SK13002000A3 - Meta-azacyclic amino benzoic acid compounds and derivatives thereof being integrin antagonists - Google Patents

Abstract

The present invention is directed to compounds of formula (1) and pharmaceutically acceptable salts and isomers thereof useful as alpha v beta 3 integrin antagonists.

Description

META-AZACYCLIC AMINOBENZOIC ACID DERIVATIVES AND THEIR DERIVATIVES THAT ARE ANTAGONISTS OF INTEGRINE

Technical field

The present invention relates to drugs (compounds) which are useful as antagonists of integrin _α ν β3, and which are used in pharmaceutical compositions and in methods for treating conditions mediated by the _α ν β3, inhibition or antagonism of _α ν β3 integrin.

BACKGROUND OF THE INVENTION

Integrins are a family of cell surface glycoproteins that mediate cell adhesion, and thus are useful mediators of the cell adhesion interaction that is present in various biological processes. Integrins are heterodimers composed of non-covalently bound α and β polypeptide subunits. Currently, 11 different subunits and 6 different β subunits have been recognized. To form different integrins, different subunits can associate with different β subunits.

The integrin designated α _ν β3 (also known as the vitronectin receptor) has been recognized as an integrin that plays a role in a variety of conditions or diseases including tumor metastasis, solid tumor growth (neoplasia), osteoporosis, Paget's disease, humoral hypercalcemia in malignancy, angiogenesis including tumor angiogenesis, retinopathy including macular degeneration, arthritis including rheumatoid arthritis, periodontal disease, psoriasis, and smooth muscle cell migration (e.g. restenosis). In addition, these drugs have been found to be useful as antiviral, antifungal and antimicrobial agents. Thus, compounds that selectively inhibit or antagonize α _ν β3 will be useful in the treatment of such conditions.

It was found that integrin _α ν β3 integrin and the other containing _and bind to many of macromolecules of the matrix comprising the amino acid sequence Arg 31 517 / B

Gly-Asp (RGD). Compounds containing the RGD sequence mimic the extracellular ligands of the matrix to bind to cell surface receptors. However, it is also known that RGD peptides are usually not selective for RGD-dependent integrins. For example, most RGD peptides that bind to α _ν β3 also bind to α _ν β5, α _ν βι and <χιη> β3 · Platelet αιΐόβ3 antagonism (also known as fibrinogen receptor) is known to block platelet aggregation in humans . In order to eliminate the side effects of bleeding in the treatment of conditions or diseases associated with α _ν β3 integrin, it will be useful to develop compounds that are selective α _ν β3 antagonists over αι ^ β3 ·

Tumor cell invasion occurs in a three-step process: 1. attaching the tumor cell to the extracellular matrix, 2. proteolytic dissolution of the matrix, and 3. moving the cells across the dissolved barrier. This process may occur repeatedly and may result in metastasis at sites distant from the original tumor.

Seftor et al., Proc. Natl. Acad. Sci. USA, 89, 1557-1561 (1992) demonstrated that integrin α _ν β ₃ has a biological function in the invasion of melanoma cells. Montgomery et al. in Proc. Natl. Acad. Sci. USA, 91, 8856-8860 (1993) showed that integrin α _ν β ₃ expressed on human melanoma cells stimulates a signal for the survival of the protective cell against apoptosis. To prevent tumor metastasis, it will be useful to mediate the pathway of tumor cell metastasis by interfering with the integrin α _ν β3 cell adhesion receptor integrin.

Brooks et al., Cell, 79, 1157-1164 (1994) has demonstrated that antagonists of _α ν β3 provide a therapeutic approach to the treatment of neoplasia (inhibition of solid tumor growth) since systemic administration of antagonists of _α ν β3 causes dramatic regression of various histologically distinct tumors in man.

Integrin adhesion receptor _α ν β3 was identified as a marker of angiogenic blood vessels in chick and man and therefore this receptor plays a critical role in angiogenesis or neovascularization. Angiogenesis is characterized by invasion, migration and proliferation of smooth muscle cells and endothelium. Α _ν β3 antagonists inhibit this process by selectively stimulating apoptosis

517 / B cells in neovasculature. New vessel growth, or angiogenesis, also promotes pathological conditions such as diabetic retinopathy and macular degeneration (Adonis et al., Amer. J. Ophtal., 118, 445-450 (1994)) and rheumatoid arthritis (Peacock et al., J. Exp. Med., 175: 1135-1138 (1992)). Antagonists α _ν β3 will therefore be useful therapeutic targets for the treatment of these conditions associated with neovascularization (Brooks et al., Science, 264, 569-571 (1994)).

The cell surface receptor α _ν β ₃ has been reported to be the major integrin on osteoclasts responsible for adherence to bone. Osteoclasts condition bone resorption, and if this bone resorption activity outweighs bone formation activity, this results in osteoporosis (bone loss), leading to increased bone fracture, disability and increased mortality. Α _ν β ₃ antagonists have been shown to be potent inhibitors of osteoclast activity both in vitro (Sato et al., J. Cell. Biol., 111, 1713-1723 (1990)) and in vivo (Fisher et al., Endocrinology, 132 1411-1413 (1993)). Antagonism α _ν β3 leads to a decrease in bone resorption and thus re-establishes a normal balance of bone formation activity and its remodeling. Thus, it will be beneficial to provide α _ν β ₃ osteoclast antagonists which are effective inhibitors of bone resorption and are therefore useful in the treatment or prevention of osteoporosis.

The role of integrin α _ν β ₃ in smooth muscle cell migration has made it a therapeutic target for preventing or inhibiting neointima hyperplasia, which is a major cause of restenosis following vascular procedures (Choi et al., J. Vasc. Surg., 19,1, 125- 134 (1994)). The prevention or inhibition of neointima hyperplasia by the drug will be beneficial in preventing or inhibiting restenosis.

White vCurrent Biology, 3, 9, 596-599 (1993) reported that adenovirus uses α _ν β ₃ to enter host cells. Integrin appears to be desirable for endocytosis of viral particles and may be desirable for penetration of the viral genome into the cytoplasm of the host cell. Thus, compounds that inhibit α _ν β ₃ will be considered useful as antiviral drugs.

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3a

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

A represents a group of formulas (a) to (d)

which are useful as integrin antagonists α _ν β3 ·

J. Med. Chem. 40, 930 (1997), J. Med. Chem. 40, 920 (1997) and Antiv. Chem. Chemother. 8, 463 (1997) are directed to the discovery of HIV-1 integrase inhibitors in specific pharmaceutical research.

Exp. Opin. Ther. Patents 8, 633 (1998) was published after the priority claimed herein and relates to the finding that integrin antagonists can be used to inhibit metastasis.

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SUMMARY OF THE INVENTION

The present invention relates to a compound of formula

in which:

X and Y represent the same or different halogen atoms, and pharmaceutically acceptable salts thereof.

The compounds described above may exist in various isomeric forms and it is intended that all of these isomeric forms are included herein. Tautomeric forms are also included herein, as well as pharmaceutically acceptable salts of these isomers and tautomers.

More specifically, the present invention relates to the following compounds:

517 / B

517 / Β

517 / ο ο

CO 2 R

OH

Ο

517 / Β

In which:

R represents a hydrogen atom or an alkyl group, or a pharmaceutically acceptable salt thereof.

It is another object of the present invention to provide pharmaceutical compositions comprising the compounds described above. Such compounds and compositions are useful in selectively inhibiting or antagonizing integrin, and thus, in another embodiment, the invention relates to a method of selectively inhibiting or antagonizing integrin α _ν β 3. The invention further includes treating or suppressing pathological conditions associated therewith, such as osteoporosis, humoral hypercalcaemia of malignancy, Paget's disease, tumor metastasis, solid tumor growth (neoplasia), angiogenesis including tumor angiogenesis, retinopathy including diabetic retinopathy and macular degeneration, arthritis including rheumatoid arthritis, periodontal disease, psoriasis, smooth muscle cell migration, restorative cervical cell migration such treatment. In addition, such drugs are useful as antiviral and antimicrobial drugs.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a group of compounds represented by the formulas I to XVI shown above.

Preferred embodiments of the invention are compounds of formulas

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517 / Β

(XXľV)

517 / B ο

(XXVII (XXVII (XXIX) (XXX) (XXXI)

517 / Β

O (XXXII)

every ₂ h .OH

The present invention further relates to pharmaceutical compositions comprising a therapeutically effective amount of the compounds described above.

The present invention also provides a method of selectively inhibiting or antagonism of an integrin _α ν β _3, and more specifically relates to a method of inhibiting bone resorption, periodontal disease, osteoporosis, humoral hypercalcemia of malignancy, Paget's disease, tumor metastasis, solid 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 the compound described above to achieve such inhibition together with a pharmaceutically acceptable carrier.

The following is a list of definitions of the various terms used here:

As used herein, the terms "alkyl" and "lower alkyl" refer to straight or branched chain hydrocarbon radicals of from about 1 to about 10 carbon atoms, and more preferably from 1 to about 6 carbon atoms. Examples of such alkyl groups are methyl, ethyl, norm. propyl, isopropyl, norm. butyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl, hexyl, isohexyl and the like.

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

As used herein, the term "haloalkyl" refers to an alkyl group, as defined above, with one or more, same or

517 / B with different halogen atoms on one or more carbon atoms. Examples of such haloalkyl groups include trifluoromethyl, dichloroethyl, fluoropropyl and the like.

As used herein, the term "composition" refers to a product that results from mixing or combining more than one component or component.

The term "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable substance, mixture or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulant, involved in carrying or transporting a chemical drug.

The term "therapeutically effective amount" shall mean that amount of drug substance or drug that elicits the biological or therapeutic response of a tissue, system or animal as sought by a scientist or physician.

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

¹ H-NMR = proton nuclear magnetic resonance AcOH = acetic acid Ar = argon

CH 3 CN = acetonitrile

CHN analysis = elemental carbon / hydrogen / nitrogen analysis

CHNCI analysis = elemental carbon / hydrogen / nitrogen / chlorine analysis

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

D1 water = deionized water

DMA = N, N-dimethylacetamide

DMAP = 4- (N, N-dimethylamino) pyridine

DMF = Ν, Ν-dimethylformamide

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

EtOAc = ethyl acetate

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EtOH = ethanol

FAB MS = fast atom bombardment mass spectrometry g = gram (s)

HOBT = 1-hydroxybenzotriazole hydrate

HPLC = high performance liquid chromatography

IBCF = isobutyl chloroformate

KSCN = potassium thiocyanate

I = liter

LiOH = lithium hydroxide

MEM = methoxyethoxymethyl

MEMCI = methoxyethoxymethyl chloride

MeOH = methanol mg = milligram

MgSO4 = magnesium sulfate ml = milliliter

MS - Mass Spectrometry

MTBE = methyl tert-butyl ether

N ₂ = nitrogen

NaHCO3 = sodium bicarbonate

NaOH = sodium hydroxide

Na ₂ SO ₄ = sodium sulphate

NMM = N-methylmorpholine

NMP = N-methylpyrrolidone

NMR = nuclear magnetic resonance

P ₂ O ₅ = phosphorus pentoxide

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PTSA = para-toluenesulfonic acid

RPHPLC = high performance reverse phase liquid chromatography

RT = room temperature

TFA = trifluoroacetic acid

THF = tetrahydrofuran

TMS - trimethylsilyl

Δ = heating of the reaction mixture

The compounds described above may be present in various isomeric forms, and it is intended that all of these isomeric forms are included herein. Also included are tautomeric forms, as well as pharmaceutically acceptable salts of these isomers and tautomers.

In the structures and formulas herein, the cyclic ring linkages cross-linked may bind to any of the suitable cyclic ring atoms.

The term "pharmaceutically acceptable salt" refers to a salt prepared by combining the above compound with an acid, the anion of which is generally believed to be suitable for human consumption. Examples of pharmaceutically acceptable salts include hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate, acetate, propionate, lactate, maleate, malate, succinate, tartrate, and the like salts. All of these pharmaceutically acceptable salts can be prepared by conventional methods (for further examples of pharmaceutically acceptable salts see Berge et al., J. Pharm. Sci., 66, 1, 1-19 (1977)).

For the selective inhibition or antagonism of _α ν β3 integrins, compounds of the invention may be administered orally, parenterally or by inhalation spray, or topically in unit-dose formulations containing conventional pharmaceutically acceptable carriers, adjuvants and vehicles. The term parenteral as used herein includes, for example, the subcutaneous, intravenous, intramuscular, intrasternal, infusion or intraperitoneal route.

The compounds of the invention are administered by any suitable route in the form of a pharmaceutical composition adapted for such use

517 / B method and at a dose effective for the intended treatment. Therapeutically effective dosages of the compounds required to prevent or arrest the development or treatment of a medical condition will be readily appreciated by those of ordinary skill in the art using preclinical and clinical approaches conventional in the medical sciences.

Accordingly, the invention provides a method of treating conditions mediated by selective inhibition or antagonism of the receptor cell surface _α ν β3, which method comprises administering a therapeutically effective amount of a compound selected from the group of compounds described above, wherein one or more compounds is administered in association with one or more non-toxic , pharmaceutically acceptable carriers and / or diluents and / or adjuvants (collectively referred to herein as "carriers") and, if desired, other active ingredients. More particularly, the present invention provides a method of inhibiting the cell surface receptor _α ν β3. More preferably, the invention provides a method of inhibiting bone resorption, treating osteoporosis, inhibiting humoral hypercalcaemia in malignancy, treating Paget's disease, inhibiting tumor metastasis, inhibiting neoplasia (solid tumor growth), inhibiting angiogenesis including tumor angiogenesis, treating diabetic retinopathy and macular degeneration, psoriasis and pontitis, and inhibition of smooth muscle cell migration, including restenosis.

