KR20050009742A - Prodrugs of Excitatory Amino Acids - Google Patents

Abstract

The present invention relates to synthetic excitatory amino acid prodrugs and methods for their preparation. The invention also relates to pharmaceutical compositions comprising compounds for the treatment of neurological disorders and psychiatric disorders, and methods of using them.

Description

Prodrugs of Excitatory Amino Acids

Treatment of neurological or psychiatric disorders (eg anxiety disorders) is associated with selective activation of metabotropic excitatory amino acid receptors. For example, (+)-4-amino-2-sulfonylbicyclo [3.1.0] hexane-4,6-dicarboxylic acid is disclosed in US Pat. No. 5,688,826 ('826 Patent; Nov. 18, 1997). It is disclosed as an active mGluR2 receptor agonist. In addition, (+)-2-amino-4-fluorobicyclo [3.1.0] hexane-2,6-dicarboxylic acid is active in US Pat. No. 5,958,960 ('960 patent; 28 September 1999). It is disclosed as a receptor agonist.

The present invention provides prodrug forms of mGluR2 receptor agonist compounds, which increase the in vivo efficacy of each parent compound and increase the degree of oral exposure of the parent compound. The compounds of the present invention represent the best approach to maintain the stability and efficacy of the mGluR2 receptor agonists with increased oral bioavailability already disclosed.

Synthetic excitatory amino acid prodrugs and methods for their preparation are disclosed in PCT Application Serial Nos. PCT / USO1 / 45866 and PCT / USO2 / 00488.

<Overview of invention>

The present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof.

Where

A is H- (Q) _p- ;

Each Q is independently selected from an amino acyl group;

p is an integer from 1 to 10;

X is O, S, SO, SO ₂ or CR ³ R ⁴ ;

R ³ is fluoro, X'OR ⁵ , SO ₃ H, tetrazol-5-yl, CN, PO ₃ R ⁶ ₂ , hydroxy or NO ₂ , and R ⁴ is hydrogen; R ³ and R ⁴ each represent fluoro; R ³ and R ⁴ together represent = O, = NOR ⁷ or = CR ⁸ R ⁹ ; Or one of R ³ or R ⁴ represents amino and the other represents carboxyl; R ³ represents N ₃ , (CH ₂ ) _m COOR ^5a , (CH ₂ ) _m PO ₃ R ^6a ₂ , NHCONHR ^5b or NHSO ₂ R ^5c , and R ⁴ represents hydrogen; Or R ³ and R ⁴ together represent = CHCOOR ^5b , = CHPO ₃ R ^6a ₂ or = CHCN;

X 'represents a bond, CH ₂ or CO;

m is an integer from 1 to 3;

R ⁵ , R ^5a , R ^5b , R ^5c , R ⁷ , R ⁸ and R ⁹ are each independently a hydrogen atom; (1-6C) alkyl groups which may be optionally substituted; (2-6C) alkenyl groups which may be optionally substituted; Optionally substituted (2-6C) alkynyl groups; Aromatic groups which may be optionally substituted; Heteroaromatic groups which may be optionally substituted; Non-aromatic carbocyclic groups; Non-aromatic heterocyclic groups; Non-aromatic monocyclic carbocyclic groups fused with one or two monocyclic aromatic or heteroaromatic groups; Or a non-aromatic monocyclic heterocyclic group fused with one or two monocyclic aromatic or heteroaromatic groups;

R ⁶ and R ^6a independently represent a hydrogen or (1-6C) alkyl group;

R ¹⁰ is hydrogen or fluoro;

R ¹¹ is hydrogen, fluoro or hydroxy.

In addition, the present invention relates to compounds of formula I wherein the substituents are as defined above, provided that X is CR ³ R ⁴ , wherein R ³ is fluoro and R ⁴ is hydrogen, p is 1 and Q is Or pharmaceutically acceptable salts thereof.

In addition, the present invention provides that A is H- (Q) _p- ; Each Q is independently selected from an amino acyl group; p is an integer from 1 to 3; X is O, S, SO, SO ₂ or CR ³ R ⁴ ; R ³ is fluoro or hydroxy and R ⁴ is hydrogen; Or R ³ and R ⁴ together represent = O; R ¹⁰ is hydrogen or fluoro; Provided is a compound of Formula I, or a pharmaceutically acceptable salt thereof, wherein R ¹¹ is hydrogen, fluoro or hydroxy.

In addition, the present invention provides that A is H- (Q) _p- ; Q is L-alanyl; p is 1; X is CR ³ R ⁴ ; R ³ is fluoro and R ⁴ is hydrogen; R ¹⁰ is hydrogen; Provided is a compound of Formula I or a pharmaceutically acceptable salt thereof, wherein R ¹¹ is hydrogen.

It will be understood that compounds of formula I contain four or more asymmetric carbon atoms. The present invention relates to each individual enantiomer and mixtures thereof (e.g. prodrug forms of the compounds disclosed in the '826 patent, for example 1SR, 4RS, 5RS, 6RS-4-amino- (2-sulfo). All stereoisomeric forms of the compound of formula I, including nibicyclo [3.1.0] hexane) -4,6-dicarboxylic acid).

A further aspect of the present invention provides a pharmaceutical formulation comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof in combination with a pharmaceutically acceptable carrier, diluent or excipient.

A further aspect of the present invention provides a method of affecting a cAMP-associated metabotropic glutamate receptor in a patient comprising administering a pharmaceutically effective amount of a compound of Formula (I) to a patient in need of controlled excitatory amino acid neurotransmission. The present invention also provides the use of a compound of formula I in the manufacture of a medicament that affects a cAMP-associated metabotropic glutamate receptor in a patient.

A further aspect of the present invention provides a method of administering an effective amount of a compound of Formula II comprising administering a pharmaceutically effective amount of a compound of Formula I to a patient in need of controlled excitatory amino acid neurotransmission. The present invention also provides the use of a compound of formula I in the manufacture of a medicament for administering an effective amount of a compound of formula II.

A further aspect of the present invention provides a method of treating a neurological disorder in a patient comprising administering a pharmaceutically effective amount of a compound of formula (I) to a patient in need thereof. The present invention also provides the use of a compound of formula I in the manufacture of a medicament for the treatment of neurological disorders in a patient.

A further aspect of the present invention provides a method of treating a psychiatric disorder in a patient comprising administering a pharmaceutically effective amount of a compound of formula (I) to a patient in need thereof. The present invention also provides the use of a compound of formula I in the manufacture of a medicament for the treatment of psychiatric disorders in a patient.

Compounds of formula (I) can be prepared by methods analogous to those known in the chemical art for the preparation of structurally similar heterocyclic compounds, or by the novel methods described herein. Useful methods and intermediates for the preparation of the compounds of formula (I) as defined above are described by the following procedure, and unless otherwise indicated, the meaning of the general radicals is as defined herein.

The present invention

Acylating the compound of formula (ii) with the corresponding amino acyl of formula III

<Formula (ii)>

Wherein Pg ^N is a nitrogen-protecting group;

Then, in any of the above procedures, removing the protecting group if the functional group is protected using a protecting group;

Then, in any of the above procedures, i) if a pharmaceutically acceptable salt of the compound of formula I is required, reacting the compound of formula I in base form with an acid to obtain a pharmaceutically acceptable counter ion; ii) for compounds of formula (I) having acidic moieties, the compound of formula (I) in acid form is reacted with a base to obtain a pharmaceutically acceptable cation; Or iii) for the zwitterionic compound of formula I, neutralizing the compound of formula I in acid-addition salt form or base-addition salt form; Or any other conventional procedure

Provided is a method of preparing a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

The present invention also provides compounds of formula I, wherein X is CH ₂ , R ¹⁰ is fluoro and other substituents as defined above.

The present invention provides synthetic excitatory amino acid prodrugs (compounds of Formula I) and methods for their preparation. The invention also relates to methods of using the compounds of formula (I) for the treatment of neurological disorders and psychiatric disorders and to pharmaceutical compositions comprising them.

The compounds of the present invention have been found to be useful prodrugs of compounds that are selective agonists of metabotropic glutamate receptors and are therefore useful for the treatment of central nervous system diseases such as neurological diseases (e.g. neurodegenerative diseases), Useful as anxiolytics, drug-withdrawal development agents, antidepressants, antispasmodics, analgesics and anti-emetic agents.

It will be understood that compounds of formula I have four or more asymmetric carbon atoms, three of which are located in the cyclopropane ring and the other is located in the α-carbon of the amino acid group. Accordingly, the compounds of the present invention may exist in enantiomerically pure forms, racemic forms or diastereomeric mixtures and may be isolated in these forms.

The amino acid residues preferably have a natural amino acid sequence (ie, L-array for D-glycerol aldehyde).

The present invention includes pharmaceutically acceptable salts of compounds of formula (I). These salts may be present with the acidic or basic portion of the molecule and may be present as acid addition salts, primary, secondary, tertiary or quaternary ammonium salts, alkali metal salts or alkaline earth metal salts. In general, acid addition salts can be prepared by reacting an acid with a compound of formula (I). Alternatively, acid addition salts can be prepared by reacting a penultimate compound (protected intermediate) with an appropriate amount of acid to produce the corresponding salt form, which is then reacted to produce a compound of formula (I) or another salt. can do. Alkali metal salts and alkaline earth metal salts can generally be prepared by reacting the hydroxide form of the desired metal salt with a compound of formula (I).

Some specific salts are advantageous for the formulation of certain agents because of their crystalline form. Compounds in non-crystalline amorphous form can be easily wetted. Sometimes pharmaceutical compounds in crystalline form may be more desirable because they exhibit advantageous solid-state properties.

Acids commonly used to form the salts include inorganic acids (eg hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid or phosphoric acid), or organic carboxylic acids (eg glycolic acid, maleic acid, hydroxy). Maleic acid, fumaric acid, malic acid, tartaric acid, citric acid, salicylic acid, o-acetoxybenzoic acid or eutectic acid (e.g. 2-hydroxyethane sulfonic acid, toluene-p-sulfonic acid, methane-sulfonic acid, naphthalene-2-sulfonic acid, Organic acids) such as benzenesulfonic acid or ethanesulfonic acid.

In addition to pharmaceutically acceptable salts, other salts are included in the present invention. They can be used as intermediates in the purification of compounds, or in the preparation of other pharmaceutically acceptable acid addition salts, or are useful for identification, characterization or purification processes.

In the present invention, the compound of formula (I) includes solvates thereof. In particular, the compounds of formula (I) include their hydrates.

The present invention also contemplates prodrugs of the fluorinated compounds disclosed in International Application Nos. PCT / JP99 / 03984, PCT / JP99 / 00324 and PCT / JPO1 / 05550. See International Application Nos. WO / 0012464, WO / 9938839 and WO / 0200605, respectively. For example, the present invention provides 1S, 2R, 5S, 6S-2-amino-6-fluoro-4-oxobicyclo [3.1.0] hexane-2,6-dicarboxylic acid; 1S, 2R, 4S, 5S, 6S-2-amino-6-fluoro-4-hydroxybicyclo [3.1.0] hexane-2,6-dicarboxylic acid; 1S, 2R, 3R, 5S, 6S-2-amino-3-fluorobicyclo [3.1.0] hexane-2,6-dicarboxylic acid; And prodrugs of 1S, 2R, 3S, 5S, 6S-2-amino-6-fluoro-3-hydroxybicyclo [3.1.0] hexane-2,6-dicarboxylic acid.

It has been found that various physiological functions are affected by excessive or inappropriate stimulation of excitatory amino acid delivery. The compounds of formula (I) of the present invention can be used in various nerves, including acute neurological disorders such as brain injury, stroke, cerebral ischemia, spinal cord trauma, head trauma, perinatal hypoxia, heart failure and hypoglycemic nerve damage associated with cardiovascular bypass surgery and heart transplantation. It is believed that mammals suffering from the disorder have the ability to treat a variety of neurological disorders, including these symptoms. Compounds of formula (I) include Alzheimer's Disease, Huntington's Chorea, Amyotrophic Lateral Sclerosis, AIDS-Induced Dementia, Vision Impairment and Retinopathy, Cognitive Impairment, and Idiopathic and Drug-Induced Parkinson's Disease It is believed to be capable of treating various chronic neurological disorders such as). The present invention also provides a method of treating a disorder comprising administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof to a patient in need thereof.

Formula I compounds of the present invention may be used in patients with glutamate dysfunction, muscle spasms, cramps, migraine, incontinence, pain, premenstrual anxiety disorders (PDD), psychosis (eg, schizophrenia), drug resistance, drug withdrawal symptoms Signs of discontinuation of the drug and various other neurological disorders, including drug addiction (e.g. nicotine, opium, cocaine, benzodiazepines and ethanol), anxiety disorders and anxiety related disorders, vomiting, brain edema, chronic pain and delayed motor disorders. Treat. In addition, the compounds of formula (I) are useful as antidepressants and analgesics. Accordingly, the present invention also provides a method of treating a disorder comprising administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof to a patient in need thereof.

The following definitions enumerate the meaning and scope of the various terms used herein. As used herein, the generic terms have their conventional meanings.

The term "influencing" denotes a compound of formula II which acts as an agonist at the excitatory amino acid receptor. The term “excited amino acid receptor” refers to a metabolic glutamate receptor, which binds to intracellular effectors via a GTP-binding protein. The term “cAMP-associated metabotropic glutamate receptor” refers to a metabolic receptor associated with inhibition of adenylate cyclase activity.

The term "neurological disorder" refers to brain injury associated with cardiovascular bypass surgery and heart transplantation, cerebral ischemia (eg, stroke resulting from heart failure), spinal cord trauma, head trauma, Alzheimer's disease, Huntington's chorea, muscular dystrophy , AIDS-induced dementia, perinatal hypoxia, hypoglycemic nerve damage, visual impairment and retinopathy, cognitive impairment, idiopathic and drug-induced Parkinson's disease, and both acute and chronic neurodegenerative symptoms. The term also refers to muscle spasms, migraine headaches, incontinence, drug resistance, drug withdrawal symptoms, signs of drug discontinuation and drug addiction (ie opiates, benzodiazepines, nicotine, cocaine or ethanol), signs of smoking cessation, vomiting, brain edema, chronic pain, Other neurological symptoms caused by glutamate dysfunction, including sleep disorders, convulsions, Tourette's syndrome, attention deficit disorders, and delayed motor disorders.

The term "psychiatric disorders" refers to schizophrenia, anxiety disorders and anxiety related disorders (eg panic attacks and stress-related cardiovascular disorders), depression, bipolar disorder, psychosis, obsessive compulsive disorder, general anxiety disorder, acute stress disorder and panic attacks Both acute and chronic psychiatric symptoms, including disability.

As used herein, the term “effective amount” refers to an amount or dosage of a compound that provides the desired effect in a patient diagnosed or treated when administered to a patient in a single or multiple doses.

Effective amounts can be readily determined by observing the results obtained by a person skilled in the art under known techniques and similar circumstances. In determining the effective amount or effective dosage of a compound administered, the species, size, age and general state of health of the mammal; Related specific diseases; The degree or severity of the disease; Response of individual patients; The specific compound administered; Mode of administration; Bioavailability of the administered agent; Selected dosage regimen; Use of medicaments in combination; And a number of factors are considered by the diagnoser, including other related situations. For example, typical daily dosages may contain about 5 mg to about 300 mg of active ingredient. The compound can be administered by a variety of routes of administration, including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, oral or intranasal routes of administration. Alternatively, the compound may be administered by continuous infusion.

As used herein, the term “patient” refers to a mammal such as a mouse, guinea pig, rat, dog or human. Preferred patients are understood to be humans.

As used herein, the term "treating" (or "treatment") includes the general meaning encompassing the inhibition, prevention, elimination and delay, discontinuation or reverse progression of the symptoms that have occurred. As above, the methods of the present invention include both therapeutic and prophylactic administration.

General compound terms as used herein have their conventional meanings. For example, the term "(1-6C) alkyl" refers to a straight or branched chain group. Examples of useful (1-6C) alkyl groups are (1-4C) alkyl such as methyl, ethyl, propyl, isopropyl, butyl and isobutyl. The term "(2-6C) alkenyl" includes (2-4C) alkenyl, such as allyl. The term "(2-6C) alkynyl" includes (2-4C) alkenyl, such as propynyl.

The terms "optionally substituted (1-6C) alkyl group", "optionally substituted (2-6C) alkenyl group" and "optionally substituted (2-6C) alkynyl group" As used, the term “optionally substituted” indicates that one or more (preferably 1 to 3) substituents may be present, wherein the substituents are metabolized when present in the compound of formula (I) It is selected from atoms and groups that do not interfere with modulating the function of the affinity glutamate receptor.

Examples of the atoms and groups which may be present on an optionally substituted (1-6C) alkyl group, an optionally substituted (2-6C) alkenyl group or an optionally substituted (2-6C) alkynyl group may optionally Aromatic groups which may be substituted, heteroaromatic groups which may be optionally substituted, non-aromatic carbocyclic groups, non-aromatic heterocyclic groups, non-aromatic mono fused with one or two monocyclic aromatic or heteroaromatic groups Cyclic carbocyclic groups, and non-aromatic monocyclic heterocyclic groups fused with one or two monocyclic aromatic or heteroaromatic groups.

The term “heteroaromatic group” refers to aromatic 5- to 6-membered rings containing 1 to 4 heteroatoms selected from oxygen, sulfur and nitrogen, and benzene rings or 1 to 4 heteroatoms selected from oxygen, sulfur and nitrogen. Aromatic bicyclic groups consisting of 5 to 6 membered rings containing 1 to 4 heteroatoms selected from oxygen, sulfur and nitrogen fused with containing 5 to 6 membered rings. Examples of heteroaromatic groups are furyl, thiophenyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, imidazolyl, pyrimidyl, benzofuryl, benzothiophenyl, benzimidazolyl, benzoxazolyl, benzothia Zolyl and indolyl.

The term "aromatic group" includes phenyl and polycyclic aromatic carbocyclic rings (eg, naphthyl).

As used herein, the terms "optionally substituted heteroaromatic group" and "optionally substituted aromatic group", the term "optionally substituted", indicate that one or more substituents may be present, wherein the substituents are of the formula When present in a compound of I, the compound of formula I is selected from atoms and groups that do not interfere with modulating the function of the metabolic glutamate receptor.

Examples of atoms and groups that may be present in an optionally substituted heteroaromatic or optionally substituted aromatic group include amino, hydroxy, nitro, halogeno, (1-6C) alkyl, (1-6C) alkoxy, (1 -6C) alkylthio, carboxy, (1-6C) alkoxycarbonyl, carbamoyl, (1-6C) alkanoylamino, (1-6C) alkylsulfonyl, (1-6C) alkylsulfonylamino, optionally Phenyl, phenoxy, phenylthio, phenylsulfonyl, phenylsulfonylamino, toluenesulfonylamino, (1-6C) fluoroalkyl and (1-6C) fluoroalkoxy which may be substituted. Particularly useful examples are amino, hydroxy, fluoro, chloro, bromo, iodo, methyl, methoxy, methylthio, carboxy, acetylamino, methanesulfonyl, nitro, acetyl, phenoxy, phenylthio, phenylsulfonyl , Methanesulfonylamino and trifluoromethyl.

Useful examples of aromatic groups which may be optionally substituted include 1-naphthyl, 2-naphthyl, phenyl, 2-biphenyl, 3-biphenyl, 4-biphenyl, 2-hydroxyphenyl, 3-hydroxyphenyl, 4-hydroxyphenyl, 2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl, 2,4-difluorophenyl, 3,4-difluorophenyl, pentafluorophenyl, 2-chloro Phenyl, 3-chlorophenyl, 4-chlorophenyl, 2,4-dichlorophenyl, 3,4-dichlorophenyl, 3-chloro-4-fluorophenyl, 3,5-dichlorophenyl, 2-bromophenyl, 3 Bromophenyl, 4-bromophenyl, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, 2,3-dimethoxyphenyl, 2 , 5-dimethoxyphenyl, 3,4-dimethoxyphenyl, 3,5-dimethoxyphenyl, 2-trifluoromethylphenyl, 3-trifluoromethylphenyl, 4-trifluoromethylphenyl, 2-fluoro-3 -Trifluoromethylphenyl, 3-trifluoromethyl-4-fluorophenyl, 3-trifluoromethyl -5-fluorophenyl, 2-fluoro-5-trifluoromethylphenyl, 2-phenoxyphenyl, 3-phenoxyphenyl, 3-carboxyphenyl and 4-carboxyphenyl.

The term "non-aromatic carboxyl group" refers to monocyclic groups, such as (3-10C) cycloalkyl groups (eg cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclo Nonyl or cyclodecyl) and fused polycyclic groups (eg 1-adamantyl or 2-adamantyl, 1-decalyl, 2-decalyl, 4a-decalyl, bicyclo [3,3, 0] oct-1-yl, -2-yl or -3-yl, bicyclo [4,3,0] non-1-yl, -2-yl, -3-yl or -7-yl, bicyclo [5,3,0] dec-1-yl, -2-yl, -3-yl, -4-yl, -8-yl or -9-yl and bicyclo [3.3.1] non-1-yl , -2-yl, -3-yl or -9-yl).

The term "non-aromatic heterocyclic group" refers to a 4- to 7-membered ring containing one or two heteroatoms selected from oxygen, sulfur and nitrogen, for example azetidin-1-yl or -2-yl, Pyrrolidin-1-yl, -2-yl or -3-yl, piperidin-1-yl, -2-yl, -3-yl or -4-yl, hexahydroazin-1-yl, -2-yl, -3-yl or -4-yl, oxetan-2-yl or -3-yl, tetrahydrofuran-2-yl or -3-yl, tetrahydropyran-2-yl, -3 -Yl or -4-yl, hexahydrooxepin-2-yl, -3-yl or -4-yl, thiethan-2-yl or -3-yl, tetrahydrothiophen-2-yl or -3 -Yl, tetrahydrothiopyran-2-yl, -3-yl or -4-yl, hexahydrothiopin-2-yl, -3-yl or -4-yl, piperazin-1-yl or -2 -Yl, morpholin-1-yl, -2-yl or -3-yl, thiomorpholin-1-yl, -2-yl or -3-yl, tetrahydropyrimidin-1-yl, -2- 1, -4-yl or -5-yl, imidazolin-1-yl, -2-yl or -4-yl, imidazolidine -1-yl, -2-yl or -4-yl, oxazolin-2-yl, -3-yl, -4-yl or -5-yl, oxazolidin-2-yl, -3-yl, -4-yl or -5-yl, thiazolin-2-yl, -3-yl, -4-yl or -5-yl, or thiazolidin-2-yl, -3-yl, -4-yl Or -5- days.

The term "non-aromatic monocyclic carbocyclic group fused with one or two monocyclic aromatic or heteroaromatic groups" refers to an aromatic 5- containing 1 to 4 heteroatoms selected from benzene rings or oxygen, sulfur and nitrogen. To (6-C) cycloalkyl groups fused with 6-membered rings, such as indanyl, 1,2,3,4-tetrahydronaphth-1-yl or -2-yl, 5,6,7 , 8-tetrahydroquinolin-5-yl, -6-yl, -7-yl or -8-yl, 5,6,7,8-tetrahydroisoquinolin-5-yl, -6-yl, -7 -Yl or -8-yl, 4,5,6,7-tetrahydrobenzothiophen-4-yl, -5-yl, -6-yl or -7-yl, dibenzo [2,3,6, 7] cycloheptan-1-yl or -4-yl, dibenzo [2,3,6,7] cyclohept-4-en-1-yl or -4-yl, or 9-fluorenyl .

The term “non-aromatic monocyclic heterocyclic group fused with one or two monocyclic aromatic or heteroaromatic groups” refers to an aromatic 5-containing 1 to 4 heteroatoms selected from benzene rings or oxygen, sulfur and nitrogen. 4- to 7-membered rings containing 1 or 2 heteroatoms selected from oxygen, sulfur and nitrogen fused to 6-membered rings, for example 2,3-dihydrobenzopyran-2-yl, -3 -Yl or -4-yl, xanthen-9-yl, 1,2,3,4-tetrahydroquinolin-1-yl, -2-yl, -3-yl or -4-yl, 9,10- Dihydroacridin-9-yl or -10-yl, 2,3-dihydrobenzothiopyran-2-yl, -3-yl or -4-yl, or dibenzothiopyran-4-yl .

The term “nitrogen-protecting group” as used herein and expressed as “Pg ^N ” refers to a group that protects or blocks a nitrogen group against undesirable reactions during the course of the synthesis. The choice of suitable nitrogen-protecting groups to be used will depend on the conditions to be used in future reaction steps in which a protective reaction is required, which is within the knowledge of the skilled person. Commonly used nitrogen-protecting groups are described in TW Greene and PGM Wuts, Protective Groups In Organic Synthesis, 3 ^rd Ed. (John Wiley & Sons, New York (1999)). Preferred nitrogen-protecting groups are tert-butoxycarbonyl.

The term "carboxy-protecting group" as used herein and expressed as "Pg ^C " is one of the ester derivatives of carboxylic acid groups which are commonly used to block or protect carboxylic acid groups during the reaction on other functional groups of the compound. Indicates. Particularly useful groups are, for example, methyl, ethyl, tert-butyl, benzyl, methoxymethyl, trimethylsilyl and the like. Further examples of such groups are described in TW Greene and PGM Wuts, Protecting Groups in Organic Synthesis, 3 ^rd Ed. (John Wiley & Sons, New York (1999)). Preferred carboxy-protecting groups are methyl and ethyl. Esters are degraded using conventional procedures that do not affect other parts of the molecule.

The term “hydroxyl protecting group” refers to a group understood by one skilled in the art of organic chemistry, of the type described in Chapter 2 of Greene and Wuts. Representative hydroxyl protecting groups include, for example, ether groups, substituted ethyl ether groups, isopropyl ether groups, phenyl and substituted phenyl ether groups, benzyl and substituted benzyl ether groups, alkylsilyl ether groups, ester protecting groups and the like. have. The species of hydroxyl protecting group used is such that the derived hydroxyl group is stable to subsequent reaction conditions on different positions of the intermediate molecule and at an appropriate point in time without destroying the rest of the molecule, including any other hydroxyl protecting group. It does not matter as long as it can be selectively removed.

The term "amino acyl" refers to amino acyl derived from an amino acid selected from the group consisting of natural and unnatural amino acids as defined herein. Natural amino acids can be neutral, positive or negative, depending on the substituents on the side chain. "Neutral amino acid" means an amino acid containing an uncharged side chain substituent. Examples of neutral amino acids include alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine, glycine, serine, threonine, cysteine, glutamine and asparagine. "Positive amino acid" means an amino acid whose side chain substituents are positively charged at physiological pH. Examples of positive amino acids include lysine, arginine and histitin. "Negative amino acid" means an amino acid whose side chain substituents are negatively charged at physiological pH. Examples of negative amino acids are aspartic acid and glutamic acid. Preferred amino acids are α-amino acids. Most preferred amino acids are α-amino acids with L stereochemistry at α-carbons. Examples of natural α-amino acids are valine, isoleucine, proline, phenylalanine, tryptophan, methionine, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, lysine, arginine, hydridine, aspartic acid and glutamic acid.

