NO315703B1 - Meta azacyclic aminobenzoic acid compounds and derivatives thereof which are integrin antagonists - Google Patents

Description

The present invention relates to pharmaceutical agents (compounds) which are usable as OvPa integrin antagonists and which are thus usable in pharmaceutical compositions and in methods for treating conditions mediated by OvP3 by inhibiting or antagonizing av|33 integrins.

The background of the invention

Integrins are a group of cell surface glycoproteins that mediate cell adhesion and are therefore useful mediators of cell adhesion interactions that occur during various biological processes. Integrins are heterodimers composed of non-covalently bound α and β polypeptide subunits. Currently, 11 different a subunits have been identified and 6 different p subunits have been identified. The different a subunits can combine with different p subunits to form different integrins.

The integrin identified as Ovp3 (also known as the vitronectrin receptor) has been identified as an integrin that plays a role in various conditions or disease states including tumor metastasis, hard tumor growth (neoplasia), osteoporosis, Pagef's disease, humoral hypercalcemia of ma-lingnancy, angiogenesis, including tumor angiogenesis, retinopathy, including macular degeneration, arteritis, including rheumatoid arteritis, gum disease, psoriasis and smooth muscle cell migration (eg, restenosis). In addition, it has been found that such agents would be useful as antivirals, antifungals and antimicrobials. Therefore, compounds that selectively inhibit or antagonize O 3 O 3 would be beneficial for treating such conditions.

It has been shown that the avp3 integrin and other Oy containing integrins bind to a number of Arg-Gly-Asp(RGD) containing matrix macromolecules. Compounds containing the RDG sequence mimic extra cellular matrix ligands to bind to cell surface receptors. However, it is also known that RGD peptides are generally non-selective for RGD-dependent integrins. For example, most RGT peptides bind to Ovp3, bind to OvPsi ctvPi, and otiibP3. Antagonism of platelet anbp3 (also known as the fibrinogen receptor) is known to block platelet aggregation in humans. In order to avoid bleeding side effects when treating the conditions or disease states associated with the integrin OvP3, it would be advantageous to develop compounds that are selective antagonists of otvp3 as opposed to aiIbp3.

Cancer cell invasion occurs by a three-step process: 1) cancer cell attachment to extracellular matrix; 2) proteolytic

dissolution of the matrix, and 3) movement of the cells through the dissolved barrier. This process can occur several times and can result in metastasis at sites distant from the original tumor.

Seftor et. eel. (Proe. Nati. Acad. Sei. USA, Vol. 89 (1992) 1557-1561) have shown that the avp3 integrin has a biological function in melonoma cell invasion. Montgomery et al. eel. (Proe. Nati. Acad. Sei. USA, Vol. 91 (1994) 8856-60) has shown that the integrin avp3 expressed on human melanoma cells promotes a survival signal, and protects the cells against apoptosis. Mediating the cancer cell's metastatic pathway by interacting with the Ovp3 integrin cell adhesion receptor to prevent tumor metastasis would be beneficial.

Brooks et al. al., (Cell, Vol. 79 (1994) 1157-1164) have shown that antagonists of avp3 provide a therapeutic approach for the treatment of neoplasia (inhibition of hard tumor growth) since systemic administration of ct^^ j antagonists causes dramatic regression of various histologically diverse human tumors.

The addition receptor integrin OvP3 was identified as a marker for angiogenic blood vessels in chicken and humans and therefore such a receptor plays a critical role in angiogenesis or neovacucularization. Angiogenesis is characterized by the invasion, movement and proliferation of smooth muscle and endothelial cells. Antagonists of Ovp3 inhibit this process by selectively promoting apoptosis of cells in the neo-vasculature. The growth of new blood vessels, or angiogenesis, also contributes to pathological conditions such as diabetic retinopathy and macular degeneration. (Adonis et. al., Amer. J. Opthal., Vol. 118 (1994) 445-450) and rheumatoid arthritis (Peacock et. al., J. Exp. Med., Vol. 175 (1992), 1135-1138). Therefore, Ovp3 antagonists would be useful therapeutic targets to treat such conditions associated with neovascularization (Brooks et. al., Science, Vol. 264,

(1994), 569-571).

It has been reported that the cell surface receptor OvP3 is the main osteoclast integrin responsible for attachment to bone. Osteoclasts cause bone resorption and when such bone resorption activity exceeds bone formation activity it results in osteoporosis (a loss of bone substance), which leads to an increased number of bone fractures, disability and increased mortality. Antagonists of Ovp3 have been shown to be potent inhibitors of osteoclastic activity both in vitro (Sato et. al., J. Cell. Biol., Vol. 111 (1990) 1713-1723) and in vivo (Fisher et. al., Endocrinology, Vol. 132

(1993) 1411-1413). Antagonists of Ovp3 lead to reduced bone resorption and therefore restore a normal balance of bone formation and "resoround bottoming" activity. Therefore, it would be advantageous to provide antagonists of osteoclast otvp3 which are effective inhibitors of bone resorption and therefore useful in the treatment or prevention of osteoporosis.

The role of the OvP3 integrin in smooth muscle cell migration also creates a therapeutic target for the prevention or inhibition of neointimal hyperplasia which is a leading cause of restenosis after vascular procedures (Choi et.al., J.Vasc. Surg. Vol. 19(1) (1994) 125-34). Prevention or inhibition of neointimal hyperplasia by pharmaceutical agents to prevent or inhibit restenosis would be beneficial.

White (Current Biology, Vol. 3(9) (1993) 596-599) has reported that adenoviruses use Ovp3 to enter

host cells. The integrin appears to be required for endocytosis of the virus particle and may be required for penetration of the viral genome into the host cell's cytoplasm. Therefore, compounds that inhibit OvP3 will find utility as

antiviral agents.

WO 97/08145 discloses meta-guanidine, urea, thiourea and azacyclic aminobenzoic acid derivatives of formula (I)

where A is

which are useful as Ovps integrin antagonists.

J. Med. Chem. 40. 930 (1997), J. Med. Chem. 40. 920 (1997) and Antiv.Chem.Chemother. 8, 463 (1997) deals with the discovery of HIV-1 integrase inhibitors by specific pharmacophore studies.

Exp.Opin.Ther. patents 8, 633 (1998) have been published after the present claimed priority and deal with the development that integrin antagonists could be used to inhibit metastasis.

Summary of the invention

The present invention relates to a compound with the following general formula

wherein X and Y are the same or different halo group and pharmaceutically acceptable salts thereof.

The compounds described above may exist in various isomeric forms and all such isomeric forms are intended to be included. Tautomeric forms are also included in addition to pharmaceutically acceptable salts of such isomers and tautomers.

More specifically, the present invention deals with the following compounds:

wherein R is H or alkyl;

or pharmaceutically acceptable salts thereof.

It is another object of the invention to provide pharmaceutical compositions comprising compounds described above. Such compounds and compositions are useful in selective inhibition or antagonism of the integrin and therefore another embodiment of the present invention deals with a method for selective inhibition or antagonism of the Ovpa integrin. The invention further involves the use of the compound to prepare a drug for the treatment or inhibition of pathological conditions associated therewith such as osteoporosis, humoral hypercalcemia of malignancy, Pagef's disease, tumor metastases, hard tumor growth (neoplasia), angiogenesis, including tumor angiogenesis, retinopathy including diabetic retinopathy and macular degeneration, arteritis, including rheumatoid arthritis, gum disease, psoriasis, smooth muscle cell migration and restinosis in mammals in need of such treatment. In addition, such pharmaceutical agents are useful as antiviral agents and antimicrobial agents.

Detailed description

The present invention relates to a class of compounds represented by the formulas I-XVI, described above.

Preferred embodiments of the present invention are compounds with the following formulas

The invention further relates to pharmaceutical compositions containing therapeutically effective amounts of the compounds described above.

The invention also relates to the use of the compounds to produce drugs for selective inhibition or antagonism of the Ovp3 integrin, and more specifically relates to the above application for inhibiting bone resorption, gum disease, osteoporosis, humoral hypercalcemia in malignancy, Pagef's disease, tumor metastases, hard tumor growth (neoplasia ), angiogenesis, including tumor angiogenesis, retinopathy including diabetic retinopathy and macular degeneration, arthritis, including rheumatoid arthritis, smooth muscle cell migration and restenosis by administering a therapeutically effective amount of a compound described above to achieve such inhibition together with a pharmaceutically acceptable carrier .

The following is a list of definitions of various terms used herein: As used herein, the term "alkyl" or "lower alkyl" refers to a linear or branched hydrocarbon chain radical having 1-10 carbon atoms, and more preferably 1-6 carbon atoms . Examples of such alkyl radicals are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, pentyl, neopentyl, hexyl, isohexyl, and the like.

As used herein, the term "halo" or "halogen" refers to bromine, chlorine, or iodine.

As used herein, the term "haloalkyl" refers to alkyl groups as defined above substituted with one or more of the same or different halo groups at one or more carbon atoms. Examples of haloalkyl groups include trifluoromethyl, dichloroethyl, fluoropropyl, and the like.

The term "composition" as used herein means a product resulting from the mixture or combination of more than one element or ingredient.

The term "pharmaceutically acceptable carrier", as used herein, means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating agent, involved in carrying or transporting a chemical medium.

The term "therapeutically effective amount" shall mean the amount of drug or pharmaceutical agent that will elicit the biological or medical response in a tissue, system or animal sought by a researcher or clinician.

The following is a list of abbreviations and corresponding meanings used interchangeably herein:

<L>H-NMR = proton nuclear magnetic resonance

AcOH = acetic acid

Ar = argon

CH3CN = acetonitrile

CHN analysis = carbon/hydrogen/nitrogen elemental analysis CHNC1 analysis = carbon/hydrogen/nitrogen/chlorine elemental analysis

CHNS analysis = carbon/hydrogen/nitrogen/sulfur elemental analysis

DI water = deionized water

DMA = N. N dimethylacetamide

DMAP = 4-( N,N dimethylamino)pyridine

DMF = N,N dimethylformamide

EDC1 = 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

EtOAc = ethyl acetate

EtOH = ethanol

FAB MS = fast atomic bombardment mass spectroscopy

g = grams

HOBT = 1-hydroxybenzotriazole hydrate

HPLC = high performance liquid chromatography

IBCF = isobutyl chloroformate

KSCN = potassium thiocyanate

1 = liters

LiOH = lithium hydroxide

MEM = methoxyethoxymethyl

MEMC1 = methoxyethoxymethyl chloride

MeOH = methanol

mg = milligrams

MgS04 = magnesium sulfate

ml = milliliters

MS = mass spectroscopy

MTBE = methyl tert-butyl ether

N2 = nitrogen

NaHCO3 = sodium bicarbonate

NaOH = sodium hydroxide

NaaS04 = sodium sulfate

NMM = N-methylmorpholine

NMP = N-methylpyrrolidinone

NMR = nuclear magnetic resonance

P205 = phosphorus pentoxide

PTSA = para-toluenesulfonic acid

RPHPLC = reversed phase high performance liquid chromatography RT = room temperature

TFA = trifluoroacetic acid

THF = tetrahydrofuran

TMS = trimethylsilyl

A = heating of the reaction mixture

The compounds described above may exist in various isomeric forms and all such isomeric forms are intended to be included. Tautomeric forms are also included in addition to pharmaceutically acceptable salts of such isomers and tautomers.

In the structures and formulas herein, a bond drawn across a bond or a ring may be to any available atom on the ring.

The term "pharmaceutically acceptable salt" refers to a salt prepared by contacting a compound described above with an acid whose anion is generally considered suitable for human consumption. Examples of pharmacologically acceptable salts include the hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate, acetate, propionate, lactate, maleate, malate, succinate, tartrate salts and the like. All the pharmacologically acceptable salts can be prepared by conventional methods. (See Berge et. al., J. Pharm. Sei., 66(1), 1-19

(1977) for additional examples of pharmaceutically acceptable salts.)

For the selective inhibition or antagonism of otvp3 integrins, compounds of the present invention can be administered orally, parenterally, or by inhalation spray, or topical unit dosage formulations containing common pharmaceutically acceptable carriers, excipients and vehicles. The term parenteral as used herein includes, for example, subcutaneous, intravenous, intramuscular, intrasternal, infusion techniques, or intraperitoneal.

The compounds of the present invention are administered in any suitable manner in the form of pharmaceutical compositions adapted to such manner, and in a dose effective for the intended treatment. Therapeutically effective doses of the compounds required to prevent or slow the development of, or treat, the medical condition are readily available to those skilled in the art by using preclinical and clinical methods known in the medical arts.

Accordingly, the present invention provides use of the compounds for the manufacture of a medicament for treating conditions mediated by selectively inhibiting or antagonizing the OvP3 cell surface receptor. The treatment comprises the administration of a therapeutically effective amount of a compound selected from the class of compounds described above, wherein one or more compounds are administered together with one or more non-toxic, pharmaceutically acceptable carriers and/or diluents and/or excipients (collectively referred to herein as "carrier" materials) and, if desired, other active ingredients. More specifically, the OvP3 cell surface receptor is inhibited. Most preferably, the compounds of the invention are used for the production of drugs to inhibit bone resorption, treat osteoporosis, inhibit humoral hypercalcemia in malignancy, treat Paget's disease, inhibit tumor metastasis, inhibit neoplasia (hard tumor growth), inhibit angiogenesis including tumor angiogenesis, treat diabetic retinopathy and macular degeneration, inhibit arthritis, psoriasis and periodontal disease, and inhibit smooth muscle cell migration including restinosis.

