KR20010041584A - Meta-azacyclic amino benzoic acid compounds and derivatives thereof being integrin antagonists - Google Patents

Abstract

The present invention relates to a compound of the formula and pharmaceutically acceptable salts and isomers thereof used as α _v β ₃ integrin antagonists.

Description

META-AZACYCLIC AMINO BENZOIC ACID COMPOUNDS AND DERIVATIVES THEREOF BEING INTEGRIN ANTAGONISTS}

Intigreens are a group of glycoproteins on the cell surface that mediate cell adhesion, thereby mediating cell adhesion interactions that occur during various biological interactions. Intigreens are heterodimers consisting of uncovalent α and β polypeptide subunits. Today eleven different α subunits and six different β subunits have been identified. The various α subunits may form intigreens by combining with the various β subunits.

Intigreens, identified as α _v β ₃ (also known as the vitronectin receptor), are known to have tumor metastasis, solid tumor growth (dysplasia), osteoporosis, Paget's disease, malignant humoral calcium hyperemia, angiogenesis (tumor angiogenesis). Play a role in various conditions or diseases, including retinal pain (including degeneration), arthritis (including rheumatoid arthritis), periodontal disease, psoriasis, and smooth muscle cell migration (eg, restenosis) It was confirmed to occupy. In addition, these intigreens have been found to be used as antiviral, antifungal and antibacterial agents. Thus, compounds capable of selectively inhibiting or antagonizing α _v β ₃ may be beneficial for the treatment of this condition.

α _v β ₃ intigrins and other α _v containing intigrins have been found to bind to many Arg-Gly-Asp (RGD) containing substrate polymers. Compounds containing the RGD sequence bind to cell surface receptors by metabolizing extracellular matrix ligands. In general, however, RGD peptides are also known to be nonselective for RGD dependent intigrins. For example, most RGD peptides that bind α _v β ₃ also bind α _v β ₅ , α _v β ₁ , and α _IIb β ₃ . Antagonism of platelet α _IIb β ₃ (also known as fibrinogen receptors) is known to inhibit platelet aggregation in humans. In order to eliminate the bleeding side-effects when treating the conditions or diseases associated with the integrated green α _v β _3, it would be desirable to develop an α _v β ₃ selective antagonist compound of as opposed to α _IIb β _3.

Tumor invasion occurs following a three step process: 1) tumor cell adhesion to extracellular matrix; 2) proteolytic dissolution of the substrate; And 3) migration of cells through the lysed barrier. This process can occur repeatedly and cause metastases at sites away from the original tumor.

See Seftor et al., Proc. Natl. Acad. Sci. USA, Vol. 89 (1992) 1557-1561 reported that α _v β ₃ intigrins have a biological effect on melanoma cell invasion. Montgomery et al., Proc. Natl. Acad. Sci. USA, Vol. 91 (1994) 8856-60, demonstrated that intigrin α _v β ₃ expressed on human melanoma cells promotes survival signals and prevents cell apoptosis. It may be beneficial to mediate tumor cell metastasis pathways using α _v β ₃ intigrin cell adhesion receptors to inhibit tumor metastasis.

Brooks et al., Cell, Vol. 79 (1994) 1157-1164], since systemic administration of antagonists of α _v β ₃ dramatically regresses various histologically obvious human tumors, the antagonists of α _v β ₃ treat abnormal growth (faithful tumor growth). Can be used for).

Adhesion receptors intigrin α _v β ₃ have been found to be markers of angiogenic vessels in chicks and males, so these receptors play an important role in angiogenesis and neovascularization. Vascular formation is characterized by invasion, migration and proliferation of smooth muscle and endothelial cells. Antagonists of α _v β ₃ inhibit the process by selectively promoting apoptosis of cells in the neovascular structure. In addition, growth of new blood vessels or vasculature leads to pathological conditions such as diabetic retinopathy, spot degeneration and rheumatoid arthritis (Adonis et al., Amer. J. Ophthal., Vol. 118, (1994) 445- 450; and Peacock et al., J. Exp. Med., Vol. 175, (1992), 1135-1138). Thus, the α _v β ₃ antagonist may be a useful therapeutic agent for treating such conditions associated with neovascularization (Brooks et al., Science, Vol. 264, (1994), 569-571).

It has been reported that cell surface receptors α _v β ₃ are the major intigrins for osteoclasts that cause adhesion to bone. Bone destructive cells cause bone resorption, and when this bone resorption activity exceeds bone formation activity, it leads to osteoporosis (bone loss), increasing fracture, lethargy and mortality. Antagonists of α _v β ₃ are both ex vivo (Sato et al., J. Cell. Biol., Vol. 111 (1990) 1713-1723) and in vivo (Fisher et al., Endocrinology, Vol. 132 (1993) 1411-1413). It has been shown that it may be an inhibitor of bone destruction cell activity in Antagonism of α _v β ₃ restores the normal balance between bone formation and bone resorption activity by reducing bone resorption. Thus, it may be desirable to provide an antagonist of bone fracture cells α _v β ₃ that can be used in the treatment or prevention of osteoporosis by becoming an effective inhibitor of bone resorption.

In addition, α _v β ₃ intigrins are a therapeutic target for preventing or preventing aberrant proliferation, which is a major cause of restenosis after vasculitis, due to its role in smooth muscle cell migration (Choi et al., J. Vasc. Surg. Vol. 19 (1) (1994) 125-34. It may be desirable to inhibit or inhibit neointimal hyperplasia using drugs to prevent or inhibit restenosis.

Next Biography, Vol. 3 (9) (1993) 596-599, it has been reported that adenoviruses use α _v β ₃ to enter host cells. The intigreens are believed to be necessary for viral particles to absorb foreign bodies and also for the viral genome to penetrate into the host cytoplasm. Therefore, compounds that inhibit α _v β ₃ can be usefully used as antiviral agents.

The present invention is α _v β ₃ integrated as green antagonist drug (compound) is used as (integrine antagonist), α _v β ₃ integrated by Green inhibit or antagonize α _v β ₃ that are used in pharmaceutical compositions and methods for treating mediated conditions It relates to a drug (compound).

The present invention relates to compounds of the formula and pharmaceutically acceptable salts thereof:

Wherein X and Y are the same or different and are halo groups.

The above compounds may exist in various isomeric forms and all such isomeric forms are included within the scope of the present invention. In addition to the tautomeric forms, pharmaceutically acceptable salts of the isomers and tautomers are also included in the scope of the present invention.

More specifically, the invention relates to compounds of formulas (1) to (16) and pharmaceutically acceptable salts thereof:

Wherein R is H or alkyl.

It is another object of the present invention to provide a pharmaceutical composition comprising the above compound. Since these compounds and compositions are useful for selectively inhibiting or antagonizing intigrins, another embodiment of the present invention relates to a method for selectively inhibiting or antagonizing α _v β ₃ intigrins. In addition, the present invention provides osteoporosis, malignant humoral hypercalcemia, faithful tumor growth (digestion), malignant humoral calcium hyperemia, Paget's disease, tumor metastasis, angiogenesis (tumor) in mammals in need of such treatment. Associated with intigreens, such as angiogenesis), retinopathy (including diabetic retinopathy and spot degeneration), arthritis (including rheumatoid arthritis), periodontal disease, psoriasis, and smooth muscle cell migration and restenosis Treating or inhibiting the pathological condition. In addition, the present invention can be usefully used as an antiviral agent and an antimicrobial agent.

The present invention relates to compounds represented by the above formulas (1) to (16).

Preferred compounds of the invention are compounds of the formulas (17) to (32):

The present invention also relates to pharmaceutical compositions containing a therapeutically effective amount of said compound.

The present invention also relates to a method for selectively inhibiting or antagonizing α _v β ₃ intigrins, and more particularly, by administering a therapeutically effective amount of the above compound together with a pharmaceutically acceptable carrier, Absorption, Osteoporosis, Malignant Humoral Calcium Hyperemia, Solid Tumor Growth (Aberrant Proliferation), Malignant Humoral Calcium Hyperemia, Paget's Disease, Tumor Metastasis, Angiogenesis (including Tumor Angiogenesis), Retinopathy (Diabetes Retinopathy) And spot degeneration), arthritis (including rheumatoid arthritis), periodontal disease, psoriasis, and striated muscle cell migration and restenosis.

The following defines various terms used herein.

The term "alkyl" or "lower alkyl" as used herein refers to a straight or branched chain hydrocarbon group having from about 1 to about 10 carbon atoms, preferably from 1 to about 6 carbon atoms. Examples of such alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, pentyl, neopentyl, hexyl, isohexyl and the like.

The term "halo" or "halogen" as used herein refers to bromo, chloro or iodo.

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

The term "composition," as used herein, means a product obtained by mixing or combining one or more components.

As used herein, “pharmaceutically acceptable salt” is used to convey a chemical drug and means a pharmaceutically acceptable material, composition or excipient such as a liquid or solid filler, diluent, excipient, solvent or encapsulating agent. .

As used herein, the term “therapeutically effective amount” means an amount of a drug that can induce a biological or medical response in the tissue, lineage or animal that the researcher or clinician is looking for.

The following describes the abbreviations or their equivalents used interchangeably herein.

¹ H-NMR = proton nuclear magnetic resonance

AcOH = acetic acid

Ar = argon

CH ₃ CN = acetonitrile

CHN analysis = carbon / hydrogen / nitrogen element analysis

CHNCl analysis = carbon / hydrogen / nitrogen / chlorine element analysis

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

DI water = deionized water

DMA = N, N-dimethylacetamide

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

DMF = N, N-dimethylformamide

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

EtOAc = ethyl acetate

EtOH = ethanol

FAB MS = Rapid Atomic Impact Spectroscopy

g = grams

HOBT = 1-hydroxybenzotriazole hydrate

HPLC = high performance liquid chromatography

IBCF = isobutylchloroformate

KSCN = sodium thiocyanate

L = liter

LiOH = lithium hydroxide

MEM = methoxyethoxymethyl

MEMCl = methoxyethoxymethyl chloride

MeOH = Methanol

mg = milligrams

MgSO ₄ = magnesium sulfate

ml = milliliters

mL = milliliters

MS = mass spectrometry

MTBE = methyl tert-butyl ether

N ₂ = nitrogen

NaHCO ₃ = sodium bicarbonate

NaOH = sodium hydroxide

Na ₂ SO ₄ = sodium sulfate

NMM = N-methylmorpholine

NMP = N-methylpyrrolidone

NMR = nuclear magnetic resonance

P ₂ O ₅ = phosphorus pentoxide

PTSA = para-toluenesulfonic acid

RPHPLC = reversed phase high performance liquid chromatography

RT = room temperature

TFA = trifluoroacetic acid

THF = tetrahydrofuran

TMS = trimethylsilyl

Δ = heating of the reaction mixture

The above compounds exist in various isomeric forms and all such isomeric forms are included within the scope of the present invention. In addition to the tautomeric forms, pharmaceutically acceptable salts of the isomers and tautomers are included within the scope of the present invention.

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

The term "pharmaceutically acceptable salts" refers to salts prepared by contacting such compounds with acids having anions which are generally considered suitable for human consumption. Examples of such pharmaceutically acceptable salts are hydrochloride, bromate, iodide, sulfate, phosphate, acetate, propionate, lactate, maleate, succinate, tartarate and the like. All such pharmaceutically acceptable salts can be prepared according to conventional methods (see, eg, Berge et al., J. Pharm. Sci., 66 (1), 1-19 (1977), for additional examples of pharmaceutically acceptable). salts).

For selective inhibition of α _v β ₃ intigrins, the compounds of the present invention are administered orally, parenterally, inhalationally sprayed or topically in the form of dosage unit formulations containing conventionally acceptable pharmaceutically acceptable carriers, adjuvants and excipients. Can be. As used herein, examples of parenteral administration include subcutaneous, intravenous, intramuscular, intrasternal, infusion or intraperitoneal administration.

The compounds of the present invention are administered via any route in the form of a suitable pharmaceutical composition and at a dose effective for the intended treatment. The therapeutically effective dose of a compound necessary to prevent the progression of or to treat the medical condition can be readily determined using preclinical or clinical methods well known in the medical arts.

Accordingly, the present invention provides a method of treating a mediated condition by selectively inhibiting or antagonizing α _v β ₃ cell surface receptors, the method comprising administering a therapeutically effective amount of a compound selected from the above compounds. do. The compound on one species is administered together with a pharmaceutically acceptable non-toxic carrier and / or diluent and / or adjuvant (collectively referred to herein as a "carrier") on one species and, if necessary, the other active ingredient. More specifically, the present invention provides a method of inhibiting α _v β ₃ cell surface receptors. More preferably, the present invention provides for bone resorption, osteoporosis, malignant humoral calcium hyperemia, faithful tumor growth (abnormal proliferation), malignant humoral calcium hyperemia, Paget's disease, tumor metastasis, angiogenesis (including tumor angiogenesis) ), Retinal pain (including diabetic retinopathy and spot degeneration), arthritis (including rheumatoid arthritis), periodontal disease, psoriasis, and smooth muscle cell migration and restenosis.

Based on standard laboratory methods and procedures well known to those skilled in the art, and in comparison with useful compounds known to the art, these compounds may be used for the treatment of patients with such pathological conditions. One skilled in the art can select the most appropriate compound of the present invention and it will also be appreciated that this choice will depend on a number of factors including the evaluation of results obtained through standard assays and animal experiments.