"Non-natural amino acid" means an amino acid without a nucleic acid codon. Examples of non-natural amino acids include, for example, D-isomers of the natural α-amino acids shown above; Aib (aminobutyric acid), βAib (3-aminoisobutyric acid), Nva (norvaline), β-Ala, Aad (2-aminoadipic acid), βAad (3-aminoadipic acid), Abu (2-amino Butyric acid), Gaba (γ-aminobutyric acid), Acp (6-aminocaproic acid), Dbu (2,4-diaminobutyric acid), α-aminopimelic acid, TMSA (trimethylsilyl-Ala), alle (allo-isoleucine ), Nle (norleucine), tert-Leu, Cit (citrulline), Orn, Dpm (2,2'-diaminopimelic acid), Dpr (2,3-diaminopropionic acid), α- or β-Nal, Cha (cyclohexyl-Ala), hydroxyproline, Sar (sarcosine), O-methyl tyrosine, phenyl glycine and the like; Cyclic amino acids; N ^a - alkylated amino acids (at this time, N ^a - alkylated amino acids MeGly (N ^a - methyl glycine), EtGly (N ^{a-ethyl-glycine)} and EtAsn (N ^a - ethyl asparagine) and N ^a like (1 -10C) alkyl amino acids and amino acids where the α-carbon has two side chain substituents Examples of non-natural α-amino acids include D-alanine, D-leucine and phenylglycine. The names of natural amino acids and residues thereof are given in the IUPAC-IUB Biochemical Nomenclature Union Committee, listed in "Nomenclature and Symbolism for Amino Acid and Peptides (Recommendation, 1983)", European Journal of Biochemistry, 138, 9-37 (1984). In accordance with the name arrangement proposed by Joint commission on Biochemical Nomenclature (JCBN), where names and abbreviations of amino acids and residues thereof used in the present specification and the appended claims are different from those mentioned, other names and abbreviations will be apparent.

All compounds of formula (I) are useful active mGluR2 receptor agonists, but certain compounds are preferred. The following paragraphs define the preferred group.

A) A group of compounds of formula (I) wherein Q is glycyl, alanyl, valeyl, leucil, isoleucyl, prolyl, phenylalanyl, tyrosyl, tryptiphyl, methionyl, lysyl or serinyl.

B) Group of compounds of formula (I) wherein Q is alanyl.

C) Group of compounds of formula (I) wherein Q is methionyl.

D) Group of compounds of formula (I) wherein p is 1.

E) Group of compounds of formula (I) wherein p is 2.

F) Group of compounds of formula (I) wherein X is SO ₂ .

G) Group of compounds of formula (I) wherein X is CR ³ R ⁴ .

H) A group of compounds of Formula (I) wherein R ³ is fluoro and R ⁴ is hydrogen.

I) A group of compounds of Formula (I) wherein R ³ is hydroxy and R ⁴ is hydrogen.

J) A group of compounds of Formula I, wherein R ³ and R ⁴ together represent = O.

K) A group of compounds of Formula (I) wherein R ¹⁰ is hydrogen.

L) A group of compounds of Formula I, wherein R ¹⁰ is fluoro.

M) Group of compounds of formula (I) wherein R ¹¹ is hydrogen.

N) group of compounds of formula (I) which are free bases.

O) A group of compounds of formula I that are salts.

P) group of compounds of formula I which are hydrochloride salts.

Q) A group of compounds of formula I that are mesylate salts.

R) A group of compounds of formula (I) which are ecylate salts.

S) A group of compounds of formula I that are tosylate salts.

The contents of the above paragraphs can be combined to define further preferred groups of compounds.

Compounds of formula (I) are useful for the treatment of mammalian disorders, with the preferred mammal being human.

Compounds of the invention can be prepared by a variety of procedures, some of which are described in the following schemes. The particular order of steps necessary to produce the compound of formula I depends on the relative reactivity of the particular compound being synthesized, the starting compound and the substituted moiety. Some substituents may be excluded from the scheme below to clarify the reaction, but this does not limit the teachings of the scheme in any way. As will be appreciated by those skilled in the art, the substituents R ¹⁵ and R ¹⁶ represent suitable side chains which form the preferred amino acyl.

Essential starting materials used in the following schemes, if not commercially available, are standard techniques of organic and heterocyclic chemistry, techniques analogous to the synthesis of known structurally similar compounds, and procedures described in Preparations and Examples. (Including new procedures).

As shown in Scheme 1, the compound of formula I is converted in vivo via an enzyme or hydrolysis method to form a compound of formula II. In particular, crystalline forms of the compounds of formula I can be prepared according to the route outlined in Scheme 2 below.

The di-ester protected peptidyl compounds of formula (iii) are hydrolyzed with suitable bases (eg lithium hydroxide or sodium hydroxide) in a suitable solvent (eg THF or THF / water) to form a compound of formula (iv) It produces a di-acid protected peptidyl compound. The compound of formula (iv) may be deprotected with a suitable acid in a suitable solvent. The conditions can produce the corresponding acid salt of the di-acid peptidyl compound (represented by Formula I salt) as an amorphous solid, or directly as a crystalline solid, wherein X ″ represents the corresponding anion. The carboxylate salt can then be formed by introducing a cationic species with a reagent such as sodium acetate, Finally, the zwitterionic by treating the crystalline salt compound with an appropriate base. Compounds can be obtained.

For example, di-acid protected peptidyl compounds of formula (iv) provide deprotected hydrochloride salts as amorphous solids when treated with hydrogen chloride gas in a suitable solvent. The amorphous hydrochloride compound can then be crystallized from acetone and water to produce the crystalline hydrochloride salt compound. If a crystalline solid is formed directly, the reaction mixture can be filtered to afford crystalline salts. Treating the crystalline hydrochloride salt compound with sodium hydroxide to obtain a zwitterionic compound; Alternatively, treatment of the mesylate salt compound or tosylate salt compound with sodium hydroxide will produce a zwitterionic compound. Those skilled in the art will understand that compounds of formula I may be prepared in a procedure in which the indicated intermediates are not isolated.

The di-esters of formula (ii) are acylated with compounds of formula III using suitable coupling agents to give the di-ester protected peptidyl compounds of formula (iii). Alternatively, this conversion process can be accomplished using acid chlorides of the compound of formula III.

Suitable peptide coupling agents include dicyclohexylcarbodiimide (DCC), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (EDC), isobutyl chloroformate, diphenyl chlorophosphate, 2-chloro -4,6-dimethoxy-1,3,5-triazine (CDMT), bis (2-oxo-3-oxazolidinyl) phosphinic chloride and benzotriazol-1-yloxytris (dimethylamino) force Phonium hexafluorophosphate.

In Scheme 4 above, the compound of formula II (di-acid) is treated with a suitable carboxy-protecting material (e.g., catalytic hydrochloric acid or thionyl chloride and methanol or ethanol) to give the corresponding di-ester of formula (ii) To obtain. Alternatively, the compound of formula II may first be treated with a nitrogen-protecting substance (eg BOC ₂ O) to obtain a nitrogen-protected compound of formula (i). The compound of formula (i) is then treated with a carboxy-protecting substance (eg methyl iodide) in the presence of a base (eg potassium carbonate) followed by a nitrogen deprotection substance (eg hydrochloric acid or Trifluoroacetic acid) to give the compound of formula (ii).

Those skilled in the art will also recognize whether or not an appropriate protective agent may be necessary depending on the type of X. For example, if X represents CR ³ R ⁴ , where R ³ represents hydroxy and R ⁴ represents hydrogen, a person of ordinary skill in the art would be required before a suitable hydroxyl protecting group proceeds to any of the above schemes. I will understand.

Compounds of formula (II) are known in the art. For example, the preparation of these compounds can be found in US Pat. Nos. 5,688,826 ('826 patent) and 5,958,960 (' 960 patent).

A variety of improvements have been suggested that are superior to the methods disclosed above for the synthetic route of the compound of Formula II. This improvement involves the optical division of sulfur and alcohol oxidation as well as the various intermediates described below.

The first improvement involves the conversion process associated with oxidizing the compound of formula VII of the '826 patent to form a compound of formula V of the' 826 patent (lines 22-34 of column 8 of the '826 patent, and line 33 of column 7 Compounds of V).

Formula (VII) of the '826 Patent>

Formula (V) of the '826 Patent

Sulfur trioxide-pyridine complexes or trifluoroacetic anhydride in combination with DMSO have been found to be preferred for many oxidation methods known in the art.

The second improvement relates to the cleavage of the compound of formula III of the '826 patent mentioned after line 3 to 7 of the' 826 patent column 8 and line 1 of the column 6 (Formula III),

<Formula (III) of the '826 Patent>

Wherein R ² represents a carboxyl group

(R) -α-methylbenzylamine and quinoline have been found to be preferred. Particularly preferred is (R) -α-methylbenzylamine.

Also, as mentioned in lines 39 to 53 of the '826 patent column 8, when the sulfides of the formula III compound of the' 826 patent where X is sulfur are oxidized to produce the formula III compound of the '826 patent where X is sulfonyl It has been found preferred to use a basic aqueous system and hydrogen peroxide with the catalyst.

The following examples further illustrate the compounds of the present invention and methods of their synthesis. The following examples are not intended to limit the scope of the invention in any aspect and should not be considered as such. All experiments were performed under a positive pressure of anhydrous nitrogen or argon. All solvents and reagents were purchased from commercial sources unless otherwise indicated and used as purchased. Anhydrous tetrahydrofuran (THF) can be obtained by distillation from sodium or sodium benzophenone ketyl before use. Proton nuclear magnetic resonance ( ¹ H NMR) spectra are available on Bruker Avance II bay-500 (500 MHz), Bruker Avance I bay-200 (200 MHz) or Varian Inova / fold. Obtained on Ryan 300 / Varian 400 (500 MHz). Electrospray mass spectroscopy (ESI) was performed on an Agilent MSD / B instrument using acetonitrile / aqueous ammonium acetate as the mobile phase. Free Atomic Impact Mass Spectroscopy (FABMS) was performed on a VG ZAB-2SE instrument. Field desorption mass spectroscopy (FDMS) was performed using a VG 70SE or Varian MAT 731 instrument. Optical rotation was measured with a Perkin-Elmer 241 polarimeter. Chromatographic separations on Waters Prep 500 LCs were generally performed using a linear gradient of solvents described herein. Thin layer chromatography (TLC) was generally used to monitor for reaction completion. Thin layer chromatography provides two. E. Merck Kieselgel 60 F ₂₅₄ plates (5 cm × 10 m, 0.25 mm thick) were performed. Spots were detected using a combination of UV and chemical detection methods (plates were immersed in a solution of cerium ammonium molybdate [75 g ammonium molybdate and 4 g of cerium (IV) sulfate in 500 mL of 10% aqueous sulfuric acid) Heated at). Flash chromatography is described in Still, et al. Still, Kahn, and Mitra, J. Org. Chem., 43, 2923 (1978). Elemental analysis of carbon, hydrogen, and nitrogen can be measured on a Control Equipment Corporation 440 Elemental Analyzer, or by the Universal Complutense Analytical Center (Pacaltard, Madrid, Spain). Carried out by Facultad de Famacia. Melting points were measured with open glass capillaries on the Gallenkamp hot air melting point apparatus or the Buechi melting point apparatus and were not calibrated.

Abbreviations, symbols and terms used in the examples have the following meanings.

Ac = acetyl

Anal. Elemental Analysis

ATR = attenuated total internal reflection

Bn or Bzl = benzyl

Bu = Butyl

BOC = t-butoxycarbonyl

calcd = calculated value

D ₂ O = deuterium oxide

DCC = dicyclohexylcarbodiimide

DCM = 1,2-dichloromethane

DIBAL-H = Hydrogenated Diisobutyl Aluminum

DMAP = 4-dimethylaminopyrimidine

DMF = dimethylformamide

DMSO = dimethyl sulfoxide

DSC = differential scanning calorimetry

EDC = N-ethyl-N'N'-dimethylaminopropyl carbodiimide hydrochloride

ES = Electrospray

Et = ethyl

EtOH = ethanol

FAB = Fast Atomic Impact (Mass Spectroscopy)

FDMS = Field Tally Mass Spectrum

FTIR = Fourier transform infrared spectroscopy

HOAt = 1-hydroxy-7-azabenzotriazole

HOBt = 1-hydroxybenzotriazole

HPLC = high performance liquid chromatography

HRMS = high resolution mass spectrum

i-PrOH = isopropanol

IR = infrared spectrum

L = liter

Me = methyl

MeOH = Methanol

MPLC = medium pressure liquid chromatography

Mp = melting point

MTBE = t-butyl methyl ether

NBS = N-bromosuccinimide

NMR = nuclear magnetic resonance

PC-TLC = preparative centrifugation thin layer chromatography

Ph = phenyl

p.o. = Oral administration

i-Pr = isopropyl

Rocelle's salt = potassium sodium tartrate

rt = room temperature

SM = starting material

TBS = tert-butyldimethylsilyl

TEA = triethylamine

Temp. = Temperature

TFA = trifluoroacetic acid

THF = tetrahydrofuran

TLC = thin layer chromatography

t-BOC = tert-butoxycarbonyl

General Procedure A

EDC coupling reaction between amines and N-BOC- (L) -amino acids

The starting amino dialkyl ester (compound of formula ii, scheme 3) (1.0 equiv) was suspended in anhydrous dichloromethane under nitrogen. The corresponding N-Boc- (L) -amino acids (1.5-2.0 equiv), EDC (1.5-2.0 equiv), HOBt (1.5-2.0 equiv) and dimethylaminopyridine (DMAP, 0.1-0.2 equiv) were added sequentially . Unless otherwise stated, the reaction mixture was stirred at room temperature until complete reaction was determined by TLC. The reaction mixture was diluted with ethyl acetate and washed sequentially with saturated aqueous NaHCO ₃ and / or aqueous NaHSO ₄ and brine. After drying over sodium sulfate and evaporation in vacuo, the crude residue (compound of formula iii) was purified by silica gel chromatography using a suitable eluent (typically ethyl acetate / hexanes).

General procedure B

Anhydrous Coupling Reaction Between Amines and N-BOC- (L) -Isobutyl Anhydride

To a solution of the corresponding N-Boc- (L) -amino acid (1.5 equiv) in anhydrous dichloromethane (10 mL) in N-methyl morpholine (NMM, 1.5 equiv, 1 mL CH ₂ Cl ₂ under nitrogen at −20 ° C. ) Iso-butyl chloroformate (IBCF, 1.5 equiv, in 5 mL CH ₂ Cl ₂ ) was added dropwise at an internal reaction temperature not exceeding -15 ° C. The resulting reaction mixture was stirred at -20 ° C. for 30 minutes before (1S, 2S, 4S, 5R, 6R) -2- (2'-amino-propionylamino) -4-hydride in dichloromethane (10 mL) A -20 ° C solution of oxy-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester hydrochloride (1.0 equiv) was added at a rate such that the internal reaction temperature did not exceed -15 ° C. Once the reaction was complete, the cooling bath was removed and the reaction mixture was stirred at room temperature until the completion of the reaction was determined by TLC. The reaction mixture was diluted with ethyl acetate and washed sequentially with saturated aqueous NaHCO ₃ , aqueous NaHSO ₄ and brine. After drying over magnesium sulfate and evaporation in vacuo, the crude residue was purified by silica gel chromatography using an appropriate eluent (usually hexane / ethyl acetate).

General Procedure C

Sequential Removal of N-Boc and Ester Protective Groups

The corresponding N-Boc diester peptide derivative (Compound of Formula iii, Scheme 2) (1.0 equiv) was stirred in a 1: 1 mixture of THF / 2.5N LiOH (10-20 equiv) at room temperature until 4 h. The reaction was diluted with H ₂ O and washed with ethyl acetate. The organic layer was removed. Adjust the pH of the aqueous phase to 2 with 1N HCl (increase the extractability by adding NaCl as needed in the aqueous phase), and all the N-boc dicarboxylic acid product (compound of formula iv) with ethyl acetate Extracted. All organic layers were combined, washed with brine, dried over MgSO ₄ and concentrated to dryness in vacuo to afford the desired carboxylate product as an effervescent solid. It was dissolved in ethyl acetate and refrigerated at 0 ° C. The reaction mixture was purged with anhydrous HCl gas until saturated with HCl. The resulting reaction mixture was stirred at 0 ° C. for 4 hours. The fully deprotected peptide derivative (compound of formula I) is isolated as its hydrochloride salt by filtration under N ₂ or by concentrating to dryness of the reaction mixture followed by trituration with ethyl acetate or Et ₂ O and concentration to a white powder. It was. Optionally, to remove residual solvent and excess HCl, the product was dissolved again in H ₂ O, frozen and then freeze dried to give the desired hydrochloride product.

Preparation Example 1

(1R, 4S, 5S, 6S) -4-amino-2,2-dioxo-2λ ⁶ -Thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid diethyl ester

(1R, 4S, 5S, 6S) -4-amino-2,2-dioxo-2λ ⁶ -thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid in 100 mL of 2B ethanol (10 g, 42.5 mmol, US Pat. No. 5,688,826) was added dropwise thionyl chloride (15.5 mL, 212.6 mmol) over 20 minutes at room temperature followed by washing with 40 mL of ethanol. The slurry was heated to reflux and stirred overnight. Analysis of concentrated aliquots via 500 MHz ¹ H NMR (CD ₃ OD) showed complete consumption of starting material and intermediate monoesters. The resulting solution was cooled to room temperature and then concentrated to gelatinous residue. To the gelatin was added EtOAc (50 mL), which was further concentrated to a solid and then diluted with additional EtOAc 94 mL. Final pH 7.95 was obtained by slowly adding 15% aqueous sodium carbonate (70 mL) to the mixture in manual rotation with manual rotation. The resulting sodium carbonate precipitate was filtered off and the layers extracted. The aqueous layer was extracted again with EtOAc (2 × 100 mL). The combined organic extracts were washed with brine (1 × 100 mL), dried (MgSO ₄ ), filtered and concentrated in vacuo to afford a pale yellow oil, which solidified to give the title compound as an off-white solid (11.71 g). , 95% yield).

Recrystallization

A mixture of the title compound (200 mg) in EtOAc (800 μl) was heated to 56 ° C. at which time a solution formed. After stirring at 56 ° C. for 15 minutes, heptane (1 mL) was added dropwise to the solution. The heating was stopped. The solution was cooled to 52 ° C., at which time a precipitate formed. Upon cooling and further dilution with heptane (600 μl), a slurry formed. The resulting slurry was stirred at rt for 1 h, then filtered, washed with heptane (2 × 500 μl) and dried at 45 ° C. overnight to yield 145 mg (73% recovery) of the title compound as a white solid.

Preparation Example 2

(1R, 4S, 5S, 6S) -4- (2'S-tert-butoxycarbonylamino-propionylamino) -2,2-dioxo-2λ ⁶ -Thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid diethyl ester

To a solution of N-Boc-L-alanine (43.52 g, 230 mmol) and N-methyl morpholine (25.5 mL, 232 mmol) in 457 mL methylene chloride iso-butyl chloroformate (30.4 mL) under nitrogen at −30 ° C. , 234 mmol) was added dropwise over 10 minutes. The resulting diluted slurry was stirred at -25 to -30 [deg.] C. for 30 minutes, at which time (1R, 4S, 5S, 6S) -4-amino-2,2-dioxo-2λ ⁶ -thia in 213 mL methylene chloride. A solution of -bicyclo [3.1.0] hexane-4,6-dicarboxylic acid diethyl ester (63.90 g, 219 mmol, compound of Preparation Example 1) was added over 25 minutes to give a reaction temperature of -25 ° C. Do not exceed. When the addition was complete, the cooling bath was removed and stirred at room temperature for 60 minutes, at which time the reaction temperature reached 19 ° C. and the color became pale orange. The reaction was treated with 350 mL of 1N HCl and the layers were separated. The organic layer was washed with saturated aqueous NaHCO ₃ (1 × 350 mL) and brine (1 × 350 mL), dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo to give a white foam (105.2 g, 104%).

Preparation Example 3

(1R, 4S, 5S, 6S) -4- (2'S-tert-butoxycarbonylamino-propionylamino) -2,2-dioxo-2λ ⁶ -Thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid

(1R, 4S, 5S, 6S) -4- (2'S-tert-butoxycarbonylamino-propionylamino) -2,2-dioxo-2λ ⁶ -thia-bicyclo [3.1.0 in 292 mL THF ] To a solution of hexane-4,6-dicarboxylic acid diethyl ester (181.4 g, theoretical 392 mmol, compound of Preparation 2) was added 490 mL (980 mmol) of 2N sodium hydroxide at room temperature. The biphasic mixture was vigorously stirred at room temperature for 1.25 h at which time the reaction was homogenized. The mixture was diluted with 490 mL of ethyl acetate and the layers separated. The aqueous layer was diluted with 490 mL of ethyl acetate and the pH of the mixture was lowered to 1.5 with concentrated HCl. The layers were separated and the aqueous layer was extracted again with 245 mL of ethyl acetate. The combined organic layers were dried (Na ₂ SO ₄ ), filtered and concentrated to give 167.9 g (105%) of the title compound as a white foam. This material was used in Examples 1 and 2 without characterization.

Preparation Example 4

(1R, 4S, 5S, 6S) -4-amino-2,2-dioxo-2λ ⁶ -Thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid dimethyl ester

Thionyl chloride (6.2 mL, 85.0 mmol) was dissolved in MeOH (170 mL, 5 ° C.) (1R, 4S, 5S, 6S) -4-amino-2,2-dioxo-2λ ⁶ -thia-bicyclo [3.1 .0] hexane-4,6-dicarboxylic acid (10.0 g, 42.5 mmol, US Pat. No. 5,688,826) was added dropwise to a high speed stirred suspension. When the addition was complete, the reaction mixture was allowed to warm slowly to room temperature and then for 48 hours under reflux. The volatiles were removed under reduced pressure and the residue was partitioned between saturated solution of NaHCO ₃ (200 mL) and ethyl acetate (400 mL). The layers were separated and the aqueous layer was extracted with ethyl acetate (2 × 400 mL each time). The combined organic layers were dried over K ₂ CO ₃ and concentrated under reduced pressure to yield 8.10 g (30.8 mmol) of the title compound in 72% yield.

Preparation Example 5

(1R, 4S, 5S, 6S) -4- (2'S-tert-butoxycarbonylamino-3'-phenyl-propionylamino) -2,2-dioxo-2λ ⁶ -Thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid dimethyl ester

Commercially available N-BOC- (L) -phenylalanine and (1R, 4S, 5S, 6S) -4-amino-2,2-dioxo-2λ ⁶ -thia-bicyclo [3.1.0] hexane-4 Prepared according to General Procedure A, using 6-dicarboxylic acid dimethyl ester (compound of Preparation Example 4). The reaction mixture was refluxed overnight. Purification by PC-TLC (4 mm SiO ₂ rotor) (10% ethyl acetate / hexanes → 100% ethyl acetate). 0.85 g (88%, 1.67 mmol) of a white foam were obtained.

Preparation Example 6

(1R, 4S, 5S, 6S) -4- (2'S-tert-butoxycarbonylamino-3'S-methyl-pentanoylamino) -2,2-dioxo-2λ ⁶ -thia-bicyclo [3.1.0 Hexane-4,6-dicarboxylic acid dimethyl ester

Commercially available N-BOC- (L) -isoleucine and (1R, 4S, 5S, 6S) -4-amino-2,2-dioxo-2λ ⁶ -thia-bicyclo [3.1.0] hexane-4 Prepared according to General Procedure A, using 6-dicarboxylic acid dimethyl ester (compound of Preparation Example 4). The reaction mixture was refluxed overnight. Purification by PC-TLC (4 mm SiO ₂ rotor) (10% ethyl acetate / hexanes → 100% ethyl acetate). 0.75 g (83%, 1.57 mmol) of a white foam were obtained.

Preparation Example 7

(1S, 4S, 5S, 6S) -4- (2'S-tert-butoxycarbonylamino-3'-methyl-butyrylamino) -2,2-dioxo-2λ ⁶ -Thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid dimethyl ester

Commercially available N-BOC- (L) -valine and (1R, 4S, 5S, 6S) -4-amino-2,2-dioxo-2λ ⁶ -thia-bicyclo [3.1.0] hexane-4 Prepared according to General Procedure A, using 6-dicarboxylic acid dimethyl ester (compound of Preparation Example 4). The reaction mixture was refluxed overnight. Purification by PC-TLC (4 mm SiO ₂ rotor) (10% ethyl acetate / hexanes → 100% ethyl acetate). 0.41 g (47%, 0.89 mmol) of a white foam were obtained.

Preparation Example 8

(1R, 4S, 5S, 6S) -4- (2'S-tert-butoxycarbonylamino-4'-methyl-pentanoylamino) -2,2-dioxo-2λ ⁶ -Thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid dimethyl ester

Commercially available N-BOC- (L) -leucine monohydrate and (1R, 4S, 5S, 6S) -4-amino-2,2-dioxo-2λ ⁶ -thia-bicyclo [3.1.0] hexane Prepared according to General Procedure A using -4,6-dicarboxylic acid dimethyl ester (compound of Preparation Example 4). The reaction mixture was refluxed overnight. Purification by PC-TLC (4 mm SiO ₂ rotor) (10% ethyl acetate / hexanes → 100% ethyl acetate). 0.85 g (94%, 1.78 mmol) of white foam were obtained.

Preparation Example 9

(1R, 4S, 5S, 6S) -4- (2'S.6'-bis-tert-butoxycarbonylamino-hexanoylamino) -2,2-dioxo-2λ ⁶ -Thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid dimethyl ester

Commercially available N-BOC-Lys (BOC) -OH and (1R, 4S, 5S, 6S) -4-amino-2,2-dioxo-2λ ⁶ -thia-bicyclo [3.1.0] hexane- Prepared according to General Procedure A using 4,6-dicarboxylic acid dimethyl ester (compound of Preparation Example 4). The reaction mixture was refluxed overnight. Purification by PC-TLC (4 mm SiO ₂ rotor) (10% ethyl acetate / hexanes → 100% ethyl acetate). 1.04 g (93%, 1.76 mmol) of a white foam were obtained.

Preparation Example 10

(1R, 4S, 5S, 6S) -4- [2'S-tert-butoxycarbonylamino-4 '-(trityl-carbamoyl) -butyrylamino] -2,2-dioxo-2λ ⁶ -Thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid dimethyl ester

Commercially available N-BOC- (L) -glutamine (Trt) -OH and (1R, 4S, 5S, 6S) -4-amino-2,2-dioxo-2λ ⁶ -thia-bicyclo [3.1. 0] hexane-4,6-dicarboxylic acid dimethyl ester (compound of Preparation Example 4) was used and prepared according to General Procedure A. The reaction mixture was refluxed overnight. Purification by PC-TLC (4 mm SiO ₂ rotor) (10% ethyl acetate / hexanes → 100% ethyl acetate). 0.53 g (48%, 0.72 mmol) of a white foam were obtained.

Preparation Example 11

(1R, 4S, 5S, 6S) -4-[(1'-tert-butoxycarbonyl-pyrrolidine-2'S-carbonyl) -amino] -2,2-dioxo-2-thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid dimethyl ester

Commercially available Boc- (L) -proline (0.61 g, 2.9 mmol) and (1R, 4S, 5S, 6S) -4-amino-2,2-dioxo-2λ ⁶ -thia-bicyclo [3.1. 0] hexane-4,6-dicarboxylic acid dimethyl ester (0.5 g, 1.9 mmol, compound of Preparation Example 4) was used and prepared according to General Procedure A. Purification using PC-TLC (ethyl acetate / hexane) gave 0.87 g (99.2%) of the title compound.

