HUP0100865A2

HUP0100865A2 - Meta-azacyclic amino benzoic acid compounds and derivatives thereof being integrin antagonists, pharmaceutical compositions comprising thereof and their use

Info

Publication number: HUP0100865A2
Application number: HU0100865A
Authority: HU
Inventors: Thomas E. Rogers; Peter G. Ruminski
Original assignee: G.D. Searle & Co.
Priority date: 1998-03-04
Filing date: 1999-02-22
Publication date: 2001-08-28
Also published as: CA2322207A1; AP2000001893A0; CN1214011C; NO315703B1; PL342726A1; HRP20000574A2; TR200002542T2; SK13002000A3; AP1244A; ID25591A; YU52000A; BR9908470A; BG104740A; OA11530A; MY123908A; AU753230B2; IS5582A; AR018139A1; ZA994406B; EA200000804A1

Abstract

A találmány av<3 integrin antagonistákként felhasználható (I) általános képletű vegyületekre - amelyek képletében X és Yjelentése azonos vagy egymástól eltérő halogénatom; és R jelentésehidrogénatom vagy 1-10 szénatomos alkilcsoport - és gyógyszerészetilegelfogadható sóikra, ilyen vegyületeket tartalmazógyógyszerkészítményekre, valamint a vegyületeknek av<3 integrin általmediált betegségek kezelésére szolgáló gyógyszerkészítményekelőállítására történő alkalmazására vonatkozik. ÓThe invention can be used as av<3 integrin antagonists for compounds of general formula (I) - in the formula of which X and Y mean the same or different halogen atoms; and R represents a hydrogen atom or an alkyl group with 1-10 carbon atoms - and refers to their pharmaceutically acceptable salts, pharmaceutical preparations containing such compounds, and the use of the compounds for the production of pharmaceutical preparations for the treatment of diseases mediated by av<3 integrin. HE

Description

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The invention relates to pharmaceutical active ingredients (compounds) that can be used as α _ν ββ integrin antagonists, to pharmaceutical compositions containing such active ingredients, and to the use of the active ingredients for the preparation of pharmaceutical compositions.

Integrins are a family of cell surface glycoproteins that mediate cell adhesion and are useful as mediators of cell adhesion interactions in a variety of biological processes. Integrins are heterodimers composed of non-covalently linked α- and β-polypeptide subunits. Eleven different α-subunits and six different β-subunits have been identified to date. Different α-subunits can combine with different β-subunits to form different integrins.

The integrin identified as α _v β3 (also known as the vitronectin receptor) has been identified as an integrin that plays a role in various disease states, including, but not limited to, tumor metastasis, solid tumor growth (neoplasia), osteoporosis, Paget's disease, malignant humoral hypercalcemia, angiogenesis, including tumor angiogenesis, retinopathy, including macular degeneration, arthritis, including rheumatoid arthritis, periodontal disease, psoriasis, and smooth muscle cell migration (e.g., restenosis). It has also been observed that said agents may be useful as antiviral, antifungal, and antimicrobial agents. Accordingly, compounds that selectively inhibit or antagonize α _v β3 would be advantageously used in the treatment of said diseases or conditions.

It has been previously shown that α _ν β ₃ integrin and other α _v -containing integrins bind to a variety of matrix macromolecules containing the Arg-Gly-Asp (RGD) sequence. Compounds containing the RGD sequence are similar to extracellular matrix ligands in that they bind to cell surface receptors. However, it is also known that RGD peptides are generally non-selective for RGD-dependent integrins. For example, most RGD peptides that bind to α _v β ₃ also bind to α _ν β5, α _ν β1 ^{, and α} llbP3 ^integrins . It is known that antagonizing platelet integrins blocks platelet aggregation in humans. To avoid bleeding side effects _{, it would be advantageous to develop compounds that are selective antagonists of the ανβ3} _integrin _, as opposed to the ctubPs integrin, in the treatment of _ανβ3 integrin-related conditions or diseases.

Tumor cell invasion occurs in a three-step process: 1) tumor cells attach to the extracellular matrix; 2) the matrix is proteolytically degraded; and 3) cells penetrate the degraded barrier. The process can be repeated and result in metastases at sites distant from the original tumor.

It has previously been shown that α _ν β3 integrin plays a biological role in melanoma cell invasion [Seftor et al. _Proc . Natl. Acad. Sci. USA, 89, 1557-1561 (1992)]. Others have shown that α _ν β3 integrin expressed on human melanoma cells promotes the generation of a survival signal, thereby protecting the cells from apoptosis. It would be very advantageous if the tumor cell metastatic process could be modified by interfering with the α _ν β3 integrin cell adhesion receptor in order to prevent tumor metastasis.

Brooks [Brooks et al., 79, 1157-1164 (1994)] demonstrated that α _ν β3 antagonists may offer a therapeutic solution for the treatment of neoplasia (inhibition of solid tumor growth) as systemic administration of α _ν _β3 antagonists causes dramatic regression in a variety of histologically diverse human tumors.

The α _ν β ₃ adhesion receptor integrin has been identified as a marker of angiogenic blood vessels in chicken and humans, suggesting that this receptor plays a critical role in angiogenesis and neovascularization. Angiogenesis is characterized by the rapid invasion, migration, and proliferation of smooth muscle cells and endothelial cells. α _ν β ₃ antagonists inhibit this process by selectively promoting cell apoptosis in the neovasculature. The growth of new blood vessels and angiogenesis may also contribute to pathological conditions such as diabetic retinopathy and macular degeneration [Adonis et al., Amer. J. Ophthal. , 118, 445-450 (1994)], and

...· .:. .:. *·:· rheumatoid arthritis [Peacock et al., J. Exp. Med., 175, 11351138 (1992)]. Based on this, α _ν β ₃ antagonists could be used as therapeutic targets in the treatment of conditions related to neovascularization [Brooks et al., Science, 264, 569-571 (1994)] .

It has also been previously reported that the α _ν β ₃ cell surface receptor is the most important integrin on osteoclasts during bone attachment. Osteoclasts cause bone resorption, and if bone resorption activity exceeds bone formation activity, osteoporosis (bone loss) can result, leading to a significant increase in the number of fractures, more days spent in hospital and ultimately higher mortality. α _ν β ₃ antagonists have also been shown to be potent inhibitors of osteoclastic activity both in vitro [Sato et al., J. Cell Biol., Ill, 1713-1723 (1990)] and in vivo [Fisher et al., Endocrinology, 132, 1411-1413 (1993)]. Antagonism of α _ν β ₃ leads to reduced bone resorption, thus restoring the balance between bone formation and resorption activity. Accordingly, there are significant advantages in producing antagonists of osteoclast α _ν β ₃ that are effective inhibitors of bone resorption and thus could be suitable for the treatment or prevention of osteoporosis.

The role of α _ν β ₃ integrin in smooth muscle cell migration may also be a therapeutic target in preventing or inhibiting neointimal hyperplasia leading to restenosis after vascular procedures [Choi et al., J. Vasc. Surg., 19 (1),

125-134 (1994)]. It would be advantageous if neointimal hyperplasia could be prevented or inhibited by pharmaceutical agents for the prevention or inhibition of restanosis.

It has recently been reported that adenoviruses use α _v P33 to enter host cells [White, Current Biology, 3 (9), 596-599 (1993)]. It appears that the integrin is required for endocytosis of the viral portion, and that the integrin may also be required for entry of the viral genome into the host cell cytoplasm. Accordingly, compounds that inhibit α _v P3 could be useful as antiviral agents.

International patent application WO 97/08145 discloses meta-guanidine, urea, thiourea and azacyclic aminobenzoic acid derivatives of general formula (I) which can be used as α _ν β3 integrin antagonists,

in the formula of which

The meaning

The following publications relate to the identification of HIV-1 integrase inhibitors by specific pharmacophore search: J. Med. Chem., 40, 930 (1997); J. Med. Chem., 40, 920 (1997); and Antiv. Chem. Chemother., 8^, 463 (1997).

A document published after the priority date of the present patent application [Exp. Opin. Ther. Patents, 8, 633 (1998)] discusses the development that integrin antagonists could be used to inhibit metastasis.

The present invention

refers to compounds of the general formula and pharmaceutically acceptable salts thereof, wherein in the general formula X and Y are identically or differently halogen atoms.

The foregoing compounds may exist in various isomeric forms, each of which is within the scope of the invention. The invention likewise includes the tautomeric forms, as well as the pharmaceutically acceptable salts of said isomers and tautomers.

More particularly, the invention relates to the following compounds and their pharmaceutically acceptable salts:

j. ··.?

where in the general formulas R represents a hydrogen atom or an alkyl group.

A further aspect of the invention is pharmaceutical compositions comprising the compounds described above. Such compounds and compositions can be used to selectively inhibit or antagonize integrin, whereby a further aspect of the invention is a method for selectively inhibiting or antagonizing α _ν β3 integrin. The invention further extends to the treatment or inhibition of α _ν β3 integrin-associated pathological conditions, such as osteoporosis, malignant humoral hypercalcemia, Paget's disease, tumor metastasis, solid tumor growth (neoplasia), angiogenesis, including tumor angiogenesis, retinopathy, including diabetic retinopathy and macular degeneration, arthritis, including rheumatoid arthritis, periodontal disease, psoriasis, smooth muscle cell migration, and restenosis in a mammal in need of such treatment. Such pharmaceutical agents can also be used as antiviral and antimicrobial agents.

The invention therefore relates to the class of compounds represented by the compounds of general formulae (I)-(XVI) described above.

Among the compounds of the invention, preferred are those of the following formula:

The invention also relates to pharmaceutical compositions comprising the compounds described above.

The invention also provides a method for selectively inhibiting or antagonizing α _ν ββ integrin, more particularly a method for inhibiting bone resorption, periodontal disease, osteoporosis, malignant humoral hypercalcemia, Paget's disease, tumor metastasis, solid tumor growth (neoplasia), angiogenesis, including tumor angiogenesis, retinopathy, including diabetic retinopathy and macular degeneration, arthritis, including rheumatoid arthritis, smooth muscle cell migration, and restenosis, which comprises administering a therapeutically effective amount of a compound as described above together with a pharmaceutically acceptable carrier to achieve the inhibition.

The following are definitions of terms used in this specification.

The term alkyl or lower alkyl as used herein refers to straight or branched chain hydrocarbon groups having from about 1 to about 10, more preferably from about 1 to about 6 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl, hexyl, isohexyl, etc.

The term halogen atom as used herein refers to a bromine, chlorine or iodine atom.

The term haloalkyl as used herein refers to alkyl groups as defined above which are substituted on one or more carbon atoms with one or more identical or different halogen atoms. Haloalkyl groups include, but are not limited to, trifluoromethyl, dichloroethyl, fluoropropyl, etc.

The term composition as used herein refers to products that can be prepared by mixing or combining more than one component or ingredient.

The term pharmaceutically acceptable carrier as used herein means a pharmaceutically acceptable substance, composition or vehicle, such as a liquid or solid filler, diluent, binder, solvent or encapsulating material.

The term therapeutically effective amount refers to that amount of an active ingredient that elicits a desired biological or therapeutic response in a tissue, system, or animal; the precise value of a therapeutically effective amount is determined by the researcher or physician.

The following is the meaning of the abbreviations used in this description:

¹ H-NMR = proton nuclear magnetic resonance spectroscopy

AcOH = acetic acid

Ar = argon

CH3CN = acetonitrile

CHN analysis = carbon/hydrogen/nitrogen elemental analysis

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

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

Dl water = deionized water

DMA. = N,N-dimethylacetamide

DMAP = 4-(dimethylamino)pyridine

DMF = N,N-dimethylformamide

EDC1 = 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride

EtOAc = ethyl acetate

EtOH = ethanol

FAB MS = fast atom bombardment mass spectroscopy g = gram

HOBT = 1-hydroxybenzotriazole hydrate

HPLC = high-performance liquid chromatography

IBCF = isobutyl chloroformate

KSCN = potassium thiocyanate

L = liter

LiOH = lithium hydroxide

METH = (2-methoxyethoxy)methyl group

MEMC1 = [(2-methoxyethoxy)methyl] chloride

MeOH = methanol mg = milligrams

MgSO ₄ = magnesium sulfate ml = milliliter mL = milliliter

MS = mass spectroscopy

MTBE = methyl tert-butyl ether

N2 = nitrogen

NaHCO ₃ = sodium bicarbonate

NaOH = sodium hydroxide

Na2 ^s0 4 ⁼ sodium sulfate

NMM = N-methyl-morpholine

NMP = N-methylpyrrolidinone

NMR = nuclear magnetic resonance spectroscopy

P2O5 = phosphorus(V) oxide

PTSA = p-toluenesulfonic acid

RPHPLC = reverse phase high performance liquid chromatography

RT = room temperature

TEA = trifluoroacetic acid

THE = tetrahydrofuran

TMS = trimethylsilyl group

Δ = heating of the reaction mixture

The foregoing compounds may exist in various isomeric forms, all of which are within the scope of the invention. The invention similarly includes the tautomeric forms, as well as the pharmaceutically acceptable salts of said isomers and tautomers.

In the structural and general formulas included in this specification, a bond drawn through a bond of a ring means that the given bond can be attached to any ring atom.

The term pharmaceutically acceptable salt refers to salts prepared by reacting a compound as defined above with an acid containing an anion generally accepted for human consumption. Examples of pharmaceutically acceptable salts include, but are not limited to, the hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate, acetate, propionate, lactate, maleate, malate, sulcinate, tartrate salts, etc. Each of the pharmaceutically acceptable salts can be prepared by conventional methods [for further examples of pharmaceutically acceptable salts, see, for example, Berge et al., J. Pharm. Sci., 66 (1), 1-19 (1977)].

For the selective inhibition or antagonism of α _ν β ₃ integrins, the compounds of the invention can be administered orally, parenterally, by inhalation spray or topically in unit dosage forms containing conventional pharmaceutically acceptable carriers, adjuvants and excipients. The term parenteral as used herein refers to, for example, subcutaneous, intravenous, intramuscular, intramuscular, intravenous, infusion or intraperitoneal administration.

The compounds of the invention may be administered by any suitable route, in the form of a pharmaceutical composition appropriate to the chosen route of administration, and in an amount effective for the desired treatment. The therapeutically effective amount of the compounds required to arrest or treat the progression of the disease can be readily determined by one skilled in the art using preclinical and clinical methods well known in ^the art.