Based on standard laboratory testing techniques and methods well known and recognized by those skilled in the art, in addition to comparisons with compounds of known utility, the compounds described above can be used in the treatment of patients suffering from the above pathological conditions. The person skilled in the art will recognize that the selection of the most suitable compound of the invention is within the ability of one of ordinary skill in the art and will depend on many factors including consideration of results obtained in standard assays and animal models.

Treatment of a patient afflicted with one of the pathological conditions comprises administering to such patient an amount of a compound described above that is therapeutically effective to control the condition or prolong the survival of the patient beyond that expected in the absence of such treatment. As used herein, the term "inhibition" of the condition refers to delaying, interrupting, slowing or stopping the condition and does not necessarily indicate a total elimination of the condition. It is believed that extending the survival of a patient, in addition to being a substantially beneficial effect in and of itself, also indicates that the condition is beneficially controlled to some extent. As stated earlier, the compounds of the invention can be used in many different biological prophylactic or therapeutic areas. It is expected that these compounds are useful in the prevention or treatment of any disease state or condition in which the OvP3 integrin plays a role.

The dosage regimen for the compounds and/or compositions containing the compounds is based on several factors, including the type, age, weight, gender and medical condition of the patient; the severity of the condition; the mode of administration; and the activity of the particular compound used. Therefore, the dosage regimen can vary widely. Dosage levels in the order of magnitude from 0.01 mg - 1000 mg per kg of body weight per day are applicable in the treatment of the conditions indicated above, and more preferably 0.01 mg - 100 mg per kg of body weight per day.

The active ingredient administered by injection is formulated as a composition in which, for example, salt, dextrose or water can be used as a suitable carrier. A suitable daily dose would typically be 0.01 - 10 mg/kg body weight injected per day in multiple doses depending on the factors listed above.

For administration to mammals in need of such treatment, the compounds in a therapeutically effective amount are usually combined with one or more excipients appropriate to the indicated mode of administration. The compound can be mixed together with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphorus and sulfuric acids, gelatin, acacia, sodium alginate, polyvinylpyrrolidine, and/or polyvinyl alcohol, and tableted or encapsulated for convenient administration. Alternatively, the compounds may be dissolved in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, and/or various buffers. Other excipients and modes of administration are well known in the pharmaceutical art.

The pharmaceutical compositions usable in the present invention may be subjected to normal pharmaceutical operations such as sterilization and/or may contain normal pharmaceutical auxiliaries such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, etc.

The general synthetic sequences for preparing the compounds useful in the present invention are illustrated in Figures I-III. Both an explanation and the actual method for the various embodiments of the present invention are described where appropriate. The following figures and examples are intended to be exclusively illustrative of the present invention, and not limiting thereof, either in scope or spirit. Those with knowledge of the subject. will readily understand that known variations of the conditions and methods described in the figures and examples can be used to synthesize the compound in the present invention.

Unless otherwise indicated, all starting materials and all equipment used were commercially available. Reaction scheme 1 illustrates methodology applicable to prepare the tetrahydropyrimidinebenzoic acid moiety of the present invention which can be coupled to a gly-p-amino acid ester. Briefly, in reaction core I, 3,5-dihydroxybenzoic acid is converted to 3-amino-5-hydroxy-benzoic acid using the method described in Austr. J. Chem., 34(6), 1319-24 (1981). The product is reacted with ammonium thiocyanate in warm, dilute hydrochloric acid to give 3-thiourea-5-hydroxybenzoic acid after normal work-up. This thiourea intermediate is converted to S-methyl derivatives by reaction with methyl iodide in ethanol at reflux. 1,3-diamino-2-hydroxypropane is reacted with this resulting intermediate in hot DMA. Upon cooling, forms are precipitated and the zwitterionic product is isolated by filtration. The HCl salt can be obtained by lyophilization from dilute hydrochloric acid. Alternatively, the product can be isolated from the original reaction mixture by removing volatiles and concentrating. The resulting product is first taken up in water and pH adjusted to 5-7 where the zwitterionic product precipitates and is isolated by filtration. The HCl salt can be obtained as previously stated or simply by dissolving in dilute hydrochloric acid and concentrating to a solid and drying.

Reaction Scheme IA illustrates methodology applicable to prepare the tetrahydropyrimidinebenzoic acid moiety of the present invention which can be coupled to a gly-p-amino acid ester. Briefly, in reaction core IA, 1,3-diamino-2-hydroxypropane is reacted with carbon disulfide in a suitable solvent such as ethanol-water, refluxed, cooled, hydrochloric acid added, refluxed again, cooled, and the product, 5-hydroxy -tetrahydropyrimidine-2-thione harvested by filtration and dried. This cyclic thiourea intermediate is converted to the S-methyl derivative by reaction of thione and methyl iodide in ethanol at reflux. The desired 2-methylthioether-5-hydroxypyrimidine hydroiodide is easily isolated by removing volatiles at reduced pressure. Therefore, 2-methylthioether-5-hydroxy-pyrimidine anhydride in methylene chloride: DMA (about 10:1) and an equivalent of triethylamine are cooled to about ice bath temperature and an equivalent of di-tert-butyl dicarbonate (BOC anhydride) is added. Usual work-up gives BOC-2-methylthioether-5-hydroxypyrimidine as an oil.

3,5-Dihydroxybenzoic acid is converted to 3-amino-5-hydroxybenzoic acid using the method of Aust.J.Chem., 34(6), 1319-24 (1981).

The desired end product, 3-hydroxy-5-[(5-hydroxy-1,4,5,6-tetrahydro-2-pyrimidinyl)amino]benzoic acid hydrochloride salt is prepared by reacting BOC-2-methylthioether-5-hydroxypyrimidine in warm DMA. On cooling, a precipitate forms and the zwitterion product is isolated by filtration. The HCl salt can be obtained by lyophilization from dilute hydrochloric acid, for example.

Y and X are halo groups

Reaction scheme II illustrates a methodology applicable to prepare the ethyl N-gly-amino-3-(3,5-dihalo-2-hydroxy)phenyl-propionate portion of the present invention which can be coupled with the tetrahydropyrimidinobenzoic acid group. Briefly, 3,5-halosubstituted salicylaldehydes can be prepared by direct halogenation as, for example, would be the case where 5-bromosalicylaldehyde is slurried in acetic acid and one or more equivalents of chlorine is added to give 3-chloro-5-bromo-2 -hydroxybenzaldehyde. Some products precipitate out and can be recovered by filtration. The rest can be recovered by diluting the filtrate with water and isolating the precipitate. Combination of the solids and drying gives 3-chloro-5-bromo-2-hydroxybenzaldehyde. 3-Iodo-5-chlorosalicylaldehyde can be prepared by reacting 5-chlorosalicylaldehyde with N-iodosuccinimide in DMF and subjecting the reaction mixture to normal work-up conditions. 3-Iodo-5-bromosalicylaldehyde can be prepared by reacting 5-bromosalicylaldehyde in acetonitrile with potassium iodide and chloramine T. Workup gives a material which when treated with hexane gives the desired 3-iodo-5-chlorosalicylaldehyde.

Coumarins are readily prepared from salicylaldehydes using a modified Perkin reaction (e.g., Vogel's Textbook and Practical Organic Chemistry, 5th Ed., 1989, p. 1040). The halo-substituted coumarins are converted to 3-aminohydrocoumarins (see J.G.Rico, Tett.Let., 1994, 35, 6599-6602) which are readily opened in acidic alcohol to give 3-amino-3-(3,5-halo-2 -hydroxy)phenyl propanoic acid esters.

3-amino-3-(3,5-halo-2-hydroxy)phenyl propanoic acid esters are converted to N-gly-3-amino-3-(3,5-halo-2-hydroxy)phenyl propanoic acid esters by reaction of Boc- N-gly-N-hydroxy-succinimide to give Boc-N-gly-3-amino-3-(3,5-halo-2-hydroxy)phenyl propanoic acid esters which are converted to HX salts of N-gly- 3-amino-3-(3,5-halo-2-hydroxy)phenyl propanoic acid esters (where X is a halo group) for example, by removal of the BOC protecting group using HCl in ethanol.

The amino acid compounds used to prepare the compounds in the present invention can be prepared according to the methods presented herein and below and according to the methodology described and required in the floating USSN Attorney. Docket 3076.

Y and X are halo groups

Reaction scheme III is illustrative of the methodology applicable to prepare various compounds in the present invention. 3-Hydroxy-5-[(1,4,5,6-tetrahydro-5-hydroxy-2-pyrimidinyl)amino]benzoic acid is activated for coupling using known methods. Therefore, after dissolution in a suitable solvent such as DMA, an equivalent of NMM is added. The reaction mixture is cooled to ice bath temperature and IBCF is added. To the mixed anhydride product, intermediate gly-p-amino acid ester and NMM are added. Upon completion of the reaction, the product is purified by prep HPLC and esters hydrolyzed to the acid by treatment with a base such as LiOH in a suitable solvent (dioxane/water or acetonitrile/water). Alternatively, a suitable acid, such as TFA, can be used. The product is isolated by prep hplc or by isolation of the zwitter ion at pH 5-7 and conversion to the desired salt by standard steps.

Example A

Manufacture of

Step 1 Preparation of

To a 2 L round bottom flask equipped with a mechanical stirrer and condenser were added 3,5-dichlorosalicylaldehyde (200 g, 1.05 mol, 1 equiv.) acetic anhydride (356 g, 3.49 mol) and trimethylamine (95.0 g, 0 .94 mol, 0.90 equiv.) added. The reaction solution was heated at reflux overnight. The dark brown reaction mixture was cooled to 50°C and water (1 L) was added with stirring. After one hour, the mixture was filtered and the filtrate combined with EtOH (1 L). This mixture was heated to 45°C for one hour, cooled to room temperature, filtered and the solid (fraction A) washed with EtOH (0.5 L). The combined EtOH solutions were concentrated by rotary evaporation to an oil (fraction B). The solid from fraction A was dissolved in methylene chloride (1.5 L) and the resulting solution passed through a bed of silica gel (1300 ml volume). The resulting dark brown solution was concentrated to an oil which was triturated with hexanes (1.3 L) to give a solid which was isolated by filtration and washed (hexanes) to give essentially pure 6,8-dichlorocoumarin (163 g ). A further 31 g of product was obtained by treating the oil, fraction B, in a similar manner; the oil was dissolved in methylene chloride (0.5 L) passed through a pad of silica (0.5 L by volume) and triturated with hexanes. The total isolated yield was 194 g or 86% yield of the brown solid.

MS and NMR were consistent with the desired structure.

Step 2

Manufacture of

To a 3-necked 2 L round bottom flask equipped with a mechanical stirrer was added 6,8-dichlorocoumarin (160 g, 0.74 mol) (prepared in step 1) and dry THF (375 mL, Aldrich Sure Seal). The resulting mixture was cooled to -40°C (dry ice/acetone bath) and lithium bis(trimethylsilyl)amide (0.80 mol, 800 mL 1M THF) was added while maintaining the temperature below -40°C. After completion of the addition, the cooling bath was removed. After 0.5 hour the mixture was warmed to -5°C. The reaction was quenched by the addition of a solution of HCl (0.5 L 4M dioxane) in EtOH (1.25 L). The temperature was maintained below 0°C overnight. The reaction mixture was concentrated to about half the original volume and partitioned between EtOAc (3 L) and water (2 L). The organic phase was washed with aqueous HCl (3 x 1 1 0.5 N HCl). The pH in the combined aqueous layers was adjusted to approx. 7 by adding 10% aqueous NaOH and extracting with methylene chloride (3x2 1). The combined organic phases were dried (MgSO 4 , filtered and 4M HCl in dioxane (210 mL) added with stirring. Upon completion of the precipitation, the solid was removed by filtration. The filtrate was concentrated to a small volume and methyl t-butyl ether was added. The solid obtained was combined with the original solid formed and the combined product was washed with methyl t-butyl, isolated by filtration and dried (vacuum oven over a weekend) to

obtain the desired product (172 g, 74% yield).

MS and NMR were consistent with the desired structure.

Step 3

Manufacture of

To a flame-dried round bottom flask (0.5 L) fitted with a magnetic stir bar was added N-t-Boc-glycine N-hydroxysuccinimide ester (Sigma 15.0 g, 0.055 mol), dry DMF (Aldrich Sure Seal, 200 mL) and the product from step 2 (21.67 g, 0.055 mol) under an inert atmosphere (Ar). The reaction mixture was cooled to approx. 0°C (salt-ice bath) and N-methylmorpholine (5.58 g, 0.056 mol) and a catalytic amount of DMAP were added and the reaction allowed to proceed overnight. The reaction mixture was concentrated to a "slush" and partitioned between EtOAc (0.41) and aqueous base (2 x 0.2 L, aq. saturated NaHCO 3 ). The organic phase was subsequently washed with aqueous citric acid (2 x 0.2 1, 10% w/v), again with aqueous sodium bicarbonate (2 x 0.2 1), brine and dried (Na 2 SO 4 ). Volatiles were removed under vacuum at 55°C to give an oil (22.5 g, 92% yield) which solidified on standing.

MS and MNR were consistent with the desired structure.

Step 4

Manufacture of

The product obtained in step 3 was deprotected to give the amine hydrochloride salt using the following procedure. To the product from step 3 (14.0 g, 0.032 mol) in a flame-dried round bottom flask (0.1 L) with a stir bar was added dry dioxane (40 mL). To this was added 4.0 N HCl in dioxane (2 equiv., 6.32 mL) at 0°C and the reaction allowed to proceed until gas evolution ceased and the reaction was complete. Volatiles were removed under vacuum and the residue triturated with diethyl ether (50ml). Solid was collected by filtration and washed with ether and dried to give the desired product (12.5 g).