Preparation Example 12

(1R, 4S, 5S, 6S) -4- (2'S-tert-butoxycarbonylamino-4'-methylsulfanyl-butyrylamino) -2,2-dioxo-2λ ⁶ -Thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid dimethyl ester

Commercially available Boc- (L) -methionine (0.71 g, 2.9 mmol) and (1R, 4S, 5S, 6S) -4-amino-2,2-dioxo-2λ ⁶ -thia-bicyclo [3.1. 0] hexane-4,6-dicarboxylic acid dimethyl ester (0.5 g, 1.9 mmol, compound of Preparation Example 4) was used and prepared according to General Procedure A. Purification using PC-TLC (ethyl acetate / hexane) afforded 0.85 g (90.5%) of the title compound.

Preparation Example 13

( 1R, 4S, 5S, 6S) -4- [2'S-tert-butoxycarbonylamino-3 '-(1-tert-butoxycarbonyl-1H-indol-3-yl) -propionylamino]- 2,2-dioxo-2λ ⁶ -thia -bicyclo [3.1.0] hexane-4,6-dicarboxylic acid dimethyl ester

Commercially available Boc- (L) -Tryptophan (Boc) (1.1 g, 2.8 mmol) and (1R, 4S, 5S, 6S) -4-amino-2,2-dioxo-2λ ⁶ -thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid dimethyl ester (0.5 g, 1.9 mmol, compound of Preparation Example 4) was used and prepared according to General Procedure A. Purification using PC-TLC (ethyl acetate / hexane) gave 0.7 g (56.7%) of the title compound.

Preparation Example 14

(1R, 4S, 5S, 6S) -4- [2'S-tert-butoxycarbonylamino-3 '-(4-tert-butoxycarbonyloxy-phenyl) -propionylamino] -2,2-di Oxo-2λ ⁶ -Thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid dimethyl ester

Commercially available 2S-tert-butoxycarbonylamino-3- (4-tert-butoxycarbonyloxy-phenyl) -propionic acid (1.1 g, 2.9 mmol) and (1R, 4S, 5S, 6S) -4 Using -amino-2,2-dioxo-2λ ⁶ -thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid dimethyl ester (0.5 g, 1.9 mmol, compound of Preparation Example 4) Prepared according to General Procedure A. Purification with PC-TLC (ethyl acetate / hexane) gave 0.94 g (79.0%) of the title compound.

Preparation Example 15

( 1R, 4S, 5S, 6S) -4- (3'-acetoxy-2'S-tert-butoxycarbonylamino-propionyl) amino-2,2-dioxo-2λ ⁶ -thiabicyclo [3.1. 0] hexane-4,6-dicarboxylic acid dimethyl ester

3-acetoxy-2S- (tert-butoxycarbonylamino) propionic acid (0.25 g, 1.0 mmol, compound of Preparation 44) and (1R, 4S, 5S, 6S) -4-amino-2,2-di Oxo-2λ ⁶ -thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid dimethyl ester (0.2 g, 0.8 mmol, compound of Preparation Example 4) was used, except that DMAP was not used. Prepared according to General Procedure A. Purification using PC-TLC (ethyl acetate / hexane) afforded 0.19 g (48.2%) of the title compound.

Preparation Example 16

(1R, 2S, 4R, 5R, 6R) -2-amino-4-fluorobicyclo [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester

(1R, 2S, 4R, 5R, 6R) -2-amino-4-fluorobicyclo [3.1.0] hexane-2,6-dicarboxylic acid (14.45 g, 71.12 mmol, US patent in 202 mL pure ethanol) To a slurry of No. 5,958,960) thionyl chloride (26 mL, 356 mmol) was added dropwise over 20 minutes at room temperature. The slurry was heated to reflux and stirred for 3 hours. Cool to room temperature and stir overnight. After the resulting solution was concentrated in vacuo, the residue was diluted with 136 mL of ethyl acetate and treated over 15 minutes while rotating manually with 306 mL of 10% aqueous sodium carbonate to reach a final pH of 10. The layers were separated and the aqueous layer was washed with ethyl acetate (1 × 136 mL). The combined organic extracts were washed with brine (1 × 136 mL), dried (MgSO ₄ ), filtered and concentrated in vacuo to give 17.07 g (93%) of the title compound as a white solid.

Preparation Example 17

(1R, 2S, 4R, 5R, 6R) -2- [2'S- (tert-butoxycarbonylamino) propionyl] amino-4-fluorobicyclo [3.1.0] hexane-2,6-dicarboxyl Acid diethyl ester

To a solution of N-Boc-L-alanine (38.62 g, 204 mmol) in 396 mL methylene chloride was added N-methyl morpholine (22.44 mL, 204 mmol) under nitrogen at −22 ° C., followed by isobutyl chloroformate. (26.48 mL, 204 mmol) was added dropwise over 15 minutes to ensure that the reaction temperature did not exceed -18 ° C. The resulting thin slurry was stirred at −20 ° C. for 30 minutes, at which time (1R, 2S, 4R, 5R, 6R) -2-amino-4-fluorobicyclo [3.1.0] hexane- in 247 mL of methylene chloride. A solution of 2,6-dicarboxylic acid diethyl ester (49.46 g, 191 mmol, compound of Preparation 16) was added over 40 minutes so that the reaction temperature did not exceed -16 ° C. When the addition was complete, the reaction was separated from the cooling bath and stirred at room temperature for 70 minutes, at which time the reaction temperature reached 15 ° C. and the color became pale orange. The reaction was treated with 408 mL of 1N HCl and stirred for 5 minutes before the layers were separated. The organic layer was washed with saturated aqueous sodium bicarbonate (1 × 408 mL), dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo to give a white foam (88.16 g).

Preparation Example 18

(1R, 2S, 4R, 5R, 6R) -2- [2'S- (tert-butoxycarbonylamino) propionyl] amino-4-fluorobicyclo [3.1.0] hexane-2,6-dicarboxyl mountain

(1R, 2S, 4R, 5R, 6R) -2- [2'S- (tert-butoxycarbonylamino) propionyl] -amino-4-fluorobicyclo [3.1.0] hexane-2, in 238 mL THF, To a solution of 6-dicarboxylic acid diethyl ester (88.16 g, 191 mmol, compound of Preparation 17) was added 238 mL (477 mmol) of 2N sodium hydroxide at room temperature. The two-phase mixture was stirred vigorously at room temperature for 2.5 hours, at which time the reaction was homogenized. The mixture was diluted with 238 mL of t-butyl methyl ether and then mixed and the layers separated. The aqueous layer was further diluted with 238 mL of water and filtered to remove particulate matter. The solution was treated with concentrated HCl (42.9 mL, 515 mmol) over 30 minutes and then seeded with the title compound and stirred for 1 hour. The resulting slurry was filtered, washed with water (2 × 100 mL) and dried in vacuo at 45 ° C. for 40 h to give 72.2 g of the title compound as a white solid. A portion of the solid (69.5 g) was stirred with 490 mL of acetone for 1 hour to produce a cloudy solution, which was filtered and washed with acetone (2 × 100 mL). The filtrate was concentrated in vacuo to give a white foam which was further vacuum dried at 45 ° C. for 16 hours to give 61.8 g (corrected for 86%, 12% (weight / weight) acetone) of the title compound. This material was used in Examples 14-18 without characterizing.

Preparation Example 19

( 1S, 2S, 4S, 5R, 6R) -4-acetyloxy-2- (tert-butoxycarbonyl) aminobicyclo [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester

(1S, 2S, 4S, 5R, 6R) -2- (tert-butoxycarbonyl) amino-4-hydroxybicyclo [3.1.0] hexane-2,6-dikar in dichloromethane (40 mL) To a solution of acid diethyl ester (1.50 g, 4.20 mmol, US 5958960), pyridine (0.365 mL, 4.62 mmol) and DMAP (0.513 g, 4.20 mmol) was added acetic anhydride (0.514 mL, 5.0 mmol) under nitrogen. . Stir at room temperature for 16 hours, dilute with dichloromethane and pour into 10% aqueous citric acid solution (50 mL). The organic layer was washed with water (50 mL) and brine (50 mL). Dry over MgSO ₄ , filter and concentrate in vacuo to afford the title compound as a white solid (1.295 g, 75%).

Preparation Example 20

(1S, 2S, 4S, 5R, 6R) -4-acetyloxy-2-aminobicyclo [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester

(1S, 2S, 4S, 5R, 6R) -4-acetyloxy-2- (tert-butoxycarbonyl) aminobicyclo [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester (1.25 g, 3.13 mmol, compound of Preparation 19) was dissolved in a solution of 95% TFA in dichloromethane (60 mL) and stirred at room temperature under nitrogen for 5 minutes. TFA / dichloromethane was then removed in vacuo. The crude product was dissolved in a suspension of NaHCO ₃ (1.00 g) in dichloromethane (50 mL) and stirred for 30 minutes. The suspension was filtered, washed with dichloromethane (3 × 25 mL) and concentrated in vacuo to give 916 mg (98%) of the product as a yellow oil.

Preparation Example 21

(1S, 2S, 4S, 5R, 6R) -4-acetyloxy-2- [2'S- (tert-butoxy) carbonylaminopropionyl] aminobicyclo [3.1.0] hexane-2,6-dicar Acid diethyl ester

(1S, 2S, 4S, 5R, 6R) -4-acetyloxy-2-aminobicyclo [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester (0.80) in anhydrous dichloromethane (50 mL) g, 1.94 mmol, compound of Preparation 20) and Boc-L-Ala (0.477 g, 2.52 mmol) under nitrogen at EDC (0.519 g, 2.72 mmol), HOBt (0.314 g, 2.32 mmol), catalytic DMAP (0.024 g, 0.19 mmol) and triethylamine (1.08 mL, 7.76 mmol) were added sequentially. After stirring for about 15 minutes at room temperature, the initial white suspension was completely dissolved. The reaction was stirred for 16 h, diluted with dichloromethane and washed with saturated aqueous NaHCO ₃ (50 mL), 1.0N HCl aqueous solution (3 × 20 mL) and saturated brine solution (40 mL). The organic layer was dried over MgSO ₄ , filtered and concentrated in vacuo. The resulting crude amide was purified by column chromatography (using a (4: 1) mixture of ethyl acetate and hexane as eluent) to give 638 mg (75%) of the product as a white solid.

Preparation Example 22

(1S, 2S, 4S, 5R, 6R) -2-amino-4-hydroxy-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester

(1S, 2S, 4S, 5R, 6R) -2-tert-butoxycarbonylamino-4-hydroxy-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester in ethyl acetate A solution of (5.0 g, 14.0 mmol, US 5958960) was cooled to 0 ° C. Anhydrous HCl (g) was bubbled into the solution until saturated and stirred for 0.5 h. The reaction was warmed to rt and stirred for 1 h. The reaction mixture was concentrated and partitioned between ethyl acetate and H ₂ O. The aqueous layer was treated with NaHCO ₃ (aq) and extracted with ethyl acetate. The organic layer was dried over K ₂ CO ₃ and concentrated to yield 2.2 g (59.7%) of a white solid.

Preparation Example 23

(1S, 2S, 4S, 5R, 6R) -2- (2'-tert-butoxycarbonylamino-acetylamino) -4-hydroxy-bicyclo [3.1.0] hexane-2,6-dicar Acid diethyl ester

Boc-glycine (458 mg, 2.62 mmol, Aldrich) and (1S, 2S, 4S, 5R, 6R) -2-amino-4-hydroxy-bicyclo [3.1.0] hexane-2,6- Dicarboxylic acid diethyl ester (450 mg, 1.75 mmol, compound of Preparation 22) was used and prepared according to General Procedure A. Purification was by eluting with a gradient of 50/50 → 20/80 hexanes / ethyl acetate on 35 g of silica. Yield: 560 mg (77%).

Preparation Example 24

(1S, 2S, 4S, 5R, 6R) -2- (2'S-tert-butoxycarbonylamino-3'-methyl-butyrylamino) -4-hydroxy-bicyclo [3.1.0] hexane-2 , 6-dicarboxylic acid diethyl ester

Boc-L-valine (569 mg, 2.62 mmol, Sigma) and (1S, 2S, 4S, 5R, 6R) -2-amino-4-hydroxy-bicyclo [3.1.0] hexane-2, 6-Dicarboxylic acid diethyl ester (450 mg, 1.75 mmol, compound of Preparation 22) was used and prepared according to General Procedure A. The crude material was purified by eluting over 35 g of silica with a gradient of 70/30 → 20/80 hexanes / ethyl acetate. Yield: 694 mg (87%) of white foam.

Preparation Example 25

(1S, 2S, 4S, 5R, 6R) -2- (2'S-tert-butoxycarbonylamino-4'-methyl-pentanoylamino) -4-hydroxy-bicyclo [3.1.0] hexane-2 , 6-dicarboxylic acid diethyl ester

Boc-L-leucine (606 mg, 2.62 mmol, Chemlog) and (1S, 2S, 4S, 5R, 6R) -2-amino-4-hydroxy-bicyclo [3.1.0] hexane-2 , 6-Dicarboxylic acid diethyl (450 mg, 1.75 mmol, compound of Preparation 22) was used and prepared according to General Procedure A. The crude material was purified by eluting over 35 g of silica with a gradient of 70/30 → 20/80 hexanes / ethyl acetate. 689 mg (84%) of white foam were obtained.

Preparation Example 26

(1S, 2S, 4S, 5R, 6R) -2- (2'S-tert-butoxycarbonylamino-3'S-methyl-pentanoylamino) -4-hydroxy-bicyclo [3.1.0] hexane-2, 6-dicarboxylic acid diethyl ester

Boc-L-isoleucine (606 mg, 2.62 mmol, Aldrich) and (1S, 2S, 4S, 5R, 6R) -2-amino-4-hydroxy-bicyclo [3.1.0] hexane-2, 6-Dicarboxylic acid diethyl ester (450 mg, 1.75 mmol, compound of Preparation 22) was used and prepared according to General Procedure A. The crude material was purified by eluting over 35 g of silica with a gradient of 70/30 → 20/80 hexanes / ethyl acetate. Yield: 731 mg (89%).

Preparation Example 27

(1S, 2S, 4S, 5R, 6R) -2- [2 '-(2-tert-butoxycarbonylamino-acetylamino) -acetylamino] -4-hydroxy-bicyclo [3.1.0] hexane -2,6-dicarboxylic acid diethyl ester

Boc-Gly-Gly (474 mg, 2.04 mmol) and (1S, 2S, 4S, 5R, 6R) -2-amino-4-hydroxy-bicyclo [3.1.0] hexane-2,6-dicarboxyl Prepared according to General Procedure A, using acid diethyl ester hydrochloride (400 mg, 1.36 mmol, compound of Preparation 22 without basic workup). DMAP was not used. 1 equivalent of triethylamine was added. The crude material was purified by eluting with ethyl acetate on 35 g of silica. Yield: 517 mg (81%).

HPLC: 16.755 min. Column: Symmetry C18, 3.5 μm, 4.6 × 150 mm. λ = 230 nM. Flow rate: 1 mL / min. Gradient: Water containing 10% -15% ACN / 0.1% TFA (25 min).

Preparation Example 28

(1S, 2S, 4S, 5R, 6R) -2- [2 '-(2S-tert-butoxycarbonylamino-propionylamino) -acetylamino] -4-hydroxy-bicyclo [3.1.0] Hexane-2,6-dicarboxylic acid diethyl ester

Boc-Ala-Gly (502 mg, 2.04 mmol) and (1S, 2S, 4S, 5R, 6R) -2-amino-4-hydroxy-bicyclo [3.1.0] hexane-2,6-dicarboxyl Prepared according to General Procedure A, using acid diethyl ester hydrochloride (400 mg, 1.36 mmol, compound of Preparation 22 without basic workup). DMAP was not used. 1 equivalent of triethylamine was added. The crude material was purified by eluting with ethyl acetate on 35 g of silica. Yield: 500 mg (76%).

Preparation Example 29

(1S, 2S, 4S, 5R, 6R) -2- (2'-tert-butoxycarbonylamino-3'-phenyl-propionylamino) -4-hydroxy-bicyclo [3.1.0] hexane- 2,6-dicarboxylic acid diethyl ester

Boc-L-phenylalanine (772 mg, 2.91 mmole) and (1S, 2S, 4S, 5R, 6R) -2-amino-4-hydroxy-bicyclo [3.1.0] hexane-2,6-dicarboxyl Acid diethyl ester (480 mg, 1.94 mmole, compound of Preparation 22) was used and prepared according to General Procedure A. The crude reaction mixture was concentrated, dissolved in dichloromethane, instantaneously evaporated through ethyl silica plug / DCM (1: 1) through a short silica gel plug and concentrated in vacuo to afford a yellow oil. This material was further purified by silica gel chromatography (eluted with 30% ethyl acetate / hexanes → 80% ethyl acetate / hexanes) to give 852 mg (87%) of the title compound.

Preparation Example 30

(1S, 2S, 4S, 5R, 6R) -2- [2'-tert-butoxycarbonylamino-4 '-(trityl-carbamoyl) -butyrylamino] -4-hydroxy-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester

Boc-L-Glutamine (trityl) -OH (1.40 g, 2.86 mmole) and (1S, 2S, 4S, 5R, 6R) -2-amino-4-hydroxy-bicyclo [3.1.0] hexane-2 Prepared according to General Procedure A, using 6-dicarboxylic acid diethyl ester (490 mg, 1.90 mmole, compound of Preparation 22), except not using DMAP. The crude reaction mixture was concentrated, dissolved in dichloromethane, briefly evaporated with ethyl acetate through a short silica gel plug and concentrated in vacuo to afford a yellow oil. This material was further purified by silica gel chromatography (eluted with 30% ethyl acetate / hexanes → 100% ethyl acetate / hexanes) to give 1.3 g (94%) of the title compound.

Preparation Example 31

(1S, 2S, 4S, 5R, 6R) -2- (2'S, 6'-bis-tert-butoxycarbonylamino-hexanoylamino) -4-hydroxy-bicyclo [3.1.0] hexane-2 , 6-dicarboxylic acid diethyl ester

Boc-L-lysine (Boc) -OH (910 mg, 2.63 mmole) and (1S, 2S, 4S, 5R, 6R) -2-amino-4-hydroxy-bicyclo [3.1.0] hexane-2, Prepared according to General Procedure A, using 6-dicarboxylic acid diethyl ester (450 mg, 1.75 mmole, compound of Preparation 22) and not adding DMAP to the reaction mixture. The crude reaction mixture was concentrated, dissolved in dichloromethane, briefly evaporated with ethyl acetate through a short silica gel plug and concentrated in vacuo to afford a yellow oil. This material was further purified by silica gel chromatography (eluted with ethyl acetate / hexanes (1: 1)) to give 800 mg (78%) of the title compound.

Preparation Example 32

(1S, 2S, 4S, 5R, 6R) -2- [2'-tert-butoxycarbonylamino-3 '-(trityl-carbamoyl) -propionylamino] -4-hydroxy-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester

Boc-L-asparagine (trityl) -OH (1.35 g, 2.84 mmole) and (1S, 2S, 4S, 5R, 6R) -2-amino-4-hydroxy-bicyclo [3.1.0] hexane-2 Prepared according to General Procedure A, using 6-dicarboxylic acid diethyl ester (476 mg, 1.85 mmole, compound of Preparation 22), except that no DMAP was added to the reaction mixture. The crude reaction mixture was concentrated, dissolved in dichloromethane, briefly evaporated with ethyl acetate through a short silica gel plug and concentrated in vacuo to afford a yellow oil. This material was further purified by silica gel chromatography (eluted with 20% ethyl acetate / hexanes → 50% ethyl acetate / hexanes) to yield 1.07 g (81%) of the title compound.

Preparation Example 33

(1S, 2S, 4S, 5R, 6R) -2- [2'-tert-butoxycarbonylamino-3 '-(1'-tert-butoxycarbonyl-1H-indol-3'-yl)- Propionylamino] -4-hydroxy-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester

Boc-L-Tryptophan (Boc) -OH (1.18 g, 2.91 mmole) and (1S, 2S, 4S, 5R, 6R) -2-amino-4-hydroxy-bicyclo [3.1.0] hexane-2, Prepared according to General Procedure A, using 6-dicarboxylic acid diethyl ester (520 mg, 1.90 mmole, compound of Preparation 22). The crude reaction mixture was concentrated, dissolved in dichloromethane, briefly evaporated with ethyl acetate through a short silica gel plug and concentrated in vacuo to afford a yellow oil. This material was further purified by silica gel chromatography (eluted with ethyl acetate / DCM (1: 1)) to give 1.2 g (92%) of the title compound.

Preparation Example 34

(1S, 2S, 4S, 5R, 6R) -2- (1'-tert-butoxycarbonyl-pyrrolidine-2'S-carbonyl) -4-hydroxy-bicyclo [3.1.0] hexane-2 , 6-dicarboxylic acid diethyl ester

Boc-L-proline and (1S, 2S, 4S, 5R, 6R) -2-amino-4-hydroxy-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester (Preparation Example 22 compound), and was prepared according to General Procedure A. The crude was purified on 110 g of silica on an ISCO system eluting with a gradient of 80% ethyl acetate / hexanes → 100% ethyl acetate to give 699 mg (84%) of the title compound.

Preparation 35

(1S, 2S, 4S, 5R, 6R) -2- [2'S-tert-butoxycarbonylamino-3 '-(4-tert-butoxycarbonyloxy-phenyl) -propionylamino] -4-hydrate Roxy-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester

2S-tert-butoxycarbonylamino-3- (4-tert-butoxycarbonyloxy-phenyl) -propionic acid and (1S, 2S, 4S, 5R, 6R) -2-amino-4-hydroxy-ratio The title compound was prepared according to General Procedure A using cyclo [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester (compound of Preparation 22). The crude material was purified on 110 g of silica on an ISCO system eluting with a gradient of 60% ethyl acetate / hexanes to 90% ethyl acetate / hexanes to give 1.09 g (91%) of the title compound.

Preparation Example 36

(1S, 2S, 4S, 5R, 6R) -2- (2'S-tert-butoxycarbonylamino-4'-methylsulfanyl-butyrylamino) -4-hydroxy-bicyclo [3.1.0] hexane -2,6-dicarboxylic acid diethyl ester

(Boc-L-methionine) and (1S, 2S, 4S, 5R, 6R) -2-amino-4-hydroxy-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester ( Compound of Preparation 22) was used and the title compound was prepared according to General Procedure A. The crude material was purified on 110 g of silica on an ISCO system eluting with a gradient of 80% ethyl acetate / hexanes → 100% ethyl acetate to afford 1.0 g (92%) of the title compound.

Preparation Example 37

(1S, 2S, 4S, 5R, 6R) -2- (2'S-tert-butoxycarbonylamino-propionylamino) -4-hydroxy-bicyclo [3.1.0] hexane-2,6-dicar Acid diethyl ester

Commercially available N-BOC- (L) -alanine and (1S, 2S, 4S, 5R, 6R) -2-amino-4-hydroxy-bicyclo [3.1.0] hexane-2,6-dicar Acid diethyl ester (compound of Preparation 22) was used and prepared according to General Procedure A. The reaction mixture was refluxed overnight. Purification by preparative HPLC (1 × 500 g SiO ₂ column) (10% ethyl acetate / hexanes → 100% ethyl acetate). 3.0 g (90%, 7.00 mmol) of a white foam were obtained.

Preparation Example 38

(1S, 2S, 4S, 5R, 6R) -2- (2'S-amino-propionylamino) -4-hydroxy-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester hydro Chloride

(1S, 2S, 4S, 5R, 6R) -2- (2'S-tert-butoxycarbonylamino-propionylamino) -4-hydroxy-bicyclo [3.1.0] hexane in ethyl acetate (30 mL) A solution of -2,6-dicarboxylic acid diethyl ester (2.95 g, 6.88 mmol, compound of Preparation 37) was purged with anhydrous HCl gas at 0 ° C. until the solution was saturated with HCl. The resulting reaction mixture was stirred at 0 ° C. until completion of reaction was determined by TLC. The reaction mixture was purged with N ₂ for 30 minutes to remove excess HCl gas. The resulting suspension was concentrated to dryness in vacuo to give 2.47 g (98%, 6.78 mmol) of the desired amino hydrochloride salt. There was no need for further purification.

Preparation Example 39

(1S, 2S, 4S, 5R, 6R) -2- [2'S- (2S-tert-butoxycarbonylamino-propionylamino) propionylamino] -4-hydroxy-bicyclo [3.1.0] hexane -2,6-dicarboxylic acid diethyl ester

(1S, 2S, 4S, 5R, 6R) -2- (2'S-amino-propionylamino) -4-hydroxy-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester hydro Prepared according to General Procedure A, using chloride (0.2 g, 0.55 mmol, compound of Preparation 38) and Boc- (L) -alanine (0.16 g, 0.82 mmol), without using DMAP. Purification using PC-TLC (ethyl acetate / hexane) afforded 0.13 g (47.3%) of the title compound.

Preparation Example 40

(1S, 2S, 4S, 5R, 6R) -2- [2'S- (2-tert-butoxycarbonylamino-acetylamino) -propionylamino] -4-hydroxy-bicyclo [3.1.0] hexane -2,6-dicarboxylic acid diethyl ester

Boc-glycine (0.29 g, 1.6 mmol) and (1S, 2S, 4S, 5R, 6R) -2- (2'S-amino-propionylamino) -4-hydroxy-bicyclo [3.1.0] hexane-2 Prepared according to General Procedure A, using 6-dicarboxylic acid diethyl ester hydrochloride (0.4 g, 1.1 mmol, compound of Preparation 38) and without using DMAP. Purification using PC-TLC (ethyl acetate / hexane) afforded 0.14 g (26.2%) of the title compound.

Preparation Example 41

(1S, 2S, 4S, 5R, 6R) -2- [2 '-(2S-tert-butoxycarbonylamino-4-methyl-pentanoylamino) -propionylamino] -4-hydroxy-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester

Commercially available N-BOC- (L) -leucine monohydrate and (1S, 2S, 4S, 5R, 6R) -2- (2'S-amino-propionylamino) -4-hydroxy-bicyclo [3.1. 0] hexane-2,6-dicarboxylic acid diethyl ester hydrochloride (0.2 g, 0.55 mmol, compound of Preparation 38) was used and prepared according to General Procedure B. Purification by PC-TLC (4 mm, SiO ₂ rotor) (10% ethyl acetate / hexanes → 100% ethyl acetate). 0.54 g (62%, 1.00 mmol) of a white foam were obtained.

Preparation Example 42

(1S, 2S, 4S, 5R, 6R) -2- [2 '-(2S-tert-butoxycarbonylamino-3-methyl-butyrylamino) -propionylamino] -4-hydroxy-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester

Commercially available N-BOC- (L) -valine and (1S, 2S, 4S, 5R, 6R) -2- (2'S-amino-propionylamino) -4-hydroxy-bicyclo [3.1.0] Hexane-2,6-dicarboxylic acid diethyl ester hydrochloride (0.2 g, 0.55 mmol, compound of Preparation 38) was used and prepared according to General Procedure B. Purification by PC-TLC (4 mm, SiO ₂ rotor) (10% ethyl acetate / hexanes → 67% ethyl acetate). 0.36 g (43%, 0.68 mmol) of a white foam were obtained.