In the treatment of a patient having one of the above pathological conditions, the amount of said therapeutically effective amount in inhibiting said condition or in extending the patient's life than would be expected in the absence of said treatment. Administering the compound to the patient. As used herein, the term "inhibition" of a state means to delay, stop, stop, or stop the state, and does not necessarily indicate the total elimination of the state. In addition, prolonging the life of the patient means that the condition is suppressed to an extent desirable.

As mentioned above, the compounds of the present invention can be used in various biological, prophylactic or therapeutic fields. Such compounds are believed to be useful for the prevention or treatment of certain disease states in which α _v β ₃ intigrins play a role.

The dose of the compound or the dose of the composition containing the compound depends on several factors, including the type of patient, age, weight, sex and medical condition, severity of the condition, route of administration, and activity of the compound employed. Done. Thus, the dose can vary widely. A dose of about 0.01 mg to about 1000 mg per kg body weight per day is useful for the treatment of this condition, with a more preferred dose being about 0.01 mg to about 100 mg per kg body weight per day.

The active ingredient administered by injection is formulated as a composition in which, for example, saline, dextrose or water is used as a suitable carrier. As a suitable daily dose, several injections of about 0.01 to 10 mg / kg body weight per day, depending on the above factors.

To administer to a patient in need of such treatment, a therapeutically effective amount of the compound is generally combined with a paper adjuvant as long as it is appropriate for the route of administration. The compounds include lactose, sucrose, starch powder, cellulose esters of alkanoic acid, cellulose alkyl esters, talc, stearic acid, magnesium stearate, sodium and calcium salts of phosphoric acid and sulfuric acid, gelatin, acacia, sodium alginate, polyvinylpyrroli It may be mixed with pigs, and / or polyvinyl alcohol, and may be tableted or encapsulated for proper administration. Alternatively, the compound may be dissolved in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride and / or various buffers. Other supplements and treatments are well known in the pharmaceutical art.

Pharmaceutical compositions useful in the present invention may be treated by conventional pharmaceutical manipulations such as sterilization and / or may contain conventional pharmaceutical adjuvants such as preservatives, fungicides, wetting agents, emulsifiers, buffers and the like.

General synthetic sequences for preparing compounds useful in the present invention are shown in Schemes (1) to (3) below. In addition, various embodiments of the present invention are described below. The following schemes and examples are intended to illustrate the invention and do not limit the scope of the invention. Those skilled in the art can synthesize the compounds of the present invention by modifying the conditions and methods described in the following schemes and examples.

Unless otherwise indicated, all starting materials and devices used are commercially available.

Scheme (1) above illustrates a method useful for preparing the tetrahydropyrimidinobenzoic acid moiety of the present invention that can be attached to the glyc-β-amino acid ester. In short, in Scheme (1), 3,5-dihydroxybenzoic acid is described in [Austr. J. Chem., 34 (6), 1319-24 (1981), to convert to 3-amino-5-hydroxy-benzoic acid. The product is generally treated and then reacted with aluminum thiocyanate in hot dilute hydrochloric acid to produce 3-thiourea-5-hydroxybenzoic acid. These thiourea intermediates are converted to S-methyl derivatives by reaction with methyl iodide in ethanol under reflux. 1,3-diamino-2-hydroxypropane is reacted with the intermediate in hot DMA. Upon cooling a precipitate forms and the zwitterionic product is separated by filtration. Alternatively, the product can be separated from the original reaction mixture by removing and condensing volatiles. The resulting product is dissolved in water and adjusted to a pH of about 5-7 to precipitate the amphoteric ionic product and separate by filtration. The HCl salt can be obtained as described above or by simply dissolving in dilute hydrochloric acid, then condensing to solid state and drying.

Scheme (2) above illustrates a method useful for the preparation of the tetrahydropyrimidinobenzoic acid moiety of the present invention that can be attached to the glyc-β-amino acid ester. Briefly, in Scheme (2) above, 1,3-diamino-2-hydroxypropane is reacted with carbon disulfide in a suitable solvent such as ethanol-water, refluxed and cooled, hydrochloric acid is added, and again refluxed. And after cooling, 5-hydroxytetrahydropyrimidine-2-thione is obtained by filtration and then dried. These thiourea intermediates are converted to S-methyl derivatives by reacting with thion and methyl iodide under reflux in ethanol. The volatiles are removed under reduced pressure to readily separate the desired 2-methylthioether-5-hydroxypyrimidine hydroiodo. Next, 2-methylthioether-5-hydroxypyrimidine hydroiodo dissolved in methylene chloride and DMA (about 10: 1) and triethylamine was cooled to ice bath temperature and di-tert-butyl dicarbon Add ate (BOC anhydride). Treatment is usually carried out to obtain BOC-2-methylthioether-5-hydroxypyrimidine as an oil.

3,5-dihydroxybenzoic acid is described in Austin. J. Chem., 34 (6), 1319-24 (1981), to convert to 3-amino-5-hydroxy-benzoic acid.

BOC-2-methylthioether-5-hydroxypyrimidine and 3-amino-5-hydroxybenzoic acid were reacted in hot DMA to yield 3-hydroxy-5-[(5-hydroxy-1,4,5, 6-tetrahydro-2-pyrimidinyl) amino] benzoic acid is obtained as the final desired product. For example, freeze drying with dilute hydrochloric acid is carried out to obtain HCl salt.

Scheme (3) prepares the ethyl N-gly-amino-3- (3,5-dihalo-2-hydroxy) phenyl propionate moiety that can be linked to a tetrahydropyrimidinobenzoic acid moiety. Illustrate the method. Briefly, 3,5-halo substituted salicylic acid using direct halogenation, such as slurrying 5-bromosalylsilaldehyde in acetic acid and adding chlorine to give 3-chloro-5-bromo-2-hydroxybenzaldehyde. Aldehydes can be prepared. Some of the product can precipitate out and be recovered by filtration. The remainder can be recovered by diluting the filtrate with water and separating the precipitate. The solids are combined and dried to give 3-chloro-5-bromo-2-hydroxybenzaldehyde. 3-Iodo-5-chlorosalicylaldehyde can be prepared by reacting 5-chlorosalicylaldehyde with N-iodosuccinimide in DMF and treating the mixture according to conventional operating conditions. 3-iodo-5-bromosalylsilaldehyde can be prepared by reacting 5-bromosalylsilaldehyde with potassium iodide and chloramine in acetonitrile. Treatment with hexanes affords the desired 3-iodo-5-chlorosalicylaldehyde.

Coumarins can be prepared from salicylicaldehyde using a modified Perkin reaction (Vogel's Textbook of Practical Organic Chemistry, 5th edition, 1989, p. 1040). The halo-substituted coumarins are converted to 3-aminohydrocomarins (see JG Rico, Tett. Let., 1994, 35, 6599-6620), which 3-aminohydrocomarins are readily ring-opened in acidic alcohols. 3-amino-3- (3,5-halo-2-hydroxy) phenyl propanoic acid ester is obtained.

3-amino-3- (3,5-halo-2-hydroxy) phenyl propanoic acid ester with N-gly-3-amino-3- (3,5-halo-2-hydroxy) phenyl propanoic acid ester Conversion and reaction with Boc-N-gly-N-hydroxysuccinimide to convert to Boc-N-gly-3-amino-3- (3,5-halo-2-hydroxy) phenyl propanoic acid ester This is converted to the HX salt of N-gly-amino-3- (3,5-halo-2-hydroxy) phenyl propanoic acid ester, where X is a halo group, by removing the BOC-protecting group with HCl in ethanol do.

The amino acid compounds used to prepare the compounds of the present invention may be prepared according to the methods described below and according to the methods described in co-pending USSN Attorney Docket 3076 filed in the United States with the same herein. The disclosure of such co-pending patent application is incorporated herein by reference.

Scheme (4) illustrates the methods useful for preparing the various compounds of the present invention. 3-hydroxy-5-[(1,4,5,6-tetrahydro-5-hydroxy-2-pyrimidinyl) amino] benzoic acid is activated and coupled using known methods. Next, NMM is added after dissolving in a suitable solvent such as DMA. The reaction mixture is cooled to ice bath temperature and then IBCF is added. Add the glyc-β-amino acid ester and NMM to the mixed anhydride intermediate. After completion of the reaction, the product is purified by PREP HPLC and the resulting ester is hydrolyzed with acid by treatment with a base such as LiOH in a suitable solvent (dioxane / water or acetonitrile / water). Alternatively, a suitable acid such as TFA may be used. The product is separated by PREP HPLC, or the zwitterion is separated at pH 5-7 and converted to the desired salt according to standard procedures.

Example A: Preparation of Compounds Having the Formula

Step 1: Preparation of a Compound Having the Formula

In a 2-liter round bottom flask with mechanical stirrer and cooler, 3,5-dichlorosalicylaldehyde (200 g, 1.05 mol, 1 equiv), acetic anhydride (356 g, 3.49 mol) and triethylamine (95.0 g, 0.94 mol) , 0.90 equiv) was added. The reaction solution was heated at reflux overnight. The dark brown reaction mixture was cooled to 50 ° C. and water (1 L) was added with stirring. After 1 h, the mixture was filtered and the filtrate was combined with ethanol (1 L). The mixture was heated to 45 ° C. for 1 h, cooled to rt, filtered and the solid (fraction A) washed with ethanol (1.5 L). The combined ethanol solution was condensed by rotary evaporation to give an oil (fraction B). The solid from fraction A was dissolved in methylene chloride (1.5 L) and the resulting solution was passed through a pad of silica gel (1300 ml volume). The resulting dark brown solution was condensed to give an oil which was triturated with hexane (1.3 L) to give a solid which was separated by filtration and washed with hexane to give practically pure 6,8-dichlorocoumarin (163 g). In an analogous manner, the oil of fraction B was treated to yield an additional 31 g of product. That is, the oil was dissolved in methylene chloride (0.5 L), passed through a silica pad (0.5 L), and then triturated with hexane. Overall 194 g or 86% yield of a brown solid was obtained.

Step 2: Preparation of a Compound Having the Formula

To a 2 L three-neck round bottom flask with mechanical stirrer was added 6,8-dichlorocomarin (160 g, 0.74 mol) and dry THF (375 ml, Aldrich Sure Seal) prepared in step 1 above. The resulting mixture was cooled to −40 ° C. (dry ice / acetone bath), and the temperature was maintained at −40 ° C. or lower while adding lithium bis (trimethylsilyl) amide (0.80 mol, 800 ml, 1M in THF). After the addition was completed, the ice bath was removed. The reaction was cooled by adding a solution of HCl (0.5 L, 4M in dioxane) dissolved in ethanol (1.25 L). The temperature was kept below 0 ° C. overnight. The reaction mixture was condensed to about 1/2 of its original volume and partitioned between ethanol (3 L) and water (2 L). The organic layer was washed with aqueous HCl (3 × 1 L 0.5 N HCl). 10% aqueous NaOH was added to adjust the pH of the combined aqueous layers to about 7 and extracted with methylene chloride (3 x 2 L). The combined organic layers were dried (MgSO ₄ ), filtered and stirred with the addition of 4M HCl dissolved in dioxane (210 ml). After completion of the precipitation, the solid was removed by filtration. The filtrate was condensed in small volumes and methyl t-butyl ether was added. The obtained solid was combined with the first formed solid and the combined product was washed with methyl t-butyl ether, separated by filtration and dried under vacuum for a week to give the desired product (172 g, 74% yield).

MS and NMR results were consistent with the desired structure.

Step 3: Preparation of a Compound Having the Formula

In a flame dried 0.5 L round bottom flask with magnetic stir bar, Nt-Boc-glycine N-hydroxysuccinimide ester (Sigma, 15.0 g, 0.055 mol), dry DMF (Aldrich Sure Seal, 200 ml), And the product obtained in step 2 (21.67 g, 0.055 mol) was added under inert atmosphere (argon). The reaction mixture was cooled to about 0 ° C. (salt-ice bath), N-methylmorpholine (5.58 g, 0.056 mole) and catalytic amount of DMAP were added and then reacted overnight. The reaction mixture was condensed in slush and partitioned between ethyl acetate (0.4 L) and aqueous base (2 x 0.2 L, aqueous saturated NaHCO ₃ ). The organic layer was washed sequentially with aqueous citric acid (2 x 0.2 L, 10% w / v), aqueous sodium bicarbonate (2 x 0.2 L) and brine and then dried (sodium sulfate). The volatiles were removed under vacuum at 55 ° C. to give an oil (22.5 G, 92% yield) which was left to solidify to solidify.

MS and NMR results were consistent with the desired structure above.

Step 4: Preparation of a Compound Having the Formula

The product prepared in step 3 was removed to protect the amine hydrochloride salt in the following procedure. The product obtained in step 3 (14.0 g, 0.032 mole) was charged to a flame dried round bottom flask (0.1 L) and dry dioxane (40 ml) was added. To this was added 4.0N HCl dissolved in dioxane (2 equiv., 6.32 ml) at 0 ° C., followed by reaction until gas evolution stopped. After the reaction, the volatiles were removed in vacuo and the residue was triturated with diethyl ether (50 ml). Filtration recovered the solid, washed with ether and dried to afford the desired product (12.5 g).

The results of MS and NMR analysis were consistent with the desired structure.

