JP2002505323A - Meta-azacyclic aminobenzoic compounds and their derivatives as integrin antagonists - Google Patents

Abstract

(57) SUMMARY The present invention relates to compounds of formula (1) and pharmaceutically acceptable salts thereof, which are useful as α _v β ₃ integrin antagonists, and isomers thereof. Embedded image

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]Field of the invention  The present invention relates to α_vβ₃Pharmaceutical compositions useful as integrin antagonists
And α_vβ₃A disease condition mediated by α_vβ₃Inhibits or antagonizes integrin
A pharmaceutical agent (compound) that is also useful for treating by antagonizing.

[0002]Background of the Invention  Integrins mediate cell adhesion and thus occur in a variety of biological processes
A group of cell surface glycoproteins that are useful mediators of cell adhesion interactions.
Integrins are non-covalently linked α polypeptide subunits and β
It is a heterodimer consisting of a peptide subunit. Currently, 11 different types
Α subunits have been identified, and six different β subunits have been identified.
ing. Different α subunits can bind to different β subunits
A distinct integrin results.

[0003] Integrins identified as α _v β ₃ (also known as vitronectin receptor) have been identified as tumor metastases, solid tumor growth (neoplasia), osteoporosis, Paget's disease, malignant fluid hypercalcemia, tumors As an integrin that plays a role in a variety of and disease states including angiogenesis including angiogenesis, retinopathy, macular degeneration, arthritis including rheumatoid arthritis, periodontal disease, psoriasis and smooth muscle cell migration (eg, restenosis) confirmed. In addition, such agents have been found to be useful as antiviral, antifungal and antibacterial. Thus, compounds that selectively inhibit or antagonize α _v β ₃ are beneficial for treating such conditions.

[0004] α _v β ₃ Integrin and α _v containing other integrins have been shown to bind to many Arg-Gly-Asp (RGD) containing matrix macromolecules. Compounds containing the RGD sequence mimic extracellular matrix ligands and bind to cell surface receptors. However, it is also known that RGD peptides are generally non-selective for RGD-dependent integrins. For example, many RGD peptides bind to alpha _v beta _3, or alpha _v beta _5, binds to alpha _v beta ₁ and α _{II b} β _3. Antagonists of platelet α _IIb β ₃ (also known as fibrinogen receptor) are known to block the aggregation of human platelets. To avoid bleeding side effects in treating conditions and disease states associated with integrin α _v β ₃ , it would be beneficial to develop compounds that are selective antagonists of α _v β ₃ as opposed to α _IIb β ₃ It is.

[0005] Tumor cell invasion occurs in three steps: 1) attachment of tumor cells to the extracellular matrix; 2) proteolytic lysis of that matrix; and 3) migration of cells through a lysis barrier. Occur. This process repeats and metastasizes at a distance from the original tumor.

[0006] Seftor et al. (Proc. Natl. Acad. Sci. USA, Vol. 89 (1992) 1557-1561) _v β₃Integrin has biological function in melanoma cell invasion
Indicated. Montgomery et al. (Proc. Natl. Acad. Sci. USA, Vol. 91 (1994) 8856-
60), α expressed in human melanocytes_vβ₃Integrin is a survival signal
And protect cells from apoptosis. Inhibits tumor metastasis
Α_vβ₃Tumor cells by interference with integrin cell adhesion receptor
Mediation of metastasis is beneficial.

[0007] Brooks et al. (Cell, Vol. 79 (1994) 1157-1164) show that systemic administration of α _v β ₃ antagonists causes dramatic regression of various histologically distinct human tumors, _v demonstrated that therapeutic approaches treatment of neoplasia (solid tumor growth) is provided by an antagonist of beta _3.

[0008] The adhesion receptor integrin α _v β ₃ has been identified as a vascular marker for angiogenesis in children and adults, and thus such receptors play an important role in angiogenesis or neovascularization. Angiogenesis is characterized by invasion, migration, smooth muscle proliferation, and endothelial cells. Antagonists of α _v β ₃ inhibit this process by selectively promoting apoptotic cells in neovascularization. New blood vessel growth,
In other words, angiogenesis is also diabetic retinopathy, macular degeneration (Adonis et al., Amer. J. Opthal.,
Vol. 118, (1994) 445-450), rheumatoid arthritis (J. Exp. Med by Peacock et al.).
, Vol. 175, (1992), 1135-1138). Therefore,
α _v β ₃ antagonists are useful therapeutic targets for treating such conditions associated with neovascularization (Brooks et al., Science, Vol. 264, (1994), 569-571).

The cell surface receptor α _v β ₃ has been reported to be the major osteoclast integrin responsible for bone attachment. If osteoclasts cause bone resorption and the resorbing activity of the affected bone exceeds the osteogenic activity, osteoporosis (bone loss) occurs, increasing the number of fractures, disability and mortality. Antagonists of α _v β ₃ are available in vitro (Sato et al., J. Cell. Bio., Vol., 111 (1990) 1713-1723) and in vivo (Fisher et al., Endocrinology, Vol. 132 (1993) 1411-1413). Both have been shown to be potential inhibitors of osteoclast activity. Antagonism of α _v β ₃ reduces bone resorption, thus increasing osteogenic activity and resound round bottoming (resoroun
d bottoming) restore normal balance of activity. Thus, it would be beneficial to provide an antagonist of osteoclast α _v β ₃ that is an effective inhibitor of bone resorption,
Therefore, it is useful for treating or preventing osteoporosis.

The role of α _v β ₃ integrin in smooth muscle cell migration targets the prevention or inhibition of neointimal hyperplasia that is responsible for restenosis following vascular treatment (Ch
J. Vasc. Surg. Vol. 19 (1) (1994) 125-34 by oi et al. Prevention or inhibition of neointimal hyperplasia to prevent or inhibit restenosis is beneficial.

[0011] White (Current Biology, Vol. 3 (9) (1993) 596-599) reports that adenovirus utilizes α _v β ₃ to enter host cells. The integrin is required for endocytosis of the virus and for penetration of the viral genome into the host cytoplasm. Thus, compounds that inhibit α _v β ₃ may prove useful as antiviral agents.

[0012]Summary of the Invention  The present invention relates to compounds of the following general formula and pharmaceutically acceptable salts thereof,

[0013]

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

There are various isomers of the above compounds, and all such isomers are included in the present invention. Not only tautomers, but also pharmaceutically acceptable salts of such isomers and tautomers are encompassed by the present invention.

More specifically, the present invention relates to the following compounds or pharmaceutically acceptable salts thereof:

[0016]

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[0017]

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[0018]

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[0019]

Embedded image Wherein R is H or alkyl.

[0020] Another object of the present invention is to provide a pharmaceutical composition comprising the above compound. Such compounds and compositions are useful in selectively inhibiting or antagonizing integrins, and therefore, another embodiment of the present invention relates to a method for selectively inhibiting or antagonizing α _v β ₃ integrin. In addition, the present invention relates to osteoporosis, malignant humoral hypercalcemia, Paget's disease, tumor metastasis, solid tumor growth (tumorigenesis), angiogenesis including tumor angiogenesis, diabetic retina in mammals in need of treatment Retinopathy including macular degeneration and macular degeneration, arthritis including rheumatoid arthritis, periodontal disease,
It includes treating or inhibiting psoriasis, disease states associated with smooth muscle cell migration and restenosis. In addition, such agents are useful as antiviral and antibacterial agents.

[0021]Detailed description  The present invention relates to compounds of the type represented by formulas I to XVI described above.
.

Preferred embodiments of the present invention are compounds of the formula:

[0023]

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[0024]

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[0025]

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[0026]

Embedded image Furthermore, the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of said compound.

Further, the present invention relates to a method for selectively inhibiting or antagonizing α _v β ₃ integrin, more specifically, bone resorption, periodontal disease, osteoporosis, malignant fluid hypercalcemia. Disease, Paget's disease, tumor metastasis, solid tumor growth (tumorigenesis), angiogenesis including tumor angiogenesis, retinopathy including diabetic retinopathy and macular degeneration, arthritis including rheumatoid arthritis, smooth muscle cell migration and restenosis And a pharmaceutically acceptable carrier, together with a therapeutically effective amount of said compound to achieve such inhibition.

The following is a list of definitions of various terms used in this application.

As used herein, the term “alkyl” or “lower alkyl” refers to about 1 to about 1
A straight or branched chain hydrocarbon group having 0 carbon atoms, preferably 1 to about 6 carbon atoms. Examples of such alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl
-Butyl, pentyl, neopentyl, hexyl, isohexyl and the like.

As used herein, the term “halo” or “halogen” refers to bromo, chloro or iodo.

The term “haloalkyl,” as used herein, refers to an alkyl group, as defined above, wherein one or more carbon atoms have been replaced with one or more same or different halo groups.
Examples of haloalkyl groups include trifluoromethyl, dichloroethyl, fluoropropyl, and the like.

As used herein, the term “composition” refers to the product resulting from mixing or combining one or more elements or components.

As used herein, the term “pharmaceutically acceptable carrier” refers to pharmaceutically acceptable liquid or solid fillers, diluents, excipients involved in carrying or delivering a chemical agent. A material, composition or vehicle, such as a solvent or an encapsulating material.

The term “therapeutically effective amount” means the amount of a drug or agent that elicits the biological or medical response of a tissue, system or animal that a researcher or clinician seeks.

The following is a list of the abbreviations used interchangeably herein and their corresponding meanings. ¹ H-NMR is hydrogen magnetic resonance, AcOH is acetic acid, CH ₃ CN is acetonitrile, CHN analysis is elemental analysis of carbon / hydrogen / nitrogen, CHNCl analysis is carbon / hydrogen CHNS analysis is elemental analysis of carbon / hydrogen / nitrogen / sulfur, Di water is deionized water, DMA is N, N-dimethylacetamide DMAP is 4- (N, N-dimethylamino) pyridine, DMF is N, N-dimethylformamide, and EDCl is 1- (3-dimethylaminopropyl) -3. -Ethyl carbodiimide hydrochloride, EtOAc refers to ethyl acetate, EtOH refers to ethanol, FAB MS refers to fast atom bombardment mass spectrometry G is the number of grams, HOBT is 1-hydroxybenzotriazole hydrate, HPLC is high performance liquid chromatography, and IBCF is isobutyl chloroformate. KSCN is potassium thiocyanate, L is liter, LiOH is lithium hydroxide, MEM is methoxyethoxymethyl, MEMCl is methoxyethoxy MeOH is methanol, mg is milligrams, MgSO ₄ is magnesium sulfate, ml is milliliter, mL is milliliter MS is mass spectrometry, MTBE is methyl t And that the ether is that of nitrogen and _{N 2} refers to a sodium carbonate and NaHCO _3, and NaOH is that of sodium hydroxide, by sodium sulphate and _Na 2 SO ₄ NMM stands for N-methylmorpholine, NMP stands for N-methylpyrrolidinone, NMR stands for nuclear magnetic resonance, and P ₂ O ₅ stands for phosphorus pentoxide. , PTSA is para-toluenesulfonic acid, RPHPLC is reversed phase high performance liquid chromatography, RT is room temperature, TFA is trifluoroacetic acid, THF is Refers to tetrahydrofuran, TMS refers to trimethylsilyl, and Δ refers to heating the reaction mixture.

The compounds described above exist in various isomers, and all such isomers are included in the present invention. Not only tautomers but also pharmaceutically acceptable salts of such isomers and tautomers are included in the present invention.

In the structures and formulas presented herein, the bond pulled through the bond of the ring is the available atom of the ring.

The term “pharmaceutically acceptable salt” refers to a salt prepared by contacting the compound with an anionic acid, which is usually considered suitable for human consumption. Examples of pharmacologically acceptable salts include hydrochloride, hydrobromide, hydroiodide,
Sulfate, phosphate, acetate, propionate, lactate, maleate, malate, succinate, tartrate and the like. All pharmacologically acceptable salts can be prepared by conventional methods (further examples of pharmacologically acceptable salts can be found in Berge et al., J. Ph.
arm. Sci., 66 (1), 1-19 (1977)).

For selective inhibition or antagonism of α _v β ₃ integrin, the compounds of the invention may be administered orally, parenterally, or by inhalation spray, or by conventional pharmaceutically acceptable carriers, adjuvants and vehicles. Is administered locally in a dosage formulation containing The term “parenteral” as used herein refers to, for example, subcutaneous, intravenous, muscular,
Includes administration by intrasternal, infusion or intraperitoneal.

The compounds of the present invention are administered in pharmaceutical compositions suitable for the route of administration, and in dosages effective for therapeutic purposes and by routes suitable for the purpose. Therapeutically effective dosages of the compounds required to prevent or arrest or treat the progress of the medical condition can be readily determined by one of ordinary skill in the art using preclinical and clinical approaches commonly practiced in the medical field. Can be confirmed.

Accordingly, the present invention provides a method of treating a condition mediated by selectively inhibiting or antagonizing an α _v β ₃ cell surface receptor, the method comprising a compound selected from a class of compounds described above. One or more non-toxic, pharmaceutically acceptable carriers and / or diluents and / or adjuvants (collectively referred to herein as one or more nontoxic, pharmaceutically acceptable carriers). (Referred to as "carrier" materials) and, if necessary, other active ingredients. More specifically, the present invention provides a method for inhibiting an α _v β ₃ cell surface receptor. More preferably, the invention relates to inhibiting bone resorption, treating osteoporosis, inhibiting malignant fluid hypercalcemia, treating Paget's disease, inhibiting tumor metastasis, inhibiting tumor formation (solid tumor growth), Methods of inhibiting angiogenesis including formation, treating diabetic retinopathy and macular degeneration, inhibiting arthritis, psoriasis and periodontal disease, inhibiting smooth muscle cell migration including restenosis.

Based on standard laboratory techniques and treatments that are well known and understood by those skilled in the art, and based on comparisons with compounds of arable land utility, the compounds can be used in patients suffering from the above mentioned disease states. It can be used for treatment. It will be understood by those skilled in the art that the selection of the most suitable compound of the present invention is within the skill of the art and will depend on many factors, including the evaluation of results obtained in standard assays and animal models. Is done.

The treatment of a patient suffering from a disease condition may include administering the compound to such patient in that it controls the condition or prolongs the patient's viability beyond what would be expected without such treatment. Administering an effective amount to treat a subject.