Preparation Example 43

(1S, 2S, 4S, 5R, 6R) -2- (2'S-acetylamino-propionylamino) -4-hydroxy-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester

Triethylamine (0.16 g, 1.6 mmol) followed by acetyl chloride (0.10 g, 1.18 mmol) was added sequentially with (1S, 2S, 4S, 5R, 6R) -2- in CH ₂ Cl ₂ (30 mL). (2'S-Amino-propionylamino) -4-hydroxy-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester hydrochloride (0.30 g, 0.82 mmol, compound of Preparation 38) The solution of was stirred at 0 ° C. The reaction mixture was warmed with stirring overnight. The reaction mixture was diluted with ethyl acetate (700 mL) and washed with aqueous NaHSO ₄ and brine. The organic layer was dried over magnesium sulfate. Purification by PC-TLC (4 mm, SiO ₂ rotor) (10% ethyl acetate / hexanes → 100% ethyl acetate) gave 0.24 g (79%, 0.65 mmol) of white foam.

Preparation Example 44

3-acetoxy-2S- (tert-butoxycarbonylamino) propionic acid

N-methyl morpholine (2.8 g, 27.2 mmole) and di-tert-butyl dicarbonate (5.8 g, 25.2 mmole) were added to O-acetyl-L-serine (3.7 g, 25.2 mmole) in 1: 1 dioxane: water. ) Solution. The reaction was stirred for 24 hours before partitioning between ethyl acetate and water. The aqueous layer was extracted with ethyl acetate and the organic layer was removed. The pH was adjusted to 0-1 with aqueous NaHSO ₄ . The aqueous layer was extracted with ethyl acetate, dried over Na ₂ S0 ₄ , filtered and concentrated in vacuo. Purification via flash chromatography yielded 2.6 g (41.7%) of compound. This material was used in Preparation 15 without characterizing.

Preparation Example 45

(1S, 5R) -3- (tert-butoxycarbonylamino) -3- (tert-butoxycarbonyl) -6-oxabicyclo [3.1.0] hexane

A solution of t-butyl methylmalonate (129 g, 0.75 mol) in THF (385 mL) was transferred to LiH (14.9 g, THF (900 mL) and N, N-dimethylpropylene urea (DMPU, 155 g, 1.2 mol). 1.875 mol) was added to the slurry over 30 minutes while maintaining the temperature at 0-5 ° C. The reaction mixture is heated to 65 ° C. and a solution of cis-1,4-dichloro-2-butene (95%, 100 g, 0.8 mol, 1.08 equiv) in THF (100 mL) is maintained at 63-67 ° C. Was added over 5.5 hours. The reaction was stirred at 65 ° C for 4 h. The reaction mixture was worked up with water / MTBE to give 1- (methoxycarbonyl) -1- (tert-butoxycarbonyl) cyclopent-3-ene.

The reaction solution containing 1- (methoxycarbonyl) -1- (tert-butoxycarbonyl) cyclopent-3-ene is cooled to 10-15 ° C. and 1N in a cooled solution (10-15 ° C.) NaOH (1.3 L, 1.3 mol) was added over 30 minutes. The reaction solution was stirred at 25 ° C. for 24 h and monitored by GC analysis. Once the hydrolysis reaction was complete, MTBE (645 mL) was added to the reaction mixture and the solution was stirred for 5 minutes. The layers were allowed to settle and separate. The organic layer was removed. 1.5M NaHSO ₄ solution (1470 mL) was added to make the aqueous layer acidic (pH 2-3). MTBE (1.3 L) was added and the layers were separated. The aqueous layer was extracted with MTBE (385 mL) and the combined organic layers were washed with 5% LiCl solution. The organic layer was concentrated in vacuo and diluted with heptane (780 mL). The solution was concentrated to approximately 500 mL and the resulting slurry was stirred at room temperature for 1 hour. The solid was filtered off, washed with heptane (250 mL) and dried in vacuo at 35 ° C. to yield 103.29 g (61% yield) of 1- (carboxylic acid) -1- (tert-butoxycarbonyl) cyclopent-3-ene. ) Was obtained as a white solid.

1- (carboxylic acid) -1- (tert-butoxycarbonyl) cyclopent-3-ene (50 g, 236 mmol) was added to 850 mL of 70:30 toluene: MTBE with a mechanical stirrer in a 1 L flask under nitrogen. Dissolved. Thionyl chloride (33.6 g, 283 mmol, 1.2 equiv) was added to the stirred reaction mixture while maintaining the temperature at 23 ° C. The reaction solution was cooled to 0-5 ° C and triethylamine (32.2 g, 318 mmol, 1.35 equiv) was added dropwise over 30 minutes. The reaction mixture was warmed to 23 ° C and stirred for 1 hour. The reaction mixture was added dropwise at high speed while maintaining the temperature at 20-25 ° C. to deionize water (625 mL). The layers were separated and the organic layer was washed with 500 mL of 1M NaHCO ₃ solution. The organic layer was concentrated to isolate 1- (chlorocarbonyl) -1- (tert-butoxycarbonyl) cyclopent-3-ene as a light yellow liquid.

A solution of ammonium bisulfate tetrabutyl (0.81 g, 2.4 mmol) in deionized water (700 mL) was added to sodium azide (20.16 g, 310 mmol). A solution containing 1- (chlorocarbonyl) -1- (tert-butoxycarbonyl) cyclopent-3-ene in MTBE / toluene was added to the azide solution over 45 minutes. The reaction mixture is stirred at 23 ° C. for 3 hours until 1- (chlorocarbonyl) -1- (tert-butoxycarbonyl) cyclopent-3-ene is consumed, which is confirmed by GC analysis of the reaction solution It was. The layers were separated and the organic layer was washed with 1M NaHCO ₃ (540 mL) and deionized water (540 mL then 270 mL). The organic layer was dried over Na ₂ S0 ₄ and filtered. The solution was concentrated in vacuo to afford 1- (acyl azide) -1- (tert-butoxycarbonyl) cyclopent-3-ene as an oil.

A solution of 1- (acyl azide) -1- (tert-butoxycarbonyl) cyclopent-3-ene was added to 110 mL of toluene at 95 ° C. over 1 hour. The release of nitrogen gas was controlled by the rate of addition under these conditions. MTBE was distilled from the reaction during the addition. The reaction was stirred at 95 ° C for 1 h and cooled to 23 ° C overnight. The solvent was concentrated in vacuo to afford 1- (isocyanate) -1- (tert-butoxycarbonyl) cyclopent-3-ene as an oil.

t-butanol (35 g, 471 mmol) was added to a solution of potassium t-butoxide (1M in THF, 471 mL, 471 mmol) under nitrogen. The reaction solution was cooled to 0 to 5 ° C., and the toluene solution containing 1- (isocyanate) -1- (tert-butoxycarbonyl) cyclopent-3-ene was kept for 60 minutes while maintaining the temperature at 0 to 10 ° C. Added over. The reaction was warmed to 23 ° C., stirred for 2 hours, and analyzed for scavenging of isocyanate starting materials by GC. The reaction mixture was added to a mixture of deionized water (1.2 L) and MTBE (1.2 L) at 15 ° C. The solution was stirred for 20 minutes and the layers separated. The organic layer was washed with 20% brine solution (250 mL) and the layers were separated. Distillation delivered the concentrated organic layer to approximately 250 mL. Heptane (500 mL) was added and the solution was concentrated to a total volume of 250 mL. The resulting slurry solution was cooled to 0 ° C., stirred for 2 hours, and filtered. The filter cake was washed with cold heptane (2 × 100 mL) and dried in vacuo to give 34.54 g (1) of 1- (tert-butoxycarbonylamino) -1- (tert-butoxycarbonyl) cyclopent-3-ene (1 -(Carboxylic acid) -1- (tert-butoxycarbonyl) cyclopent-3-ene) was obtained as a white solid).

A solution of 1- (tert-butoxycarbonylamino) -1- (tert-butoxycarbonyl) cyclopent-3-ene (20.00 g, 71 mmol) in 100 mL EtOAc was added with ammonium bisulfate tetrabutyl (4.08 g). , 10 mmol, 0.17 equiv), magnesium monoperoxyphthalate hydrate (MMPA, 52.4 g, 4.76% active oxygen = 2.1 equiv) and deionized water (100 mL) were added. The reaction was stirred for 19 hours with a mechanical stirrer. A solution of sodium sulfite (18 g) in deionized water (100 L) was added to the reaction mixture over 30 minutes. The mixture was diluted with 100 mL of EtOAc and the layers separated. The organic layer was washed with deionized H ₂ O (100 mL), 2N NaOH (2 × 100 mL) and deionized H ₂ O (2 × 100 mL). The organic layer was distilled at 75-90 ° C. under atmospheric pressure until the volume reached 40 mL. The solution was cooled to 75 ° C. and hot heptane (120 mL) was added. The solution was cooled to 65 ° C and optionally seeded with the desired product. The resulting slurry was cooled to room temperature and then cooled in an ice bath for 1 hour. The product was filtered off, washed with 80 mL of cold 4: 1 heptane: EtOAc and then dried in vacuo at 40 ° C. to give 15.45 g (73% yield) of the title compound as a white solid. This compound can be further purified by recrystallization from 2: 1 isopropanol: water.

Preparation Example 46

(1R, 3R) -1,3-di (methylbenzylamino) propane dihydrochloride

(R) -α-methylbenzylamine (effective efficiency of 98% (ee), 121 g, 1 mol) was heated to 100 ° C. under nitrogen. Dibromopropane (25.4 mL, 50.5 g, 250 mmol) was added dropwise over 70 minutes. The mixture was heated for an additional 3 hours and then cooled to 80 ° C. Concentrated (50%) NaOH solution (30 mL) was added dropwise over 10 minutes. Water (30 mL) was added to dissolve the solids and the mixture was cooled to room temperature over 30 minutes. MTBE (100 mL) was added. 100 mL of water was added to dissolve the precipitate and the layers separated. The organic layer was washed with 50 mL brine, dried (Na ₂ SO ₄ ) and concentrated in vacuo to give 126.0 g of a light yellow oil. Excess (R) -α-methylbenzylamine was removed by distilling the oil under vacuum at 70 ° C. (heating temperature) using a 12-inch column. The vessel residue (68 g of crude diamine) was dissolved in 1 L of i-PrOH using a mechanical stirrer in a 2 L flask. Concentrated HCl (12 M, 45 mL, 540 mmol) was added dropwise over 10 minutes. Further 100 mL of i-PrOH was added and mixed to form a thick slurry. The mixture was stirred for 90 minutes and filtered. The cake was washed with i-PrOH and vacuum dried at 40 ° C. to give 64.32 g (72% yield based on dibromopropane) as a white solid.

Preparation 47

(1S) -1- (tert-butoxycarbonylamino) -1- (tert-butoxycarbonyl) -cyclopent-4-en-3-one

(1R, 3R) -1,3-di (methylbenzylamino) propane dihydrochloride (24.65 g, 69.3 mmol, compound of Preparation 46) was dissolved in 150 mL of water with stirring. To this solution was added 35 mL (175 mmol) of 5N NaOH solution followed by 150 mL of MTBE. After stirring for 15 minutes, the layers were separated and the aqueous layer was extracted with 100 mL of MTBE. The combined organic layers were washed with 100 mL brine, dried over Na ₂ SO ₄ and concentrated in vacuo to give 18.06 g (92% yield) of 1R, 3R-di (methylbenzylamino) propane as a colorless oil.

In a 500 mL four-neck flask equipped with a stirrer at the top, 20.59 g (75.3 ml) of 1R, 3R-di (methylbenzylamino) propane was dissolved in 30 mL of each dry MTBE and THF under N ₂ flow. The solution was cooled to -45 ° C and 59 mL (147 mmol) of n-BuLi solution (2.5M in hexane) was added dropwise over 17 minutes. The light yellow solution was stirred at -45 ° C for 2 hours. (1S, 3S, 5R) -3- (tert-butoxycarbonylamino) -3- (tert-butoxycarbonyl) -6-oxabicyclo [3.1.0] hexane (10.00 g) in 55 mL of dry THF , 33.4 mmol) was added dropwise over 28 minutes while maintaining the temperature below -40 ° C. The addition funnel was washed with additional 5 mL of THF. The reaction mixture was stirred at −45 ° C. for 18 hours and then quenched by the dropwise addition of 4N aqueous sulfuric acid (75 mL). The cooling bath was removed after the acid addition was complete. The layers were separated and the aqueous layer was extracted with 50 mL of MTBE. The combined organic layers were washed with 50 mL of each water and brine, dried over Na ₂ S0 ₄ and concentrated in vacuo to afford 9.78 g of crude 578242 as a white solid. The solid was dissolved in 33 mL of hot MTBE and heptane (66 mL) was added in several portions. After cooling the stirred solution to room temperature, the mixture was stirred at 0 ° C for 1 h. The solid was filtered off, washed with 2 × 10 mL of chilled 2: 1 heptane: MTBE, and dried in vacuo at 35 ° C. for 4 h (1S, 3R) -1- (tert-butoxycarbonylamino) -1- ( 8.52 g (85% yield) of tert-butoxycarbonyl) -3-hydroxycyclopent-4-ene were obtained as a white solid.

2,2,6,6-tetramethyl-1-piperidinyloxy (free radical) (TEMPO) (0.84 g, 5.3 mmol) and KBr (0.63 g, 5.3 mmol, 2 mL in water) were added to MTBE (200 mL Solution of (1S, 3R) -1- (tert-butoxycarbonylamino) -1- (tert-butoxycarbonyl) -3-hydroxycyclopent-4-ene (20 g, 66.8 mmol) in Was added. The reaction mixture was cooled to 0 ° C and a solution of NaOCl (3.14%, 240 g, 100 mmol) containing NaHCO ₃ (8.4 g) was added dropwise while maintaining the temperature below 5 ° C. The reaction was stirred at 0 ° C for 1 h and allowed to warm to rt. The layers were separated and the aqueous layer was extracted with MTBE (2 × 200 mL). The combined organic layers were washed with 200 mL of 1N HCl solution containing 2.21 g of KI, followed by 10% Na ₂ SO ₃ solution (200 mL). The organic layer was washed with water (2 x 200 mL) and concentrated to dryness in vacuo. The crude product was dissolved in MTBE (60 mL) at 50 ° C. and crystallized over 1 hour by addition of heptane (200 mL). The mixture was cooled to 0 ° C. over 2 hours and then filtered. The filter cake was washed with 100 mL of cold heptane: MTBE (65:35) and dried in vacuo to give 16.99 g (89% yield) of the title compound as a white solid.

Preparation Example 48

1- (2-tert-butoxy-2-oxoethyl) tetrahydrothiophenium bromide

t-Butyl bromoacetate (2.44 L, 16.52 mole, 1 equiv) in a 22 L flask using a water bath while maintaining the temperature at 15-25 ° C. in tetrahydrothiophene (2.19 L, 24.8 mole) in acetone (11.38 L) , 1.5 equivalent) over 30-60 minutes. The reaction was stirred for 22 hours and the sample was analyzed by ¹ H NMR to confirm that the reaction was complete. The precipitate was filtered off, washed with acetone (2 L) and dried in vacuo at 28-33 ° C. for 3 days to give 4.328 Kg (92.5% yield) of the title compound.

Preparation 49

(1S, 2S, 5R, 6R) -2- (tert-butoxycarbonylamino) -4-oxo-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid di-tert-butyl ester

KOtBu (42 mL, 42 mmol, 1M solution in THF, 2.5 equiv) in 1- (2-tert-butoxy-2-oxoethyl) in acetonitrile (30 mL) while maintaining the temperature below 5 ° C. under N ₂ To a 0 ° C. solution of tetrahydrothiophenium bromide (11.9 g, 42 mmol, 2.5 equiv, compound of Preparation 48) was added over 10 minutes. The cooled milky solution was stirred for 1.5 hours. Trifluoroethanol (6.9 g, 69 mmol, 4.1 equiv) was added dropwise. (1S) -1- (tert-butoxycarbonylamino) -1- (tert-butoxycarbonyl) -cyclopent-4-en-3-one (5 g, 16.8 mmol in acetonitrile (20 mL) , Compound of Preparation 47) and trifluoroethanol (13.3 g, 132 mmol, 7.9 equiv) were added over 5 minutes while maintaining the temperature at 3-5 ° C. The solution was stirred at 0-5 ° C. for 4.5 h. MTBE (155 mL) and H ₂ O (80 mL) were added to the cold reaction mixture. The layers were separated and the organic layer washed with H ₂ O (50 mL) followed by 20% brine (50 mL). The organic layer was concentrated to approximately 30 mL via atmospheric distillation. Heptane (100 mL) was added and the solution was concentrated. If necessary, additional heptane was added until the evaporation temperature of the distillate reached 93 ° C. THF (65 mL) was added to give a solution of the title compound.

Preparation 50

(1S, 2S, 5R, 6R) -2- (tert-butoxycarbonylamino) -4-oxo-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid di-tert-butyl ester

1.7 L of saturated K ₂ CO ₃ aqueous solution was added with 1- (2-tert-butoxy-2-oxoethyl) tetrahydrothiophenium bromide in 2.2 L of CH ₂ Cl ₂ (757 g, 2.67 mol, compound of Preparation 48). Was added to a 0 ° C. solution while maintaining the temperature below 10 ° C. After the two-phase mixture was stirred for 1.5 hours, 223 mL of 50% NaOH solution was added in several portions while maintaining the temperature below 5 ° C. The mixture was stirred for 3 hours and filtered. The salt was washed with CH ₂ Cl ₂ . The layers were separated and the aqueous layer was extracted with 600 mL of CH ₂ Cl ₂ . The combined organic layers were dried over solid K ₂ CO ₃ and concentrated in vacuo to give 533.3 g (98% yield) of (2-tert-butoxy-2-oxoethyl) tetrahydrothiophenium as a pale yellow oil. While stored in the freezer, the oil crystallized to yield an off-white solid.

474 mL (6.5 mol, 10 equiv) of trifluoroethanol was added to (1S) -1- (tert-butoxycarbonylamino) -1- (tert-butoxycarbonyl) -cyclopentane in 650 mL of CH ₂ Cl _2. To a 0 ° C. solution of 4-en-3-one (194 g, 653 mmol) was added. A solution of (2-tert-butoxy-2-oxoethyl) tetrahydrothiophenium (396 g, 1.96 mol) in 325 mL of CH ₂ Cl ₂ was added dropwise over 40 minutes while maintaining the temperature below 10 ° C. After 1 hour the ice bath was removed. Deionized water (680 mL) was added and the layers separated. The aqueous layer was extracted with 400 mL of CH ₂ Cl ₂ . The combined organic layers were washed with 500 mL brine, dried over Na ₂ SO ₄ and concentrated in vacuo to give 587 g of an amber oily solid. This solid was dissolved in CH ₂ Cl ₂ 400 mL, 5 as eluent: 1: 1 hexane: CH ₂ Cl ₂ and the elution through a silica gel plug using a 1.6 kg Total 13.2 L of eluent: methyl t- butyl ether Obtained. The eluate was concentrated to give 398.7 g of a white solid. The solid was dissolved in 3 L of refluxed 70:30 hexanes: MTBE. The solution was cooled to room temperature overnight then cooled in an ice bath for 1 hour. The solid was filtered off, washed with cold solvent (approximately 700 mL) and dried in vacuo at 35 ° C. (1S, 2S, 5R, 6R) -2- (tert-butoxycarbonylamino) -4-oxo-bicyclo 173 g (64% yield) of [3.1.0] hexane-2,6-dicarboxylic acid di-tert-butyl ester were obtained as a white solid.

Preparation Example 51

(1S, 2S, 4S, 5R, 6R) -2- (tert-butoxycarbonylamino) -4-hydroxy-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid di-tert- Butyl ester

The solution from preparation 49 was cooled to 0 ° C. under nitrogen and lithium tri-sec-butylborohydride (1-selectride ^™ Selectride ^™ , 1M in THF, 21 mL, 21 mmol) was added dropwise. . The reaction mixture was stirred at 0 ° C for 45 minutes. 2M sodium carbonate solution (31 mL) was added keeping the temperature below 8 ° C. A solution of 30% H ₂ O 2 (7.15 g, 63 mmol) in 20 mL of water was added dropwise while maintaining the temperature below 15 ° C. After stirring for 10 minutes, MTBE (210 mL) and deionized water (100 mL) were added. The layers were separated and the organic layer was washed with saturated Na ₂ SO ₃ solution (40 mL) and 1M NaHSO ₄ solution (40 mL). The organic layer was concentrated to approximately 90 mL volume by distillation at atmospheric pressure. Distillation was continued and heptane was added to maintain a constant volume. When the evaporation temperature of the distillate reached 93 ° C, the solution was cooled to 70 ° C and 7 mL of THF was added. The solution was cooled to 0 ° C and stirred for 1 hour. The solid was filtered off, washed with cold heptane (10 mL) and dried in vacuo at 50 ° C. (1S, 2S, 4S, 5R, 6R) -2- (tert-butoxycarbonylamino) -4-hydroxy- 4.68 g (67% yield) of bicyclo [3.1.0] hexane-2,6-dicarboxylic acid di-tert-butylester were obtained as a white solid.

Preparation Example 52

(1R, 2S, 4R, 5R, 6R) -2- (2'S-2 '-(tert-butoxycarbonylamino) propionyl) amino-4-fluoro-bicyclo [3.1.0] hexane-2, 6-dicarboxylic acid

(1S, 2S, 4S, 5R, 6R) -2- (tert-butoxycarbonylamino) -4-hydroxy-bicyclo [3.1.0] hexane-2,6 in CH ₂ Cl ₂ (230 mL) A solution of dicarboxylic acid di-tert-butyl ester (25.00 g, 60.5 mmol, compound of Preparation 51) was added to deoxofluor (trade name; Deoxofluor ^™ ) in CH ₂ Cl ₂ (105 mL) (16.05 g, 72.6 mmol, 1.2 equiv) to a -78 ° C solution over 2 hours. The reaction mixture was stirred for 1.5 hours before further 1.66 g of Deoxofluor® was added. The solution was stirred for 30 minutes and warmed to -10 ° C. Saturated NaHCO ₃ (105 mL) solution was added dropwise over 20 minutes while maintaining the temperature below 5 ° C. The pH of the aqueous layer was adjusted to 7 by adding 160 mL of saturated NaHCO ₃ solution. The mixture was allowed to warm to room temperature and the layers separated. The aqueous layer was extracted with 100 mL of CH ₂ Cl ₂ . The combined organic layers were washed with 250 mL brine and then dried over Na ₂ SO ₄ . The solvent was removed in vacuo and heptane (75 mL) was added to give the crude product. The resulting mixture was heated to 50 ° C. until all solids dissolved and stirred at room temperature for 24 hours. The mixture was cooled to 0 ° C. in an ice bath and filtered. The product was washed with cold heptane and dried in vacuo (1R, 2S, 4R, 5R, 6R) -2- (tert-butoxycarbonylamino) -4-fluoro-bicyclo [3.1.0] hexane-2 20.23 g (80% yield) of, 6-dicarboxylic acid di-tert-butyl ester were obtained as a white solid.

SOCl ₂ (21.29 g, 13.05 mL, 0.179 mole, 5 equiv) was dissolved in EtOH (abs, 149 mL) (1R, 2S, 4R, 5R, 6R) -2- (tert-butoxycarbonylamino) -4- A solution refluxed appropriately by dropwise addition over 10 minutes without cooling to a solution of fluoro-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid di-tert-butyl ester (14.87 g, 0.036 mole) Produced. The solution was refluxed overnight. The solvent was removed from the reaction under vacuum. The residue was dissolved in EtOAc (150 mL) and 10% Na ₂ CO ₃ solution (75 mL) was added dropwise over 5-10 minutes with stirring. The layers were separated and the aqueous layer was extracted with EtOAc (50 mL). The combined organic extracts were washed with brine (1 × 50 mL), dried over Na ₂ SO ₄ , filtered and the product (1R, 2S, 4R, 5R, 6R) -2-amino-4-fluoro-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester was concentrated in vacuo to give a thick liquid which was left to solidify (11.02 g).

N-methylmorpholine (22.44 mL, 204 mmol) is added to a solution of N-Boc-L-alanine (38.62 g, 204 mmol) in 396 mL methylene chloride at -22 ° C. under nitrogen followed by iso-butyl chloroformate (26.48 mL, 204 mmol) was added dropwise over 15 minutes to ensure that the reaction temperature did not exceed -18 ° C. The resulting thin slurry was stirred at −20 ° C. for 30 minutes before (1R, 2S, 4R, 5R, 6R) -2-amino-4-fluoro-bicyclo [3.1.0] hexane- in 247 mL of methylene chloride. A solution of 2,6-dicarboxylic acid diethyl ester (49.46 g, 191 mmol) was added over 40 minutes so that the reaction temperature did not exceed -16 ° C. The reaction was separated from the cooling bath and stirred at room temperature for 70 minutes. 408 mL of 1N hydrochloric acid was added, stirred for 5 minutes, and the layers separated. The organic layer was washed with saturated aqueous sodium bicarbonate (1 × 408 mL), dried (Na ₂ SO ₄ ), filtered and the product (1R, 2S, 4R, 5R, 6R) -2- (2'S-2 ' -(tert-butoxycarbonylamino) propionyl) amino-4-fluoro-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester was concentrated in vacuo to give a white foam (88.16 g Obtained).

46.7 mL (93.4 mmol) of 2N sodium hydroxide was added to crude (1R, 2S, 4R, 5R, 6R) -2- (2'S-2 '-(tert-butoxycarbonylamino) propionyl) amino in 46.6 mL of tetrahydrofuran. To a solution of -4-fluoro-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester (17.5 g, 37.3 mmol in theory) was added at room temperature. The two-phase mixture was stirred vigorously at room temperature until homogenized and then stirred for additional time (total 3 hours). The mixture was diluted with 46 mL of t-butyl methyl ether and then mixed for 10 minutes and the layers separated. In a separate flask, 93 mL of water was added followed by 8.4 mL (101 mmol) of concentrated HCl. Optionally (1R, 2S, 4R, 5R, 6R) -2- (2'S-2 '-(tert-butoxycarbonylamino) propionyl) amino-4-fluoro-bicyclo [3.1.0] hexane- The 2,6-dicarboxylic acid seed crystals are added to the acid solution followed by the aqueous layer from above. The aqueous layer was first added slowly to form a moderately thick slurry. At this time, the rate of addition was increased (total addition time 40 minutes). The addition funnel was washed with water (16 mL). The resulting slurry was stirred for 2 hours, filtered, washed with water (2 × 32 mL) and dried in vacuo at 45 ° C. to constant weight to give 13.9 g (99%) of the title compound as a white solid.

Preparation Example 53

(1R, 4S, 5S, 6S) -4-amino-2,2-dioxo-2λ ⁶ -Thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid diethyl ester

Thionyl chloride was added to (1R, 4S, 5S, 6S) -4-amino-2,2-dioxo-2λ ⁶ -thia-bicyclo [3.1.0] hexane-4,6-dicar in 100 mL of 2B ethanol. To a slurry of acid (10 g, 42.5 mmol, US Pat. No. 5,688,826) was added dropwise over 20 minutes at room temperature (15.5 mL, 212.6 mmol) followed by washing with 40 mL of ethanol. The slurry was heated to reflux and stirred overnight. The resulting solution was cooled to room temperature and concentrated to gelatinous residue. Ethyl acetate (50 mL) was added to the residue, followed by further dilution with 94 mL of ethyl acetate. 15% aqueous sodium carbonate (70 mL) was added slowly to the mixture while rotating manually to dissolve stepwise to give a final pH 7.95. Filter and separate layers. The aqueous layer was extracted with ethyl acetate (2 × 100 mL). The combined organic extracts were washed with brine (1 × 100 mL), dried (MgSO ₄ ), filtered and concentrated in vacuo to yield the title compound as a pale yellow oil which solidified to an off-white solid (11.71 g, 95% yield). .