MS and MNR were consistent with the desired structure.

Example B

Manufacture of

Step 1 Preparation of

To a suspension of 3-bromo-5-chlorosalicylaldehyde (175.0 g, 743.2 mmol) in acetic anhydride (280.5 mL, 3.0 mol) was added triethylamine (103.6 mL, 743.2 mmol). The reaction solution was mixed at reflux for 4.5 hours. The solution was cooled and concentrated in vacuo. Absolute ethanol (730 mL) was added to the brown residue. The mixture was stored at 0°C for 14 hours. The brown solid was collected by filtration and washed with cold ethanol. The solid was dried in vacuo to give the desired product (123.0 g, 64% yield).

<X>H NMR was consistent with the proposed structure.

Step 2

Manufacture of

To a suspension of the coumarin (40.0 g, 154.1 mmol) in THF (400 mL) at -76 °C, lithium bis(trimethylsilyl)amide (154.1 mL 1M solution in THF) was added dropwise with stirring . The addition was completed in 10 minutes. The reaction mixture was then stirred for 5 minutes, warmed to -20°C and stirred for 15 minutes. To this solution was added acetic acid (9.25 g, 154.1 mmol) in THF (28 mL) over 5 minutes. The mixture was warmed to room temperature and volatiles were removed in vacuo. The residue was dissolved in ether (850 mL), washed with saturated aqueous NaHCO 3 (2 x 100 mL), brine (2 x 40 mL), and dried (MgSO 4 ). The ether solution was concentrated to approx. 160 ml and cooled to 0°C. To this solution was added 4M HCl in dioxane (56.3 mL, 225 mmol) and the mixture was stirred at 0°C for 30 minutes. The suspension was filtered and the filter cake washed with ether. The solid was dried in vacuo to give the desired product as HCl salt, dioxane solution, (45.0 g).

<*>H NMR was consistent with the desired structure.

Step 3

Manufacture of

To a suspension of the lactone (142.2 g, 354.5 mmol) in absolute ethanol (533 mL) was added 4M HCl in dioxane (157.8 mL, 631.1 mmol) over 10 min. The reaction mixture was stirred at room temperature for 2.5 hours. Volatile components were removed in vacuo. The precipitate was dissolved in ethyl acetate (450 ml) and the solution kept at 0°C for 15 hours. The leather colored precipitate was collected by filtration and washed with cold ethyl acetate. The solid was dried in vacuo to give the desired product as the hydrochloride salt (100.4 g, 79% yield).

<X>H NMR was consistent with the proposed structure.

Step 4

Manufacture of

To a flame-dried round-bottom flask (0.1 L) fitted with a magnetic stir bar was added N-t-Boc-glycine N-hydroxysuccinimide ester (Sigma, 2.72 g, 0.010 mol), dry THF (Aldrich Sure Seal, 50 mL) and the product from Step 3 (3.10 g, 0.01 mol, vacuum-dried overnight over P2O5) added under an inert atmosphere (Ar). The reaction mixture was cooled to approx. 0°C (salt-ice bath) and triethylamine (1.01 g, 0.010 mol) was added. The reaction was allowed to proceed overnight. The reaction mixture was concentrated to a semi-solid and worked up in a manner similar to Example A, Step 3. Volatiles were removed from the organic phase under vacuum at 55°C to give an oil (4 g, 83% yield), which solidified at standstill.

MS and MNR were consistent with the desired structure.

Step 5

Manufacture of

The product obtained in step 4 was deprotected to give the amine hydrochloride salt using the following procedure. To the product from step 4 (4.0 g, 0.0084 mol) in a flame-dried round bottom flask (0.1 L) with a stir bar was added dry dioxane (20 mL). To this 4.0 N HCl in dioxane (20 ml) was added and the reaction was allowed to proceed until gas evolution stopped and the reaction was complete (approx. 1 hour). Volatiles were removed under vacuum and the precipitate triturated with diethyl ether (50 mL). Solid was collected by filtration and washed with ether and dried to give a light brown solid (2.7 g, 78% yield).

MS and NMR were consistent with the desired structure.

Example C

Manufacture of

Step l

To a suspension of 3,5-dibromosalicylaldehyde (100 g, 357 mmol) in acetic acid (164.8 mL, 1.8 mol) was added triethylamine (45 mL, 375 mmol). The reaction solution was heated overnight at reflux under argon. The solution was cooled to room temperature and a solid mass formed. The dark brown reaction mixture was washed with hot hexanes (3 x 300 mL) and aqueous saturated bicarbonate. The resulting solid was dissolved in EtOAc (2 L) and washed with water. The organic phase was dried (sodium sulfate) and concentrated to give a brown solid which was collected by filtration. The solid was dried in vacuo to give essentially pure 6,8-dibromocoumarin (94.2 g, 87 yield).

MS and <X>H NMR were consistent with the desired structure.

Step 2

To 6,8-dibromocoumarin (20.0 g, 0.066 mol) (prepared in step 1) in THF (100 mL) at -78°C was added lithium bis(trimethylsilyDamide (66 mL 1M solution in THF) dropwise at stirring. The addition was completed in 10 minutes. The reaction mixture was then stirred for five minutes, warmed to 0°C, and stirred for 15 minutes. To this solution, acetic acid was added

(3.95 g) added over 1 minute. The mixture was warmed to room temperature and volatiles were removed in vacuo. The precipitate was dissolved in hexanes (500 mL), washed with saturated aqueous NaHCO 3 (2 x 100 mL) and dried (Na 2 SO 4 ). The organic solution was concentrated to give an oil which was immediately taken up in ethyl ether (400 ml) and 4M HCl in dioxane (30 ml) was added with stirring at 0°C for 30 minutes. Excess HCl was removed in vacuo, the suspension filtered and the filter cake washed with ether. The solid became

dried in vacuo to give the desired product as the HCl salt, dioxane solvate (19.9 g).

MS and AH NMR were consistent with the desired structure.

Step 3

The lactone prepared in step 2 above (15 g) was dissolved in absolute ethanol (400 mL) and anhydrous HCl gas was passed through for one minute. The reaction mixture was stirred at room temperature for 2.5 hours. RPHPLC showed complete reaction. The volatiles were removed in vacuo to give a dark residue. The residue was triturated with diethyl ether (500 mL) and the mixture stirred overnight. The leather colored precipitate was collected by filtration and washed with diethyl ether. The solid was dried in vacuo to give the desired product as the hydrochloride salt (15.2 g).

MS and ^NMR were consistent with the desired structure.

Step 4

To a flame-dried round bottom flask (0.2 L) equipped with a magnetic stir bar was added N-t-Boc-glycine N-hydroxysuccinimide ester (Sigma, 8.1 g, 0.030 mol), dry DMF (Aldrich Sure Seal, 50 mL) and the product from step 3 (12 g, 0.03 mol, vacuum dried overnight over P2O5) under an inert atmosphere

(Year). The reaction mixture was cooled to approx. 0°C (salt-ice bath) and N-methyl morpholine (3.03 g, 0.030 mol) and catalytic DMAP were added. The reaction was allowed to proceed overnight warming to room temperature. The reaction mixture was concentrated to a semi-solid and worked up in a manner similar to Example A, Step 3. Volatiles were removed from the organic phase under vacuum at 55°C to give an oil (15.7 g, 93% yield) which solidified at standstill.

MS and NMR were consistent with the desired structure.

Step 5

The product obtained in step 4 was deprotected to give the amine hydrochloride salt using the following procedure. Dry dioxane (40 ml) was added to the product from step 4 (13.0 g, 0.0084 mol) in a flame-dried round bottom flask (0.1 L) with a stirring rod. To this was added 4.0 N HCl in dioxane (30 ml) and the reaction allowed to proceed until gas evolution stopped and the reaction was complete (about one hour). The volatiles were removed under vacuum and the precipitate triturated with diethyl ether (50 ml). Solid was collected by filtration and washed with ether and dried to give a solid (10.6 g, 93% yield).

MS and NMR were consistent with the desired structure.

Example D

Manufacture of

Step l

Preparation of 3-chloro-5-bromosalicylaldehyde

To a 5 L round bottom flask equipped with a mechanical stirrer and gas supply tube, 5-bromosalicylaldehyde (495 g, 2.46 mol) and acetic acid were added at room temperature to form a slurry. To this mixture chlorine gas was added at a moderate rate until a slight molar excess of chlorine (183 g, 1.05 mol) had dissolved. After the addition was stopped, the reaction was allowed to proceed overnight. The solid formed was recovered by filtration and the filtrate diluted in water (2.5 L). The mixture was stirred vigorously for 20 minutes, the product collected by filtration and washed with water. The combined solids were vacuum dried to give the desired 3-chloro-5-bromosalicylaldehyde (475 g, 82% yield).

MS and <X>H NMR were consistent with the desired structure.

Step 2

Preparation of 6-bromo-8-chlorocoumarin

To a 5 L round bottom flask equipped with a mechanical stirrer and condenser was added 3-chloro-5-bromosalicylaldehyde {554.1 g, 2.35 mol, 1 equiv.), acetic acid {1203 g, 11.8 mol, 5 equiv.) and triethylamine (237.4 g, 2.35 mol, 1 equiv) added The reaction solution was heated at reflux (131-141°C) overnight. The dark brown reaction mixture was cooled to 50°C and ice {2 1) added (ice bath cooling) with stirring. After one hour the mixture was filtered and the filtrate combined with EtOH (11). To this mixture EtOH (300 mL) was added and the reaction mixture was stirred for one hour. The precipitate that formed was collected by filtration and washed with water:EtOH (3 x 1.3 L), vacuum and dried, then dried on a fluid bed dryer. The total isolated yield is 563 g or 92%.

MS and <1>H NMR were consistent with the desired structure.

Step 3

Preparation of 3-amino-3-(2-hydroxy-3-chloro-5-bromo)phenylpropanoic acid ethyl ester

To a 3-necked 5 L round bottom flask equipped with a mechanical stirrer was added 6-bromo-8-chlorocoumarin (300 g, 1.16 mol) {prepared in step 2) and dry THF (900 mL, Aldrich Sure Seal).

The resulting mixture was cooled to less than -45°C (dry ice/acetone bath) and lithium bis(trimethylsilyl)amide (0.80 mol, 800 mL IM in THF and 0.6 L in hexanes, 1.2 equiv) added while maintaining a temperature below -45°C for 0.5 hour. In a separate 5 L bottle, EtOH (2.5 L) and HCl (4 N HCl in dioxane, 1 L) were combined at -15°C. The coumarin reaction was stopped by adding the cooled HCl/EtOH solution. After 0.5 hours, the resulting reaction mixture temperature was -8.3°C. The reaction mixture was kept at 0°C overnight, concentrated to approx. 2.5 L and partitioned between EtOAc (3 L) and water (4 L). The organic phase was washed with aqueous HCl (4 x 1.2 L, 0.5 N HCl). The pH of the combined aqueous layers was adjusted to approx. 8 by adding 10% aqueous NaOH and extracting with methylene chloride (1 x 7 1 and 3 x 2 1). The combined organic phases were dried (MgSO 4 , 900 g), filtered and 4M HCl in dioxane (400 mL) added with stirring. On completion of the precipitation, the solid was removed by filtration. The mixture was concentrated to 2.5 L, hexanes added (2.5 L) and the precipitate isolated by filtration. The filter cake was washed with methylene chloride/hexanes (1:2), suction dried and vacuum oven dried at 40°C to obtain the desired product (251 g, 60% yield).

MS and <X>H NMR were consistent with the desired structure.

Step 5

Manufacture of

The above compound was prepared using essentially the same procedure and relative amounts as specified for its isomers in Example B, Step 4.

MS and <X>H NMR were consistent with the desired structure.

Step 6

Manufacture of

This compound was prepared using essentially the same procedure and relative amounts as specified for its isomer in Example B, Step 5.

MS and ^NMR were consistent with the desired structure.

Example E

Manufacture of

Step 1

Preparation of 3-iodo-5-chlorosalicylaldehyde

N-Iodosuccinimide {144.0 g, 0.641 mol) was added to a solution of 5-chlorosalicylaldehyde (100 g, 0.638 mol) in dimethylformamide (400 mL). The reaction mixture was stirred for two days at room temperature. Additional N-iodosuccinimide (20.0

g) was added and the stirring was continued for a further two days. The reaction mixture was diluted with ethyl acetate

(1 1) washed with hydrochloric acid {300 mL, 0.1 N) water (300 mL), sodium thiosulfate (5%, 300 mL), brine (300 mL), dried (MgSO 4 ) and concentrated to dryness to give the desired aldehyde (162 g, 90% yield) as a pale yellow solid.

MS and <*>H NMR were consistent with the desired structure.

Step 2

Preparation of 6-chloro-8-iodocoumarin

A mixture of 3-iodo-5-chlorosalicylaldehyde (100 g, 0.354 mol), acetic anhydride (300 mL) and triethylamine (54 mL) was heated to reflux for 18 hours. On cooling, the desired coumarin precipitated as a dark brown crystalline material. It was filtered, washed with hexane/ethyl acetate (4:1, 200 mL), and air dried. Yield: 60 g (55%).

MS and H1 NMR were consistent with the desired structure.