According to standard laboratory experimental methods and procedures well known and appreciated by those skilled in the art, as well as by comparison with compounds of known utility, the compounds described above can be used to treat patients suffering from the above pathological conditions. One of ordinary skill in the art will recognize that the selection of the most suitable compound of the invention is within the skill of the art and will depend on a variety of factors including evaluation of results obtained in standard assays and animal models.

Treatment of a patient afflicted with one of the pathological conditions comprises administering to said patient an amount of the above-described compound that is therapeutically effective in controlling such condition or prolonging the survival of the patient as expected in the absence of such treatment.

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As used herein, the term "inhibition" of a condition refers to slowing, interrupting, preventing, or stopping a condition and does not necessarily indicate complete elimination of the condition.

It is believed that, in addition to the highly beneficial effect itself, the prolongation of the patient &apos; s survivability shows that this condition is to some extent favorably controlled.

As previously stated, the compounds of the invention can be used in various fields of biology, prophylaxis or therapy. These compounds are believed to be useful in the prevention or treatment of a disease state or condition in which integrin α _ν β3 plays a role.

The dosage regimen for the compounds and / or compositions comprising such compounds depends on various factors including the type, age, weight, sex and condition of the patient, the severity of the condition, the mode of administration, and the effect of the particular compound employed. Thus, the dosage regimen may vary widely. Dosage levels ranging from about 0.01 mg to about 1000 mg per kilogram of body weight per day, and more preferably from about 0.01 mg to about 100 mg per kilogram of body weight per day, are useful in the treatment of the above conditions.

The active ingredient by injection is formulated into a formulation in which, for example, sodium chloride solution, dextrose or water can be used as a suitable carrier. A suitable daily dose injected in several doses depending on the above factors would usually be from about 0.01 to about 10 mg / kg body weight.

For administration to a mammal in need of such treatment, the compounds in a therapeutically effective amount are usually combined with one or more adjuvants suitable for the mode of administration used. The compounds may be admixed with lactose, sucrose, starch powder, alkanoic acid esters and cellulose, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acid, gelatin, alginate, polyvinyl, alginate, polyvinyl, alginate and / or polyvinyl alcohol and tablet or

517 / B encapsulated for appropriate 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 solution and / or in various buffers. Other adjuvants and routes of administration are well and well known in the pharmaceutical arts.

The pharmaceutical compositions useful in the present invention may be subjected to conventional pharmaceutical methods such as sterilization and / or may contain conventional pharmaceutical adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, etc.

Schemes I to III outline general synthesis methods for preparing compounds useful in the present invention. In various aspects, both explanations and current methods are described in the appropriate place. The following schemes and examples are given merely to illustrate the invention and do not limit its scope or substance. One of skill in the art will readily understand that known variations of the conditions and methods described in the Schemes and Examples can be used to synthesize the compounds of this invention.

Unless otherwise stated, all starting materials and equipment used were commercially available.

Scheme I

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Scheme I illustrates a methodology useful in preparing a portion of tetrahydropyrimidine benzoic acid of the invention that can condense with a gly-p-amino acid ester. Briefly, in Scheme I, 3,5-dihydroxybenzoic acid is converted to 3-amino-5-hydroxybenzoic acid using the method described in Aust. J. Chem., 34, 6, 1319-1324 (1981). The product is reacted with ammonium stiocyanate in hot dilute hydrochloric acid to give 3-thiourea-5-hydroxybenzoic acid after normal work-up. This thiourea intermediate is converted to the S-methyl derivative by treatment with methyl iodide in ethanol at reflux. 1,3-Diamino-2-hydroxypropane is reacted with this resulting intermediate in hot Ν, Ν-dimethylacetamide. Upon cooling, a precipitate formed and the zwitterion product was collected by filtration. The hydrochloric acid salt can be obtained by lyophilization from dilute hydrochloric acid. Alternatively, the product may be separated from the original reaction mixture by evaporation of the solvents. The resulting product is taken up in water and the pH is adjusted to about 5-7, where the zwitterion product precipitates and is collected by filtration. The hydrochloric acid salt can be obtained by the above-mentioned process or by simple dissolution in dilute hydrochloric acid and evaporation to a solid and drying.

Scheme IA

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1. NH 3 / NH 3 and Δ

From HCl / H 2 O Δ

Scheme IA illustrates a methodology useful in preparing a portion of tetrahydropyrimidine benzoic acid of the present invention that can condense with a gly-p-amino acid ester. Briefly, in Scheme IA, 1,3-diamino-2-hydroxypropane is reacted with carbon disulphide in an appropriate solvent, such as a mixture of ethanol and water, refluxed, cooled, hydrochloric acid added, refluxed again, cooled and the product, 5-hydroxytetrahydro-2-pyrimidinethion is obtained by filtration and dried. This cyclic thiourea intermediate is converted to the S-methyl derivative by reaction of thion and methyl iodide in ethanol under reflux. By evaporating the volatile solvents under reduced pressure, the desired 2-methylthioether 5-hydroxypyrimidine hydroiodide is immediately isolated. A solution of 2-methylthioether 5-hydroxypyrimidine hydroiodide in a mixture of dichloromethane and Ν, Ν-dimethylacetamide (approximately 10: 1) and an equivalent amount of triethylamine is cooled to approximately an ice bath temperature and an equivalent amount of di-tert-butyl dicarbonate anhydride is added. Conventional workup affords di-tert-butyldicarbonate-2-methylthioether-5-hydroxypyrimidine as an oil.

517 / B

The 3,5-dihydroxybenzoic acid is converted to 3-amino-5-hydroxybenzoic acid using the method of Aust. J. Chem., 34, 6, 1319-1324 (1981).

The final desired product, 3-hydroxy-5 - [(5-hydroxy-1,4,5,6-tetrahydro-2-pyrimidinyl) amino] benzoic acid, is prepared by subjecting di-tert-butyldicarbonate-2-methylthioether-5-hydroxypyrimidine to acid 3 amino-5-hydroxybenzoic acid in hot Ν, Ν-dimethylacetamide. Upon cooling, a precipitate formed and the zwitterion product was isolated by filtration. For example, the hydrochloric acid salt can be obtained by lyophilization from dilute hydrochloric acid.

M

X ₂ or N-X succinimide I or other source of "X" EtOH, HCl

Y and X represent a halogen atom.

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Scheme II illustrates a process useful for preparing a portion of the ethyl N-glyamino-3- (3,5-dihalo-2-hydroxy) phenylpropionate of the invention that can condense with a portion of tetrahydropyrimidine benzoic acid. Briefly, salicylaldehydes substituted at the 3,5-position by a halogen atom can be prepared by direct halogenation, as will be the case, for example, when 5-bromosalicylaldehyde is suspended in acetic acid and an equivalent amount or more of chlorine is added to extract 3-chloro-5- bromo-2-hydroxybenzaldehyde. A small amount of product precipitates and can be collected by filtration. The residue is recovered by diluting the filtrate with water and separating the precipitate. Combine the solids and dry to give 3-chloro-5-bromo-2-hydroxybenzaldehyde. 3-Iodo-5-chlorosalicylaldehyde can be prepared by subjecting 5-chlorosalicylaldehyde to N-iodosuccinimide in dimethylformamide and subjecting the reaction mixture to conventional processing conditions. 3-Iodo-5-bromosalicylaldehyde can be prepared by reacting 5-bromosalicylaldehyde in acetonitrile with potassium iodide and chloramine T. Workup gives a compound which, after treatment with hexanes, gives the desired 3-iodo-5-chlorosalicylaldehyde.

Coumarins are readily prepared from salicylaldehydes using a modified Perkin reaction (see Vogels Textbook of Practical Organic Chemistry, 5th Ed., P. 1040 (1989)). Halogen-substituted coumarins are converted to 3-aminohydrocoumarins (see Rico, JG, Tett. Let., 35, 6599-6602 (1994)), which are readily opened in the acidic alcohol to give the 3 amino-3- (3) acid esters. , 5-halo-2-hydroxy) phenylpropanoic.

3-Amino-3- (3,5-halo-2-hydroxy) phenylpropanoic esters are converted to N-gly-3-amino-3- (3,5-halo-2-hydroxy) phenylpropanoic esters by reaction with di- tert-butyl dicarbonate-N-gly-N-hydroxysuccinimide to give di-tert-butyl dicarbonate-Ngly-3-amino-3- (3,5-halo-2-hydroxy) phenylpropanoic acid esters which are converted to the HX salts of the acid esters N-gly-3-amino-3- (3,5-halo-2-hydroxy) phenylpropanoic (where X represents a halogen atom) by removal of the di-tert-butyl dicarbonate protecting group, for example using hydrochloric acid in ethanol.

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The amino acid compounds used in the preparation of the compounds of the invention can be prepared according to this site and the methods described below, and according to the method described and claimed in the co-pending USSN Attorney Docket 3076 co-pending with this application.

Scheme III

Y and X are halo.

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Scheme III illustrates a method useful for preparing various compounds of the invention. 3-Hydroxy-5 - [(1,4,5,6-tetrahydro-5-hydroxy-2-pyrimidinyl) amino] benzoic acid is activated to form a bond using known methods. Thus, after dissolution in a suitable solvent such as Ν, Ν-dimethylacetamide, an equivalent amount of N-methylmorpholine is added. The reaction mixture was cooled to an ice bath temperature and isobutyl chloroformate was added. To the stirred intermediate anhydride is added the gly-p-amino acid ester and N-methylmorpholine. After completion of the reaction, the product is purified by preparative HPLC and the ester is hydrolyzed to the acid by treatment with a base such as lithium hydroxide in a suitable solvent (a mixture of dioxane and water or acetonitrile and water). Alternatively, a suitable acid, such as trifluoroacetic acid, may be used. The product was separated by preparative HPLC or zwitterion separation at pH 5-7 and converted to the desired salt by standard methods.

DETAILED DESCRIPTION OF THE INVENTION

Example A

preparation

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Step 1

preparation

ABOUT

Cl

To a 2 L round bottom flask equipped with a mechanical stirrer and a condenser was added 200 g (1.05 mol, 1 equivalent) of 3,5-dichlorosalicylaldehyde, 356 g (3.49 mol) acetic anhydride and 95.0 g (0.94 mol) , 0.90 equivalents) of triethylamine. The reaction solution was heated to reflux overnight. The dark brown reaction mixture was cooled to 50 ° C and 1 L of water was added with stirring. After one hour, the mixture was filtered and the filtrate was mixed with 1 L of ethanol. The mixture was heated at 45 ° C for 1 hour, cooled to room temperature, filtered and the solid (fraction A) was washed with 0.5 L of ethanol. The combined ethanolic solutions were evaporated by rotary evaporation to an oily substance (fraction B). The solid from fraction A was dissolved in 1.5 L of dichloromethane and the resulting solution was passed through a silica gel septum (1300 mL volume). The resulting dark brown solution was evaporated to an oily substance which was triturated with 1.3 L of hexane to give a solid which was collected by filtration and washed with hexanes to afford 163 g of substantially pure 6,8-dichlorocoumarin. An additional 31 g of product was obtained by treating the oily substance (Fraction B) in a simple manner, the oily substance was dissolved in 0.5 L of dichloromethane, passed through a silica pad (0.5 L) and triturated with hexanes. A total of 194 g (86% yield) of a brown solid was obtained.

MS and NMR were consistent with the desired structure.

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Step 2

preparation

C

* CO ₂ Et

OH • HCl

Cl

To a 2 L 3-necked flask equipped with a mechanical stirrer was added 160 g (0.74 mol) of 6,8-dichlorocoumarin (prepared in Step 1) and 375 mL of anhydrous tetrahydrofuran (Aldrich Sure Seal). The resulting mixture was cooled to -40 ° C (dry ice-acetone bath) and 800 mL of a 1M solution (0.80 mol) of lithium bis (trimethylsilyl) amide in tetrahydrofuran was added while maintaining the temperature below -40 ° C. After the addition was complete, the cooling bath was removed. After 0.5 h, the mixture was warmed to -5 ° C. The reaction mixture was quenched by the addition of a solution of hydrochloric acid (0.5 L of a 4M solution in dioxane) in 1.25 L of ethanol. The temperature was kept below 0 ° C overnight. The reaction mixture was evaporated to approximately half the original volume and partitioned between 3 L of ethyl acetate and 2 L of water. The organic layer was washed three times in succession with 1 L of a 0.5 M aqueous hydrochloric acid solution. The pH of the combined aqueous layers was adjusted to about 7 by addition of 10% aqueous sodium hydroxide solution and extracted three times in succession with 2 L of dichloromethane. The combined organic layers were dried over magnesium sulfate, filtered, and 210 mL of a 4M solution of hydrochloric acid in dioxane was added with stirring. After the precipitate had formed, the solid was collected by filtration. The filtrate was evaporated to a small volume and methyl tert-butyl ether was added. The obtained solid was combined with the initially formed solid and the combined product was washed with methyl tert-butyl ether, collected by filtration and dried (vacuum drying over the weekend) to afford 172 g (yield 74%) of the desired product.

MS and NMR were consistent with the desired structure.