Accordingly, the invention provides a method for treating conditions mediated by selectively inhibiting or antagonizing the α _ν β ₃ cell surface receptor, comprising administering a therapeutically effective amount of a compound selected from the class of compounds described above, wherein one or more of the compounds are administered together with one or more non-toxic, pharmaceutically acceptable carriers and/or diluents and/or adjuvants (hereinafter collectively referred to as "carriers") and optionally other active ingredients. More particularly, the invention provides a method for inhibiting the α _ν β ₃ cell surface receptor. Most preferably, the invention relates to a method for inhibiting bone resorption, treating osteoporosis, inhibiting malignant humoral hypercalcemia, treating Paget's disease, inhibiting tumor metastasis, inhibiting neoplasia (solid tumor growth), inhibiting angiogenesis, including tumor angiogenesis, treating diabetic retinopathy and macular degeneration, inhibiting arthritis, sporiasis and periodontal disease, and inhibiting smooth muscle cell migration, including restenosis.

Based on standard laboratory experimental methods and procedures well known to those skilled in the art, and comparison with known active ingredients, the compounds of the invention can be used to treat patients suffering from the pathological conditions described above. Various factors, including the evaluation of results obtained in standard tests and animal models, are taken into account20

...· .:. j.-r.

With this, a person skilled in the art can easily select the compound of the invention most suitable for a specific case.

In treating a patient affected by one of the pathological conditions, the patient is administered an amount of a compound as described above that is therapeutically effective in controlling the condition and prolonging the survival of the patient compared to the survival time that would be expected in the absence of such treatment. As used herein, the term "inhibiting a condition" includes slowing, interrupting, arresting or stopping the condition/process, but does not necessarily mean completely eliminating the condition. In our opinion, prolonging the survival of patients, in addition to its significance in itself, also means that the condition has been advantageously controlled to some extent.

As previously mentioned, the compounds of the invention can be used in various biological, prophylactic and therapeutic fields. It is expected that the compounds can be used for the prevention or treatment of any disease or condition in which the α _ν β ₃ integrin plays a role.

Dosage regimens for the compounds and/or compositions containing the compounds will depend on various factors, including, but not limited to, the type, age, weight, sex, and health of the patient; the severity of the condition; the route of administration; and the specific drug used.

activity of the group. For the foregoing reasons, the doses employed may vary widely. In the treatment of the conditions indicated above, the daily dose per kilogram of body weight ranges from about 0.01 mg to about 1000 mg, more preferably from about 0.01 mg to about 100 mg.

The active ingredient for injection is formulated into a composition in which a suitable carrier, for example, physiological sodium chloride solution (saline), dextrose or water, is used. The daily dose for injection is about 0.01 to 10 mg per kilogram of body weight, depending on the factors listed above, which can be administered in several doses throughout the day.

When administered to mammals in need of such treatment, a therapeutically effective amount of the compounds is usually combined with one or more adjuvants appropriate to the chosen route of administration. For example, the compounds may be mixed with the following materials and the resulting mixtures tableted or encapsulated for ease of administration: lactose, sucrose, starch powder, cellulose esters of alkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gelatin, acacia, sodium alginate, polyvinylpyrrolidone and/or polyvinyl alcohol. Alternatively, the compounds are dissolved in, for example, water, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride solution ²² .: ·: : .. · and/or various buffers. Additional adjuvants and administration routes suitable for the present invention are well known in the pharmaceutical art.

Pharmaceutical preparations suitable for the present invention may be subjected to conventional pharmaceutical procedures, for example, the preparations may be sterilized and/or the pharmaceutical preparations may also contain conventional pharmaceutical adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, etc.

The following Schemes I-III. show general syntheses for the preparation of compounds useful in the present invention. The Schemes and the Examples that follow are for illustrative purposes only. The Schemes and Examples are not intended to limit the scope of the invention. It will be apparent to those skilled in the art that variations of the conditions and procedures described in the Schemes and Examples can be used to prepare the compounds of the present invention.

Unless otherwise noted, all starting materials and equipment are commercially available.

.Λ.* * · · *·» t·* ⁵

Reaction scheme I

Scheme I shows the synthesis of the tetrahydropyrimidinylbenzoic acid moiety of the compounds of the invention which can subsequently be coupled to a glycyl-p-amino acid ester. According to Scheme I, 3,5-dihydroxybenzoic acid is converted to 3-amino-5-hydroxybenzoic acid by a known procedure [Austr. J. Chem. , 34 (6), 1319-1324 (1981)]. The product can be reacted with ammonium thiocyanate in hot dilute hydrochloric acid, and then, after conventional work-up, 5-thioureido-5-hydroxybenzoic acid is obtained. The thiourea intermediate is converted to the S-methyl derivative by refluxing with methyl iodide in ethanol. The intermediate thus obtained is reacted with 1,3-diamino-2-hydroxypropane in hot N,N-dimethylacetamide. After cooling, a precipitate forms and the zwitterionic product is filtered off. The hydrochloride salt can be prepared by lyophilizing the filtrate from dilute hydrochloric acid. Alternatively, the product is isolated by removing the volatile components from the original reaction mixture by concentration. The product obtained as a residue is dissolved in water, the pH of the solution is adjusted to about 5-7, and the precipitated zwitterionic product is filtered off. The hydrochloride salt can be prepared as described above, or by simply dissolving the product in dilute hydrochloric acid, concentrating the solution, and drying the residue.

Scheme IA

Scheme IA also illustrates the synthesis of the tetrahydropyrimidinylbenzoic acid moiety of the compounds of the invention, which moiety can later be coupled to a gly-p-amino acid ester. Scheme IA involves reacting 3,5-dihydroxybenzoic acid with carbon disulfide in a suitable solvent, such as ethanol/water, at reflux temperature, cooling the reaction mixture, adding hydrochloric acid, refluxing the reaction mixture again, cooling, and filtering off and drying the 5-hydroxy-2-thioxotetrahydropyrimidine product. The resulting cyclic thiourea intermediate is converted to the S-methyl derivative by reacting with methyl iodide in ethanol at reflux temperature. The desired 2-(methylthio)-5-hydroxypyrimidine hydroiodide can be readily isolated by removing the volatile components under reduced pressure. Subsequently, a mixture of 2-(methylthio)-5-hydroxypyrimidine hydroiodide, an approximately 10:1 volume ratio of methylene dichloride/N,N-dimethylacetamide solvent mixture and an equivalent amount of triethylamine is cooled in an ice bath, and an equivalent amount of di(tert-butyl)dicarbonate (BOC anhydride) is added to the mixture. After conventional work-up, 1-(tert-butoxycarbonyl)-2-(methylthio)-5-hydroxypyrimidine is obtained as an oil.

3,5-Dihydroxybenzoic acid is converted to 3-amino-5-hydroxybenzoic acid by a known procedure [Austr. J. Chem. , 34 (6), 1319-1324 (1981)]. The desired end product, i.e. 3-hydroxy-5-[(5-hydroxy-1,4,5,6-tetrahydro-2-pyrimidinyl)amino]benzoic acid hydrochloride, is prepared by reacting 1-(tert-butoxycarbonyl)-2-(methylthio)-5-hydroxypyrimidine with hot N,N-dimethyl-

-reacted with 3-amino-5-hydroxybenzoic acid in acetamide. After cooling, a precipitate forms, and the zwitterionic product is filtered off. The hydrochloride salt can also be prepared by lyophilizing the product from a dilute hydrochloric acid solution.

% or NX-succinimide or other X* source

Y and X are halogen atoms

Reaction Scheme II

Scheme II shows a method for preparing the ethyl 3-(glycylamino)-3-(3,5-dihalo-2-hydroxyphenyl)propionate moiety of the compounds of the invention, which moiety can subsequently be coupled to the tetrahydropyrimidinylbenzoic acid moiety. The 3,5-dihalo-salicylaldehydes can be prepared by direct halogenation; for example, 5-bromo-salicylaldehyde is suspended in acetic acid and at least an equivalent amount of chlorine is added to the suspension to give 3-chloro-5-bromo-2-hydroxybenzaldehyde. A portion of the product precipitates and can be easily removed by filtration. The residue can be recovered by diluting the filtrate with water and filtering off the precipitate thus formed. 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 N,N-dimethylformamide and working up the reaction mixture in the usual manner. 3-Iodo-5-bromosalicylaldehyde can be prepared by reacting 5-bromosalicylaldehyde with potassium iodide and chloramine (chloramine) T (N-chloro-4-toluenesulfonamide sodium salt) in acetonitrile. After working up, the material obtained is treated with hexane to give the desired 3-iodo-5-chlorosalicylaldehyde.

Coumarins can be readily prepared from the corresponding salicylaldehydes by a modified Perkin reaction [see, for example, Vogel's Textbook of Practical Organic Chemistry, 5th Ed., 1040 (1989)]. Halogen-substituted coumarins are converted to 3-aminohydrocoumarins [see, for example, JG Rico, Tetrahedron Letters, 35, 6599-6602 (1994)], which readily open in acidic alcohols to give 3-amino-3-(3,5-dihalo-2-hydroxyphenyl)propionic acid esters.

The 3-amino-3-(3,5-dihalo-2-hydroxyphenyl)propionic acid esters can be converted to 3-{[(tert-butoxycarbonyl)glycyl]oxy}-succinimide by reaction with N-{[(tert-butoxycarbonyl)glycyl]amino}-3-(3,5-dihalo-2-hydroxyphenyl)propionic acid esters, and the latter compounds are then converted to the HX salts of the 3-(glycylamino)-3-(3,5-dihalo-2-hydroxyphenyl)propionic acid esters, for example by removing the (tert-butoxycarbonyl)-protecting group using an ethanolic solution of hydrogen chloride.

The amino acid compounds used in the preparation of the compounds of the invention may be prepared according to the methods described hereinafter and those described and claimed in co-pending patent document USSN Attorney Docket No. 3076, filed concurrently with this patent application.

Reaction Scheme III

Y and X are halogen atoms

Scheme III illustrates a method for preparing various compounds of the invention. The coupling involves activating 3-hydroxy-5-[(1,4,5,6-tetrahydro-5-hydroxy-2-pyrimidinyl)amino]benzoic acid by known methods. For example, the starting compound is dissolved in a suitable solvent, such as N,N-dimethylacetamide, and one equivalent of N-methylmorpholine is added to the solution. The reaction mixture is cooled in an ice bath, and isobutylchloroformate (IBCF) is added. The glycyl-p-amino acid ester and N-methylmorpholine are added to the mixed anhydrous intermediate. After the reaction is complete, the product is purified by preparative HPLC, and the ester is hydrolyzed to the acid by reaction with a base, such as lithium hydroxide, in a suitable solvent, such as dioxane/water or acetonitrile/water. Alternatively, a suitable acid, such as trifluoroacetic acid, can be used. The product is isolated by preparative HPLC, or the zwitterion is isolated at a pH-η of 5-7 and converted to the desired salt by standard methods.

EXAMPLE A

Step 1

In a two-liter flask equipped with a mechanical stirrer and a condenser, 200 g (1.05 mol, 1 equivalent) of 3,5-dichloro

-salicylaldehyde, 356 g (3.49 mol) acetic anhydride and 95.0 g (0.94 mol, 0.90 equivalent) triethylamine. The solution was refluxed overnight, then the dark brown reaction mixture was cooled to 50 °C, then 1 liter of water was added with stirring. One hour later, the mixture was filtered, then 1 liter of ethanol was added to the filtrate. The resulting mixture was heated at 45 °C for one hour, then cooled to room temperature, filtered, and the filtered solid (fraction A) was washed with 0.5 liter of ethanol. The combined ethanol solutions were concentrated using a rotary evaporator, resulting in an oily residue (fraction B). The solid obtained as fraction A was dissolved in 1.5 liters of methylene chloride and the resulting solution was passed through a 1300 ml plug of silica gel. The dark brown solution obtained as a filtrate was concentrated and the residual oil was triturated with 1.3 liters of hexane. The resulting solid was filtered and washed with hexane. 163 g of essentially pure 6,8-dichlorocoumarin was obtained. An additional 31 g of product was obtained when the oil isolated as fraction B was treated in a similar manner. The oil was dissolved in 0.5 liters of methylene chloride, the solution was filtered through a 0.5 liter plug of silica gel and the crude product was triturated with hexane. The desired compound was obtained as a brown solid and in a total amount of 194 g (86% yield).

MS and NMR data were consistent with the desired structure.

Step 2

A 2-liter, three-necked flask equipped with a mechanical stirrer was charged with 160 g (0.74 mol) of 6,8-dichlorocoumarin prepared in step 1 and 375 ml of anhydrous tetrahydrofuran (Aldrich Sure Seal). The mixture was cooled to -40 °C in a dry ice/acetone bath, and then 800 ml (0.80 mol) of 1 M lithium bis(trimethylsilyl)amide in tetrahydrofuran was added, while the temperature was kept below -40 °C throughout. After the addition was complete, the cooling bath was removed. After 30 minutes, the reaction mixture warmed to -5 °C. The reaction was then quenched by the addition of 0.5 L of 4 M hydrogen chloride in dioxane and 1.25 L of ethanol. The temperature was kept below 0 °C overnight. The reaction mixture was concentrated to approximately half its original volume and partitioned between 3 liters of ethyl acetate and 2 liters of water. The organic phase was washed three times with 1 liter of 0.5 M hydrochloric acid solution, and the combined aqueous solutions were adjusted to pH approximately 7 with 10% aqueous sodium hydroxide solution. The neutralized mixture was extracted three times with 2 liters of methylene chloride, the organic solutions were combined, dried over anhydrous magnesium sulfate, filtered, and 210 ml of 4 M hydrogen chloride in dioxane were added to the filtrate with stirring33

...· .:. ·:. ····

solution. After the precipitation was complete, the solid was filtered off. The filtrate was concentrated to a small volume and methyl tert-butyl ether was added to the residue. The resulting solid was added to the initially formed solid and the combined product was washed with methyl tert-butyl ether, filtered off and dried in a vacuum oven over a weekend to give 172 g of the desired product in 74% yield.

MS and NMR data were consistent with the desired structure.

Step 3

A flame-dried 0.5 L round-bottomed flask was equipped with a stirring magnet under an argon atmosphere, and 15.0 g (0.055 mol) succinimido-[Δ7-(tert-butoxycarbonyl)glycinate] (Sigma), 200 mL anhydrous N,N-dimethylformamide (Aldrich Sure Seal), and 21.67 g (0.055 mol) of the product from Step 2 were weighed into a 0 °C salted ice bath, and 5.58 g (0.56 mol) N-methylmorpholine and a catalytic amount of 4-(dimethylamino)pyridine were added. The reaction mixture was stirred overnight, and the residue was partitioned between 0.4 L ethyl acetate and twice 0.2 L saturated aqueous sodium bicarbonate solution. The organic phase was washed twice with 0.2 liters of 10% w/v aqueous citric acid solution, twice with 0.2 liters of saturated aqueous sodium bicarbonate solution, then with saturated aqueous sodium chloride solution and dried over anhydrous sodium sulfate. The volatile components were removed in vacuo at 55 °C to give 22.5 oil in 92% yield. The oil solidified on standing.

MS and NMR data were consistent with the desired structure.