Step 3

Preparation of (R,S)-4-amino-3,4-dihydro-6-chloro-8-iodocoumarin hydrochloride

Lithium hexamethyldisilazane {21.62 mL, 1M, 21.62 mmol) was added to a solution of 6-chloro-8-iodocoumarin (6.63 g, 21.62 mmol) in tetrahydrofuran (100 mL) at -78°C . The reaction mixture was stirred at this temperature for 30 minutes, then at 0°C for one hour. Acetic acid (1.3 g, 21.62 mmol) was added to the reaction mixture. The reaction mixture was poured into ethyl acetate (300 mL) and saturated sodium carbonate (200 mL) solution. The organic phases were separated, washed with brine (200 mL), dried (MgSO 4 ) and concentrated to give a residue. The residue was added to anhydrous ether (200 mL) followed by dioxin/HCl (4N, 30 mL) at 0°C. The reaction mixture was stirred for one hour at room temperature, filtered and was dried in vacuo to give the desired product {4.6 g, 59% yield) as a powder. (RPHPLC: Rf 6.8 minutes; Gradient 10% acetonitrile -90% acetonitrile over 15 minutes then to 100% acetonitrile over the next 6 minutes. Both water and acetonitrile contain 0.1% TFA. Vydac C18 protein peptide columns, 2 ml/minute flow rate, monitored at 254 nm).

MS and <1>H NMR were consistent with the desired structure.

Step 4

Preparation of (R,S)-ethyl 3-amino-3-(5-chloro-2-hydroxy-3-iodo)phenylpropionate hydrochloride.

Hydrochloric acid gas was bubbled into a solution of 4-amino-3,4-dihydro-6-chloro-8-iodocoumarin hydrochloride (22.0 g, 61.09 mmol) in ethanol (250 mL) maintaining the reaction mixture at 0-10 °C to saturation. After 6 hours at reflux, most of the solvent was removed by distillation. The cooled residue was added to anhydrous ether and stirred for two hours. The original rubber changed into a crystalline material. The crystalline product was filtered and dried to give the desired product (20 g, 81% yield) as an off-white crystalline powder. (Rf 7.52 minutes, conditions as step 3).

MS and <*>H NMR were consistent with the desired structure.

Step 5

Preparation of (R,S)-ethyl 3-(N-BOC-gly)amino-3-(5-chloro-2-hydroxy-3-iodo)phenylpropionate.

A mixture of BOC-gly (2.16 g, 12.31 mmol), HOBT (1.67 g, 12.31 mmol), EDC1 (2.36 g, 12.31 mmol) and DMF (50 mL) was stirred at 0°C for 1 hour. Ethyl 3-amino-3-(5-chloro-2-hydroxy-3-iodo)propionate hydrochloride (5.0 g, 12.31 mmol) was added to the reaction mixture followed by triethylamine (3.5 mL). The reaction mixture was stirred for 18 hours at room temperature. The DMF was removed in vacuo and the residue was partitioned between ethyl acetate (300 mL) and sodium bicarbonate (200 mL). The organic phase was washed with hydrochloric acid (1 N, 100 mL), brine (200 mL), dried (MgSO 4 ) and concentrated to give the desired product as a solid (6 g, 93% yield).

MS and <X>H NMR were consistent with the desired structure.

Step 6

Preparation of (R,S)-ethyl 3-(N-gly)-amino-3-(5-chloro-2-hydroxy-3-iod)phenylpropionate hydrochloride.

Dioxane/HCl (4N, 20 mL) was added to ethyl 3-(N-BOC-gly)-amino-3-(5-chloro-2-hydroxy-3-iodo)propionate (6.0 g, 11.39 mmol) at 0°C and was stirred at room temperature for three hours. The reaction mixture was concentrated and concentrated once more after addition of toluene (100 mL). The obtained residue was suspended in ether and filtered and dried to give the desired product as a crystalline powder (5.0 g, 95% yield). (RPHPLC: Rf 8.3 minutes, conditions as in step 3).

MS and <*>H NMR were consistent with the desired structure.

Example F

Manufacture of

Step 1

Preparation of 3-iodo-5-bromosalicylaldehyde.

To a solution of 5-bromosalicylaldehyde {20.0 g, 0.1 mol) and potassium iodide {17 g, 0.1 mol) in acetonitrile (150 mL) and water (50 mL) in a 500 mL round-bottom flask with a magnetic stirrer was added chloramine T (23 g, 0.1 mol) added. The mixture was allowed to react for one hour. The reaction mixture was partitioned between hydrochloric acid (10%, 200 mL) and ethyl acetate. The organic phase was dried (Na 2 SO 4 ), filtered and concentrated in vacuo. To the residue, hexanes were added and the reaction mixture heated to 50°C for 15 minutes. The undissolved material was removed by filtration. The filtrate was concentrated in vacuo to leave canary yellow 3-iodo-5-bromosalicylaldehyde (26 g).

MS and <X>H NMR were consistent with the desired structure.

Step 2

The above compound was prepared using essentially the same procedures as Example E, steps 2-6 where in step 2, an equivalent amount of product from step 1, 3-iodo-5-bromo-salicylaldehyde, was substituted with 3- iodo-5-chlorosalicylaldehyde.

MS and <*>H NMR were consistent with the desired structure.

Example H

Manufacture of

Step 1

Ethanol (375 mL) and deionized water (375 mL) were added to a 2 1 3-necked round bottom flask equipped with a mechanical stirrer, Claisen adapter, addition funnel, reflux condenser, and thermocouple. 1,3-Diamino-2-hydroxypropane (125.04 g, 1.39 mol) (Aldrich) was added to the reaction flask and stirred to dissolve. Carbon disulfide (84 mL, 1.39 mol) was added dropwise via addition funnel at 25-33°C over a 35 minute period to give a milky white mixture. The temperature was maintained with an ice bath. The reaction mixture was refluxed at 73.4°C for two hours to give a yellow solution. The reaction mixture was cooled with an ice bath to 25°C and concentrated HCl (84 mL) was added dropwise while maintaining the temperature at 25-26°C. The reaction mixture was refluxed for 21 hours at 78.4°C. The reaction solution was cooled to 2°C and the product collected via vacuum filtration. The white solid was washed three times with ice-cold ethanol:water (1:1) (50 mL) and dried in vacuo at 40°C to give 5-hydroxytetrahydropyrimidine-2-thione (63.75 g, 34.7% yield ) as a white solid.

MS and NMR were consistent with the desired structure.

Step 2

5-Hydroxytetrahydropyrimidine-2-thione (95 g, 0.72 mol) prepared in Step 1, absolute ethanol (570 mL), and methyl iodide (42 mL, 0.72 mol) were added to a 2-1 round bottom flask equipped with a mechanical stirrer and thermocouple. The reaction mixture was refluxed at 78°C for five hours and then cooled to room temperature. The reaction mixture was concentrated in vacuo to give a white solid (194.72 g). The white solid was triturated three times with ethyl ether (500 mL) and dried in vacuo to give 2-methylthioether-5-hydroxypyrimidine hydroiodide (188.22 g, 95.4% yield) as a white solid.

MS and H1 NMR were consistent with the desired structure.

Step 3

2-Methyl thioether-5-hydroxypyrimidine hydroiodide (150.81 g, 0.55 mol) methylene chloride (530 mL), dimethylacetamide (53 mL) and triethylamine (76.7 mL, 0.55 mol) were added to a 2 1 3-necked round bottom flask equipped with a reflux condenser, mechanical stirrer and a static atmosphere of nitrogen. The mixture was cooled with an ice bath and di-tert-butyl dicarbonate (120.12 g, 0.55 mol) was added at 4°C. The reaction mixture was heated at 42.5°C for 18 hours to give a light yellow solution. The reaction solution was transferred to a 2 L separatory funnel and washed three times with DI water (200 mL), dried with MgSO 4 , filtered and concentrated in vacuo to give Boc-2-methylthioether-5-hydroxypyrimidine (134.6 g, 99, 35% yield) as a light yellow viscous oil.

MS and H1 NMR were consistent with the desired structure.

Step 4

Boc-2-methylthioether-5-hydroxypyrimidine (50.3 g, 0.204 mol), 3-amino-5-hydroxybenzoic acid (Aust. J. Chem. (1981) 34(6), 1319-24) (25.0 g , 0.1625 mol) and 50 ml of anhydrous DMA were heated at 100°C with stirring for two days. A slurry precipitate resulted. The reaction was cooled to room temperature and the precipitate was filtered, washed with CH 3 CN, then ethyl ether and dried. This solution was slurried in H 2 O, acidified with concentrated HCl resulting in a solution. This was frozen and lyophilized to give the desired product as a white solid (14.4 g).

MS and <X>H NMR were consistent with the desired structure.

Example I

Manufacture of

Step 1

Preparation of Reformatsky reagent

A 4 L flask equipped with a cooler, temperature gauge and mechanical stirrer was charged with Zn metal (180.0 g, 2.76 mol -30

-100 mesh) and THF (1.25 L). With stirring, 1,2-dibromoethane (4.47 mL, 0.05 mol) [alternatively, TMS Cl (0.1 equiv) at room temperature for one hour can be substituted]. After inert gas flushing (3 N 2 /vacuum cycles), the suspension of zinc in THF was heated to reflux (65°C) and maintained at this temperature for one hour. The mixture was cooled to 50 °C before charging tert -butyl bromoacetate (488 g, 369 mL, 2.5 mol) via 50 mL syringe and syringe pump (set to deliver 4.1 mL/min) over 1.5 h . Reaction temperature of

50°C +/-5°C was maintained throughout the addition. The reaction mixture was allowed to stir at 50°C for one hour after the addition was complete. Subsequently, the mixture was allowed to cool to 25°C and the precipitated product was allowed to settle to the bottom. The THF mother liquor was decanted into a 2 L round bottom flask using a coarse fritted filter plate and partial vacuum transfer {20 mm Hg). This removed approx. 65% of THF from the mixture, 1-methyl-2-pyrrolidone (NMP, 800 mL) was added and stirring resumed for five minutes. The reaction mixture can be filtered to remove any remaining zinc. Analysis indicated a titer of the desired Reformatsky reagent of 1.57 M with a molar yield of 94%. Alternatively, the solid reagent can be isolated by filtration from the original reaction mixture. The cake can be washed with THF until a white solid is obtained and dried under N2 to obtain the desired product as a mono THF solvate which can be stored at 20°C (dried) for extended periods. Typical recovery rates are 85-90%.

Step 2

2A: Preparation of

Potassium carbonate (powder, oven dried at 100°C under vacuum, 8.82 g, 60 mmol) was added to a solution of 3,5-dichlorosalicyaldehyde (11.46 g, 60 mol) in DMF (40 mL) at room temperature in a clear yellow slurry. MEMCl (pure, 7.64 g, 61 mmol) was then added while maintaining the bath temperature at 20°C. The mixture was then stirred at 22°C for 6 h and MEMCl (0.3 g, 2.4 mmol) was added. The mixture was stirred for another 0.5 h and the reaction mixture was poured into cold water (200 mL) to precipitate the product. The slurry was filtered on a pressure filter and the cake was washed with water (2 x 50 mL) and was dried under N 2 /vacuum to give the product (14.94 g, 89%) as an off-white solid. <*>H NMR (CDCl 3 TMS) 3.37 (s, 3H), 3.54 to 3.56 (m, 2H), 3.91 to 3.93 (m, 2H), 5.30 (s, 2H), 7.63 (d, 1H), 7.73 (d, 1H), 7.73 (d, 1H), 10.30 (s, 1H); <13>C NMR (CDCl3 , TMS) d (ppm): 59.03, 70.11, 99.57, 126.60, 129.57, 130.81, 132.07, 135.36, 154.66, 188.30, . DSC: 48.24°C (endo 90.51 J/g);

Microanalytical: calculated for CnH12Cl204:

C:47.33%; H:4.33%; Cl:25.40%

found: C:47.15%, H:4.26%, Cl:25.16%

2B: Production of

The product from step 2A (35.0 g, 0.125 mol) was charged into a

l 1 3-necked round bottom flask equipped with a mechanical stirrer and an addition funnel followed by the addition of THF (200 mL). The solution was stirred at 22°C and (S)-phenylglyquinol (17.20 g, 0.125 mol) was then added in one portion. After 30 minutes at 22°C, MgSO 4 (20 g) was added. The mixture was stirred for one hour at 22°C, and filtered on a coarse fritted filter. The filtrate was concentrated under reduced pressure. No further purification was performed and the crude imine was used directly in the coupling reaction, step 2, C.

2C: Production of

A 1-1 3-necked round bottom flask equipped with a mechanical stirrer and an addition funnel was charged with the solid reagent prepared in Step 1 (91.3 g, 0.275 mol) and NMP (200 mL) under nitrogen. The solution was then cooled to -10°C and stirred at 350 rpm. A solution of imine (prepared in step 2B) in NMP was prepared under nitrogen and then added over 20 minutes to the above reaction mixture while temperatures were maintained at -5°C (mantle temperature -10°C). The mixture was stirred for an additional 1.5 hours at -8°C and 1 hour at -5°C after the addition was complete. After cooling to -10°C, a mixture of concentrated HCl/saturated solution of NH 4 Cl (8.1 mL/200 mL) was added over 10 minutes. MTBE (200 mL) was added and the mixture was stirred for 15 min at 23°C and 200 rpm. Stirring was stopped and the layers separated. The aqueous phase was extracted with MTBE (100 mL). The two organic phases were combined, washed successively with saturated solution of NH 4 Cl (100 ml), water (100 ml) and brine (100 ml). The solution was dried with MgSO 4 (30 g), filtered and concentrated to give an orange oil (66.3 g) (solidifies on standing) containing the desired product as a single diastereoisomer (confirmed by proton and carbon NMR). A sample was purified for analysis by recrystallization from heptane to give the product as an off-white solid.