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Step 3

preparation

coze

OH

CI

Cl

15.0 g (0.055 mol) of N-tert-butyldicarbonate-gly-N-hydroxysuccinimide ester (Sigma), 200 ml of anhydrous dimethylformamide (Aldrich) were added to a 0.5 L round-bottomed flame flask equipped with a magnetic stirring bar under an inert argon atmosphere. Seal Seal) and 21.67 g (0.055 mol) of the product of Step 2. The reaction mixture was cooled to approximately 0 ° C (ice-salt bath) and 5.58 g (0.056 mol) of N-methylmorpholine and catalytic amount were added. Of 4- (N, N-dimethylamino) pyridine and the reaction was allowed to proceed overnight. The reaction mixture was evaporated to a slurry and partitioned between 0.4 L of ethyl acetate and aqueous base (0.2 L of saturated aqueous sodium bicarbonate twice). The organic layer was washed successively with 0.2 L of aqueous citric acid (10% w / v) twice in succession, again with 0.2 L of saturated aqueous sodium bicarbonate solution twice, successively with sodium chloride solution and dried over sodium sulfate. The volatile solvents were evaporated in vacuo at 55 ° C to give 22.5 g (92% yield) of an oily substance which solidified on standing.

MS and NMR were consistent with the desired structure.

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Step 4

preparation

Using the following method, the product obtained in step 3 was deprotected to obtain the amine hydrochloride salt. To a 0.1 L round bottom flame flask equipped with a stirrer with 14.0 g (0.032 mol) of the product of Step 3 was added 40 mL of anhydrous dioxane. To this was added, at 0 ° C, 6.32 mL (2 equivalents) of 4.0 N hydrochloric acid in dioxane and the reaction was allowed to proceed until gas evolution ceased and the reaction was complete. The volatile solvents were evaporated in vacuo and the residue was triturated with 50 mL diethyl ether. The solids were collected by filtration and washed with diethyl ether and dried to give 12.5 g of the desired product.

MS and NMR were consistent with the desired structure.

Example B

preparation

HCl

517 / B

Step 1

Preparation ci

'Br

To a suspension of 175.0 g (743.2 mmol) of 3-bromo-5-chlorosalicylaldehyde in 280.5 mL (3.0 mol) of acetic anhydride was added 103.6 mL (743.2 mmol) of triethylamine. The reaction solution was heated under reflux for 4.5 hours. The solution was cooled and evaporated in vacuo. To the brown residue was added 730 mL of absolute ethanol. 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 under vacuum to give 123.0 g (64% yield) of the desired product. ¹ H NMR was consistent with the proposed structure.

Step 2

preparation

br

To a suspension of 40 g (154.1 mmol) of coumarin in 400 mL of tetrahydrofuran was added dropwise 154.1 mL of a 1M solution of lithium bis (trimethylsilyl) amide in tetrahydrofuran under stirring at -76 ° C. Addition completed in 10 minutes. Then the reaction mixture was stirred for 5 minutes, warmed to -20 ° C and 15 ° C

517 / B minutes. To this solution was added a solution of 9.25 g (154.1 mmol) of acetic acid in 28 mL of tetrahydrofuran over 5 minutes. The mixture was warmed to room temperature and the volatile solvents were evaporated in vacuo. The residue was dissolved in 850 ml of diethyl ether, washed twice with 100 ml of saturated aqueous sodium bicarbonate solution, twice with 40 ml of sodium chloride solution each time and dried over magnesium sulfate. The diethyl ether solution was evaporated to approximately 160 mL and cooled to 0 ° C. To this suspension was added 56.3 mL (225 mmol) of a 4 M hydrochloric acid solution in dioxane and the mixture was stirred at 0 ° C for 30 min. The suspension was filtered and the filter cake was washed with diethyl ether. The solid was dried under vacuum to give 45.0 g of the desired product as the hydrochloride salt, dioxane solvate.

'H NMR was consistent with the proposed structure.

Step 3

preparation

To a suspension of 142.2 g (354.5 mmol) of lactone in 533 mL of absolute ethanol was added 157.8 mL (631.1 mmol) of a 4M solution of hydrochloric acid in dioxane over 10 minutes. The reaction mixture was stirred at room temperature for 2.5 hours. The volatile solvents were evaporated in vacuo. The residue was dissolved in 450 mL of ethyl acetate and the solution was kept at 0 ° C for 15 hours. The yellow-brown precipitate was collected by filtration and washed with cold ethyl acetate. The solid was dried in vacuo to give 100.4 g (79% yield) of the desired product as the hydrochloride salt.

¹ H NMR was consistent with the proposed structure.

517 / B

Step 4

preparation

2.72 g (0.010 mol) of N-tert-butyldicarbonate-gly-N-hydroxysuccinimide ester (Sigma), 50 ml of anhydrous tetrahydrofuran (Aldrich) were added to a 0.1 L round-bottomed flame flask equipped with a magnetic stirring bar under an inert argon atmosphere. Seal (Seal) and 3.10 g (0.01 mol) of the product of Step 3, dried overnight under vacuum with phosphorus pentoxide. The reaction mixture was cooled to about 0 ° C (ice-salt bath) and 1.01 g (0.010 mol) of triethylamine was added. The reaction was allowed to proceed overnight. The reaction mixture was evaporated to a semi-solid and worked up in a manner similar to Example A, Step 3. The volatile solvents were evaporated from the organic layer in vacuo at 55 ° C to give 4 g (83% yield) of an oily substance which solidified on standing.

MS and NMR were consistent with the desired structure.

Step 5

preparation

HCl

517 / B

Using the following method, the product obtained in step 4 was deprotected to obtain the amine hydrochloride salt. 20 ml of anhydrous dioxane was added to 4.0 g (0.0084 mol) of the product from Step 4 to a 0.1 L round-bottom flame flask equipped with a stirrer. To this was added 20 mL of a 4N solution of hydrochloric acid in dioxane and the reaction was allowed to continue until gas evolution ceased and the reaction was complete (approximately 1 hour). The volatile solvents were evaporated in vacuo and the residue was triturated with 50 mL diethyl ether. The solids were collected by filtration and washed with ether and dried to give 2.7 g (78% yield) of a light brown solid.

MS and NMR were consistent with the desired structure.

Example C

preparation

br

Step 1

Br ₂ CO ₂ Et

OH

br

br

HCl

517 / B

To a suspension of 100 g (357 mmol) of 3,5-dibromosalicylaldehyde in 164.8 ml (1.8 mol) of acetic anhydride was added 45 ml (375 mmol) of triethylamine. The reaction solution was heated at reflux under argon overnight. The solution was cooled to room temperature and a solid formed. The dark brown reaction mixture was washed three times in succession with 300 mL of hot hexane and saturated aqueous sodium bicarbonate. The resulting solid was dissolved in 2 L of ethyl acetate and washed with water. The organic layer was dried over sodium sulfate and evaporated to give a brown solid, which was collected by filtration. The solid was dried under vacuum to give 94.2 g (87% yield) of substantially pure 6,8-dibromocoumarin.

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

Step 2

br

br

To a solution of 20.0 g (0.066 mol) of the 6,8-dibromocoumarin prepared in Step 1 in 100 mL of tetrahydrofuran at -78 ° C was added dropwise with stirring 66 mL of a 1 M solution of lithium bis (trimethylsilyl) amide in tetrahydrofuran. Addition was complete in 10 minutes. Then the reaction mixture was stirred for 5 minutes, warmed to 0 ° C and stirred for 15 minutes. To this solution was added 3.95 g acetic acid over 1 minute. The mixture was warmed to room temperature and the volatile solvents were evaporated in vacuo. The residue was dissolved in 500 mL hexanes, washed twice with 100 mL saturated aqueous sodium bicarbonate solution and dried over sodium sulfate. The organic solution was evaporated to give an oily substance which was immediately taken up in 400 ml diethyl ether and

517 / B was added with stirring 30 ml of a 4M solution of hydrochloric acid in dioxane and the mixture was stirred at 0 ° C for 30 minutes. Excess hydrochloric acid was evaporated in vacuo, the suspension was filtered and the filter cake was washed with diethyl ether. The solid was dried under vacuum to give 19.9 g of the desired product as the hydrochloride salt, dioxane solvate.

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

Step 3

The HCl g of the lactone prepared in Step 2 was dissolved in 400 ml of absolute ethanol and gaseous anhydrous hydrogen chloride was passed through for 1 minute. The reaction mixture was stirred at room temperature for 2.5 hours. Reversed phase HPLC showed completion of the reaction. The volatile solvents were evaporated in vacuo to give a dark residue. The residue was triturated with 500 mL of diethyl ether and stirred overnight. The yellow-brown precipitate was collected by filtration and washed with diethyl ether. The solid was dried under vacuum to give 15.2 g of the desired product as the hydrochloride salt.

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

Step 4

517 / B

To a 0.2 L round-bottomed flame flask equipped with a magnetic stir bar was added, under an inert argon atmosphere, 8.1 g (0.030 mol) of N-tert-butyldicarbonate-gly-N-hydroxysuccinimide ester (Sigma), 50 ml of anhydrous dimethylformamide (Aldrich). Seal (Seal) and 12 g (0.03 mol) of the product of Step 3, dried overnight under vacuum over phosphorus pentoxide. The reaction mixture was cooled to about 0 ° C (ice-salt bath) and 3.03 g (0.030 mol) of N-methylmorpholine and a catalytic amount of 4- (N, N-dimethylamino) pyridine were added. After warming to room temperature, the reaction was allowed to proceed overnight. The reaction mixture was evaporated to a semi-solid and worked up in a manner similar to that of Example A, Step 3. The volatile solvents were evaporated from the organic layer under vacuum at 55 ° C to give 15.7 g (93% yield) of an oily substance which standing stiff.

MS and NMR were consistent with the desired structure.

Step 5

HCl

Using the following method, the product obtained in step 4 was deprotected to obtain the amine hydrochloride salt. 40 ml of anhydrous dioxane was added to the 13.0 g (0.0084 mol) of the product from Step 4 into a 0.1 L round bottom flame flask equipped with a stirrer. To this was added 30 mL of a 4.0 N hydrochloric acid solution in dioxane and the reaction was allowed to continue until gas evolution ceased and the reaction was complete (approximately 1 hour). The volatile solvents were evaporated in vacuo and the residue was triturated with 50 mL diethyl ether. The solids were collected by filtration and washed with diethyl ether and dried to give 10.6 g (93% yield) of the desired product.

MS and NMR were consistent with the desired structure.

517 / B

Example D

preparation

• HCl

Step 1

Preparation of 3-chloro-5-bromosalicylaldehyde

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

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

517 / B

Step 2

Preparation of 6-bromo-8-chlorocoumarin

br

Cl

To a 5 L round bottom flask equipped with a mechanical stirrer and a condenser was added 554.1 g (2.35 mol, 1 equivalent) of 3-chloro-5-bromosalicylaldehyde, 1 203 g (11.8 mol, 5 equivalents) of acetic anhydride and 237 1.4 g (2.35 mol, 1 equivalent) of triethylamine. The reaction solution was heated to reflux at 131-141 ° C overnight. The dark brown reaction mixture was cooled to 50 ° C and 2 L of ice was added with stirring (cooling in an ice bath). After one hour, the mixture was filtered and the filtrate was mixed with 1 L of ethanol. To this mixture was added 300 mL of ethanol, and the reaction mixture was stirred for 1 hour. The precipitate formed was collected by filtration and washed three times in succession with 1.3 L of a mixture of water and ethanol, evaporated in vacuo and dried in a fluid bed dryer. In total, a yield of 563 g (92%) was isolated.

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

CO ₂ Et

OH · _HC |

br

Cl

517 / B

To a 5 L three necked flask equipped with a mechanical stirrer was added 300 g (1.16 mol) of 6-bromo-8-chlorocoumarin prepared in step 2) and 900 mL of anhydrous tetrahydrofuran (Aldrich Sure Seal). The resulting mixture was cooled to less than -45 ° C (dry ice-acetone bath) and while maintaining the temperature below -45 ° C for 0.5 hour, 800 mL of a 1M solution (0.80 mol, 1.0 mL) was added, 2 equivalents) of lithium bis (trimethylsilyl) amide in tetrahydrofuran in 0.6 L of hexane. In another 5 L flask, 2.5 L of ethanol and 1 L of 4N hydrochloric acid solution in dioxane were mixed at -15 ° C. The coumarin reaction was quenched by the addition of a cooled solution of ethanol and hydrochloric acid. After 0.5 h, the temperature of the resulting reaction mixture was -8.3 ° C. The reaction mixture was kept at 0 ° C overnight, evaporated to approximately 2.5 L and partitioned between 3 L of ethyl acetate and 4 L of water. The organic layer was washed four times in succession with 1.2 L of a 0.5 N aqueous hydrochloric acid solution. The pH of the collected aqueous layers was adjusted to about 7 by the addition of 10% aqueous sodium hydroxide solution and the layers were extracted once with 7 L and 2 L of dichloromethane three times in succession. The collected organic layers were dried over 900 g magnesium sulfate, filtered, and 400 ml of 4M hydrochloric acid in dioxane was added with stirring. After the precipitate had formed, the solid was collected by filtration. The mixture was evaporated to a volume of 2.5 L, 2.5 L of hexanes were added and the precipitate was collected by filtration. The filter cake was washed with a 1: 2 mixture of dichloromethane and hexanes, dried under vacuum and in a vacuum oven at 40 ° C to afford 251 g (yield 60%) of the desired product.

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

Step 5

preparation

517 / B

The above compound was prepared using essentially the same method and relative amounts as indicated for their isomer in Example B, Step 4.

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

Step 6

preparation

HCl

This compound was prepared using essentially the same method and relative amounts as indicated for their isomer in Example B, Step 5.