Step 4

The product of Step 3 was deprotected using the following procedure to give the amine hydrochloride. 14.0 g (0.032 mol) of the product of Step 3 was weighed into a 0.1 L round bottom flask containing a stirrer and 40 mL of anhydrous dioxane was added. The mixture was cooled to 0 °C and 6.32 mL (2 equiv) of 4.0 M hydrogen chloride in dioxane was added. The reaction mixture was allowed to stir until gas evolution ceased, and the volatiles were removed in vacuo. The residue was triturated with 50 mL of diethyl ether, the solid was filtered off, washed with diethyl ether, and dried. 12.5 g of the desired product was obtained.

MS and NMR data were consistent with the desired structure.

EXAMPLE B

Step 1

To a suspension of 175.0 g (743.2 mol) of 3-bromo-5-chlorosalicylaldehyde and 280.5 ml (3.0 mol) of acetic anhydride was added 103.6 ml (743.2 mmol) of triethylamine. The reaction mixture was refluxed for 4.5 h, then the solution was cooled and concentrated in vacuo. To the brown residue was added 730 ml of absolute ethanol, and the mixture was kept at 0 °C for 14 h. The brown solid was filtered off, washed with cold ethanol, and dried in vacuo. 123.0 g of the desired product was obtained in 64 % yield. NMR data were consistent with the desired structure.

Step 2

A suspension of 40.0 g (154.1 mmol) of coumarin from step 1 in 400 ml of tetrahydrofuran was cooled to -76 °C, and then 154.1 ml of 1 M lithium bis(trimethylsilyl)amide solution in tetrahydrofuran was added dropwise over 10 min with stirring. The reaction mixture was stirred for 5 min, then warmed to -20 °C, and stirred for 15 min. To the resulting solution was added 9.25 g (154.1 mmol) of acetic acid over 5 min. The mixture was warmed to room temperature, and the volatile components were removed in vacuo. The residue was dissolved in 850 ml of diethyl ether, the solution was washed twice with 100 ml of saturated aqueous sodium bicarbonate solution and twice with 40 ml of saturated aqueous sodium chloride solution, then dried over anhydrous magnesium sulfate, then the diethyl ether solution was concentrated to a volume of about 160 ml. The residue was cooled to 0 °C, then 56.3 ml (225 mmol) of 4 M hydrogen chloride in dioxane was added to the suspension. The mixture was stirred for 30 minutes at 0 °C, then the suspension was filtered, and the filtered material was washed with diethyl ether. The solid was dried in vacuo to give 45.0 g of the desired product as the hydrochloride salt, dioxane solvate. ¹ H-NMR data were consistent with the desired structure.

Step 3

To a suspension of 142.2 g (354.5 mmol) of the lactone from step 2 in 533 ml of absolute ethanol was added 57.8 ml (631.1 mmol) of 4 M hydrogen chloride in dioxane over 10 min. The reaction mixture was stirred at room temperature for 2.5 h, then the volatile components were removed in vacuo. The residue was dissolved in 450 ml of ethyl acetate, then the solution was kept at 0 °C for 15 h. The tan precipitate was filtered off, washed with cold ethyl acetate, and the solid was dried in vacuo. The desired product was obtained as the hydrochloride salt and in an amount of 100.4 g (79 % yield). H-NMR data were consistent with the desired structure. Step 4

Into a flame-dried 100 ml round-bottomed flask containing a stirring magnet, 2.72 g (0.010 mol) succinimido-[N-(tert-butoxycarbonyl)glycinate] (Sigma), 50 ml anhydrous tetrahydrofuran (Alfrich Sure Seal) and 3.10 g (0.01 mol) of the product from step 3, which had been previously dried in a vacuum desiccator over phosphorus(V) oxide overnight, were weighed under argon atmosphere. The mixture was cooled in a salted ice bath to approximately 0 * * * *

...· .:. 4. ·τ -·· °C, then 1.01 g (0.010 mol) of triethylamine was added. The reaction mixture was stirred overnight and then concentrated to a semi-solid residue. The residue was worked up similarly to that described in Example A, step 3. The volatile components were removed from the organic phase in vacuo at 55 °C, resulting in 4 g of an oil in 83% yield, which solidified on standing.

MS and NMR data were consistent with the desired structure.

Step 5

The product of Step 4 was deprotected using the following procedure to give the amine hydrochloride. 4.0 g (0.0084 mol) of the product of Step 4 was placed in a flame-dried 0.1 L round-bottomed flask containing a magnetic stirrer, and 20 mL of anhydrous dioxane was added. 20 mL of 4.0 M hydrogen chloride in dioxane was added to the mixture. The reaction mixture was stirred until gas evolution ceased and the reaction was complete, and the volatiles were removed in vacuo. The residue was triturated with 50 mL of diethyl ether, and the solid was filtered off, washed with diethyl ether, and dried. 2.7 g of a light brown solid was obtained in 78% yield.

MS and NMR data were consistent with the desired structure.

EXAMPLE C

Step 1

To a suspension of 100 g (357 mmol) of 3,5-dibromosalicylaldehyde and 164.8 ml (1.8 mol) of acetic anhydride was added 45 ml (357 mmol) of triethylamine. The reaction mixture was refluxed overnight and then cooled to room temperature, resulting in the formation of a solid mass. The dark brown reaction mixture was washed three times with 300 ml of hot hexane and then with saturated aqueous sodium bicarbonate solution. The solid thus obtained was dissolved in 2 liters of ethyl acetate, the solution was washed with water, and the organic phase was dried over anhydrous sodium sulfate and concentrated. The brown solid was filtered off and dried in vacuo to give 94.2 g of essentially pure 6,8-dibromocoumarin in 87% yield.

MS and H-NMR data were consistent with the desired

with structure.

Step 2

A mixture of 20.0 g (0.066 mol) of 6,8-dibromocoumarin prepared in step 1 and 100 ml of tetrahydrofuran was cooled to -78 °C, then 66 ml of 1 M lithium bis(trimethylsilyl)amide solution in tetrahydrofuran was added dropwise over 10 min with stirring. The reaction mixture was stirred for 5 min, then warmed to 0 °C, and stirred for 15 min. To the resulting solution was added 3.95 g of acetic acid over 1 min. The mixture was warmed to room temperature, and the volatile components were removed in vacuo. The residue was dissolved in 500 ml of hexane, the solution was washed twice with 100 ml of saturated aqueous sodium bicarbonate solution, then dried over anhydrous sodium sulfate, and the organic solution was concentrated. The oil obtained as a residue was immediately dissolved in 400 ml of diethyl ether, the solution was cooled to 0 °C, then 30 ml of 4 M hydrogen chloride in dioxane solution was added over 30 minutes with stirring. The mixture was stirred for 30 minutes at 0 °C, then the excess hydrogen chloride was removed in vacuo, the suspension was filtered, and the filtered material was washed with diethyl ether. The solid was dried in vacuo to give 19.9 g of the desired product as the hydrochloride salt, dioxane solvate.

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

Step 3

g of the lactone prepared in step 2 was dissolved in 400 absolute ethanol, and anhydrous hydrogen chloride gas was bubbled into the solution for one minute. The reaction mixture was stirred at room temperature for 2.5 hours. At this time, RPHPLC indicated that the reaction was complete. The volatile components were removed in vacuo, and the residue was triturated with 500 mL of diethyl ether, and the mixture was stirred overnight. The tan precipitate was filtered off, washed with diethyl ether, and the solid was dried in vacuo to give 15.2 g of the desired product as the hydrochloride salt.

MS and H-NMR data were consistent with the desired structure.

Step 4

Into a flame-dried 100 mL round bottom flask containing a stirring magnet were added 8.1 g (0.030 mol) succinimido-[N-(tert-butoxycarbonyl)glycinate] (Sigma), 50 mL anhydrous tetrahydrofuran (Alfrich Sure Seal), and 12 g (0.03 mol) of the product from step 3, which had been previously dried over phosphorus(V) oxide in a vacuum desiccator overnight, under an argon atmosphere. The mixture was cooled to approximately 0 °C in a salted ice bath, and 3.03 g (0.030 mol) triethylamine was added. The reaction mixture was stirred overnight while allowing it to warm to room temperature, and then concentrated to a semi-solid residue. The residue was worked up as described in Example A, Step 3. The organic phase was evaporated in vacuo at 55°C to give 15.7 g of an oil in 93% yield, which solidified on standing.

MS and NMR data were consistent with the desired structure.

Step 5

HCI

The product of Step 4 was deprotected using the following procedure to give the amine hydrochloride. 13.0 g (0.0084 mol) of the product of Step 4 was placed in a flame-dried 0.1 L round-bottomed flask containing a magnetic stirrer, and 40 mL of anhydrous dioxane was added. To the mixture was added 30 mL of a 4.0 M solution of hydrogen chloride in dioxane. The reaction mixture was stirred until gas evolution ceased and the reaction was complete (approximately one hour), and the volatiles were removed in vacuo. The residue was triturated with 50 mL of diethyl ether, after which the solid was filtered off and washed with diethyl ether, then dried. 10.6 g of solid was obtained in 93% yield.

MS and NMR data were consistent with the desired structure.

EXAMPLE D

• HC1

Step 1

3-Chloro-5-bromosalicylaldehyde

A 5-liter round-bottomed flask equipped with a mechanical stirrer and a gas inlet tube was charged with 495 g (2.46 mol) of 5-bromosalicylaldehyde and suspended in acetic acid at ambient temperature. Chlorine gas was bubbled through the mixture at a moderate rate until a slight molar excess (183 g, 1.05 mol) dissolved. After the gas was introduced, the reaction mixture was stirred overnight. The solid formed was filtered off and the filtrate was diluted by pouring it into 2.5 liters of water. The resulting mixture was stirred vigorously for 20 minutes, then the product was filtered off and washed with water. The solids were combined and dried in vacuo. As a result, 475 g (82% yield) of the desired 3-chloro-5-bromosalicylaldehyde was obtained.

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

Step 2

6-Bromo-8-chlorocoumarin

Into a 5-liter round-bottomed flask equipped with a mechanical stirrer and condenser were weighed 554.1 g (2.35 mol, 1 equiv) of 3-chloro-5-bromo-salicylaldehyde, 1203 g (11.8 mol, 5 equiv) of acetic anhydride and 237.4 g (2.35 mol, 1 equiv) of triethylamine. The reaction mixture was heated at reflux temperature (131-141 °C) overnight. The dark brown reaction mixture was then cooled to 50 °C, and 2 liters of ice were added while stirring and cooling with an ice bath. After one hour, the mixture was filtered, and 1 liter of ethanol was added to the filtrate. 300 ml of ethanol was added to the resulting mixture and stirred for one hour. The precipitate formed was filtered off, washed three times with 1.3 liters of water/ethanol, and dried first under vacuum and then in a fluid bed dryer. A total of 563 g of product (92% yield) was obtained.

MS and H-NMR data were consistent with the desired structure.

Step 3

Ethyl-3-amino-3-(2-hydroxy-3-chloro-5-bromophenyl)propionic acid

In a three-necked 5-liter round-bottomed flask equipped with a mechanical stirrer, 300 g (1.16 mol) of 6-bromo-8-chlorocoumarin prepared in step 2 and 900 ml of anhydrous tetrahydrofuran (Aldrich Sure Seal) were weighed. The resulting mixture was cooled to below -45 °C in a dry ice/acetone bath, and then 800 ml (0.80 mol, 1.2 equiv) of a 1 M solution of lithium bis(trimethylsilyl)amide in tetrahydrofuran and 600 ml of hexane were added over 0.5 h. In another 5-liter flask, 2.5 liters of ethanol and 1 liter of a 4 M solution of hydrogen chloride in dioxane were mixed and the mixture was then cooled to -15 °C. The coumarin reaction was quenched by the addition of cooled ethanol/dioxane hydrogen chloride solution. After 0.5 h, the temperature of the reaction mixture was -8.3 °C. The reaction mixture was kept at 0 °C overnight, then concentrated to approximately 2.5 liters, and the residue was partitioned between 3 liters of ethyl acetate and 4 liters of water. The organic phase was washed four times with 1.2 liters of 0.5 M hydrochloric acid solution. The ·**· · · ··/· ·.

··· ··· ·· · · · · · · · The combined aqueous solutions were adjusted to approximately 8 by adding 10% by weight aqueous sodium hydroxide solution, and then extracted once with 7 liters and then three times with 2 liters of methylene chloride. The organic solutions were combined, dried over 900 g of anhydrous magnesium sulfate, filtered, and 400 ml of 4 M dioxane hydrogen chloride solution was added to the filtrate while stirring. After the precipitation was complete, the solid was filtered off. The mixture was concentrated to 2.5 liters, 2.5 liters of hexane were added to the residue, and the precipitate was filtered off. The filtered material was washed with a 1:2 volume methylene chloride/hexane solvent mixture, the filter was vacuum-dried, and then dried in a vacuum oven at 40 °C. As a result, 251 g of the desired product was obtained in a yield of 60%.

MS and H-NMR data were consistent with the desired structure.

Step 5

The desired product was prepared by essentially the same procedure and relative amounts as the isomer of the compound in Example B, Step 4.

MS and H-NMR data were consistent with the desired structure.

Step 6

• HCI

The desired product was prepared by essentially the same procedure and relative amounts as the isomer of the compound in Example B, Step 5.

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

EXAMPLE E

Step 1

3-Iodo-5-chlorosalicylaldehyde

To a solution of 100 g (0.638 mol) of 5-chlorosalicylaldehyde in 400 ml of N,N-dimethylformamide was added 144.0 g (0.641 mol) of N-iodosuccinimide. The reaction mixture was stirred at room temperature for 2 days, then another 20.0 g of N-iodosuccinimide was added, and stirring was continued for another 2 days. Finally, the reaction mixture was diluted with 1 liter of ethyl acetate, then the solution was washed with 300 ml of 0.1 M hydrochloric acid solution, 300 ml of water, 300 ml of 5% by weight aqueous sodium thiosulfate solution and 300 ml of saturated aqueous sodium chloride solution, dried over anhydrous magnesium sulfate and evaporated to dryness. The desired aldehyde was obtained as a pale yellow solid in an amount of 162 g (90% yield).

MS and NMR data were consistent with the desired structure.

Step 2

6-Chloro~8-iodo-coumarin

A mixture of 100 g (0.354 mol) of 3-iodo-5-chlorosalicylaldehyde, 300 ml of acetic anhydride and 54 ml of triethylamine was refluxed for 18 hours. After cooling, the desired coumore precipitated as a dark brown crystalline solid. The solid was filtered off, washed with 200 ml of 4:1 hexane/ethyl acetate and air dried. As a result, 60 g of the desired product was obtained in a 55% yield.

MS and H-NMR data were consistent with the desired structure.