Proton and carbon NMR and IR spectra were consistent with the desired structure. [α]<D>25 = + 8.7° (c = 1.057, MeOH). Microanalytical: calculated for C25H33C12N06: C: 58.77%, H: 6.74%, N: 2.72%, Cl: 13.78% found: C: 58.22%, H: 6.54%, N: 2 .70%, Cl: 13.66%

Step 3

Manufacture of

3A:

A solution of the crude ether prepared in step 2 [17.40 g, 0.033 mol (theory)] and EtOH (250 mL) was charged to a 1 1 3-necked jacketed reactor. The solution was cooled to 0°C and Pb(OAc) 4 (14.63 g, 0.033 mol) was added in one portion. After two hours, a 15% solution of NaOH (30 mL) was added and ethanol was removed under reduced pressure. Another portion of 15% NaOH (100 mL) was added and the mixture extracted with MTBE (2 x 100 mL), washed with H 2 O (2 x 100 mL) and brine (50 mL), dried with Na 2 SO 4 , filtered on celite and concentrated under reduced pressure to give an orange oil (12.46 g). The oil was homogeneous by thin-layer chromatography (tic) and was used without further purification.

3B:

The oil from 3A was diluted with EtOH (30 mL) and paratoluenesulfonic acid (1.3 equiv, 0.043 mol, 8.18 g) was added. The solution was heated to reflux for 8 hours, cooled to ambient temperature and concentrated under reduced pressure. The residue was treated with THF (20 mL) and heated to reflux to form a solution. The solution was cooled to room temperature and the compound crystallized. Heptane (30 mL) and THF (10 mL) were added to form a liquid slurry which was filtered. The cake was washed with THF/heptane (40 mL, 1/1) and vacuum dried for two hours in a pressure filter under nitrogen to give a white solid (7.40 g).

Proton and carbon NMR and IR spectra were consistent with the desired product being essentially a single enantio-

more.

Microanalytical: calculated for Ci8H2iCl2N06S, 0.25 C4H80:

C: 48.73%, H: 4.95%, N: 2.99%, Cl: 15.14% found: C: 48.91%, H: 4.95%, N: 2.90%, Cl: 14.95%

Step 4

Manufacture of:

To a 500 mL round bottom flask equipped with a magnetic stir bar and nitrogen bubbles were added the free base of the product formed in step 3 (21.7 g, 0.065 mol), N-t-Boc-glycine N-hydroxysuccinimide ester (17.7 g, 0.065 mol) and DMF (200 mL). The reaction mixture was stirred under nitrogen at room temperature for 3.25 hours and a pale orange solution formed. The reaction mixture was poured into ice-cold ethyl acetate (1.2 L). The organic solution was washed with 1M HCl (250 mL) and brine (500 mL), dried (MgSO 4 ) and concentrated under vacuum to near dryness to afford an oil which was then dried at 50°C to afford the product as a colorless oil (28.12 g 99%). Rim crystals were prepared from ethyl acetate/hexanes. The product (about 28 g) was dissolved in ethyl acetate (35 ml) and hexanes (125 ml). The solution was seeded with the seed crystals and a precipitate formed. The solids were filtered and dried overnight under vacuum at 55°C to give a colorless solid (27.0 g, 95%).

MS and <X>H NMR were consistent with the desired structure.

Step 5

Manufacture of

The Boe-protected glycinamide prepared in step 4 (27.0 g, 0.062 mol) was dried overnight over P 2 O 5 and NaOH pellets. The solid was dissolved in dioxane (40 ml) and the solution cooled to 0°C. An equivalent volume of 4N HCl/dioxane (0.062 mol) was added and the reaction was cooled for two hours. At this point the conversion was 80% at RPHPLC. The reaction mixture was allowed to warm to room temperature for 4 hours. The reaction mixture was concentrated at 40°C to a foam which was triturated with ether (200 mL). The white solid that formed was filtered and dried over P 2 O 5 to give the desired glycine beta-amino acid ethyl ester compound as an HCl salt (20.4 g, 88.5% isolated yield).

MS and <X>H NMR were consistent with the desired structure.

Example J

Manufacture of

Step 1 Preparation of

MEM protected 3-bromo-5-chlorosalicylaldehyde (129.42 g, 0.4 mol), prepared according to the procedure of Example I, step 2A. An equivalent amount of 3-bromo-5-chlorosalicylaldehyde was substituted for 3,5-chlorosalicylaldehyde which was charged to a 2 1 3-necked round bottom flask equipped with a mechanical stirrer, followed by the addition of THF (640 mL) and (S)- phenylglyquinol (54.86 g, 0.4 mol). After 30 minutes at 22°C, MgSO 4 (80 g) was added. The mixture was stirred for two hours at 22°C, and filtered on a coarse fritted filter. The filtrate was concentrated under reduced pressure to give a pale yellow oil (180.0 g) containing the desired imine. No further purification was performed and the crude product was used directly in the coupling reaction, step 2.

Microanalytical: calculated for Ci9H2iBrClN04:

C:51.54%, H:4.78%, N:3.16%, Br:18.04%, Cl:8.00% found: C:50.22%, H:4.94%, N:2.93%, Br:17.15%, Cl:7.56%

Step 2

Manufacture of

In a 5 L 3-necked round bottom flask equipped with a mechanical stirrer, the reagent from Example I, Step 1 (332.0 g, 0.8 mol) was taken up in NMP (660 mL) under nitrogen. The solution was then cooled to -10°C. A solution of the imine from step 1 in NMP (320 mL) was prepared under nitrogen and then added over 30 minutes to the above reaction mixture while maintaining the temperature at -5°C. The mixture was stirred for an additional hour at -8°C and -5°C for two hours after the addition was complete and then cooled to -10°C. A mixture of concentrated HCl/saturated solution of NH 4 Cl (30 mL/720 mL) was added over 10 minutes. MTBE (760 mL) was added and the mixture was stirred for 30 minutes at 23°C. Stirring was stopped and the layers separated. The aqueous phase was extracted with MTBE (320 mL) the organic phases were combined, washed successively with saturated aqueous NH 4 Cl (320 mL), DI water (320 mL) and brine (320 mL). The solution was dried with MgSO 4 (60 g), filtered and concentrated to give a yellow oil (221.0 g) containing the desired product as a single diastereoisomer as determined by proton NMR.

DSC: 211.80°C (endo. 72.56 J/g), 228.34°C (98.23 J/g);

Microanalytical: calculated for C25H33BrClN06:

C: 53.72%; H:5.95%; N:2.50%; Br:14.29%; Cl:6.33% found: C:52.11%; H:6.09%; N:2.34%; Br: 12.84%; Cl:6.33%

Step 3

Manufacture of

A solution of the crude ester, prepared in step 2 (~111 g), in ethanol (1500 mL) was charged under an argon atmosphere into a 3 1 3-necked round bottom flask equipped with a mechanical stirrer. The reaction mixture was cooled to 0°C and lead tetraacetate (88.67 g, 0.2 mol) was added in one portion. The reaction mixture was stirred for 3 hours at 0°C and then 15% aqueous NaOH (150 mL) was added to the reaction mixture below 5°C. Methanol was removed under reduced pressure on a rotary evaporator. An additional 150 mL of 15% aqueous NaOH was added and the reaction mixture was extracted with ethyl acetate (3 x 300 mL) and washed with DI water (2 x 100 mL) and brine (2 x 100 mL) and dried over aqueous MgSO4 (30 g ). It was then filtered over celite and concentrated under reduced pressure to give the desired product (103 g) as a red oil.

Step 4

Manufacture of

The above compound was prepared according to the procedure used for Example I, Step 4 and Step 5 by substituting an equivalent amount of the product from Step 3 into Example I, Step 4.

MS and <*>H NMR were consistent with the desired structure.

Example K

Alternative preparation<g> of the compound in Example J

Step 1

Manufacture of

To the product from Example B, Step 3, (50.0 g, 139.2 mmol) and NaHCO 3 (33.5 g, 398.3 mmol) was added CH 2 Cl 2 (500 mL) and water (335 mL). The mixture was stirred at room temperature for 10 minutes. A solution of benzyl chloroformate (38.0 g, 222.8 mmol) in CH 2 Cl 2 (380 mL) was added over 20 min with rapid stirring. After 50 minutes, the reaction mixture was emptied into a separatory funnel and the organic phase collected. The aqueous phase was washed with CH 2 Cl 2 (170 mL). The combined organic phases were dried (MgSO 4 ) and concentrated in vacuo. The resulting gummy solid was triturated with hexane and collected by filtration. The leather-colored solid was dried in vacuo to give the desired racemic product (61.2 g, 96% yield). This material was subjected to reverse phase HPLC using a chiral column to give each pure enantiomer. The column used was a Whelk-0 (R,R), 10 µm particle size using a 90:10 heptane:ethanol mobile phase. Optical purity was determined to be greater than 98% using analytical HPLC using similar column and solvent conditions.

<X>H NMR was consistent with the desired structure.

Step 2

To a solution of the compounds obtained in step 1 (48.5 g, 106.2 mmol) in CH 2 Cl 2 (450 mL) was added trimethylsilyl iodide via cannula. The orange solution was stirred at room temperature for one hour. Methanol (20.6 mL, 509.7 mmol) was added dropwise and the solution stirred for 5 minutes. The reaction solution was concentrated in vacuo to give an orange oil. The residue was dissolved in methyl t-butyl ether (500 mL) and extracted with 1N HCl (318 mL) and water (1 x 200 mL, 1 x 100 mL). The aqueous extracts were backwashed with MTBE (100 mL). To the aqueous solution was added solid NaHCO 3 (40.1 g, 478 mmol) in small portions. The basic aqueous mixture was extracted with MTBE (lx 11, 2 x 200 mL). The combined organic solution was washed with brine and concentrated in vacuo to give the desired product (23.3 g, 68% yield).

<X>H NMR was consistent with the proposed structure.

Step 3

Manufacture of

To a solution of the product from step 2 (23.3 g, 72.1 mmol) in DMF (200 mL) was added N-t-Boc-glycine N-hydroxysuccinimide ester (17.9 g, 65.9 mmol). The reaction mixture was stirred at room temperature for 20 hours. The mixture was poured into

ethyl acetate {1.2 L) and washed with 1M HCl (2 x 250 mL), saturated aqueous NaHCO 3 solution (2 x 250 mL) and brine (2 x 250 mL). The solution was dried (MgSO 4 ) and concentrated to give the desired product {32.0 g, 100% yield).

Anal. calculated for Ci8H24BrClN206:

C, 45.06; H, 5.04; N, 5.84.

Found: C, 45.17; H, 5.14; N, 6,12.

<*>H NMR was consistent with the desired structure.

Step 4

To a solution of the product from Step 3 (31.9 g, 66.5 mmol), in absolute ethanol (205 mL) was added an ethanolic HCl solution (111 mL of a 3M solution, 332.4 mmol). The reaction solution was heated at 58°C for 30 minutes. The solution was cooled and concentrated in vacuo, the residue was dissolved in ethyl acetate (250 ml) and stirred at 0°C for two hours. A white precipitate was collected by filtration and washed with cold ethyl acetate. The solution was dried in vacuo to give the desired product (23.5 g, 85% yield).

Anal. calculated for C13Hi6BrClN204 + 1.0 HCl:

C, 37.53; H, 4.12; N, 6.73.

Found: C, 37.29; H, 4.06; N, 6.68.

<X>H NMR was consistent with the desired structure.

Example L

Manufacture of

Step 1 Preparation of

Potassium carbonate (powder, oven dried at 100°C under vacuum, 22.1 g, 0.16 mol) was added to a solution of 3-chloro-5-bromosalicylaldehyde (35.0 g, 0.15 mol) in DMF (175 ml) at room temperature to give a clear yellow slurry. MEMCl (pure, 25.0 g, 0.2 mol) was then added while maintaining the bath temperature at 20°C. The mixture was then stirred at 22°C for 6 hours and was poured into DI water (1200 mL) to precipitate the product. The slurry was filtered on a pressure filter and the cake was washed with DI water (2 x 400 mL) and then dried under N 2 /vacuum to give the product (46.0 g, 95% yield) as an off-white solid. <X>H NMR (CDCl 3 , TMS) 3.35 (s, 3H), 3.54 to 3.56 (m, 2H), 3.91 to 3.93 (m, 2H), 5.30 (s , 2H), 7.77 (d, 1H), 7.85 (d, 1H), 10.30 (s, 1H); 13 C NMR (CDCl 3 , TMS)(ppm): 59.05, 70.11, 71.49, 99.50, 117.93, 129.69, 129.78, 132.37, 138.14, 155, 12,188.22. DSC: 48.24°C (endo 90.51 J/g);

Microanalytical : calculated for CuHuBrClNO*:

C: 40.82%; H:3.74%; Cl:10.95% Br:24.69%; found: C:40.64%; H:3.48%; Cl:10.99% Br:24.67%. Step 2 Preparation of

The product from step 1 (32.35 g, 0.1 mol) was charged to a 500 mL 3-necked round bottom flask equipped with a mechanical stirrer, followed by the addition of THF (190 mL) and (S)-phenylglyquinol (13.71 g, 0.1 mol). After 30 minutes at 22°C, MgSO4 (20 g) was added. The mixture was stirred for one hour at 22°C and filtered on a coarse fritted filter. The filtrate was concentrated under reduced pressure to give a pale yellow oil (48.0 g) containing the desired imine. No further purification was carried out and the crude product was used directly in the next reaction step.

