This invention relates to a new class of chemical compounds and to their use in medicine In particular the invention concerns novel phenethanolamine derivatives, methods for their preparation, pharmaceutical compositions containing them and their use as agonists at atypical beta-adrenoceptors (also known as beta-3- adrenoceptors) Such receptors have been described for example by J R S Arch et al , Nature 309, 163-165 (1984), C Wilson et al , Eur J Pharmacol , 100, 309- 319 (1984), L J Emoπne ef al , Science, 245, 1 1 18-1 121 (1989), and A Bianchetti et al Br J Pharmacol , 100, 831-839 (1990) Phenethanolamine derivatives having activity at atypical beta-adrenoceptors are disclosed in, for example, European Patent Applications EP-A-0455006 and EP-A-0543662

Atypical beta-adrenoceptors belong to the family of adrenoceptors which mediate the physiological actions of the hormones adrenaline and noradrenaline Sub¬ types of the adrenoceptors, ₁ -, α ₂ -, β , β ₂ - and β ₃ -(atypιcal) can be identified on the basis of their pharmacological properties and physiological effects Chemical agents which stimulate or block these receptors (but not β ₃ ) are widely used in clinical medicine More recently, emphasis has been placed upon specific receptor selectivity in order to reduce side effects caused, in part, by interactions with other receptors

Atypical beta-adrenoceptors are known to occur in adipose tissue and the gastrointestinal tract

Atypical beta-adrenoceptor agonists have been found to be particularly useful as thermogenic anti-obesity agents and as anti-diabetic agents Compounds having atypical beta-adrenoceptor agonist activity have also been described as being useful in the treatment of hyperglycaemia, as animal growth promoters, as blood platelet aggregation inhibitors, as positive inotropic agents and as antiatherosclerotic agents, and as being useful in the treatment of glaucoma

We have now found a novel class of phenylethanolamine derivatives which act as agonists at atypical beta-adrenoceptors GB 9525177 3 which is the priority document for the present application, describes the syntheses of the compounds of the invention W095/33724, which was unpublished at the priority date of the present application, describes the syntheses of compounds which are also of use as agonists at atypical beta-adrenoceptors

The invention therefore provides in a first aspect compounds of formula (I)

wherein

R1 represents an aryl group optionally substituted by one or more substituents selected from halogen, hydroxy, C-| _6alkoxy, C -μøalkyl, nitro, cyano, hydroxymethyl and trifluoromethyl,

R ² represents hydrogen or C _h alky!,

R ³ represents a group A

where the aromatic ring may be optionally substituted by up to four substituents selected from Cι_ ₆ alkyl, halogen, trifluoromethyl, and d ₆ alkoxy,

R ⁴ represents hydrogen, or dialkyi,

R ⁵ represents C0 ₂ R ⁸ , C ₁ . ₆ alkylC0 ₂ R ^{1 6} , CONR ⁹ R ^{1 0} , NHCONR ^{, 1} R ¹² , S0 ₂ NHR ¹³ , P(0)(OR ¹⁴ ) ₂ , S0 ₃ H, C,^alkylS0 ₃ H, NHS0 ₂ R ^{1 5} , NHCOR ^{1 7} or tetrazol-5-yl,

R ⁶ and R ⁷ independently represent hydrogen, C , ₆ alkyl, trifluoromethyl, CN, OH C0 R ¹⁸ , CH C0 R ^{1 9} , or R ⁶ and R ⁷ form a 5-6 membered cycloalkyl ring,

R ⁸ represents hydrogen, or d-e alkyl,

R ⁹ represents hydrogen, d_ ₆ alkyl, or C^ alkylOR ²⁰ ,

R ¹⁰ R R ¹² , R ¹ -R ^{1 6} and R ^{, 8} -R ²⁰ each independently represent hyαrogen or C _{1 6} alkyl

R ¹³ represents Ci ₆ alkyl or trifluoromethyl,

R ^{1 ?} represents Ci ₆ alkyl or trifluoromethyl,

with the proviso that when R ⁶ and R ⁷ are both hydrogen, R ⁸ is other than hydrogen

and physiologically acceptable derivatives thereof

Referπng to the general formula (I), alkyl includes both straight and branched chain saturated hydrocarbon groups Similarly, alkoxy includes both straight and branched chain groups

Referring to the general formula (I), aryl includes monocyclic or bicyclic aromatic carbocyclic groups such as phenyl and naphthyl

Preferably R represents phenyl optionally substituted by one, two or three substituents selected from halogen, hydroxy, Cι_ ₆ alkoxy, dialkyi, nitro, cyano, hydroxymethyl and trifluoromethyl More preferably R ¹ represents phenyl substituted by a chlorine, fluoπne or bromine atom or a methyl or trifluoromethyl group, which atom or group is preferably located in the meta position Most preferably R ¹ represents phenyl substituted by a chlorine atom located in the meta position

R ² is preferably hydrogen or methyl

Where R ³ is further substituted on the aromatic ring, preferred substituents are selected from halogen, e g fluoro, methyl, and trifluoromethyl Most preferably, substitution by two adjacent flouπne groups is preferred

R ⁴ is preferably hydrogen, or methyl

R ⁵ is preferably S0 ₃ H

R ⁶ is preferably hydrogen or Ci ₆ alkyl, e g methyl

R ⁷ is preferably hydrogen or Ci ₆ alkyl, e g methyl

A preferred sub-class of compounds of formula (I) are those where R ¹ represents an aryl group optionally substituted by one or more substituents selected from halogen hydroxy C -| _6alkoxy C -μ _β alkyl, nitro, cyano hydroxymethyl and tπfluorometnyl,

R ² represents hydrogen or Cι- ₆ alkyl,

R ^J represents a group A

where the aromatic ring may be optionally substituted by substituents selected from dialkyi, halogen, trifluoromethyl, and C, ₆ alkoxy,

R ⁴ represents hydrogen, or Cι_ ₆ alkyl;

R ⁵ represents C0 ₂ R ⁸ , CONR ⁹ R ^{1 0} , CONHNR ¹¹ R ¹² , S0 ₂ NHR ¹³ P(0)(OR ¹⁴ ) ₂ , S0 ₃ H, or tetrazol-5-yl,

R ⁶ and R ⁷ independently represent hydrogen, dialkyi, trifluoromethyl, CN, OH , C0 ₂ R ¹⁵ , CH C0 R ¹⁶ , or R ⁶ and R ⁷ form a 5-6 membered cycloalkyl ring,

R ⁶ represents hydrogen, or Cι _-6 alkyl,

R ⁹ , R ¹⁰ R", R ¹² , R ¹⁴ , and R ¹⁵ each independently represent hydrogen, or C ,. ₆ alkyl;

R ¹³ represents Ci-β alkyl or trifluoromethyl,

with the proviso that when R ⁶ and R ⁷ are both hydrogen, R ^δ is other than hydrogen,

and physiologically acceptable derivatives thereof

A further preferred sub-class of compounds of formula (I) are those where R ¹ represents phenyl substituted by a chlorine atom located in the meta position; R ² represents hydrogen or methyl, R^ IS optionally substituted by halogen, R^ represents hydrogen or methyl, R^ represents S0 ₃ H, R ⁶ represents hydrogen or d

₆ alkyl, R ⁷ represents hydrogen or d ₆ alkyl and physiologically acceptable derivatives thereof

It will be appreciated that the above compounds of formula (I) are optically active The individual isolated isomers and mixtures thereof, including racemates, are within the scope of the present invention Particularly preferred compounds of formula (II) are those wherein the asymmetric carbon atoms in the -CH(OH)- group and the -CH(CH ₃ )- group are in the (R)-confιguratιon

Suitable compounds of formula (I) of the invention are

2-(4-{2R-[2-(3-Chloro-phenyl)-2R-hydroxy-ethylamιπo]-pr opylamιno}-phenyl)- propionic acid,

2-(4-{2R-[2-(3-Chloro-phenyl)-2R-hydroxy-ethylamιno]-pro pylamιno}-phenyl)-2- methyl-propioπic acid,

(4-{2-[2-(3-Chloro-phenyl)-2R-hydroxy-ethylamιno]-ethyla mιno}-phenyl)-hydroxy- acetic acid,

(4-{2R-[2-(3-Chloro-phenyl)-2R-hydroxy-ethylamιno]-propy lamιno}-beπzyl)- phosphonic acid diethyl ester,

2-(4-{2R-[2-(3-Chloro-phenyl)-2R-hydroxy-ethylamιno]-pro pylamιno}-phenyl)-3,3,3- tπfluoro-2-hydroxy-propιonιc acid ethyl ester,

2-(4-{2R-[2-(3-Chloro-phenyl)-2R-hydroxy-ethyiamιno]-pro pylamιno}-phenyl)-2- methyl-malonic acid diethyl ester,

1 R-(3-Chloro-phenyl)-2-{1 R-methyl-2-[4-(1 H-tetrazol-5-ylmethyl)-pheπylamιno]- ethylamιno}-ethanol,

1 -(4-{2R-[2-(3-Chloro-phenyl)-2R-hydroxy-ethylamιno]-propyla mιno}-pneπyl)-1 - cyclopentaπecarboxylic acid,

4-[2-[2R-Hydroxy-2-(3-chloro-phenyl)ethylamιno]propylam� �no)]-phenylmethyl trifluoroacetamide,

4-[2-[2R-Hydroxy-2-(3-chlorophenyl)ethylamιno]propylamι no)]-phenylmethyl-N,N- dimethylurea

(4-{2-[2R-(3-chloro-phenyl)-2R-hydroxy-ethylamιno]-propylam ιno}-2 3-dιfluoro- pheπyl)-acetιc acid methyl ester

4-[2-[2R-Hydroxy-2-(3-chloro-phenyl)ethylamιno]propylam� �no)]-phenvlmethyl methanesulfonamide,

4-[2R-[2R-Hydroxy-2-(3-chloro-phenyl)ethylamιno]propylam ιno)]-phenylmethane sulfonic acid,

4-[2R-[2R-Hydroxy-2-(3-chloro-pheπyl)ethylamιno]propyla mιno)]-phenylethane sulfonic acid,

4-[2R-{2-(3-chloro-pheπyl)-2R-hydroxy-ethylamιno}-propy iamιno]-benzyl phosphonic acid,

2-(4-{2R-[2-(3-Chloro-Dhenyl)-2R-hydroxy-ethylamιno]-pro pylamιno}-2 3-dιfluoro- phenyl)-N-(2-methoxy-ethyl)-acetamιde,