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

Example E

preparation

HCl

517 / B

Step 1

Preparation of 3-iodo-5-chlorosalicylaldehyde

Cl

OH

To a solution of 100 g (0.638 mol) of 5-chlorosalicylaldehyde in 400 ml of dimethylformamide was added 144.0 g (0.641 mol) of N-iodosuccinimide. The reaction mixture was stirred at room temperature for 2 days. An additional 20.0 g of N iodosuccinimide was added and stirring was continued for a further 2 days. The reaction mixture was diluted with 1 L of ethyl acetate, washed with 300 mL of 0.1 N hydrochloric acid, 300 mL of water, 300 mL of 5% sodium thiosulfate solution, 300 mL of sodium chloride solution, dried with magnesium sulfate and evaporated to dryness to give 162 g ( yield 90%) of the desired aldehyde as a pale yellow solid.

MS and NMR were consistent with the desired structure.

Step 2

Preparation of 6-chloro-8-iodocoumarin

Cl

A mixture of 100 g (0.354 mol) of 3-iodo-5-chlorosalicylaldehyde, 300 ml of acetic anhydride and 54 ml of triethylamine was heated under reflux for 18 hours. After cooling, the desired coumarin precipitated as a dark brown crystalline solid.

517 / B

This was filtered, washed with 200 mL of 4: 1 hexane: ethyl acetate and air dried. Yield: 60 g (55%).

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

Step 3

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

To a solution of 6.63 g (21.62 mmol) of 6-chloro-8-iodocoumarin in 100 mL of tetrahydrofuran at -78 ° C was added 21.62 mL (21.62 mmol, 1M) of lithium hexamethyldisilazane. The reaction mixture was stirred at this temperature for 30 minutes and then at 0 ° C for 1 hour. To the reaction mixture was added 1.3 g (21.62 mmol) of acetic acid. The reaction mixture was added to 300 mL of ethyl acetate and 200 mL of saturated sodium carbonate solution. The organic layer was separated, washed with 200 ml of brine, dried over magnesium sulfate and evaporated to give a residue. The residue was added to 200 ml of anhydrous diethyl ether, followed by the addition of 30 ml of a 4N solution of hydrochloric acid in dioxane. The reaction mixture was stirred at room temperature for 1 hour, filtered and dried in vacuo to give 4.6 g (59% yield) of the desired product as a powder (reverse phase HPLC: Rf 6.8 min, solvent gradient) 10% acetonitrile to 90% acetonitrile in 15 minutes, then up to 100% acetonitrile in 6 additional minutes, both water and acetonitrile contained 0.1% trifluoroacetic acid, Vydac C ₁₈ protein-peptide column, 2 ml / min flow rate, measured at 254 nm).

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

517 / B

Step 4

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

Cl‘COjEt OH · HCl

Hydrogen chloride gas was bubbled into a solution of 22.0 g (61.09 mmol) of 4-amino-3,4-dihydro-6-chloro-8-iodocoumarin hydrochloride in 250 mL of ethanol while maintaining the temperature of the reaction mixture at 0-10 ° C. saturation. After 6 hours at reflux, most of the solvent was distilled off. The cooled residue was added to anhydrous diethyl ether and stirred for 2 hours. The initially formed rubber-like substance was converted to a crystalline substance. The crystalline product was filtered and dried to give 20 g (yield 81%) of the desired product as an off-white crystalline powder; _(Rf: 52 minutes, the same conditions as in Step 3).

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

Step 5

Preparation of (R.S) -ethyl 3- (N-di-tert-butyldicarbonate-gly) -amino-3- (5-chloro-2-hydroxy-3-iodo) phenylpropionate as a.

517 / B

A mixture of 2.16 g (12.31 mmol) of N-di-tert-butyldicarbonate-gly, 1.67 g (12.31 mmol) of 1-hydroxybenzotriazole hydrate, 2.36 g (12.31 mmol) of 1- (3-dimethylaminopropyl) 3-ethylcarbodiimide hydrochloride and 50 ml of dimethylformamide were stirred at 0 ° C for 1 hour. 5.0 g (12.31 mmol) of ethyl 3-amino-3- (5-chloro-2-hydroxy-3-iodo) propionate hydrochloride was added to the reaction mixture, followed by 3.5 ml of triethylamine. The reaction mixture was stirred at room temperature for 18 hours. The dimethylformamide was evaporated in vacuo and the residue was partitioned between 300 mL of ethyl acetate and 200 mL of sodium bicarbonate solution. The organic layer was washed with 100 ml of 1N hydrochloric acid, 200 ml of brine, dried over magnesium sulfate and evaporated to give 6 g (yield 93%) of the desired product as a solid.

MS and ¹ 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-iodo) phenylpropionate hydrochloride

HCl of 4N HCl in dioxane was added to 6.0 g (11.39 mmol) of ethyl 3- (N-di-tert-butyldicarbonate-gly) amino-3- (5-chloro-2) at 0 ° C. of hydroxy-3-iodo) propionate and stirred at room temperature for 3 hours. The reaction mixture was evaporated and evaporated once more after addition of 100 ml of toluene. The resulting residue is suspended in diethylether, filtered and dried to give 5.0 g (yield 95%) of the desired product as a crystalline powder; (silica reverse phase HPLC _Rf: 8.3 minutes, conditions the same as in step 3).

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

517 / B

Example F

preparation

Br • HCl

Step 1

Preparation of 3-iodo-5-bromosalicylaldehyde

To a 500 ml round bottom flask equipped with a magnetic stir bar, a solution of 20.0 g (0.1 mol) of 5-bromosalicylaldehyde and 17 g (0.1 mol) of potassium iodide in 150 ml of acetonitrile and 50 ml of water was added 23 g (0.1 mol) of chloramine T. The mixture was allowed to react for 1 hour. The reaction mixture was partitioned between 200 mL of 10% hydrochloric acid and ethyl acetate. The organic layer was dried over sodium sulfate, filtered and evaporated in vacuo. Hexanes were added to the residue and the reaction mixture was heated at 50 ° C for 15 min. The insoluble material was collected by filtration. The filtrate was evaporated in vacuo to give 26 g of canary yellow 3-iodo-5-bromosalicylaldehyde.

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

Step 2

coje

OH • HCl

517 / B

The above compound was prepared using essentially the same method as in Example E, Steps 2 to 6, wherein in Step 2 an equivalent amount of the product of Step 1, 3-iodo-5-chlorosalicyladedehyde was replaced with 1,3-iodo-5-bromosalicylaldehyde.

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

Example H

preparation

HO

• HCl

Step 1

A 2 L round bottom flask equipped with a mechanical stirrer, a Claisen adapter, a dosing funnel, a reflux condenser, and a thermocouple was charged with 375 mL of ethanol and 357 mL of deionized water. 125.04 g (1.39 mol) of 1,3-diamino-2-hydroxypropane (Aldrich) was added to the reaction flask and stirred until dissolved. 84 ml (1.39 mol) of carbon disulfide was added dropwise over 35 minutes at 25-33 ° C via a addition funnel to obtain a milky white mixture. The temperature was maintained with an ice bath. The reaction mixture was refluxed at 73.4 ° C for 2 hours to give a yellow solution. The reaction mixture was cooled to 25 ° C with an ice bath, and 84 ml of concentrated hydrochloric acid was added dropwise while maintaining the temperature between 25 and 26 ° C. The reaction mixture was refluxed at 78.4 ° C for 21 hours. The reaction solution was cooled to 2 ° C and the product was collected by vacuum filtration. The white solid was washed three times in succession with 50 ml of a 1: 1 mixture of ethanol and water cooled with an ice bath and

517 / B was dried under vacuum at 40 ° C to give 63.75 g (34.7% yield) of 5-hydroxy-tetrahydro-pyrimidine-2-thione as a white solid.

MS and NMR were consistent with the desired structure.

Step 2

To a 2 L round bottom flask equipped with a mechanical stirrer and thermocouple was added 95 g (0.72 mol) of 5-hydroxy-tetrahydro-pyrimidine-2-thione prepared in Step 1, 570 ml of absolute ethanol and 45 ml (0.72 mol) of methyl iodide. The reaction mixture was refluxed for 5 hours at 78 ° C and then cooled to room temperature. The reaction mixture was evaporated in vacuo to give 194.92 g of a white solid. The white solid was triturated three times in succession with 500 ml diethyl ether and dried in vacuo to give 188.22 g (yield 95.4%) of 2-methylthioether-5-hydroxytetrahydropyrimidine hydroiodide as a white solid.

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

Step 3

To a 2 L 3-necked, round-bottomed flask equipped with a reflux condenser, mechanical stirrer and static nitrogen atmosphere was added 150.81 g (0.55 mol) of 2-methylthioether 5-hydroxy-pyrimidine hydroiodide, 530 mL of dichloromethane, 53 mL of dimethylacetamide and 76.7 ml (0.55 mol) of triethylamine. The mixture was cooled in 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 in succession with 200 mL of deionized water, dried over magnesium sulfate, filtered and evaporated in vacuo to give 134.6 g (99.35%) of di-tert-butyl dicarbonate-2. 5-hydroxypyrimidine methylthioether as a pale yellow viscous oil.

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

517 / B

Step 4

50.3 g (0.204 mol) of 5-hydroxy-pyrimidine di-tert-butyl dicarbonate-2-methylthioether, 25.0 g (0.1625 mol) of 3-amino-5-hydroxybenzoic acid (Aust. J. Chem., 34, 6, 1319-1324 (1981)) and 50 ml of anhydrous N, N-dimethylacetamide were heated with stirring at 100 ° C for 2 days. A precipitate suspension formed. The reaction mixture was cooled to room temperature and the precipitate was filtered off, washed with acetonitrile, then with diethyl ether and dried. This solid was suspended in water and acidified with concentrated hydrochloric acid to form a solution. This was frozen and lyophilized to yield 14.4 g of the desired product as a white solid.

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

Example I

preparation

Step 1

Preparation of Reformant Reagent

Zn-Br

NAV-tBu

A 4 L flask equipped with a condenser, thermometer and mechanical stirrer was charged with 180.0 g (2.76 mol, 0.147-0.542 mm particle size) of zinc metal and 1.25 L of tetrahydrofuran. With stirring, use a syringe

517 / B added 4.74 ml (0.05 mol) of 1,2-dibromoethane (alternatively, it can be replaced with 0.1 equivalent of trimethylsilyl chloride at room temperature for 1 hour). After purging with inert gas (three cycles of a mixture of nitrogen and vacuum), a suspension of zinc in tetrahydrofuran was heated to reflux at 65 ° C and held at this temperature for 1 hour. The mixture was cooled to 50 ° before the addition of 488 g (369 ml, 2.5 mol) over 1.5 hours of tert-butyl bromoacetate with a 50 ml syringe and a syringe pump (4.1 ml / min dropper). C. During the addition, the reaction mixture was maintained at 50 ° C ± 5 ° C. After the addition was complete, the reaction mixture was allowed to stir at 50 ° C for 1 hour. The mixture was then allowed to cool to 25 ° C and the resulting precipitate was allowed to settle. The tetrahydrofuran mother liquor was decanted into a 2 L round bottom flask using a coarse filter and partial vacuum transfer (20 mm Hg). This separated from the mixture approximately 65% tetrahydrofuran. 800 mL of 1-methyl-2-pyrrolidinone was added and stirring was continued for 5 minutes. The reaction mixture can be filtered to remove residual zinc. The analysis showed a titer of the desired Reformant reagent of 1.57 M with a molar yield of 94%. Alternatively, the solid reagent may be separated by filtration from the original reaction mixture. The cake can be washed with tetrahydrofuran until a white solid is obtained and dried under a nitrogen atmosphere to obtain the desired product as tetrahydrofuran monosolvate, which can be stored at -20 ° C (dried) for extended periods. Typical yields range from 85 to 90%.

Step 2

2A Preparation

517 / B

To a solution of 11.46 g (60 mmol) of 3,5-dichlorosalicylaldehyde in 40 mL of dimethylformamide was added at room temperature 8.82 g (60 mmol) of potassium carbonate (powder dried in an oven at 100 ° C in vacuo) to a light yellow suspension was formed. 7.64 g (61 mmol) of pure methoxyethoxymethyl chloride were then added while maintaining the bath temperature at 20 ° C. The mixture was then stirred at 22 ° C for 6 hours and 0.3 g (2.4 mmol) of methoxyethoxymethyl chloride was added. The mixture was stirred for an additional 0.5 h and the reaction mixture was poured into 200 mL of cold water to precipitate the product. The suspension was filtered through a pressure filter and the cake was washed twice with 50 ml of water and dried under an atmosphere of a mixture of nitrogen and vacuum to give 14.94 g (yield 89%) of the product as a white-gray solid.

1 H NMR (CDCl 3, TMS): 3.37 (s, 3H), 3.54-3.56 (m, 2H), 3.91-3.93 (m, 2H), 5.30 (s, 2H), 7.63 (d, 1H), 7.73 (d, 1H), 10.30 (s, 1H).

¹³ C NMR (CDCl 3, TMS) d (ppm): 59.03, 70.11, 99.57, 126.60, 129.57, 130.81, 132.07, 135.36, 154.66, 188 , 30th

DSC: 48.24 ° C (endo 90.51 J / g).

Microanalysis for C11H12Cl2O4 (%):

H, 4.33; Cl, 25.40

Found: C, 47.15; H, 4.26; Cl, 25.16

2B Preparation

A 1-L three-necked, round-bottomed flask equipped with a mechanical stirrer and dosing funnel was charged with 35.0 g (0.125 mol)

517 / B of the product of Step 2A, followed by the addition of 200 mL of tetrahydrofuran. The solution was stirred at 22 ° C, and 17.20 g (0.125 mol) of (S) phenylglycinol was added in one portion. After 30 minutes at 22 ° C, 20 g of magnesium sulfate was added. The mixture was stirred at 22 ° C for 1 hour and filtered on a coarse filter. The filtrate was evaporated under reduced pressure. No further purification was performed and the crude imine was directly used in the coupling reaction, step 2C.