Microanalytical: calculated for Ci9H2iBrClN04:

C: 51.54%; H:4.78%; N:3.16%; Br:18.04%; Cl:8.00% found: C:51.52%; H:5.02%; N:2.82%; Br:16.31%; Cl: 7.61%.

Step 3

Manufacture of

In a 5 L 3 neck round bottom flask equipped with a mechanical stirrer, reagent from Example I, Step 1, (332 g, 0.8 mol) was taken up in NMP (660 mL) under nitrogen. The solution was then cooled to -10 °C. A solution of the imine prepared in step 2, in NMP (320 mL) was prepared under nitrogen and then added over 30 minutes to the above reaction mixture while maintaining the temperature at -5 °C. The mixture was stirred for an additional 1 hour after the addition was complete and cooled to -10 °C. A mixture of concentrated HCl/saturated solution of NH 4 Cl (30 mL/720 mL) was added over 10 minutes. MTBE (760 ml) was added and the mixture was stirred for 1 hour at 23°C. Stirring was stopped and the phases were separated. The aqueous phase was extracted with MTBE (320 mL). The two organic phases were combined, washed successively with a saturated solution of NH 4 Cl (320 mL), DI water (320 mL) and brine (320 mL). The solution was dried with MgSO 4 {60 g), filtered and concentrated to give a yellow oil (228 g) containing the desired product as a single diastereoisomer. DSC: 227.54°C (endo.61.63 J/g);

Microanalytical: calculated for C25H33BrClN06:

C: 53.72%; H:5.95%; N:2.50%; Br:14.29%; Cl:6.33% found: C:53.80%; H:6.45%; N:2.23%; Br: 12.85%; Cl: 6.12%.

Step 4

Manufacture of

A solution of crude ester, prepared in step 3 (~111

g), in ethanol (1500 mL) was filled under a nitrogen atmosphere into a 3 1 3-necked round-bottom flask equipped with a mechanical

stirring. The reaction mixture was cooled to 0°C and lead tetraacetate (88.67 g, 0.2 mol) was added in one portion. The reaction mixture was stirred for 3 hours at 0°C and then 15%

aqueous NaOH (150 mL) added to the reaction mixture below 5°C. The ethanol was removed under reduced pressure on a rotary evaporator. An additional 600 mL of 15% aqueous NaOH was added and the reaction mixture was extracted with ethyl acetate (2 x 300 mL), MTBE (2 x 200 mL) and ethyl acetate (2 x 200 mL). The organic phases were combined and washed with DI water (2 x 200 ml) and brine (2 x 100 ml) and dried over anhydrous MgSO 4 (30 g). The solution was then filtered over celite and concentrated under reduced pressure to give the product as an orange oil (96 g) which was used in the next step without further purification.

DSC: 233.60°C (endo. 67.85 µg);

Microanalytical: calculated for C24H29BrClN05:

C:54.71%; H:5.54%; N:2.65%; Br:15.16%; Cl:6.72% found: C:52.12%; H:5.40%; N:2.47%; Br:14.77%; Cl: 6.48%.

Step 5

Manufacture of

The crude product from step 4 (~94 g) was taken up in absolute ethanol (180 mL) and para-toluenesulfonic acid monohydrate (50.0 g, 0.26 mol) was added. The reaction mixture was then heated to reflux for 8 hours after which the solvent was removed under reduced pressure. The residual solid was taken up in THF (100 mL) and the THF was then stripped off under reduced pressure. The residue was dissolved in ethyl acetate (500 mL) and cooled to -5°C. The solid was filtered and washed with heptane (2 x 50 mL) to give a white solid. The solid was then air dried to give the desired product as a white solid (38 g) as a single isomer. H1 NMR (DMSO, TMS)(ppm) 1.12 (t,3H), 2.29 (s,3H) , 3.0 (m,2H), 4.05 (q,2H),

4.88 (t,lH), 7.11 (d,2H), 7.48 (d,2H), 7.55 (D,1H), 7.68 (lH,d), 8.35 (br .s,3); <13>C NMR (DMSO, TMS) (ppm) : 13.82, 20.75, 37.13, 45.59, 60.59, 110.63, 122.47, 125.44, 127.87, 128.06, 129.51, 131.95, 137.77, 145.33, 150.14, 168.98;

DSC: 69.86°C (end.,406.5 J/g), 165.72°C (end. 62.27 J/g), 211.24°C (ex.20.56 j/g) [a] D25 = + 4.2° (c=0.960, MeOH) ; IR (MIR) {cm-D 2922, 1726, 1621, 1591, 1494, 1471, 1413, 1376, 1324, 1286, 1237, 1207;

Microanalytical: calculated for CiBH21BrClN06S:

C:43.69%; H:4.27%; N:2.83%; Br:16.15%; Cl:7.16%; S:6.48% found: C:43.40%; H:4.27%; N:2.73%; Br.-16.40%; Cl:7.20%; S:6, 54%

Step 6

Manufacture of

The above compound was prepared according to the procedures set forth in Example I, Step 4 and Step 5 where an equivalent amount of the intermediate prepared in Step 5 as the free base is substituted in Example I, Step 4.

MS and <X>H NMR were consistent with the desired structure.

Example M

Manufacture of

Step 1

Preparation of 3-iodo-5-chlorosalicylaldehyde.

N-iodosuccinimide (144.0 g, 0.641 mol) was added to a solution of 5-chlorosalicylaldehyde (100 g, 0.638 mol) in dimethylformamide (400 mL). The reaction mixture was stirred for two days at room temperature. Additional N-iodosuccinimide (20.0 g) was added and stirring continued for another two days. The reaction mixture was diluted with ethyl acetate (1 L), washed with hydrochloric acid (300 mL, 0.1N), water (300 mL), sodium thiosulfate (5%, 300 mL), brine (300 mL), dried (MgSO 4 ), and concentrated to dryness to give the desired aldehyde as a pale yellow solid (162 g, 90% yield).

MS and <X>H NMR were consistent with the desired structure.

Step 2

Preparation of 2-O-(MEM)-3-iodo-5-chlorosalicylaldehyde.

Potassium carbonate (41.4 g, 0.30 mol) was added to a solution of 3-iodo-5-chlorosalicylaldehyde (84.74 g, 0.30 mol) in DMF (200 mL) at 20°C. This resulted in a yellow slurry and MEM-C1 (38.2 g, 0.305 moi) was added while maintaining the reaction temperature. After two hours, additional MEMCl (1.5 g) was added. After stirring for one hour, the reaction mixture was poured into an ice-water mixture and stirred. The precipitate that formed was filtered and dried in vacuo to give the desired protected aldehyde. Yield: 95 g (85%).

MS and <X>H NMR were consistent with the desired structure.

Step 3

Manufacture of

(S)-Phenylglycinol {15.37 g, 0.112 mol) was added to a solution of 2-O-(MEM)-3-iodo-5-chlorosalicylaldehyde (41.5 g, 0.112 mol) in THF (200 mL) at room temperature. After one hour of stirring, MgSO 4 (16 g) was added and stirring was continued for two hours. The reaction mixture was filtered and the filtrate was concentrated and dried in vacuo for two hours to give the desired intermediate imine. A 2-necked round bottom flask was charged with the Reformatsky reagent from Example I, Step 1, (81.1 g, 0.2464 mol), and stirred at -10°C. A solution of the imine in N-methylpyrrolidone (100 mL) was slowly added while maintaining the temperature at -10°C. The mixture was maintained at this temperature for two hours and for one hour at -5°C. After cooling the reaction mixture to -10°C, a solution of concentrated HCl in saturated ammonium chloride (16 mL/200 mL) was added. Ethyl ether (500 ml) was added and stirred for two hours at room temperature. The ether phase was separated and the aqueous phase was further extracted with ether (300 nil). The combined ether layers were washed with saturated ammonium chloride (200 mL), water (200 mL), brine (200 mL), dried (MgSO 4 ) and concentrated to give an oil (61.0 g, 90% yield). H1 NMR indicated that the desired structure was essentially a diastereomer and MS was consistent with the desired structure. Step 4 Preparation of

A solution of the crude ester prepared in step 3 (48.85 g, 80.61 mol) was dissolved in ethanol (500 mL) and cooled to 0°C. Lead (35.71 g, 80.61 mmol) was added. After 3 hours, 15% solution of NaOH (73 mL) was added to the reaction mixture. Most of the ethanol was removed under reduced pressure. To the residue a 15% solution of NaOH (200 ml) was added and this was then extracted with ether (400 ml). The ether phase was washed with water (100 mL), brine (100 mL), dried and concentrated to give an orange oil. The oil was dissolved in ethanol (100 ml) and para-toluenesulfonic acid (19.9 g) was added. The solution was heated at reflux for 8 hours and concentrated under reduced pressure. The residue was diluted with THF (60 mL) and heated at reflux and cooled. The precipitate was filtered, washed with hexane (300 mL, 1:1) and dried to give the desired product.

MS and <*>H NMR were consistent with the desired structure.

Step 5

S-Ethyl 3-(N-BOC-gly)-amino-3-(S)-(5-chloro-2-hydroxy-3-iodo)phenylpropionate

To a mixture of BOC-gly-OSu (9.4 g, 34.51 mmol), ethyl 3-(S)-amino-3-(5-chloro-2-hydroxy-3-iodo)propionate PTSA salt (17.0 g, 31.38 mol) in DMF (200 mL) triethylene (4.8 mL) was added. The reaction mixture was stirred for 18 hours at room temperature. DMS was removed in vacuo and the residue was partitioned between ethyl acetate (600 mL) and dilute hydrochloric acid (100 mL). The organic phase was washed with sodium bicarbonate (200 mL), brine (200 mL), dried (MgSO 4 ) and concentrated to give the desired product as a solid.

(14.2 g, 86% yield).

MS and <*>H NMR were consistent with the desired structure.

Step 6

S-ethyl 3-(N-gly)-amino-3-(5-chloro-2-hydroxy-3-iodo)phenylpropionate hydrochloride.

Dioxane/HCl (4N, 70 mL) was added to ethyl 3-(S)-(N-BOC-gly)-amino-3-(5-chloro-2-hydroxy-3-iodo)phenylpropionate (37.20 g , 70.62 mmol) at 0°C and stirred at room temperature for three hours. The reaction mixture was concentrated and concentrated once more after addition of toluene (100 mL). The residue obtained was suspended in ether, filtered and dried to give the desired product as a crystalline powder (32.0 g, 98% yield).

MS and <X>R NMR were consistent with the desired structure.

Example N

Manufacture of

Step 1 Preparation of The above compound was prepared according to Example I, Step 2A, substituting an equivalent amount of 2-hydroxy-3,5-dibromobenzaldehyde for 3,5-dichlorosalicylaldehyde. Yield: 88%; pale yellow solid; m.p. 46-47°C; Rf = 0.6 (EtOAc/hexane 1:1 v/v); H1-NMR (CDCl 3 ) d 3.37 (s, 3H), 3.56 (m, 2H), 3.92 (m, 2H), 5.29 (s, 2H), 7.91 (d, 1H , J = 2.4 Hz), 7.94 {d, 1H, J = 2.4 Hz), 10.27 <S, 1H); FAB-MS m/z 367 (M<*>)

MS and <X>H NMR were consistent with the desired structure.

Step 2

The above compound was prepared using the procedure of Example I, Step 2B and Step 2C, substituting an equivalent amount of the compound in Step 1 into Example I, Step 2B.

Yield 90%; yellow solid; m.p. 57-59°C; Rf = 0.46 (EtOAc/hexane 1:1 v/v); <*>H-NMR (CDCl 3 ) d 1.45 (s, 9H); 2.1 {br, 1H exchangeable), 2.51 {d, 1H, Jj. = 9.9 Hz, J2 = 15.3 Hz), 2.66

(d, 1H, Ji = 4.2 Hz, J2 = 15.3 Hz), 3.02 {br, 1H, exchangeable), 3.39 {s, 3H), 3.58-3.62 (m, 4H), 3.81 (m, 1H), 3.93 (m, 2H), 4.63 {dd, 1H, J = 4.2 Hz), 5.15 (s, 2H), 7.17- 7.25 (m, 6H), 7.49 (d, 1H); FAB-MS m/z 602 ((M + H)

MS and <X>H NMR were consistent with the desired structure.

Step 3

Manufacture of

The above compound (p-toluenesulfonate salt) was prepared according to Example I, Step 3 by substituting an equivalent amount of the product prepared in Step 2 into Example I, Step 3A. Yield 62%; white solid; <*>H-NMR (DMSO-de) d 1.09 (t, 3H, J = 7.2 Hz) , 2.27 (s, 3H) , 2.97 (dd, 2H, Ji = 3.0 Hz, J2 = 7.2 Hz), 4.02 {q, 2H, J = 7.2 Hz), 4.87 (t, 1H, J = 7.2 Hz), 7.08 (d, 2H, J = 4.8 Hz), 7.45 {m, 3H), 7.57 {d, 1H, J = 2.4 Hz), 8.2 (br, 3H); FAB-MS m/z 365 (M + H)

MS and <X>H NMR were consistent with the desired structure.

Step 4

Manufacture of

The above compound was prepared using the procedure of Example I, Step 4 substituting the compound prepared in Step 3. The resulting BOC-protected intermediate was converted to the desired compound using the procedure of Example I, Step 5.

MS and ^NMR were consistent with the desired structure.