3-[4-{2R-[2-(3-Chlorophenyl)-2R-hydroxyl-ethylamιno]-pro pylamιπo}-ρhenyl] -(2,2- dιmethyl)-propιonιc acid,

and physiologically acceptable salts and solvates thereof

Preferred compounds of the invention include

4-[2R-[2R-Hydroxy-2-(3-chloro-phenyl)ethylamιπo]propyla mιno)]-phenylethane sulfonic acid,

and physiologically acceptable derivatives thereof

By "a physiologically acceptable derivative" is meant any physiologically acceptable salt, ester, or salt of such ester, of a compound of formula (I) or any other compound which, upon administration to the recipient, is capable of providing (directly or indirectly) a compound of formula (I) or an active metabolite or residue thereof

It will be appreciated by those skilled in the art that the compounds of formula (I) may be modified to provide physiologically acceptable derivatives thereof at any of the functional groups in the compounds of formula (I) Of particular interest as such derivatives are compounds modified at the carboxyl function, hydroxyl functions or at ammo groups

It will be appreciated by those skilled in the art that the physiologically acceptable derivatives of the compounds of formula (I) may be deπvatised at more than one position

Preferred physiologically acceptable derivatives of the compounds of formula (I) are physiologically acceptable salts thereof

Physiologically acceptable salts of the compounds of formula (!) include those derived from pharmaceutically acceptable inorganic and organic acids and bases Examples of suitable acids include hydrochloric, hydrobromic, sulphuric, nitric, perchloric fumaric, maleic, phosphoric, glycollic, lactic, salicylic, succinic, toluene- p-sulphonic, tartaric, acetic, citric, methanesulphonic, formic, benzoic, malonic, naphthalene-2-sulphonιc and benzenesulphonic acids Other acids such as oxalic while not in themselves pharmaceutically acceptable may be useful in the preparation of salts useful as intermediates in obtaining compounds o the invention and their pharmaceutically acceptable acid addition salts

Salts derived from appropriate bases include alkali metal (e g. sodium), alkaline earth metal (e g magnesium), ammonium and NR ⁺ (where R is dialkyi) salts

The compounds of formula (I) act as agonists at atypical beta -adrenoceptors and as such are useful in the treatment of clinical conditions susceptible to amelioration by administration of an atypical beta-adrenoceptor agonist Such conditions include hyperglycaemia, obesity, hyperlipemia, irritable bowel syndrome and its associated pain, motility dysfunction, excessive gastrointestinal secretion non-specific diarrhoea, neurogenic inflammation, regulation of intraocular pressure, tπglyceπdemia, diabetes, e g non-insuhn-dependent diabetes mellitus (NIDDM or Type II), such as obese NIDDM and non-obese NIDDM, diabetic complications such as retinopathy, nephropathy, neuropathy, cataracts, coronary heart diseases and arteriosclerosis, osteoporosis, and gastrointestinal disorders particularly inflammatory gastrointestinal disorders

Accordingly the present invention provides a method of treatment of a mammal, including man, suffering from condition susceptible of amelioration by an atypical beta-adrenoceptor agonist which method comprises administering to the subject an effective amount of a compound of general formula (I) or a physiologically acceptable derivative thereof

References in this specification to treatment include prophylactic treatment as well as the alleviation of symptoms

In a further aspect the invention provides the use of a compound of general formula (I) or a physiologically acceptable salt or solvate thereof, for the manufacture of a medicament for the treatment of a condition susceptible of amelioration by an atypical beta-adrenoceptor agonist

While it is possible that, for use in therapy, a compound of the invention may be administered as the raw chemical it is preferable to present the active ingredient as a pharmaceutical formulation

The invention thus further provides a pharmaceutical formulation comprising a compound of formula (I) or a physiologically acceptable derivative thereof together with one or more physiologically acceptable earners therefor and, optionally, other therapeutic and/or prophylactic ingredients The carrιer(s) or excιpιent(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof

Thus the compounds for use according to the present invention may be formulated for oral, buccal, parenteral, rectal or transdermal administration or in a form suitable for administration by inhalation or insufflation (either through the mouth or the nose)

For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with physiologically acceptable excipients such as binding agents (e g pregelatinised maize starch, polyvinylpyrrolidoπe or hydroxypropyl methylcellulose), fillers (e g lactose, microcrystalline cellulose or calcium hydrogen phosphate), lubricants (e g magnesium stearate, talc or silica), disintegrants (e g potato starch or sodium starch glycollate), or wetting agents (e g sodium lauryl sulphate) The tablets may be coated by methods well known in the art Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions or they may be presented as a dry product for constitution with water or other suitable vehicle before use Such liquid preparations may be prepared by conventionar means with physiologically acceptable additives such as suspending agents (e g sorbitσl syrup, cellulose derivatives or hydrogenateα edible fats), emulsifying agents (e g lecithin or acacia), non-aqueous vehicles (e g almond oil oily esters ethyl alcohol or fractionated vegetable oils) and preservatives (e g

methyl or propyl-p_-hydroxybenzoates or sorbic acid) The preparations may also contain buffer salts flavouring colouring and sweetening agents as appropriate

Preparations for oral administration may be suitably formulated to give controlled release of the active compound

For buccal administration the compositions may take the form of tablets or lozenges formulated in conventional manner

The compounds according to the present invention may be formulated for parenteral administration by injection e g by bolus injection or continuous ^' infusion Formulations for injection may be presented in unit dosage form e g in ampoules or in multi-dose containers, with an added preservative The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilising and/or dispersing agents Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e g sterile pyrogen-free water, before use

The compounds according to the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, e g containing conventional suppository bases such as cocoa butter or other glycerides

In addition to the formulations described previously, the compounds may also be formulated as a depot preparation Such long acting formulations may be administered by implantation (for example subcutaneously, transcutaneously or intramuscularly) or by intramuscular injection Thus, for example, the compounds according to the present invention may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example as a sparingly soluble salt

A proposed dose of the compounds according to the present invention for administration to a human (of approximately 70kg body weight) is 0 1 mg to 1 g, preferably to 1 mg to 100mg of the active ingredient per unit dose, expressed as the weight of fre.e base The unit dose may be administered, for example 1 to 4 times per day The dose will depend on the route of administration It will be appreciated that it may be necessary to make routine variations to the dosage depending on the age and weight of the patient as well as the seventy of the condition to be treated

The precise dose and route of administration will ultimately be at the discretion of the attendant physician or veterinarian

The compounds of the invention may be prepared by any of the processes known in the art for the preparation of similar compounds For example, according to a first process (A) wherein R ¹ , R ² , and R ^J are as defined as for formula (I) compounds of formula (I) may be prepared from a compound of formula (II)

R'

R '

R-

N R ^{α R}

(H) wherein R ³ represents R ³ where any carboxylic acid group/groups is su'tably protected, and R ^a and R ^b are protecting groups, by deprotection in a suitable mixture such as 6M hydrochloric acid in tetrahydrofuran

Compounds of formula (II) may be prepared by reaction of a compound of formula (III) with a compound of formula (IV)

R

R '

CHO H-R ^"

_R ³ ° R>

(IV)

(III)

wherein R ³ , R ^a and R ^b are as defined as for formula (II), in the presence of a reducing agent It will be appreciated that compounds of formula (II) may be prepared by interconversion utilising compounds of formula (II) as precursors

Compounds of formula (III) may be prepared by reaction of compounds of formula (V) with an amine acid salt of formula (VI)

(V) (VI)

wherein R ^b is as defined herein before, and R ^c is a suitable alkyl group for protection, in the presence of a reducing agent Following protection of the nitrogen, the ester is reduced by a suitable reducing agent such as di-isobutyl aluminium hydride

According to a second process (B) wherein R ¹ , R ² , and R ³ are as defined as for formula (I), compounds of formula (I) may be prepared by reaction of a compound of formula (V) with a compound of formula (VII)

(V) (v»)

wherein R ³ represents R ³ where any carboxylic acid group(s), or the anilino nitrogen wnen R ⁴ represents hydrogen, are suitably protected, and R ^a is a protecting group, in the presence of a reducing agent, followed by removal of protecting groups

Compounds of formula (VII) may be prepared from compounds of formula (VIII)

(VIII)

where R ³ is as defined as for formula (VII), and R° is a protecting group, by treatment with a suitable reducing agent

Compounds of formula (VIII) may be prepared by the reaction of compounds of formula (IX) with compounds of formula (X)

(IX)

wherein R ³ , R ^a and R ^b are as defined as for formula (VIII), in the presence of a suitable reducing agent It will be appreciated that compounds of formula (VIII) may be prepared by intercoπversion, utilising compounds of formula (VIII) as precursors

According to a third process (C) wherein R ¹ , R ² , and R ³ are as defined as for formula (I), compounds of formula (I) may be prepared by reaction of a compound of formula (XI) with a compound of formula (VII) o

(XI)

at reflux in a suitable solvent such as nitromethane.

Suitable reducing agents of use in the reactions include hydrogen in the presence of a catalyst, such as a noble metal catalyst, for example palladium, platinum or platinum oxide, Raney-nickel or hydride reducing agents such as borohydπdes, for example sodium borohydride sodium tπacetoxyborohydπde or sodium cyanoborohydride Suitable reaction conditions will be readily apparent to those skilled in the art and are further illustrated by the accompanying examples

Compounds of formula (IV), (V), (VI), (IX), (X) and (XI) are known compounds or may be prepared by processes well known in the art.

The protecting groups used in the preparation of compounds of formula (I) may be used in conventional manner See for example 'Protective Groups in Organic Chemistry' Ed. J F W McOmie (Plenum Press 1973) or 'Protective Groups in Organic Synthesis' by Theodora W Greene and P M G Wuts (John Wiley and Sons 1991 )

Conventional ammo protecting groups may include for example aralkyl groups, such as benzyl, diphenylmethyl or tπphenylmethyl groups, and acyl groups such as N- benzyloxycarbonyl or t-butoxycarbonyl

Conventional oxygen protecting groups may include for example alky silyl groups, such as trimethylsilyl, or tert-butyldimethylsilyl alkylethers such as tetrahydropyraπyl, or tert-butyl, or esters such as acetate

Removal of any protecting groups present may be achieved by conventional procedures

Atypical beta-adrenoceptor agonists are compounds which demonstrate a pharmacological response mediated at atypical beta-adrenoceptors This activity has been be measured as the ability to stimulate polysis by rat adipocytes at sub- micromolar concentrations, in a response that is resistant to blockade by standard beta-adrenoceptor blocking drugs such as propranolol

A particularly useful method for determining agoinst activity at human atypical beta- adrenoceptors involves the use of Chinese hamster ovarian (CHO) cells transfected with the human beta-3-adrenoceptor according to Method 2 The cell lines may also be transfected with human beta-1 - and beta-2- adrenoceptor in a similar manner to provide a method of determining the selectivity of the compounds of the invention at the three receptors