Preparation Example 54

(1R, 4S, 5S, 6S) -4- (2'S-4'-methylthio-2 '-(tert-butoxycarbonyl) aminobutanyl) amino-2,2-dioxo-2λ ⁶ -Thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid diethyl ester

N-methyl morpholine (14.4 mL, 130.9 mmol) is added to a clear solution of N-Boc-L-methionine (32.64 g, 130.9 mmol) in 110 mL of methylene chloride under nitrogen at -22 ° C, followed by iso-butyl chloroform Mate (17 mL, 130.9 mmol) was added over 7 minutes to maintain the reaction temperature at -22 ° C. When the addition was complete, a thin slurry formed as a result. Stir at -22 to -26 ° C for 30 minutes. (1R, 4S, 5S, 6S) -4-amino-2,2-dioxo-2λ ⁶ -thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid diethyl ester in 107 ml methylene chloride A solution of (35.65 g, 122.4 mmol, compound of Preparation 53) was added for 15 minutes and then washed with 36 mL of methylene chloride. The reaction was separated from the cooling bath and stirred at room temperature for 70 minutes. 51 mL of 5N hydrochloric acid was added to the solution, and then the layers were separated. The aqueous layer was extracted again with methylene chloride (2 x 107 mL). The combined organic layers were washed with saturated aqueous sodium bicarbonate (1 × 10 7 mL), dried (MgSO ₄ ), filtered and concentrated in vacuo to give 65.82 g (103 wt% yield) of the title compound as a white foam.

Preparation Example 55

(1R, 4S, 5S, 6S) -4- (2'S-4'-methylthio-2 '-(tert-butoxycarbonyl) aminobutanyl) amino-2,2-dioxo-2λ ⁶ -Thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid monosodium salt

141 mL (282 mmol) of 2N sodium hydroxide was dissolved in 141 mL of tetrahydrofuran (1R, 4S, 5S, 6S) -4- (2'S-4'-methylthio-2 '-(tert-butoxycarbonyl) aminobuta Nonyl) amino-2,2-dioxo-2λ ⁶ -thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid diethyl ester (58.95 g, theoretical 112.8 mmol, compound of Preparation 54) To a solution of was added at room temperature. The mixture was stirred vigorously for 2 minutes at room temperature. The solution was diluted with 141 mL tert-butyl methyl ether and the layers separated. The aqueous layer was further diluted with 141 mL of water and the pH was lowered to 4.46 by dropwise addition of concentrated hydrochloric acid. Stirring for 10 minutes gave a thin slurry. Further concentrated hydrochloric acid was added to the slurry to drop the pH to 1.4 (using a total of 17 mL of concentrated HCl, 204 mmol). After stirring for 2 hours, the slurry was filtered. The cake was washed with water (2 × 118 mL) and dried under vacuum at 45 ° C. for 1 hour before transferring to a weighing pan. The cake was again dried in vacuo at 45 ° C. for 16 h and at 58 ° C. for 52.96 g (96 wt% yield) of the title compound as a white solid.

Preparation Example 56

(1R, 4S, 5S, 6S) -4- (2'S-4'-methylthio-2 '-(tert-butoxycarbonyl) aminobutanyl) amino-2,2-dioxo-2λ ⁶ -Thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid

397 mL (795 mmol) 2N sodium hydroxide (1R, 4S, 5S, 6S) -4- (2'S-4'-methylthio-2 '-(tert-butoxycarbonyl) aminobuta) in 480 mL tetrahydrofuran Nonyl) amino-2,2-dioxo-2λ ⁶ -thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid diethyl ester (166.15 g, 318 mmol, compound of Preparation 54) To the solution was added at room temperature. The mixture was stirred vigorously for 2 minutes at room temperature, at which time the reaction homogenized to form a clear pale yellow / green solution. Further stirred at room temperature for 2 hours. The solution was diluted with 480 mL of tert-butyl methyl ether and the layers separated. The aqueous layer was added dropwise to a solution of concentrated hydrochloric acid (71.5 mL, 858 mmol) in water (960 mL). Ethyl acetate (500 mL) was added followed by addition of the remaining aqueous layer to form an emulsion, which was further diluted with ethyl acetate (460 mL). The emulsion was stirred for 40 minutes, filtered and washed with water (2 × 250 mL). The layers of the filtrate were separated and the aqueous layer was extracted again with ethyl acetate (500 mL). The combined organic layers were washed with brine (75 mL), dried (MgSO ₄ ), filtered and concentrated in vacuo to yield 125.26 g (84%, calibrated yield) of the title compound as a white foam.

Preparation Example 57

Ethyl 2-bromo-2-fluoro-2- (3-oxocyclopetyl) acetate

8.45 mL (50.4 mmol) of triethylsilyl chloride was added to a suspension of activated Zn (45.8 mmol) in anhydrous acetonitrile (100 mL) at -20 ° C and the mixture was stirred for 5 minutes. 8.0 mL (57.3 mmol) of ethyl 2,2-dibromo-2-fluoroacetate was added and the mixture was stirred at -20 ° C for 90 minutes. 1.86 mL (22.9 mmol) of 2-cyclopenten-l-one were added and the reaction mixture was stirred overnight and the temperature was slowly raised to room temperature. 1N HCl (125 mL) and EtOAc (100 mL) were added. The organic layer was washed with saturated NaHCO ₃ (2 × 150 mL), water (2 × 150 mL) and brine (2 × 150 mL), dried over anhydrous MgSO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography (using EtOAc / hexane (1: 8) as eluent) to give the title compound (5.36 g, 88% overall yield) as a colorless oil as a mixture of diastereomers.

Zn activation: 10 mL of concentrated HCl was added to a suspension of 100 g of Zn dust in water (900 mL). The mixture was stirred for 20 minutes at room temperature. The water was decanted and the residue was washed with water (3 × 250 mL), acetone (3 × 150 mL) and ether (2 × 100 mL). The residue was dried overnight at 35 ° C. under reduced pressure.

Preparation 58

Ethyl (1RS, 5RS, 6RS) -6-fluoro-2-oxobicyclo [3.1.0] hexane-6-carboxylate

13.0 mL (75.0 mmol) of ethyldiisopropylamine was added 2.0 g (7.5 of ethyl 2-bromo-2-fluoro-2- (3-oxocyclopentyl) acetate (compound of Preparation 57) in DMF (8 mL). mmol) was added at 0 ° C and the mixture was stirred overnight at room temperature. A solution of 1N HCl (20 mL), water (15 mL) and EtOAc (75 mL) was added. The organic layer was extracted, washed with saturated NaHCO ₃ (2 × 100 mL), water (2 × 100 mL) and brine (2 × 100 mL), dried over anhydrous MgSO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography (using EtOAc / hexane (1: 4) as eluent) to give the title compound (1.17 g, 84% yield) as a colorless oil as a trans: cis 5: 1 mixture of isomers.

Preparation Example 59

(1RS, 2SR, 5RS, 6RS) -2-Spiro-5'-hydantoin-6-fluoro-bicyclo [3.1.0] hexane-6-carboxylic acid

Ethyl (1RS, 5RS, 6RS) -6-fluoro-2-oxobicyclo [3.1.0] hexane-6-carboxylate (0.1 g, 0.54 mmol, compound of Preparation 58) in EtOH (1 mL) And 1N NaOH (0.55 mL, 0.55 mmol) was stirred while cooling with ice for 10 minutes. 1N HCl was added dropwise to the mixture until the pH was 1 and the resulting mixture was partitioned between EtOAc and brine. The aqueous phase was extracted twice with EtOAc and the combined organic layers were dried over MgSO _{4 and} then concentrated under reduced pressure. A mixture of EtOH and residue in water (1: 1) (2 mL), (NH ₄ ) ₂ CO ₃ (0.31 g, 3.2 mmol) and KCN (0.11 g, 1.62 mmol) was stirred at 60 ° C. overnight. Cool in an ice bath and treat with 1N KHSO ₄ to acidify the mixture. The solvent was removed under reduced pressure and the residue was dissolved again in MeOH, filtered and concentrated in vacuo. The crude material could be used without further purification.

Preparation Example 60

(1R, 2S, 5R, 6R) -Diethyl-2- (2'R-tert-butoxycarbonylamino-propionylamino) -6-fluorobicyclo [3.1.0] hexane-2,6-dica Leboxylate and (1S, 2R, 5S.6S) -diethyl-2- (2'R-tert-butoxycarbonylamino-propionylamino) -6-fluoro-bicyclo [3.1.0] hexane -2,6-dicarboxylate

1.59 g (5.0 mmol) of Ba (OH) _{2 was} dissolved in (1R ^* , 2S ^* , 5R ^* , 6R ^* )-2-spiro-5'-hydantoin-6-fluoro-bicyclo [1] in water (10 mL). 3.1.0] to a solution of 0.32 g (1.5 mmol) of hexane-6-carboxylic acid (compound of Preparation Example 59), and the mixture was stirred at 105 ° C overnight. The pH of the solution was acidified to 1 with 1N HCl at 0 ° C. and then concentrated under reduced pressure. The residue was redissolved and concentrated several times until the solid was completely dried under pure EtOH under reduced pressure. 0.37 mL (5.0 mmol) of SOCl ₂ was added to the residue dissolved in pure EtOH (20 mL) at 0 ° C., and then the mixture was stirred at reflux for 5 hours. The solution was basified with saturated NaHCO ₃ and then EtOAc (25 mL) was added. The organic layer was separated and the aqueous layer extracted with additional EtOAc (2 × 25 mL). The combined organic layers were dried over anhydrous MgSO ₄ , filtered and concentrated under reduced pressure. The residue was dissolved in 20 mL of a mixture of DCM-DMF (4: 1), 0.73 g (1.9 mmol) of HATU, 0.26 g (1.9 mmol) of HoAt, and 0.35 g (1.8 mmol) of L-Ala. 2.7 mL (15.3 mmol) of diisopropylethylamine were added and stirred overnight under the mixture Ar. DCM (15 mL) was added, separated and washed with saturated NaHCO ₃ (2 × 25 mL), water (2 × 25 mL) and brine (2 × 25 mL). The organic layer was dried over anhydrous MgSO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography (using EtOAc / hexane (1: 2) as eluent) to give the title compound (0.33 g, 51% overall yield) as a colorless oil as a 1: 1 mixture of diastereomers.

The mixture of diastereomers was separated by chiral HPLC using the following analytical method: Chiralpak AD 10 m, 4.6 × 250 mm; Eluent: 10% IPA in hexane; Flow rate: 1.0 mL / min; UV: 215 nm. Isomer A retention time = 5.9 minutes. Isomer B retention time = 9.2 minutes.

Isomer A: (1R, 2S, 5R, 6R, 2'R) -diethyl-2- (2'-tert-butoxycarbonylamino-propionylamino) -6-fluoro-bicyclo [3.1.0 ] Hexane-2,6-dicarboxylate

Isomer B: (1S, 2R, 5S, 6S, 2'R) -diethyl-2- (2'-tert-butoxycarbonylamino-propionylamino) -6-fluoro-bicyclo [3.1.0 ] Hexane-2,6-dicarboxylate

Preparation Example 61

(1R, 2S, 5R, 6R) -2-Amino-6-fluoro-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid

A solution of 30 mg (0.07 mmol) of Isomer A of Preparation 60 compound in 6N HCl (2 mL) was refluxed overnight. The solvent was removed under reduced pressure, the residue was washed with ether, dissolved in MeOH (1 mL) and propylene oxide (2 mL) was added. The mixture was stirred at rt overnight. The solvent was decanted and the residue was washed with ether and dried over an Ar stream to give the title compound (12 mg, 85%) as a white solid.

Preparation Example 62

(1S, 2R, 5S, 6S) -2-Amino-6-fluoro-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid

Starting from the Isomer B solution of Preparation 60, the title compound was prepared essentially as Preparation 61.

Preparation Example 63

Ethyl (6S) -4-thiabicyclo [3.1.0] hex-2-ene-6-carboxylate

A 3-neck, 5-liter flask was equipped with a stirrer, thermocouple and Teflon addition tube, and 2000 ml of thiophene was added to the N ₂ inlet (d = 1.05 g / mL, ethyl diazoacetate solution in thiophene starting at 25.0 mol). Increased to 45 mol by adding 17.1 equivalents based on the total amount of ethyl diazoacetate, not corrected for the efficacy of EDA, 19.0 equivalents based on ethyl diazoacetate corrected for efficacy). Here, under N ₂ , 0.968 g (1.24 mmole) Rh ₂ (octanoate) ₄ , 0.0472 mol% based on ethyl diazoacetate not corrected for potency, 0.052 mole based on the number of moles of pure ethyl diazoacetate %, Johnson Mathey: Lot No. 059255001) was added. This suspension was heated to 46 ° C. and stirred at 46 ° C. for 10 minutes to dissolve to yield a green solution. Ethyl diazoacetate (90% purity, 2.63 mole, purely 2.37 mole, 1.00 equiv., Aldrich: Lot No. 17603PI) dissolved in 1600 mL of thiophene in this solution was charged with 300 g of a positive displacement pump. Added by. The addition rate is such that the total addition time is 8 hours; The rate of addition was lowered to allow maleate and fumarate ethyl esters to form. If no ethyl diazoacetate remained (approximately 30 minutes), the dark amber reactant (3,985 g) was cooled to 23 ° C. The reaction mixture was divided into portions and more portions (3240 g out of 3,985 g = 81.3%) were concentrated directly to oil. The crude product was passed through a washed film distillation apparatus at 1.5 Torr and 23 ° C. to degas the product and remove residual thiophene. The product was then distilled at 120 ° C. and 1.3 Torr. The title compound (pale yellow) was collected in 2 fractions (155.0 g and 32.2 g). As a result of analysis by HPLC, the efficacy for the two fractions was determined to be 78% and 76%, respectively. The distillate from above was dissolved in methanol (2 mL per 1 g of distillate), cooled to -10 ° C and crystallized by optionally seeding the solution if no growth of crystals was observed. Once crystal growth begins, the mixture is further cooled to -45 ° C, stirred for 2-3 hours, filtered and washed with cold (-45 ° C) methanol (1 x 1 mL per 1 g of distillate) It was. This material was vacuum dried at 24 ° C. to give the title compound as a white to off-white solid (80-85% recovery, and> 98% efficacy).

Preparation Example 64

Ethyl (4S, 6S-4-hydroxy-2-thiabicyclo [3.1.0] hexane-6-carboxylate

Boran-THF at 0 ° C. under nitrogen to a solution of ethyl (6S) -4-thiabicyclo [3.1.0] hex-2-ene-6-carboxylate (22.2 g, 131 mmol) in 136 mL tetrahydrofuran. The complex (98 mL, 98 mmol) was added over 15-20 minutes. After stirring for 30 min at 0 ° C, the reaction was warmed to 15 ° C and stirred until complete by HPLC (for 1.5-2 hours). The reaction was cooled to 0 ° C. and transferred with pre-cooled (0 ° C.) 1N pH 7 buffer solution over 10-15 minutes while maintaining the temperature at 0 ° C. To the mixture was added sodium perborate monohydrate (15.6 g, 157 mmol) as a solid five times to keep the temperature below 20 ° C. The solution was allowed to warm to room temperature and stirred for 1 hour before adding 222 mL of water. After stirring for 2 hours, the peroxide was dissolved in 24 mL of water, quenched by addition of sodium thiosulfate pentahydrate (9.7 g), followed by stirring for 10 minutes. The mixture was extracted with ethyl acetate (2 × 222 mL). The combined organic extracts were washed with saturated aqueous sodium bicarbonate (1 × 222 mL) and brine (1 × 222 mL) and then concentrated to dryness in vacuo. The crude product is dissolved in 1,2-dichloroethane (1 mL per gram crude oil) and onto a silica gel column (4 g silica gel per gram of crude oil packed and slurried in 15% ethyl acetate-heptane) Loaded. The column was eluted with 15% ethyl acetate-heptane until the product was visualized by TLC, at which time the solvent was replaced with 50% ethyl acetate-heptane. All fractions containing the title product were combined and concentrated to oil in vacuo. Overall yield was in the range of 55-65%.

Preparation 65

Ethyl (6S) -4-oxo-2-thiabicyclo [3.1.0] hexane-6-carboxylate

To a solution of dimethylsulfoxide (33.4 mL, 471 mmol) in 194 mL methylene chloride was slowly added trifluoroacetic anhydride (33.2 mL, 235 mmol) in 73 mL of methylene chloride at −70 ° C. over 30 minutes (temperature − Kept below 66 ° C). After stirring for 20 minutes, a solution of ethyl (4S, 6S) -4-hydroxy-2-thiabicyclo [3.1.0] hexane-6-carboxylate (34.1 g, 181 mmol) in 194 mL methylene chloride Was added over 60 minutes to keep the temperature below -60 ° C. After stirring for 1 hour, the reaction was treated with triethylamine (75.7 mL, 543 mmol) over 35 minutes to keep the temperature below -50 ° C. The reaction was stirred for an additional 1 hour at which time the cooling bath was removed and 400 mL of 2N hydrochloric acid was added. The layers are separated while warming to 0 ° C. and the organic layer is washed with 2N hydrochloric acid (1 × 300 mL), 1N aqueous sodium bicarbonate (1 × 670 mL) and water (1 × 300 mL) and then dried over sodium sulfate, It was filtered, concentrated in vacuo to a red oil, and left to solidify. The crude product was applied to a pad of silica gel (2 g per g of starting alcohol packed with methylene chloride) and eluted with methylene chloride (200-300 mL). All fractions containing the product were collected and concentrated to give the title compound as an orange / brown solid. Typical recoveries ranged from 85 to 90%.

Preparation 66

(6S, 11S) -8,10-dioxo-2-thiaspiro [bicyclo [3.1.0] hexane-4,5'-imidazolidine] -6-carboxylic acid

Ammonium carbonate (2.46 g, 25.6 mmol) and potassium cyanide (0.817 mg, 12.5 mmol) were combined in 19.9 mL of methanol and stirred for 30 minutes. The mixture was treated with a solution of ethyl (6S) -4-oxo-2-thiabicyclo [3.1.0] hexane-6-carboxylate (2.39 g, 12.8 mmol) in 19.9 mL methanol and the reaction was heated to 30 ° C. Heat and stir for 23 hours. The volatiles were evaporated and the residue was dissolved in 2.75 N sodium hydroxide (13.1 mL) and stirred for 1 hour. After dilution with 13.1 mL of water, the pH is lowered to 3.1 with concentrated hydrochloric acid and optionally (6S, 11R) -8,10-dioxo-2-thiaspiro [bicyclo [3.1.0] hexane-4 , 5'-imidazolidine] -6-carboxylic acid. The pH was lowered to 1.0 and the suspension was cooled to 0 ° C and stirred for 1.25 hours. The dark brown solid is collected, washed with cold water (2.3 mL and 0.8 mL) and dried overnight under vacuum at 40 ° C. (6S, 11R) -8,10-dioxo-2-thiaspiro [bicyclo [3.1.0 2.00 g (55%, corrected for purity) of] hexane-4,5'-imidazolidine] -6-carboxylic acid were obtained. The filtrate was diluted with 50 mL of ethyl acetate and treated with 18 g of sodium chloride. After stirring for 15 minutes, the layers were separated and the aqueous layer was further washed with ethyl acetate (2 x 50 mL). The combined organic extracts were dried over sodium sulfate, filtered, concentrated in vacuo to a slurry (about 5 mL), to which tert-butyl methyl ether (25 mL) was added and stirred overnight. The solid was collected, washed with tert-butyl methyl ether and dried under vacuum at 40 ° C. for 2 hours to convert 0.64 g (10%, corrected for purity) of the title compound into a 1: 1 mixture of diastereomeric hydantoins. Obtained. The second yield of this hydantoin was combined with the first yield and used for the next step.

Division

(R) -phenylglycinol (9.0 g, 65.7 mmol) was added to a slurry of racemic acid (15 g, 65.7 mmol, about 6: 1 ratio of diastereomeric hydantoin) in 300 mL of ethanol and 75 mL of water. Added. The mixture was heated to about 80 ° C. to dissolve. The dark solution was cooled slowly and a precipitate was observed at 40 to 45 ° C. The slurry was further cooled to 0 ° C and held for 1 to 1.5 hours. The solid was collected, washed with 4: 1 ethanol: water (1 × 60 mL, pre-chilled to 0 ° C.) (stirring) and dried under vacuum at 65 ° C. for 12-24 hours. Typical yields of split salts ranged from 37 to 45%, with> 98% degradation (de) and> 98% effective efficiency (ee) observed. The partitioned salt was dissolved in 6 volumes (mL / g) of water and then treated with 1.1 equivalents of concentrated HCl. The slurry was cooled to 0 ° C., stirred for 1 hour, then filtered, washed with 1 volume of cold water and dried at 60 ° C. under vacuum. Typical yields of the title compound were> 90% (both% degradation rate and% effective efficiency were> 99%).

Preparation Example 67

(6S, 11S) -2,2,8,10-tetraoxo-2-thiaspiro [bicyclo [3.1.0] hexane-4,5'-imidazolidine] -6-carboxylic acid

To a mixture of 6.8 mL of water, 0.7 mL of 50% aqueous sodium hydroxide and 186 mg (0.74 mmol) of tungstic acid, (6S, 11S) -8,10-dioxo-2-thiaspiro [bicyclo [3.1.0] hexane- 4,5'-imidazolidine] -6-carboxylic acid (3.4 g, 14.9 mmol) was added. The resulting solution was heated to 50 ° C. and treated slowly with 35% hydrogen peroxide (7.7 mL, 74.5 mmol) over 66 minutes. The reaction was then stirred at 47-48 ° C. for 5 hours, cooled to 0 ° C., filtered over a thin pad of celite and washed with cold water (1 × 2 mL). The filtrate was heated to 50 ° C. and treated with concentrated hydrochloric acid to a pH of 1.5. The slurry was cooled to room temperature and stirred overnight. While cooling to 0 ° C., the slurry was filtered, washed with cold water (2 × 2 mL) and dried in vacuo at 55 ° C. to constant weight to give 3.19 g (82%) of the title compound as a white solid:

Preparation Example 68

(1R, 4S, 5S, 6S) -4-[(2'S)-(2'-amino) -propionyl] amino- (2-sulfonylbicyclo [3.1.0] hexane) -4,6-dicarboxyl mountain

(6S, 11S) -2,2,8,10-tetraoxo-2-thiaspiro [bicyclo [3.1.0] hexane-4,5'-imidazolidine]-in stainless steel Parr reactor 6-carboxylic acid (2.50 g, 9.60 mmol) and 2N sodium hydroxide (24.0 mL, 48.0 mmol) were added. After the mixture was heated to 95 ° C. and stirred for 21 hours, the mixture was cooled to room temperature and treated with activated charcoal (1.25 g). The mixture was filtered through celite, the filtrate was concentrated to 17 g and diluted with H ₂ O to give 24 g weight. The pH was lowered to 6.5 with concentrated HCl and the mixture was heated to 62 ° C. Crystallization occurred after the pH was lowered to 2.5 using concentrated HCl. After cooling the suspension to 30 ° C, the pH was adjusted to 1.7 and the temperature was lowered to 5 ° C. After the suspension was maintained at this temperature for 18 hours, the solids were collected and washed with cold H ₂ O (2 × 2.9 mL). The white solid was dried under vacuum at 45 ° C. to give the title compound (1.81 g, 80%). The title compound was slurried in 10 volumes of water, heated to 85 ° C. for 3-4 hours, cooled to room temperature, stirred for 2-3 hours, filtered and washed with water (1 × 1 volume). The recovery was> 95%.

Preparation Example 69

Ethyl (6S) -2-oxobicyclo [3.1.O] acid-6-carboxylate

87.4 mL (585) of 1,8-diazabicyclo [5.4.0] undes-7-ene at room temperature in a suspension of (ethoxycarbonylmethyl) dimethyl sulfonium bromide (134 g, 585 mmol) in 486 mL of acetonitrile mmol) was added dropwise over 15 minutes. After stirring for 1 hour, the yellow mixture was treated with 2-cyclopenten-l-one (40 g, 487 mmol) over 10 minutes. The mixture was stirred overnight, at which point 480 mL of tert-butyl methyl ether was added and then washed with 1N hydrochloric acid (1 × 240 mL). The aqueous layer was washed with tert-butyl methyl ether (1 x 240 mL). The combined organic extracts were washed with brine (1 × 400 mL), dried (MgSO ₄ ), filtered and concentrated in vacuo to afford crude ethyl (6S) -2-oxobicyclo [3.1.0] hexane-6- Carboxylate was obtained as an orange solid (84.8 g). After distilling the crude material (˜138 ° C., 10 mmHg), the solidified distillate may be purified by slurrying in heptane, filtered and dried.

Preparation 70

(±) (6S) -2-oxobicyclo [3.1.0] hexane-6-carboxylic acid

In a solution of crude ethyl (6S) -2-oxobicyclo [3.1.0] hexane-6-carboxylate (30.2 g, 180 mmol, uncorrected) in 30 mL of ethanol, 89 mL of 2N sodium hydroxide at room temperature ( 178 mmol) was added. With stirring for 80 minutes, the reaction mixture was washed with tert-butyl methyl ether (1 × 90 mL) and the aqueous layer was treated with concentrated hydrochloric acid (18 mL) to reach a pH of 1.0. The mixture was treated with 15 g sodium chloride and then washed with ethyl acetate (3 x 90 mL). The combined organic extracts were dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo to yield 23.8 g (94%, uncorrected) of the title compound as an off-white solid.

Preparation Example 71

(+) (6S) -2-oxobicyclo [3.1.0] hexane-6-carboxylic acid N-benzyl-α-methylbenzylamine salt

Crude (±) (6S) -2-oxobicyclo [3.1.0] hexane-6-carboxylic acid in 119 mL 6: 1 ethyl acetate: ethanol (assuming 9.9 g, 84.9 mmol, 100% effect) To the solution of was added 18 g (85.1 mmol) of (S) -N-benzyl-α-methylbenzyl-amine at reflux temperature. While dissolving, the mixture was cooled and seeded at 52 ° C. optionally. While cooling to room temperature and stirring for an additional 13.5 hours, the crystals were collected and washed with 6: 1 ethyl acetate: ethanol (2 x 48 mL). Drying in vacuo gave 10.8 g (36%, 77% degradation) of the split salt as a solid.

The rate of degradation of the salts was determined by chiral GC analysis from methyl esters prepared as follows: 150 mg of divided salts were dissolved in 5 mL of methylene chloride and washed with 1N sulfuric acid (2 × 1 mL). The organic layer was dried, filtered, diluted with 2 mL of methanol and treated with 1 mL of 2 M trimethylsilyl diazomethane in hexanes. After stirring at room temperature for 15 minutes, the mixture was concentrated in vacuo to yield a methyl ester suitable for GC analysis. GC conditions: 30 m × 0.25 mm × 0.25 μβ-DEX 325 column, 140 ° C. oven temperature, helium carrier gas @ 1 mL / min, FID detection 250 ° C., 1 μl split 1: 100, sample in methylene chloride @ 1 mg / mL.

