Ham- sters, Brain Res., 359:194-207, 1985.

F. In vitro ADME Assays

1. Microsomal Stability Experimental Procedure

Pooled human liver microsomes (pooled male and female) and pooled rat liver microsomes (male Sprague Dawley rats) are prepared. Microsomes are stored at -80⁰C prior to use.

Microsomes (final concentration 0.5 mg/ml), 0.1 M phosphate buffer pH7.4 and test compound (final substrate concentration = 3 μM; final DMSO concentration = 0.25%) are pre-incubated at 37°C prior to the addition of NADPH (final concentration = 1 mM) to initiate the reaction. The final incubation volume is 25 μl. A control incubation is included for each compound tested where 0.1 M phosphate buffer pH7.4 is added instead of NADPH (minus NADPH). Two control compounds are included with each species. All incubations are performed singularly for each test compound.

Each compound is incubated for 0, 5, 15, 30 and 45 min. The control (minus NADPH) is incubated for 45 min only. The reactions are stopped by the addition of 50 μl methanol containing internal standard at the appropriate time points. The incubation plates are centrifuged at 2,500 rpm for 20 min at 4°C to precipitate the protein.

Quantitative Analysis

Following protein precipitation, the sample supematants are combined in cassettes of up to 4 compounds and analysed using generic LC-MS/MS conditions.

Data Analysis

From a plot of the peak area ratio (compound peak area/internal standard peak area) against time, the gradient of the line is determined. Subsequently, half-life and intrinsic clearance are calculated using the equations below:

Elimination rate constant (k) = (- gradient)

0.693 Half life (t_1/2) (min) = k

Vx 0.693 t

Intrinsic Clearance (CL_int) (μl/min/mg protein) = 1/2 where V = Incubation volume μl/mg microsomal protein.

Two control compounds are included in the assay and if the values for these com- pounds are not within the specified limits the results are rejected and the experiment repeated.

2. Hepatocyte Stability

Experimental Procedure

Suspensions of cryopreserved hepatocytes are used for human hepatocyte stability assay (pooled from 3 individuals). All cryopreserved hepatocytes are purchased from In

Vitro Technologies, Xenotech or TCS.

Incubations are performed at a test or control compound concentration of 3 μM at a cell density of 0.5x10⁶ viable cells/mlL. The final DMSO concentration in the incubation is

0.25%. Control incubations are also performed in the absence of cells to reveal any non-enzymatic degradation.

Duplicate samples (50 μl) are removed from the incubation mixture at 0, 5, 10, 20, 40 and 60 min (control sample at 60 min only) and added to methanol, containing internal standard (100 μl), to stop the reaction.

Tolbutamide, 7-hydroxycoumarin, and testosterone are used as control compounds.

The samples are centrifuged (2500 rpm at 4°C for 20 min) and the supernatants at each time point are pooled for cassette analysis by LC-MS/MS using generic methods.

Data Analysis

From a plot of In peak area ratio (compound peak area/internal standard peak area) against time, the gradient of the line is determined. Subsequently, half-life and intrinsic clearance are calculated using the equations below:

Elimination rate constant (k) = (- gradient)

0.693 Half life (t_1/2)(min) = k

Vx 0.693 Intrinsic Clearance (CLj_nt)(μl/min/million cells) = ^l/2 where V = Incubation volume (μl)/number of cells

3. Caco-2 Permeability (Bi-directional)

Experimental Procedure

Caco-2 cells obtained from the ATCC at passage number 27 are used. Cells (passage number 40-60) are seeded on to Millipore Multiscreen Caco-2 plates at 1 x 105 cells/cm². They are cultured for 20 days in DMEM and media is changed every two or three days. On day 20 the permeability study is performed.

Hanks Balanced Salt Solution (HBSS) pH7.4 buffer with 25 mM HEPES and 10 mM glucose at 37⁰C is used as the medium in permeability studies. Incubations are carried out in an atmosphere of 5% CO₂ with a relative humidity of 95%. On day 20, the monolayers are prepared by rinsing both basolateral and apical sur- faces twice with HBSS at 37°C. Cells are then incubated with HBSS in both apical and basolateral compartments for 40 min to stabilize physiological parameters. HBSS is then removed from the apical compartment and replaced with test compound dosing solutions. The solutions are made by diluting 10 mM test compound in DMSO with HBSS to give a final test compound concentration of 10 μM (final DMSO concentration 1%). The fluorescent integrity marker lucifer yellow is also included in the dosing solution. Analytical standards are made from dosing solutions. Test compound perme- ability is assessed in duplicate. On each plate compounds of known permeability char- acteristics are run as controls. The apical compartment inserts are then placed into 'companion' plates containing fresh HBSS. For basolateral to apical (B-A) permeability determination the experiment is initiated by replacing buffer in the inserts then placing them in companion plates con- taining dosing solutions. At 120 min the companion plate is removed and apical and basolateral samples diluted for analysis by LC-MS/MS. The starting concentration (C₀) and experimental recovery is calculated from both apical and basolateral compartment concentrations.