Example B: Preparation of Compounds Having the Formula

Step 1: Preparation of a Compound Having the Formula

Triethylamine (103.6 ml, 743.2 mmol) was added to a suspension of 3-bromo-5-chlorosalicylaldehyde (175.0 g, 743.2 mmol) dissolved in acetic anhydride (280.5 ml, 3.0 mol). The reaction solution was heated to reflux for 4.5 hours. The solution was cooled and condensed in vacuo. Anhydrous ethanol (730 ml) was added to the resulting brown residue. The mixture was kept at 0 ° C. for 14 hours. Filtration recovered the brown solid and washed with cold ethanol. The solid was dried in vacuo to afford the desired product (123.0 g, 64% yield). ^{The 1} H NMR analysis result was in agreement with the desired structure.

Step 2: Preparation of a Compound Having the Formula

At −76 ° C., to a suspension of coumarin (40.0 g, 154.1 mmol) dissolved in THF (400 ml) was added dropwise over 10 minutes with stirring lithium bis (trimethylsilyl) amide (154.1 ml, 1M solution in THF). . The reaction mixture was then stirred for 5 minutes, warmed to -20 ° C and stirred for 15 minutes. To this solution, acetic acid (9.25 g, 154.1 mmol) dissolved in THF (28 ml) was added over 5 minutes. The resulting mixture was warmed to room temperature and the volatiles were removed under vacuum. The residue was dissolved in ether (850 ml), washed with saturated aqueous sodium bicarbonate solution (2 × 100 ml), brine (2 × 40 ml) and dried (sodium sulfate). The resulting ether solution was condensed to about 160 ml and cooled to 0 ° C. To this suspension, 4 M HCl dissolved in dioxane (56.3 ml, 225 mol) was added and the mixture was stirred at 0 ° C. for 30 minutes. The resulting suspension was filtered and the filter cake washed with ether. The resulting solid was dried in vacuo to give the desired product as an HCl salt and a dioxane solvate (45.0 g). ¹ H NMR analysis results were in agreement with the desired structure.

Step 3: Preparation of a Compound Having the Formula

To a suspension of lactone (142.2 g, 354.5 mmol) dissolved in anhydrous ethanol (533 ml), 4 M HCl dissolved in dioxane (157.8 ml, 631.1 mmol) was added over 10 minutes. The resulting reaction mixture was stirred at rt for 2.5 h. Volatile material was removed under vacuum. The residue was dissolved in ethyl acetate (450 ml) and the resulting solution was stirred at 0 ° C. for 15 h. The resulting tan precipitate was collected by filtration and washed with cold ethyl acetate. The resulting solid was dried in vacuo to afford the desired product in the form of hydrochloride (100.4 g, 79% yield). ^{The 1} H NMR analysis result was in agreement with the desired structure.

Step 4: Preparation of a Compound Having the Formula

In a flame dried round bottom flask (0.1 L) with a magnetic stir bar, Nt-Boc-glycine N-hydroxysuccinimide ester (Sigma, 2.72 g, 0.010 mole), dry THF (Aldrich Sure Seal, 50 ml) And the product obtained in step 3 (3.10 g, vacuum dried overnight using phosphorus pentoxide). The resulting reaction mixture was cooled to about 0 ° C. (salt-ice bath) and triethylamine (1.01 g, 0.010 mole) was added. Then, it was reacted overnight. The resulting reaction mixture was condensed in half and treated in the same manner as in step 3 of Example A above. The volatiles were removed from the organic layer in vacuo at 55 ° C. to obtain an oil (4 g, 83% yield) which solidified by standing.

The results of MS and NMR analysis were consistent with the desired structure above.

Step 5: Preparation of a Compound Having the Formula

The product obtained in step 4 was removed with the protecting group according to the following procedure to obtain amine hydrochloride. The product obtained in step 4 (4.0 g, 0.084 mole) was charged to a flame dried round bottom flask (0.1 L) equipped with a stirring rod, and dried dioxane (20 ml) was added. To this was added 4.0 N HCl dissolved in dioxane (20 ml) and then reacted until gas evolution stopped (about 1 hour). After the reaction, the volatiles were removed in vacuo and the residue was triturated with diethyl ether (50 ml). Filtration recovered the solid, washed with ether and dried to afford the desired product (2.7 g, 78% yield).

The results of MS and NMR analysis were consistent with the desired structure.

Example C: Preparation of Compounds Having the Formula

Step 1: Preparation of a Compound Having the Formula

Triethylamine (45 ml, 375 mmol) was added to a suspension of 3,5-dibromosalicylatealdehyde (100 g, 357 mmol) dissolved in acetic anhydride (164.8 ml, 1.8 mol). The reaction mixture was heated to reflux overnight under argon atmosphere. The resulting solution was cooled to room temperature to form a solid material. The resulting dark brown reaction mixture was washed with hot hexane (3 × 300 ml) and washed with saturated sodium bicarbonate solution. The resulting solid was dissolved in ethyl acetate (2 L) and washed with water. The organic layer was dried (sodium sulfate), condensed and filtered to give a brown solid. The solid was dried in vacuo to give practically pure 6,8-dibromocoumarin (94.2 g, 87% yield).

The results of MS and ¹ H NMR analysis were consistent with the desired structure.

Step 2: Preparation of a Compound Having the Formula

Lithium bis (trimethylsilyl) amide (66 ml, THF) in a suspension of 6,8-dibromocomarin (20.0 g, 0.066 mol) (prepared in step 1 above) dissolved in THF (100 ml) at −78 ° C. 1M solution) was added dropwise over 10 minutes with stirring. The reaction mixture was then stirred for 5 minutes, warmed to 0 ° C. and stirred for 15 minutes. To this solution, acetic acid (3.95 g) was added over 1 minute. The resulting mixture was warmed to room temperature and the volatiles were removed in vacuo. The residue was dissolved in hexane (500 ml), washed with saturated aqueous sodium bicarbonate solution (2 × 100 ml) and dried (sodium sulfate). The resulting organic solution was condensed to give an oil, which was then stirred at 0 ° C. for 30 minutes while adding 4M HCl dissolved in diethyl ether (400 ml) and dissolved in dioxane (30 ml). Excess HCl was removed in vacuo, the remaining suspension was filtered, and the filter cake was washed with ether. The resulting solid was dried in vacuo to give the desired product in the form of a HCl salt, a dioxane solvate (45.0 g). The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Step 3: Preparation of a Compound Having the Formula

The lactone (15 g) prepared in step 2 was dissolved in anhydrous ethanol (400 ml) and anhydrous HCl gas was passed for 1 minute. The resulting reaction mixture was stirred at rt for 2.5 h. RPHPLC showed a complete reaction. The volatiles were removed under vacuum to give a dark residue. The residue was dissolved in diethyl ether (500 ml) and the resulting solution was stirred overnight. The resulting tan precipitate was collected by filtration and washed with diethyl ether. The resulting solid was dried in vacuo to afford the desired product in the form of hydrochloride (15.2 g). The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Step 4: Preparation of a Compound Having the Formula

Flame-dried round bottom flask (0.2 L) with magnetic stir bar in Nt-Boc-glycine N-hydroxysuccinimide ester (Sigma, 8.1 g, 0.030 mole), dry THF (Aldrich Sure Seal, 50 ml) And the product obtained in step 3 (12 g, 0.03 mole, vacuum dried overnight using phosphorus pentoxide) was added under argon atmosphere. The resulting reaction mixture was cooled to about 0 ° C. (salt-ice bath), and N-methyl morpholine (3.03 g, 0.030 mole) and a catalytic amount of DMAP were added. Then, it was warmed to room temperature and reacted overnight. The resulting reaction mixture was condensed in half and treated in the same manner as in step 3 of Example A above. The volatiles were removed from the organic layer in vacuo at 55 ° C. to give an oil (15.7 g, 93% yield) which was allowed to settle and solidify.

The results of MS and NMR analysis were consistent with the desired structure above.

Step 5: Preparation of a Compound Having the Formula

The product obtained in step 4 was removed with the protecting group according to the following procedure to obtain amine hydrochloride. The product (13.0 g, 0.084 mole) obtained in step 4 above was charged to a flame dried round bottom flask (0.1 L) equipped with a stirring rod, and dried dioxane (40 ml) was added. To this was added 4.0 N HCl dissolved in dioxane (30 ml) and then reacted until gas evolution stopped (about 1 hour). After the reaction, the volatiles were removed in vacuo and the residue was triturated with diethyl ether (50 ml). Filtration recovered the solid, washed with ether, and dried to give a solid (10.6 g, 93% yield).

The results of MS and NMR analysis were consistent with the desired structure.

Example D: Preparation of Compounds Having the Formula

Step 1: Preparation of 3-chloro-5-bromosalicylaldehyde having the formula

To a 5L round bottom flask equipped with a mechanical stirrer and a gas supply tube, 5-bromosalicylicaldehyde (495 g, 2.46 mol) and acetic acid were added at room temperature to form a slurry. To this mixture, chlorine gas was added at a suitable rate to dissolve a slight excess of chlorine (183 g, 1.05 mol). After stopping this addition, it was allowed to react overnight. The solid formed was collected by filtration and the filtrate was diluted in water (2.5 L). The resulting mixture was stirred vigorously for 20 minutes and the product was collected by filtration and washed with water. The combined solids were dried in vacuo to afford the desired 3-chloro-5-bromosalylsilaldehyde (475 g, 82% yield).

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Step 2: Preparation of 6-Bromo-8-chlorocoumarin having the formula

In a 5L round bottom flask with mechanical stirrer and cooler, 3-chloro-5-bromosalicylicaldehyde (554.1 g, 2.35 mol, 1 equiv), acetic anhydride (1203 g, 11.8 mol., 5 equiv) and triethylamine (237.4 g, 2.35 mol, 1 equiv) was charged. The reaction solution was heated to reflux (131-141 ° C.) overnight. The resulting dark brown reaction mixture was cooled to 50 ° C, and ice (2 L) was added and stirred while cooling with ice bath. After 1 hour, the resulting mixture was filtered and the filtrate was combined with ethanol (1 L). To this mixture, ethanol (300 ml) was added and the reaction mixture was stirred for 1 hour. The precipitate formed was collected by filtration, washed with water: ethanol (3 x 1.3 L), dried in vacuo and dried in a fluid bed drier. Total yield was 563 g or 92%.

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Step 3: Preparation of 3-amino-3- (2-hydroxy-3-chloro-5-bromo) phenyl propanoic acid ethyl ester having the formula

To a 5L three neck round bottom flask with a mechanical stirrer was added 6-bromo-8-chlorocoumarin (300 g, 1.16 mole) (prepared in step 2 above) and dry THF (900 ml, Aldrich Sure Seal). . The resulting mixture was cooled to -45 DEG C or lower (dry ice / acetone bath) and the temperature was increased while adding lithium bis (trimethylsilyl) amide (0.80 mol, 800 ml, 0.1 M in THF and 0.6 M in hexane, 1.2 equiv). The temperature was kept below -45 ° C for 0.5 hours. In another 5 L flask, ethanol (2.5 L) and HCl (4N HCl dissolved in dioxane, 1 L) were combined at -15 ° C. The resulting coumarin reactant was cooled by adding a cooled HCl / ethanol solution. After 0.5 hour, the temperature of the reaction mixture obtained was -8.3 ° C. The reaction mixture was kept at 0 ° C. overnight, condensed to about 2.5 L and partitioned between ethanol (3 L) and water. The resulting organic layer was washed with aqueous HCl (4 × 1.2 L, 0.5 N HCl). The pH of the combined aqueous layers was adjusted to about 8 by addition of 10% aqueous NaOH and extracted using methylene chloride (1 × 7 L and 3 × 2 L). The combined organic layers were dried using sodium sulfate (900 g), filtered and stirred with the addition of 4M HCl dissolved in dioxane (400 ml). A precipitate was formed and then filtered to separate the solids. The resulting mixture was condensed with 2.5 L, hexane (2.5 L) was added, and the precipitate was separated by filtration. The resulting filter cake was washed with methylene chloride / hexane (1: 2), suction dried and vacuum oven dried at 40 ° C. to afford the desired product (251 g, 60% yield).

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Step 5: Preparation of a Compound Having the Formula

The above compounds were prepared using substantially the same procedures and relative amounts as provided for the preparation of the isomers in step 4 of Example B.

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Step 6: Preparation of a Compound Having the Formula

The above compounds were prepared using substantially the same procedures and relative amounts as provided for the preparation of the isomers in step 5 of Example B.

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Example E: Preparation of Compounds Having the Formula

Step 1: Preparation of 3-iodo-5-chlorosalicylaldehyde having the formula

N-iodosuccinimide (144.0 g, 0.641 mole) was added to a solution of 5-chlorosalicylaldehyde (100 g, 0.638 mole) dissolved in dimethylformamide (400 ml). The reaction mixture was stirred for 2 days at room temperature. Additional N-iodosuccinimide (20.0 g) was added and stirred for 2 days. The reaction mixture was diluted with ethyl acetate (1 L), washed with hydrochloric acid (300 ml, 0.1 N), water (300 ml), sodium thiosulfate (5%, 300 ml), brine (300 ml) and dried over sodium sulfate. Condensation was carried out to dryness to afford the desired aldehyde (162 g, 90% yield) as a pale yellow solid.