As used herein, the term “inhibiting” a condition slows, interrupts,
Prevent or stop, but does not necessarily mean that the condition is completely eliminated. Prolonging the patient's viability beyond its own significant and beneficial effect may mean that the condition can be controlled to some extent.

As mentioned above, the compounds of the present invention may be used in various biological, prophylactic or therapeutic fields. The compounds are believed to be useful in preventing or treating the disease states or conditions in which α _v β ₃ integrin plays a role.

The regimen upon administration of the compound and / or the composition containing the compound is based on a number of factors, including the patient's type, age, weight, sex, and medical condition. Mimicking also depends on the severity of the condition, the route of administration, and the activity of the particular compound employed. Thus, the regimen of administration varies widely. Approximately 0.1 kg / kg of body weight per day.
Dosages of from 01 mg to about 1000 mg are useful for treating the above-mentioned disease states, more preferably from about 0.01 mg to about 10 mg / kg of body weight per day.
0 mg dose.

The active ingredient to be administered by injection may be formulated as a composition, for example using saline, dextrose or water as a suitable carrier. In general, a suitable daily injection will be in a number of doses, from about 0.01 mg to 10 mg per kg of body weight, depending on the factors mentioned above.

For administration to a mammal in need of such treatment, a therapeutically effective amount of the compound is usually combined with one or more adjuvants suitable for the route of administration. The compounds are 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 acid and sulfuric acid,
Mix with gelatin, acacia, sodium alginate, polyvinylpyrrolidone and / or polyvinyl alcohol and tablet or encapsulate for conventional administration.
Alternatively, the compound is water, polyethylene glycol, polypropylene glycol,
Dissolve in ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride and / or various buffers. Other adjuvants and modes of administration are well known in the pharmaceutical art.

[0049] Useful pharmaceutical compositions of the present invention may perform conventional pharmaceutical operations such as sterilization and / or use conventional pharmaceutical adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, buffers and the like. Including.

The general synthetic sequence for preparing useful compounds of the present invention is outlined in Schemes 1 to III. Descriptions of various aspects of the invention and actual synthetic procedures
It will be described as appropriate. The following schemes and examples are for illustrative purposes only and are not intended to limit the scope or spirit of the present invention. Those skilled in the art will readily understand that known variations of the conditions and methods described in the schemes and examples below are available for synthesizing the compounds of the present invention.

Unless otherwise noted, all starting materials and equivalents utilized were commercially available.

[0052]

[Table 1] Scheme I illustrates a method useful for synthesizing the tetrahydropyrimidinobenzoic acid moieties of the present invention that can be attached to gly-β-amino acids. Briefly, in Scheme I, using the method described in Austr. J. Chem., 34 (6), 1319-24 (1981), 3, 5
-Dihydroxybenzoic acid is converted to 3-amino-5-hydroxy-benzoic acid. The product is reacted with ammonium thiocyanate in hot dilute hydrochloric acid to give, after usual work-up, 3-thiourea-5-hydroxybenzoic acid. Reflux in ethanol and react with methyl iodide to convert the thiourea intermediate to the S-methyl derivative. 1,3-Diamino-2-hydroxypropane is reacted with the intermediate obtained above in hot DMA. Upon cooling the precipitate, the zwitterion product is separated by filtration. Dry-freeze from dilute hydrochloric acid to obtain the HCl salt. Or,
The volatiles are distilled off and concentrated to separate the product from the original reaction mixture. The resulting product is taken up with water, the pH is adjusted to about 5-7, and the zwitterion product is precipitated and separated by filtration. The HCl salt is obtained by simply dissolving in dilute hydrochloric acid, concentrating to a solid and drying, as described above.

[0053]

[Table 2] Scheme 1A shows a method useful for synthesizing tetrahydropyrimidinobenzoic acids of the invention that can be coupled to gly-β-amino acid esters. Briefly, in Scheme IA, 1,3-diamino-2- in a suitable solvent such as ethanol-water
The hydroxypropane is reacted with carbon disulfide, refluxed, cooled, refluxed again with the addition of hydrochloric acid, cooled and filtered and dried to give the 5-hydroxytetrahydropyrimidine-2-thione product. This cyclic thiourea intermediate is converted to the S-methyl derivative by refluxing with methyl iodide in ethanol. The desired 2-methylthioether-5-hydroxypyrimidine hydroiodic acid is easily separated off by removing the volatiles under reduced pressure. Therefore, 2-methylene chloride: DMA (about 10: 1)
Methylthioether-5-hydroxypyrimidine hydroiodide and an equivalent of triethylamine are cooled to ice bath temperature and an equivalent of di-t-butyl dicarbonate (BO
C anhydride). BOC-2-methylthioether-
5-Hydroxypyrimidine is obtained as an oil.

Using the method described in Aust. J. Chem., 34 (6), 1319-24 (1981), 3,
Convert 5-dihydroxybenzoic acid to 3-amino-5-hydroxybenzoic acid.

BOC-2-methylthioether-5-hydroxypyrimidine and 3-amino-
The 5-hydroxybenzoic acid is reacted in hot DMA to give the final desired product, 3-hydroxy-5-[(5-hydroxy-1,4,5,6-tetrahydro-2-pyrimidinyl) amino The benzoic acid hydrochloride is obtained. Upon cooling, the precipitate and the zwitterion product are separated by filtration. The HCl salt is obtained, for example, by freeze-drying from diluted hydrochloric acid.

[0056]

[Table 3] Scheme II illustrates a method useful for synthesizing ethyl N-gly-amino-3- (3,5-dihalo-2-hydroxy) phenylpropionates of the present invention that can be coupled to a tetrahydropyrimidinobenzoic acid moiety. Is shown. Briefly, it is synthesized by directly halogenating a 3,5-halo-substituted salicylaldehyde, for example, by slurrying 5-bromosalicylaldehyde in acetic acid and adding an equivalent or more of chlorine to 3-chloro-5-.
Bromo-2-hydroxybenzaldehyde is obtained. The product precipitates and can be recovered by filtration. The remainder is collected by diluting the filtrate with water and separating from the precipitate. The solid is collected and dried to give 3-chloro-5-bromo-2-hydroxybenzaldehyde. 3-Iodo-5-chlorosalicylaldehyde can be obtained by reacting 5-chlorosalicylaldehyde and N-iodosuccinimide in DMF and subjecting the reaction mixture to ordinary work-up. 3-Iodo-5-bromosalicylaldehyde is synthesized by reacting 5-bromosalicylaldehyde, potassium iodide and chloroamine T in acetonitrile. After work-up and treatment with hexane, 3-iodo-5-chlorosalicylaldehyde is obtained.

Coumarins are easily synthesized from saltyl aldehyde using a modified Parkin reaction (for example, Vogel's Textbook of Practical Organic Chemistry, 5 ^th).
Ed., 1989, p. 1040). Halo-substituted coumarins were converted to 3-aminohydrocoumarins (JG Rico, Tett. Let., 1994, 35, 6599-6602), and the ring was easily opened in acidic alcohol to give 3-amino-3-amine. (3,5-Halo-2-hydroxy) phenylpropionate is formed.

[0058] 3-Amino-3- (3,5-halo-2-hydroxy) phenylpropanoic acid ester is reacted with Boc-N-gly-N-hydroxysuccinimide to form Bo
to give c-N-gly-3-amino-3- (3,5-halo-2-hydroxy) phenylpropanoate and further add N-gly-3-amino-3- (3,5-halo-
Converted to the HX salt of 2-hydroxy) phenylpropanoic acid ester and the BOC protecting group deprotected using HCl in ethanol to give N-gly-3-amino-3-
Converted to (3,5-halo-2-hydroxy) phenylpropanoate.

The amino acid compounds utilized in synthesizing the compounds of the present invention may be prepared according to the methods described herein, and in the United States Patent Application Ser. Synthesized according to the claimed method as described in needet 3076.

[0060]

[Table 4] Scheme III illustrates a method that is useful for synthesizing various compounds of the present invention. 3-Hydroxy-5-[(1,4,5,6-tetrahydro-5-hydroxy-2-pyrimidyl) amino] benzoic acid is activated and coupled using known methods. Thus, after dissolving in a suitable solvent such as DMA, an equivalent amount of NMM is added. Cool the reaction mixture to ice bath temperature and add IBCF. The gly-β-amino acid ester and NMM are added to the mixed anhydrous intermediate. After completion of the reaction, the product is purified by prep hplc and the ester is hydrolyzed to an acid with a base such as LiOH in a suitable solvent (dioxane / water or acetonitrile / water). Alternatively, a suitable acid such as TFA can be utilized. The product is
Separate by prep hplc or separate zwitterions at pH 5-7 and convert to desired salt by standard methods.

Example A

[0062]

Embedded image Synthesis process 1

[0063]

Embedded image In a 2 L round bottom flask equipped with a stirrer and a condenser, 3,5-dichlorosalicylaldehyde (200 g, 1.05 mol, 1 equivalent) and acetic anhydride (356 g,
3.498 mol) and triethylamine (95.0 g, 0.94 mol, 0.90 equiv). The reaction solution was heated at reflux overnight. 5 dark brown reaction solutions
Cool to 0 ° C. and add water (1 L) with stirring. After 1 hour, the reaction mixture was filtered and the filtrate was combined with EtOH (1 L). The mixture was heated at 45 ° C. for 1 hour, cooled to room temperature, filtered, and the solid was washed with EtOH (0.5 L) (fraction A). The combined EtOH solution was concentrated by a rotary evaporator to obtain an oil (fraction B). The solid from fraction A was washed with methylene chloride (1.5 L
) And the resulting solution was passed through a pad of silica gel (1300 mL volume). The resulting dark brown solution was concentrated to an oil, triturated with hexane (1.3 L) to give a solid, separated by filtration, and washed (hexane) to give substantially pure 6,8-dichlorocoumarin. (163 g). An additional 31 g of product was obtained by kneading the oil of fraction B in a similar manner. Oil dissolved in methylene chloride (0.5 L) was passed through a silica gel pad (0.5 L volume) and kneaded with hexane. The total separation yield was 194 g, a brown solid, and the yield was 85%.

MS and NMR were consistent with the desired structure. Step 2

[0065]

Embedded image Synthesis of 6,8-chlorocoumarin (16 liters) in a three-neck 2 L round bottom flask equipped with a stirrer
0 g, 0.74 mol) (synthesized in step 1) and dry THF (375 mL, Aldrich)
Sure Seal) was added. The resulting mixture was cooled to -40 ° C (dry ice / acetone bath) and lithium bis (trimethylsilyl) amide (0.80 mol, 800 mL of 1M in THF was maintained while maintaining the temperature below -40 ° C). ) Was added. After the addition was completed, the cooling bath was removed. After 0.5 hour, the mixture was warmed to -5 ° C. The reaction was stopped by adding a station for HCl in EtOH (1.25 L). The temperature was maintained below 0 ° C. overnight. Concentrate the reaction mixture to approximately half of the original volume and add EtOAc (
(3 L) and water (2 L). The organic layer was washed with aqueous HCl (3 × 1 L, 0.1 mL).
5N HCl). The pH of the combined aqueous layers was adjusted to about 7 by adding 10% aqueous sodium hydroxide and extracted with methylene chloride (3 × 2 L).
. The combined organic layers were dried (MgSO ₄ ), filtered, and 4M HCl in dioxane (210 mL) was added with stirring. After the precipitation was completed, the solid was removed by filtration. The filtrate was concentrated to a small volume and methyl t-butyl ether was added. The resulting solid was combined with the first formed solid, and the combined products were washed with methyl tert-butyl ether, separated by filtration and dried (over a week or more under vacuum) to give the desired product. (172 g, 74% yield).

MS and NMR were consistent with the desired structure. Step 3

[0067]

Embedded image In a flame-dried round-bottomed flask (0.5 L) equipped with a stirrer, Nt-Boc-glycine N-hydroxysuccinimide ester (Sigma, 15 2.0 g, 0.055 mol) and dry DMF (Aldrich Sure S
eal, 200 mL) and the product of step 2 (21.67 g, 0.055 mol). The reaction mixture was cooled to about 0 ° C. (salt-ice bath) and N-methylmorpholine (5
. (58 g, 0.056 mol) and a catalytic amount of DMAP were added, and the reaction solution was reacted overnight. The reaction mixture was concentrated to slush and partitioned between EtOAc (0.4 L) and aqueous base (2 × 0.2 L, aqueous saturated NaHCO 3). The organic layer was washed successively with aqueous citric acid (2 × 0.2 L, 10% w / v), then with aqueous sodium bicarbonate (2 × 0.2 L), brine and dried (Na ₂ SO 4). ₄ ). Volatile substances are distilled off at 55 ° C. under reduced pressure to give an oil,
Allowed to solidify (22.5 g, 92% yield).

MS and NMR were consistent with the desired structure. Step 4

[0069]

Embedded image According to the following procedure, the product obtained in Step 3 was deprotected to obtain an amine hydrochloride.
Dry dioxane (40 mL) was added to the product obtained in step 3 of a flame-dried round bottom flask (0.1 L) equipped with a stir bar. This was added to 4.0 in dioxane.
The reaction was terminated by adding N HCl (2 eq, 6.32 mL) at 0 ° C. and continuing the reaction until gas evolution ceased. Volatiles were removed in vacuo and the residue was triturated with diethyl ether (50 mL). The solid was collected by filtration, washed with ether and dried to give the desired product (12.5g).