Step 3 (R,S}-4-Amino-3,4-dihydro-6-chloro-8-iodocoumarin hydrochloride

To a solution of 6.63 g (21.62 mmol) of 6-chloro-8-iodocoumarin in 100 ml of tetrahydrofuran cooled to -78 °C was added 21.62 ml (21.62 mmol) of 1 M lithium-(hexamethyldisilazane) solution. The reaction mixture was stirred at -78 °C for 30 min and then at 0 °C for 1 h, and then poured into a mixture of 300 ml of ethyl acetate and 200 ml of saturated aqueous sodium carbonate solution. The organic phase was separated, washed with 200 ml of saturated aqueous sodium chloride solution, dried over anhydrous magnesium sulfate and concentrated. 200 ml of diethyl ether were added to the residue, the mixture was cooled to 0 °C, and then 30 ml of 4 M hydrogen chloride in dioxane were added. The reaction mixture was stirred at room temperature for one hour, then filtered and dried in vacuo. The desired product was obtained as a powder and in an amount of 4.6 g (59 % yield). (RPHPLC: Rt = 6.8 min; gradient: 10% v/v acetonitrile —» 90% v/v acetonitrile over 15 min, then 100% v/v acetonitrile over the next 6 min. Both water and acetonitrile contained 0.1% v/v trifluoroacetic acid. Vydac C18 protein peptide column, flow rate 2 ml/min, detection at 254 nm.)

MS and ¹ H-NMR data were consistent with the desired structure. Step 4 (R, S')-Ethyl-3-amino-3-(5-chloro-2-hydroxy-3-iodophenyl)-propionate50 —hydrochloride

Hydrogen chloride gas was bubbled into a solution of 22.0 g (61.09 mmol) of 4-amino-3,4-dihydro-6-chloro-8-iodo-coumarin hydrochloride in 250 ml of ethanol, while the temperature of the reaction mixture was maintained between 0 °C and 10 °C until the solution was saturated. The reaction mixture was then refluxed for 6 hours, and most of the solvent was removed by distillation. Anhydrous diethyl ether was added to the cooled residue, and the mixture was stirred for 2 hours. The initial gum turned into a crystalline substance. The crystalline product was filtered off and dried. The desired product was obtained as an off-white, crystalline powder in an amount of 20 g (81% yield). (RPHPLC: _RT = 7.52 min; conditions: same as described in step 3.)

MS and H-NMR data were consistent with the desired structure.

Step 5 (R,S)-Ethyl 3-{[N-(tert-butoxycarbonyl)glycyl]amino}-3-(5-chloro-2-hydroxy-3-iodophenyl)propionate

A mixture of 2.16 g (12.31 mmol) of N-(tert-butoxycarbonyl)glycine, 1.67 g (12.31 mmol) of 1-hydroxybenzotriazole hydrate, 2.36 g (12.31 mmol) of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride and 50 ml of N,N-dimethylformamide was stirred for one hour at 0 °C. Subsequently, 5.0 g of ethyl 3-amino-3-(5-chloro-2-hydroxy-3-iodophenyl)propionate hydrochloride was added to the mixture, followed by 3.5 ml of triethylamine. The reaction mixture was stirred at room temperature for 18 hours, then the N,N-dimethylformamide was removed in vacuo and the residue was partitioned between 300 mL of ethyl acetate and 200 mL of saturated aqueous sodium bicarbonate. The organic layer was washed with 100 mL of 1 M hydrochloric acid and 200 mL of saturated aqueous sodium chloride, dried over anhydrous magnesium sulfate, and concentrated to give the desired product as a solid (6 g, 93% yield).

MS and H-NMR data were consistent with the desired structure.

Step 6 (R,S)-Ethyl-3-(glycylamino)-3-(5-chloro-2-hydroxy-3-iodophenyl)propionate—hydrochloride

6.0 g (11.39 mmol) of (R,S)-ethyl-3-{[N-(tert-butoxycarbonyl)-glycyl]-amino}-3-(5-chloro-2-hydroxy-3-iodophenyl)-propionate52 • ·· · ··· · ...· .:. .t. 'Ν' was added with 20 ml of 4 M dioxane hydrogen chloride solution at 0 °C. The reaction mixture was stirred at room temperature for 3 hours, then concentrated, 100 ml of toluene was added to the residue, and the mixture was concentrated again. The residue thus obtained was suspended in diethyl ether, and the solid was filtered off. The filtered off material was dried to give the desired product in the form of a crystalline powder and 5.0 g (95 % yield) was obtained. (RPHPLC: _RT = 8.3 min; conditions: same as described in step 3.)

MS and IR-NME data were consistent with the desired structure.

EXAMPLE F

Step 1

3-Iodo-5-bromosalicylaldehyde

In a 500 ml round bottom flask containing a stirring magnet, 150 ml acetonitrile and 50 ml water were weighed, 17 g (0.1 mol) potassium iodide were added, then 20.0 g (0.1 mol) 5-bromosalicylaldehyde was added to the solution, and then 23 g (0.1 mol) chloramine (chloramine) Tt (N-chloro-4-toluenesulfonamide sodium salt) was added to the resulting solution. The reaction mixture was stirred for one hour and then partitioned between 200 ml of 10% hydrochloric acid solution and ethyl acetate. The organic phase was washed with anhydrous sodium sulfate. The reaction mixture was concentrated in vacuo to give 100 ml of water. The organic phase was washed with anhydrous sodium sulfate. The reaction mixture was stirred for 1 hour and partitioned between 200 ml of 10% hydrochloric acid solution and ethyl acetate. The organic phase was washed with anhydrous sodium sulfate. The reaction mixture was concentrated in vacuo to give 100 ml of 10% hydrochloric acid solution. The reaction mixture was then separated into 200 ml of 10% hydrochloric acid solution and ethyl acetate. The reaction mixture was then washed with 100 ml of 10% hydrochloric acid solution

The residue was dried over water, filtered and concentrated in vacuo. Hexane was added to the residue, the mixture was heated at 50 °C for 15 minutes, and the insoluble material was filtered off. The filtrate was concentrated in vacuo to give 26 g of canary yellow 3-iodo-5-bromosalicylaldehyde.

MS and ¹ R-NMR data were consistent with the desired structure.

step

• HCI

The above compound was prepared in essentially the same manner as described in Example E, steps 2-6, except that in step 2, an equivalent amount of 3-iodo-5-bromosalicylaldehyde from step 1 was used instead of 3-iodo-5-chlorosalicylaldehyde.

MS and H-NMR data were consistent with the desired structure.

EXAMPLE H

• HCI

Step 1

A mechanical mixer with a Claisen attachment, from a feeder54 '“· .:. J. l· .:·.

375 ml of ethanol and 375 ml of deionized water were weighed into a two-liter three-necked flask equipped with a tern, reflux condenser and thermocouple, then 125.04 g (1.39 mol) of 1,3-diamino-2-hydroxypropane was added. The mixture was stirred until complete dissolution, then 84 ml (1.39 mol) of carbon disulfide was added dropwise from the addition funnel at a temperature between 25-33 °C, as a result of which the mixture turned milky white. The temperature was maintained in the specified range with an ice bath. The reaction mixture was then refluxed at 73.4 °C for two hours, then the resulting yellow solution was cooled to 25 °C in an ice bath, and finally 84 ml of concentrated hydrochloric acid was added dropwise, while the temperature was maintained at 25-26 C throughout. The reaction mixture was refluxed at 78.4 °C for 21 hours, then cooled to 2 °C, and the product was isolated by vacuum filtration. The white solid was washed with 50 ml of ice-cold ethanol/water (1:1) and dried under vacuum at 40 °C. 5-hydroxy-2-thioxo-tetrahydropyrimidine was obtained as a white solid (63.75 g, 34.7% yield).

MS and NMR data were consistent with the desired structure.

Step 2

In a 2-liter round-bottomed flask equipped with a mechanical stirrer and a thermocouple, 95 g (0.72 mol) of 5-hydroxy-2-thioxotetrahydropyrimidine prepared in step 1, 570 ml of absolute ethanol and 45 ml (0.72 mol) of methyl iodide were weighed. The reaction mixture was refluxed at 78 °C for 5 hours, ··· « · · * · ...· .f.-r .:..

The residue was cooled to room temperature and concentrated. The residue (194.72 g) was triturated with 3 x 500 ml of diethyl ether and dried in vacuo to give 2-(methylthio)-5-hydroxypyrimidine hydroiodide (188.22 g, 95.4% yield) as a white solid.

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

Step 3

Into a two-liter three-necked flask equipped with a reflux condenser and mechanical stirrer and placed under a constant nitrogen atmosphere were weighed 150.81 g (0.55 mol) of 2-(methylthio)-5-hydroxypyrimidine hydroiodide, 530 ml of methylene chloride, 100 ml of N,N-dimethylacetamide and 76.7 ml (0.55 mol) of triethylamine. The flask containing the mixture was placed in an ice bath, and 120.12 g (0.55 mol) of di(tert-butyl)dicarbonate was added at 4 °C. The reaction mixture was heated at 42.5 °C for 18 hours. The light yellow solution thus obtained was poured into a 2-liter separatory funnel, washed three times with 200 ml of deionized water, dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo to give 1-(tert-butoxycarbonyl)-2-(methylthio)-5-hydroxypyrimidine as a light yellow, viscous oil (134.6 g, 99.35% yield).

MS and H-NMR data were consistent with the desired structure.

Step 4

A mixture of 50.3 g (0.204 mol) of 1-(tert-butoxycarbonyl)-2-(methylthio)-5-hydroxypyrimidine, 25.0 g (0.1625 mol) of 3-amino-5-hydroxybenzoic acid [Aust. J. Chem., 34 (6), 1319-1324 (1981)] and 50 ml of anhydrous N, N-dimethylacetamide was heated at 100 °C with stirring for 2 days. The suspension formed by this time was cooled to room temperature, the precipitate was filtered off, washed first with acetonitrile and then with diethyl ether, and finally dried. The solid thus obtained was suspended in water, then acidified with concentrated hydrochloric acid. The resulting solution was frozen and then lyophilized. 14.4 g of the desired product was obtained as a white solid. 1 _z

MS and H-NMR data were consistent with the desired structure.

EXAMPLE I

(S-isomer)

Step 1

Reformatsky reagent ,—CO ₂ -t-Bu Br-Zn

In a four-liter flask equipped with a condenser, contact thermometer, and mechanical stirrer, 180.0 g (2.76 mol,

30-100 mesh) zinc metal and 1.25 liters of tetrahydrofuran. While stirring, 4.74 ml (0.05 mol) of 1,2-dibromoethane was added via syringe [alternatively, 0.1 equivalent of (trimethylsilyl) chloride applied at room temperature for one hour was substituted]. After the flask was inertly purged with three nitrogen/vacuum cycles, the zinc-tetrahydrofuran suspension was heated to reflux temperature (65 °C) and then refluxed for one hour. The mixture was cooled to 50 °C, and 488 g (2.5 mol, 369 ml) of tert-butyl-(bromoacetate) was added over 1.5 h using a 50 ml syringe and syringe pump (at a feed rate of 4.1 ml/min). The temperature of the reaction mixture was maintained at 50 ± 5 °C during the addition. After the addition was complete, the reaction mixture was stirred at 50 °C for one hour, then allowed to cool to 25 °C, and the precipitated product was allowed to settle. The tetrahydrofuran mother liquor was decanted into a 2-liter round-bottom flask using a coarse sintered glass filter and partial vacuum [2.7 kPa (20 mmHg)]. In this way, approximately 65% of the tetrahydrofuran was removed from the mixture. To the residue was added 800 ml of N-methyl-2-pyrrolidinone, the mixture was stirred for 5 minutes, and the reaction mixture was filtered to remove the remaining zinc. According to the analysis, the titer of the desired Reformatsky reagent was 1.57 M (molar yield: 94 %). Alternatively, the solid reagent can be isolated by filtration from the original reaction mixture. The filtered material is washed with tetrahydrofuran until a white solid is obtained. After drying under nitrogen58 ·*· · · · · ra, the desired product is obtained as a mono-tetrahydrofuran solvate, which can be stored in a desiccator at -20 °C for a long time. The typical yield is 85-90 %.

step

Step 2A

To a solution of 11.46 g (60 mmol) of 3,5-dichlorosalicylaldehyde and 40 ml of N,N-dimethylformamide at room temperature was added 8.82 g (60 mmol) of potassium carbonate (dried in a vacuum oven at 100 °C and pulverized). The resulting bright yellow suspension was placed in a bath at 20 °C, and 7.64 g (61 mmol) of [(2-methoxyethoxy)methyl] chloride was added. The reaction mixture was stirred for 30 minutes and then poured into 200 ml of cold water to precipitate the product. The suspension was filtered using a filter press, after which the filtered material was washed twice with 50 ml of water and then dried under nitrogen/vacuum. It is a grayish-white solid and contains 14.94 g (89% yield).

The product was obtained in 0% yield. H-NMR (CDCl ₃ , tetramethylsilane) δ (ppm): 3.37 (s, 3H), 3.54-3.56 (m, 2H), 3.91-3.93 (m, 2H), 5.30 (s, 2H), 7.63 (d, 1H), 7.73 (d, 1H), 10.30 (s, 1H). ¹³ C-NMR (CDCl ₃ , tetramethylsilane) δ (ppm): 59.03, 70.11, 99.57, 126.60, 129.57, 130.81, 132.07, 135.36, 154.66, 188.30. DSC: 48.24 °C (endo 90.51 J/g).

Microanalysis for the formula CnH^C^: calculated (%): C 47.33; H 4.33; Cl 25.40; found (%): C 47.15; H 4.26; Cl 25.16.

Step 2B

A 1-liter, three-necked round-bottomed flask equipped with a mechanical stirrer and a dropping funnel was charged with 35.0 g (0.125 mol) of the product from Step 2A, followed by 200 mL of tetrahydrofuran. The solution was stirred at 22 °C, and then 17.20 g (0.125 mol) of (S)-phenylglycinol was added all at once. The reaction mixture was stirred at 22 °C for 30 min, and then 20 g of magnesium sulfate was added. The mixture was stirred at 22 °C for 1 h, and then filtered through a coarse sintered glass filter. The filtrate was concentrated under reduced pressure. No further purification was performed; the crude imine was used directly in the coupling reaction of Step 2C. Step 2C

««· · · · · · .:. u. **r.:..