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Step 2: Preparation of 3-chloro-8-iodokoumarin having the formula

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

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Step 3: Preparation of (R, S) -4-amino-3,4-dihydro-6-chloro-8-iodocomarin hydrochloride having the formula

Lithium hexamethyldisilaazane (21.62 ml, 1 M, 21.62 mol) was added to a solution of 6-chloro-8-iodocomarin (6.63 g, 21.62 mmol) dissolved in tetrahydrofuran (100 ml) at -78 ° C. It was. The reaction mixture was stirred for 30 minutes at this temperature and for 1 hour at 0 ° C. Acetic acid (1.3 g, 21.62 mmol) was added to the reaction mixture. The resulting reaction mixture was added to ethyl acetate (300 ml) and saturated sodium carbonate solution (200 ml). The organic layer was separated and washed with brine (200 ml), dried over sodium sulfate and condensed to give a residue. The residue was added to anhydrous ether (200 ml) at 0 ° C. and then to dioxane / HCl (4N, 30 ml). The reaction mixture was stirred at rt for 1 h, filtered and dried in vacuo to afford the desired product (4.6 g, 59% yield) as a powder. (RPHPLC: Rf 6.8 min; gradient 15 min 10% acetonitrile-90% acetonitrile for 6 min 100% acetonitrile; water and acetonitrile contain 0.1% TFA; Vydac C18 protein peptide column, 2 ml / min flow rate , Measured at 254 nm).

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Step 4: Preparation of (R, S) -ethyl 3-amino-3- (5-chloro-2-hydroxy-3-iodo) phenyl propionate hydrochloride having the formula

The reaction mixture was saturated while bubbling hydrogen chloride gas into a solution of 4-amino-3,4-dihydro-6-chloro-8-iodokoumarin (22.0 g, 61.09 mmol) dissolved in ethanol (250 ml). The temperature was kept at 0-10 ° C. until After refluxing for 6 hours, most of the solvent was distilled off. The resulting cold residue was added to anhydrous ether and stirred for 3 hours. The initial gum state changed to crystalline material. The crystalline product was filtered and dried to afford the desired product (20 g, 81% yield) as a white nodular powder. (Rf 7.52 min, same conditions as Step 3 above).

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Step 5: Preparation of (R, S) -ethyl 3- (N-BOC-gly) -amino-3- (5-chloro-2-hydroxy-3-iodo) phenyl propionate having the formula

BOC-gly (2.16 g, 12.31 mmol), HOBT (1.67 g, 12.31 mmol), EDCl (2.36 g, 12.31 mmol) and DMF (50 ml) were stirred at 0 ° C for 1 h. To the reaction mixture, ethyl 3-amino-3- (5-chloro-2-hydroxy-3-iodo) propionate hydrochloride (5.0 g, 12.31 mmol) was added, followed by triethylamine (3.5 ml). Added. The reaction mixture was stirred at rt for 18 h. The DMF was removed in vacuo and the residue was partitioned between ethyl acetate (300 ml) and sodium bicarbonate (200 ml). The resulting organic layer was washed with hydrochloric acid (1N, 100 mL), brine (200 ml), dried over sodium sulfate and condensed to afford the desired product (6 g, 93% yield) in the solid state.

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Step 6: Preparation of (R, S) -ethyl 3- (N-gly) -amino-3- (5-chloro-2-hydroxy-3-iodo) phenyl propionate hydrochloride having the formula

Dioxane / HCl (4N, 20 ml) was added to ethyl 3- (N-BOC-gly) -amino-3- (5-chloro-2-hydroxy-3-iodo) propionate (6.0 g, 11.39 mmol). It added at 0 degreeC, and stirred at room temperature for 3 hours. The reaction mixture was condensed and toluene (100 ml) was added and then condensed once more. The resulting residue was suspended in ether, filtered and dried to afford the desired product in the form of crystalline powder (5.0 g, 95% yield).

(RPHPLC: Rf 8.3 min, same conditions as in Step 3 above).

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Example F: Preparation of Compounds Having the Formula

Step 1: Preparation of 3-iodo-5-bromosalicylaldehyde

In a 500 ml round bottom flask with a magnetic stirrer, chlorine was dissolved in 5-bromosalicylicaldehyde (20.0 g, 0.1 mole) and potassium iodide (17 g, 0.1 mole) dissolved in acetonitrile (150 ml) and water (50 ml). Amine T (23 g, 0.1 mole) was added. The mixture was reacted for 1 hour. The reaction mixture was partitioned between hydrochloric acid (10%, 200 ml) and ethyl acetate. The organic layer was dried over sodium sulfate, filtered and vacuum condensed. To the residue was added hexane and the resulting mixture was heated to 50 ° C. for 15 minutes. Undissolved material was removed by filtration. The filtrate was condensed in vacuo to yield a yellow 3-iodo-5-bromosalylsilaldehyde (26 g).

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Step 2: Preparation of a Compound Having the Formula

Step 2 of Example E, except that the same amount of 3-iodo-5-bromo-salicylaldehyde was used instead of 3-iodo-5-chlorosalicylaldehyde in Step 2 of Example E The compound was prepared by the same procedure as the procedure of −6.

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Example H: Preparation of Compounds Having the Formula

Step 1

Ethanol (375 ml) and deionized water (375 ml) were added to a 2 L three necked round bottom flask equipped with a mechanical stirrer, Klisen adapter, addition funnel, reflux condenser and thermocouple. 1,3-Diamino-2-hydroxypropane (125.04 g, 1.39 mol) (Aldrich) was added to the reaction flask and stirred to dissolve. Carbon disulfide (84 ml, 1.39 mol) was added dropwise through an addition funnel at 25-33 ° C. for 35 minutes to obtain a milky mixture. The temperature was maintained using 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. using an ice bath and then the temperature was maintained at 25-26 ° C. with concentrated dropwise HCl (84 ml). The reaction mixture was refluxed at 78.4 ° C. for 21 hours. The resulting reaction solution was cooled to 2 ° C. and vacuum filtered to recover the product. The resulting gray solid was washed three times with ice-cold cooled ethanol: water (1: 1) (50 ml) and dried under vacuum at 40 ° C. to 5-hydroxytetrahydropyrimidine-2-thione (63.75 g, 34.7% yield). Was obtained in the form of a white solid.

The results of MS and NMR analysis were consistent with the desired structure above.

Step 2

5-hydroxytetrahydropyrimidine-2-thione (95 g, 0.72 mol), anhydrous ethanol (570 ml) and methyl iodide (45 ml, 0.72 mol) prepared in step 1, 2 L with a mechanical stirrer and thermocouple Added to round bottom flask. The reaction mixture was refluxed at 78 ° C. for 5 hours and then cooled to room temperature. The reaction mixture was vacuum condensed to give a white solid (194.72 g). The white solid was triturated three times with ethyl ether (500 ml) and dried in vacuo to afford 2-methylthioether-5-hydroxypyrimidine hydroiode (188.22 g, 95.4% yield) in the form of a white solid.

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Step 3

In a 2-liter three-neck round bottom flask with reflux cooler, mechanical stirrer and static nitrogen atmosphere, 2-methyl thioether-5-hydroxypyrimidine hydroiodo (150.81 g, 0.55 mol), methylene chloride (530 ml), dimethylacet Amide (53 ml) and triethylamine (76.7 ml, 0.55 ml) were added. The mixture was cooled using an ice bath and di-tert-butyl dicarbonate (120.12 g, 0.55 mol) was added at 4 ° C. The reaction mixture was heated to 42.5 ° C. for 18 hours to give a pale yellow solution. The solution was transferred to a 2 L separating funnel, washed three times with deionized water (200 ml), dried over sodium sulfate, filtered and vacuum condensed to give Boc-2-methylthioether-5-hydroxypyrimidine (134.6 g). , 99.35% yield) in the form of a pale yellow sticky oil.

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Step 4

Boc-2-methylthioether-5-hydroxypyrimidine (50.3 g, 0.204 mol), amino-5-hydroxybenzoic acid (Aust. J. Chem. (1981) 34 (6), 1319-24) (25.0 g, 0.1625 mole) and 50 ml anhydrous DMA were stirred with heating to 100 ° C. for 2 days. A slurry precipitate was obtained. The reaction was cooled to room temperature and the precipitate was filtered off, washed with CH ₃ CN, washed with ethyl ether and dried. The resulting solid was slurried in water and acidified with concentrated HCl to give a solution. The solution was freeze dried to afford the desired product (14.4 g) as a white solid.

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Example I: Preparation of Compounds Having the Formula

Step 1: Preparation of Reformatski Compound Having Formula

To a 4L flask with cooler, temperature probe and mechanical stirrer, Zn metal (180.0 g, 2.76 mol, -30-100 mash) and THF (1.25 L) were added. While stirring, 1,2-dibromomethane (4.74 ml, 0.05 mol) was added via injector. Or else, THS Cl (0.1 equiv) may be added at RT for 1 h. After being purged with an inert gas (three N ₂ / vacuum cycles), the suspension of zinc dissolved in THF was heated to reflux at 65 ° C. and maintained at this temperature for 1 hour. The mixture was cooled to 50 ° C. and tert-butyl bromoacetate (488 g, 369 ml, 2.5 mol) was charged over a period of 1.5 hours through a 50 ml injector and an infusion pump (set up at 4.1 ml / min). A temperature of 50 ° C. + / − 5 ° C. was maintained during the addition. After the addition was completed, the reaction mixture was stirred at 50 ° C. for 1 hour. The mixture was then cooled to 25 ° C. to precipitate. The THF mother liquor was decanted into a 2 L round bottom flask using a thick glass filter rod and a partial vacuum transfer (20 mmHg). Thus, about 65% of the THF was removed from the mixture. 1-methyl-2-pyrrolidinone (NMP, 800 ml) was added and stirred for 5 minutes. The mixture can be filtered to remove any remaining zinc. As a result, a titration of the desired Reformatski with 1.57M and 94% yield was confirmed. Otherwise, the solid material can be separated from the original reaction mixture by filtration. The resulting filter cake is washed with THF to give a white solid which is then dried under nitrogen atmosphere to give the desired product in the form of a mono THF solvate. The solvate can be stored at 20 ° C. (dry) for a long time. Typical yields are 85-90%.

Step 2

2A. Preparation of Compounds Having the Formula

To a solution of 3,5-dichlorosalicylaldehyde (11.46 g, 60 mole) dissolved in DMF (40 ml), potassium carbonate (powder, oven dried at 100 ° C. under vacuum, 8.82 g, 60 mmol) was added at room temperature to pale yellow color. A slurry was obtained. Next, the bath temperature was maintained at 20 ° C while adding MEMCl (pure water, 7.64 g, 61 mmol). The mixture was then stirred at 22 ° C. for 6 hours and MEMCl (0.3 g, 2.3 mmole) was added. The mixture was further stirred for 0.5 h and added to cold water (200 ml) to precipitate the product. The resulting slurry was filtered over a pressure filter and the filtrate was washed with water and dried under N ₂ / vacuum to afford the desired product (14.94 g, 89% yield) in the form of a white 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 1 H); 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, 188.30. DSC: 48.24 ° C. (endothermic 90.51 J / g);

Microanalysis: C ₁₁ H ₁₂ Cl ₂ O ₄

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

Found: C: 47.15%; H: 4.26%; Cl: 25.16%.

2B. Preparation of Compounds Having the Formula

The product obtained in step 2A (35.0 g, 0.125 mol) was charged to a 1 L three necked round bottom flask equipped with a mechanical stirrer and addition funnel and then THF (200 ml) was added. The solution was stirred at 22 ° C. and then (S) -phenylglycinol (17.20 g, 0.125 mol) was added all at once. After holding at 22 ° C. for 30 minutes, sodium sulfate 922 g) was added. The mixture was stirred at 22 ° C. for 1 hour and filtered over a coarse glass filter. The filtrate was condensed under reduced pressure. No further purification was carried out and the crude imine obtained was used directly for the coupling reaction in step 2C below.

2C. Preparation of Compounds Having the Formula

To a 1 L three necked round bottom flask equipped with a mechanical stirrer and an addition funnel, the solid material (91.3 g, 0.275 mol) and NMP (200 ml) prepared in step 1 were added under a nitrogen atmosphere. The solution was then cooled to -10 ° C and stirred at 350 rpm. The solution of the imine prepared in step 2B dissolved in NMP was prepared under a nitrogen atmosphere, and then maintained at −5 ° C. (jacket temperature −10 ° C.) while being added to the reaction mixture over 20 minutes. The mixture was then further stirred at -8 ° C for 1.5 h and at -5 ° C for 1 h. After cooling to −10 ° C., a mixture of concentrated HCl / saturated NH ₄ Cl solution (8.1 ml / 200 ml) was added over 10 minutes. MTBE (200 ml) was added and the resulting mixture was stirred at 200 rpm at 23 ° C. for 15 minutes. Agitation was stopped and the layers were separated. The aqueous layer was extracted using MTBE (100 ml). The two organic layers were combined and washed successively with saturated NH ₄ Cl solution (100 ml), water (100 ml) and brine (100 ml). The resulting solution was dried using sodium sulfate (30 g) and condensed to give an orange oil (66.3 g). The oil contains the desired product in one diastereoisomeric form as determined by proton and carbon nmr and solidifies upon standing. The analytical sample was purified by crystallization from heptane to give a gray solid. Proton and carbon NMR and IR spectra were consistent with the desired structures above. [α] ^D ₂₅ = + 8.7 ° (c = 1.057, MeOH).