Example B

[0071]

Embedded image Synthesis process 1

[0072]

Embedded image Synthesis of 3-bromo-5-chlorosalicylaldehyde (175.0 g, 7 in acetic anhydride)
43.2 mmol) in a suspension of triethylamine (103.6 mL, 743.
2 mmol) was added. The reaction solution was heated to reflux for 4.5 hours. The solution was cooled and concentrated in vacuo. Absolute ethanol (730 mL) was added to the brown residue. The mixture was stored at 0 ° C. for 14 hours. The brown solid was collected by filtration and washed with cold ethanol. The solid was dried under vacuum to give the desired product (123.
0 g, yield 64%). ¹ H-NMR was consistent with the desired structure. Step 2

[0073]

Embedded image A suspension of coumarin (40.0 g, 154.1 mmol) in THF (400 mL) was added with stirring at −76 ° C. with lithium bis (trimethylsilyl) amide (
154.1 mL of a 1 M solution in THF) was added dropwise. The addition was completed in 10 minutes. Thereafter, the reaction mixture was allowed to stir for 5 minutes, warmed to -20 ° C and stirred for 15 minutes. Acetic acid (9.25 g, 154.1 mmol) in THF (28 mL) was added to the solution over 5 minutes. The mixture was warmed to room temperature and volatiles were evaporated under vacuum. The residue was dissolved in ether (850 mL) and the aqueous saturated
Washed with O ₃ (2 × 100 mL), brine (2 × 40 mL) and dried over MgSO 4. The ether solution was concentrated to about 160 mL and cooled to 0 ° C. To the above suspension was added 4M HCl in dioxane (56.3 mL, 255 mmol) and the mixture was allowed to stir at 0 ° C. for 30 minutes. The suspension was filtered and the filter cake was washed with ether. The solid was dried under vacuum to give the desired salt as a HCl salt in dioxane (45.0 g). ¹ H-NMR was consistent with the desired structure. Step 3

[0074]

Embedded image To a suspension of lactone (142.2 g, 354.5 mmol) in absolute ethanol (533 mL) was added 4 mL of dioxane (157.8 mL, 631.1 mmol).
M HCl was added over 10 minutes. The reaction mixture was allowed to stir at room temperature for 2.5 hours. Volatiles were distilled off under vacuum. The residue was dissolved in ethyl acetate (450 mL) and the solution was kept at 0 ° C. for 15 hours. The tan precipitate was collected by filtration and washed with cold ethyl acetate. The solid was dried under vacuum to give the desired product as hydrochloride (100.4 g, 79% yield). ¹ H-NMR was consistent with the desired structure. Step 4

[0075]

Embedded image In a flame-dried round-bottomed flask (0.1 L) equipped with a stir bar, an inert atmosphere (
Under Ar), Nt-Boc-glycine N-hydroxysuccinimide ester (Sigma, 2.72 g, 0.010 mol) and dry THF (Aldrich Sure Sea
, 50 mL) and the product of step 3 (3.10 g, 0.01 mol dried in vacuo over P ₂ O ₅ overnight). The reaction mixture was cooled to about 0 ° C. (salt-ice bath) and triethylamine (1.01 g, 0.010 mol) was added. The reaction was continued overnight. The reaction mixture was concentrated to a semi-solid and worked up in the same manner as in Step 3 of Example A. The volatiles were distilled off from the organic layer under vacuum at 55 ° C. to give an oil which solidified on standing (4 g, 83% yield).

MS and NMR were consistent with the desired structure. Step 5

[0077]

Embedded image According to the following procedure, the product obtained in Step 4 was deprotected to obtain an amine hydrochloride.
Dry dioxane (20 mL) was added to the product of Step 4 (4.0 g, 0.0084 mol) in a flame-dried round bottom flask (0.1 L) equipped with a stir bar. To this was added 4.0 N HCl in dioxane (20 mL), the reaction continued until gas evolution ceased, and the reaction was terminated (about 1 hour). Volatiles were removed in vacuo and the residue was triturated with diethyl ether (50 mL). The solid was collected by filtration, washed with ether and dried to give a light brown solid (2.7 g, 78% yield).

MS and NMR were consistent with the desired structure.

Example C

[0080]

Embedded image Synthesis process 1

[0081]

Embedded image Synthesis of To a suspension of 3,5-dibromosalicylaldehyde (100 g, 357 mmol) in acetic anhydride was added triethylamine (45 mL, 375 mmol).
The reaction solution was heated to reflux overnight under argon. The solution was cooled to room temperature and a solid formed. The dark brown reaction mixture was washed with hot hexane (3 × 300 mL) and water-soluble saturated sodium bicarbonate. The resulting solid was dissolved with EtOAc (2L) and washed with water. The organic layer was dried (sodium sulfate) and concentrated to give a brown solid, which was collected by filtration. The solid is dried under vacuum to give substantially pure 6,8
-Dibromocoumarin (94.2 g, 87% yield) was obtained.

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

[0083]

Embedded image At −78 ° C., 6,8-dibromocoumarin (20.0 g, 0.066 mol) (synthesized in step 1) in THF (100 mL) was added with stirring to lithium bis (trimethylsilyl) amide (1M in THF). (66 mL of solution) was added dropwise.
The addition was completed in 10 minutes. Thereafter, the reaction mixture was stirred for 5 minutes, warmed to 0 ° C,
Stir for 15 minutes. Acetic acid (3.95 g) was added to the solution over 1 minute. The mixture was warmed to room temperature and volatiles were removed in vacuo. The residue was treated with hexane (5
00 mL), washed with saturated aqueous NaHCO ₃ (2 × 100 mL) and dried (Na ₂ SO ₄ ). Concentration of the organic solution gave an oil that was immediately dissolved in diethyl ether (400 mL) and 4M HCl in dioxane (30 mL)
) Was added over 30 minutes with stirring at 0 ° C. Excess HCl was distilled off under vacuum, the suspension was filtered and the filter cake was washed with ether. The solid was dried under vacuum to give the desired product as HCl salt, dioxane lysate (19.
9g).

MS and ¹ H NMR were consistent with the desired structure. Step 3

[0085]

Embedded image The lactone (15 g) synthesized in the above step was dissolved in anhydrous ethanol (400 mL), and anhydrous HCl was passed through the solution for 1 minute. The reaction mixture was allowed to stir at room temperature for 2.5 hours. The completion of the reaction was confirmed by RPHPLC. Volatiles were removed under vacuum to give a dark residue. The residue was diethyl ether (500 mL)
The mixture was allowed to stir overnight. The tan precipitate was collected by filtration and washed with diethyl ether. The solid was dried under vacuum to give the desired product as the hydrochloride salt (15.2 g). MS and ¹ H-NMR were consistent with the desired structure. Step 4

[0086]

Embedded image In a flame-dried round bottom flask (0.2 L) equipped with a stir bar, under an inert atmosphere (Ar), Nt-Boc-glycine N-hydroxysuccinimide ester (Sigma, 8.1 g, 0.030 mol), dry DMF (Aldrich Sure Seal, 50 mL) and the product of step 3 (12 g, 0.03 mol, dried in vacuo over P ₂ O ₅ overnight) were added. The reaction mixture was cooled to about 0 ° C. (salt-ice bath),
Methylmorpholine (3.03 g, 0.030 mol) and the catalyst DMAP were added.
The reaction was continued overnight and warmed to room temperature. The reaction mixture was concentrated to a semi-solid and worked up in the same manner as in Step 3 of Example A. The volatiles were distilled off from the organic layer under vacuum at 55 ° C. to give an oil which was allowed to solidify (15.7 g, 93% yield).
.

MS and NMR were consistent with the desired structure. Step 5

[0088]

Embedded image The product obtained in Step 4 was deprotected to obtain an amine hydrochloride by the following method. The product of Step 4 (1 L) was placed in a flame-dried round bottom flask (0.1 L) equipped with a stir bar.
(3.0 g, 0.00084 mol) was added dry dioxane (40 mL).
To this solution was added 4.0N HCl in dioxane (30 ml) and the reaction continued until gas evolution ceased, ending the reaction (about 1 hour). Volatiles were removed in vacuo and the residue was triturated with diethyl ether (50 mL). The solid was collected by filtration, washed with ether and dried to give a solid (10.6 g, 93% yield).

MS and NMR were consistent with the desired structure.

Example D

[0091]

Embedded image Synthesis process 1

[0092]

Embedded image Synthesis of 3-chloro-5-bromosalicylaldehyde in a 5-L round bottom flask equipped with a stirrer and a gas addition tube, 5-bromosalicylaldehyde (495 g, 2.46 mol) and acetic acid were added at room temperature, A slurry formed. Chlorine gas was added to the mixture at a moderate rate until a slight molar excess of chlorine was dissolved. After stopping the addition, the reaction was allowed to continue overnight. The resulting solid was collected by filtration, and the filtrate was diluted with water (2.5 L). Mix 20
After vigorous stirring for minutes, the product was collected by filtration and washed with water. The combined solids were dried under vacuum to give 3-chloro-5-bromosalicylaldehyde (475
g, 82% yield).

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

[0094]

Embedded image Synthesis of 6-bromo-8-chlorocoumarin In a 5 L round bottom flask equipped with a stirrer and a condenser, 3-chloro-5-bromosalicylaldehyde (554.1 g, 2.35 mol, 1 equivalent) and acetic anhydride (
1203 g, 11.8 mol, 5 equivalents) and triethylamine (237.4 g, 2
. 35 mol, 1 equivalent). The reaction solution was heated to reflux overnight (1
31-141 ° C). The dark brown reaction mixture was cooled to 50 ° C. (ice bath cooling) and ice (2
L) was added with stirring. After 1 hour, the mixture was filtered and the filtrate was treated with EtOH (
1l). To this mixture was added EtOH (300 mL) and the reaction mixture was stirred for 1 hour. The resulting precipitate was collected by filtration, and water: EtOH (3 × 1.
3L), dried under vacuum, and dried with a fluidized bed drier. The overall separation yield was 563 g, a 92% yield.

MS and ¹ H-NMR were consistent with the desired structure. Step 3

[0096]

Embedded image Synthesis of 3-Amino-3- (2-hydroxy-3-chloro-5-bromo) phenylpropanoic acid ethyl ester 6-bromo-8-chlorocoumarin in a three-necked 5 L round bottom flask equipped with a stirrer (300 g, 1.16 mol) (synthesized in step 2) and dry THF (900 mL,
Aldrich Sure Seal). The resulting mixture was cooled to -45 ° C or less (dry ice / acetone bath) and maintained at a temperature of -45 ° C or less in 0.5 hours with lithium (trimethylsilyl) amide (0.80 mol, 1M in THF). 800m
L and 0.6 L in hexane, 1.2 eq.). In a separate 5L flask, Et
OH (2.5 L) and HCl (4 N HCl in dioxane, 1 L) were combined at -15 ° C. The coumarin reaction was stopped by the addition of a cold HCl / EtOH solution. 0. After 5 hours, the temperature of the resulting reaction mixture was -8.3 ° C. The reaction mixture was maintained at 0 ° C. overnight, concentrated to about 2.5 L, and partitioned between EtOAc (3 L) and water (4 L). The organic layer was washed with aqueous HCl (4 × 1.2 L, 0.5N HCl). The pH of the combined aqueous layers was adjusted to about 8 by the addition of 10% aqueous NaOH and extracted with methylene chloride (1 × 7 L and 3 × 2 L). The combined organic layers were dried (MgSO ₄ , 900 g), filtered and stirred with dioxane (400 m
4M HCl in L) was added. After the precipitation was completed, the solid was distilled off by filtration. The mixture was concentrated to 2.5 L, hexane (2.5 L) was added, and the precipitate was separated by filtration. The filter cake was washed with methylene chloride / hexane (1: 2), suction dried and vacuum dried at 40 ° C. to give the desired product (25
1 g, 60% yield).

MS and ¹ H-NMR were consistent with the desired structure. Step 5

[0098]

Embedded image The above compound was synthesized in essentially the same manner as in Step 4 of Example B, utilizing the relative amounts to identify the isomer of Step 4 of Example B.

MS and ¹ H-NMR were consistent with the desired structure. Step 6

[0100]

Embedded image This compound was synthesized in essentially the same manner as in Step B of Example B, utilizing the relative amounts to identify the isomer of Step 5 of Example B.

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

Example E

[0103]

Embedded image Synthesis process 1

[0104]

Embedded image Synthesis of 3-iodo-5-chlorosalicylaldehyde N-iodosuccinimide (144.0 g, 0.641 mol) was prepared by adding 5-chlorosalicylaldehyde (100 g,
0.638 mol). The reaction mixture was allowed to stir at room temperature for 2 days. Additional N-iodosuccinimide (20.0 g) was added and stirring continued for another 2 days. Dilute the reaction mixture with ethyl acetate (1 L) and add hydrochloric acid (300 mL, 0.1 mL).
N), water (300 mL), sodium thiosulfate (5%, 300 mL), washed with brine (300 mL), dried (MgSO ₄ ), concentrated to dryness and the desired aldehyde as a pale yellow solid ( 162 g, yield 90%).

MS and NMR were consistent with the desired structure. Step 2

[0106]

Embedded image Synthesis of 6-chloro-8-iodocoumarin A mixture of 3-iodo-5-chlorosalicylaldehyde (100 g, 0.354 mol), acetic anhydride (300 mL) and triethylamine (54 mL) was heated to reflux for 18 hours. I let it. After cooling, the desired coumarin precipitated as dark brown crystalline material. This was filtered, washed with hexane / ethyl acetate (4: 1, 200 mL) and air dried. Yield: 60 g (55%).

MS and ¹ H-NMR were consistent with the desired structure. Step 3

[0108]

Embedded image Synthesis of (R, S) -4-amino-3,4-dihydro-6-chloro-8-iodocoumarin hydrochloride Lithium hexamethyldisilazane (21.62 mL, 1 M, 21.62 mmol) was added at -78 ° C. Was added to a solution of phosphorous 6-chloro-8-iodine (6.63 g, 21.62 mmol) in tetrahydrofuran (100 mL). The reaction solution was stirred at the above temperature for 30 minutes and then at 0 ° C. for 1 hour. Acetic acid (1.3 g,
21.62 mmol) was added to the reaction solution. The reaction mixture was treated with ethyl acetate (300
mL) and saturated sodium carbonate (200 mL) solution. The organic layer was separated, washed with brine (200 mL), dried (MgSO _4), and concentrated to give a residue. The residue was added to anhydrous ether (200 mL), followed by dioxane / HCl (4N, 30 mL) at 0 ° C. The reaction mixture was stirred at room temperature for 1 hour, filtered and dried under vacuum to give the desired product as a powder (4.6 g, yield 5).
9%). (RPHPLC: Rf 6.8 min; gradient, 1 over 15 min)
From 0% acetonitrile to 90% acetonitrile, then 100 minutes over 6 minutes
% Acetonitrile. Both water and acetonitrile involve 0.1% TFA VydacC 18 protein peptide, monitoring at 2 ml / min flow rate, 254 nm).

MS and ¹ H-NMR were consistent with the desired structure. Step 4

[0110]

Embedded image Synthesis of hydrochloride salt of (R, S) -ethyl 3-amino-3- (5-chloro-2-hydroxy-3-iodo) phenylpropionate Hydrochloric acid gas was added to 4-amino-ethanol in ethanol (250 mL). To a solution of 3,4-dihydro-6-chloro-8-iodocoumarin hydrochloride flame, add the reaction mixture to 0-10
While maintaining the temperature of ° C., it was blown in until it became saturated. After 6 hours at reflux, most of the solvent was distilled off. The cooling residue was added to anhydrous ether and allowed to stir for 2 hours.
Early rubber turned into crystalline material. The crystalline product was filtered and dried to give the desired product as off-white crystalline powder (20 g, 81% yield). (Rf: 7.52 minutes under the condition of step 3).