The integrity of the monolayers throughout the experiment is checked by monitoring lucifer yellow permeation using fluorimetric analysis. Lucifer yellow permeation is low if monolayers have not been damaged. Test and control compounds are quantified by LC-MS/MS cassette analysis using a 5-point calibration with appropriate dilution of the samples. Generic analytical conditions are used.

If a lucifer yellow P_app value is above QC limits in one individual test compound well, then an n=1 result is reported. If lucifer yellow P_app values are above QC limits in both replicate wells for a test compound, the compound is re-tested. Consistently high lucifer yellow permeation for a particular compound in both wells indicates toxicity. No further experiments are performed in this case.

Data Analysis

The permeability coefficient for each compound (P_app) is calculated from the following equation:

Papp = dQ/dt C₀ X A

Where dQ/dt is the rate of permeation of the drug across the cells, Co is the donor compartment concentration at time zero and A is the area of the cell monolayer. Co is obtained from analysis of donor and receiver compartments at the end of the incuba- tion period. It is assumed that all of the test compound measured after 120 min incubation was initially present in the donor compartment at 0 min. An asymmetry index (Al) is derived as follows:

Al = Papp (B-A)

An asymmetry index above unity shows efflux from the Caco-2 cells, which indicates that the compound may have potential absorption problems in vivo.

The apparent permeability (P_app (A-B)) values of test compounds are compared to those of control compounds, atenolol and propranolol, that have human absorption of approximately 50 and 90% respectively (Zhao, Y.H., et al., (2001). Evaluation of Human Intestinal Absorption Data and Subsequent Derivation of a Quantitative Structure- Activity Relationship (QSAR) with the Abraham Descriptors. Journal of Pharmaceutical Sciences. 90 (6), 749-784). Talinolol (a known P-gp substrate (Deferme, S., MoIs, R., Van Driessche, W., Augustijns, P. (2002). Apricot Extract Inhibits the P-gp-Mediated Efflux of Talinolol. Journal of Pharmaceutical Sciences. 91(12), 2539-48)) is also included as a control compound to assess whether functional P-gp is present in the Caco-2 cell monolayer.

4. Cytochrome P450 Inhibition (5 lsoform IC₅O Determination))

Experimental Procedure

CYP1A Inhibition Six test compound concentrations (0.05, 0.25, 0.5, 2.5, 5, 25 μM in DMSO; final DMSO concentration = 0.35%) are incubated with human liver microsomes (0.25 mg/ml) and NADPH (1 mM) in the presence of the probe substrate ethoxyresorufin (0.5 μM) for 5 min at 37°C. The selective CYP1A inhibitor, alpha-naphthoflavone, is screened alongside the test compounds as a positive control. CYP2C9 Inhibition

Six test compound concentrations (0.05, 0.25, 0.5, 2.5, 5, 25 μM in DMSO; final DMSO concentration = 0.25%) are incubated with human liver microsomes (1 mg/ml) and NADPH (1 mM) in the presence of the probe substrate tolbutamide (120 μM) for 60 min at 37°C. The selective CYP2C9 inhibitor, sulphaphenazole, is screened alongside the test compounds as a positive control. CYP2C19 Inhibition

Six test compound concentrations (0.05, 0.25, 0.5, 2.5, 5, 25 μM in DMSO; final DMSO concentration = 0.25%) are incubated with human liver microsomes (0.5 mg/ml) and NADPH (1 mM) in the presence of the probe substrate mephenytoin (25 μM) for 60 min at 37°C. The selective CYP2C19 inhibitor, tranylcypromine, is screened alongside the test compounds as a positive control. CYP2D6 Inhibition

Six test compound concentrations (0.05, 0.25, 0.5, 2.5, 5, 25 μM in DMSO; final DMSO concentration = 0.25%) are incubated with human liver microsomes (0.5 mg/ml) and NADPH (1 mM) in the presence of the probe substrate dextromethorphane (5 μM) for 30 min at 37°C. The selective CYP2D6 inhibitor, quinidine, is screened alongside the test compounds as a positive control. CYP3A4 Inhibition

Six test compound concentrations (0.05, 0.25, 0.5, 2.5, 5, 25 μM in DMSO; final DMSO concentration 0.26%) are incubated with human liver microsomes (0.25 mg/ml) and NADPH (1 mM) in the presence of the probe substrate midazolam (2.5 μM) for 5 min at 37°C. The selective CYP3A4 inhibitor, ketoconazole, is screened alongside the test compounds as a positive control.