Microanalysis: C ₂₅ H ₃₃ Cl ₂ NO ₆

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 of a Compound Having the Formula

3A. A solution of the crude ester (17.40 g, 0.033 mole (theoretical value)) prepared in step 2 dissolved in ethanol (250 ml) was charged to a 1 L three-neck jacket reactor. The solution was cooled to 0 ° C. and Pb (OAc) ₄ (14.63 g, 0.033 mole) was added all at once. After 2 hours, 15% solution of NaOH (30 ml) was added and ethanol was removed under reduced pressure. Another 15% NaOH (100 ml) was added, then the resulting mixture was extracted with MTBE (2 x 100 ml), washed with water (2 x 100 ml) and brine (50 ml), dried over sodium sulfate, Filtration over celite and condensation under reduced pressure gave an orange oil (12.46 g). The oil was homogenized by thin layer chromatography (TLC) and used without further filtration.

3B. The oil obtained in step 3A was diluted with ethanol (30 ml) and paratoluene sulfonic acid (1.3 equiv, 0.043 mol, 8.18 g) was added. The solution was heated to reflux for 8 hours, cooled to room temperature and condensed under reduced pressure. The residue was treated with THF (20 ml) and heated to reflux to form a solution. The solution was cooled to room temperature and crystallized. Heptane (30 ml) and THF (10 ml) were added to form a fluid slurry, which was filtered. The filter cake was washed with THF / heptane (40 ml, 1/1) and vacuum dried for 2 hours in a pressure filter under a nitrogen atmosphere to give a white solid (7.40 g). Proton and carbon NMR and IR spectra were consistent with the desired product, which is actually present in enantiomer form.

Microanalysis: C ₁₈ H ₂₁ Cl ₂ NO ₆ S, 0.25 C ₄ H ₈ O

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 of a Compound Having the Formula

In a 500 ml round bottom flask equipped with a magnetic stir bar and a nitrogen bubble generator, free base (21.7 g, 0.065 mole), Nt-Boc-glycine N-hydroxysuccinimide ester (17.7) of the product prepared in step 3 g, 0.065 mole) and DMF (200 ml) were added. The reaction mixture was stirred for 3.25 hours in a nitrogen atmosphere and at room temperature to form a pale yellow solution. The reaction mixture was added to ice cooled ethyl acetate (1.2 L). The organic solution was washed with 1M HCl (250 ml) and then brine (500 ml), dried over sodium sulfate, condensed to dryness under reduced pressure to obtain an oil, and dried at 50 ° C. to give a product as a colorless oil ( 28.12 g, 99%). Seed crystals were obtained from ethyl acetate / hexanes. The product (28 g) was dissolved in ethyl acetate (35 ml) and hexane (125 ml). The seed crystals were added to the solution to form a precipitate. The solid was filtered and dried under vacuum at 55 ° C. overnight to give a colorless solid (27.0 g, 95% yield).

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Step 5: Preparation of a Compound Having the Formula

Boc-protecting group glycine amide (27.0 g, 0.062 mole) prepared in step 4 was dried overnight using P ₂ O ₅ and NaOH pellets. The obtained solid was dissolved in dioxane (40 ml), and the solution was cooled to 0 deg. An equal volume of 4N HCl / dioxane (0.062 mole) was added and reacted for 2 hours. At this time, the conversion rate measured by RPHPLC was 80%. The reaction mixture was kept at room temperature for 4 hours. The reaction mixture was condensed at 40 ° C. to form a foam, which was triturated with ether (200 ml). The white solid formed was dried over P ₂ O _{5 to} give the desired glycine-beta-amino acid ester compound in the form of an HCl salt (20.4 g, 88.5% yield).

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Example J: Preparation of Compounds Having the Formula

Step 1: Preparation of a Compound Having the Formula

A MEM protecting group 3-bromo-5-chlorosalicylaldehyde (129.42 g, 0.4 mol) prepared according to step 2A of Example I was prepared. Instead of 3,5-dichlorosalicylaldehyde, use an equal amount of 3-bromo-5-chlorosalicylaldehyde and fill it into a 2 L round bottom flask equipped with a mechanical stirrer, then THF (640 ml) and (S) -Phenylglycinol (54.86 g, 0.4 mol) was added. After holding at 22 ° C. for 30 minutes, sodium sulfate (80 g) was added. The mixture was stirred at 22 ° C. for 2 hours and filtered over a coarse glass filter. The filtrate was condensed under reduced pressure to give a pale yellow oil (180.0 g) containing the desired imine. No further purification was carried out and the crude product obtained was used directly for the coupling reaction in step 2 below.

Microanalysis: C ₁₉ H ₂₁ BrClNO 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 of a Compound Having the Formula

In a 5 L three-neck round bottom flask with a mechanical stirrer, the material (332.0 g, 0.8 mol) prepared in step 1 of Example I was dissolved in NMP (660 ml) under nitrogen atmosphere. The solution was then cooled to -10 ° C. A solution of the imine prepared in step 1 dissolved in NMP (320 ml) was prepared under nitrogen atmosphere, and then the temperature was maintained at -5 ° C while being added to the reaction mixture over 30 minutes. The mixture was then stirred further at -8 ° C for 1 hour and at -5 ° C for 2 hours. After cooling to −10 ° C., a mixture of concentrated HCl / saturated NH ₄ Cl solution (30 ml / 720 ml) was added over 10 minutes. MTBE (760 ml) was added and the resulting mixture was stirred at 23 ° C. for 30 minutes. Agitation was stopped and the layers were separated. The aqueous layer was extracted using MTBE (320 ml). The organic layers were combined and washed successively with saturated NH ₄ Cl solution (320 ml), deionized water (320 ml) and brine (320 ml). The resulting solution was dried using sodium sulfate (60 g) and condensed to give a yellow oil (221.0 g). The oil contains the desired product in one diastereoisomeric form as determined by proton NMR.

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

Microanalysis: C ₂₅ H ₃₃ BrClNO 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%; Cl: 6.33%

Step 3: Preparation of a Compound Having the Formula

To a 3 L three neck round bottom flask equipped with a mechanical stirrer, a solution of the crude ester (111 g) prepared in step 2 dissolved in ethanol (1500 L) was charged under a nitrogen atmosphere. The reaction mixture was cooled to 0 ° C. and lead tetraacetate (88.67 g, 0.2 mol) was added all at once. The reaction mixture was stirred at 0 ° C. for 3 hours, and then 15% aqueous NaOH solution (150 ml) was added to the reaction mixture at a temperature below 5 ° C. Methanol was removed under reduced pressure on rotavap. Another 150 ml of 15% NaOH aqueous solution was added, then the reaction mixture was extracted with ethyl acetate (3 x 300 ml), washed with deionized water (2 x 100 ml) and brine (2 x 100 ml), It was dried over anhydrous magnesium sulfate (30 g). Then filtered over celite and condensed under reduced pressure to afford the desired product (103 g) in the form of a red oil.

Step 4: Preparation of a Compound Having the Formula

The compound was prepared using the product of Step 3 of Example I and using the procedure used in Steps 4 and 5 of Example I.

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Example K

Modifications for the Preparation of Compounds of Example J

Step 1: Preparation of a Compound Having the Formula

To the product (50.0 g, 139.2 mmol) and NaHCO ₃ (33.5 g, 398.3 mmol) prepared in step 3 of Example B, CH ₂ Cl ₂ (500 ml) and water (335 ml) were added. The mixture was stirred at rt for 10 min. A solution of benzyl chloroformate (38.0 g, 222.8 mmol) dissolved in CH ₂ Cl ₂ (380 ml) was stirred rapidly while adding over 20 minutes. After 50 minutes, the reaction mixture was poured into a separate funnel and the organic layer was recovered. The aqueous layer was washed with CH ₂ Cl ₂ (170 ml). The combined organic layers were dried over magnesium sulfate and condensed in vacuo. The obtained crude solid was triturated with hexane and recovered by filtration. The resulting tan solid was vacuum dried to afford the desired racemic product (61.2 g, 96% yield). This material was purified by reverse phase chromatography using a chiral column to obtain each pure enantiomer. The column used above was Whelk-O (R, R) with a 10 micron particle size using a 90:10 heptane: ethanol mobile phase. Optical purity measured using analytical HPLC using similar column and solvent conditions was at least 98%.

^{The 1} H NMR analysis result was in agreement with the desired structure.

Step 2: Preparation of a Compound Having the Formula

Trimethylsilyl iodo (25.5 g, 127.4 mmol) dissolved in CH ₂ Cl ₂ (100 ml) in the compound solution (48.5 g, 106.2 mmol) prepared in step 1 dissolved in CH ₂ Cl ₂ (450 ml) Was added via canula. The resulting 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 resulting reaction solution was vacuum condensed to give an orange oil. The residue was dissolved in methyl t-butyl ether (500 ml) and extracted with 1N HCl (318 ml) and water (1 × 200 ml, 1 × 100 ml). The resulting aqueous extract was washed again with MTBE (100 ml). Solid NaHCO ₃ (40.1 g, 478 mmol) was added little by little to the obtained aqueous solution. The resulting basified aqueous mixture was extracted using MTBE (1 × 1 L, 2 × 200 ml). The combined organic solution was washed with brine and vacuum condensed to afford the desired product (23.3 g, 68% yield). ^{The 1} H NMR analysis result was in agreement with the desired structure.

Step 3: Preparation of a Compound Having the Formula

To the product solution (23.3 g, 72.1 mmol) prepared in step 2 dissolved in DMF (200 ml) was added NT-Boc-glycine N-hydroxysuccinimide ester (17.9 g, 65.9 mmol). The reaction mixture was stirred at rt for 20 h. The mixture was poured into ethyl acetate (1.2 L) and washed with 1M HCl (2 x 250 ml), saturated aqueous NaHCO ₃ (2 x 250 ml) and brine (2 x 250 ml). The solution was dried over magnesium sulfate and condensed to afford the desired product (32.0 g, 100% yield).

Elemental Analysis: C ₁₈ H ₂₄ BrClN ₂ O ₆

Calculated: C: 46.06; H: 5.04; N: 5.84;

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

^{The 1} H NMR analysis result was in agreement with the desired structure.

Step 4: Preparation of a Compound Having the Formula

To a solution of the product (31.9 g, 66.5 mmol) prepared in step 3 dissolved in anhydrous ethanol (205 ml) was added ethanol HCl solution (3M solution, 332.4 mmol). The reaction solution was stirred at 58 ° C. for 30 minutes. The solution was cooled and condensed in vacuo. The resulting residue was dissolved in ethyl acetate (250 ml) and stirred at 0 ° C. for 2 hours. Filtration recovered the white precipitate and washed with cold ethyl acetate. The solid obtained was dried in vacuo to afford the desired product (23.5 g, 85% yield).

Elemental Analysis: C ₁₃ H ₁₆ BrClN ₂ O ₄ + 1.0HCl

Calculated: C: 37.53; H: 4. 12; N: 6.73;

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

Example L: Preparation of Compounds Having the Formula

Step 1: Preparation of a Compound Having the Formula

To a solution of 3-chloro-5-bromosalicylaldehyde (35.0 g, 0.15 mole) dissolved in DMF (175 ml), potassium carbonate (powder, oven dried at 100 ° C. under vacuum, 22.1 g, 0.16 mole) at room temperature In addition, a pale yellow slurry was obtained. Next, the bath temperature was maintained at 20 ° C. while adding MEMCl (pure water, 25.0 g, 0.2 mole). The mixture was then stirred at 22 ° C. for 6 hours and added to deionized water (1200 ml) to precipitate the product. The resulting slurry was filtered on a pressure filter and the filter cake was washed with deionized water (2 x 400 ml) and dried under N ₂ / vacuum to afford the desired product (46.0 g, 95% yield) in the form of a gray 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, 2H), 7.77 (d, 1H) 7.85 (d 1H ), 10.30 (s 1 H); ¹³ C NMR (CDCl ₃ , TMS) d (ppm): 59.05, 70.11, 71.49, 99.50, 117.93, 129.78, 132.37, 138.14, 155.12, 188.22. DSC: 48.24 ° C. (endothermic 90.51 J / g);

Microanalysis: C ₁₁ H ₁₂ BrClO ₄

Calculated value: C: 40.82%; H: 3.74%; Cl: 10.95%; Br: 24.69%

Found: C: 40.64%; H: 3.48%; Cl: 10.99%; Br: 24.67%

Step 2: Preparation of a Compound Having the Formula

Into a 500 ml three necked round bottom flask equipped with a mechanical stirrer, the product (32.35 g, 0.1 mol) prepared in step 1 was charged, followed by THF (160 ml) and (S) -phenylglycinol (13.71). g, 0.1 mol) was added. After holding at 22 ° C. for 30 minutes, magnesium sulfate (20 g) was added. The mixture was stirred at 22 ° C. for 1 hour and filtered over a coarse glass filter. The filtrate obtained was condensed under reduced pressure to give a pale yellow oil (48.0 g) containing the desired imine. No further purification was done and the crude product was used directly in the next reaction step.