Example P

Manufacture of

The above compound was prepared according to the procedure of Example I by substituting an equivalent amount of 3-iodo-5-bromosalicylaldehyde prepared in Example F, Step 1 with 3,5-dichlorosalicylaldehyde in Example I, Step 2A.

Example 1

( ±) 3- bromo- 5- chloro- 2- hydroxy- p- r\ 2 - f fr3- hydroxy- 5 r( 1. 4, 5, 6-tetrahydro- 5- hydroxypyrimidin- 2- yl) amino] phenvl ] carbonyl] amino]- acetyl] amino] benzenepropanoic acid. trifluoroacetate salt Preparation of

To the product of Example H (0.4 g, 0.0014 mol), the product of Example B (0.58 g, 0.0014 mol), triethylamine (0.142 g, 0.0014 mol), DMAP (17 mg) , and anhydrous DMA (4 mL) added EDC1 (0.268 g, 0.0014 mol) at ice bath temperature. The reaction was stirred overnight at room temperature. The resulting ester intermediate was isolated by reverse phase preparative HPLC. To this ester in H 2 O (10 mL) and CH 3 CN

(5 mL) LiOH (580 mg, 0.0138 mol) was added. After stirring at room temperature for one hour, the pH was lowered to 2 with TFA and the product was purified by reverse phase preparative HPLC to give (after lyophilization) the desired product as a white solid (230 mg).

MS and <*>H NMR were consistent with the desired structure.

Example 2

Manufacture of

The above compound was prepared according to the methodology of Example 1, by exchanging an equivalent amount of the product from Example A with the product from Example B. The yield after lyophilization was 320 mg as a white solid.

MS and <X>H NMR were consistent with the desired structure.

Example 3

Manufacture of

The above compound was prepared according to the methodology of Example 1, by exchanging an equivalent amount of the product from Example F with the product from Example B. The yield (after lyophilization) was 180 mg as a white solid.

MS and <X>H NMR were consistent with the desired structure.

Example 4

Manufacture of

The above compound was prepared according to the methodology of Example 1, by exchanging an equivalent amount of the product of Example D with the product of Example B. The yield (after lyophilization) was 180 mg as a white solid.

MS and <X>H NMR were consistent with the desired structure.

Example 5

Manufacture of

The above compound was prepared according to the methodology of Example 1, by exchanging an equivalent amount of the product from Example E with the product from Example B. The yield (after lyophilization) was 250 mg as a white solid.

MS and H1 NMR were consistent with the desired structure.

Example 6

Manufacture of

The above compound was prepared according to the methodology of Example 1, by exchanging an equivalent amount of the product of Example C with the product of Example B. The yield (after lyophilization) was 220 mg as a white solid.

MS and <X>H NMR were consistent with the desired structure.

Example 7

Manufacture of

To the product of Example H (7.8 g, 0.027 mol) dissolved in anhydrous DMA (50 mL) in a flame-dried flask under N 2 and at ice bath temperature, isobutyl chloroformate (3.7 g, 0.027 mol) was slowly added followed by N-methylmorpholine ( 2.73 g, 0.027 mol). The solution was stirred at ice bath temperature for 15 minutes. To the reaction mixture was then added the product from Example 1 (10.0 g, 0.024 mol) at ice bath temperature followed by N-methylmorpholine (2.43 g, 0.024 mol). The reaction was then stirred at room temperature overnight. The resulting ester intermediate was isolated by reverse phase preparative HPLC. To esters in H 2 O (60 mL) and CH 3 CN (30 mL) was added LiOH (10 g, 0.238 mol). The reaction mixture was stirred at room temperature for one hour. The pH was then lowered to 2 with TFA. The product was purified by reverse phase preparative HPLC to give (after lyophilization) the desired product as a white solid (9.7 g).

MS and <X>H NMR were consistent with the desired structure.

Example 8

( S ) 3, 5- dichloro- 2- hydroxy-( 3- Tf2- [ [ r3- hydroxy- 5- [( 1. 4. 5. 6-tetrahydro- 5- hydoxypyrimidin- 2- vi) amino] phenyl] carbonyl] amino] acetyl] amino]- benzenepropanoic acid, monohydrochloride monohydrate

Manufacture of

Step A

To the product from Example H (9.92 g, 0.0345 mol) dissolved in anhydrous DME (200 mL) is added N-methylmorpholine (4.0 mL, 0.0362 mol). The reaction mixture was cooled to -5°C (salt-ice bath). Isobutylchloroformate, IBCF (4.48 mL, 4.713 g, 0.0345 mol) was added over one minute and the reaction mixture stirred at ice bath temperature for 12 minutes. To the reaction mixture was then added the product from Example I (11.15 g, 0.030 mol) at ice bath temperature followed by N-methylmorpholine (4.0 ml, 0.0362 mol). The reaction mixture was allowed to warm to room temperature and go to completeness, then concentrated under vacuum at 50°C to give a dark residue. The residue was dissolved in acetonitrile: H 2 O (approx. 50 ml). The pH was made acidic by adding small amounts of TFA. The residue was placed on a 10 x 500 cm C-18 (50 µm particle size) column and esters of the desired product isolated. (Solvent program: 100% H 2 O + 0.05% TFA to 30:70 H 2 O + 0.05% TFA: acetonitrile + 0.05% TFA over 1 hour at 100 ml/min: the solvent program was initiated after solvent -front is eluted) Preparative RPHPLC purification resulted in a white solid (10.5 g) after lyophilization (50%).

MS and <*>H NMR were consistent with the desired structure.

Step B

The product produced in step A {approx. 11 g) was dissolved in dioxane water and the pH of the solution adjusted to approx. 11.5 (pH meter) by adding 2.5 N NaOH. The reaction mixture was stirred at room temperature. Periodically, the pH was re-adjusted to

>11 by further addition of base. After 2-3 hours, the conversion of ester to acid was assumed to be complete by RPHPLC. The pH of the reaction mixture was adjusted to about 6 and a viscous oil precipitated from the solution. The oil was isolated by decantation and washed with hot water (200 ml). The resulting aqueous mixture was allowed to cool and the solid was collected by filtration to give the above compound (2.6 g after lyophilization from HCl solution). The residue, which was a dark viscous oil, was treated with hot water to give on cooling a leather colored powder (4.12 g after lyophilization from HCl solution).

MS and H1 NMR were consistent with the desired structure.

Example 9

(S) 3- bromo- 5- chloro- 2- hydroxy- p-[\ 2 -\ f[ 3- hydroxys- 5[( 1, 4, 5, 6-tetrahydro- 5- hydroxypyrimidine- 2- vi) amino ] phenyl] carbonyl] amino] acetyl] amino]- benzenepropanoic acid, trifluoroacetate salt Step 1

Manufacture of

To a suspension of the product of Example J (1.0 g, 2.4 mmol), the product of Example H (0.75 g, 2.6 mmol) and 4-diethylaminopyridine (40 mg) in N,N-dimethylacetamide (10 mL) added triethylamine (0.24 g, 2.4 mmol). The mixture was stirred at room temperature for 15 minutes and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.60 g, 3.1 mmol) was added. The reaction mixture was stirred at room temperature overnight. The mixture was concentrated in vacuo and purified by reverse phase HPLC (elution gradient 90:10 H 2 O/TFA:MeCN, residence time 22 minutes) to give the desired product, (1.6 g, 52% yield).

H1 NMR was consistent with the desired structure.

Step 2

To a solution of esters prepared in step 1 (800 mg, 1.2 mmol) in a 1:4 MeCN:H 2 O solution (7 mL) was added lithium hydroxide (148 mg, 6.2 mmol). The reaction mixture was stirred at room temperature for two hours. TFA (0.71 mL, 9.2 mmol) was added and the mixture purified by reverse phase HPLC (elution gradient 95:5 H 2 O/TFA:MeCN, residence time 20 min) to give the desired product (860 mg, 83% yield) .

Analytical calculated for C22H23BrClN507 + 1.7 TFA:

C, 39.18; H: 3.20; N, 8.99

found C, 39.11; H: 3.17; N, 9.07

MS and H1 NMR were consistent with the desired structure.

Step 3

Preparation of the hydrochloride salt

The product from step 2 was dissolved in a suitable solvent (acetonitrile:water) and the solution was slowly passed through a Bio-Rad AG2-8X (chloride form, 200-400 mesh, >5 equivalents) ion exchange column. Lyophilization gives the desired product as an HCl salt.

Example 10

Manufacture of

The above compound was prepared using the procedure of Example 8 by exchanging the product of Example N for the product of Example I in Example 8, Step A. The product was isolated by preparative RPHPLC and lyophilized to give the desired product as a TFA salt.

MS and <*>H NMR were consistent with the desired structure.

Example 11

Manufacture of

The above compound was prepared using essentially the same procedures as Example 8 and by exchanging the product of Example M with the product of Example I in Example 8, Step A. The product was isolated by preparative RPHPLC and lyophilized to give the desired product as a TFA salt.

MS and <1>H NMR were consistent with the desired structure.

Example 12

Manufacture of

The above compound was prepared using the procedures of Example 8 and by exchanging the product of Example P with the product of Example I in Example 8, Step A. The product is isolated by preparative RPHPLC and lyophilized to give the desired product as a TFA salt .

Example 13

Manufacture of

Preparation of 2-O-(MEM)-3,5-diiodosalicylaldehyde

Potassium carbonate (18.5 g, 0.134 mol) was added to a solution of 3,5-diiodosalicylaldehyde (50.0 g, 0.134 mol) in DMF (150 mL) at 20°C. This resulted in a yellow slurry and MEMCl (15.8 mL, 0.134 mol) was added while maintaining the reaction temperature. After two hours, additional MEM-C1 (1.5 g) was added. After stirring for a further hour, the reaction mixture was poured into ice water and stirred. The precipitate that formed was filtered, and dried in vacuo to give the desired protected aldehyde (61 g, 99% yield).

H1 NMR was consistent with the desired structure.

Step 2

Manufacture of

(S)-Phenyl glycinol (17.9 g, 0.13 mol) was added to a solution of 2-O-MEM-3,5-diiodosalicylaldehyde (41.5 g, 0.112 mol) in THF (150 mL) at room temperature. After one hour of stirring, MgSO 4 (20.7 g) was added and stirring was continued for two hours. The reaction mixture was filtered and the filtrate was concentrated and dried in vacuo for two hours. A 2-necked round bottom flask was charged with the Refomatsky reagent (96 g, 0.289 mol) and N-methylpyrrilidone (250 mL) and stirred at -10°C. A solution of the imine in N-methylpyrrilidone (100 mL) was slowly added

maintaining the temperature at -10°C. The mixture was maintained at this temperature for two hours and one hour at -5°C. After cooling the reaction mixture to -10°C, a solution of concentrated HCl in saturated ammonium chloride (16 mL/200 mL) was added. Ethyl ether (500 mL) was added and the mixture was stirred for two hours at room temperature. The ether phase was separated and the aqueous phase further extracted with ether (300 ml). The combined ether layers were washed with saturated ammonium chloride (200 mL), water (200 mL), brine (200 mL), dried (MgSO 4 ) and concentrated to give an oil (90.0 g, 99% yield).

NMR indicated the desired product and a diastereomer.

Step 3

Manufacture of

A solution of the crude ester from Step 2 (14.0 g, 20.1 mmol) was dissolved in ethanol (100 mL) and cooled to 0 °C. Lead tetraacetate (9.20 g, 20.75 mmol) was added in one portion. After three hours, 15% solution of NaOH (73 mL) was added to the reaction mixture. Most of the ethanol was removed under reduced pressure. The residue was added to a 15% solution of NaOH (200 mL) which was extracted with ether (400 mL). The ether phase was washed with water (100 mL), brine (100 mL), dried and concentrated to give an orange oil. This was dissolved in ethanol (100 ml) and para-toluenesulfonic acid (6.08 g) was added. The solution was heated at reflux for 8 hours and concentrated under reduced pressure. The residue was diluted with THF (60 mL), heated at reflux and cooled. No precipitate was formed during storage. The reaction mixture was concentrated and purified by preparative HPLC to give the amino acid as its PTSA salt. The solid obtained was dissolved in ethanol and saturated with HCl gas. The reaction mixture was heated at reflux for 6 hours. The reaction mixture was concentrated to give the PTSA salt of the desired amino acid (12.47 g).

Step 4

Preparation of ethyl 3-(N-BOC-gly)-amino-3-(S)-(3,5-diiodo-2-hydroxyphenyl)propionate

To the mixture of BOC-gly-OSu (7.48 g, 27.04 mmol), ethyl 3-(S)-amino-3-(3,5-diiodo-2-hydroxyphenyl)propionate PTSA salt (12, 47 g, 27.04 mmol) in DMF (100 mL) was added triethylamine (3.8 mL). The reaction mixture was stirred for 18 hours at room temperature. The DMS was removed in vacuo and the residue partitioned between ethyl acetate (500 mL) and dilute hydrochloric acid (100 mL). The organic phase was washed with sodium bicarbonate (200 mL), brine (200 mL), dried (MgSO 4 ) and concentrated to give the desired product as a solid (17.0 g, 96% yield).

H1 NMR was consistent with the desired structure.

Step 5

Preparation of ethyl 3-(N-gly)-amino-3-(3,5-diiodo-2-hydroxyphenyl)-propionate hydrochloride

To dioxane/HCl (4N, 40 mL) was added ethyl 3-(N-BOC-gly)-amino-3-(S)-(3,5-diiodo-2-hydroxyphenyl)propionate (17.0 g, 25, 97 mmol) added at 0°C and the reaction mixture was stirred at room temperature for three hours. The reaction mixture was concentrated, and concentrated once more after the addition of toluene (100 mL). The residue obtained was dried to give the desired product as a crystalline powder (8.0 g, 56% yield).