MS and ¹ H-NMR were consistent with the desired structure. Step 5

[0112]

Embedded image (R, S) -ethyl 3- (N-BOC-gly) -amino-3- (5-chloro-
Synthesis of 2-hydroxy-3-iodo) phenylpropionate BOC-gly (2.16 g, 12.31 mmol) and HOBT (1.67 g,
12.31 yield), EDCl (2.36 g, 12.31 mmol) and DMF (
(50 mL) was allowed to stir at 0 ° C. for 1 hour. Hydrochloride of ethyl 3-amino-3- (5-chloro-2-hydroxy-3-iodo) propionate (5.0 g, 12
. 31 mmol) was added to the reaction mixture, followed by triethylamine (3.5 mL).
) Was added. The reaction mixture was stirred at room temperature for 18 hours. DMF was distilled off under vacuum and the residue was partitioned between ethyl acetate (300 mL) and sodium bicarbonate (200 mL). The organic layer was washed with hydrochloric acid (1N, 100 mL), brine (200 mL), dried (MgSO ₄ ) and concentrated to give the desired product as a solid (6 g)
, Yield 93%).

MS and ¹ H-NMR were consistent with the desired structure. Step 6

[0114]

Embedded image Synthesis of hydrochloride salt of (R, S) -ethyl 3- (N-gly) -amino-3- (5-chloro-2-hydroxy-3-iodo) phenylpropionate dioxane / HCl (4N, 20 mL) Add to ethyl 3- (N-BOC-gly) -amino-3- (5-chloro-2-hydroxy-3-iodo) propionate (6.0 g, 11.39 mmol) at 0 ° C. and add Allowed to stir for hours. The reaction mixture was concentrated and concentrated once more in toluene addition. The residue obtained was suspended in ether, filtered and dried to give the desired product as a crystalline powder (
5.0 g, 95% yield). (RPHPLC: Rf is 8.3 minutes under the conditions of step 3).

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

Example F

[0117]

Embedded imageSynthesis ofStep 1  Synthesis of 3-iodo-5-bromosalicylaldehyde Acetonitrile (150 mL) and 500 mL round bottom flask equipped with a stirrer
5-bromosalicylaldehyde (20.0 g, 0.1 mol) in water and water (50 mL)
) And potassium iodide (17 g, 0.1 mol) were added to a solution of chloroamine T (23
g, 0.1 mol). The mixture was reacted for 1 hour. The reaction mixture was treated with hydrochloric acid (
(10%, 200 mL) and ethyl acetate. Dry the organic layer (Na ₂ SO₄), Filtered and concentrated in vacuo. Add hexane to the residue and mix
The liquid was heated at 50 ° C. for 15 minutes. Undissolved material was removed by filtration. The filtrate
Concentrate under vacuum to give a canary yellow 3-iodo-5-bromosalicylaldehyde
(26 g) remained.

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

[0119]

Embedded image The above compound was synthesized using the same method as Steps 2 to 6 of Example E,
Used an equivalent of 3-iodo-5-bromo-salicylaldehyde of the product of step 1 instead of 3-iodo-5-chlorosalicylaldehyde.

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

Example H

[0122]

Embedded imageSynthesis ofStep 1  Mix ethanol (375 mL) and deionized water (375 mL) with a stirrer,
2L 3-port round bottom fan equipped with adapter, dropping funnel, reflux condenser and thermocouple
Added to Lasco. 1,3-diamino-2-hydroxypropane (125.04
g, 1.39 mol) was added to the reaction flask and stirred to dissolve. Carbon disulfide
(84 mL, 1.39 mol) at 25-33 ° C. via a dropping funnel for 35 minutes.
The mixture was added drop by drop to obtain a milky white mixture. Keep the temperature in an ice bath.
I held it. The reaction mixture was refluxed at 73.4 ° C. for 2 hours to obtain a yellow solution. reaction
The mixture was cooled to 25 ° C. in an ice bath and the concentrated HC
1 (84 mL) was added dropwise. The reaction mixture was refluxed at 78.4 ° C. for 21 hours.
I let you. The reaction solution was cooled to 2 ° C. and the product was collected by vacuum filtration. Ice white solid
Wash with ethanol-water (1: 1) (50 mL) three times in a bath, and wash at 40 ° C.
Dry under air to remove 5-hydroxytetrahydroxypyrimidine-2-thione
Obtained as a color solid (63.75 g, 34.7% yield).

MS and NMR were consistent with the desired structure.Step 2  The 5-hydroxytetrahydropyrimidine-2-thione synthesized in Step 1 (95
g, 0.72 mol), anhydrous ethanol (570 mL), and methyl iodide.
And a 2 L round bottom flask equipped with a thermocouple. Reaction mixture at 78 ° C for 5 hours
Reflux and cool to room temperature. The reaction mixture was concentrated in vacuo to a white solid (194.
72 g) were obtained. Knead the white solid three times with ethyl ether (500 mL),
Dry under vacuum to give 2-methylthioether-5-hydroxypyrimidine iodide
The hydrochloride was obtained as a white solid (188.22 g, 95.4% yield).

MS and¹1 H-NMR was consistent with the desired structure.Step 3  2-methylthioether-5-hydroxypyrimidine hydroiodide (150
. 81 g, 0.55 mol), methylene chloride (530 mL), dimethylacetate
Amide (53 mL) and triethyl "amine (76.7 mL, 0.55 mol)
, A reflux condenser, and a stirrer.
Added to Lasco. The mixture was cooled in an ice bath and di-t-butyl dicarbonate (12
(0.12 g, 0.55 mol) at 4 ° C. The reaction mixture is heated at 42.5 ° C for 18
Reflux for hours to give a pale yellow solution. Transfer the reaction solution to a 2 L separation funnel,
Wash three times with DI water (200 mL),₄Dried, filtered and under vacuum
Concentrate to give Boc-2-methylthioether-5-hydroxypyrimidine light yellow
(134.6 g, yield 99.35%).

MS and¹1 H-NMR was consistent with the desired structure.Step 4  Boc-2-methylthioether-5-hydroxypyrimidine (50.3 g,
0.204 mol) and 3-amino-5-hydroxybenzoic acid (Aust. J. Chem.
1981) 34 (6), 1319-24) (25.0 g, 0.1625 mol) and 50 mL of anhydrous DMA were heated at 100 ° C. for 2 days with stirring. Slurry precipitate formed
. The reaction was cooled to room temperature, the precipitate was filtered and CH₃CN and then ethyl ether
And dried. This solid is H₂Slurry in O and acidified with concentrated HCl
To form a solution. This can be frozen and lyophilized to give the desired
(14.4 g).

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

Example I

[0128]

Embedded image Synthesis process 1

[0129]

Embedded image Synthesis of Reformazki Reagent A metal zinc (180.0%) was placed in a 4 L flask equipped with a condenser, a thermometer, and a stirrer.
g, 2.76 mol, 30-100 mesh) and THF (1.25 L). While stirring, 1,2-dibromomethane (4.74 mL, 0.05 mol)
Was added via syringe (alternatively, TMS Cl (0.1
Equivalent) can be substituted). Pass inert gas to a suspension of zinc in THF.
And heated to reflux (65 ° C.) and maintained at that temperature for 1 hour. 1.5 o'clock
Over a period of time, add 50 mL of the mixture before charging t-butyl bromoacetate via a 50 mL syringe and syringe pump (transport rate set to 4.1 mL / min).
Cooled to ° C. During the addition, the reaction temperature was maintained at 50 ° C. ± 5 ° C. After the addition is complete, react
The mixture was stirred at 50 ° C. for 1 hour. Thereafter, the mixture is cooled to 25 ° C.
Settled. Decant THF mother liquor into 2L round bottom flask using coarse filter
And a partial vacuum was applied (20 mmHg). About 65% of THF is distilled off from the mixture
Was. Add 1-methyl-2-pyrrolidone (NMP, 800 mL) and stir for 5 minutes
Went again for a while. The reaction mixture was filtered to remove the remaining zinc. From analysis,
It was found to be 1.57 M of the desired Lihomatsu key reagent at a rate of 94%. There
The solid reagent was separated from the original reaction mixture by filtration. White solid bud
The cake is washed with THF until₂Dry under the mono THF solution
The desired product was obtained and stored at 20 ° C. (dried). Typical
Recoveries are between 85 and 90%.Step 2  2A

[0130]

Embedded image Synthesis of potassium carbonate (powder dried at 100 ° C. under vacuum, 8.82 g, 60 mmol) was obtained by adding 3,5-dichlorosalicylaldehyde (40 mL) in room temperature DMF (40 mL).
11.46 g, 60 mmol) to give a bright yellow slurry.
Thereafter, while maintaining the bath temperature at 20 ° C., MEMCl (neat, 7.46 g, 6
1 mmol) was added. The mixture was further stirred at 22 ° C. for 6 hours and MEMCl (
(0.3 g, 2.4 mmol). The mixture was stirred for another 0.5 h, the reaction mixture was poured into cold water (200 mL) to precipitate the product. The slurry was filtered on a pressure filter and the cake was washed with water (2 × 50 mL) and dried under N ₂ / vacuum to give the product as an off-white solid. ¹ H-NMR (CDCl ₃ , TMS
) 3.37 (s, 3H), 3.54 to 3.56 (m, 2H), 3.91 to 3,
93 (m, 2H), 5.30 (s, 2H), 7.63 (d, 1H), 7.73 (
d, 1H), 10.30 (s, 1H); ¹³ C-NMR (CDCl ₃ , TMS)
d (ppm): 59.03, 70.11, 99.57, 126.60, 129.
57, 130.81, 132.07, 135.36, 154.66, 188.3
0. DSC: 48.24 ° C (endothermic 90.51 J / g). Micro elemental analysis C ₁₁ H ₁₂ Cl ₂ O ₄ Calculated: C: 47.33%; H4.
33%; Cl: 25.40% Result: C: 47.15%; H 4.26%; Cl: 25.16% 2B.

[0131]

Embedded imageThe product from step 2A (35.0 g, 0.125 mol) was added to the stirrer
Into a 1-L three-necked round-bottomed flask, and then add THF (200 mL).
Added. The solution was allowed to stir at 22 ° C. and then (S) -phenylglycinol (1
(7.20 g, 0.125 mol) was added in one portion. After 30 minutes at 22 ° C., MgSO ₄ (20 g) was added. Stir the mixture for 1 hour at 22 ° C and filter with a coarse filter
Filtered. The filtrate was concentrated under reduced pressure. Crude imine without further purification
Was used directly in the coupling reaction of step 2C. 2C.

[0132]

Embedded image Synthesis of

In a 1 L 3-neck round bottom flask equipped with a stirrer and a dropping funnel, the solid product of Step 1 (91.3 g, 0.275 mol) and NMP (200 mL) were charged under a nitrogen atmosphere. . The solution was cooled to -10 ° and stirred at 350 rpm. A solution of imine (synthesized in step 2B) in NMP was prepared under a nitrogen atmosphere and added to the above reaction mixture over 20 minutes while maintaining the temperature at -5 ° C (jacket temperature -10 ° C). Was. After completion of the addition, the mixture was further stirred at -8 ° C for 1.5 hours, and further stirred at -5 ° C for 1 hour. After cooling to −10 ° C., a saturated solution of concentrated HCl / NH ₄ Cl (8.1 mL / 200 mL) was added in 10 minutes. MTBE (200 mL) was added and the mixture was stirred at 23 ° C. at 200 rpm for 15 minutes. The stirring was stopped and the layers were separated. The aqueous layer was extracted with MTBE (100 mL). Put together two organic layers,
Sequentially washed with a saturated solution of NH ₄ Cl (100 mL), water (100 mL) and brine (100 mL). The solution was dried over MgSO ₄ (30 g), filtered,
Concentration gave an orange oil containing the desired product as a single diastereomer (confirmed by NMR of H and C) which solidified on standing (66)
. 3g). The sample was recrystallized from heptane for elemental analysis to give the product as an off-white solid.

The 1 H and C NMR and IR spectra were consistent with the desired structure. [Α] ^D ₂₅ =
+ 8.7 ° (C = 1.057, MeOH).

Microelemental analysis of C ₂₅ H ₃₃ Cl ₂ NO ₆ Calculated: C 58.77%; H: 6.47%; N: 2.72%; Cl: 13.78
% Result: C 58.77%; H: 6.47%; N: 2.72%; Cl: 13.78
% Process 3

[0136]

Embedded image Synthesis of 3A.

A solution of the crude ester synthesized in step 2 (17.40 g, 0.033 mol (theoretical) and EtOH (250 mL) was charged to a 1 L 3-neck jacketed reactor. And mPb (OAc) ₄ (14.63 g, 0.033 mol) was added in one portion.After 2 hours, a 15% NaOH solution (30 mL) was added and the ethanol was distilled off under reduced pressure. Another 15% NaOH (100 mL) was added and the mixture was extracted with MTBE (2 × 100 mL) and H ₂ O ( ₂ × 100 mL)
) And brine (50 mL), dried over _Na 2 SO _4, filtered through Celite like to give an orange oil and concentrated under reduced pressure (12.46 g). The oil was uniform by thin layer chromatography (tic) and was not further purified. The oil from 3B 3A was diluted with EtOH (30 mL) and para-toluenesulfonic acid (1.3 eq, 0.043 mol, 8.18 g) was added. Heat the solution,
Reflux for 8 hours, cool to room temperature, and concentrate under reduced pressure. The residue was washed with THF (20 mL
) And heated to reflux to form a solution. The solution was cooled to room temperature and the compound crystallized. Heptane (30 mL) and THF (10 mL) were added to form a liquid slurry and filtered. The cake was washed with THF / heptane (40 mL, 1 /
1), dried under vacuum for 2 hours, and pressure filtered under nitrogen to give a white solid (
7.40 g).