Preparation Example 72

Ethyl (6S) -2-oxobicyclo [3.1.0] hexane-6-carboxylate

2N sodium hydroxide 198 in a suspension of (6S) -2-oxobicyclo [3.1.0] hexane-6-carboxylic acid N-benzyl-α-methylbenzylamine salt (46.3 g, 132 mmol) in 200 mL of ethyl acetate. mL (198 mmol) was added. After mixing well, the layers were separated and the aqueous layer was washed with ethyl acetate (1 × 200 mL). The aqueous layer was treated with 18 mL (211 mmol) concentrated hydrochloric acid and 100 g sodium chloride. The mixture was stirred for 30 minutes and then washed with ethyl acetate (2 x 200 mL). The combined organics were dried (MgSO ₄ ), filtered and concentrated in vacuo to 18.3 g (99) partitioned acid [(+) (6S) -2-oxobicyclo [3.1.0] hexane-6-carboxylic acid] %) Was obtained as a white solid.

Then 10 g (71 mmol) of crude partitioned acid product from above were dissolved in 42 mL of ethanol and 4 mL (71 mmol) of concentrated sulfuric acid were added dropwise. The mixture was heated to 45 ° C and stirred for 75 minutes. While cooling to room temperature, 42 mL of water were added together with 20 mL of ethyl acetate and 12 g of sodium bicarbonate. While stirring for a few minutes, the mixture was washed with ethyl acetate (2 × 50 mL). The combined organics were dried (MgSO ₄ ), filtered and concentrated in vacuo to afford 11 g (92%) of crude ethyl (6S) -2-oxobicyclo [3.1.0] hexane-6-carboxylate as a white solid. Obtained as Crystallization from 6: 1 / heptane: tert-butyl methyl ether (3.5 mL per g substrate) gave the title compound in approximately 80% yield (> 98% effective efficiency; determined by chiral GC analysis).

Preparation Example 73

(6S) -6- (ethoxycarbonyl) bicyclo [3.1.0] hex-2-en-2-yl acetate

Of ethyl (6S) -2-oxobicyclo [3.1.0] hexane-6-carboxylate (380.1 g, 2.26 mol) and sulfuric acid (18M, 6.3 mL, 0.11 mol) in isopropenyl acetate (2.26 L) The mixture is heated at reflux for 2.5 hours using a Dean-Stark apparatus, where GC analysis shows the title compound to ethyl (6S) -2-oxobicyclo [3.1.0] hexane-6- A 9: 1 mixture of carboxylates is shown. After removing 950 mL of solvent by distillation over 1 hour, GC showed a product / starting material ratio of 17: 1. Additional isopropenyl acetate (900 mL) and concentrated H ₂ S0 ₄ (3.15 mL) were added and the mixture was stirred at reflux for an additional 15 h, at which time GC gave 27: 1 product / starting material. Indicated. After further 1.35 L of solvent was removed by distillation, the mixture was cooled to room temperature and then diluted with MTBE (2 L), H ₂ O (250 mL) and aqueous saturated NaHCO ₃ (600 mL). The layers were separated and the organic layer was washed with brine (400 mL). The combined aqueous layers were extracted with MTBE (400 mL) and the combined organic layers were dried (Na ₂ SO ₄ ), filtered and concentrated to dark red / brown oil (540 g). The crude oil was divided into two equal parts and filtered (eluted with 10: 1 / heptane: ethyl acetate) through a pad of flash SiO ₂ (713 g for each batch). Fractions containing product from both plugs were combined and concentrated to give the title compound as a yellow oil (460 g, 97%; corrected for solvent by 90% NMR). Purification by column chromatography on silica gel (eluted with ethyl acetate / hexanes (1: 5)) gave an analytically pure sample of the title compound as a colorless oil.

Preparation Example 74

Ethyl (3S, 1R, 6R) -7-oxa-5-oxotricyclo [4.1.0.0 <2,4>] heptan-3-carboxylate

(6S) -6- (ethoxycarbonyl) bicyclo [3.1.0] hex-2-en-2-yl acetate (212.2 g, 1.01 mol) and 2,3- in 2.02 L of 1,4-dioxane The mixture of dichloro-5,6-dicyano-1,4-benzoquinone (252.0 g, 1.11 mol) is heated to reflux and stirred for 17 hours, at which time GC analysis yields ethyl (6S) -4-jade It showed that sorbcyclo [3.1.0] hex-2-ene-6-carboxylate was completely converted. The mixture was cooled to rt and diluted with THF (564 mL). The mixture was cooled to 8 ° C and 1,8-diazabicyclo [5.4.0] undes-7-ene (377 mL, 2.52 mol) was added over 30 minutes to maintain the reaction temperature below 10 ° C. The mixture was then cooled to 5 ° C. and tert-butyl hydroperoxide (70% by weight in water, 210 mL, 1.51 mol) was added over 50 minutes while maintaining the reaction temperature below 9 ° C. After the mixture was stirred for an additional 50 minutes, the reaction was filtered and the brown cake was washed with MTBE (2 x 800 mL). 1.20 L of 1N HCl was added to the filtrate, and after mixing well, the layers were separated. The organic layer was washed sequentially with aqueous saturated NaHCO ₃ (1.20 L), aqueous saturated Na ₂ S ₂ O ₃ (1.20 L) and brine (600 mL). After the solution was dried (Na ₂ SO ₄ ), it was concentrated to orange sludge and diluted with 200 mL of heptane. The volatiles were evaporated to yield an orange solid, which was triturated with 350 mL of heptane, filtered and the cake washed with additional heptane (2 x 175 mL). The collected solid was dried under vacuum at rt for 17 h to give 138.7 g (75%) of the title compound as a brown-yellow solid. Crystallization from MTBE gave analytical pure sample of the title compound as a white solid.

Preparation 75

Ethyl (6S) -4-oxobicyclo [3.1.0] hex-2-ene-6-carboxylate

The title compound is commonly used in the preparation of ethyl (3S, 1R, 6R) -7-oxa-5-oxotricyclo [4.1.0.0 <2,4>] heptane-3-carboxylate in situ, but this mixture The reaction mixture containing was filtered and the filtrate was evaporated to give a brown solid to give an analytically pure sample of the title compound. The solid was resuspended in ethyl acetate, the suspension was filtered and the filtrate was concentrated. The residue was purified by chromatography on silica gel (ethyl acetate / hexane (1: 5 → 1: 2)) to afford the title compound, which was recrystallized from hot ethyl acetate and again using the above conditions. Purification by gave the title compound as a white solid.

Preparation Example 76

Ethyl (4S, 6S) -4-hydroxy-2-oxobicyclo [3.1.0] hexane-6-carboxylate

Stirred solution of ethyl (3S, 1R, 6R) -7-oxa-5-oxotricyclo [4.1.0.0 <2,4>] heptan-3-carboxylate (36.3 g, 0.20 mol) in 667 mL acetone Was treated sequentially with sodium acetate (36.1 g, 0.44 mol), sodium iodide (65.8 g, 0.44 mol) and acetic acid (27.5 mL, 0.48 mol). After the mixture was stirred at 30 ° C. for 15 h, acetone was removed in vacuo to yield a brown solid, which was partitioned between ethyl acetate (323 mL) and H ₂ O (323 mL). The layers were separated and the aqueous layer was washed with ethyl acetate (3 × 323 mL). The combined organics were washed sequentially with aqueous saturated Na ₂ S ₂ 0 ₃ (364 mL) and aqueous saturated NaHCO ₃ (364 mL). Each aqueous wash was extracted again with ethyl acetate (323 mL). The combined organics were dried (Na ₂ SO ₄ ), filtered and concentrated to red-brown oil, which was dissolved in 300 mL of ethanol. The volatiles were evaporated to give the title product as red-brown oil (41.8 g, 114%). Purification by column chromatography on silica gel (using ethyl acetate / hexanes (1: 2 → 2: 1)) followed by crystallization from hot MTBE yielded an analytically pure sample of the title compound as a white solid.

Preparation Example 77

Ethyl [2-[((1R) -1-phenylethyl) amino] (2S, 4S, 6R) -2-cyano-4-hydroxybicyclo [3.1.0] hexane-6-carboxylate

Ethyl (4S, 6S) -4-hydroxy-2-oxobicyclo [3.1.0] hexane-6-carboxylate (68.2 g ethanol contamination 60.0 in ethanol (332 mL) and H ₂ O (332 mL) Corrected with g, 0.326 mol) was added (R) -methylbenzylamine (46.3 mL, 0.359 mol) and NaCN (20.8 g, 0.424 mol) while maintaining the temperature at 20-25 ° C. Concentrated HCl (35.3 mL, 0.424 mol) was then added over 10 minutes while maintaining the reaction temperature. The dark brown mixture was stirred for 1 hour and then seeded optionally with the title compound, at which time it began to crystallize. The suspension was stirred for 1 hour before H ₂ O (664 mL) was added. After stirring the suspension for an additional 1.75 h, the title compound was collected as a tan solid, which was washed with H ₂ O (332 mL). Air was injected through the wet cake and filtered for 25 minutes before the material was used for nitrile hydrolysis directly (wet cake weight 145 g). The title compound does not degrade rapidly during vacuum drying at temperatures above 25 ° C., but small amounts of sample can be dried at room temperature under vacuum without decomposition.

Preparation Example 78

2-[((1R) -1-phenylethyl) amino] (2S, 4S, 6R) -4-hydroxybicyclo [3.1.0] hexane-2,6-dicarboxylic acid

Ethyl 2-[((1R) -1-phenylethyl) amino] (2S, 4S, 6R) -2-cyano-4-hydroxybicyclo [3.1.0] hexane-6- in DMSO (220 mL) To the solution of the carboxylate wet cake (0.326 mmol theoretical) was slowly added 30% H ₂ O ₂ (44.5 mL, 0.426 mol) while maintaining the temperature below 27 ° C. The temperature was lowered to 19 ° C and 5N NaOH (52.3 mL, 0.262 mol) was added slowly and carefully over the first 15 minutes while maintaining the temperature at 22-27 ° C. Appropriate capacity of ice bath was needed to handle the exothermic reaction of this reaction. After stirring for 20 minutes with a brown heterogeneous mixture in this temperature range, HPLC showed that the starting material was consumed to produce an amide intermediate. After the reaction was stirred for an additional 1.5 hours, Na ₂ SO ₃ (13.7 g, 0.109 mol) was added and the mixture was stirred for 15 minutes, at which time the mixture was peroxed with starch-iodide paper. Seeds were tested negatively. After addition of 3N NaOH (291 mL, 0.873 mol), the mixture was heated to 85 ° C. and stirred for 18 hours. The homogeneous brown mixture was cooled to 30 ° C and the pH was lowered to 3.6 by addition of concentrated HCl while maintaining the temperature at 30 to 35 ° C. After starting crystallization at pH 3.6, the suspension was stirred for 15 minutes and then the pH was lowered to 2.5. The mixture was stirred for an additional 10 minutes, then cooled to 2 ° C. and stirred for 2 hours before the gray solid was collected and washed with cold H ₂ O (400 mL) and EtOH (300 mL). The collected solid was dried in vacuo at 45 ° C. for 17 hours to give 42.9 g (43%, starting from the compound of Preparation 18) of the title compound. All of the title compound produced in the reaction was recovered from the mother liquor in the following manner to process. The ethanol portion of the mother liquor was evaporated and the residue was combined with the aqueous portion of the mother liquor. After distilling H ₂ O (485 mL) under reduced pressure, the pH of the mother liquor was adjusted to 12.9 using 70 mL of 5N NaOH and 5 mL of 50% NaOH. After washing the solution with n-BuOH (3 × 800 mL), its pH was adjusted to 2.5 using concentrated HCl and the solution was concentrated. The residue was diluted with EtOH (100 mL) and the volatiles were evaporated (2 ×). The residue was diluted with EtOH (150 mL) and the tan solid containing additional title compound and salt was washed with EtOH (75 mL) and dried in vacuo at 50 ° C. to give 102 g. The yield of both of the title compounds was used in the subsequent esterification process.

Preparation Example 79

Ethyl 2-[((1R) -1-phenylethyl) amino] (2S, 4S, 6R) -2-carbamoyl-4-hydroxybicyclo [3.1.0] hexane-6-carboxylate

The title compound is typically 2-[((1R) -1-phenylethyl) amino] (2S, 4S, 6R) -4-hydroxybicyclo [3.1.0] hexane-2,6-dicarboxyl in situ Although used in the preparation of acids, compounds can be isolated although some yields are lost because of the ester hydrolysis involved in nitrile hydrolysis. In the isolation process, the nitrile hydrolysis reaction mixture is partitioned between CH ₂ Cl ₂ and H ₂ O and directly ethyl 2-[((1R) -1-phenylethyl) amino] (2S, 4S, 6R) -2- Cyano-4-hydroxybicyclo [3.1.0] hexane-6-carboxylate was consumed. After the organic layer was dried (MgSO ₄ ) and concentrated, the residue was purified by silica gel column chromatography (using EtOAc / hexanes (2: 1) → EtOAc) to afford the title compound as a white foam.

Preparation Example 80

Ethyl 2-[((1R) -1-phenylethyl) amino] (2S, 4S, 6R) -2- (ethoxycarbonyl) -4-hydroxybicyclo [3.1.0] hexane-6-carboxyl Rate

2-[((1R) -1-phenylethyl) amino] (2S, 4S, 6R) -4-hydroxybicyclo [3.1.0] hexane-2,6-dicarboxylic acid in 48 mL of ethanol (4 g, 13 mmol) was added acetyl chloride (11.2 mL, 157 mmol) via an addition funnel at room temperature to maintain the proper reflux temperature. The resulting mixture was stirred for additional 16 hours at reflux temperature and concentrated in vacuo while cooling to room temperature to give a solid residue. The solid was slowly treated with a solution of sodium bicarbonate (6.6 g) in 100 mL of water and then washed with ethyl acetate (2 × 100 mL). The combined organics were dried (MgSO ₄ ), filtered and concentrated in vacuo to give 4.7 g (99%) of the title compound as a solid. Purification by column chromatography on silica gel (eluted with CH ₂ Cl ₂ / MeOH (95: 5)) followed by crystallization from Et ₂ O yielded an analytically pure sample of the title compound as a white solid.

Preparation Example 81

Ethyl 2-[((1R) -1-phenylethyl) amino] (2S, 4R, 6R) -2- (ethoxycarbonyl) -4-fluorobicyclo [3.1.0] hexane-6-carboxylate

Ethyl 2-[((1R) -1-phenylethyl) amino] (2S, 4S, 6R) -2- (ethoxycarbonyl) -4-hydroxybicyclo [3.1 in CH ₂ Cl ₂ (690 mL) .0] Deoxo-Fluor® (45.1 mL, 0.245 mol) in a solution of hexane-6-carboxylate (59.0 g crude, 0.163 mol) at -20 ° C. Add over 15 minutes while maintaining at -20 ° C. The mixture was stirred at this temperature for 20 minutes and at 0 ° C. for 15 minutes before aqueous 15% Na ₂ CO ₃ (650 ml) was added slowly while maintaining the temperature below 10 ° C. The layers were separated and the aqueous layer was extracted again with CH ₂ Cl ₂ (150 mL). The combined organic layers were dried (Na ₂ SO ₄ ) and concentrated to brown oil (73 g). The oil was purified on a pad of silica gel (400 g) (eluted with EtOAc / heptanes (1: 6)) to afford the title compound as a yellow oil (49.7 g, 84%).

Preparation Example 82

1R, 2S, 4R, 5R, 6R-2-amino-4-fluorobicyclo [3.1.0] hexane-2,6-dicarboxylic acid hydrochloride

Ethyl 2-[((1R) -1-phenylethyl) amino] (2S, 4R, 6R) -2- (ethoxycarbonyl) -4-fluorobicyclo [3.1.0] hexane in EtOH (400 mL) A mixture of -6-carboxylate (68.4 g, 0.188 mol), concentrated HCl (15.7 mL, 0.188 mol) and 10% Pd / C (anhydrous, 13.7 g) was left under hydrogen (50 psi) for 18 hours. It was. The crystals were filtered off and the filtrate was evaporated to give the title compound as an off white foam (59.2 g, 106%, calibrated to 97% due to EtOH contamination). Crystallization from EtOAc / MTBE gave an analytical pure sample of the title compound as a white solid.

Preparation Example 83

1R, 2S, 4R, 5R, 6R-2-amino-4-fluorobicyclo [3.1.0] hexane-2,6-dicarboxylic acid

3N NaOH solution (251 mL, 0.753 mol) was charged with 1R, 2S, 4R, 5R, 6R-2-amino-4-fluorobicyclo [3.1.0] hexane-2,6-dicarboxylic acid hydrochloride (59.2 g Crude, 0.188 mol in theory) was added slowly, maintaining the temperature below 26 ° C. The mixture was stirred for 10 minutes to homogenize it. The mixture was stirred at room temperature for 1.25 hours and then the pH was slowly lowered to 2.8 using concentrated HCl while maintaining the temperature at 20-26 ° C. At pH 2.8, the mixture began to crystallize and the suspension was stirred at this pH for 10 minutes before the pH was lowered to 2.1 using concentrated HCl. After stirring for an additional 15 minutes, i-PrOH (67 mL) was added and the suspension was cooled to 0 ° C and stirred for 2 hours. The solid was collected and washed with 37 mL of cold H ₂ O / i-PrOH (4: 1). The collected solid was dried under vacuum at 40 ° C. for 18 hours to afford the title compound as a white solid (33.1 g, 87%. Starting from compound 23.).

Preparation Example 84

Reslurry of 1R, 2S, 4R, 5R, 6R-2-amino-4-fluorobicyclo [3.1.0] hexane-2,6-dicarboxylic acid

Of 1R, 2S, 4R, 5R, 6R-2-amino-4-fluorobicyclo [3.1.0] hexane-2,6-dicarboxylic acid (33.0 g, 0.162 mmol) in H ₂ O (165 mL) The stirred suspension was warmed to 89 ° C. over 1 h and i-PrOH (41 mL) was added. The mixture was then stirred for 5 minutes at reflux (83 ° C.), then cooled to room temperature and stirred for 4 hours. The product was collected, washed with i-PrOH / H ₂ O (1: 4, 40 mL) and i-PrOH (25 mL) and dried for 18 h at 40 ° C. under vacuum to afford the title compound as a white solid. (30.6 g, 93%).

Preparation Example 85

1R, 2S, 4R, 5R, 6R-2-amino-4-fluorobicyclo [3.1.0] hexane-2,6-dicarboxylic acid ethyl ester

Room temperature in a slurry of 1R, 2S, 4R, 5R, 6R-2-amino-4-fluorobicyclo [3.1.0] hexane-2,6-dicarboxylic acid (14.45 g, 71.12 mmol) in 202 mL of pure ethanol. Thionyl chloride (26 mL, 356 mmol) was added dropwise over 20 minutes. The slurry was heated to reflux, stirred for 3 hours and then cooled to room temperature overnight. The resulting solution was concentrated in vacuo to give a residue, which was diluted with 136 mL of ethyl acetate and treated with 306 mL of 10% aqueous sodium carbonate for 15 minutes with manual rotation to a final pH of 10. The layers were separated and the aqueous layer was washed with ethyl acetate (1 × 136 mL). The combined organic extracts were washed with brine (1 × 136 mL), dried (MgSO ₄ ), filtered and concentrated in vacuo to give 17.07 g (93%) of the title compound as a white solid.

Preparation 86

1R, 2S, 4R, 5R, 6R-2- [2'S-2 '-(tert-butoxycarbonylamino) propionyl] amino-4-fluorobicyclo [3.1.0] hexane-2,6-dicar Acid ethyl ester

To a solution of N-Boc-L-alanine (38.62 g, 204 mmol) in 396 mL methylene chloride was added N-methyl morpholine (22.44 mL, 204 mmol) under nitrogen at -22 ° C., followed by iso-butylchloroformate. (26.48 mL, 204 mmol) was added dropwise over 15 minutes to ensure that the reaction temperature did not exceed -18 ° C. The resulting thin slurry was stirred at −20 ° C. for 30 minutes, at which time 1R, 2S, 4R, 5R, 6R-2-amino-4-fluorobicyclo [3.1.0] hexane-2, in 247 mL of methylene chloride A solution of 6-dicarboxylic acid ethyl ester (49.46 g, 191 mmol) was added over 40 minutes so that the reaction temperature did not exceed -16 ° C. When the addition was complete, the reaction was separated from the cooling bath and stirred at room temperature for 70 minutes, at which time the reaction temperature reached 15 ° C. and the color became pale orange. The reaction was treated with 408 mL of 1N hydrochloric acid and then stirred for 5 minutes and the layers separated. The organic layer was washed with saturated aqueous sodium bicarbonate (1 × 408 mL), dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo to give a white foam (88.16 g).

Preparation 87

1R, 2S, 4R, 5R, 6R-2- [2'S-2 '-(tert-butoxycarbonylamino) propionyl] amino-4-fluorobicyclo [3.1.0] hexane-2,6-dicar Acid

1R, 2S, 4R, 5R, 6R-2- [2'S-2 '-(tert-butoxycarbonylamino) propionyl] amino-4-fluorobicyclo [3.1.0] hexane- in 238 mL tetrahydrofuran To a solution of 2,6-dicarboxylic acid ethyl ester (88.16 g, 191 mmol) was added 238 mL (477 mmol) of 2N sodium hydroxide at room temperature. The two-phase mixture was stirred vigorously at room temperature for 2.5 hours, at which time the reaction was homogenized. The mixture was diluted with 238 mL of t-butyl methyl ether and then mixed and the layers separated. The aqueous layer was further diluted with 238 mL of water and filtered to remove particulate matter. This solution was treated with concentrated HCl (42.9 mL, 515 mmol) over 30 minutes, seeded with the title compound and stirred for 1 hour. The resulting slurry was filtered, washed with water (2 × 100 mL) and dried in vacuo at 45 ° C. for 40 h to give 72.2 g of the title compound as a white solid. A portion of the solid (69.5 g) was stirred with 490 mL of acetone for 1 hour to produce a cloudy solution, which was filtered and washed with acetone (2 × 100 mL). The filtrate was concentrated in vacuo to a white foam, which was further vacuum dried at 45 ° C. for 16 h to give 61.8 g (corrected for 12% (weight / weight) acetone) of the title compound.

Example 1

(1R, 4S, 5S, 6S) -4- (2'S-aminopropionyl) amino-2,2-dioxo-2λ ⁶ -Thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid hydrochloride

(1R, 4S, 5S, 6S) -4- (2'S-tert-butoxycarbonylamino-propionylamino) -2,2-dioxo-2λ ⁶ -thia-bicyclo [3.1. In 563 mL ethyl acetate. To a suspension of 0] hexane-4,6-dicarboxylic acid (110.0 g, 271 mmol, compound of Preparation 3) was added a solution of hydrogen chloride in ethyl acetate (3.7 M, 514 mL) over 20 minutes. After the suspension was stirred for 2.5 hours, it was filtered and the cake was washed with ethyl acetate (1 × 200 mL, 1 × 115 mL). After vacuum drying at 46 ° C. for 18 hours, the title compound as a white solid was collected (85.77 g, 92%).

Example 2

(1R, 4S, 5S, 6S) -4- (2'S-2'-aminopropionyl) amino-2,2-dioxo-2λ ⁶ -Thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid tosylate

(1R, 4S, 5S, 6S) -4- (2'S-tert-butoxycarbonylamino-propionylamino) -2,2-dioxo-2λ ⁶ -thia-bicyclo [3.1 in toluene (3 mL) .0] Hexane-4,6-dicarboxylic acid (300 mg, 0.738 mmol, compound of Preparation Example 3) and toluenesulfonic acid monohydrate (140 mg, 0.738 mmol) was heated to 75 ° C. and stirred for 45 minutes Then cooled to room temperature and stirred for 16 hours. The suspension was filtered and the cake washed with toluene (2 × 1 mL). After vacuum drying at 45 ° C. for 1 hour, 307 mg (87%) of the title compound as a white solid were collected.

Example 3

(1R, 4S, 5S, 6S) -4- (2'S-amino-3'-hydroxy-propionyl) -amino-2,2-dioxo-2λ ⁶ -Thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid hydrochloric acid

(1R, 4S, 5S, 6S) -4- (3'-acetoxy-2'S-tert-butoxycarbonylamino-propionyl) -amino-2,2-dioxo-2λ ⁶ -thiabicyclo [3.1 .0] hexane-4,6-dicarboxylic acid dimethyl ester (380 mg, 0.77 mmole, compound of Preparation 15) was used and prepared according to General Procedure C.

Example 4

(1R, 4S, 5S, 6S) -4-[(pyrrolidine-2'S-carbonyl) -amino] -2,2-dioxo-2λ ⁶ -Thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid hydrochloride

(1R, 4S, 5S, 6S) -4-[(1'-tert-butoxycarbonyl-pyrrolidine-2'S-carbonyl) -amino] -2,2-dioxo-2λ ⁶ -thia-ratio Prepared according to General Procedure C using cyclo [3.1.0] hexane-4,6-dicarboxylic acid dimethyl ester (0.8 g, 1.7 mmol, compound of Preparation 11) to afford 0.42 g (67.0%) of the title compound. Obtained.

Example 5

(1R, 4S, 5S, 6S) -4- (2'S-amino-4'-methylsulfanyl-butyrylamino) -2,2-dioxo-2λ ⁶ -Thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid hydrochloride

(1R, 4S, 5S, 6S) -4- (2'S-tert-butoxycarbonylamino-4'-methylsulfanyl-butyrylamino) -2,2-dioxo-2λ ⁶ -thia-bicyclo [ 3.1.0] hexane-4,6-dicarboxylic acid dimethyl ester (0.77 g, 1.6 mmol, compound of Preparation 12) and prepared according to General Procedure C to give 0.41 g (54.9%) of the title compound. .

Example 6

(1R, 4S, 5S, 6S) -4- [2'S-amino-3'-1 H-indol-3-yl) -propionylamino) -2,2-dioxo-2λ ⁶ -Thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid hydrochloride

(1R, 4S, 5S, 6S) -4- [2'S-tert-butoxycarbonylamino-3 '-(1-tert-butoxycarbonyl-1H-indol-3-yl) -propionylamino]- General procedure C using 2,2-dioxo-2λ ⁶ -thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid dimethyl ester (0.54 g, 0.83 mmol, compound of Preparation Example 13) Prepared according to yield 0.28 g (73.6%) of the title compound.

Example 7

(1R, 4S, 5S, 6S) -4- [2'S-amino-3 '-(4-hydroxy-phenyl) -propionylamino] -2,2-dioxo-2λ ⁶ -Thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid hydrochloride

(1R, 4S, 5S, 6S) -4- [2'S-tert-butoxycarbonylamino-3 '-(4-tert-butoxycarbonyloxy-phenyl) -propionylamino] -2,2-di Prepared according to General Procedure C using oxo-2λ ⁶ -thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid dimethyl ester (0.33 g, 0.53 mmol, compound of Preparation 14) and titled 0.13 g (56.4%) of compound was obtained.

Example 8

(1R, 4S, 5S, 6S) -4- (2'S-amino-3'-phenyl-propionylamino) -2,2-dioxo-2λ ⁶ -Thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid hydrochloride

(1R, 4S, 5S, 6S) -4- (2'S-tert-butoxycarbonylamino-3'-phenyl-propionylamino) -2,2-dioxo-2λ ⁶ -thia-bicyclo [3.1. 0] hexane-4,6-dicarboxylic acid dimethyl ester (compound of Preparation Example 5) was used and prepared according to General Procedure C. 0.55 g (85%, 1.31 mmol) of a white solid were obtained.