Into a 1-liter three-necked round-bottomed flask equipped with a mechanical stirrer and a dropping funnel, 91.3 g (0.275 mol) of the solid reagent prepared in step 1 and 200 ml of N-methyl-2-pyrrolidinone were weighed under a nitrogen atmosphere. The solution was cooled to -10 °C and stirred at 350 rpm. A solution of the imine prepared in step 2B with N-methyl-2-pyrrolidinone was prepared and the solution was added to the previous reaction mixture over 20 minutes while maintaining the temperature of the mixture at -5 °C (external temperature: -10 °C). After the addition was complete, the reaction mixture was stirred first for 1.5 hours at -8 °C and then for one hour at -5 °C. The mixture was then cooled to -10 °C, and a mixture of 8.1 ml of concentrated hydrochloric acid and 200 ml of saturated aqueous ammonium chloride solution was added over 10 minutes. 200 ml of methyl (tert-butyl) ether was added to the mixture obtained in this way, and the mixture was stirred at 200 rpm for 15 minutes at 23 °C. The stirrer was stopped and the phases were separated. The aqueous layer was extracted with 100 ml of methyl (tert-butyl) ether. The two organic solutions were combined and washed successively with 100 ml of saturated aqueous ammonium chloride solution, 100 ml of water and 100 ml of saturated aqueous sodium chloride solution. The solution was dried over 30 g of anhydrous magnesium sulfate, filtered and the filtrate was concentrated. The residue was 66.3 g of an orange oil that solidified on standing, which contained the desired product in the form of a single diastereoisomer according to proton and carbon nmr. For analysis, a portion of the product was purified by recrystallization from heptane. The off-white solid formed61

we obtained the product.

¹ H-NMR, ¹³ C-NMR and IR data were consistent with the desired structure, [α] ^δ = +8.7° (c = 1.057; methanol).

Microanalysis for the formula C25H33CI2NO6:

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

CH ₃

Step 3A

A solution of 17.40 g [0.033 mol (theoretical)] of the crude ester from step 2 in 250 ml of ethanol was charged into a 1-liter, three-necked reactor with a temperature-controlled jacket. The solution was cooled to 0 °C, and 14.63 g (0.033 mol) of lead(IV) acetate was added all at once. Two hours later, 30 ml of 15% aqueous sodium hydroxide solution was added to the reaction mixture, and the ethanol was removed under reduced pressure. An additional 100 ml of 15% aqueous sodium hydroxide solution was added to the residue, and the mixture was extracted twice with 100 ml of methyl tert-butyl ether. The organic solutions were combined, washed twice with 100 ml of water and 50 ml of saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered through a Celite pad, and the filtrate was concentrated under reduced pressure. 12.46 g of an orange oil was obtained as a residue. The oil was homogeneous according to thin layer chromatography. The product was used in the next step without further purification.

Step 3B

The oil from Step 3A was diluted with 30 mL of ethanol and 8.18 g (0.043 mol, 1.3 equiv) of p-toluenesulfonic acid was added. The solution was refluxed for 8 h, cooled to ambient temperature, and concentrated under reduced pressure. To the residue was added 20 mL of tetrahydrofuran and the mixture was heated to reflux to obtain a solution. The solution was cooled to room temperature; the compound crystallized. To form a fluid suspension, 30 mL of heptane and 10 mL of tetrahydrofuran were added and the suspension was filtered. The filtered material was washed with 40 ml of a 1:1 tetrahydrofuran/heptane solvent mixture and then vacuum dried on a pressure filter under a nitrogen atmosphere for 2 hours. As a result, 7.40 g of a white solid was obtained.

113

H-NMR, C-NMR and IR data were consistent with the desired compound consisting essentially of a single enantiomer.

Microanalysis for the formula C ₁₈ H ₂₁ C1 ₂ NO ₆ S:

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

A 500 mL round bottom flask equipped with a stirring magnet and nitrogen inlet was charged with 21.7 g (0.065 mol) of the free base product from step 3, 17.7 g (0.065 mol) of succinimido-[N-(tert-butoxycarbonyl)glycinate], and 200 mL of N,N-dimethylformamide. The reaction mixture was stirred at room temperature under nitrogen for 3.25 h, and the resulting pale orange solution was poured into 1.2 L of ice-cold ethyl acetate. The organic solution was washed with 250 mL of 1 M hydrochloric acid, then with 500 mL of saturated aqueous sodium chloride, and then evaporated to near dryness in vacuo. The resulting oil was dried at 50°C to give 28.12 g of the product as a colorless oil (99% yield). Seed crystals were prepared from ethyl acetate/hexane solvent system. Approximately 28 g of the product was dissolved in 35 mL of ethyl acetate and 125 mL of hexane. The solution was seeded with the seed crystals to form a suspension. The solid was filtered off and dried under vacuum at 55°C overnight. This gave 27.0 g of a colorless solid (95% yield).

MS and H-NMR data were consistent with the desired structure.

Step 5

27.0 g (0.062 mol) of the (tert-butoxycarbonyl)-protected glycinamide prepared in step 4 was dried over phosphorus(V) oxide and sodium hydroxide pellets overnight. The solid was then dissolved in 40 mL of dioxane and the solution was cooled to 0 °C. An equivalent amount (0.062 mol) of 4 M hydrogen chloride in dioxane was added to the solution and the reaction mixture was stirred for 2 h. At this point, the conversion was 80% by RPHPLC. The reaction mixture was allowed to warm to room temperature over 4 h and concentrated at 40 °C. The resulting foam was triturated with 200 ml of diethyl ether, and the resulting white solid was filtered off and dried over phosphorus(V) oxide to give the desired glycyl-p-amino acid ethyl ester as the hydrochloride salt (20.4 g, 88.5% yield).

MS and H-NMR data were consistent with the desired structure.

EXAMPLE J.

• HCI

Following the procedure of Example I, Step 2A, 129.42 g (0.4 mol) of [(2-methoxyethoxy)methyl]-protected 3-bromo-5-chlorosalicylaldehyde was prepared. The 3,5-dichlorosalicylaldehyde was replaced with an equivalent of 3-bromo-5-chlorosalicylaldehyde, which was weighed into a 2-liter, 3-necked round-bottomed flask equipped with a mechanical stirrer, followed by the addition of 640 mL of tetrahydrofuran and 54.86 g (0.4 mol) of (S)-phenylglycinol. The reaction mixture was stirred at 22 °C for 30 min, followed by the addition of 80 g of magnesium sulfate, continued stirring at 22 °C for 2 h, and then filtered through a coarse sintered glass filter. The filtrate was concentrated under reduced pressure. The residue obtained was 180.0 g of a pale yellow oil containing the desired imine. No further purification was performed, and the crude product was used directly in the coupling reaction of step 2.

Microanalysis for the formula C11BrClNO3: 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

In a three-necked five-liter round-bottomed flask equipped with a mechanical stirrer, 332.0 g (0.8 mol) of the reagent prepared in step 1 of Example I was weighed under a nitrogen atmosphere, then the reagent was taken up in 660 ml of N-methyl-2-pyrrolidinone. The solution thus obtained was cooled to -10 °C. The imine obtained in step 1 was dissolved in 320 ml of N-methyl-2-pyrrolidinone under a nitrogen atmosphere, then the solution thus obtained was added to the former mixture over 30 minutes, while the temperature was maintained at -5 °C throughout. After the addition was complete, the reaction mixture was stirred first for one hour at -8 °C and then for 2 hours at -5 °C. The mixture was then cooled to -10 °C, and a mixture of 30 ml of concentrated hydrochloric acid and 720 ml of saturated aqueous ammonium chloride solution was added over 10 minutes. 760 ml of methyl (tert-butyl) ether was added to the resulting mixture, and the mixture was stirred at 23 °C for 30 minutes. The stirrer was stopped and the phases were separated. The aqueous layer was extracted with 320 ml of methyl (tert-butyl) ether. The organic solutions were combined and washed successively with 320 ml of saturated aqueous ammonium chloride solution, 320 ml of deionized water and 320 ml of saturated aqueous sodium chloride solution. The solution was dried over 60 g of anhydrous magnesium sulfate, filtered and the filtrate was concentrated. As a residue, 221.0 g of yellow oil was obtained, which according to proton nmr contained the desired product in the form of a single diastereoisomer.

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

Microanalysis for the formula C ₂ 5H ₃ 3BrClNO 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

chapter ₃

Step 3

A solution of approximately 111 g of the crude ester from step 2 in 1500 mL of ethanol was charged under argon into a 3-liter, three-necked flask equipped with a mechanical stirrer. The mixture was cooled to 0 °C, and 88.67 g (0.2 mol) of lead(IV) acetate was added all at once. The reaction mixture was stirred at 0 °C for 3 h, and then 150 mL of 15% aqueous sodium hydroxide solution was added, keeping the temperature below 5 °C. The ethanol was then removed under reduced pressure using a rotary evaporator. An additional 150 mL of 15% aqueous sodium hydroxide solution was added to the residue, and the mixture was extracted three times with 300 mL of ethyl acetate. The organic solutions were combined, washed twice with 100 ml of deionized water and twice with 100 ml of saturated aqueous sodium chloride solution, dried over 30 g of anhydrous magnesium sulfate, and then filtered through a Celite pad68

J. .:. ··:* needles and then concentrated under reduced pressure. 103 g of the desired product was obtained as a red oil.

Step 4

The above compound was prepared according to the procedure used in steps 4 and 5 of Example I, whereby in step 4 the product of step 3 of Example I was replaced with an equivalent amount of the product of step 3 of Example J. The MS and H-NMR data were consistent with the desired structure.

EXAMPLE K

Alternative method for preparing the product of Example J Step 1

To 50.0 g (139.2 mmol) of the product from Example B, Step 3 and 33.5 g (398.3 mmol) of sodium bicarbonate were added 500 mL of methylene chloride and 335 mL of water. The mixture was stirred at room temperature for 10 min, and then a solution of 38.0 g (222.8 mmol) of benzyl (chloroformate) in 380 mL of methylene chloride was added over 20 min with rapid stirring. The reaction mixture was stirred for 50 min and then poured into a separatory funnel. The organic phase was separated and the aqueous layer was extracted with 170 mL of methylene chloride. The organic solutions were combined, dried over anhydrous magnesium sulfate, and concentrated in vacuo. The resulting gummy solid was triturated with hexane and filtered. The tan solid was dried in vacuo to give the desired racemic compound (61.2 g, 96%). This material was separated into the pure enantiomers by reverse phase HPLC using a chiral column. A 10 pm Whelk-0 (R,R) column and a 90:10 heptane/ethanol mobile phase were used for the separation. Analytical HPLC using a similar column and solvent mixture showed an optical purity of >98%. H-NMR data were consistent with the expected structure. Step 2

To a solution of 48.5 g (106.2 mmol) of the product obtained in step 1 in 450 ml of methylene chloride was added via syringe a solution of 25.5 g (127.4 mmol) of (trimethylsilyl) iodide in 100 ml of methylene chloride. The orange solution was stirred at room temperature for 1 hour, then 20.6 ml (509.7 mmol) of methanol was added dropwise, the solution was stirred for 15 minutes, and finally concentrated in vacuo. The orange oil obtained as a residue was dissolved in 500 ml of methyl (tert-butyl) ether, after which the solution was extracted with 318 ml of 1 M hydrochloric acid solution and once with 200 ml and once with 100 ml of water. The aqueous solutions were combined and washed with 100 mL of methyl tert-butyl ether. 40.1 g (478 mmol) of solid sodium bicarbonate was added to the aqueous solution in small portions. The basified aqueous mixture was extracted with 1 L and 200 mL of methyl tert-butyl ether. The organic solutions were combined, washed with saturated aqueous sodium chloride solution, and concentrated in vacuo to give 23.3 g of the desired product in 1% yield. H-NMR data were consistent with the desired structure.

Step 3

To a solution of 23.3 g (72.1 mmol) of the product from step 2 in 200 ml of N,Nd±methylformamide was added 17.9 g (54.9 mmol) of succinimido-[N-(tert-butoxycarbonyl)glycinate]. The reaction mixture was stirred at room temperature for 20 hours and then poured into 1.2 liters of ethyl acetate. The solution was washed twice with

The mixture was washed with 250 ml of 1 M hydrochloric acid solution, twice with 250 ml of saturated aqueous sodium bicarbonate solution and twice with 250 ml of saturated aqueous sodium chloride solution. The organic phase was dried over anhydrous magnesium sulfate and concentrated. As a result, 32.0 g (100% yield) of the desired product was obtained.

Microanalysis for the formula C ₁₈ H24BrClN ₂ O4:

Calc'd (%): C 45.06; H 5.04; N 5.84; Found (%): C 45.17; H 5.14; N 6.12.

¹ H-NMR data were consistent with the desired structure.

Step 4

To a solution of 31.9 g (66.5 mmol) of the product from step 3 in 205 ml of absolute ethanol was added 111 ml (332.4 mmol) of ethanolic hydrogen chloride solution. The reaction mixture was heated at 58 °C for 30 min, then cooled and concentrated in vacuo. The residue was dissolved in 250 ml of ethyl acetate and the mixture was stirred at 0 °C for 2 h. The white precipitate was filtered off and washed with cold ethyl acetate. The solid was dried in vacuo to give 23.5 g of the desired product in 85% yield.

Microanalysis for the formula _{Cj3Hi8BrClN2O4} · _1.0HCl : calculated (%): C 37.53; H 4.12; N 6.73;

Found (%): C 37.29; H 4.06; N 6.68.

H-NMR data were consistent with the desired structure.

.t. ··*

EXAMPLE L.

COjEt η^ ^νη · ^OH *HC1

Step 1

To a solution of 35.0 g (0.15 mol) of 3-chloro-5-bromo-salicylaldehyde in 175 ml of N,N-dimethylformamide at room temperature was added 22.1 g (0.16 mol) of potassium carbonate (dried in a vacuum oven at 100 °C and pulverized). The resulting bright yellow suspension was placed in a bath at 20 °C, and 25.0 g (0.2 mmol) of [(2-methoxyethoxy)methyl] chloride was added. The reaction mixture was stirred for 6 hours and then poured into 1200 ml of deionized water to precipitate the product. The suspension was filtered using a filter press, after which the filtered material was washed twice with 400 ml of deionized water, and then dried under nitrogen/vacuum. The product was obtained as an off-white solid in an amount of 46.0 g (95% yield). ¹ H-NMR (CDCl ₃ , tetramethylsilane) δ (ppm): 3.35 (s, 3H), 3.54 to 3.56 (m, 2H), 3.91-3.93 (m, 2H), 5.30 (s, 2H), 7.77 (d, 1 H), 7.85 (d, 1 H), 10.30 (s, 1 H). ¹³ C-NMR (CDCl 3 , tetramethylsilane) δ (ppm): 59.05, 70.11, 71.49, 99.50, 117.93, 129.69, 129.78,

132.37, 138.14, 155.12, 188.22. DSC: 48.24 °C (endo 90.51 J/g).