1 H and C NMR and IR spectra were consistent with the desired product, substantially as a single enantiomer. Calculated micro elemental analysis _{C 18 H 21 C l2 NO 6} S C: 48.73%; H: 4.95%; N: 2.99%; Cl: 15.14% Result C: 48.91%; H: 4.95%; N: 2.90%; Cl: 14.95%. Step 4

[0139]

Embedded image In a 500 mL round bottom flask equipped with a stirrer and a nitrogen inlet tube, a free base of the product synthesized in Step 3 and Nt-Boc-glycine N-hydroxysuccinimide ester (17.7 g, 0.065 mol) and DMF (200 mL). The reaction mixture was at room temperature under nitrogen. Stirring for 3.25 hours resulted in a light orange solution. The reaction mixture was poured into ice-cooled ethyl acetate (1.2 L). The organic solution was washed with 1M HCl (250 mL), then brine (500 mL), dried (MgSO ₄ ), concentrated in vacuo and dried to neat to give an oil, which was then dried at 50 ° C. to colorless (28.12 g, 99)
%). Seed crystals were prepared from ethyl acetate / hexane. The product (about 28 g) was dissolved in ethyl acetate (35 mL) and hexane (125 mL). The solution was seeded with seed crystals to form a precipitate. The solid was filtered and dried under vacuum at 55 ° C. overnight to give a colorless solid (27.0 g, 95%).

MS and 1H-NMR were consistent with the desired structure. Step 5

[0141]

Embedded image Synthesis of

The Boc-protected glycinamide synthesized in step 4 was dried with a P ₂ O ₅ and NaOH palette overnight. The solid was dissolved in dioxane (40 mL) and the solution was cooled to 0 ° C. An equivalent volume of 4N HCl / dioxane (0.062 mol) was added and the reaction was run for 2 hours. The reaction mixture was allowed to warm to room temperature over 4 hours. The reaction mixture was concentrated at 40 ° C. to form a foam, which was triturated with ether (200 mL). The resulting white solid was filtered and dried over P ₂ O ₅ to give the desired glycine β-amino acid ethyl ester compound as hydrochloride (20.4 g, 88.5% separation yield).

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

Example J

[0145]

Embedded image Synthesis of Step 1

[0146]

Embedded image Synthesis of

MEM protected 3-bromo-5-chlorosalicylaldehyde (129.42 g, 0.4 mol) was synthesized according to the method of Step 2A of Example I. An equivalent of 3-bromo-5-chlorosalicylaldehyde was used in place of 3,5-dichlorosalicylaldehyde and charged to a 2 L 3-neck round bottom flask equipped with a stirrer, followed by THF (640 m
l) and (S) -phenylglycinol (54.86 g, 0.4 mol) were added. After 30 minutes at 22 ° C., MgSO ₄ (80 g) was added. The mixture was allowed to stir at 22 ° C. for 2 hours and filtered through a coarse filter. The filtrate was concentrated under reduced pressure to give a pale yellow oil containing the desired imine (180.0 g). The crude product was used directly for the Step 2 coupling without further purification. Micro Elemental Analysis Calculated for C ₁₉ H ₂₁ BrClNO ₄ C: 51.45%; H: 4.78%; N: 3.16%; Br: 18.04%; Cl: 8.00% Result C: 50 H: 4.94%; N: 2.93%; Br: 17.15%; Cl: 7.56% Step 2

[0148]

Embedded image Synthesis of

In a 5 L 3-neck round bottom flask equipped with a stirrer, place the product of Step I of Example I (332.
(0 g, 0.8 mol) was dissolved in NMP (660 mL) under nitrogen. Thereafter, the solution was cooled to -10 ° C. A solution of the imine from step 1 in NMP (320 ml) was prepared under nitrogen and added to the above reaction mixture over 30 minutes while maintaining the temperature at -5 ° C. After the addition is completed, the mixture is cooled to -10 ° and the reaction mixture is cooled to -8
The mixture was further stirred for 1 hour at -5 ° C and further 2 hours at -5 ° C. A mixture of saturated HCl / MH ₄ Cl (30 mL / 720 mL) was added over 10 minutes. MTBE (760 mL) was added and the mixture was allowed to stir at 23 ° C. for 30 minutes. The stirring was stopped and the layers were separated. The aqueous layer was extracted with MTBE (320 ml). The organic layers were combined, and saturated aqueous NH ₄ Cl (320 ml), DI water (320 ml)
) And brine (320 ml). The solution was diluted with MgSO ₄ (60 g
), Filtered and concentrated to give a yellow oil containing the desired product as a single diastereomer by H-NMR (221.0 g). DSC: 211.80 ° C. (endothermic; 72.56 J / g), ”228.34 ° C. (98.23 J / g); microanalysis: calculated for C ₂₅ H ₃₃ BrClNO ₆ ; C: 53.72%; H : 5.95%; N: 2.50%; Br: 14.29%; C
l: 6.33% Result: C: 52.11%; H: 6.09%; N: 2.34%; Br: 12.84
%; Cl: 6.33%. Step 3

[0150]

Embedded image Synthesis of an ester solution (about 111 g) of the crude product synthesized in step 2 in ethanol (15
00m) was charged to a 3 L 3-neck round bottom flask equipped with a stirrer under an argon atmosphere. The reaction mixture was cooled to 0 ° C. and lead tetraacetate (88.67 g, 0.2 mol) was added at once. The reaction mixture was stirred at 0 ° C. for 3 hours, after which 15% aqueous N
aOH (150 mL) was added to the reaction mixture below 5 ° C. The methanol was distilled off under reduced pressure using Rotavapo. An additional 150 mL of 15% aqueous NaOH was added and the reaction mixture was extracted with ethyl acetate (3 × 300 mL) and DI water (2 × 100 mL).
L) and brine (2 × 100 mL) and dried over anhydrous MgSO ₄ (30 g). Filtration through celite and concentration under reduced pressure gave the desired product (103 g) as a red oil. Step 4

[0151]

Embedded image The above compound was prepared according to the procedure utilized in Steps 4 and 5 of Example I, Step 3 of Example I
An equivalent amount of product from was exchanged for synthesis. MS and ¹ H-NMR were consistent with the structure as desired.

[0152]Example K  Alternative Synthesis of the Compound of Example J Step 1

[0153]

Embedded image To the product of Step 3 of Example B (50, 0 g, 139.2 mmol) and NaHCO ₃ (33.5 g, 398.3 mmol) were added methylene chloride (500 mL) and water (3
35 mL) was added. The mixture was stirred at room temperature for 10 minutes. A solution of benzyl chloroformate (38.0 g, 222.8 mmol) in methylene chloride (380 mL) was added over 20 minutes with high speed stirring. After 50 minutes, the reaction mixture was poured into a separating funnel and the organic layer was collected. The water bath was washed with methylene chloride (170 mL). The combined organic layers were dried (MgSO ₄ ) and concentrated in vacuo. The resulting gummy solid was kneaded with hexane and collected by filtration. The tan solid was dried under vacuum to give the desired racemic product (61.2 g, 96% yield). This material was subjected to reverse phase HPLC using a chiral column to obtain each pure enantiomer. The column used was Whelk-O (R, R), a 10 micron particle size, 90:10 heptane: ethanol as mobile phase. The optical purity was found to be 98% or more using an analytical hplc with the same column and solution conditions. ¹ H-NMR was consistent with the desired structure. Step 2

[0154]

Embedded image To a solution of the compound obtained in step 1 (48.5 g, 106.2 mmol) in methylene chloride (450 mL) was added via a cannula trimethylsilyl iodide (25.5 g, 127.4 mmol) in methylene chloride (100 mL). Was added. The organic solution was allowed to stir at room temperature for 1 hour. Methanol (20.6 mL, 509.7 mmol) was added dropwise and the solution was allowed to stir for 15 minutes. The reaction solution is concentrated under vacuum,
An orange oil was obtained. The residue was methyl t-butyl ether (500 mL)
And 1N HCl (318 mL) and water (1 × 200 mL, 1 × 100 m
L). The water-soluble extraction layer was extracted again with MTBE (100 mL). To the aqueous layer was added solid NaHCO ₃ (40.1 g, 478 mmol) in small portions. The basified aqueous mixture was extracted with MTBE (1 × 1 L, 2 × 200 mL). The combined organic solution was washed with brine and concentrated under vacuum to give the desired product (23.3 g, 68% yield). ¹ H-NMR was consistent with the structure as desired. Step 3

[0155]

Embedded image Of the product obtained in step 2 (23.3 g, 72.1 mmol) in DMF (200
To the (mL) solution was added Nt-Boc-glycine N-hydroxysuccinimide ester (17.9 g, 65.9 mmol). The reaction mixture was allowed to stir at room temperature for 20 hours. The mixture was poured into ethyl acetate (1.2 L) and 1N HCl (2 × 250
NaHCO ₃ solution (2 × 250 mL) and brine (2 × 250 m
L). The solution was dried over MgSO ₄ and concentrated to give the desired product (32.
0 g, 100% yield). Calculated elemental analysis for C18H24BrClN2O6 C, 45.06; H, 5.04 ; N, 5.84 the result C, 45.17; H, 5.14; N, 6.12 1 H-NMR is desired structure And matched. Step 4

[0156]

Embedded image Synthesis of the product of Step 3 (31.9 g, 66.5 mmol) in anhydrous ethanol (205
solution of ethanolic HCl (111 mL of a 3M solution, 332.4).
Mmol). The reaction solution was heated at 58 ° C. for 30 minutes. Cool the solution,
Concentrated under vacuum. Dissolve the residue in ethyl acetate (250 mL) and add
Allowed to stir for hours. The white precipitate was collected by filtration and washed with cold ethyl acetate. The solid was dried under vacuum to give the desired product (23.5 g, 85% yield). Calculated for elemental analysis of C13H16BrClN2O4. C, 37.53; H, 4.12; N, 6.73. Results C, 37.29; H, 4.06; N, 6.68.

¹ H-NMR was consistent with the desired structure.

Example L

[0159]

Embedded image Synthesis process 1

[0160]

Embedded image Synthesis of potassium carbonate (of powder dried at 100 ° C. under vacuum at 22.1 g, 0.16 g)
Mol) at room temperature with 3-chloro-5-bromosalicylaldehyde (35.0 g, 0
. 15 mol) in DMF (175 ml) to give a light yellow slurry. While maintaining the bath temperature at 20 ° C., then add MEMCl (neat, 25.0 g).
, 0.2 mol). Next, the mixture was stirred at 22 ° C. for 6 hours, and DI water (
1200 mL) and the product precipitated. The slurry was filtered through a pressure filter and the cake was washed with DI water (2 × 400 mL) and dried under N 2 / vacuum to give the product as a white solid (46.0 g, 95% yield). 1H-NMR (CDC
_{l 3, TMS) 3.35 (s} , 3H), 3,54~3.56 (m, 2H) 3.
91-3.93 (m, 2H), 5.30 (s, 2H), 7.77 (d, 1H),
7.85 (d, 1H), 10.30 (s, 1H); 13C-NMR (CDCl ₃ , TMS) (ppm): 509.05, 70.11, 71.49, 99.50,
117.93, 129.69, 129.78, 132.37, 138.14, 1
55.12, 188.22. DSC: 48.24 ° C. (endotherm, 90.51 J / g); microelemental analysis: calculated value of C11H12BrClO4 C: 40.82%; H: 3.74%; Cl: 10.95%; Br: 24.69 %
Results C: 40.64%; H: 3.48%; Cl: 10.99%; Br: 24.
67%. Step 2

[0161]

Embedded imageSynthesis of the product of step 1 (32.35 g, 0.1 mol) in 500 ml with stirrer
Charge into a three-necked round bottom flask, THF (160 mL) and (S) -phenylglycino
(13.71 g, 0.1 mol) was added. After 30 minutes at 22 ° C., MgSO ₄ (20 g) was added. Stir the mixture at 22 ° C for 1 hour,
The mixture was filtered with a filter. The filtrate is concentrated under reduced pressure to give a light yellow solution containing the desired imine.
An yl was obtained (48.0 g). The crude product is directly transferred to the next reaction step without further purification
used.

Micro Elemental Analysis Calculated for C ₁₉ H ₂₁ BrClNO ₄ C: 51.54%; H: 4.78%; N: 3.16%; Br: 18.04%; C
1: 8.00% Result C: 51.52%; H: 5.02%; N: 2.82%; Br: 16.31
%; Cl: 7.61%. Step 3

[0163]

Embedded image In a 5 L 3-neck round bottom flask equipped with a stirrer, the reagent of Step 1 of Example I (332 g, 0.8 mol) was dissolved in NMP (660 mL) under nitrogen. The solution was cooled to -10 °. NMP of the imine compound obtained in step 2 (320 ml of a solution was prepared in a nitrogen atmosphere and added to the above reaction mixture over 30 minutes while maintaining the temperature at -5 ° C. After cooling to −10 ° C., the mixture was stirred for another hour, and a mixed solution of a concentrated solution of concentrated HCl / NH ₄ Cl (30 mL / 720 ml) was added over 10 minutes.MTBE (760 ml) was added. Then, the mixture was stirred for 1 hour at 23 ° C. After completion of the stirring, the layers were separated.
(320 ml). The two organic layers are combined and a saturated solution of NH ₄ Cl (3
20 ml), DI water (320 ml) and brine (320 ml). The solution was washed with MgSO ₄ (60 g), filtered and concentrated to a yellow oil containing the desired product as a single diastereomer (228 g).
. DSC: 227.54 ° C (endotherm, 61.63 J / g). C ₂₅ H ₃₃ BrClNO Calculated micro elemental analysis of _{6 C: 53.72%; H:} 5.95%; N, 2.50%; Br: 14.29%; C
l: 6.33% Result C: 53.80%; H: 6.45%; N, 2.23%; Br: 12.85
%; Cl: 6.12%. Step 4

[0164]

Embedded image The crude ester compound obtained in Step 3 in ethanol (1500 ml) was charged to a 3 L three-necked round bottom flask equipped with a stirrer. Reaction mixture at 0 ° C
And lead tetraacetate (88.67 g, 0.2 mol) was added at once. The reaction mixture was allowed to stir at 0 ° C. for 3 hours, after which 15% aqueous NaOH (150 ml) was added.
Added to the reaction mixture below 5 ° C. Ethanol was distilled off under reduced pressure using Rotavapo. An additional 600 mL of a 15% aqueous solution of NaOH was added and the reaction mixture was combined with ethyl acetate (2 × 300 mL), MTBE (2 × 200 mL) and ethyl acetate (2 × 20 mL).
0 mL). The combined organic layers were washed with DI water (2 × 200 mL) and brine (2 × 100 mL) and dried over anhydrous MgSO ₄ (30 g). The solution was filtered through celite and concentrated under reduced pressure to give an orange oil (96 g), which was used for the next step without further purification. DSC: 233.60 ° C (endotherm, 67.85 J / g); Calculated value for microelement analysis of C24H29BrClNO5 C: 54.71%; H 5.54%; N: 2.65%; Br: 15.16 %; C
16.72% result C: 52.12%; H, 5.40%; N: 2.47%; Br: 14.77.
%; Cl 6.48%. Step 5