Microanalysis: C ₁₉ H ₂₁ BrClNO ₄

Calculated: C: 51.54%; H: 4.78%; N: 3.16% Br: 18.04%; Cl: 8.00%

Found: C: 51.52%; H: 5.02%; N: 2.82% Br: 16.31%; Cl: 7.61%

Step 3: Preparation of a Compound Having the Formula

In a 500 ml three neck round bottom flask equipped with a mechanical stirrer, the material (332.0 g, 0.8 mol) prepared in step 1 of Example I was dissolved in NMP (660 ml) under nitrogen atmosphere. The solution was then cooled to -10 ° C. The solution of the imine prepared in step 2 dissolved in NMP (320 ml) was prepared under nitrogen atmosphere, and then the temperature was maintained at -5 ° C while being added to the reaction mixture over 30 minutes. The mixture was then stirred further at -8 ° C for 1 hour and at -5 ° C for 2 hours. After cooling to −10 ° C., a mixture of concentrated HCl / saturated NH ₄ Cl solution (30 ml / 720 ml) was added over 10 minutes. MTBE (760 ml) was added and the resulting mixture was stirred at 23 ° C. for 1 hour. Agitation was stopped and the layers were separated. The aqueous layer was extracted using MTBE (320 ml). The organic layers were combined and washed successively with saturated NH ₄ Cl solution (320 ml), deionized water (320 ml) and brine (320 ml). The resulting solution was dried using magnesium sulfate (60 g) and condensed to give a yellow oil (228 g). The oil contains the desired product in one diastereoisomeric form as determined by proton NMR.

DSC: 227.54 ° C. (endotherm. 61.63 J / g);

Microanalysis: C ₂₅ H ₃₃ BrClNO ₆

Calculated: C: 53.72%; H: 5.95%; N: 2.50% Br: 14.29%; Cl: 6.33%

Found: C: 53.80%; H: 6.45%; N: 2.23% Br: 12.85%; Cl: 6.12%

Step 4: Preparation of a Compound Having the Formula

To a 3 L three neck round bottom flask equipped with a mechanical stirrer, a solution of the crude ester (111 g) prepared in step 2 dissolved in ethanol (1500 L) was charged under a nitrogen atmosphere. The reaction mixture was cooled to 0 ° C. and lead tetraacetate (88.67 g, 0.2 mol) was added all at once. The reaction mixture was stirred at 0 ° C. for 3 hours, and then 15% aqueous NaOH solution (150 ml) was added to the reaction mixture at a temperature below 5 ° C. Ethanol was removed under reduced pressure on rotavap. Another 600 ml of 15% NaOH aqueous solution was added and then the reaction mixture was extracted with ethyl acetate (2 × 300 ml), MTBE (2 × 200 ml) and ethyl acetate (2 × 200 ml). The organic layers were combined, washed with deionized water (2 x 100 ml) and brine (2 x 100 ml) and dried over anhydrous magnesium sulfate (30 g). The solution was then filtered over celite and condensed under reduced pressure to afford the desired product (96 g) in the form of an orange oil. The oil was used directly in the next step without further purification.

DSC: 233.60 ° C. (endotherm 67.85 J / g);

Microanalysis: C ₂₄ H ₂₉ BrClNO ₅

Calculated: C: 54.71%; H: 5.54%; N: 2.65% Br: 15.16%; Cl: 6.72%

Found: C: 52.12%; H: 5.04%; N: 2.47% Br: 14.77%; Cl: 6.48%

Step 5: Preparation of a Compound Having the Formula

The crude product obtained in step 4 (about 94 g) was dissolved in anhydrous ethanol (180 ml) and para toluenesulfonic acid monohydrate (50.0 g, 0.26 mol) was added. The reaction mixture was then heated to reflux for 8 hours and then the solvent was removed under reduced pressure. After the remaining solid was dissolved in THF (100 ml), the THF was stripped off under reduced pressure. The residue was dissolved in ethyl acetate (500 ml) and cooled to about 5 ° C. The solid was then filtered and washed with heptane (2 x 50 ml) to give a white solid. The solid was then air dried to afford the desired product in the form of a white solid (38 g) in the form of a single isomer.

¹ H NMR (DMSO, 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 (1H, d), 8.35 (br.s, 3H); ¹³ C NMR (DMSO, TMS) (ppm): 13.82, 20.75, 37.13, 45.59, 60.59, 110.63, 122.47, 125.44, 127.87, 128.06, 129.51, 131.95 137.77, 145.33, 150.14 168.98: DSC: 69.86 ° C. (endothermic 406.5J / g) 165.7 ° C. (endothermic 62.27 J / g), 211.24 ° C. (exothermic 20.56 J / g)

[a] ^D ₂₅ = + 4.2 ° (c = 0.960, MeOH); 1R (MIR) (cm-1) 2922, 1726, 1621, 1591, 1494, 1471, 1413, 1376, 1324, 1286, 1237, 1207;

Microanalysis: C ₁₈ H ₂₁ ClNO ₆ S

Calculated: C: 43.69%; H: 4.27%; N: 2.83%; Br: 16.15%, Cl: 7.16%; S: 6.48%

Found: C: 43.40%; H: 4.24%; N: 2.73%; Br: 16.40%, Cl: 7.20%; S: 6.54%

Step 6: Preparation of a Compound Having the Formula

The compound was prepared according to the procedure described in steps 4 and 5 of Example I, except that the same amount of intermediate prepared in Step 5 was used in place of the free base in Step 4 of Example I above. .

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Example M: Preparation of Compounds Having the Formula

Step 1: Preparation of 3-iodo-5-chlorosalicylaldehyde

N-iodosuccinimide (144.0 g, 0.641 mole) was added to a solution of 5-chlorosalicylaldehyde (100 g, 0.638 mole) dissolved in dimethylformamide (400 ml). The reaction mixture was stirred for 2 days at room temperature. Additional N-iodosuccinimide (20.0 g) was added and stirred for 2 more days. The resulting reaction mixture was diluted with ethyl acetate (1 L), washed with hydrochloric acid (300 ml, 0.1 N), water (300 ml), sodium thiosulfate (5%, 300 ml), brine (300 ml), and using magnesium sulfate After drying, it was condensed to dryness to obtain the desired aldehyde as a pale yellow solid (162 g, 90% yield).

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

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

At 20 ° C., potassium carbonate (41.4 g, 0.30 mole) was added to a solution of 3-iodo-5-chlorosalicylaldehyde (84.74 g, 0.30 mole) dissolved in DMF (200 ml) to obtain a yellow slurry. . The reaction temperature was maintained while adding MEM-Cl (38.2 g, 0.305 mole). After 2 hours, additional MEM-Cl (1.5 g) was added. After stirring for 1 hour, the resulting reaction mixture was poured into an ice-water mixture and stirred. The precipitate formed was filtered and dried in vacuo to afford the desired protected aldehyde. Yield: 95 g (85%)

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Step 3: Preparation of a Compound Having the Formula

At room temperature, (S) -phenyl glycinol (15.37 g) in a solution of 2-O- (MEM) -3-iodo-5-chlorosalicylaldehyde (41.5 g, 0.112 mole) dissolved in THF (200 ml). , 0.112 mole) was added. After stirring for 1 hour, magnesium sulfate (16 g) was added and stirred for 12 hours. The resulting reaction mixture was filtered and the filtrate was condensed and dried in vacuo for 2 hours to afford the desired intermediate imine. In a two-necked round bottom flask, Reformasky reagent (81.8 g, 0.2464 mole) and N-methylpyrrolidone (100 ml) obtained in step 1 of Example I were charged and stirred at -10 ° C. The mixture was held at this temperature for 2 hours and then at -5 ° C for 1 hour. The mixture was cooled to −10 ° C. and then a solution of concentrated HCl (16 ml / 200 ml) dissolved in saturated ammonium chloride was added. Ethyl ether (500 ml) was added and stirred at rt for 2 h. The ether layer was separated and the aqueous layer was extracted with ether (300 ml). The combined ether layers were washed with saturated ammonium chloride (200 ml), water (200 ml), brine (200 ml), dried over magnesium sulfate and condensed to give an oil (61.0 g, 90% yield). ¹ H NMR analysis confirmed that the desired structure was actually one diastereoisomer, and the MS analysis result was consistent with the desired structure.

Step 4: Preparation of a Compound Having the Formula

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

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Step 5: Preparation of S-ethyl 3- (N-BOC-gly) -amino-3- (S)-(5-chloro-2-hydroxy-3-iodo) phenyl propionate

Ethyl 3- (S) -amino-3- (5-chloro-2-hydroxy-3-iodo) propionate PTSA dissolved in BOC-gly-OSu (9.4 g, 34.41 mmol) in DMF (200 ml) Salt (17.0 g, 31.38 mmol) was added and triethylamine (4.8 ml) was added. The reaction mixture was stirred at rt for 18 h. The DMF was removed in vacuo and the residue was partitioned between ethyl acetate (600 ml) and dilute hydrochloric acid (100 ml). The organic layer was washed with sodium bicarbonate (200 ml), brine (200 ml), dried over magnesium sulfate and condensed to afford the desired product as a solid (14.2 g, 86% yield).

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Step 6: Preparation of S-ethyl 3- (N-gly) amino-3- (5-chloro-2-hydroxy-3-iodo) phenyl propionate hydrochloride

At 0 ° C., dioxane / HCl (4N, 70 ml) was added ethyl 3- (S)-(N-BOC-gly) -amino-3- (5-chloro-2-hydroxy-3-iodo) phenyl propion Add to ate (37.20 g, 70.62 mmol) and stir at room temperature for 3 hours. The resulting reaction mixture was condensed and toluene (100 ml) was added and then condensed again. The resulting residue was suspended in ether, filtered and dried to afford the desired product in the form of crystalline powder (32 g, 98% yield).

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Example N: Preparation of a Compound Having the Formula

Step 1: Preparation of a Compound Having the Formula

The above compound was prepared using the procedure of Step 2A of Example I, except that the same amount of 2-hydroxy-3,5-dibromobenzaldehyde was used instead of 3,5-dichlorosalicylaldehyde It was. Yield 88%; Pale yellow solid; mp46-47 ° C .; R _f = 0.6 (ethyl acetate / hexane 1: 1 v / v); 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 ⁺ )

HR-MS: C ₁₁ H ₁₂ Br ₂ O ₄

Calculation: 367.9083

Found: 367.9077

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Step 2: Preparation of a Compound Having the Formula

The compound was prepared according to the procedure of Step 2B and 2C of Example I, except that the compound of Step 1 was used in the same amount in Step 2B of Example I. Yield 90%; Yellow solid; mp 57-59 ° C .; R _f = 0.46 (ethyl acetate / hexane 1: 1 v / v); ¹ H NMR (CDCl ₃ ) d 1.45 (s, 9H); 2.1 (br, 1H, interchangeable), 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 ( br, 1H, interchangeable), 3.39 (s, 3H), 3.58-3.63 (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, 6 H), 7.49 (d, 1 H); FAB-MS m / z 602 (M + H)

HR-MS: C ₂₅ H ₃₄ NBr ₂ O ₆

Calculation: 602.0753

Found: 602.0749

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Step 3: Preparation of a Compound Having the Formula

The compound (p-toluenesulfonate) was prepared according to the procedure of Example 3, except that the compound of Step 2 was used in the same amount in Step 3A of Example I. 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 ₁ = 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 (br, 3 H); FAB-MS m / z 365 (M + H)

HR-MS: C ₁₁ H ₁₄ NBr ₂ O ₃

Calculation: 365.9340

Found: 365.9311

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Step 4: Preparation of a Compound Having the Formula

The compound was prepared according to the procedure of Step 4 of Example I using the compound of Step 3. The resulting BOC protecting group intermediate was converted to the desired compound using the procedure of Step 5 of Example I.

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Example P: Preparation of Compounds Having the Formula

Example I, using the same amount of 3-iodo-5-bromosalicylaldehyde prepared in Step 1 of Example F, instead of 3,5-dichlorosalicylaldehyde, prepared in Step 2A of Example I According to the procedure of the above compound was prepared.

Example 1

(±) 3-bromo-5-chloro-2-hydroxy-β-[[2-[[[3-hydroxy-5-[(1,4,5,6-tetrahydro-5-hydroxy Pyrimidin-2-yl) amino] phenyl] carbonyl] amino] acetyl] amino] benzenepropanoic acid, trifluoroacetate salt

Preparation of Compounds Having the Formula

At ice bath temperature, the product of Example H (0.4 g, 0.0014 mole), the product of Example B (0.58 g, 0.0014 mole), triethylamine (0.142 g, 0.0014 mole), DMAP (17 mg), and anhydrous To DMA (4 ml), EDCl (0.268 g, 0.0014 mole) was added. The reaction mixture was stirred at rt overnight. The resulting ester intermediate was separated by reverse phase purification HPLC. This ester was dissolved in water (10 ml) and CH ₃ CN (5 ml) to add LiOH (580 mg, 0.0138 mole). After stirring for 1 hour at room temperature, the pH was lowered to 2 using TFA, purified by reverse phase purification HPLC and freeze-dried to give the desired product in the form of a white solid (230 mg).

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Example 2 Preparation of a Compound Having the Formula

Instead of the product obtained in Example B, using the same amount of product obtained in Example A, the above compound was prepared according to the method of Example 1. The yield obtained as a white solid after freeze drying was 320 mg.

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Example 3 Preparation of a Compound Having the Formula

Instead of the product obtained in Example B, using the same amount of product obtained in Example F, the above compound was prepared according to the method of Example 1. The yield obtained as a white solid after freeze drying was 180 mg.

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Example 4 Preparation of a Compound Having the Formula

Instead of the product obtained in Example B, using the same amount of product obtained in Example D, the above compound was prepared according to the method of Example 1. The yield obtained as a white solid after freeze drying was 180 mg.

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Example 5 Preparation of a Compound Having the Formula

The above compound was prepared according to the method of Example 1, using the same amount of product obtained in Example E instead of the product obtained in Example B. The yield obtained as a white solid after freeze drying was 250 mg.