Example 9

(1R, 4S, 5S, 6S) -4- (2'S-amino-3'S-methyl-pentanoylamino) -2,2-dioxo-2λ ⁶ -Thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid hydrochloride

(1R, 4S, 5S, 6S) -4- (2'S-tert-butoxycarbonylamino-3'S-methyl-pentanoylamino) -2,2-dioxo-2λ ⁶ -thia-bicyclo [3.1.0 ] Hexane-4,6-dicarboxylic acid dimethyl ester (compound of Preparation Example 6) was used and prepared according to General Procedure C. 0.43 g (80%, 1.12 mmol) of a white solid were obtained.

Example 10

(1R, 4S, 5S, 6S) -4- (2'S-amino-3'-methyl-butyrylamino) -2,2-dioxo-2λ ⁶ -Thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid hydrochloride

(1R, 4S, 5S, 6S) -4- (2'S-tert-butoxycarbonylamino-3'-methyl-butyrylamino) -2,2-dioxo-2λ ⁶ -thia-bicyclo [3.1. 0] hexane-4,6-dicarboxylic acid dimethyl ester (compound of Preparation Example 7) was used and prepared according to General Procedure C. 0.18 g (88%, 0.49 mmol) of a white solid were obtained as the HCl salt.

Example 11

(1R, 4S, 5S, 6S) -4- (2'S-amino-4'-methyl-pentanoylamino) -2,2-dioxo-2λ ⁶ -Thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid hydrochloride

(1R, 4S, 5S, 6S) -4- (2'S-tert-butoxycarbonylamino-4'-methyl-pentanoylamino) -2,2-dioxo-2λ ⁶ -thia-bicyclo [3.1. 0] hexane-4,6-dicarboxylic acid dimethyl ester (compound of Preparation Example 8) was used and prepared according to General Procedure C. 0.50 g (76%, 1.30 mmol) of a white solid was obtained as an HCl salt.

Example 12

(1R, 4S, 5S, 6S) -4- (2'S, 6'-diamino-hexanoylamino) -2,2-dioxo-2λ ⁶ -Thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid bis hydrochloride

(1R, 4S, 5S, 6S) -4- (2'S, 6'-bis-tert-butoxycarbonylamino-hexanoylamino) -2,2-dioxo-2λ ⁶ -thia-bicyclo [3.1. 0] hexane-4,6-dicarboxylic acid dimethyl ester (compound of Preparation Example 9) was used and prepared according to General Procedure C. 0.56 g (86%, 1.28 mmol) of a white solid were obtained as a bis hydrochloride salt.

Example 13

(1R, 4S, 5S, 6S) -4- (2'S-amino-4'-carbamoyl-butyrylamino) -2,2-dioxo-2λ ⁶ -Thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid hydrochloride

(1R, 4S, 5S, 6S) -4- [2'S-tert-butoxycarbonylamino-4 '-(trityl-carbamoyl) -butyrylamino] -2,2-dioxo-2λ ^6- Thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid dimethyl ester (0.48 g, 0.65 mmol, compound of Preparation 10) was mixed with a 1: 1 mixture of 2.5N LiOH and THF (6 mL total volume). Was stirred at room temperature for 4 hours. The pH of the reaction mixture was adjusted to 2 with 1N HCl and the product was extracted with ethyl acetate. All organic layers were combined, washed with brine, dried over MgSO ₄ and concentrated to (1R, 4S, 5S, 6S) -4- [2'S-tert-butoxycarbonylamino-4 '-(trityl-car Barmoyl) -butyrylamino] -2,2-dioxo-2λ ⁶ -thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid was obtained as a white foam. The acid was dissolved in DCM and stirred at room temperature with the addition of anisole (0.28 g, 2.6 mmol) followed by TFA (3.70 g, 32.5 mmol). The resulting reaction mixture was stirred at rt for 2 h and concentrated in vacuo. The resulting yellow oil was triturated in Et ₂ O until free, forming a flowing white precipitate. TFA salt was collected under N ₂ blanket by vacuum filtration. The product was dissolved in 1 mL of 1N HCl and lyophilized to give the desired product as an HCl salt. 0.16 g (62%, 0.40 mmol) of a white solid were obtained.

Example 14

(1R, 2S, 4R, 5R, 6R) -2- (2'S-amino-propionyl) amino-4-fluorobicyclo [3.1.0] hexane-2,6-dicarboxylic acid hydrochloride

(1R, 2S, 4R, 5R, 6R) -2- (2'S-2 '-(tert-butoxycarbonylamino) propionyl) amino-4-fluoro-bicyclo [3.1.0] in 447 mL acetone A slurry of hexane-2,6-dicarboxylic acid (corrected for 53.0 g acetone, 142 mmol) was stirred at 50 ° C. for 35 minutes. The cloudy solution was filtered to clear the solution and then washed with 100 mL of acetone. 22.1 mL (265 mmol) of concentrated hydrochloric acid was added dropwise to the clear off-white filtrate over 5 minutes. The mixture was warmed to 45-50 ° C. and stirred for 90 minutes. Optionally with a mixture of (1R, 2S, 4R, 5R, 6R) -2- (2'S-aminopropionyl) amino-4-fluorobicyclo [3.1.0] hexane-2,6-dicarboxylic acid hydrochloride After seeding together the heating was stopped and cooled stepwise to room temperature. Two hours after the temperature reached 25 ° C. acetone (942 mL) was added to the slurry over 90 minutes. The slurry was stirred for an additional 16 hours before filtration, washed with acetone (2 × 200 mL), vacuum dried at 45 ° C. for 9 hours and vacuum dried at room temperature for an additional 64 hours to give the title compound 40.2 as a white solid. g (91%) was obtained.

Recrystallization

1.06 g was dissolved in 0.5 mL of water and 2.12 mL of acetone while heating at 50 ° C., then diluted with additional 5.3 mL of acetone and optionally seeded. 4.2 mL of acetone was further added to the pale, turbid mixture, optionally seeded again, the heating was stopped and gradually cooled gradually to room temperature over 1 hour. The resulting slurry was diluted with additional 9.5 mL of acetone over 30 minutes and then stirred for 15 hours. Upon filtration, washing with acetone (2 × 5 mL) and vacuum drying at 45 ° C. for 10 hours and at room temperature for 60 hours yielded 0.905 g (85% recovery) of the title compound as a white solid.

Example 15

(1R, 2S, 4R, 5R, 6R) -2- (2'S-amino-propionyl) amino-4-fluorobicyclo [3.1.0] hexane-2,6-dicarboxylic acid mesylate

(1R, 2S, 4R, 5R, 6R) -2- [2'S- (tert-butoxycarbonylamino) propionyl] -amino-4-fluorobicyclo [3.1.0] hexane-2 in 16.8 mL of acetone, A slurry of 6-dicarboxylic acid (1.87 g correction, 4.98 mmol, compound of Preparation 18) was stirred at 50 ° C. for 15 minutes. The cloudy solution was filtered to clear the solution, and then washed with acetone (3 × 1.25 mL). The clear filtrate was diluted with 0.935 mL of water, placed in a 50 ° C. heating bath and treated with dropwise addition of 0.647 mL (9.97 mmol) of methanesulfonic acid (observe gas evolution). After 25 minutes a white slurry was produced. After stirring for a total of 2 hours, 35.5 mL of acetone was added over an additional 5-10 minutes. The heating was stopped, the slurry was gradually cooled to room temperature over 2 hours, then filtered, washed with acetone (2 × 8 mL) and vacuum dried at 45 ° C. for 14 hours to afford 1.77 g (95%) of the title compound. Obtained as a pale pink solid. A sample of this material was crystallized as follows: 1.65 g was dissolved in 1.16 mL of water and 4.95 mL of acetone with heating at 50 ° C., followed by dilution with additional 1.65 mL of acetone and optionally seeding. The heating was stopped and the mixture was cooled stepwise to room temperature. Acetone (26.4 mL) was added simultaneously over 40 minutes. The resulting slurry was stirred for an additional 3 hours. On filtration, washing with acetone (2 × 6 mL) and vacuum drying at 45 ° C. for 6 hours and at room temperature for 60 hours yielded 1.59 g (96% recovery) of the title compound as a white solid.

mp (DSC) 206 ° C.

Example 16

(1R, 2S, 4R, 5R, 6R) -2- (2'S-amino-propionyl) amino-4-fluorobicyclo [3. 1.0] hexane-2,6-dicarboxylic acid acrylate

(1R, 2S, 4R, 5R, 6R) -2- [2'S- (tert-butoxycarbonylamino) propionyl] -amino-4-fluorobicyclo [3.1.0] hexane-2 in 1.8 mL acetone, A slurry of 6-dicarboxylic acid (0.2 g, 0.534 mmol, compound of Preparation 18) was stirred at 50 ° C. for 5 minutes. The cloudy solution was filtered to clear the solution and then washed with acetone (1 × 0.4 mL). The clear filtrate was diluted with 0.1 mL of water, placed in a 50 ° C. heating bath and instilled with 0.124 mL (1.07 mmol) of ethanesulfonic acid (gas evolution was observed). After 90 minutes a white slurry was produced. After stirring for a total of 2 hours, additional 1.8 mL of acetone was added over 5 minutes. The heating was stopped and the slurry was gradually cooled to room temperature over 1 hour and then stirred for an additional 2 hours. Filter, wash with acetone (2 × 1 mL) and vacuum dry for 4 h at 45 ° C. and 60 h at room temperature to give 0.173 g (84%) of the title compound as a white solid.

Example 17

(1R, 2S, 4R, 5R, 6R) -2- (2'S-amino-propionyl) amino-4-fluorobicyclo [3.1.0] hexane-2,6-dicarboxylic acid besylate

(1R, 2S, 4R, 5R, 6R) -2- [2'S- (tert-butoxycarbonylamino) propionyl] -amino-4-fluorobicyclo [3.1.0] hexane-2 in 3.6 mL acetone, A slurry of 6-dicarboxylic acid (0.402 g 1.07 mmol, compound of Preparation Example 18) was stirred at 50 ° C. for 10 minutes. The turbid solution was treated with a small amount of celite and filtered to clear the solution and then washed with acetone (2 × 0.4 mL). The clear filtrate was placed in a 50 ° C. heating bath and treated with 226 mg (90%, 1.29 mmol) of benzenesulfonic acid as a solution in 0.113 mL of water followed by washing with 0.4 mL of acetone (gas evolution was observed). After stirring for 4 hours at a suitable reflux temperature, heating was stopped and the reaction was optionally seeded after treatment with 8 mL of acetone for 10 minutes. The slurry formed over 1 hour was diluted with 3.2 mL of acetone and then stirred at room temperature for an additional 15.5 hours. Filter, wash with acetone (2 × 10 mL) and dry for 24 h at 45 ° C. under vacuum to afford 313 mg (62%, corrected for 10% by weight acetone) of the title compound as a white solid.

Example 18

(1R, 2S, 4R, 5R, 6R) -2- (2'S-amino-propionyl) amino-4-fluorobicyclo [3.1.0] hexane-2,6-dicarboxylic acid tosylate

(1R, 2S, 4R, 5R, 6R) -2- [2'S- (tert-butoxycarbonylamino) propionyl] -amino-4-fluorobicyclo [3.1.0] hexane-2, in 9.36 mL acetone, A slurry of 6-dicarboxylic acid (1.04 g correction, 2.78 mmol, compound of Preparation 18) was stirred at 50 ° C. for 15 minutes. The turbid solution was treated with a small amount of celite and filtered to clear the solution and then washed with acetone (1 × 1.04 mL after 1 × 2.08 mL). The clear filtrate was placed in a 50 ° C. heating bath and treated with 634 mg (3.33 mmol) of p-toluenesulfonic acid monohydrate as a solution in 0.317 mL of water followed by washing with 0.317 mL of acetone (gas release was observed). After stirring for 4 hours at a suitable reflux temperature, the reaction was separated from the heating bath and treated with 10.4 mL of acetone over 10 minutes. A clear colorless solution was optionally seeded, and a precipitate formed over 30 minutes, with additional 10.4 mL of acetone introduced over 20 minutes. The slurry was stirred for an additional 4 hours, then filtered, washed with acetone (2 × 10 mL) and dried in vacuo at 45 ° C. for 14 hours to 995 mg (78%, 3% by weight acetone) as the white solid. Corrected for).

Example 19

(1R, 2S, 4R, 5R, 6R) -2- (2'S-amino-propionyl) amino-4-fluorobicyclo [3.1.0] hexane-2,6-dicarboxylic acid

(1R, 2S, 4R, 5R, 6R) -2- (2'S-amino-propionyl) amino-4-fluorobicyclo [3.1.0] hexane-2,6-dicarboxylic acid mesylate in 1 mL of water To a solution of (0.5 g, 1.35 mmol, Example 15) was added 5 mL of 3A ethanol at 50 ° C., and a few minutes later 0.27 mL (1.35 mmol) of 5N aqueous sodium hydroxide was added. The heating was stopped and the clear colorless solution was diluted with 2.5 mL of ethanol, optionally seeded and diluted with additional 7.5 mL of ethanol over 30 minutes. The resulting slurry was then stirred over 1 hour while cooling to room temperature followed by 2 hours at room temperature. The solids were collected, washed with ethanol (1 × 10 mL) and dried in vacuo at 45 ° C. for 18.5 hours to yield 0.301 g (78% yield, 1.6 wt% sodium methanesulfonate and 3 wt% ethanol as white solid). Corrected for).

Example 20

(1R, 2S, 4R, 5R, 6R) -2- (2'S-amino-propionyl) amino-4-fluorobicyclo [3.1.0] hexane-2,6-dicarboxylic acid disodium salt

(1R, 2S, 4R, 5R, 6R) -2- (2'S-amino-propionyl) amino-4-fluorobicyclo [3.1.0] hexane-2,6-dicarboxylic acid mesylate in 420 μl of methanol To a solution of (70 mg, 0.19 mmol, Example 15) was added a warm solution of sodium acetate (46.5 mg, 0.57 mmol) in 470 μl methanol at 60 ° C. and washed with 230 μl methanol. The heating was stopped. The stirred turbid solution was diluted with 280 μl of methanol and then optionally seeded to crystallize. The resulting slurry was slowly cooled to room temperature over 1 hour and stirred at room temperature for 2 hours. The product was isolated by filtration, filtered, washed with methanol (2 × 280 μl) and dried under vacuum at 45 ° C. for 15 hours to give 52.5 mg (85% yield, 2.3% by weight sodium methanesulfo) of the title compound as a white solid. Nate and corrected for 0.2 wt% methanol).

Example 21

(1S, 2S, 4S, 5R, 6R) -2- (2'S-aminopropionyl) amino-4-hydroxy-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid hydrochloride

(1S, 2S, 4S, 5R, 6R) -4-acetyloxy-2- [2'S- (tert-butoxy) carbonylaminopropionyl] -aminobicyclo in THF (30 mL) and water (30 mL) Lithium hydroxide monohydrate (0.535 g, 12.8 mmol) was added to a solution of [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester (0.600 g, 1.28 mmol, compound of Preparation 21). After stirring for 3 hours at room temperature, the reaction was diluted with water and washed with ethyl acetate (2 × 25 mL). The aqueous layer was acidified to pH 1 with aqueous 1N HCl solution and extracted with ethyl acetate (3 × 30 mL). The combined organic extracts were concentrated and redissolved in 1N HCl in ethyl acetate (60 mL) and then stirred for 16 hours at room temperature under nitrogen. The solvent was removed in vacuo to yield 400 mg (99%) of the product as a white solid.

Example 22

(1S, 2S, 4S, 5R, 6R) -2- (2'-amino-acetylamino) -4-hydroxy-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid hydrochloride

(1S, 2S, 4S, 5R, 6R) -2- (2'-tert-butoxycarbonylamino-acetylamino) -4-hydroxy-bicyclo [3.1.0] hexane-2,6-dicar Acid diethyl ester (300 mg, 0.84 mmole, compound of Preparation 23) was used and prepared according to General Procedure C except removing the tert-butoxycarbonyl protecting group by treatment with 4M HCl in dioxane. Yield: 156 mg (63%).

Example 23

(1S, 2S, 4S, 5R, 6R) -2- (2'S-amino-3'-methyl-butyrylamino) -4-hydroxybicyclo [3.1.0] -hexane-2,6-dicarboxyl Acid hydrochloric acid

(1S, 2S, 4S, 5R, 6R) -2- (2'S-tert-butoxycarbonylamino-3'-methyl-butyrylamino) -4-hydroxy-bicyclo [3.1.0] hexane-2 General procedure C, except for removing the tert-butoxycarbonyl protecting group with 4M HCl in dioxane using 6-dicarboxylic acid diethyl ester (480 mg, 1.19 mmole, compound of Preparation 24) It was prepared according to. Yield 307 mg (76%).

Example 24

(1S, 2S, 4S, 5R, 6R) -2- (2'S-amino-4'-methyl-pentanoylamino) -4-hydroxy-bicyclo [3.1.0] -hexane-2,6-dicar Acid hydrochloric acid

(1S, 2S, 4S, 5R, 6R) -2- (2'S-tert-butoxycarbonylamino-4'-methyl-pentanoylamino) -4-hydroxy-bicyclo [3.1.0] hexane-2 General procedure C, except for removing the tert-butoxycarbonyl protecting group with 4M HCl in dioxane using 6-dicarboxylic acid diethyl ester (460 mg, 1.11 mmole, compound of Preparation 25) It was prepared according to. Yield: 371 mg (95%).

Example 25

(1S, 2S, 4S, 5R, 6R) -2- (2'S-amino-3'S-methyl-pentanoylamino) -4-hydroxy-bicyclo [3.1.0] -hexane-2,6-dicarboxyl Acid hydrochloric acid

(1S, 2S, 4S, 5R, 6R) -2- (2'S-tert-butoxycarbonylamino-3'S-methyl-pentanoylamino) -4-hydroxy-bicyclo [3.1.0] hexane-2, In General Procedure C, except using 6-dicarboxylic acid diethyl ester (410 mg, 0.99 mmole, compound of Preparation 26) and removing the tert-butoxycarbonyl protecting group with 4M HCl in dioxane. Prepared accordingly. Yield: 330 mg (95%).

Example 26

(1S, 2S, 4S, 5R, 6R) -2- [2 '-(2-amino-acetylamino) -acetylamino] -4-hydroxy-bicyclo [3.1.0] hexane-2,6-dica Carboxylic acid

(1S, 2S, 4S, 5R, 6R) -2- [2 '-(2-tert-butoxycarbonylamino-acetylamino) -acetylamino] -4-hydroxy-bicyclo [3.1.0] hexane -2,6-dicarboxylic acid diethyl ester (360 mg, 0.77 mmole, compound of Preparation 27) was treated with excess of 4M HCl in dioxane, stirred for 40 minutes, and concentrated in vacuo. Ethyl acetate was added and concentrated. The solid was treated with THF (3M) and 1N LiOH (3.0 equiv). After stirring for 1 hour, 1N HCl was added until the pH became 3. The reaction was concentrated in vacuo. Purification via Dowks® DOWEX® 50WX8-100 ion-exchange resin. Yield: 188 mg (78%).

Example 27

(1S, 2S, 4S, 5R, 6R) -2- [2 '-(2S-amino-propionylamino) -acetylamino] -4-hydroxy-bicyclo [3.1.0] hexane-2,6- Dicarboxylic acid

(1S, 2S, 4S, 5R, 6R) -2- [2 '-(2S-tert-butoxycarbonylamino-propionylamino) -acetylamino] -4-hydroxy-bicyclo [3.1.0] Hexane-2,6-dicarboxylic acid diethyl ester (410 mg, 0.84 mmole, compound of Preparation 28) was used and prepared according to the general procedure outlined in Example 26. Yield: 200 mg (72%).

Example 28

(1S, 2S, 4S, 5R, 6R) -2- (2'S-amino-3'-phenyl-propionylamino) -4-hydroxy-bicyclo [3.1.0] hexane-2,6-dicarboxyl Acid hydrochloride

(1S, 2S, 4S, 5R, 6R) -2- (2'-tert-butoxycarbonylamino-3'-phenyl-propionylamino) -4-hydroxy-bicyclo [3.1.0] hexane- Prepared according to General Procedure C using 2,6-dicarboxylic acid diethyl ester (400 mg, 0.89 mmole, compound of Preparation 29). Yield: 290 mg (85%).

Example 29

(1S, 2S, 4S, 5R, 6R) -2- (2'S-amino-4'-carbamoylamino) -4-hydroxy-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid Hydrochloride

(1S, 2S, 4S, 5R, 6R) -2- [2'-tert-butoxycarbonylamino-4 '-(trityl-carbamoyl) -butyrylamino] -4-hydroxy-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester (1.18 mmole, compound of Preparation 30) was dissolved in THF (10 mL) and the solution was treated with 2.5N LiOH (12 mL). The title compound was prepared. The solution was stirred for 3 hours and then the pH was adjusted to 2 with 2.5N HCl. The product was extracted four times with ethyl acetate, dried over anhydrous sodium sulfate and concentrated in vacuo to afford a white powder. Di-acid product (0.74 mmol) was dissolved in 1,2-dichloroethane (3.7 mL) and treated with anisole (0.3 mL) and trifluoroacetic acid (3.72 mL). The reaction mixture was stirred at 23 ° C. for 2.5 h and then concentrated in vacuo to give a brown oil. The brown oil was dissolved in water, extracted five times with dichloromethane, the aqueous layer was concentrated in vacuo, treated with 1N HCl (0.74 mL) and freeze dried. The solid was treated with an additional portion of 1N HCl (2 mL) and lyophilized again to give 446 mg (quantity) of the title compound.

Example 30

(1S, 2S, 4S, 5R, 6R) -2- (2'S, 6'-diamino-hexanoylamino) -4-hydroxy-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid Bis hydrochloride

(1S, 2S, 4S, 5R, 6R) -2- (2'S, 6'-bis-tert-butoxycarbonylamino-hexanoylamino) -4-hydroxy-bicyclo [3.1.0] hexane-2 Prepared according to General Procedure C, using 6-dicarboxylic acid diethyl ester (compound of Preparation 31).

Example 31

(1S, 2S, 4S, 5R, 6R) -2- (2'S-amino-3'-carbamoyl-propionylamino) -4-hydroxy-bicyclo [3.1.0] hexane-2,6-dica Carboxylic acid

(1S, 2S, 4S, 5R, 6R) -2- [2'-tert-butoxycarbonylamino-3 '-(trityl-carbamoyl) -propionylamino] -4-hydroxy-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester (1.07 mmole, compound of Preparation 32) was dissolved in THF (12 mL) and the solution was treated with 2.5N LiOH (12 mL). The title compound was prepared. The solution was stirred for 3 hours and the pH was adjusted to 2 with 2.5N HCl. The product was extracted four times with ethyl acetate, dried over anhydrous sodium sulfate and concentrated in vacuo to afford a white powder. The di-acid product (1.22 mmol) was dissolved in 1,2-dichloroethane (6 mL) and treated with anisole (1 mL) and trifluoroacetic acid (6 mL). The reaction mixture was stirred at 23 ° C. for 10 hours and then concentrated in vacuo to give a brown oil. The brown oil was diluted with diethyl ether and the product crystallized as a white solid. Filtered and washed with abundant portions of ether. The solid was dissolved in a minimum amount of water, treated with 0.5N HCl (5 mL) and lyophilized three times to give 386 mg (90%) of the title compound.

Example 32

(1S, 2S, 4S, 5R, 6R) -2- [2'S-amino-3 '-(1H-indol-3-yl) -propionylamino] -4-hydroxy-bicyclo [3.1.0] hexane -26-dicarboxylic acid

2- [2'-tert-butoxycarbonylamino-3 '-(1'-tert-butoxycarbonyl-1H-indol-3'-yl) -propionylamino] -4-hydroxy-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester (0.93 mmole, compound of Preparation 33) was dissolved in THF (11 mL) and the solution was treated with 2.5N LiOH (11 mL). Prepared. The solution was stirred for 3 hours and the pH was adjusted to 2 with 2.5N HCl. The product was extracted four times with ethyl acetate, dried over anhydrous sodium sulfate and concentrated in vacuo to afford a white powder. The di-acid product (0.78 mmol) was dissolved in a solution of 4M HCl in dioxane (20 mL) and the reaction mixture was stirred at 23 ° C. for 3 hours. The reaction mixture was concentrated, diluted with dichloromethane and concentrated again in vacuo. The crude product is dissolved in methanol, applied to a spin chromatography (silica gel) plate, rotary dried, MeOH (10%) / NH ₄ OH (1%) / CHCl ₃ → MeOH (60%) / NH ₄ OH Eluted with (2%) / CHCl ₃ . The product was dissolved in water and lyophilized twice to give 139 mg (46%) of the title compound.

Example 33

(1S, 2S, 4S, 5R, 6R) -4-hydroxy-2-[(pyrrolidine-2'S-carbonyl) -amino] -bicyclo [3.1.0] hexane-2,6-dicarboxyl Acid hydrochloride salt

(1S, 2S, 4S, 5R, 6R) -2- (1'-tert-butoxycarbonyl-pyrrolidine-2'S-carbonyl) -4-hydroxy-bicyclo [3.1.0] hexane-2 Prepared according to General Procedure C, except that, tert-1-butoxycarbonyl protecting group was removed by treatment with 4M HCl in dioxane, using 6-dicarboxylic acid diethyl ester (compound of Preparation 34). .

Example 34

(1S, 2S, 4S, 5R, 6R) -2- [2'S-Amino-3 '-(4-hydroxy-phenyl) -propionylamino] -4-hydroxy-bicyclo [3.1.0] hexane- 2,6-dicarboxylic acid hydrochloride salt

(1S, 2S, 4S, 5R, 6R) -2- [2'S-tert-butoxycarbonylamino-3 '-(4-tert-butoxycarbonyloxy-phenyl) -propionylamino] -4-hydrate Treatment of tert-1-butoxycarbonyl protecting groups with 4M HCl in dioxane using oxy-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester (compound of Preparation 35) It was prepared according to the general procedure C except for the removal. Yield: 199 mg (50%).

Example 35

(1S, 2S, 4S, 5R, 6R) -2- (2'S-amino-4'-methylsulfanyl-butyrylamino) -4-hydroxy-bicyclo [3.1.0] hexane-2,6-dica Bis ammonium salt

(1S, 2S, 4S, 5R, 6R) -2- (2'S-tert-butoxycarbonylamino-4'-methylsulfanyl-butyrylamino) -4-hydroxy-bicyclo [3.1.0] hexane In accordance with General Procedure C, except using -2,6-dicarboxylic acid diethyl ester (compound of Preparation 36) and removing the tert-1-butoxycarbonyl protecting group with 4M HCl in dioxane. Prepared. The final compound was loaded with MeOH on 2 mm Chromatotron plate and eluted with 50 / 49.5 / 0.5 CHCl ₃ / MeOH / NH ₄ OH. The amount of NH ₄ OH was increased to 1% throughout the elution process to yield a zwitterionic compound. Yield 136 mg (37%).