2C Preparation

A 1-L three-necked, round-bottomed flask equipped with a mechanical stirrer and addition funnel was charged with 91.3 g (0.275 mol) of the solid reagent produced in Step 1 and 200 mL of N-methylpyrrolidinone under a nitrogen atmosphere. Then, the solution was cooled to -10 ° C and stirred at 350 rpm. A solution of the imine (prepared in step 2B) in N-methylpyrrolidinone was prepared under nitrogen and then added to the above reaction mixture over 20 minutes while maintaining the temperature at -5 ° C (jacket temperature -10 ° C). . After the addition was complete, the mixture was stirred for an additional 1.5 hours at -8 ° C and 1 hour at -5 ° C. After cooling to -10 ° C, a solution of 8.1 ml of concentrated hydrochloric acid in 200 ml of saturated ammonium chloride solution was added over 10 minutes. 200 ml of methyl tert-butyl ether was added and the mixture was stirred at 200 rpm for 15 minutes at 23 ° C. Stirring was complete and the layers were separated. The aqueous layer was extracted with 100 mL of methyl tert-butyl ether. The two organic layers were combined, washed successively with 100 mL of saturated ammonium chloride solution, 100 mL of water and 100 mL of sodium chloride solution. The solution was dried with 30 g magnesium sulfate, filtered and evaporated to give 66.3 g

517 / B of an orange oil (solidified on standing) containing the desired product as a single diastereomer (proton and carbon NMR verified). The sample for analysis was purified by recrystallization from heptane to give the product as a gray-white solid.

Proton and carbon spectra NMR and IR spectra were consistent with the desired structure. [a] ° 5 = ₂ + 8.7 ° (c = 1.057, methanol).

Microanalysis for C ₂₅ H ₃₃ CI ₂ NO6 (%): calculated: C, 58.77 H, 6.47 N, 2.72 Cl, 13.78 found: C, 58.22 H, 6.54 N, 2.70 Cl, 13.66 Step 3 preparation CO ₂ Ht SO ₃ H 1 X- · OH ή xX Cl e M XI 1 ch ₃

3A

A three-necked 1 L jacketed reactor equipped with a mechanical stirrer was charged with a solution of 17.40 g (0.033 mol (in theory)) of the crude ester prepared in step 2 and 250 ml of ethanol. The solution was cooled to 0 ° C and 14.63 g (0.033 mol) of lead acetate was added in one portion. After 2 hours, 30 mL of 15% sodium hydroxide solution was added and the ethanol was removed under reduced pressure. An additional 100 mL of 15% sodium hydroxide solution was added and the mixture was extracted twice with 100 mL of methyl tert-butyl ether, washed twice with 100 mL of water and 50 mL of brine, dried over sodium sulfate, filtered through celite and evaporated under reduced pressure. pressure to give 12.46 g of an orange oil. The oily substance in the thin layer chromatography was homogeneous and was used without further purification.

517 / B

3Β

The oily substance prepared in Method 3A was diluted with 30 mL of ethanol and 8.18 g (0.043 mol, 1.3 equivalents) of p-toluenesulfonic acid was added. The solution was heated at reflux for 8 hours, cooled to room temperature and evaporated under reduced pressure. The residue was treated with 20 mL of tetrahydrofuran and heated to reflux to form a solution. The solution was cooled to room temperature and the compound crystallized. 30 ml of heptane and 10 ml of tetrahydrofuran were added to form a liquid suspension, which was filtered. The cake was washed with 40 mL of a 1: 1 mixture of tetrahydrofuran and heptane and vacuum dried on a pressure filter under a nitrogen atmosphere for 2 hours to give 7.40 g of a white solid.

Proton and carbon NMR and IR spectra were identical to the desired product as essentially one enantiomer.

Microanalysis for C18H21Cl2NO6S. 0,25 C4H8O (%):

calculated: 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 preparation

A 500 ml round bottom flask equipped with a magnetic stir bar and a nitrogen gas inlet was charged with 21.7 g (0.065 mol) of the free base of the product produced in step 3, 17.7 g (0.065 mol) of N-tert-butyldicarbonate-gly-N- hydroxysuccinimide ester and 200 ml dimethylformamide. The reaction mixture was stirred under a nitrogen atmosphere at 3.25 h

517 / B of room temperature and a light orange solution formed. The reaction mixture was poured into 1.2 L of ice cold ethyl acetate. The organic solution was washed with 250 ml of 1M hydrochloric acid solution and then with 500 ml of sodium chloride solution, dried over magnesium sulfate and evaporated to dryness under reduced pressure to give an oily substance which was subsequently dried at 50 ° C to give 28 ° C. 12 g (99% yield) of the product as a colorless oil. The mother crystals were prepared from a mixture of ethyl acetate and hexanes. Approximately 28 g of the product was dissolved in 35 mL of ethyl acetate and 125 mL of hexanes. The solution was seeded with the mother crystals and a precipitate formed. The solids were filtered and dried overnight under vacuum at 55 ° C to yield 27.0 g (95% yield of a colorless solid).

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

Step 5

preparation

27.0 g (0.062 mol) of di-tert-butyl dicarbonate protected glycamide prepared in step 4 were dried overnight with phosphorus pentoxide and sodium hydroxide pellets. The solid was dissolved in 40 mL of dioxane and the solution was cooled to 0 ° C. An equivalent volume of 4N hydrochloric acid in dioxane (0.062 mol) was added and the reaction was allowed to proceed for 2 hours. At this point, the conversion (reversed phase HPLC) was 80%. The reaction mixture was allowed to warm to room temperature over 4 hours. The reaction mixture was evaporated to a foam at 40 ° C which was triturated with 200 mL diethyl ether. The white solid that formed was filtered and dried with phosphorus pentoxide to yield 20.4 g (88.5% isolated yield).

517 / B of the desired glycine-β-amino acid ethyl ester compound as the hydrochloride salt.

MS and HH NMR were consistent with the desired structure.

Example J

preparation

• HCl

Step 1

preparation

129.42 g (0.4 mol) of methoxyethoxymethyl protected 3-bromo-5-chlorosalicylaldehyde were prepared according to the method of Example I, Step 2A. 3,5-Dichlorosalicylaldehyde was replaced with an equivalent amount of 3-bromo-5-chlorosalicylaldehyde to fill a 2-L three-necked round-bottomed flask equipped with a mechanical stirrer, followed by 640 mL of tetrahydrofuran and 54.86 g (0.4 mol) (S). ) -fenylgiycinolu. After 30 minutes at 30 ° C, 80 g magnesium sulfate was added. The mixture was stirred at 22 ° C for 1 hour and filtered on a coarse filter. The filtrate was evaporated under reduced pressure to give 180.0 g of a pale yellow oily substance

517 / B desired imine. No further purification was performed and the crude imine was directly used in the coupling reaction, step 2.

Microanalysis for C 19 H ₂₁ iBrCINO ₄ (%):

calculated: 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 preparation

In a 5 L three-necked, round-bottomed flask equipped with a mechanical stirrer, 332.0 g (0.8 mol) of the reagent produced in Example 1, Step 1, was dissolved in 660 mL of N-methylpyrrolidinone under nitrogen. Then the solution was cooled to -10 ° C. A solution of the imine (prepared in Step 1) in 320 mL of N-methylpyrrolidinone was prepared under nitrogen atmosphere and then added to the above reaction mixture over 30 minutes while maintaining the temperature at -5 ° C. After complete addition the mixture was stirred at-8 ^and C for 1 hour and at -5 ° C for 2 hours, then cooled to -10 ° C. A mixture of 30 ml of concentrated hydrochloric acid and 720 ml of saturated ammonium chloride solution was added over 10 minutes. 760 ml of methyl tert-butyl ether was added and the mixture was stirred at 23 ° C for 30 minutes. Stirring was complete and the layers were separated. The aqueous layer was extracted with 320 ml of methyl tert-butyl ether. The two organic layers were combined, washed successively with 320 mL saturated ammonium chloride solution, 320 mL deionized water, and 320 mL sodium chloride solution. The solution was dried with 60 g magnesium sulfate, filtered and evaporated to give 221.0 g of a yellow oil containing the desired product as

517 / B single diastereoisomer as determined by proton NMR.

DSC: 211.80 ° C (endo. 72.56 J / g), 228.4 ° C (98.23 J / g). Microanalysis for C25H33BrCINO ₆ (%): calculated: 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 C: 6.33 Step 3 preparation Shoah 1 I OH ABOUT M FROM · br 1 ch ₃

A 3-L three-necked round-bottomed flask equipped with a mechanical stirrer was charged under a nitrogen atmosphere with a solution of about 111 g of the crude ester prepared in step 2 in 1500 ml of ethanol. The reaction mixture was cooled to 0 ° C and 88.67 g (0.2 mol) of lead acetate was added in one portion. The reaction mixture was stirred at 0 ° C for 3 hours and then 150 mL of 15% aqueous sodium hydroxide solution was added to the reaction mixture below 5 ° C. The methanol was rotovapped under reduced pressure. An additional 150 ml of 15% aqueous sodium hydroxide solution was added and the reaction mixture was extracted three times in succession with 300 ml of ethyl acetate and washed twice in succession with 100 ml of deionized water and twice with 100 ml of sodium chloride solution and dried with 30 g of anhydrous magnesium sulfate. It was then filtered through celite and evaporated under reduced pressure to give 103 g of the desired product as a red oil.

517 / B

Step 4

preparation

Cl c

Br .OH · HCl

The above compound was prepared according to the method used in Example 1, step 4 and step 5, substituting an equivalent amount of the product of step 3 in example 1, step 4. MS and ¹ H NMR were consistent with the desired structure.

Example K

Alternative Preparation of Example J

Step 1: Preparation

COjEt .OH

br

To 50.0 g (139.2 mmol) of the product of Example B, Step 3 and 33.5 g (398.3 mmol) of sodium bicarbonate were added 500 mL of dichloromethane and 335 mL of water. The mixture was stirred at room temperature for 10 minutes. A solution of 38.0 g (222.8 mmol) of benzyl chloroformate in 380 ml of dichloromethane was added with rapid stirring over 20 minutes. After 50 minutes, the reaction mixture was introduced into a separatory funnel and the organic layer was collected. The aqueous phase was washed with 170 ml of dichloromethane. The combined organic layers were dried over sulfate

517 / B magnesium and evaporated in vacuo. The resulting gummy solid was triturated with hexane and collected by filtration. The tan solid was dried under vacuum to give 61.2 g (96% yield). This material was subjected to reverse phase HPLC using a chiral column to obtain each of the enantiomers in pure form. A Whelk-0 (R, R) column, particle size 10 µηη was used, using a mobile phase of a 90: 10 mixture of heptane and ethanol. An optical purity of> 98% was determined using analytical HPLC using similar column and dissolution conditions. . ¹ H NMR was consistent with the proposed structure.

Step 2

To a solution of 48.5 g (106.2 mmol) of the compound obtained in Step 1 in 450 mL of dichloromethane was added via cannula 25.5 g (127.4 mmol) of trimethylsilyl iodide in 100 mL of dichloromethane. The orange solution was stirred at room temperature for 1 hour. Methanol (20.6 mL, 509.7 mmol) was added dropwise and the solution was stirred for 15 minutes. The reaction solution was evaporated in vacuo to give an orange oil. The residue was dissolved in methyl tert-butyl ether (500 ml) and extracted with 1N hydrochloric acid (318 ml) and water (200 ml) and water (100 ml). The aqueous extracts were backwashed with 100 mL of methyl tert-butyl ether. To the aqueous solution was added 40.1 g (478 mmol) of sodium bicarbonate in small portions. The basic aqueous mixture was extracted once with 1 L and twice with 200 ml of methyl tert-butyl ether. The combined organic solution was washed with brine and evaporated in vacuo to give 23.3 g (68% yield) of the desired product.

¹ H NMR was consistent with the proposed structure.

517 / B

Step 3

preparation

To a solution of 23.3 g (72.1 mmol) of the product of Step 2 in 200 mL of DMF was added 17.9 g (65.9 mmol) of N-di-tert-butyldicarbonate glycine N-hydroxysuccinimide ester. The reaction mixture was stirred at room temperature for 20 hours. The mixture was poured into 1.2 L of ethyl acetate and washed twice with 250 ml of 1M hydrochloric acid, twice with 250 ml of saturated sodium bicarbonate solution and twice with 250 ml of sodium chloride solution. The solution was dried with magnesium sulfate and evaporated to give 32.0 g (100% yield) of the desired product.

Analysis for C ₁₈ H ₂₄ BrClN ₂ O6 (%):

H, 5.04; N, 5.84

Found: C 45.17 H 5.14 N 6.12 ¹ H NMR was consistent with the desired structure.

Step 4

HCl

517 / B

To a solution of 31.9 g (66.5 mmol) of the product of Step 3 in 205 mL of absolute ethanol was added 111 mL (332.4 mmol) of a 3M ethanolic hydrochloric acid solution. The reaction solution was heated at 58 ° C for 30 minutes. The solution was cooled and evaporated in vacuo. The residue was dissolved in 250 mL of ethyl acetate and stirred at 0 ° C for 2 hours. The white precipitate was collected by filtration and washed with cold ethyl acetate. The solid was dried under vacuum to give 23.5 g (85% yield) of the desired product.