■""H NMR was consistent with the desired product.

Step 6

A solution of ml-(5-hydroxypyrimidine)hippuric acid (3.74 g, 12.98 mmol) in dimethylacetamide (25 mL) was heated until all the material was dissolved. It was then cooled to 0°C and isobutyl chloroformate (1.68 ml) was added in one portion followed by N-methylmorpholine (1.45 ml). After 10 min, ethyl 3-(N-gly)-amino-3-(3,5-diiodo-2-hydroxyphenyl)-propionate hydrochloride (6.0 g, 10.82 mmol) was added in one portion followed by N -methylmorpholine (1.45 ml). The reaction mixture was stirred for 18 hours at room temperature. The reaction mixture was concentrated, the residue dissolved in tetrahydrofuran/water (1:1, 20 mL), and chromatographed (reverse phase, 95:5 water:acetonitrile over 60 minutes to 30:70 water:acetonitrile containing 0.1% TFA). The combined fractions were concentrated. The residue was dissolved in acetonitrile, water and lithium hydroxide were added until the solution was basic. The solution was stirred for two hours. The reaction mixture was concentrated and was purified as above by HPLC to give the desired acid as the TFA salt. The TFA salt was converted to the corresponding hydrochloride salt by passing through an ion exchange column followed by lyophilization.

<X>H NMR was consistent with the desired product.

Examples 14-18

The compounds of formula VII, VIII, IX, XIII and XIV and isomers thereof may be prepared according to the methodology set forth herein by substituting the appropriate starting material and reagents as would be apparent to those skilled in the art.

The activity of the compound of the present invention was tested in the following assays. The results of the investigations in the analyzes are shown in table 1.

VITRONETIN ADHESION ANALYSIS

MATERIALS

Human vitronectin receptor (avp3) was purified from human placenta as previously described [Pytela et al., Methods in Enzymology. 144: 475-489 (1987)]. Human vitronectin was purified from fresh frozen plasma as previously described [Yatogho et al., Cell Structure and Function. 13: 281-292

(1988)]. Biotinylated human vitronectin was prepared by coupling NHS biotin from Pierce Chemical Company (Rockford, IL) with purified vitronectin as previously described [Charo et al., J. Biol. Chem.. 266(3): 1415-1421 (1991)]. Assay buffer, OPD substrate tablets, and RIA grade BSA were obtained from Sigma (St.Louis, MO). Anti-biotin antibody was obtained from Calbiochem (La Jolla, CA). Linbro microtiter plates were obtained from Flow Labs (McLean, VA). ADP reagent was obtained from Sigma (St. Louis, MO).

METHODS OF PROCESSING

Solid-phase receptor assays

This assay was essentially the same as previously reported [Niiya et al., Blood, 70:475-483 (1987)]. The purified human vitronectin receptor (Ovp^) was diluted from stock solutions to 1.0 g/ml in Tris-buffered saline containing 1.0 mM Ca<++>, Mg<++> and Mn<++>, pH 7, 4 (TBS+++) . The diluted receptor was immediately transferred to Linbro microtiter plates at 100 µl/well (100 ng receptor/well). The plates were sealed and incubated overnight at 4°C to allow the receptor to bind to the wells. All further steps were at room temperature. The assay plates were emptied and 200 µl of 1% RIA grade BSA in TBS<+++> (TBS<+++>/BSA) was added to block exposed plastic surfaces. After two hours of incubation, the assay plates were washed with TBS<+*+> using a 96 well plate washer. Serial logarithmic dilution of the test compound and the control was done starting at a working concentration of 2 nM and using 2 nM biotinylated vitronectin in TBS<+++>/BSA as the diluent. This premixing of labeled ligand with test (or control) ligand, and subsequent transfer of 50 µl aliquots to the assay plate was performed with a CETUS Propette robot; the final concentration of the labeled ligand was 1 nM and the highest concentration of the test compound was 1.0 x 10"<4> M. The competition took place over two hours after which all wells were washed with a plate washer as before. Affinity purified horseradish peroxidase labeled shepherd anti- biotin antibody was diluted 1:3000 in TBS<+++>/BSA and 125 L was added to each well. After 30 minutes, the plates were washed and incubated with 0PD/H 2 O and substrate in 100 mM/L Citrate buffer, pH 5, 0. The plate was read with a microtiter plate reader at a wavelength of 450 nm and when the maximum binding control wells reached an absorbance trough of about 1.0, the last A450 was recorded for analysis. The data was analyzed using a macro written for use with EXCEL spreadsheet program. The mean, standard deviation, and %CV were determined for duplicate concentrations. The mean of the A450 values was normalized to the mean of four maximum-binding controls (no competitor added)( B-MAX). The normalized values were subjected to a four parameter curve fitting algorithm [Rodbard et al., Int. Atomic Energy A<g>ency. Vienna, pp 469 (1977)], plotted on a semi-logarithmic scale, and the calculated concentration corresponding to inhibition of 50% of the maximal binding of biotinylated vitronectin (IC50) and the corresponding R<2> were reported for those compounds that showed more than 50% inhibition at the highest concentration tested; otherwise, the IC50 is reported as being greater than the highest concentration tested. 0-[[2-[[5-£(aminoiminomethyl)-amino]-1-oxopentyl]amino]-1-oxoethyl]amino]-3-pyridinepropanoic acid [USSN 08/375,338, Example 1] which is a strong OvP3 antagonist (IC50 in the range 3-10 nM) was included on each plate as a positive control.

PURIFIED Ilb/ Illa RECEPTOR ANALYSIS

MATERIALS

Human fibrinogen receptor (ctnbPs) was purified from outdated platelets. (Pytela, R., Pierschbacher, M.D., Argraves, S., Suzuki, S., and Rouslahti, E. "Arginine-Glycine-Aspartic acid adhesion receptors", Methods in Enzvmolocrv 144(1987):475-489). Human vitronectin was purified from fresh frozen plasma as described in Yåtohgo, T., Izumi, M., Kashiwagi, H., and Hyashi, M., "Novel purification of vitronectin from human plasma by heparin affinity chromatograp-hy," Cell Structure and Function 13(1988):281-292. Biotinylated human vitronectin was prepared by coupling NHS boitin from Pierce Chemical Company (Rockford, IL) with purified vitronectin as described previously. (Charo, I.F., Nan-nizzi, 1., Phillips, D.R., Hsu, M.A., Scaround bottomo-rough, R.M., "Inhibition of fibrinogen binding to GP Hb/Illa by a GP Illa peptide", J. Biol. Chem. 266(3)(1991):1415-1421). Assay buffer, OPD substrate tablets, and RIA grade BSA were obtained from Sigma (St. Louis, MO). Anti-biotin antibody was obtained from Calbiochem (La Jolla, CA). Linbro microtiter plates were obtained from Plow Labs (McLean, VA). ADP reagent was obtained from Sigma (St. Louis,

MO).

METHODS OF PROCESSING

Solid phase receptor assay

This analysis is essentially the same as reported in Niiya, K., Hodson, E-, Bader, R., Byers-Ward, V. Koziol, J.A., Plow, E.F. and Ruggeri, Z.M., "Increased surface expression of the membrane glycoprotein Ilb/IIIa complex induced by platelet activation: Relationship to the binding of fibronogen and platelet aggregation" Blood 70(1987):475-48. The purified human fibrinogen receptor (ctnbpa) was diluted from the working solution to 1.0 µg/mL in Tris-buffered saline containing 1.0 mM Ca<*+>, Mg<++>, and Mn<++>, pH 7, 4 (TBS+++) . The pre-diluted receptor was immediately transferred to Linbro microtiter plates at 100 ul/well (100 ng receptor/well). The plates were sealed and incubated overnight at 4°C to allow the receptor to bind to the wells. All remaining steps were at room temperature. The assay plates were emptied and 200 µl of a 1% RIA grade BSA in TBS<+++>

(TBS<+++>/BSA) was added to block exposed plastic surfaces. Following a two hour incubation, the assay plates were washed with TBS<+++> using a 96 well plate washer. Logarithmic serial dilution of the test compound and controls were made starting at a working concentration of 2 mM and using 2 nM biotinylated vitronectin in TBS<+++>/BSA as the diluent. This premixing of labeled ligand with test (or control) ligand, and subsequent transfer of 50 µl aliquots to the assay plate was performed with a CETUS Propette robot; the final concentration of the labeled ligand was nM and the highest concentration of the test compound was 1.0 x 10 "<4> M. Competition occurred for two hours, then all wells were washed with a plate washer as before. Affinity-purified horseradish peroxidase labeled sheep anti-biotin antibody was diluted 1:3000 in TBS<+++>/BSA and

125 µl was added to each well. After 30 minutes, the plates were washed and incubated with OD/H 2 O 2 substrate in 100 mM/l citrate buffer, pH 5.0. The plate was read with a microtiter plate reader at a wavelength of 450 nm and when the maximum binding control wells reached an absorbance trough of about 1.0, the last A450 was recorded for analysis. The data were analyzed using a macro written for use with the EXCEL™ spreadsheet program. The mean of the standard deviation and %CV were determined for duplicate concentrations.

Average A450 values were normalized to the average of four maximum-binding controls {no competitor added)(B-MAX). The normalized values were subjected to a four parameter curve fitting algorithm, [Robard et al., Int. Atomic Energy Agency. Vienna. pp 469(1977)], plotted on a semilogarithmic scale, and the calculated concentration corresponding to inhibition of 50% of the maximal binding of biotinylated vitronectin {IC50) and corresponding R<2> was reported for those compounds showing greater than 50% inhibition at the highest concentration tested; or the IC50 is reported as being greater than the highest concentration tested. |3- [ [2- [ [5- [ (aminoimino-methyl)-amino]-1-oxopentyl]amino]-1-oxethyl]amino]-3-pyridinepropanoic acid [USSN 08/375,338, Example 1] as is a potent Ovp3 antagonist (IC50 in the range 3-10 nM) was included on each plate as a positive control.

Human platelet rich plasma assay

Healthy aspirin-free donors were selected from a pool of volunteers. The fixation of platelet rich plasma and subsequent ADP induced platelet aggregation assays were performed as described in Zucker, M.B., "Platelet Aggregation Measured by the Phtometric Method", Methods in Enzymology 169(1989):117-133. Standard venipuncture techniques using a butterfly allowed the withdrawal of 45 ml of whole blood into a 60 ml syringe containing 5 ml of 3.8% trisodium citrate. Following thorough mixing in the syringe, the anticoagulated whole blood was transferred to a 50 ml conical polyethylene tube. The blood was centrifuged at room temperature for 12 minutes at 200 mg to pellet non-platelet cells. Platelet-rich plasma was removed into a polyethylene tube and stored at room temperature until use. Platelet-poor plasma was obtained from a second centrifugation of the remaining blood at 2000 xg for 15 minutes. Platelet count is typically 300,000 - 500,000 per microliter. Platelet-rich plasma (0.45 mL) was aliquoted into siliconized cuvettes and stirred (1100 rpm) at 37°C for one minute before addition of 50 µl of prediluted test compound. After one minute of mixing, agglomeration was initiated by the addition of 50 µl of 200 µl (Ml) ADP. Aggregation was recorded for three minutes in a Payton dual-channel aggrometer (Payton Scientific, Buffalo, NY). Percent inhibition of maximal response (saline control) for a series of test compound dilutions was used to determine a dose response curve. All compounds were tested in duplicate and the concentration of half-maximal inhibition (IC50) was calculated graphically from the dose-response curve for those compounds that showed 50% or greater inhibition at the highest concentration tested; otherwise, the IC50 is reported as being higher than the highest concentration tested.

Claims (15)

1. Connection by formula wherein X and Y are the same or different halo group; R is H or C 1 -C 10 alkyl; and pharmaceutically acceptable salts thereof. 2. Connection according to claim 1 selected from the group consisting of: wherein R is H or alkyl; or pharmaceutically acceptable salts thereof. 3. Connection according to claim 1 selected from the group consisting of 4. Pharmaceutical composition comprising a therapeutically effective amount of a compound according to claim 1 and a pharmaceutically acceptable carrier. 5. Pharmaceutical composition comprising a therapeutically effective amount of a compound according to claim 2 and a pharmaceutically acceptable carrier. 6. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to claim 3 and a pharmaceutically acceptable carrier. 7. Use of the compound according to claim 1, 2 or 3 to prepare a drug to treat conditions mediated by the Ovp3 integrin in a mammal in need of such treatment. 8. Use of the compound according to claim 1, 2 or 3 to prepare a drug to treat tumor metastasis. 9. Use of the compound according to claim 1, 2 or 3 to prepare a drug to treat hard tumor growth. 10. Use of the compound according to claim 1, 2 or 3 to prepare a drug to treat angiogenesis. 11. Use of the compound according to claim 1, 2 or 3 to prepare a drug to treat osteoporosis. 12. Use of the compound according to claim 1, 2 or 3 to prepare a medicament for treating humoral hypercalcemia of malignancy. 13. Use of the compound according to claim 1, 2 or 3 to prepare a drug to treat smooth muscle cell migration. 14. Use of the compound according to claim 1, 2 or 3 to prepare a drug to treat rheumatoid arthritis. 15. Use of the compound according to claim 1, 2 or 3 to prepare a drug to treat macular degeneration.