[0165]

Embedded image The crude product of Step 4 (about 94 g) was dissolved in absolute ethanol (180 mL) and para-toluenesulfonic acid monohydrate (50.0 g, 0.26 mol) was added. The reaction mixture was heated to reflux for 8 hours and the solvent was distilled off under reduced pressure. Remaining solids in TH
F (100 mL) and THF was distilled off under reduced pressure. The residue was treated with ethyl acetate (
(500 mL) and cooled to about 5 ° C. The solid was filtered and heptane (2 × 50
mL) to give a white solid. The white solid was air dried to give the desired product as a single isomer of the white solid (38 g). ¹ H-NMR (DMSO, TM
S) (ppm) 1.12 (t, 3H), 2.29 (s, 3H), 3.0 (m, 2
H), 4.05 (q, 2H), 4.48 (t, 1H), 7.11 (d, 2H),
7.48 (d, 2H), 7.55 (d, 1H), 7.68 (1H, d), 8.3
5 (br.s, 3H); ^13C NMR (DMSO, TMS) (ppm): 13. 82, 20.75, 37.13, 45.590.59, 110.63, 122
. 47, 125.44, 127.87, 128.06, 129.51, 131.
95, 137.77, 145.33, 150.14, 168.98; DSC: 6
9.86 ° C (endotherm: 406.5 J / g), 165.72 ° C (endotherm: 62.27 J / g)
g), 211.24 ° C (exotherm, 20.56 J / g). [Α] ^D ₂₅ = + 4.2 ° (
C = 0.960, MeOH); IR (MIR) (cm-1) 2922, 1726
, 1621, 1591, 1494, 1471, 1413, 1376, 1324,
1286,1237,1207; _C 18 _H 21 BrClNO ₆ Calculated micro elemental analysis S C43.69%; H: 4.27% ; N: 2.38%; Br: 16.15%; Cl
7.16%; S: 6.48%. Results C 43.40%; H: 4.24%; N: 2.73%; Br: 16.40%
Cl; 7.20%; S: 6.54%. Step 6

[0166]

Embedded image The above compound was synthesized according to the procedure outlined in Steps 4 and 5 of Example I, utilizing the equivalent of the intermediate synthesized in Step 5 as the free base in place of Step 4 of Example I.

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

Example M

[0169]

Embedded imageSynthesis ofStep 1  Synthesis of 3-iodo-5-chlorosalicylaldehyde N-iodosuccinimide (144.0 g, 0.641 mol) was added to dimethylphos
5-chlorosalicylaldehyde (100 g, 0.
638 mol). The reaction mixture was stirred at room temperature for 2 days. further,
N-Iodosuccinimide (20.0 g) was added and stirring was continued for another 2 days.
Was. The reaction mixture was diluted with ethyl acetate (1 L) and hydrochloric acid (300 mL, 0.1).
N), water (300 mL), sodium thiosulfate (5%, 300 mL), brine
(300 mL), dried (MgSO 4)₄), Concentrate and dry, light yellow
The desired aldehyde was obtained as a colored solid (162 g, 90% yield).

MS and¹1 H-NMR was consistent with the desired structure.Step 2  Synthesis of 2-O- (MEM) -3-iodo-5-chlorosalicylaldehyde Potassium carbonate (41.1 g, 0.30 mol) was added to DMF (200 mL) at 20 ° C.
)) (84.74 g, 0.30 mol
). The result is a yellow slurry that, while maintaining the reaction temperature,
MEM-Cl (38.2 g, 0.305 mol) was added. 2 hours later, additional M
EM-Cl (1.5 g) was added. After stirring for 1 hour, mix the reaction mixture with an ice-water mixture
And stirred. The precipitate formed is filtered and dried under vacuum to give the desired protection
An aldehyde having a group was obtained. Yield: 95 g (85%).

MS and ¹ H-NMR were consistent with the desired structure. Step 3

[0172]

Embedded image Synthesis of (S) -phenylglycol (15.37 g, 0.112 mol) at room temperature with T
Added to a solution of 2-O- (MEM) -3-iodo-5-chlorosalicylaldehyde (41.5 g, 0.112 mol) in HF (200 mL). After stirring for 1 hour, MgSO ₄ (16 g) was added and stirring was continued for a second time. The reaction mixture was filtered and the filtrate was concentrated and dried under vacuum for 2 hours to give the desired intermediate, imine. A two-necked round-sided flask was charged with Rihomatsuki's reagent (81.8 g, 0.246 mol) synthesized in Step 1 of Example I and N-methylpyrrolidone (300 mL), and stirred at -10 ° C. A solution of the imine in N-methylpyrrolidone (100 mL) was added slowly, keeping the temperature at -10 ° C. The mixture was maintained at the above temperature for 2 hours and at -5 ° C for 1 hour. After cooling the reaction mixture to −10 ° C., a solution of concentrated hydrochloric acid (16 ml / 200 mL) in saturated ammonium chloride was added. Ethyl ether (500 mL) was added and allowed to stir at room temperature for 2 hours. The ether layer was separated, and the aqueous layer was further extracted with ether (300 mL). The combined ether layers were washed with saturated ammonium chloride (200 mL), water (200 mL), brine (200 mL), dried (MgSO ₄ ) and concentrated to give an oil (6
1.0 g, 90% yield). ¹ H-NMR indicated that the desired structure was substantially one diastereomer, consistent with the MS desired structure. Step 4

[0173]

Embedded image Synthesis of ester of crude product synthesized in Step 3 (48.85 g, 80.61 mmol)
Was dissolved in ethanol (500 mL) and cooled to 0 ° C. Lead tetraacetate (35.71 g, 80.61 mmol) was added. After 3 hours, NaOH 15
% Solution (73 mL) was added to the reaction mixture. Most of the ethanol was distilled off under reduced pressure. To the residue was added a 15% solution of NaOH followed by ether (
400 mL). The ether layer was washed with water (100 mL), brine (100 m
L) and concentrated to give an orange oil. The oil was dissolved in ethanol (10 mL) and para-toluenesulfonic acid (19.9 g) was added. The solution was heated to reflux for 8 hours and concentrated under reduced pressure. The residue was washed with THF (60
mL), heated to reflux and then cooled. The precipitate was filtered, washed with hexane / THF (300 mL, 1: 1) and dried to give the desired product.

MS and¹1 H-NMR was consistent with the desired structure.Step 5  S-ethyl 3- (N-BOC-gly) -amino-3- (S)-(5-chloro
-2-Hydroxy-3-iodo) phenylpropionate BOC-gly-OSu (9.4 g, 34.51 mm) in DMF (200 mL)
Mol) and ethyl 3- (S) -amino-3- (5-chloro-2-hydroxy-3
Triethylamine (-iodo) propionate PTSA salt mixture
4.8 mL) was added. The reaction mixture was stirred at room temperature for 18 hours. Vacuum DMF
Evaporate under reduced pressure and remove the residue between ethyl acetate (600 mL) and dilute hydrochloric acid (100 mL).
And separated. The organic layer was washed with sodium bicarbonate (200 mL), brine (200 m
L) and dried (MgSO 4)₄), Concentrate to desired product as a solid
Was obtained (14.2 g, yield 86%).

MS and¹1 H-NMR was consistent with the desired structure.Step 6  S-ethyl 3- (N-gly) -amino-3- (5-chloro-2-hydroxy
-3-Iodo) failpropionic acid ester At 0 ° C, dioxane / HCl (4N, 70 mL) was added to ethyl 3- (S)-(N-BOC-gly) -amino-3- (5-chloro-2-hydroxy- 3-Iodine)
Failpropionate (37.20 g, 70.62 mmol)
At room temperature for 3 hours. Concentrate the reaction mixture and add toluene (100 mL)
It was concentrated once more later. The residue obtained is suspended in ether, filtered and dried
This gave the desired product as a crystalline powder (32.0 g, 98% yield).

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

Example N

[0178]

Embedded image Synthesis process 1

[0179]

Embedded image The above compound was synthesized according to Step 2A of Example I using an equivalent of 2-hydroxy-3,5-dibromobenzaldehyde instead of 3,5-dichlorosalicylaldehyde.

Yield 88%; light yellow solid; m.p. p. 46-47 ° C; Rf = 0.6 (EtOA
c / hexane 1: ¹ v / v); ¹ H-NMR (CDCl ₃ ) d 3.37 (s, 3
H), 3.56 (m, 2H), 3.92 (m, 2H), 5.29 (s, 2H),
7.91 (d, 1H, J = 2.4 Hz), 7.94 (d, 1H, J = 2.4 Hz)
), 10.27 (s, 1H); FAB-MS m / z 367 (M +). C ₁₁ H ₁₂ Br ₂ O ₄ of the HR-MS Calculated 367.9083 result 367.9077 MS and ¹ H-NMR was consistent with the desired structure. Step 2

[0181]

Embedded image The above compound was synthesized according to the procedure of Step 2B and Step 2C of Example I, except that the equivalents of the compounds of Step 1 and Step 2B of Example I were exchanged.

Yield 90%; yellow solid; m.p. p. 57-59 ° C; Rf = 0.46 (EtOA
c / hexane 1: ¹ v / v); ¹ H-NMR (CDCl ₃ ) d 1.45 (s, 9
H), 2.1 (br, 1H , _{exchangeable), 2.51 (d, 1H,} J 1 = 9.9H
z, J ₂ = 15.3 Hz), 2.66 (d, 1H, J ₁ = 4.2 Hz, J ₂ = 1
5.3Hz), 3.02 (br, 1H, replaceable), 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). _{C 25 H 34 NBr 2 O 6} HR-MS Calculated 602.0753 result of 602.0749 MS and ¹ H-NMR was consistent with the desired structure. Step 3

[0183]

Embedded imageThe above compound (p-toluenesulfonate) was prepared according to Step 3 of Example I
The synthesis was performed by exchanging the equivalents of the products synthesized in Steps 2 and 3A. Yield 62%; white
Solid color;¹H-NMR (DMSO-d₆) D 1.09 (t, 3H, J = 7.2)
Hz), 2.27 (s, 3H), 2.97 (dd, 2H, J₁= 3.0 Hz, J ₂ = 7.2 Hz), 4.02 (q, 2H, J = 7.2 Hz), 4.87 (t, 1
H, J = 7.2 Hz), 7.08 (d, 2H, J = 4.8 Hz), 7.45 (m
, 3H), 7.57 (d, 1H, J = 2.4 Hz), 8.2 (br, 3H); F
AB-MS m / z 365 (M + H). C₁₁H₁₄NBr₂O₃HR-MS calcd for 365.9340 Results 365.9311 MS and¹1 H-NMR was consistent with the desired structure.Step 4

[0184]

Embedded image The above compound was synthesized using the procedure of Step 4 of Example I, replacing the compound synthesized in Step 3. The resulting BOC protected intermediate was converted to the desired compound by the procedure of Step 5 of Example I.

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

Example P

[0187]

Embedded image The above compound is replaced with the equivalent of 3-iodo-5-bromosalicylaldehyde synthesized in Step 1 of Example F, replacing the 3,5-dichlorosalicylaldehyde utilized in Step 2A of Example I, Synthesized according to the procedure.

[0188]Example 1  (±) 3-Bromo-5-chloro-2-hydroxy-β-[[2-[[[3-H
Droxy-5-[(1,4,5,6-tetrahydro-5-hydroxypyrimidine
-2-yl) amino] phenyl] carbonyl] amino] -acetyl] amino] be
Nsenpropanoic acid trifluoroacetate

[0189]

Embedded image Synthesis of the product of Example H (0.4 g, 0.0014 mol) and the product of Example B (0.58 g
, 0.0014 mol), triethylamine (0.142 g, 0.0014 mol), DMAP (17 mg) and anhydrous DMA (4 ml) in EDCl (0 ml) at ice bath temperature.
. 268 g, 0.0014 mol). The reaction was allowed to stir at room temperature overnight.
The resulting ester intermediate was separated by reverse phase synthesis HPLC. H ₂ O (10m
l) and this ester in CH ₃ CN (5 ml) were added to LiOH (580 mg, 0.0
138 mol) was added. After stirring at room temperature for 1 hour, the pH was reduced to 2 with TFA and the product was purified by reverse-phase synthetic HPLC to give (after lyophilization) the desired product as a white solid (230 mg). . MS and ¹ H-NMR were consistent with the structure as desired.

Example 2

[0191]

Embedded image The above compound was synthesized according to the method of Example 1 by replacing the product of Example B with the equivalent of the product of Example A.

The yield after lyophilization was 320 mg as a white solid.

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

Example 3

[0195]

Embedded image The above compound was synthesized according to the method of Example 1, substituting the product of Example B for the equivalent of the product of Example F. The yield (after lyophilization) was 180 m as a white solid.
g.

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

Example 4

[0198]

Embedded image The above compound was synthesized according to the method of Example 1, substituting the product of Example B for the equivalent of the product of Example D. The yield (after lyophilization) was 180 mg as a white solid.

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

Example 5

[0201]

Embedded image The above compound was synthesized according to the method of Example 1, substituting the product of Example B for the equivalent of the product of Example E. The yield (after lyophilisation) was 250 mg as a white solid.
Met.

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

Example 6

[0204]

Embedded image The above compound was synthesized according to the method of Example 1, replacing the product in Example B with the equivalent of the product in Example C. Yield (after lyophilization) was 220 m
g.

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

Example 7

[0207]

Embedded imageSynthesis of anhydrous DMA (50 mL) in a flame-dried flask under a nitrogen atmosphere
The product from Example H (7.8 g, 0.027 mol) was added with isobutyl at ice bath temperature.
Chloroformate (3.7 g, 0.027 mol) was added slowly followed by N
-Methylmorpholine (2.73 g, 0.027 mol) was added. Ice the solution
Stirred at bath temperature for 15 minutes. The product from Example L (10.0 g) was added to the reaction mixture.
, 0.024 mol) at ice bath temperature, followed by N-methylmorpholine (2.4
(3 g, 0.024 mol). The reaction was allowed to stir at room temperature overnight. Resulting
The ester intermediate was separated by reverse phase prep HPLC. H₂O (60 mL) and CH ₃ LiOH (10 g, 0.238 mol) was added to the ester in CN (30 mL).
Added. The reaction mixture was allowed to stir at room temperature for 1 hour. pH was reduced to 2 by TFA
I let you. The product was purified by reverse phase prep HPLC to give a white solid (after lyophilization).
To give the desired product (9.7 g).