Analysis for C 13 H ₁₆ BrClN ₂ O ₄ + 1.0 HCl (%):

H, 4.12; N, 6.73

Found: C 37.29 H 4.06 N 6.68 ¹ H NMR was consistent with the desired structure.

Example L

preparation

COnEt .OH • HCl

br

Cl

Step 1

preparation

br

Cl

517 / B

To a solution of 35.0 g (0.15 mol) of 3-chloro-5-bromosalicylaldehyde in 175 ml of dimethylformamide at room temperature was added 22.1 g (0.16 mol) of potassium carbonate (powder, dried in an oven under vacuum at 100 ° C) to obtain a light yellow suspension. 25.0 g (0.2 mol) of methoxyethoxymethyl chloride (neat) was then added while maintaining the bath temperature. The mixture was then stirred at 22 ° C for 6 hours and poured into 1200 mL deionized water to precipitate the product. The suspension was filtered with a pressure filter and the cake was washed twice with 400 ml of deionized water and dried under an atmosphere of a mixture of nitrogen and vacuum to give 46.0 g (95% yield) of the product as a gray-white solid.

¹ H NMR (CDCl ₃ , TMS): 3.35 (s, 3H), 3.54-3.56 (m, 2H), 3.91-3.93 (m, 2H), 5.30 (s 2 H, 7.77 (d, 1 H), 7.85 (d, 1 H), 10.30 (s, 1 H).

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).

Microanalysis for C 11 H 12 BrClO 4 (%): Calculated: C 40.82 H 3.74 Cl 10.95

Found: C 40.64 H 3.48 Cl 10.99

Br, 24.69

Br, 24.67

Step 2

preparation

517 / B

To a 500 ml three-necked round-bottomed flask equipped with a mechanical stirrer was added 32.35 g (0.1 mol) of the product of Step 1, followed by 160 ml tetrahydrofuran and 13.71 g (0.1 mol) (S). phenylglycinol. After 30 minutes at 22 ° C, 20 g of magnesium sulfate was added. The mixture was stirred at 22 ° C for 1 hour and filtered on a coarse filter. The filtrate was evaporated under reduced pressure to give 48.0 g of a pale yellow oil containing the desired imine. No further purification was performed and the crude product was directly used in the next reaction step.

Microanalysis for C ^ bhiBrCINO (%):

Calculated: C 51.54 Found: C 51.52

Step 3

preparation

H, 4.78

H, 5.02

N 3.16 N 2.82

Br, 18.04

Br, 16.31

Cl 8.00 Cl 7.61

In a 5 L three-necked round-bottomed flask equipped with a mechanical stirrer, 332 g (0.8 mol) of the reagent produced in Example I, Step 1, was dissolved in 660 mL of N-methylpyrrolidinone. The solution was cooled to -10 ^{& C.} under nitrogen, a solution of imine (prepared in Step 2) in 320 ml Nmetylpyrolidinónu and while maintaining the temperature at -5 ° C, over 30 minutes was added to the above of the reaction mixture. After the addition was complete, the mixture was stirred for an additional 1 hour and cooled to -10 ° C. A mixture of 30 ml of concentrated acid was added over 10 minutes

517 / B hydrochloric acid and 720 ml of saturated ammonium chloride solution. 760 ml of methyl tert-butyl ether was added and the mixture was stirred at 23 ° C for 1 hour. Stirring was complete and the layers were separated. The aqueous layer was extracted with 320 mL of methyl tert-butyl ether. The two organic layers were combined, washed successively with 320 mL saturated ammonium chloride solution, 320 mL deionized water, and 320 mL sodium chloride solution. The solution was dried with 60 g magnesium sulfate, filtered and evaporated to give 228 g of a yellow oil containing the desired product as one diastereomer.

DSC: 227.54 ° C (endo 61.63 J / g). Microanalysis for C ₂ 5H33BrCINO6 (in%):

H, 5.95

Found: C, 53.80; H, 6.45

N 2.50 Br 14.29 Cl 6.33

N 2.23 Br 12.85 Cl 6.12

Step 4

preparation

A 3-L three-necked round-bottomed flask equipped with a mechanical stirrer was charged with a solution of about 111 g of the crude ester prepared in step 3 in 1500 ml of ethanol under a nitrogen atmosphere. The reaction mixture was cooled to 0 ° C and 88.67 g (0.2 mol) of lead acetate was added in one portion. The reaction mixture was stirred at 0 ° C for 3 hours and then 150 ml of 15% aqueous sodium hydroxide solution was added to the reaction mixture below 5 ° C. Ethanol was evaporated on a rotary evaporator under reduced pressure. An additional 600 mL of a 15% aqueous hydroxide solution was added

The reaction mixture was extracted twice with 300 ml of ethyl acetate and twice with 200 ml of methyl tert-butyl ether and twice with 200 ml of ethyl acetate. The organic layers were combined and washed twice with 200 ml of deionized water and twice with 100 ml of brine and dried with 30 g of anhydrous magnesium sulfate. Then the solution was filtered through Celite and evaporated under reduced pressure to give 96 g of the desired product as an orange oil which was used in the next step without further purification.

DSC: 233.60 ° C (endo 67.85 J / g). Microanalysis for C24H29BrCINOs (%):

calculated: 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

preparation

About 94 g of the crude product from step 4 was dissolved in 180 mL of absolute ethanol and 50.0 g (0.26 mol) of p-toluenesulfonic acid monohydrate was added. Then the reaction mixture was heated under reflux for 8 hours, then the solvent was evaporated under reduced pressure. The solid residue was added to 100 mL of tetrahydrofuran and then the tetrahydrofuran was removed under reduced pressure. The residue was dissolved in 500 mL of ethyl acetate and cooled to about 5 ° C. The solid was filtered and washed twice with 50 mL of heptane to give a white solid. The solid was air dried to give 38 g of a white solid as one isomer.

517 / B ¹ H NMR (CDCl 3, TMS) (ppm): 1.12 (t, 3H), 2.29 (s, 3H), 3.0 (m, 2H), 4.05 (q, 2H) 4.88 (t, 1H); 7.11 (d, 2H); 7.48 (d, 2H); 7.55 (d, 1H); 7.68 (d, 1H); 8.35 ( broad s, 3H).

¹³ C NMR (CDCl 3, 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 C (end. 406.5 J / g), 165.72 ’C (end. 62.27 J / g), 211.24 ° C (exo. 20.56 J / g).

[α] D 25 = +4.2 ’(c = 0.960, methanol).

IR: (MIR) (cm ^-1 ): 2922, 1726, 1621, 1591, 1494, 1471, 1413, 1376, 1324, 1286, 1237, 1207.

Microanalysis for C ₁₅ H ₂ iBrCINO ₆ S (in%): calculated: 6.48 C, 43.69 H, 4.27; N, 2.83 Br, 16.15 Cl 7.16 S found: 6.54 Step 6 C, 43.40 H, 4.24; N, 2.73 Br, 16.40 Cl 7.20 S preparation COjEf Ä • HCI

The above compound was prepared according to the methods described in Example I, step 4 and step 5, replacing the equivalent amount of the intermediate prepared in step 5 as the free base in example I, step 4.

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

517 / B

Example M

preparation

H

N

'CO2 = t

OH

Cl • HCl

Step 1

Preparation of 3-iodo-5-chlorosalicylaldehyde

To a solution of 100 g (0.638 mol) of 5-chlorosalicylaldehyde in 400 ml of dimethylformamide was added 144.0 g (0.641 mol) of N-iodosuccinimide. The reaction mixture was stirred at room temperature for 2 days. An additional 20 g of iodosuccinimide was added and stirring was continued for a further 2 days. The reaction mixture was diluted with 1 L of ethyl acetate, washed with 300 mL of 0.1 N hydrochloric acid, 300 mL of water, 300 mL of 5% sodium thiosulfate solution, 300 mL of sodium chloride solution, dried with magnesium sulfate and evaporated to dryness to give 162 g ( yield 90%) of the desired aldehyde as a pale yellow solid.

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

Step 2

Preparation of 2-O-methoxyethoxymethyl-3-iodo-5-chlorosalicylaldehyde

To a solution of 84.74 g (0.30 mol) of 3-iodo-5-chlorosalicylaldehyde in 200 ml of dimethylformamide was added 41.4 g (0.30 mol) of potassium carbonate at 20 ° C. A yellow suspension formed and 38.2 g (0.305 mol) of methoxyethoxymethyl chloride was added while maintaining the reaction temperature. After 2 hours an additional 1.5 g of methoxyethoxymethyl chloride was added. After stirring for 1 hour, the reaction mixture was poured into a mixture of water and ice and stirred. A precipitate formed, which was filtered and dried in vacuo to give 95 g (85% yield).

517 / B of the desired protected aldehyde.

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

Step 3

preparation

To a solution of 41.5 g (0.112 mol) of 2-O-methoxyethoxymethyl-3-iodo-5-chlorosalicylaldehyde in 200 ml of tetrahydrofuran at room temperature was added 15.37 g (0.112 mol) of (S) -phenylglycinol. After stirring for 1 hour, 16 g of magnesium sulfate was added and stirring was continued for 2 hours. The reaction mixture was filtered and the filtrate was evaporated and dried in vacuo for 2 hours to give the desired intermediate imine. A two-necked round-bottomed flask was charged with 81.8 g (0.2464 mol) of the Reformant reagent of Example I, Step 1 and 300 ml of N-methylpyrrolidone and stirred at -10 ° C. While maintaining the temperature at -10 ° C, a solution of the imine in 100 mL of N-methylpyrrolidone was added slowly. The mixture was kept at this temperature for 2 hours and at -5 ° C for 1 hour. After cooling the reaction mixture to -10 ° C, a solution of 16 ml of concentrated hydrochloric acid in 200 ml of saturated ammonium chloride solution was added. 500 ml diethyl ether was added and the mixture was stirred at room temperature for 2 hours. The ether layer was separated and the aqueous layer was further extracted with 300 mL diethyl ether. The combined ether layers were washed with 200 mL saturated ammonium chloride solution, 200 mL water and 200 mL sodium chloride solution, dried over magnesium sulfate and evaporated to give 61.0 g (90% yield) of an oily substance.

1 H NMR showed that the desired structure was essentially one diastereomer and MS was identical to the desired structure.

517 / B

Step 4

preparation

H2I

SO ₃ H ch ₃

A solution of 48.85 g (80.61 mmol) of the crude ester prepared in Step 3 was dissolved in 500 mL of ethanol and cooled to 0 ° C. 35.71 g (80.61 mmol) of lead acetate was added. After 3 hours, 73 mL of 15% sodium hydroxide solution was added to the reaction mixture. Most of the ethanol was evaporated under reduced pressure. To the residue was added 200 mL of 15% sodium hydroxide solution, which was then extracted with 400 mL of diethyl ether. The ether layer was washed with 100 mL of water, 100 mL of brine, dried and evaporated to give an orange oil. The oily substance was dissolved in 100 ml of ethanol and 19.9 g of p-toluenesulfonic acid was added. The solution was heated at reflux for 8 hours and evaporated under reduced pressure. The residue was diluted with 60 mL of tetrahydrofuran, heated to reflux and cooled. The precipitate was filtered, washed with 300 mL of 1: 1 hexane: tetrahydrofuran and dried to give the desired product.

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

Step 5 (S) -ethyl 3- (N-di-tert-butyldicarbonate-gly) -amino-3- (S) - (5-chloro-2-hydroxy-3-iodo) phenylpropionate

To a mixture of 9.4 g (34.51 mmol) of di-tert-butyl dicarbonate-gly-O-succinate and 17.0 g (31.38 mmol) of p-toluenesulfonic acid salt with ethyl-3- (S) -amino- 3- (5-chloro-2-hydroxy-3-iodo) propionate in 200 ml of dimethylformamide was added 4.8 ml of triethylamine. The reaction mixture was stirred at room temperature for 18 hours. Dimethylformamide was evaporated in vacuo and the residue was partitioned between 600 mL

517 / B ethyl acetate and 100 ml dilute hydrochloric acid. The organic layer was washed with 200 mL of sodium bicarbonate solution, 200 mL of sodium chloride solution, dried over magnesium sulfate and evaporated to give 14.2 g (yield 86%) of the desired product as a solid.

MS and ¹ 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

To 37.20 g (70.62 mmol) of ethyl [3- (S) - (N-di-tert-butyldicarbonate-gly) -amino-3- (5-chloro-2-hydroxy-3-iodo) phenylpropionate was added at 70 ml of a 4N solution of hydrochloric acid in dioxane was added at 0 ° C and stirred at room temperature for 3 hours. The reaction mixture was evaporated and re-evaporated after addition of 100 ml of toluene. The obtained residue was suspended in diethyl ether, filtered and dried to give 32.0 g (yield 98%) of the product as a crystalline powder.

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

Example N

preparation

NH

CO 3 Et, OH • HCl

br

517 / B

Step 1

preparation

The above compound was prepared according to the method of Example I, step 2A, substituting 3,5-dichlorosalicylaldehyde with an equivalent amount of 2-hydroxy-3,5-dibromobenzaldehyde. Yield 88%, light yellow solid, mp: 46-47 ° C. _Rf = 0.6 (ethyl acetate-hexane = 1: 1 vol.).

¹ H 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 &lt; ^{+ &gt;} ).

HR-MS calcd for C ₁₁ H ₁₂ Br ₂ O 4: 367.9083 found: 367.9077

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

2B and Step 2C, substituting an equivalent amount of the compound of Step 1 in Example I, Step 2B.

517 / B

Yield 90%, yellow solid, mp: 57-59 ° C.

Rf = 0.46 (1: 1 v / v ethyl acetate / hexane).