For the CYP1A incubations, the reactions are terminated by the addition of methanol, and the formation of the metabolite, resorufin, is monitored by fluorescence (excitation wavelength = 535 nm, emission wavelength = 595 nm). For the CYP2C9, CYP2C19, CYP2D6, and CYP3A4 incubations, the reactions are terminated by the addition of methanol containing internal standard. The samples are then centrifuged, and the su- pernatants are combined, for the simultaneous analysis of 4-hydroxytolbutamide, 4- hydroxymephenytoin, dextrorphan, and 1-hydroxymidazolam plus internal standard by LC-MS/MS. Generic LC-MS/MS conditions are used. Formic acid in deionised water (final concentration = 0.1%) is added to the final sample prior to analysis. A decrease in the formation of the metabolites compared to vehicle control is used to calculate an IC₅₀ value (test compound concentration which produces 50% inhibition).

5. Plasma Protein Binding (10%)

Experimental Procedure

Solutions of test compound (5 μM, 0.5% final DMSO concentration) are prepared in buffer (pH 7.4) and 10% plasma (v/v in buffer). The experiment is performed using equilibrium dialysis with the two compartments separated by a semi-permeable mem- brane. The buffer solution is added to one side of the membrane and the plasma solu- tion to the other side. Standards are prepared in plasma and buffer and are incubated at 37^CC. Corresponding solutions for each compound are analyzed in cassettes by LC- MS/MS.

Quantitative Analysis

After equilibration, samples are taken from both sides of the membrane. The solutions for each batch of compounds are combined into two groups (plasma-free and plasma- containing) then cassette analyzed by LC-MS/MS using two sets of calibration stan- dards for plasma-free (7 points) and plasma-containing solutions (6 points). Generic LC-MS/MS conditions are used. Samples are quantified using standard curves prepared in the equivalent matrix. The compounds are tested in duplicate. A control compound is included in each experiment.

Data Analysis

fu = fraction unboun PC = sample concentration in protein containing side PF = sample concentration in protein free side fu at 10% plasma is converted to fu 100% plasma using the following equation:

Examples of a Pharmaceutical Composition

As a specific embodiment of an oral composition of a compound of the present invention, 25 mg of Example 1 is formulated with sufficient finely divided lactose to provide a total amount of 580 to 590 mg to fill a size 0 hard gelatin capsule.

As another specific embodiment of an oral composition of a compound of the present invention, 35 mg of Example 29 is formulated with sufficient finely divided lactose to provide a total amount of 580 to 590 mg to fill a size 0 hard gelatin capsule.

As a further specific embodiment of an oral composition of a compound of the present invention, 26 mg of Example 18 is formulated with sufficient finely divided lactose to provide a total amount of 580 to 590 mg to fill a size 0 hard gelatin capsule.

As another specific embodiment of an oral composition of a compound of the present invention, 34 mg of Example 56 is formulated with sufficient finely divided lactose to provide a total amount of 580 to 590 mg to fill a size 0 hard gelatin capsule. As a specific embodiment of an oral composition of a compound of the present inven- tion, 28 mg of Example 24 is formulated with sufficient finely divided lactose to provide a total amount of 580 to 590 mg to fill a size 0 hard gelatin capsule.

As another specific embodiment of an oral composition of a compound of the present invention, 32 mg of Example 90 is formulated with sufficient finely divided lactose to provide a total amount of 580 to 590 mg to fill a size 0 hard gelatin capsule.

While the invention has been described and illustrated in reference to certain preferred embodiments thereof, those skilled in the art will appreciate that various changes, modifications and substitutions can be made therein without departing from the spirit and scope of the invention. For example, effective dosages, other than the preferred doses as set forth above, may be applicable as a consequence of the specific pharma- cological responses observed and may vary depending upon the particular active com- pound selected, as well as from the type of formulation and mode of administration employed, and such expected variations or differences in the results are contemplated in accordance with the objects and practices of the present invention. It is intended, therefore, that the invention be limited only by the scope of the claims which follow and that such claims be interpreted as broadly as is reasonable.