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

[0209]Example 8  (S) 3,5-dichloro-2-hydroxy-β-[[2-[[[3-hydroxy
-5-[(1,4,5,6-tetrahydro-5-hydroxypyrimidine-2-i
L) amino] phenyl] carbonyl] amino] acetyl] amino] -benzenep
Lopanic acid monohydrochloride monohydrate

[0210]

Embedded imageSynthesis ofStep A  The product from Example H dissolved in anhydrous DME (200 mL) (9.92 g, 0.0
345 mol) with N-methylmorpholine (4.0 mL, 0.0362 mol).
Added. The reaction mixture was allowed to cool to -5 ° C (in a salt-ice bath). Isobutylchloroformate, IBCF (4.48 mL, 4.714 g, 0.0345 mol) in 1
The reaction mixture was allowed to stir at the greedy temperature for 12 minutes. The reaction mixture
To the combined solution, the product from Example I (11.15 g, 0.030 mol) was added at ice bath temperature.
Followed by N-methylmorpholine (4.0 mL, 0.0362 mol).
The reaction mixture was warmed to room temperature and concentrated in vacuo at 50 ° C. to give a black residue.
The residue is acetonitrile: H₂Dissolved in O (about 50 mL). Small amount of TF
The pH was made acidic by the addition of A. Residue is 10x500cmC-1
8 (50μ particle size) column to separate the desired product ester.
(Solvent program: 100% H₂O + 0.05% TFA over 1 hour, 30: 70H₂O + 0.05% TFA: to acetonitrile + 0.05% TFA
Flow rate 100 mL / min: The solvent program was started after the solvent front eluted. ) Synthetic (preparatory) RHPCL purification yielded 10.5 g (50% yield) as a white solid after lyophilization.

MS and¹1 H-NMR was consistent with the desired structure.Step B  The product synthesized in step A (about 11 g) is dissolved in dioxane: water, and the chicken
PH was adjusted to about 11.5 by adding 2.5N NaOH (to pH meter).
Measurement). The reaction mixture was stirred at room temperature. Regularly add more base
The pH was readjusted to 11 or more. After 2-3 hours, the ester
Conversion to the acid was deemed complete by ROHPLC. PH of reaction mixture
Was adjusted to about 6, and a viscous oil precipitated from the solution. Decant the oil
And washed with hot water (200 mL). The resulting water-soluble mixture
The liquid is cooled and the solid is collected by filtration and (after lyophilization from a HCl solution) the compound
2.6 g of the product were obtained. The black viscous residue is treated with hot water and allowed to cool to a tan powder.
A powder was obtained (4.12 g after freeze-drying from an HCl station).

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

[0213]Example 9  (S) 3-bromo-5-chloro-2-hydroxy-β-[[2-[[[3-hydr
Roxy-5-[(1,4,5,6-tetrahydro-5-hydroxypyrimidine-
2-yl] amino] phenyl] carbonyl] amino] acetyl] amino] -ben
Sempropanoic acid trifluoroacetateStep 1

[0214]

Embedded image Synthesis of the product from Example J (1.0 g, N, N-dimethylacetamide (10 mL)
2.4 mmol), a suspension of the product from Example H (0.75 g, 2.6 mmol) and 4-dimethylaminopyridine (40 mg) in triethylamine (0.24 mmol).
g, 2,4 mmol). The mixture was stirred at room temperature for 15 minutes and 1- (3-
Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.60 g, 3
. 1 mmol) was added. The reaction mixture was stirred overnight at room temperature. The mixture was concentrated in vacuo, reverse phase HPLC purification (starting gradient _{90:: 10H 2 O / TFA} MeO H, retention time 22 minutes) gave the desired product (1.6 g, yield 52
%).

¹ H-NMR was consistent with the desired structure. Step 2

[0216]

Embedded image 1: 4 MeCN: H₂The ester synthesized in step 1 in an O solution (7 mL) (8
00 mg, 1.2 mmol) in a solution of lithium hydroxide (148 mg, 6.2 mmol).
Lmol) was added. The reaction mixture was allowed to stir at room temperature for 2 hours. TFA (0.71
mL, 9.2 mmol) was added and the mixture was subjected to reverse phase HPLC (starting gradient 9
5: 5 H₂O / TFA: MeCN, retention time 24 min) to give the desired product (860 mg, 83% yield). C₂₂H₂₃BrClN₅O₇Calculated for elemental analysis of C, 39.18; H, 3.20; N, 8.99. Results C, 39.11; H, 3.17; N, 9.07 MS and¹1 H-NMR was consistent with the structure as desired.Step 3  Synthesis of hydrochloride The product of Step 2 is dissolved in a suitable solvent (acetonitrile: water), and
Slowly pass through Bio-Rad AG2-8X (chlorine ion form, 200-400 mesh, 5 equivalents or more) ion exchange column and freeze-dry to obtain the desired hydrochloride.
A product was obtained.

Example 10

[0218]

Embedded image The above compound was synthesized utilizing the procedure of Example 8 and substituting the product of Example N for the product of Step A of Example I, Example 8. The product was separated by prepRPHPLC and lyophilized to give the desired product as a TFA salt.

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

Example 11

[0221]

Embedded image The above compound was synthesized utilizing substantially the procedure of Example 8 and substituting the product of Example M for the product of Example I of Step A of Example 8. The product was separated by synthetic RPHPLC and lyophilized to give the desired product as a TFA salt.

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

Example 12

[0224]

Embedded image The above compound was synthesized utilizing the procedure of Example 8 and substituting the product of Example P for the product of Example I of Step A of Example 8. The products are separated by prep HPLC,
Lyophilization provided the desired product as a TFA salt.

Example 13

[0226]

Embedded image Synthesis of 2-O- (MEM) -3,5-diiodosalicylaldehyde

[0227]

Embedded image Potassium carbonate (18.5 g, 0.134 mol) was added to DMF (150 m
3,5-Diiodosalicylaldehyde in L) (50.0 g, 0.134 mol)
Was added to the solution. This resulted in a yellow slurry and MEM-Cl (15.8 mol, 0.134 mol) was added while maintaining the reaction temperature. After 2 hours, additional MEM-Cl (1.5 g) was added. After stirring for an additional hour, the reaction mixture was poured into ice water and stirred. A precipitate formed and was filtered and dried under vacuum to give the aldehyde with the desired protecting group (61 g, 99% yield). ¹ H-NMR was consistent with the desired product. Step 2

[0228]

Embedded image Synthesis of (S) -phenylglycinol (17.9 g, 0.13 mol) at room temperature in TH
To a solution of 2-O- (MEM) -3,5-diiodosalicylaldehyde (41.5 g, 0.112 mol) in F (150 mL). After 1 hour, MgSO ₄ (
20.7 g) was added and stirring continued for 2 hours. The reaction mixture was filtered, the filtrate was concentrated and dried under vacuum for 2 hours. Rehomatsu key reagent (2 bottle round bottom flask)
96 g, 0.289 mol) and N-methylpyrrolidone (250 mL) were stirred and stirred at -10 ° C. While maintaining the temperature at −10 ° C., a solution of the imine in N-methylpyrrolidone (100 mL) was added slowly. Bring the mixture to the above temperature
And maintained at -5 ° C for 1 hour. After cooling the reaction mixture to −10 ° C., a solution of concentrated HCl (16 ml / 200 mL) in saturated ammonium chloride was added. Ethyl ether (500 mL) was added and the mixture was stirred at room temperature for 2 hours. The ether layer was separated and the aqueous layer was extracted with ether (300 mL). The combined ether layers were washed with saturated ammonium chloride (200 mL), water (200 mL), brine (200 mL), dried (MgSO ₄ ) and concentrated to give an oil (90.0 g, 99% yield). NMR analysis showed the desired product and one diastereomer. Step 3

[0229]

Embedded imageThe solution of the crude ester obtained in Step 2 was dissolved in ethanol (100 mL),
Allowed to cool to ° C. Add lead tetraacetate (9.20 g, 20.75 mmol) in one lot
Added. After 3 hours, a 15% solution of NaOH was added to the reaction mixture. Most of d
Tanol was distilled off under reduced pressure. Residue in 15% chicken raising in NaOH (200 mL)
Added and extracted with ether (400 mL). The ether layer was washed with water (100 mL),
Wash with brine (100 mL), dry and concentrate to remove the orange oil
Obtained. This was dissolved in ethanol (100 mL), and para-toluenesulfonic acid was dissolved.
(6.08 g) was added. Heat the solution to reflux for 8 hours and concentrate under reduced pressure
I let it. Dilute the residue with THF (60 mL) and heat to reflux, then
Cool. After storage, no precipitate formed. The reaction mixture is concentrated and
To give an amino acid as a PTAS salt. Dissolve the obtained solid in ethanol
HCl and saturated HCl. The reaction mixture was heated to reflux for 6 hours. Anti
The reaction mixture was concentrated to obtain a PTSA salt of a desired amino acid (12.47 g).Step 4  Ethyl 3- (N-BOC-gly-)-amino-3- (S)-(3,5-diyo
Synthesis of (do-2-hydroxyphenyl) propionate

[0230]

Embedded image BOC-gly-OSu (7.48 g, 27.04 mi) in DMF (100 mL)
Remol), ethyl 3- (S) -amino-3- (3,5-diiodo-2-hydro
PTSA salt of (xyphenyl) propionic acid ester (12.47 g, 27.04
Mmol) was added triethylamine (3.8 mL). Reaction mixture
Was allowed to stir at room temperature for 18 hours. DMF is distilled off under vacuum and the residue is washed with ethyl acetate.
(600 mL) and dilute hydrochloric acid (100 mL). Organic layer with bicarbonate
Wash with thorium (200 mL), brine (200 mL), dry (MgS
O₄) And concentrated to give the desired product as a solid (17.0 g, 96% yield)
).¹1 H-NMR was consistent with the desired product.Step 5  Ethyl 3- (N-gly) -amino-3- (3,5-diiodo-2-hydroxy
Synthesis of hydrochloride of (phenyl) -propionate.

[0231]

Embedded image Ethyl 3- (N-BOC-gly) -amino-3- (S)-(3,5-diiodo-2-hydroxyphenyl) at 0 ° C. in dioxane / HCl (4N, 40 mL)
) Propionate (17.0 g, 25.97 mmol) was added and the reaction was mixed.
The combined solution was allowed to stir at room temperature for 3 hours. Concentrate the reaction mixture and add toluene (100 mL)
Was concentrated once more after the addition of. The residue obtained is dried,
This gave the desired product (8.0 g, 56% yield).¹H-NMR is the desired raw
Matched with adult.Step 6  M- (5-Hydroxypyrimidino) horse in dimethylacetamide (25 mL)
A solution of uric acid (3.74 g, 12.98 mmol) was added while all materials were in solution.
Heated. Thereafter, this was cooled to 0 ° C. and isobutyl chloroformate (1.6
8 mL) at once, followed by N-methylmorpholine (1.45 mL)
did. After 10 minutes, ethyl 3- (N-gly) -amino-3- (3,5-diiodo
2-hydroxyphenyl) -propionic acid ester hydrochloride (6.0 g, 10 g
. 82 mmol) at once, followed by N-methylmorpholine (1.45 mL).
) Was added. The reaction mixture was allowed to stir at room temperature for 18 hours. Concentrate the reaction mixture
The residue was dissolved in tetrahydrofuran / water (1: 1, 20 mL) and separated by chromatography (reverse phase, containing 0.1% TFA, 95: 5 water: acetate).
The tonitrile was converted to 30:70 water: acetonitrile over 60 minutes.
). The combined fractions were concentrated. The residue was dissolved in acetonitrile / water and lithium hydroxide was added until basic. The solution was allowed to stir for 2 hours
. The reaction mixture is concentrated and purified by hplc to give the TFA salt as described above.
To give the desired acid. The TFA salt is passed through an ion exchange column and then lyophilized
To give the corresponding hydrochloride.¹1 H-NMR was consistent with the desired product.

[0232]Examples 14 to 18  Compounds of formulas VII, VIII, IX, XIII and XIV and their isomers
In accordance with the methods disclosed herein, and as will be apparent to those skilled in the art,
It can be synthesized by replacing with a reagent.

The activity of the compounds of the present invention was tested in the following assay. Assay testing
The results are summarized in Table 1.Vitronectin adhesion assay material  Human vitronectin receptor (α_vβ₃) As previously described (Pytela et al.
Methods in Enzymology, 144: 475-489 (1987)). Hi
As described above (Toygogo et al., Cell Structure and
Function, 13: 281-292 (1988)). Biotinylated arsenic
Tovitronectin is a Pearce Chemical Company (Rockford, Illinois)
Prepared by coupling NHS-biotin commercially available from
As described above (Charo et al., J. Bio. Chem., 266 (3): 1415-1421 (1991)).
Purified vitronectin was obtained. Assay buffer, OPD substrate tablet and RIA
Grade BSA was purchased from Sigma (St. Louis, MO). Anti-
Biotin antibody was purchased from Calbiochem (La Jolla, CA)
. Linbro microtiter plates were purchased from Florabo (McLean, VA). ADP reagents are available from Sigma
(St. Louis, Li).