¹ H NMR (CDCl ₃ ): d 1.45 (s, 9H), 2.1 (broad, 1H, exchangeable), 2.51 (d, 1H, J 1 = 9.9 Hz, J ₂ = 15, 3 Hz), 2.66 (d, 1H, J 1 = 4.2 Hz, J ₂ = 15.3 Hz), 3.02 (broad, 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 2 H, 7.17-7.25 (m, 6H), 7.49 (d, 1H).

FAB-MS mlz: 602 (M + H).

HR-MS calcd for C 25 H 34 N ₂ O ₂ O ₆ : 602.0753 found: 602.0749

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

Step 3

preparation

The above compound (p-toluenesulfonic acid salt) was prepared by the method of Example I, step 3, replacing the equivalent amount of the product prepared in step 2 of Example I, step 3A.

Yield 62%, white solid.

¹ H NMR (DMSO-d 6): d 1.09 (t, 3H, J = 7.2 Hz), 2.27 (s, 3H), 2.97 (dd, 2H, J) = 3.0 Hz J ₂ = 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 (broad, 3H).

FAB-MS mlz: 365 (M + H).

HR-MS calc for C ₁₁ H 14 Br ₂ O ₃ : 365.9340

517 / B found: 365.9311

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

Step 4

preparation

HCl

The above compound was prepared by the method of Example I, step 4, replacing the compound prepared in step 3. The resulting di-tert-butyl dicarbonate protected intermediate was converted to the desired compound using the method of Example I, step 5.

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

Example P

preparation

HCl

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

517 / B

Example 1

(±) -3-Bromo-5-chloro-2-hydroxy-β - [/ 2 - ([(3-hydroxy-5 [(1,4,5,6-tetrahydro-5-hydroxy-2)] - trifluoroacetate salt pyrimidinyl) amino] phenyl) carbonyl] amino) acetyl / aminojbenzénpropánovej

preparation

At an ice bath temperature, 0.4 g (0.0014 mol) of the product of Example H, 0.58 g (0.0014 mol) of the product of Example B, 0.142 g (0.0014 mol) of triethylamine, 17 mg of 4- (N, N-dimethylaminopyridine) and 4 ml of anhydrous N-dimethylacetamide added 0.268 g (0.0014 mol) of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride. The reaction was stirred overnight at room temperature. The resulting ester intermediate was isolated by preparative reverse phase HPLC. To this ester was added 580 mg (0.0138 mol) of lithium hydroxide in 10 mL of water and 5 mL of acetonitrile. After stirring at room temperature for 1 hour, the pH was lowered to 2 with trifluoroacetic acid and the product was purified by preparative reverse phase HPLC to afford (after lyophilization) 230 mg of the product as a white solid.

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

517 / B

Example 2

preparation

The above compound was prepared by the method of Example 1, substituting the product of Example B with an equivalent amount of the product of Example A. The yield after lyophilization was 320 mg of a white solid.

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

Example 3

preparation

The above compound was prepared according to the method of Example 1, substituting the product of Example B with an equivalent amount of the product of Example F. The yield after freeze-drying was 180 mg of a white solid.

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

517 / B

Example 4

preparation

ABOUT

The above compound was prepared by the method of Example 1, substituting the product of Example B with an equivalent amount of the product of Example D. The yield after lyophilization was 180 mg of a white solid.

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

Example 5

preparation

The above compound was prepared by the method of Example 1, substituting the product of Example B with an equivalent amount of the product of Example E. The yield after lyophilization was 250 mg of a white solid.

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

Example 6

preparation

517 / B

The above compound was prepared according to the method of Example 1, substituting the product of Example B with an equivalent amount of the product of Example C. The yield after lyophilization was 220 mg of a white solid.

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

Example 7

preparation

3.7 g (0.027 mol) of isobutyl chloroformate were slowly added to a 7.8 g (0.027 mol) product of Example H dissolved in 50 ml of anhydrous N, N-dimethylacetamide in a flame annealed flask under an ice bath temperature. , 73 g (0.027 mol) of N-methylmorpholine. The solution was stirred at ice bath temperature for 15 minutes. Then, 10.0 g (0.024 mol) of the product of Example L was added to the reaction mixture at an ice bath temperature, followed by 2.43 g (0.024 mol) of N-methylmorpholine. Then the reaction was stirred overnight at room temperature. The resulting ester intermediate was isolated by preparative reverse phase HPLC. To this ester in 60 mL of water and 30 mL of acetonitrile was added 10 g (0.238 mol) of lithium hydroxide. The reaction mixture was stirred at room temperature for 1 hour. Then the pH was lowered

517 / B to 2 with trifluoroacetic acid. The product was purified by reverse phase preparative chromatography to afford (after lyophilization) 9.7 g of the desired product as a white solid.

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

Example 8

(S) -3,5-Dichloro-2-hydroxy-p - [/ 2 - ([(3-hydroxy-5 - [(1,4,5,6-tetrahydro-5-hydroxy-2-pyrimidinyl) monohydrochloride)] monohydrate amino] phenyl) carbonyl] amino) acetyl / amino] benzenepropanoic acid

preparation

• HCl

Step A

To 9.92 g (0.0345 mol) of the product of Example H dissolved in 200 ml of anhydrous Ν, Ν-dimethylacetamide was added 4.0 ml (0.0362 mol) of N-methylmorpholine. The reaction mixture was cooled to -5 ° C (ice-salt bath), 4.48 mL (4.773 g, 0.0345 mol) of isobutyl chloroformate was added over one minute, and the reaction mixture was stirred for 12 minutes at an ice bath temperature. 11.15 g (0.030 mol) of the product of Example I was then added to the reaction mixture at an ice bath temperature, followed by 4.0 ml (0.0362 mol) of N-methylmorpholine. The reaction mixture was allowed to warm to room temperature and complete, then evaporated at 50 ° C under vacuum to give a dark residue. The residue was dissolved in approximately 50 mL of acetonitrile / water. The pH was adjusted to the acid side by adding a small amount

517 / B of trifluoroacetic acid. The residue was placed on a 10 x 500 cm C-18 column (50 µm particle size) and the ester of the desired product was isolated (solvent plan: 100% water + 0.05% trifluoroacetic acid to a mixture of water with 0.05% trifluoroacetic acid and acetonitrile). with 0.05% trifluoroacetic acid at a ratio of 30:70 over 1 hour at a rate of 100 ml / min, the solvent schedule started after washing the forehead with solvent). Purification by preparative reverse phase HPLC afforded 10.5 g (50%) of a white solid after lyophilization.

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

Step B

Approximately 11 g of the product produced in Step A was dissolved in a mixture of water and dioxane and the pH of the solution was adjusted to about 11.5 (pH meter) by addition of 2.5N sodium hydroxide solution. The reaction mixture was stirred at room temperature. The pH was repeatedly adjusted to > 11 by further addition of a base. After 2-3 hours, the conversion of the ester to the acid by reverse phase HPLC was judged complete. The pH of the reaction mixture was adjusted to about 6, and a viscous oil precipitated out of solution. The oily substance was isolated by decantation and washed with 200 ml of hot water. The resulting aqueous mixture was allowed to cool and the solid was collected by filtration to give 2.6 g of the title compound after lyophilization from a hydrochloric acid solution. The residue, which was a dark viscous oily substance, was treated with hot water to give (after lyophilization from hydrochloric acid solution) 4.12 g of a tan powder.

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

Example 9

(S) -3-Bromo-5-chloro-2-hydroxy-p - [[2 - ([(3-hydroxy-5 - [(1,4,5,6-tetrahydro-5-hydroxy-2- pyrimidinyl) amino] phenyl) carbonyl] amino) acetyl / aminojbenzénpropánovej

517 / B

Step 1: Preparation

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

MS and ¹ H NMR were consistent with the proposed structure.

Step 2

To a solution of 800 mg (1.2 mmol) of the ester produced in Step 1 in 7 mL of a 1: 4 acetonitrile / water solution was added 148 mg (6.2 mmol) of lithium hydroxide. The reaction mixture was stirred at room temperature for 2 hours. Added

517 / B

0.71 mL (9.2 mmol) of trifluoroacetic acid and this mixture was purified by reverse phase HPLC (starting solvent gradient of water: trifluoroacetic acid / acetonitrile 95: 5, retention time 24 min) to give 860 mg (83% yield) of the desired product. Analysis for C ₂₂ H ₂₃ BrClN ₅ O ₇ + 1.7 TFA (%):

H, 3.20; N, 8.99

Found: C, 39.11; H, 3.17; N, 9.07.

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

Step 3

Preparation of the hydrochloride salt

The product of step 2 was dissolved in a suitable solvent (a mixture of water and acetonitrile) and this solution was slowly passed through a BioRad AG2-8X ion exchange column (chloride form, 0.038-0.074 mm,> 5 equivalents). Lyophilization gave the desired product as the hydrochloride salt.

Example 10

preparation

The above compound was prepared by the method of Example 8, substituting the product of Example N for the product of Example I in Example 8, Step A, with the product of Example N. The product was isolated by preparative reverse phase HPLC and lyophilized to afford the desired product as the trifluoroacetate salt.

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

517 / B

Example 11

preparation

ABOUT

HO

CQjH

OH

The above compound was prepared essentially according to the methods of Example 8, replacing the product of Example I in Example 8, Step A with the product of Example M. The product was isolated by preparative reverse phase HPLC and lyophilized to afford the desired product as the trifluoroacetate salt.

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

Example 12

preparation

HO

CQzH

OH

The above compound was prepared using the methods of Example 8, replacing the product of Example I in Example 8, Step A with the product of Example P. The product was isolated by preparative reverse phase HPLC and lyophilized to afford the desired product as the trifluoroacetate salt.

517 / B

Example 13

preparation

COOH

OH

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

To a solution of 50.0 g (0.134 mol) of 3,5-diiodosalicylaldehyde in 150 ml of dimethylformamide was added 18.5 g (0.134 mol) of potassium carbonate at 20 ° C. This resulted in a yellow suspension and 15.8 ml (0.134 mol) of methoxyethoxymethyl chloride was added while maintaining the reaction temperature. After stirring for an additional hour, the reaction mixture was poured into ice water and stirred. A precipitate formed, filtered and dried in vacuo to give 61 g (yield 99%) of the desired protected aldehyde.

¹ H NMR was consistent with the desired structure.

517 / B

Step 2

preparation

To a solution of 41.5 g (0.112 mol) of 2-O-methoxyethoxymethyl-3,5-diiodosalicylaldehyde in 150 ml of tetrahydrofuran at room temperature was added 17.9 g (0.13 mol) of (S) -phenylglycinol. After stirring for 1 hour, 20.7 g of magnesium sulfate was added and stirring was continued for 2 hours. The reaction mixture was filtered and the filtrate was evaporated and dried in vacuo for 2 hours. A two-necked round-bottomed flask was charged with 96 g (0.289 mol) of Reformant reagent and 250 ml of N-methylpyrrolidone and stirred at -10 ° C. While maintaining the temperature at -10 ° C, 100 mL of a solution of imine in N-methylpyrrolidone was added slowly. The mixture was kept at this temperature for 2 hours and at -5 ° C for 1 hour. After cooling the reaction mixture to -10 ° C, a solution of 16 ml of concentrated hydrochloric acid in 200 ml of saturated ammonium chloride solution was added. 500 ml diethyl ether was added and the mixture was stirred at room temperature for 2 hours. The ether layer was separated and the aqueous layer was further extracted with 300 mL diethyl ether. The combined ether layers were washed with 200 mL of saturated ammonium chloride solution, 200 mL of water and 200 mL of brine, dried over magnesium sulfate and evaporated to give 90.0 g (yield 99%) of an oily substance.

NMR showed the desired structure and one diastereomer.

517 / B

Step 3

preparation

14.0 g (20.1 mmol) of the crude ester from step 2 was dissolved in 100 mL of ethanol and cooled to 0 ° C. 9.20 g (20.75 mmol) of lead acetate was added in one portion. After 3 hours, 73 mL of 15% sodium hydroxide solution was added to the reaction mixture. Most of the ethanol was evaporated under reduced pressure. To the residue was added 200 mL of 15% sodium hydroxide solution, which was then extracted with 400 mL of diethyl ether. The ether layer was washed with 100 mL of water, 100 mL of brine, dried and evaporated to give an orange oil. This was dissolved in 100 ml of ethanol and 6.08 g of p-toluenesulfonic acid was added. The solution was heated at reflux for 8 hours and evaporated under reduced pressure. The residue was diluted with 60 mL of tetrahydrofuran, heated to reflux and cooled. No precipitate formed upon storage. The reaction mixture was evaporated and purified by preparative HPLC to give the amino acid as a p-toluenesulfonic acid salt. The solid obtained was dissolved in ethanol and saturated with hydrogen chloride gas. The reaction mixture was heated under reflux for 6 hours. The reaction mixture was evaporated to give 12.47 g of the desired amino acid as the ptoluenesulfonic acid salt.

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Step 4

Preparation of ethyl [3- (N-di-tert-butyldicarbonate-gly) amino-3- (S) - (3,5-diiodo-2-hydroxyphenyl) propionate]

To a mixture of 7.48 g (27.04 mmol) of di-tert-butyldicarbonate-gly-O-succinate and 12.47 g (27.04 mmol) of p-toluenesulfonic acid salt and ethyl- [3- (S) -amino 3- (3,5-diiodo-2-hydroxyphenyl) propionate] in 100 mL of dimethylformamide was added 3.8 mL of triethylamine. The reaction mixture was stirred at room temperature for 18 hours. The dimethylformamide was evaporated in vacuo and the residue was partitioned between 600 mL of ethyl acetate and 100 mL of dilute hydrochloric acid. The organic layer was washed with 200 mL of sodium bicarbonate solution, 200 mL of brine, dried over magnesium sulfate and evaporated to give 17.0 g (96% yield) of the desired product as a solid.