Method 1

Cell culture

General cell culture guidelines are observed (Fershney, R A ( 1 987) Culture of animal cells A manual of basic technique Wiley-Liss, Inc , N Y ) A standard cell culture incubator is used (37°C, 5% C0 ₂ in air, 95% relative humidity) H β ₃ CHO cells are grown in 75ml flasks in MEMα medium containing 9% FCS & 125μg/ml G418 One confluent flask of cells is trypsinised and resuspended in 80ml of culture medium, 1 ml of the cell suspension is added to each well of three 24-well plates The plates are then incubated for 1 day

Experimental method

The medium is aspirated from each well, and the well rinsed with phosphate- buffered saline (PBS, this is then aspirated) 1 ml of MEMα (no FCS or G418 300μ M IBMX) is added to each well Antagonists, if required, are added at this stage The plate is then placed back in the incubator for 30mιn Drugs are then added to the wells (1 Oμl, 100x required final concentration), the plate gently swirled to mix the

drugs, and the plate placed back in the incubator for 30 mm The medium is then aspirated from each well the well rinsed with PBS and 0 5ml perchloric acid (6% v/v in distilled water 2-5°C) The plate is left on ice for 30mιn The perchloric aciα (containing cAMP) is transferred to a clean 24-well plate and the aciα neutralised by addition of saturated KHC0 ₃ solution (200ul) to each well The plate is then swirled and frozen (-20°C) until cAMP is assayed cAMP is assayed using an enzyme- immunoassay kit (Amersham)

The relative potency of each test agonist (EPMR) is compared to isopreπalme as follows EC ₅ 0 agonist

EPMR =

EC ₅₀ isoprenaiine

wherein EC ₅₀ is the molar concentration of agonist which produces 50% of the maximum possible response for that agonist

Using the non-selective beta-adrenoceptor agonist isoprenaiine as a reference agonist, compounds selective for atypical beta-adrenoceptors should preferably be a minimum of 10-30 times less potent than isoprenaiine at β,- or β ₂ -adrenoceptors and, more preferably, 300-1000 times less potent than isoprenaiine at β,- or β ₂ - adrenoceptors

An experimental model in which atypical beta-adrenoceptor agonists may be shown to be of use in the treatment of gastrointestinal disorders is described below as Method 3 The procedure is based upon that described by H Satoh et_ al Gastroenterotogy, 8J_, 719-725 (1981 ) in which the effect of compounds on indomethacin-induced gastric antral lesions in the re-fed rat is investigated Indo ethacin is an example of the class of compound known as πon-steroidal anti- inflammatory drugs (NSAIDs), tne use of which is frequently associated with gastrointestinal ulcers

Method 2

Food (but not water) is withheld from female random hooded rats (70-120g) for 24 hours and then the rats are re-fed with Rat and Mouse No 1 Maintenance Diet After 1 hour of access to fooα the rats are dosed orally with either the test

compound or solvent (0 5% w/v methyl cellulose in water) 30 minutes later, indomethacin (60mg/kg dissolved in 1 % w/v NaHC0 ₃ in saline) is administered as a single subcutaneous injection at the back of the neck Subsequently the rats are allowed food but water is withheld, and the animals are humanely killed by cervical dislocation at 6 hours post dose Control animals received a single subcutaneous dose of the appropriate solvent

The rat's stomach is removed (with a small amount of duodenum attached), opened along the greater curvature and the contents removed by washing with 0 9% w/v sodium chloride solution (saline) The opened stomach is pinned out- (mucosal surface uppermost) on a polystyrene mat and the area of damage assessed by placing a grid (composed of 1mm squares) over the antral region Antral damage appears as discrete black or dark brown ulcers The total area of antral damage is then expressed as a percentage of the total surface area of the antrum

The protective effect of the test compound on indomethacin-iπduced antral damage is calculated as a percentage using the following equation

% area of damage NSAID - %area of damage NSAID + test compound 1 00 χ

% area of damage with NSAID

The invention is further illustrated by the following intermediates and examples All temperatures are in degrees centigrade

Intermediate 1

(R)-(3-chloro-phenyl)-hvdroxy-acetιc acid methyl ester

A solution of (R)-(3-chloro-phenyl)-hydroxy-acetιc acid (19 98g) in methanol (250ml) containing concentrated sulphuric acid (1 ml) was heated under reflux for 6 5h The solution was cooled, neutralised with aqueous sodium bicarbonate solution, and concentrated The residue, dissolved in ethyl acetate, was washed with aqueous sodium bicarbonate solution, dried, and evaporated to give the title compound (21 13g) as a oale-yellow oil

[a] _D -104° (c 1 OO MeOH)

Intermediate 2

(R)-(3-chloro-phenyl)-(tert-butyl-dιmethyl-sιlanoxy)-ac etιc acid methyl ester

A solution of (R)-(3-chloro-phenyl)-hydroxy-acetιc acid methyl ester (21 Og), imidazole (14 25g), and tert-butyldimethylsilyl chloride (25 Og) in N,N- dimethyiformamide (250ml) was stirred at room temperature for 18h The mixture was poured into water and extracted with ethyl acetate The combined extracts were washed with water and saturated brine, dried, and concentrated The residue was purified by chromatography, eluting with cyclohexane ethyl acetate (9 1 ) to give the title compound as a colourless oil (32 63g)

[a] _D -55 4° (c 1 21 MeOH)

Intermediate 3

(R)-(3-chloro-phenyl)-(tert-butyl-dιmethyl-sιlanoxy)-ac etaldehvde

To a stirred solution of (R)-(3-chloro-phenyl)-(tert-butyl-dιmethyl-sιlaπoxy)-acet ιc acid methyl ester (4 Og) in anhydrous ether (10ml) and maintained at <-65° was added dropwise a 1 5M solution of di-isobututylaluminium hydride in toluene (10ml) When addition was complete the solution was stirred at -65° for a further hour, then quenched with methanol (10ml) The mixture was allowed to attain room temperature, was adsorbed onto silica and purified by chromatography eluting with cyclohexane ethyl acetate (9 1 ) to give the title compound as a colourless liquid (3 09g)

[a] _D -45 3° (c 1 50 MeOH)

Intermediate 4

2R-r2R-(tert-Butyl-dιmethyl-sιlanyloxy)-2-(3-chioro-phe nyl)-ethviamιno1-propιonιc acid methyl ester

(R)-(3-chloro-phenyl)-(tert-butyl-dιmethyl-sιlanoxy)-aceta ldehyde (2 32g) was added to a stirred solution of (R)-2-amιnopropιonιc acid methyl ester hydrochloride ( 1 14g) in dichloromethane (50ml) The solution was stirred 15mιn, then sodium tπactetoxyborohydnde (3 45g) was added, and the mixture was stirred a further 18h The solution was washed with aqueous sodium bicarbonate solution then the organic phase was dried and concentrated The residue was chromatographed on silica eluting with cyclohexane ethyl acetate (9 1 ) Evaporation of the appropriate fractions gave the title compound as a colourless oil (2 42g)

[a] _D -29 4° (c 1 36 MeOH)

Similarly prepared was

Intermediate 5

f2R-(tert-Butyl-dιmethyl-sιlanyloxy)-2-(3-chloro-phenyl )-ethylamιnol-acetιc acid methyl ester as a colourless oil (1 26g)

Assay Found C 56 9, H 7 95, N 3 9%

C ₁ 7H2ΘCINO3S1 requires C 57 1 , H 7 8, N 3 9%

from amino-acetic acid methyl ester hydrochloride (1 05g) and (R)-(3-chloro- phenyl)-(tert-butyl-dιmethyl-sιlanoxy)-acetaldehyde (2 16g)

Intermediate 6

{2R-(tert-Butoxycarbonyl)-f2R-(tert-butyl-dιmethyl-sιla nyloxy)-2-(3-chloro-phenyl)- ethyll-aminol-propionic acid methyl ester

A mixture of 2R-[2R-(tert-butyl-dιmethyl-sιlanyloxy)-2-(3-chloro-phenyl )-ethylamιno]- propionic acid methyl ester (2 15g) and di-t-butyl pyrocarbonate ( 1 37g) was heated at 80-100° for 1 h Chromatography of the residue on silica (50g) and elution with cyclohexane ethyl acetate gave the title compound as a colourless oil (2 70g)

Assay Found C 58 4, H 8 1 , N 3 0%

C ₂₃ H ₃₈ CINO ₅ S1 requires C 58 5, H 6 1 N 3 0%

Similarly prepared was

Intermediate 7

((tert-Butoxycarbonyl)-f2R-(tert-butyl-dιmethyl-sιlanyl oxy)-2-(3-chioro-phenyl)-ethyl]- aminol-acetic acid methyl ester as a colourless oil (1 55g) from [2R-(tert-butyl- dιmethyl-sιlanyloxy)-2-(3-chloro-phenyl)-ethylamιno]-acet ιc acid methyl ester (1 20g)

[α] _D -25 2° (c 1 3 MeOH)

Intermediate 8

{2R-(tert-Butoxycarbonyl)-[2R-(tert-butyl- ιmethyl-sιlanyloxy)-2-(3-chloro-phenyl)- ethyll-aminol-propionaldehyde

1 5M di-isobutylalumimum hydride in toluene (8 1 ml) was added dropwise to a stirred, cooled solution of the {2R-(tert-butoxycarbonyl)-[2R-(tert-butyl-dimethyl- sιlanyioxy)-2-(3-chloro-phenyl)-ethyl]-amιno}-ρropιonιc acid methyl ester (2 296g) in toluene (50ml) at such a rate that tne reaction temperature did not rise above -70° The solution was stirred 1 h at this temperature, then quenched with methanol (10ml) The mixture was adsorbed onto silica, then purified by chromatography eluting with cyclohexane ethyl acetate (9 1 ) to give the title compound as a colourless oil (1 45g)

Similarly prepared was

Intermediate 9

((tert-Butoxycarbonyl)-r2R-ftert-butyl-dιmethyl-sιlanyloxy -2-f3-chloro-phenyl )-ethyl1- aminoj-acetaldehyde as a colourless gum (0 371 g)

n m r (CDCI ₃ ) d values include -0 1 5 (d 3H), 0 05 (d, 3H), 0 90 (s 9H) 1 45 (d 9H ) 2 9-3 2 (m 1 H), 3 4-3 65 (m, 1 H) 3 70 (s, 3H) 3 75-4 15 (m 2H) 4 8-5 0 (m 1 H) 7 05-7 35 (m 4H)

from {(tert-butoxycarboπyl)-[2R-(tert-butyl-dιmethyl-sιlaπylo xy)-2-(3-chlorophenyl)- ethyl]-amιno}-acetιc acid methyl ester (0 50g)

Intermediate 10

1 -(4-Amιno-phenyl)-cyclopentanecarboxylιc acid methyl ester

A stirred solution of 1 -(4-amιno-phenyl)-cyclopentanecarboxylιc acid (0 45g) in methanol (30ml) containing concentrated sulphuric acid (0 4ml) was heated under reflux for 4h, cooled, and concentrated The residue was distributed between ethyl acetate and aqueous sodium bicarbonate solution Concentration of the organic phase gave the title compound as light brown crystals

n m r (CDCI3) d values include 1 54-1 94 (m, 6H), 2 58 (m, 2H), 3 60 (s, 3H), 6 63 (d, 2H), 7 15 (d 2H)