...· .:. .:. ··:*

Microanalysis for the formula CiiHi2BrClC>4:

calculated (%): C 40.82; H 3.74; Br 24.69; Cl 10.95; found (%): C 40.64; H 3.48; Br 24.67; Cl 10.99. Step 2

Into a 500 ml three-necked round-bottomed flask equipped with a mechanical stirrer was first weighed 32.35 g (0.1 mol) of the product of step 1, followed by 160 ml of tetrahydrofuran. The solution was stirred at 22 °C, then 13.71 g (0.1 mol) of (S)-phenylglycinol was added. The reaction mixture was stirred for 30 min at 22 °C, then 20 g of magnesium sulfate was added. The mixture was stirred for one hour at 22 °C, then filtered through a coarse sintered glass filter. The filtrate was concentrated under reduced pressure. No further purification was performed; the crude imine was used directly in the next reaction step.

Step 3

A five-liter three-necked .:. Λ. ^: ·/·.:.. equipped with a mechanical mixer.

332 g (0.8 mol) of the reagent prepared in step 1 of Example I and 660 ml of N-methyl-2-pyrrolidinone were weighed into a round-bottomed flask under a nitrogen atmosphere. The solution was cooled to -10 °C and stirred at 350 rpm. A solution of the imine prepared in step 2 was prepared with N-methyl-2-pyrrolidinone, and the solution was then added to the previous reaction mixture over 30 minutes, while the temperature of the mixture was maintained at -5 °C (external temperature: -10 °C). After the addition was complete, the reaction mixture was stirred for one hour, then cooled back to -10 °C, and then a mixture of 30 ml of concentrated hydrochloric acid and 720 ml of saturated aqueous ammonium chloride solution was added over 10 minutes. To the resulting mixture was added 760 ml of methyl (tert-butyl) ether, and the mixture was stirred at 23 °C for 60 minutes. The stirrer was stopped and the phases were separated. The aqueous layer was extracted with 320 ml of methyl (tert-butyl) ether. The two organic solutions were combined and washed successively with 320 ml of saturated aqueous ammonium chloride solution, 320 ml of deionized water and 320 ml of saturated aqueous sodium chloride solution. The solution was dried over 60 g of anhydrous magnesium sulfate, filtered and the filtrate was concentrated. As a residue, 228 g of a yellow oil were obtained, which contained the desired product in the form of a single diastereoisomer. DSC: 227.43 °C (endo. 61.63 J/g).

Microanalysis for the formula C25H33BrClNOg:

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

...· .:. .x. t

A solution of approximately 111 g of the crude ester prepared in step 3 in 1500 ml of ethanol was weighed into a 3-liter, three-necked round-bottomed flask equipped with a mechanical stirrer under nitrogen. The mixture was cooled to 0 °C, and then 88.67 g (0.2 mol) of lead(IV) acetate was added all at once. The reaction mixture was stirred at 0 °C for 3 h, and then 150 ml of 15% aqueous sodium hydroxide solution was added, while maintaining the temperature below 5 °C throughout. The ethanol was then removed under reduced pressure using a rotary evaporator. A further 600 ml of 15% aqueous sodium hydroxide solution was added to the residue, and the mixture was extracted twice with 300 ml of ethyl acetate, twice with 200 ml of methyl tert-butyl ether and twice with 200 ml of ethyl acetate. The organic solutions were combined, washed twice with 200 ml of deionised water and twice with 100 ml of saturated aqueous sodium chloride solution, dried over 30 g of anhydrous magnesium sulphate, filtered through a pad of Celite® and concentrated under reduced pressure. 96 g of the desired product was obtained as an orange oil, which was used in the next step without further purification.

DSC: 233.60 °C (endo. 67.85 J/g).

Microanalysis for the formula C24H2gBrClNO5:

...· .:. 4. *v .:..

calculated (%) : C

54.71; H

5.54; N 2.65; Hebrews 15:16; Cl 6.72;

52.12; H

5.40; N 2.47; Br 14.77; Cl 6.48.

Step 5

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chapter ₃

Approximately 94 g of the crude product from Step 4 was taken up in 180 ml of absolute ethanol, followed by the addition of 50.0 g (0.26 mol) of p-toluenesulfonic acid monohydrate. The reaction mixture was refluxed for 8 hours, and the solvent was removed under reduced pressure. The resulting solid was taken up in 100 ml of tetrahydrofuran, and the tetrahydrofuran was removed under reduced pressure. The residue was dissolved in 500 ml of ethyl acetate, and cooled to approximately 5 °C. The solid was filtered off and washed twice with 50 ml of heptane. The resulting white solid was air-dried. 38 g of the desired product was obtained as a white solid, as a single isomer. ¹ H-NMR (DMSO-d ₆ , tetramethylsilane) δ (ppm): 1.12 (t, 3H), 2.29 (s, 3H), 3.0 (m, 2H), 4.05 (g, 2H), 4.88 (t, 1H), 7.11 (d, 2H), 7.48 (d, 2H) , 7.55 Ί T (d, 1H) , 7.68 (d, 1H) , 8.35 (broad s, 3H) . C-NMR (DMSO-d ₆ , tetramethylsilane) δ (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 (endo., 406.5 J/g) ,

165.72 (endo., 62.27 J/g), 211.24 °C (exo., 20.56 J/g). [ ^a ]|^ +4.25° (c = 0.960, methanol). IR (MIR) (cm ¹ ): 2922, 1726, 1621,

1591, 1494, 1471, 1413, 1376, 1324, 1286, 1237, 1207. Microanalysis for the molecular formula C ₁₈ H ₂ iBrClNO 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

The above compound was prepared according to the procedures described in Steps 4 and 5 of Example I, substituting an equivalent amount of the intermediate prepared in Step 5 for the intermediate of Step 4 of Example I.

MS and H-NMR data were consistent with the desired structure.

EXAMPLE M.

Step 1

3-Iodo-5-chlorosalicylaldehyde :::-1

MS and ¹ H-NMR data were consistent with the desired structure. Step 2

2-0-[(2-Methoxyethoxy)methyl]-3-iodo-5-chlorosalicylaldehyde 84.74 g (0.30 mol) of 3-iodo-5-chlorosalicylaldehyde in 200 ml of N, N-dimethylformamide at 20 °C was added 41.4 g (0.30 mol) of potassium carbonate. To the resulting yellow suspension, while maintaining the reaction temperature, was added 38.2 g (0.305 mol) of [(2-methoxyethoxy)methyl] chloride (MEM-C1). The reaction mixture was stirred for 2 hours, after which another 1.5 g of [(2-methoxyethoxy)methyl] chloride was added, stirring was continued for one hour, and the reaction mixture was poured into ice water. The aqueous mixture was stirred, then the precipitate formed was filtered off and dried in vacuo. As a result, 95 g (85% yield) of

(by addition) we obtained the desired protected aldehyde.

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

Step 3

To a solution of 41.5 g (0.112 mol) of 2-O-[(2-methoxyethoxy)methyl]-3-iodo-5-chlorosalicylaldehyde in 200 ml of tetrahydrofuran at room temperature was added 15.37 g (0.112 mol) of (S)-phenylglycinol. The reaction mixture was stirred for one hour, then 16 g of magnesium sulfate was added, stirring was continued for another two hours, and the reaction mixture was filtered. The filtrate was concentrated and dried under vacuum for 2 hours to give the desired imine intermediate. 81.8 g (0.2464 mol) of Reformatsky reagent obtained in Step 1 of Example I and 300 ml of N-methylpyrrolidone were weighed into a two-necked round-bottomed flask. The mixture was stirred at -10 °C, then a solution of the imine in 100 ml of N-methylpyrrolidone was slowly added, while maintaining the temperature at -10 °C throughout. The reaction mixture was kept at -10 °C for 2 hours, then at -5 °C for one hour. Finally, the reaction mixture was cooled back to -10 °C, and a mixture of 16 ml of concentrated hydrochloric acid and 200 ml of saturated aqueous ammonium chloride solution was added. The thus **”* · .» M· .j»· * ΐ · '* 3 · 500 ml of diethyl ether were added to the obtained mixture, and the mixture was stirred for 2 hours. The diethyl ether phase was separated, and the aqueous layer was extracted with 300 ml of diethyl ether. The diethyl ether solutions were combined, washed with 200 ml of saturated aqueous ammonium chloride solution, 200 ml of water and 200 ml of saturated aqueous sodium chloride solution, dried over anhydrous magnesium sulfate and concentrated. 61.0 g of an oil were obtained in 90% yield, which contained the desired product in the form of a single diastereoisomer according to H-NMR. The MS data were consistent with the desired structure.

Step 4

48.85 g (80.61 mmol) of the crude ester obtained in step 3 was dissolved in 500 mL of ethanol. The solution was cooled to 0 °C, and then 35.71 g (80.61 mmol) of lead(IV) acetate was added. The reaction mixture was stirred for 3 h, then 73 mL of 15% aqueous sodium hydroxide solution was added, and most of the ethanol was removed under reduced pressure. 200 mL of 15% aqueous sodium hydroxide solution was added to the residue, and the mixture was extracted with 400 mL of diethyl ether. The diethyl ether phase was washed with 100 mL of water and 100 mL of saturated aqueous sodium chloride solution, dried, and concentrated. The orange residue

- 82 - :··' .: .: : --.

Λ Λ *·*.:.

oil was dissolved in 100 ml of ethanol, 19.9 g of p-toluenesulfonic acid was added to the solution, the reaction mixture was refluxed for 8 hours, and then concentrated under reduced pressure. The residue was diluted with 60 ml of tetrahydrofuran, the mixture was refluxed, and then cooled. The precipitate was filtered off, washed with 300 ml of a 1:1 hexane/tetrahydrofuran solvent mixture, and dried to give the desired product.

MS and H-NMR data were consistent with the desired structure.

Step 5 (5)-Ethyl 3-{[N-(tert-butoxycarbonyl)glycyl]amino}-3-(S)-(5-chloro-2-hydroxy-3-iodophenyl)propionate

To a mixture of 9.4 g (34.51 mmol) of BOC-gly-OSu, 17.0 g (31.38 mmol) of ethyl 3-(S)-amino-3-(5-chloro-2-hydroxy-3-iodophenyl)propionate-(p-toluenesulfonic acid) salt and 200 ml of N,N-dimethylformamide was added 4.8 ml of triethylamine. The reaction mixture was stirred at room temperature for 18 hours, then the N,N-dimethylformamide was removed in vacuo. The residue was partitioned between 600 ml of ethyl acetate and 100 ml of dilute hydrochloric acid. The organic phase was washed with 200 ml of saturated aqueous sodium bicarbonate solution and 200 ml of saturated aqueous sodium chloride solution, dried over anhydrous magnesium sulfate and concentrated. The desired product was obtained as a solid in an amount of 14.2 g (86% yield).

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

Step 6 (S)-Ethyl-3-(glycylamino)-3-(S)-(5-chloro-2-hydroxy-3-iodophenyl)propionate—hydrochloride

To 37.20 g (70.62 mmol) of (S)-ethyl-3-{[N-(tert-butoxycarbonyl)glycyl]amino}-3-(S)-(5-chloro-2-hydroxy-3-iodophenyl)propionate at 0 °C was added 70 ml of a 4 M solution of hydrogen chloride in dioxane. The reaction mixture was stirred at room temperature for 3 hours and then concentrated. 100 ml of toluene was added to the residue and the concentration was repeated. The residue was suspended in diethyl ether, the solid was filtered off and then dried. The desired product was obtained as a crystalline powder and in an amount of 32.0 g (98% yield).

MS and H-NMR data were consistent with the desired structure.

EXAMPLE N

Step 1

The above compound was prepared according to the procedure of Example I, Step 2A, in which 3,5-dichloro

-Salicylaldehyde was replaced by an equivalent amount of 2-hydroxy-3,5-dibromobenzaldehyde. The desired product was obtained as a pale yellow solid in 88% yield. Melting point: 46-47 °C. TLC R _f 0.6 (1:1 ethyl acetate/hexane). ¹ H-NMR (CDCl ₃ ) δ (ppm): 3.37 (s, 3H), 3.56 (m, 2H), 3.92 (m, 2H), 5.29 {s, 2H), 7.91 (d, 1 H, J = 2.4 Hz), 7.94 (d, 1 H, J = 2.4 Hz), 10.27 (s, 1H). FAB-MS m/z ISI (M ⁺ ) .

HR-MS: calculated for CnH]_2Br2O4: 367.9083;

measured: 367.9077.

MS and Y-NMR data were consistent with the desired structure.

Step 2

The above compound was prepared using the procedure described in Example I, Steps 2B and 2C, using an equivalent amount of the compound from Step 1 in Example I, Step 2B. The compound of the formula was obtained as a yellow solid in 90% yield. Melting point: 57-59 °C. TLC: Rf 0.46 (1:1 ethyl acetate/hexane). ¹ H-NMR (CDCl3) δ (ppm): 1.45 (s, 9H); 2.1 (broad, 1H, deuterated), 2.51 (d, 1H, J1 - 9.9 Hz, _J2 - 15.3 Hz), 2.66 (d, 1H, = 4.2 Hz, _J2 = 15.3 Hz), 3.02 (broad, 1H, deuterated), 3.39 (s, 3H), 3.58-3.62 (m, 4H), 3.81 (m, 1H), 3.93 (m, 2H), 4.63 (dd, 1H, J = 4.2 Hz), 5.15 (s, 2H), 7.17-7.25 (m, 6H), 7.49 (d, 1H). FAB-MS m/z 602 (M+H).

HR-MS: calculated _{for C25H34NBr20g} _: _602.0753 ;

measured: 602.0749.

MS and H-NMR data were consistent with the desired structure.

Step 3

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chapter ₃

The above compound (p-toluenesulfonic acid salt) was prepared according to the procedure of Example I, Step 3, using an equivalent amount of the product of Step 2 in Example I, Step 3A. The desired product was obtained as a white solid in 62% yield. ¹ H-NMR (DMSO-d ₆ ) δ (ppm): 1.09 (t, 3H, J = 7.2 Hz), 2.27 (s, 3H), 2.97 (dd, 2H, Ji = 3.0 Hz, J ₂ = 7.2 Hz), 4.02 (q, 2H, J = 7.2 Hz), 4.87 (t, 1H, J = 7.2 Hz), 7.08 (d, 2H, J = 4.8 Hz), 7.45 (m, 3H), 7.57 (d, 1H, J = 2.4 Hz), 8.2 (broad, 3H). FAB-MS m/z 365 (M+H).

HR-MS: calculated _for _C11H14NBr2O3 : _365.9340 _;

measured: 365.9311.

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

Step 4

The above compound was prepared according to the procedure described in Example I, Step 4, using the compound of Step 3 in Step 3. The thus obtained (tert-butoxycarbonyl)-protected intermediate was converted to the desired compound according to the procedure described in Example I, Step 5.

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

EXAMPLE P.

The above compound was prepared according to the procedure of Example I, whereby in Example I, Step 2A, an equivalent amount of F was substituted for 3,5-dichlorosalicylaldehyde.

Example 3-Iodo-5-bromosalicylaldehyde prepared in step 1 was used.