Claims

Claims:

1. A compound according to formula (I)

and enantiomers, diastereomers, tautomers, solvates and pharmaceutically acceptable salts thereof, wherein

R¹ is -(C(R⁸)₂),-T,

-O-(C(R⁸)₂)_m-T,

T is N NRR⁵⁵RR⁶⁶,, morpholine,

R⁵ and R⁶ are independently

H

C₁-₆-alkyl,

C_2-6-alkenyl,

C_2-6-alkinyl, C_2-6-alkylene-O-C_1-6-alkyl wherein each alkyl, alkenyl and alkinyl is optionally substituted by one or more substituents selected from halogen atoms, CN and OH,

R⁷ is halogen, CN₁

OH,

C_1-6-alkyl, optionally substituted with 1 to 3 substituents selected from halogen, CN and OH, O-C_1-6-alkyl optionally substituted with 1 to 3 substituents selected from halogen, CN and OH,

C

_1-6-alkylene-O-C_1-6-alkyl optionally substituted with 1 to 3 substituents selected from halogen, CN and OH,

R⁸ is independently H,

F,

OH,

OMe₁

C_3-6-cycloalkyl, optionally substituted with 1 to 3 substituents selected from halogen, CN, OH and OMe,

R⁹ is independently

H, C_1-6-alkyl, optionally substituted with 1 to 3 substituents selected from halogen, CN, OH and OMe,

X is CH₁ N,

Y is CH₁ N,

Z is CH₁ N,

R² is F,

Cl, methyl,

R³ is Cl, methyl,

Ms 3, 4, m is 2, 3, 4, nis 0,1,2,3,4, ois 0,1,2, pis 0,1,2,3,4.

2. The compound of claim 1 according to formula (I¹)

wherein R1,R2 R3,R9 and n are as defined in claim 1.

3. The compound of claim 1 or 2, wherein R² is Cl,

R³ is Cl.

4. The compound of any of claims 1 to 3, wherein

I is 3, and m is 2 or 3.

5. The compound of any of claims 1 to 4, wherein R⁸ and R⁹ are both hydrogen,

R⁵ and R⁶ are independently C_1-6-alkyl,

C_2-6-alkenyl, C_2-6-alkinyl, C_2-6-alkylene-O-C_1-6-alkyl, R⁷ is halogen, CN₁ OH,

C_{1 -6}-alkyl, optionally substituted with 1 to 3 substituents selected from halo- gen, CN and OH,

O- C_{1 -6}-alkyl optionally substituted with 1 to 3 substituents selected from halogen, CN and OH.

6. The compound of any of claims 1 to 4, wherein R⁸ is independently

H,

F,

OH,

OMe,

C_{1 -6}-alkyl, optionally substituted with 1 to 3 substituents selected from halogen, CN, OH and OMe,

C_3-6-cycloalkyl, optionally substituted with 1 to 3 substituents selected from halogen, CN, OH and OMe, provided that at least one R⁸ is not hydrogen, and R⁹ is hydrogen.

7. The compound of any of claims 1 to 4, wherein

R⁸ is independently H, F,

OH, OMe,

C_{1 -6}-alkyl, optionally substituted with 1 to 3 substituents selected from halo- gen, CN, OH and OMe, C₃.₆-cycloalkyl, optionally substituted with 1 to 3 substituents selected from halogen, CN, OH and OMe, and

R⁹ is independently H₁ C_1-6-alkyl, optionally substituted with 1 to 3 substituents selected from halogen, CN, OH and OMe,

C_3-6-cycloalkyl, optionally substituted with 1 to 3 substituents selected from halogen, CN, OH and OMe with the proviso that at least one R⁹ is not hydrogen.

8. The compound of any of claims 1 to 7 as medicament.

9. Use of the compound of any of claims 1 to 7 for the preparation of a medicament for the treatment or prophylaxis of disorders, diseases or conditions responsive to the inactivation of the melanocortin-4 receptor in a mammal.

10. Use according to claim 9 for the preparation of a medicament for the treatment or prophylaxis of cancer cachexia.

11. Use according to claim 9 for the preparation of a medicament for the treatment or prophylaxis of muscle wasting.

12. Use according to claim 9 for the preparation of a medicament for the treatment or prophylaxis of anorexia.

13. Use according to claim 9 for the preparation of a medicament for the treatment or prophylaxis of anxiety and/or depression.

14. A pharmaceutical composition comprising a compound of any of claims 1 to 7 and a pharmaceutically acceptable carrier.