Intermediate 1 1

{4-[2R-(N, N-Dιbenzylamιno)-propylamιno1-phenyl-)acetιc acid ethyl ester

A solution of 4-amιnophenyl acetic acid ethyl ester (6 77g) and acetic acid (2 16ml) in dichloromethane (200ml) was stirred for 5mιn, then cooled to 5° and treated with (2R-N,N-dιbenzylamιno)-propaπal (10 37g) in dichloromethane (60ml) followed by sodium tnacetoxyborohydride (16 02g) The mixture was stirred at ambient temperature for 18h, diluted with dichloromethane and washed twice with aqueous sodium bicarbonate solution The organic phase was separated dried, and concentrated Chromatography of the residue eluting with cyclohexane ethyl acetate (9 1 ) gave the title compound as a yellow oil (1 3 45g)

C27H32N2O2 MH 17

Intermediate 12

(4-[tert-Butcxycarbonyl-(2R-dιbenzy amιno-ρropyl)-amιnol-pnenyl)-acetιc acid ethyl ester

A mixture of {4-[2R-(N, N-dιbenzylamιno)-propylamιno]-phenyl-}acetιc acid ethyl ester (1 Og) and di-tert-butyl-dicarbonate (0 6g) was heated at 125° for -1 5h The mixture was cooled and chromatographed on silica Elution with cyclohexane ethyl acetate (9 1 ) and concentration of the appropriate fractions gave the title compound (1 Og) as a pale yellow oil

C32H40N2O4 MH ⁺ 517

Intermediate 13

3 1 Mixture Of 2-f4-ftert-Butoxycarbonyl-(2R-dιbenzylamιno-propyl)-amιno ]-phenyl)- propionic acid ethyl ester and 2-f4-.tert-Butoxycarbonyl-(2R-dιbenzylamιno-propyl)- amιno1-ρhenyl)-2-methyl-propιonιc acid ethyl ester

To a stirred solution of {4-[tert-bu;oxycarbonyl-(2R-dιbenzylamιno-propyI)-amιno]- phenyl}-acetιc acid ethyl ester (0 50g) in anhydrous THF (9ml), maintained at -50°, was added dropwise a 1 5M solution of lithium di-isopropylamide in cyclohexane (1 55ml) The solution was stirred 0 5h at -50° when lodomethaπe (0 06ml) was added After 0 5h at -50° the mixture was allowed to warm to ambient temperature, and stirred a further 2h The mixture was chromatographed eluting with cyclohexane ethyl acetate (9 1 ) to give the title mixture as a yellow oil (0 32g) The relative ratio was determined by nmr

C ₃₃ H ₄ 2N2O ₄ MH ⁺ 531

C34H ₄₄ N2O ₄ -MH ^{^} 545

Intermediate 14

Mixture Of 2-r4-(tert-Butoxycarbonyl)-(2R-r2R-(tert-butyl-dιmethyl-sι ianyloxy)-2-(3- chloro-phenyl)-ethyl1-amιno)-ethylamιno)-phenyl1-propιon� �c acid ethyl ester and 2-[4- (tert-Butoxycarbonyl)-(2R- 2R-ftert-butyl-dιmethyl-sιlanyloxy)-2-(3-chloro-phenyl)- ethvn-amιno)-ethylamιnoftphenyll-2-methyl-propιonιc acid ethyl ester

5 A stirred mixture of the 3 1 mixture of 2-{4-[tert-butoxycarbonyl-(2R-dιbenzylamιno- proρyl)-amιno]-phenyl}-propιonιc acid ethyl ester and 2-{4-[tert-butoxycarbonyl-(2R- dιbenzylamιno-propyl)-amιno]-phenyl}-2-methyl-propιonιc acid ethyl ester (0 32g), ammonium formate (0 39g), 10% palladium on charcoal (0 10g) and methanol ( 1 0ml) was heated under reflux under an atmosphere of nitrogen for 2h The mixture

10 was cooled, filtered, and concentrated to give a yellow foam (0 23g) which was dissolved ιn_acetιc acid (0 035ml) in dichloromethane (10ml) was stirred for 5 mm, then cooled to 5° To this solution was added (R)-(tert-buty!-dιmethyl-sιlanoxy)-(3- chloro-phenyl)-acetaldehyde (0 44g) in dichloromethane (2ml), followed by sodium tnacetoxyborohydride (0 27g) The mixture was stirred at ambient temperature for

15 1 8h, then washed with aqueous sodium bicarbonate solution The organic phase was dried and concentrated The residue was chromatographed eluting with cyclohexane ethyl acetate (3 1 ) to give the title mixture as a yellow-brown gum (0 21 g)

C ₃ 3H5 ₁ CIN2O5S1 MH ⁺ 619

0 C ₃ 5H ₅ 5CIN2O5S1 MH ⁺ 633

Intermediate 15

[4-(2-{tert-Butoxycarbonyl-[2R-(tert-butyl-dιmethyl-sιl anyloxy)-2-(3-chloro-phenyl)- ethyl1-amιno}-ethylamιno)-phenyl1-oxo-acetιc acid methyl ester

5 A solution of (4-amιno-phenyl)-oxo-acetιc acid methyl ester (0 585g) in dichloromethane (10ml) containing acetic acid (2 24ml) was treated with a solution of {(tert-butoxycarbonyl)-[2R-(tert-butyl-dιmethyl-sιlanyloxy )-2-(3-chloro-phenyl)- ethyl]-amιno}-acetaldehyde (1 352g) in dichloromethane ( 10ml) After 0 5h the solution was cooled to 5° and sodium tnacetoxyborohydride ( 1 34g) was added The 0 mixture was stirred for 1 8h, then washed with aqueous sodium bicarbonate solution The organic phase was separated, dried, and concentrated The residue was

chromatographed on silica, elution with cyclohexane ethyl acetate (3 1 ) and concentration of the appropriate fractions gave the title compound (0 55g)

Assay Found C 60 8 H 7 6, N 4 4 C 6 3%

C ₃ 0H ₄₃ CIN2O ₆ S1 requires C 61 0 H 7 3 N 4 7 Cl 6 0%

Similarly prepared were

Intermediate 16

f4-(2R-{tert-Butoxycarbonyl-f2R-(tert-butyl-dιmethyl-sι lanyloxy)-2-(3-chloro-phenyl)- ethyll-aminol-propylamino .-benzyll- phosphonic acid diethyl ester as a colourless gum (0 365g)

Assay Found C 59 1 , H 8 55, N 4 0%

C ₃ 3H54C! 2θ ₆ PSι requires C 59 2, H 8 1 , N 4 2%

from {2R-(tert-butoxycarbonyl)-[2R-(tert-butyl-dιmethyl-sιlanyl oxy)-2-(3-chloro- phenyl)-ethyl]-amιno}-propιonaldehyde (0 382g) and (4-amιno-beπzyl)-phosphoπιc acid diethyl ester (0 21 g)

Intermediate 17

f4-f2R-jtert-Butoxycarbonyl-[2R-(tert-butyl-dιmethyl-sι lanyloxy)-2-(3-chloro-phenyl)- ethyll-amιno}-propylamιno)-phenyll-3 3 3-trιfluoro-2-hydroxy-propιonιc acid methyl ester as a colourless oil (0 1 9g)

C ₃₃ H48F ₃ CIN2O ₆ S1 MH ⁺ 689

from {2R-(tert-butoxycarbonyl)-[2R-(tert-butyl-dιmethyl-sιlanyl oxv)-2-(3-chloro- phenyl)-ethyl]-amιno}-propιoπa!dehyde (0 16g) and (4-amιno-phenyl -3 3 3-tπfluoro- 2-hydroxy-propιonιc acid ethyl ester (0 097g)

Intermediate 18

f4-(2R-(tert-Butoxycarbonyl-f2R-(tert-butyl-dιmethyl-sιlan yloχy)-2-(3-chloro-phenyl)- ethyl1-amino}-propylamino)-phenyll-2-methyl-malonic acid diethyl ester as a colourless gum (0 061 g)

C ₃₆ H55CIN O7S1 MH ⁺ 691

from {2R-(tert-butoxycarbonyl)-[2R-(tert-butyl-dιmethyl-sιlanyi oxy)-2-(3-chioro- pheπyl)-ethyl]-amιno}-propιonaldehyde (0 068g) and (4-amιno-phenyl)-2-methyl- malonic acid diethyl ester (0 040g)

Intermediate 1 9

[4-(2R-ftert-Butoxycarbonvi-[2R-(tert-butyl-dιmethyl-sι lanyloxy)-2-(3-chloro-phenyl)- ethyll-amιno)-propyiamιno)-phenyl)-acetonιtrιle as a pale yellow oil (0 99g)

C ₃₀ H4 ₄ CIN ₃ O ₃ S1 MH ⁺ 558

from {2R-(tert-butoxycarbonyl)-[2R-(tert-butyl-dιmethyl-silanylo xy)-2-(3-chloro- phenyl)-ethyl]-amιno}-propιonaldehyde (0 82g) and (4-amιno-phenyl)-acetonιtπle (0 245g)

Intermediate 20

f4-(2R-{tert-Butoxycarbonyl- 2R-(tert-butyl-dιmethyl-sιlanyloxy)-1 -(3-chloro-phenyl)- ethvπ-amιno}-propylamιno -phenyl1-cvclopentanecarboxyltc acid methyl ester as a colourless oil (1 Og)

C35H52CIN2 S1O5 MH ⁺ 645

from {2R-(tert-Butoxycarbonyl)-[2R-(tert-Butyl-dιmethy!-sιlaπy loxy)-2-(3-chloro- phenyl)-ethyl]-amιno}-propιonaldehyde (0 094g) and 1 -(4-Amιno-phenyl)- cyclopentanecarboxylic acid methyl ester (0 35g)

Intermediate 21

[4-(2R-(tert-Butoxycarbonyl-r2R-tert-butyl-dιmethyl-sιlano xy)-2-(3-chloro-phenyl)- ethyl1-amιno}-propylamιno)-benzyl-phospnonιc acid diethyl ester as a tan oil (465 mg)

n m r (CDCI ₃ ) includes d -0 08 (s, 3H), C 07 (m, 3H), 0 93 (s, 9H) 1 29 (t, 6H) 1 53 (s, 9H), 3 10-3 35 (m, 5H), 3 97-4 10 (m 4H), 6 56 (d, 2H), 7 1 1 (d, 2H) 7 1 1 -7 38 (m, 4H)

from {2R-(tert-butoxycarbonyl)-[2R-(tert-butyl-dιmethyl-sιlanyl oxy)-2-(3-chloro- phenyi)-ethyt]-amιno}-propιonaldehyde (380 mg, 0 86 mmol) and 4-amιπobeπzyl- phosphonic acid diethyl ester (310 mg, 1 27 mmol) and sodium tnacetoxyborohydride (451 mg, 2 12 mmol)