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Example 6: Preparation of a Compound Having the Formula

Instead of the product obtained in Example B, using the same amount of product obtained in Example C, the above compound was prepared according to the method of Example 1. The yield obtained as a white solid after freeze drying was 220 mg.

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Example 7: Preparation of Compounds Having the Formula

To a product of Example H (7.8 g, 0.027 mole) dissolved in anhydrous DMA (50 ml) in a flame-dried flask at nitrogen atmosphere and ice bath temperature, isobutylchloroformate (3.7 g, 0.027 mole) was slowly added. Next, N-methylmorpholine (2.73 g, 0.027 mole) was added. The solution was stirred for 15 min at ice bath temperature. To the reaction mixture, the product of Example L (10.0 g, 0.024 mole) was added at ice bath temperature followed by N-methylmorpholine (2.73 g, 0.027 mole). The reaction mixture was stirred at rt overnight. The resulting ester intermediate was separated by reverse phase purification HPLC. This ester was dissolved in water (60 ml) and CH ₃ CN (30 ml) to add LiOH (10 g, 0.238 mole). The reaction mixture was stirred for 1 hour at room temperature, then the pH was lowered to 2 using TFA, purified by reverse phase purification HPLC and freeze-dried to afford the desired product in the form of a white solid (9.7 g).

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Example 8

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

Preparation of Compounds Having the Formula

Step A

To the product of Example H (9.92 g, 0.0345 mole) dissolved in anhydrous DME (200 ml), N-methylmorpholine (4.0 ml, 0.0362 mole) was added. The reaction mixture was cooled to -5 ° C (salt-ice bath). Isobutyl chloroformate, IBCF (4.48 ml, 4.713 g, 0.0345 mole) was added over 1 minute and the reaction mixture was stirred at ice bath temperature for 12 minutes. Next, the product of Example I (11.15 g, 0.030 mole) was added to the reaction mixture at an ice bath, followed by N-methylmorpholine (4.0 ml, 0.0362 mole). The reaction mixture was allowed to warm to room temperature and then vacuum condensed at 50 ° C. to give a dark residue. The residue was dissolved in acetonitrile: water (about 50 ml). A small amount of TFA was added to adjust the pH to acidic. The residue was placed on a 10 x 500 cm C-18 (50 micron particle size) column to separate the ester of the desired product. (Solvent program: 100% water + 0.05% TFA to 30:70 water + 0.05% TFA: acetonitrile + 0.05% TFA; 1 hour; 100 ml / min; the solvent program was started after the solvent advance point eluted). After RPHPLC purification and freeze drying, a white solid (10.5 g, 50%) was obtained.

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Step B

The product obtained in step A (about 11 g) was dissolved in dioxane: water and the pH of the solution was adjusted to about 11.5 by adding 2.5N NaOH. The reaction mixture was stirred at room temperature. Periodically, the pH was adjusted back to 11 or higher by addition of base. After 2-3 hours, the conversion of the ester to acid was determined to be complete. The pH of the reaction mixture was adjusted to about 6 and a sticky oil precipitated out of the solution. The oil was removed by decantation and washed with hot water (200 ml). The resulting aqueous mixture was cooled and filtered to afford the compound (2.6 g, after freeze drying from HCl solution) in the solid state. The residue, a dark sticky oil, was treated with hot water and cooled to give a tan powder (4.12 g, after freeze drying from HCl solution).

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Example 9

(S) 3-Bromo-5-chloro-2-hydroxy-β-[[2-[[[3-hydroxy-5-[(1,4,5,6-tetrahydro-5-hydroxy Pyrimidin-2-yl) amino] phenyl] carbonyl] amino] acetyl] amino] benzenepropanoic acid, trifluoroacetate salt

Step 1: Preparation of a Compound Having the Formula

The product of Example J (1.0 g, 2.4 mmol) dissolved in N, N-dimethelacetamide (10 ml), the product of Example H (0.75 g, 2.6 mmol), and 4-dimethylaminopyridine (40 mg) To this, triethylamine (0.24 g, 2.4 mmol) was added. The mixture was stirred at rt for 15 min and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.60 g, 3.1 mmol) was added. The reaction mixture was stirred at rt overnight. The resulting mixture was vacuum condensed and reversed phase HPLC purification (starting gradient 90:10 water / TFA: MeCN, retention time 22 minutes) to afford the desired product (1.6 g, 52% yield).

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Step 2

To a solution of the ester (800 mg, 1.2 mmol) prepared in step 1 dissolved in a 1: 4 MeCN: water solution (7 ml), lithium hydroxide (148 mg, 6.2 mmol) was added. The reaction mixture was stirred at rt for 2 h. TFA (0.71 ml, 9.2 mmol) was added and the resulting mixture was reversed-phase HPLC purification (starting gradient 95: 5 water / TFA: MeCN, retention time 24 minutes) to afford the desired product (860 mg, 83% yield).

Elemental Analysis: C ₂₂ H ₂₃ BrClN ₅ O ₇ + 1.7 TFA

Calculated: C 39.18; H 3.20; N 8.99

Found: C 39.11; H 3.17; N 9.07

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Step 3

Preparation of Hydrochloride Salts

The product of Example 2 was dissolved in a suitable solvent (acetonitrile: water) and the solution was passed slowly through a Bio-Rad AG2-8X (chloride form, 200-400 mash, 5 equivalents or more) ion exchange column. Freeze drying to afford the desired product in the form of HCl salt.

Example 10 Preparation of a Compound Having the Formula

In Step A of Example 8, the above compound was prepared according to the procedure of Example 8 using the product of Example N instead of the product of Example I. The product was separated by purified RPHPLC and freeze dried to afford the desired product in the form of a TFA salt.

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Example 11 Preparation of a Compound Having the Formula

In Step A of Example 8, the above compound was prepared according to the procedure of Example 8 using the product of Example M instead of the product of Example I. The product was separated by purified RPHPLC and freeze dried to afford the desired product in the form of a TFA salt.

The results of MS and ¹ H NMR analysis were consistent with the desired structure above.

Example 12 Preparation of a Compound Having the Formula

In Step A of Example 8, the above compound was prepared according to the procedure of Example 8 using the product of Example P instead of the product of Example I. The product was separated by purified RPHPLC and freeze dried to afford the desired product in the form of a TFA salt.

Example 13: Preparation of a Compound Having the Formula

Step 1: Preparation of 2-O- (MEM) -3,5-dioodosalicylaldehyde having the formula

At 20 ° C., potassium carbonate (18.5 g, 0.134 mole) was added to a solution of 3,5-dioodosalicylaldehyde (50.0 g, 0.134 mole) dissolved in DMF (150 ml) to obtain a yellow slurry. MEM-Cl (15.8 ml, 0.134 mole) was added while maintaining the reaction temperature. After 2 hours, additional MEM-Cl (1.5 g) was added. After further stirring for 1 hour, the reaction mixture was poured into ice water and stirred. The precipitate formed was filtered and dried in vacuo to afford the desired protected aldehyde (61 g, 99% yield).

^{The 1} H NMR analysis result was in agreement with the desired structure.

Step 2: Preparation of a Compound Having the Formula

At room temperature, (S) -phenyl glycinol (17.9 g, 0.13) in a solution of 2-O- (MEM) -3,5-dioodosalicylaldehyde (41.5 g, 0.112 mole) dissolved in THF (150 ml). mole) was added. After stirring for 1 hour, magnesium sulfate (20.7 g) was added and stirred for 12 hours. The resulting reaction mixture was filtered and the filtrate was condensed and vacuum dried for 2 hours. In a two-necked round bottom flask, Reformasky reagent (96 g, 0.289 mole) and N-methylpyrrolidone (250 ml) were charged and stirred at -10 ° C. The temperature was maintained at −10 ° C. while slowly adding a solution of the imine dissolved in N-methylpyrrolidone (100 ml). The mixture was held at this temperature for 2 hours and then at -5 ° C for 1 hour. The mixture was cooled to −10 ° C. and then a solution of concentrated HCl (16 ml / 200 ml) dissolved in saturated ammonium chloride was added. Ethyl ether (500 ml) was added and stirred at rt for 2 h. The ether layer was separated and the aqueous layer was extracted with ether (300 ml). The combined ether layers were washed with saturated ammonium chloride (200 ml), water (200 ml), brine (200 ml), dried over magnesium sulfate and condensed to give an oil (90.0 g, 99% yield). NMR analysis confirmed that the desired structure was in fact one diastereoisomer.

Step 3: Preparation of a Compound Having the Formula

The solution of crude ester (14.0 g, 20.1 mmol) prepared in step 2 was dissolved in ethanol (100 ml) and cooled to 0 ° C. Lead tetraacetate (9.20 g, 20.75 mmol) was added all at once. After 3 h, 15% NaOH (73 ml) solution was added to the mixture. Most of the ethanol was removed under reduced pressure. To the resulting residue, 15% NaOH solution (200 ml) was added and extracted using ether (400 ml). The ether layer was washed with water (100 ml), brine (100 ml), dried and condensed to give an orange oil. The oil was dissolved in ethanol (100 ml) and para-toluenesulfonic acid (6.08 g) was added. The resulting solution was heated to reflux for 8 hours and condensed under reduced pressure. The resulting residue was diluted with THF (60 ml), heated to reflux and then cooled. No deposits formed upon storage. The reaction mixture was purified by condensation and HPLC to obtain the amino acid in the form of its PTSA salt. The resulting solid was dissolved in ethanol and saturated with HCl gas. The reaction mixture was heated to reflux for 6 hours. The reaction mixture was condensed to give PTSA salt (12.47 g) of the desired amino acid.

Step 4: Preparation of ethyl 3- (N-BOC-gly) -amino-3- (S)-(3,5-dioodo-2-hydroxyphenyl) propionate

Ethyl 3- (S) -amino-3- (3,5-dioodo-2-hydroxyphenyl) propionate PTSA salt dissolved in BOC-gly-OSu (7.48 g, 27.04 mmol) in DMF (200 ml) (12.47 g, 27.04 mmol) was added and triethylamine (3.8 ml) was added. The reaction mixture was stirred at rt for 18 h. The DMF was removed in vacuo and the residue was partitioned between ethyl acetate (600 ml) and dilute hydrochloric acid (100 ml). The organic layer was washed with sodium bicarbonate (200 ml), brine (200 ml), dried over magnesium sulfate and condensed to afford the desired product as a solid (17.0 g, 96% yield).

^{The 1} H NMR analysis result was in agreement with the desired structure.

Step 5: Preparation of ethyl 3- (N-gly) -amino-3- (3,5-dioodo-2-hydroxyphenyl) -propionate hydrochloride having the formula

Ethyl 3- (N-BOC-gly) -amino-3- (S)-(3,5-dioodo-2-hydroxyphenyl) propionate at 0 ° C. in dioxane / HCl (4N, 40 ml) (17.0 g, 25.97 mmol) was added and the resulting reaction mixture was stirred at rt for 3 h. The reaction mixture was condensed, toluene (100 ml) was added and then condensed again. The residue obtained was dried to afford the desired product in the form of crystalline powder (8.0 g, 56% yield).

^{The 1} H NMR analysis result was in agreement with the desired structure.

Step 6

M- (5-hydroxypyrimidino) hypuric acid (3.74 g, 12.98 mmol) was dissolved in dimethylacetamide (25 ml) by heating. Next, it was heated to 0 ° C. and isobutylchloroformate (1.68 ml) was added at once, followed by N-methylmorpholine (1.45 ml). After 10 minutes, ethyl 3- (N-gly) -amino-3- (3,5-dioodo-2-hydroxyphenyl) -propionate hydrochloride (6.0 g, 10.82 mmol) was added in one portion followed by N-methyl Morpholine (1.45 ml) was added. The reaction mixture was stirred at rt for 18 h. The reaction mixture is condensed and the residue is dissolved in tetrahydrofuran / water (1: 1, 20 ml) and chromatographed (inverted phase, 60 min, 95: 5 water: acetonitrile containing 0.1% TFA). To 30: 70 water: acetonitrile). The combined fractions were condensed. The resulting residue was dissolved in acetonitrile: water and made basic by addition of lithium hydroxide. The resulting solution was stirred for 2 hours. The resulting reaction mixture was condensed and purified by HPLC as described above to afford the desired acid in the form of the TFA salt. The TFA salt was passed through an ion exchange column and then freeze dried to convert to the corresponding hydrochloride salt. ^{The 1} H NMR analysis result was in agreement with the desired structure.

Example 14-18

As will be apparent to one skilled in the art, compounds of formulas (7), (8), (11), (13) and (14) can be prepared according to the methods described herein using suitable starting materials and reagents. have.

The activity of the compounds of the present invention was tested by the following assays. The results of the tests according to such evaluation analysis are shown in Table 1.

VITRONECTIN ADHESION ASSAY

MATERIALS

Human vitronectin receptor (α _v β ₃ ) was purified from human placenta, as described in Methods in Enzymology, 144: 475-489 (1987), by Pytela et al. Human vitronectin was purified from fresh frozen plasma as described in Yatohgo et al. Cell Structure and Function, 13: 281-292 (1988). Vitronectin of biotinylated humans is described in Charo et al. J. Biol. Chem., 266 (3): 1415-1421 (1991), was prepared by binding the purified Vitronectin with NHS-biotin obtained from Pierce Chemical Company (Rockford, IL). Evaluation Assay Buffer, OPD Substrate Purification, and RIA Grade BSA were obtained from Sigma, St. Louis, Maryland, USA. Anti-biotin antibodies were obtained from Calbiochem, La Jolla, California, USA. Linbro microtiters were obtained from Flow Labs of Maclean, Virginia, USA. ADP reagents were obtained from Sigma, St. Louis, Maryland, USA.