15. A compound according to formula (II)

R¹ is -(C(R⁸)₂),-T,

-O-(C(R⁸)₂)_m-T,

T is NR⁵R⁶, morpholine,

,

R⁵ and R⁶ are independently

H

C_1-6-alkyl.

C_2-6-alkenyl,

C₂-₆-alkinyl,

C_2-6-alkylene-O-C_1-6-alkyl wherein each alkyl, alkenyl and alkinyl is optionally substituted by one or more substituents selected from halogen atoms, CN and OH₁

R⁷ is halogen, CN, OH, C_1-6-alkyl optionally substituted with 1 to 3 substituents selected from halo gen, CN and OH,

O- C_{1 -6}-alkyl optionally substituted with 1 to 3 substituents selected from halogen, CN and OH,

C_{1 -6}-alkylene-O- C_{1 -6}-alkyl optionally substituted with 1 to 3 substituents selected from halogen, CN and OH,

R⁸ is independently H, F, OH,

OMe,

C_{1 -6}-alkyl, optionally substituted with 1 to 3 substituents selected from halogen, CN, OH and OMe, C₃-₆-cycloalkyl, optionally substituted with 1 to 3 substituents selected from halogen, CN, OH and OMe,

R⁹ is independently H₁

C_1-6-alkyl, optionally substituted with 1 to 3 substituents selected from halogen, CN, OH and OMe₁ C_3-6-cycloalkyl, optionally substituted with 1 to 3 substituents selected from halogen, CN, OH and OMe

X is CH, N₁ Y is CH, N, Z is CH, N, R² is F,

Cl₁ methyl,

R³ is H, F, Cl, with the proviso that R³ and R⁴ cannot simultaneously be Cl, R⁴ is H, Cl₁ F₁

I is 3, 4, m is 2, 3, 4, nis 0,1,2,3,4, o is 0,1,2, pis 0,1,2,3,4.

16. The compound of claim 15 according to formula (M')

wherein R₁, R² , R³ , R₄ , R9 and n are defined as in claim 15.

17. The compound of claim 15 or 16, wherein

R² is F or Cl, and R³ is H or F.

18. The compound of any of claims 15 to 17, wherein

I is 3, and m is 2 or 3.

19. The compound of any of claims 15 to 18, wherein R⁸ and R⁹ are both hydrogen,

R⁵ and R⁶ are independently C_1-6-alkyl, C₂-₆-alkenyl, C_2-6-alkinyl, C_2-6-alkylene-O- C_{1 -6}-alkyl ,

R⁷ is halogen,

CN, OH,

C₁-₆-alkyl, optionally substituted with 1 to 3 substituents selected from halo- gen, CN and OH,

R³ is H,

F,

R⁴ is Cl,

F.

20. The compound of any of claims 15 to 18, wherein R⁸ is independently

H,

F,

OH, OMe,

C_1-6-alkyl, optionally substituted with 1 to 3 substituents selected from halogen, CN, OH and OMe₁

C_3-6-cycloalkyl, optionally substituted with 1 to 3 substituents selected from halogen, CN, OH and OMe, provided that at least one R⁸ is not hydrogen, and

R⁹ is hydrogen.

21. The compound of any of claims 15 to 18, wherein

R⁸ is independently H,

F, OH, OMe,

C_1-6-alkyl, optionally substituted with 1 to 3 substituents selected from halogen, CN₁ OH and OMe,

C_3-6-cycloalkyl, optionally substituted with 1 to 3 substituents selected from halogen, CN, OH and OMe, and

R⁹ is independently H,

C_{1 -6}-alkyl, optionally substituted with 1 to 3 substituents selected from halogen, CN, OH and OMe, C_3-6-cycloalkyl, optionally substituted with 1 to 3 substituents selected from halogen, CN, OH and OMe with the proviso that at least one R⁹ is not hydrogen.

22. The compound of any of claims 15 to 21 as medicament.

23. Use of the compound of any of claims 15 to 21 for the preparation of a medicament for the treatment or prophylaxis of disorders, diseases or conditions responsive to the activation of the melanocortin-4 receptor in a mammal.

24. Use according to claim 23 for the preparation of a medicament for the treatment or prophylaxis of obesity.

25. Use according to claim 23 for the preparation of a medicament for the treatment or prophylaxis of diabetes mellitus.

26. Use according to claim 23 for the preparation of a medicament for the treatment or prophylaxis of male or female sexual dysfunction.

27. Use according to claim 23 for the preparation of a medicament for the treatment or prophylaxis of erectile dysfunction.

28. A pharmaceutical composition comprising a compound of any of claims 15 to 21 and a pharmaceutically acceptable carrier.