Intermediate 22

1 -f3-(2R-(tert-Butoxycarbonyl-r2R-(tert-butyldιmethyl-sιlan oxy)-2-(3-chloro-phenyl)- ethvπ-amιno)-propylamιno)-phenylH2,2-dιmethyl)-propιon� �c acid ethyl ester

C ₃ 5H55N2O5CIS1 M + Na ⁺ 669

from {2R-(tert-butoxycarbonyl)-[2R-(tert-butyld:methyl-sιlanoxy) -2-(3-ch!oro-phenyl)- ethyl]-amιno}-propιonaldehyde (0 30g) and 1 -(4-amιnoρhenyl)-2,2-dιmethyl- propionic acid ethyl ester (0 30 g)

Intermediate 23

{2R-r2-(3-Chloro-phenyl)-2R-hvdroxy-ethylamιnol-propylH4 -(1 H-tetrazoi-5- ylmethvP-phenyll-carbamic acid tert-butyl ester

A mixture of [4-(2R-{tert-butoxycarbonyl-[2R-(tert-butyl-dιmethyl-sιlan yloxy)-2-(3- chloro-phenyl)-ethylj-amιπo}-propylamιno)-phenyl]-aceton� �trιle (0 249g), sodium azide (0 087g) and triethylamine hydrochloride (0 092g) in N-methyl-pyrrolidinone (5ml) was heated at 150° for 18h under an atmosphere of nitrogen The mixture was cooled, diluted with water, and partioned between 0 1 N hydrochloric acid and diethyl ether The organic phase was separated off and concentrated, and the residue was

chromatographed on silica Elution with dichloromethane ethanol 0 880 ammonia solution (200 8 1 ) gave the title compound (0 049g) as an orange gum

C ₂₄ H ₃ CIN ₆ 0 ₃ MH ⁺ 444

Intermediate 24

14-(2R-ftert-Butoxycarbonyl- 2R-(tert-butyl-dιmethyl-sιlanyloxy)-2-f3-chloro-phenyl)- ethvπ-amιno)-ethylamιno)-phenyll-hydroxy-acetιc acid methyl ester

A solution of [4-(2R-{tert-butoxycarbonyl-[2R-(tert-butyl-dιmethyl-sιlan yloxy)-2-(3- chloro-phenyl)-ethyl]-amιπo}-ethylamιno)-phenyl]-oxo-acet ιc acid methyl ester (0 35g) in methanol (15ml) was treated with sodium borohydride (0 022g), and stirred for one hour The solvent was evaporated, and the residue was chromatographed on silica, eluting with cyciohexane ethyl acetate (3 1 ) to give the title compound (0 102g)

C ₃ oH ₄ 5CIN ₂ 0 ₆ Sι MH ⁺ 593 5

Intermediate 25

(2, 3-dιfiuoro-4-nιtro-phenyl)-benzyloxy-acetιc acid methyl ester (1 )

Sodium hydride (60% dispersion in mineral oil, 4 76 g) was added over 10 minutes to a suspension of benzyl methyl malonate (21 7 mLeq) in dimethylformamide (25 mL) After stirring 30 minutes 1 ,2,3-trιfluoro-4-πιtrobenzene (21 1 g) was dissolved in dimethylformamide (10 mL) and added to the reaction mixture The mixture was heated to 90°C for 4 5 hours, then cooled to room temperature overnight The reaction mixture was added dropwise to deionized water (350mL) and extracted with ethyl acetate The organic layers were combined dried over sodium sulfate filtered and evaporated in-vacuo The organic residue was purified by column chromatography with silica gel eluting with hexane ethyl acetate (9 1 -8 2) to obtain the title compound as an orange oil (10 8 g) as a mixture of desired product and (3,4-dιfluoro-2-nιtro-phenyl)-beπzyloxy-acetιc acid methyl ester

d ₇ H ₁₃ F ₂ N0 ₆ M-Na 388

Intermediate 26

(4-amιno-2 3-dιfluorσ-phenyl)-acetιc acid me'nyl ester (2)

Cyclohexene (2 2 mL) was added to (2,3-oιfluoro-4-nιtro-pπenyl)-benzyloxy-acetιc acid methyl ester (1 Og) in methanol (15 mL; 1 0% palladium on caroon (0 5 g) was added to the reaction which was hydrogeπated overnight Filtered The reaction was filtered through celite and concentrated to yield a purple oil which was purified on silica gel eluting with hexane ethyl acetate (S 1 -4 1 ) to yield the title compound (160 mg)

C ₉ H ₉ F ₂ N0 ₂ MH ⁺ 202

Intermediate 27

(2R)-(-)-te ^r t-butoxycarbonyl-amιno-1 -propanol

(R)-(-)-2-amιno-1 -propanol (9 ml) was treated with methylene chloride (200mL) Di- tert-butyl dicarbonate (27 8g) was added slowly to the reaction and stirred at room temperature for 1 5 hours The reaction was quenched by the addition of deionized water (100mL) The layers were separated and the organic layer was dried over sodium sulfate, filtered and concentrated to yield a clear liquid The liquid was purified by silica gel chromatography eluting with hexane ethyl acetate (4 1 -1 1 ) to obtain the title compound as a white solid (17 8g)

C ₈ H ₁₇ N0 ₃ MH ⁺ 176

Intermediate 28

(2R)-(-)-tert-butoxycarbonyl-amιno-1 -propιonaldehyde

Triethylamine (6 3 mL) was added to (2R)-(-)-tert-butoxycaroonyl-ammo-1 -propanol (2 6g) in anhydrous dimethyl sulfoxide (20mL) The reaction was cooled in a ice bath and suifur tπoxide (7 2g) in anhydrous dimethyl sulfoxide (20mL) was added dropwise After stirring for twenty minutes tne reaction was poureα into ice water

and stirred The mixture was extracted with hexane and methylene cnloπde The methylene chloride layers were combined and washed with 10% citric acid, deionized water, and saturated sodium bicarbo ⁿ ate (50mL) The organic layer was dried over sodium sulfate filtered and concentrated to yield the title compound as a white solid (1 55g)

mp= 74-75°C

Intermediate 29

4-[(2RH-)-tert-butoxycarbony]-amιno-1 -propylamιno)-2,3-dιfluoro-phenyl) , -acetic acid methyl ester

(4-amιno-2,3-dιfluoro-phenyl)-acetιc acid methyl ester (1 05g) in anhydrous methylene chloride (20mL) was treated with (2R)-(-)-tert-butoxycarbonyl-amιno-1 - propionaldehyde (900 mg) in anhydrous methylene chloride (25 mL) Acetic acid (3 drops) and sodium tnacetoxyborohydride (2 49g) were added to the reaction and stirred overnight The reaction was partitioned between ehtyl acetate and water The layers were separated and the aqueous layer was extracted with more ethyl acetate The organic layers were combined and dried over sodium sulfate, filtered and concentrated to of a crude orange oil (2 5 g) This was combined with a second batch (total 3 4g) which was purified by silica gel column eluting with hexane ethyl acetate (9 1 ) to afford the title compound as a clear oil (2g)

d ₇ H ₂₄ F ₂ N ₂ 0 ₄ MH ⁺ 359 1

Intermediate 30

4-f(2R)-amιno-1 -propylamιno)-2 3-dιfluoro-phenyl)]-acetιc acid methyl ester

4-[(2R)-(-)-tert-butoxycarbonyl-amιno-1 -propylamιno)-2,3-dιfluoro-phenyl)]-acetιc acid methyl ester (1 95g) in anhydrous methylene chloride (30mL) was treated with trifluoroacetic acid (3 mL) The reaction was quenched after 6 hours with saturated sodium acetate The layers were separated and the aqueous layer pH was adjusted to 8 with saturated sodium bicarbonate The aqueous was extracted with

chloroform, the chloroform layer dried over sodium sulfate filtered and concentrated to give the title compound as an oil (410mg)

C, ₂ H ₁₆ F ₂ N ₂ 0 ₂ MH ⁺ 259 1

Intermediate 31

2-ftert-Butoxycarbonylamιnol-propιonaldehyde

Di-tert-butyldicarbonate (218g) was added to a methylene chloride (800ml) solution of dl-2-amιnopropanol (75 1 g) at 0°C After 3h at rt the reaction was extracted twice with water, dried, concentrated to a colorless viscous liquid (185g) The resiαue was dissolved in DMSO (400ml), treated with triethylamine (202g), followed by pyπdine sulfur tπoxide, while at 0°, for 4 h Reaction was diluted with methylene cnloπde, extracted with 10% aq citric acid, dried, concentrated to give the title compound as an off-white powder (1 10g).

Assay Found C 54 6, H 8 7, N 7.9%

C ₈ H ₁₅ N0 ₃ 0 2 H ₂ 0 requires C 54 3, H 8 8, N 7 9%

Intermediate 32

4-Nιtrophenylmethyl N N-dimethylurea

A suspension of 4-nιtrobeπzylamιne hydrochloride ( 18 8 g) in dry dioxane ( 1 50ml) was stirred at ambient temp for 3 h with triethylamine (20.0g) and dimethylcarbamyl chloride ( 10 Og) The mixture was diluted with ethyl acetate, extracted with water, dried, concentrated to give the title compound as a pale yellow solid (10 Og)

C10H 13N3O3 MH ⁺ 224

Intermediate 33

4-Nιtrophenylmethyl methanesulfonamide

Methanesulfonyl chloride ( 1 1 4g) was added to a suspension of 4-nιtrobenzylamιne hydrochloride ( 1 8 8 g) in 1 N NaOH (200ml) and stirred at ambient temp for 5 m then diluted with ethyl acetate, extracted with water dried concentrated to give the title compound as a pale yellow solid (23 Og)

C ₈ H ₁₀ N ₂ O ₄ S MH ⁺ 229

Intermediate 34

4-Nιtrophenylmethyl trifluoroacetamide

Tπflouroacetic anhydride (21 Og) was added to a suspension of 4-nιtrobeπzylamιne hydrochloride (18 8 g) in acetonitrile and potassium carbonate (27 6g) and was stirred at ambient temp for 24 h then diluted with ethyl acetate, extracted with water, dried, concentrated to give the title compound as a pale yellow solid (24. Og).