[0234]Method  Solid phase receptor assay  This assay was essentially as described previously (Blood, 70: 475-4 by Niiya et al.).
83 (1987)). Purified human vitronectin receptor (α_vβ₃)
Diluted from stock solution, pH 7.4 (TBS +++), 1.0 mM Ca⁺⁺ , Mg⁺⁺And Mn⁺⁺To 1.0 g / mL in Tris-buffered saline containing Dilution
The receptor was immediately transferred to a Limbro microtiter plate as 100 L / well (100 ng receptor / well). The plate was sealed and allowed to incubate at 4 ° C. overnight to allow the receptor to bind to the wells. All remaining
The steps performed were at room temperature. Empty the assay plate and add TBS⁺⁺⁺200 L of 1% RIA grade BSA in TBS (TBS⁺⁺⁺/ BSA) was added and exposed
The plastic surface was blocked. After 2 hours incubation, assay
The plate was washed with TBS using a 96-well plate washing solution.⁺⁺⁺And washed. Serial dilutions of test compound and control are made starting from a 2 mM stock concentration.
Used 2 nM biotinylated vitronectin in TBS +++ / BSA as diluent
I went for it. Ligand labeled with test (or control) ligand
Premixing and subsequent transfer of a 50 L aliquot to the assay plate
Performed by CETUSPropett robot. The final concentration of labeled ligand was 1 nM
, The highest concentration of test compound is 1.0 × 10^-4M. As mentioned earlier,
After washing all wells with the rate wash, a competitive reaction occurred for 2 hours. Affi
Niti-purified horseradish peroxidase-labeled goat antibiotin antibody was obtained from TBS ⁺⁺⁺ Diluted 1: 3000 in / BSA and 125 L was added to each well. 3
After 0 min, the plate is washed and OPD / H in 100 mM / L pH 5.0 citrate buffer.₂Incubated with O substrate. The plate was read at a wavelength of 450 nm with a microtiter reader and the maximum binding control well was about 1.
When the absolute bottomance of zero is reached, the final
A₄₅₀Was recorded for analysis. The data is used in an Excel spreadsheet program
Analyzed using the written macro. Mean, standard deviation and% CV are
And calculated twice. Average A₄₅₀Values are for the four maximum binding controls (no competition).
Normalized to the mean of (B-MAX) (no titer added). Normalized value
Is fitted by a four parameter curve fitting algorithm (Robert et al.)
Atomic Energy Agency, Vienna, pp. 469 (1977)), plotted on a semi-log scale and showed a 50% inhibition of maximal binding of biotinylated vitronectin (IC₅₀ ), And R²The calculated concentrations corresponding to are summarized for the compounds and
The highest concentration showed more than 50% inhibition. Alternatively, IC₅₀Is the most tested
Are also reported as higher concentrations. Potential α_vβ₃Antagonist (3ino
IC in the range of 10 nM₅₀) Is β-[[2- [5-[(aminoiminomethyl
Ru) -Amino] -1-oxopentyl] amino] -1-oxoethyl] amino]
-3-Pyridinepropanoic acid (US patent application Ser. No. 08 / 375,338, Example 1) was included in each plate as a positive control.

[0235]Purified IIb / IIIa Receptor Assay  Materials Human fibrinogen receptor (α_IIbβ₃) Purified from old platelets
(Pytela, R., Pierschbacher, MD, Argraves, S., Suzuki, S., and Rouslahti
, E. `` Arginine-Glycine-Aspartic acid adhesion receptors '' Methods in E
nzymology 144 (1987): 475-489). Human vitronectin is obtained from fresh frozen plasma, Y
atohgo, T., Izumi, M., Kashiwagi, H., and Hayashi, M., `` Novel purification
 of vitronectin from human plasma by heparin affinity chromatography '' C
Purified as described in ell Structure and Function 13 (1988): 281-292. As described above, biotinylated human vitronectin is added to purified vitronectin.
Cups NHS-Biotin commercially available from Pierce Chemical Company
(Charo, IF, Nannizzi, L., Philips, D
R., Hsu, MA Scaround bottomorough, R.M., "Inhibition of fibrinogen b
inding to GP Iib / IIIa by a GP IIIa peptide ", J. Biol. Chem. 266 (3) (1991
): 1415-1421). Assay buffer, OPD substrate tablet and RIA grade BSA are available from Sigma (
(St. Louis, Mo.). Anti-biotin antibody
(La Jolla, CA). Limbo micro titer pre
The kits were purchased from FlowLab (McLean, VA). ADP reagent
Purchased from Bear (St. Louis, MO).

[0236]Method Solid phase receptor assay  This assay was performed by Niiya, K., Hodson, E., Bader, R., Byers-Ward, V. Koziol,
 J.A., Plow, E.F. and Ruggeri, Z.M., `` Increased surface expression of the
 membrane glycoprotein Iib / IIIa complex induced by platelet activation:
Relationship to the binding of fibrinogen and platelet aggregation '', Bl
ood 70 (1987): 475-483. Purified human fib
Linogen receptor (α<sub>IIb</sub>β<sub>3</sub>) Is diluted from a stock solution and has a pH of 7.
4 (TBS<sup>+++</sup>), 1.0 mM Ca<sup>++</sup>, Mg<sup>++</sup>And Mn<sup>++</sup>To 10 μg / mL in Tris-buffered saline containing 100 μL / well diluted receptor
Immediately transferred to a Limbo microtiter plate (100 ng receptor / well). Seal plate, incubate at 4 ° C overnight,
Was combined with the wells. All remaining steps were at room temperature. Assay plate
Empty and TBS<sup>+++</sup>200 microL of 1% RIA grade BSA (TBS<sup>+++</sup>/ BSA) was added to block the exposed plastic surface. 2
After a period of incubation, the assay plate is washed with 96 well plate washes.
Using TBS<sup>+++</sup>And washed. Serial log dilutions of test compounds and controls were started from a stock concentration of 2 mM and were diluted to 2 mM in TBS +++ / BSA as diluent.
Performed using nM biotinylated vitronectin. Test (or control)
Premixing of ligand labeled with ligand and 50 μL aliquots
Subsequent transfer to the assay plate was performed with a CETUSPropette robot. The final concentration of labeled ligand was 1 nM and the highest concentration of test compound was 1.0 × 10 5<sup>-4</sup>M
Met. 2:00 after all wells have been washed with the plate wash solution as described above
Competition took place between them. Affinity purified horseradish peroxidase labeled ya
Giant biotin antibody to TBS<sup>+++</sup>1: 3000 dilution in BSA / 125 μl
L was added to each well. After 30 minutes, the plate is washed and pH 5.0, 100m
ODO / H in M / L citrate buffer<sub>2</sub>O<sub>2</sub>Incubated with substrate. play
Read at 450 nm with a microtiter plate reader for maximum binding
When the control well reaches an absolute round bottom of about 1.0,
A<sub>450</sub>Was read for analysis. Excel (registered trademark) spreadsheet program
Analyzed using the written macro using ram. Mean, standard deviation and% C
V was calculated twice for the concentration. Average A<sub>450</sub>Values are the four maximum join controls
(B-MAX) normalized to the mean (no added competitor)
I let you. The normalized values are fitted by a four parameter curve fitting algorithm.
(Robart et al., Int. Atomic Energy Agency Vienna, pp 469 (1977), semi-logarithmic
Plotted on a scale, 50% inhibition of maximal binding of biotinylated vitronectin (
IC<sub>50</sub>), And R<sup>2</sup>The calculated concentration corresponding to is reported for the compound,
The highest concentration tested shows 50% or more inhibition and IC<sub>50</sub>Is the highest concentration tested
Report as more than. Effective α in the range of 3-10 nM<sub>v</sub>β<sub>3</sub>Antago
Β-[[2-[[5- (aminoiminomethyl) amino]
] -1-oxopentyl] amino] -1-oxoethyl] amino] -3-pyridi
Impropanic acid (US Application No. 08 / 375,338, Example 1) was included in each plate as a positive control.Abundant plasma assays in human platelets  Healthy aspirin-free donors were selected from volunteers. Rich in platelets
Harvesting of enriched plasma followed by ADP-induced platelet aggregation assay was performed by Zuck
er, M.B., `` Platelet Aggregation Measured by the Photometric Method '' Me
thods in Enzymology 169 (1989): 117-133. By standard venipuncture technique using a butterfly, 45 mL of the whole blood was 3.8% quenched.
Dispensed into a 60 mL syringe containing 5 mL of trisodium nitrate. In the syringe
After thorough mixing with anti-coagulated whole blood, add 50 mL of conical polyethylene
Moved to. Blood is centrifuged at room temperature for 12 minutes at 200 mg and non-platelet cells
The cells were allowed to settle. Transfer the platelet-rich plasma to a polyethylene tube and use it.
At room temperature. Platelet-poor plasma is blood remaining at 2000 xg for 15 minutes.
The liquid was obtained from a second centrifugation. Platelet counts are usually
300,000 to 500,000. Platelet-rich plasma (0.45
mL) of siliconized cuvettes and split of pre-diluted test compound
Stirring (1100 rpm) at 37 ° C. for 1 minute before adding 50 μL. One minute
After mixing during, aggregation was initiated by the addition of 50 μL of 200 μM ADP.
Agglomeration with Payton dual channel aggr
egometer) (Payton Scientific, Buffalo, NY)
) For 3 minutes. Percentage inhibition of the maximal response to a series of test compound dilutions
A dose response curve was determined using this. All compounds tested in duplicate, half maximal inhibition
(IC₅₀) Was calculated from the dose response curve and for the tested compound,
Inhibition of 50% or more at highest concentration, IC₅₀Above the highest concentration tested
Report.

[0237]

[Table 5]

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] March 7, 2000 (200.3.7)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claim 1

[Correction method] Change

[Correction contents]

Embedded image Wherein X and Y are the same or different halo groups, and R is hydrogen or C ₁ -C ₁₀ alkyl, and pharmaceutically acceptable salts thereof.

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0011

[Correction method] Change

[Correction contents]

[0011] White (Current Biology, Vol. 3 (9) (1993) 596-599) reports that adenovirus utilizes α _v β ₃ to enter host cells. The integrin is required for endocytosis of the virus and for penetration of the viral genome into the host cytoplasm. Thus, compounds that inhibit α _v β ₃ may prove useful as antiviral agents. WO 97/08145 discloses meta-guanidine, urea-, thiourea- and azacyclic aminobenzoic acid derivatives of the following formula (I).

Embedded image J. Med. Chem. 40, 930 (1997), J. Med. Chem. 40, 920 (1997) and Antiv. Chem.
Chemother. 8, 463 (1997) describes the discovery of HIV-1 integrase inhibitors by specific macrophage studies. Exp. Opin. Ther. Patents 8, 633 (1998), issued after the current priority claim, relates to the development of integrin antagonists used to inhibit cancer cell metastasis.

[Procedure amendment]

[Submission date] November 22, 2000 (200.11.22)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

Embedded image Wherein, X and Y are the same or different halo groups, R represents A <br/> compounds and salts may be their pharmaceutically acceptable an alkyl hydrogen also C ₁ to C _10.

Embedded image

Embedded image

Embedded image

Embedded image 2. The compound of claim 1, wherein R is hydrogen or alkyl, or a pharmaceutically acceptable salt thereof.

Embedded image

Embedded image

Embedded image

Embedded image The compound of claim 1 selected from the group consisting of:

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) A61P 19/08 A61P 19/08 29/00 101 29/00 101 35/00 35/00 35/04 35 / 04 43/00 111 43/00 111 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT , SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH , CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK , SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor Luminsky, Peter, G 63366 Dardeen Prairie Pierside Drive 7687F, Missouri, United States Term (reference) 4C086 AA01 AA02 AA03 BC42 GA16 MA01 MA02 MA03 MA04 MA05 NA14 ZA33 ZA36 ZA67 ZA89 ZA94 ZB15 ZB21 ZB26 ZB33 ZB35 ZC21 ZC41

Claims (36)

[Claims] 1. A compound represented by the following formula: Wherein X and Y are the same or different halo groups, and R is hydrogen or lower alkyl, and pharmaceutically acceptable salts thereof. ## STR2 ## Embedded image Embedded image Embedded image 2. The compound of claim 1, wherein R is hydrogen or alkyl, or a pharmaceutically acceptable salt thereof. (3) Embedded image Embedded image Embedded image The compound of claim 1 selected from the group consisting of: 4. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to claim 1 and a pharmaceutically acceptable carrier. 5. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to claim 2 and a pharmaceutically acceptable carrier. 6. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to claim 3 and a pharmaceutically acceptable carrier. 7. A method of treating a condition in need of treatment and mediated by α _v β ₃ integrin in a mammal, which comprises administering an amount of the compound according to claim 1 which effectively inhibits α _v β ₃ integrin. A therapeutic method comprising: 8. A method for treating a condition requiring treatment and mediated by α _v β ₃ integrin in a mammal, wherein the compound according to claim 2 is administered in an amount that effectively inhibits α _v β ₃ integrin. A therapeutic method comprising: 9. A method of treating a condition requiring treatment and mediated by α _v β ₃ integrin in a mammal, which comprises administering an amount of the compound according to claim 3 which effectively inhibits α _v β ₃ integrin. A therapeutic method comprising: 10. The method according to claim 7, wherein the treated condition is tumor metastasis. 11. The method according to claim 8, wherein the treated condition is tumor metastasis. 12. The method according to claim 9, wherein the treated condition is tumor metastasis. 13. The method of claim 7, wherein the condition treated is solid tumor growth. 14. The method of claim 8, wherein the condition treated is solid tumor growth. 15. The method of claim 9, wherein the condition treated is solid tumor growth. 16. The method according to claim 7, wherein the treated condition is angiogenesis. 17. The method according to claim 8, wherein the treated condition is angiogenesis. 18. The method according to claim 9, wherein the treated condition is angiogenesis. 19. The method according to claim 7, wherein the treated condition is osteoporosis. 20. The method according to claim 8, wherein the treated condition is osteoporosis. 21. The method according to claim 9, wherein the treated condition is osteoporosis. 22. The method according to claim 7, wherein the treated condition is malignant fluid hypercalcemia. 23. The method according to claim 8, wherein the treated condition is malignant fluid hypercalcemia. 24. The method according to claim 9, wherein the treated condition is malignant fluid hypercalcemia. 25. The method according to claim 7, wherein the treated condition is smooth muscle cell migration. 26. The treatment method according to claim 8, wherein the treated condition is smooth muscle cell migration. 27. The method according to claim 9, wherein the treated condition is smooth muscle cell migration. 28. The method according to claim 7, wherein restenosis is inhibited. 29. The method according to claim 8, wherein restenosis is inhibited. 30. The method according to claim 9, wherein restenosis is inhibited. 31. The method according to claim 7, wherein the treated condition is rheumatoid arthritis. 32. The method according to claim 8, wherein the treated condition is rheumatoid arthritis. 33. The method according to claim 9, wherein the condition treated is rheumatoid arthritis. 34. The method according to claim 7, wherein the treated condition is macular degeneration. 35. The method according to claim 8, wherein the treated condition is macular degeneration. 36. The method according to claim 9, wherein the treated condition is macular degeneration.