EXAMPLE 1 (±)-3-(3-Bromo-2-hydroxy-5-chlorophenyl)-3-[{[{3-hydroxy-5-[(1,4,5,6-tetrahydro-5-hydroxy-2-pyrimidinyl)amino]benzoyl}-amino]-acetyl}-amino]-propionic acid-(trifluoroacetic acid) salt

To a mixture of 0.4 g (0.0014 mol) of the product of Example H, 0.58 g (0.0014 mol) of the product of Example B, 0.142 g (0.0014 mol) of triethylamine, 17 mg of 4-(dimethylamino)pyridine and 4 ml of N,N-dimethylacetamide cooled in an ice bath was added 0.268 g (0.0014 mol) of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide—hydrochloride. The reaction mixture was stirred overnight at room temperature, and the ester intermediate formed was isolated by reverse phase preparative HPLC. To a mixture of the ester thus obtained, 10 ml of water and 5 ml of acetonitrile was added 580 mg (0.0138 mol) of lithium hydroxide. The reaction mixture was stirred at room temperature for one hour and then acidified with trifluoroacetic acid to pH 2. The crude product was purified by reverse phase preparative HPLC to give (after lyophilization) 230 mg of the desired product as a white solid.

MS and H-NMR data were consistent with the desired structure.

EXAMPLE 2

The above compound was prepared according to the procedure of Example 1, substituting an equivalent amount of the product of Example A for the product of Example B. After lyophilization, 320 mg of the desired product was obtained as a white solid.

MS and H-NMR data were consistent with the desired structure.

The above compound was prepared according to the procedure of Example 1, substituting an equivalent amount of the product of Example F for the product of Example B. After lyophilization, 180 mg of the desired product was obtained as a white solid.

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

EXAMPLE 4

The above compound was prepared according to the procedure of Example 1, using an equivalent amount of the product of Example D instead of the product of Example B. After lyophilization, it was obtained as a white solid.

180 mg of the desired product was obtained.

MS and H-NMR data were consistent with the desired structure.

The above compound was prepared according to the procedure of Example 1, using an equivalent amount of the product of Example E instead of the product of Example B. After lyophilization, it was obtained as a white solid.

250 mg of the desired product was obtained.

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

EXAMPLE 6

The above compound was prepared according to the procedure of Example 1, using an equivalent amount of the product of Example C instead of the product of Example B. After lyophilization, it was obtained as a white solid.

220 mg of the desired product was obtained.

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

EXAMPLE 7

In a flame-dried flask, 7.8 g (0.027 mol) of the product of Example H was dissolved in 50 ml of anhydrous N, N-dimethylacetamide under a nitrogen atmosphere, the flask was placed in an ice bath, and after the solution had cooled, 3.7 g (0.027 mol) of isobutyl-(chloroformate) was added first, followed by 2.73 ··· · · · · · ...· .:. .:. **:* g (0.027 mol) of N-methylmorpholine. The solution was stirred in the ice bath for 15 minutes, and then at the same temperature, 10.0 g (0.024 mol) of the product of Example L was added first, followed by 2.43 g (0.024 mol) of N-methylmorpholine. The reaction mixture was stirred overnight at room temperature, and the ester intermediate formed was isolated by reverse phase preparative HPLC. To a mixture of the ester thus obtained, 60 mL of water, and 30 mL of acetonitrile was added 10 g (0.238 mol) of lithium hydroxide. The reaction mixture was stirred at room temperature for one hour, and then acidified with trifluoroacetic acid to pH 2. The crude product was purified by reverse phase preparative HPLC to give 9.7 g of the desired product as a white solid (after lyophilization).

MS and H-NMR data were consistent with the desired structure.

EXAMPLE 8 (+)-3-(3,5-Dichloro-2-hydroxyphenyl)-3-[{[{3-hydroxy-5-[(1,4,5,6-tetrahydro-5-hydroxy-2-pyrimidinyl)amino]benzoyl}-amino]-acetyl}-amino]-propionic acid monohydrochloride monohydrate

Step A

To a solution of 9.92 g (0.0345 mol) of the product of Example H in 200 ml of anhydrous 1,2-dimethoxyethane was added 4.0 ml (0.0362 mol) of N-methylmorpholine. The reaction mixture was cooled to -5 °C in a salted ice bath, and then 4.48 ml (4.713 g, 0.0345 mol) of isobutyl-(chloroformate) was added over one minute. The reaction mixture was stirred for 12 minutes in an ice bath, and then at the same temperature, first 11.15 g (0.030 mol) of the product of Example I was added, followed by 4.0 ml (0.0362 mol) of N-methylmorpholine. The reaction mixture was warmed to room temperature, stirred until the reaction was complete, and concentrated in vacuo at 50 °C. The dark residue was dissolved in approximately 50 ml of acetonitrile/water and the mixture was acidified by adding a little trifluoroacetic acid. The residue was applied to a 10 x 500 cm C-18 (particle size: 50 pm) column, and the desired product ester was isolated. [Solvent program: (100 vol% water + 0.05 vol% trifluoroacetic acid) 30:70 vol% water/(0.05 vol% trifluoroacetic acid + acetonitrile)/(0.05 vol% trifluoroacetic acid) over one hour; flow rate: 100 ml/water; the solvent program was started after the solvent front had eluted.] After preparative RPHPLC purification and lyophilization, 10.5 g of white solid was obtained in 50% yield.

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

Step B

Approximately 11 g of the product from Step A was dissolved in a dioxane/water solvent mixture, and the pH of the solution (measured with a pH meter) was adjusted to approximately 11.5 by adding 2.5 M aqueous sodium hydroxide solution. The reaction mixture was stirred at room temperature. The pH was occasionally raised above 11 by further addition of base. According to RPHPLC, the conversion of the ester to the acid was complete after 2-3 hours. The pH of the reaction mixture was then adjusted to approximately 6, as a result of which a viscous oil separated from the solution. The oil was isolated by decantation and then washed with 200 ml of hot water. The aqueous mixture thus obtained was allowed to cool, and the solid formed was filtered off. After lyophilization from hydrochloric acid solution, 2.6 g of the above compound was obtained. The dark, viscous oil obtained as a residue was treated with hot water and then cooled to obtain a tan powder (4.12 g after lyophilization from hydrochloric acid solution).

MS and H-NMR data were consistent with the desired structure.

EXAMPLE 9 (S)-3-(3-Bromo-2-hydroxy-5-chlorophenyl)-3-[{[{3-hydroxy~5-[(1,4,5,6-tetrahydro-5-hydroxy-2-pyrimidinyl)amino]benzoyl}-amino]-acetyl}-amino]-propionic acid (trifluoroacetic acid) salt

Step 1

To a suspension of 1.0 g (2.4 mmol) of the product of Example J, 0.75 g (2.6 mmol) of the product of Example H and 40 mg of 4-(dimethylamino)pyridine in 10 ml of N,N-dimethylacetamide was added 0.24 g (2.4 mmol) of triethylamine. The reaction mixture was stirred at room temperature for 15 minutes, then 0.60 g (3.1 mmol) of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride was added. The reaction mixture was stirred at room temperature overnight and then concentrated in vacuo. The residue was purified by reverse phase HPLC [starting gradient: 90:10 (v/v) (water/trifluoroacetic acid)/acetonitrile; retention time = 22 minutes]. As a result, 1.6 g of the desired product was obtained in 51% yield.

H-NMR data were consistent with the desired structure.

Step 2

To a solution of 800 mg (1.2 mmol) of the ester prepared in step 1 in 7 mL of 1:4 acetonitrile/water was added 148 mg (6.2 mmol) of lithium hydroxide. The reaction mixture was stirred at room temperature for 2 h, and then 0.71 mL (9.2 mmol) of trifluoroacetic acid was added. The mixture was purified by reverse-phase HPLC [starting gradient: 95:5 (water/trifluoroacetic acid)/acetonitrile; retention time = 24 min]. As a result, 860 mg of the desired product was obtained in 83% yield.

Microanalysis for the formula C ₂ 2H 23BrClN ₅ O ₇ · 1.7 CF 3 COOH: calculated (%): C 39.18; H 3.20; N 8.99;

Found (%): C 39.11; H 3.17; N 9.07.

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

Step 3

Preparation of the hydrochloride salt

The product of step 2 was dissolved in a suitable solvent (acetonitrile/water) and the solution was then slowly passed through a Bio-Rad AG2 - 8X (chloride form, 200-400 mesh, > 5 equivalents) ion exchange column. After lyophilization, the desired product was obtained as the hydrochloride salt.

EXAMPLE 10

HN

CO2H OH

The above compound was prepared by the method of Example 8, wherein in Step A of Example 8, Example I '*'· .:. .:. ^: ·ί·.:..

The product of Example N was substituted for the product of Example 1. The product was isolated by preparative RPHPLC and then lyophilized to give the desired product as the trifluoroacetic acid salt.

MS and H-NMR data were consistent with the desired structure.

EXAMPLE 11

The above compound was prepared by the procedure of Example 8, whereby in Step A of Example 8, the product of Example I was replaced by the product of Example M. The product was isolated by preparative RPHPLC and then lyophilized to give the desired product as the trifluoroacetic acid salt.

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

EXAMPLE 12

The above compound was prepared by the procedure of Example 8, whereby in Step A of Example 8, the product of Example I was replaced by the product of Example P. The product was isolated by preparative RPHPLC and then lyophilized to give the desired product as the trifluoroacetic acid salt.

EXAMPLE 13

Step 1

2-O-[(2-Methoxyethoxy)methyl]-3,5-diiodosalicylaldehyde

To a solution of 50.0 g (0.134 mol) of 3,5-diiodosalicylaldehyde in 150 ml of N,N-dimethylformamide at 20 °C was added 18.5 g (0.134 mol) of potassium carbonate. To the yellow substrate thus obtained was added 15.8 ml (0.134 mol) of [(2-methoxyethoxy)methyl] chloride, while the temperature of the reaction mixture was maintained at 20 °C throughout. The reaction mixture was stirred for 2 hours, after which another 1.5 g of [(2-methoxyethoxy)methyl] chloride was added, stirring was continued for another hour, and the reaction mixture was poured into ice water. The aqueous mixture was stirred, then the precipitate formed was filtered off and dried in vacuo. As a result, 61 g (99.1% yield) of the desired protected aldehyde was obtained. H-NMR data were consistent with the desired structure.

Step 2

To a solution of 41.5 g (0.112 mol) of 2-0-[(2-methoxyethoxy)methyl]-3,5-diiodosalicylaldehyde in 150 ml of tetrahydrofuran at room temperature was added 17.9 g (0.13 mol) of (S)-phenylglycinol. The reaction mixture was stirred for one hour, then 20.7 g of magnesium sulfate was added, stirring was continued for another two hours, and the reaction mixture was filtered. The filtrate was concentrated and dried under vacuum for 2 hours. 96 g (0.289 mol) of Reformatsky reagent and 250 ml of N-methylpyrrolidone were weighed into a two-necked round-bottomed flask. The mixture was stirred at -10 °C, then a solution of the imine in 100 ml of N-methylpyrrolidone was slowly added, while the temperature was maintained at -10 °C throughout. The reaction mixture was kept at -10 °C for *·· · · · · ♦ · hours, then at -5 °C for one hour. Finally, the reaction mixture was cooled back to -10 °C, and a mixture of 16 ml of concentrated hydrochloric acid and 200 ml of saturated aqueous ammonium chloride solution was added. To the resulting mixture was added 500 ml of diethyl ether, and the mixture was stirred for 2 hours. The diethyl ether phase was separated, and the aqueous layer was extracted with 300 ml of diethyl ether. The diethyl ether solutions were combined, washed with 200 mL of saturated aqueous ammonium chloride solution, 200 mL of water, and 200 mL of saturated aqueous sodium chloride solution, dried over anhydrous magnesium sulfate, and concentrated to give 90.0 g of an oil (99% yield) which contained the desired product as a single diastereoisomer by NMR.

Step 3

14.0 g (20.1 mmol) of the crude ester prepared in step 2 was dissolved in 100 ml of ethanol. The solution was cooled to 0 C, then 9.20 g (20.75 mmol) of lead(IV) acetate was added. The reaction mixture was stirred for 3 h, then 73 ml of 15% aqueous sodium hydroxide solution was added, and most of the ethanol was removed under reduced pressure. To the residue was added 200 ml of 15% aqueous sodium hydroxide solution.

100 rium hydroxide solution, then the mixture was extracted with 400 ml diethyl ether. The diethyl ether phase was washed with 100 ml water and 100 ml saturated aqueous sodium chloride solution, dried and concentrated. The orange oil obtained as a residue was dissolved in 100 ml ethanol, 6.08 g p-toluenesulfonic acid was added to the solution, then the reaction mixture was refluxed for 8 hours and then concentrated under reduced pressure. The residue was diluted with 60 ml tetrahydrofuran, the mixture was refluxed and then cooled. No precipitate formed during standing. The reaction mixture was concentrated and then purified by preparative HPLC, as a result of which the amino acid was obtained in the form of the p-toluenesulfonic acid salt. The resulting solid was dissolved in ethanol and saturated with hydrogen chloride gas. The reaction mixture was refluxed for 6 hours and then concentrated to give 12.47 g of the desired amino acid p-toluenesulfonic acid salt. Step 4

Ethyl 3-{[N-(tert-butoxycarbonyl)glycyl]amino}-3-(S)-(3,5-diiodo-2-hydroxyphenyl)propionate

7.48 g (27.04 mmol) BOC-gly-OSu, 12.47 g (27.04 mmol)

To a mixture of 101 ethyl 3-(S)-amino-3-(3,5-diiodo-2-hydroxyphenyl)propionate-(p-toluenesulfonic acid) salt and 100 ml N,N-dimethylformamide was added 4.8 ml triethylamine. The reaction mixture was stirred at room temperature for 18 hours, then the N,N-dimethylformamide was removed in vacuo. The residue was partitioned between 600 ml ethyl acetate and 100 ml dilute hydrochloric acid. The organic phase was washed with 200 ml saturated aqueous sodium bicarbonate solution and 200 ml saturated aqueous sodium chloride solution, dried over anhydrous magnesium sulfate and concentrated. The desired product was obtained as a solid and in an amount of 17.0 g (96% yield).

¹ H-NMR data were consistent with the desired structure.

Step 5

Ethyl 3-(glycylamino)-3-(3,5-diiodo-2-hydroxyphenyl)propionate—hydrochloride

To 17.0 g (25.97 mmol) of ethyl 3-{[N-(tert-butoxycarbonyl)glycyl]amino}-3-(S)-(3,5-diiodo-2-hydroxyphenyl)propionate at 0 °C was added 40 ml of 4 M dioxane-hydrogen chloride solution. The reaction mixture was stirred at room temperature for 3 hours and then concentrated. To the residue was added 100 ml of toluene and then the concentration was repeated. The residue was dried, resulting in the form of a crystalline powder and 8.0 g of

102 • 4« * · · · ·

...» .:. .rv:..