TLC (EtOAc Hexane/1 1 ) Rf=0 25

Intermediate 35

4-(2-(tert-Butoxycarbonviamιno)propylamιno)-phenylmethy l N N-dimethylurea

4-Nιtrophenylmethyl N.N-dimethylurea (10g) was agitated in dioxane ethanol (2 1 ) (120ml) under 45 lbs hydrogen pressure on Parr apparatus with 20%> Pd(OH) ₂ /carbon (0 4g) for one hour, then filtered through celite and the filtrate was concentrated The residue was stirred with glacial acetic acid (4 ml), 2-[tert- Butoxycarbonylammoj-propionaldehyde (8 Og) and sodium cyanoborohydπde (2 79g) at rt for 4 h Diluted the reaction with ethyl acetate, extracted with aq 1 N sodium hydroxide, dried, filtered, concentrated to give the title compound as a viscous near colorless oil (15g)

Similarly prepared were

Intermediate 36

4-(2-(tert-Butoxycarbonylamιno)propylamιno)-phenylmethy l methane sulfonamide as a viscous colorless oil (27g)

n m r (DMSO-d6) delta values include 0 95 (m, 9H) 1 10 (m, 3H) 2 61 -3 00 (m, 5H), 2 72 (s, 3H), 3 95 (s, 2H), 5 65 (m 1 H), 6 50 (d, 2H), 7 00 (d, 2H)

from 4-Nιtrophenyimethyl methanesulfonamide

Intermediate 37

4-(2-(tert-Butoxycarbonylamιno)propylamιno)-phenylmethy l tπfluoroacetamide as a pale yellow solid (28g)

TLC (EtOAc Hexane/1 1 ) Rf=0 33

from 4-Nιtrophenylmethyl tπflouroacetamide

Intermediate 38

4-[2-Amιnopropylamιno1-phenylmethyl tπfluoroacetamide

A solution of 4-(2-(tert-Butoxycarboπylamιno)propylamιno)-phenyimethyl tπfluoroacetamide (28 g) and 50 ml tπflouroacetic acid was stirred at room temperature for 30 m , then the reaction mixture was concentrated and potassium carbonate (250 g) was slowly added The mixture was diluted with methylene chloride (400ml) and water (20ml), stirred at room temperature for 0 5 h, filtered, dried, concentrated to give the title compound as a golden viscous oil (15 4g)

Intermediate 39

4-(2-Amιnopropylamιno)-phenylmethyl N N-dimethylurea

4M HCI/dioxane (40ml) was added to 4-(2-(tert-Butvoxycarbonyl- amιno)propylamιno)-phenylmethyl-N,N-dιmethylurea (15gj in dioxane fSO l) for 90 mm Mixture was concentrated diluted with ethyl acetate made casrc with 1 N aqueous sodium hydroxide (80ml) and extracted several times with et ^{^} vl acetate All ethyl acetate extracts were combined, dried, filtered, concentratec to a golden viscous oil (4 Og)

C ₁₃ H ₂₂ N ₄ 0 MH ⁺ 251

Similarly prepared was

Intermediate 40

4-[2-Amιnopropylamιnol-phenylmethyl methanesulfonamide as a golden viscous oil (13 3g)

C ₁₁ H ₁₉ N ₃ 0 ₂ S MH ⁺ 258

from 4-(2-(tert-Butoxycarbonylamιno)propylamιno)-phenylmethyl methane sulfonamide

Intermediate 41

4-Nιtrobenzylthιoacetate

A suspension of 4-nιtrobenzyl bromide (21 6g) and potassium thioacetate (22 8g) was stirred in DMSO (75 ml) at rt 24 h, diluted with ethyl acetate extracted with water, dried, concentrated to give the title compound as a deep yellow solid ( 1 9 Og)

C9H9N0 ₃ S M" 212

Similarly prepared was

lntermedιate-42

4-Nιtrophenethylthιoacetate as a deep yellow solid (10 4g)

n m r (DMSO-d6) delta values include 2 27 (s 3H) 2 90 (m 2H) 3 18 ( 2H) 7 50 (d 2H) 8 1 9 (d 2H)

from 4-Nιtrophenethy! bromide

Intermediate 43

4-Nιtrobenzylsulfonιc acid

4-Nιtrobenzylthιoacetate (10 4g) was dissolved in glacial acetic acid (75ml) and 25ml 32%o H ₂ 0 ₂ After 24 h the reagents were evaporated under vacuum with repeated additions of water to give the title compound as a pale yellow solid (10 8g)

Similarly prepared was

Intermediate 44

4-Nιtrophenethylsulfonιc acid as a deep yellow solid ( 1 1 5g)

C8H ₉ NO ₅ S M-H 230

from 4-Nιtrophenethylthιoacetate

Intermediate 45

4-Amιnobenzylsulfonιc acid

4-Nιtrobenzylthιoacetate (10 8g) was shaken with 25%. aq EtOH, 1 7g NaOH and 0 5g 20% Pd(OH) ₂ /carbon for 3 h Reaction was filtered through celite concentrated and lyophilized to give the title compound a tan powder (9 10g)

n m r (DMSO-d6) delta values include 3 44 (m, 2H), 4 98 (br 2H), 6 42

(d, 2H), 6 90 (d 2H)

Similarly prepared was

Intermediate 46

4-Amιnophenethylsulfonιc acid as a golden solid ( 12 9g)

n m r (DMSO-d6) delta values include 2 50-2 60 (m, 2H), 2 60-2 70 , m 2H) 4 80 (br, 2H) 6 42 (d, 2H), 6 80 (d, 2H)

from 4-Nιtrophenethylsulfonιc acid

Intermediate 47

(4-j2R-[5-(3-Chloro-phenyl)-2-oxo-oxazolιdιn-3-yll-prop ylamιno}-2 3-dιfluoro- phenyQ-acetic acid methyl ester

To a solution of (4-{2R-[5-(3-chloro-phenyl)-2R-hydroxy-ethylamιno]-propylam ιno}- 2,3-dιfluoro-phenyl)-acetιc acid methyl ester (159 mg) in tetrahydrofuran (2 mL) was added 1 , 1 '-carbonyldιιmιdazole (69 mg) The mixture was stirred at ambient temperature for 18 h The mixture was partitioned between water and ethyl acetate The ethyl acetate layer was separated, dried over sodium sulfate filtered and concentrated to afford a residue which was dissolved in tetrahydrofuran (1 mL) and treated with 1 , 1 '-carbonyldnmιdazole (120 mg) After stirring for 2^ n additional 1 , T-carbonyldιmιdazole (60 mg) was added, and the mixture was stirred for 2 days The reaction was partitioned between water and ethyl acetate, the organic layer was separated dried over sodium sulfate, filtered and concentrated to a- ^' ord a residue which was purified by flash chromatography eluting with ethyl aceta'e followed by ethyl acetate methanol (1 0 1 ) to afford after concentration of the relε . ant fractions the title compound as a tan oil (1 17 mg)

n m r (CDCI ₃ ) d 1 29 (d, 3H) 3 18-3 31 (m 3H) 3 54 (s, 2H), 3 68 (s 3H) 3 89 (t, 1 H) 4 07-4 1 3 (m 1 H) 4 19-4 24 (m 1 H), 5 44 (m 1 H), 6 36 (t, 1 H 6 77 (t 1 H), 7 16 (d 1 H) 7 24-7 29 (m 3H )

Intermediate 48

(4-{2R-[5-(3-Chloro-phenyl)-2-oxo-oxazolιdιn-3-yl]-prop ylamιno}-2 3-dιfluoro- phenyl)-acetιc acid

A mixture of (4-{2R-[5-(3-chloro-phenyi)-2-oxo-oxazo!ιdιn-3-yl]-propyla mιno}-2,3- dιfluoro-phenyl)-acetιc acid methyl ester (52 2 mg) and lithium hydroxide (21 mg) was stirred in 1 1 THF water (2 mL) for at ambient temperature for 1 h The mixture was partitioned between ethyl acetate and 1 M aqueous hydrochloric acid The organic layer was separated, dried over Na ₂ S0 ₄ , filtered and concentrated to afford the title compound as a semi-solid residue (50 mg)

n.m r (CDCI ₃ ) d 1 27 (d, 3H), 3 18-3 32 (m, 2H), 3 41 (m, 1 H) 3 51 (s 2H), 4 00 (t, 1 H), 4 14-4 22 (m, 1 H), 5 53 (m, 1 H), 6 49 (t, 1 H), 6 78 (t, 1 H), 7 20 (d, 1 H), 7 24- 7.33 (m 3H)

Intermediate 49

(4-f2R-[5-(3-Chloro-phenyl)-2-oxo-oxazolιdin-3-yπ-propy lamιno}-2 3-dιfluoro- phenyl)- N-(2-methoxy-ethyl)-acetamιde

To a solution of 2-methoxy-1 -ethylamιne (17 mL), ethyl dnsopropylamine (67 mL), 1 - (3-dιmethylamιnopropyl)-3-ethylcarbodιιmιde hydrochloride (36 mg) in methylene chloride ( 1 mL) was added a solution of (4-{2R-[5-(3-chloro-phenyl)-2-oxo- oxazolιdιn-3-yl]-propylamιno}-2,3-dιfluoro-phenyl)-acet� �c acid (49 6 mg) in methylene chloride ( 1 mL) The mixture was stirred at ambient temperature for 18 h The mixture was diluted with ethyl acetate and washed with 2 N aquoues hydrochloric acid and brine The organic layer was dried over sodium sulfate, filtered and concentrated to afford a residue which was purified by silica gel chromatography eluting with ethyl acetate to afford, after concentration of the relevant fractions, the title compound as a colorless oil (27 8 mg)

C ₂₃ H ₂₆ ClιN ₃ F ₂ 0 ₄ MH ⁺ 482

Intermediate 50

1 -(4-Nιtrophenyl)-2 2-dιmethyl-propιonιc acid ethyl ester

To a solution of ethyl isobutyrate (1 79 g) in THF (5 ml) and HMPA (2 7 ml) was added lithium diisopropylamide (2M, 8 0 ml) at -78 °C The mixture was then stirred at 0 °C for 30 m Para-nitrobenzyl bromide (3 28 g) in THF (8 ml) was added at 0 °C The reaction mixture was allowed to come to RT and stirred for 2 h To the mixture was added 40 ml aqueous ammonium acetate and extracted three times with ether The organic extracts were dried, concentrated and the residue loaded onto a silica gel column The product was eiuted with 5%o ethyl acetate/petroleum ether giving the title compound (1 54 g)

NMR (CDCI ₃ ) d values include 8 13 (d, 2H), 7.26 (d, 2H), 4 1 1 (q, 2H) 2 94 (s, 2H), 1 25 (t 3H), 1 19 (s, 6H)

Intermediate 51

1 -(4-Amιnophenyl)-2 2-dιmethyl-propιonιc acid ethyl ester

To a solution of From 1 -(4-Nιtrophenyl)-2,2-dιmethyl-propιonιc acid ethyl ester (1 .54 g) in ethanol (40 ml) was added tιn(ll) chloride (1 1 3 g) and the mixture refluxed 16 h To the cooled reaction mixture was added aqueous sodium carbonate and extracted three times with chloroform The organic layer was dried and concentrated giving the title compound (1 21 g)