Example 36

(1S, 2S, 4S, 5R, 6R) -2- [2'S- (2S-amino-propionylamino) -propionylamino] -4-hydroxy-bicyclo [3.1.0] hexane) -2,6 Dicarboxylic acid

(1S, 2S, 4S, 5R, 6R) -2- [2'S- (2S-tert-butoxycarbonylamino-propionylamino) propionylamino] -4-hydroxy-bicyclo [3.1.0] hexane The -2,6-dicarboxylic acid diethyl ester (0.045 g, 0.1 mmol, compound of Preparation 39) was stirred in 1 ml of 4 HCl in dioxane for 40 minutes before the reaction was concentrated in vacuo. The crude material was stirred in THF (5 ml) and 0.5N LiOH (0.35 mmol) for 1 hour. The pH was adjusted to 2 with 0.5N HCl and the reaction concentrated. The crude product was purified using cation exchange chromatography (Doxx® 50X8-100; eluted with 10% pyridine / H ₂ O) to give 19 mg (55.3%) of the title compound.

Example 37

(1S, 2S, 4S, 5R, 6R) -2- [2'S- (2-amino-acetylamino) -propionylamino] -4-hydroxy-bicyclo [3.1.0] hexane-2,6-dica Carboxylic acid

(1S, 2S, 4S, 5R, 6R) -2- [2'S- (2-tert-butoxycarbonylamino-acetylamino) -propionylamino] -4-hydroxy-bicyclo [3.1.0] hexane -2,6-dicarboxylic acid diethyl ester (0.12 g, 0.25 mmol, compound of Preparation 40) was stirred in THF (5 ml) and 1N LiOH (1.0 mmol) for 1 hour. The pH of the mixture was adjusted to 2 with 1N HCl and concentrated. The crude material was stirred at 0 ° C. in saturated HCl (g) ethyl acetate solution. Excess HCl (g) was removed by purging with N ₂ and the reaction was concentrated. The crude product was purified using cation exchange chromatography (Dosex® 50X8-100; eluted with 10% pyridine / H ₂ O) to afford 0.06 g (72.9%) of the title compound.

Example 38

(1S, 2S, 4S, 5R, 6R) -2- [2'S- (2S-amino-4-methyl-pentanoylamino) -propionylamino] -4-hydroxy-bicyclo [3.1.0] hexane -2,6-dicarboxylic acid

(1S, 2S, 4S, 5R, 6R) -2- [2 '-(2S-tert-butoxycarbonylamino-4-methyl-pentanoylamino) -propionylamino] -4-hydroxy-bicyclo Prepared according to General Procedure C using [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester (compound of Preparation Example 41). 0.21 g (57%, 0.50 mmole) of a white solid were obtained.

Example 39

(1S, 2S, 4S, 5R, 6R) -2- [2'S- (2-Amino-3-methyl-butyrylamino) -propionylamino] -4-hydroxy-bicyclo [3.1.0] hexane- 2,6-dicarboxylic acid

(1S, 2S, 4S, 5R, 6R) -2- [2 '-(2S-tert-butoxycarbonylamino-3-methyl-butyrylamino) -propionylamino] -4-hydroxy-bicyclo Prepared according to General Procedure C, using [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester (compound of Preparation 42). 0.15 g (88%, 0.37 mmole) of a white solid was obtained.

Example 40

(1R, 2S, 4R, 5R, 6R) -2- (2'S-2'-aminopropionyl) amino-4-fluoro-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid mesylate Monohydrate

(1R, 2S, 4R, 5R, 6R) -2- (2'S-2 '-(tert-butoxycarbonylamino) propionyl) amino-4-fluoro-bicyclo [3.1.0] in 295 mL acetone To a turbid solution of hexane-2,6-dicarboxylic acid (32.8 g, 87.5 mmol, compound of Preparation 52) was added 16 mL of water at a suitable reflux temperature followed by 11.4 mL (175 mmol) of methanesulfonic acid to give a pale yellow The solution was produced and after 5 minutes a slurry was obtained. Stir for 130 minutes, remove the heat source and add 295 mL of acetone over 30 minutes. The slurry was cooled to room temperature and then stirred for an additional 2 hours. The slurry was filtered, washed with acetone (2 × 82 mL) and vacuum dried at 45 ° C. for 16 h to give 31.5 g (93%) of the title compound as a white solid.

This material can be further recrystallized. 30.5 g of the crude product were mixed with 152.5 mL of acetone and 35 mL of water. Heat to 55 ° C. and dissolve completely by adding water (3.66 mL). The solution was diluted with 61 mL of acetone and optionally seeded. The heat source was removed and the mixture was cooled stepwise until nuclei were formed. 396 mL of acetone was added to the slurry over 70 minutes and then stirred at room temperature for an additional 3 hours. Filter, wash with acetone (3 × 91 mL) and vacuum dry at 45 ° C. for several hours (usually overnight) to give 27.7 g (91% recovery) of the title compound as a white solid.

Example 41

(1R, 4S, 5S, 6S) -4- (2'S-4'-methylthio-2'-aminobutanyl) amino-2,2-dioxo-2λ ⁶ -Thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid monohydrate

(1R, 4S, 5S, 6S) -4- (2'S-4'-methylthio-2 '-(tert-butoxycarbonyl) aminobutanyl) amino- in acetone (110 mL) and water (550 mL) A slurry of 2,2-dioxo-2λ ⁶ -thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid monosodium salt (110.04 g, 225.3 mmol, compound of Preparation 55) was heated to 55 ° C. Heated to. Concentrated hydrochloric acid (56 mL, 675.8 mmol) was added dropwise to the stirred slurry and dissolved in stages. Once the addition was complete, the solution was stirred at 55 ° C. for 2 hours. The heat source was removed and the solution cooled to room temperature. The solution was filtered and washed with 20 mL of water. 2N sodium hydroxide (165 mL, 330 mmol) was added slowly to the solution to raise the pH to 1.71, at which time a precipitate formed. Stir for 10 minutes to form a thin slurry and drop the pH to 0.98. Additional 2N sodium hydroxide (62 mL, 124 mmol) was added to the resulting slurry to raise the pH to 3.06 and then stirred for 3 hours to give a final pH of 3.24. The slurry was filtered, washed with water (2 × 165 mL) and dried at 45 ° C. for 15 h to give 73.27 g (85 wt% yield) of the title compound as a white solid.

mp (DSC) 203 ° C.

Example 42

(1R, 4S, 5S, 6S) -4- (2'S-4'-methylthio-2'-aminobutanyl) amino-2,2-dioxo-2λ ⁶ -Thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid tosylate

(1R, 4S, 5S, 6S) -4- (2'S-4'-methylthio-2 '-(tert-butoxycarbonyl) aminobutanyl) amino-2,2-dioxo in toluene (1180 mL) -2λ ⁶ -thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid (118.09 g correction, 253 mmol, compound of Preparation 56) and p-toluenesulfonic acid monohydrate (54 g, 278 mmol) was heated to 75 ° C. to produce a thick slurry. The slurry was stirred at reflux for 165 minutes. The heat source was removed and the slurry was cooled to room temperature and then stirred overnight. The slurry was filtered, washed with toluene (3 × 240 mL) and dried for 22 h at 45 ° C. in vacuo to yield 134.92 g (98% yield) of the title compound.

Example 43

(1R, 4S, 5S, 6S) -4- (2'S-4'-methylthio-2'-aminobutanoyl) amino-2,2-dioxo-2λ ⁶ -Thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid mesylate

(1R, 4S, 5S, 6S) -4- (2'S-4'-methylthio-2 '-(tert-butoxycarbonyl) aminobutanyl) amino-2,2-dioxo in 13 mL propionitrile A slurry of -2λ ⁶ -thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid (1.08 g, 2.31 mmol, compound of Preparation 56) was heated to 85 ° C. Water (540 μl) was added to the slurry followed by dropwise addition of methanesulfonic acid (225 μl, 3.47 mmol). The slurry was stirred for 90 minutes. The heat source was removed and propionitrile (30 mL) was added. The slurry was cooled to room temperature and stirred for 90 minutes. Filter, wash with propionitrile (3 × 2.7 mL) and dry at 45 ° C. overnight to afford 1.04 g (97%) of the title compound.

Example 44

(1R, 2S, 5R, 6R) -2- (2'R-aminopropionyl) amino-6-fluoro-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid hydrochloride

1.13 mL (2.8 mmol) of 2.5N LiOH was added to a solution of 0.20 g (0.47 mmol) of isomer A from Preparation 60 compound in THF (1.2 mL) and the resulting mixture was stirred at rt overnight. The solution was washed with ether, neutralized with 1N HCl at 0 ° C. and extracted with EtOAc (3 × 3 mL). The combined organic layers were dried over anhydrous MgSO ₄ and concentrated under reduced pressure. The crude material was dissolved in 3.76 mL of 1N HCl in EtOAc and stirred overnight. The solvent was decanted, washed with ether and the product dried over an Ar stream to give the title compound as a white solid (83 mg, 57% yield).

Example 45

(1S, 2R, 5S, 6S) -2- (2'R-aminopropionyl) amino-6-fluoro-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid hydrochloride

Preparation 60 Starting with the Isomer B solution of the compound, the title compound was prepared essentially as in Example 44.

Example 46

(1S, 2S, 4S, 5R, 6R) -2- (2'S-aminopropionyl) amino-4-hydroxy-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid hydrochloride

(1S, 2S, 4S, 5R, 6R) -2- (2'S-tert-butoxycarbonylamino-propionylamino) -4-hydroxy-bicyclo [3.1.0] hexane-2,6-dicar Acid diethyl ester (0.47 g, 1.1 mmol, compound of Preparation 37) was used and prepared according to general procedure C to give 0.23 g (67.7%) of the title compound.

Prodrug compounds of the invention can be evaluated for corresponding parent compounds through various intracellular uptake assays. Such assays can provide comparative data that allows those skilled in the art to identify compounds that are readily absorbed into cells and provide a predominant degree of exposure. Two such assays include the Gly-Sar uptake assay and Caco-2 assay described below.

Gly-Sar Absorption Analysis

Several orally administered peptide mimetic drugs have been found to be absorbed through the enteric peptide delivery system (Yang, et al., Pharm. Res. 16 (9) (1999)). In particular, enteric peptide transport Chain hPepT1 has been studied for the expression of its peptidyl uptake inhibition and the corresponding level of recognition in cells (Meredith, et al, Eur. J. Biochem., 267, 3723-3728 (2000)). In addition, characterizing the intestinal absorption mechanism of amino acids in hPepT1 transporters has been adopted as an effective strategy for identifying improved oral drug absorption (Han, et al., Polym. Prepr. (Am. Chem. Soc., Div. Polym. Chem) 40 (1): 259-260 (1999); Sawada, et al., J. Pharmacol.Exp. Ther., 291 (2): 705-709 (1999)] .

US Pat. No. 5,849,525 describes a method that can be used to determine the level of affinity of a compound of the invention with a hPepT1 transporter.

For example, stably transfected Chinese Hamster Ovary (CHO) cells that overexpress hPepT1 transporters can be used to test the compounds of the invention. CHO cells will be monitored for uptake of Gly-Sar, wherein the amount absorbed in the presence of the prodrug compound of the invention is greater than the uptake if the cell lacks the prodrug compound of the invention, The amount absorbed in the presence of the prodrug compound of the present invention is less than the amount absorbed in the absence of the prodrug compound of the present invention indicates inhibitory activity. Compounds of the invention generally exhibited an EC ₅₀ value of less than 5 mM.

Caco-2 analysis

Another particular method of measuring uptake of the compounds of the present invention in cells is to study peptide transport carriers of the human enteric cell line Caco-2. Human adenocarcinoma cells (Memorial Sloan-Kettering Cancer Center, Rye, NY and / or ATCC, Rockville, Md.) Were treated with 10% fetal bovine serum and 1% minimum essential medium non-essential amino acids. Passage was performed in Dulbecco's Modified Eagle medium containing the solution (no sodium pyruvate or antibiotics added). These cells lacked mycoplasma and were used at 28 to 40 passages. To measure the flow rate, 5-10 × 10 ⁴ cells were grown for 13-18 days in collagen-coated multiwell dishes, and the medium was changed every 2-3 days.

Drug uptake was measured at 37 ° C. using cluster-tray technology and using test compounds (see Gazzola, et al., Anal. Biochem. 115, 368-74 (1981)). Flow buffer was 25 mM Mes titrated to 6.0 using KOH, and a bicarbonate-free Earle's balanced salt solution containing choline chloride instead of sodium chloride. The osmotic concentration of the flow buffer was adjusted to 300 ± 5 mosmol / kg using choline chloride. In order to estimate the zero time for measuring the rate of absorption, [ ³ H] inulin was used as a marker for extracellular fluids attached to the cells during the wash procedure. Fresh solutions of test compounds and dipeptides were prepared daily. At the end of the experiment, cells were lysed in water and test compounds could be detected in cell lysates using LC / MS / MS. Proteins are described in Smith, et al., Anal. Biochem. 150, 76-85 (1985).

Absorption was measured over 40 minutes. The initial absorption rate was calculated in the linear region of the regression line over time and the reference time was estimated as described above using linear regression analysis. Inhibition rates were calculated based on the control uptake rate measured in the absence of dipeptides. For example, the Caco-2 assay is described by Dantzig & Bergin, Biochim. Biophys. Acta 1027, 211-17 (1990).

In vivo exposure as measured by rat plasma concentration

To study the extent of in vivo exposure of compounds of formula II after oral administration of compounds of formula I as compared to compounds of formula II, studies were conducted to determine the plasma concentration of each compound of formula II in rats. Mature Fischer 344 male rats (190-270 g) were obtained from Harlan Sprague-Dawley (Cumberland, Indiana) and acclimated for three days in a kennel. On day 4, the test compound was dissolved in buffered water (1 mg / ml = test compound / 20 mM potassium hydrogen phosphate, pH = 2) and a single dose of 5 mg / kg was administered orally. Blood samples were collected via orbital sinus or cardiac puncture (last time) at 0.5 and 1 hour, or alternatively at 1 and 3 hours. Plasma samples were stored at −20 ° C. in the presence of the protease inhibitor phenylmethylsulfonyl fluoride before analysis. Plasma samples and internal standard compounds were pretreated by solid phase extraction (SAX support, methanol / water / dilute acetic acid).

As shown in Tables 1A and 1B below, the plasma concentration (ng / ml) of each Formula II compound for each test compound is determined by LC / MS / MS, and is measured at 0.5 and 1 hour, or alternatively 1 and Sum of concentrations at 3 hour sample time point.

In Vivo Exposure Analysis compound Rat exposure ((1R, 4S, 5S, 6S) -4- (2'S-aminopropionyl) amino-2,2-dioxo-2λ ⁶ -thia-bicyclo [3.1.0] hexane-4,6-dica Carboxylic acid hydrochloride ng / ml) Compound of Example 1 2251 ng / ml (after oral administration of 10 mg / kg) Example 1 Non-Prodrug Forms of Compounds 1581 ng / ml (after 5 mg / kg oral administration) 3981 ng / ml (after 10 mg / kg oral administration)

In Vivo Exposure Analysis compound Rat exposure ((1R, 2S, 4R, 5R, 6R) -2- (2'S-aminopropionyl) amino-4-fluorobicyclo [3.1.0] hexane-2,6-dicarboxylic acid hydrochloride ng / ml) Compound of Example 14 5271 ng / ml (after 5 mg / kg oral administration) Example 14 Non-Prodrug Forms of Compounds 1162 ng / ml (after oral 5 mg / kg) 1342 ng / ml (after oral 10 mg / kg)

As shown in Tables 1A and 1B, when administered orally to rats, the compounds of the present invention showed plasma concentrations comparable to the parent compound. This demonstrates that the compounds of the present invention are converted to the parent compound (compound of formula II) in vivo.

Compounds of the invention are preferably formulated prior to administration. Accordingly, another aspect of the present invention relates to a pharmaceutical formulation comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient. Pharmaceutical formulations may be prepared by methods known by those skilled in the art. In preparing the compositions of the present invention, the active ingredient will usually be mixed with the carrier, diluted by the carrier, or included in the carrier, and may be in the form of a capsule, sachet, paper or other container. When the carrier is used as a diluent, it may be a solid, semi-solid or liquid substance which acts as a vehicle, excipient or medium for the active ingredient. The compositions are tablets, pills, powders, lozenges, cachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, ointments, soft and hard gelatin containing up to 10% by weight of active compound. Capsules, suppositories, sterile injectable solutions, and sterile packaged powders.

Some examples of suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starch, rubber, acacia, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pi Ralidone, cellulose, water syrup, methyl cellulose, methyl and propyl hydroxybenzoate, talc, magnesium stearate, and mineral oils. In addition, the formulations may further comprise lubricants, wetting agents, emulsifiers and suspending agents, preservatives, sweeteners, or flavoring agents. The compositions of the present invention can be formulated to provide fast, sustained or delayed release of the active ingredient after administration to a patient by using procedures known in the art.

The composition is preferably formulated in unit dosage form, each dosage containing from about 5 mg to about 500 mg, preferably from about 25 mg to about 300 mg of the active ingredient. As used herein, the term "active ingredient" refers to a compound within the scope of the compound of formula (I).

The term “unit dosage form” refers to physically discrete units suitable for single administration to human subjects and other mammals, each unit formulating a predetermined amount of the active ingredient in a suitable pharmaceutical carrier, diluent to achieve the desired therapeutic effect. Or in combination with excipients.

Claims (27)

A compound of formula (I) or a pharmaceutically acceptable salt thereof. <Formula I> (Wherein A is H- (Q) _p- ; Each Q is independently selected from an amino acyl group; p is an integer from 1 to 10; X is O, S, SO, SO ₂ or CR ³ R ⁴ ; R ³ is fluoro, X'OR ⁵ , SO ₃ H, tetrazol-5-yl, CN, PO ₃ R ⁶ ₂ , hydroxy, NO ₂ , N ₃ , (CH ₂ ) _m COOR ^5a , (CH ₂ ) _m PO ₃ R ^6a ₂ , NHCONHR ^5b or NHSO ₂ R ^5c , R ⁴ is hydrogen; R ³ and R ⁴ each represent fluoro; R ³ and R ⁴ together represent = O, = NOR ⁷ , = CR ⁸ R ⁹ , = CHCOOR ^5b , = CHPO ₃ R ^6a ₂ or = CHCN; Or one of R ³ or R ⁴ represents amino and the other represents carboxyl; X 'represents a bond, CH ₂ or CO; m is an integer from 1 to 3; R ⁵ , R ^5a , R ^5b , R ^5c , R ⁷ , R ⁸ and R ⁹ are each independently a hydrogen atom; (1-6C) alkyl groups which may be optionally substituted; (2-6C) alkenyl groups which may be optionally substituted; Optionally substituted (2-6C) alkynyl groups; Aromatic groups which may be optionally substituted; Heteroaromatic groups which may be optionally substituted; Non-aromatic carbocyclic groups; Non-aromatic heterocyclic groups; Non-aromatic monocyclic carbocyclic groups fused with one or two monocyclic aromatic or heteroaromatic groups; Or a non-aromatic monocyclic heterocyclic group fused with one or two monocyclic aromatic or heteroaromatic groups; R ⁶ and R ^6a independently represent a hydrogen or (1-6C) alkyl group; R ¹⁰ is hydrogen or fluoro; R ¹¹ is hydrogen, fluoro or hydroxy). The compound or salt of claim 1, wherein X is CR ³ R ⁴ , wherein R ³ is fluoro and R ⁴ is hydrogen, p is 1 and Q is L-alanyl , Or pharmaceutically acceptable salts thereof. The method according to claim 1 or 2, A is H- (Q) _p- ; Each Q is independently selected from an amino acyl group; p is an integer from 1 to 3; X is O, S, SO, SO ₂ or CR ³ R ⁴ ; R ³ is fluoro or hydroxy and R ⁴ is hydrogen; Or R ³ and R ⁴ together represent = O; R ¹⁰ is hydrogen or fluoro; Or a compound or salt wherein R ¹¹ is hydrogen, fluoro or hydroxy. The compound or salt according to any one of claims 1 to 3, wherein Q is amino acyl derived from a natural amino acid. The compound or salt according to any one of claims 1 to 4, wherein X is SO ₂ . The compound or salt according to any one of claims 1 to 4, wherein X is CR ³ R ⁴ , wherein R ³ is fluoro and R ⁴ is hydrogen. The compound or salt according to any one of claims 1 to 4, wherein X is CR ³ R ⁴ , wherein R ³ is hydroxy and R ⁴ is hydrogen. The compound of claim 1, further comprising: an acid-addition salt prepared from an acid that provides a pharmaceutically acceptable anion; Base-addition salts prepared with bases that provide pharmaceutically acceptable anions for compounds containing acidic moieties; Or a zwitterionic compound containing an oppositely charged group. The method of claim 1, A is H- (Q) _p- ; Q is L-alanyl; p is 1; X is SO ₂ or CR ³ R ⁴ ; R ³ is fluoro and R ⁴ is hydrogen; R ¹⁰ is hydrogen; The R ¹¹ is hydrogen, or its hydrochloride salt, tosylate salt, mesylate salt, esil acrylate salt, besylate salt or a monosodium salt. A compound according to claim 9, wherein (1R, 4S, 5S, 6S) -4- (2'S-aminopropionyl) amino] -2,2-dioxo-2λ ⁶ -thia-bicyclo [3.1.0] hexane-4 , 6-dicarboxylic acid hydrochloride or (1R, 4S, 5S, 6S) -4- (2'S-2'-aminopropionyl) amino-2,2-dioxo-2λ ⁶ -thia-bicyclo [3.1 .0] pharmaceutically acceptable salts which are hexane-4,6-dicarboxylic acid tosylate. The compound of claim 1, wherein (1R, 4S, 5S, 6S) -4- (2'S-4'-methylthio-2'-aminobutanyl) amino-2,2-dioxo-2λ ⁶ -thia-bicyclo [3.1.0] A compound which is hexane-4,6-dicarboxylic acid or a pharmaceutically acceptable salt thereof. 12. The compound of claim 11, wherein (1R, 4S, 5S, 6S) -4- (2'S-4'-methylthio-2'-aminobutanyl) amino-2,2-dioxo-2λ ⁶ -thia-bicyclo [3.1.0] Hexane-4,6-dicarboxylic acid monohydrate. The method of claim 1, wherein 1S, 2S, 4S, 5R, 6R-2- (2'S-aminopropionyl) amino-4-hydroxy-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid hydro Pharmaceutically acceptable salts which are chlorides. The method of claim 1, A is H- (Q) _p- ; Q is L-alanyl; p is 1; X is CR ³ R ⁴ ; R ³ is fluoro and R ⁴ is hydrogen; R ¹⁰ is hydrogen; The R ¹¹ is hydrogen, or a pharmaceutically acceptable salt thereof. The method of claim 14, a) 1R, 2S, 4R, 5R, 6R-2- (2'S-2'-aminopropionyl) amino-4-fluorobicyclo [3.1.0] hexane-2,6-dicarboxylic acid hydrochloride; b) 1R, 2S, 4R, 5R, 6R-2- (2'S-2'-aminopropionyl) amino-4-fluorobicyclo [3.1.0] hexane-2,6-dicarboxylic acid mesylate; c) 1R, 2S, 4R, 5R, 6R-2- (2'S-2'-aminopropionyl) amino-4-fluorobicyclo [3.1.0] hexane-2,6-dicarboxylic acid esylate; d) 1R, 2S, 4R, 5R, 6R-2- (2'S-2'-aminopropionyl) amino-4-fluorobicyclo [3.1.0] hexane-2,6-dicarboxylic acid besylate; e) 1R, 2S, 4R, 5R, 6R-2- (2'S-2'-aminopropionyl) amino-4-fluorobicyclo [3.1.0] hexane-2,6-dicarboxylic acid tosylate; f) 1R, 2S, 4R, 5R, 6R-2- (2'S-2'-aminopropionyl) amino-4-fluorobicyclo [3.1.0] hexane-2,6-dicarboxylic acid; And g) 1R, 2S, 4R, 5R, 6R-2- (2'S-2'-aminopropionyl) amino-4-fluorobicyclo [3.1.0] hexane-2,6-dicarboxylic acid monosodium salt Compound or salt selected from the group consisting of. 16. The compound of claim 15, wherein (1R, 2S, 4R, 5R, 6R) -2- (2'S-2'-aminopropionyl) amino-4-fluoro-bicyclo [3.1.0] hexane-2,6- Pharmaceutically acceptable salt which is dicarboxylic acid mesylate. 18. The compound of claim 16, wherein (1R, 2S, 4R, 5R, 6R) -2- (2'S-2'-aminopropionyl) amino-4-fluoro-bicyclo [3.1.0] hexane-2,6- Pharmaceutically acceptable salts which are dicarboxylic acid mesylate monohydrate. Acylating the compound of formula (ii) with the corresponding amino acyl of formula III <Formula (ii)> <Formula III> Wherein Pg ^N is a nitrogen-protecting group; Then, in any of the above procedures, removing the protecting group if the functional group is protected using a protecting group; Then, in any of the above procedures, i) if a pharmaceutically acceptable salt of the compound of formula I is required, reacting the compound of formula I in base form with an acid to obtain a pharmaceutically acceptable counter ion; ii) for compounds of formula (I) having acidic moieties, the compounds of formula (I) in acid form are reacted with a base to obtain a pharmaceutically acceptable cation; Or iii) for the zwitterionic compound of formula I, neutralizing the compound of formula I in acid-addition salt form or base-addition salt form; Or any other conventional procedure A process for preparing a compound of formula (I) according to claim 1 or a pharmaceutically acceptable salt thereof. A method of influencing a cAMP-associated metabotropic glutamate receptor in a patient comprising administering a pharmaceutically effective amount of the compound of claim 1 to a patient in need of neurotransmission of modulated excitatory amino acids. A method of administering an effective amount of a compound of Formula II comprising administering a pharmaceutically effective amount of the compound of claim 1 to a patient in need of neurotransmission of modulated excitatory amino acids. <Formula II> Wherein X and R ¹⁰ are as defined in claim 1. A method of treating a neurological disorder in a patient comprising administering a pharmaceutically effective amount of the compound of claim 1 to a patient in need thereof. 22. The method of claim 21, wherein said neurological disorder is a brain defect associated with cardiovascular bypass surgery and heart transplantation; Cerebral ischemia; Spinal cord trauma; Head trauma; Alzheimer's Disease; Huntington's Chorea; Amyotrophic lateral sclerosis; AIDS-induced dementia; Perinatal hypoxia; Hypoglycemic nerve injury; Vision impairment and retinopathy; Cognitive impairment; idiopathic and drug-induced Parkinson's Disease; crick; migraine; Urinary incontinence; Drug resistance, drug withdrawal symptoms, signs of drug discontinuation and drug intoxication; No smoking signs; throw up; Brain edema; Chronic pain; Sleep disorders; convulsion; Tourette's syndrome; Attention deficit disorder; And impaired exercise impaired method. The method of claim 22, wherein the neurological disorder is selected from the group consisting of drug resistance, drug withdrawal symptoms, drug discontinuation signs, and drug addiction; Or signs of smoking cessation. A method of treating a psychiatric disorder in a patient comprising administering a pharmaceutically effective amount of the compound of claim 1 to a patient in need thereof. The method of claim 24, wherein the psychiatric disorder is schizophrenia, anxiety disorder and anxiety related disorder, depression, bipolar disorder, psychosis and obsessive compulsive disorder. The method of claim 25, wherein the psychiatric disorder is anxiety disorder and anxiety related disorder. A pharmaceutical formulation comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof in combination with a pharmaceutically acceptable carrier, diluent or excipient.