The desired product was obtained in large quantities (56% yield).

¹ H-NMR data were consistent with the desired structure.

Step 6

A mixture of 3.74 g (12.98 mmol) of m-(5-hydroxypyrimidino)hippuric acid and 25 ml of N,N-dimethylacetamide was heated until the solid was completely dissolved. The solution was then cooled to 0 °C, 1.68 ml of isobutyl-(chloroformate) was added all at once, followed by 1.45 ml of N-methylmorpholine. Ten minutes later, 6.0 g (10.82 mmol) of ethyl-3-(glycylamino)-3-(3,5-diiodo-2-hydroxyphenyl)propionate hydrochloride was added all at once, followed by

1.45 ml of N-methylmorpholine. The reaction mixture was stirred at room temperature for 18 hours and then concentrated. The residue was dissolved in 20 ml of a 1:1 tetrahydrofuran/water solvent mixture and the solution was chromatographed (reverse phase, 95:5 -> 30:70 water/acetonitrile solvent gradient containing 0.1% trifluoroacetic acid by volume over 60 minutes). The appropriate fractions were combined and concentrated. The residue was dissolved in aqueous acetonitrile and the solution was basified by addition of lithium hydroxide. The solution was stirred for 2 hours and then concentrated. The residue was purified by HPLC as above to give the desired acid as the trifluoroacetic acid salt. The trifluoroacetic acid salt was converted to the corresponding hydrochloride salt by passing it through an ion exchange column and then lyophilizing. H-NMR data were consistent with the desired structure.

EXAMPLE 14-18

103 By the methods described above, using the appropriate starting compounds and reagents during the processes, the person skilled in the art can easily prepare the compounds of general formulae (VII), (VIII), (IX), (XIII) and (XIV) and their isomers.

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

- 104 - :··' u í .

·** · * · · _ ♦*** *3^ ··*'

VITRONECTIN ADHESION TEST

MATERIALS

Human vitronectin receptor (α _ν β ₃ ) was purified from human placenta by a known method [Pytela et al., Methods in Enzymology, 144, 475-489 (1987)]. Human vitronectin was purified from fresh frozen plasma by a known method [Yatohgo et al., Cell Structure and Function, 13, 281-292 (1988)]. Biotinylated human vitronectin was prepared by coupling NHS-biotin [Pierce Chemical Company (Rockford, Illinois, United States)] to vitronectin purified by a known method [Charo et al., J. Biol. chem. , 266 (3), 1415-1421 (1991)]. Assay buffer, OPD substrate tablets, and RIA grade bovine serum albumin (BSA) were purchased from Sigma (St. Louis, Missouri, USA). Anti-biotin antibody was purchased from Calbiochem (La Jolla, California, USA). Linbro microtiter plates were purchased from Flow Labs (McLean, Virginia, USA). ADP reagent was purchased from Sigma (St. Louis, Missouri, USA). METHODS Solid-phase receptor assay

The assay was performed essentially in the same manner as the known procedure [Niiya et al., Blood, 70, 475-483 (1987)]. A stock solution of purified human vitronectin receptor (α _ν β3) was diluted to a concentration of 1.0 pg/ml in Tris buffer containing 1.0 mM Ca ⁺⁺ , Mg ⁺⁺ and Mn ⁺⁺ , pH 7.4 (TBS ⁺⁺⁺ ). The diluted receptor was immediately

We transferred the samples to Linbro microtiter plates (100 μΐ/well; 100 ng receptor- 105

tor/well). The plates were sealed and incubated overnight at 4 °C to allow receptor binding to the wells. All further steps were performed at room temperature. The assay plates were emptied and 200 μΐ 1 % RIA grade BSA/TBS solution (TBS/BSA) was added to the wells to block the plastic surfaces. After a 2-hour incubation, the assay plates were washed using a 96-well plate washer. Starting from a 2 mM + + + stock solution, a logarithmic dilution series of the test compound and controls was prepared using 2 nM biotinylated vitronectin in TBS/BSA as diluent. 50 μΐ aliquots of the premix of the labeled ligand with the test (or control) ligand were applied to the assay plate using a CETUS Propette robot; The final concentration of the labeled ligand was 1 nM, while the highest concentration of the test compound was 1.0 * 10 M. The competitive reaction was carried out for 2 hours, after which all wells were washed with a plate washer as above. The affinity purified horseradish peroxidase-labeled goat anti-biotin antibody was diluted 1:3000 in TBS/BSA, and 125 μΐ of the dilution was added to each well. Thirty minutes later, the plates were washed and then incubated with OPD/H ₂ O ₂ substrate (100 mM/liter in citrate buffer, pH 5.0). The plate was read at 450 nm using a microtiter plate reader, and when the maximally bound control wells reached a value of approximately 1.0, the final A ₄₅₀ value was recorded for analysis. The data were analyzed using a custom-written macro in the EXCEL spreadsheet program. The mean, standard deviation

106 and % CV values were determined with duplicate concentrations. The mean A ₄₅₀ values were normalized to the mean of four maximally bound controls [(without competitor)(B-MAX)]. The normalized values were substituted into a four-parameter curve fitting algorithm [Rodbard et al., Int. Atomic Energy Agency, Vienna, 469 (1977)], and the values were plotted on a semi-logarithmic scale as shown in Fig. 2 , .

was shaken; the calculated concentration corresponding to 50% inhibition of maximal binding of biotinylated vitronectin and the corresponding R was recorded for compounds that showed more than 50% inhibition at the highest concentration tested or for compounds with an IC50 greater than the highest concentration tested. β-{[2—{[5—{[(aminoimino)methyl]amino)-1-oxopentyl]amino}-1-oxoethyl]amino}-3-pyridylpropionic acid (USSN 08/375,338, Example 1) was used as a positive control on each plate; the compound is a potent α _ν β3 antagonist ( _IC50 = 3-10 nM).

PURIFIED Ilb/lIIa RECEPTOR ASSAY

MATERIALS

Human fibrinogen receptor ( _α Ibβ3) was purified from expired platelets [Pytela, R., Pierschbacher, MD, Argraves, S., Suzuki, S., and Rouslahti, E., Arginine—Glycine— -Aspartic acid, adhesion receptors, Methods in Enzymology, 144, 475-489 (1987)]. Human vitronectin was purified by a known method [Yatohgo, T., Izumi, M. , Kashiwagi, H., and Hayashi, M., Novel purification of vitronectin from human plasma by heparin affinity chromatography, Cell Structure and Function, 13, 281-292 (1988)] fresh

107 were purified from frozen plasma. Biotinylated human vitronectin was prepared by coupling NHS-biotin [Pierce Chemical Company (Rockford, Illinois, United States)] to vitronectin purified by a known procedure [Charo, IF, Nannizzi, L., Phillips, DR, Hsu, MA, Scaround bottomorough, RM, Inhibition of fibrinogen binding to GP IIb/IIIa by a GP IIa peptide, J. Biol, chem., 266 (3), 1415-1421 (1991)]. Assay buffer, OPD substrate tablets, and RIA-grade bovine serum albumin (BSA) were purchased from Sigma (St. Louis, Missouri, United States). Anti-biotin antibody was purchased from Calbiochem (La Jolla, California, United States). Linbro microtiter plates were purchased from Flow Labs (McLean, Virginia, USA). ADP reagent was purchased from Sigma (St. Louis, Missouri, USA).

METHODS

Solid-phase receptor assays

The assay was performed in essentially the same manner as the known method [Niiya, K., Hodson, E., Bader, R., Byers-Ward, V., Koziol, JA, Plow, EF, and Ruggeri, ZM, Increased surface expression of the membrane glycoprotein lib/Illa complex induced by platelet activation: Relationships to the binding of fibrinogen and platelet aggregation”, Blood, 70, 475-483 (1987)]. The purified human vitronectin receptor (α _ν β ₃ ) + 4- + + + 4- 4-4-4 solution was diluted to a concentration of 1.0 pg/ml in 1.0 mM Ca , Mg and Mn-containing tris buffer, pH 7.4 (TBS ). The diluted receptor was immediately transferred to Linbro microtiter plates (100

108 μΐ/well; 100 ng receptor/well). The plates were sealed and incubated overnight at 4 °C to allow receptor binding to the wells. All further steps were performed at room temperature. The assay plates were emptied and 200 μΐ 1 % RIA grade 1%

BSA/TBS ⁺⁺⁺ solution (TBS ⁺⁺⁺ /BSA) was added to the wells. After a 2-hour incubation, the assay plates were washed using a 96-well plate washer. Starting from a 2 mM stock solution, a logarithmic dilution series of the test compound and controls was prepared using 2 nM biotinylated vitronectin prepared in TBS ⁺⁺⁺ /BSA as diluent. 50 μΐ aliquots of the premix of the labeled ligand with the test (or control) ligand were applied to the assay plate using a CETUS Propette robot; the final concentration of the labeled ligand was 1 -4 nM, while the maximum concentration of the test compound was 1.0 χ 10 M. The competitive reaction was carried out for 2 hours, and then all wells were washed using a plate washer as described above. The affinity purified horseradish peroxidase-labeled goat anti-biotin antibody was diluted 1:3000 in TBS ⁺⁺⁺ /BSA, and 125 μΐ of the dilution was added to each well. Thirty minutes later, the plates were washed and incubated with OPD/H ₂ O ₂ substrate (100 mM/liter in citrate buffer, pH 5.0). The plate was read at 450 nm using a microtiter plate reader, and when the maximally bound control wells reached a value of approximately 1.0, the final A ₄₅ q value was recorded for analysis. The data were analyzed using a macro written for us in the EXCEL spreadsheet program. The trans

109 lag, standard deviation and % CV values were determined with duplicate concentrations. The mean A450 values were normalized to the mean of four maximally bound controls [(without competitor)(B-MAX)]. The normalized values were substituted into a four-parameter curve fitting algorithm [Rodbard et al., Int. Atomic Energy Agency, Vienna, 469 (1977)], the values were plotted on a semi-logarithmic scale; the calculated concentration corresponding to 50% inhibition of maximal binding of biotinylated vitronectin 2 and the corresponding R was recorded for compounds that showed more than 50% inhibition at the highest concentration tested or for compounds that had an IC50 greater than the highest concentration tested. β-{[2-{[5-{[(aminoimino)methyl]amino}-1-oxopentyl]amino}-1-oxoethyl]amino}-3-pyridylpropionic acid (USSN 08/375,338, Example 1) was used as a positive control on each plate; the compound is a potent α _ν β3 antagonist (IC50 = 3-10 nM).

Human platelet-rich plasma assay

Healthy, aspirin-free donors were selected from volunteers. Platelet-rich plasma was prepared using a known method [Zucker, Μ. B., Platelet Aggregation Measured by the Photometric Method, Methods in Enzymology, 166, 117-133 (1989)], and ADP-induced platelet aggregation assays were performed. Using a standard venipuncture technique using a butterfly ligature, 45 ml of whole blood was collected into a 60 ml syringe containing 5 ml of 3.8% trisodium citrate solution. The contents of the syringe were shaken, and the anti-coagulated whole blood was transferred into a 50 ml conical poly110 ...» .:. .:. ··:· -·.

was transferred to an ethylene tube. To sediment non-platelet cells, the blood was centrifuged at 200 mg/mL for 12 minutes at room temperature. The platelet-rich plasma was placed in a polyethylene tube and stored until use. A second centrifugation of the remaining blood at 2000 g for 15 minutes yielded platelet-poor plasma. The platelet count per microliter typically ranged from 300,000 to 500,000. The platelet-rich plasma was measured in 0.45 ml portions into siliconized cuvettes, mixed at 1100 rpm for one minute at 37 °C, and then 50 μΐ of pre-diluted test compound was added. After mixing for one minute, aggregation was initiated by adding 50 μΐ of 200 μΜ ADP. Aggregation was recorded for 3 min in a Payton dual-channel aggregometer (Payton Scientific, Buffalo, New York, USA). The percent inhibition of the maximal response (saline) obtained with test compound dilution series was used to determine dose/response curves. All compounds were tested in duplicate. The half-maximal inhibitory concentration (IC ₅₀ ) was determined graphically from the dose/response curves for compounds that showed at least 50% inhibition at the highest concentration tested; otherwise, those with an IC 50 greater than the highest concentration tested.

- Ill

Table 1

Example ανβ₃ IC₅₀(nM) Ilb/Illa IC₅₀ (nM) 1. 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.·HC1 0.82 226.2 10. 2.26 641 11. 12. 9.59 1060

Claims

112 PATENT CLAIMS

1. One

a compound with the general formula — in the formula of which

X and Y are the same or different halogen atoms; and

R represents a hydrogen atom or an alkyl group of 1-10 carbon atoms - and pharmaceutically acceptable salts thereof.

2. A compound according to claim 1 selected from the following compounds of the general formula:

- 113

114 -

- 115

...· .:. 4. ··*·.:·.

— where in the general formulas R represents a hydrogen atom or an alkyl group — or pharmaceutically acceptable salts thereof.

3. A compound according to claim 1 selected from the following compounds:

116 -

117 -

118 and

119 ...· -ί. 4.—*^»·

A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a pharmaceutically effective amount of a compound according to claim 1.

A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound according to claim 2.

6. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound according to claim 3.

7. Use of a compound according to any one of claims 1-3 for the preparation of a pharmaceutical composition for the treatment of α _ν β ₃ integrin mediated conditions in mammals in need thereof.

8. Use of a compound according to any one of claims 1-3 for the preparation of pharmaceutical compositions for the treatment of tumor metastasis.

Use of a compound according to any one of claims 1-3 for the preparation of pharmaceutical compositions for the treatment of solid tumor growth.

10. Use of a compound according to any one of claims 1-3 for the preparation of pharmaceutical compositions for the treatment of angiogenesis.

- 120 -

11. Use of a compound according to any one of claims 1-3 for the preparation of pharmaceutical compositions for the treatment of osteoporosis.

12. Use of a compound according to any one of claims 1-3 for the preparation of pharmaceutical compositions for the treatment of malignant humoral hypercalcemia.

13. Use of a compound according to any one of claims 1-3 for the preparation of pharmaceutical compositions for the treatment of smooth muscle cell migration.

14. Use of a compound according to any one of claims 1-3 for the preparation of pharmaceutical compositions for the treatment of rheumatoid arthritis.

15. Use of a compound according to any one of claims 1-3 for the preparation of pharmaceutical compositions for the treatment of macular degeneration.

The authorized person:

•st S.B.G. & ÍC International

Member of the Szabaíshni irala 11-1062 Budapest, Andií&y ót .113. Phone; 34-24-950, Fax: .34-24-323