Cι ₃ H ₁₉ N0 ₂ MH ⁺ 222

Example 1

2-(4-(2R-| ^' 2-(3-Chloro-phenyl)-2R-hvdroxy-ethylamιno1-propylamι no}-pι ιenyl)- propionic acid

A solution of the mixture of Intermediate 14 (0 21 g) in THF (5ml) containing 6M hydrochloπc acid (5ml) was stirred 19h , then evaporated to dryness The residue was chromatographed on silica, eluting with chloroform methanol 0 880 ammonia solution (60 10 1 -30 15 3) to elute initially 2-(4-{2R-[2-(3-Chloro-pheπyl)-2R-

hydroxy-ethylamιno]-propy!amιno}-phenyl)-2-methyl-propιon ιc acid ethyl ester (0 042g) as a brown gum

C2 ₃ H ₃₁ CIN ₂ 0 ₃ MH ⁺ 419

Further elution gave the title compound (0 10g) as a colourless solid

Assay Found C 37 7 H 6 9, N 13 4, Cl 31 9%

C2 ₀ H2 ₅ CIN2O ₃ 5NH ₄ CI requires C 37 3, H 7 0, N 15 2, Cl 33 0%

n m r (DMSO-d ₆ ) d values include 1 14 (d, 3H), 1 28 (d, 3H), 3 44 (q, 1 H), 4 83 (dd, 1 H), 6 56 (d, 2H), 6 99 (d, 2H), 7 44 (s, 1 H)

Example 2

2-(4-(2R-[2-(3-Chioro-phenyl)-2R-hydroxy-ethylamιno1-pro pylamιno)-phenyl)-2- methyi-propionic acid

A solution of 2-(4-{2R-[2-(3-chloro-phenyl)-2R-hydroxy-ethylammoj-propylam ιno}- phenyl)-2-methyf-propιonιc acid ethyl ester (0 042g) (from Example 1 ) in THF (2ml) containing 12M hydrochloric acid (5ml) was stirred for three days, then diluted with water and evaporated to dryness The residue was purified by chromatography eluting with chloroform-methanol 0 880 ammonia solution (60 10 1 -30 15 3) to give the title compound (0 030g) as a brown solid

Assay Found C 33 6, H 6 8, N 14 4%

C ₂ ιH ₂₇ CIN ₂ θ ₃ 6 5NH ₄ CI 0 5H ₂ 0 requires C 33 7, H 7 3, N 1 5 9%

π m r (DMSO-d ₆ ) d values include 1 40 (s, 6H), 4 91 (broad d, 1 H) 6 58 (d, 2H), 7 07 (d, 2H)

Example 3 ^'

(4-{2-[2-(3-Chloro-phenyl)-2R-hydroxy-ethylamιno1-ethyla mιno)-phenyl)-hydroxy- acetic acid

A solution of [4-(2R-{tert-butoxycarbonyl-[2R-(tert-butyl-dιmethyl-sιlan yloxy)-2-(3- chloro-phenyl)-ethyl]-amιno}-ethylamιno)-phenyl]-hydroxy-a cetιc acid methyl ester (0 092g) in THF (5ml) and 6M hydrochloric acid (5ml) was stirred at ambient temperature for 18h The solution was concentrated and residue was purified by chromatography eluting with chloroform methanol 0 880 ammonia solution (30 10 1 ) to give the title compound as a colourless solid (0 035g)

C ₁₈ H ₂₁ CIN ₂ θ ₄ MH ⁺ 365 126483 (error 0 9ppm)

n m r (DMSO-d ₆ ) d values include 4 89 (broad d 1 H), 6 44 (d, 2H) 7 10 (d, 2H), 7 30-7 41 (m, 3H), 7 44 (s, 1 H)

Similarly prepared were

Example 4

4(4-{2R-r2-f3-Chloro-phenvπ-2R-hvdroxy-ethylamιnol-prop yiamιno)-benzyl)- phosphonic acid diethyl ester as a pale yellow gum (0 19g)

C22H32CIN2O4P MH ⁺ 455 1843 (calc 455 1845)

n r (DMSO-d ₆ ) d values include 1 20 (t, 6H) 1 31 (d, 3H) 5 08 (dd 1 H), 6 67 (d, 2H), 7 04 (dd, 2H), 7 32-7 58 (m, 4H)

from [4-(2R-{tert-butoxycarbonyl-[2R-(tert-butyl-dιmethyl-sιlan yloxy)-2-(3-chloro- phenyl)-ethyl]-amιno}-propylamιno)-benzyl]- phosphonic acid diethyl ester (0 21 g)

Example 5

2-(4-(2R-f2-(3-Chloro-ρhenyl)-2R-hvdroχy-ethylamιno1-p ropylamιno)-D enyl)-3 3.3- trιfluoro-2-hydroxy-propιonιc acid ethyl ester as a purple gum (0 10g)

Assay Found C 55 2, H 5 5, N 5 75%

C22H2 ₆ CIF ₃ N2O ₄ requires C 55 6, H 5 5 N 5 9%

n m r (DMSO-d ₆ ) d values include 1 02 (t 3H) 1 22 (t, 3H) 4 42 (q 2H) 6 56 (d 2H), 7 22 (d, 2H)

from [4-(2R-{tert-butoxycarbonyl-[2R-(tert-butyl-dιmethyl-sιlan yloxy)-2-(3-chloro- phenyl)-ethyl]-amιno}-propylamιno)-phenyl]-3,3 3-trιfluoro-2-hydroxy-propιonιc acid methyl ester (0 19g)

Example 6

2-('4-(2R-r2-f3-Chloro-phenyl)-2R-hvdroxy-ethylamιnol-pr opylamιno)-phenyl)-2- methyl-malonic acid diethyl ester as a pale purple gum (0 027g)

C25H34CIN2O5 MH ⁺ 477 215428 (error 0 4ppm)

π m r (DMSO-d ₆ ) d values include 1 16 (d, 3H), 1 25 (t, 6H), 1 82 (s 3H), 4 21 (q, 4H), 4 68 (m, 1 H), 6 60 (d, 2H), 7 20 (d, 2H), 7 38 (s, 1 H)

from [4-(2R-{tert-butoxycarboπyl-[2R-(tert-butyl-dιmethyl-sιla nyloxy)-2-(3-chloro- phenyl)-ethyl]-amιno}-propylamιπo)-phenyl]-2-methyl-malon ιc acid diethyl ester (0 061 g)

Example 7

1 R-(3-Chloro-phenyl)-2-(1 R-methyl-2-[4-, 1 H-tetrazol-5-ylmethyl)-phenyiamιno1- ethylamιno}-ethanol dihydrochloride as a pale brown solid (0 047g)

C ₁₉ H ₂₃ CIN ₆ 0 MH ⁺ 387 169996 (calc 387 170012)

π m r (DMSO-d ₆ ) d values include 1 25 (d 3H), 3 0-3 50 (m, 5H), 5 10 (d, 1 H), 6 60 (d, 2H), 7 10 (d, 2H), 7 30-7 58 (m, 4H), 8 78 (broad s, 1 H), 9 38 (broad s 1 H)

from {2R-[2-(3-chioro-phenyl)-2R-hydroxy-ethylamιno]-propyl}-[4- ( 1 H-tetrazol-5- ylmethyl)-phenyl]-carbamιc acid tert-butyl ester (0 049g)

Example 8

1 -(4-(2R-f2-(3-Chloro-phenyl)-2R-hvdroxy-ethylamιnoi-propyla mιno)-pηenyl)-1 - cyclopentanecarboxylic acid from [4-(2R-{tert-Butoxycarbonyl-[2R-(tert-butyl- dιmethyl-sιlanyloxy)-1 -(3-chioro-phenyl)-ethyl]-amιno}-propylamιno )-pnenyl]- cyclopentanecarboxylic acid methyl ester

Example 9

4-[2-[2R-Hydroxy-2-{3-chloro-phenyl)ethylamιno,propylam� �no)]-phenylmethyl trifluoroacetamide

A suspension of 4-[2-Amιnopropylamιnoj-phenylmethyl trifluoroacetamide (15 4 g) in nitromethane (150 ml) and (R)-(+)-3-chlorostyrene oxide was heated under reflux for 24 h The solution was evaporated to dryness, dissolved in ethyl acetate (30 ml), purified by silica gel chromatography eluting with ethyl acetate ethanol (4 1 ) to give the title compound as a golden oil (1 2 g)

C ₂₀ H ₂₃ CIF ₃ N ₃ O ₂ MH+ 430

n m r (DMSO-d6) delta values include 1 00 (m, 3H), 2 61 -3 00 (m, 5H), 4 20 (m, 2H), 4 60 (m, 1 H), 5 10-5 50 (m, 2H), 6 41 (m, 2H),6 85-6 95 (m, 2H) 7 24-7 45 (m, 4H), 7 40 (s, 1 H), 9 80 (m 1 H)

Similarly prepared was

Example 10

4-r2- 2R-Hvdroxy-2-(3-chlorophenyl)ethylamιno1propylamιno)1-phen ylr ethyl N N- dimethylurea as a golden viscous oil (0 70g)

C1 1 H13N02 MH+ 405

Assay Found C 60 3 H 7 3, N 12 1 %

d , H ₁₃ N0 ₂ 0 5 EtOAC 0 5 H ₂ 0 requires C 60 3 H 7 5, N 12 2%

from 4-(2-Amιnopropylamιno)-phenylmethyl N, N-dιmethylurea

Examole 1 1

(4-(2-[2R-(3-chloro-phenyl)-2R-hyαroxy-ethylamιno|-prop ylamιno)-2 3-dιfluoro- phenyl . -acetic acid methyl ester as a white solid ( 160mg)

Assay Found C 58 1 1 , H 5 61 , N 6 79%

5 C ₂₀ H ₂₃ N ₂ O ₃ F ₂ CI requires C 58 18 H 5 61 N, 6 79%

[a] ^D ₂₀ =-20 30 (C=0 5, MeOH)

from 4-[(2R)-amιno-1 -propylamιno)-2,3-dιfluoro-phenyl)]-acetιc acid methyl ester (380mg) and (R)-(+)-3-chlorostyrene oxide (120ml_)

1 0 Example 12

4-r2R-r2R-Hvdroxy-2-(3-chloro-phenyl)ethylamιnolpropylam ιno)1-phenyimethyl methanesulfonamide

A solution of 4-[2-Amιnopropylamιno]-phenylmethyl methanesulfonamide (13.3g) and (R)-(+)-3-chlorostyrene oxide was refluxeα in absolute ethanol After 5 h 15 reaction was concentrated and chromatographed using EtOAc 2% NH40H / EtOH (20 1 ) to give the title compound as a golden yellow gum (2 08g)