Method (METHODS)

Solid Phase Receptor Assays

This evaluation analysis is basically the same as that described in [Niiya et al., Blood, 70: 475-483 (1987)]. Stock of purified human vitronectin receptor (α _v β ₃ ) in tris-buffered saline containing 1.0 mM Ca ⁺⁺ , Mg ⁺⁺ , and pH7.4 (TBS ⁺⁺⁺ ) Dilute to 1.0 g / mL from stock solution. Diluted receptors were immediately delivered to the LINBRO titration plate at a rate of 100 L / well (100 ng receptor / well). The microtiter plate is sealed and incubated overnight at 4 ° C. to allow the receptor to bind to the wells. All subsequent steps were carried out at room temperature. After the evaluation assays were empty, 200 L of 1% RIA grade BSA in TBS ⁺⁺⁺ (TBS ⁺⁺⁺ / BSA) was added to block the exposed plastic sides. After incubation for two hours, the assay plate was washed using a 96 well plate washer. Logarithmic serial dilution of test compounds and controls was performed using 2 nM biotinylated ^Vitronectin in TBS ⁺⁺⁺ / BSA as diluent, starting at a stock concentration of 2 mM. . This pre-mixing of the test (or control) ligand with the labeled ligand, and subsequent delivery of the 50 L fraction to the evaluation assay was performed by the CETUS Propette robot. The final concentration of labeled ligand was 1 nM and the highest concentration of test compound was 1.0 × 10 ⁻⁴ M. Competition took place for two hours, after which all wells were washed by a plate washer, as described above. Affinity purified horseradish peroxidase labeled goat anti-biotin antibody was diluted 1: 3000 in TBS ⁺⁺⁺ / BSA, and 125 L of each affinity purified horseradish peroxidase labeled goat anti-biotin antibody. Was added. After 30 minutes, the plates were washed and then incubated as OPD / H ₂ 0 in 100 mM / L citrate buffer at pH 5.0. The plate was read by a microtiter plate reader at a wavelength of 450 nm, when the maximum-binding control wells reached an absolute surround bottom of about 1.0. ₄₅₀ was recorded for analysis. This data was analyzed using recorded macros for use in the Excel spreadsheet program. Mean, standard deviation, and% CV were determined for duplicate concentrations. Mean A ₄₅₀ values were normalized to the mean of the four maximum-binding comparisons (no addition of competing elements; B-MAX). The normalized values are described in Rodman et al., Int. Atomic Energy Agency, Vienna, pp469 (1977)], applied to a four parameter curve fit algorithm, constructed on a semi-log scale, and biotinylated Vitronectin (IC ₅₀ ) were calculated for the calculated concentrations corresponding to 50% inhibition of the maximum binding of the compounds and the corresponding R ² at least 50% inhibition at the highest concentration tested. Alternatively, IC ₅₀ was reported to be above the highest concentration tested. Β-[[2-[[5-[(aminoiminomethyl) -amino] -1-oxopentyl] amino] -1-oxopentyl, a potent α _v β ₃ antagonist (IC _{50 in the} range of 3-10 nM) ] Amino] -3-pyridinepropane acid [USSN 08 / 375,388, see Example 1] was included in each plate as a positive control factor.

Purified IIb / IIIa Receptor Assessment Assay (PURIFIED IIb / IIIa RECEPTOR ASSAY)

MATERIALS

Human fibrinogen receptors (α _IIb β ₃ ) were purified from conventional platelets (Pytela, R., Pierschbacher, MD, Argraves, S., Suzuki, S., and Rouslahti, E., “Arginine-Glycine-Aspartic”). acid adhesion receptors "Methods in Enzymology, 144 (1987): 475-489). Human vitronectin from Yatohgo, T., Izumi, M., Kashiwagi, H., and fresh frozen plasma as described in Hayashi, M., Cell Structure and Function, 13 (1988): 281-292. Purified. Vitronectin of biotinylated humans (Charo, IF, Nannizzi, L., Phillips, DR, Hsu, MA, Scaround bottomorough, RM, J. Biol. Chem., 266 (3) (1991): 1415-1421 To vitrified vitronectin, as described in US), by binding NHS-biotin obtained from Pierce Chemical Company (Rockford, IL). Evaluation Assay Buffer, OPD Substrate Purification, and RIA Grade BSA were obtained from Sigma, St. Louis, Maryland, USA. Anti-biotin antibodies were obtained from Calbiochem, La Jolla, California, USA. Linbro microtiters were obtained from Flow Labs of Maclean, Virginia, USA. ADP reagents were obtained from Sigma, St. Louis, Maryland, USA.

Method (METHODS)

Solid Phase Receptor Assays

This evaluation analysis is described by Niiya, K., Hodson, E., Bader, R., Byers-Ward, V. Koziol, JA, Plow, EF, and Ruggeri, ZM, in Blood 70 (1987): 475-483. It is essentially the same as reported in Increased surface expression of the membrane glycoprotein IIb / IIIa complex induced by platelet activation: Relationships to the binding of fibrinogen and platelet aggregation. Stock solution of purified human fibrinogen receptor (α _IIb β ₃ ) in tris-buffered saline containing 1.0 mM Ca ⁺⁺ , Mg ⁺⁺ , and pH7.4 (TBS ⁺⁺⁺ ) from stock solution to 1.0 μg / mL. Diluted receptors were immediately delivered to the LINBRO titration plate at a rate of 100 μL / well (100 ng receptor / well). The microtiter plate is sealed and incubated overnight at 4 ° C. to allow the receptor to bind to the wells. All subsequent steps were carried out at room temperature. After the evaluation assays were empty, 200 μL of 1% RIA grade BSA in TBS ⁺⁺⁺ (TBS ⁺⁺⁺ / BSA) was added to block the exposed plastic sides. After incubation for two hours, the evaluation assay plates were washed using a 96 well plate washer. Logarithmic serial dilution of test compounds and controls was performed using 2 nM biotinylated ^Vitronectin in TBS ⁺⁺⁺ / BSA as diluent, starting at a stock concentration of 2 mM. . This preliminary mixing of the test (or control) ligand with the labeled ligand, and subsequent delivery of the 50 μL fraction to the evaluation assay was performed by a CETUS Propette robot, where the final concentration of labeled ligand was achieved. Was 1 nM and the highest concentration of the test compound was 1.0 × 10 ⁻⁴ M. Competition took place for two hours, after which all the wells were washed by a plate washer, as described above. Affinity purified horseradish peroxidase labeled goat anti-biotin antibody was diluted 1: 3000 in TBS ⁺⁺⁺ / BSA and 125 μL was added to each well. After 30 minutes, the plates were washed and incubated with ODD / H ₂ O ₂ in 100 mM / L citrate buffer at pH 5.0. The plate was read by a microtiter plate reader at a wavelength of 450 nm, when the maximum-binding control wells reached an absolute surround bottom of about 1.0, the final A ₄₅₀ was recorded for analysis. . The data were analyzed using a macro written for use in brand names Excel spreadsheet programs (EXCEL ^TM spreadsheet program). Mean, standard deviation, and% CV were determined for double concentrations. Mean A ₄₅₀ values were normalized to the mean of the four maximum-binding comparisons (no addition of competing elements; B-MAX). The normalized values are described in Int. Atomic Energy Agency, Vienna, pp469 (1977)], applied to a four parameter curve fit algorithm, constructed on a semi-log scale, and biotinylated Vitronectin (IC ₅₀ ) were calculated for the calculated concentrations corresponding to 50% inhibition of the maximum binding of the compounds and the corresponding R ² at least 50% inhibition at the highest concentration tested. Alternatively, IC ₅₀ was reported to be above the highest concentration tested. Β-[[2-[[5-[(aminoiminomethyl) -amino] -1-oxopentyl] amino] -1-oxopentyl, a potent α _v β ₃ antagonist (IC _{50 in the} range of 3-10 nM) ] Amino] -3-pyridinepropane acid [USSN 08 / 375,388, see Example 1] was included in each plate as a positive control factor.

HUMAN PLATELET RICH PLASMA ASSAYS

Healthy aspirin free donors are selected from a pool of volunteers. Collection of high platelet plasma and subsequent analysis of ADP induced platelet aggregation is performed as described in "Platelet Aggregation Measured by the Photometric Methods" of Methods in Enzymology 169 (1989): 117-133 of Zucker, MB. A standard venipuncture technique using a butterfly is to draw 45 mL of whole blood into a 60 mL syringe containing 5 mL of 3,8% trisodium citrate. Made it possible. After thorough mixing in the syringe, anti-coagulated whole blood was transferred to a 50 mL conical polyethylene tube. The blood was centrifuged into sediment non-platelet cells at 200 mg for 12 minutes at room temperature. Platelet high concentration plasma was removed through polyethylene tubes and stored at room temperature until available. Platelet low concentration plasma was obtained from the second centrifugation for 15 minutes at 2000 × g for the remaining blood. Platelet counts are approximately 300,000 to 500,000 pieces per microliter. Platelet high concentration plasma (0.45 mL) was fractionated into siliconized cuvettes, then stirred at 1100 rpm at 37 ° C. for 1 minute, and then 50 uL of pre-diluted test compound was added. After mixing for 1 minute, aggregation was initiated by adding 50 uL of 200 uM ADP. Aggregation was recorded for 3 minutes in Payton Scientific's Payton dual channel aggregometer, Buffalo, New York, USA. Percent inhibition of maximal response to a series of test compound dilutions (salt control) was used to determine the dose response curve. All compounds were tested in duplicate, and the concentration of half-maximal inhibition (IC ₅₀ ) was calculated from the dose response curve, from the dose response curve, for the compound showing 50% or more inhibition at the highest concentration tested. Alternatively, the IC ₅₀ is reported to be greater than the highest concentration tested.

Example α _v β ₃ IC ₅₀ (nM) IIb / IIIaIC ₅₀ (nM) One 0.88 310 2 1.04 430 3 23.7 2440 4 2.02 575 5 2.13 744 6 6.46 919 7 1.01 262 8 0.40 131 9. 0.37 388 9. HCL 0.82 226.2 10 2.26 641 11 12 9.59 1060

Claims (36)

Compounds of the formula and pharmaceutically acceptable salts thereof Wherein X and Y are halo groups as being the same as or different from each other, and R is H or lower alkyl. A compound according to claim 1, or a pharmaceutically acceptable salt thereof, which is selected from compounds having the formula: A compound according to claim 1, or a pharmaceutically acceptable salt thereof, which is selected from compounds having the formula: A pharmaceutical composition comprising a therapeutically effective amount of a compound according to claim 1 and a pharmaceutically acceptable carrier. 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 according to claim 3 and a pharmaceutically acceptable carrier. A method of treating said mediated condition in a mammal in need of treatment of an α _v β ₃ integrin mediated condition comprising administering a compound according to claim 1 in an effective inhibitory amount of α _v β _3. . A method of treating said mediated condition in a mammal in need of treatment of an α _v β ₃ integrin mediated condition, comprising administering a compound according to claim 2 in an effective amount of an α _v β ₃ inhibitory effect. . A method of treating said mediated condition in a mammal in need of treatment of an α _v β ₃ integrin mediated condition, comprising administering a compound according to claim 3 in an effective inhibitory amount of α _v β _3. . 8. The method of claim 7, wherein the condition to be treated is tumor metastasis. The method of claim 8, wherein the condition to be treated is tumor metastasis. The method of claim 9, wherein the condition to be treated is tumor metastasis. 8. The method of claim 7, wherein said condition being treated is solid tumor growth. The method of claim 8, wherein the condition to be treated is solid tumor growth. The method of claim 9, wherein the condition to be treated is solid tumor growth. 8. The method of claim 7, wherein the condition to be treated is angiogenesis. The method of claim 8, wherein the condition to be treated is angiogenesis. The method of claim 9, wherein the condition to be treated is angiogenesis. 8. The method of claim 7, wherein the condition to be treated is osteoporosis. The method of claim 8, wherein the condition to be treated is osteoporosis. The method of claim 9, wherein the condition to be treated is osteoporosis. 8. The method of claim 7, wherein the condition to be treated is malignant humoral calcium hyperemia. 9. The method of claim 8, wherein said condition to be treated is malignant humoral calcium hyperemia. 10. The method of claim 9, wherein said treated condition is malignant humoral calcium hyperemia. 8. The method of claim 7, wherein said condition to be treated is smooth muscle cell migration. The method of claim 8, wherein the condition to be treated is smooth muscle cell migration. The method of claim 9, wherein said condition to be treated is smooth muscle cell migration. 8. The method of claim 7, which is used for the inhibition of restenosis. The method of claim 8, which is used for the inhibition of retinopathy. 10. The method according to claim 9, which is used for the inhibition of restenosis. 8. The method of claim 7, wherein the condition being treated is rheumatoid arthritis. The method of claim 8, wherein the condition to be treated is rheumatoid arthritis. The method of claim 9, wherein the condition to be treated is rheumatoid arthritis. 8. The method of claim 7, wherein the condition to be treated is macular degeneration. The method of claim 8, wherein the condition to be treated is spot degeneration. The method of claim 9, wherein the condition to be treated is spot degeneration.