¹ H NMR was consistent with the desired product.

Step 5

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

517 / B

To 40 ml of a 4N solution of hydrochloric acid in dioxane was added 17.0 g (25.97 mmol) of 3- (S) - (N-di-tert-butyldicarbonate-gly) amino-3- (S) at 0 ° C. Of (3,5-diiodo-2-hydroxyphenyl) propionate and the reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was evaporated and re-evaporated after addition of 100 ml of toluene. The obtained residue was dried to give 8.0 g (56% yield) of the product as a crystalline powder.

¹ H NMR was consistent with the desired product.

Step 6

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

¹ H NMR was consistent with the desired product.

517 / B

Examples 14 to 18

Compounds of formulas VII, VIII, IX, XIII and XIV and their isomers may be prepared at this site as described above, substituting the appropriate starting materials and reagents, as will be apparent to one of ordinary skill in the art.

The activity of the compounds of this invention was tested by the following tests. The results of testing in the tests are summarized in Table 1.

Adhesion tests with vitronectin

Materials

The human vitronectin receptor was isolated from the human placenta as described above (Pytela et al., Methods in Enzymology, 144, 475-489 (1987)). Human vitronectin was isolated from freshly frozen plasma as described above (Yatohgo et al., Cell structure and function, 13, 281-292 (1988)). Human biotinylated vitronectin was prepared by condensing NHS-biotin obtained from Pierce Chemical Company (Rockford, II) with isolated vitronectin as previously described (Charo et al., J. Biol. Chem., 266, 3, 1415-1421 (1991)). )). Test buffer, POD substrate tablets, and RIA grade BSA were obtained from Sigma (St. Louis, MO). Antibiotin antibody was obtained from Calbiochem (La Jolla, CA). Linbro microtiter plates were obtained from Flow Labs (McLean, VA). ADP reagent was purchased from Sigma (St. Louis, MO).

ways

Solid phase receptor assays

This assay is essentially the same as previously reported (Niiya et al., Blood, 70, 475-483 (1987)). Purified human vitronectin receptor (α _ν β ₃ ) in sodium chloride buffered with TRIS buffer containing 1.0 mM

517 / B

Ca ⁺⁺ , Mg ⁺⁺ and Mn ⁺⁺ at pH = 7.4 (TBS ⁺⁺⁺ ) were diluted from the stock solutions to a content of 1.0 g / ml. The diluted receptor was immediately transferred to Libro microtiter plates at 100 μΙ / well (100 ng receptor / well). Plates were capped and incubated at 4 ° C overnight to allow receptor binding to wells. All remaining steps were performed at room temperature. The test plates were emptied and 200 μ11% of RIA in TBS ⁺⁺⁺ (TBS ⁺⁺⁺ / BSA) was added to block exposed plastic surfaces. After 2 hours of culture, the assay plates were washed with TBS ⁺⁺⁺ using a 96 well plate washer. A log dilution serial dilution of test compound and controls produced a starting solution at a stock concentration of 2 mM using 2 nM biotinylated vitronectin in TBS ⁺⁺⁺ / BSA as the diluent. This premixing of the labeled ligand with the test (or control) ligand and the sequential transfer of aliquots of 50 μΙ onto the assay plate was performed by CETUS Propette, the final labeled ligand concentration was 1 nM and the highest test compound concentration was 1.0 x 10 ⁴ M. For 2 hours, all wells were washed with a plate washer as above. Affinity purified horseradish peroxidase labeled goat antibody was diluted 1: 3,000 in TBS ⁺⁺⁺ / BSA and 125 μΙ was added to each well. After 30 minutes, the plates were washed and incubated with OPD / H ₂ O substrate in 100 mM / Ι citrate buffer at pH = 5.0. The plate was detected with a microtiter plate detector at 450 nm and a final A450 for analysis was recorded when the absorbance value of the control wells with a maximum binding of approximately 1.0 was reached. Data were analyzed using a macro program written for use with the EXCEL program. The mean value, standard deviation and% CV were determined for the double concentration. Mean A450 values were normalized to the average of 4 maximal binding controls (without the addition of competitor) as B-MAX. Normalized values were subjected to a four-parameter algorithmic approximation curve (Rodbar et al., Int. Atomic Energy Agency, Vienna, p. 469 (1977)) assembled on a semi-logarithmic scale and calculated to correspond to 50% inhibition of maximum biotinylated vitronectin binding (IC50). R ² was recorded for compounds exhibiting greater

517 / B as a 50% inhibition at the highest concentration tested, or IC ₅₀ of greater than the highest concentration tested is recorded. Β - [[2 - ([5 [(aminoiminomethyl) amino] -1-oxopentyl] amino) -1-oxoethyl / amino] -3-pyridinepropanoic acid (USSN 08/375 338, Example 1), which is a potential α antagonist _ν β3 (IC 50 in the range of 3 to 10 nM) was included as a positive control on each plate.

Isolated Ib / IIIfa receptor assays

Materials

Human fibrinogen receptor (α ^ ββ) was isolated from old platelets (Pytela, R., Pierschbacher, MD, Argraves, S., Suzuki, S. and Rouslahti, E., Arginine-Glycine-Aspartic acid adhesion receptors, in Methods in Enzymology, 144: 475-489 (1987)). Human vitronectin was isolated from fresh frozen plasma as described by Yatohgo, T., Izumi, M., Kashiwagi, H., and Hayashi, M., Novel purification of vitronectin from human plasma by heparin affinity chromatography, in Cell Structure and Function, 13 281-292 (1988). Human biotinylated vitronectin was prepared by condensing NHS-biotin from Pierce Chemical Company (Rockford, II) with isolated vitronectin as described above (Charo, IF, Nannizzi, L., Phillips, DR, Hsu, MA, Scaround Bottomough, RM, Inhibition of fibrinogen binding to GP IIb / IIIa and GP Hla peptide, in J. Biol. Chem., 266, 3, 1415-1421 (1991)). Assay buffer, OPD substrate tablets, and RIA grade BSA were obtained from Sigma (St. Louis, MO). Antibiotin antibody was obtained from Calbiochem (La Jolla, CA). Linbro microtiter plates were obtained from Flow Labs (McLean, VA). ADP reagent was purchased from Sigma (St. Louis, MO).

517 / B

ways

Solid phase receptor assays

This assay is essentially the same as previously reported (Niiya, K., Hodson, E., Bader, R., Byers-Ward, V., Koziol, JA, Plow, EF and Ruggeri, ZM, Increased surface expression of the membrane. glycoprotein IIb / IIIa complex induced by platelet activation: Relationships to the binding of fibrinogen and plateled aggregation in Blood, 70, 475-483 (1987)). Purified human fibrinogen receptor (cxiibPs) was diluted from stock solutions in saline buffered with TRIS buffer containing 1.0 mM Ca ⁺⁺ , Mg ⁺⁺ and Mn ⁺⁺ at pH = 7.4 (TBS ⁺⁺⁺ ). to 1.0 pg / ml. The diluted receptor was immediately transferred to Linbro microtiter plates at a concentration of 100 μΙ per well (100 ng receptor / well). Plates were capped and incubated at 4 ° C overnight to allow receptor binding to wells. All remaining steps were performed at room temperature. The test plates were emptied and 200 μ11% of RIA in TBS ⁺⁺⁺ (TBS ⁺⁺⁺ / BSA) was added to block exposed plastic surfaces. After 2 hours of culture, the assay plates were washed with TBS ⁺⁺⁺ using a 96 well plate washer. A log dilution serial dilution of test compound and controls produced a starting solution at a stock concentration of 2 mM using 2 nM biotinylated vitronectin in TBS ⁺⁺⁺ / BSA as the diluent. This premixing of the labeled ligand with the test (or control) ligand and successive transfer of aliquots of 50 μΙ to the assay plate was performed by CETUS Propette, the final labeled ligand concentration was 1 nM, and the highest test compound concentration was 1.0 x 10 ⁴ M. For 2 hours, all wells were washed with a plate washer as above. Affinity purified horseradish peroxidase-labeled goat antibody was diluted 1: 3,000 in TBS ⁺⁺⁺ / BSA and 125 μΙ was added to each well. After 30 minutes, the plates were washed and incubated with ODD / H ₂ O ₂ substrate in 100 mM / Ι citrate buffer at pH = 5.0. The plate was detected with a microtiter plate detector at 450 nm and reaching the absorbance value of the control wells with a maximum

517 / B binding of approximately 1.0, the final A450 for analysis was recorded. Data were analyzed using the program as written for use with the EXCEL program. The mean value, standard deviation and% CV were determined for double concentrations. Mean A450 values were normalized to the average of 4 maximum binding controls (no competitor added) as BMAX. Normalized values were subjected to a four-parameter algorithmic approximation curve (Rodbard et al., Int. Atomic Energy Agency, Vienna, p. 469 (1977)), assembled on a semi-logarithmic scale, and calculated to correspond to 50% inhibition of maximum biotinylated vitronectin binding (IC50). R ² is reported for those compounds exhibiting greater than 50% inhibition at the greatest concentration tested and is recorded on the IC _5, which is greater than the highest concentration tested. P - [[2 - ([(5 [(aminoiminomethyl) amino] -1-oxopentyl] amino) -1-oxoethyl / amino] -3-pyridinepropanoic acid) (USSN 08/375 338, Example 1), which is a potential antagonist and _in p3 (IC50) ranging from 3 to 10 nM, was included as a positive control on each plate.

Assays for plasma rich in human platelets

The volunteers were selected from healthy donors who did not take aspirin. Platelet-rich plasma culture and ADP-induced platelet aggregation assays were performed as described by Zucker, M. B., Platelet Aggregation Measured by the Photometric Method in Methods in Enzymology, 169, 117-133 (1989). Standard tourniquet venipuncture methods allowed 45 mL of whole blood to be withdrawn into a 60 mL syringe containing 5 mL of 3.8% sodium citrate. After thoroughly mixing the contents of the syringe, non-clotting whole blood was transferred to a 50 ml conical polyethylene tube. Blood was centrifuged for 12 minutes at room temperature at 200 x mg to sediment non-platelet cells. Platelet-rich plasma was separated from the polyethylene tube and stored at room temperature until use. Platelet-free plasma was obtained after a second centrifugation of the remaining blood for 15 minutes at 2000 x g. Usually the number

517 / B plates from 300,000 to 500,000 per microliter. 0.45 ml of platelet-rich plasma was dispensed into siliconized cuvettes and stirred for 1 minute at 37 ° C at 1100 rpm before addition of 50 μΙ of the pre-diluted test compound. After stirring for 1 minute, aggregation was started by adding 50 μΙ 200 μΜ ADP. Aggregation was recorded for 3 minutes on a Payton duplex channel aggregometer (Payton Scientific, Buffalo, New York). To determine the dose response curve, percent inhibition of the maximal response (sodium chloride control) was used for a series of dilutions of the test compound. All compounds were tested in duplicate and the half maximal inhibition concentration (IC ₅₀ o) was calculated graphically from the dose response curve for those compounds that showed 50% or greater inhibition at the highest test concentration, or an IC ₅₀ greater than the highest tested was recorded. concentration.

Table I

Example α _ν β3 IC 50 (nM) IIb / IIIa _IC50 (nM) 1 0.88 310 2 1.04 430 3 23.7 2 440 4 2.02 575 5 2.13 744 6 6.46 919 7 1.01 262 8 0.40 131 9 0.37 338 9.HCI 0.82 226.2 10 2.26 641 11 - - 12 9.59 1 060

517 / B

Claims (15)

PATENT CLAIMS A compound of the general formula wherein: X and Y represent the same or a different halogen atom, R represents a hydrogen atom or a lower alkyl group and pharmaceutically acceptable salts thereof. A compound according to claim 1 selected from the group consisting of 31,517 / B 31,517 / B 31,517 / B 31 517 / B in which: R represents a hydrogen atom or an alkyl group, or a pharmaceutically acceptable salt thereof. A compound according to claim 1 selected from the group consisting of 31,517 / B 31,517 / B 31,517 / B 100 31,517 / B 101 4. A pharmaceutical composition comprising a therapeutically effective amount of a compound of claim 1 and a pharmaceutically acceptable carrier. 5. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to claim 2 and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising a therapeutically effective amount of a compound of claim 3 and a pharmaceutically acceptable carrier. 7. The method of claim 1, 2 or 3 for the manufacture of a medicament for the treatment of conditions mediated by the integrin? 3, and _the mammal in need of such treatment. Use of a compound according to claim 1, 2 or 3 in the manufacture of a medicament for the treatment of tumor metatase. Use of a compound according to claim 1, 2 or 3 in the manufacture of a medicament for the treatment of solid tumor growth. Use of a compound according to claim 1, 2 or 3 in the manufacture of a medicament for the treatment of angiogenesis. Use of a compound according to claim 1, 2 or 3 in the manufacture of a medicament for the treatment of osteoporosis. Use of a compound according to claim 1, 2 or 3 for the manufacture of a medicament for the treatment of humoral hypercalcemia in malignancy. 31,517 / B 102 Use of a compound according to claim 1, 2 or 3 in the manufacture of a medicament for the treatment of smooth muscle cell migration. Use of a compound according to claim 1, 2 or 3 in the manufacture of a medicament for the treatment of rheumatoid arthritis. Use of a compound according to claim 1, 2 or 3 in the manufacture of a medicament for the treatment of macular degeneration.