Cι ₉ H ₂₆ CIN ₃ 0 ₃ S MH 12

n m r (DMSO-d6) delta values include 1 00 (m, 3H), 1 65-2 05 (br I H), 2 72 (s, 3H), 2 61 -3 00 (m, 5H), 3 98 (m, 2H), 4 60 (m, 1 H), 5 30-5 60 (m, 2H) 6 51 (m, 2H), 20 7 00 (m, 2H), 7 20-7 40 (m 4H), 7 40 (s, 1 H)

Example 13

4-f2R-f2R-Hvdroxy-2-(3-chloro-phenyl)ethylamιno|propylam ιno)1-phenylmethane sulfonic acid-

25 A suspension of 4-Amιnobenzylsulfonιc acid (0 36g), {2R-(tert-Butoxycarbonyl)-[2R- (tert-Butyl-dιmethyl-sιlanyloxy)-2-(chlorophenyl)-ethyl]-a mιno}-propιonaldehyde

(0 44g), sodium cyanoborohydπde ( 18g) 0 5ml glacial acetic acid and MeOH was stirred at rt 24 hrs Reaction was filtered, concentrated, stirred with 4M HCI/dioxane for 4 h The precipitate was filtered to give the title compound as a white solid (0 28g)

5 C, ₈ H ₂₃ CI0 ₄ N ₂ S M-H 397

π m.r (DMSO-d6) delta values include 1 30 (m, 3H), 2.95-3.60 (m 5H) 3 59 (s, 2H), 5 10 (m, 2H), 6 70 (d, 2H), 7 05 (d, 2H), 7.20-7.70 (m, 7H)

Similarly prepared was-

10 Example 14

4-f2-12R-Hvdroxy-2-(3-chloro-phenyl)ethylamιno]propylami no)1-phenylethane sulfonic acid as a white solid (0 28g)

C ₁₉ H ₂₅ CI0 ₄ N ₂ S. M-H 41 1

n.m.r. (DMSO-d6)- delta values include 1 30 (m, 3H), 2.60-3.40 (m, 9H), 5 10 (m, 1 5 2H), 6.90 (d, 2H), 7.05 (d, 2H), 7.20-7.70 (m, 7H).

from 4-Amιnophenethylsulfonιc acid.

Example 1 5

4-f2R-(2-(3-chloro-phenyl)-2R-hydroxy-ethylamιno)-propyl amino1-benzyl phosphonic

[4-(2R-{tert-butoxycarbonyl-[2R-tert-butyl-dιmethyl-sιl anoxy)-2-(3-chloro-phenyl)- ethyl]-amιno}-propylamιno)-benzyl-phosphonιc acid diethyl ester) was dissolved in methylene chloride (10 mL) and trifluoroacetic acid (1 mL) and stirred at ambient temperature for 5 h The mixture was concentrated , and the residue was heated at 25 65 °C in 20 mL 6 N aqueous hydrochloric acid ^• tetrahydrofuran ( 1 1 ) for 24 h The mixture was reconcentrated and heated in 6 N aqueous hydrochloric acid for 48 h The mixture was concentrated and the residue was purified oy silica gel chromatography eluting with chloroform-methanol. concentrated ammonium

hydroxide (30 20 5) to afford after concentration or the relevant fractions the title compound as an off-white powder (125 mg)

C ₁₈ H ₂₄ CI, N ₂ 0 ₄ P, MH ⁺ 399

n m r (CDCI ₃ ) d 1 31 (d, 3H), 2 83 (d, 2H) 3 00 (m 1 H) 3 10-3 50 (m 4H) 4 95 (dd 1 H) 6 54 (d, 2H) 7 10 (d, 2H) 7 28-7 33 (m 3H) 7 45 f s 1 H)

Example 16

2-(4-{2R-f2-(3-Chloro-phenyl)-2R-hydroxy-ethyiamιno1-pro pylamιno}-2 3-dιfluoro- phenyl)-N-(2-methoxy-ethyl)-acetamιde hydrochloride salt

A mixture of 2-(4-{2-[5-(3-chloro-phenyl)-2-oxo-oxazolιdιn-3-yl]-propyl amιno}-2,3- dιfluoro-pheπyl)-N-(2-methoxy-ethyl)-acetamιde (27 8 mg) and 1 0 N aqueous sodium hydroxide in 1 ,4-dιoxane (1 mL) was stirred at ambient temperature for 8 h, then heated at 80-90 °C for 24 h The mixture was partitioned between water and ethyl acetate The organic layer was dried over sodium sulfate, filtered and concentrated to afford a residue which was purified by reverse phase HPLC (C-18 column eluting with 80 20 0 1 % aqueous trifluoroacetic acid 0 1 % trifluoroacetic acid in acetonitrile to 50 50 0 1 % aqueous trifluoroacetic acid 0 1 %> trifluoroacetic acid in acetonitrile (30 minute gradient)) to afford after concentrating the major UV active peak (wavelength = 215 nm) the trifluoroacetic acid salt as a residue This material was converted to the titie compound by partitioning between ethyl acetate and saturated aqueous sodium bicarbonate separating the organic layer, separating and concentrating the organic layer and treating the residue with a mixture of dilute aqueous hydrochloric acid and acetonitrile The solution was lyophilized to afford the title compound as a tan powder (8 5 mg)

π m r (CDCI ₃ ) d 1 10 (d, 3H), 2 75 (bs, 2H), 2 80-3 25 (m 5H) 3 29 (s 3H), 3 40 (s 4H), 3 44 (s, 2H) 4 50 (bs, 1 H), 6 40 (t, 1 H) 6 81 (t 1 H), 7 18-7 28 (m 3H), 7 35 (s, 1 H)

Example 1 7

3-f4-{2R-[2-f3-Chlorophenyl)-2R-hydroxyl-etnylamιno !-propylamιno}-onenyl1 -(2 2- dimethyQ-propionic acid

To a solution of 1 -[3-(2R-{tert-butoxycarbonyl-[2R-(tert-butyldιmethyl-sιlan oxy)-2- (3-chloro-pheπyl)-ethyl]-amιno}-propylamιno)-phenyl]-(2,2 -dιmethyl)-propιonιc acid ethyl ester (0 32 g) in tetrahydrofuran (3 ml) was added 6 N hydrochloric acid (3 ml) The mixture was stirred at 60 °C for 9 h The solvents were evaporated, and the residue loaded onto a silica gel column The product was eiuted with methaπol/chloroform/ammonium hydroxide ( 15 60 2) giving the title compound (0 12 g)

C22H29N2O ₃ CI MH ⁺ 405

Assay found C, 55 00 H 7 62, N 6 25%

C22H2 ₉ N2O ₃ CI -4 2H ₂ 0 requires C, 54 98, H, 7 84 , N, 5 83%

TABLETS FOR ORAL ADMINISTRATION

Tablets may be prepared by the normal methods such as direct compression or wet granulation

The tablets may be film coated with suitable film forming materials, such as hydroxypropyl methylcellulose using standard techniques Alternatively the tablets may be sugar coated

Direct Compression Tablet

mg/tablet

i) Active Ingredient 4 688

Calcium Hydrogen Phosphate BP ^* 83 06

Croscarmellose Sodium NF 1 8

Magnesium Stearate BP 0_

Compression weight 90 0

^* of a grade suitable for direct compression

The active ingredient is passed through a 60 mesh sieve blenαed with the calcium hydrogen phosphate, croscarmellose sodium and magnesium stearate The resultant mix is compressed into tablets using a Manesty F3 tablet machine fitted with 5 5mm, flat bevelled edge punches

mg/tablet

(n) Active Ingredient 0 31

Anhydrous Lactose USNF 131 99

Pregelatinised Starch USNF 7 0

Magnesium Stearate BP 0 7

Compression weight 140 0

The active ingredient is passed through a 60 mesh sieve, and blended with the lactose, pregelatinised starch and magnesium stearate The resultant mix is compressed into tablets using a Manesty F3 tablet machine fitted with 7 5mm normal concave punches

SYRUP

This may be either a sucrose or sucrose free presentation

A Sucrose Syrup mq/5ml dose

Active Ingredient 2 5

Sucrose BP 2750 0

Glycerine BP 500 0

Buffer )

Flavour )

Colour ) as required

Preservative )

Purified Water BP to 5 Oml

The active ingredient, buffer, flavour, colour and preservative are dissolved in some of the water and the glycerine is added The remainder of the water is heated to dissolve the sucrose and is then cooled The two solutions are combined, adjusted to volume and mixed The syrup is clarified by filtration

B Sucrose-free Syrup mq/5ml dose

Active Ingredient 2.5

Hydroxypropylmethylcellulose USP

(viscosity type 4000) 22.5

Buffer )

Flavour )

Colour ) as required

Preservative )

Sweetener )

Purified Water BP to 5 Oml

The hydroxypropylmethylcellulose is dispersed in hot water, cooled and then mixed with an aqueous solution containing the active ingredient and the other components of the formulation The resultant solution is adjusted to volume and mixed

The syrup is clarified by filtration

INJECTION FOR INTRAVENOUS ADMINISTRATION

μq/ml

(i) Active Ingredient 800

Dilute Hydrochloric Acid BP to pH 3 5

Sodium Chloride Injection BP to 1 ml

The active ingredient is dissolved in a suitable volume of Sodium Chloride Injection BP, the pH of the resultant solution is adjusted to pH3 5 with dilute hydrochloric acid BP then the solution is made to volume with sodium chloride injection BP and thoroughly mixed The solution is filled into Type I clear glass 5ml ampoules which are sealed under a headspace of air, by fusion of the glass then sterilised by autoclavmg at 120° for not less than 15 minutes

μq/ml

(II) Active ingredient 56 2

Sodium Chloride BP as required

Water for Injection BP to 1 Oml

Sodium chloride may be added to adjust the tonicity of the solution and the pH may be adjusted, using acid or alkali, to that of optimum stability and/or facilitate solution of the active ingredient Alternatively, suitable buffer salts may be used

The solution is prepared, clarified and filled into appropriate size ampoules sealed by fusion of the glass The injection is sterilised by heating in an autoclave using one of the acceptable cycles Alternatively, the solution may be sterilised by filtration and filled into sterile ampoules under aseptic conditions The solution may be packed under an inert atmosphere of nitrogen or other suitable gas

SUPPOSITORY FOR RECTAL ADMIN ISTRATION

Active ingredient 49 0 mg

Witepsol ^* H 1 5 to 1 .0g

^* a proprietary grade of Adeps Solidus Ph. Eur

A suspension of the active ingredient in molten Witepsol is prepared and filled using suitable machinery, into 1 g size suppository moulds.

The compounds of Examples 1 and 14 were tested for beta-3-adreπoceptor activity using above described Method 1 with the following results: